Hydroxyether rapamycin, the method of production thereof and methods of treatment

 

(57) Abstract:

Describes new compounds of formula I, where each of R1and R2independently, represents hydrogen or-CO(CR3R4)b(CR5R6)dCR7R8R9each of R3and R4independently, represent hydrogen, alkyl with 1 to 6 carbon atoms; each of R5and R6independently, represent hydrogen, alkyl with 1 to 6 carbon atoms, or R5and R6may together form a group X or cycloalkyl ring with 3 to 8 carbon atoms; R7represents hydrogen, alkyl with 1 to 6 carbon atoms; each of R8and R9independently, represents hydrogen, alkyl with 1 to 6 carbon atoms, -(CR3R4)fOR10or R8and R9may together form a group X or cycloalkyl ring with 3 to 8 carbon atoms; R10represents hydrogen, alkyl with 1 to 6 carbon atoms, three(alkyl) of 1 to 6 carbon atoms silyl, three(alkyl) of 1 to 6 carbon atoms, silylated or tetrahydropyranyl; X represents 5-(2,2-di(alkyl) of 1 to 6 carbon atoms 1,3 dioxane, 5-(2-Spiro-cycloalkyl with 3 to 8 carbon atoms (1,3)dioxane, 4-(2,2-di(alkyl) of 1 to 6 atoms operor>1and R2do not simultaneously represent hydrogen, and provided that either R1or R2contains at least one (CR3R4)fOR10X or cycloalkyl with 3 to 8 carbon atoms, or a pharmaceutically acceptable salt of this compound, used as an immunosuppressive, antiinflammatory, antifungal, antiproliferative, and antitumor agent. Also describes a method of obtaining compounds of formula I, having immunosuppressive activity, and a method of treatment using compounds of formula I. 5 C. and 17 C.p. f-crystals.

The invention relates to hydroxyethyl, to a method of using such compounds for inducing immunosuppression, and for treating rejection in the transplantation or incompatibility reaction autoimmune diseases, inflammatory diseases, leukemia/lymphoma adult T-cell, solid tumors, fungal infections, and disorders associated with vascular hyperproliferative.

Rapamycin is a macrocyclic Tranby antibiotic, the producer of which is Streptomyces hygroscopicus, found that rapamycin has antifungal activity, particularly against Candida albicans, and the; is the U.S. Patent 3993749).

Found that rapamycin (U.S. Patent 4885171) or in combination with picibanil (U.S. Patent 4401653) has antitumor activity. In the work of R. Martel et al., (Can. J. Physiol. Pharmacol. 55, 48 (1977)) indicated that rapamycin has been demonstrated in an experimental model of allergic encephalomyelitis, a model of multiple sclerosis, adjuvant arthritis, rheumatoid arthritis, as well as the suppression of antibody formation, similar to IgE.

Immunosuppressive effects of rapamycin are described in the work of the FASEB 3, 3411 (1989). Found that cyclosporine A and FK-506, other macrocyclic molecules, are also effective as immunosuppressive agents, and are therefore used to prevent rejection of transplants (FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978); and U.S. Patent 5100899).

Found that rapamycin may also be used for prevention or treatment of systemic lupus erythematosus (U.S. Patent 5078999), pneumonia (U.S. Patent 5080899), diabetes mellitus with insulin-dependent (Fifth Int. Conf. Inflamm. Res. Assoc. 121 (abstract), (1990)), cell proliferation of smooth muscles and thickening of the inner lining as a result of defeat of vessels (Morris, R. J. Heart Lung Transplant 11 (pt 2 Europatent 532862 A1).

Found that mono - and diallylamine derivatives of rapamycin (estetizirovannye 28 and 43 positions) can be used as antifungal agents (U.S. Patent 4316885) and used to prepare water-soluble aminoaniline prodrugs of rapamycin (U.S. Patent 4650803). Recently the rules numbering of provisions in respect of rapamycin have been changed; therefore, in accordance with the Nomenclature of "Chemical Abstracts" the above esters will be 31 and 42 positions.

The present invention provides derivatives of rapamycin which are applicable as immunosuppressive, antiinflammatory, antifungal, antiproliferative and anticancer agents, and which have the following structure:

< / BR>
where each of R1and R2independently, represents hydrogen or

-CO(CR3R4)b(CR5R6)dCR7R8R9;

each of R3and R4independently, represents hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, quinil with 2-7 carbon atoms, trifluoromethyl or-F;

each of R5and R6independently, represent hydrogen, alkyl with 1-6 carbon atoms, alkenyl from 2-7 at the 11 or R5and R6may together form X or cycloalkyl ring of 3-8 carbon atoms, which may be mono-, di - or tizanidine -(CR3R4)fOR10;

R7represents hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, quinil with 2-7 carbon atoms, (CR3R4)fOR10, -CF3, -F, or CO2R11;

each of R8and R9independently, represent hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, quinil with 2-7 carbon atoms, -(CR3R4)fOR10, -CF3, -F, or R8and R9may together form X or cycloalkyl ring of 3-8 carbon atoms, which may be mono-, di - or tizamidine radical -(CR3R4)fOR10;

R10represents hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, quinil with 2-7 carbon atoms, three(alkyl with 1-6 carbon atoms) silyl, three(alkyl with 1-6 carbon atoms) silylated, triphenylmethyl, benzyl, alkoxymethyl with 2-7 carbon atoms, three(alkyl with 1-6 carbon atoms) ciliatoxic, chloroethyl or tetrahydropyranyl;

R11represents hydrogen, alkyl with 1-6 erode;

X represents 5-(2, 2-di(alkyl with 1-6 carbon atoms))[1,3]dioxane, 5-(2-Spiro(cycloalkyl with 3-8 carbon atoms))[1,3]dioxane, 4-(2,2-di(alkyl with 1-6 carbon atoms))[1,3]-dioxane, 4-(2-Spiro(cycloalkyl with 3-8 carbon atoms))[1,3] -dioxane, 4-(2,2-di-(alkyl with 1-6 carbon atoms))[1,3]-DIOXOLANYL, or 4-(2-Spiro(cycloalkyl with 3-8 carbon atoms))[1,3]DIOXOLANYL;

b = 0-6;

d = 0-6;

f = 0-6,

provided that R1and R2do not simultaneously represent hydrogen, and provided that either R1or R2contains at least one (CR3R4)fOR10X or cycloalkyl with 3-8 carbon atoms, substituted (CR3R4)fOR10or pharmaceutically acceptable salt of this compound.

Pharmaceutically acceptable salts are those salts which are derived from such inorganic cations such as sodium, potassium and so on; organic bases, such as mono-, di - and trialkylamines with 1-6 carbon atoms in the alkyl group, and mono-, di - and trihydroxystilbene with 1-6 carbon atoms in the alkyl group, etc.

The terms "alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms and quinil with 2-7 carbon atoms" include compounds with direct C is PI - (CR3R4)fOR10the radicals R3, R4and R10can have the same or different values. Similarly, when other substituents are repeated in the same structural formula, these substituents may be the same or different.

For compounds where R1contains R8and R9together form X, where X represents 5-(2,2-di(alkyl with 1-6 carbon atoms))[1,3] dioxane, an alkyl group, X contains 1 carbon atom, and d = O, R1will have the following structure:

< / BR>
Similarly, for compounds where R1contains R8and R9together form X, where X represents 4-(2-Spiro(cycloalkyl with 3-8 carbon atoms))[1,3] dioxane, cycloalkyl group X contains 6 carbon atoms, a d = O,R1will have the following structure:

< / BR>
From among compounds containing X, preferred are those compounds in which the alkyl group in X, if present, represents a methyl group, and cycloalkyl group in X, if present, represents cyclohexyl.

When R10is not hydrogen, alkyl, alkenyl or quinil, it is assumed that R10represents the breaking the hydroxylated compounds, they are biologically active by themselves. R10includes three(alkyl with 1-6 carbon atoms) silyl, three(alkyl with 1-6 carbon atoms) silylated, triphenylmethyl, benzyl, alkoxymethyl with 2-7 carbon atoms, three(alkyl with 1-6 carbon atoms) silyl-ethoxymethyl, chloroethyl and tetrahydropyranyl. Specialists and other known sportsedan group, which also are within the scope of the present invention.

From among the compounds of the present invention preferred are those compounds in which R2represents hydrogen; compounds wherein R2represents hydrogen, b=0, d=0; compounds where R2represents hydrogen, b= 0, d=0, and each of R8and R9independently, represents hydrogen, alkyl or -(CR3R4)fOR10or taken together form X.

Compounds of the present invention, having the ether group-CO(CR3R4)b(CR5R6)dCR7R8R942 or 31, 42 positions can be obtained by acylation of rapamycin using protected hydroxy acids or PolicyKit, alkoxycarbonyl or polyalkoxyalkyl acids are first activated, and then, when gelan Stateline methods based on the use of carbodiimides, mixed anhydrides or acid chlorides. For example, the appropriately substituted carboxylic acid can be activated as a mixed anhydride, with such alleluya group as 2,4,6-trichlorobenzoyl. Treatment of rapamycin mixed anhydride in slightly alkaline conditions allows to obtain the required connections. Otherwise, the acylation reaction can be performed using 1-(3-dimethylaminopropyl)-3-tilcara-deemed-hydrochloride and dimethylaminopyridine. A mixture of compounds containing esters 42 position and compounds containing esters 31, 42 provisions can be separated by chromatography.

The present invention also provides a method of obtaining derivatives of rapamycin. In particular, the method of producing hydroxyamino rapamycin, including the above-mentioned compounds of formula involves the following stages:

a) acylation of rapamycin or its functional derivative, or analog using Alliluyeva agent;

or

b) sequential acylation of rapamycin or its functional derivative, or analog of the two allerease agents; and specified allerease agent (or agents) selected from acids of the formula:

HO-CO(CR3R4)bis s, where R3-R9, b and d have the above values, provided that the free hydroxyl group is protected, and if desired, you can protect the team in 42 the position of the rapamycin or its functional derivative, using the appropriate protective group, and after the reaction to remove any protective groups, whose presence was necessary.

The reaction can be carried out in the presence of binders, such as appropriately substituted carbodiimide derivative. The above compounds can also be obtained by acylation using reactive derivatives of acids of formula II, such as anhydrides, mixed anhydrides or galodamadruga, such as chloride.

Compounds according to the invention, containing the ester in 31 position and a hydroxyl group at the 42 position can be obtained by protection of the alcohol group of rapamycin 42 position by means of a protective group such as tert-butyl dimethylsilicone group, followed by esterification of the group at 31 position using the methods mentioned above. Receiving rapamycin with silylating group 42 the situation described in U.S. patent B1 5120842, which is mentioned here for information. Remove protection of the l-dimethylsilicone protective group, removing the protective groups can be carried out in weakly acidic conditions, for example, in a mixture of acetic acid/water/tetrahydrofuran. The method of removal of the protective groups described in example 15 of U.S. Patent 5118678, which is mentioned here for details.

After esterification of the group at 31 position and removal of the protective groups in 42 position, group 42 position can be atrificial using Alliluyeva agent other than Alliluyeva agent, which is used to interact with the alcohol group in the 31st position, to obtain compounds having different ester groups 31 and 42 positions. Otherwise compounds having esterified group 42 position and obtained in the above way, you can interact with another allermuir agent to produce compounds having different ester groups 31 and 42 positions.

The present invention also covers analogous esters of hydroxy acids, obtained by using other rapamycins, such as 29-dimethoxyaniline (U.S. Patent 4375464, 32-dimethoxyaniline nomenclature S. A.); derivatives of rapamycin, in which the double bond in the 1-, 3 - and/or 5 positions were restored (see U.S. Patent 5023262); 29-desmethyltramadol (see U.S. Patent 5093339, i.e. 32-desmethyltramadol on the nomen is 15-hydroxyapatite (see U.S. patent 5102876). These patents are mentioned here for information.

Immunosuppressive activity of individual compounds according to the invention was determined by standard pharmacological test in vitro, during which measured the inhibition of lymphocyte proliferation test (LAF), and using two standard pharmacological tests in vivo. Test using a skin graft allows you to measure IMMUNOSUPRESSIVE activity of the test compound and the ability of the test compound to inhibit or treat graft rejection. Standard pharmacological test on adjuvant arthritis allows you to measure the ability of test compounds to inhibit inflammation, passed by the immune system. The test adjuvant arthritis is a standard pharmacological test for rheumatoid arthritis. The procedure of these standard pharmacological tests described below.

The proliferation of thymocytes induced by somitogenesis (i.e., LAF), was used to measure the in vitro immunosuppressive effects of representative compounds according to the invention.

Cells from the thymus of normal BALB/C mice were cultured for 72 hours in the United. Cells were cultured with rapamycin, cyclosporin A or the test compound in various concentrations, and without them. Then, the collected cells grown in culture, and determine the added radioactivity. Inhibition of lymphoproliferative was defined as the percentage change in counts per minute in comparison with the control cells grown without treatment drugs. Performed data mapping for rapamycin with data on each of the above compounds. IC50was determined for each test compound, and rapamycin. In comparison with representative compounds according to the invention rapamycin had IC50in the range of 0.6 to 1.5 nm. The results presented in figures IC50and as percentage of inhibition of proliferation of T cells at 0.1 μm. The results obtained for representative compounds according to the invention, were also expressed in comparison with data on rapamycin. A positive value for this ratio indicates immunosuppressive activity. A ratio above 1 indicates that the test compound inhibits proliferation of thymocytes stronger than rapamycin. The calculation of the specified ratio pok is statiuni compounds according to the invention were also carried out in vivo tests, designed to determine the duration of survival of skin grafts taken from male BALB/c mice and transplanted into male recipients C3H(H-2K). The method was an adaptation of the method described by R. E. Billingham and Medawar, P. B. J. Exp.Biol. 28:385-402, (1951). Briefly, a skin graft for transplantation from a donor were transplanted on the back of the recipient as an allogeneic transplant, and istranslated was used as a control on the same plot. Recipients were injected with different concentrations of the tested compounds intraperitoneally or orally. As control was used rapamycin. Recipients not receiving treatment, was used as a control graft rejection. The graft was observed daily and the observations recorded up until the graft has not dried up and were not formed a dark scab. This day was considered a day of rejection. The average term survival of transplant (number of days standard deviation) in the group exposed to treatment, compared with the control group. The following table shows the results obtained. The results are expressed in the form of medium term vigilan is a group of 6-7 days. Compound was administered at a dose of 4 mg/kg

Standard pharmacological test on adjuvant arthritis allows you to measure the ability of test compounds to prevent allergic inflammation and to inhibit or cure rheumatoid arthritis. Used testing method described below. Rats (male insidiously rats Wistar Lewis) had previously introduced the compound to be tested (1 hour before antigen), and then in the right hind paw of each rat was injected full adjuvant Freud (FCA) to induce arthritis. Then on Mondays, Wednesdays and Fridays rats orally was administered the drug, starting in the 0-14 day and have introduced a total of 7 doses. Measurements of both hind paws was carried out on 16, 23 and 30 days. Was determined by the difference in paw volume (ml) from the 16th on day 0, and then were compared with the control and displays the percentage change in volume. Inflammation in the left hind paw (where injections were not) caused by inflammation of the transferred T-cells; the results are shown in the above table (% change compared to control). Inflammation in the right rear paw, on the other hand, are caused by nonspecific inflammation. Compounds were tested at a dose of 5 mg/kg Results above negative value percentage change, especially strong is the connection. Rapamycin gave values from -70% to -90% change in comparison with control, and this suggests that rats which were administered rapamycin, 70-90% less than it was contracted allergic inflammation than rats in the control group.

The results of these standard pharmacological tests, are presented after the description of the method of obtaining the individual connections that have been checked. The results of these standard pharmacological tests show that the compounds according to the invention possess immunosuppressive activity both in vitro and in vivo.

The results obtained in the tests LAF indicate suppression of proliferation of T cells, demonstrating the immunosuppressive activity of the compounds according to the invention. Further, the possibility of using compounds according to the invention as immunosuppressive agents showed the results obtained in the standard pharmacological tests on skin grafts and adjuvant arthritis. Test results grafts demonstrated once again the ability of the compounds according to the invention to treat or suppress rejection after transplantation. Raza is soedinenii according to the invention to treat or suppress rheumatoid arthritis.

Based on the results of these standard pharmacological tests, the compounds according to the invention should be considered as suitable for the treatment or inhibition of rejection in the transplantation of such organs as the kidneys, heart, liver, lungs, bone marrow, pancreas (islet cells), cornea, small intestine, and skin grafts and heart valve xenografts; in the treatment or suppression of autoimmune diseases such as lupus, rheumatoid arthritis, diabetes, myasthenia gravis and multiple sclerosis; and inflammations, such as psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel, inflammation of the lung (including asthma, chronic obstructive pulmonary disease, emphysema, acute respiratory diseases, bronchitis, etc), and uveitis.

Based on the obtained profile activity is also considered that the compounds of the present invention have antitumor and antifungal activity, and antiproliferative action. Therefore, the compounds according to the invention are suitable for the treatment of solid tumors, leukemia/lymphoma adult T-cell, fungal infections, and hyperproliferative vascular diseases such as restenosis and placed restenosis, which occurs after angioplasticheskih operations. In this case, the compounds according to the invention can be entered before surgery, during surgery, immediately after surgery, or to use any combination of the above techniques.

In the treatment or inhibition of the above illnesses compounds according to the invention it is possible to enter the mammal orally, parenterally, through the nose, vnutribronhialno, transdermally, topically, vnutrivaginalno or rectally.

It is assumed that when the compounds according to the invention are used as immunosuppressive or anti-inflammatory agents, they can be administered in combination with one or more other immunoregulatory. Among these immunoregulation azathioprine, corticosteroids such as prednisolone and methylprednisolone, cyclophosphamide, rapamycin, cyclosporin A, FK-506, OCT-3, antithymocyte globulin (ATG). Combining the compounds according to the invention with such drugs or agents to cause immunosuppression or for the treatment of inflammatory processes, smaller amounts of each agent will be required to achieve the desired result. The basics of combinatorial therapies have been developed Stepko is their doses significantly increased the lifespan of a heart transplant (Transplantation Proc. 23:507 (1991)).

Compounds according to the invention it is possible to enter a mammal in pure form or together with pharmaceutical carriers. The pharmaceutical carrier may be solid or liquid. It was found that for the preparation of oral compositions can use a mixture of 0.01% Tween 80 in PHOSAL, PG-50 (phospholipid concentrate 1,2-propylene glycol, manufactured by "A. Nattermann &Cie. GmbH".

A solid carrier can include substances that also act as flavoring agents, lubricants, diluents, suspendida agents, fillers, substances that enhance the slide, auxiliary substances, for pressing, binding agents or agents stimulating the dissolution of the tablets; the solid support may be a material for the manufacture of capsules. In powder compositions, the carrier is a finely ground substance, mixed with finely ground active ingredient. In tablets, the active ingredient in appropriate proportions mixed with a carrier, which has the properties required for pressing, and compressed into tablets of desired shape and size. The powders and tablets preferably contain up to 99% of active ingredient. Suitable tomal, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, waxes with low melting temperature and ion-exchange resin.

Liquid media used for preparation of solutions, suspensions, emulsions, syrups, elixirs and compositions under pressure. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture or pharmaceutically acceptable oils or fats. The liquid carrier can contain other pharmaceutically acceptable additives such as diluents, emulsifiers, buffers, preservatives, classtitle, flavorings, suspendresume agents, thickeners, dyes, viscosity regulators, stabilizers or osmoregulatory. Suitable examples of liquid carriers for oral and parenteral products include water (partially containing the above additives, e.g., derivatives of cellulose, preferably a solution of sodium carboxymethylcellulose), alcohols (including monatomic and polyatomic alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut butter). Carrier for parenteral purposes also can is used in a sterile liquid compositions for parenteral purposes. The liquid carrier for the compositions under pressure can be a halogenated hydrocarbon or other dispersant.

Liquid pharmaceutical compositions representing sterile solutions or suspensions can be used, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously.

The connection in the form of liquid or in the form of solid compositions can be assigned for oral administration. Compounds of the present invention can be entered rectally in the form of regular candles. For insertion through the nose or intrabronchial inhalation or dowani, the compounds according to the invention can be used in the form of an aqueous or partially aqueous solution, which is used in the form of aerosols. Compounds according to the invention can also enter through percutaneous applying to the skin a composition containing the active compounds and the carrier that is inert to the active compound, is non-toxic to the skin and ensures the delivery agent for absorption into the bloodstream through the skin. The carrier may have a different shape, for example, be a cream, ointment, paste, gel and a device for delivery of the composition into the blood through the skin. The crea "water-in - oil". Suitable pastes consisting of absorptive powders dispersed in petroleum jelly, or hydrophilic petrolatum containing the active ingredient. Various devices can be used to release the active ingredient in the blood, such as palanimanickam membrane covering a reservoir containing the active ingredient with the carrier or without it, or a matrix containing the active ingredient. Described in literature and other devices.

In addition, the compounds according to the invention can be applied in the form of solutions, creams or lotions, which are prepared in compositions with pharmaceutically acceptable carriers, containing 0.1-5%, preferably 2% active compounds, which compositions are applied to areas affected by fungi.

Requirements dosages are different for each specific composition and depend on the method of administration, the severity of the disease and the patient's condition. On the basis of the results obtained in the standard pharmacological tests, the suggested daily dose is 0.1 mg/kg to 100 mg/kg, preferably in the range of 0.001-25 mg/kg, more preferably in the range of 0.001-5 mg/kg Treatment usually begin with small doses, below the optimal dose is eskers for oral, injecting, injection through the nose or intrabronchial injection are set by the physician based on experience of treating the individual patient. It is preferable to apply the pharmaceutical composition in the form of individual doses, e.g., in the form of tablets or capsules. In this form, the composition is divided into individual doses, containing the desired amount of the active ingredient; single dose can be in separate packages, for example, powders, vials, ampoules, prefilled syringes or sachets containing liquids. One dose may be a single capsule or tablet, or you can assign the desired number of capsules or tablets.

The following examples illustrate the preparation of representative compounds of the present invention and their biological activity.

Example 1

42-ester of rapamycin (tetrahydropyran-2-yloxy) acetic acid

2,4,6-trichlorobenzoyl (0,55 ml, 3,51 mmol) was added via syringe to the solution tetrahydropyranol ether of glycolic acid (0,562 g, 3,51 mmol) and triethylamine (0,49 ml, 3,51 mmol) in 10 ml of tetrahydrofuran at 0oC in nitrogen atmosphere. The mixture was stirred for 4 hours at room temperature the ke of nitrogen in a warm water bath. The residue was dissolved in 10 ml of benzene, and then added rapamycin (2,92 g, 3,19 mmol) and dimethylaminopyridine (0,429 g, 3,51 mmol) and the mixture was stirred over night at room temperature. The mixture was diluted with EtOAc, washed with cold 1N HCl solution (water), saturated NaHCO3(water) and saline solution, dried MgSO4, filtered and concentrated to obtain an oily solid yellow color. After flash chromatography (2X with 65% EtOAc-hexane) was obtained the desired compound (1,114 g, 33%) as a white solid.

(-) FAB-MS) m/z 1055,5 (M-), 590,3 (southern fragment), 463,2 (Northern fragment).

1H NMR (400 MHz, d-6 sulfoxide 4,60 (m, 1H, C(42)H), of 4.66 (m, 1H), 4,14 (s, 2H), to 3.73 (m, 1H), 3,42 (m, 1H).

13C NMR (100,6 MHz, d-6 sulfoxide 169,2, 97,4, 63,5, 61,2, 29,7, 24,8, 18,8.

Example 2

42-ester of rapamycin with exucuse acid

p-Tolilsulfonil (10 mg) was added to a solution of the product from Example 1 (306 mg, 0.29 mmol) in 10 ml of CH3OH at 0oC. the Solution was stirred 2 hours at room temperature, then stopped the reaction with a saturated solution of NaHCO3. The aqueous phase was extracted 3X EtOAc and the combined organic phases are washed with brine, dried MgSO4590 (southern fragment), 379,1 (Northern fragment).

1H NMR (400 MHz, d-6 sulfoxide) 4,60 (m, 1H, C(42)H), 3,98 (s, 2H).

13C NMR (100,6 MHz, d-6 sulfoxide) 172,1, 59,7.

The results obtained in the standard pharmacological tests:

LAF IC50: 1,80 nm

The ratio LAF: 0,83

The percentage change in the test on adjuvant arthritis in comparison with the control:

- 88%

Example 3

42-ester of rapamycin with 2,2-dimethyl-3-(tetrahydropyran-2-yloxy)propionic acid

In the solution tetrahydropyranol ether of 2,2-dimethyl-3-oxopropanoic acid (0,319 g, was 1.58 mmol) and triethylamine (0,22 ml, was 1.58 mmol) in 5 ml dry tetrahydrofuran at 0oC in an atmosphere of nitrogen was added 2,4,6-trichlorobenzoyl (0.25 ml, was 1.58 mmol), additive produced drops from a syringe. The mixture was stirred for 4.5 hours at room temperature. A white precipitate was removed by vacuum filtration and the filtrate was concentrated using a stream of nitrogen in a warm water bath. The residue was dissolved in 5 ml of benzene are then added rapamycin (1.31 g, 1,43 mmol) and dimethylaminopyridine (0,193 g, was 1.58 mmol). The mixture was stirred over night at room temperature, diluted with EtOAc, washed with 1N HCl (aqueous), a saturated solution of NaHCO3(water), HGogo substances yellow. After flash chromatography (1X with 60% EtOAc-hexane, 1X 55% EtOAc-hexane) was obtained the desired compound (0,356 g, 23%) as a white solid.

(-) FAB-MS m/z 1097,7 (M-), 590,4 (southern fragment).

1H NMR (400 MHz, d-6 sulfoxide) 4,55 (m, 1H, C(42)H) 4,55 (m, 1H), 3,69 (m, 1H), 3,60 (m, 2H), 3,42 (m, 1H), 1,13 (s, 3H), 1,11 (s, 3H).

13C NMR (100,6 MHz, d-6 sulfoxide) 175,0, 98,0, 73,8, 60,7, 42,6, 30,0, 24,9, 22,0, 21,6, 18,7.

The results obtained in the standard pharmacological tests:

LAF IC50: 7,10 nm

The ratio of LAP: 0,34

Example 4

42-ester of rapamycin with 3-hydroxy-2,2-dimethylpropionic acid

p-Toluensulfonate (10 mg) was added to a solution of the product from Example 3 (250 mg, 0.23 mmol) in 10 ml of CH3OH at 0oC. the Solution was stirred 2 hours at room temperature, then stopped the reaction with a saturated solution of NaHCO3. The aqueous phase was extracted 3X EtOAc and the combined organic phases are washed with brine, dried MgSO4, was filtered and was concentrated to yield a white solid. After purification with flash chromatography (2X using 75% EtOAc-hexane) was obtained the desired compound (103 mg, 45%) as a white solid.

(-) FAB-MS m/z 1013,3 (M-), 590,2 (South s, 6H).

13C NMR (100,6 MHz, d-6 sulfoxide) 175,5, 68,0, 44,1, 21,7.

The results obtained in the standard pharmacological tests:

LAF IC50: 0,80 nm

The ratio LAF: 1,25

The time of survival of skin graft: 10,70,5 days.

Examples 5 and 6

42-ester of rapamycin with 2,2-dimethyl(1,3)dioxolan-4-carboxylic acid (Example 5)

31,42-W rapamycin with 2,2-dimethyl(1,3)dioxolan-4-carboxylic (Example 6)

2,4,6-trichlorobenzoyl high (0.56 ml, 3.61 mmol) was added via syringe to a solution of isopropylidene 2,3-dissipational acid (0,527 g, 3.61 mmol) and triethylamine (0,50 ml, 3.61 mmol) in 10 ml of tetrahydrofuran at 0oC in nitrogen atmosphere. The mixture was stirred for 4 hours at room temperature. A white precipitate was removed by vacuum filtration and the filtrate was concentrated using a stream of nitrogen in a warm water bath. The residue was dissolved in 15 ml of benzene are then added rapamycin (3.00 g, or 3.28 mmol) and dimethylaminopyridine (0,441 g, 3.61 mmol) and the mixture was stirred over night at room temperature. The mixture was diluted with EtOAc, washed with cold 1N HCl solution (water), saturated NaHCO3(water) and saline solution, dried MgSO4, filtered and concentrated to obtain San) has received the required connections. The less polar compound with an ether group at 31, 42 positions (0,415 g) was suirable first, and the more polar compound with monoufia 42 position (0,601 g, 16%) was suirable second, and identified these compounds as white solids.

Example 5

(-) FAB-MS) m/z 1041,4 (M-), 590,3 (southern fragment), 449,2 (Northern fragment).

1H NMR (400 MHz, d-6 dimethyl sulfoxide) to 4.6 (m, 1H, C(42)H), and 4.6 (m, 1H), 4,20 (dd, 1H), 3.96 points (m, 1H), 1,36 (s, 3H), of 1.30 (s, 3H).

13C NMR (100,6 MHz, d-6 sulfoxide) 170,5, 110,2, 73,4, 66,6, 25,7, 25,4.

Example 6

(-) FAB-MS m/z 1169,6 (M-).

1H NMR (400 MHz, d-6 dimethyl sulfoxide) to 5.3 (m, 1H, C(31)H), and 4.6 (m, 1H, C(42)H), and 4.6 (m, 2H), 4,19 (t, 1H), 4,13 (t, 1H), 3,9 (m, 2H), 1,36 (s, 3H), of 1.33 (s, 3H), of 1.30 (s, 3H), of 1.28 (s, 3H).

13C NMR (100,6 MHz, d-6 dimethysulfoxide) 170,5, 169,2, 110,3, 110,2, 73,4, 66,6, 66,5, 25,8, 25,7, 25,4, 25,1.

The results obtained in the standard pharmacological tests:

Example 5

LAF IC50: 1,20 nm

The ratio LAF: 0,74

Example 6

LAF IC50: 1,30 nm

The ratio LAF: 0,5.

Example 7

42-ester of rapamycin with 2,3-dissipational acid

A solution of the product from Example 5 (351 mg, 0.34 mmol) in 10 ml of tetrahydrofuran and 10 ml of 1N HCl was stirred at room temperature for 6 hours. A mixture of RA and concentrated to obtain oil. After purification with flash chromatography (1X using EtOAc, 1X using a 10% MeOH-CH2Cl2, 1X with 5% MeOH-EtOAc) was obtained the desired compound (78 g, 23%) as a white solid.

(-) PAB-MS m/z 1001,2 (M-), 590,2 (southern fragment), 409,1 (Northern fragment).

1H NMR (400 MHz, d-6 dimethyl sulfoxide) to 4.5 (m, 1H, C(42)H), of 3.60 (m, 1H), of 3.45 (m, 2H).

The results obtained in the standard pharmacological tests:

LAF IC50: 1,4 nm

The ratio LAF: 0,40

Example 8

42-ester of rapamycin with 2,2-dimethyl(1,3)dioxane-5-carboxylic acid

2,4,6-trichlorobenzoyl (0,98 ml, 6,27 mmol) was added via syringe to a solution of isopropylidene 2-(hydroxymethyl)-3-oxopropanoic acid (1,000 g, 6,24 mmol) and triethylamine (0,90 ml, 6,46 mmol) in 20 ml of tetrahydrofuran at 0oC in nitrogen atmosphere. The mixture was stirred for 4 hours at room temperature before the formation of white precipitate. A white precipitate was removed by vacuum filtration and the filtrate was concentrated using a stream of nitrogen in a warm water bath. The residue was dissolved in 20 ml of benzene are then added rapamycin (5,70 g, 6,24 mmol) and dimethylaminopyridine (0,762 g, 6,24 mmol) and the mixture was stirred over night at room temperature. The mixture was diluted with EtOAc, washing the solid substance. After flash chromatography (75% EtOAc-hexane) was obtained the desired compound (4,17 g, 63%) as a white solid.

(-) FAB-MS m/z 1055,8 (M-), 590,5 (southern fragment), 463,4 (Northern fragment).

1H NMR (400 MHz, d-6 sulfoxide) 4,55 (m, 1H, C(42)H) 3,95 (m, 4H), of 1.30 (s, 6H).

13C NMR (100,6 MHz, d-6 sulfoxide) 170,1, 97,4, 59,5, 24,8, 22,5.

The results obtained in the standard pharmacological tests:

LAF IC50: 0,76 nm

The ratio LAF: 0,45

Example 9

42-ester of rapamycin with 3-hydroxy-2-ximetilpropan acid

A solution of the product from Example 8 (3,30 g of 3.12 mmol) in 50 ml of tetrahydrofuran and 25 ml of 1N HCI was stirred at room temperature for 2 hours. The mixture was diluted with saturated solution of NaHCO3and was extracted with EtOAc (3X). The combined organic phases are washed with saturated NaCl (aqueous), dried MgSO4, was filtered and was concentrated to obtain a yellow foam. After purification with flash chromatography (1X using EtOAc, 2X using 5% EtOH-EtOAc) was obtained the desired compound (1.68 g, 53%) as a white solid.

(-) FAB-MS, m/z 1015,5 (M-), 590,3 (southern fragment), 423,3 (Northern fragment).

1H NMR (400 MHz, d-6 sulfoxide) 4,6 (brs, Results, obtained in the standard pharmacological tests:

LAF IC50: 0,84 nm

The ratio LAF: 0,57.

Example 10

42-ester of rapamycin with 2,2,5-trimethyl(1,3)dioxane-5-carboxylic acid

In the solution isopropylidenebis 2,2-bis(oxymethyl)propionic acid (1.041 g, 5,98 mmol) (prepared according to the method described by Bruice, J. Am. Chem. Soc. 89: 3568 (1967)) and triethylamine (0,83 ml, 5,98 mmol) in 20 ml of anhydrous tetrahydrofuran at 0oC in an atmosphere of nitrogen was added 2,4,6-trichlorobenzoyl (0,93 ml, 5,98 mmol) and the resulting white suspension was stirred for 5 hours at room temperature. The precipitate was removed by vacuum filtration, rinsing the flask and the precipitate on the filter with additional quantity (10 ml) of dry tetrahydrofuran. The filtrate was concentrated in a rotary evaporator to yield a white solid. The residue was dissolved in 20 ml of dry benzene was then added rapamycin (vs. 5.47 g, 5,98 mmol) and dimethylaminopyridine (0,731 g, 5,98 mmol). After stirring over night at room temperature the mixture was diluted with EtOAc, washed with H2O and saturated NaCl (aqueous), dried MgSO4, filtered and evaporated to obtain a yellow oil. After flash chromatography (5X 60% EtOAc-hexane) received the claim 477,2 (Northern fragment).

1H NMR (400 MHz, d-6 sulfoxide) of 4.57 (m, 1H, C(42)H), was 4.02 (d, 2H), 3,60 (d, 2H), of 1.34 (s, 3H), 1,24 (s, 3H), 1.06 a (s, 3H).

13C NMR (100,6 MHz, d-6 sulfoxide) 173,2, 99,0, 65,0, 22,2, 18,1.

The results obtained in the standard pharmacological tests:

LAF IC50: 4,90 nm

The ratio LAF: 0,41

Term survival of skin graft: 11,01,3 day.

Example 11

42-ester of rapamycin with 2,2-bis-(oxymethyl)propionic acid

A solution of the product from Example 10 (2.8 g, to 2.65 mmol) in 50 ml of tetrahydrofuran and 25 ml of 1N HCl was stirred at room temperature for 4 hours. The mixture was diluted with water and three times was extracted with EtOAc. The combined organic phases are washed with saturated solution of NaHCO3saturated NaCl solution, dried MgSO4, was filtered and evaporated to obtain a yellow oily solid. After purification with flash chromatography (3X using EtOAc) was obtained the desired compound (1.6 g, 59%).

(-) FAB-MS m/z 1029,6 (M-), 590,4 (southern fragment), 437,3 (Northern fragment).

1H NMR (400 MHz, d-6 dimethyl sulfoxide) to 4.5 (m, 1H, C(42)H) to 3.45 (s, 4H), was 1.04 (s, 3H).

13C NMR (100,6 MHz, d-6 sulfoxide) 174,2, 63,7, 63,6, 49,9, 16,8.

The results obtained in the standard farm is transplantat: 11,41,5 and 12,01,1 days.

Percentage change in adjuvant arthritis in comparison with the control: -88%.

Example 12

42-ester of rapamycin with 2,2-dimethyl-5-(2-trimethylsily-dilatometer) (1,3)dioxane-5-carboxylic acid

2,4,6-trichlorobenzoyl (0,14 ml, 0.86 mmol) via syringe is added into the solution isopropylidenebis 2,2-bis-(hydroxymethyl)-2-(2-trimethylsilyloxy) propionic acid (0,250 g, 0.86 mmol) and triethylamine (of 0.12 ml, 0.86 mmol) in 2 ml of tetrahydrofuran at 0oC in nitrogen atmosphere. The mixture was stirred for 4 hours at room temperature before the formation of white precipitate. A white precipitate was removed by vacuum filtration, the filtrate was concentrated using a stream of nitrogen in a warm water bath. The residue was dissolved in 20 ml of benzene was then added rapamycin (of 0.786 g, 0.86 mmol) and stirred the mixture overnight at room temperature. The mixture was diluted with EtOAc, washed with H2O and brine solution, dried MgSO4was filtered and concentrated to obtain a yellow solid. After flash chromatography (gradient elution: 40-60-80-100% EtOAc-hexane) was obtained the desired compound (0,559 g, 54%) as a white solid.

(-) FAB-MS m/z 1185,2 (M-), 590,1 (southern fragment), 593 (Northern fragment).

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13C NMR (100,6 MHz, d-6 sulfoxide) 171,1, 97,7, 70,2, 68,1, 61,3, 46,0, 24,6, 22,1, 14,6, -1,3.

The results obtained in the standard pharmacological tests:

LAF IC50: 7,20 nm

The ratio LAF: 0,05.

Examples 13 and 14

42-ester of rapamycin with 3-methyl-1,5-dioxaspiro(5,5)undecane-3-carboxylic acid (Example 13) and

31,42-W rapamycin with 3-methyl-1,5-dioxa - Spiro(5,5)undecane-3-carboxylic acid (Example 14)

2,4,6-trichlorobenzoyl (0.16 ml, 1.0 mmol) via syringe is added into the solution cyclohexylidene 2,3-dissipational acid (0,214 g, 1.0 mmol) and triethylamine (of 0.14 ml, 1.0 mmol) in 2.5 ml of tetrahydrofuran at 0oC in nitrogen atmosphere. The mixture was stirred for 4 hours at room temperature. A white precipitate was removed by vacuum filtration, the filtrate was concentrated using a stream of nitrogen in a warm water bath. The residue was dissolved in 3 ml of benzene was then added rapamycin (0,457 g, 0.5 mmol) and dimethylaminopyridine (0,061 g, 0.5 mmol) and stirred the mixture overnight at room temperature. The mixture was diluted with EtOAc, cold IN HCl solution (of waves.), a saturated solution of NaHCO3(water), and with brine, dried MgSO4, filtered and concentrated to obtain a yellow foam. On the deposits (has 0.168 g, 26%) were suirable first, and the more polar compound with monoamino group 42 position (0,301 g, 52%) was suirable second, and highlight the products as white solids.

Example 13

(-) FAB-MS m/z 1109.5 (M-), 590,3 (southern fragment), 517,3 (Northern fragment).

1H NMR (400 MHz, d-6 sulfoxide) 4,55 (m, 1H, C(42)H), 3,61 (t, 4H), was 1.04 (s, 3H).

13C NMR (100,6 MHz, d-6 dimethyl sulfoxide) to 173.3, 97,2, 64,2.

Example 14

(-) FAB-MS m/z 1305,6 (M-).

1H NMR (400 MHz, d-6 sulfoxide) a 5.25 (m, 1H, C(31)H) 4,55 (m, 1H, C(42)H), 3,64-of 3.54 (m, 8H), of 1.05 (s, 3H), of 0.97 (s, 3H).

13C NMR (100,6 MHz, d-6 sulfoxide) 173,2, 172,1, 97,3, 97,2, 64,3, 64,2, 63,9.

The results obtained in the standard pharmacological tests:

Example 13

LAF IC50: 0,6 nm

The ratio LAF: 2,00.

Example 14

LAF: inhibited the proliferation of T-cells by 43% at 0.1 ám.

1. Hydroxyether rapamycin General formula I

< / BR>
where each of R1and R2independently represent hydrogen or- (CR3R4)b(CR5R6)dCR7R8R9;

each of R3and R4independently, represent hydrogen, alkyl with 1-6 carbon atoms;

each of R
R7represents hydrogen, alkyl with 1-6 carbon atoms, (CR3R4)fOR SIG10;

each of R8and R9independently, represent hydrogen, alkyl with 1-6 carbon atoms, -(CR3R4)fOR SIG10or R8and R9may together form a group X or cycloalkyl ring of 3-8 carbon atoms;

R10represents hydrogen, alkyl with 1-6 carbon atoms, three(alkyl with 1-6 carbon atoms) silyl, three(alkyl with 1-6 carbon atoms) silylated, or tetrahydropyranyl;

X represents 5-(2,2-di(alkyl with 1-6 carbon atoms)) (1,3) dioxane, 5-(2-Spiro(cycloalkyl with 3-8 carbon atoms)) (1,3) dioxane, 4-(2,2-di(alkyl with 1-6 carbon atoms)) (1,3)-dioxane, 4-(2-Spiro(cycloalkyl with 3-8 carbon atoms)) (1,3) dioxane;

b = 0-6;

d = 0-6;

f = 0-6,

provided that R1and R2do not simultaneously represent hydrogen, and provided that either R1or R2contains at least one (CR3R4)fOR SIG10X or cycloalkyl with 3-8 carbon atoms, or a pharmaceutically acceptable salt of this compound.

2. Connection on p. 1, where R2represents hydrogen, or its pharmaceutically acceptable with whom soedineniya.

4. Connection on p. 3, where each of R8and R9independently, represent hydrogen, alkyl, or -(CR3R4)fOR SIG10or together form X, or pharmaceutically acceptable salt of this compound.

5. Connection on p. 1, representing rapamycin, with 42 position ether (tetrahydropyran-2-yloxy) acetic acid, or a pharmaceutically acceptable salt of this compound.

6. Connection on p. 1, representing rapamycin, with 42 position the ether exucuse acid, or a pharmaceutically acceptable salt of this compound.

7. Connection on p. 1, representing a 42-ester of rapamycin 2,2-dimethyl-3-(tetrahydropyran-2-yloxy) propionic acid, or a pharmaceutically acceptable salt of this compound.

8. Connection on p. 1, representing a 42-ester of rapamycin with 3-hydroxy-2,2-dimethyl-propionic acid or pharmaceutically acceptable salt of this compound.

9. Connection on p. 1, representing a 42-ester of rapamycin with 2,2-dimethyl-(1,3)dioxolan-4-carboxylic acid, or a pharmaceutically acceptable salt of this compound.

10. Connection on p. 1, representing 31,41-W rapamycin with 2,2-dimethyl-(1,3) is tion under item 1, representing a 42-ester of rapamycin with 2,3-dihydroxypropane acid, or a pharmaceutically acceptable salt of this compound.

12. Connection on p. 1, representing a 42-ester of rapamycin with 2,2-dimethyl-(1,3)dioxane-5-carboxylic acid, or a pharmaceutically acceptable salt of this compound.

13. Connection on p. 1, representing a 42-ester of rapamycin with 3-hydroxy-2-hydroxymethylpropane acid, or a pharmaceutically acceptable salt of this compound.

14. Connection on p. 1, representing a 42-ester of rapamycin 2,2,5-trimethyl-(1,3)dioxane-5-carboxylic acid, or a pharmaceutically acceptable salt of this compound.

15. Connection on p. 1, representing a 42-ester of rapamycin with 2,2-bis(hydroxymethyl)propionic acid, or a pharmaceutically acceptable salt of this compound.

16. Connection on p. 1, representing a 42-ester of rapamycin with 2,2-dimethyl-5-(2-trimethylsilylethynyl) (1,3) dioxane-5-carboxylic acid, or a pharmaceutically acceptable salt of this compound.

17. Connection on p. 1, representing a 42-ester of rapamycin with 3-methyl-1,5-dioxa-Spiro(5,5)undecane-3-carboxylic acid, or a pharmaceutically acceptable salt of this with(5,5)undecane-3-carboxylic acid, or pharmaceutically acceptable salt of this compound.

19. The method of treatment of a condition associated with the incompatibility reaction in transplantation in a mammal in need of such treatment, comprising assigning the specified mammal an effective amount of the compounds of formula I under item 1 or a pharmaceutically acceptable salt of this compound.

20. A method of treating rheumatoid arthritis in a mammal in need of such treatment, which involves assigning a specified mammal an effective amount of the compounds of formula I under item 1 or a pharmaceutically acceptable salt of this compound.

21. The method of producing hydroxyamino rapamycin General formula I on p. 1 consists in the fact that rapamycin or its functional derivative is subjected to interaction with allermuir agent of formula II

BUT-WITH(CR3R4)b(CR5R6)dCR7R8R9,

in which R3- R9, b and d have the meanings given in paragraph 1,

provided that the free hydroxyl group is protected and optionally protected group under 42 position rapamycin or its functional derivative, using correspond to the lo must.

 

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