Chloro-1,4-dimethyl-9H-carbazole Derivatives Displaying Anti-HIV Activity

Background: Despite the progress achieved by anti-retroviral drug research in the last decades, the discovery of novel compounds endowed with selective antiviral activity and reduced side effects is still a necessity. At present, the most urgent requirement includes the improvement of HIV (Human Immunodeficiency Virus) prevention and sexual transmission and the development of new drugs to treat the chronic lifelong infection. Methods: Six chloro-1,4-dimethyl-9H-carbazoles (2a,b–4a,b) have been prepared following opportunely modified known chemical procedures and tested in luciferase and Escherichia coli β-galactosidase expressing CD4+, CXCR4+, CCR5+ TZM-bl cells. Results and Conclusion: a preliminary biological investigation on the synthesized small series of chloro-1,4-dimethyl-9H-carbazoles has been carried out. Among all tested compounds, a nitro-derivative (3b) showed the most interesting profile representing a suitable lead for the development of novel anti-HIV drugs.


Introduction
Nowadays, according to the National Institute on Drug Abuse (NIDA), part of the National Institutes of Health, approximately 37 million people are infected with HIV worldwide. HIV, the etiologic agent of AIDS (Acquired Immune Deficiency Syndrome), is a retrovirus belonging to the subclass of lentiviruses that thus shows an RNA genome, which guarantees a higher genetic variability and rapid adaptability [1].
At present, 25 anti-HIV drugs targeting reverse transcriptase, protease, integrase and viral entry and a pharmacokinetic enhancer are available. The most clinically adopted treatment consists in a combination of three anti-HIV drugs from at least two different classes in order to inhibit viral replication and diminish the onset of drug resistance [2]. This approach has resulted in a significant decrease of viral replication in HIV-infected individuals as well as a reduction of the risk of viral transmission [3,4]. Despite the encouraging results of the most recent treatments, in a considerable number of cases a therapeutic failure occurred mostly due to the virus capability to remain in a quiescent form in infected cells without being completely eradicated [5]. Considering the unremitting spread of HIV along with unpredictable outbreak of old or new virus strains, it is desirable to possess
Briefly, starting from the commercially available indoles 1a-b, the corresponding carbazole derivatives 2a-b were synthesized according to the method of Cranwell and Saxton [38]. These intermediates were transformed into the nitro derivatives 3a-b that were in turn reduced by stannous chloride to furnish the 3-amino-1,4-dimethyl-9H-carbazoles 4a-b with good yields (Scheme 1) [39].
In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle.
Briefly, starting from the commercially available indoles 1a-b, the corresponding carbazole derivatives 2a-b were synthesized according to the method of Cranwell and Saxton [38]. These intermediates were transformed into the nitro derivatives 3a-b that were in turn reduced by stannous chloride to furnish the 3-amino-1,4-dimethyl-9H-carbazoles 4a-b with good yields (Scheme1) [39]. In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4-or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle. In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4-or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle. In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4-or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle. In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4-or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle. In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4-or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle. In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4-or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle. In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4-or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle. In order to understand their mechanism of action, these compounds have been tested in CD4 + , CXCR4 + , CCR5 + TZM-bl cells [40] and some of them showed a moderate antiviral activity, although no significant differences have been observed when compounds were tested against CXCR4-or CCR5-using viruses (Table 1). These results suggest that the antiviral activity is probably also due to the inhibition of a different stage of HIV replication cycle. The obtained outcomes led us to the following remarks. The compounds bearing as R substituent a chlorine at position 7 (2-4b) showed the greater activity against HIV. On the contrary, the compounds functionalized with the chlorine at position 8 (2-4a) showed only a moderate antiviral activity, indicating that the position of the halogen plays a crucial role in the preservation of the activity. In particular, the nitro compound 3b showed the higher activity against HIV demonstrating that not only the chlorine position on the carbazole moiety is crucial for activity but it can be improved by the concomitant presence of an electro-attractor group. In conclusion, among all tested compounds, the 7-chloro-3-nitro derivative 3b resulted in the most promising compound and warrants further investigation.

General Procedure for the Preparation of 1,4-Dimethyl-9H-carbazole Derivatives (2a-b)
Acetonylacetone (0.70 mL, 6.00 mmol) and p-toluenesulphonic acid were added dropwise to a stirred solution of indole derivative 1a-b (6.00 mmol) in ethanol (10 mL). This reaction mixture was maintained under reflux for 6 h and then concentrated in vacuo. The crude product was purified by flash chromatography. then, pH solution was adjusted at 9 with 1 M solution of NaOH. After filtration, the crude product was purified by flash chromatography.

General Procedure for the Preparation of 3-Amino-1,4-dimethyl-9H-carbazole Derivatives (4a-b)
A stirred solution of 3-nitro-1,4-dimethyl-9H-carbazole derivatives 3a-b (3.6 mmol) in N,N-dimethylformamide (2 mL) was heated at 100 • C. The resulting solution was poured into a mixture containing 37% hydrochloride acid (2.6 mL), acetic acid (0.78 mL) and stannous chloride (18 mmol). The solution was stirred at 100 • C for 3 h and cooled to room temperature. The obtained suspension was poured into 20% stirred solution of sodium hydroxide and filtered. The solid was washed with water, air-dried and purified by flash chromatography.

Conclusions
The preliminary biological study of a small series of chloro-1,4-dimethyl-9H-carbazoles for their anti-HIV-1 activity is here reported. Among all tested compounds, the nitro-derivative 3b showed the most interesting profile indicating that the chlorine position on the carbazole scaffold could be important for its antiviral activity. In particular, the R 1 substituent seems to affect the antiviral activity in the following order: R 1 = H < NH 2 < NO 2 . Overall, the obtained data indicate that this class of compounds represents suitable starting points for the development of selective and perhaps alternative tools for the treatment of chronic infections induced by HIV.