CN104000807B - Phenyl-amides compound is in the application of preparing in anti-HIV-1 virus drugs - Google Patents

Abstract

The invention discloses phenyl-amides compound in the application of preparing in anti-HIV-1 virus drugs. Phenyl-the amides compound using has following general formula:

Description

Phenyl-amides compound is in the application of preparing in anti-HIV-1 virus drugs

Technical field

The present invention relates to a kind of new application of compound, particularly phenyl-amides compound is being prepared anti-HIV-1 virus medicineApplication in thing.

Background technology

HIV-1 virus is found at first 1981 and finds in the U.S., by a series of unknown causes, taking cellular immunity deficiency asAfter the syndrome of feature occurs, researcher has started the searching to its etiology. Nineteen eighty-three, research group of France was from a lymphIn enlargement syndrome patient's lymph node, isolate HIV-1 virus. It is a kind of slow virus that infects human immunity system cells,This virus is destroyed the immunity of human body, causes immune system to lose resistance, and causes various diseases and cancer to be able at human bodyInterior existence, thus aids caused---AIDS. At present, due to the universal of AIDS education do not filledPoint, the HIV in the whole world infects still in rising trend, has especially entered especially the rapid growth phase in China. Stop as early as possible AIDS to existChina popular become an instant major issue. Therefore, continue to expand our understanding to HIV-1 mechanism of causing a disease, openSend more effectively, more economical, still less the antiviral drugs of side effect copies to remove remaining HIV-1 completely, and developsThe vaccine of strong and long-acting anti-HIV-1, with protection Susceptible population, will be the key that can we finally defeat AIDS.

There is the phenyl-amides compound of following general formula:

In formula, X is halogen, be positioned on phenyl ring 1～In No. 3 positions on any, the chain alkylene that R is C1～C4, n1, n2 are independently 1～3 integer, as(N-(2-acetylamino ethyl)-2-(4-Iodine phenoxy group) acetamide, N-(2-acetamidoethyl)-2-(4-iodophenoxy) acetamide) can be purchased from Enamine companyOr manually synthesize on its basis, do not report that this compounds is for antiviral experiment or other possible effects at present.

Summary of the invention

The object of the present invention is to provide phenyl-amides compound to apply preparing in anti-HIV-1 virus drugs.

Phenyl-amides compound used in the present invention has following general formula:

In formula, X is halogen, is arranged on phenyl ring 1～No. 3 position on any, the chain alkylene that R is C1～C4, and n1, n2 are independently1～3 integer.

As a further improvement on the present invention, the alkenyl group that the R in above-claimed cpd is C1～C3.

As a further improvement on the present invention, the X in above-claimed cpd is I, Cl.

As a further improvement on the present invention, the X in above-claimed cpd is positioned on No. 2 positions of phenyl ring.

As a further improvement on the present invention, the X in above-claimed cpd is I, is positioned on No. 2 positions of phenyl ring; N1, n2 are1, R is methyl.

Inventor uses HIV-1Vif albumen by the degrade functional characteristic of A3G albumen of ubiquitination, to confirm above-mentioned general formulaPhenyl-amides compound, particularly N-(2-acetylamino ethyl)-2-(4-iodine phenoxy group) acetamide can suppress VifThe activity of PD A3G, causes the expression of screening system Green fluorescin to go up. By the primary CD4+ peopleIn the CD4+T lymphocytic series such as T lymphocyte and H9, SupT1, carry out the infection experiment confirmation of wild type HIV-1 virus,N-(2-acetylamino ethyl)-2-(4-iodine phenoxy group) acetamide has good antivirus action, for further antiviralStrong theoretical foundation and practical basis that medicament research and development provides, have important research and development and be worth and develop meaning.

Brief description of the drawings

Fig. 1: the structure schematic diagram of cell screening model;

Fig. 2: N-(2-acetylamino ethyl)-2-(4-iodine phenoxy group) acetamide has the effect that suppresses Vif activity;

Fig. 3: N-(2-acetylamino ethyl)-2-(4-iodine phenoxy group) acetamide is being expressed the H9 cell of A3G albumen and is not expressingIn the SupT1 cell of A3G albumen, suppress the print effect of wild type HIV-1;

Fig. 4: N-(2-acetylamino ethyl)-2-(4-iodine phenoxy group) acetamide suppresses copying of wild type HIV-1 in H9 cellThe titration of IC50.

Detailed description of the invention

Vif is one of indispensable protein of HIV, and its major function is antiviral agent APOBEC3G natural in antagonism host.APOBEC3G is that one of HIV-1 virus threatens greatly, it be present in the natural host cell of HIV-1 (as CD4+T cell andMacrophage) in, can be wrapped the virion into HIV-1, in HIV-1 reverse transcription process, bring into play its extremely strong antiviral workWith. For this reason, encoded Vif albumen of HIV-1 self carrys out the antiviral activity of specificity antagonism APOBEC3G, and it can be byAPOBEC3G imports ubiquitin system degraded (Figure 1A). Therefore, how deactivation Vif, is research and development anti-HIV-1 virusA very important target of medicine.

According to the molecule mechanism of Vif antagonism APOBEC3G, the Vif that filters out some HIV of allowing cannot degradeThe small-molecule drug of APOBEC3G. We will set up a kind of easy living cells screening system for this reason, as shown in Figure 1B, and willThe plasmid co-transfection of expressing APOBEC3G-GFP fusion and express Vif is in cell. Melt with APOBEC3G-GFPWhether hop protein is degraded to index. As long as certain compound can also make APOBEC3G-GFP merge in the situation that Vif existsAlbumen is not degraded (being that GFP fluorescence also exists), and this compound is exactly the inhibitor of Vif. By to Vif target furtherQualification, confirms that compound acts on host cell or acts on the Vif of virus protein.

In order to understand better essence of the present invention, below in conjunction with experiment and result, N-(2-acetylamino ethyl)-2-is described(4-iodine phenoxy group) acetamide is applied preparing in anti-HIV-1 virus drugs.

Experiment one

1) get cell line 239t cell on well-grown people's kidney, be inoculated in the 96 transparent flat undersides in hole every hole 5 × 10⁴Cell.The culture medium using is complete medium: DMEM in high glucose, and 10% hyclone and 1% dual anti-, condition of culture is 5% dioxyChange carbon, 37 DEG C;

2) 24h adherent after, two kinds of plasmids of cotransfection PEGFP-A3G and pcDNA3.1-Vif. Transfection adopts liposome to turnDye, reagent uses lipo2000, transfection liquid 20 μ l;

3) after transfection 4h, add compound to be screened, every hole 2 μ l, final concentration is 50 μ M;

4) cultivate after 48h, detect the expression of green fluorescent protein GFP. Express if there is green fluorescent protein GFPThe situation rising, this compound may become antiviral drug candidate.

Experimental result as shown in Figure 2, can be found out from experimental result, N-(2-acetylamino ethyl)-2-(4-iodine phenoxy group)Acetamide all has the effect that suppresses Vif activity.

Experiment two

Vif albumen has important function in HIV-1 virus replication, the HIV virus of vif defect can not CD4+T cell,In macrophage, copy, can not be in above-mentioned " non-permission " time multiplexed cell system; And the wild strain virus that contains vif gene can be upperThe time multiplexed cell system of stating. After some target cell of Vif gene-deleted strain cell entry, reverse transcription can be carried out, but proviral DNA can not be synthesized.Studies show that HIV copies appearance or the disappearance in a kind of cytostatic factor, this endogenic inhibiting factor isAPOBEC3G, it belongs to intracellular rna editing enzymes, can make the cytimidine deaminizating in mRNA form uracil, causesThe accumulation of G and A mutant, and then be viral DNA degraded, vif forms compound blocking-up by being combined with APOBEC3GThe inhibition activity of APOBEC3G. APOBEC3G exist clone as H9 cell in, APOBEC3G and vif eggWhite in conjunction with degrading by ubiquitination system, if compound can suppress APOBEC3G by vif PD, host cell willCan not be infected by HIV-1; And the non-existent clone of APOBEC3G as SupT1 cell in, HIV-1 albumen can be justNormal this host cell that infects; This compound will likely become the compound of anti-HIV-1 Vif albumen so.

APOBEC3G is the self-protective mechanism of cell, but vif is the albumen of HIV-1 virus antagonism APOBEC3G function,Cause HIV-1 virus to escape self-reset procedure in cell. Utilize the permission cell of HIV-1 and do not allow antiviral in cellThe effect comparison of experiment, thus the Vif that target compound can antagonism HIV-1 can be further in wild type Viral experiment, be determinedAlbumen, suppresses copying of HIV-1 virus.

1) get well-grown lymphocytic series H9 and Supt1, cell consumption is 2 × 10⁵/ hole, infects respectively packagingGood HIV-1 virus supernatant, viral consumption is 10ng/1 × 10⁶Cell; (when infection, using the short reagent polybrene that infects);

2) after infection 3h, change liquid, use PBS to clean three times, abandon supernatant, use 1640 culture mediums (10% hyclone, 1%Dual anti-) cultivate the every hole 200 μ l of culture medium, the every hole 2 μ l of compound (final concentration 50 μ M);

3) use 2%TritonX-100 to process the supernatant of receiving sample, receive the cell conditioned medium of having cultivated 4day, survey P24Elisa.

Experimental result as shown in Figure 3. Can find out N-(2-acetylamino ethyl)-2-(4-iodine phenoxy group) from experimental resultAcetamide has good anti-HIV-1 virus function in H9 cell, and there is no effect in SupT1 cell.

Experiment three

1) get well-grown lymphocytic series H9, cell consumption is 2 × 10⁵/ hole, infects packaged HIV-1 virus supernatant,Virus consumption is 10ng/1 × 10⁶Cell; (when infection, using the short reagent polybrene that infects);

2) after infection 3h, change liquid, use PBS to clean three times, abandon supernatant, use 1640 culture mediums (10% hyclone, 1%Dual anti-) cultivate, the every hole 200 μ l of culture medium, add the every hole 2 μ l of compound, and final concentration is respectively 50 μ M, 5 μ M, 0.5μM，0.05μM，0.005μM，0.0005μM，0μM；

3) use 2%TritonX-100 to process the supernatant of receiving sample, receive sample 4day, survey P24Elisa.

Experimental result as shown in Figure 4. Can find out N-(2-acetylamino ethyl)-2-(4-iodine phenoxy group) from experimental resultThe IC of acetamide in H9 cell₅₀Be 2 μ M, there is the viral effect of good inhibition.

Claims (5)

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

In formula, X is halogen, is arranged on phenyl ring 1～No. 3 position on any, the chain alkylene that R is C1～C4, and n1, n2 are independently 1～3 integer.

2. application according to claim 1, is characterized in that: the alkenyl group that R is C1～C3.

3. application according to claim 1 and 2, is characterized in that: X is I, Cl.

4. application according to claim 1 and 2, is characterized in that: X is positioned on No. 2 positions of phenyl ring.

5. application according to claim 1, is characterized in that: X is I, is positioned on No. 2 positions of phenyl ring; N1, n2 are that 1, R is methyl.