WO2024103979A1 - Compound for inhibiting herpes virus - Google Patents

Abstract

The present invention relates to a compound for inhibiting the herpes virus, a preparation method therefor, a pharmaceutical composition comprising the compound, and use of the pharmaceutical in treating or preventing CMV infection diseases. The compound has good solubility in water, and can provide unexpected increases in drug exposure (AUC) and maximum plasma concentration (Cmax), thus obtaining a longer in-vivo drug release time, extending the medication interval.

Description

A compound that inhibits herpes virus Technical Field

The invention belongs to the field of pharmaceutical chemistry, and specifically relates to a compound for inhibiting herpes virus, a preparation method thereof, a pharmaceutical composition containing the compound, and application of the drug in treating or preventing CMV infection diseases.

Background technique

Among DNA viruses, the herpes group is the source of the most common viral diseases in humans. This group consists of herpes simplex virus (HSV) types 1 and 2, varicella zoster (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV).

Like other herpes viruses, CMV infection can lead to lifelong infection of the host. After primary infection, hCMV can exist in a latent state in specific host cells of the bone marrow cell system, replicate and spread in many different types of cells (hematopoietic cells, epithelial cells, endothelial cells or fibroblasts), and avoid the host's immune system. hCMV is usually asymptomatic in healthy people because hCMV infection and spread are under the control of the immune system, but it is rare to achieve complete clearance of hCMV.

hCMV causes only mild or asymptomatic infection in immunocompetent adults but can cause severe morbidity in neonates and immunocompromised individuals (e.g., transplant recipients or AIDS patients). In neonates, infection causes severe neurologic deficits, and common symptoms in adults may be severe mononucleosis, pneumonia, hepatitis, gastroenteritis, and chorioretinitis. The virus is spread through frequent or prolonged close contact, usually breastfeeding or sexual intercourse. (Schooley in Harrison’s Principles of Internal Medicine (13th Ed.) (Isselbacher et al., eds.) Mc Graw-Hill, Inc., New York, 1994, pp. 794-796).

The hCMV virus consists of an icosahedral nucleocapsid containing a linear, 230kb long double-stranded DNA genome. The expression of the hCMV genome is controlled by a complex cascade transcription, which involves the synthesis of more than 200 proteins related to the biological activities of viral infection, latency and replication (Britt W and Mach M, 1996). The virus replicates in the nucleus and causes cell lysis followed by a tendency or latent infection. Once an individual is infected, unless the infected person's T cells are immune damaged, the infected person will carry the virus for life in a latent form. hCMV has carcinogenicity, causing changes in human and non-human cells and stimulating cell proliferation. Preventive measures to prevent hCMV infection include: tissue transplantation, transfusion of blood from seronegative donors, or transfusion of blood treated with freeze-thaw deglycerolization (reducing transplant-related infections). It has been shown that alpha interferon can prevent recurrence of reactivated hCMV syndrome and delay hCMV release in high-risk renal transplant recipients (Schooley in Harrison, s Principles of Internal Medicine (Bth Ed.) (Isselbacher et al. ed.) McGow-Hill, Inc. New York, 1994, pp. 794-796). Prophylactic non-cyclic guanosine is a nucleoside analog that has been shown to reduce hCMV infection in seronegative renal transplant recipients. Other preventive measures include the development of vaccines against human cytomegalovirus. However, vaccines cannot completely prevent hCMV infection or recurrence (EP 0277773; Elek et al. (1974) Lancet 1: 1-5; Neff et al. (1979 (Proc. Soc. Exp. Biol. Med. 160: 32-37). US 5,273,876 discloses a vaccine composed of attenuated hCMV. The attenuated virus has a DNA sequence necessary for hCMV replication and at least one foreign DNA sequence capable of expressing an antigenic polypeptide.

The prospect of hCMV treatment is not optimistic. The known antiviral drugs such as nucleoside analogs acyclovir (non-cyclic guanosine) and ara-A (adenine arabinoside) have no significant effect on active hCMV infection. It has now been found that cyclopropylguanosine (9-(1,2-dihydroxy-2-propoxymethylguanine) can act as an inhibitor of hCMV polymerase after being converted into its triphosphate form in cells. Continuous use of this drug by AIDS patients carrying hCMV can control hCMV infection, but patients who receive treatment for more than three months usually develop peripheral blood neutropenia and produce strains resistant to this drug. Drugs that have been proven to be effective in treating certain herpes virus infections, such as acyclovir, are not sufficiently effective in treating hCMV. Ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) and phosphonoformic acid (phosphonoformic acid) have large side effects and cannot obtain the safety of drugs similar to those approved for the treatment of other herpes viruses.

Treatment of hCMV infection is difficult because few therapeutic options are available. Currently available agents that inhibit viral replication (Ganciclovir, Cidofivir, Foscarnet, Maribavir and others under development) have made significant clinical progress, but may suffer from low oral bioavailability, poor activity, the development of hCMV resistant strains (due to mutations in the viral target) and dose-limiting toxicity (DeClercq E, 2003; Baldanti F and Gerna G, 2003; Gilbert C and Boivin G, 2005).

On May 21, 2021, Takeda announced that it had submitted an application for Maribavir to the FDA for the treatment of refractory, with or without drug resistance (R/R) post-transplant cytomegalovirus (hCMV). If approved, Maribavir will become the first and only small molecule compound for the treatment of post-transplant cytomegalovirus (hCMV) infection.

Maribavir is an hCMV antiviral drug that targets and inhibits UL97 protein kinase and its natural substrates, and has an innovative mechanism of inhibiting hCMV viral replication. Compared with existing therapies, maribavir has higher safety when used as a first-line therapy to prevent hCMV recurrence; maribavir achieves a 66.7% remission rate in the treatment of drug-resistant or refractory hCMV infection.

Maribavir (5,6-dichloro-2-(isopropylamino)-1-(β-L-ribofuranosyl)-1H-benzimidazole) is a benzimidazole derivative that can be used for medical treatment. It has very low solubility and is almost insoluble in water (0.019 g/L) (25°C). U.S. Patent 6077832 discloses the structure and its use for treating or preventing viral infections such as those caused by herpes viruses.

Studies have shown (US10765692) that maribavir can isomerize into one or more configurational stereoisomers or structural isomers under in vivo conditions. Maribavir compounds contain four chiral carbon centers in the furanose moiety, so maribavir may form one of 16 potential stereoisomers under various in vivo conditions.

Under in vivo conditions, maribavir can isomerize into other compounds that may (or may not) have the same or similar chemical, physical, and biological properties. The in vivo isomerization of maribavir results in the conversion of maribavir into other isomers that have the same molecular formula but different molecular structures. The different molecular structures can be combined into isomers that have different connectivity of the constituent atoms (configurational isomers) or into isomers that have the same "connectivity" but differ in the way the atoms and groups of atoms are oriented in space (configurational isomers). This in vivo molecular transformation is believed to result in a reduction in the effective maribavir concentration in vivo after treatment of a host with maribavir. In vivo isomerization converts and partitions the administered maribavir into other molecular entities that may or may not have the same or similar biological activity. If the isomerization results in the formation of an isomer that has a lower degree of corresponding biological activity relative to the activity of maribavir, then the isomerization will reduce the effective biological activity of the dose of maribavir administered to the host.

A Phase 3 maribavir clinical trial conducted by ViroPharma Incorporated evaluating maribavir for the prevention of hCMV in allogeneic stem cell or bone marrow transplant (SCT) patients did not meet its primary endpoint. In the primary analysis, there was no statistically significant difference between maribavir and placebo in reducing the incidence of hCMV disease. In addition, the study failed to meet its key secondary endpoint (ViroPharma press release dated February 9, 2009). The Phase III study results, at first glance, appear inconsistent with an earlier proof-of-concept (POC) maribavir Phase 2 clinical trial, in which ViroPharma reported positive preliminary results indicating that maribavir inhibited hCMV reactivation in SCT patients. Data from this study demonstrated that maribavir prophylaxis demonstrated potent antiviral activity, as measured by a significant reduction in the rate of CMV reactivation in allogeneic stem cell (bone marrow) transplant recipients, and that maribavir was well tolerated in this sick patient population when administered for up to 12 weeks (ViroPharma press release dated March 29, 2006).

However, the Phase III study results could be explained by the theory/discovery of immediate maribavir isomerization. A key unrecognized difference between the Phase II and Phase III studies is that the former provided a fasting dosing regimen of maribavir, whereas the latter allowed dosing under fasting or fed conditions (at the clinician’s discretion). The nature of the patient population in the Phase III study suggests that it is possible that few patients were dosed on the strict fasting dosing regimen previously used in the Phase II study. The change in dosing regimen in the Phase III study not only altered the in vivo dosing conditions of maribavir, but also altered the nature and/or extent of isomerization that occurred in vivo in maribavir, such that more maribavir was isomerized to other, less potent compounds, thereby reducing the effective bioavailable concentration of the maribavir drug to below the level necessary to adequately prevent hCMV infection and/or hCMV reactivation in the host.

The extent and nature of maribavir isomerization depends on the specific in vivo conditions to which the drug is exposed, which are variable. Potential mechanisms for isomerization of maribavir in vivo are through chemical isomerization (acid, base and/or metal-catalyzed isomerization), microbial-mediated isomerization, and/or host metabolism-induced isomerization. See, e.g., Okano, Kazuya, Tetrahedron, 65: 1937-1949 (2009); Kelly, James A. et al. J. Med; Chem., 29: 2351-2358 (1986); and Ahmed, Zakaria et al., Bangladesh J. Sci; Ind. Res., 25(1-4): 90-104 (2000).

Undesirable in vivo isomerization of maribavir can be prevented or at least reduced, thereby improving the bioavailability and efficacy of the drug, and/or counteracting potential side effects.

Reducing the effect of maribavir isomerization in vivo can improve the therapeutic effect of maribavir. The method used in US10765692 is: administering the drug under fasting conditions, or increasing the dose of maribavir; or using appropriate pharmaceutical technology, such as rapid Release formulations, delayed/controlled release formulations, combinations with antacids, intravenous (IV) formulations, combination formulations with antibiotics to prevent microbial isomerization.

However, due to the extremely poor solubility of maribavir, the dosage is large. When using a rapid release and increased maribavir dosage regimen, the dosage needs to reach 3200 mg/time, twice a day, and the daily dosage is as high as 6400 mg; even if a delayed release preparation or a regimen of combined use with antacids is used, the daily dosage is as high as 1600 mg, and the minimum is 800 mg.

Summary of the invention

The object of the present invention is to provide a maribavir prodrug. The technicians of the present invention have found that the maribavir prodrug of the present invention has significantly better solubility (such as water solubility) and bioavailability than the parent molecule; and after taking it, it can effectively avoid the in vivo isomerization of maribavir caused by the influence of gastric acid factors, which has very obvious medication advantages.

In one or more embodiments, the compounds of the present application have increased solubility.

In one or more embodiments, after administration of the compounds of the present application, such prodrugs can provide higher drug exposure (AUC) and maximum blood concentration (Cmax).

In one or more embodiments, the compounds of the present application maintain high blood concentration values for a longer period of time.

Specifically, the present invention relates to compounds with the following structures:

One or more embodiments of the present application provide salts formed by the compounds of the present application and alkaline earth metals, alkali metals, lysine, and tris(hydroxymethyl)aminomethane.

In one or more embodiments, the alkaline earth metal is beryllium, magnesium, calcium.

In one or more embodiments, the alkali metal is lithium, sodium, potassium.

One or more embodiments of the present application provide a pharmaceutical composition, which contains the compound described in the present application or its stereoisomer, solvate, deuterated substance or pharmaceutically acceptable salt, and a pharmaceutically acceptable excipient.

One or more embodiments of the present application provide the use of the compound of the present application or its stereoisomer, solvate, deuterated substance or pharmaceutically acceptable salt, or the pharmaceutical composition of the present application in the preparation of a drug for treating or preventing a disease caused by cytomegalovirus infection.

Another aspect of the present invention relates to a composition containing the compound of the present invention, and the composition further contains a pharmaceutically acceptable excipient.

The pharmaceutical compositions of the present disclosure may be administered orally, parenterally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal and intralesional. Regional injection or infusion technique.

The pharmaceutical composition can be in the form of a sterile injectable preparation, for example, in the form of a sterile injectable aqueous or oily suspension. Such suspensions can be configured using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The details of the preparation of these compounds are known to those skilled in the art.

When administered orally, the pharmaceutical compositions of the present disclosure may be administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, useful carriers/diluents include lactose, high and low molecular weight polyethylene glycols, and dry corn starch. When aqueous suspensions are administered orally, the active ingredient is mixed with an emulsifier and a suspending agent. If desired, certain sweeteners and/or flavoring agents and/or coloring agents may be added.

Other suitable carriers for the above compositions can be found in standard pharmaceutical textbooks, for example in "Remington's Pharmaceutical Sciences", 19th ed., Mack Publishing Company, Easton, Penn., 1995. Further details regarding the design and preparation of suitable delivery forms of pharmaceutical compositions are known to those skilled in the art from the disclosure.

On the other hand, the present invention provides a method for treating, preventing or delaying an infection caused by a virus, the method comprising administering a therapeutically effective amount of the above-mentioned compound, or its stereoisomer or a pharmaceutically acceptable salt thereof to a patient in need of treatment. Wherein the virus is a herpes virus, such as CMV. In addition, the above-mentioned compound, or its stereoisomer or a pharmaceutically acceptable salt thereof provided by the present invention can be co-administered with other therapies or therapeutic agents. The administration method can be performed simultaneously, sequentially or at certain time intervals.

In the present invention, in addition to the compound of the present invention or its pharmaceutically acceptable salt, other antiviral compounds may also be included, such as natural antiviral proteins, interferons, nucleoside analogs, cytosine-arabinoside, adenine-arabinoside, iodouridine, acyclovir, ganciclovir, sodium phosphate formate, Ganciclovir, Cidofivir or Foscarnet.

The dosage of the compound or pharmaceutical composition required for the treatment, prevention or delay is usually determined by the specific compound, patient, specific disease or condition and its severity, route of administration and frequency, etc., and needs to be determined by the attending physician according to the specific circumstances. For example, when the compound or pharmaceutical composition provided by the present invention is administered by intravenous route, it can be administered once a week or even at longer time intervals. The dosage level of the compound in the disclosure of the present invention is typically about 1 to about 500 milligrams per kilogram (mg/kg) of body weight per day, more specifically, about 1 to about 50 mg/kg of body weight per day. Typically, the pharmaceutical composition in the disclosure of the present invention can be administered about 1 to about 3 times a day, preferably before or after viral infection occurs. Or it can be administered in the form of continuous infusion, which can be used as a chronic or acute therapy. The number of active ingredients that can also be mixed with a carrier material to prepare a single dosage form will change with the host treated and the specific mode of administration.

The present invention also includes a method for treating CMV infection using an isotope-labeled compound of the present invention. The compounds described herein are identical in structure to those described herein, but one or more atoms are replaced by atoms whose atomic mass or mass number is different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, phosphorus, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³¹ P, ³⁵ S, ¹⁸ F and ³⁶ Cl, respectively. Compounds of the invention, prodrugs thereof, and pharmaceutically acceptable salts of the compounds or prodrugs thereof containing the above isotopes and/or isotopes of other atoms are within the scope of the invention. Certain isotopically labeled compounds of the invention, such as those incorporating radioactive isotopes such as ³ H and ¹⁴ C, can be used in drug and/or substrate tissue distribution assays, with tritiated (i.e. ³ H) and carbon-14 (i.e. ¹⁴ C) isotopes being particularly preferred because they are easy to prepare and detectable. In addition, substitution with lighter isotopes (e.g., deuterium and ² H) may offer certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some circumstances. Isotopically labeled compounds and prodrugs thereof used in the methods of the present invention can generally be prepared by performing procedures for preparing compounds disclosed in the art using readily available isotopically labeled reagents in place of non-isotopically labeled reagents.

On the other hand, the present invention relates to a product or a kit, comprising a container and a package insert, wherein the container contains a compound or a pharmaceutically acceptable salt thereof, or a composition containing the compound or a pharmaceutically acceptable salt thereof, and the package insert carries instructions for use of the drug. In a preferred embodiment, the product or kit further comprises one or more containers, which contain one or more other antiviral drugs for preventing or treating herpes virus (such as CMV) infection. In a preferred embodiment, the other drugs can be: such as natural antiviral proteins, interferons, and nucleoside analogs, cytosine-arabinoside, adenine-arabinoside, ioduridine, acyclovir, ganciclovir, sodium phosphate formate, Ganciclovir, Cidofivir or Foscarnet. 。

Physiologically or pharmaceutically acceptable salts of the prodrug compounds disclosed herein are within the scope of the present invention. The term "pharmaceutically acceptable salt" as used herein and in the claims is intended to include non-toxic base addition salts, and also includes acid salts, such as carboxylates or phosphates or phosphate monoesters containing such counterions such as ammonium; alkali metal salts, especially sodium or potassium salts; alkaline earth metal salts, especially calcium or magnesium salts; transition metal salts, such as zinc salts and salts containing suitable organic bases, such as lower alkylamines (methylamine, ethylamine, cyclohexylamine, etc.) or substituted lower alkylamines (such as hydroxy substituted alkylamines, such as diethanolamine, triethanolamine or monotromethamine, lysine, arginine, histidine, N-methylglucamine or bases, such as piperidine or morpholine. It should be understood that when isolated as a solid or crystalline form, pharmaceutically acceptable salts also include hydrates or water molecules trapped in the resulting compound substance.

In the present invention, "effective amount" refers to the amount of active ingredient required to cure or control the disease to a certain extent in the treatment of herpes virus (such as CMV virus).

Detailed ways

Reagent Abbreviation:

BSA: N,O-bis(trimethylsilyl)acetamide;

TMSOTf: trimethylsilyl trifluoromethanesulfonate

TsOH: p-toluenesulfonic acid; MsOH: methanesulfonic acid

Example 1: Preparation of (((2S,3R,4S,5S)-5-(5,6-dichloro-2-(isopropylamino)-1H-benzo[d]imidazol-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl dihydrogen phosphate

Step 1: (2S,3S,4S,5S)-2-(acetoxymethyl)-5-(2-bromo-5,6-dichloro-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-3,4-diacetic acid diester

Under _N2 , BSA (11.5 g, 56.4 mmol) was added to a solution of 2-bromo-5,6-dichloro-1H-benzo[d]imidazole (10 g, 37.6 mmol) in MeCN (1 L), and the reaction mixture was stirred at 70 °C for 1 hour and then cooled to room temperature. TMSOTf (12.77 g, 56.408 mmol) was added to the reaction. The reaction mixture was stirred at room temperature for 15 minutes. (2R, 3S, 4S, 5S)-5-(acetoxymethyl)tetrahydrofuran-2,3,4-triacetic acid triester (12.6 g, 39.5 mmol) was added. Thereafter, the reaction mixture was stirred at room temperature for 16 hours. The mixture was concentrated to remove the solvent and diluted with EtOAc (500 ml), washed with NaHCO ₃ (300 mL*2), brine (300 mL*2), dried over anhydrous sodium sulfate and concentrated to give (2S, 3S, 4S, 5S)-2-(acetoxymethyl)-5-(2-bromo-5,6-dichloro-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-3,4-diacetic acid diester (15 g, 28.6 mmol, yield: 76.1%) as a yellow solid.

LC/MS(ESI)m/z:523,525[M+H] ⁺

Step 2: (2S,3S,4R,5S)-2-(2-Bromo-5,6-dichloro-1H-benzo[d]imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol A solution of (2S,3S,4S,5S)-2-(acetoxymethyl)-5-(2-bromo-5,6-dichloro-1H-benzo[d]imidazol-1-yl)tetrahydrofuran-3,4-diacetate (15 g, 28.6 mmol) in MeOH (20 mL) was added to a solution of 7 M NH3 in MeOH (100 mL). The reaction mixture was stirred at room temperature for 16 h. The mixture was filtered and the filter cake was washed with MeOH (50 ml) to give (2S,3S,4R,5S)-2-(2-bromo-5,6-dichloro-1H-benzo[d]imidazol-1)-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (8 g, 20.1 mmol, yield: 70.2%) as a white solid.

LC/MS(ESI)m/z:397,399[M+H] ⁺

Step 3: ((3aS,4S,6S,6aS)-6-(2-bromo-5,6-dichloro-1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxol-4-yl)methanol

To a solution of (2S,3S,4R,5S)-2-(2-bromo-5,6-dichloro-1H-benzo[d]imidazol-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (8 g, 20.1 mmol) in acetone (80 mL) were added 4-methylbenzenesulfonic acid (0.69 g, 4.02 mmol) and 2,2-dimethoxypropane (10.5 g, 100 mmol). The reaction mixture was stirred at room temperature for 2 hours. The mixture was concentrated to remove the solvent and dissolved in EtOAc (100 ml). The organic phase was washed with brine (60 mL*2), dried over anhydrous sodium sulfate and concentrated to remove the solvent. The residue was purified by silica gel column chromatography (EtOAc in PE from 30% to 40%) to give ((3aS,4S,6S,6aS)-6-(2-bromo-5,6-dichloro-1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxan-4-yl)methanol (7 g, 16.0 mmol, yield: 79.5%) as a yellow solid.

LC/MS(ESI)m/z:437,439[M+H] ⁺

Step 4: ((3aS,4S,6S,6aS)-6-(5,6-dichloro-2-(isopropylamino)-1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxol-4-yl)methanol

To a solution of ((3aS,4S,6S,6aS)-6-(2-bromo-5,6-dichloro-1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxan-4-yl)methanol in EtOH (30 mL) was added propan-2-amine (5.49 mL, 63.9 mmol). The reaction mixture was stirred at 90 °C for 16 h. The mixture was concentrated and purified by silica gel column chromatography (EtOAc in PE from 60% to 70%) to give ((3aS,4S,6S,6aS)-6-(5,6-dichloro-2-(isopropylamino)-)1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxan-4-yl)methanol (2 g, 4.80 mmol, yield: 75.2%) as a yellow solid.

LC/MS(ESI)m/z:416,418[M+H] ⁺

Step 5: Di-tert-butyl ((((3aS,4S,6S,6aS)-6-(5,6-dichloro-2-(isopropylamino)-1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxol-4-yl)methoxy)methyl) phosphate

To a solution of ((3aS,4S,6S,6aS)-6-(5,6-dichloro-2-(isopropylamino)-1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxol- ₄ -yl)methanol (2.3 g, 5.53 mmol), NaI (1.66 g, 11.1 mmol), Ag ₂ O (2.54 g, 11.1 mmol) in DMF (100 mL) was added di-tert-butyl (chloromethyl)phosphate (4.29 g, 16.6 mmol) under N 2 conditions. The reaction mixture was stirred at room temperature for 3 hours. The mixture was diluted with EtOAc (100 mL) and filtered through celite. The filtrate was washed with brine (100 mL×3), dried over anhydrous sodium sulfate and concentrated. The residue was purified by reverse phase C-18 chromatography (aq. MeOH, 90% to 95%) to give di-tert-butyl ((((3aS,4S,6S,6aS)-6-(5,6-dichloro-2-(isopropylamino)-1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxol-4-yl)methoxy)methyl)phosphate (650 mg, 1.02 mmol, yield: 18.4%) as a white solid.

¹ H NMR (400 MHz, DMSO) δ7.53 (s, 1H), 7.41 (s, 1H), 6.82 (d, J = 7.5 Hz, 1H), 6.05 (s, 1H), 5.24 (dd, J = 9.3, 5.7 Hz, 1H), 5.12 (dd, J = 12.6, 5.6 Hz, 1H), 4.98 (s, 2H), 4.21 (d, J = 2.1 Hz, 1H), 4.08-4.00 (m, 1H), 3.96 (d, J = 2.5 Hz, 2H), 1.58 (s, 3H), 1.41 (d, J = 5.3 Hz, 18H), 1.30 (s, 3H), 1.22 (dd, J = 6.5, 2.1 Hz, 6H).

LC/MS(ESI)m/z:638,640[M+H] ⁺

Step 6: (((2S,3R,4S,5S)-5-(5,6-dichloro-2-(isopropylamino)-1H-benzo[d]imidazol-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl dihydrogen phosphate

A solution of di-tert-butyl ((((3aS,4S,6S,6aS)-6-(5,6-dichloro-2-(isopropylamino)-1H-benzo[d]imidazol-1-yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxol-4-yl)methoxy)methyl)phosphate (20 mg, 0.031 mmol) in MeCN (0.5 mL) was added to a solution of 0.4% MsOH in water (0.5 mL). The reaction mixture was stirred at room temperature for 3 days. 33% ammonium hydroxide (0.5 mL) was added to the solution, the mixture was concentrated, and the residue was purified by HPLC to give (((2S,3R,4S,5S)-5-(5,6-dichloro-2-(isopropylamino)-1H-benzo[d]imidazol-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)methyl dihydrogen phosphate (0.5 mg) as a white solid.

¹ H-NMR (400 MHz, D2O) δ 7.47-7.37 (m, 1H), 7.29-7.14 (m, 1H), 5.71-5.60 (m, 1H), 5.18-5.11 (m, 1H), 4.98 (dd, J = 11.3, 5.5 Hz, 1H), 4.46-4.35 (m, 1H), 4.30-4.24 (m, 1H), 4.19 (s, 1H), 3.95-3.73 (m, 3H), 1.15 (d, J = 6.0 Hz 6H).

LC/MS(ESI)m/z:486,488[M+H] ⁺

Example 2 Solubility determination of maribavir and compound 1

(a) Kinetic solubility:

(1) Weigh the test compound to measure solubility (approximately 1.0 mg in each of three separate 1.5 mL glass vials);

(2) One of the vials is used as a standard, and the other two are used to measure solubility in duplicate;

(3) Use a pipette to add an appropriate volume (~1000 μL) of PBS pH 2.0, pH 6.5, or pH 7.4 to each vial of the solubility sample plate, depending on the amount used. Add a stir bar to each vial and seal the vial using a molded PTFE/silicone stopper;

(4) Transfer the solubility sample plate to a shaker and shake at 1100 RPM at 25°C for 24 hours;

(5) After 24 hours, remove the stir bar using a large magnet and transfer the sample from the solubility sample plate to the filter plate using a pipette. Filter all compounds using a vacuum manifold. Dilute the filtrate 1000-fold with a mixture of H ₂ O and acetonitrile (1:1 v/v). The dilution factor varies depending on the solubility value and LC-MS signal response;

(6) The solubility sample plate and standard plate were placed in a well plate autosampler at 4°C and evaluated by LC-MS/MS analysis.

(b) Thermodynamic solubility:

(1) Stock solutions of test compounds and control compounds were prepared in DMSO at a concentration of 10 mM;

(2) 30 μL of stock solution (10 mM) of each sample was placed in order into its appropriate 96-well rack. 970 μL of PBS pH 2.0, pH 6.5, or pH 7.4 was added to each vial of the solubility sample plate. The assay was performed in duplicate. A stirring bar was added to each vial and sealed with a molded PTFE/silicone plug. The solubility sample plate was then transferred to an oscillator and shaken at 1100 RPM for 2 hours at 25°C. After 2 hours, the plug was removed and the stirring bar was removed using a large magnet, and the sample from the solubility sample plate was transferred to the filter plate. All samples were filtered using a vacuum manifold. A 5 μL aliquot was taken from the filtrate, and then 5 μL DMSO and 490 μL H ₂ O and acetonitrile mixture (1:1 v/v) were added. The dilution factor varied depending on the solubility value and LC-MS signal response;

(3) Place the plate into a well plate autosampler and evaluate the samples by LC-MS/MS analysis.

(c) Experimental results and conclusions:

The experimental results show that under different pH conditions, the solubility of compound 1 is greatly improved compared with Maribavir, with the increase being at least 10 times.

Example 3 Pharmacokinetic Test

(a) Drugs and reagents: The compounds to be tested were prepared into solutions using the following solvents (the preparation process of the drug solution is as follows: at room temperature, weigh appropriate amounts of maribavir and compound 1, add them to a container of appropriate size, add an appropriate volume of solvent, and perform vortexing or ultrasonic treatment when necessary to obtain a suspension of the target concentration), see Table 1.

Table 1 Preparation of test compound solutions

(b) Test animals: male SPF grade SD rats (3 in each group), purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

(c) Dosage and Animal Grouping: The test compound solution was administered to rats by oral gavage. All animals ate a normal diet before and after administration. The dosage of each group of animals is shown in Table 4.

Table 4 Grouping and dosage

(d) Pharmacokinetic testing:

Blood samples from each group of animals were collected through the jugular vein. About 0.20 mL of whole blood was collected from each animal at each time point. EDTA-K2 was used for anticoagulation. The blood collection time points were as follows: 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h after administration. After blood collection, the blood collection tube containing the anticoagulant was repeatedly inverted several times to fully mix. After blood samples were collected, they were placed in a low-temperature storage box. After all samples within this time point were collected, the plasma was centrifuged to separate the plasma (centrifugation conditions: 4000 rpm, 5 minutes, 4°C). The collected plasma was stored at -75±15°C until analysis. All plasma samples were analyzed by LC-MS/MS. The concentration of the drug to be tested in the sample was determined by the standard curve. The pharmacokinetic parameters of the plasma determination results were calculated using the software WinNonlin5.2 (PhoenixTM). The blood concentration (Cmax), peak time (Tmax), and area under the curve (AUC) of the test compound were calculated respectively. (e) Analysis of pharmacokinetic experimental results

The results showed that at each dose, the AUC and Cmax values of compound 1 were significantly improved compared with Maribavir. The AUC value could be increased by as little as 10 times, or even more than 30 times, and the Cmax value could be increased by as little as 21 times, or even more than 65 times.

Moreover, at high doses (groups not less than 200 mg/kg), the blood drug concentrations at each time point (the measured blood drug concentrations are all blood drug concentrations of Maribavir) are significantly higher than the blood drug concentrations of Maribavir, indicating that the release rate of compound 1 has a certain delaying effect.

Claims (4)

A compound having the following structure, or a stereoisomer or a pharmaceutically acceptable salt thereof, selected from the following structures:
A pharmaceutical composition comprising the compound according to claim 1 or its stereoisomer or pharmaceutically acceptable salt, and a pharmaceutically acceptable excipient. Use of the compound according to claim 1 or its stereoisomer or pharmaceutically acceptable salt in the preparation of a medicament for treating or preventing herpes virus infection. The use according to claim 3, wherein the herpes virus is CMV virus.