Neoflavonoids as Inhibitors of HIV-1 Replication by Targeting the Tat and NF-κB Pathways

Twenty-eight neoflavonoids have been prepared and evaluated in vitro against HIV-1. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase reporter gene. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the HIV-LTR and Tat protein. Seven 4-phenylchromen-2-one derivatives showed HIV transcriptional inhibitory activity but only the phenylchrome-2-one 10 inhibited NF-κB and displayed anti-Tat activity simultaneously. Compounds 10, 14, and 25, inhibited HIV replication in both targets at concentrations <25 μM. The assays of these synthetic 4-phenylchromen-2-ones may aid in the investigation of some aspects of the anti-HIV activity of such compounds and could serve as a scaffold for designing better anti-HIV compounds, which may lead to a potential anti-HIV therapeutic drug.


Introduction
UNAIDS report in 2016 indicated that tuberculosis remains the leading cause of death among people living with HIV, accounting for around one in three AIDS-related deaths. In 2014, the percentage of identified HIV-positive tuberculosis patients who started or continued on ART reached 77% [1]. This relevant fact should promote further studies to take advantage of this therapeutic ambivalence and to evaluate the possibility of using 4-phenylchromene-2-ones for treating patients suffering from both AIDS and tuberculosis.
Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS) which is one of the leading causes of 2.9% of mortality in the world [2]. Although modern antiretroviral therapy (ART) using a combination of anti-HIV drugs has been highly effective in suppressing HIV load and decreasing mortality in AIDS patients, the emergence of drug resistances in HIV and the toxicity of the therapies currently in use have made the continued search for novel anti-HIV drugs necessary [3,4]. On the other hand, failures in efforts to develop an effective vaccine against HIV-1 infection [5] have emphasized the importance of ART in treating HIV-1-infected patients. Therefore, medicinal chemists are interested in the development of novel anti-HIV agents that might be particularly effective in controlling strains of HIV that are resistant to the current drugs [6].
The HIV viral cycle can be divided into early and late stages. Early stages comprise several steps, from viral attachment on the cell surface to integration in the host genome. Late stages include the processes of HIV mRNA synthesis, protein expression and morphogenesis. Once integrated, HIV can remain in a latent state in resting lymphocytes or undergo active replication. Transition from latency to HIV expression occurs mainly when cells are activated and requires the concerted action of cellular transcription factors and regulatory HIV proteins [7,8]. Among the transcription factors involved in LTR transactivation, the HIV proximal enhancer contains three binding sites for SP1 transcription factor and two binding sites for NF-κB. The NF-κB/Rel family of transcription factors represents a major inducible regulatory element involved in HIV transcription [9]. Located downstream of the basal promoter TAR sequence is the RNA target for the viral protein Tat, which acts in concert with other cellular factors [10], to generate full-length RNA transcripts [11]. Furthermore, NF-κB and Tat cooperate in driving HIV replication from the state of latency. Therefore, inhibition of the activity of these critical proteins should result in an effective blocking of viral replication [12][13][14].
In a previous paper we reported the anti-HIV activity of natural 4-phenylcoumarins isolated from Marila pluricostata. They were structurally related to Inophyllum coumarins series, but with one prenyl and other cyclized group across the hydroxyl group at position C-7 [43]. Furthermore, these compounds showed moderate anti-mycobacterial activity. This relevant fact induced us to prepare new similar, but simpler, derivatives with the idea in mind to obtain compounds with activity against both HIV and tuberculosis, and also to increase the structural diversity. With that, a better structure-activity relationship could be established. In this paper we reported the preparation and the anti-HIV activity of several neoflavone derivatives that showed anti-mycobacterial activity [48].

Chemistry
The 4-phenylchromen-2-ones or neoflavones 1-7 have been obtained by the Peckman condensation between ethyl benzoylacetate and different phenol derivatives that is, resorcinol for 1, phloroglucinol for 2, hydroxyhydroquinone for 3, 3,4-methylenedioxyphenol for 4, 3-methoxycatechol for 5, 2,3-dimethoxyphenol for 6 and β-naphthol for 7 in presence of concentred H 2 SO 4 as condensing agent (Scheme 1). Some differences in the yield of the different compounds can be appreciated depending on the phenol derivative used (see the Experimental Section). Phloroglucinol, as the starting material (Scheme 1), was treated with ethyl benzoylacetate by the Pechmann-Duisberg reaction giving 5,7-dihydroxy-4-phenylcoumarin (2). Friedel-Craft acylation or benzoylation of compound 2, by refluxing the phenol derivative with the corresponding acylchloride in a carbon disulfide/nitrobenzene mixture, and in the presence of aluminum trichloride, followed by Fries rearrangement provided mixtures of the 6-, 8-monoacylated and benzoylated neoflavones and 6,8-diacylated or dibenzoylated neoflavones [49][50][51][52]. Workup of the crude reaction products led to the isolation of neoflavones 8-21 whose spectroscopic properties were consistent with the structures shown in the Scheme 2. On the basis of detailed analysis of the H-H COSY (Correlation SpectroscopY), H-C HMQC (Heteronuclear Single Quantum Correlation), and H-C HMBC (Heteronuclear Multiple-Bond Correlation) 2D-NMR spectra, all of the compounds described were correctly characterized and their 13 C-NMR data will be introduced in NAPROC-13 RMN spectroscopic database [53], for posterior online identification of natural products and their analogs and derivatives. Phloroglucinol, as the starting material (Scheme 1), was treated with ethyl benzoylacetate by the Pechmann-Duisberg reaction giving 5,7-dihydroxy-4-phenylcoumarin (2). Friedel-Craft acylation or benzoylation of compound 2, by refluxing the phenol derivative with the corresponding acylchloride in a carbon disulfide/nitrobenzene mixture, and in the presence of aluminum trichloride, followed by Fries rearrangement provided mixtures of the 6-, 8-monoacylated and benzoylated neoflavones and 6,8-diacylated or dibenzoylated neoflavones [49][50][51][52]. Workup of the crude reaction products led to the isolation of neoflavones 8-21 whose spectroscopic properties were consistent with the structures shown in the Scheme 2. On the basis of detailed analysis of the H-H COSY (Correlation Spectroscopy), H-C HMQC (Heteronuclear Single Quantum Correlation), and H-C HMBC (Heteronuclear Multiple-Bond Correlation) 2D-NMR spectra, all of the compounds described were correctly characterized and their 13 C-NMR data will be introduced in NAPROC-13 RMN spectroscopic database [53], for posterior online identification of natural products and their analogs and derivatives. Phloroglucinol, as the starting material (Scheme 1), was treated with ethyl benzoylacetate by the Pechmann-Duisberg reaction giving 5,7-dihydroxy-4-phenylcoumarin (2). Friedel-Craft acylation or benzoylation of compound 2, by refluxing the phenol derivative with the corresponding acylchloride in a carbon disulfide/nitrobenzene mixture, and in the presence of aluminum trichloride, followed by Fries rearrangement provided mixtures of the 6-, 8-monoacylated and benzoylated neoflavones and 6,8-diacylated or dibenzoylated neoflavones [49][50][51][52]. Workup of the crude reaction products led to the isolation of neoflavones 8-21 whose spectroscopic properties were consistent with the structures shown in the Scheme 2. On the basis of detailed analysis of the H-H COSY (Correlation SpectroscopY), H-C HMQC (Heteronuclear Single Quantum Correlation), and H-C HMBC (Heteronuclear Multiple-Bond Correlation) 2D-NMR spectra, all of the compounds described were correctly characterized and their 13 C-NMR data will be introduced in NAPROC-13 RMN spectroscopic database [53], for posterior online identification of natural products and their analogs and derivatives. Neoflavones 22-28 were obtained by the condensation of substituted cinnamic acids with the corresponding phenols, that is, phenol for 22, resorcinol for 23 and 24, 3,5-dichlorophenol for 25, 3,4-methylenedioxyphenol for 26 and 27, and 3-methoxybenzene-1,2-diol for 28. The reaction occurred in the presence of milder Friedel-Crafts catalyst BF 3 -Et 2 O and POCl 3 in 54%-75% yields (Scheme 3). Previous attempts towards condensation of substituted cinnamic acids with corresponding phenols for the preparation of 4-phenylchromen-2-ones 22-28 in the presence of concentrated HCl and HCl gas were unsuccessful. Additionally, some of these compounds were synthesized in low yields by microwave irradiation.
Neoflavones 22-28 were obtained by the condensation of substituted cinnamic acids with the corresponding phenols, that is, phenol for 22, resorcinol for 23 and 24, 3,5-dichlorophenol for 25, 3,4-methylenedioxyphenol for 26 and 27, and 3-methoxybenzene-1,2-diol for 28. The reaction occurred in the presence of milder Friedel-Crafts catalyst BF3-Et2O and POCl3 in 54%-75% yields (Scheme 3). Previous attempts towards condensation of substituted cinnamic acids with corresponding phenols for the preparation of 4-phenylchromen-2-ones 22-28 in the presence of concentrated HCl and HCl gas were unsuccessful. Additionally, some of these compounds were synthesized in low yields by microwave irradiation.

Scheme 3. Preparation of neoflavanones 22-28.
Some compounds prepared in this study have been described already in the literature (see the Experimental Section) and their structures were verified from an iterative search by 13 C-NMR chemical shifts carried out within our NAPROC-13 RMN spectroscopic database [53], and later identified unambiguously. The rest of the prepared 4-phenylcoumarins were established on the basis of 1 H-and 13 C-NMR spectra. A combination of COSY, HMQC, and NOE experiments was utilized when necessary for a correct assignment of 1 H and 13 C chemical shifts.
The synthesized neoflavonoids 1-8 were evaluated in vitro against HIV-1 with the results shown in Table 1.

Evaluation of Antiviral Activity
The neoflavonoids synthesized (1-28) belong to three groups: simple 4-phenylchromen-2-ones, acyl-4-phenylchromen-2-ones and 3,4-dihydro-4-phenylchromen-2-ones, which have been evaluated in the anti HIV bioassay. The analysis of the results of inhibitory activity of NF-κB indicates that 4-phenylchromenones 9, 10, 13, 14, and 15 show a fair inhibitory activity at 50 μM. Regarding the specific HeLa-Tat-Luc assay results, compounds 9, 13, and 15 were nonspecific, whereas compounds 10 and 14 showed specificity. Furthermore compound 14 is only slightly toxic at 50 μM, but not toxic at 25 μM, and their activity as NF-κB and tat inhibitors is still strong (83.06% for NF-κB and 41.87% for Tat). Compound 14 turned out to be identical to the natural neoflavonoid Isodispar B previously isolated from Marila pluricostata [43]. Compound 10 NF-κB activity is also strong (70.53 at 25 μM) and it is nontoxic at 10 μM. Interestingly, the dichlorinated 3,4-dihydroflavonoid 25 showed specific anti-Tat activity, whereas all other 3,4-dihydroanalogs resulted inactive in this assay. It must be noted that simple structural differences within this series of 4-phenylchromen-2-ones and acyl 4-phenylchromen-2-ones, determine substantial changes in activity and selectivity. As an example, we could compare the specific NF-κB inhibitor 8-isovaleroyl-4-phenylchromen-2-one 14 and the specific Tat inhibitor 3,4-dihydro-5,7-dichloro-4-phenyl-chroman-2-one 25, also a 4-phenylchromen Some compounds prepared in this study have been described already in the literature (see the Experimental Section) and their structures were verified from an iterative search by 13 C-NMR chemical shifts carried out within our NAPROC-13 RMN spectroscopic database [53], and later identified unambiguously. The rest of the prepared 4-phenylcoumarins were established on the basis of 1 Hand 13 C-NMR spectra. A combination of COSY, HMQC, and NOE experiments was utilized when necessary for a correct assignment of 1 H and 13 C chemical shifts.
The synthesized neoflavonoids 1-8 were evaluated in vitro against HIV-1 with the results shown in Table 1.

Evaluation of Antiviral Activity
The neoflavonoids synthesized (1-28) belong to three groups: simple 4-phenylchromen-2-ones, acyl-4-phenylchromen-2-ones and 3,4-dihydro-4-phenylchromen-2-ones, which have been evaluated in the anti HIV bioassay. The analysis of the results of inhibitory activity of NF-κB indicates that 4-phenylchromenones 9, 10, 13, 14, and 15 show a fair inhibitory activity at 50 µM. Regarding the specific HeLa-Tat-Luc assay results, compounds 9, 13, and 15 were nonspecific, whereas compounds 10 and 14 showed specificity. Furthermore compound 14 is only slightly toxic at 50 µM, but not toxic at 25 µM, and their activity as NF-κB and tat inhibitors is still strong (83.06% for NF-κB and 41.87% for Tat). Compound 14 turned out to be identical to the natural neoflavonoid Isodispar B previously isolated from Marila pluricostata [43]. Compound 10 NF-κB activity is also strong (70.53 at 25 µM) and it is nontoxic at 10 µM. Interestingly, the dichlorinated 3,4-dihydroflavonoid 25 showed specific anti-Tat activity, whereas all other 3,4-dihydroanalogs resulted inactive in this assay. It must be noted that simple structural differences within this series of 4-phenylchromen-2-ones and acyl 4-phenylchromen-2-ones, determine substantial changes in activity and selectivity. As an example, we could compare the specific NF-κB inhibitor 8-isovaleroyl-4-phenylchromen-2-one 14 and the specific Tat inhibitor 3,4-dihydro-5,7-dichloro-4-phenyl-chroman-2-one 25, also a 4-phenylchromen related. The drastic difference in bioactivity for these compounds should be due either to the presence of an isovaleroyl or heptanoyl group in C-6 or C-8, which potentiates the activity in both targets. However, regarding the comparison of compounds 7 and 14, it is worth noting that the presence of a benzene ring fused to the chromenone moiety, increases the anti-Tat activity, but the introduction 8-isovaleroyl or 6,8-diacetyl groups enhances the NF-κB inhibitory activity. Compound 10, when compared to Disparinol A, showed higher anti-HIV potency than this natural neoflavonoid. The acyl derivatives of 4-phenylchromen-2-ones are the most potent dual-target inhibitors.
The assays of these 4-phenyl-chromen-2-one derivatives may aid in the investigation of some aspects of the anti-HIV activity of this kind of compound that inhibited the transcription and could serve as a scaffold for designing better anti-HIV compounds, which may lead to a potential HIV therapeutic drug.

General Information
All of the reagents for synthesis were commercially available and either used without further purification or purified by standard methods prior to use. Melting points were determined on a Büchi 510-K melting point apparatus (Büchi Labortechnik AG, Flawil, Switzerland) and are uncorrected. IR spectra were recorded (KBr 1%) in a Nicolet Impact 410 spectrophotometer. 1 H-, 13 C-NMR, COSY, HMQC, and HMBC were recorded on Brüker AC 200 (200 MHz) and Brüker DRX 400 (400 MHz) instruments. Chemical shifts (δ) are expressed in parts per million (ppm) relative to the residual solvent peak, and coupling constants are reported in Hertz (Hz). All signals assigned to hydroxyl groups were exchangeable with D 2 O. Reaction progress was monitored using analytical thin-layer chromatography (TLC) on precoated Merck silica gel Kiesegel 60 F 254 plates, and the spots were detected under UV light (254 nm). The flash chromatography was conducted using silica gel 230-400 mesh. For EIMS and HRFABMS analysis, a VG-TS250 mass spectrometer (70 eV) was used. Elementary analyses were obtained with a LECO CHNS-932 and were within ±0.4% of the theoretical values.

General Procedures II: Synthesis of Compounds 8-22
Anhydrous aluminum trichloride (0.4 mmol) was added to a stirred suspension of compound 2 (0.1 mmol) in carbon disulfide (6 mL). Nitrobenzene (2 mL) was then added over 40 min, forming a homogeneous solution with evolution of HCl. The solution was heated under reflux for 30 min, appropriate acyl chloride (0.1 mmol) in nitrobenzene (1 mL) was added over 40 min before allowing it to cool with stirring. The mixture was poured onto ice/water and aqueous HCl and was extracted with ethyl acetate (25 mL, twice). Workup of the crude product by chromatography on silica gel led to the isolation of the different acyl derivatives products.
Following the general procedure II using benzoyl chloride, the crude reaction product was chromatographed and eluted with hexane/EtOAc, to 11 and 16.

General Procedures III: Synthesis of Compounds 22-28
To a mixture of POCl 3 (10 mmol) and BF 3 -Et 2 O (20 mmol) at 0 • C, appropriated cinnamic acid (5 mmol) was added and the reaction mixture stirred for 15 min at 0 • C. Phenol (5 mmol) was added to the above reaction mixture in small portions and stirring continued at 25-28 • C for 4-12 h. The reaction mixture was poured on to ice-water; sodium acetate (1 g) was added and the mixture was warmed on a water bath for 2 min. It was cooled, extracted with ethyl acetate (2 × 150 mL), washed with water (150 mL), dried, and the solvent removed under reduced pressure to obtain the crude product, which was purified by column chromatography using acetone-chloroform as eluent to afford pure 4-phenyldihydro-coumarins 22-28 in 60%-75% yields.

Antiviral Activity Assays
The anti-HIV activity of these neoflavonoids on Tat and NF-κB functions has been evaluated. To this aim, we have used two stably transfected cell lines. The previously described 5.1 cell line [55] is a Jurkat-derived clone stably transfected with a plasmid containing the luciferase gene under the control of HIV-LTR. In this cell clone, activation with TNFα induces NF-κB activation and subsequent HIV-1 expression. We have also analysed the anti-HIV activity in HeLa-Tat-Luc cells, in which the HIV-1 LTR is directly activated by the HIV-1 Tat protein. A compound was considered active in one assay if it inhibited the target function by more than 50% (NF-κB) or 30% (Tat) at either 25 or 50 µM concentration. The active compounds were submitted for further evaluation through a HeLa-Tet-ON assay, as previously described [56]. In the Hela-Tet-ON cells the luciferase expression is under control of an artificial promoter that can be activated by tetracycline. Therefore, compounds that inhibit tetracycline-induced luciferase activity were considered non-specific for luciferase-based anti-HIV assays.
Cell viability was evaluated in non-infected treated cultures following the same protocol as in the recombinant virus assay and measuring cell toxicity with a classical MTT assay. IC 50 were calculated using GraphPad Prism software (non-linear regression, log (inhibitor) vs. response).

Conclusions
A series of twenty-eight neoflavonoids have been synthesized and evaluated against HIV-1 in vitro. Ten 4-phenylchromen-2-one derivatives displayed HIV specific transcriptional inhibition and five displayed nonspecific mechanisms of action. The heptanoylchromen-one 10 was the more potent Tat antagonist, while compound 14 showed high inhibition of the NF-κB pathway. A preliminary SAR analysis established that the presence of the acyl group is essential for the anti HIV in both targets.