HU195819B - Process for producing 2-furyl-butyrolactone derivatives and pharmaceutics comprising these compounds - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to novel compounds of formula (IVa) wherein RJ is C1-C4 alkyl and n is 1-3 and adduct with acid anhydride or acid amide. The compounds of the present invention can be used as an active ingredient of anticancer drugs due to their effect on imnutine. 195819 -1-

Description

This invention relates to novel 2-furylbutyrolactone derivatives and to pharmaceutical compositions containing them as active ingredients. The pharmaceutical compositions of the present invention have an immunomodulatory effect.

Diseases and disorders of the immune system are a major problem in modern medical practice as they cause serious illnesses in humans and animals. Immune system disorders, if not all, are thought to be associated with some cancers.

The 2-furyl-butyrolactone-8 derivatives obtained by the process of the present invention, which are:

2,5-Dihydrofuran derivatives and 2,3-dihydro-butyrolactone derivatives, in particular ascorbic acid, are capable of modulating immune system activity.

The cyclic acetals described in U.S. Patent No. 4,287,205, some of which were prepared in a manner similar to the process of the present invention, have a hypotensive and analgesic effect. The compound described in Example 3 of the above-mentioned patent was prepared by reacting 2-methyl-2,5-dimethyl-2,5-dihydrofuran with L-ascorbic acid. This step is similar to the preferred first step of the process of the invention, but the reaction product is structurally different from the compounds of the present invention which are prepared or isolated in the absence or in the absence of the cyclic acetals described in the above patent.

More particularly, the process of the invention is a compound of formula IVa

R 'is C 1 -C 4 alkyl and n is 1-3 and is intended for the preparation of their adducts with anhydride or eamamide.

The compounds of formula (IVa) are prepared so that preferably the same amount of the compounds of formula (II)

R ¹ and R ² are independently hydrogen or C1-4 alkilceoport, with the proviso that at least one is hydrogen, and

R ³ and R ⁴ are independently C 1 -C 4 alkyl with a compound of formula III wherein

^R7 is -CH (OH) - (-CON) nOH, where n is 1-3 is reacted, and the product is optionally treated with an acid anhydride or acid amide;

separating the product obtained as an acid anhydride or acid amide adduct from the reaction mixture;

and, if desired, dissociating the product to a compound of formula IVa and the corresponding acetic anhydride or acid amide.

The purification of the pure product can be facilitated by treatment or purification of the crude product in a manner known per se, for example by charcoal filtration, ion exchange or gel chromatography.

The group represented by R 'in formula (III) contains a hydroxy group at the α, β or i position and thus R ⁷ can form a hainetal ring with the carbon at the 3-position of butyrolactone, also protonating the carbonyl at the 3-position.

The resulting hemiketals of formula (IVa) are usually partially rearranged, equilibrating the tautomeric forms of formulas (IVa) and (IVb) in aqueous or highly polar media.

Preference is given to compounds of formula II in which

R * is hydrogen,

R ² is C 1 -C 3 alkyl, and

R ³ and h ⁴ are independently C 1-3 alkyl.

Even more preferred are compounds of formula II wherein R ^{2 is} methyl.

Most preferred is 2-methyl-2,5-dimethoxy-2,5-dihydrofuran.

The preferred compounds (111) of the formula are those wherein ^R7 is -CH (OH) wherein - (-CON) nOH is a group of formula wherein n is the first

Most preferably, the compound of formula (III) is L-ascorbic acid.

C 1 -C 4 alkyl groups as described herein may be straight or branched chain, preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, more preferred 1-3 heteroatom groups such as methyl, ethyl, propyl and isopropyl, and most preferably methyl.

The molar ratio of the compounds of formula II to the compounds of formula III is preferably from 2: 1 to 1: 2, more preferably 1; Value around 1.

The acid anhydride or acid amides mentioned herein is preferably succinic anhydride or amide, more preferably succinic anhydride, succinimide or N-methyl succinimide, preferably at least 0.5 mole per mole of the compound of formula (I) more preferably about 0.5 mol.

The compounds of the invention may be formulated in a pharmaceutical composition suitable for human or veterinary use in admixture with carriers or diluents commonly used in the manufacture of a medicament.

The pharmaceutical compositions of the present invention are immuno-modulating and cytotoxic, having very low acute toxicity values.

It is believed that the cytotoxic effect of the above pharmaceutical compositions is due either to the immunomodulatory effect of the active compounds of formula IVa, or to the active ingredients alone having a cytotoxic effect or to a combination of the two.

Due to their cytotoxic activity, the pharmaceutical compositions of the present invention are useful in the field of cancer. The pharmaceutical compositions may also be used in immunotherapy to enhance the activity of the immune system, for example in the case of AIDS (Acquired Immune Deficiency Syndrome).

In higher animal organisms, the immune system is one of the primary defense mechanisms against disease germs and other foreign proteins. The immune response is mediated by specific antibody proteins that react with specific antigens. Antigens are substances of relatively high molecular weight, usually proteins, which are foreign to the body. In most cases, they are localized on the outer surface of the cells. Antigens are found, inter alia, in pollen particles, transplanted tissues, synthetic materials, animal parasites, viruses, and bacteria.

In the human body, many potential antigens never cross the two lines of defense in the body, so they never activate the immune system. The anterior line of defense consists of skin, mucous membranes, carna, and gastric acid, and the second line is specialized white blood cells (granulocytes and monocytes) and macrophages, which, by phagocytosis, kill palogens and other potential antigens, that is, absorb. The above white blood cells and macrophages are called phagocytes. When pathogens or other foreign substances cross the first two lines of defense in the body, the immune system is activated.

There are basically two immune defense systems, the humoral and the cellular, both reacting with antigens. Humoral immunity is due to circulating antibodies in the gamma-globulin fraction of plasma proteins. When plasma is centrifuged at high speed, its protein components are fractionated according to their molecular weights. Antibodies are generally found in the fraction containing the 156,000 average relative molecular weight components. This fraction is called the gamma globulin fraction. Humoral immunity is the main defense against bacterial infections.

Cellular immunity is due in part to a lymphocyte product called lymphocytosis. This type of immunity is responsible for delayed allergic reactions, prevention of foreign tissue implantation and protection against tumor cells. This is the main defense against infections caused by viruses, fungi and certain bacteria such as TB.

Specialized white blood cells, called lymphocytes, are responsible for both humoral and cellular immunity. The lymphocyte precursor cells are produced in the adult human bone marrow and migrate from there to the various organs, or into the ovum of the developing fetus, then into the fetus and then into the various organs. In the human body, some of these precursor cells migrate to the thymus, a glandular lobe located beneath the sternum in the upper chest, where it is transformed into T-lymphitis. T-lymphocytes play a role in cellular immunity. In the human body, the remainder of the precursor cells migrate to the spleen, where it is converted to B-lymphitis, which is involved in humoral immunity. T and B lymphocytes are structurally indistinguishable, although they function differently and are distinguished by various chemical means. Mature lymphocytes circulate in the blood and are also found in lymph nodes, spleen, and thymus.

Humoral immunity is mediated by B lymphocytes, which have receptors on their cell surface for each antigen. B lymphocytes are highly specific, each type of B lymphocyte reacting with only one antigen. For example, when bacteria or viruses attack the body, 3-lymphocytes on the surface of the bacterium or virus react and bind to the antigen of the year, causing the lymphocyte to divide. Girl cells transform into special cells called plasma cells. These cells produce large amounts of antibodies and are released into the general circulation system. Antibodies are specific for the antigen that caused their production and react only with these antigens. Antibodies known as agglutinants agglutinate or cause adhesion of antigen-containing material. This prevents the substances from being dispersed in the tissues and giving the phagocytes a chance to be absorbed or the lymph nodes to filter out the substance that is attacking the body. The action of other antibodies is based on piercing the bacterial cell wall, thereby killing the bacterium. The above antibodies are called lysines-37. The antibodies known as antitoxins bind to the toxins produced by the bacteria, thereby neutralizing them.

When the pathogen attacks the body and the immune response is triggered, the antibodies develop within a few hours. This initial reaction is called the primary response or primary immunization. However, pathogens also divide over time and occasionally produce toxins, for the above reasons, a variety of patientbone symptoms can be observed. It may take days or weeks for the amount of antibodies produced to kill all of the pathogen, but if the pathogens disappear from the body, the patient's symptoms also disappear. The lymphocytes, plasma cells and antibodies circulate there and remain in the bloodstream, and when the same pathogen mogoze enters the body, the lymphocytes are immediately activated and the antibody is produced. The response of sensitized lymphocytes is called the secondary response. The secondary response results in higher antibody production than the primary response. So many antibodies are produced in such a short time that microbes cannot divide and cause disease. Humoral immunity is characterized by immediate hypersensitivity, due to the ability of an organism that has previously been exposed to an infection to respond to an antigen within minutes, such as hay fever. Another example of immediate hypersensitivity is anaphylactic Bokk, arai, an extreme allergic reaction that is rarely seen when an individual is exposed to an antigen to which he or she has been sensitive. This antigen-induced humoral immune response can sometimes be fatal.

Humoral immunity can be induced both naturally and artificially. In the case of active natural immunity, the individual's lymphocytes remain in circulation and, following infection, initiate antibody production. This active natural immunity can last for years, sometimes working throughout life. The baby receives the antibody from breast milk during the first few days after birth, providing immunity during the first year of life. This immunity is called passive natural immunity because the antibody is not actually produced in the baby's body. Active artificial immunity can be induced by injecting dead or weakened microbes into the body. These microbes no longer cause sickle-cell glands like their more virulent forms, but their surface antigens can still trigger lymphocyte antibody production. Later, when a virulent microbe enters the body, it is already sensitive to Ιβεζ and responds immediately to the infection with strong antibody production. The effect of active artificial immunization lasts for years and can be sustained with booster vaccinations. There are also examples of passive artificial immunity, which provides protection for about a month. The above transient immunity is developed by injecting an antibody produced in another animal or human body into the body to be immunized. This method is usually used only in crisis or epidemic situations. As the lymphocytes are not involved in the immunization process in this case, they do not produce antibodies or 'remember the antigen', therefore only the temporary immunity can be achieved by the above method.

In contrast to humoral immunity, no cellular antibodies are detected in cellular immunity. T-lymphocytes, which mediate this type of immunity, are activated when they encounter an antigen on the surface of a cell from another organism, such as a transplanted tissue, tumor, or virus. Like B-lymphocytes, T-lymphocytes are specific and each lymphocyte responds to only one type of antigen. Lymphocytes grow, divide, and produce lymphokines, which are involved in attacking alien adrenal glands and stimulate the phagocyte activity of macrophages. Although immunological memory exists as it does with humoral immunity, the response is much slower. Many times it takes 10 to 12 hours for the response to develop in the previously sensitized organism, so cellular immunity is considered to be delayed hypersensitivity. Examples of a cellular immune response include an allergic reaction to poison ivy, a red spot on a positive tuberculin skin test, or rejection of transplanted tissues.

Azers modulating the immune system either activate or inhibit the lymphocyte proliferation process. Normal lymphocyte proliferation is the result of various interactions between antigens, macrophages, T- and B-lymphocytes, and certain chemicals. For example, the presence of a particular antigen certainly activates T or B lymphocytes. In addition, some B lymphocytes can be activated by active T lymphocytes, while others are independent of T lymphocytes and can only be activated by antigens. Activated T lymphocytes may induce macrophage production of a molecule called interleukin 1 (IL-1), which in turn activates both T and B lymphocytes. Activated T lymphocytes may also produce a molecule known as interleukin 2 (IL-2), which induces further T lymphocyte activation. Chemicals known as nitrites can trigger DNA synthesis and mitosis, which are signs of T and O lymphocyte activation. Some mitogens are only one type of lymphocyte

195619 six while the meme practiced fish on a wide variety of lymphocytes. Depending on their concentration, various immune modulating agents of various species interfere with the complex interactions between the immune system's ÖBBβ feeders. As shown below, the compounds and pharmaceutical compositions of the present invention exhibit dose-dependent stimulatory or esuppressive effects on both T and B lymphocytes.

Although the immune system is the most important defense system against disease-causing substances, it cannot distinguish between foreign substances that are beneficial and harmful to the body, and destroys both. In many cases, it would be helpful to have an agent that can regulate the immune system without damaging the body. The compounds of this invention have such modulating and regulatory activity and are therefore useful in the treatment of various immunological disorders.

The immune system is also related to aging in some respects and may also play a role in protecting against cancer. The immune system is necessary for the recognition and subsequent destruction of changing or aging cells, such as aging red blood cells, and is therefore essential for the normal functioning of the body. One theory of cancer development is that cells become quite abnormal in the body, but these altered cells are recognized and destroyed by the body as 'foreign cells'. The effect of some carcinogens is probably based on suppressing this immune response and not causing an abnormal change in the cell itself. As a result of suppression of the immune system, the body can no longer destroy naturally transformed cells, which can lead to cancerous growth. Immune-stimulating agents may be used to treat the cancerous diseases formed as described above. The compounds or pharmaceutical compositions of the present invention, when used in combination with chemotherapeutic or chemo-immunotherapeutic agents, may affect the growth of cancerous tumors.

Some of the methods used to treat the cancer, such as surgery, chemotherapy or radiation, can suppress or cause severe changes in the immune system. Immune stimulating agents, such as the compounds or pharmaceutical compositions of the present invention, can be very useful in preventing and / or combating various infections due to the dysfunction of the immune system. Among the immunosuppressive agents useful in the above case, cyclosporin A, levamisole, and iso6 prinosine (inosiplex) are currently being studied. The compounds and pharmaceutical compositions of the present invention are novel therapeutic agents that can be used in the above case and in other cases involving the immune system.

In some cases, the body's immune response can cause more damage or discomfort than ingested microbes or foreign matter, such as allergic reactions. In these cases, suppression of the immune system would be desirable.

Immunomechanisms may be sensitized to some part of your body, in which case that part may be disturbed or even damaged by the immune system. The body's ability to differentiate between "its" and "its own" is impaired and begins to destroy itself. Some examples of the above human autoimmune systemic disorders are rheumatoid arthritis, types of haemolytic anemia, rheumatic fever, thyroiditis, ulcerative colitis, myasthenia gravis, glomerulonephritis (one kidney disease), allergic encephalomyelitis and hepatitis externa, likely Multiple Sclerosis. Autoimmunity develops in some cases as a result of some trauma in areas that are not normally exposed to lymphocytes, such as nerve tissue or the lens of the eye. When the tissues of such areas come into contact with lymphocytes, their surface proteins may act as antigens and trigger antibody formation and cellular immune response, which then begins to destroy the tissues. Other autoimmune diseases develop after the body has been exposed to an antigen that is antigenically similar, that is, it cross-reacts with the body's own tissue. An example of this disease is rheumatic fever when the antigen of the streptococcal bacterium, which triggers rheumatic fever, cross-reacts with parts of the human heart tissue. Antibodies cannot differentiate between bacterial antigens and myocardial antigens, and cells containing 'antigen' are destroyed equally. Inhibition of the immune system can reduce or eliminate these diseases.

Circulating antibodies and cellular immune responses are also implicated in the rejection of organ transplanted tissues. Unless the donor is an identical twin sister to the recipient, or the donor itself, the recipient's lymphocytes "will not immediately recognize the transplanted tissue or organ and act immediately to destroy it. Exceptions to transplantation in non-vascularized areas (exceptional sites), such as cornea and the eye, are the only exceptions, since lymphocytes do not circulate and are not sensitized

-5n give an immediate immune response. There is currently no known method of suppressing the immune response to prevent rejection of the transplanted organ or tissue without seriously compromising the patient in any other way. High doses of antibiotics should be given to the patient at the same time as their own defense mechanism is suppressed against infections. The compounds and pharmaceutical compositions of this invention provide for tolerance to transplants through controlled modulation of the immune system.

Because of the vital role of the immune system in defense and in the normal functioning of the body, compounds and pharmaceutical compositions, such as the compounds and compositions of the present invention, which are capable of increasing or decreasing this role are very important. Particularly useful when the system malfunctions or destroys harmless foreign matter. They can be used to enhance the immune response against harmful antigens and thus to shorten the course of the disease.

The pharmaceutical compositions of the present invention affect mammalian lymphocytes in a way that strongly affects their reproductive capacity, which is measured by their ability to synthesize DNA. The above effect is bidirectional. The formulations have a higher concentration of immune suppressant and a lower concentration of the immune system.

Immune modulating activity was detected in vitro from human blood lymphocytes, in vivo and in vitro mouse spleen lymphocytes.

The pharmaceutical compositions of the present invention have been subjected to numerous immunoassays. In the assays, T and B lymphocytes were isolated from human blood in mouse spleen. Five mouse strains, BALB / C C57BL / 6, BDFi SJL / J and DBA / 2 were used as lymphocyte donors. The lymphocytes were then treated with a plant protein called mitectic activity called lectin.

Mitogens are substances that stimulate DNA synthesis and mitosis. In our study, phytohemagglutinin (PHA) isolated from red kidney bean and concavalin A (Con-A) isolated from bean (jack bean) were used as mitogenic material. Concavalin A binds to the Bpecellis receptor, a glycoprotein containing a mannosyl or glucosyl moiety, and stimulates each mouse T-cell for DNA synthesis, division and lymphokine release. In soluble Con-A, it is possible to distinguish between T and B cells in mice since, although T and B cells are capable of binding 10 µ molecules of Con-A per cell, only T cells are stimulated. when this lectin is present in soluble form. PHA only stimulates a subset of T and B cells (Ib cells). In mouse, PHA stimulates a subpopulation of T cells, but not B cells. It is likely to stimulate both T and B cells in humans. B-cell activation is likely to occur indirectly through the mediation of soluble mediators released from PHA-activated T cells.

The compounds of the present invention strongly influence the mitogenesis of lymphocytes by direct in vitro experiments with human lymphocytes at doses ranging from 1 ng to 10 ⁵ ng. Lower dose range 1-10- ⁵ ng exhibit stimulélást above the control level by stimulated by PHA or Con-A Mitr genesis. Higher, 10 * -10 ⁵ ng dose e. The mitogenetic activity of lymphocytes treated with PHA or Con-A mitogenic agents is strongly inhibited.

Compounds of the present invention were administered intravenously in different doses to C57B1 / 6 mice for 4-12 days, followed by treatment with spleen lymphocytes treated with PHA-cut Con-A, as in the in vitro assay. mg / kg and 400-800 mg / kg, respectively. There was a statistically significant difference between suppression at higher doses and stinulation at lower doses, p <0.05.

In other experiments, we investigated how the compounds of the present invention affect the antibody response to specific antigens, bovine serum albumin, and hippocampus hemocyanin. It has been found that, at higher doses, the compounds do not inhibit the secondary immune response to the antigen but, on the contrary, significantly enhance it. Doses that inhibit the cell-mediated response to autogens both in vitro and in vivo did not significantly reduce humoral immunity. These data demonstrate that specific antibody production can be regulated by immunostimulation.

In the experiments described below, there is a tendency for compounds to behave at higher and lower doses. However, lymphocytes stimulated with PHA or Con-A show differences in the magnitude of a given immune response. This may be due to the fact that PHA and Con-A stimulate different types of lymphocytes. Using different types of mice can give different results. These differences reflect genetic alterations in the response of lymphocytes to mitogens. Differences in in vivo and in vitro responses may be due to differences in in vivo metabolism and in human and mouse lymphocytes, respectively.

In all experiments, it was observed that control cultures not stimulated with mitogens or specific antigens, which are thus considered as dormant cells, were not affected by the compounds of the present invention. Significant atimulatory or inhibitory activity can be detected only in cells undergoing active DNA synthesis, i.e., in the division.

Immunomodulatory effects on the cytotoxic lymphocyte activity of sensitized lymphocytes from resting tumor bearing DBA / 2 mice were also investigated with the compounds of the present invention. During the experiment, DBA / 2 mice were developed for resting tumor status and then tested for effects of the compounds of the present invention on the resting tumor status. The above animal experiment can be used to model the suspected dormant tumor state that can be inferred from the clinical detection of recurrent human breast cancers and melanomas years after the apparent healing of primary tumors.

A. Method of the Lymphocyte-Bimulation Test}

The aim of the assay is to induce mitogenesis by treating lymphocytes in microcultures with one or more appropriate concentrations of polyclonal mitogen within 72 hours of treatment.

In a mouse model, the spleen of the various mouse strains was removed, the spleen cells were released and resuspended in RPMI-1640 tissue culture medium at a concentration of 1 x 10 8 cells / ml. The suspension is dispensed into wells of a 96-well plain tissue culture plate at 5.0 x 10® cells / well. Compounds of the present invention and mitogenic agents were assayed in 10 replicates. Cells are treated with PHA, Con-A, specific antigens, or buffer alone. In the in vitro assays, the compounds of the present invention are added to the cells at various concentrations, and only the cells are treated with buffer as a control. After addition of mitogens and / or test substance, the cells are incubated for 72 hours. Response to mitogens and / or test compound was determined with ¹⁴ C-labeled thymidine at 0.01 microCi / well on day four.

The labeled lymphocyte cells are harvested by suitable means and placed on a fibrous paper disk. Scintillation fluid mixtures were added to the tubes containing the disks and radioactivity was determined using an LKB liquid scintillation counter (Model 1216 / Rackbeta II). Data are expressed as the ratio of the number of minutes per minute (cpm) for the mitogen-stimulated group to the number of minutes per minute for the control (non-mitogen-treated) group and is called the Lymphocyte Stimulation Index (LSI).

The lymphocyte-stimulating activity of the compounds of the present invention can also be tested directly on untreated spleen cells from various strains of mice or on lirafocytes obtained from human plasma. In the above experiments, the compounds of the present invention are added directly to spleen lymphocytes in vitro, and the assay and evaluation of results are performed as described above. The assays can be performed by in vivo intraperitoneally treating the mice with the compounds of the invention at multiple dose levels, and then removing the spleen and collecting the spleen lymphocytes. The response of the resulting cells to polyclonal initogens or to specific antigens commonly used to immunize mice is then determined as described above. In this case, the test compounds are no longer added to the in vitro stimulation assay renderer.

B. Secondary Response Examination

The effect of the compounds of the present invention on the immune response to a specific antigen is tested by immunizing mice with specific antigens, such as bovine serum albumin (BSA) or hippopotamus hemocyanin (KLH). Following immunization, cell-mediated antibody response to the above antigens is determined by various methods.

1) One possible way of assaying lymphocyte stimulation is to add the specific antigen as a mitogen to various concentrations of the lymphocyte stimulation microculture described above. The only difference is that the incubator is fed for five days instead of three days for polyclonal mitogens.

2) Antibody response to soluble antigens such as BSA or KLH can also be determined by a micro-ELISA solid phase heterogeneous immunoassay. The assay may also be carried out by measuring the amount of oocyte-specific immunoglobulins (i.e., IgG or IgM).

Humor response (antibody) responses to BSA were determined using a solid phase heterogeneous sandwich E1, ISA microarray in a 96-well Immulon microtiter plate. The titer limit was the highest dilution of mouse serum with an optical density (O.D.) of 0.1, as measured by an MR-600 Dynatech micro-ELISA reader.

C. Method for measuring cytotoxic lymphocyte activity

Mixed lymphocytic tumor lymphocyte (MLTC) immunoassays used to determine lymphocyte cytotoxic activity (CTL) are used either in immunized (non-sensitized) mouse lymphocytes or in lymphocytes (TDS) from DBA / 2 mice carrying the resting tumor at different stages. The assay uses ^sl chromium labeled L5178Y target cells and effector cells (sensitized cells) from the above hot cells. The ratio of effector to target cells will vary depending on the different experimental conditions and may be performed in the presence or absence of irradiated stimulator L5178Y cells. Results are expressed as% lysis of target cells, calculated as the ratio of cpm obtained with treated ceoport to total (maximal) release, corrected for spontaneous release of Cr ⁾ . The assay can be performed as an 8 or 18 hour release assay.

Resting Tumor Status (TDS) was induced by subcutaneously injecting a large group of DBA / 2 mice with 1 ® 10 ® viable L 5178Y leukemia cells around the center of the abdomen. 10 days later the received - approx. 1 cm - tumor node surgically excised. If surgery is Bikeres, no more subcutaneous tumors develop at the implant site. 7 days after excision, the mice are infected with 50,000 live L5178Y leukemia cells, this dose is normally 100% likely to cause fatal ascites tumor within 14 days in normal DBA / 2 mice. Immunized mice at a given dose of L5178Y prevent rapid tumor growth and remain clinically asymptomatic for several weeks after challenge. These mice are considered to be animals with resting tumors.

D) Test method for anticancer activity

Mice belonging to the BDF strain are intraperitoneally given 10® L-1210 leukemia cells on day 0. Twenty-four hours later, the animals were divided into groups of 7 mice, and each group was treated with the compounds of the present invention in different doses for 9 days. The experiment was performed according to the National Cancer Institute screening protocol for screening for new anti-cancer agents, and the T / C ratio, i.e., the mean survival time of the treated group and the mean survival time of the control group was calculated. A compound having a T / C ratio of> 1.25 (or 125%) is considered to have significant anticancer activity.

The preparation of the compounds is described in more detail in Examples 1-6 below. Examples are provided by way of example and without limitation. The other examples illustrate the biological activity of the compounds.

Example 1 2 g of 2-methyl-2,5-dimethoxy-2,5-dihydrofuran, obtained by N. Clauson-Kass and F. Lindborg in Acta Chem. Scand. 1, 619 (1947) was added to a solution of 75 g of L-ascorbic acid in 750 ml of water with vigorous stirring at room temperature. The furan derivative was used in an excess of 50%. After 15-20 minutes, a homogeneous solution was obtained. The reaction was monitored by high performance liquid chromatography with a UV detector. The 3.6 peak of L-ascorbic acid, measured at 254 nm in original 20% aqueous methanol solution, disappears and another peak of 5.9-6.0 Rf appears at 64 atmospheric pressure. At the same time, it is observed that the solution does not consume more iodine, which leads to a complete conversion of L-ascorbic acid.

The aqueous solution was allowed to stand at 20 ° C overnight and then freeze-dried. 2- (5-Methyl-2-furyl) -3-oxo-L-gulonolactone-3,6-mercuric ketal (4) is obtained in the form of a pale yellow foam.

IR (KBr) 3600-3100 (s, broad OH), 1785 (s,

Lakion, CO), 1700 (m, CO), 1610 and 1540 (W, furan

C = C),

1 H-NMR (acetone-d 6) δ

6.57 (d, 1H),

6.20 (m, 1H),

4.87 (s, 1H),

4.63 (m, 1H),

4.25 (ra, 2H),

2.22 (s, 3H);

^{, 3} C NMR (DMSO-d 6)?

206.8; 173.0; 151.7; 148.5; 109.2; 107.0; 106.0; 87.6; 77, S;

74.7; 73.7; 13.2 ppm.

Mass spectrum (ra / e): 269.1; 238.1;

155.0; 138.0; 109.0 (base peak); 85.0.

64.0 g (0.25 mole) of the above crude product (m.p. 55 ° C) and 25.0 g (0.25 mole) of succinic anhydride are weighed into a 1 L round bottom flask equipped with a reflux condenser and nitrogen inlet. 200 ml of HPLC grade ethyl acetate were added and the reaction mixture was refluxed for 4 hours. The homogeneous solution was cooled to room temperature and placed in an ice bath. The white precipitate was filtered off with suction and washed with a few ml of cold ethyl acetate.

-817

29.9 g (46.6%) of crude product are obtained. The filtrate was concentrated to half volume and cooled in an ice bath. The precipitate formed during the second cryopolyification is also filtered.

13.5 g of a solid are obtained which also contains a small amount of unconverted boronic acetic anhydride.

The product was recrystallized from ethyl acetate-chloroform (20:80 by volume) to give a total of 22.5 g (35.2%) of pure product as long needle crystals, m.p. 134-134.5 ° C.

Elemental analysis results based on the CjelinOn formula:

Calculated: C, 50.99; H, 4.61; 0, 44.41; Found: C, 50.67; H, 4.52; 0, 44.58.

X-ray diffraction analysis of the compound suggests the structure of formula (1) and indicates the presence of a 2: 1 2-furyl-butyrolactone-boroethane-sulfuric acid anhydride in the elemental cell [J. Org. Chem. 49, 5064 (1984)].

The compound prepared above was dissolved in 2.85 mmol of 0.85% hydrochloric acid buffered bicarbonate to give succinic anhydride.

Example 2

186.0 g of 2-methyl-2,5-dimethoxy-2,5-dihydrofurene are added to a solution of 150 g of L-ascorbic acid in 1.5 L of water at room temperature with vigorous mechanical stirring. The furan derivative is used in an excess of 50%.

After 15-20 minutes, a homogeneous solution was obtained. The reaction was monitored by high performance liquid chromatography with a UV detector. The L-ascorbic acid in its original 20% aqueous methanol peak at 1.6 Rf at 254 nm disappears and a new cbubb of 5.9 to 6.0 Rr appears at 64 atmospheric pressure.

At the same time, it is observed that the solution does not consume more iodine, indicating complete disappearance of L-ascorbic acid.

The aqueous solution was allowed to stand at 20 ° C overnight and then frozen. The product (4) is obtained in the form of a pale yellow foam having IR, ⁿ C-NMR and ¹ H-NMR spectra and mass spectra similar to those of the corresponding product of Example 1.

119.65 g (100% purity 0.467 mol) of the crude product, m.p. 55 ° C, was dissolved in 216 ml of HPLC grade ethyl acetate. Succinimide (23.3 g, 0.235 mol) was added and the mixture was stirred under nitrogen at high pressure. After stirring for a few minutes, a white precipitate formed. The reaction mixture was then heated in an oil bath for 30 minutes, during which time the white precipitate dissolved. The heating was stopped and the solution was allowed to cool to room temperature and then cooled in an ice bath for 2 hours. The resulting precipitate was filtered off with suction, washed with cold chloroform (150 mL) and dried in vacuo.

67.4 g (56.3%) of crude product are obtained.

The product was recrystallized from 60:40 ethyl acetate / chloroform to give 52.2 g (43.6%) of pure product as long needle crystals, m.p. 132-133 ° C.

Elemental results based on the formula C2 «H2eNOi»:

Found: C, 51.07; H, 4.78; 0, 41.86; N, 2.92.

Found: C, 51.175; H, 4.935; 0, 41.815.

N, 2.21.

X-ray diffraction analysis of the compound suggests the structure as shown in Formula 2 and indicates the presence of a 2: 1 2-furyl-butyrolactone-Gsuccinimide molecular complex in the elemental cell.

The compound thus prepared was dissolved in 2.38 mmol of bicarbonate-buffered 0.85% hydrochloric acid to give 4 and succinimide.

Example 3 To a solution of g of L-ascorbic acid in 750 ml of water was added 93 g of 2-methyl-2,5-dimethoxy-2,5-dihydrofurene at room temperature with vigorous stirring. The furan derivative is used in a 50% excess. A homogeneous solution is obtained within 15-20 minutes. The reaction was monitored by high performance liquid chromatography with a UV detector. The original peak of L-ascorbic acid, 1.6 Rf at 254 nm in 20% aqueous methanol, disappears and a new peak of '5.9-6.0 Rf appears at 64 atmospheric pressure. At the same time, it is observed that the solution does not consume more iodine, indicating complete conversion of L-ascorbic acid.

The aqueous solution was allowed to stand overnight at 20 ° C and then freeze-dried. Compound (4) is obtained in the form of a pale yellow foam having IR, ¹³ C-NMR and 1 H-NMR spectra and mass spectra similar to that of Example 1 (4).

Subsequently, 10.24 g (0.04 mol) of crude product (m.p. 55 'C) and 2.5 g (0.022 mol) of N-methyl-esuccinimide are weighed into a 1 L round bottom flask equipped with a reflux condenser and a nitrogen inlet tube. After adding 20 ml of HPLC grade ethyl acetate, the reaction mixture was refluxed for 4 hours. The homogeneous solution was cooled to room temperature and then placed in an ice water bath.

The white precipitate is filtered off with suction and washed with a few ml of cold ethyl acetate.

5.20 g (50.8%) of crude product are obtained. The filtrate was concentrated to half volume and cooled in an ice-water bath.

-919

Recrystallization from 1: 1 ethyl acetate-chloroform gave 3.21 g (31.3%) of pure product as long white needle crystals, mp 105-106.5 ° C.

Elemental results for CnHjjNOio:

Calculated: C, 51.84; 11, 5.00; N, 2.24; 0, 40.92;

Found: C, 51.98; H, 5.12; N, 2.08.

0% = 40.63.

X-ray diffraction analysis of the compound suggests structure 3 and indicates the presence of a 2: 1 2-furyl-butyrolactone-N-methyl-succinimide molecular complex in the elemental cell.

The compound thus prepared was dissolved in 2.38 mmol of hydrogen carbonate-buffered 0.85% hydrochloric acid to give 4 and N-methylsuccinimide.

Example 4

ED The ED50 value of the compound prepared in Example 1 was determined in a single dose using the method of Spearman and Karber (Basic Exercises in Immunochemintry, 1979) of 5 71) 1/ (mouse strain). individual doses were administered, the number of surviving animals was determined after 3 days, and the LD50 was calculated using the following formula:

hangs LD 50 - hangs (highest dose tested) + + (hangs D) x (1/2 - R / N), where D is the ratio of doses, R is the number of animals killed, N is the number of animals tested.

The results are shown in Table 1.

Table 1

Single i.p., LD50 value of Example 1 compound with mouse strain C57BL / 6

Dose of compound

Number of dead animals on day 3

272 mg / 17 g mouse (16,000 mg / kg) 10

136 mg / 17 g mouse (8,000 mg / kg) 10 mg / 17 g mouse (4,000 mg / kg) 7 mg / 17 g mouse (2,000 mg / kg) 0 mg / 17 g mouse (1,000 mg / kg) 0

LD 50 = 59.2 mg / 17 g mouse = 3.480 mg / kg

The purified compound of Example 1 had a LD50 of one i.p. injection in C57BL / 6 mice was greater than 3 g / kg.

Example 5

The LD »value of the compound prepared in Example 1 was also determined after single oral administration in CD-I mice and CD rats (Charles River U.K. Ltd.). The principle of the assay is to dissolve the test compound in 0.0238 mol / L bicarbonate buffer, pH 6.8 containing 0.85% sodium chloride at 5 g / kg, but 10 ml / kg. administered orally at a constant dose volume. 14 days after oral administration of the compound prepared in Example 1 or the buffer itself, mice and rats were sacrificed and observed for necroscopic evidence of toxic signs.

No mortality or toxic symptoms were observed in the assay, suggesting that the compound of Example 1 had an LD 50 of greater than 5 g / kg following a single oral administration.

6 '. example

C57BL / 6 and BDF strain mice were treated intraperitoneally for 7 days with 25 mg, 50, 100, 200 or 400 mg / kg of the compound prepared in Example 1 or buffer. On day 8, a lymphocyte stimulation assay is performed. The results are shown in Figure 2A. and 21). table.

-1 021

2Α. spreadsheet

Examination of the in vivo lymphocyte stimulating effect of the compound prepared in Example 1 in C57BI./6 mouse

Concentration of compound

Lymphocyte Stimulation Index PHA Con-A Control

400 mg / kg 3.2 20.7 1.0 200 mg / kg 21.3 34.0 1.0 100 mg / kg 17.7 40.0 1.3 50 mg / kg * 26.0 1.0 0 mg / kg 17.3 16.7 -

* no results

2.His table.

Examination of the in vivo lymphocyte-stimulatory effect of the compound prepared in Example 1 in a BDF mouse

Concentration of compound Linifocil stimulation index PHA Con-A control * 400 mg / kg 1.1 0.9 0.9 200 mg / kg 7.8 2.9 0.8 100 mg / kg 48.7 17.9. 0.7 50 mg / kg £ 2.1 21.6 0.9 25 mg / kg 13.8 4.3 0.8 0 mg / kg 6.5 1.6 -

Buffer pH 8.05

The compound was highly inhibited at 400 mg / kg in high doses in both C57BL / 6 and BDFi mice! PHA response, weakly inhibited the Con-A response in BDFi mice, and inhibited the Con-A response when compared to the 200 mg / kg dose in C57BI./6 mice. In contrast, stimulation of T-linifocyte proliferation was evident in PHA at 50 and 100 mg / kg in BDF mice and 200 mg / kg in C57BL / 6 mice. The Con-A response was significantly increased with the compound of Example 1 at a dose of 50 and 100 mg / kg in a BDF mouse, whereas at 50, 100 and 200 mg / kg in a C57BL / 6 mouse.

7 · example

C57BL / 6 mice were compared with lymphocyte responders at 50, 100, 200, 400 or

800 mg / kg of the compound prepared according to Example 1, 1, 50, 100, 200, 400 or 800 mg / kg of ascorbinea (AA) or buffer for 4 days i.p. treatment. The lymphocyte stimulation assay was performed on day 5. The results are shown in Figures 3 / .. and 3B.

in Table.

3A. spreadsheet

Investigation of the in vivo lymphocyte stimulatory effect of ascorbineav in C57BL / 6 mice

Concentration of compound Lymphocyte Stimulation Index PHA Con-A control 800 mg / kg 12.1 23.9 0:50 400 mg / kg 14.3 17.8 1.3 200 mg / kg 10.0 21.1 1.3 100 mg / kg 17.1 19.8 1.8 50 mg / kg 17.1 25.5 0:56 0 mg / kg 14.2 17.1 -

-1 123

3Β. spreadsheet

Hydrolysis of the in vivo lymphocyte-stimulatory effect of the compound prepared in Example 1 in C 57BL / 6 mice

Concentration of compound Lymphocyte stimulation index PHA Con-A control 800 mg / kg 0408 0612 0:31 400 mg / kg 4.8 40.0 1.0 200 mg / kg 42.8 67.4 1.6 100 mg / kg 34.8 80.0 1.6 50 mg / kg * 52.8 1.2 0 mg / kg 33.0 32.6 -

* no results

Ascorbic acid showed no statistically significant measurable effect in the 20 PHA-stimulated groups, whereas in the Con-A-stimulated group at 50 mg / kg, it had a mild proliferative stimulating effect.

In PHA-treated spleen cells, the compound prepared according to Example 1, 25, exhibited lymphocyte DNA synthesis at doses of 100 and 200 mg / kg. However, at doses of 400-800 mg / kg it inhibited spleen lymphocyte DNA synthesis.

In spleen cells treated with Con-A, 50-30

Significant stimulatory effects were observed at -200 mg / kg, while significant proliferation was observed at 400-800 mg / kg. inhibitory effect was found, especially at 800 mg / kg. The above data demonstrate that the compound prepared in Example 1 has a low to moderate concentration of immunostimulatory activity, whereas at higher concentrations it exhibits immunosuppressive activity. However, the experimental data did not demonstrate that ascorbic acid alone could produce an effect similar to that of Example 1.

fi. example

C57BL / 6 and BALB / c mice were treated intraperitoneally with 50, 100, 200 or 400 mg / kg of the compound of Example 1 or buffer for 4 days. On the fifth day, a lymphocyte stimulation assay was performed. The results are shown in Figure 4A. 4B and 4B. in Table.

The table

Determination of the in vivo lymphocyte-stimulatory effect of the compound prepared in Example 1 in C57BL / 6 mice

Compound concentration Lymphocyte stimulation index

PHA Con-A Control

400 mg / kg 14.8 30.9 0.61 200 mg / kg 25.2 41.1 0.93 100 mg / kg 17.0 22.3 1.3 50 mg / kg 13.6 20.6 0.87 0 mg / kg 7.85 11.4 -

4B. spreadsheet

Determination of the in vivo lymphocyte-stimulatory effect of the compound prepared in Example 1 in BALB / c mice

Compound Concentration Lymphocyte Stimulation Index

PHA Con-A Control

400 mg / kg 6.71 18.7 0.90

200 mg / kg 7.65 20.5 0.76

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Concentration of compound Lymphocyte stimulation index PHA Con-A control 100 mg / kg 5:24 21.8 1.0 50 mg / kg 6:44 20.5 1.4 0 mg / kg 1.76 9.87 - Spleen cells treated with PHA or Con-A- Example 9

In both mice, 50, 100, and 200 mg / kg of the compound of Example 1 produced lymphocyte DNA synthesis in both mouse strains. At a dose of 400 mg / kg, it inhibited DNA synthesis of splenocytes compared with the activity measured at the dose of 200 mg / kg.

Five normal spleen lymphocytes obtained from non-normal C57BL / 6 mice were subjected to direct in vitro lymphocyte stimulation assay at 1, 10, ΙΟ ² , 10 ³ or 10 x ng / well (5 x 10 ⁵ cells / well). in the presence of a compound prepared according to example 1b or buffer. After three days of incubation, the study was terminated. The results are reported in Table 5.

Table 5

Example 1 In vitro study of the lymphocyte stimulating effect of a compound produced by bergin on C57BL / 6 mouse spleen cell lymphocytes

Concentration of compound Lymphocyte stimulation index PHA Con-A control 10 pg / well 0920 2:15 1.9 1 pg / well 53.8 178 2.6 0.1 pg / well 83.1 198 3.2 0.01 pg / well 87.5 198 3.2 0.001 pg / well 80.4 198 3.8 0 pg / well 21.9 126th -

The results show that both PHA and Con-A mitogen exhibit significant atimulation at 1.0 to 10 ³ ng / well, with significant inhibition at 10 x ng / well. The in vitro effective low concentration range is probably due to the fact that the metabolic system of the host cannot influence drug metabolism.

Example 10 • 10 Lymphocytes isolated from volunteers in vitro with PHA or Con-A and 10 ² , 10 ³ , 10 *, 5 x 10 * or 10 ^s ng / well of compound prepared in Example 1, ', 10', 10 *, 5 * 10 * or 10 ⁵ ng / well were treated with ascorbic acid 45 or buffer. The results are shown in Figure 6A. 6B and 6B. Table.

6A. spreadsheet

In vitro study of ascorbinBav lymphocyte stimulation in human lymphocytes

Concentration of compound Lymphocyte stimulation index PHA Con-A 100 pg / well 34.23 25.18 50 pg / well 35.93 23:31 10 pg / well 35.10 27.35 1.0 pg / well 34.69 24.76 0.1 pg / well 38.29 24.37 0 pg / well 31.25 25.86

-1 327

Table 611

Example 1 Hypersensitivity of in vitro lymphocyte-stimulating activity of a compound produced by ezerine to human lymphocytes

Concentration of compound himphocil ligation index PHA Con-A 100 Mg / well 0544 0:44 50 Mg / well 0:40 1:33 10 jug / hole 34.37 23.82 1.0 Mg / well 48.60 32.63 0.1 Mg / well 56.67 38.06 0 Mg / well 39.82 26.62

Ascorbic acid alone does not exert a significant stimulatory or significant inhibitory effect on proliferation. However, the compound prepared in Example 1 exhibited strong proliferation inhibitory activity at 10 ^s and 5 x 10 ⁴ ng / well, whereas it produced a lower n-increased lymphocyte response expressed at 10 ³ and 10 ^J mg / well. Who. Similar results were obtained with both PHA and Con-A mitogen.

J Example I

C57B1./6 mice were treated intraperitoneally with 50, 10G, 200 or 400 mg / kg of the compound prepared in Example 1 or buffer for four days. On the fifth day, a lymphocyte stimulation assay was performed. The result is given in Table 7.

Table 7

Investigation of the in vivo effect of the compound prepared according to Example 1 on the C57BL / 6 mouse

Concentration of compound t is a cell proliferative index

PHA Con-A Control

400 mg / kg 18.7 26.4 0:31 200 mg / kg 23.2 26.3 0:22 100 mg / kg 23.8 25.4 0:40 50 mg / kg 17.0 23.0 1.1 0 mg / kg 14.9 19.9 -

The response to polyclonal mitogens was enhanced by the compound prepared in Example 1 at a dose of 50-400 mg / kg, with maximal responses being induced at 100-200 mg / kg in both PHA and Con-A mitogen assays. It can also be shown that the PHA-induced response was less potentiated by the compound of the present invention at 400 mg / kg than at 200 mg / kg. From this it can be concluded that the compound according to the invention causes immunosuppression in high doses.

Example 12

The immunoregulatory activity of the compound prepared in Example 1 was studied in vivo in C57BL / 6 mice.

Mice were treated intraperitoneally with 100, 200, 400, or 600 mg / kg of compound or buffer. After treatment, the spleen was authorized and the spleen lymphocyte pellet treated with PHA and Con-A. The results are shown in Table 8.

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Table 8

In vivo study of the in vivo lymphocyte stimulation effect of the compound prepared in Example 1 in C57BL / 6 mice

Concentration of compound Linocyte stimulation index PHA Con-A control 600 mg / kg 17.6 26.5 1.7 400 mg / kg 44.9 42.4 1:49 200 mg / kg 48.2 36.6 1.62 100 mg / kg 51.1 43.9 1.B0 0 mg / kg 13.1 26.7 -

The compound prepared in Example 1, at doses of 100-400 mg / kg, had a significant stimulatory effect on both PHA and Con-A treated lymphocytes. The higher 600 mg / kg dose achieved less stimulation than the 100-400 mg / kg dose and was comparable to the control. The 600 mg / kg dose did not cause any toxic symptoms in animals over a five day period.

9th

Example 13

Lep20 lymphocytes from normal C57BL / 6 mice were subjected to an in vitro lymphocyte stimulation assay. The lymphocytes are 10 ³ , 5 x ³ , 10 ⁴ , 5 x 10 ⁴ or 10 ^s ng / well 1.

or a buffer. The results are shown in Table 9 of Table 9.

spreadsheet

In vitro study of the lymphocyte stimulating effect of the compound prepared in Example 1 on C57BL / 6 mouse lymphocytes

Concentration of compound Lymphocyte stimulation index PHA Con-A control 100 pg / well 0292 0308 0:20 50 pg / well 0261 3.78 0:20 10 pg / well 126 194 0.66 5 pg / well 157 162 1.8 1 pg / well 80.4 123 1.2 0 pg / well 59.5 122 - EXAMPLE 1 The compound produced by eserine 45 Example 14

x 10 ³ and 10 ⁴ ng / well dose significantly increased both the PHA and Con-A-treated lymphocytes response compared to 5 x 10 ⁴ and 10 ^s ng / well dose significantly reduced where the autogénekre lymphocyte response.

The in vivo lymphocyte-stimulating effect of the compound prepared in Example 2 was tested in C57BL / 6 mice by treatment with the test compound or buffer for four consecutive days, followed by a lymphocyte-stimulating assay on day 5. The results are shown in Table 10.

Table 10

Determination of In vivo Effect of Compound Prepared in Example 2 on Lymphocyte Stimulating Mouse C57BL / 6

Concentration of compound

Lymphocyte Stimulation Index PHA Con-A Control

600 mg / kg 400 mg / kg

15.8 94.0 1.5

12.9 109 1.3

-1 531

Concentration of compound Lymphocyte stimulation index PHA Con-A control 200 mg / kg 11.3 99.3 0.69 100 mg / kg 15.4 105 1.5 50 mg / kg 15.5 96.1 0.88 0 mg / kg 11.0 75.1 - J5. example We examined. THE investigation 10 ', 5 X 10 ³ , 10 «, 5 x 10 ⁴ or 10 ⁵ ng / well Concentration 2. The compound prepared in Example 2 was in as in the example produced ' and lymphocyte stimulating effect in vitro buffer. The results are shown in Figure 11. Spleen lymphocytes from C57BL / 6 mice 15 are given in the table.

Jl. spreadsheet

In vitro study of the lymphocyte stimulating effect of the compound prepared in Example 2 on C57BL / 6 mouse lymphocytes

Compound concentration Lymphocyte stimulation index

PHA Con-A Control

100 pg / well 3.83 144 0:49 50 pg / well 45.8 218 0.80 10 pg / well 38.8 164 1.1 5 pg / well 29.8 148 1.2 1 pg / well 25.4 147 0.92 0 pg / well 23.5 175 -

In PHA-treated spleen cells, the compound prepared in Example 2 stimulated lymphocyte DNA synthesis at concentrations of 5 x 10 ⁴ and 10 ⁵ ng / well. A compound at 10 ng / well inhibited DNA synthesis in equine lymphocytes. Con-A-treated spleen cells exhibited a stimulatory effect at 2 x 10 ⁵ ng / well, whereas 4 x 10 ⁵ ng / well exhibited a proliferative inhibitory effect.

Jf. example

Spleen cells from C57BL / 6 mice were treated with in vitro crystallization reagents, namely succinimide and succinimide. Since the molecule complex spontaneously releases 2 moles of crystallization reagent per 1 mole of the compound prepared according to Examples 1, 2 or 3, the reagents were tested at the respective molar concentration for the lymphocyte-stimulating effect. The results are shown in Figure 12A. 12B and 12B. table.

J2a. spreadsheet

In vitro study of lymphocyte stimulating activity of succinic acid on C57BL / 6 mouse lymphocytes.

Concentration of compound

Lymphocyte Stimulation Index PHA Con-A Control

6.6 pg / well 11.5 38.5 1.1 8.3 pg / well 12.4 45.6 0.91 4.15 pg / well 14.0 42.3 0.91 2.08 pg / well 15.3 51.1 1.0 1.04 pg / well 15.7 64.6 0.94 0 pg / well 15.9 83.5 -

-1 633

12Β. spreadsheet

In vitro study of the lymphocyte-stimulatory effect of succinimide on C57BL / 6 mouse lymphocytes

Concentration of compound Lymphocyte stimulation index PHA Con-A control 13.92 pg / well 17.1 92.2 1.4 6.96 pg / well 17.2 71.9 1.1 3.48 pg / well 18.1 85.1 0.98 1.74 pg / well 17.5 83.4 1.1 0.87 pg / well 19.5 76.0 0.91 0 pg / well 19.1 100 -

Lymphocytes treated with succinic acid exhibited reduced levels of mitogenesis induced by PHA or Con-A mitogen with increasing concentrations of succinic acid. Succinimide showed no significant inhibitory or significant enhancement effect on any mitogen-induced lymphocyte response.

Example 17

Spleen cell lymphocytes from C57BL / 6 mice were treated in vitro with the compound prepared in Example 3. Lymphocytes were treated with 10 ³ , 5 x 10 ³ , 10 ⁴ , 4 x 10 ⁴ or 10 ^s ng / well of the compound prepared in Example 3 or buffer and subjected to a lymphocyte B stimulation assay. The results are shown in Table 13.

13th signboard

In vitro study of the lymphocyte-stimulatory effect of the compound prepared in Example 3 on C57BL / 6 mouse lymphocytes

Concentration of compound

Lymphocyte Stimulation Index PHA Con-A Control

100 pg / well 0263 2.88 0:25 50 pg / well 4:08 97.2 0:27 10 pg / well 21.0 89.6 0:47 5 pg / well 13.5 80.9 0.64 1 pg / well 12.5 73.2 0.60 0 pg / well 12.2 94.6 -

The results of the experiment show that the compound of Example 3 significantly increased where the PHA response at 10 * ng / well, and at 5 x 10 ^s and IQ ⁴ ng / well inhibited it. At a concentration of 5 x 10 ⁴ ng / well, it slightly increased the response to Con-A, but at a concentration of 10 ng / well.

Example 18

Three-month-old SJL / J strain T-cell deficient mice were treated intraperitoneally for four days with 100, 200 or 400 mg / kg of the compound prepared in Example 1 or buffer. A lymphocyte stimulation assay was then performed. The results are shown in Table 14.

Table 14

Investigation of the in vivo inhibitory effect of the compound prepared according to Example 1 on SJL / J mouse

Concentration of compound

Lymphocyte Stimulation Index P HA Con-A Control

400 mg / kg 2:31 3:30 0693 200 mg / kg 1.79 4:57 0732 100 mg / kg 1.85 5:01 0573

-1 735

Concentration of compound Lymphocyte Atimulation Index PHA Con-A control 0 mg / kg 1.11 "2.4 -

Example 19

As SJL / J mice grow older, they appear to lose their immune regulatory function as a result of being hypersensitive to alloantigens and increasing the production of circulating antibodies to nuclear or synthetic double-stranded RNA and poly / C. In mice of the SJL / J strain, the steady increase in immune competence from birth to death indicates a lack of a regulatory T-cell subpopulation (probably suppressor and / or enhancer). Because the dynamics of the T-lymphocyte population changes with age, it is to be assumed that the age of the animals influences the experimental results obtained with the SJL / J mice treated with the compound prepared in Example 1.

The in vivo lymphocyte stimulation assay was performed on 15 SJL / J mice of various ages - 2.5 or 10 months. The animals were administered intraperitoneally 100 mg / kg of the compound prepared in Example 1 or buffer. The results are shown in Table 15.

Table 15

Examination of the in vivo lymphocyte stimulating effect of the compound prepared in Example 1 in SJL / J mice

Mice age (month) Lymphocyte stimulation index

PHA Con-A Control

2 0 mg / kg 40.8 103 - 100 mg / kg 32.6 167 2.3 5 0 mg / kg 2:09 19.5 - 100 mg / kg 1:16 12.9 1.9 10 0 mg / kg 3.97 21.9 - 100 mg / kg 9:18 14.5 2.1

Results from untreated control animals demonstrate that the lymphocyte proliferation response induced by PHA or Con-A is age dependent.

It can also be shown that a compound of Example 1 at 100 mg / kg in a 2-month-old animal reduces the PHA response while increasing the Con-A response. In contrast, in a 5-month-old animal, the compound prepared in Example 1 reduces the mitogenesis response induced by both PHA and Con-A. In 10-month-old SJL / J mice, a fully reversible effect was observed and the mitogenic response induced by both PHA and Con-A was significantly increased by the same dose of the compound prepared in Example 1.

Example 20

4J C57BL / 6 mice intraperitoneally

50, 100, 200 or 400 mg / kg of the compound prepared in Example 1 or buffer were injected for 12 days (day 1-12). On days 1, 7, and 14 of the experiment, bovine rum altumin (BSA) and Freund's complete adjuvant (FAC) were injected intraperitoneally into the animals. Antibody response was determined in blood samples on days 0, 5, 13 and 19. On days 13 and 19, lymphocyte50 proliferation assays were also performed on spleen lymphocytes from the same mice as specific booster antigen (mitogen) at 10, 10 ^. , 5 x 10 ^! obsession

10 ³ ng / well using BSA antigen or buffer. The results are shown in Figures 16A, 16B, and 16C. table.

-1 837

16Α. spreadsheet

Enhancement of the secondary immune response to BSA with the compound prepared in Example 1 as measured by microELISA titer First concentration of the compound of example Micro-ELISA titres Day 0 5 days Day 13 Day 19 400 mg / kg N / R N / R 1:80 1: 320 200 mg / kg N / R N / R 1:80 1: 1280 100 mg / kg N / R N / R 1:40 1: 1280 60 mg / kg N / R N / R 1:40 1: 640 0 mg / kg N / R N / R 1:20 1: 320 s N / R = No Answer 16B. spreadsheet The compound prepared in Example 1 is an in vivo secondary immune response to BSA lymphocyte -stimuláló effect study, C57BL / 6 mouse, Figure 13. on the sun First Example 1Berzine Compound Lymphocyte tn stimulation index BSA {pg / lyu k) concentration 0 0.01 0.1 0.5 1 400 mg / kg 10.9 13.7 12.8 12.3 13.8 200 mg / kg 10.6 34.1 15.8 13.8 14.0 100 mg / kg 3.22 9.8 11.9 12.3 10.2 50 mg / kg 16.5 19.9 23.1 22.9 18.2 0 mg / kg 14.0 14.6 12.8 13.0 16C. spreadsheet The compound prepared in Example 1 is BSA given in vivo nder's immune response, lymphocyte -stimuláló effect study, C57BL / 6 in mouse, Figure 19. on the sun

Concentration of the compound of Example 1 Lymphocyte Stimulation Index BSA (jug / well) 0 0.01 0.1 0.5 1 400 mg / kg 5.0 3.26 2.89 1.94 1:04 200 mg / kg 0.42 2.94 2.82 2:56 2:12 100 mg / kg 4.1 3.79 2.68 2:03 0:26 50 mg / kg 0.26 2.33 2:27 2:37 2.63 0 mg / kg 1:58 1:56 2:07 1:02

By day 13, no antibody response was detected. On day 13, significant (fourfold) stimulation was observed with 200 and 400 mg / kg of the compound prepared in Example 1, whereas at 50 and 100 mg / kg, only a slight stimulatory effect was observed. On day 19, we experienced a greater change. In this case, the antibody response to BSA at 0 and 400 mg / kg of the compound prepared in Example 1 was the same and greater than that observed on day 13. 50 mg / kg of the compound of Example 1 produced a slightly higher antibody titer than the control, while the compound of Example 1 at 100 and 200 mg / kg produced an extremely high 16-fold sulfur stimulating effect on the control.

In the second half of Example 20, the effect of the compound prepared in Example 1 on lymphocyte proliferation of spleen cells was tested in the presence of BSA immunizing antigen. Compared to the control, the compound prepared in Example 1 showed a significant DNA synthesis stimulating effect only at 50 mg / kg on day 13. This effect disappears at day 19, indicating that continuous treatment is very important for enhancing the immune response (T-cell) -mediated component. The above antibody response is likely to be sustained after treatment has been completed since the IgG (antibody) half life in vivc is approximately 5-7 days.

-1 939

Example 21

C57BL / 6 mice were injected intraperitoneally with 50, 100, 200 or 400 mg / kg of the compound of Example 1 for 10 days (day 1-10). On days 1, 7, and 13, hippocampus hemocyanin alligen (KLH) was administered intraperitoneally with Freund's Complete Adjuvant (FCA). 0, 5, 12 and

On day 17, blood samples were taken to determine the antibody response. The spleen was removed, the lymphocytes were isolated, and the lymphocyte stimulation assay was performed on day 17 of the experiment using PHA, Con-A or KLH antigen mitogens. The results are shown in Figure 17A. 17B. table.

17A. spreadsheet

ELISA Enhancement of the KLH-Induced Secondary Immune Response ELISA with the Compound Prepared in Example 1 by Microtiter Assay

Concentration of the compound of Example 1 Micro-ELISA titer Sun Day 0 5 days 12th Sun 17th 400 mg / kg N / R N / R 1: 5120 1: 10.240 200 mg / kg N / R N / R 1: 5120 1: 10.240 100 mg / kg N / R N / R 1: 2560 1: 5120 50 mg / kg N / R N / R 1: 2560 1: 5120 0 mg / kg N / R N / R 1: 2560 1: 1280

N / R = no answer

17B. spreadsheet

Effect of Compound Prepared in Example 1 on In vivo Response to KLH, PHA, or Con-A in a Lymphocyte-Stimulating Hatae Study in C57BL / 6 Mice

Example 1 Compound Lymphocyte Stimulation Index KLH (pg / well)

k once n tréció ja 1.0 0.5 0:05 0025 400 mg / kg 0589 0232 0667 1:03 200 mg / kg 7:45 4:26 1:02 3.91 100 mg / kg 0898 0734 0526 0401 50 mg / kg 0536 0415 0357 0.0386 0 mg / kg 0715 0614 0739 1:06

Concentration of the compound of Example 1 PHA Con-A control 400 mg / kg 2:32 35.5 0840 200 mg / kg 12.4 51.7 1.73 100 mg / kg 9:12 15.6 0961 50 mg / kg 7.65 19.0 0976 0 mg / kg 11.0 34.1 -

No antibody response was observed by day 12. On day 12, the groups treated with the compound of Example 1 at doses of 200 mg / kg and 400 mg / kg exhibited significant quadrupling of stimulation compared to the control group. On day 17 of the experiment, 7 days after completion of treatment with the compound prepared according to Example 1, a similar difference was found between the control and treated groups, with an increase of 8-fold in immune response to untreated antigen-sensitized control group at 200 mg / kg. in comparison.

The lymphocyte β-proliferation assay was performed only on day 17, more than 5 days after completion of treatment with the compound prepared in Example 1. Statistically significant increases in lymphocyte DNA synthesis were observed in Con-A or KLH mitogen-stimulated animals treated with 200 mg / kg. The lymphocyte mitogen response to Con-A (T-cell mitogen) showed a good correlation with the positive response of KLH-sensitized visceral lymphocytes to KLH.

-2 041

Example 22

Example 1 Cytotoxic lymphocyte activity (ΟΤΙ.) Of a compound produced by ezerine was determined in DBA / 2 mice, 10 days after infection with viable L5178Y tumor cells, in the early TDS section. EXAMPLE 1 From a compound produced from ezerine 1, 10,

10 ', 10', 10 * or 10 ^s ng / ml solutions were prepared and the DBA / 2 mouse

ISA.

MLTC microorganisms containing washed spleen lymphocytes, effector cells, and various E / T-targeted L5178Y target cells were assayed with 0.1 ml of the test compound containing the test compound (assays and buffer solution). The results are shown in Tables 18A and 18B.

board Zfit

Effect of Compound Example 1 on CTL Activity of DBA / 2 Mouse TDS Equine Lymphocytes by MLTC Assay Using Effect Cells Cultured with Stimulant Cells

Concentration of the compound of Example 1 % lysis E / T ratio 00: 1 30: 1 10: 1 100 pg / ml 87.3 54.2 21.2 10 pg / ml 74.3 42.0 15.9 1.0 pg / tnl 71.5 46.0 15.6 0.1 pg / ml 79.0 51.0 20.2 0.01 pg / ral 77.1 55.8 20.6 0001 71.7 40.2 16.7 0 pg / ml 55.2 23.0 8.2

18B. spreadsheet The effect of the compound prepared in Example 1 on the CTL activity of TDS spleen lymphocytes from DBA / 2 burn by MLTC assay in the absence of stimulatory cells using effector cells Concentration of the compound of Example 1 %-ancestor Taste E / T ratio 100: 1 30: 1 10: 1 100 pg / ml 42.0 18.7 8.6 10 pg / ml 36.3 16.8 4.6 1.0 pg / ml 40.1 20.0 4.6 0.1 pg / ml 31.6 14.8 3.5 0.01 pg / ml 26.0 15.7 3.5 0.001 pg / ml 23.4 12.2 3.9 0 pg / ml 22.7 11.0 • 2.2

The compound prepared in Example 1 significantly enhanced the degree of cytotoxic activity expressed by X-lysis at various concentrations, whether in the presence or absence of E / T fluorescence used.

According to the above data, the compound prepared in Example 1 potently enhances the specific cytotoxic response of TDS mouse spleen cells clocked with L5178Y tumor cells.

Example 23

Five cytoplasmic DBA / 2 mice 10 days after challenge with viable L5178Y tumor cells were tested for the cytotoxic activity of the compound prepared in Example 1. The microbial culture containing washed spleen lymphocytes, effector cells, and various E / T target L5178Y target cells from DBA / 2 mice was plated in 0.1 ml of 10 ⁴ or 10 ⁵ ng / ml of the solution of Example 1 or clear buffer. . The MLTC reaction is performed in the presence or absence of irradiated L5178Y stimulator cells. The results are shown in Figure 19A. 19B and 19B. in Table.

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Effect of Compound Example 1 on CTL Activity of DBA / 2 Mouse TDS Equine Lymphocytes by MLTC Assay Using Effector Cells Cultured with Stimulant Cells

Concentration of the compound of Example 1 50 :! % lysis E / T ratio 25: 1 12.5: 1 6.25: 1 100 pg / ml 71.6 79.8 75.7 56.3 10 pg / ml 75.7 72.0 67.1 60.5 0 pg / ml 63.9 61.9 45.0 35.5 Table 19.1) Example 1 Compound effect of DBA / 2 mouse TDS equine lymphocytes CTL activity according to MLTC test, stimulator cells in absentia i, using effector cells The compound of Example 1 % lysis E / T ratio concentration 50: 1 25: 1 12.5: 1 G.25 1 100 pg / ml 21.8 16.6 11.1 6.1 10 pg / ml 12.8 8.1 6.9 0.7 0 pg / ml -7.5 -3.0 -3.5 -1.3

The compound prepared in Example 1, as shown by α% -b lysis values, significantly increased CTL activity at many concentrations, regardless of E / T ratio and presence or absence of stimulator cells.

Example 24

Prior to provoking challenge with viable L5178Y tumor cells in mice in the early TDS stage, the effect of the compound prepared in Example 2 on CTL activity was determined. MLTC microculture wells containing washed spleen lymphocytes, effector cells, and various E / T ratios of L5178Y target mice from DBA / 2 contained 0.1 ml of the compound of Example 2 in a concentration of ΙΟ ² , ΙΟ ² , 10 ⁴ or 10 ¹ ng / ml. solution or pure buffer. The MLTC reaction was performed in the presence or absence of irradiated L5178Y '10 stimulatory cells. The results are shown in Figure 20A. 20B and 20B. Table.

20A. spreadsheet.

EXAMPLE 2 Effect of a Berserine Compound on the Cytotoxic Activity of DBA / 2 Untreated TDS Spleen Lymphocytes by MLTC Assay Using Effector Cells Cultured with Stimulus Cells

Concentration of the compound of Example 2 100: 1 % lysis E / T ratio 12.5: 1 50: 1 25: 1 100 pg / ml 78.5 73.5 65.0 47.0 10 pg / ml 73.5 72.5 72.5 58.5 1 pg / ml 75.5 72.5 C9.5 58.0 0.1 pg / ml 74.5 69.5 64.0 47.5 0 pg / ml 68.5 67.0 57.0 37.5

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20/1. signposted

Effect of Compound Preparation 2 on the Cytotoxic Activity of DBA / 2 Untreated TDS Spleen Lymphocytes by ML'1'C in the absence of Stimulant Cells Using Effector Cells

Concentration of the compound of Example 2 100: 1 % lysis E / T ratio 50: 1 25: 1 12.5: 1 10 mg / ml "1.5 56.5 40.0 26.5 0.1 pg / rnl 48 0 43.5 29.5 16.5 0 Mg / ml 58.5 49.0 32.5 19.5

The compound prepared in Example 2 increased the cytotoxic activity expressed as% lysis at various concentrations, regardless of the E / T ratio used and whether the assay was performed in the presence or absence of stimulator cells. One exception was observed at 10 ² ng / ml when tested in the presence of stimulator cells.

Example 25

MLTC was used to study the specificity of the compound prepared in Example 1, CTL activiv20 ', and anti-tumor target cell specificity. As effector cells, resting tumors from DBA / 2 mice were used as spleen cell brush and normal: normal cells, and L5178Y cells irradiated as stimulator cells. The target cells were ⁵¹ Cr-labeled L5178Y cells and FLC-745 cells. The effector / target cell ratios in the assays were 100: 1, 30: 1 and 10: 1. The results are shown in Table 21.

Table 21

Effect of Compound Prepared on Example 1 on DBA / 2 Mouse TDS and Normal Spleen Cells by MLTC Assay

%: isis E / T ratio Target cell L5178Y 100: 1 30: 1 10: 1 E + S + D 82.3 69.2 34.9 E + S 55.5 39.3 11.4 E + D 52.8 29.8 9.0 E 50.9 29.2 7.2 N + S + D -9.5 -9.7 -10.9 N + S -14.2 -10.5 -11.2 N + D -12.4 -11.4 -10.3 N -11.2 -11.0 -10.9

Table 21 (continued)

Target cell: FLC-745 % n'zis E / T ratio 100: 1 30: 1 10: 1 E + S + B 13.4 Fi 3 0.5 E + S 0.9 1.4 -4.0 E + I) 0.0 4.0 0.7 E 11.8 0.0 -1.3 N + S + D 5.8 -0.8 -1.1 N + S 5.8 1.1 -2.6

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Target cell: PLC-745 100: 1 % lysis E / T ratio 30: 1 10: 1 N + D 13.9 0.9 -3.5 N 1.7 -1.8 -2.9

For the development of luminal cells, forty DBA / 2 mice were treated with the compound prepared in Example 1 after the animals were subjected to the TDS-inducing operation. Animals were intraperitoneally injected with 50,000 viable L5178Y tumor cells, and two days later, 20 of the animals received 100 mg / kg of the compound of Example 1 for seven consecutive days, and the remaining 20 animals were given a clear buffer intraperitoneally. On the 25th day following the administration of the last 20 doses of the compound of Example 1 Ezerine, a partial peritoneal wash was performed and the number of tumor cells was determined from the smear of the peritoneal exudate fluid (tumor cells for quantitative assay). At 90 days after provocation with viable L5178Y tumor cells, survival time was determined. Percent mortality was calculated for each group. The results are shown in Figure 22A. 22B and 22B. table.

Ε = effector, resting tumor spleen cell S - stimulator, irradiated I.5178Y cell] Q

D = EXAMPLE 1 A compound produced by ezerine

N = normal effector cell, normal spleen cell

In the presence of the compound prepared in Example 1, in the presence of the target cell L5178Y, in the control system, the cytotoxic activity of the effector cells showed a significant specific increase. The above effect was not observed in the KLC-745 target cell control system, nor did the effector cells alone increase the effect. The above results show that the compound prepared in Example 1 increases the activity of the stimulated effector cells, the activity of the T cell memory of the system, and the specificity of the stimulated effector cells.

Example 26

The compound of Example 1 was exposed to the L5178Y at rest

22A. signposted

Tumor cell distribution in TDS DBA / 2 mice by quantitative tumor cell assay

Number of tumor cells Control group Managed group Frequency % Of total Frequency % Of total 0 1 6 4 22 1000 6 35 6 33 1000 - 100,000 8 47 7 39 100,000 2 12 1 6

Table 2211

Mortality data of TDS DBA / 2 mice at 90 days post challenge

Concentration Survivors% survival

100 mg / kg 10 55.6 mg / kg 3 17.6

A significant reduction in the number of tumor cells was observed in the group treated with the compound prepared in Example 1 compared to the control group. Λ In the treatment group, several mice with lower numbers of tumor cells in the peritoneal transducer were found. The survival time of mice treated with the compound prepared in Example 1 was significantly higher than that of untreated ceoport GO.

2 R. example

Assay of the compound prepared according to Example I at rest

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L.5178Y tumor cells were formed by treating 40 DBA / 2 mice with the compound of Example 1 after TDS iridation. The animals were ululated peritoneally with 50,000 L5I78Y lifestyles; and two days later, 20 animals for 7 consecutive days. 100 mg / kg was treated intraperitoneally with the compound of Example 1, while the remaining 20 animals received a clear buffer. On the 25th day after the last dose of the test compound, a partial perilone wash was performed and the number of tumor cells (quantitative assay for tumor cells) was determined from the smear taken from the perilone exudate fluid. Survival times were recorded on day 114 after provocation with viable L5178Y tumor cells. Percent mortality was calculated for each group. The results are shown in Figure 23A. 23B and 23B. Table 10 gives them.

The table

Tumor cell distribution in TDS DBA / 2 mice by quantitative tumor cell assay

Number of tumor cells Control group Frequency% of total Managed group Frequency Ötíszee% -i 0 3 13.0 16 69.6 1000 8 34.8 2 8.7 1000 - 100,000 43.5 4 17.4 100,000 2 8.7 1 4.3

Table 238

Mortality data for TDS DBA / 2 mice at 114. najion after rovocation

Concentration

You will survive today with% survival

100 mg / kg 0 mg / kg

82.6

47.8

The number of tumor cells was significantly reduced after treatment with the compound prepared in Example 1 compared to the control group. In the treatment group, several mice were found to have fewer tumor cells in the perilone wash. Mice treated with the compound prepared in Example 1 had a significantly longer survival time than untreated controls.

Example 28

Mice belonging to the BDF1 strain inoculated with 1 to 1210 leukemia cells were treated for 7 days with 25, 50, 100, 200 or 400 mg / kg of the compound prepared according to Example 1 or with clear buffer. The test is conducted by National Cancer

Institute performed the screening method for new anticancer agents and calculated T / C ratios. The results are reported in Table 24.

Table 24

Activity of Example 1 Compound against L-1210 Leukenia Cells Based on T / C Ratios in BDFi Mouse

Concentration Median survival time (days) T / C 400 mg / kg 25 1:25 200 mg / kg 311 1:50 100 mg / kg 30 1:50 50 mg / kg 21 1:05 25 mg / kg 20 1:00 0 mg / kg 20 -

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At a dose of 400, 200 and 100 mg / kg, the T / C ratios were 1.25, 1.50 and 1.50, respectively, which are considered significant by the National Canoer Inetitute Standard.

Example 29

BDF mice inoculated with L-1210 leukemia cells 100 mg / kg for seven days Example 1

25th

The compound prepared as in Example 1 and the compound prepared as described in 5-FU were treated with 5-fluorouracil (5-FU), 20 mg / kg as a known anticancer chemical, or buffer. The examination. National Cancer Institute performed the screening procedures for new cancer drug screening methods and calculated T / C ratios. The results are shown in Table 25.

effect of Table 1, -1210 leukemia cells, on the '’/ C ratio sheet, in BDF mouse

Compound Concentration Median survival time T / C Example 1 100 mg / kg 28 1:40 5-FU 20 mg / kg 30 1:50 Buffer - 20 -

In mice treated with 100 mg / kg of the compound prepared in Example 1, the T / C ratio was 1.40, compared with 1.50 for the positive controls treated with 5-FU. Based on these data, the effect of the compound prepared in Example 1 can be considered positive and the results obtained with the compound prepared in Example 1 can be compared with those obtained with the accepted positive control compound, 5-FU.

Claims (9)

A process for the preparation of a compound of formula IVa: wherein R ² is a C 1 -C 4 alkyl group and n is 1 to 3 and their adducts with ac anhydride or acid amide, characterized in that a compound of the formula II in which R ¹ and R ² are each independently hydrogen or C 1-4 alkyl, with the proviso that at least one is hydrogen, R ³ and R ⁴ are each independently C 1 -C 4 alkyl with a compound of formula III wherein R 'is -CH (OH) - (CH 2) n OH wherein n is 1-3 and the resulting treating the product, if desired, with an acid anhydride or an acid amide, separating the resulting adduct from the reaction mixture and, if desired, dissociating the resulting product with a compound of formula IVa and an acid anhydride or eavamide. 2. A process according to claim 1 wherein the starting compound of formula (II) is a compound wherein R 'is hydrogen, R * is C 1-3 alkyl, and R ³ and 1 ' ⁴ are independently C 1-3 alkyl. 3. A process according to claim 2 wherein the starting compound is 35 is a compound of formula 5 wherein R ³ is methyl. 4. The process of claim 3 wherein the compound of formula (II) is 2-methyl-2,5-dimethoxy-2,5-dihydrofuran. 5. A process according to any one of claims 1 to 3, wherein the starting compound of formula (III) is a compound wherein R ⁷ is 1. 6. A process according to claim 5 wherein the starting compound of formula III is L-aBzcorbic acid. 7. A process according to any one of claims 1 to 3, wherein the compound of formula (II) is used in a molar ratio of 2: 1 to 1: 2 in relation to the compound of formula (III). The process according to claim 7, wherein the starting compounds are used in a molar ratio of θ 1: 1. 9. A process according to any one of claims 1 to 4, characterized in that it is an eahyanhid liloolite. '. or acid amide.i l.i'.vostyánk \ - «nhihrid' or amide use In ni ..