UA73761C2 - Carboxamidine for treatment of viral hepatitis - Google Patents

Abstract

Novel nucleoside analog compounds are disclosed. The novel compounds or pharmaceutically acceptable esters or salts thereof may be used in pharmaceutical compositions, and such compositions may be used to treat an infection, an infestation, a neoplasm, or an autoimmune disease. The novel compounds may also be used to modulate aspects of the immune system, including modulation of Type 1 and Type 2 activity. EMBED ISISServer

Description

Description of the invention

This application claims the priority of prior US Patent Application No. 60/182676, filed Feb. 15, 2000, and US Application No. 09/595365, filed June 16, 2000, each of which is incorporated herein by reference in its entirety.

This invention relates to the field of nucleoside analogues.

Ribavirin (1-4-O-ribofuranosyl-1,2,4-triazole-Z-carboxamide) is a nucleoside analog that has demonstrated efficacy in the treatment of viral diseases as monotherapy (respiratory syncytial virus, Nayi S.V., MeVgide 9. T., Muaiivy E.E., Ve OO. M., Saia S. M., Niyidgesfy 5., Tep EusK Y. 0., MU. 3.

NaiYi Aegozoiilei hiramihip igeaitepi oi iptapiv m/y gezrigaogu zupsuya! miga! ipbesiop M. Eepoii. U. Mea. 1983, 308, 1443-1447), and during therapy in combination with interferon-alpha (hepatitis C virus, Keisnaga,

O., MogKkgape, O., Egudep, A., Vgasopieg, U-N., boppegrogu, A., UUeyapa, O. Kapdotigea, docsbie bBiiipa, riasero sopigoMPei giai oi ipiepiegop airpa 28V myy apa myypoci hiramigiy og spgopis Pperaiiv S. Iapseyi 1998, 351, 83-87). Recently reported studies indicate that the benefit of ribavirin i.p. may be due not only to direct inhibition of viral replication, but may also be due to its ability to enhance T-cell-mediated immunity (Niyogep, C, Miiisiai, 0. К.

In apa S migiv-vresiis ittype gezropvev. in). (bep. Migoii. 1998, 79, 2381 -2391; Mipo, 9O., Vhom/p, O., Ragodo,

U., SatsgaIm, M., Rypo, H., SogsgupeKi, K., SoYie, E., Rypo, H., Oipo, U. MU., ish M. RE., Koivivip, O., Raiyirz

М.

TV2 suioKkipe gezropve. U. Ittipoi. 1998, 160, 3487-3493; Magiip, M. 9U., Mamavz, 5., Ocigoda, 9. A., Ragdo, M,.,

Satepo, M. EPesiv oyi (Pe hiramihip-ipiepegop airpa sotbripayop op syMigei rehirpega! Biood topopisivag s 29 se|v pot spgopis Peraiiz S rayepiv. Suiokipe 1998, 79, 2381-2391). This immunomodulatory effect of ribavirin can be demonstrated by measuring the levels of Type 1 cytokines produced by activated T cells of both humans and mice (Tatp, K. S., Rai, V., Vaga, 9U., et al., S, Amegay, 0. K., Rpap,

OO. To, Maiomapomis, T. Kiramihip roiagilgev Ppitap TT sei gezropvzez isulagaz a Toure 1 suyuKipe rgoye. 4.

NerayI. 1999, 30, 376-382), as well as by using other methods of measurement. Cytokine shift induction -

Type 1 ribavirin is a functionally essential ip mimo in mouse systems (Tat, K. S, it, S, Vaga, 9U., Ra), V..Ts«Oo

Sopiasi Ppuregzepziyimyu gevzropvez oomipud hiramigipy (geaytepi ip mimo age ipysepsey Bu Toure 1 suyukKipe royagigayop, gedsatsyop oi II-10 ekhrezviop apa soziyiti|agu zidpaiipa. 9. Ittipoi. 1999, 163, 3709-3717). -

Mammalian immune systems contain two major classes of lymphocytes: B lymphocytes (B cells), which originate in the bone marrow, and T lymphocytes (T cells), which originate in the thymus. B-cells are mostly responsible for

Humoral immunity (ie, production of antibodies), while T cells are responsible mostly for T cell-mediated immunity.

T cells are generally considered to be divided into two subclasses, helper and cytotoxic T cells.

T cells. Heller T cells activate other lymphocytes, including B cells and cytotoxic T cells, and macrophages by secreting soluble protein mediators called cytokines that participate in C7Z 10 cell-mediated immunity. As used herein, lymphokines are a subpopulation of cytokines. It is also generally believed that helper T cells are divided into two subclasses, Type 1 and Type 2. Type 1 cells produce interleukin 2 (1-2), tumor necrosis factor (TME) and interferon-gamma (IEMU). and are primarily responsible for cell-mediated immunity, such as delayed-type hypersensitivity and antiviral immunity.In contrast, Type 2 cells produce interleukins, 1-4, 1/-5, 11 -6, 11 -9, - 15 HER/-10 and 1/-13, and, above all, participate in the promotion of such humoral immune reactions that are observed in response to allergens, for example, switching the isotope of the antibody ZE and Id04 (Moztapp, 1989, (95) Appy Kem Ittipoi, 7: 145-173).- As used herein, the terms "Type 1 and Type 2 reactions" are intended to include the full range of effects that result from the induction of Type 1 and Type 2 lymphocytes, respectively. Such reactions include (o) 50 include changes in the production of relevant cytokines through transcription, translation, secretion, and possibly mechanisms, increased proliferation of relevant lymphocytes, and other effects associated with increased cytokine production, including motility effects.

Prior US applications (09/462714, 09/291097,09/291093, 09/471513, 60/164365, 60/164366, 60/172097, 60/175111), each of which is incorporated herein by reference, relate to aspects of our recent discoveries , which apply the action of various nucleosides (which, as defined here, include derivatives and analogs of natural

HF) nucleosides) on the reactions of selective modulation of lymphocytes relative to each other. Among other things, we 7 have demonstrated that either a Type 1 or a Type 2 reaction can be selectively inhibited while the other of the two reactions is induced or relatively unaffected, and that either Type 1 or Type 2 reactions can be selectively induced 2, while the other of these two reactions is inhibited or relatively unaffected. We also discovered the surprising fact that some nucleosides that are effective in selectively modulating Type 1 and Type 2 reactions relative to each other tend to produce a bimodal effect.

Among other things, some nucleosides that tend to generally inhibit or induce both Type 1 and Type 2 activity at a relatively higher dose tend to selectively modulate Type 1 and Type 2 relative to each other at relatively lower doses. It has been demonstrated that Migatidipetm (1-8-O-ribofuranosyl-1,2,4-triazole-3-carboxamidine, hydrochloride)

is active against ten different viruses, which is worthy of comparison with ribavirin (U.T. MUykomuvKi, K.K.

Kobrypz, village R. Kpage, K. MU. biimeiY, U. Mei Speth , 16, 935 -937, 1973; MK. MU. Biaimzhei, U. N. Nyitap, 0. GG.

Vagpaga, Y. M. Rigai, Apiimigai Kezeagsp, 10, 193 - 208, 1988; V. SaBbgieizep, M. 9. RIeiap, III. Vapipe!I-Koza, S

Zee, U. MU. Niddipe, O. R. Keiaimeg, T. R. Mopayi, M. A. Ovvegu, b. M. Sptigpu, E. M. Zsp!, beti, K. Oranpua, S

Kumopoa, O. A. Sapeg, U. A. Besgivi P, 9. 9. Kigvi, MU. M. 5papop, K. MU. Bidmzhei!I, p. O. Kipi, K. K. Kobipv, /. Honey. Spet., 35, 3231 -3238, 1992). In addition, Migatidipetm, like Ribavirin, is an inhibitor

IMR-dehydrogenases (KK. S. Muyiv, K. K. Kobypv, U. E. Zeedtysheg, MoiIesciag РНаптасоиоду, 18, 287 - 295, 1980).

In addition, preliminary toxicological studies suggest that Migatidipetm is less toxic than 70 ribavirin (0. M. Rai, MK. MU. Ziyzhei, R. s. Sapopiso, Apiimigai Kezeagsp, 9, 136, 1988). Also, recent experiments in our laboratory (K. Tat, K. Katazatu, ISM RNaptaseciisa!5, Ips., unpublished results, 1999) revealed that Migatidipetm and Ribavirin demonstrated similar immunomodulatory properties. These results, together with the low bioavailability and toxicity of ribavirin, encourage us to develop not only

Migatidipetm for other viral diseases, but also to prepare other Migatidipetm derivatives, including the synthesis of 75 Migatidipetm prodrugs, and to screen them as possible antiviral agents.

The effect of other nucleoside analogue compounds on the responses of selective modulation of lymphocytes relative to each other has not been previously studied or documented. We have discovered that a bimodal effect, or selective modulation of Type 1 and Type 2 responses relative to each other, also occurs after administration of other nucleoside analogue compounds, such as prodrug forms of these compounds.

There are many hurdles to overcome in converting biologically active compounds into clinically useful agents. Many effective biologically active compounds never become clinically useful agents because they have undesirable biopharmaceutical properties, which include low bioavailability, which is a consequence of low ability to penetrate biological barriers such as the blood-brain barrier and the intestinal barrier. Although many factors affect the bioavailability of drugs, undesirable CM physicochemical properties (eg, charge, lipophilicity, hydrogen bonding potential, size) of many drugs are perhaps one of the common factors encountered and which prevent the penetration of drugs through biological barriers. Therefore, optimization of the physicochemical characteristics (charge, lipophilicity, hydrogen bond formation potential, size) of drugs is perhaps the most common strategy for promoting drug transport across such membrane barriers. --

In order to optimize the physicochemical properties of drugs, one possible strategy is to optimize these properties for prodrugs. (N. Vypddaaga, YuOevidp oiRgodgoidv, E|Izemieeg,

Atviegdat, 1985, M. Vodog, I. Rgokai, MU. M. UUm, N. Ragad, 5. dopaizvdadaa, M. Kazhatiga, U. ZitrKipv, Zsiepse, - 257. 1698-1700, 1992, N. E. Tauyug, KK. V. Bioap, /. RNAgpt. zZei., 87, 5-20, 1998). The term "prodrug" is used to describe an agent that must undergo chemical or enzymatic transformation into the active, or parent, drug after administration so that the metabolite or parent drug can further exhibit the desired pharmacological response. Through the temporary and biologically reversible formation of certain polar functional groups in small organic molecules, undesirable physico-chemical characteristics (for example, charge, hydrogen bond formation potential) of these groups were "masked" without changing at "

This constant pharmacological properties of molecules. This strategy has been very successfully applied in cases where the conversion of a carboxyl or hydroxyl functional group into an ester is used to obtain prodrug derivatives, which can be easily hydrolyzed by chemical or enzymatic methods. h Proposing the concept of prodrugs, we anticipate that the introduction of other components into the initial drugs will increase » bioavailability, adsorption and antiviral effects.

Despite the existence of as-yet-undefined mechanisms, we have discovered that enormous potential benefits can be gained by selectively modulating Type 1 and Type 2 responses relative to each other. We have concluded, for example, that specific modulation of Type 71 relative to Type 2 may be useful in the treatment of a variety of conditions and diseases, from infections, invasions, tumors and hypersensitivity to autoimmune diseases.

These discoveries are of particular importance because current treatments for many of the diseases listed above have limited efficacy, significant side effects, or both. Treatment of an autoimmune disease, b50 for example, is often limited to palliative measures, removal of toxic antibodies (as in the case of myasthenia gravis), and the administration of risky drugs, which include corticosteroids, chloroquine derivatives, and antimetabolic "6 and antitumor drugs, and drugs such as cyclosporins, which affect cells of the immune system.

This invention is directed to novel nucleoside analog compounds and related compounds such as prodrugs, their therapeutic use and synthesis. 22 In one aspect of the invention, there are provided compounds of nucleoside analogues of formula 1:

GF! Mn. NSi ko nd

M on on

Formula 1 - Migatidipetm (ISM 3142)

According to another aspect of the invention, the pharmaceutical composition contains a therapeutically effective amount of carboxamidine according to formula 1, or its pharmaceutically acceptable ester or salt in admixture with at least one pharmaceutically acceptable carrier.

According to a further aspect of the invention, the pharmaceutical composition contains a prodrug form of carboxamidine according to formula 1, or its pharmaceutically acceptable ester or salt in admixture with at least one pharmaceutically acceptable carrier.

According to yet another further aspect of the invention, the compound of formula 1 is used in the treatment of any condition which responds positively to the administration of this compound and which, according to any pharmaceutical formulation or administration protocol, achieves a positive response. Among other things, it is envisaged that the compounds of formula 1 can be used for the treatment of infections, invasions, cancer, tumors or other neoplasms, giant cell arteritis or autoimmune diseases. 70 Figure 1 - an example of the synthesis scheme of a compound according to formula 1.

Figure 2 is a graph showing the effect of the predicted and other compounds on the synthesis of Type 1 cytokines in human staphylococcal enterotoxin B (SEB) activated T cells.

Figure C are graphs demonstrating the effect of a concentration of 0.625-10 µM of the intended compound on the synthesis of Type 1 cytokines in activated staphylococcal enterotoxin B human T-cells.

Figure 4 is a graph showing the effect of the subject compound on contact hypersensitivity reactions in VAI V/s mice.

Figure 5 is a graph showing the maximum response and maximum range for the predicted and other compounds in relation to the synthesis of Type 1 cytokines in activated staphylococcal enterotoxin B human T cells.

Where the following terms are used in this description of the invention, they are used as defined below.

The terms "nucleoside" and "nucleoside analog compound" ("nucleoside analog compound") are interchangeable and refer to a compound consisting of any pentose or modified pentose moiety attached to a specific position of a heterocycle, an aromatic heterocycle, or to a naturally occurring position of ov purine (9-position) or pyrimidine (1-position), or to the equivalent position in the analog.

The term "nucleotide" denotes a phosphate ester of a nucleoside substituted at the 5'-position. and)

The term "heterocycle" refers to a monovalent saturated or unsaturated carbocyclic radical having at least one heteroatom, such as M, O or 5, each available position within the ring can be arbitrarily substituted, independently, for example, hydroxy-, oxo-, amino -, imino-l. lower alkyl, bromo-, chloro-"pezo and/or cyano group. Purines, pyrimidines are included in this class of substituents.

The term "purine" refers to nitrogenous bicyclic heterocycles. at

The term "pyrimidine" refers to nitrogenous monocyclic heterocycles. uh-

The term "O-nucleosides" refers to nucleoside compounds having a Y-ribose sugar component (eg, adenosine). and,

The term "I-nucleosides" refers to nucleoside compounds having an I-ribose sugar component. uh-

The terms "I-configuration" and "O-configuration" are used throughout this specification to describe the chemical configuration of the ribofuranosyl component of the compound which is linked to the pyrrolo-pyrimidine portion of the molecule.

The term "C-nucleosides" is used throughout this specification to describe the type of bond formed between a ribose sugar moiety and a heterocyclic base. In C-nucleosides, the bond originates from the position Pd) with C-1 of the ribose sugar component and attaches the carbon of the heterocyclic base. The bond formed in C-nucleosides is of the carbon-carbon type. "" The term "M-nucleosides" is used throughout this specification to describe the type of bond formed between the ribose sugar moiety and the heterocyclic base. In M-nucleosides, the bond originates from the position

C-1 of the ribose sugar component and attaches the nitrogen of the heterocyclic base. The bond formed in -I M-nucleosides is of the carbon-nitrogen type.

The term "protecting group" denotes a chemical group that is added to an oxygen or nitrogen atom to prevent it from further reacting during the formation of derivatives of other components in the molecule in which oxygen or -I nitrogen is located. A wide variety of oxygen and nitrogen protecting groups is known to specialists in the field of organic synthesis.

The term "lower alkyl" means methyl, ethyl, H-propyl, isopropyl, n-butyl, N-butyl, i-butyl or n-hexyl. b This term is also an example of a cyclic, branched or straight chain of one to six carbon atoms.

The term "aryl" refers to a monovalent unsaturated aromatic carbocyclic radical having a single ring (e.g., phenyl) or two fused rings (e.g., naphthyl), which may be arbitrarily

Substituted by hydroxyl, lower alkyl, chloro- and/or cyano group.

The term "heterocycle" refers to a monovalent saturated or unsaturated carbocyclic radical having, (F) at least one heteroatom, such as M, O, 5, Ze or P, within the ring, each available position of which may be arbitrarily substituted or an unsubstituted, independently, hydroxy-, oxo-, amino-, imino-l lower alkyl, bromo-, chloro- and/or cyano group. The term "monocyclic" refers to a monovalent saturated carbocyclic radical having at least one heteroatom, such as o, M, 5, Ze or P, within a ring, each accessible position of which may be optionally substituted, independently, by a sugar moiety or any other groups such as bromo-, chloro- and/or cyano, so , so that the monocyclic core system is eventually aromatized (e.g., thymidine). 65 The terms "immunomodulator" and "modulator" as used herein are used interchangeably to refer to natural or synthetic products capable of modifying the normal or aberrant immune system through stimulation or oppression.

The term "effective amount" refers to an amount of a compound of formula 1 that restores immune function to normal levels or increases immune function compared to normal levels to prevent infection.

Compounds according to formula 1 can have multiple asymmetric centers. Therefore, they can be prepared both in an optically active form and as a racemic mixture. The scope of the invention, as described and claimed in the claims, covers individual optical isomers and their non-racemic mixtures, as well as racemic forms of compounds according to formula 1.

The terms "y" and "b" indicate the specific stereochemical configuration of the substituent on the asymmetric carbon atom in the chemical structure as depicted. 70 The term "enantiomers" refers to a pair of stereoisomers that are non-superimpossible mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture.

The term "isomers" refers to different compounds that have the same formula. "Stereoisomers" are isomers that differ only in the arrangement of atoms in space. "Pharmaceutically acceptable salts" can be any salts derived from inorganic and organic 7/5 Acids or bases.

Compounds

Nucleoside analog compounds of the present invention are generally described by formula 1:

Plural of the era "M on Kv): | see

And what about

Formula 1 - Migatidipetm (ISM 3142) where the chemical configuration of the compound is I-configuration or Y-configuration. An example of the synthesis of the intended compounds (here: Migatidipetm) can be carried out according to the method described below and depicted in Figure 1.

3-Cyano-1-(2,3,5-tri-O-acetyl-pD-B-ribofuranosyl)-1,2,4-triazole (7) --

A mixture of 3-cyano-1,2,4-triazole (18.8 g, 20 mmol) (6), 1,2,3,5-tetra-O-acetyl-D-O-ribofuranose (63.66 g, so 200 mmol) and bis(p-nitrophenyl)phosphate (1g) were placed in a round-bottomed flask (50O0ml). The flask was placed on a preheated oil bath at 165-17592C under vacuum conditions created by a water jet pump, and - and stirred for 25 minutes. The extruded acetic acid was collected by an ice-cooled trap located between a water jet pump and a round bottom flask. The flask was removed from the oil bath and

They let her cool down. When the temperature of the flask reached approximately 60-70 C, EtOAc (300 ml) was introduced and saturated Manso" (150 ml) and extracted with EUAs. The aqueous layer was extracted again with EtOAc (200 mL). The combined EtOAc extract was washed with sat. . The residue was dissolved in ether (10 Oml), as a result of which, after cooling at 02 for 12 hours, colorless 50 crystals were obtained. The solid substance was filtered, washed with a minimum amount of cold EUN (20 ml) and dried with s under high vacuum conditions over solid Mahon. Yield: 56.4g (8095). T.pl. 96-97260, :z» "NMR (SOSIv): 5 2.11 (s, ZN, SOSNU"), 2.13 (s, ZN, SOSNU"), 2.14 (s, ZN, SOSNU"), 4.22 (dd, 1H), 4.46 (m, 2H), 5.52 (t, 1TN, U - 6.0 Gu), 5.70 (m, 1H), 6.01 (d, IN , CH, y - 3.6 Hz) and 8.39 (s, IN, CH). Elemental analysis.

Calculated for C44Ni6MAO (352.30): C, 47.73; N, 4.58; M, 15.90. Found: C, 47.70; H, 4.63; M, 16.01. и , и и - 1-4-O-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (Migathiadipetm) (8)

A mixture of compound 7 (14.08g, 40.0mmol), MNACI (2.14g, 40.0mmol) and anhydrous ammonia (150ml) was heated in a steel bomb at 85923 for 18 hours. The steel bomb was cooled, opened, and its contents evaporated to dryness. The residue was crystallized from MesSM-EUN, as a result of which 10.6 bg (95965) of compound 8 were obtained. 177-179960, b" 50 "NMR (DMSO-аv): 5 3.44-4.2 (m, ЗН), 4.40 (m, 2Н), 5.04 (t, 1Н), 5.29 (m, 1H), 5.74 (m, 1H), 5.87 (d, 1H, CH), 8.96 (s.s., ЗН) and 9.17 (c, 1H, C5H). analysis. Calculated for C8NiISIM5O, (279.68): C, 34.35, H, 5.05, M, -. 25.04, SI, 12.69. Found: C, 34.39, H, 5, 10, M, 25.14, SI 12.71.

Alternatively, the synthesis can be performed from commercially available Kiramihypm according to the following method. 2,3,5-Tri-acetyl-1-4-YOO-ribofuranosyl-1,2,4-triazole-3-carboxamide (9) 29 Suspension of 1-4-O-ribofuranosyl-1,2,4-triazole- Z-carboxamide (Kibramigptm) (28.4g, 116.4mmol) (5) in

HF) acetic anhydride (200ml) and pyridine (HOml) were stirred at room temperature overnight. The resulting clear solution was concentrated under vacuum and a clear foam was obtained (43.1 g, quantitative). This foam was homogenous on thin layer chromatography and was used directly for the next step without prior purification. A small amount was purified by flash chromatography to give an analytical 60 sample. "IN nMR (300 MHz, DMSO-iv) 5 2.01, 2.08, 2.09 (Zs, UZN, SOSNU), 4.10 (m, 1H), 3.52 (m, 2H), 5 ... ), 8.86 (c, 1H, C5H triazole).

Elemental analysis (C40Niv8MaOv) С, Н, М.

Z-Cyano-2",3,5-tri-O-acetyl-1-D-O-ribofuranosyl-1,2,4-triazole (10) 65 In a solution of compound 9 (43.1g, 11b,4mmol) in to chloroform (500 mL) was added triethylamine (244 mL) and the mixture was cooled to 09 C in an ice-salt bath. Phosphoric acid chloride (30.7 mL, 30 mmol) was added dropwise while stirring and the solution was allowed to warm to room temperature. After the mixture was stirred at at room temperature for 1 hour, thin layer chromatography (hexane/acetone, 3:1) indicated complete disappearance of the starting material. The brown reaction mixture was concentrated to dryness under vacuum and the residue was dissolved in chloroform (500 mL). The organic solution was washed with saturated aqueous sodium bicarbonate (3x200ml), dried over anhydrous sodium sulfate and concentrated under vacuum conditions.

The residue was chromatographed on silica gel (flash chromatography), 20950 acetone in hexane to give 33.14 g (8195 from ribavirin) of pure compound 10 as an amorphous solid. This solid 70 was identical in all respects to the genuine sample: M.pl. 101-103 2С, insoluble residue (potassium bromide) at 2250 (СМ), 1750 (С-О), cm"; 1H NMR (300 MHz, SOSI»5) 65 2.04, 2.06, 2.07 ( Cs, 9H, acetylmethyls), 4.15 (dd, 1H), 4.40 (m, 1H), 5.47 (t, 1H), 5.63 (dd, 1H), 5.95 (d, 1H , U - 3.2 Hz, S.H), 8.34 (c, 1H, CH triazole).

1-3-O-ribofuranosyl-1,2,4-triazole-Z3-carboxamidine hydrochloride (8)

A molar solution of methanolic sodium methoxide (12ml) was added to a suspension of compound 10 (4.0g, 11.4mmol) in methanol (100ml) and the mixture was stirred at room temperature overnight. The solution was acidified to pH 4 with methanol-washed Yuozeh Nya polymer, the polymer was filtered off, and the filtrate was concentrated to dryness under vacuum conditions. The residue was dissolved in a minimum amount of methanol (15 ml) and transferred to a beaker under pressure. Ammonium chloride (0.61g, 11.4mmol) and a solution of methanol saturated at 02С with dry ammonia gas (75ml) were added, the beaker was sealed and the solution was stirred at room temperature overnight. The solution was concentrated to dryness under vacuum and the resulting residue was crystallized from acetonitrile/ethanol to give compound 8 as a crystalline solid (2.95g, 9395). This specimen was identical in all respects to the genuine specimen.

In certain pharmaceutical dosage forms, prodrug forms of the compounds, especially including acylated c (acetylated or other) derivatives, pyridine esters and various salt forms of these compounds, are particularly preferred, (3) and can be administered according to the method of treatment of the patient's condition. One of ordinary skill in the art will determine how these compounds can be readily modified into prodrug forms to facilitate delivery of the active compounds to a target site in the host or patient.One of ordinary skill in the art will also take advantage of the beneficial pharmacokinetic parameters of the prodrug forms, where applicable, to deliver these (77 compounds to the target site in the host organism or in the patient in order to achieve the maximum predicted He) effect of the compound.

An intended example of the formation of a prodrug form of the compounds described here is as follows. One of the simplest prodrugs of Migatidipetm is the tri-O-acetyl derivative Migatidipetm. The tri-O-acetyl derivative is obtained as shown in scheme 1: i - shk A

NIM M nm M 7 ud U zadeyutu "my « oi i NSUet o. o) vh ii. ter p, s :z" po on As Odes

Scheme 1 5-Retinoyl derivative Migatidipetm is another simple prodrug form, which was obtained by the following method:

Retinoic acid Ж---6 t ok Mo (is) ii. Migatidips LOBSTER o a iy. NSUeter Retinoic acid hu no on

Scheme 2

Other 5-derivatives of Migatidipetm include the following compounds depicted in Scheme 3:

F) name) 60 b5

He nok M (V; iv) kso- o) no on ke SNIYASNIz-SNU p- basen. St son zhk MSN, SNI SNU em che

SeNe k- to syu

Scheme Z

Most of these compounds can be obtained as described in S. Zegopegaeg, S. Riegioi, A. Tapag, MU. Nepip, M.

Y etaiige, /. Honey. Spet., 36, 826 - 830, 1993.

The synthesis of prodrug forms of Migatidipetm based on coumarin can be obtained by the following method:

MnN.Ms

Х A and СООН ЕОСОМАРГипилидипе вк у у "а Mo y. NSuvler Zh o M c) s no on o

Scheme 4

It is believed that amino acid esters are the best prodrug forms due to the possible involvement of a stereoselective transporter. Amino acid derivatives of Migatidipetm can be synthesized as shown below:

MS --

Zo Te) and NIM M

And and. EPSOM Rigattdipe Y r) and -amino acids str y y y. FIG. NCIDioxane

Amipoic acid - 5 He) but he and -

Scheme 5

For the specific delivery of drugs to the liver and biliary system, the endogenous bile acid transport system is an attractive candidate. The synthesis of bile acid conjugates of Migatidipetm can be carried out as shown below:

Bile acid ---inn in M r: y. NSIiDyukshn oi

Bile acid B) -I still no on me Scheme 6 - I Nucleotide derivatives are another class of prodrugs or prodrug forms. The preparation of the b-protected 50 5-Monophosphate derivatives is shown below. Due to the protection of negative phosphate charges by neutral substituents, more lyophilic derivatives are formed, which are expected to revert back to the corresponding monophosphates when they are inside the cell. Chnma and. 1H-Tetrazol/T HF/Uigatidium n hr shchi (ny it eto" tia tt y MU 5 2 ii. Sisnenisoi NISNISI; zm (F. y. NS/Dioxane (ya-e a Ua o ime) 5 1 no on 60 Scheme 7 where Ku are alkyl groups, such as CH 3С(0)5-СНХСН»-, (СН3)ЛСсСнНе()5-СНХСНО-, (СНХ3)-СС(О0)5-СНХСН»-, (Снеазсе( )osn»o-, SenS(O)5-СНСНо- or НОСНСНХ5ВЗЗ-СНоСН»-.

Amino acid phosphoramidates are another class of prodrugs that can be synthesized as described below: b5

"MS vrchn "MS nm in M ii Mayo, OSSIVchON, BUT wl in M ; Z syu Ya Ya t s; o o ii. NSuDioxane o

What ani on op no on , meo so no on

K - anything, except hydrogen

Scheme 8

Other derivatives of monophosphate prodrugs are shown below: nm M, -6.

Its i.p-5-5-P-CH.OH/ what M, carbenyldiimidazole F is not Id; there are alert Mio inn and ni o; and. NSuDioxane B-5-5-RIASNO-O R

E: alkyl, lipids, vitamins, bile acids, cholesterol derivatives

Scheme 8A

Migatidipetm prodrugs based on salicylate can be obtained according to the following scheme:

Ma n.Ms 7 se o vk u U 1 sok: rosevyon, NI nku u pon s o PV o o her. NSDioxan o Mom no M o-- - on SH in Son soov, NO Son ish

Kich SN, -

KyeRЬ (ze) ven son 7 m. on on

Scheme 9 "

Prodrugs of nucleoside 5'-di- or triphosphates will be more interesting because they will avoid more metabolic steps. - c The following examples are potential nucleotide lipophilic prodrugs that are prepared as shown below. . and? -I (95) -I b 50 -

F) name) 60 b5 o o

Mys SNU KhStdgtTORO - rom Me o prichi de se

M

; b tn 2 t "S "5 XX oi nltF M och y. NSUDioxan my Ve) "M" o io; Hy no on snu xSninsto-Ro-/-0 no on o omom x-snyMieMvsh

ХхеО; МмяМВ

Scheme I0 n; X, nak M them

Sho b ormhoyues "U o, shchi

Ru Mini ru or rud: in od (Rv); sch o sv) i) be in ra n. Her! t 7 D I M i nim ROYSLYASIDI gu o i) M u LSIDioxan o, y H M no-' SCHO r o no su (vno); Me, so no Son ochi te

Scheme || "

The following examples are another class of potential phosphonate prodrugs of Migatidipetm, i.e. "NS with X on

NM M -SK(0)- - ; z» o 2 K U k-(WHAT)-(SNU DON what h i o / r no-R o h M

NO Su (v-F(O (SNU Ou no, -i no on no on io Scheme 12 -i Other possible prodrugs include possible combinations of groups depicted in PCT patent applications UUO 98/39342, bu 50 2 .MO 98/39343 , UMO 98/39344 and UMO 99/45016.

Migatidiletm prodrugs can be obtained not only by modifying the sugar part of the initial -Z molecule, but also by obtaining functional amidine derivatives. The following examples are several classes of prodrugs that can be prepared by modifying the amidine group as shown below:

F) name) 60 b5

Kh. Kh NneI7 ni M "from ooo r

M sn in y,

S, y

SL, in ov on it

Scheme 13 n

X Z Dt

NIM M n v; Bosnian OSS F-nm Y I o. "y . osn, their va y, NS Dioksan m) pe no ok vo on

Scheme 14

MmN. nei vn Zh. vo on nsnm cm 2

KV: osyusn". OSS S nm lah (o)

M mn nia OosOos Kk r ka? her. RIS/NSUDioxane o no ko ok no ch- on (Se)

Scheme I5 i- (ze) and -

Kh. n.ne nn M iK-SN.ONI vk-sn A, De schy Mk. then nm |; 3 « Yab iy. NSuDioxane Z ; pe with

Vo ok s no Son " i z»Ssn,. and K- fenil ve N.R ta

I re alkyl, lipids, vitamins, bile acids, cholesterol derivatives - and d) Scheme 16 - I Application

It is envisaged that the compounds of formula 1 will be used for the treatment of a wide variety

Me. conditions and, in fact, any condition that responds positively to the administration of one or more of these compounds.

Kk Among other things, it is particularly contemplated that the compounds of the invention can be used for the treatment of infections, invasions, cancer, or tumors, or autoimmune diseases. It is further contemplated that the compounds of the invention may be used to treat conditions or diseases in specific organs of the patient, such as the liver or the heart.

Infections that are intended to be treated with the compounds of the present invention include respiratory syncytial virus (RSV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex types 1 and 2, genital herpes,

F) herpetic keratitis, herpetic encephalitis, herpes zoster, human immunodeficiency virus (HIV), influenza A virus, papiapp virus (hemorrhagic fever), human papilloma virus (HPV), measles and fungi.

Infestations intended to be treated with the compounds of the present invention include protozoan infestations, as well as helminth infestations and infestations by other parasites.

The types of cancer or tumors intended to be treated include those caused by a virus, and the effect may include inhibiting the transformation of virus-infected cells to a neoplastic state, inhibiting the spread of viruses from transformed cells to other normal cells, and/or stopping the growth of virus-transformed cells. 65 Autoimmune and other diseases intended to be treated include arthritis, psoriasis, intestinal disease, juvenile diabetes, lupus, multiple sclerosis, gout and gouty arthritis, rheumatoid arthritis, transplant rejection, giant cell arteritis, allergies, and asthma.

Still other contemplated uses of the compounds of this invention include their use as intermediates in the chemical synthesis of other nucleoside or nucleotide analogs, which in turn are useful as therapeutic agents or for other purposes.

According to yet another aspect, the method of treating a mammal comprises the administration of a therapeutically and/or prophylactically effective amount of a pharmaceutical agent containing a compound of the present invention. In this aspect, the effect may relate to the modulation of some part of the immune system of the mammal, especially the modulation of lymphokine profiles

Type 1 and Type 2 relative to each other. Where modulation of Type 1 and Type 2 lymphokines occurs, it is particularly contemplated 70 that the modulation may involve suppression of both Type 1 and Type 2, and more preferably stimulation of Type 1 lymphokines, or enhancement of the Type 1 response relative to the Type 2 response.

In particular, Migathiadipe tm (1.39 µg/ml) is predicted to increase the expression and synthesis of Type 1 cytokines in (predominantly activated) T-lymphocytes, and the results of various experiments are shown in Figures 2-5. Figure 2 depicts the effect of 5 mM viramidine (compounds of formula 1), ribavirin, and levavirin on the synthesis of Type 1/5 cytokines in staphylococcal enterotoxin-activated human T-cells (n - 5 donors), with viramidine demonstrating an obvious increase in the Type 1 response, compared to triazole control experiments.

Figure C is a graph of the effect-dose dependence of the effect of viramidine in the range of 0.625-10mM on cytokine synthesis

Type 1 in human T-cells activated by staphylococcal enterotoxin B (data are representative of 4 individual donors). The effect of ip on the increased Type 1 reaction in the contact hypersensitivity (CH) assay of viramidine is clearly demonstrated in Figure 4, and Figure 5 shows the comparison between viramidine and levavirin/ribavirin with respect to the nucleoside concentration for the maximum response and the maximum range of responses (the "y" axis represents the number of patients , which responded in a certain experiment).

Preparation of human T cells and activation of migo

Peripheral blood mononuclear cells from healthy donors or patients with rheumatoid arthritis were isolated by density gradient centrifugation followed by T-cell enrichment using

GutrpokmikK (Ope IGatraa, Sapoda Rak SA). Contaminating monocytes were removed by sticking to the plastic. Purified T cells were greater than 9995 CO2g, less than 195 ng A-OCg, and less than 590 sr25, and were maintained in KRMI-ARB5 (KRMI-1640 medium containing 20 mM HERE5 buffer, pH 7.4, 590 autologous plasma, 195 I -glutamine, 190 penicillin/streptomycin and 0.0595 2-mercaptoethanol). "-

To determine the protein levels of cytokines, T-cells (1 x 109 cells in a volume of 1 ml) were activated by adding 10 ng of PMA (FMA) and 00.5 μg of ionomycin (both obtained from SaiIriospet, Ia KhdoiYiIa, CA) and incubated in plates with sho 24 wells in the presence of 0-20 µM nucleoside for up to 48 hours at a temperature of 379C and 595 CO05 in a mhmlr-humidified incubator. After activation, the supernatants were analyzed for the production of cell-derived cytokines. For studies of proliferation and viability, the above-described method was modified to the format of tablets with 96 wells, using 0.2x105 cells in a volume of 0.2 ml, and activation of ng PMA (FMA) and its

About 1 kg of inomycin. In separate experiments, 5x109 cells in 2ml were activated with 2O0ng of FMA and mcg of ionomycin.

Alternatively, cells can be activated i.p. with staphylococcal enterotoxin B according to published procedures. At the same time, all RNA was isolated from T-cells after incubation for 6-24 hours and analyzed by reverse-tase PCR in order to determine the mRNA levels of various cytokines and inflammatory mediators. Also, in separate experiments, human T-cells were further purified (using cell-enriching reagents obtained from Ziet Seiyi Tespoiodieez, Mapsohmeg, VS) and obtained pure populations of СО4- (less than 195 СОвхк, с» using a reagent for isolation CO4- human T cells from Kozeceber) and a subpopulation of COvzh T cells (less than 195 CO4, using a reagent for the isolation of CO4- human T cells from KozeCeser), after which 1x105 cells per ml were activated with FMA and ionomycin, as in in all T-cell experiments.

Analyzes of extracellular cytokines

Levels of human cytokines were determined in cell supernatants after appropriate dilution using solid-phase enzyme-linked immunosorbent assay (ELISA) kits specific for 1-2, IEMU, TMEOY, II-4 and

I/-5 (Viozotsgse Ipiegpaiopa!I, Satagyo, SA). Cytokine levels of mice were determined using solid-phase enzyme-linked immunosorbent assay kits that are specific for IEM and I/-4 mice (Kapa O Zuvietv,

Ge) 20 Mippearoiis, MM). All results of the solid-phase enzyme-linked immunosorbent assay were expressed in pg/ml. Some T cell data are plotted as a percentage of activated control cells, which was calculated as the ratio of the cytokine levels of activated T cells in the presence of test nucleoside to the cytokine levels of untreated activated T cells

of T cells multiplied by 10095. A zero effect on test nucleoside cytokine levels would give a percent value of activated control cells of 10095. Alternative data was demonstrated as a percentage change from activated control cells ((pg/ml test - pg/ml activated control) /pg/ml activated

HF) control) X 10095). A null effect on the cytokine levels of the test nucleosides would be 090. Contact hypersensitivity (CH)

Reactivity to the contact allergen, OMEV, was determined in VAI V/s mice, as previously described (Izpii, M., K.

TaKanagzni, N. MakarYita, 5. TapaKa, R. MU. AvzKepaze. 1994. YUOMEV sopiasi zepzyimyu (C5) ip VAG V/s apa szn/Ne 60 tise. in. Ipmevi Oegtayu)i. 102:321). Briefly, mice were sensitized by applying 20 μl of 0.390 OMEV in acetone:olive oil, 471, to the shaved abdomens of uninfected mice. For optimal determination of contact hypersensitivity, mice were injected with 20 μl of 0.1296 OMEV on both sides of each ear 5 days after sensitization.

Non-sensitized mice were also sensitized and used as controls in each experiment.

After 24 hours, the ear thickness was measured and the response to OMEV was analyzed by subtracting the post-allergization values from the pre-allergization values. Where indicated,

7-8-O-ribofuranosyl-4-oxopyrrolo|2,3-4)pyrimidine-5-carboxamidine in a dose of 2.2 μg in 5 μl of physiological solution with phosphate buffer (0.3 μg/kg) or 12.4 μg in 100 μl of physiological solution with phosphate buffer (0.bmg/kg), administered by intraperitoneal injection during OMEV allergy. These doses of 7-8-YOO-ribofuranosyl-4-oxopyrrolo|2,3-4|pyrimidine-5-carboxamidine gave the maximum effect in previous optimization experiments. After the last ear thickness measurements, mice were sacrificed by cervical spine displacement and axillary/lateral axillary lymph nodes were removed. After the isolation of total cellular RNA from the cells of the isolated lymph nodes, PCR reversetase analysis and Southern blotting were performed to determine the levels

IRMA mRNA, 1-2 and 1-10.

Further experiments

In general, it is assumed that a shift of the immune response to a Type 1 response is predominant. Therefore, it is envisaged that the compounds according to the essence of the invention may be particularly useful in the treatment of viral diseases (predominantly viral infections in which the type 1 response is reduced or suppressed). In order to confirm the effectiveness of immune response modulation, various experiments were performed, and the following description is an example of the essence of several 75 experiments performed with the predicted compounds:

Ip migo - viramidine inhibited the infection of Rypia Togo virus GII C-MK2 (kidney cells of rhesus macaques with EC5O of 8mg/ml (Adatez strain) and 12mg/ml (Vaille strain) - CC5O was 320mg/ml (1.0-1.2 virus rating).

IP mimo - subcutaneous or oral administration of viramidine caused 10,095 survival (10 C57VI /6 mice/group) of mice injected subcutaneously with the Ripia Togo virus (Adatezv strain).

Within 24 hours after infection with Ripia virus Togo i.p. mimo, the minimum effective subcutaneous dose was

C2mg/kg for ribavirin, and for viramidine it was 9bmg/kg under the condition of subcutaneous administration of B. i. and. within 5 days. Within 24 hours after infection with Ripia Togo ip mimo, the minimum effective oral dose was 20mg/kg for ribavirin and 4Omg/kg for viramidine under the condition of oral administration b. and. and. within 5 days. Ge

In general, the most preferred embodiments of the present invention are those in which the active compounds are relatively less cytotoxic to non-target host cells and relatively more active on the target. In this regard, it may also be advantageous that I-nucleosides may have increased stability compared to UO-nucleosides, which may lead to better pharmacokinetics. This result can be achieved because I -nucleosides may not be recognized by enzymes and, therefore, may have a longer half-life.

It is contemplated that the compounds of the present invention will be administered ip mimo, ip migo, or ex mimo in any suitable pharmaceutical formulation and by any suitable protocol. Therefore, the administration of h- can be carried out orally, parenterally (including subcutaneous injections, intravenous injections, intramuscular injections, intrathoracic injections or infusion methods), by inhalation or rectally, topically, etc., and in formulations dosage forms that contain conventional non-toxic pharmaceutically acceptable carriers, fillers and adjuvants.

By way of example, it is contemplated that a pharmaceutically acceptable carrier may be added to the compounds of the present invention. For example, the compounds of the present invention can be administered orally as "pharmaceutically acceptable salts." Since the compounds of this invention are mostly water-soluble, they can be administered intravenously in physiological saline (eg, buffered to a pH of about -7.2 to about 7.5). For this purpose, common buffers such as phosphates, bicarbonates or a citrates can be used. Of course, any person skilled in the art will be able to modify the pharmaceutical formulations according to the "" recommendations of the description to obtain multiple formulations for a certain route of administration, but not at the expense of the instability of the formulations of the present invention and their therapeutic activity. In particular, the modification of these compounds, in order to make them more soluble in water or other excipients, can be easily accomplished by minor modifications (salt composition, esterification, etc.), which is common practice in the industry.It is also common in the industry to modify the route of administration and dosage regimen of a particular compound to provide the pharmacokinetics of those compounds maximally beneficial effect in the patient. - I In addition, the compounds of the present invention may be administered alone or in combination with other agents for the treatment of the infections or conditions listed above. Combination therapy according to the present invention comprises the administration of at least one compound of this of the invention or its functional derivative and at least one other - and pharmaceutical but the active ingredient. The active ingredient(s) and pharmaceutically active agents may be administered separately or together, and when administered separately, the administration may be performed simultaneously or separately in any order. The amount of active ingredient(s) and pharmaceutically active agent(s) and relative timing of administration will be chosen to achieve the desired "combined therapeutic effect."

Preferably, the combined therapy includes the administration of one compound of the present invention or its physiologically functional derivative and one of the agents mentioned below in this description. Examples of other drugs or active ingredients that are believed to be effective in combination with a modulator of formula 1 are antiviral agents such as interferon, including but not limited to interferon o, and y, ribavirin, acyclovir, and amoxicillin. , antifungal agents such as tolnaftate, Rypdigope m,

Antiparasitic drugs such as Mipiegoi tm, Misiosidetm,

Meptochtm and Riaduitm, intestinal agents such as Ittodicttm, | Antitumor agents such as interferon A and G, Aagatusiptm, Suohaptm, |tihaptm, methotrexate, Mi(ygaciptm, Thiagoytsiptm, Tachoim, dermatological agents such as Acioma(e tm, Susiosogitm, Yuepogehtm, Riogopetm, Oxoghaieptm, coal tar and bo salicylic acid, antimigraine drugs such as ergotamine compounds, steroids and immunosuppressants not listed above, which include cyclosporins, Origozope tm, hydrocortisone, Biooptm, |idechtm, topicort and valizone, and metabolic agents such as insulin, and other medications that may not fall into the categories listed above include cytokines such as 112, 114, 1/6, 1/8, 11/10, and 11/12. guanosine analogs, 2,3-dideoxynucleosides, interleukin I, interferons such as IAB interferons, tucaresol, levamisole, isoprinosine, and cyclolignans.

Examples of such further therapeutic agents include agents effective in modulating the immune system or related conditions, such as A/LT, ZTS, 8-substituted guanosine analogs, 2,3'-dideoxynucleosides, interleukin II, interferons such as o -interferon, tucaresol, levamisole, isoprinosin 70 and cyclolignans. Certain compounds of the present invention may be effective in enhancing the biological activity of certain agents of the present invention by reducing the metabolism or deactivation of other compounds and as such are administered together to achieve this particular effect.

With respect to dosage, one of ordinary skill in the art will appreciate that a therapeutically effective amount will vary for the particular infection or condition to be treated and will depend on its severity, the regimen to be administered, the pharmacokinetics of the agent being administered, and the patient. (animal or human) that should be treated. It is believed that various alternative doses are also acceptable, including doses between 0.0 mg/kg and 0.0 mg/kg or less, and doses from 0.0 mg/kg to 1.0 mg/kg or more. It is further contemplated that although successful treatment may be achieved with some viral infections at relatively low plasma concentrations of the compounds of formula 1, other viral infections may require

Relatively high doses. It is believed, however, that a suitable regimen can be developed by administering a small amount and then increasing the amount until the side effects become harmful or until the desired effect is achieved.

Administration of the active compound may vary from continuous administration (intravenous drip) to multiple oral administrations per day (eg, O.I.) and may include, among other routes of administration, oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may contain a penetration-enhancing agent), transbuccal administration, and suppository administration. and)

In order to prepare compositions according to the present invention, a therapeutically effective amount of one or more compounds according to the present invention is preferably thoroughly mixed with a pharmaceutically acceptable carrier according to conventional methods of preparing pharmaceutical compounds to obtain a dose. The carrier can have a wide variety of forms depending on the form of the drug to be administered, for example, orally or parenterally. Any conventional pharmaceutical medium can be used to prepare pharmaceutical compositions in dosage form for oral administration. Therefore, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives can be used, which include water, glycols, oils, alcohols, flavorings, preservatives, dyes and the like. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives may be used which include starches, sugar carriers such as dextrose, mannitol, lactose and related carriers. , thinners, granulators, lubricants, binders, splitting agents and similar substances. If desired, tablets or capsules may have an enteric coating or be sustained-release by standard methods.

For parenteral pharmaceutical formulations, the carrier will usually include sterile water or an aqueous solution of sodium chloride, but other dispersing ingredients may be included. Of course, where sterile water should be used and kept sterile, formulations and media should also be sterilized. It is also possible to prepare suspensions for injections, in which case suitable liquid carriers, suspending agents, etc. can be used. treatment of viral infection of the hepatitis C virus (HCV) or viral infection of the virus

Me hepatitis B (NVM)-3 formula 1,

MN. Not 95 I o M;

I ho) "M no o on an where the compound is in the U-configuration. c5 2. Application according to item 1, where the pharmaceutically acceptable salt is a hydrogen chloride salt according to formula 1. 3. Application according to item 1 or item 2, where the viral infection is hepatitis C virus (HCV) infection.

Claims (1)

4. Use according to any of claims 1-3, where the medicinal product is intended for oral administration. 5. Use according to any one of claims 1-4, where the medicinal product contains a compound of formula 1 in a dose between 0.1 mg per kg of the patient's body weight and 40 mg per kg of the patient's body weight. 6. Application according to any of claims 1-5, where the medicinal product additionally contains interferon. 7. Application according to claim 6, where the interferon is interferon alpha. s schi 6) "- (Se) cha so i - - with . and? -I (95) -I b 50 - F) ime) 60 b5