Highly Efficient One-pot Synthesis , Antimicrobial and Docking Studies of Newer β-amino Carbonyl Derivatives Catalyzed by Silica Sulfuric Acid

Mannich reaction was applied between 4-fluorobezaldehyde, selected acetophenone and several anilines, catalyzed by silica sulfuric acid for the synthesis of β-amino carbonyl derivatives. Reaction time and yield of the products depended on the nature of acetophenone and aniline subsituents. Using aliphatic amines instead of aromatic amines under same reaction conditions, afforded aldol condensation products without yielding the expected β-amino ketones. Replacing the acetophenone derivatives with rhodanine yielded 5-(4-fluorobenzylidene)-thioxothiazolidin-4-one. Using 2-aminothiophenol instead of the aniline derivatives, 2-(4-fluorophenyl)benzothiazole was obtained without isolation of the expected (mercaptophenylamino)-1-(4-substitutedphenyl)propan-1-ones. A proposed reaction mechanism was suggested. Docking studies were designed to gain clear picture of the high active compound(s). A model of high active molecules was mapped for the antimicrobial screening and compared with least active compound(s). (doi: 10.5562/cca1983)


INTRODUCTION
Fluorine has been reported to exert many pronounced effects on the properties of organic compounds when employed as a substituent.As a result, in many areas of applied organic chemistry, including polymer chemistry and biological or medicinal chemistry, fluorinated compounds are attractive synthetic targets and the effects of fluorination on structure-activity relationships received much scrutiny. 1Moreover, owing to the growing applications of fluorinated molecules in agro-and pharmaceutical chemistry, several groups have proposed new multifunctional fluorinated synthons, which are very useful as building blocks. 2 For example, the initial biological studies of a new 2'-deoxy-2'-fluoro-2'-C-methyl nucleoside analogue that was designed and synthesized indicated that this compound showed promising activity against hepatitis C virus (HCV) replication. 3hree-component coupling Mannich reactions are among the most important carbon-carbon bond forming reactions in organic synthesis. 4These reactions provided β-amino carbonyl compounds, which are important synthetic intermediates for various pharmaceuticals and natural products. 5Therefore, the development of new synthetic methods leading to β-amino carbonyl com-pounds has attracted much attention.Due to the limited applications of the classic Mannich reaction, numerous versions of this reaction were developed using performed electrophiles, such as imines and stable nucleophiles, enolates, enol ethers, and enamines. 6Hence, there is high interest in developing convenient methods for the synthesis of β-amino ketone nucleus.Some of these achievements in the efficient construction of this nucleus included the development of Lewis acid catalysts; however, few useful Lewis acid catalysts were developed in the past years. 7Also, several one-pot procedures on the use of unmodified aldehydes or ketones were reported with a variety of catalysts such as SDA-HCl, 8−10 PS-SO 3 H, 11 and chiral Brønsted acid. 12,13−25 Therefore, the present work is intended for applying a facile and efficient one-pot three-component reac-

Synthesis of Compounds (4a-n)
General Procedure: A mixture of 4-flourobenzaldehyde (2.0 mmol), aniline (2.0 mmol), aromatic ketone (2.0 mmol), and SSA (0.11 g, 0.04 mol %) was stirred in EtOH (5 ml) at room temperature for 6-13 h.The reaction was monitored on TLC.After completion of the reaction, CH 2 Cl 2 was added and the catalyst removed by filtration.The obtained filtrate washed with saturated NaHCO 3 (aqueous) and brine, dried over anhydrous Na 2 SO 4 , filtered and evaporated to dryness under reduced pressure.The obtained residue purified by silicagel chromatography to obtain compounds (4a-n).

Anti-microbial Screening
The anti-bacterial activity of the synthesized compounds was tested against two Gram-negative bacteria: Escherichia coli NCTC 10416, Pseudomonas aeruginosa NCIB 9016, and two Gram-positive bacteria: Bacillus subtilis NCIB 3610, Staphylococcus aureus NCTC 7447, and one fungus namely: Candida albicans IMRU 3669 using nutrient agar medium.

Paper-disc Diffusion Technique
A sterilized (autoclaved at 120 °C for 30 min) medium (40−50 °C) was incubated (1 ml/100 ml of medium) with the suspension (10 5 cfu ml −1 ) of the microorganism (matched to McFarland barium sulphate standard) and poured into a petri-dish to give a depth of 3-4 mm.The paper impregnated with the test compounds (µg/ml −1 in methanol) was placed on the solidified medium.The plates were pre-incubated for 1 h at room temperature, incubated at 37 °C for 24 and 48 hr for anti-bacterial and anti-fungal activities respectively.Ampicillin (mg/disc) was used as a standard for antibacterial and anti-fungal activity respectively.The observed inhibition zones are depicted in (Table 3).

Conformational Search and Flexible Alignment
Conformational analysis and flexible alignment of the tested compounds were carried out using the software (MOE 34 of Chemical Computing Group Inc., on a Core 2 duo 3.00 GHz workstation).The molecules were built by the builder module of MOE.The geometry optimized using the MMFF94 force-field followed by a flexible alignment with systematic conformational search.Lowest energy aligned conformation(s) identified through the analysis module of DSV.

RESULTS AND DISCUSSION
In continuation of our studies on silica reagents, 35−37 the present work was directed towards using silica sulfuric acid (SSA) catalyst for the synthesis of new β-amino carbonyls derivatives.Initial studies were performed between 4-fluorobenzaldehyde (1), acetophenones (2), and anilines (3) in the presence of this catalyst at room temperature (Scheme 1) and TLC plates monitored the reaction progress.
For optimization, the reaction was carried out under various conditions using different catalysts and solvents to probe the role played by SSA to find out the best catalyst and solvent as shown in Table (1).From the obtained results, the best catalyst and solvent in terms of yield and time was silica sulfuric acid (0.04 mol) and EtOH (Table 1, entry 4).Therefore, EtOH (solvent) and SSA (catalyst) were used throughout the present study.
Substrate scope investigations revealed that a variety of aromatic ketones 2 and amines 3 reacted smoothly with 4-fluorobenzaldehyde 1 to afford the corresponding β-amino fluorinated ketones (4a-n) and the aldol products (5a-e) (Scheme 1 and Table 2).
From the obtained results, it was noticed that, both yield and reaction time depended on the nature of substituent.Thus, the presence of the electron-withdrawing group on the aromatic ketones (entry 10) increased the yield of β-amino ketones with reducing the reaction time (Table 2).Noteworthy to observe that when using aliphatic amines such as methylamine and n-butyl amine instead of aromatic amines under the same reac-  The present work was continued to explore replacing of acetophenone derivatives with other reagents.Thus, when the reaction was carried out using rhodanine under the same reaction conditions 5-(4-fluorobenzylidene)-2-thioxothiazolidin-4-one ( 6) 38 (yield 80 %) was obtained as a sole product rather than the expected product 5-((4-fluoro-phenyl)(phenyl amino)methyl)-2thioxothiazolidin-4-one (7).Structure of product (6) was elucidated chemically by reacting rhodanine with 4fluorobenzaldehyde under the same reaction conditions, which gave the same product (m.p and mixed m.p.).Also, upon using 2-aminothiophenol instead of aniline derivatives 2-(4-fluorophenyl)benzo-thiazole (8) 39 (obtained as Scheme 1. One-pot synthesis of β-amino fluorinated carbonyl compounds (4a-n).Unequivocal proofs for the role played by steric hindrance could come from the reaction of rhodanine and 2-aminothiophenol, which gave products ( 6) and ( 8) respectively as sole products.Structure of products ( 6) and ( 8) was confirmed by spectroscopic methods and elemental analyses.

Antibacterial Activity
−44 The tested microorganism strains were (Gram-negative bacteria: Escherichia coli NCTC 10416, Pseudomonas aeruginosa NCIB 9016 and Gram-positive bacteria: Bacillus subtilis NCIB 3610, Staphylococcus aureus NCTC 7447), Ampicillin was used as standard drug control.The results of antimicrobial activity values are furnished in (Table 3); and depicted graphically in (Figure 1).The data presented in (Table 3) cleared that, compound (4n) was the most active compound against all the tested bacterial strains; but compound (4m) was more potent against E. coli and S. aureus than the standard drug Ampicillin.Other synthesized compounds showed weak activity and / or loss activity against the tested bacterial strains.

Antifungal Activity
The in vitro antifungal studies of all the synthesized compounds (4a-n, 6 and 8) were tested against Candida albicans IMRU 3669, Ampicillin was used as reference drug and their inhibition zone diameter values were depicted in (Table 3) and graphically in (Figure 1).In general, all the synthesized compounds exerted weak to inactive in vitro antifungal activity against all the tested organisms.Moreover, compounds (4m and 4n) were more effective against the tested fungus than the standard drug Ampicillin.

Table 3. Anti-microbial activity of the synthesized compounds
In vitro activity-zone of inhibition / mm (a)

Structural Activity Relationship
To obtained clear picture about the structure activity relationship (SAR) for the synthesized compounds, the target compounds were numbered (Figure 2).The activity of the tested compounds must be correlated with structure modifications and variation.The obtained screening results showed that, compounds (4m and 4n) are the most active members.
A close survey in vitro antibacterial and antifungal activity profile of the new β-amino ketones against the tested bacterial and fungal organisms, gave a clear idea about the structure-activity correlations among the studied compounds.The presence of a fluoropheny moiety (phenyl ring C) at N-11 with the presence of bromo substitution attached to phenyl ring (A) at the C-6 position compound (4n) exerted a significant varied range of biological activities.In addition, the presence of bromine moiety at C-6 position (ring A) without substitution (ring C) showed significant activity of compound (4n) against E.coli and C. albicans, which revealed moderate potency against other microorganisms.For comparison, compounds (4d and 4k), showed weak activity against all microorganisms and the other tested compounds do not show any activity against any microorganisms.Furthermore, the observed marked antibacterial and antifungal activities could be considered as key steps for the building of novel therapeutic compound with comparable pharmacological features with that of the standard drugs after considering results from toxicology studies.

Absorption, Distribution, Metabolism, Elimination(ADME) Toxic Factors Profiling
Oral bioavailability considerably plays an important role for the development of bioactive molecules as therapeutic agents.Several potential therapeutic agents fail to reach the clinic, because of ADME toxic Factors.Therefore, a computational study for prediction of ADME properties of the molecules was performed for antimicrobial compounds (4d, k, m and 4n), by the deter-  6) and ( 8) against bacterial strains (Gram negative and positive bacteria), and fungal strain.mination of topological polar surface area (TPSA), C log P and ''rule of five'' which have been formulated by Lipinski. 45The same author established that, a chemical compound could be an orally active drug in humans, if no more than one violation of the following rule: i) C log P < 5, ii) number of hydrogen bond donors sites ≤ 5, iii) number of hydrogen bond acceptors sites ≤ 10, iv) and molecular weight <500.In addition, the total polar surface area (TPSA) is another key property linked to drug bioavailability, the passively absorbed molecules with (TPSA>140) have low oral bioavailability. 46Calculations were performed using MOE program, 47 the results were disclosed in (Table 4).The present results revealed that, the C Log P is the partition coefficient between water and octanol (factor of lipophilicity). 48Lipophilicity of most compounds is more than 5.0; the molecular weight (MW< 500), hydrogen bond acceptors between (1 and 2) and hydrogen bond donors (1) which fulfill Lipinski's rule.These data may suggest that, these compounds of good passive oral absorption.

Conformational Analysis
In order to gain a better insight into the molecular structures of the active compounds (4m and 4n) and low active compounds (4d and 4k).The conformational analysis of the target compounds were performed using MMFF94 force-field 49,50 (calculations in vacuum, bond dipole option for electrostatics, Polake Ribiere algorithm, RMS gradient of 0.01 kcal/A mol) implemented in MOE 2009.10.).The most stable conformer was fully optimized by AM1 49 semi-empirical molecular orbital calculation (Figure 3).
The calculation results showed that, the lowest minimization energy structures of the compounds under investigation exhibited a common arrangement and critical distance of the aryl moiety in (ring C) from the fluoropheny core (ring B).The optimal distance for the best biological activity was found in 3.12 Å and 3.13 Å for (4m and 4n) respectively, and the higher distance values in rang (3.16−3.18)lead to decrease in activity, as indicated in Figure 4.

Flexible Alignment
To understand similarity between the threedimensional structures of active compounds (4m and 4n) a flexible alignment was employed (Figure 5), using MOE/MMFF94, 50 and 200 conformers of each compound were generated and minimized with a distance-dependant dielectric model.A low energy set of 100 was selected for further analysis.Conformations of compound (4m) were generated using distance geometry and optimized with MMFF94.Five low energy, maximally dissimilar structures were selected for comparison to the other compounds.After assigning MMFF94 charges to all molecules, flexible alignment was ranked overlays of compounds (4m and 4n) based on electrostatic, steric filed, hydrophobic areas overlap, hydrogen bond acceptors and donors overlap.From the highest scoring superposition, the limited set conformers was used in the analysis for molecules with high flexibility capable for achieving complete atom-to-atom superposition (Figure 5a).A common feature of the MOE-generated alignments showed, comply of the two structures (4m and n) with: i) three phenyl rings (A, B and C), ii) COCH 2 NH spacer between rings (Figure 5a).With the same way, antimicrobial of compound (4n) and the lowest active compounds (4d and 4k) were subjected to flexible alignments.Analysis of weak active molecules (Figure 5b) is an important way to gain clear vision of the essential features for a given activity.It is clear that compounds (4d and 4k) were flexibly aligned in a different manner when compared with the active compound (4n).Common feature of the MOE-generated alignments showed, the superposition of the COCH 2 NH fragments in (4k) have a slight deviation (0.5 Å) compared with high active compound (4n), the three phenyl rings arrangement are in similar manner to all investigated compounds.These features explaining why such compound was the least active as antimicrobial agents and showing the importance of both COCH 2 NH moiety and the aromatic ring attached to it (Figure 5b).

Electrostatic and Hydrophobic Mappings
In an attempt to understand the lowest activity represented by (4k) and the highest antimicrobial activity represented by (4n), electrostatic and hydrophobic mappings was preformed for the lowest energy conformers, to examine the comply and incompliance in electronic, electrostatic binding characteristics of the surface of the molecules and conformational properties (Figure 6).First: comparison of the electrostatic mappings of lowest active compounds, represented by compound (4k)  (Figure 6a) showed common features: i) negative charge located on the carbonyl (in red), ii) non-polar area located on the aryl moiety (in green), these features explain biological similarity among the low active analogues compounds.On the contrary, the high activity of compounds (4m and n), represented by (4n, Figure 6) showed decrees distribution of negative charge (in red) on carbonyl group.From the above features, the low activity may be due to increasing negative charge located on the carbonyl group region representing hydrogen bond acceptor (in red) (Figure 6a).As the same analogy, hydrophobic mappings of the most active compound (4n) showed that, the hydrophobic region (in green) was distributed on all sides of aromatic rings (A, B and C) while the hydrophilic region (in red) was located mainly on carbonyl fragments (Figure 6b).On the other hand the hydrophobic distributions of the inactive compounds (4k) occupying aromatic ring, indicating dissimilarity hydrophobic distributions of such compound (Figure 6b).It is clear that the related charge distribution, electrostatic and hydrophobic mappings suggest a similar interaction and orientations of the molecules with a potential protein-binding site.

Pharmacophore Prediction
The aim of this approach is generation and prediction a pharmacophore model (hypothesis) depending on the most active compound.These steps are employed for 3D pharmacophore applications: i) Compound 4n (Figure 4) used as reference to other conformations of each compounds.ii) Assignment of the pharmacophoric features.ii) Application method for undertaking conformational searching of databases for new structures matching to generated pharmacophoric features.These steps will be explained (hypothetical) pharmacophoric building of antimicrobial compounds.Molecular Operating Environment (MOE) programme was used for pharmacophore building. 51The calculated conformational models were preformed with 15 kcal energy cut off (minimization convergence criteria during conformational analysis: energy convergence = 0.01 kcal/mol, gradient convergence = 0.01 kcal/mol).The number of conformers generated for each substrate was limited to a maximum of 500.All molecules with their associated conformations were regrouped depending biological data.Hypothesis generation carried out with low energy conformers of the molecules.The calculation and analysis were preformed.After assignment of possible pharmacophore elements for each analogue using MOE program, then superposition of the molecules including the assigned elements, was attempted (Figure 7), to generate Pharmacophore maps, many runs of calculation were repeated.For each run, a distinct number of specified pharmacophore elements were generated.All appropriate models showed that, the donor atoms of the NH fragments (as hydrophilic) element and the aryl moieties (as hydrophobic) were well superimposed within the set distance tolerance, which confirms the important role of the hydrophilic and hydrophobic moieties for antimicrobial activity.The initial pharmacophore query was carried out, and introducing of 5 features, as summarized in (Table 5) and illustrated graphically in (Figure 7).
From (Figure 7a), Models for (4n) and its analogs (4m) possess pharmacophore elements in the aromatic (ring C) attached to NH group (as a hydrogen bond donor) which is out of plane with aromatic ring (B), which parallel with aromatic ring (A).In contrast, model for (4d) and its analogs (4k) (Figure 7b) showed common elements, the aromatic ring (B) coplanar with aromatic ring (A), and aryl amino part out of plane with aromatic ring (A) (Figure 7b).Since the arylamino-part of compound (4n) plays an important role in activity.According to the pharmacophore generated by MOE 51 the minimal structural requirements for antimicrobial activity consist of an aromatic ring (hydrophobic re-  gion) attached to NH fragment (H-bonding donor region), and carbonyl fragment (H-bonding acceptor region) (Figures 4 and 7).This pharmacophoric assumption was in consistence with biological data.

CONCLUSIONS
A facile and efficient one-pot three-component reaction between p-fluorobezaldehyde, selected acetophenone and several aniline derivatives, catalyzed by silica sulfuric acid (SSA) for the synthesis of new β-amino carbonyl compounds (4) was investigated via Mannich reaction to yield products (4) in good to excellent yields and in short time.Flexible alignment was conducted and justified the importance of the main molecular groups (particularly, the carbonyl fragment and the aryl rings (A and C) as well as of their relative distance.In fact, the substitution patterns on the phenyl ring (A or C) considerably affect the activity.The overall outcome of this model revealed that: i) the phenyl rings (A, B and C) are a satisfactory backbone for antimicrobial activity, (ii) the aryl (ring A) should be about less than 3.16 Å apart from the (ring C) (Figure 2).iii) Absence of bromine as hydrophobic moiety at position 6 decreased the activity, such as 4-(fluoro, chloro and methyl) phenyl moiety, this indicted the importance of position 6 in the activity.These modeling studies could be very useful for the virtual screening in the development of the presently studied β-amino carbonyl compounds as antimicrobial agents.Therefore, the present work led to the development of novel antimicrobial agents especially in case of the two novel molecules (4m and 4n) for their noticed higher activity than all the other tested compounds.
(b)  Detected by TLC.(c)  Isolated yields after silica-gel chromatography.tion conditions, the corresponding aldol condensation products (5a and c) were isolated as clean cut compounds (100 % yield) without isolating the expected β-amino fluorinated ketones of type 4 (Scheme 2).A possible mechanism of the Mannich-type reaction that involves the use of SSA as catalyst in ethanol is proposed (Scheme 3).When SSA was added to the reaction mixture, the carbonyl oxygen atom of the aldehyde becomes activated and this lead to formation of the intermediate I.Then, aniline (as a nucleophile) attacks the activated carbonyl group of benzaldehyde forming a hydrogen bond (between H and O) with SiO 2 -SO 3 H, affording the intermediate II with splitting of a water molecule through intramolecular rearrangement to the intermediate III.In the mean time the carbonyl oxygen atom of acetophone is activated by SSA, forming (enol form) intermediate IV.In the reaction medium, the π-electrons of the activated (or non-stabilized) imine delocalized towards the activated enol to form a new carbon-carbon bond through the reface of the double bond plane.The π-electrons of the activated enol redistribute to give keto form (accompanied by the transfer of hydrogen atom to nitrogen atom).Finally, the Mannich adduct is formed and catalyst (SSA) renewed again (Scheme 3).

Scheme 2 .
Scheme 2. Reaction of methylamine or n-butyl amine with 4-fluorobenzaldehyde 1, aromatic ketones 2 in EtOH in presence of SSA at room temperature.

Scheme 3 .Scheme 4 .
Scheme 3. Proposed mechanism of the direct Mannich reaction catalysed by silica sulfuric acid.

Figure 3 .
Figure 3. Lowest energy conformers of the most active compounds (4m) and (4n) as representative examples in CPK rendering.

Figure 4 .
Figure 4. 2D-pharmacophoric design of β-amino ketone (red colors represent the HB regions, green colors represent hydrophobic regions and blue colors represent spacer moiety).

Figure 5 .
Figure 5. Flexible alignments of the most active compounds (Left panel): 4d (in green), 4k (in red) and 4n (in blue).Right panel showed flexible alignment of the high active compound 4n (in green) and lowest active compounds 4d (in red) and 4k (in blue).

Figure 6 .
Figure 6.Electrostatic maps (left panels a) and hydrophobic panels (right panels b) of the lowest energy conformers for the most active compounds (4n) and the least active compounds (4k); maps are color coded: red for the hydrogen bond and a hydrophilic region, cyan for a medium polar region, and green for a hydrophobic region.

Figure 7 .
Figure 7. a) Showed the best predicted pharmacophore features and geometries which are required for antimicrobial activity.b) Showed the most active compounds 4m and 4n mapped to the pharmacophore model for antibicrobial activity.c) Showed the lowest active compounds (4d and 4k) mapped to the pharmacophore model for antimicrobial activity.Pharmacophore features are color coded: yellow for hydrophobic aromatics, cyane for a hydrogen bond donor, and blue for a hydrogen bond acceptor feature.

Table 1 .
Optimization of reaction conditions for the formation of representative compound (4a) Reaction carried out in the appropriate solvent using 4-fluorobenzaldehyde (2 mmol), acetophenone (2 mmol) and aniline (2 mmol) at room temperature.

Table 4 .
Molecular parameters of the active compounds

Table 5 .
Pharmacophoric and structure features of the most active compounds