Methods of treating viral infection

CMX157 can also be used to treat HIV and HBV and / or inhibit the development of resistance to other antiviral compounds. (See PCT Publication Nos. WO 2009/094191 and WO 2009/094190). The construction of CMX157 is presented below:

<img file="PL2534150T3_D0001.tif" />

[0002] Recent studies have shown that gamma retrovirus - a related xenotropic murine leukemia virus (XMRV) and viruses similar to another murine leukemia virus (MLV) are associated with prostate cancer and / or chronic fatigue syndrome. (See, e.g., R. Schlaberg et al., PNAS, 106 (38): 6351-6 (2009), and VC Lombardi et al., Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome , Science, 326 (5952): 585-9, (October 2009), and Lo et al., PNAS early edition, published online before printing, August 23, 2010, doi: 10.1073 / pnas.1006901107). So far, the treatment options for prostate cancer are limited, and there is a lack of effective treatment of chronic fatigue syndrome. That's why new drugs are needed to treat viral diseases.

[0003] CN 1 810 816 A discloses compounds that are salts of tenofovir monoester.

Summary of the invention [0004] In a first aspect, the invention provides a compound of the formula

<img file="PL2534150T3_D0002.tif" />

in which M⁺ means K⁺.

[0005] In another aspect, the invention provides pharmaceutical compositions, compositions in a tablet or capsule dosage form, intravenous formulation, solutions, or suspensions containing the compounds described herein.

[0006] An aspect of the invention provides methods of making the compounds described herein. The methods include dissolving Compound I in a solvent,

<img file="PL2534150T3_D0003.tif" />

adding the base to the solvent mixture and Compound I and removing the solvent.

[0007] Another aspect of the invention provides methods of treating or preventing a viral disease. The methods include administering to the subject a subject an effective amount of the compounds described herein (e.g., CMX157 or a compound of Formula I). In one embodiment of the invention, the virus is a retrovirus, e.g. it is a related murine leukotropic virus of murine leukemia (XMRV).

[0008] Another aspect of the invention provides methods for treating or preventing chronic fatigue syndrome. Another aspect of the invention provides methods of treating or preventing prostate cancer.

[0009] In yet another aspect, the invention provides methods of treating a subject infected with at least one retrovirus, wherein the subject has not been administered an active antiviral agent for the retrovirus.

[0010] The methods include administering to the infected subject an amount described herein in an amount effective to treat a viral infection.

[0011] In another aspect, the invention provides methods of treating a subject infected with at least one retrovirus when the subject has developed resistance or toxic reaction to at least one other antiviral compound in response to prior administration to said subject in connection with a retroviral infection of said at least one member. one other antiviral compound. The methods include administering to the infected subject the compounds described herein in an amount effective to treat the viral infection.

[0012] Yet another aspect of the invention provides methods for inhibiting sexual transmission of HIV. The methods include topical application to the skin or human epithelial tissue of a therapeutically effective amount of a composition comprising the compound described herein as an antiviral agent. The methods further include administering to the subject one or more additional antivirally active compounds together with the compounds described herein.

[0013] Yet another aspect of the invention provides a pharmaceutical composition comprising the compound / salt described herein and a pharmaceutically acceptable carrier. Yet another aspect of the invention provides a pharmaceutical composition comprising the compound / salt described herein and at least one additional antivirally active agent and a pharmaceutically acceptable carrier.

Detailed Description of the Invention [0014] Described above, as well as other aspects of the present invention will now be described in more detail with reference to descriptions and methodologies provided herein. It will be appreciated that the invention may be embodied in various forms and its scope should not be limited only to the embodiments described herein. Rather, these variants are given so that the invention is accurate and complete and that the skilled person will be fully aware of the scope of the invention.

[0015] The terminology used in the present invention is intended to refer only to the description of specific embodiments and can not be regarded as a limitation of the invention. As used in the description of embodiments of the invention and in the appended claims, singular forms also include plural forms unless the opposite is clearly evident from the context. And also, the expression "and / or" as used herein refers to and includes all possible combinations of one or more of the terms mentioned. Furthermore, as used herein, the term "about" in relation to measurable values, such as the amount of compound, dose, time, temperature and the like, is considered to include deviations of 20%, 10%, 5%, 1% 0.5% or as much as 0.1% from the given amount.

[0016] It is also to be understood that the term "comprising" and / or "comprising" as used in the present specification defines the occurrence of certain features, numbers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other characteristics, numbers, stages, operations, elements, components and / or groups thereof. Unless otherwise stated, all terms, including technical and scientific terms used in this specification, have meanings as commonly accepted by those skilled in the art to which they refer.

[0017] The phrase "essentially consists of" (and its grammatical variants) with respect to the compositions of the present invention means that the composition may contain additional ingredients, provided that the additional components do not substantially change the composition. The expression "substantial alteration" with respect to compositions means increasing or decreasing the therapeutic effectiveness of the composition by at least 20% or more, compared to the effectiveness of the composition consisting of the given ingredients. [0018] Unless the context provides otherwise, it is in particular that the various aspects of the invention described herein can be used in any combination.

[0019] Furthermore, the present invention also provides that according to some embodiments of the invention, any of the aspects set forth herein, or combinations thereof, may be turned off or omitted.

A. Definitions [0020] As used herein, the term "alkali metals" means chemical elements from group 1 of the Periodic Table of the Elements, for example lithium (Li), sodium (Na) and potassium (K).

[0021] Individuals that can be treated by the methods of the present invention are generally mammalian and non-human primates (e.g., human, monkey, ape, chimpanzee). The subjects may be males or females of any age, including pre-birth (i.e. in the uterus), newborns, children, juveniles, adolescents, adults and old people. Thus, in some cases, individuals may be pregnant women. The reasons for treatment may vary, including treatment of previously infected individuals, as well as prophylactic treatment of uninfected individuals (e.g., individuals considered to be at high risk of infection).

[0022] As used herein, the term "Human immunodeficiency virus" (or "HIV") includes all of its subtypes, including the subtypes A, B, C, D, E, F, G and O, and HIV-2.

[0023] As used herein, the term "Hepatitis B virus" (or "HBV") includes all of its subtypes (adw, adr, ayw and ayr) and / or genotypes (A, B, C, D, E, F, G and H).

[0024] As used herein, the term "therapeutically effective amount" means an amount that will cause the subject to have some relief, alleviation and / or weakening of at least one of the clinical symptoms. Experts in the field understand that therapeutic effects do not have to be complete, nor do they have to cause a complete cure, provided that the subject does, however, feel some improvement.

[0025] As used herein, the term "specificity" or "specifically against" refers to a compound that has the ability to selectively inhibit the metabolic activity and / or replication of DNA of a certain type of virus-infected cells. The specificity can be tested using any method known to those skilled in the art, for example by determining IC90 and / or IC50. In some embodiments, the compounds described herein may exhibit IC90 and / or IC50 against virus-infected cells at least about three times lower than IC90 and / or IC50 against normal (non-infected) cells. In some embodiments, the compounds described herein may exhibit IC90 and / or IC50 against virus-infected cells at least about three to ten times lower than IC90 and / or IC50 against normal (non-infected) cells. In some variants, the compounds described herein may exhibit IC90 and / or IC50 against virus-infected cells at least ten times lower than IC90 and / or IC50 against normal (non-infected) cells. In some embodiments, the compounds described herein may exhibit specific cytotoxicity against cells infected and / or transformed by the virus. Cytotoxicity can be determined using any method known to those skilled in the art.

feline leukemia virus, feline sarcoma virus and reticuloendiosiosis avian virus. Many gamma retroviruses share the conserved RNA structural elements known as the encapsidation core signal.

As used herein, the terms "treating", "treating" refer to reversing, alleviating, inhibiting the progress of the disease or disorder described herein, or delaying, suppressing, or reducing the occurrence or appearance of the disorder or disease described herein compared to the situation, which would have happened if the treatment had not been taken. In some embodiments, the treatment may be administered after the onset of one or more symptoms. According to other variants, the treatment can be administered in the absence of symptoms. For example, the treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., taking into account symptom and / or genetic history, or other factors affecting susceptibility). Treatment may also continue after symptoms have resolved, for example to prevent or delay their relapse.

As used herein, the words "prevention", "prophylactic" or "prophylaxis" mean that the clinical manifestations of a disease or condition do not occur, i.e. inhibiting the occurrence of a disease or condition in a subject that may be exposed or predestined to a disease or condition but he has not yet suffered from a disease or condition or his symptoms have not yet revealed. Preventative administration means that a compound of the invention is administered to a subject before observing the symptoms and / or presumed exposure to the causative agent (e.g., pathogen or carcinogen). In general, preventive administration can reduce (a) the likelihood that a subject will receive a condition and / or (b) duration and / or severity of symptoms when a subject experiences a disease state.

[0030] The active compound of the present invention may optionally be administered in combination (or in combination) with other active compounds and / or agents useful in the treatment of viral infections, as described herein. Administration of two or more compounds "in combination" or "in combination" means that the two compounds are administered at close in time to provide a combined effect, e.g. additive and / or synergistic. Both compounds may be administered simultaneously (at the same time), or sequentially, or they may be two or more administrations occurring in a short interval of time, one before or the other. Simultaneous administration can be carried out by mixing the compounds with each other before administration, or by administering the compounds at the same time, but at different anatomically different sites, or by using different routes of administration.

[0031] As used herein, the term "parenterally" refers to injection, or subcutaneous, intravenous, intraarterial, intramuscular or ocular techniques.

[0032] As used herein, the term "topical" includes rectal and spray inhalation as well as more common dermal and oral mucous membranes of the mouth, nose and toothpaste.

B. Compounds [0033] The present inventors have found that CMX157 in the free acid form is quite unstable (see Example 2). It has surprisingly been found that CMX157 salts are much more stable than CMX157 and have more favorable physical properties, such as hygroscopicity and behavior at all manipulations, which makes their processing and formulating easier.

[0034] In particular, one aspect of the invention provides a compound of the formula

<img file="PL2534150T3_D0004.tif" />

in which M⁺ means K⁺. The salt may be in various forms and they are all within the scope of the present invention. These forms include anhydrous forms or solvates. According to other embodiments, the salt may be crystalline.

C. Method of production [0035] In general, the compounds of the present invention can be prepared by standard techniques known in the art using known processes analogous thereto. For example, CMX157 can be made according to known procedures, or using their modifications, obvious to those skilled in the art. See, e.g., Painter et al., Antimicrobial Agents and Chemotherapy 51, 3505-3509 (2007), and U.S. Patent Application Publication No. 2007/0003516 to Almond et al.

[0036] General methods for the preparation of compounds of the present invention are set out below. In the following description, all variables, unless otherwise stated, are as shown in the formulas described herein. The following descriptions, but not limiting, illustrate the general methodologies that can be used to obtain the compounds described herein.

[0037] In one embodiment, the compound described herein can be obtained by dissolving compound 1 in a suitable solvent

<img file="PL2534150T3_D0005.tif" />

adding a suitable base to the solvent mixture and compound 1 and removing the solvent to obtain a compound of formula I.

[0038] Any solvent known to those skilled in the art may be used as the solvent for the preparation, or a combination of solvents that will provide the product with the proper performance. In one embodiment of the invention, the solvent is a mixture of at least two solvents. Examples of solvent combinations, however, not limiting, are dichloromethane and methanol, as well as dichloromethane and ethanol. In one embodiment of the invention, the molar ratio of dichloromethane and methanol is in the range of from 7: 3 to 9: 1. In another embodiment of the invention, the molar ratio of dichloromethane and methanol is 9: 1.

[0039] The base used for the preparation can be any one known to those skilled in the art, or a combination of bases that will ensure satisfactory product performance. In some embodiments, the base is an alkali metal alkoxide. Exemplary bases include, but are not limited to, potassium methoxide, sodium methoxide, lithium tert-butoxide, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0040] The process described herein may also include a recrystallization step to remove impurities, byproducts and unreacted starting material. The recrystallization step comprises the step of dissolving the product in a suitable solvent at a suitable temperature, cooling to a suitable temperature for a time sufficient to precipitate the compound of formula I and filtration to obtain a compound of formula I. In some embodiments, the temperature in the dissolving step is in the range of about 50^aboutC to 80^aboutC.

D. Additional Antiviral Agents / Compounds [0041] Additional antiviral agents that can be used in the present invention include HIV protease inhibitors, nucleoside reverse transcriptase inhibitors (the term includes nucleotide reverse transcriptase inhibitors herein), non-nucleoside reverse transcriptase inhibitors, inhibitors integrase, entry inhibitors, fusion inhibitors, maturation inhibitors, and combinations thereof. Numerous examples are known and described, e.g., U.S. Patent Application Publication No. 2006/0234982 to Dahl et al. In Table A thereunder, and Table A below.

[0042] Additional examples are, without limitation, the Isentress integrase inhibitor, or raltegravir (MK-0518; Merck), the Maraviroc CCR5 or selzentry inhibitor (and K427857, Pfizer), and others of that class.

[0043] Additional examples are disclosed in U.S. Patent Nos. 7,094,413 to Buelow et al .; U.S. Patent No. 7,250,421 by Nair et al .; in U.S. Patent Application Publication No. 2007/0265227 to Heneine et al. and U.S. Patent Application Publication No. 2007/0072831 to Cai et al.

[0044] A non-nucleoside reverse transcriptase inhibitor ("NNRTI") 6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro = 2H3,1-benzoxazin-2-one and its pharmaceutically acceptable salts are described in, e.g. U.S. Patent No. 5,511,921. The examples herein are efavirenz.

[0045] The nucleoside reverse transcriptase inhibitor ("NRTI") 2-hydroxymethyl-5- (5-fluocytosin-1-yl) -1,3-oxatiolane ("FTC") and its pharmaceutically acceptable salts are described in, e.g., US Patent No. 6 642 245 for Liotta et al. Emtricitabine is one of the examples in the present invention.

Examples of integrase inhibitors include, but are not limited to, those described in U.S. Patent Application Publication No. 2007/0072831, WO 02/30426, WO 02/30930, WO 02/30931, WO 02/055079, WO 02/36734, patents U.S. Patent No. 6,395,743;

U.S. Patent No. 6,245,806; U.S. Patent No. 6,271,402; WO 00/039086; WO 00/075122; WO 99/62513; WO 99/62520; WO 01/00578; Jing, et al., Biochemistry, 41, 5397-5403, (2002); Pais, et al., J. Med. Chem., 45, 3184-94 (2002); Goldgur et al. Proc. Natl. Acad. Sci. USA, 96, 13040-13043 (1999); Espeseth, et al. Proc. Natl. Acad. Sci. USA 97.11244-11249, (2000); WO 2005/016927, WO 2004/096807, WO 2004/035577, WO 2004/035576 and US 2003/0055071.

Table A

5.6 dihydro-5-azacytidine

5-aza-2'-deoxycytidine

5-azacytidine

5-yl-carbocyclo-2'-deoxyguanosine (BMS200,475)

9- (arabinofuranosyl) guanine; 9- (2'-deoksyrybofuranozylo) guanine

9- (2'-deoxy-2'-fluororybofuranozylo) -2,6-diaminopurine

9- (2'-deoxy-2'-fluororybofuranozylo) guanine

9- (2'-deoksyrybofuranozylo) -2,6-diaminopurine

9- (arabinofuranosyl) -2,6-diaminopurine Abacavir, Ziagen®

Aciclovir, ACV; 9- (2-hydroxyethoxylmethyl) guanine Adefovir dipipoxil, Hepsera®

Amdoxivir, DAPD Amprenavir, Agenerase®

<td>araa; 9-β-D-arabinofuranosyloadenine (Vidarabine)</td>

<td>Atazanivir sulphate (Reyataz®)</td>

<td>AZT; 3'-Azido-2 ', 3'-dideoxytimidine, Zidovudine, (Retrovir®)</td>

<td>BHCG; (+ -) - (1a, 2b, 3a) -9- [2,3-bis (hydroxymethyl) cyclobutyl] guanine</td>

<td>BMS200,475; 5-yl-karbcyklo-2'-deoxyguanosine</td>

<td>Bucyclovir, (R) 9- (3,4-dihydroxybutyl) guanine</td>

<td>BvaraU; 1-β-D-arabinofuranosyl-E-5- (2-bromovinyl) uracil; (Sorivudine)</td>

<td>Kalanolid A</td>

<td>capravirine</td>

<td>CDG; 2'-deoxyguanosine carboxy</td>

<td>cidofovir, HPMPC; (S) -9- (3-hydroxy-2-fosfonylometoksypropylo) cytosine</td>

<td>Klewudyna, L-FMAU; 2'-Fluoro-5-methyl-β-L-arabinofuranosyluracil</td>

<td>Combivir® (lamivudine / zidovudine)</td>

<td>Cytallene; [1- (4'-hydroxy-1 ', 2'-butadienylo) cytosine]</td>

<td>DAPD; dioxolane (-) - β-2,6-diaminopurine</td>

<td>ddA; 2 ', 3'-dideoxyadenosine</td>

<td>ddAPR; 2,6-diaminopurine 2 ', 3'-dideoxyriboside</td>

<td>ddC; 2 ', 3'-dideoxycytidine (Zalcitabine)</td>

<td>ddI; 2 ', 3'-dideoxyinosine, didanosine (Videx®, Videx® EC)</td>

<td>Delavirdine, Rescriptor®</td>

<td>Didanosine, ddI, Videx®, 2 ', 3'-dideoxyinosine</td>

<td>DXG; Guanosine dioxolate</td>

<td>E-5- (2-bromovinyl) -2'-deoxyuridine</td>

<td>Efawirenz, Sustiva®,</td>

<td>Enfuwirtyd, Fuzeon®,</td>

<td>F-ara-A; Fluoroarabinosyl adenosine (Fludarabine)</td>

<td>FDOC; (-) - β-D-5-fluoro-1- [2- (hydroxymethyl) -1,3-dioxolane] cytosine</td>

<td>FEAU; 2'-deoxy-2'-fluoro-1-β-D-arabinofuranosyl-5-etylouracyl</td>

<td>FIAC; 1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-jodocytozyna</td>

FIAU; 1- (2-deoxy-2-fluoro-eD-arabinofuranosyl - (5-iodorridine FLG; 2 ', 3'-dideoxy-3'-fluoroguanosine FLT; 3'-deoxy-3'-fluorothymidine

<td>fludarabine; F-ara-A; fluoroarabinozyloadenozyna</td>

<td>FMAU; 2'-Fluoro-5-methyl-EL-arabinofuranosyluracil</td>

<td>FMdC</td>

<td>foscarnet; phosphonomethasic acid, PFA</td>

<td>FPMP; 9- (3-fluoro-2-fosfonylometoksypropylo) adenine</td>

<td>ganciclovir; GCV; 9- (1,3-dihydroxy-2-propoxymethyl) guanine</td>

<td>GS-7340; 9- [R-2 - [[(S) - [[(S) -1- (isopropoxycarbonyl) ethyl] amino] phenoxyphinylpine methoxy] propyl] adenine</td>

<td>HPMP; (S) -9- (3-hydroxy-2-fosfonylometoksypropylo) adenine</td>

<td>HPMPC; (S) -9- (3-hydroxy-2-phosphonylmethoxypropyl) cytosine (Cidofovir)</td>

<td>Hydroxycarbamide, Droxia®</td>

<td>Indinavir, Crixivan®</td>

<td>Kaletra®, (lopinavir / ritonavir)</td>

<td>Lamivudine, 3TC, Epivir ™; (2R, 5S, cis) -4-amino-1- (2-hydroxymethyl-1,3-oxathiolan-5-yl) (1H) -pyrimidin-2-one</td>

<td>L-D4C; L-3'-deoxy-2 ', 3'-didehydrocytydyna</td>

<td>L-ddC; L-2 ', 3'-dideoxycytidine</td>

<td>L-Fd4C; L-3'-deoxy-2 ', 3'-didehydro-5-fluorocytidine</td>

<td>L-FddC; L-2 ', 3'-dideoxy-5-fluorocytidine</td>

<td>lopinavir</td>

<td>Nelfinavir, Viracept®</td>

<td>Nevirapine, Viramune®</td>

<td>Oxetanocin A; 9- (2-deoxy-2-hydroxymethyl-β-D-erythro-oksetanozylo) adenine</td>

<td>Oxetanocin G; 9- (2-deoxy-2-hydroxymethyl-β-D-erythro-oxetanosyl) guanine</td>

<td>penciclovir</td>

<td>PMEDAP; 9- (2-phosphonylmethoxyethyl) -2,6-diaminopurine</td>

<td>PMPA, tenofovir; (R) -9- (2-fosfonylometoksypropylo) adenine</td>

<td>PPA; phosphonoacetic acid</td>

<td>ribavirin; 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide</td>

<td>Ritonavir, Norvir®</td>

<td>Saquinavir, Invirase®, Fortovase®</td>

<td>Sorywudyna, BvaraU; 1-β-D-arabinofuranosyl-E-5- (2-bromovinyl) uracil</td>

<td>Stavudyna, d4T, Zerit®; 2 ', 3'-didehydro-3'-deoxythymidine</td>

Trifluorothymidine, TFT;

Trizivir®, (abacavir sulphate / lamivudine / zidovudine)

Vidarabine®, araA; 9-β-D-arabinofuranosyl

Viread®, tenofovir diisoproxil fumarate (DF), Bis POC PMPA, TDF;

2,4,6,8-tetraoxa-5-phosphono-anionic acid, 5 - [[(1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy] methyl] -, bis (1-methylethyl ) ester, 5-oxide, (2E) -2-butenedioate (1: 1) Zalcitabine, Hivid®, ddC; 2 ', 3'-dideoxycytidine Zidovudine, AZT, Retrovir®, 3'-azido-2', 3'-dideoxytimidine Zonavir; 5-propynyl-1-arabinosyluracil [0047] In another embodiment of the invention, the compositions of the present invention may comprise the active compounds described herein in combination with one or more (e.g., 1, 2, 3) additional active agents described above, analogously to methods known in the art. For example, the combinations of efavirenz (NRTI), emtricitabine (NNRTI) and tenofovir DF (NRTI) are described, for example in Dahl et al. U.S. Patent Application Publication No. 2007/0099902 by Dahl et al. For specific examples of such combinations,

(a) FTC / Efavirenz;

(b) 3TC / Efavirenz;

(c) AZT / 3TC;

(d) FTC;

(e) 3TC;

(f) FTC / Isentress;

(g) 3TC / Isentress;

(h) PPL-100;

(i) FTC / TMC278;

(k) 3TC / TMC278;

(l) FTC / TMC125; or (m) 3TC / TMC125.

E. Pharmaceutical Formulations and Administration [0048] In one embodiment of the invention, the present invention is a pharmaceutical composition comprising a compound of the present invention. In another embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to any substance that as such is not a therapeutic agent, but is intended to provide a subject with a therapeutic agent. Examples of pharmaceutically acceptable carriers and methods for the preparation of various compositions are, but not limiting, those described in Remington's

Pharmaceutical Sciences, 18th Ed., Mack Publishing Co. (1990) (see also Patent Application US 2007/0072831).

[0049] The compounds of the invention may be formulated using common carriers, diluents and excipients selected in accordance with common practice in pharmacy. The tablets will contain excipients, lubricants, fillers, binders, diluents and the like. The aqueous formulations are prepared in a sterile form, and if they are intended for administration by a non-oral route, they are generally isotonic. The formulations optionally contain excipients such as those listed in the "Handbook of Pharmaceutical Excipients" (1986) and contain ascorbic acid and other antioxidants; chelating agents such as EDTA; hydrocarbons, such as dextrin, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose; stearic acid and the like.

[0050] A compound of the invention (hereinafter referred to as an active ingredient) may be administered by any route appropriate to the condition to be treated and such suitable routes include oral, rectal, nasal, topical (including ophthalmic, buccal and sublingual), vaginal and parenteral routes. (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural). The preferred route of administration may vary depending on, e.g., the condition of the recipient.

[0051] Although it is possible to administer the active ingredient alone, it is more preferred that it is in the form of a pharmaceutical formulation. The formulations of the present invention, both for veterinary and human use, contain at least one active ingredient as defined above, together with one or more pharmaceutically acceptable carriers (excipients, diluents etc.) and optionally other therapeutic ingredients. The carrier (s) must be &amp; quot; acceptable &amp; quot; in the sense of being compatible with the other ingredients of the formulation, and also not deleterious to the recipient. Formulations include those suitable for oral, rectal, nasal, topical (including on the cheek and under tongue), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural). The formulations may conveniently be in the form of dosage units and may be prepared by any of the methods well known in the art of pharmacy. These methods involve combining the active ingredient with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, or finely divided solid carriers, or with each other, and then, if necessary, forming the product.

[0053] Formulations of the present invention suitable for oral administration may be in the form of discrete units, such as capsules, cachets, or tablets, each comprising a predetermined amount of the active ingredient; a powder or granules form; a solution or suspension in an aqueous or non-aqueous fluid; or an oil-in-water or water-in-oil emulsion. The active ingredient may be present in a bolus, electuary or paste.

[0054] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated or scored and may be in a form that provides a slow or controlled release of the active ingredient contained therein.

[0055] Formulations for use in cases of ocular infections or other external tissues, e.g. mouth or skin, in some embodiments of the invention are in the form of an ointment or cream that is topically applied and which contain the active ingredient in an amount, e.g. 0.005 to 20 % by mass (which includes the content of the active ingredient in the range from 0.05% to 20% ascending 0.05% by mass, such as 0.6% by mass, 0.65% by mass, 0.7% by mass, etc.), in some variants from 0.05 to 15 mass%, and in other variants from 0.05 to 10 mass%. In the case of an ointment form, the active ingredient can be used in a paraffin base or in a base mixed with water. Optionally, the active ingredient may be formulated in a cream with an oil-in-water cream base.

[0056] If desired, the aqueous phase of the cream base may contain, for example, at least 30% by weight polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol. , glycerol and polyethylene glycol (including PGE400) and mixtures thereof. The topical formulations may desirably include a compound that increases the absorption or penetration of the active ingredient through the skin, or through other diseased areas. Examples of such skin penetration enhancers are dimethyl sulfoxide and other similar compounds.

[0057] The oily phase of the emulsions of the present invention can be prepared by known methods from commonly known ingredients. As the phase may contain an emulsifying agent (also known as an emulsifier), it preferably contains a mixture of at least one emulsifier with a fat or oil or with a fat and with oil. In some embodiments, the hydrophilic emulsifier is present together with a lipophilic emulsifier that acts as a stabilizer. In some embodiments, the invention contains both oil and fat. The emulsifier (s) with or without the stabilizer (s) together form a so-called emulsifying wax, and the wax together with the oil and fat forms a so-called emulsifying ointment base which is an oil-dispersed phase of the cream type formulation.

[0058] Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include Tween® 60, Span®80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.

[0059] The choice of oils or fat suitable for formulation is determined by the cosmetic properties desired, since the solubility of the active compound in most oils suitable for emulsion pharmaceutical formulations is low. In some embodiments of the invention, the cream should preferably be a non-greasy, non-staining and washable product, with a consistency that does not leak from the tube or other containers. Straight or branched chain mono- or dibasic esters can be used, such as diisocyanate, isocetyl stearate, diesters of coco fatty acids with propylene glycol, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a mixture thereof. branched chain esters known as Crodamol CAP, the last three of which are preferred esters. They can be used alone or in combination depending on the properties that are desired. Alternatively, fats with a high melting point, such as white soft paraffin and / or liquid paraffin, or other mineral oils can be used.

[0060] Formulations suitable for topical application to the eye also include droplets in which the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. In some embodiments of the invention, the active ingredient is present in such formulations at a concentration of 0.1 to 20%. In some embodiments of the invention, the active ingredient is present at a concentration of 0.1 to 10%. In some embodiments of the invention, the active ingredient is present at a concentration of about 1.5% by weight.

[0061] Formulations suitable for topical application in the mouth include lozenges comprising the active ingredient in a flavoring base, usually sucrose and acacia or tragacanth gum; pastilles containing the active ingredient in an inert support such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0062] Formulations for rectal administration may be in the form of suppositories with a suitable base comprising, for example, cocoa butter, or a salicylate.

[0063] For formulations suitable for nasal use in which the carrier is solid, powders having a particle size of, for example, 20 to 500 microns (including a particle size in the range of 20 to 500 microns, ascending 5 microns, such as 30 microns, 35 microns, etc.), which are drawn in quickly through the nasal passages with a powder of a container adhering closely to the nose through a rapid inspiration. Formulations in which the carrier is fluid, intended for administration in the form of a spray or nasal drops contain aqueous or oily solutions of the active ingredient. Formulations for administration in the form of an aerosol can be prepared by commonly known methods and can be administered together with other therapeutic agents, such as pentamidine in the treatment of pneumocystosis.

[0064] Formulations for vaginal administration may be in the form of pessaries, rings, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, suitable carriers known in the pharmaceutical field.

[0065] Formulations suitable for parenteral administration include aqueous or non-aqueous sterile injectable solutions containing antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the subject to which it is administered; and aqueous and non-aqueous sterile suspensions which may contain suspending and thickening agents. The formulations may be in single dose or multiple dose containers, e.g., they may be embedded in ampoules, or vials that may be stored in lyophilized form requiring only the addition of a sterile vehicle, e.g. water for injection, immediately prior to administration. Such solutions and suspensions prepared immediately prior to administration can be prepared from sterile powders, granules and tablets as previously described. Dosage formulations comprising a daily dose, or a daily dose as mentioned above, or an appropriate part of the dose of the active ingredient are preferred.

[0066] It is understood that in addition to the ingredients listed above, the formulations of the invention may contain other ingredients known in the pharmaceutical field that are suitable for the type of formulation in question, for example, formulations for oral administration may contain flavors.

[0067] The present invention also provides veterinary compositions comprising at least an active ingredient as mentioned above together with a veterinary carrier. Veterinary carriers are materials suitable for administering the composition; they may be solid, liquid or gaseous and which are inert and acceptable in the veterinary field, and compatible with the active ingredient. These veterinary compositions can be administered orally, parenterally, or any other appropriate route.

[0068] The compounds described herein can be used for the preparation of controlled release pharmaceutical formulations containing as a active ingredient a compound of the invention ("controlled release formulations") in which the release of the active ingredient can be controlled or adjusted to provide slower release, or improving the pharmacokinetic or toxic profile of a compound of the invention. Controlled release formulations for oral administration in which one or more compounds of the invention are present in individual dosage units can be made by methods well known in the art of pharmacy. Controlled release formulations can be used in the treatment or prophylaxis of various infections caused by microorganisms, in particular bacterial infections in humans, parasitic protozoal infections in humans, human viral infections caused by microorganisms, including Plasmodium, Pneumocystis, herpes viruses (CMV, HSV 1, HSV 2, VZV and the like), retroviruses, adenoviruses and the like. Controlled release formulations can be used to treat HIV infection and associated conditions such as tuberculosis, malaria, pneumocystosis, cytomegalovirus-induced retinal inflammation, AIDS, AIDS-related syndrome (ARC) and progressive generalized lymphadenopathy (PGL) and neurological conditions associated with AIDS such as multiple sclerosis and tropical spastic paraparesis. Other human retroviral infections that can be treated with the controlled release formulations of the invention include human Tlimotropin virus and HIV 2. Thus,

[0069] Pharmacokinetic enhancers. The compounds described herein can be used in combination with pharmacokinetic enhancers (sometimes also referred to as "enhancers"). In one aspect, the invention provides the use of an effective dose of an enhancer to increase or "enhance" the pharmacokinetics of a compound of the invention. An effective amount of an enhancer, e.g. the amount necessary to increase the activity of a compound of the invention, is the amount necessary to improve the pharmacokinetic profile or activity of the compound compared to its profile when used alone. The compound has a more effective pharmacokinetic profile than it would have without the addition of an enhancer. The amount of pharmacokinetic enhancer that is used to enhance the effect of the drug is preferably lower than the therapeutic (e.g. doses lower than the amount of enhancer that is usually used to treat infection in a patient). The enhancing dose for the compounds of the invention is lower than the therapeutic used to treat the infection, however high enough to cause a change in the metabolism of the compounds of the invention to enhance patient interaction by increasing bioavailability, increasing blood concentration, increasing half-life, increasing the duration of maximal concentration in plasma, increase / accelerate the inhibition of HIV integrase, RT, or protease and / or reduce clearance. One example of a pharmacokinetic enhancer is RITONAVIR ™ (Abbott Laboratories). however, high enough to cause a change in the metabolism of the compounds of the invention, enhancing patient interaction by increasing bioavailability, increasing blood concentration, increasing half-life, increasing the duration of maximum plasma concentration, increasing / accelerating inhibition of HIV integrase, RT, or protease and / or restriction of clearance. One example of a pharmacokinetic enhancer is RITONAVIR ™ (Abbott Laboratories). however, high enough to cause a change in the metabolism of the compounds of the invention, enhancing patient interaction by increasing bioavailability, increasing blood concentration, increasing half-life, increasing the duration of maximum plasma concentration, increasing / accelerating inhibition of HIV integrase, RT, or protease and / or restriction of clearance. One example of a pharmacokinetic enhancer is RITONAVIR ™ (Abbott Laboratories).

F. Methods of Treatment [0070] According to one of its aspects, the invention provides methods for treating disorders caused by viral infections. According to some aspects, the virus is a retrovirus. In one embodiment of the invention, the virus is a gamma retrovirus. The term "retrovirus" as used herein means an RNA virus that replicates in host cells by the reverse transcriptase enzyme to give DNA from its RNA genome. The DNA is then incorporated into the host genome by the enzyme integrase. The virus then replicates as part of the host cell DNA. Retroviruses are coated viruses belonging to the Retroviridae family of viruses. Examples of retroviruses include, but are not limited to, human immunodeficiency virus and a related murine leukotriese virus (XMRV) related virus. In addition, there is evidence to show that that XMRV may be associated with chronic fatigue syndrome (CFS). (See, e.g., Lombardi et al., Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome, Science, vol. 326, P 585-589 (October 2009).) [0071] In one of of the invention, the compound used to treat the subject is potassium salt CMX157. In another embodiment, the compound used to treat the subject is free CMX157.

[0072] In one embodiment of the invention, the subject is a human. In one embodiment of the invention, the subject is immunodeficient and / or has been immunosuppressed. In some embodiments of the invention, toxic side effects in an immunodeficient are reduced compared to toxic side effects when using tenofovir or other antiviral agents. In one embodiment of the invention, the subject suffers from chronic fatigue syndrome. In one embodiment of the invention, the subject suffers from prostate cancer.

[0073] The symptoms of chronic fatigue (CFS) may include, for example, fatigue impeding the performance of ordinary activities of daily living, including syncope, chest pain, muscle pain, palpitations, irritated throat, low-grade fever, inability to exercise without deterioration. symptoms including the next day, cervical lymphadenopathy, cognitive impairment and the resulting depression, and intolerance to alcohol. There may also be excessive activity of the immune system.

[0074] In one embodiment of the invention, the methods object of the invention alleviate one or more symptoms associated with chronic fatigue syndrome.

[0075] As used herein, "immunodeficiency" is a condition in which the immune system's ability to fight an infectious disease is impaired or completely absent. A subject with immunodeficiency means a subject with immunodeficiency of any type or degree. Examples of immunocompromised individuals include, but are not limited to, individuals with primary immunodeficiency (a subject who was born with a defect in the immune system) and a subject with secondary (acquired) immunodeficiency. In addition, the usual causes of secondary immunodeficiency include, but are not limited to, malnutrition, aging and certain therapies (e.g., immunosuppressive therapy such as chemotherapy, disease-suppressing anti-rheumatic drugs, immunosuppressive drugs after organ transplants, glucocorticoids).

The antiviral effects of CMX157 have been described in U.S. Patent Nos. 6,716,825,744,014, 7,094,772, 7,098,197, 7,452,898 and PCT Publication WO 2008/133966.

2. Treatment of infection in the main compartment [0077] It has also been found that the compounds described herein can bind to or bind to virus particles. The virus particles migrate or penetrate into the cellular or tissue compartments and hence the active therapeutic agents generally do not reach them (in this way a "reservoir" of infection is formed, which is virtually untreated when patients are administered such devices systemically). This finding enables (a) the treatment of infections in such major compartments and (b) the use of active ingredients for the prophylaxis or treatment of microbial infections (it is beneficial from a therapeutic point of view if the active substance is combined or binds to the virus before infection occurs) ).

[0078] In general, the main compartment is a cellular or tissue compartment into which said virus is infused in vivo and to which said active substance does not penetrate sufficiently in vivo, in the absence of said virus, and to which said active substance is introduced in vivo. by said virus in the situation where said active substance binds to said virus. For example, when the main compartment is a tissue compartment, it can be the brain (the central nervous system), the lymph gland, or the testes. Examples of major cell compartments include, but are not limited to, dendritic cells, microglia, monocytes / macrophages, and combinations thereof. Compositions for treating infections of major compartments can be prepared and treated by the methods described above. Prophylactic compositions,

[0079] The treatment of infection of major compartments using CMX157 has been described in PCT publication numbers WO 2009/094191 and WO 2009/094190.

F. Topical Compositions and Methods for Combating Infections Caused By Microbes [0080] The subject of the present invention may be in the form of a topical composition comprising an active ingredient described herein that is capable of inhibiting or combating a viral infection, e.g. for prophylactic use. Such compositions (containing active ingredients other than those disclosed herein) are known and are described, for example, in US Patent No. 6,545, 007.

[0081] The compositions may exist in several forms. Thus, in one embodiment of the invention, the composition is in the form of a cream, lotion, gel, or foam, which is applied to the changed skin or epithelium in the body cavity, and preferably spreads over the entire skin or epithelial surface at risk. contact with body fluids. Such formulations that are suitable for vaginal or rectal administration may be in the form of aqueous or oily suspensions, solutions, or emulsions (liquid formulations) containing, in addition to the active ingredient, carriers that are known in the pharmaceutical field to be suitable for the purpose. As lubricants that occur separately (ie substances that are not packaged together with condoms), they are generally beneficial for many reasons (both scientific and economic), gels and similar aqueous formulations, and these reasons are known to those skilled in the art. These formulations are useful not only because they prevent sexual transmission of the retrovirus, but also because they prevent infection of the child while crossing the genital tract. Vaginal delivery may take place before sexual intercourse, during intercourse, and just before the birth of the child.

[0082] One method of applying the genitalia of an antiviral lubricant formulation, for the reasons disclosed herein, is to take a small amount (such as a teaspoon or a few milliliters) of a gel, cream, ointment, emulsion, or similar formulation from a plastic or metal tube. , a jar, or similar container, or from a sealed plastic, metal, or other sachet containing a single dose of such a composition, and spreading the composition on the surface of the penis just prior to intercourse. Alternative application methods include (1) spreading the composition on accessible surfaces within the vagina or anus shortly before intercourse; and (2) applying a condom, disk, or similar device that has been previously coated, or otherwise contacted with an antiviral lubricant preparation, on the penis or vagina. In a preferred variant of the invention, each of these methods for spreading the antiviral lubricant preparation on the surfaces of the genitals causes that during the ratio the lubricant covers and remains in contact with the genitals or epithelial surfaces.

[0083] In one embodiment of the invention, the compositions are used in conjunction with condoms to increase the effectiveness of condoms in reducing risk and providing users with maximum protection. The composition may be coated with condoms at the stage of their production and then sealed in ordinary watertight plastic or foil packages containing individual condoms, or the composition may be applied by the user, either inside or outside of the condom, just prior to use.

[0084] The term "condom" as used herein means a barrier device that is a waterproof physical barrier separating male and female genital relationship and which is removed after intercourse. The term covers ordinary condoms that cover the penis; it also includes so-called "female condoms" that are placed in the vaginal cavity before intercourse. The term "condom" does not include discs, caps on the cervix and other divisions that cover only part of the mucosa in the vaginal cavity. Preferably, the condoms should be made of latex or other synthetic plastic material, such as polyurethane, because they provide a high degree of protection against viruses.

[0085] In another embodiment of the invention, the composition is in the form of an intravaginal or rectal pill or suppository. The suppository or pill should be placed in the vaginal or rectal cavity in a way that allows the walls of the vagina or the anus to be covered with a protective layer of the antiviral agent, after dissolving or erosion of the suppository or pill.

[0086] In yet another embodiment of the invention, the composition is applied topically as a result of its release from an intravaginal device. Such devices include vaginal rings, vaginal sponges, discs, caps on the cervix, female condoms and the like, which can be easily adjusted to release the composition into the vaginal cavity after it has been inserted.

[0087] The compositions used in the methods of the present invention may also contain additional active agents, such as another anti-retroviral agent (s) and agents to prevent fertilization, as well as protection against other sexually transmitted diseases. Thus, in another embodiment of the invention, the compositions further comprise one or more additional antiviral, virucidal agents effective against viral infections and / or spermicides.

[0088] In one particular embodiment of the invention, the composition comprises nonoxynol, a commonly used spermicide surfactant. The composition formed can be treated as a bifunctional composition, because it contains two active ingredients that performs two different desirable functions in a relatively inert fluid carrier; nonoxynol will be a spermicidal contraceptive, and the dihydroalkoxybenzyloxopyrimidine derivative (DABO) provides antiviral properties. Nonoxynol may cause at least some users some degree of irritation; this is, unfortunately, a well-known side effect of spermicidal surfactants, such as nonoxynol and octoxynol, which attack and destroy the lipid bilayer membrane surrounding sperm cells and other mammalian cells.

[0089] The compositions may also include a lubricant that facilitates application of the composition to the appropriate surfaces of the epithelial skin and tissue and reduces friction during intercourse. In the case of a pill or suppository, the lubricant may be applied to the outer surface of the dosage form to facilitate its introduction.

[0090] In yet another variant of the invention, the invention provides a device for inhibiting sexually-transmitted retrovirus comprising (a) a kind of barrier intended to be placed in a vaginal cavity and (b) a composition comprising an active agent described herein. As stated above, preferred devices that act as a barrier and that can be adapted to the delivery of the antiviral agent are the vaginal sponge, the disc, the cap on the cervix and the condom (male or female).

[0091] The methods, compositions and devices can be adapted in general so that the release of the active ingredient occurs at a time consistent with sexual activity. When applied topically in the form of a lotion or gel, the compositions are preferably applied just before sexual activity. Other methods of use, such as devices and suppositories, can be designed such that the active ingredient is released over a prolonged period of time at a predetermined rate depending on the needs of the user.

[0092] Topical compositions and antimicrobial methods using CMX157 have also been described in PCT Publication Nos. WO 2009/094191 and WO 2009/094190.

G. Examples [0093] The present invention will now be described in more detail in the following examples. Examples that are not covered by the claims should be regarded as comparative examples. An example of the invention is given by way of illustration only and does not limit the scope of the invention.

EXAMPLE 2. SCREENING AND SOLAR PRODUCTION TEST

1. Preliminary salt screening [0094] Preliminary salt screening was performed on CMX157. Co-crystals were selected from the following list: citric acid, fumaric acid, gentitic acid, hippuric acid, maleic acid, L-mandonic acid, orotic acid, oxalic acid, saccharin, succinic acid, L-tartaric acid, toluenesulphonic acid, ammonia , L-arginine, calcium hydroxide, dimethylamine, diethylaminoethanol, ethylenediamine, 1Himidazole, L-lysine, 2-hydroxyethylmorpholine, N-methylglucamine, potassium methoxide, zinc tertboxylate. The salt / co-crystal sieve screen included evaporation from four solvents, after which phase equilibration was carried out in four successive solvents. The dry residues after each experiment were examined by Raman microscopy.

[0095] Comparison of the Raman spectra showed that the salt / co-crystal formation takes place in the case of the gezsinate (crystalline) sample. The samples of fumarate and hypuran were mixtures of crystalline and amorphous material. The Raman spectrum obtained for these samples indicated the formation of salt. The Raman spectra of the following amorphous samples differed from the free drug spectrum and the corresponding salt-forming compound: a calcium sample, a diethylamino sample, a diethylaminoethanol sample, an imidazole sample, a potassium sample. Raman spectra for L-amnionate and amber samples showed only minor differences compared to the samples of free drug and the corresponding salt-forming compound. Examination of the plate after storage at room temperature for twelve days showed a partially crystalline material in the case of N-methylglucamine, ethylenediamine and imidazole. Records of Raman spectra for these samples showed the possibility of salt formation.

[0096] Based on the results of the initial screening, further optimization of the crystallization was carried out for the following salts: gentisate samples, fumarate samples, hippurate samples, ethylene diamine samples, imidazole samples, N-methylglucamine samples, potassium sample and succinate sample.

2. Preparation of Salts [0097] CMX157 in free acid form can be prepared by methods known to those skilled in the art (see, e.g., Painter et al., Antimicrob. Agents Chemother. 51: 3505-9 (2007), and Painter, et al., Trends Biotechnol. 22: 423-7 (2004).) CMX157 sodium salt: (55.0 g, 96.5 mmol) CMX157 free acid form was dissolved in DCM: MeOH (9: 1, 550 mL) in room temperature. Sodium methoxide (0.5 m solution in methanol, 193.1 ml, 96.5 mmol) was added to the solution and stirred at room temperature for 30 minutes. The reaction mixture was concentrated in vacuo to dryness (water bath 50^aboutC). The almost white foam formed was dissolved in ethanol (200 ml) at 60^aboutC, diluted with acetone (200 mL), cooled to room temperature and stirred for 18 h. The suspension was allowed to stand at^about for 48 h, filtered, washed with acetone (200 ml) and dried under vacuum at 35^aboutC for 48 h; obtaining the sodium salt CMX157 54.4 g (95.2%) as a white solid. HPLC purity (AUC) 99.6%.

[0099] Potassium salt CMX157: (55.0 g, 96.5 mmol) CMX157 as the free acid was dissolved in DCM: MeOH (9: 1, 550 mL) at room temperature. Potassium methoxide (25% solution in methanol, 28.5 ml, 96.5 mmol) was added to the solution and stirred at room temperature for 30 minutes. The reaction mixture was concentrated in vacuo to dryness (water bath at 50^aboutC). The almost white foam formed was dissolved in ethanol (200 ml) at 60^aboutC, diluted with acetone (200 mL), cooled to room temperature and stirred for 18 h. The suspension was allowed to stand at^aboutC for 48 h, filtered, washed with acetone (200 ml) and dried under vacuum at 35^aboutC for 48 h; the potassium salt CMX157 was obtained 48.4 g (82.4%) as a white solid. HPLC purity (AUC) 97.4%.

[0100] CMX157 Lithium salt: (55.0 g, 96.5 mmol) CMX157 as the free acid was dissolved in DCM: MeOH (9: 1, 550 mL) at room temperature. Lithium tert-butoxide (7.73 g, 96.5 mmol) was added to the solution and stirred at room temperature for 30 minutes. The reaction mixture was concentrated in vacuo to dryness (water bath 50^aboutC). The almost white precipitate formed was dissolved in ethanol (800 ml) at 70^aboutC, cooled to room temperature and stirred for 16 h. The fine suspension was filtered, washed with acetone (200 ml) and dried under vacuum in vacuo.^aboutC for 48 h; the lithium salt CMX157 51.2 g (92.1%) was obtained as a white solid. HPLC purity (AUC) 95.7%.

Ammonium salt CMX157: (55.0 g, 96.5 mmol) CMX157 in free acid form was dissolved in 2-propanol (220 ml) at 78 ° C.^aboutC in the presence of ammonium hydroxide (2830% solution 13.54 ml, 96.5 mmol). The reaction mixture was cooled to room temperature and left to stand for 18 h. The suspension was left at 5^aboutC for 48 h, filtered and air dried for approximately 48 h; the ammonium salt CMX157 was obtained, 51.7 g (91.3%) as a white solid. HPLC purity (AUC) 98.7%.

EXAMPLE 2. SOLAR PROPERTIES

1. Stability of CMX157 and CMX157 salt [0102] Stability studies on CMX157 and its salts were performed to assess the quality of the compound over time and under different environmental conditions. Accelerated aging studies were conducted using the Isotemp Vacuum Oven vacuum furnace at or 40^aboutC or 60^aboutC. If high humidity was needed, a saturated sodium chloride solution was used to provide about 75% air humidity.

[0103] In the course of stability studies, a reference standard of known purity was used in the samples to determine CMX157 content. It was found that the reference standard is stable at room temperature and under accelerated aging conditions. For analysis, 40 mg of test samples and standards were dissolved in 100.0 mL of diluent (80 H2O: 20 methanol: 2 NH4OH). The separation of drug substance and impurities was obtained using a mobile gradient phase (Table 1) on a Shimadzu A20 HPLC apparatus with a Phenomenex Synergi Polar RP column 4μ 150x3 mm. The percentage content of CMX157 was determined by comparing surface areas for the test samples at 259 nm (Amax) with the fields obtained for the reference standard.

Table 1. Gradient of the mobile phase for HPLC

<td>Time (Min)</td><td>% 50 mmol Ammonium acetate with 50 μΜ EDTA</td><td>% methanol</td><td>Speed flow (Ml / min)</td>

<td>0.00</td><td>35</td><td>65</td><td>0.8</td>

<td>12.00</td><td>20</td><td>80</td><td>0.8</td>

<td>18.00</td><td>5</td><td>95</td><td>0.8</td>

<td>18.01</td><td>35</td><td>65</td><td>0.8</td>

<td>22,00</td><td>end</td><td>end</td><td>0.8</td>

[0104] The results of the studies on the stability of CMX157 and various salts of CMX157 under various conditions are shown in Table 2. As shown in Table 2, about 30% of the free acid undergoes degradation upon storage at 60^aboutC for 4 weeks (28 days). On the contrary, the CMX157 sodium, potassium, lithium and ammonium salts showed stability at room temperature, or at room temperature.^aboutC after 2 or 4 weeks.

Table 2. Stability of various CMX157 salts

<td>Type CMX157</td><td></td><td></td><td></td><td></td>

<td>Free acid</td><td></td><td>output:</td><td colspan="2">94.5%</td>

<td rowspan="3"></td><td></td><td></td><td>40/75</td><td>60^aboutC</td>

<td></td><td>9 days</td><td>91.5%</td><td>81.4%</td>

<td></td><td>4th week</td><td>93.9%</td><td>70.2%</td>

<td rowspan="4">Na +</td><td></td><td>output:</td><td colspan="2">99.6%</td>

<td></td><td></td><td>temp.pokoj.</td><td>40/75</td>

<td></td><td>2 weeks</td><td>95.5%</td><td>96.9%</td>

<td></td><td>4th week</td><td>99.5%</td><td>99.9%</td>

<td rowspan="4">K +</td><td></td><td>output:</td><td colspan="2">97.4%</td>

<td></td><td></td><td>room temp.</td><td>40/75</td>

<td></td><td>2 weeks</td><td>98.3%</td><td>98 2%</td>

<td></td><td>4th week</td><td>99.6%</td><td>99.4%</td>

<td>Type CMX157</td><td></td><td></td><td></td><td></td>

<td rowspan="4">Li +</td><td></td><td>output:</td><td colspan="2">95.7%</td>

<td></td><td></td><td>temp.pokoj.</td><td>40/75</td>

<td></td><td>2 weeks</td><td>97.1%</td><td>97.3%</td>

<td></td><td>4th week</td><td>97.5%</td><td>99.0%</td>

<td rowspan="4">NH4 +</td><td></td><td>output:</td><td colspan="2">98.7%</td>

<td></td><td></td><td>temp.pokoj.</td><td>40/75</td>

<td></td><td>2 weeks</td><td>98.9%</td><td>98.5%</td>

<td></td><td>4th week</td><td>100.3%</td><td>101.6%</td>

<td colspan="4">Reference: LJK017p. 136.139 148, 152, 157.160</td><td></td>

2. Salt Hygroscopicity [0105] It is apparent from the observation of the inventors of the present invention that the CMX157 potassium salt is less hygroscopic than other salts.

3. Salt properties during processing [0106] It is apparent from the inventors of the present invention that the CMX157 potassium salt is less viscous during filtration than other salts.

EXAMPLE 3. EVALUATION OF CMX157 WITH REGARD TO ANTI-XMRV ACTIVITY [0107] The next example shows the results of in vitro studies of CMX157 activity against xenotropic mouse retrovirus (XMRV) compared to tenofovir and other antiviral agents. The test materials were evaluated on PG-4 cells against XMRV from a non-cell supernatant of 22Rv1 prostate cancer cells using a full dose response curve. A detailed description of the experiments and results obtained is presented below.

1. Compounds [0108] Test materials (salt K⁺ CMX157 and tenofovir) were dissolved in water at 40 mM and 10 mM, respectively, and stored at -20^aboutC. The materials tested were evaluated using 1 micromolar at a high test concentration and a half-log dilution series was run for the in vitro antiviral assay (SOW104-1101). A second sample (SOW104-11-02) was performed using 1 micromolar at the upper assay concentration for CMX157, and for tenofovir the upper test concentration was increased to 100 micromolar. Ribavirin used as a positive control was provided by Sigma. AZT and indinavir were obtained from NIH AIDS Repository and used as additional controls.

2. Antiv-XMRV cytoprotection assay (1) Cell preparation [0109] PG cells (ATCC # CRL-2032; cat's astrocytes) were passaged in T-75 bottles in McCoy's 5A medium supplemented before being used in an antiviral assay with 10% heat inactivated serum bovine fetuses, 2 mmol / L L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin. On the day before the test, the cells were split 1: 2 to ensure that they were in the exponential growth phase at the time of infection. Quantitative evaluation of cells and their viability was carried out using a hemocytometer with exclusion of trypan blue. The cell viability of the sample used was greater than 95%. Cells were resuspended at 5x10⁴ cells per ml (SOW104-11-01), or 1x10⁵cells per ml (SOW104-11-02) in tissue culture medium and plated 100 μl on flat bottom microtiter plates for incubation at 37^aboutC / 5% CO2.

(2) Diluting the compound [0110] A series of half-log dilutions were performed. The medium was removed from the single cell layer and 100 μl of medium was transferred to a 96-well microtiter plate (DMEM supplemented with 2% FBS, 2 mmol / L L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin for SOW104-11-01, or a culture medium for PG-4 cells for SOW104-11-02) containing double concentrations of the compound. Ribavirin and AZT were evaluated in parallel as control compounds for SOW104-11-01. Indinavir and AZT were evaluated in parallel as control compounds for SOW104-11-02.

(3) Virus preparation [0111] XMRV virus was harvested from the cell-free supernatant of human prostate cancer cells 22Rv1 (ATCC # CRL-2505). A titrated previously measured portion of the virus was removed from the freezer (-80^aboutC) and allowed to thaw in a bio-protected room. The virus was diluted in 2% FBS test medium so that the amount of virus added to each well in a 100 μl volume was an amount that gave 85 to 95% killed cells six days after infection (optical density score) for SOW104-11-01. For virus dilution for SOW104-11-02 in a volume of 100 μl giving 85 to 95% killed cells six days after infection, a culture medium for PG-4 cells was used.

(4) XTT staining of microtiter plates [0112] Inhibition of virus-induced cytopathic effects (CPE) was quantified by measuring the reduction of the XTT tetrazolium dye (2,3-bis (2-methoxy-4nitro-5-sulfophenyl) -5 - [( phenylamino) carbonyl] -2H-tetrazolium). In cells with active metabolism, XTT is metabolized by the mitochondrial NADPH oxidase enzyme to a soluble formazan. The XTT solution was prepared daily as a stock solution in PBS 1 mg / ml. Phenazine metasulfate (PMS) solution was prepared in PBS 0.15 mg / ml and stored in the dark at -20^aboutC. XTT / PMS was prepared just before use by adding 40 μl PMS per milliliter of XTT solution. 50 μl of XTT / PMS was added to each well and the plate was incubated for 4 h at 37^aboutC. Incubation 4 h was determined empirically as appropriate for a linear response to reduction of the XTT dye for the indicated number of cells in each sample. The plate was sealed using adhesives instead of lids and such a closed plate was inverted several times to mix the soluble formazan. The plate was then read at 450 nm (650 nm reference wavelength) using a spectrophotometer with a Molecular Devices SpectraMax Plus 96- and 384-well plate reader.

(5) Data analysis [0113] Raw data was obtained from the Softmax Pro 4.6 software and entered into a Microsoft Excel XLfit4 spreadsheet for analysis (analysis of curve fit of four parameters) and presentation of data on the graph. Using Microsoft Excel, EC50 and EC90 were obtained (50% and 90% inhibition of viral replication), TC50 and TC90 (50% and 90% reduction of cell viability) and therapeutic index (TI, TC50 / EC50 and TC90 / EC90). EC50

EC90, TC50 and TC90 are expressed as means + standard deviation. Three digits are significant.

Raw data were provided for both antiviral activity and toxicity in graphical form, as a printout presenting total activity for individual compounds.

3. Results (1) Evaluation of anti-XMRV activity [0114] Salt K⁺ CMX157 and tenofovir were evaluated for anti-XMRV activity obtained from 22Rv1 prostate cancer cells in PG-4 cells using a dose response curve at six concentrations. The results of the XMRV tests are shown in Table 3.

[0115] In parallel with the test materials, AZT was evaluated as a control compound and EC50 values ​​of 0.8 and 0.07 micromolar were obtained. The increased potency observed in SOW104-11-02 indicates that higher cell density and the use of PG-4 culture medium provide better testing conditions than those used in SOW104-11-01. In parallel with the tested materials, ribavirin and indinavir were also evaluated as controls and EC50 values ​​of 10.1 μg / ml and 0.8 micromolar were obtained respectively and the calculated therapeutic indices were 6.5 and 2.7 respectively.

[0116] Tenofovir was inactive up to 1 micromolar when evaluated in SOW 104-11-01 when for K salt⁺ CMX157 gave an EC50 value of 0.04 micromolar. The second trial was performed using the increased high test concentration for tenofovir and resulted in EC50 values ​​of 2.4 micromolar and 0.003 micromolar for CMX157.

Table 3: Activity of Compounds in the Anti-XMRV Cytoprotection Assay

<td>Relationship</td><td>PG-4 / XMRV EC50 (micromolar)</td><td>PG4 TC50 (micromolar)</td><td>Therapeutic Index</td>

<td>Ribavirin ^ g / ml</td><td>10.1</td><td>65.2</td><td>6.5</td>

<td>IDV (indinavir)</td><td>0.8</td><td>2.2</td><td>2.7</td>

<td rowspan="3">AZT (Retrovir)</td><td>0.8</td><td>&gt; 1.0</td><td>&gt; 1.2</td>

<td>0.07</td><td>&gt; 10.0</td><td>&gt; 143.0</td>

<td>0.6</td><td>&gt; 1.0</td><td>&gt; 15.6</td>

<td rowspan="3">TFV (Tenofovir)</td><td>&gt; 1.0</td><td>&gt; 1.0</td><td>-</td>

<td>2.4</td><td>&gt; 100.0</td><td>&gt; 41.5</td>

<td>2.6</td><td>&gt; 100.0</td><td>&gt; 38.6</td>

<td rowspan="2">CMX157</td><td>0.04</td><td>0.4</td><td>10.0</td>

<td>0.003</td><td>&gt; 1.0</td><td>&gt; 333.0</td>

<td></td><td>0.06</td><td>5.9</td><td>97.8</td>

[0117] As shown in Table 3, K salt⁺ CMS157 has about 60 to 800 times more antiviral activity than tenofovir in PGM-4 cells against XMRV obtained from 22Rv1 prostate cancer cells.

[0118] CMX157 has been shown to be a potent inhibitor of XMRV in vitro with EC50 approximately 20-fold lower than

AZT and 800 times lower than TFV. A higher EC 50 at higher passages of PG-4 cells was obtained for all compounds.

EXAMPLE 4. ANTI-XMRV ACTIVITY IN CELLS [0119] Table 4 provides data from an anti-XMRV activity assay. More specifically, anti-XMRV activity was performed in LNCaP cells (6-day trial) by the methods described in PLoS ONE, volume 5 (4), April 21010, e9948, pp. 1-7.

Table 4. Anti-XMRV activity

<td colspan="3">Anti-XMRV activity on LNCaP cells (6-day trial)</td><td>cytotoxicity (IC50, μΜ)</td>

<td>Relationship</td><td>EC50 (ΜΜ)</td><td>EC90 (ΜΜ)</td><td>LNCaP cells</td>

<td>AZT</td><td>0.0081 + 0.011</td><td>0.055 + 0.051</td><td>&gt; 100</td>

<td>CMX157</td><td>0.0031 + 0.0021</td><td>0.037 + 0.016</td><td>&gt; 100</td>

<td>Diisoproxil fumarate fumarate</td><td>0.029 + 0.026</td><td>0.21 + 0.15</td><td>87.4</td>

<td>tenofovir</td><td>4.5 + 3.8</td><td>17.4 + 9.0</td><td>&gt; 100</td>

<td>raltegravir</td><td>0.00054 + 0.00057</td><td>0.0032 + 0.0029</td><td>&gt; 100</td>

The data provided indicate that the EC50 and EC90 values ​​for CMX157 are comparable or better than for other active compounds such as AZT, tenofovir diisoproxil fumarate, tenofovir and raltegravir. Cytotoxicity of CMX157 in LNCaP cells was also investigated. [0120] The foregoing is illustrative of the present invention and can not be construed as a limitation thereof. The invention is further elucidated by the following claims.

Chimerix, Inc., United States of America Plenipotentiary:

EP 2 534 150 B1