New pyridinone derivatives as potent HIV-1 nonnucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jm801438e

Several 5-ethyl-6-methyl-4-cycloalkyloxy-pyridin-2(1H)-ones were synthesized and evaluated for their anti HIV-1 activities against wild-type virus and clinically relevant mutant strains. A racemic mixture (10) with methyl substituents at positions 3 and 5

Full text of DOI:10.1021/jm801438e

3636
J. Med. Chem. 2009, 52, 3636–3643
New Pyridinone Derivatives as Potent HIV-1 Nonnucleoside Reverse Transcriptase Inhibitors
Kiet Le Van,†,§ Christine Cauvin,*,‡,§ Ste´phane de Walque,|,§ Benoˆıt Georges,† Sandro Boland,‡ Vale´rie Martinelli,|
Dominique Demonte´,| Franc¸ois Durant,‡,# La´szlo´ Hevesi,†,# and Carine Van Lint|,#
Laboratoire de Chimie des Mate´riaux Organiques and Laboratoire de Chimie Mole´culaire Structurale, Faculte´s UniVersitaires Notre-Dame de
la Paix (FUNDP), B-5000 Namur, Belgium, and Laboratoire de Virologie Mole´culaire, Institut de Biologie et de Me´decine Mole´culaires,
UniVersite´ Libre de Bruxelles (ULB), B-6041 Gosselies, Belgium
ReceiVed NoVember 14, 2008
Several 5-ethyl-6-methyl-4-cycloalkyloxy-pyridin-2(1H)-ones were synthesized and evaluated for their anti
HIV-1 activities against wild-type virus and clinically relevant mutant strains. A racemic mixture (10) with
methyl substituents at positions 3 and 5 of the cyclohexyloxy moiety had potent antiviral activity against
wild-type HIV-1. Subsequent stereoselective synthesis of a stereoisomer displaying both methyl groups in
equatorial position was found to have the best EC50. Further modulations focused on position 3 of the
pyridinone ring improved the antiviral activity against mutant viral strains. Compounds bearing a 3-ethyl
(22) or 3-isopropyl group (23) had the highest activity against wild-type HIV-1 and displayed low-nanomolar
potency against several clinically relevant mutant strains.
Introduction
Reverse transcriptase (RTa) is a key enzyme of the human
immunodeficiency virus type 1 (HIV-1) life cycle. It is
responsible for the transformation of HIV genomic single-
stranded RNA into double-stranded proviral DNA. RT has been
recognized as an attractive target for drug development for a
number of years, and numerous RT inhibitors have been
developed.1 Nucleoside reverse transcriptase inhibitors (NRTIs),
such as azidothymidine (AZT), are activated in cells and then
act as decoy substrates and chain terminators, thereby preventing
the synthesis of a functional double-stranded DNA. Nonnucleo-
side reverse transcriptase inhibitors (NNRTIs) are a structurally
diverse class of compounds that inhibit the enzyme by binding
to a specific allosteric pocket located some 10 Å from the
polymerase active site.2,3 To date, four NNRTIs (nevirapine,
delavirdine, efavirenz, and etravirine) have been approved for
clinical use (Figure 1).4 Combination of NRTIs and NNRTIs
with other antiviral agents, such as viral protease inhibitors (e.g.,
saquinavir, indinavir, amprenavir, lopinavir, darunavir,...) or
Figure 1. Chemical structures of commercial NNRTIs.
fusion inhibitors (enfuvirtide) or integrase inhibitors (raltegravir),
led to the development of highly active antiretroviral therapy
(HAART) that allows an important decrease in viral load and
a significant improvement of the patient’s health.5 In spite of
these major advances, new and potent anti-HIV compounds are
still needed because of the continued emergence of mutant viral
strains that are resistant to current antiretroviral agents.6
Pyridinone derivatives were first described as NNRTIs in
1991.7 More recently, several NNRTIs based on the pyridinone
Figure 2. General structure of 4-cycloalkyloxypyridin-2(1H)-one
derivatives.
* To whom correspondence should be addressed. Phone: +32 81 72 45
69. Fax: +32 81 72 54 40. E-mail: christine.cauvin@fundp.ac.be.
†
scaffold have been reported such as 4-arylthiopyridin-2(1H)-
one compounds.8 Despite several studies pointing out compound
flexibility as an important parameter for conserving potency on
various mutant viral strains, the pyridinone compounds previ-
ously described only include arylthio, aryloxy, or benzyl
moieties at position 4 of the pyridinone ring.9,10 We now report
in this study the design, synthesis, and HIV-1 inhibiting
properties of a new series of potent 4-cycloalkyloxypyridin-
2(1H)-one derivatives (Figure 2). Optimization of this series
led to the identification of compounds combining high potency
Laboratoire de Chimie des Mate´riaux Organiques, Faculte´s Universi-
taires Notre-Dame de la Paix.
§
These authors contributed equally to this work.
Laboratoire de Chimie Mole´culaire Structurale, Faculte´s Universitaires
‡
Notre-Dame de la Paix.
|
Universite´ Libre de Bruxelles.
These authors codirected the present study. C.V.L. is Directeur de
#
Recherches of the Belgian “Fonds de la Recherche Scientifique-FNRS”
(FRS-FNRS).
a Abbreviations: RT, reverse transcriptase; HIV-1, human immunode-
ficiency virus type 1; NNRTI, nonnucleoside reverse transcriptase inhibitor;
EC50, 50% effective concentration; CC50, 50% cytotoxic concentration; SI,
selectivity index.
10.1021/jm801438e CCC: $40.75
2009 American Chemical Society
Published on Web 05/26/2009

Pyridinone DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3637
Scheme 1. Synthesis of Derivative 23a
Table 1. In Vitro RT Assays, Antiviral Activity against Wild-Type
HIV-1 Virus, and Selectivity Index of Pyridinone Compounds with
Various Alkyl Rings
compd
n
% RT residual activitya
EC50 (µM)b
SIc
1
2
3
4
1
2
3
4
58.6
44.7
34.6
24.0
0.74
0.23
0.39
0.70
>135
>435
205
83
a Percentage of RT residual activity after addition of 10 µM compound.
b Effective concentration 50%. c Selectivity index or ratio of CC50 to EC50.
a Reagents and reaction conditions: (a) PhCH2Br, Ag2CO3, PhH, 60 °C,
20 h; (b) MeLi (3.5 equiv), THF, -78 °C (1 h), then 0 °C (3 h); (c) SOCl2,
PhH, 5 °C to room temp, 2 h; (d) 3,5-dimethylcyclohexanol (R isomer),
DIAD, PPh3, THF, room temp, 20 h; (e) H2, 1 atm, Pd/C, EtOH, room
temp, 36 h.
yielding C. Finally, debenzylation by catalytic hydrogenation
led to 22 in high yield.
Results and Discussion
Scheme 2. Synthesis of Derivative 22a
In the first step, impact of the cycloalkyl ring size was
evaluated. Compounds 1-4 containing rings ranging from
cyclopentyl to cyclooctyl were prepared from B0 (Table 1).
These four pyridinone derivatives effectively inhibited RT at
10 µM in an in vitro enzymatic assay. All compounds
subsequently displayed moderate antiviral activity with EC50
values ranging from 0.2 to 0.7 µM.
Molecular modeling of compounds into the allosteric site was
performed using a RT structure extracted from RT-TNK-651
complex (PDB code 1RT211). Molecular docking algorithms
implemented in Gold12 were used in order to provide binding
mode hypotheses, which were then refined using Accelrys
Discover 3 module.13,14 Figure 3 displays a schematic drawing
of the best-ranking binding mode of 2. It is consistent with
binding modes experimentally observed for similar derivatives.15
The pyridinone ring is pinned between Leu100 and Val106,
allowing the lactam NH to form a hydrogen bond with Lys101.
The hydrophobic 5-Et group faces Tyr181, favoring the con-
formational change required for efficient inhibition.11 The
4-cycloalkoxy group of the inhibitor is located in the most
hydrophobic part of the binding pocket, interacting with Tyr181,
Tyr188, Phe227, and Trp229. Finally, the ester moiety of the
compound is located near Pro236, in the most flexible part of
the NNRTI binding pocket. From modeling results and
structure-activity relationships, it can be pointed out that the
cyclohexyl ring (2) was the best compromise between occupa-
tion of the hydrophobic pocket and conformational energy
penalties.
a Reagents and reaction conditions: (a) NaOEt, EtOH, toluene, reflux,
60 h; (b) PhCH2Br, Ag2CO3, C6H6, 80 °C, 6 h; (c) 3,5-dimethylcyclohexanol
(R isomer), DIAD, PPh3, THF, room temp, 24 h; (d) cyclohexene, Pd/C,
i-Pr2O, 80 °C, 6 h.
against wild-type HIV-1 and a panel of single and double mutant
strains with very low cytotoxicity.
Chemistry
Almost all the compounds studied have been synthesized from
the common intermediate B0, which is easily obtained from ethyl
2-acetylbutyrate, ammonia, and diethyl malonate. Scheme 1
shows a typical example in the case of compound 23. The
synthetic route began by the O-benzylation of B0 to give B1.
This compound was subjected to reductive methylation yielding
B2. Dehydration was performed with thionylchloride to afford
B3. Then Mitsunobu reaction with 3,5-dimethylcyclohexanol
gave B4. This last compound led to 23 after extensive catalytic
hydrogenation.
Replacement of the hydrophobic cyclohexyl ring by a more
hydrophilic piperidine ring resulted in a dramatic loss of activity,
consistent with SAR from other NRRTI series, where hydro-
phobic substituents are also favored in this position.
In the second step, the effect of substitution on this cyclohexyl
ring was investigated either in the P4 cell line or in the TZM-
bl cell line (compounds 5-10 in Table 2). These compounds
were synthesized as mixtures of stereoisomers. From comparison
of the antiviral activity of these compounds, it can be observed
that “meta” (i.e., 3,5) substitution was favored over “ortho” and
“para” substitutions. Compound 10 with methyl substituents in
positions 3 and 5 had an antiviral potency (0.043 µM), similar
to nevirapine (0.039 µM, Table 2). This mixture of isomers was
separated by chiral HPLC, giving two main fractions 10A
A slight modification of the general synthetic pathway is
illustrated for compound 22 (Scheme 2). Intermediate A was
obtained by condensation of ethyl 3-amino-2-ethylbutenoate
with diethyl 2-ethylmalonate. O-Benzylation of A gave B, which
underwent Mitsunobu reaction with 3,5-dimethylcyclohexanol

3638 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Le Van et al.
Figure 3. Stereoscopic view of the binding mode of 2 (only main surrounding residues are shown in green).
Table 2. Antiviral Activity against Wild-Type HIV-1 Virus in TZM-bl
Cells and Selectivity Index of Compounds with Substituent Variations at
Positions 3 and 4 of the Pyridinone Ring: Comparison with the
Reference Compounds Nevirapine and Efavirenz
compd
R1
Y
R2
EC50 (µM)
SI
<33
192
<14
2615
78
ND
ND
ND
1778
112
5
6
7
8
9
2-Cla
3-CH3
O
O
O
O
O
O
O
O
O
O
S
O
O
O
O
O
O
O
O
O
O
O
O
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
H
>3.0c
a
0.520c
>4.0c
a
4-CH2CH3
a
2,3-diCH3
2,6-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,3,5,5-tetraCH3
3,5-diCH3
3,5-diCH3
0.013c
0.955
0.043c
0.236c
0.085c
0.036c
0.258c
0.116
0.275
>10c
a
a
10
10A
10B
10C
11
12
13
14
15
16
17
18
19
20
21
22
23
24
b
b
862
200
ND
a
H
CO2H
CH2OH
NO2
b
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
0.121
<0.001
<0.001
1.2c
<0.001
<0.001
0.002
<0.001
<0.001
<0.001
0.039
<0.001
>826
>37000
>28000
83
>66000
>54000
40000
>67000
>82000
>100000
2564
>58000
b
b
Br
b
NH2
Figure 4. X-ray structures of compounds 10A (a) and 10C (b) (ORTEP
b
NMe2
CH2N3
Me
view with displacement ellipsoids drawn at the 50% probability level).24
b
b
b
Et
between axial substituents and residues Tyr181/Tyr188 and (ii)
favorable van der Waals contacts between equatorial substituents
and residue Trp229. Figure 5 shows the optimal fit of 10C into
the allosteric pocket, leading to an excellent inhibitory activity
against wild-type HIV-1.
The advantageous equatorial substitution vs axial substitution
was also demonstrated by comparing the antiviral activities of
10C and 11. Compound 11 with its 3,3,5,5-tetramethyl sub-
stituent pattern had a lower antiviral activity than 10C (Table
2, EC50 of 0.258 µM vs 0.036 µM). Replacement of 3,5-
dimethylcyclohexyloxy by 3,5-dimethylcyclohexylthio moiety
in position 4 of the pyridinone ring did not improve the antiviral
activity (compound 12 vs 10C).
While compound 10C was effective against wild-type HIV-
1, it only displayed moderate to poor antiviral activity against
Y181C and K103N, two HIV-1 mutant strains often observed
in patients treated with NNRTIs (Table 3). Further optimization
of 10C was undertaken in order to replace the hydrolyzable
ester group and to obtain inhibitors with a better activity against
mutant viral strains.
b
3,5-diCH3
3,5-diCH3
i-Pr
CMe2OH
b
nevirapine
efavirenz
a Mixture of stereoisomers. b Stereoisomer with Me substituents in
equatorial position. c P4 cells.
(∼70%) and 10B (∼30%). X-ray diffraction allowed the
determination of the structure of the major stereoisomer 10A;
it corresponded to the isomer having the oxypyridinone moiety
and methyl substituents in the trans position (Figure 4a). A
stereoselective Mitsunobu reaction of 3,5-dimethylcyclohexanol
(“R isomer”; see Note in the Experimental Section) with B0
produced 10C, a diastereoisomeric form of 10A, whose structure
was confirmed by XRD (Figure 4b). This stereoisomer with all
groups in a cis position had a stronger antiviral activity than
10A (Table 2). This increased potency of 10C was in agreement
with molecular modeling studies. Indeed, docking studies and
interaction energy calculations showed that equatorial substitu-
tion was more favorable than the axial one (-35.5 kcal/mol
for 10C vs -32.2 kcal/mol for 10A). The differences between
stereoisomers were essentially explained by (i) steric clashes
Structural modifications on position 3 (R2) of the pyridin-
2(1H)-one ring were performed. Table 2 discloses the activity

Pyridinone DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3639
Figure 5. View of the optimal fit of 10C (with methyl groups in equatorial position; yellow arrows) into the allosteric site of RT.
Table 3. Antiviral Activity (EC50 (µM)) in TZM-bl Cells, against a
Panel of Single-Mutant Strains, of Some Compounds with Substituent
Variations at Position 3 of Pyridinone Ring: Comparison with the
Reference Compounds Nevirapine and Efavirenz
amino acid substitutions at residues 100, 103, 108, 181 showed
only minimally reduced sensitivities to 22 or 23.
It is now established that a key factor in overcoming viral
resistance mutations is the ability of a compound to adapt to
the structural changes caused in the binding site by these
mutations. While compound flexibility is of prime importance
for some mutations,17 it has also been suggested that the K103N
mutation causes resistance by stabilizing the unliganded form
of RT.18 In order to retain activity, RT inhibitors should be able
to form additional interactions with the mutated side chain,
thereby compensating this stabilization of the unliganded
enzyme. In this context, it is especially interesting to note that
some pyridinone derivatives might adopt an alternative binding
mode when facing mutated binding sites, as suggested by
molecular docking results. These binding modes were associated
with comparable interaction energies and are illustrated in Figure
6 for compound 22. The first binding mode (Figure 6a), has
been discussed above and pointed the ethyl group in position 3
of the pyridin-2(1H)-one ring toward the Pro236 hydrophobic
pocket. The second binding mode (Figure 6b) was only
favorable in the case of compounds displaying a small and
lipophilic group on position 3 and pointed this group toward
Tyr181. The preference for small and lipophilic groups in this
region has been documented for several series of RT inhibitors,
including HEPT derivatives such as TNK-651.11,19 In the case
of the K103N mutation, this alternative binding mode allowed
positioning of the carbonyl of the pyridinone ring in the vicinity
of the Asn amide group as shown in Figure 6c, suggesting
favorable electrostatic interactions between the pyridinone and
the mutated side chain. This ability to adopt different binding
modes, depending on the structure of the binding site, might
explain the ability of compounds 17, 19, 22, and 23 to retain
activity against K103N and, possibly, other mutant viral strains
(as observed in Table 3).
compd
10C
16
17
19
20
21
22
23
Y181C
2.0a
K103N
L100I
V108I
Y188L
>10
2.2
0.009
ND
0.028
0.034
0.203
0.006
0.002
0.252
0.017
5.9
>10
>10
6.3
>10
>10
>10
5.4
1.1
>10
0.235
0.279
0.138
0.177
0.206
0.893
0.015
0.006
>10
0.177
0.009
0.014
0.074
0.142
0.005
0.002
3.3
0.002
0.001
0.006
0.011
0.121
<0.001
<0.001
0.792
<0.001
nevirapine
efavirenz
<0.001
0.031
a P4 cells.
of the resulting compounds against wild-type virus in cell culture
(P4 or TZM-bl cells). Structure-activity relationships on
position 3 (R2) of the pyridine-2(1H)-one ring suggested a
compromise between occupancy of the binding pocket and the
overall lipophilic character of this site. As shown in Table 2,
compound 13, which is not substituted in position 3, was less
active than 10, confirming the importance of filling the
hydrophobic pocket. Charged groups appeared clearly unfavor-
able for activity (14 vs 2), suggesting replacement of the
hydrolyzable ester moiety of the initial derivatives. Polar neutral
groups were better tolerated in this position (15, 18), and
compounds displaying weakly polar groups (16, 20) possessed
low nanomolar activity. Increased lipophilicity and occupancy
of the binding site were clearly beneficial for activity, as
demonstrated by compounds 19 and 21-24 and by the
substantial difference of antiviral potency between compounds
15-24 and 18-19. Among lipophilic derivatives, ethyl (22) or
propyl substituents (23) were more favorable than a methyl
group (21).
A selectivity index (cytotoxicity vs antiviral activity) was also
measured for the studied compounds. As shown in Table 2,
several derivatives displayed very low toxicity (CC50 > 50 µM)
while showing antiviral activity in the subnanomolar range,
leading to selectivity indexes over 50 000, which were signifi-
cantly higher than for nevirapine (2564) and comparable to that
for efavirenz (58 000).
The activity of the current compounds against the Y188L
strain was only in the micromolar range. From Figure 6a, it
can be seen that Tyr188 faced the 3,5-dimethylcyclohexyl
moiety of the inhibitor and that the position of this group was
not modified in the alternative binding mode (Figure 6b). The
alternative binding mode was therefore unable to compensate
the loss of lipophilic interactions resulting from the Y188L
mutation.
Mutant HIV-1 strains were generated by introducing RT
mutations that are commonly observed in clinical practice into
a wild-type HIV-1 strain.16 The antiviral activity in a single-
round replication assay (TZM-bl cells) against single mutant
strains of compounds substituted on position 3 of the pyridin-
2(1H)-one ring is disclosed in Table 3 and compared with that
observed for nevirapine and efavirenz. Mutant strains with single
We subsequently evaluated several derivatives for their
antiviral activity against four clinically relevant double mutant
strains using TZM-bl cell assay allowing single-round infection
experiment (Table 4). The conclusions from these assays were
essentially in line with the results obtained using the single
mutant strains. Compounds 22 and 23 displayed the best profile

3640 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Le Van et al.
Figure 6. Binding modes of 22 into wild-type RT (a and b) and K103N mutant (c).
Table 4. Antiviral Activity (EC50 (µM)) in TZM-bl Cells, against a
Panel of Double Mutant Strains, of Some Compounds with Substituent
Variations at Position 3 of Pyridinone Ring: Comparison with the
Reference Compounds Nevirapine and Efavirenz
Cell culture selection of mutant virus with greatly reduced
susceptibility to the inhibitor 22 resulted in a variant that
expressed a combination of substitution (Val106Ala and
Phe227Phe/Leu). Both residues are located in the NNRTI
binding site (Figure 3). It was previously reported that a high
dose of other NNRTIs (S-1153 and UC781) also selected for
the V106A plus F227L double mutant.20,21 The mutations
V106A and F227L conferred little resistance to efavirenz as
single-point mutations but enhanced the resistance level of
viruses carrying these mutations in combination with K103N
or Y188L.
compd K103N/V108I K103N/Y181C K103N/L100I L100I/V108I
16
17
19
20
21
22
23
ND
>10
0.780
>10
4.4
5.1
0.107
0.105
.10
0.034
>10
0.068
2.9
0.828
1.4
0.081
0.044
>10
8.3
0.038
1.6
0.107
0.833
0.115
0.010
>10
0.523
0.245
0.689
1.2
0.359
0.642
6.2
nevirapine
efavirenz
0.525
3.7
0.014
Conclusion
In summary, we have identified a new series of 4-cycloalky-
loxypyridin-2(1H)-one derivatives as NNRTIs. The best com-
pounds, 22 and 23, displayed high potency against wild-type
HIV-1 and a panel of single and double mutant strains often
observed in patients treated with NNRTIs. Further investigation
is ongoing in order to characterize the pharmacokinetic profile
of these compounds and to obtain the crystallographic structure
of such compounds in complex with RT and mutants.
Table 5. Antiviral Activity (EC50 (µM)) of Compounds 22 and 23,
against Wild-Type HIV-1 and a Panel of Relevant Mutant Strains, in a
Multiple-Round Replication Assay (MT-4 cells): Comparison with the
Reference Compound Efavirenz
compd
22
WT
Y181C
K103N
L100I
K103N/L100I
0.002
0.002
0.003
0.049
0.019
0.004
0.026
0.007
0.320
0.027
0.006
0.167
0.145
0.084
>10
23
efavirenz
Experimental Section
against double mutant strains and had an effect comparable or
superior to that of efavirenz.
Chemistry. 1H NMR spectra were recorded on a Jeol JNM EX-
400 spectrometer. Chemical shifts (δ) are reported in parts per
million (ppm) downfield from tetramethylsilane (TMS) used as an
internal standard. Melting points were determined with a Totolli-
Buchi melting point B-545 apparatus. Analytical thin-layer chro-
matography (TLC) was done on Merck silica gel 60 F254 plates.
Mass spectra were taken on an ion trap mass spectrometer (ITMS).
Elemental analyses (C, H, N, S) were performed on a ThermoFinni-
gan Flash EA 1112 elemental analyzer at the Pharmacy Department,
FUNDP, Namur. HPLC analyses were realized on a Varian 940-
LC apparatus with a Varian 380-LC detector.
Because of the attractive profile of compounds 22 and 23,
we next decided to test these compounds in parallel with
efavirenz in a multiple-round replication assay (MT-4 cells) for
their antiviral activity against wild-type HIV-1 as well as several
single or double mutant viral strains (Y181C, K103N, L100I,
and K103N + L100I). As shown in Table 5, the biological
results observed very nicely confirmed those we obtained in
the TZM-bl cell assays.

Pyridinone DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12 3641
1
Note. Commercial 3,5-dimethylcyclohexanol (Fluka, purum,
97%) is a mixture of stereoisomers from which the “R isomer”
(i.e., equatorial methyl groups and axial OH group) can be separated
(∼10%) by column chromatography (eluent ether/pentane, 1:9 v/v).
The remaining mixture is subjected to a Mitsunobu reaction (DIAD,
PPh3) with acetic acid, leading to a mixture of acetates in which
the “R′ isomer” (i.e., equatorial methyl groups and axial OAc group)
is predominating. After basic hydrolysis of the mixture of acetates,
chromatographic separation allows recovery of a substantial amount
(∼60%) of the desired “R isomer” of 3,5-dimethylcyclohexanol.
4-(3′,5′-Dimethylcyclohexyloxy)-5-ethyl-3-isopropyl-6-methyl-
1H-pyridin-2-one (23). Amounts of 2.25 g (10 mM) of B0, 1.45 g
(5.25 mM) of silver carbonate, and 1.25 mL (10.5 mM) of benzyl
bromide were placed under argon in a 100 mL two-necked round-
bottom flask equipped with a condenser, with a magnetic stirring
bar, and with a rubber septum. Then an amount of 30 mL of dry
benzene was introduced, and the mixture was heated to 60 °C with
stirring for 20 h. After cooling to room temperature, the mixture
was diluted with 30 mL of pentane, filtered through a filter agent
pad (Celite 521, Aldrich), and evaporated to dryness. The residue
was purified by column chromatography (silicagel, eluent pentane/
dichloromethane, 1/1 v/v) to give 2.64 g of B1 as a white solid
(83% yield).
An amount of 3.22 g (10.2 mM) of B1 was dissolved in 35 mL
of dry THF placed under argon in a 200 mL two-necked round-
bottom flask fitted with rubber septa. After cooling to -78 °C, a
total of 21 mL (33.6 mM) of methyllithium (1.6 M in ether) was
added dropwise by means of a syringe. The reaction mixture was
kept at -78 °C for 1 h and at 0 °C for 3 h. Then the mixture was
diluted with 40 mL of cold ether and slowly neutralized with cold
1 N HCl. The organic layer was separated, and the aqueous phase
was extracted twice with 40 mL of ether. The combined organic
solution was washed with a saturated sodium bicarbonate solution
followed by washing with a saturated sodium chloride solution.
After evaporation of the solvents, the residue was purified by
column chromatography (silica gel, eluent ether/pentane, 10/90 v/v)
to give 2.98 g (97% yield) of alcohol B2.
An amount of 2.39 g (7.94 mM) of B2 was dissolved under argon
in 25 mL of dry benzene in a 100 mL two-necked round-bottom
flask fitted with a rubber septum. The solution was cooled to 5 °C,
and 0.8 mL of thionyl chloride (SOCl2, 10.9 mM) was added
dropwise. After 0.5 h of reaction at 5 °C and 2 h at room
temperature, the benzene was evaporated and the residue was
dissolved in 40 mL of dichloromethane. This solution was basified
with a cold sodium bicarbonate solution, washed with a saturated
sodium chloride solution, and dried over magnesium sulfate.
Evaporation of the solvent and purification by column chromatog-
raphy (silica gel, eluent ether/pentane, 5/95 v/v) led to 1.85 g of
B3 (82% yield).
Amounts of 294 mg (1.0 mM) of B3, 524 mg (2.0 mM) of
triphenylphosphine, and 256 mg (2.0 mM) of 3,5-dimethylcyclo-
hexanol (“R isomer”) were dissolved under argon in 6 mL of dry
THF in a 25 mL two-necked round-bottom flask fitted with a rubber
septum and cooled to 0 °C. A total of 406 mg of DIAD (diisopropyl
azodicarboxylate, 2.0 mM) dissolved in 2 mL of THF was added
dropwise by means of a syringe. The reaction mixture was stirred
for 1 h at 0 °C, then for 20 h at room temperature. After evaporation
of the volatiles, the residue was triturated in 20 mL of pentane for
0.5 h and filtered and the pentane solution was washed with 5 mL
of sodium hydroxide (0.5 N), with water and with saturated sodium
chloride. After drying over magnesium sulfate and evaporation of
the solvent, the product was purified by column chromatography
(silica gel, eluent ether/pentane, 2.5/97.5 v/v) to give 340 mg (84%
yield) of B4.
4.53 g (97% yield) of 23. H NMR (CDCl3, 400 MHz): δ 1.0 (d,
6H, CH3 and CH35′), 1.1 (m, 3H, 5-CH2CH3), 1.4 (d, 6H,
3′
3-CH(CH3)2), 0.6-2.0 (m, 8H, cyclohexyl), 2.3 (s, 3H, 6-CH3),
2.45 (q, 2H, 5-CH2CH3), 3.2 (m, 1H, 3-CH(CH3)2), 3.85 (m, 1H,
H1′), 12.4 (s, 1H, NH). Mp 184 °C. MS: (M + H)+ 306.0. Anal.
(C19H31NO2) C: calcd, 74.71; found, 75.67. H: calcd, 10.23; found,
10.24. N: calcd, 4.59; found: 4.31. Purity by HPLC, 99%.
4-(3′,5′-Dimethylcyclohexyloxy)-3,5-diethyl-6-methyl-1H-py-
ridin-2-one (22). A solution of diethyl 2-ethylmalonate (13.16 g,
0.07 M) in 15 mL of toluene was added to 20 mL of a 3.75 M
solution of EtONa in EtOH freshly prepared, and the mixture was
heated to 80 °C for 30 min. Then a solution of ethyl 2-ethyl-3-
aminocrotonate (11 g, 0.07 M) in 20 mL of toluene was added,
and the mixture was refluxed for 60 h. After the mixture was cooled,
an amount of 150 mL of water was added under thorough mixing,
leading to a two phase system. The water layer was separated (it
contained the sodium salt of A), washed twice with 20 mL of
toluene, and neutralized with concentrated hydrochloric acid to pH
7. The precipitate was filtered, washed with water, and dried to
give 8 g (60% yield) of intermediate A.
Amounts of 3.62 g (0.02 M) of intermediate A, 5.5 g (0.02 M)
of silver carbonate, and 3.42 g (0.02 M) of benzyl bromide were
placed in 50 mL of benzene and heated to 80 °C for 6 h. After
cooling to room temperature, the mixture was filtered and the
solvent was evaporated. The crude product was purified by column
chromatography (silica gel, eluent pentane/dichloromethane, 50/
50 v/v %) to give 4.33 g (80% yield) of intermediate B.
To a solution of 2.71 g (0.01 M) of compound B, 2.56 g (0.02
M) of 3,5-dimethylcyclohexanol (“R isomer”), and 5.26 g (0.02
M) of triphenylphosphine in 50 mL of THF, 4 g (0.02 M) of
diisopropyl azodicarboxylate (DIAD) was added dropwise at room
temperature, and the mixture was stirred overnight. The solvent
THF was evaporated, and the pasty residue was triturated in 100
mL of hexane. The undissolved solid (triphenylphosphine oxide)
was filtered off, and the solvents were evaporated. The crude
product was purified by column chromatography (silica gel, eluent
pentane/dichloromethane, 50/50 v/v %) to give 2.65 g (70% yield)
of intermediate C.
Amounts of 0.381 g (0.001 M) of intermediate C, 4 mL of
cyclohexene, and 0.2 g of Pd/C 10% were placed in 12 mL of
diisopropyl ether, and the mixture was heated to 80 °C for 6 h.
The mixture was filtered, and the volatiles were evaporated. The
residue was purified by column chromatography (silica gel, eluent
ethanol/dichloromethane, 5/95 v/v %) to give 0.262 g (90% yield)
of compound 22. 1H NMR (CDCl3, 400 MHz): δ 0.9 (d, 6H, CH3
3′
and CH35′), 1.05 (m, 3H, 5-CH2CH3), 1.15 (m, 3H, 3-CH2CH3),
0.6-2.1 (m, 8H, cyclohexyl), 2.35 (s, 3H, 6-CH3), 2.5 (q, 2H,
5-CH2CH3), 2.6 (q, 2H, 3-CH2CH3), 3.9 (m, 1H, H1′), 12.8 (s, 1H,
NH). Mp 160-161 °C. MS: (M + H)+ 292.41. Anal. (C18H29NO2)
C: calcd, 74.18; found, 73.94. H: calcd, 10.03; found, 9.86. N: calcd,
4.81; found, 4.58. Purity by HPLC, 99.0%.
Molecular Modeling. Docking Studies. Docking of the inhibi-
tors into RT was carried out using GOLD (version 1.1) programs.12
GOLD uses a genetic algorithm and performs flexible ligand
docking into proteins with partial flexibility in the neighborhood
of the active site. Some limited flexibility of the binding site is
taken into account by optimizing the torsion angles of Ser, Thr,
and Tyr hydroxyl groups as well as Lys NH3+ moieties during the
docking run so that hydrogen bond formation is favored. Default
settings were used for the genetic algorithm parameters.
Display and examination of the docking poses were carried out
using the Insight II environment.14
Energy Minimization. In order to better take into account
protein flexibility, docking poses were refined with Discover 3
(CVFF force field, dielectric constant 1*r).13 Our energy minimiza-
tion process included two sequential steps: a “steepest descent”
algorithm, reaching a convergence of 10.0 kcal mol-1 Å-1, followed
by a “conjugate gradient” minimization to reach a final convergence
of 0.01 kcal mol-1 Å-1. All protein atoms were first kept fixed,
while the orientation of the inhibitor was optimized. Receptor side
chains within 12 Å of the inhibitor were then progressively relaxed,
An amount of 6 g (15.3 mM) of B4 was dissolved in 100 mL of
absolute ethanol in a 250 mL two-necked round-bottom flask fitted
with rubber septa. Then 1 g of palladium on carbon (10 wt %) was
introduced, and the mixture was placed under hydrogen (1 atm)
and stirred at room temperature for 36 h. After filtration through a
filter agent pad (Celite 521, Aldrich), the solvent was evaporated
and the solid residue was crystallized from absolute ethanol to give

3642 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 12
Le Van et al.
using degressive restraints (quadratic form, initial force constant
of 20.0 kcal mol-1 Å-2).
assay based on the cleavage of the tetrazolium salt WST-1 (Roche)
by mitochondrial dehydrogenases in viable cells. Absorbance of
converted dye was measured in an ELISA plate reader at a
wavelength of 570 nm. The cytotoxicity of the compounds was
calculated as the reduction (in %) of viable cells compared with
the drug-free control culture. The drug concentration required to
reduce cell viability by 50% was named CC50.
Activity of Compounds in the MT-4 Assay. The most active
compounds 22 and 23 were tested in a multiple-round replication
assay using the MT-4 human T-cell line. MT-4 cells were infected
with wild-type HIV-1 (NL4-3 strain) or mutant HIV-1 strains and
then exposed to different concentrations of antiviral compounds.
After 5 days of incubation, the viability of HIV-infected and mock-
infected cells was determined by a colorimetric assay based on the
cleavage of the tetrazolium salt WST-1 (Roche). This method, based
on the protocol originally described by Pauwels et al.,26 allows
simultaneous determination of the 50% inhibitory concentration
(EC50) for inhibiting viral cytopathicity and the 50% cytotoxic
concentration (CC50).
Production of Viral Mutants. In vitro mutagenesis was applied
to the RT-coding region of the infectious molecular clone
pNL4-3 to obtain drug-resistant mutant strains. The following
mutations were separately introduced in pNL4-3: L100I, K103N,
V108I, Y181C, Y188L, and the double mutations K103N/L100I,
K103N/V108I, K103N/Y181C, L100I/V108I. These mutations
were chosen because of the resistance they confer to existing
NNRTIs. Plasmids were checked by sequencing to confirm that
they contained the desired mutations. The mutant clones were
transfected in HEK293 cells. The virus stocks were prepared
from the cell-free supernatant at day 2 or 3 posttransfection.
In Vitro Selection of HIV-1 Mutant Strains. CEM cells were
infected with a highly infectious HIV-1 clone, HIV-1 IIIB, at
high MOI and were exposed to 22 or 23 initially at 5-fold the
50% effective concentration (EC50). Infected cells were incubated
for 4 days. The RT activities in the culture supernatants were
assayed, and then the cells were diluted 1:4 in fresh medium
containing the antiviral agents. The dose of 22 was modified
according to the CPE observed in the cells. If the virus was
replicating (as evidenced by a visible CPE or a high dose of RT
activity), the dose of the agent was increased 5-fold in the
following 1:4 dilution. Cells were diluted as described above
twice a week.27
X-ray Diffraction. Crystals were obtained by slow evaporation
of a cyclohexane solution for 10A and an isooctane solution for
10C at room temperature. X-ray intensities were collected on a
graphite-monochromated CAD-4 Enraf-Nonius diffractometer with
Cu KR radiation (λ ) 1.54178 Å) using θ-2θ scan technique.
Lorentz, polarization, and analytical absorption corrections were
applied. The structure was solved using PLATON (combining
SIR97 software for solving and SHELXL97 for refinement).24 Non-
hydrogen atoms were refined anisotropically, while hydrogen atoms
were calculated geometrically (except NH hydrogen refined on
position and with isotropic displacement parameters).
Biological Assays. Cells and Viruses. The following reagents
were obtained through the AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, NIH. The TZM-bl cell line
was from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme
Inc., and the MT-4 cell line was from Dr. Douglas Richman.
The TZM-bl cells were cultured in DMEM medium (Invitrogen)
supplemented with 5% (v/v) heat-inactivated fetal bovine serum,
50 U of penicillin/mL, and 50 µg of streptomycin/mL (Invitrogen).
Exponentially growing cells were trypsinized, centrifuged, and split
twice weekly. The HTLV-I transformed T4 cell line (MT-4) was
cultured in RPMI 1640 medium (Invitrogen) supplemented with
10% (v/v) heat-inactived fetal bovine serum, 50 U of penicillin/
mL, and 50 µg of streptomycin/mL (Invitrogen). All cells were
grown at 37 °C in a 5% CO2 atmosphere. The HIV strain used in
this study is HIV-1 NL4-3 derived from the infectious molecular
clone pNL4-3. HIV-1 stocks were stored as cell-free supernatants
at -80 °C.
Reverse Transcriptase Assays. Enzyme activities were deter-
mined by following the incorporation of [32P]dGTP into a ho-
mopolymeric template/primer substrate, poly(rC)-oligo(dG)12-18
.
The RT assays using recombinant HIV-1 RT (Calbiochem) were
performed in a final volume of 50 µL. Briefly, the reaction mixture
contained 50 mM Tris-HCl, pH 8.3, 50 mM KCl, 10 mM MgCl2,
2 mM DTT, 10 µg of BSA, 0.01% Triton X100, 20 µg/mL
poly(rC)-oligo(dG)12-18 (Amersham), a fixed concentration of the
labeled substrate (1 µCi per assay [32P]dGTP), a fixed concentration
of the inhibitor (10 µM, dissolved in 1 µL of dimethyl sulfoxide).
After a 10 min of incubation at 37 °C, the reaction mixtures were
chilled on ice and collected on a DEAE-Filtermat (Perkin-Elmer).
After washing with 5% Na2HPO4 and H2O, the amount of
radioactivity was determined by phosphorimager counting. The in
vitro activity of the compounds, at a final concentration of 10 µM,
on the HIV-1 RT activity was derived from the relative reduction
in RT activity. The RT activity in the absence of any compound
was arbitrarily assigned a value of 100% of activity.
Determination of the Amino Acid Sequence of the RT of
Drug-Resistant Virus Strains. CEM cells infected with the HIV-1
mutant strains were subjected to total DNA isolation. The RT
regions in the resistant variants were PCR-amplified, purified, and
sequenced as previously described.27
Acknowledgment. This work was supported by grants from
the DGTRE (Direction Ge´ne´rale des Technologies, de la
Recherche et de l’Energie, Re´gion Wallonne) (Programs WA-
LEO). S.B. thanks F.R.I.A. for financial support. The authors
thank Pr. Dr. Christophe Pannecouque (Rega Institute for
Medical Research, Katholieke Universiteit Leuven, Leuven,
Belgium) for performing the HIV-1 mutant strain selection
experiments and the RT sequence determination of drug-resistant
virus strains.
Anti-HIV in Cultured Cell Lines. Antiviral activity of the
compounds was tested either in the P4 cell line22 or in the TZM-bl
cell line.23 Both cell lines use a Tat protein-induced transactivation
of the ꢀ-galactosidase gene driven by the HIV-1 LTR promoter.
In brief, exponentially growing cells were plated at 5000 cells/
well in flat-bottom 96-well microtiter plates. After 48 h of
incubation, the medium was removed and replaced by 100 µL of
medium containing the pyridinone derivatives at concentrations
ranging from 0.00128 to 4 µM. Four hours after drug addition,
cells were infected with an equal amount of cell-free virus (NL4-3
strain) corresponding to 100 ng of HIV p24 antigen. At 48 h
postinfection, the medium was removed and cells were lysed in
PBS containing 0.1% NP-40 and 5 mM MgCl2. ꢀ-Galactosidase
activity was measured using chlorophenol red ꢀ-D-galactopyranoside
(Roche) assays. The 50% effective concentration (EC50) was
calculated from each dose-response curve using the
CurveExpert1.3 software.25
Supporting Information Available: Experimental and spectro-
scopic data for compounds 1-21 and 24 and HPLC data verifying
the purities of compounds 1-24. This material is available free of
charge via the Internet at http://pubs.acs.org.
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