Substituted 2-Styrylquinazoline Derivatives : Preparation and Their Biological Activities

In this study, a series of five ring-substituted 2 -styrylquinazolin-4(3H)-one and five ring-substituted 4-chloro-2-styrylquinazoline deriv ati es were prepared. The procedures for synthesis of the compounds are presented. The compo unds 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 pe rf rmed against four mycobacterial strains. Several compounds showed bio logical activity comparable with or higher than the standard isoniazid. For all the com p unds, the relationships 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].Some quinoline-based compounds also showed antineoplastics, antiasthmatic and antiplatelet activity [7][8][9][10][11][12].A series of compounds derived from 8-hydroxyquinoline and styrylquinoline derivatives as potential HIV-1 integrase inhibitors were recently synthesized [13][14][15][16].These compounds showed a significant similarity to some novel antifungal agents, namely homoallylamines.[17].Our previous study dealing with 8-hydroxyquinoline and styrylquinoline derivatives showed that they could also possess strong antifungal activity [18,19].According to the results reported recently, some new hydroxyquinoline derivatives also possess interesting herbicidal activities [18,[20][21][22].In addition, some of the quinoline derivatives investigated also showed antineoplastic activity [20,23].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 an 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 very 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 or postsurgical infections [27].The above mentioned nontuberculous strains are sometimes resistant to commonly used drugs (isoniazid, rifampicin, pyrazinamide) and other antituberculous drugs [24], therefore systematic development of new effective compounds is necessary.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 [28] and thereby inhibit photosynthesis [29][30][31].Some organic compounds, e.g.substituted benzanilides [32] or substituted anilides of 2,6-disubstituted pyridine-4-thiocarboxamides [33] or pyrazine-2-carboxylic acids [34] 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.This is a follow-up paper to our previous articles [6,[13][14][15][16][18][19][20][21][22][23] dealing with synthesis and biological activities of ring-substituted quinazolinone derivatives.Primary in vitro screening of the synthesized compounds was performed against four mycobacterial strains.The compounds were also 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 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.

RESULTS AND DISCUSSION
All studied compounds were prepared according to Scheme 1. Microwave-assisted synthesis facilitated the process of obtaining quinazoline-related structures.2-Methyl-4Hbenzo[d] [1,3]oxazin-4-one (1) was synthesized from anthranilic acids and acetic anhydride.A further reaction with ammonia afforded 2-methylquinazolin-4(3H)-one (2).Compounds 3a-e were obtained from appropriate aldehydes using neat microwave-assisted synthesis [35].Further aromatization with POCl 3 yielded 4-chloro-2-styrylquinazoline derivatives (4a-e).Hydrophobicities (log P/Clog P) of compounds 3a-e and 4a-e 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  The evaluated quinazoline derivatives showed relatively low activity related to inhibition of photosynthetic electron transport (PET) in spinach chloroplasts, see Table 2. Compounds 4a and 4c expressed the highest PET-inhibiting activity (IC 50 : 285 and 303 µmol/L, respectively).PET inhibition by several compounds (3a-c and 4b) could not be determined due to precipitation of the compounds during the experiment.The PET-inhibiting activity was expressed by negative logarithm of IC 50 value (compound concentration in mol/L causing 50% inhibition of PET).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 log k or Hammett's parameters (σ) of the R substituent for compounds 4a-d were performed.σ values [36,37] mentioned in Table 1 were used for calculations.The σ value for R: 2,4-OCH 3 was calculated from the sum of corresponding σ values for R: 2-OCH 3 and 4-OCH 3 .The inhibitory activity of compounds 4a-e depended predominantly on the Hammett's constants (σ) of R substituents: log (1/IC 50 ) = 3.507 (± 0.020) + 0.324 (± 0.056) σ r = 0.971, s = 0.033, F = 33.24,n = 4 The introduction of the lipophilicity of the whole molecule (expressed by log k) in the above equation for compounds 4a-e partially improved the results of statistical analysis: log (1/IC 50 ) = 4.122 (± 0.579) -0.537 (± 0.506) log k + 0.298 (± 0.060) σ r = 0.987, s = 0.032, F = 18.24, n = 4 Thus, it could be concluded that the electronic properties of the R substituent were decisive for photosynthesis-inhibiting activity.The dependences of PET-inhibiting activity on the Hammett's constants (σ) of the R substituent of the studied compounds are shown in Figure 2.There the PET-inhibiting activity was expressed by negative logarithm of IC 50 value.IC 50 values related to PET-inhibition determined for both compounds 3d and 3e do not enable us to make conclusions about structure-activity relationships.All compounds were evaluated for their in vitro antimycobacterial activity against four mycobacterial strains and the results are shown in Table 2.All the evaluated compounds except for 3a (H) showed biological activity comparable with or higher than the standard pyrazinamide (PZA).Nevertheless it can be stated that compounds 3b (2-OCH 3 ), 3c (3-OCH 3 ), 3d (4-OCH 3 ), 4a (H), 4d (3-OCH 3 ) and 4e (2,4-OCH 3 ) did not exhibit any significant antimycobacterial activity.Compound 4b (2-OCH 3 ) had an interesting MIC especially against M. absessus, M. kansasii and M. avium complex and likewise compound 4c (3-OCH 3 ) against M. kansasii.Both compounds were either more active than or comparable with the standard pyrazinamide (PZA) in all cases.Compound 4b was more active in case of M. absessus as its activity was higher than the standard isoniazid (INH).2-[(E)-2-(2,4-Dimethoxyphenyl)vinyl]quinazolin-4(3H)-one (3e) expressed the highest activity against M. kansasii, M. avium complex and M. absessus.This compound is more active than PZA and in the case of M. absessus it is more active than INH.Due to the medium and/or moderate activity of the compounds 3a-e and 4a-e, it is difficult to determine simple structure-activity relationships.However some observations seem to be interesting.The position and the number of substitutions on the benzylidene part of the molecule is important for antimycobacterial activity; for example, if one compares the activity of compounds 3a/3e (the unsubstituted compound without any activity / the disubstituted compound with the highest activity) or 4b/4a,4d,4e (2-OCH 3 isomer showed the highest activity within this series).Bulk parameters (volume/number of the substituents on the benzylidene part) MR [36] are also very important for activity, especially again for series 3.

Synthesis
General method for synthesis of styryl-compounds 3a-e: A mixture of compound 2 (0.01 mol) and the appropriate aldehyde (0.02 mol) was mixed thoroughly and irradiated in monomode cavity of microwave reactor using pulse sequence (3×5 minutes with 30 sec.intervals) at 250 W.During irradiation, the temperature was controlled between the range 150-180 °C.
After the reaction, the mixture was cooled and washed with boiling ether.The product was crystallized from acetic acid.

Study of inhibition photosynthetic electron transport (PET) in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and Kralova [41].The inhibition of oxygen evolution rate (inhibition of photosynthetic electron transport, PET) in spinach chloroplasts was determined spectrophotometrically (Genesys 6, Thermo Scientific, U.S.A.) using the artificial electron acceptor 2,6-dichlorophenol-indophenol (DCIPP) according to Kralova et al. [42] 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 [43].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.300 µL of each dilution was incubated with 300 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 2.

Figure 1 .
Figure 1.Comparison of the log P/Clog P values computed using two the programs with the calculated log k values.Compounds 3a-4e and 4a-e are ordered according to the increase in log k values.

Figure 2 .
Figure 2. The relationships between the PET-inhibiting activity log (1/IC 50 ) [mol/L] in spinach chloroplasts and the electronic Hammett's constants (σ) of the R substituent of the studied compounds 4a-e.

data were calculated for 3b-d and 4b-d. The results are shown in Table 1 and illustrated in Figure
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 (H < 4-OCH 3 < 2-OCH 3 < 3-OCH 3 < 2,4-OCH 3 ), see Figure1.

Table 2 .
IC 50 [µmol/L] values related to PET inhibition in spinach chloroplasts and All 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 F 254 plates (Merck, Darmstadt, Germany).The plates were illuminated under UV (254 nm) and evaluated in iodine vapour.The melting points were determined on a Boetius PHMK 05 instrument (VEB Kombinat Nagema, Radebeul, Germany) and are uncorrected.The purity of the final compounds was checked by HPLC.A detection wavelength of 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 methanolic solution (ca.6×10 -4 mol) and log ε (the logarithm of molar absorption coefficient ε) was calculated for the absolute maximum λ max of individual target compounds.Infrared spectra were recorded using KBr pellets on the FT-IR spectrometer Nicolet 6700 (Nicolet -Thermo Scientific, U.S.A.).All 1 H NMR spectra were recorded on a Bruker AM-500 (499.95MHz for 1 H), Bruker BioSpin Corp., Germany.Chemical shifts are reported in ppm (δ) against the internal standard, Si(CH 3 ) 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.
[39]8]Yield 33% of a white crystalline compound.Mp 280-281 °C (Mp 284-285 °C[39]).HPLC purity: 94.36%.UV (nm), λ max /log ε: 322.7/3.59.1HNMR (DMSO-d 6 , 500 MHz), δ: 3.80 (s, 3H, OCH 3 ),6.84 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 of 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 the log P scale.The log k values of the individual compounds are shown in Table1.
calculations Log P, i.e. the logarithm of the partition coefficient for n-octanol/water, was calculated using the programmes CS ChemOffice Ultra ver.10.0 (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 the CS ChemOffice Ultra ver.10.0 (CambridgeSoft, Cambridge, MA, U.S.A.) software.The results are shown in Table