2-(Arylamino)-6-(trifluoromethyl)nicotinic acid derivatives: New HIV-1 RT dual inhibitors active on viral replication

Article abstract of DOI:10.3390/molecules25061338

The persistence of the AIDS epidemic, and the life-long treatment required, indicate the constant need of novel HIV-1 inhibitors. In this scenario the HIV-1 Reverse Transcriptase (RT)-associated ribonuclease H (RNase H) function is a promising drug target. Here we report a series of compounds, developed on the 2-amino-6-(trifluoromethyl)nicotinic acid scaffold, studied as promising RNase H dual inhibitors. Among the 44 tested compounds, 34 inhibited HIV-1 RT-associated RNase H function in the low micromolar range, and seven of them showed also to inhibit viral replication in cell-based assays with a selectivity index up to 10. The most promising compound, 21, inhibited RNase H function with an IC₅₀ of 14 μM and HIV-1 replication in cell-based assays with a selectivity index greater than 10. Mode of action studies revealed that compound 21 is an allosteric dual-site compound inhibiting both HIV-1 RT functions, blocking the polymerase function also in presence of mutations carried by circulating variants resistant to non-nucleoside inhibitors, and the RNase H function interacting with conserved regions within the RNase H domain. Proving compound 21 as a promising lead for the design of new allosteric RNase H inhibitors active against viral replication with not significant cytotoxic effects.

Full text of DOI:10.3390/molecules25061338

molecules
Article
2-(Arylamino)-6-(trifluoromethyl)nicotinic Acid
Derivatives: New HIV-1 RT Dual Inhibitors Active on
Viral Replication
Angela Corona 1 , Valentina Onnis 1 , Claudia Del Vecchio 2, Francesca Esposito 1,
Yung-Chi Cheng 3 and Enzo Tramontano 1,2,3,4,
*
1
Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di
Monserrato, 09042 Monserrato, Cagliari, Italy; angela.corona@unica.it (A.C.); vonnis@unica.it (V.O.);
francescaesposito@unica.it (F.E.)
Department of Molecular Medicine, University of Padova, 35121 Padova, Italy; claudia.delvecchio@unipd.it
Department of Pharmacology, Yale University Medical School, New Haven, CT 06520-8066, USA;
yccheng@yale.edu
2
3
4
Genetics and Biomedical Research Institute, National Research Council, 09042 Monserrato, Italy
Correspondence: tramon@unica.it; Tel.: +39-070-675-4538
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 28 February 2020; Accepted: 12 March 2020; Published: 15 March 2020
Abstract: The persistence of the AIDS epidemic, and the life-long treatment required, indicate the
constant need of novel HIV-1 inhibitors. In this scenario the HIV-1 Reverse Transcriptase
(RT)-associated ribonuclease H (RNase H) function is a promising drug target. Here we report a
series of compounds, developed on the 2-amino-6-(trifluoromethyl)nicotinic acid scaffold, studied
as promising RNase H dual inhibitors. Among the 44 tested compounds, 34 inhibited HIV-1
RT-associated RNase H function in the low micromolar range, and seven of them showed also to
inhibit viral replication in cell-based assays with a selectivity index up to 10. The most promising
compound, 21, inhibited RNase H function with an IC50 of 14 µM and HIV-1 replication in cell-based
assays with a selectivity index greater than 10. Mode of action studies revealed that compound 21
is an allosteric dual-site compound inhibiting both HIV-1 RT functions, blocking the polymerase
function also in presence of mutations carried by circulating variants resistant to non-nucleoside
inhibitors, and the RNase H function interacting with conserved regions within the RNase H domain.
Proving compound 21 as a promising lead for the design of new allosteric RNase H inhibitors active
against viral replication with not significant cytotoxic effects.
Keywords: HIV-1 therapeutic agents; RT dual inhibitors; HIV-1 ribonuclease H; nicotinic acid esters;
nicotinic acid amide
1. Introduction
The acquired immunodeficiency syndrome (AIDS) pandemic is still one of the major world health
concerns. Presently approved treatments do not allow immunization of people or the eradication of
the infection once acquired, and among the estimated 37.9 million people living with HIV worldwide
in 2019, only 24.5 million people were accessing the current antiretroviral therapy [
highly-active antiretroviral therapy (HAART) allows patients who receive optimum treatment a
life-expectation comparable to that of the uninfected population [ ], the sub-optimal adherence
and coverage of seropositive people, coupled with life-long treatment, still cause emergence of
drug-resistant variants, that could lead to therapy failure [ ], and whose transmission to naïve patient
is a major concern and requires an optimized treatment [ ]. These data remark the constant need
to expand the antiretroviral armamentarium with new drugs, with novel targets, to be employed on
1]. While the
2,3
4
5–7
Molecules 2020, 25, 1338; doi:10.3390/molecules25061338
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1338
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salvage therapy. HIV-1 reverse transcriptase (RT) has been the first and most exploited target. RT is
a heterodimeric (p66/p51) multifunctional enzyme that acts at the early stage of viral infection by
converting the viral single-stranded RNA genome into a double-stranded DNA, with the concerted
and dynamic interplay of its enzymatic functions RNA- and DNA-dependent DNA polymerase (RDDP
and DDDP, respectively), and RT- associated ribonuclease H (RNase H) [8,9].
HIV-1 RT-associated RNase H is an enzymatic function present in all the members of the
Ortervirales order [10] and it belongs to the nucleotidyltransferase family, characterized by a highly
conserved Asp-Asp-Glu (DDE) motif, essential for coordination of two Mg2+ ions, and by a tertiary
structure [11] conserved among its members [12–15]. HIV-1 RNase H function is essential for viral
replication [16] and; therefore, is a good target for inhibitors [17]. Two decades of research identified
many classes of compounds able to block its activity, that could be grouped in four big families
according to their mode of action [17–
19]: (i) Active site inhibitors, which chelate the two Mg2+
ions coordinated in the enzyme site by the DDE motif; (ii) RNase H selective allosteric inhibitors;
(iii) allosteric inhibitors that bind at the p66/p51 interface; (iv) dual allosteric inhibitors of both the
RDDP and RNase H functions. None of them has reached the clinical trial so far, enlightening the level
of difficulty represented by the target [20].
The RNase H active-site inhibitors take advantage of the driving force represented by their
chelating moiety to stably interact with the catalytic domain [21
often hampered by off-target toxicity and competition with nucleic acid substrates [23
,
22]; however, their development is
24], and by
,
the difficulties to establish strong additional interactions within the RNase H domain, although the
possibility to target highly conserved residues in the surrounding molecular space has been successfully
reported, with particular regard to the residues involved in the RNase H primer grip motif [25,26].
The allosteric inhibition of the polymerase function of RT was achieved in the 1990s [27] with
non-nucleoside inhibitors (NNRTI) that are still in the first-line therapy [28], although displaying a
fairly low genetic barrier to selection of resistant variants, among which K103N, Y181C, and Y188L are
the most common [29]. Differently, the first approaches to allosteric RNase H inhibition attempted to
disrupt the enzyme stability by inhibiting the dimerization [30], and several efforts have been made to
develop ligands able to bind at the interface between the heterodimer subunits [31
RT heterodimer stability and flexibility, critical for its activity, as it is done by vinylogous ureas that
bind in a site located in p51 subunit involved Cys280 and Lys281, located in -helix H [34]. During the
–33], compromising
α
years also two other allosteric pockets have been identified within the p66 RT domain. The first
one, firstly reported for the binding of the hydrazone DHBNH [35], it is located >50 Å away from
the RNase H active site, between the RT polymerase active site and the polymerase primer grip,
and partially overlaps with the NNRTI binding site. The second one, located between the RNase H
active site and the region encompassing
α-helices B and D in the RNase H domain, is centered on
the residue Q507 [36 37]. Structure–activity relationship revealed that these two sites share common
,
pharmacophoric requirements that have been summarized in two diverse substituted aromatic portions,
bridged by a central core, either linear or made by heterocyclic rings, containing donors and acceptors
of hydrogen bonds [38–43]. These features lead to the design of compound RMNC6 [42] (Figure 1) that
has been proved to be a dual-site dual RT-functions inhibitor, able to block both RNase H and RDDP
functions. More recently another compound, A15 (Figure 1), showed the same dual inhibitor profile,
being also active against viral replication [40], hence embracing the promising polypharmacology
approach to develop effective drugs less susceptible to the selection of resistant variants [44].
Taking into account this information, we explored the 2-amino-6-trifluoromethyl-3-
pyridinecarboxylic acid as a central core to design new allosteric RNase H inhibitors endowed
with potential dual-target ability. Nicotinic and isonicotinic acid derivatives have been exploited as
antimicrobial agents [45,46], although none of the molecules was tested on the RNase H function
or was reported to be able to inhibit the viral replication. In the presented series of compounds,
various aromatic portions are introduced as esters or amide of the nicotinic acid function, with an
optional benzyl or piperazine linker (Scheme 1). Among the new 44 compounds, 34 inhibited HIV-1

Molecules 2020, 25, 1338
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RT-associated RNase H function in the low micromolar range, and seven of them showed also to
inhibit viral replication in cell-based assays with a selectivity index up to 10, exhibited by compound
21 (Figure 1), the most promising derivative, that was investigated for its mode-of-action, displaying a
dual-site, dual inhibition profile.
Figure 1. Structure of dual-site dual inhibitors. In red, the functionalized spacer; in black, the aromatic
side portions. Summary of the 2-(arylamino)-6-(trifluoromethyl)nicotinic acid derivatives, in blue
squares; the aromatic side portions, within red square.
CN
NH
Cl
NH2
EtO
COCF3
4
NC
H3C
NC
i
F3C
N
ii
CH3
H2N
N
Cl
H2N
OEt
H
CH3
5
1
2
Cl
3
iv
iii
CONH2
NH
COOH
NH
COOAr
NH
v
F3C
N
F3C
N
F3C
N
CH3
CH3
CH3
8
10
Cl
Cl
Cl
11-24
vi
CONHR
NH
F3C
N
CH3
Cl
25-52
Scheme 1. Synthetic routes to compounds 6, 11–52. (i) MeCN, r.t.; (ii) MeCN reflux; (iii) 20% aq
NaOH, 100 ◦C; (iv) 50% aq H2SO4, 100 ◦C; (v) ArOH, EDCI, OHBt, MeCN, r.t.; vi) RNH2, EDCI, OHBt,
MeCN, r.t.

Molecules 2020, 25, 1338
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2. Results
2.1. Synthesis of 2-amino-6-(trifluoromethyl)nicotinate derivatives
The target derivatives of 2-(2-methyl-5-chloro)-6-trifluoromethyl-3-pyridinecarboxylic acid
52 (Table 1) were synthesized as shown in Schemes 1 and 2. We have previously reported an easy
and convenient method for the synthesis of 2-arylamino-6-trifluoromethyl-3- pyridinecarboxylic
acids [47]. According to Scheme 1, 3-amino-3-ethoxypropenenitrile ( ) was first treated with
the 2-methyl-5-chloroaniline ( ) in MeCN solution to give the non-isolable 3-amino-3-(2-methyl-5-
chlorophenyl)aminopropenenitrile ( ) and then with 1,1,1-trifluoro- 4-iso-butoxy-3-buten-2-one ( ).
The temperature was gradually allowed to reach 60 ◦C. After 3 h, pyridinecarbonitrile
was obtained
in good yields. Sodium hydroxide catalyzed hydrolysis of nitrile afforded pyridine-3-carboxamide
. High yield of 6-trifluoromethyl-3-pyridinecarboxylic acid 10 was achieved by hydrolysis of
6, 9,
11
–
1
2
3
4
5
5
6
derivative
5
in 50% aqueous sulfuric acid. Chemistry similar to that described in the synthesis of
was used for the preparation of ethyl ester 9 [48]. As reported in Scheme 2, ethyl
) was sequentially treated with the amine , then with enol ether in
MeCN solution to give the ester . Treatment of acid 10 with the appropriate phenol in the presence
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and hydroxybenzotriazole
(HOBt) in MeCN solution gave esters 11 24 (Table 1) in 52$%–98% yields. Amide derivatives 25 52
compound
5
3-amino-3-ethoxypropenoate (
7
2
4
9
–
–
(Table 1) were obtained in 39%–96% yields by the reaction of acid 10 with the appropriate amine by
EDCI method [48]. All the newly synthesized compounds gave corrected analytical data. The IR and
NMR spectral data were consistent with the assigned structure.
Scheme 2. Synthetic route to compound 9. (i) MeCN, r.t.; (ii) MeCN, reflux.
2.2. Evaluation of RNase H Inhibitory Activity
To establish the potential biologic effect of the synthesized compounds we tested them on the
HIV-1 RT-associated RNase H activity, using as a control the RNase H inhibitors RDS1759 [25] and
beta-Thujaplicinol (BTP) [49] (Table 1). The ethyl nicotinate
9 showed promising inhibitory activity
(IC50 24 M) of the RNase H function. The replacement of ethoxy group with 4-substituted aryl rings,
µ
produced changes in potency, with the 4-chlorine (13), 4-methoxy (18) and 4-thiomethyl (18) aryl esters
being the most potent of the ester series. Interestingly the shift of the 4-chlorine atom into 2- or 3-position
led to drop in activity (esters 11 and 12). The shift of the 4-hydroxy group of ester 18 into 2-position to
give the analog 16 produced a small increase in inhibition potency, while the 3-hydroxyaryl ester 17 was
less potent as compared to compounds 16 and 18. The methylation of the hydroxy group of compound
18, as well as its replacement with a thiomethyl group, (esters 23 and 24, respectively), produced about
two-fold enhancement of potency. On the contrary, the introduction of further methoxy groups, as in
compounds 20–22, slightly reduced the potency of inhibition. The introduction of a 2-chlorine atom on
compound 13 to give the ester 14 abolished the activity, while the 2,4,6-trichlorophenyl ester 15 showed
about four-fold reduction in potency as compared to ester 13. The 3,5-dimethoxyphenyl ester 21
resulted about 1.5-fold more potent on the RNase H function as compared to the 3,4-dimethoxyphenyl
analog 20.

Molecules 2020, 25, 1338
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Table 1. Biological effects of derivatives on HIV-1 Reverse Transcriptase (RT)-associated RNase
H function.
Compound
R
R’
RNase H IC50 (µM) a
>100 (87%) b
24.0 ± 1.7
>100 (92%)
>100 (98%)
9.3 ± 2.5
6
9
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
H
OEt
2-Cl-phenyl
3-Cl-phenyl
4-Cl-phenyl
2,4-Cl2-phenyl
2,4,6-Cl3-phenyl
2-OH-phenyl
3-OH-phenyl
4-OH-phenyl
>100 (98%)
31.8 ± 3.5
16.2 ± 6.6
26.6 ± 5.6
20.0 ± 1.3
>100 (80%)
21 ± 11
3-OCH3-phenyl
3,4-(OCH3)2-phenyl
3,5-(OCH3)2-phenyl
3,4,5-(OCH3)3-phenyl
4-OCH3-phenyl
4-SCH3-phenyl
14.7 ± 1.4
17.3 ± 1.3
11.6 ± 4.3
9.6 ± 0.8
OH
CH2COOEt
3-pyridyl
4-SCH3-phenyl
3-CH3-phenyl
3-Cl-phenyl
0.77 ± 0.06
>100 (100%)
25.0 ± 4.9
5.9 ± 0.9
8.9 ± 0.1
4.0 ± 2.2
2,4-Cl2-phenyl
3-Cl, 4-OCH3
8.8 ± 0.5
>100 (100%)
>100 (54%)
17.5 ± 3.9
22.4 ± 4.0
19.4 ± 1.5
7.8 ± 1.2
3-OCH3-phenyl
3,5-(OCH3)2-phenyl
3,4-(OCH3)2-phenyl
3,4,5-(OCH3)3-phenyl
4-CF3-phenyl
2-Cl-benzyl
2-OCH3-benzyl
4-Cl-benzyl
4-F-benzyl
4-CF3-benzyl
19.4 ± 1.1
18.7 ± 1.7
6.4 ± 0.5
23.3 ± 3.1
21.9 ± 0.2
30.4 ± 3.9
21.3 ± 0.5
19.3 ± 0.6
25.6 ± 3.4
18.2 ± 1.7
>100 (100%)
18.0 ± 5.7
>100 (100%)
20.8 ± 2.5
7.5 ± 2.0
42
43
44
45
46
47
48
49
4-OCH3-benzyl
4-CH3-benzyl
2,4-Cl2-benzyl
3,4-Cl2-benzyl
3-OCH3, 4-OH-benzyl
4-phenylpiperazino
4-(3-Cl-phenyl)piperazino
4-(4-F-phenyl)piperazino
4-(4-OCH3-phenyl)piperazino
4-(3,4-Cl2-phenyl)piperazino
-
50
51
52
RDS1759
BTP
-
-
8.5 ± 0.5
-
0.19 ± 0.03
a
Compound concentration ( standard deviation) required to inhibit HIV-1 Reverse Transcriptase (RT)-associated
±
b
RNase H activity in cells by 50%. Numbers in parenthesis represent the percentage of cells viability in presence of
the highest concentration of inhibitor tested.

Molecules 2020, 25, 1338
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The amides showed a RNase H inhibitory activity rather different from esters. The nicotinamide
was completely inactive, while the oxime 25 showed more potent anti-RNase H activity, being
6
the best inhibitor with an IC50 value of 0.7
inhibited RNase H activity with IC50 in the 5.6-20
of amide 29 with a 3-methoxy group to give compound 33 abolished the activity, while the presence
of two or three methoxy groups as in compounds 34 36 repristinated the anti-RNase H efficacy.
The benzylamides 38 47 showed RNase H inhibitory activity (IC50 in the 9–24 M concentration
range). The arylpiperazino amides 49 and 51 displayed some RNase H inhibitory potency (IC50
values = 18 and 20 M, respectively). The introduction of a second chlorine atom on amide 49, to give
µM. The monosubstituted aryl amides 28–30 and 37
µM range. The replacement of the 3-methyl group
–
–
µ
µ
the 3,4-dichlorophenyl piperazine analog 52, produced an increase in potency (IC50 7 µM).
2.3. Evaluation of Antiviral Activity
The 28 compounds that were able to inhibit the HIV-1 RNase H activity with an IC50 value below
30
ester 13 showed to be active on the HIV replication in the same range of concentration of the IC50 value,
with an EC50 value of 10 M, with a selectivity index (SI) of 3.6. The 3,4-dimethoxyphenyl ester 21
resulted as the best ester inhibitor of the HIV replication activity with an EC50 value of 5 M and a SI
greater than 10. Amides 34 and 36 showed to be able to inhibit HIV-1 replication (EC50 values = 2 and
1.8 M, respectively) although residual cytotoxic effect activity caused a poor selectivity (SI = 2.6 and
1.8, respectively), and also the amides 49 and 52 displayed HIV inhibitory activity (EC50 values = 10
µM were tested for the ability to inhibit HIV-1 replication in TZMbl cells (Table 2). The 4-chlorophenyl
µ
µ
µ
µM
and SI = 2.7 and 1.6, respectively). Noteworthy, the most active RNase H inhibitor in biochemical
assays, compound 25, was inactive in the HIV replication assay.
Table 2. Biological effects of derivatives on HIV-1 replication.
HIV-1 a EC50 (µM)
b
CC50 (µM)
SI d
Compoud
TZMbl Cells
TZMbl
CEM Cells
9
>10
10
>10
>50 (75%) c
36 ± 1
>50 (100%)
>50 (70%)
>12.5 (100%)
>50 (100%)
>50 (100%)
>50 (100%)
>50 (100%)
35
-
3.6
-
-
>10
-
-
-
-
1.6
-
-
2.6
1.2
-
-
-
-
-
-
-
-
-
-
-
13
16
17
21
23
24
25
27
29
30
31
34
36
37
38
39
40
41
42
43
44
45
46
47
49
12.2 ± 2.1
>50 (62%)
>50 (59%)
>50 (70%)
50
>50
5 ± 0.8
>10
>10
>10
14 ± 2.8
5.8 ± 0.8
3.9 ± 0.9
3.9 ± 0.9
4.7 ± 1.7
5.3 ± 0.6
2.2 ± 0.4
2.2 ± 0.2
5.7 ± 0.7
14.8 ± 3.1
4.7 ± 0.4
6 ± 0.6
>5.8
2.5 ± 0.4
>3.9
>4.7
2 ± 0.3
1.8 ± 0.6
>2.2
>5.7
>10
>4.7
>6
>3.6
>10
16.2
6.3
8.4
7.8
10.4
10.2
4.3
12.5
>12.5 (55%)
7.75
11.6
5
20
3.5
9.15
10.6
17.4
3.6 ± 1.2
13.2 ± 1.3
1.1 ± 0
>1.1
>10
>6.3
>10
5 ± 0.6
6.3 ± 1.3
15 ± 3
10 ± 1.5
27 ± 3
>50 (82%)
2.7

Molecules 2020, 25, 1338
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Table 2. Cont.
HIV-1 a EC50 (µM)
b
CC50 (µM)
SI d
Compoud
TZMbl Cells
TZMbl
CEM Cells
51
52
EFV
>10
27.7 ± 12.8
15.7 ± 5.1
>50 (81%)
>50 (76%)
>50 (100%)
-
10 ± 1.2
1.6
357
0.15 ± 0.02
>50 (100%)
a
b
c
Effective concentration 50 (EC50): compound concentration required to decrease viral replication in cells by 50%.
Citotoxic concentration 50 (CC50): ompound concentration required to reduce infected cells viability by 50%.
Numbers in parenthesis represent the percentage of cells viability in presence of the highest concentration of
d
inhibitor tested. Selectivity index.
2.4. Mode of Action Studies
In order to confirm our hypothesis of allosteric binding mode, we firstly examined the possible
interaction between selected compounds and the magnesium, used as a cofactor by both the
RT-associated enzymatic functions. The UV spectra of the seven compounds able to inhibit viral
replication were measured in the absence and in the presence of magnesium ions, using as a positive
control the active site inhibitor RDS1759. Results (Figure 2) showed that, while the positive control
displayed a typical shift in the maximum of absorbance of the spectra, among the tested compounds
only amide 29 showed a shift of 17 nm in the peak of absorbance, while amides 34 and 52 showed
a moderate hyperchromic effect, but not a shift. Differently, the spectra of compounds 13, 21, 36,
and 49 were not affected by the presence of the Mg2+. Overall, the data seem to exclude that Mg2+
coordination might be involved in the mechanism of action of the selected compounds.
Figure 2. Effect of MgCl2 on the absorbance spectra of 2-(arylamino)-6-(trifluoromethyl)nicotinic
acid derivatives. Mg2+ chelation UV-Vis spectrum was measured with 100
µM of compound alone
(unbroken line) or in the presence of 6 mM MgCl2 (dotted line). The active site RNase H inhibitor
RDS1759 was used as reference.

Molecules 2020, 25, 1338
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In order to further dissect the mode of action the most promising compound, we selected ester 21
that inhibited the RNase H function with an IC50 value of 14 M, the HIV-1 replication in cell culture
with EC50 value of 5 M and showed a SI 10, along with the amide derivative 49, that was also active
against HIV-1 replication but showed a higher toxicity in TZMbl cells. Both compounds were tested
against the RDDP function (Table 3), showing IC50 values (16.1 and 22.9 M, respectively), very similar
µ
µ
≥
µ
to the ones obtained on the RNase H function, indicating a dual-function allosteric inhibitory profile.
Table 3. Inhibition of HIV-1 RDDP activity of mutated HIV-1 RTs.
21
IC50 (µM) a
49
IC50 (µM) a
EFV
IC50 (nM) a
RT
Foldc
Fold
Fold
WT
16.1 ± 1.2
16.5 ± 4.8
22.5 ± 5.3
82.8 ± 6.3
17.0 ± 4.5
8.0 ± 0.7
22.9 ± 1.4
68.6 ± 3.0
46.4 ± 2.4
>100 (54%) b
57.7 ± 2.1
27.0 ± 1.7
23 ± 4.1
176 ± 25
49.7 ± 9.1
21.3 ± 3.6
34.5 ± 2.3
24.7 ± 2.4
1
K103N
Y181C
V108A
Q475A
A502F
1.0
1.4
5.1
1.0
0.5
2.5
2.0
> 4.3
2.5
7.6
2.2
0.9
1.5
1.1
1.2
a
Inhibitory Concentration 50 (IC50): compound concentration ( standard deviation) required to inhibit HIV-1
Reverse Transctiptase (RT)-associated RDDP activity in cells by 50%. Numbers in parenthesis represent the
percentage of cells viability in presence of the highest concentration of inhibitor tested. Fold: ratio between the
±
b
c
IC50 showed by the compound against the mutated RT and the IC50 against the wt RT.
To clarify their binding mode, and also ascertain the possibility that these compounds could
be active against circulating drug-resistant variants, compounds 21 and 49 were tested against both
RT-associated functions of a panel of enzymes carrying single amino acid substitutions in the polymerase
domain: mutations K103N, Y181C that are related to resistance to the NNRTIs [29], and mutation
V108A that is related to the binding of the allosteric RNase H inhibitors on the pocket close to the
NNRTI binding site [42]. Besides, both compounds were tested also on RTs mutated in two amino
acid residues within the RNase H domain, Q475A and A502F, part of conserved regions of RNase H
domain [50], that have been shown to be involved in the binding of RNase H inhibitors into a pocket
close to the RNase H catalytic site but acting as allosteric RNase H inhibitors [25,37,40].
We tested the inhibition of the HIV-1 RDDP activity of mutated HIV-1 RTs by compounds 21 and
49 (Table 3), using as control the NNRTI efavirenz (EFV). Interestingly, the two compounds displayed
a profile very different from EFV. Indeed, compounds 21 and 49 were able to inhibit the K103N and
Y181C RT-associated RDDP activities. In particular, compound 21 showed no difference in potency of
inhibition towards the two enzymes, while compound 49 showed a 2.5-fold decrease in potency of
inhibition for K103N RT and a decrease of two-fold against Y181C RT. As expected, the IC50 value
of EFV showed an increase of 7.6-fold against K103N, while it did not show change in potency of
inhibition when tested against the V108A RT RDDP activity. Differently, compounds 21 and 49 showed
a consistent increase in IC50 value (5.1- and >4.3-fold for compound 21 and 49, respectively) on the
V108A RT RDDP function. Of note, all the inhibitors were able to inhibit the RT-associated RDDP
activity of A502F and Q475A RTs, except for 49 that showed to increase its RDDP IC50 value on Q475A
RT of 2.5-fold.
The HIV-1 RNase H activity of the mutated HIV-1 RTs in the presence of compounds 21 and 49
was analyzed using as control the RNase H active site inhibitor BTP (Table 4). Results showed that the
positive control equally inhibited all the tested enzymes, while compound 21 had a 3.7-fold increase in
IC50 value on the K103N RT RNase H function, and it completely lost its RNase H inhibitory efficacy
towards V108A, Q475A, and A502F RTs. Differently, compound 49 showed a moderate decrease in its
potency of inhibition on the RNase H activity of the K103N, Y181C, and A502F RTs, being completely
ineffective against the RNase H function of V108A and Q475A RTs.

Molecules 2020, 25, 1338
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Table 4. Inhibition of HIV-1 RNase H activity of mutated HIV-1 RTs.
21
IC50 (µM) a
49
IC50 (µM)
BTP
IC50 (µM)
RT
Fold
Fold
Fold
WT
K103N
Y181C
V108A
Q475A
A502F
a
14.7 ± 1.4
53.9 ± 2.2
18.0 ± 5.7
41.0 ± 2.2
0.19 ± 0.03
0.22 ± 0.08
0.23 ± 0.05
0.34 ± 0.4
0.19 ± 0.03
0.17 ± 0.03
1
3.7
0.8
>6.8
>6.8
>6.8
2.3
3.1
>5.5
>5.5
2.6
1.2
1.2
1.8
1.0
0.9
11.9 ± 1.4
55.4 ± 12.6
>100 (100%)
>100 (100%)
47.7 ± 5.4
>100 (100%) b
>100 (100%)
>100 (55%)
Compound concentration (
±
standard deviation) required to inhibit HIV-1 RT-associated RNase H activity in cells
by 50%.b Numbers in parenthesis represent the percentage of cells viability in presence of the highest concentration
of inhibitor tested.
Overall, results obtained on the V108A RT, whose RDDP and RNase H functions were both
non-susceptible to inhibition by compounds 21 and 49, indicate a major role played by the pocket close
to the NNRTI binding site on inhibition of these compounds of both the enzymatic activities, with a
combination of short- and long-range effects, and suggest a key role of residue V108 for the correct
interaction between the inhibitors and the binding pocket. On the second hand, the fact that amino
acidic substitutions Q475A and A502F caused a loss of potency only for inhibition of their RNase
H functions suggest that this second allosteric pocket exerts a short-range influence only within the
RNase H domain.
3. Discussion
Dual inhibitors are a new class of molecules that aim to increase the efficacy of the drug and raise
the genetic barrier to overcome the selection of resistant strains by hitting two targets at the same
time [44
against circulating drug-resistant variants, this approach has been applied to RDDP/Integrase and
RNase H/Integrase dual inhibitors [53 61], that target two different viral enzymes, and to RNase
,51,52]. In the constant need to identify and develop new anti-HIV drugs, possibly active
–
H/RDDP dual inhibitors, that target two distinct, but strongly interconnected, enzymatic functions
located in the same enzyme [9,36,62].
In this work, we designed a set of 44 new compounds following the pharmacophoric requirements
previously identified for RNase H/RDDP dual-site dual inhibitors [38
core was represented by the 6-(trifluoromethyl)nicotinic acid, and, differently from the previous
attempt to design nicotinate-based antimicrobial agents that had two aromatic functions [45 46], we
decorated the core, making ester or amide of the nicotinic acid and introducing an arylamino moiety at
the 2-position. The scaffold maintained a sp2 hybridization state of the linker [40 42], that in this case
–42,63]. In this case, the central
,
,
involved the aryl ring, with a steric hindrance slightly bigger than previously reported compounds.
For the synthesis of the compounds, we successfully applied our previously reported method for
the synthesis of 2-arylamino-6-trifluoromethyl-3-pyridine carboxylic acids [47], and the final esters
and amides were obtained by EDCI method in good yields and high purity without use of a tedious
purification procedure [48].
The designed molecules showed a consistent inhibition of the HIV-1 RT-associated RNase H
function, with the amide derivatives being overall slightly more potent than the ester counterparts.
Cell-based assays revealed that the scaffold is more promising than the previous reported attempts
of RNase H/RDDP dual inhibitors. Among the 44 2-(arylamino)-6-(trifluoromethyl)nicotinic acid
derivatives, 7 compounds inhibited viral replication, while no active compound has been identified so
far among the isatin-derivatives [41,42]. Furthermore, among the pyrrole and pyrazole derivatives,
only one dual inhibitor was active against viral replication [40], being less active (EC50 = 25
more toxic (CC50 = 44 µM).
µM) and
The 3,5-dimethoxyphenyl ester 21 was able to block HIV-1 replication with a SI > 10. The mode
of action studies revealed that the compound acts differently form NNRTI inhibitors, with an

Molecules 2020, 25, 1338
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activity profile against RDDP activity different from EFV on K103N and Y181C RTs, since these
amino acid substitutions had a lower impact on its efficacy as compared to previously reported
compounds [40,42,64]. Site-directed mutagenesis experiments showed that compound 21 binds
independently to two different allosteric sites previously described: S first pocket (pocket 1),
contiguous with the NNRTI binding pocket, was described for hydrazones [35], isatines [41
and pyrazoles [40]. A second pocket (pocket 2), within the RNase H domain, was firstly reported
for selective allosteric RNase H inhibitors, such as hydrazones derivatives [36 37], and later for
isatines [41 42] and pyrazoles [40]. For years the contribution of the two pockets to the inhibition of
,42]
,
,
the two enzymatic functions was controversial. Our results confirmed that pocket 2 is an allosteric
site for selective inhibition of HIV-1 RT-associated RNase H, since mutations A502F and Q475A
completely abrogated the inhibitory effect of compound 21 on that sole function, proving also that
these two conserved residues [50] play a key role on compound activity. Moreover, we showed that
binding to pocket 1 is sufficient for inhibition of both enzymatic activities, with a combination of long-
and short-range effects, as previously hypothesized [36,42], since the introduction of amino acidic
substitution V108A had a strong impact on the inhibition of both RDDP and RNase H functions by
compound 21.
These results provide useful insights for the design of novel and more potent HIV-1 RT RNase
H/RDDP allosteric dual inhibitors, active against selected drug-resistant variants. With this aim,
a major focus should be pointed on pocket 1 as the most suitable to be exploited for drug design,
and on compound 21 as a promising good hit to be developed.
4. Materials and Methods
4.1. Chemistry
All commercially available solvents and reagents were used without further purification. NMR
spectra were recorded on an Inova 500 spectrometer (Varian, Palo Alto, CA, USA). The chemical
shifts (δ) are reported in parts per million downfield from tetramethylsilane (TMS), which was used
as internal standard, and the spectra were recorded in hexadeuteriodimethylsulphoxide (DMSO-d6).
Infrared spectra were recorded on a Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol
mulls. The main bands are given in cm−1. Positive-ion electrospray ionization (ESI) mass spectra were
recorded on a double-focusing MAT 95 instrument (Finnigan, Waltham, MA, USA) with BE geometry.
Melting points (mp) were determined on a SMP1 Melting Point apparatus (Stuart Scientific, Stone, UK)
and were uncorrected. All reported products showed NMR spectra in agreement with the assigned
structures. The purity of the tested compounds was determined by combustion elemental analyses
conducted by the Microanalytical Laboratory of the Department of Chemical and Pharmaceutical
Sciences of the University of Ferrara with a MT-5 CHN recorder elemental analyzer (Yanagimoto,
Kyoto, Japan), and the values found were within 0.4% of theoretical values. Compounds 6, 9 [47]
11–15, 19, 21, 23, 27, 29–37 and 49–51 were prepared as previously described [48].
4.1.1. General Procedure for the Synthesis of Esters (11–24)
A mixture of acid 10 (0.33 g, 1 mmol), EDCI (1.92 g, 1.1 mmol), and HOBt (0.13 g, 1 mmol) in dry
MeCN (10 mL) was stirred at room temperature for 30 min and then treated with the appropriate
phenol (1 mmol). The mixture was stirred at room temperature for an additional 24 h. Then the
solution was evaporated to dryness in vacuo. The residue was dissolved in ethyl acetate (20 mL)
and washed with brine (2
×
5 mL), 5% aqueous sodium hydroxide (2
×
5 mL), and water (2
×
5 mL).
The organic layer was dried over anhydrous sodium sulfate. Concentration of the dried extract yielded
a residue which was triturated with di-isopropyl ether. The formed precipitate was filtered off and
purified by crystallization from the adequate solvent to give the ester derivatives 11–24.
2-Hydroxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (16). Yield 86%. Mp
179–180 ◦C (MeCN). 1H NMR (DMSO-d6):
δ 2.38 (s, 3H, CH3), 6.98–7.41 (m, 5H, aryl), 7.52 d, 1H, J = 8.1

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Hz, pyridyl), 7.46 (d, 1H, J = 8.4 Hz, aryl), 8.26 (d, 1H, J = 8.4 Hz, aryl), 8.88 (d, 1H, J = 8.1 Hz, pyridyl),
9.98 (s, 1H, NH), 10.06 (s, 1H, OH). IR (Nujol) 3330, 1712, 1615, 1590 cm−1. m/z 423 (M + H)+. Anal.
Calcd for C20H14ClF3N2O3 (422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.87, %H 3.32, %N 6.61.
3-Hydroxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (17). Yield 87%. Mp
◦
1
188-190 C (MeCN). H NMR (DMSO-d6):
δ 2.27 (s, 3H, CH3), 7.30-7.66 (m, 7H, aryl), 8.11 (d,
1H, J = 8.5 Hz, aryl), 8.79 (d, 1H, J = 8.0 Hz, pyridyl), 9.88 (s, 1H, NH), 10.05 (s, 1H, OH).
IR (Nujol) 3294, 1712, 1620, 1590 cm−1. m/z 423 (M + H)+. Anal. Calcd for C20H14ClF3N2O3
(422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.78, %H 3.33, %N 6.60. 4-Hydroxyphenyl
2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (18). Yield 63%. Mp 184-186 ◦C (EtOH).
1H NMR (DMSO-d6):
δ 2.27 (s, 3H, CH3), 6.83 (d, 2H, J = 9.0 Hz, aryl), 7.13 (d, 2H, J = 7.0 Hz, aryl),
7.37 (m, 3H, aryl and pyridyl), 8.14 (d, 1H, J = 9.0 Hz, aryl), 8.73 (d, 1H, J = 7.5 Hz, pyridyl), 9.55 (s, 1H,
NH), 9.86 (s, 1H, OH). IR (Nujol) 3474, 3345, 1687, 1617, 1589 cm−1. m/z 423 (M + H)+. Anal. Calcd for
C20H14ClF3N2O3 (422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.79, %H 3.36, %N 6.66.
3,4-Dimethoxyphenyl-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (20). Yield 52%. Mp
1
127-129 ◦C (2-propanol). H NMR (DMSO-d6):
δ 2.30 (s, 3H, CH3), 3.79 (s, 3H, CH3), 3.86 (s, 3H, CH3),
6.85–7.98 (m, 6H, aryl and pyridyl), 8.24 (d, 1H, J = 8.5 Hz, aryl), 8.76 (d, 1H, J = 8.0 Hz, pyridyl), 10.51
(s, 1H, NH). IR (Nujol) 3345, 3278, 1748, 1699, 1622, 1608, 1591 cm−1. m/z 467 (M + H)+. Anal. Calcd
for C22H18ClF3N2O4 (466.84) %C 56.60, %H 3.89, %N 6.00. Found %C 56.65, %H 3.90, %N 6.02.
3,4,5-Trimethoxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (22). Yield 98%.
1
Mp 138-140 ◦C (2-propanol). H NMR (DMSO-d6):
δ 2.26 (s, 3H, CH3), 3.67 (s, 3H, CH3), 3.76 (s, 6H,
CH3), 6.73 (s, 2H, aryl), 7.28 (d, 1H, J = 8.5 Hz, aryl), 7.35–7.40 (m, 2H, aryl and pyridyl), 8.08 (d, 1H,
J = 8.5 Hz, aryl), 8.72 (d, 1H, J = 7.5 Hz, pyridyl), 9.89 (s, 1H, NH). IR (Nujol) 3331, 3293, 1697, 1618,
1590 cm−1. m/z 497 (M + H)+. Anal. Calcd for C23H20ClF3N2O4 (496.86) %C 55.60, %H 4.06, %N 5.64.
Found %C 55.54, %H 4.07, %N 5.66.
4-(Methylthio)phenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (24). Yield 63%. Mp
1
125–127 ◦C (2-propanol). H NMR (DMSO-d6):
δ (s, 3H, CH3), 7.41–7.52 (m, 10H, aryl and pyridyl),
8.24 (d, 1H, J = 8.8 Hz, aryl), 8.87 (d, 1H, J = 8.1 Hz, pyridyl), 10.03 (s, 1H, NH). IR (Nujol) 3294, 1712,
1620, 1590 cm−1. m/z 453 (M + H)+. Anal. Calcd for C21H16ClF3N2O2S (452.88) %C 55.69, %H 3.56,
%N 6.19. Found %C 55.74, %H 3.58, %N 6.17.
4.1.2. General Procedure for the Synthesis of Amides (25–52)
A mixture of acid 10 (0.33 g, 1 mmol), EDCI (1.92 g, 1.1 mmol), and HOBt (0.13 g, 1 mmol) in dry
MeCN (10 mL) was stirred at room temperature for 30 min and then treated with the appropriate
amine (1 mmol). The mixture was stirred at room temperature for an additional 24 h. Then the solution
was evaporated to dryness in vacuo. The residue was dissolved in ethyl acetate (20 mL) and washed
sequentially with brine (2
(2 5 mL), and water (2
Concentration of the dried extracts yielded a solid residue which was washed with ethyl ether, filtered
off, and dried to give the amide derivatives in analytically pure form without additional purification
by crystallization if not elsewhere indicated.
×
5 mL), 10% aqueous sodium carbonate (2
5 mL). The organic layer was dried over anhydrous magnesium sulfate.
×
5 mL), 10% aqueous citric acid
×
×
2-((5-Chloro-2-methylphenyl)amino)-N-hydroxy-6-(trifluoromethyl)nicotinamide (25). Yield 61%. Mp 154–155
◦C. 1H NMR (DMSO-d6):
δ 2.37 (s, 3H, CH3), 7.36–7.68 (m, 3H, aryl and pyridyl), 8.34 (d, 1H, J = 8.8
Hz, aryl), 8.86 (d, 1H, J = 7.7 Hz, pyridyl), 9.87 (s, 1H, NH), 10.59 (s, 1H, NH) 12.01 (s, 1H, OH). IR
(Nujol) 3350, 1686, 1618, 1589 cm−1. m/z 346 (M + H)+. Anal. Calcd for C14H11ClF3N3O2 (345.70) %C
48.64, %H 3.21, %N 12.15. Found %C 48.69, %H 3.20, %N 12.18.
Ethyl 2-(2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamido)acetate (26). Yield 96%. Mp
128-130 ◦C. 1H NMR (DMSO-d6):
δ 1.22 (t, J = 7.1 Hz, 3H, CH3), 2.39 (s, 3H, CH3), 4.27 (q, J = 7.1 Hz,

Molecules 2020, 25, 1338
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2H, CH2), 4.15 (d, J = 4.8 Hz, 2H, CH2), 7.25–7.39 (m, 3H, aryl and pyridyl), 8.20 (d, 1H, J = 8.0 Hz,
aryl), 8.56 (d, 1H, J = 7.3 Hz, pyridyl), 9.59 (s, 1H, NH), 10.69 (s, 1H, NH). IR (Nujol) 3429, 1737, 1722,
1655 cm−1. m/z 416 (M + H)+. Anal. Calcd for C18H17ClF3N3O3 (415.79) %C 52.00, %H 4.12, %N 10.11.
Found %C 52.05, %H 4.10, %N 10.08.
2-((5-Chloro-2-methylphenyl)amino)-N-(4-(methylthio)phenyl)-6-(trifluoromethyl)nicotinamide (28). Yield
64%. Mp 152-154 ◦C. 1H NMR (DMSO-d6):
δ 2.27 (s, 3H, CH3), 2.48 (s, 3H, CH3), 7.23 (d, 1H, J = 8.5
Hz, aryl), 7.28 (m, 3H, aryl), 7.36 (d, 1H, J = 7.5 Hz, pyridyl), 7.65 (d, 2H, J = 7.5 Hz, aryl), 8.15 (d, 1H,
J = 8.5 Hz, aryl), 8.46 (d, 1H, J = 7.5 Hz, pyridyl), 10.35 (s, 1H, NH), 10.64 (s, 1H, NH). IR (Nujol) 3380,
3300, 1639, 1588 cm−1. m/z 452 (M + H)+. Anal. Calcd for C21H17ClF3N3OS (451,89) %C 55.82, %H
3.79, %N 9.30.
N-(2-chlorobenzyl)-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamide (38). Yield 93%. Mp
◦
169-170 C (2-propanol). 1H NMR (DMSO-d6):
δ 2.39 (s, 3H, CH3), 4.72 (d, J = 5.2 Hz, 2H, CH2),
7.36–7.61 (m, 6H, aryl and pyridyl), 8.35 (d, 1H, J = 8.8 Hz, aryl), 8.56 (d, 1H, J = 8.0 Hz, pyridyl), 9.68
(s, 1H, NH), 10.90 (s, 1H, NH). IR (Nujol) 3300, 1636, 1613, 1597 cm−1. m/z 450 (M + H)+. Anal. Calcd
for C21H16ClF3N3O (449.85) %C 58.74, %H 4.26, %N 9.34. Found %C 58.69, %H 4.27, %N 9.30.
2-((5-Chloro-2-methylphenyl)amino)-N-(2-methoxybenzyl)-6-(trifluoromethyl)nicotinamide (39). Yield 53%.
1
Mp 148-150 ◦C (2-propanol). H NMR (DMSO-d6):
δ 2.40 (s, 3H, CH3), 3.96 (s, 3H, CH3), 4.63 (d, J = 5.1
Hz, 2H, CH2), 7.02–7.48 (m, 6H, aryl and pyridyl), 8.37 (d, 1H, J = 8.6 Hz, aryl), 8.55 (d, 1H, J = 7.7
Hz, pyridyl), 9.52 (s, 1H, NH), 11.00 (s, 1H, NH). IR (Nujol) 3315, 1639, 1617, 1599 cm−1. m/z 450 (M +
H)+. Anal. Calcd for C21H16ClF3N3O (449.85) %C 58.74, %H 4.26, %N 9.34. Found %C 58.79, %H 4.24,
%N 9.37.
N-(4-chlo◦robenzyl)-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamide (40). Yield 46%. Mp
δ 2.28 (s, 3H, CH3), 4.52 (d, J = 5.5 Hz, 2H, CH2),
130–131 C (2-propanol). 1H NMR (DMSO-d6):
7.24–7.40 (m, 7H, aryl and pyridyl), 8.23 (d, 1H, J = 9.0 Hz, aryl), 8.40 (d, 1H, J = 7.5 Hz, pyridyl), 9.60 (t,
J = 5.5 Hz, 1H, NH), 10.96 (s, 1H, NH). IR (Nujol) 3283, 1634, 1612, 1599 cm−1. m/z 452 (M + H)+. Anal.
Calcd for C21H16ClF3N3O (451.89) %C 55.52, %H 3.55, % N 9.25. Found %C 55.58, %H 3.54, % N 9.22.
2-((5-Chloro-2-methylphenyl)amino)-N-(4-fluorobenzyl)-6-(trifluoromethyl)nicotinamide (41). Yield 43%. Mp
◦
184–185 C (2-propanol). 1H NMR (DMSO-d6):
δ 2.40 (s, 3H, CH3), 4.63 (d, J = 5.5 Hz, 2H, CH2),
7.25–7.54 (m, 7H, aryl and pyridyl), 8.35 (d, 1H, J = 8.7 Hz, aryl), 8.51 (d, 1H, J = 7.5 Hz, pyridyl), 9.69
(s, 1H, NH), 10.97 (s, 1H, NH). IR (Nujol) 3276, 1635, 1613 cm−1. m/z 438 (M + H)+. Anal. Calcd for
C21H16ClF4N3O (437.82) %C 55.52, %H 3.55, %N 9.25. Found %C 55.57, %H 3.54, %N 9.23.
2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)-N-(4-(trifluoromethyl)benzyl)nicotinamide (42). Yield
1
62%. Mp 170–171 ◦C (2-propanol). H NMR (DMSO-d6):
δ 2.38 (s, 3H, CH3), 4.74 (d, J = 5.5 Hz, 2H,
CH2), 7.37 (d, 1H, J = 8.8 Hz, aryl), 7.43 (s, 1H aryl), 7.84 (d, 1H, J = 7.8 Hz, pyridyl), 7.70 (d, 2H, J = 8.3
Hz, aryl), 7.82 (d, 2H, J = 8.3 Hz, aryl), 8.34 (d, 1H, J = 8.8 Hz, aryl), 8.54 (d, 1H, J = 7.8 Hz, pyridyl),
9.78 (s, 1H, NH), 10.92 (s, 1H, NH). IR (Nujol) 3325, 1635, 1613, 1597 cm−1. m/z 488 (M + H)+. Anal.
Calcd for C21H16ClF4N3O (487.83) %C 54.17, %H 3.31, %N 8.61.
2-((5-Chloro-2-methylphenyl)amino)-N-(4-methoxybenzyl)-6-(trifluoromethyl)nicotinamide (43). Yield 39%.
1
Mp 126-128 ◦C (2-propanol). H NMR (DMSO-d6):
δ 2.40 (s, 3H, CH3), 3.84 (2, 3H, CH3), 4.58 (d, J = 5.3
Hz, 2H, CH2), 7.02 (d, 1H, J = 7.1 Hz, aryl), 7.43 (s, 1H aryl), 7.34–7.45 (m, 6H, aryl and pyridyl), 8.35
(d, 1H, J = 8.8 Hz, aryl), 8.48 (d, 1H, J = 7.8 Hz, pyridyl), 9.63 (s, 1H, NH), 11.00 (s, 1H, NH). IR (Nujol)
3292, 1634, 1612, 1598 cm−1. m/z 450 (M + H)+. Anal. Calcd for C22H19ClF3N3O2 (449.85) %C 58.74,
%H 4.86, %N 9.34. Found %C 58.68, %H 4.88, %N 9.37.
2-((5-Chloro-2-methylphenyl)amino)-N-(4-methylbenzyl)-6-(trifluoromethyl)nicotinamide (44). Yield 95%.
Mp 189–190 ◦C (2-propanol). 1H NMR (DMSO-d6):
δ
2.39 (s, 3H, CH3), 2.43 (s, 3H, CH3), 4.60 (d,
J = 5.3 Hz, 2H, CH2), 7.25–7.47 (m, 6H, aryl and pyridyl), 9.66 (s, 1H, NH), 10.99 (s, 1H, NH). IR (Nujol)

Molecules 2020, 25, 1338
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3277, 1634, 1612, 1598 cm−1. m/z 434 (M + H)+. Anal. Calcd for C22H19ClF3N3O (433.85) %C 60.90,
%H 4.41, %N 9.69. Found %C 60.97, %H 4.43, %N 9.66.
2-((5-Chloro-2-methylphenyl)amino)-N-(2,4-dichlorobenzyl)-6-(trifluoromethyl)nicotinamide (45). Yield 65%.
1
Mp 153–155 ◦C (2-propanol). H NMR (DMSO-d6):
δ 2.38 (s, 3H, CH3), 4.69 (d, J = 5.5 Hz, 2H, CH2),
7.36–7.57 (m, 5H, aryl and pyridyl), 7.76, (s, 1H, aryl), 8.34 (d, 1H, J = 8.7 Hz, aryl), 8.54 (d, 1H, J = 7.8
Hz, pyridyl), 9.69 (s, 1H, NH), 10.88 (s, 1H, NH). IR (Nujol) 3304, 1635, 1614, 1598 cm−1. m/z 489 (M +
H)+. Anal. Calcd for C21H15ClF3N3O (488.72) %C 51.91, %H 3.09, %N 8.60. Found %C 51.96, %H 3.09,
%N 8.64.
2-((5-Chloro-2-methylphenyl)amino)-N-(3,4-dichlorobenzyl)-6-(trifluoromethyl)nicotinamide (46). Yield 79%.
1
Mp 179–180 ◦C (2-propanol). H NMR (DMSO-d6):
δ 2.39 (s, 3H, CH3), 4.65 (d, J = 5.5 Hz, 2H, CH2),
7.27–7.82 (m, 6H, aryl and pyridyl), 8.34 (d, 1H, J = 8.8 Hz, aryl), 8.53 (d, 1H, J = 7.3 Hz, pyridyl), 9.80
(s, 1H, NH), 10.92 (s, 1H, NH). IR (Nujol) 3283, 1644, 1615, 1597 cm−1. m/z 489 (M + H)+. Anal. Calcd
for C21H15ClF3N3O (488.72) %C 51.91, %H 3.09, %N 8.60. Found %C 51.85, %H 3.10, %N 8.63.
2-((5-Chloro-2-methylphenyl)amino)-N-(4-hydroxy-3-methoxybenzyl)-6-(trifluoromethyl)nicotinamide (47).
1
Yield 95%. Mp 142–143 ◦C (2-propanol). H NMR (DMSO-d6):
δ 2.33 (s, 3H, CH3), 3.85 (s, 3H, CH3),
4.53 (d, J = 5.4 Hz, 2H, CH2), 6.83–7.50 (m, 6H, aryl and pyridyl), 8.34 (d, 1H, J = 8.8 Hz, aryl), 8.54 (d,
1H, J = 7.8 Hz, pyridyl), 9.04 (s, 1H, OH), 9.60 (s, 1H, NH), 11.01 (s, 1H, NH). IR (Nujol) 3305, 1645,
1616 cm−1. m/z 466 (M + H)+. Anal. Calcd for C22H19Cl3F3N3O (465.85) %C 56.72, %H 4.11, %N 9.02.
Found %C 56.67, %H 4.12, %N 9.06.
(2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)pyridin-3-yl)(4-phenylpiperazin-1-yl)methanone (48).
1
Yield 81%. Mp 118–120 ◦C (n-hexane). H NMR (DMSO-d6):
δ 2.25 (s, 3H, CH3), 3.17–3.21 (m, 4H,
CH2), 3.36–3.41 (m, 4H, CH2), 6.80–7.32 (m, 8H, aryl and pyridyl), 7.55 (d, 1H, J = 8.5 Hz, aryl), 7.84 (d,
1H, J = 7.0 Hz, pyridyl), 8.55 (s, 1H, NH). IR (Nujol) 3365, 1630, 1614, 1599 cm−1. m/z 475 (M + H)+.
Anal. Calcd for C24H22ClF3N4O (474.91) %C 60.70, %H 4.67, %N 11.80. Found %C 60.64, %H 4.69,
%N 11.84.
(2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)pyridin-3-yl)(4-(3,4-dichlorophenyl)piperazin-1-yl)methanone
(
52). Yield 87%. Mp 165–167◦C (MeCN). 1H NMR (DMSO-d6):
δ 2.27 (s, 3H, CH3), 3.41–3.45 (m, 4H,
CH2), 3.73–3.75 (m, 4H, CH2), 7.05–7.55 (m, 6H, aryl and pyridyl), 7.65 (d, 1H, J = 8.4 Hz, aryl), 7.96 (d,
1H, J = 7.7 Hz, pyridyl), 8.65 (s, 1H, NH). IR (Nujol) 3323, 1638, 1609 cm−1. m/z 544 (M + H)+. Anal.
Calcd for C24H20Cl3F3N4O (543.80) %C 53.01, %H 3.71, %N 10.30. Found %C 53.07, %H 3.70, %N 10.27.
4.2. Biology
4.2.1. Expression and Purification of Recombinant HIV-1 RTs Group M Subtype B
p6HRT-prot vector was kindly provided by Dr. Stuart Le Grice Laboratory. Heterodimeric RT was
expressed essentially as described [42]. Briefly, Escherichia coli strain M15 containing the p6HRT-prot
vector were grown up to an OD600 of 0.7 and induced with isopropyl β-D-1-thiogalactopyranoside
(IPTG) 1.7 mM for 4 h. Cell pellets were resuspended in Lysis buffer (50 mM sodium phosphate pH 7.8,
0.5 mg/mL lysozyme), incubated on ice for 20 min, added 0.3 M final NaCl, sonicated and centrifuged
at 30,000
g for 1 h. The supernatant was loaded into a Ni2+-sepharose column pre-equilibrated with
×
loading buffer (50 mM sodium phosphate pH 7.8, 0.3 M NaCl, 10% glycerol, 10 mM imidazole) and
washed thoroughly with wash buffer (50 mM sodium phosphate pH 6.0, 0.3 M NaCl, 10% glycerol,
80 mM imidazole). RT was gradient-eluted with elute buffer (wash buffer with 0.5 M imidazole).
Fractions were collected, protein purity was checked by SDS-PAGE and found to be higher than 90%.
RT containing fractions were pooled and diluted 1:1 with dilute buffer (50 mM sodium phosphate pH
7.0, 10% glycerol) then loaded into a Hi-trap Heparine HP GE (Healthcare Lifescience) pre-equilibrated
with 10-column volume of loading buffer 2 (50 mM sodium phosphate pH 7.0, 10% glycerol, 150 mM
NaCl). Column was then washed with loading buffer 2 and RT was gradient-eluted with elute buffer 2

Molecules 2020, 25, 1338
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(50 mM sodium phosphate pH 7.0, 10% glycerol, 150 mM NaCl). Fractions were collected, protein
was dialyzed and stored in buffer containing 50 mM Tris HCl pH 7.0, 25 mM NaCl, 1 mM EDTA, 50%
glycerol. Catalytic activities and protein concentration were determined. Enzyme-containing fractions
were pooled and aliquots were stored at −80 ◦C.
4.2.2. Mg2+ Coordination
The inhibitors were diluted in water to a final concentration of 100 µM. UV spectra recorded in
the absence and after addition of 6 mM MgCl2 in a water solution, using a Nano Drop ONE reader
(Thermo Scientific). Results are plotted as absorbance vs wavelength using Prisme 6, version 6.01.
4.2.3. Site-Directed Mutagenesis
Amino acid substitutions were introduced into the p66 HIV-1 RT subunit coded in a p6HRT-prot
plasmid using the QuikChange protocol (Agilent Technologies Inc., Santa Clara, CA).
4.2.4. HIV-1 DNA Polymerase-Independent RNase H Activity Determination
The RT-associated RNase H activity of wt and mutated HIV RTs was measured as
described [65], in 100 µL reaction volume containing 50 mM Tris HCl pH 7.8, 6 mM MgCl2, 1 mM
dithiothreitol (DTT), 80 mM KCl, hybrid RNA/DNA (5’-GTTTTCTTTTCCCCCCTGAC-3’-Fluorescein,
5’-CAAAAGAAAAGGGGGGACUG-3’-Dabcyl), and different amounts of enzymes according to a
linear range of dose-response curve—20 ng of wt RT; 50 ng K103N RT; 100 ng V108A RT; 4 ng Y181C RT;
100 ng Q475A RT; 100 ng A502F RT. The reaction mixture was incubated for 1 h at 37 ◦C, the reaction
was stopped by addition of EDTA and products were measured with a Victor 3 (Perkin) at 490/528 nm.
4.2.5. HIV-1 RNA-Dependent DNA Polymerase Activity Determination
The HIV-1 RT-associated RDDP activity was measured as described [66]. Briefly, in 25
µL volume
containing 60 mM Tris-HCl pH 8.1, 8 mM MgCl2, 60 mM KCl, 13 mM DTT, poly(A)-oligo(dT), 100
µM
dTTP, and 6 ng wt RT (or 21 ng K103N RT; 30 ng V108A RT; 1,5 ng Y181C RT; 21 ng Q475A RT; 15
ng A502F RT). After enzyme addition, the reaction mixture was incubated for 30 min at 37 ◦C and
enzymatic reaction was stopped by addition of EDTA. Reaction products were detected by PicoGreen
addition and measured with a Victor 3 (Perkin) at 502/523 nm.
4.2.6. Antiviral Assay
Drug-mediated inhibition of virus-induced cytotoxicity was assayed in TZM-BL cells. Triplicate
wells of 96-well plates containing 1
multiplicity of infection of 0.1. Serial dilutions of drugs were added immediately after infection.
Luciferase activity was measured as reported [67]. Two days after infection, the culture medium was
×
104 TZMbl cells were infected with HIV-1 IIIB strain at a
removed from each well and 100 µl of Bright Glo reagent (Promega, Luis Obispo, CA) was added to
the cells for measurement of luminescence using a Victor 2 luminometer (Perkin). The 50% effective
concentration (EC50) was defined as the concentration that caused a 50% reduction of luciferase activity
(relative light units) compared to virus control wells. Cell viability in CEM cells was quantified 5 days
after infection with the MTT-dye reduction method.
5. Conclusions
We identified a new promising 2-amino-6-(trifluoromethyl)nicotinic acid scaffold for compounds
that can inhibit both HIV-1 RT activities and are also active against viral replication. Compound 21
active against viral replication in low micromolar range with no significant cytotoxic effects, showed
to be a dual inhibitor active also against the RNA dependent DNA polymerase activity of HIV-1
RT, being active against RTs carrying NNRTI-resistant mutations. Mode-of-action and single-site
mutagenesis studies indicated that compound 21 potency of inhibition is strongly impaired by the
,

Molecules 2020, 25, 1338
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substitutions of amino acidic residues in two different pockets, located in two different domains
of RT enzyme, hence supporting the hypothesis of a dual-site binding mode. Overall compound
21 is a promising lead for the design of new allosteric RNase H inhibitors more potent against
HIV-1 replication.
Author Contributions: Conceptualization, A.C., V.O.; methodology, F.E.; formal analysis, A.C., V.O., C.D.V.;
resources, E.T., Y.-C.C.; data curation, A.C., V.O.; writing—original draft preparation, A.C., V.O.; writing—review
and editing, A.C., C.D.V., V.O., E.T.; supervision, E.T., Y.-C.C.; funding acquisition, E.T. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the Sardinian Regional Government grant LR07/17 (F76C18000800002).
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds are available from V.O. the authors.
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