Synthesis and microbiological evaluation of mannich bases derived from 4,6-diacetylresorcinol

Article abstract of DOI:10.4067/S0717-97072010000300012

In the present investigations 4,6-diacetyl resorcinol 1 on condensation with formaldehyde and some selected amines following the Mannich reaction conditions yielded eight new Mannich bases 2a-h. It was observed that the reaction did not take place at the acetyl function but occurred at the 2-position into the aromatic ring. The compounds were characterized on the basis of elemental analysis as well as ¹H NMR and Mass spectral data. The antibacterial and antifungal activities of the title compounds were tested by the disc diffusion method using nutrient agar medium against various microorganisms such as gram positive Staphylococcus aureus and Bacillus subtilis, gram negative Escherichia coli and the fungi Aspergillus flavus and Candida albicans. Ofloxacin and voriconazole at 20 μg/mL were used as standard drugs for antibacterial and antifungal activities, respectively.

Full text of DOI:10.4067/S0717-97072010000300012

J. Chil. Chem. Soc., 55, Nº 3 (2010)
SYNTHESIS AND MICROBIOLOGICAL EVALUATION OF MANNICH BASES DERIVED FROM
4,6-DIACETYLRESORCINOL
ASIF HUSAIN1*, MOHAMMAD MAAZ2, KHURSHEED AHMAD ANSARI2, AUSAF AHMAD3, MOHD. RASHID1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi- 110 062, India; 2Faculty of Medicine
(Unani), Jamia Hamdard (Hamdard University), New Delhi-110 062, 3Department of Biochemistry and Biophysics,
University of Rochester Medical Center, Rochester, NY 14642, USA
(Received: September 14, 2009 - Accepted: June 15, 2010)
SUMMARY
In the present investigations 4,6-diacetyl resorcinol 1 on condensation with formaldehyde and some selected amines following the Mannich reaction conditions
yielded eight new Mannich bases 2a-h. It was observed that the reaction did not take place at the acetyl function but occurred at the 2-position into the aromatic
ring. The compounds were characterized on the basis of elemental analysis as well as 1H NMR and Mass spectral data. The antibacterial and antifungal activities
of the title compounds were tested by the disc diffusion method using nutrient agar medium against various microorganisms such as gram positive Staphylococcus
aureus and Bacillus subtilis, gram negative Escherichia coli and the fungi Aspergillus flavus and Candida albicans. Ofloxacin and voriconazole at 20 μg/mL were
used as standard drugs for antibacterial and antifungal activities, respectively.
Keywords : Mannich base, resorcinol, antibacterial, antifungal.
It was synthesized according to the method reported in literature6.
Preparation of Mannich bases (2a-h)
INTRODUCTION
Compound 1 (0.002 mol) was dissolved in chloroform (20 ml) and to it
were added formaldehyde (0.004 mol), an amine (0.004 mol) and tetrabutyl
ammonium bromide (0.002 mol) followed by distilled water (10 ml). After
stirring the contents for 24 h, chloroform layer was separated and washed with
aqueous sodium bicarbonate (5% w/v) followed by washing with water. The
organic layer was then dried over anhydrous sodium sulphate, filtered and
evaporated to dryness. The residue was crystallized from an appropriate solvent
to give TLC pure crystals of 2a-h (Table I). Each one of these compounds gave
a violet colour with ethanolic ferric chloride solution (test for phenol).
1,1’-{5-[(Dimethylamino)methyl]-4,6-dihydroxy-1,3-phenylene}
diethanone (2a): Yield: 63 %; m.p. 148-150 °C; 1H-NMR (DMSO-d , δ, ppm):
1.32 (s, 6H, 2x-CH3), 2.61 (s, 6H, 2x-COCH3), 4.13 (s, 2H, -CH2-, 6benzylic),
8.27 (s, 1H, aromatic proton), 13.18 (s, 2H, 2xOH); MS (m/z): 251 (M+), 207,
193, 190, 44; Anal. C13H17NO4, Calcd. C, 62.14; H, 6.82; N, 5.57; found C,
62.18; H, 6.86; N, 5.45.
The presence of nitrogen atom alongwith other features impart interesting
biological activities to the parent compounds1. Mannich bases, including
those derived from different acetophenones, possess diverse biological
activities including potent antibacterial2,4, antimycobacterial3, antifungal2,4
and anti-HIV4 activities. Resorcinol is a simple and important aromatic
chemical (1,3-benzenediol) that has been chemically incorporated into various
compounds to enhance their pharmacological profile5.
The essential feature of Mannich reaction is the replacement of the active
hydrogen by an aminomethyl or substituted aminomethyl group. However, it is
also well known that with phenols the reaction proceeds on a nuclear position.
Hence it was considered worthwhile to study this reaction on 4,6-diacetyl
resorcinol having both COCH3 and nuclear position available. This moiety
was used earlier to build benzodipyrone derivatives and found to have a good
antibacterial activity6.
Over the past few decades the bacterial resistance to antibiotics has
become one of the most important problems of infections treatment. Although
there are antimicrobial agents having different structures and mechanisms
are frequently used in the treatment of microbial infections, even then these
agents are associated with resistance. To overcome the development of drug
resistance, it is necessary to synthesize a new class of antimicrobial compounds
possessing different mechanism or chemical properties from those that are used
commonly. Derivatives of 4,6-diacetyl resorcinol show potential antibacterial
activity6. In view of these points and in continuation of our work on novel
resorcinol derivatives6, it was planned to synthesize Mannich bases derived
from 4,6-diacetyl resorcinol.
1,1’-{5-[(Diethylamino)methyl]-4,6-dihydroxy-1,3-phenylene}diethanone
1
(2b): Yield: 60 %; m.p. 135-137 °C; H-NMR (DMSO-d6, δ, ppm): 1.35 (t,
6H, 2x-CH CH3), 2.87 (q, 4H, 2x-CH2CH ), 2.60 (s, 6H, 2x-COCH3), 4.08 (s,
2H, -CH2-, 2benzylic), 8.35 (s, 1H, aromati3c proton), 13.25 (s, 2H, 2xOH). MS
(m/z): 279 (M+), 207, 193, 190, 72. Anal. C15H21NO4, calcd. C, 64.50; H, 7.58;
N, 5.01; found C, 64.62; H, 7.54; N, 5.07.
1,1’-{5-[(4-Methylphenylamino)methyl]-4,6-dihydroxy-1,3-phenylene}
diethanone (2c): Yield: 52 %; m.p. 167-169 °C; 1H-NMR (DMSO-d , δ, ppm):
1.35 (s, 3H, -CH ), 2.67 (s, 6H, 2x-COCH3), 4.14 (s, 2H, -CH2-, 6benzylic),
7.03 & 7.46 (d, e3ach, A2B2, p-substituted phenyl ring), 8.37 (s, 1H, aromatic
proton), 9.02 (s, 1H, -NH-), 13.18 (s, 2H, 2xOH); MS (m/z): 313 (M+), 207,
91, 77; Anal. C18H19NO4, calcd. C, 69.00; H, 6.11; N, 4.47; found C, 68.88; H,
6.20; N, 4.48.
1,1’-{5-[(4-Methoxylphenylamino)methyl]-4,6-dihydroxy-1,3-phenylene}
diethanone (2d): Yield: 58 %; m.p. 172-174 °C; 1H-NMR (DMSO-d , δ, ppm):
2.73 (s, 6H, 2x-COCH3), 3.91 (s, 3H, -OCH ), 4.18 (s, 2H, -CH2-, 6benzylic),
7.14 & 7.57 (d, each, A2B2, p-substituted ph3enyl ring), 8.52 (s, 1H, aromatic
proton), 9.06 (s, 1H, -NH-), 13.22 (s, 2H, 2xOH); MS (m/z): 329 (M+), 207,
193, 190, 122; Anal. C H19NO5, calcd. C, 65.64; H, 5.81; N, 4.25; found C,
65.57; H, 5.76; N, 4.31.18
1,1’-{5-[(3-Methoxylphenylamino)methyl]-4,6-dihydroxy-1,3-phenylene}
diethanone (2e): Yield: 55 %; m.p. 160-162°C; 1H-NMR (DMSO-d , δ, ppm):
2.69 (s, 6H, 2x-COCH3), 3.88 (s, 3H, -OCH ), 4.16 (s, 2H, -CH2-,6benzylic),
7.08-7.33 (m, 4H, m-substituted phenyl ring3), 8.29 (s, 1H, aromatic proton),
8.97 (s, 1H, -NH-), 13.17 (s, 2H, 2xOH); MS (m/z): 329 (M+), 207, 193, 190,
122, 121; Anal. C18H19NO5, calcd. C, 65.64; H, 5.81; N, 4.25; found C, 65.66;
H, 5.78; N, 4.22.
In the present work we report the Mannich reaction proceeds at the
2-position of 4,6-diacetyl resorcinol and several new such compounds have
been prepared by varying amine component and the products have been
evaluated for their antibacterial and antifungal activities.
EXPERIMENTAL
Chemistry
Melting points were determined in open capillary tubes and are uncorrected.
Purity of the compounds was checked by thin layer chromatography (TLC)
on silica gel G plates, with the solvent system: toluene-ethyl acetate-formic
acid (5:4:1, v/v/v). The spots were located under iodine vapours and UV light.
1H NMR spectra were recorded on a Bruker 300 MHz NMR spectrometer
(internal reference-tetramethyl silane) and Mass spectra were recorded on a
JEOL JMS-D 300 instrument. Elemental analyses were performed on a Perkin-
Elmer 240 analyzer and the values were in range of ±0.4% for each element
analyzed (C, H, N).
1,1’-{5-[(Morpholin-4-yl)methyl]-46-dihydroxy-1,3-phenylene}
Preparation of 1,1´-(4,6-dimethyl-1,3-phenylene)diethanone (1)
e-mail: drasifhusain@yahoo.com
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J. Chil. Chem. Soc., 55, Nº 3 (2010)
diethanone (2f): Yield: 64 %; m.p. 134-136 °C; 1H-NMR (DMSO-d6, δ, ppm):
2.68 (b, 10H, 2´-COCH3 and 2x-CH2-, a to nitrogen), 3.87 (b, 6H, 2´-CH2-, a to
oxygen + -CH2-, benzylic), 8.35 (s, 1H, aromatic proton), 13.16 (s, 2H, 2xOH);
MS (m/z): 293 (M+), 207, 193, 190, 86; Anal. C15H19NO5, calcd. C, 61.42; H,
6.53; N, 4.78; found C, 61.40; H, 6.51; N, 4.83.
1,1’-{5-[(Piperidin-1-yl)methyl]-4,6-dihydroxy-1,3-phenylene}
diethanone (2g): Yield: 66 %; m.p 138-140 °C; 1H-NMR (DMSO-d6, δ, ppm):
1.67 (bs, 6H, 2´-CH , meta to N + -CH , para to N), 2.82 (s, 4H, 2´-CH2, ortho
to N), 2.66 (s, 6H,22´-COCH3), 3.97 2(s, 2H, -CH2-, benzylic), 8.34 (s, 1H,
aromatic proton), 13.23 (s, 2H, 2xOH); MS (m/z): 291 (M+), 207, 193, 190,
84. Anal. C16H21NO4, calcd. C, 65.96; H, 7.26; N, 4.81; found C, 65.92; H,
7.25; N, 4.86.
1,1’-{5-[(4-Methylpiperazin-1-yl)methyl]-4,6-dihydroxy-1,3-phenylene}
diethanone (2h): Yield: 57 %; m.p. 162-164 °C; 1H-NMR (DMSO-d , δ, ppm):
2.33 (s, 3H, -CH ), 2.51 (bs, 8H, 4x CH2), 2.72 (s, 6H, 2´-COCH 6), 4.09 (s,
2H, -CH2-, benzy3lic), 8.24 (s, 1H, aromatic proton), 13.86 (s, 2H, 23xOH); MS
(m/z): 306 (M+), 207, 193, 190, 99. Anal. C16H22N2O4, calcd. C, 62.73; H, 7.24;
N, 9.14; found C, 62.65; H, 7.26; N, 9.12.
Antibacterial and antifungal activity
All the synthesized compounds were screened for in vitro antibacterial and
antifungal activities at the concentration of 100 and 200 µg/mL by cup-plate
method7 in nutrient agar (Hi-media) (antibacterial activity) and Sabouraud
dextrose agar (Hi-media) (antifungal activity). The bacterial strains gram
positive (Bacillus subtilis ATCC 6633 & Staphylococcus aureus ATCC
25923), gram negative (Escherichia coli ATCC 8739) and fungal strains
(Candida albicans NCIM 300 & Aspergillus flavus NCIM 524) were used.
The sterilized media was poured into sterile petridishes and allowed to solidify
for 30 minutes. On the surface of the media, 0.1 ml microbial suspension was
spread and after 10 minutes, cups were made by punching into agar surface
with a sterile cork borer (6 mm diameter) and scooping out the punched part of
the agar. Four cups were made in each petridish and into these cups was added
0.1 ml of the drug solution. The drug solution was allowed to diffuse for about
an hour and plates were then incubated at 37 ± 0.5 °C for 24 h (antibacterial
activity) and at 28 °C for 48-72 h (antifungal activity). A solvent control
dimethylformamide (DMF) was also run to know the activity of the blank. The
diameter of zone of inhibition (mm) was measured. Ofloxacin (20 µg/mL) and
voriconazole (20 µg/mL) were used as reference drugs for comparison. The
results are presented in Table-II.
Table-I: Physical data of the compounds 2a-h.
M.P.
(°C)
Yield
(%)
Molecular
Formula
Molecular
Weight
Compd
R
2a
-N(CH3)2
-N(C2H5)2
148-50
135-137
166-68
172-174
160-62
134-36
138-40
162-64
63
60
52
58
55
64
66
57
C13H17NO4
C15H21NO4
C18H19NO4
C18H19NO5
C18H19NO5
C15H19NO5
C16H21NO4
C16H22N2O4
251.28
279.33
313.35
329.35
329.35
293.32
291.34
306.36
2b
RESULTS AND DISCUSSION
Chemistry
2c
-NH
-NH
CH3
Titled compounds (2a-h) were synthesized through one-pot reaction as
depicted in Scheme-1. The starting material 4,6-diacetyl resorcinol 1 was
OCH3
OCH3
2d
2e
-NH
2f
-N
O
synthesized by the reported procedure6.
It (1) was condensed with different amines and formaldehyde, however,
the yields were poor and hence the reaction was carried out in the presence of
tetrabutyl ammonium bromide, a phase transfer catalyst, in chloroform solution
to give Mannich bases 2a-h in satisfactory yields. The structures assigned to
the compounds were supported by the results of elemental analysis as well as
1H NMR and Mass spectral data (Table-I).
The formation of Mannich bases could be inferred by the presence of a
methylene signal around d 4.0±0.2 instead of the signals for CH -CH2 and the
presence of acetyl signal consistently around d 2.6±0.2. Further 2mass spectral
data of all these compounds showed molecular ion peaks of reasonable intensity
besides the diagnostic peaks at m/z 207, 193 and 190 arising from diacetyl
resorcinol moiety and can be formulated as shown in Fig. 1. Beside these, there
was a fragment arising from the amine moiety, which was dependent on the
amine used.
2g
-N
2h
-N
N
CH3
Antibacterial and antifungal activity
All the synthesized compounds were screened for in vitro antibacterial and
antifungal activities at the concentration of 100 and 200 µg/mL. The bacterial
333

J. Chil. Chem. Soc., 55, Nº 3 (2010)
strains gram positive (Bacillus subtilis ATCC 6633 & Staphylococcus aureus
ATCC 25923), gram negative (Escherichia coli ATCC 8739) and fungal
strains (Candida albicans NCIM 300 & Aspergillus flaveous NCIM 524) were
used. Ofloxacin and voriconazole were used as reference drugs for comparison.
Among the tested compounds, 1,1’-{5-[(4-methylpiperazin-1-yl)methyl]-46-
dihydroxy-1,3-phenylene}diethanone (2h) showed very good activity against
S. aureus, C. albicans and A. flaveous, similar type of activity exhibited by
1,1’-{5-[(4-methoxylphenylamino)methyl]-46-dihydroxy-1,3-phenylene}
diethanone (2d) against S. aureus and A. flaveous. Both the compounds, 2h
and 2d, also showed good activity against E. coli and B. subtilis. Rests of the
compounds were moderate to low in their antimicrobial action (Table-II).
An analysis of results showed that the Mannich bases derived from
methylpiperazine and p-anisidine (2h 2d) were excellent in their
&
antimicrobial action against the tested organisms. Highest activity was observed
in the methylpiperazine derivative (2h), while replacing methylpiperazine with
p-anisidine (2d) slightly decreased the activity and it was further observed
that replacing it with m-anisidine (2e), morpholine (2f), piperidine (2g) and
p-toludene (2c) the activity decreased significantly. Among the synthesized
Mannich bases, the bases derived from dimethylamine (2a) and diethylamine
(2b) showed lowest antibacterial activity. However, the activities of tested
compounds are less than that of the standard agents used.
Table-II: Antibacterial and antifungal activities of the compounds 2a-h.
Diameter of zone of inhibition (mm)
Conc. (mg\
Compounds
mL)
E. coli
S. aureus
B. subtilis
C. albicans
A. flaveous
Voriconazole
20
20
nt
nt
nt
27
nt
31
nt
Ofloxacin
27
30
29
100
200
11
16
13
18
10
13
12
16
9
12
2a
2b
2c
2d
100
200
10
13
12
16
9
11
8
12
6
10
100
200
100
200
100
200
100
200
100
200
100
200
10
16
17
22
15
20
12
18
14
17
18
21
12
15
20
24
18
21
15
19
15
19
23
26
11
14
18
21
14
19
13
17
12
15
15
22
12
16
19
25
16
22
12
18
16
21
20
27
10
13
18
23
15
18
14
20
11
16
21
25
2e
2f
2g
2h
nt = not tested
CONCLUSIONS
REFERENCES
As concluding remarks we obtained herein eight new Mannich bases from
4,6-diacetyl resorcinol. Findings revealed that Mannich reaction did not take
place at the acetyl function but occurred at the 2-position into the aromatic
ring. Among the synthesized compounds, two compounds (2h and 2d) showed
significant antibacterial and antifungal activity against the tested microbes.
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