Newer aminopyrimidinimino isatin analogues as non-nucleoside HIV-1 reverse transcriptase inhibitors for HIV and other opportunistic infections of AIDS: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.farmac.2005.03.005

Human immuno deficiency virus (HIV) weakens the immune system so that many opportunistic infections (OIs) like tuberculosis, hepatitis, bacterial infections etc can develop. In this paper, we designed aminopyrimidinimino isatin lead compound as a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) with broad-spectrum chemotherapeutic properties for the effective treatment of AIDS and AIDS-related OIs. Compound 1-cyclopropyl-6-fluoro-1,4- dihydro-4-oxo-7-[[N⁴-[3′-(4′-amino-5′- trimethoxybenzyl pyrimidin-2′-yl)imino-1′-(5-methylisatinyl)]methyl] -N¹-piperazinyl]-3-quinoline carboxylic acid (10) emerged as the most potent broad-spectrum chemotherapeutic agent active against HIV, HCV, Mycobacterium tuberculosis and various pathogenic bacteria.

Full text of DOI:10.1016/j.farmac.2005.03.005

Il Farmaco 60 (2005) 377–384
http://france.elsevier.com/direct/FARMAC/
Newer aminopyrimidinimino isatin analogues as non-nucleoside
HIV-1 reverse transcriptase inhibitors for HIV and other opportunistic
infections of AIDS: design, synthesis and biological evaluation
D. Sriram *, T.R. Bal, P.Yogeeswari
Medicinal Chemistry Research Laboratory, Pharmacy Group, Birla Institute of Technology and Science, Pilani 333031, India
Received 24 September 2004; received in revised form 8 February 2005; accepted 5 March 2005
Available online 03 May 2005
Abstract
Human immuno deficiency virus (HIV) weakens the immune system so that many opportunistic infections (OIs) like tuberculosis, hepa-
titis, bacterial infections etc can develop. In this paper, we designed aminopyrimidinimino isatin lead compound as a novel non-nucleoside
reverse transcriptase inhibitor (NNRTI) with broad-spectrum chemotherapeutic properties for the effective treatment of AIDS and AIDS-
related OIs. Compound 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-[[N4-[3’-(4’-amino-5’-trimethoxybenzyl pyrimidin-2’-yl)imino-1’-(5-
methylisatinyl)]methyl]-N1-piperazinyl]-3-quinoline carboxylic acid (10) emerged as the most potent broad-spectrum chemotherapeutic agent
active against HIV, HCV, Mycobacterium tuberculosis and various pathogenic bacteria.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Anti-HIV; Anti-HCV; Antituberculosis; Antibacterial; Isatin
1. Introduction
in some individuals [3].An ideal drug for HIV/AIDS patients
should suppress HIV replication thereby acting as anti-HIV
drug and also should treat OIs like TB, hepatitis and other
bacterial infections. Earlier works in our laboratory have iden-
tified various isatinimino derivatives exhibiting broad-
spectrum chemotherapeutic properties [4]. As a continuation
to our effort in developing broad-spectrum chemotherapeu-
tics, we undertook the present study to design, synthesize and
evaluate aminopyrimidinimino isatin analogs, which could
suppress HIV replication and also inhibit the opportunistic
microorganisms.
Human immuno deficiency virus (HIV) has been identi-
fied as the probable causative agent forAIDS. The HIV infec-
tion, which targets the monocytes expressing surface
CD4 receptors, eventually produces profound defects in cell-
mediated immunity [1]. Overtime infection leads to severe
depletion of CD4 T-lymphocytes (T-cells) resulting in oppor-
tunistic infection (OIs) like tuberculosis (TB), fungal, viral,
protozoal and neoplastic diseases and ultimately death. TB is
the most common OI in people with AIDS and it is the lead-
ing killer of people with AIDS. The co-infection by hepatitis
C virus (HCV) and HIV is quite common, mainly because
these infections share the same parenteral, sexual and verti-
cal routes of transmission [2].Although classical OIs are now
rarely seen, the toxicity of antiretroviral drugs as well as liver
diseases caused by HCV represent an increasing cause of mor-
bidity and mortality among HIV-positive persons. Predispos-
ing liver damage favors a higher rate of hepatotoxicity of anti-
retroviral drugs, which can limit the benefit of HIV treatment
2. Experimental
Melting points (m.p.) were determined in one end open
capillary tubes on a Büchi 530 m.p. apparatus and are uncor-
rected. Infrared (IR) and proton nuclear magnetic resonance
(1H-NMR) spectra were recorded for the compounds on Jasco
IR Report 100 (KBr) and Brucker Avance (300 MHz) instru-
ments, respectively. Chemical shifts are reported in parts per
million (ppm) using tetramethyl silane (TMS) as an internal
standard. Elemental analyses (C, H, and N) were undertaken
* Corresponding author. Tel.: +91 1596 24 4684; fax: +91 1596 24 4183.
E-mail address: dsriram@bits-pilani.ac.in (D. Sriram).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.03.005

378
D. Sriram et al. / Il Farmaco 60 (2005) 377–384
with Perkin–Elmer model 240C analyzer. The homogeneity
of the compounds was monitored by ascending thin layer
chromatography (TLC) on silica gel-G (Merck) coated alu-
minium plates, visualized by iodine vapor. Developing sol-
vents were chloroform/methanol (9:1). The pharmacophoric
distance map and log P values were determined using
Alchemy-2000 and Scilog P softwares (Tripos Co.).
(s, 9H, –OCH3), 3.9–4.1 (m, 8H, piperazine-H), 5.2 (s, 2H,
–NCH2N–), 5.65 (s, 2H, NH2), 6.67–7.82 (m, 11H, Ar–H);
Calculated for C34H37N7O4: C, 67.2; H, 6.14; N, 16.13; found:
C, 67.02; H, 6.20; N, 16.32.
2.2.3. 1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-[[N4-
[3’-(4’-amino-5’-trimethoxybenzyl pyrimidin-2’-yl)imino-
1’-(5-methylisatinyl)] methyl] N’-piperazinyl]-3-quinoline
carboxylic acid (10)
Yield: 70.5%; m.p.: 164 °C; IR (KBr): 3010, 2850, 2840,
1736, 1620, 1506, 1236, 1125, cm–1; 1H-NMR (CDCl3) d
(ppm): 0.88–1.12 (m, 4H, cyclopropyl-H), 1.84 (s, 3H, CH3)
3.3 (s, 2H, CH2 of benzyl), 3.5 (m, 1H, cyclopropyl-H), 3.62
(s, 9H, –OCH3), 3.7–4.1 (m, 8H, –piperazine-H), 5.16 (s, 2H,
–NCH2N), 5.8 (s, 2H, NH2), 6.58–8.40 (m, 9H, Ar–H), 8.6
(s, 1H, C2-H); Calcd. for C41H41N8O7F: C, 63.39; H, 5.32;
N, 14.42; found: C, 63.28; H, 5.36; N, 14.40.
2.1. Synthesis of (3-{[4’-amino-5(3″,4″,5″-trimethoxyben-
zyl) pyrimidin-2’-yl]} imino}-5-methyl-1,3-dihydro-
2H-indol-2-one)
Equimolar quantities (0.01 mol) of 5-methylisatin and
5-(3’,4’,5’-trimethoxybenzyl)-2,4-diaminopyrimidine were
dissolved in warm ethanol containing 1 ml of glacial acetic
acid. The reaction mixture was irradiated in an unmodified
domestic microwave oven at 80% intensity with 30 s per cycle
for 3 min and set aside. The resultant solid was washed with
dilute ethanol dried and recrystallized from ethanol–chloro-
form mixture. Yield: 81.4%; m.p.: 232 °C; IR (KBr): 3300,
2045, 1660, 1620, 1580, cm–1; 1H-NMR (CDCl3) d (ppm):
1.82 (s, 3H, CH3), 3.22 (s, 2H, CH2), 3.7 (s, 9H, –OCH3),
5.46 (s, 2H, NH2), 6.8–7.6 (m, 6H,Ar–H), 10.68 (s, 1H, –NH).
2.3. Anti-HIV screening
2.3.1. In MT-4 cells
The compounds were tested for anti-HIV activity against
replication of HIV-1 (III B) in MT-4 cells. The MT-4 cells
were grown in RPMI-1640 DM (Dutch modification) medium
(Flow lab, Irvine Scotland), supplemented with 10% (v/v)
heat-inactivated calf serum and 20-µg ml–1 gentamicin (E.
Merck, Darmstadt, Germany). HIV-1 (III B) was obtained
from the culture supernatant of HIV-1 infected MT-4 cell lines
and the virus stocks were stored at –70 °C until used. Anti-
HIV assays were carried out in microtitre plates filled with
100 µl of medium and 25 µl volumes of compounds in tripli-
cate so as to allow simultaneous evaluation of their effects on
HIV and mock-infected cells. Fifty microlitres of HIV at
100 CCID50 medium were added to either the HIV-infected
or mock-infected part of the microtitre tray. The cell cultures
were incubated at 37 °C in a humidified atmosphere of 5%
CO2 in air. Five days after infection the viability of mock and
HIV-infected cells were examined spectrophotometrically by
the MTT method.
2.2. General procedure for the preparation of Mannich
bases
To a suspension of 3-{[4’-amino-5-(3″,4″,5″-trimethoxy-
benzyl) pyrimidin-2’-yl]} imino}-5-methyl-1,3-dihydro-2H-
indol-2-one (0.02 mol) in ethanol was added appropriate sec-
ondary amines (0.02 mol) and 37% formaldehyde (0.5 ml)
and irradiated in a microwave oven at an intensity of 80%
with 30 s per cycle. The number of cycle in turn depended on
the completion of the reaction, which was checked by TLC.
The reaction timing varied from 1.5 to 3 min. The solution
obtained after the completion of the reaction was kept at 0 °C
for 30 min and the resulting precipitate was recrystallized
from a mixture of DMF and water.
2.2.1. (3-{[4’-Amino-5’-(3″,4″,5″-trimethoxybenzyl)
pyrimidin-2’-yl]} imino}-5-methyl-1-[(4-chlorophenyl pip-
erazinyl) methyl]-1,3-dihydro-2H-indol-2-one) (2)
Yield: 65.6%; m.p.: 79 °C; IR (KBr): 3010, 2850, 2830,
2.3.2. In CEM cells
Candidate agents were dissolved in dimethylsulfoxide, and
then diluted 1:100 in cell culture medium before preparing
serial half-log10 dilutions. T4 lymphocytes (CEM cell-line)
were added and after a brief interval HIV-1 was added, result-
ing in a 1:200 final dilution of the compound. Uninfected
cells with the compound served as a toxicity control, and
infected and uninfected cells without the compound served
as basic controls. Cultures were incubated at 37 °C in a 5%
carbon dioxide atmosphere for 6 days. The tetrazolium salt,
XTT was added to all the wells, and cultures were incubated
to allow formazan color development by viable cells. Indi-
vidual wells were analyzed spectrophotometrically to quan-
titative formazan production, and in addition were viewed
microscopically for detection of viable cells and confirma-
tion of protective activity.
1
1730, 1620, 1500, 1240, cm–1; H-NMR (CDCl3) d (ppm):
1.8 (s, 3H, CH3), 3.18 (s, 2H, CH2 of trimethoxybenzyl), 3.6
(s, 9H, –OCH3), 3.9–4.2 (m, 8H, piperazine-H), 5.2 (s, 2H,
–NCH2N–), 5.65 (s, 2H, NH2), 6.7–7.82 (m, 10H, Ar–H);
Calculated for C34H36N7O4Cl: C, 63.59; H, 5.65; N, 15.27;
found: C, 63.60; H, 5.60; N, 15.12.
2.2.2. (3-{[4’-Amino-5’-(3″,4″,5″-trimethoxybenzyl)
pyrimidin-2’-yl]} imino}-5-methyl-1-[(4-phenyl piperazi-
nyl) methyl]-1,3-dihydro-2H-indol-2-one) (5)
Yield: 64.5%; m.p.: 97 °C; IR (KBr): 3010, 2850, 2840,
1
1730, 1616, 1500, 1240, cm–1; H-NMR (CDCl3) d (ppm):
1.8 (s, 3H, CH3), 3.17 (s, 2H, CH2 of trimethoxybenzyl), 3.65

D. Sriram et al. / Il Farmaco 60 (2005) 377–384
379
2.3.3. HIV-1RT assay
cellular gene GAPDH, respectively. First, the blot was hybrid-
ized with two riboprobes directed against the negative strand
of HCV RNA and the GAPDH mRNA, respectively. After
one night of hybridization at 68 °C, the membrane was washed
then exposed to X-ray film and a phosphor screen for quan-
titative analysis. The amount of GAPDH mRNA was used as
an internal loading control to standardize the amount of HCV
RNA detected. The same membrane was subsequently hybrid-
ized with a negative-sense riboprobe to determine the level
of HCV-positive strand RNA using the same approach.
The reaction mixture (50 µl) contained 50 mM Tris–HCl
(pH 7.8), 5 mM dithiothreitol, 30 mM glutathione, 50 µM
EDTA, 150 mM KCl, 5 mM MgCl2, 1.25 µg of bovine serum
albumine, an appropriate concentration of the radiolabeled
substrate [3H] dGTP, 0.1 mM poly(mC)·oligo(dG) as the
template/primer, 0.06% Triton X-100, 10 µl of inhibitor solu-
tion (containing various concentrations of compounds), and
1 µl of RT preparation. The reaction mixtures were incubated
at 37 °C for 15 min, at which time 100 µl of calf thymus
DNA (150 µg ml–1), 2 ml of Na4P2O7 (0.1 M in 1 M HCl),
and 2 ml of trichloroacetic acid (10% v/v) were added. The
solutions were kept on ice for 30 min, after which the acid-
insoluble material was washed and analyzed for radioactiv-
ity. For the experiments in which 50% inhibitory concentra-
tion (IC50) of the test compounds was determined, fixed
concentration of 2.5 µM [3H] dGTP was used.
2.5. Antimycobacterial screening
Primary screening was conducted at 6.25 µg ml–1 against
Mycobacterium tuberculosis strain H37Rv (ATCC 27294) in
BACTEC 12B medium using a broth micro dilution assay,
the Microplate Alamar Blue Assay (MABA) [14].
2.4. Antiviral and cytotoxicity assays for HCV
2.6. In vitro antibacterial activity
Compounds were evaluated for their in vitro antibacterial
activity against 28 pathogenic bacteria procured from the
Department of Microbiology, Institute of Medical Sciences,
Banaras Hindu University, India. The agar dilution method
was performed using Mueller-Hinton agar (Hi-Media) me-
dium. Suspensions of each microorganism were prepared to
contain approximately 106 colony forming units (cfu ml–1)
and applied to plates with serially diluted compounds in DMF
to be tested and incubated at 37 °C overnight (approximately
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 inoculums.
2.4.1. Cell culture
Huh-7 cells the subgenomic HCV replicon BM4-5 cells
were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) (Life Technologies) supplemented with 10% fetal
bovine serum, 1% L-glutamine, 1% L-pyruvate, 1% penicil-
lin and 1% streptomycin supplemented with 500 mg ml–1
G418 (Geneticin, Invitrogen). Cells were passaged every
4 days.
2.4.2. Cytotoxicity
Huh-7 cells were, respectively, seeded at a density of 3 ×
10–4 cells per well in 96-well plates for the cell viability assay,
or at a density of 6 × 10–5 cells per well in six-well plates for
the antiviral assay. Sixteen hours post seeding, cells were
treated with the compounds at 50 µg ml–1 for 3 days. The
administration of each drug was renewed each day. Other
drugs, including ribavirin (ICN Pharmaceuticals, USA),
mycophenolic acid (Sigma, USA), and interferon alpha-2b
(IntronA) were used in the same conditions as positive con-
trols. At the end of treatment, cell viability assays were per-
formed with the 96-well plates using Neutral Red assay
(Sigma).
2.7. In vivo antibacterial activity (mouse protection test)
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 intraperitoneally 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 interpolation among survival rates
in each group after a week. They express the total dose of
compound (mg kg–1) required to protect 50% of the mice
from an experimentally induced lethal systemic infection of
the indicated organism.
2.4.3. Antiviral assay
Total RNA (tRNA) was extracted from six-well plates with
the ’Extract All’ reagent (Eurobio), which is a mixture of
guanidinium thiocyanate–phenol–chloroform. Northern Blot
analysis was then performed using the NorthernMaxTM-Gly
(Ambion) kit, following manufacturer’s instruction. Ten
micrograms of tRNA was denatured in glyoxal buffer at 50 °C
for 30 min and separated by agarose gel electrophoresis, then
transferred for 12 h onto a charged nylon membrane (Bio-
dyne B, Merck Eurolab). Hybridization was carried out with
three different [32P] CTP-labeled riboprobes obtained by in
vitro transcription (Promega). These probes were comple-
mentary to the NS5 A region of the HCV genome, and to the
3. Results and discussion
3.1. Design
To qualify as a non-nucleoside reverse transcriptase inhibi-
tors (NNRTI), the compound should interact specifically with

380
D. Sriram et al. / Il Farmaco 60 (2005) 377–384
a non-substrate binding site of the reverse transcriptase (RT)
of HIV-1, and inhibit the replication of HIV-1 at a concentra-
tion that is significantly lower than the concentration required
to affect normal cell viability [5]. Based on these premises,
more than thirty different classes of NNRTI’s could be con-
sidered [6]. Although the NNRTI’s seemingly belong to
widely diverging classes of compounds, closer inspection
reveals that most have some features in common, that is a
carboxamide, or (thio) urea entity (’body’), surrounded by
two hydrophobic, mostly aryl moieties (’wings’), one of which
is quite often substituted by a halogen group (Fig. 1). Thus,
the overall structure may be considered reminiscent of a but-
terfly with hydrophilic center (’body’) and two hydrophobic
outskirts (’wing’). In the present study, the aminopyrimidin-
imino isatin analogs are designed in accord to this hypoth-
esis. The iminocarbamoyl moiety (–N=C–CO–N–) consti-
Fig. 3. Protocol for the synthetic compounds.
tutes the ’body’ and the aryl ring of isatin and the pyrimidine
derivative constitute the ’wings’ as depicted in Fig. 1. The
distance between the hydrophilic center (A) and hydropho-
bic outskirts (B and C) and the angle between the two aryl
rings (B and C) were measured using Tripos Alchemy-
2000 software [7] from the energy minimized structures using
MM3 program. The lead compound was found to comply
within the specification of the pharmacophoric distance map
(Fig. 2 and Table 1).
Fig. 1. Existing NNRTI’s and lead compound.
3.2. Synthesis
The synthesis of various aminopyrimidinimino isatin
derivatives was achieved in two steps (Fig. 3) [8]. 5-Methyli-
satin was condensed with 5-trimethoxybenzyl-2,4-diamino
pyrimidine in the presence of glacial acetic acid to form
Schiff’s base. The N-Mannich bases of the above Schiff’s base
were synthesized by condensing acidic imino group of isatin
with formaldehyde and various secondary amines. All com-
pounds (Tables 2,3) gave satisfactory elemental analysis. IR
and 1H-NMR spectra were consistent with the assigned struc-
tures.
Fig. 2. Schematic representation of a butterfly-like configuration of NNRTI’S
and the pharmacophoric distance map.
Table 1
The distance between the pharmacophoric functional groups of anti-HIV drugs and the lead compound
Drugs
AB (in Å)
4.36–5.19
4.22–6.67
4.20–6.72
4.25–6.60
4.20–6.72
4.23–5.34
BC (in Å)
4.25–6.25
6.53–7.6
4.44–7.08
4.86–7.02
4.25–7.6
4.07–6.63
CA (in Å)
9.4
Angle Bac
119°
Loviride
Trovirdine
9.44
117.5°
Indole carboxamide
Benzothiadiazine-1-oxide
Range
9.44
118°
9.12
114.9°
9.12–9.44
9.113
114.9–119°
116.7°
Lead compound

D. Sriram et al. / Il Farmaco 60 (2005) 377–384
381
Table 2
Physical constants of the synthesized compounds 1–12
Compound
R’
Molecular formula
Molecular weight
531.60
Yield (%)
65.60
M.p. (°C)
162
log P
1
C28H33N7O4
C34H36N7O4Cl
C35H39N7O5
6.12
2
3
642.14
637.72
60.25
57.85
79
8.04
7.99
132
4
C34H36N8O6
652.7
66.70
80
7.01
5
6
C
34H37N7O4
33H36N8O4
607.70
608.69
64.50
68.00
97
87
7.84
7.54
C
7
8
C
35H36N7O4F3
675.70
516.59
65.30
60.50
64
9.29
4.01
C28H32N6O4
117
9
C
29H34N6O4
530.61
776.81
61.40
70.50
60
4.52
5.69
10
C41H41N8O7F
164
11
12
C
41H42N8O7F2
796.819
820.86
69.00
72.50
147
141
6.29
6.20
C43H45N8O8F
3.3. Biological activities
mately 3. In the T4 lymphocytes (CEM cell lines), tested com-
pounds showed marked anti-HIV activity (10–20%) at a con-
centration below their toxicity threshold. The loss of activity
might be due to degeneration/rapid metabolism in the culture
conditions used in the screening procedure.
Compound 10 was evaluated for the inhibitory effects on
HIV-1 RT enzyme [10] and their IC50 value was found to be
28.4 3.4 µM. The in vitro IC50 values for HIV-1 RT with
Poly (mC) oligo (dG) as the template/primer were signifi-
cantly higher than the corresponding EC50 values for inhibi-
The synthesized compounds were evaluated for their
inhibitory effect on the replication of HIV-1 in MT-4 and CEM
cell lines (Table 4) [9]. In the MT-4 cell lines, compound 10
was found to be the most active against replication of HIV-
1 with EC50 of 11.6 µM and their selectivity index
(SI = CC50/EC50) was found to be more than 7 with maxi-
mum protection of 84.9%. Other compounds (3, 11 and 12)
showed maximum protection of 64–72% with SI of approxi-

382
D. Sriram et al. / Il Farmaco 60 (2005) 377–384
Table 3
Anti-HIV, anti-HCV and antimycobacterial activity
Compound
Anti-HIV activity (µM)
Anti-HCV activity at
50 µg ml–1
Antimycobacterial
activity at
6.25 µg ml–1
Cell growth Viral RNA % Inhibition
MT-4 cell line
(%)
replication
(%)
96
a
b
a
EC50
CC50
94.2
49.2
32.1
% Protection
19.2
EC50
1
2
> 94.2
17.2
> 89.2
> 36.1
> 11.9
> 37.4
> 121.0
> 121.0
> 118.0
> 122.0
> 105.0
NT
57
67
15
74
78
78
81
93
74
81
68
76
24
72.6
90
28
3
4
5
6
7
8
9
10
11
12
> 32.1
> 47.96
> 116.1
> 119.6
> 106.2
> 124
> 124.6
11.6
26.94
16.4
100
93
15
47.96
116.1
119.6
106.2
124
30
26.2
91
18
14.20
26.4
72
25
87
21
34.6
90
19
124.6
86.1
24.6
99
22
84.9
97
100
100
100
28.4
92.6
64.0
NT
92
21.2
81.4
66.8
NT
65
NT indicates not tested.
a 50% effective concentration, or concentration required to inhibit HIV-1 induced cytopathicity in cell lines by 50%.
b 50% cytotoxic concentration, or concentration required to reduce the viability of mock-infected cell lines by 50%.
tion of the cytopathic effect of HIV-1 in MT-4 cell culture.
This discrepancy is not unusual for NNRTI’s as it may reflect
the differences between the in vitro HIV-1 RT assay, in which
a synthetic template/primer is used, and the cellular systems
[11].
RNA replication at about 90–100%. Compound 8 was found
to be most active with inhibition on viral replication of 90%
and non-toxic to Huh-7 cells (93% cell growth). This paper is
first of its kind in which isatin derivatives are reported to pos-
sess anti--HCV activity.
All the synthesized compounds were also evaluated for
their inhibition of HCV viral RNA replication in HUH-
7 cells at 50-µg ml–1 [12], and the results are presented in
Table 4. Among these, eight compounds inhibited HCV viral
The synthesized compounds were also screened against
M. tuberculosis strain H37Rv (ATCC 27294) in BACTEC 12B
medium initially at 6.25 µg ml–1 (Table 4) [13]. Three com-
pounds (10–12) showed complete inhibition (100%) of
Table 4
In vitro antibacterial activity (MIC’s in µM)
Microorganism
K. ozaenae
1
2
3
4
5
6
7
8
9
0.0459
11.7562
0.0115
0.0115
11.7562
11.7562
0.0229
47.025
0.0115
23.5125
23.5125
0.0115
0.1837
11.7562
11.7562
11.7562
0.0459
11.7562
2.9390
11.7562
2.9390
2.9390
2.9390
11.7562
0.0095
38.95
0.0095
0.1521
38.95
38.95
0.0380
77.9
0.0096
19.6
0.0094
9.575
0.0748
0.0374
19.15
19.15
0.0094
38.3
0.0100
20.5675
0.0803
0.0201
20.5675
20.5675
0.0100
41.135
0.0402
20.5675
10.2838
0.1607
0.0803
20.5675
20.5675
20.5675
0.0100
20.5675
20.5675
20.5675
20.5675
5.1418
20.5675
10.2838
0.0090
20.535
0.1604
0.1604
20.535
20.535
0.0090
41.07
0.0118
18.5
0.0236
0.0118
0.0945
0.0945
48.39
0.1840
1.4723
0.0115
0.0115
1.4723
23.5575
0.0920
23.5575
0.0460
1.4723
1.4723
0.0460
0.0460
23.5575
23.5575
0.0115
0.0115
0.0115
5.8894
0.0115
23.5575
23.5575
11.7788
11.7788
K. pneumoniae
S. sonnei
0.0096
0.0766
19.6
0.0301
0.0301
18.5
Plesiomonas
S. boydii
M. morganii
S. aureus
19.6
18.5
48.39
0.0096
78.4
0.0118
37.00
0.0722
37
0.1890
48.39
P. aeruginosa
V. mimicus
0.0190
77.9
0.0096
19.6
0.0374
19.15
38.3
0.0802
41.07
0.0473
48.39
V. fluvialis
V. cholerae 0139
V. cholerae 01
V. parahaemolyticus
E. Coli NCTC10418
E. tarda
38.95
0.0095
0.0380
38.95
1.2172
19.475
0.0761
38.95
0.0095
38.95
19.475
2.4343
2.4343
38.95
19.6
41.07
37
0.0118
0.0236
0.0945
48.39
0.1531
0.1531
19.6
0.1496
0.0374
19.15
19.15
19.15
0.0094
19.15
4.7875
19.15
19.15
4.7875
19.15
4.7875
0.1604
0.0401
41.07
0.0722
0.0181
18.5
19.6
20.535
20.535
0.0090
20.535
10.2675
20.535
20.535
20.535
20.535
10.2675
18.5
24.195
48.39
P. vulgaris
19.6
18.5
P. mirabilis
0.0383
19.6
0.1445
37.00
9.25
0.1890
48.39
S. typhimurium
S. paratyphi A
S. typhi
9.8
1.5122
48.39
19.6
18.5
S. enteritidis
C. ferundii
9.8
18.5
24.195
24.195
1.5122
24.195
4.9
18.5
Enterobacter
B. megatherius
1.225
0.6125
18.5
9.25

D. Sriram et al. / Il Farmaco 60 (2005) 377–384
383
Table 5
In vitro antibacterial activity (MIC’s in µM)
Microorganism
K. ozaenae
10
11
12
Cipro
Lome
Gati
0.0002
0.0002
0.0079
0.0079
0.0079
0.0079
0.0002
0.0002
0.0157
0.0157
0.0157
0.0005
0.0002
0.0002
0.0002
0.0002
0.01571
0.0002
0.0002
0.0002
0.0002
0.0002
0.0005
0.0157
0.0613
0.0002
0.0002
0.0002
0.0005
0.0002
0.0002
0.0613
0.0306
0.0613
0.0153
0.0153
0.0002
0.0010
0.0005
0.0002
0.0002
0.0077
0.0002
0.0002
0.0002
0.0002
0.0002
0.0002
0.0149
0.0074
0.0149
0.0019
0.0019
0.0019
0.0297
0.0074
0.0074
0.0037
0.0074
0.0297
0.0005
0.0005
0.0009
0.0009
0.0019
0.0037
0.0074
0.0149
0.0074
0.0037
0.0018
0.0009
0.0092
0.0023
0.0023
0.0023
0.0023
0.0023
0.0023
0.0092
0.0023
0.0023
0.0023
0.0023
0.0023
0.0011
0.0023
0.0023
0.0023
0.0023
0.0023
0.0023
0.0023
0.0023
0.0023
0.0023
0.0629
0.1259
2.0156
0.0629
0.5039
0.0629
0.0314
0.2519
0.0629
0.0629
0.1259
0.0009
2.0156
0.0314
0.2519
0.0314
0.1259
0.2519
0.0314
0.5039
1.0078
1.0078
1.0078
1.0078
0.0037
0.0037
0.0037
0.1182
0.0590
0.0009
0.0009
0.0074
0.0074
0.0009
0.0009
0.0009
0.4727
0.0009
0.0009
0.0009
0.0009
0.0009
0.0009
0.0009
0.0009
0.0037
0.0009
0.0037
K. pneumoniae
S. sonnei
Plesiomonas
S. boydii
M. morganii
S. aureus
P. aeruginosa
V. mimicus
V. fluvialis
V. cholerae 0139
V. cholerae 01
V. parahaemolyticus
E. Coli NCTC10418
E. tarda
P. vulgaris
P. mirabilis
S. typhimurium
S. paratyphi A
S. typhi
S. enteritidis
C. ferundii
Enterobacter
B. megatherius
M. tuberculosis in the primary screening. In the secondary
level screening the actual MIC and cytotoxicity inVERO cells
of these three compounds were determined. The MIC’s of
these compounds were found to be 3.13 µg ml–1 and they
were not cytotoxic upto 62.5 µg ml–1 to VERO cells.
N-1 position was found to be equipotent against 4 and more
active against 5 tested bacteria, when compared to gatifloxa-
cin. These data are in consistent with our earlier results [15].
In vivo antibacterial activity of some selected compounds
against an experimentally induced infection of mice after oral
administration [15] are presented in Table 6, along with the
in vitro activity against the infecting organism E. coli NCTC
10418. Ciprofloxacin and lomefloxacin were used as refer-
ence compounds. Compound 10 was twice more active than
ciprofloxacin with ED50 of 0.62 mg kg–1 and compound 11
was equally active as lomefloxacin (ED50: 1.87 mg kg–1)
against the tested bacteria.
All the compounds were evaluated for their in vitro anti-
bacterial activity against 24 pathogenic bacteria by conven-
tional agar dilution procedures [14] and the results of these
assays are summarized in Tables 5,6. The data for ciprofloxa-
cin, lomefloxacin and gatifloxacin were included for compari-
son. The antibacterial activity data revealed that all the test
compounds showed mild to moderate activity against tested
bacteria. The most sensitive organisms for the tested com-
pounds were Kleb. Ozaenae, Shig. Sonnei, Plesiomonas,
Staph. aureus, Vibrio cholerae 01, V. mimicus, V. para-
haemolyticus, and Prot. mirabilis, as these compounds inhib-
ited them at a concentration less than 1 µM. Compound 10,
which containing ciprofloxacin moiety at N-1 position was
found to be more active than ciprofloxacin against 15 tested
bacteria. When compared to lomefloxacin, compound 11
(lomefloxacin derivative) was found to be more active against
23 tested bacteria. Compound 12 bearing gatifloxacin at
The enhanced in vitro and in vivo antibacterial activity of
the fluoroquinolone derivatives 10–12 might be due to the
presence of bulky lipophilic isatinyl moiety at the C-7 pip-
erazine.
To conclude, four compounds of the 12 new derivatives
developed in this work showed inhibition against replication
of HIV-1 in MT-4 cells with EC50 ranging from 11.6 to
28.4 µM. All compounds were active against HCV RNA rep-
lication showing > 65% inhibition at 50 µg ml–1. Three com-
pounds inhibited M. tuberculosis H37Rv with MIC of
3.13 µg ml–1. Three compounds showed very good activity
against various pathogenic bacteria. Among the synthesized
compounds, compound 10 emerged as more promising broad-
spectrum chemotherapeutic agents.
Table 6
In vivo antibacterial study on E.coli NCTC 10419 strain
Compound
In vitro MIC
(in µM)
0.0002
In vivo EC50
(in mg per kg body wt.)
10
11
0.62
1.87
1.25
1.87
Acknowledgements
0.0010
Ciprofloxacin
Lomefloxacin
0.0011
One of the authors Ms. Tanushree Bal deeply acknowl-
edges the Council of Scientific and Industrial Research, India,
0.0314

384
D. Sriram et al. / Il Farmaco 60 (2005) 377–384
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