Abstract
The synthesis of a series of 2,3-diaryl-7-methyl-4,5,6,7-tetrahydroindazole and 3,3a,4,5,6,7-hexahydroindazole derivatives substituted with various biologically-active function groups with anticipated chemotherapeutic activity is described. 4-(7-methyl-3-aryl-3,3a,4,5,6,7-hexahydro-indazol-2-yl)benzenesulfonamides 2a–c, which were employed as key intermediates in this study, were synthesized by cyclocondensation of 6-arylidene-2-methylcyclohexanones 1a–c with 4-hydrazinobenzenesulfonamide hydrochloride. A detailed discussion of the reactions utilized in the preparation of the intermediate and target compounds is reported, and the structures of the newly synthesized compounds were substantiated with IR, 1H and 13C NMR spectral data and elementary microanalyses. Twenty of the newly synthesized compounds were selected by National Cancer Institute (NCI), Maryland, USA, to be evaluated for their antitumor activity and the results revealed that six compounds 3c, 4d,e, 5a,d and 8c exhibited broad spectrum of antitumor activity against most of the tested tumor cell lines. In addition, the in vitro antibacterial and antifungal activities of a number of the target compounds were also tested using the Agar-diffusion method. Some of these compounds have shown significant antibacterial and mild to moderate antifungal activities.
Introduction
Over the past two decades, much evidence has been accumulated on the importance of pyrazole derivatives and their related fused heterocycles as chemotherapeutic agents. The pyrazole moiety is an important pharmacophore showing a multitude of biological and pharmacological properties. However, benzo-fused derivatives of pyrazole (indazole) are probably one of the least studied nuclei owing to its scarcity in nature. The indazole ring system is regarded as a bioisostere of indole. However, a large number of synthetically prepared indazoles are known to show a variety of biological activities, such as high binding affinity for estrogen receptorCitation1,Citation2, inhibition of protein kinase C-βCitation3, 5-HT2 receptor agonismCitation4, human immunodeficiency virus (HIV) protease inhibitionCitation5, anti-leukemic activityCitation6, and non-steroidal anti-inflammatory activityCitation7. Other compounds of interest are YC-1, a benzylindazole which has been shown to activate soluble guanylate cyclaseCitation8, nitric oxide synthase inhibition activityCitation9,Citation10, to induce cellular apoptosisCitation11 and a MCHr1 antagonism for the treatment of obesityCitation12. The most notable one is Lonidamine which was found to induce apoptosis via a direct effect on the mitochondrial permeability transition poreCitation11.
Similarly, tetrahydroindazoles are known to show activity against cancerCitation13 and inflammationCitation14 and hexahydroindazoles, a novel heterocyclic nucleus has been reported to have antimicrobialCitation15,Citation16 and anti-inflammatory activitiesCitation17,Citation18. In a molecular docking study of 4,5,6,7-tetrahydro-2H-indazoles when position-2 is substituted with 4-(trifluoromethyl)phenylhydrazine group it was found to display more anti-inflammatory activity when compared with 2-Hydrazino-4-(trifluoromethyl)pyrimidine group. The difference in the anti-inflammatory activity of these two compounds may be attributed to an interplay between hydrogen bonding and hydrophobic interactions at the catalytic site of COX-214. Minu et al. reported in a QSAR study of hexahydroindazoles as antimicrobial agents when substituted with an electron withdrawing group in position-3 of the indazole ring, the compounds were found more active than other derivativesCitation19. In another study by Gokhan-Kelekci et al. substitution of a furan ring in position-3 of the indazole ring, produces a MAO inhibitorCitation20. Compounds carrying phenyl substituents on nitrogen atom (position-2 in indazole) were found to be highly potent MAO inhibitors. Further, it was also noted that the presence of longer and voluminous groups on thiocarbamoyl nitrogen was selectively active towards MAO-A isoform while in case of MAO-B inhibitors the length of the lateral chain of the aryl diazo derivatives should not be longer than two atoms.
In view of the aforementioned findings, it was conferred that the position-2 and position-3 are crucial in the activity modulation of tetrahydro and hexahydroindazoles. Therefore, in continuation of our ongoing interest in the synthesis novel heterocycles with chemotherapeutic activitiesCitation21–30, it was considered of interest to prepare some new tetrahydro- and hexahydroindazole derivatives, bearing functionalities that are reported to contribute to potential chemotherapeutic activities such as the sulfonamide, sulfonylurea and thiourea groups. Besides incorporating an electron withdrawing group at position-3 we have also introduced a methyl substituent in position-7. Some target compounds were planned to comprise other heterocyclic rings that are known to possess significant antibacterial and anticancer activities such as the thiazole and the thiazine ring systems.
The structures of the newly synthesized compounds were confirmed by IR, 1H and 13C NMR data. The target compounds have been subjected to the National Cancer Institute NCI in vitro disease-oriented human cells screening panel assay, Maryland, USA, to screen their anticancer activityCitation31–33. In addition, in vitro antibacterial and antifungal activities were also evaluated using the Agar-diffusion methodCitation34. Finally, owing to the increase of reported cases of tuberculosis even in developed countries, along with the emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis strains, it was thought of interest to screen the in vitro antimycobacterial activity of the new analogsCitation35.
Experimental
Chemistry
Melting points were determined in open glass capillaries, on a Gallenkamp melting point apparatus, and were uncorrected ()Citation36. The infrared (IR) spectra were recorded on Perkin-Elmer 297 infrared spectrophotometer, using the NaCl plate technique. The 1H-NMR and 13C-NMR spectra were recorded on Bruker DPX-400-FT spectrometer using CDCl3 and DMSO-d6 as a solvent and tetramethylsilane as the internal standard (Chemical shifts in δ, ppm). Splitting patterns were designated as follows: s: singlet; d: doublet; m: multiplet. Elemental analyses were performed at the Microanalytical Unit, Faculty of Science, King Abdul-Aziz University, Jeddah, Saudi Arabia, and the found values were within ±0.4% of the theoretical values. Follow up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminium sheets (Type 60 F254, Merck) and the spots were detected by exposure to UV-lamp at λ 254. The starting chalcone 1 was prepared according to a literature procedure.
Table 1. Physicochemical and analytical data of compounds 2–10.
4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)benzenesulfonamide (2a–c)
A solution of the appropriate chalcone 1a–c (20 mmol) in ethanol (25 mL) was refluxed with 4-hydrazinobenzenesulfonamide hydrochloride (4.9 g, 22 mmol) for 4 h. On concentration, the separated product was filtered, washed with cold ethanol and recrystallized from a mixture of benzene-ethanol (1:1). 1H NMR data are listed in . 13C NMR (DMSO/CDCl3) 2a δ = 14.0 (CH3), 25.9, 27.6, 29.9, 33.3 (cyclohexyl-C), 42.6 (C-3a), 56.0 (C-3), 112.6, 125.7, 126.3, 127.7, 128.3, 139.4, 146.7, 155.6 (Ar-C). 2b δ = 14.5 (CH3), 26.9, 29.7, 32.8 (cyclohexyl-C), 43.2 (C-3a), 56.6 (C-3), 113.0, 126.8, 127.4, 128.2, 129.7, 131.0, 137.8, 147.3, 154.6 (Ar-C). 2c δ = 13.9 (CH3), 20.9 (CH3), 25.2, 27.2, 29.4, 33.4 (cyclohexyl-C), 42.8 (C-3a), 56.8 (C-3), 112.2, 126.1, 127.3, 128.4, 129.0, 134.7, 135.9, 146.5, 152.8 (Ar-C).
Table 2. Citation1H-NMR spectral data of compoundsa (2–10).
4-(7-Methyl-3-aryl-4,5,6,7-tetrahydroindazol-2-yl)benzenesulfonamide (3a–c)
To a stirred suspension of the indazoline derivative 2 (10 mmol) in water (10 mL), bromine water (5%, 15 mL) was gradually added over a period of 30 min. at 25°C. After stirring for 3 h at room temperature, the pyrazole derivatives thus formed, were collected by filtration, thoroughly washed with water and dried. They were recrystallized from ethanol. 1H NMR data are listed in . 13C NMR (DMSO/CDCl3) 3a δ = 21.9 (CH3), 18.1, 31.5, 32.2, 41.3 (cyclohexyl-C), 117.7, 119.1, 126.1, 127.0, 128.5, 129.0, 136.5, 136.8, 142.9, 145.4, 152.5 (Ar-C). 3c δ = 20.2 (CH3), 21.6 (CH3), 18.4, 31.2, 32.6, 41.4 (cyclohexyl-C), 117.5, 118.8, 126.0, 126.9, 129.7, 133.5, 136.0, 137.7, 141.8, 144.6, 151.9.
N1-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)benzenesulfonyl]- N3-substituted ureas (4a-f) and N1-[4-(7-Methyl-3-aryl-4,5,6,7-tetrahydroindazoline-2-yl)benzenesulfonyl]-N3-substituted ureas (5a–f)
A mixture of the appropriate pyrazoline 2 or pyrazole 3 (10 mmol) and anhydrous potassium carbonate (2.8 g, 20 mmol) in dry acetone (25 mL) was heated under reflux with stirring with the appropriate isocyanate (11 mmol) for 18 h. The solvent was removed under reduced pressure and the remaining solid residue was dissolved in water (30 mL). After acidification of the resulting solution with 2N hydrochloric acid, the precipitated crude product was filtered, washed with water, dried and recrystallized from ethanol. 1H NMR data are listed in . 13C NMR (DMSO/CDCl3) 4a δ = 14.3 (CH3), 21.6, 25.7, 27.1, 27.6, 29.2, 32.7, 33.4, 46.9 (2 cyclohexyl-C), 41.8 (C-3a), 55.7 (C-3), 119.1, 121.2, 124.6, 126.3, 128.5, 136.7, 138.4, 142.4, 155.2 (Ar-C), 162.6 (CO). 4b δ = 14.2 (CH3), 25.6, 27.3, 29.4, 33.2 (cyclohexyl-C), 42.0 (C-3a), 56.8 (C-3), 119.3, 120.4, 124.1, 126.0, 127.2, 128.3, 128.7, 129.0, 129.3, 136.5, 136.7, 142.0, 155.1 (Ar-C), 161.3 (CO). 4d δ = 14.4 (CH3), 20.4 (CH3), 21.2, 25.6, 26.9, 27.2, 29.4, 32.3, 33.2, 46.4 (2 cyclohexyl-C), 42.6 (C-3a), 55.8 (C-3), 118.8, 124.8, 126.4, 126.9, 129.7, 133.2, 137.0, 142.9, 155.6 (Ar-C), 163.1 (CO). 5a δ = 15.2 (CH3), 18.1, 22.0, 27.3, 31.6, 32.2, 33.5, 41.6, 44.4 (2 cyclohexyl-C), 117.7 (C-3a), 119.7, 126.8, 127.1, 128.5, 129.3, 135.9, 136.3, 145.2 (C-3), 145.4 (Ar-C), 150.6 (C-7a), 163.0 (CO). 5b δ = 21.6 (CH3), 18.4, 31.6, 32.8, 41.2 (cyclohexyl-C), 117.2 (C-3a), 119.3, 120.2, 124.6, 126.1, 127.2, 128.3, 128.7, 129.1, 136.4, 136.6, 139.0, 142.4, 145.0 (C-3), 152.5 (C-7a), 159.3 (CO). 5d δ = 14.6 (CH3), 20.7 (CH3), 18.6, 31.2, 32.1, 40.9 (cyclohexyl-C), 118.3 (C-3a), 119.0, 126.1, 126.8, 127.0, 128.2, 129.0, 130.1, 133.1, 135.8, 136.7, 137.9, 142.6, 144.2 (C-3), 152.0 (C-7a), 160.3 (CO).
N1-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)benzenesulfonyl]- N3-substituted thioureas (6a–h)
A solution of the appropriate isothiocyanate (11 mmol) in dry acetone (5 mL) was added to a stirred mixture of the suitable indazoline 2 (10 mmol) and anhydrous potassium carbonate (2.8 g, 20 mmol) in dry acetone (25 mL), and the mixture was heated under reflux with stirring for 10 h. The solvent was removed under reduced pressure and the remaining solid residue was dissolved in water (30 mL) and acidified with 2N hydrochloric acid. The precipitated product was filtered, washed with water, dried and recrystallized from ethanol. 1HNMR data are listed in . 13C NMR (DMSO/CDCl3) 6a δ = 14.8 (CH3), 25.6, 27.4, 29.7, 33.1 (cyclohexyl-C), 42.6 (C-3a), 55.8 (C-3), 119.1, 124.5, 125.3, 126.0, 127.2, 128.4, 128.8, 129.0, 136.5, 137.2, 140.0, 143.1, 155.2 (Ar-C), 186.3 (CS). 6b δ = 14.2 (CH3), 25.9, 27.9, 29.7, 33.2 (cyclohexyl-C), 42.6 (C-3a), 56.0 (C-3), 55.5 (CH2), 119.3, 126.1, 126.5, 127.2, 127.5, 128.3, 128.6, 129.0, 136.8, 137.1, 141.3, 142.9, 154.8 (Ar-C), 188.2 (CS).
2-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)benzenesulfonylimino]-3-substituted thiazolidin-4-ones (7a–f)
To a solution of the appropriate thiourea derivative 6 (10 mmol) in absolute ethanol (20 mL) was added ethyl bromoacetate (1.84 g, 11 mmol) and anhydrous sodium acetate (1.64 g, 20 mmol), and the reaction mixture was heated under reflux for 2 h. The mixture was left to attain room temperature then poured into ice-cold water (30 mL), and the solid product thus formed was filtered, washed with water, dried and recrystallized from ethanol-benzene mixture (1:1). 1H NMR data are listed in . 13C NMR (DMSO/CDCl3) 7a δ = 14.0 (CH3), 32.8 (CH2), 26.0, 27.7, 30.1, 33.5 (cyclohexyl-C), 42.6 (C-3a), 56.0 (C-3), 113.3, 120.4, 124.2, 125.7, 126.9, 127.1, 128.3, 128.9, 139.4, 140.8, 148.5, 155.8 (Ar-C), 163.3 (C=N), 168.8 (CO). 7b δ = 14.1 (CH3), 33.0 (CH2), 25.8, 27.6, 29.9, 33.2 (cyclohexyl-C), 41.8 (C-3a), 55.9 (C-3), 45.5 (Ph-CH2), 112.9, 125.5, 126.4, 126.9, 127.1, 127.4, 128.3, 128.5, 128.7, 138.8, 142.4, 146.9, 154.2 (Ar-C), 164.0 (C=N), 169.9 (CO). 7c δ = 14.6 (CH3), 32.8 (CH2), 25.7, 27.8, 29.6, 33.1 (cyclohexyl-C), 42.0 (C-3a), 56.2 (C-3), 113.4, 125.6, 126.8, 127.3, 128.2, 128.3, 128.6, 129.7, 131.6, 133.5, 137.5, 139.2, 148.4, 155.6 (Ar-C), 163.0 (C=N), 167.7 (CO-Ph), 172.3 (CO).
2-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)benzenesulfonylimino]- 4-phenyl-3-substituted thiazolines (8a–c)
A solution of the appropriate thiourea derivative 6 (10 mmol) in absolute ethanol (20 mL) was refluxed with ω-bromoacetophenone (2.2 g, 11 mmol) and anhydrous sodium acetate (1.64 g, 20 mmol) for 3 hr during which the solid product was partially crystallized out. The mixture was left to attain room temperature then filtered, washed with cold ethanol, dried and recrystallized from ethanol. 1H NMR data are listed in . 13C NMR (DMSO/CDCl3) 8c δ = 14.3 (CH3), 20.7 (CH3), 25.8, 27.7, 29.4, 33.3 (cyclohexyl-C), 42.3 (C-3a), 56.0 (C-3), 88.4 (C-2, thiazoline), 115.9, 120.4, 124.8, 125.7, 126.2, 126.8, 127.3, 127.8, 128.3, 128.5, 128.6, 134.9, 136.2, 140.2, 141.3, 148.5, 152.8 (C-3, thiazoline), 155.4 (Ar-C), 163.8 (C=N).
2-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)benzenesulfonylimino]-3-substituted-[1,3]thiazinan-4-ones (9a–c)
To a solution of the appropriate thiourea derivative 6 (10 mmol) in absolute ethanol (20 mL) was added ethyl 3-bromopropionate (2 g, 11 mmol) and anhydrous sodium acetate (1.64 g, 20 mmol) and the reaction mixture was heated under reflux for 4 h. After the mixture was cooled to room temperature, it was poured into ice-cold water (30 mL). The solid product thus formed was filtered, washed with water, dried and recrystallized from ethanol-benzene mixture (1:1). 1H NMR data are listed in . 13C NMR (DMSO/CDCl3) 9a δ = 14.2 (CH3), 24.8 (C-6, thiazine), 35.7 (C-5, thiazine), 25.7, 27.6, 29.5, 33.2 (cyclohexyl-C), 42.6 (C-3a), 56.3 (C-3), 114.3, 120.4, 124.1, 125.7, 126.6, 127.2, 128.2, 128.3, 128.7, 139.2, 140.8, 148.5, 155.6 (Ar-C), 163.0 (C=N), 173.4 (CO). 9b δ = 14.8 (CH3), 21.2 (CH3), 25.6 (C-6, thiazine), 36.2 (C-5, thiazine), 43.0 (C-3a), 55.2 (C-3), 114.0, 120.3, 124.6, 125.2, 126.7, 127.0, 128.2, 128.3, 128.8, 138.9, 140.6, 149.4, 154.9 (Ar-C), 164.8 (C=N), 174.3 (CO).
2-[4-(7-Methyl-3-aryl-3,3a,4,5,6,7-hexahydroindazol-2-yl)benzenesulfonylimino]-3-substituted-[1,3]thiazinan-5-ones (10a–c)
To a solution of the appropriate thiourea derivative 6 (10 mmol) in absolute ethanol (20 mL) was added 1,3-dichloroacetone (1.4 g, 11 mmol) and anhydrous sodium acetate (2 g, 25 mmol) and the reaction mixture was heated under reflux for 5 h. After the mixture was cooled to room temperature, it was poured into ice-cold water (30 mL). The solid product thus formed was filtered, washed with water, dried and recrystallized from ethanol-benzene mixture (1:1). 1H NMR data are listed in . 13C NMR (DMSO/CDCl3) 10a δ = 14.1 (CH3), 25.8, 27.6, 29.7, 33.1 (cyclohexyl-C), 37.4 (C-6, thiazine), 59.4 (C-4, thiazine), 112.3, 115.8, 116.8, 126.0, 126.8, 127.0, 127.4, 128.3, 129.2, 137.4, 143.5, 148.1, 155.2 (Ar-C), 164.6 (C=N). 10c δ = 14.3 (CH3), 20.8 (CH3), 25.7, 27.8, 30.0, 33.2 (cyclohexyl-C), 36.9 (C-6, thiazine), 58.1 (C-4, thiazine), 113.4, 114.2, 117.1, 125.4, 126.7, 127.1, 128.1, 128.3, 129.4, 137.1, 138.2, 148.4, 155.9 (Ar-C), 163.3 (C=N).
Biological activity
In vitro antitumor screening
Twenty of the newly synthesized compounds have been selected by the NCI in vitro disease-oriented human cells screening panel assay to be evaluated for their in vitro antitumor activity. Primary in vitro one dose anticancer assay was performed using the 3-cell line panel consisting of NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS) according to the protocol of the Drug Evaluation Branch, NCI, BethesdaCitation31–33. All the compounds that reduced the growth of any one of the cell lines to 32% or less namely 3c, 4d,e, 5a,d and 8c were passed on for evaluation in the full panel of 60 human tumor cell lines of 9 tumor subpanels including leukemia, non-small cell lung cancer, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast cancer cell lines. These cell lines were incubated with five concentrations (0.01–100 μM) for each compound. A 48h continuous drug exposure protocol was used, and a SRB protein assay was employed to estimate cell viability or growth. Subpanel and full panel mean-graph midpoint values (MG-MID) for certain agents are the average of individual real and default GI50, TGI, or LC50 values of all cell lines in the subpanel or the full panel, respectively.
Antimicrobial screening
Inhibition zone (IZ) measurement
Standard sterilized filter paper discs (5 mm diameter) impregnated with a solution of the test compound in DMSO (1 μg/mL) was placed on an agar plate seeded with the appropriate test organism in triplicates. The utilized test organisms were Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6051) as examples of Gram positive bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) as examples of Gram negative bacteria, Candida albicans (ATCC 10231) and Aspergillus niger (recultured) as representatives of fungi. Ampicillin trihydrate and Clotrimazole were used as standard antibacterial and antifungal agents, respectively. DMSO alone was used as control at the same above-mentioned concentration. The plates were incubated at 37°C for 24 h for bacteria and 72 h for fungi. The results were recorded for each tested compound as the average diameter of inhibition zones of bacterial growth around the discs in mm ().
Minimal inhibitory concentration (MIC) measurement
MICs were measured for compounds that showed significant growth inhibition zones (≥ 12 mm) using the two-fold serial dilution techniqueCitation34. The micro-dilution susceptibility test in Muller-Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid) was used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the tested compounds, ampicillin trihydrate and Clotrimazole were prepared in DMSO at concentration of 1600 µg/mL followed by two-fold dilution at concentrations of 800, 400, 6.25 µg/mL). The microorganism suspensions at 106 CFU/mL (Colony Forming Unit/mL) concentration were inoculated to the corresponding wells. Plates were incubated at 36°C for 24 h to 48 h and the minimal inhibitory concentrations (MIC) were determined. Control experiments were also done.
Antimycobacterial screening
The available Mycobacterium tuberculosis strain was cultured in tubes with 10 µL of a suspension of the strain in physiological saline. The strain was collected from the slant after four weeks incubationCitation33. The inoculum was prepared with 3–5 weeks old M. tuberculosis colonies from Loewenstein-Jensen slants, emulsified in dilution fluid containing 2% fatty acid free albumin and 0.02% Tween 80, pH 6.9. Suspensions were then diluted in saline to a turbidity of McFarland no.1 standard and then diluted to obtain inocula of 3 × 105 cells per well. The MICs measurement were determined by agar dilution technique Agar supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase) enrichment, was used to prepare quadrant plates with serial DMSO two-fold dilutions of the test compounds. The following concentrations were used: 1000, 500, 250, 125, 62, 32, 16, 8, 4 µg/mL. A 100 µL sample of the mycobacterial suspension was inoculated onto each compound-containing quadrant. Control quadrant consisted of agar alone, culture medium with DMSO and culture medium with reference antimycobacterial drugs; rifampicin and 1HN was performed. All plates were then incubated at 37°C in a CO2 (5% CO2 / 95% humidified air) incubator, for 3–4 weeks. The MIC was defined as the lowest chemical dilution associated with at least a 99% reduction in the number of visible colonies.
Results and discussion
Chemistry
The starting compounds in this study are 6-arylidene-2-methylcyclohexanones 1a–c could be synthesized via Claisen-Schmidt condensation of aromatic aldehydes with 2-methylcyclohexanone. These chalcones were reacted with 4-hydrazinobenzenesulfonamide hydrochloride to afford the desired hexahydroindazole derivatives 2a–c (). Their IR spectra revealed two bands at 3365–3388 cm−1 and 3220–3258 cm−1 corresponding to the amino group and two bands at 1350–1378 cm−1 and 1180–1195 cm−1 attributed to the SO2N function. The structures of these compounds were further confirmed by the 1H-NMR which exhibited a characteristic doublet at δ 4.95–5.38 ppm attributed to the H-3 proton and a multiplet at δ 3.64–3.70 ppm due to the H-3a proton which substantiate the closure of the indazoline ring (). Mild oxidation of the indazoline 2a–c with bromine water led to the formation of the corresponding indazoles 3a–c. Such oxidation was confirmed by the 1H-NMR spectra, which lacked the characteristic doublet and multiplet of H-3 and H-3a protons of the parent indazolines (). The ureido derivatives 4a–f and 5a–e were prepared in good yields by reacting the indazoline 2a–c or the indazole 3a–c derivatives with the appropriate isocyanate in the presence of anhydrous potassium carbonate as a mild basic catalyst. The IR spectra of these compounds showed beside the absorption bands corresponding to the NH group at 3216–3288 cm−1, two bands at 1345–1372 cm−1 and 1177–1190 cm−1 for the SO2N function, in addition to a characteristic urea carbonyl band at 1645–1665 cm−1. In a similar fashion, the synthesis of the thioureido derivatives 6a–h was achieved by refluxing 2a–c with the appropriate isothiocyanate in dry acetone in the presence of anhydrous potassium carbonate as a mild basic catalyst (). Their IR spectra revealed beside the characteristic absorption bands corresponding to the amino group and the SO2N function lying at the same range of their structurally-related derivatives, a characteristic C=S band at 1155–1165 cm−1. Cyclization of the thioureido derivatives 6a–h with ethyl bromoacetate afforded the corresponding 4-oxothiazolidines 7a–f in good yields. The IR of these compounds was characterized by the appearance of a new band at 1720–1735 cm−1 due to the carbonyl group at the thiazol-C4 and two bands at 1338–1365 cm−1 and 1169–1188 cm−1 for the SO2N moiety. Refluxing 6 with phenacyl bromide afforded the targeted thiazolines 8a–c. Whereas, reacting the same compounds 6a–c with ethyl 3-bromopropionate or 1,2-dichloroacetone resulted in the formation of the corresponding 4-oxo-5,6-dihydrothiazines 9a–c and the 5-oxo-5,6-dihydrothiazines 10a–c, respectively. Their IR spectra were characterized by the presence of a carbonyl band at 1720–1730 cm−1 as well as two bands at 1340–1373 cm−1 and 1174–1185 cm−1 for the SO2N group. In addition, their 1H-NMR spectra showed the appearance of new methylene groups of the thiazine rings at a range of δ 3.19–5.42 ppm ().
Scheme 1. Scheme 1 Reagents and reaction conditions: (i) ethanol, reflux, 4 h; (ii, iv) Br2 water, r.t., 3 h; (iii, v) RNCO, anhyd.K2CO3, acetone, reflux, 18 h; (vi) RNCS, anhyd. K2CO3, acetone, reflux, 10 h.
Scheme 2. Reagents and reaction conditions: (i) ethyl bromoacetate, anhyd. Na acetate, ethanol, reflux, e h; (ii) phenacyl bromide, anhyd. Na acetate, ethanol, reflux, 3 h; (iii) ethyl 3-bromopropionate, anhyd. Na acetate, ethanol, reflux, 4 h; (iv) 1,3-dichloroacetone, anhyd. Na acetate, ethanol, areflux, 5 h.
In vitro antitumor evaluation
Primary in vitro 3-cell line assay
Twenty of the newly synthesized compounds were chosen by the NCI to be evaluated for their in vitro antitumor activity. Primary in vitro anticancer assay was performed using the 3-cell line panel consisting of NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS) in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, BethesdaCitation31–33. The compounds were added at a single concentration (10−4 M) and the culture was incubated for 48 h. Compounds which reduced the growth of any one of the cell lines to 32% or less, were passed on for the full panel 60-cell lines assay. Six compounds 3c, 4d, 4e, 5a, 5d and 8c out of the 16 tested compounds have been selected for a 60-cell panel assay.
In vitro full panel 60-cell line assay
About 60 cell lines of nine tumor subpanels including leukemia, non-small cell lung cancer, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast cancer cell lines were incubated with five concentrations (0.01–100 μM) for each compound and were used to create log concentration-% growth inhibition curves. Three response parameters (GI50, TGI, and LC50) were calculated for each cell line. The GI50 value corresponds to the concentration of the compounds causing 50% decrease in net cell growth, the TGI value is the concentration of the compounds resulting in total growth inhibition and the LC50 value is the concentration of the compounds causing net 50% loss of initial cells at the end of the incubation period (48 h). Subpanel and full panel mean-graph midpoint values (MG-MID) for certain agents are the average of individual real and default GI50, TGI, or LC50 values of all cell lines in the subpanel or the full panel, respectivelyCitation31–33.
From the results obtained it could be noticed that, only six compounds, namely; 3c, 4d,e, 5a,d and 8c exhibited remarkable antitumor activity against most of the tested subpanel tumor cell lines (GI50 and TGI values < 100 μM), whereas the rest of the compounds proved to be totally inactive. Those six compounds showed an obvious sensitivity against some individual cell lines (). Compound 3c proved to be significantly active towards leukemia CCRF-CEM, colon SW-620 and renal A498 cell lines with GI50 values of 3.65, 4.82 and 6.28 μM respectively. However, compound 4d was found to be active against leukemia RPMI-8226 and non-small cell lung NCI-H322 cell lines with GI50 values of 5.56 and 4.98 μM, respectively and compound 4e exhibited distinct activity against the colon HCT-116 cancer cells (GI50 3.40 μM). Furthermore, 5d displayed potential activity against all the leukemia cell lines with a GI50 values range of 2.46–24.62 μM. A special activity pattern was shown by compound 5a which was remarkably active against most of the tested 60 cell lines with GI50 values range of 0.02–28.4 μM. It revealed super potent activity towards leukemia CCRF-CEM, SR, melanoma SK-MEL-5 and renal A498, TK-10, UO-31 with GI50 values of 0.55, 0.80, 1.36, 0.02, 1.72 and 1.35 μM, respectively ().
Table 3. Growth inhibitory concentration (GI50, μM) of the active compounds.
Figure 1. Leukaemia Growth inhibitory concentration (GI50, μM) of the active compounds.
Figure 2. Melanoma Growth inhibitory concentration (GI50, μM) of the active compounds.
Figure 3. Renal Cancer Growth inhibitory concentration (GI50, μM) of the active compounds.
Concerning the broad spectrum of antitumor activity, the active compounds displayed effective growth inhibition GI50 (MG-MID), total growth inhibitory TGI (MG-MID) and cytotoxic LC50 (MG-MID) activities (<100 μM) (). Compound 5a having GI50, TGI, and LC50 MG-MID values of 9.22, 26.61 and 69.80 μM, respectively, proved to be the most active member in this study. It revealed potential activity against all the tested subpanel tumor cell lines with special high potency on the leukemia subpanel at the GI50 level (2.45 μM) (). Furthermore, the compound showed almost the same level of antitumor activity against the non-small cell lung, colon, melanoma, renal and prostate cancer subpanels (GI50 range 10.41–14.20 μM). Moderate activity has been shown by this compound against the CNS, ovarian and breast subpanel tumor cell lines (). On the other hand, compounds 3c and 4d were almost equipotent at the GI50 level (15.60 and 15.91 μM, respectively), while they showed some difference in the TGI and LC50 MG-MID values (). Moreover, compounds 4e, 5d and 8c displayed appreciable antitumor activity towards most of the tested tumor cell lines at the GI50, TGI, and LC50 MG-MID levels ().
Table 4. Median growth inhibitory concentrations (GI50, μM), total growth inhibitory concentrations (TGI, μM) and lethal concentrations (LC50, μM) of in vitro subpanel tumor cell lines.
A close examination of the structures of the active compounds revealed that, while the hexahydroindazole derivative 2c was totally inactive, its corresponding tetrahydroindazole analog 3c showed significant antitumor activity against most of the tested subpanel tumor cell lines with GI50, TGI and LC50 MG-MID values of 15.6, 39.6 and 78.3 μM, respectively. Condensation of 3c with different isocyanates to produce the dihydrosulfonylureas 4d (X = CH3, R1 = cyclohexyl) and 4e (X = CH3, R1 = phenyl), did not significantly improve the anticancer activity at the GI50 MG-MID (15.91 and 14.40 μM, respectively), TGI MG-MID (38.4 and 49.62 μM, respectively) and the LC50 MG-MID levels (77.80 and 88.23 μM, respectively). On the contrary, oxidation of compound 4d resulted in a highly active indazolesulfonylurea 5a (X = H, R1 = cyclohexyl), which is proved to be the most active member in this study with potential activity against all the tested subpanel tumor cell lines (GI50, TGI and LC50 MG-MID values 9.22, 26.61 and 69.80 μM, respectively). In addition, 5a was able to exert potential inhibitory activity against the leukemia subpanel (GI50 2.45 μM, ), when compared with sulofenur; (NSC 656667; GI50 1.27 μM, ); a known sulfonylurea derivative with potential antineoplastic activity ().Replacing the cyclohexyl moiety in 5a with a phenyl moiety and introduction of a methyl group as in 5d (X = CH3, R1 = phenyl), resulted in about 2-fold decrease in activity at the GI50 and TGI levels (16.64 and 54.22, respectively). However, to substantiate the postulate a detailed protein assay is required in order to outline the mechanism of their anticancer activity. Moreover, in a related study 3-substituted-phenyl-1H-indole-5-sulfonamidesCitation37 have shown inhibitions of some of the isoforms of carbonic anhydrase enzyme. This involves interfering with pH regulations of various forms of tumours and represents a new anticancer drug discovery strategy. Finally, although the thiourea derivatives 6f and 6g were inactive in this test, one of the cyclized products namely 8c showed some moderate antitumor activity (GI50, TGI and LC50 MG-MID values 20.04, 51.63 and 90.11 μM, respectively).
Figure 4. Structures of sulofenur A, its congeners B–D and the general structures of the new compounds E and F.
Antimicrobial screening
Compounds 2a-c, 3a-c, 4a,b,d,e, 5a,b,d, 6a-c,f, 7a-d, 8a,c, 9a,b and 10a,c were evaluated for their in vitro antimicrobial activityCitation34 against Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6051) as examples of Gram positive bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) as examples of Gram negative bacteria, Candida albicans (ATCC 10231) and Aspergillus niger (recultured) as representatives of fungi.
The results revealed that most of the tested compounds displayed greater inhibitory effect on the growth of the tested Gram positive strain compared to Gram negative ones. Most of the compounds showed weak or no antibacterial activity against the Gram negative, P. aeruginosa. Moreover, few compounds were able to exert mild to moderate antifungal activity against C. albicans, while, all the tested compounds lacked antifungal activity against Aspergillus niger. A close examination of the structures of the active compounds revealed that the antimicrobial profile of the tetrahydroindazole compounds 3 seemed to be more interesting than their corresponding hexahydroindazole derivatives 2, as evidenced by their IZ diameters and MIC values recorded in .
Table 5. Antimicrobial activity of the target compounds 2–10.
Among the hexahydroindazole series, compound 2a showed weak antimicrobial activity against all the tested microbial strains (IZ ≤ 12 mm). Introduction of chlorine atom or methyl group in the phenyl ring in position 3 of the hexahydroindazole derivative as in 2b and 2c resulted in a slight improvement in the activity against the Gram positive S. aureus and B. subtilis (MIC values 100 and 200 µg/mL, respectively). Whereas, replacement of the hexahydroindazole moiety in 2a–c with tetrahydroindazole resulted in more potent compounds 3a–c, with an appreciable broad spectrum of antibacterial activity against the tested Gram positive, Gram negative bacteria (MIC values 100–200 µg/mL), and moderate antifungal activity towards C. albicans (MIC 100 µg/mL). The replacement of amino group in 3a with a urea moiety as in 5a produced the most potent antimicrobial activity in the current series of compounds which was as potent as ampicillin (MIC 12.5 µg/mL) against S. aureus, whereas its activity against B. subtilis and E.Coli was 50% lower than that of ampicillin (MIC 50 νs 25 µg/mL, respectively). It displayed a moderate antifungal activity towards C. albicans, which was about 50% of that of Clotrimazole (MIC 25 νs 12.5 µg/mL, respectively). However, it is worthy to mention that, structure modification of the urea derivatives to thiourea ones led to almost complete abolishment of the antimicrobial activity (IZ ≤ 10 mm).
Antimycobacterial screening
Determination of in νitro antimycobacterial activity of the target compounds 2a–c, 3a–c, 4a,b,d,e, 5a,b,d, 6a–c,f, 7a–d, 8a,c, 9a,b and 10a,c was performed by employing the two-fold agar dilution method slightly modified from that described by Mamolo and VioCitation34. A strain of Mycobacterium tuberculosis (locally isolated, Alexandria, Egypt) was utilized in this assay. Rifampicin and isonicotinic acid hydrazide (INH) were used as reference antimycobacterial drugs. The MIC was defined as the lowest concentration of the tested compound that yielded no visible growth on the plate. Among the compounds tested, only compound 7a was able to exert weak growth inhibitory effect (MIC 250 µg/mL) against the Mycobacterium tuberculosis used in this screening, while the rest of the synthesized compounds were totally inactive.
Conclusion
In conclusion, the objective of the present study was to synthesize and investigate the antitumor, antimicrobial and antimycobacterial activities of new compounds incorporating the sulfonamido, N1,N3-disubstituted sulfonylurea and thiourea pharmacophores structurally related to a well-documented sulfonylurea anticancer agent Sulofenur A and its structure congeners B-D (). This aim has been verified by the synthesis of hybrid compounds comprising the above mentioned pharmacophores substituted essentially with a 3-(4-toly)-[1,2-c]pyrazol(in)e counterpart at the N3 aryl moiety having the general structure E and F (), for synergistic purpose. The results revealed that six compounds namely, 3c, 4d,e, 5a,d and 8c have exhibited broad spectrum of antitumor activity against most of the tested tumor cell lines. Compound 5a proved to be the most active antitumor agent in the present study with GI50, TGI and LC50 MG-MID values of 9.22, 26.61 and 69.80 μM, respectively, with high sensitivity towards some leukemia, melanoma and renal cell lines. The other five active compounds showed variable degrees of appreciable antitumor activity (GI50 and TGI MG-MID values range 14.40–20.04 and 38.40–54.22 μM, respectively). In addition, the in vitro antibacterial and antifungal activities of a number of the target compounds revealed that some of them have significant antibacterial and mild to moderate antifungal activities. Compound 5a produced the most potent antimicrobial activity in the current series of compounds with equipotency to ampicillin (MIC 12.5 µg/mL) against S. aureus, and 50% of ampicillin’s activity against B. subtilis and E.coli (MIC 50 νs 25 µg/mL, respectively), beside a moderate antifungal activity against C. albicans, which was about 50% of that of Clotrimazole (MIC 25 νs 12.5 µg/mL, respectively).
The broad spectrum antitumor activity displayed by these compounds will be of interest for future derivatization in the hope of finding more active and selective antitumor and/or antimicrobial agents.
Declaration of interest
The authors report no conflicts of interest.
References
- Cerecetto H, Gerpe A, González M, Arán VJ, de Ocáriz CO. Pharmacological properties of indazole derivatives: recent developments. Mini Rev Med Chem 2005;5:869–878.
- De Angelis M, Stossi F, Carlson KA, Katzenellenbogen BS, Katzenellenbogen JA. Indazole estrogens: highly selective ligands for the estrogen receptor beta. J Med Chem 2005;48:1132–1144.
- Zhang HC, Derian CK, McComsey DF, White KB, Ye H, Hecker LR et al. Novel indolylindazolylmaleimides as inhibitors of protein kinase C-beta: synthesis, biological activity, and cardiovascular safety. J Med Chem 2005;48:1725–1728.
- May JA, Dantanarayana AP, Zinke PW, McLaughlin MA, Sharif NA. 1-((S)-2-aminopropyl)-1H-indazol-6-ol: a potent peripherally acting 5-HT2 receptor agonist with ocular hypotensive activity. J Med Chem 2006;49:318–328.
- Han W, Pelletier JC, Hodge CN. Tricyclic ureas: a new class of HIV-1 protease inhibitors. Bioorg Med Chem Lett 1998;8:3615–3620.
- Showalter HD, Angelo MM, Berman EM, Kanter GD, Ortwine DF, Ross-Kesten SG et al. Benzothiopyranoindazoles, a new class of chromophore modified anthracenedione anticancer agents. Synthesis and activity against murine leukemias. J Med Chem 1988;31:1527–1539.
- Gugliemotti A, Capezzone de Joannon A, Cazzolla N, Marchetti M, Soldo L, Cavallo G, Pinza M. Radical scavenger activity of bendazac, an anticataract non-steroidal antiinflammatory agent. Pharmacol Res 1995; 32:369–373.
- Makino R, Obayashi E, Homma N, Shiro Y, Hori H. YC-1 facilitates release of the proximal His residue in the NO and CO complexes of soluble guanylate cyclase. J Biol Chem 2003;278:11130–11137.
- Claramunt RM, López C, Pérez-Medina C, Pérez-Torralba M, Elguero J, Escames G et al. Fluorinated indazoles as novel selective inhibitors of nitric oxide synthase (NOS): synthesis and biological evaluation. Bioorg Med Chem 2009;17:6180–6187.
- Elguero J, Alkorta I, Claramunt RM, López C, Sanz D, María DS. Theoretical calculations of a model of NOS indazole inhibitors: interaction of aromatic compounds with Zn-porphyrins. Bioorg Med Chem 2009;17:8027–8031.
- Ravagnan L, Marzo I, Costantini P, Susin SA, Zamzami N, Petit PX et al. Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene 1999;18:2537–2546.
- Souers AJ, Gao J, Brune M, Bush E, Wodka D, Vasudevan A et al. Identification of 2-(4-benzyloxyphenyl)-N- [1-(2-pyrrolidin-1-yl-ethyl)-1H-indazol-6-yl]acetamide, an orally efficacious melanin-concentrating hormone receptor 1 antagonist for the treatment of obesity. J Med Chem 2005;48:1318–1321.
- Xia M, Zhang T, Wang Y, Xing G. Preparation of tetrahydroindole and tetrahydroindazole derivatives as anticancer agents (PRC). 2006; WO: 2006133634.
- Rosati O, Curini M, Marcotullio MC, Macchiarulo A, Perfumi M, Mattioli L et al. Synthesis, docking studies and anti-inflammatory activity of 4,5,6,7-tetrahydro-2H-indazole derivatives. Bioorg Med Chem 2007;15:3463–3473.
- Raikova SV, Shub GM, Golikov AG, Kriven’ko AP. [Antimicrobial activity of new original 2-aryliden-6-furfuryliden cyclohexanones and hexahydroindazoles on their basis]. Antibiot Khimioter 2004;49:21–24.
- Raikova SV, Shub GM, Luneva IO, Golikov AG, Bugaev AA, Kriven’ko AP. [Chemotherapeutic effect of 3-(5-nitrofuryl)-7-(5-nitrofurfurylidene)-3,3a,4,5,6,7-hexahydro-2H-indazole in experimental staphylococcal infection]. Antibiot Khimioter 2005;50:18–21.
- Nagakura M, Ota T, Shimidzu N, Kawamura K, Eto Y, Wada Y. Syntheses and antiinflammatory actions of 4,5,6,7-tetrahydroindazole-5-carboxylic acids. J Med Chem 1979;22:48–52.
- Nasr MN, Said SA. Novel 3,3a,4,5,6,7-hexahydroindazole and arylthiazolylpyrazoline derivatives as anti-inflammatory agents. Arch Pharm (Weinheim) 2003;336:551–559.
- Minu M, Thangadurai A, Wakode SR, Agrawal SS, Narasimhan B. Synthesis, antimicrobial activity and QSAR studies of new 2,3-disubstituted-3,3a,4,5,6,7-hexahydro-2H-indazoles. Bioorg Med Chem Lett 2009;19:2960–2964.
- Gökhan-Kelekçi N, Simsek OO, Ercan A, Yelekçi K, Sahin ZS, Isik S et al. Synthesis and molecular modeling of some novel hexahydroindazole derivatives as potent monoamine oxidase inhibitors. Bioorg Med Chem 2009;17:6761–6772.
- Faidallah HM, Al-Saadi MS, Rostom Sh AF. Synthesis of new coumarin-3-carbonylurea, thiourea analogs and some derived ring systems of possible therapeutic interest. J Saudi Chem Soc 2003; 7:367–372.
- Al-Saadi MSM, Rostom Sh AF, Faidallah HM. Synthesis and Biological Evaluation of Some 3-Cyano-4-(1-methyl-1H-pyrrol-2-yl)-6-substituted-2(1H)-pyridinones and Their 2-imino isosters. Alex J Pharm Sci 2005; 19:15–21.
- Al-Saadi MSM, Rostom Sh AF, Faidallah HM. In vitro antitumor screening of some polysubstituted pyrazole analogs. Saudi Pharm J 2005; 13:89–96.
- Al-Saadi MS, Rostom SA, Faidallah HM. 3-methyl-2-(4-substituted phenyl)-4,5-dihydronaphtho[1,2-c]-pyrazoles: synthesis and in-vitro biological evaluation as antitumour agents. Arch Pharm (Weinheim) 2008;341:181–190.
- Al-Saadi MS, Faidallah HM, Rostom SA. Synthesis and biological evaluation of some 2,4,5-trisubstituted thiazole derivatives as potential antimicrobial and anticancer agents. Arch Pharm (Weinheim) 2008;341:424–434.
- Faidallah HM, Al-Saadi MS, Rostom SAF. Synthesis and biological evaluation of certain 2H-pyran-2-ones and some derived 1Hpyridin-2-one aanalogs as antimicrobial agents. Saudi Pharm J 2008; 16:33–42.
- Rostom SA, Ashour HM, El Razik HA, El Fattah Ael F, El-Din NN. Azole antimicrobial pharmacophore-based tetrazoles: synthesis and biological evaluation as potential antimicrobial and anticonvulsant agents. Bioorg Med Chem 2009;17:2410–2422.
- Rostom SA, Ashour HM, Abd El Razik HA. Synthesis and biological evaluation of some novel polysubstituted pyrimidine derivatives as potential antimicrobial and anticancer agents. Arch Pharm (Weinheim) 2009;342:299–310.
- Rostom SA, Hassan GS, El-Subbagh HI. Synthesis and biological evaluation of some polymethoxylated fused pyridine ring systems as antitumor agents. Arch Pharm (Weinheim) 2009;342:584–590.
- Rostom SA. Polysubstituted pyrazoles, part 6. Synthesis of some 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-3-carbonyl derivatives linked to nitrogenous heterocyclic ring systems as potential antitumor agents. Bioorg Med Chem 2010;18:2767–2776.
- Grever MR, Schepartz SA, Chabner BA. The National Cancer Institute: cancer drug discovery and development program. Semin Oncol 1992;19:622–638.
- Boyd MR, Paull KD. Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen. Drug Dev Res 1995; 34:91–109.
- Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 1991;83:757–766.
- Conte JE, Barriere SL. Manual of antibiotics and infectious disease. 1st ed., Lea and Febiger, USA, 1988: 135–138.
- Mamolo MG, Vio L, Banfi E, Predominato M, Fabris C, Asaro F. Synthesis and antimycobacterial activity of some 2-pyridinecarboxyamidrazone derivatives. Farmaco 1992;47:1055–1066.
- Hassan SY, Basaif SA, Faidallah HM. Synthesis of novel indan [1,2-c] pyrazole benzenesulfonylureas, thioureas and their cyclized derivatives. Egyptian J Chem 1999; 42:213–220.
- Güzel O, Innocenti A, Vullo D, Scozzafava A, Supuran CT. 3-phenyl-1H-indole-5-sulfonamides: structure-based drug design of a promising class of carbonic anhydrase inhibitors. Curr Pharm Des 2010;16:3317–3326.