INTRODUCTION:

Treatment with antiretroviral therapy (ART) became widely available for patients with HIV. ART generally can suppress viral load and helps keep patients healthy. However, treatment only works until the virus is resistant to the used drugs. When the first antiretroviral drugs appeared in the 1980s, drug resistance was a definite result for all patients, and the duration of successful treatment was limited.

Currently, some patients receive treatment for many years without any problems with resistance, while for others it is harmful to their health. Thus, there is a need in new compounds for antiviral drugs.

The three pyrimidines namely uracil, thymine and cytosine are found in all living systems as components of nucleic acids¹, and also play an important role in cell metabolism and treatment. Several pyrimidine analogues have been developed as nucleoside reverse transcriptase inhibitors (NRTIs), which are currently used in the treatment of HIV. However, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have also been developed. NNRTIs are chemically distinct from nucleosides and, unlike the NRTIs, do not require intracellular metabolism for activity. Although NNRTIs are generally well tolerated, a major limitation for all currently available NNRTIs is the low genetic barrier to resistance. Thus, the drug resistance occur quickly due to a small number of point mutations in the target location. To improve the NNRTI drug design for further clinical development, a number of compounds (uracil derivatives) were synthesized and tested for antiviral activity against HIV-1. The study presents a long-term culture experiment with HIV-1-infected MT-4 cells with escalating concentrations of the uracil compounds.

However, with serial passages of the infected cells, escape viruses were obtained, which displayed complete resistance to these compounds. The sequential analysis of RT from escape viruses, in our opinion, will contribute to the development of a new generation of compounds effective against different viruses.

LITERATURE REVIEW:

The human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of AIDS. Replication of this virus requires the activity of a retrovirus encoded RNA-dependent DNA polymerase, or reverse transcriptase (RT). HIV-1 RT is required for the synthesis of the double-stranded proviral DNA from the single-stranded retroviral RNA genome. HIV-1 RT has two subunits of 66 kDa and 51 kDa¹. The 66-kDa subunit contains the DNA polymerase and RNase H domains whereas the 51-kDa subunit, obtained by proteolytic maturation of the former subunit, has only the DNA synthetic activity². Two recently reported crystal structures of HIV-1 RT have revealed the very asymmetric structure of this molecule³. In addition to providing information concerning the mechanism of nucleic acid polymerization, biochemical and biophysical studies of this enzyme are providing key insights for the design of selective antiviral agents. The multiple activities displayed by reverse transcriptase in the replication of the retroviral genome ensure that this enzyme will remain at the forefront of antiviral strategies in the fight against AIDS and other retrovirus-related pathologies.

The polymerase domain is currently targeted by two classes of antiretroviral drugs, nucleoside reverse transcriptase inhibitors (RT NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). NNRTIs are important components of antiretroviral therapy currently used to treat HIV-1. Currently, more than 50 structurally diverse classes of compounds have been reported as genuine NNRTIs. Earlier NNRTIs include nevirapine (NVP), delavirdine (DLV), and efavirenz (EFV), and recent NNRTIs include etravirine (ETR)⁴. Several other NNRTIs, such as emivirine (MKC-442), underwent clinical trials, and yet they were not approved due to unfavourable pharmacokinetics, insufficient efficacy, and/or safety concerns. Recently, rilpivirine (RPV) has been formally licensed for clinical use in treatment-naive adult patients, while other NNRTIs, including # lersivirine, are currently under clinical development⁵.

Although NNRTIs are generally well tolerated, a major limitation for all currently available NNRTIs is the low genetic barrier to resistance. Thus, the drug resistance occur quickly due to a small number of point mutations in the target location. Mutations of drug resistance HIV-1 in the RT structure are widely described for NRTIs and NNRTIs. NNRTIs inhibit HIV-1 by allosteric binding to a hydrophobic pocket in RT behind the catalytic centre⁶.

Mutations that are selected after failure of treatment with NNRTIs are all located in the enzyme pocket targeted by these compounds, and they reduce the binding affinity of the inhibitors for the enzyme^7,8. A single mutation in the NNRTI-binding pocket may confer high-level resistance to one or more NNRTIs. Since efavirenz (EFV), delavirdine (DLV) and nevirapine (NVP) have similar binding modes to RT, viruses resistant to one of these compounds develop cross-resistance to the others⁴. Reverse transcriptase maintains its activity against NVP-, DLV-, or EFV-resistant mutants due to its ability of multiple binding modes to RT.

In order to improve the design of novel NNRTIs for future clinical development, in vitro isolation and analyses of drug-resistant viruses are necessary to obtain valuable information on the resistance patterns of novel compounds. This article describes nine new uracil analogues that we previously synthesized and evaluated as NNRTIs, including four 1-substituted 3-(3,5-dimethylbenzyl)-5-fluorouracils and five 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl) uracils. Two of these compounds—6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil were found to be highly active and selective inhibitors of HIV-1 replication in vitro.

The study presents a long-term culture experiment with HIV-1-infected MT-4 cells with escalating concentrations of the above-described compounds. After serial passages of the infected cells, escape viruses were obtained, which displayed complete resistance to these compounds. Sequence analysis of RT from escape viruses was performed to determine the mutations related to the acquisition of resistance which, in our opinion, in our opinion, will contribute to the development of a new generation of compounds effective against different viruses, considering their quickly mutation and increased resistance.

PROBLEM STATEMENT:

The issues of antiviral drug resistance become quite important considering viruses ultra-fast ability to mutate. Even though the new drugs appear every year, their efficiency is not always satisfactory or they are poorly tolerated. Although NNRTIs are generally well tolerated, a major limitation for all currently available NNRTIs is the low genetic barrier to resistance. Thus, the drug resistance occur quickly due to a small number of point mutations in the target location. Therefore, we previously synthesized and evaluated nine novel uracil analogues as NNRTIs, including four 1-substituted 3-(3,5-dimethylbenzyl)-5-fluorouracils and five 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl) uracils. Two of these compounds—6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil were found to be highly active and selective inhibitors of HIV-1 replication in vitro. The aim of the research is to study the effect of the synthesis of uracil derivatives on the HIV-1 activity. To achieve the goal, the following tasks were determined:

· To study the specificity of possible compounds for HIV-1 treatment;

· To synthesize uracil derivatives in compliance with all standards to reduce the error of the results;

· To study the effect of the compounds on HIV-1 replication in vitro and select the most optimal concentrations, considering the cytotoxic effect;

· To determine the most effective anti-HIV-1 compounds for further research.

METHODS AND MATERIALS:

Compounds:

Uracil was chosen as one of the aromatic systems, since it is a convenient basis for the development of molecular structures. A benzene ring with methyl substituents in Meta positions was used as the second system. In our opinion, it can enhance the binding of these derivatives to the enzyme.

The 1-substituted 6-azidouracil derivatives and 1-substituted 6-aminouracil derivatives were synthesized by alkylation at the 1-position of the uracil skeleton of the readily available 6-chlorouracil using a conventional method⁹. Other studied compounds are 1-benzyl-3-(3,5-dimethylbenzyl) uracil (BBF-29), 6-benzyl-1-ethoxymethyl-5-isopropyluracil (MKC-442), and the nucleoside analogue 2′,3′-didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), they were synthesized based on the methods of our colleagues¹⁰. All compounds were dissolved in dimethyl sulfoxide at 100 mM and stored at −20°C until use.

HIV test:

MT-4 cells were maintained in RPMI 1640 medium with L-glutamine (Roswell Park Memorial Institute medium) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100u/ml penicillin g and 0.10g/ml of streptomycin. The HIV-1 strain III_Bwas used throughout the antiviral experiments. The virus was propagated and titrated in MT-4 cells. Virus stocks were stored at −80°C until use. The antiviral activity of test compounds against HIV-1 was determined by the inhibition of virus-induced cytopathic effect (CPE) in MT-4 cells in accordance to our colleagues’ method¹¹. Thus, MT-4 cells (1 × 105 cells/ml) were infected with HIV-1 at a multiplicity of infection (MOI) of 0.1 and were cultured in the presence of various concentrations of the tested compounds. After a 4-day incubation at 37°C in 5% CO2, the number of viable cells was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. The cytotoxicity of the compounds was evaluated in parallel with their antiviral activity, based on the viability of mock-infected cells, as determined by the MTT method. All experiments were performed in triplicate.

Long-term culture of infected cells MT-4:

MT-4 cells were infected with the HIV-1 strain III_B and incubated at 37°C for 4 days in the presence of compounds. The initial concentration of 1-substituted 6-azidouracil derivatives and 1-substituted 6-aminouracil derivatives corresponded to 1.9-fold higher than their 50% effective concentrations (EC50), i.e. 0.180 and 0.115μM, respectively. As control cultures, exactly identical passages of the infected MT-4 cells in the absence of the compounds were carried out in parallel with the cultures exposed to the compounds. At each passage, virus-induced CPE of the cells was monitored to confirm virus replication. The concentration of each compound was escalated 2-fold, when the CPE in the compound-treated culture exceeded 70%. The escape viruses when the CPE in the culture treated with the compound exceeded 70%.

Statistical analysis:

Statistical analysis for the EC50 of the test compounds against the wild-type and resistant virus was performed using an unpaired two-tailed Student t-test. P values of <0.01 were considered statistically significant.

RESULTS:

At the initial stage of the experiment, 1-substituted derivatives of 6-azidouracil and 1-substituted derivatives of 6-aminouracil were tested for their inhibitory effects on the replication of HIV-1 III_B, HIV-1 III_B-R. These compounds showed high activity against HIV-1 III_B with similar EC50 values (0.090±0.008 and 0.059±0.010μM, respectively) and 50% cytotoxic concentrations (CC50s) (39.8±6.5 and 49.0±1.2)μM, respectively). 1-substituted 6-aminouracil derivatives showed a higher selectivity index than 1-substituted 6-azidouracil derivatives (SI = 829 versus SI = 482) (Fig. 1). Although these compounds showed higher anti-HIV-1 activity against III_B compared to BBF-29 (0.29±0.03μM), they were not active against the NNRTI-resistant HIV-1 stain III_B-R. Moreover, 4'-Ed4T was equally active against HIV-1 III_B, HIV-1 III_B-R. Therefore, they were active only against HIV-1 III_B at the next stages of the experiment.

Figure 1: Antiviral activity of 1,3-disubstituted uracil derivatives against HIV-1

Note: A1 - 1-substituted derivatives of 6-azidouracil; A2 - 1-substituted derivatives of 6-aminouracil; BBF-29 - 1-benzyl-3-(3,5-dimethylbenzyl) uracil; MKC-442 - 6-benzyl-1-ethoxymethyl-5-isopropyluracil; 4'-Ed4T - 4′-ethynylthymidine; * - values for HIV-1 III_B; ** - values for HIV-1 III_B-R.

The 6-substituted 1-benzyl-3- (3,5-dimethylbenzyl) uracil and 1-substituted 3- (3,5-dimethylbenzyl) -5-fluorouracil (10a-10d) were studied for their inhibitory effects on HIV-1 induced sectional view of antiviral activity tests. Moreover, the comparative analysis for MKC-442 (emivirin) and 4'-ethynylstavudine (4'-Ed4T) was conducted. In a series of 6-substituted analogues, 6-methoxy derivative provided a comparable result to BBF-29, while in 6-thiomethoxy analogue, anti-HIV-1 activity was reduced as compared to BBF-29, suggesting that smaller electronegativity in the chalcogen atom at the 6-position in the uracil skeleton caused reduced anti-HIV-1 activity.

Next, introduction of linear group, such as cyano and azido groups at 6-carbon, was investigated for the structure-activity relationship. It is interesting that the 6-azido derivative showed excellent anti-HIV-1 activity and emerged as the most potent inhibitor in this series with 50% effective concentration (EC50) of 0.081± 0.023μM (50% concentration cytotoxicity [CC50] 48.7 ± 0.8μM and selectivity index [SI] 679). By contrast, the 6-cyano compound showed only moderate anti-HIV-1 activity. These wildly divergent results could be explained by the following. Since the steric effect is similar for both groups, the difference presumably comes from the anti-HIV-1 activity of their metabolites. To investigate the possibility that 6-azido derivative (3e) showed strong antiviral activity after converting metabolically to the 6-amino congener, the anti-HIV-1 activity of compound 3f was evaluated and found that it had comparable potency to that of 6-azido derivative (3e) with an EC50 of 0.069 ± 0.006μM (CC50 of 45.6 ± 0.9μM and SI 658). This result supports our assumption that 3e is reduced to 3f in cell cultures, and the metabolite (3f) inhibits HIV-1 RT. To prove this hypothesis, it is necessary to perform the anti-HIV-1 assay for 3e and 3f in a cell-free system.

5-Fluorouracil analogues (10a–10d) had reduced anti-HIV-1 activity and cytotoxicity in MT-4 cell (Fig. 2), suggesting that the introduction of fluorine into C5-position of uracil ring results in reduced anti-HIV-1 activity, presumably because the strong electron-withdrawing effect of fluorine causes electron deficiency of uracil ring and reduced affinity to the HIV-1 RT.

Figure 2: Studied 1,3-disubstituted uracil derivatives

DISCUSSION:

The use of purine and pyrimidine derivatives is widespread as antiviral drugs in the medical and pharmaceutical industry, but it would be a mistake to say that only high molecular compounds are effective. Significant progress in the development of antiviral drugs has come by targeting viral proteins with small molecules. or examples, compounds like aciclovir and zidovudine block viral reverse transcriptase to treat herpes simplex virus and HIV infections, respectively, and RNA-dependent RNA polymerase (RdRp) inhibitors like dasabuvir and sofosbuvir are used to treat hepatitis C virus infections¹². Thus, the properties of a compound with low-molecular-weight, which inhibits a wide range of viruses, have been described¹³. There are at least eight strongly inhibited virus families, including RNA-containing viruses (Orthomyxoviridae, Togaviridae, Flaviviridae, Paramyxoviridae, Rhabdoviridae), retroviruses (Retroviridae) and even coronavirus¹⁴, as well as DNA-containing viruses (Pox, Adenoviridae). According to Hoffmann et al., compound A3 was identified in a high-throughput screen for inhibitors of influenza virus replication. It displays broad-spectrum antiviral activity, and at noncytotoxic concentrations it is shown to inhibit the replication of negative-sense RNA viruses (influenza viruses A and B, Newcastle disease virus, and vesicular stomatitis virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, West Nile virus, and dengue virus), DNA viruses (vaccinia virus and human adenovirus), and retroviruses (HIV)^13,15,16.

In particular, the use of modified uracil derivatives is widespread not only against the AIDS, but they are also quite effective against other viruses, including flu. Therefore, inhibition of influenza A virus infection by conjugates of uridine and 2-deoxy sugars is a new promising approach for the development of new derivatives with anti-influenza activities¹⁷. A series of 1,6-bis[(benzyloxy)methyl] uracil derivatives combining structural features of both diphenyl ether and pyridone types of NNRTIs were synthesized by colleagues from Russia, Belgium and the United States. The target compounds were found to inhibit HIV-1 reverse transcriptase at micro- and submicromolar levels of concentrations and exhibited anti-HIV-1 activity in MT-4 cell culture, demonstrating resistance profile similar to first generation NNRTIs¹⁸. The synthesized compounds also showed profound activity against influenza virus (H1N1) in cell culture without detectable cytotoxicity.

The study results demonstrated that some new 1,3-disubstituted uracils selectively inhibit HIV-1 replication in cell cultures, which is also confirmed by other researchers^19,20. It should be noted that the hydrogen bonding interaction (H-bond) between ligand and amide group of Lys 101 residue as well as the hydrophobic interaction is important for the binding of uracil derivatives to HIV-1 RT²¹. Strong anti-HIV-1 activity of the 6-amino derivative (3f) can be explainable by the H-bond formed between 6-amino group of 3f and amide group of HIV-1 RT. Accordingly, the discrepancy of anti-HIV-1 activity between the 6-azido (3e) and 6-cyano analogues (3d) is caused by the antiviral activity of their metabolite²².

The 6-azido analogue (3e) may be converted to 6-amino congener (3f) and exert the anti-HIV-1 activity²³. The antiviral activity against drug-resistant mutants is important for developing these compounds for the treatment of HIV-1. Therefore, it is interesting to know whether 3e and 3f inhibit the replication of NNRTI-resistant strains. The ability of 3,4'-substituted derivatives of 1- (4'-hydroxy-2'-cyclopentenyl) uracil to act as non-nucleoside inhibitors against wild-type HIV-1, as well as mutant forms corresponding to HIV-1 strains, resistant to non-nucleoside reverse transcriptase inhibitors of the first generation was shown for the first time by Matyugina E.S and other in the post-Soviet space²³. Similar results were also obtained in Japan, Sakakibara N. et al. also found that, in comparison with emivirin, the compound 6-amino-3- (3,5-dimethylbenzyl) -1- (4-aminobenzyl) uracil significantly activity against HIV-1 with an EC50 value of 10 nM and a high selectivity index of 1896^24,25. In conclusion, the two 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl) uracils as novel anti-HIV agents were discovered. These compounds should be further pursued for their toxicity and pharmacokinetics in vivo as well as antiviral activity against NNRTI-resistant strains.

CONCLUSIONS:

According to study results, nine new uracil analogues were synthesized and evaluated as inhibitors of HIV-1. Key structural modifications included replacement of the 6-chloro group of 1-benzyl-6-chloro-3-(3, 5-dimethylbenzyl) uracil by other functional groups or N(1)-alkylation of 3-(3,5-dimethylbenzyl)-5-fluorouracil. These compounds showed only micromolar potency against HIV-1 in MT-4, though two of them; 6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil were highly potent (half maximal effective concentration =0.081 and 0.069μM) and selective (selectivity index =679 and 658), respectively. This result supports our assumption that 3e is reduced to 3f in cell cultures, and the metabolite (3f) inhibits HIV-1 RT. The anti-HIV-1 activity of the test compounds was determined by the inhibition of virus-induced cytopathogenicity in MT-4 cells. The cytotoxicity of the compounds was evaluated based on the viability of mock-infected cells. The structure–activity relationships among the newly synthesized uracil analogues suggest the importance of the H-bond formed between 6-amino group of 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil and amide group of HIV-1 reverse transcriptase. Two 6-substituted 1-benzyl-3-(3, 5-dimethylbenzyl) uracils, (6-azido-1-benzyl-3-(3, 5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3, 5-dimethylbenzyl) uracil) were discovered as novel anti-HIV agents.

When 1-substituted 6-azidouracil derivatives and 1-substituted 6-aminouracil derivatives were examined for their activity against the escape viruses obtained at passages 12 and 24, the compounds did not show any significant inhibition at their nontoxic concentrations. Thus, the isolates were more than 490 times more resistant to 1-substituted 6-azidouracil derivatives and 1-substituted 6-aminouracil derivatives compared to the wild type. The compound BBF-29 was also inactive against the escape viruses. The viruses had partial cross-resistance to MKC-442, probably due to its structural similarity. Meanwhile, 4′-Ed4T was equally inhibitory to the replication of the escape viruses and the wild type. These compounds should be further pursued for their toxicity and pharmacokinetics in vivo as well as antiviral activity against non-nucleoside reverse transcriptase inhibitor-resistant strains. Thus, it will contribute to the development of a new generation of compounds effective against different viruses, considering their quickly mutation and increased resistance. Therefore, in the future, we plan to adapt the results obtained for in vivo experiments.

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Received on 30.07.2020 Modified on 11.09.2020

Research J. Pharm. and Tech. 2021; 14(5):2723-2728.

DOI: 10.52711/0974-360X.2021.00480