Microwave-assisted Synthesis, Structural Characterization of Amino Pyridines, Pyrrolidine, Piperidine, Morpholine, Acetamides, and Assessment of Their Antibacterial Activity

A sequence of new acetamide derivatives 9-15 of primary, secondary amine, and para-toluene sulphinate sodium salt have been synthesized under microwave irradiation and assessed in vitro for their antibacterial activity against one Gram-positive and two Gram-negative bacterial species such as S. pyogenes, E. coli, and P.mirabilis using the Mueller-Hinton Agar diffusion (well diffusion) method. The synthesized compounds with significant differences in inhibition diameters and MICs were compared with those of amoxicillin, ampicillin, cephalothin, azithromycin and doxycycline. All of the evaluated acetamide derivatives were used with varying inhibition concentrations of 6.25, 12.5, 37.5, 62.5, 87.5, 112.5 and 125 µg/ml. The results show that the most important antibacterial properties exercised by the synthetic compounds 9 and 11 bearing para-chlorophenyl moiety incorporated into the 2-position moiety of acetamide 2. The molecular structures of the new compounds were determined using FT-IR, 1H-NMR techniques.

The compounds atorvastatin [12] 16, lidocaine [13] 17, paracetamol [14] 18, amoxicillin [15] 19, levobupivacaine [16] 20 are among the acetamide derivatives of Synthesis of broad spectrum compounds with antibacterial activity is required because of the ex-tense use of antibiotics in medicine and prevailing resistance in microorganism. Conventional organic preparation methods are sluggish to meet the rapid need for the synthesis of these compounds. Nowadays microwave plays an appealing role in organic synthesis. Herein, we report the use of microwave in organic synthesis of seven newly acetamides derivatives 9-15 form reaction of chloroacetyl chloride 1, with primary, secondary amines and para toluene sulphinate sodium salts. Also, the aim of this study is also to assess the susceptibility profile of bacterial isolates species to synthesized compounds 9-15, reference antibiotics, and to determine minimum concentrations of inhibition (MIC's). Compounds 9-15 were structurally determined by 1 H-NMR, and FT-IR and elemental analysis. Scheme 1 shows a central aspect of that approach.

Chemistry
We are engaged in intensive efforts to synthesize nitrogen-heterocyclic compounds with high yields. Following a previously described study [21], the acetamide intermediates 3-8 and the new targeted derivatives 9-15 synthesized in this work are depicted in Scheme (1). Over the past thirty years, microwave irradiation was used to enhance reaction rates [22,23]. We have thus exploited the advance of microwave to accelerate the creation of C-N bonds. For the synthesis of the precursors 3-8, the process was performed under mild conditions involving the addition of chloroacetyl chloride 1 to a mixture of amines and para-toluene sulphinate sodium salt in dry CH2Cl2 at 0 ºC and the isolated yields of these compounds range from 60 to 90% see Table 1. The synthesis of targeted compounds 9-15 was achieved through two routes: conventional heating and microwave irradiation methods. The heating process involved precursor 3-8, amine, and sodium p-toluene sulphinate reactions in a CH3CN solution containing Et3N as a catalyst giving the compounds 9-15 in moderate yields reached of 60% at 70 o C see Scheme 1 and Table 1, On the other hand, treatment of the precursors 3-8 with amines as well as sodium p-toluene sulphinate in a dry CH3CN provided the desired compounds 9-15 at 65-70 ºC in good yields under microwave irradiation. Reactions took 5-10 min to complete and one of the advantages of this technique is allowed us to isolate products with high purity and without side products, see Tables 1, 2, and Table 3 for 1 H-NMR details.

In vitro Antibacterial activity testing
Instructive bacterial infections such as tuberculosis, urinary tract infection, pneumonia, brain abscess, phyaryngitis, and tonsillitis are a series of life-threatening diseases commonly recognized in immunocompromised patients. Such infectious diseases are becoming more and more resistant to antibiotics in human patients.
Resistance to microbial drugs is an inevitable consequence of the use of antibacterial drugs. Antimicrobial resistance to antibiotics has arisen due to overuse of these antibacterial compounds. Susceptibility to antibiotics by pathogens has now become a crucial factor in the effectiveness of antibacterial choice. Amoxicillin, ampicillin, doxycycline, azithromycin and cephalothin, the five reference antibiotics used in this research, are commonly used for the treatment of infections. Researchers continue to discover new antimicrobial agents as a high-resistance drug organism. This research was carried out to determine the susceptibility of seven newly synthesized compounds to certain bacterial organisms. Synthesized compounds 9-15 have been evaluated for antibacterial activity in comparison with the mentioned reference antibiotics.
Acetamide derivatives are well known to have a broad range of antibacterial action [24]. With regard to our recent research [21] on the synthesis of acetamide derivatives and the assessment of their biological role, in this in vitro study, three species of microorganisms were included, all isolates of the three species were susceptible to synthesized compounds 9-15 at MICs greater than 6.25 μg/ml.
Tests for the gram-negative species, E. coli and P. mirabilis cause urinary tract infections (UTI) in patients exhibited inhibition zones for compounds 9 and 10 ranging from 6.0 to 8.4 mm at MIC of 12.5 μg/ml. At MIC of 37.5 μg/ml, isolates of these bacterial species were not susceptible to compounds 13 and 15, while inhibiting zones of other compounds ranging from 6-14 mm Table 4. The results for these two gram-negative species exhibited intermediate inhibition zone diameters are in agreement with other authors who reported 11-12 mm using amoxicillin (20 μg) and ampicillin (20 μg) [25,26,27] antimicrobial disks Tables 4 and 5. Gram-positive species, S. pyogenes are one of the main causes of urinary tract infection and pose a significant health concern and cause a number of human diseases [28,29]. Also, S. pyogenes considered to be the most common and significant cause of bacterial tonsillitis in children and adults, and isolates this bacterial species were obtained from Tikrit hospital, Tikrit, Iraq. All isolates at MICs more than 6.25 μg/ml were susceptible to synthesized compounds 9-15, and compounds 14 and 15 did not display any inhibition zones in this test, even at MIC of 37.5 μg/ml Table 6.  Table 6.
However, all species included in this study were more susceptible to synthesized compounds 9-15 with a range of 37.5-125 μg/ml higher MICs relative to the related antibiotics, but this low activity could be caused by resistance-acquiring bacteria as a result of patients arbitrary use of antibiotics.
The aim of this analysis was also to observe any relation between the structure of the synthesized compounds 9-15 and the susceptibility of the bacterial isolates to these compounds. Thus, as described earlier, the antibacterial susceptibility of compounds 9-15 and antibiotics given as MIC values in μg/ml is used to achieve this goal. For compounds 9-15, the figures 4, 5, and 6, clearly indicated that E. coli is more susceptible to compounds 9, 10, and 12, as P. mirabilis which is more susceptible to compounds 9 and 11 so does S. pyogenes see figures 7, 8, and 9. The presence of para -chlorophenyl moiety at 2-position 2 of these compounds appears to be due to this effect.. Presence of chlorine atom presumably makes such compounds more active than other compounds against isolates of the three bacterial species. In case of antibiotics, E. coli tends to be more susceptible to these five antibiotics, and there were few variations among isolates towards antibiotics as it can be seen see figures 7, 8, 9. * means there are significant differences, n.s. means there are no significant differences

Chemistry
On  Table 2.

Bacterial species
Isolates of the three different bacterial species, mamely, two Gram-negative bacteria E. coli plus P. mirabilis and one Gram-positive S. pyogenes bacteria were collected from Tikrit teaching hospital, Tikrit, Iraq. All the isolates of E. coli and P. mirabilis were collected from urine samples of urinary tract infection (UTI) patients, and the isolates of S. pyogenes were collected from tonsillitis patients.

Antibacterial susceptibility Testing
Antibacterial resistance to seven new synthesized compounds 9-15 of the acetamide class and five antibiotics, including amoxicillin, ampicillin, cephalothin, azithromycin and doxycycline, was evaluated using the standard disk diffusion method [33]  Statistically standard deviation, variations are calculated relative to normal antibiotics and the amount of dispersion between them.