Synthesis, Bacteriostatic and Anticancer Activity of Novel Phenanthridines Structurally Similar to Benzo[c]phenanthridine Alkaloids

In this study, we report the synthesis, antibacterial and anticancer evaluation of 38 novel phenanthridines that were designed as analogs of the benzo[c]phenanthridine alkaloids. The prepared phenanthridines differ from the benzo[c]phenanthridines in the absence of a benzene A-ring. All novel compounds were prepared from 6-bromo-2-hydroxy-3-methoxybenzaldehyde in several synthetic steps through reduction of Schiff bases and accomplished by radical cyclization. Twelve derivatives showed high antibacterial activity against Bacillus subtilis, Micrococcus luteus and/or Mycobacterium vaccae at single digit micromolar concentrations. Some compounds also displayed cytotoxicity against the K-562 and MCF-7 cancer cell lines at as low as single digit micromolar concentrations and were more potent than chelerythrine and sanguinarine. The active compounds caused cell-cycle arrest in cancer cells, increased levels of p53 protein and caused apoptosis-specific fragmentation of PARP-1. Biological activity was connected especially with the presence of the N-methyl quaternary nitrogen and 7-benzyloxy substitution (compounds 7i, 7j, 7k, and 7l) of phenanthridine.


Introduction
Benzo[c]phenanthridine alkaloids contain a chrysene-skeleton-based heterocyclic core and are classified as isoquinoline alkaloids (Figure 1).They are distributed especially in the plant family Papaveraceae and display a broad spectrum of biological activities, including anti-inflammatory, antimicrobial, antifungal and antitumor effects [1][2][3][4][5][6][7].Sanguinarine and chelerythrine are the best-known benzo[c]phenanthridine alkaloids, most frequently studied for their antitumor effects [8].As flat polyaromatic compounds they directly interact with DNA [9].However, cell cycle arrest and induction of cell death caused by these alkaloids probably occurs not only due to DNA damage alone, but as a combined result of targeting other cellular structures, including topoisomerases, tubulin and antiapoptotic protein Bcl-X L [10,11].Importantly, these alkaloids act at concentrations comparable to those of cytostatics used as anticancer drugs.Benzo[c]phenanthridine alkaloids have been subjected to chemical modifications with the aim of understanding their structure-activity relationships and to improve their biological functions.Early studies indicated that the activity of benzo[c]phenanthridine alkaloids can be linked to the presence of their cationic quaternary nitrogen [12].The quaternary iminium may undergo nucleophilic addition to biological amines or thiols, modify DNA and proteins and as a consequence induce cell death.In contrast, a recent report suggests that the iminium group is not essential for cytotoxicity in cancer cell lines [13].Other systematic studies examined the correlation between the type of substituents at positions 2, 3, 7, 8 and 9 of their skeletons and antiproliferative activity [14] and revealed that cationic iminium alkaloids displayed stronger activity than their corresponding uncharged bases.The cytotoxic activity was further increased if these quaternary bases were 7,8-oxygenated [15].
These studies led to identification of NK109, the 7-O-demethylated synthetic analogue of chelerythrine, that was shown to have submicromolar dose growth-inhibitory activity against cancer cell lines, i.e., significantly lower than chelerythrine [16][17][18][19] (Figure 1).Further modification of NK109, in which ring C was fused to a pyrrolidine cycle, yielded NK314 with even stronger activities in several cancer models [20][21][22].The molecular mechanism of action of these compounds has been often attributed to inhibition of topoisomerases, which are also targeted by the related compounds nitidine and fagaronine [23].
To our knowledge, there have not been many attempts to modify the heterocyclic skeleton of benzo[c]phenanthridines up until now in order to modify their biological activity.With the exception of benzo[h]quinolones, which are structurally related to the benzo[c]phenanthridines but lack the D-ring [24], and phenanthridines lacking the A-ring [25], most other reports describe the synthesis of various isomeric and aza-analogous structures such as benzo[i]phenanthridines and dibenzo[c,h]cinnolines [26], benzo[c]phenanthrolines [27], pyridophenanthrolines and azapyrimido-phenanthrolines [28] or compounds containing the 3,4-dihydroisoquinolin-2-ium scaffold [29,30].
We therefore decided to prepare novel phenanthridines related to benzo[c]phenanthridines lacking the benzene A-ring (Figure 2), because previous work has suggested that phenanthridines may retain biological activity [25].The series contains derivatives differing in the presence and position of hydroxy and methoxy groups, 2,3-or 3,4-methylenedioxy bridges and 7-benzyloxy groups, but all compounds contain an 8-methoxy group.Some derivatives have been prepared either as free bases or protonated salts.In some compounds, the heterocyclic nitrogen is available in a quaternary form with methyl substitution.Thus, such variability allowed us to build a preliminary structure-activity relationship for antibacterial and antiproliferative activity, and to compare the phenanthridines with known benzo[c]phenanthridines (Figure 1).Desired phenanthridines for biological evaluation (for details see Table 1).
For the preparation of novel phenanthridine derivatives we adapted methodologies from benzo[c]phenanthridine chemistry.Many synthetic approaches to the benzo[c]phenanthridine nucleus have been reported, with most routes involving the construction of either the B or C ring in the final or semifinal stage.These synthetic methods are summarized in reviews [31,32].Our synthesis of desired phenanthridine compounds is based on our previous experience with the synthesis of isodecarine [33] and metabolites of benzo[c]phenanthridines [34], where a radical cyclization leading to ring C closure was used to accomplish the construction of the benzo[c]phenanthridine heterocyclic system.By this method we prepared phenanthridine derivatives where the B ring is formed.
Reductive amination of aldehydes 2a-2c with selected anilines 1a-1d by NaBH(OAc)3 in toluene proceeded quantitatively and afforded the corresponding novel secondary amines 4. We have also verified an alternative two-step route leading to the same secondary amines through their isolated Schiff bases 3. The preparation of Schiff bases 3a, 3c, and 3d possessing unsubstituted hydroxyl groups proceeded smoothly providing products that readily precipitated from the reaction mixture with excellent nearly quantitative yields.Facile reduction of their iminium double bonds by NaBH4 in ethanol led to the corresponding secondary amines 4a, 4c, and 4d.Surprisingly, the condensation leading to the formation of Schiff bases with substituted hydroxyl group (R 4 = benzyl or methyl) under the same reaction conditions was not quantitative (conversions ranged from 50-75%) and subsequent isolation attempts by conventional methods were complicated.Therefore, we tried to influence the reaction equilibrium by increasing the ratio of aniline to aldehyde, prolonging the reaction time, performing the reactions in alternative solvents or by use of a microwave reactor, but unfortunately, without any satisfactory improvement.In summary, reductive amination seems to be a more suitable method for the preparation of these sorts of amines 4.

Compound Number
Type of Substitution • Newly prepared compounds (for general structures see Scheme 1).To obtain compounds possessing a phenanthridine core, amines 4 needed to be cyclized.For this purpose, several synthetic approaches including palladium-catalyzed cross-coupling cyclization [37][38][39][40][41] or base-promoted homolytic aromatic substitution [42,43] were tested with amines 4a and 4e, unfortunately, the reactions failed or yields were very low.During these experiments debromination was the main reaction observed.Finally, radical cyclization by Bu3SnH using AIBN (azobisisobutyronitrile) as a radical initiator in toluene and subsequent aromatization by activated

Compound Number
Type of Substitution • Newly prepared compounds (for general structures see Scheme 1).
For the preparation of novel phenanthridine derivatives we adapted methodologies from benzo[c]phenanthridine chemistry.Many synthetic approaches to the benzo[c]phenanthridine nucleus have been reported, with most routes involving the construction of either the B or C ring in the final or semifinal stage.These synthetic methods are summarized in reviews [31,32].Our synthesis of desired phenanthridine compounds is based on our previous experience with the synthesis of isodecarine [33] and metabolites of benzo[c]phenanthridines [34], where a radical cyclization leading to ring C closure was used to accomplish the construction of the benzo[c]phenanthridine heterocyclic system.By this method we prepared phenanthridine derivatives where the B ring is formed.
Reductive amination of aldehydes 2a-2c with selected anilines 1a-1d by NaBH(OAc) 3 in toluene proceeded quantitatively and afforded the corresponding novel secondary amines 4. We have also verified an alternative two-step route leading to the same secondary amines through their isolated Schiff bases 3. The preparation of Schiff bases 3a, 3c, and 3d possessing unsubstituted hydroxyl groups proceeded smoothly providing products that readily precipitated from the reaction mixture with excellent nearly quantitative yields.Facile reduction of their iminium double bonds by NaBH 4 in ethanol led to the corresponding secondary amines 4a, 4c, and 4d.Surprisingly, the condensation leading to the formation of Schiff bases with substituted hydroxyl group (R 4 = benzyl or methyl) under the same reaction conditions was not quantitative (conversions ranged from 50-75%) and subsequent isolation attempts by conventional methods were complicated.Therefore, we tried to influence the reaction equilibrium by increasing the ratio of aniline to aldehyde, prolonging the reaction time, performing the reactions in alternative solvents or by use of a microwave reactor, but unfortunately, without any satisfactory improvement.In summary, reductive amination seems to be a more suitable method for the preparation of these sorts of amines 4.
To obtain compounds possessing a phenanthridine core, amines 4 needed to be cyclized.For this purpose, several synthetic approaches including palladium-catalyzed cross-coupling cyclization [37][38][39][40][41] or base-promoted homolytic aromatic substitution [42,43] were tested with amines 4a and 4e, unfortunately, the reactions failed or yields were very low.During these experiments debromination was the main reaction observed.Finally, radical cyclization by Bu 3 SnH using AIBN (azobisisobutyronitrile) as a radical initiator in toluene and subsequent aromatization by activated MnO 2 [44] afforded the desired novel phenanthridine derivatives 5 in various yields.During all radical cyclization reactions we observed formation of debrominated starting amines 10 as side products in amounts of 10-20% (Scheme 2).Formation of these reduced compounds is in accordance with earlier results [33].Further side products were detected when 3,4-disbstituted (R 1 and R 2 ) amines 4 were subjected to the radical cyclization conditions.Apart from reduced amines 10, cyclized regioisomers 11 were also observed as minor products.In the case of substituted amines 4, where R 4 is benzyl or methyl the amount of 11 was around 5%, whereas the ratio of these regioisomers was much higher (up to 30%, based on HPLC analysis) for the unsubstituted hydroxylamines 4a-4d (Scheme 2).
Molecules 2018, 23, x FOR PEER REVIEW 5 of 24 with earlier results [33].Further side products were detected when 3,4-disbstituted (R 1 and R 2 ) amines 4 were subjected to the radical cyclization conditions.Apart from reduced amines 10, cyclized regioisomers 11 were also observed as minor products.In the case of substituted amines 4, where R 4 is benzyl or methyl the amount of 11 was around 5%, whereas the ratio of these regioisomers was much higher (up to 30%, based on HPLC analysis) for the unsubstituted hydroxylamines 4a-4d (Scheme 2).Crystallization was utilized here as a useful method for the separation of most phenanthridines 5 from their reaction by-products.Unfortunately, a problem concerning the isolation of pure compounds was observed with hydroxyphenanthridines 5a-5d.The byproducts formed had similar properties to the targeted molecules, with only silica gel column chromatography providing pure compound 5d, but again with difficulties.For these reasons the remaining hydroxyphenanthridines 5a-5c were obtained by catalytic hydrogenation of the corresponding benzyl derivatives 5i-5k.This benzyl group deprotection of proceeded under atmospheric pressure of hydrogen on 10% Pd/C.Under these conditions reduction of the iminium double bond can occur, so subsequent stirring of the reaction mixture in air or by addition of MnO2 was necessary for rearomatization.Alternatively, to avoid the need for this re-oxidation step benzyl group removal could also be achieved by acid hydrolysis with HCl followed by treatment with aqueous NH3 to obtain the free bases (Scheme 3).
Phenanthridine derivatives 5 were converted to their hydrochlorides 6 by addition of HCl into dioxane solutions of the free bases (Scheme 1).Surprisingly these compounds have very low solubility in water, and even in other polar solvents (DMSO, EtOH, DMF) which hindered biological testing.
N-methylation of phenanthridines 5 was carried out with methyl iodide in acetonitrile under mild conditions to give N-methylphenanthridinium iodides 7.These conditions enabled the selective methylation, even of the phenanthridines possessing a free hydroxyl group.Similarly to the hydrochlorides 6, the prepared quaternary iodides 7 were also surprisingly very poorly soluble in water.To verify the possibility if the anion exchange can influence the solubility of these compounds, selected quaternary iodides were transformed in a NaOH/EtOH solution into their unisolated colorless pseudobases 8. Different salts (chloride, hydrogensulfate, perchlorate, and nitrate) were obtained by addition of large excess of corresponding acids to provide the anion modified Nmethylphenanthridine precipitates.It was found that anion exchange did not improve solubility of these mentioned compounds significantly.For illustration, we demonstrate experimentally the preparation of various salts of compound 9, which were confirmed by elemental analysis.Crystallization was utilized here as a useful method for the separation of most phenanthridines 5 from their reaction by-products.Unfortunately, a problem concerning the isolation of pure compounds was observed with hydroxyphenanthridines 5a-5d.The byproducts formed had similar properties to the targeted molecules, with only silica gel column chromatography providing pure compound 5d, but again with difficulties.For these reasons the remaining hydroxyphenanthridines 5a-5c were obtained by catalytic hydrogenation of the corresponding benzyl derivatives 5i-5k.This benzyl group deprotection proceeded under atmospheric pressure of hydrogen on 10% Pd/C.Under these conditions reduction of the iminium double bond can occur, so subsequent stirring of the reaction mixture in air or by addition of MnO 2 was necessary for rearomatization.Alternatively, to avoid the need for this re-oxidation step benzyl group removal could also be achieved by acid hydrolysis with HCl followed by treatment with aqueous NH 3 to obtain the free bases (Scheme 3).
Phenanthridine derivatives 5 were converted to their hydrochlorides 6 by addition of HCl into dioxane solutions of the free bases (Scheme 1).Surprisingly these compounds have very low solubility in water, and even in other polar solvents (DMSO, EtOH, DMF) which hindered biological testing.
N-methylation of phenanthridines 5 was carried out with methyl iodide in acetonitrile under mild conditions to give N-methylphenanthridinium iodides 7.These conditions enabled the selective methylation, even of the phenanthridines possessing a free hydroxyl group.Similarly to the hydrochlorides 6, the prepared quaternary iodides 7 were also surprisingly very poorly soluble in water.To verify the possibility if the anion exchange can influence the solubility of these compounds, selected quaternary iodides were transformed in a NaOH/EtOH solution into their unisolated colorless pseudobases 8. Different salts (chloride, hydrogensulfate, perchlorate, and nitrate) were obtained by addition of large excess of corresponding acids to provide the anion modified N-methylphenanthridine precipitates.It was found that anion exchange did not improve solubility of these mentioned compounds significantly.For illustration, we demonstrate experimentally the preparation of various salts of compound 9, which were confirmed by elemental analysis.

Antibacterial Activity
Prepared derivatives were screened for antibacterial activity against representative Grampositive bacteria (Bacillus.subtilis, ATCC 6633; Micrococcus luteus, ATCC 10240; Mycobacterium vaccae, DSM 43514; Staphylococcus aureus, CCM 2524) and Gram-negative bacteria (Pseudomonas aeruginosa, CCM 3955; Escherichia coli, CCM 3954) using agar diffusion assays [45].Derivatives with zones of inhibition ≥20 mm were subjected to a further assay [46] that determines their minimum inhibitory concentrations (MIC).From the measured data (Table 2) it is possible to make a few general conclusions regarding their structure-activity relationships: (1) Antibacterial activity was observed only for derivatives containing a phenanthridine skeleton.
No tested strain was susceptible to representative intermediates 3 and 4.
(2) Derivatives with benzyl substituent as R 4 (5k, 5l, 7i, 7j, 7k, and 7l) showed high antibacterial activity against B. subtilis, M. luteus and/or M. vaccae with MIC in single digit micromolar values.(3) Compounds with a charged nitrogen bearing methyl group (7i, 7j, 7k and 7l) demonstrated high activity as well.To summarize, the most active compounds have a similar structural motif-a phenanthridine skeleton with a charged N-methyl nitrogen and a benzyl group as a R 4 substituent.
These points are noteworthy for potential structural exploitation in further antibacterial agent development.To compare the antibacterial activity results for well-known natural (chelerythrine, sanguinarine, isodecarine, norchelerythrine) or synthetic compounds (NK-109, 13, 14) with similar structure motifs are presented in Table 2 as well.From the measured results it is evident that some prepared derivatives provided similar or even better activity than these compounds (MIC in micromolar or submicromolar values).All newly reported compounds were less active than a previously developed analogue 14, with 7j performing better than that reference compound only against M. luteus.No compound displayed any relevant activity against Gram-negative bacteria.If the structures are compared the importance of the presence of a charged N-methyl nitrogen in the molecule is confirmed.

Antibacterial Activity
Prepared derivatives were screened for antibacterial activity against representative Gram-positive bacteria (Bacillus.subtilis, ATCC 6633; Micrococcus luteus, ATCC 10240; Mycobacterium vaccae, DSM 43514; Staphylococcus aureus, CCM 2524) and Gram-negative bacteria (Pseudomonas aeruginosa, CCM 3955; Escherichia coli, CCM 3954) using agar diffusion assays [45].Derivatives with zones of inhibition ≥20 mm were subjected to a further assay [46] that determines their minimum inhibitory concentrations (MIC).From the measured data (Table 2) it is possible to make a few general conclusions regarding their structure-activity relationships: (1) Antibacterial activity was observed only for derivatives containing a phenanthridine skeleton.
No tested strain was susceptible to representative intermediates 3 and 4.
(2) Derivatives with benzyl substituent as R 4 (5k, 5l, 7i, 7j, 7k, and 7l) showed high antibacterial activity against B. subtilis, M. luteus and/or M. vaccae with MIC in single digit micromolar values.(3) Compounds with a charged nitrogen bearing methyl group (7i, 7j, 7k and 7l) demonstrated high activity as well.To summarize, the most active compounds have a similar structural motif-a phenanthridine skeleton with a charged N-methyl nitrogen and a benzyl group as a R 4 substituent.
These points are noteworthy for potential structural exploitation in further antibacterial agent development.To compare the antibacterial activity results for well-known natural (chelerythrine, sanguinarine, isodecarine, norchelerythrine) or synthetic compounds (NK-109, 13, 14) with similar structure motifs are presented in Table 2 as well.From the measured results it is evident that some prepared derivatives provided similar or even better activity than these compounds (MIC in micromolar or submicromolar values).All newly reported compounds were less active than a previously developed analogue 14, with 7j performing better than that reference compound only against M. luteus.No compound displayed any relevant activity against Gram-negative bacteria.If the structures are compared the importance of the presence of a charged N-methyl nitrogen in the molecule is confirmed.

Anticancer Activity In Vitro
Benzo[c]phenanthridine alkaloids are known to display antiproliferative and anticancer activities [8,18].Due to their structural analogy, the preliminary in vitro anticancer activity of the newly prepared compounds was evaluated on two established cancer cell lines, MCF-7 (breast carcinoma) and K-562 (chronic myelogeneous leukemia).The resulting data are presented in Table 3 and show that several of the new compounds have significant activity in both cell lines, with single digit micromolar EC 50 values.Derivative 7j reached submicromolar values in K-562 cells and was even more potent than chelerythrine and sanguinarine.The most active compounds bear 7-benzyloxy substitution and either 2,3-dimethoxy or an isosteric 2,3-methylenedioxy bridge and are either N5-methylated (7j, 7k) or contain an unsubstituted N5 (5k).Compounds without a benzyl moiety or with 3,4-dimethoxy/3,4-methylenedioxy substitution were less potent.Interestingly, removal of the methyl from N5 of 7j, that also uncharged the nitrogen, decreased potency (5j).In contrast, presence of the quaternary nitrogen (methylated) significantly reduced the activity of several compounds (compare especially pairs 5f and 7f, 5g and 7g, 5h or 7h).According to previous studies, the activity of benzo[c]phenanthridine alkaloids can be explained by the presence of a cationic quaternary nitrogen [12,15].These conclusions however probably cannot be applied to phenanthridines, because many of these compounds with a quaternary nitrogen have poor activity and one of the most potent compounds (5k) does not contain a quaternary nitrogen at all.
With the aim of directly comparing the activity of phenanthridines with the corresponding benzo[c]phenanthridines, we prepared also four variously substituted benzo[c]phenanthridines-13 [16,33], 14 [33], isodecarine [33] and norchelerythrine (Figure 1).In line with the abovementioned findings for phenanthridines, the presence of the benzyloxy functionality at position 7 correlated with in vitro anticancer activity for compounds 13 and 14.However, previous studies indicated that the activity of benzo[c]phenanthridine alkaloids can be explained by the presence of a cationic quaternary nitrogen atom [12].We also observed lack of cellular activity with isodecarine and norchelerythrine, which were prepared as demethylated derivatives of the very potent NK-109 and chelerythrine, respectively.Compound 14, killing both cancer cell lines with EC 50 values around 1 µM, contains not only a benzyloxy functionality, but also a quaternary nitrogen that could be essential for its activity.We cannot rule out the possibility that this cation is even more important for the activity.

Mechanism of Cellular Activity
Anticancer activity of benzo[c]phenanthridine alkaloids relates at least partly to their ability to inhibit topoisomerase I or II.For example, nitidine and fagaronine inhibit the topoisomerase I-mediated DNA relaxation [23].
Their synthetic derivatives NK109 and NK314 exerted their cytotoxic activity through inhibition of topoisomerase II, followed by DNA breaks [19,22].DNA damage usually results in a cell cycle arrest and eventually leads to apoptosis.We therefore treated MCF-7 cells with several compounds and NK109 as a control.Interestingly, these compounds influenced the cell cycle profile in different manners (Figure 3).The most potent 7k reduced S and G2/M phases, 7g reduced S phase and caused slight accumulation in G2/M, whereas the only weakly cytotoxic 6g resulted in strong G2/M arrest.NK109 used as a control also arrested cells in G2/M.These differences suggest that the compounds may have different molecular targets.
DNA damage induces various responses, including stabilization and activation of tumor suppressor protein p53 [47].In the following experiments, we analyzed levels and activities of tumor suppressor protein p53, which is typically activated in cells upon topoisomerase inhibition.We performed immunoblotting of proteins extracted from MCF-7 cells treated for 24 h with compounds 6g, 7k and 7g.Levels of p53 and its typical downstream target p21 waf1 were both increased (Figure 4).In addition, fragmentation of PARP producing a 89 kDa band was observed in cells treated with 7k and NK109, indicating ongoing apoptosis.This fragmentation was dose-dependent for compound 7k.In contrast, neither 6g nor 7g triggered PARP fragmentation, which is in line with their weaker cytotoxicities.the activity.

Mechanism of Cellular Activity
Anticancer activity of benzo[c]phenanthridine alkaloids relates at least partly to their ability to inhibit topoisomerase I or II.For example, nitidine and fagaronine inhibit the topoisomerase Imediated DNA relaxation [23].Their synthetic derivatives NK109 and NK314 exerted their cytotoxic activity through inhibition of topoisomerase II, followed by DNA breaks [19,22].DNA damage usually results in a cell cycle arrest and eventually leads to apoptosis.We therefore treated MCF-7 cells with several compounds and NK109 as a control.Interestingly, these compounds influenced the cell cycle profile in different manners (Figure 3).The most potent 7k reduced S and G2/M phases, 7g reduced S phase and caused slight accumulation in G2/M, whereas the only weakly cytotoxic 6g resulted in strong G2/M arrest.NK109 used as a control also arrested cells in G2/M.These differences suggest that the compounds may have different molecular targets.
DNA damage induces various responses, including stabilization and activation of tumor suppressor protein p53 [47].In the following experiments, we analyzed levels and activities of tumor suppressor protein p53, which is typically activated in cells upon topoisomerase inhibition.We performed immunoblotting of proteins extracted from MCF-7 cells treated for 24 h with compounds 6g, 7k and 7g.Levels of p53 and its typical downstream target p21 waf1 were both increased (Figure 4).In addition, fragmentation of PARP producing a 89 kDa band was observed in cells treated with 7k and NK109, indicating ongoing apoptosis.This fragmentation was dose-dependent for compound

General Information
All commercially available reagents were used without further purification and purchased from standard chemical suppliers.Reactions were monitored by LC/MS analyses on a UHPLC-MS system (Thermo Scientific, Waltham, MA, USA) consisting of a UHPLC chromatograph equipped with a photodiode array detector and a triple quadrupole mass spectrometer using a C18 column at 30 °C and flow rate of 800 µL/min. 1 H and 13 C-NMR spectra were measured on an ECA 400II ( 1 H: 399.78 MHz, 13 C: 100.53 MHz) NMR spectrometer (JEOL Resonance, Tokyo, Japan).Samples were dissolved and subsequently measured in DMSO-d6 or CDCl3.Chemical shifts (δ) are reported in ppm and referenced to the middle of the solvent signal (DMSO-d6: 2.50 ppm, 39.51 ppm; CDCl3: 7.27 ppm, 77.00 ppm.Data are reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), doublet of

General Information
All commercially available reagents were used without further purification and purchased from standard chemical suppliers.Reactions were monitored by LC/MS analyses on a UHPLC-MS system (Thermo Scientific, Waltham, MA, USA) consisting of a UHPLC chromatograph equipped with a photodiode array detector and a triple quadrupole mass spectrometer using a C18 column at 30 • C and flow rate of 800 µL/min. 1 H and 13 C-NMR spectra were measured on an ECA 400II ( 1 H: 399.78 MHz, 13 C: 100.53 MHz) NMR spectrometer (JEOL Resonance, Tokyo, Japan).Samples were dissolved and subsequently measured in DMSO-d 6 or CDCl 3 .Chemical shifts (δ) are reported in ppm and referenced to the middle of the solvent signal (DMSO-d 6 : 2.50 ppm, 39.51 ppm; CDCl 3 : 7.27 ppm, 77.00 ppm.Data are reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartet (q), multiplet (m), broad resonance (br)], coupling constants [Hz], integration).All the NMR spectra were acquired at ambient temperature.All recorded 1 H and 13 C-NMR spectra are available as Supplementary material.High resolution mass spectra (HRMS) measurements were performed on an Orbitrap mass analyzer (Thermo Scientific, Waltham, MA, USA) equipped with Heated Electrospray Ionization (HESI).The spectrometer was tuned to obtain a maximum response for m/z 70-700.Elemental analyses were performed on an EA 1108 Elemental Analyser (Fisons Instruments, Thermo Scientific, Waltham, MA, USA).Thin layer chromatography (TLC) were performed on pre-coated silica gel 60 F254 plates (Merck, Prague, Czech Republic) and visualized by exposure to UV light (254 or 366 nm).Melting points were measured on a Boetius stage apparatus (WEB Analytik, Dresden, Germany) and are uncorrected.

Agar Diffusion Test
Overnight cultures of test organisms were grown in LB broth for 18-24 h and standard suspensions of 1.5 × 10 8 CFU/mL were prepared in a sterile saline solution (0.9% NaCl) according to a BaSO 4 0.5 McFarland Standard.The standardized suspension (0.1 mL) was added to 34 mL of sterile, melted and tempered (47-50 • C) Mueller-Hinton No. 2 agar.After gentle mixing, the inoculated melted agar was poured into a sterile Petri dish (145 mm × 20 mm, Greiner Bio-One, Kremsmünster, Austria) and allowed to solidify next to the flame with lids slightly ajar.Wells of 9 mm diameter were cut from the Petri dish agar and filled with 50 µL of the test sample solution.Solutions were made at 20 mM in DMSO and diluted in MeOH to 2 mM.The Petri dish was incubated at 37 • C for 18-24 h and the inhibition zone diameters were measured (mm) with an electronic caliper after 24-48 h.

MIC Determination
Antibacterial activity of the compounds was determined by measuring their minimum inhibitory concentrations (MIC) using the broth microdilution method.Each well of a 96-well microtiter plate was filled with 50 µL of sterile iron deficient MH2 broth.Each test compound was dissolved in DMSO making a 10 mM solution, then diluted with sterile MH2 broth to 800 µM.Exactly 50 µL of the compound solution was added to the first well of the microtiter plate and 2-fold serial dilutions were made down each row of the plate.A pre culture of bacteria was grown in Luria-Bertani broth overnight at 37 • C.This was diluted to McFarland standard 0.5 (1.5 × 10 8 CFU) with saline.100 µL of the bacterial suspension was further diluted with 14.9 mL of MHII broth.Exactly 50 µL of bacterial (in broth) inoculum (1 × 10 6 CFU/mL) was then added to each well giving a total volume of 100 µL/well and 5 × 10 5 CFU/mL and a compound concentration gradient of 200-0.1 µM.The plate was incubated at 37 • C.After 18 h, each well was examined visually for bacterial growth.The MIC was recorded as the lowest compound concentration required to inhibit bacterial growth as judged by turbidity relative to a row of wells diluted with a the solvent DMSO as a growth control.Ciprofloxacin was included in a control row at a concentration gradient of 5 µg/mL-0.0025µg/L.

Anticancer Activity In Vitro
The in vitro anticancer activity was determined using MCF-7 (breast adenocarcinoma) and K-562 (chronic myelogeneous leukemia) cell lines as described earlier [48].Briefly, cells were treated in triplicate with three different doses of each compound for 72 h.After treatments, Calcein AM solution was added, and fluoresence from live cells was measured at 485 nm/538 nm (excitation/emission) using a Fluoroskan Ascent microplate reader (Thermo Scientific, Waltham, MA, USA).The EC 50 value, that is, the drug concentration reducing number of live cells to 50%, was calculated from the dose response curves that resulted from the assays.The cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) and cultivated at 37 • C in 5% CO 2 .

Cell Cycle Analysis
Cell cycle analysis was performed as described earlier [48].Briefly, subconfluent cells were treated with different concentrations of test compound for 24 h.The cultures were pulse-labeled with 10 µM 5-bromo-2 -deoxyuridine (BrdU) for 30 min at 37 • C prior to harvesting.The cells were then washed in PBS, fixed with 70% ethanol, and denatured in 2 M HCl.Following neutralization, the cells were stained with anti-BrdU fluorescein-labeled antibodies, washed, stained with propidium iodide, and analyzed by flow cytometry using a 488 nm laser (FACSVerse, Becton Dickinson, Franklin Lakes, NJ, USA).

Immunoblotting
Immunoblotting was performed as described earlier [48].Briefly, cellular lysates were prepared by harvesting cells in Laemmli sample buffer.Proteins were separated on SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes.After blocking, the membranes were incubated with specific primary antibodies overnight, washed and then incubated with peroxidase-conjugated secondary antibodies.Finally, peroxidase activity was detected with ECL+ reagents (AP Biotech, Prague, Czech Republic) using a LAS-4000 CCD camera (AP Biotech, Prague, Czech Republic).Specific antibodies against PARP were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) peroxidase-labeled secondary antibodies was from Sigma Aldrich (Prague, Czech Republic, peroxidase-labeled secondary antibodies) or were generously gifted by Dr. B. Vojtěšek (p53, p21WAF1, PCNA).

Conclusions
In this work we extended a previously developed method for the preparation of novel phenanthridine derivatives, which were derived from benzo[c]phenanthridines by deletion of the A-ring.Derivatives were prepared with substituents on the formed skeleton that mimic biologically active benzo[c]phenanthridines.The main principle for the preparation of the aforementioned compounds was based on the radical cyclization of reduced Schiff bases prepared by the condensation of appropriate aromatic aldehydes and amines.The prepared compounds were tested for antibacterial and anticancer cytotoxic effects and compared with several compounds containing a benzo[c]phenanthridine skeleton (e.g., chelerythrine, sanguinarine, isodecarine, norchelerythrine, NK-109).Several derivatives displayed antibacterial activity against Bacillus subtilis, Micrococcus luteus and/or Mycobacterium vaccae and cytotoxicity against K-562 and MCF-7 cancer cell lines at micromolar concentrations; these compounds typically contained a N-methylated quaternary nitrogen and

Molecules 2018 ,
23,  x FOR PEER REVIEW 3 of 24 structure-activity relationship for antibacterial and antiproliferative activity, and to compare the phenanthridines with known benzo[c]phenanthridines (Figure1).

Figure 3 .
Figure 3. Cell cycle effects of compounds 7k, 7g, 6g and NK-109 in MCF-7 cells treated for 24 h with doses corresponding to 3 × EC50 values (60, 5.4, 33 and 13.8 µM, respectively).Cells were harvested and then flow cytometric analysis of DNA stained by propidium iodide (10,000 counts) was performed as described in the Materials and Methods section.

Figure 3 . 24 7k.
Figure 3. Cell cycle effects of compounds 7k, 7g, 6g and NK-109 in MCF-7 cells treated for 24 h with doses corresponding to 3 × EC 50 values (60, 5.4, 33 and 13.8 µM, respectively).Cells were harvested and then flow cytometric analysis of DNA stained by propidium iodide (10,000 counts) was performed as described in the Materials and Methods section.Molecules 2018, 23, x FOR PEER REVIEW 10 of 24

Figure 4 .
Figure 4. Dose-dependent effect of compounds 7k, 7g, 6g and NK-109 on p53 and its activity in MCF-7 cells.The cells were treated for 24 h with indicated doses of compounds (given in µM) and then specific proteins were analyzed by immunoblotting as described in the Materials and Methods section.C.f., 89 kDa cleavage fragment of PARP.

Figure 4 .
Figure 4. Dose-dependent effect of compounds 7k, 7g, 6g and NK-109 on p53 and its activity in MCF-7 cells.The cells were treated for 24 h with indicated doses of compounds (given in µM) and then specific proteins were analyzed by immunoblotting as described in the Materials and Methods section.C.f., 89 kDa cleavage fragment of PARP.

Diameter of Inhibition Zone in Agar Diffusion Assay (mm) (MIC) (µmol L −1 )
* used as a standard.

Table 3 .
Anticancer activity in vitro.