Guido Hesselmann

André Knops
email: andre.knops@cnrs.fr

No conclusive evidence for number-induced attentional shifts in a temporal order judgment task

Keywords: attentional SNARC, numerical cognition, spatial attention, prior entry, spatial numerical association

No conclusive evidence for number-induced attentional shifts in a temporal order judgment task

Guido Hesselmann, André

Introduction

According to an influential notion, numerical magnitude information is mentally represented along a spatially oriented one-dimensional manifold, often referred to as the mental number line (MNL). In Western societies with left-to-right reading direction, smaller numbers are represented left from larger numbers on the MNL. The congruency between the spatial position of an internally represented number and external factors such as position of the response hand has been used to explain empirical phenomena such as the Spatial Numerical Association of Response Codes (SNARC). The SNARC effect refers to the observation that relatively small (e.g., 1) and large numbers (e.g., 9; small and large within the context of a given experiment using only one-digit numbers) elicit faster left-sided and right-sided manual responses, respectively [START_REF] Dehaene | The mental representation of parity and number magnitude[END_REF]. In this framework, the congruency between the response side and the spatial position of the presented number on the MNL facilitates responses, whereas an incongruency between both spatial codes slows down responses. However, different theoretical approaches have been proposed to account for the SNARC effect that do not predominantly call on the congruence between spatial semantic and motor codes.

For example, Santens and Gevers (p. 269 Santens & Gevers, 2008) question a direct mapping between semantic and motor codes and instead argue for "intermediate categorization of numbers as relatively small (-polarity) or large (+ polarity) […] that are subsequently linked with their corresponding alternatives on a response dimension (left/right, bottom/top, close/far,.

. .)." Alternatively, others argue against an association of response location with a location on a long-term memory representation and instead stress the ad-hoc, task-dependent serial position in working memory as the main determinant of spatial-numerical associations [START_REF] Fias | How is Number Associated with Space? The Role of Working Memory[END_REF][START_REF] Van Dijck | A working memory account of the interaction between numbers and spatial attention[END_REF]. To further probe the assumption of a spatial layout of the mental number representation, Fischer and colleagues [START_REF] Fischer | Perceiving numbers causes spatial shifts of attention[END_REF]) used a target detection paradigm. They centrally presented small (i.e., 1 or 2) or large (i.e., 8 or 9) numbers as non-informative cues prior to laterally presented targets. Participants were required to indicate target appearance as fast and accurately as possible via button press with the preferred hand. In line with the idea that the spatial position of the numbers deviates spatial attention to the left for smaller numbers and to the right for larger numbers, target detection was facilitated when the spatial position of the number on the MNL was congruent with the side at which the target would appear. That is, left targets were responded to faster after the presentation a smaller number, while right targets were responded to faster after the presentation of larger numbers. This attentional SNARC effect has proven hard to replicate though -despite multiple attempts and a large number of participants [START_REF] Colling | Registered Replication Report on Fischer, Castel, Dodd, and Pratt[END_REF][START_REF] Fattorini | Perceiving numbers does not cause automatic shifts of spatial attention[END_REF][START_REF] Zanolie | Number-induced shifts in spatial attention: a replication study[END_REF]. Hence, both the SNARC and the attentional SNARC effect do not unanimously provide evidence for the assumption of a spatially organized mental number representation.

Another frequently used paradigm to probe attentional prior-entry biases is the temporal order judgment (TOJ) paradigm. In a typical TOJ paradigm participants are sequentially presented with two target stimuli that are presented to the left and right of a central fixation stimulus. The stimulus onset asynchrony (SOA) between both targets is manipulated such that the left target is preceding the right one by a varying duration in half of the trials and vice versa. The TOJ paradigm builds on a long-standing stance in experimental psychology that goes back to [START_REF] Titchener | Lectures on the elementary psychology of feeling and attention[END_REF] and is known as the prior-entry hypothesis: "the object of attention comes to consciousness more quickly than the objects which we are not attending to." Applied to a TOJ paradigm, the target that is in the focus of attention enters the cognitive system first. This is even the case if the attended target is lagging behind in time. In a sense, the attentional benefit would compensate the temporal lag effect up to a certain degree (i.e., a certain temporal lag): the attended stimulus needs to be presented later in time in order to be perceived as simultaneous. A parameter that is often used to quantify the attentional benefit is the point of subjective simultaneity (PSS). It illustrates the estimated point in time where the probability of saying that the right stimulus appeared first equals the probability of saying that the left stimulus appeared first (i.e., 50 % each). TOJs have been deployed in a number of previous studies probing the attentional deflection following from presenting single digits [START_REF] Casarotti | Temporal order judgment reveals how number magnitude affects visuospatial attention[END_REF][START_REF] Galarraga | Is the attentional SNARC effect truly attentional? Using temporal order judgements to differentiate attention from response[END_REF] or arithmetic operations [START_REF] Glaser | When adding is right: Temporal order judgements reveal spatial attention shifts during two-digit mental arithmetic[END_REF]. Casarotti and colleagues (2007) found that smaller numbers that were centrally presented before target onset induced a bias favouring left targets while larger numbers induced a bias favouring right targets. These results are in line with the assumption that numbers induce attentional biases as a function of their position on the MNL (i.e., their numerical magnitude).

However, there remained a concern due to the fact that response side codes and the attentional effects of the number cues were confounded in [START_REF] Casarotti | Temporal order judgment reveals how number magnitude affects visuospatial attention[END_REF]. That is, participants verbally indicated which target appeared first by using non-words (fulpo/pingo) that were associated with left and right side (counterbalanced across participants). According to a prominent theory, the brain organizes the processing of visual input along two principal pathways; a ventral pathway that elaborates on object identity is contrasted with a dorsal pathway that operates on spatial attributes [START_REF] Milner | Two visual systems re-viewed[END_REF]; but see [START_REF] Schenk | Do we have independent visual streams for perception and action?[END_REF]. Task designs that explicitly associate responses with spatial codes may preactivate the spatial processing stream (e.g., in parietal cortex) which then "contaminates response times even in tasks with arbitrary stimulus-response association rules" [START_REF] Fias | Irrelevant digits affect feature-based attention depending on the overlap of neural circuits[END_REF]. That is, in tasks that require the processing of spatial stimulus properties or refer to the spatial layout of responses may facilitate the activation of the spatial dimension of numerical magnitude which may then lead to congruency effects such as the (attentional) SNARC.

To orthogonalize response dimension from the stimulus and cue dimensions, participants are asked to report which stimulus appeared second rather than which appeared first. If a left target appears first and benefits from an attentional bias (e.g., due to a small number cue), under this instruction it would not be referred to in the response. That is, the cued target is not identical with the response code which refers to the uncued target. Galarraga and colleagues (2022) used this procedure to disentangle spatial response codes from attentional prior-entry biases. They observed a reversed spatial bias because "participants were more likely to report that the right target appeared first (left-side response) following a low digit and that the left target appeared first (right-side response) following a high digit (p. 813; [START_REF] Galarraga | Is the attentional SNARC effect truly attentional? Using temporal order judgements to differentiate attention from response[END_REF])." The authors concluded that the attentional SNARC effect that is observed in TOJ tasks is not induced by attentional shifts. However, [START_REF] Galarraga | Is the attentional SNARC effect truly attentional? Using temporal order judgements to differentiate attention from response[END_REF] used manual button presses with laterally organized response buttons (left vs. right response button) which leads to the activation of spatial response codes and thereby may pre-activate the spatial processing of the number cues. This, in turn may have led to the current effects that can alternatively be explained by a congruency between numerical magnitude and response side. Under this alternative assumption, smaller numbers would pre-activate left sided responses while larger numbers would pre-activate right sided responses. Hence, the current evidence does not allow to decide whether or not numbers do induce spatial biases.

The current study was designed to probe the notion that numbers induce shifts of spatial attention in accordance with their position on a MNL in the absence of any putative spatial response code that may contaminate the responses. Preceding a classical TOJ task, participants were presented with non-informative number cues (experiments 1 and 2) or arrows (experiment 3). We used a square and a tilted square as targets, thereby situating the decisive response dimension in the ventral, non-spatial processing stream. To further eliminate putative response biases, participants reported which target form appeared first in one half of the experiments and which target appeared last on the other. This procedure is commonly argued to isolate the attentional prior-entry effect [START_REF] Spence | Prior-entry: a review[END_REF].

Methods

Participants

We report how we determined our sample size, all data exclusions, all inclusion/exclusion criteria, whether inclusion/exclusion criteria were established prior to data analysis, all manipulations, and all measures in the study. No part of the study procedures or analyses was pre-registered prior to the research being conducted.

Sample size was determined apriori based on the study by [START_REF] Casarotti | Temporal order judgment reveals how number magnitude affects visuospatial attention[END_REF].

In experiment 6, the small digit (1) induced a positive temporal bias of 5.7 ms. Using WebPlotDigitizer (https://apps.automeris.io/wpd/), we extracted the standard error of the mean (SE) from Figure 5, and calculated the corresponding standard deviation (SD) as 2.6 * sqrt(10) = 8.2 ms. Using GPower 3.1.9.7 [START_REF] Faul | G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences[END_REF] we then determined that for effect size d = 0.70, and alpha = 0.05, a sample of N = 24 was necessary to achieve a power of 0.95 (test family: t-test; means: difference from constant, one sample case; one-tailed).

Twenty-five participants took part in experiment 1 (mean age: 23 years, range: 19-32; 16 female; right handed: 21). Participant #14 was excluded from group analysis due to a poor psychometric fit (Figure S1; see below for exclusion criteria). Thirty-two new participants took part in experiment 2 (mean age: 23 years, range: 18-46; 25 female; right handed: 27). The data from participant #1 were discarded because the participant did not follow the instructions, but named the number before the performing the ba/bo task. The data from participant #32 were not included in the analysis because the desired N had already been reached. Note that the reported results do not change substantially when including participant #32. Participants #5, #11, #15, #19, #25, and #26 were excluded from group analysis due to poor psychometric fits (Figure S2). Twenty-seven new participants took part in experiment 3 (mean age: 25 years, range: 18-50; 13 female; right handed: 25). The data from participant #19 were discarded due to technical problems during data acquisition. Participants #11 and #16 were excluded from group analysis due to poor psychometric fits (Figure S3).

Participants were recruited via student mailing lists and personal contact (exp.3). All participants had normal or corrected-to-normal vision and no history of neurological or psychiatric disorders. All participants were naïve to the purpose of the experiments, provided written informed consent and received monetary compensation (€8/h) or course credit for their participation. The total duration of the experiments was approximately 60 minutes. The experiments were conducted at the Psychologische Hochschule Berlin (PHB), and were approved by the local ethics committee (approval number PHB10032019).

Setup and stimuli

The experiment was conducted in a darkened, sound-attenuated room, with indirect light coming from the experimenter's PC screen. Participants viewed a 19″ CRT monitor (SAMTRON 98PDF; 1280 x 1024 pixels; refresh rate 85 Hz) using a chinrest to stabilize head position. The viewing distance was 57 cm. All stimuli were created using Matlab 2019a (MathWorks, Natick, MA), and the Psychophysics toolbox 3.0.12 [START_REF] Brainard | The Psychophysics Toolbox[END_REF], on a PC with Windows 10 and an AMD Radeon 6450 graphics card. Participants provided verbal responses, and used the PC keyboard to navigate through the experiments (e.g., to terminate a pause). Verbal responses were recorded using a Behringer XM8500 microphone and a Focusrite Scarlett Solo audio-interface, and stored as wav files.

Figure 1 illustrates the display sequence used in all experiments. In experiment 1, each trial began with a centrally presented fixation circle (494 ms; diameter 0.5°), followed by a digit cue (247 ms; 0.5° x 1°, width x height). Two digits were used: 1 and 9. After a jittered random delay (250 or 500 ms ± 10%), the first target stimulus was presented, either to the left or to the right of the fixation (eccentricity 4°). The second target stimulus was presented to the other side of fixation. Two different target stimuli were used on each trial: a square, and a tilted square (both 1° x 1°). The fixation circle and target stimuli were white (101 cd/m 2 ) on a uniformly grey background (31 cd/m 2 ). The first target stimulus was presented for 12, 24, 35, or 71 ms, resulting in 2*4 = 8 different stimulus onset asynchronies (SOA) between the left and the right target stimulus. Following Casarotti et al. [START_REF] Casarotti | Temporal order judgment reveals how number magnitude affects visuospatial attention[END_REF], negative SOAs refer "left target first" trials, and positive SOAs refer to "right target first" trials. Please note that all presentation durations are multiples of 11.8 ms (i.e., the duration of one screen frame at 85 Hz refresh rate). The second target stimulus was presented until a verbal response was successfully recorded, which started the next trial. In one block of trials, the participant's task was to identify and name the target stimulus that was presented first (square = "bo", tilted square = "ba"). In another block of trials, the task was to identify and name the stimulus that was presented last.

Each block consisted of 192 trials in random order (12 trials per condition; 8 SOAs and 2 cues = 16 conditions). In each condition, both locations of the first stimulus (left/ right of fixation) and both shapes of the first stimulus (square/ tilted square) were equally likely. There were additional 30 training trials at the beginning of each block, the first 10 trials used the longest SOAs, followed by 20 trials with random SOAs. The order of the two blocks was counterbalanced across participants.

Experiment 2 was identical with experiment 1, with the following exception. In randomly selected 33% of all trials, participants had a second task. After providing the first verbal response ("ba", or "bo"), participants were instructed to also name the presented digit ("1", or "9"). When this second verbal response was successfully recorded, the next trial began.

Experiment 3 was identical with experiment 2, with the following exception. Instead of digits, arrow cues were presented (2° x 1°), either pointing to the left, or to the right (Figure 1). After providing the first verbal response ("ba", or "bo"), participants were instructed to name the direction of the arrow cue ("left", or "right").

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Data analysis

Data pre-processing, descriptive and inferential statistics were performed using R 4.1.1 (R Core Team, 2021) and RStudio 2021.09.0, Build 351 (RStudio Team, 2021). Data visualizations were created with the R package ggplot2 [START_REF] Wickham | ggplot2: Elegant graphics for data analysis[END_REF]. Trial-by-trial data in csv-format and R scripts for data analysis are available at an online repository (https://doi.org/10.17605/OSF.IO/T4J7W).

The recorded verbal responses were decoded manually (experiments 1-3: "ba", "bo"; experiment 2: "1", "9"; experiment 3: "left", right"). In the rare case that the audio file contained more than one response (e.g., when a participant corrected the first response), only the first response was decoded. Training trials were discarded. Trials without audible verbal responses, and trials with incomprehensible verbal responses were marked and removed from the analysis (experiment 1: 0%; experiment 2: 0.7%; experiment 3: 1.4%; mean percentage of trials). For each participant and condition, the proportion of "left stimulus first" responses were calculated based on the verbal "ba/bo" responses. In experiment 2, the proportion of correct responses in the number identification task was calculated for each participant. In experiment 3, the proportion of correct responses in the arrow identification task was calculated for each participant.

A logistic model was fitted to the proportion data of each participant as a function of the SOA (in frames), separately for digit 1 and digit 9 (experiment 3: arrow left and arrow right). The data were collapsed across both instructions (i.e., name the stimulus that appeared first/last).

The logistic model allowed us to determine the point of subjective simultaneity (PSS) for each cue condition. Using the psignifit4 toolbox [START_REF] Schutt | Painfree and accurate Bayesian estimation of psychometric functions for (potentially) overdispersed data[END_REF] in Matlab 2019a we verified the PSS estimates (see supplementary tables S1 and S2). The PSS represents the SOA for which the observer perceives the stimuli as simultaneous. A shift in the PSS in either direction thus indicates that the cue induced a temporal bias [START_REF] Casarotti | Temporal order judgment reveals how number magnitude affects visuospatial attention[END_REF]. When piloting the first experiment, we noticed that poor psychometric fits were associated with extreme PSS values. Therefore, we decided to exclude the data from participants showing poor psychometric fits. For each participant's proportion data (collapsed across both cue conditions), we calculated McFadden's pseudo-R 2 as a goodness-of-fit measure; participants with pseudo-R 2 < 0.18 were excluded from group analysis [START_REF] Louviere | Stated Choice Methods: Analysis and Applications[END_REF].

For each experiment, we computed a repeated-measures ANOVA with cue (digit 1 versus digit 9, or arrow left versus arrow right) and SOA (-6, -3, -2, -1, 1, 2, 3, 6; screen refresh frames), following Casarotti et al. [START_REF] Casarotti | Temporal order judgment reveals how number magnitude affects visuospatial attention[END_REF]. Degrees of freedom were Greenhouse-Geissercorrected; we report uncorrected degrees of freedom, the correction value epsilon (eps), and the effect size partial eta-squared (ηp 2 ). A paired samples two-tailed t-test was then performed on the individual PSS to compare the temporal biases induced by each cue. To quantify the evidence in favour of the alternative hypothesis over the null hypothesis (or, vice versa), a Bayesian equivalent of the paired t-test was calculated using the BayesFactor package 0.9.12-4.4 in R. Bayes factors describe the relative probability of data under competing positions (e.g., a null model H0 and one alternative model H1). The Bayes factor (BF) refers to the ratio of marginal likelihoods of different models under consideration, and quantify the change from prior to posterior model odds. The prior odds describe the beliefs about the models before observing the data. The BF thus describes how the evidence from the data should change beliefs. The subscripts on BFs refer to the models being compared, with the first and second subscript referring to the model in the numerator and denominator, respectively. For example, a BF10 of 5 indicates that the data are five times more likely under H1 than under H0. We used the ttestBF and extractBF functions from the BayesFactor package. TtestBF uses a Cauchy prior (scale parameter: sqrt(2)/2 = .707). Models, priors, and methods of computation are provided in Rouder et al. [START_REF] Rouder | Default Bayes factors for ANOVA designs[END_REF]. Classification schemes can be used to group Bayes factors into different categories. According to [START_REF] Lee | Bayesian cognitive modeling: A practical course[END_REF], BFs between 1 and 3 indicate anecdotal evidence, BFs larger than 3 moderate evidence, and BFs larger than 10 strong evidence.

Results -experiment 1

Figure 2A shows the mean percentage of left-first responses as a function of SOA in experiment 1, separately for digits 1 and 9. As expected, both psychometric curves show a decreasing shape, but they overlap to a large degree, irrespective of SOA. Accordingly, the rm-ANOVA revealed that only the main effect of SOA was significant (F(7,161) = 141.95, p < .001, ηp 2 = .86, eps = .31). Neither the main effect of digit magnitude (F(1,23) < 1, p = .421, ηp 2 = .03, eps = 1), nor the interaction were significant (F(7,161) < 1, p = .690, ηp 2 = .03, eps = .75).

In order to measure the amount of temporal bias induced by each number, we calculated the PSS for each individual psychometric curve, i.e., the point at which the observer perceived the stimuli as simultaneous. Figure 3A shows the mean temporal biases induced by digits 1 and 9.

Digit 1 resulted in a negative bias of -2.9 ms (i.e., in the wrong direction), and digit 9 induced a negative bias of -1.9 ms (i.e., in the predicted direction). The statistical comparison between the temporal biases turned out to be not significant (t(23) = -0.40, p = .694; paired two-tailed ttest), with a Bayes Factor favouring the null over the alternative hypothesis (BF01 = 4.35).

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Discussion

Experiment 1 aimed at probing the notion that numbers induce shifts of spatial attention in accordance with their position on a MNL in the absence of any putative spatial response code that may contaminate the responses. The results do not support this notion. The results revealed a small overall shift of spatial attention to the right side of space. Importantly, there was no interaction with the numerical magnitude of the number cue. Rather, the Bayesian analysis revealed evidence that favoured the null hypothesis over the alternative hypotheses when comparing the number cues. In sum, when removing all spatial associations from the paradigm, the presentation of a number did not induce shifts of spatial attention. However, since the number cues in the current paradigm were task-irrelevant, one might argue that the absence of spatial cueing was due to the fact that the number cues were not actively processed by the participants. Previous studies (e.g. [START_REF] Zanolie | Number-induced shifts in spatial attention: a replication study[END_REF] suggest that the attentional SNARC arises when participants actively processed the number cues (but see [START_REF] Galarraga | Is the attentional SNARC effect truly attentional? Using temporal order judgements to differentiate attention from response[END_REF].

Indeed, the number cues were non-informative and despite the fact that adult participants cannot not read written symbols (e.g. [START_REF] Pattamadilok | Automaticity of phonological and semantic processing during visual word recognition[END_REF], we cannot know to what extent the semantic content of the numbers has been processed. We therefore replicated experiment 1 and added a secondary task that would require participants to identify and memorize the number cues. In 33% of the trials, participants were asked to name the number cue after having completed the TOJ task.

Results -experiment 2

In experiment 2, the mean performance in the number naming task was 85% (range 66-94%).

Figure 2B shows the mean percentage of left-first responses as a function of SOA, separately for digits 1 and 9. Both psychometric curves show a decreasing shape, and again overlap to a large degree, irrespective of SOA. Accordingly, the rm-ANOVA revealed that only the main effect of SOA was significant (F(7,161) = 186.69, p < .001, ηp 2 = .89, eps = .50). Neither the main effect of digit magnitude (F(1,23) = 3.78, p = .064, ηp 2 = .14, eps = 1), nor the interaction were significant (F(7,161) < 1, p = .727, ηp 2 = .02, eps = .64).

Figure 3A shows the mean temporal biases induced by digits 1 and 9. Digit 1 resulted in a positive bias of 6.9 ms (i.e., in the predicted direction), and digit 9 induced a positive bias of 4.5 ms (i.e., in the wrong direction). The statistical comparison between the temporal biases turned out to be not significant (t(23) = 1.52, p = .142; paired two-tailed t-test), with a Bayes Factor slightly favouring the null over the alternative hypothesis (BF01 = 1.70), albeit providing only anecdotal evidence. When the data from both experiments 1 and 2 were analysed together (N=48), the temporal biases turned out to be 2.0 ms and 1.3 ms for cue numbers 1 and 9, respectively, and they were statistically not different from each other (t(47) = 0.51, p = .612; BF01 = 5.64).

Discussion -experiment 2

The results of experiment replicated those from experiment 1. We did not observe a significant modulation of the locus of spatial attention as a function of the numerical magnitude of the number cue. Since participants were asked to identify and memorize the number cue, we can exclude that the absence of a number-induced attentional bias is due to the lack of cognitive resources deployed on the processing of the number cues. Instead, these results underline the notion that in the absence of any spatial codes at the task level, numbers do not induce shifts of spatial attention.

Although TOJ tasks have been used to demonstrate spatial biases in the past, we scrutinized the potential of revealing spatial biases in the current cueing set-up in a third experiment. In experiment 3, we replaced the number cues by non-informative arrows. All other details were identical to experiment 2, including the secondary task asking participants to identify and memorize the direction that the arrows pointed to.

Results -experiment 3

In experiment 3, the mean performance in the arrow naming task was 95% (range 83-100%).

Figure 2C shows the mean percentage of left-first responses as a function of SOA, separately for left and right arrows. Both psychometric curves show a decreasing shape. At short SOAs between the stimuli, however, the data points from the left arrow curve lie above the data points from the right arrow curve. The rm-ANOVA revealed a significant main effect of SOA (F(7,161) = 147.52, p < .001, ηp 2 = .87, eps = .40), but also a significant main effect of arrow direction (F(1,23) = 14.98, p < .001, ηp 2 = .39, eps = 1), and a significant interaction (F(7,161) = 4.24, p = .003, ηp 2 = .16, eps = .61).

Figure 3C shows the mean temporal biases induced by left and right arrows. Left arrows resulted in a positive bias of 6.7 ms, and right arrows induced a negative bias of -11.6 ms. Thus, both biases were in the expected direction. The statistical comparison between the temporal biases turned out to be significant (t(23) = 3.78, p < .001; paired two-tailed t-test), with a Bayes Factor strongly favouring the alternative over the null hypothesis (BF10 = 36.03).

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Discussion -experiment 3

Experiment 3 was designed to test the potential of our TOJ set-up to reveal attentional biases.

When using arrows instead of numbers as non-informative cues, we observed a reliable deflection of spatial attention in the direction that the arrows pointed to. Hence, we can exclude that the absence of spatial biases in experiments 1 and 2 were due to our task set-up.

General Discussion

The current study was designed to probe the notion that numbers induce shifts of spatial attention in accordance with their position on a MNL in the absence of any putative spatial response code that may contaminate the responses. In two experiments where numbers were used as non-informative cues preceding a TOJ task, we did not observe a deflection of the locus of spatial attention as a function of the numerical magnitude of the cue. By making the number cues task-relevant in a secondary task in experiment 2 (while still being non-informative for the primary TOJ task), we sought to guarantee that the cues were actively processed by the participants. Even under these conditions, the number cues did not influence the locus of spatial attention. Finding a significant modulation of TOJ performance as a function of the pointing direction of arrow cues in experiment 3 allowed us to rule out the possibility that the absence of any significant modulation in experiments 1 and 2 was due to a lack of sensitivity of our task set-up.

Inspired by the metaphor of the mental number line, a spatial representation of numerical magnitude, the idea that numbers can induce shift of spatial attention as a function of their position on this one-dimensional manifold has received empirical support from behavioural experiments [START_REF] Casarotti | Temporal order judgment reveals how number magnitude affects visuospatial attention[END_REF][START_REF] Fischer | Perceiving numbers causes spatial shifts of attention[END_REF][START_REF] Ristic | The number line effect reflects top-down control[END_REF], neural measures [START_REF] Knops | Recruitment of an area involved in eye movements during mental arithmetic[END_REF][START_REF] Pinto | Visualising numerals: An ERPs study with the attentional SNARC task[END_REF] and neuropsychological observations [START_REF] Zorzi | Neglect impairs explicit processing of the mental number line[END_REF][START_REF] Zorzi | Brain damage: neglect disrupts the mental number line[END_REF]. At the behavioural level, however, the attentional effects have not always been successfully replicated [START_REF] Colling | Registered Replication Report on Fischer, Castel, Dodd, and Pratt[END_REF][START_REF] Zanolie | Number-induced shifts in spatial attention: a replication study[END_REF]. Casarotti and colleagues (2007) presented number cues prior to a TOJ task to probe this notion and observed a preference for left targets following small number cues, and a preference for right targets following large number cues.

However, this study suffered from two major shortcomings that lead to confounds with nonnumerical mechanisms. First, they consistently instructed participants to report the target that appeared first. Second, although they replaced the words 'left' and 'right' by pseudowords (fulpo/pingo), participants responded by referring to the spatial position of the target that appeared first. This may have primed a spatial processing pathway and thereby paved the way for putative deflections of spatial attention [START_REF] Fias | Irrelevant digits affect feature-based attention depending on the overlap of neural circuits[END_REF].

To address the first issue, a previous study used a TOJ in combination with number cues and found that spatial response correspondences overshadow putative attentional biases induced by numbers [START_REF] Galarraga | Is the attentional SNARC effect truly attentional? Using temporal order judgements to differentiate attention from response[END_REF]. By asking participants to decide which target in a TOJ task appeared second, Galarraga and colleagues (2022) orthogonalized the assumed attentional deflection due the number cues with biases that might arise from response-based spatial correspondences. For example, with the instruction to report the first target, both the left-sided bias due to a small number cue is confounded with the correspondence between left target side and left response side. By instructing to report the second target, this confound is alleviated.

Galarraga and colleagues found an overall preference for left-first/right-second targets that was more pronounced for the large cue number (9) compared to the small cue number (1). Hence, the response bias followed the side of response rather than the spatial position of the number on the mental number representation, taking the form of a response-related SNARC effect. In the current experiments, we counter-balanced the correspondence effects between manual response side and number cues within-participant by instructing participants to report the first target in one half of the trials, while reporting the second target in the other half of the trials.

Hence, if the response-related spatial correspondence is what drives the observed biases, these effects would be cancelled out by our procedure. Our results are consistent with this interpretation since they show that (a) numbers do not elicit deflections of spatial attention when (b) no spatial correspondences are present in the experimental procedure.

The current experiments also help addressing the second issue that we identified in the study by Casarotti and colleagues (2007). We tried to eliminate a maximum of spatial codes from our task. Most crucially, instead of reporting the side which the first or the second target appeared on, participants reported the shape of the target under question (square/ tilted square). We thereby eliminated the focus on the spatial dimension of the target and primed ventral processing pathways. Under these conditions, numbers no longer induced any spatial deflections of spatial attention. Note however, that this manipulation does not prevent putative attentional biases from emerging. Provided the cue possesses sufficiently strong spatial codes, as is the case for the arrow cues, attentional biases should still be detectable by the TOJ task.

This is exactly what we observe in experiment 3 where arrow cues elicit an attentional bias over and above the counter-balanced response mapping.

While we cannot rule out that our task design is not sufficiently sensitive for detecting spatial biases for example by using online measures of brain activity such as EEG [START_REF] Pinto | Visualising numerals: An ERPs study with the attentional SNARC task[END_REF], the cognitive cascade initiated by our task requirements (identifying, maintaining in working memory, and naming) are not sufficient to elicit spatial biases that can be detected in a TOJ task.

We conclude from the current findings that the spatial codes that the perception and naming of numbers potentially elicit are not in and by themselves sufficient to elicit deflections of spatial attention. These findings add to existing evidence that question the reflexive and automatic nature of attentional shifts elicited by centrally presented number cues [START_REF] Galfano | Number magnitude orients attention, but not against one's will[END_REF][START_REF] Ristic | The number line effect reflects top-down control[END_REF]. To deflect attention in space, centrally presented numbers need to be embedded in a context that triggers spatial associations. This includes, but is not limited to, the activation of top-down mental sets as demonstrated by reversed spatial effects with imagined right-to-left number lines or clock faces [START_REF] Ristic | The number line effect reflects top-down control[END_REF] or the explicit association of number cues with spatial positions via cue validity [START_REF] Galfano | Number magnitude orients attention, but not against one's will[END_REF]. At a very minimum, the task context needs to activate spatial references at the stimulus or response level which may then coincide with the processing of numerical magnitude. This may have its origin in an overlap of processing sites in the parietal cortex [START_REF] Fias | Irrelevant digits affect feature-based attention depending on the overlap of neural circuits[END_REF]. Spatial-numerical associations are often discussed in the context of dual-route models with a "parallel activation of preexisting links between magnitude and spatial representation and shortterm links created on the basis of task instructions [START_REF] Gevers | Numbers and space: a computational model of the SNARC effect[END_REF]." The present results add to the body of evidence that either questions the existence of "pre-existing links" that are automatically activated or the degree to which those unconditional processing routes impact overt behaviour.

Figure Captions

Figure 1. Display sequence (not drawn to scale). Each trial began with a centrally presented fixation circle (494 ms), followed by a digit cue (1 or 9) in experiments 1 and 2, or by an arrow cue (left or right) in experiment 3 (247 ms). After a jittered random delay (250 or 500 ms ± 10%), the first target stimulus was presented, either to the left or to the right of the fixation (12, 24, 35, or 71 ms). The second target stimulus was presented to the other side of fixation, until the verbal response was provided. In one block of trials, the participant's task was to identify and name the target stimulus that was presented first (square = "bo", tilted square = "ba"). In another block of trials, the task was to identify and name the stimulus that was presented last. Please note that all presentation durations are multiples of 11.8 ms (i.e., the duration of one screen frame at 85 Hz refresh rate).

Figure 2 .

 2 Figure 2. Mean percentage of left-first responses in experiments 1-3 (each N=24), as a function of SOA (in ms). (A) Data from Experiment 1, separately for number magnitude (1 = green, 9 = red). (B) Data from Experiment 2, separately for number magnitude (1 = green, 9 = red). (C) Data from Experiment 3, separately for arrow direction (left = green, right = red). Negative and positive values of SOA refer to left-and right-side stimulus precedence, respectively. Error bars indicate 95% confidence intervals calculated for within-subject data using the summarySEwithin function from the Rmisc package (version 1.5.1).

Figure 3 .

 3 Figure 3. Mean temporal biases induced by digits and arrows in Experiments 1-3 (each N=24). (A) Data from Experiment 1, separately for number magnitude (1 = green, 9 = red). (B) Data from Experiment 2, separately for number magnitude (1 = green, 9 = red). (C) Data from Experiment 3, separately for arrow direction (left = green, right = red). The temporal bias is identical with the point of subjective simultaneity (PSS), and was determined by fitting a logistic model to the individual probabilities of left-first responses. Grey connected dots indicate PSS values from individual participants in both conditions. Error bars indicate 95% confidence intervals calculated for within-subject data using the summarySEwithin function from the Rmisc package (version 1.5.1).

Figure 1 Figure 3

 13 Figure 1


Seyed Gholamhossein Mansouri

Hassan Zali-Boeini

Kamiar Zomorodian

Bahman Khalvati

Razie Helali Pargali

Ali Dehshahri

Amiri Hadi

Mehdi Rudbari

Zahra Sahihi

Chavoshpour

Hassan Zali- Boeini

Rudbari

Mehdi Sahihi

Zahra Chavoshpour

Synthesis of novel naphtho[1,2-e][1,3]oxazines bearing an arylsulfonamide moiety and their anticancer and antifungal activity evaluations

Keywords:

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The development of environmentally friendly and economically inexpensive processes for the synthesis of biologically active heterocycles using readily available and cheap reagents is an important goal of current organic synthesis [START_REF] Laszlo | Organic Reactions: Simplicity and Logic[END_REF]. 1,3-Oxazine derivatives condensed with aromatic rings have gained much attention due to varied pharmacological and biological properties such as anticancer (Urbański et al., 1956, Chylińska et al., 1963[START_REF] Verma | Synthesis, antimicrobial and cytotoxicity study of 1,3disubstituted-1Hnaphtho[1,2-e][1,3]oxazines[END_REF][START_REF] Bouaziz | Synthesis of some naphthoxazinecarbolactone derivatives with in vitro cytotoxic and antifungal activities[END_REF][START_REF] Thaler | Synthesis and biological characterization of spiro[2H-(1,3)-benzoxazine-2,40 -piperidine] based histone deacetylase inhibitors[END_REF][START_REF] Benameur | Synthesis of furnaphth[1,3]oxazine and furo[,3] oxazinoquinoline derivatives as precursors for an o-quinonemethide structure and potential antitumor agents[END_REF], antibacterial [START_REF] Mathew | An eco-friendly synthesis and antimicrobial activities of dihydro2H-benzo-and naphtho-1,3-oxazine derivatives[END_REF][START_REF] Kategaonkar | An efficient synthesis of 3,4-dihydro-3-substituted-2H-naphtho[END_REF][START_REF] Mayekar | Synthesis and antimicrobial study of new 8-bromo-1,3-diaryl-2,3-dihydro-1Hnaphtho[END_REF][START_REF] Didwagh | Novel one-pot synthesis and antimicrobial activity of 6-chloro-2,4-diphenyl 3,4-dihydro-2H1,3benzoxazine derivatives[END_REF][START_REF] Abou-Elmagd | Synthesis of 1-amidoalkyl2-naphthols and oxazines derivatives with study of their antibacterial and antiviral activities[END_REF][START_REF] Verma | Synthesis, antimicrobial and cytotoxicity study of 1,3disubstituted-1Hnaphtho[1,2-e][1,3]oxazines[END_REF], antifungal [START_REF] Verma | Synthesis, antimicrobial and cytotoxicity study of 1,3disubstituted-1Hnaphtho[1,2-e][1,3]oxazines[END_REF][START_REF] Bouaziz | Synthesis of some naphthoxazinecarbolactone derivatives with in vitro cytotoxic and antifungal activities[END_REF][START_REF] Mathew | An eco-friendly synthesis and antimicrobial activities of dihydro2H-benzo-and naphtho-1,3-oxazine derivatives[END_REF][START_REF] Kategaonkar | An efficient synthesis of 3,4-dihydro-3-substituted-2H-naphtho[END_REF][START_REF] Mayekar | Synthesis and antimicrobial study of new 8-bromo-1,3-diaryl-2,3-dihydro-1Hnaphtho[END_REF], anti-HIV [START_REF] Pedersen | The flourishing syntheses of nonnucleoside reverse transcriptase inhibitors[END_REF]Pedersen, 2000, Cocuzza et al., 2001), and anti-inflammatory [START_REF] Akhter | Synthesis of some new 3,4-dihydro-2H-1,3-benzoxazines under microwave irradiation in solvent-free conditions and their biological activity[END_REF] activities.

Naphtho [1,2-e] [1,3]oxazine derivatives are also included in this category.

Owing to their medicinal importance, several methods have been reported for the preparation of these compounds in the literature. The most widely adopted route has been benefited from a Mannich-type condensation reaction between phenols, primary amines, and formaldehyde through either direct or indirect pathway. The multi-component condensation of phenols or naphthols with primary amines (or ammonia) and two equivalents of aldehydes leads to these target molecules directly [START_REF] Burke | 3,4-Dihydro-1,3,2H-benzoxazines. Reaction of psubstituted phenols with N,N-dimethylolamines[END_REF][START_REF] Burke | Condensation of naphthols with formaldehyde and primary amines[END_REF], Burke et al., 1954[START_REF] Burke | Condensation of 2-naphthol with acetaldehyde ammonia[END_REF][START_REF] Dhakane | An efficient and green method for the synthesis of [1,3]oxazine derivatives catalyzed by thiamine hydrochloride (VB1) in water[END_REF][START_REF] Sadaphal | Water mediated synthesis of various [1,3]oxazine compounds using alum as a catalyst[END_REF], whereas a Betti base as starting material represents an indirect approach to the synthesis of 1,3-naphthoxazines [START_REF] Shi | Efficient synthesis of naphtho[1,2-e][,3]oxazine derivatives via a chemoselective reaction with the aid of low-valent titanium reagent[END_REF][START_REF] Turgut | Synthesis of new 1,3-disubstituted-2,3-dihydro-1H-naphth[1,2-e][1,3]oxazines[END_REF][START_REF] Sapkal | Dual role of ammonium acetate for solvent-free synthesis of 1,3disubstituted-2,3-dihydro-1H-naphth[END_REF][START_REF] Verma | Synthesis, antimicrobial and cytotoxicity study of 1,3disubstituted-1Hnaphtho[1,2-e][1,3]oxazines[END_REF]. Another method includes using the condensation reaction of salicylaldehyde with a primary amine followed by reduction and subsequent cyclization reaction with a suitable aldehyde [START_REF] Tang | Synthesis of 2,3-diaryl-3,4-dihydro-2H-1,3-benzoxazines and their fungicidal activities[END_REF][START_REF] Tang | Synthesis and fungicidal activity of novel 2,3-disubstituted-1,3-benzoxazines[END_REF].

Furthermore, miscellaneous approaches such as reaction of oxygen-containing dihalo compounds with primary amines [START_REF] Colin | Nouvelle voied'acces aux dihydro3,4-2Hbenzoxazines-1,3[END_REF], rhodium-catalyzed reaction of 2-(alkenyloxy)benzylamines with a mixture of H2/CO [START_REF] Campi | Allylic rearrangements during the rhodium-catalysed reactions of 2allyloxybenzylamines and 2-(N-allyl-N-benzylamino)benzylamine[END_REF][START_REF] Campi | The stereochemistry of organometallic compounds. XLIII. rhodium-catalysed reactions of 2-(alkenyloxy)benzylamines and 2-(Nallyl-N-benzylamino)benzylamine[END_REF], neutral redox reaction of cyclic amines and 2-hydroxy aromatic aldehydes or ketones [START_REF] Richers | Redox-neutral a-oxygenation of amines: reaction development and elucidation of the mechanism[END_REF], and direct reaction of ortho-lithiated phenols or thiophenols with N, N-bis[(benzotriazol-1-yl)methyl]amines [START_REF] Katritzky | A novel dilithiation approach to 3,4-dihydro-2H-1,3-benzothiazines, 3,4dihydro-2H1,3-benzoxazines, and 2,3,4,5-tetrahydro-1,3-benzothiazepines[END_REF] have also been reported.

The prominent roles of this heterocyclic skeleton in pharmaceutical chemistry inspired chemists to develop novel synthetic methods for efficient preparation of these biologically important compounds. On the other hand, the value of sulfonamide moiety in medicinal chemistry cannot be disregarded, as it consists of a large class of drugs used broadly as pharmaceutical agents. Various biological properties such as antibacterial (Bekdemir et al., 2008), antiviral, (Romines et al., 1997) and carbonic anhydrase (CA) inhibitory [START_REF] Duffel | N-Substituted sulfonamide carbonic anhydrase inhibitors with topical effects on intraocular pressure[END_REF][START_REF] Modak | Structure-activity relationship for sulfonamide inhibition of Helicobacter pylori a-carbonic anhydrase[END_REF] have been reported for the sulfonamide structures as lead [START_REF] Jain | Sulphonamides: deserving class as MMP inhibitors?[END_REF].

Over the past years, dithioimidocarbonates have been identified as useful synthons in organic synthesis and in construction of diverse Heterocycles [START_REF] Zali-Boeini | One-step synthesis of azole-and benzazolebased sulfonamides in aqueous media[END_REF][START_REF] Maybhate | Regiospecific synthesis of N-sulfonyl derivatives of 3,5-diamino-1H-1, 2,4-triazole and 2,5-diamino-1,3,4thiadiazole[END_REF][START_REF] Servi | The synthesis and antimicrobial activity of some new methyl N-arylthiocarbamates, dimethyl Naryldithiocarbonimidates and 2-arylamino-2-imidazolines[END_REF][START_REF] Sato | New synthesis of dimethyl Naroylcarbimidodithioates and 3-aryl-5-methylthio-1H-1,2,4-triazoles[END_REF][START_REF] Alvarez Ibarra | A novel synthesis of 2,3-dihydrooxazoles from dimethyl N-ethoxycarbonyl methyliminodithiocarbonate and aromatic aldehydes[END_REF][START_REF] Sauter | N-[Bis (methylthio)methylene]amino ester (BMMA): Novel reagents for annelation of pyrimidine moieties[END_REF][START_REF] Sullivan | Studies in.beta.-lactams. Synthesis of.beta.lactams via cycloaddition of iminodithiocarbonate esters with azidoketene[END_REF]. Meanwhile, our preliminary plan was to extend the synthetic utility of 1-(amino(aryl)methyl)naphthalen-2-ol derivatives (Betti bases) by examining their applications in the synthesis of novel 1,3-naphthoxazine derivatives. We intended to develop a novel one-step and efficient method for synthesis of new 1-aryl-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine derivatives bearing a sulfonamide moiety via the reaction of N-sulfonyldithioimidocarbonates and Betti base salts in the presence of a base in aqueous EtOH as a green solvent. After characterization of all synthesized compounds, their anticancer and antimicrobial activities were evaluated.

Experimental

Chemistry

Reagents and solvents were obtained from commercial suppliers and used as received unless otherwise indicated. Melting points were determined with a Stuart Scientific SMP-2 apparatus and are uncorrected. Reactions were monitored by thin-layer chromatography (TLC Silica gel 60 F254). 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer referenced to residual solvent protons and signals are reported in ppm (δ). The singlet peak at 3.35 ppm in 1 H NMR spectra corresponds to protons of H2O. X-ray crystallographic data was obtained using a Bruker AXS Kappa APEX II single crystal X-ray diffraction instrument.

General procedure for the synthesis of 1-(amino(aryl)methyl)naphthalen-2-ol hydrochlorides (Betti base hydrochlorides, 3a-e)

The Betti bases (2a-e) required for the synthesis of [1,3]naphthoxazines were obtained by the Betti reaction through a known method [START_REF] Betti | b-Naphtholphenylaminomethane[END_REF] as follows: In a 50 mL round-bottomed flask was placed a cold solution of β-naphthol (1) (7.2 g, 50 mmol) in 95 per cent alcohol (10 mL). To this solution was added benzaldehyde derivative (100 mmol) and EtOH (95%, 10 mL) saturated with ammonia at room temperature. The flask was stoppered and allowed to stand for 2 h. Then the excess ammonia was removed. After about 12 h, the condensation product (2a-e), which separated as white needle crystals, was filtered with suction and washed with EtOH (95%, 50 mL). The condensation product thus obtained was introduced into a 250 mL round-bottomed flask arranged for steam distillation and treated four times its volume with a solution of HCl (20%). The mixture was steam distilled about two hours. Meanwhile, an abundant flocculent precipitate of light pink or white needles was separated. The mixture in the flask was cooled thoroughly and filtered with suction. The yields of 3a-e were 84-91%.

General procedure for the synthesis of arylsulfonamides (5a-d)

Arylsulfonyl chloride (50 mmol) and chloroform (20 mL) were placed in a 100 mL roundbottomed flask. To the mixture an aqueous solution of ammonia (25%, 20 mL) was added and the reaction mixture was stirred for 2 h at room temperature. Then, the precipitated product was filtered and washed with cold water. The crude product was recrystallized using EtOH to give pure sulfonamides (5a-d) as white crystals in excellent yields (91-98%).

General procedure for the synthesis of dimethyl arylsulfonylcarbonimidodithioates (6a-d)

Dimethyl arylsulfonylcarbonimidodithioates (6a-d) were synthesized according to a reported procedure [START_REF] Maybhate | Regiospecific synthesis of N-sulfonyl derivatives of 3,5-diamino-1H-1, 2,4-triazole and 2,5-diamino-1,3,4thiadiazole[END_REF] starting from aryl sulfonamides 5a-d with slight modifications as follows: A solution of arylsulfonamide5a-d (14 mmol) in DMF (10 mL) was cooled in an icebath. To this, a solution of NaOH (20 M, 0.8 mL, 16 mmol) was added with vigorous stirring followed by addition of CS2 (0.7 g, 9 mmol). After 20 min a second lot of NaOH solution (20 M, 0.4 mL, 8 mmol) and CS2 (0.35 g, 4.5 mmol) was added to the reaction mixture followed by a third portion of NaOH solution (20 M, 0.4 mL, 8 mmol) and CS2 (0.35 g, 4.5 mmol) after an interval of 10 min. The ice-bath was removed and the mixture was stirred at r.t. for 2 h. The mixture was cooled in an ice-bath and dimethyl sulfate (3.73 g, 29 mmol) was added dropwise during 10 min.

The reaction mixture was stirred for 2 h at r.t. and poured into H2O (50 mL). The precipitated solid was filtered, washed with H2O, and dried under vacuum. The crude product was recrystallized where necessary from a suitable solvent to obtain compounds 6a-d as odoriferous white solids in good yields (75-86%).

General procedure for the synthesis of (E)-N-(1-aryl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide derivatives (7a-o)

A mixture of 1-(amino(aryl)methyl)naphthalen-2-ol hydrochloride 3 (1 mmol), dimethyl arylsulfonylcarbonimidodithioate6 (1 mmol) and Na2CO3 (0.318 g, 3 mmol) in H2O/EtOH (1: 3; 5 mL) was refluxed for 2 h. The reaction mixture was cooled to room temperature and neutralized by addition of aqueous HCl (10%). After some dilution with water, the liquor was decanted and the residue after purification by column chromatography using silica gel and a mixture of EtOAc-Hexane (1:1) as eluent gave pure compounds 7a-o as white or off-white solids in moderate to good yields (43-78%). [1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7a) M.p.: 247-249 °C (decomp.); yield: 62%; 1 H NMR (400 MHz, DMSO-d6): δ 10.18 (s, 1H), 8.03 (d, J = 9.0 Hz, 1H), 8. 1H),7.91 (d,J = 7.1 Hz,2H),7.80 (d,J = 8.7 Hz,1H),5H),4H),7.22 (d,J = 7.4 Hz, 2H), 6.34 (s, 1H); 13 C NMR (100 MHz, DMSO-d6): δ 152. 1, 145.0, 143.2, 141.1, 131.9, 131.0, 130.7, 129.1, 128.8, 128.7, 128.4, 127.8, 126.9, 126.3, 125.8, 123.0, 115.8, 114.1, 52.0; IR (cm -1 , KBr): 3294, 3059, 1630, 1415, 1350, 1272, 1084; Anal. Calcd. for C24H18N2O3S: C, 69.55; H, 4.38; N, 6.76; S, 7.74 Found: C, 62.59; H, 4.41; N, 6.71; S, 7.77. 2.1.5.2. (E)-4-methyl-N- (1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)ylidene)benzenesulfonamide (7b) M.p.: 260-262 °C (decomp.); yield: 78%; 1 H NMR (400 MHz, DMSO-d6): δ 10.08 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 8. 1H),3H),2H),7.36 (d,J = 9.0 Hz,1H),5H),7.21 (d,J = 7.1,2H),6.33 (s,1H), 2.35 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 152. 1, 145.1, 142.1, 141.1, 140.3, 130.9, 130.7, 129.2, 129.0, 128.7, 128.40, 128.36, 127.8, 126.9, 126.4, 125.8, 123.0, 115.8, 114.1, 52.0, 20.9; IR (cm -1 , KBr): 3240, 3140, 1643, 1431, 1311, 1288, 1153; Anal. Calcd. for C25H20N2O3S: C, 70.07; H, 4.70; N, 6.54; S, 7.48 Found: C, 70.09; H, 4.68; N, 6.51; S, 7.47. 2.1.5.3. (E) -4-chloro-N-(1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H (d, J = 7.2 Hz, 2H), 6.34 (s, 1H). 13 C NMR (100 MHz, DMSO-d6): δ 152. 1, 145.1, 142.0, 141.2, 136.6, 131.0, 130.7, 129.0, 128.9, 128.7, 128.5, 128.4, 128.3, 127.7, 126.9, 125.8, 123.1, 115.8, 114.0, 52.1; IR (cm -1 , KBr): 3225, 3085, 1643, 1477, 1431, 1219, 1157; Anal. Calcd. for C24H17ClN2O3S: C, 64.21; H, 3.82; N, 6.24; S, 7.14 Found: C, 64.27; H, 3.80; N, 6.21; S, 7.11. 2.1.5.4 1, 145.0, 143.2, 138.2, 137.8, 131.9, 131.0, 130.6, 129.5, 128.8, 128.7, 128.4, 127.7, 126.8, 126.3, 125.8, 123.0, 115.7, 114.2, 51.8, 20.6; IR (cm -1 , KBr): 3245, 3078, 1650, 1446, 1423, 1223, 1130; Anal. Calcd. for C25H20N2O3S: C, 70.07; H, 4.70; N, 6.54; S, 7.48 Found: C, 70.10; H, 4.73; N, 6.50; S, 7.45. 2.1.5.5

Spectral data ( 1 H-NMR and 13 C-NMR spectra) of the synthesized compounds

2.1.5.1. (E)-N-(1-phenyl-1H-naphtho
. (E)-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide
. (E)-4-methyl-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)- ylidene)benzenesulfonamide (7e)
M.p.: 203-205 °C; yield: 43%; 1 H NMR (400 MHz, DMSO-d6): δ 10.08 (s, 1H), 8.01 (d,J = 9.0 Hz,1H),7.96 (d,J = 7.1 Hz,1H),7.76 (d,J = 8.0 Hz,3H),2H),7.33 (d,J = 9.0 Hz,1H),7.29 (d,J = 8.0 Hz,2H),4H),6.25 (s,1H), 2.34 (s, 3H), 2.22 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 151.9, 145.3, 141.7, 140.6, 138.6, 137.5, 130.8, 130.4, 129.4, 129.0, 128.7, 128.5, 127.6, 126.8, 126.5, 125.6, 123.0, 115.9, 114.4, 52.0, 20.9, 20.6; IR (cm -1 , KBr): 3244, 3067, 1651, 1435, 1219, 1161, 1134; Anal. Calcd. for C26H22N2O3S: C, 70.57; H, 5.01; N, 6.33; S, 7.25 Found: C, 70.61; H, 5.03; N, 6.30; S, 7.21. 2.1.5.6 224225226yield: 55%;1 H NMR (400 MHz,1H),7.89 (d,J = 8.7 Hz,2H),3H),2H),7.34 (d,J = 8.5 Hz,2H),7.23 (d, J = 8.9 Hz, 1H), 6.99-6.92 (m, 4H), 6.07 (s, 1H), 2.21 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 150. 8, 146.9, 143.4, 140.9, 136.2, 135.1, 130.4, 129.2, 129.1, 128.9, 128.6, 128.5, 127.8, 127.0, 126.7, 124.7, 122.9, 116.3, 115.3, 53.6, 20.6; IR (cm -1 , KBr): 3290, 3074, 1651, 1423, 1335, 1219, 1011;Anal. Calcd. for C25H19ClN2O3S: C, 64.86;H, 4.14;N, 6.05;S, 6.93 Found: C, 64.80;H, 4.11;N, 6.07;S, 6.98. 2.1.5.7

. (E)-4-chloro-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)- ylidene)benzenesulfonamide (7f) M.p.:
. (E)-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4- methylbenzenesulfonamide (7g)
M.p.: 255-257 °C; yield: 51%; 1 H NMR (400 MHz, DMSO-d6): δ 10.07 (s, 1H), 8.03 (d, J = 9.0 Hz, 1H), 8.00-7.96 (m, 1H), 7.82-7.77 (m, 3H), 7.56-7.47 (m, 2H), 7.38-7.31 (m, 3H), 7.18 (d, J = 8.3 Hz, 2H), 7.12 (d, J = 8.3 Hz, 2H), 6.29 (s, 1H), 2.81 (sep, J = 6.9 Hz, 1H), 2.35 (s, 3H), 1.13 (dd, J = 6.9 Hz, 1.9 Hz, 6H); 13 C NMR (100 MHz, DMSO-d6): δ 152. 1, 148.6, 145.1, 142.0, 140.4, 138.6, 130.9, 130.6, 129.2, 128.7, 128.4, 127.8, 126.94, 126.87, 126.4, 125.8, 123.0, 115.8, 114.3, 51.7, 33.0, 23.7, 23.6, 20.9; IR (cm -1 , KBr): 3240, 3090, 1643, 1419, 1304, 1161, 1134;Anal. Calcd. for C28H26N2O3S: C, 71.46;H, 5.57;N, 5.95;S, 6.81 Found: C, 71.49;H, 5.52;N, 5.97;S, 6.85. 2.1.5.8 (d, J = 8.3 Hz, 2H), 6.31 (s, 1H), 2.81 (sep, J = 6.9 Hz, 1H), 1.13 (dd, J = 6.9 Hz, 1.6 Hz, 6H); 13 C NMR (100 MHz, DMSO-d6): δ 152. 3, 148.6, 145.0, 142.0, 138.6, 136.7, 131.0, 130.6, 128.9, 128.7, 128.5, 128.4, 127.8, 126.95, 126.88, 125.8, 123.1, 115.7, 114.2, 51.8, 33.0, 23.65, 23.57; IR (cm -1 , KBr): 3225, 3080, 1643, 1477, 1311, 1219, 1134;Anal. Calcd. for C27H23ClN2O3S: C, 66.05;H, 4.72;N, 5.71;S, 6.53 Found: C, 66.01;H, 4.76;N, 5.67;S, 6.57. 2.1.5.9

. (E)-4-chloro-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)- ylidene)benzenesulfonamide (7h) M.
. (E)-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4- methoxybenzenesulfonamide (7i)
M.p.: 228-230 °C; yield: 46%; 1 H NMR (400 MHz, DMSO-d6): δ 10.01 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 8.00-7.97 (m, 1H), 7.84 (d, J = 8.9 Hz, 2H), 7.82-7.79 (m, 1H), 7.56-7.48 (m, 2H), 7.38 (d, J = 9.0 Hz, 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 7.05 (d, J = 8.9 Hz, 2H), 6.28 (s, 1H), 3.81 (s, 3H), 2.81 (sep, J = 6.9 Hz, 1H), 1.13 (dd, J = 6.9 Hz, 1.8 Hz, 6H); 13 C NMR (100 MHz, DMSO-d6): δ 161. 7, 152.0, 148.5, 145.1, 138.7, 135.0, 130.9, 130.6, 128.7, 128.5, 128.4, 127.8, 126.9 126.8, 125.8, 123.0, 115.9, 114.4, 113.8, 55.5, 51.7, 33.0, 23.65, 23.61 9, 145.4, 143.2, 140.3, 132.9, 131.8, 130.9, 130.7, 129.0, 128.8, 128.75, 128.69, 128.4, 127.8, 126.4, 125.8, 122.9, 115.9, 113.7, 51.5 (s, 1H), 2.34 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 152. 1, 145.2, 142.2, 140.0, 139.9, 133.0, 130.95, 130.91, 129.2, 129.1, 128.9, 128.8, 128.3, 127.9, 126.4, 125.9, 122.9, 115.9, 113.6, 51.3, 20.9; IR (cm -1 , KBr): 3294, 3047, 1635, 1411, 1377, 1234, 1080;Anal. Calcd. for C25H19ClN2O3S: C, 64.86;H, 4.14;N, 6.05;S, 6.93 Found: C, 64.82;H, 4.13; N, 6.08; S, 6.95. 9, 148.4, 144.6, 144.5, 134.1, 130.7, 130.0, 129.6, 128.8, 128.6, 128.5, 128.4, 127.8, 127.1, 126.7, 124.0, 122.6, 116.8, 115.5, 54.3 4, 151.6, 145.9, 141.0, 135.4, 132.5, 130.7, 130.4, 128.8, 128.7, 128.6, 128.5, 127.6, 125.5, 122.8, 116.1, 114.1, 113.5, 55.4, 51.9 145.3, 142.3, 136.3, 133.7, 130.9, 130.3, 128.7, 128.52, 128.49, 128.2, 127.6, 125.6, 123.0, 115.8, 114.4, 114.2, 55.0, 51.9; IR (cm -1 , KBr): 3240, 3086, 1643, 1438, 1254, 1161, 1080;Anal. Calcd. for C25H19ClN2O4S: C, 62.69; H, 4.00; N, 5.85; S, 6.69 Found: C, 62.65; H, 4.02; N, 5.87; S, 6.66. 151.7, 145.6, 135.4, 134.0, 130.8, 130.2, 128.64, 128.60, 128.5, 128.2, 127.5, 125.4, 123.0, 116.0, 114.7, 114.1, 113.6, 55.4, 55.0 51.9; IR (cm -1 , KBr): 3271, 3067, 1643, 1496, 1261, 1153, 1084; Anal. Calcd. for C26H22N2O5S: C, 65.81; H, 4.67; N, 5.90; S, 6.76 Found: C, 65.85; H, 4.63; N, 5.96; S, 6.72.

Biology

In vitro investigation of antimicrobial activity

Microorganisms

The antimicrobial activities of the synthetic compounds against four American Type Culture

Determination of minimum inhibitory concentration

MICs were determined using broth microdilution method recommended by the CLSI with some modifications (Clinical and Laboratory Standards Institute, 2006a, 2006b, 2006c). Briefly, for determination of antifungal activities against fungi, serial dilutions of the synthesized compounds 7a-o (0.25-256.0 µg/ml) were prepared in 96-well microtiter plate using RPMI-1640 media (Sigma, St. Louis, USA) buffered with MOPS (Sigma, St. Louis, USA). To determine the antibacterial activities, serial dilutions of the synthesized compounds 7a-o (0.25-256.0 µg/ml) were prepared in Muller-Hinton Broth media (Merck, Darmstadt, Germany). The test fungi or bacteria strains were suspended in the media and the cell densities were adjusted to 0.5 McFarland standards at 530 nm wavelength using a spectrophotometric method (this yields stock suspension of 1-5 × 106 cells/ml for yeast and 1-1.5 × 108 cells/ml for bacteria). One hundred microliters of the working inoculums was added to the microtiter plates which were incubated in a humid atmosphere at 30 °C for 24-48 h (fungi) or at 37 °C for 24 h (bacteria). Two hundred microliters of the uninoculated medium was included as a sterility control (blank). In addition, growth controls (medium with inoculums but without drugs) were also included. The growth in each well was compared with that of the growth control well. MICs were visually determined and defined as the lowest concentration of the synthetic compounds or antibiotics that prevents any visible growth (MIC90) of microorganisms (both bacteria and fungi). For fungi MIC50 were also determined and defined as the lowest concentration of the compounds that inhibits about half, or 50% growth of fungal cells than that of the controls. Each experiment was performed in triplicate. The reference antibiotics, fluconazole (Sigma, St.

Louis, MO, USA), griseofulvin (sigma)

and ciprofloxacin (sigma) were used as positive controls against saprophytes, dermatophytes and bacteria, respectively.

In addition, media from wells with fungi showing no visible growth were further cultured on Sabouraud Dextrose Agar (Merck, Darmstadt, Germany) and from wells with bacteria showing no visible growth on Muller-Hinton agar (Merck, Darmstadt, Germany) to determine the minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC). MBCs and MFCs were determined as the lowest concentration yielding no more than 4 colonies, which corresponds to a mortality of 99.9% of the microbes in the initial inoculums.

Determination of cytotoxicity

Cell line and culture conditions

The human breast (MCF-7), human colon (HCT116) and human B-CLL (Waco3-CD5) cancer cell lines were used in the study. We used RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin for cell growth under the condition of 37 °C in 5% CO2.

Peripheral blood mononuclear cells (PBMC) were obtained by venipuncture from healthy adult donors and mononuclear cells were isolated by Ficol1-Hypaque gradient separation. Lymphocytes were washed and resuspended in RPMI medium supplemented with 10% fetal bovine serum.

RPMI-1640 medium was used for B-CLL and DMEM medium for others. 5-Fluorouracil (5-FU)

and Rituximab were employed as positive control drugs. Rituximab (Mabthera) drug was obtained from Roche Company and 5-FU was purchased from Merck Company.

Cytotoxic activity

MTT assay

The MTT assay was used to evaluate the cytotoxicity of novel synthesized derivatives (7a-o) on cell viability (Liuet al., 1997). When cells were in exponentially growing phase, seeded at a concentration of 10,000 cells/well on 96 tissue culture plate overnight. After the culture medium replaced with a fresh one, a solution of synthesized compounds in DMSO was added to reach various concentrations. After 48 h, the drug solutions discarded and cells were washed with PBS.

Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml, in PBS)

was added into each well and cultured for another 4 h. Subsequently, the supernatant replaced with DMSO to soften the formed formazan. In parallel, we used negative control, blank and positive control (5-FU and Rituximab) in the same conditions. At the final step, absorbance at the wavelength of 540 nm was read by a microplate reader. Data was calculated and expressed as the 50% inhibitory concentrations (IC50) and presented as mean ± SD, n = 3. The MTT assay was also carried out on the non-cancer cells of PBMC as descried for the cancer cell lines. As for the PBMC cells, lymphocytes were washed, resuspended in RPMI medium supplemented with 10% fetal bovine serum and plated (1 × 10 6 cells/mL) in the presence of phytohemagglutinin. After incubation for 24 h at 37 °C and 5% CO2, the cells were treated with a solution of synthesized compounds in DMSO in various concentrations. (0.1-20 mmol L21) for 48 h. Cytotoxicity of violacein was determined by the MTT assay.

Results and discussion

Chemistry

A new route for the synthesis of novel 1-aryl-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine derivatives bearing a sulfonamide moiety was accomplished in five steps starting from readily available chemicals in EtOH-H2O as an environmentally benign medium with good to excellent yields. The synthetic approach is outlined in Scheme 1. The Betti bases 3a-e required for the synthesis of 1,3-naphthoxazines were obtained by the Betti reaction through a known method [START_REF] Betti | b-Naphtholphenylaminomethane[END_REF]. Also, N-sulfonyldithioimidocarbonates 6a-d were synthesized according to a reported procedure starting from arylsulfonamides 5a-d [START_REF] Maybhate | Regiospecific synthesis of N-sulfonyl derivatives of 3,5-diamino-1H-1, 2,4-triazole and 2,5-diamino-1,3,4thiadiazole[END_REF]. The structure of compounds 7a-o were proved by 1 H NMR, and 13 C NMR spectra, alongside the X-ray single crystallography method. 1 H NMR spectrum of 7a revealed the presence of a singlet at 10.17 ppm which could be assigned to NH and a singlet at 6.34 ppm attributed to the only aliphatic C H of the compound 7a. Absolute assignment of compound 7a was achieved by single-crystal X-ray crystallography (Fig. 2, CCDC 1405842). Single crystals of The crystallographic data reveal that compound 7a crystallized in a monoclinic system, space group P21/n. The asymmetric unit of 7a contains a whole molecule (Fig. 3). All bond lengths and angles are in the normal ranges and comparable with the reported ones [START_REF] Gelbrich | Polymorph VI of sulfapyridine: interpenetrating two-and threedimensional hydrogen-bonded nets formed from two tautomeric forms[END_REF][START_REF] Selig | 4-Chloro-1-(4-methylphenylsulfonyl)-1Hpyrrolo[2,3-b]pyridine[END_REF][START_REF] Pfaffenrot | N-[(3RS,4SR)-1-Benzyl-4-methylpiperidin-3-yl]-1-(4-methylphenylsulfonyl)-5-nitro1Hpyrrolo[2,3-b]pyridin-4-amine[END_REF].

Fig. 3. A representation of intramolecular and intermolecular hydrogen bonds in molecule of 7a.

H atoms have been omitted for clarity except hydrogens that participate in hydrogen bonds.

The hydrogen-bonding pattern of compound 7a can be described using graph theory [START_REF] Bernstein | Patterns in hydrogen bonding: functionality and graph set analysis in crystals[END_REF][START_REF] Grell | Graph-set analysis of hydrogen-bond patterns: some mathematical concepts[END_REF]. For this structure, there are a intramolecular hydrogen bond of the N H⋯O type (dashed line a in Fig. 3), which is described as S( 6) ring in the first level graph set (Fig. 3).

There are also two intermolecular hydrogen bonds of O⋯H C and O⋯H N types in the structure that create a dimer (dashed line b and c in Fig. 2) (Table 1). According to graph theory, a R 2 2(7) ring is formed by two intermolecular hydrogen bonds (Fig. 3). According to the hydrogen bonding classification (Steiner, 2002, Desiraju and[START_REF] Desiraju | The Weak Hydrogen Bond in Structural Chemistry and Biology[END_REF], these intramolecular and intermolecular hydrogen-bonding are moderate electrostatic interactions.

Biology

Antimicrobial activity

In the present study, the antibacterial and antifungal effects of newly synthesized compounds on two bacteria (one gram positive and one gram negative) and various fungi were analyzed (Table 2).

Although [START_REF] Verma | Synthesis, antimicrobial and cytotoxicity study of 1,3disubstituted-1Hnaphtho[1,2-e][1,3]oxazines[END_REF] reported antibacterial activity for their naphthoxazine derivatives, none of the examined compounds in this study exhibited antibacterial activity against the tested bacteria at the concentrations up to 256 µg/mL. The difference in antibacterial activities between their compounds and ours might be attributed to presence of an aryl group in their compounds instead of an arylsulfonamide moiety. Antibacterial inactivity of our synthesized compounds 7a-o also might be due to lack of an essential p-amino substituent in the aromatic ring at R 2 position, which is quite necessary for the antibacterial role of arylsulfonamides.

Among several tested fungi, the compounds 7a-o only exhibited notable inhibitory activities against Candida albicans (yeasts) with MICs ranging from 32-256 µg/mL.

Compounds7c and 7l with a chloro substituent as an electron withdrawing group at the R 2 position exhibited the most anti Candida activity with MIC of 32 µg/mL. A comparison between 7c, 7f, 7l, and 7n which have a chloro substituent at R 2 position and respectively an H, CH3, Cl and/or OCH3 at R 1 position, revealed that existence of an electron withdrawing group at R 1 position enhances antifungal activity of the final product compared to electron donating groups. Moreover, comparison of 7b and 7g as well as 7c and 7h revealed that placement of a bulky group such as isopropyl at R 1 position might decrease the anti-Candida activity of the title compounds. We found that, placement of a halogen such as chloro as an electron withdrawing group in R 2 position leads to more effective compounds in terms of antifungal activity. As shown in Table 3, lack of a substituent at para position of arylsulfonamide moiety (compounds 7a, 7d, and 7j) results in lessening the inhibitory activity against the Candida albicans and the best result was obtained when there is a methyl group at this position (7e). These data show that, this class of compounds could be regarded as fungistatic antifungal agents.

Table 3. Investigation of antimicrobial activities of compounds 7a-o.

Empty Cell

Minimum inhibitory concentration (MIC) (µg/ml sample)

Microorganisms E. coli S. aureus C. albicans A. fumigatus E. floccos

Entry R 1 R 2 MIC90 MBC MIC90 MBC MIC50 MIC90 MFC MIC50 MIC90 MFC MIC50 M 7a H H G G G G 32 128 G G G G G G 7b H CH3 G G G G 128 256 G G G G G G 7c H Cl G G G G 16 32 G G G G G G Microorganisms E. coli S. aureus C. albicans A. fumigatus E. floccos Entry R 1 R 2 MIC90 MBC MIC90 MBC MIC50 MIC90 MFC MIC50 MIC90 MFC MIC50 M GF - - - - - - - - - - 0.5 1 CIPX 0.025 G 0.5 G - - - - - - - -
G: >256 µg/mL; FCZ: Fluconazole; GF: Griseofulvin; CIPX: Ciprofloxacin.

Cytotoxic activity

All the compounds were tested in vitro against a 3-cell line panel consisting of HCT116, ATCC

No.: CCL-247 (human colon cancer), MCF-7, ATCC No.: HTB-22 (human breast cancer), and Waco3-CD5, NCBI No.: C547 (human chronic lymphocytic leukemia) and their IC50 values were obtained. The MTT assay was also carried out on the non-cancer cells of PBMC as descried for the cancer cell lines. Results of IC50 experiment demonstrated that all of the compounds prepared in this study showed more inhibitory effect on colon and breast cancer cell lines compared to B-CLL cell line. Among the tested compounds, 7j and 7l showed the most cytotoxic effects on breast and colon cancer cell lines whereas their cytotoxicity on Waco3-CD5 was negligible. The lowest cytotoxic effects were achieved by the compound 7f which showed the lowest toxicity on all the cell lines tested. The presence of electron withdrawing chloro group at either R 1 or R 2 position improved the anticancer activity in these structures, but the combination of CH3 and Cl at R 1 and R 2 positions might decrease the cytotoxicity of these compounds (Table 4). The results of cytotoxicity assay on the non-cancer cells of PBMC demonstrated that the compounds prepared in this investigation had less toxic effects on non-cancer cells in comparison with the cancer cell lines. The compounds of 7g and 7i exhibited the lowest toxic effects on PBMC cells whereas the 7a, 7n and 7o showed the highest toxicity on PBMC cells. However, all of the compounds prepared in this study showed higher toxicity on PBMC cells compared with 5FU. The high toxicity of rituximab on PBMC cell could be explained by the fact that the phytohemagglutinin used for the preparation of the cells results in the over expression of CD20 protein on the surface of the cells which acts as the receptor for rituximab. Therefore, the normal cells with the receptor for the cytotoxic drug could be affected. As shown in Table 4, the cytotoxic effects of 7j and 7l against breast cancer cell line as well the 7d, 7e, 7j, 7l, and 7m against colon cancer cells were comparable with the standard anticancer drug 5-FU. However, cytotoxic activities of the compounds 7a-o against human B-CLL (Waco3-CD5) were lower than the corresponding standard drug Rituximab (with minimal IC50 approximately 3 folds higher than the positive control).

Conclusion

In this study we reported the synthesis, characterization and anticancer activities evaluation of new 1-aryl-2,3-dihydro-1H-naphtho [1,2-e][1,3] showed that, all the compound were less toxic on the normal cells of PBMC rather than the cancer cell lines which make them great candidates for further investigations. However, The MTT test could not be able to show the mechanism by which the compounds induce cytotoxicity on different cells including normal or cancer cell lines. Therefore, it is suggested to carry out the investigations on the mechanism of cytotoxic activity of the compounds to determine the potential role of different functional groups on the cytotoxic mechanism.

In vitro evaluations of the compounds 7a-o revealed that they presented only notable antifungal activities against Candida albicans (yeasts). The most of compounds 7a-o showed moderate antifungal activities but among them compounds 7c, 7e, 7f, 7i, 7l, 7m, and 7n exhibited the highest activities. These results are suggesting that the synthesized compounds can be good nominees for future investigations to find new anticancer and antifungal compounds.

  .: 244-246 °C; yield: 70%; 1 H NMR (400 MHz, DMSO-d6): δ 10.27 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 8.00-7.96 (m, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.81-7.77 (m, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.55-7.47 (m, 2H), 7.37 (d, J = 9.0 Hz, 1H), 7.34-7.26 (m, 3H), 7.21

  .: 271-273 °C (decomp.); yield: 54%; 1 H NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.99-7.95 (m, 1H), 7.90 (d, J = 7.0 Hz, 2H), 7.79-7.75 (m, 1H), 7.61-7.47 (m, 5H), 7.32 (d, J = 9.0 Hz, 1H), 7.13-7.07 (m, 4H), 6.29 (s, 1H), 2.23 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 152.

  p.: 262-264 °C; yield: 55%; 1 H NMR (400 MHz, DMSO-d6): δ 10.22 (s, 1H), 8.05 (d, J = 9.0 Hz, 1H), 8.01-7.97 (m, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.56-7.48 (m, 2H), 7.37 (d, J = 9.0 Hz, 1H), 7.18 (d, J = 8.3 Hz, 2H), 7.12

  2.1.5.10. (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)ylidene)benzenesulfonamide (7j) M.p.: 226-228 °C; yield: 63%; 1 H NMR (400 MHz, DMSO-d6): δ 10.20 (s, 1H), 8.03 (d, J = 9.0 Hz, 1H), 8.00-7.96 (m, 1H), 7.90-7.87 (m, 2H), 7.78-7.74 (m, 1H), 7.59-7.47 (m, 5H), 7.37 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 9.0 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 6.35 (s, 1H); 13 C NMR (100 MHz, DMSO-d6): δ 151.

  2.1.5.11. (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4methylbenzenesulfonamide (7k) M.p.: 249-251 °C (decomp.); yield: 47%; 1 H NMR (400 MHz, DMSO-d6): δ 10.05 (s, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.77 (d, J = 7.8 Hz, 2H), 7. 1H), 2H), J = 8.4 Hz, 3H), 7.30 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 6.36

2. 1

 1 .5.12. (E)-4-chloro-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)ylidene)benzenesulfonamide (7l) M.p.: 242-244 °C (decomp.); yield: 75%; 1 H NMR (400 MHz, DMSO-d6): δ 10.25 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.66(d, J = 8.5 Hz, 2H), 2H), 7.20 (d, J = 8.5 Hz, 2H), 7.15 (m, 1H), 7.12 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 5.97 (s, 1H); 13 C NMR (100 MHz, DMSO-d6): δ 149.

2. 1

 1 .5.13. (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4methoxybenzenesulfonamide (7m) M.p.: 235 °C (decomp.); yield: 65%; 1 H NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7 3H), 2H), 3H), 7.17 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 6.28 (s, 1H), 3.79 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 161.

  ; IR (cm -1 , KBr):3290, 3070, 1643, 1497, 1338, 1257, 1084; Anal. Calcd. for C25H19ClN2O4S: C, 62.69; H, 4.00; N, 5.85; S, 6.69 Found: C, 62.66; H, 4.04; N, 5.81; S, 6.73. 2.1.5.14. (E)-4-chloro-N-(1-(4-methoxyphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)ylidene)benzenesulfonamide (7n) M.p.: 193-195 °C; yield: 48%; 1 H NMR (400 MHz, DMSO-d6): δ 10.20 (s, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.99-7.95 (m, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 7.5 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.53-7.45 (m, 2H), 7.34 (d, J = 9.0 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 6.24 (s, 1H), 3.69 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 158.9, 151.9,

2. 1

 1 .5.15. (E)-4-methoxy-N-(1-(4-methoxyphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)ylidene)benzenesulfonamide (7o) M.p.: 182-184 °C; yield: 55%; 1 H NMR (400 MHz, DMSO-d6): δ 10.02 (s, 1H), 8.01-7.97 (d, J = 9.0 Hz, 1H), 7.97-7.94 (m, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.78-7.74 (m, 1H), 7.53-7.47 (m,2H), 7.33 (d, J = 9.0 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 6.21 (s, 1H), 3.79 (s, 3H), 3.68 (s, 3H); 13 C NMR (100 MHz, DMSO-d6): δ 161.4, 158.8,

  Collection (ATCC) microorganisms, including Candida albicans (ATCC 10261), A. fumigatus (ATCC 14110), Staphylococcus aureus (ATCC 700698), and Escherichia coli (ATCC 25912) were determined. Moreover, three clinical isolates of dermatophytes including Epidermophyton flocossum (SUCC 93-552), Microsporumcanis (SUCC 93-560)and Trichophytonrubrum (SUCC 93-565) were also tested in this study. The susceptibility of all isolated bacteria and fungi against selected antibiotics were examined by microdilution methods(Clinical and Laboratory Standards Institute, 2006a, Clinical and Laboratory Standards Institute, 2006b, Clinical and Laboratory Standards Institute, 2006c).

Scheme 1 .

 1 Scheme 1. Synthetic approach for the preparation of Betti bases (A), Nsulfonyldithioimidocarbonates (B), and 1-aryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine derivatives (C).

Fig. 1 .

 1 Fig. 1. One-step preparation of 1-aryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine derivatives. ( a Isolated yields;) ( b Reaction conditions: 1-(amino(aryl)methyl)naphthalen-2-ol hydrochloride 3 (1 mmol), dimethyl arylsulfonylcarbonimidodithioate6 (1 mmol) and Na2CO3 (3 mmol) in H2O/EtOH (1: 3, 5 mL), reflux condition, 2 h.)

Fig. 2 .

 2 Fig. 2. ORTEP representation of 7a. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small spheres of arbitrary radii.

  bases in the presence of Na2CO3 as a cheap base and in aqueous ethanol as a green medium. To study the potential anticancer and antifungal activities of the synthesized compounds, the screening tests were performed for three cancer cell lines and five fungi strains. The best anticancer activity were presented by 7j and 7l against all three kinds of cancer cell lines [HCT116, ATCC No.: CCL-247 (human colon cancer), MCF-7, ATCC No.: HTB-22 (human breast cancer), and Waco3-CD5, NCBI No.: C547 (human B-CLL)]. The results

  ; IR (cm-1 , KBr): 3290, 3067, 1635, 1488, 1250, 1153, 1080; Anal. Calcd. for C24H16Cl2N2O3S: C, 59.64; H, 3.34; N, 5.80; S, 6.63 Found: C, 59.62; H, 3.31; N, 5.85; S, 6.61.

Table 1 .

 1 Intramolecular and intermolecular hydrogen bond geometries (Å, °) for 7a.

					H⋯A	D⋯A
	D	H⋯A	D	H			D	H⋯A
			0.800(18)	2.543(18)	3.2175(19)	142.9(16)
	N(1)	H(1)⋯O(3) #
			0.800(18)	2.235(17)	2.8462(18)	133.6(16)
	N(1)	H(1)⋯O(2)
			0.98		2.38	3.2780(18)	151.6
	C(11)	H(11)⋯O(2) #
	#
		Symmetry transformations used to generate equivalent atoms: -x - 1/2, y + 1/2, -z + 3/2.
	The selected crystal structure data are presented in Table 2.
	Table 2. Selected bond lengths (Å) and angles (°) for 7a.

Table 4 .

 4 Cytotoxicity evaluation of new compounds 7a-o against three different cancer cell lines and PBMC cells.

	Empty Cell	Empty Cell	PBMC	Breast	Colon	B-CLL
	Empty Cell	Empty Cell	Normal	(MCF-7)	(HCT116)	(Waco3-CD5)
	Compd.	R 1	Empty Cell	IC50(µM)
	7a	H	62.20 ± 2.9	35.11 ± 4.3	28.56 ± 2.4	53.56 ± 3.3
	7b	H	80.56 ± 1.7	47.85 ± 0.6	54.93 ± 1.0	91.29 ± 0.6.0
	7c	H	94.01 ± 3.4	38.37 ± 2.3	34.36 ± 2.3	88.11 ± 1.8
	7d	CH3	78.62 ± 1.8	37.11 ± 3.1	21.18 ± 1.8	76.15 ± 4.6
	7e	CH3	83.31 ± 3.9	37.14 ± 2.5	22.14 ± 1.1	78.12 ± 0.1
	7f	CH3	106.74 ± 2.8	96.31 ± 7.0	93.05 ± 1.7	95.57 ± 0.9
	7g	Isopropyl	127.92 ± 3.6	62.25 ± 6.1	71.92 ± 0.2	92.97 ± 0.2.7
	7h	Isopropyl	93.47 ± 4.2	46.87 ± 3.8	68.28 ± 4.6	89.96 ± 3.6
	7i	Isopropyl	129.1 ± 2.9	46.06 ± 0.5	60.41 ± 2.4	108.39 ± 5.9
	7j	Cl	82.02 ± 3.7	16.14 ± 1.4	19.72 ± 2.6	49.11 ± 2.9

Acknowledgements

We are thankful to the University of Isfahan Research Council and Shiraz University of Medical Sciences Research Council for financial support of this work.

Microorganisms E. coli S. aureus C. albicans A. fumigatus E. floccos

Conflict of interest

The authors have declared no conflicts of interest.