Ring-substituted 4-Hydroxy-1H-quinolin-2-ones: Preparation and Biological Activity

In the study, a series of twelve ring-substituted 4-hydroxy-1H-quinolin-2-one derivatives were prepared. The procedures for synthesis of the compounds are presented. The compounds were analyzed using RP-HPLC to determine lipophilicity and tested for their photosynthesis-inhibiting activity using spinach (Spinacia oleracea L.) chloroplasts. All the synthesized compounds were also evaluated for antifungal activity using in vitro screening with eight fungal strains. For all the compounds, the relationships between the lipophilicity and the chemical structure of the studied compounds are discussed, as well as their structure-activity relationships (SAR).

Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. Binding of herbicides to photosystem II inhibits the electron transfer from Q A to Q B due to competition of herbicides with plastoquinone bound at the Q B site. Thus, the Q B quinone-binding site of photosystem II is an important target for herbicides, including herbicides based on phenylurea moieties. It was found that a tail can be attached to the para position of phenylurea-type herbicides without any loss of binding, provided that the tail is hydrophobic. This indicates that the herbicides must be oriented in the Q B site so that these positions point toward the natural isoprenyl tail-binding pocket that extends out of the Q B site. In turn, the requirement that the tail must extend out of the Q B site constrains the size of the other herbicide substituents in the pocket [15]. In addition to phenylurea-type herbicides, various other compounds possessing an amide -NHCO-moiety were also found to inhibit the photosynthetic electron transport [16][17][18][19][20][21]. Better understanding of these SAR relationships are not only important for the design of modern agricultural agents, but can also provide remarkable insights into the photosynthetic mechanisms of green cells.
Over the last three decades there has been a dramatic increase in the incidence of fungal infections, and the discovery of new drugs for the treatment of systemic mycoses is a major challenge in infectious disease research. There is an intensified need for new antifungal remedies with novel modes of action due to the rapid growth of the immunocompromised patient population, the development of resistance to the present azole therapies, and high toxicity of polyenes [22][23][24].
Compounds bearing a quinoline moiety are well known due to their broad biological activity [6]. In particular, hydroxyquinoline and its derivatives were introduced as antifungal agents in clinical practice and the novel compounds of this type are still investigated [25,26]. This paper deals with synthesis, herbicidal and antifungal activity of ring-substituted 4-hydroxy-1H-quinolin-2-one derivatives. All the compounds were tested for their photosynthesis-inhibiting activity (the inhibition of photosynthetic electron transport in spinach chloroplasts (Spinacia oleracea L.). Primary in vitro screening of all synthesized compounds was evaluated against eight fungal strains by means of the broth microdilution test in RPMI 1640 medium [27]. Lipophilicity (log k) of the compounds was determined using RP-HPLC. The procedure was performed under isocratic conditions with methanol as an organic modifier in the mobile phase using end-capped non-polar C 18 stationary RP column. The structure-activity relationships of the compounds are also discussed.

Chemistry
In most of the synthesis protocols, aniline derivatives were used as the starting materials due to their convenient availability from chemical providers. Microwave assisted synthesis with malonic acid or its esters, was used to make compounds 1-4. Further nitration and reduction according to established procedures were used to make compounds 5 and 6. Acylation of 6 with cinnamoyl chloride provided compound 7. Diazo derivative 8 was made by means of a two-step synthesis from 4-aminobenzoic acid and diethyl malonate and gave 4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid, which was coupled with the freshly prepared diazo salt derived from 4-nitro-2,5-dichloroaniline. Quinolines functionalized with carboxylic acid groups at C (3) 9-12 were obtained in neat microwave assisted synthesis in moderate or good yield. Synthesis of all the above compounds is depicted in Scheme 1.

Lipophilicity
Hydrophobicities (log P/Clog P values) of the compounds 1-12 were calculated using two commercially available programs and also measured by means of the reversed phase high performance liquid chromatography (RP-HPLC) method for lipophilicity measurement. 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 capacity factors k were determined and subsequent log k values were calculated.
The results are summarized in Table 1 and illustrated in Figure 1. The results obtained with all the compounds show that the experimentally-determined lipophilicities (log k values) are lower than those indicated by the calculated log P/Clog P, as shown in Figure 1, indicating relatively poor correlation between the experimentally-determined log k values and the calculated values. As expected, compound 8 showed the highest lipophilicity, while compound 3 possessed the lowest hydrophobicity, which was unexpected. Compound 7 showed less hydrophobicity contrary to all the results of the lipophilicity calculated by software. Comparing the lipophilicity data log k of both position analogues 3 and 4, it can be stated that the 7-hydroxy derivative 4 possessed higher hydrophobicity than 5-hydroxy analogue 3. The salicylic acid derivative 12 showed higher lipophilicity than benzoic derivative 11. These facts are caused by intramolecular interactions [28].

Oxygen evolution rate inhibition in spinach chloroplasts
All compounds were evaluated for their in vitro herbicidal efficiency. The results are listed in Table  2. Quinoline derivatives 1-12 showed a wide range of activity related to inhibition of oxygen evolution rate (OER) in spinach chloroplasts. Two compounds showed interesting IC 50 (half maximal inhibitory concentration) values: 126 µmol/L (8) and 157 µmol/L (2); nevertheless the studied activity of all the other compounds was very low.
Due to the moderate and/or low activity of compounds 1-12, it is difficult to determine simple structure-activity relationships. However some interesting observations can be made: in the case of compound 1, an unsubstituted structure did not have any effect on OER in chloroplasts. The remaining compounds could be divided into two groups according to their chemical structure. Group 1 includes compounds 2-4, 8, 11 and 12, and Group 2 compounds 5-7, 9 and 10.
Group 1 showed higher biological activity than Group 2. The activity related to OER inhibition seems to be positively influenced by substitution of ring B: especially the C (6) position (see compounds 2-4, 11, 12). Comparison of the OER-inhibiting activities of compounds 2-4, 8, 11 and 12 also indicated that the lipophilicity increase is connected with the quasi-parabolic increase of biological activity ( Figure 2). It is noteworthy that there are great differences in OER inhibition levels caused by positional isomers 3 (6-COOH-5-OH) and 4 (6-COOH-7-OH). Introducing a further phenolic moiety in compound 12 (salicylic derivative) positively influenced OER inhibition. The higher inhibitory effect of compound 8 compared with compound 2 may have resulted from higher lipophilicity (easier penetration of the compound through cell walls) and/or redox properties of the nitro moiety of the 2,5-dichloro-4-nitrophenyldiazenyl substituent.  Generally, Group 2 compounds only caused slight inhibition of OER; nevertheless compounds 5 and 9 were approximately twice as effective as compound 1. All these compounds possess the substituted position C (3) of ring A, which caused the decrease in OER inhibition compared with Group 1 compounds. The most active compound from Group 2 was the ester 9.

In vitro antifungal susceptibility testing
All the compounds were tested for their in vitro antifungal activity. Compounds 1-3, 5-7, 9-11 did not show any activity and compounds 4, 8 and 12 showed only a moderate activity, especially against Candida albicans ATCC 44859. Compound 4 showed medium activity against Candida glabrata 20/I, and compound 8 against Trichophyton mentagrophytes 445. The activities of the compounds are shown in Table 2.
Generally, it can be stated that in vitro antifungal activity depends on heteroaromatic ring A. Hydrogenation of ring A and introduction of keto group resulted in the loss of the antifungal effect compared with hydroxyquinoline derivatives [6,7]. Substitution of the C (3) position by various moieties did not have a significant influence on the activity. Nevertheless salicylic acid derivative 12 showed a higher activity compared with benzoic derivative 11, probably due to the substitution of the C (3)´ position by phenolic moiety.
Unsubstituted ring B or C (6) substitution by a methyl moiety did not results in any activity. Substitution of phenyl ring B by 6- COOH (compounds 3, 4, 8 and 11, 12) caused the activity to increase. Position of the phenolic moiety seems to be a very important parameter for antifungal activity. While a 6-COOH-5-OH substitution pattern (compound 3) did not show any activity increase, introduction of 6-COOH along with a 7-OH moiety (compound 4) increased the activity significantly. The antifungal activity of compounds 8 and 12 was connected with 2,4-dichloro-4-nitrophenyldiazenyl and 3-(4-carboxy-3-hydroxyphenylcarbamoyl) substituents, respectively. According to the results, it can be assumed that lipophilicity is only of secondary importance for antifungal activity.

3-(4-Carboxyphenylcarbamoyl)-4-hydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic
acid (11). 4-Aminobenzoic acid (0.7 g, 0.005 mol) was mixed with triethyl methanetricarboxylate (2.12 mL, 0.01 mol) and heated in microwave reactor at 50% of power during 15 min and 3 min at 90%. The temperature reached 231 °C during heating. Et 2 O was added to the cooled mixture and the precipitate was washed with hot (55 °C) MeOH to obtain the pure product as a yellow crystalline compound. Yield 62%; Mp 340-350 °C; Anal. calc. for C 18 (12). 4-Aminosalicylic acid (0.7 g, 0.005 mol) was mixed with triethyl methanetricarboxylate (2.12 mL, 0.01 mol) and heated in microwave reactor at 50% of power for 15 min and 3 min at 90%. ) was used as a mobile phase. The total flow of the column was 0.9 mL/min, injection 30 μL, column temperature 30 °C and sample temperature 10 °C. The detection wavelength 210 nm was chosen. The KI methanolic solution was used for the dead time (T D ) determination. Retention times (t R ) were measured in minutes. 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. 9.0 (CambridgeSoft, Cambridge, MA, U.S.A.) and ACD/LogP ver. 1.0 (Advanced Chemistry Development Inc., Toronto, Canada). Clog P values (the logarithm of n-octanol/water partition coefficient based on established chemical interactions) were generated by means of CS ChemOffice Ultra ver. 9.0 (CambridgeSoft, Cambridge, MA, U.S.A.) software. The results are shown in Table 1.

Study of inhibition of oxygen evolution rate (OER) in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and Kralova [36]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was determined spectrophotometrically (Genesys 6, Thermo Scientific, U.S.A.) using an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCPIP) according to Kralova et al. [37] 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 [38]. The results are summarized in Table 2.