Synthesis, biological evaluation and molecular modeling of 4,6-diarylpyrimidines and diarylbenzenes as novel non-nucleosides HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.10.036

A series of novel 4,6-diarylpyrimidines (4,6-DAPY) and diarylbenzenes (DABE) compounds were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 8b, 8d, 14b and 18 (EC₅₀ = 0.049, 0.381, 0.599 and 0.398 μM, respectively), with HIV-1 inhibitory activity improved or similar to nevirapine (NVP, EC₅₀ = 0.097 μM) and delavirdine (DEV, EC ₅₀ = 0.55 μM). The other compounds displayed moderate activity (8c, EC₅₀ = 5.25 μM) or were inactive (8a and 14a) against HIV-1 replication. Molecular modeling studies were performed with the synthesized compounds in complex with the wild-type reverse transcriptase (RT). A correlation was found between the anti-HIV activity and the electrostatic energy of interaction with Lys101 residue. These findings enrich the SAR of these Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) families.

Full text of DOI:10.1016/j.ejmech.2012.10.036

European Journal of Medicinal Chemistry 58 (2012) 485e492
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and molecular modeling of 4,6-diarylpyrimidines
and diarylbenzenes as novel non-nucleosides HIV-1 reverse transcriptase
inhibitors
Sergio R. Ribone a, Volker Leen b, Marcela Madrid c, Wim Dehaen b, Dirk Daelemans d,
**
,
a,
*
Christophe Pannecouque d , Margarita C. Briñón
a Departamento de Farmacia, Facultad de Ciencias Químicas, Ciudad Universitaria, Universidad Nacional de Córdoba, X5000HUA Córdoba, Argentina
b Pittsburgh Supercomputing Center, Pittsburgh, PA 15213, USA
c Chemistry Department, KU Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium
d Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of novel 4,6-diarylpyrimidines (4,6-DAPY) and diarylbenzenes (DABE) compounds were
Received 21 September 2012
Received in revised form
15 October 2012
Accepted 20 October 2012
Available online 29 October 2012
synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them,
the most potent HIV-1 inhibitors were 8b, 8d, 14b and 18 (EC50 ¼ 0.049, 0.381, 0.599 and 0.398
respectively), with HIV-1 inhibitory activity improved or similar to nevirapine (NVP, EC50 ¼ 0.097
m
M,
m
M)
and delavirdine (DEV, EC50
(8c, EC50 ¼ 5.25
¼
0.55 mM). The other compounds displayed moderate activity
M) or were inactive (8a and 14a) against HIV-1 replication. Molecular modeling studies
m
were performed with the synthesized compounds in complex with the wild-type reverse transcriptase
(RT). A correlation was found between the anti-HIV activity and the electrostatic energy of interaction
with Lys101 residue. These findings enrich the SAR of these Non-Nucleoside Reverse Transcriptase
Inhibitors (NNRTIs) families.
Keywords:
HIV-1
NNRTIs
4,6-Diarylpyrimidines
Diarylbenzenes
Molecular modeling
SAR
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Since their discovery, NNRTIs have received special attention due
to their high antiviral activity and low cytotoxicity [5]. However, the
Human immunodeficiency virus (HIV) is the causative virus of
acquired immunodeficiency syndrome (AIDS). At present, there is
no effective vaccine against HIV, and the generally adopted highly
active antiretroviral therapy (HAART) has significantly reduced the
morbidity and mortality of HIV-infected people [1]. Reverse Tran-
scriptase (RT) of the HIV-1, is an enzyme essential for the replica-
tion of the virus, and is one of the main targets for antiretroviral
chemotherapy [2]. Non-Nucleoside Reverse Transcriptase Inhibi-
tors (NNRTI), a class of components of HAART, elicit viral reverse
transcription inhibition by binding to an allosteric pocket located
rapid emergence of viral drug resistance prompted the development
of diverse structures of NNRTI, such us 2-[(2-acetyl-5-methylphenyl)
amino]-2-(2,6-dichlorophenyl)acetamide (a-APA) [6,7], benzophe-
nones [8,9], dihydro-alkoxybenzyl-oxopyrimidines (DABOs) [10,11],
diaryltriazines (DATAs) [12,13], and diarylpyrimidines (DAPYs)
[14e16]. In particular, DAPYs have been regarded as one of the most
successful scaffolds for NNRTIs because of their potent antiviral
activity against wild-type and mutant strains of the HIV-1. Their
molecular structure consists of three rings bound by rotatable
bonds with a NH moiety linker between wing II and the central ring
(Fig. 1) [3,4].
ꢀ
10 A away from the DNA polymerase active site, the NNRTI Binding
Pocket (NNIBP) [3,4].
Because of their high antiviral potency, in previously published
work, we carried out molecular modeling of DAPYs compounds and
other different NNRTI, among them, those of first generation, in
order to reveal the structural bases for the design of novel highly
potent NNRTIs [17]. We concluded that a potent inhibitor of wild-
type RT (wtRT) must possess the following structural properties:
a) maximized van der Waals interactions in the NNIBP, b) hydrogen
bond interaction with Lys101 residue and c) high molecular
Abbreviations: 4,6-DAPY, 4,6-diarylpyrimidines; DABE, diarylbenzenes.
* Corresponding author. Tel.: þ54 0351 5353865x53355; fax: þ54 0351 5353865
x53364.
** Corresponding author. Tel.: þ32 016 332171; fax: þ32 016 332131.
E-mail addresses: christophe.pannecouque@rega.kuleuven.be (C. Pannecouque),
macribri@fcq.unc.edu.ar (M.C. Briñón).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.036

486
S.R. Ribone et al. / European Journal of Medicinal Chemistry 58 (2012) 485e492
procedure described before, to obtain the desired diarylbenzenes,
14a,b (Scheme 2).
When the linker moieties were only amine groups, the synthesis
started with the SNAr reaction between 1,3-diaminobenzene (15)
and 4-fluorobenzonitrile (16) leading to the corresponding inter-
mediate (17) using K2CO3 in DMF at 130 ꢀC during 72 h. The next
step was a BuchwaldeHartwig [18] reaction between 17 and 2,4,6-
trimethylbromobenzene (13a), affording the diarylbenzene 18,
under the same condition as described above (Scheme 3).
Spectral data of all compounds are in full agreement with the
proposed structures.
I
II
I
II
2
2, Etravirine
(TMC125)
1, TMC120
3, R185545
Fig. 1. Chemical structures of DAPYs compounds.
2.2. Biological activity
flexibility between the central ring and the lateral wings (Fig. 1, I
and II in compounds 2 and 3). Following these guidelines, we
designed novel NNRTIs which kept the overall molecular structure
of DAPYs, but incorporated 4,6-pyrimidine and benzene as central
ring (Schemes 1e3).
The aim of the present work was to examine the influence of
4,6-pyrimidine and benzene as central rings, leading to novel
structural scaffolds (4,6-diarylpyrimidines, 4,6-DAPYs and diary-
lbenzenes, DABEs, respectively). We report the synthesis, anti-HIV
activity and molecular modeling of 4,6-DAPY and DABE derivatives.
All 4,6-DAPY and DABE compounds were evaluated for antiviral
activity and cytotoxicity using the MTT [(3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide)] method [19,20] in MT-4
cells infected with wild-type HIV-1 strain IIIB, double mutant
HIV-1 strain RES056 (K103N þ Y181C) and HIV-2 strain ROD.
Zidovudine (AZT), delavirdine (DEV), nevirapine (NVP) and etra-
virine (ETV), four drugs currently used in clinical treatment of HIV-
1 infection, were also included as reference compounds. The bio-
logical evaluation is summarized in Table 1.
Except for compounds 8a and 14a, all the novel synthesized
NNRTI exhibited from moderate (compound 8c, EC50 ¼ 5.25
excellent potency (compounds 8b, 8d, 14b and 18, EC50 from 0.049
to 0.599 M) against wild-type virus. Of this series, compound 8b
was identified as the most active derivative against wild-type HIV-1
M and selectivity index (SI) of 870).
mM) to
2. Results and discussion
m
2.1. Chemistry
(EC50 ¼ 0.049
m
Table 1 shows that derivatives 8d, 14b and 18 also displayed
Synthesis of 4,6-DAPYs. The synthesis of 4,6-diarylpyrimidine
compounds is described in Scheme 1. The first step takes place by
a Nucleophilic Aromatic Substitution (SNAr) reaction between
commercially available 4,6-dichloropyrimidine (4) and substituted
phenols (5a,b) in dimethylformamide (DMF), K2CO3 and 18-crown-
6 ether at 90 ꢀC during 3 h, to provided 6a,b, respectively. Then,
compounds 6a,b were coupled with different substituted anilines
(7a,b) using palladium acetate as catalyst (BuchwaldeHartwig [18]
reaction) to afford the corresponding target compounds 8ae
d (Scheme 1).
Synthesis of DABEs. These diarylbenzene derivatives were
formed by different synthetic procedures depending on the linker
moiety between the central ring and the wing I. For the compounds
with an oxygen atom as linker group, the first step also involves
a SNAr reaction between the 3-fluoronitrobenzene (9) and 2,4,6-
trimethylphenol (10) in DMF, K2CO3 and 18-crown-6 at 100 ꢀC
during 24 h to provided 11 (Scheme 2). Then, the nitro group of
compound 11 was reduced to the amine leading to derivative 12,
which finally was coupled with substituted bromobenzenes
(13a,b), according to BuchwaldeHartwig reaction, by the same
activity against wild-type HIV-1 at micromolar concentration level
(EC50 ¼ 0.381, 0.599 and 0.398
mM, respectively). Compound 8b was
2-fold and 11-fold more potent than NEV and DEV against HIV-1 IIIB
strain, respectively. Meanwhile, compounds 8d and 18, were 1.5-fold
more potent than DEV against the same strain. Unfortunately, the
tested compounds were inactive against the double RT mutant
(K103N þ Y181C) HIV-1 RES056 strain and HIV-2 strain ROD.
The molecular structure of the synthesized compounds shows
that derivatives with a 2,4,6-trimethyl substituent in the wing II
displayed moderate or no activity against the HIV-1 IIIB strain
(compounds 8a, 14a and 8c); while compounds with a 4-nitrile
substituent in the wing II exhibited an excellent potency against
this strain.
2.3. Molecular modeling
Molecular docking was carried out for compounds 8aed; 14a,b
and 18 into the NNIBP of wtRT where these NNRTI are complexed
(NNRTI-RT), using the software Autodock3 [21]. Then, NNRTI-RT
Scheme 1. Reagents and conditions: i) DMF, K2CO3, 18-crown-6, 90 ꢀC, 3 h; ii) Pd(OAc)2, Xantphos, Cs2CO3, xylene, 140 ꢀC, 48 h.

S.R. Ribone et al. / European Journal of Medicinal Chemistry 58 (2012) 485e492
487
Scheme 2. Reagents and conditions: i) DMF, K2CO3, 18-crown-6, 100 ꢀC, 24 h; ii) Pd/C 10%, H2, ethanol, 24 h; iii) Pd(OAc)2, Xantphos, Cs2CO3, toluene, 100 ꢀC, 48 h.
complexes were simulated by molecular dynamics (MD) and
analyzed using Amber10 [22].
central ring. These results are in agreement with previous obser-
vations that methyl substitutions in the ortho positions of wing I
favor this spatial arrangement due to steric hindrance, without
It is important to point out that the synthesized compounds
adopted a very similar conformation of interaction in the NNIBP to
that of the reference DAPY compound 3. These positions in the
INNBP were maintained during the 3 ns MD simulations (Fig. 2).
The novel NNRTIs accommodate wing I in the hydrophobic pocket
lined by residues Pro95, Tyr181, Tyr188, Val189 and Trp229, and
wing II in the pocket lined by Lys103, Val106, Gly190, Hie235,
Pro236, Tyr318 (Figs. 2 and 3). The central ring established hydro-
phobic contacts with Leu100 and Glu138. Finally, some of the
compounds undergo a hydrogen bond with residue Lys101 through
the nitrogen atom of the central ring or the NH linker moiety with
the wing II (Fig. 3).
The interaction between the Lys101 backbone carbonyl oxygen
and the NH linker moiety present in the 4,6-DAPYs and DABEs
compounds was followed during the 3 ns MD simulation (Table 2).
Compounds with excellent potency against wild-type virus (8b,
8d, 14b and 18), showed sustained hydrogen bond interactions,
with occupancies of 90.5, 91.9, 91.9 and 78.5%, respectively
(Table 2). Compound 8c, which has moderate antiviral potency, also
established a hydrogen bond with a moderate occupancy of 61.1%.
Finally, inactive derivatives 8a and 14a, had very low hydrogen
bond occupancy with residue Lys101, of 29.0 and 14.6%, respec-
tively. In addition, compounds 8b and 8d established hydrogen
bond interactions with the NH moiety of residue Lys101 through
the nitrogen atom of the 4,6-pyrimidine central ring, with occu-
pancies of 10.6 and 17.2%, respectively. The highly potent reference
compound ETV also showed high occupancy of this interaction
(91.8%) [17]. Clearly, a critical feature for the inhibition of the wild-
type strain of the enzyme is to establish and maintain hydrogen
bond interaction with Lys101.
restraining the flexibility of the ligand, and maximizing the
pep
stacking interaction with Tyr181, Tyr188 and Trp229 [17].
The antiviral molecules had different s2 values (Table 2). Deriv-
atives with a nitrile substituent in the position 4 of the wing II (8b,
8d, 14b, 18 and ETV) adopted dihedral angles of less than 40ꢀ. The
rest of the compounds (8a, 8c and 14a) adopted dihedral angles close
to 90ꢀ, due to the presence of methyl substituents in positions 2, 4
and 6 in wing II. The trimethyl substitution in wing II forced the wing
to adopt a perpendicular orientation respect to the central ring,
similar to wing I, moving the inhibitor away from the Lys101 residue,
and therefore breaking the biologically important hydrogen bond.
In order to prove this hypothesis, an electrostatic energy analysis
between the novel compounds and the residue Lys101 was per-
formed from the MD trajectories, using the Molecular Mechanics-
Poisson Boltzmann Surface Area (MM-PBSA) approximation (Eq.
(1), Experimental section). The compound with the highest antiviral
activity (8b) maintained high electrostatic energy interaction with
the residue Lys101, while the compound with the lowest activity (8c)
showed the lowest electrostatic energy interaction (Table 3).
Fig. 4 shows the correlation between the electrostatic energy of
interaction with Lys101 and the antiviral activity. Thus, the five
active novel compounds present a significant dependency between
these two parameters, showing the importance of the hydrogen
bond interaction with Lys101 residue for the NNRTI, in order to
produce an effective inhibition against the RT wild-type.
3. Conclusion
A series of novel NNRTIs scaffolds 4,6-DAPYs and DABEs were
synthesized, evaluated for their activity against HIV and their
interaction with RT was modeled. These novel compounds were
designed based on conclusions from our previous molecular
modeling studies of established NNRTI examples [17]. Among all
tested derivatives, 8b was identified as the most active compound
In order to analyze the position of wings I and II with respect to
the central ring of the novel compounds, the dihedral angles
s
1 and
s
2 (Fig. 1) were studied during the MD trajectories (Table 2). In all
cases, the dihedral angle
s
1 adopted a value closed to 90ꢀ, indi-
cating that wing I is in a perpendicular position with respect to the
Scheme 3. Reagents and conditions: i) DMF, K2CO3, 130 ꢀC, 72 h; ii) Pd(OAc)2, Xantphos, Cs2CO3, toluene, 100 ꢀC, 48 h.

488
S.R. Ribone et al. / European Journal of Medicinal Chemistry 58 (2012) 485e492
Table 1
Biological activities, cytotoxicities and selectivity indices of new designed 4,6-DAPY and DABE compounds.
SIc
a
b
X
Y
R1
R2
EC50
(
m
M)
CC50 (mM)
HIV-1
HIV-2
RES056e
d
IIIB
8a
8b
8c
8d
14a
14b
18
AZT
DEV
NVP
ETV
N
N
N
N
C
O
O
O
O
O
O
NH
2,4,6-(CH3)3
2,4,6-(CH3)3
2,6-(CH3)2e4-CN
2,6-(CH3)2e4-CN
2,4,6-(CH3)3
2,4,6-(CH3)3
4-CN
2,4,6-(CH3)3
4-CN
2,4,6-(CH3)3
4-CN
4-CN
>38.5
>38.5
>38.5
>42.21
>37.54
>291.8
>43.62
>52.56
>220.18
0.006 ꢁ 0.001
38.2 ꢁ 2.82
42.21 ꢁ 8.91
37.54 ꢁ 4.50
>291.8
<1
870
7
0.049 ꢁ 0.005
5.251 ꢁ 0.363
0.381 ꢁ 0.117
>43.62
0.599 ꢁ 0.330
0.398 ꢁ 0.061
0.008 ꢁ 0.002
0.548 ꢁ 0.285
0.097 ꢁ 0.038
0.002 ꢁ 0.0004
>42.21
>37.54
>291.8
ꢂ740.1
43.62 ꢁ 7.25
52.56 ꢁ 15.40
ꢂ220.18
<1
C
C
2,4,6-(CH3)3
2,4,6-(CH3)3
>52.56
>220.18
65
ꢂ552
>12,034
>8
>155
12,884
0.006 ꢁ 0.0004
>93.63
>4.39
>4.39
ꢂ7.60
>14.99
27.79 ꢁ 11.54
0.034 ꢁ 0.005
>27.79
a
EC50: effective concentration of compound required to protect the cell against viral cytopathogenicity by 50% in MT-4 cells.
CC50: cytotoxic concentration of compound that reduces the uninfected MT-4 cell viability by 50%.
SI: selectivity index: ratio CC50/EC50
IIIB: HIV-1 wild-type strain.
b
c
.
d
e
RES056: HIV-1 mutated strain bearing both K103N and Y181C mutations.
Fig. 2. Binding modes and superposition of compound 3 (red) in the crystallographic complex 1SUQ with a) molecular docking conformation of compound 8a (green) and 14a
(yellow) and b) molecular docking conformation of compound 8b (blue) and 18 (purple) to the NNIBP of wtRT. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

S.R. Ribone et al. / European Journal of Medicinal Chemistry 58 (2012) 485e492
489
Fig. 3. Ligplots graphics of molecular docking conformation resulting from compounds: a) 8a, b) 8b, c) 14a and d) 18 in the NNIBP of wtRT.
against the wild-type HIV-1 strain (EC50 ¼ 0.049
m
M, SI ¼ 870),
between NNRTI and the RT for the anti-HIV activity, which is clearly
confirmed here by the correlation between biological activity and
electrostatic energy interaction with residue Lys101.
These studies enrich the SAR of these NNRTIs families, showing
that the 2,4,6-trimethyl substitution on wing II is not crucial for
future design of potent NNRTIs of HIV.
while 8a and 14a were inactive against the same virus strain.
None of the compounds exhibited activity against the double
HIV-1 mutant virus (K103N þ Y181C) and the HIV-2 ROD strain.
Compounds 8a, 8c and 14a, which have a 2,4,6-trimethyl
substitution on wing II, displayed inactivity or had moderate
activity against the wild-type HIV-1 strain. Molecular dynamics
studies show that wing II of these compounds is perpendicular to the
central ring and unable to form hydrogen bond with Lys101. These
findings underscore the importance of hydrogen bond interactions
4. Experimental section
4.1. Chemistry
Table 2
Hydrogen bond interaction, average (avg) dihedral angles and their standard devi-
ation (SD) of the 4,6-DAPY and DABE compounds, during the 3 ns of molecular
dynamics simulation. ETV was included as reference compound.
4.1.1. General procedure for the preparation of 6a,b
Potassium carbonate (10 mmol), 4,6-dichloropyrimidine
(5 mmol) and the substituted phenol (5a,b) in 10 mL of anhydrous
4
Hydrogen bond
occupancy %a
Dihedral angle (avg ꢁ SD, deg)
s1
s2
Table 3
8a
8b
8c
8d
14a
14b
18
29.0
90.5
61.1
91.9
14.6
91.9
78.5
91.8
ꢃ83 ꢁ 12
ꢃ84 ꢁ 9
120 ꢁ 21
37 ꢁ 20
77 ꢁ 10
40 ꢁ 14
120 ꢁ 10
ꢃ18 ꢁ 16
39 ꢁ 14
2 ꢁ 32
Electrostatic energy component (kcal/mol) and antiviral activity (ꢃlog EC50) of the
novel compounds.
ꢃ74 ꢁ 12
ꢃ77 ꢁ 9
a
b
ELELys101
ꢃLog EC50
ꢃ99 ꢁ 10
ꢃ99 ꢁ 11
ꢃ78 ꢁ 11
ꢃ82 ꢁ 12
8b
8c
8d
14b
18
ꢃ4.14 ꢁ 0.64
ꢃ1.96 ꢁ 0.64
ꢃ3.79 ꢁ 0.86
ꢃ2.31 ꢁ 0.62
ꢃ2.27 ꢁ 0.48
1.310
ꢃ0.720
0.419
0.223
0.400
ETV [17]
a
Hydrogen bond occupancy produced by the interaction between Lys101 back-
bone carbonyl oxygen and the NH linker moiety present in the 4,6-DAPYs and
DABEs compounds.
a
Electrostatic energy of interaction with residue Lys101.
Half effective concentration.
b

490
S.R. Ribone et al. / European Journal of Medicinal Chemistry 58 (2012) 485e492
2.0
1.5
84.79, 129.38, 129.51, 130.42, 131.79, 135.06, 136.38, 137.53, 147.52,
158.77, 164.43, 170.11; HR-MS calcd. for C22H25N3O m/z 347.19976,
found: 337.19973.
1.0
4.1.2.2. 4-[4-(2,4,6-Trimethylphenoxy)pyrimidin-6-ylamino]benzoni-
trile (8b). Yield 89%; yellow solid; mp 197.5e201.7 ꢀC; 1H NMR
(300 MHz, CDCl3):
d
2.09 (s, 6H, CH3-700, CH3-800), 2.29 (s, 3H, CH3-
0.5
900), 6.19 (s, 1H, NH-70), 6.91 (s, 2H, CH-300, CH-500), 7.26 (s, 1H, CH-2),
7.58 (d, J ¼ 8.7 Hz, 2H, CH-20, CH-60), 7.63 (d, J ¼ 8.7 Hz, 2H, CH-30,
0.0
CH-50), 8.44 (s, 1H, CH-4); 13C NMR (75 MHz, CDCl3):
d 16.47, 21.00,
-0.5
-1.0
-1.5
-2.0
88.75, 106.22, 119.10, 119.95, 129.69, 130.35, 133.63, 135.67, 143.19,
147.24, 159.06, 161.62, 170.13; HR-MS calcd. for C20H18N4O m/z
330.14806, found: 330.14792.
4.1.2.3. 2,6-Dimethyl-4-[4-(2,4,6-trimethylaniline)pyrimidin-6-
yloxy]benzonitrile (8c). Yield 21%; white solid; mp: 166.0e168.5 ꢀC;
1H NMR (300 MHz, CDCl3): 2.11 (s, 6H, CH3-80, CH3-90), 2.22 (s, 6H,
d
CH3-700, CH3-800), 2.33 (s, 3H, CH3-100), 5.49 (s,1H, NH-70), 6.79 (s,1H,
CH-2), 6.99 (s, 2H, CH-30, CH-50), 7.38 (s, 2H, CH-300, CH-500), 8.19
-6
-5
-4
-3
-2
-1
0
ELELys101 (kcal/mol)
(s, 1H, CH-4). 13C NMR (75 MHz, CDCl3):
d 16.53, 18.28, 21.13, 84.93,
Fig. 4. Biological activity (ꢃlog EC50) vs. electrostatic energy interaction between
109.50, 118.89, 129.65, 131.45, 132.58, 132.96, 136.36, 137.89, 153.63,
158.61, 164.67, 169.19; HR-MS calcd. for C20H18N4O m/z 358.17936,
found: 358.17954.
residue Lys101 and the novel active compounds.
DMF were added to a dried flask of 50 mL and then, under
continuing stirring, 18-crown-6 (0.25 mmol) was also added. The
reaction mixture was heated to 90 ꢀC under nitrogen atmosphere
for 3 h. Next, the resulting reaction mixture was extracted with
diethyl ether. The organic layer was dried over anhydrous sodium
carbonate, filtered evaporated under reduced pressure and subse-
quently purified by medium pressure chromatography to give
products 6a,b.
4.1.2.4. 4-[4-(4-Cyano-2,6-dimethylphenoxy)pyrimidin-6-ylamino]
benzonitrile (8d). Yield 24%; white solid; mp: 198.9e200.7 ꢀC; 1H
NMR (300 MHz, CDCl3):
d
2.16 (s, 6H, CH3-700, CH3-800), 6.36 (s, 1H,
NH-70), 7.18 (s, 1H, CH-2), 7.43 (s, 2H, CH-300, CH-500), 7.61
(d, J ¼ 8.4 Hz, 2H, CH-20, CH-60), 7.67 (d, J ¼ 8.4 Hz, 2H, CH-30, CH-50),
8.38 (s, 1H, CH-4); 13C NMR (75 MHz, CDCl3):
d 16.58, 89.06, 106.68,
109.90, 118.73, 118.97, 120.27, 132.76, 132.94, 133.70, 142.88, 153.30,
158.87, 161.84, 169.02; HR-MS calcd. for C20H15N5O m/z 341.12766,
found: 341.12893.
4.1.1.1. 4-Chloro-6-(2,4,6-trimethylphenoxy)pyrimidine (6a). Yield 84%;
white crystals; mp 64.4e66.9 ꢀC; 1H NMR (300 MHz, CDCl3):
d
2.07 (s,
6H, CH3-70, CH3-80), 2.31 (s, 3H, CH3-90), 6.88 (s, 1H, CH-2), 6.93 (s, 2H,
CH-30, 50), 8.56 (s, 1H, CH-4); 13C NMR (75 MHz, CDCl3):
16.18, 20.76,
4.1.3. 3-(2,4,6-Trimethylphenoxy)-1-nitrobenzene (11)
To a dried flask of 50 mL, 2,4,6-trimethylphenol (10) (10 mmol),
potassium carbonate (20 mmol) and 20 mL of DMF were added. Then,
3-fluoro-1-nitrobenzene (9) (10 mmol) was added to the flask via
a syringe, followed by 18-crown-6 (0.5 mmol) in continuing stirring.
The reaction was heated at 90 ꢀC under nitrogen atmosphere during
24 h. Next, the resultingreactionwas extractedwithdiethyl ether. The
organic layer was dried over anhydrous sodium carbonate, filtered
and evaporated under reduced pressure before purification by
medium pressure chromatography to obtain 11. Yield 54%; orange
d
106.72, 129.57, 129.80, 135.85, 146.74, 158.82, 161.92, 169.77; HR-MS
calcd. for C13H13ClN2O m/z 248.07164, found: 248.07268.
4.1.1.2. 4-Chloro-6-(4-cyano-2,6-dimethylphenoxy)pyrimidine (6b).
Yield 80%; white crystals; mp: 135.2e139.1 ꢀC; 1H NMR (300 MHz,
CDCl3):
8.52 (s, 1H, CH-4); 13C NMR (75 MHz, CDCl3):
118.48,132.61,132.85,152.75,158.80,162.58,168.81; HR-MS calcd. for
13H10ClN3O m/z 259.05124, found: 259.05119.
d
2.14(s, 6H, CH3-70, 80), 7.06(s,1H, CH-2), 7.45(s, 2H, CH-30, 50),
d
16.49, 107.49, 110.41,
solid;mp: 65.3e70.5 ꢀC;1HNMR (300 MHz, CDCl3):
d 2.07 (s, 6H, CH3-
C
70, CH3-80), 2.31 (s, 3H, CH3-90), 6.93 (s, 2H, CH-30, CH-50), 7.12
(d, J ¼ 8.1 Hz,1H, CH-6), 7.41(t, J ¼ 8.1Hz,1H, CH -5), 7.56(s,1H, CH -2),
4.1.2. General procedure for the preparation of 8aed
7.83 (d, J ¼ 7.8, 1H, CH-4); 13C NMR (75 MHz, CDCl3):
d 16.28, 20.92,
In a dried screw tube of 10 mL, 4-chloropyrimidine (6a,b)
(0.5 mmol), substituted anilines (7a,b) (0.6 mmol), Pd(OAc)2
(0.025 mmol), Xantphos (0.05 mmol), Cs2CO3 (1.0 mmol) and
xylene (5 mL) were added, under nitrogen atmosphere and
continuous stirring at 100e140 ꢀC until the chloropyrimidine was
consumed. The course of the reaction was followed by thin layer
chromatography (TLC) analysis. Next, the resulting reaction was
extracted with diethyl ether, dried over anhydrous sodium
carbonate, filtered and purified by medium pressure chromatog-
raphy to give products 8aed.
109.71, 116.42, 121.14, 130.10, 130.37, 130.62, 135.51, 148.11, 149.61,
158.82; HR-MS calcd. for C15H15NO3 m/z 257.10519, found: 257.10419.
4.1.4. 3-(2,4,6-Trimethylphenoxy)aniline (12)
To a dried flask of 50 mL, compound 11 (2.1 mmol), 21 mg of
Pd/C 10% and 20 mL of ethanol were added under hydrogen
atmosphere at room temperature during 24 h. After this time, the
resulting reaction was extracted with diethyl ether, and the organic
layer was dried over anhydrous sodium carbonate, filtered and
purified by medium pressure chromatography to obtain 12. Yield
93%, uncolored oil; 1H NMR (300 MHz, CDCl3): 2.09 (s, 6H, CH3-70,
d
4.1.2.1. 6-(2,4,6-Trimethylaniline)-4-(2,4,6-trimethylphenoxy)pyrimi-
CH3-80), 2.29 (s, 3H, CH3-90), 3.59 (s, 2H, NH-7), 6.08 (s, 1H, CH-2),
6.16 (d, J ¼ 8.1 Hz, 1H, CH-6), 6.27 (d, J ¼ 7.8 Hz, 1H, CH-4), 6.88 (s,
2H, CH-30, CH-50), 6.99 (t, J ¼ 7.8 Hz, 1H, CH-5). 13C NMR (75 MHz,
dine (8a). Yield 29%; yellow solid; 1H NMR (300 MHz, CDCl3):
d
2.04 (s, 6H, CH3-80, CH3-90), 2.20 (s, 6H, CH3-700, CH3-800), 2.26
(s, 3H, CH3-100), 2.31 (s, 3H, CH3-900), 5.43 (s, 1H, NH-70), 6.73 (s, 1H,
CDCl3): d 16.38, 20.92, 101.53, 105.23, 108.37, 129.56, 130.34, 131.27,
CH-2), 6.86 (s, 2H, CH-30, CH-50), 6.96 (s, 2H, CH-300, CH-500), 8.23
134.38, 148.00, 148.98, 159.34; HR-MS calcd. for C15H15NO3 m/z
227.13101, found: 227.12980.
(s, 1H, CH-4). 13C NMR (75 MHz, CDCl3):
d
16.45, 18.27, 20.95, 21.08,

S.R. Ribone et al. / European Journal of Medicinal Chemistry 58 (2012) 485e492
491
4.1.5. General procedure for the preparation of 14a,b
(75 MHz, CDCl3): d 18.16, 20.87, 101.11, 105.16, 109.26, 110.27, 114.97,
In a dried screw tube, 12 (0.6 mmol), bromobenzene compounds
(13a or 13b) (0.4 mmol), Pd(AcO)2 (0.03 mmol), Xantphos
(0.06 mmol), Cs2CO3 (0.8 mmol) and toluene (5 mL) were added
under nitrogen atmosphere and heated with stirring to 100 ꢀC until
the bromide compound disappeared, which was monitored by TLC
analysis. Next, the resulting reaction mixture was extracted with
diethyl ether, dried over anhydrous sodium carbonate, filtered and
purified by medium pressure chromatography to obtain 14a,b.
119.92, 129.22, 130.21, 133.59, 134.91, 135.87, 136.09, 141.01, 147.96,
148.15; HR-MS calcd. for C22H21N3 m/z 327.17355, found: 327.17333.
4.2. Anti-HIV activity assay
The anti-HIV activity and cytotoxicity of the compounds 8aed,
14a,b and 18 were evaluated against wild-type HIV-1 strain IIIB,
a double RT mutant (K103N þ Y181C) HIV-1 strain and HIV-2 strain
ROD in MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazoliumbromide (MTT) method [19,20]. Briefly,
stock solutions (10ꢄ final concentration) of test compounds were
4.1.5.1. N-[3-(2,4,6-Trimethylphenoxy)phenyl]-N-(2,4,6-
trimethylphenyl)amine (14a). Yield 27%; white solid, mp: 122.2e
124.1 ꢀC; 1H NMR (300 MHz, CDCl3):
d
2.09 (s, 6H, CH3-80, CH3-
added in 25 mL volumes to two series of triplicate wells so as to
90), 2.16 (s, 6H, CH3-700, CH3-800), 2.28 (s, 3H, CH3-100), 2.29 (s, 3H,
CH3-900), 5.04 (s,1H, NH-70), 6.01 (d, J ¼ 9.0 Hz,1H, CH-4), 6.02 (s,1H,
CH-2), 6.04 (d, J ¼ 9.0 Hz, 1H, CH-6), 6.87 (s, 2H, CH-30, CH-50), 6.91
(s, 2H, CH-300, CH-500), 6.93 (t, J ¼ 7.8 Hz, 1H, CH-5). 13C NMR
allow simultaneous evaluation of their effects on mock-and HIV-
infected cells at the beginning of each experiment. Serial 5-fold
dilutions of test compounds were made directly in flat-bottomed
96-well microtiter trays using a Biomek 3000 robot (Beckman
instruments). Untreated control HIV- and mock-infected cell
(75 MHz, CDCl3): d 16.42, 18.36, 20.91, 21.02, 100.39, 104.10, 106.58,
129.31, 129.52, 130.05, 131.28, 134.25, 135.57, 135.58, 136.11, 148.48,
149.20, 159.52; HR-MS calcd. for C24H27NO m/z 345.20926, found:
345.20968.
samples were included for each sample. Virus stock (50 mL) at 100e
300 CCID50 (50% cell culture infectious dose) or culture medium
was added to either the virus-infected or mock-infected wells of the
microtiter tray. Mock-infected cells were used to evaluate the effect
of test compounds on uninfected cells in order to assess the cyto-
toxicity of the test compounds. Exponentially growing MT-4 cells
were centrifuged for 5 min at 220 g and the supernatant was dis-
carded. The MT-4 cells were resuspended at 6 ꢄ 105 cells/mL and
4.1.5.2. 4-[3-(2,4,6-Trimethylphenoxy)phenyl-1-ylamino]benzonitrile
(14b). Yield 49%; white solid; mp: 162.0e163.5 ꢀC; 1H NMR
(300 MHz, CDCl3):
d
2.10 (s, 6H, CH3-700, CH3-800), 2.29 (s, 3H, CH3-
900), 6.05 (s, 1H, NH-70), 6.49 (d, J ¼ 8.4 Hz, 1H, CH-4), 6.54 (s, 1H, CH-
2), 6.78 (d, J ¼ 8.4 Hz, 1H, CH-6), 6.90 (s, 2H, CH-300, CH-500), 6.96
(d, J ¼ 8.8 Hz, 2H, CH-20, CH-60), 7.19 (t, J ¼ 8.0 Hz, 1H, CH-5), 7.46
50 mL volumes were transferred to the microtiter tray wells. Five
days after infection, the viability of mock- and HIV-infected cells
was examined spectrophotometrically by the MTT assay.
(d, J ¼ 8.8 Hz, 2H, CH-30, CH-50). 13C NMR (75 MHz, CDCl3):
d 16.35,
20.90, 101.93, 107.15, 110.19, 113.46, 115.40, 119.93, 129.76, 130.63,
131.00, 133.84, 134.81, 141.65, 147.70, 148.73, 159.42; HR-MS calcd.
for C22H20N2O m/z 328.15756, found: 328.15643.
The MTT assay is based on the reduction of yellow colored MTT
(Acros Organics) by mitochondrial dehydrogenase activity of
metabolically active cells to a blue-purple formazan that can be
measured spectrophotometrically. The absorbances were read in an
eight-channel computer-controlled photometer (Infinite M1000,
Tecan), at two wavelengths (540 and 690 nm). All data were
calculated using the median OD (optical density) values of tree
wells. The 50% cytotoxic concentration (CC50) was defined as the
concentration of the test compound that reduced the absorbance
(OD540) of the mock-infected control sample by 50%. The
concentration achieving 50% protection from the cytopathic effect
of the virus in infected cells was defined as the 50% effective
concentration (EC50).
4.1.6. 4-(3-Aminophenyl-1-yl-amino)benzonitrile (17)
Potassium carbonate (40 mmol), 1,3-diaminobenzene (15)
(30 mmol) and 4-fluorobenzonitrile (16) (20 mmol) were added to
a dried flask with 50 mL of anhydrous DMF. The reaction mixture
was heated to 130 ꢀC under nitrogen atmosphere and continuing
stirring for 72 h. The resulting reaction mixture was extracted with
diethyl ether. The organic layer was dried over anhydrous sodium
carbonate, filtered and purified by medium pressure chromatog-
raphy to obtain 14. Yield 5.0%; brown solid; mp: 150.9e153.8 ꢀC; 1H
NMR (300 MHz, CDCl3): d
3.70 (s, 2H, NH-7), 5.95 (s,1H, NH-70), 6.41
(d, J ¼ 7.8 Hz, 1H, CH-4), 6.46 (s, 1H, CH-2), 6.51 (d, J ¼ 7.8 Hz, 1H,
CH-6), 6.94 (d, J ¼ 8.7 Hz, 2H, CH-20, CH-60), 7.09 (t, J ¼ 7.8 Hz, 1H,
CH-5), 7.44 (d, J ¼ 8.7 Hz, 2H, CH-30, CH-50). 13C NMR (75 MHz,
4.3. Molecular modeling
The novel NNRTIs were built using the Gabedit software [23],
after which conformational searches were performed using
Gaussian03 [24], applying the AM1 semi-empirical method [25].
The minimum energy conformation was further optimized at the
ab-initio (HF6-31G*) level, with the goal of obtaining a minimum
energy conformation that allowed a representative charge assign-
ment over each atom. In this way, restrained electrostatic potential
(RESP) fitted charges were computed using Gaussian03. Atom
parameters are added using the ff99 force field with the module
xLeap of Amber10 [22].
As in previous work [17], the crystallographic structure of the
wtRT in complex with the reference compound 3 (PDB ID: 1SUQ
[26]) was used to performed all the molecular modeling analysis.
First, the crystallographic structure was subjected to energy mini-
mization with the sander module of Amber10 [22]. Affinity grids
were constructed on the minimized structures using AutoGrid [27].
Docking assays were carried out using the AutodockTools and
Autodock3 [21]. The lowest-energy and most populated clusters of
docked conformations were selected for the MD simulations.
Intermolecular interactions were visualized with LigPlot [28].
CDCl3):
d 101.49, 107.50, 110.97, 111.43, 115.33, 120.08, 130.60,
133.84, 141.19, 147.84, 148.12.
4.1.7. 4-[3-(2,4,6-Trimethylaniline)phenyl-1-ylamino]benzonitrile (18)
To
a dried screw tube of 10 mL, 17 (0.6 mmol), 2,4,6-
trimethylbromobenzene (13a) (0.5 mmol), Pd(OAc)2 (0.025 mmol),
Xantphos (0.05 mmol), Cs2CO3 (1.0 mmol) and toluene (5 mL) were
added. Then, the reaction mixture was stirred under nitrogen
atmosphere at 100 ꢀC, until the brominated compound was
consumed, which was monitored by TLC analysis. Then, the resulting
reaction mixture was extracted with diethyl ether. The organic layer
was dried over anhydrous sodium carbonate, filtered, evaporated
under reduced pressure and purified by medium pressure chroma-
tography to obtain 18. Yield 75.8%; white solid, mp: 218.6e222.4 ꢀC;
1H NMR (300 MHz, CDCl3): 2.19 (s, 6H, CH3-800, CH3-1000), 2.30 (s, 3H,
d
CH3-900), 5.13 (s, 1H, NH-70), 5.90 (s, 1H, NH-700), 6.15 (s, 1H, CH-2),
6.30 (d, J ¼ 7.8 Hz, 1H, CH-4), 6.55 (d, J ¼ 7.8 Hz, 1H, CH-6), 6.93
(d, J ¼ 8.7 Hz, 2H, CH-20, CH-60), 6.94 (s, 2H, CH-300, CH-500), 7.11
(t, J ¼ 7.8 Hz,1H, CH-5), 7.44 (d, J ¼ 8.7 Hz, 2H, CH-30, CH-50). 13C NMR

492
S.R. Ribone et al. / European Journal of Medicinal Chemistry 58 (2012) 485e492
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The wtRT-NNRTI complexes were solvated using
a pre-
equilibrated TIP3P water model, applying a solvent box with
ꢀ
a minimum distance from the solute of 8 A in each direction. After
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M.P. De Béthune, H. Azijn, R. Pauwels, H.E.L. Moereels, J. Heeres,
L.M.H. Koymans, M.R. De Jonge, K.J.A. Van Aken, F.F.D. Daeyaert, P.J. Lewi, K. Das,
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J. Heeres, L.M.H. Koymans, M.R. De Jonge, K.J.A. Van Aken, F.F.D. Daeyaert, K. Das,
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minimization and equilibration, MDs were carried out at 300 K for
3 ns, using the pmemd module of Amber10 [22]. Hydrogen bonds
and dihedral angles analyses were studied with the ptraj module of
Amber10 [22]. The trajectories were processed using VMD software
[29]. The electrostatic energy component analysis was estimated by
the Molecular Mechanics-Poisson Boltzmann Surface Area (MM-
PBSA) method [30], integrated in Amber10 [22]:
G ¼ EMM þ Gsolv ꢃ T
D
S
(1)
where, G is the Gibbs free energy, T is the absolute temperature,
D
S
is the entropic changes, EMM is the total molecular mechanic
energy, which includes internal (bond, angle, dihedral), van der
Waals and electrostatic terms; Gsolv ¼ Gpol þ Gnp; Gpol is the
electrostatic component of the solvation free energy, which was
computed by the Poisson-Boltzmann approximation [31]; and Gnp
is the non-polar contribution to the solvation free energy, calcu-
lated by an empirical model. The solvent was treated as
a continuum model of high-dielectric (ε ¼ 80) and the solute as
a low-dielectric media (ε ¼ 1) with embedded charges [32].
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J. Desmyter, E. De Clercq, Rapid and automated tetrazolium-based colori-
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Acknowledgments
[21] G.M. Morris, D.S. Goodsell, R. Huey, W.E. Hart, S. Halliday, R. Belew, A.J. Olson,
Autodock3.0, in: Molecular Graphics Laboratory, Department of Molecular
Biology, The Scripps Research Institute, 1999.
[22] D.A. Case, T.A. Darden, T.E. Cheatham, C.L. Simmerling, J. Wang, R.E. Duke,
R. Luo, M. Crowley, W. Walker, K.M. Zhang, K.M. Merz, B. Wang, S. Hayik,
A. Roitberg, G. Seabra, I. Kolossvary, K.F. Wong, F. Paesani, J. Vanicek, X. Wu,
S.R. Brozell, T. Steinbrecher, H. Gohlke, L. Yang, C. Tan, J. Mongan, V. Hornak,
G. Cui, D.H. Matthews, M.G. Seetin, C. Sagui, V. Babin, P.A. Kollman, AMBER10,
University of California, San Francisco, 2008.
The authors gratefully acknowledge financial support from the
Secretaria de Ciencia y Técnica of the Universidad Nacional de
Córdoba (SECYT-UNC) and the Ministerio de Ciencia y Tecnología
(MINCYT) of Córdoba, as well as the Consejo Nacional de Inves-
tigaciones Científicas y Técnicas (CONICET), the Agencia Nacional
de Promoción Científica y Tecnológica (FONCyT) of Argentina and
grant GOA 10/14 from KU Leuven. This research was supported in
part by the National Science Foundation through XSEDE resources
provided by the Pittsburgh Supercomputing Center (TG-
MCB070064). Sergio R. Ribone also acknowledges fellowships from
CONICET and Erasmus Mundus External Cooperation Windows
(EADIC Lot 16).
[23] A.R. Allouche, Gabedit e a graphical user interface for computational chem-
istry softwares, J. Comput. Chem. 32 (2011) 174e182.
[24] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb,
J.R. Cheeseman, J. Montgomery, J.A.,T. Vreven, K.N. Kudin, J.C. Burant,
J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara,
K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken,
C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin,
R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth,
P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels,
M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman,
J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu,
A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-
Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson,
W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian03, Gaussian Inc.,
Wallingford CT, 2004.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.10.036.
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