From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1 ribonuclease H inhibitors

Article abstract of DOI:10.1080/14756366.2018.1523901

The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC₅₀s of 0.53 and 2.90 μM against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre.

Full text of DOI:10.1080/14756366.2018.1523901

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
From cycloheptathiophene-3-carboxamide to
oxazinone-based derivatives as allosteric HIV-1
ribonuclease H inhibitors
Serena Massari, Angela Corona, Simona Distinto, Jenny Desantis, Alessia
Caredda, Stefano Sabatini, Giuseppe Manfroni, Tommaso Felicetti, Violetta
Cecchetti, Christophe Pannecouque, Elias Maccioni, Enzo Tramontano &
Oriana Tabarrini
To cite this article: Serena Massari, Angela Corona, Simona Distinto, Jenny Desantis,
Alessia Caredda, Stefano Sabatini, Giuseppe Manfroni, Tommaso Felicetti, Violetta Cecchetti,
Christophe Pannecouque, Elias Maccioni, Enzo Tramontano & Oriana Tabarrini (2019) From
cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1
ribonuclease H inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 55-74, DOI:
10.1080/14756366.2018.1523901
To link to this article: https://doi.org/10.1080/14756366.2018.1523901
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 26 Oct 2018.
Submit your article to this journal
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 34, NO. 1, 55–74
https://doi.org/10.1080/14756366.2018.1523901
RESEARCH PAPER
From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as
allosteric HIV-1 ribonuclease H inhibitors
a
, Angela Coronab
, Simona Distintob, Jenny Desantisa,c, Alessia Careddab, Stefano Sabatinia,
ꢀ
ꢀ
Serena Massari
Giuseppe Manfronia, Tommaso Felicettia, Violetta Cecchettia, Christophe Pannecouqued, Elias Maccionib, Enzo
Tramontanob,e and Oriana Tabarrinia
b
aDepartment of Pharmaceutical Sciences, University of Perugia, Perugia, Italy; Department of Life and Environmental Sciences, University of
c
Cagliari, Cittadella Universitaria di Monserrato, Monserrato, Cagliari, Italy; Department of Chemistry, Biology and Biotechnology, University of
d
Perugia, Perugia, Italy; Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, K.U. Leuven, K.U. Leuven, Leuven,
e
Belgium; Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche (CNR), Monserrato, Italy
ABSTRACT
ARTICLE HISTORY
Received 10 August 2018
Revised 11 September 2018
Accepted 11 September 2018
The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide
derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibi-
tor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thor-
ough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based
compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of
the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA
dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative
22 resulted the most potent dual inhibitor exhibiting IC50s of 0.53 and 2.90 lM against the RNase H and
RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pock-
ets within the RT, one located between the RNase H active site and the primer grip region and the other
close to the DNA polymerase catalytic centre.
KEYWORDS
Allosteric inhibitors; antiviral
agents; thieno[2,3-d][1,3]
oxazin-4-one derivatives;
HIV-1 ribonuclease H
GRAPHICAL ABSTRACT
antiretroviral treatment–naïve patients4. The resistance can affect
multiple classes of drugs5, dramatically impairing the outcome of
cART. Thus, the discovery of new agents is crucial. One approach
to minimise the development of drug resistance may rely on com-
pounds endowed with alternative mechanisms of action or new
binding sites on traditional targets to complement/enrich the cur-
rent drugs cocktails6.
Introduction
Despite many efforts in declining morbidity and mortality in AIDS
patients through the combination antiretroviral therapy (cART),
controlling HIV infection remains a global health priority. cART sig-
nificantly suppresses viral load, thus preventing the progression to
advanced AIDS and improving both the quality and expectancy of
patients life1,2. However, the benefits of such cocktail of drugs are
often compromised by severe side effects and the need for a life-
long treatment. Most important, neither a cure nor eradication of
The reverse transcriptase (RT) represents the most exploited
target within the anti-HIV therapy, with more than half of the
HIV infection is possible yet3, due to the development of latent approved drugs inhibiting this enzyme. RT is a heterodimeric
but replication-competent viral forms. The number of newly (p66/p51) multifunctional enzyme responsible for the conversion
infected people per year is constant and there is an overall of the viral single-stranded RNA genome into the double-stranded
increase of detected drug-resistant variants transmitted among DNA. This activity is essentially accomplished by the RNA- and
CONTACT Enzo Tramontano
tramon@unica.it
Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato,
serena.massari@unipg.it Department of Pharmaceutical Sciences, University of Perugia, Perugia 06123, Italy
Monserrato, Cagliari 09042, Italy; Serena Massari
ꢀ
S.M. and A.C. equally contributed to this work
ß 2018 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.

56
S. MASSARI ET AL.
DNA-dependent DNA polymerase (RDDP and DDDP) and ribo- Q500 was reported as responsible for the RNA:DNA hybrid binding
nuclease H (RNase H) functions, whose active sites reside on the
p66 subunit. While the polymerase catalyses nucleotidyl transfer
reactions for the DNA synthesis, the RNase H hydrolyses the RNA
strand within the RNA:DNA hybrid generated during the reverse
transcription. Both functions are necessary for successful reverse
transcription, but while the polymerase function has been tar-
geted by all the RT inhibitors in therapy, no drug inhibiting the
RT-associated RNase H function has entered clinical development
up to now, despite the fact that its abrogation7 and selective
inhibition8,9 has been proved to compromise the viral infectivity.
Since 1990 several RNase H inhibitors (RNHIs) have been identified
that10,11, based on their mechanism of action, can be classified in
active site and allosteric inhibitors. The wider class of RNHIs is rep-
resented by compounds acting through the chelation of the diva-
lent cations present at the RNase H active site and required as
cofactors in enzyme catalysis. Some of them were originally identi-
fied as inhibitors of the influenza virus endonuclease or HIV inte-
grase (IN)12. Given the structural similarities between RNase H and
IN active sites, they both can be targeted by some classes of com-
pounds that act as metal coordinating agents13–15. Less populated
is the class of allosteric RNHIs, which were identified through vari-
ous approaches and include structurally different compounds, of
which some endowed with dual activity against both RT-associ-
ated functions16–19. Targeting allosteric sites could be more advan-
tageous to avoid the inhibition of related host enzymes.
thus compounds interacting with this residue or residues in its
vicinity could potentially interfere with duplex accommodation in
the RNase H active site30.
Having in hand good and selective RNase H allosteric inhibitors
that act by recognising a new allosteric site, in this work we
decided to further exploit the cHTC scaffold designing and synthe-
sising a wide series of analogues.
Materials and methods
Chemistry
Starting materials, reagents, and solvents that were commercially
available were used as supplied. The reactions were monitored by
TLC on silica gel 60F254 (Merck) and visualised by UV and/or iod-
ine. Flash chromatography columns were performed on Merck sil-
ica gel 60 (mesh 230–400). After extraction, organic solutions were
dried by using anhydrous Na2SO4, filtered, and evaporator to dry-
€
ness at reduced pressure with a Buchi rotary. Yields are of pure
products and were not optimised. HRMS spectra were registered
on Agilent Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS,
HPLC 1290 Infinity. Purities of target compounds were determined
by UHPLC on Agilent Technologies 6540 UHD Accurate-Mass Q-
TOF LC/MS, HPLC 1290 Infinity with DAD detector and evaluated
As a part of our work aimed at identifying innovative anti-HIV to be ꢁ95%. HPLC conditions to assess the purity of final com-
compounds20–23, we have recently contributed to the allosteric
pounds were as follows: column, Phenomenex AERIS Widepore
RNHIs class reporting a series of cycloheptathiophene-3-carboxa-
C4, 4.6 mm ꢂ100 mm (6.6 lm); flow rate, 0.85 ml/min; acquisition
mide (cHTC) derivatives24, which were identified through a repur-
posing approach. Indeed, they were born as influenza virus
inhibitors with the ability to disrupt the PA-PB1 subunits inter-
action of the viral RNA polymerase25–28, and then, based on the
strict structural similarity with a series of known RNHIs29, they
were assayed for this activity. From the initial screening, com-
pounds endowed with good inhibitory activity emerged whose
structural optimization led to the catechol derivative 1 (Figure 1),
which, showing an IC50 value of 0.84 mM, ranked from the most
active allosteric RNHIs.
time, 10 min; DAD 254 nm; oven temperature, 30 ꢃC; gradient of
acetonitrile in water containing 0.1% of formic acid (0 ꢄ 100% in
10 min). Nuclear magnetic resonance (NMR) spectra were recorded
on Bruker Avance DRX-400 MHz using residual solvents such as
dimethylsulphoxide (d ¼ 2.48) or chloroform (d ¼ 7.26) as an
internal standard. Chemical shifts (d) are reported in parts per mil-
lion (ppm), and peak multiplicity are reported as s (singlet), d
(doublet), t (triplet), q (quartet), p (pentet), hept (heptet), m (multi-
plet), or brs (broad singlet). Compounds 225, 331, and 528 were
prepared as previously described.
Many other cHTCs showed IC50 values in the low micromolar
range, confirming the cycloheptathiophene ring particularly suit-
able to inhibit this function of the RT enzyme. They were unable
to chelate the Mg2þ ions excluding any interaction with the
RNase H catalytic centre. In addition, they did not inhibit RT-asso-
ciated RDDP function with the only exception of compound 1,
which, however, showed a 20-fold weaker potency against the
polymerase activity with respect to RNase H activity. Docking sim-
ulations performed to achieve insights on these selective inhibi-
tors showed that they recognised a new allosteric binding site
including residue Q500 that played a key role in the binding.
General procedure for demethylation (method A)
To a solution of the appropriate methoxy derivative (1.0 equiv) in
dry CH2Cl2, a 1 M solution of BBr3 in CH2Cl2 (6.0 equiv) was added
dropwise maintaining the temperature at 0 ꢃC. The reaction mix-
ture was stirred at room temperature (r.t.) until no starting mater-
ial was detected by TLC, and then quenched with MeOH and
water. The organic solvent was removed under vacuum affording
a residue, which was filtered and purified as described below.
2-Amino-N-(2-hydroxyphenyl)-5,6,7,8-tetrahydro-4H-cyclohep-
ta[b]thiophene-3-carboxamide (4). The title compound was pre-
pared starting from 331 through Method A (16 h) and purified by
crystallization from EtOH, in 65% yield as light-yellow crystals; 1H
NMR (DMSO-d6, 400 MHz): d 1.45–1.60 (m, 4H, cycloheptane CH2),
1.70–1.80 (m, 2H, cycloheptane CH2), 2.50–2.60 and 2.70–2.80 (m,
each 2H, cycloheptane CH2), 6.20 (bs, 2H, NH2), 6.70 (t, J ¼ 7.6 Hz,
1H, aromatic CH), 6.80–6.90 (m, 2H, aromatic CH), 7.90 (d,
J ¼ 7.9 Hz, 1H, aromatic CH), 8.60 (s, 1H, NH), 9.90 (s, 1H, OH); 13 C
NMR (DMSO-d6, 101 MHz): d 27.3, 28.0, 28.5, 28.7, 32.0, 113.6,
115.5, 119.5, 121.0, 121.7, 124.0, 127.4, 136.3, 147.2, 154.6, 164.1;
HRMS: calcd for C16H18N2O2S 303.1168 (M þ H)þ, found 303.1169.
Figure 1. Chemical structure of cHTC compound 1.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
57
General procedure for carbodiimide formation (method B)
aromatic CH), 7.30–7.50 (m, 3H, aromatic CH and NH2), 7.90 (dd,
J ¼ 1.6 and 7.8 Hz, 1H, aromatic CH), 11.75 (s, 1H, OH), 12.10 (s, 1H,
NH); 13 C NMR (DMSO-d6, 101 MHz): d 27.6, 28.2, 28.6, 28.7, 32.2,
117.1, 117.7, 120.0, 122.0, 130.7, 131.3, 134.1, 135.1, 136.5, 156.7,
161.8, 167.5; HRMS: m/z calcd for C17H18N2O3S 331.1117 (M þ H)þ,
found 331.1146.
Ethyl 2-[(3-methoxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclo-
hepta[b]thiophene-3-carboxylate (40). The title compound was
prepared starting from 3333 by Method B (4 h) and purified by
treatment with cyclohexane, in 44% yield; 1H-NMR (CDCl3,
400 MHz) d 1.40 (t, J ¼ 7.1 Hz, 3H, CH2CH3), 1.60–1.70 (m, 4H, cyclo-
heptane CH2), 1.75–1.85, 2.65–2.75, and 3.00–3.10 (m, each 2H,
cycloheptane CH2), 3.80 (s, 3H, OCH3), 4.40 (q, J ¼ 7.0 Hz, 2H,
CH2CH3), 7.70–7.80 (m, 3H, aromatic CH), 12.15 (s, 1H, NH).
A solution of the appropriate synthone (1.0 equiv) in dry pyridine
was added to the suitable benzoyl chloride (2.0 equiv). The reac-
tion mixture was maintained at r.t. until no starting material was
detected by TLC. After cooling, the reaction mixture was poured
into ice/water, obtaining a precipitate which was filtered and puri-
fied as described below.
2-[(4-Chlorobenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclohepta[b]-
thiophene-3-carboxamide (9). The title compound was prepared
starting from 3132 through Method B (16 h) using 4-chlorobenzoyl
chloride, and purified by flash chromatography eluting with cyclo-
hexane/EtOAc (7:3), in 35% yield as light-yellow solid; 1H NMR
(DMSO-d6, 400 MHz): d 1.50–1.60 (m, 4H, cycloheptane CH2),
1.70–1.80 (m, 2H, cycloheptane CH2), 2.65–2.70 and 2.75–2.80 (m,
each 2H, cycloheptane CH2), 7.50 (bs, 2H, NH2), 7.60 (d, J ¼ 8.5 Hz,
2H, aromatic CH), 7.80 (d, J ¼ 8.5 Hz, 2H, aromatic CH), 11.75 (s,
1H, NH); 13 C NMR (DMSO-d6, 101 MHz): d 27.5, 27.9, 28.4, 28.6,
31.9, 122.2, 129.4, 129.4, 131.5, 131.8, 135.7, 137.4, 137.4, 162.1,
167.8; HRMS: calcd for C17H17ClN2O2S 349.0778 (M þ H)þ,
found 349.0774.
Ethyl
2-[(3,4-dimethoxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxylate (42). The title compound
was prepared starting from 3333 by Method B (1 h) and purified
by treatment with a Et2O/cyclohexane (1:2) mixture, in 100% yield;
1H NMR (DMSO-d6, 400 MHz) d 1.25 (t, J ¼ 7.0 Hz, 3H, CH2CH3),
1.50–1.60 (m, 4H, cycloheptane CH2), 1.75–1.80, 2.20–2.25, and
3.00–3.05 (m, each 2H, cycloheptane CH2), 3.80 (s, 6H, OCH3), 4.30
(q, J ¼ 7.0 Hz, 2H, CH2CH3), 7.10–7.15 (m, 1H, aromatic CH),
7.45–7.50 (m, 2H, aromatic CH), 11.75 (s, 1H, NH).
Ethyl 2-[(3,4-dimethoxybenzoyl)amino]-4,5,6,7-tetrahydro-1-ben-
zothiophene-3-carboxylate (43). The title compound was prepared
starting from 3434 by Method B (1 h) and purified by treatment
with a Et2O/cyclohexane (1:2) mixture, in 96% yield; 1H NMR
(DMSO-d6, 400 MHz) d 1.25 (t, J ¼ 7.0 Hz, 3H, CH2CH3), 1.60–1.70
(m, 4H, cyclohexane CH2), 2.55–2.60 and 2.65–2.70 (m, each 2H,
cyclohexane CH2), 3.75 (s, 6H, OCH3), 4.25 (q, J ¼ 7.0 Hz, 2H,
CH2CH3), 7.05–7.10 (m, 1H, aromatic CH), 7.40–7.45 (m, 2H, aro-
matic CH), 12.00 (s, 1H, NH).
Ethyl 2-[(3,4-dimethoxybenzoyl)amino]-5,6-dihydro-4H-cyclopen-
ta[b]thiophene-3-carboxylate (44). The title compound was pre-
pared starting from 3534 by Method B (1 h) and purified by
treatment with a Et2O/cyclohexane (1:2) mixture, in 71% yield; 1H
NMR (CDCl3, 400 MHz) d 1.25 (t, J ¼ 7.0 Hz, 3H, CH2CH3), 2.30–2.40
(m, 2H, cyclopentane CH2), 2.80–2.90 (m, 4H, cyclopentane CH2),
3.90 and 3.95 (s, each 3H, OCH3), 4.30 (q, J ¼ 7.0 Hz, 2H, CH2CH3),
6.90 (d, J ¼ 8.3 Hz, 1H, aromatic CH), 7.50 (dd, J ¼ 2.2 and 8.3 Hz, 1H,
aromatic CH), 7.65 (d, J ¼ 2.2 Hz, 1H, aromatic CH), 12.00 (s, 1H, NH).
2-[(3,4-Dimethoxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclohepta
[b]thiophene-3-carboxamide (32). The title compound was pre-
pared starting from 3132 through Method B (4 h) using 3,4-
methoxybenzoyl chloride, and purified by treatment with Et2O, in
32% yield as white solid. 1H NMR (DMSO-d6, 400 MHz):
d
1.50–1.60 (m, 4H, cycloheptane CH2), 1.70–1.80 (m, 2H, cyclohep-
tane CH2), 2.60–2.70 and 2.75–2.85 (m, each 2H, cycloheptane
CH2), 3.80 (s, 6H, OCH3), 7.00–7.10 (m, 1H, aromatic CH),7.30–7.60
(m, 3H, aromatic CH and NH2), 11.75 (s, 1H, NH).
2-[(2-Methoxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclohepta
[b]thiophene-3-carboxamide (7). The title compound was prepared
starting from 3132 through Method B (16 h) using 2-methoxyben-
zoyl chloride, and purified by flash chromatography eluting with
cyclohexane/EtOAc (5:5), in 57% yield as white solid; 1H NMR
(DMSO-d6, 400 MHz): d 1.50–1.60 (m, 4H, cycloheptane CH2),
1.65–1.85 (m, 2H, cycloheptane CH2), 2.60–2.70 and 2.75–2.85 (m,
each 2H, cycloheptane CH2), 4.00 (s, 3H, OCH3), 7.00–7.10 (m, 1H,
aromatic CH), 7.20 (d, J ¼ 7.6 Hz, 1H, aromatic CH), 7.30–7.45 (m,
1H, aromatic CH), 7.60 (bs, 2H, NH2), 8.05 (d, J ¼ 6.3 Hz, 1H, aro-
matic CH), 12.10 (s, 1H, NH); 13 C NMR (DMSO-d6, 101 MHz): d 27.6,
28.0, 28.5, 28.6, 32.1, 56.4, 112.8, 119.4, 121.4, 121.6, 130.8, 131.8,
134.4, 135.2, 137.0, 157.8, 160.8, 167.7; HRMS: m/z calcd for
C18H20N2O3S 345.1274 (M þ H)þ, found 345.1269.
General procedure for hydrolysis (method C)
2-[(3,4-Dihydroxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclohep-
ta[b]thiophene-3-carboxamide (6). The title compound was pre-
pared starting from 32 through Method A (4 h) and purified by
flash chromatography eluting with CHCl3/MeOH (95:5), in 49%
A suspension of the appropriate ethyl thiophene-3-carboxylate
(1.0 equiv) and LiOH (4.0 equiv) in a mixture H2O/THF (1:1) was
maintained at 50 ꢃC until no starting material was detected by
TLC. After cooling the reaction mixture was acidified (pH 4–5)
with 2 N HCl and the precipitate was filtered and purified as
described below.
yield as light-yellow solid; 1H NMR (DMSO-d6, 400 MHz,):
d
1.50–1.65 (m, 4H, cycloheptane CH2), 1.70–1.85 (m, 2H, cyclohep-
tane CH2), 2.60–2.70 and 2.75–2.85 (m, each 2H, cycloheptane
CH2), 6.85 (d, J ¼ 8.1 Hz, 1H, aromatic CH), 7.15 (d, J ¼ 8.1 Hz, 1H,
aromatic CH), 7.25–7.30 (m, 1H, aromatic CH), 7.40 (bs, 2H, NH2),
9.40 and 9.60 (s, each 1H, OH), 11.70 (s, 1H, NH); 13 C NMR (DMSO-
d6, 101 MHz): d 27.5, 27.9, 28.6, 31.9, 114.8, 115.9, 119.4, 120.2,
123.8, 130.5, 135.2, 139.4, 145.9, 150.1, 162.7, 168.4; HRMS: m/z
calcd for C17H18N2O4S 347.1066 (M þ H)þ, found 347.1061.
2-(Benzoylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-
3-carboxylic acid (47). The title compound was prepared starting
from 3833 by Method C (overnight) and purified by treatment
1
with Et2O, in 81% yield; H-NMR (CDCl3, 400 MHz) d 1.55–1.70 (m,
4H, cycloheptane CH2), 1.75–1.90, 2.70–2.75 and 3.05–3.15 (m,
each 2H, cycloheptane CH2), 7.40–7.55 (m, 3H, aromatic CH),
7.90–7.95 (m, 2H, aromatic CH), 12.00 (s, 1H, NH).
2-[(3-Methoxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclohepta
[b]thiophene-3-carboxylic acid (49). The title compound was pre-
pared starting from 40 by Method C (overnight) and purified by
2-[(2-Hydroxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclohepta
[b]thiophene-3-carboxamide (8). The title compound was prepared
starting from 7 through Method A (4 h) and purified by flash chro-
matography eluting with cyclohexane/EtOAc (6:4), in 74% yield as
white solid; 1H NMR (DMSO-d6, 400 MHz,): d 1.50–1.65 (m, 4H, treatment with Et2O, in 61% yield; 1H-NMR (DMSO-d6, 400 MHz) d
cycloheptane CH2), 1.70–1.85 (m, 2H, cycloheptane CH2), 2.60–2.70 1.50–1.60 (m, 4H, cycloheptane CH2), 1.65–1.75, 2.75–2.85, and
and 2.70–2.80 (m, each 2H, cycloheptane CH2), 6.90–7.00 (m, 2H, 3.05–3.10 (m, each 2H, cycloheptane CH2), 3.80 (s, 3H, OCH3), 7.25

58
S. MASSARI ET AL.
(d, J ¼ 8.0 Hz, 1H, aromatic CH), 7.40–7.45 (m, 1H, aromatic CH), aromatic CH), 8.05 (d, J ¼ 7.4 Hz, 2H, aromatic CH); 13 C NMR
7.55 (t, J ¼ 7.7 Hz, 1H, aromatic CH), 12.20 (s, 1H, NH), 13.50 (s,
(DMSO-d6, 101 MHz): d 27.0, 27.6, 27.8, 29.5, 32.0, 117.3, 128.0,
1H, COOH).
129.5, 129.9, 133.1, 137.5, 139.1, 155.2, 158.3, 159.8. HRMS: m/z
calcd for C17H15NO2S 298.0902 (M þ H)þ, found 298.0899.
2-[(3,4-Dimethoxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclohepta
[b]thiophene-3-carboxylic acid (51). The title compound was pre-
pared starting from 42 by Method C (12 h) and purified by treat-
ment with Et2O, in 90% yield; 1H NMR (DMSO-d6, 400 MHz) d
1.45–1.55 (m, 4H, cycloheptane CH2), 1.70–1.75, 2.60–2.65, and
3.00–3.05 (m, each 2H, cycloheptane CH2), 3.75 (s, 6H, OCH3), 7.10
(d, J ¼ 8.1 Hz, 1H, aromatic CH), 7.35–7.40 (m, 2H, aromatic CH),
12.20 (s, 1H, NH).
2-[(3,4-Dimethoxybenzoyl)amino]-4,5,6,7-tetrahydro-1-benzo-
thiophene-3-carboxylic acid (52). The title compound was pre-
pared starting from 43 by Method C (24 h) and purified by
2–(2-Fluorophenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]-
thieno[2,3-d][1,3]oxazin-4-one (15). The title compound was pre-
pared starting from 4825 by Method
D and purified by
crystallization by petroleum ether, in 20% yield; 1H NMR (DMSO-
d6, 400 MHz): d 1.50–1.70 (m, 4H, cycloheptane CH2), 1.75–1.85 (m,
2H, cycloheptane CH2), 2.80–2.90 and 3.05–3.15 (m, each 2H,
cycloheptane CH2), 6.85–6.95 (m, 2H, aromatic CH), 7.15 (t,
J ¼ 8.8 Hz, 2H, aromatic CH), 7.35 (q, J ¼ 7.6 Hz, 2H, aromatic CH),
7.60–7.70 (m, 1H, aromatic CH), 8.00 (t, J ¼ 7.7 Hz, 1H, aromatic
CH); 13 C NMR (DMSO-d6, 101 MHz): d 27.0, 27.6, 27.7, 29.5, 32.1,
117.5, 117.7 (d, JCꢄF ¼ 21 Hz), 118.4 (d, JCꢄF ¼ 9 Hz), 125.3 (d,
JCꢄF ¼ 3 Hz), 131.3, 135.0 (d, JCꢄF ¼ 9 Hz), 137.5, 139.8, 155.1, 155.8
(d, JCꢄF ¼ 5 Hz), 159.3 (d, JCꢄF ¼ 19 Hz), 162.0. HRMS: m/z calcd for
C17H14FNO2S 316.0808 (M þ H)þ, found 316.0805.
1
treatment with Et2O, in 56% yield; H NMR (DMSO-d6, 400 MHz) d
1.60–1.70 (m, 4H, cyclohexane CH2), 2.55–2.60 and 2.65–2.70 (m,
each 2H, cyclohexane CH2), 3.75 (s, 6H, OCH3), 4.25 (q, J ¼ 7.0 Hz,
2H, CH2CH3), 7.05–7.10 (d, J ¼ 8.1 Hz, 1H, aromatic CH), 7.40–7.45
(m, 2H, aromatic CH), 12.30 (s, 1H, NH), 13.25 (bs, 1H, COOH).
2-[(3,4-Dimethoxybenzoyl)amino]-5,6-dihydro-4H-cyclopenta[b]-
thiophene-3-carboxylic acid (53). The title compound was prepared
starting from 44 by Method C (24 h) and purified by treatment
with Et2O, in 96% yield; 1H NMR (DMSO-d6, 400 MHz) d 2.70–2.75
(m, 4H, cyclopentane CH2), 3.25–3.30 (m, 2H, cyclopentane CH2),
3.75 (s, 6H, OCH3), 7.05 (d, J ¼ 8.5 Hz, 1H, aromatic CH), 7.40–7.45
(m, 2H, 1H, aromatic CH), 12.00 (s, 1H, NH), 13.20 (bs, 1H, COOH).
2–(3-Methoxyphenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]-
thieno[2,3-d][1,3]oxazin-4-one (18). The title compound was pre-
pared starting from 49 by Method
D and purified by
crystallization by EtOH, in 50% yield; 1H-NMR (CDCl3, 400 MHz) d
1.70–1.80 (m, 4H, cycloheptane CH2), 1.89–1.95, 2.80–2.85, and
3.10–3.20 (m, each 2H, cycloheptane CH2), 3.85 (s, 3H, OCH3),
7.00–7.10 (m, 1H, aromatic CH), 7.35 (t, J ¼ 8.1 Hz, 1H, aromatic
CH), 7.70 (t, J ¼ 2.4 Hz, 1H, aromatic CH), 7.80 (dt, J ¼ 1.2 and
2.5 Hz, 1H, aromatic CH); 13 C NMR (DMSO-d6, 101 MHz): d 26.9,
27.5, 27.7, 29.4, 32.0, 55.7, 112.2, 117.3, 119.3, 120.4, 130.6, 131.1,
137.4, 139.2, 155.1, 158.0, 159.6, 159.9; HRMS: m/z calcd for
C18H17NO3S 328.1008 (M þ H)þ, found 328.1005.
General procedure for cyclisation (Method D)
A mixture of the appropriate thiophene-3-carboxylic acid (1.0
equiv) and acetic anhydride (10.5 equiv) was irradiated in a micro-
wave oven at 100 ꢃC for 30 min. The reaction mixture was then
evaporated to dryness obtaining a residue, which was treated
with hot petroleum ether and then purified as described below.
2–(2-Methoxyphenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]-
thieno[2,3-d][1,3]oxazin-4-one (11). The title compound was pre-
2–(4-Methoxyphenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]-
thieno[2,3-d][1,3]oxazin-4-one (19). The title compound was pre-
pared starting from 5025 by Method
D and purified by
crystallization by EtOH, in 44% yield; 1H-NMR (CDCl3, 400 MHz) d
1.55–1.65 (m, 4H, cycloheptane CH2), 1.80–1.85, 2.80–2.85, and
3.10–3.15 (m, each 2H, cycloheptane CH2), 3.80 (s, 3H, OCH3), 7.05
and 8.05 (d, J ¼ 9.1 Hz, each 2H, aromatic CH); 13 C NMR (DMSO-d6,
101 MHz): d 26.9, 27.5, 27.7, 29.3, 32.0, 55.9, 114.9, 116.6, 122.0,
130.1, 137.3, 138.1, 155.3, 158.4, 160.3, 163.2; HRMS: m/z calcd for
C18H17NO3S 328.1008 (M þ H)þ, found 328.1004.
pared starting from 4528 by Method
D and purified by
crystallization by EtOH, in 55% yield; 1H-NMR (CDCl3, 400 MHz) d
1.60–1.75 (m, 4H, cycloheptane CH2), 1.85–2.00, 2.75–3.00, and
3.10–3.25 (m, each 2H, cycloheptane CH2), 4.00 (s, 3H, OCH3),
6.90–7.10 (m, 2H, aromatic CH), 7.45 (dt, J ¼ 5.7 and 6.5 Hz, 1H,
aromatic CH), 7.85 (dd, J ¼ 1.8 and 7.6 Hz, 1H, aromatic CH); 13 C
NMR (DMSO-d6, 101 MHz): d 26.9, 27.5, 27.7, 29.3, 32.0, 55.9, 116.6,
118.2, 120.3, 122.0, 128.8, 135.3, 137.4, 139.2, 155.4, 159.4, 160.4,
163.1; HRMS: m/z calcd for C18H17NO3S 328.1008 (M þ H)þ,
found 328.1005.
2–(3,4-Dimethoxyphenyl)-6,7,8,9-tetrahydro-4H,5H-cyclo-
hepta [4,5]thieno[2,3-d][1,3]oxazin-4-one (54). The title compound
was prepared starting from 51 by Method D and purified by treat-
ment with Et2O, in 89% yield; 1H NMR (DMSO-d6, 400 MHz) d
1.50–1.60 (m, 4H, cycloheptane CH2), 1.80–1.90, 2.80–2.90, and
3.10–3.20 (m, each 2H, cycloheptane CH2), 3.85 (s, 6H, OCH3), 7.05
(d, J ¼ 8.6 Hz, 1H, aromatic CH), 7.50 (d, J ¼ 2.0 Hz, 1H, aromatic
CH), 7.70 (dd, J ¼ 2.0 and 8.5 Hz, 1H, aromatic CH).
2–(4-Chlorophenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]-
thieno[2,3-d][1,3]oxazin-4-one (13). The title compound was pre-
pared starting from 4625 by Method
D and purified by
1
2–(3,4-Dimethoxyphenyl)-5,6,7,8-tetrahydro-4H-[1]benzothieno
[2,3-d][1,3]oxazin-4-one (55). The title compound was prepared
starting from 52 by Method D and purified by treatment with
Et2O, in 61% yield; 1H NMR (DMSO-d6, 400 MHz) d 1.65–1.75
and 2.65–2.75 (m, each 4H, cyclohexane CH2), 3.75 (s, 6H,
OCH3), 7.05 (d, J ¼ 8.6 Hz, 1H, aromatic CH), 7.55 (d, J ¼ 2.0 Hz,
1H, aromatic CH), 7.70 (dd, J ¼ 2.0 and 8.5 Hz, 1H, aro-
matic CH).
crystallization by EtOH, in 68% yield; H NMR (DMSO-d6, 400 MHz):
d 1.50–1.70 (m, 4H, cycloheptane CH2), 1.75–1.85 (m, 2H, cyclohep-
tane CH2), 2.80–2.90 and 3.05–3.15 (m, each 2H, cycloheptane
CH2), 7.55 (d, J ¼ 8.6 Hz, 2H, aromatic CH), 8.05 (d, J ¼ 8.6 Hz, 2H,
aromatic CH), 13 C NMR (DMSO-d6, 101 MHz): d 27.0, 27.6, 27.7,
29.5, 32.0, 120.4, 129.7, 129.8, 137.6, 137.9, 139.5, 155.4, 158.8,
160.7, 162.3; HRMS: m/z calcd for C17H14ClNO2S 332.0513
(M þ H)þ, found 332.0511.
2–(3,4-Dimethoxyphenyl)-6,7-dihydro-4H,5H-cyclopenta[4,5]-
thieno[2,3-d][1,3]oxazin-4-one (56). The title compound was pre-
pared starting from 53 by Method D and purified by flash
2-Phenyl-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]thieno[2,3-d]
[1,3]oxazin-4-one (14). The title compound was prepared starting
from 47 by Method D and purified by crystallization by EtOH, in
1
1
chromatography eluting with CHCl3, in 56% yield; H NMR (CDCl3,
68% yield; H NMR (DMSO-d6, 400 MHz): d 1.50–1.65 (m, 4H, cyclo-
400 MHz) d 2.45 (quin, J ¼ 7.0 Hz, 1H, cyclopentane CH2), 2.95
heptane CH2), 1.75–1.85 (m, 2H, cycloheptane CH2), 2.80–2.90 and
3.05–3.15 (m, each 2H, cycloheptane CH2), 7.50–7.65 (m, 3H, (quin, J ¼ 7.0 Hz, 2H, cyclopentane CH2), 3.90 and 3.95 (s, each 3H,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
59
1
OCH3), 6.85 (d, J ¼ 8.5 Hz, 1H, aromatic CH), 7.70 (d, J ¼ 2.0 Hz, 1H, chromatography eluting with MeOH/CHCl3 (7.5%), in 61% yield; H
aromatic CH), 7.90 (dd, J ¼ 2.0 and 8.5 Hz, 1H, aromatic CH).
NMR (DMSO-d6, 400 MHz) d 2.35 (quin, J ¼ 7.1 Hz, 1H, cyclopentane
2–(3,4-Dihydroxyphenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta CH2), 2.90 (quin, J ¼ 7.1 Hz, 2H, cyclopentane CH2), 6.85 (d,
[4,5]thieno[2,3-d][1,3]oxazin-4-one (10). The title compound was
J ¼ 8.3 Hz, 1H, aromatic CH), 7.45 (dd, J ¼ 2.0 and 8.3 Hz, 1H, aro-
prepared starting from 54 by Method A and purified by crystalliza-
matic CH), 7.50 (d, J ¼ 2.0 Hz, 1H, aromatic CH), 9.5 and 9.95 (s,
tion by a cyclohexane/EtOAc mixture, in 65% yield; 1H NMR each 1H, OH); 13 C NMR (DMSO-d6, 101 MHz) d 27.7, 28.7, 29.5,
(DMSO-d6, 400 MHz) d 1.60–1.70 (m, 4H, cycloheptane CH2),
1.80–1.90, 2.80–2.90, and 3.10–3.20 (m, each 2H, cycloheptane
112.8, 115.0, 116.4, 120.6, 121.0, 138.9, 140.6, 146.0, 150.9, 155.1,
158.8, 167.7; HRMS: m/z calcd for C15H11NO4S 302.0488 (M þ H)þ,
CH2), 6.90 and 7.45 (d, J ¼ 8.4 Hz, each 1H, aromatic CH), 7.50 (s, found 302.0493.
1H, aromatic CH), 9.5 and 9.95 (bs, each 1H, OH); 13 C NMR
Ethyl
2-[(3,4-dimethoxybenzoyl)amino]-1-benzothiophene-3-
(DMSO-d6, 101 MHz) d 27.0, 27.6, 27.8, 29.4, 32.1, 115.0, 116.1, carboxylate (57). A mixture of 43 (0.54 g, 1.38 mmol), and DDQ
116.3, 116.4, 120.6, 120.9, 137.2, 137.7, 146.0, 150.9, 155.4, 158.8, (0.47 g, 2.0 mmol) in benzene (15 ml) was refluxed for 24 h. The
160.8; HRMS: m/z calcd for C17H15NO4S 330.0801 (M þ H)þ, reaction mixture was then poured into a saturated solution of
found 330.0809.
NaHCO3 and extracted with EtOAc. The organic layers were evapo-
rated to dryness obtaining a solid which was purified by flash
2–(2-Hydroxyphenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]-
thieno[2,3-d][1,3]oxazin-4-one (12). The title compound was pre- chromatography eluting with EtOAc/petroleum ether (15%), to
pared starting from 11 by Method and purified by
crystallization by EtOH, in 83% yield; H NMR (DMSO-d6, 400 MHz):
give 57 (0.5 g, 93%); 1H NMR (DMSO-d6, 400 MHz)
(t, J ¼ 7.0 Hz, 3H, CH2CH3), 3.80 (s, 3H, OCH3), 4.45 (q, J ¼ 7.0 Hz,
d 1.45
A
1
d 1.55–1.70 (m, 4H, cycloheptane CH2), 1.80–1.90 (m, 2H, cyclohep- 2H, CH2CH3), 7.15–7.50 (m, 5H, aromatic CH), 7.90 and 8.20
tane CH2), 2.80–2.90 and 3.05–3.15 (m, each 2H, cycloheptane
(d, J ¼ 7.8 Hz, each 1H, aromatic CH), 12.40 (s, 1H, NH).
CH2), 6.90–7.05 (m, 2H, aromatic CH), 7.45 (dt, J ¼ 1.6 and 7.8 Hz,
2-[(3,4-Dimethoxybenzoyl)amino]-1-benzothiophene-3-carbox-
1H, aromatic CH), 7.85 (dd, J ¼ 1.6 and 8.4 Hz, 1H, aromatic CH), ylic acid (58). The title compound was prepared starting from 57
7.60–7.70 (m, 1H, aromatic CH), 11.50 (s, 1H, OH); 13 C NMR by Method C (overnight) and purified by flash chromatography
(DMSO-d6, 101 MHz): d 27.0, 27.6, 27.7, 29.4, 32.0, 118.1, 120.2, eluting with MeOH/CHCl3 (2%), in 92% yield; 1H NMR (DMSO-d6,
128.7, 135.2, 137.5, 139.2, 155.1, 155.8, 159.4, 162.0. HRMS: m/z 400 MHz) d 3.80 (s, 3H, OCH3), 7.15–7.45 (m, 3H, aromatic CH),
calcd for C17H15NO3S 314.0852 (M þ H)þ, found 314.0851.
2–(3-Hydroxyphenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]-
thieno[2,3-d][1,3]oxazin-4-one (16). The title compound was pre-
7.50–7.60 (m, 2H, aromatic CH), 7.90 (d, J ¼ 7.7 Hz, each 1H, aro-
matic CH), 8.40 (d, J ¼ 8.1 Hz, each 1H, aromatic CH), 12.40 and
12.85 (s, each 1H, NH and COOH).
pared starting from 18 by Method
A
and purified by
2–(3,4-Dimethoxyphenyl)-4H-[1]benzothieno[2,3-d][1,3]oxazin-4-
1
crystallization by EtOH, in 91% yield; H NMR (DMSO-d6, 400 MHz): one (59). The title compound was prepared starting from 58 by
d 1.50–1.70 (m, 4H, cycloheptane CH2), 1.75–1.85 (m, 2H, cyclohep- Method D and purified by treatment with Et2O, in 77% yield; 1H
tane CH2), 2.80–2.90 and 3.05–3.15 (m, each 2H, cycloheptane NMR (DMSO-d6, 400 MHz) d 3.80 (s, 3H, OCH3), 7.15 (d, J ¼ 8.6 Hz,
CH2), 7.00 (dd, J ¼ 2.5 and 8.2 Hz, 1H, aromatic CH), 7.35 (t, 1H, aromatic CH), 7.50–7.70 (m, 3H, aromatic CH), 7.80
J ¼ 7.9 Hz, 1H, aromatic CH), 7.45–7.55 (m, 2H, aromatic CH), 9.90 (d, J ¼ 8.5 Hz, 1H, aromatic CH), 8.15 and 8.25 (d, J ¼ 7.3 Hz, each
(s, 1H, OH); 13 C NMR (DMSO-d6, 101 MHz): d 27.0, 27.6, 27.7, 29.5, 1H, aromatic CH).
2–(3,4-Dihydroxyphenyl)-4H-[1]benzothieno[2,3-d][1,3]oxazin-4-
32.1, 114.3, 117.3, 118.9, 120.3, 130.6, 131.0, 137.5, 139.0, 155.2,
158.2, 158.3, 159.8. HRMS: m/z calcd for C17H15NO3S 314.0852 one (22). The title compound was prepared starting from 59 by
(M þ H)þ, found 314.0855.
Method A (8 h) and purified by flash chromatography eluting with
2–(4-Hydroxyphenyl)-6,7,8,9-tetrahydro-4H,5H-cyclohepta[4,5]-
MeOH/CHCl3 (2%), in 80% yield; 1H NMR (DMSO-d6, 400 MHz) d
thieno[2,3-d][1,3]oxazin-4-one (17). The title compound was pre- 6.95 (d, J ¼ 8.2 Hz, 1H, aromatic CH), 7.50–7.70 (m, 4H, aromatic
pared starting from 19 by Method
A
and purified by CH), 8.15 and 8.25 (d, J ¼ 7.8 Hz, each 1H, aromatic CH), 9.65 and
1
crystallization by EtOH, in 64% yield; H NMR (DMSO-d6, 400 MHz): 10.20 (s, each 1H, OH); 13 C NMR (DMSO-d6, 101 MHz) d 110.4,
d 1.50–1.65 (m, 4H, cycloheptane CH2), 1.75–1.85 (m, 2H, cyclohep- 115.5, 116.4, 120.2, 121.9, 123.4, 123.7, 126.7, 126.9, 133.2, 134.9,
tane CH2), 2.75–2.85 and 3.05–3.15 (m, each 2H, cycloheptane 146.1, 151.8, 154.6, 161.7, 167.0; HRMS: m/z calcd for C16H9NO4S
CH2), 6.85 (d, J ¼ 8.6 Hz, 2H, aromatic CH), 7.95 (d, J ¼ 8.6 Hz, 2H, 312.0331 (M þ H)þ, found 312.0336.
2-Chlorocyclohept-1-ene-1-carbonitrile (61). A solution of 6035
aromatic CH), 10.50 (s, 1H, OH); 13 C NMR (DMSO-d6, 101 MHz): d
27.0, 27.6, 27.8, 29.4, 32.1, 116.3, 120.4, 130.3, 137.2, 137.8, 155.4, (3.15 g, 19.85 mmol) in NMP (10 ml) was added of NH2OH hydro-
chloride (1.65 g, 28.83 mmol) and it was maintained at 115 ꢃC for
8 h. After cooling, the reaction mixture was poured into ice/water
and extracted with EtOAc. The organic layers were evaporated to
158.8, 160.7, 162.3. HRMS: m/z calcd for C17H15NO3S 314.0852
(M þ H)þ, found 314.0848.
2–(3,4-Dihydroxyphenyl)-5,6,7,8-tetrahydro-4H-[1]benzothieno
[2,3-d][1,3]oxazin-4-one (20). The title compound was prepared dryness obtaining a residue that was purified by flash chromatog-
starting from 55 by Method A and purified by flash chromatog- raphy eluting with EtOAc/petroleum ether (5%), to give 61 (1.5 g,
raphy eluting with MeOH/CHCl3 (7.5%); in 30% yield; 1H NMR 50%); 1H NMR (CDCl3, 400 MHz) d 1.55–1.65 (m, 4H, cycloheptane
(DMSO-d6, 400 MHz) d 1.70–1.80 and 2.70–2.80 (m, each 4H, cyclo- CH2), 1.70–1.75 (m, 2H, cycloheptane CH2), 2.35–2.40 and
hexane CH2), 6.85 (d, J ¼ 8.3 Hz, 1H, aromatic CH), 7.45 (dd, J ¼ 2.0 2.65–2.70 (m, each 2H, cycloheptane CH2); 13 C NMR (CDCl3,
and 8.3 Hz, 1H, aromatic CH), 7.50 (d, J ¼ 2.0 Hz, 1H, aromatic CH), 101 MHz) d 24.4, 25.5, 30.6, 31.3, 39.4, 114.0, 118.0, 152.4.
9.5 and 9.95 (s, each 1H, OH); 13 C NMR (DMSO-d6, 101 MHz) d
Ethyl 3-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-2-
21.9, 22.7, 24.8, 25.2, 115.0, 115.4, 116.4, 120.6, 121.0, 131.7, 133.6, carboxylate (62). A mixture of 61 (1.5 g, 9.6 mmol) and K2CO3
(1.3 g, 9.6 mmol) in MeOH (25 ml)/THF (5 ml) mixture was added of
ethyl thioglycolate (1.05 ml, 9.6 mmol) and it was refluxed for 16 h.
After cooling, the reaction mixture was poured into ice/water
146.0, 151.0, 154.9, 159.1, 162.6; HRMS: m/z calcd for C16H13NO4S
316.0644 (M þ H)þ, found 316.0651.
2–(3,4-Dihydroxyphenyl)-6,7-dihydro-4H,5H-cyclopenta[4,5]thieno
[2,3-d][1,3]oxazin-4-one (21). The title compound was prepared obtaining a precipitate that was filtered to give 1.2 g of a mixture
starting from 56 by Method and purified by flash of 62 and a side product (ratio 2:1), which could not be easily
A

60
S. MASSARI ET AL.
separated by chromatography. While only a small sample of pure
3-[(3,4-Dimethoxybenzoyl)amino]-1-benzothiophene-2-carbox-
compound 62 was obtained and used for its characterisation, ylic acid (68). The title compound was prepared starting from 67
by Method C (4 h) and purified by treatment with Et2O, in 58%
yield; 1H NMR (DMSO-d6, 400 MHz) d 3.90 (s, 6H, OCH3), 7.10 (d,
J ¼ 8.6 Hz, 1H, aromatic CH), 7.40–7.80 (m, 5H, aromatic CH), 8.00
(d, J ¼ 7.9 Hz, 1H, aromatic CH), 10.30 (s, 1H, NH), 13.50 (bs,
1H, COOH).
2–(3,4-Dimethoxyphenyl)-4H-[1]benzothieno[3,2-d][1,3]oxazin-4-
one (69). The title compound was prepared starting from 68 by
Method D and purified by treatment with Et2O, in 88% yield; 1H
NMR (DMSO-d6, 400 MHz) d 3.95 and 3.90 (s, each 3H, OCH3), 7.20
(d, J ¼ 8.6 Hz, 1H, aromatic CH), 7.60–7.80 (m, 3H, aromatic CH),
7.85 (dd, J ¼ 1.9 and 8.5 Hz, 1H, aromatic CH), 8.20 (d, J ¼ 8.0 Hz,
1H, aromatic CH), 8.35 (d, J ¼ 7.6 Hz, 1H, aromatic CH).
impure compound 62 was used in the next step without further
purification; 1H NMR (CDCl3, 400 MHz) d 1.30 (t, J ¼ 7.0 Hz, 3H,
CH2CH3), 1.60–1.75 (m, 4H, cycloheptane CH2), 1.80–1.90,
2.40–2.45, and 3.70–3.75 (m, each 2H, cycloheptane CH2), 4.25 (q,
J ¼ 7.0 Hz, 2H, CH2CH3), 5.45 (bs, 2H, NH2).
General procedure for carbodiimide formation (method E)
A solution of the appropriate synthones (1.0 equiv) and Et3N (1.2
equiv) in dry CH2Cl2 was added of 3,4-dimethoxybenzoyl chloride
(1.5 equiv). The reaction mixture was maintained at r.t. until no
starting material was detected by TLC. After cooling, the reaction
mixture was poured into ice/water and extracted with CH2Cl2. The
organic layers evaporated to dryness, obtaining a residue that was
purified as described below.
3-[(3,4-Dimethoxybenzoyl)amino]-5,6,7,8-tetrahydro-4H-cyclohepta
[b]thiophene-2-carboxylic acid (64). The title compound was pre-
pared starting from 62 by Method E (24 h) to give compound 63
(analogously to compound 62, compound 63 was obtained in
mixture with a side product that could not be purified by chroma-
tography and was used in the next step without further purifica-
tion) followed by Method C (8 h) but by applying a different
workup. In particular, after cooling, the reaction mixture was
poured into ice/water and extracted with EtOAc, in order to
extract the side compound. The water layer was then acidified
with 2 N HCl obtaining a precipitate, which was filtered and
treated with Et2O, to give 64 (0.85 g, 23% overall yield); 1H NMR
(CDCl3, 400 MHz) d 1.50–1.70 (m, 4H, cycloheptane CH2), 1.75–1.85,
2.40–2.50, and 2.75–2.85 (m, each 2H, cycloheptane CH2), 3.80 (s,
6H, OCH3), 7.10 (d, J ¼ 8.4 Hz, 1H, aromatic CH), 7.50 (s, 1H, aro-
matic CH), 7.65 (d, J ¼ 8.4 Hz, 1H, aromatic CH), 9.80 and 12.75 (s,
each 1H, NH and COOH).
2–(3,4-Dihydroxyphenyl)-4H-[1]benzothieno[3,2-d][1,3]oxazin-4-
one (24). The title compound was prepared starting from 69 by
Method A (overnight) and purified by flash chromatography elut-
ing with MeOH/CHCl3 (2%), in 38% yield; 1H NMR (DMSO-d6,
400 MHz) d 6.95 (d, J ¼ 8.3 Hz, 1H, aromatic CH), 7.60–7.75 (m, 4H,
aromatic CH), 8.20 and 8.30 (d, J ¼ 8.0 Hz, each 1H, aromatic CH),
9.60 and 10.10 (s, each 1H, OH); 13 C NMR (DMSO-d6, 101 MHz) d
115.0, 115.3, 116.3, 120.6, 121.4, 124.1, 124.5, 126.4, 130.5, 133.3,
141.9, 146.0, 151.2, 153.0, 156.1, 161.8; HRMS: m/z calcd for
C16H9NO4S 312.0331 (M þ H)þ, found 312.0336.
Ethyl 2-aminocyclohept-1-ene-1-carboxylate (71). A mixture of
7037 (1.0 g, 5.4 mmol) and ammonium acetate (2.0 g, 27.1 mmol) in
MeOH (15 ml) was maintained at r.t. overnight. Then, the reaction
mixture was evaporated to dryness, obtaining a white solid, which
was treated with CH2Cl2, filtered, and washed with ample CH2Cl2.
The combined CH2Cl2 was washed with water and evaporated to
1
dryness, to give 71 (0.88 g, 88%); H NMR (CDCl3, 400 MHz) d 1.25
(t, J ¼ 7.0 Hz, 3H, CH2CH3), 1.40–1.50 (m, 2H, cycloheptane CH2),
1.60–1.70 (m, 4H, cycloheptane CH2), 2.25–2.30 and 2.45–2.50 (m,
each 2H, cycloheptane CH2), 4.10 (q, J ¼ 7.0 Hz, 2H, CH2CH3).
Ethyl 2-[(3,4-dimethoxybenzoyl)amino]cyclohept-1-ene-1-carb-
oxylate (72). The title compound was prepared starting from 71
by Method E (24 h) and purified by flash chromatography eluting
with EtOAc/petroleum ether (5%), in 32% yield; 1H NMR (CDCl3,
400 MHz) d 1.25 (t, J ¼ 7.0 Hz, 3H, CH2CH3), 1.50–1.60 (m, 2H, cyclo-
heptane CH2), 1.70–1.80 (m, 4H, cycloheptane CH2), 2.55–2.60 and
3.20–3.25 (m, each 2H, cycloheptane CH2), 3.90 (s, 6H, OCH3), 4.20
(q, J ¼ 7.0 Hz, 2H, CH2CH3), 6.90–6.95 (m, 1H, aromatic CH),
7.55–7.60 (m, 2H, aromatic CH), 12.25 (bs, 1H, NH).
2–(3,4-Dimethoxyphenyl)-7,8,9,10-tetrahydrocyclohepta[4,5]-
thieno[3,2-d][1,3]oxazin-4(6H)-one (65). The title compound was
prepared starting from 64 by Method D and purified by treatment
with Et2O, in 61% yield; 1H NMR (DMSO-d6, 400 MHz) d 1.60–1.75
(m, 4H, cycloheptane CH2), 1.80–1.90, (m, 2H, cycloheptane CH2),
2.90–3.00 (m, 4H, cycloheptane CH2), 3.85 (s, 6H, OCH3), 7.15 (d,
J ¼ 8.6 Hz, 1H, aromatic CH), 7.70 (d, J ¼ 2.0 Hz, 1H, aromatic CH),
7.80 (dd, J ¼ 2.0 and 8.6 Hz, 1H, aromatic CH).
2–(3,4-Dihydroxyphenyl)-7,8,9,10-tetrahydrocyclohepta[4,5]-
thieno[3,2-d][1,3]oxazin-4(6H)-one (23). The title compound was
prepared starting from 65 by Method A (6 h) and purified by flash
chromatography eluting with MeOH/CHCl3 (2%), in 67% yield; 1H
NMR (DMSO-d6, 400 MHz) d 1.50–1.70 (m, 4H, cycloheptane CH2),
1.80–1.90, 2.80–2.85, and 2.85–2.90 (m, each 2H, cycloheptane
CH2), 6.80 (d, J ¼ 8.3 Hz, 1H, aromatic CH), 7.50 (dd, J ¼ 2.1 and
8.3 Hz, 1H, aromatic CH), 7.60 (d, J ¼ 2.1 Hz, 1H, aromatic CH), 9.50
and 9.90 (s, each 1H, OH); 13 C NMR (DMSO-d6, 101 MHz) d 26.2,
27.1, 27.6, 31.3, 32.2, 110.9, 115.2, 116.3, 120.9, 121.0, 137.2, 145.9,
150.9, 154.4, 155.2, 156.2, 161.6; HRMS: m/z calcd for C17H15NO4S
330.0801 (M þ H)þ, found 330.0806 (M þ Hþ).
2-[(3,4-Dimethoxybenzoyl)amino]cyclohept-1-ene-1-carboxylic
acid (73). The title compound was prepared starting from 72 by
Method C (overnight) and purified by treatment with Et2O, in
100% yield; 1H NMR (DMSO-d6, 400 MHz) d 1.40–1.50 (m, 2H,
cycloheptane CH2), 1.55–1.75 (m, 4H, cycloheptane CH2), 2.45–2.50
and 3.05–3.10 (m, each 2H, cycloheptane CH2), 3.75 (s, 6H, OCH3),
7.05 (d, J ¼ 8.3 Hz, 1H, aromatic CH), 7.40–7.50 (m, 2H, aromatic
CH), 12.10 (s, 1H, NH).
2–(3,4-Dimethoxyphenyl)-6,7,8,9-tetrahydrocyclohepta[d][1,3]ox-
azin-4(5H)-one (74). The title compound was prepared starting
from 73 by Method D and purified by treatment with Et2O, in
1
35% yield; H NMR (DMSO-d6, 400 MHz) d 1.50–1.65 (m, 4H, cyclo-
Ethyl
3-[(3,4-dimethoxybenzoyl)amino]-1-benzothiophene-2-
heptane CH2), 1.70–1.80 (m, 2H, cycloheptane CH2), 2.60–2.65 and
2.70–2.75 (m, each 2H, cycloheptane CH2), 3.80 (s, 6H, OCH3), 7.10
(d, J ¼ 8.6 Hz, 1H, aromatic CH), 7.55 (d, J ¼ 2.0 Hz, 1H, aromatic
CH), 7.70 (dd, J ¼ 2.0 and 8.6 Hz, 1H, aromatic CH),
carboxylate (67). The title compound was prepared starting from
6636 by Method E (48 h) and purified by treatment with Et2O, in
89% yield; 1H NMR (CDCl3, 400 MHz) d 1.40 (t, J ¼ 7.0 Hz, 3H,
CH2CH3), 3.95 (s, 6H, OCH3), 4.40 (q, J ¼ 7.0 Hz, 2H, CH2CH3), 6.95
(d, J ¼ 8.2 Hz, 1H, aromatic CH), 7.40–7.55 (m, 2H, aromatic CH),
7.65–7.80 (m, 3H, aromatic CH), 8.25 (d, J ¼ 7.7 Hz, 1H, aromatic
CH), 10.50 (s, 1H, NH).
2–(3,4-Dihydroxyphenyl)-6,7,8,9-tetrahydrocyclohepta[d][1,3]oxa-
zin-4(5H)-one (25). The title compound was prepared starting
from 74 by Method A (overnight) and purified by flash chroma-
tography eluting with MeOH/CHCl3 (2%), in 39% yield; 1H NMR

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
61
(DMSO-d6, 400 MHz) d 1.50–1.65 (m, 4H, cycloheptane CH2), a linear range of dose-response curve: 6 ng wt RT; 19 ng V108F RT;
1.70–1.80 (m, 2H, cycloheptane CH2), 2.60–2.65 and 2.70–2.75 (m, 45 ng Y188A RT; 30 ng W229A RT; A502F RT. The Yonetani-
Theorell analysis was performed as previously described41,42
.
each 2H, cycloheptane CH2), 6.80 (d, J ¼ 8.3 Hz, 1H, aromatic CH),
7.45 (d, J ¼ 8.3 Hz, 1H, aromatic CH), 7.50 (s, 1H, aromatic CH), 9.50
and 9.90 (s, each 1H, OH); 13 C NMR (DMSO-d6, 101 MHz) d 24.5,
24.8, 26.0, 31.6, 37.4, 115.0, 115.8, 116.3, 119.7, 120.8, 145.9, 151.0,
160.1, 160.6, 167.3; HRMS: m/z calcd for C15H15NO4 274.1080
(M þ H)þ, found 274.1085.
Site-directed mutagenesis
The QuikChange mutagenesis kit (Agilent Technologies Inc., Santa
Clara, CA)was used to introduce amino acid substitutions into the
p66 HIV-1 RT subunit coded in a p6HRT-prot plasmid by following
the manufacturer’s instructions.
2–(3,4-Dihydroxyphenyl)-4H-3,1-benzoxazin-4-one (26). The title
compound was prepared starting from 7538 by Method A, with
the exception that toluene was used as a solvent and the reaction
was refluxed for 1 h. The compound was purified by flash chroma-
tography eluting with MeOH/CHCl3 (2%) and obtained in 24%
yield; 1H NMR (DMSO-d6, 400 MHz) d 6.85 (d, J ¼ 8.3 Hz, 1H, aro-
matic CH), 7.45–7.50 and 7.55–7.60 (m, each 2H, aromatic CH),
7.85 (dt, J ¼ 1.2 and 8.3 Hz, 1H, aromatic CH), 8.05 (dd, J ¼ 1.2 and
7.8 Hz, 1H, aromatic CH), 9.50 and 9.90 (s, each 1H, OH); 13 C NMR
(DMSO-d6, 101 MHz) d 115.2, 116.2, 116.8, 120.9, 121.1, 126.9,
128.2, 128.4, 137.2, 145.9, 147.2, 150.8, 157.1, 159.9; HRMS: m/z
calcd for C14H9NO4 256.0611 (M þ H)þ, found 256.0618.
Detection of protein inhibitor interactions by differential
scanning fluorimetry
Differential Scanning Fluorimetry assay was used to determine the
thermal stability of the protein according to Nettleship et al.43. In
a MJ Research Opticon 2 qPCR (Bio-Rad) 96-well plate 100 ng/ml of
HIV-1 RT enzyme was incubated alone or in presence of increasing
concentrations of compound 22 or 24, using the interface inhibi-
tor compound 7644 as a control in 50 ml of final volume of reac-
tion buffer containing 20 mM HEPES, pH 7.5, 10 mM MgCl2,
2–(3,4-Dihydroxyphenyl)quinazolin-4(3H)-one (27). A mixture of
2-aminobenzamide (0.5 g, 3.6 mmol), 3,4-dihydroxybenzaldehyde
(0.6 g, 4.4 mmol), and I2 (1.2 g, 4.7 mmol) in CH3CN (25 ml) was
maintained at r.t. overnight. Then, the reaction mixture was
poured into a solution of 5% Na2S2O3 obtaining a precipitate that
was filtered and purified by flash chromatography eluting with
MeOH/CHCl3 (5%), to give 27 (0.23 g, 22%); 1H NMR (DMSO-d6,
400 MHz) d 6.80 (d, J ¼ 8.3 Hz, 1H, aromatic CH), 7.40 (t, J ¼ 7.5 Hz,
1H, aromatic CH), 7.50 (dd, J ¼ 2.0 and 8.3 Hz, 1H, aromatic CH),
7.60–7.65 (m, 2H, aromatic CH), 7.70–7.80 (m, 1H, aromatic CH),
8.05 (d, J ¼ 7.5 Hz, 1H, aromatic CH), 9.30 and 9.65 (bs, each 1H,
OH), 12.20 (s, 1H, NH); 13 C NMR (DMSO-d6) d 115.5, 115.6, 119.9,
120.9, 123.9, 126.1, 126.2, 127.5, 134.8, 145.7, 149.4, 152.5, 162.6,
165.1; HRMS: m/z calcd for C14H10N2O3 255.0770 (M þ H)þ,
found 255.0778.
100 mM NaCl, and
a 1:1000 dilution of Sypro Orange dye
(Invitrogen). The mixture was heated from 30 to 90 ꢃC in incre-
ments of 0.2 ꢃC. Fluorescence intensity was measured using excita-
tion and emission wavelengths of 490 and 565 nm, respectively.
Changes in protein thermal stability (DTm) upon inhibitor binding
were analysed using the Opticon MonitorTM Software. All assays
were performed in triplicate.
Interaction between Mg21 and HIV-1 RT inhibitors
The effects of the magnesium ions were evaluated by mean of
spectrophotometric method as reported45, using a Ultrospec 2100
pro UV-Vis spectrophotometer (Amersham Biosciences) and
Hellma quartz cuvette with 1 cm optical path. MgCl2 1 M solution
was prepared from powder (Carlo Erba Reagents, Milano, Italy).
Each studied compound was dissolved in DMSO to a final concen-
tration of 10 mM and diluted in milliQ water to a final concentra-
tion of 100 mM. Each obtained solution was placed in a cuvette
and the UV-Vis and the spectra were recorded between 200 and
700 nm using water milliQ (with a percentage of DMSO equal to
the one present in the sample solution) as reference. Thereafter,
small volumes of MgCl2 aqueous solution were added both in the
sample and in the reference cuvettes to obtain a series of solu-
tions containing increasing concentrations of MgCl2, up to
1.25 mM, carefully pipetting to mix, and the UV-Vis spectra were
repeated. Each experiment was performed in triplicate.
HIV-1 RT expression and purification: HIV-1 RT group M
subtype B
Heterodimeric RT was expressed essentially as described19,39
.
Using a BioLogic LP system (Biorad), using a combination of
immobilised metal affinity (Ni-Sepharose high performance,
Healthcare Lifescience) and ion exchange chromatography (Hi-trap
heparin HP GE). The protein was dialysed and stored in buffer
containing 50 mM Tris HCl pH 7.0, 25 mM NaCl, 1 mM EDTA, and
50% glycerol. Catalytic activities and protein concentrations were
determined. Enzyme-containing fractions were pooled and ali-
quots were stored at ꢄ80 ꢃC.
In vitro antiviral assays
HIV-1 DNA polymerase-independent RNase H activity
determination
The evaluation of the antiviral activity of the target compounds
against HIV-1 strain IIIB in MT-4 cells was performed using the
HIV RT-associated RNase H activity was measured as described40
using as a control the RNase H selective inhibitor RDS17599, and
using different amount of enzymes according to a linear range of
dose-response curve: 20 ng of WT RT; 75 ng V108F RT; 50 ng
Y188A RT; 30 ng W229A RT; 100 ng A502F RT.
MTT assay as previously described46,47
.
Ligand preparation
Theoretical 3 D model of compound 22 was built with the
Maestro software48. Then, the compound was subjected to a con-
formational search protocol with MacroModel version 9.249, con-
sidering Merck molecular force fields (MMFFs)50 as force field and
HIV-1 RNA-dependent DNA polymerase activity determination
RNA-dependent DNA polymerase (RDDP) activity was measured as the implicit solvation model Generalised Born/Surface Area (GB/
described17 using and different amounts of enzymes according to SA) water51. All conformational search parameters were left as

62
S. MASSARI ET AL.
default. Therefore, the compound geometry was energy mini- anti-RNase H activity shown by the o-hydroxy derivative 12
mised using Polak-Ribier Conjugate Gradient (PRCG) method, 5000
iterations and a convergence criterion of 0.05 kcal/(mol Å).
(IC50¼18.2 lM), the meta- and para-hydroxyphenyl derivatives 16
and 17 were synthesised, also evaluating the methoxy analogues
18 and 19, respectively.
Since the biological evaluation confirmed the catechol deriva-
tive 10 as the most potent in inhibiting the RNase H function
within the cHTO series, further compounds (derivatives 20–27,
Table 2) were synthesised by fixing the catechol moiety at C-2
position of the oxazinone ring, while widely modifying the cyclo-
heptathieno portion. In particular, the cycloheptane ring was
reduced to a six- and five-membered rings in compounds 20 and
21, respectively, and was also replaced by a benzene ring (com-
pound 22). Then, the cycloheptathiophene and benzothiophene
moieties were reversed in the cycloeptathieno[3,2-d]oxazinone
and benzothieno[3,2-d]oxazinone derivatives 23 and 24, respect-
ively. Moreover, the size of the scaffold was further reduced
by deleting the thiophene ring, as in bicyclic derivatives 25 and
26. In the case of compound 26, the corresponding derivative 27
was also synthesised by replacing the oxygen with a nitro-
gen atom.
Protein preparation
The coordinates for RTs were taken from the RCSB Protein Data
Bank52 (PDB codes 1vrt53, 2zd154, 1ep455, 3qo954, 1rti53, 1tv656,
3lp257. The proteins were prepared by means of Maestro Protein
Preparation Wizard default setting. Original water molecules were
removed. A502F-RT mutated enzyme was generated starting from
wt protein and optimized17.
Docking protocol
Molecular docking studies were performed using QMPL workflow
protocol58,59 applying the same settings previously reported19.
The same protocol was applied for all simulations.
Post-docking protocol
In order to better take into account the induced fit phenomenon
occurring at the ligand binding domain, the most energy favoured
generated complexes were fully optimised with 10,000 steps of
Chemistry
The synthesis of the cHTC derivatives 225, 331, 4, and 528, was
accomplished as reported in Scheme 1, by applying
a
the Polak-Ribier conjugate gradient (PRCG) minimisation method
considering OPLS_2005 force field and GB/SA implicit water. The
optimisation process was performed up to the derivative conver-
Knoevenagel condensation. In particular, reaction of cyclohepta-
none with 2-cyanoacetamides 2861, 2962, and 3062, prepared by
reaction of 3–(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile61
with the appropriate amines in toluene, followed by cyclisation
performed in the presence of sulphur and N,N-diethylamine in
EtOH, provided compounds 2, 3, and 5. Compound 4 was
obtained by O-demethylation of the methoxy derivative 3.
Starting from synthone 3132, synthesised by reacting cyclohep-
tanone with 2-cyanoacetamide, the coupling reaction with the
appropriate benzoyl chloride led to the target compounds 7 and
9, and intermediate 32, of which methoxy derivatives 7 and 32
were O-demethylated yielding hydroxy compounds 6 and 8
(Scheme 2).
ꢀ
gence criterion equal to 0.1 kJ/(mol Å).
Graphics
The resulting complexes were considered for the binding modes
graphical analysis with Pymol60 and Maestro Ligand Interaction
visualization48.
Results and discussion
Design
The synthesis of cycloheptathieno[2,3-d][1,3]oxazin-4-one deriv-
atives 10–19, tetrahydrobenzothieno[2,3-d][1,3]oxazin-4-one 20,
and cyclopentathieno[2,3-d][1,3]oxazin-4-one 21 was accom-
plished, as outlined in Scheme 3, starting from synthones 3333,
3434, and 3534, respectively. Synthones 33–35 were obtained by
applying the one-pot Gewald reaction by reacting the appropriate
cycloalkyl ketones with ethyl cyanoacetate in the presence of sul-
fur and N,N-diethylamine in EtOH. Coupling reaction of synthones
33–35 with the appropriate acyl chlorides in pyridine furnished
intermediates 3628, 3733, 3833, 3925, 40, 4125, and 42–44, which
were then hydrolysed under basic conditions to give correspond-
ing acids 4528, 4625, 47, 4825, 49, 5025, and 51–53. The successive
cyclisation in acetic anhydride at 100 ꢃC under MW irradiations fur-
nished compounds 11, 13–15, 18, 19, and 54–56. O-demethyla-
tion of monomethoxy derivatives 11, 18, 19 and dimethoxy
derivatives 54–56 by using BBr3 in CH2Cl2 gave hydroxy deriva-
tives 10, 12, 16, 17, 20, and 21.
Compound 1 and most of the cHTCs previously reported were
characterised by two aromatic rings at both C-2 and C-3 positions
of the cHTC scaffold, but they could not be essential for the activ-
ity as compared to known inhibitors29. Thus, the two aromatic
moieties were alternatively removed by exploring substituents
endowed with different electronic and steric characters (see
Table 1 for the structures). In particular, by removing the catechol
ring and fixing the amino group at the C-2 position, the C-3 pyri-
dine moiety was maintained in compound 2 and then replaced
by o-methoxyphenyl, o-hydroxyphenyl, and p-chlorophenyl rings
(compounds 3–5, respectively). The same three substituents along
with the catechol moiety were explored as C-2 substituents in 3-
carboxamide derivatives 6–9. Then, the presence of only one aro-
matic ring was also studied in cycloheptathienooxazinone (cHTO)
derivatives 10–13, a set of rigid analogues.
The biological evaluation of the three series of derivatives con-
firmed the catechol moiety as particularly useful to impart RNase
H inhibitory activity, especially when placed at C-2 position of the
cHTO scaffold as in compound 10 (IC50 ¼ 1.1 lM). Given the good
anti-RNase H activity of 10, the cHTO core was maintained in a
successive set of derivatives (compounds 14–19, Table 1). In par-
ticular, over the non-substituted phenyl ring (compound 14), the
Benzothieno[2,3-d][1,3]oxazin-4-one derivative 22 was synthes-
ised, as reported in Scheme 4, starting from tetrahydrobenzo-
thieno intermediate 43, which was oxidised by using 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (DDQ) in benzene to give benzo-
thieno derivative 57. The successive basic hydrolysis of compound
57 to intermediate 58, cyclisation to compound 59, and demethy-
2-fluorine derivative 15 was prepared. Moreover, based on the lation, furnished target compound 22.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
63
Table 1. Effect of cHTC and cHTO derivatives on the HIV-1 RT-associated RNase H and RDDP functions.
HIV-1 RNase H
IC50, µMa
HIV-1 RDDP
IC50, µMb
Compd
R
1
2
0.84 0.10
20.1 2.81
20.9 1.70
>100 (74%)c
3
91.3 1.5
51.0
78.0
7
9
4
5
6
>100
16.5 1.25
2.60 0.4
39.0 7
>100 (60%)c
7
14.0 6.6
>100 (51%)c
>100 (61%)c
8
9
66.4 4.7
62.7 18.2
1.10 0.3
56.0
4
10
9.10 1.14
>100
11
12
100
5
18.2 2.6
>100
13
14
97.0 11
>100
20.7 1.00
37.1 2.70
15
34.9 0.01
81.0 13.00
16
17
18
33.9 4.60
21.0 1.60
21.9 7.40
54.2 7.96
21.1 2.19
67.3 2.90
>100
19
14.1 3.03
8.06 0.57
-
RDS1759 9
efavirenz
0.023 0.0016
aIC50: compound concentration required to inhibit the HIV-1 RT-associated RNase H activity by 50%. The table reports
the average and standard deviation of three independent experiments, made in duplicate.
bIC50: compound concentration required to inhibit the HIV-1 RT-associated RNA dependent DNA polymerase activity by
50%. The table reports the average and standard deviation of three independent experiments, made in duplicate;
cPercentage of residual enzymatic activity in the presence of 100 mM of compound.
The synthesis of cycloheptathieno[3,2-d][1,3]oxazin-4-one was used for the preparation of compound 61 by reaction
derivative 23 entailed the preparation of key intermediate 62, with hydroxylamine hydrochloride in N-methyl-2-pyrrolidinone
as shown in Scheme 5. In particular, cycloheptanone was (NMP). Intermediate 61 was then reacted with ethyl thioglyco-
reacted with DMF and POCl3 to give intermediate 6035, which late in presence of K2CO3 in a MeOH/THF mixture to give

64
S. MASSARI ET AL.
Table 2. Effect of oxazinone-based derivatives on the HIV-1 RT-associated RNase H and RDDP functions.
HIV-1 RNase H
IC50, µMa
HIV-1 RDDP
IC50, µMb
Compd
10
Core
1.10 0.3
3.35 0.63
1.00 0.50
0.53 0.03
9.10 1.14
13.1 2.66
27.2 2.85
2.90 0.20
20
21
22
23
24
4.86 0.57
0.96 0.02
22.6 0.17
14.6 1.93
25
26
27
4.36 0.03
3.61 1.09
3.46 0.47
>100
>100
>100
RDS1759 9
efavirenz
8.06 0.57
-
0.023 0.0016
aIC50: compound concentration required to inhibit the HIV-1 RT-associated RNase H activity by 50%. The table reports
the average and standard deviation of three independent experiments, made in duplicate.
bIC50: compound concentration required to inhibit the HIV-1 RT-associated RNA dependent DNA polymerase activity by
50%. The table reports the average and standard deviation of three independent experiments, made in duplicate.
synthone 62. Coupling reaction of 62 with 3,4-di-methoxyben- 70 with ammonium acetate in MeOH furnished compound
71, which in turn was subjected to coupling reaction with 3,4-
dimethoxybenzoyl chloride, basic hydrolysis, cyclisation, and
demethylation, furnishing target compound 25, through inter-
mediates 72–74.
zoyl chloride in CH2Cl2 in presence of Et3N furnished inter-
mediate 63, which was in turn hydrolysed, cyclized, and
demethylated to target compound 23, through intermediates
64 and 65.
As reported in Scheme 8, benzoxazin-4-one derivative 26 was
synthesised by demethylation of intermediate 7538, which was
obtained by reaction of 2-aminobenzoic acid and 3,4-dimethoxy-
benzoyl chloride in pyridine. On the other hand, quinazolin-4-one
27 was synthesised, differently from the procedure reported in lit-
erature63, by reaction of 2-aminobenzamide with 3,4-dihydroxy-
benzaldehyde in presence of I2 in CH3CN (Scheme 8).
By using an analogous procedure, benzothieno[3,2-d][1,3]oxa-
zin-4-one derivative 24 was synthesised starting from synthone
66 (Scheme 6), prepared as reported in literature36 by reaction
of 2-chlorobenzonitrile and ethyl thioglycolate in presence
of KOH in DMF. Coupling reaction of synthone 66 with 3,4-dime-
thoxybenzoyl chloride, basic hydrolysis, cyclisation, and demethy-
lation,
furnished
target
compound
24,
through
intermediates 67–69.
Cyclohepta[d][1,3]oxazin-4-one derivative 25 was synthesised,
as reported in Scheme 7, starting from compound 7037,
which was prepared by reaction of cycloheptanone and diethyl
Inhibition of RT-associated activities
The whole set of derivatives were initially evaluated for their abil-
carbonate in presence of NaH in toluene. Reaction of intermediate ity to inhibit the HIV-1 RNase H function (Table 1 and Table 2).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
65
Table 1 showed that the mono-aromatic substituted derivatives above all that the aromatic derivatives are more suitable inhibitors
are in general less potent than the previous di-substituted than the corresponding cycloheptathiophene analogues. For
cHTCs24. However, compounds 5 (IC50¼16.5 mM), 6 (IC50¼2.60 mM), the bicyclic derivatives 25 and 26, a reduced ability to inhibit the
RNase H function was observed. Finally, quinazolinone derivative
and 7 (IC50¼14.0 mM) showed a good activity with the catechol
27 showed an IC50 comparable to that of the benzoxazine ana-
logue 26, indicating that the nature of the heteroatom does not
play essential role in the interaction to the target domain.
From the biological evaluation of the oxazinone-based com-
pounds, clear SAR insights emerged: (i) the catechol resulted in a
critical moiety to achieve potent anti-RNase H activity; (ii) the
modifications made on the scaffold, such as size reduction, geom-
etry inversion, and nature of heteroatom, seem to be not critical
for the target binding; (iii) benzene derivatives emerged more
active than the cycloheptane counterparts.
To examine their specificity of action, all the compounds were
tested against the RDDP function, using as a positive control
the non-nucleoside RT inhibitor (NNRTI) efavirenz (Table 1 and
Table 2). From this assay, some active compounds emerged along
with others that did not recognise this RT function at all, leading
to add another clear structural information. Indeed, all the cat-
echol derivatives characterised by a tricyclic scaffold (compounds
10 and 20–24) showed the ability to inhibit also the RDDP activity
resulting dual-acting compounds. Derivative 22 emerged again
the best inhibitor with an IC50 of 2.90 mM. Interestingly, its struc-
tural isomer 24, which exhibited a similar anti-RNase H potency,
was significantly less potent against the RDDP function
(IC50¼14.6 mM) suggesting that the RDDP binding is more suscep-
tible to the molecular geometry. On the other hand, the bicyclic
catechol derivatives 25–27 were completely inactive against
RDDP, thus emerging as selective RNHIs.
3-carboxamide derivative 6 that emerged as the most active. With
the only exception of o-methoxyphenyl derivative 11 and p-chlor-
ophenyl derivative 13, all the tricyclic cHTOs showed RNase H
inhibitory activity with IC50 values ranging from 1.10 and 34.9 mM.
Once again, the presence of the catechol moiety granted the best
anti-RNase H activity, with derivative 10 that showed an IC50
of 1.10 mM.
The suitability of this peculiar moiety was further confirmed
and even improved by the successive series of derivatives, all
bearing the catechol moiety at C-2 position of different oxazi-
none-based scaffolds (Table 2). In particular, when compared to
compound 10, the reduction of cycloheptane ring to cyclohexane
(compound 20) slightly decreased the anti-RNase
H activity
(IC50¼3.35 mM), while its further reduction to cyclopentane
(compound 21) permitted to maintain the same biological activity
(IC50¼1.00 mM). The replacement of the cycloheptane with a ben-
zene ring improved the activity, as shown by compound 22 that
emerged as the most potent derivative of the series, with IC50 of
0.53 mM. When the geometry of the core was changed by revers-
ing the thiophene portion in compound 23, a slight decreased
anti-RNase H activity emerged, while the aromatic derivative 24
showed a potent inhibitory activity (IC50¼0.96 mM) similarly to its
analogue 22. These results suggested how the geometry of the
scaffold does not influence the interaction with the target and
Mode of action studies
Investigation of Magnesium-Complexation
Firstly, the involvement of the Mg2þ cofactors in the mechanism
of inhibition of the new RNHIs was studied by recording the UV
spectra for compound 22 together with the structurally-related
24, alone and in the presence of increasing concentrations of
MgCl2 (Figure 2(A) and B). Results showed that, differently from
RNase H active site inhibitors8, compounds 22 and 24 did not
show any shift in the maximum of absorbance (hypsochromic
effect), neither the presence of an isosbestic point. In both cases,
we observed a slight increase in absorbance across the analysed
UV-Vis spectra for concentrations of MgCl2 equal or greater than
750 lM, which could be attributed to the moderate increase of
the ionic strength of the solution.
Scheme 1. Synthetic pathway for preparation of 2–5. Reagents: (i) suitable
amine, toluene, reflux; (ii) ammonium acetate, glacial acetic acid, benzene, reflux;
(iii) sulphur, N,N-diethylamine, EtOH, 40–50ꢃC; (iv) BBr3, dry CH2Cl2, rt.
Scheme 2. Synthetic pathway for preparation of 6–9. Reagents: (i) sulphur, N,N-diethylamine, EtOH, rt; (ii) suitable benzoyl chloride, dry pyridine, rt; (iii) BBr3, dry
CH2Cl2, rt.

66
S. MASSARI ET AL.
Scheme 3. Synthetic pathway for preparation of 11–21. Reagents: (i) sulphur, N,N-diethylamine, EtOH, rt; (ii) suitable benzoyl chloride, dry pyridine, rt; (iii) LiOH, H2O/
THF (1:1), 50 ꢃC; (iv) acetic anhydride, 100 ꢃC MW; (v) BBr3, dry CH2Cl2, rt.
subunits have been shown to negatively impact on RT Tm44. Hence,
to investigate the effect of our compounds on the RT stability, differ-
ential scanning fluorimetry analysis was performed in presence of
compounds 22 and 24, using the previously reported interface
inhibitor
benzyl
2–(9-(4-ethoxyphenyl)-1,7-dimethyl-2,4-dioxo-
1,4,4a,6,7,8,9,10a-octahydropyrimido[2,1-f]purin-3(2H)-yl)acetate (com-
pound 76)44 as control (Figure 3). A dose-dependent increase in the
RT Tm was observed in presence of increasing concentration of
both the compounds, differently to compound 76 that decreased
the Tm. These data clearly indicated that the nature of the interac-
tions occurring in the RT-inhibitor complex does not destabilise the
RT conformation.
Docking studies
Scheme 4. Synthetic pathway for preparation of 22. Reagents: (i) DDQ, benzene,
reflux; (ii) LiOH, H2O/THF (1:1), 50 ꢃC; (iii) acetic anhydride, 100 ꢃC MW; (iv) BBr3, In order to investigate the possible binding mode of the newly
dry CH2Cl2, rt.
identified compounds, derivative 22 was subjected to blind dock-
ing studies by using the whole structure of the wt HIV-1 RT. In par-
ticular, the QM-polarized ligand docking protocol was applied58,59
.
Differential scanning fluorimetry
The interaction between RT and an inhibitor can be investigated by
differential scanning fluorimetry since RT inhibitors can either stabil-
ise or destabilise the RT heterodimer64 so causing either an increase
Due to flexibility of the target and the different shapes of
known inhibitors, we decided to carry out ensemble docking
experiments using seven different crystal structures19.
or a decrease in its melting temperature (Tm)65. In particular, com- The obtained compound-RT complexes showed that the
pounds reported to bind close to the interface between the two ligand could bind two different pockets within the RT structure

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
67
Scheme 5. Synthetic pathway for preparation of 23. Reagents: (i) DMF, POCl3, from 0 ꢃC to rt; (ii) NMP, NH2OH hydrochloride, 115 ꢃC; (iii) K2CO3, MeOH/THF (5:1), ethyl
thioglycolate, reflux; (iv) 3,4-dimethoxybenzoyl chloride, Et3N, dry CH2Cl2, rt; (v) LiOH, H2O/THF (1:1), 50 ꢃC; (vi) acetic anhydride, 100 ꢃC MW; (vii) BBr3, dry CH2Cl2, rt.
Scheme 6. Synthetic pathway for preparation of 24. Reagents: (i) ethyl thioglycolate, DMF, KOH, from 0 ꢃC to 80 ꢃC; (ii) 3,4-dimethoxybenzoyl chloride, Et3N, dry
CH2Cl2, rt; (iii) LiOH, H2O/THF (1:1), 50ꢃC; (iv) acetic anhydride, 100 ꢃC MW; (v) BBr3, dry CH2Cl2, rt.
(Figure 4 and Figure 5). The first binding site (pocket 1) is Site-directed mutagenesis
located close to the DNA polymerase catalytic centre partially
With the aim of verifying the insights emerged by the computa-
overlapping the binding pocket of the NNRTIs. The pocket, that
tional studies on the binding mode of the compounds, the HIV-
possess a “L shape”, could possibly accommodate the com-
1 wt RT structure was modified introducing amino acidic substitu-
pound in two different orientations (Figure 4). According to the
tions in the putative binding pocket.
docking results, it appears that compound 22 in pose A (Figure
4(a)) could make critical interactions with Y188, W229, H235,
In order to evaluate the impact of the disruption of the main
hypothesised interactions for compound 22 in pocket 1, residues
K103, and K101. Furthermore, it is stabilised by several hydro-
Y188 and W229 were substituted with an alanine residue.
phobic interactions with F227, V106, Y318, Y181, and P236. In
Moreover, V108 within the site described by Himmel et al.66 was
pose B (Figure 4(b)), compound 22 putative binding mode
replaced by a phenylalanine, with the aim to reduce the space
involves hydrogen bonds with amino acids K223, D186, and
at disposal of the compound for the binding in pose B.
W229 and many hydrophobic residues such as L228, V108,
Analogously, to investigate the effect of mutation occurring in
F227, L234, Y188, and Y183.
pocket 2, the residue A502 located in the alpha helix close to the
The second putative binding pocket (pocket 2) is in the RNase
putative binding pocket, was substituted with phenylalanine resi-
H domain, between the RNase H active site and the primer grip
due, since it was reported that such change causes a movement
of alpha helix that reduces the space between the two subunits
p51 and p66, thus hindering the entrance of the compound in
the pocket17.
Compound 22 was then tested against both RT-associated
enzymatic functions of the mutated RTs, using as comparison the
region, close to the interface of subunits p66 and p51 (Figure 5).
Docking experiments suggest that, in this site, the complex is sta-
bilised by a cation-p interaction with K424, and two hydrogen
bonds with W535 and Y532. Furthermore, other interactions,
mainly with hydrophobic residues of both chains A (p66) and B
(p51), contribute to anchoring the compound in this site.

68
S. MASSARI ET AL.
Scheme 7. Synthetic pathway for preparation of 25. Reagents: (i) NaH, diethyl carbonate, toluene, from 0 ꢃC to 100 ꢃC; (ii) ammonium acetate, MeOH, rt; (iii) 3,4-dime-
thoxybenzoyl chloride, Et3N, dry CH2Cl2, rt; (iv) LiOH, H2O/THF (1:1), 50ꢃC; (v) acetic anhydride, 100 ꢃC MW; (vi) BBr3, dry CH2Cl2, rt.
Scheme 8. Synthetic pathway for preparation of 26 and 27. Reagents: (i) 3,4-dimethoxybenzoyl chloride, dry pyridine, rt; (ii) BBr3, toluene, reflux; (iii) I2, CH3CN, rt.
Figure 2. Investigation of Magnesium-Complexation. UV–vis spectra of compounds (a) 22 and (b) 24 measured alone or in the presence of increasing concentration
of MgCl2.
wt enzyme (Figure 6). Regarding the mutations introduced within values<.0001) not significantly affecting the inhibition of the
the pocket 1, results showed that W229A did not significantly RDDP activity.
affect compound 22 inhibition of neither the RDDP or the RNase
Overall, these results support the possibility that oxazinone-
H activity. On the other hand, Y188A and V108F substitutions based compounds bind two different pockets in the RT enzyme,
impaired the inhibition of the RDDP (IC50¼9.64 mM and 10.57 mM, inhibiting the RDDP function binding on pocket 1 and the RNase
respectively, p values¼.0039 and .0074, respectively), while no sig- H function binding on pocket 2. Moreover, the results obtained
nificant effect was observed on the inhibition of the RNase H on the inhibition of the RDDP activity of the RT mutated on
function. On the contrary, the A502F mutation totally impaired pocket 1 suggest the possibility that the binding of compound 22
the RNase
H
inhibition by compound 22 (IC50>100 mM,
p
on this pocket occurs preferentially with the orientation A,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
69
partially overlapped with the NNRTIs binding site. Nevertheless, belong to the Himmel and NNRTI sites in the L-shaped cavity,
the comparable loss of RDDP activity shown by compound 22 in
the presence of V108F and Y188A substitutions (amino acids that
respectively) suggests that the compound may assume both the
poses, depending on the amino acids substitution. The hypothes-
ised binding in pocket 2 is confirmed by biochemical data on the
A502F RT, in which the amino acidic substitution prevents the
proper binding within the allosteric pocket as suggested by com-
putational modelling results. Successive docking experiments into
the mutated enzyme confirm that, in presence of this mutation,
the compound cannot be accommodated in pocket 2 (Figure 7).
This can explain the drastic decrease in RNase
tory potency.
H inhibi-
Yonetani–Theorell
In order to confirm the possibility of binding of compound 22
on the NNRTI binding pocket, we performed a Yonetani–Theorell
analysis41 on the combined effects of compound 22 and EFV
on RDDP function. Such an analysis reveals whether the binding
of the two inhibitors is or not kinetically mutually exclusive, prov-
ing if the simultaneous binding (or inhibition) of two compounds
is possible or not. Results showed that the RDDP inhibition of
compound 22 and EFV are kinetically mutually exclusive
(Figure 8).
Figure 3. Effect of compounds 22, 24, and reference 76 on the thermal stability
of p66/p51 HIV-1 RT.
Figure 4. Three-dimensional representation of putative binding modes in (a) pose A and (b) pose B of compound 22 into RT obtained by blind docking experiments
and relative 2D representation of the complexes stabilising interactions with binding pocket 1.

70
S. MASSARI ET AL.
Figure 5. Three-dimensional representation of the putative binding mode obtained by blind docking experiments of compound 22 into RT and relative 2D representa-
tion of the complexes stabilising interactions with binding pocket 2.
In our study, the intrinsic fluorescence of all the tested com-
pounds have been initially determined to exclude an interference
in the readout of the assay used for the determination of the
RNase H inhibitory activity. All the compounds emerged no fluor-
escent at the excitation/emission wavelength (490/528 nm) ana-
lysed for the product quantification of the assay. Moreover, as
reported above, the magnesium-complexation ability has been
investigated for catechol derivatives 22 and 24. Both the com-
pounds arose unable to chelate Mg2þ ions (Figure 2) ruling out
both RNase H active site inhibition and an interference with the
anti-RNase H activity assay that requires MgCl2. Finally, to discard
the hypothesis of chemical aggregation, all the compounds were
examined by using the ZINC15 remover filter (http://zinc15.dock-
ing.org/patterns/home)71 and none of them were found as poten-
tial aggregator.
Figure 6. Effects of selected amino acid substitutions in pocket 1 and 2 of HIV-1
RT in the susceptibility to compound 22 in RNase H and RDDP activity
ꢀ
ꢀꢀ
ꢀꢀꢀ
assays. p values <0.05 ( ); p values <0.01 ( ); p values <0.001 ( );
ꢀꢀꢀꢀ
p values <0.0001(
).
Conclusions
Inhibition of viral replication
Starting from derivative 1 previously reported by us as an allo-
steric RNHI, new series of compounds based on cHTC and bicyc-
lic/tricyclic oxazinone scaffolds have been developed in this study.
While in the cHTC series the deletion of one of the two aromatic
moieties of hit compound 1 was detrimental, oxazinone-based
compounds showed an efficient RNase H inhibition when func-
tionalised at the C-2 position with a catechol moiety. Among
them, the tricyclic derivatives showed, although to a different
extent, the ability to inhibit also the RDDP activity, thus resulting
dual-acting compounds. On the other hand, the bicyclic deriva-
tives were completely inactive against RDDP emerging as selective
RNHIs. Overall, the most interesting compounds were cyclohepta-
tienooxazinone derivative 10 and benzothienooxazinones 22 and
24, of which compound 22 resulted in the most potent inhibitor
of both RNase H and RDDP functions, with IC50 values of 0.53 lM
and 2.90 lM, respectively. Spectrophotometric and differential
scanning fluorimetry assays showed that the compounds do not
chelate the Mg2þ nor destabilise the RT conformation, respect-
ively, thus indicating that they are not active site neither RT heter-
odimer interface inhibitors. Docking studies suggested that they
could bind two different pockets within the RT: the first located
close to the DNA polymerase catalytic centre partially overlapping
the binding pocket of the NNRTIs, and the second in the RNase H
Compounds 10 and 20–27 were tested for their ability to inhibit
HIV-1 (IIIB strain) and HIV-2 (ROD strain) replication in acutely
infected MT-4 cells, evaluating in parallel their cytotoxicity in the
same cells. Unfortunately, none of the compounds affected the
viral replication at concentrations lower than those cytotoxic (CC50
values ranging from 27 to 300 mM), independently from their abil-
ity to inhibit only the RNase H or both the RT functions.
Liability assays
For the best inhibitors identified in this study, the presence of the
catechol moiety emerged as particularly critical in imparting
RNase H inhibitory activity. Since catechol has been counted as
one among the well-known Pan-Assay Interference Compounds
(PAINS) motifs68,69, focused studies have been performed in order
to reject the possibility that the inhibitory potency detected in the
in vitro assays could be related to an artefact. Various mechanisms
of assay interference or promiscuous behaviour have been
described as responsible for PAINS activity, including metal chela-
tion, compound fluorescence effect, redox activity, cysteine oxida-
tion, and chemical aggregation68,70
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
71
Figure 7. (a) Comparison of wt-RT and A502F-RT; (b) Three-dimensional representation of putative binding mode obtained by docking experiments of compound 22
into A502F-RT.
Figure 8. (a) Yonetani-Theorell plot of the combination of EFV and 22 on the HIV-1 RT RNA-dependent DNA polymerase activity of HIV-1 RT wild type. The enzyme
was incubated in the presence of EFV alone (᭹) or in presence of different concentrations of compound 22: 10 l (᭢) and 30 lM (w); (b) Superimposition of 22 puta-
67
tive binding mode (in green) with experimental EFV (in orange) binding mode reported in pdb 1fk9
.
domain, between the RNase H active site and the primer grip several chemotypes have been reported to inhibit HIV RNase H at
region, close to the interface of subunits p66 and p51. Site- low micromolar or even nanomolar level, but only a few of them
directed mutagenesis studies confirmed the docking results. showed significant antiviral activity in cell-based assays10. They are
Indeed, A502F substitution in pocket 2 completely impaired RNase mainly metal-chelating active site RNHIs which however inhibited
H inhibition by compound 22, and a comparable loss of RDDP also other essential enzymes within HIV replicative cycle (IN or RT
activity was observed with V108F and Y188A substitutions within DNA polymerase), thus not permitting to evaluate the exact contri-
the pocket 1 suggesting that the compound may assume two dif- bution of the RNase H inhibition to the antiviral activity. Recently,
ferent poses within NNRTI site and that described by Himmel in Vernekar et al. demonstrated that an ultrapotent RNase H biochem-
the L-shaped cavity.
ical inhibition (low nanomolar) is required to effectively compete
Unfortunately, even exhibiting the ability to inhibit RNase H against RNA:DNA duplex substrate while achieving only a moderate
activity in the sub-micromolar range, the compounds failed in level of antiviral activity (low micromolar to sub-micromolar)72. For
inhibiting the HIV replication in cellular context. This issue and the a potent RNase H active-site inhibitor devoid of anti-HIV activity,
crucial presence of the catechol moiety to achieve potent anti- the authors proposed two different binding modes, in the presence
RNase H compounds led us to determine their potential as PAINS, or absence of the substrate, with a loss of a key interaction in the
which was however ruled-out through appropriate liability assays.
Despite many hypotheses may be postulated on the lack of anti-
presence of the duplex.
We also observed a weaker anti-RNase H activity when the
HIV activity shown by the compounds herein identified, it is worth compounds were added after the RNA:DNA duplex substrate, as
to remark that this behaviour characterises most of the RNHIs iden- measured for compound 10 that was tenfold less active
tified so far10. Since 1990, when the earliest RNHIs can be traced, (IC50¼11 lM vs 1.1 lM). However, for our allosteric inhibitors, we

72
S. MASSARI ET AL.
cannot properly evaluate the binding mode in the presence/
absence of the substrate by docking experiments. Indeed, despite
the efforts made in past73,74 and the recent mechanistic studies
published75, the crystal structures of RT-DNA-RNA hybrid com-
plexes are artefacts obtained with artificial crosslinkers, nicked
nucleic acids, ion substitutions and NNRTI addiction to lock the
structure in a conformation compatible with RNase H cleavage. In
fact, complexes without a NNRTI show the duplex trajectory far
from the RNase H catalytic site. At the moment, the most prob-
able hypothesis is that the RT could switch between two struc-
tural states, one competent for polymerisation and the other for
RNA degradation activity74. Certainly, it is known that conform-
ational flexibility of both enzyme and RNA/DNA hybrid does not
help structural studies and therefore also computational studies.
Although the inhibitors until now reported clearly highlighted
that it is extremely difficult to achieve potent antiviral activity, the
RNase H remains a valid target that is worth to be further investi-
gated to achieve for an alternative anti-HIV treatment.
6. Zhan P, Pannecouque C, De Clercq E, Liu X. Anti-HIV drug
discovery and development: current innovations and future
trends. J Med Chem 2016;59:2849–78.
7. Schatz O, Cromme FV, Naas T, et al. Inactivation of the
RNase H domain of HIV-1 reverse transcriptase blocks viral
infectivity. Gene regulation and AIDS. Houston: Portfolio;
1990.
8. Tramontano E, Esposito F, Badas R, et al. 6-[1-(4-
Fluorophenyl)Methyl-1H-Pyrrol-2-Yl)]-2,4-Dioxo-5-Hexenoic
acid ethyl ester a novel diketo acid derivative which select-
ively inhibits the HIV-1 viral replication in cell culture and
the ribonuclease H activity in vitro. Antiviral Res 2005;65:
117–24.
9. Corona A, Di Leva FS, Thierry S, et al. Identification of highly
conserved residues involved in inhibition of HIV-1 RNase H
function by diketo acid derivatives. Antimicrob Agents
Chemother 2014;58:6101–10.
10. Wang X, Gao P, Menendez-Arias L, et al. Update on recent
developments in small molecular HIV-1 RNase H inhibitors
(2013–2016): opportunities and challenges. Curr Med Chem
2018;25:1682–702.
Acknowledgments
11. Corona A, Masaoka T, Tocco G, et al. Active site and allo-
steric inhibitors of the ribonuclease H activity of HIV reverse
transcriptase. Future Med Chem 2013;5:2127–39.
12. Esposito F, Tramontano E. Past and future. Current drugs
targeting HIV-1 integrase and reverse transcriptase-associ-
ated ribonuclease H activity: single and dual active site
inhibitors. Antivir Chem Chemother 2014;23:129–44.
We thank the COST action CA15135 (Multi-Target Paradigm for
Innovative Ligand Identification in the Drug Discovery Process,
MuTaLig) for support. We thank Prof Patrizia Zavattari from the
Department of Scienze e Tecnologie Biomediche of the University
of Cagliari for the logistic support to perform the Thermal stabil-
ity assays.
ꢀ
13. Cuzzucoli Crucitti G, Metifiot M, Pescatori L, et al. Structure-
activity relationship of pyrrolyl diketo acid derivatives as
dual inhibitors of HIV-1 integrase and reverse transcriptase
ribonuclease H domain. J Med Chem 2015;58:1915–28.
Disclosure statement
No potential conflict of interest was reported by the authors.
ꢀ
14. Costi R, Metifiot M, Chung S, et al. Basic quinolinonyl diketo
acid derivatives as inhibitors of HIV Integrase and their activ-
ity against RNase H function of reverse transcriptase. J Med
Chem 2014;57:3223–34.
ORCID
Serena Massari
Angela Corona
Enzo Tramontano
http://orcid.org/0000-0002-9992-6318
http://orcid.org/0000-0002-6630-8636
http://orcid.org/0000-0002-4849-0980
15. Corona A, di Leva FS, Rigogliuso G, et al. New insights into
the interaction between pyrrolyl diketoacids and HIV-1 inte-
grase active site and comparison with RNase H. Antiviral Res
2016;134:236–43.
References
16. Distinto S, Esposito F, Kirchmair J, et al. Identification of
HIV-1 reverse transcriptase dual inhibitors by a combined
shape-, 2D-fingerprint- and pharmacophore-based virtual
screening approach. Eur J Med Chem 2012;50:216–29.
17. Corona A, Onnis V, Deplano A, et al. Design, synthesis and
antiviral evaluation of novel heteroarylcarbothioamide deriv-
atives as dual inhibitors of HIV-1 reverse transcriptase-associ-
ated RNase H and RDDP functions. Pathog Dis 2017;75:6.
18. Poongavanam V, Corona A, Steinmann C, et al. Structure-
guided approach identifies a novel class of HIV-1 ribonucle-
ase H inhibitors: binding mode insights through magnesium
complexation and site-directed mutagenesis studies. Med
Chem Comm 2018;9:562–75.
19. Corona A, Meleddu R, Esposito F, et al. Ribonuclease H/DNA
polymerase HIV-1 reverse transcriptase dual inhibitor: mech-
anistic studies on the allosteric mode of action of isatin-
based compound RMNC6. PLoS One 2016;11:e0147225.
20. Tabarrini O, Massari S, Cecchetti V. 6-Desfluoroquinolones as
HIV-1 tat-mediated transcription inhibitors. Future Med.
Chem 2010;2:1161–80.
1. Murray AJ, Kwon KJ, Farber DL, Siliciano RF. The latent reser-
voir for HIV-1: how immunologic memory and clonal expan-
sion contribute to HIV-1 persistence. J Immunol 2016;197:
407–17.
2. May MT, Gompels M, Delpech V, et al. Impact on life expect-
ancy of HIV-1 positive individuals of CD4þ cell count and
viral load response to antiretroviral therapy. AIDS 2014;28:
1193–202.
3. Stein J, Storcksdieck Genannt Bonsmann M, Streeck H.
Barriers to HIV cure. HLA 2016;88:155–63.
4. Margot NA, Wong P, Kulkarni R, et al. Commonly transmit-
ted HIV-1 drug resistance mutations in reverse-transcriptase
and protease in antiretroviral treatment-naive patients and
response to regimens containing tenofovir disoproxil fumar-
ate or tenofovir alafenamide. J Infect Dis 2017;215:920–7.
€
5. Schneider A, Corona A, Sporing I, et al. Biochemical charac-
terization of a multi-drug resistant HIV-1 subtype AG reverse
transcriptase: antagonism of AZT discrimination and excision
pathways and sensitivity to RNase H inhibitors. Nucleic
Acids Res 2016;44:2310–22.
21. Tabarrini O, Massari S, Sancineto L, et al. Structural investi-
gation of the naphthyridone scaffold: identification of a 1,6-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
73
Naphthyridone derivative with potent and selective anti-HIV 38. Wu X-F, Schranck J, Neumann H, Beller M. A convenient and
activity. Chem Med Chem 2011;6:1249–57.
general palladium-catalyzed carbonylative coupling for the
synthesis of 2-arylbenzoxazinones. Chem - A Eur J 2011;17:
12246–9.
22. Tabarrini O, Massari S, Daelemans D, et al. Studies of Anti-
HIV transcription inhibitor quinolones: identification of
potent N1-vinyl derivatives. Chem Med Chem 2010;5:
1880–92.
39. Le Grice SF, Cameron CE, Benkovic SJ. Purification and char-
acterization of human immunodeficiency virus type
1
23. Sancineto L, Iraci N, Massari S, et al. Computer-aided design,
synthesis and validation of 2-phenylquinazolinone fragments
as CDK9 inhibitors with anti-HIV-1 tat-mediated transcription
activity. Chem Med Chem 2013;8:1941–53.
24. Corona A, Desantis J, Massari S, et al. Studies on
Cycloheptathiophene-3-Carboxamide derivatives as allosteric
HIV-1 ribonuclease H inhibitors. Chem Med Chem 2016;11:
1709–20.
reverse transcriptase. Meth. Enzymol 1995;262:130–44.
40. Corona A, Schneider A, Schweimer K, et al. Inhibition of
foamy virus reverse transcriptase by human immunodefi-
ciency virus type 1 RNase H inhibitors. Antimicrob Agents
Chemother 2014;58:4086–93.
41. Yonetani T. The yonetani-theorell graphical method for
examining overlapping subsites of enzyme active centers.
Methods Enzymol 1982;87:500–9.
25. Massari S, Nannetti G, Goracci L, et al. Structural investiga-
tion of Cycloheptathiophene-3-carboxamide derivatives tar-
geting influenza virus polymerase assembly. J Med Chem
2013;56:10118–31.
26. Massari S, Nannetti G, Desantis J, et al. A broad anti-influ-
enza hybrid small molecule that potently disrupts the inter-
action of polymerase acidic protein-basic protein 1 (PA-PB1)
subunits. J Med Chem 2015;58:3830–42.
27. Massari S, Goracci L, Desantis J, Tabarrini O. Polymerase
acidic protein-basic protein 1 (PA-PB1) protein-protein inter-
action as a target for next-generation anti-influenza thera-
peutics. J Med Chem 2016;59:7699–718.
28. Desantis J, Nannetti G, Massari S, et al. Exploring the
Cycloheptathiophene-3-Carboxamide scaffold to disrupt the
interactions of the influenza polymerase subunits and obtain
potent anti-influenza activity. Eur J Med Chem 2018;156:
302–3.
29. Wendeler M, Lee HF, Bermingham A, et al. Vinylogous ureas
as a novel class of inhibitors of reverse transcriptase-associ-
ated ribonuclease H activity. ACS Chem Biol 2008;3:635–44.
30. Felts AK, Labarge K, Bauman JD, et al. Identification of alter-
native binding sites for inhibitors of HIV-1 ribonuclease H
through comparative analysis of virtual enrichment studies.
J Chem Inf Model 2011;51:1986–98.
31. Roopa HR, Saravanan J, Mohan S, Parmesh R. Synthesis,
characterization and anti-fungal activity of some novel thio-
semicarbazides. IJRPC 2014;4:445–56.
42. Martini R, Esposito F, Corona A, et al. Natural product kuwa-
non-L inhibits HIV-1 replication through multiple target
binding. Chembiochem 2017;18:374–7.
43. Nettleship JE, Brown J, Groves MR, Geerlof A. Methods for
protein characterization by mass spectrometry, thermal shift
(ThermoFluor) assay, and multiangle or static light scatter-
ing. Methods Mol Biol 2008;426:299–318.
44. Tintori C, Corona A, Esposito F, et al. Inhibition of HIV-1
reverse transcriptase dimerization by small molecules. Chem
biochem 2016;17:683–8.
45. Meleddu R, Distinto S, Corona A, et al. (3Z)-3-(2-[4-(aryl)-1,3-
thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one
derivatives as dual inhibitors of HIV-1 reverse transcriptase.
Eur J Med Chem 2015;93:452–60.
46. Pannecouque C, Daelemans D, De Clercq E. Tetrazolium-
based colorimetric assay for the detection of HIV replication
inhibitors: revisited 20 years later. Nat Protoc 2008;3:427–34.
47. Pauwels R, Balzarini J, Baba M, et al. Rapid and automated
tetrazolium-based colorimetric assay for the detection of
anti-HIV compounds. J Virol Methods 1988;20:309–21.
€
48. Schrodinger LLC, Maestro GUI, New York, NY, USA, 2016.
49. Mohamadi F, Richards NGJ, Guida WC, et al. Macromodel?
An integrated software system for modeling organic and
bioorganic molecules using molecular mechanics. J Comput
Chem 1990;11:440–67.
50. Halgren TA. Merck molecular force field. II. MMFF94 van der
waals and electrostatic parameters for intermolecular inter-
actions. J Comput Chem 1996;17:520–52.
51. Kollman PA, Massova I, Reyes C, et al. Calculating structures
and free energies of complex molecules: combining molecu-
lar mechanics and continuum models. Acc Chem Res 2000;
33:889–97.
52. Berman HM, Westbrook J, Feng Z, et al. The protein data
bank. Nucleic Acids Res 2000;28:235–42.
32. Wang YD, Johnson S, Powell D, et al. Inhibition of tumor
cell proliferation by thieno[2,3-d]pyrimidin-4(1H)-one-based
analogs. Bioorg Med Chem Lett 2005;15:3763–6.
33. Perrissin M, Luu DC, Narcisse G, et al. 4,5,6,7-
Tetrahydrobenzo[b]-
and
5,6,7,8-Tetrahydro-4H-
Cyclohepta[b]Thiophenes. Eur J Med Chem 1980;15:413–8.
34. Gewald K, Schinke E, Bottcher H. Heterocyclenaus CH-
acidenNitrilen, VII. 2-Amino-thiophene aus methylenaktiven
nitrilen, carbonylverbindungen und schwefel. Chem Ber
1966;99:94–100.
35. Usui K, Tanoue K, Yamamoto K, et al. Synthesis of substi-
tuted azulenes via Pt(II)-catalyzed ring-expanding cycloiso-
merization. Org Lett 2014;16:4662–5.
53. Ren J, Esnouf R, Garman E, et al. High resolution structures
of HIV-1 RT from four RT-inhibitor complexes. Nat Struct Biol
1995;2:293–302.
54. Das K, Bauman JD, Clark AD, Jr, et al. High-resolution struc-
tures of HIV-1 reverse transcriptase/TMC278 complexes: stra-
tegic flexibility explains potency against resistance
mutations. Proc Natl Acad Sci USA 2008;105:1466–71.
55. Ren J, Nichols C, Bird LE, et al. Binding of the second gener-
ation non-nucleoside inhibitor S-1153 to HIV-1 reverse tran-
scriptase involves extensive main chain hydrogen bonding. J
Biol Chem 2000;275:14316–20.
36. Romagnoli R, Baraldi PG, Kimatrai Salvador M, et al.
Synthesis and biological evaluation of 2-(Alkoxycarbonyl)-3-
Anilinobenzo[b]Thiophenes and Thieno[2,3-b]Pyridines as
new potent anticancer agents.
J Med Chem 2013;56:
2606–18.
37. Darses B, Michaelides IN, Sladojevich F, et al. Expedient con-
struction of the [7-5-5] all-carbon tricyclic core of the daph-
niphyllum alkaloids daphnilongeranin B and daphniyunnine
D. Org Lett 2012;14:1684–7.
56. Pata JD, Stirtan WG, Goldstein SW, Steitz TA. Structure of
HIV-1 reverse transcriptase bound to an inhibitor active
against mutant reverse transcriptases resistant to other

74
S. MASSARI ET AL.
nonnucleoside inhibitors. Proc Natl Acad Sci USA 2004;101:
10548–53.
dihydroxy benzoyl naphthyl hydrazone bound at a novel
site. ACS Chem Biol 2006;1:702–12.
67. Ren J, Milton J, Weaver KL, et al. Structural basis for the
resilience of Efavirenz (DMP-266) to drug resistance muta-
tions in HIV-1 reverse transcriptase. Structure 2000;8:
1089–94.
57. Himmel DM, Maegley KA, Pauly TA, et al. Structure of HIV-1
reverse transcriptase with the inhibitor beta-thujaplicinol
bound at the RNase
H active site. Structure 2009;17:
1625–35.
68. Baell JB, Holloway GA. New substructure filters for removal
of pan assay interference compounds (PAINS) from screen-
ing libraries and for their exclusion in bioassays. J Med
Chem 2010;53:2719–40.
69. Baell J, Walters MA. Chemistry: chemical con artists foil drug
discovery. Nature 2014;513:481–3.
70. Baell JB. Feeling nature’s PAINS: natural products, natural
product drugs, and pan assay interference compounds
(PAINS). J Nat Prod 2016;79:616–28.
71. Sterling T, Irwin JJ. ZINC 15-ligand discovery for everyone.
J Chem Inf Model 2015;55:2324–37.
72. Vernekar SKV, Tang J, Wu B, et al. Double-winged 3-
Hydroxypyrimidine-2,4-Diones: potent and selective inhib-
ition against HIV-1 RNase H with significant antiviral activity.
J Med Chem 2017;60:5045–56.
73. Das K, Martinez SE, Bandwar RP, Arnold E. Structures of HIV-
1 RT-RNA/DNA ternary complexes with DATP and nevirapine
reveal conformational flexibility of RNA/DNA: insights into
requirements for RNase H cleavage. Nucleic Acids Res 2014;
42:8125–37.
74. Lapkouski M, Tian L, Miller JT, et al. Complexes of HIV-1 RT,
NNRTI and RNA/DNA hybrid reveal a structure compatible
with RNA degradation. Nat Struct Mol Biol 2013;20:230–6.
75. Tian L, Kim M-S, Li H, et al. Structure of HIV-1 reverse tran-
scriptase cleaving rna in an RNA/DNA Hybrid. Proc Natl
Acad Sci USA 2018;115:507–12.
58. Cho AE, Guallar V, Berne BJ, Friesner R. Importance of accur-
ate charges in molecular docking: quantum mechanical/
molecular mechanical (QM/MM) approach. J Comput Chem
2005;26:915–31.
59. Schroedinger Suite. Portland, USA: Schrodinger Inc; 2016.
60. The PyMOL Molecular Graphics System, Version 1.7
€
Schrodinger, LLC.
61. Gorobets NY, Yousefi BH, Belaj F, Kappe CO. Rapid micro-
wave-assisted solution phase synthesis of substituted 2-pyri-
done libraries. Tetrahedron 2004;60:8633–44.
62. Schellhase M, Boehm R, Pech R. Thieno compounds. part 4:
3-Aryl-3,4-Dihydro-4-Oxobenzo[b]Thieno[2,3-d]pyrimidines.
Pharmazie 1984;39:19–21.
63. Khan KM, Saad SM, Shaikh NN, et al. Synthesis and
b-Glucuronidase inhibitory activity of 2-Arylquinazolin-4(3H)-
Ones. Bioorg Med Chem 2014;22:3449–54.
64. Chung S, Wendeler M, Rausch JW, et al. Structure-activity
analysis of vinylogous urea inhibitors of human immunodefi-
ciency virus-encoded ribonuclease H. Antimicrob Agents
Chemother 2010;54:3913–21.
65. Cummings MD, Farnum M, Nelen MI. Universal screening
methods and applications of ThermoFluor. J Biomol Screen
2006;11:854–63.
66. Himmel DM, Sarafianos SG, Dharmasena S, et al. HIV-1
reverse transcriptase structure with RNase
H inhibitor