Design, Synthesis and Anti-HIV Integrase Evaluation of N-(5-Chloro-8-Hydroxy-2-Styrylquinolin-7-yl)Benzenesulfonamide Derivatives

Styrylquinoline derivatives are demonstrated to be HIV-1 integrase inhibitors. On the basis of our previous CoMFA analysis of a series of styrylquinoline derivatives, N-[(2-substituted-styryl)-5-chloro-8-hydroxyquinolin-7-yl]-benzenesulfonamide derivatives were designed and synthesized, and their possible HIV IN inhibitory activity was evaluated.


Introduction
There has been an increasing attention in the development of HIV integrase (IN) as a promising anti-HIV target, due to the fact that HIV IN is essential in the replication of HIV-1 and there are no similar enzymes involved in human cellular functions [1,2]. Therefore, extensive efforts have been made, resulting in a large number of HIV IN inhibitors [3,4], among which polyhydroxylated styrylquinolines have displayed an antiviral activity in a de novo infection assay of CEM4 cells, thereby opening an exciting structural platform for the design of new anti-HIV drugs [5][6][7][8]. The structure-activity-relationship of these compounds reveals that for in vitro activity a carboxyl group at C-7, a hydroxyl group at C-8 (salicylic acid structure) in the quinoline subunit and an ancillary phenyl OPEN ACCESS ring are required. For example, FZ-41 in Figure 1 is one of the typical styrylquinoline-type HIV IN inhibitors [5][6][7][8]. To better understand the pharmacophore properties of styrylquinoline derivatives and to further design potential HIV-IN inhibitors, we recently investigated 38 styrylquinoline derivatives employing a comparative molecular field analysis (CoMFA) method [9]. The results indicated that inhibitory activity should be increased if a bulky group was near the carboxyl group at C-7 in the quinoline ring. Simultaneously, the presence of H-bonding donor is favorable near the C-7 atom, which might form a stable H-bond with some protein residues.
On the basis of the above information, we decided to modify the basic scaffold of styrylquinolinetype HIV IN inhibitors by replacing the carboxylic functionality at the C-7 position with an aromatic sulfonamide as its bioisosteric functionality, bearing a bulky aromatic group and, meanwhile, maintaining the feature of the H-bonding donor. In addition, to increase the acidic properties of the C-8 phenolic OH, a chloride atom was introduced at the C-5 of the quinoline ring. Such a design resulted in the target N-phenyl benzenesulfonamides III (Figure 1). Very recently, we have reported the synthesis and HIV IN screening of type II compounds [10]. In this work, we further describe the synthesis of type III compounds and their inhibitory activity against HIV IN. It should be pointed out that most of the work on the modification of styrylquinoline-type HIV IN concentrated on the modification of ancillary phenyl ring and linker unit. To the best of our knowledge, this is the first report concerning the replacement of salicyclic acid moiety of styrylquinoline HIV-IN inhibitors modified by sulfonamide.

Scheme 2.
Reaction between 5-chloro-2-methyl-7-nitroquinolin-8-ol and aldehydes. Selective sulfonylation reaction was also observed to be essential for the synthesis of title products III. In the presence of either pyridine or triethylamine, reaction of 2-styryl-7-amino-5-chloroquinolin-8-ols 6 and sulfonyl chloride always generated a mixture of N-and O-sulfonylation products, that proved to be hard to separate by conventional techniques. These results are quite different from the case of o-aminophenol, which was reported to give selective N-tosylation or O-tosylation by using 1 equiv. of pyridine or triethylamine, respectively [12]. Finally, we found that utilizing DMAP as a catalyst and pyridine as the solvent, the desired sulfonamide analogues could be synthesized smoothly [13]. The structures of styrylquinolin-7-yl-benzenesulfonamide derivatives III were confirmed by 1 H-NMR, 13 C-NMR, IR, and ESI-MS. Taking compound IIIa as an example, in its 1 H-NMR spectrum, there are two AA'BB' systems coupling in the range of 7.02 and 8.23 ppm, attributed to the proton signals of two benzene rings. The ethenyl linker shows two signals at 7.29 and 8.23 ppm (AB system) with a coupling constant of 16.0 Hz. This observation indicates that the styrylquinoline scaffold is in a trans configuration. In addition, the signals of three protons in quinoline ring (C-3, C-4 and C-6) exhibited a couple of doublets and a singlet at slightly low field at 7.75 (doublet), 8.34 (doublet) and 7.56 (singlet) ppm, respectively. The structure of IIIa (as well as for all compounds) was also characterized by ESI-MS. Strong peaks at 496.8 and 518.8 were recorded, which correspond to [M+H] + and [M+Na] + .

HIV IN inhibitory activity
All title compounds IIIa-q were preliminarily tested against purified HIV IN to determine any inhibitory activity possessed on the strand transfer reaction of IN. Using the high-throughput format assay approach developed by us [14], the inhibition percentages of styrylquinolin-7-ylbenzenesulfonamide derivatives IIIa-q were calculated based on the positive (baicalein) and negative (10% DMSO) controls and are listed in Table 2. For comparison, the IC 50 data of baicalein and FZ-41 were also included.
As shown in Table 2, compounds IIIn-IIIq, in which a free para-hydroxy group is present, showed higher inhibitory activity than that of the positive control, whereas, when the hydroxyl group was replaced by an electron-donating group (such as methoxy, compounds IIIa-IIId), hydrogen (IIIe-IIIi), or an electron-withdrawing group (such as bromide, compounds IIIj-IIIm), only a moderate inhibitory rate was observed. This observation means that the free hydroxyl moiety of styrylquinolin-7-yl-benzenesulfonamide derivatives is required for the inhibitory activity against HIV-IN. In addition, it was observed that the electron-withdrawing group at the para-position of benzenesulfonamide, such as nitro, may favor the inhibitory activity. For example, compound IIIi exhibits 96.7% inhibitory rate which decreases to 82.0% and 72.9% when the nitro was replaced by methyl (IIIf) and methoxy groups (IIIg), respectively. This results is not surprising because the electron-withdrawing group at the para-position of benzenesulfonamide moiety will increase the acidity of the benzenesulfonamide and lead to easier chelating with co-enzyme (mostly metallic ions), which is essential for the HIV IN inhibitory activity [15].  [14]; b Value of IC 50 cited from reference [6].

General
All solvents were of commercial quality and were dried and purified by conventional methods. Melting points (mp) were determined on an XT4A Electrothermal apparatus equipped with a microscope and are uncorrected. Infrared spectra (IR) were recorded as thin films on KBr plates with a Bruker IR spectrophotometer and are expressed in v (cm -1 ). The 1 H-and 13 C-NMR spectra were obtained using an AV 400M Bruker spectrometer in CDCl 3 or DMSO-d 6 with TMS as internal reference. The MS spectra (ESI) were recorded on a Bruker Esquire 6000 mass spectrometer.

Conclusions
In summary, on the basis of our previous CoMFA analysis of styrylquinoline derivatives, we have designed and synthesized for the first time a series of N-[ (2-substituted-styryl)-5-chloro-8hydroxyquinolin-7-yl]-benzenesulfonamide derivatives. The structures of these compounds were characterized and their HIV IN inhibitory activities were evaluated. Results indicate that most of the title compounds exhibit moderate inhibitory activity. Improved inhibitory activity can be achieved when free hydroxyl at the styryl moiety and nitro group at the benzenesulfonamide moiety are present.