Inhibitors of impdh enzyme

Patent Office of the Republic of Poland (21) Application number: 350911 (22) Application date: 17.03.2000 (86) Date and number of the international application:

March 17, 2000, PCT / US00 / 007129 (87) International application publication number and date:

28/09/2000, WO00 / 56331 (51) Int.Cl.

C07D 263/32 (2006.01) C07C 275/34 (2006.01) C07C 275/40 (2006.01) C07C 275/42 (2006.01) A61K 31/277 (2006.01) A61K 31/421 (2006.01) A61P 31/12 (2006.01) A61P 37/00 (2006.01) A61P 35/00 (2006.01) A61P 29/00 (2006.01) A61P 11/06 (2006.01) (54)

Compounds, compositions containing them and their uses (73) Patent holder:

(30) Priority:

1999-03-19, US, 60 / 125,507 Jan 7, 2000, US, 60 / 174,882

VERTEX PHARMACEUTICALS

INCORPORATED, Cambridge, US (72) Inventor (s):

(43) Application announced:

10.02.2003 BUP 03/03

DEAN STAMOS, Framingham, US

MARTIN TRUDEAU, Tewksbury, US SCOTT BETHIEL, Cambridge, US MICHAEL BADIA, Bedford, US

JEFFREY SAUNDERS, Acton, US (45) The grant of the patent was announced:

May 31, 2012 WUP 05/12 (74) Plenipotentiary:

item. stalemate. Janina Kossowska

PL 211 562 B1

Description of the invention

The invention relates to compounds, compositions containing them, and their uses. The compounds of the invention are inhibitors of the enzyme IMPDH and, consequently, can be conveniently used as therapeutics in processes involving IMPDH.

Nucleotide synthesis is required for cell division and replication in organisms. Nucleotide synthesis in mammals can take place in two ways: de novo synthesis or via a reserve pathway. Different types of cells use these routes to a varying extent.

Inosine-5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is an enzyme that is involved in the de novo synthesis of guanine nucleotides. IMPDH catalyzes the NAD-dependent oxidation of inosine-5'-monophosphate (IMP) to xanthosine-5'-monophosphate (XMP) [RC Jackson et al., Nature, 256, pp. 331-333, (1975)].

IMPDH is present in eukaryotic cells, bacteria and protozoa [Y. Natsumeda and SF Carr, Ann. NY Acad., 696, pp. 88-93 (1993)]. The prokaryotic species have sequences 30-40% identical to the human enzyme. Two isoforms of human IMPD, designated type I and type II, have been identified and sequenced. [FR Collart and E. Huberman, J. Biol. Chem., 263, pp. 15769-15772 (1988); Y. Natsumeda et al., J. Biol. Chem., 265, pp. 5292-5295 (1990)]. They each contain 514 amino acids and share 84% sequence identity. Both types of IMPDH, I and II, form active tetramers in solution with a 56 kDa subunit [Y. Yamada et al., Biochemistry, 27 pp. 2737-2745 (1988)].

De novo synthesis of guanosine nucleotides, and hence the activity of IMPDH, is particularly important in B and T lymphocytes. The generation of sufficient levels of nucleotides necessary to initiate a proliferative response to mitogen or antigen in these cells depends on de novo synthesis rather than on a reserve pathway [AC Allison et al., Lancet II, 1179, (1975) and AC Allison et al., Ciba Found Symp. 48, 207 (1977)]. Thus, IMPDH is an attractive target for selectively suppressing the immune system without simultaneously inhibiting the proliferation of other cells.

Immunosuppression can be achieved by inhibiting various enzymes including, for example, calcineurin phosphatase (inhibited by cyclosporin and FK-506); dihydroorotanate dehydrogenase, an enzyme involved in the biosynthesis of pyrimidines (inhibited by leflunomide and brequinar); FRAP kinase (inhibited by rapamycin); and the heat shock protein hsp70 (inhibited by deoxyspergualin). [See BD Kahan, Immunological Reviews, 136, pp. 29-49 (1993); RE Morris The Journal of Heart and Lung Transplantation, 12 (6), pp. S275-S286 (1993)].

IMPDH inhibitors are also known. U.S. Patent Nos. 5,380,879 and 5,444,072 and PCT publications WO 94/01105 and WO 94/12184 describe mycophenolic acid (MPA) and some of its derivatives as potent, incompetent, reversible inhibitors of human IMPDH type I (Ki = 33 nM) and type II (Ki = 9 nM). MPA has been shown to block the response of B and T cells to a mitogen or antigen [AC Allison et al., Ann. NY Acad. Sci., 696, 63 (1993).

Immunosuppressants, such as MPA, are drugs useful in the treatment of transplant rejection and autoimmune diseases. [RE Morris, Kidney Intl., 49, Suppl. 53, S-26 (1996)]. However, MPA has undesirable pharmacological properties such as gastrointestinal toxicity. [LM Shaw, et al., Therapeutic Drug Monitoring, 17, pp. 690-699 (1995)].

IMPDH is also inhibited by nucleoside analogs such as thiazofurin, ribavirin and misoribine [L. Hedstrom et al., Biochemistry, 29, pp. 849-854 (1990)]. However, these compounds are not specific for IMPDH.

Mycophenolate mofetil, a prodrug that rapidly releases free MPA in vivo, has recently been shown to prevent acute renal allograft rejection following kidney transplantation [LM Shaw et al., Therapeutic Drug Monitoring, 17, pp. 690-699 (1995); HW Sollinger, Transplantation, 60, pp. 225-232 (1995)]. However, some clinical observations limit the therapeutic effect of this drug. [LM Shaw et al., Therapeutic Drug Monitoring, 17, pp. 690-699 (1995)]. MPA is rapidly metabolized in vivo to an inactive glucuronide [AC Allison and EM Eugui, Immunological Reviews, 136, pp. 5-28 (1993)]. The glucuronide then returns to the enterohepatic system, causing MPA to accumulate in the gastrointestinal tract where it cannot exert an IMPDH inhibitory effect on the immune system. This effectively reduces the effect of the drug in vivo, while increasing its undesirable side effects in the digestive system.

Various classes of IMPDH inhibitors have recently been described in PCT publications WO 97/40028 and WO 98/40381.

PL 211 562 B1

IMPDH is also known to play a role in other metabolic events. Increased IMPDH activity has been observed in rapidly proliferating human leukemia cell lines and other cancer cell lines, indicating IMPDH as a target for anti-cancer as well as immunosuppressive chemotherapy [M. Nagai et al., Cancer Res., 51, pp. 3886-3890,1991)]. IMPDH has also been shown to play a role in smooth muscle cell proliferation, indicating that IMPDH inhibitors such as MPA or rapamycin may be useful in preventing restenosis or other hyperproliferative vascular disease [CR Gregory et al., Transplantation, 59, p. 655-61 (1995); PCT Publication WO 94/12184; and PCT Publication WO 94/01105].

Furthermore, IMPDH has been shown to play a role in viral replication in some virus-infected cell lines. [SF Carr et al., J. Biol. Chem., 268, pp. 27286-27290 (1993)]. By analogy with lymphocytes and lymphocytic and tumor cell lines, the conclusion is that the de novo pathway rather than the back-up pathway is critical to viral replication.

Thus, there is still a need for potent IMPDH inhibitors with improved pharmacological properties.

Such inhibitors would have therapeutic effects as immunosuppressants, anti-cancer agents, anti-vascular hyperproliferation agents, anti-inflammatory, anti-fungal, anti-psoriatic and anti-viral agents.

The invention relates to compounds that are useful as inhibitors of IMPDH. These compounds can be used alone or in combination with other therapeutic or prophylactic agents, such as antivirals, anti-inflammatory agents, antibiotics and immunosuppressants, for the treatment and prophylaxis of transplant rejection and autoimmune diseases.

These compounds, alone or in combination with other agents, are further useful as therapeutic and prophylactic agents with antiviral, antitumor, anticancer, anti-inflammatory, anti-fungal, anti-psoriatic agents, in immunosuppressive chemotherapy and in the treatment of restenosis.

The invention also relates to pharmaceutical compositions containing the compounds of the invention.

For a better understanding of the invention, the following detailed description is provided below. The description uses the following abbreviations:

Reagent designation or fragment

Ac acetyl

Me methyl

Et ethyl

Bn benzyl

CDI carbonyldiimidazole

DBU 1,8-diazabicyclo [5,4.0] undec-7-ene

DIEA diisopropylethylamine

DMAP dimethylaminopyridine

DMF dimethylformamide

DMSO Dimethyl Sulfoxide

DPPA diphenylphosphorylic acid

EDC 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride

EtOAc ethyl acetate

IPA isopropyl alcohol

MeCN acetonitrile

THF tetrahydrofuran

TEA triethylamine t-bu tert-butyl

Boc butyloxycarbonyl

The term "immunosuppressant" refers to a compound or drug that exhibits immune response suppression activity. Examples of such agents include cyclosporin A, FK506, rapamycin, leflunomide, deoxyspergualin, prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG, interferon, and mizoribine.

The term "interferon" refers to all forms of interferons, including, but not limited to, the alpha, beta, and gamma forms.

PL 211 562 B1

The term "IMPDH-associated diseases" refers to any disease state in which the metabolic pathway of the enzyme IMPDH plays a regulatory role. Examples of diseases associated with IMPDH include transplant rejection and autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma and inflammatory bowel disease, as well as inflammation, cancer, viral replication diseases, and vascular diseases.

For example, the compounds and compositions of the present invention can be used to treat transplant rejection (e.g. kidney, liver, heart, lung, pancreas (pancreatic islet cells), bone marrow, cornea, small intestine and skin allografts as well as heart valve xenografts), rheumatoid arthritis, multiple sclerosis, juvenile diabetes mellitus, asthma, inflammatory bowel disease ( Crohn's disease, ulcerative colitis), lupus, diabetes, myasthenia gravis, psoriasis, dermatitis, eczema, seborrhea, pneumonia, uveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis, Behcet's or Sjorgen's syndrome (dry eyes / mouth), pernicious or immunohemolytic anemia, idiopathic adrenal insufficiency, multi-gland autoimmune syndrome, scleroderma, lichen planus, viteligo (skin depigmentation), autoimmune thyroiditis, alveolitis, inflammatory diseases such as osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma and respiratory distress syndrome in adults, as well as in the treatment of cancer and neoplasms such as solid tumors, lymphomas and leukemia, vascular diseases such as restenosis, stenosis and atherosclerosis, and diseases associated with the replication of viral DNA and RNA such as retroviral and herpes infections.

Moreover, it is known that IMPDH enzymes are also present in bacteria and therefore can regulate the growth of bacteria. Thus, the IMPDH inhibitor compounds described herein, alone or in combination with other antibiotics, and compositions may be useful in the treatment or prevention of bacterial infections.

The term "treatment as used herein" refers to the alleviation of the symptoms of a particular disorder in a patient or to the measurable improvement associated with the particular disorder. The term "patient as used herein" refers to a mammal, including a human.

The terms "HBV," HCV, and "HGV" refer to hepatitis B, type C, and type G, respectively.

<img file="PL211562B1_D0001.tif" />

and compositions containing these compounds, ie, compound 181 or 169, respectively, as the active ingredient together with a pharmaceutically acceptable carrier, excipient or vehicle.

The compounds of the invention may be synthesized by conventional methods. Preferably, these compounds are prepared from commercially available starting materials. More specifically, the compounds of the invention can be synthesized by the methods set forth in the schemes in Example 1, with modifications apparent to those skilled in the art.

In order to improve the selective biological properties, the compounds according to the invention can be modified by adding appropriate functional groups. These modifications are known in the art and include those modifications which increase penetration into a given biological system (e.g. blood, lymphatic system, central nervous system), increase oral bioavailability, increase solubility, which enables administration by injection, alter metabolism or alter excretion rate.

The novel compounds of the invention are excellent ligands for IMPDH. Thus, these compounds are capable of reaching and inhibiting the enzyme IMPDH. Inhibition can be measured in various ways, for example in the IMP dehydrogenase assay by HPLC (measurement of the enzymatic production of XMP and NADH from IMP and NAD) and in the spectrophotometric assay of IMP dehydrogenase (measurement of the enzyme NADH production from NAD). [See C. Montero et al., Clinica Chimica Acta, 238, pp. 169-178 (1994)].

The compositions of the invention contain a compound of the invention and a pharmaceutically acceptable carrier, excipient or vehicle. Preferably the compositions of the invention are pharmaceutical compositions.

The term "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that can be administered to a patient together with a compound of the invention without compromising its pharmacological activity, and is nontoxic when administered in doses sufficient to provide a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, excipients and vehicles that can be used in the compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as α-tocopherol, polyethylene glycol 1000 succinate, surfactants used in pharmaceutical formulations such as Tween's or other similar polymeric matrices for drug delivery, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, chloride sodium, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. Cyclodextrins such as α-, β- and γ-cyclodextrin, or chemically modified derivatives thereof such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrins, or the other thereof may also be advantageously used to increase the availability of the compounds of the invention. solubilized derivatives.

The pharmaceutical compositions of the invention may be administered orally, parenterally, by inhalation inhalation, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral or injection is preferred. The pharmaceutical compositions of the invention may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or diluents. In some cases, the pH of the composition may be adjusted by the addition of pharmaceutically acceptable acids, bases, or buffers to increase the stability of the compound or formulation thereof. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intrathecal and intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, in the form of a sterile injectable aqueous or oleaginous suspension. These suspensions may be prepared according to known techniques, using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be in the form of a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Acceptable diluents and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils may also be conveniently used as a solvent or suspending medium. For this purpose any liquid oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, and natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially polyoxyethylated versions thereof, are also useful in the preparation of injectables. Such oily solutions or suspensions may also contain a long chain alcohol diluent or dispersant, as described in Parmacopeia Helvetica, Ph. Helv., Or a similar alcohol or carboxymethylcellulose or similar dispersants, which are commonly used in the preparation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants such as Tween or Span and / or other similar emulsifying or bioavailability enhancing substances can also be used to prepare pharmaceutically acceptable solid, liquid or other dosage forms.

The pharmaceutical compositions of the invention may be orally administered in any orally suitable form, including, but not limited to, capsules, tablets, emulsions and

And aqueous suspensions, dispersions and solutions. In the case of tablets for oral administration, carriers that are commonly used are lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. In the case of aqueous suspensions and / or emulsions for oral administration, the active ingredient suspended or dissolved in the oily phase is combined with emulsifying and / or suspending agents. If desired, sweeteners and / or flavors and / or dyes can also be added.

The pharmaceutical compositions of the invention may be administered in the form of suppositories for rectal administration. Such compositions are prepared by mixing a compound of the invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and thus will melt in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

Pharmaceutical compositions for topical administration are particularly useful when the desired treatment involves areas or organs readily accessible by topical administration. The pharmaceutical composition for topical administration to the skin should be formulated with a suitable ointment containing the active ingredients suspended or dissolved in a carrier. Carriers for topical administration of the compounds of the invention include, but are not limited to, mineral oil, liquid kerosene, petroleum jelly, propylene glycol, polyoxyethylene-polyoxypropylene, emulsifying waxes, and water. Alternatively, the pharmaceutical composition may be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester waxes, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions according to the invention may also be applied topically to the lower gastrointestinal tract as suppository compositions or in the form of a suitable infusion. Patches for topical administration through the skin are also within the scope of the invention.

The pharmaceutical compositions of the invention may also be administered intranasally in the form of aerosol formulations or by inhalation. Such compositions are prepared by methods well known in the pharmaceutical formulation art and may be formulated as saline solutions using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, hydrofluorocarbons and / or other solubilizing or dispersing agents. which are known in the art.

As monotherapy or combination therapy, dosage levels ranging from about 0.01 to about 100 mg / kg body weight per day are used to prevent and treat IMPDH related diseases, preferably in the range of between about 0.5 and about 75 mg of the described herein. here, IMPDH inhibitory compounds per kg of body weight per day. The pharmaceutical compositions of the invention are typically administered from about 1 to about 5 times a day or, alternatively, by continuous infusion. Such administration is used in chronic or acute therapy. The amount of active ingredient which is combined with the carrier materials to produce a unit dose will vary depending upon the host treated and the particular mode of administration. A typical composition will typically contain from about 5% to about 95% active ingredient (w / w). Preferably, such compositions contain from about 20% to about 80% active ingredient.

Additional therapeutic or prophylactic agents may be administered separately in a multiple dosing regimen with the compositions of the present invention. Examples of such additional measures are provided below.

Examples of additional immunosuppressants include, but are not limited to, cyclosporin A, FK506, rapamycin, leflunomide, deoxyspergualin, prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG, interferon, and mizoribine.

Examples of additional anti-neoplastic agents include, but are not limited to, cisplatin, actinomycin D, doxorubicin, vincristine, vinblastine, etoposide, amsacrine, mitoxantrone, tenipazide, taxol, colchicine, cyclosporin A, phenothiazines, interferon, and thioxanthers.

Examples of additional antiviral agents include, but are not limited to, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, d4T, ddl, AZT, and acyclovir.

Examples of additional anti-vascular hyperproliferation agents include, but are not limited to, HGM Co-A reductase inhibitors such as lovastatin, thromboxane A2 synthetase inhibitors,

Eicosapentane, ciprosten, trapidil, ACE inhibitors, low molecular weight heparin, mycophenolic acid, rapamycin and 5- (3'-pyridinylmethyl) benzofuran-2-carboxylate.

After improvement of the patient's condition, a maintenance dose of a compound or composition according to the invention or a combination thereof with an additional agent may be administered as needed. Thereafter, the dose or frequency of administration, or both, may be reduced, depending on the symptoms, to a level where the improvement is maintained, and when the symptoms have improved to the desired level, treatment may be discontinued. However, when disease symptoms reappear, patients may require continued long-term treatment.

One skilled in the art will understand that doses lower or higher than those set forth herein may be used. The specific dosage and treatment regimen of a particular patient will depend on a number of factors, including the activity of the particular compound employed, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, severity and course of the infection, susceptibility of the patient to infection. and judgment of the attending physician.

Also included within the scope of the invention are compositions as defined above for the treatment or prevention of a disease or condition associated with IMPDH in a mammal selected from the group consisting of transplant rejection, graft versus host disease, and autoimmune disease. The invention also includes compositions as defined above for the treatment or prevention of viral infection in mammals caused by viruses selected from the group consisting of orthomyxoviruses, paramyxoviruses, herpesviruses, retroviruses, flaviviruses, pestiviruses, hepatotrophic viruses, bunyaviruses, Hantaan virus, Caraparavirus virus, human papillomavirus, brain, arenaviruses, reoviruses, foot-and-mouth disease virus, rhinoviruses, enteroviruses, Lassa fever virus, togaviruses, poxviruses, adenoviruses, erysipelas, or rubella, and used to treat or prevent restenosis, stenosis, atherosclerosis, or other vascular hyperproliferative disease in mammals.

Also within the scope of the invention are compositions as defined above for inhibiting cancer or tumors in mammals and for treating or preventing lymphoma, leukemia and other forms of cancer, and for inhibiting inflammation or an inflammatory disease in mammals, and for treating or preventing osteomyelitis. -articular acute pancreatitis, chronic pancreatitis, asthma or respiratory distress syndrome in adults.

The methods of treating or preventing diseases associated with IMPDH include the step of administering to the mammal any of the pharmaceutical compositions or combinations described herein. When used in combination, such methods further include the step of administering to the mammal an agent selected from anti-inflammatory agents, immunosuppressants, anti-neoplastic, anti-viral, and anti-vascular hyperproliferation agents. Such additional agent may be administered to mammals prior to, concurrently with, or subsequent to administration of a composition containing an IMPDH inhibitor.

The compositions of the invention are useful for suppressing the immune response in a mammal. They are therefore useful in the treatment or prevention of diseases including transplant rejection (e.g. kidney, liver, heart, lung, pancreas (pancreatic islet cells), bone marrow, cornea, small intestine and skin allografts as well as heart valve xenografts), graft versus host disease, autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatory disease of the large intestine (Crohn's disease, ulcerative colitis), lupus, diabetes mellitus, myasthenia gravis, psoriasis, dermatitis, eczema, seborrhea, pneumonia, uveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis, Behcet's or Sjorgen's syndrome (dry eyes / mouth), pernicious or immunohemolytic anemia, idiopathic adrenal insufficiency, polygland autoimmune syndrome, scleroderma, lichen planus, viteligo (skin depigmentation), autoimmune thyroiditis, and alveolitis.

In the above use, in addition to the compositions of the invention, a composition comprising an additional immunosuppressive agent and a pharmaceutically acceptable excipient may be administered to the mammal.

The compositions of the invention are useful for inhibiting viral replication in mammals. Such methods are suitable for the treatment or prevention of viral DNA or RNA diseases caused for example by infection with orthomyxoviruses (influenza A and B viruses), paramyxoviruses (viruses causing respiratory disease (RSV), subacute atherosclerotic encephalitis virus ( SSPE)), measles and parainfluenza type 3 virus), herpes viruses (HSV-1, HSV-2, HHV-6, HHV-7, HHV-8, Epstein-Barr virus (EBV), cytomegalovirus (HCMV) and poxvirus Varicella zoster (VZV)), retroviruses (HIV-1, HIV-2, HTLV-1 and HTLV-2), flaviruses and pestiviruses (

PL 211 562 B1 yellow fever (YFV), hepatitis C virus (HCV), dengue virus, calf diarrhea rotavirus (BVDV), hepatotrophic viruses (hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis virus liver D (HDV), hepatitis E virus (HEV), hepatitis G virus (HGV), Crimean-Congo haemorrhagic fever virus (CCHF), bunyaviruses (Punta Toro virus, Rift Valley virus (RVFV), Sicilian fever virus) , Hantaan virus, Caraparu virus), human papillomaviruses, encephalitis viruses (La Crosse virus), arenaviruses (Junin and Tacaribe viruses), reoviruses, foot-and-mouth disease viruses, rhinoviruses, enteroviruses (polio virus, coxsackie viruses, encephalomyocarditis virus (EMC)), Lassa fever virus , togaviruses (Sindbis and Semlike forest virus) and poxviruses (vaccinia virus), adenoviruses, rose and rubella virus.

In such use, the compositions of the invention are preferably additionally administered to the mammal a composition comprising an additional antiviral agent and a pharmaceutically acceptable excipient.

The compositions of the invention are useful for inhibiting vascular cell hyperproliferation in mammals and are useful for treating or preventing diseases including restenosis, stenosis, atherosclerosis, and other diseases associated with vascular hyperproliferation.

Preferably, in this use, in addition to the composition of the invention, a composition is administered to the mammal which comprises an additional anti-vascular hyperproliferation agent and a pharmaceutically acceptable excipient.

To better understand the present invention, the following examples are provided.

EXAMPLE 1

Preparation of compound 169

A. Preparation of an Intermediate - Left Side of Compound 169

<img file="PL211562B1_D0002.tif" />

Copper (I) cyanide (7.2 g, 80.8 mmol) was combined with 2-bromo-5-nitroanisole (I) (15 g, 64.6 mmol) in NMP (70 ml) and heated to 150 ° C. overnight under N2 atmosphere. The mixture was treated with Celite, cooled to room temperature, then diluted with EtOAc and 1.0 N NaOH solution and stirred for 15 minutes. The homogeneous mixture was filtered through a Celite pad with EtOAc, the phases were separated and the aqueous phase was washed 3 times with EtOAc. The combined organics were washed sequentially with 1.0 N NaOH, water, and brine, then dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was dissolved in CH2Cl2, filtered through a short pad of silica gel to remove solids and most of the colored impurities, then concentrated in vacuo to give product II (10.41, 90%) as a brown orange solid.

¹H NMR (500 MHz, CDCl 3): 7.90 (d, 1H), 7.84 (s, 1H), 7.77 (d, 1H), 4.07 (s, 3H).

To a solution of product II at room temperature (7.2 g 40.4 mmol) in EtOAc-EtOH (220-15 ml) was added 10% Pd / C (1.8 g) to give a uniform black mixture. The reaction was placed under 1 atmosphere (balloon) of H 2, heated to 50 ° C, and stirred overnight. The reaction mixture was cooled to room temperature, the catalyst was removed by filtration and the filtrate was concentrated in vacuo. Product III (5.56 g, 93%) was obtained as a crystalline solid.

¹H NMR (500 MHz, CDCl 3): 7.29 (d, 1H), 6.22 (d, 1H), 6.17 (s, 1H), 4.20 (bs, 2H), 2.85 (s, 3H). For a two-phase mixture at room temperature of phenyl chloroformate (1.6 ml,

12.82 mmol) in EtOAc (20 mL) and saturated NaHCO3 solution (about 1 M, 16 mL) were added product III (950 mg, 6.41 mmol) as a solution in EtOAc (10 mL) over 10 minutes. The resulting homogeneous mixture was stirred at room temperature for 30 minutes and the phases were separated. The organic phase was washed with brine, dried over Na2SO4, filtered through a silica gel pad and concentrated in vacuo to a thick oil. This oil was diluted with toluene (30 mL) and treated with hexanes (30 mL) to give a thick precipitate. This mixture was stirred for 30 minutes, filtered, the solids washed with toluene: hexanes 1: 1, then with hexanes only, and dried to constant weight under high vacuum. Product IV (1.65 g, 86%) was obtained in the form of a white powder.

¹H NMR (500 MHz, dmso-d6): 10.76 (s, 1H), 7.69 (d, 1H), 7.44 (d, 1H), 7.40 (d, 1H), 7.26 (m, 3H), 7.15 (d, 1H), 3.85 (s, 3H).

B. Production of Intermediate Product - Right Side of Compound 169:

<img file="PL211562B1_D0003.tif" />

To a room temperature solution of product V (200 g, 1.21 mol) in EtOH (2 L), NaBH4 (50.3 g, 1.33 mol) was added portionwise over 30 minutes, controlling the internal temperature not to exceed 40 ° C. C. The reaction mixture was stirred at room temperature for 4 hours. The reaction was then quenched with water (about 100 mL), the mixture concentrated in vacuo, diluted with EtOAc, washed twice with water and once with saturated NaHCO3, dried over MgSO4, filtered and concentrated in vacuo. Product VI (191.7 g, 95%) was obtained in the form of a yellowish powder.

¹H NMR (500 MHz, CDCl 3): 8.21 (s, 1H), 8.09 (d, 1H), 7.70 (d, 1H), 7.49 (dd, 1H), 5.01 (dd , 1H), 2.45 (s, 1H), 1.52 (d, 3H).

To a room temperature solution of Product VI (181 g, 1.089 mol) was added DPPA (250 ml, 1.16 mol) at a rate slow enough to keep the reaction temperature below 45 ° C. After the addition of DPPA was complete, the mixture was treated with DBU (177 mL, 1.18 mol) at a rate slow enough to keep the reaction temperature below 45 ° C. After the addition was complete, the reaction mixture was heated to 60 ° C and held at that temperature overnight. The resulting biphasic mixture was cooled to room temperature, washed successively with water then 0.5 M HCl solution. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo to yield a yellow / green oil which was not further purified.

¹H NMR (500 MHz, CDCl 3): 8.21 (s, 1H), 8.18 (d, 1H), 7.68 (d, 1H), 7.56 (q, 1H), 4.76 (dd , 1H), 1.59 (d, 3H).

To a room temperature solution of product VII (8.17 g, 42.51 mmol) in a THF-water mixture (80 ml-10 ml) was added Ph3P (12.3 g, 46.76 mol) as a solution in 10 minutes. in THF (20 ml). During the addition, nitrogen was evolved immediately and solidly. The reaction mixture was then heated to 65 ° C overnight, then cooled to room temperature. The crude mixture was concentrated in vacuo, diluted with EtOAc, washed with brine, dried over Na2SO4, and filtered. A 1N HCl / Et2O solution was added to the resulting filtrate over 10 minutes at room temperature, resulting in the formation of a precipitate. The mixture was stirred at room temperature for 15 minutes and then filtered. The solids were washed with Et2O to give a yellow powder. The crude salt, amine hydrochloride, was suspended in brine / EtOAc and treated at room temperature with 10 N NaOH (5 mL, 50 mmol). The resulting mixture was stirred at room temperature until all solids were dissolved. The phases were separated, the aqueous phase was washed twice with EtOAc, the combined organic phases were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude amine was diluted in MeOH (50 ml) and added to a boiling solution of L - (+) - tartaric acid (5.33 g, 35.33 mmol) in MeOH (450 ml). A precipitate formed immediately which was dissolved in the MeOH mixture while refluxing for 15 minutes. The internal temperature was lowered to 50 ° C and kept at that level overnight. The internal temperature was then lowered to 30 ° C and kept at that level

After a further 24 hours, the mixture was kept for 24 hours at room temperature. The obtained crystals (spikes) were filtered, washed with MeOH and Et2O, and the mother liquor was discarded. The resulting crystals were dissolved in 200 ml of boiling MeOH, cooled slowly as described above, filtered and washed with MeOH followed by Et2O. The first crop of product VIII (2.21 g, 20%) was obtained as a white solid. The mother liquor was concentrated in vacuo, the solids were dissolved in 50 mL boiling MeOH, cooled as above, filtered and washed with MeOH and Et2O. A second crop of product VIII (1.50 g, 13%) was obtained as a white solid. The optical purity for each crop, based on the corresponding phenyl carbamate, was&gt; 97% ee.

The enantiomeric excess was determined using a Chiralcel OD column (0.45 cm x 25 cm) manufactured by Daicel Chemical Industries and purchased from Chiral Technologies. A mixture of hexane: IPA, 70:30 is used as the mobile phase in an isocratic run for up to 65 minutes at a flow rate of 0.8 ml / min, using 3-4 μl injections of 1-2 mg / ml of a solution of phenyl carbamate dissolved in the above mixture hexane : IPA. The desired S-methyl enantiomer eluted first after approximately 47.2 minutes, while the undesired R-methyl enantiomer appeared after approximately 51.7 minutes when monitored at 214, 254, 280 nm.

All samples were tested on a Hewlett Packard Series 1050 HPLC instrument with a radial diode detector.

To a homogeneous suspension of product VIII (1.11 g, 3.51 mol) in EtOAc (20 ml) and brine (20 ml) at room temperature was added 10 N NaOH solution (0.77 ml, 7.72 mmol). The resulting solution was stirred at room temperature until the salts dissolved. The phases were then separated and the aqueous phase was washed with EtOAc. The combined organics were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude nitro-benzylamine was diluted in 7M NH3-MeOH (20 mL), 20% Pd (OH) 2-C was added and placed under 45 psi H2 for 5 hours. The resulting mixture was filtered to remove catalyst, concentrated in vacuo and azeotroped with CH2Cl2 once, then placed under high vacuum. Product IX (455 mg, 95%) was obtained as a waxy white solid.

¹H NMR (500 MHz, dmso-d6): 6.91 (dd, 1H), 6.56 (s, 1H), 6.50 (d, 1H), 6.38 (d, 1H), 4.90 (bs, 2H), 3.82 (q, 1H), 3.31 (bs, 2H), 1.18 (d, 3H).

<img file="PL211562B1_D0004.tif" />

To a room temperature solution of 3- (R) -hydroxypentanitrile (212 mg, 2.14 mmol) was added CDI (521 mg, 3.21 mmol) in one portion. The resulting mixture was stirred at room temperature for 1 hour and then treated with solid silica gel. The homogeneous mixture was stirred vigorously for 10 minutes, filtered through a short silica gel pad with EtOAc: IPA, 4: 1, concentrated in vacuo, azeotroped twice with MeCN, then combined with product IX (350 mg, 2.57 mmol). ) in MeCN (2 ml) and stirred at room temperature for 1 day. The resulting mixture was diluted with EtOAc, washed with water and brine, dried over Na2SO4, concentrated, and subjected to flash chromatography (silica gel, 1/2&gt; 1/3&gt; 0/1 hexanes / EtOAc&gt; 4/1 EtOAc / IPA). Product X (472 mg, 84%) was obtained as a clear thick oil.

PL 211 562 B1 ¹H NMR (500 MHz, dmso-d6): 7.73 (d, 1H), 6.94 (dd, 1H), 6.51 (s, 1H), 6.47 (d, 1H), 6.38 (d, 1H), 4.98 (bs, 2H), 4.67 (m, 1H), 4.49 (m, 1H), 2.82 (m, 2H), 1.62 (m, 2H), 1.27 (d, 3H), 0.89 (dd, 3H).

Product TV (440 mg, 1.63 mmol) and TEA (0.23 mL, 1.63 mmol) were added to a room temperature solution of product X (470 mg, 1.80 mmol) in EtOAc (5 mL). The resulting mixture was heated to reflux and stirred at this temperature for 6 hours. The resulting crude mixture was cooled to room temperature, diluted with EtOAc, washed with brine / 1N HCl followed by brine alone, dried over Na2SO4, filtered, and concentrated in vacuo. Flash chromatography (silica gel, 1/1&gt; 1/2&gt; 1/3&gt; 1/4&gt; 0/1 hexanes / EtOAc&gt; 4/1 EtOAc / IPA) provided compound 169 (740 mg, 100%) as a white , a foamy solid.

¹H NMR (500 MHz, dmso-d6): 9.21 (s, 1H), 8.84 (s, 1H), 7.93 (d, 1H), 7.59 (d, 1H), 7.51 (s, 1H), 7.41 (s, 1H), 7.29 (d, 1H), 7.23 (dd, 1H), 7.01 (d, 1H), 6.92 (d, 1H) , 4.69 (m, 1H), 4.63 (m, 1H), 3.89 (s, 3H), 2.82 (m, 2H), 2.62 (m, 2H), 1.31 ( d, 3H), 0.90 (t, 3H).

EXAMPLE 2

Study of the inhibitory effect of IMPDH

IMP dehydrogenase activity was tested using an adaptation of the method first described by Magasanik. [B. Magasanik, HS ,. Moyed and LB Gehring (1957), J. Biol. Chem. 226, 339, which publication is hereby incorporated by reference]. The enzyme activity was measured spectrophotometrically, monitoring the increase in absorbance at 340 nm in relation to the production of NADH (λ340 means 6220 M^-1 cm^-1). The reaction mixture contained 0.1 M potassium phosphate 8.0, 0.5 mM EDTA, 2 mM DTT, 200 gM IMP, and the enzyme (human IMPDH type II) at a concentration of 15 to 50 nM. This solution was incubated at 37 ° C for 10 minutes. The reaction was started by the addition of NAD to a final concentration of 200 µM and the initial rate was measured by the linear increase in absorbance at 340 nm for 10 minutes. For reading in a standard spectrophotometer (track length 1 cm), the final volume in the cuvette is 1.0 ml. This study was also adapted to a 96-well microtiter plate; in this case the concentrations of all reagents remained the same and the final volume was reduced to 200 µl.

For inhibitor analysis, test compounds were dissolved in DMSO to a final concentration of 20 mM and added to the initial assay mixture to be pre-incubated with enzyme in a final volume of 2-5% (v / v). The reaction was started with the addition of NAD and the final rates were measured as described above. Ki constants were determined by measuring initial rates in the presence of varying amounts of inhibitor and fitting the data using tight-binding equations [PJF Henderson (1972) Biochem. J., 127, 321].

The results are shown in Table 1. Category "A is Ki = 10 mM or less.

T abl ca 1

IMPDH inhibitory activity

<td>Relationship</td><td>Ki (nm)</td>

<td> 169</td><td>AND</td>

<td> 181</td><td>AND</td>

EXAMPLE 3

Cell research

A. Isolation of human peripheral blood mononuclear cells (PBMC):

Venous blood was collected from normal healthy volunteers using heparin as an anticoagulant. PBMCs were isolated from blood by centrifugation using a Ficoll-paque gradient or CPT tubes (Becton-Dickinson) under standard conditions. PBMCs were harvested, washed and resuspended in complete RPMI, counted and diluted to 1 x 10⁶ cells / ml.

B. PBMC and Splenocyte Proliferation Assay

5 x 10 were added to the wells of a 96-well plate⁴ cells (for human T PBMC cells) or 1 x 10⁵ (for human PBMC B cells). In T cell studies, phytohaemoglutinin (PHA) was added to the cells in the well to a final concentration of 10-20 gg / ml. In B cell studies, Protein A Staphylococcal (SPAS) was added to a final concentration of 2 gg / ml.

A series of 4 dilutions of inhibitor solutions was prepared in complete RPMI medium and added to the cells such that the final concentration of compounds was between 20 gM to 20 nM and the final concentration of DMSO was 0.1%. The cells were then incubated for 3 days. All samples were tested in triplicate. Label was added for the last 24 hours of the test

PL 211 562 B1 (0.4 μCi / well). Cells were harvested on Betaplate filters and counted in a scintillation counter. Compound concentrations required to inhibit cell proliferation by 50% (IC50 values) were calculated using the SoftMax Pro computer program.^TM (Molecular Devices).

Table 2 presents the results of these tests.

"A is IC50 = 100 nM or less.

Table 2

Activity in cell research

<td>Relationship</td><td>T lymphocytes IC50</td><td>B lymphocytes IC50</td>

<td> 169</td><td>AND</td><td>AND</td>

EXAMPLE 4

Antiviral test

The antiviral activity of the compounds was evaluated in various in vitro and in vivo tests. For example, the compounds can be tested in in vitro viral replication assays. Whole cells or isolated cell components can be used in in vitro tests. In vivo studies include animal models of viral disease. Examples of such animal models include, but are not limited to, rodent HBV or HCV infection models, the Woodchuck model of HBV infection, and the chimpanzee model of HCV infection.