Estelar - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/48801/2/chapter 2.pdf · possesses...
Transcript of Estelar - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/48801/2/chapter 2.pdf · possesses...
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Chapter 2
Synthesis and antibacterial activity
of piperazine derivatives
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2.1. Introduction
Piperazine consists of a six-member ring containing the two opposing nitrogen
atoms was originally named because of its chemical similarities with piperidine, a
constituent of piperine in the black pepper plant (Piper nigrum). It is a weak base with
pka of 4.19 and freely soluble in water and ethylene glycol but insoluble in diethyl
ether.
Piperazine is an interesting heterocyclic structure present in many biologically
active molecules. The polar nitrogen atoms into the piperazine ring confer bioactivity
of molecules and enhance favorable interaction with macromolecules.1,2 Piperazine
and substituted piperazines are important pharmacophores that can be found in many
marketed drugs such as the Merck HIV protease inhibitor Crixivan.3-10 Piperazinyl-
linked ciprofloxacin dimers reported as potent antibacterial agents against resistant
strains,11 antimalarial agents
12 and potential antipsychotic agents.
13 Recently,
piperazine derivatives containing tetrazole nucleus have been reported as an
antifungal agent.14
S,S-1-adamantan-1-yl-3-[2-(5-benzyl-piperazine-2-yl)-ethyl]urea a
piperazine containing dialkyl urea exhibited good potency against human SEH
(soluble epoxide hydrolase) with an IC50 value of 1.37 µM and developed as a drug
for anti-hypertension and anti-inflammation.15 Substituted benzamide piperazine
derivatives have shown strong agonistic activity while the substituted acetamide
piperazine derivative have better dopamine D4 receptor agonist activity than
substituted benzamide piperazine derivatives.16,17
Diphenyl piperazine derivative
possesses broad pharmacological action on central nervous system (CNS), especially
on dopaminergic neurotransmission.18 Sulfonamides are among the most widely used
antibacterial agents because of their low toxicity and excellent activity against
common bacterial diseases. N-sulfonamide derivatives of 1-[bis(4-fluorophenyl)-
methyl]piperazine exhibited potent antibacterial activity against Escherichia coli,
Proteus vulgaris and Salmonella typhi.19 Most of the quinoline drugs, such as
norfloxacin and ciprofloxacin having piperazine nucleus showed broad spectrum
activity of respiratory, urinary, gastrointestinal tracts, skin and soft tissue infection
caused by either Gram-negative or Gram-positive bacteria.20 Various cyano
derivatives of piperazine are known for their uses in the synthesis of pharmaceutical
intermediates, peptide analogues and antibacterial drugs.21-23
Cyano derivatives of
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N-alkyl and N-aryl substituted piperazine derivatives exhibited potent activity against
Pseudomonas aeruginosa.24 Dihydro-1,2-oxazine and 2-pyrazoline oxazolidinones
related to linezolid and eperezolid showed potent antibacterial activity.25 A novel
series of N-aryl piperazine-1-carboxamide derivatives were found to exhibit potent
antiandrogenic activity.26 The new piperazine linked chalcone derivatives showed
potent antibacterial activity against Staphylococcus aureus, Escherichia coli, Proteus
vulgari and antifungal activity against Aspergillus fumigatus and Candida albicans.27
Many other piperazine derivatives are notably successful drugs.
In the present study, we have aimed to synthesize new piperazinyl-linked
antibacterials by nucleophilic substitution reaction of N-alkyl and N-aryl piperazine
derivatives with different p-substituted phenacyl halides.
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2.2. Experimental
2.2.1. Reaction scheme for the synthesis of piperazine derivatives (Pz 1 to
Pz 9)
N NHH3C
R
O
BrN NH3C
O
R
+
N-methyl piperazine p-substituted
phenacyl bromide
Pz 1 - Pz 3
N NH
R
O
BrN N
O
R
+
N-phenyl piperazine p-substituted
phenacyl bromide
Pz 4 - Pz 6
R
O
Br+
N-benzyl piperazine p-substituted
phenacyl bromidePz 7 - Pz 9
N
NHN
NO
R
where R= -Br, -CH3, -OCH3
Scheme 2.1. Reagent and conditions: (i) K2CO3, acetonitrile, 0 oC- rt, 8-10 h.
i
i
i
2.2.2. General experimental procedure for synthesis of piperazine
derivatives
To a solution of substituted piperazine derivatives (1.0 mequiv) in acetonitrile
(20 ml), were added powdered potassium carbonate (5.0 mequiv) at 0 oC, and stirred
the resulting solution for 15 min at 0 oC then p-substituted phenacyl bromide
derivatives (1.0 mequiv) were added at 0 oC. The reaction mixture was stirred at room
temperature for about 8-10 h, progress of reaction was monitored by TLC using 5%
methanol in DCM. After completion of reaction the reaction mixture was diluted with
distilled water (50 ml) and extracted with DCM (3×50 ml), combined organic layer
was washed with aqueous solution of sodium bicarbonate (3×50 ml), distilled water
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(3×50 ml), brine solution (3×50 ml) and dried over anhydrous sodium sulphate and
concentrate it under reduced pressure, obtained crude products. which was purified by
column chromatography using silica gel (60-120 mesh) as adsorbent with DCM:
MeOH (99:1 to 95:5) as an eluent to obtain the pure compounds in 62–71% yields.
All the synthesized compounds were characterized with the help of their IR, MS, and
NMR (1H &
13C NMR) spectral data.
2.3. Synthesis of derivatives
2.3.1. Synthesis of 1-(4-bromophenyl)-2-(4-methylpiperazin-1-yl)ethanone
(Pz 1)
The general synthetic procedure described earlier, afforded compound Pz 1
from N-methyl piperazine (1.0 g, 9.9 mmol, 1.0 mequiv) with p-bromophenacyl
bromide (2.7 g, 9.9 mmol, 1.0 mequiv) and powdered potassium carbonate (6.8 g,
49.9 mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
NNH3C
Br
O
1-(4-bromophenyl)-2-(4-methylpiperazin-1-yl)ethanone
Molecular Formula: C13H17BrN2O (M.W. 296.19); Physical state: solid; Colour:
yellow; Yield: 64%; Purity: 95%; mp: 81-83 oC; MS: [M
+1] & [M
+1+2], m/z 297.07
& m/z 299.09; IR υmax: 2938, 2799, 2696, 2475, 1693, 1513, 1376, 1172, 810 cm-1; 1H
NMR (500 MHz, CDCl3): δ 7.87 (d, 2H, J = 8.5 Hz, Ar-H), 7.60 (d, 2H, J = 8.5 Hz,
Ar-H), 3.77 (s, 2H, N-CH2), 3.07 (brs, 2H, piperazine- H), 2.64 (brd, 3H, piperazine-
H), 2.56 (brs, 3H, piperazine-H), 2.32 (s, 3H, N-CH3); 13
C NMR (125 MHz, CDCl3):
δ 195.3 (C=O), 134.4 (CAr), 131.6 (2 × CHAr), 129.5 (2 × CHAr), 128.3 (CAr), 64.3 (N-
CH2), 54.5 (2 × piperazine-CH2), 53.0 (2 × piperazine-CH2), 45.5 (N-CH3).
2.3.2. Synthesis of 2-(4-methylpiperazin-1-yl)-1-p-tolylethanone (Pz 2)
The general synthetic procedure described earlier, afforded compound Pz 2
from N-methyl piperazine (1.0 g, 9.9 mmol, 1.0 mequiv) with p-methylphenacyl
bromide (2.1 g, 9.9 mmol, 1.0 mequiv) and powderd potassium carbonate (6.8 g, 49.9
mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
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NNH3C
CH3
O
2-(4-methylpiperazin-1-yl)-1-p-tolylethanone
Molecular Formula: C14H20N2O (M.W. 232.32); Physical state: solid; Colour:
yellow; Yield: 68%; Purity: 91%; mp: 108-110 oC; MS: [M
+1], m/z 233.23; IR υmax:
2947, 2785, 2680, 2477, 1695, 1606, 1455, 1376, 1284 cm-1;
1H NMR (500 MHz,
CDCl3): δ 7.84 (d, 1H, J = 8.0 Hz, Ar-H), 7.06 (d, 3H, J = 11.0 Hz, Ar-H), 3.88 (brs,
2H, piperazine-H), 3.48 (s, 2H, N-CH2), 3.13 (brs, 2H, piperazine-H), 2.98 (brs, 2H,
piperazine-H), 2.42 (brs, 2H, piperazine-H), 2.20 (s, 3H, N-CH3), 2.18 (s, 3H, CH3);
13C NMR (125 MHz, CDCl3,): δ 195.6 (C=0), 144.5 (CAr), 132.6 (CAr), 129.3 (2 ×
CHAr), 127.8 (2 × CHAr), 63.9 (N-CH2), 54.3 (2 × piperazine-CH2), 52.8 (2 ×
piperazine-CH2), 45.5 (N-CH3), 21.6 (CH3).
2.3.3. Synthesis of 1-(4-methoxyphenyl)-2-(4-methylpiperazin-1-yl)
ethanone (Pz 3)
The general synthetic procedure described earlier, afforded compound Pz 3
from N-methyl piperazine (1.0 g, 9.9 mmol, 1.0 mequiv) with p-methoxyphenacyl
bromide (2.2 g, 9.9 mmol, 1.0 mequiv) and powderd potassium carbonate (6.8 g, 49.9
mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
NNH3C
OCH3
O
1-(4-methoxyphenyl)-2-(4-methylpiperazin-1-yl)ethanone
Molecular Formula: C14H20N2O2 (M.W. 248.32 ); Physical state: solid; Colour:
yellow; Yield: 70%; Purity: 96%; mp: 88-92 0C MS: [M
+1], m/z 249.36; IR υmax:
2947, 2875, 2799, 2686, 2471, 1690, 1460, 1284, 1147 cm-1;
1H NMR (500 MHz,
CDCl3): δ 7.96 (d, 2H, J = 7.0 Hz, Ar-H), 6.92 (d, 2H, J = 7.0 Hz, Ar-H), 3.85 (s, 3H,
OCH3), 3.77 (s, 2H, N-CH2), 2.64 (brd, 8H, piperazine-H), 2.34 (s, 3H, N-CH3); 13
C
NMR (125 MHz, CDCl3): δ 194.5 (C=O), 163.3 (CAr), 130.1 (2 × CHAr), 128.8 (CAr),
113.4 (2 × CHAr), 63.8 (N-CH2), 55.2 (OCH3), 54.3 (2 × piperazine-CH2), 52.8 (2 ×
piperazine-CH2), 45.3 (N-CH3).
2.3.4. Synthesis of 1-(4-bromophenyl)-2-(4-phenylpiperazin-1-yl)ethanone
(Pz 4)
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The general synthetic procedure described earlier, afforded compound Pz 4
from N-phenyl piperazine (1.0 g, 6.1 mmol, 1.0 mequiv) with p-bromophenacyl
bromide (1.7 g, 6.1 mmol, 1.0 mequiv) and powderd potassium carbonate (4.2 g, 30.8
mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
NN
Br
O
1-(4-bromophenyl)-2-(4-phenylpiperazin-1-yl)ethanone
Molecular Formula: C18H19BrN2O (M.W. 358.07); Physical state: solid; Colour:
off white; Yield: 62%; Purity: 97%; mp: 132-134 oC; MS: [M
+1] & [M
+1+2], m/z
359.18 & m/z 361.11; IR υmax: 3085, 3027, 2937, 2813, 2770, 1697, 1585, 1397,
1216, 987, 817 cm-1;
1H NMR (500 MHz, CDCl3): δ 7.92 (d, 2H, J = 8.5 Hz, Ar-H),
7.62 (d, 2H, J = 8.5 Hz, Ar-H), 7.25- 7.29 (m, 2H, Ar-H), 6.94 (d, 2H, J = 8.0 Hz, Ar-
H), 6.87 (t, 1H, J = 7.5 Hz, Ar-H), 3.83 (s, 2H, N-CH2), 3.27 (t, 4H, J = 5.0 Hz,
piperazine-H), 2.77 (t, 4H, J = 5.0 Hz, piperazine-H); 13
C NMR (125 MHz, CDCl3):
δ 195.4 (C=O), 151.2 (N-CAr), 134.6 (CAr), 131.9 (2 × CHAr), 129.8 (2 × CHAr), 129.1
(2 × CHAr), 128.5 (CAr), 119.8 (CHAr), 116.1 (2 × CHAr), 64.6 (N-CH2), 53.5 (2 ×
piperazine-CH2), 49.0 (2 × piperazine-CH2).
2.3.5. Synthesis of 2-(4-phenylpiperazin-1-yl)-1-p-tolylethanone (Pz 5)
The general synthetic procedure described earlier, afforded compound Pz 5
from N-phenyl piperazine (1.0 g, 6.1 mmol, 1.0 mequiv) with p-methylphenacyl
bromide (1.3 g, 6.1 mmol, 1.0 mequiv) and powderd potassium carbonate (4.2 g, 30.8
mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
NN
CH3
O
2-(4-phenylpiperazin-1-yl)-1-p-tolylethanone
Molecular Formula: C19H22N2O (M.W. 294.39); Physical state: solid; Colour: off
white; Yield: 67%; Purity: 97%; mp: 125-128 oC; MS: [M
+1], m/z 295.63; IR υmax:
3062, 3027, 2912, 2880, 2770, 1682, 1568, 1455, 1170, 978, 752 cm-1;
1H NMR (500
MHz, CDCl3): δ 7.94 (d, 2H, J = 8.5 Hz, Ar-H), 7.27- 7.30 (m, 4H, Ar-H), 6.96 (d,
2H, J = 8.0 Hz, Ar-H), 6.88 (t, 1H, J = 7.5 Hz, Ar-H), 3.88 (s, 2H, N-CH2), 3.30 (t,
4H, J = 5.0 Hz, piperazine-H), 2.80 (t, 4H, J = 5.0 Hz, piperazine-H), 2.44 (s, 3H,
CH3); 13
C NMR (125 MHz, CDCl3): δ 195.7 (C=O), 151.2 (N-CAr), 143.5 (CAr),
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133.5 (CAr), 129.2 (2 × CHAr), 129.1 (2 × CHAr), 128.2 (2 × CHAr), 119.8 (CHAr),
116.2 (2 × CHAr), 64.2 (N-CH2), 53.5 (2 × piperazine-CH2), 49.0 (2 × piperazine-
CH2), 21.7 (CH3).
2.3.6. Synthesis of 1-(4-methoxyphenyl)-2-(4-phenylpiperazin-1-yl)
ethanone (Pz 6)
The general synthetic procedure described earlier, afforded compound Pz 6
from N-phenyl piperazine (1.0 g, 6.1 mmol, 1.0 mequiv) with p-methoxyphenacyl
bromide (1.4 g, 6.1 mmol, 1.0 mequiv) and powderd potassium carbonate (4.2 g, 30.8
mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
NN
OCH3
O
1-(4-methoxyphenyl)-2-(4-phenylpiperazin-1-yl)ethanone
Molecular Formula: C19H22N2O2 (M.W. 310.39); Physical state: solid; Colour: off
white; Yield: 66%; Purity: 95%; mp: 156-158 oC; MS: [M
+1], m/z 311.29; IR υmax:
3085, 2936, 2812, 2701, 1693, 1607, 1455, 1295, 1011, 810 cm-1;
1H NMR (500
MHz, CDCl3): δ 8.02 (d, 2H, J = 7.0 Hz, Ar-H), 7.26 (m, 2H, Ar-H), 6.93 (d, 4H, J =
9.0 Hz, Ar-H), 6.85 (t, 1H, J = 7.5 Hz, Ar-H), 3.82 (s, 2H, N-CH2), 3.27 (t, 4H, J =
5.0 Hz, piperazine-H), 2.87 (s, 3H, OCH3), 2.76 (t, 4H, J = 5.0 Hz, piperazine-H);
13C NMR (125 MHz, CDCl3): δ 194.6 (C=O), 163.4 (CAr), 151.1 (N-CAr), 130.3 (2 ×
CHAr), 128.9 (3 × CHAr), 119.5 (CHAr), 115.9 (2 × CHAr), 113.5 (2 × CHAr), 64.1 (N-
CH2), 55.3 (OCH3), 53.4 (2 × piperazine-CH2), 48.8 (2 × piperazine-CH2).
2.3.7. Synthesis of 2-(4-benzylpiperazin-1-yl)-1-(4-bromophenyl)ethanone
(Pz 7)
The general synthetic procedure described earlier, afforded compound Pz 7
from N-benzylpiperazine (1.0 g, 5.6 mmol, 1.0 mequiv) with p-bromophenacyl
bromide (1.5 g, 5.6 mmol, 1.0 mequiv) and powderd potassium carbonate (3.9 g, 28.3
mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
N
N
O
Br
2-(4-benzylpiperazin-1-yl)-1-(4-bromophenyl)ethanone
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Molecular Formula: C19H21BrN2O (M.W. 372.29); Physical state: solid; Colour:
yellow; Yield: 71%; Purity: 98%; mp: 142-144 oC; MS: [M
+1] & [M
+1+2], m/z
373.22 & m/z 375.11; IR υmax: 3062, 2937, 2813, 2700, 1697, 1585, 1397, 1216, 978,
817 cm-1;
1H NMR (500 MHz, CDCl3): δ 7.89 (d, 2H, J = 8.5 Hz, Ar-H), 7.59 (d,
2H, J = 8.5 Hz, Ar-H), 7.25- 7.32 (m, 5H, Ar-H), 3.75 (s, 3H, N-CH2-Ph), 3.53 (s, 2H,
N-CH2-C=O), 2.59 (brd, 8H, piperazine-H); 13
C NMR (125 MHz, CDCl3): δ 195.4
(C=O), 137.6 (CAr), 134.5 (CAr), 131.6 (2 × CHAr), 129.6 (2 × CHAr), 129.0 (2 ×
CHAr), 128.2 (CAr), 128.0 (2 × CHAr), 126.9 (CHAr), 64.1 (N-CH2-C=O), 62.7 (N-
CH2-Ph), 53.2 (2 × piperazine-CH2), 52.6 (2 × piperazine-CH2).
2.3.8. Synthesis of 2-(4-benzylpiperazin-1-yl)-1-p-tolylethanone (Pz 8)
The general synthetic procedure described earlier, afforded compound Pz 8
from N-benzylpiperazine (1.0 g, 5.6 mmol, 1.0 mequiv) with p-methylphenacyl
bromide (1.2 g, 5.6 mmol, 1.0 mequiv) and powerd potassium carbonate (3.9 g, 28.3
mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
N
N
O
CH3
2-(4-benzylpiperazin-1-yl)-1-p-tolylethanone
Molecular Formula: C20H24N2O (M.W. 308.42); Physical state: solid; Colour:
yellow; Yield: 68%; Purity: 94%; mp: 131-135 0C: MS: [M
+1], m/z 309.31; IR υmax:
3085, 3028, 2936, 2812, 2769, 1693, 1607, 1455, 1170, 975, 746 cm-1;
1H NMR (500
MHz, CDCl3): δ 7.86 (d, 2H, J = 8.0 Hz, Ar-H), 7.28- 7.33 (m, 4H, Ar-H), 7.21-7.26
(m, 3H, Ar-H), 3.80 (s, 2H, N-CH2-Ph), 3.59 (s, 2H, N-CH2-C=O), 2.64 (brd, 8H,
piperazine-H), 2.38 (s, 3H, CH3); 13
C NMR (125 MHz, CDCl3): δ 196.3 (C=O),
144.7 (CAr), 137.5 (CAr), 134.1 (CAr), 130.1 (2 × CHAr), 129.8 (2 × CHAr), 128.9 (2 ×
CHAr), 128.7 (2 × CHAr), 127.9 (CHAr), 64.6 (N-CH2-C=O), 63.2 (N-CH2-Ph), 53.6 (2
× piperazine-CH2), 53.1 (2 × piperazine-CH2), 22.2 (CH3).
2.3.9. Synthesis of 2-(4-benzylpiperazin-1-yl)-1-(4-methoxyphenyl)
ethanone (Pz 9)
The general synthetic procedure described earlier, afforded compound Pz 9
from N-benzylpiperazine (1.0 g, 5.6 mmol, 1.0 mequiv) with p-methoxyphenacyl
bromide (1.2 g, 5.6 mmol, 1.0 mequiv) and powderd potassium carbonate (3.9 g, 28.3
mmol, 5.0 mequiv) in dry acetonitrile (20 ml) as a reaction solvent.
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N
N
O
OCH3
2-(4-benzylpiperazin-1-yl)-1-(4-methoxyphenyl) ethanone
Molecular Formula: C20H24N2O2 (M.W. 324.42); Physical state: solid; Colour:
yellow; Yield: 64%; Purity: 96%; mp: 124-128 0C; MS: [M
+1]: m/z 325.29; IR υmax:
3085, 3027, 2937, 2813, 1684, 1585, 1455, 1295, 1170, 978, 752 cm-1;
1H NMR (500
MHz, CDCl3): δ 7.97 (d, 2H, J = 8.0 Hz, Ar-H), 7.29-7.32 (m, 5H, Ar-H), 6.92 (d,
2H, J = 7.0 Hz, Ar-H), 3.84 (s, 3H, OCH3), 3.77 (s, 2H, N-CH2-Ph), 3.59 (s, 2H, N-
CH2-C=O), 2.64 (brd, 8H, piperazine-H); 13
C NMR (125 MHz, CDCl3): δ 194.6
(C=O), 163.4 (CAr), 136.8 (CAr), 130.2 (2 × CHAr), 129.3 (2 × CHAr), 128.9 (CAr),
128.1 (2 × CHAr), 127.1 (CHAr), 113.5 (2 × CHAr), 63.9 (N-CH2-C=O), 62.5 (N-CH2-
Ph), 55.3 (OCH3), 53.0 (2 × piperazine-CH2), 52.4 (2 × piperazine-CH2).
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Figure 2.1. 1H NMR spectrum of Pz 1
Figure 2.2. 13
C NMR spectrum of Pz 1
NNH3C
Br
O
NNH3C
Br
O
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Figure 2.3. 1H NMR spectrum of Pz 2
Figure 2.4. 13
C NMR spectrum of Pz 2
NNH3C
CH3
O
NNH3C
CH3
O
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Figure 2.5. 1H NMR spectrum of Pz 3
Figure 2.6. 13
C NMR spectrum of Pz 3
NNH3C
OCH3
O
NNH3C
OCH3
O
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Figure 2.7. 1H NMR spectrum of Pz 4
Figure 2.8. 13
C NMR spectrum of Pz 4
NN
Br
O
NN
Br
O
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Figure 2.9. 1H NMR spectrum of Pz 5
Figure 2.10. 13
C NMR spectrum of Pz 5
NN
CH3
O
NN
CH3
O
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Figure 2.11. 1H NMR spectrum of Pz 6
Figure 2.12. 13
C NMR spectrum of Pz 6
NN
OCH3
O
NN
OCH3
O
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Figure 2.13. 1H NMR spectrum of Pz 7
Figure 2.14. 13
C NMR spectrum of Pz 7
N
N
O
Br
N
N
O
Br
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Figure 2.15. 1H NMR spectrum of Pz 8
Figure 2.16. 13
C NMR spectrum of Pz 8
N
N
O
CH3
N
N
O
CH3
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Figure 2.17. 1H NMR spectrum of Pz 9
Figure 2.18. 13
C NMR spectrum of Pz 9
N
N
O
OCH3
N
N
O
OCH3
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2.4. Results and discussion
The nucleophilic substitution reactions of N-alkyl and N-aryl substituted
piperazine with different phenacyl bromide derivatives were carried out in presence of
mild base potassium carbonate in different solvents (N,N-dimethylformamide,
ethanol, acetone and acetonitrile) to improve the yields.28-32
Among the different
solvents used acetonitrile showed a better nucleophilic substitution reaction for the
series of compounds Pz 1 to Pz 9 [Table 2.1].
Table 2.1. Effect of solvent on the yield (%) of reaction
% Yields in different solvents used Compounds
N,N-
Dimethylformamide
Acetone Ethenol Acetonitrile
Pz 1 9% < 5% < 5% 64%
Pz 2 13% < 5% < 5% 68%
Pz 3 10% < 5% < 5% 70%
Pz 4 15% < 5% < 5% 62%
Pz 5 20% < 5% < 5% 67%
Pz 6 16% < 5% < 5% 62%
Pz 7 20% < 5% < 5% 71%
Pz 8 15% < 5% < 5% 68%
Pz 9 15% < 5% < 5% 64%
The nucleophilic substitution reaction of 1.0 mequiv of N-methyl piperazine,
N-phenyl piperazine and N-benzyl piperazine with 1.1 mequiv of 2-bromo-1-(4-
bromophenyl)ethanone, 2-bromo-1-p-tolyl ethanone, 2-bromo-1-(4-methoxyphenyl)-
ethanone and 5.0 equiv of potassium carbonate in acetonitrile at 0 oC to 25
oC for 8-10
h gave 1-(4-bromophenyl)-2-(4-methylpiperazin-1-yl)ethanone (Pz 1), 2-(4-methyl-
piperazin-1-yl)-1-p-tolylethanone (Pz 2), 1-(4-methoxyphenyl)-2-(4-methylpiperazin-
1-yl)ethanone (Pz 3), 1-(4-bromophenyl)-2-(4-phenylpiperazin-1-yl)ethanone (Pz 4),
2-(4-phenylpiperazin-1-yl)-1-p-tolylethanone (Pz 5), 1-(4-methoxyphenyl)-2-(4-
phenylpiperazin-1-yl)ethanone (Pz 6), 2-(4-benzylpiperazin-1-yl)-1-(4-bromo-
phenyl)ethanone (Pz 7), 2-(4-benzylpiperazin-1-yl)-1-p-tolylethanone (Pz 8), 2-(4-
benzylpiperazin-1-yl)-1-(4-methoxyphenyl)ethanone (Pz 9) respectively with 62-71%
yields. Purity of all the synthesized compounds were checked by TLC and HPLC. The
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reaction conditions and physicochemical properties of the compounds are summerized
in Table 2.2.
Table 2.2. Physicochemical properties of piperazine derivatives
Comp Structures Reaction
conditions
Yield
(%)
Melting
point
Physical
state
Purity
(%)
Pz 1
K2CO3,
CH3CN, rt, 8 h
64%
81-83 oC
yellow solid
95 %
Pz 2
K2CO3,
CH3CN, rt, 10h
68%
108-110 oC
yellow solid
97 %
Pz 3
K2CO3,
CH3CN, rt, 10h
70%
88-92 oC
yellow solid
96 %
Pz 4
K2CO3,
CH3CN, rt, 10h
62%
132-134 oC
off white
solid
97 %
Pz 5
K2CO3,
CH3CN, rt, 9 h
67%
125-128 oC
off white
solid
97 %
Pz 6
K2CO3,
CH3CN, rt, 10h
62%
156-158 oC
off white
solid
95 %
Pz 7
K2CO3,
CH3CN, rt, 9h
71%
142-144 oC
yellow solid
98 %
Pz 8
K2CO3,
CH3CN, rt, 10h
68%
131-135 oC
yellow solid
94 %
Pz 9
K2CO3,
CH3CN, rt, 10h
64%
124-128 oC
yellow solid
96 %
Formation of the products was confirmed by the absence of –NH stretching
absorption at 3300-3400 cm-1 and presence of stretching absorption at 1680-1700 cm
-1
for >C=O group in IR spectrum, which was further confirmed by the MS and NMR
spectrum (1H &
13C NMR) of respective compounds. The compound Pz 1 was
N NH3C
O
Br
N NH3C
O
CH3
N NH3C
O
OCH3
N N
O
Br
N N
O
OCH3
N N
O
CH3
N
N
O
Br
N
N
O
CH3
N
N
O
OCH3
Estelar
43
obtained as yellow amorphous solid. The mass of the compound displayed a
molecular ion peak at m/z 297.07 [M+1] & 299.09 [M
+1+2] corresponding to the
molecular formula C13H17BrN2O. A stretching absorption at 1693 cm-1 in IR spectrum
of compound revealed the presence of >C=O group which is confirmed by the
presence of 13C resonance at δ 195.3 in
13C NMR. Broad singlet at δ 2.56 for 3H, at δ
2.64 for 3H and at δ 3.07 for 2H showed the presence of piperazine nucleus, which is
also, supported by the 13C resonance at δ 53.0 and 54.5 in
13C NMR. A sharp singlet
at δ 2.32 in 1H NMR spectrum of compound revealed the presence of N-CH3 group.
Presence of methylene group was confirmed by the presence of singlet for two
protons at δ 3.77 in 1H NMR and
13C resonance at δ 64.3 in
13C NMR spectrum.
Doublet at δ 7.60 (d, 2H, J = 8.5 Hz) and at δ 7.87 (d, 2H, J = 8.5 Hz) confirmed the
presence of para substituted phenyl aromatic ring, which was also, supported by the
13C resonance at δ 128.3 & 134.4 for quaternary aromatic carbon, at δ 131.6 & 129.5
for four CH aromatic carbon. Similarly, all the other analogues where characterized
by their spectroscopic studies.
2.5. Antibacterial activity of piperazine derivatives
2.5.1. Bacterial culture
Standard pure cultures of bacteira were procured from the Institute of
Microbial Technology (IMTECH), Chandigarh, India as microbial type culture
collection (MTCC) and maintained in the laboratory by regular sub-culturing on the
nutrient agar. Antimicrobial assays were performed with four Gram positive
(Streptococcus mutans MTCC- 890, Staphylococcus aureus MTCC- 96, Bacillus
subtilis MTCC- 121, Staphylococcus epidermidis MTCC- 435) and one Gram
negative (Escherichia coli MTCC- 723) bacterial strains.
2.5.2. Zone of inhibition
A standarized filter paper disc-agar diffusion procedure known as Kirby-
Bauer method was used to determine the drug susceptibility of microorganism33. The
antibiotic impregnated disc absorbes moisture from agar and diffuses into the agar
medium. The rate of extraction of antibiotic from disc is greater than the rate of
diffusion. Visible growth of the bacteria occurs on the surface of agar where antibiotic
concentration has fallen below its inhibitory level for the test strain. The point at
which the sritical cell mass is reached appears as a circle of bacterial growth, with the
middle of the antibiotic disc forming centre of the circle.
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44
The zones of inhibition of the synthesized compounds were determined by the
paper disc diffusion method, performed in sterilized (autoclaved at 120 oC for 1 h)
petri dish. Bacterial inoculums were prepared from over night (16 hrs) grown culture
in nutrient broth (Himedia, India) and the turbidity was adjusted equivalent to 0.5
McFarland standard (approximetly 3 × 106 cfu ml
-1 bacteria). An aliquot (100 µl) of
cultures were spread over the surface of agar plate with sterile glass spreaded.
Compound with 100 µg/ disc concentration were impregnated on the paper disces
(diameter 5 mm, Whatman filter paper No. 3), which were placed on the surface of
agar plates already inoculated with pathogenic bacteria. The plates were incubated at
37 oC and examined after 24 h for zone of inhibition. Ampicillin/ streptomycin were
used as a standard. An additional control disc with an equivalent amount of solvent
(DMSO) was also used in the assay. The results showed that some of the compounds
exhibited significant zones.
2.5.3. Minimum inhibitory concentration
Minimum inhibitory concentrations (MICs) are considered as the ‘gold
standard’ for determining the susceptibility of organisms to antimicrobial and are
therefore used to judge the performance of all other methods of susceptibility testing.
The minimum inhibitory concentration was detrmined by the well established
microbroth dilution technique.34 The range of antibiotic concentrations used for
determining MICs is universally accepted to be in doubling dilution step up and down
for 1 mg/ L is required. Various concentrations of compounds were prepared in the
well by two-fold dilution method. The last well of micro titer plate was considered as
control, contained no test compounds. The inoculum was prepared using a 16 h broth
culture of each bacterial strains adjusted to a turbidity equivalent to a 0.5 Mc Farland
standard. The micro titer plates were incubated for 24 h at 37 oC. The MIC was
defined as the lowest concentration of compound giving complete inhibition of visible
growth.
2.5.4. Results and discussion
Antibacterial activity of all the synthesized derivatives were evaluated against
four Gram positive (S. mutans MTCC- 890, S. aureus MTCC- 96, B. subtilis MTCC-
121, S. epidermidis MTCC- 435) and one Gram negative (E. coli MTCC- 723)
bacterial strains using disc diffusion33 and microbroth dilution methods.
34 Ampicillin
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45
was used as a standard drug. The inhibition zone (IZ) and MIC of the compounds
against above bacterial strain are summerized in Table 2.3.
Results showed that most of the compounds possess potent to moderate
activity as compared to the reference drug ampicillin. Benzyl piperazine analougues
(Pz 7 to Pz 9) showed potent activity against Gram positive and significant activity
against Gram negative bacterial strains. Compound Pz 9 having benzyl group in
piperazine and methoxy group in phenacyl nucleus exhibited potent activity against
B. subtilis (IZ = 31 mm and MIC = 5.8 µg/ml), S. epidermidis (IZ = 30 mm and MIC
= 5.8 µg/ml) and S. aureus (IZ = 28 mm and MIC = 11.7 µg/ml) while significant
activity against S. mutans (IZ = 29 mm and MIC = 11.7 µg/ml) and E. coli (IZ = 25
mm and MIC = 23.4 µg/ml). When methoxy group was replaced by methyl group
(Pz 8) the activity was slightly enhanced for S. mutans (IZ = 30 mm and MIC = 5.8
µg/ml) while it was diminished for S. aureus (IZ = 26 mm and MIC = 11.7 µg/ml)
S. epidermidis (IZ = 27 mm and MIC = 11.7µg/ ml) and B. subtilis (IZ = 25 mm and
MIC = 23.4 µg/ml). Compound Pz 7 showed significant activity against only
S. epidermidis with IZ 28 mm and MIC value 7.8 µg/ ml while moderate activity
were noticed against other strains. Methyl piperazine analogues Pz 3 having methoxy
group in phenacyl nucleus was found to be active against S. aureus, B. subtilis,
S. epidermidis and E. coli bacterial strains with MIC value 23.4 to 46.8 µg/ml. All the
phenyl substituted analogues Pz 4 to Pz 6 showed poor activity against both Gram
positive and Gram negative bacterial strains. Compounds Pz 3, Pz 7, Pz 8 and Pz 9
revealed better activity in comparison to other compounds used in study, indicating
that methyl and benzyl substitution at 4-position of piperazine nucleus showed better
activity as compared to phenyl substitution. Enhanced activity of compound Pz 9 and
Pz 3 might be due to the presence of methoxy group at para position of phenacyl
nucleus.
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46
Tab
le 2
.3. A
nti
bacte
rial acti
vit
y o
f p
ipera
zin
e d
eriv
ati
ves
Pz
1 t
o P
z 9
Zone of inhibition (mm) and Minimum inhibitory concentrations (µg ml-1)
Streptococcus mutans
Staphylococcus aureus
Bacillus subtilis
Staphylococcus epidermidis
Escherichia coli
*Compounds
zone of
inhibition
MIC
zone of
inhibition
MIC
zone of
inhibition
MIC
zone of
inhibition
MIC
zone of
inhibition
MIC
Pz 1
12± 0.53
250
18 ± 0.72
62.5
18 ± 0.72 62.5
17 ± 0.81
62.5
15 ± 0.66 125
Pz 2
NA
ND
10 ± 0.44
500
8 ± 0.34
500
NA
ND
NA
ND
Pz 3
22 ± 1.03
46.8
26 ± 1.18
23.4
25 ± 1.12 23.4
24 ± 0.98
23.4
23 ± 0.87 46.8
Pz 4
7 ± 0.29
500
7 ± 0.28
500
14 ± 0.64 125
13 ± 0.58
250
11 ± 0.52 250
Pz 5
9 ± 0.37
500
12 ± 0.53
250
12 ± 0.49 250
11 ± 0.61
250
10 ± 0.44 500
Pz 6
12 ± 0.46
250
16 ± 0.76
125
13 ± 0.58 125
17 ± 0.81
62.5
NA
ND
Pz 7
18 ± 0.86
62.5
20 ± 0.89
31.2
26 ± 1.22 15.6
28 ± 1.26
7.8
22 ± 0.98 31.2
Pz 8
30 ± 1.29
5.8
26 ± 1.18
11.7
25 ± 1.12 23.4
27 ± 1.22
11.7
19 ± 0.91 93.7
Pz 9
29 ± 1.26
11.7
28 ± 1.26
11.7
31 ± 1.36
5.8
30 ± 1.32
5.8
25 ± 1.07
23.4
Ampicillin**
27± 1.21
4
22 ± 0.94
8
25 ± 1.12
4
25 ± 1.07
2
12 ± 0.49
12
values are mean of three determinations, the ranges of which are less than 5% of the mean in all cases.
*compounds (100µg/disc) were used for experiments; NA = not active; ND = not determined.
**used as positive reference (20µg/disc).
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47
2.6. Structures of potent compounds
N
N
O
Br
N
N
O
CH3
N
N
O
OCH3
N NH3C
O
OCH3
Pz 03 Pz 07
Pz 08 Pz 09
2.7. Conclusion
In conclusion, the synthesis of nine new N-substituted piperazine analogues
was carried out and their antibacterial activity has been determined. Among the
synthetic piperizine derivatives under investigation, Pz 3, Pz 7, Pz 8 and Pz 9 showed
significant activity. The benzyl piperazine derivatives Pz 8 & Pz 9 viz. 2-(4-
benzylpiperazin-1-yl)-1-p-tolylethanone Pz 8 and 2-(4-benzylpiperazin-1-yl)-1-(4-
methoxyphenyl)ethanone Pz 9, in particular, showed remarkable antibacterial activity
even at low concentration against S. epidermidis, S. mutans and B. subtilis which were
closer to ampicillin. Furthermore, benzyl substitution increased the antibacterial
activity as compared to the methyl and phenyl substituents under identical conditions
and might be of interest for developing new antibacterial molecules.
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48
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