Peptidomimetic compounds, pharmaceutical compositions thereof, and uses thereof

Abstract

This invention discloses peptide-like compounds, pharmaceutical compositions thereof, and their applications. Specifically, this invention discloses a peptide-like compound, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof, which has a novel structure and the potential to treat or prevent infections caused by orthopoxviruses such as monkeypoxvirus and vaccinia virus, and to treat or prevent related diseases caused by orthopoxvirus infections such as monkeypoxvirus and vaccinia virus.

Description

[0001] This application claims priority to Chinese patent application 2025103851884, filed on March 28, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to peptide-like compounds, pharmaceutical compositions thereof, and their applications. Background Technology

[0003] Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus belonging to the Poxviridae family. It was first discovered in monkeys in 1958, and the first human case was reported in 1970. After infection, patients often present with symptoms such as fever, headache, swollen lymph nodes, and skin blisters. In severe cases, it can lead to encephalitis, sepsis, permanent vision loss, and even death. Besides monkeypox virus, the orthopoxvirus genus also includes three other viruses that can infect humans: variola virus (VARV), cowpox virus (CPXV), and vaccinia virus (VACV).

[0004] Currently, no vaccines or drugs with proven efficacy have been approved for the prevention and treatment of monkeypox virus infection. Only two vaccines (ACAM2000 and Jynneos) and two antiviral drugs (Tecovirimat and Brincidofovir) have been approved by the U.S. Food and Drug Administration (FDA) for the prevention and treatment of smallpox viruses. Based on the safety of Tecovirimat in humans and its anti-monkeypox virus efficacy in animal models, the European Medicines Agency (EMA) approved it for the treatment of monkeypox virus infection. However, the latest clinical data show that Tecovirimat is not significantly more effective than placebo in the clinical treatment of monkeypox virus. Currently, monkeypox virus continues to cause intermittent outbreaks and circulate globally. Therefore, developing novel, low-toxicity, highly effective anti-monkeypox virus drugs with independent intellectual property rights is not only crucial for addressing the current monkeypox virus epidemic but also has significant public health and social value for responding to potential future orthopox virus outbreaks.

[0005] The life cycle of ornithopox virus is highly conserved, involving five morphologies of viral particles during replication and infection: immature virion (IV), intracellular mature virion (IMV), intracellular enveloped virion (IEV), cell-associated enveloped virion (CEV), and extracellular enveloped virion (EEV). Monkeypox virus replication primarily occurs within the "viral factory" in the host cell cytoplasm, including viral DNA replication and transcription, accumulation of structural proteins, and viral particle assembly. The accumulation of viral structural proteins depends on the proteolytic action of two proteases: the core protease and the G1L protease. The core protease is a cysteine ​​protease that mainly acts in the late stages of viral assembly. Furthermore, the G7L protein, a crucial component of the intracellular mature viral core, also relies on the hydrolytic action of the core protease to function.

[0006] Studies have shown that the core protease plays a crucial role in the formation of infectious IMV particles of orthopoxvirus. This protease is highly conserved within the orthopoxvirus genus and has no homologous proteins in humans, thus exhibiting extremely high selectivity. The core protease sequences of monkeypoxvirus, smallpoxvirus, and vaccinia virus share up to 99% homology, implying that inhibitors targeting the core protease may have broad-spectrum inhibitory effects against various orthopoxviruses. Inhibiting the activity of the core protease can effectively block IMV formation, thereby inhibiting viral replication and proliferation. Proteases have been successfully used as antiviral drug targets in the treatment of HIV, HCV, and SARS-CoV-2. Therefore, elucidating the three-dimensional structure of the core protease and designing drugs based on this structure to develop highly effective core protease inhibitors is not only of significant scientific importance for the prevention and control of monkeypoxvirus but also offers broad application prospects for addressing potential threats from the orthopoxvirus genus in the future. Summary of the Invention

[0007] This invention aims to address the technical problem of the lack of highly effective anti-monkeypox virus drugs in the prior art, and provides a peptide-like compound, its pharmaceutical composition, and its applications. Experiments show that the compound of this invention has a significant inhibitory effect on the core protease of monkeypox virus, demonstrating great potential in the treatment of monkeypox virus and other orthopoxvirus infections and related diseases. This invention provides a novel pharmaceutical strategy for addressing major infectious diseases caused by monkeypox virus and has significant clinical application value.

[0008] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0009] This invention provides a compound A, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof, characterized in that the compound A has any of the following structures: Formula (I); Formula (II); Formula (III); Formula (IV).

[0010] The present invention also provides a pharmaceutical composition comprising the aforementioned compound A, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

[0011] The present invention also provides the use of the aforementioned compound A, its pharmaceutically acceptable salt, its solvate, a solvate of its pharmaceutically acceptable salt, or the aforementioned pharmaceutical composition; The application is to prepare a drug for treating and / or preventing infection with Orthopoxvirus, or to prepare a drug for treating and / or preventing related diseases caused by Orthopoxvirus infection.

[0012] In certain preferred embodiments of the present invention, certain diseases or conditions in the application of compound A, its pharmaceutically acceptable salt, its solvate, a solvate of its pharmaceutically acceptable salt, or the pharmaceutical composition have the following definitions, and any unmentioned parts are as described in any embodiment of the present invention (hereinafter referred to as "in one embodiment of the present invention"). Other aspects of the present invention are referred to using the same abbreviations as this invention and will not be described further.

[0013] In one embodiment of the present invention, the orthopox virus is selected from one or more of mpox virus, smallpox virus, cowpox virus, and vaccinia virus.

[0014] In one embodiment of the present invention, the orthopoxvirus is a monkeypox virus, such as the C.1 subgroup of the Clade IIb branch of monkeypox virus.

[0015] In one embodiment of the present invention, the vaccinia virus is a vaccinia virus, such as vaccinia virus strain WR.

[0016] In one aspect of the present invention, the related diseases caused by orthopoxvirus infection are one or more of the following: fever, headache, back pain, myalgia, lymphadenopathy, rash, mucosal rash, and generalized papules caused by orthopoxvirus infection.

[0017] In one aspect of the present invention, the related diseases caused by the orthopoxvirus infection are one or more of the following caused by monkeypox virus infection: fever, headache, back pain, myalgia, lymphadenopathy, rash, and mucosal rash.

[0018] In one aspect of the present invention, the related diseases caused by the orthopoxvirus infection are local lymph node swelling or systemic papules caused by vaccinia virus infection.

[0019] The present invention also provides the use of the aforementioned compound A, its pharmaceutically acceptable salt, its solvate, a solvate of its pharmaceutically acceptable salt, or the aforementioned pharmaceutical composition in the preparation of a viral core protease inhibitor.

[0020] In one embodiment of the present invention, the viral core protease is a core protease of a virus belonging to the genus Orthopoxvirus; the core protease of the Orthopoxvirus virus is preferably a core protease of monkeypoxvirus, smallpoxvirus, vaccinia virus, or vaccinia virus, and more preferably a core protease of monkeypoxvirus or vaccinia virus.

[0021] In one embodiment of the present invention, the viral core protease is a monkeypox virus core protease.

[0022] In one aspect of the present invention, the viral core protease inhibitor is used in vivo or in vitro, for example, in vitro.

[0023] The present invention also provides a method for treating orthopoxvirus infection or related diseases caused by orthopoxvirus infection, comprising administering to a patient in need a therapeutically effective amount of the aforementioned compound A, a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0024] In one embodiment of the present invention, the orthopox virus is selected from one or more of mpox virus, smallpox virus, cowpox virus, and vaccinia virus.

[0025] In one embodiment of the present invention, the orthopoxvirus is a monkeypox virus, such as the C.1 subgroup of the Clade IIb branch of monkeypox virus.

[0026] In one embodiment of the present invention, the vaccinia virus is a vaccinia virus, such as vaccinia virus strain WR.

[0027] In one aspect of the present invention, the related diseases caused by orthopoxvirus infection are one or more of the following: fever, headache, back pain, myalgia, lymphadenopathy, rash, mucosal rash, and generalized papules caused by orthopoxvirus infection.

[0028] In one aspect of the present invention, the related diseases caused by the orthopoxvirus infection are one or more of the following caused by monkeypox virus infection: fever, headache, back pain, myalgia, lymphadenopathy, rash, and mucosal rash.

[0029] In one aspect of the present invention, the related diseases caused by the orthopoxvirus infection are local lymph node swelling or systemic papules caused by vaccinia virus infection.

[0030] The present invention also provides a method for inhibiting the activity of viral core protease, comprising the steps of using the aforementioned compound A, a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

[0031] In one embodiment of the present invention, the viral core protease is a core protease of a virus belonging to the genus Orthopoxvirus; the core protease of the Orthopoxvirus virus is preferably a core protease of monkeypoxvirus, smallpoxvirus, vaccinia virus, or vaccinia virus, and more preferably a core protease of monkeypoxvirus or vaccinia virus.

[0032] In one embodiment of the present invention, the viral core protease is a monkeypox virus core protease.

[0033] Unless otherwise specified, the terms used in this invention may be defined as follows: The term "pharmaceutically acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent.

[0034] The term "pharmaceutical excipients" refers to all substances contained in a pharmaceutical preparation other than the active pharmaceutical ingredient, and are generally divided into two main categories: excipients and additives. See Volume IV of the Pharmacopoeia of the People's Republic of China (2020 Edition) or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009 Sixth Edition).

[0035] The term "solvate" refers to a substance formed by the combination of a compound and a solvent (including but not limited to water, methanol, ethanol, etc.). Solvates are classified into stoichiometric solvates and non-stoichiometric solvates.

[0036] The term "solvate of a pharmaceutically acceptable salt" refers to a substance formed by the combination of a compound with a pharmaceutically acceptable acid or base and a solvent (including but not limited to water, methanol, ethanol, etc.). The amount of solvent can be stoichiometric or non-stoichiometric.

[0037] The term “treatment” refers to a therapeutic approach. When a specific condition is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; or (4) slowing the development of the condition or one or more biological manifestations of the condition.

[0038] The term "prevention" refers to a period of time during which a subject remains healthy relative to the disease or condition mentioned herein. It should be understood that this period of time depends on the amount of pharmaceutical compound administered and individual factors of the subject discussed elsewhere in this specification. It should be understood that prevention may not be effective in all subjects treated with the compound according to the invention. However, the term requires, preferably, the effective prevention of a statistically significant portion of a cohort or group of subjects from developing the disease or condition referred to herein or its accompanying symptoms. Preferably, in this case, a group or cluster of subjects is anticipated who would typically, i.e., without taking the preventive measures according to the invention, develop the disease or condition referred to herein. Those skilled in the art can readily determine whether a portion is statistically significant using various well-known statistical evaluation tools discussed elsewhere in this specification.

[0039] The term "inhibitor" refers to a class of compounds or biomolecules that interfere with the normal biological function of target molecules (such as enzymes, receptors, or signaling proteins) by specifically binding to their active or allosteric sites. Their mechanisms of action can be classified into competitive, non-competitive, and anti-competitive types, specifically by inhibiting the catalytic activity, conformational changes, or interactions with other molecules of the target molecule, ultimately leading to the obstruction of related metabolic pathways or signal transduction.

[0040] In this invention, the "inhibitor" can be used in mammalian organisms; it can also be used in vitro, mainly for experimental purposes, such as providing a standard or control sample for comparison, or preparing a kit according to conventional methods in the art.

[0041] The term "viral infection-related diseases" refers to a set of clinical symptoms caused by viral invasion of host cells and the resulting replication and proliferation, leading to direct cellular damage, immunopathological responses, or gene integration mechanisms. The pathological process involves a dynamic interplay between the virus and the host immune system, and clinical manifestations can range from asymptomatic latent infection to multiple organ failure. The apparent symptoms are essentially external manifestations of the virus-host interaction, exhibiting significant target organ specificity and diverse pathological processes.

[0042] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0043] The reagents and raw materials used in this invention are all commercially available.

[0044] The significant advancements of this invention are as follows: To date, there have been no reports on the development of drugs targeting the core protease of monkeypox virus. This invention is the first to creatively discover that a class of self-designed peptide-like small molecules can significantly inhibit the activity of the monkeypox virus core protease, possessing the potential to treat related diseases caused by monkeypox virus. Furthermore, these small molecules are safe and reliable, allowing for rapid advancement into clinical trials and providing an effective treatment for combating monkeypox virus. Attached Figure Description

[0045] Figure 1 The results showed that small molecules A3-A6 exhibited inhibitory activity against the monkeypox virus core protease. The inhibition rate curves of small molecules A3-A6 against the monkeypox virus core protease corresponded to... Figure 1 Parts a, b, c, and d; Figure 2 The results showed that small molecules A3-A6 had inhibitory activity against vaccinia virus replication, and the inhibition rate curves of small molecules A3-A6 against vaccinia virus corresponded to... Figure 2 Parts a, b, c, and d; Figure 3The results showed that small molecules A3-A6 had inhibitory activity against monkeypox virus replication, and the inhibition rate curves of small molecules A3-A6 against monkeypox virus corresponded to... Figure 3 Parts a, b, c, and d. Detailed Implementation

[0046] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0047] Example 1: Synthesis and structural characterization of the compound

[0048] The synthetic routes for compounds A3-A6 are as follows.

[0049]

[0050] Preparation of compound A3

[0051] 1. Synthesis of intermediates 1-3

[0052] Compound 1-1 (2.00 g, 7.21 mmol) was dissolved in dichloromethane (100 mL) and then cooled to -20°C. o C, then HATU (4.11 g, 10.82 mmol) was added to the reaction solution, and the mixture was stirred for 30 minutes. Compounds 1-2 (1.11 g, 7.21 mmol) and DIPEA (2.80 g, 21.64 mmol) were then added sequentially, and the reaction was maintained at this temperature overnight. After the reaction was complete, the mixture was allowed to return to room temperature naturally. The mixture was washed with 1M hydrochloric acid solution (100 mL × 3), saturated sodium bicarbonate solution (100 mL × 3), and saturated sodium chloride solution (100 mL × 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compounds 1-3. This crude compound was purified by rapid column chromatography (PE / EA = 3:1 to 1:1, v / v) to obtain compound 1-3 (2.05 g, 78%). LRMS (ESI) m / z 263.1[M – Boc + H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 8.30 (d, J = 7.3 Hz, 1H), 7.23 –7.16 (m, 4H), 4.62 – 4.54 (m, 2H), 4.40 – 4.30 (m, 1H), 4.28 – 4.15 (m, 1H), 3.57 (s, 3H), 3.08 (d,J = 6.1 Hz, 2H), 1.36 (s, 9H), 1.20 (d, J = 7.3 Hz, 3H).

[0053] 2. Synthesis of intermediates 1-4

[0054] Compounds 1-3 (2.00 g, 5.52 mmol) were dissolved in dichloromethane (30 mL), and then 20 mL of hydrochloric acid solution (4 M 1,4-dioxane solution) was added. The mixture was stirred at room temperature for 1 h. The raw materials were consumed by TLC monitoring. The solvent was removed by vacuum concentration and the mixture was used directly in the next step.

[0055] 3. Synthesis of intermediates 1-6

[0056] Compounds 1-5 (1.00 g, 3.53 mmol) were dissolved in dichloromethane (50 mL) and then cooled to -20°C. o C, then HATU (2.01 g, 5.29 mmol) was added to the reaction solution, and the mixture was stirred for 30 minutes. Compounds 1-4 (1.16 g, 3.88 mmol) and DIPEA (1.37 g, 10.59 mmol) were then added sequentially, and the reaction was maintained at this temperature overnight. After the reaction was complete, the mixture was allowed to return to room temperature. The solution was washed with 1M hydrochloric acid solution (50 mL × 3), saturated sodium bicarbonate solution (50 mL × 3), and saturated sodium chloride solution (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compounds 1-6. These crude compounds 1-6 were purified by rapid column chromatography (PE / EA = 2:1 to 1:2, v / v) to obtain compounds 1-6 (1.23 g, 66%). LRMS (ESI) m / z 528.2 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 7.64 (d, J = 9.0 Hz, 1H), 7.29 – 7.20(m, 3H), 7.19 – 7.12 (m, 2H), 7.12 – 7.06 (m, 1H), 7.06 – 7.00 (m, 2H), 5.70(d, J = 8.2 Hz, 1H), 4.75 (dd, J = 12.4, 0.9 Hz, 1H), 4.61 (dd, J= 12.4, 0.9Hz, 1H), 4.57 – 4.49 (m, 2H), 4.49 – 4.41 (m, 1H), 3.69 (s, 3H), 3.33 – 3.26(m, 1H), 3.16 – 3.02 (m, 2H), 3.02 – 2.94 (m, 1H), 1.38 (d, J = 6.1 Hz, 3H).

[0057] Compounds 1-5 here are .

[0058] 4. Synthesis of intermediates 1-7

[0059] Compounds 1-6 (1.00 g, 1.90 mmol) were dissolved in dichloromethane (15 mL), and then 15 mL of hydrochloric acid solution (4 M 1,4-dioxane solution) was added. The mixture was stirred at room temperature for 1 h. The raw materials were consumed by TLC monitoring. The solvent was removed by vacuum concentration and the mixture was used directly in the next step.

[0060] 5. Synthesis of intermediates 1-9

[0061] Crude product 1-7 (0.80 g, 1.72 mmol) was dissolved in dichloromethane (20 mL) and then cooled to 0. o C. TEA (436 mg, 4.31 mmol) and DMAP (42 mg, 0.34 mmol) were added sequentially, followed by the slow dropwise addition of compounds 1-8 (237 mg, 2.07 mmol). The system was brought to room temperature and stirred overnight. After the reaction was complete, the mixture was washed sequentially with 1M hydrochloric acid solution (20 mL × 3), saturated sodium bicarbonate solution (20 mL × 1), and saturated sodium chloride solution (20 mL × 1). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then subjected to rapid column chromatography (DCM / MeOH = 50:1 to 30:1, v / v) to give compounds 1-9 (0.72 g, 83%). LRMS(ESI) m / z 506.1 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 7.64 (d, J = 9.0 Hz,1H), 7.29 – 7.20 (m, 3H), 7.19 – 7.12 (m, 2H), 7.12 – 7.06 (m, 1H), 7.06 –7.00 (m, 3H), 4.75 (dd, J= 12.5, 0.9 Hz, 1H), 4.61 (dd, J = 12.4, 1.0 Hz, 1H), 4.53 (dd, J = 7.6, 4.9 Hz, 1H), 4.50 – 4.42 (m, 1H), 4.17 – 4.09 (m,1H), 3.69 (s, 3H), 3.32 – 3.25 (m, 1H), 3.15 – 3.08 (m, 1H), 3.08 – 2.95 (m,5H), 1.38 (s, 3H).

[0062] Compounds 1-8 here are .

[0063] 6. Synthesis of intermediates 1-10

[0064] Compounds 1-9 (0.50 g, 0.99 mmol) were dissolved in 9 mL of methanol, and then lithium hydroxide monohydrate (0.12 g, 2.97 mmol, dissolved in 5 mL of water) was added. The mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC until the starting material was fully reacted. 1 M hydrochloric acid was slowly added to adjust the pH to 1-2, followed by extraction with EA (20 mL × 2). The combined EA phases were washed with saturated sodium chloride (20 mL × 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compounds 1-10, which were used directly in the next reaction without purification. LRMS(ESI) m / z 492.1 [M + H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 7.85 (d, J = 9.0 Hz,1H), 7.29 – 7.20 (m, 3H), 7.19 – 7.13 (m, 2H), 7.13 – 7.06 (m, 1H), 7.06 –7.00 (m, 3H), 4.75 (dd, J = 12.5, 0.9 Hz, 1H), 4.64 – 4.58 (m, 1H), 4.52 (dd, J = 7.6, 4.9 Hz, 1H), 4.35 – 4.26 (m, 1H), 4.13 (dt, J= 10.8, 7.6 Hz, 1H), 3.32 – 3.25 (m, 1H), 3.15 – 3.08 (m, 1H), 3.08 – 2.95 (m, 5H), 1.38 (s, 3H).

[0065] 7. Synthesis of compound A3 ((S)-N-((S)-1-((cyanomethyl)amino)-1-oxopropane-2-yl)-2-((S)-3-(4-fluorophenyl)-2-(methylsulfonamido)propionyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide)

[0066] Compound 1-10 (100 mg, 0.20 mmol) was dissolved in dichloromethane (10 mL) and then cooled to -20°C. o C. Then, HATU (117 mg, 0.31 mmol) was added to the reaction solution, and the mixture was stirred for 30 minutes. Compounds 1-11 (20.7 mg, 0.22 mmol) and DIPEA (79 mg, 0.61 mmol) were then added sequentially, and the reaction was maintained at this temperature overnight. After the reaction was complete, the mixture was allowed to return to room temperature. The solution was washed with 1M hydrochloric acid solution (10 mL × 3), saturated sodium bicarbonate solution (10 mL × 3), and saturated sodium chloride solution (10 mL × 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound A3. This crude compound A3 was purified by rapid column chromatography (DCM / MeOH = 30:1 to 20:1) to obtain compound A3 (67 mg, yield 62%). LRMS (ESI) m / z 530.2 [M + H] + HRMS (ESI) m / z [M + H] + Calculated for C 25 H 29 FN5O5S, 530.1868, found 530.1867. HPLC purity 99.35%.

[0067] 1 H NMR (500 MHz, DMSO- d 6) δ 8.41 (t, J = 5.6 Hz, 1H), 8.09 (d, J = 7.4Hz, 1H), 7.57 (d, J = 9.1 Hz, 1H), 7.46 (dd, J= 8.5, 5.7 Hz, 1H), 7.33 –7.27 (m, 2H), 7.24 – 7.20 (m, 2H), 7.20 – 7.08 (m, 2H), 7.07 – 7.02 (m, 1H), 4.97 (d, J = 15.0 Hz, 1H), 4.80 (t, J = 6.4 Hz, 1H), 4.69 – 4.60 (m, 2H), 4.25 – 4.15 (m, 1H), 4.10 (d, J = 5.6 Hz, 2H), 3.17 – 2.97 (m, 2H), 2.89 –2.66 (m, 2H), 2.49 (s, 3H), 1.19 (d, J = 7.2 Hz, 3H). 13 C NMR (150 MHz, DMSO- d 6) δ 172.6, 170.8, 170.3, 161.1 (d, J = 242.3 Hz), 133.8, 133.2 (d, J = 3.5Hz), 133.2, 131.6 (d, J = 7.9 Hz), 127.4, 127.1, 126.3, 125.9, 117.4, 114.7(d, J = 21.0 Hz), 54.1, 53.7, 47.8, 45.2, 40.7, 36.6, 30.9, 27.0, 17.7. 19 FNMR (471 MHz, DMSO- d 6) δ -116.91 – -117.05 (m).

[0068] The structures of compounds A3-A6 are as follows: Compound A3; Compound A4; Compound A5; Compound A6.

[0069] The synthesis of compounds A4-A6 is similar to that of compound A3, differing only in the structures of intermediates 1-5 and 1-8. Specific characterization data are as follows: Structural characterization data of compound A4 ((S)-N-((S)-1-((cyanomethyl)amino)-1-oxopropane-2-yl)-2-((S)-3-(3,5-difluorophenyl)-2-(methylsulfonamido)propionyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide): LRMS (ESI) m / z 548.2 [M + H] + HRMS (ESI) m / z [M + H] + Calculated for C 25 H 28 F2N5O5S, 548.1774, found 548.1773. HPLC purity > 99.99%.

[0070] 1 H NMR (500 MHz, DMSO- d 6) δ 8.42 (t, J = 5.6 Hz, 1H), 8.14 (d, J = 7.5Hz, 1H), 7.53 (d, J = 9.1 Hz, 1H), 7.37 – 7.32 (m, 1H), 7.25 – 7.14 (m, 4H), 7.12 – 7.02 (m, 2H), 5.02 (d, J = 15.1 Hz, 1H), 4.81 – 4.72 (m, 2H), 4.66 (d, J = 15.0 Hz, 1H), 4.28 – 4.16 (m, 1H), 4.10 (d, J = 5.6 Hz, 2H), 3.19 – 2.95(m, 3H), 2.83 – 2.74 (m, 1H), 2.59 (s, 3H), 1.19 (d, J = 7.2 Hz, 3H). 13 C NMR (150 MHz, DMSO-) d 6) δ 172.6, 170.4, 170.3, 162.0 (dd, J = 245.1, 13.3 Hz), 141.4 (t, J= 9.5 Hz), 133.9, 133.6, 127.3, 127.1, 126.3, 125.9, 117.4, 112.9(dd, J = 48.7, 24.4 Hz), 102.0 (t, J = 25.6 Hz), 54.2, 53.7, 47.8, 45.2,40.7, 36.9, 31.0, 27.0, 17.7. 19 F NMR (471 MHz, DMSO- d 6) δ -111.29 – -111.41(m).

[0071] Structural characterization data of compound A5 ((S)-N-((S)-1-((cyanomethyl)amino)-1-oxopropane-2-yl)-2-((S)-2-(cyclopropanesulfonamido)-3-(4-fluorophenyl)propionyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide): LRMS (ESI) m / z 556.1 [M + H] + HRMS (ESI) m / z [M + H] + Calculated for C 27 H 31 FN5O5S, 556.2024, found 556.2024. HPLC purity 97.59%.

[0072] 1 H NMR (500 MHz, DMSO- d 6) δ 8.42 (t, J = 5.6 Hz, 1H), 8.08 (d, J = 7.4Hz, 1H), 7.54 (d, J = 9.5 Hz, 1H), 7.48 – 7.42 (m, 1H), 7.36 – 7.29 (m, 2H), 7.23 (d, J = 2.8 Hz, 2H), 7.18 – 7.03 (m, 3H), 4.94 (d, J = 15.0 Hz, 1H),4.83 – 4.72 (m, 2H), 4.59 (d, J = 15.0 Hz, 1H), 4.26 – 4.14 (m, 1H), 4.11 (d,J = 5.6 Hz, 2H), 3.19 – 3.11 (m, 1H), 3.08 – 2.72 (m, 3H), 2.10 – 2.02 (m,1H), 1.19 (d, J = 7.1 Hz, 3H), 0.88 – 0.44 (m, 4H). 13 C NMR (150 MHz, DMSO- d 6)δ 172.6, 171.0, 170.4, 161.0 (d, J = 242.0 Hz), 133.9, 133.7, 133.2 (d, J =2.9 Hz), 131.5 (d, J = 7.9 Hz), 127.3, 127.2, 126.4, 125.9, 117.4, 114.7 (d, J = 21.1 Hz), 54.6, 53.9, 47.9, 45.1, 36.7, 30.9, 30.2, 27.0, 17.7, 4.8, 4.4. 19 F NMR (471 MHz, DMSO- d 6) δ -117.06 – -117.22 (m).

[0073] Structural characterization data of compound A6 ((S)-N-((S)-1-((cyanomethyl)amino)-1-oxopropane-2-yl)-2-((S)-2-(cyclopropanecarbamoyl)-3-(4-fluorophenyl)propionyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide): LRMS (ESI) m / z 520.3 [M + H] + HRMS (ESI) m / z [M + H] + Calculated for C 28 H 31 FN5O4, 520.2355, found 520.2357. HPLC purity 98.11%.

[0074] 1 H NMR (500 MHz, DMSO- d 6) δ 8.46 (d, J = 8.8 Hz, 1H), 8.37 (t,J = 5.7Hz, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.38 – 7.32 (m, 1H), 7.29 – 7.17 (m, 2H), 7.16 – 7.06 (m, 3H), 7.03 – 6.98 (m, 2H), 5.13 – 5.05 (m, 1H), 4.91 (d, J =15.0 Hz, 1H), 4.71 – 4.64 (m, 1H), 4.48 (d, J = 15.0 Hz, 1H), 4.28 – 4.19 (m,1H), 4.18 – 3.98 (m, 2H), 3.15 – 3.09 (m, 1H), 3.03 – 2.92 (m, 2H), 2.86 –2.79 (m, 1H), 1.55 – 1.46 (m, 1H), 1.19 (d, J = 7.2 Hz, 3H), 0.73 – 0.43 (m,4H). 13 C NMR (150 MHz, DMSO- d 6) δ 172.6, 172.2, 170.9, 170.5, 160.9 (d, J =242.0 Hz), 133.9, 133.8 (d, J = 2.9 Hz), 132.0, 131.1 (d, J = 7.7 Hz), 127.1,126.3, 126.0, 125.5, 117.4, 114.6 (d, J = 20.9 Hz), 54.0, 50.5, 47.8, 45.0,36.1, 30.7, 27.0, 17.6, 13.1, 6.6, 6.2. 19 F NMR (471 MHz, DMSO- d 6) δ -117.48 –-117.62 (m).

[0075] Example 2: In vitro enzyme activity inhibition experiment

[0076] Preparation of protein purification buffer: 30 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride, CAS: 1185-53-1), 300 mM NaCl, 5% glycerol (glycerol, CAS: 56-81-5), 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl, CAS: 51805-45-9), pH 8.0.

[0077] The monkeypox virus core protease (amino acid sequence SEQ ID NO: 1) sample was expressed and purified. 40 μL of monkeypox virus core protease solution (final concentration 0.4 μM, solvent: the aforementioned protein purification buffer) was added to 4 rows of 19 columns in a black 384-well microplate (brand: PerkinElmer, catalog number: 6007270). 1 μL of each of the 14 concentration gradients of small molecule inhibitors A3-A6 (initial and final concentration 100 μM, 2-fold serial dilution, solvent: the aforementioned protein purification buffer) was added to the first 18 columns. 1 μL of DMSO was added to the 20th column as a positive control, and 40 μL of the aforementioned protein purification buffer was added to the 21st column as a negative control. Next, 10 μL of the synthetic fluorescent substrate MCA-DDLQMVIAGAKSK(Dnp) (the amino acid sequence of which is DDLQMVIAGAKSK (SEQ ID NO: 2), where MCA represents 7-methoxycoumarinyl and Dnp represents 2,4-dinitrophenyl) was added to each well (using the aforementioned protein purification buffer) to a final concentration of 50 μM. The wells were then placed in a microplate reader, and detection and readings were performed using EnVision Manager 1.13.3009.1409 software. The initial enzyme activity rate was calculated using the spot taken 4 minutes before the enzyme reaction. Small molecules A3-A6 were each subjected to four biological replicates to calculate the IC50. 50 Numerical value.

[0078] like Figure 1 As shown in this embodiment, in vitro biochemical enzyme activity inhibition experiments demonstrate that the small molecule A3-A6 has a good inhibitory effect on the core protease activity of monkeypox virus, IC50. 50 The values ​​range from 44.9 nM to 100.3 nM.

[0079] Example 3: Antiviral activity experiment

[0080] First, an experiment was conducted to investigate the anti-vaccinia virus activity in a HeLa cell infection system.

[0081] With 1.5×10 4HeLa cells were seeded at the specified cell density in 48-well plates (brand: Jetech Biotechnology, catalog number: TCP-010-048) and incubated overnight. After incubation for 1 h with different concentrations of compounds A3-A6 (starting concentration 200 μM, diluted sequentially to 0.39 μM), the cells were then inoculated with vaccinia virus (VVV) at an MOI (multiple of infection) of 0.05. vaccinia virus (belongs to the Poxviridae family) Poxviridae ), genus Orthopodovirus ( Orthopoxvirus ), vaccinia virus ( vaccinia virus The WR strain (derived from the Wuhan Institute of Virology, Chinese Academy of Sciences) was used to infect cells for 2 hours, followed by 24 hours of culture in DMEM (Thermo Fisher Scientific, catalog number: 12491015) containing the corresponding concentrations of compounds A3-A6. The supernatant was collected, and viral DNA was extracted using a viral DNA / RNA extraction kit (Novozymes, catalog number: 027E3242BA). Quantitative real-time PCR (qRT-PCR) was used to quantify the viral DNA copy number in the supernatant using the upstream primer 5′-GATGATAACACTATCATGTA-3′ (SEQ ID NO: 3) and the downstream primer 5′-GTATAATTATCAAAATACAAGACGTC-3′ (SEQ ID NO: 4). DMSO was used as the control group.

[0082] The orthopoxvirus HA gene (SEQ ID NO: 5) was cloned into the pBS vector to form a plasmid as a control. The plasmid was then subjected to a series of dilutions (10⁻¹⁰). 3 -10 9 Copy number was determined, and a standard curve was generated. Cell viability was assessed three times for each concentration of compounds A3-A6 on 96-well plates (brand: Corning, catalog number: 10023601). All compounds were serially diluted (initial concentration 400 μM, 2-fold serial dilution to 6.25 μM) in maintenance medium (DMEM containing 2% FBS) (brand: Thermo Fisher Scientific, catalog number: 8123545) at an initial concentration of 400 μM. After incubation for 24 h, the supernatant was added to 100 μL of medium containing CCK-8 (brand: Hongye Biotechnology, catalog number: GK10001). After another 2 h of incubation, absorbance at 450 nm was measured using a BioTek spectrophotometer to calculate cell viability.

[0083] like Figure 2 As shown in the figure, this embodiment demonstrates through antiviral activity experiments that the small molecule A3-A6 has a good inhibitory effect on vaccinia virus replication, EC 100%. 50 The values ​​range from 1.39 μM to 3.66 μM, CC 50 The values ​​are all greater than 200 μM.

[0084] Subsequently, an experiment was conducted to investigate the anti-monkeypox virus activity in the Velo-E6 cell infection system.

[0085] With 1.5×10 4 Cells were seeded at the specified density in 48-well plates (brand: Jetech Biotechnology, catalog number: TCP-010-048) and incubated overnight for Velo-E6 cells. After incubation for 1 h with different concentrations of compound A3-A6 (starting concentration 200 μM, diluted sequentially to 0.39 μM) and 0.5 μM CP-100356 (a P-gp inhibitor, CAS: 142716-85-6), the cells were then incubated with monkeypox virus (MSV) at an MOI (multiple of infection) of 0.05. mpox virus (belongs to the Poxviridae family) Poxviridae ), genus Orthopodovirus ( Orthopoxvirus Monkeypox virus ( mpox virus Cells from Clade IIb clade C.1 subgroup (obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences) were infected for 2 h and then cultured for another 24 h in DMEM (Thermo Fisher Scientific, catalog number: 12491015) containing the corresponding concentrations of compounds A3-A6. The supernatant was collected, and viral DNA was extracted using a viral DNA / RNA extraction kit (Novozymes, catalog number: 027E3242BA). The viral DNA copy number in the supernatant was quantified using real-time quantitative PCR (qRT-PCR) with upstream primer 5′-GATGATAACACTATCATGTA-3′ (SEQ ID NO: 3) and downstream primer 5′-GTATAATTATCAAAATACAAGACGTC-3′ (SEQ ID NO: 4). DMSO was used as the control group.

[0086] The orthopoxvirus HA gene (SEQ ID NO: 5) was cloned into the pBS vector to form a plasmid as a control. The plasmid was then subjected to a series of dilutions (10⁻¹⁰). 3 -10 9Copy number was determined, and a standard curve was generated. Cell viability was assessed three times for each concentration of compounds A3-A6 on 96-well plates (brand: Corning, catalog number: 10023601). All compounds were serially diluted (initial concentration 400 μM, 2-fold serial dilution to 6.25 μM) in maintenance medium (DMEM containing 2% FBS) (brand: Thermo Fisher Scientific, catalog number: 8123545) at an initial concentration of 400 μM. After incubation for 24 h, the supernatant was added to 100 μL of medium containing CCK-8 (brand: Hongye Biotechnology, catalog number: GK10001). After another 2 h of incubation, absorbance at 450 nm was measured using a BioTek spectrophotometer to calculate cell viability.

[0087] like Figure 3 As shown in the figure, this embodiment demonstrates through antiviral activity experiments that the small molecule A3-A6 has a good inhibitory effect on monkeypox virus replication, EC 100%. 50 The values ​​range from 1.98 μM to 7.31 μM, CC 50 The values ​​are all greater than 200 μM.

[0088] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A compound A, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof, characterized in that, Compound A has any of the following structures: Formula (I); Formula (II); Formula (III); Formula (IV).

2. A pharmaceutical composition comprising compound A as claimed in claim 1, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

3. The use of a compound A as claimed in claim 1, a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 2; The application is to prepare a drug for treating and / or preventing orthopodovirus infections, or to prepare a drug for treating and / or preventing related diseases caused by orthopodovirus infections.

4. The application as described in claim 3, characterized in that, It meets one or more of the following conditions: (1) The orthopoxvirus is selected from one or more of monkeypoxvirus, smallpoxvirus, cowpoxvirus and vaccinia virus; (2) The diseases caused by the orthopoxvirus infection are one or more of the following: fever, headache, back pain, myalgia, lymphadenopathy, rash, mucosal rash and generalized papules caused by orthopoxvirus infection.

5. The application as described in claim 3, characterized in that, It meets one or more of the following conditions: (1) The virus of the Orthopoxvirus genus is monkeypoxvirus or vaccinia virus; for example, the C.1 subgroup of the Clade IIb branch of monkeypoxvirus, or vaccinia virus WR strain; (2) The diseases caused by the orthopoxvirus are one or more of the following caused by monkeypox virus infection: fever, headache, back pain, myalgia, lymphadenopathy, rash and mucosal rash. (3) The diseases caused by the vaccinia virus infection are local lymph node swelling or systemic papules caused by vaccinia virus infection.

6. The use of a compound A as claimed in claim 1, a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 2 in the preparation of a viral core protease inhibitor.

7. The application as described in claim 6, characterized in that, The viral core protease is a viral core protease belonging to the orthopoxvirus genus.

8. The application as described in claim 7, characterized in that, The core protease of the orthopoxvirus is the core protease of monkeypoxvirus, smallpoxvirus, vaccinia virus, or vaccinia virus.

9. The application as described in claim 6, characterized in that, The viral core protease inhibitor is used in vivo or in vitro.

10. The application as described in claim 9, characterized in that, The viral core protease inhibitor is used in vitro.

Images