Structure-based approach to falcipain-2 inhibitors: Synthesis and biological evaluation of 1,6,7-Trisubstituted dihydroisoquinolines and isoquinolines

Article abstract of DOI:10.1016/S0968-0896(03)00117-2

1,4,7-Trisubstituted isoquinolines were designed, synthesized and evaluated for their inhibition against Plasmodium falciparum cysteine protease falcipain-2. The 1-benzyloxyphenyl-dihydroisoquinoline and -isoquinoline derivatives were found to exhibit better activity against falcipain-2 than their corresponding 1-hydroxyphenyl or 1-methoxyphenyl analogues. The docking scores correlate with the IC₅₀ values of compounds and give a high coefficient correlation of 0.94.

Full text of DOI:10.1016/S0968-0896(03)00117-2

Bioorganic & Medicinal Chemistry 11 (2003) 2293–2299
Structure-Based Approach to Falcipain-2 Inhibitors: Synthesis and
Biological Evaluation of 1,6,7-Trisubstituted Dihydroisoquinolines
and Isoquinolines
Sanjay Batra,a,*,y Yogesh A. Sabnis,b Philip J. Rosenthalc and Mitchell A. Averyb,d,
*
aMedicinal Chemistry Division, Central Drug Research Institute, Lucknow 226001, India
bDepartment ofMedicinal Chemistry, National Center for Natural Products Research, School ofPharmacy,
University ofMississippi, University, MS 38677-1848, USA
cDepartment ofMedicine, University ofCalifornia at San Francisco, San Francisco, CA, USA
dDepartment ofChemistry, PO Box 1848, University ofMississippi, University, MS 38677-1848, USA
Received 21 November 2002; accepted 4 February 2003
Abstract—1,4,7-Trisubstituted isoquinolines were designed, synthesized and evaluated for their inhibition against Plasmodium fal-
ciparum cysteine protease falcipain-2. The 1-benzyloxyphenyl-dihydroisoquinoline and -isoquinoline derivatives were found to
exhibit better activity against falcipain-2 than their corresponding 1-hydroxyphenyl or 1-methoxyphenyl analogues. The docking
scores correlate with the IC50 values of compounds and give a high coefficient correlation of 0.94.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
for chemotherapy has led to the identification of pro-
tease inhibitors as an attractive alternative.9À15 The
acidic food vacuole within the parasite contains two
major peptide hydrolases (proteases), namely aspartic
and cysteine proteases along with other enzymes. These
proteases are considered to be responsible for an
ordered hemoglobin degradation that produce essential
amino acids required for parasite survival.16À19 There-
fore, the disruption of the hemoglobin degradation
pathway in the malaria parasite seems to be a logical
approach to antimalarial chemotherapy.20 Though the
various steps in the degradation pathway can be tar-
geted individually, there are several reports indicating
that inhibition of the plasmepsins (aspartic protease)
and falcipains (cysteine vacuolar proteases) could prove
lethal to the parasite.21À27 In our endeavors toward
development of antimalarials following a structure-
based design approach against the cysteine proteases we
have recently reported the homology modeling of falci-
pain-2.28 This model was built with great accuracy and
validated by docking studies of the known peptidyl
vinyl sulfone inhibitors. Based on this three-dimensional
model, a new non-peptide inhibitor having isoquinoline
motif (Fig. 1) was designed, synthesized and assayed for
inhibition of falcipain-2. In our studies interaction of
1-(4-hydroxy-phenyl) group with the Asp 234 in the S-2
Malaria remains to be one of the most deadly parasitic
diseases affecting 300 million people worldwide and
leading to more than 2 million deaths per year.1 The
management of continuing upsurge of malaria is further
complicated due to widespread resistance of the parasite
to common antimalarials and cross resistance to struc-
turally unrelated drugs.2À4 To prevent resistance against
the new drugs, artemisinin and its analogues are
restricted to the institutional use in the malaria-prone
areas. In addition to overcome resistance, usage of
combination therapy5 and development of resistance
modulators6,7 have also been attempted. Recently, the
feasibility of generating populations of transgenic mos-
quitoes that have diminished potential to carry and
transmit parasite8 has been explored. Therefore, chemo-
therapy of malaria remains a challenge to medicinal
chemists. Extensive research to identify newer targets
*Corresponding authors. S. Batra: tel.: +91-522-2212411-18 ext. 4368;
fax: +91-522-2223405 or 2223938; M.A. Avery: tel.: +1-662-915-5880;
fax: +1-662-915-5638. E-mail: batra_san@yahoo.co.uk (S. Batra);
mavery@olemiss.edu (M.A. Avery).
yVisiting Research Associate from November 2000–November 2001 in
Department of Medicinal Chemeistry, School of Pharmacy, University
of Mississippi, University, MS 38677, USA.
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00117-2

2294
S. Batra et al. / Bioorg. Med. Chem. 11 (2003) 2293–2299
Table 1. Effect of substituent on the cyclization of amides to dihy-
droiso-quinolines
Entry
R
R0
Time
Yield (%)
1
2
3
4a
528
6
3,4-(OMe)2
3-OMe-4-OBn
4-OBn
4-Br
4-Bn
3,4-(OMe)2
3-OMe-4-OBn
3,4-OCH2O
OMe
OMe
OMe
OMe
OMe
OBn
OBn
OBn
1 h
3 h
27 h
85
68
30
—
25
72
26
43
No reaction
3 days
1.5 h
Figure 1. Structures of reported isoquinolines active at micromolar
concentration.28
7
8h
16 h
8
pocket29 was considered essential, therefore it was deci-
ded to synthesize and evaluate simpler 1,6,7-trisub-
stituted isoquinoline analogue against falcipain-2 as
prelude to discover new antimalarial agents. The details
of this study are presented herein.
aNo data since it did not gave the corresponding dihydroisoquinoline.
(8b–c). These isoquinolines could not be regioselectively
demethylated at position 4 of the phenyl ring present at
position 1 of isoquinolines as any such attempt led to
polymeric material. Therefore, in an improved synthetic
methodology instead of p-anisoyl chloride, p-benzy-
loxy benzoyl chloride was used to obtain the ethana-
mides 5a–b, d followed by cyclization to furnish the
dihydroisoquinolines (7a–b, d). These were then dehydro-
genated to afford the quinolines (9a–b, d) (Table 2). The
deprotection of the benzyl group in the dihydro-
isoquinolines 7a led to 10a while similar deprotection in
isoquinolines 9a, d yielded the corresponding hydroxy
derivatives 11a, d in good yields. However deprotection in
9b resulted in mixture of 11b and 11f. Thus, the hydroxy
quinolines obtained herein were in better yields than that
obtained through deprotection of methoxy group.28
Chemistry
The synthetic strategy adopted to obtain various iso-
quinoline derivatives is described in Scheme 1. The
substituted benzaldehydes (1a–e) used as starting sub-
strates were converted to corresponding nitroalkenes
(2a–e) via Henry reaction under reported conditions.30
These nitroalkenes (2a–e) were then reduced to benzene-
ethanamines (3a–e) in the presence of LiAlH4. These
amines (3b–c, e) were converted to amides (4b–c, e)
using p-anisoyl chloride followed by cyclization in the
presence of phosphorous oxychloride to afford 3, 4-dihy-
dro-isoquinolines (6b–c). Interestingly the cyclizations
to dihydroisoquinolines depend on the substitution on
phenyl ring (Table 1) as 4-bromo derivative failed to
cyclize (entry 4). The 3,4-dihydoisoquinolines were then
subjected to dehydrogenation in the presence of palla-
dium–carbon to furnish the isoquinoline analogues
Results and Discussion
The dihydroisoquinoline and isoquinoline analogues
synthesized were subjected to in vitro evaluation for
Scheme 1. Reagents and conditions; (i) CH3NO2, N HOAc, AcOH, ultrasound, 3 h; (ii) LiAlH4, dry THF, 65 ꢀC, 1.5 h; (iii) dry DCM, 6 h, rt;
(iv) POCl3, dry toluene; (v) 10% Pd/C, dry decalin; (vi) 10% Pd/C, H2, 30 psi, 8–12 h.
4

S. Batra et al. / Bioorg. Med. Chem. 11 (2003) 2293–2299
2295
Table 3. In vitro activity against Falcipain-2 along with the docking
scores in the homology model
Table 2. Effect of substituents on the dehydrogenation of dihydroiso-
quinolines to quinolines
R0
Time (h)
Yield (%)
Compd
Compd
IC50 (mM)
Docking scores
(kcal/mol)
Entry
R
1
2
3
428
5
3,4-(OMe)2
3-OMe-4-OBn
4-OBn
OMe
OMe
OMe
OMe
OBn
OBn
OBn
3
4.5
15
16
30
38
36
57
48
39
35
78
76
72
R
R’
6b
6c
7a
7b
7d
8b
9a
9b
3-OMe-4-OBn
4-OBn
3,4-(OMe)2
OMe
OMe
OBn
OBn
OBn
OMe
OBn
OBn
OBn
OH
10
10
10
4
3
10
3
À200
À233
À216
À328
À406
À178
À401
À399
À408
4-Bn
3,4-(OMe)2
3-OMe-4-OBn
3,4-OCH2O
3-OMe-4-OBn
3,4-OCH2O
3-OMe-4-OBn
3, 4-(OMe)2
3-OMe-4-OBn
3,4-OCH2O
3-OMe-4-OBn
3,4-(OMe)2
6
7
3
3
inhibition of protease activity. The results of biological
activity are summarized in Table 3. The activities of
these new analogues were comparable to that of the
previous compounds (Fig. 1) with a few analogues
showing better potency.
9d
10b
11a
11a
11b
11f
11d
NS
10
10
8
10
N
OH
OH
OH
OH
À176
À176
À298
À244
3,4-(OMe)2
3-OMe-4-OBn
3-OMe-4-OH
3,4-OCH2O
OH
S
The docking scores for these compounds were calcu-
lated as described in the experimental section and are
summarized in Table 3. From the graph between the log
IC50 values and docking scores, it is evident that dock-
ing scores indeed correlate to the IC50 values of the
compounds, giving a high coefficient correlation of 0.94
(Fig. 3). This trend was analogous with that of vinyl
sulfone inhibitors, which were originally used to vali-
date our homology model.28 From results of the activity
it could be speculated that substitutions on 6, 7 posi-
tions of the isoquinoline does not play a significant role
toward the activity. All compounds with p-methoxy
phenyl substitution at position 1 of the isoquinoline
were almost equipotent as the earlier reported ligands.28
On the other hand compounds with p-benzyloxy phenyl
group were found to be more potent as compared to
Figure 3. Correlation between log IC50 versus docking scores.
Figure 2. Sequences having significant scores.

2296
S. Batra et al. / Bioorg. Med. Chem. 11 (2003) 2293–2299
their corresponding hydroxyl analogues suggesting that
S-2 pocket prefer to accommodate hydrophobic groups.
Also the planarity of the isoquinolines is not important
since both the dihydroisoquinoline and isoquinoline
were found to have almost similar IC50 values. The
narrow range of IC50 of all compounds described herein
may be from the fact that all these inhibitors share com-
mon skeleton with little variation on their side chains. On
the basis of these results the new ligand having benzyloxy
phenyl group at position 1 and 6, 7 disubstitution in the
isoquinoline moiety was docked into the active pocket of
the falcipain homology model (Figs 4 and 5). The results
from the assay of these compounds could be used to fur-
ther gain insight of the nature of binding interactions
they have with the enzyme, which may promote the
development of better inhibitors. Future efforts will
focus on the synthesis and evaluation of other hetero-
cyclic derivatives with similar structural framework with
an objective to have better inhibition against falcipain-2
correlating to the antimalarial activity.
Experimental
Melting points were determined in capillary tubes on a
hot stage apparatus and are uncorrected. 1H N MR
spectra were recorded on a Bruker 400 FT spectrometer,
using TMS as an internal reference; chemical shifts
values are in d ppm and J values are in Hz. Mass spectra
were recorded on a Waters-Micromass LCMS system
using direct flow injections. Reactions were run in oven-
dried glassware and dried solvents that were prepared
by distillation over calcium hydride.
Nitroalkenes (2a–e). A mixture of appropriately sub-
stituted benzaldehyde (10 mmol) ammonium acetate
(1.76 g, 22 mmol), nitromethane (7.0 mL, 100 mmol),
acetic acid (1.7 mL) was sonicated for 3 h at room tem-
perature. The separated solid was filtered off and
washed with ethanol–water (50:50) mixture and air
dried to obtain the product in 76–88% yields.
Benzeneethanamines (3a–e). To the stirred suspension of
LiAlH4 (470 mg, 12.2 mmol) in THF (3 mL) was added
dropwise,
a solution of appropriately substituted
nitroalkene (3.5 mmol) in THF (25 mL) and the reac-
tion mixture was stirred at 65 ꢀC for 6 h. It was then
cooled down to 0 ꢀC and decomposed with 30% aq
sodium hydroxide solution. To this solution ethyl ace-
tate was added and the mixture was stirred for another
30 min. The organic layer was separated, dried over
sodium sulfate and evaporated to obtain pure amines as
oils in 70–82% yields. These amines were used for fur-
ther reaction without any purification.
Benzeneethanamides (4a–c, e and 5a–b, d). To the stirred
solution of amine (6.8mmol) in dry dichloromethane (15
mL) was added freshly baked potassium carbonate (2.25
g, 13.6 mmol) and the reaction mixture was cooled to
0 ꢀC. A solution of acid chloride (6.8 mmol) in di-
chloromethane (15 mL) was added dropwise and the
reaction was continued to stir at room temperature
for 8 h. Thereafter the reaction mixture was extracted
with water and dichloromethane. The organic layer
was separated, washed with brine dried over sodium
sulfate and evaporated to obtain a residue. This upon
column chromatography over silica gel using di-
chloromethane–methanol (99:1) furnished the pure
product.
Figure 4. 1,4,7-Substituted isoquinoline docked in the active pocket.
Dihydroisoquinolines (6b–c, 7a–b, d). To an appropriate
solution of substituted ethanamide (1.3 mmol) in tolu-
ene (10 mL) was added phosphorous oxychloride (3.37
mL, 3.62 mmol) and the reaction mixture was refluxed
under stirring at 110 ꢀC as per the time indicated in
Table 1. The reaction was cooled down and was poured
onto ice and decomposed using 20% aq sodium
hydroxide solution. It was then extracted with ethyl
acetate (2ꢁ50 mL) and water. The organic layers were
combined, dried over sodium sulfate and evaporated to
obtain a residue. This residue upon column chromato-
graphy over silica gel employing dichloromethane–
methanol (98:2) furnishes the pure product.
Figure 5. The falcipain-2 active pocket with OBn group in the S-2
subsite.

S. Batra et al. / Bioorg. Med. Chem. 11 (2003) 2293–2299
2297
1-(4-Methoxyphenyl)-6-methoxy-7-benzyloxy-3, 4-di-hy-
droisoquinoline (6b). Mp 188–189 ꢀC; H NMR (CDCl3,
1-(4-Methoxyphenyl)-6-methoxy-7-benzyloxy-isoquino-
line (8b). Mp 167–168 ꢀC; H NMR (CDCl3, 400 MHz):
1
1
400 MHz): 3.06 (t, 2H, J=6.0 Hz, –CH2), 3.91 (s, 3H,
OMe), 3.95 (m, 2H, J=6.0 Hz, –NCH2), 4.06 (s, 3H,
OMe), 5.09 (s, 2H, –OCH2), 6.95 (s, 3H, Ar–H), 7.30 (s,
5H, Ar–H), 7.51, 7.53(d, 2H, J=8.0 Hz, Ar–H); mass
(ESMS+) 374.17 (calcd), 374.25 (found).
3.90, 4.05 (2s, 6H, 2ꢁOMe), 5.06 (s, 2H, –OCH2), 6.96,
6.98 (d, 2H, J=8.0 Hz, Ar–H), 7.37 (m, 7H, Ar–H),
7.43, 7.45 (d, 1H, J=8.0 Hz, Ar–H), 8.41, 8.42 (d, 1H,
J=6.0 Hz, Ar–H); mass (ESMS+) 372.11 (calcd),
372.27 (found).
1-(4-Methoxyphenyl)-7-benzyloxy-3, 4-dihydroisoquino-
1-(4-Methoxyphenyl)-6-methoxy-7-hydroxy-isoquinoline
(8e). Mp 100–102 ꢀC; 1H NMR (CDCl3, 400 MHz):
3.86, 4.06 (2s, 6H, 2ꢁOMe), 7.01, 7.03 (d, 2H, J=8.0
Hz, Ar–H), 7.12 (s, 1H, Ar–H), 7.45, 7.47 (d, 1H, J=6.0
Hz, Ar–H), 7.51 (s, 1H, Ar–H), 7.58, 7.60 (d, 2H, J=8
Hz, Ar–H), 8.40, 8.42 (d, 1H, J=8.0 Hz, Ar–H); mass
(ESMS+) 282.11 (calcd), 282.19 (found).
ꢀ
1
line (6c). Mp 98–99 C; H NMR (CDCl3, 400 MHz):
2.71(t, 2H, J=6.0 Hz, –CH2), 3.79 (m, 2H, J=6.0 Hz,
–NCH2), 3.86 (s, 3H, OMe), 4.99 (s, 2H,–OCH2), 6.90,
6.92(d, 2H, J=8.0 Hz, Ar–H), 7.01, 7.03 (d, 1H, J=8.0
Hz, Ar–H), 7.17, 7.19 (d, 1H, J=8.0 Hz, Ar–H), 7.34 (s,
5H, Ar–H), 7.51, 7.53 (d, 2H, J=8.0 Hz, Ar–H); mass
(ESMS+) 356.16 (calcd), 356.00 (found).
1-(4-Benzyloxyphenyl)-6, 7-dimethoxy-isoquinoline (9a).
Mp oil; 1H NMR (CDCl3, 400 MHz): 3.88 (s, 3H,
OMe), 4.05 (s, 3H, OMe), 5.17 (s, 2H, –OCH2), 7.14 (m,
4H, Ar–H), 7.43 (m, 5H, Ar–H), 7.65, 7.67 (d, 2H,
J=8.0 Hz, Ar–H), 8.44, 8.46 (d, 1H, J=8.0 Hz, Ar–H);
mass (ESMS+) 372.15 (calcd), 372.23 (found).
1-(4-Methoxyphenyl)-6-methoxyꢀ-7-hydroxy-3, 4-dihy-
1
droisoquinoline (6f). Mp 206–207 C; H NMR (CDCl3,
400 MHz): 2.71 (t, 2H, J=6.0 Hz, –CH2), 3.78 (t, 3H,
J=6.0 Hz, –NCH2), 3.85 (s, 3H, OMe), 4.02 (s, 3H,
OMe), 6.76 (s, 1H, Ar–H), 6.90 (s, 1H, Ar–H), 6.92, 6.94
(d, 2H, J=8.0 Hz, Ar–H), 7.54, 7.56 (d, 2H, J=8.0 Hz,
Ar–H); mass (ESMS+) 284.12 (calcd), 284.13 (found).
1-(4-Benzyloxyphenyl)-6-methoxy-7-benzyloxy-isoquino-
line (9b). Mp oil; 1H NMR (CDCl3+DMSO-d6,
400 MHz): 4.05 (s, 3H, OMe), 5.18 (s, 4H, OCH2O),
7.03, 7.05 (d, 2H, J=8.0 Hz, Ar–H), 7.12 (s, 1H, Ar–H),
7.34 (m, 12H, Ar–H), 7.49, 7.51 (d, 2H, J=8.0 Hz, Ar–
H), 8.41, 8.42 (d, 1H, J=6.0 Hz, Ar–H); mass
(ESMS+) 448.19 (calcd), 448.27 (found).
1-(4-Benzyloxyphenyl)-6, 7-dimethoxy-3, 4-dihydroiso-
quinoline (7a). Mp 147–149 ꢀC; 1H NMR (CDCl3,
400 MHz): 2.71 (t, 2H, J=7.2 Hz,–CH2), 3.76 (m, 5H,
–NCH2 and OMe), 3.95 (s, 3H, OMe), 5.12 (s, 2H,
–OCH2), 6.78 (s, 1H, Ar–H), 6.84 (s, 1H, Ar–H), 7.02,
7.04 (d, 2H, J=8.0 Hz, Ar–H), 7.39 (m, 5H, Ar–H),
7.56, 7.58 (d, 2H, J=8.0 Hz, Ar–H), 8.39, 8.40 (d, 1H,
J=5.6 Hz, Ar–H); mass (ESMS+) 374.17 (calcd),
374.38 (found).
1-(4-Benzyloxyphenyl)-6, 7-methylenedioxy-isoquinoline
(9d). Mp 96–97 ꢀC; 1H NMR (CDCl3+DMSO-d6,
400 MHz): 5.18 (s, 2H, –OCH2), 5.83 (s, 2H, OCH2O),
6.87, 6.89 (d, 2H, J=8.0 Hz, Ar–H), 7.02 (m, 3H, Ar–
H), 7.21 (s, 5H, Ar–H), 7.68, 7.70 (d, 2H, J=8.0 Hz,
Ar–H), 8.31, 8.32 (d, 1H, J=6.0 Hz, Ar–H); mass
(ESMS+) 356.38 (calcd), 356.57 (found).
1-(4-Benzyloxyphenyl)-6-methoxy-7-benzyloxy-3, 4-dihy-
droisoquinoline (7b). Mp oil; 1H NMR (CDCl3
+DMSO-d6, 400 MHz): 2.68 (t, 2H, J=7.2 Hz,–CH2),
3.75 (t, 2H, J=7.2 Hz, –NCH2), 3.96 (s, 3H, OMe), 5.03
(s, 2H, –OCH2), 5.13 (s, 2H, –OCH2), 6.78 (s, 1H, Ar–
H), 6.83 (s, 1H, Ar–H), 6.91, 6.93 (d, 2H, J=8.0 Hz,
Ar–H), 7.36 (m, 10H, Ar–H), 7.46, 7.48 (d, 2H, J=8.0
Hz, Ar–H), 8.39, 8.40 (d, 1H, J=5.6 Hz, Ar–H); mass
(ESMS+) 450.20 (calcd), 450.45 (found).
Debenzylation (10a–b, d, f and 11a–b, d, f). A mixture of
appropriate compound (0.1 g) in methanol (5 mL) and
10% palladium (20 mg) on carbon was hydrogenated
at 40 psi for 8–12 h. The catalyst was filtered off
through a bed of Celite and the filtrate was evaporated
to obtain a residue. This residue upon column chro-
matography over silica gel using dichloromethane:
methanol (95:5) furnished the pure product in 90–95%
yields.
1-(4-Benzyloxyphenyl)-6, 7-methylenedioxy-isoquinoline
(7d). Mp 175–176 ꢀC; 1H NMR (CDCl3+DMSO-d6,
400 MHz): 2.91 (t, 2H, J=6.0 Hz, –CH2), 3.83 (t, 2H,
J=6.0 Hz, –NCH2), 5.18 (s, 2H, –OCH2), 5.88 (s, 2H,
OCH2O), 6.58 (s, 1H, Ar–H), 6.93 (s, 1H, Ar–H), 7.01,
7.03 (d, 2H, J=8.0 Hz, Ar–H), 7.34 (s, 5H, Ar–H), 7.74,
7.76 (d, 2H, J=8.0 Hz, Ar–H); Mass (ESMS+) 358.40
(calcd), 358.29 (found).
1-(4-Hydroxyphenyl)-6, 7-dimethoxy-3, 4-dihydroisoqui-
120–121 ꢀC;
noline
(10a).
Mp
1H
N
MR
(CDCl3+DMSO-d6, 400 MHz): 2.79 (t, 2H, J=7.2
Hz,–CH2–), 3.64 (s, 3H, OMe), 3.76 (m, 3H, –NCH2),
3.96 (s, 3H, OMe), 6.64, 6.66 (d, 2H, J=8.0 Hz, Ar–H),
6.78 (s, 1H, Ar–H), 6.86 (s, 1H, Ar–H), 7.35, 7.37 (d,
2H, J=8.0 Hz, Ar–H); Mass (ESMS+) 284.12 (calcd),
284.35 (found).
Isoquinolines (8b–c and 9a–b, d). A mixture of appro-
priate dihydroisoquinoline (0.3 g) and 10% palladium
on carbon in 2 mL of decalin was heated at 230 ꢀC
under stirring for a period of time as indicated in Table
2. To the reaction mixture dichloromethane was added
and the catalyst was filtered off over Celite. The filtrate
was evaporated and subjected to column chromato-
graphy over silica gel. Elution with dichloromethane–
methanol (98:2) furnished the pure product.
1-(4-Hydroxyphenyl)-6, 7-dimethoxy-isoquinoline (11–a).
Mp 212–214 ꢀC; 1H NMR (CDCl3+DMSO-d6,
400 MHz): 3.87 (s, 3H, OMe), 4.05 (s, 3H, OMe), 6.78,
6.80 (d, 2H, J=8.0 Hz, Ar–H), 7.13 (s, 1H, Ar–H), 7.36
(s, 1H, Ar–H), 7.37, 7.39 (d, 2H, J=8.0 Hz, Ar–H),

2298
S. Batra et al. / Bioorg. Med. Chem. 11 (2003) 2293–2299
7.51, 7.52 (d, 2H, J=5.2 Hz, Ar–H), 8.41, 8.42 (d, 2H,
J=5.2 Hz, Ar–H); Mass (ESMS+) 282.11 (calcd),
282.11 (found).
manually refined to optimize the matching of several
characteristics including (a) conserved protease hydro-
phobic side chains and buried positions in the template
structures, (b) functionally important sites such as the
active site cysteine, and (c) insertions/deletions.
1-(4-Hydroxyphenyl)-6-methoxy-7-benzyloxy-isoquino-
212–214 ꢀC;
line
(11b).
Mp
1H
N
MR
(CDCl3+DMSO-d6, 400 MHz): 4.06 (s, 3H, OMe),
5.16 (s, 2H, –OCH2), 6.65, 6.67 (d, 2H, J=8.0 Hz, Ar–
H), 7.13 (s, 1H, Ar–H), 7.32 (m, 5H, Ar–H), 7.49, 7.51
(d, 2H, J=5.6 Hz, Ar–H), 8.39, 8.40 (d, 1H, J=5.6
Hz, Ar–H); mass (ESMS+) 358.14 (calcd), 358.31
(found).
Five SCRs were conceived based on this alignment and
are shown in Fig. 2. Cruzain, having an overall percent
identity of 55% and more than 90% in the SCRs with
falcipain-2 (Fig. 1), was chosen to derive coordinates for
building the SCRs of falcipain-2.
The last step in the generation of the 3-D model was
building the structurally variable regions (SVRs) or the
loops onto the SCRs. The Tweak Loop approach34,35
was used to build loops onto the SCRs. The loop resi-
due torsion angles were then manually adjusted to
achieve an exact fit to the model anchor coordinates.
1-(4-Hydroxyphenyl)-6, 7-methylenedioxy-isoquinoline
(11d). Mp 66–67 ꢀC; 1H NMR (CDCl3+DMSO-d6,
400 MHz): 5.88 (s, 2H, OCH2O), 6.69, 6.71 (d, 2H,
J=8.0 Hz, Ar–H), 7.03 (m, 3H, Ar–H), 7.66, 7.68 (d,
2H, J=8.0 Hz, Ar–H), 8.40, 8.41 (d, 1H, J=5.6 Hz,
Ar–H); mass (ESMS+) 266.07 (calcd), 266.09 (found).
Further refinement of the model was performed using
Kollman charges36 by first minimizing the backbone
atoms to a gradient of 0.01 A followed by complete
side-chain minimization. All molecular dynamic (MD)
simulations assisted by simulated annealing (SA) were
performed at a constant temperature of 300 K. A
time step of 1 fs (10À15) for a total length of 1000 fs,
with a snapshot at every 5 fs, a long non-bonding
cutoff distance of 12 A, and a distance dependent
dielectric of e=4 were used. The non-bonding pair list
was updated every 25 fs. In SA, the protein was heated
at 700 K for 10,000 fs and annealed to 200 K for
10,000 fs for a total of 10 cycles. The different
conformations generated by MD-SA were further
minimized and the structure with lowest energy was
validated using the PROTABLE and was then used for
docking studies.
1-(4-hydroxyphenyl)-6-methoxy-7-hydroxy-isoquinoline
(11f). Mp 175–176 ꢀC; H NMR (CDCl3+DMSO-d6,
1
400 MHz): 3.93 (s, 3H, OMe), 6.82, 6.84 (d, 2H, J=8
Hz, Ar–H), 6.99 (s, 1H, Ar–H), 7.31, 7.33 (d, 2H, J=5.6
Hz, Ar–H), 7.40 (m, 3H, Ar–H), 8.24, 8.25 (d, 2H,
J=5.6 Hz, Ar–H); mass (ESMS+) 268.09 (calcd),
268.08 (found).
Computational method
Construction of homology model, docking, molecular
dynamics and energy minimization studies were per-
formed on a Silicon Graphics Octane 2 workstation
with dual R12000 processors. Homology modeling was
performed within the COMPOSER module of SYBYL
6.7 (Tripos Asso. Inc. St. Louis, MO, USA). The model
was theoretically validated by the PROTABLE module
of BIOPLOYMER. DOCK 4.031 was employed for
docking the designed inhibitors into the active pocket of
falcipain-2.
Docking studies/protocol
In order to further validate the falcipain-2 model, the
designed inhibitors (Table 3) were docked into the
active site of falcipain-2. MD-SA was performed on
these inhibitors using a protocol similar to the one
described for protein refinement above, except that the
snapshots were taken at every 25 fs and length of simu-
lation was 5000 fs. This resulted in 20 conformations for
each of the inhibitor, which were then subjected to rigid
docking. For docking, a Connolly surface of the pro-
tein’s active site (all residues within a radius 5 A around
Cys 42) was created using a 1.4 A probe radius and
further used to generate a cluster of 46 overlapping
spheres. To compute interaction energies, a 3-D grid of
0.3 A resolution was centered on the active site. Gastei-
ger–Huckel charges for the ligands and Kollman char-
ges for the protein were employed. A 6-12 Lennard–
Jones potential was used for the vander Waals compo-
nent of the scoring function. A distance-dependent
dielectric function (e=r), energy cutoff distance of 10.0
A and contact cutoff distance of 4.5 A were used. A 0.75
A of van der Waals overlap was allowed.15 All other
parameters were set to their default values. Different
orientations of the ligands were searched and ranked
based on their energy scores.
Homology modeling
The falcipain-2 sequence was obtained from SWISS-
PROT and TrEMBL databases of ExPASy Molecular
Biology Server.32 Only the mature sequence (Q9N6S8)
was considered for deriving the homology model. A
WU-BLAST 2.0 search was performed on the mature
falcipain-2 sequence with default parameters of
BLASTP gapped alignment. All the homologous pro-
teins identified by this search (a total of 13 sequences),
belonged to the cysteine protease family. The sources
for these homologs included Homo sapiens, Carica
papaya, Trypanosoma cruzi and Leishmania major.
The model sequence was aligned against each sequence
in the database using the reported method of Needle-
man and Wunsch.33 Gaps were inserted into either
sequence to find an optimal alignment. A significance
score in the range of 19.8–27.6 was obtained for the
query sequence. Only those sequences having a sig-
nificance score of 25 or more were considered for struc-
ture alignment (Fig. 2). The resulting alignment was

S. Batra et al. / Bioorg. Med. Chem. 11 (2003) 2293–2299
12. Rosenthal, P. J. Adv. Parasitol. 1999, 43, 105.
2299
Assay for protease activity
13. Berry, C. In Proteases ofInfectious Agents ; Dunn, B. M.,
Ed.; Proteases as Drug Targets for the Treatment of Malaria;
Academic: San Diego, 1999; p 165.
14. Bell, A. Curr. Opin. Anti-infect. Invest. Drugs 2000, 2, 310.
15. McKerrow, J. H. J.; Engel, C.; Caffrey, C. R. Bioorg.
Med. Chem. 1999, 7, 639.
16. Eggelson, K. K.; Duffin, K. L.; Goldberg, D. E. J. Biol.
Chem. 1999, 274, 32411.
17. Shenai, B. R.; Sijwali, P. S.; Singh, A.; Rosenthal, P. J.
J. Biol. Chem. 2000, 275, 29000.
18. Gavigan, C. S.; Dalton, J. P.; Bell, A. Mol. Biochem.
Parasitol. 2001, 117, 37.
19. Humphreys, M. J.; Moon, R. P.; Klinder, A.et al. FEBS
Lett. 1999, 463, 43.
20. Francis, S. E.; Sullivan, D. J.; Goldberg, D. E., Jr. Ann.
Rev. Microbiol. 1997, 51, 97.
21. Rosenthal, P. J. Emerg. Infect. Dis. 1998, 4, 49.
22. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
23. Ring, C. S.; Sun, E.; McKerrow, J. H.; Lee, G. K.;
Rosenthal, P. J.; Kuntz, I. D.; Cohen, F. E. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 3583.
24. Li, Z.; Chen, X.; Davidson, E.; Zwang, O.; Mendis, C.;
Ring, C. S.; Roush, W. R.; Fegley, G.; Li, R.; Rosenthal, P. J.;
Lee, G.; Kenyon, G. L.; Kuntz, I. D.; Cohen, F. E. Chem.
Biol. 1994, 1, 31.
Assays of the hydrolysis of the fluorogenic substrate
Z-Phe-Arg-AMC (Enzyme Systems Products, Liver-
more, CA, USA), by soluble parasite extracts contain-
ing falcipain were performed as previously described.
For all assays, 4 nM falcipain was incubated with 10
nM dithiothreitol and compounds added from 100ꢁ
stocks (in DMSO) in 0.1 M sodium acetate, pH 5.5, for
30 min at room temperature before the Z-Phe-Arg-
AMC substrate (final concentration 50 mM) was added.
Fluorescence caused by the cleavage of the substrate
(excitation 380 nm, absorbance 460 nm) was then mon-
itored continuously over a period of 30 min. The rate of
substrate (increase in fluorescence over time) in the pre-
sence of isoquinolines inhibitors was compared with the
rate of hydrolysis in controls incubated with an equiva-
lent volume of DMSO. In each experiment, multiple
concentrations of dihydroisoquinolines and iso-
quinolines were evaluated in duplicate or triplicate, and
IC50 values were extrapolated from curves of percent
control activity over concentration.
25. Domy´ ’nguez, J. N.; Charris, J. E.; Lobo, G.; Gamboa de
Domy’nguez, N.; Moreno, M. M.; Riggione, F.; Sanchez, E.;
Acknowledgements
´
Olson, J.; Rosenthal, P. J. Eur. J. Med. Chem. 2001, 36, 555.
26. Scheidt, K. A.; Roush, W. R.; McKerrow, J. H.; Selzer,
P. M.; Hansell, E.; Rosenthal, P. J. Bioorg. Med. Chem. 1998,
6, 2477.
This work was supported by a CDC Cooperative
agreement number U50/CCU418839.
27. Olson, J. E.; Lee, G. K.; Semenov, A.; Rosenthal, P. J.
Bioorg. Med. Chem. 1999, 7, 633.
References and Notes
28. Sabnis, Y.; Rosenthal, P. J.; Desai, P.; Avery, M. A.
J. Biomol. Str. Dyn. 2002, 19, 765.
29. Sajid, M.; McKerrow, J. H. Mol. Biochem. Parasitol.
2002, 120, 1.
30. McNulty, J.; Steere, J. A.; Wolf, S. Tetrahedron Lett.
1998, 39, 8013.
31. Kuntz, I. D.; Blaney, J. M.; Oatley, S. J.; Langridge, R. L.
J. Mol. Biol. 1982, 161, 269.
32. Bairoch, A.; Apweiler, R. Nucleic Acids Res. 2000, 28, 45.
33. Needleman, S.; Wunsch, C. J. Mol. Biol. 1970, 48, 443.
34. Shenkin, P. S.; Yarmush, D. L.; Fine, R. M.; Wang, H.;
Levinthal, C. Biopolymers 1987, 26, 2053.
1. Breman, J. G.; Am, J. Trop. Med. Hyg. 2001, 64s, 1.
2. Wongsrichanalai, C.; Pickard, A. L.; Wernsdorfer, W. H.;
Meshnick, S. R. Lancet Infect. Diseases 2002, 2, 209.
3. Hyde, J. E. Microbes Infect. 2002, 4, 165.
4. Olliaro, P.; Cattani, J.; Wirth, D. JAMA 1996, 275, 230.
5. Batra, S.; Bhaduri, A. P. Adv. Drug Res. 1997, 30, 202.
6. Batra, S.; Srivastava, P.; Roy, K.; Pandey, V. C.; Bhaduri,
A. P. J. Med. Chem. 2000, 43, 3428.
7. Nosten, F.; Brasseur, P. Drugs 2002, 62, 1315.
8. Ito, J.; Ghosh, A.; Moriera, L. A.; Winnes, E. A.; Jacobs-
Lorena, M. Nature 2002, 417, 452.
9. Olliaro, P. L.; Yuthavong, Y. Pharmacol. Ther. 1999, 81, 91.
10. Coombs, G. H.; Mottram, J. C. Parasitology 1997, 114, S61.
11. Coombs, G. H.; Goldberg, D. E.; Klemba, M.; Berry, C.;
Kay, J.; Mottram, J. C. Trends Parasitol. 2001, 17, 532.
35. Shenkin, P. S.; Yarmush, D. L.; Fine, R. M.; Wang, H.;
Levinthal, C. Proteins 1986, 1, 342.
36. Cieplak, P.; Cornell, W.; Bayly, C.; Kollman, P. J. Com-
put. Chem. 1995, 16, 1357.