Design and development of some thiazole-based flavanoids as novel antibacterial against pathogens causing surgical site infection for possible benefit in bone trauma via inhibition of DNA gyrase

Article abstract of DOI:10.1111/cbdd.12999

In this study, a novel class of hybrid thiazole-based flavanoid derivatives were synthesized and characterized by FT-IR, ¹H-NMR, ¹³C-NMR, mass and elemental analysis. These derivatives were evaluated for antibacterial activity for possible benefit in bone trauma via inhibition of DNA gyrase enzyme. Results suggested that compounds 9n, 9o, and 9p showed considerable inhibition of DNA gyrase with considerable activity against tested forty strains of Staphylococcus aureus clinical isolates. Moreover, compound 9n showed hydrogen bonding with LYS460 along with low binding free energy of ?4.36?kcal/mol against DNA gyrase enzyme. The hemolytic activity of the potent compounds showed mild to no activity together with excellent pharmacokinetics, suggesting to have a potential for the development of designed compounds as novel antibacterial agents.

Full text of DOI:10.1111/cbdd.12999

DR. GANG ZHAO (Orcid ID : 0000-0003-2624-3003)
Article type : Research Article
Design and development of some thiazole-based flavanoids as novel antibacterial
against pathogens causing surgical site infection for possible benefit in bone trauma via
inhibition of DNA gyrase
Gang Zhao1#*, Dengzhe Lan2#, Changfeng Yue3, Guobao Qi4*
1Department of Traumatic Orthopedics, Dongying People's Hospital, Dongying, Shandong
257091, China
2Department of Hand and Foot Surgery, Dongying People's Hospital, Dongying, Shandong
257091, China
3Department of Orthopaedics, Dongying People's Hospital, Dongying, Shandong 257091,
China
4Department of Spinal Surgery, Dongying People's Hospital, Dongying, Shandong 257091,
China
#: These two authors contribute this work equally
*Corresponding author:
Gang Zhao,
Department of Traumatic Orthopedics,
Dongying People's Hospital, Dongying, Shandong 257091, China
Tel/Fax: 0086- 546-8331577
Email: gangzhao134@hotmail.com
Running Head title: Antibacterial activity of hybrid thiazole-based flavanoids derivatives.
Abstract
In the present study, a novel class of hybrid thiazole based flavanoids derivatives were
1
synthesized and charactered by FT-IR, H-NMR, 13C-NMR, mass and elemental analysis.
These derivatives were evaluated for antibacterial activity for possible benefit in bone trauma
via inhibition of DNA gyrase enzyme. Results suggested that compounds 9n, 9o and 9p
showed considerable inhibiton of DNA gyrase with considerable activity against tested
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12999
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fourty-strains of S. aureus clinical isolates. Moreover, compound 9n showed hydrogen
boinding with LYS460 along with low binding free energy of -4.36 kcal/mol against DNA
gyrase enzyme. The haemolytic activity of the potent compounds showed mild to no activity
together with excellent pharmacokinetics, suggesting to have a potential for the development
of designed compounds as novel antibacterial agents.
Keywords: Thiazole, flavanoid, docking, antibacterial activity, DNA gyrase .
1. Introduction
The danger of injuries has resulted now into global burden and estimated as the major cause
of the mortality or disability of the affected person either for the short-period or for the whole
life [1,2]. As per the estimates, the injuries in the developing nations responsible for more the
10% of the disease burden of the world [3]. It was also estimated that, in next 10 years, this
figure rose to 60-million affected people together with mortality of more than 5-million
people [4]. The majority of deaths in injuries are deemed to be as a result of sepsis, which
further causes infection [5]. It followed by the traumatic injury with an increase in mortality
and morbidity, and often leads to longer hospital residence time [6].
The use of antibiotics in the treatment of open fractures is frequently used as a first line
medication to prevent sepsis in both developed and developing countries [7]. In contrast to
the developed nation; in resource-poor countries, the antibiotics may not be used because of
various factors, such as; higher costs, availability and lack of proper knowledge which
collectively aggravate the complications of the injury [8]. Antibiotic resistance is another
cause which greatly contributes to the improper management of traumatic injuries and often
compromise the treatment of open fracture [9].
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Therefore, the discovery of novel antibacterial agents could serve the purpose. The use of
plant based medication against the various ailments have been well documented in many
civilizations across the world, and increases its acceptability among the common masses of
the peoples [10, 11].
Flavnoids are class of natural compound which classified as polyphenolic derivative that
found in many plant as low-molecular weight metabolites [12]. These are known as
aglycones, glycosides, and methylated analogues which reported to exhibit numerous
pharmacological properties, for instance, anticancer [13], anti-inflammatory [14], antioxidant
[15] and more importantly antibacterial activity [16]. Thiazoles are also showed to exhibit
neumerous pharmacological activities [17]. Concerning this, in the present study, we wish to
evaluate the effect of combination of some thiazole with flavanoids as antibacterial agents for
possible benefit in bone trauma.
2. Material and methods
2.1.Chemistry
The chemicals used in the present study were procured from Sigma Aldrich (USA). The
spectra of 1H NMR and 13C NMR were recorded on Bruker Avance 400 and Bruker Avance
100 Spectrophotometer, respectively. The chemical shifts are expressed in parts per million
(ppm), and coupling constants are expressed in Hertz (Hz). The Low-resolution mass
spectrum (MS) was recorded on a Waters ZQ LC/MS single quadrupole system equipped
with an electrospray ionization (ESI) source. The elemental analysis of the final derivatives
was performed via Vario Elemental analyser. The Thin-layer chromatography was
performed on 0.25 mm Merck silica gel plates (60F-254) and visualized under UV light.
2.1.1. Synthesis of 3-Hydroxy-2-methyl-4-(3-oxo-3-phenylpropanoyl)phenyl benzoate (2)
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1-(2,4-dihydroxy-3-methylphenyl)ethanone (1) (20 mmol) was dissolved in acetone (60 mL)
and benzoyl chloride (20 mmol) was added in above solution using K2CO3 as base. After that
the resulting mixture was refluxed for 12 hr. The mixture was filtered and resulting
precipitate was decomposed with 10% CH3COOH to afford compound 2.
o
Yield: 71%; MP: 145-146 C; MW: 374.39; Rf: 0.61; FT-IR (νmax; cm-1 KBr): 3583 (OH),
3084 (aromatic C-H), 2981 (aliphatic C-H), 1719 (C=O), 1622 (C=C), 1132 (aromatic C-C)
734 cm -1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 8.21 (d, 2H, J = 8.4 Hz, Ar-H), 7.48 (d,
2H, J = 7.5 Hz, Ar-H), 7.63 (d, 1H, J = 7.2 Hz, Ar-H), 7.94(d, 1H, J = 8.6 Hz, Ar-H), 7.54 (d,
1H, J = 7.1 Hz, Ar-H), 7.61 (d, 1H, J = 6.8 Hz, Ar-H), 7.65 (d, 1H, J = 8.2 Hz, Ar-H), 7.35
(d, 1H, J = 7.8 Hz, ArH), 5.36 (s, 1H, Ar-OH), 3.89 (s, 2H, Methylene), 2.16 (s, 3H, CH3) ;
13C-NMR (100MHz, CDCl3) δ,ppm:201.2 (Ar-OH-C=O), 190.4 (Ar-C=O), 165.2 (Ar-COO),
163.2 (Ar-OH-C), 156.3 (Ar-COO-C), 136.8 (Ar-CO-C), 133.9 (Ar-4C), 133.2 (Ar-4C),
130.4 (Ar-2,6C), 130.1 (Ar-1C), 128.9 (Ar-2,6C), 128.4 (Ar-3,5C), 127.6 (Ar-OH 5C), 118.6
(Ar-OH 4C), 114.2 ((Ar-OH 2,6C), 52.1 (CH2), 10.2 (CH3); Mass :375.46 (M+1); Elemental
analysis for C23H18O5: Calculated: C, 73.79; H, 4.85; Found: C, 73.72; H, 4.92.
2.1.2. Synthesis of 8-Methyl-4-oxo-2-phenyl-4H-chromen-7-yl benzoate (3)
The compound 2 (20 mmol) was treated with NaOAc/HOAc (8g/40mL) and resulting
mixture was refluxed for 12 h and cooled to room temperature. The water was added to the
above mixture and product was precipitated, filtered and washed with saturated solution of
NaHCO3 and cold water to furnish compound 3.
o
Yield: 68%; MP: 153-154 C; MW: 356.37; Rf: 0.65; FT-IR (νmax; cm-1 KBr): 3087
(aromatic C-H), 2978 (aliphatic C-H), 1723 (C=O), 1628 (C=C), 1137 (aromatic C-C) 1145
-1
(C-O-C), 738 cm ; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 8.23 (d, 2H, J = 8.4 Hz, Ar-
H), 8.02 (d, 1H, J = 7.5 Hz, Ar-H), 7.78 (d, 2H, J = 7.8 Hz, Ar-H), 7.71 (d, 1H, J = 7.3 Hz,
Ar-H), 7.61 (d, 2H, J = 7.4 Hz, Ar-H), 7.43 (d, 1H, J = 7.1 Hz, ArH), 7.42 (d, 2H, J = 7.2 Hz,
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Ar-H), 7.34 (d, 1H, J = 1.4 Hz, Ar-H), 6.73 (s, 1H, ArH), 2.18 (s, 3H, CH3); 13C-NMR
(100MHz, CDCl3) δ,ppm:178.2 (flavone 4C=O), 165.3 (Ar-COO), 163.7 (flavone 2C), 157.8
(flavone 9C), 156.9 (flavone 7C), 133.8 (Ar-4C), 130.4 (flavone Ar-1C; Ar-2,6C), 130.1 (Ar-
1C), 129.9 (flavone 5C), 128.7 (flavone-Ar-3,5 C), 127.8 (flavone Ar-2,4,6C), 122.5 (flavone
8C), 120.7 (flavone 10C), 116.3 (flavone 6C), 104.7 (flavone 3C), 10.2 (CH3); Mass :357.28
(M+1); Elemental analysis for C23H16O4: Calculated: C, 77.52; H, 4.53; Found: C, 77.59; H,
4.48.
2.1.3. Synthesis of 7-Hydroxy-8-methyl-2-phenyl-4H-chromen-4-one (4)
Compound 3 (20 mmol) was dissolved in 40 mL methanol. The resulting solution was stirred
at room temperature for 5-6 h and then 30 mL water was added. The product was
precipitated, filtered and recrystallized from ethanol to give product 4
o
Yield: 74%; MP: 134-135 C; MW: 252.26; Rf: 0.69; FT-IR (νmax; cm-1 KBr): 3587 (OH),
3089 (aromatic C-H), 2972 (aliphatic C-H), 1728 (C=O), 1632 (C=C), 1139 (aromatic C-C)
1143 (C-O-C), 731 cm -1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 7.76 (d, 2H, J = 7.5 Hz,
Ar-H), 7.47 (d, 2H, J = 7.2 Hz, Ar-H), 7.38 (d, 1H, J = 8.4 Hz, Ar-H), 7.31 (d, 1H, J = 7.1
Hz, ArH), 7.02 (d, 1H, J = 8.1 Hz, Ar-H), 6.71 (s, 1H, ArH), 5.36 (s, 1H, Ar-OH), 2.18 (s,
3H, CH3) ; 13C-NMR (100MHz, CDCl3) δ,ppm:178.2 (flavone 4C=O, 166.1 (flavone 7C),
163.7 (falvone 2C), 158.9 (flavone 9C), 130.4 (flavone-Ar-1C), 128.7 (flavone-Ar-3,5C),
127.8 (flavone-Ar-2,4,6C), 125.2 (flavone 5C), 116.5 (flavone 10C), 110.6 (flavone 6C),
107.4 (flavone 8C), 104.5 (flavone 3C), 10.2 (CH3); Mass :253.34 (M+1); Elemental analysis
for C16H12O3: Calculated: C, 76.18; H, 4.79; Found: C, 76.23; H, 4.68.
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2.1.4. Synthesis of 7-(2-bromoethoxy)-8-methyl-2-phenyl-4H-chromen-4-one (5)
Compound 4 (5 mmol) was dissolved in DMF 30 mL and 1,2,-dibromoethane (5 mmol) was
added to above solution using K2CO3 (5 mmol) as base to maintain the alkaline medium. The
resulting mixture was stirred at room temperature for 4-5 h the product was precipitated,
filtered and recrystallized from ethanol to afford compound 5.
o
Yield: 66%; MP: 182-183 C; MW: 359.21; Rf: 0.77; FT-IR (νmax; cm-1 KBr): 3083
(aromatic C-H), 2975 (aliphatic C-H), 1732 (C=O), 1628 (C=C), 1141 (aromatic C-C) 1137
-1
1
(C-O-C), 708 (C-Br), 726 cm ; H-NMR (400MHz, DMSO, TMS) δ ppm: 7.75 (d, 2H, J =
7.5 Hz, Ar-H), 7.45 (d, 2H, J = 7.2 Hz, Ar-H), 7.43 (d, 1H, J = 8.2 Hz, Ar-H), 7.32 (d, 1H, J
= 6.9 Hz, ArH), 6.98 (d, 1H, J = 7.7 Hz, Ar-H), 6.73 (s, 1H, ArH), 4.34 (d, 2H, 1H, J = 6.4
Hz, CH2-CH2 linkage), 3.78 (d, 2H, 1H, J = 6.1 Hz, CH2-CH2 linkage), 2.18 (s, 3H, CH3);
13C-NMR (100MHz, CDCl3) δ,ppm:177.6 (flavone 4C=O), 164.1 (flavone 7C), 163.8
(flavone 2C), 158.6 (flavone 9C), 130.4 (flavone-Ar-1C), 128.7 (flavone-Ar-3,5C), 127.8
(flavone-Ar-2,4,6C), 124.9 (flavone 5C), 115.6 (flavone 10C), 115.2 (flavone 8C), 109.2
(flavone 6C), 104.6 (flavone 3C), 68.6 (CH2), 28.2 (CH2-Br), 9.8 (CH3); Mass :360.25
(M+1); Elemental analysis for C18H15BrO3: Calculated: C, 60.18; H, 4.21; Found: C, 60.21;
H, 4.26.
2.1.5. General procedure for synthesis of 4-(4-subsituted phenyl)thiazole-2-yl)amine 8 (a-p)
Diversly substituted acetophenone 6 (a-p) (110 mmol) and thiourea (7) (220 mmol) were
mixed, after that bromine (110 mmol) was added in above mixture in small fractions (0.5
mL) over 2 h with stirring. The resulting mixture was heated on a water bath for 12 h. After
that 20 mL water was added and heated until most of the solid had gone into solution and
filtered while hot. The filtrate was cooled to room temperature and ammonia solution
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(concentrated) was added till the precipitation ceases off. The resulting product was
recrystallized with pyridine–water (1:1) mixture to give products 8 (a-p) [18].
2.1.6. General procedure for synthesis of title compounds 9 (a-p).
Corresponding derivative 8 (a-p) (0.1 mol), compound 5 and K2CO3 (0.1 mol) were added
o
into 40 mL of 1,4-dioxane. The resulting mixture was refluxed at 135–145 C for 9 h. The
product was filtered and washed with water and recrystallized with ethanol to afford the title
compounds 9 (a-p).
2.1.6.1.8-Methyl-2-phenyl-7-(2-((4-phenylthiazol-2-yl)amino)ethoxy)-4H-chromen-4-one
(9a)
o
Yield: 69%; MP: 221-223 C; MW: 454.54; Rf: 0.72; FT-IR (νmax; cm-1 KBr): 3398 (N–H),
3083 (aromatic C-H), 2971 (aliphatic C-H), 1725 (C=O), 1627 (C=C), 1511 (N–H), 1141
-1
1
(aromatic C-C) 1144 (C-O-C), 1032 (C–N, aromatic-NH-aromatic), 637 (C–S) cm ; H-
NMR (400MHz, DMSO, TMS) δ ppm: 7.82 (d, 2H, J = 7.7 Hz, Ar-H), 7.76 (d, 2H, J = 7.5
Hz, Ar-H), 7.55 (d, 2H, J = 7.4 Hz, Ar-H), 7.45 (d, 1H, J = 8.4 Hz, Ar-H), 7.40 (d, 2H, J =
7.1 Hz, Ar-H), 7.38 (d, 1H, J = 6.9 Hz, Ar-H), 7.32 (s, 1H, Thiazole), 7.31 (d, 1H, J = 6.7 Hz,
ArH), 6.94 (d, 1H, J = 7.3 Hz, Ar-H), 6.72 (s, 1H, ArH), 4.32 (d, 2H, J = 5.7 Hz,
13
Methylene), 3.98 (s, 1H, NH), 3.72 (d, 2H, J = 5.4 Hz, Methylene), 2.16 (s, 3H, CH3); C-
NMR (100MHz, CDCl3) δ,ppm:177.6 (flavone 4C=O), 164.8 (thiazole 2C), 163.8 (flavone
7C), 163.7 (flavone 2C), 158.2 (flavone 9C), 150.3 (thiazole 4C), 133.3 (thiazole Ar-1C),
130.4 (flavone Ar-1C), 129.3 (thiazole Ar-3,5C), 128.7 (thiazole Ar 4C), 128.5 (flavone Ar-
3,5C), 127.8 (flavone Ar-2,4,6C), 127.4 (thiazole Ar-2,6C), 124.9 (flavone 5C), 115.6
(flavone 10C), 115.3 (flavone 8C), 109.3 (flavone 6C), 105.1 (thiazole 3C), 104.6 (flavone
3C), 68.9 (CH2), 45.2 (CH2) 9.8 (CH3); Mass :455.59 (M+1); Elemental analysis for
C27H22N2O3S: Calculated: C, 71.34; H, 4.88; N, 6.16; Found: C, 71.38; H, 4.92; N, 6.14.
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2.1.6.2.7-(2-((4-(4-Chlorophenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9b)
o
Yield: 74%; MP: 234-235 C; MW: 488.99; Rf: 0.79; FT-IR (νmax; cm-1 KBr): 3396 (N–H),
3087 (aromatic C-H), 2979 (aliphatic C-H), 1723 (C=O), 1630 (C=C), 1508 (N–H), 1145
(aromatic C-C) 1141 (C-O-C), 1037 (C–N, aromatic-NH-aromatic), 1092 (aromatic-Cl), 638
-1
(C–S) cm ; 1H-NMR (400MHz, DMSO, TMS) δ ppm:7.98 (d, 2H, J = 8.8 Hz, Ar-H), 7.77
(d, 2H, J = 7.8 Hz, Ar-H), 7.56 (d, 2H, J = 7.2 Hz, Ar-H), 7.46 (d, 1H, J = 8.4 Hz, Ar-H),
7.41 (d, 2H, J = 7.1 Hz, Ar-H), 7.32 (s, 1H, Thiazole), 7.32 (d, 1H, J = 6.5 Hz, ArH), 6.92 (d,
1H, J = 7.4 Hz, Ar-H), 6.73 (s, 1H, ArH), 4.31 (d, 2H, J = 5.6 Hz, Methylene), 3.98 (s, 1H,
13
NH), 3.71 (d, 2H, J = 5.2 Hz, Methylene), 2.17 (s, 3H, CH3); C-NMR (100MHz, CDCl3)
δ,ppm:177.6 (flavone 4C=O), 164.7 (thiazole 2C), 163.9 (flavone 7C), 163.7 (flavone 2C),
158.2 (flavone 9C), 150.4 (thiazole 4C), 134.3 (thiazole Ar-4C), 131.2 (thiazole Ar-1C),
130.4 (flavone Ar-1C), 129.3 (thiazole Ar-3,5C), 128.7 (thiazole Ar-2,6C), 128.9 (thiazole
Ar-2,6C), 127.8 (flavone Ar-2,4,6C), 124.8 (flavone 5C), 115.6 (flavone 10C), 115.3 (flavone
8C), 109.1 (flavone 6C), 105.1 (thiazole 5C), 104.7 (flavone 3C), 68.5 (CH2), 43.2 (CH2),
9.7 (CH3); Mass :489.94 (M+1); Elemental analysis for C27H21ClN2O3S: Calculated: C,
66.32; H, 4.33; N, 5.73; Found: C, 66.41; H, 4.29; N, 5.78.
2.1.6.3.7-(2-((4-(3-Chlorophenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9c)
o
Yield: 64%; MP: 243-245 C; MW: 488.99; Rf: 0.73; FT-IR (νmax; cm-1 KBr): 3395 (N–H),
3088 (aromatic C-H), 2979 (aliphatic C-H), 1725 (C=O), 1632 (C=C), 1507 (N–H), 1147
(aromatic C-C) 1142 (C-O-C), 1039 (C–N, aromatic-NH-aromatic), 1097 (aromatic-Cl), 632
(C–S) cm -1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 8.02 (d, 2H, J = 1.4 Hz, Ar-H), 7.78
(d, 2H, J = 7.8 Hz, Ar-H), 7.68 (d, 1H, J = 7.9 Hz, Ar-H), 7.53 (d, 1H, J = 8.1 Hz, Ar-H),
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7.40 (d, 2H, J = 7.2 Hz, Ar-H), 7.36 (d, 1H, J = 7.8 Hz, Ar-H), 7.31 (s, 1H, Thiazole), 7.32
(d, 1H, J = 6.7 Hz, ArH), 6.92 (d, 1H, J = 7.3 Hz, Ar-H), 6.72 (s, 1H, ArH), 4.32 (d, 2H, J =
5.7 Hz, Methylene), 3.98 (s, 1H, NH), 3.73 (d, 2H, J = 5.1 Hz, Methylene), 2.18 (s, 3H,
13
CH3); C-NMR (100MHz, CDCl3) δ,ppm:177.8 (flavone 4C=O), 164.7 (thiazole 2C), 163.8
(flavone 7C), 163.5 (flavone 2C), 158.2 (flavone 9C), 150.2 (thiazole 4C), 134.7 (thiazole
Ar-3C), 134.4 (thiazole Ar-1C), 130.3 (flavone Ar 1C), 129.6 (thiazole Ar-2C), 129.5
(thiazole Ar-5C), 128.7 (thiazole Ar-4C), 128.5 (flavone Ar- 3,5C), 127.9 (flavone Ar-
2,4,6C), 125.6 (thiazole Ar-6C), 124.8 (flavone 5C), 115.5 (flavone 10C), 115.3 (flavone
8C), 109.3 (flavone 6C), 105.3 (thiazole 5C), 104.6 (flavone 3C), 68.9 (CH2), 43.4 (CH2),
9.7 (CH3); Mass :489.92 (M+1); Elemental analysis for C27H21ClN2O3S: Calculated: C,
66.32; H, 4.33; N, 5.73; Found: C, 66.38; H, 4.35; N, 5.78.
2.1.6.4.7-(2-((4-(2-Chlorophenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9d)
o
Yield: 67%; MP: 239-240 C; MW: 488.99; Rf: 0.71; FT-IR (νmax; cm-1 KBr): 3399 (N–H),
3086 (aromatic C-H), 2972 (aliphatic C-H), 1728 (C=O), 1636 (C=C), 1509 (N–H), 1143
(aromatic C-C) 1147 (C-O-C), 1031 (C–N, aromatic-NH-aromatic), 1096 (aromatic-Cl), 637
(C–S) cm -1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 7.78 (d, 2H, J = 7.8 Hz, Ar-H), 7.73
(d, 1H, J = 7.5 Hz, Ar-H), 7.55 (d, 1H, J = 8.2 Hz, Ar-H), 7.41 (d, 2H, J = 7.3 Hz, Ar-H),
7.39 (d, 1H, J = 7.6 Hz, Ar-H), 7.36 (d, 1H, J = 2.4 Hz, Ar-H), 7.32 (s, 1H, Thiazole), 7.31
(d, 1H, J = 6.7 Hz, ArH), 6.92 (d, 1H, J = 7.1 Hz, Ar-H), 6.73 (s, 1H, ArH), 6.58 (d, 1H, J =
8.1 Hz, ArH), 4.31 (d, 2H, J = 5.6 Hz, Methylene), 3.97 (s, 1H, NH), 3.72 (d, 2H, J = 5.2 Hz,
13
Methylene), 2.17 (s, 3H, CH3); C-NMR (100MHz, CDCl3) δ,ppm:177.6 (flavone 4C=O),
164.9 (thiazole 2C), 163.9 (flavone 7C), 163.7 (flavone 2C), 158.2 (flavone 9C), 147.9
(thiazole 4C), 132.5 (thiazole Ar-3C), 132.2 (thiazole Ar-2C), 130.7 (thiazole Ar-5C), 130.5
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(flavone Ar-1C), 130.1 (thiazole Ar-4C), 129.8 (thiazole Ar-1C), 128.9 (thiazole Ar-6C),
128.4 (flavone Ar-3,5C), 127.8 (flavone Ar-2,4,6C), 124.8 (flavone 5C), 115.4 (flavone 10C),
115.3 (flavone 8C), 109.1 (flavone 6C), 105.2 (thiazole 5C), 104.7 (flavone 3C), 68.5 (CH2),
43.1 (CH2), 9.7 (CH3); Mass :489.97 (M+1); Elemental analysis for C27H21ClN2O3S:
Calculated: C, 66.32; H, 4.33; N, 5.73; Found: C, 66.36; H, 4.29; N, 5.78.
2.1.6.5.7-(2-((4-(4-Fluorophenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9e)
o
Yield: 77%; MP: 248-249 C; MW: 472.53; Rf: 0.64; FT-IR (νmax; cm-1 KBr): 3396 (N–H),
3089 (aromatic C-H), 2978 (aliphatic C-H), 1718 (C=O), 1637 (C=C), 1504 (N–H), 1148
(aromatic C-C) 1149 (C-O-C), 1035 (C–N, aromatic-NH-aromatic), 1157 (aromatic-F), 634
(C–S) cm -1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 8.15 (d, 2H, J = 8.8 Hz, Ar-H), 7.78
(d, 2H, J = 7.8 Hz, Ar-H), 7.58 (d, 1H, J = 8.4 Hz, Ar-H), 7.42 (d, 2H, J = 7.4 Hz, Ar-H),
7.36 (d, 1H, J = 7.6 Hz, Ar-H), 7.30 (d, 2H, J = 4.8 Hz, Ar-H), 7.28 (s, 1H, Thiazole), 6.72 (s,
1H, ArH), 6.60 (d, 1H, J = 8.2 Hz, ArH), 4.30 (d, 2H, J = 5.4 Hz, Methylene), 3.87 (s, 1H,
13
NH), 3.71 (d, 2H, J = 5.1 Hz, Methylene), 2.16 (s, 3H, CH3); C-NMR (100MHz, CDCl3)
δ,ppm:177.8 (flavone 4C=O), 164.7 (thiazole 2C), 163.9 (flavone 7C), 163.5 (flavone 2C),
162.8 (thiazole Ar-4C), 158.2 (flavone 9C), 150.2 (thiazole 4C), 130.4 (thiazole Ar-2,6C),
130.2 (flavone Ar-1C), 128.7 (flavone Ar-3,5C;thiazole Ar-1C), 127.8 (flavone Ar-2,4,6C),
124.9 (flavone 5C), 116.1 (thiazole Ar-3,5C), 115.5 (flavone 10C), 115.2 (flavole 8C), 109.2
(flavone 6C), 105.2 (thiazole 5C), 104.8 (flavone 3C), 68.9 (CH2), 43.3 (CH2), 9.8 (CH3);
Mass :473.56 (M+1); Elemental analysis for C27H21FN2O3S: Calculated: C, 68.63; H, 4.48;
N, 5.93; Found: C, 68.71; H, 4.42; N, 5.98.
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2.1.6.6.7-(2-((4-(3-Fluorophenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9f)
o
Yield: 62%; MP: 253-254 C; MW: 472.53; Rf: 0.67; FT-IR (νmax; cm-1 KBr): 3397 (N–H),
3091 (aromatic C-H), 2972 (aliphatic C-H), 1715 (C=O), 1639 (C=C), 1506 (N–H), 1149
(aromatic C-C) 1143 (C-O-C), 1037 (C–N, aromatic-NH-aromatic), 1152 (aromatic-F), 637
(C–S) cm -1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 7.77 (d, 2H, J = 7.8 Hz, Ar-H), 7.58
(d, 1H, J = 8.7 Hz, Ar-H), 7.52 (d, 1H, J = 1.5 Hz, Ar-H), 7.59 (d, 1H, J = 8.2 Hz, Ar-H),
7.49 (d, 1H, J = 7.4 Hz, Ar-H), 7.40 (d, 2H, J = 7.5 Hz, Ar-H), 7.34 (d, 1H, J = 7.2 Hz, Ar-
H), 7.26 (s, 1H, Thiazole), 7.21 (d, 1H, J = 7.1 Hz, Ar-H), 6.71 (s, 1H, ArH), 6.61 (d, 1H, J
= 8.1 Hz, ArH), 4.28 (d, 2H, J = 5.5 Hz, Methylene), 3.91 (s, 1H, NH), 3.73 (d, 2H, J = 5.2
Hz, Methylene), 2.18 (s, 3H, CH3);13C-NMR (100MHz, CDCl3) δ,ppm:177.6 (flavone
4C=O), 164.8 (thiazole 2C), 163.8 (flavone 7C), 163.5 (flavone 2C), 162.1 (thiazole Ar-3C),
158.4 (flavone 9C), 150.3 (thiazole 4C), 134.7 (thiazole Ar-1C), 130.4 (flavone Ar-1C),
128.7 (flavone Ar-3,5C), 127.8 (flavone Ar-2,4,6C), 127.5 (thiazole Ar-5C), 124.7 (flavone
5C), 123.2 (thiazole Ar-6C), 115.9 (thiazole Ar-2C), 115.6 (thiazole Ar-4C), 115.3 (flavone
10C), 115.1(flavone 8C), 109.1 (flavone 6C), 105.2 (thiazole 5C), 104.8 (flavone 3C), 68.7
(CH2), 43.5 (CH2), 9.7 (CH3); Mass :473.49 (M+1); Elemental analysis for C27H21FN2O3S:
Calculated: C, 68.63; H, 4.48; N, 5.93; Found: C, 68.71; H, 4.54; N, 5.89.
2.1.6.7.7-(2-((4-(2-Fluorophenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9g)
o
Yield: 67%; MP: 258-259 C; MW: 472.53; Rf: 0.69; FT-IR (νmax; cm-1 KBr): 3395 (N–H),
3093 (aromatic C-H), 2976 (aliphatic C-H), 1714 (C=O), 1636 (C=C), 1504 (N–H), 1147
(aromatic C-C) 1142 (C-O-C), 1039 (C–N, aromatic-NH-aromatic), 1156 (aromatic-F), 639
(C–S) cm -1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 7.79 (d, 1H, J = 8.1 Hz, Ar-H), 7.76
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(d, 2H, J = 7.8 Hz, Ar-H), 7.71 (d, 1H, J = 7.1 Hz, Ar-H), 7.59 (d, 1H, J = 8.2 Hz, Ar-H),
7.48 (d, 1H, J = 7.5 Hz, Ar-H), 7.41 (d, 2H, J = 7.3 Hz, Ar-H), 7.33 (d, 1H, J = 7.2 Hz, Ar-
H), 7.27 (s, 1H, Thiazole), 7.24 (d, 1H, J = 6.9 Hz, Ar-H), 6.73 (s, 1H, ArH), 6.62 (d, 1H, J
= 8.2 Hz, ArH), 4.27 (d, 2H, J = 5.4 Hz, Methylene), 3.94 (s, 1H, NH), 3.72 (d, 2H, J = 5.1
Hz, Methylene), 2.16 (s, 3H, CH3); 13C-NMR (100MHz, CDCl3) δ,ppm:177.6 (flavone
4C=O), 164.8 (thiazole 2C), 163.9 (flavone 7C), 163.6 (flavone 2C), 158.3 (thiazole Ar-2C),
158.2 (flavone 9C), 147.9 (thiazole 4C), 130.4 (thiazole Ar-4C; flavone Ar-1C), 129.2
(thiazole Ar-6C), 128.7 (flavone Ar-3,5C), 127.8 (flavone Ar-2,4,6C), 124.9 (thiazole Ar-
5C), 124.4 (flavone 5C), 123.5 (thiazole Ar-1C), 115.5 (flavone 10C), 115.3 (flavone 8C),
114.8 (thiazole Ar-3C), 109.1 (flavone 6C), 105.1 (thiazole 5C), 104.5 (flavone 3C), 68.9
(CH2), 43.7 (CH2), 9.8 (CH3); Mass :473.57 (M+1); Elemental analysis for C27H21FN2O3S:
Calculated: C, 68.63; H, 4.48; N, 5.93; Found: C, 68.59; H, 4.56; N, 6.89.
2.1.6.8.7-(2-((4-(4-Hydroxyphenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9h)
o
Yield: 78%; MP: 264-265 C; MW: 470.54; Rf: 0.83; FT-IR (νmax; cm-1 KBr): 3404
(aromatic-OH),3397 (N–H), 3095 (aromatic C-H), 2973 (aliphatic C-H), 1717 (C=O), 1638
(C=C), 1502 (N–H), 1149 (aromatic C-C) 1145 (C-O-C), 1037 (C–N, aromatic-NH-
-1
1
aromatic), 637 (C–S) cm ; H-NMR (400MHz, DMSO, TMS) δ ppm: 7.78 (d, 2H, J = 7.8
Hz, Ar-H), 7.58 (d, 1H, J = 8.2 Hz, Ar-H), 7.53 (d, 2H, J = 8.8 Hz, Ar-H), 7.41 (d, 2H, J =
7.3 Hz, Ar-H), 7.31 (d, 1H, J = 7.1 Hz, Ar-H), 7.29 (s, 1H, Thiazole), 6.92 (d, 2H, J = 2.8 Hz,
Ar-H), 6.72 (s, 1H, ArH), 6.64 (d, 1H, J = 8.3 Hz, ArH), 5.28 (s, 1H, Ar-OH), 4.29 (d, 2H, J
= 5.6 Hz, Methylene), 3.95 (s, 1H, NH), 3.73 (d, 2H, J = 5.2 Hz, Methylene), 2.18 (s, 3H,
CH3);13C-NMR (100MHz, CDCl3) δ,ppm:177.6 (flavone 4C=O), 164.7 (thiazole 2C), 163.8
(flavone 7C), 163.6 (flavone 2C), 158.7 (thiazole Ar-4C), 158.2 (flavone 9C), 150.3 (thiazole
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4C), 130.4 (flavone Ar-1C), 128.8 (thiazole Ar-2,6C), 128.7 (flavone Ar-3,5C), 127.8
(flavone Ar-2,4,6C), 125.7 (thiazole Ar-1C), 124.9 (flavone 5C), 116.5 (thiazole Ar-3,5C),
115.4 (flavone 10C), 115.2 (flavone 8C), 109.2 (flavone 6C), 105.2 (thiazole 5C), 104.6
(flavone 3C), 68.5 (CH2), 43.4 (CH2), 9.7 (CH3); Mass :471.57 (M+1); Elemental analysis
for C27H22N2O4S: Calculated: C, 68.92; H, 4.71; N, 5.95; Found: C, 68.95; H, 4.77; N, 5.98.
2.1.6.9.7-(2-((4-(3-Hydroxyphenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9i)
o
Yield: 62%; MP: 267-268 C; MW: 470.54; Rf: 0.79; FT-IR (νmax; cm-1 KBr): 3407
(aromatic-OH),3394 (N–H), 3098 (aromatic C-H), 2979 (aliphatic C-H), 1719 (C=O), 1632
(C=C), 1508 (N–H), 1142 (aromatic C-C) 1147 (C-O-C), 1032 (C–N, aromatic-NH-
-1
1
aromatic), 639 (C–S) cm ; H-NMR (400MHz, DMSO, TMS) δ ppm: 7.77 (d, 2H, J = 7.8
Hz, Ar-H), 7.59 (d, 1H, J = 8.3 Hz, Ar-H), 7.41 (d, 2H, J = 7.4 Hz, Ar-H), 7.35 (d, 1H, J =
7.7 Hz, Ar-H), 7.34 (d, 1H, J = 7.5 Hz, Ar-H), 7.32 (d, 1H, J = 7.2 Hz, Ar-H),7.31 (d, 1H, J =
6.9 Hz, Ar-H), 7.27 (s, 1H, Thiazole), 6.91 (d, 1H, J = 7.1 Hz, Ar-H), 6.73 (s, 1H, ArH),
6.63 (d, 1H, J = 8.2 Hz, ArH), 5.32 (s, 1H, Ar-OH), 4.26 (d, 2H, J = 5.4 Hz, Methylene), 3.97
(s, 1H, NH), 3.71 (d, 2H, J = 5.1 Hz, Methylene), 2.17 (s, 3H, CH3);13C-NMR (100MHz,
CDCl3) δ,ppm:177.6 (flavone 4C=O), 164.8 (thiazole 2C), 163.8 (flavone 7C), 163.5 (flavone
2C), 158.4 (flavone 9C), 157.7 (thiazole Ar-3C), 150.2 (thiazole 4C), 134.5 (thiazole Ar-1C),
130.7 (thiazole Ar-5C), 130.4 (flavone Ar-1C), 128.7 (flavone Ar-3,5C), 127.8 (flavone Ar-
2,4,6C), 124.8 (flavone 5C), 120.2 (thiazole Ar-6C), 115.9 (thiazole Ar-2,4C), 115.3 (flavone
10C), 115.1 (flavone 8C), 109.1 (flavone 6C), 105.1 (thiazole 5C), 104.6 (flavone 3C), 68.9
(CH2), 43.7 (CH2), 9.7 (CH3); Mass :471.58 (M+1); Elemental analysis for C27H22N2O4S:
Calculated: C, 68.92; H, 4.71; N, 5.95; Found: C, 68.97; H, 4.68; N, 5.92.
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2.1.6.10. 7-(2-((4-(2-Hydroxyphenyl)thiazol-2-yl)amino)ethoxy)-8-methyl-2-phenyl-4H-
chromen-4-one (9j)
o
Yield: 75%; MP: 264-266 C; MW: 470.54; Rf: 0.71; FT-IR (νmax; cm-1 KBr): 3409
(aromatic-OH),3391 (N–H), 3094 (aromatic C-H), 2972 (aliphatic C-H), 1715 (C=O), 1638
(C=C), 1504 (N–H), 1142 (aromatic C-C) 1148 (C-O-C), 1039 (C–N, aromatic-NH-
-1
1
aromatic), 641 (C–S) cm ; H-NMR (400MHz, DMSO, TMS) δ ppm: 8.24 (d, 1H, J = 7.9
Hz, Ar-H), 7.78 (d, 2H, J = 7.8 Hz, Ar-H), 7.58 (d, 1H, J = 8.2 Hz, Ar-H), 7.40 (d, 2H, J =
7.3 Hz, Ar-H), 7.32 (d, 1H, J = 7.4 Hz, Ar-H), 7.24 (d, 1H, J = 8.1 Hz, Ar-H), 7.28 (s, 1H,
Thiazole), 7.08 (d, 1H, J = 7.9 Hz, Ar-H), 6.98 (d, 1H, J = 6.2 Hz, Ar-H), 6.72 (s, 1H, ArH),
6.62 (d, 1H, J = 8.3 Hz, ArH), 5.34 (s, 1H, Ar-OH), 4.28(d, 2H, J = 5.6 Hz, Methylene), 3.98
(s, 1H, NH), 3.72 (d, 2H, J = 5.2 Hz, Methylene), 2.18 (s, 3H, CH3);13C-NMR (100MHz,
CDCl3) δ,ppm:177.6 (flavone 4C=O), 164.8 (thiazole 2C), 163.8 (flavone 7C), 163.5 (flavone
2C), 158.2 (flavone 9C), 155.3 (thiazole Ar-2C), 147.9 (thiazole 4C), 131.5 (thiazole Ar-6C),
130.4 (flavone Ar-1C), 130.2 (thiazole Ar-4C), 128.7 (flavone Ar-3,5C), 127.9 (flavone Ar-
2,4,6C), 124.8 (flavone 5C), 121.8 (thiazole Ar-5C), 120.5 (thiazole Ar-1C), 117.9 (thiazole
Ar-3C), 115.4 (flavone 10C), 115.2 (flavone 8C), 109.2 (flavone 6C), 105.1 (thiazole 5C),
104.5 (flavone 3C), 68.6 (CH2), 43.9 (CH2), 9.7 (CH3); Mass :471.59 (M+1); Elemental
analysis for C27H22N2O4S: Calculated: C, 68.92; H, 4.71; N, 5.95; Found: C, 68.86; H, 4.78;
N, 5.98.
2.1.6.11. 8-Methyl-2-phenyl-7-(2-((4-(p-tolyl)thiazol-2-yl)amino)ethoxy)-4H-chromen-
4-one (9k)
o
Yield: 79%; MP: 258-259 C; MW: 468.57; Rf: 0.82; FT-IR (νmax; cm-1 KBr): 3394 (N–H),
3091 (aromatic C-H), 2978 (aliphatic C-H), 2921 (CH3), 1719 (C=O), 1636 (C=C), 1506 (N–
H), 1143 (aromatic C-C), 1147 (C-O-C), 1035 (C–N, aromatic-NH-aromatic), 645 (C–S) cm -
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1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 7.79 (d, 2H, J = 7.9 Hz, Ar-H), 7.69 (d, 2H, J =
8.2 Hz, Ar-H), 7.58 (d, 1H, J = 8.4 Hz, Ar-H), 7.42 (d, 2H, J = 7.4 Hz, Ar-H), 7.32 (d, 1H, J
= 7.2 Hz, Ar-H), 7.29 (d, 2H, J = 8.3 Hz, Ar-H), 7.28 (s, 1H, Thiazole), 6.71 (s, 1H, ArH),
6.62 (d, 1H, J = 8.1 Hz, ArH), 4.27 (d, 2H, J = 5.5 Hz, Methylene), 3.97 (s, 1H, NH), 3.71 (d,
13
2H, J = 5.1 Hz, Methylene), 2.38 (s, 3H, Ar-CH3),2.16 (s, 3H, CH3); C-NMR (100MHz,
CDCl3) δ,ppm:177.6 (flavone 4C=O), 164.8 (thiazole 2C), 163.8 (flavone 7C), 163.4 (flavone
2C), 158.2 (flavone 9C), 150.3 (thiazole 4C), 131.8 (thiazole Ar-4C), 130.4 (flavone Ar-1C),
130.1 (thiazole Ar-1C), 129.6 (thiazole Ar-3,5C), 128.7 (flavone Ar-3,5C), 127.8 (flavone
Ar-2,4,6C), 125.9 (thiazole Ar-2, 6C), 124.6 (flavone 5C), 115.4 (flavone 10C), 115.2
(flavone 8C), 109.1 (flavone 6C), 105.2 (thiazole 5C), 104.6 (flavone 3C), 68.7 (CH2), 43.6
(CH2), 21.4 (Ar-CH3), 9.8 (CH3); Mass :469.62 (M+1); Elemental analysis for
C28H24N2O3S: Calculated: C, 71.77; H, 5.16; N, 5.98; Found: C, 71.83; H, 5.14; N, 5.99.
2.1.6.12. 8-Methyl-2-phenyl-7-(2-((4-(m-tolyl)thiazol-2-yl)amino)ethoxy)-4H-chromen-
4-one (9l)
o
Yield: 71%; MP: 268-269 C; MW: 468.57; Rf: 0.76; FT-IR (νmax; cm-1 KBr): 3392 (N–H),
3089 (aromatic C-H), 2974 (aliphatic C-H), 2925 (CH3), 1715 (C=O), 1637 (C=C), 1508 (N–
H), 1147 (aromatic C-C), 1149 (C-O-C), 1037 (C–N, aromatic-NH-aromatic), 648 (C–S) cm -
1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 7.85 (d, 1H, J = 8.2 Hz, Ar-H), 7.77 (d, 2H, J =
7.6 Hz, Ar-H), 7.63 (d, 1H, J = 7.4 Hz, Ar-H), 7.59 (d, 1H, J = 8.3 Hz, Ar-H), 7.41 (d, 2H, J
= 7.3 Hz, Ar-H), 7.38 (d, 1H, J = 7.9 Hz, Ar-H), 7.33 (d, 1H, J = 7.1 Hz, Ar-H), 7.28 (s, 1H,
Thiazole), 7.19 (d, 2H, J = 7.8 Hz, Ar-H), 6.72 (s, 1H, ArH), 6.61 (d, 1H, J = 8.2 Hz, ArH),
4.27 (d, 2H, J = 5.6 Hz, Methylene), 3.98 (s, 1H, NH), 3.72 (d, 2H, J = 5.2 Hz, Methylene),
13
2.36 (s, 3H, Ar-CH3),2.18 (s, 3H, CH3); C-NMR (100MHz, CDCl3) δ,ppm:177.7 (flavone
4C=O), 164.8 (thiazole 2C), 163.8 (flavone 7C), 163.5 (flavone 2C), 158.2 (flavone 9C),
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150.3 (thiazole 4C), 138.9 (thiazole 3C), 132.8 (thiazole Ar- 1C), 130.5 (thiazole Ar- 2C),
130.3 (flavone Ar-1C), 129.3 (thiazole Ar-5C), 129.1 (thiazole Ar-4C), 128.7 (flavone Ar-
3,5C), 127.8 (flavone Ar-2,4,6C), 124.6 (flavone 5C), 124.3 (thiazole Ar-6C), 115.5 (flavone
10C), 115.2 (flavone 8C), 109.2 (flavone 6C), 105.1 (thiazole 5C), 104.6 (flavone 3C), 68.9
(CH2), 43.5 (CH2), 21.7 (Ar-CH3), 9.8 (CH3); Mass :469.63 (M+1); Elemental analysis for
C28H24N2O3S: Calculated: C, 71.77; H, 5.16; N, 5.98; Found: C, 71.83; H, 5.13; N, 5.92.
2.1.6.13. 8-Methyl-2-phenyl-7-(2-((4-(o-tolyl)thiazol-2-yl)amino)ethoxy)-4H-chromen-
4-one (9m)
o
Yield: 82%; MP: 281-283 C; MW: 468.57; Rf: 0.79; FT-IR (νmax; cm-1 KBr): 3394 (N–H),
3081 (aromatic C-H), 2975 (aliphatic C-H), 2928 (CH3), 1717 (C=O), 1639 (C=C), 1505 (N–
H), 1149 (aromatic C-C), 1142 (C-O-C), 1039 (C–N, aromatic-NH-aromatic), 646 (C–S) cm -
1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 7.78 (d, 2H, J = 7.8 Hz, Ar-H), 7.58 (d, 1H, J =
8.2 Hz, Ar-H), 7.54 (d, 1H, J = 7.6 Hz, Ar-H), 7.42 (d, 2H, J = 7.3 Hz, Ar-H), 7.33 (d, 1H, J
= 7.1 Hz, Ar-H), 7.31 (d, 1H, J = 7.7 Hz, Ar-H), 7.30 (d, 1H, J = 7.5 Hz, Ar-H), 7.29 (d, 1H,
J = 7.2 Hz, Ar-H), 7.29 (s, 1H, Thiazole), 6.71 (s, 1H, ArH), 6.63 (d, 1H, J = 8.1 Hz, ArH),
4.29 (d, 2H, J = 5.4 Hz, Methylene), 3.97 (s, 1H, NH), 3.71 (d, 2H, J = 5.1 Hz, Methylene),
13
2.58 (s, 3H, Ar-CH3),2.16 (s, 3H, CH3); C-NMR (100MHz, CDCl3) δ,ppm:177.6 (flavone
4C=O), 164.8 (thiazole 2C), 163.8 (flavone 7C), 163.6 (flavone 2C), 158.2 (flavone 9C),
147.8 (thiazole 4C), 136.9 (thiazole Ar-2C), 130.4 (flavone Ar-1C), 129.8 (thiazole Ar-1C),
129.6 (thiazole Ar-3C), 128.7 (flavone Ar-3,5C;thiazole Ar-4C), 127.8 (flavone Ar-2,4,6C),
126.2 (thiazole Ar-5C), 124.9 (flavone 5C), 122.8 (thiazole Ar-6C), 115.4 (flavone 10C),
115.2 (flavone 8C), 109.1 (flavone 6C), 105.2 (thiazole 5C), 104.6 (flavone 3C), 68.7 (CH2),
43.6 (CH2), 18.7 (Ar-CH3), 9.8 (CH3); Mass :469.54 (M+1); Elemental analysis for
C28H24N2O3S: Calculated: C, 71.77; H, 5.16; N, 5.98; Found: C, 71.75; H, 5.21; N, 5.97.
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2.1.6.14. 8-Methyl-7-(2-((4-(4-nitrophenyl)thiazol-2-yl)amino)ethoxy)-2-phenyl-4H-
chromen-4-one (9n)
o
Yield: 69%; MP: 284-286 C; MW: 499.54; Rf: 0.86; FT-IR (νmax; cm-1 KBr): 3392 (N–H),
3083 (aromatic C-H), 2985 (aliphatic C-H), 1714 (C=O), 1636 (C=C), 1526 (NO2), 1508 (N–
H), 1148 (aromatic C-C), 1145 (C-O-C), 1038 (C–N, aromatic-NH-aromatic), 647 (C–S) cm -
1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 8.32 (d, 2H, J = 8.8 Hz, Ar-H), 8.05 (d, 2H, J =
8.3 Hz, Ar-H), 7.78 (d, 2H, J = 7.8 Hz, Ar-H), 7.59 (d, 1H, J = 8.1 Hz, Ar-H), 7.41 (d, 2H, J
= 7.3 Hz, Ar-H), 7.34 (d, 1H, J = 7.1 Hz, Ar-H), 7.29 (s, 1H, Thiazole), 6.73 (s, 1H, ArH),
6.64 (d, 1H, J = 8.3 Hz, ArH), 4.26 (d, 2H, J = 5.7 Hz, Methylene), 3.98 (s, 1H, NH), 3.73 (d,
2H, J = 5.2 Hz, Methylene), 2.18 (s, 3H, CH3); 13C-NMR (100MHz, CDCl3) δ,ppm:177.6
(flavone 4C=O), 164.8 (thiazole 2C), 163.9 (flavone 7C), 163.6 (flavone 2C), 158.2 (flavone
9C), 150.2 (thiazole 4C), 147.8 (thiazole Ar-4C), 139.2 (thiazole Ar-1C),130.4 (flavone Ar-
1C), 128.7 (flavone Ar-3,5C), 127.8 (flavone Ar-2,4,6C), 126.3 (thiazole Ar-2,6C), 124.6
(flavone 5C), 124.5(thiazole Ar-3,5C), 115.4 (flavone 10C), 115.2 (flavone 8C), 109.1
(flavone 6C), 105.2 (thiazole 5C), 104.5 (flavone 3C), 68.9 (CH2), 43.7 (CH2), 9.8 (CH3);
Mass :500.59 (M+1); Elemental analysis for C27H21N3O5S: Calculated: C, 64.92; H, 4.24; N,
8.41; Found: C, 64.95; H, 4.29; N, 8.39.
2.1.6.15. 8-Methyl-7-(2-((4-(3-nitrophenyl)thiazol-2-yl)amino)ethoxy)-2-phenyl-4H-
chromen-4-one (9o)
o
Yield: 64%; MP: 291-292 C; MW: 499.54; Rf: 0.71; FT-IR (νmax; cm-1 KBr): 3394 (N–H),
3085 (aromatic C-H), 2987 (aliphatic C-H), 1719 (C=O), 1631 (C=C), 1528 (NO2), 1509 (N–
H), 1151 (aromatic C-C), 1142 (C-O-C), 1036 (C–N, aromatic-NH-aromatic), 645 (C–S) cm -
1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 8.65 (d, 1H, J = 1.6 Hz, Ar-H), 8.24 (d, 1H, J =
8.4 Hz, Ar-H), 8.16 (d, 1H, J = 7.9 Hz, Ar-H), 7.78 (d, 2H, J = 7.8 Hz, Ar-H), 7.74 (d, 1H, J
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= 7.2 Hz, Ar-H), 7.58 (d, 1H, J = 8.2 Hz, Ar-H), 7.42 (d, 2H, J = 7.2 Hz, Ar-H), 7.32 (d, 1H,
J = 7.1 Hz, Ar-H), 7.27 (s, 1H, Thiazole), 6.72 (s, 1H, ArH), 6.62 (d, 1H, J = 8.1 Hz, ArH),
4.28 (d, 2H, J = 5.6 Hz, Methylene), 3.97 (s, 1H, NH), 3.72 (d, 2H, J = 5.1 Hz, Methylene),
2.17 (s, 3H, CH3); 13C-NMR (100MHz, CDCl3) δ,ppm:177.5 (flavone 4C=O), 164.8
(thiazole 2C), 163.8 (flavone 7C), 163.5 (flavone 2C), 158.2 (flavone 9C), 150.3 (thiazole
4C), 148.5 (thiazole Ar-3C), 133.8 (thiazole Ar-1C), 133.2 (thiazole Ar-6C), 130.8 (thiazole
Ar-2C), 130.2 (flavone Ar-1C), 128.7 (flavone Ar-3,5C), 127.8 (flavone Ar-2,4,6C), 124.6
(flavone 5C), 123.8 (thiazole Ar-4C), 122.7 (thiazole Ar-5C), 115.5 (flavone 10C),115.2
(flavone 8C), 109.1 (flavone 6C), 105.1 (thiazole 5C), 104.6 (flavone 3C), 68.8 (CH2), 43.5
(CH2), 9.7 (CH3); Mass :500.49 (M+1); Elemental analysis for C27H21N3O5S: Calculated: C,
64.92; H, 4.24; N, 8.41; Found: C, 64.96; H, 4.18; N, 8.45.
2.1.6.16. 8-Methyl-7-(2-((4-(2-nitrophenyl)thiazol-2-yl)amino)ethoxy)-2-phenyl-4H-
chromen-4-one (9p)
o
Yield: 68%; MP: 295-296 C; MW: 499.54; Rf: 0.68; FT-IR (νmax; cm-1 KBr): 3392 (N–H),
3087 (aromatic C-H), 2989 (aliphatic C-H), 1716 (C=O), 1635 (C=C), 1523 (NO2), 1507
(N–H), 1153 (aromatic C-C), 1147 (C-O-C), 1038 (C–N, aromatic-NH-aromatic), 647 (C–S)
cm -1; 1H-NMR (400MHz, DMSO, TMS) δ ppm: 8.31 (d, 1H, J = 8.5 Hz, Ar-H), 7.95 (d, 1H,
J = 7.6 Hz, Ar-H), 7.84 (d, 1H, J = 7.8 Hz, Ar-H), 7.78 (d, 2H, J = 7.8 Hz, Ar-H), 7.67 (d,
1H, J = 7.5 Hz, Ar-H), 7.57 (d, 1H, J = 8.1 Hz, Ar-H), 7.41 (d, 2H, J = 7.2 Hz, Ar-H), 7.34
(d, 1H, J = 7.2 Hz, Ar-H), 7.32 (s, 1H, Thiazole), 6.71 (s, 1H, ArH), 6.64 (d, 1H, J = 8.2 Hz,
ArH), 4.26 (d, 2H, J = 6.3 Hz, Methylene), 3.98 (s, 1H, NH), 3.71 (d, 2H, J = 5.6 Hz,
Methylene), 2.18 (s, 3H, CH3); 13C-NMR (100MHz, CDCl3) δ,ppm:177.7 (flavone 4C=O),
164.8 (thiazole 2C), 163.8 (flavone 7C), 163.5 (flavone 2C), 158.2 (flavone 9C), 148.9
(thiazole Ar-2C), 147.8 (thiazole 4C), 135.4 (thiazole Ar-5C), 132.6 (thiazole Ar-6C), 130.4
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(flavone Ar-1C), 129.6 (thiazole Ar-4C), 128.8 (flavone Ar-3,5C), 127.9 (flavone Ar-2,4,6C),
125.2 (thiazole Ar-1C), 124.6 (flavone 5C), 124.3 (thiazole Ar-3C), 115.4 (flavone 10C),
115.2 (flavone 8C), 109.1 (flavone 6C), 105.1 (thiazole 5C), 104.6 (flavone 3C), 68.6 (CH2),
43.4 (CH2), 9.8 (CH3); Mass :500.56 (M+1); Elemental analysis for C27H21N3O5S:
Calculated: C, 64.92; H, 4.24; N, 8.41; Found: C, 64.98; H, 4.21; N, 8.37.
2.2.Antibacterial activity
Total number of forty-clinical S. aureus strains (MRSA and MSSA) were isolated from the
blood of the individual patients enrolled at Dongying People's Hospital for bone traumatic
injury for the duration of 2014-16 after written consent. The experimental protocol was duly
approved via Human Ethical Committiee of Dongying People's Hospital, Dongying,
Shandong, China. The identity of the strains were ascertained to the species level by
conventional method, such as, colony morphology, gram strain characterstics and cogulase
reaction. The MRSA strains were defined on the basis of the occurrence of the mecA gene
and resistance to the methicillin and oxacillin, according to the guidelines of the National
Committee for Clinical Laboratory Standards (M100-S9). The mecA gene was believed to be
responsible for the generation of resistance against widely used antibiotics such as
methicillin, penicillin and other penicillin-like antibiotics. Out of the forty strains, thirty-five
were found as mecA-positive and remaining five were mecA-negative. The ATCC 29213 (a
MSSA strain) and ATCC 43300 (a MRSA strain) were used as the reference control strains.
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2.3. S. aureus DNA gyrase supercoiling
The S. aureus DNA gyrase were purified from a crude extract of S. aureus and cultivated
with medium B, which was composed of 10 g of polypeptone, 2 g of yeast extract, 8 g of
Na2HPO4, 2 g of KH2PO4, 1.2 g of (NH4)2SO4, 0.2 g of MgSO4, 4 g of glucose perliter of
distilled water as suggested by Blanche et al [19]. Supercoiling and decatenation were
performed according to Blanche et. al. [19].
2.4.MIC determination
To estimate the antibacterial activity of individual polyphenol, a MIC was determined by the
agar dilution method in Müller-Hinton (MH) agar plates, as recommended by the Clinical and
Laboratory Standards Institute [20]. In brief, two-fold serial dilutions of the test drugs were
totally solubilized in DMSO, and then prepared to afford initial concentrations corresponding
to four times the MICs of the respective polyphenol alone as determined in individual
susceptibility tests. The media, inocula and conditions were the same as those used for MIC
tests. In the control experiments, the same amount of DMSO that was contained in these
agents was added into the MH agar to check the antibacterial effect of DMSO in the absence
of polyphenols. All experiments were performed in triplicate.
2.5.Hemolytic activity
Human red blood cells (hRBCs) were diluted with PBS to 1%. Depending on solubility,
analogues 9b, 9c, 9d, 9n, 9o and 9p were dissolved in (5%) DMSO/H2O (v/v) solvent mixture
to concentrations of 8–256 µg mL-1. Into each well of the clear, flat-bottom 96-well plate, 50
mL of the hRBCs were placed followed by addition of 50 mL analogue solution to final
concentrations of compounds. Assays were performed in triplicate, and each experiment was
repeated twice. To determine the potential effect of DMSO on haemolytic activity, control
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containing 5% DMSO in H2O (v/v) were added to the assay setup. As a negative control, 50
mL H2O and PBS was used. Plates were incubated for 1 h at 37°C. To each well 100 mL of
PBS was added, and the plates were centrifuged for 10 min at 1000 xg. Resulting
supernatants (150 mL) were transferred into a new plate, and absorbance at 405 nm was
measured. Within the tested concentration range, the effect of DMSO on hemolysis was
minimal, and was subtracted to obtain solely hemolytic activity of the compounds. The
degree of hemolysis was expressed in percent relative to total hemolysis [21].
2.6.Docking Study
Docking calculations were carried out using DockingServer using 3D crystal structure of S.
aureus complex with ciprofloxacin and DNA (pdb: 2xct) [22]. Gasteiger partial charges were
added to the ligand atoms. Non-polar hydrogen atoms were merged, and rotatable bonds were
defined. Docking calculations were carried out on voltage-dependent calcium channel beta
subunit functional core in complex with alpha1 interaction domain protein model. Essential
hydrogen atoms, Kollman united atom type charges [23], and solvation parameters were
added with the aid of AutoDock tools. Affinity (grid) maps of 60×60×60 Å grid points and
0.375 Å spacing were generated using the Autogrid program . AutoDock parameter set- and
distance-dependent dielectric functions were used in the calculation of the van der Waals and
the electrostatic terms, respectively. Docking simulations were performed using the
Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method [24]. Initial
position, orientation, and torsions of the ligand molecules were set randomly. All rotatable
torsions were released during docking. Each docking experiment was derived from 10
different runs that were set to terminate after a maximum of 250000 energy evaluations. The
population size was set to 150. During the search, a translational step of 0.2 Å, and
quaternion and torsion steps of 5 were applied.
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2.7. In vivo Pharmacokinetic Assessment
For this, Sprague-Dawley rats (male, 200 20 g) were obtained from animal house of the
institute and kept in a laboratory atmosphere with free access to food and water. The rats
were fasted overnight before the test. The compound 4k was intragastrically administered at
the dose of 10 mg/kg in rats. The 100 μL of heparinized blood samples were collected at the
regular interval till 24 h from the ocular veins followed by subsequent centrifugation at 5,000
rpm for 10 min. The resulting supernatant was collected and subjected to validated HPLC
method using a noncompartmental method. Animal welfare and experimental procedures
were strictly in accordance with the guide for the care and use of laboratory animals.
3. Result and discussion
3.1.Chemistry
The target derivatives were synthesized in excellent yield via facile synthetic route as shown
in Scheme 1. Initially, compound 2 was obtained by modified Baker–Venkataraman
transformation method, 1 was condensed with benzoyl chloride using K2CO3 as base in
acetone. In the second stage, resulting compound 2 was allowed to react with NaOAc/HOAc
to obtain compound 3. In next step, compound 4 was obtained by removing the benzoyl ring
of compound 3 in good yield. The compound 4 was allowed to react with 1,2-dibromoethane
to afford corresponding compound 5. The next part of the synthesis is deals with the
development of various substituted phenyl thiazole derivatives 8(a-p). These compounds
were obtained by reacting with various acetophenone 6 (a-p) and thiourea (7) in ethanol.
Finally, the title derivatives 9 (a-p) were obtained by the reaction of above phenyl thiazole
derivative 8(a-p) with compound 5 in dimethylformamide (DMF). The entire reaction were
12
monitored by TLC and structure of derivatives were ascertained by the Mass, 1H-NMR, C-
NMR, FT-IR and elemental analysis. The FT-IR spectra of all synthesized derivatives 9(a-p)
were characterized by the appearance of a strong bands at 3087-3083 cm-1 which is attributed
to C-H group of aromatic ring. Another strong band at 3391-3398 cm-1 was attributed to
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stretching vibrations corresponding to secondary amine group. Furthermore, another strong
band at 1714-1725 cm-1 confirmed the presence of C=O group in title compounds. The
aromatic NO2 group appears at 1523-1528 cm-1. Another strong band at 1141-1149 cm-1
attributed to the stretching vibrations of the ether (C-O-C) group. Moreover, the appearance
of band at 642-648 cm-1 showed presence of C–S group in thiazole ring. Compound 9 (h-j)
showed the band at 3404-3409 cm-1 which confirmed the presence of aromatic hydroxyl
group. The strong band at 2971-2989 cm-1 corresponds to the presence of aliphatic C-H
1
group. The H NMR spectrums of synthesized derivatives 9(a-p) revealed a doublet
corresponding to flavone ring at 7.58–6.64 ppm. The flavone phenyl ring appears at 7.78-732
ppm as doublet. Furthermore, the resonance at 7.32-7.26 ppm confirmed the presence of
methylene proton with single peak of thiazole ring. The side chain of aliphatic methylene
peak appeared at 4.32-3.71 ppm as doublet peak. The resonance due to methyl proton of
flavones ring showed at 2.16-2.18 ppm as singlet peaks. The NH proton of thiazole side chain
appeared at 3.96-3.98 ppm as singlet peaks. Finally, all the synthesized derivatives structure 9
(a-p) were confirmed by mass spectroscopy and elemental analysis.
3.2.DNA Gyrase Inhibitory Activity
The DNA gyrase inhibition is a critical step to control the bacterial infection by preventing its
supercoiling. Thus, in the present study, we wish to elucidate the effect of designed target
compounds agains the two strains of the bacterial organism isolated from the clinical samples
from the patients of bone traumatic injury. The compounds DNA gyrase inhibitory activity
was performed against one mecA-positive one mecA-negative strain of the clinical isolate.
The mecA gene was believed to be responsible for the generation of resistance against widely
used antibiotics such as methicillin, penicillin and other penicillin-like antibiotics. As shown
in Table 1, entire set of target compounds showed significant activity against the both tested
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strains as compared to Novobiocin as standard. For instance, compound 9a showed moderate
activity against the strains with no substitution at the flanked phenyl rings. However, the
activity was greatly influenced by the presence of substitution, viz. compound 9b, 9c and 9d
showed considerably enhanced activity revealing p-substituted compound more active than
other two isomers (9c and 9d). Surprisingly, the activity was reported to decline in the next
instance on replacement of chloro with fluro in the case of compound 9e, 9f and 9g against
both the strains. The next part of the study was aimed to elucidate the effect of electron
donating group on the bioactivity. Thus we have synthesized compounds containing isomeric
substituents of hydroxyl and methyl group. The compound 9h, 9i and 9j showed very mild
activity against the tested strain with most prominent activity in p-substitution than other two
strains. In the next instance, the methyl substituted compounds showed no activity in the case
of compounds 9k, 9l with mild activity in 9m. It is marked to note that, remaining compounds
9n, 9o and 9p found to exhibit most potent activity against the tested bacterial strains among
the designed compounds.
The detailed bioactivity profile of the designed compounds suggested that, the inhibitory
activity was greatly influenced by the minor structural modifications. The compounds
containing electron withdrawing group found to be more potent than compounds containing
electron donating counterparts. The activity was further influenced by the para-positions than
other isomeric counterparts.
3.3.
3.4.Antibacterial activity against the panel of clinical isolates
Encouraged by the considerable DNA gyrase of compounds 9b, 9c, 9d, 9n and 9o and 9p, it
was imperative to determine the effect of these analogues against the panel of fourty-one
clinical isolates from the patients having bone traumatic injury. For this study we have used
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thirty-five were mecA-positive and remaining five are mecA-negative. The ATCC 29213 (a
MSSA strain) and ATCC 43300 (a MRSA strain) were used as the reference control strains.
As shown in the Table 2, the tested compound showed considerable activity against the tested
strain with more prominent activity against the mecA-negative strains. This observation was
further found in accordance with inhibitory profile observed in the case of ATCC 29213 (a
MSSA strain). On the contrary, as supposed, these compounds have failed to provide any
significant benefit against the mecA-positive strains with similar observation in ATCC43300
(a MRSA strain). The study also revealed greater potency by compounds containing electron
withdrawing group than electron donating congeners as observed in previous study [18].
Thus, results of the study showed compounds have the mild to moderate ability against the
clinical isolates of bacrerial organisms.
3.5.Heamolytic activity of compounds
The most active compound as found in above study were further subjected to determine its
hemolytic activity against human erythrocytes (0.5% in PBS) to define its possible safety
profile. The PBS and 0.5% Triton X-100 were used as references for 0 and 100% hemolysis,
respectively. As shown in Table 3, the compounds showed no hemolytic activity till the
tested dose of 64 µg/mL. However, the hemolytic activity was found to be well correlated
with structural features. As found in Table 3, compounds having electron withdrawing group
showed haemolytic activity at the maximum tested dose of 256 µg/mL, while no activity was
reported by other compounds at the maximum tested dose.
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3.6.Docking study of compound 9n
In the above synthesized derivatives 9n showed heighted antibacterial activity which is
further evaluated by docking study as shown in the Figure 1 and 2, the compound 9n has been
found highly oriented in the active site of the DNA gyrase receptor. The kinase domain of
the DNA gyrase receptor has been shown in the typical bilobal structure. It has been found
that, the highly conserved residue of the ATP binding site of the the protein were involved in
making the prolific interaction with the 9n. As presented in fig 1, the nitro fragment of the
ligand shown to engage the LYS 460 via the formation of polar bond. Whereas, the chromen-
4-one moiety of the 9n shown to engage the for polar interaction with ARG458, ASN475 and
GLU477. Furthermore the introducation of nitro group at fourth position of phenyl ring show
hydrogen boinding with LYS460 along with having low binding free eregy -4.36 kcal/mol.
Compound 9n showed similar value of interaction on close inspection of the HB plot as
shown in Figure 2. The results were found in accordance with previous study [25]. The
compound 9n was further evaluated for its pharmacokinetics in the Sprague-Dawley rats and
the results were presented Table 4. It has been found the compound 9n showed considerable
pharmacokinetic bioaviliability.
4. Conclusion
The present revealed a novel series of thiazole-based flavanoids via economical and short
synthetic route. These derivatives showed considerable inhibiton of DNA gyrase with
significant activity against thirty-five mecA-positive and five mecA-negative S. aureus
clinical isolates causing traumatic bone infections. The excellent pharmacokinetics together
with no haemolytic activity render designed analogues as lead for discovery of novel
antibacterial agents.
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Conflicts of Interest
The authors declare no conflict of interest.
Figures, Tables and Schemes captions
Figure 1: Docked orientiention of compound 9n in the active site of DNA gyrase .
Figure 2: HB Plot value of compound 9n.
Table 1: DNA Gyrase inhibitory activity of compounds.
Table 2: Antibacterial activity of designed compounds
Table 3: Hemolytic activity of target compounds
Table 4 In-vivo pharmacokinetic assessment of compound 9n in Sprague-Dawley rats.
Scheme 1: Reagents and conditions: (i) benzoyl chloride, K2CO3, acetone, reflux; (ii)
NaOAc/HOAc, reflux; (iii) K2CO3, MeOH/CH2Cl2, rt; (iv) Br(CH2)2Br, K2CO3, DMF; (v)
ethanol, reflux; (vi) reflux, K2CO3, DMF.
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List of Tables
Table 1
Compounds
IC50 (in µg/mL)
DPHS001
54.03
DPHS0036
45.23
27.03
30.03
31.34
47.11
50.42
52.28
65.33
71.65
74.07
>100
9a
9b
33.22
9c
9d
37.11
38.00
9e
9f
9g
54.31
55.01
55.61
9h
9i
73.18
78.54
9j
9k
84.23
>100
9l
>100
>100
9m
9n
9o
9p
89.01
23.45
27.22
33.02
70.32
12.21
18.13
21.34
0.8
Novobiocin
3.23
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Table 2
Bacteria
MIC (in µg/mL)
9d 9n 9o
MRSA ATCC43300 > 1000 250 500 62.5 > 1000 > 1000
9b
9c
9p
DPHS001
DPHS002
DPHS003
DPHS004
DPHS005
DPHS006
DPHS007
DPHS008
DPHS009
DPHS010
DPHS011
DPHS012
DPHS013
DPHS014
DPHS015
DPHS016
DPHS017
DPHS018
DPHS019
DPHS020
DPHS021
DPHS022
DPHS023
DPHS024
DPHS025
DPHS026
DPHS027
DPHS028
DPHS029
DPHS030
DPHS031
DPHS032
DPHS033
DPHS034
DPHS035
> 1000 62.5 500 62.5 > 1000 > 1000
> 1000 250 500 62.5 > 1000 > 1000
> 1000 250 500 125 > 1000 > 1000
> 1000 250 500 62.5 > 1000 > 1000
> 1000 125 500 62.5 > 1000 > 1000
> 1000 125 500 62.5 > 1000 > 1000
> 1000 250 500 62.5 > 1000 > 1000
> 1000 250 500 125 > 1000 > 1000
> 1000 125 500 125 > 1000 > 1000
> 1000 250 500 125 > 1000 > 1000
> 1000 62.5 500 62.5 > 1000 > 1000
> 1000 250 500 62.5 > 1000 > 1000
> 1000 125 250 62.5 > 1000 > 1000
> 1000 125 500 62.5 > 1000 > 1000
> 1000 125 500 62.5 > 1000 > 1000
> 1000 250 250 125 > 1000 > 1000
> 1000 250 250 125 > 1000 > 1000
> 1000 125 250 125 > 1000 > 1000
> 1000 62.5 250 31.2 > 1000 > 1000
> 1000 250 250 62.5 > 1000 > 1000
> 1000 62.5 500 31.2 > 1000 > 1000
> 1000 62.5 500 125 > 1000 > 1000
> 1000 250 500 31.2 > 1000 > 1000
> 1000 62.5 500 62.5 > 1000 > 1000
> 1000 250 500 62.5 > 1000 > 1000
> 1000 125 500 62.5 > 1000 > 1000
> 1000 125 500 62.5 > 1000 > 1000
> 1000 250 500 62.5 > 1000 > 1000
> 1000 250 500 125 > 1000 > 1000
> 1000 250 500 31.2 > 1000 > 1000
> 1000 500 500 31.2 > 1000 > 1000
> 1000 500 500 31.2 > 1000 > 1000
> 1000 500 500 125 > 1000 > 1000
> 1000 500 500 125 > 1000 > 1000
> 1000 250 500 125 > 1000 > 1000
MSSA ATCC29213 > 1000 125 125 31.2 > 1000 > 1000
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