Scaffold hopping and optimisation of 3’,4’-dihydroxyphenyl- containing thienopyrimidinones: synthesis of quinazolinone derivatives as novel allosteric inhibitors of HIV-1 reverse transcriptase-associated ribonuclease H

Article abstract of DOI:10.1080/14756366.2020.1835884

Bioisosteric replacement and scaffold hopping are powerful strategies in drug design useful for rationally modifying a hit compound towards novel lead therapeutic agents. Recently, we reported a series of thienopyrimidinones that compromise dynamics at the p66/p51 HIV-1 reverse transcriptase (RT)-associated Ribonuclease H (RNase H) dimer interface, thereby allosterically interrupting catalysis by altering the active site geometry. Although they exhibited good submicromolar activity, the isosteric replacement of the thiophene ring, a potential toxicophore, is warranted. Thus, in this article, the most active 2-(3,4-dihydroxyphenyl)-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one 1 was selected as the hit scaffold and several isosteric substitutions of the thiophene ring were performed. A novel series of highly active RNase H allosteric quinazolinone inhibitors was thus obtained. To determine their target selectivity, they were tested against RT-associated RNA-dependent DNA polymerase (RDDP) and integrase (IN). Interestingly, none of the compounds were particularly active on (RDDP) but many displayed micromolar to submicromolar activity against IN.

Full text of DOI:10.1080/14756366.2020.1835884

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Scaffold hopping and optimisation of 3’,4’-
dihydroxyphenyl- containing thienopyrimidinones:
synthesis of quinazolinone derivatives as novel
allosteric inhibitors of HIV-1 reverse transcriptase-
associated ribonuclease H
Graziella Tocco , Francesca Esposito , Pierluigi Caboni , Antonio Laus , John
A. Beutler , Jennifer A. Wilson , Angela Corona , Stuart F. J. Le Grice & Enzo
Tramontano
To cite this article: Graziella Tocco , Francesca Esposito , Pierluigi Caboni , Antonio Laus ,
John A. Beutler , Jennifer A. Wilson , Angela Corona , Stuart F. J. Le Grice & Enzo Tramontano
(2020) Scaffold hopping and optimisation of 3’,4’-dihydroxyphenyl- containing thienopyrimidinones:
synthesis of quinazolinone derivatives as novel allosteric inhibitors of HIV-1 reverse transcriptase-
associated ribonuclease H, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
1953-1963, DOI: 10.1080/14756366.2020.1835884
To link to this article: https://doi.org/10.1080/14756366.2020.1835884
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1953–1963
https://doi.org/10.1080/14756366.2020.1835884
RESEARCH PAPER
Scaffold hopping and optimisation of 3’,4’-dihydroxyphenyl- containing
thienopyrimidinones: synthesis of quinazolinone derivatives as novel allosteric
inhibitors of HIV-1 reverse transcriptase-associated ribonuclease H
Graziella Toccoa , Francesca Espositoa , Pierluigi Cabonia , Antonio Lausa, John A. Beutlerb
Jennifer A. Wilsonb, Angela Coronaa , Stuart F. J. Le Gricec and Enzo Tramontanoa
,
b
aDepartment of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, Cagliari, Italy; Molecular Targets
c
Program, National Cancer Institute, Frederick, MD, USA; Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA
ABSTRACT
ARTICLE HISTORY
Received 20 August 2020
Revised 8 October 2020
Accepted 8 October 2020
Bioisosteric replacement and scaffold hopping are powerful strategies in drug design useful for rationally
modifying a hit compound towards novel lead therapeutic agents. Recently, we reported a series of thie-
nopyrimidinones that compromise dynamics at the p66/p51 HIV-1 reverse transcriptase (RT)-associated
Ribonuclease H (RNase H) dimer interface, thereby allosterically interrupting catalysis by altering the active
site geometry. Although they exhibited good submicromolar activity, the isosteric replacement of the thio-
phene ring, a potential toxicophore, is warranted. Thus, in this article, the most active 2-(3,4-dihydroxy-
phenyl)-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one 1 was selected as the hit scaffold and several
isosteric substitutions of the thiophene ring were performed. A novel series of highly active RNase H allo-
steric quinazolinone inhibitors was thus obtained. To determine their target selectivity, they were tested
against RT-associated RNA-dependent DNA polymerase (RDDP) and integrase (IN). Interestingly, none of
the compounds were particularly active on (RDDP) but many displayed micromolar to submicromolar
activity against IN.
KEYWORDS
Bioisosters; RNase H
allosteric inhibitors; HIV-1
virus; RNase H; integrase
Introduction
Many site-directed mutagenesis studies have demonstrated the
essential role of the RNase H domain in virus infectivity, also
indicating that the retrovirus-associated activity is unlikely to be
supplemented by a host enzyme9,10. This observation suggests RT-
associated RNase H as an attractive target that may open opportu-
nities for new systematic HIV-drug discovery efforts11–15. In fact,
several RNase H inhibitors have been developed over the last two
decades, with promising results against drug-resistant variants16–20
the majority of which belonging to the class of active site inhibi-
tors21. Unfortunately, many representatives of this class can be
easily displaced by the nucleic acid substrate22 and this drawback
has made the progression of RNase H active site inhibitors
towards clinical trials challenging. Consequently, attention moved
to a less populated class of compounds able to allosterically tackle
the viral RNase H function. Such an approach appeared extremely
advantageous since the inhibition of related host enzymes, espe-
cially those of the polynucleotidyl phosphotransferase class, might
be avoided. In this regard, we have recently reported a series of
differently decorated thienopyrimidinones, that compromise
dynamics at the p66/p51 HIV-1 RT-RNase H dimer interface and
consequently interrupting catalysis by altering the active
site geometry.11
Since the beginning of the AIDS epidemic, almost 80 million peo-
ple have been infected with human immunodeficiency virus, type
1 (HIV-1), and currently an estimated 38 million people are
infected worldwide1. Unfortunately, a treatment that eradicates
the virus from infected people2 or a vaccine3 is still not available.
At present, the challenge of a successful antiretroviral therapy is
to convert a terminal disease into a manageable chronic infection
by reducing HIV-1 levels in the blood (viral load). In this regard,
the advent of the combination active antiretroviral therapy (cART)
represents a major achievement in reducing mortality and morbid-
ity of HIV-1 infected patients4. Nowadays, although 27 FDA-
approved drugs are available for the treatment of AIDS5, antiviral
drug discovery has not waned. In fact, the selection and spread of
HIV-1 variants resistant to current therapies represent the major
clinical problem in the fight against AIDS6–8, justifying the increas-
ing demand for new drugs to reinforce the cART arsenal. For that
reason, the design of new molecules that target key enzymes of
the viral lifecycle with an innovative mode of action could have a
tremendous impact on the fight against HIV/AIDS. Reverse tran-
scriptase (RT) together with integrase (IN) and protease (PR), is
one of the three viral enzymes essential for HIV-1 replication. DNA
polymerase and Ribonuclease H (RNase H) are the two enzymatic
activities of RT which catalyse conversion of single-stranded viral
Interestingly, all these compounds, in contrast to active site
RNase H inhibitors, were able to destabilise HIV-1 RT both in the
RNA into integration-competent, double-stranded linear DNA. absence and presence of the nucleic acid substrate, decreasing
CONTACT Graziella Tocco
toccog@unica.it
Department of Life and Environmental Sciences, Unit of Drug Science, University of Cagliari, Cittadella Universitaria
di Monserrato, Monserrato, Cagliari 09042, Italy
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1954
G. TOCCO ET AL.
Table 1. Inhibition of wild type and p66/p51C280A mutant HIV-1 RNase H activ-
the melting temperature of the enzyme in some cases by more
than 5 ꢀC. In addition, by using a panel of drug-resistant and
-sensitive recombinant enzymes we could identify several thieno-
pyrimidinone derivatives which retained activity against drug-
resistant variants and 2–(3,4-dihydroxyphenyl)-5,6-dimethylth-
ieno[2,3-d]pyrimidin-4(3H)-one 1 emerged as the best compound
(wt RT RNase H IC50 ¼ 0.26 lM and C280A RT RNase H IC50
¼ 0.32 lM)12.
ity by compounds 2–17.
OH
CORE
OH
IC50 (mM)
Compound
CORE
WT
C280A
212
0.41 0.01
0.50 0.02
0.57 0.02
0.73 0.02
0.37 0.01
0.37 0.01
O
Since bioisosterism is a well-known strategy for rational lead
optimisation in drug discovery23 particularly to improve pharma-
cological activity, we applied it in an effort to enhance the thieno-
pyrimidinone 1 biological effects. The initial candidate, 2–(3,4-
dihydroxyphenyl)-6-methylquinazolin-4(3H)-one 2, showed promis-
ing activities, inhibiting HIV-1 RNase H in a submicromolar range
(IC50 ¼ 0.41 mM) (Figure 1 and Table 1). From a structural perspec-
tive, our studies indicated that 3,4-dihydroxyphenyl moiety was
crucial for activity of every inhibitor, while the bioisosteric substi-
tution of the thiophene core with a 6-methyl phenyl unit as in
compound 2 slightly affected the activity12 (Figure 1). In fact, since
the thiophene ring is a potential toxicophore24, with a strict trop-
ism for liver, kidneys, and immune system25, its isosteric replace-
ment in biologically active molecules is desirable.
Similar to thienopyrimidinones, we speculated that decorations
of the benzene ring might positively affect inhibitory activity since
the recently reported26 2–(3,4-dihydroxyphenyl)-quinazolin-4(3H)-
one (17) is an 8-fold less potent HIV-1 RNase H inhibitor. Based
on this evidence, we elected to investigate the introduction of dif-
ferent groups on the phenyl ring in order to establish their inhibi-
tory role. A total of 22 new compounds were synthesised and
tested against wt and C280A RT, also investigating their mode of
action by means of magnesium-complexation and differential
scanning fluorimetry experiments. Furthermore, since many com-
pounds are reported to be dual inhibitors of different HIV-1 func-
tions27,28, to determine target specificity, we tested the effect of
the entire set of quinazolinone-based derivatives on RDDP and IN
activities. We also tested the compounds in a cell-based anti-
viral system.
H3C
NH
NH
N
O
3
4
5
6
0.23 0.02
0.41 0.01
0.33 0.003
0.29 0.006
H3C
N
O
H3CO
NH
N
O
H3CO
H3CO
NH
NH
N
O
N
O
7
1.1 0.04
0.51 0.05
0.12 0.001
0.40 0.01
0.20 0.01
0.66 0.05
0.84 0.03
0.17 0.004
0.40 0.03
NH
N
O
8
0.15 0.01
0.45 0.008
0.31 0.01
0.43 0.01
0.52 0.06
0.31 0.01
0.56 0.25
I
NH
N
O
9
Br
Cl
NH
NH
NH
NH
Material and methods
N
O
Chemistry
10
11
12
13
14
All reagents were purchased from commercial sources and used
without further purification. TLC performed on Aldrich silica gel 60
F254 (0.25 mm) with detection by UV was used for reaction moni-
toring. 1H and 13 C NMR spectra were recorded on a Varian Unity
Inova 500 MHz spectrometer. High-resolution mass spectra were
recorded using an Agilent 6520 LC-QTOF-MS system.
N
O
Cl
F
N
O
General procedure for arylpyrimidinone core formation
A mixture of the appropriate 2-amino arylamide (1.14 mmol),
molecular iodine (1.48 mmol), and the appropriate aromatic alde-
hyde (1.37 mmol) in the presence of acetonitrile as solvent (20 ml)
was stirred at room temperature for 6 h (TLC). The reaction was
N
O
O2N
Cl
NH
NH
N
O
O
H3C
H3C
H3C
O
NH
NH
OH
OH
OH
OH
S
N
N
N
H
2
1
15
0.64 0.03
0.39 0.01
(continued)
Figure 1. Bioisosteric core replacement.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1955
13C NMR (500 MHz, DMSO): d 162.21, 154.45, 151.16, 148.12,
135.89, 129.09, 122.23, 119.00, 118.34, 116.78, 115.57, 114.38,
57.08 ppm. HRMS calculated for C16H14N2O5 314.09, found 314.10.
2–(3,4-dihydroxyphenyl)-6-phenylquinazolin-4(3H)-one (6). Yield
Table 1. Continued.
IC50 (mM)
Compound
CORE
WT
C280A
O
1
(%) ¼ 77. H NMR (500 MHz, DMSO): d 12.35 (s, 1H), 9.69 (bs, 1H),
O
NH
9.29 (bs, 1H), 8.37 (s, 1H), 8.07 (d, J ¼ 7.0 Hz, 1H), 7.67–7.56 (m,
3H), 7.45 (s, 1H), 7.37–7.28 (m, 3H), 6.83 (dd, J ¼ 6.7 Hz, 2H) ppm.
13C NMR (500 MHz, DMSO): d 165.00, 155.61, 148.23, 141.09,138.12,
135.64, 135.20, 134.88, 133.56, 129.76,128.23, 127.22, 126.88,
124.67, 122.34, 120.54, 118.22, 117.11 ppm. HRMS calculated for
C20H14N2O3 330.10, found 330.07.
N
O
16
2.4 1.6
2.0 0.1
NH
N
N
O
2–(3,4-dihydroxyphenyl)benzo[g]quinazolin-4(3H)-one (7). Yield
1727
3.46 0.47
–
1
(%) ¼ 80. H NMR (500 MHz, DMSO): d 12.02 (s, 1H), 9.52 (bs, 1H),
9.23 (bs, 1H), 8.43 (d, J ¼ 7.2 Hz, 2H), 8.23 (d, J ¼ 7.5 Hz, 1H), 8.11
(d, J ¼ 7.5 Hz, 1H), 7.32 (s, 1H), 7.03 (t, J ¼ 7.5 Hz, 1H), 6.98 (t,
J ¼ 7.5 Hz, 1H), 6.84 (d, J ¼ 8.1 Hz, 1H), 6.73 (d, J ¼ 8.1 Hz, 1H) ppm.
13C NMR (500 MHz, DMSO): d 160.00, 155.66, 153.09, 150.12,
147.89, 145.43, 140.06, 138.24, 133.56, 132.16, 130.87, 126.11,
122.34, 120.56, 114.98, 111.22 ppm. HRMS calculated for
C18H12N2O3 304.08, found 304.07.
NH
N
quenched with 40 ml of 5% Na2S2O3 solution, and the resulting
precipitate was filtered off. The crude product was washed with n-
hexane/ethyl acetate (1:1) and recrystallised from ethanol to give
the final arylpyrimidinone as pure product.
For compounds 4 and 5, the corresponding 2-amino arylamide
was prepared as follow.
2–(3,4-dihydroxyphenyl)-6-iodoquinazolin-4(3H)-one (8). Yield (%)
1
¼ 87. H NMR (500 MHz, DMSO): d 11.56 (s, 1H), 9.45 (bs, 1H), 9.27
(bs, 1H), 8.35 (s, 1H), 8.03 (d, J ¼ 8.0 Hz, 1H), 7.67(d, J ¼ 8.0 Hz, 1H),
7.13 (s, 1H), 6.83 (d, J ¼ 7.0 Hz, 1H), 6.78 (d, J ¼ 7.0 Hz, 1H) ppm.
13 C NMR (500 MHz, DMSO):
d 162.00, 160.54,154.27, 147.12,
To a magnetically stirred suspension of stannous chloride
(24.68 mmol) in concentrated HCl (37%) (9.7 ml), the appropriate
2-nitrobenzamide (8.52 mmol) was added in about 1 h, maintain-
ing the temperature of the slurry below 5 ꢀC. After addition was
complete, the mixture was stirred for 30 h at room temperature. A
white slurry was obtained which was diluted with cold water
(130 ml) and then treated with aqueous NaOH 40% (40 ml) to dis-
solve the tin salt. The mixture was extracted with ethyl acetate
(4 ꢁ 100 ml), and the extracts dried on Na2SO4 and concentrated
under vacuum to obtain the pure 2-amino arylamide.
146.04, 145.76,138.59, 134.73, 127.17, 122.22, 119.19, 116.15,
110.90 ppm. HRMS calculated for C14H9IN2O3 379.97, found 379.97.
2–(3,4-dihydroxyphenyl)-6-bromoquinazolin-4(3H)-one (9). Yield
1
(%) ¼ 79. H NMR (500 MHz, DMSO): d 12.11 (s, 1H), 9.56 (bs, 1H),
9.33 (bs, 1H), 8.15 (s, 1H), 7.98 (d, J ¼ 8.2 Hz, 1H), 7.87 (d,
J ¼ 8.2 Hz, 1H), 7.22 (s, 1H), 6.95 (d, J ¼ 7.5 Hz, 1H), 6.76 (d,
J ¼ 7.5 Hz, 1H) ppm. 13 C NMR (500 MHz, DMSO): d 162.00, 155.21,
148.22, 147.04, 145.15, 144.17, 137.09, 134.07, 125.13, 121.22,
118.00, 116.00, 100.10 ppm. HRMS calculated for C14H9BrN2O3
331.98 found 331.97.
2–(3,4-dihydroxyphenyl)-6-methylquinazolin-4(3H)-one (2). Yield
2–(3,4-dihydroxyphenyl)-6-chloroquinazolin-4(3H)-one (10). Yield
1
1
(%) ¼ 78. H NMR (500 MHz, DMSO): d 12.12 (s, 1H), 9.59 (bs, 1H),
(%) ¼ 90. H NMR (500 MHz, DMSO): d 12.34 (s, 1H), 9.47 (bs, 1H),
9.29 (bs, 1H), 7.91 (s, 1H), 7.67(s, 1H), 7.61 (d, J ¼ 8.0 Hz, 1H),
7.57–7.52 (dd, J ¼ 7.1 Hz, 2H), 6.83 (d, J ¼ 8.0 Hz, 1H), 2.44 (s, 3H)
ppm. 13 C NMR (500 MHz, DMSO): d 161.17, 155.29, 148.12, 147.01,
146.76, 145.18, 137.56, 133.71, 128.17, 122.43,120.11, 117.17,
116.15, 21.01 ppm. HRMS calculated for C15H12N2O3 268.08,
found 268.07.
9.38 (bs, 1H), 8.02(s, 1H), 7.78 (d, J ¼ 8.0 Hz, 1H), 7.67 (s, 1H), 7.54
(d, J ¼ 7.5 Hz, 1H), 6.83 (d, J ¼ 8.0 Hz, 1H), 6.79 (d, J ¼ 7.5 Hz, 1H)
ppm. 13 C NMR (500 MHz, DMSO): d 162.34, 156.27, 151.01, 149.04,
147.66, 145.89, 137.12, 135.77, 123.00, 121.45, 118.50, 111.76 ppm.
HRMS calculated for C14H9ClN2O3 288.03 found 288.02.
2–(3,4-dihydroxyphenyl)-7-chloroquinazolin-4(3H)-one (11). Yield
1
2–(3,4-dihydroxyphenyl)-7-methylquinazolin-4(3H)-one (3). Yield
(%) ¼ 87. H NMR (500 MHz, DMSO): d 12.45 (s, 1H), 9.82 (bs, 1H),
1
(%) ¼ 68. H NMR (500 MHz, DMSO): d 12.06 (s, 1H), 9.11 (bs, 1H),
9.42 (bs, 1H), 8.20 (d, J ¼ 10 Hz, 1H), 7.79 (d, J ¼ 8.9 Hz, 1H), 7.77 (s,
1H), 7.67 (d, J ¼ 10 Hz, 1H), 7.60 (d, J ¼ 8.9 Hz, 1H), 6.95 (d,
J ¼ 8.9 Hz, 1H) ppm. 13 C NMR (500 MHz, DMSO): d 162.31, 156.12,
150.99, 149.54, 147.16, 145.87, 137.22, 135.58, 123.16, 121.75,
118.50, 110.60 ppm. HRMS calculated for C14H9ClN2O3 288.03
found 288.03.
8.48 (bs, 1H), 7.98 (d, J ¼ 7.5 Hz, 1H), 7.66 (s, 1H), 7.53 (d,
J ¼ 8.0 Hz, 1H), 7.46 (s, 1H), 7.27 (d, J ¼ 8.0 Hz, 1H), 6.83 (d,
J ¼ 7.5 Hz, 1H), 2.45 (s, 3H) ppm. 13 C NMR (500 MHz, DMSO): d
161.23, 155.45 148.67, 147.11, 146.56, 145.01, 137.58, 133.88,
128.16, 122.42,120.01, 117.00, 116.15, 20.56 ppm. HRMS calculated
for C15H12N2O3 268.08, found 268.09.
2–(3,4-dihydroxyphenyl)-6-fluoroquinazolin-4(3H)-one (12). Yield
1
2–(3,4-dihydroxyphenyl)-6-methoxyquinazolin-4(3H)-one (4). Yield
(%) ¼ 93. H NMR (500 MHz, DMSO): d 12.46 (s, 1H), 9.76 (bs, 1H),
1
(%) ¼ 73. H NMR (500 MHz, DMSO): d 12.16 (s, 1H), 9.56 (bs, 1H),
9.39 (bs, 1H), 7.88 (d, J ¼ 9.0 Hz, 1H), 7.85 (d, J ¼ 8.0 Hz, 1H), 7.79
(s, 1H), 7.77 (d, J ¼ 9.0 Hz, 1H), 7.66 (d, J ¼ 8.0 Hz, 1H), 6.94 (d,
J ¼ 8.0 Hz, 1H) ppm. 13 C NMR (500 MHz, DMSO): d 160.11, 157.22,
151.39, 149.67, 147.28, 145.88, 137.02, 136.56, 123.12, 121.79,
119.00, 111.00 ppm. HRMS calculated for C14H9FN2O3 272.06
found 272.05.
9.23 (bs, 1H), 7.65 (s, 1H), 7.61 (d, J ¼ 8.5 Hz, 1H), 7.51 (d,
J ¼ 6.0 Hz, 1H), 7.40 (d, J ¼ 8.5 Hz, 1H), 6.82 (d, J ¼ 6.0 Hz, 1H), 3.88
(s, 3H) ppm. 13C NMR (500 MHz, DMSO): d 162.11, 153.99, 151.14,
148.78, 135.88, 129.00, 122.33, 119.10, 118.36, 116.11, 115.57,
114.38, 57.04 HRMS calculated for C15H12N2O4 284.08, found
284. 10.
2–(3,4-dihydroxyphenyl)-6-nitroquinazolin-4(3H)-one (13). Yield
1
2–(3,4-dihydroxyphenyl)-6,7-dimethoxyquinazolin-4(3H)-one
(5).
(%) ¼ 84. H NMR (500 MHz, DMSO): d 12.66 (s, 1H), 9.49 (bs, 1H),
1
Yield (%) ¼ 74. H NMR (500 MHz, DMSO): d 12.07 (s, 1H), 9.60 (bs, 8.78 (bs, 1H), 8.42 (d, J ¼ 7.5 Hz, 1H), 8.09 (d, J ¼ 8.0 Hz, 1H), 7.80
1H), 9.19 (bs, 1H), 7.63 (s, 1H), 7.50 (d, J ¼ 7.0 Hz, 1H), 7.43 (s, 1H), (s, 1H), 7.78 (d, J ¼ 7.5 Hz, 1H), 7.64 (d, J ¼ 8.0 Hz, 1H), 6.86 (d,
7.10 (s, 1H), 6.81 (d, J ¼ 7.0 Hz, 1H), 3.90 (s, 3H), 3.85 (s, 3H) ppm. J ¼ 7.5 Hz, 1H) ppm. 13 C NMR (500 MHz, DMSO): d 162.01, 155.12,

1956
G. TOCCO ET AL.
151.67, 150.53, 148.12, 146.88, 139.14, 136.58, 123.00, 121.00, dihydroxybenzaldehyde (0.91 mmol) were dissolved in a mixture
118.10, 115.00 ppm. HRMS calculated for C14H9N3O5 299.05 of dichloromethane (10 ml) and acetonitrile (7 ml) and refluxed for
found 299.05.
40 h. After completion, the solvent was removed under vacuum.
The solid residue was treated with water, filtered and then
washed again with water to give compound 14 as a pale yellow
crystal. Yield (%) ¼ 82. 1H NMR (500 MHz, DMSO): d 9.42 (s, 1H),
8.47 (bs, 1H), 8.38 (bs, 1H), 7.99 (s, 1H), 7.85 (d, J ¼ 7.0 Hz, 1H),
7.54 (d, J ¼ 7.5 Hz, 1H), 6.93 (s, 1H), 6.77 (d, J ¼ 7.5 Hz, 1H), 6.44 (d,
J ¼ 7.5 Hz, 1H),6.29 (bs, 1H), 6.13 (s, 1H) ppm. 13 C NMR (500 MHz,
DMSO): d 159.78 149.27, 147.11, 145.64, 137.76, 135.99, 132.00,
124.75, 122.10, 121.00, 116.70, 110.75, 80.05 ppm. HRMS calculated
for C14H11ClN2O3 290.05 found 290.05.
2–(3,4-dihydroxyphenyl)benzofuro[3,2-d]pyrimidin-4(3H)-one (15).
1
Yield (%) ¼ 91. H NMR (500 MHz, DMSO): d 12.73 (s, 1H), 9.65 (bs,
1H), 9.31 (bs, 1H), 8.08 (d, J ¼ 8.2 Hz, 1H), 7.82 (d, J ¼ 7.5 Hz, 1H),
7.69–7.65 (m, 2H), 7.52 (s, 1H), 6.96 (d, J ¼ 7.5 Hz, 1H), 6.85 (d,
J ¼ 8.2 Hz, 1H) ppm. 13 C NMR (500 MHz, DMSO): d 162.26, 149.89,
146.12, 135.86, 133.22, 123.36, 122.12, 121.09, 120.14, 118.90,
118.21, 113.45 ppm. HRMS calculated for C16H10N2O4 294.06
found 294.05.
2–(3,4-dihydroxyphenyl)pyrido[2,3-d]pyrimidin-4(3H)-one
(16).
1
Yield (%) ¼ 81. H NMR (500 MHz, DMSO): d 12.45 (s, 1H), 9.57 (bs,
1H), 9.01 (bs, 1H), 8.77 (d, J ¼ 7.8 Hz, 1H), 8.32 (d, J ¼ 7.8 Hz, 1H),
7.70 (s, 1H), 7.58 (t, J ¼ 7.8 Hz, 1H), 7.12 (d, J ¼ 8.0 Hz, 1H), 6.86 (d,
J ¼ 8.0 Hz, 1H) ppm. 13 C NMR (500 MHz, DMSO): d 160.91, 159.14,
155.87, 146.22, 139.88, 138.15, 135.76, 133.33, 122.10, 120.00,
117.45, 114.10 ppm. HRMS calculated for C13H9N3O3 255.06
found 255.06.
2–(3,4-dihydroxyphenyl)quinazolin-4(3H)-one (17). Yield (%) ¼ 93.
1H NMR (500 MHz, DMSO): d 12.20 (s, 1H), 9.63 (bs, 1H), 9.28 (bs,
1H), 8.11 (d, J ¼ 9.0 Hz, 1H), 7.79 (d, J ¼ 9.0 Hz, 1H), 7.67(s, 1H), 7.65
(t, J ¼ 9.0 Hz, 2H), 7.43 (d, J ¼ 8.0 Hz, 1H), 6.85 (d, J ¼ 9.0 Hz, 1H),
ppm. 13 C NMR (500 MHz, DMSO): d 161.00, 159.17, 157.12, 149.72,
145.43, 138.12, 134.96, 133.31, 122.33, 120.00, 116.42, 114.15 ppm.
HRMS calculated for C14H10N2O3 254.07 found 254.06.
2-hydroxy-4-(4-oxo-3,4-dihydroquinazolin-2-yl)benzoic acid (18).
Yield (%) ¼ 89. 1H NMR (500 MHz, DMSO): d 13.12 (bs, 1H), 12.15
(s, 1H), 11.06 (bs, 1H), 8.22 (d, J ¼ 8.0 Hz, 1H), 7.91 (d, J ¼ 8.0 Hz,
1H), 7. 71 (s, 1H), 7.54 (t, J ¼ 8.0 Hz, 2H), 7.33 (d, J ¼ 7.5 Hz, 1H),
6.98 (d, J ¼ 7.5 Hz, 1H), ppm. 13 C NMR (500 MHz, DMSO): d 172.00,
160.17, 159.24, 151.15, 148.63, 145.12, 139.64, 133.96, 133.11,
121.33, 119.00, 116.22, 111.78 ppm. HRMS calculated for
C15H10N2O4 282.06 found 282.04.
2-(4-amino-3-hydroxyphenyl)quinazolin-4(3H)-one (19). Yield (%)
¼ 83. 1H NMR (500 MHz, DMSO): d 12.34 (s, 1H), 10.03 (bs, 1H),
8.03 (d, J ¼ 7.5 Hz, 1H), 7.67 (d, J ¼ 7.5 Hz, 1H), 7.54 (s, 1H), 7.11 (t,
J ¼ 7.5 Hz, 1H), 7.02 (t, J ¼ 7.5 Hz, 1H), 6.81 (d, J ¼ 8.0 Hz, 1H), 6.76
(d, J ¼ 8.0 Hz, 1H), 5.90 (bs, 2H) ppm. 13 C NMR (500 MHz, DMSO): d
159.20, 157.19, 155.32, 147.79, 144.67, 141.12, 135.00, 122.33,
121.14, 118.65, 116.22, 113.17 ppm. HRMS calculated for
C14H11N3O2 253.09 found 253.10.
2–(3,4-diaminophenyl)quinazolin-4(3H)-one (20). Yield (%) ¼ 82.
1H NMR (500 MHz, DMSO): d 11.98 (s, 1H) 8.25 (d, J ¼ 8.0 Hz, 1H),
8.09 (d, J ¼ 8.0 Hz, 1H), 7.68 (s, 1H), 7.43 (t, J ¼ 8.0 Hz, 2H), 7.04 (d,
J ¼ 7.4 Hz, 1H), 6.76 (d, J ¼ 7.4 Hz, 1H), 5.45 (bs, 2H), 5.34 (bs, 2H)
ppm. 13 C NMR (500 MHz, DMSO): d 163.09, 160.00, 159.12, 152.07,
148.41, 139.12, 136.00, 135.31, 123.13, 121.00, 117.33, 113.18 ppm.
HRMS calculated for C14H12N4O 252.10 found 252.09.
2–(3-hydroxy-4-nitrophenyl)quinazolin-4(3H)-one (21). Yield (%) ¼
92. 1H NMR (500 MHz, DMSO): d 12.21 (s, 1H), 8.13 (d, J ¼ 7.5 Hz,
1H), 7.97 (d, J ¼ 7.5 Hz, 1H), 7.34 (s, 1H), 7.15 (t, J ¼ 7.5 Hz, 1H),
7.00 (t, J ¼ 7.5 Hz, 1H), 6.91 (d, J ¼ 8.0 Hz, 1H), 6.80 (d, J ¼ 8.0 Hz,
1H), 6.25 (bs, 2H) ppm. 13 C NMR (500 MHz, DMSO): d 159.70,
156.13, 154.32, 147.43, 145.68, 142.24, 135.10, 126.37, 122.56
117.85, 115.00, 111.17, 110.67 ppm. HRMS calculated for
C14H10N4O3 282.08 found 282.08.
Synthesis of 4-(quinolin-2-yl)benzene-1,2-diol (22). To a mixture
of (3,4-dimethoxyphenyl)boronic acid (3.29 mmol), K2CO3
(3.29 mmol), and Pd(OAc)2 (0.027 mmol) in ethanol (9 ml) and
water (3 ml) was slowly added 2-bromoquinoline (2.74 mmol). The
mixture was stirred at room temperature for 18 h. To the resulting
mixture was added water (50 ml), and then extracted with
dichloromethane. The organic phase was separated, dried over
Mg2SO4 and concentrated under reduced pressure to give a solid
residue which was purified by flash chromatography (silica gel
and hexane/acetone 3/1). The pure 2–(3,4-dimethoxyphenyl)quino-
line was obtained as light grey solid and used for the next reac-
tion. Yield (%) ¼ 92. 1H NMR (500 MHz, DMSO): d 8.76 (d,
J ¼ 8.0 Hz, 1H), 8.11 (d, J ¼ 7.0 Hz, 1H), 7.93 (d, J ¼ 7.0 Hz, 1H), 7.77
(t, J ¼ 7.0 Hz, 1H), 7.68 (s, 1H), 7.59 (d, J ¼ 7.5 Hz, 1H), 7.55 (t,
J ¼ 7.0 Hz, 1H),7.29 (d, J ¼ 8.0 Hz, 1H), 7.02 (d, J ¼ 7.0 Hz, 1H), 3.85
(s, 6H) ppm. 13 C NMR (500 MHz, DMSO): d 156.00, 153.12, 145.12,
135.82, 130.27, 128.00, 125.26, 122.30, 119.33, 117.01, 115.40,
113.13, 57.89 ppm. HRMS calculated for C17H15NO2 265.11
found 265.10.
To
a stirred solution of 2–(3,4-dimethoxyphenyl)quinoline
(0.604 mmol) in dichloromethane (4 ml) at 0 ꢀC was added boron
tribromide 1 M solution in dichloromethane (9.66 mmol). After
1.5 h, the resulting mixture was stirred for 4 more hours at room
temperature. The resulting mixture was the poured into ice water
and the yellow residue obtained was dissolved in ethanol. The
mixture was then extracted with dichloromethane and the organic
phase, previously dried over anhydrous MgSO4, was evaporated to
give compound 22 as a yellow solid. Yield (%) ¼ 91. 1H NMR
(500 MHz, DMSO): d 9.09 (bs, 1H), 8.87 (bs, 1H), 8.73 (d, J ¼ 8.0 Hz,
1H), 8.09 (d, J ¼ 7.0 Hz, 1H), 7.89 (d, J ¼ 7.0 Hz, 1H), 7.80 (t,
J ¼ 7.0 Hz, 1H), 7.64 (s, 1H), 7.53 (d, J ¼ 7.5 Hz, 1H), 7.51 (t,
J ¼ 7.0 Hz, 1H),7.29 (d, J ¼ 8.0 Hz, 1H), 7.12 (d, J ¼ 7.0 Hz, 1H) ppm.
13 C NMR (500 MHz, DMSO): d 155.20, 153.44, 144.23, 136.81,
133.17, 127.06, 125.97, 122.32, 118.56, 117.15, 116.40, 112.99,
110.78 ppm. HRMS calculated for C15H11NO2 237.08 found 237.08.
Synthesis of 4-(naphthalen-2-yl)benzene-1,2-diol (23). To a mix-
ture of 2-naphtylboronic acid (5.81 mmol), K2CO3 (5.81 mmol), and
Pd(OAc)2 (0.048 mmol) in ethanol (15 ml) and water (5 ml) was
slowly added 4-bromo-1,2-dimethoxybenzene (4.84 mmol). The
mixture was stirred at room temperature for 18 h. To the resulting
mixture was added water (80 ml), and then extracted with
dichloromethane. The organic phase was separated, dried over
Mg2SO4 and concentrated under reduced pressure to give a solid
residue which was purified by flash chromatography (silica gel,
hexane/ethyl acetate 5/1). The pure 2–(3,4-dimethoxyphenyl)naph-
thalene was obtained as white solid and used for the next reac-
tion. Yield (%) ¼ 93. 1H NMR (500 MHz, DMSO): d 8.29 (d,
J ¼ 8.0 Hz, 1H), 8.12 (d, J ¼ 8.0 Hz, 1H), 7.98 (d, J ¼ 7.4 Hz, 1H), 7.82
(t, J ¼ 8.0 Hz, 1H), 7.75 (t, J ¼ 8.0 Hz, 1H), 7.61 (s, 1H), 7.45 (d,
Synthetic protocols for the preparation of compounds (14),
(22), and (23)
Synthesis of 6-chloro-2–(3,4-dihydroxyphenyl)-2,3-dihydroquinazolin- J ¼ 8.2 Hz, 1H), 7.39 (d, J ¼ 7.4 Hz, 1H), 7.12 (s, 1H), 7.02 (d,
4(1H)-one (14). 2-amino-5-chlorobenzamide (0.7 mmol) and 3,4- J ¼ 8.2 Hz, 1H), 3.85 (s, 3H), 3.78 (s, 3H) ppm. 13 C NMR (500 MHz,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1957
DMSO): d 151.20, 148.19, 139.45, 135.45, 132.13, 127.00, 124.15, incubated at 37 ꢀC for 90 min. After incubation, 4 nM of
119.32, 117.56, 116.03, 113.40, 112.26, 110.67, 108.65, 57.23 ppm. Europium–Streptavidin was added to the reaction mixture and the
HRMS calculated for C18H16O2 264.12 found 264.12.
HTRF signal was recorded using a Perkin Elmer Victor 3 plate
reader using a 314 nm for excitation wavelength and 668 and
620 nm for the wavelength of the acceptor and the donor sub-
strates emission, respectively.
To a stirred solution of 2–(3,4-dimethoxyphenyl)naphthalene
(0.36 mmol) in dichloromethane (3 ml) at 0 ꢀC was added boron
tribromide 1 M solution in dichloromethane (6.00 mmol). After 1 h,
the resulting mixture was stirred for 3 more hours at room tem-
perature. The resulting mixture was the poured into ice water and
treated with methanol (20 ml). The pale pink mixture was then
extracted with dichloromethane and the organic phase, previously
dried over anhydrous MgSO4, was evaporated to give compound
23 as a grey solid. Yield (%) ¼ 89. 1H NMR (500 MHz, DMSO): d
9.56 (bs, 1H), 9.38 (bs, 1H), 8.31 (d, J ¼ 8.1 Hz, 1H), 8.15 (d,
J ¼ 8.1 Hz, 1H), 7.88 (d, J ¼ 7.5 Hz, 1H), 7.83 (t, J ¼ 8.1 Hz, 1H), 7.75
(t, J ¼ 8.1 Hz, 1H), 7.62 (s, 1H), 7.47 (d, J ¼ 8.0 Hz, 1H), 7.39 (d,
J ¼ 7.5 Hz, 1H), 7.22 (s, 1H), 7.00 (d, J ¼ 8.0 Hz, 1H) ppm. 13 C NMR
(500 MHz, DMSO): 149.80, 148.00, 140.46, 136.41, 133.16, 128.00,
125.16, 118.82, 117.76, 116.27, 112.47, 112.16, 111.57, 108.65 ppm.
HRMS calculated for C18H12O2 236.08 found 236.08.
HIV-1 RNA-dependent DNA polymerase activity determination
HIV-1 RT-associated RDDP activity was measured as described17,38
.
Briefly, in 25 lL volume containing 60 mM Tris-HCl pH 8.1, 8 mM
MgCl2, 60 mM KCl, 13 mM DTT, poly(A)-oligo(dT), 100 lM dTTP,
and 6 ng wt RT. After enzyme addition, the reaction mixture was
incubated for 30 min at 37 ꢀC and stopped by addition of EDTA.
Reaction products were detected by PicoGreen addition and
measured with a Victor 3 (Perkin) at 502/523 nm. IC50 values were
determined using GraphPad Prism version 6.01 software
(GraphPad Software, Inc.; San Diego, CA). Figures were prepared
with GraphPad Prism 6 version 6.01. All assays were performed
in triplicate.
Biology
Evaluation of MgCl2 coordination
Expression and purification of recombinant HIV-1 RT
The coordination properties for the compounds were determined
as reported previously39–41. Briefly, compounds were solubilised in
1 ml of 96% ethanol at a final concentration of 100 lM. The
UV–Vis spectrum was recorded from 200 to 600 nm before and
after titration with increasing final concentrations of MgCl2, from
100 to 10 mM. UV–Vis spectra were recorded from 200 to 600 nm
using an Ultrospec 2100 pro (Amersham Biosciences, Little
Chalfont, UK) and spectra were plotted using SigmaPlot for
Windows version 11.0 (Systat Software Inc., San Jose, CA). Colour
legend indicates the final Mg2þ micromolar concentration within
the sample.
Wt and mutant heterodimeric RT were expressed essentially as
previously described29,30
.
RNase H inhibitor analysis
IC50 values were determined as previously reported31 using an 18-
nt 30-fluorescein-labeled RNA annealed to a complementary 18-nt
50-dabcyl-labeled DNA. To a 96-well plate was added 1 lL of each
inhibitor (in DMSO), followed by 10 lL of the appropriate RT
(15ꢂ80 ng/mL) in reaction buffer. Hydrolysis was initiated by add-
ing 10 lL of RNA/DNA hybrid (2.5 lM). Final assay conditions were
50 mM TrisꢃHCl, pH 8.0, 60 mM KCl, 10 mM MgCl2, 1% DMSO,
150ꢂ800 ng RT, 250 nM substrate, and increasing concentrations
of inhibitor. Wells containing only DMSO was used as negative
control. Plates were incubated at 37 ꢀC in a Spectramax Gemini
EM fluorescence spectrometer for 10 min, and fluorescence (lex¼
485 nm; lem ¼ 520 nm) was measured at 1-min intervals such that
linear initial rates could be measured in the presence (vi) and
absence (vo) of inhibitor. Percent inhibition was calculated as 100
ꢁ (vo–vi)/vo and plotted against log[I]. IC50 values were deter-
mined using Prism5 (GraphPad Software, San Diego, CA). All
assays were performed in triplicate.
Differential scanning fluorimetry (ThermoFluor)
Thermal stability assays were performed according to Nettleship
R
et al.42 To
a
LightCyclerV480 96-well plate (Roche, Basel,
Switzerland) was added 1 lL of 500 lM inhibitor in DMSO, fol-
lowed by 49 lL of 300 nM HIV-1 RT in reaction buffer containing
20 mM HEPES, pH 7.5, 10 mM MgCl2, 100 mM NaCl, and a 1:1000
R
dilution of SyproV Orange dye (Invitrogen, Carlsbad, CA). The mix-
ture was heated from 30 to 90 ꢀC in increments of 0.2 ꢀC.
Fluorescence intensity was measured using excitation/emission
wavelengths of 483 and 568 nm, respectively. Changes in protein
thermal stability (DTm) upon inhibitor binding were analysed by
R
using LightCyclerV 480 Software. All assays were performed
Expression and purification of recombinant HIV-1 in and LEDGF
HIV IN was expressed as previously described32–34
.
in triplicate.
Yonetani–Teorell analysis
HTRF in LEDGF-dependent assay
Yonetani–Theorell analysis43 was performed as reported44. Briefly,
HIV RT-associated RNase H activity was measured in 100 mL reac-
tion volume containing 50 mM Tris–HCl buffer pH 7.8, 6 mM
MgCl2, 1 mM dithiothreitol (DTT), 80 mM KCl, 0.25 mM hybrid RNA/
DNA 50-GAUCUGAGCCUGGGAGCU-Fluorescin-30 (HPLC, dry, QC:
The IN LEDGF/p75-dependent assay measures inhibition of IN
activity in the presence of LEDGF/p75 protein35–37. Briefly, 50 nM
IN was pre-incubated with increasing concentration of compounds
for 1 h at room temperature in reaction buffer containing 20 mM
HEPES pH 7.5, 1 mM DTT, 1% Glycerol, 20 mM MgCl2, 0.05% Brij-
35 and 0.1 mg/ml BSA. To this mixture, 9 nM DNA donor substrate
(50-ACAGGCCTAGCACGCGTCG-Biotin-30 annealed with 50-CGACGC
GTGGTAGGCCTGT-Biotin3’) and 50 nM DNA acceptor substrate (50-
Mass
Check)
(available
from
Metabion)
50-Dabcyl-
AGCTCCCAGGCTCAGATC-30(HPLC, dry, QC: Mass Check), and differ-
ent amount of enzymes according to a linear range of dose-
Cy5-ATGTGGAAAATCTCTAGCAGT-30 annealed with 50-Cy5- TGAGCT response curve: 20 ng of WT RT, and increasing concentrations of
CGAGATTTTCCACAT-30) and 50 nM LEDGF/p75 were added and inhibitor (diluted) in water and incubated for 1 h at 37 ꢀC, at

1958
G. TOCCO ET AL.
O
O
N
CHO
R3
R
R
i
NH2
NH2
NH
+
R2
R3
R1
X
R1
X
R2
2,3, 6 - 13, 16 - 21
2 R = CH3, R1 = H, R2 = OH, R3 = OH, X = C
3 R = H, R1 = CH3, R2 = OH, R3 = OH, X = C
6 R = Ph, R1 = H, R2 = OH, R3 = OH, X = C
7 R- R1 = -Ph-, R2 = OH, R3 = OH, X = C
8 R = I, R1 = H, R2 = OH, R3 = OH, X = C
9 R = Br, R1 = H, R2 = OH, R3 = OH, X = C
10 R = Cl, R1 = H, R2 = OH, R3 = OH, X = C
11 R =H, R1 = Cl, R2 = OH, R3 = OH, X = C
12 R = F, R1 = H, R2 = OH, R3 = OH, X = C
13 R = NO2, R1 = H, R2 = OH, R3 = OH, X = C
16 R = R1 = H, R2 = OH, R3 = OH, X = N
17 R = R1 = H, R2 = OH, R3 = OH, X = C
18 R = R1 = H, R2 = OH, R3 = COOH, X = C
19 R = R1 = H, R2 = OH, R3 = NH2, X = C
20 R = R1 = H, R2 = R3 = NH2, X = C
21 R = R1 = H, R2 = OH, R3 = NO2, X = C
O
O
CHO
O
NH
O
i
NH2
NH2
OH
+
N
OH
OH
OH
15
O
O
O
R
R
R
R1
NH2
NO2
NH2
NH2
NH
ii
i
OH
OH
R1
R1
N
4, 5
4 R = OCH3, R1 = H
5 R = OCH3, R1 = OCH3
Scheme 1. Synthetic protocol for compounds 2–13 and 15–21. Reagents and conditions: i) I2/CH3CN, rt, 6 h. ii) SnCl2/HCl 37%,ꢂ5 ꢀC (1 h), rt (30h).
200 rpm. Products were measured with a multilabel counter plate good yields. Synthesis of quinazolinones 4 and 5 required synthe-
reader Victor 3 (Perkin Elmer model 1420-051) equipped with fil- sising the 2-amino-5-methoxybenzamide and 2-amino-4,5-dime-
thoxybenzamide.
Starting
from
the
corresponding
2-
ters for 490/528 nm (excitation/emission wavelength). A calibration
curve for the fluorescein signal was generated in parallel, calculat-
ing pmols of product generated by the reaction. The reciprocal of
the reaction product was plotted vs. the compound concentration
a different condition using the Sigma-Plot software.
nitrobenzamide, we carried out a reduction of the nitro group47 in
concentrated HCl in the presence of SnCl2, obtaining the corre-
sponding mono or dimethoxy ꢂ2-amino arylamide as
pure product.
In contrast, cyclisation48 (Scheme 2) of 2-amino-6-chlorobenza-
mide with 3,4-dihydroxybenzaldehyde carried out at reflux in a mix-
ture of dichloromethane/acetonitrile gave the racemic 6-chloro-
2–(3,4-dimethoxyphenyl)-2,3-dihydroquinazolin-4(1H)-one 14, which
is more flexible than the corresponding derivative 10. Moreover, it
adds a stereocenter on C-2, making the molecule chiral. Synthesis
of compounds 22 and 23 started with a Suzuki C-C coupling reac-
tion between the appropriate arylboronic acid and the proper aryl
bromide under basic conditions and in the presence of Pd(OAc)2 as
catalyst49. Subsequent demethylation was performed using BBr3 as
demethylating agent in dichloromethane50.
Antiviral activity
Antiviral activity of selected thienopyrimidinones was determined
via the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)-
carbonyl]-2H tetrazolium hydroxide (XTT)-based cell viability assay
of Weislow et al.45, using the HIV-1RF isolate and human T-cell
line CEM-SS.
Results and discussion
Chemistry
Biology
As reported in Scheme 1, compounds 2, 3, 6 2 13, 15 2 21 were
synthesised by a one-pot direct oxidative condensation of the
appropriate commercially available 2-amino arylamide with the
3,4-disubstituted benzaldehyde in the presence of molecular iod-
All compounds were tested for inhibitory activity against both wt
and p66/p51 C280A RT. In fact, p51 residue Cys280 is crucial for
binding of thienopirimidinones51. Table 1 provides IC50 values for
ine46. After 6 h, the reaction, carried out in acetonitrile at room the first panel of quinazolinone derivatives. It is immediately evi-
temperature, afforded the final aryl pyrimidinone derivative in dent that every substitution gave rise to compounds with

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1959
O
O
CHO
OH
Cl
Cl
i
NH
NH2
+
OH
N
H
14
NH2
OH
OH
B(OH)2
ii
iii
+
OCH3
OCH3
OH
OH
N
N
OCH3
OCH3
N
Br
22
Br
ii
iii
+
OCH3
OCH3
OH
OH
B(OH)2
OCH3
OCH3
23
Scheme 2. Synthetic protocol for compounds 14, 22, and 23. Reagents and conditions: i) CH2Cl2/CH3CN, reflux, 40 h. ii) Pd(OAc)2/K2CO3, EtOH/H2O 3/1, rt (18h). iii)
BBr3 1 M in CH2Cl2, CH2Cl2, 0 ꢀC (1h), rt (3 h).
submicromolar activity, equally effective on both RT wild type and
p66/p51 C280A mutant.
Table 2. Inhibition of HIV-1 RNase H activity by compounds 18–21.
O
Interestingly, steric hindrance of the chosen substituents
seemed not to affect RNase H activity. In fact, compounds 6 (IC50
¼ 0.37 lM) and 2 (IC50 ¼ 0.41 lM) share the same potency.
Conversely, compound 7 (IC50 ¼ 1.10 lM) demonstrated that add-
ing rigidity to the molecule by introducing another aromatic ring
ortho condensed to the quinazolinone core impaired the activity
while replacing naphthalene with benzofuran as in 15
(IC50 ¼ 0.64 lM) was only slightly detrimental to inhibitory
potency. Noteworthy, electron withdrawing groups, and in par-
ticular halogens in position 6, provided the most active com-
pounds, with 8 being the best compound (IC50 ¼ 0.15 lM) of the
entire series of derivatives. Finally, the 2–(3,4-dihydroxyphenyl)
unit was demonstrated to be crucial for the inhibitory activity
similarly to thienopyrimidinones. In fact, as reported in Table 2,
replacing one or both OH groups with –COOH, NH2, or NO2 led to
a complete loss of activity.
NH
N
Ar
IC50 (mM)
WT
Compound
Ar
OH
18
13.6 0.3
COOH
OH
19
20
21
>50
NH2
NH2
>50
NH2
OH
>50
We also explored the role of the pyrimidinone moiety. First, in
order to determine the importance of the rigidity of the quinazoli-
none core, we decided to synthesise compound 14, in which
removal of the double bond between N-1 and C-2 gave more
flexibility to the pyrimidinone ring and introduced a stereocenter
on C-2. Since the synthetic pathway was not stereoselective, we
obtained and subsequently tested, compound 14 as a racemic
mixture. As shown in Table 1, this structural modification did not
affect RNase H inhibitory activity on both wt and p66/p51 C280A
mutant RTs. Encouragingly, this modification rendered quinazoline
14 more selective for RNase H activity than 9 (Table 4) and
prompted us to explore a stereoselective synthetic approach that
will be the subject of a future paper.
NO2
Table 3. Inhibition of wild type and p66/p51C280A mutant HIV-1 RNase H activ-
ity by compounds 22 and 23.
IC50 (mM)
Compound
WT
C280A
22
2.9 0.89
0.61 0.22
As shown in Table 3, removing the amide unit gave rise to
2–(3,4-dihydroxyphenyl) quinoline 22 with almost the same inhibi-
tory potency of 2–(3,4-dihydroxyphenyl)- quinazolin-4(3H)-one 17
against wt RT (IC50 ¼ 2.9 uM)26. Noteworthy, 22 inhibited p66/p51
C280A mutant RT RNase H activity 4-fold more potently than wt
RT (IC50 ¼ 0.61 lM). Substituting the pyridine core with its sim-
plest bioisoster, a benzene ring, generated compound 23, 5-fold
more potent than the reported compound 17. However, 23
OH
OH
N
23
0.71 0.03
1.2 0.04
OH
OH

1960
G. TOCCO ET AL.
2
1
Table 4. Effect of compounds 2–17 on HIV-1 RDDP and IN functions.
IC50 (mM)
0
Compound
RDDP
IN
15
14
6
13
23
22
7
-1
-2
-3
-4
-5
-6
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
EFV
RAL
>100
4.3 1.5
2.35 0.44
8.5 0.3
6.5 2.5
89 10
31.5 6.5
3.4 0.83
7 2
3.4 0.6
7.2 0.7
6.61 0.93
23.18 0.8
>100
5.35 0.35
0.69 0.19
1.44 0.40
ND
12
16
60
57
9
3
10 11
9
8
2
5
4
3
>100
63 11
23.4 0.5
26.7 2.2
17
46
3
Compound
30.5 0.4
>100
>100
Figure 2. Effect of compounds 2–17, 22, and 23 on the thermal stability of p66/
p51 HIV-1 RT. Tm values are the average of triplicate analysis.
99
1
>100
25.3 6.3
O
O
38
70
4
1
NH
NH
OH
OH
OH
OH
0.010 0.005
S
Cl
N
N
ND
0.055 0.002
11
24
inhibited p66/p51 C280A mutant RT RNase H activity with the
same potency of the wt enzyme (Table 3).
Inhibitory activity against RNA-dependent DNA polymerase
(RDDP) and integrase (IN)
In order to determine the specificity of our quinazolinone-based
derivatives, we tested their effect on RDDP and IN activities. With
respect to RDDP function, all compounds were assayed using efa-
virenz, a non-nucleoside RT inhibitor, as positive control. Data of
Table 4 indicate that all the quinazolinone derivates, albeit with a
different degree, were poorly active against RDDP with IC50 values
ranging from 23 to more than 100 mM. Thus, they are highly
selective RNase H inhibitors.
Since the catechol moiety has been identified in a variety of
HIV-1 IN inhibitors52,53, we also tested our compounds against this
target, using the strand-transfer inhibitor raltegravir, as positive con-
trol. Almost all quinazolinones were moderately active on HIV-1 IN,
displaying, and IC50 range of 2.3–89 mM, showing high selectivity
for RNase H. Remarkably, the pyridine core was subtly deleterious
for RT activity but powerful in IN function suppression. In fact, com-
pounds 16 and 17 showed a different trend. In particular, these
compounds inhibited IN activities in the presence of LEDGF/p75
protein with IC50 values of 0.69 and 1.44 mM, respectively.
Moreover, these derivatives exhibited a higher selectivity against IN.
Compound 24 [mM]
Figure 3. Yonetani–Theorell analysis. Combination of compound 11 and 24 on
HIV-1 RNase H activity. HIV-RT was incubated in the presence of 24 alone (᭹) or
combined with increasing concentrations of compound 11: 2,5 mM (O); 5 mM(D)
and 10 mM (᭿).
Results graphically reported in Figure 2, indicate that almost all
tested inhibitors destabilise the RT heterodimer stability, i.e. they
decrease the Tm. Since it had been demonstrated that compounds
that bind in the proximity of the p66/p51 interface usually reduce
RT Tm11, we can assume that present quinazolinones, similarly to
thienopyrimidinones, might act as allosteric inhibitors.
To confirm this hypothesis, we performed a Yonetani-Theorell
analysis of simultaneous action of compound 11 vs. the 2–(3,4-
dihydroxyphenyl)-3,5,6,7,8,9-hexahydro-4H-cyclohepta[4,5]thieno[2,
3-d]pyrimidin-4-one 2412 on HIV-1 RT-associated RNase H activity.
Such an analysis reveals whether simultaneous binding (or inhib-
ition) of two compounds is possible or not. Results showed that
compound 11 and compound 24 inhibition are not kinetically
mutually exclusive (Figure 3), suggesting that they may bind sim-
ultaneously to RT. However, it is worth noting that the calculated
interaction constant a had a value of greater than 1, suggesting a
negative interference between the two compounds (i.e. com-
Investigation of mode of action
Typically, RNase H active site inhibitors chelate the catalytically cru-
cial Mg2þ ions. Since catechol-containing compounds usually display
metal-chelating properties; we performed UV spectrometric analysis
to determine the possible involvement of Mg2þ cofactors in the
inhibition mode of our quinazolinones. In this respect, a magnesium
effect was examined at increasing concentrations of the divalent
metal (Supplementary Material). Results showed that the presence of
the Mg2þ failed to shift the peak of absorbance of the compounds
spectra, excluding the involvement of Mg2þ cofactor coordination
on the mode-of-action of compounds. Only compound 2 showed a
hyperchromic effect at the highest concentration tested.
To gain additional evidence, thermal denaturation experiments
were carried out. Differential scanning fluorimetry was the tech-
nique of choice, as can determine the effect of an inhibitor on RT pound binding 11 has a negative influence on compound 24
stability by measuring changes in its melting temperature (Tm)54. binding, and vice versa).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1961
Antiviral activity
the manuscript apart from those disclosed. No writing assistance
was utilised in the production of this manuscript.
The ability of all compounds to inhibit HIV-1 virus replication was
evaluated. Only compounds 3, 8, 10, 11, and 23, showed a mod-
est antiviral activity within low micromolar range. Unfortunately,
they displayed very low SI values, demonstrating to be highly
cytotoxic. In fact, the most active compound in cell-based assays,
Funding
A.C. research was funded by the Sardinian Regional Government
grant LR07/17 [F76C18000800002]. SFJLG and JAB were supported
by the Intramural Research Program of the National Cancer
Institute, National Institutes of Health, Department of Health and
Human Services.
compound 8, showed a very small selectivity index (SI) (EC50
¼
8.3 mM, CC50 ¼ 17 mM, SI ¼ 2). Similar results were obtained for 3
(EC50 ¼ 12 mM, CC50 ¼ 25 mM, SI ¼ 2) and 23 (EC50 ¼ 14 mM, CC50
¼ 26 mM, SI ¼ 2). Interestingly, the simple substitution of an iod-
ine atom with a chlorine in 10 displayed a slightly better SI (EC50
¼ 5.4 mM, CC50 ¼ 22 mM, SI ¼ 4). Ring walking of the chlorine
from position 6–7 as in 11, reduced the SI (EC50 ¼ 1.8 mM, CC50
¼
ORCID
6 mM, SI
not shown).
¼
3). All other compounds were inactive (data
Graziella Tocco
http://orcid.org/0000-0003-0081-5704
http://orcid.org/0000-0001-9725-7977
http://orcid.org/0000-0003-2448-3767
http://orcid.org/0000-0002-4646-1924
http://orcid.org/0000-0002-6630-8636
http://orcid.org/0000-0001-5818-8796
http://orcid.org/0000-0002-4849-0980
Francesca Esposito
Pierluigi Caboni
John A. Beutler
Angela Corona
Stuart F. J. Le Grice
Enzo Tramontano
Conclusions
The isosteric replacement approach allowed us to synthesise a ser-
ies of 22 quinazolinone-based derivatives. All compounds were
equally effective on both wt RT and p66/p51 C280A mutant,
unable to chelate the Mg2þ ions, and in most cases destabilised
the RT heterodimer by decrease its Tm. These combined findings
prompted us to hypothesise that they are not active site, but
most likely allosteric, and in particular RT heterodimer interface
inhibitors. Yonetani–Theorell analysis showed that compound 11
and the reference thienopyrimidinone 24 are not kinetically mutu-
ally exclusive and the value of the calculated interaction constant
a indicated a negative interference between the two compounds.
Thus, they may bind simultaneously to different, but probably
close, RT allosteric sites since compound 11 binding has a nega-
tive influence on compound 24 binding, and vice versa.
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The authors have no other relevant affiliations or financial involve-
ment with any organisation or entity with a financial interest in or 11. Masaoka T, Chung S, Caboni P, et al. Exploiting drug-resist-
financial conflict with the subject matter or materials discussed in ant enzymes as tools to identify thienopyrimidinone

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