Novel 2H-isoquinolin-3-ones as antiplasmodial falcipain-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.08.013

A series of 1-aryl-6,7-disubstituted-2H-isoquinolin-3-ones (2-10) was synthesized and evaluated for their inhibition against Plasmodium falciparum cysteine protease falcipain-2, as well as against cultured P. falciparum strain FCBR parasites. All compound

Full text of DOI:10.1016/j.bmc.2009.08.013

Bioorganic & Medicinal Chemistry 17 (2009) 6505–6511
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 2H-isoquinolin-3-ones as antiplasmodial falcipain-2 inhibitors
a
b
c
c
Nicola Micale a, , Roberta Ettari , Tanja Schirmeister , Astrid Evers , Christoph Gelhaus ,
*
Matthias Leippe c, Maria Zappalà a, Silvana Grasso a
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata 98168, Messina, Italy
b Institute of Pharmacy and Food Chemistry, University of Würzburg Am Hubland, 97074 Würzburg, Germany
c Zoological Institute, University of Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-aryl-6,7-disubstituted-2H-isoquinolin-3-ones (2–10) was synthesized and evaluated for
their inhibition against Plasmodium falciparum cysteine protease falcipain-2, as well as against cultured
P. falciparum strain FCBR parasites. All compounds displayed inhibitory activity against recombinant fal-
cipain-2 and against in vitro cultured intraerythrocytic P. falciparum, with the exception of 9. The new
compounds exhibited no selectivity against human cysteine proteases such as cathepsins B and L. The
inhibitory activity of the synthesized compounds was also evaluated against another protozoal cysteine
protease, namely rhodesain of Trypanosoma brucei rhodesiense.
Received 16 June 2009
Revised 29 July 2009
Accepted 6 August 2009
Available online 12 August 2009
Keywords:
Cysteine proteases
Falcipain-2 inhibitors
2H-Isoquinolin-3-ones
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
infections.8 Several plants, traditionally used against malaria, con-
tain alkaloids with an isoquinoline core which have a broad-spec-
Malaria remains to be one of the most deadly parasitic diseases
affecting about 250 million people worldwide and leading to more
than 1 million deaths per year.1 The spread of multi-resistant Plas-
modium falciparum strains combined with the lack of effective vac-
cines, represents therefore a serious concern for the population in
malaria endemic regions and creates a great urge to the develop-
ment of new and effective antimalarial agents.2 Currently, artemis-
inin-based combination therapy seems to be the most safe and
efficacious solution, thus strictly recommended from WHO for
treatment of uncomplicated and severe malaria.3 The recently dis-
covered Plasmodium genome sequences have opened new perspec-
tives for malaria research. Although no licensed therapies have yet
resulted from genome-dependent experiments, it is likely that this
discovery can lead to new therapeutic approaches.4
Cysteine protease falcipain-2 (FP-2) of P. falciparum emerged as
a valid target for antimalarial drugs.5 FP-2 plays an essential role
for the parasite survival during the blood stage as a major peptide
hydrolase within the hemoglobin degradation pathway along with
other enzymes.5,6 Moreover, it is responsible for the erythrocyte
rupture by cleaving ankyrin and protein 4.1, the cytoskeletal ele-
ments vital to the stability of the red cell membrane.7
trum antimicrobial activity and many other pharmacological
effects.9 Several of these isoquinoline alkaloids with remarkable
antimalarial activity have been already identified and reported in
literature, including nitidine, duguevalline, berberine and allied
protoberberines such as palmatine and jatrorrhizine.10
Moreover, synthetic derivatives containing the isoquinoline
core, for example, 1-aryl-6,7-disubstituted isoquinolines 1 (Fig. 1)
designed on the basis of homology modeling studies,11 were found
to exhibit a promising inhibitory activity against FP-2 at micromo-
lar level.12
On these basis and in connection with our ongoing efforts to de-
velop FP-2 inhibitors for the treatment of malaria,13 we sought to
test 1-(4-aminophenyl)-3-methyl-6,7-methylenedioxy-isoquino-
line 2, previously synthesized in our research laboratories (Fig. 1)
against FP-2 and cultured P. falciparum.14
The results of the biological evaluation prompted us to synthe-
size a series of analogues of compound 2 in order to identify new
antimalarial lead compounds and find preliminary SAR trends.
CH3
O
O
3
6
7
R
1
N
N
Plants are still important resources for the discovery of new
drugs and are employed nowadays in folk medicine in poverty-
stricken regions of Africa, Asia and South America against parasitic
R= 6-OCH , 7-OCH ,
3
3
6-OCH3, 7-OCH Ph
2
6,7-OCH2O
OR'
NH2
R'= CH , CH2Ph
3
2
1
* Corresponding author. Tel.: +39 090 6766466; fax: +39 090 355613.
E-mail address: nmicale@pharma.unime.it (N. Micale).
Figure 1. Structures of isoquinolines 1 and 2.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.013

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N. Micale et al. / Bioorg. Med. Chem. 17 (2009) 6505–6511
RO
In particular, in order to explore the effects of structural varia-
RO
a
COOR''
O
COOCH3
c
tions at positions 2 and 3, the methyl group of 2 has been substi-
tuted with an ester moiety (3), a carbonyl group has been
introduced at position 3 and an amine function at position 2 (6),
which has been further functionalized (4–5, 7–10). In derivatives
4–10, the 40-NH2 group has been replaced by alkoxy groups,
according to the work of Batra et al., where it has been evidenced
that the S2 pocket of FP-2 prefers to accommodate hydrophobic
groups.12
All compounds were tested against recombinant FP-2 as well as
cultured P. falciparum strain FCBR parasites. Moreover, the inhibi-
tory activity of the synthesized compounds was also evaluated
against rhodesain, the major cysteine protease of Trypanosoma bru-
cei rhodesiense. Finally, selectivity against the target enzyme was
estimated by testing the new compounds against human cysteine
proteases of the papain-family, that is, cathepsins B and L.
R'O
R'O
13-14
OMe
13, 15, 17, 19, 4 RR'=CH2
14, 16, 18, 20, 5 R=R'=CH3
15-16, R'' = CH3
17-18, R'' = H
b
RO
O
N
CONHNHBoc
RO
d
O
R'O
R'O
NHBoc
OMe
OMe
19-20
4-5
Scheme 2. Reagents and conditions: (a) anisic acid, P2O5, (CH2)2Cl2, 16 h, room
temperature; (b) LiOH 1 N, THF/CH3OH (4/1), 4 h, room temperature; (c)
BocNHNH2, EDCI, CH2Cl2/DMF (1/1), 12 h, room temperature; (d) NaH, THF, 2 h,
room temperature.
2. Results and discussion
2.1. Chemistry
Target compounds were prepared following the synthetic strat-
egies outlined in Schemes 1–4.
O
O
O
O
O
O
O
Methylester 11,14 was treated with formamide to give 2H-iso-
quinolin-3-one 12 which, by reaction with acetyl chloride and sub-
sequent catalytic reduction of the nitro group afforded compound
3 (Scheme 1).
a
b
4
N
N
N
R
NH2
H
Ketoesters 15–16 were obtained via acylation of commercially
available methyl phenylacetates 13–14 with 4-methoxybenzoic
acid, in the presence of an excess of phosphorus pentoxide
(Scheme 2).15 The alkaline hydrolysis (LiOH) of 15–16 led the cor-
responding acids 17–18 which, by treatment with N-Boc-hydra-
zine, afforded the hydrazides 19–20. The cyclization to N-
substituted isoquinolin-3-ones 4–5 required the treatment with
NaH in THF.
OMe
OMe
7, R=OC2H5
8, R=NHPh-4-COCH3
9, R=NHPh-4-S-CH3
6
Scheme 3. Reagents and conditions: (a) TFA (25% in CH2Cl2), 5 h, room temper-
ature; (b) ethyl chloroformate/Et3N (for 7), or ArNCO (for 8–9), CH2Cl2, 3–6 h, room
temperature.
The removal of the N-Boc-protecting group of 4 was realized by
treatment with trifluoroacetic acid (TFA) to afford 6 which was
made to react with ethyl chloroformate in presence of triethyl-
amine or with aryl isocyanates to give derivatives 7 and 8–9,
respectively (Scheme 3).
HO
BnO
BnO
COOH
O
a
b
COOR
O
d
15
HO
According to the previous work of Batra,12 we decided to more
closely investigate the modifications at position 40 of the phenyl
ring at C-1 by introducing a benzyloxy group in this position.
Firstly, we established a convenient way to remove the O-methyl
group of 15 with boron tribromide. However, the treatment of 15
with the harsh Lewis acid BBr3 caused the methylenedioxy ring
cleavage and the hydrolysis of the ester moiety, giving [4,5-dihy-
droxy-2-(4-methoxybenzoyl)-phenyl]-acetic acid 21 as the sole
product. Direct and long-range heteronuclear chemical shift corre-
lation experiments (HETCOR and LR-HETCOR) have been per-
formed to confirm the structure of the obtained compound 21.
The key to the structure elucidation lies in a three-bond correlation
of the MeO protons (3.88 ppm) with the carbon resonating at
164.97 ppm, assigned as C-40 on the basis of long-range coupling
OMe
OMe
21
22, R =Bn
23, R =H
c
O
BnO
BnO
BnO
BnO
CONHNHBoc
O
e,f
N
NH2
OMe
OMe
24
10
Scheme 4. Reagents and conditions: (a) BBr3, CH2Cl2, 0 °C, then 3 h, room
temperature; (b) BnBr, K2CO3, acetone, 5 h, reflux; (c) LiOH 1 N, THF/CH3OH (4/1),
8 h, room temperature; (d) BocNHNH2, EDCI, CH2Cl2/DMF (1/1), 12 h, room
temperature; (e) NaH, THF, 2 h, room temperature; (f) TFA (25% in CH2Cl2), 5 h,
room temperature.
O
O
OCOCH3
O
COOCH3
O
O
O
O
b, c
O
a
NH
N
with H-20,60 (7.72 ppm), thus confirming that the O-demethylation
failed. In spite of this result, we moved forward with the synthesis
and derivative 21 was then reacted with benzyl bromide in alka-
line medium to effect the O-benzylation of both hydroxy groups
and esterification of carboxylic group to give intermediate 22
which was converted into the corresponding acid 23 by treatment
with LiOH. With the same procedure depicted in Scheme 1, target
NO2
NO2
12
NH2
11
3
Scheme 1. Reagents and conditions: (a) HCONH2, AcOH, reflux, 6 h; (b) AcCl,
CH2Cl2, 2 h, room temperature; (c) H2, 5% Pd–C, CHCl3, 3 h, room temperature.

N. Micale et al. / Bioorg. Med. Chem. 17 (2009) 6505–6511
6507
compound 10 was obtained by sequential treatment with N-Boc-
hydrazine, NaH and TFA (Scheme 4).
phenyl ring which promotes hydrophobic interactions. Unexpect-
edly, compound 9 turned out to be inactive against the cultured
P. falciparum whereas compound 8 displays a significant activity
2.2. Pharmacology
(IC50 = 11.8
The most potent compound in the enzymatic assay resulted to
be 10 (Ki = 2.3 M) underlying the importance of hydrophobic
lM) in the same assay.
Compounds 2–10 were tested for their inhibitory activity
against recombinant FP-216 using Cbz-Phe-Arg-AMC as fluorogenic
substrate. First, a preliminary screening with inhibitor concentra-
l
and bulky groups at positions 6 and 7 of the isoquinoline scaffold.
The presence of an amino group at position 40 (compounds 2–3)
tions of 100
l
M was performed. An equivalent volume of DMSO
seems to be basically favorable for FP-2 inhibition (Ki = 7.3
for 2 and Ki = 19.7 M for 3). It could be speculated that their fair
antiplasmodial activity (IC50 = 38.8 M for 2 and IC50 = 58.8 lM
for 3) could be explained in terms of the difficulty for this class
of derivatives to cross the biological membranes of the parasites
and reach the target enzyme.
lM
was used as negative control, and the irreversible standard inhibi-
tor of clan CA family C1 cysteine proteases (papain-family), namely
E-64,17 was used as positive control. The screening showed all
compounds to abolish enzyme activity in a reversible manner. Con-
tinuous assays (progress curve method) were then performed to
l
l
determine the constants of inhibition Ki
Standard units of
All the synthesized compounds were also tested against rhodes-
ain, the major cysteine protease of Trypanosoma brucei rhodesiense
(Table 1). With the exception of compound 5, all derivatives
showed to inhibit this enzyme at micromolar level. The trend of
the inhibition potency against rhodesain is somehow different
from that against FP-2, however, also against rhodesain, the most
potent inhibitor was again compound 10.
Selectivity against the target enzyme was also estimated, test-
ing inhibitors against papain-family human cysteine proteases
such as cathepsins B and L (Table 1). Overall, these data demon-
strate no selectivity towards FP-2 of the compounds under study:
in general, this new class of cysteine protease inhibitors are
slightly more potent towards human cathepsins with respect to
parasitic FP-2 and rhodesain.
(lM).
FP-2 were added to the reaction mixture containing the fluorogenic
substrate and increasing from 0 to 100 M concentrations of com-
l
pounds. FP-2 activity results in the cleavage of Cbz-Phe-Arg-AMC,
thus releasing the fluorescent AMC group. Hence, fluorescence of
AMC in a sample reflects actual activity of the enzyme.
In parallel, all compounds were tested against P. falciparum
strain FCBR. Dose-dependent effects of compounds on parasite
development were quantified using a previously published assay.18
Data reported in Table 1 show that this structural class pos-
sesses an inhibitory activity towards the target enzyme in the
micromolar range. Compounds 2 and 8–10 most potently inhibit
FP-2. What does attract attention from a rough survey of these re-
sults is that the trend of the inhibitory potency does not match
with the one of the antiplasmodial activity and in some cases it
is even reversed. Compounds 4–5 and 7 possess an alkyloxycar-
bonylamino motif at position 2 of the isoquinoline core which
seems to negatively affect the interaction with the enzyme differ-
ently to what observed for derivative 6 bearing a free amino group
3. Conclusion
In conclusions, new synthesized 2H-isoquinolin-3-ones
showed a good inhibitory activity against FP-2 as well as against
cultured P. falciparum strain FCBR parasites. New synthesized
compounds also inhibited rhodesain of Trypanosoma brucei rho-
desiense in the micromolar range suggesting a potential use for
treatment of sleeping sickness. Unfortunately, these isoquinoline
derivatives did not show selectivity towards FP-2. Further work
is required to improve the inhibitory properties as well as the
antiplasmodial activity and to enhance selectivity towards the
target enzyme.
at the same position (i.e.,
4 Ki = 24.4 lM, 5 Ki = 40.3 lM, 7
Ki = 62.7 M vs 6 Ki = 15.0 M). In compounds 4–5 and 7, the func-
l
l
tionalization of the amino group at C-2 reasonably increases the
lipophilicity of these compounds which turn out to be the three
most potent against cultured parasites (i.e., 4 IC50 = 7.7
IC50 = 9.3 M, 7 IC50 = 8.4 M). On the contrary, compounds with
an arylureido motif at C-2 inhibit more efficiently FP-2 (i.e., 8
Ki = 5.9 M and 9 Ki = 6.5 M). This could be possibly due to the
lM, 5
l
l
l
l
presence of an additional hydrogen donor group (NH) or of the
Table 1
Inhibition of FP-2, antiplasmodial activity, inhibition of rhodesain, human cathepsins B and L of compounds 2–10a
R
O
O
O
R'O
N
N
R''O
NHR
NH2
OMe
4-10
M)
2-3
Compd
R
R0
R00
Falcipain-2 Ki (
l
M)
P. falciparum IC50
(l
Rhodesain Ki (
lM)
Cathepsin B Ki (
lM)
Cathepsin L Ki (lM)
2
3
4
5
6
7
8
9
CH3
OCOCH3
Boc
Boc
7.3 0.3
19.7 1.9
24.4 0.2
40.3 4.9
15.0 0.6
62.7 1.2
5.9 0.4
6.5 1.9
2.3 0.5
0.29 0.09
38.8 0.6
58.8 5.8
7.7 0.4
9.3 0.3
25.5 3.3
8.4 0.8
11.8 1.5
>100
19.4 3.8
38.3 4.6
19.4 6.3
ni
6.3 0.6
9.4 0.2
14.0 1.7
18.7 7.2
4.0 0.2
6.9 0.1
6.7 0.1
7.5 1.5
ni
8.7 0.9
13.6 0.1
4.1 0.2
39.7 9.5
7.0 0.5
7.7 0.8
CH2
CH3
CH3
H
CH2
CH2
CH2
CH2
16.3
3
CO2C2H5
16.7 0.1
3.7 0.6
4.0 0.3
CONHPh-4-COCH3
CONHPh-4-SCH3
H
2.1 0.1
0.85 0.02
4.2 1.2
10
E-64
Bn
Bn
16.9 1.6
5.3 1.05
3.3 0.2
16 (IC50, nM)22
47 (IC50, nM)22
a
All results include standard deviations from two independent measurements, each performed in duplicate.

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N. Micale et al. / Bioorg. Med. Chem. 17 (2009) 6505–6511
rous pentoxide (2.0 g). The mixture was stirred at room tempera-
4. Experimental
4.1. Chemistry
ture for 16 h, then water (30 mL) was cautiously added and the
mixture was extracted with CHCl3 (2 ꢀ 30 mL). The organic layer
was separated and sequentially treated with 10% NaOH (30 mL),
brine (30 mL) and water (2 ꢀ 30 mL). The organic phase was dried
(Na2SO4) and the solvent removed under reduced pressure to yield
crude which was purified by column chromatography (Et2O/light
petroleum 60:40), white needles. Yield: 64% (1.15 g). Mp: 100–
102 °C. Rf = 0.31 (Et2O/light petroleum, 50:50). 1H NMR
(300 MHz, CDCl3) d 3.57 (s, 3H, COOCH3), 3.74 (s, 2H, CH2), 3.85
(s, 3H, OCH3), 5.99 (s, 2H, OCH2O), 6.82 (s, 1H, H-6), 6.86 (s, H-3),
6.92 (d, J = 8.8 Hz, 2H, H-30,50), 7.77 (d, J = 8.8 Hz, 2H, H-20,60).
4.1.1. Instruments and analyses
Melting points were determined on a Kofler hot stage apparatus
and are uncorrected. Elemental analyses were carried out on a C.
Erba Model 1106 (Elemental Analyzer for C, H and N) and the re-
sults are within 0.4% of the theoretical values. Merck Silica Gel
60 F254 plates were used as analytical TLC; column chromatogra-
phy was performed on Merck Silica Gel 60 (70–230 mesh). 1H
NMR spectra were recorded in CDCl3, CD3OD, or DMSO-d6 by
means of a Varian Gemini 300 spectrometer. Chemical shifts are
expressed in d (ppm) relative to TMS as internal standard and cou-
pling constants (J) in hertz. For compounds 6 and 21, complete 1H
and 13C assignments were made by means of HETCOR and LR-HET-
COR spectra, carried out by using the standard software package.
4.1.5. Methyl 2-[4,5-dimethoxy-2-(4-methoxybenzoyl)-
phenyl]acetate (16)
With a similar procedure, 16 was prepared from 14 (1.072 g,
5.10 mmol) by treatment with p-methoxybenzoic acid (1.008 g,
6.63 mmol) and phosphorous pentoxide (2.0 g). Eluent: Et2O/light
petroleum (60:40), whitish prisms. Yield: 62% (1.09 g). Mp: 112–
114 °C. Rf = 0.31 (Et2O/light petroleum, 60:40). 1H NMR
(300 MHz, CDCl3) d 3.60 (s, 3H, COOCH3), 3.79 (s, 2H, CH2), 3.81
(s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 6.85 (s, 1H,
H-6), 6.93 (s, 1H, H-3), 6.95 (d, J = 7.7 Hz, 2H, H-30,50), 7.80 (d,
J = 7.7 Hz, 2H, H-20,60).
4.1.2. 6,7-Methylenedioxy-1-(4-nitrophenyl)-2H-isoquinoline-
3-one (12)
Methyl 2-[4,5-methylenedioxyphenyl-4-(nitrobenzoyl)]acetate
11 (103 mg, 0.3 mmol), obtained as previously described,14 was
dissolved in AcOH (15 mL) and then formamide (3 mL) was added.
The solution was stirred for 6 h at reflux, then cooled and water
(50 mL) was added. The mixture was extracted with CHCl3
(3 ꢀ 10 mL) and the combined organic layers washed with water
(3 ꢀ 20 mL), aq Na2CO3 (2 ꢀ 10 mL), then dried (Na2SO4). After re-
moval of the solvent, the product was purified by column chroma-
tography (CHCl3/EtOH 90:10), yellowish powder. Mp >300 °C
(60 mg, 64%) Rf = 0.27 (CHCl3/EtOH 90:10). 1H NMR (300 MHz,
DMSO-d6), d 5.99 (s, 2H, OCH2O), 6.21 (s, 1H, H-4), 6.73 (s, 1H, H-
5), 6.77 (s, 1H, H-8), 7.69 (d, J = 8.2 Hz, 2H, H-20,60), 8.52 (d,
J = 8.2 Hz, 2H, H-30,50).
4.1.6. 2-[2-(4-Methoxybenzoyl)-4,5-methylenedioxyphenyl]-
acetic acid (17)
Compound 15 (1.15 g, 3.5 mmol) was dissolved in a mixture of
THF/MeOH (4:1, 25 mL), then 1 N LiOH (5 mL) was added and the
solution was stirred at room temperature for 4 h. The mixture
was diluted with H2O and extracted with diethyl ether. The aque-
ous phase was acidified with 2 N HCl and extracted with EtOAc
(2 ꢀ 50 mL). The combined organic layers were dried (Na2SO4)
and the solvent removed under reduced pressure. The crude was
triturated with diethyl ether to afford the acid 17 as a light-brown
powder. Yield: 96% (1.06 g). Mp: 216–220 °C. Rf = 0.31 (Et2O/light
petroleum, 60:40). 1H NMR (300 MHz, CDCl3) d 3.66 (s, 2H, CH2),
3.92 (s, 3H, OCH3), 6.08 (s, 2H, OCH2O), 6.92 (s, 1H, H-6), 6.97 (d,
J = 8.8 Hz, 2H, H-30,50), 7.00 (s, 1H, H-3), 7.85 (d, J = 8.8 Hz, 2H, H-
20,60).
4.1.3. 3-Acetoxy-1-(4-aminophenyl)-6,7-methylenedioxy-
isoquinoline (3)
To a stirred solution of 12 (60 mg, 0.19 mmol) in CH2Cl2 (10 mL),
acetyl chloride (15 ll, 0.22 mmol) was added dropwise over a few
min. The reaction mixture was stirred for further 2 h at room tem-
perature, then was poured into aq NaHCO3 (20 mL, 5%) and extracted
with CHCl3 (3 ꢀ 10 mL). The combined organic layers were dried
(Na2SO4), filtered and concentrated in vacuo. The crude product
was purified by column chromatography (CH2Cl2/MeOH 95:5) to af-
ford nitroderivative as a yellowish powder. Yield: 77% (52 mg). Mp:
262–266 °C. Rf = 0.71 (CH2Cl2/MeOH, 95:5). 1H NMR (300 MHz,
CDCl3) d 2.06 (s, 3H, CH3), 5.95 (s, 2H, OCH2O), 6.30 (s, 1H, H-4),
6.72 (s, 1H, H-5), 6.79 (s, 1H, H-8), 7.74 (d, J = 8.5 Hz, 2H, H-20,60),
8.50 (d, J = 8.5 Hz, 2H, H-30,50). Anal. Calcd for C18H12N2O6: C,
61.37; H, 3.43; N, 7.95. Found: C, 61.29; H, 3.58; N, 8.07. A solution
of nitroderivative intermediate (52 mg, 0.15 mmol) in CHCl3
(10 mL) was stirred under H2 (1 atm) for 3 h, in the presence of a cat-
alytic amount of 5% Pd/C (10 mol %). The mixture was then filtered
through Celite, and the solvent was evaporated under reduced pres-
sure. Column chromatography (EtOAc/MeOH 98:2) afforded pure
compound 3 as a brownish powder. Yield: 80% (38 mg). Mp
>300 °C (dec.). Rf = 0.58 (EtOAc/MeOH, 98:2). 1H NMR (300 MHz,
CDCl3) d 2.02 (s, 3H, CH3), 5.98 (s, 2H, OCH2O), 6.28 (s, 1H, H-4),
6.69 (s, 1H, H-5), 6.84 (d, J = 8.8 Hz, 2H, H-30,50), 7.01 (s, 1H, H-8),
7.18 (d, J = 8.8 Hz, 2H, H-20,60). Anal. Calcd for C18H14N2O4: C,
67.07; H, 4.38; N, 8.69. Found: C, 67.22; H, 4.50; N, 8.47.
4.1.7. 2-[4,5-Dimethoxyphenyl-2-(4-methoxybenzoyl)]acetic
acid (18)
With a similar procedure, 18 was prepared from 16 (500 mg,
1.45 mmol) and 1 N LiOH (2.5 mL). Whitish powder. Yield: 87%
(420 mg). Mp: 134–136 °C. Rf = 0.78 (CHCl3/EtOH, 90:10). 1H
NMR (300 MHz, CDCl3) d 3.73 (s, 2H, CH2), 3.80 (s, 3H, OCH3),
3.89 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.95–6.99 (m, 4H, H-6, H-
3, H-30,50), 7.83 (d, J = 8.5 Hz, 2H, H-20,60).
4.1.8. N0-{2-[4,5-Methylenedioxy-2-(4-methoxybenzoyl)-
phenyl]acetyl}-hydrazine carboxylic acid tert-butyl ester (19)
Compound 17 (250 mg, 0.79 mmol) was dissolved in CH2Cl2/
DMF (1:1, 10 mL), then tert-butyl carbazate (136 mg, 1.03 mmol),
and EDCI (230 mg, 1.18 mmol) were added and the reaction mix-
ture was stirred at room temperature. After being stirred for 12 h
it was neutralized with 2% HCl and then extracted with chloroform
(2 ꢀ 15 mL). The combined organic layers were treated with brine
and water then dried over Na2SO4 and the solvent removed under
reduced pressure to yield a crude that was purified by column
chromatography (CHCl3/EtOAc 80:20), clear solid residue. Yield:
82% (280 mg). Mp: 116–120 °C. Rf = 0.41 (CHCl3/EtOAc, 80:20). 1H
NMR (300 MHz, CDCl3) d 1.42 (s, 9H, t-Bu), 3.56 (s, 2H, CH2), 3.88
(s, 3H, OCH3), 6.01 (s, 2H, OCH2O), 6.42 (br s, 1H, NH), 6.82 (s,
1H, H-6), 6.94 (d, J = 8.8 Hz, 2H, H-30,50), 7.01 (s, 1H, H-3), 7.79
(d, J = 8.8 Hz, 2H, H-20,60). 9.29 (br s, 1H, NH).
4.1.4. Methyl 2-[2-(4-methoxybenzoyl)-4,5-methylenedioxy-
phenyl] acetate (15)
To a solution of 13 (1.056 g, 5.46 mmol) in CH2Cl2 (10 mL), were
added 4-methoxybenzoic acid (1.081 g, 7.1 mmol) and phospho-

N. Micale et al. / Bioorg. Med. Chem. 17 (2009) 6505–6511
6509
4.1.9. N0-{2-[4,5-Dimethoxy-2-(4-methoxybenzoyl)phenyl]-
acetyl}-hydrazine carboxylic acid tert-butyl ester (20)
With a similar procedure, 20 was prepared from 18 (420 mg,
1.27 mmol) using tert-butyl carbazate (218 mg, 1.65 mmol), and
EDCI (365 mg, 1.91 mmol). Eluent: CHCl3/EtOAc (80:20), whitish
solid residue. Yield: 89% (500 mg). Mp: 161–163 °C. Rf = 0.43
(CHCl3/EtOAc, 80:20). 1H NMR (300 MHz, CDCl3) d 1.43 (s, 9H, t-
Bu), 3.60 (s, 2H, CH2), 3.79 (s, 3H, OCH3–40), 3.90 (s, 3H, OCH3),
3.96 (s, 3H, OCH3), 6.51 (br s, 1H, NH), 6.89 (s, 1H, H-6), 6.97 (d,
J = 8.8 Hz, 2H, H-30,50), 7.05 (s, 1H, H-3), 7.82 (d, J = 8.8 Hz, 2H, H-
20,60), 9.38 (br s, 1H, NH).
Na2SO4 and the solvent was removed under reduced pressure. The
crude was purified by a chromatography column (EtOAc/c-hexane/
i-PrOH 60:30:10) to afford desired compound 7 as a dark yellow
powder. Yield: 72% (89 mg). Mp: 224–227 °C (dec.) Rf = 0.39
(EtOAc/c-hexane/i-PrOH, 60:30:10). 1H NMR (300 MHz, CDCl3) d
1.25 (t, 3H, J = 6.9 Hz, COOCH2CH3), 3.89 (s, 3H, OCH3), 4.02 (q, 2H,
J = 6.9 Hz, COOCH2CH3), 5.93 (s, 2H, OCH2O), 6.30 (s, 1H, H-8), 6.57
(s, 1H, H-5), 6.69 (s, 1H, H-4), 6.97–7.05 (m, 2H, Ar-H), 7.28–7.45
(m, 2H, Ar-H), 8.15 (br s, NH). Anal. Calcd for C20H18N2O6: C, 62.82;
H, 4.74; N, 7.33. Found: C, 62.96; H, 4.61; N, 7.12.
4.1.14. 2-(4-Acetylphenylureido)-1-(4-methoxyphenyl)-6,7-
methylenedioxy-2H-isoquinolin-3-one (8)
4.1.10. 2-tert-Butyloxycarbonylamino-6,7-methylenedioxy-1-
(4-methoxyphenyl)-2H-isoquinolin-3-one (4)
4-Acetylphenylisocyanate (93.5 mg, 0.58 mmol) was added to a
solution of 6 (90 mg, 0.29 mmol) in dry CH2Cl2 The mixture was
stirred under nitrogen at room temperature until the disappearing
of the starting material, then the solution was diluted with CHCl3
and washed with water. The organic layer was dried over Na2SO4
and the solvent removed in vacuo. The crude was purified by a
chromatographic column (EtOAc/c-hexane/i-PrOH, 60:30:10) to af-
ford the desired compound 8 as a yellowish powder. Yield: 68%
(93 mg). Mp: 211–215 °C. Rf = 0.40 (EtOAc/c-hexane/i-PrOH
60:30:10). 1H NMR (300 MHz, CDCl3) d 2.39 (s, 3H, COCH3), 3.85
(s, 3H, OCH3), 5.98 (s, 2H, OCH2O), 6.37 (s, 1H, H-8), 6.67 (s, 1H,
H-5), 6.77 (s, 1H, H-4), 6.97–7.56 (m, 8H, Ar-H), 9.12 (br s, NH),
9.65 (br s, NH). Anal. Calcd for C26H21N3O6: C, 66.24; H, 4.49; N,
8.91. Found: C, 66.04; H, 4.66; N, 8.81.
Compound 19 (280 mg, 0.65 mmol) dissolved in dry THF
(20 mL) was added dropwise to
a solution of NaH (18 mg,
0.75 mmol) in dry THF (10 mL). The mixture was stirred at room
temperature for 2 h then the precipitate obtained was filtered off
and dissolved in EtOAc and washed with brine. The organic phase
was dried (Na2SO4) and the solvent removed under reduced pres-
sure. The crude was purified by column chromatography (CHCl3/
EtOH 95:5), bright-yellow powder. Yield: 91% (243 mg). Mp:
208–210 °C. Rf = 0.32 (CHCl3/EtOH, 95:5). 1H NMR (300 MHz,
CDCl3) d 1.41 (s, 9H, t-Bu), 3.93 (s, 3H, OCH3), 5.95 (s, 2H, OCH2O),
6.33 (s, 1H, H-8), 6.59 (s, 1H, H-5), 6.72 (s, 1H, H-4), 7.00–7.47 (m,
4H, Ar-H) 7.76 (br s, 1H, NH). Anal. Calcd for C22H22N2O6: C, 64.38;
H, 5.40; N, 6.83. Found: C, 64.23; H, 5.48; N, 6.97.
4.1.11. 2-tert-Butyloxycarbonylamino-6,7-dimethoxy-1-(4-
methoxyphenyl)-2H-isoquinolin-3-one (5)
4.1.15. 1-(4-Methoxyphenyl)-6,7-methylenedioxy-2-(4-
methylthiophenyl-ureido)-2H-isoquinolin-3-one (9)
With the same procedure, 9 was obtained from 6 (113 mg,
With a similar procedure, 5 was prepared from 20 (500 mg,
1.12 mmol) and 30 mg (1.25 mmol) of NaH in dry THF (50 mL).
Eluent: CHCl3/EtOH (95:5), yellow soft powder. Yield: 88%
(422 mg). Mp: 228–230 °C Rf = 0.32 (EtOAc/c-hexane/i-PrOH,
60:30:10). 1H NMR (300 MHz, CDCl3) d 1.40 (s, 9H, t-Bu), 3.64 (s,
3H, OCH3), 3.90 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 6.24 (s, 1H, H-
8), 6.52 (s, 1H, H-5), 6.71 (s, 1H, H-4), 6.96–7.04 (m, 2H, Ar-H),
7.25–7.53 (m, 3H, 2H Ar-H and NH). Anal. Calcd for C23H26N2O6:
C, 64.78; H, 6.15; N, 6.57. Found: C, 64.93; H, 6.08; N, 6.69.
0.36 mmol)
and
4-methyltiophenylisocyanate
(0.73 mmol,
0.1 mL). In this case, the solvent was removed in vacuo and the res-
idue triturated with Et2O. The obtained solid was filtered off to af-
ford pure 9 as a yellow powder. Yield: 73% (126 mg). Mp: 228–
232 °C. Rf = 0.72 (EtOAc/c-hexane/i-PrOH, 60:30:10). 1H NMR
(300 MHz, CDCl3) d 2.36 (s, 3H, SCH3), 3.86 (s, 3H, OCH3), 5.97 (s,
2H, OCH2O), 6.37 (s, 1H, H-8), 6.67 (s, 1H, H-5), 6.81 (s, 1H, H-4),
6.98–7.59 (m, 8H, Ar-H), 8.62 (br s, 1H, NH), 9.43 (br s, 1H, NH).
Anal. Calcd for C25H21N3O5S: C, 63.15; H, 4.45; N, 8.84. Found: C,
62.88; H, 4.28; N, 9.03.
4.1.12. 2-Amino-1-(4-methoxyphenyl)-6,7-methylenedioxy-
2H-isoquinolin-3-one (6)
Compound 4 (243 mg, 0.59 mmol) was dissolved in a mixture of
CH2Cl2 and TFA (3:1, 10 mL). The resulting solution was stirred at
room temperature for 5 h, then diluted with CHCl3 and washed
with a saturated solution of NaHCO3, dried over Na2SO4 and evap-
orated under reduced pressure to yield a crude which was purified
by column chromatography (CHCl3/EtOH, 90:10), bright-yellow
powder. Yield: 87% (160 mg). Mp: 188–190 °C Rf = 0.31 (CH2Cl2/
EtOH 95:5). 1H NMR (300 MHz, CDCl3) d 3.91 (s, 3H, OCH3), 5.79
(s, 2H, NH2), 5.93 (s, 2H, OCH2O), 6.34 (s, 1H, H-8), 6.64 (s, 1H,
H-5), 6.71 (s, 1H, H-4), 7.11 (d, J = 8.5 Hz, 2H, H-30,50), 7.34 (d,
J = 8.5 Hz, 2H, H-20,60). 13C NMR (75 MHz, CDCl3) d 55.39 (OCH3),
99.30 (C-5), 101.10 (C-8), 101.23 (OCH2O), 106.31 (C-4), 113.88
(C-8a), 114.66 (C-30,50), 123.64 (C-10), 130.46 (C-1), 130.69 (C-
20,60), 140.10 (C-6), 145.54 (C-7), 151.81 (C-4a), 156.80 (CO),
160.69 (C-40). Anal. Calcd for C17H14N2O4: C, 65.80; H, 4.55; N,
9.03. Found: C, 65.67; H, 4.64; N, 9.16.
4.1.16. [4,5-Dihydroxy-2-(4-methoxybenzoyl)-phenyl]acetic
acid (21)
An ice-cold dichloromethane solution (15 mL) of 15 (250 mg,
0.76 mmol) was charged dropwise with BBr3 (2.28 mL of 1 M solu-
tion in CH2Cl2), and equipped with a reflux condenser. The mixture
was stirred at room temperature for 3 h, forming a viscous, deep-
red solution; at this time, the reaction was cautiously quenched
with MeOH (5 mL) and the solvent was removed in vacuo. This
process was repeated until a clear red color solution was restored.
The resulting sticky residue was purified by column chromatogra-
phy (1% HCOOH in EtOAc/c-hexane (50:50), light-brown residue.
Yield: 68% (156 mg). Mp: 167–170 °C Rf = 0.40 (1% HCOOH in
CHCl3/EtOAc, 50:50). 1H NMR (300 MHz, CD3OD) d 3.71 (s, 2H,
CH2), 3.89 (s, 3H, OCH3), 6.84 (s, 1H, H-6), 6.90 (s, 1H, H-3), 7.01
(d, J = 8.5 Hz, 2H, H-30,50), 7.76 (d, J = 8.5 Hz, 2H, H-20,60). 13C NMR
(75 MHz, CD3OD) d 39.35 (CH2), 56.05 (OCH3), 114.58 (C-30,50),
119.38 (C-3), 119.94 (C-6), 128.60 (C-1), 130.84 (C-2), 132.33 (C-
10), 133.72 (C-20,60), 144.31 (C-4), 149.37 (C-5), 165.03 (C-40),
175.52 (COOH), 198.48 (CO).
4.1.13. 2-Ethyloxycarbonylamino-1-(4-methoxyphenyl)-6,7-
methylene-dioxy-2H-isoquinolin-3-one (7)
Ethylchloroformate (0.64 mmol, 0.062 mL) was added to a stir-
red CH2Cl2 solution (10 mL) of 6 (100 mg, 0.32 mmol) in the pres-
ence of Et3N (excess). The mixture was further stirred at room
temperature for 3 h (TLC monitoring), then was diluted with CHCl3
(10 mL) and washed with water. The organic layer was dried over
4.1.17. Benzyl [4,5-dibenzyloxy-2-(4-methoxybenzoyl)-
phenyl]acetate (22)
To a solution of 21 (156 mg, 0.52 mmol) in acetone (50 mL) was
added K2CO3 (862 mg, 6.24 mmol) and benzyl bromide (0.2 mL,

6510
N. Micale et al. / Bioorg. Med. Chem. 17 (2009) 6505–6511
1.98 mmol). The reaction mixture was heated at reflux for 5–6 h,
then cooled to 0 °C to ensure complete precipitation of the base.
The supernatant was isolated by filtration, and concentrated under
reduced pressure. The residue was dissolved with ethyl acetate and
washed with 10% HCl. The organic layer was dried (Na2SO4) and re-
duced under vacuum to a residue which furnished compound 22
by triturating with Et2O. Light-yellow powder. Yield: 55%
(164 mg). Mp: 185–187 °C. Rf = 0.36 (Et2O/light petroleum,
80:20). 1H NMR (300 MHz, CDCl3) d 3.81 (s, 2H, CH2), 3.87 (s, 3H,
OCH3), 5.02 (s, 2H, OCH2Ph), 5.09 (s, 2H, OCH2Ph), 5.19 (s, 2H,
OCH2Ph), 6.82 (d, J = 8.5 Hz, 2H, H-30,50), 6.93 (s, 1H, H-6), 6.98 (s,
1H, H-3), 7.19–7.53 (m, 15H, Ar-H), 7.63 (d, J = 8.5 Hz, 2H, H-
20,60). Anal. Calcd for C37H32O6: C, 77.60; H, 5.63. Found: C, 77.42;
H, 5.76.
tive control. Product release from substrate hydrolysis (Cbz-Phe-
Arg-AMC, 40 M for FP-2 and 10 M for rhodesain) was deter-
mined continuously over a period of 10 min. Compounds showing
at least 50% inhibition were subjected to detailed assays. These
were performed in a 100 mM sodium acetate buffer, pH 5.5 con-
l
l
taining 10 mM DTT with Cbz-Phe-Arg-AMC (40 or 10
strate.17 The Km values used to correct Kiapp values was
determined to 21.5 M (FP-2) and 0,9
M (rhodesain).19 Inhibitor
solutions were prepared from stocks in DMSO. Each assay was per-
formed twice in 96-well plates in a total volume of 300 L. A Var-
lM) as sub-
l
l
l
ian Cary Eclipse spectrofluorometer Varian, Darmstadt, Germany
with a microplate reader (excitation 365 nm, emission 460 nm)
was used. Standard units of FP-2 were added to the reaction mix-
ture containing the fluorogenic substrate and increasing from 0 to
100 lM concentrations of compounds. Kinac was obtained by a Dix-
4.1.18. [4,5-Dibenzyloxy-2-(4-methoxybenzoyl)-phenyl]acetic
acid (23)
on plot20 using equation [E]0/[E]a = 1+[I]/Kiapp and correction to
zero substrate concentration from Ki ¼ Kiapp=ð1 þ ½SꢁKꢂ1
Þ
with
m
Following the same procedure employed for the synthesis of
ketoacid 17, 23 was obtained from 22 (164 mg, 0.29 mmol) and
1 N LiOH (2 mL). In this case, the reaction was prolonged for 8 h.
Eluent: CHCl3/MeOH (90:10), light-brown powder. Yield: 95%
(131 mg). Mp: 226–229 °C. Rf = 0.34 (CHCl3/MeOH, 90:10). 1H
NMR (300 MHz, CDCl3) d 3.65 (s, 2H, CH2), 3.91 (s, 3H, OCH3),
5.11 (s, 2H, OCH2Ph), 5.25 (s, 2H, OCH2Ph), 6.88 (d, J = 8.8 Hz, 2H,
H-30,50), 6.99 (s, 1H, H-6), 7.10 (s, 1H, H-3), 7.27–7.55 (m, 10H,
Ar-H), 7.67 (d, J = 8.8 Hz, 2H, H-20,60).
[E]0 as enzyme activity in the absence, and [E]a as residual enzyme
activities in the presence of the inhibitor. Assays with cathepsins B
and L were performed as described previously.21 Cbz-Phe-Arg-AMC
was used as substrate (80
L). The Km values used to correct Kiapp values were 150
sin B) and 6.5 M (cathepsin L).
l
M for cathepsin B, 5
l
M for cathepsin
l
M (cathep-
l
4.2.2. Drug screening on P. falciparum cultures
The compounds were screened in quadruplicates against the
human malaria pathogen P. falciparum at concentrations between
100 and 0.0488 lM. P. falciparum (strain FCBR) was maintained
4.1.19. N0-{2-[4,5-Dibenzyloxy-2-(4-methoxybenzoyl)-phenyl]-
acetyl}hydrazine carboxylic acid tert-butyl ester (24)
in continuous culture basically according to Trager and Jensen.23
Parasites were cultured in human red blood cells (RBC) (blood
group ARh+25) in RPMI 1640 medium supplemented with
25 mM HEPES (Molecular Probes, Invitrogen), 20 mM sodium
bicarbonate, and 0.5% AlbuMAX I (Molecular Probes, Invitrogen)
at 2.5% (v/v) hematocrit. Cultures were maintained at 37 °C with
a gaseous phase of 94% N2, 1% O2, and 5% CO2. Synchronized ring
stages of P. falciparum strain FCBR were plated in 96-well-plates
at a parasitemia of 1%, in the presence of the compounds (dissolved
in DMSO and diluted 10-fold in 50% ethanol before added to the
cells). Incubation of parasites with an equal concentration of 50%
ethanol alone was used as a negative control. To kill the plasmodia
completely for the positive control, the parasites were incubated in
With the same procedure employed for the synthesis of inter-
mediate 19, 24 was obtained from 23 (131 mg, 0.27 mmol) using
tert-butyl carbazate (46 mg, 0.35 mmol), and EDCI (79 mg,
0.41 mmol). Eluent: CHCl3/EtOAc (80:20), yellowish solid residue.
Yield: 79% (128 mg). Mp: 189–193 °C. Rf = 0.29 (CHCl3/EtOAc,
80:20). 1H NMR (300 MHz, CDCl3) d 1.42 (s, 9H, t-Bu), 3.56 (s, 2H,
CH2), 3.88 (s, 3H, OCH3), 5.07 (s, 2H, OCH2Ph), 5.22 (s, 2H, OCH2Ph),
6.44 (br s, 1H, NH), 6.85 (d, J = 9.0 Hz, 2H, H-30,50), 6.94 (s, 1H, H-6),
7.16 (s, 1H, H-3), 7.25–7.53 (m, 10H, Ar-H), 7.64 (d, J = 9.0 Hz, 2H,
H-20,60), 9.31 (br s, 1H, NH).
4.1.20. 2-Amino-6,7-dibenzyloxy-1-(4-methoxyphenyl)-2H-
isoquinolin-3-one (10)
presence of 2 lM chloroquine. The viability of the parasites was
With a similar procedure employed for the synthesis of com-
pounds 4–5, 10 was prepared from 24 (128 mg, 0.21 mmol) and
NaH (6 mg, 0.25 mmol) in dry THF (15 mL). Yellow powder. Yield:
82% (102 mg). Mp: 199–202 °C. Rf = 0.47 (CHCl3/MeOH, 95:5). 1H-
NMR (300 MHz, CDCl3) d 1.43 (s, 9H, t-Bu), 3.90 (s, 3H, OCH3),
5.09 (s, 2H, OCH2Ph), 5.24 (s, 2H, OCH2Ph), 6.05 (s, 1H, H-8), 6.41
(s, 1H, H-5), 6.70 (s, 1H, H-4), 6.94 (d, J = 8.8 Hz, 2H, H-30,50),
7.27–7.50 (m, 7H, Ar-H). The obtained intermediate was then
deprotected with TFA as previously described, and purified by
chromatographic column (EtOAc/c-hexane/i-PrOH 70:20:10) to af-
ford the desired compound 10 as a dark yellow powder. Overall
yield: 75% (75 mg). Mp: 175–177 °C. Rf = 0.32 (EtOAc/c-hexane/i-
PrOH, 70:20:10). 1H NMR (300 MHz, CDCl3) d 3.94 (s, 3H, OCH3),
4.94 (s, 2H, OCH2Ph), 5.23 (s, 2H, OCH2Ph), 5.78 (s, 2H, NH2), 6.35
(s, 1H, H-8), 6.66 (s, 1H, H-5), 6.67 (s, 1H, H-4), 7.07 (d, J = 8.5 Hz,
2H, H-30,50), 7.20–7.48 (m, 12H, Ar-H). Anal. Calcd for
C30H26N2O4: C, 75.30; H, 5.48; N, 5.85. Found: C, 75.16; H, 5.61;
N, 5.91.
screened subsequently using the assay according to Evers et al.
(2008).18
Acknowledgments
We would like to thank the DAAD Vigoni project (2009/10) for
partial support of this work. T.S. thanks the German Research Soci-
ety (DFG, SFB630) for financial support.
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The preliminary screening was performed with 100
tor concentrations using an equivalent amount of DMSO as nega-
lM inhibi-

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6511
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