Preliminary Safety Assessment of New Azinesulfonamide Analogs of Aripiprazole using Prokaryotic Models

 Quinoline-sulfonamides  Vibrio harveyi assay Abstract Purpose : Determination of the mutagenic potential of new biologically active compounds is of great concern for preliminary toxicity testing and drug development. Methods: The mutagenic and antimutagenic effects of some quinoline- and isoquinoline-sulfonamide analogs of aripiprazole ( 1 - 8 ), which display potent antidepressant, anxiolytic, and antipsychotic properties, were evaluated using the Vibrio harveyi assay and OSIRIS Property Explorer software. Additionally, the Ames test was used as the reference.


Introduction
5][6] Furthermore, in patients with depression the elevated plasma ROS levels were observed to effectively amplify oxidative stress. 7,8ll of these observations solidified the oxidative stress hypothesis of depression.As the removal of ROS is a vital element of antimutagenesis strategy, it was further suggested that the improvement in antimutagenic defense system may be one of the mechanisms underlying the neuroprotective effects of antidepressant drugs during depressive disorder therapies.Therefore, the development of new compounds with antidepressant properties which additionally display antimutagenic and possibly chemopreventive properties is of great practical and therapeutic significance. 9o evaluate mutagenic activity of new compounds many short-term and highly sensitive tests were introduced. 10,11 general strategy behind mutagenicity testing is to apply a diversified set of tests to cover all of the main mutagenicity endpoints.Another big challenge for mutagenicity assessment is the prospect for using alternative assays to animal testing i.e. in vitro and in silico test methods.The in vitro Ames/Salmonella test is a key tool for mutagenicity assessment and a substantial element of the official genotoxicity testing package 12 required for accomplishing the preclinical evaluation.The test is suitable for the detection of molecules that cause mutations such as frame-shifts or base-pair substitutions. 13In the last several decades, several rapid bacterial mutagenicity tests have been developed and optimized, such as the Vibrio harveyi assay. 14,15The test involves a series of genetically modified strains of a marine bacterium Vibrio harveyi.The sensitivity of this assay was found to be similar to, or even somewhat higher than, that of the commonly used the Ames test. 16o prevent genotoxic risk it is pertinent to identify potential mutagens in order to minimize human exposure to them, as well as to enhance the exposure to antimutagenic agents.8][19] Focusing on the application of alternative in vitro and in silico test methods to predict compounds mutagenicity in the present study the Vibrio harveyi assay and OSIRIS Property Explorer software were employed.Additionally, the Ames test was used as the reference.

Tested compounds and chemicals
The tested compounds were synthesized in the Department of Medicinal Chemistry, Jagiellonian University Medical College by Zajdel et al. 17,18 The compounds structures were established previously on the basis of CHN elemental analysis and spectral data ( 1 HNMR and mass spectra).Dimethylsulfoxide (DMSO) of high grade purity (99,9%) was obtained from Merck, (Darmstadt, Germany).4nitroquinoline-N-oxide (NQNO) chosen as positive control, was purchased from Sigma (Seelze, Germany).The examined compounds were dissolved in DMSO to obtain the desired final test concentrations (i.e.40 ng/ml for the Vibrio harveyi assay; 40 and 500 ng/ml for the Ames test) which were established in pilot experiments.It was demonstrated previously that in the Vibrio harveyi assay very low concentrations of mutagens could be detected. 20As regards the Ames test, two compounds concentrations were employed, namely the one used in the Vibrio harveyi assay and the higher one commonly used in the assay. 10,21,225][16] These strains were kindly provided by Prof. G. Węgrzyn (University of Gdańsk, Poland).Salmonella typhimurium tester strain TA100, as described by Maron and Ames 13 and Mortelmans and Zeiger, 10 was kindly provided by Dr. T. Nohmi (National Institute of Hygienic Sciences, Tokyo, Japan).

Culture media and growth conditions
In the Ames test the minimal medium described previously by Maron and Ames 13 was used.In case of the Vibrio harveyi assay BOSS medium was employed. 23ll strains were stored at _ 80°C in 10% glycerol (final concentration).

Mutagenicity assays
Mutagenicity assays were performed as described previously by Maron and Ames 13 and Czyż et al. 16 All experiments were carried out in triplicate and the results were expressed as mutagenic index (M.I. = the number of revertant colonies induced in the tested sample/ the number of spontaneous revertants in the negative control). 14,15,24A compound was considered mutagenic when the M.I. was equal or greater than 2.

Antimutagenicity assays
15]25 Triplicate plates were set up with each test compound concentration and the entire experiment was repeated twice.The inhibition of mutagenicity was expressed as percentage decrease of mutant colonies and calculated using the following equation: where R 1 is the number of mutants per plate induced by test compound plus mutagen and R 2 is the number of mutants per plate induced by mutagen alone. 26,27A 25-40% inhibition was defined as moderate antigenotoxicity, 40% or higher inhibition as strong antigenotoxicity, and 25% or less inhibition as no antigenotoxicity. 24

Mutagenic activity
Table 2 presents a number of mutants per plate and mutagenic index (M.I.) for selected quinoline-and isoquinoline-sulfonamide analogs of aripiprazole (1-8) evaluated in V. harveyi strains.Additionally, Table 2 was supplemented with the results of in silico mutagenicity prediction of the tested compounds.On the basis of the OSIRIS prediction results it was noted that two of the tested compounds, namely 5 and 6 trigger a mutagenic structural alert.The current in vitro study demonstrated that in a concentration of 40 ng/ml all of the tested compounds exhibited no mutagenic activity in V. harveyi BB7, BB7M, BB7X and BB7XM strains.Similarly, as shown in Table 3 the tested compounds were non mutagenic in the Ames TA100 mutagenicity assay when tested in two concentrations i.e. 40 and 500 ng/ml.

Antimutagenic activity
The antimutagenic effects of the selected quinoline-and isoquinoline-sulfonamide derivatives (1-8) were examined against NQNO in V. harveyi BB7, BB7M, BB7X, BB7XM and in S. typhimurium TA100 strains.All of the tested compounds effectively reduced a number of mutations induced by a direct acting agent NQNO in all bacterial strains used in the experiment (Tables 4 and 5).In the Vibrio harveyi antimutagenicity assay compounds 4 and 5 demonstrated the highest antimutagenic activity against NQNO induced mutagenicity.The inhibition rates for these compounds were between 40% and 84%.It is worth noting, that only compound 5 exhibited strong antimutagenic properties in all tested V. harveyi strains.Aripiprazole also strongly inhibited the mutagenicity induced by NQNO in V. harveyi BB7XM assay system.In the remaining three strains the compound demonstrated moderate antimutagenic potential.Three of the tested compounds i.e. 2, 3 and 8 strongly reduced NQNO mutagenicity in V. harveyi strains BB7 and BB7XM.The inhibition percentages of these substances ranged from 41 to 65.For the remaining two strains these compounds demonstrated moderate antimutagenic activity (inhibition percentages between 28 and 38), except for compound 3 which showed weak antimutagenic potency in BB7M strain.Finally, compounds 6 and 7 strongly suppressed mutagen activity in one tested strain and moderately inhibited mutagenicity in the other three V. harveyi strains (Table 4).The results presented in Table 5 show that in S. typhimurium TA100 strain the strongest antimutagenic effect was observed for compound 5 with inhibition percentages of 48% (40 ng/ml) and 43% (500 ng/ml).

Safety assessment of azinesulfonamides
Advanced Pharmaceutical Bulletin, 2016, 6(3), 377-384 Three other compounds 1, 4 and 8 were strong inhibitors of the mutagenicity induced by a direct-acting mutagen NQNO only in one tested concentration.Compound 1 in a lower concentration inhibited NQNO mutagenicity by 48%, whereas compound 4 when tested in higher concentration reduced NQNO -induced mutagenic effect by 51%.Aripiprazole used as a reference psychotropic drug inhibited the mutations induced by NQNO ranging from 27% to 30%, which indicates a moderate antimutagenic effect.Additionally, compounds 2, 3 and 7 displayed moderate antimutagenic activity in both tested concentrations.The inhibition percentages for these compounds were between 25 and 37.Only compound 6 when tested in lower concentration exhibited weak antimutagenic effect with inhibition rate of 18%.

Discussion
Determination of a mutagenic potential of new compounds is an essential component of regulatory toxicology. 12,31,32Its highly recommended to perform genetic toxicology screening studies in the early stage of product development to minimize potential mutagenic activity of new molecules and to prioritize structural modifications.
In the present study we evaluated mutagenic and antimutagenic properties of the new bioactive quinolineand isoquinoline-sulfonamide derivatives of long-chain arylpiperazines using the combination of both in silico and in vitro methods.With a view to develop new alternative approaches to safety testing we employed the Vibrio harveyi assay to assess compounds muta-and antimutagenicity.This assay, based on the detection of colonies of neomycin-resistant mutants appearing frequently after a contact with mutagens, was shown to be of sensitivity equal to or higher than that of the Ames test, depending on the nature of a tested mutagen. 20sing the V. harveyi assay it is possible to detect significantly lower concentrations (such as 40 ng/ml) of typical chemical mutagens than when employing the Ames test.Additionally, in the present study the standard Ames test was used to check the reliability of OSIRIS Property Explorer and Vibrio harveyi prediction.Structural modifications within evaluated compounds (1-8) comprised diversification of the kind of azinyl moiety, introduction of a flexible (three-and four methylene groups spacer) and semi-rigid alkylene linker, and variation of the position of the halogen atoms in the phenylpiperazine moiety.Quinoline-and isoquinolinesulfonamides of LCAPs containing monochlorosubstituted phenylpiperazine (1, 2) were classified as multireceptor 5-HT 1A /5-HT 2A /5-HT 7 /D 2 /D 3 ligands, which behaved as 5-HT 2A /5-HT 7 /D 2 antagonists.Compound 3 was classified as 5-HT 2A /5-HT 7 /D 2 /D 3 ligand.However, azinesulfonamides containing 2,3-diClPhP (4-8) were classified as potent, multireceptor 5-HT 1A /5-HT 2A /5-HT 7 /D 2 /D 3 ligands and behaved as 5-HT 1A receptor partial agonist/5-HT 2A and 5-HT 7 receptor antagonist/D 2 receptor partial agonists.Such receptor profile and the functional properties of the investigated agents were similar to those reported for aripiprazole.It's worth noting, that aripiprazole, a reference drug approved for the treatment of schizophrenia and depression, was classified as a neuroprotective agent based on non-clinical studies using transformed cell lines and in vivo stress and lesion paradigms. 33,34Additionally, aripiprazole inhibited chronic mild stress induced accumulation of ROS. 4 Firstly, the tested compounds were submitted to in silico toxicity screening using the OSIRIS program.It was found that compounds containing 7-quinolinyl fragment, namely 5 (N-(3-(4-(2,3-dichlorophenyl)piperazin-1yl)propyl)quinoline-7-sulfonamide) and 6 (N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-7sulfonamide) trigger a mutagenic structural alert and should be consider potentially hazardous.Subsequently, all the tested compounds were evaluated in vitro using the Vibrio harveyi assay and the Ames test.Contrary to the preliminary in silico data, none of the compounds showed mutagenic activity on V. harveyi BB7, BB7M, BB7X and BB7XM and S. typhimurium TA100 strains.
Interestingly, in general mutagenic indexes was lower in S. typhimurium TA100 strain than in V. harveyi strains.This phenomenon could be explained in terms of a higher sensitivity of the Vibrio harveyi mutagenicity assay in comparison to the Ames test.All of the tested azinesulfonamide derivatives of longchain arylpiperazines displayed a protective effect against the mutagenicity induced by a direct acting mutagen NQNO in the Vibrio harveyi and the Ames assays with inhibition percentages ranging from 22 to 84 in V. harveyi assay and from 18 to 51 in the Ames test.The most beneficial antimutagenic properties showed compound 5 which exhibited strong antimutagenic properties in all tested V. harveyi strains.High antimutagenic potency of this compound was confirmed in the Ames TA100 assay system.Moreover, antimutagenic effects of compounds 7 and 8 obtained in Vibrio harveyi were closely related to the Ames test.In can be concluded that antimutagenicity data are comparable between Vibrio harveyi and Salmonella assays.Only in case of compounds 3, 6 and aripiprazol small discrepancy exists between the data obtained in these two antimutagenicity tests.The inhibitory effects of tested azinesulfonamides 1-8 on the mutagenicity of NQNO indicated that these compounds may protect the bacterial genome against genotoxicity induced by directly acting mutagens.Although the tested compounds may exert their antimutagenic actions by more than one mechanism, it seems probable that tested compounds facilitate or stimulate the bacterial transmembrane export system to eliminate the mutagen.Alternative mechanism may involve uptake of mutagen into bacteria. 35In addition, as NQNO is an oxidative mutagen that undergoes redox recycling to generate ROS 36 the antimutagenic action of tested compounds may be attributed to the inhibition of free radicals formation.However, further studies are required in order to establish the exact mechanism of these compounds action.

Conclusion
Newly synthesized azinesulfonamide analogs of aripiprazole may be considered as genotoxically safe as they do not display mutagenic activity on the tester strains.Moreover, the tested compounds demonstrated significant antimutagenic properties that can be valuable for prevention of the NQNO genotoxicity.The present study showed that although the results of in silico analysis are informative and accurate for some structural classes, they have often limited application for prediction of mutagenic properties of the novel classes of compounds.Thus, experimental verification of structural alerts for such compounds should be always considered.Additionally, it appears that the Vibrio harveyi assay can be applied for primary mutagenicity and antimutagenicity assessment of chemical substances, thus, representing a useful alternative tool for compounds safety evaluation.Finally, the obtained preliminary mutagenicity and antimutagenicity results encourage further search in the group of quinoline-and isoquinoline-sulfonamide derivatives of long-chain arylpiperazines as potential psychotropic agents that additionally display antimutagenic properties.

a
Number of revertants; b NQNO (nitroquinoline-N-oxide, 40 ng/ml) -positive control; DMSO -negative control; c The values in parenthesis are the inhibition rates (%) of mutagenicity.

Table 2 .
Mutagenic activity of azinesulfonamides in the Vibrio harveyi test and by using OSIRIS Property Explorer.

Table 3 .
Mutagenic activity of azinesulfonamides in the Ames test.
a Mean ± S.D. M.I. c DMSO

Table 4 .
Antimutagenic activity of azinesulfonamides in the Vibrio harveyi test.

Table 5 .
Antimutagenic activity of azinesulfonamides in the Ames test.