Identification and biological evaluation of a series of 1H-benzo[de]isoquinoline-1,3(2H)-diones as hepatitis C virus NS5B polymerase inhibitors

Article abstract of DOI:10.1021/jm900517t

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for inhibition. Herein, we present 1H-benzo

Full text of DOI:10.1021/jm900517t

J. Med. Chem. 2009, 52, 5217–5227 5217
DOI: 10.1021/jm900517t
Identification and Biological Evaluation of a Series of 1H-Benzo[de]isoquinoline-1,3(2H)-diones as
Hepatitis C Virus NS5B Polymerase Inhibitors†,‡
Jesus M. Ontoria,*,§ Edwin H. Rydberg,§ Stefania Di Marco,§ Licia Tomei,§ Barbara Attenni,§ Savina Malancona,§
§
§
§
§
§
Jose I. Martin Hernando, Nadia Gennari, Uwe Koch, Frank Narjes, Michael Rowley, Vincenzo Summa,
§
ꢀ
Steve S. Carroll, David B. Olsen, Raffaele De Francesco, Sergio Altamura, Giovanni Migliaccio, and Andrea Carfi*
§
§
§
,§
ꢁ
§Istituto Di Ricerche Di Biologia Molecolare, P. Angeletti, S.p.A. (IRBM-MRL Rome), Via Pontina Km 30,600, I-00040 Pomezia, Italy, and
Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19846
Received April 23, 2009
The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in
virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an
attractive target for inhibition. Herein, we present 1H-benzo[de]isoquinoline-1,3(2H)-diones as a new
series of selective inhibitors of HCV NS5B polymerase. The HTS hit 1 shows submicromolar potency in
two different HCV replicons (1b and 2b) and displays no activity on other polymerases (HIV-RT, Polio-
pol, GBV-b-pol). These inhibitors act during the pre-elongation phase by binding to NS5B non-
nucleoside binding site Thumb Site II as demonstrated by crystal structure of compound 1 with the
ΔC55-1b and ΔC21-2b enzymes and by mutagenesis studies. SAR in this new series reveals inhibitors,
such as 20, with low micromolar activity in the HCV replicon and with good activity/toxicity window in
cells.
Introduction
genotypes 1b, 2a and 2b have been determined alone or in
complex with a short RNA template or with NTPs.5 The
enzyme shares the same general right-handed fingers, thumb,
and palm domain structure of other single-chain polynucleo-
tide polymerase. However, the HCV polymerases has an
elongated loop that extends from the fingers domain to make
contact with the thumb domain, enclosing the active site cleft
and resulting in a spherical appearance for the enzyme.
NS5B is crucial for viral infectivity and is a validated target
for the development of therapeutics against HCV. Screening of
small molecule libraries has resulted in the discovery of several
classes of non-nucleoside inhibitors (NNIs). At least four
binding sites have been identified:6 Thumb Site I7 and Thumb
Site II,8 located on the surface of the thumb domain at about
Hepatitis C virus (HCV) is a (þ)-stranded RNA virus
belonging to the Flaviviridae family of enveloped viruses. It
is estimated that 180 million people worldwide have been
infected by HCV, and two-thirds of them are at risk for
developing chronic liver disease, cirrhosis, and hepatocellular
carcinoma.1 Currently the only approved therapy against
HCV is pegylated interferon IFN-R (IFN), either as mono-
therapy or in combination with ribavirin. However, this
therapy is poorly tolerated and of limited efficacy.2
HCV exhibits a high degree of genetic variability. To date,
six genotypes and more than 50 subtypes have been identified,
with genotypes 1 and 2 being the most prevalent forms and
accounting for more than 13 million cases of disease in the U.
S. and Europe combined. HCV genotype is one of the major
determinants of the efficacy of treatment. In addtion, it has
been found that inherent genotypic differences are responsible
for different sensitivities to several small molecule inhibitors.3
The HCV RNA genome is 9.6 kb long and codes for a single
polyprotein. Polyprotein processing by viral and cellular host
factors resultsin fourstructuralproteins (Core-E1-E2-p7)and
six nonstructural (NS) proteins.4 NS5B is the RNA-depen-
dent RNA polymerase (RdRp) at the core of the HCV
replicative complex. The three-dimensional structures of C-
terminally truncated forms of NS5B (ΔC21 or ΔC55) from
30-35 A from the active site; Palm Site I5f,9 and Palm Site II10
˚
are two adjacent and partially overlapping binding sites
located in proximity to the enzyme active site cavity. Point
mutations conferring resistance to inhibitors binding to each of
these sites have been described. Replacement of Pro495 with
Ala and Leu, or Leu392 with Ile in binding Thumb Site I have
been shown to confer resistance to the Thumb Site I specific
indole-acetamide class of inhibitors.5g,7a,7d Mutation of
Met423 has been shown to confer resistance of the NS5B
enzyme to inhibition by compounds binding to Thumb Site
II,11 whereas mutation of Met414 to Thr or Val gave resistance
to compounds binding to the internal Palm Site I11 and Palm
Site II,10a respectively.
Herein, we report the identification and biochemical char-
acterization of a series of 1H-benzo[de]isoquinoline-1,3(2H)-
diones as selective inhibitors of the NS5B polymerase, some of
which are effective inhibitors of HCV replication in 1b and 2b
replicon assays with no associated toxicity. Several studies on
the genotype selectivity, mechanism of inhibition, and binding
† PDB codes for the X-ray structures of the ΔC21-2b and ΔC55-1b
NS5B polymerase bound to 1 are 3HVO and 2WHO, respectively.
‡ Dedicated to the memory of Giovanni Migliaccio.
*To whom correspondence should be addressed. For J.M.O.: phone,
þ39-0691093520; fax, þ39-0691093654; e-mail, jesus_ontoria@merck.
com. For A.C.: phone, þ39-0691093550; fax, þ39-0691093654; e-mail,
andrea_carfi@merck.com.
r
2009 American Chemical Society
Published on Web 07/24/2009
pubs.acs.org/jmc

5218 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ontoria et al.
mode are presented. The high resolution crystal structures of
one such compound in complex with the NS5B polymerases
from genotypes 1band 2bare alsodescribed. Finally, an initial
SAR for this class of compounds is discussed in light of the
structural data.
Lead Identification
Figure 1. Structure of compound 1 and inhibition potency on the
purified NS5B ΔC21-1b and ΔC21-2b enzymes and on the replicon
HEPREP 1b and 2b assays.
In order to identify compounds that inhibit the HCV NS5B
polymerase from more than one genotype, we initiated a high
throughput screen (HTS) of our compound collection using
two enzyme assays, which utilize the isoforms ΔC21-1b
(BK strain) for genotype 1 and ΔC21-2b (2b.2 strain) for
genotype 2.7a This HTS campaign led to the identification of 1
as a submicromolar inhibitor of both genotypes 1 and 2 NS5B
polymerase. The compound was also an effective inhibitor of
HCV replication in the Huh7 clone HBI10A expressing the
1b-Con1 replicon (HEPREP 1b)12 and in the H2B2 clone
expressing a chimeric replicon carrying the NS5B-2b enzyme
in the context of the 1b (BK) replicon (HEPREP 2b) with a
10-fold activity/toxicity window in cells (Figure 1). Com-
pound 1 represented an HCV NS5B polymerase inhibitor
with a novel chemotype and prompted us to characterize its
biological profile and mechanism of inhibition.
Physicochemical and Pharmacokinetic Profile
The lipophilic structure of 1 was reflected in the slightly
high logD (log D = 3.45) and low solubility (0.44 μg/mL) of
the compound. However, when 1 was dosed in rat at 3 mg/kg
orally (po), an excellent oral bioavailability (F = 83%)
togetherwithhighexposure(AUC=9.7μM h) was observed.
3
Peak plasma concentrations, Cmax=4.0 μM, occurred after
0.7 h from administration. Moreover, despite a short elimination
half-life (t1/2 = 0.7 h), a low clearance (Cl=12 (mL/min)/kg) and
a good volume of distribution (Vdss=1.1 L/kg) were observed
after iv administration. Globally these results showed that
compound 1 had a good overall profile and represented a valid
lead for a drug discovery optimization program.
Chemistry
Mechanism of Inhibition of NS5B Polymerase
Activity validation for our hit compound 1 was accom-
plished via resynthesis and retesting. The synthesis of 1 was
based on the condensation of commercially available 6-bro-
mo-1H,3H-benzo[de]isochromene-1,3-dione 2a with 3-bro-
moaniline. When this reaction was performed by heating in
NMP at 145 °C for 24 h to obtain the completion of the
reaction, the intermediate 3a was obtained with moderate
yields.13 Use of microwave irradiation increased the yield and
shortened the reaction time (200 °C, 30 min). Introduction of
the 2-hydroxyethylamine fragment was achieved by Ullman
reaction of intermediate 3a with ethanolamine under micro-
wave irradiation, affording 1.14 Compounds 4-9 and 12-20
were prepared by using the synthetic route described in
Scheme 1.
The 2,3-dihydro-1H-benzo[de]isoquinolin-1-one 10 was
prepared by treatment of compound 1 with NaBH4 that led
tothe selective reductionofthe carbonylgroupin position1 of
the tricyclic scaffold.15 Reaction of 1 with BH3 in THF at
reflux gave 11 by reduction of both carbonyl groups
(Scheme 2).
The study of the mechanism of inhibition indicated that
compound 1 acts during the pre-elongation phase, similar to
that previously observed for several other NNIs of the HCV
polymerase.7a,7e,16 In fact, complete inhibition of the ΔC21-1b
enzymatic activity could not be achieved if the inhibitor was
added to a preformed enzyme-RNA complex and a signifi-
cant fraction of the polymerase activity could not be inhibited
even at very high compound concentrations (Figure 2A). This
result suggests that the fraction of NS5B engaged with the
RNA in a pre-elongation complex is protected from the action
of the inhibitor. This hypothesis is further supported by the
observation that increasing the preincubation time of the
enzyme with the RNA template previous to the addition of
compound 1, at more than 30-fold of its IC50, resulted in an
increased residual polymerase enzymatic activity (Figure 2B).
As previously observed,16 the enzyme activity in the absence
of inhibitor also increased as preincubation time with the
RNA template increased, likely reflecting the formation of a
productive pre-elongation complex (data not shown). Pre-
sumably, the residual inhibition after longer preincubation
time results from those polymerase molecules that dissociated
from the template during the elongation reaction and thus
become susceptible to inhibition. Indeed, in reactions per-
formed in the presence of heparin as a trapping agent for free
enzyme, preincubation with template completely protected
the NS5B RdRp from inhibition (Figure 2C). Equivalent
results were obtained for the HCV NS5B enzyme from
genotype 2b (data not shown).
HCV and Genotype Selectivity
When tested on purified NS5B polymerases from all the
major HCV genotypes, compound 1 appeared active in the
submicromolar range on all the enzymes. Differences were
observed among the genotypes with a major shift (38-fold) on
genotypes 2a and 3a with respect to genotype 1b and only a
10-fold difference for genotype 2b (Table 1).
Compound 1 was also shown to be strictly selective for the
HCV polymerase, since it was unable to inhibit at concentra-
tions up to 50 μM the in vitro activity of closely related
polymerases such as HIV-RT, GBVa-b-NS5B, BVDV RdRp,
and Polio-pol (Table 1). Altogether, these results indicate that
compound 1 is an HCV selective inhibitor able to affect viral
replication of 1a, 1b, and 2b genotypes.
Elucidation of Binding Site of Compound 1
In order to identify the binding site of compound 1, its
efficiency was evaluated initially on ΔC21-1b RdRp carrying
mutations able to confer resistance to inhibition by molecules
known to interact with different sites on the enzyme. As
reported in Table 2, the Thumb Site II mutants M423K and
R501E, but not the Palm Sites I and II mutant M414T,
appeared highly resistant to compound 1 as well as to
a Abbreviations: GBV, GB virus; BVDV, bovine diarrhea virus.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5219
Scheme 1. Synthesis of Compounds 1, 4-9, and 12-20a
a Reagents and conditions: (a) ArNH2, NMP, microwave, 200 °C, 30 min; (b) R0NH2, NMP, microwave, 200 °C.
Scheme 2. Synthesis of Compounds 10 and 11a
a Reagents and conditions: (a) NaBH4, EtOH/H2O, room temp; (b) BH3, THF, reflux.
Table 1. Activity of Resynthesized Compound 1 on the Purified NS5B ΔC21 Enzymes of Different HCV Genotypes (First Three Columns), on Huh7
Clones Carrying Replicons from the Indicated Genotypes (Middle Two Columns), and on HCV-Unrelated Polymerases (Last Two Columns)
NS5B ΔC21a
replicon cell, ELISAa
selectivity
genotype
IC50 (μM)
shift vs 1b
genotype
EC50 (μM)
polymerase
IC50 (μM)
1a RB5
1b (BK)
2a.4
0.064
0.020
0.77
0.19
0.76
0.54
0.49
3.2
1
H77-1a
1b-BK
1b-Con1
2b.2
4.7
4.6
3Dpol
Klenow
HIV-RT
>50
>50
>50
>50
>50
39
9.5
38
27
25
6.4
11.1
2b.2
3a.7
GBV-b NS5B
BVDV RdRp
4a
6a
a Values are the mean of two or more experiments. SD values are (30% of the mean value.
compound 21, a NS5B inhibitor witha thiophenescaffoldthat
has been shownto bind to Thumb Site II.5f,8a Ofnote, whereas
compound 21 showed a 4-fold loss of enzymatic inhibition to
the Thumb Site I P495A mutation, compound 1 potency was
not affected. Finally, titration experiments showed that com-
pound 21 was able to compete for inhibition by compound 1
(not shown), confirming that the thiophene inhibitor 21 and
compound 1 share the same binding site, Thumb Site II.
Importantly, compound 1 represents the first Thumb Site II
NS5B inhibitor with a non-acid chemotype.
soaking the crystals with the inhibitor for several hours prior
to data collection. The structures were determined by the
˚
molecular replacement method and refined up to 2.0 A
resolution (Supporting Information Table S1). Both struc-
tures indicated that compound 1 binds at the polymerase
Thumb Site II in proximity of Met423 (Figure 3A,B).
In both structures, the inhibitor binds the enzyme in a
similar fashion and establishes very similar interactions
(Figure 3B). Indeed, only four conservative amino acid
differences between genotypes 1b and 2b HCV polymerase
are located at this site: Ile419Leu, Thr476Ser, Leu482Ile, and
Lys501Arg (2b/1b; Figure 3B). Furthermore, a superposition
of these inhibited structures with those of the respective
noninhibited enzymes reveals no major changes induced by
compound 1 binding except for the displacement of few
Crystal Structures of Genotypes 1b and 2b NS5B Bound to
Compound 1
The structures of the genotypes ΔC55-1b and ΔC21-2b
polymerase in complex with compound 1 were obtained by

5220 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ontoria et al.
Figure 2. (A) Order of addition. Increasing amounts of compound 1 (3 nM to 6.4 μM) were added to ΔC21-1b polymerase (20 nM) and polyA/
oligoU18 RNA that were (b) or were not (9) preincubated 15 min at room temperature. (B) Preincubation time course. The NS5B ΔC21-1b and
the polyA/oligoU18 RNA were preincubated from 0 to 120 min before the addition of 1 μM compound 1. (C) Inhibition curves by compound 1
in single cycle conditions. The NS5B ΔC21-1b and the polyA/oligoU18 RNA were preincubated 5 min before the addition of compound 1 from
0.1 nM to 1 μM. Elongation was started by the addition of 3H-UTP together with (9) or without (b) heparin (50 ng/μL).
Table 2. Structure and Activity of Compounds 1 and 21 on the Wild-Type (wt) NS5B ΔC21-1b Enzyme and Mutants Resistant to Inhibitors Binding at
Thumb Site I (P495A), Thumb Site II (R501E and M423K) and Either Palm Site I or II (M414T)a
a Values are the mean of two or more experiments. SD values are (30% of the mean value.
ordered water molecules (Figure 3A) and the repositioning of
the side chains of Arg498 and Arg501 in the 1b enzyme (not
shown).
(Figure 3B). The inhibitor establishes a large number of van
der Waals and hydrophobic interactions with the protein
mainly via the tricyclic moiety, the phenyl group, and the
bromine substituent. In particular, the bromine atom and the
phenyl ring fill completely a small deep pocket formed by the
side chains of Trp528, Ile419, Tyr477, Met423, and Arg422
(Figure 3C). The interaction between compound 1 and the
enzyme is also characterized by the presence of several
H-bonds, most of which are water-mediated (Figure 3A). In
the 2b structure, one carbonyl oxygen of the tricyclic moiety
forms awater-mediated H-bondwiththe sidechainofLys501,
The inhibitor binds at the center of a long cleft that is
˚
˚
˚
approximately 30 A long, 10 A wide, and 10 A deep. The side
chainsof Arg422, Trp528, Met423, Ile419 (Leu in 1b), Tyr477,
and Leu497 contribute to the formation of the floor of the
inhibitor binding cleft, whereas the rims are formed in the
upper part by the side chains of Lys501 (Arg in 1b) and
Arg498 and at the bottom by the side chains of Leu482 and
Ala486 and the backbone atoms of residues 474-477

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5221
Figure 3. Structure of NS5B-1 complex. (A) The inhibitor binding cleft in the ΔC21-2b polymerase. The inhibitor and residues forming the
binding site are in stick representation. Carbon, oxygen, and nitrogen for the inhibitor are yellow, red, and blue, respectively. Carbon, oxygen,
and nitrogen for the protein are gray, red, and blue, respectively. Water molecules in the inhibited ΔC21-2b and ΔC55-1b polymerase structures
are shown as red and blue spheres, respectively. Water molecules in the noninhibited ΔC21-2b polymerase are shown as magenta spheres.
H-Bonds are represented by dashed, yellow lines. (B) Comparison of inhibited ΔC55-1b and ΔC21-2b polymerase structures. Water molecules
in the 1b and 2b polymerase structures are shown as red and green spheres, respectively. (C) Picture of compound 1 docked in the Thumb Site II
binding pocket of the ΔC21-2b polymerase. The interactions of compound 1 with the lipophilic pocket on the inhibitor binding cleft and the
water-mediated H-bonds formed with Lys501 and Arg498 are shown. (D) Compound 21 docked on the structure of compound 1/ΔC21-2b
complex. Water molecules are shown as red spheres.
whereas in the 1b structure, this water molecule is absent and
the same oxygen establishes a direct H-bond with the bulkier
sidechainofArg501 (Figure3B). The second carbonyl oxygen
of the inhibitor forms in both structures water-mediated
H-bonds with the main chain nitrogens of Tyr477 and
Thr476 (Ser476 in 1b). Finally, the -NH and -OH groups
of the ethanolamine extension form H-bonds with the side
chain guanidinium groupand the main chaincarbonyl oxygen
of Arg498 through three bridging water molecules (Figure 3B,
C). We also calculated the intermolecular binding energy of
our compound 1 with the NS5B enzyme using the MMFF
forcefield,17 as implemented in Macromodel 7.0,18 obtaining a
value of ΔEinter = -271 kJ/mol. Calculation of the intermo-
lecular binding energy for analogue 4, which is devoid of the
bromine atom, gave a value of ΔEinter = -196 kJ/mol.
Moreover, the value obtained for the molecule without the
bromophenyl group is ΔEinter = -31 kJ/mol. These value’s
differences reflect the importance of the lipophilic interactions
of the bromine atom and the phenyl ring with the side chains of
the amino acids that form the small deep pocket. To compare
the interactions established by compounds 1 and 21 with the
NS5B polymerase, we docked the latter compound into the
Thumb Site II pocket of the ΔC21-2b enzyme. The docking of
21 was performed using the crystal structure of the ΔC21-2a
polymerase bound to a structurally closely related compound
(PDB ID code 1YVX) as template. The superposition reveals
that the methylcyclohexyl group of 21 binds to the same small
lipophilic pocket as the bromophenyl group of compound
1 (Figure 3D). Moreover, the two oxygen atoms of the
carboxylate of 21 occupy the same position occupied in
structure of the ΔC21-2b/1 complex by two water molecules
that form hydrogen bonds with one of the carbonyl oxygens
of the inhibitor and with the main chain nitrogens of Tyr477
and Thr476.
SAR Studies
In parallel with the structural and functional characteriza-
tion of the hit compound 1, we initiated SAR studies to
identify the pharmacophoric elements of this class of com-
pounds that are essential for NS5B polymerase binding and

5222 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Table 3. Enzymatic Activity of Compounds 1 and 4-11
Ontoria et al.
a Values are the mean of two or more experiments. SD values are (30% of the mean value.
inhibition. With this purpose, we initially prepared a series of
analoguesof 1 thatwere devoidof one of itsstructuralfeatures
(Table 3).
bromine atom, the carbonyl groups, and the alkylamine frag-
ment is determinant for the interaction between the lead com-
pound 1 and the HCV NS5B polymerase and is in agreement
with the crystal structure of compound 1bound to ΔC55-1b and
ΔC21-2b enzymes. With the aim of increasing the inhibitory
activity against both genotypes, we performed additional SAR
studies by exploring alternative substituents in the phenyl ring
and in the amino group of the tricyclic core (Table 4).
In agreement with the X-ray structures, the debrominated
analogue 4 displayed a loss of 2 orders of magnitude in
potency, highlighting the relevance of the bromine atom for
the lipophilic interaction of 1 with the binding pocket formed
by Trp528, Ile419, Tyr477, Met423, and Arg422. Similarly,
removal of the ethanolamine fragment as in 5, which in the
structure ofthe complex establishes both apolar and polar and
H-bond interactions with residues forming the cleft, led to a
complete loss of inhibition. An important fraction of this
inhibitory activity was recovered by introduction of an amino
group in position 6 of the tricyclic system, as demonstrated by
compound 6, in line with the H-bond observed in the crystal
structure between the NH group of 1 and Arg498. A further
improvement in inhibition potency, particularly against the
genotype 1b, was observed by the introduction of an ethyl
chain as in compound 7. The N-methylated analogue 8
showed a 25-fold loss of enzymatic activity, further confirm-
ing the importance of the H-bond with Arg498. Methylation
of the hydroxyl group, as in 9, did not affect significantly
potency, and it led to a moderate decrease in genotype
selectivity. The importance of the H-bonds between the
compound carbonyl oxygens and the side chains of Lys501
(Arg501 in 1b) and the main chain nitrogens of Tyr477 and
Thr476 (Ser476 in 1b) was demonstrated by compounds 10
and 11, where elimination of one or two of the carbonyl
groups led to a very significant or complete loss of inhibition.
Overall, the SAR demonstrated that the presence of the
As expected, the replacement of the bromine atom by
chlorine as in 12 did not affect inhibition, indicating that the
chlorine atom retains most of the lipophilic interactions with
the small deep pocket on the NS5B Thumb Site II surface
(Figure 3C). In contrast, introduction of smaller halogen such
as fluorine (13) resulted in a 10-fold loss of potency, and the
introduction of polar groups such as the sulfonamide (14) led
to a relevant decrease of potency on both genotypes of NS5B
polymerase (IC50 = 1,600 nM, ΔC21-1b). The latter result is
not surprising and is consistent with the hydrophobic nature
of the bromine binding pocket. As demonstrated by analogue
9 (Table 3), where the aliphatic ethylamine maintains a good
level of intrinsic potency, branched alkylamines such as
isobutylamine or cyclohexylamine, as in compounds 15 and
16, showed a little loss of potency against genotype 1 enzyme
while maintaining a similar potency against genotype 2, thus
resulting in molecules that inhibit both genotypes similarly.
Introduction of a basic center in the amino substituent, as in
17, led toa 7-fold loss of inhibitory activity against genotype 1,
which was partially recovered by inclusion of the amino group
in a piperidine ring (18), probably due to additional lipophilic
interaction with the protein established by the six-membered

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5223
Table 4. Enzymatic and Cell-Based Activity of Compounds 1 and 12-21
a Values are the mean of two or more experiments. SD values are (30% of the mean value. b Structure of compound 21 is shown in Table 2.
ring. Introduction in the piperidine ring of a bulky group such
as benzyl (19) led to a very potent inhibitor with low nano-
molar potency against genotype 1 enzyme. This finding is
consistent with the X-ray structures that suggest that addi-
tional lipophilic contacts can be established with residues
forming the elongated inhibitor binding cleft by further
extending the inhibitor on the position 6 of the tricyclic
system. Unfortunately, the compounds bearing a basic center
in their structure were cytotoxic in cells. Compounds that
possess a 1H,3H-benzo[de]isochromene-1,3-dione core and
bear a basic amine in their structures are reported to be good
DNA intercalators,19 and this could be the reason for the
toxicity in the replicon cells. This finding prompted us to look
for alternative groups that could abolish binding to DNA.
One approach was the introduction of a carboxylate group to
reduce the intercalation properties of these compounds by
electrostatic repulsion with the phosphate groups of the DNA
chain. Introduction of a carboxylic group in the amino
substituent, as in 20, resulted in a compound that, similar to
analogue 16, maintained nanomolar activity against both
genotypes and displayed low micromolar inhibition in cells
and showed at least a 13-fold window respect to cytotoxicity.
When compound 21 was tested on the ΔC21-2b enzyme, no
inhibiton was observed up to 10 μM, showing a more than 3
orders of magnitude difference in inhibitory activity com-
pared to genotype 1 (Table 4). Interestingly, although com-
pounds 1 and 21 establish some common interactions with the
NS5B polymerase, they showed very different genotype se-
lectivity profile on genotypes 1b and 2b.
Conclusion
A HTS campaign led to the identification of compound 1, an
HCV inhibitor with a novel chemotype, which inhibits NS5B
polymerases from all the major HCV genotypes at submicro-
molar concentrations. Compound 1 is an effective inhibitor of
HCV replication in 1b and 2b replicons with a 10-fold activity/
toxicity window in cells and excellent selectivity over a panel of
closely related polymerases. Experiments aimed at clarifying the
mechanism of inhibition showed that 1 acts by binding to NS5B
polymerase during the pre-elongation phase. X-ray structures of
1 with NS5B ΔC55-1b and ΔC21-2b show that this compound
binds to the non-nucleoside binding site Thumb Site II establish-
ing a large number of van der Waals and hydrophobic interac-
tions with the protein via the tricyclic core and the phenyl group
with the bromine substituent filling a small deep pocket. In
addition, the polymerase establishes a number of direct and
water-mediated H-bonds, where the number varies depending
on genotype, with the carbonyl oxygens of the inhibitor. The
relevance of these interactions was confirmed by the preparation
of analogues that lacked some of these key structural motifs
leading to analogues that showed a relevant loss of enzymatic
activity. Further SAR in the aromatic ring and in the amino
group led to compounds with slightly improved activity in the
replicon assay and good activity/toxicity window in cells.
Experimental Section
Chemistry. Solvents and reagents were obtained from com-
mercial suppliers and were used without further purification.

5224 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ontoria et al.
Organics extracts were dried over anhydrous sodium sulfate
(Merck). Flash chromatography purifications were performed
on Merck silica gel (200-400 mesh) as the stationary phase or
were conducted using prepacked cartridges on a Biotage system,
eluting with petroleum ether/ethyl acetate or toluene/acetone
mixtures. Microwave irradiation was performed in a Personal
Chemistry (now Biotage) optimizer, model Emrys. Purity of the
compounds was determined by analytical RP-HPLC, data were
obtained by two methods on an Acquity Waters UPLC using a
flow rate of 0.5 mL/min, an Acquity UPLC BEH C18, 1.7 μm,
2.1 mm ꢀ 50 mm column as the stationary phase, and a mobile
phase comprising MeCN þ 0.1% HCO2H (solvent A) and H2O
þ 0.1% HCO2H (solvent B). Method 1 parameters are 30%
solvent A (0.0 min) to 100% solvent A (2.6 min) and then 100%
solvent A (0.3 min). Method 2 parameters are 0% solvent A (0.0
min) to 100% solvent A (2.6 min) and then 100% solvent A (0.3
min). All the compounds described in this article showed
purities higher than 95% in both analytical methods. HPLC-
MS was performed on a Waters Alliance 2795 apparatus
equipped with a diode array and a ZQ mass spectrometer using
an X-Terra C18 column (5 μm, 4.6 mm ꢀ 50 mm). Mobile phase
comprised a linear gradient of binary mixtures of H2O contain-
ing 0.1% formic acid (solvent A) and MeCN containing 0.1%
formic acid (solvent B). Nuclear magnetic resonance spectra (1H
NMR recorded at 500, 400, or 300 MHz, 13C NMR recorded at
100 or 75 MHz) were obtained on Bruker AMX spectrometers
and are referenced in ppm relative to TMS. Unless indicated,
spectra were acquired at 300 K. High resolving power accurate
mass measurement electrospray (ES) mass spectral data were
acquired by use of a Bruker Daltonics apex-Qe Fourier trans-
form ion cyclotron resonance mass spetrometer (FT-ICR MS).
External calibration was accomplished with oligomers of poly-
propylene.
2-aminoethanol (0.05 mL, 0.9 mmol) and stirred at 200 °C under
microwave irradiation for 30 min. After cooling down, the
reaction mixture was purified by RP-HPLC (conditions: Waters
X-TERRA MS C18, 5 μm, 19 mm ꢀ 150 mm; flow of 20 mL/min;
gradient, (A) H2O þ 0.1% TFA, (B) MeCNþ 0.1% TFA, 99% A
isocratic for 2 min, linear to 1% A in 10 min, 1% A isocratic for
5 min) to give 5 (61 mg, 38%) as a solid. 1H NMR (400 MHz,
DMSO-d6) δ 8.76 (d, J = 8.5 Hz, 1H), 8.44 (d, J = 7.2 Hz, 1H),
8.26 (d, J = 8.5 Hz, 1H), 7.79-7.76 (m, 1H), 7.72 (t, J = 7.9 Hz,
1H), 7.52-7.48 (m, 2H), 7.43 (t, J = 7.2 Hz, 1H), 7.30 (d, J =
7.4 Hz, 2H), 6.85 (d, J = 8.5 Hz, 1H), 4.91 (br s, 1H), 3.74-3.71
(m, 2H), 3.52-3.49 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ
163.4, 162.6, 150.4, 136.0, 133.7, 130.2, 129.4, 128.7, 128.1, 127.2,
123.7, 121.7, 119.7, 107.3, 103.3, 58.3, 45.0; HRMS (ESI) m/z
calcd for C20H17N2O3 333.1233, found 333.1234; RP-HPLC
method 1, tR = 0.77 min; method 2, tR = 1.32 min.
2-(3-Bromophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (5).
In a manner identical to that described above for the preparation
of 3a, 1,8-naphthalic anhydride 2b (324 mg, 1.63 mmol) was
treated with 3-bromoaniline (0.27 mL, 2.45 mmol) to give 5
1
(383 mg, 67%) as a solid. H NMR (400 MHz, DMSO-d6) δ
8.86 (d, J = 8.3 Hz, 1H), 8.48 (d, J = 8.1 Hz, 1H), 8.23 (d, J = 8.3
Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.90-7.86 (m, 1H), 7.82-7.78
(m, 2H), 7.67 (d, J = 7.2 Hz, 1H), 7.55-7.53 (m, 2H); 13C NMR
(75 MHz, DMSO-d6) δ 167.8, 167.0, 136.2, 135.7, 133.4, 131.0,
130.7, 130.0, 129.2, 129.0, 127.3, 126.4, 123.9, 121.1, 118.6; HRMS
(ESI) m/z calcd for C18H11BrNO2 351.9970, found 351.9962; RP-
HPLC method 1, tR = 1.86 min; method 2, tR = 2.35 min.
6-Amino-2-(3-bromophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (6). A solution of 6-amino-1H,3H-benzo[de]isochromene-
1,3-dione 2c (50 mg, 0.23 mmol) and TEA (0.16 mL, 1.16 mmol)
in DMF (1 mL) was treated with 3-bromoaniline (0.13 mL,
1.17 mmol). The reaction mixture was stirred at 230 °C under
microwave irradiation for 3 h. The resulting precipitate was
filtered, washed with Et2O, and dried to give 6 (18 mg, 21%) as
6-Bromo-2-(3-bromophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (3a). 3-Bromoaniline (1.2 mL, 10.82 mmol) was added to a
solution of 2a (2.0 g, 7.21 mmol) in NMP (10 mL). The reaction
mixture was stirred at 200 °C under microwave irradiation for
30 min. The resulting precipitate was filtered, washed subse-
quently with EtOH and Et2O, and dried to give 3a (2.0 g, 64%)
as a pale-brown solid. 1H NMR (300 MHz, DMSO-d6) δ 8.64-
8.58 (m, 2H), 8.36 (d, J = 7.7 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H),
8.07-8.02 (m, 1H), 7.70-7.67 (m, 2H), 7.53-7.43 (m, 2H);
HRMS (ESI) m/z calcd for C18H10Br2NO2 429.9073, found
429.9079.
1
a pale-yellow powder. H NMR (400 MHz, DMSO-d6) δ 8.67
(d, J = 8.4 Hz, 1H), 8.44 (d, J = 6.8 Hz, 1H), 8.20 (d, J = 8.4 Hz,
1H), 7.69 (dd, J = 8.4, 6.8 Hz, 1H), 7.66-7.62 (m, 2H), 7.52-7.45
(m, 3H), 6.88 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6) δ 163.9, 162.9, 152.9, 138.2, 134.0, 132.2, 131.1, 130.8,
130.5, 130.2, 129.6, 128.6, 123.9, 122.1, 121.0, 119.5, 108.1,
107.6; HRMS (ESI) m/z calcd for C18H12BrN2O2 367.0077, found
367.0071; RP-HPLC method 1, tR = 1.22 min; method 2, tR
1.70 min.
=
General Procedure for the Synthesis of 7-9, 15-17, and 19. A
solution of 3a (80 mg, 0.19 mmol) in NMP (0.9 mL) was treated
with the appropriate amine (10 equiv). The reaction mixture was
stirred at 200 °C under microwave irradiation for 30 min.
Addition of water (2 mL) afforded a precipitate that was isolated
by filtration to give 7-9, 15-17, and 19.
2-(3-Bromophenyl)-6-[(2-hydroxyethyl)amino]-1H-benz[de]-
isoquinoline-1,3(2H)-dione (1). A solution of 3a (0.7 g,
2.52 mmol) and 2-aminoethanol (1.5 mL, 25.26 mmol) in
12.5 mL of NMP was stirred at 200 °C under microwave irradia-
tion for 30 min. Addition of water gave a precipitate that was
filtered and washed subsequently with EtOH and Et2O. The crude
was crystallized from acetone to give 1 (0.28 g, 27%) as a yellow
solid. 1H NMR (300 MHz, DMSO-d6) δ 8.77 (d, J = 8.4 Hz, 1H),
8.45 (d, J = 7.2 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 7.84-7.79 (m,
1H), 7.66-7.64 (m, 2H), 7.50-7.46 (m, 1H), 7.37 (d, J = 7.6 Hz,
1H), 7.31 (dd, J = 8.4, 7.2 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H),
2-(3-Bromophenyl)-6-(ethylamino)-1H-benzo[de]isoquinoline-1,
3(2H)-dione (9). Yield 57%; 1H NMR (300 MHz, DMSO-d6) δ
8.76 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 7.1 Hz, 1H), 8.27 (d, J = 8.6
Hz, 1H), 7.83-7.79 (m, 1H), 7.71 (dd, J = 8.2, 7.1Hz, 1H), 7.46-
7.44 (m, 3H), 7.38-7.35 (m, 1H), 7.37 (d, J =8.0Hz, 1H), 6.82 (d,
4.92-4.90 (m, 1H), 3.75-3.71 (m, 2H), 3.53-3.49 (m, 2H); 13
C
J = 8.8 Hz, 1H), 3.46-3.42 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H); 13
C
NMR (100 MHz, DMSO-d6) δ 163.8, 162.9, 151.0, 138.2, 134.3,
132.2, 130.8, 130.5, 129.9, 128.8, 128.6, 124.2, 122.1, 121.0, 120.2,
107.7, 103.9, 58.8, 45.9; HRMS (ESI) m/z calcd for
C20H16BrN2O3 411.0339, found 411.0332; RP-HPLC method 1,
NMR (100 MHz, DMSO-d6) δ 163.8, 162.9, 150.7, 138.2, 134.3,
132.2, 130.8, 130.5, 129.9, 128.8, 128.6, 124.1, 122.1, 121.0, 120.2,
107.6, 103.7, 37.6, 13.6; HRMS (ESI) m/z calcd for
C20H16BrN2O2 395.0386, found 395.0390; RP-HPLC method 1,
tR = 1.07 min; method 2, tR = 1.63 min.
6-[(2-Hydroxyethyl)amino]-2-phenyl-1H-benzo[de]isoquinoline-
t
R = 1.54 min; method 2, tR = 2.01 min.
2-(3-Bromophenyl)-6-[(2-hydroxyethyl)(methyl)amino]-1H-
1,3(2H)-dione (4). A solution of 4-bromo-1,8-naphthalic anhy-
dride 2a (330 mg, 1.20 mmol) in NMP (2 mL) was treated with
aniline (0.16 mL, 1.80 mmol). The reaction mixture was stirred at
200 °C under microwave irradiation for30 min. Addition of water
gave a precipitate that was purified by silica gel chromatography
(5-10% acetone/toluene gradient), giving 210 mg of 6-bromo-2-
phenyl-1H-benzo[de]isoquinoline-1,3(2H)-dione 3b. A solution
of this intermediate (0.6 mmol) in NMP (2 mL) was treated with
benzo[de]isoquinoline-1,3(2H)-dione (8). Yield 45%; 1H NMR
(400 MHz, DMSO-d6) δ 8.78 (d, J = 8.6 Hz, 1H), 8.46 (d, J = 6.8
Hz, 1H), 8.35 (d, J = 8.2 Hz, 1H), 7.78 (dd, J = 8.6, 6.8 Hz, 1H),
7.66-7.65 (m, 2H), 7.50-7.40 (m, 2H), 7.33 (d, J = 8.4 Hz, 1H),
4.89-4.87 (m, 1H), 3.79-3.77 (m, 2H), 3.48-3.45 (m, 2H), 3.30 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ163.8, 163.1, 156.9, 137.8,
132.2, 132.1, 131.8, 130.9, 130.6, 130.1, 128.5, 124.9, 124.7, 122.7,
121.1, 114.1, 113.7, 59.3, 58.3; HRMS (ESI) m/z calcd for

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5225
C21H18BrN2O3 425.0491, found 425.0495; RP-HPLC method 1,
tR = 1.22 min; method 2, tR = 1.79 min.
dried and filtered, and the filtrate was concentrated in vacuo to
yield the crude product that was purified by RP-HPLC
(conditions: Waters X-TERRA MS C18, 5 μm, 19 mm ꢀ 150
mm; flow of 20 mL/min; gradient, (A) H2O þ 0.1% TFA, (B)
MeCN þ 0.1% TFA, 90% A isocratic for 2 min, linear to 30% A
in 10 min, then linear to 0% A in 2 min) to give 10 (13 mg, 26%) as
a solid. 1H NMR (300 MHz, DMSO-d6) δ 8.13 (m, 1H), 8.06 (d, J
= 8.4 Hz, 1H), 7.74 (s, 1H), 7.53-7.40 (m, 5H), 6.91 (br s, 1H),
6.68 (d, J = 8.4 Hz, 1H), 5.29 (s, 2H), 4.83 (t, J = 5.5 Hz, 1H),
3.73-3.67 (m, 2H), 3.41 (m, 2H); 13C NMR (100 MHz, DMSO-
d6) δ 162.8, 148.0, 144.5, 130.3, 129.2, 128.7, 128.5, 124.9, 124.1,
123.1, 121.1, 120.5, 120.1, 111.4, 102.3, 58.9, 52.5, 45.5; HRMS
(ESI) m/z calcdfor C20H18BrN2O2 397.0546, found 397.0540; RP-
HPLC method 1, tR = 1.16 min; method 2, tR = 1.73 min.
2-{[2-(3-Bromophenyl)-2,3-dihydro-1H-benzo[de]isoquinolin-
6-yl]amino}ethanol (11). BH3 (0.93 mL, 1 M solution in THF)
was added dropwise to a solution of 1 (50 mg, 0.12 mmol) in
THF (1.2 mL) under nitrogen atmosphere. The reaction mix-
ture was stirred to reflux for 5 h. After cooling to 0 °C, the
reaction was carefully quenched by dropwise addition of
MeOH until hydrogen evolution ceased. The mixture was
concentrated in vacuo to give a residue that was diluted with
6 N HCl (0.5 mL) and stirred to reflux for 30 min. After cooling
to room temperature, the mixture was basified with 2 N NaOH.
The aqueous phase was extracted with EtOAc. The combined
organic layers were dried and filtered and the filtrate was
concentrated in vacuo to yield the crude product that was
purified by silica gel chromatography (10-70% EtOAc/petro-
2-(3-Bromophenyl)-6-[(2-methoxyethyl)amino]-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (9). Yield 36%; 1H NMR (400 MHz,
DMSO-d6 þ TFA) δ 8.77 (d, J = 8.4 Hz, 1H), 8.45 (d, J = 7.2
Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 7.86 (br s, 1H), 7.73 (dd, J =
8.4, 7.2 Hz, 1H), 7.66-7.64 (m, 2H), 7.50-7.46 (m, 1H), 7.37 (d,
J = 8.0 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 3.67-3.61 (m, 4H),
3.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 163.9, 163.0,
150.8, 138.2, 134.3, 132.2, 130.8, 130.5, 130.0, 128.9, 128.6,
124.3, 122.2, 121.0, 120.2, 107.9, 104.0, 69.7, 58.1, 42.6; HRMS
(ESI) m/z calcd for C21H18BrN2O3 425.0495, found 425.0502;
RP-HPLC method 1, tR = 1.37 min; method 2, tR = 1.91 min.
2-(3-Bromophenyl)-6-[(2-methylpropyl)amino]-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (15). Yield 48%; 1H NMR (300 MHz,
DMSO-d6) δ8.80 (d, J= 8.4 Hz, 1H), 8.44 (d, J = 7.1 Hz, 1H), 8.25
(d, J = 8.4 Hz, 1H), 7.86-7.82 (m, 1H), 7.72 (dd, J = 8.4, 7.1 Hz,
1H), 7.64-7.60 (m, 2H), 7.48-7.45 (m, 1H), 7.38 (d, J = 8.0 Hz,
1H), 6.83 (d, J = 8.6 Hz, 1H), 3.24-3.22 (m, 2H), 2.18-2.02 (m,
1H), 0.99 (d, J = 6.4 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ
163.9, 162.9, 151.0, 138.2, 134.3, 132.1, 130.7, 130.5, 130.0, 128.8,
128.6, 124.2, 122.2, 121.0, 120.2, 107.5, 103.9, 50.4, 26.8, 20.3; HRMS
(ESI) m/z calcd for C22H20BrN2O2 422.0708, found 422.0724; RP-
HPLC method 1, tR = 1.79 min; method 2, tR = 2.24 min.
2-(3-Bromophenyl)-6-(cyclohexylamino)-1H-benzo[de]isoq-
1
uinoline-1,3(2H)-dione (16). Yield 40%; H NMR (300 MHz,
DMSO-d6) δ 8.85 (d, J = 8.4 Hz, 1H), 8.43 (d, J = 7.3 Hz, 1H),
8.24 (d, J = 8.6 Hz, 1H), 7.70 (dd, J = 8.4, 7.3 Hz, 1H), 7.54-7.46
(m, 2H), 7.49-7.34 (m, 3H), 6.89 (d, J = 8.8 Hz, 1H), 3.67 (br s,
1H), 2.10-1.99 (m, 2H), 1.82-1.78 (m, 3H), 1.42-1.15 (m, 5H);
13C NMR (100 MHz, DMSO-d6) δ 163.9, 162.9, 149.9, 138.2,
134.3, 132.2, 130.8, 130.5, 130.1, 129.2, 128.6, 132.9, 122.0, 121.0,
120.2, 107.4, 104.2, 51.5, 31.8, 25.3, 24.7; HRMS (ESI) m/z calcd
for C24H22BrN2O2 449.0854, found 449.0856; RP-HPLC method
1, tR = 1.96 min; method 2, tR = 2.38 min.
1
leum ether gradient) to give 11 (16 mg, 34%) as a solid. H
NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 7.8 Hz, 1H), 7.37
(m, 2H), 7.23-7.20 (m, 2H), 7.12-7.10 (m, 2H), 6.86-6.84 (m,
1H), 6.49 (d, J = 7.6 Hz, 1H), 5.89-5.86 (m, 1H), 4.78 (s, 2H),
4.69 (s, 2H), 3.70-3.66 (m, 2H), 3.28-3.24 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 151.8, 143.0, 132.1, 130.6, 128.4,
123.7, 123.4, 122.6, 122.4, 120.7, 120.1, 118.7, 118.1, 114.9,
102.5, 59.2, 51.7, 51.3, 45.9; HRMS (ESI) m/z calcd for
C20H20BrN2O 381.0680, found 381.0679; RP-HPLC method
1, tR = 0.71 min; method 2, tR = 1.29 min.
General Procedure for the Synthesis of 12-14. A solution of 2a
(100 mg, 0.36 mmol) in EtOH (2 mL) was treated with the
appropriate aniline (6 equiv). The reaction mixture was stirred
at 150 °C under microwave irradiation for 4 h. The resulting
precipitate was filtered, affording a crude that was diluted in
NMP (1 mL). After addition of 2-aminoethanol (0.066 mL,
1.08 mmol) the resulting solution was stirred at 200 °C under
microwave irradiation for 30 min and purified by RP-HPLC
(conditions: Waters X-TERRA MS C18, 5 μm, 19 mm ꢀ
150 mm; flow of 20 mL/min; gradient, (A) H2O þ 0.1% TFA;
(B) MeCN þ 0.1% TFA, 99% A isocratic for 2 min, linear to 1%
A in 10 min, 1% A isocratic for 5 min) to give 12-14.
2-(3-Chlorophenyl)-6-[(2-hydroxyethyl)amino]-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (12). Yield, 20%; 1H NMR (400
MHz, DMSO-d6) δ 8.75 (d, J = 7.9 Hz, 1H), 8.44 (d, J = 7.2
Hz, 1H), 8.26 (d, J = 8.6 Hz, 1H), 7.80-7.76 (m, 1H), 7.74-
7.70 (m, 1H), 7.54-7.50 (m, 3H), 7.34-7.31 (m, 1H), 6.85 (d,
J = 8.6 Hz, 1H), 3.74-3.71 (m, 2H), 3.52-3.48 (m, 2H); 13C
NMR (75 MHz, DMSO-d6) δ 163.8, 162.9, 151.0, 138.0, 134.3,
132.8, 130.8, 130.2, 130.0, 129.4, 128.9, 128.2, 127.9, 124.2,
122.2, 120.2, 107.7, 103.9, 58.8, 45.6; HRMS (ESI) m/z calcd for
C20H16ClN2O3 367.0844, found 367.0836; RP-HPLC method
1, tR = 1.02 min; method 2, tR = 1.58 min.
2-(3-Fluorophenyl)-6-[(2-hydroxyethyl)amino]-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (13). Yield, 61%; 1HNMR(400MHz,
DMSO-d6) δ 8.75 (d, J = 8.6 Hz, 1H), 8.44 (d, J = 7.4 Hz, 1H),
8.26 (d, J = 8.5 Hz, 1H), 7.80-7.76 (m, 1H), 7.74-7.70 (m, 1H),
7.54-7.50 (m, 1H), 7.30-7.28 (m, 2H), 7.19 (d, J = 7.9 Hz, 1H),
6.85 (d, J = 8.5 Hz, 1H), 3.74-3.71 (m, 2H), 3.52-3.48 (m, 2H);
13C NMR (75 MHz, DMSO-d6) δ 163.8, 162.9, 151.0, 138.2 (d, J
= 10.5 Hz), 134.2, 130.8, 130.1, 130.0, 129.9, 128.8, 125.6, 124.2,
122.1, 120.2, 116.7 (d, J = 22.5 Hz), 114.7 (d, J = 20.0 Hz), 107.7,
2-(3-Bromophenyl)-6-{[2-(methylamino)ethyl]amino}-1H-ben-
zo[de]isoquinoline-1,3(2H)-dione (17). Yield 71%; 1H NMR
(400 MHz, DMSO-d6) δ 8.70 (d, J = 8.4 Hz, 1H), 8.63 (br s,
2H), 8.48 (d, J = 7.2 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.82-7.70
(m, 2H), 7.67-7.65 (m, 2H), 7.51-7.47 (m, 1H), 7.38 (d,
J = 7.6 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 3.74-3.72 (m, 2H),
3.38-3.29 (m, 2H), 2.68 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ163.8, 163.0, 150.2, 138.0, 134.0, 132.2, 130.9, 130.5, 129.8, 129.0,
128.6, 124.5, 122.2, 121.1, 120.5, 108.9, 104.1, 46.4, 32.6; HRMS
(ESI) m/z calcd for C21H19BrN3O2 424.0651, found 424.0659; RP-
HPLC method 1, tR = 0.3 min; method 2, tR = 1.15 min.
1-Benzyl-4-{[2-(3-bromophenyl)-1,3-dioxo-2,3-dihydro-1H-ben-
zo[de]isoquinolin-6-yl]amino}piperidinium Trifluoroacetate (19).
After cooling down, the reaction mixture was purified by RP-
HPLC (conditions: Waters X-TERRA MS C18, 5 μm, 19 mm ꢀ
150 mm; flow of 20 mL/min; gradient, (A) H2O þ 0.1% TFA, (B)
MeCN þ 0.1% TFA, 99% A isocratic for 2 min, linear to 1% A in
1
10 min, 1% A isocratic for 5 min) to give 5 (17 mg, 14%). H
NMR (300 MHz, DMSO-d6 þ TFA) δ 9.70 (br s, 1H), 8.84 (d,
J = 8.2 Hz, 1H), 8.45 (d, J = 7.3 Hz, 1H), 8.25 (d, J = 8.6 Hz,
1H), 7.71 (dd, J = 8.2, 7.3 Hz, 1H), 7.69-7.64 (m, 2H), 7.53-7.43
(m, 7H), 7.35 (d, J = 7.9 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 4.37
(br s, 2H), 3.96-3.80 (m, 1H), 3.58-3.50 (m, 2H), 3.22-3.10 (m,
2H), 2.31-2.22 (m, 2H), 1.98-1.89 (m, 2H); 13C NMR (100
MHz, DMSO-d6) δ 163.8, 162.9, 149.6, 138.0, 134.0, 132.1, 121.5,
130.9, 130.5, 129.9, 129.6, 129.4, 129.3, 128.8, 128.6, 124.3, 122.2,
121.0, 120.3, 108.4, 104.6, 59.0, 50.6, 47.6, 28.3; HRMS (ESI) m/z
calcd for C30H27BrN3O2 540.1281, found 540.1296; RP-HPLC
method 1, tR = 0.61 min; method 2, tR = 1.40 min.
2-(3-Bromophenyl)-7-[(2-hydroxyethyl)amino]-2,3-dihydro-1H-
benzo[de]isoquinolin-1-one (10).
A solution of 1 (50 mg,
0.12 mmol) in EtOH/H2O (10:1) (6 mL) was treated with sodium
borohydride (23 mg, 0.61 mmol) at room temperature. The
reaction mixture was stirred overnight, quenched with 1 N HCl,
and extracted with EtOAc. The combined organic layers were

5226 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Ontoria et al.
103.9, 58.8, 45.6; HRMS (ESI) m/z calcd for C20H16FN2O3
351.1139, found 351.1133; RP-HPLC method 1, tR = 0.86 min;
method 2, tR = 1.42 min.
References
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(2) Field, J. J.; Hoofnagle, J. H. Mechanism of Action of Interferon and
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3-{6-[(2-Hydroxyethyl)amino]-1,3-dioxo-1H-benzo[de]iso-
quinolin-2(3H)-yl}benzenesulfonamide (14). Yield, 75%; 1H
NMR (400 MHz, DMSO-d6) δ 8.77 (d, J = 8.6 Hz, 1H), 8.45 (d, J
= 7.2 Hz, 1H), 8.27 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H),
7.82 (br s, 1H), 7.78 (br s, 1H), 7.75-7.69 (m, 2H), 7.58 (d, J = 7.9
Hz, 1H), 7.48 (br s, 2H), 6.87 (d, J = 8.5 Hz, 1H), 3.74-3.71 (m,
2H), 3.52-3.48 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 164.0,
163.0, 151.1, 144.8, 136.9, 134.3, 132.9, 130.8, 130.0, 129.4, 128.9,
126.5, 125.2, 124.2, 122.1, 120.3, 107.6, 103.9, 58.8, 45.6; HRMS
(ESI) m/z calcd for C20H18BrN3O5S 412.0962, found 412.0957;
RP-HPLC method 1, tR = 0.54 min; method 2, tR = 1.04 min.
4-{[2-(3-Bromophenyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]-
isoquinolin-6-yl]amino}piperidinium Trifluoroacetate (18). A
solution of 3a (70 mg, 0.162 mmol) in NMP (0.8 mL) was
treated with tert-butyl 4-aminopiperidine-1-carboxylate (228
mg, 1.14 mmol). The reaction mixture was stirred at 250 °C
under microwave irradiation for 20 min. Addition of water (2
mL) gave a precipitate that was filtered, affording a crude that
was diluted in 0.5 mL of CH2Cl2/TFA (2:1) (0.5 mL), and the
resulting solution was stirred at room temperature for 15 min
and concentrated in vacuo to obtain a residue that was purified
by RP-HPLC (conditions: Waters X-TERRA MS C18, 5 μm,
19 mm ꢀ 150 mm; flow of 20 mL/min; gradient, (A) H2O þ
0.1% TFA, (B) MeCN þ 0.1% TFA, 90% A isocratic for
2 min, linear to 30% A in 10 min, then linear to 0% A in 2 min)
to give 18 (16 mg, 39%) as a solid. 1H NMR (500 MHz,
DMSO-d6) δ 8.87 (d, J = 8.5 Hz,1H), 8.60 (br s, 1H), 8.47
(d, J = 7.0 Hz, 1H), 8.42 (br s, 1H), 8.28 (d, J = 8.5 Hz, 1H),
7.77 (dd, J = 8.4, 7.0 Hz, 1H), 7.67-7.64 (m, 2H), 7.57-7.55
(m, 1H), 7.50-7.47 (m, 1H), 6.99 (d, J = 8.8 Hz, 1H), 4.08-
3.98 (m, 1H), 3.45-3.42 (m, 2H), 3.14-3.08 (m, 2H), 2.20-
2.18 (m, 2H), 1.88-1.82 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 163.8, 162.9, 149.5, 138.0, 134.0, 132.2, 130.8,
130.5, 130.0, 129.3, 128.6, 124.3, 121.0, 120.4, 108.4, 104.5,
47.3, 42.5, 27.9; HRMS (ESI) m/z calcd for C23H21BrN3O2
450.0811, found 450.0812; RP-HPLC method 1, tR = 0.30
min; method 2, tR = 1.22 min.
cis-4-{[2-(3-Bromophenyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]-
isoquinolin-6-yl]amino}cyclohexanecarboxylic Acid (20). In a
manner identical to that described above for the preparation
of 1, reaction of 3a (43 mg, 0.1 mmol) and cis-4-aminocyclo-
hexanecarboxylic acid (43 mg, 0.3 mmol) gave 20 (5 mg, 10%).
1H NMR (400 MHz, DMSO-d6) δ 12.22 (br s, 1H), 8.86 (d, J =
8.5 Hz, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.24 (d, J = 8.7 Hz, 1H),
7.74-7.59 (m, 3H), 7.50-7.34 (m, 3H), 6.90 (d, J = 8.7 Hz,
1H), 3.81-3.70 (m, 1H), 2.63-2.56 (m, 1H), 2.16-2.03 (m,
2H), 1.94-1.82 (m, 2H), 1.76-1.62 (m, 3H); 13C NMR (100
MHz, DMSO-d6) δ 175.9, 163.9, 162.9, 150.0, 138.2, 134.3,
132.2, 130.8, 130.5, 130.1, 129.3, 128.6, 124.1, 122.1, 121.0,
120.3, 107.6, 104.3, 50.3, 38.3, 28.3, 25.4; HRMS (ESI) m/z
calcd for C25H22BrN2O4 493.0757, found 493.0765; RP-
HPLC method 1, tR = 1.44 min; method 2, tR = 1.93 min.
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Acknowledgment. We thank Maria-Cecilia Palumbi for
automatedRP-HPLC purification, Cinzia Volpari for crystal-
lization experiments, Dr. Odalys Gonzalez Paz for pharma-
cokinetic studies, Monica Bisbocci for biological testing, Dr.
Silvia Pesci for NMR spectrometry, Dr. Charles W. Ross and
Claudio Giuliano for HRMS determinations, and Dr. Jorg
Habermann for preparation of 21.
Supporting Information Available: Enzyme and cell-based
assay protocols, pharmacokinetic studies, soaking experiments,
and X-ray data collection, structure determination, and refine-
ment description. This material is available free of charge via the
Internet at http://pubs.acs.org.
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