Synthesis, absorption properties and biological evaluation of some novel disazo dyes derived from pyrazole derivatives

Article abstract of DOI:10.14233/ajchem.2015.18769

In this study, 20 novel disazo dyes containing pyrazole derivatives were synthesized by a convenient method. Diazotized aniline and some aniline derivatives were reacted with malononitrile to give 2-arylazomalononitrile derivatives 1(a-e). The synthesized 2-arylazomalononitrile derivatives were reacted with hydrazine and phenyl hydrazine to afford the corresponding 3,5-diamino-4-arylazo-1 H-pyrazole 2(a-e) and 3,5-diamino-4-arylazo-1-phenylpyrazole 3(a-e). Diazotized compounds of 2(a-e)and 3(a-e) reacted with ethylacetoacetate to give 4-arylazo-3-amino-1 H-pyrazole-5-ylazo-ethylacetoacetate 4(a-e) and 4-arylazo-3-amino-1-phenylpyrazole-5-ylazo-ethylacetoacetate 7(ae). The obtained 4(a-e) and 7(a-e) were reacted with hydrazine and phenyl hydrazine to give disazo dyes 5(a-e), 6(a-e), 8(a-e) and 9(a-e), respectively. The synthesized disazo dyes were characterized by spectroscopic techniques as well as elemental analysis. The solvatochromic behaviours of these dyes in various solvents were examined. Acid-base effects on the absorption maxima of the dyes were also reported. Antimicrobial activities of the synthesized novel disazo dyes were investigated.

Full text of DOI:10.14233/ajchem.2015.18769

Asian Journal of Chemistry; Vol. 27, No. 8 (2015), 3003-3012
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2015.18769
Synthesis, Absorption Properties and Biological Evaluation of
Some Novel Disazo Dyes Derived from Pyrazole Derivatives
1,*
1
2
1
NESRIN SENER , IZZET SENER , SERKAN YAVUZ and FIKRET KARCI
1Department of Chemistry, Faculty of Science-Arts, Pamukkale University, 20017 Denizli, Turkey
2Department of Chemistry, Faculty of Science, Gazi University, 06500 Ankara, Turkey
*Corresponding author: Fax: +90 258 2963723; Tel: +90 258 2963603; E-mail: nesrinburukoglu_sener@hotmail.com.tr
Received: 20 September 2014; Accepted: 16 December 2014; Published online: 27 April 2015;
AJC-17181
In this study, 20 novel disazo dyes containing pyrazole derivatives were synthesized by a convenient method. Diazotized aniline and some
aniline derivatives were reacted with malononitrile to give 2-arylazomalononitrile derivatives 1(a-e). The synthesized 2-arylazomalononitrile
derivatives were reacted with hydrazine and phenyl hydrazine to afford the corresponding 3,5-diamino-4-arylazo-1H-pyrazole 2(a-e) and
3,5-diamino-4-arylazo-1-phenylpyrazole 3(a-e). Diazotized compounds of 2(a-e)and 3(a-e) reacted with ethylacetoacetate to give 4-
arylazo-3-amino-1H-pyrazole-5-ylazo-ethylacetoacetate 4(a-e) and 4-arylazo-3-amino-1-phenylpyrazole-5-ylazo-ethylacetoacetate 7(a-
e). The obtained 4(a-e) and 7(a-e) were reacted with hydrazine and phenyl hydrazine to give disazo dyes 5(a-e), 6(a-e), 8(a-e) and 9(a-e),
respectively. The synthesized disazo dyes were characterized by spectroscopic techniques as well as elemental analysis. The solvatochromic
behaviours of these dyes in various solvents were examined. Acid-base effects on the absorption maxima of the dyes were also reported.
Antimicrobial activities of the synthesized novel disazo dyes were investigated.
Keywords: Pyrazolone, Diazotization, Solvatochromism, Pyrazole, Disazo dyes, Antimicrobial activity.
by considering the fact that many of pyrazole derivatives show
antimicrobial activity, disazo dyes containing pyrazole ring
can also show antimicrobial activity.
In the present study, 20 novel disazo dyes containing
pyrazole derivatives were synthesized and investigated for their
absorption properties and antimicrobial activity. The structural
characterization, absorption properties and preliminary biolo-
gical evaluation of these novel compounds could be interesting
for screening potent dyes having antimicrobial activity.
INTRODUCTION
Azo dyes and pigments generate the largest and most
varied group of synthetic organic colorants in use today, having
wide applications in textile, food, paper printing, ink, bio-
medical and cosmetics industries. They have characteristically
good tinctorial strength as well as stability. Their preparation
procedures by the classic diazotization and coupling reactions,
is very simple and low cost1-7.
Pyrazole and their substituted derivatives are used as
potential pharmaceuticals and intermediates in dye industry.
Azo pyrazoles were exhibited a wide variety of biological and
pharmaceutical activities and therefore they play important
role in medicinal chemistry. An exciting development in the
synthesis of nitrogen heterocycles like azopyrazoles has com-
menced in last few years8,9. The pyrazole nucleus has been
reported to possess a wide spectrum of biological properties
such as anti-inflammatory, antibacterial, analgesic, antifungal
and antiviral10-14. Pyrazoles having azo group have been found
to exhibit a wide range of biological activities like antibac-
terial, CNS depressant, antitumor, potent local anesthetics and
EXPERIMENTAL
The chemicals were purchased from Aldrich, Sigma and
Merck Chemical Company without further purification. Solvents
were of spectroscopic grade. Melting points of the synthesized
dyes were determined by using stuart smp 30 melting point
apparatus (UK). Nuclear magnetic resonance (1H NMR) spectra
were recorded on a Bruker (Germany) spectrosp in avance DPX
400 ultra-shield 400 MHz spectrometer at room temperature by
using tetramethylsilane (TMS) as the internal standard. Chemical
shifts were (δ) given in ppm. IR spectra were recorded on a
Perkin Elmer FT-IR spectrometer (USA). Mass spectra were
obtained from waters LCT premier XE LTOF (TOF MS) instru-
ments (USA). Elemental analyses were done on a Leco CHNS-
932 analyzer (USA). Absorption spectra were recorded on an
dyes9,15,16
.
Diazo compounds are widely used as coloring materials.
The antimicrobial activity of these compounds can provide a
great advantage in practice. This study has been carried out

3004 Sener et al.
Asian J. Chem.
ATI (UK) Unicam UV-100 spectrophotometer over the range
of λ between 300-700 nm. The wavelengths of maximum
absorption (λmax) were investigated in various solvents such as
dimethylsulfoxide (DMSO), dimethylformamide (DMF),
acetonitrile, methanol, acetic acid and chloroform at various
concentrations (1 × 10-6-1 × 10-8 M). λmax change of 1 mL the
dye solution in methanol due to addition of 0.1 mL of base
(potassium hydroxide, 0.1 M) or 0.1 mL of acid (hydrochloric
acid, 0.1 M) was investigated.
Synthesis: 2-Arylazomalononitriles 1(a-e) were synthe-
sized as described by reported method17. The general route
for the synthesis of 2-arylazomalononitriles 1(a-e) is showed
in Fig. 1.
in a mixture of glacial acetic acid (10 mL) and concentrated
hydrochloric acid (5 mL).The solution was cooled to 0-5 °C.
Sodium nitrite 0.35 g (4.95 mmol) was dissolved in water (10
mL) then added to this solution dropwise with vigorous stirring,
during about 1 h, while cooling at 0-5 °C. The clear diazonium
salt solution was poured in portions over 0.5 h. into well-cooled
(0-5 °C) and stirred solution of ethylacetoacetate 0.64 g (4.95
mmol) in pyridine. Stirring continued for 2 h at 0-5 °C and the
precipitated products separated upon dilution with cold water
(50 mL) were filtered off, washed with water several times
and dried. The obtained product 4-phenylazo-3-amino-1H-
pyrazole-5-ylazo-ethylacetoacetate (4a) was dissolved in
ethanol (50 mL) and then hydrazine monohydrate 0.74 g (14.85
mmol) was added into this solution. The reaction mixture was
heated under reflux for 3-4 h, then cooled up to the room
temperature and the precipitated products were separated upon
dilution with water (50 mL), filtered off, washed with water
several times and dried. The obtained product was crystallized
from DMF-H2O mixture (2:3 by volume) to give 4-(4'-phenyl-
azo-3'-amino-1'H-pyrazole-5-ylazo)-3-methyl-1H-pyrazole-5-
on (5a) as dark red solid crystals, yield 1.17 g (76 %), m.p. >
291 °C dec. Anal. Calcd. for C13H13N9O; C: 50.16 %; H: 4.18
%; N: 40.51 %; found: C: 50.21 %; H:4.21 %; N: 40.54 %.
FT-IR (νmax, cm-1): 3251 (-NH2); 3147 (-NH); 3020 (Ar-H);
CN
NC
NaNO2 /HCl
N
C
X
NH2
X
NH
CH2
+
CN
NC
1(a-e)
X: H, p-NO2 , p-OCH , p-Cl, p-CH3
3
Fig. 1. Synthesis of 2-arylazomalononitriles 1(a-e)
Synthesis of 3,5-diamino-4-arylazo-1H-pyrazoles and
3,5-diamino-4-arylazo-1-phenylpyrazoles: 3,5-Diamino-4-
arylazo-1H-pyrazole 2(a-e) and 3,5-diamino-4-arylazo-1-
phenylpyrazole 3(a-e) were prepared according to the proce-
dures given in literatures18-20. The general route for the synthesis
of 3,5-diamino-4-arylazo-1H-pyrazoles and 3,5-diamino-4-
arylazo-1-phenylpyrazoles is outlined in Fig. 2.
1
2990 (Aliphatic C-H); 1672 (C=O); 1532, 1482 (N=N). H
NMR (400 MHz, DMSO-d6, 25 °C) δ (ppm): 2.18 (s, 3H,
pyrazolone-CH3); 6.79 (s, 2H, -NH2); 7.31-8.19 (m, 5H, Ar-
H); 11.62-11.89 (g, -NH); 13.70-14.48 (s, -OH). HR-MS:
311.3026 ([M + H]+, calcd. 311.3024.
General synthesis of disazo dyes 5(a-e), 6(a-e), 8(a-e) and
9(a-e)
H
Synthesis of 4-(4'-phenylazo-3'-amino-1'H-pyrazole-
5-ylazo)-3-methyl-1H-pyrazole-5-one (5a): 3,5-Diamino-4-
phenylazo-1H-pyrazole (2a) 1 g (4.95 mmol) was dissolved
N
O
N
X
N = N
CH3
N
N
NH2NH2.H2
O
O
OEt
NH
H
H2
N
N
X
N = N
NH2
X
N = N
H2N
N
N
NH2NH2.H2
O
CH3
C
5(a-e)
NaNO2 / HCl / CH3COOH
CH3COCH2CO2C2H5
NH
O
NH
H2
N
N
N
2(a-e)
4(a-e)
N
O
N
PhNHNH2
CH3
X
N = N
N = N
NH
H2
N
N
CN
6(a-e)
X
N
N
C
H
CN
1(a-e)
H
N
O
N
CH3
X
NH2NH2.H2
O
N = N
N = N
O
OEt
N
H
N
H2
N
N
X
N = N
N
X
N = N
NH2
CH3
C
8(a-e)
NaNO2 / HCl / CH3COOH
CH3COCH2CO2C2H5
O
N
N
PhNHNH2
H2
N
H2N
N
N
3(a-e)
7(a-e)
N
O
N
PhNHNH2
N = N
CH3
X
N = N
N
N
H2
N
9(a-e)
Fig. 2. General route of synthesized dyes

Vol. 27, No. 8 (2015)
Synthesis, Absorption Properties and Biological Evaluation of Some Novel Disazo Dyes 3005
The above procedure was also used to synthesize dye
5(b-e). The general route of synthesized dyes is outlined in
Fig. 2.
4-[4'-(4''-Nitrophenyl)azo-3'-amino-1'H-pyrazole-5-
ylazo]-3-methyl-1-phenylpyrazole-5-one (6b): Red solid
crystals, yield 1.21 g (69 %), m.p. > 330 °C dec. Anal. Calcd.
for C19H16N10O3; C: 52.278 %; H: 3.70 %; N: 32.41 %; found:
C: 52.90 %; H: 3.71 %; N: 32.43 %. FT-IR (νmax, cm-1): 3241
(-NH2); 3120 (-NH); 3048 (Ar-H); 2960 (Aliphatic C-H); 1672
(C=O); 1515, 1324 (NO2); 1531, 1498 (N=N). 1H NMR (400
MHz, DMSO-d6, 25 °C) δ (ppm): 2.29 (s, 3H, pyrazolone-
CH3); 6.95 (s,pyrazole-NH2); 7.21-8.28 (m, 9H, Ar-H); 11.75
(b, pyrazole-NH); 13.20 (s, -OH). HR-MS: 432.3954 ([M +
H]+, calcd. 432.3955.
4-[4'-(4''-methoxyphenyl)azo-3'-amino-1'H-pyrazole-
5-ylazo]-3-methyl-1-phenylpyrazole-5-one (6c): Light orange
solid crystals, yield 1.26 g (70 %), m.p.: 305-306 °C. Anal.
Calcd. for C20H19N9O2; C: 57.55 %; H: 4.56 %; N: 30.21 %;
found: C: 57.57 %; H: 4.57 %; N: 30.23 %. FT-IR (νmax, cm-1):
3230 (-NH2); 3105 (-NH); 3065 (Ar-H); 2914 (Aliphatic
C-H); 1670 (C=O); 1551-1496 (N=N). 1H NMR (400 MHz,
DMSO-d6, 25 °C) δ (ppm): 2.28 (s, 3H, pyrazolone-CH3); 3.58
(s, 3H, p-OCH3); 6.69 (s, pyrazole-NH2); 7.01-8.01 (m, 9H,
Ar-H); 11.88 (b, pyrazole-NH); 14.15 (s, -OH). HR-MS:
417.4239 ([M + H]+, calcd. 417.4240.
4-[4'-(4''-Nitrophenyl)azo-3'-amino-1'H-pyrazole-5-
ylazo]-3-methyl-1H-pyrazole-5-one (5b): Dark-orange solid
crystals, yield 1.05 g (73 %), m.p. > 340 °C dec. Anal. Calcd.
for C13H12N10O3; C: 43.82 %; H: 3.37 %; N: 39.325 %; found:
C: 43.83 %; H:3.36 %; N: 39.37 %. FT-IR (νmax, cm-1): 3255
(-NH2); 3187 (-NH); 3081 (Ar-H); 2980 (Aliphatic C-H); 1667
1
(C=O); 1547,1413 (N=N); 1513,1336 (NO2). H NMR (400
MHz, DMSO-d6, 25 °C) δ (ppm): 2.18 (s, 3H, pyrazolone-
CH3); 7.15 (s, 2H, -NH2); 8.01-8.65 (m, 4H, Ar-H); 11.95 (b,
-NH); 14.27 (s, -OH). HR-MS: 356.310 ([M + H]+, calcd.
356.2996.
4-[4'-(4''-Methoxyphenyl)azo-3'-amino-1'H-pyrazole-
5-ylazo]-3-methyl-1H-pyrazole-5-one (5c): Burgundy red
solid crystals, yield 1.03 g (70 %), mp: 188 °C. Anal. Calcd.
for C14H15N9O2; C: 49.26 %; H: 4.40 %; N: 36.95 %; found:
C: 49.27 %; H:4.41 %; N: 36.96 %. FT-IR (νmax, cm-1): 3256
(-NH2); 3195 (-NH); 3054 (Ar-H); 2997 (Aliphatic C-H); 1670
1
(C=O); 1542, 1491 (N=N). H NMR (400 MHz, DMSO-d6,
25 °C) δ (ppm): 2.15 (s, 3H, pyrazolone-CH3); 3.78 (s, 3H,
p-OCH3); 7.05 (s, 2H, -NH2); 7.01-8.10 (m, 4H,Ar-H); 11.51-
11.67 (b, -NH); 13.71-14.26 (s, -OH). HR-MS: 341.3165 ([M
+ H]+, calcd. 341.3284.
4-[4'-(4''-Chlorophenyl)azo-3'-amino-1'H-pyrazole-5-
ylazo]-3-methyl-1-phenylpyrazole-5-on (6d): Orange solid
crystals, yield 1.28 g (72 %), m.p.: 288-289 °C. Anal. Calcd.
for C19H16N9OCl; C: 54.09 %; H: 3.795 %; N: 29.89 %; found:
C: 54.11 %; H: 3.80 %; N: 29.95 %. FT-IR(νmax, cm-1): 3235
(-NH2); 3104 (-NH); 3064 (Ar-H); 2920 (Aliphatic C-H); 1671
4-[4'-(4''-Chlorophenyl)azo-3'-amino-1'H-pyrazole-5-
ylazo]-3-methyl-1H-pyrazole-5-on (5d): Dark-brown solid
crystals, yield 1.07 g (73 %), m.p. > 340 °C dec. Anal. Calcd.
for C13H12N9OCl; C: 45.15 %; H: 3.47 %; N: 36.47 %; found:
C: 45.17 %; H:3.48 %; N: 36.47 %. FT-IR (νmax, cm-1): 3245
(-NH2); 3086 (-NH); 3040 (Ar-H); 2918 (Aliphatic C-H); 1670
(C=O); 1550,1409 (N=N). 1H NMR (400 MHz, DMSO-d6, 25
°C) δ (ppm): 2.17 (s, 3H, pyrazolone-CH3); 6.95 (s, 2H, -NH2);
7.63-8.01 (m, 4H, Ar-H); 11.89 (b, -NH); 14.21 (s, -OH). HR-
MS: 345.7472 ([M + H]+, calcd. 345.7471.
1
(C=O); 1553-1499 (N=N). H NMR (400 MHz, DMSO-d6,
25 °C) δ(ppm): 2.29 (s, 3H, pyrazolone-CH3); 6.79 (s, pyrazole-
NH2); 7.22-8.05 (m, 9H,Ar-H); 14.01 (b, pyrazole-NH); 14.28
(s, -OH). HR-MS: 421.8431 ([M + H]+, calcd. 421.8430.
4-[4'-(4''-Methylphenyl)azo-3'-amino-1'H-pyrazole-5-
ylazo]-3-methyl-1-phenylpyrazole-5-one (6e): Brick red solid
crystals, yield 1.32 g (71 %), m.p.: 309-310 °C. Anal. Calcd.
for C20H19N9O; C: 59.85 %; H: 4.738 %; N: 31.42 %; found:
C: 59.88 %; H: 4.74 %; N: 31.45 %. FT-IR (νmax, cm-1): 3240
(-NH2); 3110 (-NH); 3002 (Ar-H); 2918 (Aliphatic C-H); 1651
4-[4'-(4''-Methylphenyl)azo-3'-amino-1'H-pyrazole-5-
ylazo]-3-methyl-1H-pyrazole-5-one (5e): Dark-red solid
crystals, yield 1.06 g (70 %), m.p. > 278 °C dec. Anal. Calcd.
for C14H15N9O; C: 51.69 %; H: 4.61 %; N: 38.77 %; found: C:
51.70 %; H:4.63 %; N: 38.79 %. FT-IR (νmax, cm-1): 3230 (-
NH2); 3115 (-NH); 3050 (Ar-H); 2948 (Aliphatic C-H); 1673
1
(C=O); 1541-1498 (N=N). H NMR (400 MHz, DMSO-d6,
25 °C) δ (ppm): 2.31 (s, 3H, pyrazolone-CH3); 2.35 (s, 3H,
p-CH3); 6.82 (s, pyrazole-NH2); 7.21-7.95 (m, 14H, Ar-H);
13.33 (b, pyrazole-NH); 14.29 (s, -OH). HR-MS: 401.4245
([M + H]+, calcd. 401.4246.
1
(C=O); 1540, 1420 (N=N). H NMR (400 MHz, DMSO-d6,
25 °C) δ (ppm): 2.17 (s, 3H, pyrazolone-CH3); 2.31 (s, 3H, p-
CH3); 7.05 (s, 2H, -NH2); 7.22-7.45 (m, 4H, Ar-H); 11.54 (b,
-NH); 13.45 (s, -OH). HR-MS: 325.3290 ([M + H]+, calcd.
325.3286.
Synthesis of 4-[4'-(phenyl)azo-3'-amino-1'-phenylpyrazole-
5-ylazo]-3-methyl-1H-pyrazole-5-one (8a)
Compounds 8(a-e) were prepared as described above for
5a using 3,5-diamino-4-arylazo-1-phenylpyrazole 3(a-e):
Silvery red solid crystals, yield 1.02 g (73 %), m.p.: 276-
277 °C. Anal. Calcd. for C19H17N9O; C: 58.91 %; H: 4.39 %;
N: 32, 558 %; found: C: 58.88 %; H: 4.41 %; N: 32.539 %.
FT-IR (νmax, cm-1): 3310 (-NH2); 3191 (-NH); 3054 (Ar-H);
Synthesis of 4-(4'-[phenyl)azo-3'-amino-1'H-pyrazole-5-
ylazo]-3-methyl-1-phenylpyrazole-5-one (6a)
Compounds 6(a-e) were prepared as described above for
5a using phenylhydrazine: Dark-red solid crystals, yield 1.38
g (72 %), m.p.: 276-277 °C. Anal. Calcd. for C19H17N9O; C:
58.91 %; H: 4.39 %; N: 32.558 %; found: C: 58.92 %; H: 4.41
%; N: 32.56 %. FT-IR (νmax, cm-1): 3261 (-NH2); 3113 (-NH);
3064 (Ar-H); 2959 (Aliphatic C-H); 1672 (C=O); 1531-1491
(N=N). 1H NMR (400 MHz, DMSO-d6, 25 °C) δ (ppm): 2.29
(s, 3H, pyrazolone-CH3); 6.85 (s,pyrazole-NH2); 7.25-8.05 (m,
10H, Ar-H); 11.95 (b,pyrazole-NH); 14.10 (s, -OH). HR-MS:
387.3981 ([M + H]+, calcd. 387.3980.
1
2996 (Aliphatic C-H); 1664 (C=O); 1528-1497 (N=N). H
NMR (400 MHz, DMSO-d6, 25 °C) δ (ppm): 2.14 (s, 3H,
pyrazolone-CH3); 7.17 (g, 2H, pyrazole-NH2); 7.35-8.09 (m,
10H, Ar-H); 11.71 (b, pyrazolone-NH); 14.21 (s, -OH). HR-
MS: 387.3981 ([M + H]+, calcd. 387.3980.
4-[4'-(4''-Nitrophenyl)azo-3'-amino-1'-phenylpyrazole-
5-ylazo]-3-methyl-1H-pyrazole-5-one (8b): Dark red solid

3006 Sener et al.
Asian J. Chem.
crystals,yield 0.92 g (69 %), m.p. > 280 °C dec. Anal. Calcd.
for C19H16N10O3; .C: 52.278 %; H: 3.70 %; N: 32.41 %; found:
C: 52.28 %; H: 3.73 %; N: 32.42 %. FT-IR (νmax, cm-1): 3381
(-NH2); 3231 (-NH); 3174 (Ar-H); 2960 (Aliphatic C-H); 1669
(C=O); 1535-1521 (N=N); 1504 and 1337 (-NO2). 1H NMR
(400 MHz, DMSO-d6, 25 °C) δ (ppm): 2.14 (s, 3H, pyrazolone-
CH3); 7.17 (g, 2H, pyrazole-NH2); 7.58-8.37 (m, 9H, Ar-H);
11.77 (b, pyrazolone-NH); 14.25 (s, -OH). HR-MS: 432.3954
([M + H]+, calcd. 432.3955
C: 58.97 %; H: 3.95 %; N: 27.53 %. FT-IR (νmax, cm-1): 3353
(-NH2); 3063 (Ar-H); 2930 (Aliphatic C-H); 1661 (C=O);
1
1553-1493 (N=N); 1537, 1324 (NO2). H NMR (400 MHz,
DMSO-d6, 25 °C) δ (ppm): 2.26 (s, 3H, pyrazolone-CH3); 7.26
(b, 2H, pyrazole-NH2); 7.48-8.38 (m, 14H, Ar-H); 14.29 (s,
-OH). HR-MS: 508.4916 ([M + H]+, calcd. 508.4915.
4-[4'-(4''-Methoxyphenyl)azo-3'-amino-1'-phenyl-
pyrazole-5-ylazo]-3-methyl-1-phenylpyrazole-5-one (9c):
Light pinksolid crystals, yield 1.12 g (70 %), m.p.: 231-232
°C. Anal. Calcd. for C26H23N9O2; C: 63.28 %; H: 4.66 %; N:
25.558 %; found: C: 63.25 %; H: 4.69 %; N: 25.56 %. FT-IR
(νmax, cm-1): 3360 (-NH2); 3062-3024 (Ar-H); 2964 (Aliphatic
4-[4'-(4''-Methoxyphenyl)azo-3'-amino-1'-phenylpyra-
zole-5-ylazo]-3-methyl-1H-pyrazole-5-one (8c): Dark red
crystals, yield 0.95 g (70 %), m.p.: 285-286 °C. Anal. Calcd.
for C20H19N9O2; C: 57.55 %; H: 4.56 %; N: 30.21 %; found:
C: 57.56 %, H: 4.58 %; N: 30.20 %. FT-IR (νmax, cm-1): 3307
(-NH2); 3171 (-NH); 3094 (Ar-H); 2990 (Aliphatic C-H); 1671
1
C-H); 1671 C=O); 1525-1498 (N=N). H NMR (400 MHz,
DMSO-d6, 25 °C) δ (ppm): 2.27(s, 3H, pyrazolone-CH3); 3.85;
(s, 3H, p-OCH3); 7.00 (g, 2H, pyrazole-NH2); 7.08-8.10 (m,
14H, Ar-H); 14.20 (s, -OH). HR-MS: 493.5198 ([M + H]+,
calcd. 493.5199.
1
(C=O); 1523-1496 (N=N). H NMR (400 MHz, DMSO-d6,
25 °C) δ (ppm): 2.14 (s, 3H, pyrazolone-CH3); 3.84 (s, 3H,
p-OCH3); 7.00 (g, 2H, pyrazole-NH2); 7.06-8.09 (m, 9H, Ar-
H); 11.69 (b, pyrazolone-NH); 14.20 (s, -OH). HR-MS:
417.4239 ([M + H]+, calcd. 417.4240.
4-[4'-(4''-Chlorophenyl)azo-3'-amino-1'-phenylpyra-
zole-5-ylazo]-3-methyl-1-phenylpyrazole-5-one (9d): Claret
red solid crystals, yield 1.15 g (72 %), m.p.: 252-253 °C.Anal.
Calcd. for C25H20N9OCl; C: 60.36 %; H: 4.02 %; N: 25.35 %;
found: C: 60.35 %; H: 4.05 %; N: 25.37 %. FT-IR (νmax, cm-1):
3349 (-NH2); 3059 (Ar-H); 2923 (Aliphatic C-H); 1663 C=O);
1538-1493 (N=N). 1H NMR (400 MHz, DMSO-d6, 25 °C) δ
(ppm): 2.27 (s, 3H, pyrazolone-CH3); 7.29 (g, 2H, pyrazole-
NH2); 7.43-8.13 (m, 14H, Ar-H); 14.22 (s, -OH). HR-MS:
497.9395 ([M + H]+, calcd. 497.939.
4-[4'-(4''-Chlorophenyl)azo-3'-amino-1'-phenylpyrazole-
5-ylazo]-3-methyl-1H-pyrazole-5-one (8d): Orange solid
crystals, yield 0.97 g (72 %), m.p. > 325 °C. Anal. Calcd. for
C19H16N9OCl; C: 54.09 %; H: 3.795 %; N: 29.89 %. found: C:
54.11 %; H: 3.78 %; N: 29.86 %. FT-IR (νmax, cm-1): 3304
(-NH2); 3108 (-NH); 3079 (Ar-H); 2929 (Aliphatic C-H); 1669
1
(C=O); 1521-1502 (N=N). H NMR (400 MHz, DMSO-d6,
25 °C) δ (ppm): 2.14 (s, 3H, pyrazolone-CH3); 6.85 (g, 2H,
pyrazole-NH2); 7.22-8.11 (m, 9H,Ar-H); 11.70 (b, pyrazolone-
NH); 14.22 (s, -OH). HR-MS: 421.8431 ([M + H]+, calcd.
421.8430.
4-[4'-(4''-Methylphenyl)azo-3'-amino-1'-phenylpyrazole-
5-ylazo]-3-methyl-1-phenylpyrazole-5-one (9e): Dark red
solid crystals, yield 1.16 g (71 %), m.p.: 240 °C. Anal. Calcd.
for C26H23N9O; C: 65.408 %; H: 4.82 %; N: 26.415 %; found:
C: 65.410 %; H: 4.83 %; N:26.42 %. FT-IR (νmax, cm-1): 3419
(-NH2); 3023 (Ar-H); 2917 (Aliphatic C-H); 1668 C=O); 1525-
1496 (N=N). 1H NMR (400 MHz, DMSO-d6, 25 °C) δ (ppm):
2.27 (s, 3H, pyrazolone-CH3); 2.39; (s, 3H, p-CH3); 7.12 (g,
2H, pyrazole-NH2); 7.27-8.03 (m, 14H,Ar-H); 14.21 (s, -OH).
HR-MS: 477.522 ([M + H]+, calcd. 477.520
4-[4'-(4''-Methylphenyl)azo-3'-amino-1'-phenylpyrazole-
5-ylazo]-3-methyl-1H-pyrazole-5-one (8e): Brown solid
crystals, yield 0.98 g (71 %), m.p.: 283-284 °C. Anal. Calcd.
for C20H19N9O; C: 59.85 %; H: 4.738 %; N: 31.42 %; found:
C: 59.82 %; H: 4.74 %; N: 31.43 %. FT-IR (νmax, cm-1): 3370
(-NH2); 3191 (-NH); 3062 (Ar-H); 2980 (Aliphatic C-H); 1671
1
(C=O); 1528-1404 (N=N). H NMR (400 MHz, DMSO-d6,
Antimicrobial evaluation: Newly synthesized compounds
were individually tested against a panel of Gram-positive and
Gram-negative bacterial pathogens, yeast and fungi. Anti-
microbial tests were carried out by the agar well diffusion
method21 using 100 µL of suspension containing 1 × 106 CFU/
mL of pathological tested bacteria, 1 × 106 CFU/mL of yeast
spread on nutrient agar (NA) and Sabouraud dextrose agar
(SDA), respectively.After the media had cooled and solidified,
wells (10 mm in diameter) were made in the solidified agar
and loaded with 100 µL of tested compound solution prepared
by dissolving 100 µg of the chemical compound in one mL of
DMSO. The inculcated plates were then incubated for 24 h. at
37 °C for bacteria and 48 h. at 28 °C for fungi. Negative controls
were prepared using DMSO employed for dissolving the tested
compound. Ciprofloxacin (50 µg/mL) and ketoconazole (50
µg/mL) were used as standard for antibacterial and antifungal
activities, respectively. After incubation time, antimicrobial
activity was evaluated by measuring the zone of inhibition against
the test organisms and compared with that of the standard.
Antimicrobial activities were expressed as inhibition diameter
zones in millimeters (mm). The experiment was carried out in
triplicate and the average zone of inhibition was calculated.
25 °C) δ (ppm): 2.14 (s, 3H, pyrazolone-CH3); 2.38 (s, 3H,
p-CH3); 7.22 (g, 2H, pyrazole-NH2); 7.30-8.02 (m, 9H, Ar-
H); 11.70 (b, pyrazolone-NH); 14.20 (s, -OH). HR-MS:
401.4245 ([M + H]+, calcd. 401.4246.
Synthesis of 4-[4'-(phenyl)azo-3'-amino-1'-phenylpyrazole-
5-ylazo]-3-methyl-1-phenylpyrazole-5-one (9a)
Compound 9(a-e) were prepared as described above for
5a using 3,5-diamino-4-arylazo-1-phenylpyrazole 3(a-e) and
phenylhydrazine: Light orange solid crystals, yield 1.18 g (71
%), m.p.: 220-221°C. Anal. Calcd. for C25H21N9O; C: 64.79
%; H: 4.53 %; N: 27.21 %; found: C: 64.81 %; H: 4.57 %; N:
27.24 %. FT-IR (νmax, cm-1): 3338 (-NH2); 3063 (Ar-H); 2980
1
(Aliphatic C-H); 1667 (C=O); 1538-1494 (N=N). H NMR
(400 MHz, DMSO-d6, 25 °C) δ (ppm): 2.29 (s, 3H, pyrazolone-
CH3); 7.22 (b, 2H, pyrazole-NH2); 7.26-8.13 (m, 15H, Ar-H);
14.23 (s, -OH). HR-MS: 463.4940 ([M + H]+, calcd. 463.4939.
4-[4'-(4''-Nitrophenyl)azo-3'-amino-1'-phenylpyrazole-
5-ylazo)-3-methyl-1-phenylpyrazole-5-one (9b): Red solid
crystals, yield 1.07 g (68 %), m.p.: 282-283 °C. Anal. Calcd.
for C25H20N10O3; C: 59.05 %; H: 3.93 %; N: 27.56 %; found:

Vol. 27, No. 8 (2015)
Synthesis, Absorption Properties and Biological Evaluation of Some Novel Disazo Dyes 3007
The minimum inhibitory concentration (MIC) measure-
ment was determined for compounds using twofold serial
dilution method22.
RESULTS AND DISCUSSION
In continuation of our previous works23-25, we report here
the synthesis of new disazo dyes 5(a-e), 6(a-e), 8(a-e), 9(a-e)
in this study. By purification of the reaction mixtures, 20 new
disazo dyes were obtained in 68-76 % yield. These compounds
were characterized by elemental analysis, absorption spectra
analysis, FT-IR, 1H NMR and mass spectral data. The synthe-
sized new dyes 5(a-e) may exist in eight possible tautomeric
forms and 6(a-e) in six possible tautomeric forms. Similarly,
8(a-e) may exist in four possible tautomeric forms and 9(a-e)
in three possible tautomeric forms. These tautomeric forms
are disazo-keto form, disazo-enol form, azo-hydrazo-keto form,
azo-hydrazo-enol form, dishydrazo-keto form, dishydrazo-
enol form, hydrazo-azo-keto form and hydrazo-azo-enol form,
respectively. Typical example for dyes 5(a-e) is given in Fig. 3.
The infrared spectra of all the compounds showed azo
band located at 1550-1409 cm-1 and carbonyl bands at 1672-
1651 cm-1. These results confirm that all the dyes in the solid
state exist as disazo-keto form, azo-hydrazo-keto form and
hydrazo-azo-keto form. The values of νmax of the others are
The microdilution susceptibility test in Müeller Hinton
Broth (Oxoid) was used for the determination of antibacterial
activity and sabouraud liquid medium (Oxoid) was used for
the determination of antifungal activity. Stock solutions of the
tested compounds, ciprofloxacin and ketoconazole were
prepared in DMF at concentration of 1000 µg/mL. Two-fold
serial dilutions of the tested compounds solutions were pre-
pared using the proper nutrient broth. The final concentration
of the solutions was 132, 66, 33, 16.5 and 8.25 µg/mL. The
tubes were then inoculated with the test organisms, grown in
their suitable broth at 37 °C for 24 h. for bacteria (about 1 ×
106 CFU/mL), each 5 mL received 0.1 mL of the previous
inoculums and incubated at 37 °C for 24 h. The lowest concen-
tration showing no growth was taken as the minimum inhibi-
tory concentration (MIC). Control experiments with DMF and
uninoculated media were run parallel to the test compounds
under the same conditions. The MIC (µg/mL) and inhibition
zone diameters values are recorded.
H
H
N
O
N
N
N
HO
N
N
CH3
CH3
X
N=N
H2N
N=N
X
N=N
H2N
N=N
NH
NH
N
N
disazo-keto
disazo-enol
H
N
O
HO
H
N
N
H
X
N=N
X
N=N
H2N
N
N
CH3
N
N
CH3
NH
NH
N
N
H2N
azo-hydrazo-keto
azo-hydrazo-enol
H
N
O
HO
N
N
H
N
H
N
H
H
X
N
X
N
CH3
N
N
CH3
N
N
N
N
N
N
H2N
H2N
dishydrazo-keto
dishydrazo-enol
H
N
H
HO
N
O
N
N
H
N
H
N
X
N
N
N
CH3
X
N
CH3
N
N
N
N
N
N
H2N
H2N
hydrazo-azo-enol
hydrazo-azo-keto
Fig. 3. Tautomeric forms of dyes 5(a-e)

3008 Sener et al.
Asian J. Chem.
located at 3419-3245 cm-1 assigned to NH2, 3231-3086 cm-1
assigned to NH except for 9(a-e), 3174-3002 cm-1 assigned to
aromatic C-H, 2990-2917 cm-1 assigned to aliphatic C-H and
a band located at 1537-1324 cm-1 assigned to (NO2) for the
dyes 5b, 6b, 8b and 9b were also recorded.
dishydrazo-enol form and hydrazo-azo-enol form in DMSO-
d6, as depicted in Fig. 3.
Absorption spectra analysis: In general, tautomeric
equilibrium strongly depends on the nature of the media.
Therefore, the behaviours of disazo dyes were studied in various
solvents. The absorption spectra of disazo dyes were measured
in various solvents at a concentration of (10-6-10-8 M). Solvents
used for the absorption spectra measurements have different
dielectric constants (ε), i.e. DMSO (ε, 46.45), DMF (ε, 36.71),
acetonitrile (ε, 35.94), methanol (ε, 32.66), acetic acid (ε, 6.17)
and chloroform (ε, 4.89)26.
The 1H NMR spectra of all compounds showed a singlet
peak for methyl protons (pyrazolone-CH3) at between 2.14-
1
2.31 ppm. The H NMR spectra of dyes 5c, 6c, 8c and 9c
showed a singlet peaks for methoxy protons (Ph-OCH3) at
between 3.78-3.85 ppm and 5e, 6e, 8e and 9e, showed a singlet
peaks for methyl protons (Ph-CH3) at between 2.31-2.39 ppm.
1H NMR spectra of the all compounds demonstrated a broad
peak for NH2 protons at between 6.69-7.29 ppm. The 1H NMR
spectra of the all compounds showed a multiple peak at
between 7.01-8.65 ppm for aromatic protons (Ar-H). The 1H
NMR spectra of all the dyes showed a broad peak for hydroxyl
protons (pyrazole-OH) at between 13.20-14.48 ppm. The 1H
NMR spectra of the all dyes showed a broad peak for -NH
protons except for 9(a-e). The structure of compounds 9(a-e)
was confirmed by 1H-NMR spectra through the loss of -NH
protons signal belonging to other compounds. As a typical
UV spectra of dye 6b in DMF (2 × 10-3 mol L-1) titrated
with diluted NaOH (3 × 10-3 mol L-1) and HCl (3 × 10-3 mol L-1)
at room temperature. The color changes are depicted in
Fig. 5.
The dye 6b has responded to H+ in a wide pH range from
1.84 to 12.96. The reversible conversion may be attributed to
the protonation or deprotonation27. The cationic and anionic
form of dye 6b is given in Fig. 6. as a typical example.
Usually, the ground state nearly all of molecules is less
polar, than excited state so that a polar solvent will tend to
stabilize in the excited state more than ground state. It was
determined that, meanwhile the polarity of the solvents was
increased with the increasing dielectric constant of the solvents,
the absorption maximum of the dyes mostly shows small batho-
chromic shifts28,29. The obtained results from the absorption
measurements are given in Table-1.
1
example, the H-NMR spectra of 9c in DMSO-d6, is shown
in Fig. 4. The 1H-NMR spectra of dyes showed only -NH
proton, but did not show hydrazo -NH proton except for
5a and 5c. According to 1H-NMR results, all of the dyes have
enol tautomeric forms, but not keto tautomeric forms. The dyes
named as 5a and 5c can be present asazo-hydrazo-enol form,
0.04
8.1
0.04
0.04 0.04 0.04 0.02
0.02
0.04 0.04
8.2
8.0
7.9
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
6.9
0.02
0.04 0.04 0.04
0.06 0.36
0.060.06
14.0 13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5
Chemical shift (ppm)
Fig. 4. 1H NMR spectra of the dye 9c
1.84
2.41
3.10
3.73
4.48
8.23
9.67
10.56
11.12
11.42
12.05
12.96
Fig. 5. Color changes corresponding to different pH values

Vol. 27, No. 8 (2015)
Synthesis, Absorption Properties and Biological Evaluation of Some Novel Disazo Dyes 3009
-
N
HO
N
+
O
N
HO
H
N
N
N
+
+ H
- H+
CH3
CH3
O2N
N=N
H2N
N=N
O2N
N=N
H2N
N=N
NH
CH
O N
N=N
H2N
N=N
3
2
NH
NH
N
N
N
anionic form
cationic form
Fig. 6. Cationic and anionic form of dye 6b
TABLE-1
INFLUENCE OF SOLVENT ON λmax (nm) OF DYES
Dye
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
DMSO
360, 451
350, 480
DMF
364, 460, 564s
366, 430
345, 461
348, 469
362, 456
341, 411
337, 456
383, 489
342, 508
403, 528
352, 438
379, 470
361, 443
382, 468
356, 438
351, 431
384, 464
363, 438
356, 434
355, 433
Acetonitrile
353, 435
341, 469
425, 355s
333, 438
352, 428
360, 434
346, 470
423, 358s
330,426
398
347, 429
388, 447
356, 432
374, 452
350, 429
346, 417
375, 448
358, 428
352, 421
352, 416
Methanol
350, 433
342, 466
433
334, 434
355, 437
347, 414
338, 451
435
Acetic acid
Chloroform
342, 434
343, 470
433, 348s
339, 417
426
343, 414
342, 455
433
355, 442
341, 464
437
334, 405
357, 434
338, 416
337, 446
425
432, 360s
342, 464
362, 455
355, 438
355, 483
469, 410s
337, 447
407
353, 447
387, 471
350, 429
384, 473
356, 444
384, 470
385, 473
365, 442
357, 438
360, 438
332, 414
400
335, 405
405
337, 428
435
344, 429
378, 448
356, 435
369, 453
351, 430
346, 416
373, 456
356, 426
350, 419
348, 420
346, 441
373, 448
357, 446
370, 451
351, 443
346, 423
370, 446
357, 434
350, 420
351, 426
350, 429
382, 443
361, 437
375, 450
353, 429
351, 416
351, 414
361, 426s
355, 423
352, 421
s : shoulder
According to the absorption results, it is concluded that
the absorption spectra of the dyes have not correlated with the
polarity of solvents. It was observed that λmax of the dyes 5e
and 6e shifted bathochromically in chloroform with respect
to the λmax in DMSO and DMF. The explanation for this
irregular behaviour may be due to presence of nonbonding
electron pairs of the carbonyl oxygen and nitrogen atoms in
the molecule ring29. The molecular structure of the dyes with
intramolecular hydrogen bonding, have great potential of
interacting with the solvent molecules through non-covalent
or non-conventional interactions28. In polar protic solvents the
lone pair of electron is engaged in hydrogen bonding and the
promotion of these electrons to a π* orbital requires energy
to weaken or break the hydrogen bond in addition normal
transition energy. This results in absorption spectra at shorter
wavelength or rather a blue shift in going from polar to non-
polar solvents29, 30. Absorption measurements showed that the
λmax of dye 5e did not change in DMSO and DMF. We can
conclude from the absorption measurements that the stability
of excited state in going from DMSO to DMF is not change
remarkably. From the absorption spectra of dyes 5(b,d), 6d,
8(a-d) and 9(c-e) in DMSO and DMF, little bathochromic
shifting with respect to the absorption spectra in chloroform
has been recorded. For example for dye 5b, 29, λmax is 343
and 470 nm in CHCl3, 350 and 480 nm in DMSO. For dye 9e,
max is 352 and 421 nm in CHCl3, 360 and 438 nm in DMSO,
λ
355 and 433 nm in DMF. It may conclude that there is no
significant change in stability of excited state in going from
DMSO to DMF and CHCl3. We can say that the stabilization
is less in going from DMSO, DMF to CHCl3. The absorption
spectra of dyes 5(a,b,d), 6(a,b,d), 8(a-e), 9(a,b,d,e) showed
two absorbance. Dye 6e showed single absorbance in all used
solvents. It can be suggested that all of the dyes except for 6e
may be a mixture of tautomeric forms in various solvents.
Dye 6e is predominantly in single tautomeric form in all sol-
vents. The absorption spectra of dye 5a showed two maximum
absorption peaks with a shoulder in DMF. The dye 5c showed
only one absorption peak in all used solvents except for DMF.
Dye 5c has one maximum absorption peak with a shoulder in
DMSO, acetonitrile and chloroform. The absorption spectra
of dye 6c showed one maximum absorption peak in all used
solvents except for DMF. Dye 6c has one maximum absorption
peak with a shoulder in DMSO and acetonitrile. The dye 9c
showed a maximum absorption peak with a shoulder in
chloroform. There is no significant change in the absorption
spectra of all the dyes in acetonitrile, methanol and acetic acid.
The spectral shifts of dye 5b in various solvents are depicted
in Fig. 7.

3010 Sener et al.
Asian J. Chem.
hypsochromic shift with a shoulder at longer wavelength in
acidic solution. These results indicate that the tautomeric form
in methanol changed with another tautomeric form in acidic
and basic solution. Typical example is given in Fig. 8 for the
dye 6b.
DMSO
DMF
Acetonitrile
Methanol
Acetic acid
Chloroform
1.0
0.8
0.6
0.4
0.2
Methanol
1.0
0.8
0.6
0.4
0.2
0.0
Methanol + HCl
Methanol + KOH
0
300
400
500
600
700
Wavelength (nm)
Fig. 7. Absorption spectra of dye 5b in various solvents
The effects of the acid and base on the absorption spectra
of the dye solutions were investigated and the results are
depicted in Table-2.
300
400
500
600
700
Wavelength (nm)
The absorption spectra of the dyes in methanol were
sensitive to the addition of base (potassium hydroxide, 0.1
M). Therefore, λmax of all the dyes except for 6c and 6e showed
a bathochromic shifts with the addition of base to methanol.
For example; λmax of 5e was recorded at 355 and 437 nm in
methanol, 461 and 453 nm in methanol + KOH. The λmax of 6e
showed a hypsochromic shift with a shoulder at longer wave-
length in basic solution.
When hydrochloric acid (0.1 M) was added to the dye
solutions in methanol hypsochromic shifts were detected,
except for 5a, 5c, 5d, 6e and 8b. The λmax of 5b in methanol
did not change when 0.1 M HCl was added. The λmax of 6b
was observed at 338 and 451 nm in methanol and 333 and
443 nm in methanol + HCl. The λmax of 5b and 5d showed a
bathochromic shift with a shoulder at shorter wavelength, 9d
Fig. 8. Absorption spectra of dye 6b in acidic and basic solutions
The effects of substituent on the absorption spectra of
the dye solution were investigated and the results are given
in Table-1. The electron-donating groups (-OCH3, CH3) are
attached to the benzene ring. Dye 5c resulted in bathochromic
shifts with a shoulder in shorter wavelength in DMSO, aceto-
nitrile and chloroform when compared with dye 5a. For
example, for dye 5c λmax is 432 nm (∆λ:72) in DMSO, 425 nm
(∆λ:72) in acetonitrile, 433 nm (∆λ:91) in chloroform with a
shoulder. Dye 5c resulted in bathochromic shifts in methanol
and acetic acid and has only one maximum absorption peak
when compared with dye 5a. For example, 5c λmax is 433 nm
(∆λ:73) in methanol, λmax is 437 nm (∆λ:82) in acetic acid.As
TABLE-2
ABSORPTION MAXIMA OF DYES IN ACIDIC AND BASIC SOLUTIONS
Dye
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
Methanol
350, 433
342, 466
433
334, 434
355, 437
347, 414
338, 451
435
Methanol + HCl
356, 435
342, 466
458, 359s
351, 414s
342, 438
332, 400
333, 443
428
Methanol +KOH
373, 480
380, 506
379, 415
353, 467
361, 453
351, 465
379, 515
379, 488
352, 488
380, 494s
357, 435
391, 458
362, 437
385, 462
358, 432
352, 435
382, 464
357, 439
358, 434
352, 439
Chloroform
342, 434
343, 470
433, 348s
339, 417
426
343, 414
342, 455
433
Acetic acid
355, 442
341, 464
437
334, 405
357, 434
338, 416
337, 446
425
332, 414
400
329, 403
402
332, 447
387
348, 471
357, 448
335, 452
337, 420
365, 433s
347, 453
346, 432
342, 435
337, 428
435
335, 405
405
344, 429
378, 448
356, 435
369, 453
351, 430
346, 416
373, 456
356, 426
350, 419
348, 420
350, 429
382, 443
361, 437
375, 450
353, 429
351, 416
351, 414
361, 426s
355, 423
352, 421
346, 441
373, 448
357, 446
370, 451
351, 443
346, 423
370, 446
357, 434
350, 420
351, 426
s : shoulder

Vol. 27, No. 8 (2015)
Synthesis, Absorption Properties and Biological Evaluation of Some Novel Disazo Dyes 3011
seen in Table-1, dye 5a showed two absorption peaks in all
solvents. Dye 5e resulted in bathochromic shift in DMSO and
methanol when compared with 5a. Dye 5e showed batho-
chromic shift and has only one absorption peak in chloroform.
In contrast, dye 5b which include electron-withdrawing groups
(NO2), resulted in hypsochromic shifts in shorter wavelength
absorption peak on the other hand bathochromic shift in longer
wavelength in all solvents except for DMF when compared
with dye 5a. Dye 5d which include electron-withdrawing
groups (Cl), resulted in hypsochromic shift in acetic acid and
chloroform with respect to dye 5a. Dye 5d showed hypso-
chromic shift in shorter wavelength absorption peak and batho-
chromic shift in longer wavelength in all solvents except for
acetic acid and chloroform with respect to dye 5a. It was
observed that λmax of dyes have electron-donating groups 6c
and 6e resulted bathochromic shifts in all used solvents except
for DMF with respect to dye 6a.Additionally, λmax is 6c showed
a bathochromic shift with a shoulder at longer wavelength
in DMSO and acetonitrile and has one absorption peak in
methanol, acetic acid and chloroform. On the other hand, λmax
of dyes, 6b and 6d, have electron-withdrawing groups showed
generally hypsochromic shift when compared with dye 6a.
For example, dye 6b showed hypsochromic shift in shorter
wavelength absorption peak and bathochromic shift in longer
wavelength absorption peak in DMF, acetonitrile, methanol,
acetic acid and chloroform when compared with 6a. Dye 6d
showed hypsochromic shift in acetonitrile and acetic acid. Dye
6d showed hypsochromic shift in shorter wavelength absor-
ption peak and bathochromic shift in longer wavelength absor-
ption peak in DMSO, DMF and chloroform. On the contrary
the absorption maxima of dyes 8(b-e) have electron-donating
groups and electron-withdrawing groups showed bathochromic
shifts in all solvents except for 8c and 8e in DMSO when
compared with dye 8a. The λmax of 9(b-e) have electron-donating
groups and electron-withdrawing groups showed bathochromic
shift in all solvents except for DMSO when compared with
dye 9a. The λmax of 9(c-e) have electron-donating groups and
electron-withdrawing groups showed hypsochromic shift in
DMSO when compared with dye 9a. On the other hand, λmax
of dye 9b which include electron-withdrawing groups (NO2),
resulted bathochromic shift in DMSO when compared with
dye 9a. The dye 9c showed a bathochromic shift with a shoulder
at longer wavelength in chloroform.
Antimicrobial activity: Synthesized 20 novel dyes were
evaluated for their in vitro antimicrobial activities at 100 mg/
mL concentration against Staphylococcus aureus ATCC 29213
and Bacillus subtilis ATCC 6633 as examples of Gram-positive
bacteria, Klebseilla pneumonia ATCC13883 and Escherichia
coli ATCC 25922 as examples of Gram-negative bacteria and
Saccharomyces cerevisiae and Candida albicans NRRLY-477
fungal strains. Agar-diffusion method was used for the deter-
mination of the preliminary antibacterial and antifungal
activity. Also the minimum inhibitory concentration (MIC)
measurement was determined for compounds using twofold
serial dilution method. Ciprofloxacin and ketoconazole were
used as reference drugs. The results of antimicrobial screening
of newly prepared compounds are summarized in Table-3.
Table-3 revealed that the majority of the synthesized
compounds showed variable inhibitory effects on the growth
of the tested Gram-positive and Gram-negative bacterial strains
and also against antifungal strains. In general, most of the test
compound revealed better antibacterial potency than the
TABLE-3
MINIMAL INHIBITORY CONCENTRATIONS (MIC, µg/mL) AND
INHIBITION ZONE (MM) OF SOME NEW SYNTHESIZED COMPOUNDS
MIC in µg/mL, and zone of inhibition (mm)
Compound
Gram-positive bacteria
Gram-negative bacteria
Fungi
S. aureus
B. subtilis
132 (16)
132 (16)
66 (21)
132 (14)
132 (14)
66 (20)
132 (14)
66 (20)
132 (16)
33 (24)
132 (18)
33 (24)
132 (14)
132 (18)
132 (16)
33 (23)
16.5 (28)
8.25 (30)
33 (25)
16.5 (29)
8.25 (31)
NT
K. pneumoniae
E. coli
33 (24)
132 (18)
33 (26)
16.5 (28)
132 (15)
16.5 (28)
8.25 (33)
8.25 (32)
8.25 (30)
16.5 (29)
132 (14)
33 (24)
8.25 (30)
33 (26)
132 (18)
33 (26)
16.5 (28)
8.25 (34)
8.25 (30)
33 (25)
16.5 (28)
NT
S. cerevisiae
132 (16)
132 (14)
66 (22)
132 (15)
132 (15)
132 (14)
33 (25)
132 (16)
132 (17)
132 (18)
132 (15)
66 (18)
66 (20)
33 (26)
66 (21)
132 (18)
33 (24)
33 (26)
132 (19)
66 (21)
NT
C. albicans
132 (19)
132 (12)
66 (20)
132 (15)
132 (15)
132 (13)
16.5 (27)
16.5 (28)
33 (25)
66 (21)
33 (24)
66 (22)
16.5 (28)
33 (24)
33 (25)
132 (16)
66 (22)
8.25 (30)
132 (18)
16.5 (28)
NT
66 (22)
132 (18)
132 (17)
132 (18)
66 (22)
132 (18)
16.5 (28)
16.5 (28)
66 (23)
66 (19)
66 (20)
33 (25)
33 (24)
33 (24)
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
8a
8b
8c
8d
8e
9a
33 (24)
33 (26)
8.25 (30)
8.25 (29)
16.5 (28)
132 (18)
132 (15)
16.5 (27)
66 (20)
66 (22)
132 (17)
66 (21)
33 (24)
132 (16)
132 (13)
66 (20)
8.25 (31)
16.5 (28)
66 (25)
16.5 (30)
8.25 (32)
NT
33 (23)
16.5 (28)
16.5 (29)
8.25 (33)
8.25 (30)
16.5 (28)
8.25 (29)
NT
9b
9c
9d
9e
Ciprofloxacin
Ketoconazole
8.25 (30)
8.25 (31)
Experiment was carried out in triplicate and the average zone of inhibition was calculated; NT: Not tested

3012 Sener et al.
Asian J. Chem.
antifungal potency. Furthermore, among all the studied com-
pound, 9c displayed the highest antibacterial and antifungal
activities. In case of Gram-negative bacteria, compounds 6c,
6d, 9c and 9d were found to be most effective against K.
pneumonia ATCC13883 with zone of inhibition ranging
between 29 and 33 mm and the compounds 6b, 6c, 6d, 6e, 8c,
9c and 9e were most effective against E. coli ATCC 25922
with zone of inhibition ranging between 29 and 34 mm.
Compound 9c inhibited the growth of B. subtilis ATCC6633,
K. pneumonia ATCC13883, E. coli ATCC 25922 and C. albicans
NRRLY-477 with inhibition zones 30, 33, 34 and 30 mm, respec-
tively. Also, compound 9b showed highest activity against S.
aureus ATCC 29213, with inhibition zone 30 mm. Compounds
9c exhibited low MIC 8.25 mg/mL against B. subtilisATCC6633,
K. pneumonia ATCC13883, E. coli ATCC 25922 and C. albicans
NRRL Y-477. Compounds 6c, 6d and 9b showed MIC 8.25
µg/mL against K. pneumonia ATCC13883 and E. coli ATCC
25922. Additionally, compounds 6b and 8c exhibited MIC
8.25 µg/mL against E. coli ATCC 25922 and also compound
9b showed MIC 8.25 µg/mL against Staphylococcus aureus
ATCC 29213.
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ACKNOWLEDGEMENTS
The authors are grateful to the Scientific Research Projects
Council of Pamukkale University (PAU.BAP, 2013FBE012).