Investigating the Spectrum of Biological Activity of Substituted Quinoline-2-Carboxamides and Their Isosteres †

In this study, a series of thirty-five substituted quinoline-2-carboxamides and thirty-three substituted naphthalene-2-carboxamides were prepared and characterized. They were tested for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Primary in vitro screening of the synthesized compounds was also performed against four mycobacterial species. N-Cycloheptylquinoline-2-carboxamide, N-cyclohexylquinoline-2-carboxamide and N-(2-phenylethyl)quinoline-2-carboxamide showed higher activity against M. tuberculosis than the standards isoniazid or pyrazinamide and 2-(pyrrolidin-1-ylcarbonyl)quinoline and 1-(2-naphthoyl)pyrrolidine expressed higher activity against M. kansasii and M. avium paratuberculosis than the standards isoniazid or pyrazinamide. The most effective antimycobacterial compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. The PET-inhibiting activity expressed by IC50 value of the most active compound N-benzyl-2-naphthamide was 7.5 μmol/L. For all compounds, the structure-activity relationships are discussed.

Both pharmaceuticals and pesticides are designed to target particular biological functions, and in some cases these functions overlap in their molecular target sites, or they target similar processes or molecules. Modern herbicides express low toxicity against mammals and one of the reasons is that mammals lack many of the target sites for herbicide action. At present, approximately 20 mechanisms of action of herbicides are known. It was determined that inhibitors of protoporphyrinogen oxidase, 4-hydroxyphenylpyruvate dioxygenase and glutamine synthetase inhibit these enzymes both in plants and mammals. However, the consequences of inhibition of the overlapping target site can be completely different for plants and animals. Therefore a compound that has lethal action on plants may be beneficial for mammals [32]. Such chemical compounds are characterized by low toxicity on mammals as a result of quick metabolism and/or elimination of herbicide from the mammal system. Taking into consideration that mammals may also have molecular sites of action of herbicides, most pharmaceutical companies until recently had pesticide divisions, sometimes with a different name. All compounds generated by either division of the company were evaluated for both pesticide and pharmaceutical uses. In the past, some leading pesticides have become pharmaceuticals and vice versa. However, little information of this type was published and must usually be deduced from patent literature. One of the exceptions is fluconazole, a fungicide product discovered by the pharmaceutical sector that is now used both as a pharmaceutical and patented as a crop production chemical [32][33][34].
In the context of the previously-described azanaphtalenes [13,[15][16][17][18]26] or various amides [3][4][5][6][7][8][9], new simple modifications of quinoline and naphtalene as quinoline isosteres 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.). The compounds were also assessed for activity against various mycobacterial species. Relationships between the structure and their in vitro antimycobacterial activities or/and activity related to inhibition of photosynthetic electron transport (PET) in spinach chloroplasts are discussed.

Chemistry
All the studied compounds were prepared according to Scheme 1. Condensation of the chlorides of 2-quinaldic and 2-naphthoic acids with commercially available substituted amines yielded a series of thirty-five substituted quinoline-2-carboxamides 1-19c and thirty-three substituted naphthalene-2-carboxamides 20-38c. Quinoline-2-carbonyl chloride was prepared using oxalyl chloride to ensure mild conditions and prevent quinoline nucleus chloration, whereas 2-naphtoyl chloride was obtained by the classical procedure using thionyl chloride. Hydrophobicities (log P) of all compounds 1-38c were calculated using the commercially available program ACD/LogP (ACD/LogP ver. 1.0, Advanced Chemistry Development Inc., Toronto, Canada). The results are shown in Tables 1 and 2. Compounds show a wide range of lipophilicities, with log P (ACD/LogP) values from 1.15 (compound 3, pyrrolidinyl) to 6.98 (compound 2, octyl) within the series of quinolinecarboxamides and from 2.10 (compound 22, pyrrolidinyl) to 7.94 (compound 21, octyl) within the series of naphthalenecarboxamides. Individual substituents in the amide part of the discussed compounds also result in a wide range (from −0.39 to 1.26) of electronic properties expressed as σ parameters [35][36][37][38].

Biological Activities
The compounds under investigation could be divided into two groups based on their chemical structure: Group 1 included quinoline-2-carboxamides 1-19c; Group 2 contained naphthalene-2-carboxamides 20-38c. Compounds within both series can be also divided according to whether they contain an aromatic or a non-aromatic amine. The compounds showed a wide range of biological activities and some interesting structure-activity relationships were observed. All the results are summarized in Tables 1-3. Generally, all the discussed compounds exhibited problematic solubility, but quinoline derivatives generally possess better aqueous solubility in comparison to the naphthamides. The PET-inhibiting activity of the evaluated quinoline derivatives (Group 1) is summarized in Table 1. According to both Figure 1, where all compounds except substituted phenyl rings are illustrated, it can be stated that the dependence of PET-inhibiting activity on the lipophilicity showed a parabolic course. Cyclic non-aromatic N-substituents are illustrated in Figure 1a, where the most active compound N-cyclooctyl 8 has a lipophilicity optimum at log P = 4.33. The dependence of PET-inhibiting activity on electronic constants σ of non-aromatic as well as phenyl 9 and benzyl 10 substituents obtained from literature [36] seems to also follow a parabolic course, see Figure 1b. Benzyl derivative 10 showed an optimum of weak electron-withdrawing effect influencing the electronic density of the amido moiety. It is evident that bulkiness of the N-substituents did not influence PET-inhibiting activity.
On the other hand, the biological activity was also affected by electronic σ properties of these anilide substituents, see Figure 2b.  In general, the dependence of log (1/IC 50 ) on σ reflects two trends. The first, i.e., compounds with extreme electron σ effects, show a similar trend as in case of lipophilicity and thus, the activity decreases with increasing σ value. The second trend is a parabolic course with an optimum activity for compound 16a (2-Cl-Ph; σ = 0.20). In this case the σ values of the corresponding compounds ranged from −0.07 to 0.37, but the differences between activities of the compounds expressed as IC 50 values were relatively low and they ranged from 100.6 (14b) to 56.3 μmol/L (16a). However, the importance of the lipophilicity of the anilide substituent was unambiguously much more significant for the inhibitory activity (IC 50 [mol/L]) of the studied compounds than the electronic properties of the substituent.
The most active compound from the series Group 1 was 12a (R = 2-OH, IC 50 = 16.3 μmol/L). The result indicates that PET inhibition could be associated with additional interaction of the phenol moiety with photosynthetic proteins. This compound can be understood as a bioisoster of 2-[(2-hydroxyphenylimino)methyl]quinolin-8-ol that expresses high herbicide effect [18].
Generally, the activity of the evaluated naphthalene derivatives (Group 2) related to PET inhibition in spinach (Spinacia oleracea L.) chloroplasts seems to be higher than that of the corresponding quinoline isosters ( Table 2). The PET inhibition of 34 of the 68 compounds could not be determined due to their precipitation during the experiments. With respect to these small but closed specifically substituted groups of compounds some structure-activity relationships (SAR) can be proposed. Figure 3a illustrates the exponential decay of PET-inhibiting activity on the lipophilicity increase of all naphthalene derivatives (compounds 20, 21, 26-29) except substituted phenyl moieties. Dependence of the activity on the electronic properties could not be performed due to small amount of data. As mentioned above, PET-inhibiting activity is not influenced by bulkiness of the N-substituents. Benzyl derivative 29 showed higher activity (IC 50 = 7.5 μmol/L) than unsubstituted phenyl derivative 28 (IC 50 = 20.7 μmol/L), similarly to quinolinecarboxamides [IC 50 = 59.4 μmol/L (10) and 85.1 μmol/L (9), respectively].
Dependence of PET inhibition on the lipophilicity of all compounds with the aromatic N-substituents is shown on Figure 3b. It is evident that unsubstituted phenyl 28 derivative expressed the highest PET inhibition; and this decreases as lipophilicity increases. This is observed for lipophilic meta-substituted derivatives 35b (3-Cl), 36b (3-Br) and 37b (3-CF 3 ), contrary to the quinaldinanilides, and it is not valid for methoxy and nitro moieties (32a/b, 38b/c). This lower inhibitory activity of compounds 32a (2-OCH 3 -Ph), 32b (2-OCH 3 -Ph) and 38c (4-NO 2 -Ph) could be a result of the limited solubility of the compounds, which is typical for -OCH 3 and -NO 2 substituents. Figure 3c shows PET inhibition of naphtanilides on Hammett's σ parameters of the individual substituents. Based on the results from Figure 3c it can be concluded that PET-inhibiting activity is strongly decreased by the electron-withdrawing effect of substituents in the anilide part of the molecule: , especially in meta-position, contrary to the SAR of quinaldinanilides discussed above.

In Vitro Antimycobacterial Evaluation
Although all the compounds were evaluated for their in vitro antimycobacterial activity against M. tuberculosis (clinical isolate with partial INH and PZA resistance) and other atypical mycobacterial strains, most compounds did not show any activity due to their low solubility and precipitation during the experiments. Only the 10 compounds presented in Table 3 showed actimycobacterial activities. N-Cycloheptylquinoline-2-carboxamide (7) and N-(2-phenylethyl)quinoline-2-carboxamide (11) showed high activity against M. tuberculosis, whereas 2-(pyrrolidin-1-ylcarbonyl)quinoline (3) and 1-(2-naphthoyl)pyrrolidine (22) expressed high activity against M. avium paratuberculosis and M. kansasii. Compounds 6, 1 and 32a also showed noteworthy activity. Nevertheless, no thorough structure-activity relationships could be established.
According to the results, it can be generally concluded that quinaldinamides (Group 1) possessed higher activity than corresponding naphtamides (Group 2), and anilides seem to be less effective than amides, e.g., highly effective compound 11 and non-active phenyl derivative 9 (not-discussed). Figure  4 shows dependence of the average of antituberculosis/antimycobacterial activities expressed as log 1/MIC [mol/L] on lipophilicity expressed as log P. Based on these results, it can be concluded that activity increases as lipophilicity increases. Also, it seems that the increase in antituberculosis activity is connected with the increase in the bulkiness of individual N-substituents within the series of cycloalkane, (i.e., cycloheptyl 7 > cyclohexyl 6 > cyclopentyl 5). As cyclooctyl derivative 8 demonstrated no activity, it can be concluded that cycloheptyl compound 7 showed the maximum antituberculosis efficacy within this type of compound and under these testing conditions. This decrease might have also actually been caused by decreased solubility of compound 8 in the test media (precipitation occurred). It can be speculated that substituents which are bulkier than cyclooctyl could further potentiate the antimycobacterial activity. But the testing conditions for these more lipophilic compounds would have to be changed to prevent the precipitation during the dilution of samples.  Branching within the N-substituents seems also very important, especially from the point of view of antitubercular versus antimycobacterial activity. Unbranched long-chain alkyl dodecyl compound 2 or ethylphenyl one 11 as well as isopropyl compound 1 and other cycloalkyl moietis branched in the α position of individual N-substituents, seem to be the most advantageous for antituberculosis activity, while N,N-disubstitution, e.g., compounds 3 and 22, is fundamental for high efficacy against the atypical mycobacterial strains M. avium paratuberculosis and M. kansasii.

In Vitro Cytotoxicity Assay
The most effective antimycobacterial compounds 11, 7, 3 and 22 were tested for their in vitro cytotoxicity LD 50 (µmol/L), and subsequently the Selectivity Index, i.e., the ratio of cell toxicity (LD 50 ) to activity (MIC), was obtained; the results are presented in Table 3. The LD 50 exact values of compounds 3, 11 and 22 could not be determined due to their limited solubility and their precipitation from solution during the tests in concentration higher than 100 µmol/L, but the highest dose achieved in the medium (100 µmol/L) did not lead to the 100% lethal effect on THP-1 cells. All the evaluated compounds demonstrated low toxicity against the human monocytic leukemia THP-1 cell line (e.g., LD 50 of oxaliplatin 1.7 ± 6.4 and camptothecin 0.16 ± 0.07 assessed in this line formerly showed much lower values). It can be drawn from Table 3 that compounds 3, 7 and 11 from Group 1 and compound 22 from Group 2 showed the highest inhibition activity and also moderate cytotoxicity against THP-1 cells; compound 7 expressed the highest toxicity within the series of compounds, but it can not be considered toxic as its LD 50 value (62 ± 4.5) is fairly high. Based on these observations it can be concluded that the discussed amides 11, 3 and 22 can be considered as promising agents for subsequent design of novel antitubercular/antimycobacterial agents.

General
All reagents were purchased from Aldrich. Kieselgel 60, 0.040-0.063 mm (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 Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and are uncorrected. Infrared (IR) spectra were recorded on a Smart MIRacle™ ATR ZnSe for Nicolet™ Impact 410 FT-IR spectrometer (Thermo Scientific, USA). The spectra were obtained by accumulation of 256 scans with 2 cm −1 resolution in the region of 4,000-600 cm −1 . All 1 H and 13 C NMR spectra were recorded on a Bruker Avance III 400 MHz FT-NMR spectrometer (400 MHz for 1 H and 100 MHz for 13 C, Bruker Co., Karlsruhe, Germany). Chemicals shifts are reported in ppm () using internal Si(CH 3 ) 4 as the reference with diffuse, easily exchangeable signals being omitted. Mass spectra were measured using a LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron Corporation, USA) with direct injection into an APCI source (400 °C) in the positive mode.

General Procedure for Synthesis of Carboxamide Derivatives 1-19c
2-Quinaldic acid (1 g, 5.8 mmol) was suspended in dry toluene (15 mL) at room temperature and oxalyl chloride (1 mL, 1.61 g, 12.7 mmol, 2.2 eq.) was added dropwise. The reaction mixture was stirred for 30 min at the same temperature and then DMF (2 drops) was added. The mixture was stirred for 24 h and then evaporated to dryness. The residue was washed with petroleum ether and used directly in the next step. Into the solution of 2-quinaldic acid chloride in dry toluene (15 mL), triethylamine (4.5 mL, 2.92 g, 32.5 mmol) and corresponding substituted aniline (5.8 mmol) were added dropwise. The mixture was stirred at room temperature for 24 h after which the solvent was removed under reduced pressure. The residue was extracted with CHCl 3 . Combined organic layers were washed with water and saturated aqueous solution of NaHCO 3 and dried over anhydrous MgSO 4 . The solvent was evaporated to dryness under reduced pressure. The crude product was recrystallized from isopropanol or EtOAc. The studied compounds 1-19c are presented in Table 1.

Conclusions
A series of thirty-five substituted quinoline-2-carboxamides and thirty-three substituted naphthalene-2-carboxamides were prepared and characterized. The prepared compounds were tested for their ability to inhibit photosynthetic electron transport (PET) in spinach chloroplasts (Spinacia oleracea L.) and for their antituberculosis/antimycobacterial activity. Two compounds, N-benzyl-2naphthamide (29) and N-(2-hydroxyphenyl)quinoline-2-carboxamide (12a) showed relatively high PET inhibition. N-(2-Phenylethyl)quinoline-2-carboxamide (11), N-cycloheptylquinoline-2-carboxamide (7) and N-cyclohexylquinoline-2-carboxamide (6) expressed high activity against Mycobacterium tuberculosis. 1-(2-Naphthoyl)pyrrolidine (22) and 2-(pyrrolidin-1-ylcarbonyl)quinoline (3) showed high activity against M. kansasii and M. avium paratuberculosis. All five compounds exhibited activity comparable with or higher than the standards isoniazid or pyrazinamide. Lipophilicity was fundamental for the biological activities of all compounds in both biological assays. It can be stated that the dependence of PET-inhibiting activity on the lipophilicity decreases with increasing lipophilicity, while antimycobacterial activity increases with lipophilicity increase. Highly effective compounds against M. tuberculosis were detected, namely quinaldinamides, while naphtamides were more active against the other mycobacterial species. Substituted N-quinaldinanilides and/or N-naphtanilides seem to be less effective than other discussed N-nonaromatic amide derivatives. The most effective antimycobacterial compounds 11, 7, 3 and 22 were tested for their in vitro cytotoxicity against THP-1 cells. According to the calculated selectivity index of compounds 11, 3 and 22 it can be concluded that the discussed amides can be considered as promising agents for subsequent design of novel antitubercular/ antimycobacterial agents.