Allosteric inhibitors of hepatitis C virus NS5B polymerase thumb domain site II: Structure-based design and synthesis of new templates

Article abstract of DOI:10.1016/j.bmc.2010.03.024

Chronic hepatitis C virus (HCV) infections are a significant medical problem worldwide. The NS5B Polymerase of HCV plays a central role in virus replication and is a prime target for the discovery of new treatment options. We recently disclosed 1H-benzo[d

Full text of DOI:10.1016/j.bmc.2010.03.024

Bioorganic & Medicinal Chemistry 18 (2010) 2836–2848
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Allosteric inhibitors of hepatitis C virus NS5B polymerase thumb domain site
II: Structure-based design and synthesis of new templates
*
Savina Malancona , Monica Donghi, Marco Ferrara, Josè I. Martin Hernando, Marco Pompei, Silvia Pesci,
Jesus M. Ontoria, Uwe Koch, Michael Rowley, Vincenzo Summa
IRBM - Merck Research Laboratories Rome, Via Pontina km 30,600, Pomezia (Rome) 00040, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Chronic hepatitis C virus (HCV) infections are a significant medical problem worldwide. The NS5B Poly-
merase of HCV plays a central role in virus replication and is a prime target for the discovery of new treat-
ment options. We recently disclosed 1H-benzo[de]isoquinoline-1,3(2H)-diones as allosteric inhibitors of
NS5B Polymerase. Structural and SAR information guided us in the modification of the core structure
leading to new templates with improved activity and toxicity/activity window.
Received 5 August 2009
Revised 8 March 2010
Accepted 11 March 2010
Available online 15 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HCV
NS5B polymerase
Non nucleoside inhibitors
1. Introduction
for this class of enzymes with finger, thumb and palm domains
that fully encircle the active site cavity.7 Several small molecules
The hepatitis C virus (HCV) is the principal etiological agent of
chronic hepatitis C infection,1 affecting 170 million people world-
wide. If untreated, more than 60% of these individuals will develop
chronic liver disease that leads to chronic hepatitis, liver cirrhosis
acting as allosteric inhibitors of NS5B have been disclosed during
the past years reflecting the major effort in this area from pharma-
ceutical industries and academia.8
Recently, we disclosed a series of 1H-benzo[de]isoquinoline-
1,3(2H)-diones as allosteric inhibitors of NS5B polymerase9 (Table
1). The lead compound 1a showed nanomolar activity on the main
NS5B polymerase genotypes (1b, 2b), while activity was in the
micromolar range in the cell based assays (Table 1). X-ray analysis
of 1a bound to both DC55_1b and DC21_2b enzymes revealed that
the inhibitor binds to the thumb site II. The tricyclic core interacts
with a flat lipophilic surface, the bromophenyl substituent makes
extensive Van der Waals and hydrophobic interactions, whereas
the imide oxygens and heteroatoms of the ethanolamine side chain
are involved in direct or water mediated H-bonds.9
The bromine present in 1a can be replaced with a chlorine as
in 1b without changing the activity profile both on the enzyme
and in the cell based assay. The structural information gained
with X-ray guided the SAR studies that culminated in the identi-
fication of the more active compound 2 in which the ethanol-
amine side chain had been substituted by a benzylpiperidine
moiety. Comparing 2 with the lead compounds 1a or 1b, it
showed a significant improvement in enzymatic activity for both
genotypes. Unfortunately, the improvement of the enzymatic
activity corresponded to a poor cellular response with slight sep-
aration (<3-fold) between EC50 and CC50. In this article we report
the work which has been done in our laboratories to overcome
the limitations of this series of HCV polymerase inhibitors via
modification of the core scaffold.
and hepatocellular carcinoma.2 The current therapy, pegylated
a-
interferon (IFN) alone or in combination with ribavirin, is poorly
tolerated. Additionally, it is of limited efficacy in patients infected
with HCV genotype 1, which accounts for about 70% of the infec-
tions in the western world.3 In response to these limitations, more
efficacious and better tolerated agents are needed.
HCV is a linear single-stranded enveloped RNA virus classified
in the hepacivirus genus of the flaviviridae family.4 Similarly to
other single-stranded RNA viruses, HCV lacks the ability to proof-
read and correct errors during replication. This accounts for the
highly heterogeneous viral genome classified in six major geno-
types (1–6) and a variety of subtypes. The expected onset of resis-
tance upon treatment with a single agent targeting a viral protein
and the possibility of pre-resistance in patients that harbor multi-
ple quasi-species, suggests the need for multiple drugs to effica-
ciously treat the viral infection.5 The NS5B RNA-dependent RNA
polymerase plays a central role in virus replication and is consid-
ered an attractive target for drug discovery efforts because it has
no counterpart in mammalian cells and so its inhibition is not ex-
pected to cause target-related side effects.6 The three-dimensional
structure of NS5B reveals the characteristic right hand architecture
* Corresponding author. Tel.: +39 06 89910222; fax: +39 06 91093756.
E-mail address: savinamal@libero.it (S. Malancona).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.024

S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
2837
Table 1
1H-Benzo[de]isoquinoline-1,3-diones as HCV polymerase inhibitors: activity profile of 1a,b and 2
O
O
Bn
N
Br
N
X
N
HO
N
H
O
O
N
H
2
1
a X:Br
b X:Cl
SIb
a
a
a
a
a
Compd
D
C21-1b IC50
(
lM)
D
C21-2b IC50
(l
M)
HBI10A EC50
(lM)
H2B2 EC50
(l
M)
HBI10A CC50
(lM)
1a
1b
2
0.020
0.039
<0.014
0.18
0.21
0.043
7.1
8.4
2.2
18
35
1.7
68
57
5.2
9.6
6.8
2.4
a
Values are means of two or more experiments with standard deviations <30% of the mean value.
SI = CC50 (HBI10A)/EC50 (HBI10A).
b
M423K. These results meant that the mechanism of action in the
enzyme assays is different for the two classes of compounds (1a,
2 vs 3, 4). In the cell based assay 3 and 4 were active in the low
micromolar range and they did not show a window between cyto-
toxicity and activity suggesting that their intercalation properties
are responsible for both activity and toxicity. In contrast the exis-
tence of a window between CC50 and EC50 in our compounds, could
offer the possibility to dial out the toxicity. For the reasons men-
tioned above going from 1a to 2 the observed increase in cytotox-
icity could be related to the introduction of the basic residue. In
order to improve the activity-toxicity window we designed tem-
plates able to maintain the key interactions of 1 with HCV Poly-
merases: the general strategy was to reduce the lipophilic
contact surface, the aromatic character to have less flat systems,
and introduce more polar fragments (Scheme 1). In order to iden-
tify an inhibitor with a broad spectrum of applicability we moni-
tored the activities for genotypes 1b and 2b both in enzymes and
cellular assays. The cytotoxicity was measured for genotype 1b
and the Selectivity Index was calculated as the ratio between
CC50 and EC50. This parameter was used to evaluate the activity-
toxicity window: when SI < 3 the compound is considered toxic.13
2. Results and discussion
2.1. Rationale for core structures modifications
We hypothesized that the observed cytotoxicity of 1H-
benzo[de]isoquinoline-1,3-(2H)-diones could be due to DNA inter-
calation properties of the scaffolds.10 Generally DNA intercalators
are characterized by typical features like a flat bi- or tricyclic frag-
ment acting as the intercalating part and the presence of a positive
charge (Fig. 1). The positive charge can be located on the ring itself
as for Ethidium bromide 3, giving interactions with the negative p-
electron density of the base pairs, or it can be located on a side
chain of the intercalating fragment, establishing interactions with
the sugar-phosphate backbone as for Doxorubicin 4 and Quinine
5. The intercalation induces local structural changes to DNA such
as lengthening of the DNA strand or twisting of the base pairs, thus
interfering with replication and leading to adverse biological ef-
fects like mutagenesis and cytotoxicity.11 Nevertheless, if the side
effects are tolerated and inferior to the benefit coming from the
treatment, molecules acting as DNA-intercalators are used in ther-
apies, like Quinine 5 as an antimalarial or Doxorubicin 4 used in
cancer therapy.12 We evaluated these known intercalators in our
assays and compared them with our inhibitors 1a and 2 (Table
2). The parameters considered were IC50, EC50 and CC50 for geno-
type 1b. We also included a very specific mutation M423K that
abolishes the activity of this class of compounds because it is lo-
cated in the pocket were the 3-bromophenyl substituent is deeply
buried. Compound 5 was inactive in all assays, proving that the
ability to intercalate DNA alone is not sufficient to cause toxicity
in the HCV cell based assay. Compounds 3 and 4 were both active
in the enzymatic assay, moreover they were active on the mutant
2.2. Chemistry
Quinazolinediones14 6a–e were synthesized according to the
route reported in Scheme 2 from the commercially available anthra-
nilic acid 12a. This was first reacted with phenyl chloroformate fol-
lowed by amide bond formation with 3-chloroaniline. Ring closure
to 13a was achieved in DMF under microwave irradiation in good
yield.15 After standard alkylation at N-1 (base/R1X) to furnish 14a,
the side chain was introduced by aromatic nucleophilic substitution
with amines under microwave irradiation. This methodology al-
lowed the synthesis of a number of analogs in moderate to good
yields. For the synthesis of 6f 2-amino-4-fluoro-3-methylbenzoic
acid16 12b was first converted to the methyl ester by treatment with
SOCl2 in MeOH and then reacted with 3-chlorophenylisocyanate.
The resulting intermediate was cyclised in basic media to afford
13b.17 Methylation at N-1 and introduction of the ethanolamine side
chain as previously described gave 6f.
Pyridoquinazolinedione 7a was prepared starting from 5-flu-
oroquinoline-8-carboxylic acid18 15 by reduction with H2/PtO2 in
acetic acid (Scheme 3). Unfortunately dehalogenation and decom-
position were prevalent side reactions that drastically reduced the
yield of the desired product. Attempts to optimize the reaction
conditions varying the solvent, the acid source and the catalyst,
did not improve the yield. In the next step esterification of the acid
gave 16. Ring formation to 17 was obtained by reaction with 3-
chlorophenylisocyanate followed by treatment with sodium
hydroxide.17 The ethanolamine side chain was introduced via
O
OH
O
Br
OH
OH
N
NH2
OH
O
OMe O
OH
O
H2N
4 Doxorubicin
3 Ethidium Bromide
Cl
NH3
N
HO
MeO
5 Quinine
N
Figure 1. Known DNA-intercalators.

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S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
Table 2
Profiling of Known DNA-intercalators in HCV assays
SIb
a
a
a
a
Compd
D
C21-1b IC50
(l
M)
D
C21 M423K IC50
(lM)
HBI10A EC50
(
lM)
HBI10A CC50 (lM)
1a
2
3
4
5
0.020
<0.014
0.81
NA at 2
ND
3.8
l
M
7.1
2.2
0.6
68
9.6
2.4
0.85
<1.0
—
5.2
0.7
<0.3
2.6
12
<0.3
NAc up to 50
lM
NAc up to 50
lM
NAc up to 20
lM
NAc up to 20
l
M
a
b
c
Values are means of two or more experiments with standard deviations <30% of the mean value.
SI = CC50 (HBI10A)/EC50 (HBI10A).
NA = Not active.
O
Ar
N
R
N
H
N
R2 R1
6a-f
O
R3
O
N
O
Ar
N
Cl
N
R
R
N
O
N
H
N
N
H
O
R2
N
7a-d
Ar
O
11a-b
N
R
N
H
1, 2
O
N
O
N
Ar
O
Ar
N
N
R
N
N
H
O
N
R
8a-b
Ar
10a,b
N
N
H
O
R
N
H
O
9a,b
Scheme 1. Core structure modifications.
O
N
N
O
Cl
a,b,c
CO2H
NH2
N
Cl
g
or d,e
O
X
O
X
N
H
X
R2 R1
R2
R2
14a R2 = H, R1 = Me, X = Br
14b R2 = Me, R1 = Me, X= F
12a X = Cl, R2 = H
12b X = F, R2 = Me
13a X = Cl, R2 = H
13b X = F, R2 = Me
f or h
f
6b-e R2 = H, R1 = Me
6f R2 = Me, R1 = Me
6a R2 = H
Scheme 2. Synthesis of compounds 6a–f. Reagents and conditions: (a) Phenylchloroformate, Py, THF, 0 °C to rt; (b) 3-chloroaniline, HOBt, DCC, NMM, THF; (c) DMF,
l
l
W
W
150 °C, 15 min; (d) SOCl2, MeOH, rt; (e) 3-chlorophenylisocyanate, Et2O, then NaOH, EtOH, rt; (f) RNH2, NMP,
200 °C, 30 min.
lW 170–200 °C; (g) MeI, DBU, DMF, rt; (h) RNH2, DBU, NMP,
fluorine displacement under microwave irradiation to afford 7a. An
alternative approach was explored to improve the low yielding
reduction step. 5-Bromoquinoline-8-carboxylic acid 18 was first
coupled with 3-chloroaniline, the amide was then reduced to
19a,b in the presence of sodium cyanoborohydride and BF3ÁEt2O
in high yield.19 A two step sequence furnished 20a,b. The introduc-
tion of the side chains by aromatic nucleophilic substitution gave
unsatisfactory results. Alternatively the side chain were introduced
using the more general palladium catalyzed amination in moder-
ate-low yield.
2,3-Dihydro-1H,5H-pyrazino[3,2,1-ij]quinazoline-5,7(6H)-
diones 8a,b were prepared starting from quinoxaline-5-carboxylic
acid20 21, which was first coupled with 3-chloroaniline to furnish
the corresponding amide and then partial ring reduction (NaBH4/
AcOH) gave 22.21 The side chains were introduced at this stage
by reductive amination. The tricyclic core was established as usual

S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
2839
O
O
CO2H
OMe
NH
N
Cl
a, b
c, d
F
N
e
F
7a
F
N
O
15
16
17
R3
R3
O
O
CO2H
N
H
Cl
f, g
N
Cl
h, i
Br
N
j, k
Br
NH
7b-d
Br
N
O
18
19a R3 = H;
19b R3 = CO2Me
20a R3 = H;
20b R3 = CO2Me
Scheme 3. Synthesis of compounds 7a–d. Reagents and conditions: (a) H2/PtO2, AcOH; (b) SOCl2, MeOH, reflux; (c) 3-chorophenylisocyanate, toluene, 90 °C; (d) NaOH, EtOH,
rt; (e) RNH2, NMP,
lW 120 °C, 2 h; (f) 3-chloroaniline, HATU, DIPEA, DCM, 16 h; (g) NaCNBH3, BF3ÁEt2O, 80 °C; (h) phenylchloroformate, DCE, 80 °C; (i) TEA, MeOH; (j) RNH2,
NaOtBu, BINAP, Pd2(dba)3, toluene, 80 °C, 16 h; (k) NaOH, dioxane, 40 °C.
upon treatment with phenylchloroformate and ring closure in ba-
sic media (Scheme 4).
against both genotypes. In the cell based assay 6b showed mar-
ginal activity only for genotype 1b. Other changes to the ethanol-
amine side chain or substitutions on N-1 with ethyl, benzyl, and
branched alkyl did not improve the activity (data not shown).
Keeping the methyl group at N-1, the introduction of the benzylpi-
peridine moiety, derived from the SAR on the lead 1, gave 6c with a
significant improvement in enzymatic activity showing nanomolar
activity for genotype 1b and low micromolar on genotype 2b. In
contrast the cellular assays gave a toxic response. Since the
increased toxicity could be related to the introduction of the basic
amino group, in compounds 6d and 6e the basic nitrogen was re-
moved and the lipophilicity reduced by the truncation of the sub-
stituent. Compared with 6c both compounds showed the expected
improvement in the selectivity index (more than fourfold for 6e)
but they lost in the enzymatic activity. These results showed that
the drastic reduction of the lipophilic surface area is responsible
for the loss in potency. We postulated that introduction of a methyl
substituent in the 8-position of the quinazoline ring as in 6f could
restore part of the surface area, and consequently restore the activ-
ity on the enzymes. Moreover steric strain between the two methyl
groups (at N-1 and C-8) should twist the molecule reducing the
planarity as evidenced in the superimposition of 1a and 6f
(Fig. 2). Unfortunately the designed molecule 6f exhibit an activity
profile similar to 6b.
The two methyl substituents of 6f were then constrained into a
saturated ring as outlined in compounds 7a–d: this modification
was made to increase the lipophilic contact surface area respect
to 6f and reduce the aromatic character of the original tricyclic core
scaffold 6b. Compound 7a with an ethanolamine side chain re-
sulted 12-fold more active than the analog 6f in the enzymatic as-
say for genotype 1b and eightfold for genotype 2b. Unfortunately,
this significant improvement in enzymatic potency for 7a did not
translate into an improved cellular response. In fact, this relatively
minor structural change led to an unexpected increase in cellular
toxicity. When the benzylpiperidine side chain was introduced
on this scaffold (7b), as expected we gained an excellent improve-
ment of the enzymatic activities comparing with 7a. More interest-
ingly the compound is in the low micromolar range in the cellular
assays and the selectivity index around six, meaning that in this
case we were able to separate the activity from cytotoxicity. To fur-
ther improve the window between EC50 and CC50, in compound 7c
the basic amine was removed from the side chain and the benzyl
moiety replaced by a carboxylic group.9 While 7c showed modest
potency, loosing both in enzymatic and cells assays and mainly
with respect to genotype 1b, it was less cytotoxic (SI = 8). The ben-
eficial effect of the carboxylic acid presence was not general since
Substituted anthranilic acids22 23a,b were converted to inter-
mediates 24a,b according to the chemistry previously described.
Side chains were introduced via transamidation of the ester 24a,
or upon methyl ester hydrolysis followed by amine coupling to
give 9a,b (Scheme 5).
In Scheme 6 is reported the synthesis we followed to prepare
compounds 10a,b and 11a,b. Substituted benzoic acids16 25a,b
were first coupled with 3-chloroaniline to give the amide followed
by nitro-reduction for 25b to furnish 26a,b. Ring closure with car-
bon disulfide gave the mercaptoquinazolones 27a,b. Compound
27a, upon treatment with SOCl2 followed by displacement of the
chlorine intermediate with dimethoxyethylamine and ring closure
in acid media, afforded 28 and then the desired side chains were
introduced (10a,b). Compound 27b was reacted with hydrazine
followed by ring closure upon heating in formic acid to give 29;
introduction of the side chains produced 11a,b.
2.3. Biological results
Quinazolinediones 6a–f (Table 3) were designed to have a re-
duced lipophilic surface area compared to the tricyclic lead 1a.
Modeling studies (data not shown) pointed out that 6b and 1b
adopt the same conformation and are engaged in the same interac-
tions. The main difference between the two compounds is the re-
duced lipophilic contact surface area. By calculation for genotype
1b, the total contact surface area of 6b is reduced by 16 Å2 with re-
spect to 1b.
In the SAR study compounds 6a–e maintain the meta-chloro-
phenyl substitution, while substitution at N-1 as well as the side
chains were varied. The N-1 unsubstituted 6a showed modest
enzymatic activity only on genotype 1b, whereas the N-methyl
analog 6b showed enzymatic activity in the micromolar range
O
CO2H
N
H
NH
Cl
a, b
N
c, d, e
8a-b
N
HN
21
22
Scheme 4. Synthesis of compounds 8a,b. Reagents and conditions: (a) 3-Chloro-
aniline, HATU, DIPEA, DCM; (b) NaBH4, AcOH; (c) RCHO, NaCNBH3, MeOH, AcOH;
(d) phenylchloroformate, DCE, 80 °C; (e) TEA, MeOH.

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S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
O
a,b,c
CO2H
NH2
f or g,h
or d,e
N
Cl
9a-c
O
N
H
R
R
23a R = CO2Me
23b R = H
24a R = CO2Me
24b R = H
Scheme 5. Synthesis of compounds 9a–d. Reagents and conditions: (a) 3-Chloroaniline, Et2O, rt; (b) phenylchloroformate, DCE, 80 °C, 2 h; (c) TEA, MeOH; (d) 3-
chlorophenylisocyanate, PE, reflux; (e) HCl (concd), EtOH, reflux; (f) RNH2, NMP,
lW 170–200 °C; (g) NaOH, MeOH; (h) RNH2, HATU, DIPEA, DCM.
O
O
O
Ar
Ar
S
OH
N
H
N
a or a+ b
c
Br
X
Br
NH2
Br
N
H
R2
R2
R2
26a R2 = H
26b R2 = Me
25a R2 = H, X = NO2
25b R2 = Me, X = NH2
27a R2 = H
27b R2 = Me
O
Ar
N
d, e, f
g
27a
10a,b
Br
N
N
28
O
Ar
N
g
h, i
Ar =
Cl
27b
11a,b
Br
N
N
N
29
Scheme 6. Synthesis of compounds 10a,b and 11a,b. Reagents and conditions: (a) 3-Chloroaniline, HATU, DIPEA, DCM; (b) Fe, AcOH, EtOH/H2O; (c) CS2, DBU, DMF; 45 °C; (d)
SOCl2, CHCl3, 80 °C; (e) 2,2-dimethoxyethylamine, DMF, 80 °C; (f) HCl, DMF; (g) RNH2, NMP,
THF, EtOH, 80 °C; (i) HCO2H, 120 °C.
lW 170 °C or RNH2, XANTPHOS, Cs2CO3, Pd2(dba)3, toluene, 80 °C; (h) NH2NH2/
other analogs in the series exhibited a lower selectivity index (data
not shown). The introduction of the carboxylic acid substituent on
the phenyl ring in the right portion of the molecule (7d) gave a
beneficial effect regarding the toxicity issue. Compared to 7b, com-
pound 7d was less potent in enzyme (20-fold for genotype 1b and
fivefold for 2b) and cellular assays (twofold for genotype 1b and
fourfold for 2b), but more importantly the window between toxic-
ity and activity was improved (SI = 10).
ethanolamine side chain attached to the amide nitrogen was
essentially inactive in all assays. Also for this series the benzylpi-
perazine analogue 9b showed a restored activity in enzymes, but
the compound was toxic.
All compounds discussed so far maintained the two carbonyl
groups of the imide portion of the lead. From the X-ray analysis
of 1a we knew that they are involved in H-bonds. We evaluated
structures like 10a,b and 11a,b in which an H-bond accepting car-
bonyl oxygen was replaced by a nitrogen still capable of forming
H-bonds but incorporated into a five membered ring. The modeling
of 10a (Fig. 3) suggested that two possible superimpositions with
compound 1a were possible. The red lines indicate unfavorable
interactions. In one orientation one of the two water molecules
can not be bound by the carbonyl O-atom and in the other orienta-
tion the imidazole C-H comes close to the Lys (501) side chain. In-
deed compounds 10a,b showed weak inhibitory activity: probably
this could be explained by the unfavorable interaction we have
postulated. To overcome this problem we investigated structures
like 11a,b in which the unfavorable interaction of the C–H of the
imidazole with the Lys residue has been suppressed by replacing
the imidazole with a triazole and a methyl substituent at C-8 has
been introduced to increase the lipophilic contact surface with
the protein. In fact 11a showed an increased enzymatic activity re-
spect to 10a for both genotypes. The compound is moderate active
in the cellular assay only for genotype 1b. The benzylpiperidine
analog 11b showed a peculiar profile in the enzymatic assays
showing a inverted selectivity between genotype 1b and 2b respect
to the compounds described in this article. 11b gave a toxic re-
sponse in cellular assays.
In a different design of the core structure, the nitrogen of the side
chain was incorporated into the saturated ring and the side chain
attached to it (compounds 8a–b). The first compound in this class,
8a having a propanol side chain to maintain the same chain length
of the lead, showed modest affinity for both enzymes but it was
essentially inactive in the cellular assays. Other analogs such as
8b were prepared but biological results were disappointing and
the scaffold did not show any advantages over the original lead.
Another designed template was based on the possibility that an
internal H-bond between the carbonyl moiety of an amide and the
N1–H of the quinazolinedione could mimic the second aromatic
ring of the original tricyclic scaffold. 2,4-Dioxo-1,2,3,4-tetrahydro-
quinazoline-8-carboxamides 9a,b were considered as hybrid struc-
tures between 6f and 1b. More specifically, the hypothesis was that
the amide in position 8 could form an intramolecular H-bond with
the N1–H of the quinazoline core ring, directing as a consequence
the amide side chain in the same direction as the ethanolamine
substituent does in 1a. This hypothesis was confirmed by evalua-
tion of the temperature coefficient (Dd/D
T) of N1–H.23 In spite of
the fact that compounds 9a,b have a similar conformation com-
pared to the lead 1a, the first analog 9a, which bears the classical

S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
2841
Table 3
Biological evaluation of the new scaffolds 6a–f, 7a–d, 8a–b, 9a–b, 10a–b and 11a–b
SIb
a
a
a
a
a
Compound
D
C21-1b IC50
M)
D
C21-2b IC50
M)
HBI10A EC50
M)
H2B2 EC50
M)
HBI10A CC50
M)
(l
(l
(
l
(
l
(l
O
N
N
Cl
Cl
6a
6b
6c
5.5
>50
8.2
1.3
>100
>100
>100
>25
>100
>100
9.9
—
HO
N
H
N
H
O
O
2.0
40
—
HO
Bn
N
H
N
O
N
O
N
N
Cl
Cl
0.075
11
0.9
N
O
H
O
N
6d
6e
5.2
3.0
2.3
5.0
22
24
16
36
1.6
>4
N
N
O
H
O
N
N
Cl
>100
>100
N
H
O
O
N
N
Cl
Cl
Cl
6f
2.4
5.3
40
>100
>100
>2.5
HO
N
H
N
O
O
O
7a
7b
0.20
0.67
0.14
15
26
12
10
0.8
5.9
HO
Bn
N
H
N
O
N
N
0.008
1.7
4.9
N
N
O
H
O
HO2C
N
Cl
7c
1.1
0.49
0.72
13
17
20
>100
7.8
N
N
O
H
CO2H
O
N
Bn
N
7d
0.16
4.1
42
10
N
Cl
N
H
O
O
N
N
Cl
8a
5.2
7.7
<100
>100
NAc
—
O
HO
N
(continued on next page)

2842
S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
Table 3 (continued)
SIb
a
a
a
a
a
Compound
D
C21-1b IC50
M)
D
C21-2b IC50
M)
HBI10A EC50
M)
H2B2 EC50
M)
HBI10A CC50
M)
(l
(l
(
l
(
l
(l
O
N
N
Cl
8b
0.13
2.0
27
>100
>100
>3.7
O
N
HO2C
O
N
Cl
9a
>10
>10
<100
NAc
NAc
—
N
H
O
N
HO
N
H
O
O
Cl
Cl
9b
Bn
0.076
0.73
4.0
6.6
7
1.8
N
N
H
O
N
O
H
O
N
N
10a
10b
>10
>10
>100
3.7
NAc
2.6
NAc
3.2
—
HO
Bn
N
H
N
O
N
N
Cl
0.84
>5.0
0.9
N
H
N
N
O
N
N
Cl
11a
11b
0.38
6.6
5.3
14
NAc
NAc
—
3
HO
Bn
N
H
N
N
O
N
N
Cl
0.40
3.0
<100
9.0
N
H
N
N
N
a
b
c
Values are means of two or more experiments with standard deviations <30% of the mean value.
SI = CC50 (HBI10A)/EC50 (HBI10A).
NA = Not active.
achieved. A beneficial effect on the selectivity index is observed
when a carboxylic acid is introduced (7c and 7d) in the right or left
hand side of the molecules. The encouraging results obtained could
be used to further develop this class of compounds.
3. Conclusions
In conclusion we have illustrated the work done to overcome
the limitations of 1H-benzo[de]isoquinoline-1,3(2H)-diones as
allosteric inhibitors of NS5B polymerase. The goals we wanted to
achieve were the increase of the activities and the dialing out the
toxicity related to the series. Several core scaffolds were synthe-
sized and evaluated. As observed for the original series the highest
affinity for the enzyme is conferred by the benzylpiperidinyl frag-
ment giving inhibitors in the nanomolar range (6c, 7b, 9b) on
genotype 1b. The activities for genotype 2b are generally lower
(exception 11b) than for 1b and the most active compound 7b is
threefold more active than the reference compound 2. The same
trend was observed for the activities in the cell based assays for
both genotypes. The structural changes we introduced did not im-
proved the potency in cell based assays: 7b is equipotent with 2,
but an improvement in the activity-toxicity window has been
4. Experimental
4.1. Chemistry
4.1.1. General
Solvents and reagents were obtained from commercial sources
and were used without further purification. With the exception of
routine deprotection and coupling steps, reactions were carried
out under an atmosphere of nitrogen in oven dried (110 °C) glass-
ware. Organic extracts were dried over sodium sulfate, and were
concentrated (after filtration of the drying agent) on rotatory

S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
2843
Figure 2. Superimposition of compounds 1a and 6f.
Figure 3. Superimpositions of compounds 1a and 10a.
evaporators operating under reduced pressure. Flash chromatogra-
phy was carried out on silica gel or on commercial flash chromatog-
raphy systems (Biotage corporation and Jones Flashmaster) utilising
pre-packed columns. Microwave irradiation was performed in a Per-
sonal Chemistry (now Biotage) Optimizer, Model Emrys. 1H NMR
spectra were recorded on Bruker Avance series spectrometers oper-
ating at 300 K and at (reported) frequencies between 300 and
600 MHz. Chemical shifts (d) for signals corresponding to non-
exchangeable protons (and exchangeable protons where visible)
are referenced to the residual proton signal of the deuterated solvent
(CDCl3 at 7.26 ppm; DMSO-d6 at 2.50 ppm; CD3CN at 1.94 ppm). Sig-
nals are tabulated in the order: multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; b, broad, and combinationsthereof);
coupling constant(s) in Hertz; number of protons. Mass spectral
(MS) data were obtained on a Perkin Elmer API 100 operating in neg-
ative (ESÀ) or positive (ES+) ionization mode and results are reported
as the ratio of mass over charge (m/z) for the parent ion only. Detec-
tion of High-Resolution Mass (HR-MS) was carried out using a SYN-
APT High Definition Mass Spectrometer equipped with an
electrospray interface (ESI). Source temperature was set at 120 °C.
Desolvation gas flow was set at 600 L/h. Identical source conditions
were used for all analytes. Detection of the ions was performed in the
TOF MS mode with a scan time of 100 ms. UPLC components were a
Waters binary pump, an Acquity UPLC Sample Manager, a PDA
Detector and an Acquity UPLC Sample Organizer kept refrigerated
at 4 °C during analysis, (Waters Corporation, USA). Both UPLC pump,
detector and autosampler were controlled by MassLynx version 4.1.
The same software was interfaced for subsequent data acquisition
and data analysis.
Automated (mass-triggered) RP-HPLC purifications were per-
formed using a Waters Micromass system incorporating a 2525
pump module, a Micromass ZMD detector, and a 2767 collection
module, operating under Fraction Lynx software. The stationary
phases used were Waters Symmetry C18
5
lm, 19 Â 100 mM

2844
S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
(Column A); Waters XTerra 5
l
m, 19 mm  100 mm (Column B).
(92 lL, 1.5 mmol). The reaction mixture was stirred at rt for 1 h.
The mobile phase comprised a linear gradient of binary mixtures
of MeCN (containing 0.1% TFA) (solvent A) and H2O (containing
0.1% TFA) (solvent B). The conditions used were as follows: 20% sol-
vent A (2 min) to 80% solvent A over 12 min, solvent A to 100% over
2 min, Column A (method 1); 40% solvent A (2 min) to 90% solvent
A over 12 min, solvent A to 100% over 2 min, Column A (method 2);
20% solvent A (2 min) to 80% solvent A over 12 min, solvent A to
100% over 2 min, Column B (method 3); 40% solvent A (2 min) to
90% solvent A over 12 min, solvent A to 100% over 2 min, Column
B (method 4). HPLC-MS were performed on a Waters Alliance
2795 apparatus, equipped with a diode array and a ZQ mass spec-
After dilution with water, the resulting precipitate was filtered
off affording the title compound (350 mg, 90%) as a white solid.
1H NMR (400 MHz, DMSO-d6) d 8.04 (d, J = 8.4 Hz, 1H), 7.65 (d,
J = 1.5 Hz, 1H), 7.53–7.49 (m, 3H), 7.39 (dd, J = 8.4, 1.5 Hz, 1H)
7.34–7.30 (m, 1H), 3.53 (s, 3H); MS (ES+) m/z 365, 367 (M+H)+.
4.1.2.4. 3-(3-Chlorophenyl)-7-[(2-hydroxyethyl)amino]-1-meth-
ylquinazoline-2,4(1H,3H)-dione (6b). Compound 14a (80 mg,
0.25 mmol) and 2-aminoethanol (0.15 mL, 2.55 mmol) in NMP
(0.8 mL) were irradiated in a microwave apparatus at 200 °C for
30 min. The reaction was diluted with MeCN, followed by purifica-
tion by RP-HPLC (method 1) affording the title compound (15 mg,
18%). 1H NMR (400 MHz, DMSO-d6) d 7.71 (d, J = 8.8 Hz, 1H), 7.51–
7.45 (m, 2H), 7.43–7.41 (m, 1H), 7.27–7.24 (m, 1H), 6.88 (bt,
J = 5.5 Hz, 1H), 6.58 (dd, J = 8.8, 1.9 Hz, 1H), 6.39 (d, J = 1.9 Hz,
1H), 4.80 (t, J = 5.3 Hz, 1H), 3.61 (td, J1 = J2 = 5.7 Hz, J3 = 5.2 Hz,
2H), 3.46 (s, 3H), 3.30–3.25 (m, 2H); HRMS (ES+) m/z calcd for
C17H17ClN3O3 (M+H)+: 346.0958; found: 346.0955.
trometer using an X-Terra C18 column (5
l
, 4.6 Â 50 mm). The mo-
bile phase comprised a linear gradient of binary mixtures of H2O
containing 0.1% formic acid (solvent A) and MeCN containing
0.1% formic acid (solvent B).
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 Acuity UPLC BEH C18
1.7
l
m, 2.1 mm  50 mm column as the stationary phase, and a
mobile phase comprising MeCN + 0.1% HCO2H (solvent A) and
H2O + 0.1% HCO2H (solvent B). Method 1: 30% solvent A to 100%
solvent A over 2.6 min, then 100% solvent A for 0.3 min. Method
2: 0% solvent A to 100% over 2.6 min and then 100% solvent A for
0.3 min. All the compounds described in this article showed puri-
ties higher than 95% in both analytical methods.
4.1.2.5. 7-[(1-Benzylpiperidin-4-yl)amino]-3-(3-chlorophenyl)-
1-methylquinazoline-2,4(1H,3H)-dione (6c). Prepared as de-
scribed for 6b from 14a (50 mg, 0.16 mmol), purification by RP-
HPLC (method 3) affording the title compound (8 mg, 11%) as a so-
lid. 1H NMR (400 MHz, DMSO-d6 + TFA) d 9.84 (br s, 1H), 7.93 (d,
J = 8.7 Hz, 1H), 7.76–7.64 (m, 10H), 7.49–7.41 (m, 1H), 6.78 (bd,
J = 8.7 Hz, 1H), 6.58 (s, 1H), 4.60–4.47 (m, 2H), 3.92 (m, 1H),
3.74–3.61 (m, 5H), 3.38–3.22 (m, 2H), 2.45–2.37 (m, 2H), 1.94–
1.80 (m, 2H); HRMS (ES+) m/z calcd for C27H28ClN4O2 (M+H)+:
475.1901; found: 475.1898.
4.1.2. General procedure for the synthesis of compounds 6a–f
4.1.2.1. 7-Chloro-3-(3-chlorophenyl)quinazoline-2,4(1H,3H)-dione
(13a). 2-Amino-4-chlorobenzoic acid (2.0 g, 11.7 mmol) was dis-
solved in dry THF (40 mL) and cooled to 0 °C. Pyridine (1.3 mL,
16.3 mmol) and phenyl chloroformate (1.8 mL, 14.0 mmol) were
added dropwise. The reaction mixture was stirred at 0 °C for 2 h.
After dilution with EtOAc the organic phase was filtered and washed
with 1 N HCl, brine and evaporated. The resulting crude was dis-
solved in THF (40 mL) and cooled to 0 °C. The solution was treated
with 3-chloroaniline (1.5 g, 11.9 mmol), HOBt (3.6 g, 23.8 mmol)
and NMM (1.3 mL, 11.9 mmol). After 30 min of stirring, DCC (2.5 g,
11.9 mmol) was added and the reaction mixture was stirred at
60 °C for4 h. Aftercoolingdown, thereactionwas dilutedwith water
and the resulting precipitate was filtered off and washed with Et2O,
affording phenyl (5-chloro-2-{[(3-chlorophenyl)amino]carbonyl}-
phenyl)carbamate as a pale yellow powder (1.65 g, 35%); MS (ES+)
m/z 401, 403 (M+H)+. This crude (0.4 g, 1.0 mmol) was dissolved in
DMF (5 mL) and irradiated in a microwave apparatus at 150 °C for
15 min. Water was added and the resulting precipitate was isolated
by filtration affording the title compound (250 mg, 80%) as an off-
white powder. 1H NMR (300 MHz, DMSO-d6) d 11.71 (s, 1H), 7.93
(d, J = 8.4 Hz, 1H), 7.52–7.50 (m, 3H), 7.35–7.25 (m, 3H); MS (ES+)
m/z 307, 309 (M+H)+.
4.1.2.6. 3-(3-Chlorophenyl)-1-methyl-7-[(4-methylcyclohexyl)-
amino]quinazoline-2,4(1H,3H)-dione (6d). Compound 14a
(70 mg,
0.22 mmol),
4-methylcyclohexanamine
(0.23 mL,
1.74 mmol) and DBU (0.039 mL, 0.26 mmol) in NMP (0.8 mL)
were irradiated in a microwave apparatus at 200 °C for 30 min.
The reaction was diluted with MeCN and was purified by RP-
HPLC (method 2) affording the title compound (26 mg, 30%) as
a solid in a mixture cis/trans (c/t) = 60/40. 1H-NMR (600 MHz,
DMSO-d6) d 7.70 (d, J = 8.7 Hz, 0.6H, c), 7.68 (d, J = 8.7 Hz, 0.4H,
t), 7.50–7.46 (m, 2H), 7.42–7.40 (m, 1H), 7.26–7.24 (m, 1H),
6.77–6.71 (br s, 1H), 6.61 (dd, J = 8.7, 1.7 Hz, 0.6H, c), 6.53 (dd,
J = 8.7, 1.7 Hz, 0.4H, t), 6.43 (d, J = 1.7 Hz, 0.6H, c), 6.34 (d,
J = 1.7 Hz, 0.4H, t), 3.69 (m, 0.6H, c), 3.44 (s, 3H), 3.46 (m, 0.4H,
t), 2.00–1.96 (m, 0.8H, t), 1.74–1.66 (m, 2H), 1.65–1.59 (m,
1.2H, c), 1.58–1.49 (m, 1.8H, c), 1.40–1.31 (m, 1.6H), 1.24–1.18
(m, 0.8H, t), 1.13–1.07 (m, 0.8H, t), 0.93 (d, J = 6.6 Hz, 1.8H, c),
0.09 (d, J = 6.6 Hz, 1.2H); HRMS (ES+) m/z calcd for C22H25ClN3O2
(M+H)+: 398.1635; found: 398.1637.
4.1.2.7. 3-(3-Chlorophenyl)-7-(isobutylamino)-1-methylquinaz-
oline-2,4(1H,3H)-dione (6e). Prepared as described for 6d from
14a (50 mg, 0.16 mmol); purification by RP-HPLC (method 2)
affording the title compound (12 mg, 22%) as a solid. 1H NMR
(300 MHz, DMSO-d6) d 7.76 (d, J = 8.7 Hz, 1H), 7.58–7.51 (m, 2H),
7.50–7.46 (m, 1H), 7.33–7.29 (m, 1H), 6.97 (bt, J = 5.8 Hz, 1H),
6.63 (dd, J = 8.7, 1.8 Hz, 1H), 6.41 (d, J = 1.8 Hz, 1H), 3.51 (s, 3H),
3.07 (bt, J = 5.8 Hz, 2H), 1.96 (m, 1H), 1.03 (d, J = 6.7 Hz, 6H); HRMS
(ES+) m/z calcd for C19H21ClN3O2 (M+H)+: 358.1322; found:
358.1323.
4.1.2.2. 3-(3-Chlorophenyl)-7-[(2-hydroxyethyl)amino]quinazo-
line-2,4(1H,3H)-dione (6a). Compound 13a (50 mg, 0.16 mmol)
and 2-aminoethanol (29 lL, 0.5 mmol) in NMP (0.5 mL) were irra-
diated in a microwave apparatus at 200 °C for 30 min. The reaction
was diluted with MeCN, followed by purification by RP-HPLC
(method 1) affording the title compound (3 mg, 5%). 1H NMR
(400 MHz, DMSO-d6) d 11.16 (s, 1H), 7.59 (d, J = 8.8 Hz, 1H),
7.50–7.42 (m, 3H), 7.28–7.24 (m, 1H), 6.81 (bt, J = 5.4 Hz, 1H),
6.50 (dd, J = 8.8, 1.8 Hz, 1H), 6.21 (d, J = 1.8 Hz, 1H), 4.79 (br s,
1H), 3.61–3.56 (m, 2H), 3.18–3.13 (m, 2H); HRMS (ES+) m/z calcd
for C16H15ClN3O3 (M+H)+: 332.0802; found: 332.0809.
4.1.2.8. 3-(3-Chlorophenyl)-7-fluoro-8-methylquinazoline-2,4-
(1H,3H)-dione (13b). 2-Amino-4-fluoro-3-methylbenzoic acid15
12b (130 mg, 0.77 mmol) was dissolved in MeOH (8 mL) and trea-
ted with SOCl2 (0.084 mL, 1.15 mmol). The reaction was stirred at
RT overnight, then the solvent was partially evaporated and the
4.1.2.3. 7-Chloro-3-(3-chlorophenyl)-1-methylquinazoline-2,4-
(1H,3H)-dione (14a). A solution of 13a (0.38 g, 1.2 mmol) in
DMF (5.5 mL) was treated with DBU (0.22 mL, 1.5 mmol) and MeI

S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
2845
residue partitioned between NaHCO3 (saturated solution) and
EtOAc. Separated organic layer was washed with NaHCO3 (satu-
rated solution), brine, dried and evaporated to afford the interme-
diate methyl 2-amino-4-fluoro-3-methylbenzoate (109 mg, 77%);
1H NMR (400 MHz, CDCl3) d 7.75 (dd, J = 8.7, 8.2 Hz, 1H), 6.34 (t,
J = 8.7 Hz, 1H), 6.71 (br s, 2H), 3.83 (s, 3H), 2.02 (s, 3H). The latter
compound (54 mg, 0.29 mmol) was dissolved in Et2O (1.0 mL)
and 3-chlorophenylisocyanate was added dropwise. The reaction
mixture was stirred 18 h at rt. Evaporation of the solvent gave a
residue that was disolved in EtOH (1.0 mL) and treated with 1 M
NaOH (0.5 mL). After stirring for 2 h at RT, the reaction was diluted
with water, acidified with 6 N HCl and the precipitate isolated by
filtration affording the title compound (85 mg, 94%) as a solid. 1H
NMR (400 MHz, DMSO-d6) d 11.06 (br s, 1H), 7.87 (dd, J = 8.8,
6.6 Hz, 1H), 7.54–7.49 (m, 3H), 7.36–7.32 (m, 1H), 7.08 (t,
J = 8.8 Hz, 1H), 2.30 (s, 3H); MS (ES+) m/z 304, 306 (M+H)+.
7.36 (m, 1H), 7.14 (t, J = 8.9 Hz, 1H), 3.90–4.00 (m, 2H), 2.82–2.91
(m, 2H); MS (ES+) m/z 331, 333 (M+H)+.
4.1.3.3. 2-(3-Chlorophenyl)-8-[(2-hydroxyethyl)amino]-6,7-di-
hydro-1H,5H-pyrido[3,2,1-ij]quinazoline-1,3(2H)-dione
(7a). Intermediate 17 (30 mg, 0.09 mmol) was dissolved in NMP
(1 mL) and treated with ethanolamine (33 lL, 0.54 mmol). The
reaction mixture was irradiated in a microwave apparatus at
200 °C for 2 h. The solution was purified by RP-HPLC (method 3)
affording the title compound (17 mg, 35%) as a white solid. 1H
NMR (300 MHz, DMSO-d6) d 7.72 (d, J = 8.8 Hz, 1H), 7.51–7.46
(m, 2H), 7.44–7.41 (m, 1H), 7.29–7.22 (m, 1H), 6.62 (d, J = 8.8 Hz,
1H), 5.90 (br s, 1H), 4.79 (br s, 1H), 3.92–3.87 (m, 2H), 3.59 (t,
J = 5.9 Hz, 2H), 3.39 (t, J = 5.9 Hz, 2H), 2.54–2.52 (m, 2H), 2.05–
1.96 (m, 2H); HRMS (ES+) m/z calcd for C19H19ClN3O3 (M+H)+:
372.1115; found: 372.1121.
4.1.2.9. 3-(3-Chlorophenyl)-7-fluoro-1,8-dimethylquinazoline-2,4-
(1H,3H)-dione (14b). Prepared as described for 14a from 13b
(82 mg, 0.27 mmol) to afford the title compound (80 mg, 93%) as
a solid. 1H NMR (300 MHz, DMSO-d6) d 7.95 (dd, J = 8.7, 6.6 Hz,
1H), 7.53–7.47 (m, 3H), 7.33–7.28 (m, 1H), 7.18 (t, J = 8.7 Hz, 1H),
3.58 (s, 3H), 2.46 (s, 3H); MS (ES+) m/z 318, 320 (M+H)+.
4.1.4. General procedure for the synthesis of compounds 7b–d
4.1.4.1. 5-Bromoquinoline-8-carboxylic acid (18). A solution of
8-methylquinoline (4.0 g, 28.0 mmol) in H2SO4 (80 mL) was trea-
ted with Ag2SO4 (13.1 g, 42 mmol) then bromine (4.5 g, 28 mmol)
was added. The reaction mixture was stirred at rt for 5 h and then
poured into ice and filtered. The resulting filtrate was basified with
aqueous Na2CO3 (saturated solution) and the aqueous phase was
extracted with EtOAc. The combined organic layers were evapo-
rated affording 5-bromo-8-methylquinoline (5.0 g, 71%) as a solid,
MS (ES+) m/z 222, 224 (M+H)+. The latter was dissolved in H2SO4/
H2O (2:3, 200 mL) and treated with CrO3 (22.4 g, 22 mmol) at
90 °C. The reaction mixture was heated at 90 °C for 2 h. Then, the
mixture was poured into ice and the precipitate was filtered
affording the title compound (3.54 g, 62%) as an orange solid. 1H
4.1.2.10. 3-(3-Chlorophenyl)-7-[(2-hydroxyethyl)amino]-1,8-di-
methylquinazoline-2,4(1H,3H)-dione (6f). Prepared as described
for 6b from 14b (86 mg, 0.27 mmol), purification by RP-HPLC
(method 1) afforded the title compound (4 mg, 4%) as a solid. 1H
NMR (600 MHz, DMSO-d6 + TFA) d 7.70 (d, J = 8.7 Hz, 1H), 7.49–
7.41 (m, 3H), 7.28–7.25 (m, 1H), 6.65 (d, J = 8.7 Hz, 1H), 3.60 (t,
J = 6.0 Hz, 2H), 3.45 (s, 3H), 3.31 (t, J = 6.0 Hz, 2H), 2.20 (s, 3H);
HRMS (ES+) m/z calcd for C18H19ClN3O3 (M+H)+: 360.1115; found:
360.112.
NMR (400 MHz, DMSO-d6)
d 9.25–9.13 (m, 1H), 8.82 (d,
J = 7.7 Hz, 1H), 8.47–8.37 (m, 1H), 8.23 (d, J = 7.7 Hz, 1H), 8.01–
7.91 (m, 1H); MS (ES+) m/z 252, 254 (M+H)+.
4.1.3. Synthesis of 7a
4.1.3.1. Methyl 5-fluoro-1,2,3,4-tetrahydroquinoline-8-carbox-
ylate (16). 5-Fluoroquinoline-8-carboxylic acid17 15 (612 mg,
3.22 mmol) in acetic acid (13 mL) was treated with PtO2 (219 mg,
0.97 mmol) and stirred under hydrogen at atmospheric pressure
for 3 h. The catalyst was filtered off and the solution was concen-
trated to dryness under reduced pressure to give a residue that
after purification by RP-HPLC (method 3) afforded 5-fluoro-
1,2,3,4-tetrahydroquinoline-8-carboxylic acid (253 mg, 40%) as a
white solid; 1H NMR (300 MHz, DMSO-d6) d 12.34 (br s, 1H), 8.06
(br s, 1H), 7.62 (dd, J = 8.7, 7.1 Hz, 1H), 6.26 (t, J = 8.9 Hz, 1H),
3.31–3.36 (m, 2H), 2.61–2.69 (m, 2H), 1.71–1.85 (m, 2H); MS
(ES+) m/z 196 (M+H)+. The latter compound (137 mg, 0.7 mmol)
was dissolved in MeOH (18 mL), treated with SOCl2 (1.4 mL,
19.6 mmol) and heated to reflux for 24 h. Volatiles were evapo-
rated under reduced pressure and the residue partitioned between
CH2Cl2 and aqueous NaHCO3 (saturated solution). The organic
layer was separated and dried. Evaporation of the solvent afforded
the title compound (118 mg, 80%) as a solid; MS (ES+) m/z 210
(M+H)+.
4.1.4.2. 5-Bromo-N-(3-chlorophenyl)-1,2,3,4-tetrahydroquinoline-
8-carboxamide (19a). Compound 18 (1.0 g, 3.94 mmol) in CH2Cl2
(40 mL) was treated with DIPEA (1.5 mL, 8.7 mmol), 3-chloroani-
line (0.13 mL, 4.33 mmol), and HATU (1.6 g, 4.33 mmol). The reac-
tion mixture was stirred at rt for 16 h. Then, it was treated with
aqueous NaHCO3 (saturated solution) and the organic phase sepa-
rated. The organic phase was washed with 1 N HCl, aqueous NaH-
CO3 (saturated solution) and dried. Evaporation of the solvent gave
a crude that was treated with Et2O. The solid obtained (1 g,
2.75 mmol) was filtered and dissolved in THF (30 mL). The result-
ing solution was treated with NaCNBH3 (0.52 g, 8.26 mmol) and
BF3ÁEt2O (1.05 mL, 8.26 mmol). The reaction mixture was heated
at 80 °C for 1 h, left to cool down and then treated with NH3 and
Et2O. The aqueous phase was extracted with Et2O. The combined
organic layers were dried and evaporated to give a crude which
was resubmitted to the same reaction conditions to afford after
workup the title compound (1.00 g, 70%) as a solid. 1H NMR
(400 MHz, DMSO-d6) d 11.18 (s, 1H), 7.90 (t, J = 2.1 Hz, 1H), 7.80
(br s, 1H), 7.61 (dd, J = 8.2, 2.1 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H),
7.36 (t, J = 8.3 Hz, 1H), 7.14 (dd, J = 8.2, 2.1 Hz, 1H), 6.78 (d,
J = 8.5 Hz, 1H), 3.30 (bt, J = 6.8 Hz, 2H), 2.74 (t, J = 6.4 Hz, 2H),
1.86–1.78 (m, 2H); MS (ES+) m/z 365, 367 (M+H)+.
4.1.3.2. 2-(3-Chlorophenyl)-8-fluoro-6,7-dihydro-1H,5H-pyrido-
[3,2,1-ij]quinazoline-1,3(2H)-dione (17). A solution of 16
(118 mg, 0.56 mmol) in toluene (2 mL) was treated with 1-
chloro-3-isocyanatobenzene (206
l
L, 1.68 mmol) and heated at
4.1.4.3. 8-Bromo-2-(3-chlorophenyl)-6,7-dihydro-1H,5H-pyrido-
[3,2,1-ij]quinazoline-1,3(2H)-dione (20a). To a solution of 19a
(1.00 g, 2.72 mmol) in 1,2-dicloroethane (25 mL) was added phenyl
chloroformate (0.34 mL, 2.72 mmol) and the reaction mixture was
stirred at 80 °C. After 1 h the solvent was evaporated and the crude
was dissolved in MeOH (25 mL). The resulting solution was treated
with Et3N (7.6 mL, 54.4 mmol) and heated at 80 °C for 30 min. The
solvent was evaporated and the crude washed with Et2O and
90 °C for 20 h. Solvent was evaporated under reduced pressure to
give a residue that was dissolved in ethanol/1 N NaOH (2:1,
3 mL). The resulting solution was stirred at rt for 3 h, and concen-
trated to dryness under reduced pressure to afford a residue which
was purified by RP-HPLC (method 2) affording the title compound
(65 mg, 35%) as a white solid. 1H NMR (300 MHz, DMSO-d6) d 7.94
(dd, J = 8.9 Hz, 6.4, 1H), 7.46–7.56 (m, 3H), 1.97–2.08 (m, 2H), 7.28–

2846
S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
filtered off affording the title compound (300 mg, 28%) as a solid.
1H NMR (400 MHz, DMSO-d6) d 7.81 (d, J = 8.5 Hz, 1H), 7.58–7.48
(m, 5H), 3.96 (dd, J = 7.5, 5.9 Hz, 2H), 2.93 (t, J = 6.7 Hz, 2H),
2.10–2.01 (m, 2H); MS (ES+) m/z 391, 393 (M+H)+.
for 2 h, then the mixture was diluted with EtOAc and H2O. The
aqueous phase was separated and the organic phase was washed
with brine and dried. Evaporation of the solvent afforded the title
compound (613 mg, 78%) as an orange solid. 1H NMR (400 MHz,
DMSO-d6) d 10.32 (s, 1H), 7.95 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H),
7.44 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 8.1 Hz, 1H), 7.16 (d, J = 7.8 Hz,
1H), 7.07 (d, J = 7.8 Hz, 1H), 6.71 (t, J = 7.8 Hz, 1H), 3.50–3.43 (m,
2H), 3.41–3.36 (m, 2H); MS (ES+) m/z 287, 289 (M+H)+.
4.1.4.4. 8-[(1-Benzylpiperidin-4-yl)amino]-2-(3-chlorophenyl)-
6,7-dihydro-1H,5H-pyrido[3,2,1-ij]quinazoline-1,3(2H)-dione
(7b). To a solution of 20a (20 mg, 0.05 mmol) in toluene (1 mL), 4-
amino-benzyl piperidine (0.012 mL, 0.06 mmol), NaOtBu (6.7 mg,
0.07 mmol), BINAP (0.93 mg, 0.0015 mmol) and Pd2(dba)3
(0.92 mg, 0.001 mmol) were added. The reaction mixture was
heated at 80 °C for 16 h. The solvent was evaporated and the resi-
due purified by RP-HPLC (method 3) affording the title compound
(5.1 mg, 20%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d 9.54
(br s, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.55–7.47 (m, 6H), 7.44–7.40 (m,
1H), 7.28–7.23 (m, 2H), 6.71 (d, J = 8.8 Hz, 1H), 5.77 (bd, J = 8.1 Hz,
1 H), 4.33 (d, J = 4.2 Hz, 2H), 3.92–3.88 (m, 2H), 3.71 (m, 1H), 3.46
(bd, J = 13.2 Hz, 2H), 3.14–3.06 (m, 2H), 2.53–2.51 (m, 2H), 2.12
(bd, J = 13.1 Hz, 2H), 2.04–1.96 (m, 2H), 1.83–1.73 (m, 2H); HRMS
(ES+) m/z calcd for C29H30ClN4O2 (M+H)+: 501.2057; found:
501.2064.
4.1.5.2. 6-(3-Chlorophenyl)-1-(3-hydroxypropyl)-2,3-dihydro-1H,-
5H-pyrazino[3,2,1-ij]quinazoline-5,7(6H)-dione (8a). A solution
of 22 (72 mg, 0.25 mmol) in MeOH (5 mL) was treated with 3-
{[tert-butyl(dimethyl)silyl]oxy}propanal (0.052 mL, 0.25 mmol),
NaBH3CN (15.7 mg, 0.25 mmol) and catalytic amount of AcOH.
The reaction mixture was stirred at rt overnight. Then, aqueous
NaHCO3 (saturated solution) was added and the aqueous phase ex-
tracted with EtOAc. The combined organic layers were dried and
evaporated to give a solid which was purified to give N-(3-chloro-
phenyl)-1-(3-hydroxypropyl)-1,2,3,4-tetrahydroquinoxaline-5-
carboxamide (MS (ES+) m/z 346,348 (M+H)+). The latter (20 mg,
0.058 mmol) was dissolved in 1,2-dichloroethane (5 mL). The
resulting solution was treated with phenyl chloroformate
(0.008 mL, 0.058 mmol). The reaction mixture was stirred at
80 °C for 2 h. Evaporation of the solvent gave a crude which was
dissolved in MeOH (5 mL) and treated with Et3N (0.08 mL,
0.58 mmol). The reaction mixture was heated at 80 °C for 30 min.
The solvent was evaporated and the residue purified by RP-HPLC
(method 3) affording the title compound (8 mg, 20%) as a white so-
lid. 1H-NMR (400 MHz, DMSO-d6) d 7.55–7.46 (m, 3H), 7.34–7.30
(m, 2H), 7.14 (t, J = 7.9 Hz, 1H), 7.08 (dd, J = 8.4, 1.2 Hz, 1H), 4.58
(br s, 1H), 4.02 (bt, J = 5.4 Hz, 2H), 3.51 (t, J = 6.1 Hz, 2H), 3.46–
3.42 (m, 4H), 1.76–1.69 (m, 2H); HRMS (ES+) m/z calcd for
C19H19ClN3O3 (M+H)+: 372.1115; found: 372.112.
4.1.4.5. cis-4-{[2-(3-Chlorophenyl)-1,3-dioxo-2,3,6,7-tetrahydro-
1H,5H-pyrido[3,2,1-ij]quinazolin-8-yl]amino}cyclohexanecar-
boxylic acid (7c). Prepared as described for 7b from intermediate
20a (100 mg, 0.25 mmol), affording after RP-HPLC purification
(method 3) the title compound (14 mg, 12%) as a solid. 1H NMR
(600 MHz, DMSO-d6) d 12.10 (s, 1H), 7.70 (d, J = 8.7 Hz, 1H),
7.50–7.47 (m, 2H), 7.43–7.41 (m, 1H), 7.26–7.24 (m, 1H), 6.67 (d,
J = 8.7 Hz, 1H), 5.60 (d, J = 8.3 Hz, 1H), 3.90–3.88 (m, 2H), 3.54
(m, 1H), 2.53–2.51 (m, 2H), 2.42 (tt, J1 = J2 = 12.2 Hz,
J3 = J4 = 3.3 Hz, 1H), 2.11 (bd, J = 12.7 Hz, 1H), 2.01–1.97 (m, 2H),
1.92–1.88 (m, 2H), 1.79 (dt, J1 = 13.5 Hz, J2 = J3 = 3.3 Hz, 1H),
1.45–1.39 (m, 2H), 1.35–1.19 (m, 2H); HRMS (ES+) m/z calcd for
C24H25ClN3O4 (M+H)+: 454.1534; found: 454.1547.
4.1.5.3. 3-{[6-(3-Chlorophenyl)-5,7-dioxo-2,3,6,7-tetrahydro-1H,-
5H-pyrazino[3,2,1-ij]quinazolin-1-yl]methyl}benzoic
acid
4.1.4.6. 3-[8-[(1-Benzylpiperidin-4-yl)amino]-1,3-dioxo-6,7-di-
hydro-1H,5H-pyrido[3,2,1-ij]quinazolin-2(3H)-yl]-5-chloroben-
zoic acid (7d). Prepared according to the procedure for 7b, from
20b (52 mg, 0.115 mmol) to afford methyl 3-(8-(1-benzylpiperi-
din-4-ylamino)-1,3-dioxo-6,7-dihydropyrido[3,2,1-ij]quinazolin-
2(1H,3H,5H)-yl)-5-chlorobenzoate. This was dissolved in dioxane
(1 mL) and treated with 2 M NaOH (0.2 mL, 0.4 mmol). After stir-
ring for 8 h at 40 °C, solvents were evaporated and the resulting
crude was purified by RP-HPLC (method 3) affording the title com-
pound (15 mg, 24%) as a solid. 1H NMR (400 MHz, DMSO-d6) d
13.40 (br s, 1H), 7.95 (s, 1H), 7.80 (s, 1H), 7.74 (s, 1H), 7.71 (d,
J = 8.9 Hz, 1H), 7.55–7.48 (m, 5H), 6.72 (d, J = 8.9 Hz, 1H), 5.79 (br
s, 1H), 4.43 (s, 2H), 3.96–3.87 (m, 2H), 3.72 (m, 1H), 3.50–3.41
(m, 2H), 3.16–3.03 (m, 2H), 2.12 (bd, J = 13.4 Hz, 2H), 2.06–1.97
(m, 2H), 1.86–1.71 (m, 2H); HRMS (ES+) m/z calcd for C30H30ClN4O4
(M+H)+: 545.1956; found: 545.1965.
(8b). Prepared according to the procedure described for 8a from
intermediate 22 (100 mg, 0.35 mmol) and methyl-3-formylbenzo-
ate (57 mg, 0.35 mmol) to obtain the intermediate methyl 3-{[6-
(3-chlorophenyl)-5,7-dioxo-2,3,6,7-tetrahydro-1H,5H-pyrazino[3,-
2,1-ij]quinazolin-1-yl]methyl}benzoate MS (ES+) m/z 462, 465
(M+H)+. This was dissolved in dioxane (3 mL) and treated with
2 M NaOH (0.2 mL, 0.4 mmol). After stirring for 8 h at rt, solvents
were evaporated and the resulting crude was purified by RP-HPLC
(method 3) affording the title compound (11 mg, 20%) as a white
solid. 1H NMR (400 MHz, DMSO-d6) d 12.98 (br s, 1H), 7.93 (s,
1H), 7.85 (d, J = 7.8 Hz, 1H), 7.64–7.43 (m, 5H), 7.40–7.26 (m,
2H), 7.11–7.01 (m, 2H), 4.69 (s, 2H), 4.15–4.05 (m, 2H), 3.62–3.52
(m, 2H); HRMS (ES+) m/z calcd for C24H19ClN3O4 (M+H)+:
448.1064; found: 448.1077.
4.1.6. General procedure for the synthesis of compounds 9a,b
4.1.6.1. Methyl 3-(3-chlorophenyl)-2,4-dioxo-1,2,3,4-tetrahy-
droquinazoline-8-carboxylate (24a). A solution of 2-amino-3-
(methoxycabonyl)benzoic acid21 23a (2.00 g, 10.2 mmol) in CH2Cl2
(60 mL) was treated with DIPEA (2.5 mL, 14.28 mmol), 3-chloroan-
iline (1.5 mL, 14.28 mmol), and HATU (4.26 g, 11.2 mmol). The
reaction mixture was stirred at rt for 16 h. Then, it was treated
with aqueous NaHCO3 (saturated solution) and the organic phase
separated. The organic phase was washed with 1 N HCl, aqueous
NaHCO3 (saturated solution) and dried. Evaporation of the solvent
gave a crude that was treated with PE-CH2Cl2. The precipitate was
filtered to give methyl 2-amino-3-{[(3-chlorophenyl)amino]car-
bonyl}benzoate (2.45 g, 77%); MS (ES+) m/z 305, 307 (M+H)+. This
intermediate (2.45 g, 8 mmol) was dissolved in 1,2-dicloroethane
(50 mL) and phenylchloroformate (1 mL, 8 mmol) was added. The
4.1.5. General procedure of compounds 8a–b
4.1.5.1. N-(3-Chlorophenyl)-1,2,3,4-tetrahydroquinoxaline-5-car-
boxamide (22). A solution of quinoxaline-5-carboxylic acid19 21
(500 mg, 2.87 mmol) in CH2Cl2 (30 mL) was treated with DIPEA
(1.1 mL, 6.32 mmol), 3-chloroaniline (0.33 mL, 3.16 mmol) and
HATU (1.20 g, 3.16 mmol). The reaction mixture was stirred at rt
overnight. The solution was diluted with EtOAc and the organic
phase was washed with aqueous NaHCO3 (saturated solution)
and dried. Evaporation of the solvent gave a crude that was washed
with MeCN and filtered off affording N-(3-chlorophenyl)quinoxa-
line-5-carboxamide (775 mg, 93%), MS (ES+) m/z 284, 286
(M+H)+. The latter was dissolved in AcOH (30 mL) and treated with
NaBH4 (201 mg, 5.46 mmol). The reaction mixture was stirred at rt

S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
2847
reaction mixture was stirred at 80 °C. After 2 h the solvent was
evaporated and the crude was dissolved in MeOH (50 mL). The
resulting solution was treated with Et3N (11 mL, 80 mmol). The
reaction mixture was heated at 80 °C for 30 min. The solvent was
evaporated and the crude washed with Et2O-PE and filtered off
affording the title compound (2.95 g, 100%) as a solid. 1H-NMR
(400 MHz, DMSO-d6) d 10.81 (s, 1H), 8.35 (dd, J = 7.8, 1.3 Hz, 1H),
8.28 (dd, J = 7.9, 1.4 Hz, 1H), 7.58–7.53 (m, 3H), 7.42–7.36 (m,
2H), 3.97 (s, 3H); MS (ES+) m/z 332, 334 (M+H)+.
4.1.7. General procedure for the synthesis of compounds 10a,b
and 11a,b
4.1.7.1. 2-Amino-4-bromo-N-(3-chlorophenyl)benzamide (26a).
To a solution of 4-bromo-2-nitrobenzoic acid 25a (2.10 g, 8.5 mmol)
in CH2Cl2 (40 mL), 3-chloroaniline (0.99 mL, 9.39 mmol), DIPEA
(3.25 mL, 18.7 mmol) and HATU (3.60 g, 9.39 mmol) were added.
The reaction mixture was stirred at rt for 16 h and then treated with
aqueous NaHCO3 (saturated solution). The organic phase was sepa-
rated, washed with 1 N HCl and dried. Evaporation of the solvent
gave a solid that was washed with Et2O affording 4-bromo-N-(3-
chlorophenyl)-2-nitrobenzamide (2.00 g, 67%) as a white solid. 1H
NMR (400 MHz, DMSO-d6) d 10.89 (s, 1H), 8.40 (s, 1H), 8.13 (d,
J = 8.1 Hz, 1H), 7.85 (s, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.51 (d,
J = 8.2 Hz, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H); MS
(ES+)m/z355, 357, 359(M+H)+. Thelatter(2.00 g, 5.6 mmol)wasdis-
solved in EtOH/H2O/AcOH (12:1:0.008) (130 mL). Iron (6.25 g,
112 mmol) was added and the resulting suspension was heated to
66 °C for 5 h. The resulting precipitated was filtered and the filtrated
concentrated. The residue was dissolved in EtOAc and the resulting
organic phase was washed with aqueous NaHCO3 (saturated solu-
tion), brine and dried over Na2SO4. Evaporation of the solvent gave
the title compound (1.37 g, 76%) as a solid; MS (ES+) m/z 325, 327,
329 (M+H)+.
4.1.6.2. 3-(3-Chlorophenyl)-N-(2-hydroxyethyl)-2,4-dioxo-1,2,3,4-
tetrahydroquinazoline-8-carboxamide (9a). A solution of 24a
(50 mg, 0.15 mmol) in NMP (3 mL) was treated with 2-aminoeth-
anol (92.4 mg, 1.51 mmol). The reaction mixture was irradiated
in a microwave apparatus at 170 °C for 300 s. The solution was
purified by RP-HPLC (method 1) affording of the title compound
(6 mg, 11%) as a solid. 1H NMR (600 MHz, DMSO-d6) d 11.85 (s,
1H), 9.02 (t, J = 5.8 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.14 (d,
J = 7.8 Hz, 1H), 7.56–7.50 (m, 3H), 7.39–7.32 (m, 2H), 4.82 (br
s, 1H), 3.56 (t, J = 5.8 Hz, 2H), 3.39 (q, J = 5.8 Hz, 2H); HRMS
(ES+) m/z calcd for C17H15ClN3O4 (M+H)+: 360.0751; found:
360.0754.
4.1.6.3. N-(1-Benzylpiperidin-4-yl)-3-(3-chlorophenyl)-2,4-dioxo-
1,2,3,4-tetrahydroquinazoline-8-carboxamide (9b). To a solu-
tion of 24a (55 mg, 0.17 mmol) in MeOH (3 mL) was added 2 N
NaOH (0.17 mL, 0.34 mmol). The reaction mixture was stirred at
rt for 6 h. After evaporation of the solvent the residue was dis-
solved in EtOAc and the resulting solution was washed with 1 N
HCl, dried. Evaporation of the solvent gave 3-(3-chlorophenyl)-
2,4-dioxo-1,2,3,4-tetrahydroquinazoline-8-carboxylic acid, MS
(ES+) m/z 317, 319 (M+H)+. This intermediate (50 mg, 0.16 mmol)
was dissolved in CH2Cl2 (5 mL) and treated with 4-amino benzylpi-
peridine (0.039 mL, 0.19 mmol), DIPEA (0.033 mL, 0.19 mmol) and
HATU (66.2 mg, 0.17 mmol). The reaction mixture was stirred at rt
for 48 h. After dilution with CH2Cl2, the resulting solution was
washed with water and dried. Evaporation of the solvent gave a
crude that was purified by RP-HPLC (method 1) affording the title
compound (33 mg, 35%) as a solid. 1H NMR (400 MHz, DMSO-d6) d
11.60 (s, 1H), 9.54 (br s, 1H), 9.01 (d, J = 7.6 Hz, 1H), 8.24 (dd,
J = 7.8, 1.3 Hz, 1H), 8.17 (d, J = 7.8 Hz, 1H), 7.56–7.49 (m, 8H),
7.39–7.33 (m, 2H), 4.32 (s, 2H), 4.07 (m, 1H), 3.44 (bd,
J = 12.2 Hz, 2H), 3.20–3.10 (m, 2H), 2.13 (bd, J = 12.3 Hz, 2H),
1.87–1.76 (m, 2H); HRMS (ES+) m/z calcd for C27H26ClN4O3
(M+H)+: 489.1693; found: 489.1702.
4.1.7.2. 7-Bromo-3-(3-chlorophenyl)-2-thioxo-2,3-dihydroqui-
nazolin-4(1H)-one (27a). Compound 26a (1.37 g, 4.21 mmol)
was dissolved in DMF (4 mL) and added slowly to a solution of
CS2 (6 mL) and DBU (626 mL, 4.2 mmol) in DMF (9 mL) at rt. The
resulting suspension was heated at 45 °C for 30 min and then at
rt for 16 h. The reaction mixture was poured in 1 N HCl and the
resulting precipitated was collected and washed with EtOH and
PE affording of the title compound (920 mg, 60%) as a yellow solid.
1H NMR (400 MHz, DMSO-d6) d 13.11 (s, 1H), 7.87 (d, J = 8.3 Hz,
1H), 7.62 (s, 1H), 7.53–7.47 (m, 4H), 7.30 (d, J = 6.8 Hz, 1H); MS
(ES+) m/z 367, 369, 371 (M+H)+.
4.1.7.3. 8-Bromo-4-(3-chlorophenyl)imidazo[1,2-a]quinazolin-
5(4H)-one (28). To a solution of 27a (320 mg, 0.87 mmol) in
CHCl3 (5 mL) at rt, SO2Cl2 (0.035 mL, 0.43 mmol) was added and
the resulting solution was heated at 80 °C. After 3 h the solution
was poured into ice, CH2Cl2 was added and the organic phase
was separated, washed with water and dried over Na2SO4.
Evaporation of the solvent gave 7-bromo-2-chloro-3-(3-chloro-
phenyl)quinazolin-4(3H)-one (150 mg, 46%), MS (ES+) m/z 369,
371, 373 (M+H)+. The latter was dissolved in EtOH (2 mL). The
resulting solution was treated with 2,2-dimethoxyethanamine
(0.26 mL, 2.4 mmol) and Et3N (0.33 mL, 2.4 mmol). The reaction
mixture was heated at 80 °C. After 30 min the solvent was evapo-
rated, water was added and the aqueous phase extracted with
EtOAc. The combined organic phase was dried and evaporated to
give a residue which was disolved in DMF/HClconc (2:1) (3 mL).
The resulting solution was heated at 80 °C for 1 h. After cooling
down it was purified by RP-HPLC (method 3) affording the title
compound (50 mg, 33%) as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 8.52 (br s, 1H), 8.22 (br s, 1H), 8.12 (d, J = 8.6 Hz,
1H), 7.73 (d, J = 8.6 Hz, 1H), 7.64 (s, 1H), 7.60–7.40 (m, 3H), 7.03
(s, 1H); MS (ES+) m/z 374, 376, 378 (M+H)+.
4.1.6.4. 3-(3-Chlorophenyl)quinazoline-2,4(1H,3H)-dione (24b).
Methyl 2-aminobenzoate (9.05 g, 66.2 mmol) were dissolved in PE
(50 mL) at rt and 1-chloro-3-isocyanatobenzene (15.1 g, 99.3 mmol)
was added dropwise. The mixture was then stirred overnight at
reflux. The reaction was cooled down and the solid filtered off. The
residue was triturated with clean PE and dried overnight under
vacuum affording methyl 2-({[(3-chlorophenyl)amino]carbonyl}-
amino)benzoate (19.0 g, 95%); MS (ES+) m/z 305, 307 (M+H)+. The
latter was dispersed in absolute EtOH (90 mL) and concentrated
HCl (60 mL) were added dropwise at rt. The mixture was stirred at
reflux for 6 h, a clear solution was obtained. The mixture was cooled
down and a solid started to precipitate. The precipitate was filtered
off, washed extensively with Et2O and dried at vacuum overnight,
affording the title compound (13.8 g, 77%) as off-white solid. 1H
NMR (600 MHz, DMSO-d6) d 11.59 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H),
7.71 (t, J = 8.1 Hz, 1H), 7.53–7.49 (m, 3H), 7.35–7.32 (m, 1H), 7.25–
7.21 (m, 2H); MS (ES+) m/z 273, 275 (M+H)+).
4.1.7.4. 4-(3-Chlorophenyl)-8-[(2-hydroxyethyl)amino]imidazo-
[1,2-a]quinazolin-5(4H)-one (10a). A solution of 28 (15 mg,
0.04 mmol) in NMP (0.5 mL) was treated with 2-aminoethanol
(24.4 mg, 0.4 mmol). The reaction mixture was irradiated in a
microwave apparatus at 170 °C 70 min. The solution was purified
by RP-HPLC (method 3) affording the title compound (2 mg, 14%)

2848
S. Malancona et al. / Bioorg. Med. Chem. 18 (2010) 2836–2848
as a white solid. 1H NMR (400 MHz, CD3CN) d 7.97 (d, J = 8.7 Hz,
1H), 7.70 (d, J = 1.5 Hz, 1H), 7.58–7.48 (m, 3H), 7.42–7.39 (m,
1H), 7.01 (d, J = 1.5 Hz, 1H), 6.82 (d, J = 1.9 Hz, 1H), 6.77 (dd,
J = 8.7, 1.9 Hz, 1H), 3.78–3.72 (m, 3H), 3.42 (t, J = 5.4 Hz, 1H),
3.07–3.05 (m, 2H); HRMS (ES+) m/z calcd for C18H16ClN4O2
(M+H)+: 355.0962; found: 355.0965.
3.49–3.47 (m, 2H), 3.14–3.08 (m, 2H), 2.50 (s, 3H; overlapped by
residual protonic DMSO signal), 2.19–2.14 (m, 2H), 1.86–1.79 (m,
2H); HRMS (ES+) m/z calcd for C28H28ClN6O (M+H)+: 499.2013;
found: 499.2006.
Acknowledgments
4.1.7.5. 8-[(1-Benzylpiperidin-4-yl)amino]-4-(3-chlorophenyl)-
imidazo[1,2-a]quinazolin-5(4H)-one (10b). To a solution of 28
(35 mg, 0.089 mmol) in toluene (1 mL) were added 4-amino-
benzylpiperidine (0.022 mL, 0.107 mmol), NaOtBu (12 mg,
0.125 mmol), XANTPHOS (15.5 mg, 0.027 mmol) and Pd2(dba)3
(16.3 mg, 0.018 mmol). The reaction mixture was heated at 80 °C
for 2 h. After evaporation of the solvent the residue was purified
by RP-HPLC (method 3) affording the title compound (30 mg,
70%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d 9.71 (br s,
1H), 8.04 (br s, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.61–7.49 (m, 8H),
7.44–7.41 (m, 1H), 7.14 (bd, J = 7.1 Hz, 1H), 7.04 (br s, 1H), 6.91
(d, J = 1.8 Hz, 1H), 6.76 (dd, J = 8.8, 1.8 Hz, 1H), 4.38 (s, 2H), 3.70
(m, 1H), 3.53 (bd, J = 12.1 Hz, 2H), 3.10–3.01 (m, 2H), 2.23 (bd,
J = 13.2 Hz, 2H), 1.72–1.62 (m, 2H); HRMS (ES+) m/z calcd for
C28H27ClN5O (M+H)+: 484.1904; found: 484.1908.
We thank Maria Cecilia Palumbi for automated RP-HPLC purifi-
cation; Nadia Gennari and Monica Bisbocci for biological testing;
Giacomo Paonessa and Sergio Altamura for the experiment on
the mutat; Maria Vittoria Orsale and Claudio Giuliano for HR-MS
analysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.024.
References and notes
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7.28 (m, 1H), 2.61 (s, 3H); MS (ES+) m/z 381, 383, 385 (M+H)+.
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