Carlos Fernando

Crispim Junior

Francois Bremond
email: francois.bremond@inria.fr

Veronique Joumier
email: veronique.joumier@inria.fr

A Multi-Sensor Approach for Activity Recognition in Older Patients

Keywords: Event Streams Fusion, Instrumental Activities of Daily Living, Dementia, Elderly I

Existing surveillance systems for older people activity analysis are focused on video and sensors analysis (e.g., accelerometers, pressure, infrared) applied for frailty assessment, fall detection, and the automatic identification of self-maintenance activities (e.g., dressing, self-feeding) at home. This paper proposes a multi-sensor surveillance system (accelerometers and video-camera) for the automatic detection of instrumental activities of daily living (IADL, e.g., preparing coffee, making a phone call) in a lab-based clinical protocol. IADLs refer to more complex activities than self-maintenance which decline in performance has been highlighted as an indicator of early symptoms of dementia. Ambient video analysis is used to describe older people activity in the scene, and an accelerometer wearable device is used to complement visual information in body posture identification (e.g., standing, sitting). A generic constraint-based ontology language is used to model IADL events using sensors reading and semantic information of the scene (e.g., presence in goal-oriented zones of the environment, temporal relationship of events, estimated postures). The proposed surveillance system is tested with 9 participants (healthy: 4, MCI: 5) in an observation room equipped with home appliances at the Memory Center of Nice Hospital. Experiments are recorded using a 2D video camera (8 fps) and an accelerometer device (MotionPod®). The multisensor approach presents an average sensitivity of 93.51% and an average precision of 63.61%, while the vision-based approach has a sensitivity of 77.23%, and a precision of 57.65%. The results show an improvement of the multi-sensor approach over the vision-based at IADL detection. Future work will focus on system use to evaluate the differences between the activity profile of healthy participants and early to mild stage Alzheimer's patients.

INTRODUCTION

The growth of older people population in past years has highlighted the importance of the development of tools that could extend their independent living, and to improve the diagnosis and treatment of age-related diseases. Information and Communication Technologies (ICT) have proposed wearable sensors [START_REF] Maki | A daily living activity remote monitoring system for solitary older people[END_REF][START_REF] Yang | Development of a fall detection algorithm based on a tri-axial accelerometer[END_REF], smart homes, and video surveillance systems [START_REF] Foroughi | Intelligent video surveillance for monitoring fall detection of older people in home environments[END_REF][START_REF] Nasution | Video surveillance for older people monitoring and safety[END_REF][START_REF] Nasution | Intelligent video surveillance for monitoring older people in home environments[END_REF][START_REF] Wang | An intelligent surveillance system based on an omnidirectional vision sensor[END_REF][START_REF] Fleck | Smart camera based monitoring system and its application to assisted living[END_REF] to address older people surveillance, detect emerging patterns of frailty and falling down events, and to support the early diagnosis of aging related diseases (e.g., Alzheimer, Parkinson, Mild Cognitive Impairment -MCI). People surveillance is generally based in smart home approaches, which analyzes older people daily living activities using a set of heterogeneous sensors placed at home appliances (e.g., infrared presence sensors, contact sensors, temperature sensors, microphones, and video cameras). Their goal is generally to identify falling down events or emerging patterns of frailty by the detection and analysis of a set of self-maintenance activities known as Activities of Daily Living (ADL [START_REF] Monaci | Neuropsychological screening performance and the association with activities of daily living and instrumental activities of daily living in dementia: baseline and 19-to 24-month follow-up[END_REF], e.g., hygiene, toilet use, eating, resting, and dressing) [START_REF] Fleury | SVM-based multimodal classification of activities of daily living in health smart homes: sensors, algorithms, and first experimental results[END_REF][START_REF] Fleury | Introducing knowledge in the process of supervised classification of activities of Daily Living in Health Smart Homes[END_REF][START_REF] Zhang | Decision support for alzheimer's patients in smart homes[END_REF]. Although a multi-sensor approach enriches the quantity of data about the person daily routine, the multiple sources of readings also increase the complexity of data analysis process, as it is necessary to choose the relevant sensor to detect the activity of interest (disregarding the existent data storage issues).

Video-monitoring systems are an alternative to the fully sensor equipped smart-homes, as they can replace or be used in parallel to reduce the number of sensors necessary to describe the overall activity of a person. Existing applications vary from fall detection to ADL detection at constrained environments [START_REF] Foroughi | Intelligent video surveillance for monitoring fall detection of older people in home environments[END_REF][START_REF] Nasution | Video surveillance for older people monitoring and safety[END_REF][START_REF] Nasution | Intelligent video surveillance for monitoring older people in home environments[END_REF][START_REF] Wang | An intelligent surveillance system based on an omnidirectional vision sensor[END_REF][START_REF] Fleck | Smart camera based monitoring system and its application to assisted living[END_REF]. An ontology-based approach was shown for modeling the context of human status (e.g., body posture) and the environment context (semantic information about the scene). The models use information provided by a set of cameras for person detection, and accelerometer devices attached to objects of daily living for environment events triggering (e.g., TV remote control or doors use). A rule-based reasoning engine is used for processing and combining both models types at activity detection level. The ontology tries to solve the semantic gap among the human activities (and scene context) and the sensors raw signals [START_REF] Cao | An event-driven context model in elderly health monitoring[END_REF]. A Fuzzy logic scheme [START_REF] Medjahed | A pervasive multi-sensor data fusion for smart home healthcare monitoring[END_REF] was also proposed to cope with multiple sensor fusion at activity analysis in a smart home. Audio, infrared sensors and a wearable device (acquiring physiological signals, like, ECG, and body posture) are combined to infer ADLs events.

Although daily activity surveillance can support the analysis of medium-to long-term patterns of activity, in-lab clinical protocols supported by ICT have been recently proposed to analyze a person performance in specific activities (like ADL), that could highlight emerging symptoms of a certain diseases. For example, wearable devices have been used to assess older people motor functions performance in a gait analysis test. Patients wear a chest or fist sensor which automatically extracts parameters about their gait performance (e.g., stride length and cadence, vital signals). These parameters are used to identify disturbances in gait patterns that could be associated to emerging symptoms of Dementia (e.g., Alzheimer, MCI, and Parkinson) [START_REF] Gillain | The value of instrumental gait analysis in older people healthy, MCI or Alzheimer's disease subjects and a comparison with other clinical tests used in single and dual-task conditions[END_REF][START_REF] Cancela | Gait assessment in Parkinson's disease patients through a network of wearable accelerometers in unsupervised environments[END_REF][START_REF] Manap | Anomaly gait classification of parkinson disease based on ann[END_REF][START_REF] Auvinet | Dual tasking and gait in people with mild cognitive impairment according to amnesic and non-amnesic subgroups, preliminary results[END_REF].

This paper proposes a multi-sensor surveillance system using heterogeneous sensors data (video and accelerometer recordings) to identify activities during a medical clinical protocol. An ontology approach is used to model the semantic information about activities and the clinical protocol tasks. Accelerometer data is combined with visual information to identify the body posture of the person in situations where video camera data is not sufficient due to occlusion or scene characteristics that affect people detection on the scene.

The set of activities analyzed differ from previous studies as Instrumental Activities of Daily living (IADL) are used instead of ADL. IADL set of activities have been recently discussed as a better estimator for the evaluation of emerging neuropsychiatric symptoms, as they are associated with tasks that require independence, organization, judgment and sequencing abilities (e.g., using the phone, shopping groceries, organizing medications, and managing personal finances) [START_REF] Monaci | Neuropsychological screening performance and the association with activities of daily living and instrumental activities of daily living in dementia: baseline and 19-to 24-month follow-up[END_REF].

The system prototype is tested in an ongoing medical clinical protocol under development by the Memory Center of Nice Hospital, which intends to quantitatively and objectively assess older people executive functions and functional abilities. The long-term goal of this approach and the clinical protocol is to differentiate early stage Alzheimer's patients from healthy participants.

The main contributions of the present work are the use of accelerometer-based and visual-based information for posture recognition. The sensors data are combined using an ontology language similarly to [START_REF] Cao | An event-driven context model in elderly health monitoring[END_REF], but the set of sensors used is different. The IADLs recognition instead of ADLs as previous works discussed is stated as a contribution for the clinical domain. Next sections will present the proposed multi-sensor approach, the clinical protocol where the proposed system is tested, and the preliminary results.

II. MATERIALS AND METHODS

A. Clinical Protocol

Participants aged more than 65 years are recruited by the Memory Center (MC) of the Nice Hospital. Inclusion criteria of the AD group are: diagnosis of AD according to NINCDS-ADRDA criteria and a Mini-Mental State Exam (MMSE) [START_REF] Romdhane | Automatic video monitoring system for assessment of alzheimers disease symptoms[END_REF][START_REF] Folstein | The mini-mental state examination[END_REF] score above 15. AD participants which have significant motor disturbances (per the Unified Parkinson's Disease Rating Scale) are excluded. Controls participants were healthy in the sense of behavioral and cognitive disturbances.

In the designed clinical protocol the participants have to undertake a set of physical activities and IADLs in a Hospital observation room furnished with home appliances. The clinical protocol activities are divided in three scenarios are as follows:

(1) Scenario 01 -Directed activities (10 minutes): it intends to assess kinematic parameters about the participant gait profile (e.g., static and dynamic balance test, walking test); (2) Scenario 02 -Semi-directed activities (15 minutes): it aims to evaluate the degree of independence of the participant by taking and organizing a list of daily living activities (IADLs) within 15 minutes. Participant is alone in the room with the list of activities to perform, and he/she is advised to leave the room only when he/she has felt that the required tasks are completed; (3) Undirected ("free") activities (5 minutes): it aims to assess how the participant spontaneously initiates activities and organize his/her time. The proposed monitoring system is tested with the video recordings of the Semi-directed scenario (Scenario 02). Table 1 shows the set of IADLs that the participant is instructed to perform. Results using a previous version of the clinical and a video-based approach for activity detection be seen in [START_REF] Romdhane | Automatic video monitoring system for assessment of alzheimers disease symptoms[END_REF].

B. Data recording

Experimental data is recorded using a 2D video camera (AXIS®, Model P1346, 8 frames per second), a 3D camera (Kinect® sensor), a motion sensor (MotionPod®), and ambient audio microphone (Tonsion, Model TM6, Software Audacity, WAV file format, 16bit PCM/16kHz). The proposed approach is tested with 9 participants (healthy: 4, MCI: 5).

MotionPod® is a proprietary device that provides estimation about a person posture based on the readings coming from accelerometers and gyroscopes sensors. MotionPod® is attached to Patient chest using a chest strap accessory. As Video and MotionPod® recordings are acquired using different computers, these computers had their internal clock reference automatically synchronized using a network service (Network Time Protocol) to avoid time issues.

C. Multi-sensor surveillance system

The proposed multi-sensor system is divided in two main components: the vision and the event detection components. The video record is processed by the Vision Component which is responsible to detect mobile objects and classify them according to a set of objects of interest (e.g., person). Once a person is detected on the video frame under analysis, the event module component analyzes his/her activity in relation to the activity models previously defined by an expert using an ontology language. Posture events coming from MotionPod® data are taken in account only at the event component level. Fig. 1 illustrates a diagram of the system architecture and sensors contribution for each module.

1) Vision Component

The vision component has been developed using a modular vision platform locally developed that allows the test of different algorithms for each step of the computer vision chain (e.g., video acquisition, image segmentation, physical objects detection, physical objects tracking, actor identification, and actor events detection). The vision component extracts the objects to track from the current frame using an extension of the Gaussian Mixture Model algorithm for background subtraction [START_REF] Nghiem | Controlling background subtraction algorithms for robust object detection[END_REF]. People tracking is performed by a multi-feature tracking algorithm presented in [START_REF] Chau | A multi-feature tracking algorithm enabling adaptation to context variations[END_REF], using the following features: 2D size, 3D displacement, color histogram, and dominant color.

2) Event Detection Component

Event detection component uses a generic constraint-based ontology language to model and recognize activity events. Event models are described using a priori knowledge about the experimental scene (scene model), the clinical protocol activities (event models), attributes of the physical object tracked by the vision component (e.g., spatial, temporal, kinematic and appearance properties), and posture estimations coming from MotionPod® sensor.

The a priori knowledge about the experimental scene consists of the decomposition of a 3D projection of the scene floor plan in a set of spatial zones which represent contextual information about the clinical scenario (e.g., zoneTV, zoneArmChair, zoneOfficeDesk). The constraint-based ontology is declarative and intuitive language as it is based in natural terminology, allowing domain experts to easily define and modify the event models.

Event models are hierarchically categorized according to their complexity (primitive or complex) and the information they model (the current state or time-based event). Primitive states refer to a specific value in the property(ies) of a tracked physical object (e.g., the participant). Primitive events refer to a change(s) in the value(s) of an object property(ies) (e.g., change of posture). Composite/Complex events are defined as a set of primitive events and/or states. More details about the generic ontology language used are seen in [START_REF] Zaidenberg | A generic framework for video understanding applied to group behavior recognition[END_REF]. Table 2 shows an example of the complex event called "Person standing and using OfficeDesk". The ontology event description is divided among four components: the Physical objects involved in the event model (an object classified as a Person -p1, a contextual zone -z1, and a contextual objecteq. 1); the Components (or sub-events) that must be detected for the Event Model identification (e.g., Person_using_OfficeDesk); the constraints which components and physical objects attributes must hold (e.g., minimal duration of the event "Person using OfficeDesk"); and an Alarm category used as reference to a specific treatment that could be performed in case of the event is detected. Table 3 describes the primitive state called "Person using OfficeDesk". "Person using Office Desk" event is modeled based on the participant presence inside the contextual zone "OfficeDesk", and in the distance between participant 3D bounding box projection on the scene floor and the parallelepiped modeling the real desk.

3) Multi-sensor event fusion

Sensor fusion can be performed at different levels, e.g., data level (raw data combination), feature level (fusion of observation data from different sensors), or at decision level (fusion of events detected by different sensors) [START_REF] Zouba | Multisensor fusion for monitoring elderly activities at home[END_REF]. The multi-sensor fusion herein presented is performed at decision level. One advantage of sensor fusion at this level is the possibility of changing the sensor type without the need of change of event models, e.g., postures could be defined as an attribute or a subevent, the source of which could be replaced as soon as it keeps its label. Herein the posture status coming from MotionPod® and the Visual component are defined as attributes of the ontology model called "Person", and the specific changes in these attribute values are modeled as primitive events.

Raw MotionPod® is post-processed using MotionPod® manufacturer software which provides the set of participant postures during the experiment (e.g., standing, sitting, lying down, changing from a posture to another). The vision component estimates standing and sitting postures based on a sitting threshold applied over the person 3D height. The sitting threshold is manually defined per video (and patient) based on the visualization of the participant average sitting height. A time synchronization step is performed based on sensors (video and accelerometer) readings timestamps.

The choice of which sensor posture estimation is used per activity model was based in the analysis of the system activity recognition performance in the defined contextual zones. For example, goal-oriented events that are performed farther from the camera viewpoint, where the Person remains immobile used accelerometer posture estimation, and Event models that requires response in posture estimation (Sitting to Standing transfer tests) were modeled in respect to the vision module posture estimation, as the accelerometer standard data acquisition rate is around 1 data acquisition/sec.

4) Events Models and IADLs

Event models are described taking in account a person presence in a contextual zone, his/her proximity to a contextual objects, and his/her current body posture (e.g., standing, bending). These constraints pose a limitation to some IADLs models as their target actions that cannot be directly detected (e.g., writing a check to pay electricity bill, reading a magazine, organizing a set of cards) and they have the same contextual information.

In these cases, a few IADLs of the Clinical protocol are merged in a single activity. For example, answering and calling someone is a voice-based behavior in which the person interacts with an object (the phone), and, as there is none pressure sensor in the phone nor audio recognition in this approach, these two models are simplified to "using phone" activity. Table 4 lists the activity models of the clinical protocol (Scenario 02) and their simplified version.

D. Evaluation

Evaluation results are presented for event models based only on visual constraints and the Multi-sensor approach to evaluate the improvements in event detection performance. Vision-based models consist of the same activity models but only taking in account posture estimation obtained from visual information.

Activity recognition performance is assessed using the performance indices precision and sensitivity (Equations 1 and 2, respectively), where TP refers to True Positive events; FP to False Positive events, TN to True Negative events, and FN to False Negative events. The evaluation is performed for the 15 min. of each video sequence, and not in the form of previously extracted video chunks containing only IADL events.

(1)

(2)

III. RESULTS AND DISCUSSION

Table 5 shows the events detection performance rate for the vision system approach about 9 participants (15 min. each). GT states for ground-truth, and it specifies the total number of events inside the video sequences of all participants. The term "Person" was omitted from the event model name to improve the table readability. Table 6 shows the event detection rate for the multi-sensor approach. The vision system has an average precision of 57.65% and a sensitivity of 77.23%. The multi-sensor system performance is higher at the two indices, 63.61% at precision and 93.51% at sensitivity, highlighting that the use of accelerometer data in posture estimation has increased the overall system performance by improving the precision of the first three events of Table 6.

Factors like occlusion, illumination changes, and color clothing similar to background have reduced person body detection at visual component, increasing the difficult at determining posture. Accelerometer data acquisition resolution is sometimes not sufficient to cope with person speed, e.g., in changing postures activities. But, the use of both sensors, each one applied in the situation (event model) where it provides better estimation, have improved the overall system performance without incorporating noisy in the event estimation framework.

Part of the FP events that caused lower values of precision could be explained by unexpected behaviors of the participants that are not predicted in the event models. For example, events based on the spatial zone "OfficeDesk" are affected by this zone proximity to the "Watching TV" zone. FP events of "Using pharmacy" events are explained by a few patients placing a chair into this zone to watch the TV (located in the opposite side of the room). These inconsistences will be future addressed by refining the event and scene models and by the adoption of new features (e.g., adding an orientation attribute to the "Person" model and place constraints avoiding the cited FP event).

IV. CONCLUSION AND FUTURE WORK

The preliminary results of the proposed system shows that the adoption of a multi-sensor approach (a video data complemented by a wearable accelerometer) has improved the event detection performance of the activity recognition system in comparison to the same system based only on video data.

Future work will focus at analyzing an unsupervised way of choosing among the posture attributes based on reliability measurement. A reliability measurement could support the automatic choice of one sensor over the others or the calculation of the likelihood of events based on multiple sensors reading. A broader validation is also planned to evaluate the reproducibility of results shown in a larger quantity of patients. The proposed multi-sensor approach also added new posture types (e.g., bending, lying down) that will be considered in the next evaluation. The long-term goal of the proposed approach is to support clinicians in the identification of emerging symptoms that could early diagnose Alzheimer patients at mild to moderate stages in a quantitative and objective way.

Figure 1 .

 1 Figure 1. System architecture

Fig. 2

 2 shows an example of the vision component output. Rectangle envelope highlights the detection of a person in the scene. Blue dots represent previous positions of the detected person in the scene.

Figure 2 .

 2 Figure 2. Multi-sensor activity recognition system screen. Rectangle envelope highlights the detection of a person in the scene. Blue dots represent previous positions of the detected person in the scene.

TABLE 2 .

 2 EVENT MODEL OF PERSON_STADING_AND_USING_OFFICEDESK CompositeEvent(Person_standing_ and_using_OfficeDesk, PhysicalObjects( (p1 : Person), (z1 : Zone), (eq1 : Equipment)) Components( (c1 : CompositeEvent Person_using_OfficeDesk(p1, z1, eq1)) (c2: PrimitiveState Person_standing_MP ( p1) ) ) Constraints((duration(c1) > 2) Alarm ((Level : NOTURGENT)) )

TABLE 1 .

 1 CLINICAL PROTOCOL -SCENARIO 02

	Watch TV,
	Make tea/coffee,
	Write the shopping list of the lunch ingredients,
	Answer the Phone,
	Read the newspaper/magazine,
	Water the plant
	Organize the prescribed drugs inside the drug box according to the
	daily/weekly intake schedule,
	Write a check to pay the electricity bill,
	Call a taxi,
	Get out of the room.

TABLE 3 .

 3 EVENT MODEL OF PERSON_USING_OFFICEDESK

	CompositeEvent(Person_using_OfficeDesk,
	PhysicalObjects( (p1 : Person), (z1 : Zone), (eq1 : Equipment))
	Components(
	(c1: PrimitiveState Person_Inside_Zone_OfficeDesk(p1, z1))
	(c2: PrimitiveState Person_closeTo_OfficeDesk(p1, eq1))
	)
	Alarm ((Level : URGENT))
	)

TABLE 4 .

 4 COMPARISON BETWEEN EVENT MODELS AND PROTOCOL IADLS

	EVENT MODEL		MODELED EVENT
	Person using OfficeDesk	 Make a shopping list of the
			ingredients for lunch
		 Write a check to pay the
			electricity bill.
	Person watching TV	 Watch TV
	Person preparing Coffee	 Make Tea/Coffee.
	Person using Pharmacy		Organize	the
		prescribed drugs inside the

TABLE 5 .

 5 ACTIVITY DETECTION RATE OF THE VISION SYSTEM

	Activity	GT	Precision (%)	Sensitivity (%)
	Standing and preparing Coffee	13	37.5	37.50
	Sitting and using OfficeDesk	8	5.80	100.00
	Standing and using OfficeDesk	12	43.37	100.00
	Standing and using Pharmacy	8	75.92	100.00
	Standing and using Phone	19	83.33	25.92
	Standing and watering Plant	10	100.00	100.00
	Average performance	70	57.65	77.23
	TABLE 6. ACTIVITY DETECTION RATE OF THE FUSION SYSTEM
	Activity	GT	Precision (%)	Sensitivit y (%)
	Standing and preparing Coffee	13	51.85	100
	Sitting and using OfficeDesk	8	27.22	100
	Standing and using OfficeDesk	12	43.37	100
	Standing and using Pharmacy	8	75.92	100
	Standing and using Phone	19	83.33	100
	Standing and watering Plant	10	100.00	61.11
	Average performance	70	63.61	93.51

V. ACKNOWLEDGEMENTS

We would like to thank the CEA for providing us the MotionPod® accelerometer data used in the experiments.

VI. REFERENCES


Sarah Zeghada

Ghenia Bentabed-Ababsa

Aïcha Derdour

Safer Abdelmounim

Luis R Domingo

José A Sáez

Thierry Roisnel

Ekhlass Nassar

Florence Mongin

A combined experimental and theoretical study of the thermal cycloaddition of aryl azides with activated alkenes

Reactions were performed from aryl azides on the one hand, and activated alkenes coming from dicarbonyl compounds or malonodinitrile on the other hand, either with recourse to conventional heating or to microwave activation, to afford 1-aryl-1H-1,2,3-triazoles. The mechanism and the regioselectivity of the reactions involving -dicarbonyl compounds have been theoretically studied using DFT methods at the B3LYP/6-31G* level: they are domino processes comprising a tautomeric equilibrium of the dicarbonyl compounds with their enol forms, a 1,3-dipolar cycloaddition of the enol forms with the aryl azides (high activation energy), and a dehydration process (lower activation energy). The effect of nonconventional activation ways on the degradation of 1,2,3-triazolines was next studied experimentally. Finally, some of the 1,2,3-triazoles such synthesized were evaluated for their bactericidal and cytotoxic activities.

Introduction

Cycloaddition reactions are one of the most important synthetic processes, with both synthetic and mechanistic interest in organic chemistry. Among them, 1,3-dipolar cycloadditions (13DCs), whose general concept was introduced by Huisgen and coworkers in 1960s, 1 are versatile tools for building five-membered heterocycles. 2 1,2,3-Triazole occupies a special place in heterocyclic chemistry because it is the core structure of many agents of various interests (use as pharmaceuticals, agrochemicals, dyes, corrosion inhibitors, sensors, photostabilizers…), and 13DC between azides and alkynes or enolizable compounds are important ways to generate this heterocycle. 3 We focused on the synthesis of 1-aryl-5-methyl-1H-1,2,3triazoles bearing a ketone or an ester function at the 4 position, these compounds being both appreciated for their biological 4 and photophysical 5 properties, and we decided to attempt their syntheses with recourse to less conventional activating systems 35 than oil bath heating such as microwaves and ultrasounds. The study was then extended to reactions with other dipolarophiles, and the mechanism of these reactions was investigated using DFT calculations. Finally, some of the triazoles such synthesized were evaluated for their bactericidal and cytotoxic activities.

40

Results and discussion

Synthetic aspects

We first considered 13DC reactions between different aromatic azides on the one hand, and acetylacetone or methyl acetoacetate on the other hand. Since the first 1,2,3-triazole formations by 45 reaction between aryl azides and methylene-activated compounds in the early 1900s, 6 chemists have extended the scope of the reaction. 5,7 The latter proceeds in good yields at room temperature but requires long reaction times, and higher temperatures lead to reduced conversions. 7k In addition, the 50 efficiency of the reaction is lowered when electron-donating groups are present on the aryl azide. 5 When 4-nitro-, 4-chloro-, 4-bromo-, and 3-nitrophenyl azides (1a-d) were treated with acetylacetone (2a, 2 equiv) in the presence of triethylamine (2 equiv), and using DMF as solvent, 55 the 1,2,3-triazoles 3a-d were isolated in high yields (Table 1, entries 1-4). As expected, a lower yield was noted when the nitro group was present at the 3 position instead of the 4 position. Similar results were recorded using methyl acetoacetate (2b) instead of acetylacetone to afford the derivatives 3e-h satisfactorily (Table 1, entries 5-8).

A rising number of articles have advocated the use of microwave technology in organic synthesis. Harsh conditions such as high temperatures and long reaction times often required for cycloaddition reactions were generally improved using this technique. 8 In order to reduce the reaction times, we thus decided to examine the effect of microwave irradiation on the reactions. Solvents being optional using this technology, the aryl azides were simply microwave-heated in the presence of 2 equiv of 10 acetylacetone or methyl acetoacetate, and 2 equiv of triethylamine at 160 W. The precipitate formed after 5 min was then collected and washed with water. Whereas good results were obtained under these conditions from nitrophenyl azides 1a and 1d (Table 2, entries 1, 4, 5 and 8), moderate to low yields were observed using halogen-substituted phenyl azides 1b and 1c (Table 2, entries 2, 3, 6 and 7), and extended reaction times (15 min) and higher microwave powers did not allow to improve them.

The reactions under both types of conditions are regioselective. 20 The nature of the regioisomer formed was established by NMR techniques: HMBC for the assignments of the methyl signals, and NOESY to confirm an interaction between the methyl group at the 5 position of the 1H-1,2,3-triazole ring and the phenyl ring. In addition, the structures of the cycloadducts 3b, 3c, and 3f were 25 identified unequivocally by X-ray diffraction analysis. -Benzoylacetophenone (4a) and malonodinitrile (4b)

similarly reacted with 4-nitrophenyl azide (1a) to afford the compounds 5a and 5b, respectively (Table 3). The former was identified by X-ray diffraction analysis. It was isolated in 40 and 30 85% yield, respectively at room temperature and under microwave irradiation, under the conditions employed before (entries 1 and 2). Using a catalytic amount of copper(I) chloride 9 allowed to perform the reaction at room temperature in water in a satisfying 57% yield (entry 3). The same three sets of conditions 35 were similarly employed to furnish the derivative 7 in 61, 59 and 70% yield, respectively (entries 4-6).

In the previous reactions, the 1,2,3-triazolines 6 were not observed due to elimination of water after the cycloaddition step according to the mechanism depicted in Scheme 1. More 40 generally, the isolation of 4,5-dihydro-1H-1,2,3-triazoles, which are compounds of biological interest, 10 is not obvious, 11 either due to subsequent elimination, or to cleavage with for example conversion to aziridines or diazo compounds. 7h,12 In order to study the effect of non-conventional activation 45 ways on the degradation of 1,2,3-triazolines, we chose N-(2hydroxyphenyl)acrylamide (7a), its 2-allyl derivative 7b and Nvinylpyrrolidinone (7c), and examined their behavior toward 4nitrophenyl azide (1a) (Table 4). At the reflux temperature of an apolar solvent such as toluene, the triazoline 8 was obtained from 50N-(2-hydroxyphenyl)acrylamide (7a) in a low 10% yield after 24 h reaction time (entry 1). Turning to acetone as solvent allowed the yield to reach 23% (entry 2). The reaction was also attempted without solvent, with recourse to microwave irradiation, but without success, the starting materials being recovered (entry 3).

55

We thus decided to turn to ultrasound activation. [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] Sonicating the acetone reaction mixture for 24 h resulted in an improved but still moderate 34% yield (entry 4), due to the formation of other derivatives. By replacing the sonication by an activation based on the catalytic use of a montmorillonite clay containing copper(II), 60 Bentonite-Cu 2+ , [START_REF] Caillère | Minéralogie des argiles[END_REF] and by using toluene as solvent, the side reactions were partly discarded, and the yield was improved to 68% (entry 5).

N-(2-Allyloxyphenyl)acrylamide (7b) regioselectively reacted at the conjugated double bond with 4-nitrophenyl azide (1a). The 65 reaction performed at the reflux temperature of acetone worked in a moderate 20% yield (entry 6), as observed with N-(2hydroxyphenyl)acrylamide (7a) (entry 2). A similar yield being obtained using a catalytic amount of Bentonite-Na + in toluene at

R R N 3 N N N R' R + 1a 5 4 NO 2 NO 2 entry R (4) product (5) R' yield 1 a C(O)Ph (4a) 5a Ph 40% 2 b C(O)Ph (4a) 5a Ph 85% 3 c C(O)Ph (4a) 5a Ph 57% 4 a CN (4b) 5b NH2 61% 5 b CN (4b) 5b NH2 59% 6 c CN (4b) 5b NH2 87%

R' Me

N 3 O OH N N N Me R' O + R 1 R 3 2' R' Me O O 2 N N N Me R' O 6 H HO -H 2 O R Scheme 1
Mechanism for the 1H-1,2,3-triazole formation. 5 room temperature under ultrasound activation (entry 7), we turned to the use of Bentonite-Cu 2+ : under the same reaction conditions, the triazoline 9 was isolated in 33% yield (entry 8). Kadaba has shown that N-vinylamides can be used as dipolarophiles in reactions with aryl azides, and that the subsequent elimination of carboxamide largely depends on the reaction conditions. 15 Compared with N-(2hydroxyphenyl)acrylamide, N-vinylpyrrolidinone (7c) (dominant HOMO olefin -LUMO azide interactions) 15 proved to react more efficiently with 4-nitrophenyl azide (1a). When acetone was used as solvent, the cycloadduct 10 was provided in 63% yield after 24 h reflux (entry 9). In contrast, recourse to microwave irradiation without solvent resulted in the elimination of 2-pyrrolidinone, giving 1-(4-nitrophenyl)-1H-1,2,3-triazole (11) in 58% yield (entry 10). These results are different from those reported by Kadaba, where elimination takes place at the reflux temperature of ethanol, and formation of triazolines occurs in the absence of solvent at room temperature. 15 Calculations 1) DFT study of the mechanism of the domino reactions of dicarbonyl compounds 2 with 4-nitrophenyl azide (1a). The thermal reactions between the -dicarbonyl compounds 2 and 4-nitrophenyl azide 1a are domino processes that comprise three consecutive reactions: i) a tautomeric equilibrium between the -dicarbonyl compounds 2 and their enolic forms 2', ii) a 13DC reaction between the corresponding enols 2' and 4nitrophenyl azide (1a) to give the 4,5-dihydro-1,2,3-triazoles 6, and iii) an acid-base catalyzed dehydration process to yield the final 1,2,3-triazoles 3 (see Scheme 2). In order to obtain mechanistic details, as well as to explain the complete regioselectivity experimentally observed in the formation of the 1,2,3-triazoles 3, the two regioisomeric channels involved in the 13DC reactions were studied using DFT calculations at the B3LYP/6-31G* level.

The first reaction of this domino process is a tautomeric equilibrium between the keto and the enol forms of the dicarbonyl compounds 2. The enols 2' can adopt an E or Z configuration at the C4-C5 double bond. The Z isomers are 6.2 kcal/mol (2'a-Z) and 4.5 kcal/mol (2'b-Z) more stable than the corresponding E isomers, due to the formation of an 40 intramolecular hydrogen bond in the Z isomers (see Table 5). The formation of the 2'a-Z and 2'b-Z enols is exothermic by -5.2 and -0.2 kcal/mol, respectively. (As some species involved in these domino reactions have ionic character, the discussion is performed using the energies in acetonitrile).

45

Due to the asymmetry of the enols 2' and 4-nitrophenyl azide (1a), the subsequent 13DC reactions can take place through two regioisomeric channels associated with the approach of the N1 nitrogen of 4-nitrophenyl azide (1a) to the C5 or the C4 carbon atoms of the enols 2', named 1 and 2, respectively. In order to 50 analyze the role of the electron-withdrawing (EW) substitution in the aryl azides, the 13DC reaction of phenyl azide (1e) with the enol 2'a was also studied. In addition, the two regioisomeric channels associated with these 13DC reactions were studied in order to explain the total regioselectivity experimentally observed 55 (see Scheme 2).

The activation energies for the 13DC reactions of the enols 2' with the aryl azides 1 through the more favorable regioisomeric channels 1 via TS11 present very high values, between 22.2 to 24.7 kcal/mol (see Table 5). The 13DC reaction of the enol 2'a-Z 60 with phenyl azide (1e) has the largest activation energy, 24.7 kcal/mol (TS11ea) (the anachronism "e" is used to distingue the species involved in the reactions of phenyl azide (1e)). A similar activation energy of 25.1 kcal/mol was obtained by single point energy calculations at the MP3/6-31G* level. The inclusion of an 65 EW nitro group on the aryl substituent decreases slightly this unfavorable energy, 22.2 kcal/mol (TS11a). The similar

10 N N N O 2 N N O 63% 0% a a 1-(4-Nitrophenyl)-1H-1,2,3-triazole (11) was isolated in 58% yield. N N N O 2 N 11 1a: Ar = 4-NO 2 C 6 H 4 1e: Ar = Ph 2a: R = Me 2b: R= OMe TS11 TS12 O O R 2 R O OH 2' 1 4 N N N Ar O R N N N Ar O R 31 32 N N N Ar OH O R 61 N N N Ar HO O R 62 N N N 1 2 3 Ar -H 2 O 5 -H 2 O
Scheme 2 Regioisomeric channels 1 and 2 associated with the thermal reactions of the -dicarbonyl compounds 2 with the aryl azides 1.

activation energy obtained for the 13DC reaction between the enol 2'b-Z and 4-nitrophenyl azide (1a), 22.2 kcal/mol (TS11b), accounts for a similar reactivity for the enols 2'b-Z and 2'a-Z.

The activation energy for the 13DC reaction of the enol 2'a-Z with phenyl azide (1e) via TS11ea is ca. 10 kcal/mol higher than that for the 13DC reaction of phenyl azide (1e) with methyl vinyl ether, as a model of electron-rich olefins, 16 and higher than that obtained for the 13DC reaction of hydrazoic acid, N 3 H, with ethylene, 19.5 kcal/mol, 17 and with vinyl alcohol, 20.2 kcal/mol. 18 The activation energies associated with the more unfavorable regioisomeric channels 2 via TS12 are between 7.0 and 10.5 kcal/mol higher in energy that those associated with the channels 1 (see Table 5). These high energy differences, which are higher than that found by Nguyen for the regioisomeric TSs associated with the 13DC reactions between hydrazoic acid and vinyl alcohol, E 5.1 kcal/mol, 18 are in complete agreement with the total regioselectivity experimentally observed.

From the enols 2' and the aryl azides 1, the formation of the cycloadducts (CA) 61 and 62 is slightly exothermic (see Table 5).

The last reaction of these domino processes is the acid-base 20 catalyzed dehydration of the CA 61. These dehydration reactions proceed in three steps: i) a protonation at the keto or ester carbonyl oxygen atom of 61, which correspond to the most basic centers, ii) an E1 elimination of water at the hydroxy group of the protonated 61, and iii) a hydrogen elimination at the vicinal C5 carbon atom to rebuild the C4-C5 double bond of the enol, thus yielding the final 1,2,3-triazoles 31 (see Scheme 3). We selected trimethylammonium cation as model of the acid catalyst.

The elimination process was studied for the thermal reactions of the -dicarbonyl compounds 2a,b with 4-nitrophenyl azide (1a). Because similar results were obtained for both -dicarbonyl compounds, only the results obtained for 1a will be discussed for simplicity. Anyway, the conclusions obtained for this catalyzed dehydration process could be applied to the rest of the reactions. Protonation of 61a by trimethylammonium cation takes place at Similar energy results were obtained for the water elimination in the regioisomeric CA 62a (see Table 5). Because the activation energies involved at the elimination reaction are lower than those involved in the 13DC reaction, it makes possible to discard a 50 thermodynamic equilibration between the regioisomeric CAs 61a and 62a. Consequently, the formation of the final product 31a is kinetically controlled in the 13DC reaction. Finally, similar results were obtained for the elimination reaction at the regioisomeric CAs 61b and 62b associated with the reaction of 55 methyl acetoacetate (2b) (see Table 5).

The geometries of the TSs involved in the 13DC reaction between the enol 2'a-Z and 4-nitrophenyl azide (1a) are given in Figure 1, while the geometrical and electronic parameters of the TSs involved in the 13DC reactions are given in Table 6. The 60 lengths of the forming bonds at the TSs are 1.974 Å (N1-C5) and 2.251 Å (N3-C4) at TS11a, and 1.906 (N1-C4) and 2.150 (N3-C5) Å at TS12a. At these TSs the N-C bond formation at the unsubstituted N1 nitrogen atom of the aryl azide is slightly more advanced than that at the substituted N3 nitrogen atom. 18 Similar 65 bond formation is observed at the other 13DC reactions (see Table 6). The asynchronicity on the bond-formation in the cycloaddition reactions can be measured by the difference of the lengths of the  forming bonds, l. The corresponding values are given in Table 6. These values are in the range of 0.1 to 0.3, 70 indicating that the TSs correspond to concerted but asynchronous bond formation processes. For the 13DC reaction involving phenyl azide (1e), the TS12ea associated with the more unfavorable regioisomeric channel 2 is more asynchronous than the TS11ea associated with the channel 1, while for the reactions 75 involving 4-nitrophenyl azide (1a), TS11a associated with the more favorable regioisomeric channels 1 are more asynchronous. The extension on bond-formation at the TSs is provided by the bond order (BO). 19 The BO values at the TSs associated with the 13DC reactions are given in Table 6. At the TSs associated with the more favorable regioisomeric channels 1, the BO values of the N1-C5 forming bonds are in the range of 0.45 -0.48, while those for the N3-C4 forming bonds are in the range of 0.25 -0.29. The bond formation at the unsubstituted N1 is more advanced than at the substituted N3 atom. At the most favorable regioisomeric channels 1, the TSs involving 4-nitrophenyl azide (1a) are more advanced and more asynchronous. At the TSs associated with the more unfavorable regioisomeric channels 2, the BO values of the N1-C4 forming bonds are in the range of 0.45 -0.49, while those for the N3-C5 forming bonds are in the range of 0.33 -0.34. These TSs are slightly more advanced and more synchronous than the regioisomeric TSs 1.

The natural population analysis (NPA) allows us to evaluate the charge transfer (CT) at the TSs. The natural charges at the TSs were shared between the aryl azides 1 and the enols 2'. The CT, which takes place from the enols 2' to the azides 1, is given in Table 6. The CT at the TSs associated with the more favorable regioisomeric channels 1, between 0.17e and 0.26e, is larger than that at the TSs associated with the regioisomeric channels 2, between 0.08e and 0.15e. The more polar TSs are those associated with the 13DC reactions between the electron-rich enols 2'a-Z and 2'b-Z with electron-deficient (ED) 4-nitrophenyl azide (1a).

Finally, the geometries of the TSs involved in the elimination reaction of the CAs 61a and 62a are given in Figure 1. The lengths of the breaking and forming bonds at the TSs involved in the water elimination step are given in Table 7. At TS21a, the 30 length of the C4-O6 breaking bond is 1.627 Å, while the lengths of the O8-H7 breaking and H7-O6 forming bonds are 1.305 and 1.129 Å, respectively. The length of the N10-H9 hydrogen bond is 1.997 Å. At TS22a, the length of the C4-O6 breaking bond is 1.740 Å, while the lengths of the O8-H7 breaking and H7-O6 35 forming bonds are 1.599 and 1.016 Å, respectively. The length of the N10-H9 hydrogen bond is 2.196Å. At TS22a the water elimination process is more advanced than at TS21a.

Similar geometrical parameters were obtained for the water elimination step of the domino reaction between the enol 2'b and 40 the aryl azide 1a (see Table 7).

2) Analysis based on the global and local reactivity indexes at

the ground state of the reagents. Recent studies carried out on cycloaddition reactions 20 towards ED 4-nitrophenyl azide (1a), in clear agreement with the CT analysis performed at the TSs.

The electrophilicity and nucleophilicity indices of hydrazoic acid, N 3 H, have low values,  = 1.37 and N = 1.62 eV. This means that the simplest azide does not participate in polar processes. Phenyl azide (1e) shows a slight electrophilic deactivation,  = 1.27 eV, and a slight nucleophilic activation, N = 2.92 eV (see Table 8). The inclusion of an halogen atom in the 4 position of benzene produces a slight electrophilic activation,  = 1.42 eV for 1b and 1c. A more clear electrophilic activation is found at ED 4-nitrophenyl azide (1a), which has an  = 2.66 eV. On the other hand, the electrophilicity and nucleophilicity indices of the enols are  = 1.35 and N = 2.65 eV for 2'a-Z, and  = 1.11 and N = 2.74 eV for 2'b-Z. Therefore, they will act as moderate nucleophiles.

Analysis of the global reactivity indices indicates that these reactions will take place through cycloadditions with low polar character. Only the 13DC reactions involving the EW 4nitrophenyl group will present a large polar character.

Finally, the analysis of the local electrophilicity  k at the electrophilic azides and the local nucleophilicity N k at the nucleophilic enols allows for a rationalization of the regioselectivity of cycloadditions. 22 The values of the corresponding local indices are given in Table 9. The enols 2'a,b present the largest nucleophilic activation at the C5 carbon atom, which corresponds with the  carbon atom of the enols. On the other hand, 4-nitrophenyl azide (1a) presents the largest electrophilic activation at the terminal N1 nitrogen atom. [START_REF]For 4-nitrophenyl azide (3n), the k f  Fukui functions for 115 nucleophilic attacks were evaluated using the LUMO + 1, since those obtained from the LUMO are mainly concentrated to the aromatic ring[END_REF] Note that the N2 Nitrogen also presents an electrophilic activation. Consequently, the more favorable regioisomeric channel will correspond with the earlier formation of the N1-C5 bonds than the N3-C4 one, in clear agreement with the energy results and with the asynchronicity found at the TSs.

3) Conclusions.

The thermal reactions between acetylacetone (2a) and methyl acetoacetate (2b) with ED 4-nitrophenyl azide (1a) have been theoretically studied using DFT methods at the B3LYP/6-31G* level. They are domino processes that comprise three consecutive reactions: i) a tautomeric equilibrium of the -dicarbonyl compounds 2 with their enol forms 2', ii) a 13DC reaction of the 40 enol forms 2' with 4-nitrophenyl azide (1a) to yield the 4,5dihydro-1,2,3-triazoles 6, and iii) an acid-base catalyzed dehydration process in 6 to yield the final 1,2,3-triazoles 3. For 13DC reactions, the two regioisomeric channels have been studied.

45

The 13DC reactions present high activation energies, which are only slightly reduced by the use of ED 4-nitrophenyl azide (1a). The higher activation energies associated with the regioisomeric channels 2 prevent the formation of the corresponding cycloadducts, these 13DC reactions being 50 completely regioselective.

The lower activation energies of the water elimination step, respect to the 13DC reaction, together with the irreversible character of this dehydration process make possible to displace the reaction toward the formation of the final 1,2,3-triazoles 3.

55

Pharmacology

Applying the agar plate diffusion technique, [START_REF] Bauer | [END_REF] the newly synthesized compounds 3a-h and 5a were screened in vitro for their bactericidal activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria 60 (Escherichia Coli and Pseudomonas aeroginosa), and for their fungicidal activity towards Fusarium oxysporium, Aspergillus niger and Candida albicans (Table 10). The compounds 3a-c and 3e-g showed moderate bactericidal activities against Staphylococcus Aureus and Escherichia Coli compared to that of 65 ciprofloxacin as a reference drug, and the compounds 3d, 3h and 5a showed significant activities against Staphylococcus Aureus. All the compounds tested showed moderate fungicidal activities towards Fusarium oxysporium and Aspergillus niger compared to that of nystin as a reference, except the compounds 3b-d that 70 showed significant activities against Fusarium oxysporium. All the compounds tested showed no activity against Candida albicans.

The same compounds were also tested against a human liver carcinoma cell line (HEPG2) (Table 11). Moderate cytotoxic 75 activities were observed, except for the compounds 3d and 5a (entries 4 and 9), which were found to have significant activities, compared to a reference drug (doxorubicin).

Conclusions

Reactions performed between aryl azides on the one hand, and activated alkenes coming from -dicarbonyl compounds or malonodinitrile on the other hand showed that microwave activation allowed the reaction times to be considerably reduced in the case of nitrophenyl azides. With the help of DFT methods at the B3LYP/6-31G* level, the mechanism of the reactions involving -dicarbonyl compounds was studied, and their regioselectivity explained. They are domino processes that comprise a tautomeric equilibrium of the -dicarbonyl compound with its enol form, a 13DC with the aryl azide, and a dehydration process. The lower activation energy of the last step compared with the previous one helps to explain why 1,2,3-triazolines were not obtained, but the corresponding 1,2,3-triazoles. The effect of non-conventional activation ways on the degradation of other 1,2,3-triazolines was next studied experimentally. Finally, some among the 1,2,3-triazoles evaluated exhibited interesting bactericidal and cytotoxic activities.

Experimental

Syntheses: general methods 20 Liquid chromatography separations were achieved on silica gel Merck-Geduran Si 60 (40-63 m). Petrol refers to petroleum ether (bp 40-60 °C). Melting points were measured on a Kofler apparatus. Infrared spectra were recorded on a JASCO FT/IR-4200 apparatus, and main wavenumbers are given in cm -1 .

25

Nuclear Magnetic Resonance spectra were acquired using a Bruker AC-300 spectrometer (300 MHz and 75 MHz for 1 H and 13 C respectively). 1 H chemical shifts () are given in ppm relative to the solvent residual peak, and [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] C chemical shifts relative to the central peak of the solvent signal. Assignments of protons and carbons could be made on the basis of bidimensional techniques (NOESY, HMBC and HMQC experiments) using a Avance 500 spectrometer (500 MHz and 125 MHz for 1 H and 13 C respectively). High resolution mass spectra measurements were recorded at the Centre Régional de Mesures Physiques de l'Ouest Bentonite-Cu 2+14 was obtained as follows. Deposit bentonite (60 g) was dried (80 °C, 18 h), crushed and sieved (200 μm) before treatment in order to remove carbonates (calcite and 40 dolomite) at pH 2-3 (addition of 0.1 N HCl to a suspension in water) for 12 h, and adjustment at pH 7. The clay was then scattered in aqueous 1 M NaCl; three stirring of the suspension for 12 h, centrifugation (4500 t.min -1 ) and washing sequences were then performed. After two drying (80 °C and 105 °C)-45 crushing steps, resulting bentonite-Na + was suspended in water (50 g.l -1 ); the suspension was stirred and treated over 12 h by an aqueous 1 N CuCl 2 solution (0.5 L). This was repeated three times after decantation and removal of the supernatant liquid. Bentonite-Cu 2+ was washed using water, centrifuged (5000 50 t.min -1 ), and dried at 60 °C. After crushing and sieving (60 μm), it was dried again at 105 °C for 12 h.

Aryl azides were prepared according to the method of Nolting and Michel. 25 N-(2-Hydroxyphenyl)acrylamide (7a) was prepared as described previously. 26 N-(2-Allyloxyphenyl)acrylamide (7b) 55 was prepared using a described procedure, 27 General procedure 1. To the required methylene-activated compound (10 mmol) and Et 3 N (10 mmol) in DMF (2 mL) was added the required azide (5 mmol), and the mixture was stirred at room temperature for 24 h. The precipitate was filtrated, washed with H 2 O, dried, and recrystallized from i PrOH. 70 General procedure 2. To the required methylene-activated compound (10 mmol) and Et 3 N (10 mmol) was added the required azide (5 mmol), and the mixture was microwave-heated at 160 W for 5 min. The precipitate was washed with H 2 O and dried.

75 General procedure 3. To the required methylene-activated compound (10 mmol) and Et 3 N (10 mmol) in DMF (2 mL) was added the required azide (5 mmol) and CuCl (1 mmol), and the mixture was stirred at room temperature for 17 h. The precipitate was filtrated, washed with H 2 O, dried, and recrystallized from 80 i PrOH.

5-Methyl-1-(4-nitrophenyl)-1H-1,2,3-triazole-4-ethanone (3a).

Yield: 98% (general procedure 1), 72% (general procedure 2). Yellow needles, mp 146 °C (lit. 28 132-133 °C). IR (KBr): 1520, 1680, 3102. 1 H NMR (300 MHz, CDCl 3 ): 2.71 (s, 3H), 2.78 (s, 85 3H), 7.76 (d, 2H, J = 8.8 Hz), 8.50 (d, 2H, J = 8.8 Hz). [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF]

5-Methyl-1-(4-bromophenyl)-1H-1,2,3-triazole-4-ethanone (3c).

Yield: 90% (general procedure 1), 46% (general procedure 2). White powder, mp 116 °C (lit. 29 108-110 °C. IR (KBr): 645, 1549, 1688, 3092. 1 H NMR (300 MHz, CDCl 3 ): 2.63 (s, 3H), 2.78 (s, 3H), 7.38 (d, 2H, J = 8.6 Hz), 7.75 (d, 2H, J = 8.6 Hz). [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] C NMR (75 MHz, CDCl 3 ): 10.6 (CH 3 ), 28.3 1-(4-Nitrophenyl)-5-phenyl-1H-1,2,3-triazole-4-(phenylketone) (5a). 7i Yield: 40% (general procedure 1), 85% (general procedure 2), 57% (general procedure 3). Beige powder, mp 172 °C. 1 H NMR (300 MHz, (CD 3 ) 2 CO): 7.45 (m, 5H), 7.55 60 (m, 2H), 7.67 (tt, 1H, J = 7.4 and 1.4 Hz), 7.76 (d, 2H, J = 9.3 Hz), 8.23 (dd, 1H, J = 8.4 and 1.2 Hz), 8.37 (d, 2H, J = 9.3 Hz). [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] 5-Amino-1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbonitrile (5b). 31 Yield: 61% (general procedure 1), 59% (general procedure 2), 70% after 2 h reaction time and 87% after 24 h reaction time (general procedure 3). Beige powder, mp 204 °C. 1 H NMR (300 70 MHz, (CD 3 ) 2 CO): 6.82 (br s, 2H), 8.01 (d, 2H, J = 8.7 Hz), 8.50 (d, 2H, J = 8.7 Hz). [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] (7a, 3 mmol) in toluene (25 mL) was added 4-nitrophenyl azide (1a, 3 mmol) and bentonite-Cu 2+ (0.1 g), and the mixture was heated under reflux for 24 h. The precipitate was filtrated, and 80 extracted with acetone. After removal of the solvent, the product was recrystallized from EtOH. Yield: 68%. Yellow powder, mp 196 °C. IR (KBr): 1095, 1596, 1671, 3079, 3198, 3388. 1 H NMR (200 MHz, (CD 3 ) 2 SO): 3.40 (d, 2H, J = 11 Hz), 5.83 (t, 1H, J = 11 Hz), 6.88 (m, 3H), 7.49 (d, 2H, J = 8.6 Hz), 7.90 (s, 1H), 8.26 85 (d, 2H, J = 8.6 Hz), 9.81 (s, 1H), 10.0 (s, 1H). [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] C NMR (50 MHz, (CD 3 ) 2 SO): 43.8 (CH 2 ), 81.4 (CH), 114. 5, 115.4, 115.6, 119.3, 122.2, 125.4, 125.9, 126.1, 135.9, 141.9, 145.5, 165.4 (C=O). (ddd, 1H, J = 5.6, 10 and 17 Hz), 6.50 (s, 1H), 6.63 (d, 2H, J = 8.8 Hz), 6.90-7.20 (m, 4H), 8.10 (dd, 2H, J = 8.8 Hz). [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] [START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] C NMR (50 MHz, CDCl 3 ): 17.8 (CH 2 ), 30.7 (CH 2 ), 41.7 (CH 2 ), 58.9 (CH 2 ), 69.7 (CH), 108. 5, 114.9, 126.4, 143.4, 143.8, 155.4, 175.9 (C=O). 1-(4-Nitrophenyl)-1H-1,2,3triazole (11) was obtained instead in 58% yield using the following procedure: To N-vinylpyrrolidinone (5 mmol) was added 4-nitrophenyl azide (1a, 5 mmol), and the mixture was microwave-heated at 350 W for 10 min. The product collected with EtOH was washed and dried. Mp 204 °C (lit. 32 205-207 °C).

N-(2-

Crystallography

Single crystals suitable for X-ray diffraction were grown after slow evaporation (several days at room temperature) of solutions of 3b, 3c, 3f and 5a in AcOEt. The samples were studied with graphite monochromatized Mo K radiation ( = 0.71073 Å). Xray diffraction data were collected at T = 100(2) K for 3c and 5a, 20 and at T = 150(2) K for 3b and 3f, using APEXII Bruker-AXS diffractometer. The structure was solved by direct methods using the SIR97 program, 33 and then refined with full-matrix leastsquare methods based on F 2 (SHELX-97) 34 with the aid of the WINGX program. 35 All non-hydrogen atoms were refined with g.cm -3 , μ = 3.72 mm -1 . A final refinement on F 2 with 2494 unique intensities and 147 parameters converged at ωR(F 2 ) = 0.0446 programs 37 . DFT calculations were carried out using the B3LYP 38 exchange-correlation functionals, together with the standard 6-31G(d) basis set. 39 The optimizations were carried out using the Berny analytical gradient optimization method. 40 The stationary points were characterized by frequency calculations in 60 order to verify that TSs have one and only one imaginary frequency. The intrinsic reaction coordinate (IRC) 41 paths were traced in order to check the energy profiles connecting each TS to the two associated minima of the proposed mechanism using the second order González-Schlegel integration method. 42 The 65 stationary points were also optimized using the 6-311+G** basis set. The energy and geometry results are given in Supplementary Material. No significant changes in relative energies and geometries were found. The electronic structures of stationary points were analyzed by the natural bond orbital (NBO) 70 method. 43 Solvent effects have been considered at the same level of theory by single point energy calculations of the gas-phase structures using a self-consistent reaction field (SCRF) 44 based on the polarizable continuum model (PCM) of the Tomasi's group. 45 75 As polar solvent we selected acetonitrile ( = 35.69), which has a dielectric constant similar to the experimental solvent, dimethylformamide ( = 36.7).

The global electrophilicity index, 46 , has been given by the simple expression  = ( 2 /2) (eV), in terms of the electronic 80 chemical potential  and the chemical hardness . Both quantities may be calculated in terms of one of the electron HOMO and LUMO energies,  H and  L , as   ( H +  L )/2 and   ( L - H ), respectively. 47

Pharmacology

Applying the agar plate diffusion technique, [START_REF] Bauer | [END_REF] the compounds were screened in vitro for their bactericidal activity against Gram 95 positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia Coli and Pseudomonas aeroginosa), and for their fungicidal activity against Fusarium oxysporium, Aspergillus niger and Candida albicans. In this method, a standard 5 mm diameter sterilized filter paper disc impregnated 100 with the compound (0.3 mg/0.1 ml of DMF) was placed on an agar plate seeded with the test organism. The plates were incubated for 24 hours at 37 °C for bacteria and 28 °C for fungi. The zone of inhibition of bacterial and fungi growth around the disc was observed.

105

The compounds were tested against a liver carcinoma cell line (HEPG2). The method applied is similar to that reported by Skehan et al. 53 using 20 Sulfo-Rhodamine-B stain (SRB). Cells were plated in 96-multiwell plate (104 cells/well) for 24 h before treatment with the test compound to allow attachment of cell to 110 the wall of the plate. Different concentrations of the compound under test (0, 1.0, 2.5, 5.0, and 10 μg/ml) were added to the cell monolayer in triplicate wells individual dose, and monolayer cells were incubated with the compounds for 48 h at 37 °C and in atmosphere of 5% CO 2 . After 48 h, cells were fixed, washed and stained with SRB stain, excess stain was washed with acetic acid and attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader, and the relation between surviving fraction and drug concentration is plotted to get the survival curve of each tumor cell line after the specified compound and the IC50 was calculated.

The authors thank Asmaa Atrous for her contribution to this study. We are grateful to the Spanish Government (project CTQ2009-11027/BQU).

Notes and references

a Laboratoire de Synthèse Organique Appliquée, Faculté des Sciences de l' Université, BP 1524 Es-Senia, Oran 31000, Algeria. Fax: +213-04158-2540; Tel: +213-79175-8768; E-mail: badri_sofi@yahoo.fr b Chimie et Photonique Moléculaires, UMR 6510 CNRS, Université de Rennes 1, Bâtiment 10A, Case 1003, Campus Scientifique de Beaulieu, 35042 Rennes Cedex, France. Fax: +33-2-2323-6955; Tel: +33-2-2323-20 6931; E-mail: florence.mongin@univ-rennes1.fr c Departamento de Química Orgánica, Universidad de Valencia, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain. Fax: +34-9-6354-4328; Tel: +34-9-6354-3106; E-mail: domingo@utopia.uv Graphical and textual abstract for the contents pages.

Cycloadditions between aryl azides and activated alkenes e.g. coming from -dicarbonyl compounds were performed; their mechanism and regioselectivity were theoretically studied using DFT methods at the B3LYP/6-31G* level.

  35 the carbonyl O8 oxygen atom, preserving the hydrogen bond between both oxygen atoms at the intermediate IN11a (see Scheme 3). Once protonated, IN11a loses a water molecule through an E1 mechanism via TS21a with a very low activation energy of 5.4 kcal/mol. After passing TS21a, the subsequent 40 proton elimination to yield the final adduct 31a is downhill, being unfeasible to obtain the carbocation intermediate IN21a as a stationary point. This elimination reaction is very exothermic, -18.0 kcal/mol. This fact together with the favorable entropy associated with the loss of a water molecule, makes irreversible 45 the domino reaction.

Scheme 3

 3 Scheme 3 Regioisomeric channels 1 and 2 associated with the elimination reactions of the CA 6.

  have shown that the analysis of the reactivity indices defined within 45 the conceptual DFT 21 is a powerful tool to study the reactivity in polar cycloadditions. In Table 8, the static global properties (electronic chemical potential, , chemical hardness, , global electrophilicity, , and global nucleophilicity, N) of the enols 2' and the aryl azides 1 are presented. The global properties of 4-50 chlorophenyl azide (1b), 4-bromophenyl azide (1c) and hydrazoic acid, N 3 H are also included for comparative purposes. The electronic chemical potential of 4-nitrophenyl azide (1a),  = -0.1771 au, is lower than those for the enolates,  = -0.1396 (2'a) and -0.1306 (2'b) au, indicating thereby that along a polar 55 13DC reaction the net CT will take place from these enolates

Fig. 1

 1 Fig. 1 Transition states involved in the regioisomeric channels 1 and 2 associated with the 13DC reaction and water elimination steps of the thermal domino reaction between the -dicarbonyl compound 2a with 4nitrophenyl azide (1a).

35(

  CRMPO) in Rennes. Microwave reactions were performed in a Whirpool M571 domestic oven using open glass containers.

  and recrystallized from 1:1 EtOH/H 2 O: yield: 79%; yellow powder, mp 37 °C; IR (KBr): 1542, 1668, 3018, 3223; 1 H NMR (300 MHz, CDCl 3) 4.78 (d, 2H, J = 5.7 Hz), 5.47 (dd, 1H, J = 1.3 and 17 Hz), 5.56 (dd, 1H, J = 1.3 and 10 Hz), 5.91 (d, 1H, J = 10 Hz), 6.27 (ddd, 1H, J 60 = 5.7, 10 and 17 Hz), 6.44 (dd, 2H, J = 10 and 17 Hz), 6.55 (d, 1H, J = 17 Hz), 7.00-7.25 (m, 4H), 8.03 (s, 1H); 13 C NMR (75 MHz, CDCl 3 ) 69.6 (OCH 2 ), 113.7 (CH 2 ), 115.5 (CH 2 ), 120.0 (CH), 121.5 (CH), 122.4 (CH), 127.7 (CH), 130.1 (CH), 132.0 (CH), 135.6 (C), 153.5 (C), 168.0 (C=O).

  65

  C NMR (75 MHz, CDCl 3 ): 10.8 (CH 3 ), 28.4 (CH 3 ), 125.7 (2 CH), 126.2 (2 CH), 137.8 (C), 140.6 (C), 144.5 (C), 148.6 (C), 194.5 (C=O). HRMS (ESI): calcd for C 11 H 10 N 4 NaO 3 [(M+Na) +• ] 269.0651, found 269.0653.

  Yield: 85% (general procedure 1), 37% (general procedure 2). Pale yellow powder, mp 112 °C (lit.29 108-110 °C. IR (KBr): 836, 1550, 1687, 3104.1 H NMR (300 MHz, CDCl 3 ): 2.60 (s, 3H), 2.76 (s, 3H), 7.41 (d, 2H, J = 8.7 Hz), 7.57(d, 2H, J = 8.7 95 Hz).[START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] C NMR (75 MHz, CDCl 3 ): 10.1 (CH 3 ), 27.9 (CH 3 ), 126.5 (2 CH), 130.0 (2 CH), 133.8 (C), 136.3 (C), 137.4 (C), 143.8 (C),

25 = 9 . 1

 2591 1 H NMR (300 MHz, CDCl 3 ): 2.73 (s, 3H), 4.04 (s, 3H), 7.78(d, 2H, J = 9.1 Hz), 8.51 (d, 2H, J Hz).[START_REF] Fillion | Synthetic Organic Sonochemistry[END_REF] C NMR (75 MHz, CDCl 3 ): 10.2 (CH 3 ), 52.3 (CH 3 ), 125.2 (2 CH), 125.9 (2 CH), 137.2 (C), 139.0 (C), 140.2 (C), 148.3 (C), 161.8 (C=O). HRMS (ESI): calcd for C 11 H 10 N 4 NaO 4 [(M+Na) +• ] 285.0600, found 285.0604.

  Yield: 71% (general procedure 1), 75% (general procedure 2). Yellow powder, mp 140 °C. IR (KBr): 50 1092, 1561, 1736, 3104. 1 H NMR (300 MHz, CDCl 3 ): 2.70 (s, 3H), 4.01 (s, 3H), 7.90 (m, 2H), 8.44 (m, 2H). 13 C NMR (75 MHz, CDCl 3 ): 10.5 (CH 3 ), 52.6 (CH 3 ), 120.7 (CH), 125.2 (CH), 131.4 (CH), 131.4 (CH), 136.7 (C), 137.4 (C), 139.5 (C), 149.1 (C), 162.2 (C=O). HRMS (ESI): calcd for C 11 H 10 N 4 NaO 4 55 [(M+Na) +• ] 285.0600, found 285.0600.

  C NMR (75 MHz, (CD 3 ) 2 CO): 125.1 (2 CH), 126.4 (C), 127.1 (2 CH), 128.7 (2 CH), 128.9 (2 CH), 130.4 (CH), 130.8 (2 CH), 130.8 (2 CH), 133.5 (CH), 137.8 (C), 141.3 (C), 141.9 (C), 144.2 65 (C), 148.5 (C), 186.4 (C=O).

  C NMR (75 MHz, (CD 3 ) 2 CO): 101.9 (C), 105.0 (C), 114.1 (C), 127.1 (2 CH), 127.4 (2 CH), 141.4 (C), 149.9 (C). HRMS (EI): calcd for C 9 H 8 N 6 O 2 [M +• ] 232.0709, found 232.0709. 75 N-(2-Hydroxyphenyl) 1-(4-nitrophenyl)-4,5-dihydro-1H-1,2,3triazole-4-carboxamide (8). To N-(2-hydroxyphenyl)acrylamide

  carboxamide(9).To N-(2-allyloxyphenyl)acrylamide (7b, 2 mmol) in toluene (15 mL) was added 4-nitrophenyl azide (1a, 2 mmol) and bentonite-Cu 2+ (0.1 g), and the mixture was sonicated for 7 h. The precipitate was filtrated, and extracted with acetone. After removal of the solvent, the product was 95 recrystallized from EtOH. Yield: 33%. Yellow powder, mp 220 °C. IR (KBr): 1115, 1597, 1679, 3080, 3368. 1 H NMR (200 MHz, (CDCl 3 ): 3.35 (2 d, 2H, J = 18 and 19 Hz), 3.55 (d, 1H, J = 18 Hz), 3.91 (d, 1H, J = 19 Hz), 4.65 (d, 2H, J = 5.6 Hz), 5.34 (dd, 1H, J = 1.3 and 10 Hz), 5.48 (dd, 1H, J = 1.3 and 17 Hz), 6.09 100

25 anisotropic

 25 atomic displacement parameters. H atoms were finally included in their calculated positions. Molecular diagrams were generated by ORTEP-3 (version 1.08). 36 Crystal data for 3b: C 11 H 10 ClN 3 O, M = 235.67, triclinic, P-1, a = 5.5353(2), b = 6.5391(3), c = 15.0596(7) Å, α = 85.657(1), β 30 = 81.972(1),  = 89.189(2) °, V = 538.20(4) Å 3 , Z = 2, d = 1.45 g.cm -3 , μ = 0.335 mm -1 . A final refinement on F 2 with 2440 unique intensities and 147 parameters converged at ωR(F 2 ) = 0.0817 (R(F) = 0.0302) for 2317 observed reflections with I > 2σ(I). 35 Crystal data for 3c: C 11 H 10 BrN 3 O, M = 280.13, triclinic, P-1, a = 5.6105(2), b = 6.4884(3), c = 15.2886(7) Å, α = 86.338(2), β = 81.785(2),  = 89.249(2) °, V = 549.71(4) Å 3 , Z = 2, d = 1.69

  Recently, we have introduced an empirical (relative) nucleophilicity index,  based on the HOMO energies 85 obtained within the Kohn-Sham scheme, 48 defined as  = E HOMO(Nu) -E HOMO(TCE) (eV).49 This nucleophilicity scale takes tetracyanoethylene (TCE) as a reference. Local electrophilicity[START_REF]mm -1 . A final refinement on F 2 with 3847 unique intensities and 253 parameters converged at ωR(F 2 ) = 0.0905 (R(F) = 0.037) for 3284 observed reflections with I > 2σ(I). Computational methods All calculations were carried out with the Gaussian 03 suite of 2 (a)[END_REF] and nucleophilicity 51 indices,  k and N k were evaluated using the following expressions: are the Fukui functions for a nucleophilic and electrophilic attacks, respectively.52

Table 1

 1 Cycloaddition reaction of aryl azides 1a-d with acetylacetone (2a) and methyl acetoacetate (2b) performed at room temperature

								R'
								O
		N 3								N
		+	Me	O	O	R'	Et 3 N, DMF rt, 24 h	Me	N	N
		R
		1		2
								R
									3
	entry R (1)		R' (2)		product (3)	yield
	1	4-NO2 (1a)	Me (2a)	3a		98%
	2	4-Cl (1b)		Me (2a)	3b		85%
	3	4-Br (1c)		Me (2a)	3c		90%
	4	3-NO2 (1d)	Me (2a)	3d		65%
	5	4-NO2 (1a)	OMe (2b)	3e		80%
	6	4-Cl (1b)		OMe (2b)	3f		92%
	7	4-Br (1c)		OMe (2b)	3g		78%
	8	3-NO2 (1d)	OMe (2b)	3h		71%

Table 2

 2 Cycloaddition reaction of aryl azides 1a-d with acetylacetone (2a) and methyl acetoacetate (2b) performed under microwave irradiation

									R'
									O
		N 3									N
	R	+	Me	O	O	R'	160 W, 5 min MW Et 3 N	Me	N	N
		1		2
									R	3
	entry R (1)		R' (2)		product (3)	yield
	1	4-NO2 (1a)	Me (2a)		3a		72%
	2	4-Cl (1b)		Me (2a)		3b		37%
	3	4-Br (1c)		Me (2a)		3c		46%
	4	3-NO2 (1d)	Me (2a)		3d		70%
	5	4-NO2 (1a)	OMe (2b)	3e		93%
	6	4-Cl (1b)		OMe (2b)	3f		16%
	7	4-Br (1c)		OMe (2b)	3g		20%
	8	3-NO2 (1d)	OMe (2b)	3h		75%

Table 3

 3 Cycloaddition

reaction of 4-nitrophenyl azide (1a) with benzoylacetophenone (4a) and malonodinitrile (4b) performed at room temperature (with or without CuCl) or under microwave irradiation

Table 4

 4

	Cycloaddition reaction of 4-nitrophenyl azide (1a) with N-(2-hydroxyphenyl)acrylamide (7a), N-(2-allyloxyphenyl)acrylamide (7b) and N-
	vinylpyrrolidinone (7c) (1 equiv)
	entry dipolarophile	conditions	product				yield
	1				toluene, reflux, 24 h						10%
	2 3 4 5	7a	HO N H O	acetone, reflux, 24 h MW, 600 W, 10 min acetone, US, 24 h bentonite-Cu 2+ (cat.), toluene, reflux, 24 h	8	O 2 N	N N N	O	HO N H	23% 0% 34% 68%
	6 7 8	7b	O	O N H	acetone, reflux, 24 h bentonite-Na + (cat.), toluene, US, 7 h bentonite-Cu 2+ (cat.), toluene, US, 7 h	9	O 2 N	N N N	O	O N H	20% 22% 33%
	9 10	7c	O N		acetone, reflux, 24 h MW, 350 W, 10 min

Table 5

 5 B3LYP/6-31G* relative energies (E, in kcal/mol, relative i to 2, ii to 1+2', and iii to IN11 or IN12) in gas phase and in solution, of the stationary points involved in the thermal domino reactions of the dicarbonyl compounds 2 with the aryl azides 1

		E	Esolv
	i) enolization reaction
	2'a-Z	-10.0	-5.2
	2'a-E	1.7	1.0
	2'b-Z	-1.9	-0.2
	2'b-E	7.8	4.3
	ii) 13DC reaction
	TS11ea	23.6	24.7
	TS12ea	31.9	31.7
	61ea	-3.3	-3.0
	62ea	-0.4	-4.3
	TS11a	20.9	22.2
	TS12a	32.4	32.7
	61a	-4.6	-4.1
	62a	-1.7	-5.4
	TS11b	21.2	22.2
	TS12b	30.0	30.8
	61b	-5.7	-5.5
	62b	-5.1	-7.0
	iii) elimination reaction
	TS21a	4.1	5.4
	TS22a	4.8	3.7
	31a	-11.1	-18.0
	32a	-3.8	-11.6
	TS21b	3.3	5.5
	TS22b	2.8	4.4
	31b	-9.7	-17.9
	32b	-6.9	-14.3

Table 6

 6 Lengths of the C-N forming bonds (in angstroms), asynchronicity (l), charge transfer (CT, in e), and bond orders (BO) of the C-N forming bonds involved in the 13DC reactions between the enols 2' and the aryl azides 1

		N1-C5(4)	N3-C4(5)	l	CT	BO N1-C5(4)	BO N3-C4(5)
	TS11ea	2.010	2.190	0.18	0.17	0.45	0.29
	TS12ea	1.905	2.172	0.27	0.08	0.49	0.33
	TS11a	1.974	2.251	0.28	0.26	0.48	0.25
	TS12a	1.906	2.150	0.24	0.13	0.48	0.34
	TS11b	1.977	2.264	0.29	0.25	0.48	0.25
	TS12b	1.951	2.139	0.19	0.15	0.45	0.34

Table 7

 7

	TS21a	1.627	1.305	1.129	1.997
	TS22a	1.740	1.599	1.016	2.196
	TS21b	1.619	1.280	1.145	1.941
	TS22b	1.941	1.578	1.022	2.078

Lengths of the breaking and forming bonds (in angstroms) in the TSs involved in the water elimination step C4-O6 O8-H7 H7-O6 H9-N10

Table 9

 9 Local electrophilicity (k, in eV) and nucleophilicity (Nk, in eV) indices of the enols 2' and the aryl azides 1 The diameters of zones of inhibition are given in mm. Stock solution: 5 g in 1 mL of DMF. 0.1 mL of stock solution in each hole of each paper disk. +: < 15 mm; ++: 15-24 mm; +++: 25-34 mm; ++++: 35-44 mm, etc.

		N1 N2 N3	NC4 NC5
	1e	0.62 0.44 0.13 2'a 0.32 1.32
	1a	1.30 0.92 0.27 2'b 0.37 1.39
	Table 10 Bactericidal and fungicidal activity a of the compounds 3a-h, 5a, and ciprofloxacin and nystin
	entry compound	Staphylococcus aureus Escherichia coli	Pseudomonas aeroginosa	Fusarium oxysporium	Aspergillus niger	Candida albicans
	1	3a	22 (++)	18 (++)	-	19 (++)	19 (++)	-
	2	3b	19 (++)	16 (++)	-	32 (+++)	19 (++)	-
	3	3c	22 (++)	17 (++)	-	25 (+++)	20 (++)	-
	4	3d	31 (+++)	-	-	26 (+++)	18 (++)	-
	5	3e	16 (++)	15 (++)	-	22 (++)	17 (++)	-
	6	3f	22 (++)	17 (++)	-	21 (++)	17 (++)	-
	7	3g	21 (++)	20 (++)	-	21 (++)	17 (++)	-
	8	3h	31 (+++)	15 (++)	16 (++)	18 (++)	18 (++)	-
	9	5a	28 (+++)	-	15 (++)	20 (++)	18 (++)	-
	10	ciprofloxacin ++++	++++	++++	-	-	-
	11	nystin	-	-	-	++++	++++	++++
	a

Table 11

 11 In vitro cytotoxic activity (IC50) a of the compounds 3a-h, 5a, and doxorubicin against a liver carcinoma cell line (HEPG2) IC50 is defined as the concentration which results in a 50% decrease in cell number as compared with that of the control structures in the absence of an inhibitor. 194.3 (C=O). HRMS (ESI): calcd for C 11 H 10 35 ClN 3 NaO [(M+Na) +• ] 258.0410, found 258.0413.

	entry	compound	HEPG2 (g.mL -1 )
	1	3a	3.34
	2	3b	2.88
	3	3c	2.92
	4	3d	0.53
	5	3e	2.46
	6	3f	3.11
	7	3g	2.24
	8	3h	3.74
	9	5a	0.81
	10	doxorubicin	0.60

a

1-(4-Nitrophenyl)-4,5-dihydro-1H-1,2,3-triazolyl)-2- pyrrolidinone (10).

  To N-vinylpyrrolidinone (7c, 5 mmol) in acetone(30 mL) was added 4-nitrophenyl azide (1a, 5 mmol), 110 and the mixture was heated under reflux for 24 h. The precipitate was filtrated, and the product was recrystallized from EtOH. Yield: 63%. Yellow powder, mp 184 °C. IR (KBr): 1047, 1599, 1685, 3113. 1 H NMR (200 MHz, CDCl 3 ): 1.90 (m, 2H), 2.35 (m, 2H), 2.62 (m, 1H), 2.96 (m, 1H), 4.44 (t, 2H, J = 2.1 Hz), 6.38 (dd, 1H, J = 4.0 and 8.3 Hz), 7.45 (d, 2H, J = 9.2 Hz), 8.22 (d, 2H, J = 9.2 Hz).

		C NMR
		(50 MHz, CDCl 3 ): 46.9 (CH 2 ), 69.2 (CH 2 ), 69.3 (CH), 117.6
		(CH 2 ), 119.2 (2 CH), 121.4 (CH), 121.5 (CH), 124.1 (2 CH),
	105	124.1 (CH), 124.9 (CH), 129.2 (CH), 147.1 (C), 147.5 (C), 149.0
		(C), 163.2 (C), 181.8 (C=O). HRMS (EI): calcd for C 18 H 17 N 5 O 4 [M +• ] 367.1281, found 367.1281.
		1-(

  .es d Centre de Diffractométrie X, Sciences Chimiques de Rennes, UMR 6226 25 CNRS, Université de Rennes 1, Bâtiment 10B, Campus Scientifique de Beaulieu, 35042 Rennes Cedex, France. e Department of Chemistry, Faculty of Women for Arts, Science and Education, Ain Shams University, Asma Fahmy Street, Heleopolis (El-Margany), Cairo, Egypt † Electronic Supplementary Information (ESI) available: CIF file of 3b (CCDC 806634), 3c (CCDC 806635), 3f (CCDC 806636), and 5a (CCDC 806637). See DOI: 10.1039/b000000x/ 1 R. Huisgen, R. Grashey and J. Sauer, in The Chemistry of Alkenes, Interscience: New York, 1964.

Acknowledgment