Synthesis and antiviral activity of 4-benzyl pyridinone derivatives as potent and selective non-nucleoside human immunodeficiency virus type 1 reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jm0009437

Several 4-benzyl analogues of 5-ethyl-6-methyl-4-(phenylthio)pyridin-2(1H)-ones were synthesized and evaluated for their anti-HIV-1 activities. Key transformations include metalation at the 4-C-position of 5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridin

Full text of DOI:10.1021/jm0009437

J . Med. Chem. 2000, 43, 3949-3962
3949
Syn th esis a n d An tivir a l Activity of 4-Ben zyl P yr id in on e Der iva tives a s P oten t
a n d Selective Non -Nu cleosid e Hu m a n Im m u n od eficien cy Vir u s Typ e 1 Rever se
Tr a n scr ip ta se In h ibitor s
Vale´rie Dolle´,† Chi Hung Nguyen,*,† Michel Legraverend,† Anne-Marie Aubertin,‡ Andre´ Kirn,‡
Marie Line Andreola,§ Michel Ventura,§ Laura Tarrago-Litvak,§ and Emile Bisagni†
UMR 176 CNRS-Institut Curie, Section de Recherche, Bitiment 110, 15 rue Georges Climenceau, 91405 Orsay, France,
INSERM U74, Institut de Virologie, 3 rue Koeberle´, 67000 Strasbourg, France, and EP-REGER, CNRS-Universite´ Victor
Segalen Bordeaux 2, IFR 66 Pathologies Infectieuses, 1, rue Camille Saint Sae¨ns, 33077 Bordeaux Cedex, France
Received March 20, 2000
Several 4-benzyl analogues of 5-ethyl-6-methyl-4-(phenylthio)pyridin-2(1H)-ones were synthe-
sized and evaluated for their anti-HIV-l activities. Key transformations include metalation at
the 4-C-position of 5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridine (5) and its coupling
with benzyl bromide or benzaldehyde derivatives. Biological studies revealed that some of the
new 4-benzylpyridinones show potent HIV-1 specific reverse transcriptase inhibitory properties.
Compounds 14, 19, and 27, which inhibit the replication of HIV-1 in CEM-SS cells, with IC50
values ranging from 0.2 to 6 nM are the most active compounds in this series. Biochemical
studies showed that compound 27 strongly inhibited the activity of a recombinant HIV-1 RT.
Moreover, the infectivity of isolated HIV-1 particles was severely decreased after exposure to
compound 27. Although cross resistance is frequently observed between non-nucleoside reverse
transcriptase inhibitors, compound 27 was capable of inhibiting a virus resistant to nevirapine
with an IC50 of 40 nM.
In tr od u ction
Our continuing interest in the search for new non-
nucleoside human immunodeficiency virus type 1 (HIV-
1) specific reverse transcriptase inhibitors led us re-
cently to describe the synthesis and potent biological
activities of a new series of variously substituted 4-
(phenylthio)pyridin-2(1H)-ones 3a ,1 which can be con-
sidered as HEPT-pyridinone2-9 hybrid molecules. How-
ever, chemical instability of 4-(phenylthio)pyridin-2(1H)-
ones derivatives 3a , under basic conditions (for example,
CH3ONa/CH3OH), leads to complex degradation mix-
tures and the loss of the thiophenyl group. Therefore,
our concern was to obtain more chemically stable
analogues in which the sulfur atom would be replaced
by a methylene group (Figure 1, 3b). Since for the
4-phenylthio series, the structure-activity relationship
(SAR) studies showed that the 5-ethyl and 6-methyl
substituents led to the best anti-HIV-1 activities (IC50
F igu r e 1.
values ranging from 6 to 90 nM), 3-substituted-4-benzyl-
described7,10 and a 14% overall yield. Our preliminary
5-ethyl-6-methylpyridinones were selected as new target
molecules.
In this paper we report the synthesis and biological
properties of these 4-benzylpyridinone derivatives 3b.
studies showed that treatment of 5 with n-butyllithium
at 0 °C in the presence of TMEDA (Scheme 1) led to
the lithiated intermediate 6 in quantitative yield.
Surprisingly it did not give efficient condensation with
benzyl bromide. Then we turned to the transformation
of the lithiated derivative 6 into the organocopper
derivative with the CuII:dimethyl sulfide11 complex. The
reaction with benzyl bromide and 3,5-dimethylbenzyl
bromide12,13 generated the expected 4-benzylpyridinones
7a ,b in 53 and 37% yields, respectively. Hydrolysis in
hydrochloric acid at reflux cleaved both methoxy and
pivaloyl groups, giving the pyridinones 8a ,b in good
yields.
Ch em istr y
Starting from pentan-2-one (4), the synthesis of the
intermediate substituted-pyridine 5, bearing a pivaloyl-
amino ortho directing metalation group at its 3-position,
was performed with a 8-step sequence as previously
* Corresponding author: Chi Hung Nguyen. Phone: 33 (0)1 69 86
30 89. Fax: 33 (0)1 69 07 53 81. E-mail: chi.hung@curie.u-psud.fr.
† UMR 176 CNRS-Institut Curie.
Fisher et al.14 reported that organozinc species coupled
efficiently with benzyl bromide in the presence of Pd-
‡ INSERM U74, Institut de Virologie.
§ EP-REGER, CNRS-Universite´ Victor Segalen Bordeaux 2.
10.1021/jm0009437 CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/03/2000

3950 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dolle´ et al.
Sch em e 1a
a
Reagents: (a) n-BuLi (3.5 equiv)/TMEDA (3.5 equiv)/THF/-78 °C to 0 °C; (b) CuII:DMS (3.5 equiv)/THF/-78 °C to 0 °C; (c*)
BrCH2C6H3R1R2 (4.5 equiv)/THF/-78 °C to r.t.; (c**) 3,5-dimethylbenzaldehyde (4.5 equiv)/THF/-78 °C to r.t.; (d) HCl 3 M/H2O/∆reflux to
r.t.; (e) MnO2 (5.0 equiv)/toluene/∆reflux; (f) HCl 3 M/H2O/∆reflux to r.t.
Ta ble 1. Attempts To Improve the Synthesis of 7b from 5
% yield
% recovered
experimental conditions
(expected compound)
starting material
a. n-BuLi (3.5 equiv)/TMEDA (3.5 equiv)/THF/-78 °C to 0 °C
b. ZnCl2 (0.50 equiv)
31
46
c. 3,5-dimethylbenzyl bromide (4.5 equiv)/Pd(PPh3)4 (0.02 equiv)
a. n-BuLi (3.5 equiv)/TMEDA (3.5 equiv)/THF/-78 °C to 0 °C
b. ZnCl2 (1.00 equiv)
c. 3,5-dimethylbenzyl bromide (4.5 equiv)/Pd(PPh3)4 (0.02 equiv)
11.5
30
70.5
67
a. n-BuLi (3.5 equiv)/TMEDA (3.5 equiv)/THF/-78 °C to 0 °C
b. ZnCl2 (0.25 equiv)
c. 3,5-dimethylbenzyl bromide (4.5 equiv)/Pd(PPh3)4 (0.02 equiv)
a. n-BuLi (3.5 equiv)/TMEDA (3.5 equiv)/THF/-78 °C to 0 °C
b. ZnCl2 (0.25 equiv)
7.5
72.5
c. 3,5-dimethylbenzyl bromide (4.5 equiv)/Pd(PPh3)4 (0.06 equiv)
(PPh3)4. Different experiments (summarized in Table 1
and see Experimental Section) were performed from 5
using various proportions of ZnCl2 and Pd(PPh3)4, but
the resulting yield did not exceed 31%. On the contrary,
we observed that our lithiated intermediate 6 coupled
efficiently with 3,5-dimethylbenzaldehyde15 to give 7c
with a 71% yield, avoiding either the use of highly toxic
dimethyl sulfide for the preparation of the organocopper
intermediate or the use of Pd(PPh3)4 for the coupling
with the organozinc compound (Scheme 1). This result
gave an alternative way for obtaining 4-benzylpyridi-
none 7b since it could be prepared from the benzyl
alcohol 7c. For this purpose, several attempts were
performed from 7c. First, treatment of 7c with trieth-
ylsilane and trifluoroacetic acid16 did not afford the
expected compound 7b but the oxazines 9 and 10,
probably resulting from an acid-catalyzed intramolecu-
lar reaction (Scheme 2). The use of zinc powder in acetic
acid17 afforded only a partial deprotection of 7c, giving
compound 11 in a very low yield (5%). Acetylation of 7c
gave acetate 12 in good yield (95%), and an attempt to
replace the acetoxy group by a hydrogen atom was
performed in the presence of triethylsilane in dichlo-
romethane but totally failed (starting material 12 was
recovered). Finally when 12 was subjected to hydro-
genolysis18,19 in the presence of 30% Pd/C under a 10
atm hydrogen pressure, the expected 4-benzyl derivative
7b was obtained almost quantitatively.
To undertake SAR studies in this new series of
3-amino-4-benzylpyridin-2(1H)-one derivatives, the ben-
zyl alcohol and ketone derivative 8c and 14 have been
synthesized. Thus, deprotection of 7c in hydrochloric
acid at reflux led to the pyridinone 8c and oxidation of
7c in the presence of manganese dioxide,20 followed by
deprotection in acidic conditions, provided compound 14
via 13.
With respect to the model of 3-amino-substituted
pyridinone 2b, modifications of the amino function of

Activity of 4-Benzyl Pyridinone Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3951
Sch em e 2a
a
Reagents: (a) Et3SiH (1.2 equiv)/CF3CO2H/r.t.; (b) Zn (6.0 equiv)/AcOH/∆reflux; (c) 1. Ac2O (10.0 equiv)/pyridine/r.t. to 60 °C; 2. Et3SiH
(5.0 equiv)/CH2Cl2/r.t. to ∆reflux; (d) 1. Ac2O (10.0 equiv)/pyridine/r.t. to 60 °C; 2. H2/P ) 10 atm/Pd/C(30%)/AcOH/H2O/dioxane/r.t.
Sch em e 3a
a
Reagents: (a) ClCOCH2CH3 (1.03 equiv)/Et3N (0.95 equiv)/CH2Cl2/0 °C to r.t.; (b) (CH3CO)2O (1.4 equiv)/AcOH/∆reflux; (c) HCO2H/
HCO2Et/∆reflux; (d) LiAlH4 (5.0 equiv)/THF/∆reflux
.
3-amino-4-(3,5-dimethylbenzyl)-5-ethyl-6-methylpyridin-
2(H)-one (8b) have been performed. Thus, under the
usual acylation conditions, using propionyl chloride,
acetic anhydride, and ethyl formate, the amides 15, 17,
and 19 were obtained in 44-66% yields. Reduction of
the formamidopyridinone 19 in the presence of lithium
aluminum hydride led to the methylaminopyridinone
21 in 57% yield (Scheme 3). The carbamate 23 was
synthesized with ethyl chloroformate in 43% yield
(Scheme 4).
We previously describedl that in various conditions
no condensation occurred from 3-amino-4-phenylthiopy-

3952 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dolle´ et al.
Sch em e 4a
a
Reagents: (a) ClCO2CH2CH3 (30.0. equiv)/Et3N (2.5 equiv)/EtOH/∆reflux, to r.t.; (b) 1. 2-methoxy-4,5-dimethylbenzaldehyde (1.0 equiv)/
AcOH/MeOH/r.t.; 2. NaBH4 (2.0 equiv)/MeOH/CHCl3/r.t.; (c) HCHO 37% aq (10.0 equiv)/NaBH3CN (3.0 equiv)/AcOH glacial (6.0 equiv)/
CH3CN/r.t.; (d) BocNHCH2CO2H (7.0 equiv)/DCC (7.0 equiv)/HOBt (7.0 equiv)/NMM (7.7 equiv)/CH2Cl2/0 °C to r.t.; (e) HCI/EtOAc/r.t.
ridinones 3a and 4,5-dimethyl-2-methoxybenzaldehyde.
On the contrary, compound 25 was obtained under the
usual conditions6,21 in 60% yield. In the same way, the
methylation of 8b by the Eschweiler-Clarke reaction22
using formaldehyde and sodium cyanoborohydride, which
was unsuccessful with the 3-amino-4-phenylthiopyridi-
none derivative 3a , afforded the dimethylamino deriva-
tive 27 in 91% yield. Condensation of N-Bocglycine on
amine 8b followed by deprotection gave compound 30
with a 70% overall yield (Scheme 4).
Treatment of diaryl ketone 13 with methyllithium led
to compound 31 (72% yield) which could not be dehy-
drated under the following conditions: when acetic
anhydride23 was used at reflux only the acetamidoace-
tate 33 was obtained with a 9% yield beside 45% of
starting material 31; treatment of 31 with p-toluene-
sulfonic acid led to a partial deprotection to give 34 in
18-66% yields (Scheme 5); treatment of 31 in hydro-
chloric acid at reflux did not afford the expected product
32 but a mixture of the totally deprotected compound
35 and aminopyridinone 36.4 Moreover, we also tried
to prepare compound 32 by using a Wittig reaction from
ketone 13 under various conditions,24-26 but no reaction
occurred and 13 was totally recovered.
respectively (Table 2). Some of the best inhibitors were
tested on a Nevirapine resistant strain (Table 3). Thus
compound 27 exhibited a good anti-HIV activity on this
resistant strain (IC50 ) 40 nM). This last result can be
compared to the activity on the same Nevirapine
resistant strain found for 5-ethyl-6-methyl-3-nitro-4-
[(3,5-dimethylphenyl)thio]pyridin-2(1H)-one,1 which was
6-fold less active (IC50 ) 260 nM). The evaluation of the
antiviral activities was extended to different HIV-1
isolates including an AZT resistant strain, lymphotropic,
and macrophage tropic viruses infecting human primary
cells, peripheral blood mononuclear cells (PBMC), or
monocyte-derived macrophages (Table 4). Compound 27
has, in all cases, the highest activity, inhibiting HIV-1
multiplication in primary cultures at subnanomolar
concentrations. However, none of the molecules were
active against HIV type 2 and, except compound 29,
were not toxic at the highest concentrations tested (100
µM to 1 µM).
Retr ovir u cid a l Effect. It has been reported that
retrotranscription can occur not only in the host cell
cytoplasm but also in the extracellular virion.27-29 This
endogenous reverse transcription seems to be corre-
lated with an increased level of infectivity. Therefore,
inhibition of extracellular retrotranscription would im-
ply that RT inhibitors enter the plasma virions. Recent
work also shows that HIV-1 virions are present in the
blood plasma during all stages of the AIDS disease and,
more particularly, that the life span of a free virion
reaches 7 to 8 h.30 This new set of data strongly suggests
that in addition to HIV-1 infected cells, free virions may
Biologica l Resu lts
In h ibition of HIV-1 Replication . Twenty-five newly
synthesized pyridinones were studied for their anti-
HIV-l activity. Several molecules showed significant
antiviral properties. The most active ones are com-
pounds 27, 19, and 14 with IC50s of 0.2, 3, and 6 nM,

Activity of 4-Benzyl Pyridinone Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3953
Sch em e 5a
a
Reagents: (a) MeLi (3.0 equiv)/Et2O/0 °C to r.t.; (b) Ac2O/∆reflux; (c) p-TsOH (2.5 equiv)/toluene/∆reflux; (d) p-TsOH (10.0 equiv)/xylene/
∆
reflux/Dean-Stark; (e) HCl 3 M/H2O/∆reflux
.
Ta ble 2. Anti-HIV-1 Activity of the Pyridinones on HIV-1 Lai Wild Type in CEM-SS
compd IC50 (nM) CC50 (nM)
SI
compd
IC50 (nM)
CC50 (nM)
SI
compd
IC50 (nM)
CC50 (nM)
SI
AZT
5
3
>100 000
>1 000
>100 000
>1 000
730
>100 000
>100 000
>1 000
>33 333
13
14
>10 000
6
4 600
41 400
100
50
>10 000
>10 000
>10 000
>100 000
>10 000
80 000
>10 000
>100 000
>10 000
207 000
>10 000
-
241
25
7 500
1 000
8
>100 000
>1 000
>13
-
>1 666
-
7a
7b
7c
8a
8b
8c
9
-
-
-
15
>2
>2
2620
27
>100 000
>1 000
161
17
0.2
0.6
>1 000
43 000
86 000
>100 000
>10 000
>10 000
>100 000
>100 000
>5 000
72 000
12
>100
1 600
>3 333
>15
2818
29
30
31
33
>100 000
>10 000
>10 000
>10 000
>10 000
>10 000
>136
>588
>10
-
>2
>1
181
19
6 700
18
>10 000
2 500
12 000
30 000
17
1000
3
>5 555
201
21
6 600
30
1.6
460
-
>10 000
>333
130 000
>21
>4
10
12
4 200
2218
23
34
36
>8
10 000
>3
Ta ble 3. Anti-HIV-1 Activity of the Pyridinones on HIV-1
infectivity of the viral suspension was severely affected,
in a concentration-dependent manner, by compound 27
as shown in Figure 2. In contrast, TIBO R82913, HEPT,
and nevirapine were unable to produce an antiviral
effect even at concentrations as high as 10 µM. Similar
results were recently described by Borkow et al.31 and
by our group32 showing that the infectivity of isolated
HIV-1 particles was inhibited by a non-nucleoside
reverse transcriptase inhibitor.
In h ibition of RT. The effect of these antiviral agents
on recombinant HIV-1 RT activity was analyzed. Reac-
tions were carried out in the presence of poly(C)-oligo-
(dG) as template-primer. The concentration inhibiting
50% of the RT activity (IC50) for each compound is given
in Table 5. The best inhibitor was compound 27 with
an IC50 value very similar to that of the best compound
of the pyridin-2(1H)-ones 3.1 Compounds 8b, 14, and
19 were also able to inhibit HIV-1 RT, but at higher
concentrations, giving IC50 values comparables to those
of nevirapine and 1-[(benzyloxy)methyl]-6-(phenylthio)-
thymine (BPT).
Nevirapine Resistant Strain1b
compd
IC50 (nM)
CC50 (nM)
SI
TIBO R82913
>10 000
1 000
4 500
1 300
>10 000
2 900
1 700
40
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>1 000
-
8b
8c
14
17
19
21
27
>10
>2
>7
-
>3
>5.5
>25
be considered as a potential target for HIV-1 RT
inhibitors.
Therefore, it seemed interesting to investigate whether
4-benzyl pyridinone derivatives 8b, 14, 19, and 27 were
active against free virions. In parallel experiments,
different well-known described nonnucleoside reverse
transcriptase inhibitors (HEPT, TIBO R82913, nevi-
rapine) were also used. Viral preparations were incu-
bated with different concentrations of each inhibitor as
described in the Experimental Section. Although sig-
nificant inhibition was observed with the four pyridi-
none derivatives, compound 27 was the most active. The
It has been previously shown that nonnucleoside RT
inhibitors present different levels of inhibition depend-

3954 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dolle´ et al.
Ta ble 4. Anti-HIV-1 Activity of the Pyridinones on Various HIV Strains and Primary Cell Cultures
IC50 (nM)/CC50 (nM)
compd
HIV-1 IIIB/MT4
HIV-1 AZTres./MT4
HIV-1 IIIB/PBMC
HIV-2 D 194/PBMC
HIV-1 Bal/Mono/Macrophages
8b
8c
14
17
19
21
27
500/>10 000
3 400/10 000
85/>10 000
900/>10 000
26/>10 000
310/>10 000
2.4/>1 000
4 350/>10 000
420/>10 000
25/>10 000
440/>10 000
71/>10 000
76/>10 000
0.2/>1 000
310/>10 000
690/>10 000
20/>10 000
67/>10 000
1.8/>10 000
16/>10 000
0.58/>1 000
>10 000/>10 000
>10 000/>10 000
>10 000/>10 000
>10 000/>10 000
>10 000/>10 000
>10 000/>10 000
>1 000/>1 000
130/>10 000
180/>10 000
17/>10 000
200/>10 000
21/>10 000
720/>10 000
0.004/>1 000
Ki for 27, determined from the replot of the slopes
(Figure 4, inset) was 35 nM.
All nonnucleoside inhibitors thus far identified on the
basis of their activities against HIV-1 do not inhibit RT
from HIV-2. Nevertheless all the compounds were tested
with both RTs. At concentrations where HIV-1 RT was
well inhibited, the activity of HIV-2 RT was not affected
(results not shown). This discriminatory behavior to-
ward HIV-1 vs HIV-2 RT makes these compounds share
another common property of non-nucleoside inhibitors.
To determine a possible synergism in the inhibition
of RT, we investigated the effect of combining 4-benzyl
pyridinone derivatives and nucleoside analogues. Syn-
ergistic inhibition by combination of compound 27 and
AZT-triphosphate was analyzed as described by Caroll
et al.,33 using the method of fractional inhibitory
concentrations.
F igu r e 2. Effect of compound 27 on free virions. The infectiv-
ity of HIV-1 in the presence of the inhibitor was determined
as described in the Experimental Section. 0% represents the
infectivity of virions in the absence of inhibitor.
Preliminary results showed synergistic inhibition
when both inhibitors were used at low concentrations
(Figure 5): an important percentage of the data points
fall below and to the left of the line of additivity.
Ta ble 5. Inhibition of HIV-1 Reverse Transcriptase (RT)a
compd
IC50 (nM)
Discu ssion
nevirapine
200
30
In this work, biological studies were carried out on
various 3,4-disubstituted 5-ethyl-6-methylpyridin-2(1H)-
one derivatives of general formula 3b. Results are
reported in Table 2.
5-ethyl-6-methyl-3-nitro-4-[(3,5-
dimethylphenyl)thio]pyridin-2(1H)-one1
BPT
8b
14
19
27
300
100
100
200
20
From these first SAR studies limited to 25 new 4-C-
aralkylpyridinone derivatives, some general comments
can be made. (a) As previously described1 by us and as
in the HEPT series,18 the methyl groups at the 3- and
5-positions of the 4-benzyl substituent play an impor-
tant role for biological activity. Thus, the nonmethylated
4-benzyl compound 8a displayed a moderate activity
(IC50 ) 730 nM), whereas the 3,5-dimethylbenzyl de-
rivative 8b exhibited a pronounced inhibitory effect (IC50
) 17 nM). (b) When an hydroxy group is present on the
methylene linking group, a striking decrease of biologi-
cal activity was observed: thus, compound 8c is 60-fold
less active than its analogue 8b. On the contrary, when
a ketone function is introduced at this position as it is
the case for ketone 14, the IC50 is improved and reached
6 nM. (c) In all cases, when pyridinones are protected
(2-OMe and 3-NHPiv), no activity was observed (7a , 7b,
7c, 12, 13, 31, 33, 34). (d) Concerning the replacement
of the sulfur atom by a methylene group, when similar
compounds, bearing the same substituent at their
3-position, are compared (compound 8b vs 3-amino-5-
ethyl-6- methyl-4-[(3,5-dimethylphenyl)thio]pyridin-
2(1H)-one,l 15 vs 16, 19 vs 20, and 23 vs 24), the
biological activity is generally maintained or improved.
In the case of the formamido derivative 19, IC50 reached
3 nM compared to 6600 nM for its corresponding
4-thiophenyl analogue 20. This replacement of the
a
RT activity was measured in the presence of poly(C)-oligo(dG),
as described in the Experimental Section.
ing on the template-primer used to measure the RT ac-
tivity. We determined the inhibition produced by these
compounds using different template-primers. The best
inhibition was obtained in the presence of poly(C)-oligo-
(dG) as compared to that obtained with poly(A)-oligo-
(dT).
Besides utilizing synthetic template-primers to mea-
sure the inhibitory effect of compound 27, a system
closer to the native one was used, where the tem-
plate was a fragment of HIV-1 RNA containing the
primer binding site (PBS) sequence, and the primer was
either tRNALys3, the natural primer of HIV-1, or an
oligodeoxynucleotide complementary to the PBS (anti
PBS). In both cases, the addition of compound 27
produced a strong inhibitory effect in a concentration-
dependent manner (Figure 3) with IC50 values of 6-8
nM.
As all derivatives of the present work were targeted
at HIV-1 RT, inhibition studies, carried out with 27,
(Figure 4), show that this optimized compound is a
linear, noncompetitive inhibitor against dGTP in reac-
tions with poly (C)-oligo(dG) as template-primer. The

Activity of 4-Benzyl Pyridinone Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3955
F igu r e 3. Inhibition of cDNA synthesis by HIV-1 RT in the presence of compound 27. Reverse transcription was performed as
described in the Experimental Section. Reactions primed with tRNALys3 yielded a cDNA product of 254 nucleotides; those primed
with anti PBS, a cDNA of 196 nucleotides. Products were analyzed by denaturing polyacryl amide gels. Lanes 1 and 2: complete
system primed with anti PBS, in the absence of compound 27. Lanes 3 to 7: 2, 4, 6, 8, and 10 nM, respectively, of compound 27.
Lanes 8 and 9: complete system primed with tRNALys3. Lanes 10 to 14: 2, 4, 6, 8, and 10 nM, respectively, of compound 27.
F igu r e 4. Inhibition by 27 of reverse transcriptase-catalyzed synthesis. The activity was measured with poly(C)-oligo(dG) as
template-primer and dGTP as substrate. RT was incubated, as described in the Experimental Section, in the absence (0), or in
the presence of 4 nM (b), 10 nM (O), or 20 nM (4) of 27. Data were fit as a double-reciprocal plot according to Lineweaver-Burk.
Inset: the apparent Ki was determined from the secondary plot of slopes versus inhibitor concentration.
arylthio group by an arylmethyl substituent, leading to
more stable pyridinones and to the more reactive
3-amino function in these 4-benzyl analogues, allowed
us to obtain the 3-dimethylaminopyridinone 27 which
is the best compound in this series. (e) When an amino
acid is present on the 3-amino function in the case of
compound 30, the activity is maintained (IC50 ) 18 nM).
On the contrary, when this last compound is still
protected by a tert-butoxycarbonyl group (compound 29),
the biological activity is completely abolished (IC50
reached a value 400-fold higher than that of compound
30). (f) Since these new pyridinones are considered as
[1a ,b (HEPT derivatives)-2a ,b (pyridinones)] hybrid
molecules, we tried to compare activities of some of these
pyridinones with related HEPT or Merck-pyridinone
compounds. Activities of the monomethylamino and
dimethylamino pyridinones 21 and 27 were thus com-
pared to those of the HEPT derivatives 22 and 28. As

3956 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dolle´ et al.
which was capable of inhibiting a virus resistant to
nevirapine with an IC50 of 40 nM, can be considered as
a potent HIV-1 inhibitor which has the unusual capabil-
ity to reach the reverse transcription complex inside the
extracellular virions.
Exp er im en ta l Section
Ch em istr y. TLC were carried out on precoated plates of
silica gel 60F254 (Merck). To reveal the compounds, TLC
plates were exposed to UV-light. Purifications were performed
on silica gel (40-60 µm, SDS) columns by medium pressure
chromatography. In all experiments involving lithium deriva-
tives or amino acid coupling, the glassware was dried in the
oven for a 24 h period before use. Tetrahydrofuran (THF) was
systematically freshly distilled from sodium/benzophenone. All
melting points were measured on an Electrothermal 9200
apparatus and were uncorrected. 1H NMR spectra were
recorded in the given solvents at 294 K (except when mention-
ing) with a Bruker AC 200 or a Bruker AMX 300 apparatus
using the hydrogenated residue of deuterated solvents (CHCl3,
δ ) 7.25 ppm and DMSO, δ ) 2.54 ppm) as internal standards
(*, # ) interchangeable assignments). Chemical shifts (δ) were
reported in ppm units, downfield from TMS (s, d, t, q, m, br
for singlet, doublet, triplet, quadruplet, multiplet, and broad,
respectively), and coupling constants (J ) were given in hertz
(Hz). Elemental analyses, performed by the “Service Central
de Microanalyses du CNRS”, 91190 Gif-sur-Yvette, France,
were within 0.3% of the theoretical values calculated for C,
H, N, O, and Cl. Mass spectra (MS) were obtained on a
NERMAG R10-10-C by direct introduction for the chemical
ionization (IC).
F igu r e 5. Effect of combination of compound 27 and AZT-
triphosphate on HIV-1 RT inhibition. Inhibition of RT activity
by the combination of compound 27 and AZT-TP was deter-
mined by the method of fractional inhibitory concentrations
(FIC). The FIC of each inhibitor was calculated as the
concentration of the inhibitor present in the reaction divided
by the concentration required to give the same degree of
inhibition when the inhibitor was used alone. The right line
indicates additivity of inhibition.
it can be seen for these compounds, IC50 values are of
the same order of magnitude. On the other hand,
compound 25 is 125-fold less active than the pyridinone
26. The bulkiness of the two groups at 3- and 4-positions
probably prevents compound 25 to fit in the allosteric
site of the RT and accounts for this result.
Due to a limited solubility in the culture medium and
as the highest concentrations tested were not toxic, the
selectivity indexes could not be determined.
CAUTION: Since dimethyl sulfide is very toxic and very
irritating for eyes, this reagent must be used very cautiously
with an appropriate respirator.
4-(3,5-Dim eth ylben zyl)-5-eth yl-2-m eth oxy-6-m eth yl-3-
p iva loyla m in op yr id in e (7b). (i) By Lith ia tion of 5: The
starting material 5 and the 3,5-dimethylbenzyl bromide were
dried in the presence of phosphorus pentoxide under vacuum
at room temperature during 24 h. Copper iodide (CuII) was
dried in the presence of phosphorus pentoxide under vacuum
at 50 °C for 24 h. 5-Ethyl-2-methoxy-6-methyl-3-pivaloylami-
nopyridine10 (5) (1.06 g, 4.23 mmol) and freshly distilled (on
calcium hydride) TMEDA (2.24 mL, 14.82 mmol) were dis-
solved in dry THF (26 mL), and the mixture was cooled at -78
°C under a nitrogen atmosphere. n-Butyllithium (1.6 M in
hexane, 9.26 mL, 14.82 mmol) was added dropwise. The
mixture was stirred for 1 h at 0 °C. An orange-yellow
precipitate was observed. On another hand, the CuII:dimethyl
sulfide complex was prepared at -78 °C under a nitrogen
atmosphere by addition of dimethyl sulfide (14.00 mL, 190.54
mmol) to a suspension of copper iodide (2.82 g, 14.82 mmol)
in dry THF (52 mL). The CuII:dimethyl sulfide complex was
then added dropwise to the mixture at -78 °C. The black
mixture was stirred at 0 °C for a 30 min period and cooled
again at -78 °C to allow the addition of 3,5-dimethylbenzyl
bromide4,5 (3.81 g, 19.05 mmol) dissolved in THF (4 mL). The
resulting mixture was stirred at 0 °C for 3 h and at room
temperature for 12 h. Water (16 mL) and 28% aqueous
ammonium hydroxide solution (20 mL) were added. The blue
aqueous layer was extracted with 3 × 80 mL of ether. The
combined organic layers were washed with 40 mL of brine,
dried over magnesium sulfate, and concentrated under reduced
pressure. The residue was purified by column chromatography
using cyclohexane-ethyl acetate (1:0 to 8:2) as eluent, giving
7b (577 mg, 37%) as a white solid and 544 mg (51%) of
Con clu sion
This work led us to elaborate two different ways to
obtain 4-benzyl-3,5,6-trisubstituted pyridin-2(1H)-one
derivatives. These compounds constitute a new series
of potent non-nucleoside HIV-1 RT inhibitors related
both to HEPT and Merck-pyridinone series and chemi-
cally more stable than their 4-phenylthio analogues.
Biological studies revealed that some of new 4-ben-
zylpyridinones show potent HIV-1 specific reverse tran-
scriptase inhibitory properties. Compounds 14, 19, and
27, which inhibit the replication of HIV-1 in CEM-SS
cells, with IC50 values ranging from 0.2 to 6 nM are the
most active compounds in this series. They are very
efficient in blocking the replication of lymphotropic and
macrophage tropic isolates in primary lymphocytes or
monocyte-derived macrophages. Biochemical studies
showed that compound 27 strongly inhibited the activity
of a recombinant HIV-1 RT, with IC50 values of 6-8 nM.
The new 4-benzyl derivatives showed different levels of
inhibition depending on the synthetic template-primer
used. Whereas better levels of inhibition were obtained
with poly(C)-oligo(dG) as template-primer, as compared
to poly(A)-oligo(dT), enzyme kinetic analysis of RT
inhibition by these compounds indicated that they were
noncompetitive with respect to the substrate dGTP, and
the compounds were shown to be specifically active
against HIV-1 RT, but not against HIV-2 RT. These
4-benzylpyridinones are therefore HIV-1 specific non-
competitive RT inhibitors which share similar properties
with other non-nucleoside inhibitors of this enzyme.
Moreover, besides inhibiting HIV-1 replication in cell
culture, compound 27 is able to strongly decrease the
infectivity of isolated HIV-1 particles. This compound,
1
starting material 5. 7b: mp 138-139 °C; H NMR (CDCl3) δ
1.02 (3H, t, J ) 7.4 Hz, CH3CH2), 1.19 (9H, s, COC(CH3)3),
2.21 (6H, s, CH3-3′ and 5′), 2.46 (3H, s, CH3-6), 2.57 (2H, q, J
) 7.6 Hz, CH3CH2), 3.89 (5H, s, CH3O and CH2C6H5), 6.56 (2H,
s, H-2′ and 6′), 6.78 (s, 1H, H-4′). Anal. (C23H32N2O2‚0.25H2O)
C, H, N.
(ii) By Hyd r ogen olysis of 12: A mixture of (+,-)-1-(5-
ethyl-2-methoxy-6-methyl-3- pivaloylaminopyridin-4-yl)-1-(3,5-

Activity of 4-Benzyl Pyridinone Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3957
dimethylphenyl)methyl acetate (12) (850 mg, 1.99 mmol) and
Pd-C (30%, 850 mg) in acetic acid-water-dioxane (42.5 mL,
2:1:2, v/v/v) was stirred at room temperature for 24 h under
10 atm of hydrogen. The catalyst was removed by filtration
and washed with ethanol. The combined filtrates were con-
centrated to dryness under reduced pressure, giving 7b (726
mg, 99%) as a white solid which was identical to that described
above.
2-methoxy-6-methyl-3-pivaloylaminopyridin-4-yl)-methanol (7c)
(200 mg, 0.52 mmol), triethylsilane (0.10 mL, 0.62 mmol), and
trifluoroacetic acid (1 mL) was stirred at room temperature
for 27 h (the flask was fitted with a calcium chloride drying
tube) and evaporated to dryness under reduced pressure. To
the residue were added 10 mL of water. The aqueous layer
was basified by addition of concentrated ammonium hydroxyde
and extracted with 3 × 20 mL of dichloromethane. The
combined organic layers were washed with 10 mL of brine,
dried over magnesium sulfate, and concentrated under reduced
pressure. The residue was purified by column chromatography
using dichloromethane as eluent to give 53 mg (26.5%) of 10
as a yellow oil and 125 mg (65%) of 9 as a yellow solid. 9: mp
111-1 12 °C; 1H NMR (CDCl3) δ 0.87 (3H, t, J ) 7.5 Hz, CH3-
CH2), 1.03 (9H, s, C(CH3)3), 2.18-2.30 (8H, m, CH3CH2, CH3-
3′ and CH3-5′), 2.40 (3H, s, CH3-6), 4.03 (3H, s, OCH3), 6.14
(1H, s, H-4), 6.78 (2H, s, H-2′ and 6′), 6.91 (1H, s, H-4′); MS
(IC): 384 [M + NH4]+, 367 [M + H]+; 309. Anal. (C23H30N2O2‚
0.25H2O) C, H, N, 0. 10: 1H NMR (CDCl3) δ 1.05 (3H, t, J )
7.2 Hz, CH3CH2), 1.29 (9H, s, C(CH3)3), 2.27 (6H, s, CH3-3′
and 5′), 2.42 (3H, s, CH3-6), 2.57-2.80 (2H, m, CH3CH2), 3.96
(4H, s, OCH3 and OH), 6.79 (2H, s, H-2′ and 6′), 6.92 (1H, s,
H- 4′), 7.18 (1H, s, H-4); MS (IC): 385 [M + H]+; 367; 283.
Anal. (C23H32N2O3) C, H, N.
(+,-)-1-(3,5-Dim et h ylp h en yl)-1-(5-et h yl-2-m et h oxy-6-
m eth yl-3-p iva loyla m in op yr id in -4-yl)m eth a n ol (7c). The
starting materials were dried in the presence of calcium
chloride under vacuum at room temperature during 12 h.
5-Ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridine10 (5) (620
mg, 2.47 mmol) and freshly distilled (on calcium hydride)
TMEDA (1.31 mL, 8.67 mmol) were dissolved in dry THF (15
mL), and the mixture was cooled at -78 °C under a nitrogen
atmosphere. n-Butyllithium (1.6 M in hexane, 5.42 mL, 8.67
mmol) was added dropwise. The mixture was stirred for 1 h
at 0 °C. The formation of an orange precipitate was observed.
The mixture was cooled again at -78 °C to allow the addition
of 3,5-dimethylbenzaldehyde15 (1.49 g, 11.14 mmol), dissolved
in dry THF (30 mL). The resulting mixture was stirred at 0
°C for 2 h and at room temperature for 12 h. The organic layer
was washed with 30 mL of water. The aqueous layer was
extracted with 3 × 60 mL of ether. The combined organic
layers were washed with 30 mL of brine, dried over magne-
sium sulfate, and concentrated under reduced pressure. The
residue was washed with 10 mL of cyclohexane to give the
product 7c (570 mg) after filtration. The filtrate was evapo-
rated, and the residue was purified by column chromatography
using cyclohexanes-ethyl acetate (1:0 to 8:2) as eluent giving
the product 7c (104 mg, total yield ) 71%) as a white solid
and 72 mg (12%) of starting material 5. 7c: mp 184-185 °C;
1H NMR (CDCl3) δ 1.00 (3H, t, J ) 7.4 Hz, CH3CH2), 1.09 (9H,
s, COC(CH3)3), 2.24 (6H, s, CH3-3′ and 5′), 2.47 (3H, s, CH3-
6), 2.68 (2H, m, CH3CH2), 3.90 (3H, s, CH3O), 4.94 (1H, d, J )
7.7 Hz, OH), 5.94 (1H, d, J ) 7.4 Hz, CH), 6.81 (4H, br s, H-2′,
4′, 6′ and NH). Anal. (C23H32N2O3) C, H, N, O.
3-Am in o-4-(3,5-d im eth ylben zyl)-5-eth yl-6-m eth ylp yr i-
d in -2(1H)-on e (8b). A 3 M aqueous hydrochloric acid solution
(150 mL) was added to a suspension of 4-(3,5-dimethylbenzyl)-
5-ethyl- 2-methoxy-6-methyl-3-pivaloylaminopyridine (7b) (2.36
g, 6.41 mmol) in water (300 mL). The mixture was refluxed
for 3.5 h and then stirred at room temperature for 12 h. The
solution was basified by addition of concentrated ammonium
hydroxyde and extracted with 3 × 800 mL of ethyl acetate.
The combined organic layers were washed with 110 mL of
brine, dried over magnesium sulfate, and concentrated under
reduced pressure giving 8b (1.79 g, ≈100%) as a light orange
solid: mp 204-205 °C; 1HNMR (CDCl3) δ 1.00 (3H, t, J ) 7.5
Hz, CH3CH2), 2.25 (6H, s, CH3-3′ and 5′), 2.27 (3H, s, CH3-6),
2.39 (2H, q, J ) 7.5 Hz, CH3CH2), 3.81 (4H, br s, CH2C6H5
and NH2), 6.72 (2H, s, H-2′ and 6′), 6.83 (1H, s, H-4′), 10.65
(1H, br s, NH-1). Anal. (C17H22N2O‚0.2H2O) C, H,N,O.
4-(3,5-Dim eth ylben zyl)-5-eth yl-6-m eth yl-3-pivaloylam i-
n op yr id in -2(1H)-on e (11). Zinc powder (127 mg, 1.95 mmol)
was added to a solution of (+,-)-l-(3,5-dimethylphenyl)-1-(5-
ethyl-2- methoxy-6-methyl-3-pivaloylaminopyridin-4-yl)-metha-
nol (7c) (300 mg, 0.78 mmol) in acetic acid (2.50 mL). The
heterogeneous mixture was refluxed for 4.5 h. An additional
127 mg of zinc powder (1.95 mmol) was still added, and the
mixture was again refluxed for 22 h, then filtered. The solvent
was evaporated under reduced pressure and the residue was
taken up in 20 mL of ethyl acetate. The organic layer was
washed with 10 mL of an aqueous saturated sodium bicarbon-
ate solution, 10 mL of water, and 10 mL of brine, dried over
magnesium sulfate, and concentrated under pressure. The
residue was purified by column chromatography using dichlo-
romethane-ethanol (100:0 to 95:5) to give 11 (40 mg, 5%) as
1
a yellow solid: mp 237-238C; H NMR (CDCl3) δ 0.95 (3H, t,
J ) 7.4 Hz, CH3CH2), 1.23 (9H, s, COC(CH3)3), 2.23 (6H, s,
CH3-3′ and 5′), 2.33 (3H, s, CH3-6), 2.41 (2H, q, J ) 7.6 Hz,
CH3CH2), 3.90 (2H, s, CH2), 6.60 (2H, s, H-2′ and 6′), 6.80 (1H,
s, H-4′), 7.17 (1H, br s, NH-1). Anal. (C22H30N2O2‚0.9H2O‚0.9
EtOH) C, N, O.
(+,-)-1-(5-Eth yl-2-m eth oxy-6-m eth yl-3-pivaloylam in opy-
r id in -4-yl)-1-(3,5- d im eth ylp h en yl)m eth yl a ceta te (12). A
total of 8.34 g (21.70 mmol) of (+,-)-1-(3,5-dimethylphenyl)-
1-(5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridin-4-yl)-
methanol (7c) was dissolved in pyridine (200 mL) and added
to acetic anhydride (10.24 mL, 108.51 mmol), and the solution
was stirred for 1.5 h at room temperature and for 60 h at 60
°C. An additional 10.24 mL of acetic anhydride (108.51 mmol)
was added, and heating was continued at 60 °C for 24 h. The
pyridine was evaporated under reduced pressure, and the
residue was taken up in 500 mL of ethyl acetate. The organic
layer was washed with 170 mL of an aqueous saturated
sodium bicarbonate solution, 170 mL of water, and 170 mL of
brine, dried over magnesium sulfate, and concentrated to
dryness. The residue was purified by column chromatography
using dichloromethane-ethanol (100:0 to 95:5) to give 12 (8.78
g, 95%) as a white mass: mp 70-71 °C; 1H NMR (CDCl3) δ
0.88 (3H, t, J ) 7.4 Hz, CH3CH2), 1.11 (9H, s, COC(CH3)3),
2.17 (3H, s, OCOCH3), 2.24 (6H, s, CH3-3′ and 5′), 2.45 (3H, s,
CH3-6), 2.52-2.68 (2H, m, CH3CH2), 3.92 (3H, s, OCH3), 6.75
(2H, s, H-2′ and 6′), 6.87 (1H, s, H-4′), 7.15 (1H, br s, NH),
7.18 (1H, s, CH). Anal. (C25H34N2O4) C, H, N.
(+,-)-3-Am in o-4-[1-(3,5-d im et h ylp h en yl)-1-h yd r oxy]-
m eth yl-5-eth yl-6-m eth ylp yr id in -2(1H)- on e (8c). Starting
from (+,-)-1-(3,5-dimethylphenyl)-1-(5-ethyl-2-methoxy-6-
methyl-3- pivaloylaminopyridin-4-yl)-methanol (7c) (150 mg,
0.39 mmol) and 3 M aqueous hydrochloric acid (9 mL), the
reaction was performed as above (for 8b). The residue was
purified by column chromatography using dichloromethane-
ethanol (100:0 to 95:5) as eluent giving the starting material
7c (70 mg, 47%) and 8c (19 mg, 17%) as white crystals (after
1
recrystallization from dichloromethane): mp 238-239 °C; H
NMR (DMSO-d6) δ 0.94 (3H, t, J ) 7.0 Hz, CH3CH2), 2.13 (3H,
s, CH3-6), 2.25 (6H, s, CH3-3′ and 5′), 2.32 (2H, m, CH3CH2),
4.84 (2H, br s, NH2), 5.80 (1H, s, CH), 6.08 (1H, s, OH), 6.87
(1H, s, H-4′), 6.96 (2H, s, H-2′ and 6′), 11.26 (1H, br s, NH-1).
Anal. (C17H22N2O2‚0.25H2O) C, H, N, O.
1-(3,5-Dim eth ylp h en yl)-l-(5-eth yl-2-m eth oxy-6-m eth yl-
3-p iva loyla m in op yr id in -4-yl)m eth a n on e (13). A solution
of (+,-)-1-(3,5-dimethylphenyl)-1-(5-ethyl-2-methoxy-6-meth-
yl-3-pivaloylaminopyridin-4-yl)-methanol (7c) (1.66 g, 4.32
mmol) in 80 mL of toluene was heated at reflux. Manganese
dioxide (1.88 g, 21.58 mmol) in suspension in 40 mL of toluene
was added, and the mixture was then stirred at reflux for 6 h.
(+,-)-2-t er t -Bu t yl-4-(3,5-d im e t h ylp h e n yl)-5-e t h yl-8-
m eth oxy-6-m eth yl-4H-p yr id o[3,4-d ]-m -oxa zin e (9) a n d
(+,-)-2-ter t-Bu tyl-4-(3,5-d im eth ylp h en yl)-5-eth yl-8-m eth -
oxy-6-m et h yl-2,4-d ih yd r o-1H -p yr id o[4,3-d ]-m -oxa zin -2-
ol (10). A mixture of (+,-)-1-(3,5-dimethylphenyl)-1-(5-ethyl-

3958 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dolle´ et al.
The catalyst was filtered off, and the solvent was evaporated
under reduced pressure. The residue was purified by column
chromatography using dichloromethane-ethyl acetate (100:0
to 95:5) as eluent giving 13 (1.48 g, 89.5%) as a white solid:
(0.39H, d, J ) 12.0 Hz, NHCHO strans), 9.23 (0.61H, s,
1
NHCHO scis), 11.71-11.81 (1H, m, NH-1); H NMR (DMSO-
d6; 360 K) δ 0.89 (3H, t, J ) 6.0 Hz, CH3CH2), 2.25 (9H, s,
CH3-3′, 5′ and 6), 2.35 (2H, q, J ) 6.0 Hz, CH3CH2), 3.88 (1H,
s, CH2), 6.74 (2H, s, H-2′ and 6′), 6.84 (1H, s, H-4′), 8.16 (1H,
s, NHCHO), 8.67 (1H, br s, NHCHO), 11.38 (1H, br s, NH-1).
Anal. (C18H22N2O2‚0.25H2O) C, H, N.
1
mp 143-144 °C; H NMR (CDCl3) δ 0.88 (9H, s, COC(CH3)3),
0.97 (3H, t, J ) 7.3 Hz, CH3CH2), 2.27 (6H, s, CH3-3′ and 5′),
2.28-2.45 (2H, m, CH3CH2), 2.46 (3H, s, CH3-6), 3.92 (3H, s,
CH3O), 6.78 (1H, s, NH), 7.14 (1H, s, H-4′), 7.38 (2H, s, H-2′
and 6′). Anal. (C23H30N2O3‚0.25H2O) C, H, N.
4-(3,5-Dim eth ylben zy)-5-eth yl-3-m eth yla m in o-6-m eth -
ylp yr id in -2(1H)-on e (21). Lithium aluminum hydride (25
mg, 0.67 mmol) was cautiously added to a suspension of 4-(3,5-
dimethylbenzyl)-5-ethyl-3-formamido-6-methylpyridin-2(1H)-
one (19) (100 mg, 0.33 mmol) in dry THF (6 mL). The mixture
was heated at reflux for 2 h. An additional 38 mg (1.00 mmol)
of lithium aluminum hydride was added, and stirring was
continued for 2 h (the flask was fitted with a calcium chloride
drying tube). A total of 10 mL of water was slowly added. The
two layers were separated and the aqueous layer was acidified
by addition of a 3 M aqueous hydrochloric acid solution and
then extracted with 3 × 10 mL of ether. The combined organic
layers were washed with 5 mL brine, dried over magnesium
sulfate, and concentrated under reduced pressure. The residue
was purified bycolumn chromatographyusingdichloromethane-
ethanol (1:0 to 95:5) as eluant, giving 21 (54 mg, 57%) as a
light brown solid (after washing with cyclohexane and drying):
3-Am in o-4-(3,5-d im eth ylben zoyl)-5-eth yl-6-m eth ylp y-
r id in -2(1H)-on e (14). Starting from 1-(3,5-dimethylphenyl)-
1-(5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridin-4-yl)-
methanone (13) (400 mg, 1.05 mmol) and 3 M aqueous
hydrochloric acid (24 mL), the reaction was performed as above
(for 8b). The residue was purified by column chromatography
using dichloromethane-ethanol (100:0 to 95:5) as eluent giving
75 mg (19%) of starting material 13 and 191 mg (64%) of 14
as a yellow solid: mp 212-213 °C; 1H NMR (CDCl3) δ 0.89
(3H, t, J ) 7.2 Hz, CH3CH2), 2.19 (2H, q, J ) 7.5 Hz, CH3CH2),
2.30 (3H, s, CH3-6), 2.34 (6H, s, CH3-3′ and 5′), 4.11 (2H, br s,
NH2), 7.24 (1H, s, H-4′), 7.50 (2H, s, H-2′ and 6′), 12.85 (1H,
br s, NH-1). Anal. (C17H20N2O2‚0.45H2O) C, H, N.
4-(3,5-Dim e t h ylb e n zyl)-5-e t h yl-6-m e t h yl-3-p r op ion -
a m id op yr id in -2(1H)-on e (15). To a solution of 3-amino-4-
(3,5-dimethylbenzyl)-5-ethyl-6-methylpyridin-2(1H)-one (8b )
(200 mg, 0.74 mmol) and triethylamine (97.9 µL, 0.70 mmol)
in dichloromethane (9 mL) was added freshly distilled propio-
nyl chloride (65.9 µL, 0.76 mmol) dropwise at 0 °C (the flask
was fitted with a calcium chloride drying tube). The mixture
was stirred at room temperature during 3.5 h. The solvent was
evaporated under reduced pressure, 10 mL of water was added,
and the solid was filtered off. After washings with 10 mL of
cyclohexane and drying in the presence of calcium chloride
under vacuum, 133 mg (55%) of 15 was obtained as a light
beige solid: mp 246-247 °C; 1H NMR (CDCl3) δ 0.95 (3H, t, J
) 7.4 Hz, CH3CH2), 1.16 (3H, t, J ) 7.1 Hz, CH3CH2CO), 2.23-
2.38 (13H, m, CH3CH2, CH3CH2CO, CH3-3′, 5′ and 6), 3.91 (2H,
s, CH2), 6.59 (2H, s, H-2′ and 6′), 6.80 (1H, s, H-4′), 6.89 (1H,
br s, NHCOCH2CH3), 11.60 (1H, br s, NH-1). Anal. (C20H26N2O2‚
0.25H2O) C, H, N, O.
3-Acetyla m in o-4-(3,5-d im eth ylben zyl)-5-eth yl-6-m eth -
ylp yr id in -2(1H)-on e (17). A solution of the 3-amino-4-(3,5-
dimethylbenzyl)-5-ethyl-6-methylpyridin-2(1H)-one (8b) (200
mg, 0.74 mmol) and acetic anhydride (106 mg, 1.03 mmol) in
acetic acid (40 mL) was heated at reflux for 2 h. After
evaporation of the volatile materials under reduced pressure,
20 mL of ice water was added, and the mixture was neutralized
at 0 °C with diluted ammonium hydroxyde. After filtration,
the residue was washed with 10 mL of cyclohexane giving 17
(101 mg, 44%) as a beige solid: mp 234-235 °C; 1H NMR
(CDCl3) δ 0.94 (3H, t, J ) 7.2 Hz, CH3CH2), 2.08 (3H, s, CH3-
CO), 2.23 (9H, s, CH3-6, 3′ and 5′), 2.31 (2H, q, J ) 7.3 Hz,
CH3CH2), 3.90 (2H, s, CH2C6H5), 6.60 (2H, s, H-2′ and 6′), 6.81
(1H, s, H-4′), 6.88 (1H, br s, NHCOCH3), 10.90 (1H, br s, NH-
1). Anal. (C19H24N2O2‚0.25H2O) C, H,O.
4-(3,5-Dim et h ylb en zyl)-5-et h yl-3-for m a m id o-6-m et h -
ylp yr id in -2(1H)-on e (19). To a solution of 3-amino-4-(3,5-
dimethylbenzyl)-5-ethyl-6-methylpyridin-2(1H)-one (8b) (400
mg, 1.47 mmol) in ethyl formate (previously distilled over
calcium hydride) (30 mL) was added formic acid (7 mL). The
mixture was heated under reflux for 3 h. After evaporation of
the volatile materials, the residue was washed with ethanol
and dried in the presence of calcium chloride during 12 h under
vacuum at room temperature to give 255 mg of the expected
product 19. The filtrate was concentrated under reduced
pressure, and the resulting residue was purified by column
chromatography using dichloromethane-ethyl acetate-etha-
nol (100:0:0 to 47.5:47.5:5) as eluent to give 35 mg (total yield
) 66%) of 19 as white crystals (after recrystallization from
ethanol): mp 246-247 °C; 1H NMR (DMSO-d6; 294 K) δ 0.83-
0.85 (3H, m, CH3CH2), 2.20-2.29 (11H, m, CH3CH2, CH3-3′,
5′ and 6), 3.78 (0.61H, s, CH2), 3.89 (0.39H, s, CH2), 6.71-
6.74 (2H, m, H-2′ and 6′), 6.83 (1H, s, H-4′), 8.08 (0.39H, d, J
) 12.0 Hz, NHCHO strans), 8.17 (0.61H, s, NHCHO scis), 8.94
1
mp 153-154 °C; H NMR (CDCl3) δ 0.93 (3H, t, J ) 7.4 Hz,
CH3CH2), 2.25-2.30 (11H, m, CH3CH2, CH3-3′, 5′ and 6), 2.66
(3H, s, NHCH3), 3.98 (2H, s, CH2), 6.60 (1H, br s, NHCH3),
6.72 (2H, s, H-2′ and 6′), 6.81 (1H, s, H-4′), 12.84 (1H, br s,
NH-1). Anal. (C18H24N2O‚0.2H2O) C, H, N.
4-(3,5-Dim eth ylben zyl)-3-eth oxycar bon ylam in o-5-eth yl-
6-m eth ylp yr id in -2(1H)-on e (23). Triethylamine (0.26 mL,
1.85 mmol) was added to a solution of 3-amino-4-(3,5-dimeth-
ylbenzyl)-5-ethyl-6-methylpyridin-2(1H)-one (8b) (200 mg, 0.74
mmol) in ethanol (7 mL). To this mixture, cooled in ice water,
was added dropwise freshly distilled ethyl chloroformate (2.12
mL, 22.19 mmol), and the mixture was stirred at room
temperature for 3 h, at reflux for 4 h, and then at room
temperature for 96 h. After evaporation of the solvent under
reduced pressure, 10 mL of water was added. After filtration
of the precipitate and washings with 10 mL of cyclohexane,
the solid was purified by column chromatography using
dichloromethane-ethanol (100:0 to 95:5) as eluent to give the
recovered amine 8b (10 mg, 5%) and 23 (108 mg, 43%) as a
1
yellow light solid: mp 192-193 °C; H NMR (CDCl3) δ 0.92
(3H, t, J ) 7.3 Hz, CH3CH2), 1.24 (3H, t, J ) 7.0 Hz, CH3CH2-
CO2), 2.23-2.36 (11H, m, CH3CH2, CH3-3′, 5′ and 6), 3.95 (2H,
s, CH2), 4.14 (2H, d, J ) 7.4 Hz, CH3CH2CO2), 6.15 (1H, br s,
NHCO2CH2CH3), 6.62 (2H, s, H-2′ and 6′), 6.80 (1H, s, H- 4′),
12.25 (1H, br s, NH-1). Anal. (C20H26N2O3‚0.1H2O) C, H, N.
4-(3,5-Dim eth ylben zyl)-3-[N-(4,5-d im eth yl-2-m eth oxy-
b en zyl)a m in o]-5-et h yl-6-m et h ylp yr id in -2(1H )-on e (25).
To a solution of 3-amino-4-(3,5-dimethylbenzyl)-5-ethyl-6-
methylpyridin-2(1H)-one (8b) (200 mg, 0.74 mmol) and of 4,5-
dimethyl-2-methoxybenzaldehyde6,21 (121.5 mg, 0.74 mmol) in
10 mL of methanol was added a drop of glacial acetic acid.
The mixture was stirred at room temperature for 2.4 h. The
yellow precipitate was filtered off and dissolved in 10 mL of a
mixture of methanol-chloroform (1:1, v/v), and 56 mg of
sodium borohydride (1.48 mmol) was added. A discoloration
of the solution was observed. The solvents were evaporated
under reduced pressure, and the resulting residue was taken
up in 50 mL of dichloromethane. The organic layer was washed
with 2 × 10 mL of water and 10 mL of brine, dried over
magnesium sulfate, and concentrated under reduced pressure
to give 25 (185 mg, 60%) as a yellow light solid: mp 179-180
1
°C; H NMR (DMSO-d6) δ 0.86 (3H, t, J ) 7.1 Hz, CH3CH2),
2.10-2.22 (17H, m, CH3CH2, CH3-3′, 4", 5′, 5" and 6), 3.68 (3H,
s, CH3O), 3.87 (2H, s, CH2), 3.96 (2H, d, J ) 6.9 Hz, NHCH2),
4.53 (1H, t, J ) 7.0 Hz, NHCH2), 6.70 (2H, s, H-2′ and 6′),
6.74 (1H, s, H-6′′), 6.84 (2H, s, H-3′′ and 4′), 11.30 (1H, br s,
NH-1). Anal. (C27H32N2O2‚1.75H2O) C, H, N, O.
3-Dim e t h yla m in o-4-(3,5-d im e t h ylb e n zyl)-5-e t h yl-6-
m eth ylp yr id in -2(1H)-on e (27). To a stirred solution of
3-amino-4-(3,5-dimethylbenzyl)-5-ethyl-6-methylpyridin-2(1H)-

Activity of 4-Benzyl Pyridinone Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3959
one (8b) (200 mg, 0.74 mmol) and 37% aqueous formaldehyde
(0.60 mL, 7.39 mmol) in 5 mL of acetonitrile was added 139
mg (2.22 mmol) of sodium cyanoborohydride. Glacial acetic acid
(0.07 mL, 2.22 mmol) was added dropwise, and the reaction
mixture was stirred at room temperature for 2 h. An additional
0.07 mL (2.22 mmol) of glacial acetic acid was added, and
stirring was continued for 30 min. The solvent was evaporated,
and 15 mL of ether was added to the resulting residue. The
organic layer was washed with 3 × 30 mL of 1 N aqueous
potassium hydroxide and 3 mL of brine, dried over magnesium
sulfate, and concentrated under reduced pressure to give 27
(200 mg, 91%) as a yellow solid: mp 229-230 °C; 1H NMR
(CDCl3) δ 0.89 (3H, t, J ) 7.3 Hz, CH3CH2), 2.24-2.29 (11H,
m, CH3CH2, CH3-3′, 5′ and 6), 2.73 (6H, s, N(CH3)2), 4.10 (2H,
s, CH2), 6.66 (2H, s, H-2′ and 6′), 6.78 (1H, s, H-4′), 11.42 (1H,
br s, NH-1). Anal. (C19H26N2O‚0.3H2O) C, H, N.
mmol) in 20 mL of anhydrous ether was added methyllithium
(1.6 M in ether, 1.96 mL, 3.14 mmol) dropwise under a nitrogen
atmosphere at 0 °C. The mixture was stirred at this temper-
ature during 2 h and then at room temperature for 12 h. The
reaction was quenched by addition of 20 mL of water. The two
layers were separated, and the aqueous layer was extracted
with 3 × 40 mL of ether. The combined organic layers were
washed with 20 mL of brine, dried over magnesium sulfate,
and concentrated under reduced pressure. The residue was
purified by column chromatography using dichloromethane-
ethanol (99:1 to 95:5) as eluent giving 31 (302 mg, 72.5%) as
1
a white solid: mp 159-160 °C; H NMR (CDCl3) δ 0.96 (9H,
s, COC(CH3)3), 1.02 (3H, t, J ) 7.4 Hz, CH3CH2), 2.17 (3H, s,
CH3-3′ or 5′*), 2.28 (3H, s, CH3-5′ or 3′*), 2.39-2.48 (8H, m,
CH3-6, CH3CH2 and CH3C(OH)), 3.93 (3H, s, OCH3), 6.77 (1H,
s, H-4′), 6.97 (1H, s, H-2′ or 6′#), 7.08 (1H, s, H-6′ or 2′#). Anal.
(C24H34N2O3) C, H, N, O.
3-[N-(ter t-Bu toxyca r bon yl)glycyl]a m in o-4-(3,5-d im eth -
ylb en zyl)-5-et h yl-6-m et h ylp yr id in -2(1H )-on e (29). The
starting materials were dried in the presence of calcium
chloride under vacuum at room temperature, and the 1-hy-
droxybenzotriazole hydrate was dehydrated in the presence
of calcium chloride at 70 °C under vacuum for 12 h. The 1,3-
dicyclohexylcarbodiimide (4.81 g, 23.30 mmol) was added to
the mixture, cooled at 0 °C, of dehydrated 1-hydroxybenzo-
triazole (3.15 g, 23.30 mmol, commercial) and N-(tert-butoxy-
carbonyl)glycine (4.08 g, 23.30 mmol) in 25 mL of anhydrous
dichloromethane. A slight exothermic reaction occurred, and
the formation of a white precipitate was observed. The mixture
was stirred at 0 °C under a nitrogen atmosphere for 30 min.
Then the 3-amino-4-(3,5-dimethylbenzyl)-5-ethyl-6-methylpy-
ridin-2(1H)-one (8b) (0.90 g, 3.33 mmol), dissolved in 25 mL
of dichloromethane, was added dropwise. After 5 min, freshly
distilled (barium oxide), N-methylmorpholine (2.82 mL, 25.63
mmol) was added. The resulting mixture was stirred at 0 °C
for 1.5 h and at room temperature for 3.5 h. The 1,3-
dicyclohexylurea was filtered off, and the solvent was evapo-
rated under reduced pressure. The residue was taken up in
250 mL of ethyl acetate. This organic layer was washed
successively with a saturated aqueous sodium bicarbonate
solution (2 × 50 mL), a saturated aqueous citric acid solution
(50 mL), water (50 mL), and brine (50 mL). It was then dried
over magnesium sulfate and concentrated in vacuo. The
residue was purified by column chromatography using dichlo-
romethane-ethanol (98:2 to 95:5) as eluent giving 29 (1.00 g,
70.5%) as a yellow light solid: mp 184-185 °C; 1H NMR
(CDCl3) δ 0.92 (3H, t, J ) 7.0 Hz, CH3CH2), 1.38 (9H, s, COOC-
(CH3)3), 2.22 (6H, s, CH3-3′ and 5′), 2.31 (3H, s, CH3-6), 2.35
(2H, q, J ) 7.0 Hz, CH3CH2), 3.88 (4H, s, 2 CH2), 5.29 (1H, br
s, NH-3), 6.59 (2H, s, H-2′ and 6′), 6.79 (1H, s, H-4′), 7.52 (1H,
br s, NH-Boc), 12.58 (1H, br s, NH-1). Anal. (C24H33N3O4‚
0.25H2O) C, H, N.
(+,-)-1-(3-Acetyla m in o-5-eth yl-2-m eth oxy-6-m eth ylp y-
r id in -4-yl)-1-(3,5-d im eth ylp h en yl)eth yl a ceta te (33). A
solution of (+,-)-1-(3,5-dimethylphenyl)-1-(5-ethyl-2-methoxy-
6-methyl-3-pivaloylaminopyridin-4-yl)ethan-l-ol (31) (100 mg,
0.25 mmol) in 5 mL of acetic anhydride was heated at reflux
for 72 h. The solvent was evaporated under reduced pressure,
and the resulting residue was taken up in 10 mL of water and
10 mL of dichloromethane. The aqueous layer was basified by
addition of 1 N aqueous potassium hydroxide and extracted
twice with 10 mL of dichloromethane. The combined organic
layers were washed with 5 mL of brine, dried over magnesium
sulfate, and concentrated under reduced pressure. The residue
was purified by column chromatography using dichlorometh-
ane-ethyl acetate (99:1 to 95:5) as eluent to give 33 (9 mg,
9%) as a yellow light oil and 45 mg (45%) of the recovered
1
pyridine 31: H NMR (CDCl3) δ 1.01 (3H, t, J ) 7.5 Hz, CH3-
CH2), 2.07 (6H, s, CH3-3′ and 5′), 2.19 (3H, s, CH3-6), 2.30 (3H,
s, CH3COO), 2.34-2.48 (2H, m, CH3CH2), 2.43 (3H, s, COCH3),
2.54 (3H, s, CH3), 3.93 (3H, s, OCH3), 7.01 (1H, s, H-2′ or 6′*),
7.11 (1H, s, H-6′ or 2′*), 7.37 (1H, s, H-4′); MS (IC): 399 [M +
H]+, 355 [M-COCH3]+. Anal. (C23H30N2O4‚0.5H2O) C, H.
(+,-)-4-[1-(3,5-Dim eth ylph en yl)-1-h ydr oxy]eth yl-5-eth yl-
6-m eth yl-3-p iva loyla m in op yr id in -2(1H)-on e (34). To a
solution of (+,-)-1-(3,5-dimethylphenyl)-1-(5-ethyl-2-methoxy-
6-methyl-3-pivaloylaminopyridin-4-yl)ethan-l-ol (31) (100 mg,
0.25 mmol) in 10 mL of toluene was added p- toluenesulfonic
acid (119 mg, 0.63 mmol). The mixture was stirred at reflux
for 21 h. The toluene was evaporated under reduced pressure,
and 10 mL of water was added to the resulting residue. The
aqueous layer was extracted with 3 × 15 mL of dichlo-
romethane. The combined organic layers were washed with
10 mL of a saturated aqueous sodium bicarbonate solution,
dried over magnesium sulfate and concentrated under reduced
pressure. The residue was purified by column chromatography
using dichloromethane-ethanol (1:0 to 95:5) as eluent giving
4-(3,5-Dim et h ylb en zyl)-5-et h yl-3-(N-glycyl)a m in o-6-
m eth ylp yr id in -2(1H)-on e (30). To a suspension of 3-[N-(tert-
butoxycarbonyl)glycyl]amino-4-(3,5-dimethylbenzyl)-5-ethyl-6-
methylpyridin-2(1H)-one (29) (500 mg, 1.17 mmol) in 40 mL
of ethyl acetate was added concentrated aqueous hydrochloric
acid (2.70 mL). The solution obtained was stirred at room
temperature for 10 min and basified with concentrated am-
monium hydroxyde. Water (60 mL) was added, and the
aqueous layer was extracted with ethyl acetate (3 × 120 mL).
The combined organic layers were washed with 40 mL of brine,
dried over magnesium sulfate, and concentrated under reduced
pressure to give 380 mg (99%) of the deprotected product 30
1
34 (64 mg, 66%) as a white solid: mp 257-258 °C; H NMR
(CDCl3) δ 0.96 (9H, s, COC(CH3)3), 1.03 (3H, t, J ) 7.1 Hz,
CH3CH2), 2.20 (3H, s, CH3-3′ or 5′*), 2.27 (3H, s, CH3-5′ or
3′*), 2.31-2.41 (5H, m, CH3-6 and CH3CH2), 2.48 (3H, s, CH3C-
(OH)), 6.98 (1H, s, H-2′ or 6′#), 7.08 (1H, s, H-6′ or 2′#), 7.40
(1H, s, H-4′), 12.36 (1H, br s, NH-1). Anal. (C23H32N2O3‚0.5H2O)
C, H, N.
(+,-)-3-Am in o-4-[1-(3,5-d im et h ylp h en yl)-1-h yd r oxy]-
eth yl-5-eth yl-6-m eth ylpyr idin -2(1H)-on e (35) an d 3-am in o-
5-eth yl-6-m eth ylp yr id in -2(1H)-on e4 (36). Starting from
(+,-)-1-(3,5-dimethylphenyl)-1-(5-ethyl-2-methoxy-6-methyl-
3-pivaloylaminopyridin-4-yl)ethan-1-ol (31) (100 mg, 0.25 mmol)
and 3 M aqueous hydrochloric acid (6 mL), the reaction was
performed as above (for 8b). The residue was purified by
column chromatography using dichloromethane-ethanol (100:0
to 95:5) as eluent giving 9 mg (9%) of the recovered pyridine
31, 45 mg (60%) of 35 as a yellow-brown solid and 7 mg (18%)
of 36 as a violet solid. 35: mp 144-145 °C; 1H NMR (CDCl3) δ
0.81 (3H, t, J ) 7.4 Hz, CH3CH2), 2.13 (2H, q, J ) 7.5 Hz,
CH3CH2), 2.23-2.34 (9H, m, CH3-3′, 5′ and 6), 2.41 (3H, s,
CH3C(OH)), 4.71 (2H, br s, NH2), 7.05 (2H, s, H-2′ and 6′), 7.12
(1H, s, H-4′), 12.66 (1H, br s, NH-1). Anal. (C18H24N2O2‚
1
as a white solid: mp 148-149 °C; H NMR (DMSO-d6) δ 0.82
(3H, t, J ) 6.9 Hz, CH3CH2), 2.22 (11H, br s, CH3-3′, 5′, 6 and
CH3CH2), 3.23 (2H, s, CH2-4), 3.38 (2H, br s, NH2), 3.77 (2H,
s, CH2NH2), 6.73 (2H, s, H-2′ and 6′), 6.81 (1H, s, H-4′), 6.86
(1H, br s, NH-3). Anal. (C19H25N3O2‚0.7 H2O) C, H.
(+,-)-1-(3,5-Dim et h ylp h en yl)-1-(5-et h yl-2-m et h oxy-6-
m eth yl-3-p iva loyla m in op yr id in -4-yl)eth a n -1-ol (31). The
starting material 13 was dried in the presence of calcium
chloride at room temperature under vacuum for 12 h. To a
solution of 1-(3,5-dimethylphenyl)-1-(5-ethyl-2-methoxy- 6-meth-
yl-3-pivaloylaminopyridin-4-yl)methanol (13) (400 mg, 1.05

3960 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dolle´ et al.
1
0.4EtOH) C, N, O. 36: mp 169-170 °C; H NMR (CDCl3) δ
ranoside (MUG). After 3 h at 37 °C, the level of the reaction
was measured in a fluorescence microplate reader.
Exp r ession a n d P u r ifica tion of th e Recom bin a n t
HIV-1 RT En zym e. Yeast cells, transformed with the vector
pAB 24/RT-4, were used to purify the recombinant HIV-1 RT
enzyme as described previously.37
The system for the expression of recombinant HIV-2 RT in
Escherichia coli was a kind gift of Dr. R. Goody. HIV-2 RT
was purified as HIV-1 RT.38
Rever se Tr a n scr ip ta se Assa ys. Incubation was carried
out at 37 °C for 10 min in the presence of different template-
primers. (a) Poly(C)-oligo(dG): the reaction mixture contained
in a final volume of 0.05 mL, 50 mM Tris-HCl pH 8.0, 5 mM
MgCl2, 4 mM dithiothreitol, 0.48 A260 units/mL of poly(C)-oligo
(dG) (5:1), 1.0 µCi [3H]dGTP (28 Ci/mmol), 2 µM dGTP, 80 mM
KC1, 1 µg bovine serum albumin and 20-50 nM RT. (b) Poly-
(A)-oligo(dT): same conditions as in (a), except that 0.48 A260
units/mL of poly(A)-oligo(dT) (5:1), 0.5 µCi of [3H]dTTP (46 Ci/
mmol), 20 µM dTTP were used.
Reactions were stopped by the addition of 1 mL of cold 10%
trichloroacetic acid plus 0.1 M sodium pyrophosphate. The
precipitates were filtered through nitrocellulose membranes,
washed with 2% trichloroacetic acid, dried, and counted in a
PPO/POPOP/toluene scintillation mixture.
1.07 (3H, t, J ) 7.5 Hz, CH3CH2), 2.21 (3H, s, CH3-6), 2.32
(2H, q, J ) 7.5 Hz, CH3CH2), 3.93 (2H, br s, NH2), 6.54 (1H, s,
H-4), 11.81 (1H, br s, NH-1); MS (IC): 153 [M + H]+. Anal.
(C8H12N2O‚0.01H2O) C, H, O.
Biology. Eva lu a tion of An tivir a l Activity of th e Com -
p ou n d s. The effects of the compounds on the replication of
HIV-1 were evaluated (Table 2), as previously described, in
CEM-SS cells (a cell line of the lymphocytic lineage) acutely
infected with HIV-1 LAI.34 CEM-SS cells were obtained from
Peter Nara, the nevirapine resistant HIV-1 (N119) strain
bearing a point mutation at RT codon 181 and AZT resistant
HIV-1 (A018 G910-6) ) AZT res. from D. Richman, HIV-1 Bal
is a macrophage tropic virus and received from S. Gartner,
M. Popovic, and R. Gallo through the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, NIH.
The compounds were solubilized in DMSO at an initial
concentration of 10-2 M. The solubility in aqueous phase was
tested with a dilution 1:100 in culture medium. When neses-
sary, in case of insolubility, a 10-3 M solution in DMSO, or
lower concentration, was diluted at 1% in culture medium.
This solution, as well as serial dilutions in culture medium,
were used in the antiviral assays.
The production of virus was measured by quantification of
the RT activity associated with the virus particles released in
the culture supernatant. Briefly, cells were infected with 100
TCID50 for 30 min; after virus adsorption, unbound particles
were eliminated by two washes, and cells were cultured in the
presence of different concentrations of test compounds for 5
days before virus production determination. The 50% inhibi-
tory concentration of virus multiplication (IC50) was derived
from the computer-generated median effect plot of the dose-
effect data.35 In parallel experiments, cytotoxicity of the
molecules for uninfected cells was measured after an incuba-
tion of 5 days in their presence using a colorimetric assay (MTT
test) based on the capacity of mitochondrial dehydrogenases
of living cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-
yltetrazolium bromide into formazan.35 The 50% cytotoxic
concentration (CC50) is the concentration at which OD540 was
reduced by one-half and was calculated using the program
mentioned above. The assay procedures for measuring the
anti-HIV activity of the compounds in the different cells
Rever se Tr a n scr ip tion . The plasmid pAV4 containing the
50-997 HIV-1 nucleotide fragment (MAL strain) in pSP64,
under the control of the bacteriophage T7 promoter, was a kind
gift from Dr. J .-L. Darlix (INSERM-Lyon, France). E. coli HB
101 recA- was used for plasmid amplification. After digestion
of this clone with PstI and in vitro transcription using T7 RNA
polymerase, a HIV-1 genomic RNA fragment starting at
position +50 of the MAL sequence was obtained. In vitro
transcription using T7 RNA polymerase was performed as
follows. Three micrograms of linearized plasmid DNA were
transcribed in 100 µL of 40 mM Tris-HCl pH 8.0, 8 mM MgCl2,
10 mM spermidine, 25 mM NaCl, 10 mM dithiothreitol, 0.5
mM of each ribonucleoside triphosphate, with 100 units of T7
RNA polymerase and in the presence of 20 units of human
placenta ribonuclease inhibitor, for 2 h at 37 °C. After
treatment with 12 units of RNase-free DNase I (for 10 min at
37 °C), the RNA transcripts were extracted with 1 volume of
phenol/chloroform/isoamyl alcohol (24:24:1) and with chloro-
form and precipitated in 2.5 volumes of ethanol and 0.3 M
ammonium acetate (pH 5.5).
infected with other virus isolates were also based on
a
quantitative detection of reverse transcriptase activity associ-
ated with the virus particles liberated in the culture super-
natant, except for HIV-1 IIIB tested in MT4 cells for which
the assay is based on the virus-induced cytopathogenicity (test
MTT). The experimental protocoles have been detailed previ-
ously, note that monocyte-derived macrophages were main-
tained, in absence of serum, in medium AIM V (Gibco)
supplemented with 100 U/mL GM-CSF (Genzyme, TEBU,
France).1b,34
Reverse transcription was performed in a total volume of
50 µL containing 50 mM Tris-HC1 pH 8.0, 6 mM MgCl2, 2
mM dithiothreitol, 12 mM NaCl, 150 nM HIV-1 RNA, and
either 200 nM of a synthetic oligodeoxynucleotide primer (18-
mer ODN) complementary to the PBS of HIV-1 RNA, or 200
nM tRNA.Lys3 When the 18-mer ODN was used as primer,
incubation was carried out at 37 °C with the template and
300 nM RT. After 30 min, 10 µCi[(R-32P]dGTP (3000 Ci/mmol)
and 0.1 mM of each dNTP were added, and the incubation
proceeded for 30 min at 37 °C. With tRNALys3 as primer, the
same conditions were used except that tRNA and RNA were
prehybridized by heating for 2 min at 90 °C and then slowly
cooled. Samples were extracted with phenol-chloroform and
collected by ethanol precipitation. Reaction products were
analyzed on 8% polyacrylamide-TBE (90 mM Tris pH 8.3, 90
mM borate, 2 mM EDTA)-7 M urea gels.
Retr ovir u cid a l Effect. HIV-1 viral suspensions were
obtained by coculture of MT4 cells and H9 cells chronically
infected by HIV-1Lai isolate. A cell supernatant (200 µL)
containing viral particles (HIV-1Lai: 100 TCID50) was incu-
bated at room temperature with various concentrations of
different inhibitors. After 3 h, virions were washed through
0.02 µm Anopore membrane in 1.5 mL Vectaspin tube (What-
man) for 10 min at 5 000g. Each of the three subsequent
washes was performed in the same conditions after the viral
concentrate was refilled with 500 µL of RPMI medium. Then,
the viral concentrate was readjusted to the initial volume with
RPMI plus 10% fetal calf serum (FCS). The residual infectivity
was assayed on P4 cells as described by Charneau et al.36
Briefly, P4 cells were plated using 100 µL of DMEM medium
plus 10% FCS in 96 plate multi-wells at 20 × 105 cells per
milliliter. After overnight incubation at 37 °C, the supernatant
was discarded, and the viral preparation (200 µL) was added.
One day later the wells were washed three times in PBS. Each
well was refilled with 200 µL of a reaction buffer containing
50 mM Tris-HC1 pH 8.5, 100 mM 2-mercaptoethanol, 0.05%
Triton X-100, and 5 mM 4-methylumbelliferyl â-D-galactopy-
In h ibition Exp er im en ts. All compounds were dissolved
in dimethylsulfoxide. Controls were made in the presence of
the same final concentration of DMSO.
Ack n ow led gm en t. The authors thank Drs. Claude
Monneret and David Grierson for many fruitful discus-
sions and advice. We thank the French Agency for
Research Against AIDS (ANRS) for financial support
including fellowships to V.D. We thank Dr. Daniel
Dauzonne and the Drug Synthesis and Chemistry
Branch, Development Therapeutics Program, Division
of Cancer Treatment, National Cancer Institute, Be-
thesda, Maryland, for supplying us with 2-methoxy-4,5-

Activity of 4-Benzyl Pyridinone Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3961
(13) Olah, G. A.; Balaram Gupta, B. G.; Malhotra, R.; Narang, S. C.
Chlorotrimethylsilane/Lithium Bromide and Hexamethyl disi-
lane/Pyridinium Bromide Perbromide: Effective and Selective
Reagents for the Conversion of Alkyl (Cycloalkyl and Aralkyl)
Alcohols into Bromides. J . Org. Chem. 1980, 45, 1638-1639.
(14) Fisher, L. E.; Labadie, S. S.; Reuter, D. C.; Clark, R. D.
Palladium-Mediated Benzylation of Dimetalated 1-Butylindole-
3-carboxylic Acid. J . Org. Chem. 1995, 60, 6224-6225.
(15) Schaefer, T.; Takeuchi, C. S. A Positive 6J (H, CHO) in some
meta Derivatives of Benzaldehyde. A Simple Model. Can. J .
Chem. 1989, 67, 827-830.
dimethylbenzaldehyde and nevirapine, respectively.
This work was also supported by the Conseil Regional
d'Aquitaine. The expert technical assistances by Ms.
Christina Calmels (Supported by SIDACTION), Sylvie
Schmidt, and Ge´raldine Albrecht are acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(16) Bisagni, E.; Rautureau, M.; Huel, C. 1-Functionalized 5,6-
dimethyl-6H-pyrido[4,3-b]carbazoles (Ellipticines) and Ana-
logues: A New Rapid Synthesis. Heterocycles 1988, 27 (7), 1671-
1678.
Refer en ces
(17) Komatsu, Y.; Minami, N. Synthesis of a Novel Dual Inhibitor of
Thromboxane A2 Synthetase and 5-Lipoxygenase (E3040) via the
Direct Coupling Reaction of Hydroquinone with 3-Pyridine
carboxaldehyde. Chem. Pharm. Bull. 1995, 43 (10), 1614-1616.
(18) Tanaka, H.; Takashima, H.; Ubasawa, M.; Sekiya, K.; Inouye,
N.; Baba, M.; Shigeta, S.; Walker, R. T.; De Clercq, E.; Miyasaka,
T. Synthesis and Antiviral Activity of 6-Benzyl Analogues of
1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as
Potent and Selective Anti-HIV-1 Agents. J . Med. Chem. 1995,
38, 2860-2865.
(19) Suzuki, K.; Tatsuoka, T.; Murakami, T.; Ishihara, T.; Aisaka,
K.; Inoue, T.; Ogino, R.; Kuroki, M.; Miyazaki, T.; Satoh, F.;
Miyano, S.; Sumoto, K. 2-Arylmethyl-1,4-benzoquinones. I. Novel
Inhibitors of Platelet Aggregation: Synthesis and Pharmacologi-
cal Evaluation. Chem. Pharm. Bull. 1996, 44, (1), 139-144.
(20) (a) Highet, R. J .; Wildman, W. C. Solid Manganese Dioxide as
an Oxidizing Agent. J . Am Chem. Soc. 1955, 77, 4399-4401.
(b) Gritter, R. J .; Wallace, T. J . The Manganese Dioxide
Oxidation of Allylic Alcohols. J . Org. Chem. 1959, 24, 1051-
1056. (c) Harfenist, M.; Bavley, A.; Lazier, W. A. The Oxidation
of Allyl and Benzyl Alcohols to the Aldehydes. J . Org. Chem.
1954, 19, 1608-1616.
(21) The 2-methoxy-4,5-dimethylbenzaldehyde was provided by Dr.
Dauzonne, Institut Curie, Section de Recherche, Paris.
(22) Borch, R. F.; Hassid, A. I. A New Method for the Methylation of
Amines. J . Org. Chem. 1972, 37, 1673-1674.
(23) Nguyen, C. H.; Bisagni, E.; Lhoste, J .-M. Synthesis of N-5
substituted 5H-pyrido[4,3-b]benzo[f]indole derivatives, isomers
of 6H-pyrido[4,3-b]carbazoles (ellipticines). Can. J . Chem. 1986,
64, (3), 545-551.
(24) Nicolaou, K. C.; Barnette, W. E.; Ma, P. A Remarkably Simple,
Highly Efficient, and Stereoselective Synthesis of Steroids and
Other Polycyclic Systems. Total Synthesis of Estra-1,3,5(10)-
trien-17-one via Intramolecular Capture of o-Quinodimethanes
Generated by Cheletropic Elimination of SO2. J . Org. Chem.
1980, 45, 1463-1470.
(25) Dolle´, F.; Hetru, C.; Rousseau, B.; Sobrio, F., Blais, C.; Lafont,
R.; Descamp, M.; Luu, B. Synthesis of a Tritiated 3-Dehydro-
ecdysteroid Putative Precursor of Ecdysteroid Biosynthesis.
Tetrahedron 1993, 49, 2485-2498.
(26) Wittig, G.; SchoellKopf, U. Methylenecyclohexane. Org. Synth.
1973, V, 751-754.
(27) Trono, D. Partial Reverse Transcripts in Virions from Human
Immunodeficiency and Murine Leukemia Viruses. J . Virol. 1992,
66, 4893-4900.
(28) Lori, F.; Veronese, F. M.; DeVico A. L.; Lusso, P.; Reitz, M. S.;
Gallo, R. C. Viral DNA Carried by Human Immunodeficiency
Virus Type 1 Virions. J . Virol. 1992, 66, 5067-5074.
(29) Zhang, H.; Zhang Y.; Spicer, T. P.; Abbott, L. Z. Abbott, M.;
Poiesz, B. J . Reverse Transcription Takes Place within Extra-
cellular HIV-1 Virions: Potential Biological Significance. AIDS
Res. Hum. Retroviruses 1993, 9, 1287-1296.
(1) (a) Bisagni, E.; Dolle, V.; Nguyen, C.-H.; Legraverend, M.;
Aubertin, A.-M.; Kirn, A.; Andreola, M. L.; Tarrago-Litvak, L.
4-Aryl-thio-pyridin-2(1H)-ones, medecines containing them and
their uses in the treatment of illnesses linked to HIV-1 and 2.
Fr. Demande Appl. 9509323, 30 J ul 1995. (b) Dolle, V.; Fan, E.;
Nguyen, C. H.; Aubertin, A.-M.; Kirn, A.; Andreola, M. L.;
J amieson, G.; Tarrago-Litvak, L.; Bisagni, E. A New Series of
Pyridinone Derivatives as Potent Non-Nucleoside Human Im-
munodeficiency Virus Type 1 Specific Reverse Transcriptase
Inhibitors. J . Med. Chem. 1995, 38, 4679-4686.
(2) Goldman, M. E.; O’Brien, J . A.; Ruffing, T. L.; Schleif, W. A.;
Sardana, V. V.; Byrnes, V. W.; Condra, J . H.; Hoffman, J . M.;
Emini, A. E. A Nonnucleoside Reverse Transcriptase Inhibitor
Active on Human Immunodeficiency Virus Type 1 Isolates
Resistant to Related Inhibitors. Antimicrob. Agents Chemother.
1993, 37, 947-949.
(3) Saari, W.8.; Hoffman, J . M.; Wai, J . S.; Fisher, T. E.; Rooney, C.
S.; Smith, A. M.; Thomas, C. M.; Goldman, M. E.; O’Brien, J .
A.; Nunberg, J . H.; Quintero, J . C.; Schleif, W. A.; Emini, A. E.;
Stern, A. M.; Anderson, P. S. 2-Pyridinone Derivatives: A New
Class of Nonnucleoside, HIV-1-Specific Reverse Transcriptase
Inhibitors. J . Med Chem. 1991, 34, 2922-2925.
(4) Hoffman, J . M.; Wai, J . S.; Thomas, C. M.; Levin, R. B.; O’Brien,
J . A.; Goldman, M. E. Synthesis and Evaluation of 2-Pyridinone
Derivatives as HIV-1 Specific Reverse Transcriptase Inhibitors.
1. Phthalimido alkyl and -alkylamino Analogues. J . Med. Chem.
1992, 35, 3784-3791.
(5) Saari, W. S.; Wai, J . S.; Fisher, T. E.; Thomas, C. M.; Hoffman,
J . M.; Rooney, C. S.; Smith, A. M.; J ones, J . H.; Bamberger, D.
L.; Goldman, M. E.; O’Brien, J . A.; Nunberg, J . H.; Quintero, J .
C.; Schleif, W. A.; Emini, A. E.; Anderson, P. S. Synthesis and
Evaluation of 2-Pyridinone Derivatives as HIV-1-Specific Re-
verse Transcriptase Inhibitors. 2. Analogues of 3-Aminopyridin-
2(1H)-one. J . Med. Chem. 1992, 35, 3792-3802.
(6) Wai, J . S.; Williams, T. M.; Bamberger, D. L.; Fisher, T. E.;
Hoffman, J . M.; Hudcosky, R. J .; MacTough, S. C.; Rooney, C.
S.; Saari, W. S.; Thomas, C. M.; Goldman, M. E.; O’Brien, J . A.;
Emini, E. A.; Nunberg, J . H.; Quintero, J . C.; Schleif, W. A.;
Anderson, P. S. Synthesis and Evaluation of 2-Pyridinone
Derivatives as Specific HIV-1 Reverse Transcriptase Inhibitors.
3. Pyridyl and Phenyl Analogues of 3-Aminopyridin-2(1H)-one.
J . Med. Chem. 1993, 36, 249-255.
(7) Hoffman, J . M.; Smith, A. M.; Rooney, C. S.; Fisher, T. E.; Wai,
J . S.; Thomas, C. M.; Bamberger, D. L.; Barnes, J . L.; Williams,
T. M.; J ones, J . H.; Olson, B. D.; O’Brien, J . A.; Goldman, M. E.;
Nunberg, J . H.; Quintero, J . C.; Schleif, W. A.; Emini, A. E.;
Anderson, P. S. Synthesis and Evaluation of 2-Pyridinone
Derivatives as HIV-1-Specific Reverse Transcriptase Inhibitors.
4. 3-[2-(Benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-one
and Analogues. J . Med. Chem. 1993, 36, 953-966.
(8) Saag, M. S.; Emini, A. E.; Laskin, O. L.; Douglas, J .; Lapidus,
W. I.; Schleif, W. A.; Whitley, R. J .; Hildebrand, C.; Byrnes, V.
W.; Kappes, J . C.; Anderson, K. W.; Massari, F. E.; Shaw, G. M.
The L-697,661, Working Group. A Short-Term Clinical Evalu-
ation of L-697, 661, A Non-Nucleoside Inhibitor of HIV-1 Reverse
Transcriptase. N. Engl. J . Med. 1993, 329, 1065-1072.
(9) Houpis, I. N.; Molina, A.; Lynch, J .; Reamer, R. A.; Volante, R.
P.; Reider, P. J . Condensation of 2-Methylbenzoxazole with
Aromatic Aldehydes Bearing Acidic Protons. A Convenient
Coupling in the Synthesis of the HIV-Reverse Transcriptase
Inhibitor L-696,229. J . Org. Chem. 1993, 58, 3176-3178.
(10) Doll6, V.; Nguyen, C. H.; Bisagni, E. Studies towards 4-C-
Alkylation of Pyridin-2(1H)-one Derivatives. Tetrahedron 1997,
53, 12505-12524.
(11) Casas, R.; Cave´, C.; d'Angelo, J . Ortho-metalated Aromatic
Tertiary Amides: New Synthetic Applications. Tetrahedron Lett.
1995, 36 (7), 1039-1042.
(12) Kelley, J . L.; Linn, J . A.; Selway, J . W. T. Synthesis and
Antirhinovirus Activity of 6-(dimethylamino)-2-(trifluoromethyl)-
9-(substitutedbenzyl)-9H-purines. J . Med. Chem. 1989, 32,
1757-1763.
(30) Ho, D. D.; Neumann, A. U.; Perelson, A. S.; Chen, W.; Leonard,
J . M.; Markowitz, M. Rapid Turnover of Plasma Virions and CD4
Lymphocytes in HIV-1 Infection. Nature 1995, 373, 123-126.
(31) Borkow, G.; Barnard, J .; Nguyen, T. M.; Belmonte, A.; Wainberg,
M. A.; Parniak, M. A. Chemical Barriers to Human Immuno-
deficiency Virus Type 1 (HIV-1) Infection: Retrovirucidal Activity
of UC781, a Thiocarboxanilide Nonnucleoside Inhibitor of HIV-1
Reverse Transcriptase. J . Virol. 1997, 71, 3023-3030.
(32) Ventura, M.; Tarrago-Litvak, L.; Dolle´, V.; Nguyen, C. H.;
Legraverend, M.; Fleury, H. J . A.; Litvak, S. Effect of nucleoside
analogues and nonnucleoside inhibitors of HIV-1 reverse tran-
scriptase on cell-free virions. Arch. Virol. 1999, 144, 513-523.
(33) Carroll, S. S.; Stahlhut, M.; Geib, J .; Olsen, D. B. Inhibition of
HIV-1 Reverse Transcriptase by a Quinazolinone and Compari-
son with Inhibition by Pyridinones. Differences in the Rates of
Inhibitor Binding and in Synergistic Inhibition with Nucleoside
Analogues. J . Biol. Chem. 1994, 269, 32351-32357.
(34) Moog, C.; Wick, A.; Le Ber, P.; Kirn, A.; Aubertin, A. M. Bicyclic
Imidazo Derivatives, a New Class of Highly Selective Inhibitors
for the Human Immunodeficiency Virus Type 1. Antiviral Res.
1994, 24, 275-288.

3962 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Dolle´ et al.
(35) Chou, J .; Chou, T.-C. Dose-effect Analysis with Microcomput-
ers: Quantitation of ED50, LD50, Synergism, Antagonism, Low-
dose Risk, Receptor Binding and Enzyme Kinetics. Computer
Software for Apple II Series and IBM-PC and Instruction
Manual; Elsevier-Biosoft, Elsevier Science Publishers: Cam-
bridge, U.K., 1985; pp 19-28.
(38) Sallafranque-Andreola, M. L.; Robert, D.; Barr, P. J .; Fournier,
M.; Litvak, S.; Sarih-Cottin, L.; Tarrago-Litvak, L. Human
Immunodeficiency Virus Reverse Transcriptase Expressed in
Transformed Yeast Cells. Eur. J . Biochem. 1989, 184, 367-374.
(39) Mu¨ller, B.; Restle, T.; Ku¨hnel, H.; Goody, R. S. Expression of
the Heterodimeric Form of Human Immunodeficiency Virus
Type 2 Reverse Transcriptase in E. coli and Characterization of
the Enzyme. J . Biol. Chem. 1991, 266, 14709-14713.
(36) Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and
Survival: Application to Proliferation and Cytotoxicity Assays.
J . Immunol. Methods 1983, 65, 55-63.
(37) Charneau, P.; Mirambeau, G.; Roux, P.; Paulous, S.; Buc, H.;
Clavel, F. HIV-1 Reverse Transcription. A Termination Step at
the Center of the Genome. J . Mol. Biol. 1994, 241, 651-662.
J M0009437