Synthesis , Characterization , and Antibacterial and Anti-Inflammatory Activities of New Pyrimidine and Thiophene Derivatives

Substituted[4,5]thieno[2,3-d]thiazolo[3,2-a]pyrimidin-5-one (3a–b) and pyrimidin-5(6H)-imine (3c–e) were synthesized via reaction of the starting compounds, ethyl 2-amino-substituted[b]thiophene-3-carboxylate (2a–c) and 2-amino-substituted [b]thiophene-3-carbonitrile (2d–f ), respectively, with 2-bromothiazole. Synthesis of (bromo-substituted[b]thiophen-2-yl) alkanamide derivatives (4a–e) and thieno[2,3-d][1,3]oxazin-4-imine derivative (5) was accomplished via reaction of the starting compounds with bromoalkyl chloride through nucleophilic substitution; however, for the synthesis of compound 5, nucleophilic substitution was followed by nucleophilic addition to a nitrile group to form the oxazinimine ring. 1-(3-cyanosubstituted[b]thiophen-2-yl)-3-(4-(trifluoromethyl)phenyl)thiourea derivatives (6a–c) were obtained via reaction of the starting compounds (2d–f ) and 4-(trifluoromethyl phenyl)isothiocyanate. -e lead compounds (2d–f ) rapidly reacted with 4-(trifluoromethyl)benzaldehyde or 4-(2-pyridyl)benzaldehyde in acidic medium to yield compounds (7a–f) in large quantities. X-ray crystallography of compounds 4c and 7e confirmed their structures. Antimicrobial studies revealed that compound 6a was equally potent to ampicillin against Bacillus strains. Moreover, compounds 3e, 4d, and 6a possessed greater anti-inflammatory potency than that of the standard drug.


Introduction
iophene, thiazole, and pyrimidine derivatives have been used as therapeutic drugs over years.Figure 1 shows some potent drugs containing thiazole or thiophene ring.
Dasatinib is a dual Src/Abl [1] and pan-Src [2] kinase inhibitor, zopolrestat [3] and lidorestat [4] are aldose reductase inhibitors used for the treatment of diabetic complications, and raloxifene is the first clinically available selective estrogen receptor modulator (SERM) used to prevent both osteoporosis and breast cancer [5][6][7][8].In addition, clopidogrel is an antiplatelet agent used to inhibit blood clots in coronary artery disease and to prevent heart attacks and strokes in patients with heart or circulatory diseases [9][10][11].
Within the scope of these diverse synthetic methods and utility of thiophene-based systems and in continuation to our interest in the design of bioactive heterocycles [21][22][23], we aimed at developing novel heterocyclic compounds containing thiophene, thiazole, and pyrimidine rings, identifying their structures using infrared (IR), 1 H nuclear magnetic resonance ( 1 H NMR), 13 C NMR spectroscopy, and X-ray, and evaluating their biological effects, in particular, their antimicrobial and anti-inflammatory activities.

Synthetic Procedure for Compounds (4a-e, 5).
Compounds 2b-f (1.7 mmol) were refluxed in bromoalkanoyl chloride for 2-3 h.en, the mixture was left at room temperature until the product precipitated.e precipitate was filtered, washed with absolute ethanol, dried, and recrystallized from a mixture of absolute ethanol and methanol.
1 SheLXT was used to determine the structure [24,25].e final refinement was carried out by the full-matrix leastsquares technique with anisotropic thermal data for -nonhydrogen atoms on F-CCDC 1823351 and 1534088 that contain the supplementary crystallographic data for these compounds obtained free of charge from the Cambridge Crystallographic Data Centre (http://www.ccdc.cam.ac.uk/data_ request/cif).is suspension (25 mL) was placed into Petri dishes (90 mm diameter) and left to cool and solidify by placing the Petri dishes on a cool horizontal surface.A 10 mm diameter well was holed on both sides of the agar plate by using a sterilized hollow cylinder as a template.e formulations and control antibiotics (1 mg/mL) were placed into each well (50 μL) to permit diffusion.All plates were incubated at 37 ± 0.5 °C for 24 h in aerobic conditions; the test was performed in triplicate.
e antimicrobial activities of the selected formulations and control antibiotics against the tested microorganisms were compared.e diameter of the inhibition zone was measured with a gauge and expressed in mm (mean ± standard deviation (SD)).

Evaluation of Anti-Inflammatory Activity.
Fresh whole human blood was collected and mixed with equal volumes of sterilized Alsever's solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid, 0.42% sodium chloride, and 100 mL of distilled water).
is blood solution was centrifuged at 3,000 rpm for 10 min and then washed three times with an equal volume of normal saline.e volume of blood was measured, and it was reconstituted with normal saline to prepare 10% v/v suspension.
e reaction mixture consisted of 1 mL of the test sample in normal saline at different concentrations: 0.5 mL of 10% human red blood cell (HRBC) suspension, 1mL of 0.2 M phosphate buffer, and 1 mL of hypotonic saline.
ey were incubated at 37 °C for 30 min and centrifuged at 3,000 rpm for 30 min.Hemoglobin content in the supernatant was determined spectrophotometrically at 560 nm.Each experiment was performed in triplicate.Diclofenac sodium was used as a standard, and distilled water was used as a control.e blood control represented 100% lysis or zero percent stability.
is reaction involves nucleophilic substitution in bromothiazole followed by nucleophilic substitution on the ester group to form the pyrimidinone ring (compounds 3a-b).However, for compounds 3c-e, nucleophilic substitution was followed by nucleophilic addition of a cyanide group to the thiazole nitrogen to form the pyrimidinimine ring.
IR, 1 H NMR, 13 C NMR, and mass spectral data of the polycyclic compound 3a and new compounds 3b-e were consistent with the assigned structures.e IR spectrum of compound 3b showed an absorption band at 1674.41 cm −1 for the carbonyl group, whereas there was no amino group absorption band.
e 1 H NMR spectrum showed the presence of the cycloheptenyl protons and appearance of new signals: two doublet signals at δ � 6.855 and 7.984 ppm (each integrates for one proton corresponding to the two protons of the thiazole ring) with coupling constant (J) � 4.8 Hz, whereas there was no signal for the ethyl group linked to the ester group.In 13 C NMR, the three carbons of the thiazole group appeared at δ � 109.494,121.667, and 156.982 ppm, carbonyl peak appeared at δ � 162.317 ppm, and no peaks for the ethyl group.Finally, the direct analysis in real-time/time-of-flight mass spectrometry (DART-TOF-MS) spectrum showed a molecular ion peak [M + H] + at m/z � 277.05.
However, the nucleophilic substitution reaction between compound 2e and 6-bromohexanoyl chloride was followed by nucleophilic addition of a cyanide group to the carbonyl oxygen to form the oxazinimine ring of the novel compound 5 (Scheme 4).
e IR data of compound 4d showed the appearance of an NH absorption band at 3360.95 cm −1 and another absorption band with two heads at 1675.80 cm −1 , which represents the two carbonyl groups.In 1 H NMR, five new signals appeared: multiplet at δ � 1.508, 1.756, and 1.888 ppm and triplet at δ � 2.456 and 3.396 ppm (each signal integrates for two protons corresponding to the protons of the side chain).In addition, one proton singlet signal for NH appeared at δ � 11.2 ppm.
e 13 C NMR spectrum showed the appearance of the five carbons of the side chain in the aliphatic range; besides, it showed signals for the cycloheptenyl carbons in the range of δ � 24.442-36.564ppm and a peak for the amide group at δ � 166.748 ppm.Finally, the DART-TOF-MS spectrum showed a molecular ion peak [M + H] + at m/z � 416.09.
For compound 5, the disappearance of the cyanide absorption band in the IR spectrum, appearance of one proton signal for NH at δ � 12.177 ppm in the 1 H NMR spectrum, and appearance of carbon peaks at δ � 159.013 and 163.559 ppm for C4 and C2, respectively, in the 13 C NMR spectrum proved the formation of the oxazinimine ring.e six protons of the side chain appeared in the 1 H NMR spectrum as multiplet signals at δ � 1.376 and 1.641-1.816ppm for H2-H4′ and two triplet signals at δ � 2.533 and 3.508 ppm for H1′ and H5′, respectively (each signal integrates for two protons).Moreover, the 13 C NMR spectrum showed the five carbons of the side 6 Journal of Chemistry chain in the aliphatic range.Finally, the molecular weight was confirmed by the appearance of a molecular ion peak [M + H] + at m/z � 355.05 in the DART-TOF-MS spectrum.
A mixture of the appropriate lead compound (2d-f ) and 4-(trifluoromethyl phenyl) isothiocyanate in absolute ethanol was stirred at 25 °C for 2-7 h.
Structures of compounds (6a-c) were confirmed by the presence of a cyanide group and appearance of (4-(trifluoromethyl)phenyl)thiourea bands and peaks in the IR, 1 H NMR, and 13 C NMR spectra.Herein, for compound 6b, the IR spectrum showed two absorption bands for the two NH groups at 3328.78 and 3235.86 cm −1 and an absorption band for cyanide at 2210.19 cm −1 .e 1 H NMR spectrum showed a doublet signal (2 H) with J � 7.8 Hz at δ � 7.715 ppm for H2′ and H6′, doublet signal (2 H) with J � 7.8 Hz at δ � 7.838 ppm for H3′ and H5′, and two singlet signals for NH at δ � 10.765 and 11.016 ppm (each integrates for one proton).e 13 C NMR spectrum showed a peak for cyanide at δ � 114.791 ppm; additionally, the phenyl ring carbons appeared as follows: C3′ and C5′ at δ � 123.07 ppm, C2′ and C6′ at δ � 126.297 ppm, C4′ at δ � 130.988 ppm, and C1′ at δ � 142.862 ppm.CF 3 appeared at δ � 123.77 ppm, whereas the thionyl group appeared at δ � 176.728 ppm.Finally, the molecular weight was confirmed by the appearance of a molecular ion peak [M + H] + at m/z � 382.07 in the DART-TOF-MS spectrum.
Addition of few drops of sulfuric acid to a mixture of the appropriate lead compound (2d-f ) and 4-(trifluoromethyl) benzaldehyde or 4-(2-pyridyl) benzaldehyde in absolute ethanol resulted in instant precipitation of the product (Scheme 6).
All structures of the novel synthesized compounds (7a-f) were confirmed by the existence of a cyanide group, disappearance of NH 2 , and appearance of 4-(trifluoromethyl) benzylidene or 4-(pyridin-2-yl)benzylidene bands and peaks in the IR, 1 H NMR, and 13 C NMR spectra.For instance, compound 7d showed a cyanide absorption band at

X-Ray Crystallography.
e structures of three of the synthesized compounds (4c, 7e) were examined by X-ray crystallography.e crystallographic data and refinement information are summarized in Table 1.As shown in Figure 2, the asymmetric units contained one independent molecule.

Evaluation of Antimicrobial Activity. An in vitro
antimicrobial study was performed using the agar diffusion method to evaluate the ability of the synthesized compounds to inhibit microbial growth, as previously described by Scheme 6: Synthesis of thiophene-3-carbonitrile derivatives (7a-f ).
As shown in Table 2, results of the antimicrobial activity studies revealed that the test compounds displayed variable inhibitory effects against the growth of the tested bacteria.Interestingly, compound 6a was equally potent to ampicillin against Bacillus species.e antimicrobial potencies of other derivatives, particularly compound 5 (against Klebsiella and Bacillus), were potentially comparable to those of ampicillin.e antifungal activity study revealed that the tested compounds exhibited no activity against Candida albicans.

Evaluation of Anti-Inflammatory Activity.
Compounds 3a-c, 3e, 4a-e, 5, 6a-c, and 7a-f were screened in vitro for anti-inflammatory activity, using a method previously described by Mahajan et al. [29].e anti-inflammatory activity of the thiazole and thiophene derivatives (Table 3) showed that compounds 3e, 4b, and 6a possessed more potent anti-inflammatory activity than that of diclofenac sodium.Compounds 4d and 7b showed good antiinflammatory activity, whereas compounds 5, 7d, and 7f showed moderate activity.

Conclusions
In this study, we successfully synthesized some thiazole, pyrimidine, and thiophene derivatives.Among them, fifteen new target compounds were prepared.In addition, seven compounds (4c, 7e) were successfully obtained as pure crystals.Antimicrobial activity studies revealed that the test compounds displayed broad antibacterial spectrum and good potency.Interestingly, compound 6a was equally potent to ampicillin against Bacillus strains.According to the Table 2: e zones of inhibition (mm) of the synthesized compounds and standard antimicrobial drugs.
anti-inflammatory activity test, compounds 3e, 4b, and 6a possessed greater anti-inflammatory potency than that of the standard drug.erefore, compound 6a was shown to exhibit both antibacterial and anti-inflammatory activities, which could be beneficial in the treatment of various diseases.

Figure 2 :
Figure 2: ORTEP diagrams of the titled compounds 4c and 7e.Displacement ellipsoids are plotted at the 40% probability level for non-H atoms.

Table 1 :
Experimental detail of the crystalline structures of 4c and 7e.