Design and Synthesis of New 6-Nitro and 6-Amino-3,3a,4,5-Tetrahydro-2H-Benzo[g]indazole Derivatives: Antiproliferative and Antibacterial Activity

New substituted benzo[g]indazoles functionalized with a 6-nitro and 6-amino groups have been synthesized by the reaction of benzylidene tetralones with hydrazine in acetic acid. The resulting conformationally-constrained compounds were evaluated for their antiproliferative activity against selected cancer cell lines. The nitro-based indazoles 11a, 11b, 12a and 12b have shown IC50 values between 5–15 μM against the lung carcinoma cell line NCI-H460. Moreover, the nitro compounds were tested for antibacterial activity where compounds 12a and 13b have shown MIC values of 250 and 62.5 μg/mL against N. gonorrhoeae with no hemolytic activity in human red blood cells (RBC).

It should be highlighted that these fused tricyclic pyrazole derivatives are characterized by their conformational restriction [15].
Colchicine-site inhibitors based on combretastatin A-4 (1, CA-4, Figure 1A) have been extensively studied as antimitotic and vascular disrupting agents [17][18][19]. Among the different approaches followed to mimic the cis (active) configuration of CA-4, the design and synthesis of conformationally restricted ligands have led to very potent inhibitors. Among recently reported examples, the dihydronaphthalene and benzosuberene derivatives (2 and 3, respectively, Figure 1A) have shown antiproliferative activity at the sub nM range [20]. Very recently, Jiang, J. et al. [21] described a novel 1-phenyl-dihydrobenzoindazole (4, Figure 1A) with a locked conformation, that inhibited tubulin polymerization with an IC 50 of 1.6 µM and showed antitumor properties against a human colon cancer cell line with an IC 50 value of 1 nM. In both series the presence of an amino group on ring B is meant to mimic the phenolic OH of CA-4 and this amino group has been used for the synthesis of prodrugs thereof [20,21]. In addition, in all cases a trimethoxyphenyl is present as ring A (Figure 1). We have recently reported on tetrahydronaphtalene derivatives as conformational restricted mimetics of chalcone derivatives that also bind the colchicine binding site in tubulin [22]. As in the previous examples, an amino group was incorporated at position 5 on ring B and this was shown to be crucial for the antiproliferative activity and the tubulin binding capacity. Thus, compound 5 was found to inhibit CEM (human lymphoblastic leukemia) proliferation at the sub µM range.
Based on the general interest of tetrahydroindazole derivatives, we have here designed and synthesized a new series of tricyclic pyrazoline derivatives incorporating an amino group at position 6 on ring B that were obtained through reduction of their nitro precursors ( Figure 1B). Several 4-methoxy aryl rings have been incorporated as ring A. In particular, those compounds with a 3,4,5-trimethoxyphenyl as ring A and an amino group at position 6 on ring B could mimic the conformation of our colchicine-binding site inhibitor 5, as will be later discussed. The synthesized compounds have been tested for their antiproliferative activity against a wide panel of tumor cells. As a part of our continuing effort to find molecules with antibacterial activity [23][24][25][26], the nitro derivatives were also screened against wild type and multidrug-resistant bacteria, particularly those frequently found in relevant human infections.

Chemistry
The synthesis of the targeted compounds showed in Figure 1B required the 6-methoxy-5-nitro-1-tetralone (6) as a starting material that was prepared as previously described [20,27]. Reaction of 6 by Claisen-Schmidt condensation with aromatic aldehydes (Scheme 1) in the presence of NaOH in ethanol provided the corresponding chalcones 7-10 in excellent yields (82, 86, 82 and 77% yield, respectively). Scheme 1. Synthesis of the substituted chalcones 7-10. Reagents and conditions: (a) aromatic aldehyde, NaOH, EtOH, rt., 7 h. The chalcones 7-9 were then treated with hydrazine hydrate in the presence of acetic acid to give a mixture of diastereoisomers 11a-13a and 11b-13b in a 1:1 ratio that could be separated by chromatography, and with global yields of 59, 74 and 71%, respectively (Scheme 2). The structural and stereochemical assignment of each pair of enantiomers was performed based on NMR experiments. As an example, the 2D NOESY spectrum for compound 11b showed a NOE signal between the protons H-3 and H-3a, and therefore 11b was assigned as the (3R,3aR)-rel stereoisomer. In addition, the signal corresponding to H-3 in 11b has a coupling constant (J 3,3a = 11.0 Hz) larger than the one of the same signal in the diastereomer 11a (J 3,3a = 9.7 Hz). When the 13 C NMR spectra of 11a and 11b were compared, the signal corresponding to C-3a appeared at 53.8 and 46.9 ppm, respectively. Thus, there is a significant upfield shift for this signal in 11b, suggesting the cis configuration ((3R,3aR)-rel), in agreement with those reported in the literature [8,10,12,28]. Interestingly, in the whole series, one of the signals corresponding to H-4 for the (3R,3aR)-rel isomers showed a chemical shift around 0.7-0.8 ppm, probably due to the shielding effect of the aromatic ring A, while this signal for the (3R,3aS)-rel isomers is around 1.90 ppm. We consider that this difference can also be employed as useful and convenient criteria to assign both isomers. Similarly, reaction of chalcone 10 with hydrazine hydrate in the presence of acetic acid gave a mixture of the two trimethoxy derivatives 14a and 14b (Scheme 2), in a global yield of 66%, but whose separation by chromatography was very laborious. Thus, the mixture of 14a and 14b was used for the next reaction. Finally, the nitro derivatives 11-14a,b were reduced to their amino analogs by treatment with Fe in an EtOH:H 2 O mixture at reflux in the presence of HCl (Scheme 2), following the procedure previously used for the reduction of the 5-nitrotetrahydronaphthalenes [22], providing compounds 15a-18a and 15b-18b in moderate to good yields (25-75%). These compounds 15a-18a and 15a-18b showed similar spectroscopic data and therefore, we discuss here compound 15a as the representative of this series.
In the 1 H-NMR spectrum, the signal corresponding to H-3a appears as a double-double-doublet at 3.05 ppm with coupling constants 3 J = 13.8, 9.2, 4.8 Hz; the signals for H-4 appear as two multiplets at 1.83 ppm integrating for one proton and between 2.15 and 2.17 ppm for four protons corresponding to H-4 and the methyl protons of acetyl group. A broad singlet for amino protons was observed at 4.61 ppm. The 13 C-NMR spectrum showed the expected nineteen signals for the carbons of compound 15a.
As mentioned in the introduction, these compounds are characterized by a high degree of conformational restriction, thus they could represent an alternative scaffold for colchicine site binding agents. Thus, 3D structures of compounds 18a and 18b were constructed and their geometries were optimized with MOPAC2012 (AM1 method) [29]. This was followed by a manual superposition of the geometry-optimized structures with the conformation of the tetrahydronaphtalene derivative 5 for which we have recently reported its tubulin binding capacity and antiproliferative activity [22]. As shown in Figure 2, the degree of overlapping is quite relevant, the major differences being the relative orientation of ring A and the presence of the additional acetylated pyrazoline ring. Interestingly, a comparison of the 3D structures of 18a (3R,3aS) and 18b (3R,3aR) also helps to explain the differences observed in the chemical shifts in the 1 H-NMR spectrum of one of the protons H-4. As shown in Figure 3, for 18b one of the H-4 protons is facing the phenyl ring A. Thus, this shielding effect explains the differences in chemical shifts for this proton between the b-isomers (0.7-0.8 ppm) and the a-isomers (around 1.90 ppm) in the whole series.

Antiproliferative Activity
All benzo[g]indazole derivatives 11-18a,b were tested for their antiproliferative activity against a wide panel of tumor cells. As shown in Table 1, most of the 3,3a,4,5-tetrahydro-2H-benzo[g]indazole derivatives showed modest antiproliferative activity against the cell lines tested. In particular, the trimethoxy derivatives 18a and 18b showed IC 50 values around 50 µM. On the other hand, the 6-nitro derivatives 11a, 11b and 12a, 12b, showed IC 50 values in the low µM range against a lung carcinoma cell line NCI-H460. Some additional studies were performed for compounds 11a, 11b, and 12a, 12b. At 10 µM, the compounds did not affect tubulin as measured by immunostaining in HeLa cells, suggesting that tubulin is not the target for the antiproliferative activity. They were also screened for DNA intercalation or DNA damage, using standardized procedures. These protocols are included as Supplementary Materials. Also, in this case, no significant effect was observed.

Antibacterial Activity
Compounds 7-10 and 11-14a,b were investigated for their antibacterial activity against wild type and multidrug-resistant bacteria including methicillin-susceptible S. aureus ATCC 25923 (MSSA), methicillin-resistant S. aureus ATCC 43300 (MRSA), vancomycin-intermediate S. aureus (VISA), E. coli ATCC 25922, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 700603 (Extended spectrum beta-lactamase positive, ESBL+), K. pneumoniae BAA1705 (carbapenemase-positive) and N. gonorrhoeae ATCC 49226. The minimum inhibitory concentration (MIC) was determined by broth microdilution in those compounds with reproducible antimicrobial effect. Table 2 shows that N. gonorrhoeae growth was inhibited by 12a and 13b. Compound 13b was the most active of the series with a MIC value of 62.5 µg/mL, whereas 12a induced moderate growth inhibition (MIC = 250 µg/mL). Comparison of the antibacterial activity of the isomers 13a (3R,3aS)-rel-(MIC ≥ 500 µg/mL) and 13b (3R,3aR)-rel-(MIC = 62.5 µg/mL) against N. gonorrhoeae shows that the configuration of the molecule has an important effect on the activity. On the other hand, it is worth to take into account the recent emergence of resistant clinical isolates of N. gonorrhoeae to a variety of antimicrobial agents, for which the need to find new antigonococcal agents is of paramount importance [30][31][32]. Compounds 12a and 13b were further tested for hemolytic activity or toxicity against RBC. None of the active compounds induced hemolysis of the human RBC.

General Information
Reagents and solvents used were purchased from Sigma-Aldrich (St. Louis, MO, USA). Reactions were monitored by TLC on silica gel 60 F 254 aluminum plates (Merck, Dramstand, Germany). Spots were detected under UV light (254 nm) and/or charring with ninhydrin or phosphomolybdic acid. Melting points were measured on a M170 apparatus (Mettler Toledo, Columbus, Ohio, USA) and are uncorrected. The elemental analyses were performed using a CHN-O-RAPID instrument (Heraeus, Hanau, Germany). Analyses indicated by the symbols of the elements or functions were within ±0.4% of the theoretical values. 1 H and 13 C-NMR spectra were run on an INOVA-300 instrument (Varian, now Agilent, Santa Clara, CA, USA) operating at 300 ( 1 H) and 75 MHz ( 13 C) and a Varian INOVA-400 operating at 400 ( 1 H) and 100 MHz ( 13 C), respectively. Chemical shift (δ) values are reported in parts per million (ppm). Separations on silica gel were performed by preparative centrifugal circular thin-layer chromatography (CCTLC) on a Chromatotron R (Kiesegel 60 PF 254 gipshaltig, Merck), with a layer thickness of 1 and 2 mm and flow rate of 4 or 8 mL/min, respectively. Flash chromatography was performed on silica gel as stationary phase. HPLC analysis was performed on an Agilent 1120 compact LC system (Santa Clara, CA, USA), equipped with an ACE 5 C18-300 column (15 cm × 4.6 mm), UV detection, and flow rate 1 mL/min, using as mobile phase A H 2 O (containing 0.1% TFA) and as mobile phase B acetonitrile. Retention times (T R ) are reported in minutes. For all compounds, HPLC purity was determined to be greater than 95%. Compounds were also analyzed by HPLC/MS with an e2695 LC (Waters, Milford, Massachusetts, USA) coupled to a Waters 2996 Photodiode Array Detector and a Waters Micromass ZQ. The column used is a Waters SunFire C18 2.1 × 50 mm, 3.5 µm, and the mobile phases were A: acetonitrile and B: H 2 O, together with a constant 5% of C (H 2 O with 2% formic acid) to assure a 0.1% of formic acid along the run.

General Procedure for the Synthesis of Chalcones 7-10 Derived from 6-Methoxy-5-Nitro-1-Tetralone (General Procedure A)
A mixture of 6-methoxy-5-nitro-1-tetralone (6) (1.0 eq), the selected aldehyde (1.2 eq) and a pellet of NaOH (185 mg) in ethanol was stirred at room temperature for 7 h. The solid thus formed was filtered and washed with ethanol and water to provide the chalcones 7 and 8 as pure yellow solids. For 9 and 10 purifications by flash column chromatography were required and performed as specified.

Antibacterial Activity
The nitro compounds were tested to determine the antibacterial activity against gram-negative and gram-positive bacteria. Wild-type and multi-resistant strains were included as follows: methicillin-susceptible Staphylococcus aureus ATCC 25923 (MSSA), methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), Escherichia coli ATCC 25922, carbapenemase-positive Klebsiella pneumoniae BAA 1705, Klebsiella pneumoniae ATCC 700603 (extended spectrum beta lactamase, ESBL positive), Pseudomonas aeruginosa ATCC 27853 and Neisseria gonorrhoeae ATCC 49226. Stock solutions (100 mg/mL) of the compounds were prepared in dimethyl sulfoxide (DMSO) and diluted to a final screening concentration of 1 mg/mL. An initial screening of bacterial inhibition was performed by agar diffusion method. Briefly, sterile Mueller Hinton agar (MHA, BBL) was prepared in Petri dishes and inoculated with a bacterial suspension prepared in trypticase soy broth and adjusted to 1.5 × 10 8 colony forming unit CFU/mL (i.e., 0.08-0.1 OD at 600 nm) [33]. Wells (6 mm in diameter) were punched in the agar and 10 µL of each compound (stock solution) was filled into each well. Dimethyl sulfoxide and trypticase soy broth were included as negative controls (i.e., no inhibition of bacterial growth). Gentamicin and tetracycline (Sigma-Aldrich) were included as positive controls of growth inhibition. Compounds showing growth inhibition were tested at least twice before being selected for microdilution testing. For N. gonorrhoeae the agar diffusion method was also used in the screening process with some modifications. For this method, 200 µL of a bacterial suspension (1.5 × 10 8 CFU/mL) was inoculated in gonococcal (GC) agar (BBL) supplemented with 1% isovitalex (BBL), then the compounds were added to the wells as above mentioned and incubated at 35-36.5 • C in 5% CO 2 atmosphere for 48 h. Penicillin, ceftriaxone and ciprofloxacin (BBL) were used as controls [34].

Microdilution Test
Minimum inhibitory concentration (MIC) was determined in those compounds with reproducible bacterial growth inhibition at the screening. Bacterial suspensions were adjusted with Mueller Hinton broth (MHB) to a concentration of 5 × 10 5 -8 × 10 5 [33]. Stock solutions of the newly synthesized compounds were diluted in MHB containing 5% DMSO and 0.1% Tween 80 [35] and added to 90 µL of the bacterial inoculum. The microplates were incubated for 24 h at 35-37 • C. MICs were defined as the lowest concentration with visible inhibition of bacterial growth [33] and/or detection using resazurin (1 mg/mL) using a microplate spectrophotometer (Cytation 3M, Biotek, Winooski, VT, USA). Gentamicin (Sigma-Aldrich, St. Louis, MO, USA) and tetracycline were included as controls of growth inhibition; MHB and DMSO were used as a negative control. Experiments were performed in duplicate and replicated at least three times.
For N. gonorrhoeae, those compounds with visible growth inhibition in the screening test were further tested for MIC on agar plates as described by the Centers for Disease Control and Prevention [36] and the Clinical and Laboratory Standards Institute [34] with modifications. Briefly, GC agar supplemented with 1% isovitalex was prepared to contain increasing concentrations of the compounds and inoculated with 5 µL of the bacterial suspension (i.e., 1 × 10 4 CFU) [36]. The lowest concentration of the compound that inhibited bacterial growth was determined as the MIC. Bacterial growth was examined and verified using the oxidase test. Experiments were performed in duplicate and replicated at least three times.

Hemolytic Activity
The ability to induce hemolysis was evaluated to compounds that showed antibacterial activity following the method of cytotoxicity by spectrophotometry. The method was adapted from Conceição et al. [37] with modifications. Briefly, 240 µL of Human Red blood cells (huRBC) adjusted to 5% hematocrit in phosphate buffer saline (PBS) were placed into each well of 96-well plate and subsequently exposed to 200 µg/mL of the selected compounds (i.e., 10 µL of 5 mg/mL working solution of each compound in MHB with 5% DMSO 0.1% Tween-80). As positive control for hemolytic activity 10 µL SDS 1% was added. For a negative control only medium with no chemicals was added to huRBC. Free hemoglobin was measured after 24 h incubation at 37 • C by spectrophotometry (420 nm Cytation 3M, Biotek, Winooski, VT, USA). Non-specific absorbance was subtracted from a blank. Determinations were done by triplicate in at least two independent experiments.

Conclusions
A series of new benzo[g]indazole compounds 11-18a,b incorporating 6-nitro and 6-amino groups were synthesized by cyclocondensation of 2-benzylidene-1-tetralones 7-10 with hydrazine hydrate in presence of acetic acid, resulting in the formation of cis (3R,3aR-rel) and trans (3R,3aS-rel) diastereoisomeric pyrazoline derivatives. The stereochemical assignment of the obtained diasteroisomers was performed based on experimental NMR data and a convenient criterion concerning the chemical shift of the H-4 protons has been proposed for the assignment of both isomers. The antiproliferative evaluation against a panel of tumor cell lines revealed that the 6-nitro benzo[g]indazoles 11a, 11b, 12a and 12b display IC 50 values between 5-15 µM against NCI-H460 (lung carcinoma) while they were less active against other cell types. The in vitro screening for antibacterial activity showed that compounds 12a and 13b exhibited antigonococcal activity by inhibiting bacterial growth at MIC values of 250 and 62.5 µg/mL, respectively. None of the active compounds (12a and 13b) showed hemolytic activity suggesting low membrane interactions and toxicity.