Synthesis, antimalarial, antileishmanial, and antimicrobial activities of some 8-quinolinamine analogues

Article abstract of DOI:10.1016/j.bmc.2005.04.034

In the present communication, newly synthesized 8-quinolinamines (25-27) related to previously reported 2-tert-butylprimaquine (2) were evaluated for their in vitro antimalarial activity against chloroquine sensitive and resistant Plasmodium falciparum strains, in vivo antimalarial activity against P. berghei infected mice, in vitro antileishmanial activity against Leishmania donovani, in vitro antimicrobial activity against various fungi and bacteria, and cytotoxicity in a panel of mammalian cell lines. No promising cytotoxicities were observed for compounds reported herein. Analogue 25 was found to exhibit curative antimalarial activity at a dose of 25 mg/kg/day × 4 in a P. berghei infected mice model, and produced suppressive activity at a lower dose of 10 mg/kg/day × 4. In vitro antileishmanial activities (IC₅₀ and IC₉₀) comparable to standard drug pentamidine were exhibited by all synthesized 8-quinolinamines 25-27. At the same time, promising antibacterial and antifungal activities were also observed for synthesized compounds against a panel consisting of several bacteria and fungi.

Full text of DOI:10.1016/j.bmc.2005.04.034

Bioorganic & Medicinal Chemistry 13 (2005) 4458–4466
Synthesis, antimalarial, antileishmanial, and antimicrobial
activities of some 8-quinolinamine analogues
Meenakshi Jain,a Shabana I. Khan,b Babu L. Tekwani,b Melissa R. Jacob,b Savita Singh,c
Prati Pal Singhc and Rahul Jaina,*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar,
Punjab 160 062, India
bNational Center for Natural Products Research, School of Pharmacy, University of Mississippi, MS 38677, USA
cDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector 67,
S.A.S. Nagar, Punjab 160 062, India
Received 1 April 2005; revised 15 April 2005; accepted 15 April 2005
Available online 5 May 2005
Abstract—In the present communication, newly synthesized 8-quinolinamines (25–27) related to previously reported 2-tert-butylpri-
maquine (2) were evaluated for their in vitro antimalarial activity against chloroquine sensitive and resistant Plasmodium falciparum
strains, in vivo antimalarial activity against P. berghei infected mice, in vitro antileishmanial activity against Leishmania donovani, in
vitro antimicrobial activity against various fungi and bacteria, and cytotoxicity in a panel of mammalian cell lines. No promising
cytotoxicities were observed for compounds reported herein. Analogue 25 was found to exhibit curative antimalarial activity at a
dose of 25 mg/kg/day · 4 in a P. berghei infected mice model, and produced suppressive activity at a lower dose of 10 mg/kg/
day · 4. In vitro antileishmanial activities (IC50 and IC90) comparable to standard drug pentamidine were exhibited by all synthe-
sized 8-quinolinamines 25–27. At the same time, promising antibacterial and antifungal activities were also observed for synthesized
compounds against a panel consisting of several bacteria and fungi.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
novel antibacterial and antifungal agents active against
these pathogens.3,4
Protozoal infections such as malaria and leishmaniasis
remain major public health problem in large areas of
the world. Malaria is responsible for the death of
approximately three million people annually, mostly
youngsters in more than 100 countries.1 Majority of
these mortality cases occur in sub-Saharan Africa, where
malaria accounts for one in five of all childhood deaths.
Leishmaniasis is a tropical disease caused by protozoal
parasites of the genus Leishmania and it remains a signi-
ficant health issue in large part due to the lack of effec-
tive and affordable drugs and increasing resistance
against existing drugs.2 Similarly, increasing number of
multidrug-resistant microbial pathogens have become
a serious problem particularly during the last decade
and provide impetus for the search and discovery of
Rapid development of resistance by Plasmodium falcipa-
rum to the conventional drugs like chloroquine necessi-
tates search for new antimalarial drugs, and a careful
re-examination of the existing drugs. 8-Quinolinamines
constitute an interesting class of compounds because of
their versatile biological and pharmacological activities,
such as antimalarial,5 antileishmanial,6 and anticocci-
dial.7 Primaquine (PQ, 1) (Fig. 1) is the only 8-quinolin-
amine available to treat the malaria parasites in the
infections due to P. vivax and P. ovale. It has been sug-
gested that PQ has various degrees of activities against
more life cycle stages of plasmodia than any other drug
currently employed for treating malaria infection.8 How-
ever, drawbacks like toxicity, ineffectiveness as blood-
schizontocide (infection caused by P. falciparum), and
rapid metabolism have limited PQꢀs usefulness. Despite
these drawbacks, in addition to excellent radical curative
activity, PQ has broad-range of antimalarial activities
including efficacy as causal prophylactic, gametocyto-
cide, and sporontocide. These interesting pharmacological
Keywords: 8-Quinolinamine; 2-tert-Butylprimaquine; Malaria; Leish-
maniasis; Antibacterial; Antifungal; Cytotoxicity.
*
Corresponding author. Tel.: +91 0172 221 4682; fax: +91 0172 221
4692; e-mail: rahuljain@niper.ac.in
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.034

M. Jain et al. / Bioorg. Med. Chem. 13 (2005) 4458–4466
4459
globin (MetHb) toxicity associated with 1. In
continuation of our research on structural modification
of 8-quinolinamines in general,11,12 and 2-tert-butylpri-
maquine (2) in particular, we report herein synthesis,
antiprotozoal, and antimicrobial activities of analogues
25–27, in which quinoline ring is substituted with a 5-
(3-trifluoromethylphenoxy) group, while C-4 position
of the quinoline ring is substituted with a methyl or an
ethyl group or is unsubstituted. It is important to note
that 5-(3-trifluoromethylphenoxy) substituent has been
an integral part of several promising antimalarial com-
pounds and appears to be responsible for enhancement
in antimalarial activities. One such compound tafenoqu-
ine is presently undergoing clinical trial studies.13
H3CO
N
R
HN
NH2
1. Primaquine (PQ), R = H
2. 2-tert-Butylprimaquine, R = C(CH3)3
Figure 1.
attributes make PQ an ideal drug to emulate while
designing new antimalarials with improved activity.
We have recently reported that the placement of a bulky
metabolically stable tert-butyl group at the C-2 position
of quinoline ring in 1 results in tremendous improve-
ment in the blood-schizontocidal antimalarial activ-
ity.9,10 Antimalarial compound, 2-tert-butylprimaquine
(2) (Fig. 1), exhibits potent in vivo blood-schizontocidal
antimalarial activities against both drug-sensitive strain
(P. berghei) and multi-drug resistant strain (P. yoelii
nige- riensis). Furthermore, 2 also represents first re-
ported 8-quinolinamine completely devoid of methemo-
2. Chemistry
4-Alkyl-5,6-dimethoxy-8-nitroquinolines (4–6) synthe-
sized using procedures reported earlier14,15 starting from
4,5-dimethoxy-2-nitroaniline (3) upon direct ring-alkyl-
ation via a silver catalyzed radical oxidative decarboxyl-
ation of trimethylacetic acid by ammonium persulfate in
CH3CN and 10% H2SO4 at 80 ꢁC efficiently produced 4-
alkyl-2-tert-butyl-5,6-dimethoxy-8-nitroquinolines (7–9)
H3CO
H3CO
H3CO
H3CO
R
N
H3CO
H3CO
R
N
i
ii
NH2
C(CH3)3
NO2
NO2
7-9
NO2
3
4. R= H
5. R= CH3
6. R= C2H5
iii
CF3
O
R
N
OH
R
N
Cl
R
N
H3CO
v
iv
H3CO
H3CO
C(CH3)3
C(CH3)3
C(CH3)3
NO2
NO2
NO2
16-18
13-15
10-12
vi
CF3
CF3
CF3
O
R
N
O
R
N
O
R
N
vii
C(CH3)3
viii
H3CO
H3CO
H3CO
C(CH3)3
NH2
C(CH3)3
NPhth
NH2
HN
HN
19-21
22-24
25-27
Scheme 1. Reagents and conditions: (i) glycerol, H2SO4, As2O5, 110 ꢁC, 21 h or methylvinylketone, 85% o-H3PO4, As2O5, 100 ꢁC, 3 h or 1-chloro-3-
pentanone, 85% o-H3PO4, As2O5, 80 ꢁC, 3 h; (ii) (CH3)3CCO2H, AgNO3, (NH4)2S2O8, 10% H2SO4, CH3CN, 70 ꢁC, 15 min; (iii) concd HCl, 95%
EtOH, 100 ꢁC, 2 h; (iv) POCl3, 80 ꢁC, 2 h; (v) 3-(trifluoromethyl)phenol, KOH, p-xylene, 12 h, 150 ꢁC; (vi) raney nickel, H2, EtOH, 45 psi, 45 min;
(vii) 2-(4-bromopentyl)-1,3-isoindolinedione, Et3N, 120 ꢁC, 24 h; (viii) NH2NH2ÆH2O, EtOH, reflux, 8 h.

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M. Jain et al. / Bioorg. Med. Chem. 13 (2005) 4458–4466
in good yields (Scheme 1).9 Selective demethylation of
the 5-methoxy group with concd hydrochloric acid
in 95% ethyl alcohol at 100 ꢁC for 2 h easily afforded
4-alkyl-2-tert-butyl-6-methoxy-8-nitro-5-quinolinols (10–
12) in excellent yields. Reaction of the latter compounds
10–12 with phosphorous oxychloride at 80 ꢁC for 2 h
produced 4-alkyl-2-tert-butyl-5-chloro-6-methoxy-8-nitro-
quinolines (13–15) in quantitative yields. Chloro deriva-
tives (13–15) upon reaction with 3-(trifluoromethyl)phe-
nol in the presence of potassium hydroxide pallets in
p-xylene at 150 ꢁC for 12 h afforded 4-alkyl-2-tert-
butyl-6-methoxy-8-nitro-5-(3-trifluoromethylphenoxy)-
quinolines (16–18) in good yields. The latter compounds
16–18 were then efficiently converted to requisite N8-(4-
amino-1-methylbutyl)-4-alkyl-2-tert-butyl-6-methoxy-5-
(3-trifluoromethylphenoxy)-8-quinolinamines (25–27) in
three steps using the procedure reported earlier,9 and
finally isolated as hydrochloride salts for their biological
evaluation by treatment with ethereal hydrochloric acid
solution.
orally, in mice (6 mice per group). The concentrations
tested were 100, 50, 25, and 10 mg/kg/day · 4 (oral).
The compounds were administered on days 0–3 post
infection. The results were compared to a positive con-
trol group of mice treated with chloroquine (CQ) at
the suppressive dose of 10 mg/kg/day · 4 (oral) and a
negative control group of mice where no treatment for
the infection was administered, and in this case 100%
mortality is observed within 6–8 days, with a mean sur-
vival time of 6.2 days.
Analogues 25–27 produced 100% cure at the primary
tested dose of 100 mg/kg. Upon evaluation at the subse-
quent lower doses of 50 and 25 mg/kg, compound 25
again produced 100% cure with all treated animals sur-
viving on day 60 (termination of experiment). Further
antimalarial activity evaluation of 25 at a dose of
10 mg/kg produced suppressive activity with 2/6 mice
surviving on day 60. Thus, compound 25 is equipotent
to CQ and 2, and is much superior to 1 as a blood-schiz-
ontocide. In contrast, analogue 26 (curative at 50 mg/
kg) and analogue 27 (active at 50 mg/kg) were less effec-
tive. As evident from the results, placement of a 5-(3-tri-
fluoromethylphenoxy) group (compound 25) in 2-tert-
butylprimaquine (2) essentially retains the in vivo
antimalarial activity. On the other hand, simultaneous
placement of a 5-(3-trifluoromethylphenoxy) and a
methyl or an ethyl group in 2 led to reduced antimalarial
activity as evident from results obtained for analogues
26 and 27.
3. Biological activity
Determination of in vitro antimalarial activity was
based on the determination of plasmodial LDH activ-
ity.16 As shown in Table 1, antimalarial activities of ana-
logues 25–27 are reported as IC50 values for the
inhibition of growth of chloroquine-sensitive (D6) and
chloroquine-resistant (W2) strains of P. falciparum.
Among the analogues, the most effective was N8-(4-amino-
1-methylbutyl)-2-tert-butyl-6-methoxy-5-(3-trifluoromethyl-
phenoxy)-8-quinolinamine (25), which exhibited moder-
ate antimalarial activity with IC50 of 1000 ng/mL for D6
and 1800 ng/mL for W2 clone (Table 1).
Antileishmanial activity against Leishmania donovani
promastigotes was determined by Alamar Blue assay
as described earlier.17,18 It was interesting to note that
8-quinolinamines 25–27 exhibited potent antileishma-
nial activities, as shown in Table 2, with IC50 values of
3.0, 3.4, and 2.9 lg/mL, respectively, and were compar-
able to the activity of standard drug pentamidine
(IC50 = 3.4 lg/mL) used as positive control. Their IC90
values ranged from 6.5–6.7 lg/mL as compared to
The target compounds were also evaluated for the
blood-schizontocidal antimalarial activity against P.
berghei (sensitive strain) in a rodent model (Table 1).
Briefly, testing was conducted at various concentrations,
Table 1. In vitro (P. falciparum) and in vivo (P. berghei) antimalarial activities of the 8-quinolinamine analogues (25–27)
No.
P. berghei (mg/kg/day · 4, oral)
P. falciparum
(D6 clone)
IC50 (ng/mL)
P. falciparum
(W2 clone)
IC50 (ng/mL)
10
25
50
100
25
26
27
2
(2/6)
Active
—
(6/6)
Curative
(0/6)
(6/6)
Curative
(6/6)
(6/6)
Curative
(6/6)
1800
>4760
NA
39
1000
2800
NA
—
Inactive
—
Curative
(3/6)
Curative
(6/6)
—
Active
(6/6)
Curative
(6/6)
(4/6)
Active
—
(6/6)
Curative
—
Curative
—
Curative
(0/6)
Inactive
PQ
—
—
The term ꢁcurativeꢀ indicates complete elimination of malaria parasites from the body, and animals survive up to day D+60. The term ꢁactiveꢀ or
ꢁsuppressiveꢀ indicates that all of the treated animals show negative parasitaemia up to D+7. However, by D+60, some mice die, and some survive
with complete elimination of parasitaemia as indicated by numbers given in parentheses. The term ꢁinactiveꢀ indicates that the treated animals show
positive parasitaemia either on D+4 or D+7 and usually die by D+14.
Chloroquine: IC50 = 18 ng/mL (D6 strain), 135 ng/mL (W2 strain).
Artemisinin: IC50 = 11.5 ng/mL (D6 strain), 15 ng/mL (W2 strain).
NA, Not active.

M. Jain et al. / Bioorg. Med. Chem. 13 (2005) 4458–4466
4461
Table 2. In vitro antileishmanial (L. donovani) studies of the 8-
quinolinamine analogues (25–27)
Compounds 25 and 26 exhibited promising antibacterial
activities with IC50 value in the range of 1.5–3.0 lg/mL
against all three organisms and MICs and MBCs in
the range of 2.5–10 lg/mL. In contrast, analogue 27
was inactive against M. intracellulare and S. aureus
but showed moderate activity against methicillin-resis-
tant S. aureus with an IC50 value of 8.5 lg/mL (Table 3).
No.
IC50 (lg/mL)a
IC90 (lg/mL)a
25
26
3.0
3.4
2.9
3.4
0.17
6.5
6.7
6.5
8.0
1.7
27
Pentamidine
Amphotericin B
a IC50 and IC90 are the sample concentrations that kill 50% and 90%
cells compared to solvent controls.
The antifungal activities of the target compounds
against pathogenic fungi associated with opportunistic
infections, Candida albicans, Cryptococcus neoformans,
and Aspergillus fumigatus, are summarized in Table 4.
Amphotericin B was included, as a standard drug, for
comparison. IC50, MICs, and MFCs were determined
according to NCCLS methods.19–21
IC90 of 8.0 lg/mL for pentamidine. However, they were
less potent than amphotericin B (IC50 = 0.17 and
IC90 = 1.7 lg/mL).
The antibacterial activities of 8-quinolinamines 25–27
against Staphylococcus aureus, methicillin-resistant S.
aureus, and Mycobacterium intracellulare are reported
as IC50, MIC, and MBC in Table 3. Ciprofloxacin is in-
cluded as positive control for comparison. Susceptibility
of S. aureus and methicillin-resistant S. aureus to test
compounds was determined according to the procedure
as described by the National Committee for Clinical
Laboratory Standards (NCCLS).19–21 Susceptibility of
M. intracellulare was done using the modified Alamar
Blue procedure of Franzblau et al.22 The IC50 was de-
fined as the concentration that affords 50% growth of
bacteria, whereas MIC was defined as the lowest con-
centration resulting in inhibition of visible bacterial
growth after incubation at 37 ꢁC for 18–24 h, and
MBC was the minimum bactericidal concentration that
kills 100% of the organism.
Compounds 25 and 26 produced promising antifungal
effects with IC50 values in the range of 1.5–6.5 lg/mL
against all three organisms under investigation. Both
analogues also exhibited MICs of 5.0 lg/mL against C.
neoformans and A. fumigatus, however MIC for C. albi-
cans was P20 lg/mL. At the same time, analogue 27
was effective only against C. neoformans with IC50 of
3.5 lg/mL, and MIC and MFC of 10 lg/mL (Table 4).
Cytotoxicity of these compounds was tested against four
human cancer cell lines (SK-MEL, KB, BT-549, and
SK-OV-3) and two noncancerous mammalian cells
(VERO and LLC-PK1) up to a highest concentration
of 10 lg/mL using neutral red assay procedure as de-
scribed earlier.23,24 None of the analogues showed any
cytotoxic effects (data not shown).
Table 3. In vitro antibacterial activities of the 8-quinolinamine analogues (25–27)
No.
Methicillin-resistant S. aureus
(MRS)
M. intracellulare
S. aureus
a
IC50
(lg/mL)
MICb
MBCc
IC50
(lg/mL)
MIC
(lg/mL)
MBC
(lg/mL)
IC50
(lg/mL)
MIC
(lg/mL)
MBC
(lg/mL)
(lg/mL)
(lg/mL)
25
26
1.5
3.0
8.5
0.06
2.5
5.0
2.5
5.0
1.5
3.0
5.0
5.0
5.0
5.0
1.5
3.0
10
10.0
5.0
5.0
NT
0.63
27
Ciprofloxacin
20
20
NA
20
NA
0.63
NA
1.25
NA
0.06
NT
0.63
0.31
0.15
NA, Not active; NT, not tested.
a IC50, The concentration that affords 50% inhibition of bacterial growth.
b MIC, (Minimum inhibitory concentration) is the lowest test concentration that allows detectable growth of bacteria.
c MBC, Minimum bactericidal concentration (the lowest test concentration that kills 100% of the organism).
Table 4. In vitro antifungal activities of the 8-quinolinamine analogues (25–27)
No.
C. albicans
C. neoformans
A. fumigatus
a
IC50
(lg/mL)
MICb
MFCc
IC50
(lg/mL)
MIC
(lg/mL)
MFC
(lg/mL)
IC50
(lg/mL)
MIC
(lg/mL)
MFC
(lg/mL)
(lg/mL)
(lg/mL)
25
26
6.5
6.5
>20
20
>20
>20
NA
1.25
1.5
2.0
3.5
0.70
5.0
5.0
5.0
5.0
4.0
4.0
5.0
5.0
5.0
5.0
27
Amphotericin B
NA
0.40
NA
0.63
10
10
NA
NT
>20
0.63
>20
NT
1.25
1.25
NA, Not active; NT, not tested.
a IC50, The concentration that affords 50% inhibition of bacterial growth.
b MIC, (Minimum inhibitory concentration) is the lowest test concentration that allows detectable growth of bacteria.
c MFC, Minimum fungicidal concentration (the lowest test concentration that kills 100% of the organism).

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M. Jain et al. / Bioorg. Med. Chem. 13 (2005) 4458–4466
4. Conclusions
washed with NaCl solution (2 · 10 mL). It was then
dried over Na2SO4, and the solvent removed under
reduce pressure to afford oil, which upon column
chromatography over silica gel (230–400 mesh) afforded
4-alkyl-2-tert-butyl-5,6-dimethoxy-8-nitroquinolines (7–
9).
In conclusion, new 8-quinolinamines related to 2-tert-
butylprimaquine (2) have been synthesized in eight steps
from 4,5-dimethoxy-2-nitroaniline. None of the tested
compounds showed cytotoxic activity. Compound 25
was the most effective in antimalarial assay and was as
potent as 2-tert-butylprimaquine against drug-sensitive
P. berghei in mice model. Most interestingly, all three
compounds 25–27 exhibited potent in vitro antileishma-
nial activities against L. donovani promastigotes. At the
same time, target analogues have also exhibited promis-
ing in vitro antibacterial and antifungal activities
against a panel consisting of various bacteria and fungi.
These results clearly indicate that newly synthesized 8-
quinolinamines reported herein are promising com-
pounds and provide useful model for further structural
and biological optimization. It can be concluded that
8-quinolinamines provide interesting lead in our search
for newer antimicrobial and antileishmanial agents in
addition to their proven usefulness in malaria
chemotherapy.
5.1.1. 2-tert-Butyl-5,6-dimethoxy-8-nitroquinoline (7).
1
Yield: 65%; mp 79–80 ꢁC; H NMR (CDCl3): d 8.41
(d, 1H, J = 9.0 Hz), 7.89 (s, 1H), 7.60 (d, 1H,
J = 9.0 Hz), 4.07 (s, 3H), 4.02 (s, 3H), 1.42 (s, 9H);
ESI MS m/z 291 (M+1); Anal. Calcd for C15H18N2O4
(290.3): C, 62.06; H, 6.25; N, 9.65. Found: C, 62.11;
H, 6.51; N, 9.78.
5.1.2. 2-tert-Butyl-5,6-dimethoxy-4-methyl-8-nitroquino-
1
line (8). Yield: 47%; mp 72–73 ꢁC; H NMR (CDCl3):
d 7.74 (s, 1H), 7.29 (s, 1H), 4.00 (s, 3H), 3.98 (s, 3H),
2.87 (s, 3H), 1.38 (s, 9H); ESI MS m/z 305 (M+1); Anal.
Calcd for C16H20N2O4 (304.3): C, 63.14; H, 6.62; N,
9.20. Found: C, 63.11; H, 6.67; N, 9.07.
5.1.3. 2-tert-Butyl-5,6-dimethoxy-4-ethyl-8-nitroquinoline
(9). Yield: 32%; mp 75–76 ꢁC; 1H NMR (CDCl3): d 7.74
(s, 1H), 7.34 (s, 1H), 4.01 (s, 3H), 3.98 (s, 3H), 3.25 (q,
2H, J = 15.0 Hz), 1.39 (s, 9H), 1.31 (t, 3H, J =
15.0 Hz); ESI MS m/z 319 (M+1); Anal. Calcd for
C17H22N2O4 (318.4): C, 64.13; H, 6.97; N, 8.80. Found:
C, 64.09; H, 6.89; N, 8.73.
5. Experimental
Melting points were recorded on Mettler DSC 851 or
capillary melting point apparatus and are uncorrected.
1H spectra were recorded on 300 MHz Bruker FT-NMR
(Avance DPX300) spectrometer using tetramethylsilane
as internal standard and the chemical shifts are reported
in d units. Mass spectra were recorded on either GCMS
(Shimadzu QP 5000 spectrometer) auto sampler/direct
injection (EI/CI) or HRMS (Finnigan Mat LCQ spec-
trometer) (APCI/ESI). Elemental analyses were re-
corded on Elementar Vario EL spectrometer. All
chromatographic purification was performed with silica
gel 60 (230–400 mesh), whereas all TLC (silica gel)
development was performed on silica gel coated (Merck
Kiesel 60 F254, 0.2 mm thickness) sheets. All chemicals
were purchased from Aldrich Chemical Ltd. (Milwau-
kee, WI, USA). Solvents used for the chemical synthesis
acquired from commercial sources were of analytical
grade and were used without further purification unless
otherwise stated.
5.2. General method for the synthesis of 4-alkyl-2-tert-
butyl-6-methoxy-8-nitro-5-quinolinols (10–12)
The dimethoxyquinoline (7–9, 0.69 mmol) was dissolved
in 95% ethyl alcohol (4 mL) and concd HCl (0.2 mL)
was added. The reaction mixture was stirred at 100 ꢁC
for 2 h. Upon cooling, brownish crystals of 4-alkyl-2-
tert-butyl-6-methoxy-8-nitro-5-quinolinols (10–12) sepa-
rated out and used without further purification for the
next reaction.
5.2.1. 2-tert-Butyl-6-methoxy-8-nitro-5-quinolinol (10).
1
Yield: 82%; mp 184–185 ꢁC; H NMR (CDCl3): d 9.20
(d, 1H, J = 8.4 Hz), 7.82 (s, 1H), 7.45 (d, 1H, J =
8.4 Hz), 3.93 (s, 3H), 3.49 (br s, 1H, exchangeable with
D2O), 1.60 (s, 9H); APCI MS m/z 277 (M+1); Anal.
Calcd for C14H16N2O4 (276.3): C, 60.86; H, 5.84; N,
10.14. Found: C, 61.08; H, 5.93; N, 9.92.
5.1. General method for the synthesis of 4-alkyl-2-tert-
butyl-5,6-dimethoxy-8-nitroquinolines (7–9)
4-Alkyl-5,6-methoxy-8-nitroquinoline14,15 (4–6, 1 mmol)
was dissolved in CH3CN (5 mL) and the reaction mix-
ture was heated to 70 ꢁC. Silver nitrate (0.6 mmol),
trimethylacetic acid (2.5 mmol), and 10% H2SO4
(10 mL) were then added to the reaction mixture. A
freshly prepared solution of ammonium persulfate
(3 mmol) in water (10 mL) was added dropwise to the
pre-heated (70 ꢁC) mixture during 10 min. The heating
source was then removed and reaction proceeded
with the evolution of carbon dioxide. After 15 min, the
reaction mixture was poured onto ice. The resulting
mixture was made alkaline with addition of 25%
aqueous NH4OH solution. It was extracted with ethyl
acetate (4 · 50 mL), and the combined extracts were
5.2.2. 2-tert-Butyl-6-methoxy-4-methyl-8-nitro-5-quino-
linol (11). Yield: 94%; mp 162–163 ꢁC; 1H NMR
(CDCl3): d 7.74 (s, 1H), 7.15 (s, 1H), 3.90 (s, 3H), 3.13
(s, 3H), 1.57 (s, 9H); ESI MS m/z 291 (M+1); Anal.
Calcd for C15H18N2O4 (290.1): C, 62.06; H, 6.25; N,
9.65. Found: C, 62.17; H, 6.09; N, 9.43.
5.2.3. 2-tert-Butyl-4-ethyl-6-methoxy-8-nitro-5-quinolinol
1
(12). Yield: 99%; mp 186–187 ꢁC; H NMR (CDCl3): d
7.76 (s, 1H), 7.18 (s, 1H), 3.91 (s, 3H), 3.59 (q, 2H,
J = 15.0 Hz), 1.57 (s, 9H), 1.37 (t, 3H, J = 15.0 Hz);
APCI MS m/z 305 (M+1); Anal. Calcd for
C16H20N2O4 (304.1): C, 63.14; H, 6.62; N, 9.20. Found:
C, 63.37; H, 6.41; N, 9.08.

M. Jain et al. / Bioorg. Med. Chem. 13 (2005) 4458–4466
4463
5.3. General method for the synthesis of 4-alkyl-2-tert-
butyl-5-chloro-6-methoxy-8-nitroquinolines (13–15)
MS m/z 435 (M+1); Anal. Calcd for C22H21F3N2O4
(434.4): C, 60.83; H, 4.87; N, 6.45. Found: C, 61.14;
H, 5.11; N, 6.37.
POCl3 (2.5 mL) was added to 4-alkyl-2-tert-butyl-6-
methoxy-8-nitro-5-quinolinol (10–12, 0.45 mmol) and
the reaction mixture was heated at 80 ꢁC for 2 h. The
reaction mixture was poured onto crushed ice, basified
with 25% NH4OH, and extracted with CHCl3
(3 · 25 mL). Combined organic layer was dried over
Na2SO4 and removal of solvent under reduced pressure
provided 4-alkyl-2-tert-butyl-5-chloro-6-methoxy-8-nitro-
quinolines (13–15) in excellent yield.
5.4.3. 2-tert-Butyl-4-ethyl-6-methoxy-8-nitro-5-(3-trifluo-
romethylphenoxy)quinoline (18). Yield: 62%; mp 176–
177 ꢁC; 1H NMR (CDCl3): d 7.77 (s, 1H), 7.38
(m, 1H), 7.32 (s, 1H), 7.30 (m, 1H), 7.09 (s, 1H), 6.94
(m, 1H), 3.82 (s, 3H), 3.03 (q, 2H, J = 14.3 Hz), 1.41
(s, 9H), 1.27 (t, 3H, J = 14.3 Hz); APCI MS m/z 449
(M+1); Anal. Calcd for C23H23F3N2O4 (448.4): C,
61.60; H, 5.17; N, 6.25. Found: C, 61.72; H, 5.23; N,
6.47.
5.3.1. 2-tert-Butyl-5-chloro-6-methoxy-8-nitroquinoline
1
(13). Yield: 100%; mp 230–231 ꢁC; H NMR (CDCl3):
5.5. General method for the synthesis of 4-alkyl-2-tert-
butyl-6-methoxy-5-(3-trifluoromethylphenoxy)-8-quino-
linamines (19–21)
d 8.50 (d, 1H, J = 9.0 Hz), 7.79 (s, 1H), 7.71 (d, 1H,
J = 9.0 Hz), 4.07 (s, 3H), 1.42 (s, 9H); APCI MS m/z
295 (M+1); Anal. Calcd for C14H15ClN2O3 (294.7): C,
57.05; H, 5.13; N, 9.50. Found: C, 56.79; H, 4.85; N,
9.78.
A solution of 4-alkyl-2-tert-butyl-6-methoxy-8-nitro-5-
(3-trifluoromethylphenoxy)quinoline (16–18, 10 mmol)
in 95% ethyl alcohol (30 mL) was hydrogenated over ra-
ney nickel (T1 grade) at 45 psi in a parr hydrogenator for
45 min. The catalyst was removed by filtration and the
filtrate was evaporated under reduced pressure to afford
4-alkyl-2-tert-butyl-6-methoxy-5-(3-trifluoromethylphen-
oxy)-8-quinolinamines (19–21) as dark colored oil.
5.3.2. 2-tert-Butyl-5-chloro-6-methoxy-4-methyl-8-nitro-
quinoline (14). Yield: 95%; mp 137–138 ꢁC; H NMR
(CDCl3): d 7.65 (s, 1H), 7.37 (s, 1H), 4.04 (s, 3H), 3.06
(s, 3H), 1.38 (s, 9H); ESI MS m/z 309 (M+1); Anal.
Calcd for C15H17ClN2O3 (308.8): C, 58.35; H, 5.55; N,
9.07. Found: C, 58.67; H, 5.41; N, 9.18.
1
5.5.1. 2-tert-Butyl-6-methoxy-5-(3-trifluoromethylphen-
oxy)-8-quinolinamine (19). Yield: 100%; oil; H NMR
1
5.3.3.
2-tert-Butyl-5-chloro-4-ethyl-6-methoxy-8-nitro-
1
quinoline (15). Yield: 100%; oil; H NMR (CDCl3): d
7.65 (s, 1H), 7.43 (s, 1H), 4.05 (s, 3H), 3.50 (q, 2H,
J = 15.0 Hz), 1.39 (s, 9H), 1.35 (t, 3H, J = 15.0 Hz);
APCI MS m/z 323 (M+1); Anal. Calcd for
C16H19ClN2O3 (322.8): C, 59.54; H, 5.93; N, 8.68.
Found: C, 59.74; H, 5.86; N, 8.86.
(CDCl3): d 7.97 (d, 1H, J = 8.7 Hz), 7.44 (d, 1H,
J = 8.7 Hz), 7.34 (m, 1H), 7.25 (m, 1H), 7.14 (s, 1H),
6.99 (m, 1H), 6.76 (s, 1H), 5.07 (br s, 2H, exchangeable
with D2O), 3.84 (s, 3H), 1.43 (s, 9H); APCI MS m/z 391
(M+1); Anal. Calcd for C21H21F3N2O2 (390.4): C,
64.61; H, 5.42; N, 7.18. Found: C, 64.97; H, 5.16; N,
7.43.
5.4. General method for the synthesis of 4-alkyl-2-tert-
butyl-6-methoxy-8-nitro-5-(3-trifluoromethylphenoxy)-
quinolines (16–18)
5.5.2.
2-tert-Butyl-6-methoxy-4-methyl-5-(3-trifluoro-
methylphenoxy)-8-quinolinamine (20). Yield: 98%; oil;
1H NMR (CDCl3): d 7.78 (s, 1H), 7.42 (m, 1H), 7.33
(s, 1H), 7.30 (s, 1H), 7.09 (m, 1H), 6.93 (m, 1H), 4.98
(br s, 2H, exchangeable with D2O), 3.91 (s, 3H), 2.87
(s, 3H), 1.40 (s, 9H); APCI MS m/z 405 (M+1); Anal.
Calcd for C22H23F3N2O2 (404.4): C, 65.34; H, 5.73; N,
6.93. Found: C, 65.76; H, 5.92; N, 7.28.
To a mixture of 3-(trifluoromethyl)phenol (2.49 mmol)
in p-xylene (10 mL) containing KOH pallets
(2.42 mmol) was added 4-alkyl-2-tert-butyl-5-chloro-6-
methoxy-8-nitroquinoline (13–15, 2.20 mmol). The reac-
tion mixture was heated at 150 ꢁC for 12 h. The solvent
was removed under reduced pressure and crude product
was purified by chromatography on silica gel using
EtOAc–hexanes (1:1) as eluant.
5.5.3. 2-tert-Butyl-4-ethyl-6-methoxy-5-(3-trifluorometh-
ylphenoxy)-8-quinolinamine (21). Yield: 100%; oil; 1H
NMR (CDCl3): d 7.35 (s, 1H), 7.33 (m, 1H), 7.34 (m,
1H), 7.22 (m, 1H), 7.05 (s, 1H), 6.77 (s, 1H), 5.15 (br
s, 2H, exchangeable with D2O), 3.80 (s, 3H), 2.95 (q,
2H, J = 14.7 Hz), 1.43 (s, 9H), 1.25 (t, 3H,
J = 14.7 Hz); APCI MS m/z 419 (M+1); Anal. Calcd
for C23H25F3N2O2 (418.5): C, 66.02; H, 6.02; N, 6.69.
Found: C, 66.38; H, 5.71; N, 6.87.
5.4.1. 2-tert-Butyl-6-methoxy-8-nitro-5-(3-trifluorometh-
ylphenoxy)quinoline (16). Yield: 78%; mp 185–
1
186 ꢁC; H NMR (CDCl3): d 8.21 (d, 1H, J = 9.0 Hz),
7.91 (s, 1H), 7.61 (d, 1H, J = 9.0 Hz), 7.39 (m, 1H),
7.33 (m, 1H), 7.16 (s, 1H), 6.98 (m, 1H), 3.91 (s, 3H),
1.43 (s, 9H); APCI MS m/z 421 (M+1); Anal. Calcd
for C21H19F3N2O4 (420.4): C, 60.00; H, 4.56; N, 6.66.
Found: C, 60.09; H, 4.37; N, 6.73.
5.6. General method for the synthesis of 2-{4-[4-alkyl-
2-tert-butyl-6-methoxy-5-(3-trifluoromethylphenoxy)-8-
quinolylamino]pentyl}-1,3-isoindolinediones (22–24)
5.4.2. 2-tert-Butyl-6-methoxy-4-methyl-8-nitro-5-(3-tri-
fluoromethylphenoxy)quinoline (17). Yield: 68%; mp
152–153 ꢁC; 1H NMR (CDCl3): d 7.78 (s, 1H), 7.40
(m, 1H), 7.33 (s, 1H, 3), 7.30 (s, 1H), 7.09 (s, 1H), 6.93
(m, 1H), 3.84 (s, 3H), 2.69 (s, 3H), 1.40 (s, 9H); APCI
A mixture of 4-alkyl-2-tert-butyl-6-methoxy-5-(3-trifluo-
romethylphenoxy)-8-quinolinamine (19–21, 2.7 mmol),
2-(4-bromopentyl)-1,3-isoindolinedione (2.7 mmol), and

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M. Jain et al. / Bioorg. Med. Chem. 13 (2005) 4458–4466
triethylamine (2.7 mmol) was heated at 120 ꢁC with stir-
ring for 4 h. An additional quantity of 2-(4-bromopent-
yl)-1,3-isoindolinedione (2.7 mmol) and triethylamine
(2.7 mmol) was added, and stirring continued with heat-
ing for another 4 h. A third aliquot of 2-(4-bromopent-
yl)-1,3-isoindolinedione (2.7 mmol) and triethylamine
(2.7 mmol) was added, and the reaction mixture stirred
at 120 ꢁC for additional 16 h. The dark brown reaction
mixture was diluted with ethyl acetate (50 mL) and fil-
tered. The filtrate was basified with 2 N NaOH solution
and extracted with ethyl acetate (3 · 30 mL). The com-
bined extracts were washed with water (10 mL), dried
over Na2SO4, and concentrated to afford dark colored
residue. Flash column chromatography over silica using
EtOAc–hexanes (25:75) as eluant provided 2-{4-[4-alkyl-
2-tert-butyl-6-methoxy-5-(3-trifluoromethylphenoxy)-8-
quinolylamino]pentyl}-1,3-isoindolinediones (22–24) as
colorless or pale yellow viscous oil.
(25 mL) was added hydrazine hydrate (50 mmol), and
the reaction mixture was heated under reflux for 8 h.
The solvent was removed under reduced pressure and
the residue was diluted with water (25 mL). The reaction
mixture was basified with 8 N NaOH solution, extracted
with CHCl3 (3 · 25 mL), and washed with water
(15 mL). Combined organic extracts were dried over
Na2SO4 and concentrated under reduced pressure to
yield N8-(4-amino-1-methylbutyl)-4-alkyl-2-tert-butyl-
6-methoxy-5-(3-trifluoromethylphenoxy)-8-quinolinam-
ines (25–27) as oil. Treatment with ethereal hydrochloric
acid solution provided the requisite N8-(4-amino-1-meth-
ylbutyl)-4-alkyl-2-tert-butyl-6-methoxy-5-(3-trifluorometh-
ylphenoxy)-8-quinolinamines as their hydrochloride
salts.
5.7.1. N8-(4-Amino-1-methylbutyl)-2-tert-butyl-6-meth-
oxy-5-(3-trifluoromethyl-phenoxy)-8-quinolinamine (25).
Yield: 99%; mp 125–126 ꢁC; 1H NMR (free base,
CDCl3): d 7.94 (d, 1H, J = 8.7 Hz), 7.43 (d, 1H,
J = 8.7 Hz), 7.34 (m, 1H), 7.22 (m, 1H), 7.20 (m, 1H),
7.01 (m, 1H), 6.43 (s, 1H), 6.23 (br s, 1H, exchangeable
with D2O), 3.88 (s, 3H), 3.68 (m, 1H), 2.81 (m, 2H), 2.03
(m, 4H), 1.42 (s, 9H), 1.38 (d, 3H, J = 6.0 Hz); APCI
MS m/z 476 (M+1); Anal. Calcd for C26H34Cl2F3N3O2
(548.5): C, 56.94; H, 6.25; N, 7.66. Found: C, 56.87;
H, 6.13; N, 7.88.
5.6.1. 2-{4-[2-tert-Butyl-6-methoxy-5-(3-trifluoromethyl-
phenoxy)-8-quinolylamino]-pentyl}-1,3-isoindolinedione
(22). Yield: 84%; colorless oil; 1H NMR (CDCl3): d 7.98
(d, 1H, J = 8.7 Hz), 7.82 (m, 4H), 7.70 (d, 1H,
J = 8.7 Hz), 7.45 (m, 1H), 7.23 (m, 1H), 7.17 (m, 1H),
6.98 (m, 1H), 6.76 (s, 1H), 6.43 (br s, 1H, exchangeable
with D2O), 3.86 (s, 3H), 3.76 (m, 1H), 3.72 (m, 2H), 1.89
(m, 4H), 1.43 (s, 9H), 1.36 (d, 3H, J = 6.3 Hz); APCI
MS m/z 606 (M+1); Anal. Calcd for C34H34F3N3O4
(605.7): C, 67.43; H, 5.66; N, 6.94. Found: C, 67.74;
H, 5.88; N, 6.62.
5.7.2. N8-(4-Amino-1-methylbutyl)-2-tert-butyl-6-meth-
oxy-4-methyl-5-(3-trifluoromethylphenoxy)-8-quinolin-
1
amine (26). Yield: 90%; mp 115–116 ꢁC; H NMR (free
5.6.2. 2-{4-[2-tert-Butyl-6-methoxy-4-methyl-5-(3-trifluoro-
methylphenoxy)-8-quinolyl-amino]pentyl}-1,3-isoindol-
inedione (23). Yield: 81%; colorless oil; 1H NMR
(CDCl3): d 7.79 (m, 4H), 7.34 (m, 1H), 7.28 (s, 1H),
7.18 (m, 1H), 7.08 (s, 1H), 6.93 (m, 1H), 6.43 (s, 1H),
6.25 (br s, 1H, exchangeable with D2O), 3.88 (m, 1H),
3.79 (s, 3H), 3.76 (m, 2H), 2.57 (s, 3H), 1.61 (m, 4H),
1.39 (s, 9H), 1.35 (d, 3H, J = 6.3 Hz); APCI MS m/z
620 (M+1); Anal. Calcd for C35H36F3N3O4 (619.7): C,
67.84; H, 5.86; N, 6.78. Found: C, 68.15; H, 6.17; N,
6.49.
base, CDCl3): d 7.84 (s, 1H), 7.44 (m, 1H), 7.29 (m, 1H),
6.99 (m, 1H), 6.95 (s, 1H), 6.60 (s, 1H), 4.93 (br s, 1H,
exchangeable with D2O), 3.88 (m, 1H), 3.81 (s, 3H),
2.80 (m, 2H), 2.58 (s, 3H), 1.74 (m, 4H), 1.42 (s, 9H),
1.37 (d, 3H, J = 6.3 Hz); APCI MS m/z 490 (M+1);
Anal. Calcd for C27H36Cl2F3N3O2 (562.5): C, 57.65;
H, 6.45; N, 7.47. Found: C, 57.77; H, 6.51; N, 7.52.
5.7.3. N8-(4-Amino-1-methylbutyl)-2-tert-butyl-4-ethyl-6-
methoxy-5-(3-trifluoromethylphenoxy)-8-quinolinamine
1
(27). Yield: 94%; mp 112–113 ꢁC; H NMR (free base,
CDCl3): d 7.60 (s, 1H), 7.26 (m, 1H), 7.08 (m, 1H),
6.92 (m, 1H), 6.90 (m, 1H), 6.45 (s, 1H), 3.80 (s, 3H),
3.82 (m, 1H), 2.93 (q, 2H, J = 6.0 Hz), 2.80 (m, 2H),
1.76 (m, 4H), 1.42 (s, 9H), 1.34 (d, 3H, CH3,
J = 6.0 Hz) 0.88 (t, 3H, J = 7.2 Hz); APCI MS m/z 505
(M+1); Anal. Calcd for C28H38Cl2F3N3O2 (576.5): C,
58.33; H, 6.64; N, 7.29. Found: C, 58.37; H, 6.75; N,
7.43.
5.6.3. 2-{4-[2-tert-Butyl-4-ethyl-6-methoxy-5-(3-trifluoro-
methylphenoxy)-8-quinolyl-amino]pentyl}-1,3-isoindol-
inedione (24). Yield: 89%; pale yellow oil; 1H NMR
(CDCl3): d 7.83 (m, 4H), 7.35 (m, 1H), 7.32 (s, 1H),
7.22 (m, 1H), 7.19 (s, 1H), 7.09 (s, 1H), 6.95 (m, 1H),
6.45 (s, 1H), 4.18 (m, 1H), 3.80 (s, 3H), 3.72 (m, 2H),
2.92 (q, 2H, J = 7.5 Hz), 1.84 (m, 4H), 1.41 (s, 9H),
1.35 (d, 3H, J = 6.3 Hz), 1.25 (m, 3H); APCI MS m/z
634 (M+1); Anal. Calcd for C36H38F3N3O4 (633.7): C,
68.23; H, 6.04; N, 6.63. Found: C, 68.02; H, 6.37; N,
6.41.
5.8. Assay for in vitro antimalarial activity
The assay is based on the determination of plasmodial
LDH activity.16 For the assay, a suspension of red blood
cells infected with D6 or W2 strains of P. falciparum
(200 lL, with 2% parasitemia and 2% hematocrit in
RPMI 1640 medium supplemented with 10% human ser-
um and 60 lg/mL amikacin) is added to the wells of a 96-
well plate containing 10 lL of test samples diluted in
medium at various concentrations. The plate is placed
in a modular incubation chamber (Billups-Rothenberg,
5.7. General method for the synthesis of N8-(4-amino-1-
methylbutyl)-4-alkyl-2-tert-butyl-6-methoxy-5-(3-trifluo-
romethylphenoxy)-8-quinolinamines (25–27)
To a solution of 2-{4-[4-alkyl-2-tert-butyl-6-methoxy-5-
(3-trifluoromethylphenoxy)-8-quinolylamino]pentyl}-1,3-
isoindolinedione (22–24, 5 mmol) in 95% ethyl alcohol

M. Jain et al. / Bioorg. Med. Chem. 13 (2005) 4458–4466
4465
CA) and flushed with a gas mixture of 90% N2, 5% O2,
5.10. Assay for in vitro antileishmanial activity
and 5% CO2 and incubated at 37 ꢁC, for 72 h. Parasitic
LDH activity is determined by using MalstatTM reagent
(Flow Inc., Portland, OR) according to the procedure
of Makler and Hinrichs.16 Briefly, 20 lL of the incuba-
tion mixture is mixed with 100 lL of the MalstatTM reagent
and incubated at room temperature for 30 min. Twenty
microliters of a 1:1 mixture of NBT/PES (Sigma, St.
Louis, MO) is then added and the plate is further incu-
bated in the dark for 1 h. The reaction is then stopped
by the addition of 100 lL of a 5% acetic acid solution.
The plate is read at 650 nm using the EL-340 Biokinetics
Reader (Bio-Tek Instruments, Vermont). IC50 values are
computed from the dose response curves. Artemisinin
and chloroquine are included in each assay as the drug
controls. DMSO (0.25%) is used as vehicle control.
Antileishmanial activity of the compounds was tested
in vitro against a culture of L. donovani promastigotes.
They were grown in RPMI 1640 medium supplemented
with 10% fetal calf serum (Gibco Chem. Co.) at 26 ꢁC. A
3 day-old culture was diluted to 5 · 105 promastigotes/
mL. Drug dilutions (50–3.1 lg/mL) were prepared di-
rectly in cell suspension in 96-well plates. Plates were
incubated at 26 ꢁC for 48 h and growth of leishmania
promastigotes was determined by Alamar Blue assay
as described earlier.17,18 Standard fluorescence was mea-
sured on a Fluostar Galaxy plate reader (BMG Lab
Technologies) at excitation wavelength of 544 nm and
emission wavelength of 590 nm. Pentamidine and
amphotericin B were used as the standard antileishma-
nial agents. IC50 and IC90 values were computed from
dose curves generated by plotting percent growth versus
drug concentration.
5.9. Assay for blood-schizontocidal activity evaluation
against P. berghei infection in mice
The method used for screening of the synthesized com-
pounds for their blood-schizontocidal activity is based
on a comparison of responses by groups of treated
and control mice, six in each group, after infection with
P. berghei. Using a standard inoculum of P. berghei, it is
possible to produce a uniform disease that is fatal to
100% of untreated animals, within 6–8 days, with a
mean survival time of 6.2 days. Test animals (Swiss mice
of either sex, approximately 15–20 g and same age) were
housed in metal-topped cages, given a standard labora-
tory diet and water ad libitum. In order to check factors
such as changes in the infectivity of the strain or in the
susceptibility of the host or to detect technical errors,
a group of infected animals treated with chloroquine
diphosphate at dose levels (10 mg/kg/day · 4), produc-
ing definite increases in survival time, is included as a
positive control in every experiment. In each experi-
ment, the test compounds were administered in graded
doses of 100, 50, 25, and 10 mg/kg. On day ꢁ0ꢀ, groups
of 6 mice each were inoculated intraperitoneally with
1 · 107 infected-erythrocytes from a donor mouse. Four
h later, mice were administered test compounds/chloro-
quine/vehicle, orally. A total of four doses were given
orally on days D ꢁ0ꢀ, D+1, D+2, and D+3. The tail
blood smears were made on day D+4 and D+7, stained
with Giemsa and examined microscopically. The mini-
mum dose that completely suppressed parasitaemia on
days D+4 and D+7 was termed as minimum effective
dose (MED), and the minimum dose that cleared the
parasitaemia for up to 60 days was termed as curative
dose (CD). The terms ꢁcurativeꢀ, ꢁactiveꢀ, and ꢁinactiveꢀ
are used to describe the biological activities exhibited
by the tested compounds. The term ꢁcurativeꢀ indicates
complete elimination of malaria parasites from the
body, so that relapse cannot occur up to day D+60
and all mice survived. The term ꢁactiveꢀ indicates that
the treated animals show negative parasitaemia up to
D+7. However, by D+28, 50% or more mice show nega-
tive and remaining mice may show positive test result
for parasitaemia. The term ꢁinactiveꢀ indicates that the
treated animals show positive test result for parasita-
emia either on D+4 or D+7 or on both D+4 and D+7
and the animal usually dies by D+14.
5.11. Assay for in vitro antimicrobial activity
Susceptibility testing against C. albicans, C. neoformans,
S. aureus, methicillin-resistant S. aureus (MRS), and A.
fumigatus was performed using a modified version of the
NCCLS methods.19–21 Susceptibility testing against M.
intracellulare was done using the modified Alamar Blue
procedure of Franzblau et al.22 All organisms were ob-
tained from the American Type Culture Collection
(ATCC), Manassas, VA. Samples (dissolved in DMSO)
were serially diluted using 0.9% saline and transferred in
duplicate to 96-well microplates. Microbial inocula were
prepared after comparison of the absorbance at 630 nm
of cell suspensions to the 0.5 McFarland standard and
diluting the suspensions in broth [Sabouraud Dextrose
and cation-adjusted Mueller–Hinton (Difco) for the fun-
gi and bacteria, respectively, and 5% Alamar Blue (Bio-
Source International) in Middlebrook 7H9 broth with
OADC enrichment for M. intracellulare] to afford re-
commended inocula. Microbial inocula were added to
the diluted samples to achieve a final volume of
200 lL. Growth, solvent, and media controls were in-
cluded in each assay. Plates are read at either 630 nm
or 544ex/590em (M. intracellulare) prior to and after
incubation. Percent growth was plotted versus test con-
centration to afford the IC50. The minimum bactericidal/
fungicidal concentrations (MBC/MFCs) were deter-
mined by removing 5 lL from each clear well, transfer-
ring to agar and incubating until growth is seen.
5.12. Cytotoxicity assay
The in vitro cytotoxicity was determined against a panel
of cell lines that included SK-MEL, KB, BT-549, SK-
OV-3, VERO, and LCC-PK1 (obtained from ATCC).
The assay is performed in 96-well tissue culture-treated
microplates and compounds were tested up to a highest
concentration of 10 lg/mL as described earlier.24 Briefly,
cells (25,000 cells/well) were seeded to the wells of the
plate and incubated for 24 h. Samples were added and
plates were again incubated for 48 h. The number of via-
ble cells was determined according to a modified version
of neutral red assay procedure.23 IC50 values were

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M. Jain et al. / Bioorg. Med. Chem. 13 (2005) 4458–4466
11. Vangapandu, S.; Sachdeva, S.; Jain, M.; Singh, S.; Singh,
P. P.; Kaul, C. L.; Jain, R. Bioorg. Med. Chem. 2004, 12,
239.
12. Vangapandu, S.; Sachdeva, S.; Jain, M.; Singh, S.; Singh,
P. P.; Kaul, C. L.; Jain, R. Bioorg. Med. Chem. 2003, 11,
4557.
determined from logarithmic graphs of growth inhibi-
tion versus concentration. Doxorubicin was used as a
positive control, while DMSO was used as the negative
(vehicle) control.
13. LaMontagne, M. P.; Blumbergs, P.; Smith, D. C. J. Med.
Chem. 1989, 32, 1728.
Acknowledgment
14. Jain, R.; Jain, S.; Gupta, R. C.; Anand, N.; Dutta, G. P.;
Puri, S. K. Indian J. Chem. 1994, 33B, 251.
15. Carroll, F. I.; Berrang, B.; Linn, C. P. J. Med. Chem. 1985,
28, 1564.
16. Makler, M. T.; Hinrichs, D. J. Am. J. Trop. Med. Hyg.
1993, 48, 205.
Financial support from the Council of Scientific and
Industrial Research (CSIR), New Delhi, India (Grant
no. 01(1555)/98/EMR-II) for the synthesis of primaqu-
ine derivatives is gratefully acknowledged.
17. Mikus, J.; Steverding, D. Parasitol. Int. 2000, 48, 265.
18. Ma, G.; Khan, S. I.; Jacob, M. R.; Tekwani, B. L.; Li, Z.;
Pasco, D. S.; Walker, L. A.; Khan, I. A. Antimicrob.
Agents Chemother. 2004, 48, 4450.
19. NCCLS, In Reference Method for Broth Dilution Anti-
fungal Susceptibility Testing of Yeasts; Approved Standard
M27-A, National Committee on Clinical Laboratory
Standards, 1997; Vol. 17, p 9.
20. NCCLS, In Methods for Dilution Antimicrobial Suscepti-
bility Tests for Bacteria that Grow Aerobically M7-A5,
National Committee on Clinical Laboratory Standards,
2000; Vol. 20, p 2.
References and notes
´ ´
´
1. (a) Frederich, M.; Donge, J.-M.; Angenot, L.; De Mol, P.
Curr. Med. Chem. 2002, 9, 1435; (b) Boss, C.; Richard-
Bildstein, S.; Weller, T.; Fischli, W.; Meyers, S.; Binkert,
C. Curr. Med. Chem. 2003, 10, 883.
2. Davis, A. J.; Murray, H. W.; Handman, E. Trends
Parasitol. 2004, 20, 73.
3. Bren, L. FDA Consum. 2002, 36, 28.
4. Liu, J.; Balasubramanian, M. K. Curr. Drug Targets—
Infect. Disorders 2001, 1, 159.
5. Carson, P. E. In Antimalarial Drugs II; Peters, W.,
Richards, W. H. G., Eds.; Springer: New York, 1984; p 83.
6. Yeates, C. Curr. Opin. Invest. Drugs 2002, 3, 1446.
7. Armer, R. E.; Barlow, J. S.; Dutton, C. J.; Greenway, D.
H. J.; Greenwood, S. D. W.; Lad, N.; Tommasini, I.
Bioorg. Med. Chem. Lett. 1997, 7, 2585.
8. Nodiff, E. A. Prog. Med. Chem. 1991, 28, 1.
9. Jain, M.; Vangapandu, S.; Sachdeva, S.; Singh, S.; Singh,
P. P.; Jena, G. B.; Tikoo, K.; Ramarao, P.; Kaul, C. L.;
Jain, R. J. Med. Chem. 2004, 47, 285.
21. NCCLS, In Susceptibility Testing of Mycobacteria, Nocar-
dia, and Other Aerobic Actinomycetes; Tentative Stan-
dard—Second Edition, M24-T2, National Committee on
Clinical Laboratory Standards, 2000; Vol. 20, p 26.
22. Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres,
P.; Madico, G.; Hernandez, A.; Degnan, M. T.; Cook, M.
B.; Quenzer, V. K.; Ferguson, R. M.; Gilman, R. H.
J. Clin. Microbiol. 1998, 36, 362.
23. Borenfreund, E.; Babich, H.; Martin-Alguacil, N. In Vitro
Cell Dev. Biol. 1990, 26, 1030.
10. Jain, M.; Vangapandu, S.; Sachdeva, S.; Jain, R. Bioorg.
Med. Chem. 2004, 12, 1003.
24. Mustafa, J.; Khan, S. I.; Ma, G.; Walker, L. A.; Khan, I.
A. Lipids 2004, 39, 167.