Alkylamino and aralkylamino derivatives of avarone and its mimetic as selective agents against non-small cell lung cancer cells , their antibacterial and antifungal potential

In this paper, the synthesis of fourteen alkylamino and arylamino derivatives of sesquiterpene quinone avarone and its model compound tert-butylquinone is described. Branched, cyclic, allylic and benzylic alkylamino/arylamino groups were introduced into the quinone moiety. For all the obtained derivatives, their biological activity and redox properties were studied. The cytotoxic activity of the synthesized derivatives towards multidrug resistant (MDR) human non-small cell lung carcinoma NCI-H460/R cells, their sensitive counterpart NCI-H460 and human normal keratinocytes (HaCaT) was investigated. The antimicrobial activity towards Gram-positive and Gram-negative bacteria, and fungal cultures was determined. Some of the synthesized derivatives showed selectivity for cancer cells, including MDR cells. Regarding their cell death induction potential, the most promising compounds were allylamino derivatives, preferentially triggering apoptosis, with high selectivity for cancer cells, including MDR cells. Several compounds showed promising antimicrobial activity, comparable to those of commercial antibiotic and antimycotic agents.


INTRODUCTION
Cancer is one of the leading causes of death worldwide, with over 8 million deaths in 2015. 1 Therefore, a great amount of resources and effort have been

Chemistry
As a continuation of previous work, fourteen aminoquinones 3-10a and 3-10b were synthesized (preparation of compounds 6a and 6b was presented in a previous paper 20 ).Treatment of the parent quinones tert-butylquinone (1) and avarone (2) with various amines produced the corresponding two regioisomers of aminoquinones via 1,4-Michael addition reactions (Scheme 1).Scheme 1. Synthesis of the quinone derivatives.
As expected, with tert-butylquinone, the 2,6-disubstituted quinone products were dominant over the 2,5-disubstituted products.On the other hand, with avarone, only in the reaction with pyrrolidine was the 2,6-disubstituted product dominant while in the reaction with allylamine, the 2,5-disubstituted product was the main product and with the other two nucleophiles, similar amounts of two products were obtained.Racemic sec-butylamine was used in the reactions.The NMR spectra of the products obtained in its reaction with avarone indicate the presence of only one diastereoisomer.Unfortunately, the configuration of the asymmetric carbon could not be determined, since all attempts to crystallize the products were unsuccessful.
Half-wave potentials were recorded at a glassy carbon disk (3 mm diameter) in DMSO towards a silver wire immersed in the electrolyte solution containing 0.01 M silver ions as the reference electrode, and ferrocene as the reference compound.The results are given in Table I and   Typical quinone electrochemical behaviour was observed for all the synthesized compounds, i.e., two waves that could be attributed to the reduction of quinone to the semiquinone radical and, subsequently, to the hydroquinone dianion (Fig. S-1).First reduction wave was fully reversible, indicating a diffusion controlled process.However, second reduction process was not reversible, having higher peak separation potentials.The derivatization did not perturb the nature of these two redox processes, but rather shifted the peak potentials.As expected, all the derivatives had more negative reduction potential than the parent quinone.It could be assumed that amino substituents with electron-donating ability destabilize the intermediate semiquinone relative to the corresponding quinone.This effect leads to a negative shift in the peak potential.Since the inductive effect of the alkyl part of the alkylamino substituent influences the electron-donating ability of the substituent, the pyrrolidino derivatives have the most negative reduction potentials, while benzylamino and allylamino derivatives have the least negative one.As previously described, 20 the position of the substituent plays a particular role in shifting the peak potential.It was observed that the 4′-derivatives generally had a peak potential more negative by about 10 to 20 mV than the corresponding 3′-derivatives.The probable reason is that the arrangement is more favourable when the electron-donating amino and alkyl substituents in semiquinone anion radical are both in the meta position to the negative oxygen.

Anticancer activity
Derivatives of TBQ were compared with corresponding avarone derivatives regarding their growth inhibition activity against human cancer and normal cell lines.The cell growth inhibition activity was studied in non-small cell lung carcinoma cells -NCI-H460/R, their multidrug resistant (MDR) counterpart -NCI--H460/R and human keratinocytes -HaCaT cells.The differences in response between cancer and normal cells, evaluated by the MTT assay after 72 h treatment, are presented in Table II and   The growth inhibition abilities of parent compounds TBQ and avarone, as well as CDDP, an FDA-approved drug for non-small cell lung carcinoma treatment, 22 were investigated in our previous study. 20CDDP showed stronger effect in MDR cancer cells in comparison with their sensitive counterparts, but it was not selective against cancer cells due to the pronounced activity obtained in normal human keratinocytes.TBQ was largely ineffective in all cell lines while avarone showed high selectivity towards MDR cells.The derivatives had generally a higher activity than the parent compounds.Avarone 2,6-disubstituted sec-butylamino and allylamino derivatives (7a and 8a, respectively) exhibited the highest activity in all tested cell lines with IC 50 values below 5 µM and, importantly, their activity was not affected by the presence of MDR phenotype.Corresponding 2,5-disubstituted derivatives 7b and 8b showed the same pattern of activity (without selectivity against cancer cells) but their efficacy was significantly diminished as their IC 50 values were around one order of magnitude higher than those obtained by 7a and 8a.Similarly, 2,6-disubstituted allylamino derivative 4a with IC 50 values near 10 µM was more efficient than 4b but without selectivity to cancer cells.Benzylamino avarone derivative 10b was the least active compound in all cell lines, while the IC 50 values for corresponding 10a and two pyrrolidino tert-butylquinone derivatives 5a and 5b were above 25 µM in cancer cells.These four derivatives were more active in normal cells.Compounds 3a and 9b exhibited both high cytotoxicity and selectivity towards cancer cell lines and most notably MDR phenotype did not reduce their activity.Although direct correlation between redox properties and cytotoxicity of derivatives with different substituents could not be established, it should be noticed that out of two regioisomers, the isomer with a less negative cathodic potential of the first wave, i.e., the isomer that is more prone to the formation of semiquinone radicals, is more active.
In the tert-butylquinone series, the 6-derivatives were always more active to tumour cells than the 5-derivatives.The introduction of the bulky sec-butylamino group into the 6-position, i.e., closer to the tert-butyl group, did not have much influence on the activity to tumour cells, but it decreased the toxicity to normal cells, and hence, contrary to the 6-(butylamino) derivative, the corresponding sec-butylamino substituted quinone showed a pronounced selectivity to tumour cell lines.This did not apply for a more remote 5 substituent.The introduction of allylamino group led to products with higher cytotoxicity than similar derivatives with linear saturated alkylamino substituents, but the selectivity for tumour cells displayed by 6-(ethylamino) derivative was lost. 205-(Allylamino) and 5-(benzylamino) products were the only 5-substituted derivatives that showed a relatively high cytotoxicity, but only the benzylamino derivative showed selectivity for tumour cell lines.The pyrrolidino derivatives were only moderately active, without selectivity for tumour cells.If the whole set of compounds is considered, for a strong cytotoxic activity against tumour cells, an unsaturated or bulky substituent should be present in position 6.For achieving selectivity, the set of sub-stituents was narrowed to bulky groups, and even more to aralkyl groups (benzylamino, phenethylamino) if selectivity to MDR cells is considered.
As for avarone derivatives, the 3′-derivatives (2,6-disubstitution) were always much more active than the 4′-derivatives (2,5-disubstitution), with the exception of the pyrrolidino derivatives when the inverse was true.Within this series, the introduction of branching or unsaturation into alkylamino substituent increased the cytotoxicity regardless of the position of the substituent.3′-(sec--Butylamino) and 3′-(allylamino) derivatives showed IC 50 values in the range 3-4 μM.Unfortunately, the selectivity for MDR cells, shown by 3′-(butylamino)avarone, 20 was lost.The benzylamino derivatives were less active to tumour cells than the phenethylamino ones.The 4′-pyrrolidino derivative (the more active isomer) showed strong activity to the tumour cell lines, with an IC 50 value of 7.1 μM against the multi-drug resistant cells, and selectivity for tumour cells.The 3′-derivative showed moderate activity, without selectivity.Summarizing these results, the necessary prerequisite for an avarone derivative to have a strong activity against tumour cells is a substituent with a relatively low number of carbon atoms at the position 3′ or pyrrolidine at position 4′.Of these substituents, only unbranched saturated butylamino derivative and heterocyclic pyrrolidino derivative showed the desired selectivity.
Apoptosis and necrosis are the two main forms of cell death.Apoptosis is regarded as a programmed process, with minimal level of ATP required for the assembly of a apoptosome complex and activation of caspases, while necrosis is often referred to as the complete decay of cell metabolism. 23They can be distinguished from one another by various morphological and biochemical characteristics, although there is no clear-cut distinction between these two (hence, other forms of cell death, i.e., necroptosis, aponecrosis, etc.). 24The most obvious characteristics of apoptosis are cell shrinkage, DNA fragmentation, condensation of chromatin, formation of apoptotic bodies, disruption of mitochondrial redox processes and generation of ATP.Cells undergoing apoptosis do not release their constituents into the surrounding tissue because the integrity of the cell membrane is preserved.In addition, macrophages quickly phagocytose apoptotic bodies and the surrounding cells do not produce anti-inflammatory cytokines. 25herefore, no inflammatory reaction occurs during the apoptotic process.Necrosis, on the other hand, is usually followed by inflammation, because one of the main characteristics of necrosis is the loss of membrane integrity and the release of the cytoplasmic content into the surrounding area, leading to inflammation. 26nflammation, for its part, can damage cells and even cause cancer. 27Bearing this in mind, apoptosis is clearly the preferred type of cell death, considering the removal of either healthy, aging or tumour cells.Besides inflammation, the other characteristics of necrosis are cell swelling and complete abolition of ATP pro-duction.However, many types of cancer employ anti-apoptotic mechanisms for their survival, so necrosis could be useful in promoting the death of tumour cells.
The cell death inducing capacity of 25 µM TBQ and the avarone derivatives was analyzed after 72 h treatment (Table III, Fig. S-3 of the Supplementary material).Both pyrrolidino tert-butylquinone derivatives (5a and 5b) showed significant activity towards sensitive cancer cells, triggering both apoptosis and necrosis, which was not observed in normal cells.The allylamino derivatives 4a and 4b induced apoptotic type of cell death more prominently in both cancer cell lines than in HaCaT cells.The sec-butylamino derivatives 3a and 3b also induced apoptosis, most effectively in NCI-H460 cells.Cells treated with the avarone pyrrolidino derivatives 9a and 9b underwent necrosis as the predominant cell death type.Consistent with the cell growth analysis, the inverted efficacy pattern of 9a and 9b as well as the selectivity of 9b towards cancer cells was confirmed by cell death induction.Compounds 8a and 8b (allylamino derivatives of avarone) also predominantly induced necrosis in cancer cells with 8a being the significantly more potent derivative.sec-Butylamino derivative 7a was considerably more active against MDR cancer cells in triggering necrotic cell death compared to 7b.A similar but reduced effect was also observed in NCI-H460 and HaCaT cells.Importantly, according to the cell death analysis, 7a and 8a were shown to be selective against cancer cells.The cell death-inducing activity of compounds 4a and 4b significantly stand out from the others in the series.The majority of the cells treated with 4a or 4b underwent apoptotic cell death, targeting late apoptosis preferentially.Most importantly, this pair were very selective toward cancer cell lines, both sensitive and resistant ones, particularly the 4a derivative with over 60 % of viable cells in healthy HaCaT cell lines.This distinction could be attributed to the allyl moiety.The probable reason is the additional generation of ROS via allyl radicals, formed by the decomposition of the semiquinone radical generated by the one-electron reduction of allylamino quinone (the other product would be amino quinone) and/or the formation of cytotoxic acrolein by the oxidative metabolism of allylamino quinones. 28Considering that allylamino TBQ derivatives preferentially induce (late) apoptosis, and that cancer cells are usually already in a state of oxidative stress, 29 this seems a plausible explanation for the selective action.

Antimicrobial activity
Compounds 3-10a and 3-10b were tested for their antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as fungal strains, and compared to the commercial antibiotics amikacin and antimycotic nystatin.The results are given in Tables IV and V.
Most of the derivatives showed weak activity in comparison to amikacin, having at least an order of a magnitude higher MIC.The most active TBQ derivatives were allylamino derivatives 4a and 4b, with activity comparable to amikacin for E. coli.
This pair had almost identical MIC for all strains except M. luteus (ATCC 10240 and 4698) and S. enterica.The pair 3a and 3b showed generally weaker activity than 4a and 4b, with 3a being only slightly more active against B. subtilis, M. luteus (ATCC 10240 and 4698) and E. coli, and 3b for K. rhizophila.The pair 5a and 5b displayed the weakest activity of all the tested TBQ compounds, with some activity only towards K. rhizophila, E. coli and M. luteus, ATCC 4698, (only 5a).The avarone counterparts showed no activity, as expected from previous results. 20The probable reason for inactivity is insufficient hydrophilicity.This conclusion is corroborated by the fact that several amino acid derivatives of avarone showed strong antibacterial activity. 21In general, in order to show a relatively broad activity comparable to amikacin, tert-butylquinone derivatives should have a non-branched medium length alkylamino group or an aralkyl group in position 3′.It is interesting that among the avarone amino acid derivatives, the most active were those with aromatic amino acids, implying that there is an aromatic binding site in a putative target.All the tested compounds, except 10a and 10b, showed stronger activity toward C. albicans than nystatin, with 3a, 4a, 4b and 5a even having a two orders of a magnitude lower MIC (less than 50 μM).Towards A. brasiliensis, the activities were generally comparable to that of nystatin, with 4a, 4b and 5a displaying stronger activity.Considering S. cerevisiae, 4a, 5a, 7b, 9a and 9b possess stronger activity than amikacin.Of all the tested compounds, 4a showed the strongest antifungal activity, while 10a and 10b showed no activity at all.Based on these results and those from a previous paper, 20 it could be concluded that in order to achieve a strong anti-Candida activity, a compound should be a 6-substituted 2-tert-butyl-1,4-benzoquinone (exception are allylamino derivatives, where both isomers have a similar activity), ________________________________________________________________________________________________________________________ Available on line at www.shd.org.rs/JSCS/(CC) 2018 SCS.
with at least three carbon atoms in the side chain.The avarone derivatives were much less active, but in contrast to antibacterial effects, most of them had some activity, although much lower than the amino acid derivatives. 21The amino acid derivatives of TBQ had weak antimicrobial activity, probably due to their excessive hydrophilicity.

General synthetic procedure
The parent quinones were obtained from hydroquinones according to a previously described procedure. 20Quinones (300mg; 1.83 mmol 1; 0.96 mmol 2) were dissolved in ethanol (50 mL).Amine hydrochloride salts (in large excess, 22×) were prepared as aqueous solutions.The pH of the solution was adjusted to 7-8 by the addition of solid sodium bicarbonate, and the solution was added to quinone.Water and ethanol were added to the reaction mixture to a final ratio water:ethanol of 1:1, and total volume of 300 mL.The reaction mixture was stirred at 60-70 °C for 3 h.Ethanol was removed by vacuum evaporation, and the reaction mixture was extracted two times by dichloromethane, with half the volume of the aqueous phase each time.The organic phase was separated, dried with anhydrous calcium chloride, and the solvent was removed by evaporation under vacuum.The crude products were separated by column or low-bar chromatography and purified by preparative thin-layer chromatography, with the indicated solvents.Numbering scheme for assignment of signals in NMR spectra of all compounds is given in Scheme S-1.

Biological activity
Cytotoxic, antibacterial and antifungal activity of synthesized compounds were analyzed according to a previous study. 20

CONCLUSIONS
Among the 14 newly synthesized compounds, a potential to be antitumor agents was shown for sec-butylamino derivatives of tert-butylquinone (3a and b) because of their selectivity for tumour cells, allylamino derivatives of tert-butylquinone (4a and b) because of their selective induction of apoptosis in tumour cells, including MDR cells, 3′-(sec-butylamino)avarone (7a) and 3′-(allylamino)avarone (8a) because of their higher cytotoxic activity than cisplatin, as well as 4′-pyrrolidinoavarone (9b) because of selectivity to tumour cells, including MDR cells.Some derivatives showed promising antimicrobial properties: sec-butylamino (3a) and allylamino (4a and b) derivatives of tert-butylquinone, because of an activity against E. coli similar to that of amikacin, and strong antifungal activity to C. albicans, which also applies to the pyrrolidino derivative of tert--butylquinone (5a).
Fig. S-2 of the Supplementary material.

Acknowledgement.
The authors are grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia for financial support (Grant Nos.OI172055 and III41031).

TABLE I .
Voltammetric half-peak potentials and standard redox potentials of the synthesized compounds (V vs. silver/silver chloride electrode)