Use of a nourseothricin in the manufacture of a medicament for the treatment of herpes viruses

Abstract

The present application provides the use of nourseothricin in the preparation of anti-herpes virus drugs, especially against varicella-zoster virus, herpes simplex virus and human cytomegalovirus, belonging to the field of pharmacy. In addition, the present application relates to nourseothricin which can effectively inhibit the formation of plaques of these viruses and significantly reduce the production of progeny viruses. Nourseothricin exhibits significant inhibition on the growth of varicella-zoster virus, herpes simplex virus and human cytomegalovirus, so it can not only be used to prepare anti-varicella-zoster virus drugs, but also be used to prepare anti-herpes simplex virus and human cytomegalovirus drugs, and treat diseases related to herpes virus infection.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the use of norsinolate in the preparation of antiherpes virus drugs. Background Technology

[0002] Herpesviruses are a class of DNA viruses, including many types, among which common ones include varicella-zoster virus (VZV), herpes simplex virus (HSV), and human cytomegalovirus (HCMV). These viruses are widespread in humans and can cause a variety of infectious diseases.

[0003] Varicella-zoster virus (VZV) infection causes chickenpox and shingles. Chickenpox primarily occurs in children and is usually self-limiting, but it can cause serious complications in adults or individuals with compromised immune systems. After recovery from chickenpox, VZV can remain latent in the dorsal root ganglia and, upon reactivation, can cause shingles, manifesting as a painful rash along the nerve distribution. Serious complications can include neuralgia, eye infections, pneumonia, and encephalitis.

[0004] Herpes simplex virus (HSV) is divided into two types: HSV-1 and HSV-2. HSV-1 mainly causes herpes around the lips (oral herpes), while HSV-2 mainly causes herpes around the genitals (genital herpes). Both viruses often exist in a latent state in the body, but under the influence of weakened immune function or other triggering factors, they can be reactivated and cause infection, leading to herpes lesions on the skin, mucous membranes, and other sites, seriously affecting the patient's quality of life.

[0005] Human cytomegalovirus (HCMV) infection can cause mononucleosis in healthy individuals. Although most infected individuals are asymptomatic, approximately 20% may experience neurological deficits. Furthermore, primary or reactivation of HCMV in individuals with compromised immune systems, such as organ transplant recipients and HIV / AIDS patients, can lead to severe infections and serious complications such as organ damage, pneumonia, and retinitis, even threatening life.

[0006] Overall, herpesvirus infection is very common in the population, and the various diseases and complications it causes impose a significant social burden. Furthermore, some studies suggest a potential link between herpesvirus infection and the development of Alzheimer's disease. Therefore, timely prevention, diagnosis, and treatment of herpesvirus infections are of paramount importance.

[0007] Currently, vaccination is the primary measure to prevent viral infections. Of all herpes viruses, only varicella-zoster virus (VZV) has a corresponding vaccine available. However, due to issues such as vaccine availability and the target population, a large number of VZV-infected individuals still exist.

[0008] Currently, there are no effective, specific treatments for individuals with herpesvirus-related diseases. Clinically, nucleoside analog antiviral drugs are primarily used for treatment. Nucleoside analog antiviral drugs are effective for both primary and recurrent herpesvirus infections, but less effective for latent infections. Early nucleoside analogs, including iododeoxyuridine, vidarabine, and trifluorothymidine, had drawbacks such as low selectivity and high cytotoxicity to normal cells. Acyclovir, with relatively low cytotoxicity, has become a first-line drug for treating herpesvirus infections. However, acyclovir has a short half-life, requiring continuous high-dose administration. High-dose administration can lead to renal failure due to its low water solubility and precipitation in the renal tubules. Furthermore, for acyclovir and similar acyclovir-like drugs, mutations in the herpesvirus thymidine kinase (TK) or DNA polymerase can lead to viral resistance, further complicating the treatment of herpesvirus infections. Ganciclovir, a first-line treatment for human cytomegalovirus (HCMV), also presents similar problems. Therefore, the search for novel, highly effective, and low-toxicity anti-herpesvirus drugs has long been a focus of the medical community.

[0009] Nourseothricin is a broad-spectrum antibiotic produced by Streptothricum. Due to the presence of the Streptothricin (STs) chemical group in its structure, it belongs to this subfamily of antibiotics. Naturally extracted nourseothricin is generally a mixture of ST-F and ST-D, exhibiting extremely broad inhibitory activity. It can inhibit the growth of prokaryotes such as bacteria, as well as eukaryotes including various yeasts, filamentous fungi, protozoa, insects, and plants. The mechanism of action of nourseothricin is through interfering with mRNA translocation, thereby inhibiting peptide chain synthesis. In addition, nourseothricin can bind to bacterial ribosomes, triggering mRNA misreading, which introduces incorrect amino acids into the peptide chain, ultimately leading to the synthesis of non-functional proteins. These proteins accumulate in the bacteria, eventually causing bacterial death.

[0010] For a long time, the clinical application of norotrione has been limited due to its cytotoxicity and nephrotoxicity. However, in the field of biotechnology, it is often used as a highly effective reagent for screening for resistance. In recent years, the molecular modification to reduce the toxicity of these early-discovered, highly toxic antibiotics and their diversified applications have gradually become a new trend. Many antibacterial drugs recently approved by the FDA are derivatives of compounds discovered decades ago, such as plazomicin (a derivative of the aminoglycoside antibiotic sisomycin), lefamulin (a semi-synthetic derivative of the antibiotic pleuromutilin), and tetracyclines eravacycline and omadacyline (third-generation tetracycline antibiotics). With in-depth research on the properties of norotrione and the elucidation of its molecular targets, there is also great potential for its molecular modification and derivative development. However, there are currently no reports or related studies on the application of norotrione in the field of antiherpesvirus treatment.

[0011] Therefore, the search for novel, highly effective, and low-toxicity anti-herpesvirus drugs is urgent and necessary. The application of norsinosin, a drug of this invention, in the field of anti-herpesvirus therapy is expected to fill the gaps in existing treatments and provide more effective treatment options for clinical practice. Summary of the Invention

[0012] This invention provides the novel use of norovirus extract in the preparation of anti-herpesvirus drugs. Studies have shown that norovirus extract effectively inhibits the formation of herpesvirus plaques and significantly reduces the production of progeny viruses. Norovirus extract exhibits significant inhibitory effects on the growth of varicella-zoster virus, herpes simplex virus, and human cytomegalovirus. Therefore, the norovirus extract of this invention can be used not only to prepare drugs against varicella-zoster virus but also drugs against herpes simplex virus and human cytomegalovirus, possessing broad-spectrum anti-herpesvirus activity and the ability to treat diseases caused by these viral infections.

[0013] In this invention, drug screening revealed that norovirus can effectively inhibit the growth and replication of varicella-zoster virus (VZV) both in vitro and in vivo, with antiviral efficacy superior to acyclovir, currently the first-line drug. Furthermore, norovirus can also inhibit the growth of herpes simplex virus HSV-1, HSV-2, and human cytomegalovirus (HCMV) in cell infection models, demonstrating broad-spectrum anti-herpesvirus activity.

[0014] Uses of the compound shown in Formula I

[0015] I. Herpes virus infection or herpes virus-related diseases

[0016] Therefore, in a first aspect, the present invention provides the use of compounds of Formula I, or pharmaceutically acceptable salts thereof, stereoisomers thereof, crystals thereof, solvates, hydrates thereof, or derivatives thereof, in the preparation of medicaments for the prevention and / or treatment of herpes virus infection or herpes virus infection-related diseases:

[0017]

[0018] In addition, the present invention provides a method for preventing and / or treating herpes virus infection or herpes virus infection-related diseases in subjects, comprising administering to a subject in need an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystal thereof, or its derivative thereof.

[0019] In some implementations, the subject is a mammal, including bovines, equines, sheep, pigs, canines, felines, rodents, primates, such as humans, mice, or monkeys.

[0020] Secondly, the present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of herpes virus infection or herpes virus infection-related diseases, said pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a crystal thereof, a solvate thereof, a hydrate thereof, or a derivative thereof:

[0021]

[0022] In some embodiments, the pharmaceutical composition further comprises one or more nucleoside analogs, such as iodothyridine, vidarabine, trifluorothymidine, acyclovir, and ganciclovir.

[0023] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a combination preparation.

[0024] In addition, the present invention provides a method for preventing and / or treating herpes virus infection or herpes virus infection-related diseases in subjects, comprising administering an effective amount of a pharmaceutical composition to a subject in need, said pharmaceutical composition comprising a compound represented by Formula I, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a crystal thereof, a solvate thereof, a hydrate thereof, or a derivative thereof.

[0025] II. Inhibits herpesvirus replication or proliferation

[0026] Thirdly, the present invention also provides the use of compounds of Formula I, or pharmaceutically acceptable salts thereof, stereoisomers thereof, crystals thereof, solvates, hydrates thereof, or derivatives thereof, in the preparation of reagents or medicaments for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells):

[0027]

[0028] In addition, the present invention provides a method for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), comprising administering to a subject in need an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates thereof, or derivatives thereof.

[0029] Fourthly, the present invention also provides the use of pharmaceutical compositions in the preparation of reagents or medicaments for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), said pharmaceutical compositions comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a crystal thereof, a solvate thereof, a hydrate thereof, or a derivative thereof:

[0030]

[0031] In some embodiments, the pharmaceutical composition further comprises one or more nucleoside analogs, such as iodothyridine, vidarabine, trifluorothymidine, acyclovir, and ganciclovir.

[0032] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a combination preparation.

[0033] In addition, the present invention provides a method for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), comprising administering an effective amount of a pharmaceutical composition to a subject in need of such action, said pharmaceutical composition comprising a compound represented by Formula I, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a crystal thereof, a solvate thereof, a hydrate thereof, or a derivative thereof.

[0034] In some implementations, the mammals include bovines, equines, sheep, suidae, canines, felines, rodents, primates, such as humans, mice, or monkeys.

[0035] In some embodiments, the herpesviruses described in the first to fourth aspects are selected from varicella-zoster virus (VZV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), gamma herpesvirus (e.g., EBV), human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7), or Kaposi's sarcoma-associated herpesvirus (KSHV) or combinations thereof.

[0036] In some embodiments, the herpesviruses described in the first to fourth aspects are selected from varicella-zoster virus (VZV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), or combinations thereof.

[0037] In some implementations, herpesvirus infection-related diseases, as described in the first or second aspect, are diseases caused by herpesvirus infection.

[0038] In some embodiments, the herpesvirus infection-related diseases are diseases caused by varicella-zoster virus (VZV), including but not limited to chickenpox, herpes zoster, keratitis, encephalitis, myelitis, pneumonia, myocarditis, etc.

[0039] In some implementations, the herpesvirus infection-related diseases are diseases caused by herpes simplex virus (HSV), including but not limited to herpes simplex, keratitis, encephalitis, myelitis, pneumonia, myocarditis, etc.

[0040] In some implementations, the herpesvirus infection-related diseases are diseases caused by human cytomegalovirus (HCMV), including but not limited to encephalitis, myelitis, pneumonia, myocarditis, hepatitis, gastroenteritis, etc.

[0041] In some embodiments, the compounds described in the first or third aspect, or the pharmaceutical compositions described in the second or fourth aspect, have at least one of the following biological activities: (i) inhibiting the proliferation of herpesviruses; (ii) inhibiting the formation of herpesvirus plaques; and (iii) inhibiting the expression of herpesvirus proteins.

[0042] In some embodiments, the compounds described in the first or third aspect, and the pharmaceutical compositions described in the second or fourth aspect, are able to effectively inhibit the growth and replication of herpes viruses (e.g., varicella-zoster virus, herpes simplex virus, human cytomegalovirus) in vitro and in vivo, with inhibition rates as high as, for example, about 80%, 90%, or 100%.

[0043] In some embodiments, the compounds described in the first or third aspect, and the pharmaceutical compositions described in the second or fourth aspect, effectively inhibit the formation of plaques by herpesviruses (e.g., varicella-zoster virus, herpes simplex virus, human cytomegalovirus) in vitro and in vivo, with inhibition rates as high as 80%, 90%, and 100%.

[0044] In some embodiments, compared to existing antiherpesvirus drugs (e.g., acyclovir), the compounds of the present invention as described in the first or third aspect, and the pharmaceutical compositions as described in the second or fourth aspect, can increase the inhibitory effect on the growth and replication of herpesvirus by, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times.

[0045] In some embodiments, compared to existing antiherpesvirus drugs (e.g., acyclovir), the compounds of the present invention as described in the first or third aspect, and the pharmaceutical compositions as described in the second or fourth aspect, can increase the inhibitory effect on herpesvirus plaque formation by, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times.

[0046] In some embodiments, the compounds as described in the first or third aspect, and the pharmaceutical compositions as described in the second or fourth aspect, have cure rates of up to, for example, 60%, 70%, 80%, 90%, or 100% for herpesvirus infection-related diseases (e.g., diseases caused by varicella-zoster virus, diseases caused by herpes simplex virus, or diseases caused by human cytomegalovirus).

[0047] Uses of compound combinations

[0048] I. Herpes virus infection or herpes virus-related diseases

[0049] Fifthly, the present invention also provides the use of the compound combination in the preparation of a medicament for the prevention and / or treatment of herpesvirus infection or herpesvirus infection-related diseases, wherein:

[0050] The compound combination comprises: compound 1 of formula I or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; compound 2 of formula II or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; and compound 3 of formula III or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof.

[0051] Compound 1 represented by Formula I is streptothricin D (C 31 H 58 N 12 O 10 ),

[0052]

[0053] Compound 2, represented by Formula II, is streptothricin F (C 19 H 34 N8O8),

[0054]

[0055] Compound 3, represented by Formula III, is streptothricin E (C 25 H 46 N 10 O9),

[0056]

[0057] In some embodiments, the mass percentage of compound 1 is 40% to 50%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%.

[0058] In some embodiments, the mass percentage of compound 1 is 40% to 45%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%.

[0059] In some embodiments, compound 1 (Streptothricin D) accounts for 40.76% by mass, compound 2 (Streptothricin F) accounts for 54.13% by mass, and compound 3 (Streptothricin E) accounts for 5.11% by mass.

[0060] In addition, the present invention provides a method for preventing and / or treating herpes virus infection or herpes virus infection-related diseases in subjects, comprising administering an effective amount of a combination of compounds to a subject in need, the combination of compounds comprising: compound 1 of formula I or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; compound 2 of formula II or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; and compound 3 of formula III or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof.

[0061] In some embodiments, the mass percentage of compound 1 is 40% to 50%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%.

[0062] In some embodiments, the mass percentage of compound 1 is 40% to 45%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%.

[0063] In some embodiments, compound 1 (Streptothricin D) accounts for 40.76% by mass, compound 2 (Streptothricin F) accounts for 54.13% by mass, and compound 3 (Streptothricin E) accounts for 5.11% by mass.

[0064] In some implementations, the subject is a mammal, including bovines, equines, sheep, pigs, canines, felines, rodents, primates, such as humans, mice, or monkeys.

[0065] In a sixth aspect, the present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of herpes virus infection or herpes virus infection-related diseases, wherein the pharmaceutical composition comprises the compound combination described in the fifth aspect.

[0066] In some embodiments, the pharmaceutical composition further comprises one or more nucleoside analogs, such as iodothyridine, vidarabine, trifluorothymidine, acyclovir, and ganciclovir.

[0067] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a combination preparation.

[0068] In some embodiments, a method for preventing and / or treating herpes virus infection or herpes virus infection-related diseases in a subject includes administering an effective amount of a pharmaceutical composition to the subject in need, said pharmaceutical composition comprising the combination of compounds described in the fifth aspect.

[0069] II. Inhibits herpesvirus replication or proliferation

[0070] In a seventh aspect, the present invention also provides the use of the compound combination in the preparation of a reagent or medicament for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), wherein:

[0071] The compound combination comprises: compound 1 of formula I or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; compound 2 of formula II or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; and compound 3 of formula III or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof.

[0072] Compound 1 represented by Formula I is streptothricin D (C 31 H 58 N 12 O 10 ),

[0073]

[0074] Compound 2, represented by Formula II, is streptothricin F (C 19 H 34 N8O8),

[0075]

[0076] Compound 3, represented by Formula III, is streptothricin E (C 25 H 46 N 10 O9),

[0077]

[0078] In some embodiments, the mass percentage of compound 1 is 40% to 50%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%.

[0079] In some embodiments, the mass percentage of compound 1 is 40% to 45%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%.

[0080] In some embodiments, compound 1 (Streptothricin D) accounts for 40.76% by mass, compound 2 (Streptothricin F) accounts for 54.13% by mass, and compound 3 (Streptothricin E) accounts for 5.11% by mass.

[0081] In addition, the present invention provides a method for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), comprising administering an effective amount of a combination of compounds to a subject in need, the combination of compounds comprising: compound 1 of formula I or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; compound 2 of formula II or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; and compound 3 of formula III or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof.

[0082] In some embodiments, the mass percentage of compound 1 is 40% to 50%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%.

[0083] In some embodiments, the mass percentage of compound 1 is 40% to 45%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%.

[0084] In some embodiments, compound 1 (Streptothricin D) accounts for 40.76% by mass, compound 2 (Streptothricin F) accounts for 54.13% by mass, and compound 3 (Streptothricin E) accounts for 5.11% by mass.

[0085] In an eighth aspect, the present invention also provides the use of a pharmaceutical composition in the preparation of a reagent or medicament for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), wherein the pharmaceutical composition comprises the compound combination described in the seventh aspect.

[0086] In some embodiments, the pharmaceutical composition further comprises one or more nucleoside analogs, such as iodothyridine, vidarabine, trifluorothymidine, acyclovir, and ganciclovir.

[0087] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a combination preparation.

[0088] In some embodiments, a method for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells) includes administering an effective amount of a pharmaceutical composition to a subject in need, said pharmaceutical composition comprising the combination of compounds described in the seventh aspect.

[0089] In some embodiments, the herpesviruses described in aspects five through eight are selected from varicella-zoster virus (VZV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), gamma herpesvirus (e.g., EBV), human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7), or Kaposi's sarcoma-associated herpesvirus (KSHV) or combinations thereof.

[0090] In some embodiments, the herpes virus is selected from varicella-zoster virus (VZV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), or a combination thereof.

[0091] In some implementations, herpesvirus infection-related diseases, as described in aspects five or six, are diseases caused by herpesvirus infection.

[0092] In some implementations, the herpesvirus infection-related diseases are diseases caused by varicella-zoster virus (VZV), including chickenpox, herpes zoster, keratitis, encephalitis, myelitis, pneumonia, myocarditis, etc.

[0093] In some implementations, the herpesvirus infection-related diseases are diseases caused by herpes simplex virus (HSV), including herpes simplex, keratitis, encephalitis, myelitis, pneumonia, myocarditis, etc.

[0094] In some implementations, the herpesvirus infection-related diseases are diseases caused by human cytomegalovirus (HCMV), including encephalitis, myelitis, pneumonia, myocarditis, hepatitis, gastroenteritis, etc.

[0095] In some embodiments, the combination of compounds as described in the fifth or seventh aspect, and the pharmaceutical composition as described in the sixth or eighth aspect, have at least one of the following biological activities: (i) inhibiting the proliferation of herpesvirus; (ii) inhibiting the formation of herpesvirus plaques; and (iii) inhibiting the expression of herpesvirus proteins.

[0096] In some embodiments, the compound combinations as described in aspects five or seven, and the pharmaceutical compositions as described in aspects six or eight, are effective in inhibiting the growth and replication of herpesviruses (e.g., varicella-zoster virus, herpes simplex virus, human cytomegalovirus) both in vivo and in vitro, with inhibition rates as high as, for example, about 80%, 90%, or 100%. In some embodiments, the compound combinations or pharmaceutical compositions thereof exhibit an inhibition rate of up to about 84% against VZV growth and replication. In some embodiments, the norovirus-based lincomycin exhibits an inhibition rate of up to about 87% against HCMV growth and replication.

[0097] In some embodiments, the combination of compounds as described in the fifth or seventh aspect, and the pharmaceutical composition as described in the sixth or eighth aspect, effectively inhibit the formation of plaques of herpesviruses (e.g., varicella-zoster virus, herpes simplex virus, human cytomegalovirus) in vitro and in vivo, with inhibition rates as high as 80%, 90%, and 100%.

[0098] In some embodiments, the combination of compounds as described in the fifth or seventh aspect of the present invention, and the pharmaceutical composition as described in the sixth or eighth aspect, can increase the inhibitory effect on the growth and replication of herpesvirus by, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times compared to existing antiherpesvirus drugs (e.g., acyclovir).

[0099] In some embodiments, the combination of compounds as described in the fifth or seventh aspect of the present invention, and the pharmaceutical composition as described in the sixth or eighth aspect, can improve the inhibitory effect on herpesvirus plaque formation by, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times compared to existing antiherpesvirus drugs (e.g., acyclovir).

[0100] In some embodiments, the compound combinations as described in the fifth or seventh aspect, and the pharmaceutical compositions as described in the sixth or eighth aspect, have cure rates of up to, for example, about 60%, 70%, 80%, 90%, or 100% for herpesvirus infection-related diseases (e.g., diseases caused by varicella-zoster virus, diseases caused by herpes simplex virus, or diseases caused by human cytomegalovirus). In some embodiments, the pharmaceutical combinations or pharmaceutical compositions thereof have cure rates of up to 75% for HSV-1 infectious keratitis. In some embodiments, the noroside has a cure rate of up to 65% for HSV-2 infectious vaginal vesicles and mucosal lesions.

[0101] preparation

[0102] The compounds, combinations of compounds, or pharmaceutical compositions of Formula I of this invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical formulations of this invention should be sterile and stable under the conditions of production and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the required dose of the compound, combination of compounds, or pharmaceutical composition of Formula I of this invention, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) into a suitable solvent, followed by sterilization by filtration. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0103] The Formula I compounds, combinations of compounds, or pharmaceutical compositions thereof described in this invention may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the Formula I compounds, combinations of compounds, or pharmaceutical compositions thereof of this invention are administered by intraperitoneal injection or bolus.

[0104] In some exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0105] Terminology Definition

[0106] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0107] As used herein, the term "nourseothricin" is an antimicrobial agent produced by the soil bacterium *Streptothricin*, belonging to the subclass of antibiotics with the chemical structure of streptocytin (STs). Streptothricins are composed of a polymer of carboxylated glucosamine molecules with atypical amino acids (streptolidine) and 1–7 lysine residues (ST-F through ST-A and ST-X) (Khoklov & Shutova, 1972). Nourseothricin is also known as nourseothricin sulfate, nourseothricin sulfate, and Streptothricin Sulfate. The norotrione described herein comprises compound 1 of formula I, compound 2 of formula II, and compound 3 of formula III, wherein compound 1 is streptothricin D (ST-D), compound 2 is streptothricin F (ST-F), and compound 3 is streptothricin E (ST-E).

[0108] As described herein, the components of norovirus can be detected and analyzed using chemical composition testing methods known to those skilled in the art. In some embodiments, commonly used chemical composition testing methods include chromatography, mass spectrometry, fluorescence analysis, and ultraviolet-visible spectroscopy. After determining a suitable detection method, the sample is prepared in a form suitable for detection, ensuring the sample's homogeneity and stability. Depending on the requirements, methods such as extraction, dissolution, and drying can be selected. Appropriate instruments and equipment are selected based on the required analytical method; for example, for chromatography, gas chromatographs and liquid chromatographs can be selected; for mass spectrometry, mass spectrometers can be selected. During the testing process, accurate data is acquired and subjected to reasonable statistical analysis. Through data analysis, information such as the content, purity, and structure of the chemical components can be obtained.

[0109] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0110] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (e.g., survival without treatment).

[0111] As used herein, the term "subject" refers to a mammal, including bovine, equine, sheep, suidae, canine, feline, rodent, and primate animals, such as humans, mice, or monkeys. In some embodiments, the subject (e.g., human, mouse, or monkey) has a herpesvirus infection or a herpesvirus-related disease (e.g., diseases caused by varicella-zoster virus (VZV), including chickenpox, shingles, keratitis, encephalitis, myelitis, pneumonia, myocarditis, etc.), or is at risk of having such a disease.

[0112] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., herpesvirus infection, herpesvirus-related diseases) means an amount sufficient to prevent, stop, or delay the onset of said disease; an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0113] Beneficial effects of the invention

[0114] This invention discovers that noroside is a promising candidate broad-spectrum anti-herpesvirus drug prodrug with excellent clinical application potential. The application of noroside for the treatment of herpesvirus infection described in this invention is a first in the field and is of great significance for the development of novel broad-spectrum herpesvirus therapeutics.

[0115] The norovirus extract of the present invention has the following beneficial technical effects:

[0116] (1) It can effectively inhibit the formation of viral plaques of varicella-zoster virus (VZV), herpes simplex virus HSV-1, and human cytomegalovirus (HCMV) and significantly inhibit the production of progeny viruses.

[0117] (2) It has a significant inhibitory effect on the growth of varicella-zoster virus, herpes simplex virus and human cytomegalovirus.

[0118] Therefore, the norovirus of the present invention can not only be used to prepare drugs against varicella-zoster virus, but also drugs against herpes simplex virus and human cytomegalovirus, and has a certain broad-spectrum anti-herpes virus activity.

[0119] In addition, the norovirus extract of the present invention can also be used to prepare drugs for treating herpes virus infectious diseases, such as varicella caused by VZV, herpes zoster, HSV-1 infectious keratitis, etc.

[0120] Furthermore, the toxicity of the norovirus to normal cells of the present invention is within an acceptable range, and it has broad application prospects in the preparation of anti-herpes virus drugs and drugs for the prevention and treatment of herpes virus infection diseases. Attached Figure Description

[0121] Figure 1 The results of CCK8 assay for norotrione cytotoxicity were presented. The results showed that incubation with norotrione at concentrations below 50 μM for 72 hours did not produce significant cytotoxicity against the human diploid cell line ARPE-19, and treatment with norotrione at concentrations below 200 μM for 72 hours did not produce significant cytotoxicity against juvenile Syrian hamster kidney cells BHK-21.

[0122] Figure 2 The kinetics of the inhibitory effect of noroside on VZV replication levels in ARPE-19 cells were shown. The results indicated that both noroside and acyclovir inhibited the viral titer of VZV-GFP in ARPE-19 cells 72 hours after infection, and these inhibitory effects showed a dose-dependent effect, with noroside exhibiting a superior inhibitory effect compared to acyclovir.

[0123] Figure 3 The kinetics of the inhibitory effect of norotrione on HSV-1 replication in BHK-21 cells were shown. The results indicated that both norotrione and acyclovir inhibited the viral titer of HSV-1-GFP in BHK-21 cells 72 hours after infection, and these inhibitory effects showed a dose-dependent effect.

[0124] Figure 4The kinetics of the inhibitory effect of norocrifen on HSV-2 replication in BHK-21 cells were shown. The results indicated that both norocrifen and acyclovir inhibited the viral titer of HSV-2-GFP in BHK-21 cells 72 hours after infection, and these inhibitory effects showed a dose-dependent effect.

[0125] Figure 5 The kinetics of the inhibitory effect of norotrione on HCMV replication levels in ARPE-19 cells were shown. The results indicated that both norotrione and ganciclovir inhibited the viral titer of HCMV-GFP in ARPE-19 cells 96 hours after infection, and these inhibitory effects showed a dose-dependent effect, with norotrione showing a slightly better inhibitory effect than ganciclovir.

[0126] Figure 6 shows the inhibitory effect of norotrione on plaque size in VZV, HSV-1, HSV-2, and HCMV-infected cells. Cells infected with VZV-GFP, HSV-1-GFP, HSV-2-GFP, or HCMV-GFP were treated with different concentrations of norotrione, acyclovir, or ganciclovir. The viral plaque detection results after 72 hours are shown in Figure 6 (A–D). The size of GFP-containing lesions was inhibited by the drugs, and the inhibitory effect increased with increasing drug concentration. Norotrione showed better inhibitory effects on VZV-GFP plaque size than acyclovir, and norotrione showed better inhibitory effects on HCMV-GFP plaque size than ganciclovir.

[0127] Figure 7 shows the inhibitory effect of noroside on protein expression in cells infected with VZV, HSV-1, HSV-2, and HCMV. As shown in Figures 7(A-D), treatment with different concentrations of noroside in cells infected with VZV-GFP, HSV-1-GFP, HSV-2-GFP, and HCMV-GFP inhibited the expression of early proteins IE62 (VZV), RS1 (HSV-1, HSV-2), and IE1 (HCMV), as well as late proteins gE (VZV), US8 (HSV-1, HSV-2), and gB (HCMV) to varying degrees. Furthermore, the inhibitory effect gradually increased with increasing drug concentration, indicating that the inhibitory effect of noroside on viral protein expression of VZV, HSV-1, HSV-2, and HCMV has a dose-dependent effect.

[0128] Figure 8This study demonstrated the inhibitory effect of norsidinic acid on a human skin organoid model infected with VZV in vitro. Results showed that in treatment groups with different concentrations of norsidinic acid and acyclovir, Luc signaling in VZV-luc-infected human skin tissue was significantly inhibited. The inhibitory effect of norsidinic acid was similar to that of acyclovir, and the inhibitory strength increased with increasing drug concentration, exhibiting a dose-dependent effect.

[0129] Figure 9 shows the inhibitory effects of different concentrations of norotrione on human neuronal microspheres infected with VZV, HSV-1, HSV-2, and HCMV. As shown in Figures 9(A-D), in the treatment groups with different concentrations of norotrione and acyclovir or ganciclovir, the Luc signaling in human neuronal microspheres infected with VZV-luc, HSV-1-luc, HSV-2-luc, and HCMV-luc was significantly inhibited, and the inhibitory effect increased with increasing drug concentration, indicating that the inhibitory effects of these drugs on viral replication were dose-dependent. Specifically, norotrione showed a better inhibitory effect on VZV-luc replication than acyclovir, and norotrione showed a better inhibitory effect on HCMV-luc replication than ganciclovir. Detailed Implementation

[0130] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0131] Based on the content of this invention, and according to common technical knowledge and conventional methods in the art, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of this invention. The following detailed embodiments further illustrate the above-mentioned content of this invention. However, this should not be construed as limiting the scope of the above-mentioned subject matter of this invention to the following examples. All technologies implemented based on the above-mentioned content of this invention fall within the scope of this invention.

[0132] Experimental Materials and Methods

[0133] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products or by self-preparation.

[0134] I. Experimental Materials

[0135] (1) Based on the wild-type VZV-POka strain, the VZV-GFP and VZV-luc strains, which are expressed by inserting the reporter gene GFP and luciferase into the genome, were constructed in our laboratory. They were passaged by infecting ARPE-19 cells and cultured in DMEM / F12 medium supplemented with 2% fetal bovine serum, glutamine, penicillin and streptomycin. The working strains were stored at -80℃ for later use.

[0136] (2) Based on the wild-type HSV-1-KOS strain, the HSV-1-GFP strain and HSV-1-luc strain, which are expressed by inserting reporter gene GFP and luciferase into the genome, were constructed in our laboratory. They were passaged by infecting BHK-21 cells and cultured in DMEM medium supplemented with 2% fetal bovine serum, glutamine, penicillin and streptomycin. The working strains were stored at -80℃ for later use.

[0137] (3) Based on the wild-type HSV-2-MS strain, HSV-2-GFP and HSV-2-luc strains with inserted expression of reporter gene GFP and luciferase were passaged by infecting BHK-21 cells and cultured in DMEM medium supplemented with 2% fetal bovine serum, glutamine, penicillin and streptomycin. The working strains were stored at -80℃ for later use.

[0138] (4) The HCMV-GFP and HCMV-luc strains, which are based on the wild-type HCMV-AD169 strain genome with inserted expression of reporter gene GFP and luciferase, were constructed by our laboratory. They were passaged by infecting ARPE-19 cells and cultured in DMEM / F12 medium supplemented with 2% fetal bovine serum, glutamine, penicillin and streptomycin. The working strains were stored at -80℃ for later use.

[0139] (5) Human retinal epithelial cells (ARPE-19), purchased from ATCC, catalog number: CRL-2302, were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin. The working cell bank was stored in liquid nitrogen for later use.

[0140] (6) Young Syrian hamster kidney cells (BHK-21) were purchased from ATCC, catalog number: CCL-10. They were cultured in DMEM medium supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin. The working cell bank was stored in liquid nitrogen for later use.

[0141] (7) Human neural stem cells were purchased from Beijing Saibei Biotechnology Co., Ltd., catalog number: CA2301106, and cultured using NeuroEasy human neural stem cell (adherent culture) maintenance medium (catalog number: CA2312100).

[0142] (8) Human neuron differentiation kit: purchased from Beijing Saibei Biotechnology Co., Ltd., catalog number: CA2307100.

[0143] (9) Ultra-low adsorption culture plate (96-Well): purchased from Liwo Biotechnology Co., Ltd., catalog number: LV-ULA002.

[0144] (10) Nourseothricin sulfate: purchased from MedChemExpress, purity: ≥99%.

[0145] (11) Acyclovir: purchased from MedChemExpress, catalog number HY-17422, purity: ≥99%.

[0146] (12) Ganciclovir: purchased from MedChemExpress, catalog number HY-13637, purity: ≥99%.

[0147] (13) Cell lysis buffer: purchased from Beyotime, catalog number P0013.

[0148] (14) β-actin antibody: purchased from Abcam, catalog number ab6276.

[0149] (15) 3D printed skin tissue: purchased from Shanghai Sianfuno and cultured using KBM-Gold medium.

[0150] (16) Cell Luc assay reagent: One-Lite Luciferase Assay System, purchased from Novizan, product number DD1203-01.

[0151] (17) Luc substrate: D-luciferin potassium salt, purchased from Promega, catalog number E1605, purity: ≥99%.

[0152] (18) Sodium carboxymethyl cellulose: purchased from Sangon Biotech, catalog number A501427-0250.

[0153] (19) The human fetal skin, embryonic liver and thymus tissue were all obtained from the Affiliated Zhongshan Hospital of Xiamen University, and the acquisition and use of the specimens were approved by the Ethics Committee of Xiamen University.

[0154] II. Drug Preparation

[0155] (1) Noorsitin: Dissolve noorsitin in PBS and prepare a 10mM stock solution. Store it in a 20°C refrigerator for later use. Dilute it with cell culture medium to the required concentration before use.

[0156] (2) Acyclovir: Dissolve acyclovir in DMSO and prepare a 10mM stock solution. Store it in a 20°C refrigerator for later use. Dilute it with cell culture medium to the required concentration before use.

[0157] (3) Ganciclovir: Dissolve ganciclovir in DMSO and prepare a 10mM stock solution. Store it in a 20℃ refrigerator for later use. Dilute it with cell culture medium to the required concentration before use.

[0158] (4) Luc substrate: Dissolve D-luciferin potassium salt in sterile purified water to prepare a 30 mg / mL D-luciferin potassium salt stock solution (100-200×). Shake well before use. Stock solutions that cannot be used immediately can be aliquoted and stored at -20℃ in the dark to avoid repeated freeze-thaw cycles, which may affect the activity of the reagent.

[0159] III. Statistical Methods

[0160] Original records were simultaneously created using both paper-based and computer-based methods. Measurement data was collected using... This indicates that the data obtained from each group conforms to the normal distribution compared with the model group. One-way ANOVA or independent samples t-test was used in SPSS 19.0. For those that do not conform, the nonparametric rank-sum test was used (P < 0.05 was considered to be significant between sample groups).

[0161] Example 1: Component characterization of norsinolate

[0162] Nourseothricin (also known as: nourseothricin sulfate; nourseothricin sulfate; Streptothricin Sulfate; Nourseothricin sulfate): purchased from MedChemExpress.

[0163] Physicochemical properties characterization: Purity: ≥99%, stored at 4℃, it is a white to off-white solid, solubility in H2O: ≥100mg / mL (73.56mM).

[0164] Compositional Analysis: The composition of noroside was analyzed using High Performance Liquid Chromatography (HPLC). Chromatographic conditions were as follows: Hypersil GOLD column was selected. TM (Thermo Scientific) (250 × 4.6 mm, 5 μM); column temperature: 30 °C; mobile phase A: 1 L H₂O containing 0.02% sodium heptanesulfonate and 0.05% TFA; mobile phase B: acetonitrile (ACN); the volume of mobile phase B was set to 10%–100% for gradient elution; injection volume: 20 μL; sample concentration: 1 mg / mL; flow rate: 1 mL / min. Component characterization: The components of norovirin used in this invention include compound 1 of formula I, compound 2 of formula II, and compound 3 of formula III;

[0165] Compound 1 represented by Formula I is streptothricin D (C 31H 58 N 12 O 10 ),

[0166]

[0167] Compound 2, represented by Formula II, is streptothricin F (C 19 H 34 N8O8),

[0168]

[0169] Compound 3, represented by Formula III, is streptothricin E (C 25 H 46 N 10 O9),

[0170]

[0171] Of these compounds, compound 1 (Streptothricin D) accounts for 40.76% by mass, compound 2 (Streptothricin F) accounts for 54.13% by mass, and compound 3 (Streptothricin E) accounts for 5.11% by mass.

[0172] Example 2: Detection of drug cytotoxicity

[0173] 1. Experimental Methods

[0174] (1) Cell preparation: ARPE-19 cells and BHK-21 cells were digested, centrifuged, counted, and then seeded into 96-well plates and cultured at 37°C, 5% CO2 and 90% humidity for 24 hours until their confluence reached about 80%.

[0175] (2) Drug administration and culture: 200 μM solutions of norovir and acyclovir were prepared using growth medium (DMEM / F12 or DMEM containing 10% fetal bovine serum, glutamine, penicillin, and streptomycin). These solutions were then serially diluted twofold to 1.5625 μM to obtain different concentration gradients of the two drugs. A 0 μM group containing only the solvent was also set up as a control. The original culture medium was discarded, and three replicates of each concentration were added to the above 96-well plates. The plates were then incubated at 37°C, 5% CO2, and 90% humidity for 72 hours.

[0176] (3) CCK-8 assay: CCK-8 was thawed and centrifuged at room temperature before use. 10 μL of CCK-8 solution was added to each well of the 96-well plate and incubated at 37°C, 5% CO2, and 90% humidity for 0.5-4 hours. The absorbance was measured at 450 nm using a microplate reader. The data were then processed and analyzed using Graphpad Prism.

[0177] 2. Experimental Results

[0178] ARPE-19 and BHK-21 cells were incubated with different concentrations of norepinephrine for 72 hours. Microscopic examination revealed no significant difference in cell morphology compared to the untreated group. At low concentrations, no cytotoxicity was detected by the CCK-8 assay. Figure 1 ).

[0179] The results show that in this cell model, norsinolate has almost no cytotoxicity below 50 μM and does not induce cell death at higher doses.

[0180] Example 3: Plaque assay to detect the inhibitory effect of drugs on VZV-infected ARPE-19 cells

[0181] 1. Experimental Methods

[0182] (1) Cell and virus preparation: ARPE-19 cells were seeded into 6-well plates and cultured at 37°C, 5% CO2 and 90% humidity for 8 hours until they adhered to the plate. When the confluence was about 80%, VZV-GFP was infected with MOI=0.001.

[0183] (2) Drug administration and culture: 200 μM solutions of norovir and acyclovir were prepared in maintenance medium (DMEM / F12 containing 2% fetal bovine serum, glutamine, penicillin, and streptomycin). These solutions were serially diluted twofold to 1.5625 μM to obtain different concentration gradients of the two drugs. A control group containing only 0 μM solvent was also included. After incubating the virus and cells at 37°C, 5% CO2, and 90% humidity for 1 hour, the culture medium was removed, and the cells were washed three times with PBS. Subsequently, three replicates of each drug concentration were prepared, and the prepared drug solutions were added to the above-mentioned 6-well plates. The plates were then cultured at 37°C, 5% CO2, and 90% humidity for another 72 hours.

[0184] (3) Plaque assay for virus titer determination: Cells from each well were digested, resuspended in maintenance medium, and serially diluted 5-fold to obtain cell suspensions with different virus titer gradients. 100 μL of each suspension was added to a 6-well plate pre-coated with ARPE-19 cells. After culturing for 72 hours, the cells were observed under an inverted fluorescence microscope. Plaques with GFP were counted at the optimal dilution. The results were processed and analyzed using Graphpad Prism to calculate the virus titer in each well.

[0185] 2. Experimental Results

[0186] ARPE-19 cells infected with VZV-GFP were treated with different concentrations of norovir and acyclovir, and their antiviral effects were verified by detecting viral titers. The viral titer results after 72 hours of drug treatment are shown in Table 1, and the inhibition curves are shown in Figure 2. Figure 2 .

[0187] The results showed that both norovir and acyclovir inhibited the proliferation of VZV-GFP, and the inhibitory effect increased with increasing drug concentration, indicating that the inhibitory effect of both norovir and acyclovir on viral proliferation was dose-dependent. Based on the inhibition curves, the EC50 of norovir was calculated to be 2.931 μM, while the EC50 of acyclovir in the same batch of experiments was calculated to be 8.613 μM. Therefore, norovir showed a superior inhibitory effect on VZV virus growth compared to acyclovir.

[0188] Table 1. VZV virus titers after 72 hours of treatment with different concentrations of drugs.

[0189]

[0190]

[0191] Example 4: Plaque assay to detect the inhibitory effect of drugs on HSV-1 and HSV-2 infected BHK-21 cells.

[0192] 1. Experimental Methods

[0193] (1) Cell and virus preparation: BHK-21 cells were seeded into 6-well plates and cultured at 37°C, 5% CO2 and 90% humidity for 8 hours until they adhered to the plate. When the confluence was about 80%, HSV-1-GFP or HSV-2-GFP was infected with MOI=0.001.

[0194] (2) Drug administration and culture: 200 μM solutions of norovir and acyclovir were prepared in maintenance medium (DMEM containing 2% fetal bovine serum, glutamine, penicillin, and streptomycin) and serially diluted twofold to 1.5625 μM to obtain different concentration gradients of the two drug solutions. A 0 μM group containing only solvent was set up as a control. After incubating the virus and cells at 37°C, 5% CO2, and 90% humidity for 1 hour, the culture medium was removed, and the cells were washed three times with PBS. Then, according to different drug concentrations, three replicates were set up for each concentration, and the prepared drug solutions were added to the above 6-well plates. The plates were then cultured at 37°C, 5% CO2, and 90% humidity for another 72 hours.

[0195] (3) Plaque assay for virus titer determination: First, the culture supernatant from each well was collected and serially diluted 10-fold to obtain virus suspensions with different viral titer gradients. Then, 100 μL of the virus suspension was added to each well of a 6-well plate pre-coated with BHK-21 cells. After incubation at 37°C, 5% CO2, and 90% humidity for 1 hour, the culture medium was removed, and the cells were washed three times with PBS. Next, 2 mL of 2% sodium carboxymethyl cellulose was added to each well to cover the culture medium, immobilizing virus particles and promoting plaque formation. The 6-well plate was then placed in an incubator at 37°C and 5% CO2 for another 48 hours. After incubation, GFP-containing lesions were observed using a fluorescence inverted microscope and counted at the optimal dilution. Finally, the data were processed and analyzed using Graphpad Prism software, and the viral titer in each well was calculated based on the number of plaques.

[0196] 2. Experimental Results

[0197] BHK-21 cells infected with HSV-1-GFP and HSV-2-GFP were treated with different concentrations of norovir and acyclovir, and their antiviral effects were verified by detecting viral titers. The viral titer results after 72 hours of drug treatment are shown in Tables 2 and 3, and the inhibition curves are shown below. Figure 3 and Figure 4 .

[0198] The results showed that both norovir and acyclovir inhibited the proliferation of HSV-1-GFP and HSV-2-GFP, and the inhibitory effect increased with increasing drug concentration, indicating that the inhibitory effect of norovir and acyclovir on viral proliferation was dose-dependent. Based on the inhibition curves, the EC50 values ​​of norovir against HSV-1-GFP and HSV-2-GFP were calculated to be 3.312 μM and 3.128 μM, respectively. In the same batch of experiments, the EC50 values ​​of acyclovir against HSV-1-GFP and HSV-2-GFP were calculated to be 0.1483 μM and 0.1429 μM, respectively.

[0199] Table 2. HSV-1 viral titers after 72 hours of treatment with different concentrations of drugs.

[0200]

[0201] Table 3. HSV-2 viral titers after 72 hours of treatment with different concentrations of drugs.

[0202]

[0203] Example 5: Plaque assay to detect the inhibitory effect of drugs on HCMV-infected ARPE-19 cells

[0204] 1. Experimental Methods

[0205] (1) Cell and virus preparation: ARPE-19 cells were seeded into 6-well plates and cultured at 37°C, 5% CO2 and 90% humidity for 8 hours until they adhered to the plate. When the confluence was about 80%, HCMV-GFP was infected with MOI=0.001.

[0206] (2) Drug administration and culture: 200 μM solutions of norovir and ganciclovir were prepared in maintenance medium (DMEM / F12 containing 2% fetal bovine serum, glutamine, penicillin, and streptomycin). These solutions were serially diluted twofold to 1.5625 μM to obtain different concentration gradients of the two drugs. A 0 μM group containing only the solvent was set up as a control. After incubating the virus and cells at 37°C, 5% CO2, and 90% humidity for 1 hour, the culture medium was removed, and the cells were washed three times with PBS. Subsequently, three replicates were set up for each drug concentration, and the prepared drug solutions were added to the above-mentioned 6-well plates. The plates were then cultured at 37°C, 5% CO2, and 90% humidity for another 72 hours.

[0207] (3) Plaque assay for virus titer determination: First, the culture supernatant from each well was collected and serially diluted 10-fold to obtain virus suspensions with different viral titer gradients. Then, 100 μL of the virus suspension was added to each well of a 6-well plate pre-coated with ARPE-19 cells. After culturing at 37°C, 5% CO2, and 90% humidity for 1 hour, the culture medium was removed, and the cells were washed three times with PBS. Next, 2 mL of 2% sodium carboxymethyl cellulose was added to each well to cover the culture medium, immobilizing virus particles and promoting plaque formation. The 6-well plate was then placed in an incubator at 37°C and 5% CO2 for another 48 hours. After incubation, GFP-containing lesions were observed using a fluorescence inverted microscope and counted at the optimal dilution. Finally, the data were processed and analyzed using Graphpad Prism software, and the viral titer in each well was calculated based on the number of plaques.

[0208] 2. Experimental Results

[0209] ARPE-19 cells infected with HCMV-GFP were treated with different concentrations of norovir and acyclovir, and their antiviral effects were verified by detecting viral titers. The viral titer results after 96 hours of drug treatment are shown in Table 4, and the inhibition curves are shown in Figure 5. Figure 5 .

[0210] The results showed that both norovir and acyclovir inhibited the proliferation of HCMV-GFP, and the inhibitory effect increased with increasing drug concentration, indicating that the inhibitory effect of both norovir and acyclovir on viral proliferation was dose-dependent. Based on the inhibition curves, the EC50 of norovir was calculated to be approximately 1.341 μM, while the EC50 of ganciclovir in the same batch of experiments was approximately 2.15 μM. Therefore, norovir showed a superior inhibitory effect on HCMV-GFP viral growth compared to ganciclovir.

[0211] Table 4. HCMV viral titers after 96 hours of treatment with different concentrations of drugs

[0212]

[0213] Example 6: Detection of the inhibitory effect of drugs on plaque size in VZV, HSV-1, HSV-2 and HCMV infected cells

[0214] 1. Experimental Methods

[0215] (1) Cell and virus preparation: ARPE-19 or BHK-21 cells were seeded into 6-well plates and cultured at 37°C, 5% CO2 and 90% humidity for 8 hours until they adhered to the plate. When the confluence was about 80%, they were infected with VZV-GFP, HSV-1-GFP, HSV-2-GFP or HCMV-GFP with MOI=0.001.

[0216] (2) Drug administration: 200 μM solutions of norovir, acyclovir, and ganciclovir were prepared in maintenance medium (DMEM containing 2% fetal bovine serum, glutamine, penicillin, and streptomycin). These solutions were serially diluted twofold to 1.5625 μM to obtain three drug solutions of different concentrations. A 0 μM group containing only the solvent was set up as a control. Simultaneously, sodium carboxymethyl cellulose was added to each drug solution to a final concentration of 2%. After incubating the virus and cells at 37°C, 5% CO2, and 90% humidity for 1 hour, the culture medium was removed, and the cells were washed three times with PBS. Next, 2 mL of different concentrations of drug solutions containing 2% sodium carboxymethyl cellulose were added to each well of the above 6-well plates, with three replicates for each concentration. The plates were then incubated at 37°C, 5% CO2, and 90% humidity for 72 hours. For VZV-GFP, HSV-1-GFP, and HSV-2-GFP infected cells, noroside and acyclovir solutions were added; for HCMV infected cells, noroside and ganciclovir solutions were added.

[0217] (3) Monitoring of plaque size: The cytopathic effect was observed and recorded under a fluorescence microscope every day for three consecutive days. Fifteen plaques were randomly selected each day and their size was measured using ImageJ software. The results were processed and analyzed using Graphpad Prism.

[0218] 2. Experimental Results

[0219] Cells infected with VZV-GFP, HSV-1-GFP, HSV-2-GFP, or HCMV-GFP were treated with different concentrations of norovir and acyclovir or ganciclovir. The antiviral efficacy was verified by detecting the size of viral GFP plaques. The results of viral plaque detection 72 hours after drug treatment are shown in Figures 6(A-D).

[0220] The results showed that the size of GFP-containing lesions was inhibited by the drugs, and the inhibitory effect increased with increasing drug concentration, indicating that the inhibitory effect of these drugs on lesion size was dose-dependent. Among them, norovir was more effective than acyclovir in inhibiting the size of VZV-GFP lesions, and norovir was more effective than ganciclovir in inhibiting the size of HCMV-GFP lesions.

[0221] Example 7: Detection of the inhibitory effect of drugs on protein expression in cells infected with viruses VZV, HSV-1, HSV-2 and HCMV

[0222] 1. Experimental Methods

[0223] (1) Cell and virus preparation: ARPE-19 or BHK-21 cells were seeded into 6-well plates and cultured at 37°C, 5% CO2 and 90% humidity for 8 hours until they adhered to the plate. When the confluence was about 80%, they were infected with VZV-GFP, HSV-1-GFP, HSV-2-GFP or HCMV-GFP with MOI=0.001.

[0224] (2) Drug administration and culture: 200 μM solutions of norovir, acyclovir, and ganciclovir were prepared in maintenance medium (DMEM containing 2% fetal bovine serum, glutamine, penicillin, and streptomycin). These solutions were serially diluted twofold to 1.5625 μM to obtain three drug solutions of different concentrations. A 0 μM group containing only the solvent was set up as a control. After incubating the virus and cells at 37°C, 5% CO2, and 90% humidity for 1 hour, the culture medium was removed, and the cells were washed three times with PBS. Next, 2 mL of different concentrations of drug solution were added to each well of the above 6-well plate, with three replicates for each concentration, and the plates were cultured for another 72 hours at 37°C, 5% CO2, and 90% humidity. Norovir and acyclovir solutions were added to cells infected with VZV-GFP, HSV-1-GFP, and HSV-2-GFP, while norovir and ganciclovir solutions were added to cells infected with HCMV.

[0225] (3) Western blot sample collection and processing: First, the cells were washed once with PBS and scraped from the 6-well plate into 1.5 mL EP tubes. After centrifugation at 300 g / min for 10 minutes, the supernatant was removed. Then, cell lysis buffer was added to fully lyse the cells, centrifuged at 12000 rpm for 10 minutes, and the supernatant was added to loading buffer and mixed before being placed in a hot water bath for 10 minutes. Next, the processed samples were subjected to SDS-PAGE gel electrophoresis and Western blot detection, including steps such as membrane transfer, blocking, antibody incubation, and colorimetric reaction. The β-actin antibody was purchased from Abcam, while all monoclonal antibodies against VZV virus proteins (IE62, gE), HSV-1 virus proteins (RS1, US8), HSV-2 virus proteins (RS1, US8), and HCMV virus proteins (IE1, gB) were prepared in the inventor's laboratory.

[0226] 2. Experimental Results

[0227] ARPE-19 cells infected with VZV-GFP and HCMV-GFP, and BHK-21 cells infected with HSV-1-GFP and HSV-2-GFP were treated with different concentrations of norovirus extract. The antiviral effect was verified by detecting the expression of viral proteins. The Western blot results after 72 hours of drug treatment are shown in Figure 7 (A-D) (A: VZV; B: HSV-1; C: HSV-2; D: HCMV).

[0228] The results showed that the expression of viral proteins in infected cells was inhibited to varying degrees by different concentrations of the drug, and the inhibitory effect on protein expression gradually increased with the increase of drug concentration, indicating that the inhibitory effect of norovirus on the expression of VZV, HSV-1, HSV-2 and HCMV viral proteins has a dose-dependent effect.

[0229] Example 8: Detection of the inhibitory effect of the drug on a VZV-infected human skin organoid model

[0230] 1. Experimental Methods

[0231] (1) Preparation of human skin tissue and inoculation with virus: Human skin organoid tissue was divided into 4mm × 4mm pieces and placed in 96-well plates. ARPE-19 cells with more than 80% lesions after infection with VZV-luc strain were digested, centrifuged, and resuspended with PBS. Infected cells were injected into the skin model using an insulin needle, with 5 μl of cell suspension (approximately 5000 PFU) injected into each skin patch, and then cultured at 37°C, 5% CO2, and 90% humidity for 1 hour.

[0232] (2) Drug addition and culture: 100 μM solutions of norovir and acyclovir were prepared in KBM-GOLD medium and serially diluted twofold to 1.5625 μM to obtain two drug solutions of different concentration gradients. A 0 μM group containing only solvent was set up as a control. The original medium was removed, and three replicates were set up for each drug concentration. The prepared drug solutions were added to the above 96-well plates and cultured at 37°C, 5% CO2, and 90% humidity for 72 hours.

[0233] (3) Detection of viral replication level in human skin model: KBM-GOLD medium was preheated at 37℃ for 10-20 minutes, and then diluted with Luc stock solution using the preheated medium to obtain a working solution with a concentration of 0.3 mg / mL. The tissue culture medium in the 96-well plate was removed, Luc working solution was added, and the plate was incubated at 37℃ for 5-10 minutes. The Luc signal intensity was then monitored, and the inhibition rate was calculated using Graphpad Prism. Inhibition rate = (Luc value of 0 μM group - Luc value of any drug group) / Luc value of 0 μM group.

[0234] 2. Experimental Results

[0235] like Figure 8 As shown, in the treatment groups with different concentrations of noroside and acyclovir, Luc signaling in VZV-luc-infected human skin tissue was significantly inhibited. The inhibitory effect of noroside was similar to that of acyclovir, and the inhibitory intensity increased with increasing drug concentration, exhibiting a dose-dependent effect. No significant toxicity was observed in human skin tissue in any of the drug treatment groups.

[0236] Example 9: Detection of the inhibitory effect of drugs on VZV, HSV-1, HSV-2 and HCMV-infected human nerve cell microspheres

[0237] 1. Experimental Methods

[0238] (1) Cell preparation: Human nerve cells were digested and diluted to 2.5 × 10⁻⁶ using nerve cell maintenance medium. 6 200 μL / well was inoculated into a 96-well ultra-low adsorption culture plate for suspension culture.

[0239] (2) Neurosphere differentiation culture: When human nerve cells were cultured in suspension for 3 days, the cell clusters spontaneously aggregated into a relatively compact microsphere structure. The culture supernatant was aspirated, and the culture medium was replaced with 200 μL of nerve cell differentiation medium. The medium was changed every 2 days, and differentiation was completed after 10 days of culture.

[0240] (3) Virus inoculation: After the human neural cell microspheres have completed differentiation, they are infected with VZV-luc, HSV-1-luc, HSV-2-luc or HCMV-luc strains at MOI=0.1 and cultured at 37℃, 5% CO2 and 90% humidity for 1 hour.

[0241] (4) Drug administration and culture: 100 μM solutions of norovir, acyclovir, and ganciclovir were prepared in nerve maintenance medium and serially diluted twofold to 1.5625 μM to obtain three drug solutions with different concentration gradients. A 0 μM group containing only solvent was set up as a control. The original culture medium was removed, and three replicates were set up for each drug concentration. The prepared drug solutions were added to the above 96-well plates and cultured at 37°C, 5% CO2, and 90% humidity for 72 hours.

[0242] (5) Viral replication level detection: The nerve maintenance medium was preheated at 37°C for 10-20 minutes, and then the Luc substrate stock solution was diluted with the preheated medium to obtain a working solution with a concentration of 0.3 mg / mL. The tissue medium in the 96-well plate was removed, the Luc working solution was added, and the plate was incubated at 37°C for 5-10 minutes. The Luc signal intensity was then monitored, and the inhibition rate was calculated using Graphpad Prism. Inhibition rate = (Luc value of 0 μM group - Luc value of any drug group) / Luc value of 0 μM group.

[0243] 2. Experimental Results

[0244] As shown in Figures 9(A-D), in the treatment groups with different concentrations of noroside and acyclovir or ganciclovir solutions, the Luc signaling in human neurospheres infected with VZV-luc, HSV-1-luc, HSV-2-luc, or HCMV-luc strains was significantly inhibited, and the inhibitory effect increased with increasing drug concentration, indicating that the inhibitory effect of these drugs on viral replication is dose-dependent. Noroside showed a better inhibitory effect on VZV-luc replication than acyclovir, and noroside showed a better inhibitory effect on HCMV-luc replication than ganciclovir. None of the drug treatment groups showed significant toxicity to human neurospheres.

[0245] Example 10: Detection of the inhibitory effect of the drug on a VZV-infected human skin chimeric mouse model

[0246] 1. Experimental Methods

[0247] (1) Construction of the SCID-hu human-skin chimeric mouse model: Six-week-old CB-17SCID mice (purchased from Shanghai Silex Laboratory Animal Co., Ltd.) were used in the experiment and were separated into different cages to adapt to the breeding environment 7 days before the operation. Human fetal skin obtained clinically was cut into 10mm×10mm pieces with sterile surgical scissors. Then, 5-8 week old male CB-17SCID mice were anesthetized with isoflurane gas mixed with oxygen (flow rate 2mL / min). An incision of about 1cm was made on each side of the SCID mouse near the hind leg. Then, a piece of skin was inserted into each incision and flattened, with the dermis side facing inward to contact the muscle layer of the mouse and the epidermis side facing outward. BD Biosciences' SCID model was used to construct the model. Apply an autoclip (9mm) to the wound or suture it with surgical needles and sutures. It can be used for VZV infection two weeks after transplantation.

[0248] (2) Virus inoculation: VZV-luc strain was injected at multiple points on the back of infected SCID mice at a dose of 5000 PFU / 50 μL / piece using an insulin syringe into the human skin tissue graft.

[0249] (3) Drug treatment: Drug treatment was started on the 3rd day after challenge. Norisulin and acyclovir were prepared into a drug solution with PBS and administered intraperitoneally according to the weight of the mice. Different dosage groups of 0 mg / kg, 10 mg / kg and 20 mg / kg were set up, with 3 mice in each group. The drugs were administered once a day for a total of 8 days.

[0250] (4) Viral replication level detection: 14 days after challenge, in vivo imaging technology was used to determine the in vivo replication of VZV-luc. Each mouse was injected intraperitoneally with 100 μL of D-fluorescein potassium solution (Promega; 15 mg / mL, prepared in PBS). Ten minutes later, the mice were anesthetized with isoflurane, and the in vivo Luc signal was detected using a Xenogen in vivo imaging system. The level of Luc signal indirectly reflects the viral titer. The inhibition rate was calculated using Graphpad Prism: Inhibition rate = (Luc value of 0 mg / kg group - Luc value of any drug group) / Luc value of 0 mg / kg group.

[0251] 2. Experimental Results

[0252] As shown in Table 5, Luc signaling in VZV-luc-infected human skin tissue was significantly inhibited in mice treated with different concentrations of noroside and acyclovir. The viral inhibition effect in the treatment groups increased with increasing drug strength, indicating that the inhibitory effects of these drugs on viral replication were dose-dependent, and noroside and acyclovir showed similar in vivo inhibitory effects on VZV replication. No significant toxicity was observed in mice treated with any of the drugs.

[0253] Table 5. Average in vivo inhibition rate of different drug concentrations on VZV-infected human skin tissue

[0254]

[0255] Example 11: Detection of the inhibitory effect of drugs on VZV and HCMV-infected human thymus transplant mouse models

[0256] 1. Experimental Methods

[0257] (1) Construction of the SCID-hu human thymus transplantation mouse model: Human thymus and fetal liver tissue were combined in a "sandwich" structure and transplanted subrenally into the renal capsule of 5-8 week old male CB-17SCID mice. First, thymus and fetal liver tissues from embryos at 18-24 weeks of gestation were obtained, washed several times with DMEM / F12 medium supplemented with penicillin-streptomycin resistance, and then cut into small pieces of about 1 mm3 with surgical scissors. Then, the small pieces of thymus and fetal liver tissue were placed together into an 18-G transplantation needle for transplantation. Subsequently, SCID mice were anesthetized with isoflurane gas, the skin on the back of the mice was cut open with scissors, the kidneys of the mice were squeezed out, and the thymus and fetal liver were injected subrenally with the transplantation needle. Finally, the kidneys of the mice were gently placed back into the body, and the peritoneum was sutured with surgical needles and sutures. Simply clamp the outer skin wound with an autoclip (9mm). The mouse will wake up after a few minutes. Add food and water and place it in an isolation bag for rearing. Two to three months after transplantation, the thymus will be well vascularized in the renal sac and grow significantly in size (about 8mm3). At this time, the thymus can be used for VZV or HCMV inoculation.

[0258] (2) Virus inoculation: The transplanted thymus tissue was surgically exposed, and the human thymus tissue graft infected with SCID mice was injected at a single point using an insulin injector at a dose of 100 PFU / 50 μL / graft.

[0259] (3) Drug treatment: Drug treatment was started on the 3rd day after challenge. Norciclovir, acyclovir and ganciclovir were prepared into drug solutions with PBS and administered intraperitoneally according to the weight of mice. Different dosage groups of 0 mg / kg, 10 mg / kg and 20 mg / kg were set up, with 3 mice in each group. The drugs were administered once a day for a total of 8 days.

[0260] (4) Monitoring of viral growth curves in vivo: 14 days after challenge, in vivo replication of VZV-luc and HCMV-luc was determined using in vivo imaging technology. Each mouse was injected intraperitoneally with 100 μL of D-fluorescein potassium solution (Promega; 15 mg / mL, prepared in PBS). Ten minutes later, the mice were anesthetized with isoflurane, and the in vivo Luc signal was detected using a Xenogen in vivo imaging system. The level of Luc signal indirectly reflects the viral titer. The inhibition rate was calculated using Graphpad Prism, and the inhibition rate was calculated as (Luc value of 0 mg / kg group - Luc value of any drug group) / Luc value of 0 mg / kg group.

[0261] 2. Experimental Results

[0262] As shown in Table 6, Luc signaling in human thymus tissue infected with VZV-luc and HCMV-luc was significantly inhibited in mice treated with different concentrations of norovir, acyclovir, and ganciclovir. The viral inhibition effect in the treatment groups increased with increasing drug strength, indicating that the inhibitory effects of these drugs on viral replication were dose-dependent. Norovir showed comparable in vivo inhibitory effects on VZV replication to acyclovir, and comparable in vivo transplantation effects on HCMV replication to ganciclovir. No significant toxicity was observed in any of the drug treatment groups in mice.

[0263] Table 6. Mean in vivo inhibition rate of different drug concentrations on VZV and HCMV infection in human thymus tissue

[0264]

[0265] Example 12: Testing the therapeutic effect of the drug on HSV-1 infectious keratitis in mice

[0266] 1. Experimental Methods

[0267] (1) HSV-1 inoculation into mouse corneas: 6-8 week old female BALB / c mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were used in the experiment. Mice were anesthetized with isoflurane, and after scratching the corneal epithelium of the right eye of each mouse in a cross-shaped pattern, 10 μL of HSV-1 was instilled into the cornea. 5 PFU / 5μL HSV-1-(KOS strain) was left on the cornea of ​​mice for 10 seconds to complete virus inoculation. Infected eyes were treated daily with 0.5% gentamicin eye drops, while control eyes were not infected with the virus.

[0268] (2) Drug treatment: Drug treatment was started 1 day after the challenge. Norisulin and acyclovir were prepared into a drug solution with PBS and administered intraperitoneally according to the weight of the mice at a dose of 10 mg / kg. A control group of 0 mg / kg was set up. Each group consisted of 20 mice. The drugs were administered once a day for a total of 7 days.

[0269] (3) Evaluation of the therapeutic effect of HSV-1 infectious keratitis in mice: Fourteen days after challenge, the keratitis status of mice was examined, and the ratio of mice with keratitis to those that were cured was recorded. The criteria for determining the cure of keratitis were: a) disappearance of irritation symptoms and ciliary congestion; b) corneal epithelial repair; c) reduction of corneal edema and infiltration; d) negative virus isolation from corneal epithelium and tears. Mice that met the above four signs were judged to be cured of keratitis, and the remaining mice were still considered to have keratitis.

[0270] 2. Experimental Results

[0271] As shown in Table 7, most cases of keratitis in mice treated with norovir and acyclovir were effectively improved and cured. No significant toxicity was observed in any of the drug treatment groups in the mice.

[0272] Table 7. HSV-1 infectious keratitis and healing status in mice of each group after 14 days.

[0273]

[0274] Example 13: Detection of the therapeutic effect of drugs on a guinea pig model infected with HSV-2

[0275] (1) Vaginal inoculation of HSV-2 in guinea pigs: Adult female guinea pigs (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were used in the experiment. After anesthesia with isoflurane, the vulva was cleaned with physiological saline. The vagina was rubbed several times with a dry cotton swab. Then, the vulva of the guinea pig was gently tapped with a plum blossom needle to cause vulvar congestion and slight bleeding. The vulva was then wiped clean with a clean cotton swab. 10 ml of HSV-2 was drawn into a 1 ml syringe. 7 PFU / 0.2 mL of HSV-2 (MS strain) virus solution was prepared and inserted into the guinea pig's vagina 3-4 cm with a gavage needle. The virus was injected into the vaginal canal and then slowly withdrawn. A gelatin sponge was inserted into the vagina to maintain the virulence. A small amount of virulence was dripped onto the vulva and gently spread evenly with a glass rod to allow the virus to penetrate the skin.

[0276] (2) Drug treatment: Drug treatment was started 1 day after the challenge. Norisulin and acyclovir were prepared into a drug solution with PBS and administered intraperitoneally according to the weight of the mice at a dose of 10 mg / kg. A control group of 0 mg / kg was set up. Each group consisted of 12 mice. The drugs were administered once a day for a total of 5 days.

[0277] (3) Evaluation of the therapeutic effect of HSV-2 infection in guinea pigs: Seven days after the challenge, the number of guinea pigs with and without vesicles and mucosal damage in the vagina of each group was counted.

[0278] 2. Experimental Results

[0279] As shown in Table 8, in guinea pigs treated with norovir and acyclovir, most animals did not develop vaginal vesicles or mucosal damage. No significant toxicity was observed in any of the drug treatment groups in guinea pigs.

[0280] Table 8. HSV-2-infected vaginal lesions in guinea pigs of each group after 7 days.

[0281]

Claims

1. Use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates, or derivatives thereof, in the preparation of a medicament for the prevention and / or treatment of herpes virus infection or diseases related to herpes virus infection:

2. Use of the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of herpesvirus infection or herpesvirus-related diseases, said pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a crystal thereof, a solvate thereof, a hydrate thereof, or a derivative thereof: Preferably, the pharmaceutical composition further comprises one or more nucleoside analogs, such as iodothyronine, vidarabine, trifluorothymidine, acyclovir, and ganciclovir. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient; specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a compound preparation.

3. Use of the compound combination in the preparation of a medicament for the prevention and / or treatment of herpesvirus infection or herpesvirus-related diseases, wherein: The compound combination comprises: compound 1 of formula I or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; compound 2 of formula II or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; and compound 3 of formula III or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof. Preferably, the mass percentage of compound 1 is 40% to 50%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%. Preferably, the mass percentage of compound 1 is 40% to 45%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%. More preferably, the mass percentage of compound 1 is 40.76%, the mass percentage of compound 2 is 54.13%, and the mass percentage of compound 3 is 5.11%.

4. Use of the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of herpes virus infection or herpes virus infection-related diseases, wherein, The pharmaceutical composition comprises the compound combination of claim 3; Preferably, the pharmaceutical composition further comprises one or more nucleoside analogs, such as iodothyronine, vidarabine, trifluorothymidine, acyclovir, and ganciclovir. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient; specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a compound preparation.

5. The use according to any one of claims 1-4, wherein, The herpesvirus is selected from: varicella-zoster virus (VZV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), gamma herpesvirus (e.g., EB virus), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), or Kaposi's sarcoma-associated herpesvirus (KSHV) or a combination thereof; Preferably, the herpes virus is selected from varicella-zoster virus (VZV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), or a combination thereof.

6. The use according to any one of claims 1-4, wherein, The herpesvirus infection-related diseases mentioned are diseases caused by herpesvirus infection; Preferably, the herpes virus infection-related diseases are diseases caused by varicella-zoster virus (VZV), including but not limited to chickenpox, herpes zoster, keratitis, encephalitis, myelitis, pneumonia, myocarditis, etc. Preferably, the herpes virus infection-related diseases are diseases caused by herpes simplex virus (HSV), including but not limited to herpes simplex, keratitis, encephalitis, myelitis, pneumonia, myocarditis, etc. Preferably, the herpesvirus infection-related diseases are diseases caused by human cytomegalovirus (HCMV), including but not limited to encephalitis, myelitis, pneumonia, myocarditis, hepatitis, gastroenteritis, etc.

7. The use according to any one of claims 1-6, wherein the subject is a mammal, including bovines, equines, sheep, suidae, canines, felines, rodents, primates, such as humans, mice, or monkeys.

8. Use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates, or derivatives thereof, in the preparation of a reagent or medicament for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells):

9. Use of a pharmaceutical composition in the preparation of an agent or medicament for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), said pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, a crystal thereof, a solvate thereof, a hydrate thereof, or a derivative thereof: Preferably, the pharmaceutical composition further comprises one or more nucleoside analogs, such as iodothyronine, vidarabine, trifluorothymidine, acyclovir, and ganciclovir. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient; specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a compound preparation.

10. Use of the compound combination in the preparation of a reagent or medicament for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), wherein: The compound combination comprises: compound 1 of formula I or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; compound 2 of formula II or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof; and compound 3 of formula III or a pharmaceutically acceptable salt thereof, its stereoisomer, its crystals, solvates, hydrates or derivatives thereof. Preferably, the mass percentage of compound 1 is 40% to 50%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%. Preferably, the mass percentage of compound 1 is 40% to 45%, the mass percentage of compound 2 is 50% to 60%, and the mass percentage of compound 3 is 0% to 10%. More preferably, the mass percentage of compound 1 is 40.76%, the mass percentage of compound 2 is 54.13%, and the mass percentage of compound 3 is 5.11%.

11. Use of the pharmaceutical composition in the preparation of a reagent or medicament for inhibiting the replication or proliferation of herpesviruses in cells (e.g., mammalian cells), wherein, The pharmaceutical composition comprises the compound combination of claim 10; Preferably, the pharmaceutical composition further comprises one or more nucleoside analogs, such as iodothyronine, vidarabine, trifluorothymidine, acyclovir, and ganciclovir. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient; specifically, the pharmaceutical composition is a solid dosage form, an injection, a topical preparation, a spray, a liquid preparation, or a compound preparation.

12. The use according to any one of claims 8-11, wherein, The herpesvirus is selected from: varicella-zoster virus (VZV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), gamma herpesvirus (e.g., EB virus), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), or Kaposi's sarcoma-associated herpesvirus (KSHV) or a combination thereof; Preferably, the herpes virus is selected from varicella-zoster virus (VZV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), or a combination thereof.

13. The use according to any one of claims 8-11, wherein the subject is a mammal, including bovines, equines, sheep, suidae, canines, felines, rodents, primates, such as humans, mice, or monkeys.

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