Substituted Amides of Quinoline Derivatives : Preparation and Their Photosynthesis-inhibiting Activity

The series of nine amides of substituted 8-hydroxyquinolines were prepared. The synthetic procedures of compounds are presented. All the prepared quinoline derivatives were analyzed using RP-HPLC method for the lipophilicity measurement and their lipophilicity were determined. The prepared compounds were tested for the reduction of chlorophyll content in Chlorella vulgaris Beij. Several compounds showed biological activity comparable with or higher than the standard 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The relationships between the lipophilicity and the chemical structure of the studied compounds are discussed as well as structure-activity relationships (SAR) between the chemical structure and the biological activities of the evaluated compounds.


INTRODUCTION
Quinoline moiety is present in many classes of biologically active compounds.A number of them have been clinically used as antifungal, antibacterial and antiprotozoic drugs [1,2] as well as antituberculotic agents [3,4].Some quinoline based compounds showed also 2 antineoplastics activity [5].Styrylquinoline derivatives have gained strong attention recently due to their activity as perspective HIV integrase inhibitors [6,7,8,9,10].Our previous study dealing with styrylquinoline derivatives showed that they could possess also strong antifungal activity [11], the compounds containing 8-hydroxyquinoline pharmacophore seem especially interesting.According to the results reported recently some new 8-hydroxyquinoline derivatives possessed interesting antifungal and herbicidal activities [12,13,14,15].The chemistry of quinoline has been described very well.On the other hand synthetic routes of quinoline derivatives are time consuming.Thus new efficient methods of microwave assisted organic synthesis for discussed quinoline derivatives were applied [16,17,18].This interesting route to structurally diverse quinoline derivatives is now under optimisation.Wider discussion will be subsequently published.During our preliminary studies we have found that some up-to-date synthesized structures could be interesting to wider forum.Various compounds possessing -NHCO-moiety were found to inhibit photosynthetic electron transport.Amides of the substituted pyridine-4-carboxylic acids [19] as well as anilides of the substituted pyrazine-2-carboxylic acids [20,21,22,23] inhibited oxygen evolution rate in Chlorella vulgaris and they showed some antialgal properties.Therefore a new series of amides of 8-hydroxyquinoline derivatives were prepared by means of the above-discussed pathways [16,17,18] and evaluated as potential herbicides.Synthesis, lipophilicity as well as structure-activity relationships are discussed in this paper.

RESULTS AND DISCUSSION
The compounds 1-9 were synthesized according to the procedure showed below.Kolbe-Schmidt reaction was leading to carboxylic acids which further reacted with the appropriate amine in presence of DCC or ethyldimethylaminopropyl carbodiimid (EDCI) to afford an amide.In case of 8, 9 diamine and twofold of quinaldic acid were used, see Scheme 1. Hydrophobicities of the studied compounds 1-9 were measured by means of the reversed phase high performance liquid chromatography (RP-HPLC) method for the lipophilicity measurement.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 capacity factors K were determined and subsequent log K values were calculated.The results are shown in Table 1.The total lipophilicity of the studied compounds is affected by the lipophilicity of the substituents on the aromatic rings as well as by the lipophilicity of the CH 2 and other function groups (e.g.-NH-or -C=O) in the linker.Using the π-parameter the lipophilicity of the substituents on the benzene rings can be expressed [24].On the other hand the lipophilicity of CH 2 , -NH-and -C=O groups can be expressed by aliphatic lipophilic fragment constants (f) whereas for the lipophilicity of the substituents on aromatic ring, aromatic lipophilic fragment constants (π) can be used [25].The lipophilicity of phenyl substituents in position 4 increased in the following order: H (1, π = 0) < OCH 3 (4, π = -0.03)< F (2, π = 0.15) < CH 3 (3, π = 0.60), which corresponds to the experimentally determined log K values (Table 1).The lipophilicity of substituents in amide part of molecules expressed by log K values increased in the following order: benzyl (5) < phenyl (1) < n-propylphenyl (7).Lower lipophilicity of benzyl substituent than that of phenyl substituent was also described in ref [26].The compound 7 possessed the highest hydrophobicity within this series.Low lipophilicity of both dimmers 8 and 9 is connected with hydrophilic 8-hydroxy substituents, on both 2methylquinoline rings as well as by two carbonyl and -NH-groups in the spacer linking of these two ring structures (the corresponding lipophilic fragment constants (f) are -0.44 for -OH, -1.09 for -C=O and -2.15 for -NH- [25]).
All the studied compounds were handed over for herbicidal evaluation.The compound 5 was not tested due to low solubility in the testing medium.Two studied compounds (3, 7) inhibited chlorophyll production in C. vulgaris comparable with the standard DCMU and the inhibitory activity of compound 4 evenly exceeded the activity of DCMU (Table 1).The interesting IC 50 values varied in the range from 4.8 (4) to 17.2 µmol/l (1).Compound 4 (IC 50 = 4.8 µmol/l) was the most efficient inhibitor.
The compounds 1 and 7, 2 and 6 as well as 8 and 9 differ from each other by the number of CH 2 groups in the linker connecting two ring structures in the molecule.According to Hansch and Leo the hydrophobic fragment constant (f) for CH 2 group (corresponding to the contribution of CH 2 group to the compound lipophilicity) is 0.66 [25].The comparison of compound 1 and 7 showed that the increase of the compound lipophilicity caused by prolongation of the linker by two CH 2 groups led to moderate increase of inhibitory activity.
On the other hand, the prolongation of the spacer in 8-hydroxy-2-methylquinoline-7carboxylic acid 4-fluorobenzylamide (2) by one CH 2 group (6) did not practically affect the biological activity of the compound.The addition of one CH 2 group to the spacer of bis-[8hydroxy-2-methylquinoline-7-carboxylic acid]-1,2-etylamide (8) led to moderately lower activity of ( 9).It can be assumed that the biological activity of the studied compounds depends not only on the lipophilicity but also on the electron-releasing or electron-withdrawing power of the substituent on the benzene ring.Using suitable electron-withdrawing substituent on the benzene ring and/or choosing suitable lipophilicity of the linker by addition of CH 2 group(s) the biological activity of the compounds could be optimised.

Synthesis of compounds 1-9
General procedure for synthesis discussed compounds is as follows.8-hydroxy-quinaldine-7carboxylic acid, starting material for all synthesized amides was obtain according to known procedure.

Lipophilicity HPLC determination (capacity factor K / calculated log K)
The ) and H 2 O-HPLC -Mili-Q Grade (50.0%) 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 the 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.

Study of chlorophyll content reduction in Chlorella vulgaris Beij.
The green algae C. vulgaris Beij.was cultivated statically at room temperature according to Kralova et al. [28] (photoperiod 16 h light/8 h dark; photosynthetic active radiation 80 µmol/m 2 .s;pH 7.2).The effect of the compounds on algal chlorophyll (Chl) content was determined after 4-day cultivation in the presence of the tested compounds.The Chl content in the algal suspension was determined spectrophotometrically (Kontron Uvikon 800, Kontron, Muenchen, Germany) after extraction into methanol according to Wellburn [29].
The Chl content in the suspensions at the beginning of the cultivation was 0.1 mg/l.Because of the low solubility of the studied compounds in water, these were dissolved in DMSO.DMSO concentration in the algal suspensions did not exceed 0.25% and the control samples contained the same DMSO amount as the suspensions treated with the tested compounds.The antialgal activity of compounds was expressed as IC 50 .Comparable IC 50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (DIURON) was about 7.3 µmol/l.The results are summarized in Table 1.

Table 1 .
Experimentally found hydrophobicity (log K) and IC 50 values related to reduction of chlorophyll content in C. vulgaris of the compounds 1-9 in comparison with standard (DCMU).
a not tested due to precipitation of a dissolved drug.