Antiproliferative Activity of 2,3,6,7-Tetrahydro-1 H -benzo[ a ]quinolizino[1,9-hi ]phenoxazin-14(5 H )-iminium Chloride Derivatives

: Compounds based on 1,2,3,5,6,7-hexahydropyrido[3,2,1-ij ]quinolone, trivially designated as julolidine, have been reported due to their biological interest, namely as inhibitors of the amyloid-β protein self-assembly (which is associated to the pathogenesis of Alzheimer’s disease), as well as colorimetric and fluorometric probes. Taking this in account, in addition to the relevant biological importance of benzophenoxazinium salts such as Nile Blue derivatives, the present work evaluated the influence of the julolidine moiety in antimicrobial activity against Saccharomyces cerevisiae PYCC 4072 of newly synthesised fluorescent 2,3,6,7-tetrahydro-1 H -benzo[ a ]quinolizino[1,9-hi ]phenoxazin-14(5 H )-iminium chlorides, in comparison with their analogues.


Introduction
Polycyclic heteroaromatic compounds having the oxazine ring, namely phenoxazines, benzophenoxazines and their derivatives, with absorption and emission at longer wavelengths, presented various bioapplications as fluorescent probes. 1 These compounds also exhibited antiproliferative properties with potential applications as antitumor and antimicrobial agents, probably due to intercalation with DNA through the polycyclic planar system. [2][3][4] An earlier study of structure-activity performed by our research group demonstrated the antiproliferative activity (antifungal) of several 2 benzo[a]phenoxazines and naphtho [2,3-a]phenoxazines possessing different combinations of substituents on the amine side groups. 5,6 On the other hand, compounds based on the julolidine skeleton have been reported as inhibitors for the amyloid-β protein self-assembly, which is associated to the Alzheimer's disease, among other interesting applications in biological systems related to their interesting photophysical properties. [7][8][9][10] Currently, our group is studying the photophysical properties of new benzophenoxazines fused with julolidine, in order to understand the effect of the replacement of a freely rotating N-substituent group by a rigid structure. The synthesis of a new benzophenoxazinium chloride 3a, with the julolidine moiety and a N- (3-chloro)propyl group as a substituent at the 14-position was performed.
Furthermore, with the purpose of extending this work with julolidine-based compounds and in continuation of our research interests in the biological applications of Nile Blue derivatives, we evaluated their potential as antiproliferative agents. Saccharomyces cerevisiae was used as a model organism in the assessment of the activity of new benzophenoxazine derivatives, which was performed in comparison with other related compounds of this family.
The progress of reaction was monitored by TLC (n-hexane/ethyl acetate, 9:1). After completion of the reaction, the solvent was evaporated and the mixture was purified by column chromatography on silica gel using n-hexane and n-hexane/ethyl acetate, mixtures of increasing polarity, as the eluent.

Scheme 1. Synthesis of benzophenoxazinium chloride 3a
The 1 H NMR spectra showed the signals of aliphatic protons from the methylenic groups of substituent of positions 14, directly linked to the nitrogen atom NHCH 2 or to the chlorine atom CH 2 Cl that appeared as triplets (δ 3.78 and 3.83 ppm, respectively), as well as the group closed to the same atoms, NHCH 2 CH 2 , showed as quintet (δ 2.33 ppm). The duos H-2/H-6 and H-3/H-5 are methylenic protons of the julolidine moiety, having both protons in each pair the same electronic environment and so they both appear in one signal each, as multiplets (δ 2.05-2.14 and 3.60-3.69 ppm, respectively). There was also the presence of protons of the methylenic groups directly linked to the aromatic ring at position 1 and 7, which appeared in different signals as a multiplet (δ 2.83-2.92 ppm, H-1) and a triplet (δ 2.95 ppm, H-7).
In addition, spectra showed the expected aromatic protons of the polycyclic system, in particular H-15 (δ 6.73 ppm) and H-8 (δ 7.33 ppm), which appeared in the form of singlets.
Along with other structurally related benzophenoxazinium chlorides 3b-d, 4 and 5a Compound 3a with a chloride atom as terminal of the propyl substituent at 14-position of the system displays the best activity against S. cerevisiae. Moreover, this compound is 8-times more active comparatively to its corresponding non-functionalized analogue 3c, thus emphasising that the presence of this terminal is significant.
Regarding the effect of the presence of the julolidine structure, we can compare compound 3a with the related unrestricted dye 5b to observe the same increment in the activity observed for 3c, i.e. an 8-times increase. The result for compound 4 revealed that extending the aromatic ring system, by replacing the benzene ring with fused naphthalene in the phenoxazine skeleton leads to an improved activity of the compounds (compare to compound 3d). A well-established measure of the lipophilicity of a compound is the logarithm of its partition coefficient between n-octanol and water, i.e. log (c octanol /c water ) or Log P, being used to stimulate drug partitioning into membranes. 13 Drugs with higher values of Log P are more lipophilic, having lower affinity for an aqueous environment, like that existing in intracellular environment, but higher affinity for membranous systems, and therefore this property might influence the compound's absorption and permeation into cells. 14

Conclusions
New julolidine-based benzophenoxazinium chloride possessing the chloride atom as terminal in substituent at 14-amine position was successfully synthesised. Growth inhibition assays showed that this compound present the highest antifungal activity when compared to other derivatives of the same family, with or without the julolidine nucleus in their polycyclic system. All tested compounds exhibited activity against the yeast Saccharomyces cerevisiae. The results obtained show that the effect on the activity of the benzophenoxazine derivative is highly dependent on the substituent present at the 14position (3a-d and 4) or 5-position (5a,b). Further studies are required in order to better understand the effect of the julolidine structure in the biological activity of benzophenoxazinium chlorides.