In vitro biological investigations of novel piperazine based heterocycles

Article abstract of DOI:10.3184/174751914X14116443659287

Eleven N.phenyl- and 11 N.benzothiazolyl-2-(4-(2,3,4-trimethoxybenzyl)piperazin-1-yl)acetamides have been synthesised by a simple and efficient method. The 22 novel compounds were tested for their in vitro biological efficacy against two Grampositive bacteria, three Gram-negative bacteria, two fungi and Mycobacterium tuberculosis H37Rv. The bioassay results revealed that the majority of the N.benzothiazole-substituted piperazine derivatives exhibited moderate to good bioefficacies with encouraging MICs. The influence of the presence or absence of various electron-withdrawing or -donating functional groups on the aryl acetamide moiety on the different bioassay results is discussed.

Full text of DOI:10.3184/174751914X14116443659287

JOURNAL OF CHEMICAL RESEARCH 2014
VOL. 38 OCTOBER, 611–616
RESEARCH PAPER 611
In vitro biological investigations of novel piperazine based heterocycles
Nirmal M. Chhatriwalaa, Amit B. Patela,b, Rahul V. Patelc and Premlata Kumaria*
aDepartment of Applied Chemistry, S.V. National Institute of Technology, Surat – 395007, India
bH.V.H.P. Institute of Postgraduate Studies and Research, Kadi Sarva Vishwavidyalaya, Kadi – 382715, India
cOrganic Research Laboratory, Department of Bioresources and Food Science, College of Life and Environmental Sciences, Konkuk University,
Seoul – 143701, South Korea
Eleven N‑phenyl‑ and 11 N‑benzothiazolyl‑2‑(4‑(2,3,4‑trimethoxybenzyl)piperazin‑1‑yl)acetamides have been synthesised by
a simple and efficient method. The 22 novel compounds were tested for their in vitro biological efficacy against two Gram‑
positive bacteria, three Gram‑negative bacteria, two fungi and Mycobacterium tuberculosis H37Rv. The bioassay results
revealed that the majority of the N‑benzothiazole‑substituted piperazine derivatives exhibited moderate to good bioefficacies
with encouraging MICs. The influence of the presence or absence of various electron‑withdrawing or ‑donating functional
groups on the aryl acetamide moiety on the different bioassay results is discussed.
Keywords: benzothiazole, N-(2,3,4-trimethoxybenzyl)piperazine, antimicrobial, antimycobacterial
Fighting disease with drugs is a timeless struggle, with the
human survival on this planet dependent on its success.
Steadily increasing antibiotic resistance and decreasing
numbers of newer antibiotics appear to point to a post-
antibiotic period during which treatment of infections would
become increasingly difficult.1 Mycobacterium tuberculosis,
moreover, remains the primary cause of comparatively
high mortality worldwide. Due to the quiescent form of
mycobacterium tuberculosis strains, many of the currently
available antimycobacterial drugs have become ineffective by
the imminent onset of multidrug–resistance.2,3 According to
the World Health Organization (WHO), 1.3 million MDR–TB
cases will need to be treated in the 27 countries with the highest
MDR–TB burden between 2010 and 2015.4 Thus the necessity
to develop newer, potent and unique antibacterial agents is
urgent, not least to counteract widespread drug resistance.
Heterocycles play an important role in all spheres of life
including pharmaceuticals, natural resources, veterinary
products, analytical reagents, agrochemicals and dyes. The
development of new approaches for the synthesis of novel
heterocycles substituted with unique functional groups forms
the basis of an extensive research activity in synthetic organic
chemistry.5 The design of inhibitors that can show potency
towards multiple biological targets remains an intriguing
scientific endeavour. In the design of new compounds, the
developments of hybrid molecules through the combination
of different pharmacophores in one structure may lead to
increased antimicrobial activity.6 Indeed, the merging of
pharmacophores may offer important medical advances not
only to minimise the probability of resistance but to construct
scaffolds that would possess multiple biological activities on a
range of specific biological targets.7 In pursuit of this aim, we
have synthesised a number of N-phenyl- and N-benzothiazolyl-
substituted quinazoline derivatives and tested them against
various microorganisms.
with potassium thiocyanates in a satisfactory yield by a known
method. The 2–amino-6-substituted benzothiazoles were
converted to the corresponding 2-chloro-N-(6-R-benzo[d]
thiazol-2-yl)acetamides 4a–k using chloroacetyl chloride in
benzene solvent as described in the literature. All the above
mentioned intermediate derivatives (2a–k and 4a–k) were
synthesised according to a known procedure reported in the
literature and the products were characterised by FTIR and
1H NMR spectroscopy. All the analytical characterisation data
of the said intermediates were in good accordance with the
proposed structures.
The above acetamide derivatives, 2a–k and 4a–k, were then
condensed with N-(2,3,4-trimethoxybenzyl)piperazine 5 in
acetone solvent at reflux temperature to furnish products 6a–k
and 7a–k. The IR and 1H NMR spectral data of compounds 6a–k
and 7a–k were in accordance with their assumed structures.
The purity of the synthesised compounds was monitored by
TLC and confirmed by elemental analysis. The yields, melting
points and elemental analysis data of compounds 6a–k and
7a–k are listed in Table 1.
Reagents and conditions: (a) chloroacetyl chloride, acetone,
K2CO3, reflux; (b) potassium thiocyanate, bromine in glacial
acetic acid, room temperature (c) K2CO3, acetone, reflux.
Pharmacology
In vitro antimicrobial evaluation of analogues 6a–k and
7a–k: From the in vitro antimicrobial activity bioassay results
presented in Table 2 it can be seen that some of the compounds
displayed good inhibitory efficacy against several of the panel
of seven human pathogenic microorganisms. Compounds with
halogen substituents such as chloro, bromo, fluoro or iodo on
the para-position of the phenyl acetamides moiety condensed
to the piperazine ring system 6b–e showed higher inhibitory
potential than those compounds possessing alkyl or alkoxy
substituents 6h–j. In general, this series of compounds, 6a–k
and 7a–k, was found to be more efficacious in inhibiting Gram-
positive strains than Gram-negative strains as the level of
minimum inhibitory concentration was lowest at 12.5 µg mL–1
in the case of the former while in the latter it was found to
be between 25 and 50 µg mL–1. Compounds 6d, 7c, 7d and
7j with a halogen- or an alkoxy-substituted benzylpiperazine
moiety showed significant antibacterial activity against Gram-
positive strain Staphylococcus aureus at 12.5 µg mL–1. The
methoxy-substituted N-benzothiazolyl analogue 7i displayed
the highest activity against Gram-positive strain Bacillus
cereus at 12.5 µg mL–1. Analogues 6c, 6d, 7b, 7c, 7d and 7j
Result and discussion
Synthesis of intermediates and target compounds was
accomplished according to the steps illustrated in Scheme 1.
Chloroacetyl chloride was reacted with para-(substituted)
phenyl amines to give the corresponding 2-chloro-N-4-
R-phenyl acetamides 2a–k. The 2-amino-6-substituted
benzothiazoles 3a–k were synthesised by reacting aryl amines
* Correspondent. E-mail: premlatakumari1@gmail.com
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612 JOURNAL OF CHEMICAL RESEARCH 2014
O
C
a
l
C
R
NH
2a- k
R
NH2
O
C
N
S
N
S
1a–k
a
NH2
NH
Cl
b
R
R
3a–k
4a–k
H3CO
H3CO
OCH3
c
O
C
O
C
H2
C
l
C
R
NH
HN
R
N
N
H3CO
H3CO
OCH3
2a–k
6a–k
H2
C
+
N
NH
H3CO
H3CO
OCH3
O
N
S
5
c
O
C
N
H2
C
NH
C
Cl
N
N
NH
R
S
R
4a–k
7a–k
R= H, Cl, Br, F, I, NO2, CN, CH3, OCH3, OCH2CH3, NHCOCH3
Scheme 1 Synthetic method for the synthesis of 6a–k and 7a–k.
with electron-withdrawing halo substituents showed strong
inhibitory effects against Gram-positive strain B. cereus
at 25 µg mL–1. Two of the benzothiazole derivatives, one
with a highly electronegative fluorine atom 7d and the
other with a strong electron-withdrawing nitro functional
group 7f exhibited excellent level of activity against Gram-
negative bacteria Escherichia coli at 25 µg mL–1. In the case
of antibacterial efficacy of the final analogue against Gram-
negative strain Pseudomonas aeruginosa compounds with
electron-donating with methoxy and ethoxy functional groups
7i and 7j, respectively, displayed a good level of inhibitory
efficacy at 25 µg mL–1. The analogues 6f, 6g, 7c and 7g
displayed excellent inhibitory efficacy at 25 µg mL–1 against
Gram-negative Klebsiella pneumoniae. The activities of these
compounds against K. pneumoniae were equipotent with the
control drugs. Some of the compounds also had good inhibitory
potential against the above-mentioned Gram-positive and
Gram-negative strains at 25–100 µg mL–1, while many of the
others were found to be inactive at 200–500 µg mL–1.
In the two in vitro antifungal bioassays, it can be seen that
compounds with electron withdrawing halo substituents, 6d,
6e, 7b and 7d, showed good activity against Aspergillus niger at
Table 1 Yields and melting points of compounds 6a–k and 7a–k and their elemental analysis data
C/%
H/%
N/%
Entry
Yield /%
M.p./°C
Mol. wt
Calcd
Found
Calcd
Found
Calcd
Found
6a
6b
6c
6d
6e
6f
6g
6h
6i
72
56
67
75
70
64
72
73
66
68
71
67
60
61
69
74
68
55
66
59
71
65
193–195
204–205
234–236
200–202
221–223
198–199
233–235
256–257
240–242
219–221
195–197
212–214
226–227
251–252
206–208
241–243
210–211
250–251
266–268
233–235
236–238
200–203
399.48
433.93
478.38
417.47
525.38
444.48
424.49
413.51
429.51
443.54
456.53
456.56
491.00
535.45
474.55
582.45
501.56
481.57
470.58
486.58
500.61
513.61
66.14
60.89
55.24
63.29
50.29
59.45
65.08
66.81
64.32
64.99
63.14
60.51
56.26
51.59
58.21
47.73
55.08
59.86
61.26
59.24
59.98
58.46
65.98
61.04
55.09
63.56
50.12
59.58
64.92
66.90
64.48
65.11
62.98
60.33
56.44
51.02
58.40
47.55
55.22
59.99
61.12
59.11
59.76
58.55
7.32
6.50
5.90
6.76
5.37
6.35
6.65
7.56
7.27
7.50
7.06
6.18
5.55
5.08
5.73
4.67
5.43
5.65
6.43
6.21
6.44
6.08
7.54
6.72
5.78
6.68
5.22
6.51
6.47
7.43
7.21
7.73
6.97
5.98
5.76
4.91
5.60
4.79
5.22
5.47
6.66
6.44
6.31
6.23
10.52
9.68
8.78
10.07
8.00
12.60
13.20
10.16
9.78
10.29
9.51
8.65
9.96
8.26
12.38
13.02
9.98
9.86
9.31
12.21
22.02
11.57
10.25
11.77
9.47
14.09
14.33
12.07
11.33
11.34
13.75
6j
9.47
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
12.27
22.27
11.41
10.46
11.81
9.62
13.96
14.54
11.91
11.51
11.19
13.64
7j
7k
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JOURNAL OF CHEMICAL RESEARCH 2014 613
Table 2 Antimicrobial activity of compounds 6a–k and 7a–k
a
MIC/µg mL–1
Entry
R
S. aureus
B. cereus
E. coli
P. aeruginosa K. pneumoniae
A. niger
C. albicans
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
H
Cl
Br
F
500
100
25
12.5
50
100
100
200
50
200
50
25
25
50
100
250
500
100
100
200
200
25
25
25
50
50
100
250
12.5
25
200
200
100
200
100
500
200
500
250
50
500
200
200
100
200
50
200
500
250
200
500
200
50
100
50
200
50
200
500
25
100
50
50
50
100
25
500
100
100
50
250
50
100
100
250
250
200
500
50
100
200
200
100
100
50
500
200
200
500
50
50
100
–
I
50
NO2
CN
CH3
OCH3
OC2H5
NHCOCH3
H
Cl
Br
F
I
NO2
CN
CH3
OCH3
OC2H5
NHCOCH3
200
100
500
100
100
100
500
50
25
200
100
50
100
100
50
25
50
100
50
25
200
100
50
100
25
25
–
–
25
50
250
250
50
12.5
12.5
50
100
100
200
25
12.5
100
12.5
6.25
–
100
200
100
50
25
250
25
100
200
50
50
100
100
50
100
200
100
250
50
100
200
–
25
100
25
12.5
–
–
7k
Amp.
Gen.
Flu.
DMSO
12.5
6.25
–
–
6.25
12.5
–
–
6.25
–
–
12.5
–
–
–
aEach value is the mean of three independent experiments.
50 µg mL–1, whereas it was compounds with electron donating
alkoxy substituents, 6b, 6i, 7i and 7j that showed good activity
against Candida albicans, also at 50 µg mL–1. The remainder
of the compounds showed moderate activity either of 100 or
200–500 µg mL–1.
Table 3 Antimycobacterial activity of compounds 6a–k and 7a–k
L.J. MIC Methoda
Entry
R
MIC/µg mL–1
% Inhibition
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
Cl
Br
F
500
100
50
92
95
97
98
93
96
94
92
98
96
94
93
97
98
99
94
96
95
92
98
97
95
In vitro antimycobacterial activity of analogues 6a–k and
7a–k: In vitro antimycobacterial activities of compounds 6a–k
and 7a–k were assessed against M. tuberculosis H37Rv and the
results are shown in Table 3. Some of the compounds displayed
good levels of antimycobacterial activity. The results observed
by the L.J. MIC method indicated that the N-benzothiazole-
substituted piperazine analogue with an electron withdrawing
fluoro substituent 7d exhibited the highest inhibition (99%)
at a constant concentration level (12.5 µg mL–1). However,
the similar N-phenyl derivative 6d displayed slightly less
antimycobacterial activity (25 µg mL–1). Interestingly, both the
Methoxy-substituted phenyl and benzothiazolyl compounds, 6i
and 7i, exhibited good antimycobacterial activity at 25 µg mL–1.
However, many of the compounds exhibited only moderate
antimycobacterial activity at 50–100 µg mL–1 and some were
found inactive with values>100 µg mL–1.
25
I
250
62.5
200
500
25
NO2
CN
CH3
OCH3
OC2H5
NHCOCH3
H
Cl
Br
F
I
NO2
CN
CH3
OCH3
OC2H5
NHCOCH3
6j
50
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
200
250
62.5
25
12.5
250
100
200
500
25
Experimental
Melting points were determined in open capillaries on a Veego
electronic apparatus VMP-D and are uncorrected. IR spectra
(4000–400 cm–1) were recorded on a Shimadzu 8400-S FTIR
spectrophotometer using KBr pellets. TLC was performed on object
glass slides (2×7.5 cm) coated with silica gel-G and spots were
visualised under UV irradiation. 1H NMR spectra were obtained on
a Varian 400 MHz model spectrometer in DMSO-d6 using TMS as
internal standard. Chemical shifts (δ) are given in ppm and coupling
constants (J) in Hz. Elemental analyses (CHN) were performed using
a Heraeus Carlo Erba 1180 CHN analyser (Hanau, Germany).
7j
7k
Isoniazid
Rifampicin
Ethambutol
Pyrazinamide
DMSO
50
100
0.20
0.25
3.12
6.25
–
–
–
aEach value is the mean of three independent experiments.
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614 JOURNAL OF CHEMICAL RESEARCH 2014
Synthesis of 2‑chloro‑N‑aryl acetamides (2a–k);general procedure
Chloroacetyl chloride (0.06 mol) was added dropwise to a mixture
of the appropriate amine (0.05 mol) and K2CO3 (0.06 mol) in acetone
(50 mL) at room temperature. The reaction mixture was refluxed for
4 to 8 h, then, after cooling to room temperature, it was slowly poured
into 100 mL of ice water. The solid that formed was separated by
filtration and washed repeatedly with water. The product was dried
under vacuum to obtain 2a–k.8–12 The progress of the reaction was
monitored by TLC using toluene: acetone (8:2) solvent system.
1H NMR: δ 8.12 (s, 1H, –NH), 7.61–7.38 (m, 6H, ArH), 4.22 (s, 2H,
CO–CH2), 3.86 (br s, 4H, piperazine), 3.58 (s, 2H, N–CH2), 3.47 (s, 9H,
3OCH3), 3.39 (br s, 4H, piperazine).
N‑(4‑Nitrophenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl) piperazin‑1‑yl)
acetamide (6f): IR (KBr, cm–1): 3298 (N–H), 3011 (C–H), 1679 (C=O);
1H NMR: δ 8.28 (s, 1H, –NH), 7.70–7.32 (m, 6H, ArH), 4.30 (s, 2H,
CO–CH2), 3.78 (br s, 4H, piperazine), 3.63 (s, 2H, N–CH2), 3.41 (s, 9H,
3OCH3), 3.31 (br s, 4H, piperazine).
N‑(4‑Cyanophenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)piperazin‑1‑
yl)acetamide (6g): IR (KBr, cm–1): 3303 (N–H), 2961 (C–H), 2224
(C≡N), 1672 (C=O); 1H NMR: δ 8.15 (s, 1H, –NH), 7.67–7.18 (m, 6H,
ArH), 4.21 (s, 2H, CO–CH2), 3.83 (br s, 4H, piperazine), 3.52 (s, 2H,
N–CH2), 3.48 (s, 9H, 3OCH3), 3.35 (br s, 4H, piperazine).
N‑(p‑Tolyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)piperazin‑1‑yl)acetamide
(6h): IR (KBr, cm–1): 3317 (N–H), 3018 (C–H), 1679 (C=O); 1H NMR:
δ 8.41 (s, 1H, –NH), 7.84–7.32 (m, 6H, ArH), 4.20 (s, 2H, CO–CH2),
3.81 (br s, 4H, piperazine), 3.57 (s, 2H, N–CH2), 3.52 (s, 9H, 3OCH3),
3.38 (br s, 4H, piperazine), 2.56 (s, 3H, CH3).
Synthesis of 2‑amino‑6‑substituted benzothiazoles (3a–k);general
procedure
A
mixture of 4–substituted aniline (0.1 mol) and potassium
thiocyanate (0.1 mol) in glacial acetic acid (100 mL) was cooled
in an ice bath and stirred for 10 to 20 min, and then bromine
(0.1 mol) in glacial acetic acid was added dropwise at such a rate as
to keep the temperature below 10°C throughout the addition. The
reaction mixture was stirred at room temperature for 2–4 h, and
the hydrobromide salt that separated out was filtered, washed with
acetic acid, dried, dissolved in hot water and basified to pH 11.0 with
ammonia solution. The resulting precipitate was filtered, washed with
water and dried to give the products 3a–k.13–16 The progress of the
reaction was monitored by TLC using toluene: acetone (8:2) solvent
system.
N‑(4‑Methoxyphenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)piperazin‑1‑yl)
acetamide (6i): IR (KBr, cm–1): 3320 (N–H), 2961 (C–H), 1676 (C=O);
1H NMR: δ 8.38 (s, 1H, –NH), 7.67–7.28 (m, 6H, ArH), 4.19 (s, 2H,
CO–CH2), 3.85 (br s, 4H, piperazine), 3.60 (s, 2H, N–CH2), 3.48 (s,
12H, 4OCH3), 3.21 (br s, 4H, piperazine).
N‑(4‑Ethoxyphenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)piperazin‑1‑
yl)acetamide (6j): IR (KBr, cm–1): 3345 (N–H), 3008 (C–H), 1680
(C=O); H NMR: δ 8.21 (s, 1H, –NH), 7.75–7.31 (m, 6H, ArH), 4.27
(s, 2H, CO–CH2), 4.06 (q, J=6.8 Hz, 2H, –OCH2CH3), 3.91 (br s, 4H,
piperazine), 3.58 (s, 2H, N–CH2), 3.54 (s, 9H, 3OCH3), 3.29 (br s, 4H,
piperazine), 1.48 (t, J=7.3 Hz, 3H, –OCH2CH3).
Synthesis of 2‑chloro‑N‑(6‑substitutedbenzo[d]thiazol‑2‑yl)acetamides
(4a–k); general procedure
1
Chloroacetyl chloride (0.06 mol) was added dropwise to a mixture of
the appropriate 2-amino-6-substituted benzothiazole, 3a–k (0.05 mol)
and K2CO3 (0.06 mol) in benzene (50 mL) at room temperature. The
reaction mixture was refluxed for 6 to 12 h, then, after cooling to room
temperature, it was slowly poured into ice water (100 mL). The solid
that formed was separated by filtration, washed successively with
water, then dried under vacuum to obtain 4a–k.17–20 The progress of the
reaction was monitored by TLC using toluene: acetone (8:2) solvent
system.
N‑(4‑Acetamidophenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)piperazin‑
1‑yl)acetamide (6k): IR (KBr, cm–1): 3328 (N–H), 2978 (C–H), 1672
1
(C=O); H NMR: δ 8.36 (s, 1H, –NH), 8.25 (s, 1H, NHCOCH3),
7.59–7.17 (m, 6H, ArH), 4.20 (s, 2H, CO–CH2), 3.82 (br s, 4H,
piperazine), 3.65 (s, 2H, N–CH2), 3.58 (s, 9H, 3OCH3), 3.38 (br s, 4H,
piperazine), 2.32 (s, 3H, NHCOCH3).
Synthesis of 6a–k and 7a–k;general procedure
N‑(Benzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)piperazin‑
1‑yl)acetamide (7a): IR (KBr, cm–1): 3311 (N–H), 2985 (C–H), 1669
To a solution of 5 (0.01 mol) in acetone (30 mL), the appropriate
quantities of 2a–k and 4a–k (0.01 mol) was added and the reaction
mixture was refluxed for 15–25 h. Potassium carbonate (0.01 mol)
was used for neutralisation of the reaction mixture. Progress of the
reaction was monitored by TLC using toluene: acetone (8:2) as eluent.
The mixture was then treated with crushed ice. The precipitate thus
obtained was filtered off, dried and recrystallised from acetone to
afford 6a–k and 7a–k.6
N‑Phenyl‑2‑(4‑(2,3,4‑trimethoxybenzyl)piperazin‑1‑yl)acetamide
(6a): IR (KBr, cm–1): 3328 (N–H), 2990 (C–H), 1676 (C=O); 1H NMR:
δ 8.21 (s, 1H, –NH), 7.68–7.32 (m, 7H, ArH), 4.24 (s, 2H, CO–CH2),
3.88 (br s, 4H, piperazine), 3.51 (s, 2H, N–CH2), 3.37 (s, 9H, 3OCH3),
3.28 (br s, 4H, piperazine).
1
(C=O), 1507 (benzothiazole); H NMR: δ 8.13 (s, 1H, –NH), 7.67 (d,
1H, benzothiazole), 7.52–6.95 (m, 6H, ArH), 4.14 (s, 2H, CO–CH2),
3.70 (br s, 4H, piperazine), 3.61 (s, 2H, N–CH2), 3.35 (s, 9H, 3OCH3),
3.12 (br s, 4H, piperazine).
N‑(6‑Chlorobenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7b): IR (KBr, cm–1): 3367 (N–H), 2939
(C–H), 1685 (C=O), 1599 (benzothiazole), 750 (–Cl); 1H NMR: δ 8.22
(s, 1H, –NH), 7.79 (d, 1H, benzothiazole), 7.69–7.18 (m, 5H, ArH), 4.22
(s, 2H, CO–CH2), 3.86 (br s, 4H, piperazine), 3.65 (s, 2H, N–CH2), 3.50
(s, 9H, 3OCH3), 3.38 (br s, 4H, piperazine).
N‑(6‑Bromobenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7c): IR (KBr, cm–1): 3347 (N–H), 3008
(C–H), 1680 (C=O), 1532 (benzothiazole); 1H NMR: δ 8.54 (s,
1H, –NH), 7.83 (d, 1H, benzothiazole), 7.72–7.34 (m, 5H, ArH), 4.43 (s,
2H, CO–CH2), 3.81 (br s, 4H, piperazine), 3.63 (s, 2H, N–CH2), 3.37 (s,
9H, 3OCH3), 3.29 (br s, 4H, piperazine).
N‑(4‑Chlorophenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl) piperazin‑1‑yl)
acetamide (6b): IR (KBr, cm–1): 3287 (N–H), 2938 (C–H), 1688 (C=O),
737 (–Cl); 1H NMR: δ 8.43 (s, 1H, –NH), 7.71–7.26 (m, 6H, ArH), 4.13
(s, 2H, CO–CH2), 3.80 (br s, 4H, piperazine), 3.59 (s, 2H, N–CH2), 3.52
(s, 9H, 3OCH3), 3.34 (br s, 4H, piperazine).
N‑(4‑Bromophenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl) piperazin‑1‑yl)
acetamide (6c): IR (KBr, cm–1): 3349 (N–H), 3021 (C–H), 1668 (C=O);
1H NMR: δ 8.15 (s, 1H, –NH), 7.83–7.40 (m, 6H, ArH), 4.38 (s, 2H,
CO–CH2), 3.76 (br s, 4H, piperazine), 3.42 (s, 2H, N–CH2), 3.23 (s, 9H,
3OCH3), 3.12 (br s, 4H, piperazine).
N‑(4‑Fluorophenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl) piperazin‑1‑yl)
acetamide (6d): IR (KBr, cm–1): 3308 (N–H), 3032 (C–H), 1680
(C=O); 1H NMR: δ 8.39 (s, 1H, –NH), 7.67–7.18 (m, 6H, ArH), 4.08 (s,
2H, CO–CH2), 3.82 (br s, 4H, piperazine), 3.50 (s, 2H, N–CH2), 3.34 (s,
9H, 3OCH3), 3.21 (br s, 4H, piperazine).
N‑(6‑Fluorobenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7d): IR (KBr, cm–1): 3302 (N–H), 3085
(C–H), 1678 (C=O), 1564 (benzothiazole); 1H NMR: δ 8.09 (s,
1H, –NH), 7.64 (d, 1H, benzothiazole), 7.49–7.22 (m, 5H, ArH), 4.05
(s, 2H, CO–CH2), 3.73 (br s, 4H, piperazine), 3.48 (s, 2H, N–CH2), 3.33
(s, 9H, 3OCH3), 3.21 (br s, 4H, piperazine).
N‑(6‑Iodobenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7e): IR (KBr, cm–1): 3289 (N–H), 2982
(C–H), 1667 (C=O), 1585 (benzothiazole); 1H NMR: δ 8.19 (s,
1H, –NH), 7.87 (d, 1H, benzothiazole), 7.72–7.30 (m, 5H, ArH), 4.38 (s,
2H, CO–CH2), 3.78 (br s, 4H, piperazine), 3.60 (s, 2H, N–CH2), 3.45 (s,
9H, 3OCH3), 3.37 (br s, 4H, piperazine).
N‑(4‑Iodophenyl)‑2‑(4‑(2,3,4‑trimethoxybenzyl) piperazin‑1‑yl)
acetamide (6e): IR (KBr, cm–1): 3327 (N–H), 2984 (C–H), 1668 (C=O);
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N‑(6‑Nitrobenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7f): IR (KBr, cm–1): 3315 (N–H), 3042
(C–H), 1679 (C=O), 1540 (benzothiazole); 1H NMR: δ 8.02 (s,
1H, –NH), 7.69 (d, 1H, benzothiazole), 7.53–7.26 (m, 5H, ArH), 4.29
(s, 2H, CO–CH2), 3.74 (br s, 4H, piperazine), 3.58 (s, 2H, N–CH2), 3.43
(s, 9H, 3OCH3), 3.29 (br s, 4H, piperazine).
into a specified quantity of molten sterile agar, i.e. nutrient agar for
evaluation of antibacterial and sabouraud dextrose agar for antifungal
activity, respectively. The medium containing the test compound
was poured into a Petri dish at a depth of 4–5 mm and allowed to
solidify under aseptic conditions. A suspension of the respective
microorganism of approximately 105 CFU mL–1 was prepared and
applied to plates with serially diluted compounds with concentrations
in the range of 3.12–50 µg mL–1 in DMSO and incubated at (37±1)
°C for 24 h (bacteria) or 48 h (fungi). The lowest concentration of
the substance that prevented the development of visible growth was
considered to be the MIC value.
N‑(6‑Cyanobenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7g): IR (KBr, cm–1): 3348 (N–H), 3011
1
(C–H), 2229 (C≡N), 1684 (C=O), 1560 (benzothiazole); H NMR: δ
8.21 (s, 1H, –NH), 7.80 (d, 1H, benzothiazole), 7.67–7.25 (m, 5H, ArH),
4.08 (s, 2H, CO–CH2), 3.65 (br s, 4H, piperazine), 3.49 (s, 2H, N–CH2),
3.38 (s, 9H, 3OCH3), 3.26 (br s, 4H, piperazine).
In vitro evaluation of antitubercular activity evaluation: The
antitubercular screening of test compounds was achieved for M.
Tuberculosis H37Rv by adopting the L.J. (conventional Lowenstein
and Jensen) agar dilution method for the measurement of MIC, and
is defined as the lowest concentration of drug, which inhibits≥99%
of the bacterial population present at the beginning of the assay.
Stock solutions of 100, 62.5, 50, 25, 12.5, 6.25, 3.12 and 1.56 μg mL–1
dilutions of each test compound in DMSO were added into the liquid
L.J. Medium and then the media were sterilised by the inspissation
method. A culture of M. tuberculosis H37Rv growing on L.J. Medium
was harvested in 0.85% saline in bijou bottles.23 These tubes were then
incubated at 37 °C for 24 h followed by streaking of M. tuberculosis
H37Rv (5×104 bacilli per tube). These tubes were then incubated at
37 °C. Growth of bacilli was assessed after 12 days, 22 days and
finally 28 days of incubation. Tubes having test compounds were
compared with control tubes where medium alone was incubated with
M. tuberculosis H37Rv. The concentration at which no development of
colonies occurred or <20 colonies was taken as the MIC concentration
of the test compound. The standard strain M. tuberculosis H37Rv was
tested with the known drugs Isoniazid, Rifampicin, Ethambutol and
Pyrazinamide.
N‑(6‑Methylbenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7h): IR (KBr, cm–1): 3360 (N–H), 2987
(C–H), 1669 (C=O), 1587 (benzothiazole); 1H NMR: δ 8.38 (s,
1H, –NH), 7.61 (d, 1H, benzothiazole), 7.48–6.89 (m, 5H, ArH), 4.12
(s, 2H, CO–CH2), 3.71 (br s, 4H, piperazine), 3.58 (s, 2H, N–CH2), 3.30
(s, 9H, 3OCH3), 3.19 (br s, 4H, piperazine), 2.38 (s, 3H, CH3).
N‑(6‑Methoxybenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7i): IR (KBr, cm–1): 3298 (N–H), 3031
(C–H), 1674 (C=O), 1545 (benzothiazole); 1H NMR: δ 8.32 (s,
1H, –NH), 7.68 (d, 1H, benzothiazole), 7.53–7.28 (m, 5H, ArH), 4.30
(s, 2H, CO–CH2), 3.92 (br s, 4H, piperazine), 3.60 (s, 2H, N–CH2), 3.42
(s, 12H, 4OCH3), 3.27 (br s, 4H, piperazine).
N‑(6‑Ethoxybenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7j): IR (KBr, cm–1): 3330 (N–H), 2995
(C–H), 1679 (C=O), 1598 (benzothiazole); 1H NMR: δ 7.96 (s,
1H, –NH), 7.67 (d, 1H, benzothiazole), 7.53–7.20 (m, 5H, ArH), 4.25
(s, 2H, CO–CH2), 4.14 (q, J=7.6 Hz, 2H, –OCH2CH3), 3.88 (br s, 4H,
piperazine), 3.57 (s, 2H, N–CH2), 3.39 (s, 9H, 3OCH3), 3.26 (br s, 4H,
piperazine), 1.52 (t, J=7.3 Hz, 3H, –OCH2CH3).
N‑(6‑Acetamidobenzo[d]thiazol‑2‑yl)‑2‑(4‑(2,3,4‑trimethoxybenzyl)
piperazin‑1‑yl)acetamide (7k): IR (KBr, cm–1): 3321 (N–H), 3026
(C–H), 1668 (C=O), 1548 (benzothiazole); 1H NMR: δ 8.28 (s,
1H, –NH), 8.11 (s, 1H, NHCOCH3), 7.86 (d, 1H, benzothiazole),
7.58–7.21 (m, 5H, ArH), 4.35 (s, 2H, CO–CH2), 3.78 (br s, 4H,
piperazine), 3.56 (s, 2H, N–CH2), 3.49 (s, 9H, 3OCH3), 3.40 (br s, 4H,
piperazine), 2.25 (s, 3H, NHCOCH3).
Conclusion
In conclusion, this work has focused on the development of new
small molecules endowed with a piperazine nucleus bearing
various substituents in the form of aryl acetamides moieties.
In general, the results of the in vitro pharmacological activities
are encouraging, as out of two series tested, the compounds
containing a N-benzothiazolyl acetamide moiety emerged as
the class of compounds exhibiting the highest antimicrobial
activity. Based on the results described above, such compounds
can be recognised as new active leads that provide a powerful
incentive for further research in this area.
Microbiology
In vitro antimicrobial activity evaluation: Compounds 6a–k and 7a–k
were examined for their antimicrobial activity against two Gram-
positive bacteria (S. aureus and B. cereus), three Gram-negative
bacteria (E. coli, P. aeruginosa and K. pneumoniae) and two fungal
species (A. niger and C. albicans) using the paper disc diffusion
technique and the agar streak dilution method.21 The Mueller–Hinton
agar media were sterilised (autoclaved at 120 °C for 30 min), poured
at uniform depth of 5 mm and allowed to solidify. The microbial
suspension (105 CFU/mL) (0.5 McFarland Nephelometry Standards)
was streaked over the surface of media using a sterile cotton swab
to ensure even growth of the organisms. The test compounds
were dissolved in dimethylsulfoxide (DMSO) to give solutions of
3.12–50 µg mL–1. Sterile filter paper discs measuring 6.25 mm in
diameter (Whatman no. 1 filter paper), previously soaked in a known
concentration of the respective test compound in DMSO were placed
on the solidified nutrient agar medium that had been inoculated with
the respective microorganism and the plates were incubated for 24 h
at (37±1) °C. A control disc impregnated with an equivalent amount
of DMSO without any sample was also used and did not produce any
inhibition. Ampicillin and gentamicin (100 µg/disc) were used as
standard antibacterial drugs while fluconazole (100 µg/disc) was used
as an antifungal drug.
Received 21 August 2014; accepted 13 September 2014
Paper 1402842 doi: 10.3184/174751914X14116443659287
Published online: 17 October 2014
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