Ring-substituted 4-Hydroxy-1 H-quinolin-2-ones : Preparation and Their Photosynthesis-inhibiting Activity

The series of twelve ring-substituted 4-hydroxy-1H-quinolin-2-one derivatives were prepared. The synthetic procedures of the compounds are presented. All the prepared quinoline derivatives were analyzed using RP-HPLC method for the lipophilicity measurement and their lipophilicity was determined. The prepared compounds were tested for their photosynthesis-inhibiting activity (the inhibition of photosynthetic electron transport in spinach chloroplasts (Spinacia oleracea L.). The relationships between the lipophilicity and the chemical structure of the studied compounds are discussed as well as the structure-activity relationships (SAR) between the chemical structure and the biological activities of the evaluated compounds.


INTRODUCTION
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 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 [1].In addition to these herbicides various compounds possessing an amide -NHCO-functionality were found to inhibit photosynthetic electron transport as well [2][3][4][5].Better understanding of the SAR regularities are not only important for the design of modern agricultural agents but can also give the remarkable insight into the photosynthesis mechanisms of the green cells.Quinoline scaffold is present in many classes of biologically active compounds [6].A series of compounds derived from 8-hydroxyquinoline and styrylquinoline derivatives as potential HIV-1 integrase inhibitors were synthesized recently [7][8][9][10].Our study dealing with 8-hydroxyquinoline and styrylquinoline derivatives showed that they could possess also strong antifungal activity [11][12][13].According to the results reported recently some new hydroxyquinoline derivatives possess interesting herbicidal activities as well [12,[14][15][16][17][18].Some investigated compounds showed also antineoplastic activity [19].This paper deals with synthesis and herbicidal activity of ring-substituted 4-hydroxy-1Hquinolin-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.).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 structureactivity relationships of the studied compounds are discussed in the present study as well.

Chemistry
In most of described synthesis aniline derivatives were used as starting materials for their great availability from chemical vendors.Microwave assisted synthesis with malonic acid or its esters provide us compounds 1-4.Further nitration and reduction according to known procedures appeared to be succeful in obtaining compounds 6 and 7. Diazo derivative 5 was synthesised from 3 with dichloroaniline.Acylation of 7 with cinnamoyl chloride provide us compound 8. Quinolines functionalized with carboxylic group at C (3) 9, 10 and 11 were obtained in neat microwave assisted synthesis with moderate or good yield.Structure 12 was obtained according to modified procedure from 4-Hydroxyquinolin-2(1H)-one (1).Hydrophobicities (log P/Clog P values) of the studied 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 shown 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, see Figure 1.The results show that experimentally determined log k values correlate relatively poorly.As expected, compound 5 showed the highest lipophilicity, while compound 3 possessed the lowest hydrophobicity, which was unexpected.Compound 8 showed less hydrophobicity contrary to all the results of the lipophilicity calculated by software.If compared the lipophilicity data log k of both position analogues 3 and 4 it can be stated, that 7-hydroxy derivative 4 possessed higher hydrophobicity than 5-hydroxy analogue 3.This fact is caused by intramolecular interactions [20].All compounds were evaluated for their in vitro herbicidal efficiency.The results are showed in Table 1.
Quinoline derivatives 1-11 showed a wide range of activity related to OER inhibition in spinach chloroplasts activities.Two compounds showed interesting IC 50 values: 126 µmol/L (5) and 157 µmol/L (2); nevertheless the studied activity of all the other compounds was very low.
Due to the medium and/or moderate activity of all the evaluated compounds 1-11 it is difficult to determine simple structure-activity relationships.However some observations seem to be interesting.Unsubstituted structure (compound 1) did not practically affect OER in chloroplasts.The studied compounds could be divided into two groups according to their chemical structure.Group 1 includes compounds 2-5 and 11, and Group 2 compounds 6-10.
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   Chemicals shifts are reported in ppm (δ) to internal Si(CH 3 ) 4 , when diffused easily exchangeable signals are omitted.

4,5-Dihydroxy-2-oxo-1,2-dihydroquinoline-6-carboxylic acid (3).
Naphthalene (15.4 g, 0.12 mol) and malonic acid (18.7 g, 0.18 mol) were melted with stirring at temperature control (<150 °C) to avoid decarboxylation of acid.POCl 3 (32.9g, 0.36 mol) was then added dropwise during 30 min and p-aminosalicylic acid (15.3 g, 0.1 mol) was added.The resulted mixture was heated for next 30 min and left for cooling.Water (100 mL) was added to the warm mixture and the solution was alkalized with 20% NaOH to pH 9.After cooling on ice precipitated naphthalene was filtered and filtrate was acidified to pH 2. The product was filtered and crystallized from acetic acid as a bright yellow crystalline compound.Yield 36%.Mp

3-Acetyl-4-hydroxyquinolin-2(1H)-one (12).
NaH (2.4 g, 0.06 mol) was added to anhydrous benzene (180 mL) and then ethyl acetylacetate (7.6 mL, 0.06 mol) was added dropwise.The mixture was stirred for 1 h at ambient temperature and 2-methyl-3,1-benzoxazin-4-one (3.2 g, 0.02 mol) was added and stirred overnight.Water (180 mL) and Et 2 O (150 mL) was added to the resulted mixture and the product was isolated from inorganic layer as a white crystalline compound.Yield 78%  -HPLC -Mili-Q Grade (45.0%) 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 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 [28].The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was determined spectrophotometrically (Kontron Uvikon 800, Kontron, Muenchen, Germany) using an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCIPP) according to Kralova et al. [29] 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 (PET).The inhibitory efficiency of the studied compounds has been expressed by IC 50 values, i.e. by molar concentration of the compounds causing 50% decrease in the oxygen evolution relative to the untreated control.The comparable IC 50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (Diurone ® ) was about 1.9 µmol/L [30].The results are summarized in Table 1.

Figure 1 .
Figure 1.Comparison of the computed log P/Clog P values using the two programs with the calculated log k values.The discussed compounds 1-12 are ordered according to the log k values increase.
P [ChemOffice] Clog P [ChemOffice] log P [ACD/LogP] (6) position, see compounds 2-4, 11.Comparison of the OER-inhibiting activities of compounds 2-5 and 11 also indicated, that the lipophilicity increase is connected with the quasi-parabolic increase of biological activity, see Figure2.Interesting are great differences in inhibition of OER of position analogues 3 (6-COOH-5-OH) and 4 (6-COOH-7-OH).Higher inhibiting effect of 5 compared with 2 may be caused by higher lipophilicity (easier penetration of compound to cell) and/or redox properties of nitro moiety of 2,5-dichloro-4nitrophenyldiazenyl substituent.
2 inhibited OER only slightly; nevertheless the compounds 6 and 9 were approximately twice as efficient as the compound 1.All these compounds possess the substituted position C (3) of ring A, that caused decrease of OER inhibition compared to Group 1.The most active compound from Group 2 was ester 9.EXPERIMENTALGeneralAll reagents were purchased from Aldrich.Kieselgel 60, 0.040-0.063mm (Merck, Darmstadt, Germany) was used for column chromatography.TLC experiments were performed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Germany).The plates were illuminated under UV (254 nm) and evaluated in iodine vapour.The melting points were determined on Boetius PHMK 05 (VEB Kombinat Nagema, Radebeul, Germany) and are uncorrected.Elemental analyses were carried out on an automatic Perkin-Elmer 240 microanalyser (Boston, USA).The purity of the final compounds was checked by HPLC, see section 4.3.The detection wavelength 210 nm was chosen.The peaks in the chromatogram of the solvent (blank) were deducted from the peaks in the chromatogram of the sample solution.The purity of individual compounds was determined from the area peaks in the chromatogram of the sample solution.UV spectra (λ, nm) were determined on a Waters Photodiode Array Detector 2996 (Waters Corp., Milford, MA, U.S.A.) in ca 6×10 -4 mol methanolic solution and log ε (the logarithm of molar absorption coefficient ε) was calculated for the absolute maximum λ max of individual target compounds.All 1 H NMR spectra were recorded on a Bruker AM-500 (499.95MHz for 1 H), Bruker BioSpin Corp., Germany.

Table 1 .
Comparison of the calculated lipophilicities (log P/Clog P) with the determined log k values.IC 50 values related to OER inhibition in spinach chloroplasts of compounds 1-12 in comparison with standard DCMU.
a interacted with DCPIP.