Investigating the Activity Spectrum for Ring-Substituted 8-Hydroxyquinolines

In this study, a series of fourteen ring-substituted 8-hydroxyquinoline derivatives were prepared. The synthesis procedures are presented. The compounds were analyzed using RP-HPLC to determine lipophilicity. They were tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Primary in vitro screening of the synthesized compounds was also performed against four mycobacterial strains and against eight fungal strains. Several compounds showed biological activity comparable with or higher than the standards isoniazid or fluconazole. For all the compounds, the relationships between the lipophilicity and the chemical structure of the studied compounds are discussed.

Tuberculosis is a worldwide pandemic. About 1/3 of the world's population is infected with Mycobacterium tuberculosis, and every year almost 2 million people die as a result [24]. The Mycobacterium genus is composed of the M. tuberculosis complex and other species known as nontuberculous mycobacteria (NTM). In recent decades, the decrease in the prevalence of tuberculosis in developed countries has resulted in the increase in the proportion of diseases caused by NTM [25]. Among these species, the M. avium complex (MAC) has emerged as a major human pathogen, being a common cause of disseminated disease and death in patients with HIV/AIDS [26].
Chronic pulmonary disease is the most common clinical manifestation among the diseases caused by NTM, and the most common pathogens are the species belonging to the MAC, followed by M. kansasii. The clinical characteristics of NTM-related pulmonary disease are, in many cases, extremely similar to those of tuberculosis. Other clinical manifestations are caused principally by M. fortuitum, M. smegmatis and M. abscessus due to peritoneal infection as a result of catheterization, postsurgical infections, such as those following mammoplasty and heart transplant, as well as those following invasive procedures [27]. The above mentioned non-tuberculous strains are sometimes resistant to commonly used drugs (isoniazid, rifampicin, pyrazinamide) and other anti-tuberculous drugs [24], therefore systematic development of new effective compounds is necessary. Similarly, the discovery of new drugs for the treatment of systemic mycoses with novel modes of action due to the rapid growth of the immunocompromised patient population and development of resistance to the present azole therapies, and high toxicity of polyenes [28] is indispensable. It should be stressed that hydroxyquinoline and its derivatives were introduced as antifungal or antimycobacterial agents in clinical practice and novel compounds of this type are still investigated [3][4][5]29,30].
Over 50% of commercially available herbicides act by reversibly binding to photosystem II (PS II), a membrane-protein complex in the thylakoid membranes which catalyses the oxidation of water and the reduction of plastoquinone [31] and thereby inhibit photosynthesis [32][33][34]. Some organic compounds, e.g., substituted benzanilides [35] or substituted anilides of 2,6-disubstituted pyridine-4-thiocarboxamides [36] or pyrazine-2-carboxylic acids [37] were found to interact with tyrosine radicals Tyr Z and Tyr D which are situated in D 1 and D 2 proteins on the donor side of PS II and due to this interaction the photosynthetic electron transport is interrupted. This is a follow-up paper to our previous articles [12][13][14][15][17][18][19][20][21][22][23] dealing with syntheses and biological activities of ring-substituted quinoline derivatives. On the basis of formerly described azanaphtalenes we tried to search for new modifications of quinoline moiety that can trigger interesting biological activity.
Primary in vitro screening of the synthesized compounds was performed against four mycobacterial strains and against eight fungal strains. The compounds were also tested for their photosynthesisinhibiting activity (the inhibition of photosynthetic electron transport) in spinach chloroplasts (Spinacia oleracea L.). Relationships among the structure and in vitro antimicrobial activities or/and inhibitory activity related to inhibition of photosynthetic electron transport (PET) in spinach chloroplasts of the new compounds are discussed.

Chemistry
All studied compounds were prepared according to Scheme 1. Compounds 1-3 were obtained according to a previously described procedure [14]. Sulfonamides 4-7 were obtained from 8-hydroxyquinoline through chlorosulfonation and amination. Compound 8 was obtained from commercially available 8-hydroxy-2-aminoquinoline by acylation in acetic anhydride. Styryquinolines 9-14 were obtained from 5,7-dichloro-8-hydroxyquinaldine and the appropriate aldehyde in a two-step reaction in acetic anhydride followed by pyridine/water. Scheme 1. Synthesis of studied compounds.

Lipophilicity
Many low molecular weight drugs cross biological membranes through passive transport, which strongly depends on their lipophilicity. Lipophilicity is a property that has a major effect on absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties as well as pharmacological activity. Lipophilicity has been studied and applied as an important drug property for decades [38]. Hydrophobicities (log P/Clog P) of the compounds 1-14 were calculated using two commercially available programs (ChemDraw Ultra 10.0 and ACD/LogP) and also measured by means of the RP-HPLC determination of capacity factors k with subsequent calculation of log k. The procedure was performed under isocratic conditions with methanol as an organic modifier in the mobile phase using an end-capped non-polar C 18 stationary RP column. The ChemDraw program did not resolve various lipophilicity values of individual positional isomers, that is, the same log P/Clog P data were calculated for 9/10 and 11/12. The results are shown in Table 1 and illustrated in Figure 1. The results obtained with all the compounds show that the experimentally-determined lipophilicities (log k) of compounds 1-14 are lower than those indicated by the calculated log P/Clog P, as shown in Figure 1. The results indicate that experimentally-determined log k values correlate relatively poorly with the calculated values. The correlation factors R 2 yielded 0.83 for ChemOffice log P; 0.86 for ACD/LogP and 0.55 for ChemOffice Clog P respectively.
These facts are probably caused by intramolecular interactions. As expected, compound 14 showed the highest lipophilicity, while compound 2 exhibited the lowest. The presence of the sulfonic acid moiety decreased the lipophilicity. Compound 6 showed less hydrophobicity compared with 5, contrary to the lipophilicity results calculated by software. This observation has been made previously [19]. Interesting results were obtained with subseries 9-13. Comparing the lipophilicity data log k of both OH-monosubstituted isomers 9 and 10, it can be stated that 4-hydroxy derivative 10 possessed higher hydrophobicity than 2-hydroxy isomer 9. This fact was predicted only by ACD/LogP. When OH-disubstituted compounds were compared, it was found that strong intramolecular interactions play a significant role. Hydrophobicity increased in the following order: 11 (2,4-OH) < 12 (3,5-OH). These results were also predicted by ACD/LogP.
It can be assumed, that log k values specify lipophilicity within the individual series of studied compounds. Calculated log P data (ChemDraw) of compounds 1-8 corresponded with experimentallydetermined log k, while for compounds 9-14 there was better agreement between the calculated log P data (ACD/LogP) and experimentally-determined log k.

Biological activities
The compounds under investigation could be divided into two groups based on their chemical structure. Group 1 included compounds 1-8, and Group 2 compounds 9-14. The compounds showed a wide range of biological activities and structure-activity relationships observations are interesting. All the results are shown in Table 2 and Table 3. Table 2. IC 80 values related to PET inhibition in spinach chloroplasts in comparison with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) standard and in vitro antifungal activity (IC 80 ) of compounds 1-10, 12 and 14 compared with fluconazole (FLU) standard. (ND = not determined due to insufficient solubility of the compound).

Comp.
PET reduction

Inhibition of photosynthetic electron transport (PET) in spinach chloroplasts
Quinoline derivatives 1-10, 12 and 14 showed a wide range of activity related to inhibition of photosynthetic electron transport (PET) in spinach chloroplasts, see Table 2. Three compounds showed moderate inhibitory IC 50 values: 33.5 µmol/L (3), 54.4 µmol/L (5) and 78.0 µmol/L (1) while the activity of all the other studied compounds was very low. PET inhibition by 2, 4 and 9 could not be determined due to precipitation of the compounds during the experiments.
The highest PET inhibition was shown by compounds 3, 5 and 1 with suitable aqueous solubility as well as lipophilicity. The sulfonic acid moiety contributed to enhanced aqueous solubility whereas a 7-bromo moiety (compound 3) increased the lipophilicity. Herbicidal effects of organic compounds with bromo or nitro substituents were observed previously [39]. Bulkiness and rigidity of the chain in the R substituent play a fundamental role in the series of sulfonamides 4-7, in addition to lipophilicity and water-solubility. The least lipophilic morpholine derivative 4 did not show any activity. On the other hand, phenylbutyl substitution in compound 7 is probably too bulky for it to reach the site of action in the photosynthetic electron transport chain and thus the inhibitory activity of this compound is low. Within the sulfonamide series the isopropyl substituent was the most advantageous from the aspect of effective PET inhibition and further lipophilicity increase caused reduction of inhibitory activity. On the other hand, great differences in the activity of compounds characterized with rather small differences in the lipophilicity (the log k values of compounds belonging to Group 1) indicate that the position of substituents and their electronic Hammett's parameters σ (especially electronwithdrawing effect) seem to be also important for biological activity. Lipophilicity of the discussed compounds is only a secondary parameter influencing PET-inhibiting activity. In general, Group 2 compounds exhibited only slight inhibition of PET and IC 50 values determined for three compounds do not enable conclusions about structure-activity relationships.

In vitro antifungal susceptibility testing
Quinoline derivatives 1-10, 12 and 14 were tested for their in vitro antifungal activity. The results are shown in Table 2. Two compounds 3 and 14 showed very high antifungal activity, comparable or higher than the standard fluconazole.
Generally, Group 1 showed lower biological activity than Group 2, although compound 3 demonstrated high activity against all fungal strains. Compounds 1, 2, 4-7 did not show any antifungal activity. Compound 8 showed only moderate activity especially against Candida albicans ATCC 44859.
Unfortunately due to general low activity, no thorough structure-activity relationships (SAR) can be established. Nevertheless, an interesting relationship can be observed. According to the results presented in Tables 1 and 2 it can be concluded that bulky substituents and low lipophilicity decreased antifungal activity. A bromo moiety (compound 3) seems to be very important for high antifungal effect, as was reported recently [17]. Contrary to all expectations [18], compounds with a nitro moiety (i.e., 1, 2) did not show any activity. It can be concluded that relatively minor differences in lipophilicity were correlated with major differences in the activity of compounds. This suggests that for biological activity, the position of substituents and their electronic Hammett's parameters σ seem to be important. Similar observations were made with the PET-inhibiting activity.
Compound 14 showed the highest antifungal activity within Group 2. The compounds 9-12 showed only a moderate activity especially against Absidia corymbifera 272 and Trichophyton mentagrophytes 445. Contrary to all expectations [18], 9 (2-OH substitution) possessed less activity than 10 and 12 (4-OH or 3,5-OH respectively). This fact is probably caused by low solubility of 9. Lipophilicity is probably an important parameter influencing the activity.

In vitro antimycobacterial evaluation
Six compounds within Group 2 9-14 were evaluated for their in vitro antimycobacterial activity against four mycobacterial strains. The results are shown in Table 3. Compound 12 (3,5-OH) did not exhibit any significant antimycobacterial activity and compounds 9-11, 13 showed only medium and/or moderate activities. Nevertheless, compound 14 had an interesting MIC especially against M. smegmatis, M. kansasii and M. absessus. This compound was more active than the standard pyrazinamide (PZA) and in case of M smegmatis, its activity was comparable with the standard isoniazid (INH). Due to medium and/or moderate activity of the majority of evaluated compounds, it is difficult to determine simple structure-activity relationships. According to Tables 1 and 3, it can be concluded that lipophilicity (log k) and other physico-chemical parameters only influence secondary characteristics of the compounds.

Lipophilicity determination by HPLC (capacity factor k/calculated log k)
A Waters Alliance 2695 XE HPLC separation module and Waters Photodiode Array Detector 2996 (Waters Corp., Milford, MA, USA) were used. A Symmetry ® C 18  The capacity factors k were calculated using the Millennium32 ® Chromatography Manager Software according to formula k = (t R -t D )/t D , where t R is the retention time of the solute, whereas t D denotes the dead time obtained via an unretained analyte. Log k, calculated from the capacity factor k, is used as the lipophilicity index converted to log P scale. The log k values of the individual compounds are shown in Table 1.

Lipophilicity calculations
Log P, i.e. the logarithm of the partition coefficient for n-octanol/water, was calculated using the programs CS ChemOffice Ultra ver. 10 Table 1.

Study of inhibition photosynthetic electron transport (PET) in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and Kralova [46]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was determined spectrophotometrically (Genesys 6, Thermo Scientific, USA) using an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCIPP) according to Kralova et al. [47] and the rate of photosynthetic electron transport was monitored as a photoreduction of DCPIP. The measurements were carried out in phosphate buffer (0.02 mol/L, pH 7.2) containing sucrose (0.4 mol/L), MgCl 2 (0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content was 30 mg/L in these experiments and the samples were irradiated (~100 W/m 2 ) from 10 cm distance with a halogen lamp (250 W) using a 4 cm water filter to prevent warming of the samples (suspension temperature 22 °C). The studied compounds were dissolved in DMSO due to their limited water solubility. The applied DMSO concentration (up to 4%) did not affect the photochemical activity in spinach chloroplasts. The inhibitory efficiency of the studied compounds was expressed by IC 50 values, i.e., by molar concentration of the compounds causing 50% decrease in the oxygen evolution rate relative to the untreated control. The comparable IC 50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1dimethylurea, DCMU (Diurone ® ) was about 1.9 μmol/L [48]. The results are summarized in Table 2.

In vitro antimycobacterial evaluation
Clinical isolates of Mycobacterium avium complex CIT19/06, M. kansasii CIT11/06, M. absessus CIT21/06 and strain M. smegmatis MC2155 were grown in Middlebrook broth (MB), supplemented with OADC supplement (Oleic, Albumin, Dextrose, Catalase, Becton Dickinson, U.K.). Identification of these isolates was performed using biochemical and molecular protocols. At log phase growth, the 10 mL culture was centrifuged at 15,000 RPM for 20 minutes using a bench top centrifuge (Model CR 4-12 Jouan Inc U.K). Following the removal of the supernatant, the pellet was washed in fresh Middlebrook 7H9GC broth and re-suspended in 10 ml of fresh supplemented MB. The turbidity was adjusted to match McFarland standard No. 1 (3 × 10 8 cfu) with MB broth. A further 1:20 dilution of the culture was then performed in MB broth.
The antimicrobial susceptibility of all four mycobacteria was investigated in 96 well plate format. Here, 300 µL of sterile deionised water was added to all outer-perimeter wells of the plates to minimize evaporation of the medium in the test wells during incubation. 100 µL of each dilution was incubated with 100 µL of each of the mycobacterial species. Dilutions of each compound were prepared in duplicate. For all synthesized compounds, final concentrations ranged from 300 µg/mL to 10 µg/mL. All compounds were prepared in DMSO and subsequent dilutions were made in supplemented Middlebrook broth. The plates were sealed with parafilm and were incubated at 37 °C overnight in the case of M. smegmatis and M. absessus and for 5 days in the case of M. kansasii and M. avium complex. Following incubation, a 10% addition of alamarBlue (AbD Serotec) was mixed into each well and readings at 570 nm and 600 nm were taken, initially for background subtraction and subsequently after 24 hour re-incubation. The background subtraction is necessary with strongly coloured compounds which may interfere with the interpretation of any colour change. In non-interfering compounds, a blue colour in the well was interpreted as an absence of growth, and a pink colour was scored as growth. The MIC was initially defined as the lowest concentration which prevented a visual colour change from blue to pink. The results are shown in Table 3.
The MIC for mycobacteria was defined as a 90% or greater (IC 90 ) reduction of growth in comparison with the control. The MIC/IC 90 value is routinely and widely used in bacterial assays and is a standard detection limit according to the Clinical and Laboratory Standards Institute (CLSI, www.clsi.org/).