Sanjay F. Thakor

, Dinesh M. Patel, Manish P. Patel & Ranjan G. Patel
Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar-388 120, Gujarat, INDIA.

E-mail: drsanjaythakor@yahoo.co.in
Abstract
3-amino-6-methyl-1 H -pyrazolo[3,4b ]quinoline was synthesized in good yield. Monoazo compounds were prepared using this intermediate as diazo component with various heterocyclic coupling components. All the azo compounds were characterized by their percentage yield, melting point, elemental analysis, UV-visible spectra, IR-spectra and dyeing performance on nylon and polyester fibres and by their antibacterial activity against gram positive and gram negative bacteria.
Key Words: 3-amino-6-methyl-1 H -pyrazolo[3,4b ]quinoline, azo compounds, synthesis, dyeing, fastness properties, antibacterial activity.

Address correspondence to author: B/18, Shantinagar Soc., Refinery Road, Gorwa,
Vadodara-390 016. Gujarat, INDIA.
1

Introduction
The chemistry of quinoline has gained increasing attention due to its various diverse pharmacological activities (1-3). Quinoline ring fused with five or six membered ring in linear fashion is found in natural product as well as in synthetic compounds of biological interest. Dictemine and skimmianine are the examples of two naturally occurring compounds which are associated with smooth muscle contracting properties.
Biological importance of azo compounds is well known for their use as antineoplastics (4), antidiabetics (5), antiseptics (6) and other useful chemotherapeutic agents. It has been found that the activity of azo linkage increases on the incorporation of suitable heterocyclic moiety. Pyrazole derivatives are also considered as potent biologically active compounds (7,8). With this background it has been thought worth to synthesize linearly fused 3-amino-6-methyl-1 H -pyrazolo[3,4b ]quinoline and use as a diazo component to prepare some new azo compounds as possible antibacterial agents.
All the azo compounds were also applied on nylon and polyester fibres as disperse dyes and their dyeing performance have assessed. The general structure of azo compounds is as shown in Figure 1.
H
3
C
N N R
N N
H
N
6a-i
Figure 1: General structure
2

Experimental
All melting points are uncorrected and determined by an electrothermal melting point apparatus and expressed in o
C. All the coupling components used were synthesized in laboratory except 8-hydroxy quinoline which was purchased from Spectrochem Co.
The IR spectra were recorded on a Nicolet Impact-400D FT-IR spectrophotometer using
KBr pellets technique. The
1
H-NMR spectra were recorded on a Hitachi R-1500, 60MHz instrument using TMS as the internal standard. Chemical shifts are given in

(ppm). The mass spectrum was carried out on a Jeol D-300 model. The absorption spectra (
 max
) of all the azo compound solutions in DMF were recorded on a Shimadzu UV-240 spectrophotometer.
Preparation of 2-chloro-6-methyl-3-quinolinecarboxaldehyde (1)
The title compound was synthesized following a sequence of reactions according to a procedure described in the literature (9,10). Yield 69%; m.p.123-125 o
C.
Preparation of 2-chloro-6-methyl-3-quinolinecarboxaldehyde oxime (2)
The title compound was synthesized following a reaction according to a procedure described in the literature (11). Yield 75%; m.p.193-195 o
C.
Preparaton of 2-chloro-6-methyl-3-quinolinecarbonitrile (3)
The title compound was synthesized following a reaction according to a procedure described in the literature (12). Yield 70%; m.p.181-183 o
C.
3

Preparation of 3-amino-6-methyl-1H-pyrazolo[3,4-b]quinoline (4)
The title compound was synthesized following a reaction according to a procedure described in the literature (12). Yield 65%; m.p.331
o
C.
Preparation details of pyrazolo[3,4-b]quinoline based azo compounds (6a-i)
Diazotization of 3-amino-6-methyl-1 H -pyrazolo[3,4b ]quinoline (Scheme 1) was carried out with hydrochloric acid as follows.
Diazotization
Concentrated hydrochloric acid (2 ml, 0.016 mol) was added to a well stirred suspension of 3-amino-6-methyl-1 H -pyrazolo[3,4b ]quinoline 4 (0.534 g, 0.0027 mol) in water (20 ml) and the mixture was heated up to 70 o C and maintained at that temperature till a clear solution obtained. After cooling the solution to 0-5 o
C in an ice bath, a solution of sodium nitrite (0.75 g, 0.0054 mol) in water (20 ml) was added drop wise over a period of 10 minutes with stirring. The reaction was stirred at a temperature below 5 o
C for an hour. The excess of nitrous acid (tested for using starch iodide paper) was removed by adding required amount of sulphamic acid solution (10%). The clear diazonium salt solution 5 thus obtained was used immediately in the coupling reaction (Scheme 1).
3-methyl-4-((6-methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)diazenyl)-1-phenyl-1H-pyrazol-
5-ol ( 6a )
General coupling procedure
4

3-methyl-1-phenyl-5-pyrazolone (0.469 g, 0.0027mol) was dissolved in sodium hydroxide solution (7 ml, 10% W/V). The solution was cooled to 0-5 o C in an ice-bath.
To this well stirred solution, the above diazonium solution 5 was added dropwise keeping temperature below 5 o
C. The reaction mass was further stirred for 2 hrs at 0-5 o
C maintaining the pH 8.0 by adding required amount of 10% sodium carbonate solution.
The reaction mass was then heated up to 60 o
C and diluted with water (80 ml). The dye was filtered off, washed with hot water until the washings were neutral, dried and powered. The product was purified by dissolving in DMF and pouring in water.
1
H-
NMR(DMSO-d
6
):

2.48 (s, 3H, CH
3
quinoline), 2.33 (s, 3H, CH
3
pyrazole), 8.60 (s, 1H,
NH), 7.41-7.97 (m, 9H, ArH), 11.34 (s, 1H, OH). m/z 383 (M+), 384(M+1), 406(Na adduct), 276.9, 183, 142.
The following compounds were prepared in similar manner.
1-(3-chlorophenyl)-3-methyl-4-((6-methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)diazenyl)-
1H-pyrazol-5-ol ( 6b ). 1 H-NMR (DMSO-d
6
):

2.50 (s, 3H, CH
3
quinoline), 2.46 (s, 3H,
CH
3
pyrazole), 8.66 (s, 1H, NH), 7.47-7.99 (m, 8H, ArH), 11.20 (s, 1H, OH).
3-amino4-((6-methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)diazenyl)-1-phenyl-1H-pyrazol-
5-ol ( 6c ).
1
H-NMR (DMSO-d
6
):

2.55 (s, 3H, CH
3
quinoline), 6.50 (s, 2H, NH
2
), 8.59 (s,
1H, NH), 7.40-7.95 (m, 9H, ArH), 11.33 (s, 1H, OH).
5

2-amino-5-((6-methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)diazenyl)benzo[d]thiazol-6-ol
( 6d ).
1
H-NMR (DMSO-d
6
):

2.50 (s, 3H, CH
3
quinoline), 7.10 (s, 2H, NH
2
), 8.65 (s, 1H,
NH), 7.33-7.98 (m, 6H, ArH), 10.12 (s, 1H, OH).
7-((6-methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)diazenyl)quinolin-8-ol ( 6e ).
1
H-NMR
(DMSO-d
6
):

2.42 (s, 3H, CH
3
quinoline), 8.68 (s, 1H, NH), 7.10-8.85 (m, 9H, ArH),
10.26 (s, 1H, OH).
2-mercapto-6-methyl-5-((6-methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)diazenyl) pyrimidin-4-ol ( 6f ).
1
H-NMR (DMSO-d
6
):

2.42 (s, 3H, CH
3
quinoline), 8.60 (s, 1H,
NH), 2.58 (s, 3H, CH
3
pyrimidine), 7.34-7.95 (m, 4H, ArH), 10.23 (s, 1H, OH), 12.35 (s,
1H, SH).
7-hydroxy-4-methyl-8-((6-methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)diazenyl)-2Hchromen-2-one.
( 6g ).
1
H-NMR (DMSO-d
6
):

2.33 (s, 3H, CH
3
quinoline), 8.66 (s, 1H,
NH), 2.55 (s, 3H, CH
3
coumarin), 7.50-7.98 (m, 7H, ArH), 9.90(s, 1H, OH).
5-hydroxy-1,4-dimethyl-6-((6-methyl-1H-pyrazolo[3,4-b]quinolin-3-yl)diazenyl) pyrimidin-2(1H)-one.
( 6h ).
1
H-NMR (DMSO-d
6
):

2.40 (s, 3H, CH
3
quinoline), 8.60 (s,
1H, NH), 1.2 (s, 3H, CH
3
), 2.79 (s, 3H, CH
3
-N),7.45-7.96 (m, 4H, ArH), 11.45(s, 1H,
OH).
3-(4-hydroxyphenyl)-1-(4-methoxyphenyl)-2-thioxo-3-((6-methyl-1H-pyrazolo[3,4-b]
6

quinolin-3-yl)diazenyl)dihydropyrimidin-4,6(1H,5H)-dione.
( 6i ). 1 H-NMR (DMSO-d
6
):

2.48 (s, 3H, CH
3
quinoline), 8.55 (s, 1H, NH), 3.80 (s, 3H, OCH
3
), 3.20 (s, 2H, CH
2
),
6.97-7.98 (m, 10H, ArH), 10.08(s, 1H, OH).
Dyeing method
Dyeing of nylon and polyester has been carried out for 2% shade following standard procedures (13).
Fastness properties
The fastness to light, sublimation and perspiration were assessed in accordance with AATCC/15/1985. The rubbing fastness test was carried out with a crockmeter
(Atlas) in accordance with AATCC/88/1988 and the wash fastness test in accordance with IS: 765-1979. By observing the alternation of dyed pattern light fastness rating was given by grey-scale (1 to 8). Where, 1-very poor, 2-poor, 3-fair, 4-fairly good, 5-good, 6very good, 7-excellent, 8-outstanding. Rating for sublimation, perspiration and wash fastness is given by grey-scale (1 to 5). Where, 1-poor, 2-fair, 3-good, 4-very good and 5excellent. Data of fastness properties are given in Table 3.
Antibacterial activity
Antibacterial activities of synthesized compounds were examined in vitro by known agar diffusion cup method (14-17). All the compounds were tested for activity against gram-positive bacteria like Bacillus cereus ATCC 10987 , Staphylococcus aureus
ATCC 6538 and Bacillus subtilis ATCC 6633 and gram-negative bacteria Escherichia
7

coli ATCC 10536. The culture medium was nutrient agar. All the compounds were dissolved in DMF (500ppm concentration) and DMF used as control. Norfloxacin was employed as the standard drug. The results are summerised in Table 4.
Results and Discussion
The fused pyrazole intermediate, 3-amino-6-methyl-1 H -pyrazolo[3,4b ]quinoline
4 was prepared by dehydration of 2-chloro-6-methyl-3-quinolinecarboxaldehyde oxime with thionyl chloride followed by cyclization with hydrazine hydrate in ethanol. (Scheme
1). The fused pyrazole intermediate 4 was diazotized satisfactorily at 0-5 o
C by usual procedure using hydrochloric acid and sodium nitrite. The diazonium salt was used immediately since it decomposed on standing, even when cold. Subsequent coupling reactions took place readily on adding the diazonium salt continuously to the solution of coupling component in alkaline medium at 0-5 o
C maintaining the pH 8.0 by adding required amount of 10% sodium carbonate solution.
Spectral properties of the azo compounds
The absorption maxima (
 max) and logarithm of the molar extinction coefficient
(log

) of the dyes 6a-i are given Table 1. The absorption maxima of 6a-i were recorded in DMF solution. The absorption maxima were in the range of 383 nm to 523 nm. The value of lagarithm of molar extinction coefficient (log

) of the dyes 6a-i were in the range 4.15-4.68.
8

Infrared spectra of azo compounds
All the azo compounds showed a characteristic band at 1575-1594 cm -1 for the azo (-N=N-) group. The band at 2873-2920 cm
-1
is due to C-H stretching of methyl groups. The band at 1312-1318 cm
-1
can be attributed to bending of aromatic methyl group. The characteristic band at 3120-3178 cm
-1
is due to N-H stretching of secondary amine group. The band appears at 3032-3057 cm
-1
corresponding to C-H stretching of aromatic rings. The band at 742 cm -1 is due to a C-Cl stretching.
Dyeing properties of azo compounds
The azo compounds 6a-i were applied at 2% depth on polyester and nylon as disperse dyes. The properties are given in Table 1. These dyes mostly gave various brown, orange, reddish orange and yellow shades with good depth on the fibres. The variation of the shades of the dyed fibres results from the different coupling components.
The light fastness of all the disperse dyes on both the fibres is found fair to fairly good to good. The obtained results of washing fastness of the dyes for both the fibres showed that they are very good to excellent. Fastness to rubbing of dyed patterns was very good to excellent for all the dyes on both the fibres. This is attributed to good penetration and affinity of dyes to the fibres. The perspiration and sublimation fastness are very good to excellent.
9

Antibacterial Assay
The antibacterial assay indicated that none of the tested compounds showed significant activity towards selected gram positive and gram negative bacteria. The compound 6f exhibited higher activity than other against all selected microorganisms. It is observed that azo compounds 6a , 6b and 6c showed moderate activity against E.coli
and B.subtilis.
The remaining compounds displayed weak activity against all microbes under investigation. A close examination of the structure of the active compound revealed that the best activity was confined with the compound having pyrimidine as coupling component. Morever pyrazolone as coupling component in compounds 6a , 6b and 6c showed moderate activity. In conclusion, the compounds having pyrimidine and pyrazolone as coupling components could be useful for derivatization to develop more selective antibacterial agents.
Acknowledgement
The authors are thankful to Dr. K C Patel and Mr. Kishor H Chauhan, Department of Biosciences, Sardar Patel University for the antibacterial screening of the compounds.
We are also thankful to CDRI, Lucknow for recording the mass spectral data.
10

References
1.
Suresh T, Nandha Kumar R, Magesh S, Mohan PS. Synthesis, characterization and antimicrobial activities of 4-phenyl-3-thiopyrimido[4,5b ]quinolines.
Indian
Journal of Chemistry, 2003, 42B: 2133-2135.
2.
Rajendran SP, Karvembu R. Synthesis and antifungal activities of Schiff bases derived from 3-amino-2 H -pyrano[2,3b ]quinolin-2-ones. Indian Journal of
Chemistry , 2002, 41B: 222-224.
3.
Kalluraya B, Gururaja R, Rai G. One pot reaction: Synthesis, characterization and biological activity of 3-alkyl/aryl-9-substituted 1,2,4-triazolo[3,4b ][1,3,4] quinolino thiadiazepines.2003, 42B: 211-214.
4.
Child RG, Wilkinson RG, Tomcu-Fucik A. Effect of substrate orientation of the adhesion of polymer joints. Chem. Abstr., 1977, 87: 6031.
5.
Garg HG, Praksh C. Preparation of 4-arylazo-3,5-disubstituted-(2 H )-1,2,6thiadiazine-1,1-dioxides. J.Med. Chem, 1972, 15(4): 435-436.
6.
Browing CH, Cohen JB, Ellingworth S, Gulbransen R. The antiseptic properties of the aminoderivatives of styryl and anil quinoline. Journal Storage, 1926, 100:
293-325.
7.
Abadi AH, Eissa AA, Hassan GS. Synthesis of novel 1,3,4-trisubstituted pyrazole derivatives and their evaluation as antitumor and antiangiogenic agents. Chem.
Pharm. Bull, 2003, 51(7), 838-844.
11

8.
Jain R, Dixit A, Pandey P. Synthesis of some New 1-carbamoyl-3-aminophenyl- and 1-carbamoyl-3-amino-(2-chlorophenyl)-5-methyl-4-arylpyrazoles as possible potential antidiabetics.
J. Indian Chem. Soc., 1989, 66: 486-489.
9.
Horming EC. Organic synthesis, ed. John Wiley & Sons, Inc., New York, 1955,
3: 661-663.
10.
Cohn OM, Narine B and others. A versatile new synthesis of quinolines and related fused pyridines, part-5. J. Chem.Soc. Perkin Trans I, 1981, 1520-1526.
11.
Nandeeshaiah SK, Ambekar SY. Synthesis of 2-aryl-1,2,3,4tetrahydropyrido[2’,3’,4,5]thieno[2,3b ]quinoline-4-ones. Indian Journal of
Chemistry, 1994, 33B: 375-379.
12.
Malcom BR, Ackerman JH, Pyrazolo[3,4b ]quinolines and their use as antiviral agents. U.S. Patent, 1990, 4 920 128.
13.
Desai JA, Patel MH, Patel RG, Patel VS. Azodisperse Dyes with 4-Quinazolinone ring for dyeing polyester and nylon fibres. Indian J. of Textile Research, 1985, 10:
75-79.
14.
Chauhan KH, Trivedi UB. Studies on contamination management in plant
tissue culture, Dissertation Report, Sardar Patel University, V.V.Nagar, India
2001.
15.
Atlas RM. Principle of Microbiology, 1997, 10: 356-357.
16.
Perry JJ, Staley JT.
Microbiology Dynamics and Diversity, Forth Wroth:
Saunders College Pub., 1997, 7 : 165-168.
17.
Stainer RY. The Microbial World 5 th ed., 1986, 12: 16-20.
12

Scheme 1: Synthetic route of azo compounds 6a-i .
H
3
C
Ac
2
O/AcOH
NH
2
H
3
C
NH
CH
3
O
DMF/POCl
3
Reflux
H
3
C CHO
1
N Cl
H
3
C
4
N
HCl /NaNO
2
0-5 o C
N
H
N
NH
2
NH
2
NH
2
H
3
C
EtOH
H
3
C
3
N
+
N
2
Cl
Coupling with R
H
3
C
5
N N
H
N
CN
SOCl
2
H
3
C
Cl
N
6
N
H
N
NH
2
OH EtOH
CH NOH
N
N
2
N R
Cl
Wherein, R= various heterocyclic coupling components with their coupling position.
CH
3
CH
3
NH
2 a.
O
N
N b.
O
N
N c.
O
N
N
.
d
H O
S
N
NH
2 e.
OH
N f.
H
3
C i.
H O
OH
Cl
N
N
SH g.
H O
S
N N
O O
CH
3
O O h.
H O
CH
3
N
N
CH
3
O
OMe
13

Table 1: Absorption maxima, logarithm of molar extinction coefficient and dyebath exhaustion of azo compounds 6a-i on nylon and polyester.
Comp. no.
Absorption in DMF
 max log

% Exhaustion
Nylon Polyester
6a
6b
6c
6d
6e
6f
6g
6h
6i
485
481
468
383
523
394
400
477
441
4.26
4.15
4.45
4.68
4.34
4.17
4.28
4.56
4.48
74
72
73
64
73
71
66
70
68
70
68
72
62
68
70
65
68
63
14

Table 2: Physicochemical properties of azo compounds 6a-i .
Comp. Molecular no. formula
Molecular Melting point
Weight ( o C)
Yield
(%)
C% H% N%
Calcd. Found Calcd. Found Calcd. Found g/mol. (d)
6a C
21
H
17
N
7
O 383
6b C
21
H
16
N
7
OCl 417
6c C
20
H
16
N
8
O
6d C
18
H
13
N
7
SO
384
375
6e C
20
H
14
N
6
O
6f C
16
H
13
N
7
SO
6g C
21
H
15
N
5
O
3
354
351
385
6h C
17
H
15
N
7
O
2
349
6i C
28
H
21
N
7
SO
4
551
240-245
290-296
276-280
234-237
225-228
193-195
201-204
248-255
259-264
70
77
73
71
75
67
74
69
70
65.98
60.14
62.78
57.87
67.34
54.55
65.22
58.21
60.77
65.75
60.23
62.88
57.60
67.59
54.70
65.45
58.42
60.98
(d) Decomposition temperature
4.58
3.76
4.56
4.16
3.58
3.95
3.60
4.67
3.69
4.43
3.83
4.37
4.36
3.44
3.70
3.85
4.49
3.81
25.34
23.88
29.45
26.45
20.14
27.67
18.56
28.34
17.92
25.58
23.60
29.26
26.23
20.33
27.92
18.38
28.18
17.78
15

Table 3: Fastness properties of azo compounds 6a-i .
Comp. no.
Light Wash
Fastness Fastness
Rubbing
Fastness
Perspiration
Fastness
Sublimation
Fastness
Dry
N P N P N P N
Wet Acidic
P N P
Alkaline
N P N
6a 4 4 4-5 5 4 5
6b 4 4-5 5 5 4 5
5
4
6c 4 4-5 5 4-5 4 4 4
6d 3-4 4 5 5 4 4-5 5
4-5
4
4
4
5
5
5
4-5
5
5
5
4
5
5
5
4
5
5
5
4-5
5
5
5
5
6e
6f
4
3-4
3-4
4
5
5
5 5
4-5 5
4
4
6g 3-4 3 5 5 4 4
6h 4 4-5 5 5 4 4
4 4-5 5 4-5 5 4-5 4-5
4 4 5 5 5 5 5
4
4
4-5
4
4-5
5
4
5
5
5
4-5
5
5
5
6i 3 3-4 4-5 4-5 4 4-5 4 4 4 4-5 4 4 4-5
N-nylon and P-polyester
5
5
4-5
5
4
P
5
5
5
5
16

Table 4: Antibacterial activity data of compounds 6a-i .
Zone of inhibition (mm)
Compd. E. coli
6a
6b
13
15
6c
6d
18
9
10 6e
6f 20
8 6g
6h 10
13 6i
Norfloxacin 40
DMF --
B. subtilis
--
8
--
12
15
17
--
8
10
30
--
6
7
25
--
--
12
13
--
B. cereus
10
8
8
5
9
37
--
--
9
11
--
S. aureus
--
10
9
17