Novel 2-Chloro-8-arylthiomethyldipyridodiazepinone Derivatives with Activity against HIV-1 Reverse Transcriptase †

Based on the molecular modeling analysis against Y181C HIV-1 RT, dipyridodiazepinone derivatives containing an unsubstituted lactam nitrogen and 2-chloro-8-arylthiomethyl were synthesized via an efficient route. Some of them were evaluated for their antiviral activity against HIV-1 RT subtype E and were found to exhibit virustatic activity comparable to some clinically used therapeutic agents.


Introduction
The introduction of antiretroviral therapy results in delayed progression of HIV-1.The majority of existing therapy methods have targeted the viral replication at reverse transcriptase (RT) and protease enzyme [1,2].However, the emergence of drug resistance has been observed [3,4], therefore, new therapeutic agents are still needed.Recently, a new class of therapeutic agents has focused on inhibiting HIV entry into cells, CD4 binding, coreceptor binding and membrane fusion such as T-20 [5].A number of bioactive nucleoside-based compounds against HIV virus have been clinically used [6].

Biological Testing
Compounds T1 and T2, together with some intermediates, were evaluated for their virustatic and virucidal activities against HIV-1 subtype E (CRF01 AE).In addition, the toxicity of the compounds, DMSO and cell controls were also examined.The biological activity of T1 and T2 as well as of intermediates 14 and 15 is summarized in Table 2. Compound T1 exhibited virustatic activity at EC 50 ≤ 1 µg/mL for seven days.On the other hand, compound T2 exhibited virustatic activity at EC 50 ≤ 1 µg/mL during the first four days, but the activity decreased to EC 50 ≤ 10 µg/mL by the seventh day.Other intermediates did not show virustatic activity.This result suggested that thioaryl group could be involved in regulating virustatic activity.However, all compounds did not show virucidal activity.
Virustatic activity of T1 and T2 against HIV-1 subtype E observed in seven days is shown in Figure 2. T1 exhibited comparable virustatic activity to that of AZT, GPOvir and nevirapine at concentration of 1 µg/mL on the fourth day.On the seventh day, T1 exhibited comparable activity to that of nevirapine at a concentration of 1 µg/mL and to that of GPOvir at 10 µg/mL, but showed higher activity than AZT at 1 µg/mL.T2 was less potent than T1 (~6-fold) at concentration of 1 µg/mL.

Molecular Docking
In order to investigate the orientation and estimated binding energies of the inhibitors in the enzyme binding site, molecular docking by using Lamarckian genetic algorithm AutoDock 3.05 program [11] was used to dock 68nv, T1 and T2 into the binding pocket of HIV-1 RT (pdb code 1KLM) and the results were compared with that of nevirapine, as shown in Table 3.The grid size was set to 80x80x80.A grid spacing of 0.375 Å was used and the numbers of docking runs were set to 50.Superimposition of the binding pocket of HIV-1 RT with docked 68nv, T1 and T2, compared with nevirapine (green), is shown in Figure 3.The obtained results demonstrate clearly that 68nv and both T1 and T2 oriented their structures in the HIV-1 RT binding site similar to nevirapine.However, the final docked energies and free energies of binding of both T1 and T2 which are the same as those of 68nv with the value of about ± 0.5 kcal/mol are much lower than that of nevirapine by about 4 kcal/mol.The explanation for this might be the fact that rotatable thiophenyl side chain in the three compounds can interact with the residues in the binding site such as Lys103, Leu234, His235 and Tyr318.However, the docked orientations of T1 and T2 revealed that the lack of methyl group at R 2 caused a slight move upwards of the T1 and T2 structures and make the adjustment of ethyl group attached at diazepinone ring to form the attractive interaction with Val189.Furthermore, methoxy group attached in thiophenyl side chain of T2 also interacts with Val106.This can significantly contribute to the conformational change of the whole enzyme structure, and consequently reduce catalytic efficiency of the enzyme.

Conclusions
In summary, two dipyridodiazepinone derivatives, T1 and T2, were synthesized based on a molecular modeling study and were found to exhibit virustatic activity against HIV-1 RT subtype E at 2.5 µM (1 µg/mM) and 23.5 µM (10 µg/mL), respectively.

General
The 1 H-and 13 C-NMR spectra were recorded on a Varian Gemini 2000 spectrometer operating at 200 and 50 MHz, respectively.Chemical shifts were recorded as δ values in ppm referenced to the solvent.Coupling constants (J) are given in Hertz.Infrared (IR) spectra were recorded in cm -1 on a Perkin Elmer 1760x FT-IR spectrometer.Mass spectra were obtained on a Finnigan Polaris GCQ mass spectrometer and accurate masses (HRMS) were obtained on a Bruker Micro TOF in ESI positive mode.Melting points (m.p.) were determined on a SMP3 melting point apparatus and are reported in o C uncorrected.Column chromatography was performed on Scharlau silica gel 60 (70-230 mesh).

3-Amino-2,6-dichloropyridine (6)
A solution of SnCl 2 •2H 2 O (970 mg, 4.3 mmol) in concentrated HCl (0.8 mL) was added dropwise to a stirred solution of 2,6-dichloro-3-nitropyridine (250 mg, 1.3 mmol) in acetic acid (2.6 mL) and the mixture was stirred for 3 hours at room temperature.Then the mixture was cooled in ice bath, and water was added.After stirring for an additional half an hour, the mixture was made basic (pH 12) with aqueous 50% sodium hydroxide.The reaction mixture was extracted with CH 2 Cl 2 and the combined organic layers were washed with water, dried (Na 2 SO 4 ) and concentrated under reduced pressure to give 6 (200 mg, 94.8%) as white solid which could be used directly in the next step.

N-(2,6-Dichloro-3-pyridinyl)-5-bromo-2-ethylamino-3-pyridinecarboxamide (10)
A solution of Br 2 (0.06 mL, 1.17 mmol) in acetic acid (1 mL) was added dropwise to a stirred solution of 4 (355 mg, 1.14 mmol), and potassium acetate (134 mg, 1.36 mmol) in acetic acid (15 mL).After 15 minutes, to the reaction mixture was added water and the precipitate was collected by suction filtration and washed with water for several times to provide  [1,4]diazepin-6-one (11) A solution of 10 (440 mg, 1.13 mmol) in pyridine (10 mL) was heated at 90 o C under a nitrogen atmosphere.Then the solution was added 0.6 M sodium bis(trimethylsilyl)amide in toluene (6.3 mL, 3.7 mmol) and the mixture was stirred at 90 o C for 15 minutes.After cooling down, the mixture was poured into ice water and stirred for additional 2 hours.The precipitate was filtered and washed with water to afford 11 (370 mg, 92.8%) as a yellow solid, m.p. 263 [1,4]diazepin-6-one (12) A solution of 11 (500 mg, 1.4 mmol) in DMF (7 mL) was treated with tetrakis(triphenylphosphine)palladium(0) (70 mg, 0.06 mmol) followed by vinyl tributyltin (1 mL, 3.42 mmol) and heated at 100 o C under nitrogen atmosphere for half an hour.After cooling down, the reaction mixture was poured into water and extracted with ethyl acetate.The combined organic layers were washed with 15% aqueous ammonia, brine and water, then dried (Na 2 SO 4 ) and concentrated under reduced pressure.The residue was purified by silica gel column chromatography (30% EtOAc/hexane) to provide 12 (318 mg, 74.6%) as a yellow solid, m.   [1,4]diazepin -6-one (13) A cooled (-78 o C) solution of 12 (343 mg, 1.14 mmol) in 1:1 CH 2 Cl 2 -MeOH (20 mL) was treated with O 3 for 30 minutes.After the completion of reaction, the solution was purged with O 2 for 5 minutes.Then the reaction mixture was stirred with triphenylphosphine (598 mg, 2.28 mmol) for additional 1 hour at room temperature.The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (30%EtOAc/hexane) to yield 13 (295 mg, 85.4%) as a yellow solid, m.p. 216

Figure 2 .
Figure 2. Virustatic activity of T1 and T2 observed in seven days.

Table 3 .
The docked energy and free energy of binding (kcal/mol) of T1 and T2 as compared with nevirapine and 68nv.