Given the aforementioned importance and pharmaceutical applications of fused thiazole and indoline 1,3-dione derivatives, we have made an unswerving interest to synthesize the fused thiazolyl derivatives, hoping that these compounds may exhibit and enhance pivotal biological activity.
The initial experiment was focused on identifying the suitable reaction parameters (solvent, catalyst, and temperature) to obtain the 4a (Table 1) using the readily available 5-amino-2-mercaptobenzimidazole 1, phthalic anhydride 2a, and benzyl bromide 3a in DMF solvent, we found that the reaction takes longer reaction time and provides poor reaction yield (Table 1, entry-1). Then the same reaction mixture was attempted under the thermal condition at 60 ºC, the reaction yielded little improved product with 17% (Table 1, entry-2). Further, we demonstrated the same reaction in various polar protic solvents such as methanol, ethanol and glacial acetic acid to find the effect of solvent on reaction yield and timings as shown in Table 1. These findings suggest that the reaction proceeds much better in ethanol in catalytic amount of glacial acetic acid (Table 1, entry 10). Before that, we examine the same reaction in various solvents and catalysts to improve the reaction yield (Table-1, entries 1–9). Here, we demonstrated various bases such as DMF, NaOH, KOH, Na2CO3, and K2CO3 (Table 1, entries 5–9). Among the tested, we could not find any better base to improve the reaction yields. Finally, we demonstrated the reaction in catalytic amount of glacial acetic acid, fortunately there is sharp increase in reaction yields (Table-1, entry-10) and reduce the reaction timings (Table 1, entry 14). Next, we examine the temperature study demonstrated that the reaction requires a high temperature (70 ºC) for the formation of 4a in good yields as shown in Table 1, Entry-11 Vs 1–9. In addition, we have also carried out the reaction at more than 70 ºC and lesser than 70 ºC without acetic acid and we found lower yields (Table 1, entry-4). Table 1 indicates the best results were obtained when the reaction was performed using ethanol and catalytic amount of glacial acetic acid at 70 ºC (Table 1, entry 11). In order, to explore the scope of this new three-component reaction, we examined the effectiveness and tolerance of the product yields when both benzyl and phenacyl bromides contain various functional groups on the phenyl ring.
Table 1
Optimization of reaction conditions 4a.
S.No.
|
Solvent
|
Catalyst
|
Temp (ºC)
|
Time (h)
|
Yieldsb (%)
|
1
|
DMF
|
-
|
RT
|
24
|
Trace
|
2
|
DMF
|
-
|
60
|
18
|
17
|
3
|
Methanol
|
-
|
60
|
15
|
30
|
4
|
Ethanol
|
-
|
60
|
12
|
55
|
5
|
Ethanol
|
DMF
|
60
|
12
|
40
|
6
|
Ethanol
|
NaOH (1.0 mmol)
|
60
|
12
|
41
|
7
|
Ethanol
|
KOH (1.0 mmol)
|
60
|
12
|
44
|
8
|
Ethanol
|
Na2CO3 (1.0 mmol)
|
60
|
12
|
42
|
9
|
Ethanol
|
K2CO3 (1.0 mmol)
|
60
|
12
|
45
|
10
|
Ethanol
|
AcOH
|
60
|
10
|
70
|
11
|
Ethanol
|
AcOH
|
70
|
7
|
87
|
12
|
Ethanol
|
AcOH
|
80
|
8
|
80
|
13
|
AcOH
|
-
|
60
|
12
|
50
|
Reaction conditions
5-amino-2-mercaptobenzimidazole (1.0 mmol), phthalic anhydride (1.0 mmol), benzyl bromide (1.2 mmol), Ethanol (1.0 mL), Acetic acid (1.0 mL) 70 ºC. bIsolated yields.
The developed optimized conditions were applied for both diversely substituted benzyl bromide and phenacyl bromide compounds, and we observed that the benzyl and phenacyl bromides with electron-donating substituents (4-methyl benzyl bromide, 4-methyl phenacyl bromides) gave excellent yields 95% and 93% respectively as shown in Figure-2, entries-4b, 4p, and 4q. When the same reaction conditions were performed to electron withdrawing halo, nitro-substituted benzyl bromides and phenacyl bromides will reduce the product's yields (Figure-2).
Initially, the condensation between phthalic anhydride and 5-amino-2-mercaptobenzimidazoles takes place by an acid-catalysed reaction. At first, the ester carbonyl oxygen was pronated to give intermediate I. Further this intermediate I was attacked by the amino group of 5-amino-2- mercaptobenzimidazole to yield protonated amine intermediate II. Next, this intermediate undergoes deprotonated followed by ring opening and leads to the formation of elimination of water molecule gave 2-(2-mercapto-1H-benzo[d]imidazol-5-yl) isoindoline-1,3-dione intermediate III. Further, the thiol hydrogen of intermediate III was attacked by alkyl/aralkyl/phenacyl bromide to give final compounds 4a-w (Scheme 2).
The final structure of the synthesized compounds (4a-w) and (5a-f) was subjected to their spectral and analytical data. The 1H-NMR spectrum of compound 4a as a representative example showed a characteristic two singlet signals at 10.43 and 4.65 δ ppm due to presence of –NH and -S-CH2 and NH-protons respectively. The 13C-NMR spectrum displayed a significant signal at δC 167.90, and 36.44 δ ppm are assigned for isoindoline C = O, and S-CH2 carbons respectively. The infrared spectra of compound 4a show frequencies at 3390 cm− 1, 1735 cm− 1, 1602, cm− 1 of amine (-NH), isoindoline carbonyl (N-C = O), and imine (C = N) functional groups respectively. The HRMS (ESI) spectra of all the synthesized compounds are shown [M + H] + as base peck.
Anti-microbial evaluation
All the newly synthesized compounds (4a-w) were investigated for antibacterial activity. Compounds 4a, 4g, 4j, 4q, 4s, and 4t showed good activity against Gram-positive, Gram-positive bacteria had a thick cell wall, containing a high amount of peptidoglycan and Gram-negative bacteria had two layers of cell membrane: the inner membrane contains peptidoglycan and the outer membrane contains lipopolysaccharides (Table 2).
Table 2
Compounds
|
Conc. µg/ml
|
Minimum Inhibitory Concentration
|
Escherichia coli
|
Salmonella typhi
|
Staphylococcus aureus
|
Micrococcus luteus
|
4a
|
900
|
450
|
200
|
200
|
150
|
4b
|
900
|
400
|
300
|
200
|
450
|
4c
|
900
|
400
|
450
|
600
|
200
|
4d
|
900
|
700
|
600
|
170
|
300
|
4e
|
900
|
160
|
260
|
550
|
500
|
4f
|
900
|
00
|
00
|
00
|
00
|
4g
|
900
|
510
|
430
|
260
|
125
|
4h
|
900
|
700
|
650
|
600
|
640
|
4i
|
900
|
500
|
420
|
500
|
550
|
4j
|
900
|
700
|
290
|
210
|
450
|
4k
|
900
|
00
|
00
|
560
|
700
|
4l
|
900
|
700
|
500
|
400
|
440
|
4m
|
900
|
00
|
00
|
00
|
500
|
4n
|
900
|
00
|
400
|
00
|
00
|
4o
|
900
|
00
|
00
|
700
|
00
|
4p
|
900
|
00
|
700
|
00
|
120
|
4q
|
900
|
285
|
220
|
190
|
180
|
4r
|
900
|
390
|
400
|
600
|
500
|
4s
|
900
|
400
|
530
|
300
|
210
|
4t
|
900
|
515
|
320
|
200
|
330
|
4u
|
900
|
800
|
750
|
600
|
480
|
4v
|
900
|
300
|
620
|
510
|
600
|
4w
|
900
|
600
|
500
|
600
|
720
|
Standard
(streptomycin)
|
900
|
08
|
15
|
10
|
12
|
Molecular docking studies
Molecular docking results were identified basis on the ideal interacted ligands were scrutinized based on the greatest ligand binding poses were identified using the low binding energy, high docking score and the number of H-bonding, hydrophobic interactions at receptor site i.e., 4s, 4j, 4a, 4q, 4t, 4g (Figure-3). Table-3 represents the docking score, Hydrogen bond distance and interacting atoms. All the compounds were found to be buried.
Table 3
Molecular interactions with ligands against B-DNA (PDB ID: 1BNA).
Receptor
|
Ligands
|
Receptor Interaction Atoms
|
Ligand Atoms
|
Distance
(Ao)
|
Docking
Affinity
(kcal/mol)
|
1BNA
|
4g
|
NH
|
O
|
2.02
|
-7.26
|
NH
|
O
|
2.06
|
NH
|
O
|
2.07
|
4a
|
NH
|
O
|
1.55
|
-8.90
|
4j
|
O
|
NH
|
1.53
|
-8.94
|
4q
|
O
|
NH
|
1.74
|
-8.60
|
4q
|
NH
|
O
|
1.80
|
-9.50
|
4s
|
O
|
NH
|
1.44
|
-9.94
|
4t
|
O
|
NH
|
1.73
|
-8.55
|
NH
|
O
|
1.80
|
Scheme 3
Synthesis of benzo[4, 5]imidazo[2,1-b]thiazol derivatives.
Table 4
Optimization of reaction conditions 5a.
S.NO
|
Solvent
|
Catalyst
|
Temp( ºC)
|
Time(h)
|
Yieldb
|
1
|
EtOH
|
HCl (5 mol%)
|
90
|
7
|
50
|
2
|
EtOH
|
HCl (10 mol%)
|
90
|
6
|
55
|
3
|
AcOH
|
H2SO4 (5 mol%)
|
90
|
6
|
58
|
4
|
AcOH
|
H2SO4 (10 mol%)
|
90
|
5
|
62
|
5
|
POCl3
|
-
|
90
|
3
|
88
|
6
|
POCl3
|
-
|
100
|
3
|
65
|
7
|
POCl3
|
-
|
110
|
3
|
51
|
Reaction conditions
2-(2-((2-oxo-2-phenylethyl)thio)-1H-benzo[d]imidazol-5-yl)isoindoline-1,3-dione (1 mmol), solvent 2 mL. 90 ºC. b Isolated yields.
Next, we turned our attention to investigating the suitable solvent for the conversion of 2-(2-((2-oxo-2-phenylethyl)thio)-1H-benzo[d]imidazol-5-yl)isoindoline-1,3-dione derivatives to 2-(3- phenylbenzo[4,5]imidazo[2,1-b]thiazol-7-yl)isoindoline-1,3-dione derivatives 5a-h. Therefore, we examine different solvents and acid catalysts (Table-2, entries 1-7) to find the better yields a model reaction was demonstrated by using a combination of EtOH: HCl, AcOH:H2SO4 and phosphorous oxychloride (POCl3) (Table-2, entries 1-7). The findings suggest that the reaction proceeds better in POCl3 and provides good reaction yields (Table-2, entry-5). The temperature study indicates that this conversion requires a high temperature (90ºC).
Experimental section
Materials
All the chemicals and reactants were purchased from commercial sources. All the melting points were determined by open capillaries with a “Stuart” melting point apparatus (SMP 30) in Mumbai India and are uncorrected. The progress of the reaction was monitored by Thin-layer chromatography (TLC) using E. Merck 60F254 precoated silica gel plates and was detected by UV-lamp at 254 nm or iodine vapour. The products were purified by recrystallization using ethanol solvent, IR spectra (ʋ in cm− 1) were recorded on Perkin Elmer 100S instrument on KBr pellet, 1H-NMR and 13C-NMR spectra were recorded on Bruker Avance 400 MHz spectrometers in CDCl3 or DMSO-d6 as solvents and with the 1H-NMR resonant frequency of 400 MHz and 13C-NMR resonant frequency of 100 MHz. All chemical shifts values were expressed in δ (ppm) downfield from internal TMSi at δ = 0, coupling constant J measured in hertz, the peaks were presented as singlet (s), doublet (d), triplet (t), broad singlet (br s), doublet of doublet (dd) and multiplet (m). Electron spray ionization-high resolution mass spectrum (ESI-HRMS) was recorded on the Shimadzu instrument at 12.5eV.
General procedure for the synthesis of benzo[3, 4]imidazo[2,1-b]thiazole (4a-w) and (5a-h):
An equimolar mixture of 5-amino-2-mercaptobenzimidazole (1 mmol) and various substituted cyclic anhydrides (1mmol) in ethanol and catalytic amount of glacial acetic acid were used as a reaction media, then various aralkyl halides/ phenacyl bromides (1.2 mmol) were added after the disappearance of all starting compounds by TLC and continued the stirring under reflux condition for 3–4 h. After completion of the reaction (ensured by TLC), the solvent from the reaction mixture was removed under reduced pressure (5a-h). Then the reaction mixture was cooled in an ice bath and treated with an ice-cold solution of POCl3 (1 mL) was added and allowed to stir at 0–5 ºC for 10–15 minutes followed by at room temperature for another 15 minutes. Then, the reaction mixture was serried at 90 ºC for 2–3 h. After completion of the reaction, the reaction mixture was poured into crushed ice and the precipitated product was neutralized with 10 mol% of K2CO3 solution, filtered the precipitated solid, and washed several times with ice-cold water the crude product was recrystallized from ethanol and yields of the products are 88–95%.
Anti-microbial evaluation
Assay of in-vitro antibacterial activity, bacterial strains were purchased from the National Collection of industrial microorganisms, Pune, India. Antibacterial activity was tested against E. coli, S. typhi, and S. aureus, Micrococcus luteus by agar well diffusion method. The zone of inhibition was measured after 24 hrs incubation at 37 0C. The sterilized nutrient agar medium was distributed 100 mL each in two 250 mL conical flasks and allowed to cool to room temperature. To these media, 18–24 h grown bacterial sub-cultures were added and shaken thoroughly to ensure uniform distribution of organisms throughout the medium.
Then, the agar medium was distributed in equal portions, in sterilized Petri dishes, ensuring that each petri dish contains about 900 µg/ml of the medium. The medium was then allowed for solidification. The cups were made with the help of a sterile cork borer (6 mm diameter) punching into the set of agar media. The solutions of required concentrations (100 µg/ml) of test compounds were prepared by dissolving the compounds in DMSO and were filled into the cups with 1 mL of respective solution. Then, the Petri dishes were kept for incubation in an inverted position for 24–48 h at 37°C in an incubator. When growth inhibition zones were developed surrounding each cup, their diameter in mm was measured and compared with that of the standard drugs. Each experiment was made in triplicate using DMSO as a control.
Molecular docking studies
In silico docking studies are very useful to examine and to gain a wise reflection in the way of binding interactions of each ligand molecule (4a-w) with receptor structure. All the synthesized structures were drawn by using Chem Draw Ultra 13.0, Molecular energy was minimized using the Energy Minimization module of Maestro Tool (Schrodinger software). The three-dimensional structure of the receptor structure was retrieved from the RCSB database (PDB ID: 1BNA). Similar synthesized chemical compounds have shown cancer-like activity in the previous literature and the active site of the structure of a B-DNA dodecamer will have a similar binding potentiality to inhibit the DNA binding event. The target receptor was prepared by removing the structural water molecule, hetero atoms and co-factors by leaving only the residues associated with the receptor structure. Further, the grid was prepared and molecular docking was performed using the Glide docking module the results obtained were scrutinized based on the highest dock score and number of H-bonds by visualizing in Pymol.