Indian Journal of Chemistry

Vol. 52B, December 2013, pp 1531-1535

Note

Synthesis, antituberculosis and antimicrobial be the basic moiety for a number of dyes and drugs6,7
study of some new aminoacridine linked and several bioactive molecules containing pyrazole
also found to possess antimicrobial, antiviral and
pyrazoles anticancer properties have been reported earlier8.
P R Kawle*a, P P Deohatea, B N Beradb, Survey of literature revealed that when one
K K Srivastavac & P Sharmac biodynamic heterocyclic system was coupled with
a
Department of Chemistry, Shri Radhakisan Laxminarayan another, a molecule with enhanced biological
Toshniwal College of Science, Akola 444 001, India activity9,10 is produced. Synthetically pyrazole
b
Department of Chemistry, Rashstrasant Tukadoji Maharaj
derivatives can be obtained by the action of
Nagpur University, Nagpur 440 033, India hydrazine/hydrazides on 1,3-dicarbonyls11 using
c
suitable solvents or no solvents. In view of this
Department of Microbiology, CSIR-Central Drug Research
Institute, Lucknow 226 021, India
observation the synthesis of heterocycles linked to
pyrazole ring12,13 established to explore the
E-mail: pravink280685@rediffmail.com antimicrobial activity of these compounds. Although
Received 16 April 2013; accepted (revised) 10 September 2013 numerous methods are available for construction of
pyrazoles14, only little attention has been given to the
Synthesis of heterocycles containing pyrazole linked to pyrazolysis of acridine chromophore and reported
acridine chromophore has been studied and evaluated for their in
vitro antibacterial activity against Mycobacterium tuberculosis. synthetic method for pyrazole heterocycles containing
Acridin-9-yl-(5-methyl-2-aryl)-2,4-dihydropyrazol-3-ylidene)- pyrazole ring linked to acridine.
amine and [5-(acridin-9-yl-imino)-3-methyl-4,5-dihydro-pyrazol-
1-yl]-pyridin-4-yl/aryl-methanone have been prepared by Results and Discussion
cyclocondensation reaction of N-acridin-9-yl-3-oxo-butyramide
with hydrazine hydrate in ethanol at room temperature. The title N-acridin-9-yl-3-oxo-butyramide 3 was prepared
compounds have been screened for their antimicrobial potency by condensation of 9-aminoacridine 1 and ethyl
against some selected microorganisms to set-up structure activity acetoacetate 2 in ethanolic medium on water bath for
relationship. All the compounds are found to have potent
antimicrobial response. The structural elucidation of synthesised 3 hr. The N-acridin-9-yl-3-oxo-butyramide 3 was
compounds has been performed by IR, 1H NMR and mass treated with hydrazine hydrate 4a to afford acridin-9-
spectroscopic data besides elemental analysis. The products are yl-(5-methyl-4H-pyrazol-3-yl)-amine 5a. This
obtained by a simple methodology and in good yields. reaction of N-acridin-9-yl-3-oxo-butyramide 3 is
further extended with aryl hydrazine 4b-c to prepare
Keywords: Pyrazole, hydrazine, acid hydrazides, antitubercular
screening, antimicrobial acridin-9-yl-(5-methyl-2-aryl)-2,4-dihydropyrazol-3-
ylidene)-amine 5b-c. The products obtained after
Heterocycles play a vital role in pharmacological, usual work-up showed single spot in TLC. These
agricultural and synthetic field1. The assembly of were recrystallized ffom absolute alcohol in cold
molecule containing heterocyclic templates continues condition. [5-(Acrdin-9-yl-imino)-3-methyl-4,5-dihy-
to attract the attention of both academic and industrial dro-pyrazol-1-yl]-pyridin-4-yl-methanone 7a was also
communities. It is important to develop new drugs prepared from N-acridin-9-yl-3-oxo-butyramide 3 and
because most of the anti-neoplastic drugs are highly isoniazid (acid hydrazide) 6a in ethanolic medium on
toxic, the new improved potential drugs are highly water-bath for 3 hr. Then this reaction was further
superior to older drugs. Beside this pyrazole deri- extended to prepare [5-(acrdin-9-yl-imino)-3-methyl-
vative are found to acts as an analgesic, antimicrobial, 4,5-dihydro-pyrazol-1-yl]-aryl-methanone 7b-c using
hypoglycaemic agent2. Pyrazole containing N-acridin-9-yl-3-oxo-butyramide 3 and aryl acid
compounds have practical application in the hydrazide 6b-c. Homogeneity of the compounds was
medicinal and agrochemical field3,4 and many checked by TLC method (eluent: CHCl3-MeOH 7:2).
pyrazole-fused heterocyclic compounds have been to Recrystallization was carried out from absolute
exhibit biological activity widely used in pesticides ethanol in cold condition and characterised by spectral
and medicines5. The pyrazole ring has been shown to methods. The IR spectrum15 of the compounds
1532 INDIAN J. CHEM., SEC B, DECEMBER 2013

O O
H
N CH3
N H2
O O O il bath
120 0 C , 3 hr
+ H 3C O CH3
N
N
3
1 2
W aterbath
ethanolic R N H -N H 2
m edium rt, 3h 4

R
N N

N C H3
O2N

W here, 4 and 5 R = H , , NO2

a b c N
5
Scheme I

Table I — Physical characterisation data of synthesized compounds 5a-f

Compd m.p. Mol. formula Yield Elemental analysis Found (Calcd) (%)
(°C) (%) C H N
5a 208 C17H14N4 78 74.31 (74.45) 5.02 (5.10) 20.32 (20.43)
5b 118 C23H18N4 85 78.66 (78.85) 4.66 (5.14) 15.87 (16.01)
5c 188 C23H16N6O4 76 61.06 (62.72) 2.56 (3.69) 18.35 (19.09)
7a 288 C23H17N5O 70 72.07 (72.82) 3.93 (4.48) 17.89 (18.46)
7b 184 C24H18N4O 82 74.19 (76.19) 4.76 (3.98) 14.80 (13.06)
7c 224 C24H19N5O 74 71.75 (73.09) 3.03 (4.56) 12.52 (14.21)

showed characteristic peak at 1526 cm-1 which antimicrobial activity against E. coli, S. aureus,
indicated nitro group for 5c and 1660 cm-1 K. pneumonia, P. vulgerius and P. aeruginosa
characteristic peak of carbonyl group for 7a-c. 1H revealed that almost all of them exert significant
NMR spectrum16 of the compounds indicated signal at activity. All compounds 5a-c and 7a-c were found to
δ 7.9 and 8.6-8.7 for the presence of aromatic ring have an effective response against E. coli, S. aureus,
linked to pyrazole nucleus and acridinyl ring. In mass some of them are good and some are sufficiently
spectrum base peak was observed at m/z 195.904 and active against rest of the microorganisms (Table III).
elemental analysis fully agrees with the structures of
all compounds. The structure of the compounds 7a-c Experimental Section
characterised by IR, 1H NMR and mass spectral Melting points were determined on a digital
studies was found identical with already reported melting point apparatus (Veego VMP-D) and are
compounds 5a-c (Scheme I) besides differs in their uncorrected. The starting material (Sigma-Aldrich)
elemental analysis (Table I). and all chemicals used were of AR grade, synthesized
In conclusion we have synthesized pyrazoles linked compounds were monitored on silica gel-G plates
with acridines to enhance the antimicrobial activity of using chloroform-methanol (7:2) as mobile phase. IR
these compounds. Antimycobacterial screening results spectra were recorded on a Perkin-Elmer
indicated that two compounds showed promising spectrophotometer using KBr disc. 1H NMR spectra
activity against M. tuberculosis compared to standard were obtained on a Bruker DRX-600 MHz
drug rifampin (Table II). Results on their spectrophotometer in CDCl3 using tetramethyl silane
 NOTES 1533

Table II  Antituberculosis activity of compounds 7a-c

Compd Concentration in µM
MABA BACTEC
50 25 12.5 6.25 3.125 50 25 12.5 6.25 3.125
7a A A A IA IA A A A IA IA
7b A IA IA IA IA A IA IA IA IA
7c A A A IA IA A A A IA IA
A= active ; IA= Inactive
Table III  Antimicrobial activity of synthesized compounds 5a-c, 7a-c

Compd Microorganisms with inhibition zone recorded in mm

E. coli S. aureus K. pneumonia P. vulgerius P. aeruginosa
5a 22 25 17 14 13
5b 23 20 18 15 12
5c 21 19 19 13 11
7a 23 24 12 12 12
7b 21 26 - 11 11
7c 24 24 11 12 10
Streptomycin 20 22 14 18 14

as internal standard. The chemical shifts are expressed lized from absolute alcohol in cold condition and
in δ (ppm). Mass spectral measurements were carried identified as a acridin-9-yl-(5-methyl-2-phenyl)-2,4-
out by EI method on a Jeol JMC-300 spectrometer at dihydropyrazol-3-ylidene)-amine 5a. Yield: 78.12%;
70 eV. Antituberculosis activity of some selected m.p. 208°C. This reaction was further extended for
compounds were studied by micro almar blue assay preparation of 5b-c using N-acridin-9-yl-3-oxo-butyr-
method (MABA)17 and antibacterial screening of all amide 3 and aryl hydrazine hydrates 4b-c.
synthesized compounds were performed using agar
well diffusion method. Synthesis of [5-(acrdin-9-yl-imino)-3-methyl-4,5-
dihydro-pyrazol-1-yl]-pyridin-4-yl-methanone, 7a
Synthesis of N-acridin-9-yl-3-oxo-butyramide, 3 [5-(Acrdin-9-yl-imino)-3-methyl-4,5-dihydro-pyra-
A mixture of 9-aminoacridine 1 (0.02 mol) and ethyl zol-1-yl]-pyridin-4-yl-methanone 7a was prepared by
acetoacetate 2 (0.02 mol) was refluxed on oil bath at refluxing mixture of N-acridin-9-yl-3-oxo-butyramide
120°C for 3 hr to afford N-acridin-9-yl-3-oxo-butyra- 3 (0.02 mol) and isoniazid 6a (0.02 mol) in absolute
mide. Completion of the reaction was monitored with ethanol on water bath for 3 hr, to afford a solid mass,
thin layer chromatography and the crude residue was which was recrystallized from absolute alcohol in
recrystallized with absolute ethyl alcohol to get pure cold condition and identified as a [5-(acrdin-9-yl-
yellow product which was identified as an N-acridin-9- imino)-3-methyl-4,5-dihydro-pyrazol-1-yl]-pyridin-4-
yl-3-oxo-butyramide 3, yield: 84.16%; m.p. 192°C. yl-methanone, 7a yield: 82.09%; m.p. 184°C.
Reaction was monitored on silica gel-G plates by thin
Synthesis of acridin-9-yl-(5-methyl-2-phenyl) 2,4- layer chromatography. This reaction is further
dihydropyrazol-3-ylidene)-amine, 5a extended to preparation of 7b-c using N-acridin-9-yl-
Acridin-9-yl-(5-methyl-2-phenyl)-2,4-dihydropyra- 3-oxo-butyramide 3 and aryl acid hydrazides 6b-c
zol-3-ylidene)-amine prepared by mixture of N- (Scheme II, Table I).
acridin-9-yl-3-oxo-butyramide 3 (0.02 mol) and
hydrazine hydrate or aryl hydrazine hydrates 4a (0.02 Spectral data of N-substituted acridin-9-yl-(5-
mol) taken in a R. B. flask, stirred magnetically in methyl-2-aryl)-2,4-dihydropyrazol-3-lidene)-ami-
absolute ethanol, refluxed on water bath for 3 hr, the ne, 5a-c
product obtained after usual work-up showed single 5a: IR: 3615 (NH), 3019 (C-H), 1610 cm-1 (C=N);
1
spot in thin layer chromatography. It was recrystal- H NMR: δ 8.66 (m, 8H, Acridn-H), 8.25 (s, 1H, NH),
1534 INDIAN J. CHEM., SEC B, DECEMBER 2013

O O R N N
H Waterbath
N CH3 ethanolic N CH 3
O
medium
NH 2 rt, 3h
+ R N
H
N N
3 6 7

Where, 6 and 7 R = N, , NH 2

a b c

Scheme II

1.93 (s, 3H, Pyraz-CH3); ESI-MS: m/z 274.08 (M+), tubercular activity by BACTEC radiometric and by
195.04. microplate alamar blue assay (MABA) methods for
5b: IR: 3197 (C-H), 1657(Aro C=C), 1599 cm-1 direct determination of the minimum inhibitory
(C=N); 1H NMR: δ 8.7 (m, 8H, Acridn-H), 7.5-7.9(m, concentration (MIC) against M. tuberculosis. For the
5H, Ar-H), 1.92 (s, 3H, Pyraz-CH3); ESI-MS: m/z 350 BACTEC assay, the test vial sjnhjn of 7H12 medium
(M+), 195.09. containing 14C labelled palmitic acid were inoculated
5c: IR: 3133 (C-H), 1624(Aro C=C), 1692 (C=N), with mycobacteria and incubated at 37°C temperature.
1526 cm-1 (NO2); 1H NMR: δ 8.7 (m, 8H, Acridn-H), The amount of 14CO2 reflects the rate and amount of
7.9(m, 3H, Ar-H), 1.93 (s, 3H, Pyraz-CH3); ESI-MS: growth and is expressed in term of the “Growth
m/z 440 (M+), 195. Index” (GI). On addition of compound to the test vial,
suppression of growth of the test organism
Spectral data of [5-(acridin-9-yl-imino)-3-methyl- M. tuberculosis could be detected by routine
4,5-dihydro-pyrazol-1-yl]-pyridin-4-yl-/aryl-me- observation of GI output as compared to the control
thanone, 7a-c and standard drug (Rifampin 2 µg/mL). For MABA
7a: IR: 3140 (CH), 1660 (C=O), 1591 cm-1 (C=N); assay two fold serial dilutions of compounds were
1 made in Middlebrook 7H9 medium supplemented
H NMR: δ 8.9 (m, 8H, Acridn-H), 7.6-7.8(m, 4H,
Pyri-H), 1.93 (s, 3H, Pyraz-CH3); ESI-MS: m/z with 10% (v/v) ADC, in 96-well plates (Nunc) in
378.02 (M+), 194.04. duplicate. An inoculum of 105 CFU/mL was prepared
7b: IR: 3133 (CH), 1660 (C=O), 1601 (C=N), 1289 and 200 µL was added per well. Growth controls
cm-1 (C-N); 1H NMR: δ 8.9 (m, 8H, Acridn-H), containing no drug and a sterile control without
7.8(m, 5H, Aro-H), 1.94 (s, 3H, Pyraz-CH3); ESI-MS: bacteria were also prepared for each assay. The plates
m/z 393 (M+), 195.06. were incubated at 37°C for 5 days before adding 20
7c: IR: 3331 (NH), 3142 (CH), 1660 (C=O), 1601 µL of sterile 0.01% resazurin to the wells and
(C=N), 1289 cm-1 (C-N); 1H NMR: δ 8.9 (m, 8H, incubating for a further 24 hr at 37°C. A change in
Acridn-H), 8.2 (s, 1H, N-H), 6.9 (m, 4H, Aro-H), 1.93 color from blue (oxidized state) to pink (reduced
(s, 3H, Pyraz-CH3); ESI-MS: m/z 394.02 (M+), state) indicated growth of the bacteria.
195.06. The compounds showing MIC at 50 µM were
further evaluated for CFU determination using agar
Antituberculosis activity of compounds, 7a-c dilution method. Serial dilutions of compounds,
(Ref 18) prepared in 0.1 mL, 10% (v/v) DMSO, added to each
Test compounds were dissolved in 10% (v/v) well of 12 well plate (Nunc) and 1.9 mL MB7H10
DMSO at a concentration of 10 mM. All the agar medium supplemented with 10% (v/v) OADC
compounds were evaluated for their in vitro anti- were poured to respective wells and allowed to
 NOTES 1535

solidify at room temperature. For positive control Laxminarayan Toshniwal College of Science, Akola
rifampin and streptomycin were dissolved in water; for providing laboratory facilities and to the Director,
filter sterilized and used in 2 and 6 µg/mL SAIF, Lucknow for providing spectral facility and
respectively. 10µL was inoculated in each well on Head, S & T Division CDRI, Lucknow for anti-
solidified agar medium and incubated at 37°C for 4 tubercular activity.
weeks and growth was recorded. Antituberculosis
screening results indicated that two compounds References
showed promising activity against M. tuberculosis. 1 Lang S A & Lin Yi, Comprehensive Heterocyclic Chemistry,
Among these compounds 7a and 7c MIC value is 12.5 edited by A R Katritzky and C W Rees (Pergamon Press,
and 7b was sensitive at concentration of 50 µM Oxford), 6, 1984, 1.
(Table II). 2 Aboxoat A A, Abdullah W A & Sakka I A, J Drug Res, 7,
1975, 1.
3 Krikpatrick W E, Okabe I, Hillyard W, Robins R K, Dren A T
Antimicrobial activity & Novinson T J, Med Chem, 20, 1977, 386.
All the synthesized compounds 5a-c, 7a-c and 4 Elagamy A A, E1-Taweed F M A, Amer F A & Zoorobs H H,
Arch Pharm, 246, 1987, 320.
streptomycin were screened for antibacterial activity 5 Silva A M G, Tome A C, Neves M G, Tome M S & Cavaleiro
on gram positive and gram-negative bacterial strains J A S, Syn Lett, 7, 2002, 155.
using the agar well diffusion method19. With the aid 6 Kost A N & Grandberg I I, in Advances in Heterocyclic
of a sterile 1 mL pipette, about 0.2 mL of the broth Chemistry (Academic Press, New York), 1996, 347.
7 Raiziss G W, Clemence L W & Friefekder M J, J Am Chem
culture of the test organism was added to an 18 mL
Soc, 63, 1941, 2739.
sterile molten diagnostic sensitivity test agar. This 8 Greenhill J V, Comprehensive Heterocyclic Chem, 5, 1984,
was well mixed and poured into previously sterilized 305.
Petri dishes, which were properly labelled according 9 Boschail C, Cana A, Disfilo R & Frutlero A, Bioorg Med
to the test organisms. The required number of holes Chem Lett, 7, 2000, 1727.
10 Molony G P, Martin G R & Mathews N J, J Chem Soc Perkin
was bored into the medium using sterile cork borer.
Trans, 19, 1999, 2725.
The wells (diameter 10 mm) were then filled up 11 Polshettiwar V & Varma R S, Tetrahedron Lett, 49, 2008,
aseptically with the solution of the compound in 397.
DMSO using Pasteur pipettes. Streptomycin was used 12 Mariappan G, Saha B P, Sutharson L & Haldar A, Indian J
as a standard antibacterial agent at a concentration of Chem, 49B, 2010, 1671.
13 Vogel A I, Text Book of Practical Organic Chemistry (ELBS
100 µg/mL. The plates were allowed to stand for
and Longman, London), 1996, 136.
about 1 hr on the bench for proper diffusion of the 14 Jairo Q, Debora C, Braulio I, Rodrigo A, Silvia C, Manuel N
antibacterial agents into the medium and then & Justo C, J Heterocycl Chem, 45, 2008, 155.
incubated uprightly at 37°C for 24 hr. The inhibition 15 Williams & Dudley H, Spectroscopic Methods in Organic
zones recorded in mm using antibiotic zone scale Chemistry (Tata Mc Graw-Hill, UK), 2004.
16 Silverstein R M, Bassler G C & Morril T C, Spectrometric
indicated the relative susceptibility of the bacteria to Identification of Organic Compounds (John Wiley and Sons,
compounds 5a-c, 7a-c and streptomycin standard New York), 1981.
shown in the Table III. 17 Laurenco M, De Souza M, Pinhiero A, Ferreira M &
Concalves R, Arkivoc, xv, 2007, 181.
Acknowledgements 18 Babalola I B, Adelakun E A, Wang Y & Shode F O, J
Pharma Phytochem, 1, 2012, 19.
Authors are grateful to Principal and Head, 19 Carrod L P & Grady F D, Antibiotics and Chemotherapy, 3rd
Department of Chemistry, Shri Radhakisan Edn (Churchill Livingstone, Edinburgh), 1972, 477.