Synthesis and antimicrobial activity of some new N-acyl substituted phenothiazines

Article abstract of DOI:10.1016/j.ejmech.2009.07.006

A series of 2-substituted N-acylphenothiazines were synthesized by using imides, N-carboxymethyl imides and the structures of these newly synthesized compounds were confirmed by spectral and elemental analyses. All new compounds were tested for their antibacterial and antifungal activities. Some compounds showed promising antibacterial and antifungal activities.

Full text of DOI:10.1016/j.ejmech.2009.07.006

European Journal of Medicinal Chemistry 44 (2009) 5094–5098
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antimicrobial activity of some new N-acyl substituted
phenothiazines
*
Tanaji N. Bansode, Jayant V. Shelke, Vaijanath G. Dongre
Department of Chemistry, University of Mumbai, Vidyanagari, Kalina, Santacruz (East), Mumbai 400 098, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-substituted N-acylphenothiazines were synthesized by using imides, N-carboxymethyl
imides and the structures of these newly synthesized compounds were confirmed by spectral and
elemental analyses. All new compounds were tested for their antibacterial and antifungal activities. Some
compounds showed promising antibacterial and antifungal activities.
Received 16 April 2009
Received in revised form
4 June 2009
Accepted 9 July 2009
Available online 16 July 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Phenothiazines
Phthalimide
Succinimide
Maleimide
N-carboxymethyl imides
Antimicrobial activity
1. Introduction
systems connected to the N-acylphenothiazine unit are rarely
studied even though N-acylated models equipped with 1,4-ben-
Phenothiazines have found widespread use in medicinal
chemistry. Phenothiazine and related compounds have been
reported to possess various diverse biological activities including
tranquilizers [1], anti-inflammatory [2], antimalarial [3], anti-
psychotropic [4], antimicrobial [5], antitubercular [6–8], antitumor
[9–11], antihistamine [12] and analgesic [13] properties. Pheno-
thiazine derivatives having amino alkyl side chain connected to the
nitrogen atom of heterocyclic unit playing crucial role in medicinal
chemistry are reported in the literature [14,15].
Cyclic imides, such as succinimide, maleimide and phthalimide
possess structural features, which confer potential biological
activity [16] and pharmaceutical use. Their molecules contain an
imide ring and the general structure –CO–N(R)–CO–, so that they
are hydrophobic and neutral, and can therefore cross biological
membranes in vivo. The various classes of cyclic imides have
received attention due to their antibacterial, antifungal, analgesic
[17] and antitumor activities [18,19].
zodioxan [20], furan [21] or benzofuran [22] aromatic units have
shown interesting neurotropic properties. These examples
prompted us to develop a synthetic strategy for N-acylphenothia-
zines using imides and N-carboxyimides and evaluate their anti-
microbial activities.
2. Chemistry
The N-acyl derivatives of phenothiazines 3a–d, 4a–d and 5a–d
(Scheme 1) were synthesized by two different methods. In method
(A), the compounds (2a–d) were treated with different imides in
presence of anhydrous K2CO3 at room temperature and in method
(B), the compounds (1a–d) were treated with chlorides of acids 6, 7,
and 8 in xylene at 120 ꢀC. The yield of the products was found to be
excellent by method (A). The reaction sequences are outlined in
Scheme 1.
As part of our project, we have synthesized some new N-acyl-
phenothiazines with imides and subjected for antimicrobial
activity. To our knowledge, compounds in which such aromatic
3. Result and discussion
Formation of N-acyl derivatives of phenothiazine 3, 4 and 5 was
confirmed on the basis of elemental analysis, IR and NMR. The IR
spectra of compound (3a) did not show absorption in the range
3320–3340 cmꢁ1 due to the absence N–H group, but instead
* Corresponding author. Tel.: þ91 022 2652 60 91; fax: þ91 022 2652 85 47.
E-mail address: dongrevg@yahoo.com (V.G. Dongre).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.006

T.N. Bansode et al. / European Journal of Medicinal Chemistry 44 (2009) 5094–5098
5095
5. Experimental section
H
N
R
All chemicals were purchased from Aldrich and Merck Ltd,
Mumbai (India), and were used without further purification. The
melting points were determined in open capillaries using a Tosh-
niwal melting point apparatus and are uncorrected. The IR spectra
were recorded in the solid state as a KBr suspension on a Perkin–
Elmer spectrum one FT-IR spectrophotometer and 1H NMR spectra
were obtained in CDCl3 on a Brucker 400 MHz instrument using
(1a-d)
S
(i)
O
Cl
R
N
S
TMS as an internal standard (chemical shifts in d, ppm), Mass
spectra on a LCQ Adavantage Thermo Finnigen spectrometer.
Elemental analysis was performed on Carlo Erba 1108 analyzer.
(2a-d)
5.1. General procedure for synthesis of N-(2-chloroacetyl)
phenothiazines [23,24] (2a–d)
(ii)
(iii)
(iv)
N
To the solution of 10H-phenothiazines [25–27] 1a–d, (0.01 mol)
in dry benzene 50 ml, chloroacetyl chloride (0.01 mol) was added
at 0–5 ꢀC, the reaction mixture was refluxed under stirring for
3–4 h at 50–60 ꢀC temperature. The resulting mixture was distilled
off and poured on ice cold water. The solid thus obtained was
recrystallized from ether.
O
O
O
O
O
O
O
O
O
N
N
R
N
S
R
N
R
N
S
5.1.1. Compound 2a
S
White powder, yield 85%, m.p. 100–102 ꢀC, IR (KBr) cmꢁ1: 1693
(3a-d)
(4a-d)
(5a-d)
(C]O), 2952 (CH2), 3067 (Ar–H); 1H NMR (CDCl3):
d 7.08–7.82 (m,
8H, Ar–H), 4.52 (s, 2H). Anal. calcd. for C14H10ClNOS: C, 60.98; H,
3.6; N, 5.08. Found: C, 61.03; H, 3.52; N, 5.19%.
(1a-d)
(v)
(1a-d)
(v)
(1a-d)
(v)
O
O
O
5.1.2. Compound 2b
White powder, yield 63%, m.p. 115–118 ꢀC, IR (KBr) cmꢁ1: 1670
N
N
N
OH
(C]O), 2960 (CH2), 3089 (Ar–H); 1H NMR (CDCl3):
(m, 7H, Ar–H), 4.50 (s, 2H). Anal. calcd. for C14H9Cl2NOS: C, 54.19;
H, 2.90; N, 4.15. Found: C, 53.99; H, 3.12; N, 4.38%.
d 7.32–8.12
OH
OH
O
O
O
O
O
O
8
6
7
Scheme1. Synthetic route of compounds 3a–d, 4a–d and 5a–d. R: a – H, b – Cl, c – CF3,
d – COCH3. Reaction reagents and conditions: (i) ClCH2COCl, C6H6, reflux 60 ꢀC, 74%; (ii)
phthalimide, K2CO3, DMSO, rt 3–4 h, 80%; (iii) succinimide, K2CO3, DMSO, rt, 3–4 h,
82%; (iv) maleimide, K2CO3, DMSO, rt, 3–4 h, 79%; (v) (a) PCl5 in dry C6H6, reflux 80 ꢀC,
1 h, (b) 1a–d in xylene, reflux 120 ꢀC, 2–3 h, 74%.
5.1.3. Compound 2c
Greenish white powder, yield 70%, m.p. 110–112 ꢀC, IR
(KBr) cmꢁ1: 1663 (C]O), 2961 (CH2), 3040 (Ar–H); 1H NMR
(CDCl3):
d 7.14–7.89 (m, 7H, Ar-H), 4.58 (s, 2H). Anal. calcd. for
C15H9ClF3NOS: C, 52.40; H, 2.62; N, 4.07. Found: C, 52.59; H, 2.68; N,
4.13%.
showed new peak at 1698 cmꢁ1 due to amide and at 1769 cmꢁ1 due
to cyclic C]O stretch.
5.1.4. Compound 2d
The 1H NMR spectrum of 3a showed a singlet at
d
4.50 due to the
CH2 protons. It showed two multiplets resonating at (7.88–7.71)
and (7.60–7.32) due to phthalimide and phenothiazine ring
protons respectively. In case of 3d, 4d and 5d additional singlet at
Faint Green powder, yield 74%, m.p. 136–140 ꢀC, IR (KBr) cmꢁ1
:
d
1666, 1681 (C]O), 2958 (CH2), 2989 (CH3), 3059 (Ar–H); 1H NMR
(CDCl3): 7.02–7.35 (m, 7H, Ar–H), 4.48 (s, 2H), 2.28 (s, 3H). Anal.
d
d
calcd. for C16H12ClNO2S: C, 60.47; H, 3.77; N, 4.40. Found: C, 60.31;
H, 3.52; N, 4.48%.
d
2.44–2.48 showed due to acetyl protons.
Further evidence for the formation of compound (3a) was
obtained by recording the mass spectra. The mass spectrum of
compound 3a showed a molecular ion peak at m/z 386 which is in
conformity with the molecular formula C22H14N2O3S. The other
fragmentation peaks are observed at 200 (26%), 188 (60%) and 160
(86%). The characterization data of N-acyl derivatives of pheno-
thiazine 3a–d, 4a–d and 5a–d are given in Table 1.
5.2. General procedure for synthesis of compounds (3a–d,
4a–d and 5a–d)
Method (A). A mixture of N-(2-Chloroacetyl) phenothiazines
2a–d (0.125 mmol), phthalimide (0.125 mmol) and anhydrous
K2CO3 (0.125 mmol) in 20 ml DMSO was stirred at room tempera-
ture for 4–5 h. The reaction mixture was poured on water and crude
precipitate was filtered to afford 3a–d.
4. Conclusion
Similarly, compounds 4a–d and 5a–d were synthesized by using
succinimide and maleimide respectively.
In conclusion, we have developed a simple and efficient method
for the synthesis of N-acyl substituted phenothiazine derivatives.
The antibacterial and antifungal activities were observed in all the
tested compounds. Four compounds showed good bacterial inhi-
bition almost equivalent to standard drug. As far as the antifungal
screening results are concerned all compounds showed moderate
to good activity against all fungal strains.
Method (B). PCl5 (0.255 mmol) was added to 2-(1,3-dioxoi-
soindolin-2yl) acetic acid (6) (0.250 mmol) in dry benzene 20 ml
and refluxed for 1 h at 70–80 ꢀC, the solvent was removed under
vacuum. Then phenothiazines 1a–d (0.125 mmol) in dry xylene
(10 ml) was added to reaction mixture. Then reaction mixture was
refluxed further for 2–3 h at 120 ꢀC. The reaction mixture was kept

5096
T.N. Bansode et al. / European Journal of Medicinal Chemistry 44 (2009) 5094–5098
Table 1
Physical data of compounds 3a–d, 4a–d and 5a–d.
Compound no.
R
Mol. formula
M.p. (ꢀC)
Yield (%)
Method
(a)
Analysis (%)
Calculated (found)
C
(b)
H
N
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
H
Cl
CF3
Ac
H
C22H14N2O3S
C22H13ClN2O3S
C23H13F3N2O3S
C24H16N2O4S
C18H14N2O3S
C18H13ClN2O3S
C19H13F3N2O3S
C20H16N2O4S
C18H12N2O3S
C18H11ClN2O3S
C19H11F3N2O3S
C20H14N2O4S
232–234
228–230
238–240
224–226
178–182
170–172
102–106
80–82
189–192
158–162
142–146
108–112
82
76
82
80
87
85
74
82
81
69
78
86
77
72
69
77
80
84
70
72
79
66
73
77
68.38(68.48)
62.78(62.69)
60.79(60.85)
67.28(67.25)
63.89(63.78)
57.99(60.11)
56.16(56.10)
63.14(63.18)
64.27(64.08)
58.30(58.21)
56.44(56.32)
63.48(63.32)
3.65(3.89)
3.11(3.12)
2.88(2.95)
3.76(3.82)
4.17(4.28)
3.51(3.39)
3.22(3.08)
4.24(4.27)
3.60(3.66)
2.99(2.89)
2.74(2.75)
3.73(3.65)
7.25(6.99)
6.66(6.74)
6.16(6.26)
6.54(6.24)
8.28(8.19)
7.51(7.59)
6.89(6.77)
7.36(7.37)
8.33(8.23)
7.55(7.50)
6.93(6.89)
7.40(7.43)
Cl
CF3
Ac
H
Cl
CF3
Ac
Ac – COCH3.
overnight; the solid thus obtained was recrystallized from xylene
with addition of activated carbon to afford 3a–d.
Similarly, compounds 4a–d and 5a–d were synthesized by using
2-(2,5-dioxopyrrolidin-1-yl) acetic acid (7) and 2-(2,5-dioxo-2H-
pyrrol-1(5H)-yl) acetic acid (8) respectively.
5.2.6. 1-[2-(2-Chloro-10H-phenothiazin-10-yl)-2-oxoethyl]
pyrrolidine-2,5-dione (4b)
White powder, IR (KBr) cmꢁ1: 1709 (C]O), 1780 (cyclic C]O),
2938, 2967 (CH2), 1577(C]C aromatic), 3062 (Ar–H); 1H NMR
(CDCl3): d 7.81–7.35 (m, 7H, phenothiazine), 4.32 (s, 2H), 2.70 (s, 4H,
succinimide–CH2); MS: m/z (%): 374 [Mþ þ 1] (85), 375 [Mþ þ 2]
5.2.1. 2-[Oxo-2-(10H-phenothiazin-10-yl)ethyl]-1H-isoindole-
1,3(2H)-dione (3a)
(60), 140 (100), 112 (40).
White powder, IR (KBr) cmꢁ1: 1698, 1715 (C]O), 1769 (cyclic
5.2.7. 1-{2-Oxo-2-[2-(trifluoromethyl)-10H-phenothiazin-10-
yl]ethyl}pyrrolidine-2,5-dione (4c)
C]O), 2937 (CH2), 1586,1615 (C]C aromatic), 3059 (Ar–H); 1H
NMR (CDCl3):
d
7.88–7.71 (m, 4H, phthalimide), 7.60–7.32 (m, 8H,
White powder, IR (KBr) cmꢁ1: 1710 (C]O), 1781 (cyclic C]O),
phenothiazine), 4.50 (s, 2H); MS: m/z (%): 387 [Mþ þ 1] (100), 388
2943, 2970 (CH2), 1583 (C]C aromatic), 3068 (Ar–H); 1H NMR
[Mþ þ 2] (30), 188 (60), 160 (100).
(CDCl3): d 7.82–7.44 (m, 7H, phenothiazine), 4.39 (s, 2H), 2.68 (s, 4H,
succinimide–CH2); MS: m/z (%): 407 [Mþ þ 1] (100), 266 (70), 140
5.2.2. 2-[2-(2-Chloro-10H-phenothizin-10-yl)-2-oxoethyl]-1H-
isoindole-1,3(2H)-dione (3b)
(100).
White powder, IR (KBr) cmꢁ1: 1728 (C]O), 1771 (cyclic C]O),
5.2.8. 1-[2-(2-Acetyl-10H-phenothiazin-10-yl)-2-
2942 (CH2), 1587, 1614 (C]C aromatic), 3068 (Ar–H); 1H NMR
oxoethyl]pyrrolidine-2,5-dione (4d)
(CDCl3):
d
7.87–7.72 (m, 4H, phthalimide), 7.57–7.31 (m, 7H,
White powder, IR (KBr) cmꢁ1: 1709, 1673 (C]O), 1778 (cyclic
phenothiazine), 4.58 (s, 2H); MS: m/z (%): 422 [Mþ þ 1] (96), 423
C]O), 2948, 2971 (CH2), 1588 (C]C aromatic), 3058 (Ar–H); 1H
[Mþ þ 2] (65), 188 (60), 160 (95).
NMR (CDCl3): d 7.80–7.46 (m, 7H, phenothiazine), 4.41 (s, 2H), 2.75
(s, 4H, succinimide–CH2), 2.48 (s, 3H); MS: m/z (%): 381 [Mþ þ 1]
5.2.3. 2-{2-Oxo-2-[2-(trifluoromethyl)-10H-phenothiazin-10-yl]
ethyl}-1H-isoindole-1,3(2H)-dione (3c)
(100), 300 (20), 241 (90).
White powder, IR (KBr) cmꢁ1: 1723 (C]O), 1776 (cyclic C]O),
5.2.9. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl]-1H-pyrrole-2,
5-dione (5a)
2944 (CH2), 1582, 1608 (C]C aromatic), 3066 (Ar–H); 1H NMR
(CDCl3):
d
7.89–7.70 (m, 4H, phthalimide), 7.70–7.38 (m, 7H,
White powder, IR (KBr) cmꢁ1: 1686, 1708 (C]O), 1779 (cyclic
C]O), 2941, 2977 (CH2), 1628 (C]C cyclic), 1589 (C]C aromatic),
phenothiazine), 4.59 (s, 2H); MS: m/z (%): 455 [Mþ þ 1] (85), 266
(100), 247 (10), 235 (70), 198 (20), 160 (100).
3014 (C]C–H), 3054 (Ar–H); 1H NMR (CDCl3):
d 7.62–7.26 (m, 8H,
phenothiazine), 6.72 (s, 2H, olefinic–CH), 4.40 (s, 2H); MS: m/z (%):
5.2.4. 2-[2-(2-Acetyl-10H-phenothiazin-10-yl)-2-oxoethyl]-1H-
isoindole-1,3(2H)-dione (3d)
337 [Mþ þ 1] (100), 339 [Mþ þ 2] (30), 200 (35), 138 (100), 110 (35).
White powder, IR (KBr) cmꢁ1: 1721, 1686 (C]O),1774 (Cyclic
C]O), 2955 (CH2), 2997 (CH3), 1588, 1612 (C]C aromatic), 3059
5.2.10. 1-[2-(2-Chloro-10H-phenothiazin-10-yl)-2-oxoethyl]-1H-
pyrrole-2,5-dione (5b)
(Ar–H); 1H NMR (CDCl3):
d
7.91–7.69 (m, 4H, phthalimide), 7.82–
White powder, IR (KBr) cmꢁ1: 1713 (C]O), 1778 (cyclic C]O),
2938, 2967 (CH2), 1577 (C]C aromatic), 3062 (Ar–H); 1H NMR
7.48 (m, 7H, phenothiazine), 4.48 (s, 2H), 2.44 (s, 3H); MS: m/z (%):
429 [Mþ þ 1] (100), 241 (35), 160 (50).
(CDCl3):
d 7.81–7.35 (m, 7H, phenothiazine), 6.70 (s, 2H, olefinic–
CH), 4.32 (s, 2H), MS: m/z (%): 372 [Mþ þ 1] (85), 373 [Mþ þ 2] (60),
5.2.5. 1-[2-Oxo-2-(10H-phenothiazin-10-yl)ethyl] pyrrolidine-2,
5-dione (4a)
140 (100), 112 (40).
White powder, IR (KBr) cmꢁ1: 1685, 1706 (C]O), 1777 (Cyclic
5.2.11. 1-{2-Oxo-2-[2-(trifluoromethyl)-10H-phenothiazin-10-yl]
ethyl}-1H-pyrrole-2,5-dione (5c)
C]O), 2941, 2977 (CH2), 1589 (C]C aromatic), 3054 (Ar–H); 1H
NMR (CDCl3):
d
7.62–7.26 (m, 8H, phenothiazine), 4.40 (s, 2H), 2.79
White powder, IR (KBr) cmꢁ1: 1708 (C]O), 1781 (cyclic C]O),
2943, 2970 (CH2), 1618 (C]C cyclic), 1583 (C]C aromatic),
(s, 4H, succinimide–CH2); MS: m/z (%): 339 [Mþ þ 1] (100), 340
[Mþ þ 2] (20), 200 (30), 140 (100), 112 (35).
3018 (C]C–H), 3068 (Ar–H); 1H NMR (CDCl3):
d 7.82–7.44

T.N. Bansode et al. / European Journal of Medicinal Chemistry 44 (2009) 5094–5098
5097
Table 2
(m, 7H, phenothiazine), 6.69 (s, 2H, olefinic–CH), 4.39 (s, 2H); MS:
m/z (%): 405 [Mþ þ 1] (100), 266 (70), 140 (100).
Antibacterial activity of the compounds 3a–d, 4a–d and 5a–d.
Compound no.
Antibacterial activity
5.2.12. 1-[2-(2-Acetyl-10H-phenothiazin-10-yl)-2-oxoethyl]-1H-
pyrrole-2,5-dione (5d)
B. subtilis
E. coli
S. aureus
P. aeruginosa
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
17(15)
13(30)
14(25)
16(15)
18(15)
14(30)
14(25)
–
16(15)
–
17(15)
16(15)
22(10)
22(10)
23(10)
–
20(10)
19(10)
–
19(10)
–
12(30)
18(15)
–
15(15)
18(15)
–
–
White powder, IR (KBr) cmꢁ1: 1709, 1673 (C]O), 1778 (cyclic
18(10)
–
–
C]O), 2948, 2971 (CH2), 1588 (C]C aromatic), 3058 (Ar–H); 1H
21(10)
24(10)
16(15)
21(20)
24(10)
20(10)
20(10)
19(10)
18(20)
25(10)
NMR (CDCl3):
d 7.80–7.46 (m, 7H, phenothiazine), 6.72(s, 2H,
olefinic–CH), 4.41 (s, 2H), 2.44 (s, 3H); MS: m/z (%): 379 [Mþ þ 1]
–
16(15)
19(15)
–
17(15)
17(15)
19(15)
17(15)
23(10)
(100), 300 (20), 241 (90).
5.3. General procedure of N-carboxy acids [28–30]
5.3.1. 2-(1,3-Dioxoisoindolin-2yl) acetic acid (6)
16(15)
21(10)
A
mixture of equivalents amount of phthalic anhydride
Standard
(0.05 mol) and glycine (0.05 mol) were refluxed in 40 ml toluene
for 3 h. Toluene was removed under vacuum, followed by addition
of 80 ml of water and 1.5 ml of concentrated HCl. The mixture was
stirred well, filtered and recrystallized from ethanol.
‘‘–’’ Indicates bacteria are resistant to the compounds at concentration >50
mg/ml;
MIC values are given in brackets.
White powder, yield 95%, m.p, 191–192 ꢀC, IR (KBr) cmꢁ1: 1720
10
mg/ml against S. aureus and Compounds 3b is active at 10 mg/ml
(C]O), 1766 (cyclic C]O), 3040 (Ar–H); 1H NMR (CDCl3):
d 7.60–
against P. aeruginosa i.e. almost equivalent to standard. Compounds
3a, 3d, 4a, 4c, 5a, 5c, 5d are active at 15 mg/ml against B. subtilis.
7.95 (m, 4H, Ar–H), 4.57(s, 2H); MS: m/z (%): 161 [Mþ ꢁ CO2] (90),
Anal. calcd. for C10H7NO4: C, 58.54; H, 3.44; N, 6.83. Found: C, 58.59;
H, 3.32; N, 6.38%.
6.2. Antifungal studies
Similarly 7 and 8 were synthesized from succinic anhydride and
maleic anhydride respectively.
Newly prepared compounds were screened for their antifungal
activity against Aspergillus niger (NCIM no. 617), Aspergillus flavus
(NCIM no. 524), Aspergillus fumigatus (NCIM no. 902), Candida
albicans (NCIM no. 300) in DMSO by serial plate dilution method
[33,34].
5.3.2. 2-(2,5-Dioxopyrrolidin-1-yl) acetic acid (7)
White powder, yield 92%, m.p. 108–109 ꢀC, IR (KBr) cmL1: 1718
(C]O), 1768 (cyclic C]O), 2972 (CH2); 1H NMR (CDCl3):
d 2.70 (s,
4H,), 4.07(s, 2H); MS: m/z (%): 113 [Mþ ꢁ CO2] (96). Anal. calcd. for
Sabourands agar media were prepared by dissolving peptone
C6H7NO4: C, 45.86; H, 4.49; N, 8.91. Found: C, 45.49; H, 4.25; N, 9.02%.
(1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 ml) and
adjusting pH to 5.7. Normal saline was used to make a suspension of
spore of fungal strain for lawning. A loopful of particular fungal
strain was transferred to 3 ml saline to get a suspension of corre-
sponding species. Agar media (20 ml) were poured into each Petri
dish. Excess of suspension was decanted and the plates were dried
by placing in an incubator at 37 ꢀC for 1 h using an agar punch,
wells were made and each well was labeled. A control was also
prepared in triplicate and maintained at 37 ꢀC for 2–3 days. Zone of
inhibition and minimum inhibitory concentration (MIC) were
noted. The activity of each compound was compared with fluco-
nazole as the standard drug. The results of antifungal studies are
given in Table 3. The antifungal screening data showed good
5.3.3. 2-(2,5-Dioxo-2H-pyrrol-1(5H)-yl) acetic acid (8)
White powder, yield 93% m.p 112–113 ꢀC, IR (KBr) cmꢁ1: 1710
(C]O), 1776 (cyclic C]O), 3040 (C–H olefinic); 1H NMR (CDCl3):
d
6.70 (s, 2H, olefinic), 3.97(s, 2H); MS: m/z (%): 111 [Mþ ꢁ CO2] (87),
Anal. calcd. for C6H5NO4: C, 46.5; H, 3.15; N, 9.10. Found: C, 46.44; H,
3.25; N, 9.10%.
6. Biological activity
6.1. Antibacterial studies
The newly synthesized compounds were screened for their
antibacterial activity against Bacillus subtilis (ATCC-11774), Escher-
ichia coli (ATCC-25922), Staphylococcus aureus (ATCC-25923) and
Pseudomonas aeruginosa (ATCC-27853) bacterial strains by disc
diffusion method [31,32]. Discs measuring 6.25 mm in diameter
were punched from Whatman no. 1 filter paper. Batches of 100
discs were dispensed to each screw capped bottles and sterilized by
dry heat at 140 ꢀC for an hour. The test compounds were prepared
with different concentrations using dimethylsulphoxide. One
milliliter containing 100 times the amount of chemical in each disc
was added to each bottle, which contains 100 discs. The discs of
each concentration were placed in triplicate in nutrient agar
medium seeded with fresh bacteria separately. The incubation was
carried out at 37 ꢀC for 24 h. Ciprofloxacin was used as a standard
drug. Solvent and growth controls were prepared and kept. Zones
of inhibition and minimum inhibitory concentrations (MICs) were
noted. The results of antibacterial studies are given in Table 2.
The investigation of antibacterial screening data revealed that all
the tested compounds showed moderate to good bacterial inhibi-
activity. Compounds 3a, 3b, 3c, 3d are active at 10
mg/ml against
Table 3
Antifungal activity of the compounds 3a–d, 4a–d and 5a–d.
Compound no.
Antifungal activity
A. niger
A. fumigatus
A. flavus
C. albicans
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
23(10)
24(10)
21(10)
25(10)
19(20)
23(20)
21(20)
26(20)
17(20)
18(20)
19(20)
–
22(10)
21(10)
22(10)
22(10)
24(15)
12(25)
23(10)
20(25)
18(20)
15(20)
19(20)
18(25)
24(10)
20(15)
19(10)
18(10)
19(15)
19(20)
18(20)
19(15)
13(15)
15(20)
16(20)
10(30)
16(30)
20(10)
17(10)
18(10)
21(10)
14(10)
17(15)
13(25)
19(20)
–
17(20)
17(20)
19(20)
–
Standard
23(10)
21(10)
tion. Compounds 3a, 3b, 3d, 4a, 4d, 5a, 5b, 5c are active at 10
mg/ml
‘‘–’’ Indicates fungus are resistant to the compounds at concentration >50
mg/ml;
concentrations against E. coli. Compounds 3a, 3b, 3d are active at
MIC values are given in brackets.

5098
T.N. Bansode et al. / European Journal of Medicinal Chemistry 44 (2009) 5094–5098
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Acknowledgement
The authors are grateful to Dr. V.B. Patrawale, Department of
Pharmaceutical Sciences and Technology, UICT, Mumbai for
screening the newly synthesized compounds for their antimicrobial
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