Application of acridine compound in preparation of anti-human immunodeficiency virus drugs

Abstract

The invention discloses application of acridine compounds in preparation of anti-human immunodeficiency virus drugs, and belongs to the field of preparation of antiviral drugs. Experiments find that the compound can significantly improve the latent HIV-1 transcriptional activity in the body of a patient when being used for preparing drugs for treating AIDS, so that the compound can be recognized by anti-retrovirus drugs. Taking HIV-1 latent infection cells as an example, the compound enables the expression quantity of HIV-1 virus genes to be remarkably improved, and has the potential of activating a virus repository. Therefore, the compound has the potential of eliminating the virus latent library in the body of an AIDS patient, and provides a new possibility for functional cure of AIDS.

Description

Technical Field

[0001] This invention belongs to the field of antiviral drug preparation and relates to the application of an acridine compound with a general structural formula in the preparation of anti-AIDS drugs. The compound achieves the function of clearing the viral reservoir by activating the transcriptional activity of latent HIV-1. Background Technology

[0002] AIDS (Acquired Immune Deficiency Syndrome) is a serious infectious disease caused by the Human Immunodeficiency Virus (HIV). Since its first discovery in 1981, AIDS has spread rapidly, becoming a major global public health challenge. The HIV virus primarily attacks CD4+ T lymphocytes, the core cells of the human immune system, leading to a gradual decline in immune function, making patients more susceptible to other diseases, and ultimately potentially causing death from complications.

[0003] Despite significant advancements in modern medicine, with highly active antiretroviral therapy (HAART) effectively suppressing HIV replication, significantly prolonging patient survival, and improving quality of life, this therapy cannot completely eliminate HIV latent within host cells. In the early stages of infection, HIV enters resting CD4+ T cells, forming a latent viral reservoir. The viral genome in these reservoirs is in a low-level transcriptional state, producing almost no viral particles, thus evading the host's immune system and antiviral drugs. Once treatment is discontinued, the latent virus reactivates, the viral load rebounds rapidly, and the disease relapses. Therefore, the presence of latent HIV infection is a major obstacle to achieving a functional cure for AIDS.

[0004] In the research of anti-HIV drugs, the development of latency reversing agents (LRAs) has become an important research direction. These agents aim to awaken the dormant HIV virus from its resting state, exposing it to the immune system and antiretroviral drugs, thereby achieving a "Shock and Kill" strategy. However, most current latency reversing agents suffer from high cytotoxicity, limited activation effects, or unclear mechanisms of action, restricting their clinical application. Therefore, finding new and effective latency reversing agents and their targets is of great significance for achieving a functional cure for AIDS.

[0005] In antiviral drug research, small molecule compounds hold significant promise due to their diverse structures and ease of synthesis and modification. Compared to traditional antiviral drugs, small molecule compounds can target specific sites on viruses or host cells, thereby achieving highly effective antiviral effects. In recent years, with the development of medicinal chemistry and molecular biology techniques, an increasing number of small molecule compounds have been developed for the treatment of various viral diseases, including HIV, influenza virus, and hepatitis virus. In the field of HIV treatment, research on small molecule compounds mainly focuses on blocking viral entry into host cells, inhibiting viral replication enzyme activity, and regulating the immune response of host cells.

[0006] However, current research on latent HIV activators is mainly based on viral transcriptional regulation mechanisms, and includes the following categories: histone deacetylase inhibitors (HDACi, such as vorinostat, romidazine, and pabistal), protein kinase C agonists (PKC agonists, such as lichenstatin-1 and pabistalse), BET bromide domain inhibitors (such as JQ1 and I-BET), histone methyltransferase inhibitors (such as chaetocin), non-classical NF-κB pathway activators (such as SMAC analogs), and T cell activators (such as IL-15 superagonists and anti-PD-1 antibodies). Although these compounds have shown the ability to activate latent HIV in in vitro models or some animal experiments, their clinical application faces significant challenges. Most HDACi have high cytotoxicity, limited activation efficiency, and cell type selectivity; PKC agonists, while having strong activation effects, are generally accompanied by a strong risk of cytokine release syndrome and potential side effects that promote T cell exhaustion; other types of LRAs generally have complex mechanisms of action, poor pharmacokinetic characteristics, or unclear in vivo effects. To date, no latency activator has been proven in clinical trials to safely and effectively eliminate the latent viral reservoir in patients, nor has any such drug been clinically approved for use in strategies for a functional cure of HIV. Therefore, developing novel, highly effective, and low-toxicity latency activators, and elucidating their precise mechanisms of action, remains a key scientific bottleneck in achieving a cure for AIDS.

[0007] This invention is the first to discover that an acridine compound with a general structural formula can significantly activate latent HIV, demonstrating its high application value in the treatment of HIV patients. Through systematic experimental research, this invention reveals the role of this type of compound in activating latent HIV transcription, bringing new hope to the treatment of AIDS patients. Summary of the Invention

[0008] Given that existing HIV latency activators generally suffer from high cytotoxicity and limited activation efficiency, the purpose of this invention is to provide a compound that can significantly activate HIV latent transcription with minimal side effects.

[0009] To achieve the above objectives, this invention, based on the key regulatory mechanisms of latent viral infection, through large-scale drug screening combined with experimental verification, discovered a class of small molecule compounds that can significantly promote HIV latent activation, the general structural formula of which is shown in Formula I:

[0010]

[0011] R1 and R2 are independently selected from Cl, F, Br, Me, Et, Pr, OMe, OEt, and OPr, respectively; R3 is selected from substituted alkyl, alkylene, aryl, alkylaryl, heteroaryl, and alkylenearyl groups.

[0012] Specific sub-compounds include, but are not limited to, quinacrine, proflavine hemisulfate, ethacridine lactate, and quinacrine hydrochloride, with the following structures:

[0013]

[0014] These compounds can specifically activate latent HIV-1 viral transcription. Their mechanism of action is as follows: by targeting transcriptional regulatory pathways within host cells, these compounds significantly enhance the promoter activity of HIV-1 long terminal repeats (LTRs), thereby breaking the transcriptional silencing state of the viral genome.

[0015] This invention has shown that acridine-like structures and their analogues can activate the HIV latent viral reservoir and, when used in combination with antiretroviral drugs, can achieve complete eradication of HIV. Based on this, antiviral drugs are prepared using these small molecule compounds.

[0016] The acridine compounds obtained in this invention significantly enhance the transcriptional activity of firefly luciferase in NH1 cells. In the 2D10 model cells treated with the drug, p24 protein levels significantly increased, and the viral particle content in the cell supernatant also increased further, demonstrating its significant activation effect on the latent state of HIV-1 virus. Real-time PCR results showed that when this class of drugs is used in combination with antiretroviral drugs (such as lamivudine), latent virus can be permanently eliminated.

[0017] The specific research results are shown below:

[0018] The acridine compounds can efficiently induce the expression of reporter genes (such as firefly luciferase Fluc) in HIV latent cell models, causing the Flu value to increase in a time- and concentration-dependent manner.Figure 3-5 In 2D10 cells, treatment with the compound promoted the reactivation of latent virus, thereby enabling the re-expression of the viral structural protein p24 to be detected in the cells. Figure 7 Furthermore, viral particles were detected in the culture medium supernatant, further demonstrating that the expression of viral proteins in cells can effectively package viral particles and release them into the culture medium supernatant, confirming that acridine compounds can effectively activate latent viruses in cells. Figure 6 More importantly, when such activators are used in combination with antiretroviral drugs (such as lamivudine), they can trigger a "Shock and Kill" effect—first activating the latent viral reservoir and exposing it to the drug's effects, thereby achieving permanent clearance of the viral reservoir. Figure 8 The acridine compounds with the general structural formula described in this invention provide a novel approach for developing new anti-HIV therapies.

[0019] The beneficial effects of this invention are mainly reflected in:

[0020] (1) This patent provides a small molecule compound with a general structural formula that can significantly activate latent HIV transcription and utilizes these small molecule compounds to play a role in the fight against HIV-1. By activating the transcription of latent virus, the virus is exposed to the immune system and antiretroviral drugs, thereby realizing the "Shock and Kill" strategy and providing new possibilities for the functional cure of AIDS.

[0021] (2) The compounds of the present invention have the characteristics of structural diversity and easy synthesis. Among them, quinacrine and proflavin sulfate have been approved for marketing by the US FDA and are expected to play an important role in subsequent clinical trials. Attached Figure Description

[0022] Figure 1 This is the general structural formula of the acridine compounds of the present invention;

[0023] Figure 2 These are the structural formulas of four small molecule compounds used in the embodiments of the present invention based on the general structural formula; wherein, (A) is the structural formula of quinacrine, (B) is the structural formula of proflavine hemisulfate, (C) is the structural formula of ethacridine lactate, and (D) is the structural formula of quinacrine hydrochloride.

[0024] Figure 3This is a statistical graph showing the changes in Flu values ​​in NH1 cells after treatment with quinacrine, proflavine hemisulfate, ethacrine lactate, and quinacrine hydrochloride (50 nM) for 6 hours.

[0025] Figure 4 The changes in Flu values ​​in NH1 cells after treatment with two compounds over time gradients are shown; (A) is a statistical graph of the quinacrine treatment results, and (B) is the result of the proflavine hemisulfate treatment.

[0026] Figure 5 The changes in Flu values ​​in NH1 cells after treatment with two compounds at concentration gradients are shown; (A) is a statistical graph of the treatment results of ethacridine lactate, and (B) is a statistical graph of the treatment results of quinacrine hydrochloride.

[0027] Figure 6 The graph shows the changes in viral particle content in the culture supernatant of 2D10 cells after treatment with two drugs at time gradients and detection by Real-time PCR. (A) is the statistical graph of the treatment results of proflavine hemisulfate, and (B) is the statistical graph of the treatment results of quinacrine.

[0028] Figure 7 The expression of Gag in 2D10 cells was detected by Western blot after treatment with quinacrine, proflavine hemisulfate, ethacrine lactate, and quinacrine hydrochloride, respectively.

[0029] Figure 8 The expression of Gag in 2D10 cells was detected by Western blot after quinacrine, proflavine hemisulfate, ethacrine lactate, quinacrine hydrochloride, and antiretroviral drugs were used in combination. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0031] In the following examples, four small molecule compounds—quinacrine, proflavine hemisulfate, ethacrine lactate, and quinacrine hydrochloride—are used as examples to further illustrate the technical effects of the present invention. The structures of the four small molecule compounds are as follows: Figure 2 As shown.

[0032] Example 1:

[0033] NH1 cells were treated with quinacrine, proflavine hemisulfate, ethacrine lactate, and quinacrine hydrochloride (final concentration 50 nM) for 6 h, and the changes in Firefly Luciferase expression were detected by a multi-functional microplate reader. Results are as follows: Figure 3 The results showed that after treatment with the four compounds, the Firefly Luciferase value increased significantly, indicating that the compounds had a significant function in activating HIV-1 expression.

[0034] Example 2:

[0035] NH1 cells were treated with quinacrine and proflavine hemisulfate (final concentration 50 nM) for different time periods (0 h, 1 h, 2 h, 4 h, 6 h), respectively. The changes in Firefly Luciferase expression were detected using a multi-functional microplate reader. Results are as follows: Figure 4 The results show that when treated with the two compounds, the value of FireflyLuciferase increases over time, indicating that the compounds have the function of activating HIV-1 expression in a time-dependent manner.

[0036] Example 3:

[0037] NH1 cells were treated with different concentrations of ethacridine lactate and quinacrine hydrochloride for 6 hours, and the changes in Firefly Luciferase expression were detected using a multi-functional microplate reader. The results are as follows: Figure 5The results showed that when the two compounds were treated separately, the value of FireflyLuciferase increased with the concentration gradient, indicating that the compounds had the function of dose-dependent activation of HIV-1 expression.

[0038] Example 4:

[0039] 2D10 cells were treated with quinacrine and proflavine hemisulfate (final concentration 50 nM) at time gradients (0 h, 1 h, 2 h, 4 h, 6 h). Total RNA was extracted from the cell supernatant, and the content of HIV viral particles in the supernatant was detected by real-time PCR (quantitative real-time PCR). The detection targets included eGFP, p24, and Pol. The results are as follows: Figure 6 The results showed that, compared to the control group DMSO, the experimental group with added quinacrine and proflavine hemisulfate had significantly higher viral particle content, indicating that quinacrine and proflavine hemisulfate could significantly activate HIV-1 transcription.

[0040] Example 5:

[0041] 2D10 cells were treated with quinacrine, proflavine hemisulfate, ethacrine lactate, and quinacrine hydrochloride (final concentration 50 nM) for 6 h, and the expression level of Gag was detected by Western blot. Results are as follows: Figure 7 The results showed that when the four compounds were used for treatment, the expression level of Gag protein increased significantly, indicating that the compounds could significantly activate HIV-1 transcription, thereby leading to an increase in the expression level of Gag protein.

[0042] Example 6:

[0043] First, 2D10 cells were treated with quinacrine, proflavine hemisulfate, ethacrine lactate, and quinacrine hydrochloride (final concentration 50 nM) for 6 hours. Then, an antiretroviral drug (lamivudine was used in this example) was added for another 6 hours. Cells were then collected, and p24 expression was detected again by Western blot. The results are as follows: Figure 8 The results showed that the expression level of Gag in cells was significantly reduced after the use of antiretroviral drugs, indicating that the combination of antiretroviral drugs (such as lamivudine) can achieve the effect of clearing latent viruses.

[0044] The embodiments described above are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort are within the scope of protection of this invention.

Claims

1. The application of acridine compounds in the preparation of antiviral drugs, characterized in that, The general formula of the acridine compounds is shown in Formula I: R1 and R2 are independently selected from Cl, F, Br, Me, Et, Pr, OMe, OEt, and OPr, respectively; R3 is selected from substituted alkyl, alkylene, aryl, alkylaryl, heteroaryl, and alkylenearyl.

2. The application according to claim 1, characterized in that, The acridine compounds include: quinacrine, proflavine hemisulfate, ethacrine lactate, and quinacrine hydrochloride.

3. The application according to claim 1, characterized in that, The virus in question is HIV-1.

4. An HIV latency activator, characterized in that, The HIV latency activator is an acridine compound.

5. An anti-HIV drug composition, characterized in that, The pharmaceutical composition comprises the HIV latency activator and antiretroviral agent as described in claim 4.

Images