Design, synthesis and biological evaluation of novel non-nucleoside HIV-1 reverse transcriptase inhibitors with broad-spectrum chemotherapeutic properties

Article abstract of DOI:10.1016/j.bmc.2004.08.028

Aminopyrimidinimino-5-fluoroisatin compounds were designed as a novel non-nucleoside reverse transcriptase inhibitor with broad-spectrum chemotherapeutic properties that include anti-HIV, anti-HCV, anti-tuberculous and anti-bacterial activities. Acquired immunodeficiency syndrome (AIDS) results from infection by the retrovirus, human immunodeficiency virus (HIV). HIV is the most significant risk factor for many opportunistic infections like tuberculosis, hepatitis, bacterial infections, etc. In this paper, we designed aminopyrimidinimino isatin lead compound as a novel non-nucleoside reverse transcriptase inhibitor with broad-spectrum chemotherapeutic properties for the effective treatment of AIDS and AIDS-related opportunistic infections. Compound 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7[[N⁴-[3′-(4′-amino- 5′-trimethoxybenzylpyrimidin-2′-yl)imino-1′-(5-fluoroisatinyl) ]methyl]-N¹-piperazinyl]-3-quinoline carboxylic acid (12) 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.bmc.2004.08.028

Bioorganic & Medicinal Chemistry 12 (2004) 5865–5873
Design, synthesis and biological evaluation of novel non-nucleoside
HIV-1 reverse transcriptase inhibitors with broad-spectrum
chemotherapeutic properties
Dharmarajan Sriram,* Tanushree Ratan Bal and Perumal Yogeeswari
Medicinal Chemistry Research Laboratory, Pharmacy group, Birla Institute of Technology and Science, Pilani 333031, India
Received 6 August 2004; revised 20 August 2004; accepted 21 August 2004
Available online 28 September 2004
Abstract—Acquired immunodeficiency syndrome (AIDS) results from infection by the retrovirus, human immunodeficiency virus
(HIV). HIV is the most significant risk factor for many opportunistic infections like tuberculosis, hepatitis, bacterial infections,
etc. In this paper, we designed aminopyrimidinimino isatin lead compound as a novel non-nucleoside reverse transcriptase inhibitor
with broad-spectrum chemotherapeutic properties for the effective treatment of AIDS and AIDS-related opportunistic infections.
Compound
1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7[[N4-[30-(40-amino-50-trimethoxybenzylpyrimidin-20-yl)imino-10-(5-fluoroisatin-
yl)]methyl]-N1-piperazinyl]-3-quinoline carboxylic acid (12) emerged as the most potent broad-spectrum chemotherapeutic agent
active against HIV, HCV, Mycobacterium tuberculosis and various pathogenic bacteria.
ꢀ 2004 Published by Elsevier Ltd.
1.Introduction
Although classical OIs are now rarely seen, the toxicity
of anti-retroviral drugs as well as liver diseases caused
by HCV represent an increasing cause of morbidity
and mortality among HIV-positive persons. Predispos-
ing liver damage favours a higher rate of hepatotoxicity
of anti-retroviral drugs, which can limit the benefit of
HIV treatment in some individuals.6 Through logic
and orderly thinking, it appears that 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 identified various
isatinimino derivatives exhibiting broad-spectrum chemo-
therapeutic properties.7 As a continuation to our effort
in developing broad-spectrum chemotherapeutics, we
undertook the present study to design, synthesize and
evaluate aminopyrimidinimino isatin analogues, which
could suppress HIV replication and also inhibit the
opportunistic microorganisms.
Since first reported in the 1980s, acquired immunodefi-
ciency syndrome (AIDS), which is caused by the human
immunodeficiency virus (HIV) and results in life threat-
ening opportunistic infections and malignancies, has
spread rapidly through the human population and be-
come a major worldwide pandemic.1,2 The global AIDS
epidemic claimed more than three million lives in 2003,
and ca. 40 million people were living with HIV or AIDS
at the end of that same year.3 The HIV infection, which
targets the monocytes expressing surface CD4receptors,
eventually produces profound defects in cell-mediated
immunity.4 Overtime infection leads to severe depletion
of CD4T-lymphocytes (T-cells) resulting in opportunis-
tic infections (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 leading killer of people with AIDS. The coinfec-
tion by hepatitis C virus (HCV) and HIV is quite com-
mon, mainly because these infections share the same
parenteral, sexual and vertical routes of transmission.5
2.Chemistry
2.1. Design
Keywords: Anti-HIV; Anti-HCV; Anti-tuberculous; Anti-bacterial;
Isatin.
To qualify as a non-nucleoside reverse transcriptase
inhibitors (NNRTI), the compound should interact spe-
cifically with a non-substrate binding site of the reverse
*
Corresponding author. Tel.: +91 1596 244684; fax: +91 1596
244183; e-mail: dsriram@bits-pilani.ac.in
0968-0896/$ - see front matter ꢀ 2004Published by Elsevier Ltd.
doi:10.1016/j.bmc.2004.08.028

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D. Sriram et al. / Bioorg. Med. Chem. 12 (2004) 5865–5873
N
O
O
Cl
O
S
H
Cl
Br
NH2
N
NH
N
H
O
Cl
N
N
H
S
H2N
O
Indole Carboxamide
OCH3
Loviride
Trovirdine
N
N
N
CH2
OCH3
O
S
F
N
O
NH2
OCH3
N
N
H
R1
S
Lead Compound
Benzothiadiazine-1-oxide
Figure 1. Existing NNRTIꢀs and lead compound.
Table 1. The distance between the pharmacophoric functional groups
of anti-HIV drugs and the lead compound
transcriptase (RT) of HIV-1, and inhibit the replication
of HIV-1 at a concentration that is significantly lower
than the concentration required to affect normal cell via-
bility.8 Based on these premises, more than 30 different
classes of NNRTIꢀs could be considered.9 Although
the NNRTIꢀs seemingly belong to widely diverging
classes of compounds, closer inspection reveals that
most have some features in common, that is, a carboxa-
mide, or (thio) urea entity (ꢁbodyꢀ), surrounded by two
hydrophobic, mostly aryl moieties (ꢁwingsꢀ), one of
which is quite often substituted by a halogen group
(Fig. 1). Thus, the overall structure may be considered
reminiscent of a butterfly with hydrophilic centre
(ꢁbodyꢀ) and two hydrophobic outskirts (ꢁwingꢀ). In the
present study, the aminopyrimidinimino isatin ana-
logues are designed in accord to this hypothesis. The
iminocarbamoyl moiety (–N@C–CO–N–) constitutes
the ꢁbodyꢀ and the aryl ring of isatin and the pyrimidine
derivative constitute the ꢁwingsꢀ as depicted in Figure 1.
The distance between the hydrophilic centre (A) and
hydrophobic outskirts (B and C) and the angle between
the two aryl rings (B and C) were measured using Tripos
Alchemy 2000 software10 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).
Drugs
AB
(in A)
BC
(in A)
CA
(in A)
Angle
BAC
˚
˚
˚
Loviride
Trovirdine
4.36–5.19 4.25–6.25 9.4
4.22–6.67 6.53–7.6 9.44
119ꢁ
117.5ꢁ
118ꢁ
Indole carboxamide 4.20–6.72 4.44–7.08 9.44
Benzothiadiazine-1- 4.25–6.60 4.86–7.02 9.12
oxide
114.9ꢁ
Range
Lead compound
4.20–6.72 4.25–7.6 9.12–9.44 114.9ꢁ–119ꢁ
4.23–5.34 4.07–6.63 9.113
116.7ꢁ
2.2. Synthesis
The synthesis of various aminopyrimidinimino isatin
derivatives was achieved in two steps (Fig. 3).11 5-Fluor-
oisatin was condensed with 5-trimethoxybenzyl-2,4-
diaminopyrimidine in the presence of glacial acetic acid
to form Schiffꢀs base. The N-Mannich bases of the above
Schiffꢀs base were synthesized by condensing acidic imi-
no group of isatin with formaldehyde and various sec-
ondary amines. All compounds (Tables 2 and 3) gave
satisfactory elemental analysis. IR and 1H NMR spectra
were consistent with the assigned structures.
Hydrophilic site
3.Results and discussion
A
The synthesized compounds were evaluated for their
inhibitory effect on the replication of HIV-1 in MT-4
and CEM cell lines (Table 4).12 In the MT-4cell lines,
compound 12 was found to be the most active against
replication of HIV-1 with EC50 of 12.1lg/mL and their
selectivity index (SI = CC50/EC50) was found to be more
than 13 with maximum protection of 96.6%. Other com-
pounds (4, 9, 13–15) showed maximum protection of
54–86% with SI of 2–8. In the T4 lymphocytes (CEM
4.2 – 6.7 Å
9.12 – 9.44 Å
114-119o
C
B
Lipophilic site
4.25 - 7.6 Å
Lipophilic site
Figure 2. Schematic representation of a butterfly-like configuration of
NNRTIꢀS and the pharmacophoric distance map.

D. Sriram et al. / Bioorg. Med. Chem. 12 (2004) 5865–5873
5867
OCH3
OCH3
O
F
N
H2N
CH2
O
N
N
H
NH2
OCH3
CH3COOH
Schiff Reaction
OCH3
OCH3
N
N
N
CH2
NH2
F
O
OCH3
N
H
R1
,
HCHO
NH
R2
Mannich Reaction
OCH3
OCH3
N
N
CH2
F
N
O
NH2
OCH3
N
R1
CH2
N
R2
1-15
Figure 3. Protocol for the synthetic compounds.
cell lines), only compound 14 was found to be active
with EC50 of 50.8lg/mL with maximum protection of
63%. Other tested compounds showed marked anti-
HIV activity (11–47%) at a concentration below their
toxicity threshold. The loss of activity might be due to
degeneration/rapid metabolism in the culture conditions
used in the screening procedure. Moreover the bioactiv-
ity did not have any correlation with the logP.
paper is first of its kind in which isatin derivatives are
reported to possess anti-HCV activity.
The synthesized compounds were also screened against
Mycobacterium tuberculosis strain H37Rv (ATCC
27294) in BACTEC 12B medium at a initial concentra-
tion of 6.25lg/mL (Table 4).16 Four compounds (12–15)
showed complete inhibition (100%) of M. tuberculosis in
the primary screening. In the secondary level screening
the actual minimum inhibitory concentration (MIC)
and cytotoxicity in VERO cells of these three com-
pounds were determined. The MICꢀs of these com-
pounds were found to be 3.13lg/mL and they were
not cytotoxic upto 62.5lg/mL to VERO cells.
Further, compound 12 was evaluated for the inhibitory
effects on HIV-1 RT enzyme13 and their IC50 value was
found to be 32.6 6.4lM. The in vitro IC50 values for
HIV-1 RT with Poly (mC) oligo (dG) as the template/pri-
mer were significantly higher than the corresponding
EC50 values for inhibition of the cytopathic effect of
HIV-1 in MT-4cell culture. This discrepancy is not
unusual for NNRTIꢀs 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.14
All the compounds were evaluated for their in vitro anti-
bacterial activity against 24pathogenic bacteria by con-
ventional agar dilution procedures17 and the results of
the assays are summarized in Tables 5 and 6. The data
for norfloxacin, ciprofloxacin, lomefloxacin and gatifl-
oxacin were included for comparison. The anti-bacterial
activity data revealed that all the test compounds
showed mild to moderate activity against tested
bacteria. The most sensitive organisms for the tested
compounds were K. ozaenae, Vibrio mimicus, E. coli
NCTC10439, P. mirabilis, S. Typhimurium and S.
enteritidis as these compounds inhibited them at a con-
centration less than 2.5lg/mL. Compound 5, which con-
tain 4-chlorophenyl piperazinomethyl moiety at N-1
position was found to be the most active compound
All the synthesized compounds were also evaluated for
their inhibition of HCV viral RNA replication in
HUH-7 cells at 50lg/mL,15 and the results are presented
in Table 4. Among these, six compounds (1, 3, 8, 10, 11
and 15) were found to be less toxic to Huh-7 cells (cell
growth of >80%) and inhibited HCV viral RNA replica-
tion at about 82–100%. Compound 11 was found to be
the most active analogue showing 100% inhibition on
viral replication and was non-toxic to Huh-7 cells. This

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D. Sriram et al. / Bioorg. Med. Chem. 12 (2004) 5865–5873
Table 2. Physical constants of the synthesized compounds 1–10
OCH3
OCH3
N
N
N
CH2
F
O
NH2
OCH3
N
CH2 R'
Compound
R0
Molecular formula
Molecular weight
522.57
Yield (%)
62.08
Mp (ꢁC)
LogP
C2H5
C2H5
1
N
N
C27H31N6O4F
123
4.00
CH3
CH3
2
3
C25H27N6O4F
C34H36N7O4F
494.52
625.69
60.10
58.65
77
79
3.23
8.30
N
N
CH2
N
C6H5
N
4
5
C33H33N7O4ClF
C33H33N7O4ClF
646.11
646.11
65.81
66.10
112
114
8.32
8.31
Cl
N
N
Cl
N
N
6
C28H32N7O4F
549.60
69.00
111
6.28
CH3
N
N
7
8
C34H36N7O5F
C33H34N7O4F
641.70
611.67
60.50
62.50
132
60
8.09
8.01
OCH3
N
N
C6H5
N
N
9
C34H33N7O4F4
679.66
520.56
65.27
67.91
76
8.89
3.58
CF3
N
10
C27H29N6O4F
137
being more potent than lomefloxacin against 20 tested
bacteria. Compound 12 containing norfloxacin moiety
at N-1 position was found to be more active than norfl-
oxacin against 18 tested bacteria. Compound 13 con-
taining ciprofloxacin moiety at N-1 position was found
to be more active than ciprofloxacin against 13 tested
bacteria. When compared to lomefloxacin, compound
14 (lomefloxacin derivative) was found to be more active
against 23 tested bacteria. Compound 15 bearing gatifl-
oxacin at N-1 position was found to be more active than
gatifloxacin against six tested bacteria. The better anti-
bacterial activity of these compounds might be due to
the inhibitory effect of both bacterial dihydrofolate
reductase and DNA gyrase enzymes. These data are
consistent with our earlier results in which the deriva-
tives of the known fluoroquinolones were potent than
the parent drug.18
In vivo anti-bacterial activity of some selected compounds
against an experimentally induced infection of mice after
oral administration18 is presented in Table 7, along with
the in vitro activity against the infecting organism E. coli
NCTC 10418. Norfloxacin, ciprofloxacin and lomefloxa-
cin were used as reference compounds. Compound 12 was
found to be three times more active (ED50: 1.87mg/kg
body weight) than norfloxacin (ED50: 6.0mg/kg) while

D. Sriram et al. / Bioorg. Med. Chem. 12 (2004) 5865–5873
Table 3. Physical constants of the synthesized compounds 11–15
5869
Compound
R0
Molecular formula
Molecular weight
Yield (%)
65
Mp (ꢁC)
LogP
11
N
N
CH3
C29H33N6O4F
548.61
99
4.59
O
F
COOH
12
C39H38N8O7F2
768.77
63
2645.33
N
N
C2H5
O
F
COOH
13
C40H38N8O7F2
780.77
64230
5.29
N
N
N
O
F
COOH
14
C40H39N8O7F3
800.78
68
212
5.71
N
N
N
F
C2H5
CH3
O
N
F
COOH
15
C42H42N8O8F2
824.83
69
144
5.86
N
N
OCH3
CH3
Table 4. Anti-HIV, anti-HCV and anti-mycobacterial activities
Compound
Anti-HIV activity
Anti-HCV activity
at 50lg/mL
Anti-mycobacterial
activity at 6.25lg/mL
MT-4cell line
CEM cell line
Cell
growth (%)
Viral RNA
replication (%)
Inhibition (%)
a
b
a
b
EC50
CC50
Protection (%) EC50
CC50
Protection (%)
1
>141.01 141.0
30.2
12.1
34.6
46.1
59.4
42.6
22.1
21.6
54.2
40.1
23.6
99.6
86.2
61.6
56.8
>116.0 116.0
NT NT
11.28
NT
90
55
87
63
66
68
10
91
71
90
97
100
38
73
89
82
100
97
19
19
37
20
45
24
63
33
40
36
24
2
>62.7
>139.3
>121.6
36.2
62.7
139.3
121.6
90.1
3
>112.0 112.0
>104.0 104.0
11.53
15.53
47.47
13.91
NT
4
92
90
5
>95.6 95.6
>115.0 115.0
6
>106.7
>81.6
>84.7
62.1
106.7
81.6
84.7
96
7
NT
NT
NT
NT
100
98
8
NT
9
136.6
129.6
69.6
>127.0 127.0
>121.0 121.0
10.95
14.25
NT
88
88
10
11
12
13
14
15
>129.6
>69.6
12.1
NT NT
50.8 1398.0
100
160.2
141.6
130.9
91.6
63.844 95
NT
NT
17.9
57.1
NT
NT
NT
NT
100
89
100
100
100
NT
NT
25.2
NT
92
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%.

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D. Sriram et al. / Bioorg. Med. Chem. 12 (2004) 5865–5873
Table 5. In vitro anti-bacterial activity (MICꢀs in lg/mL)
Microorganism
1
2
3
4
5
6
7
8
9
10
K. ozaenae
0.31
2500
625
0.31
0.31
1250
625
0.31
625
0.1526
1.22
1.22
2.44
1.22
2.44
2.44
K. pneumoniae
S. sonnei
1250
625
2500
625
2500
625
2500
312.5
625
1250
625
2500
2500
1250
1250
625
9.765
625
625
0.3051
0.6103
0.6103
0.6103
0.6103
1.22
Plesiomonas
S. boydii
2500
2500
2500
625
625
625
625
625
625
625
1250
2500
2500
2500
1250
0.1526
2500
2500
2500
78.125
2.44
625
625
625
M. morganii
S. aureus
625
625
625
312.5
625
312.5
1250
1.22
19.531
78.125
1250
1.22
625
625
312.5
1250
0.3051
1250
1250
0.3051
78.12
0.3051
625
78.125
1250
0.31
625
1250
2500
2.44
P. aeroginosa
V. mimicus
2500
0.31
2500
1250
0.31
19.531
0.31
625
1250
1.22
0.1526
625
625
0.3051
1.22
0.1526
312.5
0.6103
39.06
39.06
1.22
V. fluvialis
0.6103
1250
1250
0.6103
0.1526
312.5
312.5
0.6103
0.6103
1.22
1250
1250
4.88
1250
1250
1250
39.06
1.22
V. cholerae 0139
V. cholerae 01
V. parahaemolyticus
E. coli NCTC10418
E. tarda
625
0.3051
0.3051
2.44
0.31
78.12
0.31
0.31
0.31
0.31
0.1526
1250
0.6103
0.31
0.61
78.12
1.22
0.3051
19.531
1.22
39.062
1.22
0.3051
0.3051
4.88
625
625
312.5
312.5
0.6103
0.6103
625
4.88
2.44
39.062
156.25
0.6103
0.6103
1250
1250
2.44
4.88
625
P. vulgaris
625
625
0.31
P. mirabilis
S. typhimurium
S. paratyphi A
S. typhi
0.31
0.31
2500
2500
0.31
625
0.3051
0.3051
625
0.3051
0.3051
0.3051
0.1526
0.3051
0.3051
4.88
2.44
1.22
0.3051
0.6103
78.125
625
0.31
1250
625
2500
2500
2.44
625
625
1.22
312.5
1.22
1.22
S. enteritidis
C. ferundii
0.3051
625
0.3051
312.5
312.5
312.5
1.22
1.22
312.5
625
1250
1250
1250
1250
312.5
2500
312.5
1250
1250
Enterobacter
B. megatherius
9.765
625
156.25
625
312.5
625
0.3051
Table 6. In vitro anti-bacterial activity (MICꢀs in lg/mL)
Microorganism
11
12
13
14
15
Cipro
Lome
Gati
Nor
K. ozaenae
K. pneumoniae
S. sonnei
1.22
1.22
0.3051
0.0190
0.1526
0.1526
0.0381
0.1526
0.0763
0.0190
0.1526
0.1526
0.6103
0.1526
0.0095
0.0095
0.3051
0.0095
0.0095
0.0095
0.0190
0.0190
0.0095
0.0095
0.0095
0.0190
0.0095
0.6103
0.6103
0.6103
0.0095
0.0095
0.0095
2.441
0.3051
0.15
0.15
0.04
1.22
2.44
0.08
0.08
1.22
2.44
2500
1250
2500
2500
2500
2500
1250
2.44
0.3051
0.1526
0.1526
1.22
0.0763
0.0381
0.01
0.0439.06
0.04
0.08
1.22
1.22
9.76
2.44
1.22
Plesiomonas
S. boydii
2.44
0.049.77
0.041.22
0.040.61
0.15
M. morganii
S. aureus
0.1526
0.3051
1.22
0.0763
0.1526
0.31
0.02
0.02
9.76
39.06
P. aeroginosa
V. mimicus
4.88
0.15
19.53
1.22
1.22
2.44
2.44
2.44
0.31
1.22
2.44
0.0095
0.0095
1.22
0.0381
0.0763
0.1526
1.22
0.04
0.04
1.22
1.22
2.44
0.02
39.06
0.61
0.15
0.02
0.02
0.02
9.77
0.02
V. fluvialis
1250
2500
2500
78.125
1.22
V. cholerae 0139
V. cholerae 01
V. parahaemolyticus
E. coli NCTC10418
E. tarda
1.22
1.22
0.04
0.04
4.882
0.0763
0.0763
0.1526
0.0190
1.22
0.6103
0.3051
0.3051
0.0095
0.0095
0.0095
0.3051
0.0095
0.0095
0.3051
0.0095
0.1526
2.44
0.15
0.04
0.02
312.5
625
1.22
0.0763 .88 0.044 0.02
0.0190 0.040.61
9.76
0.02
P. vulgaris
0.61
P. mirabilis
S. typhimurium
S. paratyphi A
S. typhi
0.0095 0.402 0.042.4 1.22
0.0381 .88 0.044 0.02
1.22
1.22
0.31
0.02
0.02
19.53
0.08
0.02
0.08 .88
2500
2500
2.44
0.1526
0.3051
1.22
0.0763
0.0381
0.0190
0.0763
0.15
0.040.61
0.049.77
0.04
0.61
1.22
S. enteritidis
C. ferundii
0.02
9.76
9.76
1250
1250
1250
1.22
0.6103
0.1526
0.0419.53
0.0419.53
0.0419.53
Enterobacter
B. megatherius
4
2.4
4
0.0190
compound 13 was twice more active than ciprofloxacin
with ED50 of 0.62mg/kg and compound 14 was slightly
more active (ED50: 1.25mg/kg) than lomefloxacin
(ED50: 1.87mg/kg) against the tested bacteria.
Table 7. In vivo anti-bacterial study on E. coli NCTC 10419 strain
Compound
In vitro MIC
(lg/mL)
In vivo EC50
(mg/kg body wt)
12
13
0.076
1.87
0.62
1.25
6.0
0.0095
0.3051
0.30
14
4.Conclusion
Norfloxacin
Ciprofloxacin
Lomefloxacin
0.0190
0.6103
1.25
1.87
Six compounds of the 15 new derivatives developed in
this work showed inhibition against replication of

D. Sriram et al. / Bioorg. Med. Chem. 12 (2004) 5865–5873
5871
HIV-1 in MT-4cells with EC
ranging from 12.1 to
30min and the resulting precipitate was recrystallized
from a mixture of DMF and water.
50
62.1lg/mL. All the compounds were active against
HCV RNA replication showing >80% inhibition at
50lg/mL. Four compounds inhibited M. tuberculosis
H37Rv with MIC of 3.13lg/mL. Generally the com-
pounds, which possessed substituted piperazines showed
good activity. Especially the quinolone containing com-
pounds (12–15) had exhibited promising activity profile.
Five compounds showed very good activity against var-
ious pathogenic bacteria. In this series compound 12
emerged as a more promising broad-spectrum chemo-
therapeutic agent.
5.2.1. (3-{[40-Amino-50-(300,400,500-trimethoxybenzyl)pyrim-
idin-20-yl]imino}-5-bromo-1-[(dimethylamino)methyl]-1,3-
dihydro-2H-indol-2-one) (2). Yield: 60.10%; mp: 77ꢁC;
IR (KBr): 3010, 2850, 2840, 1730, 1616, 1506, 1236,
1129cmꢀ1 1H NMR (CDCl3): d (ppm) 2.5 (s, 6H,
;
–N(CH3), 3.16 (s, 2H, CH2 of benzyl), 3.7 (s, 9H,
–OCH3), 5.1 (s, 2H, –NCH2N–), 5.6 (s, 2H, NH2),
6.8–7.26 (m, 6H, Ar–H); calculated for C25H27N6O4F:
C, 60.72; H, 5.55; N, 16.99; found: C, 60.13; H, 5.42;
N, 17.09.
5.Experimental
5.2.2. (3-{[40-Amino-50-(300,400,500-trimethoxybenzyl)pyrim-
idin-20-yl]imino}-5-bromo-1-[(4-chlorophenyl
piperazi-
Yield:
Melting points were determined in one end open capil-
lary tubes on a Buchi 530 melting point apparatus and
¨
nyl)methyl]-1,3-dihydro-2H-indol-2-one)
(5).
66.10%; mp: 114ꢁC; IR (KBr): 3010, 2850, 2830, 1730,
are uncorrected. Infrared (IR) and proton nuclear mag-
netic resonance (1H NMR) spectra were recorded for
the compounds on Jasco IR Report 100 (KBr) and Bruc-
ker Avance (300MHz) instruments, respectively. Chem-
ical shifts are reported in parts per million (ppm) using
tetramethyl silane (TMS) as an internal standard. Ele-
mental analyses (C, H and N) were undertaken with Per-
kin–Elmer model 240C analyzer. The homogeneity of the
compounds was monitored by ascending thin layer chro-
matography (TLC) on silica gel-G (Merck) coated alu-
minium plates, visualized by iodine vapour. Developing
solvents were chloroform–methanol (9:1). The pharma-
cophoric distance map and logP values were determined
using Alchemy-2000 and Scilog P softwares (Tripos Co.).
1620, 1500, 1240,cmꢀ1 1H NMR (CDCl3): d (ppm)
;
3.17 (s, 2H, CH2 of trimethoxybenzyl), 3.65 (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, 10H,
Ar–H); calculated for C33H33N7O4ClF: C, 61.35; H,
5.15; N, 15.17; found: C, 61.60; H, 5.20; N, 15.22.
5.2.3. (3-{[40-Amino-50-(300,400,500-trimethoxybenzyl)pyrim-
idin-20-yl]imino}-5-bromo-1-[(piperidino)methyl]-1,3-di-
hydro-2H-indol-2-one) (10). Yield: 67.91%; mp: 137ꢁC;
IR (KBr): 3010, 2858, 2842, 1730, 1622, 1506, 1236,
1129cmꢀ1 1H NMR (CDCl3): d (ppm) 2.0 (m, 4H,
;
2 · CH2), 2.6 (t, 4H, –CH2N CH2–), 3.16 (s, 2H, CH2
of benzyl), 3.72 (s, 9H, –OCH3), 5.16 (s, 2H, –
NCH2N–), 5.6 (s, 2H, NH2), 6.82–7.26 (m, 6H, Ar–H);
calculated for C27H29N6O4F: C, 62.3; H, 5.62; N,
16.14; found: C, 62.13; H, 5.58; N, 16.19.
5.1. Synthesis of (3-{[40-amino-5(300,400,500-trimeth-
oxybenzyl)pyrimidin-20-yl]imino}-5-fluoro-1,3-dihydro-
2H-indol-2-one)
Equimolar quantities (0.01mol) of 5-flouroisatin and 5-
(30,40,50-trimethoxybenzyl)-2,4-diaminopyrimidine were
dissolved in warm ethanol containing 1mL of glacial
acetic acid. The reaction mixture was irradiated in an
unmodified domestic microwave oven19 at 80% intensity
with 30s/cycle for 3min and set aside. The resultant
solid was washed with dilute ethanol dried and recrystal-
lized from ethanol–chloroform mixture. Yield 78.4%;
mp: 197ꢁC; IR (KBr): 3300, 2050, 1665, 1620,
5.2.4. 1-Ethyl-6-fluoro-1,4-dihydro-4-oxo-7-[[N4-[30-(40-
amino-50-trimethoxybenzyl
pyrimidin-20-yl)imino-10-5-
(flouroisatinyl)]methyl]-N0-piperazinyl]-3-quinoline carb-
oxylic acid (12). Yield: 63.17%; mp: 264ꢁC; IR (KBr):
3010, 2850, 2840, 1736, 1620, 1596, 1506, 1236,
1125cmꢀ1 1H NMR (CDCl3): d (ppm) 1.28 (t, 3H,
;
CH3 of C2H5), 3.3 (s, 2H, CH2 of benzyl), 3.62 (s, 9H,
–OCH3), 3.7–4.1 (m, 8H, –piperazine–H), 4.25 (q, 2H,
CH2 of C2H5), 5.1 (s, 2H, –NCH2N), 5.8 (s, 2H,
NH2), 6.58–8.60 (m, 9H, Ar–H), 8.6 (s, 1H, C2–H); cal-
culated for C39H38N8O7F2: C, 60.93; H, 4.98; N, 14.58;
found: C, 60.82; H, 4.91; N, 14.30.
1580cmꢀ1 1H NMR (CDCl3): d (ppm) 3.20 (s, 2H,
;
CH2), 3.7 (s, 9H, –OCH3), 5.5 (s, 2H, NH2), 6.8–7.5
(m, 6H, Ar–H), 10.68 (s, 1H, –NH).
5.2. General procedure for the preparation of Mannich
bases
5.2.5. 1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-[[N4-
[30-(40-amino-50-trimethoxybenzyl pyrimidin-20-yl)imino-
10-5-(flouroisatinyl)]methyl]-N0-piperazinyl]-3-quinoline
carboxylic acid (13). Yield: 64.5%; mp: 230ꢁC; IR (KBr):
3010, 2850, 2844, 1740, 1628, 1596, 1506, 1236,
To
a
suspension of 3-{[40-amino-5-(300,400,500-tri-
methoxybenzyl)pyrimidin-20-yl]}imino}-5-fluoro-1,3-di-
hydro-2H-indol-2-one (0.02mol) in ethanol was added
appropriate secondary amines (0.02mol) and 37% for-
maldehyde (0.5mL) and irradiated in a microwave oven
at an intensity of 80% with 30s/cycle. The number of
cycle in turn depended on the completion of the reac-
tion, which was checked by TLC. The reaction timing
varied from 1.5 to 3min. The solution obtained after
the completion of the reaction was kept at 0ꢁC for
1125cmꢀ1 1H NMR (CDCl3): d (ppm) 0.88–1.1 (m,
;
4H, cyclopropyl–H), 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), 5.1 (s, 2H, –NCH2N), 5.8 (s,
2H, NH2), 6.58–8.60 (m, 9H, Ar–H), 8.6 (s, 1H, C2–
H); calculated for C40H38N8O7F2: C, 61.53; H, 4.91;
N, 14.35; found: C, 61.80; H, 4.97; N, 14.33.

5872
D. Sriram et al. / Bioorg. Med. Chem. 12 (2004) 5865–5873
5.3. Anti-HIV screening
modified Eagleꢀs medium (DMEM) (Life Technologies)
supplemented with 10% fetal bovine serum, 1% L-gluta-
mine, 1% L-pyruvate, 1% penicillin and 1% streptomycin
supplemented with 500mg/mL G418 (Geneticin, Invi-
trogen). Cells were passaged every 4days.
5.3.1. In MT-4 cells. The compounds were tested for
anti-HIV activity against replication of HIV-1 (III B)
in MT-4cells. The MT-4cells were grown in RPMI-
1640 DM (Dutch modification) medium (Flow lab,
Irvine Scotland), supplemented with 10% (v/v) heat-
inactivated calf serum and 20-lg/mL gentamicin (E.
Merck, Darmstadt, Germany). HIV-1 (III B) was ob-
tained from the culture supernatant of HIV-1 infected
MT-4cell lines and the virus stocks were stored at
ꢀ70ꢁC until used. Anti-HIV assays were carried out in
microtitre plates filled with 100lL of medium and
25lL volumes of compounds in triplicate 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 cul-
tures were incubated at 37ꢁC in a humidified atmos-
phere of 5% CO2 in air. Five days after infection the
viability of mock and HIV-infected cells were examined
spectrophotometrically by the MTT method.
5.4.2. Cytotoxicity. Huh-7 cells were, respectively, seeded
at a density of 3 · 10ꢀ4 cells/well in 96-well plates for the
cell-viability assay, or at a density of 6 · 10ꢀ5 cells/well in
six-well plates for the anti-viral assay. Sixteen hours
postseeding, cells were treated with the compounds at
50lg/mL for 3days. The administration of each drug
was renewed each day. Other drugs, including ribavirin
(ICN Pharmaceuticals, USA), mycophenolic acid (Sig-
ma, USA), and interferon alpha-2b (IntronA) were used
in the same conditions as positive controls. At the end of
treatment, cell-viability assays were performed with the
96-well plates using Neutral Red assay (Sigma).
5.4.3. Anti-viral assay. Total RNA (tRNA) was ex-
tracted from six-well plates with the ꢁExtract Allꢀ reagent
(Eurobio), which is a mixture of guanidinium thiocya-
nate–phenol–chloroform. Northern Blot analysis was
then performed using the NorthernMaxTM-Gly (Am-
bion) kit, following manufacturerꢀs instruction. Ten
micrograms of tRNA was denatured in glyoxal buffer
at 50ꢁC for 30min and separated by agarose gel electro-
phoresis, then transferred for 12h onto a charged nylon
membrane (Biodyne B, Merck Eurolab). Hybridization
was carried out with three different [32P]CTP-labelled
riboprobes obtained by in vitro transcription (Promega).
These probes were complementary to the NS5A region
of the HCV genome, and to the cellular gene GAPDH,
respectively. First, the blot was hybridized with two
riboprobes directed against the negative strand of
HCV RNA and the GAPDH mRNA, respectively.
After one night of hybridization at 68ꢁC, the membrane
was washed then exposed to X-ray film and a phosphor
screen for quantitative 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 hybridized with a
negative-sense riboprobe to determine the level of
HCV-positive strand RNA using the same approach.
5.3.2. 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, resulting 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 37ꢁC in a
5% carbon dioxide atmosphere for 6days. The tetrazo-
lium salt, XTT was added to all the wells, and cultures
were incubated to allow formazan colour development
by viable cells. Individual wells were analyzed spectro-
photometrically to quantitative formazan production,
and in addition were viewed microscopically for detection
of viable cells and confirmation of protective activity.
5.3.3. HIV-1 RT assay. The reaction mixture (50lL)
contained 50mM Tris–HCl (pH7.8), 5mM dithiothrei-
tol, 30mM glutathione, 50lM EDTA, 150mM KCl,
5mM MgCl2, 1.25lg of bovine serum albumin, an
appropriate concentration of the radiolabelled substrate
[3H] dGTP, 0.1mM poly(mC)Æoligo(dG) as the template/
primer, 0.06% Triton X-100, 10lL of inhibitor solution
(containing various concentrations of compounds) and
1lL of RT preparation. The reaction mixtures were
incubated at 37ꢁC for 15min, at which time 100lL of
calf thymus DNA (150lg/mL), 2mL of Na4P2O7
(0.1M in 1M HCl) and 2mL of trichloroacetic acid
(10% v/v) were added. The solutions were kept on ice
for 30min, after which the acid-insoluble material was
washed and analyzed for radioactivity. For the experi-
ments in which 50% inhibitory concentration (IC50) of
the test compounds was determined, fixed concentration
of 2.5M [3H] dGTP was used.
5.5. Anti-mycobacterial screening
Primary screening was conducted at 6.25lg/mL against
M. tuberculosis strain H37Rv (ATCC 27294) in BAC-
TEC 12B medium using a broth microdilution assay,
the Microplate Alamar Blue Assay (MABA).14
5.6. In vitro anti-bacterial activity
Compounds were evaluated for their in vitro anti-bacte-
rial 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 Muel-
ler-Hinton agar (Hi-Media) medium. Suspensions of
each microorganism were prepared to contain approxi-
mately 106 colony forming units (cfu/mL) and applied
to plates with serially diluted compounds in DMF to
5.4. Anti-viral and cytotoxicity assays against HCV
5.4.1. Cell culture. Huh-7 cells the subgenomic HCV
replicon BM4-5 cells were maintained in Dulbecco0s

D. Sriram et al. / Bioorg. Med. Chem. 12 (2004) 5865–5873
5873
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Acknowledgements
One of the authors Ms. Tanushree Bal deeply acknowl-
edges the Council of Scientific and Industrial Research
for providing Senior Research Fellowship. The authors
also thank Dr. Robert H. Shoemaker, National Cancer
Institute, USA and Dr. Sam Ananthan, TAACF, South-
ern Research Institute, USA for their support in biolog-
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19. A domestic microwave oven with the following specifica-
tions had been used: Make LG; input 220V–50Hz, 980W,
4.7A; frequency 2450MHz.