Preparation and Herbicidal Properties of Ring-Substituted 4-Chloro-2-styrylquinazolines

In this study, a series of new seven ring-substitu ted 4-chloro-2-styrylquinazoline derivatives were prepared. The procedures for synth esis of the compounds are presented. The compounds were analyzed using RP-HPLC to determine lipophilicity. They were tested for their activity related to inhibition of photosynthe ic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. For all the compounds, the relat ionships between the lipophilicity and the chemical structure of the stu died compounds are discussed, as well as their structure-activity relationships (SAR).


INTRODUCTION
A 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][5][6][7][8]. Some quinoline-based compounds also showed antiasthmatic and antiplatelet activity [9][10][11]. A series of compounds derived from 8-hydroxyquinoline and styrylquinoline derivatives were recently synthesized as potential HIV-1 integrase inhibitors [12][13][14][15]. These compounds showed a significant similarity to some novel antifungal agents, namely homoallylamines [16]. Our previous study dealing with 8-hydroxyquinoline and styrylquinoline derivatives showed that they could also possess strong antifungal activity [6][7][8]17,18]. According to the results reported recently, some new hydroxyquinoline derivatives also possess interesting herbicidal activities [6][7][8]17,19,20]. In addition, some of the quinoline derivatives investigated also showed antineoplastic activity [19,21,22]. 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 [23] and thereby inhibit photosynthesis [24][25][26]. Some organic compounds, e.g. substituted benzanilides [27] or pyrazine-2-carboxylic acids [28] 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 interruption of the photosynthetic electron transport occurred. In the context of the previously-described azanaphtalenes [6][7][8][17][18][19][20][21][22], new modifications of quinoline moiety that can trigger interesting biological activity were investigated. The compounds were tested for their photosynthesis-inhibiting activity (the inhibition of photosynthetic electron transport in spinach chloroplasts (Spinacia oleracea L.). 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. Relationships among the structure and inhibitory activity related to inhibition of photosynthetic electron transport (PET) in spinach chloroplasts of the new compounds are discussed.
The results show that experimentally determined log k values correlate relatively poorly with the calculated log P. Compound 5 showed the highest lipophilicity, while compound 12 possessed the lowest hydrophobicity within individual series of compounds. Surprisingly, 3,5-OCH 3 derivative 6 showed much lower lipophilicity than expected on the basis of calculated log P data and compared with 2,4-OCH 3 is its hydro/lipophilic properties diametrically opposed. If the lipophilicity data log k of three position isomers 2-4, 7-9 are compared, it can be stated that 3-alkoxy derivative 3/8 possessed higher hydrophobicity than 2-alkoxy derivative 2/7 and 4-alkoxy derivative 4/9 showed the lowest lipophilicity. Due to the facts discussed above, it can be assumed that lipophilicity of individual compounds within both series is strongly influenced by intramolecular interactions. It can be assumed, that the determined log k data specify lipophilicity within the individual series of compounds (2-  The activity of the evaluated quinazoline derivatives related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts was moderate or low relative to the standard, see Table 1. The PET-inhibiting activity was expressed by negative logarithm of IC 50 value (compound concentration in mol/L causing 50% inhibition of PET). Compound 11 expressed the highest (IC 50 = 115 µmol/L) and compound 6 the lowest PET-inhibiting activity (IC 50 = 862 µmol/L). Despite the relatively low inhibitory activity of the studied compounds as well as the relative scarcity of compounds for which PET-inhibiting activity could be determined, correlations between log (1/IC 50 ) and lipophilicity (expressed as log P) or electronic properties (expressed as Hammett's σ parameters [30,31]) of the benzylidene R substituent for compounds 1-12 were performed, see Fig 2A and Fig. 2B. It is not possible to perform clear conclusions from the dependence of log (1/IC 50 ) on log k, due to large variation of biological activity for compounds within narrow range of lipophilicity expressed by log k, therefore neither dependence nor any Figure can be presented. According to Fig. 2A it can be stated that the dependence of PET-inhibiting activity on the lipophilicity expressed as log P (ACD/LogP) shows quasi-parabolic relation except compound 12, which benzylidene substituent contains by one oxygen atom more than the other studied compounds and except compound 11 (R = CH 3 ), where benzylidene substituent does not contain any oxygen atom. On the other hand, the biological activity was expressively affected by electronic σ properties of these benzylidene substituents, see Fig. 2B. The dependence of log (1/IC 50 ) on σ was bilinear, for compounds with σ in the range of -0.66 (compound 5) to -0.07 (compound 11) the activity linearly raised with increasing value of σ, however further increase of σ led to sharp activity decrease. The most active compound from the series was 11 (R = 3-CH 3 ). The results indicate that PET inhibition depends not only on the lipophilicity but also on the σ values of electronic properties of individual benzylidene substituents whereby the effect of electronic properties of benzylidene R substituent is more important. The relatively high PET-inhibiting activity of compound 12 (R = 2-OCOCH 3 ) despite of low lipophilicity of this compound (log k = 0.7016, log P = 3.8) can be associated with additional interaction of the second oxygen atom in R substituent with photosynthetic proteins.
Within a series of alkoxy derivatives it can be concluded that introduction of methoxy group to benzylidene decreased activity compared with unsubstituted compound 1 analogous to 2-ethoxy moiety introduction. Contrary, substitution by longer chain (ethoxy or propoxy moeity) especially to the position 4 of the benzylidene ring (compounds 9 and 10) caused significant PET inhibition increase.    4 . Easily exchangeable signals were omitted when diffuse. Syntheses were performed on Plazmatronika RM-800PC microwave reactor with monomode cavity, magnetic stirrer and external IR temperature measurements. Microwave power was automatically adjusted to achieve the desired temperature unless specified otherwise.
Compounds 1-5 was described in detail in ref. [7,22,29]. ( ( (12 ) were measured in minutes. The capacity factors k were calculated using the Empower™ 2 Chromatography Data 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 using 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 [32]. 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. [33], 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. 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)-1,1-