Isatin thiazoline hybrids as dual inhibitors of HIV-1 reverse transcriptase

Abstract A series of 3-3-{2-[2-3-methyl-4-phenyl-2,3-dihydro-1,3-thiazol-2-ylidene]hydrazin-1-ylidene-2,3-dihydro-1H-indol-2-one derivatives has been designed and synthesized to study their activity on both HIV-1 (Human Immunodeficiency Virus type 1) RT (Reverse Transcriptase) associated functions. These derivatives are analogs of previously reported series whose biological activity and mode of action have been investigated. In this work we investigated the influence of the introduction of a methyl group in the position 3 of the dihydrothiazole ring and of a chlorine atom in the position 5 of the isatin nucleus. The new synthesized compounds are active towards both DNA polymerase and ribonuclease H in the µM range. The nature of the aromatic group in the position 4 of the thiazole was relevant in determining the biological activity.


Introduction
HIV-1 (Human Immunodeficiency Virus type 1) is one of the major causes of death now days. There are different approaches to keep under control this infection. The current approved treatment is based on the highly active antiretroviral therapy (HAART), which associates a combination of antiviral agents, targeting different steps of the virus replication cycle [1][2][3] . This multidrug therapeutic regimen leads to the reduction of the amount of circulating virus, in some cases below the current blood testing techniques detectable level, and allows high control of the infection. Moreover, it leads to the reduction of drug resistance occurrence, decrease of mortality and morbidity rates, and an overall improvement of patients quality of life 4

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However, there is not a therapeutic regimen capable of completely eradicate the virus from the host and, therefore, due to the chronic nature of HIV infection, a lifelong therapy is required. Hence both adherence to treatment and the management of drug-related toxicities are issues to deal with. In the light of the above the design and synthesis of new and more effective antiviral agents is an attractive open field for medicinal chemists.
In this respect, the identification of a multiple-acting molecule, able to inhibit different steps of the virus replication cycle, appears as a promising approach for the design of new and likely more efficient antiviral agents. Such agent should combine a dual function targeting capability in only one molecule [5][6][7][8] . Albeit HIV-1 Reverse Transcriptase (RT) has been the first and most investigated target for the therapy of HIV-1 infected patients, RT inhibitors (RTIs) are within the most represented components of HAART 3 . RT plays a key role in the HIV-1 replication cycle and is therefore a strategic target for the design of new therapeutic agents to combat the virus infection 9 . The molecular aspects of the RT/drug interaction have been reviewed by some of us and indication for the design of dual-acting RT inhibitors outlined 10 . Moreover the identification, design and synthesis of small molecules, able to simultaneously inhibit the two RT associated enzymatic functions, namely DNA/RNA dependent polymerase (DDDP and RDDP) and ribonuclease H (RNase H) functions, has been reported [11][12][13][14] . Isatin based molecular hybrids have been reported as a valid scaffold for the design of multi-target agents [15][16][17][18][19] , and in particular for the dual inhibition of RT associated enzymatic functions 12,14 . Hence, in continuation with our previous studies, we report on the synthesis and the structure-activity relationships of a series of 3-[2-(3methyl-4-aryl-1,3-thiazol-2-ylidene)hydrazin-1-ylidene]-1H-indol-2-ones whose activity has been evaluated towards both RT associated enzymatic functions DDDP/RDDP and RNase H.

Materials and apparatus
Starting materials and reagents were obtained from commercial suppliers and were used without purification. Chemical reagents were purchased form Sigma-Aldrich (St. Louis, MO). RNA-DNA labeled sequences were purchased from Metabion international AG.
All melting points were determined on a Stuart SMP11 melting points apparatus and are uncorrected. Electron ionization mass spectra were obtained by a Fisons QMD 1000 mass spectrometer (70 eV, 200 mA, ion source temperature 200 C). Samples were directly introduced into the ion source. Found mass values are in agreement with theoretical ones. Melting points, yield of reactions and descriptive data of derivatives EMAC 3039-3064 are reported in Table 1. 1 H-NMR spectra ( Table 2) were registered on a Varian 500 MHz spectrometer (Palo Alto, CA). All samples were measured in DMSO-d 6 and CDCl 3 . Chemical shifts are reported referenced to the solvent in which they were measured. Coupling constants J are expressed in hertz (Hz). Elemental analyzes were obtained on a Perkin-Elmer 240 B microanalyser (Waltham, MA). Analytical data of the synthesized compounds are in agreement within ±0.4% of the theoretical values. TLC chromatography was performed using silica gel plates (Merck F 254, Kenilworth, NJ), spots were visualized by UV light.

HIV-1 RT-associated RNA dependent DNA polymerase activity determination
The HIV-1 RT-associated RNA-Dependent DP activity was measured as previously described 21 . Briefly, 20 ng of HIV-1 wt RT was incubated for 30 min at 37 C in 25 mL volume containing 60 mM Tris-HCl pH 8.1, 8 mM MgCl 2 , 60 mM KCl, 13 mM DTT, 2.5 mM poly(A)-oligo(dT), 100 mM dTTP. Enzymatic reaction was stopped by addition of EDTA. Reaction products were detected by picogreen addition and measured with a Perkin-Elmer Victor 3 multilabel counter plate reader at excitation-emission wavelength of 502/523 nm.

Ligand preparation
Theoretical 3D models of the compounds were built by means of Maestro GUI 22 . Considering the configuration of double bonds described in literature 23 . The lowest energy conformers were considered for the following studies. These were obtained from the starting conformations through a conformational search by means of MacroModel version 7.2 (Schr€ odinger LLC, New York, NY) 24 , considering MMFFs 25 as force field and solvent effects by adopting the implicit solvation model Generalized Born/Surface Area (GB/ SA) water 26 . The simulations were performed allowing 1000 steps Monte Carlo analysis with Polak-Ribier Coniugate Gradient (PRCG) method and a convergence criterion of 0.05 kcal/(molÅ).

Docking experiments
QMPL default settings were applied 34 . The docking grids were defined by centering on W229 and Q500. The grid boxes of the same size (46 Â 46 Â 46 Å) covered overall the whole p66 subunit. Best solutions were subjected to post-docking procedure and analysed. 5000 steps of the Polak-Ribier conjugate gradient (PRCG) minimization method were conducted on the top ranked theoretical complexes using AMBER force field 35 . The optimization process was performed up to the derivative convergence criterion equal to 0.1 kJ/(mol Ã Å) À1 . The binding free energies were computed applying molecular mechanics and continuum solvation models with the molecular mechanics generalized Born/surface area (MM-GBSA) method 36 .

Figures
The resulting best complexes were considered for the binding modes graphical analysis with LigandScout (inte:Ligand, Vienna, Austria) 37 and Pymol (Schr€ odinger LLC, New York, NY) 38 .

Results and discussion
The synthesis of the isatin derivatives EMAC 3039-3063 is illustrated in Figure 1. Firstly, the 1-amino-3-methylisothiourea ( 1 ) was obtained by direct reaction between methylisothiocyanate and hydrazine hydrate (ratio 1:1), at rt, using ethanol as solvent. Secondly, the condensation between substituted isatin and compound 1, at reflux condition, using ethanol, gave the desired thiosemicarbazones. Finally, EMAC 3039-3063 derivatives were obtained in good yields by reaction of compound 2 with differently substituted bromo or chloro acetophenones in isopropanol. All the synthesized compounds were submitted to biological assays to evaluate their ability to inhibit both RT-associated enzymatic functions.
Results indicates that most of the EMAC isatin derivatives are able to inhibit both RDDP and RNase H functions at mM concentrations, indicating that these derivatives could represent a good starting point for the design of new and more efficient dual HIV-1 RT inhibitors. With the aim of obtaining more information on the structure activity relationships and to achieve insights into the binding mode of compounds EMAC 3039-3063, we performed blind docking studies on the wt-HIV-1 RT heterodimer by applying the QM-polarized ligand docking protocol (QMPLD) 34,39 considering two grid box: one centered in W229 (NNRTIBP) and one in Q500 (RNAse domain), in order to include all the p66 subunit 39 . In particular, we carried out ensemble docking experiments 40 using seven different crystal structures to take into account the target flexibility. The same protocol was successfully applied in our previous studies 12, 13 . In fact, the high NNRTIBP plasticity allows different orientations of Y181, Y188, Y183 and primer grip b12-b13 hairpins 41 . The overall volume of this pocket is 620-720 Å 3 and is characterized by a L shape. Its volume is approximately more than twice the one occupied by most of the known NNRTIs. Thus it offers many binding possibilities to small molecules 3 . This explains the large variety of chemical scaffolds of this class of inhibitors, whose shapes was often described in a creative way: e.g. "butterfly" 42 , "horseshoe" 43 and "dragon" 31 . These inhibitors are able to lock the enzyme into an inactive conformation.
We focused our attention on the most active compounds of the two series: EMAC 2045 and EMAC 2056.
The best RT complexes obtained by the docking experiments were subjected to a post-docking procedure based on energy minimization and successive binding free energy calculation. The binding free energies were obtained applying molecular mechanics and continuum solvation models using the molecular mechanics generalized Born/surface area (MM-GBSA) method 36 .
Blind docking calculations indicated the presence of two energetically favored sites that we named as Pocket 1 and Pocket 2. Pocket 1 is located close to the polymerase triad and   (c and f) 2D depiction of EMAC2045 and its respective interactions with RT residues. pale yellow sphere indicates hydrophobic interactions with lipophilic residues. Red arrow indicates a hydrogen bond (HB) acceptor interaction, while the violet sphere represents the aromatic p À p stacking interaction.
In pocket 2 the compounds are sandwiched between the two subunits p66 and p51. In fact, the complexes are stabilized by interactions with both A (p66) and B (p51) chains ( Figure 3).

Conclusions
In this research we have designed and synthesized a library of isatin based derivatives EMAC 3039-3063 to evaluate their activity towards both RT-associated enzymatic functions RDDP and RNase H. Our data indicate that the isatin derivatives are generally able to inhibit both RDDP and RNase H functions al mM concentrations and that these molecules are a good starting point for the design of new dual HIV-1 RT inhibitors. Nevertheless, considering the high potential of the isatin scaffold we will pursue in our efforts to identify new isatin based anti-viral agents.