Ph.D Philippe Sarrazin
email: philippe.sarrazin@ujf-grenoble.fr

Keywords: pessimistic, optimistic, explanatory style, resilience, sport

Some athletes habitually explain bad events with causes that are stable in time and global in effect, and explain good events with causes that are unstable and specific. This pessimistic explanatory style constitutes a dispositional risk factor likely to lead to lower expectations of success, to increased anxiety, and to poor achievement. 62 participants (mean age 14 years) performed a basketball dribbling trial and were given false feedback indicating that they had failed. Consistent with prediction, in a second trial, the optimistic participants (N = 22) were less anxious (assessed by heart rate acceleration), more confident, and performed better than pessimistic participants (N = 20). A third group with a neutral explanatory style (N = 20) obtained scores which were between the two other groups.

Explanatory Style and Resilience after Sports Failure Why do some athletes rebound after failure, whereas others give up? What is the mechanism which explains why some athletes who have been seriously injured later become champions? Why do other athletes drop out of their sport following a setback? One key psychological factor influencing how athletes react to adversity may be explanatory style [START_REF] Peterson | Causal explanations as risk factor for depression: Theory and evidence[END_REF][START_REF] Peterson | Meaning and measurement of explanatory style[END_REF]; see [START_REF] Buchanan | Explanatory Style[END_REF] for a review).

Originally proposed in the context of the attributional reformulation of learned helplessness theory [START_REF] Abramson | Learned Helplessness in Humans: Critique and Reformulation[END_REF], explanatory style reflects the way that people usually explain disparate bad or good events (e.g., [START_REF] Peterson | The future of optimism[END_REF][START_REF] Peterson | Learned helplessness and explanatory style[END_REF][START_REF] Peterson | Explanatory style, expectations, and depressive symptoms[END_REF][START_REF] Peterson | Optimistic explanatory style[END_REF]. People who usually explain bad events by causes that are stable in time ("it's going to last forever"), global in effect ("it's going to undercut everything that I do"), and internal ("it's me") and who explain good events with unstable, specific, and external causes are said to have a pessimistic explanatory style. People with the opposite attributional pattern are said to have a optimistic explanatory style.

Explanatory style has been extensively studied as a correlate of helplessness-related outcomes such as depression, illness, and failure in academic athletic and vocational realms.

People with an optimistic explanatory style fare usually better than those with a pessimistic explanatory style [START_REF] Peterson | Learned helplessness and explanatory style[END_REF]. To our knowledge, only five studies have investigated the link between explanatory style and athletic performance. In a cross-sectional survey with 50 young elite tennis players, [START_REF] Prapavessis | Learned helplessness in sport[END_REF] found that players presenting cognitive, motivational, and emotional maladaptive achievement patterns (evaluated by a questionnaire) gave ratings that were internal, persistent, and recurrent for explaining failure performances, and were judged by their coaches to be less persistent in their matches. Nevertheless the cross-sectional nature of the design makes the inference of causality difficult between the attributional style and the maladaptive achievement patterns.

Two other studies using Content Analysis of Verbatim Explanation (CAVE; see [START_REF] Peterson | Learned helplessness and explanatory style[END_REF] were reported by Retew and Reivich (1995). Baseball and basketball teams with a more optimistic explanatory style won more games in the target season, and performed significantly better in games following a loss than teams with a pessimistic explanatory style. Nevertheless because the CAVE technique measures explanatory style only in an indirect way, and as it employs retrospective data, this design cannot unambiguously support a causal association between explanatory style and athletic performance.

In prospective (study 1) and quasi-experimental (study 2) research with members of highly ranked U.S. university swimming teams, [START_REF] Seligman | Explanatory style as a mechanism of disappointing athletic performance[END_REF] explored further the links between explanatory styles and sporting performance. Results showed that (1) attributional style and coaches' judgements of swimmers' resilience after defeat predicted how many unexpectedly poor swims the team members would go on to show over the season (study 1), and (2) after a failure feedback given by their coach (i.e., a bad swim time), times on a second swim thirty-minutes later were poorer for pessimistic, but not for optimistic swimmers (study 2). This latter study was the first attempt to test recovery after failure as a function of the explanatory style, via a quasi-experimental design. However, to our knowledge it has not been replicated. The present experiment was designed to fill that gap, and had two primary aims.

First, we wanted to extend the internal and external validity of the Seligman et al. study by (1) using ordinary high school (in competitive swimmers place) in order to test the generality of the former results on a less elite population, (2) comparing the optimistic and pessimistic pattern of outcomes with a more "neutral" attributional profile, and (3) including several variables of control likely to influence athletic performance like ability, gender, perceived ability (e.g., [START_REF] Cury | Achievement goals and perceived ability predict investment in learning a sport task[END_REF] and importance to succeed (e.g., [START_REF] Reeve | Elements of the competitive situation that affect intrinsic motivation[END_REF].

The second, and more important, aim of the present research was to more thoroughly examine the processes by which explanatory style affects performance following failure.

According to the attributional reformulation (e.g., [START_REF] Abramson | Learned Helplessness in Humans: Critique and Reformulation[END_REF] explanatory style is not itself a direct cause but rather a "distal" dispositional risk factor for helplessness following failure (Peterson et al., 2001;[START_REF] Peterson | Optimistic explanatory style[END_REF]. Several factors can be hypothesized as being the proximal causes of helplessness vs. recovery, even though research in this tradition has rarely looked at these mediating variables [START_REF] Peterson | Optimistic explanatory style[END_REF]. In the present research, we investigated the role of two potential mediator variables: success expectation and state anxiety.

Success expectation represents the perceived chances of performing well on the task.

We expect that individuals with a pessimistic explanatory style will perform more poorly after a negative outcome than people with an optimistic explanatory style, because they attribute the failure to a stable cause (such as their own lack of ability) and come to expect that negative events will be pervasive and enduring [START_REF] Helton | Optimism, pessimism, and false failure feedback effects on vigilance performance[END_REF].

As a result, their expectation of success will be lowered, and will lead to poorer performance [START_REF] Bandura | Social foundations of thought and action: A social cognitive theory[END_REF].

Explanatory styles are not just a purely cognitive disposition concerning future events; they entail an emotional component as well. Several studies found that a pessimistic explanatory style correlated positively with anxiety (e.g., [START_REF] Helton | Optimism, pessimism, and false failure feedback effects on vigilance performance[END_REF][START_REF] Mineka | Explanatory Style in Anxiety and Depression[END_REF]. The feeling of a lack of control over the situations which be characteristic of the pessimistic profile can increase the perceived threat and in turn the individual's state anxiety (e.g., McGrath, 1970), which in turn will alter the availability of certain cognitive and physiological resources to performers (e.g., [START_REF] Parfitt | Somatic anxiety and physiological arousal: their effects upon a high anaerobic, low memory demand task[END_REF].

To recapitulate, this study was patterned after the [START_REF] Seligman | Explanatory style as a mechanism of disappointing athletic performance[END_REF] swimming study in order to test resilience ability after failure in a motor task (basketball dribbling), as a function of explanatory style. We expected that after failure, pessimistic style leads (1) to lower expectations of success, (2) to more state anxiety (somatic arousal) and in turn, (3) to poorer achievement in the second test than an optimistic style, controlling for variables likely to influence motor performance such as ability, gender, perceived competence and importance to succeed. Lastly, we expected that a "neutral" explanatory style will obtain scores of expectation, anxiety and recovery located between the two other groups.

Method

Participants Students (N=278) from a suburban school near a large town in France, largely of high socio-economic status, volunteered to answer a questionnaire assessing attributional style in sport at the beginning of the year. One month later, 62 of them were selected according to the procedure explained below. These 33 boys and 29 girls were 14 to 16 years old (M = 14 years, SD = .76) and had been practicing basketball for at least one year as part of sport classes in the school. Participation was voluntary, but no pupil declined. Moreover, parental and school administrator permission was requested before starting the investigation.

Experimental task

Participants were requested to perform a basketball dribbling exercise. The course was made up of a series of obstacles that the performer had to negotiate while dribbling a basketball. The test consisted of two timed attempts interrupted with a rest and a training period.

Questionnaire Measures Explanatory Style

Although explanatory style is conceptualized as a trait, some theorists (e.g., Cutrona, Russell, & Jones, 1985) have recommended a domain-specific assessment of this construct.

Therefore, a sport-specific measure was used. Participants completed a modified French version of the Sport Attributional Style Scale [START_REF] Hanrahan | Development of a questionnaire measure of sport-related attributional style[END_REF], called the Sport Explanatory Style Questionnaire (SESQ; [START_REF] Martin-Krumm | Validation of the Sport Explanatory Style Questionnaire[END_REF]. This self-report instrument consists of 10 hypothetical situations: 5 good outcomes (e.g., 'You perform very well in a competition'; 'Your team-mates claim that you are a very good performer") and 5 bad outcomes. The positive and negative items were matched for content. Respondents are asked to imagine each event happening to them and to provide in writing the one major cause of this event, and then use seven-point bipolar scales in each case to rate the degree of stability and globality of the cause. Confirmatory factor analysis carried out on more than 600 teenagers provided strong support for the stable and global dimensions for both positive and negative events [START_REF] Martin-Krumm | Validation of the Sport Explanatory Style Questionnaire[END_REF]. Satisfactory indices of reliability (i.e., internal consistency and test-retest correlation over 6 months) and concurrent validity (e.g., significant correlations with success expectations and procrastination in sport) were also found.

In the present sample, a composite score for explanations of bad events (CN) was obtained by averaging the participant's score on the stability + globality dimensions for the bad events (α = .75). Similarly, a composite for good event explanations (CP) is acquired by averaging the respondent's score on the two dimensions for the good events (α = .71).

Finally, subtracting CN from CP yields a full scale score (CPMCN). The more this score is positive and high the more the participant is optimistic. By contrast, the more this score is negative and high the more the participant is pessimistic (see [START_REF] Peterson | Meaning and measurement of explanatory style[END_REF][START_REF] Reivich | The measurement of explanatory style[END_REF] for a more detailed explanation).

Explanatory Style and Resilience 8

Perceived basketball ability.

To assess perceived basketball ability, a 4-item questionnaire similar to the one developed by Nicholls and colleagues (e.g., [START_REF] Nicholls | Adolescents' theories of education[END_REF] was used (e.g., 'When you play basketball and you compare yourself to most friends of your age, you feel…'). The answers are indicated on an 11-point scale anchored by 'very bad' (1) and 'very good' (11). In previous research conducted on teenagers (e.g., [START_REF] Cury | Achievement goals and perceived ability predict investment in learning a sport task[END_REF], the questionnaire has shown good construct validity, internal consistency and predictive validity.

In this study, the internal consistency was high (α =.88); consequently the average was computed and used in subsequent analyses.

Success expectation and importance given to task success

Before each testing, participants rated two statements concerning their success expectation (i.e., 'in the dribble circuit, what are your chances in 100 of performing a good time?'), and the value which they assigned to the accomplishment of the course (i.e., 'For you, to succeed in this course is something that is:'). Answers were given by placing a vertical line between the two endpoints of a 10 cm horizontal bar anchored by '0%' and '100%', and 'little importance' and 'very important', respectively for the two questions. These scales allow very accurate answers, by measuring the distance in millimeters between the left anchor and the mark carried out by the participant [START_REF] Vallerand | Méthodes de recherche en psychologie[END_REF].

Perception of experimental manipulation

After the feedback of the first testing was given, participants completed a question assessing their perception of success versus failure: 'broadly, how do you consider your performance?' An answer was given by making a box between 'rather a success' or 'rather a failure'.

State Anxiety

Explanatory Style and Resilience 9

9

In this study, anxiety was conceptualized as the cardiovascular reactivity to psychological stress, and more precisely by the increase in heart rate (HR). Several studies found that anxious people have greater cardiovascular responses (e.g., [START_REF] Smith | Interpersonal control and cardiovascular reactivity: goals, behavioral expression, and the moderating effects of sex[END_REF]. The HR was recorded continuously with an ambulatory device (Polar XtrainerPlus; Polar Electro Oy, Kempele, Finland). The apparatus comprised a transmitter which was attached to a strap around the subject's chest and a receiver which was worn on his/her wrist. Data were transferred into a computer via the interface provided with the instrument. So that the HR elevation cannot be attributed to the physiological stress involved in the basketball dribbling exercise, the maximum heart rate preceding the beginning of the testing, with resting heart rate as the covariate, was retained as the anxiety indicator.

Procedure

Three experimental groups were created in using the percentile distribution of the CPMCN score, and by separating the boys from the girls to keep a balance between the sexes in each group. Participants in the upper tenth percentile were considered more optimistic in explanatory style (M = 2.02 and 1.83; SD = .77 and .43, respectively for 13 boys and 9 girls), while those in the lower tenth percentile were considered more pessimistic in explanatory style (M = -0.83 and -1.23; SD = .29 and .39, respectively for 10 boys and 10 girls); lastly, those close to the median were considered as neutral in explanatory style (M = 0.30; SD = .08

for 10 boys and 10 girls).

Each participant was carried out by an experimenter who was blind to the subject's explanatory style. Under the pretext of checking the students' health, the participants were given a cardio-frequency meter strapped on a belt (see above), and the heart rate was monitored continuously throughout the experiment. The experiment proceeded in three phases. In the first phase, participants were seated at a table and began a 10-min baseline, during which they could leaf through magazines while resting HR was taken. Next, they were Explanatory Style and Resilience 10 10 invited to fill out a questionnaire intended to measure their perceived basketball ability. In the second phase, the experimenter presented the basketball dribbling circuit. The participant was told that this course was a test to classify individuals in relation to one another according to their technical level of dribbling from the time taken on the circuit. Fifteen minutes were allocated to the participants for training.

Next, they were invited to fill out another questionnaire that measured success expectation and the value assigned to finishing the course, and they completed a first timed attempt. Time taken was not communicated to the participants, who instead were told: "You have not produced a very good time compared to the other pupils who have performed. But once that you have rested, you will have the possibility to train and test a second time."

Participants were requested to rate their perceptions of the success / failure, success expectation and value which they assigned to the accomplishment of the course. Then they were allowed five minutes to prepare for Test 2. In the third phase, participants completed a second timed attempt. One week after the end of the study, a debriefing was conducted, and the full objectives of the investigation were explained.

Results

After the feedback from the first testing was given, 2 optimistic and 1 neutral participants did not perceive themselves as having failed. They were dropped from subsequent analyses. To test the mediating hypothesis that the explanatory style would affect the recovery after failure through its effects on success expectation and state anxiety, controlling for perceived basketball ability and gender, we used regression-based path models. According to [START_REF] Judd | Estimating the effects of social interventions[END_REF], data fit a mediation model if (a) the independent variables significantly predict the outcome measure, (b) the independent variables significantly predict the hypothesized mediating variables, and (c) the mediators significantly predict the outcome measure when the effects of the independent variables are controlled.

To reduce the number of variables of the model while taking account of the initial ability, we used the percentage of progress between the two tests like a dependent variable.

Moreover, only the participants with a pessimistic style (coded 1) and optimistic style (coded 2) were retained. Table 1 shows the zero-order correlations between all variables used in the process analysis. In a test of the first requirement of the process analysis, percentage of recovery was regressed onto explanatory style, gender, and perceived basketball ability. The In the test of the final requirement, percentage of recovery was regressed onto the seven-term model (three predictors, two mediators and two control variables). Overall, the model was significant F(7, 32) = 5.60, p<.001 (R 2 =.55), with individual effects for success expectation, F(1, 32) = 4.28, p=.047 (β =.38), and state anxiety F(1, 32) = 6.60, p=.02 (β =-.41). Figure 1 shows the path model for these results.

Discussion

This study was designed to explore why some persons recover after failure in sports, whereas others give up. It is crucial to understand this phenomenon because an athlete's life-like that of anyone else-is filled with ups and downs, and the ability to recover after failure is an important part of resilience. One key psychological factor to be investigated is a personality variable: the explanatory style.

First, in accordance with former studies in the field of sports [START_REF] Prapavessis | Learned helplessness in sport[END_REF]Retew & Reivich, 1995;[START_REF] Seligman | Explanatory style as a mechanism of disappointing athletic performance[END_REF], the results showed that participants with a more optimistic style performed better at the second test after a failure feedback, whereas those with a more pessimistic style did not improve. Those results extend the external validity of the former studies to a less elite population. Moreover, the internal validity of the study is strong insofar as these results were obtained by controlling variables likely to affect motor performance like initial ability, gender, perceived ability, and importance of success.

Secondly, the results showed that explanatory style also affected two other components, one cognitive and another affective in nature: success expectation and state anxiety. Participants with the pessimistic style had a greater fall in success expectations after failure than the most optimistic; a result which is in keeping with certain former works showing that pessimism correlates negatively with expectations [START_REF] Peterson | Explanatory style, expectations, and depressive symptoms[END_REF]. It is likely that individuals with a pessimistic explanatory style usually explain their lack of control over consequences with stable causes such as their own lack of ability and expect that negative events will be pervasive and enduring in their lives [START_REF] Helton | Optimism, pessimism, and false failure feedback effects on vigilance performance[END_REF]. This attributional pattern leads to a drop in expectation of success. By contrast, it is likely that individuals with a optimistic explanatory style usually explain failure with more unstable and contextual causes such as the use of a bad strategy or the lack of effort, which allows relatively consistent success expectations for the events to come.

In addition, the results showed that participants with the pessimistic style had greater stress reactivity (assessed by an increase in heart rate before the second testing) than the one with the optimistic style; a result which is in keeping with certain former works showing that pessimism correlates positively with anxiety (e.g., [START_REF] Mineka | Explanatory Style in Anxiety and Depression[END_REF][START_REF] Helton | Optimism, pessimism, and false failure feedback effects on vigilance performance[END_REF][START_REF] Jackson | Pessimistic explanatory style moderates the effect of stress on physical illness[END_REF]. To explain the lack of control over bad events with stable and general causes can increase the perceived threat and in turn the level of anxiety. By contrast, to explain failure with more unstable and contextual causes can lead to the feeling of "keeping control" of the situation and to provoke less anxiety.

Neutral participants obtained scores which were between the two other groups for both performances, for success expectations and for state anxiety. In view of failure, optimistic explanatory style seems to be more adaptive than pessimistic explanatory style is, but also more adaptive than neutral explanatory style is. Optimistic explanatory style could lead to a better protection against adversity, whereas a pessimistic explanatory style could lead to intensify the failure's consequences (i.e., more decreased self-confidence and higher anxiety level).

The unique contribution of the results is the demonstration that the explanatory style effects on performance were mediated by expectancies and state anxiety. If explanatory style is presumed as being only a distal risk factor (Peterson et al., 2001;[START_REF] Peterson | Optimistic explanatory style[END_REF], to date only few studies (e.g., [START_REF] Peterson | Explanatory style, expectations, and depressive symptoms[END_REF]) investigated the role of potential mediators. In this study, pessimistic vs. optimistic predicted (1) positively success expectations and (2) negatively state anxiety, before the second testing. In turn, the more success expectations were high and state anxiety low, the better was the percentage of progress between the two tests. As a result, researchers working in the helplessness tradition should measure not only explanatory style and helplessness outcomes, but also expectations, and state anxiety level which seem to deserve attention, as well as other potential mediators, maybe like irrational thinking (e.g., [START_REF] Ziegler | Relation of irrational thinking and the pessimistic explanatory style[END_REF].

Most of the previous studies dealt with the relationships between pathological population's explanatory style and behavioral, cognitive, and affective variables. The concern of this study was to observe how ordinary pupils behave when facing false failure feedback in a sport task, with regard to their explanatory style, mediators, and various variables of control.

The results we obtained show that the explanatory style's construct is particularly heuristic for such a population. It could have a particular interest in sports. Currently, most high level athletes have physical training, and often psychological training. Such results could pave the way to further studies which could emphasize the importance of explanatory style's role as a personality trait on sport performances after a poor showing, and have practical implications for the managerial staff.

¦ As explanatory style research has progressed and theory has been modified, the internality dimension has become of less interest [START_REF] Peterson | Meaning and measurement of explanatory style[END_REF][START_REF] Peterson | The future of optimism[END_REF][START_REF] Peterson | Optimistic explanatory style[END_REF] and less likely to be examined in empirical studies (e.g., [START_REF] Peterson | Explanatory style, expectations, and depressive symptoms[END_REF][START_REF] Peterson | Explanatory style as a risk factor for traumatic mishaps[END_REF]. § Analyses of covariance carried out on the three interest variables, with perceived basketball ability serving as a covariate did not alter the explanatory style effect. By consequence, perceived basketball ability will not be discussed any more in the following paragraphs.

  effect of the three-term model was significant, F(3, 36) = 3.71, p=.02 (R 2 =.24), and individual effects emerged only for explanatory style, F(1, 36) = 7.09, p=.012 (β =.44). In a test of the second requirement, each possible mediator was regressed individually onto the same three-term predictor model plus one control variable: the success expectation before the first testing and the rest heart rate respectively for the success expectation model, and for the state anxiety model. In the success expectation regression, the effect of the four-term predictor model was significant overall, F(4, 35) = 8.18, p<.001 (R 2 =.48), and individual effects emerged for explanatory style, F(1, 35) = 15.38, p<.001 (β =.55), and success expectation Explanatory Style and Resilience 13 13 before first testing, F(1, 35) = 15.38, p=.02 (β =.38). In the state anxiety regression, the effect of the four-term predictor model was significant overall, F(4, 35) = 2.89, p=.036 (R 2 =.25), and individual effects emerged only for explanatory style, F(1, 35) = 7.01, p=.012 (β =-.45).

Figure 1 .

 1 Figure 1. Process model. Path values are standardized regression coefficients form the regression analyses. No influential variables and nonsignificant paths were deleted. *p<.05; **p<.01; R 2 = percentage of explained variance of each variable.

  was higher for the most pessimistic participants than the most optimistic participant [F (1, 52) = 8.50, p = .005, η 2 = .14; Ms = 145.10 vs. 132.40, respectively] or the neutral participants [F (1, 52) = 4.18, p = .045, η 2 = .07; M = 135.90]. No difference appeared between the last two groups [F (1, 52) = 0.76, p = .39, η 2 = .001].

	Explanatory Style and Resilience 11
	process model) with simultaneous regression analyses, following the Judd and Kenny (1981)
	recommendations.
	To test the hypothesis of a greater recovery after failure by the optimistic participants,
	a 3 (explanatory style) × 2 (gender) × 2 (testing) analyse of variance (ANOVA) was
	conducted, with repeated measures on the testing variable § . Results showed a significant
	explanatory style × testing interaction [F (2, 53) = 4.35, p = .017, η 2 = .14]. Contrast analyses
	revealed that progress in performance between the two tests was significant for the most
	optimistic [F (1, 53) = 19.80, p < .001, η 2 = .27; mean score progressed from 134.75s. to
	129.21s.], was almost significant for the neutral [F (1, 53) = 3.53, p = .066, η 2 = .06; mean
	score progressed from 134.75s. to 129.21s.], and was not significant for the most pessimistic
	[F (1, 53) = 0.10, most pessimistic [F (1, 53) = 40.36, p < .001, η 2 = .43; mean score down from 51.20 to
	Two sets of analyses were carried to test the hypotheses. The first set focused on the 26.90], for the neutral [F (1, 53) = 12.87, p = .001, η 2 = .20; mean score down from 56.32 to
	changes of performance, expectations and state anxiety between the tests as a function of 42.22], and was not significant for the most optimistic [F (1, 53) = 1.65, p = .20, η 2 = .03;
	explanatory style controlling for gender. Because the importance of task success was mean score remained relatively stable between the two tests: 55.70 and 50.76, respectively].
	unrelated to explanatory, gender, and performance (rs <.22, p>.11), it was excluded from To test the hypothesis of a greater stress reactivity after failure from the pessimistic
	subsequent analyses. The second set of analyses tested the mediating hypothesis (i.e., the participants, a 3 (explanatory style) × 2 (gender) ANCOVA was conducted, with resting heart

p = .75, η 2 = .002; mean scores remained stable between the two tests:

139.75s. and 139.35, respectively]

.

To test the hypothesis of a greater fall in success expectation after failure for the most pessimistic participants, a 3 (explanatory style) × 2 (gender) × 2 (expectancies) ANOVA was conducted, with repeated measures on the expectancies variable. Results showed a significant explanatory style × expectancies interaction [F (2, 53) = 6.38, p = .003, η 2 = .19]. Contrast analyses revealed that fall in success expectation between the two tests was significant for rate as the covariate. Results showed a main effect for explanatory style [F (2, 52) = 4.47, p = .016, η 2 = .15]. Contrast analyses revealed that increase in heart rate reactivity before the second test

Table 1

 1 Intercorrelation Matrix for Predictors (Variables 1 through 3), control variables (Variables 4 and 5), Possible Mediators (Variables 6 and 7), and the Outcome Measure of Percentage of Recovery (Variable 8). Pessimistic = 1, Optimistic = 2; b Girl = 1, Boy = 2. *p<.05; **p<.01; ***p<.001.

	Variable	1	2	3	4	5	6	7	8
	1. Explanatory Style a 2. Gender b 3. Perceived ability 4. Success expect 1 5. Rest heart rate 6. Success expect 2 7. State Anxiety 8. Percentage of rebound	-	.05 -	.47** .30 -	.12 .45** .50*** -	-.23 -.35* -.16 -.19 -	.57*** .25 .42** .45** -.19 -	-.45** -.11 -.17 -.27 .29 -.62*** -	.47** -.10 .24 .04 .11 .57*** .56*** -
	Note.

a

Acknowledgments

We thank the Polar Electro Oy company, Kempele, Finland for providing the apparatus for measuring heart rate reactivity.

Footnotes


Simon Rokicki

Joseph Paturel

Olivier Sentieys

Comet: a RISC-V Core Synthesized from C++ Specifications

. In this poster we present the status and roadmap around Comet.

I. INTRODUCTION

Innovations in hardware architecture are leading to better processors which can support the complex software applications being developed today. Similarly, software and programming languages have made it easier to prototype, simulate and even synthesize hardware, for example the creation of High-Level Synthesis (HLS) tools. Standard development flows for processor architecture are based around the development and maintenance of a hardware model for synthesis (usually at Register Transfer Level), and a software model (i.e. an instruction-set simulator) to validate the applications that will run on the design. Those two models have to be verified independently. To cut down the complexity of the hardware development in a processor design methodology, Comet introduces the use of a single behavioral model of the simulator, which is compiled into a cycle-accurate bit-accurate simulator by standard compilers and also synthesized into a hardware component through HLS. It is also possible to debug the behavioral model at the C/C++ level and to simply validate the behavior of the generated hardware through co-simulation and standard software development tools. The design methodology based on Comet is depicted in Figure 1.

Although high-level synthesis is making huge progress in dealing with complex structures, how far can these tools go? Can they be used to design something as complex as a microprocessor? In particular how to specify such parallel computing pipelines (e.g., core pipeline stages, cache hierarchy, communications with uncore components) with the HLS technology and to demonstrate that there is a potential high gain in design time, without jeopardizing performance and cost.

II. CONTRIBUTIONS

In this work, we present the design of Comet, a five-stage pipelined processor implementing the RISC-V Instruction Set Architecture (ISA). Comet is fully synthesized from a unique C/C++ model using state-of-the-art HLS tools. Our results show that, if the C/C++ model is written using the methodology we describe in this paper, the generated hardware is comparable or even better than other implementations when targeting an ASIC 28 nm technology node. Moreover, a fast, cycle-accurate and bit-accurate simulator of the processor can be compiled from the same C/C++ model. The resulting maximum performance of the simulator reaches 24 million cycles per second (Mcps) on integer workloads and around 15 Mcps on average, when executing floating-point and integer workloads.

The object oriented nature of C++ also greatly improves the readability and flexibility of the design description compared to classical HDL-based implementations. Therefore, the processor model can easily be modified, expanded and verified using standard software development methodologies.

Recent developments include compatibility with modern embedded operating systems such as Zephyr or RIOT. A shared memory multicore version supporting Linux is in its final step and an Out-of-Order version of Comet is on our roadmap. Comet is used in several projects around fault tolerance, intermittently-powered systems and hardware security.

Fig. 1 .

 1 Fig. 1. Comet processor design flow.


Zohreh Mehri Lighvan

Hossein Ali Khonakdar

Ali Akbari
email: akbari.a@umsu.ac.ir

Maryam Dehdashti Jahromi

Azar Ramezanpour

Anthony Kermagoret

Abolfazl Heydari

Esmaiel Jabbari

Zohreh Mehri
email: z.mehri@ippi.ac.ir

| Hossein

Ali Khonakdar1

| Ali Akbari3

Maryam Dehdashti

| Azar

Anthony Kermagoret6

Abolfazl Heydari7

| Esmaiel

Synthesis and biological evaluation of novel tetranuclear cyclopalladated complex bearing thiosemicarbazone scaffold ligand: Interactions with double

Keywords: anticancer, cyclopalladated, DNA, thiosemicarbazones scaffold, threading intercalation

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

| INTRODUCTION

The prevalence of cancer and parasitic diseases has posed a huge health risk to people in developing countries with a narrow spectrum of effective drugs available for treatment. Organometallic compounds display a range of characteristics providing pharmacological profiles which are not attainable by organic molecules. [1] In fact, many organometallic compounds have been designed to specifically bind to a well-defined target site on biomolecules. [2,3] In the bioinorganic systems, cyclopalladated complexes, due to their high lability in biological fluids, are promising antitumor drugs that are effective in both in vitro and in vivo environments, when compared with platinum group metal (PGM) as an anticancer compounds. [4,5] The cyclopalladated complexes are more stable and significantly less toxic, suggesting they could possess a more specific in vivo antitumor activity. [6][7][8] In particular, orthometallated N-donor ligands, such as thiosemicarbazones, benzylamines, and imines, have been successfully used as targeted antitumor drugs due to the presence of a strong palladium-carbon σ-bond in the "(C,N)Pd" metallacycle, which enhances the stability of the organometallic complex. [9] Some reports have shown that cyclopalladated compounds interact with thiol groups of mitochondrial membranes to induce apoptotic cell death via the intrinsic pathway. [10,11] The other reports show that the antitumor activity of cyclopalladated compounds is dependent on their interaction with DNA. [12,13] The antitumor activity of cyclopalladated compounds is believed to be due to the inhibition of DNA synthesis resulting from changes in reaction conditions in reductive conversion of ribonucleotides to deoxyribonucleotides. [14] Thiosemicarbazones (TSCs) scaffold as ligands are an important class of bioactive compounds with a wide range of properties ranging from their use as anticancer agents [15] to antiviral, [16] antibacterial, [17] antiproliferative, [18] anti-leprosy, [19] antimalarial, [20] antitubercular, [21] and antiparasitic [22][23][24] agents. The anticancer activity of thiosemicarbazones stems from their chelating ability with transition metal ions by bonding through the sulfur and the azomethinic nitrogen atoms to inhibit the biosynthesis of DNA, possibly by blocking the enzyme ribonucleotide diphosphate reductase. [25,26] Notably, chelating ligands have been used to overcome this high lability, forming thermodynamically stable and kinetically inert cyclopalladated complexes. [27,28] Demertzi et al. reported Pd(II) complexes of N4-alkyl-2-acetylpyridine thiosemicarbazones inhibits the activity of DNA synthesized in L1210 and P388 cell cultures. [29] In a study by Quiroga et al., Pd(II) and Pt(II) complexes of phenylacetaldehyde thiosemicarbazone and binuclear chloro-bridged palladated and platinated complexes derived from p-isopropylbenzaldehyde thiosemicarbazone showed activity against several human and murine cell lines (HL-60, U937, HeLa, and 3T3) that are resistant to the clinically used cisplatin. [30,31] DNA plays a crucial role in a wide variety of cellular processes [32] and serves as a superior target for the treatment of treating genetic diseases like cancer. [33] The anticancer, antiparasitic, and antimicrobial agents exert their biological response via interaction with ellipticine, which unwinds DNA and inhibits the processes of transcription, replication, and DNA repair mechanisms. [34] The COVID-19 epidemic has caused in a global health emergency and has spread to 213 countries and is uncontrollable so far. [35] CoVs (COVID-19) are positive-stranded RNA viruses with a crown-like appearance under an electron microscope due to the presence of spike glycoproteins on the envelope. Two groups of proteins characterize CoVs. The first group is structural proteins such as Spike (S) that are common in all coronaviruses, that is, Nucleocapsid (N), Matrix (M), and Envelope (E). The second group is the nonstructural proteins, such as proteases (nsp3 and nsp5) and RdRp (nsp12). [36,37] The Spike protein is a crucial recognition factor for virus attachment and entry to the host cells. It is present on the virion's outer surface in a homo-trimeric state. [36] The RNAdependent RNA polymerase (RdRp, also named nsp12) is the central component of coronaviral replication/ transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. [38] It has been reported that TSCs possess antiviral as well as antiHIV properties. [39,40] A series of isatinβ-thiosemicarbazones have been tested against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). [41] Hence, we hypothesized that the synthesis and testing of more novel TSC compounds could be beneficial to understanding the mechanism of their antiviral activity. As part of our ongoing research and interest in the structural chemistry of palladium compounds, [42][43][44][45][46][47] we have turned our attention to cyclopalladated thiosemicarbazone complexes. Herein, we report on the synthesis, structural characterization, DNA interaction, and cytotoxicity of cyclopalladated thiosemicarbazone. The molecular docking technique is used to determine the mechanism of interaction of the complex with DNA and RNA.

| Synthesis and characterization of the compounds

The synthesis process and the chemical structure of the thiosemicarbazone ligand (TSC) and cyclopalladated thiosemicarbazone (Pd-TSC) used in this study are shown in Scheme 1. The chemical structure of the compounds was characterized by elemental analysis (C, H, N, and S), IR spectroscopy, and 1D including 1H and 13C NMR modes and 2D NMR, including COSY and HMQC modes. The infrared (IR) spectra of compounds were performed for identifying the functional groups of the ligand and the complex (see Supporting Information). The IR spectrum of the ligand shows bands at 3309 and 3138 cm1, which are attributed to N-H and N-H hydrazinic stretching vibrations, respectively, and bands in the ranges 1597-1536 cm1 and 815-743 cm1 are corresponding to C=N and C=S stretching vibrations, respectively. This is in agreement with the reported values. [48] Comparison of the spectra of the complex with the free ligand shows the disappearance of N-Hhydrazinic and C=S bands, as identified by others. [49] The C=N band has shifted to lower wavenumbers upon complex formation by approximately 40 cm1 in agreement with coordination of the palladium atom to the C=N moiety through the lone nitrogen pair. [50,51] Additionally, the presence of a new band at 687 cm1 in complex, attributed to C-S stretching vibration, indicated coordination of Pd via the sulfur atom in the thiol form. [14] The electronic spectra of the complexes showed the intra-ligand bands in the 230-266 nm and 350-372 nm ranges, corresponding to the n ! π* and π ! π* transitions, respectively. The chemical structure of thiosemicarbazone ligand (TSC) and cyclopalladated thiosemicarbazone complex (Pd-TSC) were characterized by the 1H NMR, 13C NMR, COSY, and HMBC spectra in DMSO-d6. Figure 1a shows 1H NMR spectra of the TSC ligand that is exhibited the secondary = N-H-and -NHPh-proton signals at 10.16 ppm and 11.86 ppm, respectively. In addition, a singlet associated with the -CH=N-(H6) proton observed at 8.15 ppm and the signals in the range of 7.20-7.96 ppm were attributed to aromatic protons (Figure 1a) with the help of COSY experiments. No signal at around 4.00 ppm (i.e., the -SH proton) was observed in the 1H NMR spectra of the TSC, indicating that it remained solely in the thione form, even in a polar solvent like DMSO-d6, as reported by others. [52] APT 13C NMR and 2D HMQC experiment confirmed the ligand structure and permitted to identify the S=C signal at 176.7 ppm, the imine (-N=CH6-) signal at 141.9 ppm and the eight aromatic signals between 140 and 124 ppm (see Supporting Information). The reaction of potassium tetrachloropalladate and 1.1 equiv. of TSC ligand afforded the Pd-TSC complex in good yield. In contrast to the 1H NMR spectrum of TSC (Figure 1a), the proton signal of NH imine group was not observed upon formation of the complex, demonstrating the deprotonation of =N-NH group, thus confirming metalation. [49] The two singlets at 9. 61 While the NMR spectra of Pd-TSC clearly showed a strong chemical modification compared with the ligand NMR spectra and elemental analysis are in good agreement with the proposed complex, the structure of cyclopalladated thiosemicarbazone complex was ascertained by high resolution mass spectrometry. Using the negative electrospray technique, the isotopic pattern measured at m/z 1574.715 for [C56H39N12S4Cl4Pd4] was identified and confirmed the tetranuclear character of the cyclopalladated thiosemicarbazone complex (see Supporting Information).

| Nucleic acid binding

Understanding the non-covalent binding of transition metal complexes to nucleic acids are an important aspect for developing new therapeutic agents and molecular probes. Many anticancer and antiviral compounds exert their therapeutic effect through intercalation with the nucleic acids of DNA, thereby interfering with DNA. This is the basis of designing novel anticancer therapeutics with greater efficiency, where effectiveness depends on mode and affinity of DNA binding.

| Study of DNA-tetranuclear complex interaction with UV-vis spectroscopy

A novel method for characterizing the type of DNA interaction with organometallic complexes is electronic absorption spectroscopy. [33] The absorption spectra of the thiosemicarbazones scaffold ligand and its Pd(II) complex in the absence and presence of CT-DNA are shown in Figure 2. Electronic absorption of the ligand showed two bands at 376 and 324 nm that were assigned to n-π* transition and one band at 248 nm that was assigned to π-π* transition. Following the addition of CTDNA to the ligand solution, 28.05, 35.27, and 31.18% hypochromism was observed at 376, 324, and 226 nm, respectively. Further, the absorption band at 324 nm exhibited approximately 2-nm red shift due to a change of polarity in the ligand's microenvironment. [57] It is well known that the "hyperchromic effect" results from the damage to the structure DNA double helix, which leads to DNA contraction in the direction of helix axis and changes to DNA conformation. [58] Such marked hypochromic effect in spectrum of ligand confirmed the significant interaction of ligand with DNA.

Typically, the hypochromic effect occurs via noncovalent interaction including intercalation and groove binding modes. [59] The spectrum of Pd(II) complex exhibited absorption bands at 228, 256, and 350 nm with 26.53, 27.48, and 6.65% hypochromism, respectively. The absorption intensity of the complex decreased after the addition of DNA, due to noncovalent binding (hypochromism), as shown in Figure 1b. The exact mode of binding can be determined by calculating the intrinsic binding constant (Kb) between the ligand and Pd(II) complex using Equation 1. [60] where εa, εb, and εf are extinction coefficients for the apparent, bound, and free compound, respectively.

The coefficient εf was calculated from the standard curve of the isolated compound in a buffer solution using Beer's law. The coefficient εa was calculated as the ratio of the observed absorbance (Aobs) over the concentration of the complex, that is, Aobs/[complex]. A plot of [DNA]/ (εa εf) versus [DNA] gives a slope of 1/(εb εf) and a y-intercept of 1/Kb (εb εf); therefore, Kb is determined from the ratio of the slope to the y-intercept (see inset of Figure 2). Using the above approach, the Kb values of 1.50 107 and 2.68 107 M1 were obtained for the ligand and Pd(II) complex, respectively. One approach to increase DNA binding affinity to tetranuclear complexes is to reposition the active ligand for coordination with the metal. [61,62] Therefore, we used thiosemicarbazone as a ligand to improve anticancer and antiviral potential of tetranuclear complexes. Based on our calculations, the larger value of Kb indicates higher affinity for DNA binding to the Pd(II) complex as compared with the ligand alone. The Pd(II) complex contains four ligands for stronger DNA interaction. The Kb value of the complex is comparable with those complexes that interact with DNA via threading intercalation. [63,64] Therefore, it can be concluded that Pd(II) complex binds to different DNA sites on the basis of the structure. As the DNA double helix possesses many hydrogen binding sites that are accessible both in the minor and major grooves, it is likely that the -NH-group, N and Cl atoms of the Pd(II) complex formed hydrogen bonds with DNA, which led to the observed hypochromism in the absorption spectra of the Pd(II) complex. Conversely, our Pd(II) complex possesses aromatic rings that bind to DNA base pairs by intercalation. This type of Pd(II) complex binding engages more DNA bases, which, in turn, causes inhibition of replication and inefficient transcription. When synthesizing DNA-targeted drugs, high DNA binding affinity and slow DNA binding kinetics are considered important factor to enhance therapeutic effects. [65,66] These desired properties are found in compounds that interact with DNA by threading intercalation, an interaction that first requires non intercalating moieties of a ligand to pass between DNA base pairs prior to intercalative binding. [67,68] High binding affinity is related to the stability of Pd complex. The tetranuclear nature and stable Pd-S bonds contribute to the stability of Pd(II) complex even in the presence of nucleophiles like DNA bases. [53] Considering high mortality of patients inflicted with cancer and viral infection, particularly those infections transmitted via the respiratory route, we hypothesized that the thiosemicarbazones scaffold ligand and its tetranuclear cyclopalladated complex could potentially serve as an efficient carrier for cancer therapeutics and antiviral agents targeting DNA and RNA.

| Competitive displacement assay with MB

The competitive interaction assay of ligand and Pd(II) complex was performed with DNA bound Methylene Blue (MB) to determine the interaction mode of DNA with the ligand and Pd(II) complex. Figure 3 shows the emission spectra of DNA bound MB in the presence of different concentrations of the ligand and Pd complex with excitation wavelength of 630 nm. The emission intensity of MB slowly increased following the addition of ligand. This result indicated that the ligand inserts itself in the DNA double helix, which is a characteristic of intercalative mode of binding. Consequently, the addition of Pd(II) complex has resulted in a more efficient change in emission intensity. Therefore, it could be concluded that the ligand and Pd(II) complex interacted with DNA mainly by intercalative mode. [69,70] 2.5 | Electronic circular dichroism Electronic circular dichroism (ECD) is used extensively in the characterization of helical structure of DNA and its A, B, and Z-forms as well as their complexes with ligands. [71] The ECD spectrum of right handed B-DNA exhibits a positive band at 220 nm due to hydrogen bonding, a negative band at 240 nm due to handedness/polynucleotide helicity, [72] and a positive band at 275 nm due to base stacking. [73] Figure 4 depicts the ECD spectra of DNA in the presence of the ligand and Pd(II) complex are compared with that of DNA in the presence of Actinomycin D (ActD), as a threading intercalator. [74] When DNA was incubated with the Pd(II) complex at a molar ratio of 0.5, [complex]/[CT-DNA], the complex broadly induced spectral changes similar to that of DNA-ActD. Specifically, the complex and ActD binding to DNA induced a significant increase in the intensity of negative CD band at 240 nm along with a 4-nm red shift. The change in band intensity and a shift in wavelength at 240 nm can be attributed to a change in base pair geometry and a reduction in stacking interaction between the DNA bases as a result of the complex or ActD intercalation with the DNA base pairs.[75] The band attributed to right-handed helicity at 275 nm showed a large increase in intensity along with a slight red shift of 2 nm. These changes indicate alteration in duplex helicity of the DNA by interaction with the complex or ActD in which it forms a reversible, non-covalent and stable complex with DNA. [76] These observations support the threading intercalative mode of interaction between the DNA and the Pd-ligand complexes. [77,78] In addition to threading intercalation binding mode, the CD spectral changes induced by Pd-ligand complexes and ActD reveal groove binding intercalation mode that stabilizes the right handed B-DNA, indicating and that the DNA unwounds upon interaction with the compounds. [75] These results are in conformance with our UV-vis studies which show distortion in the B-DNA conformation upon binding with the compounds.

| Hyperchromic effect of melting DNA with the compounds

DNA melting refers to the separation of double strands of DNA upon heating due to the breaking of hydrogen bonds between the DNA bases, which is greatly affected by the presence of other binding molecules. [79] The intercalation leads to a considerable increase in DNA melting temperature (Tm), as measured by the temperature at which the hypochromicity at 260 nm is reduced by half, [80] due to the stabilization of DNA duplex, whereas groove binding or electrostatic interactions leads a smaller change in the value of Tm. [44,81] The melting curves of DNA in the absence and presence of compounds 1, 2, and ActD are shown in Figure 5. In the absence of compounds 1-3, the Tm of DNA was 83.01C. However, in the presence of compounds 1, 2, and 3, the Tm of DNA increased from 83.01C to 87.67, 88.86, and 88.08C, respectively, corresponding to ΔTm values of 4.66, 5.85, and 5.07C. The ΔTm values in the presence of compounds 1-3 were slightly lower than 7.1C for threading intercalating of Act-D. [80] Molecular docking is a computational procedure and aims to predict the favored orientation of a ligand to its macromolecular target (receptor), when these are bound to each other to form a stable complex. [84] After the development of the first algorithms, molecular docking has become an important common and suitable computational tool of the drug discovery toolbox.

As it has relative low-cost implications and perceived simplicity of use, the molecular docking techniques became essential tool in drug discovery. [85] Also the interaction of any complex with the target biomolecule can be predict via molecular docking. The interaction between a complex and the biomolecules plays important role in many biological processes. In this study, the molecular docking calculations were used to investigate the interaction of the simple ligand structure and the complex structure with DNA and human coronavirus Mers and coronavirus disease 2019. The optimized structure of the ligand and the complex is demonstrated at Figures 6 and7, respectively. Semiempirical methods employing PM6 functional was used for the optimization. The structure of the ligand + DNA and the interactions of ligand atoms and DNA residues are depicted in Figure 8a,b, respectively, while the structure of complex + DNA system and the interactions of complex atoms and DNA residues are depicted in Figure 9a,b, respectively. According to the calculation, the interaction between the ligand and DNA seems to be intercalation while the interaction of the complex and DNA seems to be almost threading intercalation. The calculated free binding energy of the interaction of the complex with DNA is 8.71 kcal/mol, and the calculated free binding energy of the interaction of the ligand with DNA is 6.86 kcal/mol. The structure of ligand + human coronavirus Mers system and the interactions between the ligand atoms and the binding sites of receptor are shown in Figure 10 while the structure of the complex + human coronavirus Mers system and the interactions between the complex atoms and the binding sites of receptor are shown in Figure 11. As shown in the Figures 10b and11b, the 10 residues are involved in the interaction of the ligand with human coronavirus Mers as the same as the complex with human coronavirus Mers. The calculated free binding energy of the interaction of the complex with the receptor is 5.03 kcal/ mol, and for the ligand + receptor system, the calculated free binding energy of the interaction is 5.29 kcal/mol. Figures 12a,b and13a,b demonstrate the ligand + coronavirus disease 2019 and the complex + coronavirus disease 2019 systems and the interactions between the ligand and complex atoms and the binding sites of the receptor, respectively. The calculated free binding energies of the interaction of the complex with the receptor and the ligand with the named receptor are 10.11 and 8.32 kcal/mol. As the docking calculations show, the involved residues of the receptors in the interaction with the host molecules are clearly more in the receptor + complex structure system (18 residues) than in the ligand + receptor system (11 residues).

| In vitro cytotoxicity using MTT assay

The cytotoxicity of the synthesized ligand and complex was assessed against human MCF-7 breast cancer cells and HCT116 colorectal carcinoma cells for 24, 48, and 72 h with MTT assay. Cisplatin was used as a control group. The complex showed cytotoxic effect against both cell lines and the effect were concentration dependent (Figure 14a,b). The viability of MCF-7 and HCT116 cells decreased with increasing concentration of either the complex or the ligand. In the 0.05-to 0.4-mM concentration range, the ligand toxicity against HCT116 and MCF-7 cells was 24.07%-83.26% and 1.0%-80.0% after 24 h incubation, respectively. The complex in the same concentration range showed the values of 28.9%-94.2% and 5.39%-90.23%. The ligand showed a slightly higher cytotoxicity against both MCF-7 and HCT116 cells compared with the complex, while the difference was not significant. The cytotoxicity results demonstrate that the coordination of ligand to Pd has no significant effect on cytotoxicity of the complex against both HCT116 and MCF-7 cells. Moreover, the ligand and the complex exhibited low and high toxicity, respectively, on HCT116 and MCF-7 cells compared with cisplatin.

| CONCLUSION

This work describes the synthesis, tumor toxicity, and antiviral activity of thiosemicarbazone scaffold ligands and their binding to the tetranuclear cyclopalladate complex. The structures of the synthesized compounds were elucidated spectroscopically and by diffractometry. Methylene blue displacement experiments showed support for an intercalative binding mode between DNA and the compounds. The DNA binding experiments using electronic spectral technique showed evidence for the hypochromic effect and small red shift with a DNA binding constant on the order of 107 M1, consistent with intercalation and groove binding modes. According to CD spectrometry results, the compounds stabilized stabilization of the right-handed B form of DNA and, to a lesser extent, the DNA double strand. A ΔTm value of 4.5-5.5C in the presence of the compounds was evidence for a mixed binding mode of interaction with the threading mode as the dominant mode, which was verified by docking simulations. The complex and the ligand showed cytotoxic effect against both human breast (MCF-7) and colorectal (HCT116) cancer cells and the effect was concentration dependent. The results demonstrate that the palladacycle complex [Pd(4-ClC6H4CHN=NC(S)NHPh)4] has higher potency than the standard cisplatin chemotherapeutic.

| EXPERIMENTAL SECTION

| General material and methods

These are given in the Supporting Information.

| Synthesis of ligand (compound 1)

A 140-mg 4-chlorobenzaldehyde (1 mmol) in 30ml MeOH was added to a suspension of 167-mg Nphenylhydrazinecarbothioamide (1 mmol) in 20-ml MeOH, in a round bottom flask. The reaction was proceeded under stirring at ambient temperature for 18 h. After completion of the reaction, the white precipitate was filtered, washed with cold MeOH, and dried under ambient condition. The resulting ligand was isolated and characterized as follows: Yellow solid (63.4%); mp: 207-208C (dec.). Anal. calc. for C14H12ClN3S: C, 58.03; H, 4.17; N, 14.50, Found: C, 58.0; H, 4.17

| Synthesis of [pd(4-ClC6H4CHN=NC (S)NHPh)]4 (compound 2)

To a solution of 100-mg potassium tetrachloropalladate (0.30 mmol) in 5 ml deionized water, 50-ml ethanol was added. The obtained fine yellow suspension of potassium tetrachloropalladate was treated with the 97-mg ligand (0.33 mmol). The reaction was proceeded at 40C for 4 days. The orange-yellowish product was isolated by vacuum filtration, washed with ethanol (40 ml) and dried at oven at 50C. The resulting ligand was isolated and characterized as follows: orange-yellow solid (75%); mp: 315-318C (dec.

| Culture medium and cell lines

The colorectal carcinoma cell line and the MCF7 human breast adenocarcinoma cell line were supplied by the National Cell Bank of Pasteur Institute, Tehran, Iran. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 Uml1 penicillin, 100 μgml1 streptomycin, and 5-mM L-glutamine. The cell lines were subsequently cultured at 37C in a humidified atmosphere containing 5% CO2. All reagents and cell culture medium were provided by Gibco (Germany).

| Cytotoxicity assay

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] was used to evaluate the cytotoxic activity of the complex. The test is based on the soluble MTT metabolic reduction by mitochondrial enzyme activity of viable tumor cells to produce an insoluble colored formazan that can be spectrophotometrically measured by dissolution in dimethyl sulfoxide (DMSO). Cytotoxicity assay was performed by seeding 200 μl of the cell suspension (5 104 cellsml1) in 96-well plates and incubated for 24 h (37C, 5% CO2 humidified air). Afterwards, 20 μl of the prepared solutions of each compound was added to each well. The compounds were dissolved in DMSO prior to adding to the culture medium. The control and treatment groups contained identical DMSO concentrations of 0.5% (v/v). For evaluation of cytotoxicity, 20 μl of the MTT solution (5 mgml1 in phosphate buffer saline) was added in 48 h into the incubation and the plates were incubated for another 3 h. Subsequently, 150 μl of the medium containing MTT was slowly substituted with DMSO and pipetted to dissolve any formazan crystal formed. Absorbance values at 560 nm were determined using an ELISA plate reader (Awareness Technology, Stat Fax 2100).

| Molecular docking

The geometry optimization was separately performed for the ligand structure and for the complex structure at first, prior to docking calculations. Gaussian 09 Quantum Chemistry package [86] was used for the optimization procedures and semiempirical methods employing PM6 functional [87] were carried out for them. The structure of DNA with the PDB ID: 4QLC was obtained from the Protein Data Bank (PDB). The resolution of chosen DNA was 3.5 Å. As it is explained, the complex has the antimalaria effect so the authors believe that it is interesting to investigate the interaction of the complex with the coronaviruses theoretically. So the structures of human coronavirus Mers and coronavirus disease 2019 with the PDB ID 5ZVK and 6YB7 were obtained from PDB at the resolutions of 3.31 Å and 1.25 Å, respectively. It should be noticed that it was not possible for the authors to investigate these interactions experimentally. The Autodock 4.2 software [88] was employed to perform the molecular docking calculations. The Lamarckian genetic algorithm(LGA) [89] was used during the calculations. In order to prepare the input file of DNA all the hetero atoms and the water molecules were removed primarily. The protein molecules were processed by adding all hydrogen, merging non-polar hydrogen atoms using AutoDock Tools. The charges were assigned using the Gasteiger method. The standard values were used for docking parameters. Before the docking without any prior knowledge of the target pocket, the blind docking is needed. To find a favorable protein-ligand complex pose the whole surface of the receptor (DNA, human coronavirus Mers and coronavirus disease 2019) is scanned and the possible binding sites and the highest binding affinity is identified. [90,91] After the blind docking, the focus docking was done. In the process of docking, the size of the grid map were set to 126 126 126 Å3 for DNA and 90 90 90 Å3 and 90 90 90 Å3 for the human coronavirus Mers and coronavirus disease 2019, respectively. A gridpoint spacing of 0.375 Å that is roughly a quarter of the length of a carbon-carbon single bond was used in the AutoGrid runs. The conformations were ranked according to the lowest free binding energy. It is noteworthy that the structures were considered as flexible cases during docking.

  ; N, 14.52. F I G U R E 1 4 Comparison of the cytotoxicity of the complex and the ligand with cisplatin measured with HCT116 and MCF-7 cell lines for 24, 48, and 72 h incubation IR (KBr pellet, cm1): ν (N-H) = 3309, ν (N-H) hydrazinic = 3138, ν (C=N) = 1597, ν (C=S) = 815. 1H NMR (400 MHz, DMSO-d6) δ 11.86 (s, 1H, NH, H4), 10.16 (s, 1H, NH, H5), 8.15 (s, 1H, N=CH, H6), 7.96 (d, J = 8.4, 2H, CH-CH-CCl, H8), 7.57 (d, J = 7.2, 2H, CH-CH-CNH, H3), 7.49 (d, J = 8.4, 2H, CH-CH-CCl, H7), 7.38 (t, J = 7.2, 2H, CH-CH-CNH, H2), 7.23 (t, J = 7.2, 1H, CHCH-CH, H1). 13C NMR (101 MHz, DMSO-d6) δ 176.7 (s, C5), 141.9 (s, C6), 139.5 (s, CIV), 134.9 (s, CIV), 133.5 (s, CIV), 129.7 (s, C9), 129.2 (s, C8), 128.5 (s, C2), 126.4 (s, C3), 125.9 (s, C1).

  S C H E M E 1 Synthesis of thiosemicarbazone ligand and tetranuclear cyclopalladated complex. Reaction conditions: (i) MeOH, 25C, 18 h; (ii) K2[PtCl4], EtOHH2O, 40C, 4 days F I G U R E 1 (a) H-NMR Pd-TSC and TSC, (b) APT 13C NMR Pd-TSC and TSC, (c) TSC COSY, (d) TSC HMQC, (e) Pd-TSC COSY, and (f) Pd-TSC HMQC experiments F I G U R E 2 Electronic spectra of (a) ligand and (b) palladium complex in buffer solution of 5-mM Tris-HCl/50 mM NaCl at pH = 7.2 following addition of the DNA. [Pd Complex] = 6 105 M, [DNA] = 0-6.5 105 M. Arrow shows the decrease in absorption intensity upon increasing DNA concentration (Inset: plot of [DNAεa εf] vs. [DNA] for determination of Kb) F I G U R E 4 CD spectrum of DNA (15 104 M) in 5-mM Tris-HCl buffer with 50-mM NaCl (pH = 7.3) in the presence of ligand, Pd complex, and ActD (7.5 105 M F I G U R E 3 The emission spectra of the DNA-MB, in the presence of (a) ligand and (b) palladium complex. M, [Ligand] = [Pd complex] = 0-8 105. The arrow shows the change in emission intensity upon increasing concentration of the compounds F I G U R E 5 The effect of heating on the absorbance of CT-DNA at 260 nm (7.5 106 M) in the absence and presence of ligand, Pd complex, and Act D (3.75 106 M) in 5-mM Tris-HCl with 50-mM NaCl F I G U R E 6 The structure of the ligand optimized at semiempirical methods applying PM6 F I G U R E 7 The structure of the complex optimized at semiempirical methods applying PM6 from two points of view (a and b) F I G U R E 8 Results of docking procedure. (a) Predicted orientations of the lowest docking energy conformations of ligand with the threading intercalation of DNA. (b) Twodimensional interactions between ligand . (b) Twodimensional interactions between complex and DNA F I G U R E 1 0 Results of docking procedure. (a) Predicted orientations of the lowest docking energy conformations of ligand with the human coronavirus Mers. (b) Twodimensional interactions between ligand with the human coronavirus U R E 1 2 Results of docking procedure. (a) Predicted orientations of the lowest docking energy conformations of ligand with the coronavirus disease 2019. (b) Two-dimensional interactions between ligand and coronavirus disease U R E 1 4 Comparison of the cytotoxicity of the complex and the ligand with cisplatin measured with HCT116 and MCF-7 cell lines for 24, 48, and 72 h incubation

ACKNOWLEDGMENTS

This study was supported by Iran Polymer and Petrochemical Institute and the Iran National Science Foundation under contract number 97006605.

Funding information Iran National Science Foundation, Grant/Award Number: 97006605

DATA AVAILABILITY STATEMENT Data are openly available in a public repository that issues datasets with DOIs.