5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein

Article abstract of DOI:10.1021/acsmedchemlett.9b00608

The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid 1 acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties. A bioisosteric catechol replacement approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. Hit validation efforts led to the identification of optimized analogs, as represented by compound 28, showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.

Full text of DOI:10.1021/acsmedchemlett.9b00608

Subscriber access provided by WESTERN SYDNEY U
Letter
5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein
Savina Malancona, Mattia Mori, Paola Fezzardi, Marisabella Santoriello, Andreina Basta,
Martina Nibbio, Lesia Kovalenko, Roberto Speziale, Maria Rosaria Battista, Antonella Cellucci,
Nadia Gennari, Edith Monteagudo, Annalise Di Marco, Alessia Giannini, rajhans sharma,
Manuel Pires, Eleonore Real, Maurizio Zazzi, Maria Chiara Dasso Lang, Davide De Forni,
Francesco Saladini, Yves Mély, Vincenzo Summa, Steven Harper, and Maurizio Botta
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00608 • Publication Date (Web): 19 Mar 2020
Downloaded from pubs.acs.org on March 21, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid
Protein
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Savina Malancona1*°, Mattia Mori2°, Paola Fezzardi1, Marisabella Santoriello1, Andreina Basta1,†, Martina
Nibbio1, Lesia Kovalenko3, Roberto Speziale1, Maria Rosaria Battista1, Antonella Cellucci1, Nadia
Gennari1, Edith Monteagudo1, Annalise Di Marco1, Alessia Giannini4, Rajhans Sharma3, Manuel Pires3,
Eleonore Real3, Maurizio Zazzi4, Maria Chiara Dasso Lang2, Davide De Forni5, Francesco Saladini4,Yves
Mely3, Vincenzo Summa1,††, Steven Harper1,# and Maurizio Botta2#
1IRBM S.p.A., Via Pontina Km 30.600, 00071 Pomezia (RM), Italy
2Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via Aldo Moro 2, 53100 Siena, Italy
3Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74 Route
du Rhin, 67401 Illkirch, France
4Department of Medical Biotechnologies, University of Siena, Viale Mario Bracci, 16, 50100 Siena, Italy
5ViroStatics S.r.l, Viale Umberto I 46, 07100 Sassari, Italy
#This work is dedicated to the beloved memory of Prof. Maurizio Botta (August 2, 2019) and Steven Harper (June 30, 2019)
KEYWORDS: Nucleocapsid protein, HIV, NC inhibitors, Dihydroxypyrimidine, Drug Resistance, Antiretroviral.
ABSTRACT: The HIV-1 Nucleocapsid Protein (NC) is a small basic DNA and RNA binding protein which is absolutely necessary
for viral replication and thus represent a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved
sequence offers the opportunity to escape the drug resistance (DR) emerged following the highly active antiretroviral therapy
(HAART) treatment. Based on our previous research nordihydroguaiaretic acid 1 acts as NC inhibitor showing moderate antiviral
activity and sub-optimal drug-like properties due to the presence of the catechol moieties. A bio-isosteric catechol replacement
approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as privileged scaffold of a new class of HIV-1
NC inhibitors. Hit validation efforts led to the identification of optimised analogs, as represented by compound 28, showing improved
NC inhibition and antiviral activity as well as good ADME and PK properties.
Nowadays AIDS (Acquired Immune Deficiency Syndrome)
is still a major global public health issue. According to the Joint
United Nation Program on HIV/AIDS (UNAIDS) nearly 38
million people were living with HIV in 2018.1 The current
pharmacological strategy relays on the combination of drugs
(HAART, highly active antiretroviral therapy) targeting various
steps of the viral replication: entry, fusion, reverse transcription,
integration, protein maturation. Despite successful in mortality
and morbidity reduction, HAART long-term treatment is
associated with the insurgence of drug resistance as results of
the ability of HIV-1 to mutate.2 In this context there is an urgent
need to develop new drugs to treat HIV infection based on novel
mechanism of action or targets.3 One emerging approach to
overcome drug resistance is to target HIV-1 proteins that are
highly conserved among phylogenetically distant viral strains
and currently not targeted by available therapies. The
Nucleocapsid Protein (NC) of the Human Immunodeficiency
Virus type-1 (HIV-1) is a small and basic protein with two zinc
fingers that is highly conserved among the retroviruses. This
nucleic acids (NA) binding protein is involved in both early and
late steps of the HIV-1 replication cycle, through its ability to
chaperone NAs towards their most stable conformation.4,5,6,7
Thus, NC appears as an ideal target for the development of new
drugs to prevent HIV-1 replication and complement the highly
active anti-retroviral therapy.8 Since NC is highly conserved,
anti-NC drugs are expected to provide a sustained replication
inhibition of the wild type and HIV-1 drug-resistant strains.
Three classes of NC inhibitors have been reported based on the
mechanism of action: (i) zinc ejectors,9,10 (ii) noncovalent NCIs
binding to nucleic acid partners of NC,11,12 and (iii) noncovalent
NCIs binding to NC.13,14 The development of NCIs directly
binding to NC is currently our main research area, as it is
expected to overcome the selectivity and toxicity issues
intrinsically related to NCIs belonging to the classes (i) and (ii).
In this work, we describe the most recent advances achieved,
leading to the identification of a new class of NCIs showing
both good antiviral activity as well as promising in-vitro and in-
vivo profiles.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 8
1
2
3
4
5
6
7
8
OH
OH
to the identification of compound 4 as a positive hit (Figure 1),
HO
HO
showing an IC50 below 200 M (Table 1). Despite its modest
potency, compound 4 represented an optimal starting point
from the development perspective due to the favorable drug-
like properties of the dihydroxypyrimidine carboxamide class.
This class of compounds is known to have led to the discovery
of Raltegravir23 (Figure 1), the first in class HIV integrase
inhibitor. Furthermore, in the antiviral field, the
dihydroxypyrimidine core has been investigated to target other
NA processing enzymes, such as HIV-1 RT24 or HCV NS5B
RNA-dependent RNA polymerase.25 Due to the close structural
similarity between compound 4 and Raltegravir, a possible
polypharmacology profile could be expected in the
development of this class of compounds as NC inhibitors.
Nonetheless, first rough structural comparison and lack of
activity of Raltegravir in the NC inhibition assay (data not
shown), suggested that a degree of selectivity was already
present in the hit compound 4. The structure activity
relationship (SAR) of the dihydroxypyrimidine carboxamide as
inhibitors of the HIV-integrase enzyme has been fully
elucidated and well documented in literature.26,27,28,29 Optimized
HIV-integrase inhibitors show a marked preference for the N-
methylpyrimidone core, benzylic carboxamide and neutral aryl
or alkyl substituent at the 2-position of the pyrimidine core. In
contrast, the catechol-like dihydroxypyrimidine core of the hit
compound 4, is decorated with an alkyl amide that in the HIV
integrase inhibitor abolish completely the activity against this
target, and a basic aromatic substituent in the 2-position of the
pyrimidine.28 A hit validation and optimization process was
thus conducted on 4 with the aim of improving the potency as
NC inhibitor. The compounds were also tested for cell-based
antiretroviral activity. The in-vitro and in-vivo ADME
properties were finally evaluated for the most interesting
analogs.
1
OH
NC inhibition IC50 = 26 M
6
OH
R2
5
N
1
4
R1
N
3
2
OH
3
Cl
N
OH
9
N
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
2
NC inhibition IC50 = 110 M
OH
N
OH
N
O
H
N
OH
H
N
F
N
N
N
H
N
N
O
O
N
O
O
4
NC inhibition IC50 = 167 M
Raltegravir
Figure 1. NC inhibitors based on catechol template 1 and 2. 5,6-
dihydroxypyrimidine 3 as catechol replacement and hit compound
4 active in the NC-inhibition assay. Raltegravir, first in class HIV
integrase inhibitor.
Recently, we have developed non-covalent NC inhibitors,15
by applying a computational structure-based screening of
commercial compounds collection followed by in-vitro
assessment of the NC inhibitory activity. This led to the
identification of the natural product nordihydroguaiaretic acid
1 and compound 2 as NC inhibitors sharing a catechol moiety
(Figure 1). Based on our pharmacophore, the catechol moiety is
a common structural feature that plays a critical role in binding
NC in a guanidine-like manner by means of the two hydroxyl
groups.16,17,18 Biophysical and computational studies of
compound 1 interacting with the protein confirmed the binding
postulated by modeling.15 While valuable as a tool compound,
the low metabolic profile associated with the catechol group
limits the further development of both compounds 1 and 2. The
replacement of undesirable functional groups with bioisosters is
a common strategy applied in drug discovery to modulate the
activity profile, reduce potential toxicities and circumvent
metabolic liabilities.19 In this perspective, using the protocol
previously described,16 a virtual screening campaign on the
ZINC catalog of commercial products was conducted to
identify catechol-like compounds as putative candidate
inhibitors of NC. Following virtual screening and clusterization
of the top-ranking compounds,15 the dihydroxypyrimidine core
scaffold 3 (Figure 1) emerged as a potential replacement for the
catechol moiety and thus offering the possibility to develop a
new class of NCIs.
While retaining the central dihydroxypyrimidine core,
structural modifications were introduced first in the aromatic
region at the 2-position (R2, Table 1). The 2-(pyridin-2-yl)
group emerged as the privileged substitution for the activity: the
replacement with a simple phenyl 5 or replacement with the 2-
(pyridin-3-yl) or 2-(pyridin-4-yl) isomers, respectively
compounds 6 and 7, resulted in complete loss of the activity.
Neither a 2-phenethyl, 2-(1-phenylethyl) or bulky 2-(tert-butyl)
substituents were tolerated, compounds 8,
9 and 10
respectively. The amide moiety (R1, Table 1) of compound 4 is
believed from modelling to be oriented towards the
hydrophobic binding site of the protein. The SAR in the amide
region showed that the replacement of the N-
(cyclohexylmethyl) group with N-benzyl 11, N-ethyl 12 and the
tertiary amide 13 are not tolerated. The exploration of the linker
length pointed out that anchoring the cyclohexyl directly to the
amide nitrogen as for 14 produces a 2-fold loss of activity in the
NC inhibition assay with respect to the hit compound 4, whereas
the elongation of the linker by one carbon atom 15 is beneficial
for the activity and accounts for a 4-fold improved activity.
Similar gain of potency is achieved with the branched analogs
(R)-N-(1-cyclohexylethyl) 16 and (S)-N-(1-cyclohexylethyl)
17, showing the same activity regardless of chirality. The results
obtained are in line with the proposed binding mode that
suggests the amide occupying the hydrophobic pocket and that
the lipophilic substituents are thus well tolerated, chirality is not
influencing conformation and binding of the amide moiety.
Along this line further scaffold expansion was achieved
through the design of a focused virtual library of analogs with
the scope of selecting a pool of compounds to be evaluated in-
vitro for their ability to inhibit the NC chaperone activity. To
this aim, a well-established fluorescence assay20 (NC-inhibition
assay) was used; it allows to monitor the destabilization of
cTAR DNA, the complementary sequence to the transactivation
response element of the HIV-1 genome labeled with the
Alexa488 dye and Dabcyl quencher.21,22 The protein
concentration used in the inhibition assay is 1 M and thus 0.5
M is the lowest IC50s values measurable. This effort conducted
ACS Paragon Plus Environment

Page 3 of 8
ACS Medicinal Chemistry Letters
1
2
3
Table1. In-vitro NC inhibition for the analogs exploring the amide and aromatic substituent regions
4
OH
5
6
7
8
OH
N
R1
R2
N
O
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NC inhibition
IC50 (µM)a
NC inhibition
IC50 (µM)a
ID
R1
R2
ID
R1
R2
H
N
H
N
4
5
167 ± 7
N.A.b
17
18
47 ± 5
20 ± 1
N
N
N
H
N
H
N
H
N
N.A.b
N.A.b
N.A.b
19
20
21
20c
H
N
6
7
8
N
N
H
N
H
N
8 ± 0.7
41 ± 10
N
N
N
H
N
H
N
H
N
N.A.b
N.A.b
N.A.b
N.A.b
22
23
24
25
21 ± 15
20d
H
N
9
N
N
H
N
H
N
10
11
12
CF3
H
N
H
23 ± 6
12 ± 6
N
N
N
N
F3C
F3C
H
H
N
N
N
CF3
H
N
13
14
15
16
N.A.b
300 ± 100
38 ± 10
26
27
28
29
20d
N
N
N
N
N
N
H
O
N
H
N
22 ± 7
2 ± 1
4 ± 1
N
H
Cl
N
H
N
N
H
H
N
N
42 ± 10
N
a Data are average ± SD of a least two independent experiments. bN.A. = not active. cIC40. dIC35.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 8
1
2
3
4
5
6
7
8
Preliminary SAR at the 2-position of the 5,6-
and with the side-chain of Gln45. In addition, the amide moiety
established hydrophobic interactions with the side chain of
residues from the NC hydrophobic platform17 such as Phe16,
Ala25, Trp37, and Met46 (Figure 3). Overall, this binding mode
is highly consistent with the binding mode of NA nucleotides30
and of nordihydroguaiaretic acid 115. Most notably, the
predicted binding for these dihydroxypyrimidines also overlaps
with the binding mode of the NC inhibitor characterized by
NMR spectroscopy in complex with the NC, whose structure
was used as a receptor in molecular docking simulation31
(Supporting Information, Figure S2). A good correlation
between the score of compounds 4, 20, 25, and 28 and their –
logIC50 was observed (R2 = 0.939) (Supporting Information,
Figure S3). Finally, the binding mode described above and
shown in Figure 3 and Figure S1 clearly accounts for the lack
of NC inhibition by derivatives deprived of the dihydroxyl
moiety, such as 30, and 31, or bearing the N-methyl pyrimidone
substructure such as 32.
dihydroxypyrimidine core has shown that 2-(pyridin-2-yl)
substituent is required for the activity. Further optimization was
thus conducted introducing substituents on the pyridine ring.
First modifications introduced were the methyl (18-22) and
trifluoromethyl (23-26) groups which were selected considering
the differences in the electron donating and withdrawing ability,
as well as lipophilicity. Regardless of the electronic nature, the
introduction of a substituent in position 4- and 5 is beneficial
for the activity, affording compounds 20 and 25 showing IC50s
around 10 µM. In the other positions of the pyridine ring, two-
fold activity gain was achieved with respect to the hit 4. A
variety of other substituents were evaluated, in particular, the
most relevant results were obtained with the introduction of 5-
methoxy and 5-chlorine groups on the pyridine ring
(respectively, compounds 27 IC50 =20 µM and 28 IC50 = 2 µM).
To complete the exploration at the 2- position of the pyrimidine
core, a series of heterocycles retaining the 2-nitrogen atom
present in the 2-pyridine substituent were explored. Best results
were achieved with fused bicyclic groups, such as the quinoline
analogs 29 showing more than 40-fold improvement in the NC
inhibition with respect to the original compound 4. Smaller 5-
member ring heterocycles such as thiophene or thiazole were
not tolerated (data not shown).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The antiviral activity on the whole replication cycle of HIV-1
was evaluated for selected compounds by means of the cell-
based assay named BiCycle (Table 2). The assay consists of a
first infection round of the T-cell line MT-2 using HIV-1 wild-
type reference strain NL4-3, followed by a second infection
round of the reporter cell line TZM-bl.32 Hit compound 4 shows
activity in the low micromolar range. A similar activity is found
for the analog 15, which is more active in the biochemical assay
as result of the amide elongation.
Modification at the dihydroxypyrimidine core showed that
both hydroxyl groups are essential for the activity. Indeed,
compounds 30 and 31 lacking respectively the 4- and 5-
hydroxyl substituent, were found inactive in the NC inhibition
assay (Figure 2).
The 5,6-dihydroxypyrimidine core exists in two tautomeric
forms, namely dihydroxypyrimidine and pyrimidone. A
different hydrogen bond donator/acceptor asset, potentially able
to influence the binding to the nucleocapsid protein, is
associated to each form. The N-methyl pyrimidone analog 32
resulted to be completely inactive in the NC inhibition assay,
indicating the catechol form as decisive for NC inhibition, in
contrast to the preference for the pyrimidinone form in the HIV-
1 integrase area. The SAR conducted on the hit compound 4 has
shown that it is possible to modulate NC activity and improve
80-fold its potency (cmpd 28), thus obtaining a number of
compounds in the low micromolar range of activity.
O
OH
OH
OH
N
N
N
H
H
H
Figure 3. Predicted binding mode of 28 within the hydrophobic
pocket of the NC. The protein is shown as green cartoon. Residues
within 5 Å from the ligands are shown as lines, and are labeled.
Compound 28 is shown as yellow sticks. H-bond interactions are
highlighted by black dashed lines.
N
N
N
N
N
N
N
O
N
O
N
O
32
31
30
Figure 2. Dihydroxypyrimidine core modifications leading to lack
of NC inhibition.
A beneficial effect on the antiviral activity is seen when the
branched amide is present, with both 16 and 17 enantiomers
active in the sub-micromolar range. The difference between the
R and S enantiomers in term of antiviral activity is very modest
(3-fold), confirming the behavior observed in the NC inhibition
assay. Analogs substituted in the aromatic region such as 20,
25, 27, 28 and 29 exhibit nanomolar antiviral activity with
EC50s ranging from 40 to 400 nM. In parallel, cytotoxicity was
evaluated in Hela and peripheral blood mononuclear cells
(PMBC) and the selectivity indexes (SI) were calculated as the
ratio between the CC50 and EC50 values measured in their
respective assays (see footnote of Table 2). Despite the narrow
difference between the activity and cytotoxicity found for 4 and
close analogues (SI range 2-5-fold), much higher SI values were
The binding mode of most potent NC inhibitors identified was
investigated by molecular docking simulations and reported in
Figure 3 for compound 28 and Figure S1 for compounds 4, 20
and 25. To this aim, we took advantage of the computational
protocol already refined by our research group for the NC
protein, and based on molecular docking with the FRED
program (OpenEye) against the NMR structure of the NC in
complex with a small molecule inhibitor (PDB: 2M3Z), see also
the Supporting Information.15,17,16 Overall, the molecules shared
a highly similar binding mode, and proved to fit the
hydrophobic pocket of the NC. Besides a π-stacking interaction
with the side chain of Trp37, the core dihydroxypyrimidine
established H-bonds with the backbone of Gly35, and Met46,
ACS Paragon Plus Environment

Page 5 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
found for compounds 20, 27, 28, 29 as a result of their improved
group is an issue to be considered. To overcome this issue, we
decided to search for catechol-like scaffold with improved
physicochemical properties able to bind NC protein. The
process allowed the identification of the 5,6-
dihydroxypyrimidine-4-carboxamide analog 4 as representative
of a new class of HIV-1 nucleocapsid inhibitors. Initial SAR
studies were conducted in order to prove the bona fide nature of
the hit and identify the key structural features needed to achieve
and modulate NC inhibition. Optimized analogs such as 27, 28,
29 not only showed nanomolar antiviral activity and improved
selective index, but were active against a panel of HIV-1
resistant strains in agreement with the proposed mechanism of
action. Furthermore, the compounds showed a very clean
profile against the major CYP-P450 isoforms and the hERG
channel. The pharmacokinetic profile in mice was good for all
three compounds but in particular for compound 28, which
shows 40% oral bioavailability, good half-life and moderate
clearance. In conclusion, we identified an unprecedented
structural class of NC inhibitors with good drug like properties.
In particular compound 28 represents a novel NC inhibitor lead
with low nanomolar potency in cell-based assay, low
cytotoxicity, and good PK profile. It represents a good starting
point for further characterization and optimization.
activity in cells (SI in the range 13-75). Overall, these data
clearly show that the potency and selectivity can be modulated
and improved in future investigations of the series. The
inhibition of NC is expected to overcome drug resistance. To
prove this hypothesis most potent compounds were tested
against a representative panel of drug-resistant HIV-1 strain
(Table 2). Notably, only a 1-2-fold IC50 shift was measured
compared to wild type IC50, consistent with the hypothesis that
NC inhibitors retain their full potency against resistant strains.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In order to further characterize the new class of NC inhibitors
(NCIs), a series of in-vitro and in-vivo tests were performed on
a small set of selected compounds. The metabolic stability was
evaluated in-vitro in rat and human species, plasma and
hepatocytes matrices (Table 3). Data show that compounds 25,
27, 28 and 29 were stable up to one hour or more in both plasma
and hepatocytes matrices. The same compounds were not
inhibitors of major human cytochrome P450 isoforms
(CYP1A2, CYP2D6, CYP3A4) and the hERG ion channels.
Based on the potency profile in the different assays and the in-
vitro DMPK data, compounds 27, 28 and 29 were selected for
in-vivo PK studies in C57BL/6 mice (Table 3). Compounds
were administrated IV and PO at 2 and 5 mg/kg, respectively.
Eight time points were taken up to 24h to obtain a plasma PK
profile (Table 4).
Table 4: PK parameters for compounds 27, 28 and 29 following
i.v. and p.o administration in C57BL/6 mice at 2 mg/kg and 5
mg/kg respectively
in-vivo profiling
Cl
ID
t1/2 (h)
Vdss (L/kg)
%F
(ml/min/kg)
25
27
28
29
1.4
3.1
1.1
0.8
1.2
0.8
33.1
37.1
30.2
25
40
26
Compounds 27 and 29 showed similar PK parameters:
medium-low plasma clearance (33 and 30 mL/min/kg,
respectively) and medium volume of distribution (Vdss 0.8
L/kg for both) with a corresponding half-life of 1.4 and 1.1
hours respectively for 27 and 29. The oral bioavailability was
good (25% and 26%, respectively). Compared to 27 and 29,
compound 28 showed a better profile presenting higher oral
bioavailability (40 %), prolonged half-life of 3.1 hours and
volume of distribution of 1.2 L/kg.
To assess
a
preliminary polypharmacology profile
compounds 4, 27 and 29 were tested for their ability to inhibit
the HIV Integrase enzyme by means of the well-established
LEDGF-dependent IN activity assay33 resulting not active up to
10 uM, Raltegravir used as positive control showed IC50 = 0.027
± 0.01M.
In summary, based on our previous studies the catechol
moiety is a privileged structure to achieve NC inhibition as
demonstrated by compound 1. In the prospective of drug
development, the intrinsic metabolic liability of the catechol
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 8
1
2
3
4
5
6
7
8
9
Table 2: Antiviral and cytotoxicity profile. Susceptibility to viral strains harbouring resistance to drug used in clinical practice
BiCycle
Hela
PBMC MTS
Strain 11808
(PI)e
Strain 7401
(NRTI) e
Strain 12231 Strain 11845
Wild type
strain NL4-3
(NNRTI) e
(INI) e
CC50
SI-
SI-
ID EC50 (µM)a
CC50 (µM)a
EC50 (µM)a
EC50 (µM)a
EC50 (µM)a
EC50 (µM)a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(µM)a
3.6 ± 0.7
4.2 ± 3.4
3.2 ± 0.9
0.6 ± 0.04
2.7 ± 0.4
6.0 ± 0.2
Helab
PBMCc
4
1.2 ± 0.7
2.3 ± 2
3
2
2.6 ± 1.7
11 ± 6.0
3.0 ± 1.4
2.4 ± 1
2
5
15
16
17
20
25
0.7 ± 0.1
0.2 ± 0.1
0.2 ± 0.1
0.4 ± 0.3
5
4
3
12
16
3
13
15
3.1 ± 0.8
1.1 ± 0.1
0.056 ± 0.02
(1.4)d
0.031 ± 0.02
(0.8) d
0.11 ± 0.04
(2.8) d
0.09 ± 0.04
(2.3) d
27
28
29
0.04 ± 0.03
0.1 ± 0.03
0.1 ± 0.05
3.0 ± 0.1
3.0 ± 0.5
2.1 ± 0.3
75
30
21
2.7 ± 0.1
2.5 ± 0.2
5 ± 2
75
25
50
0.02 ± 0.01
(0.2) d
0.01 ± 0.005
(0.1) d
0.08 ± 0.06
(0.8) d
0.07 ± 0.08
(0.7) d
0.16 ± 0.1
(1.6) d
0.13 ± 0.08
(1.3) d
0.21 ± 0.07
(2.0) d
0.09 ± 0.02
(0.9) d
aData are average ± SD for a least two independent experiments; bSI-Hela: HELA CC50 / BiCycle EC50; cSI-PBMC: PBMC CC50 / BiCycle
d
e
EC50; Fold change values indicate the ratio between IC50 values from drug-resistant and NL4−3 wild type reference strains. NIH AIDS
Reagent Program catalogue number of resistant strains (www.aidsreagent.org). PI, resistance to Protease Inhibitor; NRTI, resistance to
Nucleoside Reverse Transcriptase Inhibitor; NNRTI, resistance to Non-Nucleoside Reverse Transcriptase Inhibitor; INI, resistance to
Integrase Inhibitor.
Table 3: In-vitro profiling for compounds 25, 27, 28 and 29; PK parameters for compounds 27, 28 and 29 following i.v. and
p.o administration in C57BL/6 mice at 2 mg/kg and 5 mg/kg respectively
in-vitro profiling
Mouse
hepatocytes
t1/2 (h)
Human
hepatocytes
t1/2 (h)
HERG
Mouse
Human
CYP1A2
IC50 (µM)
CYP2D6
IC50 (µM)
CYP3A4
IC50 (µM)
ID
plasma t1/2 (h) plasma t1/2 (h)
IC50 (µM)
25
27
28
29
>1
>24
>6
>1
>6
2.4
1.9
1.7
1.7
>4
>4
>4
2.6
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>6
3.7
ND
ASSOCIATED CONTENT
Supporting Information
Savina Malancona: 0000-0002-5388-6533
Present Addresses
The Supporting Information is available free of charge on the ACS
Publications website.
†Tubilux Pharma spa, via Costarica 20, 00071 Pomezia (Rome)
††Department of Pharmacy, University of Naples “Federico II”,
Via D. Montesano 49, 80131 Naples, Italy
Synthetic experimental details and characterization data,
description of biological assay protocols, modeling (PDF).
Author Contributions
°These authors equally contributed to this work. All authors
have given approval to the final version of the manuscript.
AUTHOR INFORMATION
Corresponding Author
Funding Sources
* Email: s.malancona@irbm.com
This project has received funding from the European Union’s
Seventh Programme for research, technological development,
ORCID
ACS Paragon Plus Environment

Page 7 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
anthraquinones: Influence of Side Chain Length on HIV-1
and demonstration under grant agreement no. 601969.
Nucleocapsid Inhibitors. J. Med. Chem. 2016, 59, 1914–1924.
(13) Kim, M.-J.; Kim, S. H.; Park, J. A.; Yu, K. L.; Jang, S. I.; Kim,
B. S.; Lee, E. S.; You, J. C. Identification and characterization of a new
type of inhibitor against the human immunodeficiency virus type-1
nucleocapsid protein. Retrovirology 2015, 12, 90.
(14) Gamba, E.; Mori, M.; Kovalenko, L.; Giannini, A.; Sosic, A.;
Saladini, F.; Fabris, D.; Mély, Y.; Gatto, B.; Botta, M. Identification of
novel 2-benzoxazolinone derivatives with specific inhibitory activity
against the HIV-1 nucleocapsid protein. Eur. J. Med. Chem. 2018, 145,
154–164.
(15) Mori, M.; Kovalenko, L.; Malancona, S.; Saladini, F.; Forni, D.
de; Pires, M.; Humbert, N.; Real, E.; Botzanowski, T.; Cianférani, S.;
et al. Structure-Based Identification of HIV-1 Nucleocapsid Protein
Inhibitors Active against Wild-Type and Drug-Resistant HIV-1
Strains. ACS Chem. Biol. 2018, 13, 253–266.
(16) Mori, M.; Manetti, F.; Botta, M. Predicting the binding mode
of known NCp7 inhibitors to facilitate the design of novel modulators.
J. Chem. Inf. Model. 2011, 51, 446–454.
(17) Mori, M.; Nucci, A.; Lang, M. C. D.; Humbert, N.; Boudier, C.;
Debaene, F.; Sanglier-Cianferani, S.; Catala, M.; Schult-Dietrich, P.;
Dietrich, U.; et al. Functional and structural characterization of 2-
amino-4-phenylthiazole inhibitors of the HIV-1 nucleocapsid protein
with antiviral activity. ACS Chem. Biol. 2014, 9, 1950–1955.
(18) Mori, M.; Dasso Lang, M. C.; Saladini, F.; Palombi, N.;
Kovalenko, L.; Forni, D. de; Poddesu, B.; Friggeri, L.; Giannini, A.;
Malancona, S.; et al. Synthesis and Evaluation of Bifunctional
Aminothiazoles as Antiretrovirals Targeting the HIV-1 Nucleocapsid
Protein. ACS Med. Chem. Lett. 2019, 10, 463-468.
ACKNOWLEDGMENT
We acknowledge Fabrizio Colaceci (in-vivo work); Costanza
Iaccarino and Letizia Lazzaro (compounds purification and QC);
Prof. Enzo Tramontano and Francesca Esposito Dipartimento di
Scienze della Vita e dell’Ambiente Università di Cagliari for the
HIV-IN assay. The authors wish to thank the OpenEye Free
Academic Licensing Program for providing a free academic license
for molecular modeling and chemoinformatics software. YM is
grateful to the Institut Universitaire de France (IUF) for support and
providing additional time to be dedicated to research.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ABBREVIATIONS
AIDS, acquired immune deficiency syndrome; HAART, highly
active antiretroviral therapy; NC, nucleocapsid protein; HIV-1,
human immunodeficiency virus type-1; NCIs, nucleocapsid protein
inhibitors; HIV-IN, human immunodeficiency virus integrase; SI,
selectivity index; SAR, structure-activity relationship; NMR,
nuclear magnetic resonance; PK, pharmacokinetics; hERG, human
Ether-à-go-go-Related Gene.
REFERENCES
(1) UNAIDS. Global HIV & AIDS statistics - 2019 fact sheet.
https://www.unaids.org/en/resources/fact-sheet (accessed on March
19, 2020)
(2)
AIDSinfo.
Understanding
HIV/AIDS-Fact
Sheets
(19) Patani, G. A.; LaVoie, E. J. Bioisosterism: A Rational Approach
in Drug Design. Chem. Rev. 1996, 96, 3147–3176.
https://aidsinfo.nih.gov/understanding-hiv-aids (accessed on March
19, 2020)
(3) Zhan, P.; Pannecouque, C.; Clercq, E. de; Liu, X. Anti-HIV Drug
Discovery and Development: Current Innovations and Future Trends.
J. Med. Chem. 2016, 59, 2849–2878.
(4) Darlix, J.-L.; Rocquigny, H. de; Mauffret, O.; Mély, Y.
Retrospective on the all-in-one retroviral nucleocapsid protein. Virus
Res. 2014, 193, 2–15.
(5) Levin, J. G.; Mitra, M.; Mascarenhas, A.; Musier-Forsyth, K.
Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription.
RNA Biol. 2010, 7, 754–774.
(20) Shvadchak, V.; Sanglier, S.; Rocle, S.; Villa, P.; Haiech, J.;
Hibert, M.; van Dorsselaer, A.; Mély, Y.; Rocquigny, H. de.
Identification by high throughput screening of small compounds
inhibiting the nucleic acid destabilization activity of the HIV-1
nucleocapsid protein. Biochimie 2009, 91, 916–923.
(21) Bernacchi, S.; Mély, Y. Exciton interaction in molecular
beacons: a sensitive sensor for short range modifications of the nucleic
acid structure. Nucleic Acids Res. 2001, 29, E62-2.
(22) Bernacchi, S.; Stoylov, S.; Piémont, E.; Ficheux, D.; Roques,
B. P.; Darlix, J. L.; Mély, Y. HIV-1 nucleocapsid protein activates
transient melting of least stable parts of the secondary structure of TAR
and its complementary sequence. J. Mol. Biol. 2002, 317, 385–399.
(23) Summa, V.; Petrocchi, A.; Bonelli, F.; Crescenzi, B.; Donghi,
M.; Ferrara, M.; Fiore, F.; Gardelli, C.; Gonzalez Paz, O.; Hazuda, D.
J.; et al. Discovery of raltegravir, a potent, selective orally bioavailable
HIV-integrase inhibitor for the treatment of HIV-AIDS infection. J.
Med. Chem. 2008, 51, 5843–5855.
(24) Lacbay, C. M.; Menni, M.; Bernatchez, J. A.; Götte, M.;
Tsantrizos, Y. S. Pharmacophore requirements for HIV-1 reverse
transcriptase inhibitors that selectively “Freeze” the pre-translocated
complex during the polymerization catalytic cycle. Bioorg. Med.
Chem. 2018, 26, 1713–1726.
(25) Koch, U.; Attenni, B.; Malancona, S.; Colarusso, S.; Conte, I.;
Di Filippo, M.; Harper, S.; Pacini, B.; Giomini, C.; Thomas, S.; et al.
2-(2-Thienyl)-5,6-dihydroxy-4-carboxypyrimidines as inhibitors of the
hepatitis C virus NS5B polymerase: discovery, SAR, modeling, and
mutagenesis. J. Med. Chem. 2006, 49, 1693–1705.
(26) Petrocchi, A.; Koch, U.; Matassa, V. G.; Pacini, B.; Stillmock,
K. A.; Summa, V. From dihydroxypyrimidine carboxylic acids to
carboxamide HIV-1 integrase inhibitors: SAR around the amide
moiety. Bioorg. Med. Chem. Lett. 2007, 17, 350–353.
(27) Summa, V.; Petrocchi, A.; Matassa, V. G.; Gardelli, C.;
Muraglia, E.; Rowley, M.; Paz, O. G.; Laufer, R.; Monteagudo, E.;
Pace, P. 4,5-dihydroxypyrimidine carboxamides and N-alkyl-5-
hydroxypyrimidinone carboxamides are potent, selective HIV
integrase inhibitors with good pharmacokinetic profiles in preclinical
species. J. Med. Chem. 2006, 49, 6646–6649.
(6) Zgheib, S.; Lysova, I.; Réal, E.; Dukhno, O.; Vauchelles, R.;
Pires, M.; Anton, H.; Mély, Y. Quantitative monitoring of the
cytoplasmic release of NCp7 proteins from individual HIV-1 viral
cores during the early steps of infection. Sci. Rep. 2019, 9, 945.
(7) Retureau, R.; Oguey, C.; Mauffret, O.; Hartmann, B. Structural
Explorations of NCp7-Nucleic Acid Complexes Give Keys to Decipher
the Binding Process. J. Mol. Biol. 2019, 431, 1966–1980.
(8) Mori, M.; Kovalenko, L.; Lyonnais, S.; Antaki, D.; Torbett, B.
E.; Botta, M.; Mirambeau, G.; Mély, Y. Nucleocapsid Protein: A
Desirable Target for Future Therapies Against HIV-1. Curr. Top.
Microbiol. Immunol. 2015, 389, 53–92.
(9) Sancineto, L.; Mariotti, A.; Bagnoli, L.; Marini, F.; Desantis, J.;
Iraci, N.; Santi, C.; Pannecouque, C.; Tabarrini, O. Design and
Synthesis of DiselenoBisBenzamides (DISeBAs) as Nucleocapsid
Protein 7 (NCp7) Inhibitors with anti-HIV Activity. J. Med. Chem.
2015, 58, 9601–9614.
(10) Hartman, T. L.; Yang, L.; Helfrick, A. N.; Hassink, M.; Shank,
N. I.; George Rosenker, K.; Scerba, M. T.; Saha, M.; Hughes, E.;
Wang, A. Q.; et al. Preclinical evaluation of a mercaptobenzamide and
its prodrug for NCp7-targeted inhibition of human immunodeficiency
virus. Antiviral Res. 2016, 134, 216–225.
(11) Sosic, A.; Sinigaglia, L.; Cappellini, M.; Carli, I.; Parolin, C.;
Zagotto, G.; Sabatino, G.; Rovero, P.; Fabris, D.; Gatto, B. Mechanisms
of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-
Anthraquinone Conjugates. Bioconjugate Chem. 2016, 27, 247–256.
(12) Frecentese, F.; Sosic, A.; Saccone, I.; Gamba, E.; Link, K.;
Miola, A.; Cappellini, M.; Cattelan, M. G.; Severino, B.; Fiorino, F.; et
al. Synthesis and in Vitro Screening of New Series of 2,6-Dipeptidyl-
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 8
1
2
3
4
5
6
7
8
(28) Pace, P.; Di Francesco, M. E.; Gardelli, C.; Harper, S.;
structural characterization of a new inhibitor series of HIV-1
nucleocapsid function: NMR solution structure determination of a
ternary complex involving a 2:1 inhibitor/NC stoichiometry. J. Mol.
Biol. 2013, 425, 1982–1998.
(32) Saladini, F.; Giannini, A.; Boccuto, A.; Vicenti, I.; Zazzi, M.
Agreement between an in-house replication competent and a reference
replication defective recombinant virus assay for measuring phenotypic
resistance to HIV-1 protease, reverse transcriptase, and integrase
inhibitors. J. Clin. Lab. Anal. 2018, 32, e22206.
(33) Esposito, F.; Sechi, M.; Pala, N.; Sanna, A.; Koneru, P. C.;
Kvaratskhelia, M.; Naesens, L.; Corona, A.; Grandi, N.; Di Santo, R.;
et al. Discovery of dihydroxyindole-2-carboxylic acid derivatives as
dual allosteric HIV-1 Integrase and Reverse Transcriptase associated
Ribonuclease H inhibitors. Antiviral Res. 2020, 174, 104671.
Muraglia, E.; Nizi, E.; Orvieto, F.; Petrocchi, A.; Poma, M.; Rowley,
M.; et al. Dihydroxypyrimidine-4-carboxamides as novel potent and
selective HIV integrase inhibitors. J. Med. Chem. 2007, 50, 2225–
2239.
(29) Gardelli, C.; Nizi, E.; Muraglia, E.; Crescenzi, B.; Ferrara, M.;
Orvieto, F.; Pace, P.; Pescatore, G.; Poma, M.; Ferreira, M. D. R. R.; et
al. Discovery and synthesis of HIV integrase inhibitors: development
of potent and orally bioavailable N-methyl pyrimidones. J. Med. Chem.
2007, 50, 4953–4975.
(30) Guzman, R. N. de. Structure of the HIV-1 Nucleocapsid Protein
Bound to the SL3 -RNA Recognition Element. Science 1998, 279, 384–
388.
(31) Goudreau, N.; Hucke, O.; Faucher, A.-M.; Grand-Maître, C.;
Lepage, O.; Bonneau, P. R.; Mason, S. W.; Titolo, S. Discovery and
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
SYNOPSIS TOC (Word Style “SN_Synopsis_TOC”). If you are submitting your paper to a journal that requires a synopsis
graphic and/or synopsis paragraph, see the Instructions for Authors on the journal’s homepage for a description of what needs
to be provided and for the size requirements of the artwork.
ACS Paragon Plus Environment