text:
Hassiba Hadj

Youcef Taibi
email: htaibi@hotmail.fr

Noria Smail-Saadoun

Sonia Labidi

Karima Abdellaoui

Mahfoud Makhlouf

Abdelkrim Laouar

Chérifatagmount Benouaret

Lila Rezki-Sekhi

Amel Belkebir Boukais

Anissa Lounès-Hadj

Sahraoui

The Influence of No-till Farming on Durum Wheat Mycorrhization in a Semi-Arid Region: A Long-Term Field Experiment

Keywords: arbuscular mycorrhizal fungi, no-till farming, conventionnel seedling, lipid biomarkers, root endophytes, semi-arid region

Maintaining a reliable and sustainable agricultural production system has become one of the major concerns of producers in arid and semi-arid regions. Simplifying farming techniques and practicing Direct Seedling (DS) could contribute to insure the sustainability of agriculture, preserving the natural resources and the environment. Direct Seedling based on limiting soil plowing has a potential number of benefits, including reduced production costs and soil erosion. Associated with the organic mulch, this technique improves the soil fertility and favors the establishment of root symbioses. Given the importance of the no-till farming techniques, the present research work aims to compare the effects of DS and those of Conventional Seedling (CS) on the evolution of arbuscular mycorrhizal symbiosis in durum wheat (Triticum durum Desf) roots, cultivated in the field for five years. Soil and root samples were collected during three different cropping stages at two different treatments. The results of Arbuscular Mycorrhizal Fungi (AMF) root colonization kinetics have shown an increase in the percentage of arbuscules and a decrease in vesicles for plants sampled from a DS field compared to those from CS. Effects of the DS on the mycorrhizal parameters appears clearly in the fourth year of the experiment and continues in the fifth year, with an arbuscule percentage reaching 80% in a DS field and not exceeding 21% in a CS field. Soil phospholipid fatty acids (PLFA) C16:1ω5 (biomarker of AMF) and C18:2ω 6, 9 (biomarker of saprophytic/ectomycorrhizal fungi) demonstrate that no-till practice improves AMF biomass and saprotrophic/ectomycorrhizal fungal biomasses by 52 and 159%, respectively, in comparison with those found in a CS field. In both treatments, no-till farming and CS plots, the AMF biomass is higher than saprotrophic/ectomycorrhizal biomasses. The natural biodiversity of AMF is also enhanced in a no-till field. In addition, an increase in the relative abundance of six families of Glomeromycota (Gigasporaceae, Diversisporaceae, Scutellosporaceae, Entrophosporaceae, Acaulosporaceae, Dentiscutataceae) was observed. To summarize, the present study highlights the importance of no-till practice as an approach to restore the microbiome in soils disturbed by tillage in semi-arid regions.

Introduction

Maintaining a reliable and sustainable agricultural production system has become one of the major concerns of producers in arid and semi-arid regions (Lyons et al., 1996;[START_REF] Bruelle | Can conservation agriculture improve crop water availability in an erratic tropical climate producing water stress? A simple model applied to upland rice in Madagascar[END_REF]. Simplifying farming techniques and using no-till techniques contribute to ensure the sustainability of agriculture, preserving the natural resources and the environment. To cope with the various constraints, including drought, many countries around the world (The United States, Argentina, Chile, Brazil, and Australia), have adopted the simplification of cultural techniques, known as SCT [START_REF] Freitas | The transformation of agriculture in Brazil through development and adoption of Zero Tillage Conservation Agriculture[END_REF][START_REF] Bellotti | The Development of Conservation Agriculture in Australia Farmers as innovators[END_REF]. In 2007, the extension of this system was about 95 million hectares (Lal et al., 2007), and presently it exceeds 125 million hectares [START_REF] Bruelle | Can conservation agriculture improve crop water availability in an erratic tropical climate producing water stress? A simple model applied to upland rice in Madagascar[END_REF].

In Algeria, cereals are rainfed crops that occupy an annual area of 3.5 million hectares [START_REF] Rouabhi | Socioeconomic Assessment of No-Till in Wheat Cropping System: A Case Study in Algeria[END_REF]. They are mainly located in the east of the country, in the semi-arid regions of the High Plateaus, where yearly rainfall levels are less than 400mm. Soils of these regions are predominantly calcareous with low levels of soil organic matter (Batouche & Labiod, 1991). The combined effects of all these factors affect cereal production, and yields remain low even though the cultivated varieties are improved [START_REF] Lahmar | Dégradation des sols et stratégies coopérative en Méditerranée[END_REF]. Cultivation of cereals is conducted according to the soil conventional tillage system, based mainly on soil plowing to control weeds, preparing seedlings, incorporating residue crops and fertilizers, and increasing water infiltration. Conventional farming system increases grain production costs and threat of soil erosion (about 2000 to 4000 tons/km 2 of soil are lost) [START_REF] Kaci | Application of universal soil loss equation (USLE) model to estimate water erosion by GIS system and remote sensing: catchment area of oued Rhiou (Watershed of Cheliff), Algeria[END_REF][START_REF] Arabi | Influence du système de production et du sol sur l'érosion et le ruissellement en nappe en milieu montagnard méditerranéen (station d'Ouezra, Algérie)[END_REF], especially when combined with overgrazing.

Compared to other farming systems, conventional soil tillage (CT) influences the soil physical characteristics. It increases the soil pulverization at 0-10cm depth leading to lower bulk density and higher macroporosity, which facilitates water erosion. In layers below 10 cm depth, CT increases bulk density and reduces the macro-porosity to critical levels for crop growth (Tuzzin de [START_REF] Tuzzin De Moraes | Soil physical quality on tillage and cropping systems after two decades in the subtropical region of Brazil[END_REF], which increases the risk of soil compaction, penetration resistance and affects their biological activity [START_REF] Aubert | Microbial biomass and activity at various soil depths in a Brazilian oxisol after two decades of no-tillage and conventional tillage[END_REF][START_REF] Aslam | Tillage impacts on soil microbial biomass C, N and P, earthworms and agronomy after two years of cropping following permanent pasture in New Zealand[END_REF].

Conservation agriculture (CA) is a more recent innovation that combines soil cover-organic-mulch, reduced plowing or no-till practice and crop rotation. It is currently promoted as a means to reduce soil degradation in the Mediterranean semi-arid areas [START_REF] Lahmar | Du mulch terreux au mulch organique pour assurer une transition Vers l'agriculture durable dans les hautes plaines sétifiennes[END_REF]. A number of experiments and research results from Spain, Morocco and Tunisia, underlines the prominent role of the organic mulch in the improvements that occur in the soil, and suggest that no-till practice is a plausible alternative to conventional tillage (Mrabet, 2001;Lahmar & Arrúe, 2007;Lahmar, 2010;[START_REF] Vega | Comparing the effectiveness of seeding and mulching + seeding in reducing soil erosion after a high severity fire in Galicia (NW Spain)[END_REF]. However, the adoption of no-till farming should be done in an integrated approach, allowing good management of rotations, judicious control of weeds, diseases and the attack of herbivores while minimizing the use of chemical inputs (Karuma et al., 2014). This will promote biological activity and microbial biomass in soils [START_REF] Labreuche | Evaluation des impacts environnementaux des Techniques Culturales Sans Labour en France[END_REF]. [START_REF] Celik | Impacts of different tillage practices on some soil microbiological properties and crop yield under semi-arid Mediterranean conditions[END_REF] have specified that no-tillage practices under a semi-arid climate have considerably increased the number of arbuscular mycorrhizal fungi (AMF) spores in the soil. Indeed, for cultivated cereal species, direct seedling promotes interactions between the root system and the symbiotic fungi such as Dark Septated Endophytes (DSE) or AMF [START_REF] Jansa | The effects of agricultural practices on arbuscular Mycorrhizal fungi[END_REF][START_REF] Schalamuk | Fungal spore diversity of arbuscular Mycorrhizal fungi associated with spring wheat: Effects of tillage[END_REF][START_REF] Duponnois | La symbiose mycorhizienne et la fertilité des sols dans les zones arides: un outil biologique sous-exploité dans la gestion des terres de la zone sahélo-saharienne[END_REF].

AMF symbiotic interactions allow the host plant to tolerate abiotic stress through a structural change of the root system and especially by providing them better water and mineral nutritions [START_REF] Rodriguez | The role of fungal symbioses in the Adaptation of plants to high stress environments[END_REF]Soka & Ritchie, 2014;[START_REF] Wu | Arbuscular Mycorrhizas and Stress Tolerance of Plants[END_REF]. Root Dark Septate Endophytes (DSE) are fungi that locate and grow between root tissues [START_REF] Vega | Entomopathogenic fungal endophytes[END_REF]. They are characterized by having dark pigmentation, microsclerotia and melanized septated hyphae that colonizes the epidermis as well as the inter and intracellular root cortex of the host plant [START_REF] Peterson | Associations between microfungal endophytes and roots: Do structural featuresindicate function[END_REF]. Many of these fungi are able to colonize the root cells of plants, promoting growth without causing pathologies [START_REF] Clay | Fungi and the food of the gods[END_REF][START_REF] Yuan | A new species of Harpophora (Magnaporthaceae) recovered from healthy wildrice (Oryza granulata) roots, representing a novel member of abeneficial dark septate endophyte[END_REF][START_REF] Vergara | Contribution of dark septate fungi to the nutrient uptake and growth of rice plants[END_REF]. DSE are found worldwide and coexist often with different mycorrhizal fungi [START_REF] Mandyam | Seeking the elusive function of the root-colonising dark septate endophytic fungi[END_REF], 2008).

In Algeria, only 5,559 ha of cereal areas are converted to no-till farming as a result of sensitization campaigns carried out by agricultural research and development institutions. They encourage farmers to adopt the no-till farming system in order to reduce the environmental and economic costs of agricultural production [START_REF] Zeghouane | L'agriculture de conservation: Introduction et perspectives de développement du semis direct dans les systèmes de production céréaliers, une nouvelle expérience en Algérie[END_REF](Zeghouane et al., , 2016)).

Consequently, whilst aiming to compare the effect of no-till farming and conventional seedling systems on two important types of fungal root symbiosis-arbuscular mycorrhiza and dark septated endophytic fungi of durum wheat in a field experiment, the present study lasted five years.

Material and Methods

Experimental Site

This study was carried out from 2008 to 2013, at the agronomic experimental station of the Technical Institute of Field Crops (ITGC) of Sétif (latitude, 36°08′ N; longitude, 5°20′ E and altitude 962 m. The climate is Mediterranean and semi-arid, characterized by cold wet winters and hot dry summers. During the experiment, the average annual rainfall did not exceed 400 mm and presented some irregularity in distribution, with large inter-month and inter-annual variations. The dry season was usually noted from May to September (Figure 1). Soil characterization analyses of the experimental plot were made by the Technical Institute of Field Crops (ITGC) at the beginning of the project. They indicated that on the horizons which delimited the first thirty centimeters of topsoil, the soil of the experimental plot had a clay-sandy-loamy texture, with an average organic matter content of 2.6%. Basic pH (8.26) justified the strong presence of total and active limestone (51.58%; 21.25%), which weakened the availability of absorbable phosphorus (7.33 ppm) (Table 1). The soil organic carbon was determined by means of the Anne method.

The total and active carbon dosages were made by the methods of Bernard's calcimeter and that of Drouineau, respectively. Soil Granulometry by the Robinson Method.

Plant Material and Field Conditions

The studied variety of durum wheat used in the present study is named "Boussalem". It was obtained from the Sétif experimental station (Heider/Marli/Heider Cro ICD4141BLCTR4AP) on an ICARDA-CIMMYT introduced material. The experimental design was based on the application of two soil management practices: 1) No-till with seedling (SD) carried out directly in the residue of the previous crop, using a combined seeder (SEMEATO) which opened only a seed line with 2 cm of width; 2) Conventional seedling (CS), where the soil was prepared with a moldboard plough followed by a cover crop and a disc harrow. Seedling was carried out by means of a conventional seed drill (Figure 2).

Plant and Soil Sampling

The experimental trial was field-installed in 2008, but our observations started in the third year of testing (2010-2011) and proceeded as follows:

Soil Fatty Acid Analysis

Two soil samples were taken from each plot at a depth of 10 cm with an auger, following the harvest of the 2010/2011 trial (third year of application of the two different operating systems). After removing plant debris from soil samples, four replicates of 3 g of freeze-dried soil (from each soil sample) were analysed to measure the phospholipid fatty acid (PLFA) contents. Amounts of PLFA and neutral fatty acid (NLFA) specific to AMF (C16:1ω5) and to saprotrophic/ectomycorrhizal fungi (C18:2 ω 6.9) were measured.

Lipid extraction was carried out following the method suggested by [START_REF] Frostegård | Microbial biomass measured as total lipid phosphate in soils of different organic content[END_REF]. The extracted lipids were fractionated into neutral lipids, glycolipids and polar lipids on a silica acid column by successively eluting with chloroform, acetone and methanol (1:2:1, V:V:V). Chloroform fraction (containing the neutral lipids) and the methanol fraction (containing the phospholipids) were subjected to a transesterification using a base solution (KOH 0.2 M) prepared in methanol to transform the PLFA and the NLFA into free fatty acid methyl esters. Final extracts were analysed by using a PerkinElmer Auto system gas chromatograph (GC) equipped with a flame-ionization detector (Norwalk, CT) and an EC-1000 (Alltech) capillary column (30 m × 0.53 mm i.d.) with hydrogen as a carrier gas (3.6 ml min -1 ). The temperature program included a fast rising from 50 °C to 150 °C at 15 °C min -1 and then a rise from 150 to 220 °C at 5 °C min -1 . Fatty acid quantification was made by using nonadecanoic acid methyl ester (C19:0) as an internal standard. Their identification relied on the retention times of a wide range of standards (fatty acid methyl ester mixtures C4-C24:1, Sigma Aldrich) and mass analysis. The system was operated in the constant linear velocity (70 cm s -1 ) using helium as a carrier gas. Fatty acids were identified by comparing their mass spectra with the standard mass spectra in the NIST MS library.

Determination of Arbuscular Mycorrhizal Fungal Colonization

To estimate the root colonization by AMF, during three stages of the vegetative cycle of wheat (tillering, grain filling and grain maturity), 6 plants/plot/stage for each treatment were sampled in 2011, 2012 and 2013 (last three years of the experiment). The measurement was performed on fresh roots. Roots were soaked during 5 min in an H 2 O 2 (30%) bath to remove pigments, cleared in KOH (10%) and stained with Trypan blue (0.05%) [START_REF] Phillips | Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular rnycorrhizal fungi for rapid assessment of infection[END_REF]). Root mycorrhizal rates were calculated using a magnified grid line intersect method (Mc Gonigle et al., 1990). For each repetition three microscopic slides with ten randomly root fragments (1 cm) were observed under an optical microscope (× 100). Intersections were counted in the following categories: negative (no fungal material in root), arbuscules, vesicles and hyphae. This made it possible to quantify the following rates: H% = (G -p)/G × 100; Arb% = (q + s)/G × 100; Ves% = (r + s)/G × 100 [START_REF] Alvarez | Short-term effects of tillage systems on active soil microbial biomass[END_REF] where, G: total number of intersections; p: no mycorrhizal structure; q: presence of arbuscules; r: presence of vesicles; s: presence of arbuscules and vesicles.

Determination of Dark Septated Endophytic Fungal Root Colonization Rate

The estimation of root colonization was completed in 2013 by the observation of structures specific to dark septated endophytic fungi (DSE). Microscopic observations were performed on the same slides used for AMF but focused on the structures specific to DSE (dark septated hyphae, microsclerotia) in the root cortex and the central cylinder. The rate of DSE (DSE%) was calculated according to the following formula:

DSE% = E/G × 100 (2)
where, E: presence of DSE structures; G: total number of intersections, using the method suggested by [START_REF] Mcgonigle | A new method which gives and objective measure of colonization of roots by vesicular arbuscular fungi[END_REF].

Biodiversity of AMF Species

Soil samples were collected during the grain filling stage in the last year of the study. Two soil samples were taken from the rhizosphere zone per plot for each treatment. A mixed homogenized sample was made for each plot to form three samples per treatment. AMF spores were extracted from 100 g of soil using the wet-sieving method (Gerdemann & Nicolson, 1963) followed by flotation-centrifugation in 50% sucrose (Dalpé, 1993). The spores were isolated from supernatant on a filter paper and were counted using a stereomicroscope at ×40 magnification to determine the total density of AMF (density of spores per 100 g of soil collected in each studied plot). Spores were thus classified by spore morphotype (size, color, hyphae) (Gerdemann & Trappe, 1974) and then mounted in polyvinyl alcohol-lacto-glycerol (PVLG) with or without Melzer's reagent on microscopic slides. Observations were carried out under an optical microscope (× 100 magnification). Identification, based on morphological characters, was performed using the INVAM website: http://invam.wvu.edu. Following identification, AMF species richness was evaluated based on the number of species present in each treatment.

The relative abundance of each species in each treatment was calculated following the method provided by [START_REF] Johnson | Dynamics of vesicular-arbuscular mycorrhizaeduring old field succession[END_REF]:

RA% = (n/N) × 100 (3)
Where, n = number of identified spores, N = total number of spores. This made it possible to determine the relative abundance of each spore family.

Carbon and Nitrogen Stocks

An analysis of organic matter and mineral nitrogen was performed on the soil samples after harvesting during the last year of the experiment. Two soil samples per plot and replicate were taken from a 10-cm depth for each treatment. The soil organic carbon was determined by means of the Anne method [START_REF] Aubert | Microbial biomass and activity at various soil depths in a Brazilian oxisol after two decades of no-tillage and conventional tillage[END_REF]. The quantification of total nitrogen was determined according to the KJELDAHL method [START_REF] Bremner | Total nitrogen[END_REF].

Grain Yield Evaluation

The grain yield was determined after harvesting for each plot area, during the five years of testing [START_REF] Grignac | Contraintes de l'environnement et élaboration du rendement dans la zone méditerranéenne française[END_REF].

Statistical Analysis

An analysis of variance with a Newman and Keuls test for the comparison of means was applied to the data (p < 0.05) with Statbox 2012 software. The Percentage data was transformed by a square root procedure.

Results

Effect of Farming System on Wheat Mycorrhizal Colonization

The results of the three years of observation showed that the total mycorrhizal rate was about 90% at the tillering stage and during grain filling. At the grain maturity stage, this rate decreased until reaching 26% in the last year. However, during this latter stage, no-till treatment showed a higher mycorrhizal root colonization compared to conventional seedling. This increase was about 22% during the second year of observation, and it reached 45% during the last year (Figure 4). Arbuscules were higher in plant roots sampled from the no-till farming plots than those from the conventional seeding plots during all the time of the trial. At the second year of observation, the arbuscules reached in no-till plots 65% and 45% at tillering and filling stages of the grain, respectively. However, in the conventional seedling system, it did not exceed 25%. While this increase continued during the last year of the experiment until reaching 80% at tillering stage, it remained stable in conventional seedling (Figure 4).

Contrary to this evolution, the vesicles were found to have increased in the conventional seedling plots, especially in the grain filling stage (Figure 4). Note. T: tillering, GF: grain filling, GM: Grain maturity. Means were obtained from 3 replicates. Significant differences between CS and DS are reported according to the Newman-Keuls test (P < 0.05).

Effect of Cultivation System on AMF Diversity

Spores density varied from 1280 spores to 2114 spores isolated from the soil of conventional and in direct seedling plots respectively. A total of 21 AMF species belonging to 7 families were identified. The AMF richness values of direct and conventional seedling soils were estimated respectively to 85.71 and 66.67% of the total estimated richness. Seven species were found exclusively in direct seedling soil. Among them five species of Acaulosporaceae (Acaulospora scrobiculata; Acaulospora koskei; Acaulospora thomii; Acaulospora capsicula; Acaulospora denticulata) and two species of Gigasporaceae (Gigaspora albida and Gigaspora margarita) with respective relative abundance (38 and 33%) for two families. Three species of Glomeraceae (G. mossae, G. aggregates and G. ambisporum) were occurred only in the conventional seedling soil.

Eleven species were present in both treatments but more abundant in plots conducted in the no-till system (Figure 5). The respective relative abundance in DS/CS was as follows: Gigasporaceae (13.4%/7.4%) with one species (Gigaspora decipiens); Diversisporaceae (12.8%/5.5%) with the species Diversispora tortuosa; Scutellosporaceae (24%/10.5%) with the species Scutellospora scutata and Scutellospora calospora and Entrophosporaceae (15.6%/3.7%) with Entrophospora infrequens. Dentiscutataceae were rarely present in the conventional seedling plots and are poorly present in the no-till plots (7.8%/0.2%) with Dentiscutata erythropa and Dentiscutata nigra. Glomeraceae had a dominant presence in the soil of both treatments, but their relative abundance is higher in conventional seedling (58.7%/76.6%) with (G. intraradices; G. fasciculatus; G. gerdemannii and G. multicaule) (Table 3 and Figure 5). Figure 5. Influence of conventional seedling (CS) and no-till farming system (DS) on the relative abundance (RA%) of Glomales families. Means were obtained from 3 replicates

Arbuscular Mycorrhizal Fungi and Dark Septate Endophytic Root Colonization

The comparative measurement of wheat root colonization by symbiotic structures specific to AMF and those of dark septate endophytic fungi (DSE) showed that mycorrhizal colonization remained more important in both treatments in comparison with DSE root colonization. The latter reached 40% in conventional seedling and 57% in the no-till farming plots, while AMF root colonization exceeded 90% in both treatments (Figure 6).

Figure 6. Influence of conventional seedling (CS) and no-till farming system (SD) on mycorrhizal and dark septated endophytic fungi root colonization, after five years of experimentation for the principal stages of plant growth Note. T: tillering, G F: grain filling, GM: Grain maturity. Means were obtained from 3 replicates. Different letters indicate significant differences between DS and CS according to the Newman-Keuls test (P < 0.05).

Effect of the Farming System on Soil Fungal Biomass

Quantification of microbial biomass by means of specific lipid biomarkers showed that the amounts of PLFA C16:1ω5, a specific fatty acid of AMF, increased by 52% in the no-till plots (DS) compared to the conventional seedling ones with respective values of (DS 1.07/CS 0.70) nanomoles g -1 of soil. Similarly, the amount of PLFA C18:2ω6.9 used to quantify saprotrophic and ectomycorrhizal fungal biomasses increased by 159% with respective values of (DS 0.96/CS 0.37) nanomoles g -1 of soil. C16:1ω5 PLFA amounts were higher than C18:2ω6.9 PLFA both in the no-till and conventional seedling plots (Figure 7). In contrast, the content of C16:1ω5 NLFA representing mainly the storage structures of the AMF (vesicles and spores) was almost the same for both treatments: 1.6 nanomoles/g of soil.

The NLFA/PLFA ratio of C16:1ω5 fatty acid was superior to 1 indicating that the C16:1ω5 fatty acid was originating from AMF and not bacteria. Figure 7. Amounts of phospholipid fatty acid (PLFA) (C16.1w5) specific to AMF and (C18:2 w 6,9) specific to saprotrophic/ectomycorrhizal fungi in root-free soil in no-till farming system (DS) and conventional seedling (CS), after three years of experiment. Means were obtained from 6 replicates. Different letters indicate significant differences between DS and CS according to the Newman-Keuls test (P < 0.05)

Organic Matter and Nitrogen Amounts in the Soil

The results showed that after five years of no-till farming, the soil organic matter and organic carbon contents increased by 29% compared to conventional seedling. However, this increase was statistically insignificant, and the soil organic matter (SOM) remained low for respectively the no-till and conventional seedling systems (3.71 and 2.62%) (Figure 8). The mineral nitrogen content remained similar for the two cultural systems (0.17%) (Figure 8). The C/N ratio was improved by 27%.

Figure 8. The effect of conventional seedling (CS) and no-till farming (DS), after five years of experimentation, on soil organic matter organic carbon and nitrogen amounts. Means were obtained from 3 replicates. (NS) No significant differences between DS and CS according Newman-Keuls test (P < 0.05) were observed

Grain Yields

In the first three years of the experiment, the wheat grain yield was higher in plots conducted in the direct seedling system compared to the conventional seedling system (Figure 9). The recorded grains are 1.6; 3.6 and 3 t/ha respectively for the three years of trial. However, in the last two years, cereal yields were similar for both systems.

Discussion

This long-term study of durum wheat rainfed crop in the east of Algeria, in no-till farming and in the conventional seedling system, has allowed us to determine the effect of these two farming systems on the establishment of a mycorrhizal symbiosis and the diversity of AMF in the field.

After three years of no-till practice, the amounts of PLFA biomarkers (C16:1ω5 for AMF) showed an increase in AM fungal biomass in the soil compared to those found in the conventional plots (Figure 7). This reflects a better growth and establishment of extra-radical AMF mycelium [START_REF] Olsson | Growth of arbuscular mycorrhizal mycelium in calcareous dune sand and its interaction with other soil microorganisms as estimated by measurement of specific fatty acids[END_REF]. [START_REF] Alguacil | The impact of tillage practices on arbuscular mycorrhizal fungal diversity in subtropical crops[END_REF] reported that the persistence of AM fungi in the soil depends on the survival of their active propagules (e.g. spores, hyphens, colonized roots). Our findings are in agreement with those of [START_REF] Roldan | Changes in soil sustainability indicators following conservation tillage practices under subtropical maize and bean crops[END_REF] who observed the highest levels of mycorrhizal propagules in the soil of maize and bean crops without tillage compared to tillage soils.

Under the same conditions, an improvement of saprotrophic and ectomycorrhizal fungi biomass was observed through the measurements of PLFA biomarkers: C18:2ω6.9. Consequently, one may say that the no-till system promotes the presence of a mulch of crop residues on the soil surface over a thickness varying from 5 to 30 cm, which may cause the increase in the biomass of the microbial community [START_REF] Aslam | Tillage impacts on soil microbial biomass C, N and P, earthworms and agronomy after two years of cropping following permanent pasture in New Zealand[END_REF][START_REF] Balota | Long-term tillage and crop rotation effects on microbial biomass and C and N mineralization in a Brazilian Oxisol[END_REF][START_REF] Aubert | Microbial biomass and activity at various soil depths in a Brazilian oxisol after two decades of no-tillage and conventional tillage[END_REF][START_REF] Kabiri | Tillage effects on soil microbial biomass, SOM mineralization and enzyme activity in a semi-arid Calcixerepts[END_REF]. On the other hand, tillage in conventional seedling causes a dilution of the organic matter and disturbs the fungal mycelium biomass. Our results are consistent with those reached by [START_REF] Bourgeois | Positive effect of the Miscanthus bioenergy crop on microbial diversity in wastewater-contaminated soil[END_REF] and [START_REF] Lienhard | Sayphoummie S et Ranjard L Pyrosequencing evidences the impact of cropping on soil bacterial and fungal diversity in Laos tropical grassland[END_REF], who claim that stopping tillage avoids physical disruption of mycelial hyphae and preserves microbial habitat.

Mycorrhizal and saprophytic fungi constitute the major component of the microbial community of unplowed soils or those with reduced soil tillage [START_REF] Fontaine | The priming effect of organic matter: A question of Microbial competition?[END_REF]. The most intensively managed soils have the highest relative abundance of bacteria and actinobacteria [START_REF] García-Orenes | Changes in Soil Microbial Community Structure Influenced by Agricultural Management Practices in a Mediterranean Agro-Ecosystem[END_REF].

Our results showed that AMF biomass was higher than saprotrophic and ectomycorrhizal biomasses in both no-till farming and conventional seedling plots. Similar findings have been reported by [START_REF] Olsson | Estimation of the biomass of arbuscular mycorrhizal fungi in a linseed field[END_REF] in cultivated soils. [START_REF] Van Groenigen | Abundance production and stabilization of microbial biomass under conventional and reduced tillage[END_REF] have shown, using both ergosterol and PLFA analyses, that while reduced tillage increased biomass of saprotrophic fungi in the 0-5 cm soil layer, it increased AMF biomass across the whole plough layer. The aridity of the study area (a rainfall close to 400 mm) may explain the dominance of the AMF biomass [START_REF] Rodriguez | The role of fungal symbioses in the Adaptation of plants to high stress environments[END_REF][START_REF] Duponnois | La symbiose mycorhizienne et la fertilité des sols dans les zones arides: un outil biologique sous-exploité dans la gestion des terres de la zone sahélo-saharienne[END_REF][START_REF] Nadji | Diversity of arbuscular mycorrhizal fungi of durum Wheat (Triticum durum Desf.) fields of the East of Algeria[END_REF]. Similarly, some characteristics of the study area soil such as the low total phosphorus level [START_REF] Nadji | Diversity of arbuscular mycorrhizal fungi of durum Wheat (Triticum durum Desf.) fields of the East of Algeria[END_REF] and the alkaline soil pH in relation with limestone mother rock [START_REF] Djebaili | Steppes algériennes: Phytosociologie et écologie[END_REF][START_REF] Halitim | Sols des régions arides[END_REF]) also contributes to the AMF installation. [START_REF] Coutinho | Variation of arbuscular mycorrhizal fungal communities along an altitudinal gradient in rupestrian grasslands in Brazil[END_REF] reported that low levels of phosphorus allow production by AMF of a large number of propagules in an environment with few nutrients. This highlights the adaptation of endogenous mycorrhizal fungi strains to the soil physicochemical characteristics [START_REF] Meglouli | Arbuscular mycorrhizal inoculum sources influence bacterial, archaeal, and fungal communities' structures of historically dioxin/furan-contaminated soil but not the pollutant dissipation rate[END_REF]. Moreover, the monoculture facilitates the AMF installation because the presence or absence of host plant is important in the occurrence of colonization and sporulation of AMF [START_REF] Cavalcante | Aspectos da simbiose micorrízica arbuscular[END_REF][START_REF] Coutinho | Variation of arbuscular mycorrhizal fungal communities along an altitudinal gradient in rupestrian grasslands in Brazil[END_REF]. The root colonization also demonstrated a predominance of the number of intra-radicular structures specific to AM compared to the dark septated endophytic fungi (Figure 6). This is in accordance with the work of [START_REF] Jansa | The effects of agricultural practices on arbuscular Mycorrhizal fungi[END_REF], who have observed a competitive effect of mycorrhizal fungi on non-mycorrhizal fungi and have claimed that this effect is promoted by reduced tillage. [START_REF] Olsson | Growth of arbuscular mycorrhizal mycelium in calcareous dune sand and its interaction with other soil microorganisms as estimated by measurement of specific fatty acids[END_REF] maintains that there is a negative impact of AMF on the growth of saprotrophic fungi in calcareous dune sand. However, Keenan et al. (2007) andLiu et al. (2011) showed that the presence of saprotrophic fungi in Gramineae crops reduced the density of AMF spores in the soil and mycorrhizal root colonization could be decrease by 50%.

The emergence of arbuscules in the root cortex of the no-till farming plants at the fourth year of this experiment is likely to reflect a good AMF activity, as put forward by [START_REF] Jansa | The effects of agricultural practices on arbuscular Mycorrhizal fungi[END_REF] and [START_REF] Curaqueo | Tillage Effect on Soil Organic Matter, Mycorrhizal Hyphae and Aggregates in a Mediterranean Agroecosystem[END_REF]. Their significant positive evolution from the early stage to the last year of the vegetative cycle demonstrates the efficiency of no-till farming in comparison with the conventional seedling system (Figure 4). In fact, arbuscules are the main symbiotic structures ensuring the exchanges between both symbiotic partners [START_REF] Brundrett | Working with Mycorrhizas[END_REF][START_REF] Balestrini | Plant-soil biota interactions[END_REF].

In our experiment, we observed a large production of vesicles in the cortical compartments of the plant roots grown under the conventional seedling system. The vesicles are carbon storage structures in particular lipids, obtained from the host plant [START_REF] Brundrett | Working with Mycorrhizas[END_REF][START_REF] Olsson | Estimation of the biomass of arbuscular mycorrhizal fungi in a linseed field[END_REF][START_REF] Trépanier | Dependence of arbuscular-mycorrhizal fungi on their plant host for palmitic acid synthesis[END_REF]. Vesicle formation is abundant at the end of the growth cycle of the host plant (Al-Karaki et al., 2004). It is a mean of conservation and propagation of fungi [START_REF] Staddon | The differential vitality of intraradical mycorrhizal structures and its implications[END_REF]. At this stage, AMF will be disconnected from the root of their host plant and deprived of their usual source of energy, thereby catabolising the lipids stored in the vesicles [START_REF] Olsson | Growth of arbuscular mycorrhizal mycelium in calcareous dune sand and its interaction with other soil microorganisms as estimated by measurement of specific fatty acids[END_REF]. Under the conditions of the conventional sowing system, the vesicles formation occurred at the grain filling stage reflected a negative partnership between the host plant (durum wheat) and the AMF.

AMF are obligate biotrophs and constitute a strong sink for plant carbon [START_REF] Gavito | Direct evidence for modulation of photosynthesis by an arbuscular mycorrhizainduced carbon sink strength[END_REF] they take advantage of 3% to 20% of the carbon substances produced by the host plant [START_REF] Strullu | Les mycorhizes des arbres et des plantes cultivées[END_REF]Smith & Read, 2008;Garbaye, 2013). These reserves stored in the vesicles will allow the AMF to develop extended extraradical hyphal networks for efficient soil exploration [START_REF] Keymer | Lipid transfer from plants to arbuscular mycorrhiza fungi[END_REF]. The growth of the mycelial networks requires a large amount of energy and carbon building blocks, which are transported mostly as lipid droplets and glycogen to the growing hyphal tips [START_REF] Bago | Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Bago | Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid[END_REF]. The destruction of the hyphal network by tillage explains the high presence of vesicles in the roots of plants growing under the conditions of conventional seeding.

AMF biodiversity studied in the last year of the trial showed the positive effect of no-till farming on the values of spore density and total species richness. Several studies reported the effect of soil tillage on the diversity and structure of AMF in temperate [START_REF] Jansa | Diversity and structure of AMF communities as affected by tillage in a temperate soil[END_REF][START_REF] Jansa | Soil tillage affects the community structure of mycorrhizal fungi in maize roots[END_REF], tropical [START_REF] Boddington | The effect of agricultural practices on the development of indigenous arbuscular mycorrhizal fungi. I. Field studies in an Indone-sian ultisol[END_REF] subtropical [START_REF] Alguacil | The impact of tillage practices on arbuscular mycorrhizal fungal diversity in subtropical crops[END_REF] and Mediterranean soils (Brito et al., 2012). Our findings are in agreement with these studies indicating that intensification of agriculture practices affects negatively AMF abundance and diversity.

The twenty-one species belonging to the seven families of Glomales recorded in the soil of the experimental plot reflect a high AMF diversity. [START_REF] Nadji | Diversity of arbuscular mycorrhizal fungi of durum Wheat (Triticum durum Desf.) fields of the East of Algeria[END_REF] reported the importance of diversity and abundance of mycorrhizal propagules in the eastern wheat fields of Algeria. However, a specific richness is observed in the no-tillering treatment soil, linked mainly to the presence of five species of Acaulosporaceae (Acaulospora scrobiculata; Acaulospora koskei; Acaulospora thomii; Acaulospora capsicula; Acaulospora denticulata) and two species of Gigasporaceae (Gigaspora albida and Gigaspora margarita) which completely disappeared in conventional seedling. Similarly [START_REF] Jansa | The effects of agricultural practices on arbuscular Mycorrhizal fungi[END_REF] have reported that, after thirteen years of non-tillage testing, there is the presence of some species of AMF, especially those not belonging to the genus Glomus such as Acaulospora paulinae. Soka and Ritchie (2014) have explained that the hyphae of Acaulospora laevis completely lose their infectivity when the soil is disturbed.

In the Grime's classification of functional groups [START_REF] Chagnon | A trait-based framework to understand life history of mycorrhizal fungi[END_REF], the Acaulosporaceae family corresponds to the stress-tolerant AMF which produce less extraradical hyphae and intraradical root structures as compared to Glomeraceae and Gigasporaceae [START_REF] Maherali | Influence of Phylogeny on Fungal Community Assembly and Ecosystem Functioning[END_REF][START_REF] Chagnon | A trait-based framework to understand life history of mycorrhizal fungi[END_REF] which may reduce the exposure to abiotic factors (i.e. drought, salinity, heavy metals, acidity) [START_REF] Chagnon | A trait-based framework to understand life history of mycorrhizal fungi[END_REF]. These AMF are capable to complete their life cycle producing low biomass, which disappears under plowing in the conventional seedling soil.

Seven species were present in both treatments but more abundant in plots conducted in the no-till system. This is the case of (Gigaspora decipiens; Diversispora tortuosa; Scutellospora scutata; Scutellospora calospora; Entrophospora infrequens; Dentiscutata erythropa and Dentiscutata nigra). This finding is in line with the study of [START_REF] Jansa | The effects of agricultural practices on arbuscular Mycorrhizal fungi[END_REF] which reported that Scutellospora pellucida and Entrophospora infrequens were most abundant no tilled soils. The work of [START_REF] Alguacil | The impact of tillage practices on arbuscular mycorrhizal fungal diversity in subtropical crops[END_REF] showed an interaction between the tillage system and the host plant showing very significant differences between AM fungal populations for different managed tillage systems. They concluded that no-tillage system had in fact a higher number of fungal types colonizing bean and sorghum crops than the other management.

Spore diversity may also be related to root density which increases favorably under no-till conditions [START_REF] Cardoso | Distribution of Mycorrhizal fungal spores in soils under agroforestry and monoculturalcoffee systems in Brazil[END_REF]. Similarly, the more abundant weeds in direct seedling [START_REF] Karkour | Tillage Effects on Selected Soil Physical Properties in a Maize-Bean Intercropping System in Mwala District, Keny[END_REF] can be an intermediate host between AMF and the main crop [START_REF] Kabir | Tillage or no-tillage: Impact on mycorrhizae[END_REF].

The Glomeraceae are predominant in both treatments, but their relative abundance was higher in conventional seedling. Three species of Glomeraceae (G. mossae, G. aggregates and G. ambisporum) were occurred only in the conventional seedling soil. The predominance of spores belonging to the family of Glomeraccae has also been recorded in some countries of Africa, particularly in the semi-arid zones of Senegal [START_REF] Diallo | Arbuscular mycorrhizal fungi in the semi-arid areas of Senegal[END_REF] and Burkina Faso (Guisson, 2001). [START_REF] Schalamuk | Fungal spore diversity of arbuscular Mycorrhizal fungi associated with spring wheat: Effects of tillage[END_REF] have noted that Glomeraceae were more abundant in the conventional system, compared to the no-till farming system. The ubiquity of Glomeraceae family in disturbed environments is related to their ability to grow fast (de Souza et al., 2005), to sporulate rapidly and to restore a network of hyphae by quickly forming anastomoses between the different mycelial branches of the same or neighboring genotype [START_REF] Giovannetti | Anastomosis formation and nuclear and protoplasmic exchange in arbuscular mycorrhizal fungi[END_REF][START_REF] Oehl | Impact of land use intensity on the species diversity of arbuscular mycorrhizal fungi in agro-ecosystems of central Europe[END_REF]de la Providencia et al., 2005;[START_REF] Voets | Glomeraceae and Gigasporacea differ in their ability to form hyphal networks[END_REF]. They are therefore considered ruderales AMF (Van der Heijden & Scheublin, 2007).

Most Glomeraceae have been classified on the basis of their life-history strategies (LHS) as r-strategist because they invest mainly in the production of many offspring, favoring their development in unstable environments [START_REF] Ijdo | Do arbuscular mycorrhizal fungi with contrasting life-history strategies differ in their responses to repeated defoliation[END_REF]. In this sense (Johnson et al., 1992;[START_REF] Wang | Diversity and infectivity of arbuscular mycorrhizal fungi in agricultural soils of the Sichuan Province of mainland China[END_REF] reported in the tillage soil system, the plowed horizon is mainly occupied by AMF species less useful for crops and investing a large part of their resources in the production of spores.

In addition, Glomeraceae colonize via fragments of mycelium or mycorrhizal root pieces, and therefore have the ability to high root colonization and to develop larger extraradical hyphae (Declerck et al., 2001). In contrast, Gigasporaceae invest more energy in the production of extraradical biomass than in intraradical fungal structures (Hart et al., 2002;[START_REF] Maherali | Influence of Phylogeny on Fungal Community Assembly and Ecosystem Functioning[END_REF], they produce few and large spores (Johnson et al., 1992, de Souza et al., 2005;[START_REF] Wang | Diversity and infectivity of arbuscular mycorrhizal fungi in agricultural soils of the Sichuan Province of mainland China[END_REF] and propagate via sporal dispersal or infection from an intact mycelium [START_REF] Biermann | Use of vesicular-arbuscular mycorrhizal roots, intraradical vesicles and extraradical vesicles as inoculum[END_REF][START_REF] Daniell | Molecular diversity of arbuscular mycorrhizal fungi colonizing arable crops[END_REF]. Such differences clearly explain the dominance of Glomeraceae over Gigasporaceae in environments characterized by repeated severe physical disturbances, such as tillage agricultural practices and clearly explain the loss of species richness of Gigasporaceae by the deep burial of their propagules following the plowing [START_REF] Oehl | Impact of long-term conventional and organic farming on the diversity of arbuscular mycorrhizal fungi[END_REF]) [START_REF] Jasper | The effect of soil disturbance on vesicular Arbuscular mycorrhizal fungi in soil from different vegetation types[END_REF][START_REF] Hart | Do arbuscular mycorrhizal fungi recover from soil disturbance differently?[END_REF].

The presence of plant residues (mulch) contributes in the increase of the organic soil matter. This hypothesis is in line with [START_REF] Van Groenigen | Abundance production and stabilization of microbial biomass under conventional and reduced tillage[END_REF] study which has reported that, after twenty years of no-till operations, the soil organic carbon increases by 16% in humid temperate regions and by 10% in dry temperate regions, compared to conventional seedling. In our study, the soil organic matter and organic carbon contents showed a tendency increase of 29% in no-till plots (Figure 8). Contents of soil organic matter (SOM) and total nitrogen remained low for respectively the no-till and conventional seedling systems respectively (Figure 6), which reflects soil poor fertility.

This situation could be explained by the low amounts of cereal crop residues to return to the soil. In North Africa, often cereal residues are used as a pasture source for sheep farming [START_REF] Lahmar | Dégradation des sols et stratégies coopérative en Méditerranée[END_REF]. The monoculture (wheat/wheat), applied in our experiment, implies a slow-down of organic matter mineralization in comparison to crop rotation (cereal/legume).

An increase in yields observed in our experiment during the first three years of the no-tillage farming system (Figure 9), is consistent with the results reached by Angar et al. (2010), who have reported that durum wheat yields, during ten years of no tillage in a sub-humid zone of Tunisia, increased by 0.8 t/ha, compared to conventional seedling. In a semi-arid zone, this difference was lower (0.35 t/ha), and it reached 0.7 t/ha in the upper semi-arid areas.

Consequently, one may say that late sowing (12/01/2012 and 09/01/2013) (Table 3) during the last two years of experimentation does not allow a good early planting of the crop and limits the H 2 O intake of the rainfall during the autumn season. These effects are to be added to the low temperatures of January and February that occur just after sowing, which prevents an adequate growth of plants by affecting their physiological development of meristems (Chassot et al., 2002). [START_REF] Chapman | Influence of temperature and vegetation cover on soluble inorganic and organic nitrogen in a Spodosol[END_REF] have pointed the negative effect of low temperatures on the rate of mineralization of nitrogen in the soil. These factors together seem to be at the origin of the suppression of the positive effect of the no-till farming system during these two agricultural campaigns.

Conclusion

The results of our study demonstrated that the no-till farming system has a positive impact on the AMF symbiosis, as demonstrated by the increase of AMF biomass in the soil and the arbuscular root colonization percentages. Besides, in conventional seedling, root colonization was characterized by a strong presence of vesicles. The biodiversity of AMF was improved by the relative abundance of six families of Glomales and the emergence of new AMF species. These results are in favor of the adoption of the no-till system as an approach to restore the microbiome in agro-systems disturbed by tillage.

The aridity of our study area, the monoculture and the presence of a high level of soil limestone facilitated the predominance of the AMF root colonization over the other endophytic fungi.

For the sake of productivity and sustainability, it is desirable that a similar study be carried out using co-culture of wheat/legume rotation to improve nitrogen and soil organic matter levels. An economic study is also needed to assess the impact of no tillage technique on the cost of production and profitability of the crop.

Figure 1 .

 1 Figure 1. Evolution of precipitation and temperature during the five years of field experimental trial conducted at the agronomic experimental station of the Technical Institute of Field Crops (ITGC) of Setif (ONM National Office of the meteorology).

  D J F MAM J S O N D J F MAM J S O N D J F MAM J S O N D J F MAM J S O N D J F

Figure 3 .

 3 Figure 3. The experimental design applied during the five years of experimentation Note. Test with six plots with 180 m 2 (6 m × 30 m) arranged as a complete random block (CRB).

Figure 4 .

 4 Figure 4. Effect of conventional seedling (CS) and no-till farming system (DS) on total mycorrhizal root colonization (a), abuscules (b) and vesicles(c) percentage during the last three-years of the experiment for the three plant growth stages

  grain yield during the five years trials in no-till farming system and in conventional one. Means were obtained from 3 replicates. Different letters indicate significant differences between DS and CS according to the Newman-Keuls test (P < 0.05)

Table 1 .

 1 Physicals and chemicals characteristics of the soil in the experimental field

	Physical characteristics %	Chemical characteristics			Organic characteristics
	Silt	Clay	Sand	CE (mmho/cm)	pH	Total CaCo 3 (%)	Actif CaCo 3 (%)	Available P 2 O 5 (ppm)	OM (%)	C org (mg g -1 )
	34.66	34.66	30.66	0.175	8.26 51.58	21.25	7.33	2.6	1.51

Note. Available phosphorus (Olsen Phosphorus) was determined by the

Olsen et al. (1954)

method.

Table 2

 2

	. C
	Years of stu
	A1 (2008/0
	A2 (2009/1
	A3 (2010/1
	A4 (2011/1
	A5 (2012/1
	Note. DS:
	*The high
	post-emerg
	OD (anti d
	* The conv
	* The cha
	rubens) wh
	For the tw
	following

Table 3 .

 3 AMF spore species isolated from the soil of the experimental field plots, for each treatment (DS: no-till farming system; CS: conventional seedling) in the last year of the trial

	Family/Species	DS	CS
	Glomeraceae
	G. intraradices	x	x
	G. fasciculatus	x	x
	G. gerdemannii	x	x
	G. multicaule	x	x
	G. mossae		x
	G. aggregates		x
	G. ambisporum		x
	Acaulosporaceae
	A. koskei	x
	A. thomii	x
	A. capsicula	x
	A. denticulata	x
	A. scrobiculata	x
	Gigasporacae
	Gg. Albida	x
	Gg. margarita	x
	Gg. decipiens	x	x
	Dentiscutataceae
	D. erythropa	x	x
	D. nigra	x	x
	Diversisporaceae
	D. tortuosa	x	x
	Scutellosporaceae
	S. scutata	x	x
	S. calospora	x	x
	Entrophosporaceae
	E. infrequens	x	x

Acknowledgements

This work is part of the Algerian-Australian Research and Cooperation Project (CANA) (Conservation Agriculture North Africa). The experimental trials were conducted on rainfed cereals in semi-arid areas, notably at the Experimental Station of the Technical Institute of Field Crops (ITGC) of Sétif, located in eastern Algeria. Our gratitude goes to the technical team of ITGC Sétif for the installation and conduct of field trials. We are grateful to Mrs Y. Dalpé and Mrs K. Bencherif for their valuable contribution to the identification of mycorrhizal spores. We also wish to thank Mrs N. Deguiche (ITGC) and Mr R. Bradai (INRA) for having carried out the analysis of organic matter and mineral nitrogen of the soil.

This research was financed by the Ministry of Higher Education and Scientific Research of Algeria and the Ministry of Agriculture.

Journal of Agricultural Science

Vol. 12, No. 4; 2020

Copyrights

Copyright for this article is retained by the author(s), with first publication rights granted to the journal.

This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).


Abdeslem Bouzina
email: abdeslem.bouzina@univ-annaba.dz

Abdelhak Djemel

Omar Sekiou

Imededdine Kadi

Yousra Ouafa Bouone

Rachida Mansouri

Zineb Aouf

Malika Ibrahim-Ouali

Nour Eddine Aouf

Yousra Ouafa Bouone

Synthesis, in silico study, and biological evaluation of cyclosulfamide derivatives as new anticholinesterase inhibitors

Keywords: CNS: Central Nervous System Cyclosulfamides, Anticholinesterase inhibition, Molecular docking study, MD simulation

à la diffusion 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

Neurodegenerative diseases such as Alzheimer, Parkinson and Epilepsy are chronic disorders affecting the nervous system progressively. Alzheimer disease (AD) is one of the greatest challenges in medical practice inducing significant socioeconomic impacts. It is frequently reported in old people and the number of affected persons actually might triplicate by 2050 (1 for 85) [START_REF] Lolak | Sulphonamides incorporating 1,3,5-triazine structural motifs show antioxidant, acetylcholinesterase, butyrylcholinesterase, and tyrosinase inhibitory profile[END_REF]. Patients suffering from AD showed several symptoms, particularly memory decreasing and cognitive disorders [START_REF] Akıncıoğlu | Discovery of potent carbonic anhydrase and acetylcholine esterase inhibitors: Novel sulfamoylcarbamates and sulfamides derived from acetophenones[END_REF]. These neurologic dysfunctions are a result of cholinergic deficits in different brain parts, due to the alteration of cholinergic neurons [START_REF] Uddin | Anticholinesterase activity of eight medicinal plant species: in vitro and in silico studies in the search for therapeutic agents against Alzheimer's disease[END_REF]. AChE and BuChE are mainly enzymes that modulate the acetylcholine neurotransmitter levels in different neurologic conditions [START_REF] Taslimi | Benzenesulfonamide derivatives as potent acetylcholinesterase, α-glycosidase, and glutathione S-transferase inhibitors: biological evaluation and molecular docking studies[END_REF]. Consequently, the inhibition of dual AChE and BuChE is considered as a potent strategy to attenuate symptoms and the worsening of AD, and many other neuromuscular disruptions [START_REF] Lolak | Sulphonamides incorporating 1,3,5-triazine structural motifs show antioxidant, acetylcholinesterase, butyrylcholinesterase, and tyrosinase inhibitory profile[END_REF][START_REF] Göçer | Carbonic anhydrase and acetylcholinesterase inhibitory effects of carbamates and sulfamoylcarbamates[END_REF]. Until the day; few drugs approved by FDA were used in the clinical treatment of AD: aducanumab, galantamine, rivastigmine, donepezil, and tacrine which has been withdrawn from the market. However, their hepatotoxicity and gastrointestinal disorders were reported [START_REF] Akıncıoğlu | Novel sulphamides and sulphonamides incorporating the tetralin scaffold as carbonic anhydrase and acetylcholine esterase inhibitors[END_REF]. Therefore, the development of a new drug or molecule candidate for the treatment of AD has become a necessity.

Heterocyclic scaffolds present a considerable importance in medicinal chemistry constituting the building block of a remarkable number of biologically active entities including many commercialized drugs [START_REF] Heravi | Prescribed drugs containing nitrogen heterocycles: an overview[END_REF]. Cyclosulfamides are a specific class of heterocycles that incorporate the well-known pharmacophore (N-SO2-N). The capacity of such compounds to exhibit beneficial pharmacological activities was widely reported in the literature, indeed sulfamidebased heterocyclic compounds showed antibacterial [8], antiviral [START_REF] Dou | Potent Inhibition of Norwalk Virus by Cyclic Sulfamide Derivatives[END_REF], and anticancer activities [START_REF] Jun | Investigation on the anticancer activity of symmetric and unsymmetric cyclic sulfamides[END_REF]. Cyclosulfamides were also investigated for their inhibitory effects against a great number of enzymes [START_REF] Winum | Therapeutic potential of sulfamides as enzyme inhibitors[END_REF]. For example, compound 1 showed inhibition against 11β-hydroxysteroid dehydrogenase 1 inhibitor (11β-HSD1) for the potential treatment of ischemic brain injury [START_REF] Lee | Optimization of cyclic sulfamide derivatives as 11β-hydroxysteroid dehydrogenase 1 inhibitors for the potential treatment of ischemic brain injury[END_REF].

Compounds 2 and 3 were evaluated as inhibitors of aspartic proteases of types HIV-1 protease and γ-secretase respectively [START_REF] Ax | Cyclic sulfamide HIV-1 protease inhibitors, with sidechains spanning from P2/P2' to P1/P1[END_REF][START_REF] Sparey | Cyclic sulfamide gamma-secretase inhibitors[END_REF]; the inhibition of γ-secretase is known to be efficient in preventing the progression of AD [START_REF] Mekala | Recent developments of small molecule γ-secretase modulators for Alzheimer's disease[END_REF]. Inhibitory activity against serine protease of type human leukocyte elastase (HLE) was found in analogues of compound 4 [START_REF] Winum | Therapeutic potential of sulfamides as enzyme inhibitors[END_REF], this activity can result in an anti-inflammatory effect [START_REF] Marinaccio | Peptide Human Neutrophil Elastase Inhibitors from Natural Sources: An Overview[END_REF]. Many other cyclosufamide-based molecules were described as inhibitors of Protein Tyrosine Phosphatase (PTP) [START_REF] Black | Structure-based design of protein tyrosine phosphatase-1B inhibitors[END_REF], HIV-1 non-nucleoside reverse transcriptase [START_REF] Liu | Design, synthesis, and biological evaluation of novel 4-aminopiperidinyllinked 3,5-disubstituted-1,2,6-thiadiazine-1,1-dione derivatives as HIV-1 NNRTIs[END_REF], cholesteryl ester transfer protein (CETP) [START_REF] Liu | Cholesteryl ester transfer protein (CETP) inhibitors based on cyclic urea, bicyclic urea and bicyclic sulfamide cores[END_REF], and noroviruses [START_REF] Dou | Cyclosulfamide-based derivatives as inhibitors of noroviruses[END_REF]. The main focus of this study is to evaluate derivatives of cyclosulfamide for the first time as AChE and BuChE inhibitors, in order to reduce the neurologic disorders and improve the neurotransmission state in AD patients. Additionally, in-silico investigations against AChE were also performed to verify the anti-alzheimer activities.

The design strategy of this study is described in Figure 2.

Material and methods

Chemistry

Chemical methods

All chemicals and solvents were purchased from common commercial sources (Sigma Aldrich) and were used as received without any further purification. All reactions were monitored by TLC on silica Merck 60 F254 percolated aluminum plates and were developed by spraying with ninhydrin solution. Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded on a Brücker spectrometer at 400 MHz. Chemical shifts are reported in δ units (ppm) with TMS as reference (δ 0.00). All coupling constants (J) are reported in Hertz. Multiplicity is indicated by one or more of the following: s (singlet), brs (board singlet), d (doublet), dd (doublet of doublet), t (triplet), td (triplet of doublet), q (quartet), m (multiplet). Carbon nuclear magnetic resonance ( 13 C NMR) spectra were recorded on a Brücker at 100 MHz. Chemical shifts are reported in δ units (ppm) relative to DMSO-d6 (δ 39.52). Infrared spectra were recorded on a Perkin Elmer 600 spectrometer. The purity of the final compounds (greater than 95%) was determined by uHPLC/MS on an Agilent 1290 system using a Agilent 1290 Infinity ZORBAX Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 μm particle size) with a gradient mobile phase of H2O/CH3CN (90:10, v/v) with 0.1% of formic acid to H2O/CH3CN (10:90, v/v) with 0.1% of formic acid at a flow rate of 0.5 mL/min, with UV monitoring at the wavelength of 254 nm with a run time of 10 min. Microanalysis spectra were performed by Elementar Analyzer (Euro E.A. 3000-V3.0-single-2007) and the determined values were within the acceptable limits of the calculated values. Melting points were recorded on a Büchi B-545 apparatus in open capillary tubes. Microwave assisted reactions were carried out using a Biotage Initiator Microwave Synthesizer 2.0 with a nominal power of 400 W. The reactions were carried out in a reactor to M.W (volume: 10 mL) under pression at room temperature.

Typical experimental procedure for the synthesis of benzothiadiazinone dioxide

In a reactor MW (volume: 10 mL) taken a mixture of aldehyde (1 mmol), sulfamide (1 mmol), and cyclohexane-1,3-dione (1 mmol) in the presence of H2SO4 (0.1 mmol)/CH3COOH (0.9 mmol) as catalyst under solvent-free at room temperature. The reaction mixture was subjected to microwave irradiation for appropriate time. After completion of the reaction (5-10 min), as indicated by TLC, silica gel; dichloromethane:methanol (9:1), mixture of ethanol and acetone (9:1) or mixture of ethyl acetate and n-hexane (5/5) was added to the reaction mixture and pure product was crystallized to 6 °C overnight. The products were finally filtered and dried.

4-phenyl-4,6,7,8-tetrahydro-1H-benzo [c] [START_REF] Lolak | Sulphonamides incorporating 1,3,5-triazine structural motifs show antioxidant, acetylcholinesterase, butyrylcholinesterase, and tyrosinase inhibitory profile[END_REF][START_REF] Akıncıoğlu | Discovery of potent carbonic anhydrase and acetylcholine esterase inhibitors: Novel sulfamoylcarbamates and sulfamides derived from acetophenones[END_REF][START_REF] Akıncıoğlu | Novel sulphamides and sulphonamides incorporating the tetralin scaffold as carbonic anhydrase and acetylcholine esterase inhibitors[END_REF]

4-(4-chlorophenyl)-4,6,7,8-tetrahydro-1H-benzo[c][1,2,6]thiadiazin-5(3H)-one 2,2-dioxide (6a, C13H13ClN2O3S):

White powder; 92 % yield; Rf = 0.20 (CH2Cl2/CH3OH:85/15) ; 1

4-(4-bromophenyl)-4,6,7,8-tetrahydro-1H-benzo[c][1,2,6]thiadiazin-5(3H)-one 2,2-dioxide (8a, C13H13BrN2O3S):

Yellow powder; 90 % yield; Rf = 0.20 (CH2Cl2/CH3OH:85/15) ; 1 H NMR (400 MHz,

DMSOd6): δ = 1.92-2.15 (m, 2H, CH2), 2.27-2.30 (m, 1H, CH2), 2.37-2.43 (m, 1H, CH2), 2.

4-(2-hydroxyphenyl)-4,6,7,8-tetrahydro-1H-benzo[c][1,2,6]thiadiazin -5(3H)-one 2,2-dioxide (9a, C13H14N2O4S):

White powder; 91 % yield; Rf = 0.20 (CH2Cl2/CH3OH:85/15); 1

AChE and BuChE inhibitory assay

The inhibitory activity of AChE and BuChE was determined using an in vitro microplate assay developed by Ellman et al [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF]. Each molecule was dissolved in MeOH (4mM) and diluted to various concentrations before use. Briefly, 150 µL of sodium phosphate buffer (0.1M, pH 8.0)

were mixed with 10 µL of each sample and 20 µL of AChE (5.32x10 -3 U) or BuChE (6.85x10 - 3 U). The mixture was incubated at 25°C for 15 min. Then, 10 µL of DTNB (0.5 mM) and 10 µL of acetylthiocholine iodide (ACI, 0.71 mM) or butyrylthiocholine chloride (BCI, 0.2mM) were added. The absorbance was measured in 412 nm against a negative control using a microplate reader (PerkinElmer, EnSpire Multimode Plate Reader, USA) and the inhibition rate was determined for each concentration. Galantamine was used as a reference drug and all tests were evaluated in triplicate.

Statistical analysis

The results were expressed as mean±SD of three parallel measurements. The analysis and drawing were realized using GraphPad prism (software, San Diego, CA, USA). Comparison between IC50 values were analyzed by ANOVA one way followed by Tuckey post-hoc test.

The difference was considered statistically significant when the P value was less than 0.05

2.3.

In silico study

Molecular Docking

The X-ray crystal structure of AChE in complex with galantamine (Protein Data Bank ID:

4EY6) [START_REF] Cheung | Structures of human acetylcholinesterase in complex with pharmacologically important ligands[END_REF] were obtained from the RSC Protein Data Bank, and were prepared with protein preparation wizard in Schrodinger suites. The three-dimensional structures of the derivatives were constructed using Maestro software, and prepared with Ligprep using OPLS3e force field.

[23]

The final prepared PDB file of the protein and synthesized compounds were submitted in order to run docking process. Docking studies were performed by Glide software [START_REF] Friesner | Glide: A new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy[END_REF] at extra precision [START_REF] Friesner | Extra precision glide: Docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes[END_REF]. Output files of docked compounds were visualized on Chimera software [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF].

Molecular Dynamics

Molecular Dynamics simulations (MD) were performed using Desmond [27]. The best docking complex for the identified inhibitors from the biological experiments were taken as the initial coordinates for MD simulations. A 10 Å cubic water box employing the TIP3P water model was used for the solvation of the system with OPLS3e force field. Sodium ions were added as counter ions to neutralize the systems. We have then subjected the system to minimization using an energy gradient convergence threshold of 1 kcal/mol/Å and pre-equilibration using the default six-step relaxation protocol implemented in Desmond. The first two steps are minimization steps, one in which solute is kept restrained while another without restraints.

Further, steps three to six are short MD simulations of 12 ps, 12 ps, and 24 ps each using the NPT ensemble at 10, 10, 300, and 300 K, respectively. Subsequently, a 100 nanosecond (ns) MD simulation production run was performed. Remaining parameters were kept at the Desmond default values. We have visualized the protein-ligand complexes and analyzed the MD trajectory using Maestro. The detailed analyses were performed using the Simulation Event Analysis tool of Desmond.

Results and discussion

Chemistry

In order to further our research on the development and synthesis of bioactive compounds [28] using modern and efficient synthesis techniques such as ultrasound and microwave irradiation

[29], we have expanded our investigations by synthesizing heterocyclic compounds containing the sulfamide moiety.

The synthesis of these heterocyclic compounds (also called cyclosulfamides) was carried out in a single step via the Biginelli cyclo-condensation reaction.

Synthesized derivatives of cyclosulfamide are obtained from a reaction between various aromatic aldehydes, cyclohexanedione, and sulfamide in the presence of a catalytic amount of H2SO4/CH3COOH (1/9) under microwave irradiation and solvent-free condition at room temperature [START_REF] Bouzina | Microwave-accelerated multicomponent synthesis and X-ray characterization of novel benzothiadiazinone dioxide derivatives, analogues of Monastrol[END_REF].

The target compounds (1a-12a) reacted very well, giving excellent yields (82-92%) in short reaction times (5-10min) (Scheme 1).

Scheme 1. Synthesis of cyclosulfamide derivatives via Biginelli reaction under microwave irradiation.

The structures of the synthesized cyclosulfamides (1a-12a) are presented in Table 1.

In vitro Anticholinesterase activity

In parallel to cognitive and behavioral disorders, several biochemical events were observed in AD patients, decreasing in ACh levels, aggregation of β-amyloid peptide (βA), increasing in intracellular phosphorylation, dys-regulation of metallic ions and mitochondrial dysfunctions. [START_REF] Göçer | Carbonic anhydrase and acetylcholinesterase inhibitory effects of carbamates and sulfamoylcarbamates[END_REF][START_REF] Skrzypek | Synthesis and anticholinesterase activities of novel 1,3,4-thiadiazole based compounds[END_REF]. AChE (EC.3.1.1.7) is an important enzyme in the modulation of cholinergic transmission through synaptic Acetylcholine hydrolysis. BuChE (EC.3.1.1.8) participated in the stability of nerve signal by compensation of AChE activity [START_REF] Zhou | Simple analogues of natural product chelerythrine: Discovery of a novel anticholinesterase 2-phenylisoquinolin-2-ium scaffold with excellent potency against acetylcholinesterase[END_REF]. Actually, no specific treatment for this multifactorial disease, but the cholinergic hypothesis remained the most powerful theory in the development of novel anti-Alzheimer's drugs, through inhibition of both AChE and BuChE [START_REF] Omidpanah | Phytochemical investigation and anticholinesterase activity of ethyl acetate fraction of Myristica fragrans Houtt. seeds[END_REF]. Moreover, the presence of sulfamide unit in heterocyclic structures offered potent alternatives with high therapeutic efficiency in medical practice. Many reports revealed the antibacterial, antidiabetic, and antitrypanosomal activities of sulfamides [START_REF] Akıncıoğlu | Discovery of potent carbonic anhydrase and acetylcholine esterase inhibitors: Novel sulfamoylcarbamates and sulfamides derived from acetophenones[END_REF]. This study is focused on cyclosulfamide derivatives, in the first hand, due to their ability to affect the central neurons system and generating several effects such as anticonvulsion, treatment of depression and reduction of inflammatory and neurologic pain, consequently, the ability to improve some neuronal dysfunctions in AD patients [START_REF] Lolak | Synthesis, characterization, inhibition effects, and molecular docking studies as acetylcholinesterase, α-glycosidase, and carbonic anhydrase inhibitors of novel benzenesulfonamides incorporating 1,3,5-triazine structural motifs[END_REF][START_REF] Taslimi | Benzenesulfonamide derivatives as potent acetylcholinesterase, α-glycosidase, and glutathione S-transferase inhibitors: biological evaluation and molecular docking studies[END_REF]. In another hand, the enzymatic inhibitory activity of cyclosulfamides is widely reported especially against HIV-1 proteases, tyrosine kinase, human elastase and particularly serine proteases such as AChE and BuChE [START_REF] Yang | Inhibition of serine proteases by a new class of cyclosulfamide-based carbamylating agents[END_REF][START_REF] Lai | Potent inhibition of human leukocyte elastase by 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamide derivatives[END_REF]. In this context, the anticholinesterase activity of novel synthetic cyclosulfamides were evaluated.

The inhibitory activity of studied cyclosulfamide-based derivatives was determined as IC50 values (µM) and mentioned in Table 2. The structural diversity of these compounds radically influenced the activity against AChE and BuChE. For AChE inhibition, the IC50 ranged from 45.30±0.88 to >200 µM. The basic compound 1a showed a moderate activity (IC50=125.08±2.69 µM). The addition, in different positions, of halogen atoms such as fluorine in 2a, 3a, 4a and chloride in 5a and 6a reduced the activity and made them inactive. Moreover, the addition of bromine atoms in 7a slightly improved the activity (IC50=119.92±1.61µM) while, in 8a the activity was reduced. An interesting activity was recorded for compounds 10a (IC50=45.30±0.88µM) and 12a (IC50=116.34±1.37µM) which showed the greatest activity of the series. Other compounds 9a and 11a were considered inactive. Galantamine exhibited the best inhibitory effect.

In BuChE inhibitory activity, the behavior of molecules changed and the efficiency clearly appeared. The IC50 values ranged from 52.87±3.73 to >200 µM and the basic molecule 1a

showed a moderate activity (IC50=137.87±0.64µM). Compounds 2a, 4a, 6a, 9a, 11a which were inactive in AChE assay exhibited a potent BuChE inhibition. The most potent one was the fluorine substituted 4a (IC50=52.87±3.73 µM). In another hand, 7a and 12a molecules showed an opposite effect, the great efficiency against AChE has become inactive to BuChE.

Compounds 3a and 8a remained inactive against dual AChE and BuChE. In addition, the selectivity indexes of studied compounds against both enzymes showed a more important selectivity for BuChE (2a, 4a, 5a, 6a, 7a, 9a, 11a) than AChE (1a, 10a, 12a). Compounds 3a and 8a revealed inactive non-selective (Table 2). IC50 values represent the means ± SD. of three parallel measurements (p<0.05).

Galantamine: Reference drug.

The blockage of AChE improved the level of ACh in different brain parts and attenuated temporarily the cognitive disorders [START_REF] Lolak | Synthesis, characterization, inhibition effects, and molecular docking studies as acetylcholinesterase, α-glycosidase, and carbonic anhydrase inhibitors of novel benzenesulfonamides incorporating 1,3,5-triazine structural motifs[END_REF]. Molecules that inhibited the anionic catalytic site (ACS) and the peripheral anionic site (PAS) revealed as potent therapeutic drugs [START_REF] Mehrabi | Design, synthesis, molecular modeling and anticholinesterase activity of benzylidene-benzofuran-3-ones containing cyclic amine side chain[END_REF]. Several studies reported the involvement of BuChE inhibitors in the treatment of AD especially in the late stage. Then, it modulates the AChE levels in different states. Therefore, the best management of AD symptoms requires the dual inhibition of both enzymes [START_REF] Boualia | Synthesis, molecular docking studies, and biological evaluation of novel alkyl bis(4amino-5-cyanopyrimidine) derivatives[END_REF].

Many reports discussed the correlation between the inhibitory effects against cholinesterase enzymes with aromatic ring of inhibitors and the presence of aliphatic chains in active compounds [START_REF] Akıncıoğlu | Novel sulphamides and sulphonamides incorporating the tetralin scaffold as carbonic anhydrase and acetylcholine esterase inhibitors[END_REF]. Thus, the integration of halogen atoms in the aromatic ring, in different positions, improved the therapeutic effect by utilizing of tensile and steric effects of the halogen bonds to occupy the binding sites of molecular targets, as well as, the potent sensitivity of C-X (Cl, Br, F, I) to steric factors [START_REF] Burmaoglu | Synthesis of novel tris-chalcones and determination of their inhibition profiles against some metabolic enzymes[END_REF]. Additionally, due to their lipophilicity and permeability properties, the addition of halogens improved the pharmacokinetic and pharmacodynamics properties of drugs, especially those affecting the central nerves system (CNS) for AD treatment [START_REF] Burmaoglu | Synthesis of novel tris-chalcones and determination of their inhibition profiles against some metabolic enzymes[END_REF].

In silico Study

Molecular docking

To investigate and explain the binding mode and interactions between the targeted protein and synthesized ligands, a molecular docking study was carried out.

We used the Schrodinger suite (version 11.8) and UCSF Chimera (version 1.13.1) programs for our study, and Galantamine was taken as a reference ligand.

The precision of the docking protocol was evaluated by re-docking of Galantamine in the active site of AChE enzyme. The results of this study including the estimated glide score of the docked positions are provided in Figure 4 and Table 3.

According to molecular docking studies, all synthesized ligands showed significant stability in the active site due to the formation of important hydrogen bonds, hydrophobic interactions, and electrostatic attraction forces.

Compounds 10a, 11a and 5a gave a better glide score when compared with Galantamine for the target protein, with binding score of -7.82kcal/mol, -7.86kcal/mol, and -7.89 kcal/mol respectively, and the other compounds showed a docking score in the range of -6.89kcal/mol and -7.60kcal/mol which is less than the reference ligand docking score. Molecular docking analysis showed that most of studied compounds interact with the active site of AChE in a very efficient and satisfactory manner, emphasizing the importance of the presence of the cyclosulfamide moiety as well as aromatic ring substituted by nitrogen dioxide or substituted by another aromatic group.

Molecular dynamic (MD) simulation

The complex structure containing the 10a ligand bound with AChE was taken to perform the molecular dynamic (MD) simulation. The molecular dynamic simulation was conducted using the free Desmond academic software-2022 and OPLS-2005 force field implemented on the LINUX environment's Intel Xeon Octal-Core 2.88 Mhz. The AChE-10a ligand complex was solvated in an orthorhombic box with a dimension of 10.0x10.0x10.0 nm 3 . The simulation box contains 51182 Atoms, 14330 TIP3P water molecules, and 530 residues. The net charge of the complex system was neutralized by 8 molecules of Na + . All protein atoms were maintained at a distance equal to 10 Angstroms from the solvate box edges. The solvated system was subjected to energy minimization of 50000 steps. After performing the energy minimization, the minimized system was equilibrated for 100ps at 300K. Following equilibration, the system was subjected to a final production run of 100ns MD simulations at 300K temperatures.

Periodic boundary conditions were applied under isothermal and isobaric conditions with a relaxation time of 0.2ps.

Root mean square deviation (RMSD) analysis

Structural changes, specifically the deviation between two compositions such as protein and ligand, can be best interpreted by measuring the protein-ligand Root Mean Square Deviation (PL-RMSD) obtained from the MD simulation trajectories. [START_REF] Kufareva | Methods of protein structure comparison[END_REF] The PL-RMSD measures the scale of distance between the protein and its ligand throughout the simulation time. In this study, all protein frames (1000 frames) are first aligned on the reference frame backbone (frame 0), and then the RMSD is calculated based on the C-Alpha. The RMSD of the protein provide insights into the structural conformations throughout the simulation. It can indicate if the simulation is equilateral, and its fluctuations throughout the simulation are around some thermal average. Changes in the order of one to three angstroms are perfectly acceptable for small molecules [START_REF] Langarizadeh | A novel dual three and five-component reactions between dimedone, aryl aldehydes, and 1naphthylamine: synthesis and computational studies[END_REF]. Thus, a high RMSD value indicates the instability of the protein system. In this research work, the comparative RMSD pattern analysis between the 10a ligand bound to the AChE protein demonstrate that both compositions showed a regular scale of distance throughout the simulation time (Figure 6). From the first frame (0.10ns) to the last frame (100ns) of the molecular dynamic (MD) simulation trajectory, a relative deviation of around

Root mean square fluctuation (RMSF)

The RMSF is an essential result for measuring the deviation from a reference position of a ligand atom during the simulation time. This parameter represents the average deviation for each residue compared to the same atoms of the reference structure. Indeed, Figure 7 Therefore, the comparison of the RMSF curve of the alpha carbons (Cα) of the protein with the curve of the atomic displacement factor "B-factor" indicates a good simulation since the curve is parallel to that "B-factor" throughout the 100ns molecular dynamic simulation (Figure 7). (Protein residues that interact with the ligand are marked with green-colored vertical bars)

Hydrogen bond analysis

Hydrogen Bonds (H-bonds) play a significant role in ligand binding. Therefore, consideration of hydrogen-bonding properties in drug design is essential because of their strong influence on drug specificity, metabolization, and adsorption. [START_REF] Hernandes | Halogen atoms in the modern medicinal chemistry: hints for the drug design[END_REF] In our research, the number of Hydrogen bonds formed between the 10a ligand and AChE-enzyme during the 100ns of molecular dynamic simulation is summed up in Figure 8. Results analysis shows that the ligand formed 3 to 5 hydrogen bonds on average, with the residues of the active site of the AChE during the 100ns of molecular dynamic simulation (Tyr124 52%, Phe295 46%, Tyr337 55%, Tyr341 5.5%, and Asn87 0.6%). The adopted position of the 10a ligand in the active site of the AChE deployed more interactions, including the following low-energy interactions: Hydrophobic interaction (Trp86, Trp286, Phe297, Phe338, Tyr341) and a multitude of Water bridges interactions. In addition to hydrogen bonds, these low-energy interactions stabilize ligand binding in the protein's active site. This result confirms the potent inhibition of the AChE by the synthesized inhibitor 10a, thus supporting the results already obtained by the docking, having predicted four hydrogen bonds with the residues of the active site of the AChE.

Total Protein-Ligand Contacts

A timeline representation of the interactions and contacts (H-bonds, Hydrophobic, Ionic, and Water bridges) is summarized in the Figure 9. The top panel shows the total number of specific interactions the protein AChE makes with the ligand 10a over the 100ns molecular dynamic simulation trajectory. The bottom panel shows which residues interact with the ligand in each trajectory frame. Some residues, specifically: Tyr341, Phe338, Tyr337, Tyr124, and Trp86, make more than one specific interaction with the ligand 10a, which is represented by a darker orange shade, according to the scale to the right of the plot.

Secondary structural analysis during simulation

An outstretched study has been carried out to comprehend the structural evolution of the AChEligand during the 100ns simulation. The way that the ligand adheres to the inhibition site of AChE is crucial in understanding the ligands' stability. To highlight this, four snapshots of the AChE-ligand complexes have been taken at every 25ns interval (25ns, 50ns, 75ns, and 100ns).

It is observed that the secondary structure elements (helix and beta-sheets) remained conserved throughout the simulation process which highlights the stability and reliability of the AChE after binding to the ligands. A bit of fluctuation is observed in the N-terminal and the C-terminal coils, but structurally, no significant changes have been seen. It has also been observed that the ligands are constantly attached to the inhibition site without any structural modification, which implies that the ligand is highly stable. The Secondary structural analysis has shown no significant level of overall conformational change in ligand-bound to the AChE (Figure 10).

Figure 1 .

 1 Figure 1. Structures of some biologically active cyclosulfamides.

Figure 2 .

 2 Figure 2. Schematic representation of the design strategy illustration.

  2H, CH2), 5.58 (d, J = 6.4 Hz, 1H, CH*), 7.02 (td, J1 = 7.5, J2 = 1.1 Hz, 2H, H-Ar), 7.11 (ddd, J1 = 16.4, J2 = 8.5, J3 = 1.3 Hz, 1H, H-Ar), 7.27 (ddd, J1 = 15.3, J2 = 5.4, J3 = 1.8 Hz, 1H, H-Ar), 8.02 (d, J = 6.8 Hz, 1H, NH-CH*), 10.97 (br s, 1H, NH) ppm; 13 C NMR (100 MHz, DMSOd6): δ = 20.98, 28.04, 36.53, 49.89, 106.52, 114.68, 114.90, 123.30, 126.38, 126.51, 129.25, 129.88, 158.98, 193.85 ppm; IR (KBr):  = 3272 (NH), 3126 (NH), 1702 (C=O), 1606 (C=C), 1353 and 1171 (SO2) cm -1 ; MS: (m/z) = 313.0 (M+1); Anal. Calc. for C13H13ClN2O3S: C, 49.92; H, 4.19; N, 8.96; Found: C, 49.97; H, 4.25; N, 8.89.

  H NMR (400 MHz, DMSOd6): δ = 1.92-2.03 (m, 2H, CH2), 2.22-2.24 (m, 1H, CH2), 2.25-2.30 (m, 1H, CH2), 2.37-2.57 (m, 2H, CH2), 5.31 (d, J = 6.8 Hz, 1H, CH*), 7.14 (d, J = 8.4 Hz, 2H, H-Arortho), 7.42 (d, J = 8.4 Hz, 2H, H-Armeta), 8.02 (d, J = 6.8 Hz, 1H, NH-CH*), 10.97 (br s, 1H, NH) ppm; 13 C NMR (100 MHz, DMSOd6): δ = 20.99, 28.04, 36.54, 55.08, 107.32, 119.57, 130.14, 130.39, 139.02, 156.35, 193.72 ppm; IR (KBr):  = 3431 (NH), 3254 (NH), 1701 (C=O), 1601 (C=C), 1352 and 1169 (SO2) cm -1 ; MS: (m/z) = 313.0 (M+1); Anal. Calc. for C13H13ClN2O3S: C, 49.92; H, 4.19; N, 8.96; Found: C, 49.84; H, 4.23; N, 8.88.4-(2-bromophenyl)-4,6,7,8-tetrahydro-1H-benzo[c][1,2,6]thiadiazin-5(3H)-one 2,2-dioxide (7a, C13H13BrN2O3S): Yellow powder; 89 % yield; Rf = 0.20 (CH2Cl2/CH3OH:85/15) ; 1 H NMR (400 MHz, DMSOd6): δ = 1.92-2.15 (m, 2H, CH2), 2.21-2.23 (m, 1H, CH2), 2.24-2.28 (m, 1H, CH2), 2.33-2.60 (m, 2H, CH2), 5.60 (d, J = 7.2 Hz, 1H, CH*), 7.03 (td, J1 = 8.5, J2 = 1.1 Hz, 1H, H-Ar), 7.13 (tdd, J1 = 9.4, J2 = 7.9, J3 = 1.3 Hz, 2H, H-Ar), 7.28 (ddd, J1 = 15.3, J2 = 5.4, J3 = 1.8 Hz, 1H, H-Ar), 8.02 (d, J = 7.0 Hz, 1H, NH-CH*), 10.98 (br s, 1H, NH) ppm; 13 C NMR (100 MHz, DMSOd6): δ = 21.19, 28.28, 36.71, 50.11, 106.76, 114.90, 123.51, 126.62, 129.38, 130.06, 159.20, 193.72 ppm; IR (KBr):  = 3254 (NH), 3119 (NH), 1702 (C=O), 1605 (C=C), 1355 and 1171 (SO2) cm -1 ; MS: (m/z) = 357.0 (M+1), 359.0 (M+1+2); Anal. Calc. for C13H13BrN2O3S: C, 43.71; H, 3.67; N, 7.84; Found: C, 43.76; H, 3.71; N, 7.88.

50- 2 .

 2 57 (m, 2H, CH2), 5.32 (d, J = 6.4 Hz, 1H, CH*), 7.14 (d, J = 8.4 Hz, 2H, H-Ar), 7.42 (d, J = 7.2 Hz, 2H, H-Ar), 8.02 (d, J = 6.8 Hz, 1H, NH-CH*), 10.97 (br s, 1H, NH) ppm; 13 C NMR (100 MHz, DMSOd6): δ = 21.16, 27.06, 36.28, 55.08, 107.32, 119.97, 130.14, 130.39, 139.03, 156.60, 193.73 ppm; IR (KBr):  = 3253 (NH), 3128 (NH), 1703 (C=O), 1602 (C=C), 1354 and 1170 (SO2) cm -1 ; MS: (m/z) = 357.0 (M+1), 359.0 (M+1+2); Anal. Calc. for C13H13BrN2O3S: C, 43.71; H, 3.67; N, 7.84; Found: C, 43.67; H, 3.70; N, 7.86.

Figure 2

 2 Figure 2 illustrates docked Galantamine and co-crystallized one in nearly identical positions among the receptor (RMSD = 0.42), confirming the validation of docking protocol using Extra Precision scoring function in the absence of water molecules.

Figure 3 .

 3 Figure 3. Docked and co-crystalized Galantamine in AChE enzyme after self-docking calculation.

Figure 4 .Table 3 .Figure 5 .

 435 Figure 4. Super imposition of docked derivatives in the active site of AChE enzyme.

1. 25

 25 Angstroms was observed between both RMSD values which is an indicator of the stability of the receptor-ligand complex.The result indicates that the AChE protein has a periodic backbone stability throughout the MD simulation while bound with the 10a ligand.

Figure 6 .

 6 Figure 6. Protein and ligand root mean square deviation (PL-RMSD) obtained from the molecular dynamic simulation trajectories.

  shows the RMSF diagram of the amino acids of the AChE protein in the studied system. The results were compared with the atomic displacement factor "B factor" of the Cα atoms of each residue of the studied protein. On this plot, peaks indicate areas of the protein that fluctuate the most during the simulation. In addition, we observe that the tails (N-and C-terminal) fluctuate more than any other part of the protein. Indeed, the residues: Pro25 (1.77 Å), Ser57 (1.32 Å), Gln66 (1.45 Å), Pro78 (4.16Å), Asn265 (2.19Å), Trp385 (2.35Å), Leu386 (3.09Å), Asp494 (4.70Å), Lys538 (2.08Å) and Ser541 (2.75Å), have higher RMSF values of alpha carbons (Cα).

Figure 7 .

 7 Figure 7. RMSF diagram of the amino acids of the AChE protein in the studied system.

Figure 8 .

 8 Figure 8. Protein and Ligand Hydrogen bonds contacts obtained from the molecular dynamic simulation trajectories.

Figure 9 .

 9 Figure 9. Total Protein-Ligand Contacts obtained from the molecular dynamic simulation trajectories.

Figure 10 .

 10 Figure 10. The secondary structure content of the AChE protein with the inhibitor 10a during the molecular dynamic simulation.

Table 1 .

 1 Structures of synthesized derivatives of cyclosulfamide.

	Code	1a	2a	3a	4a	5a	6a
	Structure
	Code	7a	8a	9a	10a	11a	12a
	Structure

Table 2 .

 2 Anticholinesterase activity and enzymatic selectivity of tested compounds.

	Compound Code	Anticholinesterase activity	Selectivity index
		AChE	BuChE	AChE	BuChE
	1a	125.08±2.69	137.87±0.64	0.90	1.10
	2a	>200	102.96±1.76	1.21	0.82
	3a	>200	>200	2.36	0.42
	4a	>200	052.87±3.73	>1.94	<0.51
	5a	153.58±0.93	064.90±0.06	>1.19	<0.83
	6a	>200	147.67±0.88	>1.00	>1.00
	7a	119.92±1.61	098.66±1.38	3.17	0.31
	8a	>200	>200	>3.78	<0.26
	9a	>200	166.57±2.32	<0.58	>1.71
	10a	045.30±0.88	>200	<0.22	>4.41
	11a	199.01±1.01	147.67±0.88	>1.35	<0.73
	12a	116.34±1.37	>200	>1.00	>1.00
	Galantamine	002.10±0.07	020.38±2.10	0.10	9.70

Acknowledgements

This work was supported financially by The General Directorate for Scientific Research and Technological Development (DG-RSDT), Algerian Ministry of Scientific Research, Applied Organic Chemistry Laboratory (FNR 2000).

Disclosure statement

All authors declare no conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

ORCID

Abdeslem Bouzina https://orcid.org/0000-0002-8908-8912 Abdelhak Djemel https://orcid.org/0000-0001-6442-4687.

Omar Sekiou https://orcid.org/0000-0003-2727-0740.

Imededdine Kadi https://orcid.org/0000-0002-4703-5783.

Yousra Ouafa Bouone https://orcid.org/0000-0002-3202-5304.

Rachida Mansouri https://orcid.org/0000-0002-6886-9269.

Zineb Aouf https://orcid.org/0000-0002-3873-8647.

Nour Eddine Aouf https://orcid.org/0000-0001-6819-708X.

The binding interaction of synthesized cyclosulfamides (1a-12a) and Galantamine against AChE enzyme were reported in Table 4.

Table 4. Analysis of binding interaction of synthesized cyclosulfamides (1a-12a) and the reference ligand against AChE enzyme.

Comp code Hydrogen bond Hydrophobic interaction

Phe295, Phe297, Tyr124, Tyr337, Phe338, Tyr341, Trp86, 1a Following the visualization of molecular docking results using the Chimera program, most of synthesized compounds form hydrogen bonds with Tyr337, Glu202, and Ser203 as the binding of the reference ligand residues. Further, these compounds form additional critical hydrogen bond with Tyr124 residue.

The benzaldehyde-derived compound 1a shows significant stability in the active pocket of AChE enzyme due to the formation of two hydrogen bonds with Glu202 and Ser203 as well as hydrophobic interactions with Phe295, Phe297, Trp86, and His447 residues that are responsible for the inhibition of AChE enzyme.

The best in vitro inhibitor of AChE enzyme 10a (IC50=045.30±0.88µM) showed a better stability in silico (-7.82kcal/mol) than the reference ligand (-7.72kcal/mol) in the active site because of the formation of four hydrogen bonds with Tyr337, Tyr124, Tyr 341 and Ser203.

Also, 10a interacts with Phe295, Phe297, Trp86, and His447 residues in a hydrophobic manner.

The binding mode of the compound 5a positioned in the AChE active site showed that Tyr337 and Tyr124 residues form two hydrogen bonds with nitrogen atoms of the cyclosulfamide ring, and His447 residue forms a halogen bond with the chlorine of the aromatic group. Moreover, the phenyl and cyclosulfamide groups of 5a form π-π stacking with Trp86, while the residues such as Phe295, Phe297, Phe338, Tyr341, Tyr133, and Ala204 form hydrophobic interactions with the cyclohexenone and phenyl rings of compound 5a.

Compound 11a also shows significant stability in the active site of AChE and moderate in vitro inhibition (IC50=199µM) despite forming only one hydrogen bond with Glu202.

We observed that the two aromatic rings of 11a increase its stability in the active pocket and that due to the formation of two π-π staking with Tyr337 and Phe338 residues, and many crucial