Boosting the antimicrobial performance based on new fused spirothiazolidine framework analogs

New spiro[cyclohexane-1,2'-thiazolo[4,5-b]pyridine derivatives (3–23) were investigated. Then there is in vitro antimicrobial potency against possible organisms Staphylococcus aurous ATCC-47,077, Bacillus cereus ATCC-12228, Escherichia coli ATCC-25922, Salmonella typhiATCC-15566, and Candida albicans ATCC-10231 were tested utilizing commercially available antibiotics ampicillin as a reference drug. A preliminary antimicrobial test represented that derivatives: (Aldoses) 3'-(4-fluorophenyl)-5'-(methyleneamino)-7'-(p-tolyl)-3'H-spiro[cyclohexane-1,2'-thiazolo[4,5-b]pyridine]-6'-carbonitrile (16–19) and (Acetyl aldoses) 3'-(4-fluorophenyl)-5'-(methyleneamino)-7'-(p-tolyl)-3'H-spiro [cyclohexane-1,2'-thiazolo[4,5-b]pyridine]-6'-carbonitrile (20–23) exhibited higher antifungal, and antibacterial performance with minimum inhibitory concentrations values of (39–67 µg/ml) toward all pathogenic strains compared to common reference drug ampicillin.


Introduction
The design, synthesis, and manufacture of molecules with therapeutic benefits for humans are one of the fundamental goals of medicinal organic chemistry [1][2][3][4]. Chemical libraries based on favored structures have been available thanks to advances in combinatorial chemistry during the last decade [5][6][7]. Spiro-compounds are a type of naturally founding chemical with significant biological features ( Fig. 1) [8][9][10]. Because of their structure-activity relationship, are a very appealing target for combinatorial library synthesis [11][12][13]. It is a significant category of nitrogen and sulfur-containing heterocycles that are frequently used as major building blocks in the field of pharmaceuticals and pharmacological agents [14][15][16][17]. Recently; an efficient and additive-free synthesis of heterocycles via green strategy in excellent yields and short reaction time as an environmental procedure [18][19][20][21][22] The spiro-thiazolidinone nucleus is also recognized as the "wonder nucleus" since it produces a set of derivatives with various biological activities [23][24][25]. The existence of an N-C-S linkage in the compounds has been found to have antimicrobial and antithyroid properties [24]. The antifungal performance of several 4-thiazolidinones has been evaluated [26]. Antitubercular [27], antioxidant [28], analgesic, [29] anticonvulsant [30], anti-inflammatory [31], antihyperglycemic [32], diuretic [33], antihistaminic [34], antidiabetic [35,36], cyclooxygenase inhibitors [37], and lipoxygenase inhibitors [37] properties were also discovered. The presence of nitrogen and sulfur atoms is highlighting the importance of thiazolidinone which is widely used as a key building block in the scope of pharmaceutical agents and drugs [38,39].
Carbohydrates have a unique property: the electrophilic nature of the anomeric core allows the sugar to link with diverse molecules through presenting a functional group that acts as a nucleophile [40,41]. In nature, the OH; NH 2 group usually performs this linkage; however, sulfur or carbon nucleophile may also be involved, resulting in S-glycosides and C-glycosides, respectively [42]. The attention to S-and C-glycosides is attributed to the metabolic stability of the glycosidic bond, which cannot be hydrolyzed by glycosidases enzyme [43]. The metabolic stability of S-and C-glycosides is also exploited in the generation of glycomimetic drugs resistant against in vivo hydrolysis. [44] The most popular strategy for treating diseases caused by resistant bacteria is antimicrobial therapy [45]. As a result, there is a lot of demand and benefit in the discovery of novel antimicrobial compounds [46,47]. Furthermore, some Sand C-glycosides are significant pharmacophore that is integrated into various bioactive compounds, especially in antimicrobial therapy [48,49]. In the past decade, many heterocyclic compounds and their corresponding glycosides have been synthesized to obtain a novel antimicrobials drug, which would be able to treat infections caused by resistant bacteria and fungi strains. [50,51] Because of the above-mentioned, the main subject of our study is to synthesize the biologically important scaffold based on new spiro(cyclohexane-thiazolidine) derivatives and its corresponding glycosides with the assessment of their antibacterial and antifungal activity.
Moreover, the solvent effect in the organic h e t e r o c yc l i z a t i o n c a n b e i l l u s t r a t e d w h e n  [5',4':5,6] pyrido [2,3-d]pyrimidin]-8'-amine derivatives 9-15 were given, respectively (Fig. 3). The 1 H-NMR spectrum of compounds 9 displayed a new singlet signal related to pyrimidine-H at 8.20 ppm; compound 10 showed a broad singlet signal exchangeable with D 2 O at δ = 7.02 for -NHCS-group in pyrimidine ring; compound 11 showed new signals for substituted phenyl group in pyrimidine ring, compound 12 observe two signals exchangeable with D 2 O specific for two NH groups in pyrimidine ring at 9.19 ppm for C = NH and at aromatic-H range 7.21-8.41 ppm for NHCS; compound 13 showed a characteristic signal for pyrimidine-H at δ 8.28 ppm compound 14 displayed new signal related to CH 2 Cl group substituted in pyrimidine ring at δ 5.17 ppm and compound 15 presented singlet signal at δ 2.32 ppm for new methyl group substituted pyrimidine ring ( Fig. 3; c.f. experimental). While the IR and 13   Finally, the reaction of spiro[cyclohexane-1,2'thiazolo [4,5-b]pyridine]carbonitrile derivative 2 with aldoses namely; D-xylose, D-arabinose, D-glucose, and D-galactose in presences of Schiff's based reaction condition produced the corresponding sugars amides 16-19, respectively. The IR and 1 H-NMR spectrum of latter derivatives represented the disappearance of the characteristic signal of the NH 2 group besides the appearance of signal characteristics for both OH groups and sugars chain moieties. Acetylating sugars amides derivatives 16-19 leads to the formation of the corresponding acetylated derivatives 20-23, respectively; where IR and NMR spectrum of last derivatives 20-23 exhibited characteristic signals attributed to acetyl groups ( Fig. 4; c.f. experimental).

Anti-microbial activity
The antimicrobial performance for novel derivatives based on spirothiazolidine backbone, which was prepared in  Table 1.
As shown in Table 1, compounds 16-23 showed higher performance than the reference drug commonly utilized toward all strains according to the following activity order 16-19 > 20-23 > ampicillin, whereas the potency of derivatives 8-15 against all strains was the same as ampicillin (8-15 ≈ ampicillin) according to the following activity order; 8-11 > 13-15 ≥ ampicillin. Moreover, the estimation of the potency revealed that compounds 4-7 exhibited moderate performance against all strains when compared with the reference ampicillin drug with potency order; ampicillin ≥ 4-7. Additionally, all newly prepared compounds were more selective toward fungi strain Candida albicans ATCC-10231 (C. Alb.) The minimal inhibitory concentration (MIC) of all new prepared analogs 1-23 was tested by utilizing different concentrations of each compound and determining the lowest concentration showing pathogen growth inhibition. The results are illustrated in Table 2 based on the type of prepared derivative, and microbe strain. For the

Structure Activity Relationship (SAR)
The structure-activity relationship (SAR) of spirothiazolopyridine can be established from the data of the antimicrobial potency shown in Table 1. Analysis of the SAR of the spirothiazolopyridine derivatives 1-23 explains the link between the activity and the structure, which offers clues for structural modifications that can enhance antimicrobial performance. SAR analysis is important in understanding antibacterial and antifungal activities for newly prepared derivatives 1-23. First: Introduce open-chain sugar moiety shows a significant role to increase antimicrobial performance more than the common reference drug; ampicillin and the activity increased by increasing the number of -OH groups rather than -OAc groups in the open-chain sugar nucleus according to the following order: 19≈18 (5 free polar hydroxyl groups) > 17≈16 (4 free polar hydroxyl groups) > 23≈22 (5 free acetylated hydroxyl groups) > 21≈20 (4 free acetylated hydroxyl groups) > Ampicillin (Fig. 5a). Second: The presence of pyrimidine ring fused to spirothiazolopyridine backbone leads to an increase in the antimicrobial potency for derivatives 8-15 more than common reference drugs. The substitutions on position 4 and 2 in the pyrimidine nucleus play an essential role to enhance the activity; where the presence of group with high nucleophilicity (NH 2 > C = S > C = O > C = NH) in position 4 is preferred beside presence of electron-donating group in position 2 according to the following derivatives order (10 > 9 > 8 > 11) > (15 > 14 > 13 > 12) ≥ Ampicillin (Fig. 5b). Third: The presence of pyridine ring fused to spirothiazolopyridine framework leads to a decrease in the antimicrobial potency for derivatives 4-7 less than common reference drugs. The substitutions on positions 2, 3, and 4 in the pyridine ring effect directly the antimicrobial activity, the existence of an amino group in position 4 is a fundamental group with the presence of an electron-withdrawing group in position 3 (CN > CO 2 C 2 H 5 ) and presence of an electron-donating group in position 2 (NH 2 > OH > C 6 H 5 ); the derivatives 4-7 order can be represented as Ampicillin > (4 > 6 > 5 > 7) (Fig. 5c).

Conclusion
In the present work, we discovered a new antimicrobial candidate that might be utilized alone or in combination with research methods for therapeutic, preventive, and growth promoter purposes. A series of novel spirothiazolopyridine derivatives were designed and prepared. Most of the analogs exhibited excellent antibacterial and antifungal activity against all tested species. Some of the compounds displayed lower MIC values than the positive control on some of the tested species. We carried out the first SAR investigation into the antimicrobial activity of all prepared compounds. The SAR results showed that the N-nucleoside and S-nucleoside produced in spirothiazolopyridine skeleton have significant effects on the activity. All of the results revealed that the compounds are potential antimicrobial agents, which could be further optimized and developed as a new antibiotic

General
Melting points were measured using an Electro-Thermal IA 9100 digital melting point apparatus (Büchi, Flawil, Switzerland) and are uncorrected. Infrared spectra were recorded on a PerkinElmer 1600 FTIR (PerkinElmer, Waltham, MA, USA) discs. NMR spectra were determined on a Jeol-Ex-300 NMR spectrometer (JEOL, Tokyo, Japan) and chemical shifts were expressed as part

General method for synthesis of amino-sugar derivatives 16-19.
Aldoses; D-xylose, D-arabinose, D-glucose, and D-galactose (0.05 mol) in water (1 mL) was added to compound 2 (0.01 mol) in ethanol (30 mL) /glacial acetic acid (0.5 mL). The reaction mixture was refluxed for 6 h (TLC; chloroform: methanol; 95:5), then keep the reaction mixture cooled down to room temperature. The excess ethanol was evaporated under reduced pressure, and the residue was treated with diethyl ether (15 mL). The solid formed was filtered off and crystallized from ethanol/DMF (3:1) to give the corresponding amino sugar 16-19, respectively.

General method for synthesis of acetylated amino sugar derivatives 20-23.
A solution of the amino sugars 16-19 (1 mmol) in acetic anhydride/acetic acid (10 mL; 1:1) was heated at 100 °C until TLC (chloroform: methanol; 96:4) showed completion of the reaction. The resulting solution was poured onto icecold water, and then extracted with chloroform (40 mL). Sodium hydrogen carbonate was added to the organic layer and mixture was stirred for 1 h and filtered. The chloroform layer was washed with water, dried with sodium sulfate anhydrous and evaporated till dryness to afford the corresponding acetyl sugar derivatives 20-23, respectively.

Anti-microbial activity
The antimicrobial potency of the tested samples was examined against some targeted pathogenic microorganisms obtained from the American type culture collection (ATCC; Rockville, MD, USA). The tested organisms were Staphylococcus aurous ATCC-47,077 (St.), Bacillus cereus ATCC-12228 (B.C.), Escherichia coli ATCC-25922 (E.C.), Salmonella typhiATCC-15566 (Salm.) and Candida albicans ATCC-10231 (C. Alb.). The stock cultures of pathogens utilized in this study were kept on nutrient agar slants at 4 °C. The Agar well diffusion method was employed to study the antimicrobial activities of the samples according to the method described. Reference antibacterial drug ampicillin was evaluated for their antibacterial and antifungal action and compared with the tested samples. Seventy microliters of bacterial and yeast cells (10 6 CFU/mL) of each pathogen were spread on the nutrient agar plates. The wells (6 mm diameter) were dug on the inoculated agar plates and 100 µl of the samples and its derivatives suspended in DMSO, were added to the wells. The reference antibiotics disks (10 µg/ disk of ampicillin) were potted onto surface of agar inoculated plates. The plates were allowed to stand at 4 °C for 2 h before incubation to allow for diffusion. The plates were incubated at 37 °C for 24 h except yeast strain that were incubated at 28 °C for 24 h then followed by the measurement of the diameter of the inhibition zone (mm), and three replicates were averaged [53][54][55].

Minimum inhibition concentration (MIC):
The MIC calculation of the prepared materials was performed according to a slightly modified previous method. In brief, serial dilutions were prepared of the tested materials dissolved in DMSO. 150μL of double strength Mueller Hinton broth medium were loaded in each well of the 96 well micro liter plate followed by 150 μL of the twofold appropriate concentration and mixed well to obtain the final concentration. Overnight broth cultures of the tested bacterial and yeast strains prepared as an inoculums of 5% (V/V) (OD = 0.5 McFarland standard) was inoculated into the respective wells. For the positive growth control, the same inoculums size of each test strain was inoculated in wells that did not containing any of the tested materials. DMSO solution was tested as negative control. The plates were statically incubated at 37 °C for 24 h. 30 μL of prepared solution (0.18%) was added to each well to act as an electron acceptor and reduce to a pink, red or purple resourcing colored product by active micro-organisms (i.e., inhibition of bacterial growth was visible as a dark blue well and the presence of growth was detected by the presence of pink, red or purple color). The MIC was defined as the concentration at which the bacteria and yeast do not show visible growth with respect to the positive control [53][54][55].

Statistical analysis
Statistical analyses were carried out using GraphPad Prism 5.0 (Graph Pad Software, LaJolla, CA). In One-way model ANOVA, the observed variance is partitioned into components due to different explanatory variables. A level of p < 0.05 was considered to be statistically significant.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Conflict of interest
The authors declare that they have no conflict of interest.
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