Synthesis, characterization and antimicrobial screening of thiazole based 1,3,4-oxadiazoles heterocycles

Article abstract of DOI:10.1007/s11164-015-2196-x

In an effort to find a new pharmacologically dynamic molecule, we report here the synthesis and in vitro antimicrobial activity of various N-(4-methyl-5-(4-((substituted phenylamino) methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl) benzamid

Full text of DOI:10.1007/s11164-015-2196-x

Res Chem Intermed
DOI 10.1007/s11164-015-2196-x
Synthesis, characterization and antimicrobial screening
of thiazole based 1,3,4-oxadiazoles heterocycles
N. C. Desai1 Nayan Bhatt1 Amit Dodiya1
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•
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Tushar Karkar1 Bonny Patel1 Malay Bhatt1
•
•
Received: 20 June 2015 / Accepted: 24 July 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract In an effort to find a new pharmacologically dynamic molecule, we
report here the synthesis and in vitro antimicrobial activity of various N-(4-methyl-
5-(4-((substituted phenylamino) methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-
yl)thiazol-2-yl) benzamides. The structures of newly synthesized compounds were
1
assigned on the bases of IR, H NMR, 13C NMR, and mass spectral data. Synthe-
sized compounds were screened for their antibacterial and antifungal activities on
gram-positive bacteria (Staphylococcus aureus and Streptococcus pyogenes), gram-
negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and three fungi
(Candida albicans, Aspergillus niger and Aspergillus clavatus) by serial broth
dilution method. Compounds 5e and 5k were associated with considerable potential
antibacterial and compounds 5a and 5b antifungal activities.
Keywords Thiazole Á 1,3,4-Oxadiazole Á Potential antimicrobial activity Á MIC
Introduction
The thiazole ring system has nitrogen and sulfur heteroatoms at positions 1 and 3,
respectively. The thiazole ring has been widely used in several bioactive compounds
like thiamin (Formula I) and penicillin G (Formula II) [1, 2]. Due to this reason and
medicinal significance of thiazole and 1,3,4-oxadiazole, we have incorporated both
the moieties in one structure for the development of more potential antimicrobial
agents (Fig. 1).
&
N. C. Desai
dnisheeth@rediffmail.com
1
Division of Medicinal Chemistry, Department of Chemistry, Maharaja Krishnakumarsinhji
Bhavnagar University, Mahatma Gandhi Campus, Bhavnagar 364002, India
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N. C. Desai et al.
Fig. 1 Thiazole containing important motifs
The search for biologically active substances led us for the investigation of
condensed sulfur- and nitrogen-containing heterocycles. Thiazole derivatives have
been reported to possess significant and diverse biological activities such as
antimicrobial [3], analgesic [4], anti-inflammatory [5], antioxidant [6], anti HIV [7]
and antitubercular [8, 9] activities. Moreover, thiazoles are also synthetic
intermediates and substructures in several biologically active compounds [10–16].
The substituted thiazole-based compounds are associated with various pharmaco-
logical functions such as relatively stable molecules, enhanced lipophilicity and
easy metabolism.
Oxadiazole is an important class of heterocyclic compounds with a broad
spectrum of biological activities. Out of its four theoretical isomers, 1,3,4-
oxadiazole is screened for various biological activities including anti-microbial
[17], anticancer [18], anti-inflammatory [19], anti-infective [20] and anti-HIV [21].
Hence, 1,3,4-oxadiazoles have occupied a unique place in the field of medicinal
chemistry particularly in anti-edema and anti-inflammatory activity [22–26]. This
moiety is an important bioactive class of anti-HIV and angiogenesis inhibitor [27].
Nesapidil [Formula (III)] is useful as an antihypertensive agent while furamizole
[Formula (IV)] is useful as an antibiotic. Both the drugs possess an oxadiazole
nucleus in the structural framework (Fig. 2).
In continuation of our previous work [28–31] and because of the medicinal
importance of thiazole and 1,3,4-oxadiazole, we report herein synthesis of a new
class of N-(4-methyl-5-(4-((substituted phenylamino)methyl)-5-thioxo-4,5-dihydro-
Fig. 2 Oxadiazole containing marketed drugs
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Synthesis, characterization and antimicrobial screening…
1,3,4-oxadiazol-2-yl)thiazol-2-yl)benzamides (5a–k). We also tried to develop
potential antimicrobials. The structures of newly synthesized compounds were
assigned on the basis of IR, 1H NMR, 13C NMR, and Mass spectral analysis. These
compounds were evaluated for their antimicrobial screening on different strains of
bacteria and fungi.
Experimental
Materials and methods
All reactions except those in aqueous media were carried out by standard techniques
for the exclusion of moisture. Melting points were determined on an electro thermal
melting point apparatus and were reported uncorrected. TLC on silica gel plates
60
(Merck,
F
254
) was used for purity checking and reaction monitoring. Elemental
analysis (% C, H, N) was carried out on a Perkin-Elmer 2400 CHN analyzer. IR
spectra of all compounds were recorded on a Perkin-Elmer FT-IR spectrophotome-
ter in KBr. 1H NMR spectra were recorded on a Varian Gemini 300 MHz, and 13C
NMR spectra are recorded with a Varian Mercury-400 (100 MHz) NMR
spectrometer, using tetramethylsilane as the internal reference, with dimethyl
sulfoxide (DMSO-d6) as solvent. All chemical shifts were expressed in ppm. Mass
spectra were scanned on a Shimadzu LCMS 2010 spectrometer. Anhydrous
reactions were carried out in oven-dried glassware in a nitrogen atmosphere.
Chemistry
Synthetic strategies adopted to obtain the target compounds are depicted in Fig. 3.
The series of title compounds (5a–k) were synthesized in four steps. Compound (1)
was prepared by a mixture of thiourea and ethyl acetoacetate heated at 60–70 °C
and a catalytic amount of benzoyl peroxide was added and treated with N-
bromosuccinamide using benzene as solvent to give ethyl 2-amino-4-methylthia-
zole-5-carboxylate (1). In the first step, compound (1) was treated with benzoyl
chloride at 0–5 °C to give the intermediate ethyl 4-methyl-2-(phenylcarbony-
lamino)-1,3-thiazole-5-carboxylate (2). In the second step, intermediate (2) was
refluxed with hydrazine hydrate in the presence of ethanol (99 %) as a solvent to
give compound N-(5-(N-aminocarbamoyl)-4-methyl(1,3-thiazol-2-yl))benzamide
(3). In the third step, compound (3) was refluxed with carbon disulfide (CS2) in
ethanol as a solvent in the presence of potassium hydroxide to achieve N-(4-methyl-
5-(2-thioxo(1,3,4-oxadiazolin-5-yl))(1,3-thiazol-2-yl))benzamide (4). In the final
step, compounds (5a–k) were generated by Mannich condensation of the
intermediate (4) with 36 % formaldehyde and different aniline derivatives in
ethanol (95 %) used as solvent.
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N. C. Desai et al.
Synthesis of ethyl 4-methyl-2-(phenylcarbonylamino)-1,3-thiazole-carboxylate (2)
In a round bottom flask, a mixture of 2-amino-4-methyl-1,3-thiazole-5-carboxylate
(0.01 mol) and pyridine (10 mL) was placed and benzoyl chloride (0.01 mol) was
added dropwise at 0–5 °C. The mixture was stirred at a temperature not exceeding
5 °C for 3 h. The solution was poured into ice cold water. The solid product was
filtered and washed with cold dilute hydrochloric acid solution to remove access
2-amino-4-methyl-1,3-thiazole-5-carboxylate. The resulting solid was recrystallized
from ethanol (99 %). Yield: 23 %, mp 128–130 °C. IR (KBr, cm-1): 3220 (N–H
stretching, secondary amide), 2891 (C–H stretching, CH3 group), 1753–1740 (–CO
stretching, aliphatic ester), 1704 (C=O stretching, secondary amide). 1H NMR
(300 MHz, DMSO-d6, d ppm): 12.80 (s, 1H, Het–NH–CO–Ar), 8.15–7.60 (m, 5H,
Ar–H), 4.25 (q, –CH2–CH3), 2.54 (s, 3H, –CH3), 1.30 (t, –CH2–CH3). 13C NMR
(100 MHz, DMSO-d6, d ppm): 165.5, 162.7, 162.3, 156.7, 134.5, 132.3, 128.6,
128.6, 127.3, 127.3, 116.5, 60.4, 16.2, 14.3. LCMS (m/z): 290.07 (M?). Anal. calcd.
for C14H14N2O3S: C, 57.65; H, 5.02; N, 9.56 %. Found: C, 57.69; H, 5.09; N,
9.63 %.
Synthesis of N-(5-(N-aminocarbamoyl)-4-methyl(1,3-thiazol-2-yl))benzamide (3)
Compound (2) (0.01 mol) and 99 % hydrazine hydrate (0.015 mol) were placed in a
round bottom flask and the mixture was refluxed for 10 min. Alcohol was added till
both the layers were miscible and refluxing was continued for 5 h. Excess of alcohol
and unreacted hydrazine hydrate was distilled out and the contents were poured into
a beaker. The solid was recrystallized from ethanol to get pure crystalline product.
Yield: 63 %, mp 141–143 °C. IR (KBr, cm-1): 3424 (–NH stretching, primary
amine), 3310 (–NH stretching, secondary amine), 3224 (N–H stretching, secondary
amide), 2889 (C–H stretching, CH3 group), 1707 (C=O stretching, secondary
1
amide). H NMR (300 MHz, DMSO-d6, d ppm): 12.83 (s, 1H, Het–NH–CO–Ar),
9.37 (t, –NH–NH2), 8.14–7.58 (m, 5H, Ar–H), 4.57 (d, –NH–NH2), 2.52 (s, 3H,
–CH3). 13C NMR (100 MHz, DMSO-d6, d ppm): 165.5, 162.7, 160.5, 156.3, 134.5,
132.6, 132.3, 128.6, 128.6, 127.3, 127.3, 16.7. LCMS (m/z): 276.07 (M?). Anal.
Calcd. for C12H12N4O2S: C, 51.95; H, 4.22; N, 20.06 %. Found: C, 51.89; H, 4.18;
N, 20.10 %.
Synthesis of N-(4-methyl-5-(2-thioxo(1,3,4-oxadiazolin-5-yl))(1,3-thiazol-2-yl))
benzamide (4)
A mixture of compound (3) (0.01 mol), potassium hydroxide (0.01 mol), carbon
disulfide (0.02 mol), and absolute ethanol (10 mL) was refluxed for 12 h. The
excess solvent was removed by vacuum evaporation, and the residue was dissolved
in water and acidified with acetic acid to get a solid product. It was filtered, dried,
and recrystallized from water–ethanol (60–40 %). Yield: 72 %, mp 171–175 °C. IR
(KBr, cm-1): 3348 (N–H stretching, secondary amine), 3220 (N–H stretching,
secondary amide), 2982 (C–H stretching, aromatic ring), 2891 (C–H stretching, CH3
group), 1704 (C=O stretching, secondary amide), 1554 (C=C stretching, aromatic
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Synthesis, characterization and antimicrobial screening…
1
ring), 1292 (C–H bending), 1191, 1090 (C–O–C stretching). H NMR (300 MHz,
DMSO-d6, d ppm): 13.86 (s, N–NH–CS), 12.81 (s, 1H, Het–NH–CO–Ar),
8.17–7.52 (m, 5H, Ar–H), 2.51 (s, 3H, –CH3). 13C NMR (100 MHz, DMSO-d6, d
ppm): 189.7, 165.6, 162.8, 157.2, 156.7, 134.4, 132.3, 132.1, 128.9, 128.9, 127.7,
127.7, 17.3. LCMS (m/z): 318.02 (M?). Anal. Calcd. for C13H10N4O2S2: C, 48.85;
H, 3.32; N, 17.46 %. Found: C, 48.79; H, 3.27; N, 17.51 %.
General procedure for the synthesis of N-(4-methyl-5-(4-((arylamino)methyl)-5-
thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)benzamides (5a–k)
A mixture of N-(4-methyl-5-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-
yl)benzamide [compound (4)] (0.01 mol) and aryl amines (0.01 mol) was refluxed
in ethanol (30 mL) with 36 % formaldehyde (0.02 mol) for 3 h. The solid product
generated was filtered off, washed with water, and crystallized from ethanol (99 %),
yield: 58–74 %. All compounds (5a–k) of this series were prepared by using the
same method.
N-(5-(4-(((2-fluorophenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-
yl)-4-methylthiazol-2-yl)benzamide (5a)
Yield: 62 %, mp 139–143 °C. IR (KBr, cm-1): 3352 (N–H stretching, secondary
amine), 3220 (N–H stretching, secondary amide), 2891 (C–H stretching, CH3
group), 2982 (C–H stretching, aromatic ring), 2772 (C–H stretching, –CH2 group),
1704 (C=O stretching, secondary amide), 1552 (C=C stretching, aromatic ring),
1290 (C–H bending), 1194, 1088 (C–O–C stretching), 1118 (C–F stretching), 775
(1:2-substituted benzene ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.17 (s, 1H,
Het–NH–CO–Ar), 8.03–6.58 (m, 9H, Ar–H), 4.44 (s, 2H, Het–CH2), 4.02 (s, 1H,
Ar–NH–CH2), 2.47 (s, 3H, –N–C–(CH3)–C–). 13C NMR (100 MHz, DMSO-d6, d
ppm): 177.8, 166.3, 163.2, 156.2, 155.1, 154.2, 133.9, 132.5, 130.9, 130.9, 129.2,
128.0, 125.7, 120.4, 116.3, 115.1, 104.1, 70.2, 17.4. LCMS (m/z): 441.07 (M?).
Anal. Calcd. for C20H16FN5O2S2; C, 54.41; H, 3.65; N, 15.86 %. Found: C, 54.44,
H-3.60, N-15.80 %.
N-(5-(4-(((4-fluorophenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-
yl)-4-methylthiazol-2-yl)benzamide (5b)
Yield: 74 %, mp 148–152 °C. IR (KBr, cm-1): 3358 (N–H stretching, secondary
amine), 3222 (N–H stretching, secondary amide), 2893 (C–H stretching, CH3
group), 2980 (C–H stretching, aromatic ring), 2774 (C–H stretching, –CH2 group),
1705 (C=O stretching, secondary amide), 1551 (C=C stretching, aromatic ring),
1292 (C–H bending), 1193, 1086 (C–O–C stretching), 1115 (C–F stretching), 810
(1:4-substituted benzene ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.15 (s, 1H,
Het–NH–CO–Ar), 8.03–7.02 (m, 9H, Ar–H), 4.42 (s, 2H, Het–CH2), 4.0 (s, 1H,
Ar–NH–CH2), 2.45 (s, 3H, –N–C-(CH3)–C–). 13C NMR (100 MHz, DMSO-d6, d
ppm): 177.7, 166.1, 163.1, 156.2, 155.5, 154.8, 144.2, 133.9, 132.4, 129.1, 127.8,
119.0, 116.3, 103.7, 70.0, 17.2. LCMS (m/z): 441.09 (M?). Anal. Calcd. for
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N. C. Desai et al.
C20H16FN5O2S2; C, 54.40; H, 3.64; N, 15.85 %. Found: C, 54.46; H, 3.59; N,
15.78 %.
N-(5-(4-(((2-methoxyphenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-
2-yl)-4-methylthiazol-2-yl)benzamide (5c)
Yield: 70 %, mp 139-144 °C. IR (KBr, cm-1): 3357 (N–H stretching, secondary
amine), 3220 (N–H stretching, secondary amide), 2987 (C–H stretching, aromatic
ring), 2896 (C–H stretching, CH3 group), 2778 (C–H stretching, –CH2 group), 2840
(C–OCH3 stretching), 1705 (C=O stretching, secondary amide), 1553 (C=C
stretching, aromatic ring), 1296 (C–H bending), 1191, 1088 (C–O–C stretching),
773 (1:2-substituted benzene ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.13 (s,
1H, Het–NH–CO–Ar), 8.04–6.89 (m, 9H, Ar–H), 4.42 (s, 2H, Het–CH2), 4.13 (s,
1H, Ar–NH–CH2), 3.83 (s, 3H, –OCH3), 2.41 (s, 3H, –N–C–(CH3)–C–). 13C NMR
(100 MHz, DMSO-d6, d ppm): 177.4, 166.2, 163.0, 156.6, 155.1, 144.4, 137.9,
134.1, 132.1, 129.0, 127.9, 121.9, 121.2, 114.3, 113.2, 103.6, 69.9, 56.1, 17.1.
LCMS (m/z): 453.09 (M?). Anal. Calcd. for C21H19N5O3S2; C, 55.61; H, 4.22; N,
15.44 %. Found: C, 55.65; H, 4.16; N, 15.40 %.
N-(5-(4-(((3-methoxyphenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-
2-yl)-4-methylthiazol-2-yl)benzamide (5d)
Yield: 66 %, mp 153–156 °C. IR (KBr, cm-1): 3356 (N–H stretching, secondary
amine), 3222 (N–H stretching, secondary amide), 2987 (C–H stretching, aromatic
ring), 2894 (C–H stretching, CH3 group), 2840 (C–OCH3 stretching), 2777 (C–H
stretching, –CH2 group), 1703 (C=O stretching, secondary amide), 1554 (C=C
stretching, aromatic ring), 1298 (C–H bending), 1190, 1086 (C–O–C stretching),
705 (1:3-substituted benzene ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.13 (s,
1H, Het–NH–CO–Ar), 7.95–6.35 (m, 9H, Ar–H), 4.44 (s, 2H, Het–CH2), 4.14 (s,
1H, Ar–NH–CH2), 3.83 (s, 3H, –OCH3), 2.43 (s, 3H, –N–C-(CH3)–C–). 13C NMR
(100 MHz, DMSO-d6, d ppm): 177.2, 166.0, 162.8, 161.0, 156.5, 155.2, 148.7,
134.2, 132.2, 128.9, 127.8, 110.5, 108.8, 105.6, 105.2, 103.4, 69.7, 55.9, 17.1.
LCMS (m/z): 453.09 (M?). Anal. Calcd. for C21H19N5O3S2; C, 55.61; H, 4.22; N,
15.44 %. Found: C, 55.67; H, 4.28; N, 15.38 %.
N-(5-(4-(((4-methoxyphenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-
2-yl)-4-methylthiazol-2-yl)benzamide (5e)
Yield: 58 %, mp 153–157 °C. IR (KBr, cm-1): 3359 (N–H stretching, secondary
amine), 3221 (N–H stretching, secondary amide), 2986 (C–H stretching, aromatic
ring), 2895 (C–H stretching, CH3 group), 2842 (C–OCH3 stretching), 2775 (C–H
stretching, –CH2 group), 1704 (C=O stretching, secondary amide), 1554 (C=C
stretching, aromatic ring), 1294 (C–H bending), 1194, 1087 (C–O–C stretching),
807 (1:4-substituted benzene ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.13 (s,
1H, Het–NH–CO–Ar), 8.04–6.89 (m, 9H, Ar–H), 4.42 (s, 2H Het–CH2), 4.12 (s, 1H,
Ar–NH–CH2), 3.83 (s, 3H, –OCH3), 2.43 (s, 3H, –N–C–(CH3)–C–). 13C NMR
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Synthesis, characterization and antimicrobial screening…
(100 MHz, DMSO-d6, d ppm): 177.2, 166.0, 162.7, 156.6, 155.1, 152.0, 140.1,
134.1, 132.0, 128.9, 127.7, 115.6, 115.2, 103.3, 69.5, 56.0, 17.1. LCMS (m/z):
453.09 (M?). Anal. Calcd. for C21H19N5O3S2; C, 55.61; H, 4.22; N, 15.44 %.
Found: C, 55.64; H, 4.18; N, 15.40 %.
N-(4-methyl-5-(4-(((2-nitrophenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-
oxadiazol-2-yl)thiazol-2-yl)benzamide (5f)
Yield: 68 %, mp 155–160 °C. IR (KBr, cm-1): 3357 (N–H stretching, secondary
amine), 3224 (N–H stretching, secondary amide), 2988 (C–H stretching, aromatic
ring), 2896 (C–H stretching, CH3 group), 2774 (C–H stretching, –CH2 group), 1702
(C=O stretching, secondary amide), 1554 (C=C stretching, aromatic ring), 1543
(C=N stretching, aromatic ring), 1520 (N=O stretching), 1295 (C–H bending), 1195,
1086 (C–O–C stretching), 780 (1:2-di substituted benzene ring). 1H NMR
(300 MHz, DMSO-d6, d ppm): 9.12 (s, 1H, Het–NH–CO–Ar), 8.14–6.78 (m, 9H,
Ar–H), 4.49 (s, 2H, Het–CH2–NH–), 4.14 (s, 1H, Ar–NH–CH2), 2.46 (s, 3H, –N–C–
(CH3)–C–). 13C NMR (100 MHz, DMSO-d6, d ppm): 177.0, 165.8, 162.8, 156.5,
155.3, 146.2, 135.8, 134.2, 132.1, 131.5, 128.9, 127.5, 125.7, 118.2, 114.6, 103.0,
68.9, 16.9. LCMS (m/z): 468.07 (M?). Anal. Calcd. for C20H16N6O4S2; C, 51.27; H,
3.44; N, 17.94 %. Found: C, 51.24; H, 3.41; N, 17.90 %.
N-(4-methyl-5-(4-(((3-nitrophenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-
oxadiazol-2-yl)thiazol-2-yl)benzamide (5g)
Yield: 66 %, mp 164–168 °C. IR (KBr, cm-1): 3356 (N–H stretching, secondary
amine), 3223 (N–H stretching, secondary amide), 2985 (C–H stretching, aromatic
ring), 2893 (C–H stretching, –CH3 group), 2773 (C–H stretching, –CH2 group),
1701 (C=O stretching, secondary amide), 1556 (C=C stretching, aromatic ring),
1541 (C=N stretching, aromatic ring), 1522 (N=O stretching, aromatic ring), 1293
(C–H bending), 1196, 1085 (C–O–C stretching), 700 (1:3-di substituted benzene
ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.10 (s, 1H, Het–NH–CO–Ar),
8.15–7.20 (m, 9H, Ar–H), 4.50 (s, 2H, Het–CH2–NH–), 4.15 (s, 1H, Ar–NH–CH2),
2.40 (s, 3H, –N–C–(CH3)–C–). 13C NMR (100 MHz, DMSO-d6, d ppm): 176.9,
165.4, 162.9, 156.6, 155.2, 148.9, 148.4, 134.0, 132.1, 130.5, 128.8, 127.6, 119.7,
112.2, 106.6, 103.2, 69.2, 17.0. LCMS (m/z): 468.07 (M?). Anal. Calcd. for
C20H16N6O4S2; C, 51.27; H, 3.44; N, 17.94 %. Found: C, 51.31; H, 3.48; N,
17.88 %.
N-(4-methyl-5-(4-(((4-nitrophenyl)amino)methyl)-5-thioxo-4,5-dihydro-1,3,4-
oxadiazol-2-yl)thiazol-2-yl)benzamide (5h)
Yield: 65 %, mp 172–177 °C. IR (KBr, cm-1): 3358 (N–H stretching, secondary
amine), 3225 (N–H stretching, secondary amide), 2986 (C–H stretching, aromatic
ring), 2894 (C–H stretching, –CH3 group), 2775 (C–H stretching, –CH2 group),
1703 (C=O stretching, secondary amide), 1558 (C=C stretching, aromatic ring),
1542 (C=N stretching, aromatic ring), 1523 (N=O stretching, aromatic ring), 1295
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N. C. Desai et al.
(C–H bending), 1198, 1088 (C–O–C stretching), 806 (1:4-di substituted benzene
ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.12 (s, 1H, Het–NH–CO–Ar),
7.06–6.98 (m, 9H, Ar–H), 4.48 (s, 2H, Het–CH2–NH–), 4.16 (s, 1H, Ar–NH–CH2),
2.44 (s, 3H, –N–C–(CH3)–C–). 13C NMR (100 MHz, DMSO-d6, d ppm): 177.0,
165.4, 163.0, 156.6, 155.2, 153.8, 135.9, 134.1, 132.0, 128.9, 127.6, 127.4, 114.6,
103.1, 69.1, 17.0. LCMS (m/z): 468.07 (M?). Anal. Calcd. for C20H16N6O4S2; C,
51.28; H, 3.47; N, 17.91 %. Found: C, 51.25; H, 3.42; N, 17.97 %.
N-(4-methyl-5-(5-thioxo-4-((o-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-
yl)thiazol-2-yl)benzamide (5i)
Yield: 59 %, mp 143–148 °C. IR (KBr, cm-1): 3355 (N–H stretching, secondary
amine), 3223 (N–H stretching, secondary amide), 2985 (C–H stretching, aromatic
ring), 2892 (C–H stretching, –CH3 group), 2848 (C–CH3 stretching), 2773 (C–H
stretching, –CH2 group), 1703 (C=O stretching, secondary amide), 1558 (C=C
stretching, aromatic ring), 1294 (C–H bending), 1196, 1087 (C–O–C stretching),
775 (1:2-di substituted benzene ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.13
(s, 1H, Het–NH–CO–Ar), 7.92–6.63 (m, 9H, Ar–H), 4.48 (s, 2H, Het–CH2–NH–),
4.13 (s, 1H, Ar–NH–CH2), 2.41 (s, 3H, –N–C–(CH3)–C–), 2.12 (s, 3H, C–CH3). 13
C
NMR (100 MHz, DMSO-d6, d ppm): 177.1, 165.4, 163.1, 156.5, 155.0, 146.7,
134.0, 132.2, 128.9, 127.6, 127.1, 126.6, 122.2, 121.8, 110.4, 103.1, 68.8, 17.8,
17.1. LCMS (m/z): 437.10 (M?). Anal. Calcd. for C21H19N5O2S2; C, 57.65; H, 4.38;
N, 16.01 %. Found: C, 57.69; H, 4.34; N, 16.0 %.
N-(4-methyl-5-(5-thioxo-4-((m-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-
yl)thiazol-2-yl)benzamide (5j)
Yield: 60 %, mp 159–164 °C. IR (KBr, cm-1): 3354 (N–H stretching, secondary
amine), 3225 (N–H stretching, secondary amide), 2983 (C–H stretching, aromatic
ring), 2894 (C–H stretching, –CH3 group), 2850 (C–CH3 stretching), 2771 (C–H
stretching, –CH2 group), 1700 (C=O stretching, secondary amide), 1553 (C=C
stretching, aromatic ring), 1291 (C–H bending), 1194, 1083 (C–O–C stretching),
703 (1:3-di substituted benzene ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.09
(s, 1H, Het–NH–CO–Ar), 8.02–6.65 (m, 9H, Ar–H), 4.48 (s, 2H, Het–CH2–NH–),
4.14 (s, 1H, Ar–NH–CH2), 2.42 (s, 3H, –N–C–(CH3)–C–), 2.33 (s, 3H, C–CH3). 13
C
NMR (100 MHz, DMSO-d6, d ppm): 177.0, 165.5, 162.8, 156.2, 155.3, 147.7,
139.4, 134.2, 132.1, 129.3, 128.8, 127.5, 117.2, 113.6, 110.4, 103.0, 68.5, 21.5,
17.1. LCMS (m/z): 437.10 (M?). Anal. Calcd. for C21H19N5O2S2; C, 57.65; H, 4.38;
N, 16.01 %. Found: C, 57.69; H, 4.42; N, 16.06 %.
N-(4-methyl-5-(5-thioxo-4-((p-tolylamino)methyl)-4,5-dihydro-1,3,4-oxadiazol-2-
yl)thiazol-2-yl)benzamide (5k)
Yield: 60 %, mp 152–157 °C. IR (KBr, cm-1): 3357 (N–H stretching, secondary
amine), 3224 (N–H stretching, secondary amide), 2984 (C–H stretching, aromatic
ring), 2896 (C–H stretching, –CH3 group), 2850 (C–CH3 stretching), 2772 (C–H
123

Synthesis, characterization and antimicrobial screening…
stretching, –CH2 group), 1702 (C=O stretching, secondary amide), 1555 (C=C
stretching, aromatic ring), 1292 (C–H bending), 1195, 1082 (C–O–C stretching),
818 (1:4-di substituted benzene ring). 1H NMR (300 MHz, DMSO-d6, d ppm): 9.08
(s, 1H, Het–NH–CO–Ar), 8.01–6.60 (m, 9H, Ar–H), 4.49 (s, 2H, Het–CH2–NH–),
4.16 (s, 1H, Ar–NH–CH2), 2.39 (s, 3H, –N–C–(CH3)–C–), 2.35 (s, 3H, C–CH3). 13
C
NMR (100 MHz, DMSO-d6, d ppm): 177.2, 165.9, 162.8, 156.4, 155.2, 144.8,
134.3, 132.2, 129.8, 129.4, 128.7, 127.6, 113.1, 102.8, 68.7, 21.4, 17.1. LCMS
(m/z): 437.10 (M?). Anal. Calcd. for C21H19N5O2S2; C, 57.65; H, 4.38; N, 16.01 %.
Found: C, 57.68; H, 4.45; N, 16.06 %.
Result and discussion
Spectral characterization
Characterization of newly synthesized compounds of the series was carried out by
IR, NMR, and mass spectra, and the data with a detailed discussion are given in the
experimental section (Fig. 3).
Fig. 3 Synthetic pathway of compounds (5a–k)
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N. C. Desai et al.
IR DATA of compound (5g)
IR spectrum of compound 5g (molecular formula C20H16N6O4S2, M.W. 468.51
gm/mol) has a stretching vibration at 3356 cm-1 indicating the presence of N–H
stretching of secondary amine. Stretching vibration at 3223 cm-1 is an indication of
the presence of N–H stretching of secondary amide. The absorption band at
3016 cm-1 indicates the presence of Ar–H stretching vibrations. C–H stretching at
2893 cm-1 shows the presence of the –CH3 group of the thiazole ring. Stretching at
2773 cm-1 is the proof of a –CH2 group in compound 5g. Absorption bands at 1556,
1541, and 1522 cm-1 showed the presence of C=C, C=N, and N=O stretching of the
aromatic ring. Also C–H bending at 1293 cm-1 showed conformation of the
aromatic ring. Stretching at 1196 and 1085 cm-1 represent C–O–C bending
vibration. The deformation vibration at 812 and 670 is due to presence of nitro
group at the third position in our final compound 5g (Fig. 4).
1H-NMR spectra of compound (5g)
1
The H-NMR spectra of the final compound 5g showed that protons were attached
at C-15, giving a shift at d = 2.40 (–N–C–(CH3)–C–) with a singlet peak and proton
of (Ar–NH–CH2) appearing as a singlet at d = 4.15. In the heterocyclic ring, two
protons showed a chemical shift at d = 4.50 due to heterocyclic ring –CH2–NH,
while protons of the phenyl ring gave multiples at d = 7.20-8.15 (m, 9H, Ar–H) and
heterocyclic protons at position C-8 gave a singlet at d = 9.10 ppm (s, 1H, Het–
NH–CO) (Fig. 4).
13C-NMR spectra of compound (5g)
The chemical shifts of the final compound 5g has carbons that varied from
d = 176.9–17.0 ppm. The carbon nuclei under the influence of a strong elec-
tronegative environment appeared downfield. Carbon of the amide (Ar–CONH)
group has a chemical shift at d = 165.4. The carbon of phenyl group at C-5 attached
with amide group has a chemical shift value at d = 132.1. The carbons of the
thiazole ring of carbon C-9, C-11, and C-12 showed chemical shifts at d = 162.9,
103.2, and 156.6, respectively. The methyl group attached with the C-12 carbon
Fig. 4 Compound 5g
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Synthesis, characterization and antimicrobial screening…
atom in the thiazole ring system showed a chemical shift at 17.0 because of its de-
shielding effect. 1,3,4-oxadiazole ring carbons C-16 and C-18 appeared with
chemical shifts at d = 155.2 and 176.9, respectively. Methylene carbon C-22,
which is attached on both sides to nitrogen atoms, appeared as a chemical shift at
d = 69.2. Aromatic carbon (C-24 to C-29) showed a chemical shift value between
106.6 and 148.9. The nitro group at C-26 in the phenyl ring gave a chemical shift at
d = 148.9.
Discussion of antimicrobial screening
All the newly synthesized compounds (5a–k) were initially evaluated for in vitro
antibacterial activity against gram- positive bacteria [Staphylococcus aureus
(MTCC-96), Streptococcus pyogenes (MTCC-442)] and gram-negative bacteria
[Escherichia coli (MTCC-443), Pseudomonas aeruginosa (MTCC-1688)] using the
conventional broth dilution method. The individual minimal inhibitory concentra-
tion (MIC, lg/mL) values of test compounds are listed in Table 1 along with MIC
values of the reference compound ampicillin. From screening results, it was
observed that final compounds 5c (–2–OCH3–C6H4), 5i (2–CH3–C6H4) exhibited
good activity against E. coli and S. aureus. Compound 5d (–3–OCH3–C6H4)
showed good activity against P. aeruginosa. Compound 5e (–4–OCH3–C6H4),
showed excellent activity against E. coli, P. aeruginosa and S. aureus with a two to
fourfold higher MIC value (12.5–25 lg/mL) than the reference drug. Very good
activity was shown by compound 5j (3–CH3–C6H4) against P. aeruginosa and S.
aureus. Compound 5k (4–CH3–C6H4) displayed excellent activity against S. aureus
and S. pyogenes. The remaining compounds of the series possessed moderate
antibacterial activity. On the other hand, the presence of similar functional groups at
the meta position resulted in minor changes in antibacterial activity as compared to
5c, 5i, 5e and 5k.
Compounds (5a–k) were also evaluated for their in vitro antifungal activity
against three fungal strains, C. albicans, A. niger, and A. clavatus at various
concentrations of 1000, 500, 200, and 100 lg/mL as shown in (Table 1). The data
of antimicrobial evaluation of compounds (5a–k) were collected in Table 1. The
results of antifungal activity indicated that compound 5a (–2–F–C6H4) showed good
activity against C. albicans, and A. clavatus. Compound 5a (–2–F–C6H4) exhibited
very good activity against A. niger. While compound 5b (–4–F–C6H4) showed
excellent activity against C. albicans, A. niger and A. clavatus with a twofold higher
MIC value (12.5–25 lg/mL) than the reference drug. The remaining compounds of
the series showed feeble antifungal activity. Thus, we have discussed and compared
antibacterial and antifungal activities based on standard drugs ampicillin and
griseofulvin, respectively.
Determination of antibacterial activity
Antibacterial studies of newly synthesized compounds (5a–k) were carried out
against the representative panel of gram-positive [S. aureus (MTCC-96), S.
pyogenes (MTCC-442)] and gram-negative [E. coli (MTCC-443), P. aeruginosa
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N. C. Desai et al.
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Synthesis, characterization and antimicrobial screening…
(MTCC-1688)] bacteria. All MTCC cultures were collected from the Institute of
Microbial Technology, Chandigarh. The activity of compounds was determined as
per the National Committee for Clinical Laboratory Standards (NCCLS) protocol
using Mueller–Hinton Broth (Becton–Dickinson, USA) [32–35]. Primary screening
was done first for antibacterial activity in six sets against E. coli, S. aureus, P.
aeruginosa and S. pyogenes at different concentrations of 1000, 500, and
250 lg/mL. The compounds found to be active in primary screening were similarly
diluted to obtain 200, 125, 100, 62.5, 50, 25, and 12.5 lg/mL concentrations for
secondary screening to test in a second set of dilutions against all microorganisms.
Inoculum size for test strains was adjusted to 106 CFU/mL (Colony Forming Units
per milliliter) by comparing the turbidity (turbidimetric method). Mueller–Hinton
Broth was used as a nutrient medium to grow and dilute the compound suspension
for test organisms. Then 2 % DMSO was used as a diluent/vehicle to obtain the
desired concentration of synthesized compounds and standard drugs to test upon
standard microbial strains. Synthesized compounds were diluted to 1000 lg/mL
concentration as stock solution. The control tube containing no antibiotic was
immediately subcultured (before inoculation) by spreading a loopful evenly over
quarter of a plate of medium suitable for the growth of test organisms. The culture
tubes were then incubated for 24 h at 37 °C and the growth was monitored visually
and spectrophotometrically. The 10 lg/mL suspensions were further inoculated on
an appropriate media, and growth was noted after 24 and 48 h. The lowest
concentration (highest dilution) required to arrest the growth of bacteria was
regarded as the minimum inhibitory concentration (MIC), i.e., the amount of growth
from the control tube before incubation (which represents the original inoculum)
that was compared. Solvent had no influence on strain growth. The result of this was
greatly affected by the size of the inoculum. The test mixture contained 106 CFU/
mL organisms. DMSO and sterilized distilled water were used as a negative control
while ampicillin antibiotic (1 U strength) was used as a positive control. The
standard drug used in the present study was ‘ampicillin’ for evaluating antibacterial
activity which showed 100, 100, 250, and 100 lg/mL MIC against E. coli, P.
aeruginosa, S. aureus and S. pyogenes, respectively.
Determination of antifungal activity
The newly prepared compounds (5a–k) were screened for their antifungal activity
as primary screens in six sets against C. albicans, A. niger, and A. clavatus at
various concentrations of 1000, 500, and 250 lg/mL. The primary active
compounds were similarly diluted to obtain 200, 125, 100, 62.5, 50, 25, and
12.5 lg/mL concentrations for secondary screening to test in a second set of
dilutions against all fungi. The fungal activity of each compound was compared
with ‘griseofulvin’ as a standard drug, which showed 500, 100, and 100 lg/mL MIC
against C. albicans, A. niger, and A. clavatus, respectively. For fungal growth, in the
present protocol, we have used Sabouraud’s dextrose broth at 28 °C in aerobic
condition for 48 h. DMSO and sterilized distilled water were used as negative
control while griseofulvin (1 U strength) was used as a positive control.
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N. C. Desai et al.
SAR studies
The structure–activity relationships (SAR) of compounds (5a–k) were determined
on the basis of results presented in Table 1. The substitution pattern on the thiazole
based 1,3,4-oxadiazole ring system derivatives was carefully selected to confer a
different electronic environment of the molecules. SAR studies revealed that the
presence of the thiazole ring was essential for a broad range of antimicrobial
activity. From the activity data, compounds containing electron-donating substitu-
tions in a skeleton led to an increase in antimicrobial activity. Compound 5e and 5k
containing 4–OCH3 and 4–CH3 groups showed the highest inhibition at
MIC = 12.5–25 lg mL-1 against bacterial strain S. aureus and S. pyogenes, and
compound 5e 4–OCH3 group showed the highest inhibition at MIC =
12.5 lg mL-1 against E. coli. On the basis of the screening results, it has been
observed that the compound 5b exhibited excellent inhibitory action against all
fungal strains while compound 5a also showed reasonably good inhibition against
C. albicans, A. niger, and A. clavatus. The SAR study indicates that compounds
containing electron releasing groups at the para position increases bacterial activity
while the presence of electron withdrawing group at the same position lead to
enhanced fungal activity.
Conclusion
We have synthesized new thiazole-based 1,3,4-oxadiazoles derivatives with
potential antimicrobial activity from available the starting material ethyl 2-amino-
4-methylthiazole-5-carboxylate by routine methods. The new compounds were
tested for their antimicrobial activity and majority of the compounds showed
significant antimicrobial activity. The antimicrobial activity data of the prepared
compounds indicated that electron donating groups such as 5e (methoxy) and 5k
(methyl) groups increased antibacterial activity and electron withdrawing groups
such as fluoro, and nitro groups at the para position 5b increased antifungal activity.
Electron withdrawing groups such as fluoro, nitro on the aromatic ring, and the
electron donating group such as methoxy and methyl that were taken as substitutes
in the chemical structure of the target compounds increased activity against
antibacterial and antifungal strains. Results of biological activities of novel thiazole
based 1,3,4-oxadiazoles derivatives are interesting for optimization of these leading
molecules for further generation of antimicrobial agents.
Acknowledgments The authors are thankful to the University Grants Commission, New Delhi and
Department of Science and Technology, New Delhi for financial support under the NON-SAP and DST-
FIST programs, respectively.
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