CN104000816A - Application of dioxoimidazolidine-amide compounds to preparation of HIV-1 (Human Immunodeficiency Virus-1)-resistant drugs - Google Patents

Abstract

The invention discloses the application of dioxoimidazolidine-amide compounds in the preparation of anti-HIV-1 virus medicines. The general formula of the compound is , where: R is H, methyl or ethyl; R ₁ is a 4-7 membered cycloalkane group; R ₂ and R ₃ are C1-C3 alkane groups; n is an integer between 1 and 3. The inventors used the interaction of Vif-APOBEC3G to confirm that the compound of the above general formula, especially N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2, 5-dioxoimidazolidin-1-yl]acetamide can inhibit the activity of Vif protein to degrade A3G, resulting in the increase of the expression level of green fluorescent protein in the screening system. It is further confirmed that it has a better antiviral effect by conducting wild-type HIV-1 virus infection experiments in human primary CD4+T lymphocytes and CD4+T lymphocyte lines such as H9 and SupT1. It provides a strong theoretical basis and practical basis for further research and development of antiviral drugs, and has important research and development value and development significance.

Description

Application of dioxoimidazolidine-amide compounds in the preparation of anti-HIV-1 virus drugs

technical field

The invention relates to a new application of a compound, in particular to the application of dioxoimidazolidine-amide compounds in the preparation of anti-HIV-1 virus drugs.

Background technique

The HIV-1 virus was first discovered in the United States in 1981. After a series of unexplained syndromes characterized by cellular immune deficiency appeared, researchers began to search for its pathogenic source. In 1983, a French research team isolated the HIV-1 virus from the lymph nodes of a patient with lymphoma syndrome. It is a lentivirus that infects the cells of the human immune system, which destroys the body's immunity and causes the immune system to lose its resistance, causing various diseases and cancers to survive in the human body, resulting in Acquired Immunodeficiency Syndrome— -AIDS. At present, due to insufficient popularization of AIDS education, the global HIV infection is still on the rise, especially in China, which has entered a period of rapid growth. Stopping the epidemic of AIDS in our country as soon as possible has become an urgent matter. Therefore, it is imperative to continue to expand our understanding of HIV-1 pathogenic mechanisms, develop more effective, more economical, and less side-effect antiviral drugs to completely eliminate residual HIV-1 replication, and develop potent and long-acting antiviral drugs. Vaccines against HIV-1 to protect susceptible populations will remain critical to our eventual victory over AIDS.

Dioxoimidazolidine-amides having the general formula:

 

In the formula:

R is H, methyl or ethyl;

R ₁ is a cycloalkane group with 4-7 membered rings, and no more than 2 methyl or ethyl groups may be connected to the ring;

R ₂ is a C1-C3 alkane group, located in any of the 1-5 positions on the benzene ring;

R ₃ is an alkane group of C1～C3;

n is an integer between 1 and 3, such as (N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2,5-dioxoimidazolidin-1-yl]acetamide, N-cyclohexyl -N-methyl-2-(4-methyl-2,5-dioxo-4-p-tolylimidazolidin-1-yl)acetamide) can be purchased from Enamine Company or artificially synthesized on the basis of it, and no such compound has been reported so far For antiviral experiments or other similar effects.

Contents of the invention

The object of the present invention is to provide the application of dioxoimidazolidine-amide compounds in the preparation of anti-HIV-1 virus drugs.

The dioxo imidazolidine-amide compound used in the present invention has following general formula:

 

In the formula:

R is H, methyl or ethyl;

R ₁ is a cycloalkane group with 4-7 membered rings, and no more than 2 methyl or ethyl groups may be connected to the ring;

R ₂ is a C1-C3 alkane group, located in any of the 1-5 positions on the benzene ring;

R ₃ is an alkane group of C1～C3;

n is an integer between 1 and 3.

Preferably, R ₂ in the above-mentioned dioxoimidazolidine-amide compounds is methyl or ethyl, and is located at any one of the 2-4 positions on the benzene ring.

Preferably, R in the above-mentioned dioxoimidazolidine-amide compounds is methyl.

Preferably, R ₂ and R ₃ in the above-mentioned dioxoimidazolidine-amide compounds are methyl groups.

Preferably, n in the above-mentioned dioxoimidazolidine-amide compounds is 1 or 2. In particular, n is 1.

Preferably, _R in the above-mentioned dioxoimidazolidine-amide compound is a 5- or 6-membered cycloalkane group.

Preferably, the structural formula of the dioxoimidazolidine-amide compound is .

The inventor uses the interaction of Vif-APOBEC3G to confirm the dioxoimidazolidine-amide compounds of the above general formula, especially to confirm that N-cyclohexyl-N-methyl-2-[4-methyl-4-(4 -Methylphenyl)-2,5-dioxoimidazolidin-1-yl]acetamide can inhibit the activity of Vif protein in degrading A3G, resulting in a rise in the expression of green fluorescent protein in the screening system. N-cyclohexyl-N-methyl-2-[4-methanol was confirmed by wild-type HIV-1 virus infection experiments in human primary CD4+ T lymphocytes and CD4+ T lymphocyte lines such as H9 and SupT 1. Base-4-(4-methylphenyl)-2,5-dioxoimidazolidin-1-yl]acetamide has good antiviral effect and provides a strong theory for further antiviral drug development The foundation and practical basis have important research and development value and development significance.

Description of drawings

Figure 1: The construction principle of the cell screening model;

Figure 2: N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2,5-dioxoimidazolidin-1-yl]acetamide has inhibitory The role of Vif activity;

Figure 3: N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2,5-dioxoimidazolidin-1-yl]acetamide in the expression The effect of inhibiting the replication of wild-type HIV-1 in H9 cells expressing A3G protein and SupT1 cells not expressing A3G protein;

Figure 4: N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2,5-dioxoimidazolidin-1-yl]acetamide in H9 Titration of IC50 for inhibition of wild-type HIV-1 replication in cells.

Detailed ways

Vif is one of the essential proteins of HIV, and its main function is to antagonize the natural antiviral factor APOBEC3G in the host. APOBEC3G is a major threat to HIV-1 virus. It exists in HIV-1 natural host cells (such as CD4+ T cells and macrophages), can be packaged into HIV-1 virus particles, and is reverse transcribed in HIV-1 In the process, it exerts its strong antiviral effect. To this end, HIV-1 itself encodes the Vif protein to specifically counter the antiviral activity of APOBEC3G, which can import APOBEC3G into the ubiquitin system and degrade it (Fig. 1A). Therefore, how to inactivate Vif is a very important target for the development of anti-HIV-1 virus drugs.

According to the molecular mechanism of Vif antagonizing APOBEC3G, several small molecule drugs that can prevent HIV Vif from degrading APOBEC3G were screened out. To this end, we will establish a simple living cell screening system, as shown in Figure 1B, co-transfect the plasmid expressing APOBEC3G-GFP fusion protein and expressing Vif into the cells. Whether APOBEC3G-GFP fusion protein was degraded was used as an index. As long as a certain compound can prevent APOBEC3G-GFP fusion protein from being degraded in the presence of Vif (that is, GFP fluorescence is still there), the compound is an inhibitor of Vif. Through further identification of the Vif target, it is confirmed whether the compound acts on the host cell or on the Vif of the viral protein.

In order to better understand the essence of the present invention, the following combines experiments and results to illustrate N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2,5- The dioxoimidazolidin-1-yl]acetamide is used in the preparation of anti-HIV-1 virus drugs.

experiment one

Take well-grown human adrenal cell line 239t cells and inoculate them in a 96-well transparent flat-bottom plate with 5×10 ⁴ cells per well. The medium used is a complete medium: high-glucose DMEM, 10% fetal bovine serum and 1% double antibody, and the culture condition is 5% carbon dioxide, 37°C;

After 24 hours of wall attachment, two plasmids, PEGFP-A3G and pcDNA3.1-Vif, were co-transfected. For transfection, liposome-encapsulated transfection was used, the reagent used was lipo2000, and the transfection solution was 20 μl

4 hours after transfection, add the compound to be screened, 2 μl per well, the final concentration is 50 μM

After culturing for 48 h, the expression of green fluorescent protein GFP was detected. If there is an increase in the expression of the green fluorescent protein GFP, the compound may become an antiviral drug candidate.

The experimental results are shown in Figure 2. It can be seen from the experimental results that N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2,5-diox All imidazolidin-1-yl]acetamides have the effect of inhibiting the activity of Vif.

Experiment 2

Vif protein plays an important role in the replication of HIV-1 virus. Vif-deficient HIV virus cannot replicate in CD4+ T cells and macrophages, that is, it cannot replicate in the above-mentioned "non-permissive" cells; while the wild virus containing vif gene strains of virus can replicate in the above cells. After entering some target cells, the Vif-deficient virus can perform reverse transcription, but cannot synthesize proviral DNA. Studies have shown that HIV replication is determined by the appearance or absence of a cell inhibitory factor. This endogenous inhibitory factor is APOBEC3G, which belongs to the intracellular RNA editing enzyme, which can deaminate cytosine in mRNA to form uracil, resulting in G and the accumulation of A mutants, followed by viral DNA degradation, vif blocks the inhibitory activity of APOBEC3G by binding to APOBEC3G to form a complex. In cell lines where APOBEC3G exists, such as H9 cells, APOBEC3G binds to vif protein and is degraded by the ubiquitination system. If the compound can inhibit APOBEC3G from being degraded by vif protein, the host cell will not be able to be infected by HIV-1; while APOBEC3G does not exist In a cell line such as SupT1 cells, HIV-1 protein can normally infect the host cell; then this compound may become an anti-HIV-1 Vif protein compound.

APOBEC3G is the self-protection mechanism of cells, but vif is the protein of HIV-1 virus against the function of APOBEC3G, causing HIV-1 virus to escape the process of self-clearance in cells. By comparing the effects of antiviral experiments in HIV-1 permissive cells and nonpermissive cells, it can be further confirmed in wild-type virus experiments that the target compound can antagonize the Vif protein of HIV-1 and inhibit the replication of HIV-1 virus.

1) Take the well-grown lymphoid cell lines H9 and Supt 1, the cell dosage is 2×10 ⁵ /well, and infect the packaged HIV-1 virus supernatant respectively, the virus dosage is 10ng/1×10 ⁶ cells; (during infection Use pro-infection agent polybrene);

2) Change the medium after 3 hours of infection, wash with PBS three times, discard the supernatant, and culture in 1640 medium (10% fetal bovine serum, 1% double antibody) with 200 μl of medium per well and 2 μl of compound per well (final concentration 50 μM) ;

3) Use 2% Triton X-100 to treat the collected supernatant, collect the cultured cell supernatant for 4 days, and measure P24 Elisa.

The experimental results are shown in Figure 3. It can be seen from the experimental results that N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2,5-dioxoimidazolidin-1-yl] Acetamide has a good anti-HIV-1 virus effect in H9 cells, but has no effect in SupT1 cells.

the

Experiment three

1) Take the well-growing lymphoid cell line H9, the cell dosage is 2×10 ⁵ /well, and infect the packaged HIV-1 virus supernatant, the virus dosage is 10ng/1×10 ⁶ cells; polybrene);

2) Change the medium after 3 hours of infection, wash with PBS three times, discard the supernatant, and culture in 1640 medium (10% fetal bovine serum, 1% double antibody) with 200 μl of medium per well, add 2 μl of compound per well, and the final concentration is respectively 50 μM, 5 μM, 0.5 μM, 0.05 μM, 0.005 μM, 0.0005 μM, 0 μM;

3) Use 2% Triton X-100 to process the collected supernatant, collect the sample for 4 days, and measure P24 Elisa.

The experimental results are shown in Figure 4. It can be seen from the experimental results that N-cyclohexyl-N-methyl-2-[4-methyl-4-(4-methylphenyl)-2,5-dioxoimidazolidin-1-yl] The IC ₅₀ of acetamide in H9 cells is 1.26 μM, which has a good effect of inhibiting the virus.

Claims (8)

1. dioxo alkyl imidazole-amides compound is in the application of preparing in anti-HIV-1 virus drugs, and described dioxo alkyl imidazole-amides compound has following general formula:

In formula:

R is H, methyl or ethyl;

R ₁be the cycloalkyl group of 4～7 rings, the optional methyl or the ethyl that are no more than 2 of being connected with on ring;

R ₂for the alkyl of C1～C3, be arranged on phenyl ring any of 1～5;

R ₃for the alkyl of C1～C3;

N is the integer between 1～3.

2. application according to claim 1, is characterized in that: R ₂for methyl or ethyl, be arranged on phenyl ring any of 2～4.

3. application according to claim 1 and 2, is characterized in that: R is methyl.

4. application according to claim 1 and 2, is characterized in that: R ₂, R ₃for methyl.

5. application according to claim 1 and 2, is characterized in that: n is 1 or 2.

6. application according to claim 1, is characterized in that: n is 1.

7. according to the application described in claim 1,2 or 6, it is characterized in that: R ₁it is the cycloalkyl group of 5 or 6 rings.

8. application according to claim 1, is characterized in that: the structural formula of dioxo alkyl imidazole-amides compound is .