Synthesis, antibacterial, antifungal and anti-HIV activities of norfloxacin Mannich bases

Article abstract of DOI:10.1016/S0223-5234(00)00125-2

Mannich bases of norfloxacin were synthesized by reacting them with formaldehyde and several isatin derivatives. Their chemical structures have been confirmed by means of their IR, ¹H-NMR data and by elemental analysis. Investigation of in vitro antimicrobial activity of compounds was done 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 in vivo antibacterial efficacy of selected derivatives was determined using a mouse infection model. All the synthesized compounds are more active than norfloxacin against the 13 bacteria tested. The compounds are also more active than the standard drug clotrimazole against Histoplasma capsulatum. Two compounds S-8 and S-9 have shown inhibition against HIV-1 (III B) with EC₅₀ values of 11.3 and 13.9 μg/mL, respectively. In the mouse protection test, two compounds S-4 (ED₅₀: 1.25 mg/kg) and S-9 (ED₅₀: 1.62 mg/kg) are more active than norfloxacin (ED₅₀: 6mg/kg). Among the compounds tested, 1- ethyl-6-fluoro-l,4-dihydro-4-oxo- 7[[N⁴-[5'-bromo-3-(4'-amino-5- trimethoxybenzylpyrimidin-2'-yl]-imino-1'-isatinyl]methyl]N¹-piperazinyl]-3- quinoline carboxylicacid (S-9) showed promising activity in all the three tests. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00125-2

Eur. J. Med. Chem. 35 (2000) 249−255
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001252/FLA
249
Original article
Synthesis, antibacterial, antifungal and anti-HIV activities of norfloxacin
Mannich bases
Surendra N. Pandeyaa*, Dhamrajan Srirama, Gopal Nathb, Erik De Clercqc
aDepartment of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi – 221 005, India
bDepartment of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi – 221 005, India
cRega Institute for Medical Research, Kotholieke University, Leuven,
Minderbroedersstraat, B-3000 Leuven, Belgium
Received 27 May 1999; revised 10 August 1999; accepted 10 August 1999
Abstract – Mannich bases of norfloxacin were synthesized by reacting them with formaldehyde and several isatin derivatives. Their chemical
structures have been confirmed by means of their IR, 1H-NMR data and by elemental analysis. Investigation of in vitro antimicrobial activity
of compounds was done 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 in vivo antibacterial efficacy of selected derivatives was determined using a mouse infection
model. All the synthesized compounds are more active than norfloxacin against the 13 bacteria tested. The compounds are also more active
than the standard drug clotrimazole against Histoplasma capsulatum. Two compounds S-8 and S-9 have shown inhibition against HIV-1 (III
B) with EC50 values of 11.3 and 13.9 µg/mL, respectively. In the mouse protection test, two compounds S-4 (ED50: 1.25 mg/kg) and S-9
(ED50: 1.62 mg/kg) are more active than norfloxacin (ED50: 6mg/kg). Among the compounds tested, 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-
7[[N4-[5'-bromo-3'-(4'-amino-5'-trimethoxybenzylpyrimidin-2'-yl]-imino-1'-isatinyl]methyl]N1-piperazinyl]-3-quinoline carboxylicacid (S-9)
showed promising activity in all the three tests. © 2000 Éditions scientifiques et médicales Elsevier SAS
Mannich bases / norfloxacin / isatin / antimicrobial
1. Introduction
deficiency virus type 1 [8]. In the present study we have
aimed to achieve a better antimicrobial profile at lower
concentrations, by preparing Mannich bases with isatin
derivatives. This report deals with the synthesis of Man-
nich bases of norfloxacin and screening for their antibac-
terial, antifungal and anti-HIV activities.
The fluoroquinolone antibacterial agents have been
found to be one of the fastest growing group of drugs in
recent years. To date, more than 10 000 different ana-
logues have been synthesized. They are unusual in being
totally synthesized chemically. This means that various
side chains can be altered and the resulting analogues
tested for their antimicrobial properties. Most of these
agents are substituted at the 7 position by a nitrogen
heterocycle. Norfloxacin [1], characterized by having a
piperazine moiety at C-7, which represents a site ame-
nable to significant modification. We report our results
from a study of replacing the N4-hydrogen of piperazine
with various isatin derivatives. Isatin derivatives are
reported to show antibacterial [2, 3], antifungal [4], and
anti-HIV activities [5–7]. Fluoroquinolone derivatives
also exhibit inhibitory activity against human immuno-
2. Chemistry
The starting materials 3-[4-sulfadiazinimino] isatin,
3-[4(-sulfadoximino] isatin and 3-[4'-amino,5'-(3'', 4'',5''-
trimethoxybenzyl)pyrimidin-2'-yl]imino isatin and their 5
substituted derivatives were prepared according to the
literature methods [9–11] by reacting isatin and its de-
rivatives with sulfadiazine, sulfadoxine and trimethoprim
in the presence of glacial acetic acid. Mannich bases of
norfloxacin were prepared by condensing the active
hydrogen atom of isatin derivatives with formaldhyde
*Correspondence and reprints: snpande@banaras.ernet.in

250
Figure 1. Protocol for the synthetic compounds.
and the secondary amino function (piperazino moiety) of
norfloxacin (figure 1).
nas hydrophila, Shigella sonnei, Plesiomonas shigel-
loides, Proteus rettgeri, Shigella flexneri, Morgonella
morganii and Salmonella paratyphi A. All the compounds
are more active than norfloxacin (MIC: 9.76 µg/mL)
against E. tarda and P. rettgeri (0.075–1.22 µg/mL), V.
cholerae 0139 (0.018–1.22 µg/mL), S. faecalis (0.15–
2.44 µg/mL), S. sonnei (0.61–4.88 µg/mL), P. shigel-
loides (0.037–0.3 µg/mL) and S. paratyphi A (0.037–
2.44 µg/mL). The synthesized compounds are more ac-
tive than norfloxacin (2.44 µg/mL) against V. para-
haemolyticus (0.3–1.22 µg/mL), Shigella flexneri (0.018–
1.22 µg/mL) and Morgonella morganii (0.018–0.6 µg/
mL). The compounds are also more active than norfloxa-
cin (0.3 µg/mL) against Escherichia coli (≤ 0.018–
0.15 µg/mL) and A. hydrophile (≤ 0.018–0.075 µg/mL).
Seven compounds (S3–S9) are more active (312.5–
3. Biological investigation and discussion
Compounds S1–S9 were evaluated for their in vitro
antibacterial activity against 28 pathogenic bacteria by
conventional agar dilution procedures and the results of
these assays are summerized in table I. The data for
norfloxacin are included for comparison. The observation
from the antibacterial activity of norfloxacin Mannich
bases showed very good activity against tested bacteria.
All the compounds are more active than norfloxacin
against Vibrio parahaemolyticus, Edwardsiella tarda,
Vibrio cholerae 0139, Staphylococcus aureus, Escheri-
chia coli NCTC 10418, Streptococcus faecalis, Aeromo-

251
Table I. Biological testing results from antibacterial screening: MICs in µg/mL.
Drugs
Micro-organism
S-1
S-2
S-3
S-4
S-5
2.44
1.22
0.075
0.30
S-6
1.22
S-7
0.61
0.61
0.3
0.6
0.037
1 250
S-8
0.61
1.22
0.15
0.3
0.037
625
0.037
S-9
0.15
Nor-
floxacin
Salmonella typhimurium
Vibrio parahaemolyticus
Salmonella paratyphi B
Edwardsiella tarda
Vibrio cholerae 0139
Staphylococcus aureus
0.3
0.3
0.3
0.3
0.3
2.44
1.22
0.075
1.22
1.22
0.3
2.44
≤ 0.018
9.76
9.76
0.61
0.075
0.15
0.075
625
0.3
0.3
0.037
0.15
0.037
1 250
0.037
0.075
0.037
625
0.018
0.61
0.61
1 250
≤ 0.018 0.018
0.018
0.3
0.61
39.06
625
2 500
0.037
0.075
0.075
0.018
625
≤ 0.018 0.3
≤ 0.018 ≤ 0.018
0.61
1 250
0.018
1 250
0.018
0.018
1.22
0.61
78.12
625
2 500
Escherichia coli NCTC 10418 0.15
0.15
0.075
Vibrio cholerae non-01
Streptococcus faecalis
Salmonella typhi
Pseudomonas aeruginosa
Klebsiella pneumoniae
Staphylococcus albus
Salmonella enteritidis
Aeromonas hydrophila
Vibrio cholerae-01
Bacillus subtilis
Shigella sonnei
Shigella boydii
Plesiomonas shigelloides
Proteus rettgeri
Shigella flexneri
≤ 0.018 ≤ 0.018 ≤ 0.018 0.037
≤ 0.018 0.018
0.61
0.3
0.15
0.075
≤ 0.018 0.61
19.53
625
312.5
≤ 0.018 0.037
≤ 0.018 ≤ 0.018 0.037
≤ 0.018 ≤ 0.018 ≤ 0.018 ≤ 0.018 0.037
0.61
1.22
2.44
0.3
0.15
9.76
0.018
39.06
2 500
625
0.018
19.53
1 250
312.5
0.018
0.037
78.12
625
1 250
0.075
0.037
78.12
312.5
625
0.075
0.075
0.075
0.075
0.61
0.075
0.037
0.15
0.018
0.037
0.037
0.15
≤ 0.018 ≤ 0.018
78.12
625
78.12
625
2 500
19.53
1 250
312.5
≤ 0.018
0.3
≤ 0.018
1.22
9.76
2 500
0.037
625
0.037
0.075
0.018
0.018
0.018
0.61
≤ 0.018 ≤ 0.018 ≤ 0.018 0.037
0.037
1.22
4.88
0.075
0.3
0.018
1.22
4.88
0.15
4.88
0.15
0.15
1.22
1.22
0.075
0.075
0.075
0.3
0.15
4.88
0.075
0.075
0.6
0.3
2.44
0.15
1.22
0.037
0.037
0.6
0.037
0.037
0.075
0.6
0.15
0.075
0.15
0.61
0.018
0.037
0.075
0.3
0.075
0.075
0.037
0.075
0.075
0.15
0.15
≤ 0.018
9.76
9.76
0.3
0.075
0.075
0.018
≤ 0.018 ≤ 0.018
≤ 0.018 ≤ 0.018
0.075
0.075
0.037
0.15
1.22
0.61
0.61
0.61
0.61
0.6
2.44
Proteus vulgaris
Enterobacter
Morgonella morganii
Citrobacter freundii
Proteus morgonii
0.037
0.075
0.075
0.075
0.075
0.075
0.037
0.075
0.075
0.037
0.075
0.075
0.018
0.018
0.018
0.037
0.075
0.037
0.075
0.037
0.075
0.15
0.15
0.15
2.44
0.075
0.15
2.44
0.15
0.15
0.61
≤ 0.018
0.018
9.76
0.3
Salmonella paratyphi A
0.15
MIC – Minimum inhibitory concentration
1250 µg/mL) than norfloxacin (2 500 µg/mL) against
Klebsiella pneumoniae. Seven compounds (S3–S9) are
more active (0.018–0.61 µg/mL) and two compounds (S1
and S2) are equipotent to that of norfloxacin (1.22 µg/
mL) against Bacillus subtilis. In general, the order of
antibacterial activity of the substituents at the 5th position
of isatin is Br > Cl > H. In the case of the substitution of
the 3-keto derivative, order of activity is trimethoprim >
sulfadiazine > sulfadoxine. Compound S-9 showed ex-
cellent in vitro antibacterial activity. Sulfonamides and
trimethoprim showed antibacterial activity by inhibiting
the folate reductase, whereas quinolones exert their
antibacterial activity by inhibiting the subunit A of DNA
gyrase [12]. The better antibacterial activity of the syn-
thesized Mannich bases might be due to the inhibitory
effect of both the folate reductase and DNA gyrase
enzymes. In vivo antibacterial activities of the Mannich
bases against an experimentally induced infection of mice
after oral administration are given in table II, together
with the in vitro activity against the infecting strains.
Norfloxacin was used as the reference compound. Com-
pounds S-4 and S-9 are more active than norfloxacin
(ED50: 6 mg/kg body weight) with ED50’s of 1.25 and
1.62 mg/kg body weight, respectively. From the compari-
son of the activities of these compounds, S-4 is 4.8 times
Table II. In vitro and in vivo antibacterial activities and acute oral
toxicities.
E. coli NCTC 10418
Compound MIC, µg/mL ED50, mg/kg, LD50 mg/kg
T.I.a
p.o.
S-3
S-4
S-9
Norfloxacin
0.075
0.018
0.018
0.3
10.0
1.25
1.62
6.0
> 4 000
> 4 000
3 200
> 400
> 3 200
1 975
666
4 000
aRatio of LD50 to ED50

252
Table III. Antifungal activity of the compounds: MIC in µg/mL.
Micro-organism
Epidermophyton Microsporum Histoplasma
Drug
Cryptococcus Microsporum Trichophyton
Candida
albicans
Aspergillus
niger
neoformans
audouinii
mentagrophytes
floccosum
gypsum
capsulatum
S-1
S-2
S-3
S-4
S-5
S-6
S-7
S-8
2.44
2.44
2.44
1.22
2.44
1.22
2.44
0.6
9.76
19.53
9.76
0.6
0.6
0.6
0.6
0.6
0.6
19.53
4.88
156.25
156.25
156.25
39.06
19.53
39.06
19.53
9.76
4.88
4.88
2.44
2.44
4.88
2.44
4.88
1.22
1.22
19.53
2.44
9.76
4.88
2.44
1.22
1.22
0.6
2.44
9.76
2.44
19.53
2.44
4.88
2.44
2.44
1.22
2.44
1.22
4.88
1.22
2.44
19.53
19.53
156.25
156.25
156.25
156.25
156.25
312.5
156.25
156.25
78.12
156.25
156.25
78.12
9.76
39.06
19.53
78.12
78.12
19.53
156.25
2.44
S-9
Norfloxacin
Clotrimazole
0.6
4.88
2.44
4.88
156.25
2.44
312.5
0.3
MIC – Minimum inhibitory concentration
and S-9 is 3.7 times more active than norfloxacin. The in
vivo activity may be ascribed due to the increased water
solubility of the corresponding Mannich bases of nor-
floxacin.
mans, three compounds (S4–S6) are more active
(0.6–1.22 µg/mL) against Microsporum gypsum. Com-
pound S-9 showed better antifungal activity when com-
pared to other Mannich bases.
The results from the antifungal study (table III) show
that all the compounds exhibited significant antifungal
activity. All the Mannich bases are more potent than
norfloxacin against the fungi tested. When compared to
clotrimazole (MIC: 19.53 µg/mL) all the compounds are
more active (1.22–2.44 µg/mL) against Histoplasma cap-
sulatum. Six compounds (S4–S9) are more active
(0.6 µg/mL) than clotrimazole (4.88 µg/mL) against Mi-
crosporum audouinii. When compared to clotrimazole
(2.44 µg/mL), four compounds (S4, S6, S8 and S9) are
more active (0.6–1.22 µg/mL), five compounds (S1–S3,
S5 and S7) are equipotent against Cryptococcus neofor-
The compounds were evaluated for their inhibitory
effect on the replication of HIV-1 in human MT-4 cells
(table IV). Compounds S-8 and S-9 showed inhibition
with an EC50 of 11.3 and 13.9 µg/mL, respectively, and
the selectivity index is up to 5. Other compounds showed
marked anti-HIV activity at a concentration below their
toxicity threshold.
The acute toxicities of the selected compounds are
summarized in table II. It is apparent that the toxicities of
S-3 and S-4 were significantly lower than that of nor-
floxacin.
4. Experimental protocols
Table IV. Anti-HIV activity of the compounds.
SIc
Max. Prot.d
a
b
Compound
EC50
,
CC50 ,
4.1. Chemistry
µg/mL
µg/mL
S-1
S-2
S-3
S-4
S-5
S-6
S-7
S-8
S-9
> 48
> 63
> 62
> 88
> 51
> 34
> 66
11.3
13.9
48.5
63.1
61.8
88.5
51.2
33.9
66.5
57.6
54.7
< 1
< 1
< 1
< 1
< 1
< 1
< 1
5
0
1
0
4
3
2
7
95
73
Melting points were determined in open capillary tubes
on a Thomas-Hoover melting point apparatus and are
uncorrected. IR spectra were recorded for KBr pellets on
1
a Jasco IR report-100 infrared spectrophotometer. H-
NMR spectra were determined at 90 MHz on a Jeol 90Q
FT spectrometer with tetramethyl silane as an internal
standard. The purity of the compounds was checked by
TLC on silica-gel coated glass plates visualized by iodine
vapour. Microanalyses were performed by the microana-
lytical unit, Central Drug Research Institute, India. All
compounds (table V) gave satisfactory elemental analy-
4
a
Effective dose of compound achieving 50% protection in MT-4
cells against the cytopathic effect of HIV-1. bCytotoxic dose of
compound required to reduce the viability of mock infected MT-4
cells by 50%. cSelectivity index or ratio of CC50 to EC50.
dMaximum protection in %.
1
ses. IR and H-NMR spectra were consistent with the
assigned structures.

253
Table V. Physical constants of the compounds.
No.
S-1
R
H
R'
Yield (%)
76.56
M.p. (°C)
125
Mol. formula
C35H31O6N8F
Rf: C6H6:C2H5OH; 9:1
0.5950
S-2
S-3
S-4
Cl
Br
H
83.77
89.41
67.07
120
121
119
C35H30O6N8FCl
C35H30O6N8FBr
C37H35O8N8F
0.6097
0.6226
0.6078
S-5
S-6
S-7
Cl
Br
H
71.23
66.44
76.36
127
135
122
C37H34O8N8FCl
C37H34O8N8FBr
C39H39O7N8F
0.6126
0.6250
0.5869
S-8
S-9
Cl
Br
63.77
64.19
126
141
C39H38O7N8FCl
0.6216
0.6390
C39H38O7N8FBr
4.1.1. Synthesis of 3-(4'-sulfadiazinyl) isatin
Equimolar (0.06 mol) quantities of isatin and sulfadi-
azine were dissolved in warm ethanol in the presence of
two drops of glacial acetic acid and refluxed on a water
bath for 1 h. The reaction mixture was allowed to stand
for 24 h at room temperature. The precipitated product

254
was collected and recrystallized from ethanol. Yield:
81.1%; m.p.: 240 °C; IR (KBr): 3 400, 1 650, 1 320,
4.1.6. 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7[[N4-(3'-
sulfadoximino-1'-isatinyl)methyl]N1-piperazinyl]-
3-quinoline carboxylic acid S-4
1
1 156, 800, 700 cm–1; H-NMR (DMSO-d6) δ (ppm):
6.5–7.5 (m, 8H, Ar–H), 8.5 (m, 3H, pyrimidinyl), 10.4 (s,
1H, NH), 11.2 (s, 1H, SO2NH).
Yield: 64.07%; m.p.: 119 °C; IR (KBr); 2 840, 1 730,
1
1 620, 1 583, 1 167, 1 596 cm–1; H-NMR (CDCl3) δ
(ppm): 1.60 (t, 3H, CH3 of C2H5), 3.2 (s, 6H, OCH3),
3.8–4.0 (m, 8H, piperazine-H), 4.34 (q, 2H, CH2 of
C2H5), 4.9 (s, 2H, -NCH2N-), 6.6–8.35 (m, 11H, Ar–H),
8.64 (s, 1H, C2–H), 10.9 (s, 1H, -SO2NH); calculated for
C37H35O8N8F: C, 60.15; H, 5.19; N, 15.16; found: C,
60.11; H, 5.10; N, 14.92%.
4.1.2. Synthesis of 3-(4'-sulfadoximino) isatin
Equimolar quantities of isatin and sulfadoxine were
refluxed for 4 h in ethanol in the presence of 2–3 drops of
glacial acetic acid. The contents were then cooled at room
temperature and the product was obtained by filtration,
washed with ethanol, dried and recrystallized from an
ethanol and chloroform mixture. Yield: 89.72%; m.p.:
4.1.7. 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7[[N4-
[3'-(4'-amino-5'-trimethoxybenzylpyrimidin-2'-
yl)imino-1'-isatinyl]methyl]N1-piperazinyl]-3-
quinoline carboxylic acid S-7
1
196 °C; IR (KBr): 3 230, 1 642, 1 579 cm–1; H-NMR
(CDCl3) δ (ppm) 6.75–7.55 (m, 9H, Ar–H), 10.1 (s, 1H,
NH), 11.0 (s, 1H, SO2NH).
Yield: 76.36%; m.p.: 122 °C; IR (KBr): 3 010, 2 860,
1
2 840, 1 730, 1 616, 1 506, 1 236, 1 129 cm–1; H-NMR
4.1.3. Synthesis of 3-[4'-amino-5'-(3',4',5'-
trimethoxybenzyl)pyrimidinyl]imino isatin
(CDCl3) δ (ppm): 1.60 (t, 3H, CH3 of C2H5), 3.3 (s, 2H,
-CCH2C-), 3.54 (s, 9H, -OCH3), 3.7–4.1 (m, 8H,
piperazine-H), 4.2 (q, 2H, CH2 of C2H5), 5.1 (s, 2H,
-NCH2N-), 5.8 (s, 2H, NH2), 6.58–8.56 (m, 9H, Ar–H),
8.6 (s, 1H, C2–H); calculated for C39H39O7N8F: C, 62.39;
H, 5.23; N, 14.92; found: C, 62.12; H, 5.36; N, 14.73%.
Isatin and trimethoprim in equimolar quantities were
dissolved in ethanol containing 1 mL of glacial acetic
acid. The reaction mixture was refluxed for 4 h and set
aside. The resulting solid was washed with dilute ethanol,
dried and recrystallized from an ethanol and chloroform
mixture. Yield: 96%; m.p.: 185 °C; IR (KBr): 3 300,
1
3 050, 1 660, 1 620, 1 580 cm–1; H-NMR (CDCl3) δ
4.2. Biology
(ppm): 3.2 (s, 2H, CH2), 3.7 (s, 9H, -OCH3), 5.6 (s, 2H,
NH2), 6.8–7.1 (m, 7H, Ar–H), 10.6 (s, 1H, SO2NH).
4.2.1. In vitro antibacterial screening
Compounds were evaluated for their in vitro antibac-
terial activity against 28 pathogenic bacteria procured
from the Department of Microbiology, Institute of Medi-
cal Sciences, Banaras Hindu University. The agar dilution
method [13] was performed using Mueller-Hinton agar
(Hi-Media) medium. Suspensions of each micro-
organism were prepared to contain approximately 106
colony forming units (cfu/mL) and applied to plates with
serially diluted compounds in DMF to be tested and
incubated at 37 °C overnight (approx. 18–20 h). The
minimum inhibitory concentration (MIC) was considered
to be the lowest concentration that completely inhibited
growth on agar plates, disregarding a single colony or a
faint haze caused by the inoculum.
4.1.4. General procedure for the
preparation of Mannich bases
To
a solution of 1-ethyl-6-fluoro-1,4-dihydro-7-
piperazin-1-yl-4-oxo quinoline-3-carboxylic acid (nor-
floxacin, 0.02 mol) in glacial acetic acid (50 mL), was
added isatin derivatives (0.02 mol) and 37% formalin
(1 mL). The reaction mixture was refluxed over a water
bath for 1–3 h. TLC control indicated that norfloxacin
had disappeared almost completely. The reaction mixture
was concentrated to approximately half of the initial
volume, and the resulting precipitate was recrystallized
from a mixture of DMF and water.
4.1.5. 1-Ethyl-6-fluoro-1,4-dihydro-4-oxo-7[[N4-(3'-
sulfadiazinimino-1'-isatinyl)methyl]N1-piperazinyl]-
3-quinoline carboxylic acid S-1
4.2.2. In vivo antibacterial studies
(mouse protection test)
Yield: 76.56%; m.p.:125 °C; IR (KBr): 2 850, 1 730,
The in vivo antibacterial activity of the test compounds
was determined in CF-strain male mice (20–25 g body
weight, six per group). The mice were infected intraperi-
toneally with a suspension containing an amount of the
indicated organism slightly greater than its lethal dose
100 (LD100). The mice were treated orally (p.o.) with a
specific amount of the test compound administered at 1
and 4 h after infection. ED50 values were calculated by
1
1 640, 1 620, 1 325, 1 155 cm–1; H-NMR (CDCl3) δ
(ppm): 1.78 (t, 3H, CH3 of C2H5), 3.7–4.1 (m, 8H,
piperazine-H), 4.88 (q, 2H, CH2 of C2H5), 5.2 (s, 2H,
-NCH2N-), 6.7–8.5 (m, 13H, Ar–H), 9.32 (s, 1H, C2–H),
11.3 (s, 1H, -SO2NH); calculated for C35H31O6N8F: C,
61.9; H, 4.6; N, 16.5. Found: C, 61.74; H, 4.30; N,
16.41%.

255
interpolation among survival rates in each group after a
week. They express the total dose of compound (mg/kg)
required to protect 50% of the mice from an experimen-
tally induced lethal systemic infection of the indicated
organism.
body weight, six per group). Seven days later, LD50
values were determined by using the Weil method [17].
Acknowledgements
4.2.3. In vitro antifungal screening
The compounds were evaluated for their in vitro
antifungal activity against Candida albicans, Aspergillus
niger, C. neoformans, Microsporum audouinii, Tricho-
phyton mentagrophytes, Epidermophyton floccosum, Mi-
crosporum gypsum and H. capsulatum using an agar
dilution method with Sabouraud’s dextrose agar (Hi-
Media). Suspensions of each micro-organism were pre-
pared to contain 105 cfu/mL and applied to agar plates
which have been serially diluted with compounds to be
tested in DMF. The plates were incubated at 25 °C for
48–72 h and MICs were determined.
The authors are thankful to the Head of the Department
of Pharmaceutics and Department of Microbiology, Ba-
naras Hindu University for providing the necessary fa-
cilities for carrying out this work.
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