Source: http://www.google.es/patents/WO2011028779A1?cl=en
Timestamp: 2018-01-20 13:29:15
Document Index: 700089407

Matched Legal Cases: ['application no. 61', 'application no. 61', 'application no. 12', 'application no. 12', 'application no. 12', 'application no. 61', 'application no. 61', 'application no. 61', 'application no. 61', 'application no. 61', 'application no. 61', 'application No. 12']

Patente WO2011028779A1 - Iminosugars and methods of treating filoviral diseases - Google Patentes
Provided are methods of treating a disease or condition caused by or associated with a virus belonging to the Filoviridae family using iminosugars, such as DNJ derivatives....http://www.google.es/patents/WO2011028779A1?cl=en&utm_source=gb-gplus-sharePatente WO2011028779A1 - Iminosugars and methods of treating filoviral diseases
Número de publicación WO2011028779 A1
Número de solicitud PCT/US2010/047493
También publicado como CA2772813A1, CN102595895A, CN105748476A, EP2473046A1, EP2473046A4, EP2473046B1, US20110065754
Número de publicación PCT/2010/47493, PCT/US/10/047493, PCT/US/10/47493, PCT/US/2010/047493, PCT/US/2010/47493, PCT/US10/047493, PCT/US10/47493, PCT/US10047493, PCT/US1047493, PCT/US2010/047493, PCT/US2010/47493, PCT/US2010047493, PCT/US201047493, WO 2011/028779 A1, WO 2011028779 A1, WO 2011028779A1, WO-A1-2011028779, WO2011/028779A1, WO2011028779 A1, WO2011028779A1
Solicitante United Therapeutics Corporation, University Of Oxford
Citas de patentes (30), Otras citas (2), Citada por (2), Clasificaciones (4), Eventos legales (7)
WO 2011028779 A1
Provided are methods of treating a disease or condition caused by or associated with a virus belonging to the Filoviridae family using iminosugars, such as DNJ derivatives.
1. A method of treating or preventing a disease or condition caused by or associated with a virus belonging to the Filoviridae family, the method comprising administering to a subject in need thereof an effective amount of a compound of the formula,
, or a pharmaceutically acceptable salt thereof, wherein R is either selected from substituted or unsubstituted alkyl groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted aryl groups, or substituted or unsubstituted oxaalkyl groups; or wherein R is
Ri is a substituted or unsubstituted alkyl group;
Xi-5 are independently selected from H, N02, N3, or NH2;
Y is absent or is a substituted or unsubstituted Ci-alkyl group, other than carbonyl; and Z is selected from a bond or NH; provided that when Z is a bond, Y is absent, and provided that when Z is NH, Y is a substituted or unsubstituted Ci-alkyl group, other than carbonyl; and
2. The method of claim 1, wherein each of Wi, W2, W3 and W4 is hydrogen.
4. The method of claim 1, wherein R is C2-C12 alkyl group.
5. The method of claim 1, wherein said administering comprises administering B-butyl deoxynojirimycin or a pharmaceutically acceptable salt thereof.
6. The method of claim 1, wherein said administering comprises administering B-nonyl deoxynojirimycin or a pharmaceutically acceptable salt thereof.
7. The method of claim 1, wherein R is an oxaalkyl group.
8. The method of claim 1, wherein R is C2-C16 oxaalkyl group that contains from 1 to 3 oxygen atoms.
9. The method of claim 1, wherein R is C6-C12 oxaalkyl group that contains from 1 to 2 oxygen atoms.
10. The method of claim 1, wherein said administering comprises administering N-(7-oxadecyl)deoxynojirimycin or a pharmaceutically acceptable salt thereof.
11. The method of claim 1 , wherein said administering comprises administering is N-(9-Methoxynonyl)deoxynojirimycin or a pharmaceutically acceptable salt thereof.
12. The method of claim 1 , wherein R is
13. The method of claim 12, wherein Xi is N02 and X3 is N3.
14. The method of claim 12, wherein each of X2, X4 and X5 is hydrogen.
15. The method of claim 12, wherein said administering comprises administering is N-(N- {4'-azido-2'-nitrophenyl}-6-aminohexyl)deoxynojirimycin or a pharmaceutically acceptable salt thereof.
16. The method of claim 12, wherein the virus is a Marburg virus.
17. The method of claim 12, wherein the virus belongs to the Ebola virus family.
18. The method of claim 17, wherein the virus is a Zaire virus.
19. The method of claim 1 , wherein the subject is a mammal.
20. The method of claim 1 , wherein the subject is a human being.
21. The method of claim 1 , wherein the virus belongs to the Ebola virus family.
22. The method of claim 21 , wherein the virus is a Zaire virus.
23. A method of inhibiting infectivitity of a cell infected with a virus belonging to the Filoviridae family, the method comprising
contacting a cell infected with a virus belonging to the Filoviridae family with an effective amount of a compound of the formula,
a pharmaceutically acceptable salt thereof, wherein R is either selected from substituted or unsubstituted alkyl groups, substituted or unsubstituted cycloalkyl groups, substituted or unsubstituted aryl groups, or substituted or unsubstituted oxaalkyl groups; or wherein R is
X1-5 are independently selected from H, N02, N3, or NH2;
wherein W1-4 are independently selected from hydrogen, substituted or unsubstituted alkyl groups, substituted or unsubstituted haloalkyl groups, substituted or unsubstituted alkanoyl groups, substituted or unsubstituted aroyl groups, or substituted or unsubstituted haloalkanoyl groups, wherein said contacting reduces the infectivity of the cell.
IMINOSUGARS AND METHODS OF TREATING FILOVIRAL
The present application claims priority to U.S. provisional application no. 61/272,253 filed September 4, 2009 and U.S. provisional application no. 61/282,507 filed February 22, 2010, both of which are incorporated herein by reference in their entirety.
The present application relates to iminosugars and methods of treating viral infections with iminosugars and, in particular, to the use of iminosugars for treatment and prevention of viral infections caused by or associated with a virus belonging to the Filoviridae family.
One embodiment is a method of treating or preventing a disease or condition caused by or associated with a virus belonging to the Filoviridae family, which method comprises administering to a subject in need thereof an effective amount of a compound of the formula,
Another embodiment is a method of inhibiting infectivity of a cell infected with a virus belonging to the Filoviridae family, which method comprises contacting a cell infected with a virus belonging to the Filoviridae family with an effective amount of a compound of the formula,
Figures 1(A)-(E) present chemical formulas of the following iminosugars: A) N-Butyl deoxynojirimycin (NB-DNJ or UV-1); B) N-Nonyl deoxynojirimycin (NN-DNJ or UV-2); C) N-(7-Oxadecyl)deoxynojirimycin (N7-0-DNJ or UV-3); D) N-(9-Methoxynonyl)
deoxynojirimycin (N9-DNJ or UV-4); E) N-(N-{4'-azido-2'-nitrophenyl}-6- aminohexyl)deoxynojirimycin (NAP-DNJ or UV-5).
Figure 2 is a synthesis scheme for NN-DNJ.
Figures 3A-D illustrate synthesis of N7-0-DNJ. In particular, Figure 3A shows a sequence of reactions leading to N7-0-DNJ; Figure 3B illustrates preparation of 6-propyloxy-l- hexanol; Figure 3C illustrates preparation of 6-propyloxy-l-hexanal; Figure 3D illustrates synthesis of N7-0-DNJ.
Figures 4A-C relate to synthesis of N-(9-Methoxynonyl) deoxynojirimycin. In particular, Figure 4A illustrates preparation of 9-methoxy-l-nonanol; Figure 4B illustrates preparation of 9-methoxy-l-nonanal; Figure 4C illustrates synthesis of N-(9-Methoxynonyl)
deoxynoj irimycin. Figure 5 provides data for inhibition of infectivity of Ebola Zaire and Marburg viruses for N9-DNJ (UV-4) and NAP-DNJ (UV-5).
Figure 6 presents effects of 10 day administration of UV-5 on survival of mice infected with Ebola virus.
Figure 7 presents in vivo safety data for UV-4 and UV-5.
Figure 8 presents survival data for mice challenged with Ebola Zaire virus (left) and Marburg virus (right) after administering UV-5.
DETAILED DESCRIPTION Related Documents
The following patent documents, which are all incorporated herein by reference in their entirety, may be useful for understanding the present disclosure:
1) US patent no. 6,545,021;
2) US patent no. 6,809,803;
3) US patent no. 6,689,759;
4) US patent no. 6,465,487;
5) US patent no. 5,622,972;
6) US patent application no. 12/656,992 filed February 22, 2010;
7) US patent application no. 12/656,993 filed February 22, 2010;
8) US patent application no. 12/813,882 filed June 11, 2010;
9) US patent provisional application no. 61/282,507 filed February 22, 2010;
10) US patent provisional application no. 61/272,252 filed September 4, 2009;
11) US provisional application no. 61/272,253 filed September 4, 2009;
12) US provisional application no. 61/272,254 filed September 4, 2009;
13) US provisional application no. 61/282,508 filed February 22, 2010;
14) US provisional application no. 61/353,935 filed June 11, 2010.
Unless otherwise specified, "a" or "an" means "one or more." As used herein, the term "viral infection" describes a diseased state, in which a virus invades a healthy cell, uses the cell's reproductive machinery to multiply or replicate and ultimately lyse the cell resulting in cell death, release of viral particles and the infection of other cells by the newly produced progeny viruses. Latent infection by certain viruses is also a possible result of viral infection.
The present inventors discovered that certain iminosugars, such as deoxynojirimycin derivatives, may be effective against viruses belonging to the Filoviridae family, which are also known as filoviruses.
In particular, the deoxynojirimycin derivatives may be useful for treating or preventing a disease or condition caused by or associated with a virus belonging to the Filoviridae family. The Filoviridae family includes the Ebolavirus genus and the Marburgvirus genus. The Ebolavirus genus includes Zaire virus, Bundibugyo Ebola virus, Ivory Coast Ebola virus, Reston Ebola virus and Sudan Ebola virus, while the Marburgvirus genus includes Lake Victoria Marburg virus. Diseases that are caused or associated with filoviruses include Ebola hemorrhagic fever and Marburg hemorrhagic fever.
In many embodiments, the iminosugar may be N-substituted deoxynojirimycin. In some embodiments, such N-substituted deoxynojirimycin may be a compound of the following formula:
In some embodiments, R may be selected from, but is not limited to -(CH2)60CH3,
-(CH2)6OCH2CH3, -(CH2)60(CH2)2CH3, -(CH2)60(CH2)3CH3, -(CH2)20(CH2)5CH3,
-(CH2)20(CH2)6CH3;-(CH2)20(CH2)7CH3; -(CH2)9-OH; -(CH2)9OCH3.
In some embodiments, R may be branched or unbranched, substituted or unsubstituted alkyl group, which may contain up 20 carbon atoms. In some embodiments, the alkyl group may be C2-C12 or C3-C7 alkyl group.
In certain embodiments, the alkyl group may be a long chain alkyl group, which may be C6- C20 alkyl group; C8-C16 alkyl group; or C8-C10 alkyl group. In some embodiments, R may be a long chain oxaalkyl group, i.e., a long chain alkyl group, which may contain from 1 to 5 or from 1 to 3 or from 1 to 2 oxygen atoms. In some embodiments, R may have the following formula
where Ri is a substituted or unsubstituted alkyl group;
Y is absent or is a substituted or unsubstituted Ci-alkyl group, other than carbonyl; and Z is selected from a bond or NH; provided that when Z is a bond, Y is absent, and provided that when Z is NH, Y is a substituted or unsubstituted Ci-alkyl group, other than carbonyl. In some embodiments, Z is NH and Ri-Y is a substituted or unsubstituted alkyl group, such as C2-C20 alkyl group or C4-C12 alkyl group or C4-C10 alkyl group.
In some embodiments, Xi is N02 and X3 is N3. In some embodiments, each of X2, X4 and X5 is hydrogen.
In some embodiments, the iminosugar may be one of the compounds presented in Figure 1. Methods of synthesizing deoxynojirimycin derivatives are disclosed, for example, in U.S. Patent nos. 5,622,972, 5,200,523, 5,043,273, 4,994,572, 4,246,345, 4,266,025, 4,405,714, and 4,806,650 and U.S. Patent application publication no. 2007/0275998, which are all incorporated herein by reference.
In some embodiments, the iminosugar may be in a form of a salt derived from an inorganic or organic acid. Pharmaceutically acceptable salts and methods for preparing salt forms are disclosed, for example, in Berge et al. (J. Pharm. Sci. 66: 1-18, 1977). Examples of appropriate salts include but are not limited to the following salts: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,
camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate, and undecanoate.
In some embodiments, the iminosugar may also used in a form of a prodrug. Prodrug of DNJ derivatives, such as the 6-phosphorylated DNJ derivatives, are disclosed in U.S. Patents nos. 5,043,273 and 5,103,008.
In some embodiments, the iminosugar may be used as a part of a composition, which further comprises a pharmaceutically acceptable carrier and/ or a component useful for delivering the composition to an animal. Numerous pharmaceutically acceptable carriers useful for delivering the compositions to a human and components useful for delivering the
composition to other animals such as cattle are known in the art. Addition of such carriers and components to the composition of the invention is well within the level of ordinary skill in the art.
In some embodiments, the pharmaceutical composition may consist essentially of N- substituted deoxynojirimycin, which may mean that the N-substituted deoxynojirimycin is the only active ingredient in the composition.
Yet in some embodiments, N-substituted deoxynojirimycin may be administered with one or more additional antiviral compounds.
In some embodiments, the treatment or prevention of the disease or condition caused by or associated with a virus belonging to the Filoviridae family may be performed without administering N-(phosphonoacetyl)-L-aspartic acid to the subject, to whom the iminosugar is being administered. N-(phosphonoacetyl)-L-aspartic acid is disclosed, for example, in U.S. patent no. 5,491,135.
In some embodiments, the iminosugar may be used in a liposome composition, such as those disclosed in US publications nos. 2008/0138351 and 2009/0252785 as well as in US application No. 12/732630 filed March 26, 2010.
The iminosugar, such as a DNJ derivative, may be administered to a cell or an animal affected by a virus. The iminosugar may inhibit morphogenesis of the virus, or it may treat the individual. The treatment may reduce, abate, or diminish the virus infection in the animal.
Animals that may be infected with a filovirus include primates, such as monkeys and humans. The amount of iminosugar administered to an animal or to an animal cell to the methods of the invention may be an amount effective to inhibit the morphogenesis of a filovirus. The term "inhibit" as used herein may refer to the detectable reduction and/or elimination of a biological activity exhibited in the absence of the iminosugar. The term "effective amount" may refer to that amount of the iminosugar necessary to achieve the indicated effect. The term "treatment" as used herein may refer to reducing or alleviating symptoms in a subject, preventing symptoms from worsening or progressing, inhibition or elimination of the causative agent, or prevention of the infection or disorder related to the filovirus in a subject who is free therefrom.
Thus, for example, treatment of the disease caused by or associated with a virus may include destruction of the infecting agent, inhibition of or interference with its growth or maturation, and neutralization of its pathological effects. The amount of the iminosugar which may be administered to the cell or animal is preferably an amount that does not induce toxic effects which outweigh the advantages which accompany its administration.
The selected dose level may depend on the activity of the iminosugar, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound(s) at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to four doses per day. It will be understood, however, that the specific dose level for any particular patient can depend on a variety of factors, including the body weight, general health, diet, time and route of administration and combination with other therapeutic agents and the severity of the condition or disease being treated. The adult human daily dosage may range from between about one microgram to about one gram, or from between about 10 mg and 100 mg, of the iminosugar per 10 kilogram body weight. In some embodiments, a total daily dose may be from 0.1 mg/kg body weight to 100 mg/kg body weight or from 1 mg/kg body weight to 60 mg/kg body weight or from 2 mg/kg body weight to 50 mg/kg body weight or from 3 mg/kg body weight to 30 mg/kg body weight. The daily dose may be administered over one or more administering events over day. For example, in some embodiments, the daily dose may be distributed over two (BID) administering events per day, three administering events per day (TID) or four administering events (QID). In certain embodiments, a single administering event dose ranging from 1 mg/kg body weight to 10 mg/kg body weight may be administered BID or TID to a human making a total daily dose from 2 mg/kg body weight to 20 mg/kg body weight or from 3 mg/kg body weight to 30 mg/kg body weight. Of course, the amount of the iminosugar which should be administered to a cell or animal may depend upon numerous factors well understood by one of skill in the art, such as the molecular weight of the iminosugar and the route of administration.
Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in oral solid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations. For example, it may be in the physical form of a powder, tablet, capsule, lozenge, gel, solution, suspension, syrup, or the like. In addition to the iminosugar, such pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer the iminosugar. Such pharmaceutical compositions may be administered by a number of routes. The term
"parenteral" used herein includes subcutaneous, intravenous, intraarterial, intrathecal, and injection and infusion techniques, without limitation. By way of example, the pharmaceutical compositions may be administered orally, topically, parenterally, systemically, or by a pulmonary route.
These compositions may be administered a in a single dose or in multiple doses which are administered at different times. Because the inhibitory effect of the composition upon a filovirus may persist, the dosing regimen may be adjusted such that virus propagation is retarded while the host cell is minimally effected. By way of example, an animal may be administered a dose of the composition of the invention once per week, whereby virus propagation is retarded for the entire week, while host cell functions are inhibited only for a short period once per week. Embodiments described herein are further illustrated by, though in no way limited to, the following working examples.
1. Synthesis of N-Nonyl DNJ Table 1. Materials for NN-DNJ synthesis
Procedure: A 50-mL, one-necked, round-bottom flask equipped with a magnetic stirrer was charged with DNJ (500 mg), ethanol (100 mL), nonanal (530 mg), and acetic acid (0.5 mL ) at room temperature. The reaction mixture was heated to 40-45 °C and stirred for 30-40 minutes under nitrogen. The reaction mixture was cooled to ambient temperature and Pd/C was added. The reaction flask was evacuated and replaced by hydrogen gas in a balloon. This process was repeated three times. Finally, the reaction mixture was stirred at ambient temperature overnight. The progress of reaction was monitored by TLC (Note 1). The reaction mixture was filtered through a pad of Celite and washed with ethanol. The filtrate was concentrated in vacuo to get the crude product. The crude product was purified by column chromatography (230-400 mesh silica gel). A solvent gradient of methanol in dichloromethane (10-25%) was used to elute the product from the column. All fractions containing the desired product were combined, and concentrated in vacuo to give the pure product (420mg). Completion of the reaction was monitored by thin layer chromatography (TLC) using a thin layer silica gel plate; eluent; methanol : dichloromethane = 1 :2 2. Synthesis of N-7-Oxadecyl DNJ
2a. Synthesis of 6-propyloxy-l-hexanol Table 2. Materials for synthesis of 6-propyloxy-l-hexanol
Procedure: a 500-mL, one-necked, round-bottom flask equipped with a magnetic stirrer was charged with 1,6-hexanediol (6.00 g), potassium tert-butoxide (5.413 g) at room temperature. The reaction mixture was stirred for one hour, and then 1-iodopropane (8.63 g) was added. The reaction mixture was heated to 70-80 °C and stirred overnight. The progress of reaction was monitored by TLC (Note 1). After completion of the reaction, water was added to the reaction mixture, and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were concentrated in vacuo to get the crude product. The crude product was dissolved in dichloromethane and washed with water, and then brine, dried over sodium sulfate. The organic layer was concentrated in vacuo to get the crude product. The crude product was purified by column chromatography using 230-400 mesh silica gel. A solvent gradient of ethyl acetate in hexanes (10-45%) was used to elute the product from the column. All fractions containing the desired pure product were combined and concentrated in vacuo to give pure 6-propyloxy-l-hexanol (lot D-1029-048, 1.9 g, 25%>) Completion of the reaction was monitored by thin layer chromatography (TLC); (eluent: 60% ethyl acetate in hexanes).
2b. Preparation of 6-propyloxy-l-hexanal
Table 3. Materials for preparation of 6-propyloxy-l-hexanal
CH2C12 10 mL
Procedure: a 50-mL, one-necked, round-bottom flask equipped with a magnetic stirrer was charged with 6-propyloxy-l-hexanol (1.0 g), PDC (4.7 g), dichloromethane (10 mL), Celite (1.0 g), and sodium acetate (100 mg). The reaction mixture was stirred at room temperature under nitrogen for 5 minutes. PDC (4.70 g) was added to the reaction mixture, and stirred overnight. The progress of reaction was monitored by TLC (Note 1). After completion of the reaction, the reaction mixture was directly loaded on the column (230-400 mesh silica gel). A solvent gradient of dichloromethane in ethyl acetate (10-20%) was used to elute the product from the column. All fractions containing the desired pure product were combined and concentrated in vacuo to give pure 6-propyloxy-l-hexanal (lot D- 1029-050, 710 mg, 71%). Completion of the reaction was monitored by thin layer chromatography (TLC); (eluent: 60% ethyl acetate in hexanes).
Procedure: a 50-mL, one-necked, round-bottom flask equipped with a magnetic stirrer was charged with DNJ (500 mg), ethanol (15 mL), 6-propyloxy-l-hexanal (585 mg), and acetic acid (0. lmL) t room temperature. The reaction mixture was heated to 40-45 °C and stirred for 30-40 minutes under nitrogen. The reaction mixture was cooled to ambient temperature and Pd/C was added. The reaction flask was evacuated and replaced by hydrogen gas in a balloon. This process was repeated three times. Finally, the reaction mixture was stirred at ambient temperature overnight. The progress of reaction was monitored by TLC (Note 1). The reaction mixture was filtered through a pad of Celite and washed with ethanol. The filtrate was concentrated in vacuo to get the crude product. The crude product was purified by column chromatography (230-400 mesh silica gel). A solvent gradient of methanol in dichloromethane (10-40%) was used to elute the product from the column. All fractions containing the desired product were combined, and concentrated in vacuo to give the pure product. (Lot: D- 1029-052 (840 mg). Completion of the reaction was monitored by thin layer chromatography (TLC); (eluent: 50% methanol in dichloromethane).
3 a Preparation of 9-methoxy- 1 -nonanol Table 5. Materials for preparation of 9-methoxy- 1 -nonanol
Procedure: a 500-mL, one-necked, round-bottom flask equipped with a magnetic stirrer and stir bar was charged with 1 ,9-nonanediol (10.00 g, 62.3 mmol) in dimethyl sulfoxide (100 mL) and H20 (100 mL). To this was added slowly a solution of sodium hydroxide (5.0 g, 125.0 mmol) in H20 (10 mL) at room temperature. During addition of sodium hydroxide the reaction mixture generated heat and the temperature rose to ~40 °C. The mixture was stirred for one hour, and then dimethyl sulfate (16.52 g, 131 mmol) was added in four portions while maintaining the temperature of the reaction mixture at ~ 40°C. The reaction mixture was stirred at room temperature overnight. Progress of the reaction was monitored by TLC (Note 1). TLC monitoring indicated that the reaction was 25 % conversion. At this stage additional dimethyl sulfate (24.78g, 196.44 mmol) was added and the resulting mixture was stirred at room temperature for an additional 24 h. After completion of the reaction, sodium hydroxide (10% solution in water) was added to the reaction mixture to adjust the pH of the solution to 11-13. The mixture was stirred at room temperature for 2 h and extracted with
dichloromethane (3 x 100 mL). The combined organic layers were washed with H20 (200 mL), brine (150 mL), dried over anhydrous sodium sulfate (20 g), filtered and concentrated in vacuo to obtain a crude product (14 g). The crude product was purified by column
chromatography using 250-400 mesh silica gel. A solvent gradient of ethyl acetate in hexanes (10-50%) was used to elute the product from the column. All fractions containing the desired pure product were combined and concentrated in vacuo to give pure 9-methoxy- 1- nonanol (lot D-1027-155, 2.38 g, 21.9 %). Completion of the reaction was monitored by thin layer chromatography (TLC) using a thin layer silica gel plate; eluent: 60% ethyl acetate in hexanes.
3b Preparation of 9-methoxy- 1 -nonanal
Table 6. Materials for preparation of 9-methoxy-l -nonanal
Procedure: a 50-mL, one-necked, round-bottom flask equipped with a magnetic stirrer and stir bar was charged with 9-methoxy-nonanol (1.0 g, 5.9 mmol), dichloromethane (10 mL), molecular sieves (1.0 g, 3A), sodium acetate (0.1 g) at room temperature. The reaction mixture was stirred at room temperature under nitrogen for 5 minutes. The reaction mixture was charged with pyridinium dichromate (4.7 g, 12.5 mmol) and stirred overnight. The progress of reaction was monitored by TLC (Note 1). After completion of the reaction, the reaction mixture was filtered through a bed of silica gel (~15 g). The filtrate was evaporated in vacuo to obtain a crude compound. This was purified by column chromatography using silica gel column (250-400 mesh, 40 g). A solvent gradient of ethyl acetate in hexane (10- 50%) was used to elute the product from the column. All fractions containing the desired pure product were combined and concentrated in vacuo to give pure 9-methoxy-nonanal (lot D-1027-156, 553 mg, 54.4%). Completion of the reaction was monitored by thin layer chromatography (TLC) using a thin layer silica gel plate; eluent: 60% ethyl acetate in hexanes.
3 c Synthesis of N-(9-methoxy)-nonyl DNJ Table 7. Materials for synthesis of N-(9-methoxy)-nonyl DNJ
Procedure: a 50-mL, two-necked, round-bottom flask equipped with magnetic stirrer and a stir bar was charged with DNJ (300 mg, 1.84 mmol), ethanol (20 mL), 9-methoxy- 1 -nonanal (476 mg, 2.76 mmol) at room temperature. The reaction mixture was stirred for 5-10 minutes under nitrogen and Pd/C was added at room temperature. The reaction mixture was evacuated and was replaced by hydrogen gas using a balloon. This process was repeated three times and then reaction mixture was stirred under atmospheric hydrogen at room temperature. The progress of reaction was monitored by TLC (Note 1). The reaction mixture was filtered through a bed of Celite and was washed with ethanol (20 mL). The filtrate was concentrated in vacuo to get a crude product. The crude product was purified by column chromatography using 250-400 mesh silica gel (20 g). A solvent gradient of methanol in ethyl acetate (5- 25%) was used to elute the product from the column. All fractions containing the desired pure product were combined, and concentrated in vacuo to give an off white solid. The solid was triturated in ethyl acetate (20 mL), filtered and dried in high vacuum to give a white solid [lot: D-1027-158 (165.3 mg, 28.1%). Completion of the reaction was monitored by thin layer chromatography (TLC) using a thin layer silica gel plate; eluent: 50% methanol in
dichloromethane . 4. Inhibition of Ebola (Zaire) and Marburg viruses
Table 1 presents IC50 values for Ebola Zaire and Marburg viruses in μΜ. The table provides data for inhibition of infectivity of Ebola Zaire and Marburg viruses for NB-DNJ (UV-1), NN-DNJ (UV-2), N7-0-DNJ (UV-3), N9-DNJ (UV-4) and NAP-DNJ (UV-5).
Procedure. The compounds were screened for inhibition of generation of infectious virus was conducted on the UV compounds at concentrations from 6 μΜ up to 250 μΜ. The filovirus Ebola-Zaire and Marburg-Ci67 strains were evaluated for virus inhibition. Vero cells (African green monkey kidney epithelial cell line) obtained from American Type Culture Collection (ATCC, Manassas, Virginia). Cells were cultured in lx modified Eagle medium (MEM, Gibco), supplemented with 2% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin in cell culture treated 24-well flat bottom plates at 37°C in a 5% C02 incubator for 24 hr prior to assay. Cells (lxlO6 cells per well ) were pretreated with compounds in a final concentration of 1% DMSO for 1 hr followed by discarding the culture medium and addition of virus inoculums at an MOI of 0.1 in EMEM with 2% FBS. After 1 hr incubation the virus inoculums were removed and fresh media with compounds at correct dilutions were added. Three days later virus containing supernatants were collected and 10 fold dilutions of virus-containing supernatants was done in a virus plaque assay with Vero cells plated on 6 well- virus plaque assay plates. The plaque assay data were collected on day 8 for the Ebola and Marburg plates. IC 50 was determined as concentration of compound resulting in 50% virus inhibition.
Figure 5 provides data for inhibition of infectivity of Ebola Zaire and Marburg viruses for N9-DNJ (UV-4) and NAP-DNJ (UV-5).
Procedure. The virus yield assay were performed by standard plaque assay on supernatant samples generated from virus-infected cells incubated with iminosugars at concentrations from 4 μΜ up to 64 μΜ. The filovirus Ebola-Zaire and Marburg-Ci67 strains were evaluated for virus inhibition. 24-well cell culture plates were seeded with Vero cells (ATCC,
Mannassas, VA; ATCC number CCL-81) in ImL lx modified Eagle medium (MEM, Gibco), supplemented with 10% heat- inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and incubated at 37°C for 24 hours or until -80% confluency. Medium were replaced with medium supplemented with 2% fetal bovine serum and the cells were pretreated with compounds in a final concentration of 1% DMSO for lhr followed by discarding the culture medium and addition of virus inoculums at an MOI of 0.1 in triplicate and incubated for lhr at 37°C, 5% C02. After 1 hr incubation the virus inoculums were removed and fresh media with compounds at correct dilutions were added. Three days are required for the EBOV and MARV virus infection. Upon completion of infection, supernatant were collected for titering. To titer, 6-well plates with 80% confluent
Vero cells in growth medium were used. Viral supernatant were diluted from 10" to 10" and added (100 uL) to the cells and incubated at 37°C for 1 hour with shaking every 5-10 minutes. Viral infection medium (100 uL) were aspirated and replace with 1 mL pre-warmed 2% low-melt agarose mixed 1 : 1 with 2X MEM (5% fetal calf serum) and incubated at 37°C, 5% C02 for 8 days followed by plaque visualization by neutral red staining.
Ebola In Vivo Study
UV-5 was administered as a free drug dissolved in water. The compound was given at lOOmg/kg and lOmg/kg by the intraparenteral route (IP) twice daily. Balb/c mice received the compound for 10 days. Mice were infected with Ebola virus (strain Zaire) with ~5 LD50 30 minutes following the first iminosugar dose. Animals were monitored for 15 days.
Animals were weighed once per day, and given health scores 2X per day. Animals displaying severe illness (as determined by 30%> weight loss, extreme lethargy, ruffled coat, or paralysis) were euthanized.
Figure 6 presents data for the effects of 10 day administration of UV-5 on survival of mice infected with Ebola virus. Animals receiving 100 mg/kg and 10 mg/kg BID showed a 71% survival rate, versus no survival in control animals.
Conclusion: these results demonstrate that UV-5 can be used as an antiviral drug to treat Ebola. Iminosugar Safety Study
Methods and Discussion: BALB/c and C57/B1/6 mice were given oral suspensions of UV-1, UV-4, UV-5, twice a day for seven days, in lOOul per mouse at 100 and 10 mg/kg (2mg and 0.2 mg/mouse, respectively) 8 hours apart for 7 days, and then monitored for weight loss and general health. After seven days of treatment, the mice did not show any significant signs of weight loss compared to the "vehicle only" control. The results of these experiments are in Figure 7.
When the BALB/c mice were treated with UV-5 at the highest concentration, they displayed signs of diarrhea, red urine, and a ruffled appearance although they did not show signs of weight loss. The C57/B1/6 mice displayed these same symptoms but without the ruffled look, These symptoms promptly ceased when treatment was done, and by day 11 (day 4 post compound treatment) the BALB/c mice in these groups looked very healthy.
Conclusions: These compounds have shown to be relatively non-toxic in this mouse model and these concentrations of compound are deemed safe.
Filoviridae In Vivo Data
The study assessed the efficacy of the iminosugar compound UV-5 in promoting survival of mice challenged with Ebola and Marburg viruses. C57B1/6 mice were used in the Ebola experiments, while Balb/c mice were used in the Marburg virus experiments. UV-5 compound was previously tested in both in vitro (CC50 of 125-250uM) and in vivo (no weight loss or adverse effects observed in multiple mouse studies) and shown it possesses low toxicity. In this study, UV-5 compound was administered to the mice as a free drug dissolved in PBS. The compound was be given by the intraperitoneal (IP) route (2x per day IP) for a total number of 10 days after the start of the compound dosing. Study mice were infected IP with Ebola Zaire or Marburg Ravn with 1000 pfu/mouse lh before the first UV-5 dose.
Animals were monitored for 15 days. Animals were weighed once per day, and given health scores 2X per day. Animals displaying severe illness (as determined by 30% weight loss, extreme lethargy, ruffled coat, or paralysis) were euthanized. Figure 8 shows survival data (Y-axis, percent of mice in a study group survived, X-axis, the number of days post infection) for mice infected with Ebola Zaire or Marburg Ravn viruses. Each of the groups in the study, i.e. i) a control group (treated with water only) infected with the Ebola virus, ii) a control group (treated with water only) infected with the Marburg virus; iii) a treated group (treated with 100 mg/kg of UV 5, BID) infected with the Ebola virus; iv) a treated group (treated with 10 mg/kg of UV 5, BID) infected with the Marburg virus, contained 10 mice at the beginning of the study.
Survival of >60% is statistically significant. UV-5 provided significant protection against Ebola virus at dosing of 100 mg/kg IP, BID. UV-5 provided protection against Marburg virus at dosing of 10 mg/kg IP, BID.
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Clasificación internacional A61K31/445, A01N43/40
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