Asbjørn Rolstadås

Agnar Johansen

Yvonne C Bjerke

Tobias O Malvik

Managing risk and opportunities in complex projects

Keywords: Project Management, Risk Management, Opportunity Management

Projects is the preferred model for one-of-a-kind production. Projects may be difficult to manage due to complexity and many involved stakeholders. Stakeholders are a major source of uncertainty. Uncertainty may be both positive and create opportunities and negative giving risks. Risks and opportunities are either operational, strategic or contextual. The traditional approach to managing risk comprise identification and analysis of risks as well as response planning and control. There is a need for a shift in mindset for managing risks. Rather than regarding risks as "evil", they should be managed because uncertainties also create opportunities. The Bermuda Risk Triangle is the intersection between operational, strategic and contextual risks. Project risk navigation is about how project leaders can navigate in this triangle to reach their objectives. Opportunities are often more or less neglected in projects. At the most, just a few are identified. A framework for managing opportunities is suggested. It builds on the project control variables: time, cost and scope of work. It contains a classification of eight opportunity types. Using this classification in dedicated workshops has shown to produce far more opportunities than usual. The framework is verified in a case study. The case is the construction of the new National Museum in Oslo, Norway. Through the framework a total of 246 opportunities have been identified representing an estimated cost reduction of about 64.2 million USD.

Complex Projects

The classical manufacturing typology distinguishes between flow, batch and one-of-akind production. One-of-a-kind production is widely applied in the shipbuilding, oil & gas and construction industry. It is also the normal approach for developing process facilities and infrastructure (for example power plants). One-of-a-kind production is also referred to as project work. Project management is a discipline in itself with its own research community. The Project Management Institute defines a project as a temporary endeavor undertaken to create a unique product, service, or result [START_REF]Project Management Institute: A guide to the Project Management Body of Knowledge[END_REF]. Projects tend to be more difficult to manage than flow or batch production. In fact, many major projects fail to meet their objectives with respect to schedule and costs.

One of the most cited examples is the Sydney Opera House, which overran its initial budget by a factor of 14 and ran 15 years behind schedule [START_REF] Kharbanda | What made Gertie Gallup? Lessons from project failures[END_REF]. Another example is the early oil & gas development in the North Sea. The Statfjord A platform suffered a two-year delay and had a cost overrun of 222 % [START_REF] Rolstadås | Cost Study Norwegian Continental Shelf[END_REF].

There are several reasons why projects are more difficult to manage:

 They may involve substantial innovation, as there might not be any proven technology available.  As they are one-of-a-kind, there is limited experience data available for planning and execution.  They involve cooperation amongst several organizations that may not have previous experience from working together.  They have a high complexity involving many actors and challenging processes, organizational aspects, and logistics.

Many authors have discussed complexity. One of the first is Baccarini [START_REF] Baccarini | The concept of Project complexitya review[END_REF]. He defines two components of complexity: organizational and technological. Williams [START_REF] Williams | Modelling Complex Projects[END_REF] combine these into structural complexity and adds uncertainty as a second component. Brady and Davies [START_REF] Brady | Managing Structural and Dynamic Complexity: A Tale of Two Projects[END_REF] take a systems view and make a distinction between structural and dynamic complexity, where structural complexity has to do with the arrangement of components and subsystems into an overall systems architecture. This architecture includes the system produced, the producing system, and the wider system. Projects include a number of stakeholders. A stakeholder is a person or organization that either can influence the project execution or is affected by the project. The large number of stakeholders having different views on what the best solution is and what to deliver makes a project notoriously difficult to plan and manage. Consequently, stakeholders may have different motives for participating in. Some may tend to benefit their own interests above the outcome of the project. Thus, stakeholders create uncertainty making it difficult to plan and predict the execution of the project.

Project uncertainty, risk and opportunity

Uncertainty in projects is often regarded as lack of information. This means that projects carry uncertainty, and that the uncertainty will vary over the project life cycle. Some major sources of uncertainty are [START_REF] Johansen | Project Uncertainty Management a New Approach: The "Lost Opportunities[END_REF]: The uncertainty sources can be classified into controllable and non-controllable factors [START_REF] Johansen | Project Risk and Opportunity Management -An Owner's Perspective[END_REF]. The controllable factors are foreseeable events such as quality and deviation of design, change of methods and technology, etc. The non-controllable factors are beyond the control of the project organization. They fall into two categories: nature (beyond the control of man) and stakeholders. There is both negative and positive uncertainty. The negative uncertainty and its potential impact comprise a risk in the project. Likewise, a positive uncertainty and its impact, represents an opportunity for the project.


Whereas risk management has been a major focus of project managers and owners for many years, few projects has managed to exploit the opportunities that arises from positive uncertainty. Krane et al. [START_REF] Krane | Exploiting opportunities in the uncertainty management[END_REF] has shown that projects may have a balanced approach to manage both risks and opportunities at project start up. However, while projects sustain their effort in managing risks during project execution, there is almost no focus on opportunities.

Rolstadås and Johansen [START_REF] Rolstadås | From Protective to Offensive Project Management[END_REF] classifies risks and opportunities into three categories

 operational  strategic  contextual
Operational risk and opportunity is due to internal circumstances in the project that typically can be controlled by the project management team. Strategic risk and opportunity is the potential impact on the project benefits. Contextual risk and opportunity is connected to circumstances outside the project that may influence the scope of work and the performance of the organization.

Risk navigation

The Project Management Institute defines risk management as the processes concerned with conducting risk management planning, identification, analysis, responses planning, and controlling risk [START_REF]Project Management Institute: A guide to the Project Management Body of Knowledge[END_REF]. This classical approach to risk management is illustrated in Figure 1. The first step is to identify risk factors. Risk analyses includes estimating the consequences of each risk factor. The final two steps are to develop an action plan and to monitor and control it.

Identification of risk factors Risk analysis

Risk action plan Risk control This approach is based on a perception that risk is something "evil" that should be avoided or at least mitigated. This perception of risk has led to major efforts to forecast any risk and develop plans for mitigation or elimination. Despite of this large effort, cost overruns and delays still happen.

Rolstadås et al. [START_REF] Rolstadås | Risk Navigation Strategies for Major Capital Projects -Beyond the Myth of Predictability[END_REF] argues that project managers need to make a shift in mindset towards a stronger focus on managing risks rather than eliminating them:  Projects as deliverables to a means to enhance project business value.  Uncertainties as "evil" to acknowledge the project nature as unique and uncertain.  Projects as known tasks to be accomplished in known environments to embracing a continuum of tasks to be executed in turbulent business environment.  Deviations from project baselines as inaccurate planning or inappropriate control to acknowledgement of deviations as being the rule.

They argue for an extended risk concept based on the three risk and opportunity categories defined above, and launches a new approach to project risk navigation. The concept is illustrated in Figure 2 and shows the three risk categories in the intersection between the project management, the project owner and the environment. The intersection of these three risk areas is called "The Bermuda Risk Triangle". Just as travel in the area of the Atlantic Ocean known as the Bermuda Triangle requires one to accept unknown risks, so too does the multi-year duration of a major capital project. Project risk navigation is about how to navigate in the Bermuda Project Risk Triangle and reach their objectives. The navigator is a framework containing three major components: the governance system, the decision process, and the strategic planning.

Although this new approach covers risk, the principles are also valid for exploiting opportunities. This follows from the arguments for a shift in mindset.

Hunting for opportunities

According to Davies et al. [START_REF] Davies | Making innovation happen in a megaproject: London's crossrail suburban railway system[END_REF] innovation is often avoided in large projects due to uncertainty and associated cost escalations. Rather than seeking novel ideas and innovative approaches, the owner and the project management try to minimize risks. They rely on using proven technology and well-established procedures. In the end, this means choosing the lowest bid, transferring risk to contractors, and sticking to the original plan. However, in opportunity management, projects cannot be reluctant to innovation and change since change and innovation are the drivers for harvesting and exploiting the opportunities. There are several reasons why projects fail to hunt for opportunities:

 It feels safer to stick to the agreed plan rather than test new options even if there is a potential reward.  Existing tools neglect opportunities and focus on mitigating risks.  Exploiting an opportunity could be time consuming and costly. Thus exploiting the opportunity turns into a risk (to be avoided).  If the project is on track, there is little motivation for the project management to seek new innovations. The Project Management Institute defines opportunity as a condition or situation favorable to the project [START_REF]Project Management Institute: A guide to the Project Management Body of Knowledge[END_REF]. It will have a positive impact on the project objectives and represents a possibility for positive changes. Johansen et al. [START_REF] Johansen | Uncertainty Analysis: 5 Challenges with Today's Practice[END_REF] have developed a nine-step process for identifying, analyzing and following up on project uncertainty as shown in Figure 3. In steps 1 and 2 goals and key deliverables are confirmed, and stakeholders identified. Steps 3 through 7 involve workshops for identifying, analyzing and developing measures of exploiting or controlling risks and opportunities. In steps 8 and 9 risks and opportunities are followed up over the project life cycle.

To hunt for opportunities requires ways of identifying opportunities. Normally this is achieved using brainstorming techniques at workshops involving several key stakeholders. The authors have found that there has to be dedicated workshops for identifying opportunities separate from those identifying risks. It has proven helpful to develop a framework for classification of different types of opportunities.

The suggested framework is based on the three project control variables: cost, time and scope of work as well as the project's second order effect for the project owner in the operation phase. Scope of work, however, is in this context regarded as quality. It is well known that in order to meet budget requirements, the quality level may have to be adjusted (for example by simplifying the design or by using alternative, less expensive, materials). The three control variables are often illustrated by the three sides of a triangle referred to as the iron triangle [START_REF] Rolstadås | Applied Project Management: How to Organize, Plan and Control Projects[END_REF]. The idea is that the need for a change in one of the variables may be compensated by a change in one or both the other variables (the schedule may for example be compressed at a higher cost). The classification framework is shown in Table 1. It shows eight categories. The first category describes the opportunities that are beneficial for both cost, time, and quality. Categories 2 through 4 represents the opportunities that have a combination of two out of three of cost, time, and quality. Categories 5 through 7 contain the opportunities that only gives benefits in either cost, time or quality. Opportunities that will give second-order consequences for the project owner in the operation phase are placed in category 8. This category differs from the others, as it is counted regardless if it is combined with other opportunity types. Eight different opportunity properties are defined: (a) reduced cost, (b) avoid cost overruns, (c) faster deliverance, (d) avoid delays, (e) higher quality, (f) avoid unnecessary high quality, (g) increased value for the client, (h) increased value for the user.

Verification case

The suggested framework for hunting opportunities has been verified using a case from the construction industry, the new National Museum in Norway's capital, Oslo. How the project harvested opportunities was followed over a three and a half year period (2016 -2019) harvesting potential opportunities.

The new museum is built in the city center with demanding construction and site logistics and many stakeholders to be managed. Because of the art that will be exhibited and stored, the building is it equipped with advanced, state of the art alarm and monitoring systems, and lot of other sophisticated technical systems. The architectural and technical design is state of the art and includes several innovative solutions.

During spring 2016 the project faced problems. It was behind schedule and forecasted a cost overrun. In spite of a good uncertainty management system, there were only three opportunities left to harvest in 2015. Thus, the management system had to be adjusted. Two problems had to be solved: (1) How to chase opportunities during the execution phase and (2) How to achieve commitment at all levels of the organization? To get the project back on track, the project management created 11 different sources/arenas with activities that facilitated opportunity hunting such as formal workshops and informal café dialogues. Six separate opportunity studies on the largest contracts (believed to have the largest potential) were organized.

By April 2019, 246 opportunities have been identified, and the project has an estimated cost reduction of about 64.2 million USD. 175 of the 246 identified opportunities were harvested. A harvested opportunity is considered as an opportunity the project made an effort to realize. Table 2 shows the number of opportunities harvested for the first order opportunities. 56 % were assumed to have an effect on cost, 62 % on time and 32 % on quality. Based on the framework, there are two important conclusions for the museum case: 1) opportunities related to cost and time are dominant, and 2) it looks like there is less focus on opportunities that are beneficial for the National Museum in the long run. The project management rated the performance objectives as 1) cost, 2) quality, 3) time. The frequency of cost, time, and quality in the framework show that the harvested opportunities are not aligned with the performance objectives. However, the focus was on reducing time and cost at the time of implementation of the new opportunity management approach, and one could argue that the strategy was successful. The project had to get back on track with respect to cost and time as the quality standard already were set extremely high. This might explain why most of the opportunities were cost-and time-related in the construction phase and why these opportunities are so dominant in this case. Another reason is that cost and time is more straightforward to measure than quality, which could make such opportunities easier to define during identification.

Conclusion

Managing uncertainty is of crucial importance for a complex project to be successful. However, it is manly the negative uncertainty (creating risks) that is considered in most projects. The positive uncertainty (enabling opportunities) is often neglected. To remedy this, a framework for managing opportunities is suggested. It uses an opportunity classification system in a number of formal and informal workshops. In this way, almost the same number of opportunities as risks can be identified. Future project managers must pay more attention to the harvesting of opportunities, as they are crucial to enhance projects benefits or to bring projects running over time and budget back on track. However, this requires a new mindset, both with the project organization and the project owner.

Fig. 1 .

 1 Fig. 1. Classical risk management process.

Fig. 2 .

 2 Fig. 2. A new concept for risk navigation.

Fig. 3 .

 3 Fig. 3. Risk and opportunity management process.

Table 1 .

 1 Framework for classification of opportunities.

	Number Opportunity category	Control variables
	1	Multiple first order	Cost, Time, Quality
	2		Cost, Time
	3	Double first order	Cost, Quality
	4		Time, Quality
	5		Cost
	6	Single first order	Time
	7		Quality
	8	Second order	Value for user/client

Table 2 .

 2 Number of first order opportunities harvested.

		Cost		Time		Quality
	Reduced	Avoid cost	Faster	Avoid	Higher	Avoid
	cost	overruns	deliverance	delays	quality	unnecessary
						high quality
	105	33	90	62	27	51
		138		152		78


Giuseppe Ismael Fragapane
email: giuseppe.fragapane@ntnu.no

Aili Biriita Bertnum
email: aili.b.bertnum@ntnu.no

Hans-Henrik Hvolby

Jan Ola Strandhagen
email: ola.strandhagen@ntnu.no

Aili Biriita -8915-509x]

Bertnum

Medical Supplies to the Point-Of-Use in Hospitals

Keywords: Inventory control, Point-of-use, Hospital

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

Introduction

New medical advances broaden the spectre of possible treatments and the patients have higher expectations to the quality of treatment than ever [START_REF] Bacik | Pathfinder-Development of Automated Guided Vehicle for Hospital Logistics[END_REF][START_REF] Oecd | Health at a Glance[END_REF]. The trend of increasing demand in hospitals is accompanied by increasing shortages of doctors, nurses and supporting staff [START_REF] Bendavid | RFID-Enabled Traceability System for Consignment and High Value Products: A Case Study in the[END_REF]. In addition, healthcare expenditures represent an increasing share of the economy in most OECD countries [START_REF] Oecd | Health at a Glance[END_REF]. The cost of supplies and services are starting to outpace hospital budgets. In order to match financial sustainability with the delivery of high-quality healthcare hospitals need to seek efficient ways of managing inventories with a large variety of medical supplies [START_REF] Rosales | The 2Bin system for controlling medical supplies at point-of-use[END_REF][START_REF] Papanicolas | Health System Performance Comparison : An Agenda for Policy, Information and Research[END_REF].

Several challenges are present in the processes of inventory control in hospitals. Hospitals experience unpredictable demand and large varieties in the supply consumption due to varying patient mix. Fluctuating demand is especially present in the emergency department in which patients have not scheduled treatments. Hospitals need to keep an extensive number of stock keeping units (SKUs) to be prepared to treat a variety of illnesses. The variety is even further increased by individual physicians' preferences regarding specific brands of equipment or consumables they prefer using.

Traditionally, there are at least two levels of inventory in the hospital supply chain, a central warehouse and point-of-use (POU) inventories. The POU inventories are generally characterized by lack of space to store the different medical supplies needed at the point-of-care. The space restrictions combined with fluctuating demand and a large amount of different SKUs lead to the need for efficient control of available inventories. Though slightly neglected in the past, inventory management in hospitals has in recent years been recognized as one key lever towards realizing efficiency improvements with regards to cost and waste, while at the same time satisfying service levels [START_REF] Volland | Material logistics in hospitals: A literature review[END_REF]. To find a balance in the trade-off between cost and the service levels that ensures on time and high-quality patient treatment is a challenge. It is especially crucial in the hospital setting, where the consequence of a stock-out can be much more severe than lost revenue [START_REF] Moons | Measuring the logistics performance of internal hospital supply chains -A literature study[END_REF]. The process of ensuring that the required supplies are available at the right time is a particularly important supporting role of hospital logistics.

The study concerns the short-term planning and control area, which focus on coping with the actual demand and making the necessary changes in order to match efficiently to plans [START_REF] Slack | Operations management[END_REF]. The objective of this study is to investigate the challenges of inventory control in hospitals and discuss how technology can support high availability of medical supplies. The inventory control processes in hospitals will also be visualised, showing the interdependencies between the internal and external hospital supply chain.

The rest of this study is organized according to the following structure. Section 2 embodies the literature on inventory control in general, specific inventory control policies and their application in an overall inventory control system. Section 3 describes the inventory control processes of an emergency department at a large hospital in Norway, which serves as input to the following section. Section 4 discusses the different review policies in hospitals and enablers with the use of technology for inventory control. The study is concluded with a summary of major insights and an outline of future research.

Theoretical background

Inventory control is based on answering the questions of what to order, how much to order and when to order [START_REF] Waters | Inventory control and management[END_REF]. It entails both controlling the inventory in form of on-hand physical inventory, as well as record counts and monetary worth [START_REF] Muller | Essentials of Inventory Management[END_REF]. The demand is hard to predict and there is lack of information on the consumption of supplies from the POU inventories [START_REF] Moons | Measuring the logistics performance of internal hospital supply chains -A literature study[END_REF]. Non-availability of supplies may result in postponement of surgeries, and can in the worst case be critical to a patient's life. On the other side, overstocking or hidden stocks lead to supply chain inefficiencies and higher inventory costs [START_REF] De Vries | The shaping of inventory systems in health services: A stakeholder analysis[END_REF]. Demand for items of POU inventories can be characterized as independent. Demand of a product is independent when it is influenced by conditions outside the control of the organization, rather than being dependent on another product [START_REF] Muller | Essentials of Inventory Management[END_REF]. With independent demand, efforts must be made to match the inventory with demand based on historical demand, forecasts and "best guesses", while still be prepared to respond quickly when mismatches occur [START_REF] Slack | Operations management[END_REF]. According to Muller [START_REF] Muller | Essentials of Inventory Management[END_REF], independent demand calls for a replenishment approach to inventory management, where stock is replenished as it is used, in order to have items on hand to fulfill demand.

The decisions on how much to order and when to order are closely linked. How much to order depends on the frequency and time in which orders are placed, in the same way as when to order depends on the volumes ordered. In order to control inventories, a policy that is simple and efficient to use is of importance. The policy needs to be combined with identification technologies to decrease the response time. The choice of suitable inventory (replenishment) policy, is a widely discussed topic within the area of hospital inventory management [START_REF] Volland | Material logistics in hospitals: A literature review[END_REF]. Traditionally, the choice of inventory policy concerns deciding the suitable inventory review cycle and reorder quantity [START_REF] Volland | Material logistics in hospitals: A literature review[END_REF].

The review cycle can be either periodic or continuous. Slack [8 p.707] defines the periodic review as "an approach to making inventory decisions that define points in time for examining inventory levels and making decisions accordingly". Points in time can be e.g. on a daily or weekly basis. Continuous review is defined as "an approach to managing inventory that makes inventory-related decisions when inventory reaches a particular level" [8 p.702]. The review policy requires continuous tracking of inventory levels. The review cycle must be determined based on variability in demand and costs of ordering, holding and delivering supplies [START_REF] Rossetti | Medical Supply Logistics[END_REF].

The periodic review seems to be most commonly used in hospitals today, although some argue that continuous review is superior to periodic review [START_REF] Moons | Measuring the logistics performance of internal hospital supply chains -A literature study[END_REF]. This is due to the ability of continuous review to provide real-time updates on inventory levels by the use of data capturing technologies. Çakıcı et al. [START_REF] Çakıcı | Using RFID for the management of pharmaceutical inventorysystem optimization and shrinkage control[END_REF] argue that in cases where real-time information is made available without large additional cost, the continuous review is superior to periodic review. E.g., the use of RFID allows for a continuous review policy instead of the typically utilized periodic review [START_REF] Paltriccia | Supplying networks in the healthcare sector: A new outsourcing model for materials management[END_REF].

The number of shipments is expected to be higher with a continuous review policy than with a periodic review policy. This results in the continuous review being most convenient when the cost per shipment is low. However, with a period review policy it is easier to incorporate different orders in the same shipment, which will save transportation costs. The traditional periodic review policies applied in hospitals are being replaced with continuous or hybrid review policies (periodic-continuous) enabled by modern point-of-use technology, such as automated dispensing cabinet and two-bin systems in combination with RFID [START_REF] Rosales | Technology Enabled New Inventory Control Policies in Hospitals[END_REF].

Inventory control systems, also called replenishment systems or distribution systems, describe how inventory control policies are applied in practice. It defines how inventories are intentionally organized and combined with enabling identification technologies in order to enable different inventory control policies. Several methods have been applied and discussed in the literature on how to control and distribute supplies from the central warehouse to POU in hospitals [START_REF] Landry | The Challenges of Hospital Supply Chain Management, from Central Stores to Nursing Units[END_REF].

The variation of methods and inventory policies have an impact on the degree of staff involvement. The replenishment responsibility either is centralized to the logistics department or decentralized to the staff at the POU locations. The range of methods applied at POU inventories varies from manual systems like the requisition based system or exchange carts, which are rarely used today, to commonly used systems such as Periodic Automatic Replacement (PAR) or two-bin systems enabled by barcode or RFID technology.

3

Case study

Methodology

case study was carried out with a large Norwegian hospital that treats 60'000 inpatients (patients who stay overnight in the hospital during treatment) and 370'000 outpatients (patients who visit the hospital for treatment without staying overnight) yearly. The hospital has 800 beds, 8000 employees and an annual budget of 8.2 billion NOKs [START_REF] Nedland | Avanserte logistikkløsninger på St[END_REF]. The purpose of the case study was to get a holistic view of the logistic processes at a hospital in practise, and more specifically the process of inventory control at the POU inventories in the emergency department subject to fluctuating demand. The advantage of a single case study is to analyse the phenomena in greater depth [START_REF] Voss | Case research in operations management[END_REF].

Empirical data was collected through semi-structured interviews, observations, and information retrieved from field documents. Multiple semi-structured interviews were conducted both on-site and through meetings. The purpose was to interview key personnel able to provide solid descriptions of the processes inside and outside the hospital. Interviews were conducted with both a warehouse manager, purchaser, logistics manager at the hospital and a porter from the emergency & cardiothoracic centre.

A semi-structured interview is a flexible interview technique consisting of a set of predefined questions while allowing for open-ended exploration and deflection from the predefined questions [START_REF] Wilson | Chapter 2 -Semi-Structured Interviews[END_REF]. In preparation of the interviews, a brief interview guide was prepared, consisting of the purpose of the study and topics to be covered in the interview, as well as suggested questions. During the interviews, most of the topics were covered and additional information was collected. Based on notes made during interviews, reports were formulated as documentation.

Materials ordering flow

The case hospital utilizes periodic review and order-up to level as the inventory control policy. The current inventory level is decided by roughly estimating the inventory levels of the different products. However, the input provided to the handheld computer is not a count of the current levels of inventory, but rather a direct input on the quantity wished to be replenished. The disturbances that can occur during the periodic measure and order of inventory level is a combination of human and technological errors. It is up to human accuracy to scan the right barcodes, give the correct input on order quantity and make sure that every room, cabinet, and shelves are checked each round. If the order is not sent according to the schedule provided, it is also a human error. Additionally, the handheld computers are quite old, and the software operates slowly, which sometimes leads to technical errors such as hang-ups or that the computer fails to scan the barcode. The deadline for sending the order is 8:30 AM. Once the orders are sent, there is a leadtime of 2 hours (8:30-10.30AM) on replenishment between the central warehouse and goods reception in the hospital. As all supplies are delivered to the good reception at once, queues in AGV transportation often occurs. Therefore, the supplies are normally not available at the POU before around 12.00 PM. An additional disturbance to the system regarding the replenishment of supplies is observed. Several times a week the hospital get wrong or products delivered. This could be due to technological or human errors in the ordering process at the POU, or by wrongful picking of orders at the central warehouse.

The overall processes of inventory control from the central warehouse to POU in the case hospital are visualised in Fig. 1. The external supply processes are visualised in the top timeline, while the internal inventory control processes are visualised in the bottom timeline. T1, T2, and T3 represent the scheduled transportation of goods deliveries by trucks from the central warehouse to the hospital.

Discussion

Due to the demand patterns found in the emergency department (especially on a busy night) stock-outs can easily occur in between the periodic reviews of inventory. With the characteristics of demand at emergency departments, literature states that the continuous review policy is a more suitable method. New technologies enabling real-time inventory records, visibility of material flows, as well as automated order generation are already available. RFID is an enabler of continuous review that has been given attention within the field of hospital inventory management. However, hospitals have not adopted the technology as fast as in other industries. Different inventory control policies and technologies are combined with replenishment systems that control the POU inventories. The descriptive control model in Fig. 1 serves to describe the POU inventory system and support to discuss how different technological solutions support the availability of medical supplies. The storage area is very limited in the emergency department at the case hospital, meaning that not all supplies can be stored in the common two-bin systems. With a limited storage capacity, the availability of items becomes more dependent on the responsiveness of the overall supply chain. The study by Little and Coughlan [START_REF] Little | Optimal inventory policy within hospital space constraints[END_REF] shows that optimizing inventory within space constrictions requires more frequent deliveries of supplies to keep high service levels. This implies using continuous review solutions and quick replenishment systems, by either employing extra labour or implementing technological solutions. The inventory control system can support in identifying and mapping the current state of the replenishment and inventory control process. Modelling the process provides a visualization of the different flows, input and output controlling the inventory level. This can support to decide for a suitable technological solution within the internal hospital supply chain.

One of the strengths of the inventory control system at the case hospital is the central warehouse's quick response to signal and demand, and the use of order-up-to levels to decide order quantities. This solution is appropriate and efficient for limited storage space, while still providing high service levels [START_REF] Bijvank | Inventory control for point-of-use locations in hospitals[END_REF]. The responsibility and time consuming task of monitoring inventory and supplies have been outsourced from the nurses to a small group of porters. However, stock-outs and unavailability of medical supplies are unavoidable and occur weekly in the emergency department. Stock-outs can occur especially quickly in situations where several patients are treated at the emergency department due to e.g. a car crash.

With the implementation of technologies for the inventory control processes, it can support the external supply processes by providing better visibility of the inventory. With this information, the supply chain can adjust its efforts towards being more responsive to changing demand. The use of real-time information of inventory levels can be an enabler for increased responsiveness in the overall supply chain, which can result in the possibility of having better availability of a broader range of SKUs, while keeping smaller quantities of the SKUs at POU inventories in the hospital. In an emergency department, this can prove to be especially valuable due to its characteristics of fluctuating and emergent demand.

Conclusion and further research

Most hospitals have limited storage space at the POU and an increasing variety of SKUs. This provides challenges when it comes to the availability of medical supplies. Every hospital has its own individual supply chain capabilities and configurations, and space restrictions when it comes to the storage area. The infrastructure can be new or old, and the degree of technology advancement varies. This has led to a range of different ways to control POU inventories. In the case hospital, the handheld computers used for ordering are old and can cause several types of errors.

The literature has highlighted the use of periodic review in combination with either fixed quantity or order-up-to level as the traditional inventory control policy applied at POU inventories in hospitals. However, this may not be a robust enough control policy if the goal is to assure availability. The policy gives poor stock visibility in between the periodic review and lack of real-time inventory data. The case hospital experience often that the wrong quantity or product is delivered from the central warehouse. By improving stock visibility and using real-time inventory data when ordering it can be assumed that such errors can be reduced or even eliminated.

Different inventory control policies are being applied in combination with various supporting technologies, to form the replenishment systems of the POU-inventories. The inventory control process has been mapped and visualised. In order to improve the visibility of the inventory levels new replenishment systems have been introduced. One can conclude with the fact that ensuring availability of medical supplies needs a joint effort from both the supply chain and the inventory control processes. Identification technology such as RFID has shown great potential to enable continuous real-time tracking of inventory levels and automatic order generation. Several barriers are present for the successful implementation of these solutions in the hospitals. Continuous improvements in both areas, in combination with the possibilities implied by emerging technologies, can change the design of the inventory control process towards a state were better availability of medical applies could be ensured.

Future research should focus on how different technological solution and hospital supply chain configuration have an impact on the response time and inventory efficiency.

Fig. 1 .

 1 Fig. 1. The inventory control process in the case hospital

Acknowledgement

This research received funding from the strategic research area NTNU Health in 2019 at NTNU, Norwegian University of Science and Technology. The authors also gratefully acknowledge the case hospital that made it possible to carry out this study and Elise Keseler, master student at NTNU, for supporting this research.


Neusa Andrade

Pedro Luiz De Oliveira Costa Neto

De Mello Torres

Irapuan Glória Júnior

Cláudio Guimarães Scheidt

Welleson Gazel

e-Health: A framework proposal for interoperability and health data sharing. A Brazilian Case

Keywords: Health Interoperability, Health Exchange Data, Quality of Care, Action Research

Interoperability among systems is a challenge that requires several regards and infrastructure often complex. The best worldwide reports and frameworks say that this can also improve health care and bring the best outcomes for stakeholders. Implementing Interoperability in developing countries is less affordable even it can also promote quality care and save lives. The best models and guidelines could offer protocols for sharing health data allowing to build a system that can deliver at the same time quality, transparency, and social value. This paper addresses an interoperability problem providing the steps built in a pilot to enable a conceptual framework for exchange healthcare data through EHR, and presents the first step and overview of a platform build using rules of PDCA. The experiment was built in a small Brazilian town intends to be a standard for deliver interaction between local government and citizens and also to offer to patients to control own medical data records through a mobile application.

Introduction

The interoperability at the Health Sector is considerate a very complex task, but according to the American Hospital Association (AHA) can provide advances and better health care outcomes that saving lives and involving key stakeholders [START_REF]AHA-American Hospital Association -Sharing Data, Saving Lives: The Hospital Agenda for Interoperability[END_REF][START_REF] Spyrou | Information System Interoperability in a Regional Health Care System Infrastructure: a pilot study using Health Care Information Standards[END_REF].

Recent studies from The European Commission (EC) says that yet today many countries like France, Germany, and Italy are still struggling with several challenges to exchange their health data. Developing countries suffering also with a lack of infrastructure and financial resources, presenting a systematic deficit in the quality of care, costs, transparency, and deficient management [START_REF] Spyrou | Information System Interoperability in a Regional Health Care System Infrastructure: a pilot study using Health Care Information Standards[END_REF][START_REF] Greer | Health law and policy in the European Union[END_REF].

The Commission on Health System Quality (HQSS) and World Health Organization (WHO) offer some frameworks and guidelines to produce wellfunctioning and performance health system in order to ensure the use of reliable and timely information regarding also to protecting privacy and security patient data [START_REF] Kruk | High-quality health systems in the Sustainable Development Goals era: time for a revolution. The Lancet Global Health Commission[END_REF][START_REF]WHO Library Cataloguing-in-Publication Data. Everybody business: strengthening health systems to improve health outcomes: WHO's the framework for action[END_REF].

Brazil has 5,500 municipalities where the major part of the population suffers from a lack of resources and it is dependent on a public single and universal health care system (SUS). A partnership between UNIP researchers and a small town in the Paraiba Valley to develop a pilot platform, aiming to promote interoperability that intends to become a standard and scalable solution that could be implemented at other cities that faces the same problem, enabled the development of this research [6,[START_REF] Brasil | Ministério da Saúde[END_REF].

This paper is the first one of an Action Research Cycle that describes the experience and proposal to design a framework based on PDCA to implement interoperability at public Brazilian health systems. Following the best practices is possible to design a strategy to be followed, based on the premise that is possible to build a cost-effective platform to reach data integration between entities using patients record as key data. The work is competing for an international award as a solution for this Brazilian quest [START_REF] Mello | Pesquisa-ação na engenharia de produção: proposta de estruturação para sua condução[END_REF][START_REF] González | Ontology-based framework for electronic health records interoperability[END_REF][START_REF] Liang | Integrating blockchain for data sharing and collaboration in mobile healthcare applications[END_REF][START_REF] Brasil | para o SUS: Acesso Universal[END_REF].

Methodology

Action Research is a qualitative method that has been used in Production Engineering researches, and also a step-by-step method that helps to conduct interventions in improving a business situation. The term "Research" refers to the production of knowledge, and the term "Action" to an intentional modification of a given reality.

The method was chosen to drive this project because leads to collaboration and involves stakeholders in a diagnostic and active-learning [START_REF] Mello | Pesquisa-ação na engenharia de produção: proposta de estruturação para sua condução[END_REF]. This work has begun with diagnosing that lead to an action plan. Action Research is a tool whose cycles aiming to affect and change the social reality using observations, explanations, and understandings. Table 1 shows the summarized steps used to fulfill the objectives of this research providing a structure for replication [START_REF] Mello | Pesquisa-ação na engenharia de produção: proposta de estruturação para sua condução[END_REF]. The theoretical background was chosen after a detailed analysis of frameworks, interoperability ontologies, best practices and policies documents regarding the exchange of patient data health. The main concepts were pointed out by the AHA originated in countries like the USA, and by the European Commission (EC) at countries like Croatia, France, Germany, Italy, The Netherlands, and Sweden [START_REF]AHA-American Hospital Association -Sharing Data, Saving Lives: The Hospital Agenda for Interoperability[END_REF][START_REF] Greer | Health law and policy in the European Union[END_REF][START_REF]WHO Library Cataloguing-in-Publication Data. Everybody business: strengthening health systems to improve health outcomes: WHO's the framework for action[END_REF].

The final work will join the best practices and ontologies for interoperability suggested by the American Hospital Association (AHA) and Quality impacts offered by The Lancet, as shown at figure 1 indicating the guidelines for the relationship between citizens and governs referring to health data [START_REF]AHA-American Hospital Association -Sharing Data, Saving Lives: The Hospital Agenda for Interoperability[END_REF][START_REF] Greer | Health law and policy in the European Union[END_REF][START_REF]WHO Library Cataloguing-in-Publication Data. Everybody business: strengthening health systems to improve health outcomes: WHO's the framework for action[END_REF]. Figure 2 shows the suggested rules for decision makings, to access quality, transparency, use, share and support information for health professionals, and patients that encouraging government-citizen relationship and also will be used to measure the impact of the pilot project [START_REF]AHA-American Hospital Association -Sharing Data, Saving Lives: The Hospital Agenda for Interoperability[END_REF][START_REF] Greer | Health law and policy in the European Union[END_REF][START_REF]WHO Library Cataloguing-in-Publication Data. Everybody business: strengthening health systems to improve health outcomes: WHO's the framework for action[END_REF]. Thus, we will introduce a summary of the pilot that is being developed that intends to become a standard to fulfill this gap. The chosen Brazilian city to applying the pilot project was due to their number of inhabitants, relationship and proximity with health managers that facilitated the diagnoses of their health infrastructure for the first step to collect data to test the platform with patients regarding health data exchange. [START_REF]AHA-American Hospital Association -Sharing Data, Saving Lives: The Hospital Agenda for Interoperability[END_REF][START_REF] González | Ontology-based framework for electronic health records interoperability[END_REF].

Results

The public health system (SUS) offered in Brazil was designed to fulfill all kind of health care whose Information Policy offers a lot of stand-alone software free supplied to manage data in order to provide information for the government. New society goals and health systems need to produce better health outcomes, including greater social value suggesting that citizens are able to collaborate at the governmentcitizen relationship, improving quality of access, transparency, the security of the information and also support for decision making that is stickle by this disconnected technologies. [START_REF]WHO Library Cataloguing-in-Publication Data. Everybody business: strengthening health systems to improve health outcomes: WHO's the framework for action[END_REF][START_REF] Brasil | Ministério da Saúde[END_REF][START_REF] Liang | Integrating blockchain for data sharing and collaboration in mobile healthcare applications[END_REF].

A Brazilian Case

According to data of the Brazilian Statistic Institute (IBGE), Bananal is a little town in the state of São Paulo at the Metropolitan Region of Vale do Paraíba that has 10,775 inhabitants. The city presenting a health infrastructure with a single Joint Health Unit, four offices for developing family health strategy, and one basic care unit that not exchange information among themselves causing many duplicates records and unnecessary costs without any participation of their citizens [6,[START_REF] Brasil | Ministério da Saúde[END_REF].

Figure 3 shows the first process and their 3 steps that enable the platform to be implemented at the city allowing a standardized method. After this, the platform will use a single key of patient data from an Electronic Medical Record to track and exchange data through entities that also establishes a channel between the local municipality and population and given to patients the control of carrying on their medical history on palm through their own mobile [START_REF] González | Ontology-based framework for electronic health records interoperability[END_REF][START_REF] Liang | Integrating blockchain for data sharing and collaboration in mobile healthcare applications[END_REF]. The platform also offers an interface to establish a communication process between the local government with citizens through an ordinary mobile application (APP). The final procedure is consolidated sending all medical historic data to patients that used the health services provided by the city allowing transparency. The applied model at the city will be extended to other cities reaching a population of more than 2 million inhabitants within 39 municipalities [START_REF] González | Ontology-based framework for electronic health records interoperability[END_REF][START_REF] Liang | Integrating blockchain for data sharing and collaboration in mobile healthcare applications[END_REF][START_REF] Brasil | para o SUS: Acesso Universal[END_REF].

However, health data are also highly privacy-sensitive, and even if more users are complaining by not obtain control over their personal health data, governs are compelling to face regulations generating several challenges. At the platform, we are concerned to safeguarding all steps of security and authentication, in a similar model done by the Blockchain platform allowing tracking and reliability of the data. [START_REF] Liang | Integrating blockchain for data sharing and collaboration in mobile healthcare applications[END_REF].

A summary of the workflow is shown in figure 4 that illustrates since the first identification of each patient at any health facility at the city. It is required to validate data before the first use of the mobile application. [START_REF] Liang | Integrating blockchain for data sharing and collaboration in mobile healthcare applications[END_REF].

Conclusions

A new society demand is affecting the Health Sector ending up in regulations. Electronic Medical Records (EHR) and patient data are usually retained by health institutions and very critical information to be safeguarded with systems that were not designed to offer patients own data. The best practices in healthcare and patient expectations include trusting that shared data is accurate. Health systems need to be designed to produce better outcomes including greater social value [1, 2, 5, 10] The American Hospital Association (AHA) suggests an interoperability ontology through Electronic Health Records to build an efficient solution with a costeffective platform for improving health care and sharing best practices with stakeholders. The Lancet Global Health Commission allows to understanding a framework to reach transparency implementing high-quality health systems that could save 8 million lives. These frameworks were chosen at this research by offering models and guidelines to create a reliable Health Systems including delivering data on the palm of patients at their mobile phones [START_REF]AHA-American Hospital Association -Sharing Data, Saving Lives: The Hospital Agenda for Interoperability[END_REF][START_REF] Spyrou | Information System Interoperability in a Regional Health Care System Infrastructure: a pilot study using Health Care Information Standards[END_REF][START_REF] Kruk | High-quality health systems in the Sustainable Development Goals era: time for a revolution. The Lancet Global Health Commission[END_REF][START_REF]WHO Library Cataloguing-in-Publication Data. Everybody business: strengthening health systems to improve health outcomes: WHO's the framework for action[END_REF]. This work presented a proposal of interoperability that reveals possibilities of sharing data between entities through a single key acquired through Electronic Medical Record. The pilot intends to be a model to be implemented at the suggested platform allowing major safety, tracking, and reliability of data at systems. The project is competing for an international award between 1,294 practices which can generate significant savings and better health outcomes for the Brazilian population [START_REF] González | Ontology-based framework for electronic health records interoperability[END_REF][START_REF] Liang | Integrating blockchain for data sharing and collaboration in mobile healthcare applications[END_REF][START_REF] Brasil | para o SUS: Acesso Universal[END_REF].

Fig. 1 .

 1 Fig. 1. Foundations for High-Quality Health Systems Commission [3].

Fig. 2 .

 2 Fig. 2. Suggested Framework for High-Quality Health Systems Commission [3].

Fig. 3 .

 3 Fig. 3. Diagnose and a process of identification and patient authentication data.

Fig. 4 .

 4 Fig. 4. Summarized workflow process.

Table 1 .

 1 Applied Cycles of this Action Research

	Step	Main Objective	Expected Outcome
	Identify the problem and theories	Research literature and cases and concepts	Set goal and target audience
	Develop a plan	Envision Success	Invites to participate, Techniques and Analysis Units
	Collect Data	Describe actual situation	Register Data
	Analyze Data	Develop a plan for improvement	Compare Theories and Practices
	Adjust the Theory and Plan	Implement the plan	Actions Plan Improvements
	Reports and Results	Framework Guidelines	Provide structure for replication

Acknowledgments

The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their support for the development of this research, University Paulista (UNIP), and the Ministry of Health Brazilian Government.


Shaima Hkiri

Afifa Hafidh

Jean-François Cavalier

Soufiane Touil

Ali Samarat
email: ali.samarat@gmail.com

Design, synthesis, antimicrobial evaluation and molecular docking studies of novel symmetrical 2,5-difunctionalized 1,3,4-oxadiazoles

Keywords: 1,3,4-oxadiazole, amidines, symmetrical 2,5-difunctionalized 1,3,4-oxadiazoles, antibacterial activity, antifungal activity, molecular docking

A series of novel symmetrical 2,5-difunctionalized 1,3,4-oxadiazole derivatives of pharmacological significance have been synthesized. The obtained compounds were screened for their in vitro antimicrobial activities against Gram-negative (Escherichia coli and Salmonella typhimurium) and Gram-positive bacteria (Staphylococcus aureus, Enterococcus feacium and Streptococcus agalactiae or group B Streptococcus), as well as against the fungus Candida albicans.

Although the synthesized compounds showed moderate antifungal activity against C. albicans, they exhibited good to excellent antibacterial activities against several strains, compared with standard drugs Ampicillin and Nystatin. In silico molecular docking in FabI enzyme active site, gave information regarding the binding mode of the drug candidate at molecular level.

INTRODUCTION

For the past few years, there has been a growing interest in developing new antimicrobial agents from various sources to combat microbial resistance [START_REF] Mayers | Antimicrobial Drug Resistance C: Clinical and Epidemiological Aspects[END_REF]. Currently, treatment failures associated with multidrug-resistant bacteria have become a critical problem worldwide and one of the most important current threats to public health [START_REF] Guschin | [END_REF]3]. As a consequence, the discovery of new antimicrobial agents is an exclusively important objective. Nowadays, synthetic products still remain one of the major sources of new drug molecules. Sulfonamides, oxazolidinones and fluoroquinolones [4] are indeed the most frequently-used antimicrobial drugs from synthetic origin.

1,3,4-Oxadiazoles are valuable five-membered aromatic heterocycles that possess a wide range of biological properties, including antibacterial [5][6][7][8][9], antifungal [10][11][12], antiviral [13,14], antiinflammatory [15], antiparasitic [15], and anti-HIV [17,18] activities. Some examples of drugs used in clinical medicine, containing the 1,3,4-oxadiazole unit, are furamizole which is an antibiotic drug, and raltegravir, an antiretroviral medication used to treat HIV infection [19] (Fig. 1). On the other hand, the amide, amidine, imine and aminoguanidine functional groups are important antimicrobial pharmacophores, and the introduction of such functionalities to the 1,3,4-oxadiazole core could improve the antimicrobial activity of such compounds, in conformity with the active substructure combination principle. Indeed, some commercialized antimicrobial drugs, such as linezolid and pentamidine (Fig. 1), contain in their structures the amide and amidine moieties, respectively [20,21]. Furthermore, it was found that introducing an imine or aminoguanidine group into sulfonamides could significantly improve the antimicrobial activity of the target compounds by 4 to 16-fold [22,23] (Fig. 1).

In view of the above, and as a continuation of our interest to develop efficient protocols for the synthesis of heterocyclic compounds with possible biological properties [24], we now report a convenient one-step synthesis of 1,3,4-oxadiazole-2,5-diamine, through the hydrogen peroxide-promoted oxidative dimerization of urea, followed by intramolecular cyclisation. A wide variety of unprecedented symmetrical 2,5-difunctionalized 1,3,4-oxadiazoles bearing the amide, amidine, imine, or aminoguanidine functional groups, was then easily obtained from the reaction of 1,3,4-oxadiazole-2,5-diamine with different carbonyl or thiocarbonyl electrophiles such as salicylic acid, paracetamol, phenacetin, -tetralone, sorbic acid and thiosemicarbazide. All the synthesized compounds were screened for their antimicrobial activity and they showed moderate to high antibacterial and antifungal efficacy against several microbial strains.

RESULTS AND DISCUSSION

Synthesis. In the first part of this work, we focused our efforts to synthesize the 1,3,4-oxadiazole-2,5-diamine 1 (Scheme 1). Being a binucleophilic agent with two amino groups in symmetrical 2,5positions, this compound could undergo condensation reactions with carbonyl or thiocarbonyl electrophiles leading to the target structures.

A literature survey on the preparation of 1,3,4-oxadiazole-2,5-diamine 1 showed only a few specific synthetic routes, most of these involving dehydrative cyclisation of the hardly accessible hydrazine-1,2-dicarboxamide [25,26]. Therefore, a step-economic and low-cost approach to synthesize this heterocyclic scaffold from easily obtained starting materials is of great significance.

With this in mind, we developed a convenient one-step synthesis of 1,3,4-oxadiazole-2,5-diamine 1, through the hydrogen peroxide-promoted oxidative dimerization of urea, followed by intramolecular cyclisation. The reaction was achieved in a simple one-step protocol, by treating urea with excess H2O2 in the presence of a catalytic amount of sulfuric acid and heating the mixture under reflux for 24 h (Scheme 1). The desired 1,3,4-oxadiazole-2,5-diamine 1 was obtained as a white solid in 72% yield, after recrystallization from ethanol. By comparison with existing methods [25,26], our protocol, which uses commercially available and low-cost urea, hydrogen peroxide and sulfuric acid as starting materials, is endowed with several other advantages such as easy operation, satisfactory yield and mild reaction conditions.

A plausible mechanism for the formation of compound 1 is depicted in Scheme 2. The reaction is assumed to proceed, firstly, via oxidative dimerization of urea promoted by hydrogen peroxide in the presence of sulfuric acid as catalyst. This will afford the crucial intermediate hydrazine-1,2dicarboxamide, which then undergoes an acid-catalyzed intramolecular cyclisation through its reactive tautomer form. A subsequent dehydrative aromatization leads to the target compound 1,3,4-oxadiazole-2,5-diamine 1 (Scheme 2).

With the 1,3,4-oxadiazole-2,5-diamine 1 in hand, we next studied its reaction with various commercially available carbonyl or thiocarbonyl electrophiles such as salicylic acid, paracetamol, phenacetin, -tetralone, sorbic acid and thiosemicarbazide, which would allow a straightforward approach to the target symmetrical 2,5-difunctionalized 1,3,4-oxadiazoles incorporating the amide, amidine, imine, or aminoguanidine functional groups. The reactions were found to proceed efficiently by condensing compound 1 with two molar equivalents of the above-mentioned carbonyl or thiocarbonyl electrophile, in refluxing ethanol for 4-12 h, in the presence of a catalytic amount of sulfuric acid (Scheme 3). The desired 2,5-difunctionalized 1,3,4-oxadiazoles 2-7 were then obtained as solids in 75-86% yield, after recrystallization from ethanol.

It should be mentioned that in case of sorbic acid used as carbonyl electrophile (Scheme 3, product 6), an atypical reaction behaviour was observed. As shown in Scheme 4, the condensation proceeds firstly via a nucleophilic conjugate addition leading to the -amino-acid intermediate, which then undergoes an intramolecular cyclisation through a 6-exo-trig process [27], followed by spontaneous dehydration, to afford the corresponding 1,3,4-oxadiazole 6, bearing two dihydropyridinone moieties in symmetrical 2,5-positions.

Antimicrobial activity. The newly synthesized compounds 2-7 as well as the starting 1,3,4oxadiazole-1,5-diamine 1, were investigated for their in vitro antimicrobial activities against Gram negative (Escherichia coli and Salmonella typhimurium) and Gram positive bacteria (Staphylococcus aureus, Enterococcus feacium and Streptococcus agalactiae or group B Streptococcus), as well as against the fungus Candida albicans. Their antimicrobial properties were first assessed by the inhibition zone diameter using filter paper disc-diffusion method [28]. Then, their respective minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (i.e., the minimum fungicidal concentration MFC for C. albicans), were determined using the MTT assay [START_REF]European Committee for Antimicrobial Susceptibility Testing[END_REF] and the agar plating method [28], respectively. The results were compared to the commercially available Ampicillin [30] used as positive control for bacterial strains, and Nystatin [31] used as positive control for clinical strains of Candida albicans.

As indicated in Table 1, at the concentration of 15 µg/mL, the majority of tested compounds displayed good to excellent inhibitory effects against all bacterial strains with a variable inhibition zone diameter ranging from 7 to 46.5 mm. In contrast, their antifungal activity was found to be moderate against the clinical Candiat albicans, compared to standard Nystatin. Furthermore, for all examined bacterial and fungal strains, the newly synthesized 2,5-difuctionnalized 1,3,4-oxadiazoles 2-7 were found 1.1 to 5.5 fold more active than their precursor 1,3,4-oxadiazole-2,5-diamine 1 which showed the less diameter of the inhibition zone in all cases. These results suggest that the incorporation of amide, amidine, imine or aminoguanidine functional groups into the 1,3,4-oxadiazole 2,5-diamine 1, allows to obtain more active compounds.

Interestingly, 1,3,4-oxadiazoles derivatives 3 and 4 bearing two amidine moieties in symmetrical 2,5positions and resulting from the condensation of 1 with paracetamol and phenacetin, respectively, (Scheme 3, products 3 and 4), exhibited the best antibacterial activity against all microbial strains tested with inhibition zone ranging between 11.0 and 46.5 mm (Table 1). Moreover, except for compounds 6, all other tested 1,3,4-oxadiazole derivatives exhibited inhibitory effect against S. agalactiae strain, better than or comparable to that of Ampicillin used as positive control.

More accurate data on the antimicrobial activities of the synthesized compounds were obtained through their MIC (Table 2) and MBC (MFC for C. albicans) values (Table 3). For the sake of comparison, the MIC and MBC values of the positive references Ampicillin and Nystatin were also determined (Tables 2 and3). As shown in Tables 2 and3, the MIC and MBC (MFC for C. albicans) values of 1,3,4-oxadiazole derivatives 1-7 are in concordance with the obtained inhibition zones, with MIC and MBC values ranging from 0.015 to 1.000 g.mL -1 and 0.031 to 1.000 g.mL -1 , respectively.

The compounds 1-7 exhibited moderate MIC and MBC activity against Gram-negative strains (Tables 2 and3). Regarding S. aureus and E. feacium, although the compounds 1-7 showed weaker MIC activity than that of Ampicillin, compounds 2, 3, 4, 5, and 7 exhibited relatively stronger MIC activity compared to that of compounds 1 and 6. Interestingly, compounds 3, 4 and 7 showed MBC activity against S. aureus and E. feacium stronger or comparable to that of Ampicillin. In case of S.

agalactiae, compounds 3, 4 and 7 exhibited relatively stronger MIC and MBC activity compared to others. It is noteworthy that only compound 4 exhibited stronger MIC and MBC activity than others though the MIC and MFC activities of 4 were weaker than that of Nystatin.

Importantly, the ratio MBC/MIC was close to 2, indicating that the synthesized 1,3,4oxadiazole derivatives were bactericidal in nature and not bacteriostatic [START_REF]European Committee for Antimicrobial Susceptibility Testing[END_REF]. This is illustrated in Table 4, by the exclusive correlation between MIC and MBC for the -0,9292 <0,01 -0,9290 <0,01 1,0000 <0,0001 a NS: Not Significant pIZD = -log(IZD); pMIC = -log(MIC) and pMBC = -log(MBC); Pearson's correlation coefficient r was calculated to demonstrate intraspecific microbial association between pIZD, pMIC and pMBC. There was no correlation between pIZD and pMIC as well as between pIZD and pMBC for all bacterial and fungal strains tested.

In silico molecular docking. Among the molecular targets in pathogenic strains, enoyl-acyl carrier protein reductase (FabI) which catalyzes the last step of the type II fatty acid biosynthesis elongation cycle [30] has been validated as an attractive drug target [32]. This enzyme is indeed highly conserved across many clinically relevant pathogens, including E. coli and S. aureus, and represents the sole enoyl-ACP reductase present [START_REF] Gerusz | Recent advances in the inhibition of bacterial fatty acid biosynthesis[END_REF]34].

In view of the promising antibacterial activity of 1,3,4-oxadiazole derivatives 3 and 4 against Gram-positive and Gram-negative selective FabI-containing organisms, and to get insight into the putative binding mode of these latter inhibitors in the active site of this enzyme, in silico molecular docking experiments were conducted as described previously [35,36], using the reported crystal structure of S. aureus FabI (saFabI) in complex with the inhibitor CG400549 and the cofactor NADPH in its active site (PDB entry code: 4CV1 [36]).

The best scoring position obtained (i.e. lowest energy complex) showed that compounds 3 and 4 may occupy the entire saFabI active site with predicted binding energy values E3 = -9.6 kcal/mol and E4 = -9.7 kcal/mol, respectively. Moreover, a high level of concordance between the most favorable docked conformation of each compound and the crystalized CG400549 inhibitor (Fig. 2A-B) was observed. Each of the docked compounds 3-saFabI and 4-saFabI complexes was then subjected to interactions analysis using Ligplot+ v.1.4 (Fig. 2C-D) [37]. The Ligplot+ diagram thus generated schematically depicts the hydrogen bonds and hydrophobic interactions between the ligand (i.e., 1,3,4-oxadiazole 3 or 4), the cofactor (i.e., NADPH) and the active site residues of the protein (i.e., saFabI) during the binding process. First, one 4-hydroxyphenylacetimidamide group from 3 and one 4-ethoxyphenylacetimidoyl group from 4 were found to bind deeply in the hydrophobic pocket where they interact with Tyr 147 , Val 154 , Gln 155 , Tyr 157 , Ala 198 , Val 201 , Gly 203 , Phe 204 and Ile 207 . On the other side, the second amidine moiety of each compound may point towards the entrance of the active site thus interacting with Phe 96 , Ala 97 , Met 160 and Leu 196 residues. In addition, compound 4 would also be stabilized by seven H-bonding with Ala 95 (2×H bonds), Tyr 147 , and Ser 197 residues, as well as with the NADPH nicotinamide ribose 2'-OH (2×H bonds) and the NH2 group of the 3carbamoylpyridinium (Fig. 2D). In contrast, only five H-bonding would be detected in the case of compound 3 involving Tyr 147 and Ser 197 residues, as well as the NADPH nicotinamide ribose 2'-OH (2×H bonds) and the phosphate group of the adenine ribose (Fig. 2C). Taken together, these results would suggest that saFabI could be an effective target of our 1,3,4-oxadiazole derivatives 3 and 4. It is noteworthy that, in our in silico molecular model, Tyr 157 , a key residue in saFabI activity, would only be involved in hydrophobic interactions (i.e., π-stacking interaction) with the aniline cycle of both compounds, contrary to inhibitors CG400549 and Triclosan for which a strong H bond was formed [36][37][38].

Although 1,3,4-oxadiazoles 3 and 4 may be able to inhibit FabI activity in either S. aureus or E. coli strains, the fact that compound 4 also impairs the growth of C. albicans which is lacking FabI enzyme, would suggest that in the latter bacteria this inhibitor might also interact with other enzyme(s) than FabI.

CONCLUSION

In conclusion, we have successfully developed a straightforward one-step approach to 1,3,4oxadiazole-2,5-diamine, through the hydrogen peroxide-promoted oxidative dimerization of urea, followed by intramolecular cyclisation. A wide variety of unprecedented symmetrical 2,5difunctionalized 1,3,4-oxadiazoles bearing the amide, amidine, imine, or aminoguanidine functional groups, was then easily obtained from the reaction of 1,3,4-oxadiazole-2,5-diamine with different carbonyl or thiocarbonyl electrophiles such as salicylic acid, paracetamol, phenacetin, -tetralone, sorbic acid and thiosemicarbazide. All the synthesized compounds showed good to excellent antibacterial activities against several Gram positive and negative strains, and moderate antifungal activity against C. albicans. The compounds 3 and 4 bearing two amidine moieties in symmetrical 2,5-positions on the 1,3,4-oxadiazole ring, displayed the most potent antibacterial activities, in some cases better than Ampicillin control drug.

In silico molecular docking study have also brought useful and informative data regarding the potential binding mode of compounds 3 and 4 inside saFabI enzyme active site, therefore providing some clues for developing new lead compounds as potent antibacterial agents. Additionally, further investigation on the cytotoxicity of the obtained compounds against eukaryotic cells and diverse structural modifications are currently in progress through iterative synthesis in conjugation with computer modeling and the results will be reported in due course.

EXPERIMENTAL

Chemistry. Uncorrected melting points were determined on a Buchi apparatus. IR spectra (KBr Discs) were performed on a 550 Nicolet Magana Spectrometer in the range 4000 -400 cm -1 . 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were carried out on an MSL 500 Bruker spectrometer at room temperature. Chemical shifts (𝛿) were reported in parts per million (ppm), relative to tetramethylsilane (TMS) as internal reference. Elemental analyses for C, H, and N were performed on an instrument microanalyser Perkin Elmer CHNOS. All reagents and solvents were supplied by Aldrich chemical, with analytical grade, and were used as received without further purification. The chemical structures of compounds 1-7 were established based on their spectral data and they had shown purity greater than 95%. NMR abbreviations and spin multiplicities are described as: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), dd (doublet of doublet). Coupling constants (J) are reported in Hertz (Hz).

Synthesis of 1,3,4-oxadiazole-2,5-diamine 1

6 g (100 mmol) of urea was introduced into a 50 mL round bottomed flask and 10 mL of H2O2 (40%) was added with 0.15 mL of concentrated H2SO4 (5 mol%). The reaction mixture was then heated at reflux temperature with continuous stirring for 24 hours. After cooling to room temperature, the reaction mixture was left to crystallize for 24 h. Crystals were then collected by filtration and recrystallized from absolute ethanol to afford a white solid. m. p.: 164 °C. Yield: (3.6g) 72%. IR (KBr, ν (cm -1 )): 3387-3280 (N-H), 1570 (C=N), 1232 (C-O). 1 H NMR (500 MHz, DMSO-d6)  = 7.95 (br, 4H, 2NH2). 13 C NMR (125MHz, DMSO-d6)  = 160.59. Anal. Calcd. for C2H4N4O.

(100.08): C, 24; H, 4.03; N, 55.98; found: C, 24.03; H, 4.07; N, 56.01.

General procedure for the synthesis of compounds 2-7

A mixture of the carbonyl or thiocarbonyl electrophilic reagent (20 mmol, 2 equiv), 1,3,4-oxadiazole-2,5-diamine (1) (1 g, 10 mmol, 1 equiv) and a catalytic amount of concentrated sulfuric acid (0.1 mL, 15 mol%) in ethanol (10 mL) was stirred under reflux for 6 to 12 h. After completion of the reaction as indicated by TLC, the reaction mixture was concentrated under reduced pressure. The residue was poured into water (10 mL) and extracted with chloroform (3 × 15 mL). The combined organic layer was dried over anhydrous MgSO4, filtered and concentrated under vacuum to afford a solid, which was recrystallized from absolute ethanol.

N,N'-(1,3,4-oxadiazole-2,5-diyl)bis(2-hydroxybenzamide) 2

White solid. Yield: 86%; m. p.: 167 °C. IR (KBr, ν (cm -1 )): 3335 (NH), 3286 (OH), 1652 (C=O), 1600 (C=C), 1562 (C=N), 1252 (C-O). 1 H NMR (DMSO-d6)  = 10.43 (br, 4H, 2OH, 2NH), 7.70 (d, 2H, J=6.5), 7.30 (d, 2H, J=7.5), 6.80 (dd, 4H, J=7.5). 13

N,N'-(1,3,4-oxadiazole-2,5-diyl) bis(N-(4-hydroxyphenyl)acetimidamide) 3

White solid. Yield: 80%. m. p.: 128 °C. IR (KBr, ν (cm -1 )): 3452 (OH), 3340 (NH), 1659 (C=N), 1570 (C=N), 1600 (C=C). 1 H NMR (DMSO-d6)  = 9.76 (br, 2H, 2OH), 9.41 (br, 2H, 2NH), 7.39 (d, 4H, J=9), 6.75 (d, 4H, J=8.5), 2.03 (s, 6H, 2CH3). 13 C NMR (125MHz, DMSO-d6)  = 161. 13

N, N'-(4-ethoxyphenyl)bis(acetimidoyl) amino-1,3,4-oxadiazol-2-yl-acetamide 4

Brown solid. Yield: 79%. m. p.: 140 °C. IR (KBr, ν (cm -1 )): 3297 (NH), 1660 (C=N), 1560 (C=N). 1 H NMR (DMSO-d6)  = 9.80 (br, 2H, 2NH), 7.51 (d, 4H, J=9), 6.80 (d, 4H, J=9.5), 3.95 (q, 4H, 2 OCH2, J=7), 2.04 (s, 6H, 2CH3), 1.30 (t, 6H, 2 CH3, J=7). 13

N,N'-(1,3,4-oxadiazole-2,5-diyl)bis(3,4-dihydronaphthalen-1(2H)-imine) 5

Brown solid. Yield: 76%. m. p.: 242°C. IR (KBr, ν (cm -1 )): 1645 (C=N), 1557 (C=N), 1613 (C=C). 1 H NMR (DMSO-d6)  = 8.45 (d, 2H, J=9,5), 8.08 (dd, 2H, J=9), 7.59 (dd, 2H, J=9), 7.47 (d, 2H, J=9,5), 2.93 (t, 4H, J=7), 2.53 (t, 4H, J=7), 2.07 (m, 4H). 13

1,1'-(1,3,4-oxadiazole-2,5-diyl)bis(6-methyl-5,6-dihydropyridin-2(1H)-one) 6

Yellow solid. Yield: 75%; m. p.: 124°C. IR (KBr, ν (cm -1 )): 1665 (C=O), 1603 (C=C), 1563 (C=N). 1 H NMR (DMSO-d6)  = 7.51 (d, 2H; J=10,5), 6.85 (m, 2H), 3.94 (m, 2H), 2.55 (m, 2H), 2.04 (m, 2H), 1.31 (d, 6H, 2CH3, J=7,5). 13

N, N'-(1,3,4-oxadiazole-2,5-diyl)bis(hydrazinecarboximidamide) 7

Yellow solid. Yield: 84%; m. p.: 262°C. IR (KBr, ν (cm -1 )): 3344 (NH2), 3236 (NH), 1654 (C=N), 1558 (C=N). 1 H NMR (DMSO-d6)  = 8.40 (br, 4H, 2NH2), 6.00 (br, 2H, 2NH), 4.80 (br, 4H, 2NH2). Thabet, Tunis, Tunisia. Prior to use, the bacteria and fungus are subcultured and incubated under optimal growth conditions. They are conserved by transplanting on nutrient agar favorable for their growth for 24 h, in the dark and at 37 °C. Nutrient agar was used to culture the microbes. The plates were inculcated by the microbes and incubated for 24 h at 37 °C. Ampicillin and Nystatin, at a concentration of 10 g.mL -1 and 100 g.mL -1 respectively and were used as reference drugs against microbes. All the microbes were grown on Mueller-Hinton Agar (Hi-media) plates (37 °C, 24 h).

Inhibition-zone measurement. A suspension of the tested microorganisms was spread on the appropriate solid media plates and incubated overnight at 37 °C. After 1 day, 4-5 loops of pure colonies were transferred to saline solution in a test tube for each bacterial strain and adjusted to the 0.5 McFarland turbidity standard (~10 8 cells.mL -1 ). Sterile cotton dipped into the bacterial suspension and the agar plates were streaked three times, each time turning the plate at a 60° angle and finally rubbing the swab through the edge of the plate. Sterile paper discs (Glass Microfibre filters, Whatman; sterile water. Ampicillin (15 μg/disc) was used as positive control for all strains except C. albicans for which nystatin (100 μg/disc) was used. Inoculated plates with discs were placed in a 37 °C incubator. After 24 h of incubation, the results were recorded by measuring the zones of growth inhibition surrounding the filter paper disc in millimeters. Clear inhibition zones around the discs indicated the presence of antimicrobial activity. The test was run in duplicate.

Minimum inhibitory concentration (MIC) measurement. The MIC was considered to be the lowest concentration that completely inhibited the growth on agar plates, disregarding a single colony or faint haze caused by the inoculum. The MIC values were determined in 96 well-microplate using the microdilution method according to reference [START_REF]European Committee for Antimicrobial Susceptibility Testing[END_REF]. Briefly, a stock solution of each product was prepared in DMSO. Then, serial dilutions of all tested products were performed in Mueller Hinton Broth medium (MHB) to obtain final concentrations in the range of 10 g/mL to 0.0025 g/mL. The 12th well was considered as growth control (free drug control). Afterwards, 50 μL of bacterial inoculum was added to each well at a final concentration of 10 6 CFU/mL. After incubation at 37 °C for 24 h, 10 μL of MTT were added to each well as bacterial growth indicator. After further incubation at 37 °C for 2 h, the bacterial growth was revealed by the change of coloration from yellow to black.

The MIC was considered to be the lowest active compound concentration at which growth inhibition was clearly visible (absence of the color change at the bottom of the well) within a defined period of time.

Minimal bactericidal concentration (MBC) measurement. MIC tests were always extended to measure the MBC as follows: a loop-full from the tube that did not show visible growth (MIC) was spread over a quarter of Müller-Hinton agar plate. After 18 h of incubation at 37 °C, the plates were examined for CFU count. Again, the tube containing the lowest concentration of the test compound that failed to yield growth on subculture plates was considered the MBC for the respective test organism (Table 3). Thus, it reduces by 99.9% the in vitro survival of micro-organisms in a medium containing a defined inoculum of bacteria, within a defined period of time.

Statistical analysis.

Results of quantitative variables were expressed as the arithmetic mean.

Statistical analysis of the data was performed using MS Excel for descriptive statistics and Pearson's correlation between parameters. All values were considered significant at P<0.05.

Molecular docking experiments.

A computational docking and scoring procedure using the Autodock Vina program [39] was used to generate the putative binding modes of 1,3,4-oxadiazoles 3 and 4 into the active site of the S. aureus enoyl-acyl carrier protein reductase (saFabI) as previously described [35]. The PyMOL Molecular Graphics System (version 1.4, Schrödinger, LLC) was used as working environment with an in-house version of the AutoDock/Vina PyMOL plugin [40]. The X-ray crystallographic structure of saFabI in complex with the inhibitor CG400549 and the cofactor NADPH in its active site (PDB entry code: 4CV1; 1.95 Å resolution [36]) available at the Protein Data Bank was used as receptor. Docking runs were performed after removing the ligand (i.e., CG400549 inhibitor) from the enzyme active site. The box size used for the receptor was chosen to fit the whole active site cleft and to allow non constructive binding positions. The binding site region was defined by NADPH and amino acids 93-99, 102, 121, 146-147, 154-157, 160, 164, 190-193, 195, 197-204, and 207 as previously reported [36]. A model structure file was generated for the inhibitor molecules, and their geometry was refined using the Avogadro open-source program (version 1.1.1. http://avogadro.openmolecules.net/). As a first step, compound CG400549 was submitted to in silico molecular docking into saFabI active site. Comparison of the generated CG400549 docking pose with the available binding geometry observed in the 4CV1 crystal structure revealed root mean square deviations of 0.230 Å for the top ranked pose (predicted binding energy value, ECG400549 = -10.3 kcal/mol). This re-/cross-docking experiment thus confirms the validity and reliability of our computational approach. -0,9292 <0,01 -0,9290 <0,01 1,0000 <0,0001 a NS: Not Significant pIZD = -log(IZD); pMIC = -log(MIC) and pMBC = -log(MBC); Pearson's correlation coefficient r was calculated to demonstrate intraspecific microbial association between pIZD, pMIC and pMBC. There was no correlation between pIZD and pMIC as well as between pIZD and pMBC for all bacterial and fungal strains tested.

Fig. 1 .

 1 Fig. 1. Design strategy of target structures.

1, 3 , 4 -

 34 oxadiazole derivatives, and on the other hand, by the absence of correlation of these two latter parameters with the inhibition zone diameter (IZD), index of bacteriostatic activity.On the basis of obtained results from MIC and MBC values, compounds 3 and 4 bearing amidine moieties can be regarded as a new valuable source to produce new 1,3,4-oxadiazole derivatives against bacteria, especially Gram-positive as well as the fungus C. albicans. This indicates that the incorporation of significant functional unit such amidine, in symmetrical 2,5-positions, into the 1,3,4-oxadiazole 2,5-diamine 1, was as effective in obtaining of compounds 3 and 4 more active than the original precursor and which might be a potential source for synthetic products for the pharmaceutical industry against microorganisms of bacterial and fungal origin.

Figure 2 .

 2 Figure 2. (A-B) in silico molecular docking of 1,3,4-oxadiazoles 3 (panel A -yellow color) and 4 (panel B -cyan color) into the crystallographic structures of S. aureus enoyl-acyl carrier protein reductase (saFabI) in a van der Waals surface representation, and superimposed to CG400549 (salmon color) saFabI inhibitor. NADPH cofactor also present in saFabI active site is colored in green. Hydrophobic residues (alanine, leucine, isoleucine, valine, tryptophan, tyrosine, phenylalanine, proline and methionine) are highlighted in white. (C-D) Ligplot+ analyses results: 3D representation of schematic binding mode of compounds 3 (panel Cyellow color) and 4 (panel D -cyan color) in complex with saFabI and NADPH (green color) showing both hydrogen-bonds (yellow dashed lines) and hydrophobic interactions. Selected residues of the saFabI binding pocket are shown as gray sticks. The stick representation uses the following atom colorcode: nitrogen, blue; oxygen, red; sulfur, yellow; phosphorus, orange. Structures were drawn with PyMOL Molecular Graphics System (Version 1.4, Schrödinger, LLC) using the PDB file 4CV1 [36].

13 C

 13 NMR (125MHz, DMSO-d6)  = 161.90, 158.90. Anal. Calcd. for C4H10N10O (214.10): C, 22.43; H, 4.71; N, 65.39; found: C, 22.48; H, 4.74; N, 65,38. Antimicrobial evaluation. The used microorganisms; Gram(-): Escherichia coli (ATCC 8739), Salmonella typhimurium (ATCC 14028), Gram(+): Staphylococcus aureus (ATCC 6538), Enterococcus feacium (ATCC 19434), Streptococcus B (agalactiae), and the fungus Candida albicans (ATCC 10231); were from Microbiology Laboratory of National Institute INRAP Biotech Pole -Sidi

Table 1 .

 1 The in vitro antimicrobial activity of compounds 1-7 and the control drugs. At the concentration of 15g/mL, b At the concentration of 100g/mL.

	Compound a	E. coli	Inhibition zone diameter (mm) S. typhimurium S. aureus E. feacium	S. agalactiae C. albicans
	1	7.00	7.50	7.00	9.00	8.50	8.00
	2	9.25	9.50	15.25	25.50	37.75	9.25
	3	13.75	12.00	18.25	27.00	36.50	11.00
	4	12.75	11.50	16.00	25.50	46.50	11.25
	5	12.25	12.25	14.25	17.75	29.25	10.00
	6	10.00	9.50	13.25	15.00	15.50	10.50
	7	10.50	10.00	17.75	25.00	36.50	11.00
	Ampicillin a	12	14.50	34.75	37.00	28.75	-
	Nystatin b	-		-	-	-	30.75
	a

Table 2 .

 2 Minimum inhibitory concentration (MIC) of compounds 1-7

	Compound	E. coli	MIC (g.mL -1 ) S. typhimurium S. aureus E. feacium	S. agalactiae C. albicans
	1	1.000	0.500	1.000	0.500	0.250	0.250
	2	0.500	0.500	0.062	0.062	0.062	0.250
	3	0.500	0.500	0.031	0.031	0.031	0.500
	4	0.500	0.500	0.015	0.015	0.015	0.015
	5	0.500	0.500	0.062	0.125	0.250	0.250
	6	0.500	0.500	1.000	0.500	0.500	0.500
	7	0.500	0.500	0.015	0.031	0.031	0.250
	Ampicillin	0.031	0.015	0.004	0.004	0.003	-
	Nystatin	-		-	-	-	0.003

Table 3 .

 3 Minimum bactericidal concentration (MBC) of compounds 1-7

	Compound	E. coli	MBC (g/mL) S. typhimurium S. aureus E. feacium	S. agalactiae C. albicans a
	1	1.000	1.000	1.500	1.000	0.500	0.500
	2	1.000	1.000	0.125	0.125	0.125	0.500
	3	1.000	1.000	0.062	0.062	0.062	1.000
	4	0.500	0.500	0.031	0.031	0.031	0.031
	5	1.000	1.000	0.125	0.250	0.125	1.000
	6	1.000	1.000	1.500	1.000	0.500	1.000
	7	1.000	1.000	0.031	0.062	0.062	0.500
	Ampicillin	0.062	0.031	0.095	0.095	0.007	-
	Nystatin	-		-	-	-	0.007

a The minimum fungicidal concentration MFC.

Table 4 .

 4 Association between pMIC, pMBC and pIZD of 1,3,4-oxadiazole derivatives 1-7 against all bacterial and fungal strains tested.

	Compound	pIZD -pMIC r p-value a	r	pIZD -pMBC p-value *	pMIC -pMBC r p-value
	1	-0,7086	NS	-0,5336	NS	0,9120	<0,02
	2	-0,8503	<0,05	-0,8501	<0,05	1,0000	<0,0001
	3	-0,8347	<0,05	-0,8347	<0,05	1,0000	<0,0001
	4	-0,3193	NS	-0,6253	NS	0,8358	<0,05
	5	-0,2333	NS	-0,0437	NS	0,5124	NS
	6	0,2151	NS	-0,3561	NS	0,6252	NS
	7

  C NMR (125MHz, DMSO-d6)  = 161.28, 160.44, 154.80, 132.87, 121.05, 114.69, 63.48, 24.19, 15.09. Anal. Calcd. for C22H26N6O3 (422.48): C, 62.54; H, 6.20; N, 19.89; found: C, 62.51; H, 6.23; N, 19.94.

  C NMR (125MHz, DMSO-d6)  = 160.13, 151.40, 144.63, 135.74, 132.21, 128.37, 126.21, 122.19, 23.98, 27.26, 28.05. Anal. Calcd. for C22H20N4O (356.16): C, 74.14; H, 5.66; N, 15.72; found: C, 74.18; H, 5.63; N, 15.75.

  C NMR (125MHz, DMSO-d6)  = 165.28, 162.44, 132.8, 123.05, 67.48, 33.50, 20.95. Anal. Calcd. for C14H16N4O3 (288.12): C, 58.32; H, 5.59; N, 19.43; found: C,58.27; H, 5.62; N, 19.47.

Table 1 .

 1 The in vitro antimicrobial activity of compounds 1-7 and the control drugs. At the concentration of 15g/mL, b At the concentration of 100g/mL.

	Compound a	E. coli	Inhibition zone diameter (mm) S. typhimurium S. aureus E. feacium	S. agalactiae C. albicans
	1	7.00	7.50	7.00	9.00	8.50	8.00
	2	9.25	9.50	15.25	25.50	37.75	9.25
	3	13.75	12.00	18.25	27.00	36.50	11.00
	4	12.75	11.50	16.00	25.50	46.50	11.25
	5	12.25	12.25	14.25	17.75	29.25	10.00
	6	10.00	9.50	13.25	15.00	15.50	10.50
	7	10.50	10.00	17.75	25.00	36.50	11.00
	Ampicillin a	12	14.50	34.75	37.00	28.75	-
	Nystatin b	-		-	-	-	30.75
	a

Table 2 .

 2 Minimum inhibitory concentration (MIC) of compounds 1-7

	Compound	E. coli	MIC (g.mL -1 ) S. typhimurium S. aureus E. feacium	S. agalactiae C. albicans
	1	1.000	0.500	1.000	0.500	0.250	0.250
	2	0.500	0.500	0.062	0.062	0.062	0.250
	3	0.500	0.500	0.031	0.031	0.031	0.500
	4	0.500	0.500	0.015	0.015	0.015	0.015
	5	0.500	0.500	0.062	0.125	0.250	0.250
	6	0.500	0.500	1.000	0.500	0.500	0.500
	7	0.500	0.500	0.015	0.031	0.031	0.250
	Ampicillin	0.031	0.015	0.004	0.004	0.003	-
	Nystatin	-		-	-	-	0.003

Table 3 .

 3 Minimum bactericidal concentration (MBC) of compounds 1-7

	Compound	E. coli	MBC (g/mL) S. typhimurium S. aureus E. feacium	S. agalactiae C. albicans a
	1	1.000	1.000	1.500	1.000	0.500	0.500
	2	1.000	1.000	0.125	0.125	0.125	0.500
	3	1.000	1.000	0.062	0.062	0.062	1.000
	4	0.500	0.500	0.031	0.031	0.031	0.031
	5	1.000	1.000	0.125	0.250	0.125	1.000
	6	1.000	1.000	1.500	1.000	0.500	1.000
	7	1.000	1.000	0.031	0.062	0.062	0.500
	Ampicillin	0.062	0.031	0.095	0.095	0.007	-
	Nystatin	-		-	-	-	0.007

a The minimum fungicidal concentration MFC.

Table 4 .

 4 Association between pMIC, pMBC and pIZD of 1,3,4-oxadiazole derivatives 1-7 against all bacterial and fungal strains tested.

	Compound	pIZD -pMIC r p-value a	r	pIZD -pMBC p-value *	pMIC -pMBC r p-value
	1	-0,7086	NS	-0,5336	NS	0,9120	<0,02
	2	-0,8503	<0,05	-0,8501	<0,05	1,0000	<0,0001
	3	-0,8347	<0,05	-0,8347	<0,05	1,0000	<0,0001
	4	-0,3193	NS	-0,6253	NS	0,8358	<0,05
	5	-0,2333	NS	-0,0437	NS	0,5124	NS
	6	0,2151	NS	-0,3561	NS	0,6252	NS
	7

Acknowledgements

We thank the Tunisian Ministry of Higher Education and Scientific Research for financial support (LR18ES11). We would also like to thank Professor Mosadok Ben Attia (University of Carthage, Faculty of Sciences of Bizerte) for his fructuous discussion about antimicrobial activity of our products.