4-Benzyl and 4-benzoyl-3-dimethylaminopyridin-2(1H)-ones: In vitro evaluation of new C-3-amino-substituted and C-5,6-alkyl-substituted analogues against clinically important HIV mutant strains

Article abstract of DOI:10.1021/jm0408621

In a program to optimize the anti-HIV activity of the 4-benzyl and 4-benzoyl-3-dimethylaminopyridinones 9 and 10, lead compounds in a new class of highly potent non-nucleoside type inhibitors of HIV-1 reverse transcriptase, modification of the alkyl substitutents at the C-5 and C-6 positions on the pyridinone ring and of the substitutents on the C-3 amino group has been studied. Of the 17 new 5/6-modified analogues prepared, compounds 31b and 32b substituted at C-5 by an extended nonpolar chain containing an ether function and a C-6 methyl group and compound 35 bearing a C-5 ethyl/C-6 hydroxymethyl substituent pattern were selected on the basis of their in vitro activity against wild-type HIV and the three principle mutant strains, K103N, Y181C, and Y188L. When tested further, it was shown that these molecules, and in particular compound 35, are globally more active than 9, 10, and efavirenz against an additional eight single [L100I, K101E, V106A, E138K, V179E, G190A/S, and F227C] and four double HIV mutant strains [L100I + K103N, K101E + K103N, K103N + Y181C, and F227L + V106A], which are clinically relevant. Concerning modulation of the N-3 substituent, 36 new analogues were prepared. Of these, the N-methyl-N-(2-methoxyethyl)-substituted compounds 40, 42, and 62, as well as the doubly modified compounds 77a and 77b, were selected from the initial screen and were subsequently shown to be active at sub-micromolar concentrations (IC₅₀'s) against all the other mutant strains except K103N + Y181C and F227L + V106A. Two possible, but distinct, modes of binding of these analogues in RT were suggested from molecular modeling studies. The preferred mode of binding for compound 62, corresponding to the predicted "orientation 1", was revealed in the X-ray crystal structure of the compound 62-RT complex.

Full text of DOI:10.1021/jm0408621

1948
J. Med. Chem. 2005, 48, 1948-1964
4-Benzyl and 4-Benzoyl-3-dimethylaminopyridin-2(1H)-ones: In Vitro
Evaluation of New C-3-Amino-Substituted and C-5,6-Alkyl-Substituted
Analogues against Clinically Important HIV Mutant Strains
Abdellah Benjahad,† Martine Croisy,† Claude Monneret,† Emile Bisagni,† Dominique Mabire,‡ Sophie Coupa,‡
Alain Poncelet,‡ Imre Csoka,‡ Je´roˆme Guillemont,*,‡ Christophe Meyer,‡ Koen Andries,§ Rudi Pauwels,|
Marie-Pierre de Be´thune,| Daniel M. Himmel, Kalyan Das, Eddy Arnold, Chi Hung Nguyen,*,† and
David S. Grierson†
Laboratoire de Pharmacochimie, Section de Recherche, UMR 176 CNRS-Institut Curie, Batiment 110, Centre Universitaire,
91405 Orsay, France, Medicinal Chemistry Department, Johnson & Johnson Pharmaceutical Research and Development,
Campus de Maigremont BP315, Val de Reuil, France, Virology Drug Discovery, Johnson & Johnson Pharmaceutical Research
and Development, Tumhoutseweg 30 B-2340 Beerse, Belgium, TIBOTEC, General De Wittelaan L 11 B3,
B-2800 Mechelen, Belgium, and Center for Advanced Biotechnology and Medicine, Department of Chemistry and Biology,
Rutgers University, 679 Hoes Lane, Piscataway, New Jersey 08854
Received August 3, 2004
In a program to optimize the anti-HIV activity of the 4-benzyl and 4-benzoyl-3-dimethylamino-
pyridinones 9 and 10, lead compounds in a new class of highly potent non-nucleoside type
inhibitors of HIV-1 reverse transcriptase, modification of the alkyl substitutents at the C-5
and C-6 positions on the pyridinone ring and of the substitutents on the C-3 amino group has
been studied. Of the 17 new 5/6-modified analogues prepared, compounds 31b and 32b
substituted at C-5 by an extended nonpolar chain containing an ether function and a C-6 methyl
group and compound 35 bearing a C-5 ethyl/C-6 hydroxymethyl substituent pattern were
selected on the basis of their in vitro activity against wild-type HIV and the three principle
mutant strains, K103N, Y181C, and Y188L. When tested further, it was shown that these
molecules, and in particular compound 35, are globally more active than 9, 10, and efavirenz
against an additional eight single [L100I, K101E, V106A, E138K, V179E, G190A/S, and F227C]
and four double HIV mutant strains [L100I + K103N, K101E + K103N, K103N + Y181C, and
F227L + V106A], which are clinically relevant. Concerning modulation of the N-3 substituent,
36 new analogues were prepared. Of these, the N-methyl-N-(2-methoxyethyl)-substituted
compounds 40, 42, and 62, as well as the doubly modified compounds 77a and 77b, were selected
from the initial screen and were subsequently shown to be active at sub-micromolar
concentrations (IC50’s) against all the other mutant strains except K103N + Y181C and F227L
+ V106A. Two possible, but distinct, modes of binding of these analogues in RT were suggested
from molecular modeling studies. The preferred mode of binding for compound 62, corresponding
to the predicted “orientation 1”, was revealed in the X-ray crystal structure of the compound
62-RT complex.
Introduction
relevance to the development of the NNRTI class of
compounds for which it has been shown that cross
resistance is a major concern.7 Promising new molecules
in the NNRTI group, which are currently undergoing
clinical evaluation, include DPC 083, 4,8 and TMC125,
5.9,10
Nevirapine 1, delaviridine 2, and efavirenz 3 are at
present the only non-nucleoside type reverse tran-
scriptase inhibitors (NNRTIs) used in combination
(HAART) therapy for the treatment of AIDS.1 In view
of the long-term complications that arise due to toxicity,
resistance, and associated side effects (lipodystrophy,
hyperlipidaemia, etc.)2-6 using combinations of nucleo-
side inhibitors, protease inhibitors, and NNRTIs, it is
important to find new drugs which display a high level
of activity against the clinically relevant HIV single and
multiple mutant strains. This criterion is of particular
Our efforts have been directed toward the evaluation
of 4-arylthiopyridinones/benzylpyridinones of general
formula 8 as a new class of anti-HIV agents which
target reverse transcriptase at the hydrophobic pocket
common to all NNRTIs.11,12 Preliminary SAR studies led
to the identification of compounds 9 and 10 as potent
inhibitors of wild-type HIV.13 These compounds are in
many respects hybrids of the HEPT analogue GCA-186,
6,14 and the Merck pyridinone, 7,15-19 in that they
contain both a 3′,5′-dimethylphenyl group and a 5-ethyl-
6-methyl-substituted pyridinone motif in their struc-
ture. The presence of the 3-NMe2 substitution in 9 and
10, as opposed to a nonfunctionalized amino group, was
similarily inspired by the presence of an i-Pr group at
* To whom correspondence should be addressed. For C.H.N.: phone,
33-1 69 86 30 89; fax, 33-1 69 07 53 81; e-mail, chi.hung@curie.u-
psud.fr. For J.G.: phone, 33-2-32 61 74 58; fax, 33 2 32 61 72 98; e-mail,
jguillem@prdfr.jnj.com.
† UMR 176 CNRS-Institut Curie.
‡ Medicinal Chemistry Department, Johnson & Johnson Pharma-
ceutical Research and Development.
§ Virology Drug Discovery, Johnson & Johnson Pharmaceutical
Research and Development.
| TIBOTEC.
Rutgers University.
10.1021/jm0408621 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/08/2005

In Vitro Evaluation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1949
the corresponding position in the HEPT derivatives
MKC-442 and 6.20
From the outset it was clear that drug resistance
would be a major obstacle to the development of the
arylthiopyridinones/benzylpyridinones series. Indeed,
the rapid appearance of drug-resistant HIV strains
bearing the Y181C, Y188L, and K103N mutations in RT
ultimately led to the abandonment of the clinical
development of HEPT and the Merck pyridinones. The
question was, will a lack of sensitivity to these major
mutant strains and others commonly encountered in
AIDS patients also be an “inherent” limiation of our
pyridinone family?
The increase in potency of GCA-186 over HEPT
against both wild-type HIV and the Tyr-181 mutant has
been correlated with an overall tighter and/or more
complete fit in the hydrophobic pocket due to the
presence of the 3′,5′-dimethyl groups.14 The key to the
problem was thus to find the correct combination of
modifications of our initial lead molecules which would
reinforce the crucial interactions in the hydrophobic
region in the mutant forms of RT. Following this logic,
we recently prepared and evaluated more than 100 new
analogues of lead molecules 9 and 10 in which the
number, position, steric hindrance, and electronic prop-
erties of the substituent(s) on the aromatic ring were
varied.13 This study permitted the identification of two
new lead compounds 11 and 12a in the 4-benzoylpyri-
dinone series and compound 12b in the 4-benzylpyri-
dinone group. These molecules are potent inhibitors of
wild-type HIV-1, as well as a large panel of HIV-1
mutant strains which are responsible for the onset of
resistance to the NNRTIs that are currently used in
HAART therapy. Globally, the activity profile for these
new molecules, and in particular for 12a, is better in
vitro than that for efavirenz.
The next step of this SAR study involved looking
closely at the influence of modifications at the C-5 and
C-6 positions on the pyridinone ring and at the amino
substitutent at C-3 on anti-HIV activity in vitro.
In earlier studies on the arylthio- and benzylpyridi-
nones, the 5-Et/6-Me substitution pattern was generally
adopted, since this combination of alkyl groups was
optimized for the Merck pyridinones (cf. 7).18,19 To
expand the SAR at these two centers, a series of 5,6-
dialkyl-substituted pyridinones were thus prepared and
evaluated. As can be seen from Tables 1 and 3, analogue
35, bearing a polar hydroxymethyl group at C-6, and
the analogues 31b and 32b with extended nonpolar
chains containing an ether function at C-5 and a C-6
methyl group are highly active against the three prin-
ciple mutants and against the larger panel of eight
single [L100I, K101E, V106A, E138K, V179E, 190A/S,
and F227C] and four double [L100I + K103N, K101E
+ K103N, K103N + Y181C, and F227L + V106A] HIV
mutant strains.
To explore the effects of modulating the 3-amino
substituent, a wide range of 3-N,N-dialkylamino and
cyclic amine analogues were prepared. Several of these
displayed potent activity against wild-type HIV-1 and
the three major mutant strains (Tables 2 and 3). In
particular, the N-methyl-N-(2-methoxyethyl)-substi-
tuted compounds 40, 42, and 62, as well as the doubly
modified compounds 77a and 77b selected from the
initial screen, were subsequently shown to be active at
sub-micromolar concentrations (IC50’s) against all the
mutant strains except K103N + Y181C and F227L +
V106A. To gain insight as to the mode of binding of
these interesting new N-3-modified analogues in the
hydrophobic pocket of RT, a molecular modeling study
was undertaken for compounds 9 and 62 using the
HEPT-RT structure as a starting point.20 This study
revealed two possible and essentially equivalent modes
of binding of these compounds (orientations 1 and 2,
Figure 1), generated by rotation of the pyridinone ring
about an axis defined by atoms N-1 and C-4. These
orientations differ most importantly in the positioning
of the extended amino side chain substituent. Note that
in orientation 2 this side chain occupies the same space
as the N-1 substituent in HEPT and HEPT analogues.
The preferred mode of binding for compound 62, corre-
sponding to the predicted orientation 1, was revealed

1950 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Benjahad et al.
Scheme 2a
Figure 1. Orientation 1 and orientation 2 for compound 62.
Scheme 1a
a Conditions: (a) (i) 6 N HCl, reflux; (ii) HN3/H2SO4); (iii) POCl3,
BnEt3NCl, CH3CN, reflux; (iv) NaOMe, MeOH; (v) H2, Raney Ni;
(vi) t-BuCOCl, CH2Cl2, Et3N; (b) (i) n-BuLi, TMEDA, 3,5-dimethyl-
1-bromomethylbenzene; (ii) 6 N HCl; (c) (i) n-BuLi, TMEDA,
3-methylbenzaldehyde; (ii) MnO2, toluene, reflux; (iii) 3 N HCl;
(d) HCHO, NaBH3CN, HOAc.
a Conditions: (a) NaOMe, EtOH, HCO2Et, Et2O; (b) cyanoac-
etamide, piperidine, HOAc, H2O.
substituted pyridinones 24a,b. Compounds 28 were
then converted to the target molecules in a fashion
similar to the preparation of compounds 20.
Ready access to the interesting 6-hydroxymethyl
derivative 35 of lead molecule 10 was achieved by
formation of the N-oxide of 33, its reaction with Ac2O
under Boekelheide conditions, acid hydrolysis to give
34, and reductive amination (Scheme 4).
in the X-ray crystal structure of the compound 62-RT
complex.21 The results obtained for both series of
compounds broaden our view of the SAR in the 4-benzyl/
4-benzyloxypyridinone family and clearly indicate direc-
tions to be taken for further analogue synthesis.
Chemistry
As for the preparation of 3-dimethylamino-4-ben-
zylpyridinone 9,12 the synthesis of analogues 18b-i
containing different alkyl substituents at C-5 and C-6
involved four distinct stages (Schemes 1 and 2): (i)
construction of the 3-cyanopyridinones 15 by condensa-
tion of the requisite ketones 13 with ethyl formate, and
reaction of the derived intermediates 14 with cyanoac-
etamide (piperidine/HOAc),17,22-24 (ii) conversion of
intermediates 15b-i to 2-methoxy-3-N-pivaloylamino-
pyridines 16b-i in six steps according to a procedure
established for the conversion of 15a to 16a,12 (iii)
reaction of the dianion of compounds 16 with 3,5-
dimethylbenzyl bromide, followed by treatment of the
derived condensation product with 6 N HCl to effect
amide hydrolysis and liberation of the 2-pyridinone
motif, and (iv) conversion of amines 17b-i to the
corresponding N,N-dimethylamines under reductive
alkylation conditions. As described for 20a,13 compounds
20a-c,i,j were obtained by reaction of the dianion of
the requisite 2-methoxy-3-N-pivaloylaminopyridines 16
with 3-methylbenzaldehyde, followed by alcohol to
ketone oxidation, treatment with acid, and reductive
alkylation of amine intermediates 19a-c,i,j.
The “mixed” N,N-dialkylpyridinone analogues 38 and
39 and in particular compounds 40-43 in which a sulfur
or oxygen atom is present in the longer of the two alkyl
chains were obtained by N-formylation of 17a, reduction
of the N-formyl group in 36,12 and reaction of the derived
monomethylated intermediate 3712 with the requisite
aldehyde and cyanoborohydride (Scheme 5). The mono-
N-Et (47), N-Pr (48), and N-Bu (49) derivatives were
similarily prepared by LAH reduction of amides 44-
46. Reaction of 17a with a large excess of the aldehyde
component and cyanoborohydride produced the sym-
metrical dialkyl compounds 50 and 51, whereas with a
smaller amount of benzaldehyde the mono- and di-N-
benzylated compounds 52 and 53 were obtained as a
separable mixture. Reductive amination conditions were
also employed to prepare the cyclic morpholine and
piperidine based analogues 54 and 55. By the simple
reaction of 17a with 2,5-dimethoxytetrahydrofuran, the
pyrrole analogue 56 was produced (Scheme 6).
Following the successive reductive alkylation strat-
egy, we prepared the mixed dialkylpyridinones 59-63,
related to 3′-methyl-3-dimethylaminopyridinone 20a,
from intermediate 5713 (Scheme 7). However, for the
preparation of 64-66, alkylation of the initially formed
mono-N-methyl compound 58 using the requisite chloro-
alkylnitrile reagent was preferred. Through reaction of
57 with ethyl and phenyl isothiocyanate, respectively,
the thiourea analogues 67 and 68 were formed. Alterna-
tive reaction of 57 with NH4SCN and benzoyl chloride
In an initial attempt to prepare pyridinones 31a,b
and 32b (Scheme 3) it was found that intermediate 23b
was acid-sensitive, precluding sucessful hydrolysis-
decarboxylation of the 3-cyano group. An alternate route
to the pivotal compounds 28a,b was thus devised
involving in the key step a Beckmann rearrangement
of the oximes 25a,b, obtained from the 3-acetyl-

In Vitro Evaluation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1951
Scheme 3a
Scheme 5a
a Conditions: (a) HCO2Et, HCO2H; (b) LiAlH4, THF; (c) RCOCl,
Et3N; (d) R′CHO, NaBH3CN; (e) R′CHO (excess), NaBH3CN; (f)
PhCHO (1.5 equiv), NaBH3CH.
a Conditions: (a) HCO2Et, NaOMe, Et2O; (b) cyanoacetamide,
piperidine, H2O; (c) acetoacetamide, piperidine, H2O; (d) EtOH,
H2NOH‚HCl, pyr; (e) HCO2H, heat; (f) (i) 3 N HCl; (ii) Piv-Cl, Et3N,
CH2Cl2; (g) MeI, Ag2CO3, CH2Cl2; (h) n-BuLi, TMEDA, 3-meth-
ylbenzaldehyde or 3,5-dimethylbenzaldehyde; (i) (i) Jones reagent;
(ii) 3 N HCl; (iii) HCHO, NaBH3CN, HOAc.
Scheme 6
Scheme 4a
ponent in the HEPT ligand of the HEPT-RT X-ray
crystal structure (PDB code: 1RTI) using the SYBYL
modeling package.25 For each molecule, two orientations
of the pyridinone ring were considered (Figures 1 and
2). Then, with the exception of the N-3 side chain in
orientation 2 which was modeled to match the corre-
sponding HEPT side chain conformation, the substitu-
ents at the 3-, 4-, and 5-positions of the pyridinone ring
were oriented toward the hydrophobic key residues
(Y181, Y188, W229, and P95). The four resulting models
were subsequently minimized using the Tripos force
field. The protein structure was then modified to reflect
the conformational changes occurring upon binding of
potent HEPT analogues bearing bulky substituents at
C-5.20 In particular, Y181 was set in the “switched”
conformation, thus, enabling the N-side chain to extend
deeper into the hydrophobic pocket and to interact
directly with the key hydrophobic residues. A local
minimization was performed in the Y181 region to
account for the conformational adjustments of the
surrounding residues triggered by the tyrosine switch.
Finally, the four ligand models were separately merged
into the modified protein structure, and the four com-
plexes were minimized using the Tripos force field along
a Conditions: (a) (i) m-CPBA, CH2Cl2; (ii) Ac2O, reflux; (b) (i)
12 N HCl; (ii) HCHO, NaBH3CN, HOAc.
gave compound 69, and by treatment of this product
with 3 N NaOH, thiourea 70 was obtained.
In view of the interesting biological activities observed
for compounds 42 and 62, the 4-benzoylpyridinone
analogue 72 possessing the 2-methoxyethylamine side
chain was also prepared via 19a and 71 (Scheme 8).
Further, given the very potent anti-HIV properties of
compound 12b bearing a 3′-acrylonitrile function on the
phenyl ring, it was similarily pertinent to construct
compounds 77a and 77b where this modification and
the 2-methoxyethylamine motif are combined in the
same molecule (Scheme 9).
Modeling
Manual Docking. Compounds 9 and 62 were con-
structed and superimposed onto the heterocycle com-

1952 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Benjahad et al.
Scheme 7a
Scheme 9a
a Conditions: (a) HCO2Et, HCO2H; (b) LiAlH4; (c) RCH2CHO,
NaBH3CN; (d) n-BuLi, DMF; (e) (EtO)2POCH2CN, t-BuOK.
a Conditions: (a) (i) HCO2Et, HOAc; (ii) LiAlH4; (b) R′CHO,
NaBH3CN (for 59-63); (c) RCl, Et3N (for 64-66); (d) RNCS (for
67 and 68) or C6H5COCl/NH4SCN for (for 69); (e) 3 N NaOH.
Scheme 8a
Figure 2. (a) Compound 9 superimposed onto the HEPT
crystal structure. Only the six-member ring was used for the
alignment. For the sake of clarity, compound 9 was shifted to
show matching atoms: left, orientation 1; right, orientation
2. (b) Compound 62 superimposed onto the HEPT crystal
structure. Only the six-member ring was used for the align-
ment. For the sake of clarity, compound 62 was shifted to show
matching atoms: left, orientation 1; right, orientation 2.
a Conditions: (a) (i) HCO2Et, HOAc; (ii) LiAlH4; (b) MeOCH2CHO,
NaBH3CN.
with Gasteiger-Hu¨ckel charges for the ligands and
Kollman-all-atoms charges for the protein. During the
minimization, all residues included in a 10 Å sphere
around the ligand were allowed to move, while the
remaining part of the protein structure was kept rigid.
It is to be noted that the minimization process takes
into account all atoms in the complex when calculating
its internal energy.
by calculating the nonbounded interactions (steric,
electrostatic, and hydrophobic) between a C+ probe and
each atom of the binding site. Leapfrog then built new
substituents that matched the required interactions and
resulted in a decrease of the estimated binding energy.
De Novo Design. The “optimize” mode of the de novo
design module Leapfrog25 was used for this analysis. In
this mode, the software was first provided with the RT
protein structure taken from the HEPT-RT X-ray
crystal structure (PDB code: 1RTI) from which the
ligand has been removed. The protein structure was
modified in the same way as described above. In a
second step, compound 10 was constructed and manu-
ally docked within the RT protein. On the basis of the
protein alone, the process generated an interaction map
Results and Discussion
The pyridinone analogues described in Schemes 1-9
were evaluated in vitro against wild-type HIV-1 (HVTL
IIIB, LAI cell line) and against the three principle
mutant strains, K103N, Y181C, and Y188L, which
confer resistance to the NNRTIs currently used in the
clinic.26 The results are presented in Tables 1 and 2.
From the data in Table 1, one sees that the 5,6-
dimethyl compound 18b, the 6-ethyl/5-methyl compound

In Vitro Evaluation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1953
Table 1. Activity (IC50, µM) versus HIV-1
IC50 (µM)
compd
R1
R2
R3
Y
LAI
SIa
K103N
Y181C
Y188L
9
CH3
C2H5
C2H5
CH3
CH3
i-C4H9
CH3
n-C3H7
H
H
-(CH2)4-
C2H5
CH3
CH3
-(CH2)4-
-(CH2)3-
(CH2)2OCH3
(CH2)3OCH3
(CH2)3OCH3
C2H5
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3-CH3
CH2
CO
0.008
0.004
0.005
0.010
0.050
0.794
0.050
7.943
0.398
0.010
0.0025
0.016
0.100
0.063
0.016
0.004
0.008
0.002
0.001
12 589
2 512
1 995
10 000
1 995
126
1 995
10
251
158
39 811
6 310
1 000
158
3 162
7 943
1 259
12 589
12 589
0.032
0.010
0.398
0.100
0.063
ndb
0.100
0.063
0.398
0.251
0.158
ndb
0.251
0.158
3.162
5.012
0.398
ndb
10
CH3
18b
18c
18d
18e
18f
18g
18h
18i
20a
20b
20c
20i
20j
31a
31b
32b
35
CH3
C2H5
CH3
i-C3H7
CH3
H
CH3
-(CH2)4-
CH3
CH3
C2H5
-(CH2)4-
-(CH2)3-
CH3
CH3
CH3
CH2OH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CO
0.079
39.811
31.623
0.316
ndb
0.316
100
0.631
ndb
100
79.43
0.501
ndb
31.620
1.995
50.119
ndb
3-CH3
CO
0.398
2
3-CH3
CO
ndb
ndb
3-CH3
CO
ndb
ndb
ndb
3-CH3
CO
ndb
ndb
ndb
3-CH3
CO
0.158
0.126
0.01
1.26
0.160
0.05
0.072
10
3-CH3
CO
0.158
0.2
3,5-diCH3
3,5-diCH3
CO
CO
0.003
0.631
a Selectivity index or ratio of CC50 to IC50 relative to LAI (fold). b nd, not determined.
18c, and the bicyclic compound 18i are essentially
equipotent to lead molecule 9 against wild-type HIV.
However, these same molecules display a very low level
of activity toward the crucial Y188L mutant. A similar
loss in sensitivity toward the Y188L mutant was
observed for compound 20b relative to the reference
compound 20a. These findings suggested that even
minor changes in the nature of the 5- and 6-alkyl
substituents will strongly influence the positioning of
the molecule in the hydrophobic pocket of the Y188L
RT mutant. Consistent with this idea, the mono-(C-6)-
methyl-substituted analogue 18h fails against the mu-
tant strains, and the unsubstituted compound 18g is
totally inactive. However, the results for analogues 18d
and 18f show that modification at C-5 is possible
without substantial loss of activity against the Y188L
and the two other HIV mutant strains. It is probable
that the presence of the 6-methyl substituent in these
molecules is important for their activity. Consistent with
these observations and using HEPT as a model,20 the
4-benzoyl-type-pyridinone analogues 31a, 31b, and 32b
were all shown to be active at nanomolar concentrations
against wild-type HIV. Compound 31a fails against
Y181C and Y188L, but the closely related compound
31b remains active against all three mutants, and the
profile for 32b is, in fact, better than that for lead
molecule 9.
Contrary to what might be deduced from the data for
the C-6-ethyl-substituted compound 18c, incorporation
of a hydroxyl group into the C-6 methyl side chain, as
in 35, did not lead to loss of activity. In fact, this
analogue is a potent inhibitor of the three mutant
strains and in particular the crucial K103N mutant.
Conception of this molecule issues from the following
molecular modeling considerations. In HEPT, the N-1
substituent and the C-2 carbonyl are oriented toward a
Figure 3. Schematic drawing of the interaction map gener-
ated by Leapfrog (white background).
cavity lined by the three lysines K101, K102, and K103
on one hand and by the “P236 hairpin” on the other
hand. Examination of the interaction map generated by
Leapfrog clearly shows that the “lysine wall” is respon-
sible for a polar interaction surface due to a connection
of accessible NH-CdO amide bonds, while the “P236
hairpin” originates a hydrophobic interaction surface
due to the presence of hydrophobic side chains (Figure
3). Noteworthy are the electrostatic interaction fields
generated by the carbonyl and NH groups of K103 and
the carbonyl groups of K101 and P236. In light of this
information, the modeling predicted that new pyridi-
none derivatives bearing a C-6 hydroxymethyl group
may be active, since the OH group would point toward

1954 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Table 2. Activity (IC50, µM) versus HIV-1
Benjahad et al.
IC50 (µM)
compd
R1
R2
R3
Y
LAI
SIa
K103N
Y181C
Y188L
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
59
60
61
62
63
64
65
66
67
68
69
70
72
77a
77b
H
CHO
CH3
C2H5
C3H7
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3,5-diCH3
3-CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CO
0.079
0.01
1 259
5 012
3 981
6 310
7 943
398
0.794
0.079
0.079
0.1
0.079
1
0.063
0.1
19.95
100
100
0.316
1.585
1.995
0.398
100
3.162
50.12
100
7.943
1
0.316
1.995
1.259
0.398
0.251
ndb
H
CH3
CH3
CH3
CH3
CH3
CH3
H
0.008
0.016
0.006
0.025
0.002
0.004
0.398
3.981
0.316
0.398
0.398
0.501
0.316
0.126
25.12
100
100
100
15.85
5.012
1.259
100
CH(CH3)CH2OCH3
(CH2)3SCH3
CH2CH2OCH3
(CH2)5OH
COCH3
19 953
12 589
251
0.1
2.512
100
H
COC2H5
25
ndb
H
COC3H7
100
0.016
0.02
1
ndb
H
C2H5
126
100
H
C3H7
398
100
H
C4H9
0.126
0.016
50.119
0.251
398
ndb
C2H5
C4H9
H
C2H5
6 310
2
2.512
C4H9
ndb
CH2C6H5
251
10
79.43
10
ndb
CH2C6H5
CH2C6H5
100
1
ndb
(CH2CH2)2O
(CH2CH2)2O
(CH2)5
0.158
0.631
0.0126
0.005
0.003
0.01
251
ndb
(CH2)5
158
63.096
0.398
0.794
0.316
0.501
0.079
ndb
39.81
1.259
1.259
1.585
1.585
0.794
ndb
ndb
-CHdCH-CHdCH-
-CHdCH-CHdCH-
(CH2)2OH
(CH2)3OH
(CH2)5OH
(CH2)2OCH3
(CH2)2OC2H5
CH2CN
1 259
7 943
31 623
6 310
10 000
7 943
25 119
6 310
12 589
4
1.995
12.59
5.012
5.012
0.398
ndb
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
3-CH3
3-CH3
3-CH3
0.001
0.013
0.004
0.016
0.005
25.119
3.162
6.31
0.316
0.001
0.001
0.001
3-CH3
3-CH3
0.316
1.585
12.59
(CH2)2CN
(CH2)3CN
3-CH3
ndb
ndb
ndb
3-CH3
0.316
ndb
1.995
ndb
3.162
ndb
NH-CS-NHC2H5
NH-CS-NHC6H5
NH-CS-NHCOC6H5
NH-CS-NH2
(CH2)2OCH3
C2H5
3-CH3
H
3-CH3
25
ndb
ndb
ndb
H
3-CH3
16
100
100
100
H
3-CH3
316
ndb
ndb
ndb
CH3
CH3
CH3
3-CH3
79 433
10 000
10 000
0.020
0.006
0.02
0.631
0.032
0.04
1.259
0.251
0.316
3-CHdCHCN
3-CHdCHCN
CH2
CH2
(CH2)2OCH3
a Selectivity index or ratio of CC50 to IC50 relative to LAI (fold). b nd, not determined.
the polar surface and engage in a hydrogen-bonding
interaction with one of the accessible backbone amide
functions of the lysines or proline. The model was built
by complexing the RT crystal structure with the Leap-
frog orientation of compound 35. The complex was
minimized using the procedure described above for
manual docking. The minimized structure revealed that
the hydroxyl group of compound 35 engages in a direct
hydrogen bond with the backbone carbonyl of P236
(Figure 4). This became possible through a substantial
conformational change of the “P236 hairpin” which is
known to be able to enlarge or contract the binding site
in order to optimize contacts with the different RT
substrates.20 This interaction along with the crucial
pyridinone N-1-H hydrogen-bond with K101 and the
hydrophobic stacking interactions made by the dimeth-
ylphenyl substituent with the key hydrophobic residues
Y181 and W229 provides a good explanation for the
strong binding and thus the good activity of 35 against
the different HIV strains.
analogue 3612 displayed a good level of activity against
wild-type HIV (IC50 ) 0.08 µM), the corresponding
N-acetyl compound 44 is less active and the N-propionyl
compound 45 is only marginally active (IC50 ) 3.98 µM).
It was therefore not suprising that the N-butyryl
analogue 46 was inactive. Further evaluation confirmed
that none of these molecules displayed activity against
the three mutant strains. The thioureas 67-69 were
also uninteresting. These results created uncertainty as
to whether it would be possible to modify the C-3 amino
position such that more bulky aliphatic type substi-
tutents could be present. This doubt was reinforced by
the data for the corresponding monosubstituted amines
47-49 and compound 52 against the three mutant HIV
strains. Similarily, the morpholino/piperidine com-
pounds 54 and 55 and the pyrrole analogue 56 were
poorly active against the mutant strains, and the bulky
dialkyl-substituted analogues 51 and 53 were devoid of
activity even against wild-type HIV.
However, a change in potency began to make itself
noticed for the dialkyl analogues 38 and 39 wherein one
of the N-3 methyl groups in lead compound 9 was
Next, we turn to the analysis of the data in Table 2.
It was shown earlier that, whereas the formamide

In Vitro Evaluation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1955
binding (Figure 1). Indeed, although backbone hydrogen
bonding is potentially possible, it was unexpected that
compounds 59-61 with a polar hydroxyl substituent
would bind effectively in the hydrophobic pocket. The
idea was born that whereas compounds 9 and 10 most
probably bind to RT in a manner very similar to GCA-
186 [i.e., where the N-dimethyl substituent replaces the
GCA-186 5-i-Pr group (orientation 1)], the other mol-
ecules, bearing longer N-side chains, might bind in a
mode that places this N-substituent in the same orien-
tation as the N-1 side chain in HEPT (orientation 2).
In this conformation, obtained by a simple 180° rotation
about an axis through N-1 and C-4 of the pyridinone
ring, the crucial N-1-NH‚‚‚K101 carbonyl H-bond is
retained, while the carbonyl group flips to a new position
where it now functions like the C-2 carbonyl in HEPT.
These two options were studied for compounds 9 and
62 by molecular modeling using results from HEPT as
the starting template.
Visual inspection of the four minimized complexes for
9 and 62 for the two orientations showed no unrealistic
structural distortion (Figure 2). Comparison with the
starting protein structure revealed that the major
movements that occurred upon binding impact residues
Y181, Y188, and W229 as well as the position of the
“P236 hairpin”. These changes are consistent with
previous observations highlighting two key features for
inhibitory activity, namely, a discrete conformational
switch of Y181 and more continuous variations in the
position of the “P236 hairpin”.20 During the minimiza-
tion, a slight shift of the ligands occurred with respect
to the HEPT starting position. This was needed to allow
the bulkier mono- or dimethylbenzyl moiety versus the
more compact thiophenyl substituent of HEPT to fit
optimally in the hydrophobic pocket. For both com-
pounds, the protein-ligand interaction pattern in the
hydrophobic key residues area in either conformation
was found to be compatible with the observed activities.
Thus, it was not possible to choose one orientation over
the other.
A significant parameter that may help to distinguish
between ligand orientations 1 and 2 is the internal
energy of the inhibitor-RT complexes. For both com-
pounds, the lowest absolute energy, which translates
to enhanced stability, was obtained when the ligand
adopted the orientation 1 in the binding site. However,
the relative energy difference within each orientation
couple was 5.0 and 2.1 kcal/mol for compounds 9 and
62, respectively. A recent study on the energetics of the
conformational changes druglike molecules experience
upon binding revealed that approximately 60% of the
ligands were calculated to bind with strain energies
lower than 5 kcal/mol.27 Strain energies over 9 kcal/mol
were calculated in at least 10% of the cases. A clear
correlation was found between acceptable strain energy
and ligand flexibility, while there was no correlation
between strain energy and binding affinity. This indi-
cates that expensive conformational rearrangements
can be tolerated in some cases without penalizing the
tightness of the binding. The low-energy differences that
we calculated in our case do not permit us to favor one
orientation over the other. Moreover, close examination
of the X-ray crystal structure of the GCA-186-RT
complex (PDB code: 1C1B) shows that the two carbonyl
Figure 4. Binding mode of compound 35. The ligand has been
manually docked in the RT according to Leapfrog’s orientation,
and the complex was fully minimized. The Connolly surface
of the NNRTI binding pocket has been clipped off to reveal
the binding mode of compound 35. Especially interesting are
the H-bonds made with Lys101 and Pro236 (dashed yellow
lines). The molecular lipophilic potential has been calculated
and mapped onto the Connolly surface (color coding: from
brown (hydrophobic region) to blue (polar region)).
elongated by one and two carbons, respectively. With
the exception of a weak activity for 38 against the
Y188L mutant, these two compounds displayed sub-
micromolar activities against the three mutant strains.
Exploring further, we found that compounds 40 and 42
with a terminal methoxy group were also active, dis-
playing significantly better activity against the Y188L
mutant. In a subsequent effort to “adjust” the substit-
uents on the aryl group to accommodate the presence
of the large substituents on N-3, compounds 59-66 and
the 4-benzoyl analogue 72 which all have a single
methyl substituent on the aromatic ring were tested.
However, the data showed that the compounds 62, 63,
and 72 containing the ether motif offered no advantage
over 40 and 42 against the three mutant strains.
Interestingly, analogues 64-66 with a terminal cyano
substituent failed against the Y181C and Y188L muta-
tions. Similarily, although compounds 59-61 with a
polar hydroxyl substituent at the end of the alkyl chain
were highly active against wild-type HIV, they failed
against the mutants.
In view of the positive contribution to the activity
provided by the introduction of an ethyl and in particu-
lar a 2-methoxyethyl substituent to the N-3 nitrogen,
it was of interest to incorporate these modifications into
the acrylonitrile lead compound 12b. As seen, this
combination resulted in a very significant increase in
potency for compound 77a relative to 38 against all
three mutant strains and in a further 10-fold increase
in the potency of 77b relative to 42 against the Y181C
mutant.
The finding that compounds 40, 42, and 59-66 are
active against wild-type HIV at nanomolar concentra-
tions, and further that analogues 40, 42, 62, 72, and
77b with the elongated methoxy terminated N-3 side
chains display activity against the two crucial Y181C
and Y188L mutations present in the hydrophobic pocket
of RT, led us to ask whether the activities observed for
these analogues and the lead compounds 9 and 10 were
not the consequence of two entirely different modes of

1956 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Benjahad et al.
methoxy group makes with the p51 residue E138, as
well as hydrophobic contacts which the terminal N-
methyl makes with V179 and G190. From the structure,
it is evident that the Y181C mutation would have a
significant effect on an RT-bound 62. Since this part of
62 is interacting primarily with Y181, the Y181C
mutation would cause the loss of this inhibitor-protein
interaction, resulting in a significant problem of resis-
tance. Accordingly, although compounds 38, 40, 42, 59-
62, 64, and 66 tend to be potent against wild-type RT
(probably because of extensive hydrophobic interactions
with Y181), all of them are significantly vulnerable to
resistance from the Y181C mutation.
The 3′-methylbenzyl group at C-4 of the pyridinone
ring has substantial interactions with Y188, W229, and
L234. The interactions with W229 receive additional
stabilization from a contact which the 3′-methyl group
makes with P95. Consistent with this observation, a
Y188L mutation offers considerable resistance to com-
pound 62 as well as most other compounds in this series.
It is possible, however, to introduce other interactions
that compensate for the loss of contacts with Y188.
Figure 5. Crystal structure of 62-RT. Shown is the X-ray
crystal structure of compound 62 bound in the NNRTI binding
pocket of RT, near the polymerase catalytic site. The ligand
conformation, which corresponds to “orientation 1” (see text),
allows both the 3-(2-methoxyethyl)methylamino and 4-benzyl
substitutents to interact extensively with the hydrophobic core.
The (2-methoxyethyl)methylamino group interacts primarily
with Y181, while the 4-benzyl ring has extensive contacts with
Y188 and W229. Both the Y181 and Y188 side chains point
toward the active site to accommodate the ligand. The struc-
ture was determined at a resolution of 2.95 Å from a 95.5%
complete data set. The R and Rfree factors, respectively, were
24.6% and 30.7%.21
Compensatory Interactions. Structural studies30a-e
suggest that contacts with the highly conserved residue
W229 and the ability to form hydrophobic stacking
interactions with p66 amino acid residues Y181 and/or
Y188 are important for NNRTI binding. The substituent
pattern on a ligand should permit positioning in the
binding pocket so as to optimize these interactions,
while it is desirable that the backbone of the compound
be flexible enough to permit conformational and posi-
tional adaptation in response to mutations of other
residues in the pocket.10 Other compensatory interac-
tions are desirable as well, so that inhibitor activity is
not lost when either Y181 or Y188 mutates. At the
5-position of the pyridinone ring, a substituent contain-
ing an ethyl or n-Pr chain can interact with L234, as
seen in the 62-RT structure. A longer substituent (e.g.,
the 3-methoxypropyl in compounds 31b and 32b) can
interact with V106, F227, and P236, similar to what is
seen in RT complexes with HEPT-like compound TNK-
651,31 and flexibility can help redefine its orientiation
in response to mutations at these positions (106 and 236
are positions for common drug resistance mutations).
Compound 31a, whose 2-methoxyethyl substituent is
not long enough to interact extensively with residues
V106, F227, and P236, has 10-fold poorer activity
against Y181C and Y188L than compound 31b.
The 62-RT structure indicates that substituents on
the C-6 atom of the pyridinone ring will be positioned
to interact with Y318. If either this or the interactions
described above for substituents on the 5-position are
lost, then ligand binding relies more heavily on contacts
of the benzyl ring with Y188 and contacts with Y181,
so that mutations at these residues are likely to
introduce significant resistance to the inhibitor. Bulky
substituents at the 6-position may be unable to interact
with Y318 or find other compensatory contacts. Com-
pounds such as 18i, 20i, and 20j that conjoin C-5 and
C-6 with a bulky aliphatic ring reduce the backbone
flexibility of the ligand. Consequently, these compounds
are limited in their ability to adapt to changes in the
shape of the pocket such as in a Y188L mutation.
groups of GCA-186 form water-mediated H-bonds with
K101 and the K103 backbone amino groups.14 Although
no solvent molecule has been taken into account in our
calculations, it can be anticipated that when the ligands
adopt the orientation 2 the pyridinone carbonyl group
would be ideally positioned to form a similar water-
mediated H-bond with K103 lowering further, by ap-
proximately 2 kcal/mol, the energy difference between
the two orientation patterns.
On the basis of these arguments, neither the struc-
tural nor the energetic elements were significant enough
to allow us to discriminate between the two ligand
orientations. For this reason, the X-ray crystal structure
analysis of the 62-RT complex was obtained for com-
pound 62 complexed with HIV-1 RT (Figure 5), deter-
mined to a resolution of 2.95 Å.21 This structure is
consistent with the first orientation hypothesis.
Contacts with Y181 and Y188. Especially striking
is the effect of the bulky (2-methoxyethyl)methylamino
substituent in the 3-position of the pyridinone ring of
compound 62 on the conformation of the Y181 side
chain. Compared to its position in the unliganded RT,
the side chain of this tyrosine rotates ∼100° about the
side chain dihedral angle ø1.28,29 In the crystal structure
of the 62-RT complex, the (2-methoxyethyl)methy-
lamino substituent at position 3 is aligned with the
aromatic 4-benzyl ring and points toward the hydro-
phobic core of the NNRTI inhibitor binding pocket
formed by the amino acid residues Y181, Y188, W229,
P95, L100, and L234 (Figure 5).
In this orientation, this long chain nitrogen substitu-
ent makes extensive contacts with Y181 that are
stabilized by hydrophobic contacts which the terminal

In Vitro Evaluation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1957
Table 3. Activity (IC50, µM) versus HIV-1
L100I K101E K103N F227L
+
+
+
+
compd LAI
SIa
K103N Y181C Y188L L100I K101E V106A E138K V179E G190A G190S F227C K103N K103N Y181C V106A
31b
32b
35
38
40
42
62
72
0.008
1259 0.126
0.160
0.05
0.158 0.020 0.032
0.2
0.008 ndbb
0.631 0.001 0.005
1.259 0.063 0.079
0.251 0.079 0.058
0.040
ndb
0.020
ndb
0.008
ndb
0.016
ndb
0.003
0.063
0.078
0.016
0.1
0.003
0.010
ndb
0.063
0.013
7.943
0.01
0.032
ndb
0.631 0.063
ndb ndb
0.234 0.020
1.119 0.598
0.869 1.000
0.339 0.121
0.158
ndb
0.007
0.139
0.362
0.069
0.1
0.015
0.158
ndb
0.200
ndb
0.149
3.122
8.680
3.344
10
2.811
0.199
0.398
0.794
0.158
100
0.040
ndb
0.002 12 589 0.01
0.001 12 589 0.003
0.008
0.006
0.002 19 953 0.063
0.001 10 000 0.079
0.001 79 433 0.020
0.001 10 000 0.006
0.001 10 000 0.02
0.008 12 589 0.032
0.072
0.316
0.398
0.316
0.794
0.631
0.032
0.04
ndb
0.004
0.063
0.017
0.010
0.008
0.003
0.006
ndb
0.001
0.032
0.009
0.003
0.004
0.001
0.002
ndb
0.002
0.316
0.062
0.042
0.039
0.003
0.125
ndb
0.794
3981 0.079
7943 0.079
0.316
ndb
ndb
10
0.1
0.003 0.010
ndb
2.512
3.981
0.251
3.162
1
0.398 0.006 0.016
1.259 0.008 0.003
0.251 0.010 0.006
0.316 0.008 ndb
0.063
0.79
0.794
ndb
0.224 0.017
0.794 0.2
77a
77b
9
0.008
ndb
0.04
0.006
5.012
0.04
ndb
0.05
0.1
0.063
0.251 0.05
0.158 0.006 0.008
100 0.316 0.316
0.158 0.04 0.006
0.016
ndb
0.005
0.050
0.002
ndb
0.002
0.195
0.005
ndb
0.004
0.044
0.251
ndb
ndb
nd
ndb
10
0.004
2512 0.01
5012 6.310 10
0.631 0.04
0.135 1.452
0.158 10
0.013
0.509
0.158
0.398
0.163
0.025
NVPc 0.032
EFVc 0.001 10 000 0.04
0.002
0.04
a Selectivity index or ratio of CC50 to IC50 relative to LAI (fold). b nd, not determined. c NVP, nevirapine; EFV, efavirenz.
Compound 35 is 4-fold more potent than 9 against
wild-type RT (Table 1). Compounds 9 and 35 differ only
in that 9 has a methyl at the 6-position, whereas 35
has a hydroxymethyl there. The 62-RT structure
suggests that a hydroxymethyl at the 6-position can
form a hydrogen bond with the main chain carbonyl of
K101. A hydrogen bond between the hydroxyl and K101
is consistent with this improved potency. However, this
interaction is not sufficient to improve inhibitory activity
against Y181C or Y188L mutants (Table 1).
inhibitors. In contrast, a small but significant gain in
sensitivity toward this mutant was observed for com-
pound 35, relative to the lead molecule 10.
Concerning the double mutants, all the new com-
pounds were much more active than efavirenz against
the L100I + K103N double mutant, but when compared
to 10, an important loss in sensitivity was observed for
the more bulky N-3-modified analogues 38, 40, and 62.
Globally, all the N-3 analogues were “comparable” to
efavirenz and 10 in their activity toward the K101E +
K103N mutant. However, they displayed little or no
activity against one or both of the two remaining double
mutant strains. In contrast, the C-5-modified analogue
31b remains active toward these double mutants, sug-
gesting that there is still considerable opportunity to
do SAR at this position. Very interesting also was the
very pronounced gain in activity for compound 35
relative to both efavirenz and lead compound 10 against
the K101E + K103N mutant strain. This observation
is consistent with the prediction that the hydroxyl
substitutent in 35 interacts with polar residues in the
vicinity of amino acids 101-103 in RT.
In summary, the N-3-modified analogues 42, 77a,
77b, and, to a lesser degree, 62 display in vitro activity
profiles which are comparable to those for 10 and
efavirenz. However, the data for the K103N + 181C and
F227L + V106A mutants show that increasing the steric
bulk at the N-3 motif has a negative influence on
activity. Indeed, these results clearly reveal the “Achil-
les’ heal” to the development of new pyridinone ana-
logues wherein the alkyl substituents on the C-3
nitrogen are modified. More encouraging, however, is
that modification of the C-5 substituent in 9 and 10 to
give 31b did not reduce the potency toward these two
double mutants. Compound 35 is less active against the
F227L + V106A mutant, but like 31b, this molecule
offers considerable promise for future development. This
avenue of investigation is being persued.
Having determined that the C-5-modified analogues
31b and 32b, the C-6 hydroxymethyl-substituted ana-
logue 35, the N-3 ethyl/methyl analogues 38, and 77a
and the series of N-modified compounds 40, 42, 62, 72
and 77b all displayed significant activities against the
three key HIV mutant strains, these compounds were
further evaluated against the larger panel of HIV
mutants. The results are presented in Table 3 along
with the values for 9, 10, and the two clinically used
NNRTI type anti-HIV agents nevirapine and efavirenz.
From the data, one sees that nevirapine has a poor
activity profile in vitro against the three crucial mutants
K103N, Y181C, and Y188L, whereas the lead molecule
10 and efavirenz are equipotent inhibitors of nearly all
the single and double mutant strains present in the
panel. Indeed, the only major differences between these
two molecules is that the pyridinone 10 is approximately
30 times less potent than efavirenz against the Y181C
mutant and 60- and 250-fold more potent than this
clinically used NNRTI type anti-HIV agent against the
G190S and the L100I + K103N double mutant, respec-
tively. Discussion of the new pyridinone analogues will
thus focus mainly on their activity against these three
mutant strains as well as against F227C and the three
other double mutant strains (right side of Table 3).
Looking first at the IC50 values against Y181C, we
see that all the new N-3- and C-5/C-6-modified com-
pounds are sub-micromolar inhibitors of this mutant,
but only 32b, 35, and the doubly modified analogues
77a and 77b are interesting relative to efavirenz.
Against the important Y188L mutant, the N-3-modified
compounds 38 and 72 are weakly active, whereas the
other pyridinone analogues are not significantly less
potent than efavirenz. Moving further across the table,
we find that all the new molecules are equipotent or
more active than efavirenz against the G190S mutant.
Against the F227C mutant, compound 38 fails, and
compounds 40, 62, and 77a are only moderately active
Experimental Section
Chemistry. General Remarks. All solvents were reagent
grade. Diethyl ether and tetrahydrofuran (THF) were distilled
from sodium/benzophenone under argon. Acetonitrile and
dichloromethane (CH2Cl2) were distilled from calcium hydride.
Triethylamine was distilled from calcium hydride and stored
over potassium hydroxide. N,N-Dimethylformamide (DMF)
was purchased from Aldrich and used without purification
unless otherwise noted. All reactions were monitored by thin-
layer chromatography (TLC) using E. Merk 60F254 procoated

1958 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Benjahad et al.
silica gel plates. Flash column chromatography was performed
with the indicated solvents and using E. Merk silica gel 60
(particle size of 0.035-0.070 mm unless otherwise stated).
Melting points were taken on a Kofler melting point apparatus
and are uncorrected. Proton NMR spectra were recorded on a
Bruker AC-300 (300 MHz) spectrometer at ambient temper-
ature using an internal deuterium lock. Chemical shifts (δ)
were reported in ppm units (s, d, t, q, m, and br for singlet,
doublet, triplet, quadruplet, multiplet, and broad, respectively).
Elemental analyses, performed by the “Service Central de
Microanalyse du CNRS” Gif-sur-Yvette (France), were within
0.4% of the theoretical values calculated for C, H, and N.
Preparation of 2-Oxo-1,2-dihydropyridin-3-ylcarboni-
triles 15a-j. 2-Oxo-1,2-dihydropyridin-3-ylcarbonitriles 15a,23
15b,22 15c,22 15f,17 15i,24 and 15j24 have been described
previously. Compounds 15g and 15h are commercially avail-
able.
Step 5: NO2 Reduction. A mixture of 6-methoxy-3-methyl-
5-nitro-2-isopropylpyridine (20 g, 0.095 mol) in 1/1 MeOH/THF
(200 mL) was hydrogenated under 2 atm pressure at room
temperature for 1 h using Raney Ni (10 g) as catalyst.
2-Methoxy-5-methyl-6-isopropylpyridin-3-ylamine was ob-
tained as a yellow oil (16.4 g, 96%).
Step 6: N-Pivaloylation. 2,2-Dimethylpropionyl chloride
(12 g, 0.1 mol) in CH2Cl2 (100 mL) was added at 5 °C to a
mixture of 2-methoxy-5-methyl-6-isopropylpyridin-3-ylamine
(16.2 g, 0.09 mol) and triethylamine (13.9 mL) in CH2Cl2 (100
mL). The mixture was stirred at 5 °C for 1 h. Water (150 mL)
was then added, and the mixture was extracted with CH2Cl2.
The combined organic layers were dried over MgSO4 and
concentrated to give the compound 16e as a white solid (24.2
1
g, 100%): mp 45 °C (Et2O); H NMR (DMSO-d6) δ 1.16 (6 H,
d, J ) 6.7 Hz), 1.20 (9 H, s), 2.20 (3 H, s), 3.11 (1 H, m, J ) 6.7
Hz), 3.88 (3 H, s), 7.79 (1 H, s), 8.38 (1 H, s). Anal. (C15H24N2O2)
C, H, N.
6-Methyl-2-oxo-5-isobutyl-1,2-dihydropyridin-3-ylcar-
bonitrile 15d (via 14d). According to the protocol for 15e
described below, compound 15d (mp 214 °C, Et2O) was
prepared in 16% overall yield from 5-methylhexan-2-one 13d.
Preparation of 5,6-Disubstituted-3-dimethylamino-4-
(3,5-dimethylbenzyl)pyridin-2(1H)-ones 18b-i and Com-
pound 9 (via 17a-i). Compound 9 was obtained as previously
described.12
5-Methyl-2-oxo-6-isopropyl-1,2-dihydropyridin-3-yl-
carbonitrile 15e (via 14e). A solution of 2-methylpentan-3-
one 13e (100 g, 1 mol) and HCO2Et (81.4 g, 1.1 mol) was added
slowly at 5 °C to a 30% solution of MeONa in MeOH (210 mL,
1.1 mol) in Et2O (1000 mL). The mixture was stirred at room
temperature for 8 h. The mixture was then concentrated under
reduced pressure providing 14e as a yellow oil (45 g, 30%).
This intermediate, in its crude form (0.3 mol), and 2-cyano-
acetamide (84 g, 1 mol) were added to a solution of piperidine
(63.7 g, 0.75 mol) and HOAc (43 mL, 0.75 mol) in water (1000
mL), and the resultant mixture was refluxed for 8 h. Further
HOAc (165 mL) was subsequently added at 0 °C, and the
precipitate formed was collected, washed with water, and dried
in vacuo. Compound 15e crystallized from Et2O as a white
solid (44 g, 83%): mp 241 °C.
3-Dimethylamino-4-(3,5-dimethylbenzyl)-5-methyl-6-
isopropylpyridin-2(1H)-one 18e: Example of the General
Method. Step 1: Lithiation/Reaction with ArCH2Br.
n-BuLi (1.6 M, 83 mL, 132 mmol) was added dropwise at -78
°C under 1 atm of nitrogen to a solution of N-(2-methoxy-5-
methyl-6-isopropylpyridin-3-yl)-2,2-dimethylpropionamide 16e
(10 g, 38 mmol) and TMEDA (20 mL, 132 mmol) in THF (50
mL). The mixture was stirred at 0 °C for 1 h, then recooled to
-78 °C. A solution of bromomethyl-3,5-dimethylbenzene (26.3
g, 132 mmol) in THF (50 mL) was added dropwise. The
mixture was stirred at 5 °C for 3 h, then poured into water
(100 mL) and extracted with EtOAc. The combined organic
layers were dried over MgSO4 and concentrated, and the
residue was silica gel column chromatographed (CH2Cl2/EtOAc
9/1). N-[4-(3,5-Dimethylbenzyl)-6-isopropyl-2-methoxy-5-meth-
yl-pyridin-3-yl]-2,2-dimethylpropionamide (3.7 g, 25%) was
obtained as a yellow oil.
Preparation of N-(5,6-Disubstituted-2-methoxypyri-
din-3-yl)-2,2-dimethylpropionamides 16a-j. Compounds
16a,12 16g,32 and 16h33 have been previously described.
Step 2: N-Pivaloyl/O-Methyl Imidate Cleavage. A solu-
tion of the above alkylation product (3.0 g, 8.0 mmol) in 6 N
HCl (85 mL) was refluxed overnight, then poured into ice
water, basified with solid K2CO3, and extracted with CH2Cl2.
The combined organic layers were dried over MgSO4 and
concentrated under reduced pressure. The residue was silica
gel column chromatographed (CH2Cl2/MeOH/NH4OH 96/4/0.5).
3-Amino-4-(3,5-dimethylbenzyl)-5-methyl-6-isopropyl-pyridin-
2(1H)-one 18e was obtained as a white solid (0.45 g, 20%).
Step 3: N-Dimethylation. NaBH3CN (1.0 g, 15 mmol) was
added portionwise at room temperature to a solution of 17e
(1.42 g, 5 mmol) and HCHO (37% in H2O, 36 mmol) in CH3-
CN (35 mL). The mixture was stirred at room temperature
for 15 min before slow addition of HOAc (1 mL). The reaction
was stirred at room temperature for 8 h, then basified with
10% aqueous K2CO3 and extracted with CH2Cl2. The combined
organic layers were washed with H2O, dried over MgSO4, and
concentrated under reduced pressure. Crystallization of the
residue from CH3CN provided compound 18e as a white solid
(1.2 g, 77%): mp 182 °C (CH3CN); 1H NMR (CDCl3) δ 1.35 (6
H, d, J ) 7.0 Hz), 1.90 (3 H, s), 2.28 (6 H, s), 2.80 (6 H, s), 3.15
(1 H, m), 4.16 (2 H, s), 6.71 (2 H, s), 6.83 (1 H, s), 11.35 (1 H,
br s). Anal. (C20H28N2O‚0.33H2O) C, H, N.
N-(2-Methoxy-5-methyl-6-isopropylpyridin-3-yl)-2,2-
dimethylpropionamide 16e: Example of the General
Method. Step 1: Hydrolysis/Decarboxylation. A solution
of compound 15e (42 g, 0.24 mol) in 6 N HCl (400 mL) was
refluxed for 6 days, then poured into ice water and basified
with NH4OH. The precipitate was collected, washed with
water, and dried in vacuo to give 5-methyl-6-isopropylpyridin-
2(1H)-one (33 g, 91%) as a white solid.
Step 2: Nitration. Nitric acid (d 1.52, 16.6 mL, 0.4 mol)
was added slowly to a solution of 5-methyl-6-isopropylpyridin-
2(1H)-one (30 g, 0.2 mol) in 98% H2SO4 (300 mL), making sure
that the temperature did not exceed 15 °C. The mixture was
stirred at 5 °C for 1 h and poured into ice water. The
precipitate was collected, washed with water, and dried in
vacuo to give 5-methyl-3-nitro-6-isopropylpyridin-2(1H)-one (35
g, 90%) as a white powder.
Step 3: Chlorination. POCl3 (47 mL, 0.5 mol) was added
slowly at room temperature to a solution of 5-methyl-3-nitro-
6-isopropylpyridin-2(1H)-one (33 g, 0.168 mol) and benzyltri-
ethylammonium chloride (19.2 g, 0.084 mol) in CH3CN (350
mL). The mixture was stirred at 80 °C for 8 h, then concen-
trated to dryness. The residue was taken up in ice water,
basified with NH4OH, and extracted with CH2Cl2. The com-
bined organic layers were dried over MgSO4 and concentrated
to give 2-chloro-5-methyl-3-nitro-6-isopropylpyridine as an oil
(37 g, 100%).
Preparation of the Compounds 5,6-Disubstituted-3-
dimethylamino-4-(3-methylbenzoyl)pyridin-2(1H)-ones
20a-c,i,j and 10 (via 19a-c,i,j). Compounds 10 and 20a
were obtained as described previously.13
Step 4: Methoxylation. A solution of 30% NaOMe in
MeOH (98 mL, 0.517 mol) was added slowly at room temper-
ature to a solution of 2-chloro-5-methyl-3-nitro-6-isopropyl-
pyridine (37 g, 0.172 mol) in MeOH (350 mL). The mixture
was stirred at room temperature for 8 h, then poured into ice
water and extracted with CH2Cl2. The combined organic layers
were dried (MgSO4) and concentrated to give 6-methoxy-3-
methyl-5-nitro-2-isopropylpyridine (32 g, 88%) as an oil.
3-Dimethylamino-4-(3-methylbenzoyl)-5,6-dimethylpy-
ridin-2(1H)-one 20b: Example of the General Method.
Step 1: Lithiation/Condensation with ArCHO. n-BuLi
(1.6 M, 33 mL, 53 mmol) was added dropwise at -78 °C under
1 atm of nitrogen to a mixture of N-(2-methoxy-5,6-dimeth-
ylpyridin-3-yl)-2,2-dimethylpropionamide 16b (3.54 g, 15 mmol)
and TMEDA (8 mL, 53 mmol) in THF (50 mL). The mixture
was stirred at 0 °C for 1 h and cooled again to -78 °C. A

In Vitro Evaluation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1959
solution of 3-methylbenzaldehyde (5.5 g, 46 mmol) in THF (50
mL) was added dropwise. The mixture was stirred at 0 °C for
3 h, then poured into water (50 mL) and extracted with Et2O.
The combined organic layers were dried over MgSO4, filtered,
and concentrated under reduced pressure. The residue was
crystallized from cyclohexane to give N-[4-((3-methylphenyl)-
hydroxymethyl)-2-methoxy-5,6-dimethylpyridin-3-yl]-2,2-di-
methylpropionamide as a white solid (2.9 g, 40%).
Step 2: Oxidation. MnO2 (10 g) in toluene (54 mL) was
added portionwise at reflux to a solution of the above alcohol
(8.5 g, 24 mmol) in toluene (100 mL), and the mixture was
refluxed for 2 h. The solids were removed by filtration through
Celite, and the filtrate was concentrated under reduced
pressure. The residue was crystallized from Et2O providing
N-[2-methoxy-5,6-dimethyl-4-(3-methylbenzoyl)pyridin-3-yl]-
2,2-dimethylpropionamide as a white powder (8.2 g, 95%).
Step 3: N-Pivaloyl/O-Methyl Imidate Cleavage. A
mixture of the derived 4-benzoylpyridinone (7.1 g, 20 mmol)
in 6 N HCl (72 mL) was refluxed for 3 h, then poured into ice
water and basified with concentrated NH4OH. The precipitate
that formed was collected and taken up in CH2Cl2. The
combined organic layers were dried over MgSO4, filtered, and
concentrated. The residue was crystallized from Et2O providing
3-amino-5,6-dimethyl-4-(3-methylbenzoyl)pyridin-2(1H)-one 19b
as a white solid (5 g, 96%).
acetone: mp 142 °C; 1H NMR (CDCl3) δ 1.80 (2 H, m), 2.44 (3
H, s), 2.56 (2 H, t, J ) 7.8 Hz), 2.73 (3 H, s), 3.35 (3 H, s), 3.38
(2 H, t, J ) 6.0 Hz), 8.13 (1 H, s), 13.45 (1 H, s).
N-[2-Methoxy-5-(2-methoxyethyl)-6-methylpyridin-3-
yl]-2,2-dimethylpropionamide 28a. Step 1: Oximation.
A solution of 24a (8.4 g, 40 mmol) and hydroxyamine (2 g, 60
mmol) in ethanol (100 mL) was stirred at room temperature
for 2 h. A 10% solution of potassium carbonate (50 mL) was
then added, and the mixture was extracted with CH2Cl2. The
combined organic layers were dried over MgSO4 and concen-
trated in vacuo. The residue was crystallized from 2-pro-
panone/Et2O. 3-(1-Hydroxyiminoethyl)-5-(2-methoxyethyl)-6-
methylpyridin-2(1H)-one 25a was obtained as a white solid
(9 g, 100%): mp 172 °C.
Step 2: Beckmann Rearrangement. A solution of oxime
25a (8.5 g, 38 mmol) in formic acid (100 mL) was stirred at
100 °C for 1 h. The mixture was then concentrated to dryness,
redissolved in CH2Cl2, and washed with 10% aqueous K2CO3.
The organic layer was dried over MgSO4 and concentrated in
vacuo. The residue was crystallized from 2-propanone/Et2O.
N-[5-(2-Methoxyethyl)-6-methyl-2-oxo-1,2-dihydropyridin-3-yl]-
acetamide 26a was obtained as a white solid (7.1 g, 83%): mp
206 °C.
Step 3: Hydrolysis/Amine Protection. A solution of 26a
(6.7 g, 30 mmol) in 3 N HCl (50 mL) was refluxed for 2 h. The
mixture was then concentrated to dryness, and the residue
was recrystallized from 2-propanone/Et2O to give 3-amino-5-
(2-methoxyethyl)-6-methylpyridin-2(1H)-one (6 g, 52%) as a
white solid (mp 160 °C). To this 3-aminopyridinone intermedi-
ate (8.9 g, 49 mmol) in CH2Cl2 (100 mL) and Et3N (20.8 mL)
was added slowly at 5 °C a solution of 2,2-dimethylpropionyl
chloride (5.9 g, 49 mmol) in CH2Cl2 (50 mL). The mixture was
stirred at room temperature for 1 h. The resulting mixture
was washed with 10% K2CO3 solution, dried over MgSO4, and
concentrated in vacuo. The residue was crystallized from
2-propanone/Et2O. Compound 27a was obtained as a white
Step 4: N-Methylation. Intermediate 19b (6.7 g, 26.1
mmol) was dimethylated following the procedure for 18e.
Compound 20b was obtained as a white solid (2.2 g, 30%): mp
1
250 °C (Et2O); H NMR (CDCl3) δ 1.82 (3 H, s), 2.35 (3 H, s),
2.44 (3 H, s), 2.64 (6 H, s), 7.33-7.47 (2 H, m), 7.62 (1 H, d, J
) 7.0 Hz), 7.73 (1 H, s), 13.20 (1 H, br s). Anal. (C17H20N2O2‚
0.20H2O) C, H, N.
5-(3-Methoxypropyl)-6-methyl-2-oxo-1,2-dihydropyri-
din-3-ylcarbonitrile 23b (via 22b). As described for 14e,
6-methoxyhexan-2-one 21b34 (16 g, 123 mmol) was converted
to sodium 3-oxo-2-(3-methoxypropyl)but-1-en-1-olate 22b (white
solid; 6.6 g, 30%). This product was contaminated with small
amounts of sodium 3-oxo-7-methoxyhept-1-en-1-olate. The
crude mixture of 22b (13.3 g, 80 mmol) and 2-cyanoacetamide
(7.4 g, 88 mmol) in water (150 mL) was added to a solution of
piperidine (5.1 g, 60 mmol) and HOAc (3.4 mL, 60 mmol) in
water (150 mL), and the resultant mixture was refluxed for
48 h. Further HOAc (10 mL) was then added at 0 °C, and the
precipitate formed was collected, washed with H2O, and dried
in vacuo. It was dissolved in CH2Cl2, washed with 10% aqueous
K2CO3, dried over MgSO4, and concentrated. Compound 23b
crystallized from Et2O as a white solid (5.5 g, 33% yield): mp
1
solid (2.5 g, 19%): mp 175 °C; H NMR (DMSO-d6) δ 1.21 (9
H, s), 2.15 (3 H, s), 2.74 (2 H, t, J ) 6.5 Hz), 3.24 (3 H, s), 3.40
(2 H, t, J ) 6.5 Hz), 8.10 (1 H, s), 8.47 (1 H, s), 11.90 (1 H, s).
Step 4: O-Methylation. A mixture of 27a (10 g, 38 mmol),
MeI (10.6 g, 75 mmol), and Ag2CO3 (20.6 g, 75 mmol) in CH2-
Cl2 (100 mL) was stirred at room temperature for 4 days. The
mixture was then filtered through Celite, washed with CH2-
Cl2, and concentrated. The residue was silica gel column
chromatographed (cyclohexane/EtOAc 90/10). Compound 28a
crystallized from petroleum ether as a white solid (5.1 g,
1
48%): mp 85 °C; H NMR (DMSO-d6) δ 1.22 (9 H, s), 2.36 (3
1
H, s), 2.74 (2 H, t, J ) 6.5 Hz), 3.24 (3 H, s), 3.47 (2 H, t, J )
172 °C; H NMR (CDCl3) δ 1.73-1.86 (2 H, m), 2.47 (3 H, s),
6.5 Hz), 3.87 (3 H, s), 7.88 (1 H, s), 8.37 (1 H, s).
2.54 (3 H, t, J ) 7.3 Hz), 3.61 (3 H, s), 3.37 (2 H, t, J ) 5.8
N-[2-Methoxy-5-(3-methoxypropyl)-6-methylpyridin-3-
yl]-2,2-dimethylpropionamide 28b. Compound 28b (a white
solid) was prepared from 3-acetyl-5-(3-methoxypropyl)-6-me-
thylpyridin-2(1H)-one 24b by the four step procedure used to
obtain 28a. The yields for the individual steps were compa-
rable: mp 140 °C (Et2O); 1H NMR (DMSO-d6) δ 1.21 (9 H, s),
1.70 (2 H, m), 2.34 (3 H, s), 2.51 (2 H, t, J ) 6.5 Hz), 3.25 (3
H, s), 3.31 (2 H, m), 3.87 (3 H, s), 7.85 (1 H, s), 8.35 (1 H, s).
5-(2-Methoxyethyl)-6-methyl-3-dimethylamino-4-(3-me-
thylbenzoyl)pyridin-2(1H)-one 31a (via 29a). As for com-
pound 20b, the 5-(2-methoxyethyl)-substituted pyridin-2(1H)-
one 31a was prepared in four steps from 28a.
Hz), 7.73 (1 H, s), 13.6 (1 H, s).
3-Acetyl-5-(2-methoxyethyl)-6-methylpyridin-2(1H)-
one 24a (via 22a). As described for 14e, 5-methoxypentan-
2-one 21a35 (16 g, 138 mmol) was converted to sodium 3-oxo-
2-(2-methoxyethyl)but-1-en-1-olate 22a (white solid; 6.9 g,
30%). This product was contaminated with small amounts of
sodium 3-oxo-6-methoxyhex-1-en-1-olate. The crude mixture
of 22a (21 g, 126 mmol) and 2-acetoacetamide (14 g, 139 mmol)
in water (100 mL) was added to a solution of piperidine (8.1
g, 95 mmol) and HOAc (5.5 mL, 95 mmol) in water (100 mL),
and the resultant mixture was refluxed for 48 h. Further HOAc
(10 mL) was then added at 0 °C, and after 30 min, the mixture
was extracted with CH2Cl2, dried over MgSO4, and concen-
trated to afford a residue that was silica gel column chromato-
graphed (CH2Cl2/MeOH/NH4OH 97/3/0.1). Crystallization from
2-propanone afforded 24a as a white solid (1.5 g, 5%): mp 172
°C; 1H NMR (CDCl3) δ 2.46 (3 H, s), 2.71 (2 H, t, J ) 6.6 Hz),
2.72 (3 H, s), 3.34 (3 H, s), 3.52 (2 H, t, J ) 6.5 Hz), 8.14 (1 H,
s), 13.55 (1 H, s). Anal. (C11H14N2O2) C, H, N.
Step 1: Lithiation/Condensation with 3-Methylben-
zaldehyde. Intermediate 29a was obtained as a white solid
in 60% yield (15 mmol scale).
Step 2: Oxidation. Jones’ reagent (20 mmol) was added
slowly at 5 °C to a solution of 29a (4 g, 10 mmol) in
2-propanone (60 mL). The mixture was stirred at 5 °C for 1 h,
then poured into ice water, basified using solid K2CO3, and
filtered through Celite (washing with CH2Cl2), and the filtrate
was extracted with CH2Cl2. The combined organic layers were
dried over MgSO4 and concentrated in vacuo. Crystallization
of the residue from Et2O provided N-[2-methoxy-5-(2-meth-
oxyethyl)-6-methyl-4-(3-methylbenzoyl)pyridin-3-yl]-2,2-di-
methylpropionamide as a white solid (1.9 g, 47%).
3-Acetyl-5-(3-methoxypropyl)-6-methylpyridin-2(1H)-
one 24b (via 22b). Following the procedure for 24a, we
reacted sodium 3-oxo-2-(3-methoxypropyl)but-1-en-1-olate 22b
(see protocol for 23b) with 2-acetoacetamide. Compound 24b
was obtained as a white solid (5% yield) after silica gel column
chromatography (CH2Cl2/MeOH 95/5) and crystallization from

1960 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Benjahad et al.
Step 3: N-Pivaloyl/O-Methyl Imidate Cleavage. 3-Amino-
5-(2-methoxyethyl)-6-methyl-4-(3-methylbenzoyl)pyridin-2(1H)-
one was obtained as a white solid after crystallization from
CH3CN (46%, 8 mmol scale).
3712 (3.0 g, 10.5 mmol) and acetaldehyde (2.90 mL, 52 mmol)
in CH3CN (70 mL). The mixture was stirred at room temper-
ature for 15 min before slow addition of HOAc (0.1 mL). The
reaction was stirred at room temperature for 12 h, then
basified with 10% aqueous K2CO3 and extracted with CH2Cl2.
The combined organic layers were washed with H2O, dried over
MgSO4, and concentrated over reduced pressure. The residue
was silica gel column chromatographed (CH2Cl2/MeOH 98.5/
1.5). The pure fractions were collected, and the solvent was
evaporated. Crystallization of the residue from i-Pr2O provided
compound 38 as a white solid: mp 170 °C (2.30 g, 70% yield);
1H NMR (CDCl3) δ 0.93 (6 H, m), 2.27 (11 H, m), 2.71 (3 H, s),
3.09 (2 H, m), 4.15 (2 H, m), 6.69 (2 H, s), 6.81 (1 H, s), 12.80
(1H, br s). Anal. (C20H28N2O‚0.20H2O) C, H, N.
N-[4-(3,5-Dimethylbenzyl)-5-ethyl-6-methyl-2-oxo-1,2-
dihydropyridin-3-yl]butyramide 46. Butyryl chloride (0.95
g, 8.9 mmol) was added dropwise at 10 °C to a suspension of
17a (2.0 g, 7.4 mmol) and Et3N (1.55 mL, 11.1 mmol) in CH2-
Cl2 (30 mL). The mixture was stirred for 1 h and poured out
into a H2O and 10% K2CO3 solution. The precipitate was
filtered off, washed with CH2Cl2, and dried. Compound 46 was
obtained as a white powder (1.94 g, 77%): mp 250 °C (Et2O);
1H NMR (CDCl3) δ 1.01 (6 H, m), 1.76 (2 H, m), 2.36 (13 H,
m), 3.96 (2 H, s), 6.65 (2 H, s), 6.85 (1 H, s), 7.02 (1 H, br s),
12.85 (1 H, br s). Anal. (C21H28N2O2) C, H, N.
Step 4: N-Methylation. Compound 31a was obtained as a
1
white solid (33%, 8 mmol scale): mp 182 °C (Et2O); H NMR
(CDCl3) δ 1.65 (2 H, s), 2.41 (3 H, s), 2.44 (3 H, s), 2.62 (6 H,
s), 3.19 (3 H, s), 3.30 (2 H, s), 7.38-7.42 (2 H, m), 7.65 (1 H, d,
J ) 7.1 Hz), 7.72 (1 H, s), 12.75 (1 H, br s). Anal. (C19H24N2O3‚
0.50H2O) C, H, N.
5-(3-Methoxypropyl)-6-methyl-3-dimethylamino-4-(3-
methylbenzoyl)pyridin-2(1H)-one 31b (via 29b). Following
the protocol for 31a, we prepared compound 31b in four steps
(43, 95, 100, and 50% yields) from N-[2-methoxy-5-(3-meth-
oxypropyl)-6-methylpyridin-3-yl]-2,2-dimethylpropionamide
1
28b: mp 172 °C (Et2O); H NMR (DMSO-d6) δ 1.29-1.32 (2
H, m), 1.94-2.11 (2 H, m), 2.19 (3 H, s), 2.38 (3 H, s), 2.44 (6
H, s), 3.08 (3 H, s), 3.10-3.15 (2 H, m), 7.38-7.50 (2 H, m),
7.55 (1H, d, J ) 8.8 Hz), 7.62 (1 H, s), 11.75 (1 H, br s). Anal.
(C20H26N2O3) C, H, N.
5-(3-Methoxypropyl)-6-methyl-3-dimethylamino-4-(3,5-
dimethylbenzoyl)pyridin-2(1H)-one 32b (via 30b). Fol-
lowing the protocol for 31a, we prepared compound 32b in four
steps (46, 100, 60, and 22% yields) from N-[2-methoxy-5-(3-
methoxypropyl)-6-methylpyridin-3-yl]-2,2-dimethylpropiona-
1
3-Butylamino-4-(3,5-dimethylbenzyl)-5-ethyl-6-meth-
ylpyridin-2(1H)-one 49. A mixture of 46 (1.12 g, 3.3 mmol)
in THF (10 mL) was stirred at room temperature under N2
flow, and LiAlH4 (0.50 g, 13.2 mmol) was added. The mixture
was stirred for 15 h and cooled to 5 °C. Water (2 mL) was
added dropwise, then CH2Cl2 (30 mL) was added. The pre-
cipitate was filtered off and washed with CH2Cl2. The filtrate
was dried (MgSO4) and filtered, and the solvent was evapo-
rated into dryness. The residue was silica gel column chro-
matographed (CH2Cl2/CH3OH/NH4OH 98.5/1.5/0.1). Crystal-
lization of the residue from Et2O provided compound 49 as a
white solid (0.50 g, 46%): mp 106 °C (Et2O); 1H NMR (CDCl3)
δ 0.83 (3 H, t, J ) 7.3 Hz), 0.96 (3 H, t, J ) 7.3 Hz), 1.24 (2 H,
sextuplet, J ) 7.3 Hz), 1.45 (2 H, quintuplet, J ) 7.3 Hz), 2.28
(11 H, m), 2.91 (2 H, t, J ) 7.3 Hz), 3.99 (2 H, s), 4.20 (1 H, br
s), 6.75 (2 H, s), 6.84 (1 H, s), 12.80 (1 H, br s). Anal.
(C21H30N2O) C, H, N.
mide 28b: mp 210 °C (i-Pr2O); H NMR (DMSO-d6) δ 1.29-
1.57 (2 H, m), 1.81-2.17 (2 H, m), 2.18 (3 H, s), 2.33 (6 H, s),
2.44 (6 H, s), 3.02-3.20 (5 H, m), 7.29 (1 H, s), 7.38 (2 H, s),
11.70 (1 H, br s). Anal. (C21H28N2O3‚0.20H2O) C, H, N.
4-(3,5-Dimethylbenzoyl)-5-(2,2-dimethylpropionylami-
no)-3-ethyl-6-methoxypyridin-2-ylmethyl Acetate 34. To
N-[5-ethyl-2-methoxy-6-methyl-4-(3,5-dimethylbenzoyl)pyridin-
3-yl]-2,2-dimethylpropionamide 3312 (12.3 g, 32 mmol) in CH2-
Cl2 (120 mL) was added m-chloroperoxybenzoic acid (11.1 g,
64 mmol) at 5 °C. The mixture was stirred at 5 °C for 1 h,
then at room temperature for 3 days. The mixture was poured
into water, extracted with CH2Cl2, dried over MgSO4, filtered,
and evaporated. The residue was purified by chromatography
over silica gel with CH2Cl2/CH3OH/NH4OH 96/4/0.1 as eluent
to afford the intermediate N-[4-(3,5-dimethylbenzoyl)-5-ethyl-
2-methoxy-6-methyl-1-oxide-pyridin-3-yl]-2,2-dimethylpropi-
onamide (1.2 g, 9%) that was used without further purification
in the next step.
In an identical fashion, compounds 4412 and 4512 were
converted to 47 and 48, respectively.
The pyridine N-oxide obtained above (3.0 g, 7.5 mmol) in
acetic anhydride (30 mL) was refluxed for 2 h, then poured
out on ice, basified with NH4OH and extracted with CH2Cl2,
dried over MgSO4, filtered, and evaporated. The residue was
purified by chromatography over silica gel with cyclohexane/
EtOAc 75/25 as eluent to give a product, which was crystallized
from CH3CN to afford the titled compound 34 (1.1 g, 33%) that
was used without further purification or characterization in
the following steps.
3-Dimethylamino-4-(3,5-dimethylbenzoyl)-5-ethyl-6-
hydroxymethylpyridin-2(1H)-one 35. 4-(3,5-Dimethylben-
zoyl)-5-(2,2-dimethylpropionylamino)-3-ethyl-6-methoxypyridin-
2-ylmethyl acetate (34, 800 mg, 1.8 mmol) in concentrated HCl
(25 mL) was refluxed for 4 h, poured out on ice, basified with
solid K2CO3 and extracted with CH2Cl2, dried over MgSO4,
filtered, and evaporated. The residue was purified by chro-
matography over silica gel with CH2Cl2/CH3OH 98/2 as eluent
to afford 3-amino-4-(3,5-dimethylbenzoyl)-5-ethyl-6-hydroxy-
methylpyridin-2(1H)-one (54 mg, 10%) that was submitted to
N-methylation as described for 18e giving the titled compound
35 (20 mg, 34%) after chromatography over silica gel using
CH2Cl2/CH3OH 98/2 as eluent: mp > 200 °C (diethyl ether);
1H NMR (DMSO-d6) δ 0.82 (3 H, t, J ) 7.6 Hz), 2.28 (6 H, s),
2.50 (2 H, q, J ) 7.6 Hz), 3.32 (6 H, s), 4.62 (2 H, br s), 4.88 (1
H, br s), 7.22 (1 H, s), 7.41 (2 H, s), 13.14 (1 H, s). Anal.
(C19H24N2O3) C, H, N.
Compound 47. Yield 45%; mp 137 °C (Et2O); 1H NMR
(DMSO-d6) δ 0.95 (3 H, t, J ) 7.5 Hz), 1.07 (3 H, t, J ) 7.1
Hz), 2.31 (11 H, m), 2.96 (2 H, q, J ) 7.5 Hz), 3.99 (3 H, m),
6.74 (2 H, s), 6.84 (1H, s), 12.40 (1 H, br s). Anal. (C19H26N2O)
C, H, N.
Compound 48. Yield 42%; mp 108 °C (Et2O); 1H NMR
(DMSO-d6) δ 0.73 (3 H, t, J ) 7.4 Hz), 0.84 (3 H, t, J ) 7.2
Hz), 1.32 (2 H, sextuplet, J ) 7.2 Hz), 2.15 (11 H, m), 2.76 (2
H, q, J ) 7.2 Hz), 3.86 (2 H, s), 4.20 (1 H, t, J ) 7.2 Hz), 6.70
(2 H, s), 6.80 (1 H, s), 11.40 (1 H, br s). Anal. (C20H28N2O) C,
H, N.
3-Diethylamino-4-(3,5-dimethylbenzyl)-5-ethyl-6-meth-
ylpyridin-2(1H)-one 50. As for 51, compound 50 was pre-
pared from 17a (55% yield): mp 188 °C (i-Pr2O); 1H NMR
(DMSO-d6) δ 0.79 (9 H, m), 2.17 (11 H, m), 2.97 (4 H, m), 4.12
(2 H, s), 6.64 (2 H, s), 6.78 (1 H, s), 11.30 (1 H, br s). Anal.
(C21H30N2O) C, H, N.
3-Dibutylamino-4-(3,5-dimethylbenzyl)-5-ethyl-6-me-
thylpyridin-2(1H)-one 51. Sodium cyanoborohydride (1.4 g,
22.2 mmol) was added portionwise at room temperature to a
solution of 17a (2.0 g, 7.4 mmol) and butyraldehyde (5.30 g,
74 mmol) in MeOH (60 mL). The mixture was stirred at room
temperature for 15 min before slow addition of HOAc (0.1 mL).
The reaction was stirred at room temperature for 12 h, then
basified with 10% aqueous K2CO3 and extracted with CH2Cl2.
The combined organic layers were washed with H2O, dried over
MgSO4, and concentrated under reduced pressure. The residue
was silica gel column chromatographed (CH2Cl2/MeOH 97/3).
The pure fractions were collected, and the solvent was
evaporated. Crystallization of the residue from a small amount
Preparation of 3-Methylamino-Substituted Pyridino-
nes 38-43. 4-(3,5-Dimethylbenzyl)-5-ethyl-3-(ethylmethyl-
amino)-6-methylpyridin-2(1H)-one 38: Example of the
General Method. Sodium cyanoborohydride (2.0 g, 32 mmol)
was added portionwise at room temperature to a solution of

In Vitro Evaluation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1961
of CH3OH provided compound 51 as a white solid (2.1 g, 74%):
mp 109 °C (MeOH); 1H NMR (DMSO-d6) δ 0.75 (9 H, m), 1.15
(8 H, m), 2.13 (5 H, m), 2.19 (6 H, s), 2.90 (4 H, m), 4.10 (2 H,
s), 6.60 (2 H, s), 6.80 (1 H, s), 11.25 (1 H, br s). Anal.
(C25H38N2O) C, H, N.
up in CH2Cl2 while stirring. The mixture was filtered and
washed with CH2Cl2. The filtrate was dried (MgSO4) and
filtered, and the solvent was evaporated. The residue was silica
gel column chromatographed (CH2Cl2/CH3OH/NH4OH 97.5/2.5/
0.2). The desired fractions were collected, and the solvent was
evaporated. Crystallization from 2-propanone and i-Pr2O
provided compound 56 as an oil (0.87 g, 32%): 1H NMR
(DMSO-d6) δ 0.81 (3 H, t, J ) 7.3 Hz), 2.19 (11 H, m), 3.53 (2
H, s), 6.07 (2 H, s), 6.59 (4 H, m), 6.79 (1 H, s), 11.90 (1 H, br
s). Anal. (C21H24N2O) C, H, N.
3-Benzylamino-4-(3,5-dimethylbenzyl)-5-ethyl-6-meth-
ylmethylpyridin-2(1H)-one 52 and 3-Dibenzylamino-4-
(3,5-dimethylbenzyl)-5-ethyl-6-methylpyridin-2(1H)-
one 53. Sodium cyanoborohydride (1.5 g, 24 mmol) was added
portionwise at room temperature to a solution of 17a (2.2 g, 8
mmol) and benzaldehyde (1.27 g, 12 mmol) in CH3CN (50 mL).
The mixture was stirred at room temeperature for 15 min
before slow addition of HOAc (0.1 mL). The reaction was
stirred at room temperature for 12 h, then basified with 10%
K2CO3 solution. The precipitate was filtered off, washed with
H2O, and dried. The residue was silica gel column chromato-
graphed (CH2Cl2/MeOH/NH4OH 98/2/0.1). The pure fractions
were collected, and the solvent was evaporated. Crystallization
of the residue from i-Pr2O provided compound 52 as a white
solid (1.3 g, 44%): mp 137 °C (i-Pr2O); 1H NMR (DMSO-d6) δ
0.82 (3 H, t, J ) 7.2 Hz), 2.15 (11 H, m), 3.86 (2 H, s), 4.07 (2
H, d, J ) 7.2 Hz), 4.70 (1 H, t, J ) 7.2 Hz), 6.66 (2 H, s), 6.80
(1 H, s), 7.20 (5 H, m), 11.40 (1 H, br, s). Anal. (C24H28N2O‚
0.25H2O) C, H, N.
The mother layer was extracted with CH2Cl2, the organic
layer was separated, dried (MgSO4), and filtered, and the
solvent was evaporated. The residue was silica gel column
chromatographed (CH2Cl2/MeOH/NH4OH 98/2/0.1). Crystal-
lization of the residue from i-Pr2O provided compound 53 as
a white solid (0.55 g, 15%): mp 188 °C (i-Pr2O); 1H NMR
(DMSO-d6) δ 0.59 (3 H, t, J ) 7.1 Hz), 2.00 (2 H, q, J ) 7.1
Hz), 2.10 (9 H, s), 3.70 (2 H, s), 4.13 (4 H, s), 6.44 (2 H, s), 6.70
(1 H, s), 7.15 (10 H, m), 11.45 (1 H, br s). HRMS: calcd for
C31H34N2O (MH)+ m/z 451.1275; found, 451.1275.
4-(3,5-Dimethylbenzyl)-5-ethyl-6-methyl-3-(morpholin-
4-yl)pyridin-2(1H)-one 54. Sodium cyanoborohydride (2.1 g,
33 mmol) was added slowly to a solution of 17a (3.0 g, 11
mmol) and 2,2′-oxybis(acetaldehyde), prepared in situ from 1,4-
anhydroerythritol36 (1.12 g, 11 mmol; 0.4 M in H2O/CH3CN).
After addition, HOAc (0.8 mL) was added. The mixture was
stirred at room temperature for 2 h, poured out into H2O, and
extracted with CH2Cl2. The organic layer was separated, dried
(MgSO4), and filtered, and the solvent was evaporated. The
residue was silica gel column chromatographed (CH2Cl2/
MeOH/NH4OH 97/3/0.1). The pure fractions were collected,
and the solvent was evaporated. Crystallization of the residue
from Et2O/petroleum ether provided compound 54 as a white
solid (1.32 g, 35%): mp 163 °C (Et2O/petroleum ether); 1H
NMR (DMSO-d6) δ 0.81 (3 H, t, J ) 7.2 Hz), 2.18 (11 H, m),
2.25 (2 H, m), 3.60 (6 H, m), 4.00 (2 H, s), 6.65 (2 H, s), 6.76 (1
H, s), 11.40 (1 H, br s). Anal. (C21H28N2O2‚0.50H2O) C, H, N.
4-(3,5-Dimethylbenzyl)-5-ethyl-6-methyl-3-(piperidin-
1-yl)pyridin-2(1H)-one 55. Sodium cyanoborohydride (465
mg, 7.4 mmol) was added slowly to a solution of 17a (2.0 g,
7.4 mmol) and glutaraldehyde (2.22 g, 22.2 mmol) in CH3CN
(50 mL). After addition, HOAc (0.1 mL) was added. The
mixture was stirred at room temperature for 2 h, poured out
into H2O, and extracted with CH2Cl2. The organic layer was
separated, dried (MgSO4), and filtered, and the solvent was
evaporated. The residue was silica gel column chromato-
graphed (CH2Cl2/MeOH/NH4OH 97/3/0.1). The pure fractions
were collected, and the solvent was evaporated. Crystallization
of the residue from CH3OH provided compound 55 as a white
solid (0.88 g, 33%): mp 208 °C (CH3OH); 1H NMR (DMSO-d6)
δ 0.81 (3 H, t, J ) 7.1 Hz), 1.37 (6 H, m), 2.12 (3 H, s), 2.19 (6
H, s), 2.20 (2 H, m), 2.51 (2 H, m), 3.39 (2 H, m), 3.99 (2 H, s),
6.68 (2 H, s), 6.80 (1 H, s), 11.30 (1 H, br s). Anal. (C22H30N2O)
C, H, N.
5-Ethyl-6-methyl-3-(methylamino)-4-(3-methylbenzyl)-
pyridin-2(1H)-one 58. A mixture of 5713 (20 g, 78 mmol) in
ethyl formate (460 mL) was stirred at room temperature for
15 min, then HOAc (250 mL) was added, and the mixture was
refluxed for 4 h. The solvent was evaporated till dryness, and
the residue was taken up in H2O, basified with a concentrated
NH4OH solution, and extracted with CH2Cl2. The organic layer
was separated, dried (MgSO4), and filtered, and the solvent
was evaporated. The residue was silica gel column chromato-
graphed (CH2Cl2/CH3OH/NH4OH 92/8/0.1). Crystallization of
the residue from Et2O provided the formamide intermediate
as a white powder (20.4 g, 92%) which was treated by addition
portionwise at room temperature of LiAlH4 (7.7 g, 200 mmol)
in THF. The mixture was stirred at room temperature for 3
h, and H2O (8 mL) was added dropwise at 0 °C. The organic
layer was separated, washed with CH2Cl2, and dried. Crystal-
lization from Et2O provided (9.0 g, 46%) of compound 58 as a
1
white solid: mp 204 °C (Et2O); H NMR (DMSO-d6) δ 0.85 (3
H, t, J ) 7.4 Hz), 2.12 (5 H, m), 2.26 (3 H, s), 2.67 (3 H, s),
4.15 (2 H, s), 4.31 (1 H, s), 7.41 (4 H, m), 12.75 (1 H, br s).
Preparation of 3-Methylamino-Substituted Pyri-
dinones 59-63. 4-(3-Methylbenzyl)-5-ethyl-3-[N-(3-hy-
droxypropyl)-N-methyl]amino-6-methylpyridin-2(1H)-
one 60: Example of the General Method. Sodium cy-
anoborohydride (1.38 g, 22 mmol) was added portionwise at
room temperature under N2 flow to a mixture of 58 (2.0 g, 7.4
mmol) and 3-hydroxypropionaldehyde37 (1.1 g, 18.5 mmol) in
CH3CN (50 mL). The mixture was stirred for 5 h, then H2O
was added, and the mixture was extracted with AcOEt. The
organic layer was separated, dried (MgSO4), and filtered, and
the solvent was evaporated. The residue was silica gel column
chromatographed (CH2Cl2/CH3OH 94/6). Crystallization of the
residue from Et2O/i-Pr2O provided compound 60 as a white
powder (500 mg, 20%): mp 110 °C (Et2O/i-Pr2O); 1H NMR
(DMSO-d6) δ 0.79 (3 H, m), 1.43 (2 H, s), 2.16 (8 H, m), 2.54 (3
H, s), 2.98 (2 H, m), 3.32 (2 H, s), 4.14 (2 H, m), 4.26 (1 H, s),
6.82 (1 H, m), 6.88 (1 H, m), 6.95 (1 H, m), 7.14 (1 H, m), 11.30
(1 H, br s). Anal. (C20H28N2O2‚0.20H2O) C, H, N.
N-{[4-(3-Methylbenzyl)-5-ethyl-6-methyl-2-oxo-1,2-di-
hydropyridin-3-yl]}-4-(methylamino)butyronitrile 66. A
mixture of 58 (3.0 g, 11 mmol), 4-chlorobutyronitrile (1.35 g,
13 mmol), and Et3N (1.8 mL, 13 mmol) in DMF (30 mL) was
stirred at 100 °C for 48 h, poured out into H2O, and extracted
with AcOEt. The organic layer was separated, dried (MgSO4),
and filtered, and the solvent was evaporated. The residue was
silica gel column chromatographed (CH2Cl2/ 2-propanol 95/5).
Crystallization of the residue from Et2O/i-Pr2O provided
compound 66 as a white powder (100 mg, 3%): mp 130 °C
(Et2O/i-Pr2O); 1H NMR (CDCl3) δ 0.94 (3 H, t, J ) 7.4 Hz),
1.70 (2 H, m), 2.12 (2 H, m), 2.33 (8 H, m), 2.65 (3 H, s), 3.20
(2 H, m), 4.17 (2 H, s), 6.87 (2 H, m), 7.03 (1 H, d, J ) 7.5 Hz),
7.14 (1 H, m), 12.65 (1 H, br s). Anal. (C21H27N3O) C, H, N.
Compounds 64 and 65 were similarily prepared from 58
through reaction with chloroacetonitrile and 3-chloropropioni-
trile, respectively.
1
Compound 64. Yield 28%; H NMR (DMSO-d6) δ 0.78 (3
H, t, J ) 7.2 Hz), 2.16 (3 H, s), 2.24 (5 H, s), 2.61 (3 H, s), 4.04
(2 H, s), 4.11 (2 H, s), 6.84 (1 H, m), 6.99 (2 H, m), 7.13 (1 H,
m), 11.60 (1 H, br s). Anal. (C19H23N3O) C, H, N.
4-(3,5-Dimethylbenzyl)-5-ethyl-6-methyl-3-(pyrrol-1-
yl)pyridin-2(1H)-one 56. 2,5-Dimethoxytetrahydrofuran (1.36
g, 10.3 mmol) was added dropwise at 0 °C to a stirred
suspension of 17a (2.33 g, 8.6 mmol) in HOAc (40 mL). The
mixture was refluxed for 2 h, cooled, and poured out into H2O,
NH4OH, and ice. The precipitate was filtered off and taken
Compound 65. Yield 5%; mp 166 °C (Et2O/petroleum
1
ether); H NMR (DMSO-d6) δ 0.97 (3 H, t, J ) 7.4 Hz), 2.29
(10 H, m), 2.67 (3 H, s), 3.38 (2 H, m), 4.20 (2 H, m), 6.89 (2 H,
m), 7.02 (1 H, m), 7.16 (1 H, m), 13.07 (1 H, br s). Anal.
(C20H25N3O) C, H, N.

1962 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Benjahad et al.
1-Ethyl-3-[4-(3-methylbenzyl)-5-ethyl-6-methyl-2-oxo-
1,2-dihydropyridin]-3-ylthiourea 67. According to the pro-
tocol for 68, compound 67 was obtained by reaction of 57 with
ethylisothiocyanate: yield 34%; mp 112 °C (Et2O); 1H NMR
(DMSO-d6) δ 0.74 (3 H, t, J ) 7.3 Hz), 1.02 (3 H, t, J ) 7.1
Hz), 2.19 (8 H, m), 3.42 (2 H, m), 3.80 (2 H, s), 6.98 (3 H, m),
7.09 (1 H, m), 7.45 (1 H, br s), 8.45 (1 H, br s), 11.65 (1 H, br
s). Anal. (C19H25N3OS) C, H, N.
1-[4-(3-Methylbenzyl)-5-ethyl-6-methyl-2-oxo-1,2-dihy-
dropyridin]-3-yl-3-phenylthiourea 68. A mixture of 57 (1.9
g, 7.4 mmol) and phenylisothiocyanate (1.2 g, 8.9 mmol) in
THF was stirred and refluxed for 20 h, cooled to room
temperature, poured out into H2O, and extracted with EtOAc.
The precipitate was filtered off and dried. Crystallization of
the residue from H2O/EtOAc provided compound 68 as a white
solid (1.2 g, 41%): mp 176 °C (H2O/AcOEt); 1H NMR (DMSO-
d6) δ 0.78 (3 H, t, J ) 7.2 Hz), 2.25 (8 H, m), 3.90 (2 H, s), 7.02
(3 H, m), 7.25 (6 H, m), 8.80 (1 H, br s), 9.65 (1 H, br s), 11.70
(1 H, br s). Anal. (C23H25N3OS) C, H, N.
1-Benzoyl-3-[4-(3-methylbenzyl)-5-ethyl-6-methyl-2-oxo-
1,2-dihydropyridin]-3-ylthiourea 69. Benzoyl chloride (9.8
g, 70 mmol) was added on stirring at room temperature to a
solution of ammonium thiocyanate (5.32 g, 70 mmol) in
2-propanone (200 mL), and the mixture was refluxed for 30
min. Aminopyridinone 57 (15.4 g, 60 mmol) was added slowly
portionwise, and the mixture was refluxed for 3 h, then
basified with 10% K2CO3. The precipitate was filtered off,
washed with H2O, and dried. Crystallization of the residue
from 2-propanone provided compound 69 as a white solid (17.9
g, 71%): mp 237 °C (2-propanone); 1H NMR (DMSO-d6) δ 0.82
(3 H, t, J ) 7.3 Hz), 2.22 (8 H, m), 3.86 (2 H, s), 6.93 (3 H,
m),7.10 (1 H, m), 7.52 (2 H, m), 7.62 (1 H, m), 7.95 (2 H, m),
11.62 (1 H, s), 11.66 (1 H, s), 11.79 (1 H, br s). Anal.
(C24H25N3O2S) C, H, N.
for 8 h, basified with 10% K2CO3, and extracted with CH2Cl2.
The organic layer was separated, dried (MgSO4), and filtered,
and the solvent was evaporated. The residue was purified by
column chromatography over silica gel (CH2Cl2/CH3OH/NH4-
OH 95/5/0.5). The pure fractions were collected, and the solvent
was evaporated. The residue was purified again by column
chromatography over C 18 (eluent, CH3OH/H2O 65/35; column,
KROMASIL C 18 3.5 µm). The pure fractions were collected,
and the solvent was evaporated. The residue was crystallized
from petroleum ether. The precipitate was filtered off and dried
to provide compound 72 as a white solid (210 mg, 18%): mp
126 °C (petroleum ether); 1H NMR (CDCl3) δ 0.97 (3 H, t, J )
7.5 Hz), 2.37 (5 H, m), 2.43 (3 H, s), 2.61 (3 H, s), 3.15 (7 H,
m), 7.38 (2 H, m), 7.60 (1 H, d, J ) 9.0 Hz), 7.70 (1 H, s), 13.20
(1H, br s). Anal. (C20H26N2O3) C, H, N.
3-{3-[5-Ethyl-3-(ethylmethylamino)-6-methyl-2-oxo-1,2-
dihydropyridin-4-ylmethyl]-phenyl}acrylonitrile 77a.
Step 1: N-Formylation. A solution of the brominated 3-ami-
nopyridinone 7313 (20.0 g, 62 mmol) in ethyl formate (500 mL)
and formic acid (100 mL) was refluxed for 8 h, then poured
into ice water, and basified using concentrated NH4OH. The
precipitate was filtered off, providing the formamide interme-
diate (21.5 g, 98%) as a pale yellow solid.
Step 2: Reduction with LiAlH4. LiAlH4 (7.0 g, 185 mmol)
was added portionwise at 5 °C to a solution of the above
formamide intermediate (21.5 g, 62 mmol) in THF (300 mL).
The mixture was stirred at 5 °C for 2 h, then hydrolyzed with
water in the presence of EtOAc. The precipitate was filtered
off, and the filtrate was extracted with EtOAc. The combined
organic layers were dried (MgSO4), filtered, and concentrated
under vacuum. 4-(3-Bromobenzyl)-5-ethyl-6-methyl-3-methy-
laminopyridin-2(1H)-one 74 (11.0 g, 53%) was obtained as a
white solid.
Step 3: Reductive Amination. Sodium cyanoborohydride
(1.7 g, 27 mmol) was added at room temperature to a solution
of the 3-aminopyridinone 74 (3.0 g, 9 mmol) and acetaldehyde
(2.5 mL, 45 mmol) in acetonitrile (65 mL). Acetic acid (1.0 mL)
was added, and the reaction was stirred at room temperature
for 8 h. The mixture was poured into H2O, basified with 10%
aqueous K2CO3, and extracted with CH2Cl2. The combined
organic layers were dried (MgSO4), filtered, and concentrated
under vacuum. The residue was silica gel column chromato-
graphed (CH2Cl2/CH3OH 95/5) providing 4-(3-bromobenzyl)-
5-ethyl-3-(ethylmethylamino)-6-methylpyridin-2(1H)-one 75a
(2.6 g, 80%) as a white solid.
Step 4: Formylation via Metalation. n-Butyllithium (18
mmol) was added dropwise at -70 °C to a solution of 75a (2.6
g, 7.2 mmol) in THF (50 mL) under nitrogen. The mixture was
stirred at -10 °C for 30 min; DMF (5.5 mL, 72 mmol) was
added slowly. The mixture was stirred at -10 °C for 1 h. This
was followed by addition at room temperature of H2O and
extraction with EtOAc. The combined organic layers were
dried (MgSO4), filtered, and concentrated under vacuum. The
residue was silica gel column chromatographed (CH2Cl2/CH3-
OH/NH4OH 99/1/0.1 to 85/15/2) providing 3-[5-ethyl-3-(ethyl-
methylamino)-6-methyl-2-oxo-1,2-dihydro-pyridin-4-ylmethyl]-
benzaldehyde 76a (1.7 g, 79%) as a pale yellow solid.
Step 5: Wittig-Horner Reaction. Potassium terbutoxide
(0.8 g, 6.8 mmol) was added at 5 °C under nitrogen to a
solution of diethyl diethylcyanophosphonate (1.1 mL, 6.8
mmol) in THF (20 mL), and the mixture was stirred at 5 °C
for 30 min. A solution of the benzaldehyde derivative 76a (1.7
g, 5.7 mmol) in THF (20 mL) was added dropwise, and the
reaction was stirred at room temperature for 8 h before
addition of H2O and extraction with EtOAc. The combined
organic layers were dried (MgSO4) and concentrated. The
residue was silica gel column chromatographed (cyclohexane/
i-PrOH 87/13), and the concentrated product fractions were
crystallized from petroleum ether, providing the titled com-
pound 77a (0.26 g, 13%) as a pale yellow solid: mp 196 °C; 1H
NMR (DMSO-d6) δ 0.70-0.89 (6 H, m), 2.14 (3 H, s), 2.21 (2
H, t, J ) 7.1 Hz), 2.55 (3 H, s), 2.95 (2 H, m), 4.14 (2 H, s),
6.40 (1 H, d, J ) 16.7 Hz), 7.09 (1 H, d, J ) 7.5 Hz), 7.28-7.40
[4-(3-Methylbenzyl)-5-ethyl-6-methyl-2-oxo-1,2-dihy-
dropyridin]-3-ylthiourea 70. A mixture of 69 (2.05 g, 4.9
mmol) in 3 N NaOH (30 mL) was stirred and refluxed for 8 h,
poured out on ice, basified with NH4OH, and extracted with
CH2Cl2. The organic layer was separated, dried (MgSO4), and
filtered, and the solvent was evaporated. Crystallization of the
residue from pentane provided compound 70 as a white solid
1
(210 mg, 14%): mp 122 °C (pentane); H NMR (DMSO-d6) δ
0.74 (3 H, t, J ) 7.2 Hz), 2.21 (8 H, m), 3.80 (2 H, m), 6.99 (4
H, m), 7.10 (2 H, m), 8.65 (1 H, br s), 11.65 (1 H, br s). Anal.
(C17H21N3Os) C, H, N.
5-Ethyl-6-methyl-3-methylamino-4-(3-methylbenzoyl)-
pyridin-2(1H)-one 71. A mixture of 19a13 (20.3 g, 75 mmol)
in ethyl formate (460 mL) was stirred at room temperature
for 15 min. Acetic acid (248 mL) was added, then the mixture
was refluxed for 3 h. The solvent was evaporated till dryness,
and the residue was taken up in H2O. The mixture was
basified with concentrated NH4OH solution and extracted with
CH2Cl2. The organic layer was separated, dried (MgSO4), and
filtered, and the solvent was evaporated. The residue was
purified by column chromatography over silica gel (eluent:
CH2Cl2/CH3OH/NH4OH 99/1/0.1 and 80/20/0.1). The pure
fractions were collected, and the solvent was evaporated. The
residue was crystallized from 2-propanone/diethyl ether giving
the N-formyl intermediate. LiAlH4 (8.2 g, 210 mmol) was added
portionwise at room temperature to this intermediate in THF,
and the mixture was stirred at room temperature for 3 h,
cooled, poured out into H2O (24 mL) and 15% NaOH (8 mL),
then filtered over Celite, washed with CH2Cl2, dried (MgSO4),
and filtered, and the solvent was evaporated. The residue was
silica gel column chromatographed (CH2Cl2/CH3OH/NH4OH
94/5/0.5). The pure fractions were collected to provide com-
pound 71 as a white powder (1.0 g, 5%) which was directly
transformed into 72 without any further purification.
5-Ethyl-3-[(2-methoxyethyl)methylamino]-6-methyl-4-
(3-methylbenzoyl)pyridin-2(1H)-one 72. Sodium cyanoboro-
hydride (630 mg, 10 mmol) was added portionwise at room
temperature to a solution of 71 (1.0 g, 3.5 mmol) and meth-
oxyacetaldehyde (4.27 M in H2O, 4.12 mL, 17.6 mmol) in CH3-
CN (10 mL). The mixture was stirred at room temperature

In Vitro Evaluation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1963
(3) Yerly, S.; Kaiser, L.; Race, E.; Bru, J. P.; Clavel, F.; Perrin, L.
Transmission of Antiretroviral-Drug-Resistant HIV-1 Variants.
Lancet 1999, 354, 729-733.
(4) Carr, A.; Samaras, C. K.; Thorisdottir, A.; Kaufmann, G. R.;
Ghisholm, D. J.; Cooper, D. A. Diagnosis, Prediction, and Natural
Course of HIV-1 Protease Inhibitor Associated Lipodystrophy,
Hyperlipidaemia, and Diabetes Mellitus: A Cohort Study. Lancet
1999, 353, 2093-2099.
(5) Carr, A.; Cooper, D. A. Adverse Effects of Antiviral Therapy.
Lancet 2000, 356, 1423-1430
(2 H, m), 7.49 (1 H, d, J ) 7.8 Hz), 7.63 (1 H, d, J ) 16.7 Hz),
11.4 (1 H, br s). Anal. (C21H25N3O) C, H, N.
3-(3-{5-Ethyl-3-[(2-methoxyethyl)methylamino]-6-meth-
yl-2-oxo-1,2-dihydropyridin-4-ylmethyl}phenyl)-acrylonitrile
77b. This compound was obtained in three steps (36, 41, and
10% yields) from methylaminopyridinone 74 in the same
manner to that described above for the parent compound 77a:
mp 169 °C; 1H NMR (CDCl3) δ 0.93 (3 H, t, J ) 7.3 Hz), 2.28-
2.40 (5 H, m), 2.65 (3 H, s), 3.22-3.37 (7 H, m), 3.93-4.56 (2
H, m), 5.86 (1 H, d, J ) 16.7 Hz), 7.16 (1 H, d, J ) 6.6 Hz),
7.20 (1 H, s), 7.24-7.32 (2 H, m), 7.38 (1 H, d, J ) 16.7 Hz),
12.6 (1 H, br s). Anal. (C22H27N3O2) C, H, N.
Biology. Evaluation of Antiviral Activity of the Com-
pounds. Cells and Viruses. MT4 cells are human T-lym-
phoblastoid cells that are highly sensitive to HIV infection,
producing a rapid and pronounced cytopathic effect. All cells
were cultured in RPMI 1640 medium supplemented with 10%
fetal calf serum and antibiotics in a humidified incubator with
a 5% CO2 atmosphere at 37 °C.
Site-Directed Mutants. Mutant RT coding sequences were
generated from a pGEM vector containing the HIV-1 LAI
(clone HXB2) protease (PR) and the RT coding sequence, using
the QuikChange site-directed mutagenesis kit (Stratagene)
and HPLC-purified primers (Genset Oligos). Plasmids were
checked by sequencing to confirm that they contained the
desired mutations. Mutant viruses were created by recombina-
tion of the mutant PR-RT sequence with a PR-RT deleted
HIV-1 HXB2 proviral clone.26
(6) Bastard, J.-P.; Caron, M.; Vidal, H.; Jan, V.; Auclair, M.;
Vigouroux, C.; Luboinski, J.; Laville, M.; Maachi, M.; Girard,
P.-M.; Rozenbaum, W.; Levan, P.; Capeau, J. Association be-
tween Altered Expression of Adipogenic Factor SREBP1 in
Lipotrophic Adipose Tissue from HIV-1 Infected Patients and
Abnormal Adipocyte Differentiation and Insulin Resistance.
Lancet 2002, 359, 1026-1031.
(7) Hoffmann, C.; Kamps, B. S. HIV Medicine 2003. http://ww-
w.HIV.com, Flying Publisher, 2003.
(8) Ruiz, N.; Nusrat, R.; Lauenroth-Mai, E.; Berger, D.; Walworth,
C.; Bacheler, L. T.; Ploughman, L.; Tsang, P.; Labriola, D.;
Echols, R.; Levy, R. Study DPC 083-203, a Phase II Comparison
of 100 and 200 mg Once-Daily DPC 083 and 2 NRTIs in Patients
Failing a NNRTI-Containing Regimen. Presented at the 9th
Conference on Retroviruses and Opportunustic Infections, Se-
attle, WA, 2002; Abstract 6.
(9) Ludovici, D. W.; De Corte, B. L.; Kukla, M. J.; Ye, H.; Ho, C. Y.;
Lichtenstein, M. A.; Kavash, R. W.; Andries, K.; de Be´thune,
M.-P.; Azijn, H.; Pauwels, R.; Lewi, P. J.; Heeres, J.; Koymans,
L. M. H.; de Jonge, M. R.; Van Aken, K. J. A.; Daeyaert, F. F.
D.; Das, K.; Arnold, E.; Janssen, P. A. J. Evolution of Anti-HIV
Drug Candidates. Part 3: Diarylpyrimidine (DAPY) Analogues.
Bioorg. Med. Chem. Lett. 2001, 11, 2235-2239.
(10) Das, K.; Clark, A. D., Jr.; Lewi, P. J.; Heeres, J.; de Jonge, M.
R.; Koymans, L. M. H.; Vinkers, H. M.; Daeyaert, F.; Ludovici,
D. W.; Kukla, M. J.; De Corte, B.; Kavash, R. W.; Ho, C. Y.; Ye,
H.; Lichtenstein, M. A.; Andries, K.; Pauwels, R.; de Bethune,
M.-P.; Boyer, P. L.; Clark, P.; Hughes, S. H.; Janssen, P. A. J.;
Arnold, E. Roles of Conformational and Positional Adaptability
in Structure-Based Design of TMC125-R165335 (Etravirine) and
Related Non-nucleoside Reverse Transcriptase Inhibitors That
Are Highly Potent and Effective against Wild-Type and Drug-
Resistant HIV-1 Variants. J. Med. Chem. 2004, 47, 2550-2560.
(11) (a) Dolle´, V.; Fan, E.; Nguyen, C. H.; Aubertin, A.-M.; Kirn, A.;
Andreola, M. L.; Jamieson, G.; Tarrago-Litvak, L.; Bisagni, E.
A New Series of Pyridinone Derivatives as Potent Non-Nucleo-
side Human Immunodeficiency Virus Type 1 Specific Reverse
Transcriptase Inhibitors. J. Med. Chem. 1995, 38, 4679-4686.
(b) 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. WO9705113, 1997.
(12) (a) Dolle´, V.; Nguyen, C. H.; Legraverend, M.; Aubertin, A.-M.;
Kirn, A.; Andreola, M. L.; Ventura, M.; Tarrago-Litvak, L.;
Bisagni, E. Synthesis and Antiviral Activity of 4-Benzyl Pyri-
dinone Derivatives as Potent and Selective Non-Nucleoside
Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Inhibitors. J. Med. Chem. 2000, 43, 3949-3962. (b) Bisagni, E.;
Dolle´, V.; Nguyen, C.-H.; Monneret, C.; Grierson, D. S.; Aubertin.
A. M. 3-(Amino- or aminoalkyl)pyridinone Derivatives and Their
Use for the Treatment of HIV Related Diseases. WO9955676,
1999.
Drug Sensitivity Assays. The antiviral activity of com-
pounds against laboratory adapted strains, site-directed mu-
tants, and clinical sample derived recombinant viruses was
tested using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) colorimetric assay as previously de-
scribed.26,38 Briefly, various concentrations of the test com-
pounds were added to wells of a flat-bottom microtiter plate.
Subsequently, virus and MT4 cells were added to a final
concentration of 200 CCID50/well and 30 000 cells/well, re-
spectively. To determine the toxicity of the test compound,
mock-infected cell cultures containing an identical compound
concentration range were incubated in parallel with the virus-
infected cell cultures. After 5 days of incubation (37 °C, 5%
CO2), the viability of the cells was determined using MTT. The
results of drug susceptibility assays were expressed as an EC50
defined as the concentration of drug at which there was 50%
infection compared with the drug-free control. In some cases
a fold change in susceptibility was calculated by dividing the
EC50 for the tested virus by the EC50 for the wild-type virus
(HIV-1 LAI) tested in parallel. Toxicity results are expressed
as CC50, defined as the concentration of drug at which the cell
viability was reduced by 50% compared to the drug-free
control.
Acknowledgment. Financial support from “En-
semble contre le SIDAsSidaction” for the acquisition
of a 300 MHz NMR spectrometer is gratefully acknowl-
edged. The Arnold laboratory gratefully acknowledges
financial support from the Janssen Research Founda-
tion.
(13) Benjahad, A.; Courte´, K.; Guillemont, J.; Mabire, D.; Coupa, S.;
Poncelet, A.; Csoka, I.; Andries, K.; Pauwels, R.; de Be´thune,
M.-P.; Monneret, C.; Bisagni, E.; Nguyen, C. H.; Grierson, D. S.
4-Benzyl and 4-Benzoyl-3-dimethylaminopyridin-2(1H)-ones, a
New Family of Potent Anti-HIV Agents: Optimization and in
Vitro Evaluation against Clinically Important HIV Mutant
Strains. J. Med. Chem. 2004, 47, 5501-5514.
(14) Hopkins, A. L.; Ren, J.; Tanaka, H.; Baba, M.; Okamato, M.;
Stuart, D. I.; Stammers, D. K. Design of MKC-442 (Emivirine)
Analogues with Improved Activity against Drug-Resistant HIV
Mutants. J. Med. Chem. 1999, 42, 4500-4505.
(15) 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.
(16) Saari, W. S.; 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.
Supporting Information Available: Synthetic procedure
and intermediate and final product characterzation. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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JM0408621