Synthesis, characterization, antioxidant power and acute toxicity of some new azo-benzamide and azo-imidazolone derivatives with in vivo and in vitro antimicrobial evaluation

Article abstract of DOI:10.1016/j.bioorg.2019.01.014

In this research paper, a stepwise chemical reaction was conducted to synthesize and develop of a new potent azo-oxazolone, which was used as prototypical molecule for production of two series of azo-benzimide (5a–j) and azo-imidazolone (6a–j). FT-IR,

Full text of DOI:10.1016/j.bioorg.2019.01.014

Accepted Manuscript
Synthesis, characterization, antioxidant power and acute toxicity of some new
azo-benzamide and azo-imidazolone derivatives with in vivo and in vitro anti-
microbial evaluation
Mohammed kareem samad, Farouq Emam Hawaiz
PII:
S0045-2068(18)31359-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.014
YBIOO 2718
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 November 2018
17 December 2018
7 January 2019
Please cite this article as: M. kareem samad, F. Emam Hawaiz, Synthesis, characterization, antioxidant power and
acute toxicity of some new azo-benzamide and azo-imidazolone derivatives with in vivo and in vitro antimicrobial
evaluation, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.014
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, characterization, antioxidant power and acute toxicity of some new
azo-benzamide and azo-imidazolone derivatives with in vivo and in vitro
antimicrobial evaluation
Mohammed kareem samad* and Farouq Emam Hawaiz*
*Department of Chemistry, College of Education, Salahaddin University-
Hawler, Erbil-Kurdistan –Iraq
mohammed.samad@su.edu.krd
farouq.hawaiz@su.edu.krd
Abstract
In this research paper, a stepwise chemical reaction was conducted to synthesize
and develop of a new potent azo-oxazolone, which was used as prototypical
molecule for production of two series of azo-benzimide (5a-j) and azo-imidazolone
1
(6a-j). FT-IR, H-NMR, 13C-NMR and CHN analysis were used for the structural
elucidation. The high biological efficiency of newly obtained compounds was
confirmed by in vitro antioxidant efficacy and in vitro antimicrobial activity against
gram-positive and gram-negative bacteria via disc diffusion and tube dilution
techniques. In addition, in vivo anti-microbial activity of some of the synthesized
compounds was determined by using burnt rats which infected by Staphylococcus
aureus. Tested compounds have shown high anti-microbial activity and wound
healing in comparison to ucederm as a control. In vivo acute toxicity was carried out
by up and down method for the compounds 4, 5d and 6d. The limited test dose was
2000mg/kg, while the maximum tolerated dose was 5000mg/kg which has
administered no lethality recorded.
________
Keywords: Synthesis; characterization; antimicrobial; antioxidant power,
wound healing; acute toxicity.
*Corresponding author Tel.:+9647504042363; e-mail: mohammed.samad@su.edu.krd
1

1. Introduction
The 5-(4H)-oxazolones or Erlenmeyer azlactones are five membered heterocyclic
compounds conveniently accessible by condensation of substituted hippuric acids
with the corresponding substituted benzaldehydes in the presence of acetic
anhydride and sodium acetate [1,2]. In terms of the reactivity, oxazolone 4 resemble
lactone, and therefore react with diverse nucleophiles via ring opening as the initial
step of their conversion. 5-(4H)-oxazolones are serving as privilege structures in the
synthesis of various important compounds such as imidazolones [3], benzamides [4],
non-natural amino acids [5], peptides [6] and pericyclic cycloaddition reaction [7].
5-(4H)-Imidazolone and its derivatives have widely been used in drug discovery
particularly for antitumor [8],
anti-cancer [9,10], antiproliferative [11], anti-
inflammatory [12], antihypertensive [13] and antiepileptic [14] diseases. Moreover,
due to the long π-conjugated electrons their optical properties of imidazolones could
lead to potential applications in micro fabrication, such as optical imaging [15] and
for preparation of green fluorescent protein (GFP) [16,17].
In the past decades, a number of published studies show the degradation and toxicity
of the azo dyes in both aerobic and anaerobic conditions [18–20]. In contrast to these
disadvantages, there are several studies showing the low-toxicity and stability of
these dyes for long-term (72 days) incubations [21]. More specifically some
oxazolones, benzamides and imidazolones containing azo linkage have been shown
to exhibit a broad range of pharmacological and biological activities, including
antimicrobial activity [22] and tyrosinase inhibition activity [23,24].
A variety of animal models over the last decade have focused on applying well-
known treatments for traditional mechanisms of burn wound progression [25,26].
Wounds and especially infected wounds caused by S. aureus have posed an
increasingly severe threat to human’s health all over the world [27]. Thus, during the
wound repairing progress, infection control, healing promotion and defined biological
2

safety limits are equally important of these antimicrobial materials to promote the
healing of the infected wound.
In Recent decades, literature survey showed that many researchers in worldwide
focused on oxazolones and imidazolones for drug discovery [28–30], but very few of
them focused on azo-oxazolones and azo-imidazolones [22–24,31,32]. Herein, In the
light of all these facts, this work deals with the synthesis of an azo-oxazolone and
successfully modified the desired addition mode of either an efficient fast nucleophilic
ring opening of 4 by various substituted anilines to (5a-j) followed by intramolecular
cyclodehydration in acidic solution to afford azo-imidazolone derivatives (6a-j) in
excellent yields. Followed by in vivo and vitro antimicrobial activities and acute
toxicity for some of the newly produced compounds as an ideal nontoxic material for
infected wound repairing and as a preliminary studies for future engineering in the
course of our drug discovery programs.
2. Methodology
2.1. Chemistry
All the chemicals used in Scheme 1 were of analytical grade and they were
obtained from different commercial sources (Fluka, BDH, and Riedel-de Haen) and
used without any further purification.1H-NMR and 13C-NMR spectra were recorded on
Bruker spectrometer (400 MHz) with CDCl3 as a solvent at Jordan university of
science and technology. Chemical shifts are expressed in parts per million (δ) using
residual solvent protons as internal standard (δ 7.26 in 1H and δ 77.0 in 13C). Splitting
patterns are designated as s (singlet), d (doublet), t (triplet), m (multiplet). Elemental
analyses were carried out with VarioMicro V3.1.1 CHNS analyzer at University
technology of Malaysia. Infrared spectra were recorded with IRaffinity-1 (Shimadzu)
spectrometers with KBr pellets. The spectral records were performed on a Shimadzu
UV-1800 Series single beam UV/Vis scanning spectrophotometer. Melting points
3

were determined by using Electro thermal melting point apparatus model 9100
(capillary method) and are uncorrected.
2.1.1. Preparation of 4-Bromohippuric acid (1):-
According to previously published procedure [33], a solution of glycine
(8.25gm, 0.11Mol) and potassium hydroxide (0.2Mol, in 50mL) was cooled to 00C
then 4-Bromobenzoylchlororide (21.95gm, 0.1Mol) was added in two portions with
shaking. Further, the reaction mixture was stirred for 30 min. After that, the solution
was acidified with conc. HCl and the product was collected (25gm) and recrystallized
from ethanol.
Yield: 96.89 %, M.P: found (155-157) °C, IR. (KBr) (νmax, Cm-1) 3296 (NH), 2550-
3296 (acid OH), 1703 (acid C=O), 1635 (amide C=O).
2.1.2. Preparation of 5-((3-methylphenyl)diazenyl)-2-hydroxybenzaldehyde (2).
The purified compound (2) was prepared according to our developed
procedure [34] in an excellent yield. The solid product dried and recrystallized from a
mixture of toluene and ethanol (1:4) to give (brown-yellow) crystals of compound (2).
C14H12N2O2; M.P: 160-161oC; yield: 91%, IR(νmax/cm-1): 3186, 2839, 2733, 1660,
1579, 1479, 1280.
2.1.3 Preparation of (E)-2-(benzyloxy)-5-((3-methylphenyl)diazenyl)
benzaldehyde (3).
According to described procedure [35], a solution of benzylbromide (13.68gm,
0.08mol) and 5-((3-methylphenyl)diazenyl)-2-hydroxybenzaldehyde (12gm, 0.05Mol)
in (100 mL) of ethanol 96% were refluxed for 10min. Anhydrous Na2CO3 (15.9gm,
0.15Mol) was added in three portions to the reaction mixture. Further refluxed
continued for six hours, then the reaction mixture was added to cold distilled water
(150 mL) to give an orange precipitate, which was filtered off and washed with a
mixture of cold water and ethanol (1:1) to remove excess of salts and benzyl
4

bromide, then recrystallized from a mixture of toluene and ethanol (1:4) to give an
orange-brownish precipitate (3).
C21H18N2O2 Orange solid, yield: 15.5gm (93.9%); M.P: 86-88; IR (νmax/cm-1): 2864,
1
2765, 1683(C=O), 1602 (C=C ring), 1577(N=N), 1253- 1161(C-O-C). H-NMR (δ,
ppm): 2.27-2.55 (s, 3H- Ar-CH3), 5.25 (s, 2H-OCH2), 7.12-8.42 (m, 12H- Ar-H), 10.57
(s, 1H- aldehydic group). 13C-NMR (δ, ppm): (21.44:C-Ar-CH3, 71.32:C-OCH2,
113.52:C3, 120.52:C12, 123.66:C8, 123.68:C6, 125.49:C14,18, 127.43:C16, 128.56:C10,
128.90:C1, 128.99:C15,17, 129.76:C11, 131.88:C4, 135.66:C13, 139.06:C9, 146.74:C5,
152.63:C7, 162.60:C2, 189.24:C=O.
2.1.4 Synthesis of an azo-oxazolone; 4-((Z)-2-(benzyloxy)-5-((E)-m-
tolyldiazenyl)benzylidene)-2-(4-bromophenyl)oxazol-5(4H)-one (4):-
According to modified procedure [36], (6.6gm, 0.020Mol) of compound 3 and
glacial acetic acid (15 mL) stirred under reflux. Then freshly fused sodium acetate
(2.46gm, 0.030Mol), 4-bromohippuric acid (5.42gm, 0.021Mol) and acetic anhydride
(15 mL) were added. The reaction mixture immediately became soluble, after 2min.
Orange crystals appeared in the solution, during 5min large quantity of precipitate of
azo-oxazolone formed and the reaction mixture was refluxed for further 5min.,
(herein, during the reaction the volume of acetic acid and time very important
because, in the presence excess of acetic acid and extra time, the product become
soluble) the mixture was transferred in to a beaker and the flask rinsed with 20mL
ethanol then 15 mL of water added dropwise with stirring, then the reaction mixture
left in refrigerator for 30 min. The orange product was collected and washed with cold
aqueous sodium carbonate (50mL, 4%) to afford the desired compounds (4).
o
C30H22BrN3O3 Orange solid, yield: 10.5gm (95%), M.P: 180–181.2 C. IR (νmax/cm-
1): 1797(C=O) with shoulder at 1766, 1649 (C=N), 1587(C=C), 1552 (N=N), 1481,
1
1253, 1159. H-NMR (δ, ppm): 2.41 (s, 3H, Ar-CH3), 5.19 (s, 2H, -OCH2), 7.00-7.99
(m, 15H, Ar-H), 9.4 (s, 1H, vinylic H). 13C-NMR 21.55:C Ar-CH3, 70.96:C-OCH2-,
5

112.51:C3, 120.38:C=C, 123.12:C1, 123.29:C6, 124.60:C12, 125.43:C8, 127.09:C4,
127.38:C19, 128.33:C22, 128.38:C14,18, 128.44:C16, 128.92:C10, 129.09:C15,17
,
129.77:C11, 131.72:C20,24, 132.35:C=C ring, 133.23:C21,23, 135.86:C13, 139.05:C9,
146.94:C5, 152.68:C7, 160.15:C=N, 162.74:C2, 167.22:C=O. Elemental Anal. calcd
for C30H22BrN3O3: C, 65.23; H, 4.01; N, 7.61; found: C, 65.19; H, 3.98; N, 7.55.
2.1.5. Synthesis of azo benzamide derivatives
N-(-1-(2-(benzyloxy)-5-(m-tolyldiazenyl)phenyl)-3-((substitutedphenyl)-amino)-3-
oxoprop-1-en-2-yl)-4- bromobenzamide (5a-j ):-
In accordance with a previously established method [37] with some modifications, a
mixture of an Azo-oxazolone (0.83gm, 0.0015Mol) and 5mL of acetic acid were
heated in a beaker to boil, then the mixture transferred from the heating source to on
a sensitive balance (in fuming hood) and (0.0025Mol) of substituted aromatic amine
was added drop wise with stirring. Immediately the insoluble orange mixture became
liquor solution (in case 5i and 5j were refluxed for 30min), on cooling yellow-orange
crystals of (5a-j) appeared in the solution then 10 mL of D.W added. The described
product was filtered off, washed by D.W, dried and crystallized (twice) in a mixture of
toluene and ethanol in which each compound was dissolved in 10mL of toluene,
filtered (hot) and evaporated to half. After cooling ethanol was added drop wise until
nice crystals produced.
Table 1 Some physical properties and IR data for the synthesized azo-
benzamides (5a-j)
Molecular
formula
IR str.cm-1
Prod. R
M.P
%Yield
C=O N-H
5a
5b
5c
H
C36H29BrN4O3
C37H31BrN4O3
C37H31BrN4O3
211-212
192-193
168-169
83
91
95
1645 3240
1647 3240
1672, 3388
1658
2-CH3
3-CH3
6

5d
5e
5f
4-CH3
C37H31BrN4O3
C37H31BrN4O4
C37H31BrN4O4
210-212
188-189
196-197
186-187
316-317
96
92
98
82
97
98
78
1641 3219
1670 3244
1641 3224
1662 3230
1645 3240
1641 3263
1678 3236
2-OCH3
4-OCH3
5g
5h
5i
2-OCH2CH3 C38H33BrN4O4
4-OCH2CH3 C38H33BrN4O4
3-Cl-4-CH3
5-CH3-py
C37H30BrClN4O3 308-309
C36H30BrN5O3 175-176
5j
2.1.5d
N-((Z)-1-(2-(benzyloxy)-5-((E)-m-tolyldiazenyl)phenyl)-3-oxo-3-(p-
1
tolylamino)prop-1-en-2-yl)-4-bromobenzamide H-NMR (δ, ppm): 2.2 (s, 3H, p-
CH3), 2.33-.246 (s, 3H, m-CH3), 4.74 (s, 2H, -OCH2), 6.60 (s, 1H, NH-1), 6.84 (s, 1H,
NH-2), 7.11-7.94 (m, 19H, Ar-H), 9.24 (s, 1H, vinylic H). C13-NMR(δ, ppm): 20.90:C
of p-CH3, 21.39:C Ar-CH3,70.38:C-OCH2-, 112.91:C3, 119.68:C1, 121.08:C6,
121.45:C12, 122.73:C26,26`, 123.14:C17, 123.60:C8, 125.91:C4, 126.50:C14,14`
,
126.88:C10, 127.36:C23, 128.31:C16, 128.49:C18, 128.86:C15,15` 129.27:C11,
,
129.37:C27,27`, 131.65:C21,21`, 132.06:C22,22`, 132.39:C20, 135.57:C25, 135.76:C13,
136.16:C28, 138.94:C9, 146.79:C5, 152.45:C7, 157.69:C2, 163.97:C19(C=O),
165.77:C24 (C=O). Elemental Anal. Calcd. for C37H31BrN4O3: C, 67.38; H, 4.74; N,
8.49; found: C, 67.22; H, 4.66; N, 8.47.
2.1.5f N-((Z)-1-(2-(benzyloxy)-5-((E)-m-tolyldiazenyl)phenyl)-3-((4-ethoxyphenyl)
1
amino)-3-oxoprop-1-en-2-yl)-4-bromobenzamide H-NMR (δ, ppm): 2.39 (s, 3H,
m-CH3), 3.70 (s, 3H, OCH3), 4.83 (s, 2H, -OCH2), 6.64 (s,1H, NH-1), 6.70 (s,1H, NH-
2), 7.10-7.88 (m, 19H, Ar-H), 9.07 (s, 1H, vinylic H). C13-NMR(δ, ppm): 21.43:C Ar-
CH3, 55.47:OCH3, 71.53:C-OCH2-, 113.61:C3, 113.89:C27,27`, 119.58:C1, 121.29:C6,
121.72:C12, 123.10:C26,26`, 123.25:C17, 123.64:C8, 126.49:C4, 126.85:C14,14`
,
127.26:C10, 127.41:C23, 128.46:C16, 128.72:C18, 128.84:C15,15` 129.47:C11,
,
131.55:C21,21`, 131.63:C25, 131.70:C22,22`, 132.15:C20, 135.79:C13, 138.98:C9,
7

146.69:C5, 152.43:C7, 156.26:C28, 157.62:C2, 164.07:C19(C=O), 165.90:C24 (C=O).
Elemental Anal. Calcd. for C37H31BrN4O4: C, 65.78; H, 4.63N, 8.29; found: C, 65.45;
H, 4.62; N, 8.27.
2.1.5h N-((Z)-1-(2-(benzyloxy)-5-((E)-m-tolyldiazenyl)phenyl)-3-((4-ethoxyphenyl)
1
amino)-3-oxoprop-1-en-2-yl)-4-bromobenzamide H-NMR (δ, ppm): 1.24-1.41 (t,
3H, CH3 of -OCH2CH3), 2.34-2.46 (s, 3H, m-CH3), 3.83-3.86 (q, 2H, OCH2 of -
OCH2CH3), 4.69 (s, 2H, -OCH2), 6.54 (s,1H, NH-1), 6.56 (s,1H, NH-2), 7.08-7.91 (m,
19H, Ar-H), 9.37 (s, 1H, vinylic H). C13-NMR(δ, ppm):14.14:CH3 of OCH2CH3,
21.45:C Ar-CH3, 63.60:CH2 of OCH2CH3, 71.03:C-OCH2, 113.13:C3, 114.36:C27,27`
119.36:C1, 121.37:C6, 121.54:C12, 122.60:C26,26`, 123.38:C17, 123.93:C8, 126.71:C4,
126.85:C14,14`, 126.99:C10, 127.21:C23, 128.19:C16, 128.35:C18, 128.71:C15,15`
,
,
128.86:C11, 129.60:C21,21`, 131.43:C25, 131.54:C22,22`, 132.82:C20, 135.96:C13,
138.92:C9, 146.41:C5, 152.34:C7, 155.40:C28, 157.78:C2, 164.50:C19(C=O),
165.99:C24 (C=O). Elemental Anal. Calcd. for C38H33BrN4O4: C, 66.18; H, 4.82; N,
8.12; found: C, 65.81; H, 4.45; N, 7.98.
2.1.6. Synthesis of azo-imidazolone
5-((Z)-2-(benzyloxy)-5-((E)-m-tolyldiazenyl)benzylidene)-2-(4-bromophenyl)-3-(2-
ethylphenyl)-3,5-dihydro-4H-imidazol-4-one (6a-j):
Azo-imidazolone derivatives (6a-j) were synthesized as reported in procedure
[38] by mixing of azo-oxazolone (0.83gm, 0.0015Mol) with a substituted aromatic
amine (0.003Mol) in acetic acid (10 mL) and refluxed with stirring for (2-15) hrs. The
reaction mixture was poured into water and the described product was filtered off,
dried, then crystallized in a mixture of dioxane and ethanol or toluene and ethanol.
(compound was dissolved in toluene or dioxane (10mL) by slightly heating then
filtered, and toluene evaporated to the half, cooled to room temperature and 10mL of
ethanol was added, then put it in the refrigerator to produce orange or dark orange
crystals of (6a-j).
8

Table 2 Some physical properties and IR data for the synthesized azo-
imidazolones (6a-j).
Prod
.
R
Chemical
formula
Tim
e
M.P
%
IR str.cm-1
Yield
C=O C=N
hrs.
2
6a
6b
6c
6d
6e
6f
H
C36H27BrN4O2
C37H29BrN4O2
C37H29BrN4O2
C37H29BrN4O2
C37H29BrN4O3
C37H29BrN4O3
C38H31BrN4O3
C38H31BrN4O3
C37H30BrClN4O2
C36H28BrN5O2
266-267
165-166
252-253
314-315
247-248
218-219
235-237
211-212
94
83
89
94
89
92
86
98
92
81
1718 1635
1751 1680
1712 1635
1724 1635
1724 1670
1726 1635
1722 1635
1722 1627
1710 1633
1716 1674
2-CH3
3-CH3
4-CH3
2
2
2
2-OCH3
2
4-OCH3
2
6g
6h
6i
2-OCH2CH3
4-OCH2CH3
3-Cl-4-CH3
5-CH3-
2
2
10 200-201.5
15 195-196
6j
pyridine
2.1.6d
5-((Z)-2-(benzyloxy)-5-((E)-m-tolyldiazenyl)benzylidene)-2-(4-
1
bromophenyl)-3-(p-tolyl)-3,5-dihydro-4H-imidazol-4-one H-NMR (δ, ppm): 2.38
(s, 3H, p-CH3), 2.45 (s, 3H, m-CH3), 5.21 (s, 2H, -OCH2), 7.01-7.94 (m, 19H, Ar-H),
9.66 (s, 1H, vinylic H). C13-NMR(δ, ppm):21.36:C of p-CH3, 21.55:C Ar-CH3,70.79:C-
OCH2-, 112.44:C3, 120.16:C=C, 122.70:C1, 123.34:C6, 124.28:C12, 126.28:C8,
126.80:C4, 127.10:C22, 127.89:C14,14-, 128.25:C16,10, 128.37:C20,20-, 128.84:C26,30
,
129.00:C15,17, 130.30:C11, 130.90:C27,29, 131.55:C=Cring, 131.64:C21,23, 132.07:C25,
136.21:C13, 138.67:C19, 138.86:C28, 139.00:C9, 147.07:C5, 152.80:C7, 159.54:C=N,
160.34:C2, 170.49:C=O. Elemental Anal. calcd for C37H29BrN4O2: C, 69.27; H, 4.56;
N, 8.73; found: C, 69.1155; H, 4.63; N, 8.69..
9

2.1.6f
5-((Z)-2-(benzyloxy)-5-((E)-m-tolyldiazenyl)benzylidene)-2-(4-
1
bromophenyl)-3-(4-methoxyphenyl)-3,5-dihydro-4H-imidazol-4-one H-NMR (δ,
ppm): 2.41-2.47 (s, 3H, m-CH3), 3.83 (s, 3H, OCH3), 5.26 (s, 2H, -OCH2), 6.93-7.96
(m, 19H, Ar-H), 9.65 (s, 1H, vinylic H). C13-NMR(δ, ppm): 21.50:CH3 of Ar-CH3,
55.59:OCH3, 70.85:C-OCH2, 112.46:C3, 114.94:C25,25-, 120.17:C17, 122.79:C1,
123.25:C6, 124.32:C12, 126.32:C4, 126.77:C8, 127.25:C22, 127.34:C14,14-, 127.43:C16,
127.90:C10, 128.26:C20,20-, 128.33:C24,24-, 128.69:C15,15-, 128.84:C23, 128.98:C11,
130.88:C18, 131.69:C21,21-, 136.17:C13, 138.85:C19, 139.01:C9, 147.13:C5, 152.85:C7,
158.97:C26, 159.63:C=N, 160.33:C2, 170.72:C=O. Elemental Anal. Calcd. for
C37H29BrN4O3: C, 67.58; H, 4.45; N, 8.52; found: C, 67.2314; H, 4.50; N, 8.46.
2.1.6h
5-((Z)-2-(benzyloxy)-5-((E)-m-tolyldiazenyl)benzylidene)-2-(4-
bromophenyl)-3-(4-ethoxyphenyl)-3,5-dihydro-4H-imidazol-4-one 1H-NMR (δ,
ppm): 1.41-1.45 (t, 3H, CH3 of -OCH2CH3), 2.46 (s, 3H, m-CH3), 4.01-4.07 (q, 2H,
OCH2 of -OCH2CH3), 5.25 (s, 2H, -OCH2), 6.91-7.95 (m, 19H, Ar-H), 9.64 (s, 1H,
vinylic H). C13-NMR(δ, ppm): 14.56:CH3 of OCH2CH3, 21.61:C Ar-CH3, 63.37:CH2 of
OCH2CH3, 70.84:C-OCH2, 112.46:C3, 115.41:C25,25-, 120.16:C17, 122.73:C1,
123.26:C6, 124.33:C12, 126.30:C4, 126.78:C8, 127.16:C22, 127.26:C14,14-, 127.91:C16,
128.26:C10, 128.34:C20,20-, 128.64:C24,24-, 128.84:C15,15-, 128.98:C23, 130.89:C11,
131.53:C18, 131.68:C21,21-, 136.18:C13, 138.87:C19, 139.01:C9, 147.12:C5, 152.84:C7,
159.05:C26, 159.64:C=N, 160.33:C2, 170.72:C=O. Elemental Anal. Calcd. for
C38H31BrN4O3: C, 67.96; H, 4.65; N, 8.34; found: C, 67.70; H, 4.31; N, 8.27.
10

1
Note; The numbers(1,2,3,..28) indicated on the structures are used just to interpreting the H-NMR
and 13C-NMR spectra.
Scheme 1 Stepwise chemical reaction for synthesis of 4, (5a-j) and (6a-j)
11

2.2. Biological evaluation
2.2.1. Antimicrobial activity
2.2.1.1 Diffusion method
The antimicrobial activity was performed by disc diffusion method [39]. Some of the
synthesized chemicals were tested in vitro for their antimicrobial activity against two
types of bacterial strains namely Staphylococcus aureus and Escherichia coli, Known
antibiotics like Azithromycin and metronidazole were used for comparison. The
inhibition zones were measured in millimeters for different concentrations (200, 400,
600 and 800) (µg/mg in KBr) Figure (1), which were prepared under pressure for
each compound Table (3).
Table
3
preparation
of
difference concentration for each
disc.
Conc.
(µg)
Conc. Of
stock (mg)
10mg
Final wt
KBr of each
mg
disc
chemicals +
490mg KBr
10
(mg)
200
400
600
800
290
280
270
260
300
300
300
300
20
Figure 1 prepared known
30
concentration of each disc (12mm)
40
12

2.2.1.2. Dilution method for Determination of (MIC)
The tube macro dilution was used for measuring the minimum inhibition
concentration (MIC) some of the synthesized organic compounds. The procedure
was conducted by using the spectrophotometer technique at 600nm [40]. The
following dilutions were prepared for each compound (200, 400, 600, 800 and 1000)
(µg/mL) Table 4.The results were compared with metronidazole and azithromycin as
control. S. aureus and E. coil were used as the standard bacterial strains. The MIC
was measured for organic materials which inhibited bacterial growth, against control
sample which consisted of 2mL of nutrient broth and 40µL of activated culture of
bacterial suspension as clarified in Table 4, and then it was incubated at 37oC for
20hrs. The next day optical density (OD) was recorded as shown in Table 5.
Table 4 Preparation of test samples
Stock solution 8000µg/mL (0.04g in 5mL DMSO)
Concentration Chemical
Nutrient broth Bacterial suspension
NO.
(µg/mL)
200
Volume (µL) volume (mL) volume (µL)
1
2
3
4
5
50
1.91
1.86
1.81
1.76
1.71
40
40
40
40
40
400
600
100
150
200
250
800
1000
DMSO; dimethylsulfoxide
Table 5 Antimicrobial activity of the synthesized compounds against
Staphylococcus aureus and Escherichia coil.
Compounds
Concentration of chemicals (µg/mL)
(conc.vs abs.)
S.aureus
200
400
600
NG
800
NG
1000
NG
4
E.coli
S.aureus
E.coli
1.631
1.758
1.340
1.174
1.674
1.424
0.935
0.610
1.300 0.836 0.882 0.795
5a
5c
5d
0.876 0.398 0.158
0.650 NG NG
1.058 0.688 0.273
NG
NG
NG
NG
NG
S.aureus
E.coli
S.aureus
E.coli
0.762 0.375
NG NG
NG
NG
13

S.aureus
E.coli
0.747
1.128
1.525
1.038
1.481
1.070
1.725
1.256
1.341
1.524
1.114
1.574
1.375
1.203
1.400
1.384
0.075 0.123
NG
NG
5h
5i
0.983 0.631 0.358 0.269
S.aureus
E.coli
0.775 0.487 0.286
0.695 0.205 NG
0.985 0.643 0.500
0.897 0.376 NG
NG
NG
NG
NG
S.aureus
E.coli
6a
6c
6d
S.aureus
E.coli
1.075 0.817 0.404 0.005
0.745 0.234
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
S.aureus
E.coli
0.858
0.762
0.226
0.450
0.040
NG
NG
NG
NG
NG
NG
NG
NG
NG
S.aureus
E.coli
6h
Azithromycin
S.aureus
E.coli
Metronidazole
S.aureus
E.coli
0.11
0.028
NG; No growth of bacterial strain.
2.2.2. Creation of burn wounds
2.2.2.1. Animal care
The experiment and data collection were performed at the Department of biology,
University of salahaddin, albino wistar female rats weighing 200 ± 10 g were utilized
for this experimental model in which, three rats were housed in individual cage (40 ×
25 × 20) cm. The standard care and condition like photoperiod lighting conditions (12
h light/dark), temperature control (24 ± 2)oC and had free access to food and water at
all times were fixed. The experimental procedure was carried out according to the
ethics committee on animal experimentation (DIRECTIVE 2010/63/EU).
2.2.2.2 Burn procedure
The rats were acclimated to the laboratory for 7 days prior to beginning the study.
Initially, 18 rats (n=3) were weighed and anesthetized with a mixture of 10%
ketamine and 2% xylazine (90+10) mg/kg body weight intramuscularly. After all rats
accurately anesthetized the hair were removed by electrical clipper. Thermal injury
was induced by heating 10 mm a solid stainless steel bar Figure 2(e) on Bunsen
burner until the temperature reached between (160-180)oC, which was measured
with a thermometer simultaneously. Approximately 4cm2 areas on the dorsal proximal
14

region of the animal skin contacted with the bar twice side by side for 5 sec. to
produce thickness burn wounds. Figure 2(a&b). Corresponded to the high
temperature of the bar no pressure applied on the animal skin used in the burn
induction [26,41].
2.2.2.3 Infection
In order to show their inhibitory roles in vivo antimicrobial activity and in wound
healing assay of the synthesized compounds 4, 5d and 6d, after 24hrs. a burn
occurred, the burnt rats were infected by activated culture of S.aureus [42] which
were stored for 18hrs. In Müller-Hinton agar medium and then the turbidity of
bacterial suspensions were adjusted in sterile distilled water to match 0.5 McFarland
standards (108 CFU/mL) approximately, then the activated culture swabbed over the
wound of each injured rat Figure 2(c) and left to growth up for 24hrs.
Figure 2 Photographs of the appearance of different wounds A& B) are
on burnt day C) infected burn with S.aureus D) treatment of burnt wound E)
10mm bar used for making burn.
2.2.2.4. Cream preparation
Three creams of the compounds 4,5d and 6d in a concentration of (1% w/w) were
prepared by dissolving 0.05g of each compound in 2mL acetone in crucible and
adding then 5gm of pure paraffin wax added, heated on the sand bath with gentle
stirring to liquidity the wax and also to remove the acetone from the mixture, then
cooled to room temperature with stirring to produce homogenous cream, they were
sterilized in autoclave under steam heat at 121°C (15 Ib/in2) typically for 15 minutes,
then cooled with stirring Figure 3.
15

Figure 3 prepared creams
2.2.2.5. Wound healing
Burnt rats were randomly divided for four main groups test (4,5d and 6d), positive
control (pure paraffin wax), negative control (no treatment) and stander group
(Ucederm 1%) ( (n=3), the treatment started after 24 hours of infection, the injured
area in each wounded rat swabbed by these creams in a single dose during 24hrs.
the percentage of wound closure was monitored and calculated for each group and
the result shown in Figure 4 and Figure 11.
Figure 4 Differences in healing progression of wounds at days 0 (A), 4
(B), 10 (C), 15 (D) and 25 (E). Note F is representing shaved E.
2.2.3. Oral acute toxicity/single dose toxicity study
In order to examine the toxicity ability of each (4, 5d and 6d) in living system.
Wistar albino female rats (weighing between 210-230g) were selected for acute oral
toxicity test by up and down procedure (OECD Test Guidelines 425). The animals
were acclimatized for one week and fasted overnight prior to dosing.
16

2.2.3.1. Dose preparation
The required amount of the tested compounds (mg/kg) body weight, dissolved
in acetone and then the vehicle (corn oil) added, heated gently with stirring to
homogenize the mixture and removing acetone, Table 6.
2.2.3.2. Dose administration
According to (OECD/OCDE 425-27), three animals were weighed, the limited
test (2000mg/kg) body weight is administered in a single dose or twice dose in
24hrs., by gavage using a stomach tube for each compound, and corn oil was used
as vehicle and control Table 6. After dose administration, the animals were
individually monitored for first 6 hours (each 30 minutes), periodically during the first
24 hours and daily for a total of 14 days. Further observation such as behavioral
changes and Changes in eyes, skin and fur were recorded for each rat in comparison
to the control during the experiment. On day 15, the rats were intramuscularly
anesthetized with a mixture of ketamine10% and xylazine2%, sacrificed, and were
examined macroscopically for their external surface, abdominal organs and thoracic
organs.
Table 6 dose preparation and dosing per rat.
Conc.
mg/kg
2000
2500
3000
3500
4000
4500
5000
Acetone
Vehicle
No. of doses in
No. of used lethality
rat
(mL)
(mL)
24hrs.
2
2
2
4
4
4
4
3
3
3
5
5
5
5
1
1
1
2
2
2
2
1
1
1
1
1
1
3
No
No
No
No
No
No
No
2.2.4. FRAP (Ferric Reducing Antioxidant Power) assay
In order to measure the ferric reducing activity of our samples, assay was
conducted according to [43]. The assay, originally proposed by [44]. The FRAP
solution was freshly prepared by combination of acetate buffer (0.3Mol/L) (pH= 3.6),
2,4,6-tripyridyltriazine (TPTZ) (0.01Mol/L in HCl (0.04Mol/L) and FeCl3 (0.02Mol/L)
(10:1:1) by volume, respectively. The assay was carried out by placing 100 µL of the
tested chemicals (Conc. 1mg/mL) and 3 mL of FRAP reagent in a test tube, the
17

samples were continuously shaked for 15 min. Then the absorbance was recorded at
592 nm. Table 7. The standard curve of ascorbic acid was prepared using different
concentrations for comparison Figure 5. The FRAP value for each compounds
calculated by the following equation.
Table 7 Anti-oxidant activity some of the synthesized compounds
FERAP value
Comp.
Abs.
µM
µM/mL µM/gm
4
6d
6f
6h
5d
5f
5h
0.213
0.081
0.138
0.171
0.037
0.106
0.216
2.327
7.66
2.91
4.96
6.15
1.33
3.81
7.77
83.705
76.6
29.1
49.6
61.5
13.3
38.1
77.7
766
291
496
615
133
381
777
Ascorbic acid
837.05 8370.5
2.5
2
1.5
1
0.5
0
0
20
40
60
80
100
conc.
Figure 5 The standard curve of ascorbic acid.
Data analyses
Data analysis was performed using Microsoft Excel 2010, Graphpad prism6,
UVprobe 2.43 and IRsolution software.
3. Result and discussion
3.1. Chemistry
The improvement of advanced materials from simple molecules requires their
modification to develop functional properties. Which was desirable for some bio-
related uses, such as drug, biomedical, food and electronical applications.
18

Herein, we have synthesized hybrid molecules involving azo linkage and heterocyclic
moiety (oxazolones or imidazolones) Scheme 1, Oxazolone 4 was synthesized in
buffered solution via modified Erlenmeyer–Plöchl azlactone synthesis as the 4-
bromobenzoylglycine 1 is interfered to an azo-benzylated benzaldehyde 3. This
modification greatly enhanced the yield and reducing time from hours to minutes. The
resulting product was unbalanced molecule Scheme 2 due to the azo moiety and
benzyloxy group were in the same side of azlactone ring, respectively, which serving
us to synthesis a series of azo-benzamide (5a-j) in accordance to green synthesis
protocol. The reactive azo-oxazolone refluxed in acetic acid 99.9% in the presence of
various primary aromatic amines involves fast ring opening (within seconds) even
faster than under microwaves [37,45]. Finally, azo-benzamide derivatives were
refluxed in glacial acetic acid resulting azo-imidazolone derivatives (6a-j) by
intramolecular cyclodehydration. Notably, the presence of an electron-withdrawing
part in the aniline significantly decreases the rate of reaction Table 2.
Scheme 2 unbalanced azo-oxazolone 4 and approximately balanced
benzamide 5a
The structural confirmation of some synthesized compounds were made by their
1
physical properties and spectral data analysis such as (FT-IR, H-NMR, C13-NMR
and CHN analysis). The structure of the synthesized compounds (4, 5d, 5f, 5h, 6d, 6f
and 6h) adequately identical with the assumed structures. IR spectra was used to
monitor the carbonyl shifting from compound (2) to compound (6) Figure 6.
19

Figure 6 FT-IR charts of C=O str. Shifting from compound 2 to compound 6
series
The absorption spectra of the benzamide azo dyes (5a-j) Figure 7 and
imidazolone azo dyes (6a–i) Figure 8 were recorded in dioxane at room temperature
in quartz cuvette (C = 50ppm). Generally, two absorption bands at 400–410 and
290–300 nm available for azo compounds corresponding to n–π* and π–π*
transitions, respectively [46]. The UV-visible spectra azo-benzamide derivatives
significantly differ from those showed for azo-imidazolone derivatives, the azo-
imidazolone dyes show three intense bands in the (270–280, 340–360 and 410–420)
nm regions. The (410-420) nm band of the parent imidazolone ring disappeared from
the determinate region for the azo-benzamide dyes [22]. It was noted from both
figures that the approximately same spectrum observed for each compounds and the
absence of red shift due to the all substitutes far away from the azo group, which
means there is no electronic effect on the azo group. Finally, it should be
emphasized that all compounds with long π-conjugated bonds present absorption in
the visible region which may serve as an efficient photo switch in biological media
that require low-energy light, because, in case more energetic light could destroy
the system [22].
20

1.6
1.6
1.4
1.2
1
5a
1.4
5b
1.2
5c
1
5d
5e
0.8
0.6
0.4
0.2
0
0.8
5f
0.6
5g
0.4
5h
0.2
5i
250 275 300 325 350 375 400 425 450 475 500
5j
0
-0.2
λmax (nm)
Figure 7 UV-Visible spectra of azo-benzamidederivaties (5a-j)
2
4
6a
6b
6c
6d
6e
6f
1.5
1
0.5
0
6g
6h
6i
250 275 300 325 350 375 400 425 450 475 500
-0.5
6j
λmax (nm)
Figure 8 UV-Visible spectra of azo-imidazolone derivatives (6a-j) and
compound 4
3.2. Biological activity
3.2.1. Antimicrobial activity
The synthesized chemicals (4, 5d, 5f, 5h, 5i, 6d, 6f, 6h and 6i) were screened
against S.aureus as a Gram positive and E. coli as a Gram negative bacterial strains
by diffusion and dilution methods. The results of antibacterial screening as in Figure
9 & Figure 10 reveal that all tested compounds displayed better activity toward E.
coli than S. aureus, In our experiments, the tested compounds show relatively
stronger antimicrobial activity in dilution method than diffusion method. This
difference due to the large surface area available for contacting between chemicals
and bacterial strains, ultimately kill or inhibit growth maximum number of bacterial
21

strains. In both methods the antimicrobial intensity was concentration dependent.
The maximum zone inhibition activity of the tested compounds was showen at the
concentration (800µg) between (20-30) mm against S. aureus Figure 9 and (22-31)
mm against E. coil Figure 10. Compounds 4, 5h, 5i, 6d, 6f and 6i showed highest
activity (22-31) mm) against both microorganisms. The rest of the studied
compounds exhibited good to moderate activity (11-25 mm) against these
microorganisms.
40
S.aureus
35
30
25
200
20
15
10
5
400
600
800
0
Compounds
Figure 9 In vitro antimicrobial activity of screened compounds against S.
aureas
40
E.coli
35
30
200
25
20
15
10
5
400
600
800
0
Compounds
Figure 10 In vitro antimicrobial activity of screened compounds against
E. coli
22

Figure 11 Antimicrobial activities of 5i, 6d and metronidazole by disc
diffusion method with difference concentrations (200, 400, 600 and 800) µgm.
3.2.2. Wound Healing Assays
Encouraged by the in vitro results of the screened above, the skin wounds infected
with S. aureus on animal model was performed to evaluate whether these chemicals
had the competence to repair the wounds with Gram-positive bacterial infections in
vivo and on the other hand, in the last decades one of the most common pathogens
isolated from burn patients was S. aureus. The strong pathogenicity of S .aureus is
due to its ability to produce various exotoxins which cause to nonhealing residual
wounds and skin grafting failure [47]. The active compounds 4, 5d and 6d were
subjected to further study in the form of cream (1%) in comparison to ucederm (1%)
as a stander drug. First, approximately 4cm2 wound dressing were prepared, infected
with S.aureus and treatment with different creams were done in three successive
24hrs. Rats were daily treated in a single dose by approximately 2mg used in 24hrs.
for treated groups Table 8. The percentage of wound closure was monitored for each
group and the results were shown in Figure 12.
Table 8 Clarification of used mg for each treated group.
Treatment Total wt Remained
Used
cream
for
day
used
for
cream of
dose/day
mg of
each
comp.
used/day
2.3
groups
of cream
1%
after
treatment
(gm)
(gm)
treatment healing
4
5
2.02
2.98
13
0.23
23

5d
6d
+
5
5
15
1.92
1.3
4.52
3.08
3.7
10.48
16
14
25
0.19
0.26
0.42
1.9
2.6
4.2
uced.
15
7.3
7.7
22
0.35
3.5
100
80
60
40
20
0
-
+
Uced.
4
5d
6d
4
7
10
13
16
20
25
Time after injury (d)
Figure 12 the wound closure rate of different wounds on different days; - :
represent infected wounds without treatment, +: represent infected wounds
treated with pure paraffin wax, uced.: represent infected wounds treated with
Ucederm drug, (4, 5d and 6d) groups: represent infected wounds treated with
synthesized compound 4, 5d and 6d respectively.
3.2.3. Acute oral toxicity
The 4, 5d and 6d compounds were tested first for acute toxicity on rats at
concentrations (2000- 5000) mg/kg body weight to determine their 50% lethal
Concentration, during the experiment no lethality was recorded and the rats did not
show any significant evident concerning the toxicological signs, It is clear from Table
6 and Table 9 that the tested compounds were classified as potentially nontoxic [48].
At the end of the experiment, the used rats were sacrificed, the abdominal organs
and thoracic organs were weighed, the changes were not considered to be
associated with the toxicological changes between the control group and treated
group.
Table 9 Acute toxicity hazard categories and classification System for
(approximate) LD50/LC50 values defining the respective categories (OECD
1998b; updated OECD, 2001).
Acute Toxicity Toxicity
Route Class 1
Toxicity
Class 2
Toxicity
Class 3
Toxicity
Class 4
Toxicity
Class 5
24

Oral
LD50 Values
(mg/kg)
≤5
>5 ≤50
>50 ≤300 >300 ≤2000 >2000 ≤5000
[approximate]
3.2.4. FRAP reducing power
The antioxidant activity of seven synthesized compounds were measured by FRAP is
presented in Figure 13. It shows that electron donating groups enhanced reducing
power. Compounds (5h and 6h) with OCH2CH3 group indicate that they is most
effective electron donor and can reduce the oxidized intermediates highly reactive
molecules like free radicals and reactive oxygen species of peroxidation processes.
In this study, the yellow color of the test solution reduce ferric complex with TPTZ
(color less) to Ferrous complex with TPTZ (violet color) changes to various shades of
violet depending on the reducing power of each compound, In this assay. The higher
reductive potential was dermined by higher absorbance of the reaction mixture.
766
291
291
615
381
133
777
4
5d
5f
5h
6d
6f
6h
Compounds
Figure 13 Antioxidant activity of tested compounds measured by FRAP
assay.
4. Conclusion
In the present study we have showen the combination of two excellent molecular
moieties azo group and heterocyclic chromophores. The highly versatile azo-
oxazolone intermediate has been synthesized in buffer solution. It is noteworthy that
in comparison to classical method buffer solution afforded some advantages such as
excellent yield, short reaction time and easy workup. Also, it should be noted that the
azo side chain in oxazolone made extremely reactive molecule which enhance the
25

ring opening reaction for synthesis of a series of azo-benzamides and a series of
azo-imidazolone analogs, which were confirmed by proper apparatuses such as FT-
1
IR, H-NMR, C13-NMR and CHN analysis. The expected structures were in
accordance with collected spectral data. The comparable in vivo and in vitro
antimicrobial evaluated. All the screened compounds 4, 5h, 5i, 6d and 6f showed
good activity against S. aureus whereas all screened compounds were found active
toward E. coli. On the other hand, the growth inhibition of S. aureus and wounds
repair was achieved as results of treatment by 4, 5d and 6d on the rats infected
wound. Consequently, showed balanced between biological safety and antimicrobial
activity. The results obtained for the three used compounds in comparison to the
stander drug ucedem will be highly beneficial toward new powerful compounds as
novel antimicrobial and repair of the wounds. Compounds 4, 5h and 6h showed
remarkable reducing power which is significant indicator of its potential antioxidant
activity. According to acute toxicity hazard categories (OECD) the tested compounds
(4, 5d and 6d) are totally safe against living system.
Conflict of interest
Authors have no conflict of interest.
Acknowledgement
This work was supported by Salahaddin University- Hawler, Erbil-Kurdistan –Iraq as
the PhD program (No. 15738/1/1- 1/ 12/ 2015).The authors would like to thank
Rebwar Muhammad Hamasalih, Saman Muhsin Abdulkareem and Sawar
Nawzad Jafar for excellent technical assistance performed in vitro and in vivo
biochemical assays.
26

Figure 14 Represent IR Spectrum of compound 4
Figure 15 Represent 1H-NMR Specrum of (4)
Figure 16 Represent C13-NMR Spectum of copmpound 4
27

80
%T
60
40
20
4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500
1ARO3 REC (2) 1/cm
Figure 17 Represent IR Spectrum of compound 5h
Figure 18 Represent 1H-NMR Spectrum of compound 5h.
Figure 19 Represent C13-NMR Spectrum of compound 5h.
28

Figure 20 Represent IR Spectrum of compound 6g
Figure 21 Represent 1H-NMR Spectrum of compound 6g
Figure 22 Represent C13-NMR Spectrum of compound 6g
29