Composition for inhibiting herpes virus

Abstract

The present disclosure provides compounds of formula I-V, which can be used to inhibit viral diseases in subjects. In some embodiments, compounds of formula I-V can be used to inhibit herpes viruses.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Application No. 62 / 965,755, filed on January 24, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on January 22, 2021 is named 2635-000IWO0I_Sequence Listing_ST25.txt and is 1KB in size.

[0005] Statement of Government Interests

[0006] This invention was made with government support under Grant No. 1R0IDC013550 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the Invention

[0008] Infection with human cytomegalovirus (HCMV), a member of the herpes virus family, is common in humans. The seroprevalence in individuals over 80 years of age reaches 90%. 1 Although infection is usually asymptomatic, HCMV remains a serious threat to transplant recipients and HIV patients. It is also the most common congenital infection worldwide, causing hearing loss, global disabilities, and central nervous system damage in children. 2-6 .

[0009] The nucleoside analog ganciclovir (GCV) and its oral formulation val-GCV significantly improved transplant outcomes by reducing HCMV replication and sequelae of disease 7,8 GCV has also been shown to prevent hearing deterioration in children born with the infection 9 However, prolonged treatment with GCV or val-GCV can lead to severe bone marrow toxicity. 10-13 A phase III trial of oral val-GCV in infants with congenital infection concluded that 6 months of treatment may have better neurological effects than 6 weeks, but GCV-resistant mutants have emerged 14 Similarly, GCV-resistant mutants have emerged in transplant recipients 15-16When GCV resistance emerges, limited therapeutic alternatives, foscarnet and cidofovir, are highly nephrotoxic and can only be given intravenously. Their use in cases of resistant or refractory HCMV is associated with significant morbidity and mortality. 13 Some new drugs have entered FDA approval and clinical trials. The terminal enzyme inhibitor letermovir was recently approved for HCMV prevention after hematopoietic bone marrow transplantation. 17 . reported rapid selection of UL56 mutations in cell culture 16 and patients' resistance to letermovir 18 Maribavir, an inhibitor of viral UL97 kinase, is undergoing clinical trials 19 .

[0010] The identification of new herpesvirus inhibitors is an important area of ​​drug development. Although a variety of drugs are available, there is a clear need to improve existing drugs given the toxicity of available drugs and the ongoing selection of drug-resistant mutants. SUMMARY OF THE INVENTION

[0012] Disclosed herein is a method of inhibiting human herpes virus in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier:

[0013]

[0014] Wherein, R1 is a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally substituted by one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group. In some embodiments, the compound is selected from the following group:

[0015]

[0016]

[0017] Disclosed herein is a method of inhibiting human herpes virus in a subject in need thereof, the method comprising administering to the subject a compound of formula II or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier:

[0018]

[0019] wherein R1 is H, and R2 is independently H or alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group; or R1 and R2 together form a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms; and R3 is H or cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more halogen, CF3, CN and NO2 moieties. In some embodiments, R2 is a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted by one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl. In some embodiments, the compound is selected from the group consisting of:

[0020]

[0021] Disclosed herein is a method of inhibiting human herpes virus in a subject in need thereof, the method comprising administering to the subject a compound of formula III or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier:

[0022]

[0023] Disclosed herein are methods of inhibiting human herpes virus in a subject in need thereof, the method comprising administering a compound of formula IV or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, the pharmaceutical composition comprising a pharmaceutically acceptable carrier:

[0024]

[0025] Disclosed herein are methods of inhibiting human herpes virus in a subject in need thereof, the method comprising administering to the subject a compound of formula V or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier:

[0026]

[0027] Among them, R 1 is hydrogen, C1-C6 straight or branched alkyl, or C3-C6 cycloalkyl; and R 2 is unsubstituted or replaced by R 3 OR3 Substituted phenyl, wherein R 3 is hydrogen, C1-C6 straight or branched alkyl or C3-C6 cycloalkyl. 1 is a C1-C4 straight or branched chain alkyl group; and R 2 is OR 3 Substituted phenyl, wherein R 3 is a C1-C4 straight or branched alkyl.

[0028]

[0029] In some embodiments, the methods disclosed herein further comprise administering more than one biologically active agent, for example, two, three, four or more biologically active agents.

[0030] In some embodiments, the herpes virus is selected from human herpes virus type 1 (HHV-1), human herpes virus type 2 (HHV2), human herpes virus type 3 (HHV3), human herpes virus type 4 (HHV4), human herpes virus type 5 (HHV5), human herpes virus type 6 (HHV6), human herpes virus type 7 (HHV7) and human herpes virus type 8 (HHV8). In some embodiments, the herpes virus is HHV-5.

[0031] Disclosed herein is a pharmaceutical composition comprising a compound of formula I or a salt, solvate or stereoisomer thereof:

[0032]

[0033] Wherein, R1 is a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally substituted by one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group. In some embodiments, the compound is selected from the following group:

[0034]

[0035]

[0036] Disclosed herein is a pharmaceutical composition comprising a compound of formula II or a salt, solvate or stereoisomer thereof:

[0037]

[0038] wherein R1 is H, and R2 is independently H or alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group; or R1 and R2 together form a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms; and R3 is H or cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more halogen, CF3, CN and NO2 moieties. In some embodiments, R2 is a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted by one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl. In some embodiments, the compound is selected from the group consisting of:

[0039]

[0040] Disclosed herein is a pharmaceutical composition comprising a compound of formula III or a salt, solvate or stereoisomer thereof and a pharmaceutically acceptable carrier:

[0041]

[0042] Disclosed herein is a pharmaceutical composition comprising a compound of formula IV or a salt, solvate or stereoisomer thereof and a pharmaceutically acceptable carrier:

[0043]

[0044] Disclosed herein is a pharmaceutical composition comprising a compound of Formula V or a salt, solvate or stereoisomer thereof and a pharmaceutically acceptable carrier:

[0045]

[0046] Among them, R 1 is hydrogen, C1-C6 straight or branched alkyl, or C3-C6 cycloalkyl; and R 2 is unsubstituted or replaced by R 3 OR 3 Substituted phenyl, wherein R 3 is hydrogen, C1-C6 straight or branched alkyl or C3-C6 cycloalkyl. 1 is a C1-C4 straight or branched chain alkyl group; and R 2is OR 3 Substituted phenyl, wherein R 3 is a C1-C4 straight or branched alkyl.

[0047]

[0048] Disclosed herein are compounds of Formula I or salts, solvates or stereoisomers thereof:

[0049]

[0050] wherein R1 is a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected via a C1-C3 alkyl group, provided that the compound cannot be compound A:

[0051]

[0052] In some embodiments of the compound or its salt, solvate or stereoisomer, the compound is selected from:

[0053]

[0054] Disclosed herein are compounds of formula II or salts, solvates or stereoisomers thereof:

[0055]

[0056] wherein R1 is H, and R2 is independently H or alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group; or R1 and R2 together form a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms; and R3 is H or cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more halogen, CF3, CN and NO2 moieties, provided that the compound cannot be a compound selected from the group consisting of:

[0057]

[0058] In some embodiments, R2 is a C1-C6 heterocycloalkyl or C1-C6 heteroaryl group optionally substituted with one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected through a C1-C3 alkyl group.

[0059] Disclosed herein are compounds of formula V or salts, solvates or stereoisomers thereof:

[0060]

[0061] Among them, R 1 is hydrogen, C1-C6 straight or branched alkyl, or C3-C6 cycloalkyl; and R 2 is unsubstituted or replaced by R 3 OR 3 Substituted phenyl, wherein R 3 is hydrogen, C1-C6 straight or branched alkyl or C3-C6 cycloalkyl. 1 is a C1-C4 straight or branched chain alkyl group; and R 2 is OR 3 Substituted phenyl, wherein R 3 is a C1-C4 straight or branched alkyl. In some embodiments, the compound is

[0062]

[0063] Disclosed herein is a method of inhibiting or treating human herpes virus in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition comprising one or more of the above compounds and a pharmaceutically acceptable carrier.

[0064] Also disclosed herein is a method of inhibiting or treating human herpes virus in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition comprising one or more of the above compounds, at least one other biologically active agent, and a pharmaceutically acceptable carrier.

[0065] Also disclosed herein are uses of the compounds or compositions disclosed herein for treating human herpes viruses in a subject. Also disclosed herein are uses of the compounds or compositions disclosed herein for treating human herpes viruses in a subject. Also disclosed herein are uses of the compounds or compositions disclosed herein for manufacturing a medicament for treating human herpes viruses in a subject.

[0066] BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 : Flowchart of high throughput screening (HTS).

[0068] Figure 2 : Chemical structures and molecular weights (MW) of five compounds.

[0069] Figure 3 :MLS8969 inhibits HCMV replication, Figure 3 A: HFF were infected with HCMV Towne pp28 (MOI=1) and then treated with MLS8999 or GCV. Luciferase activity was measured at 72 hpi. Figure 3 B: HFF were infected with HCMV TB40 (100 PFU / well) and treated with MLS8969 or GCV. Plaques were stained and counted 10 days later. Figure 3 C: HFFs were pretreated with 8969 or GCV for 24 h and then infected with HCMV Towne at MOI 1, 0.1, and 0.01. Luciferase activity was measured at 72 hpi. Figure 3 D. Figure 3 E: HFFs were pretreated with the indicated concentrations of MLS8969 or GCV for 24 h and then infected with 100 PFU / well of HCMV TB40 ( Figure 3 D) or GCV-resistant HCMV Towne ( Figure 3 E) Plaques were stained and counted 10 days later. Figure 3 A-3E: Data are means ± SD of triplicate values ​​from a representative experiment. Figure 3 F: HFFs were pre-treated or infected after treatment. Infections were performed at MOI 1, 0.1 and 0.01 (Towne). Expression of viral proteins from cell lysates was measured at 72 hpi. WB data are from a representative experiment.

[0070] Figure 4 MLS8969 is an entry inhibitor of HCMV. HFFs were pretreated with 3 μM MLS8999 for 24 hours and then infected with HCMV Towne (MOI=1, 0.1, 0.01). Heparin (30 μM) was used as a positive control to block viral entry into cells, and GCV pretreatment (5 μM) was used as a negative control. IFA for HCMV-encoded pp65 was performed at 2 hpi to determine the amount of pp65 localized in the nucleus under different conditions. The experiment was repeated three times independently, and images from a representative experiment are shown.

[0071] Figure 5 : NFU1827 is an immediate early-early inhibitor of HCMV replication. Figure 5A: HFFs were infected with HCMV TB40 (100 PFU / cell) and treated with the indicated concentrations of compound NFU1827. Plaques were stained and counted at 8 dpi. The experiment was repeated three times, and the data shown are the mean ± SD of three values ​​from a representative experiment. Figure 5 B: HFFs were infected with GCV-resistant HCMV Towne (MOI = 0.1 PFU / cell) and treated with the indicated concentrations of NFU1827. Luciferase activity in cell lysates was measured at 72 hpi. Data shown are the mean ± SD of three values ​​from a representative experiment. Figure 5 C, 5D: HFFs were infected with HCMV Towne (MOI = 1 PFU / cell), and compounds were added or removed after infection (0, 6, 12, 24, 48 and 72 hours). Luciferase activity in cell lysates was measured at 72 hpi. The data shown are the mean ± SD of three values ​​from a representative experiment. Figure 5 E: HFF were infected with HCMV Towne (MOI = 1 PFU / cell), and the expression of viral proteins and cellular β-actin was determined at 72 hpi. The experiment was repeated twice, and WB data are from a representative experiment. Figure 5 F, 5G: HFF were infected with HCMV TB40 (MOI = 0.1) and treated with NFU1827 (5 μM) or GCV (5 μM). Cells were lysed at the indicated time points to isolate DNA. Viral DNA replication in cells was measured by real-time PCR ( Figure 5 F) and viral DNA load in the supernatant ( Figure 5 G). The experiment was repeated three times and the data shown are the mean ± SD of triplicate values ​​of a representative experiment.

[0072] Figure 6 : MLS8554 is an immediate-early inhibitor of HCMV replication. Figure 6 A: HFFs were infected with TB40 at 100 PFU / well and treated with the indicated concentrations of MLS8554. Plaques were stained and counted at 8 dpi. The experiment was repeated three times, and data from a representative experiment are shown. Figure 6 B: HFFs were infected with GCV-resistant HCMV Towne (MOI=0.1) and treated with the indicated concentrations of MLS8554. Luciferase activity was measured in cell lysates at 72 hpi. Data shown are the mean ± SD of three values ​​from a single experiment. Figure 6 C, 6D: HFFs were infected with HCMV Towne (MOI=1), and compounds were added or removed at the indicated time points after infection. Luciferase activity was measured in cell lysates at 72 hpi. The experiment was repeated twice independently, and representative data from a single experiment are shown. Figure 6E: HFF were infected with HCMV Towne (MOI=1), and the expression of viral proteins and cellular β-actin was determined after 3 days. WB data are from a representative experiment. Figure 6 F, 6G: HFF were infected with HCMV Towne (MOI = 0.1) and treated with MLS8554 (2 μM) or GCV (5 μM). The supernatant was collected and the cells were lysed at the indicated time points to isolate DNA. Viral DNA replication in the cells was determined by real-time PCR ( Figure 6 F) and viral DNA load in the supernatant ( Figure 6 G). Data shown are the mean ± SD of four values ​​from two independent experiments.

[0073] Figure 7 :MLS8091 inhibits HCMV replication at early and late stages. Figure 7 A: HFFs were infected with HCMV TB40 (100 PFU / well) and treated with the indicated concentrations of MLS8091. Plaques were stained and counted at 8 dpi. The experiment was repeated three times independently, and data from a single representative experiment are shown. Figure 7 B: HFFs were infected with GCV-resistant HCMV Towne (MOI=0.1) and treated with the indicated concentrations of MLS8091. Luciferase activity was detected in cell lysates at 72 hpi. The data shown are the mean ± SD of three values ​​from a single experiment. Figure 7 C, 7D: HFF infected with HCMV Towne (MOI = 1), and at different time points after infection (0, 6, 24, 48 and 72 hours) add or remove compounds. Luciferase activity in cell lysates was measured at 72hpi. The experiment was repeated twice independently, and representative data from a single experiment are shown. Figure 7 E: HFF were infected with HCMV Towne (MOI=1), and the expression of viral proteins and cellular β-actin was determined at 72 hpi. WB data are from a representative experiment. Figure 7 F, 7G: HFF were infected with HCMV (MOI = 0.1) and treated with MLS8091 (1.5 μM) or GCV (5 μM). The supernatant was collected and the cells were lysed at the indicated time points to isolate DNA. Viral DNA replication in cells was determined by real-time PCR ( Figure 7 F) and viral DNA load in the supernatant ( Figure 7 G). The experiment was repeated twice independently. The data shown are the mean ± SD of three values ​​from a single experiment.

[0074] Figure 8 : NCGC2955 is an early-late inhibitor of HCMV replication. Figure 8A: HFFs were infected with HCMV TB40 (100 PFU / well) and treated with the indicated concentrations of NCGC2955. Plaques were stained and counted at 8 dpi. The experiment was repeated three times independently, and data from a single representative experiment are shown. Figure 8 B: HFFs were infected with GCV-resistant HCMV Towne (100 PFU / well) and treated with the indicated concentrations of NCGC2955. Plaques were stained and counted at 8 dpi. The data shown are the mean ± SD of three values ​​from a single experiment. Figure 8 C, 8D: HFFs were infected with HCMV Towne (MOI = 1), and compounds were added or removed at different times after infection (0, 6, 24, 48 and 72 hours). Luciferase activity in cell lysates was measured at 72 hpi. The data shown are the averages of three independent experiments (mean ± SD). Figure 8 E: HFF were infected with HCMV Towne (MOI=1) and the expression of viral proteins was determined at 72 hpi. The experiment was repeated three times independently and data from a single representative experiment are shown. Figure 8 F, 8G: HFF were infected with HCMV Towne (MOI = 0.1) and treated with 2955 (3 μM) or GCV (5 μM). The supernatant was collected and the cells were lysed at the indicated time points to isolate DNA. Viral DNA replication in cells was determined by real-time PCR ( Figure 8 F) and viral DNA load in the supernatant ( Figure 8 G). Data shown are the mean ± SD of four values ​​from two independent experiments.

[0075] Fig. 9 : The combination of newly identified HCMV inhibitors and GCV is additive. ( Fig. 9 A-9E) HFF were infected with HCMV Towne (MOI = 1) and treated with each compound alone or in combination with GCV at different doses. For drug combinations, HCMV infected cells were treated with single compound EC 50 The luciferase activity in the cell lysate was detected at 72 hpi, and the antiviral activity of the compound in the drug combination was calculated by the Bliss model. The solid line represents the HCMV inhibition (dose response) observed, and the dotted line represents the expected HCMV inhibition of each dosage of the drug combination. Fig. 9 A-9D: Experiments were repeated three times independently. Data from a single representative experiment are shown. Fig. 9 E: Data shown are means ± SD of triplicate values ​​from a single experiment.

[0076] Fig.10: The combination of newly identified HCMV inhibitors and the HCMV terminal enzyme inhibitor Letermovir is additive. ( Fig.10 A-10E) HFF were infected with TB40 (100 PFU / well) and treated with each compound alone or in combination with Letermovir at different doses. For drug combinations, HCMV infected cells were treated with single compound EC 50 The number of plaques in each case was counted at 10 dpi, and the antiviral activity of the compound was calculated using the Bliss model. The solid line represents the observed HCMV inhibition (dose response), and the dotted line represents the expected HCMV inhibition of each dose of the drug combination. The data shown are the mean ± SD of three values ​​of a single experiment.

[0077] Fig.11 Table of contents: Time of maximum activity of five novel inhibitors and GCV during HCMV replication. Each of the five compounds was subjected to additional addition and removal tests as described in the Materials and Methods section and is shown in Table 1. Figure 5-8 C & D. The time for 75% reduction in luciferase activity was calculated for each compound when added and when removed and represented by a unique color. Figure 5 , 6 Separate addition and removal tests are provided in , 7, 8C and 8D. DETAILED DESCRIPTION OF THE INVENTION

[0079] There is a need to improve currently available herpes virus drugs. To this end, a highly sensitive pp28-luciferase HCMV reporter and a collection of multiple chemical libraries were used. 20 A large-scale screening campaign of approximately 400,000 compounds was conducted.

[0080] Disclosed herein are compounds and pharmaceutical compositions for preventing and treating human herpes viruses in a subject in need thereof.

[0081] The compounds described herein can inhibit human herpes when administered to a cell or cell population in an effective amount. In certain embodiments, the cell or cell population is in a host organism or subject.

[0082] As used herein, the term "human herpesvirus" refers to a DNA virus of the Herpesviridae family known to infect humans. Examples of species of the Herpesviridae family include: HSV-1, which causes cold sores on the face (HHV-1), HSV-2 (genital herpes) (HHV-2), varicella-zoster virus (HHV-3), which causes chickenpox and shingles, Epstein-Barr virus (HHV4), which causes mononucleosis, HCMV or human cytomegalovirus (CMV or HHV-5), roseola virus (HHG-6A), herpes lymphotrophic virus (HHHV-6B), pityriasis rosea (HHV-7), and Kaposi's sarcoma-associated herpesvirus (HHV-8).

[0083] Disclosed herein are compounds of formula IV that can be used to inhibit viral diseases. In some embodiments, compounds of formula IV can be used to inhibit herpes virus infection in subjects in need thereof, including mammals, such as humans, such as infants, children, and adults, including adults 40 years and older.

[0084] Also disclosed herein is a pharmaceutical composition comprising a compound of formula IV and a pharmaceutical carrier, which can be used to inhibit viral diseases. In some embodiments, the pharmaceutical composition can be used to inhibit herpes virus infection in mammals (including humans).

[0085] According to one or more embodiments, the present invention also discloses a pharmaceutical composition comprising a compound of formula IV, at least one additional bioactive agent and a pharmaceutical carrier, which can be used to inhibit viral diseases. In some embodiments, the pharmaceutical composition can be used to inhibit herpes virus infection in mammals (including humans).

[0086] According to one or more embodiments, disclosed herein is a method of inhibiting human herpes virus in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula IV or a pharmaceutically acceptable composition comprising a compound of formula IV and a pharmaceutically acceptable carrier.

[0087] Disclosed herein is a method of inhibiting human herpes virus in a subject in need thereof, the method comprising administering to the subject a compound of Formula I or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier:

[0088]

[0089] Wherein, R1 is a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally substituted by one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group. In some embodiments, the compound is selected from the following group:

[0090]

[0091]

[0092] Disclosed herein is a method of inhibiting human herpes virus in a subject in need thereof, the method comprising administering to the subject a compound of formula II or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier:

[0093]

[0094] wherein R1 is H, and R2 is independently H or alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group; or R1 and R2 together form a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms; and R3 is H or cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more halogen, CF3, CN and NO2 moieties. In some embodiments, R2 is a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted by one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl. In some embodiments, the compound is selected from the group consisting of:

[0095]

[0096]

[0097] Disclosed herein is a method of inhibiting human herpes virus in a subject in need thereof, the method comprising administering to the subject a compound of formula III or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier:

[0098]

[0099] Disclosed herein are methods of inhibiting human herpes virus in a subject in need thereof, the method comprising administering a compound of formula IV or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, the pharmaceutical composition comprising a pharmaceutically acceptable carrier:

[0100]

[0101] Disclosed herein are methods of inhibiting human herpes virus in a subject in need thereof, the method comprising administering to the subject a compound of formula V or a pharmaceutical composition thereof or a salt, solvate or stereoisomer thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier:

[0102]

[0103] Among them, R 1 is hydrogen, C1-C6 straight or branched alkyl, or C3-C6 cycloalkyl; and R 2 is unsubstituted or replaced by R 3 OR 3 Substituted phenyl, wherein R 3 is hydrogen, C1-C6 straight or branched alkyl or C3-C6 cycloalkyl. 1 is a C1-C4 straight or branched chain alkyl group; and R 2 is OR 3 Substituted phenyl, wherein R 3 is a C1-C4 straight or branched alkyl. In some embodiments, the compound is

[0104]

[0105] In some embodiments, the methods disclosed herein further comprise administering more than one biologically active agent.

[0106] In some embodiments, the herpes virus is selected from: human herpes virus type 1 (HHV-1), human herpes virus type 2 (HHV2), human herpes virus type 3 (HHV3), human herpes virus type 4 (HHV4), human herpes virus type 5 (CMV or HHV5), human herpes virus type 6 (HHV6), human herpes virus type 7 (HHV7) and human herpes virus type 8 (HHV8). In some embodiments, the herpes virus is HHV-5.

[0107] Disclosed herein is a pharmaceutical composition comprising a compound of formula I or a salt, solvate or stereoisomer thereof:

[0108]

[0109] Wherein, R1 is a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally substituted by one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group. In some embodiments, the compound is selected from the following group:

[0110]

[0111] Disclosed herein is a pharmaceutical composition comprising a compound of formula II or a salt, solvate or stereoisomer thereof:

[0112]

[0113] wherein R1 is H, and R2 is independently H or alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group; or R1 and R2 together form a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms; and R3 is H or cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more halogen, CF3, CN and NO2 moieties. In some embodiments, R2 is a C1-C6 heterocycloalkyl or C1-C6 heteroaryl group optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group. In some embodiments, the compound is selected from the group consisting of:

[0114]

[0115]

[0116] Disclosed herein is a pharmaceutical composition comprising a compound of formula III or a salt, solvate or stereoisomer thereof and a pharmaceutically acceptable carrier:

[0117]

[0118] Disclosed herein is a pharmaceutical composition comprising a compound of formula IV or a salt, solvate or stereoisomer thereof and a pharmaceutically acceptable carrier:

[0119]

[0120] Disclosed herein is a pharmaceutical composition comprising a compound of Formula V or a salt, solvate or stereoisomer thereof and a pharmaceutically acceptable carrier:

[0121]

[0122] Among them, R 1 is hydrogen, C1-C6 straight or branched alkyl, or C3-C6 cycloalkyl; and R 2 is unsubstituted or replaced by R 3 OR 3 Substituted phenyl, wherein R 3 is hydrogen, C1-C6 straight or branched alkyl or C3-C6 cycloalkyl. 1 is a C1-C4 straight or branched chain alkyl group; and R 2 is OR 3 Substituted phenyl, wherein R 3 is a C1-C4 straight or branched alkyl. In some embodiments, the compound is

[0123]

[0124] Disclosed herein are compounds of Formula I or salts, solvates or stereoisomers thereof:

[0125]

[0126] wherein R1 is a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected via a C1-C3 alkyl group, provided that the compound cannot be compound A:

[0127]

[0128] In some embodiments of the compound or its salt, solvate or stereoisomer, the compound is selected from:

[0129]

[0130] Disclosed herein are compounds of formula II or salts, solvates or stereoisomers thereof:

[0131]

[0132] wherein R1 is H, and R2 is independently H or alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected by a C1-C3 alkyl group; or R1 and R2 together form a C1-C6 heterocycloalkyl or C1-C6 heteroaryl optionally substituted with one or more amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms; and R3 is H or cycloalkyl, heterocycloalkyl, aryl or heteroaryl optionally substituted with one or more halogen, CF3, CN and NO2 moieties, provided that the compound cannot be a compound selected from the group consisting of:

[0133]

[0134] In some embodiments, R2 is a C1-C6 heterocycloalkyl or C1-C6 heteroaryl group optionally substituted with one or more alkyl, amidino, guanidino, phosphate, sulfate, tetrazole, 3-hydroxyisoxazole, secondary amide, sulfonamide, sulfonylurea, acyl amidine, acylguanidine, having 1 to 10 carbon atoms and connected through a C1-C3 alkyl group.

[0135] Disclosed herein are compounds of formula V or salts, solvates or stereoisomers thereof:

[0136]

[0137] Among them, R 1 is hydrogen, C1-C6 straight or branched alkyl, or C3-C6 cycloalkyl; and R 2 is unsubstituted or replaced by R 3 OR 3 Substituted phenyl, wherein R 3 is hydrogen, C1-C6 straight or branched alkyl or C3-C6 cycloalkyl. 1 is a C1-C4 straight or branched chain alkyl group; and R 2 is OR 3 Substituted phenyl, wherein R 3 is a C1-C4 straight or branched alkyl.

[0138]

[0139] The term "aliphatic" is an art-recognized term that includes straight-chain, branched-chain, and cyclic alkanes, alkenes, or alkynes. In certain embodiments, an aliphatic group is straight-chain or branched and has 1 to about 20 carbon atoms.

[0140] The term "alkyl" is art-recognized and includes saturated aliphatic groups including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl groups (alicyclic groups), alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl group has about 30 or fewer carbon atoms in its backbone (e.g., C1-C2 for a straight chain). 30 , for the branched chain C3-C 30 ), or about 20 or fewer carbon atoms. Similarly, cycloalkyl has about 3 to about 10 carbon atoms in its ring structure, or about 5, 6, or 7 carbon atoms in the ring structure.

[0141] In addition, the term "alkyl" (or "lower alkyl") includes "unsubstituted alkyl" and "substituted alkyl", the latter refers to an alkyl moiety having a substituent replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphonate, phosphinate, amino, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl or aromatic or heteroaromatic moieties. Those skilled in the art will appreciate that the moieties substituted on the hydrocarbon chain may themselves be substituted if appropriate. For example, the substituents of substituted alkyl groups can include substituted and unsubstituted forms of amino, azido, imino, amide, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate) and silyl, as well as ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate and ester), -CF3, -CN, etc. Exemplary substituted alkyl groups are described below. Cycloalkyl groups can be further substituted by alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF3, -CN, etc.

[0142] The term "aralkyl" is art-recognized and includes aryl groups (eg, aromatic or heteroaromatic groups).

[0143] The terms "alkenyl" and "alkynyl" are art-recognized and refer to a moiety containing at least one double or triple bond, respectively.

[0144] The term "aryl" is art-recognized and includes 5-, 6- and 7-membered monocyclic aromatic groups that may contain 0 to 4 heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, etc. The term "heteroatom" generally includes atoms of any element except carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium. The aryl group with heteroatoms in the ring structure may also be referred to as an "aryl heterocycle" or a "heteroaromatic". Aromatic rings may be substituted at one or more ring positions with substituents as described above, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, etc. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more carbon atoms are common to two adjacent rings (the rings are "fused"), in which at least one ring is aromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclyl, or rings connected by non-cyclic moieties.

[0145] The terms "ortho", "meta", and "para" are art-recognized and apply to 1,2-, 1,3-, and 1,4-disubstituted benzene rings, respectively. For example, the names 1,2-xylene and ortho-xylene are synonymous.

[0146] The term "heterocyclyl" or "heterocycloalkyl" is art recognized and includes about 3 to about 10 membered ring structures, such as 3 to about 7 membered rings, which include 1 to 4 heteroatoms in the ring structure. The heterocycle can also be polycyclic. Heterocyclic groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthin, pyrrolimidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenpyrazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams (such as azetidinone and pyrrolidone), sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with substituents as described above, such as halogen, alkyl, aralkyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic group, aromatic or heteroaromatic moiety, -CD3, -CN, etc.

[0147] The terms "polycyclic radical" and "polycyclic group" are art-recognized and include structures having two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclic radicals) wherein two or more carbons are common to two adjacent rings, e.g., the rings are "fused rings". Rings connected by non-adjacent atoms, e.g., three or more atoms are common to two rings, are referred to as "bridged" rings. Each ring of the polycyclic ring may be substituted with the above-mentioned substituents, e.g., halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic radical, aromatic or heteroaromatic moiety, -CD3, -CN, etc.

[0148] The term "carbocycle" as used herein is art-recognized and includes aromatic or non-aromatic rings in which each atom of the ring is carbon. The following art-recognized terms have the following meanings: "nitro" refers to -NO2; the term "halogen" means -F, -Cl, -Br or -I; the term "thiol" refers to -SH; the term "hydroxy" or "hydroxyl group" refers to -OH; the term "sulfonyl" refers to -SO2-.

[0149] The terms "amine" and "amino" are recognized in the art and include unsubstituted amines and substituted amines. Primary amines have two hydrogens, secondary amines have one hydrogen and another substituent, and tertiary amines have two hydrogens substituted. For example, the substituents for one or both hydrogens can be alkyl, alkenyl and aryl, cycloalkyl, cycloalkenyl, heterocyclic, polycyclic, etc. If both hydrogens are replaced by carbonyls, the carbonyls around the nitrogen will form an imide.

[0150] The term "alkylamine" embraces an amine group as defined above to which is attached a substituted or unsubstituted alkyl group.

[0151] The term "amido" is art-recognized as a carbonyl group substituted with an amino group.

[0152] The term "alkylthio" is art-recognized and includes an alkyl group as defined above to which a thiol radical is attached. In certain embodiments, the "alkylthio" moiety is represented by -S-alkyl, -S-alkenyl, -S-alkynyl, etc. Representative alkylthio groups include methylthio, ethylthio, etc.

[0153] The term "carbonyl" is art-recognized and includes a C=O structure. Carbonyl is included in esters; carboxyls; formates; thiocarbonyls; thioesters; thiocarboxylic acids; thioformates; ketones; and aldehydes.

[0154] The terms "alkoxy" and "alkoxy group" are art-recognized and include an alkyl group as defined above to which is attached an oxygen radical. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0155] An "ether" is two hydrocarbons covalently linked by an oxygen. Thus, an alkyl substituent that makes the alkyl an ether is an alkoxy or similar to an alkoxy, represented, for example, by one of -O-alkyl, -O-alkenyl, -O-alkynyl, and the like.

[0156] The term "sulfonate" is art-recognized and includes moieties in which the sulfur atom carries two doubly-bonded oxygens and one single-bonded oxygen.

[0157] The term "sulfate" is art-recognized and includes moieties similar to sulfonate but containing two singly bonded oxygens.

[0158] The terms "sulfonamide," "sulfamoyl," "sulfonyl," and "sulfoxido" are art-recognized and each encompass the various R group substituents as described herein.

[0159] The terms "phosphoramidite" and "phophonamidite" are art-recognized.

[0160] The term "selenoalkyl" is recognized in the art and includes an alkyl group having a substituted seleno group attached thereto. Exemplary "selenoethers" that may be substituted on the alkyl group are selected from one of the following: -Se-alkyl, -Se-alkenyl, -Se-alkynyl, etc.

[0161] Substitution can be made on alkenyl and alkynyl groups to produce, for example, aminoalkenyl, aminoalkynyl, amidoalkenyl, iminoalkenyl, iminoalkynyl, thioalkenyl, thioalkynyl, carbonyl substituted alkenyl or alkynyl groups.

[0162] Hydrocarbon is a term recognized in the art and includes all permissible compounds having at least one hydrogen atom and one carbon atom. For example, permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds that may be substituted or unsubstituted.

[0163] The term "protecting group" is well known in the art and includes temporary substituents that protect potential reactive functional groups from undesirable chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, acetals and ketals of aldehydes and ketones. The field of protecting group chemistry has been reviewed, for example Greene et al., Protective Groups in Organic Synthesis, 2nd edition, Wiley, New York, (1991).

[0164] Unless expressly stated otherwise or the context dictates otherwise, a definition of each expression (eg, alkyl, aryl, etc.) when it occurs more than one time in any structure is intended to be independent of definitions elsewhere in the same structure.

[0165] The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to triflyl, tosyl, mesyl, and nonaflyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to triflate, tosylate, mesylate, and nonaflate functional groups, respectively, and molecules containing said groups.

[0166] The abbreviations Me, Et, Ph, Tf, Nf, Ts and Ms are recognized in the art and represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, toluenesulfonyl and mesyl, respectively. A more comprehensive list of abbreviations used by organic chemists of ordinary skill in the art may be found in the first issue of each volume of the Journal of Organic Chemistry; the list generally appears in a table entitled "List of Standard Abbreviations".

[0167] Therefore, the compounds disclosed herein include tautomeric forms of the disclosed compounds (including isomeric forms of enantiomers, stereoisomers and diastereoisomers), as well as pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salt" includes salts that are commonly used to form alkali metal salts and to form free acid or free base addition salts. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids (such as hydrochloric acid, sulfuric acid and phosphoric acid) and organic acids (such as maleic acid, succinic acid and citric acid). Other pharmaceutically acceptable salts include salts containing alkali metals or alkaline earth metals (such as sodium, potassium, calcium and magnesium), or salts containing organic bases (such as dicyclohexylamine). For example, suitable pharmaceutically acceptable salts of the disclosed compounds include acid addition salts, for example, which can be formed by mixing a solution of the disclosed compounds with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. All of these salts can be prepared by conventional methods, for example, by reacting a suitable acid or base with the corresponding compound disclosed herein.

[0168] Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0169] For use in medicine, the salts of the compounds disclosed herein will be pharmaceutically acceptable salts. However, other salts may also be useful in the preparation of the compounds disclosed herein or their pharmaceutically acceptable salts.

[0170] In addition, embodiments disclosed herein include hydrates of the compounds disclosed herein. The term "hydrate" includes, but is not limited to, hemihydrates, monohydrates, dihydrates, trihydrates, and the like. Hydrates of compounds disclosed herein can be prepared by contacting the compound with water under appropriate conditions to produce the selected hydrate.

[0171] Embodiments disclosed herein also include methods for preparing pharmaceutical products comprising compounds. The term "pharmaceutical product" refers to a composition suitable for medical use (pharmaceutical composition) as defined herein. Pharmaceutical compositions formulated for specific applications comprising compounds disclosed herein are considered embodiments thereof.

[0172] The compositions disclosed herein may include a carrier. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered.

[0173] Relative to the pharmaceutical compositions described herein, a pharmaceutically acceptable carrier can be any carrier that is conventionally used and is limited only by physicochemical factors (e.g., solubility and lack of reactivity with the active compound), and by the route of administration. Pharmaceutically acceptable carriers described herein, such as carriers, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. Examples of pharmaceutically acceptable carriers include soluble carriers, such as physiologically acceptable known buffers (e.g., phosphate buffers), and solid compositions, such as solid carriers or latex beads. Pharmaceutically acceptable carriers are preferably those that are chemically inert to the active agent and those that have no or little harmful side effects under the conditions of use.

[0174] The carrier or diluent used herein may be a solid carrier or diluent for solid preparations, a liquid carrier or diluent for liquid preparations, or a mixture thereof.

[0175] Solid carriers or diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0176] For liquid formulations, pharmaceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, injectable organic co-solvents, surfactants and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.

[0177] Examples of oils are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower seed oil, cod liver oil, sesame oil, cottonseed oil, corn oil, olives, petrolatum and mineral oil. Suitable fatty acids for parenteral formulations include, for example, oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0178] Parenteral vehicles (for subcutaneous, intravenous, arterial or intramuscular injection) include, for example, sodium chloride solution, saline solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous solutions, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions containing suspending agents, solubilizers, thickeners, stabilizers and preservatives.

[0179] Intravenous carriers include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, etc. Examples are sterile liquids such as water and oils with or without the addition of surfactants and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose solutions and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol can be liquid carriers, particularly for injectable solutions.

[0180] In addition, in some embodiments, the compounds described herein may further include: for example, binders (e.g., acacia, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone); disintegrants (e.g., corn starch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate); buffers of different pH values ​​and ionic strengths (e.g., Tris-HCl, acetate, phosphate); additives, such as albumin or gelatin, to prevent adsorption to the surface; detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile salt); protease inhibitors; surfactants (such as sodium lauryl sulfate); penetration enhancers, solubilizers (e.g., cremophor, glycerol, polyethylene glycol, benzlkonium chloride, benzyl benzoate, cyclodextrin, sorbitan esters, stearic acid); antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole); stabilizers (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose); viscosity enhancers (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum); sweeteners (e.g., aspartame, citric acid); preservatives (e.g., thimerosal, benzyl alcohol, parabens); lubricants (e.g., fatty acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate); glidants (e.g., colloidal silicon dioxide); plasticizers (e.g., diethyl phthalate, triethyl citrate); emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate); polymer coatings (e.g., poloxamers or poloxamines); coating and film-forming agents (e.g., ethylcellulose, acrylates, polymethacrylates); and / or adjuvants.

[0181] The choice of carrier will be determined in part by the specific compound and the specific method for administering the compound. Therefore, there are a variety of suitable formulations of the pharmaceutical composition disclosed herein. The following preparations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and intraperitoneal administration are exemplary and not restrictive. More than one approach can be used to administer the compound, and in some cases, a specific approach can provide a more direct and more effective response than another approach. In some embodiments, in the methods disclosed herein, the compound or pharmaceutical composition disclosed herein is administered orally, for example, with tablets, capsules, suspensions, or intravenous administration.

[0182] Suitable soaps for parenteral formulations may include, for example, fatty alkali metal, ammonium and triethylamine salts, and suitable detergents include, for example, (a) cationic detergents, for example, dimethyldialkylammonium halides and alkylpyridinium halides, (b) anionic detergents, for example, alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglycerol sulfonates, and sulfosuccinates, (c) nonionic detergents, for example, fatty amine oxides, fatty acid alkanolamides and polyoxyethylene-polypropylene copolymers, (d) amphoteric detergents, for example, alkyl-b-aminopropionates and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0183] In solution, parenteral formulations typically contain about 0.5% to about 25% by weight of the compound. Preservatives and buffers may be used. To minimize or eliminate the irritation at the injection site, such compositions may contain one or more nonionic surfactants, for example, having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is typically about 5% to about 15% by weight. For example, suitable surfactants include: polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide and hydrophobic bases formed by the condensation of propylene oxide and propylene glycol.

[0184] Parenteral preparations can be presented in unit dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in freeze-dried (lyophilized) conditions, requiring only the addition of sterile liquid excipients for injection (such as water) prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

[0185] Injectable formulations are consistent with the disclosure herein. Those skilled in the art are familiar with the requirements for effective drug carriers for injectable compositions (see Pharmaceutics and Pharmacy Practice, JBL, Philadelphia, Pennsylvania, Banker and Chalmers, eds., pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (2009).

[0186] The amount or dosage of any of the compounds, salts, solvates or stereoisomers of Formula I, II, III, IV and V compounds as described above should be sufficient to produce, for example, a therapeutic or prophylactic response in a subject within a reasonable time frame. The dosage will be determined by the efficacy of the particular compound and the condition of the person and the weight of the person to be treated.

[0187] The dosage of any one of the compounds, salts, solvates or stereoisomers of Formula I, II, III, IV and V compounds as described above will also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of a particular compound. Typically, the attending physician will consider a variety of factors, such as age, weight, overall health, diet, sex, the compound to be administered, the route of administration and the severity of the condition being treated to determine the dosage of the compound for treating each individual patient. For example, but not intended to limit the disclosure herein, the dosage of the compound may be about 0.001 to about 1000 mg / kg body weight / day of the subject to be treated.

[0188] In some embodiments, the term "administering / dosing" refers to introducing a compound disclosed herein into a subject, e.g., a subject receiving disease treatment, and allowing the compound to contact one or more disease-related cells or cell groups in the body. In some embodiments, the host cell or host cell group in the host can be any cell or cell group that can be selectively bound by an antigen bound to the above-mentioned Formula I, II, III, IV, and V compounds. It will be understood by those skilled in the art that the host cell can be a cell infected with a virus.

[0189] According to some embodiments, the disclosure herein provides a method for inhibiting human herpes virus in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition comprising one or more of the above compounds and a pharmaceutically acceptable carrier.

[0190] According to some embodiments, the disclosure herein provides a method for treating a medical condition in a subject (including but not limited to inhibiting human herpes virus), the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition comprising one or more of the above-mentioned compounds, a pharmaceutically acceptable carrier and at least one other biologically active agent.

[0191] According to some embodiments, the disclosure herein provides a method for inhibiting or treating human herpes virus in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition comprising one or more of the above compounds and a pharmaceutically acceptable carrier.

[0192] According to some embodiments, the disclosure herein provides a method for inhibiting or treating human herpes virus in a subject (e.g., inhibiting human herpes virus replication), the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutically acceptable composition comprising one or more of the above-mentioned compounds, at least one biologically active agent, and a pharmaceutically acceptable carrier.

[0193] "Inhibiting herpes virus" or "inhibition of herpes virus" refers to reducing or preventing viral replication, treating symptoms caused by viral infection (such as hepatitis or pneumonia), reducing viral load in the blood or plasma, and / or reducing persistent viremia.

[0194] "Treating" or "treatment" are art-recognized terms that include curing as well as alleviating at least one symptom of any condition or disease. Treatment includes reducing the likelihood of a disease, disorder or condition occurring in an animal susceptible to the disease, disorder or condition but not yet diagnosed as having the disease, disorder or condition; inhibiting the disease, disorder or condition, such as arresting its progression; and relieving the disease, disorder or condition, such as causing regression of the disease to any extent; inhibiting the disease, disorder or condition, such as arresting its progression; and relieving the disease, disorder or condition, even if the underlying pathophysiology is not affected or other symptoms remain at the same level.

[0195] In some embodiments, the pharmaceutical compositions disclosed herein can be used to treat a viral infection in a subject. In some embodiments, the viral infection is a herpes virus infection, for example, a CMV infection.

[0196] In some embodiments, the pharmaceutical compositions disclosed herein can be combined with one or more additional biologically active agents, including, for example, antiviral agents.

[0197] "Preventative / prophylactic" or "therapeutic / therapeutic" treatments are art-recognized and include administering one or more of the subject compositions to a host. If administered prior to clinical manifestation of an unwanted condition (e.g., a disease or other unwanted state of the host animal), the treatment is preventative, i.e., it protects the host from developing the unwanted condition, whereas if administered after the unwanted condition has manifested, the treatment is therapeutic (i.e., its purpose is to reduce, alleviate, or stabilize the existing unwanted condition or its side effects).

[0198] Bioactive agents can vary widely depending on the intended use of the composition. The term "active" is recognized in the art and refers to any part of a biological, physiological or pharmaceutically active substance that acts locally or systemically in a subject. Examples of bioactive agents that can be referred to as "drugs" are described in well-known literature, such as Merck Index, Physicians' Desk Reference, and Pharmacological Basis of Therapeutics, and include, but are not limited to: medicaments; vitamins; mineral supplements; substances for treating, preventing, diagnosing, curing or alleviating diseases or illnesses; substances that affect body structure or function; or prodrugs that become biologically active or more active after being located in a physiological environment. Various forms of bioactive agents can be used, which can release the subject composition into, for example, adjacent tissues or fluids after being administered to the subject. In some embodiments, bioactive agents can be used in a cross-linked polymer matrix, for example, to promote cartilage formation. In other embodiments, bioactive agents may be used in the cross-linked polymer matrix to treat, alleviate, inhibit or prevent a disease or condition, for example, in conjunction with promoting cartilage formation.

[0199] Further examples of bioactive agents include, but are not limited to, enzymes, receptor antagonists or agonists, hormones, growth factors, autologous bone marrow, antibiotics, antimicrobials, and antibodies. The term "bioactive agent" is also intended to encompass various cell types and genes that can be incorporated into the compounds and compositions disclosed herein.

[0200] In certain embodiments, the subject compositions may comprise from about 1% to about 75% or more by weight of the total composition, or contain about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the bioactive agent.

[0201] Non-limiting examples of biologically active agents include the following: adrenergic blocking agents, anabolic agents, androgenic steroids, antacids, anti-asthmatic agents, antiallergenic materials, anti-cholesterolemic and anti-lipid agents, anti-cholinergics and sympathomimetics, anti-coagulants, anti-convulsants, anti-diarrheals, anti-emetics, anti-hypertensive agents, anti-infective agents, anti-inflammatory agents such as steroids, nonsteroidal anti-inflammatory agents, anti-malarials, anti-manic agents), antinauseants, antineoplastic agents, antiobesity agents, antiparkinsonian agents, antipyretic and analgesic agents, antispasmodic agents, antithromboticagents, antiuricemic agents, antianginal agents, antihistamines, antitussives, appetite suppressants, benzophenanthridine alkaloids, biologicals, cardioactive agents, cerebral dilatorsdilators, coronary dilators, decongestants, diuretics, diagnostic agents, erythropoietic agents, estrogens, expectorants, gastrointestinal sedatives, agents, hyperglycemic agents, hypnotics, hypoglycemic agents, ion exchange resins, laxatives, mineral supplements, mitotics, mucolytic agents, growth factors, neuromuscular drugs, nutritional substances, peripheral vasodilators, progestational agents, prostaglandins, psychic stimulants energizers, psychotropics, sedatives, stimulants, thyroid and anti-thyroid agents, tranquilizers, uterine relaxants, vitamins, antigenic materials and prodrugs.

[0202] Specific examples of useful bioactive agents of the above categories include: (a) antineoplastic agents, such as androgen inhibitors, antimetabolites, cytotoxic agents and immunomodulators; (b) antitussives, such as dextromethorphan, hydrobromide, noscapine, carbetapentane citrate and chlophedianol hydrochloride; (c) antihistamines, such as chlorpheniramine phenindamine tartrate, pyrilamine doxylamine succinate and phenyltoloxamine citrate. citrate; (d) decongestants, such as hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, and ephedrine; (e) various alkaloids, such as codeine phosphate, codeine sulfate, and morphine; (f) mineral supplements, such as potassium chloride, zinc chloride, calcium carbonate, magnesium oxide, and other alkali and alkaline earth metal salts; (g) ion exchange resins, such as N-acetylprocainamide; (i) antipyretics and analgesics, such as acetaminophen, aspirin, and ibuprofen; appetite suppressants, such as phenylpropanolamine; amine or caffeine; (k) expectorants, such as guaifenesin; (l) antacids, such as aluminum hydroxide and magnesium hydroxide; biological products, such as peptides, polypeptides, proteins and amino acids, hormones, interferons or cytokines and other biologically active peptide compounds, such as calcitonin, ANF, EPO and insulin;(n) anti-infective agents, such as antifungals, antivirals, antiseptics and antibiotics; and (m) desensitizing agents and antigenic materials, such as those used in vaccine applications.

[0203] More specifically, non-limiting examples of useful bioactive agents include the following therapeutic classes: analgesics, such as nonsteroidal anti-inflammatory drugs, opiate agonists, and salicylates; antihistamines, such as H1-blockers and H2-blockers; anti-infectives, such as antihelmintics, antianaerobics, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, miscellaneous antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antibiotics, antimycobacterials, antituberculosis antimycobacterials, antiprotozoals, antimalarial antiprotozoals, antiviral agents, antiretroviral agents, scabicides and urinary anti-infectives; antineoplastic agents, such as alkylating agents, nitrogen mustard alkylating agents, nitrosourea alkylating agents, antimetabolites, purine analog antimetabolites;Pyrimidine analog antimetabolites; hormonal antineoplastics, natural antineoplastics, antibiotic natural antimetabolites and vinca alkaloid natural antimetabolites; autonomic agents such as anticholinergics, antimuscarinic anticholinergics, ergot alkaloids, parasympathomimetics, cholinergic agonist parasympathomimetics, cholinesterase inhibitor parasympathomimetics, sympatholytics, a-blocker sympatholytics, sympatholytics, sympathomimetics, and adrenergic agonist sympathomimetics;Cardiovascular agents, such as antianginals, antianginals, calcium channel blocker antianginals, nitrate antianginals, antiarrhythmics, cardiac glycoside antiarrhythmics, class I antiarrhythmics, class II antiarrhythmics, class III antiarrhythmics, class IV antiarrhythmics, antihypertensive agents, a-blocker antihypertensives, angiotensin-converting enzyme inhibitors ACE inhibitors, antihypertensives, 13-blocker antihypertensives, calcium-channel blocker antihypertensives, central-acting adrenergic antihypertensives, diuretic antihypertensives, peripheral vasodilator antihypertensives, antilipemics, bile acid sequestrant antilipemics, reductase inhibitor antilipemics, inotropes, cardiac glycoside inotropes, and thrombolytic agents;Dermatological agents, such as antihistamines, anti-inflammatory agents, corticosteroid anti-inflammatory agents, anesthetics, topical anti-infectives, topical anti-infectives, antiviral topical anti-infectives, and topical antineoplastics; Electrolytic and renal agents, such as acidifying agents, alkalinizing agents, diuretics, carbonic anhydrase inhibitor diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics diuretics, electrolyte replacements, and uricosuric agents; enzymes, such as pancreatic enzymes and thrombolytic enzymes;Gastrointestinal agents, such as antidiarrheals, antiemetics, gastrointestinal anti-inflammatory agents, salicylate gastrointestinal anti-inflammatory agents, antacid anti-ulcer agents, gastric acid-pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents, H2-blocker anti-ulcer agents, cholelitholytic agents, digestants, emetics, laxatives and stool softeners, and prokinetic agents; general anesthetics, such as inhalation anesthetics, halogenated inhalation anesthetics, inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opiate agonist intravenous anesthetics;hematological agents, such as antianemia agents, hematopoietic antianemia agents, coagulation agents, anticoagulants, hemostatic coagulation agents, platelet inhibitor coagulation agents, thrombolytic enzyme coagulation agents, and plasma volume expanders; hormones and hormone modulators, such as abortifacients, adrenal agents, corticosteroid adrenal agents, androgens, anti-androgens, antidiabetic agents, sulfonylurea antidiabetic agents, antihypoglycemic agents, oral contraceptives, progestin contraceptives contraceptives, estrogens, fertility agents, oxytocics, parathyroid agents, pituitary hormones, progestins, antithyroid agents, thyroid hormones, and tocolytics; immunobiological agents, such as immunoglobulins, immunosuppressives, toxoids, and vaccines; local anesthetics, such as amide local anesthetics and ester local anesthetics;musculoskeletal agents, such as anti-gout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressive anti-inflammatory agents, nonsteroidal anti-inflammatory drugs, salicylate anti-inflammatory agents, skeletal muscle relaxants, neuromuscular blocker skeletal muscle relaxants, and reverse neuromuscular blocker skeletal muscle relaxants; neurological agents, such as anticonvulsants, barbiturate anticonvulsants, benzodiazepine anticonvulsants, anti-migraine agents, anti-parkinsonian agents, anti-vertigo agents, opiate agonists, and opiate antagonists;ophthalmic agents, such as anti-glaucoma agents, anti-glaucoma agents, mitotics, anti-glaucoma agents, mydriatics, adrenergic agonist mydriatics, antimuscarinic mydriatics, ophthalmic anesthetics, ophthalmic anti-infectives, ophthalmic aminoglycoside anti-infectives, ophthalmic macrolide anti-infectives, ophthalmic quinolone anti-infectives, ophthalmic sulfonamide anti-infectives ophthalmic tetracycline anti-infectives, ophthalmic anti-inflammatory agents, ophthalmic corticosteroid anti-inflammatory agents and ophthalmic nonsteroidal anti-inflammatory drugs;Psychotropic agents, such as antidepressants, heterocyclic antidepressants, monoamine oxidase inhibitors selective serotonin re-uptake inhibitors tricyclic antidepressants, antimanics, anti-psychotics, phenothiazine antipsychotics, anxiolytics, sedatives and hypnotics, barbiturate sedatives and hypnotics, benzodiazepine anxiolytics, sedatives and hypnotics, and psychostimulants; respiratory drugs agents, such as antitussives, bronchodilators, adrenergic agonist bronchodilators, antimuscarinic bronchodilators, expectorants, mucolytic agents, respiratory anti-inflammatory agents, and respiratory corticosteroid anti-inflammatory agents; toxicology agents, such as antidotes, heavy metal agents, substance abuse agents, deterrent substance abuse agents, and withdrawal substance abuse agents; minerals; and vitamins, for example, vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and K.;

[0204] Other classes of bioactive agents from the above classes include: (1) general analgesics, such as lidocaine, other "caine" analgesics or their derivatives, and nonsteroidal anti-inflammatory drug (NSAID) analgesics, including diclofenac, ibuprofen, ketoprofen, and naproxen; (2) opioid agonist analgesics, such as opiate agonists, analgesics, such as codeine, fentanyl, hydromorphone, and morphine; (3) salicylates, such as aspirin (ASA) (enteric-coated ASA); (4) H1-blocker antihistamines, such as clemastine and terfenadine; (5) H2-blocker antihistamines, such as cimetidine, famotidine, nizadine, and ranitidine; (6) anti-infectives, such as mupirocin; (7) anti-anaerobic anti-infectives, such as chloramphenicol and clindamycin; (8) antifungal antibiotics (10) Miscellaneous antibiotic anti-infectives, such as imipenem; (11) Antibiotic anti-infectives, such as nafcillin, oxacillin, penicillin G and penicillin V;(12) Quinolone antibiotic anti-infectives, such as ciprofloxacin and norfloxacin; (13) Tetracycline antibiotic anti-infectives, such as doxycycline, minocycline and tetracycline; (14) Antituberculosis antibiotic anti-infectives, such as isoniazid and rifampin; (15) Antiprotozoal anti-infectives, such as atovaquone and dapsone; (16) Antimalarial anti-protozoal anti-infectives. (17) anti-retroviral anti-infectives, such as ritonavir and zidovudine; (18) anti-viral anti-infectives, such as acyclovir, ganciclovir, interferon-y and rimantadine; (19) alkylating antineoplastic agents, such as carboplatin and cisplatin; (20) nitrosourea alkylating antineoplastic agents, such as carmustine (BCNU); (21) antimetabolite antineoplastic agents, such as sirolimus (sirolimus) and sirolimus (sirolimus). agents), such as methotrexate; (22) pyrimidine analog antimetabolite antineoplastic agents, such as fluorouracil (S-FU) and gemcitabine;(23) Hormonal antineoplastics, such as goserelin, leuprolide and tamoxifen; (24) Natural antineoplastics, such as aldesleukin, interleukin-2, docetaxel, etoposide, interferon; paclitaxel, other taxane derivatives and tretinoin (ATRA); (25) Antibiotic natural antineoplastics, such as leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), leukocyte antigen (VEGA), (26) vinca alkaloid natural antineoplastics, such as vinblastine and vincristine; (27) autonomic agents, for example, nicotine; (28) anticholinergic autonomic agents, such as benztropine and rihexyphenidyl; (29) anticholinergic autonomic agents, such as atropine and oxybutynin; (30) ergot alkaloid autonomic agents, such as sedrin and dapoxetine; (31) sedrin and dapoxetine; (32) sedrin and dapoxetine; (33) sedrin and dapoxetine; (34) sedrin and dapoxetine; (35) sedrin and dapoxetine; (36) sedrin and dapoxetine; (37) sedrin and dapoxetine; (38) sedrin and dapoxetine; (39) sedrin and dapoxetine; (40) sedrin and dapoxetine; (41) sedrin and dapoxetine; (42) sedrin and dapoxetine; (43) sedrin and dapoxetine; (44) sedrin and dapoxetine; (45) sedrin and dapoxetine; (46) sedrin and dapoxetine; (47) sedrin and dapoxetine; (48) sedrin and dapoxetine; (49) sedrin and dapoxetine; (50) (31) cholinergic agonist parasympathomimetics, such as pilocarpine; (32) cholinesterase inhibitor parasympathomimetics, such as pyridostigmine;(33) α-blocker sympatholytics, for example, prazosin; (34) D-blocker sympatholytics, for example, atenolol; (35) adrenergic sympathomimetics, for example, albuterol and dobutamine; (36) cardiovascular agents, such as aspirin (ASA) (enteric-coated ASA); (37) D-blocker antianginals, for example, atenolol and propranolol; (38) calcium channel blocker antianginals. (39) nitrate antianginals, for example, isosorbide dinitrate (ISDN); (40) cardiac glycoside antiarrhythmics, for example, (41) class I antiarrhythmics, for example, lidocaine, mexiletine, phenytoin, procainamide, and quinidine; (42) class II antiarrhythmics, for example, (43) class III antiarrhythmics, for example, atenolol, metoprolol, propranolol and timolol; (44) class IV antiarrhythmics, for example, diltiazem and verapamil; (45) antihypertensives, for example, prazosin;(46) Angiotensin-converting enzyme inhibitor (ACE inhibitor) antihypertensives, for example, captopril and enalapril; (47) Antihypertensives, for example, atenolol, metoprolol, nadolol and propranolol; (48) Calcium-channel blocker antihypertensive agents, for example, diltiazem and nifedipine; (49) Central-acting adrenergic antihypertensives, for example, clonidine and methyldopa; (50) Diuretic antihypertensives (51) peripheral vasodilator antihypertensives, for example, minoxidil; (52) lipid-lowering drugs, for example, gemfibrozil and probucol; (53) bile acid sequestrant antilipemics, for example, cholestyramine; (54) reductase inhibitor antilipemics, for example, thiabendazole; (55) thiabendazole; (56) oxadiazine; (57) dapoxetine; (58) thiabendazole; (59) dapoxetine; (60) dapoxetine; (61) dapoxetine; (62) dapoxetine; (63) dapoxetine; (64) dapoxetine; (65) dapoxetine; (66) dapoxetine; (67) dapoxetine; (68) dapoxetine; (69) dapoxetine; (70) dapoxetine; (71) dapoxetine; (72) dapoxetine; (73) dapoxetine; (74) dapoxetine inhibitorantilipemics), for example, lovastatin and pravastatin; (55) inotropes, for example, amrinone, dobutamine and dopamine;(56) Cardiac glycoside inotropes, for example, (57) Thrombolytic agents, for example, alteplase, anistreplase, streptokinase and urokinase; (58) Dermatological agents, for example, colchicine, isotretinoin, methotrexate, minoxidil, tretinoin; (59) Dermatological corticosteroid anti-inflammatory agents, for example, betamethasone and dexamethasone; (60) Antifungal topical anti-infectives (61) antiviral topical anti-infectives, such as acyclovir; (62) topical antineoplastics, such as dapoxetine; (63) electrolytic and renal agents, such as lactulose; (64) loop diuretics, such as furosemide; (65) potassium-sparing diuretics, such as triamterene; (66) thiazide diuretics, such as pheniramine; (67) thiazide diuretics, such as pheniramine; (68) topical anti-infectives, such as acyclovir; (69) topical anti-neoplastics, such as dapoxetine; (70) topical anti-infectives, such as pheniramine; (71) topical anti-infectives, such as acyclovir; (72) topical anti-neoplastics, such as dapoxetine; (73) electrolyte and renal agents, such as lactulose; (74) loop diuretics, such as furosemide; (75) potassium-sparing diuretics, such as triamterene; (76) thiazide diuretics, such as pheniramine; (77) topical anti-infectives, such as acyclovir; (78) topical anti-neoplastics, such as dapoxetine; (79) topical anti-infectives, such as acyclovir; (80) topical anti-neoplastics, such as dapoxetine; (81) topical anti-infectives, such as acyclovir; (82) topical anti-neoplastics, such as dapoxetine; (83) topical anti-neoplastics, such as dapoxetine diuretics, for example, hydrochlorothiazide (HCTZ); (67) uricosuric agents, for example, probenecid; (68) enzymes and (69) thrombolytic enzymes, for example, alteplase, anistreplase, streptokinase and urokinase;(70) Antiemetics, for example, prochlorperazine; (71) Salicylate gastrointestinal anti-inflammatory agents, for example, sulfasalazine; (72) Gastric acid-pump inhibitor anti-ulcer agents, for example, omeprazole; (73) H2-blocker anti-ulcer agents, such as cimetidine, famotidine, nizadine, and ranitidine; (74) Digestants, for example, pancrelipase; (75) Prokinetic agents, for example, erythromycin; (76) Opiate agonists intravenous anesthetics. agonist intravenous anesthetics, for example, fentanyl; (77) hematopoietic antianemia agents, such as G-CSF and GM-CSF; (78) coagulation agents, for example, factor 1-10 (AlIF 1-10); (79) anticoagulants, for example, warfarin; (80) thrombolytic enzyme coagulation agents, for example, alteplase, anistreplase, streptokinase and urokinase; (81) hormones and hormone modulators, for example, bromocriptine; (82) abortifacients, for example, methotrexate; (83) antidiabetic drugs (84) oral contraceptives, such as estrogen and progestin;(85) progestin contraceptives, for example, levonorgestrel and norgestrel; (86) estrogens, for example, conjugated estrogens, diethylstilbestrol (DES), estrogens (estradiol, estrone and estropipate); (87) fertility agents, for example, clomiphene, human chorionic gonadotropin (HCG) and menotropins; (88) parathyroid agents, for example, calcitonin; (89) pituitary hormones. hormones, such as desmopressin, goserelin, oxytocin and vasopressin (ADH); (90) progestins, such as medroxyprogesterone, norethindrone and progesterone; (91) thyroid hormones, such as levothyroxine; (92) immunobiologic agents, such as interferon beta-1b and interferon gamma-1b; (93) immunoglobulins, such as immunoglobulin 1M, IMIG, IGIM and immunoglobulin IVIG; (94) amide local anesthetics, such as lidocaine; (95) ester local anesthetics, such as dapoxetine; (96) sedatives, such as tadalafil; (97) steroids, such as sedatives, such as beta-1g; (98) steroids, such as tadalafil; (99) steroids, such as sedatives, such as beta-1g; (100) steroids, such as tadalafil; (101) steroids, such as tadalafil; (102) steroids, such as tadalafil; (103) steroids, such as tadalafil; (104) steroids, such as tadalafil; (105) steroids anesthetics, for example, benzocaine and procaine;(96) musculoskeletal corticosteroid anti-inflammatory agents, such as beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisone; (97) musculoskeletal anti-inflammatory immunosuppressives, such as azathioprine, cyclophosphamide, and methotrexate; (98) musculoskeletal nonsteroidal anti-inflammatory drugs. anti-inflammatory drugs, such as diclofenac, ibuprofen, ketoprofen, ketorlac, and naproxen; (99) skeletal muscle relaxants, such as diazepam; (100) reverse neuromuscular blocker skeletal muscle relaxants, such as pyridostigmine; (101) neurological drugs agents), for example, nimodipine, riluzole, tacrine and ticlopidine; (102) anticonvulsants, for example, carbamazepine, gabapentin, lamotrigine, phenytoin and valproic acid; (103) barbiturate anticonvulsants, for example, phenobarbital and primidone;(104) benzodiazepine anticonvulsants, for example, clonazepam, diazepam, and lorazepam; (105) anti-migraine agents, for example, bromocriptine, levodopa, carbidopa, and pergolide; (106) anti-vertigo agents, for example, meclizine; (107) opiate agonists, for example, codeine, fentanyl, hydromorphone, methadone, and morphine; (108) opiate antagonists, for example, antagonists, for example, naloxone; (109) anti-glaucoma agents, for example, timolol; (110) mitotic anti-glaucoma agents, for example, pilocarpine; (111) ophthalmic aminoglycoside anti-infectives, for example, gentamicin, neomycin, and tobramycin; (112) ophthalmic quinolone anti-infectives, for example, ciprofloxacin, norfloxacin, and ofloxacin; (113) ophthalmic corticosteroid anti-inflammatory drugs. agents, such as dexamethasone and prednisolone; (114) ophthalmic nonsteroidal anti-inflammatory drugs, such as diclofenac; (115) antipsychotics, such as clozapine, haloperidol, and risperidone;(116) Benzodiazepine anxiolytics, sedatives and hypnotics, for example, clonazepam, diazepam, lorazepam, oxazepam and prazepam; (117) Psychostimulants, for example, methylphenidate and pemoline; (118) For example, codeine; (119) Bronchodilators, for example, (120) Adrenergic agonist bronchodilators, for example, albuterol; (121) Respiratory corticosteroid anti-inflammatory drugs (122) anti-inflammatory agents, such as dexamethasone; (123) antidotes, such as flumazenil and naloxone; (124) heavy metal agents, such as penicillamine; (125) deterrent substance abuse agents, such as disulfiram, naltrexone, and nicotine; (126) withdrawal substance abuse agents, such as bromocriptine; (127) minerals, such as iron, calcium, and magnesium; (128) vitamin B compounds, such as cyanocobalamin (vitamin B12) and niacin (vitamin B3); (129) vitamin C compounds, such as ascorbic acid (vitamin B12); and vitamin C compounds, such as niacin (vitamin B3). acid); and (129) vitamin D, as calcitriol. ;

[0205] In addition, recombinant or cell-derived proteins may be used, such as recombinant β-glucan; bovine immunoglobulin concentrate; bovine superoxide dismutase; a preparation containing fluorouracil, epinephrine, and bovine collagen; recombinant hirudin (r-Hir), HIV-1 immunogen; recombinant human growth hormone recombinant EPO (r-EPO); genetically activated EPO (GA-EPO); recombinant human hemoglobin (r-Hb); recombinant human mecasermin (r-1GF-1); recombinant interferon alpha; lenograstim (G-CSF); olanzapine; recombinant thyroid stimulating hormone (r-TSH); and topotecan.

[0206] In addition, the peptides, proteins and other macromolecules listed below can also be used, such as interleukins 1 to 18, including mutants and analogs; interferon a, y, which can be used for cartilage regeneration, hormone releasing hormone (LHRH) and analogs, gonadotropin releasing hormone transforming growth factor (TGF); fibroblast growth factor (fibroblast growth factor) (FGF); tumor necrosis factor-α); nerve growth factor (NGF); growth hormone releasing factor (GHRF), epidermal growth factor (EGF), connective tissue activated osteogenic factor, fibroblast growth factor homologous factor (FGFHF); hepatocyte growth factor (HGF); insulin growth factor (IGF); invasion inhibitory factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-ay-globulin; superoxide dismutase (SOD); and complement factors, and biologically active analogs, fragments and derivatives of these factors (eg, growth factors).

[0207] Members of the transforming growth factor (TGF) supergene family are multifunctional regulatory proteins that can be incorporated into polymer matrices. Members of the TGF supergene family include β-transforming growth factors (e.g., TGF-131, TGF-132, TGF-133); bone morphogenetic proteins (e.g., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (e.g., fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)) (e.g., inhibin A, inhibin B), growth differentiation factors (e.g., GDF-1); and activins (e.g., activin A, activin B, activin AB). Growth factors can be isolated from natural or natural sources, such as from mammalian cells, or can be prepared by synthetic methods, such as by recombinant DNA technology or various chemical processes. In addition, analogs, fragments or derivatives of these factors may be used as long as they exhibit at least some of the biological activity of the native molecule. For example, analogs can be prepared by expressing genes altered by site-specific mutagenesis or other genetic engineering techniques.

[0208] The methods, pharmaceutical compositions and compounds disclosed herein can be combined to provide one, two or more compounds disclosed herein, optionally with, for example, maribavir, foscarnet, GCV and / or letermovir.

[0209] Various forms of bioactive agents can be used, including but not limited to uncharged molecules, molecular complexes, salts, ethers, esters, amides, prodrug forms, etc., which are bioactivated when implanted, injected or otherwise placed in a subject.

[0210] The following examples are included to guide those of ordinary skill in the art to practice representative embodiments of the disclosed subject matter. Based on the present disclosure and the general skill level in the art, it will be understood by those skilled in the art that the following examples are only for illustration, and that a variety of changes, modifications, and variations may be employed without departing from the scope of the present disclosure. The following synthesis descriptions and specific examples are only for illustrative purposes and should not be construed as limiting in any way the compounds of the present disclosure prepared by other methods. Example

[0211] Example 1

[0212] Materials and methods

[0213] Compounds: These compounds were identified by high throughput screening (HTS) and then validated using multiple HCMV strains, multiple antiviral assays, and novel compound preparation. In addition to the compounds obtained from the library used in the screen, NFU1827 was synthesized in the Department of Biochemistry, Program in Chemical Biology at the Medical College of Wisconsin, MLS8091 was purchased from Princeton Biomolecular Research, Inc., MLS8969 was purchased from ChemBridge, San Diego, CA, MLS8554 was purchased from InterBioScreen STOCK1N-59867, and NCGC2955 was synthesized by the Chemistry Core, Johns Hopkins University School of Medicine. The purity of all synthesized and purchased compounds used in bioassays was determined by HPLC using a Phenomenex Luna C 18 3.0x75mm column with a 7 minute gradient of 4-100% ACN in H2O containing 0.05% v / v TFA or a Higgins Analytical, Inc. Targa C 18 5μm 4.6x150mm column with a 30 minute gradient of 0-100% ACN in H2O containing 0.01% TFA and absorbance detection at 254nm or evaporative light scattering detection. NMR and MS data for all compounds used in bioassays were consistent with their structures and the purity determined by HPLC was >95%.

[0214] Compounds were dissolved in dimethyl sulfoxide (DMSO) and 50 and 10 mM stock solutions were stored at -80° C. Ganciclovir was purchased from Sigma Aldrich (St. Louis, MO) and a 10 mM stock solution was prepared in ddH 2 O.

[0215] Synthesis of NCGC2955: tert-butyl 4-(isopropylcarbamoyl)piperidine-1-carboxylate (1). 35N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (3.46g, 18.0mmol, 1.05 equivalents), 1-hydroxybenzotriazole monohydrate (2.83g, 18.5mmol, 1.075 equivalents) and isopropylamine (5.1mL, 60.15mmol, 3.5 equivalents) were added to a solution of CH2Cl2 (39mL) (3.94g, 17.2mmol, 1.0 equivalents). After stirring at room temperature for 21h, the reaction was diluted with CH2Cl2 and washed with 5% HCl aqueous solution. The aqueous layer was extracted with CH2Cl2 (3x20mL). The organic layers were combined, dried over anhydrous MgSO4, and concentrated in vacuo. Purified by flash chromatography (0 to 100% EtOAc / hexane) to provide 2.95g solid compound 1 in a yield of 63%. 1 H NMR (500MHz, CDCl3) δ5.28 (d, J=6.45Hz, lH), 4.01-4.25 (m, 3H), 2.73 (br.s., 2H), 2.16 (tt, J=3.69, 11. 63Hz, lH), 1.79 (d, J = 11.32Hz, 2H), 1.61 (dq, J = 4.24, 12.42Hz, 2H), 1.45 (s, 9H), 1.14 (d, J = 6.60Hz, 6H).

[0216] N-isopropylpiperidine-4-carboxamide trifluoroacetate (2). Triisopropylsilane (250 μL) and trifluoroacetic acid (3 mL) were added to a solution of compound 1 (2.95 g, 10.9 mmol) in CH2Cl2 (5 mL) at room temperature. After stirring at room temperature for 24 hours, the volatiles were removed in vacuo to provide a viscous slurry that was used without further purification. 1H NMR (500 MHz, CDC13) δ 4.06 (qd, J = 6.86, 13.38 Hz, 1H), 3.57 (d, J = 12.89 Hz, 2H), 3.10 (br. s., 2H), 2.45-2.63 (m, 1H), 2.05-2.16 (m, 4H), 1.18 (d, J = 6.60 Hz, 6H).

[0217] 4-(4-Chlorobenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid methyl ester (3). To 4H-thieno[3,2-b]pyrrole-5-carboxylic acid methyl ester in anhydrous DMF (10 mL) was added at room temperature. 364-chlorobenzyl bromide (1.97g, 9.59mmol, 1.2 equivalents) was added in a suspension of (1.44g, 7.99mmol, 1.0 equivalents) and cesium carbonate (3.91g, 12.0mmol, 1.5 equivalents) at once. After stirring at room temperature until completion (about 4 hours), the reaction was diluted with water and extracted with EtOAc (3x20 mL). The organic layers were combined, washed with brine, dried over anhydrous MgSO4, and concentrated in a vacuum. Purified by flash chromatography (3m 0%, 6m gradient 0 to 50%, 2m 50% EtOAc / hexane), 2.40g of methyl ester 3 of light yellow solid was provided in a yield of 98%. lHNMR (500MHz, CDC13) δ7.33 (d, J = 5.34Hz, 1H), 7.21-7.29 (m, 3H), 7.05 (d, J = 8.17Hz, 2H), 6.85 (d, J = 5.50Hz, lH), 5.71 (s, 2H), 3.83 (s, 3H).

[0218] 4-(4-Chlorobenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (4). To a mixture of the methyl ester 3 (3.58 g, 11.7 mmol, 1.0 equiv) in THF (1252 mL) and MeOH (12 mL) was added a solution of lithium hydroxide monohydrate in water (12 mL) in one portion. After 2 days of rapid magnetic stirring at room temperature, the starting material was consumed and the organic solvent was removed in vacuo. The remaining aqueous solution was diluted with water (about 20 mL) and the pH was adjusted to pH = 1 with concentrated aqueous HCl under strong magnetic stirring. A precipitate formed during the acidification and was collected by vacuum filtration, washed with water and dried under vacuum to provide 3.34 g of compound 4 in 98% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.57 (br. s., 1H), 7.56 (d, J = 5.19 Hz, 1H), 7.29-7.43 (m, J = 8.49 Hz, 2H), 7.17-7.29 (m, 2H), 7.05-7.17 (m, J = 8.49 Hz, 2H), 5.76 (s, 2H).

[0219] 1-(4-(4-chlorobenzyl)-4H-thieno[3-b]pyrrole-5-carbonyl)-N-isopropylpiperidine-4-carboxamide (NCGC2955). To a mixture of compound 4 (51.9 mg, 0.177 mmol, 1.0 equiv) in CH2Cl2 (1.7 mL) was added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (37.5 mg, 0.19 mmol, 1.1 equiv), 1-hydroxybenzotriazole monohydrate (31.0 mg, 0.204 mmol, 1.15 equiv) and diisopropylethylamine (123 μL, 0.71 mmol, 4 equiv). After stirring at room temperature for 5 minutes, TFA salt 2 (60.7 mg, 0.21 mmol, 1.2 equiv) was added as a solution in CH2Cl2 (0.2 mL, plus 0.2 mL rinse) and the reaction was stirred at room temperature. After 24 hours, the reaction was diluted with CH2Cl2 and washed with 5% HCl aqueous solution. The aqueous layer was extracted with EtOAc (3x10mL). The organic layers were combined, dried over anhydrous MgSO4, and concentrated in vacuo. Purified by flash chromatography (0 to 100% EtOAc / hexane), 48.9 mg of white solid NCGC2955 was provided in a yield of 62%. 1 H NMR (500MHz, CDCl3) δ7.24(d,J=8.33Hz,2H),7.18(d,J=5.19Hz,lH),7.08(d,J=8.33Hz ,2H),6.83(d,J=5.19Hz,lH),6.58(s,lH),5.43(s,2H),5.19(d,J=8.02Hz,lH),4.44(d ,J=12.89Hz,2H),4.08(qd,J=6.77,13.34Hz,lH),2.92(t,J=12.34Hz,2H),2.24(tt,J= 3.62, 11.08Hz, lH), 1.80 (d, J = 14.93Hz, 2H), 1.43-1.63 (m, 6H), 1.15 (d, J = 6.60Hz, 6H).

[0220] Synthesis of NFU1827: 4-(4-methoxyphenyl)-7-thia-2,5-diazatricyclo[6.4.0.02,6]dodecan-1(8),3,5,9,11-pentaene-10-carboxylic acid (5). 2-Aminobenzothiazole-6-carboxylic acid (2.0 g, 10.30 mmol, 1.0 equiv) and 2-bromo-4'-methoxyacetophenone (2.60 g, 11.33 mmol, 1.1 equiv) were charged into a pressure bottle and suspended in 2-methoxyethanol (50 mL). The suspension was heated to 40 °C for 120 h and at 140 °C for 19 h. The reaction was allowed to cool to room temperature and concentrated in vacuo. The crude compound was purified by flash column chromatography (5 to 75% EtOAc / hexanes) to provide 1.83 g of compound 6 in 55% yield. 1 H NMR (500MHz, DMSO-d6) δ13.17 (broads, lH), 8.69 (s, lH), 8.63 (d, J = 1.2Hz, lH), 8.10 (dd, J = 1. 6, 8.3Hz, lH), 8.01 (d, J = 8.4Hz, lH), 7.78 (d, J = 8.7Hz, lH), 6.99 (d, J = 8.8Hz, lH), 3.77 (s, 3H).

[0221] N-[3-(4-ethylpiperazin-1-yl)propyl]-4-(4-methoxyphenyl)-7-thia-2,5-diazatricyclo[6.4.0.02,6]dodecan-1(8),3,5,9,11-pentaene-10-carboxamide (NFU1827). Compound 5 (100 mg, 0.308 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (118.2 mg, 0.617 mmol) and 1-hydroxybenzotriazole hydrate (83.3 mg, 0.617 mmol) were dissolved in DMF and stirred at room temperature for 1 hour. The reaction was then treated with 3-(4-ethylpiperazin-1-yl)propan-1-amine (0.2269 mL, 211.2 mg, 1.23 mmol) and stirred at room temperature for 18 hours. The reaction was then poured into H2O (20 mL). It was extracted with EtOAc (3x20mL). The combined extracts were washed with H2O (4x20mL) and then with brine (1x20mL). The organic layer was dried (Na2SO4), filtered, and the solvent was removed in vacuo. The crude compound was purified by flash column chromatography using EtOAc / hexanes (90:10 to 0:100 gradient elution) to provide 60mg NFU1827 in 41% yield. lH NMR (500MHz, CDCl3) δ8.71(s,lH),8.21(s,lH),7.99(d,J=8.4Hz,lH),7.91(s,lH),7.80(d,J=8.7Hz,2H),7.62(d,J=8.4Hz,lH),6.96(d,J=8.7 Hz, 2H), 3.85 (s, 3H), 3.60 (q, J = 5.2Hz, 2H), 2.63 (t, J = 5.5Hz, 2H), 2.46 (q, J = 7.3Hz, 2H), 1.82 (m, 2H), 1.78 (broads, 8H), 1.10 (t, J = 7.2Hz, 3H). 13CNMR(126MHz, CDCl3)δ162.72,156.34,145.14,130.89,128.49,127.24,123.49,123.25,122.78,120.2 6,111.13,109.09,102.92,57.32,55.69,52.27,50.25,49.91,49.39,38.42,26.62,20.55,17.98,11.12.C 26 H 31 N5O2S[M+H] + HRMS (ESI + )m / z calculated as 478.2272, found as 478.2271.

[0222] Cells: Human foreskin fibroblasts (HFFs), passage 12-16; (ATCC, CRL-2088) were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco, Carlsbad, CA) containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. Mouse embryonic fibroblasts (MEF, ATCC, CRL-1658) were used to infect mice with CMV. Vero cells were obtained from the laboratory of Dr. Gary Hayward at the Johns Hopkins University School of Medicine.

[0223] Cytotoxicity assay: MTT assay was performed as described in the manufacturer's instructions (Sigma-Aldrich). Uninfected cells were treated with compounds for 72 h or 10 days (same time points for antiviral assays), and 20 μL / well of MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, 5 mg / mL in phosphate-buffered saline (PBS) solution] was added to each well. After shaking at 150 rpm for 5 min, the plate was incubated at 37°C for 2-3 h. The yellow solution was converted to dark blue formazan by mitochondrial dehydrogenases of living cells. The quantification of formazan was performed by measuring the absorbance at 560 nm.

[0224] Viral and antiviral assays: A recombinant pp28-luciferase Towne HCMV strain expressing luciferase under the control of the UL99 (pp28) late promoter was reported, providing a highly sensitive and reproducible reporter for drug screening. 20 A GCV-resistant HCMV pp28 luciferase containing the C607Y mutation in UL97 was reported. 37 . HCMV TB40 strain was obtained from ATCC (VR-1578). Clinical isolates of human herpesvirus type 1 and 2 (HSV1, HSV2) were collected from the Johns Hopkins Microbiology Laboratory without identifiers that could link them to specific patients. Mouse CMV (MCMV) Smith strain (ATCC VR-1399) was used for MEF infection.

[0225] Luciferase activity: Cell lysates were collected at 72 hpi and luciferase activity was measured using the Glomax Multi+ Assay System (Promega, Madison, WI) as described previously. 20 .

[0226] Plaque reduction assay: HFFs were seeded into 12-well plates (2x10 5Cells / well) were plated and infected with HCMV TB40 at approximately 100 plaques / well. After 90 minutes, the medium was aspirated and DMEM containing 0.5% carboxymethylcellulose (CMC), 4% fetal bovine serum (FBS) and drugs was added to two replicate wells. After 10-12 days of incubation at 37°C, the overlay was removed and plaques were counted after crystal violet staining. For HSV1 and HSV2 replication in Vero cells, the adsorption time was 60 minutes and plaques were counted after 36 hours. Plaque assays in MEFs were completed 3 days later.

[0227] Inhibition of EBV replication: Akata cells (0.5x10 cells / well plate) latently infected with EBV were induced with 100 μg / ml goat anti-human IgG (Sigma). 6 After adding human IgG, the compounds and GCV control were immediately added to three wells. Cells were collected for DNA quantification 48 hours after lytic induction. Cellular DNA was purified using Wizard SV Genomic Kit (Promega, Madison, Wisconsin). The effects of these compounds on EBV lytic induction were measured by EBV DNA polymerase gene BALF5 38 Real-time PCR analysis was performed.

[0228] Addition and removal assays: These assays were performed to identify the stage during HCMV replication at which compounds have the highest activity. In the addition group, compounds were added to infected HFFs at 0, 6, 24, and 48 hpi, and luciferase activity was measured at 72 hpi. In the removal group, compounds were added immediately after viral infection and then removed after 0, 6, 24, and 48 hours; luciferase was measured at 72 hpi.

[0229] DNA isolation and real-time quantification (qPCR): Total DNA was isolated from uninfected control and HCMV-infected HFFs using the Wizard SV Genomic DNA Isolation Kit (Promega, Madison, WI). To determine viral load in the supernatant, total DNA was isolated from the supernatant using automated DNA extraction on a BioRobot M48 instrument (Qiagen, Valencia, CA). A real-time PCR assay targeting 151 bp of the highly conserved US 17 region from the CMV genome was used. 39 The primers and probes for US 17 are:

[0230] Forward 5'-GCGTGCTTTTTAGCCTCTGCA-3' (SEQ ID NO: 1),

[0231] Reverse 5'-AAAAGTTTGTGCCCCAACGGTA-3' (SEQ ID NO: 2), and

[0232] US17 probe FAM 5'-TGATCGGGCGTTATCGCGTTCT-3' (SEQ ID NO: 3).

[0233] HCMV entry and indirect immunofluorescence assay: Inhibition of HCMV entry was determined using MLS8969, GCV, and heparin controls. Compounds were diluted in serum-free medium and added to HFFs seeded on slides 24 hours prior to infection. After 2 hours of infection (MOI 1 or 0.1 PFU / cell), cells were fixed with 100% chilled methanol and blocked for 1 hour with phosphate-buffered saline (PBS), 5% serum, 0.3% Triton X-100. Cells were then incubated with 1:50 mouse monoclonal anti-pp65 antibody (Vector Laboratories, Burlingame, CA) for 1 hour at 37°C in a humidified chamber, washed three times with TBST (0.1%), incubated with 1:500 rhodamine-conjugated anti-mouse IgG (Sigma) for 1 hour at 37°C in a humidified chamber, and washed with TBST (0.1%). A drop of immersion oil containing DAPI (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the slides prior to visualization with a Zeiss ZI fluorescence microscope. Images were taken at 40× magnification.

[0234] SDS polyacrylamide gel electrophoresis and immunoblot analysis: Cell lysates containing equal amounts of protein were mixed with an equal volume of sample buffer (125 mM Tris-HCL, pH 6.8, 4% SDS, 20% glycerol, and 5% β-mercaptoethanol) and boiled at 100°C for 10 min. Denatured proteins were resolved in Tris-tris-glycine polyacrylamide gels (8-10%) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA) by electroblotting. The membranes were incubated in blocking solution [5% w / v nonfat dry milk and PBS containing 0.1% Tween-20 (PBST)] for 1 h, washed with PBST, and incubated with primary antibodies overnight at 4°C. The membranes were washed with PBST and incubated with horseradish peroxidase-conjugated secondary antibodies in PBST for 1 h at room temperature. After washing with PBST, protein bands were visualized by chemiluminescence using SuperSignal WestDura and Pico reagents (Pierce Chemical, Rockford, IL).

[0235] Antibodies: The following antibodies were used - mouse monoclonal anti-HCMV IE1 and IE2 (MAB810, Millipore, Billerica, MA), mouse monoclonal anti-HCMV UL83 (pp65, Vector Laboratories, Burlingame, CA), mouse monoclonal anti-HCMV UL44, mouse monoclonal anti-HCMV UL84, and mouse anti-actin anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG was from GE Healthcare (Waukesha, WI). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was from Cell Signaling (Beverly, MA).

[0236] Drug combinations and analyses: These experiments were performed as previously reported 40 The combination of GCV and each compound was tested using pp28 luciferase. In brief, 2x10 6 HFF / plate were seeded in 96-well plates and infected with the pp28-luciferase Towne HCMV strain (MOI = 1). First, a dose-response curve was generated for each drug to determine its EC 50 The drug is then 50 Two-fold combinations of 50 μg / ml were diluted in DMEM with 4% FBS, then serially diluted and added together after infection. Luciferase activity of the combination and each drug alone was quantified at 72 hpi. The combination of levofloxacin and each novel compound was tested by plaque assay using TB40. The same principle applied to the test of the drug combination effect, and plaques were counted at 8 dpi. The effect of each drug combination on pp28 luciferase activity and plaque reduction was calculated using the Bliss model. In this model, the drug combination represents the product of two probabilistic independent events, as described in the following equation 41 :

[0237]

[0238] where D is the drug concentration, m is the slope, and EC 50 Is the effective concentration that causes 50% viral inhibition. The combined effect of two inhibitors (Fu, unaffected fraction) is calculated as the product of the individual effects of the two inhibitors Fu1 and Fu2. If the ratio of the observed inhibition fold divided by the expected inhibition fold is greater than 1, the compound is synergistic. If the ratio is less than 1, the combination is considered to be antagonistic, and if it is equal to 1, the combination is additive.

[0239] Statistical Analysis: Student's t-test was performed using Sigmaplot (Systat Software, San Jose, CA) and GraphPad Prism (GraphPad-Software, La Jolla, CA). All sample groups were compared to the control group using one-way ANOVA. P values ​​were adjusted for multiple comparisons. In all figures, the following conventions were followed. * indicates p value < 0.05, ** indicates p value < 0.01, and *** indicates p value < 0.001. Curve Fitting Toolbox, MATLAB software (v7.10), MathWorks (Natick, MA) was used to determine EC by four-parameter logistic regression 50 and CC 50 value.

[0240] Example 2

[0241] For high-throughput screening of HCMV inhibitors: pp28-HCMV luciferase has been shown to provide highly reproducible and sensitive results for inhibition of HCMV with compounds and was used to screen the MLSMR collection of 370,000 compounds, the NCGC Diversity Collection of 65,000 compounds, and the NCGC Drug Collection of approved and investigational drugs. Screens were performed using 2 compound doses plated pre-infection and lysates were assayed for luciferase activity at 72 hours post-infection (hpi). Compound dose-response curves were calculated using published algorithms 21 , a total of 2215 HCMV luciferase inhibitors were selected. A secondary screen was performed to remove compounds that showed toxicity in human foreskin fibroblasts (HFF), resulting in 847 confirmed non-toxic HCMV inhibitors ( Figure 1 ).

[0242] HCMV inhibition was measured by luciferase activity of pp28 recombinant Towne and pp28 GCV Towne strains of HCMV (72 hpi) and plaque reduction using HCMV Towne or TB40 (10 days). Viral protein expression was determined by Western blot analysis. A colorimetric MTT assay was performed in uninfected HFFs to determine cytotoxicity in parallel with the infectivity assay, i.e., at 72 h and 10 days, and is expressed as CC 50 All five compounds were active against all HCMV strains used at sub-μM to low μM concentrations. Based on plaque reduction, MLS8969 had the best activity against HCMV, followed by MLS8091, MLS8554, NCGC2955, and NFU1827 (Table 1).

[0243] Table 1. Anti-HCMV activity, toxicity and slope of the five compounds against different HCMV strains

[0244]

[0245] * = pretreatment followed by plaque reduction test

[0246] ** = Plaque reduction test

[0247] The slope parameter (analogous to the Hill coefficient) was calculated for each compound. A slope of 1 indicates one binding site or non-cooperativity. Positive cooperativity exists when binding at one site increases the affinity of ligand binding at another site (slope > 1). If binding at one site decreases the affinity of the ligand at another site, the compound exhibits negative cooperativity (slope < 1). For other chronic viral infections, such as HIV, the slope was found to be not only an important factor in distinguishing drug classes and known mechanisms of action, but also to contribute to reduced antiviral activity and even EC values ​​for resistant viral mutants 22, 23. 50 Based on the plaque reduction assay, all compounds except MLS8969 had a slope of 1.

[0248] Example 3

[0249] Inhibition of other herpes viruses: The five compounds were tested for inhibition of herpes simplex virus 1 and 2 (HSV1, HSV2), Epstein-Barr virus (EBV), and mouse CMV (MCMV). Plaque reduction assays were used for HSV1, HSV2, and MCMV. Inhibition of lytic EBV replication in AKATA cells was measured by real-time PCR after IgG induction. Only MLS8554 was active against HS1I, HSV2, and MCMV (Table 2).

[0250] Table 2. Activity of five compounds against other herpes viruses; NA - no activity; CI - clinical isolate

[0251]

[0252] NCGC2955 is active against MCMV (EC 50 1 μM, Table 2), but toxicity to MEFs was observed at 10 μM and higher concentrations. NFU1827 showed activity against EBV (EC50 - 1.92 ± 0.1 μM). The other compounds had specific activity against HCMV, but did not inhibit HSV1 or MCMV.

[0253] Example 4

[0254] Time of HCMV inhibition: Additional addition and removal tests were performed. Compounds were added or removed at 0, 6, 12, 24, 36 and 48 hpi, and luciferase activity was measured at 72 hpi. Based on these tests, each compound had a specific maximum activity time, which was recorded as follows based on the time of maximum viral inhibition.

[0255] Example 5

[0256] Inhibition of HCMV entry: Compound MLS8969 was first tested using a luciferase assay at 72 hpi after infection at an MOI of 1 PFU / cell, but minimal reduction in luciferase enzyme activity was observed ( Figure 3 A). However, plaque assay showed good anti-HCMV activity of MLS8969 ( Figure 3 B).

[0257] Because in the high-throughput screening, HFFs were treated with compounds before infection (pretreatment), and our plaque assay (addition of MLS8969 after infection) showed EC 50 0.12 ± 0.02 μM (Table 1), we suspect that the lack of inhibition of luciferase activity may represent an MOI-dependent phenomenon, thus suggesting entry inhibition 24 Therefore, the experiments were performed using MOIs of 0.01, 0.1, and 1 PFU / cell ( Figure 3 C) and MLS8969 was added before infection. MOI-dependent inhibition of luciferase activity was observed ( Figure 3 C). Pretreatment with MLS8969 also inhibited TB40 and GCV-resistant HCMV Towne in the plaque reduction assay ( Figure 3 D, 3E). Western blot analysis confirmed these data and showed no inhibition of viral protein expression at post- or pre-treatment at an MOI of 1 PFU / cell, whereas pre-treatment after infection at a low MOI (0.01) resulted in decreased IE1 / 2, pp65, UL84, and UL44 protein levels ( Figure 3 F). Further evidence that MLS8969 is an entry inhibitor was obtained by indirect immunofluorescence assay (IFA) of HCMV pp65 ( Figure 4 MLS8969 inhibited HCMV entry only at an MOI of 0.1; GCV did not affect pp65 nuclear staining regardless of the MOI ( Figure 4 ). Heparin (positive control) inhibits HCMV entry.

[0258] Example 6

[0259] Inhibitors of the immediate early-to-early stage of HCMV replication: Two compounds showed anti-HCMV activity from 0 to 24 hpi (NFU1827 and MLS8554). NFU1827 was active only against HCMV and GCV-R HCMV, EC 50 0.85±0.1 and 0.84±0.1 μM, respectively (Table 1, Figure 5 A and B), but had no activity against MCMV or HSV1 (Table 2). Additional addition and removal experiments indicated an early period of activity. When added to infected cells 24 hours later, it lost its ability to inhibit HCMV. Removal of NFU1827 after 6 hours already achieved significant HCMV inhibition ( Figure 5 C&D). NFU1827 reduced the levels of IE1 and IE2 ( Figure 5 E). Based on its immediate early activity, viral DNA replication and DNA yield in the supernatant were significantly reduced ( Figure 5 F&G).

[0260] MLS8554 was active in all antiviral assays against HCMV, GCV-R HCMV, MCMV, and HSV1 ( Figure 6 , Tables 1 and 2). Its maximum activity was determined at the early time after infection. When added at or after 24 hpi, its activity was significantly reduced, and when removed at 6 h, HCMV inhibition was nearly complete ( Figure 6 C&D). Western blot analysis at 72 hpi showed decreased expression of IE2, UL44, UL84, and pp65 ( Figure 6 E). Compared with NFU1827, which reduces both IE1 and IE2, MLS8554 mainly reduces IE2 expression. Viral DNA replication and DNA supernatant yield were both reduced ( Figure 6 F&G).

[0261] Example 7

[0262] MLS8091 is an inhibitor of the early-to-late stages of HCMV replication: it shows a dose-response against HCMV and GCV-resistant HCMV, and the EC 50 0.39±0.5 and 0.26±0.02 μM, respectively (Table 1, Figure 7 ), but had no activity against HSV1 or MCMV (Table 2). In the addition test, its activity time overlapped with that of GCV, but in the removal test, MLS8091 showed an earlier effect ( Figure 7 C&D). Inhibition of all viral proteins was observed at 72 hpi ( Figure 7E). Although the effect on DNA yield was not significantly different from that of GCV, the inhibition of viral DNA replication was greater than that of GCV ( Figure 7 F and 7G).

[0263] NCGC2955 inhibits HCMV and GCV-R HCMV ( Figure 8 A and 8B). For this compound, the plaque assay is preferred because it does not show the full dose response using the luciferase assay. In the addition assay, the activity time of NCGC2955 overlaps with that of GCV, but in the removal assay, NCGC2955 shows longer activity than GCV ( Figure 8 C&D). At 72 hpi, there was a significant decrease in the expression of viral proteins including IE1 and IE2 ( Figure 8 E). Despite inhibition of viral progeny and suppression of viral protein expression by plaque assay, NCGC2955 had little effect on viral DNA replication and viral DNA yield ( Figure 8 F and 8G), suggesting repression by a mechanism that does not involve the DNA replication machinery.

[0264] Example 8

[0265] Drug combination mode with GCV and Letermovir: The effect of each compound alone or in combination with GCV or Letermovir was tested in HCMV-infected HFF. For all five agents, no significant differences were observed with GCV ( Fig. 9 ) or letermovir ( Fig.10 ) additive or slightly synergistic effects. The ratio of the observed inhibition fold divided by the expected inhibition fold > 1 was considered a drug synergistic effect, < 1 was considered a drug antagonistic effect, and = 1 was considered an additive effect. The Bliss coefficients of the drug combinations are shown in Table 3. For letermovir and its combination with each of the five compounds, EC 50 Determined based on plaque assay. The combination of GCV and 2955 was tested by plaque assay. All other combinations and Bliss coefficients were determined using pp28-luciferase. All Bliss coefficients were close to 1, indicating an additive effect between the compounds.

[0266] Table 3. Bliss coefficients of drug combinations

[0267]

[0268] Example 9

[0269] Discussion and conclusions: We report the results of the largest HTS to date against HCMV inhibitors using our pp28-luciferase HCMV reporter virus. This reporter virus showed high sensitivity and reproducibility in this screen. 20The advantage is that pp28 encoded by HCMV is a true late gene, so its activation represents a nearly complete cycle of HCMV replication.

[0270] Screening campaigns may result in false positive hits that require careful validation. Our combinatorial approach using multiple HCMV strains and multiple detailed antiviral assays led to the identification and characterization of five different hits, each with its own unique features. For the five hits, MLS8969 inhibited HCMV entry, MLS8554 and NFU1827 inhibited the immediate early phase of HCMV-replication (but the effects on other herpesviruses were different from each other), MLS8091 and NCGC2955 were active in the early and late phases, but NCGC2955 did not strongly inhibit viral DNA replication. All five compounds showed additive or slightly synergistic effects when combined with GCV or temovir. No antagonism was found in all drug combination experiments. In addition, the five compounds were active against GCV-resistant HCMV, suggesting that their mechanism is different from the viral DNA polymerase. Future studies should provide insight into the mechanism of action of the five compounds. Based on the data presented here, we expect that each compound has a unique mechanism of HCMV inhibition.

[0271] Screening was performed using a library containing a limited number of small molecules, different HCMV strains, cell types and treatment durations. Based on the fluorescence intensity, multiple kinase inhibitors blocked HCMV replication. Three recombinant HCMV strains carrying enhanced yellow fluorescent protein (EYFP) fused to immediate early viral protein 2 (IE-2), ppUL32 (ppl50) and ppUL83 (pp65) were applied to the cellular kinase library (with 80 compounds) as a pilot screen. 25 The screen used a fixed drug concentration of 10 μM at the time of infection and a limited number of hits were found without overt cytotoxicity. Reporter cell lines have been generated to screen anti-HCMV compounds 26、27 In one approach using a luciferase reporter cell line, the promoter provides IE protein activation; therefore, compounds that inhibit HCMV at later stages of infection cannot be evaluated. 27 The Gray Kinase Inhibitor library of 187 compounds was successfully screened with HCMV strain AD169 and cells were stained with antibodies to detect the HCMV antigen pp28. Three kinase inhibitors were identified as inhibitors of IE2 production 28 A recent drug repurposing screen used a spectral collection of 2,560 compounds from Micro Discovery System 29,30Mercorelli et al. used a mechanism-based screen designed to identify compounds that interfere with the transactivation activity of IE2. The phenotypic assay measured the expression of proteins fused to IE2. 31 or UL99 32 The main success of HTS lies in the expression of EGFP from the compound library 33 In a screening study, letermovir (AIC246) was identified, which was subsequently shown to have a novel mechanism of action for inhibiting HCMV. 34 .

[0272] In summary, our study provides several novel compounds that inhibit HCMV replication at sub-μM to low-μM concentrations. Each of these compounds is a good candidate for future mechanistic studies and drug development.

[0273] All references cited herein (including publications, patent applications and patents) are incorporated herein by reference to the same extent as if each document were individually introduced and expressly indicated to be incorporated by reference and fully set forth herein. The terms "one" and "a kind of" and "the" and similar expressions used in the context (especially in the context of the appended claims) to describe the disclosure herein should be interpreted as covering the singular and plural, unless otherwise specified or the context clearly indicates otherwise. The terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including, but not limited to"), unless otherwise specified. Unless otherwise specified herein, the reference to the numerical range herein is only a simple representation method, each independent value falling within the range is individually represented, and each independent value is included in the specification, as if stated separately herein. All methods described herein can be performed in any suitable order, unless otherwise specified herein or clearly contradictory to the context. Unless otherwise specified, the use of any and all embodiments and examples or exemplary language (e.g., "for example / such as / such as") provided herein is only to better illustrate the disclosure herein, rather than to limit the scope of the disclosure. No language in the specification should be construed as indicating a non-claimed element as essential to the practice of the disclosure herein.

[0274] This disclosure includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. Furthermore, unless otherwise indicated herein or clearly contradicted by context, this disclosure encompasses any combination of all possible variations of the above elements.

[0275] References

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Claims

1. Use of the following compounds or their salts in the preparation of a medicament for inhibiting human herpes virus in a subject in need thereof: in, The human herpes virus is human herpes virus type 5, namely HHV-5.

2. Use of the following compounds or their salts in the preparation of a medicament for inhibiting human herpes virus in a subject in need thereof: in, The human herpes virus is human herpes virus type 5, namely HHV-5.

3. Use of the following compounds or their salts in the preparation of a medicament for inhibiting human herpes virus in a subject in need thereof: in, The human herpes virus is human herpes virus type 5 (HHV-5), human herpes virus type 1 (HHV-1), human herpes virus type 2 (HHV-2), or a combination thereof.

4. Use of the following compounds or their salts in the preparation of a medicament for inhibiting human herpes virus in a subject in need thereof: in, The human herpes virus is human herpes virus type 5, namely HHV-5.

5. Use of the following compounds or their salts in the preparation of a medicament for inhibiting human herpes virus in a subject in need thereof: in, The human herpes virus is human herpes virus type 5 (HHV-5), human herpes virus type 4 (HHV-4), or a combination thereof.

6. The use according to any one of claims 1 to 5, wherein: HHV-5 is resistant to ganciclovir.

7. The use according to any one of claims 1 to 5, wherein the medicament is also administered together with more than one biologically active agent.

8. The use according to any one of claims 1 to 5, wherein the drug is also administered together with ganciclovir or letermovir.

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