Synthesis and antimicrobial activity of Schiff and Mannich bases of isatin and its derivatives with pyrimidine

Article abstract of DOI:10.1016/S0014-827X(99)00075-0

Isatin and its derivatives have been reacted with 4-(4'-chlorophenyl)-6-(4''-methyl phenyl)-2-aminopyrimidine to form Schiff bases and the N-Mannich bases of these compounds were synthesized by reacting them with formaldehyde and several secondary amines.

Full text of DOI:10.1016/S0014-827X(99)00075-0

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 624–628
Synthesis and antimicrobial activity of Schiff and Mannich bases
of isatin and its derivatives with pyrimidine
S.N. Pandeya a,*, D. Sriram a, G. Nath b, E. De Clercq c
a Department of Pharmaceutics, Institute of Technology, Banaras Hindu Uni6ersity, Varanasi 221 005, India
b Department of Microbiology, Institute of Medical Sciences, Banaras Hindu Uni6ersity, Varanasi 221 005, India
c Rega Institute for Medical Research, Katholieke Uni6ersity-Leu6en, Minderbrodersstraat 10, B-3000 Leu6en, Belgium
Received 15 April 1999; accepted 19 July 1999
Abstract
Isatin and its derivatives have been reacted with 4-(4%-chlorophenyl)-6-(4¦-methyl phenyl)-2-aminopyrimidine to form Schiff
bases and the N-Mannich bases of these compounds were synthesized by reacting them with formaldehyde and several secondary
amines. Investigation of antimicrobial activity of the compounds was made by the agar dilution method against 28 pathogenic
bacteria, eight pathogenic fungi and anti-HIV activity against replication of HIV-1 (III B) in MT-4 cells. The compounds are
significantly active against bacteria and fungi. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Schiff bases; N-Mannich bases; Isatin; Antimicrobial activity
1. Introduction
2. Experimental
Isatin (indole-2,3-dione), its Schiff and Mannich bases
are reported to show a variety of biological activities,
such as antibacterial [1], antifungal [2] and anti-HIV [3]
activities. Pyrimidines are reported to have antibacterial
[4], antifungal [5] and anti-HIV [6] activities. In view of
these facts and as a continuation of previous efforts
carried out in our laboratory [3,7], 4-(4%-chlorophenyl)-6-
(4¦-methylphenyl)-2-aminopyrimidine has been synthe-
sized from 3-(4%-chlorophenyl)-1-(4¦-methyl phenyl)
2-propen-1-one. It was condensed with isatin and its
5-chloro and 5-bromo derivatives to form Schiff bases.
The N-Mannich bases of the above Schiff bases were
synthesized by condensing the acidic imino group of
isatin with formaldehyde and secondary amines (Scheme
1). All compounds (Table 1) gave satisfactory elemental
Melting points were determined on a Thomas–
Hoover melting point apparatus and are uncorrected. IR
spectra were recorded on a Jasco infrared spectrometer
in KBr. H NMR spectra were recorded on a Jeol FX
90Q FT-NMR spectrometer (90 MHz) employing te-
tramethyl silane as the internal reference.
1
2.1. Synthesis of 3-(4%-chlorophenyl)-1-(4¦-methyl
phenyl)-2-propen-1-one
An aqueous solution of sodium hydroxide (10% w/v,
10 ml) was added to a solution of 4-chloro benzaldehyde
(0.02 mol) and 4-methyl acetophenone (0.02 mol) in
ethanol (6 ml). The reaction mixture was stirred at room
temperature overnight and poured into water (100 ml).
After neutralization with hydrochloric acid (10% w/v), a
yellow solid was obtained which was recrystallized from
water–ethanol; yield 85%, m.p. 125°C.
1
analysis. IR and H NMR spectra were consistent with
the assigned structures. All the synthesized compounds
were screened for antibacterial, antifungal activity by the
agar dilution method and anti-HIV activity against
HIV-1 (III B) in MT-4 cells.
2.2. Synthesis of 4-(4%-chlorophenyl)-6-(4¦methyl
phenyl)-2-aminopyrimidine
A mixture of 3-(4%-chlorophenyl)-1-(4¦-methylphenyl)-
2-propen-l-one (0.01 mol) and guanidine hydrochloride
* Corresponding author. Fax: +91-542-316 427.
E-mail address: snpande@banaras.ernet.in (S.N. Pandeya)
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00075-0

S.N. Pandeya et al. / Il Farmaco 54 (1999) 624–628
625
Scheme 1. Synthetic protocol of the compounds.
Table 1
Physical constants
Code
R
R1
Yield (%)
M.p. (°C)
Mol. Formula
Rf C6H6:C2H5OH
(9.5:0.5)
S1
S2
H
H
H
72.34
79.00
135
122
C25H17ON4Cl
C28H24ON5Cl
0.73
0.63
ꢀCH2ꢀN(CH3)2
S3
S4
H
H
90.97
47.41
98
C
31H28ON5Cl
0.67
0.75
138
C30H26O2N5Cl
S5
S6
Cl
Cl
H
78.76
75.08
160
134
C25H16ON4Cl2
C28H23ON5Cl2
0.84
0.72
ꢀCH2ꢀN(CH3)2
S7
S8
Cl
Cl
78.26
76.26
101
118
C31H27ON5Cl2
C30H25O2N5Cl2
0.69
0.75
S9
S10
Br
Br
H
57.31
84.66
209
235
C25H16ON4ClBr
C28H23ON5ClBr
0.59
0.75
ꢀCH2ꢀN(CH3)2
S11
S12
Br
Br
81.11
85.52
109
115
C31H27ON5ClBr
C30H23O2N5ClBr
0.68
0.75

626
S.N. Pandeya et al. / Il Farmaco 54 (1999) 624–628
Table 2
a
Antibacterial activity MIC values (mg ml−1
)
Microorganisms/drugs
S1
156.25
78.12
56.25
1250
156.25
625
S2
156.25
156.25
156.25
312.5
78.12
312.5
19.53
2.44
9.76
156.25
5000
1250
625
S3
156.25
78.12
156.25
312.5
78.12
625
S4
S5
156.25
78.12
156.25
156.25
78.12
312.5
19.53
2.44
9.76
156.25
5000
1250
1250
S6
156.25
39.06
156.25
156.25
78.12
625
9.76
4.88
19.53
39.06
1250
625
S7
156.25
39.06
78.12
156.25
156.25
312.5
9.76
9.76
19.53
78.12
1250
625
1
2
3
4
S. typhimurium
Vibrio parahaemolyticus
Salmonella paratyphi B
E. tarda
78.12
39.06
156.25
156.25
39.06
625
5
6
V. cholerae 0139
S. aureus
7
8
9
E. coli NCTC 10418
V. cholerae non-01
E. faecalis
1953
9.76
9.76
9.76
9.76
9.76
4.88
2.44
9.76
156.25
2500
1250
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Salmonella typhi
P. aeruginosa
K. pneumoniae
S. albus
Salmonella enteritidis
A. hydrophila
V. cholerae-01
B. subtilis
Shigella sonnei
Shigella boydii
Plesiomonas shigelloides
P. rettgeri
Shigella flexneri
Proteus 6ulgaris
Enterobacter spp.
Morgonella morganii
Citrobacter freundii
Proteus morganii
S. paratyphi A
78.12
2500
1250
625
78.12
625
9.76
39.06
625
156.25
9.76
312.5
312.5
312.5
625
312.5
78.12
625
156.25
1250
1250
625
39.06
312.5
4.88
1250
625
1250
78.12
312.5
19.53
39.06
312.5
156.25
9.76
156.25
156.25
312.5
625
78.12
625
39.06
156.25
9.76
78.12
312.5
9.76
78.12
625
39.06
9.76
78.12
156.25
312.5
625
39.06
312.5
4.88
39.06
312.5
78.12
9.76
156.25
156.25
156.25
625
312.5
78.12
625
19.53
19.53
312.5
156.25
9.76
312.5
156.25
312.5
625
9.76
78.12
312.5
78.12
19.53
156.25
156.25
312.5
625
312.5
78.12
9.76
78.12
156.25
156.25
312.5
78.12
78.12
625
156.25
78.12
625
156.25
156.25
625
156.25
156.25
625
156.25
78.12
625
156.25
78.12
156.25
312.5
78.12
78.12
156.25
a MIC, minimum inhibitory concentration.
(0.015 mol) was added to sodium hydroxide (0.045 mol
in 2 ml of water) and ethanol (50 ml). The reaction
mixture was refluxed for 6 h. The mixture was concen-
trated under reduced pressure and cooled, a yellow
solid was obtained which was recrystallized from
ethanol. Yield: 82%, m.p. 190°C. IR (KBr) (cm−1):
Ar-H, tolyl), 7.75 (1H, s, H-5%), 7.9–8.1 (4H, Sym 2d,
Ar-H, p-chlorophenyl), 10.4 (1H, s, NH, D2O ex-
changeable). Anal. (C25H17ON4Cl) C, H, N.
Similarly, compounds S5 and S9 were synthesized
using 5-chloro and 5-bromo isatin.
1
3280 (NH2), 1610 (ring CꢁC, CꢁN). H NMR (CDCl3)
2.4. Synthesis of 1-piperidinomethyl-3-[4%(4¦-
chlorophenyl)-6%-(4§-methylphenyl) pyrimidin-2%-yl]
iminoisatin (S3)
l (ppm): 2.30 (3H, s, CH3), 5.5 (2H, s, NH2), 7.3–7.5
(4H, m, Ar-H of tolyl), 7.75 (1H, s, H-5), 7.9–8.0 (4H,
Sym 2d, Ar-H of p-Cl-phenyl). Anal. (C17H14N3Cl) C,
H, N.
A slurry consisting of S1 (0.005 mol), tetrahydro-
furan (5 ml) and 37% formalin (2 ml) was made. To
this, piperidine (0.005 mol) was added dropwise with
cooling and shaking. The reaction mixture was allowed
to stand at room temperature for 1 h with occasional
shaking after which it was warmed on a steam bath for
15 min. At the end of the period the contents were
cooled and the product obtained was recrystallized
from chloroform–petroleum ether. Yield: 90.97%; m.p.
2.3.
Synthesis
of
3-[4%(4¦-chlorophenyl)-6%-(4§-
methylphenyl) pyrimidin-2%-yl] iminoisatin (S1)
Equimolar quantities (0.03 mol) of isatin and 4-(4%-
chlorophenyl)-6-(4¦-methylphenyl)-2-aminopyrimidine
were dissolved in 75 ml of warm ethyl alcohol contain-
ing 1 ml of glacial acetic acid. The reaction mixture was
refluxed for 18 h and set aside. The resultant solid was
washed with dilute ethanol, dried and recrystallized
from an ethanol–chloroform mixture. Yield: 72.34%;
m.p. 135°C. IR (KBr) (cm−1): 1640 (CꢁN), 1610 (ring
1
98°C. IR (KBr) (cm−1): 2870 (CH2), 1645 (CꢁN); H
NMR (CDCl3) l (ppm): 1.8 (6H, m, (CH2)3), 2.3 (3H,
s, CH3), 2.6 (4H, t, CH2NCH2), 4.45 (2H, s, NCH2N),
6.8–7.1 (4H, m, Ar-H, isatin), 7.1–7.5 (4H, m, Ar-H,
tolyl), 7.7 (1H, s, H-5), 7.9–8.1 (4H, Sym 2d, Ar-H,
p-Cl-phenyl). Anal. (C31H28ON5Cl) C, H, N.
1
CꢁC, CꢁN). H NMR (CDCl3) l (ppm): 2.35 (3H, s,
CH3), 6.8–7.1 (4H, m, Ar-H isatin), 7.2–7.5 (4H, m,

S.N. Pandeya et al. / Il Farmaco 54 (1999) 624–628
627
Table 3
a
Antibacterial activity MIC values in (mg ml−1
)
Microorganisms/drugs
S8
S9
S10
S11
S12
Sulphametho-
xazole
Trimethoprim
1
2
3
4
5
6
7
8
S. typhimurium
V. parahaemolyticus
S. paratyphi B
E. tarda
V. cholerae 0139
S. aureus
E. coli NCTC 10418
V. cholerae non-01
E. faecalis
39.06
39.06
78.12
156.25
19.53
78.12
4.88
1.22
4.88
39.06
1250
312.5
1250
19.53
156.25
4.88
39.06
312.5
19.53
9.76
78.12
312.5
156.25
312.5
312.5
78.12
312.5
156.25
156.25
156.25
156.25
156.25
78.12
39.06
39.06
39.06
78.12
2.44
0.3
1.22
19.53
312.5
312.5
625
9.76
312.5
9.76
9.76
312.5
9.76
5000
1250
5000
5000
\5000
5000
2500
312.5
5000
2500
78.12
2500
2500
2500
2500
5000
5000
2500
2500
5000
2500
2500
2500
1250
2500
5000
5000
2500
\5000
2.44
156.25
156.25
156.25
156.25
312.5
9.76
0.61
4.88
156.25
2500
625
78.12
156.25
156.25
156.25
625
9.76
0.61
9.76
78.12
156.25
78.12
78.12
625
9.76
1.22
4.88
9.76
312.5
39.06
\5000
19.53
1.22
78.12
4.88
5000
5000
\5000
4.88
1250
5000
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
S. typhi
156.25
2500
1250
39.06
1250
625
P. aeruginosa
K. pneumoniae
S. albus
S. enteritidis
A. hydrophila
V. cholerae-01
B. subtilis
S. sonnei
S. boydii
P. shigelloides
P. rettgeri
S. flexneri
625
1250
625
78.12
156.25
19.53
39.06
312.5
39.06
9.76
78.12
156.25
312.5
625
39.06
312.5
19.53
9.76
312.5
78.12
4.88
78.12
156.25
312.5
625
156.25
156.25
312.5
156.25
39.06
312.5
9.76
19.53
312.5
39.06
4.88
156.25
156.25
312.5
625
78.12
156.25
312.5
156.25
5000
9.76
9.76
4.88
4.88
39.06
312.5
312.5
156.25
156.25
39.06
156.25
39.06
2500
156.25
156.25
156.25
156.25
19.53
156.25
156.25
P. 6ulgaris
Enterobacter spp.
M. morganii
C. freundii
P. morganii
S. paratyphi A
156.25
156.25
625
312.5
a MIC, minimum inhibitory concentration.
Similarly other Mannich bases S2, S4, S6–S8 and
the presence of various concentrations of test com-
pounds and the number of viable cells was determined
by the MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] method on day 5 after virus
infection.
S10–S12 were prepared by using appropriate Schiff
bases with formaldehyde and corresponding secondary
amines in tetrahydrofuran as a solvent.
2.5. In 6itro antimicrobial acti6ity
Evaluation of antibacterial (28-bacteria) and antifun-
gal (8-fungi) activity by the agar dilution method [8].
The microorganisms used were procured from the De-
partment of Microbiology, Institute of Medical Sci-
ences, Banaras Hindu University. All bacteria were
grown on Mueller–Hinton agar (Hi-media) plates
(37°C, 24 h) and fungi were grown on Sabouraud
dextrose agar (Hi-media) plates (26°C, 48–72 h). The
minimum inhibitory concentration (MIC) was consid-
ered to be the lowest concentration that completely
inhibited the growth on agar plates, disregarding a
single colony or faint haze caused by the inoculum.
3. Results and discussion
All the synthesized compounds were tested for in
vitro antibacterial activity by the agar dilution method.
The MIC values of the synthesized compounds against
28 pathogenic bacteria are presented in Tables 2 and 3.
Also included is the activity of reference compounds
sulphamethoxazole and trimethoprim. It has been ob-
served that all the compounds tested showed mild to
moderate activity against the tested bacteria. All the
tested compounds showed more activity (less MIC)
than sulphamethoxazole except Pseudomonas aerugi-
nosa. When compared to trimethoprim all the com-
pounds were more active against Salmonella
typhimurium, Staphylococcus aureus, Enterococcus fae-
calis, P. aeruginosa, Klebsiella pneumoniae, Staphylococ-
cus albus, Aeromonas hydrophila, Vibrio cholerae-01,
Bacillus subtilis and Proteus rettgeri; nine compounds
2.6. Anti-HIV acti6ity
The procedure to measure anti-HIV activity in MT-4
cells has been described previously [3]. Either mock-in-
fected or HIV-1 infected MT-4 cells were incubated in
.

628
S.N. Pandeya et al. / Il Farmaco 54 (1999) 624–628
Table 4
Antifungal activity MIC values in (mg ml−1
)
Drugs/
microorganism
Candida
albicans
Aspergillus
niger
Cryptococcus
neoformans
M. audouinii
T. menta- Epidermophyton
M. gypseum
Histoplasma
capsulatum
grophytes
floccosum
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
39.06
78.12
78.12
39.06
78.12
78.12
78.12
78.12
39.06
39.06
19.53
39.06
0.3
39.06
39.06
78.12
39.06
39.06
39.06
78.12
78.12
39.06
39.06
39.06
39.06
2.44
39.06
19.53
19.53
39.06
39.06
39.06
39.06
78.12
19.53
9.76
9.76
4.88
9.76
9.76
4.88
2.44
4.88
4.88
4.88
9.76
4.88
9.76
4.88
4.88
9.76
9.76
9.76
4.88
2.44
2.44
4.88
4.88
2.44
2.44
9.76
2.44
19.53
9.76
9.76
4.88
4.88
2.44
2.44
4.88
4.88
1.22
4.88
2.44
4.88
9.76
9.76
2.44
39.06
19.53
19.53
39.06
78.12
78.12
78.12
78.12
39.06
39.06
78.12
39.06
19.53
4.88
19.53
19.53
39.06
9.76
19.53
4.88
19.53
19.53
2.44
19.53
39.06
2.44
Clotrimazole
rized in Table 4. Clotrimazole was used as the reference
for inhibitory activity against fungi. All the compounds
showed significant antifungal activity. When compared
to clotrimazole, six compounds were equipotent (4.88
mg ml−1) and one compound (S6) was more active
against Microsporum audouinii. The compounds showed
good activity against dermatophytes like Microsporum
gypseum, M. audouinii and Trichophyton mentagro-
Table 5
Anti-HIV activity
SI c
a
b
Drug
ED50 (mM)
CD50 (mM)
S1
S2
S3
S4
S5
S6
S7
S8
\9
\10
\1
\3
\8
\7
\3
\3
\10
\13
\1
\2
9.4
10.1
1.1
2.7
8.2
6.8
2.6
3.0
10.4
12.9
B1
1.9
B1
B1
B1
B1
B1
B1
B1
B1
B1
B1
B1
B1
phytes with an MIC of less than 10 mg ml−1
.
The synthesized compounds were evaluated for their
inhibitory effect of the replication of HIV-1 in human
MT-4 cells. None of the compounds showed marked
anti-HIV activity at a concentration significantly below
their toxicity threshold (Table 5).
S9
S10
S11
S12
a Effective dose of compound, achieving 50% protection of MT-4
cells against the cytopathic effect of HIV.
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b Cytotoxic dose of compound, required to reduce the viability of
normal uninfected MT-4 cells by 50%.
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c Selectivity index or ratio of CD50 to ED50
.
(S4–S12) were more active against Edwardsiella tarda,
Escherichia coli NCTC 10418; and three compounds
(S9, S10, S12) were more active against V. cholerae
non-01. Compound S12 was found to be the most
active antibacterial agent. In general the order of an-
tibacterial activity of the substituents at the 5th position
is Br\Cl\H. In case of substituents at the 1st posi-
tion, the morpholinomethyl group showed good
activity.
The antifungal activity of the compounds was studied
for the eight pathogenic fungi. The results are summa-