Synthesis and biological properties of novel 2-aminopyrimidin-4(3H)-ones highly potent against HIV-1 mutant strains

Article abstract of DOI:10.1021/jm070811e

Following the disclosure of dihydro-alkoxy-, dihydro-alkylthio-, and dihydro-alkylamino-benzyl-oxopyrimidines (DABOs, S-DABOs, and NH-DABOs) as potent and selective anti-HIV-1 agents belonging to the non-nucleoside reverse transcriptase inhibitor (NNRTI)

Full text of DOI:10.1021/jm070811e

5412
J. Med. Chem. 2007, 50, 5412-5424
Synthesis and Biological Properties of Novel 2-Aminopyrimidin-4(3H)-ones Highly Potent
against HIV-1 Mutant Strains
Antonello Mai,*,† Marino Artico,† Dante Rotili,† Domenico Tarantino,† Imma Clotet-Codina,‡ Mercedes Armand-Ugo´n,‡
Rino Ragno,† Silvia Simeoni,† Gianluca Sbardella,§ Maxim B. Nawrozkij,| Alberta Samuele, Giovanni Maga, and
Jose´ A. Este´‡
Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, UniVersita` degli Studi di Roma “La Sapienza”,
P. le A. Moro 5, 00185 Roma, Italy, RetroVirology Laboratory IrsiCaixa, Hospital UniVersitari Germans Trias i Pujol, UniVersitat Auto`noma
de Barcelona, 08916 Badalona, Spain, Dipartimento di Scienze Farmaceutiche, UniVersita` degli Studi di Salerno, Via Ponte Don Melillo,
84084 Fisciano (SA), Italy, Volgograd State Technical UniVersity, pr. Lenina, 28, 400131 Volgograd, Russia, and Istituto di Genetica
Molecolare IGM-CNR, Via Abbiategrasso 207, 27100 PaVia, Italy
ReceiVed July 6, 2007
Following the disclosure of dihydro-alkoxy-, dihydro-alkylthio-, and dihydro-alkylamino-benzyl-oxopyri-
midines (DABOs, S-DABOs, and NH-DABOs) as potent and selective anti-HIV-1 agents belonging to the
non-nucleoside reverse transcriptase inhibitor (NNRTI) class, we report here the synthesis and biological
evaluation of a novel series of DABOs bearing a N,N-disubstituted amino group or a cyclic amine at the
pyrimidine-C2 position, a hydrogen atom or a small alkyl group at C5 and/or at the benzylic position, and
the favorable 2,6-difluorobenzyl moiety at the C6 position (F2-N,N-DABOs). The new compounds were
highly active up to the subnanomolar level against both wt HIV-1 and the Y181C mutant and at the
submicromolar to nanomolar range against the K103N and Y188L mutant strains. Such derivatives were
more potent than S-DABOs, NH-DABOs, and nevirapine and efavirenz were chosen as reference drugs.
The higher inhibitor adaptability to the HIV-1 RT non-nucleoside binding site (NNBS) may account for the
higher inhibitory effect exerted by the new molecules against the mutated RTs.
Introduction
infection.4 Thus, the needed long-term or permanent use of anti-
AIDS drugs induces the selection of drug-resistant viral mutants
and the emergence of undesired metabolic side effects.4
Therefore, although the HIV treating physician has a multitude
of agents to choose from, rapid development of drug resistance,
long-term reduced compliance, and toxicity problems demand
the discovery of novel anti-HIV agents effective against resistant
mutants without unpleasant side effects. Actually, the search
for novel anti-HIV drugs is pursued either by improvement of
the existing drug classes (NRTIs, NtRTIs, NNRTIs, PIs, and
FIs) or by the discovery of new agents showing different
mechanisms of action (such as inhibition of integrase, Rnase-
H, and viral entry).5
Human immunodeficiency virus (HIV)a1 is the etiological
agent of acquired immunodeficiency syndrome (AIDS), an
infection characterized by loss of helper T lymphocytes and
heavy damage of lymphatic tissues.1 The current therapy against
AIDS is based on four classes of anti-HIV drugs: the nucleoside
and nucleotide reverse transcriptase (RT) inhibitors (indicated
as NRTIs and NtRTIs, respectively), the non-nucleoside reverse
transcriptase inhibitors (NNRTIs), the protease inhibitors (PIs),
and the fusion inhibitors (FIs).2,3 NRTIs, NNRTIs, and PIs are
actually mixed in the highly active antiretroviral therapy
(HAART), which dramatically reduces the incidence of HIV
infection and death. Despite HAART combination regimens that
have significantly decreased the morbidity and mortality among
patients with HIV infections, slowing viral replication to very
low levels, they are still unable to eradicate the retroviral
The NNRTIs identified so far include more than fifty
structurally quite different classes of molecules.6 The marketed
drugs nevirapine, delavirdine, and efavirenz are significant
examples of such inhibitors (Chart 1). Despite their low toxicity
and favorable pharmacokinetic properties, the use of first-
generation NNRTIs both in monotherapy and in multidrug anti-
AIDS cocktails led to unsuccessful results due to the rapid
emergence of drug resistance.7 As a consequence, in the NNRTI
field, the efforts are now focused on the development of novel
compounds endowed with higher anti-HIV-1 activity and a
resistance profile different from that of known drugs (second
generation NNRTIs). Continuous efforts in this field are
documented by the wide number of NNRTIs described in the
recent literature,2,5,8-10 some of which, such as capravirine11
and etravirine (TMC125)12 (Chart 1) are under clinical trials.
* To whom correspondence should be addressed. Tel.: +396-4991-3392.
Fax: +396-491491. E-mail: antonello.mai@uniroma1.it.
† Universita` degli Studi di Roma “La Sapienza”.
‡ Retrovirology Laboratory IrsiCaixa.
§ Universita` degli Studi di Salerno.
| Volgograd State Technical University.
Istituto di Genetica Molecolare IGM-CNR.
a Abbreviations: DABOs, dihydro-alkoxy-benzyl-oxopyrimidines; HIV,
human immunodeficiency virus; NNRTIs, non-nucleoside reverse tran-
scriptase inhibitors; NNBS, non-nucleoside binding site; RT, reverse
transcriptase; AIDS, acquired immunodeficiency syndrome; NRTIs, nucleo-
side reverse transcriptase inhibitors; NtRTIs, nucleotide reverse transcriptase
inhibitors; PIs, protease inhibitors; FIs, fusion inhibitors; HAART, highly
active antiretroviral therapy; SAR, structure-activity relationship; S-DABOs,
dihydro-alkylthio-benzyl-oxopyrimidines; NH-DABOs, dihydro-alkylamino-
benzyl-oxopyrimidines; F2-N,N-DABOs, 5-alkyl-2-(N,N-disubstituted)-
amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)ones; NVP,
nevirapine; EFV, efavirenz; EC50, effective concentration able to protect
50% of cells from the HIV-1 induced cytopathogenicity; CC50, compound
concentration toxic for 50% of cells; WT, wild type; RCSB, research
collaboratory for structural bioinformatics; PDB, protein data bank; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Dihydro-alkoxy-benzyl-oxopyrimidines (DABOs) were dis-
closed by our group in 1992 as a novel NNRTI class.13-15 Since
then, a great number of oxopyrimidines were synthesized and
tested as anti-HIV-1 agents to obtain more potent and selective
compounds.16-28 In addition, the pyrimidin-4(3H)-one scaffold
attracted the interest of other authors for the design of new anti-
10.1021/jm070811e CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/02/2007

2-Aminopyrimidin-4(3H)-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5413
Chart 1. Known Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors
HIV agents,29-35 and some DABO-related triazines and pyri-
midines were described by the Janssen group as highly potent,
wide spectrum NNRTIs, including the clinical candidate
etravirine.36-40
hydrogen bond into the NNBS, can confer an implicit increased
capacity to re-adapt the obtained molecules into different
experimental NNBS conformations. This implies that such a
flexibility could lead to a broader range of RT inhibitors en-
dowed with high activity against both wild-type HIV-1 and
resistant mutants.
Prompted by these computational hypotheses, and to establish
further SAR on 2-amino-DABO derivatives, we prepared a
novel series of N,N-DABOs (4-7) with a C2-dialkylamino or
-aza(hetero)cycloalkyl side chain, with a hydrogen, methyl, or
ethyl substituent at the pyrimidine C5 and/or the C6-benzylic
position and with the highly favorable 2,6-difluoro substitution
at the C6-phenylalkyl group (Chart 2).
Chemistry. Title compounds were routinely synthesized by
nucleophilic displacement of the methylthio group from the
5-alkyl-6-(2,6-difluorophenylalkyl)-2-methylthiopyrimidin-4(3H)-
ones 8a-d22,26 (a: R1 ) R2 ) H; b: R1 ) Me, R2 ) H; c: R1
) H, R2 ) Me; d: R1 ) R2 ) Me) with the appropriate
secondary or cyclic amines, by heating the two starting materials
in a sealed tube at 170 °C for 8 h. Alternatively, with low-
boiling amines (N-methyl-N-ethylamine, N-methyl-N-n-propyl-
amine, N-methyl-N-iso-propylamine), some N,N-DABOs were
obtained by heating the proper 2-methylthiopyrimidinone with
the amine as an acetate salt in an oil bath at 160 °C for 12 h
(Scheme 1, A). Condensation of N,N-dimethylguanidine sulfate
with the ethyl 2,4-dialkyl-3-oxo-4-substitutedphenylbutyrates
9a-h (a: R ) 2,6-Cl2, R1 ) R2 ) Me; b: R ) 2-F, R1 ) R2
) Me; c: R ) 2,6-F2, R1 ) Me, R2 ) Et; d: R ) 2,6-F2, R1
) Et, R2 ) Me; e: R ) 2,6-F2, R1 ) R2 ) H; f: R ) 2,6-F2,
R1 ) Me, R2 ) H; g: R ) 2,6-F2, R1 ) H, R2 ) Me; h: R )
2,6-F2, R1 ) R2 ) Me) in the presence of sodium ethoxide
furnished the 2-N,N-dimethylaminopyrimidinones 4a, 5a, 6a,
6c, and 7a-e. The unknown â-oxoesters 9a-d were pre-
pared by reaction of the 2-(2,6-dichlorophenyl)propionic acid
A structure-activity relationship (SAR) profile of DABOs
together with molecular modeling investigation on their putative
binding mode have shown that the presence of a C2-alkoxy
(DABOs, 1), C2-alkylthio (S-DABOs, 2), or C2-alkylamino (NH-
DABOs, 3) side chain is a structural determinant for the antiviral
activity of these derivatives, with the length and size of the C2
side chain having only modulator effects on potency (Chart
1).16-19,22,26,28 The 2,6-difluoro substitution at the C6-benzyl
moiety of S-DABOs and NH-DABOs produced favorable
π-stacking interactions with the Tyr188 side chain into the
non-nucleoside binding site (NNBS), leading to compounds
(F2-S-DABOs and F2-NH-DABOs) active in the nanomolar
range.22,26-28 Moreover, the insertion of two methyl groups, one
at the pyrimidine C5 position, and the second one on the
methylene connecting pyrimidine and phenyl rings (benzylic
position), furnished new conformationally restricted compounds
exhibiting nanomolar activity against wild-type HIV-1 and
submicromolar activity against the Y181C mutant strain.26
Docking experiments performed on F2-NH-DABOs confirmed
that their further anchor point (hydrogen bond of C2-NH
function with Lys101) into the NNBS produced a tight ligand
binding that may compensate the incoming lack of positive
hydrophobic ligand/NNBS interactions due to Tyr181 to Cys181
mutation.27,28
On the other hand, the F2-NH-DABO further anchor point is
a source of some strictness in the ligand/enzyme interaction;
moreover, cross-docking experiments41 performed on 68 dif-
ferent NNRTI/RT complexes suggested that a double N,N
substitution on the 2-amino-DABO template, though preventing
the capability of the C2-NH group to establish a further

5414 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Mai et al.
Chart 2. Novel 5-Alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F2-N,N-DABOs) and
Related Derivatives 4-7
Scheme 1a
a (a) XYNH, 170 °C, sealed tube; (b) XYNH‚CH3COOH, 160 °C, oil bath; (c) (1) SOCl2, room temperature, (2) EtOCOCHR1COOK, (Et)3N, MgCl2,
MeCN, room temperature, (3) 13% HCl, room temperature; (d) N,N-dimethylguanidine sulfate, EtONa, EtOH, reflux; (e) 2,5-dimethoxytetrahydrofuran,
CH3COOH, reflux.
(10a),26 or 2-(2-fluorophenyl)propionic acid (10b), or 2-(2,6-
difluorophenyl)propionic acid (10c),26 or 2-(2,6-difluorophenyl)-
butyric acid (10d)26 with (i) thionyl chloride, (ii) potassium ethyl
malonate or potassium ethyl 2-methyl- or 2-ethylmalonate in
the presence of triethylamine and magnesium dichloride, and
(iii) acidic hydrolysis with 13% HCl (part B of Scheme 1).

2-Aminopyrimidin-4(3H)-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5415
Table 1. Physical and Chemical Data for 4-7
recryst
synth
wayb
%
yield
cpd
R
R1
R2
X, Y
mp, °C
solventa
formulac
4a
4b
4c
4d
4e
4f
4g
4h
4i
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me, Me
Me, Et
Me, i-Pr
Me, n-Pr
210-211
150-151
153-154
150-151
159-160
111-112
238-239
233-234
209-210
215-216
181-182
183-184
174-175
215-216
233-234
195-196
202-203
235-236
237-238
191-192
176-178
173-174
170-171
188-189
168-169
142-143
229-230
219-220
210-211
134-135
201-202
261-262
224-225
244-245
255-256
235-236
232-233
197-198
255-256
198-199
137-138
136-138
174-175
>200
A
B
C
A
B
C
B
B
B
D
B
E
B
B
B
B
B
B
B
B
B
B
B
B
B
F
B
B
B
A
B
B
B
B
B
B
B
B
G
A
H
B
H
B
B
C
I
d
b
b
b
a
a
a
a
a
a
a
a
a
a
a
a
a
e
d
b
b
b
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
d
b
d
d
d
d
d
d
b
b
b
a
a
a
a
a
a
a
85
72
64
41
87
86
23
39
51
49
53
55
48
63
57
40
54
43
68
51
86
68
44
39
82
75
8
35
53
51
47
52
56
49
56
25
48
51
46
78
31
72
73
61
65
72
84
81
58
38
32
57
48
53
55
51
55
C13H13F2N3O
C14H15F2N3O
C15H17F2N3O
C15H17F2N3O
C15H17F2N3O
C17H21F2N3O
C15H15F2N3O
C19H23FN4O
C16H17F2N3O
C17H19F2N3O
C17H19F2N3O
C17H19F2N3O
C17H19F2N3O
C15H15F2N3O2
C15H15F2N3OS
C16H18F2N4O
C18H19F2N3O3
C15H11F2N3O
C14H15F2N3O
C15H17F2N3O
C16H19F2N3O
C16H19F2N3O
C19H23F2N3O
C19H17F2N3O
C16H19F2N3O
C18H23F2N3O
C16H17F2N3O
C20H25FN4O
C17H19F2N3O
C18H21F2N3O
C18H21F2N3O
C18H21F2N3O
C18H21F2N3O
C16H17F2N3O2
C16H17F2N3OS
C17H20F2N4O
C19H21F2N3O3
C23H24F2N4O2
C21H21F2N5O
C14H15F2N3O
C16H19F2N3O
C15H17F2N3O
C15H17F2N3O
C15H17Cl2N3O
C15H18FN3O
C16H19F2N3O
C16H19F2N3O
C16H19F2N3O
C17H21F2N3O
C17H21F2N3O
C17H19F2N3O
C18H21F2N3O
C19H23F2N3O
C19H23F2N3O
C19H23F2N3O
C17H19F2N3O2
C17H19F2N3OS
Et, Et
n-Pr, n-Pr
pyrrolidin-1-yl
pyrrolidin-1-yl
piperidin-1-yl
2-Me-piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
hexahydroazepin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
4-Me-piperazin-1-yl
1,4-dioxa-8-aza spiro[4.5]decan-8-yl
1H-pyrrol-1-yl
Me, Me
Me, Et
Me, i-Pr
Me, n-Pr
Me, c-hex
Me, Ph
Et, Et
n-Pr, n-Pr
2-F-6-pyrrolidin-1-yl
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2-F-6-pyrrolidin-1-yl
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-Cl2
2-F
4j
4k
4l
4m
4n
4o
4p
4q
4r
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
pyrrolidin-1-yl
pyrrolidin-1-yl
piperidin-1-yl
2-Me-piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
hexahydroazepin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
4-Me-piperazin-1-yl
1,4-dioxa-8-aza spiro[4.5]decan-8-yl
4-(2-MeO-Ph)-1-piperazinyl
4-(2-pyridinyl)-1-piperazinyl
Me, Me
Me, n-Pr
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Et
5m
5n
5o
5p
5q
5r
5s
H
H
H
H
H
H
H
5t
5u
6a
6b
6c
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
Me
Me
Et
Me
Me
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Me
Me
Me
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
>200
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
148-149
145-146
166-167
156.5-157.0
128-130
188-189
151-152
152-153
165-166
188-189
193-194
219-220
C
D
J
B
B
C
C
B
B
B
Me, i-Pr
Me, n-Pr
pyrrolidin-1-yl
piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
hexahydroazepin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
7m
7n
7o
a A: cyclohexane/benzene; B: acetonitrile; C: n-hexane; D: benzene/acetonitrile; E: acetonitrile/diethyl ether; F: petroleum ether; G: ethanol/water;
H: cyclohexane; I: diethyl ether; J: dichloromethane/diethyl ether. b See Scheme 1. c Analytic results were within (0.40% of the theoretical values.
Table 2. Physical and Chemical Data for 9a-d and 10b
Finally, the 1H-pyrrol-1-yl derivative 4r was prepared from
the 2-amino-6-(2,6-difluorobenzyl)pyrimidin-4(3H)-one 1128
with 2,5-dimethoxytetrahydrofuran and glacial acetic acid under
Clauson-Kaas conditions (part C of Scheme 1).
Compounds 7 sharing a chiral center at the benzylic position
were prepared and tested as racemates. Chemical and physical
data of 4-7 are listed in Table 1. Chemical and physical data
of the intermediate compounds 9a-d and 10b are listed in
Table 2.
cpd
R
R1
R2
mp, °C
% yield
formulaa
9a
9b
9c
9d
10b
2,6-Cl2
2-F
2,6-F2
2,6-F2
2-F
Me
Me
Me
Et
Me
Me
Et
Me
Me
oil
oil
oil
oil
oil
69
81
75
79
76
C14H16Cl2O3
C14H17FO3
C15H18F2O3
C15H18F2O3
C9H9FO2
a Analytic results were within (0.40% of the theoretical values.
strain to evaluate their in-cell anti-HIV-1 activity in parallel
with cytotoxicity. Nevirapine (NVP) and efavirenz (EFV) were
included in the test as reference drugs. The results, expressed
as EC50 (effective concentration able to protect 50% of cells
Results and Discussion
Wild-Type HIV-1 Inhibiting Activity. The novel N,N-
DABOs 4-7 were tested against wild-type (wt) HIV-1 NL4-3

5416 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Table 3. Cytotoxicity and Anti-HIV-1 Activity of 4-7a
Mai et al.
compound
R
R1
R2
X, Y
CC50,b µM
EC50,c µM
SId
4a
4b
4c
4d
4e
4f
4g
4h
4i
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me, Me
Me, Et
Me, i-Pr
Me, n-Pr
>94e
>89
85
0.023
0.013
0.008
0.009
0.133
1.78
>4087
>6846
10 625
9444
85
Et, Et
>85
>78
>86
>73
>82
>78
>78
>78
>78
>81
>77
>78
>69
>87
>89
>85
79
>81
>72
>73
>81
>74
>82
>70
>78
75
>75
>75
>75
>78
>74
>75
>66
37
>63
89
>81
73
>639
n-Pr, n-Pr
pyrrolidin-1-yl
pyrrolidin-1-yl
piperidin-1-yl
2-Me-piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
hexahydroazepin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
4-Me-piperazin-1-yl
1,4-dioxa-8-aza spiro[4.5]decan-8-yl
1H-pyrrol-1-yl
Me, Me
Me, Et
Me, i-Pr
Me, n-Pr
Me, c-hex
Me, Ph
Et, Et
n-Pr, n-Pr
>44
0.093
0.71
>925
2-F-6-pyrrolidin-1-yl
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2-F-6-pyrrolidin-1-yl
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-Cl2
2-F
>103
0.013
0.107
0.032
0.035
0.022
0.211
0.035
6.09
>6308
>729
4j
4k
4l
>2437
>2229
>3545
>384
4m
4n
4o
4p
4q
4r
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
>2200
>13
>31
>5
H
H
2.22
17.32
0.005
0.018
0.002
0.002
0.009
0.086
0.030
2.09
0.032
0.62
0.003
0.009
0.018
0.100
0.05
0.026
0.004
0.598
0.65
0.218
0.012
0.001
0.0006
0.002
0.0003
0.03
0.006
0.007
0.002
0.0003
0.0005
0.0006
0.002
0.007
0.003
0.002
0.009
0.002
0.003
0.001
0.003
0.08
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
>17 800
>4722
39 500
>40 500
>8000
>849
>2700
>35
pyrrolidin-1-yl
pyrrolidin-1-yl
piperidin-1-yl
>2562
>113
>26 000
8333
2-Me-piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
hexahydroazepin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
4-Me-piperazin-1-yl
1,4-dioxa-8-aza spiro[4.5]decan-8-yl
4-(2-MeO-Ph)-1-piperazinyl
4-(2-pyridinyl)-1-piperazinyl
Me, Me
Me, n-Pr
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Et
5m
5n
5o
5p
5q
5r
5s
>4167
>750
H
H
H
H
H
H
H
>1500
>3000
>18 500
>125
>101
170
5t
5u
6a
6b
6c
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
2a
3a
NVP
EFV
>5250
89 000
>135 000
36 500
>56 667
1167
Me
Me
Et
Me
Me
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Me
Me
Me
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
>17
35
44
7333
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
>81
>81
81
>81
>78
>78
>75
>72
54
>72
>74
>71
>3
>11 571
>40 500
270 000
>162 000
>130 000
>39 000
>10 714
>24 000
27 000
>8000
>37 000
>23 667
>3000
11 667
>87
Me, i-Pr
Me, n-Pr
pyrrolidin-1-yl
piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
hexahydroazepin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
35
>7
>0.3
0.004
>75
a Values are means ( SD determined from at least two experiments. b Cytotoxic concentration 50, concentration to induce 50% death of noninfected
cells, evaluated with the MTT method in MT-4 cells. c Effective concentration 50, concentration needed to inhibit 50% HIV-induced cytopathic effect,
evaluated with the MTT method in MT-4 cells (HIV-1 strain: NL4-3). d Selectivity index, CC50/EC50 ratio. e Higher concentrations could not be achieved
for the precipitation of compounds in the culture medium.
from the HIV-1-induced cytopathogenicity) and CC50 (com-
pound concentration toxic for 50% of cells) values, are reported
in Table 3. As the virus strain used in this article differs from
the virus strain used by us in our earlier publications, the
measured activities of the S-DABO and NH-DABO prototypes
2a and 3a as well as those of the reference compounds NVP
and EFV differ in this and earlier articles.
Amine Substituent at the Pyrimidine-C2 Position. In the
4, 5, and 7 series, both the F2-N,N-DABOs carrying a N,N-
disubstituted amine and a cyclic amine at the pyrimidine-C2
generally displayed similar HIV-1 inhibiting activity. In some
cases, compounds bearing a pyrrolidine, 2- or 4-methylpiperi-
dine, and morpholine at C2 showed a drop of activity with a
corresponding lowering of the selectivity index (for instance,
4g, 4j, 4n, and 5n). To reach the highest potency, among the
two alkyl moieties at the C2-amino group, the first should be a
From inhibitory data, we can draw the following structure-
activity relationship (SAR).

2-Aminopyrimidin-4(3H)-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5417
EC50,b µM (fold-resistance)c
Table 4. Anti-HIV-1 Activity of 4-7 against HIV-1 Clinical Isolate and Mutant Strainsa
cpd
4a
4b
4c
4g
4i
4j
4k
4l
4m
4n
4o
5a
5b
5c
5d
5e
5i
R
R1
R2
X, Y
IRLL98d
K103N
Y181C
Y188L
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Et
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me, Me
Me, Et
Me, i-Pr
2.0 (87)
2.0 (154)
0.18 (22)
>10
2.2 (169)
3 (28)
>10
3.6 (156)
0.50 (38)
0.03 (4)
>10
>10
>10
>10
>10
pyrrolidin-1-yl
piperidin-1-yl
2-Me-piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
hexahydroazepin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
Me, Me
>10
>10
>10
>10
0.71 (55)
1.14 (11)
0.43 (13)
1.29 (37)
0.43 (19)
>10
8 (615)
>10
>10
1.8 (56)
4.6 (131)
1.4 (64)
>10
>10
>10
>10
>10
H
H
H
>10
>10
>10
>10
>10
>10
>10
>10
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
Me
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
0.5 (100)
0.42 (23)
0.08 (40)
0.055 (27)
2 (222)
2.4 (480)
4.6 (256)
1.2 (600)
0.75 (375)
4.4 (489)
>10
1.82 (364)
0.71 (39)
0.03 (15)
0.039 (19)
0.14 (16)
5.15 (161)
0.14 (47)
0.14 (16)
0.57 (32)
>10
>10
Me, Et
Me, i-Pr
8 (444)
2.3 (1150)
1.6 (800)
6.2 (689)
>10
Me, n-Pr
Me, c-hexyl
pyrrolidin-1-yl
piperidin-1-yl
2-Me-piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
hexahydroazepin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
Me, Me
>10
5k
5l
0.82 (273)
1.4 (156)
2.1 (117)
>10
>10
10 (3333)
>10
>10
5m
5n
5o
5p
5q
6a
6b
6c
7a
7d
7e
7f
7g
7h
7i
7j
7k
7l
>10
>10
>10
>10
1.7 (34)
>10
>10
>10
0.57 (11)
>10
>10
>10
>10
>10
>10
>10
0.67 (670)
0.15 (250)
1.6 (800)
0.005 (17)
1.18 (169)
0.88 (440)
0.003 (10)
0.001 (2)
0.001 (1.7)
0.2 (100)
0.06 (9)
0.2 (67)
0.3 (150)
0.05 (25)
0.06 (20)
0.12 (120)
0.09 (30)
9.9 (124)
0.08 (20)
0.12 (120)
0.08 (133)
0.18 (90)
0.05 (167)
1.83 (261)
0.25 (125)
0.02 (67)
0.006 (12)
0.01 (17)
0.155 (77)
0.52 (74)
0.193 (64)
0.322 (161)
0.39 (195)
1.24 (413)
0.225 (225)
0.37 (125)
1.8 (23)
0.09 (22)
0.26 (260)
0.05 (83)
0.43 (215)
0.014 (47)
0.29 (41)
0.10 (50)
0.002 (7)
0.0007 (1.4)
0.0006 (1)
0.071 (35)
0.07 (10)
0.014 (5)
0.01 (5)
0.09 (45)
0.03 (10)
0.097 (97)
0.06 (20)
0.87 (11)
0.006 (1.5)
>10
Me, n-Pr
>10
Me, Me
Me, Me
Me, Me
Me, Me
Me, Et
Me, i-Pr
>10
0.05 (167)
0.48 (69)
0.55 (275)
0.03 (100)
0.04 (80)
0.03 (50)
>10
Me, n-Pr
pyrrolidin-1-yl
piperidin-1-yl
3-Me-piperidin-1-yl
4-Me-piperidin-1-yl
morpholin-4-yl
thiomorpholin-4-yl
0.39 (56)
0.12 (40)
0.22 (110)
0.56 (280)
0.86 (287)
>3
>10
5.6 (70)
0.23 (57)
7n
7o
2a
3a
NVP
EFV
a Values are means ( SD determined from at least two experiments. b Effective concentration 50, concentration needed to inhibit 50% HIV-induced
cytopathic effect, evaluated with the MTT method in MT-4 cells. c Fold-resistance: ratio of EC50 value against drug-resistant strain and EC50 of the wild-
type NL4-3 strain (Table 3). d Clinical isolate resistant to NRTIs (lamivudine, emtricitabine) and NNRTIs (NVP, delavirdine, EFV).
methyl group, and the latter a C2- or better a C3-carbon unit
chain (compare 4a with 4d, and 5a with 5d). The presence of
a first alkyl group bigger than methyl (i.e., ethyl (4e, 5g) or, to
a further extent, n-propyl (4f, 5h)) strongly decreased the anti-
HIV-1 activity, as well as the introduction, in addition to the
methyl group, of a phenyl ring as a second amine substituent
(5f). Among the cyclic amines inserted at the F2-N,N-DABOs’
pyrimidine-C2, the piperidine, thiomorpholine, and, to a lesser
extent, pyrrolidine and morpholine furnished the most-active
derivatives in inhibiting wt HIV-1. The introduction of less-
hydrophobic amines, such as 1,4-dioxa-8-azaspiro[4.5]decane
and 4-methylpiperazine, as well as the insertion of the aromatic,
planar pyrrole ring, abated the inhibiting activity of the F2-N,N-
DABOs, whereas the activity was restored when the C2-
piperazine residue was substituted at N4 with a 2-pyridinyl ring
(5u, EC50 ) 12 nM).
benzylic position of the pyrimidine ring and was better if
together with a C5-methyl/ethyl substitution. By inserting the
above substitutions, highly active at subnanomolar level anti-
HIV-1 compounds (6b, 7a,f-h) were obtained, with an increase
of up to 30-fold in potency, with respect to the uracil counter-
parts. Moreover, the selectivity indexes of the above derivatives
reached very high values such as 270 000 (7f), >162 000 (7g),
and >130 000 (7h).
Substituents at the Benzene Ring. The reaction of 6-(2,6-
difluorobenzyl)-2-methylthiopyrimidin-4(3H)-ones 8a,b with
pyrrolidine to synthesize the 2-pyrrolidino analogues 4g and 5i
also yielded 6-[2-fluoro-6-(pyrrolidin-1-yl)benzyl]-2-(pyrrolidin-
1-yl)pyrimidin-4(3H)-ones 4h and 5j as side-products. Tested
against wt HIV-1 NL4-3 strain, 4h and 5j were 10- to 40-fold
less potent than the corresponding 2,6-difluoroderivatives 4g
and 5i. 7b and 7c were synthesized, respectively, as the 6-[1-
(2,6-dichlorophenyl- and 2-fluorophenyl)ethyl] analogues of the
6-[1-(2,6-difluorophenyl)ethyl]-2-dimethylamino-5-methylpyri-
midin-4(3H)-one 7a, one of the most-active derivatives. When
tested, they were 7-30 times less potent than 7a.
Pyrimidine-C5 Substituent. As already reported for the
previous series of DABOs, thymine derivatives (5,7) were, in
general, more efficient than the corresponding uracils (4,6) in
inhibiting wt HIV-1.
Substitution at the C6-Benzylic Position. The real increase
of activity in the F2-N,N-DABO series was obtained with the
introduction of small alkyl (methyl, ethyl) groups at the C6-
Activity Against a Clinical Isolate and Clinically Relevant
HIV-1 Mutant Strains. Most of the reported F2-N,N-DABOs
were tested against the HIV-1 clinical isolate IRLL98, containing

5418 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Mai et al.
Table 5. Inhibitory Potencies of Selected 4-7 in the Presence of HIV-1 RT Wild-Type and NNRTI-resistant Mutantsa
Ki,b µM (fold-resistance)c
cpd
WTIIIB
K103N
L100I
V106A
V179D
Y181I
Y188L
4a
4i
0.08
2.5
0.7
0.2
11.23
0.1
3 (37)
>20
>20
10 (125)
10 (4)
15 (21)
0.6 (3)
>20
10 (100)
18 (180)
0.9 (18)
5 (33)
0.5 (6)
0.2 (0.08)
2.9 (4)
3 (15)
>40
0.7 (7)
5 (62)
3 (37)
>20
>20
30 (375)
>20
>20
4n
4o
4p
5a
5d
5p
5q
5r
5t
5u
6c
7a
7d
7e
>40
>20
1.5 (7)
>20
1 (5)
0.4 (2)
>20
1 (5)
>20
>40
9 (90)
>20
1.6 (16)
0.2 (2)
8.3 (166)
40 (267)
>40
>20
11 (110)
>20
0.1
0.008 (0.08)
0.4 (8)
>20
0.05
0.15
2
0.25
0.05
0.1
>20
>20
>20
>20
0.09 (0.6)
0.9 (0.45)
4.1 (16)
0.01 (0.2)
0.14 (1.4)
0.1 (1)
>20
>20
NDd
ND
ND
ND
>20
>20
10 (100)
0.5 (5)
>20
2 (40)
0.5 (12)
0.08 (2)
0.17 (6)
0.6 (9)
3.3 (1100)
10 (167)
0.03 (6)
0.47 (12)
7 (17)
3 (100)
0.22 (0.9)
>20
3.3 (33)
1 (10)
>20
4 (80)
2.5 (62)
0.12 (3)
0.18 (6)
2 (29)
10 (3333)
5 (83)
0.3 (60)
0.31 (8)
9 (22)
8.5 (34)
0.01 (0.2)
0.03 (0.3)
2 (20)
>20
25.7 (103)
0.49 (10)
5 (50)
0.5 (5)
>20
10 (200)
4 (100)
1.19 (30)
1.43 (48)
3.5 (50)
5.5 (1833)
4 (67)
1.4 (280)
3.11 (78)
18 (45)
ND
>20
>20
0.1
1
4 (40)
>20
>40
>40
0.05
0.04
0.04
0.03
0.07
0.003
0.06
0.005
0.04
0.4
0.06 (1.2)
0.02 (0.5)
0.03 (0.7)
0.02 (0.7)
0.02 (0.3)
>20
0.03 (0.6)
0.02 (0.5)
ND
5 (100)
10 (250)
0.85 (21)
1.26 (42)
>20
7f
7g
7h
7j
7n
7o
2a
3a
NVP
EFV
ND
0.01 (0.14)
>20
0.02 (0.33)
0.25 (50)
ND
0.3 (0.8)
ND
>20
>20
0.9 (180)
5.02 (125)
35 (87)
0.08 (3)
0.03 (0.5)
0.5 (100)
0.04 (1)
10 (25)
0.03
ND
ND
a Values are means ( SD determined from at least three experiments. b Compound concentration required to inhibit the HIV-1 rRT activity by 50%.
c Fold-resistance: ratio of Kimut/Kiwt values. d ND, not determined.
multiple drug-resistance mutations (to both NRTIs (lamivudine,
emtricitabine) and NNRTIs (NVP, delavirdine, EFV)), as well
as against a panel of clinically relevant HIV-1 mutant strains
(K103N, Y181C, and Y188L), and the results are summarized
in Table 4. To define the resistance profile of these inhibitors,
both the absolute activity against the HIV-1 clinical isolate and
mutants (EC50 values) and the relative activity compared with
the activity against wt HIV-1 (fold-resistance) need to be
considered. In particular, in this assay, the uracil derivatives
4a-c,i-m retained in part their activities against the clinical
isolate IRLL98 and the Y181C mutant, with EC50 values at the
low micromolar (IRLL98) or submicromolar (Y181C) level,
whereas they were inactive against the HIV-1 K103N and
Y188L mutant strains. Nevertheless, their fold-resistance values
ranged from 22 to 169 (IRLL98), and from 4 to 156 (Y181C),
so that the pyrimidin-4(3H)-one 4c, carrying at the C2 position
(5d); EC50sY188L ) 2.3 (5c) and 1.6 µM (5d)), but joined to
high fold-resistance values.
The introduction of a methyl/ethyl substituent at the C6
benzylic position of the pyrimidine ring in the uracil series of
the F2-N,N-DABOs (6a-c) increased the inhibitory potency of
the derivatives against the clinical isolate IRLL98 and the
K103N mutant. However, 6a-c were not active against the
Y188L mutant strain. Differently, the introduction at the same
time of methyl groups at the pyrimidine C5 and at the benzylic
position (7a,d-l,n,o) produced the most interesting derivatives,
highly active against all of the HIV-1 mutant strains tested. In
particular, 7a, 7f, 7g, and 7h, carrying N-methyl-N-alkylamino
substituents at the C2 pyrimidine ring showed single-digit
nanomolar inhibition against the clinical isolate IRLL98 con-
taining multiple drug-resistance mutations (EC50s: 5 (7a), 3 (7f),
1 (7g), and 1 (7h) nM) and nanomolar to subnanomolar
inhibiting activity against the Y181C mutant (EC50s: 14 (7a),
2 (7f), 0.7 (7g), and 0.6 (7h) nM). The same derivatives were
able to inhibit the K103N and Y188L HIV-1 mutant strains in
the range of 50 to 6 nM. Moreover, 7g and 7h displayed very
low or no fold-resistance values (1 to 17) against the IRLL98,
K103N, and Y181C variants. When compared with the proto-
types of F2-S-DABOs (2a) and F2-NH-DABOs (3a) as well as
with EFV, 7a, 7f, 7g, and 7h, they were one or 2 orders of
magnitude more potent than the reference drugs against the
tested clinical isolate and mutant strains. The F2-N,N-DABOs
of series 7 and those bearing a cyclic amine at C2 (7i-l,n,o)
were less potent than the corresponding derivatives with a
N-methyl-N-alkylamino moiety at C2, and they were active at
the submicromolar to nanomolar level against the tested HIV-1
mutant strains. 7i-l,n,o displayed activities similar to those of
2a and 3a and lower than that of EFV. Differently from 2a and
3a, inactive against the Y188L mutant strain, the F2-N,N-DABOs
7j-l,n,o showed similar activity as EFV against the above
HIV-1 mutant. The replacement of one of the two C5/benzylic
methyl groups in the 7a structure with an ethyl group (7d, with
the ethyl group at the benzylic position, and 7e, carrying a C5-
a N-methyl-N-iso-propylamine residue, was the only real highly
IRLL98
active compound (EC50
) 0.18 µM with a 22-fold
Y181C
resistance ratio with respect to wt, and EC50
with a 4-fold resistance ratio).
) 0.03 µM
The introduction of a methyl group at the C5 position (thymine
derivatives) conferred to the C2-N-methyl-N-alkylamino-
substituted compounds (5a-e) an additional inhibitory activity
against the K103N and Y188L mutant strains (at the micromolar
range) besides that against the clinical isolate IRLL98 and the
mutant Y181C (at the submicromolar/nanomolar level). Dif-
ferently, the thymine derivatives having at C2 a cyclic amine
remained inactive against the K103N and Y188L mutants. In
this series also, the fold-resistance values of the derivatives
showed moderate to high values. The most-active compounds
were 5c and 5d, bearing an N-methyl-N-iso-propylamino (5c)
or an N-methyl-N-propylamino (5d) substituent at C2 and
endowed with nanomolar activity against IRLL98 and Y181C
mutants [EC50sIRLL98 (nM)/fold resistance: 80/40 (5c) and 55/
27 (5d); EC50sY181C (nM)/fold resistance: 30/15 (5c) and 39/
19 (5d)], and micromolar inhibiting activity against the K103N
and Y188L mutant strains (EC50sK103N ) 1.2 (5c) and 0.75 µM

2-Aminopyrimidin-4(3H)-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5419
Figure 1. Upper left: 3a conformations as docked into WT (cyan), L100I (orange), Y181C (deep pink), and Y188C (spring green). Upper right:
3a conformations as docked into V106A (cyan), Y188L (light purple) and K103N (light green). Lower left: 7h conformations as docked into WT
(cyan), L100I (orange), Y181C (deep pink), Y188C (spring green). Lower right: 7h conformations as docked into V106A (cyan), Y188L (light
purple) and K103N (light green). All of the conformations are readily comparable because the corresponding RTs were superimposed before any
docking experiments. Figures were generated with the Chimera program,51 and color names are taken directly from the all-color grid within the
program. Hydrogen atoms are not shown for sake of clarity.
ethyl substitution) was detrimental for the inhibitory potencies
of the derivatives: indeed, the EC50 values of 7d,e were 5- to
236-fold lower than that of 7a.
10 min preincubations in the presence of both wt and NNRTI-
resistant mutant RTs.
Against K103N RT, among the tested uracil and thymine
derivatives, only 2-thiomorpholin-4-yl uracil analogue 4o
retained to some extent a limited inhibitory activity. Differently,
the compounds carrying the double-methyl substitution at the
C5/benzylic positions of the pyrimidine ring and a N,N-dimeth-
ylamino (7a), N-methyl-N-alkylamino (7f-h), or piperidin-1-
yl (7j) substituent at C2 showed a submicromolar potency, lower
than that of 2a, similar or slightly higher than that of 3a, and
up to 87- and 37-fold higher than the ones exhibited by EFV
and NVP, respectively, used as reference drugs.
Enzyme Inhibiting Activity against HIV-1 RT Wild-Type
and NNRTI-Resistant Mutants. Selected F2-N,N-DABOs 4-7
were tested against a panel of recombinant HIV-1 RTs either
wild type (WTIIIB) or carrying known NNRTI-resistance muta-
tions (K103N, L100I, V106A, V179D, Y181I, and Y188L)
(Table 5). As in a kinetic study,42 a number of N,N-DABOs
belonging to the 7 series (i.e., 7a, 7f, 7j, and 7n) displayed lower
association rates (Kon) and much slower dissociation rates (Koff)
than EFV and UC781,43 used as reference compounds (thus
behaving as slow-, tight-binding inhibitors of HIV-1 RT), the
inhibitory potencies of DABO derivatives were determined with
The L100I mutant RT was inhibited at submicromolar level
by the uracil 4o, the thymine derivatives 5p and 5t, and 7g and

5420 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Mai et al.
Figure 2. 3a (left) and 7h (right) rigidly superimposed by means of surflex.50
7h carrying the two methyl groups at pyrimidine-C5 and at the
benzylic position, in addition to the S-DABO 2a and the NH-
DABO 3a. In the same assay, the thymine 5q, the ethyl-
substituted at linker position 6c, and most of the compounds
belonging to the 7 series (7a, 7e, 7f, 7i, 7n) were active at a
low micromolar range.
Against the mutant V106A and V179D RTs, an unexpected
range of activity with N,N-DABOs was observed: such mutant
RTs seem to be more susceptible toward a lot of the tested
compounds than the WT RT (for example 4i, 5d,q,r,u, and
7f-h,j,o, that were more potent against V106A than against
the RT wt, and 6c and 7a,e that, similarly to 3a, showed the
same activities against the two cited RTs). This behavior is
totally different from that of the S-DABO prototype 2a. More
intriguingly, V179D has been reported as a RT mutation elicited
from cellular chronic treatment with S-DABOs.21
Against the Y181I mutant RT, the DABOs 4o, and 7g, as
well as 2a and the reference drug EFV retained inhibitory
activity at the submicromolar level, and 4a, 7a, 7e, 7f, and 7h
together with 3a were shown to be active at the low micromolar
range. In the presence of Y188L, the uracil derivative 4o, the
thymine 5u, and all of the tested compounds 7 except for 7d
(in addition to the S-DABO 2a and the NH-DABO 3a) displayed
micromolar/submicromolar inhibitory activity.
dinates of 65 HIV-RT/ligand complexes (including wt and
K103N, Y181C, Y188L, Y188C, V106A, V108I, and L100I-
mutated NNBSs; Table A in the Supporting Information) were
retrieved from the Research Collaboratory for Structural Bio-
informatics (RCSB) Protein Data Bank (PDB).49 To check the
structural role of either the single or double substitution at the
C2-N DABO nitrogen atom, beyond the above selected 7h, a
structurally related F2-NH-DABO (3a) was also cross-docked
in the 65 NNBSs. The analyses of the docking results revealed
that monosubstituted F2-NH-DABO 3a has a common binding
mode to wt, Y181C, Y188C, and L100I NNBSs, similar to those
earlier described for other F2-NH-DABOs28 in wt and Y181C
RTs (Figure 1). These observations confirm that the introduction
of an extra anchor point in the S-DABO f NH-DABO conver-
sion was an important improvement28 for the anti-RT activity
against Y181C, Y188C, and L100I mutated forms. Regarding
the 3a binding mode into the V106A, Y188L, and K103N RTs,
the docked conformations display different arrangements into
the NNBSs (Figure A, in the Supporting Information). This
could reflect that, for such mutations, the introduced anchor
point of the F2-NH-DABO28 would not be any more sufficient
to contrast the NNBS geometry variations, and a sort of
adaptation to the mutated RTs occurred (Figure 1).
Interestingly, in the cross-docking studies, 7h (the N-methyl
F2-N,N-DABO analogue of 3a) did not display any common
binding mode within the seven docked conformations. This
higher adaptable behavior of 7h resembles that described by
Das et al.38 for the experimental conformations of etravirine
and analogues. In the cross-docking studies performed here, 7h,
when compared to 3a, seems to have a higher level of
adaptability into the NNBS binding pocket that involves a
combination of conformational adjustments (“wiggling”) and
rotational and translational shifts (“jiggling”) of the inhibitor
within the binding pocket.38 In particular, the rigidly surflex50
superimposed 3a and 7h docked conformations (Figure 2)
suggested that the conformational variations of F2-N,N-DABO
derivatives would mainly result from changes in torsion angles
τ3 and τ4 (Figure 3) because τ1 and τ2 are preset by internal
energies.26 Moreover, the 3a and 7h compact structures also
permit rotational and translational shifts within the binding
pocket. Apparently, the F2-N,N-DABO analogues can change
conformation (wiggle) and reorient and reposition (jiggle) in
response to changes in the NNBS caused by mutations, and
this adaptability may help to explain their enhanced ability to
inhibit mutated HIV-1 RTs.
Binding Mode Studies. On several occasions, we have
described the ability of the Autodock44 program as a useful tool
both to investigate the binding mode and to design new enzyme
inhibitors.27,41,45-47 In particular, when used in cross-docking
experiments, Autodock proved to be a valuable docking program
to include the ligand-induced fit in molecular-modeling-assisted
drug design.41 Therefore, we decided to employ the Autodock
suite to look into the binding mode of the newly synthesized
F2-N,N-DABOs. Derivative 7h was highly active against a panel
of NNRTI-resistant RT mutants [Ki values, µM: 0.03 (WT),
0.17 (K103N), 0.18 (L100I), 0.02 (V106A), 1.26 (Y181I), and
1.43 (Y188L)] and showed the highest inhibitory action against
HIV-1 clinical isolate and some mutant strains [EC50 values,
µM: 0.0006 (WT), 0.001 (IRLL98), 0.01 (K103N), 0.0006
(Y181C), and 0.03 (Y188L)]. Thus, 7h was taken as a
representative compound to study the F2-N,N-DABOs’ binding
mode into the experimental RT non-nucleoside binding site
(NNBS) by the means of cross-docking studies.41 7h was
modeled in its R conformation, because it was expected to be
the most-active enantiomer in analogy with that reported with
S-DABOs and NH-DABOs.26-28,48 The experimental 3D coor-

2-Aminopyrimidin-4(3H)-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5421
shifts are reported in δ units relative to the internal reference
tetramethylsilane (Me4Si). All of the compounds were routinely
checked by TLC and 1H NMR. TLC was performed on aluminum-
backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254
)
with spots visualized by UV light. All of the solvents were reagent
grade and, when necessary, were purified and dried by standard
methods. Concentration of solutions after reactions and extractions
involved the use of a rotary evaporator operating at a reduced
pressure of ca. 20 Torr. Organic solutions were dried over anhydrous
sodium sulfate. Analytical results are within (0.40% of the
theoretical values. All of the chemicals were purchased from Aldrich
Chimica, Milan (Italy), or from Lancaster Synthesis GmbH, Milan
(Italy), and were of the highest purity.
General Procedure for the Synthesis of Ethyl â-Oxoesters
(9). Example: Ethyl 4-(2,6-Difluorophenyl)-2-ethyl-3-oxopen-
tanoate (9d). Triethylamine (12.6 mL, 90.2 mmol) and magnesium
dichloride (6.71 g, 70.5 mmol) were added to a stirred suspension
of potassium 2-ethylmalonate monoethyl ester24 (11.74 g, 59.2
mmol) in dry acetonitrile (90 mL), and stirring was continued at
room temperature for 2 h. Then, a solution of the 2-(2,6-
difluorophenyl)propionic imidazolide in the same solvent (28 mL),
prepared 15 min before by reaction a between 2-(2,6-difluorophe-
nyl)propionic acid26 (10c, 5.25 g, 28.2 mmol) and N,N′-carbonyl-
diimidazole (5.49 g, 33.8 mmol) in acetonitrile (28 mL), was added.
The reaction mixture was stirred overnight at room temperature
and then was heated at reflux for 2 h. After the mixture was cooled,
13% HCl (235 mL) was cautiously added while the temperature
was kept below 25 °C, and the resulting clear mixture was stirred
for a further 15 min. The organic layer was separated and
evaporated, and the residue was treated with ethyl acetate (100 mL).
The aqueous layer was extracted with ethyl acetate (3 × 100 mL),
and the organic phases were collected, washed with a sodium
hydrogen carbonate saturated solution (3 × 100 mL) and brine (100
mL), dried, and concentrated to give pure 9d (6.00 g) as a yellow
oil, which was directly used in the following step. 1H NMR (CDCl3)
δ 0.83 (t, 3H, CHCH2CH3), 1.20 (t, 3H, COCH2CH3), 1.48 (d, 3H,
CHCH3), 1.85 (m, 2H, CH2CH3), 3.39 (m, 1H, CHCH2CH3), 4.12
(m, 3H, COCH2CH3 and CHCH3), 6.91 (m, 2H, C3,5-H Ar), 7.24
(m, 1H, C4-H Ar). Anal. C, H, F.
Figure 3. 7h and the torsional angles influencing the compound
flexibility.
Conclusion
In this article, a number of 5-alkyl-2-(N,N-disubstituted)-
amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-
ones (F2-N,N-DABOs) and related derivatives 4-7 belonging
to the DABO family14 have been reported as novel anti-HIV-1
agents. Such compounds were tested against wt HIV-1 NL4-
3-infected MT-4 cells as well as against MT-4 cells infected
with three different HIV-1 mutant strains (K103N, Y181C, and
Y188L). In addition, the effect of the F2-N,N-DABOs 4-7
against IRLL98, an HIV-1 clinical isolate resistant to NRTIs
(lamivudine, emtricitabine) and NNRTIs (NVP, delavirdine,
EFV) was determined. Cytotoxicity data of 4-7 were evaluated
on MT-4 cells with the mock-infected method. Enzyme inhibi-
tory assays were performed with selected derivatives against a
panel of recombinant HIV-1 RTs, either wild type or carrying
known NNRTI-resistance mutations (K103N, L100I, V106A,
V179D, Y181I, and Y188L).
In cellular assays, the majority of the synthesized F2-N,N-
DABOs proved to be highly active against both wt and mutant
HIV-1 strains, at the submicromolar to nanomolar range. In
some cases (6b, 7a,f-h), they showed subnanomolar potency
against wt HIV-1 and, more interestingly, against the Y181C
mutant strain (7g,h). The highest HIV-1 inhibitory activity is
associated with the presence of two methyl groups at the
pyrimidine-C5 position and at the benzylic position, together
with the insertion of a N-methyl-N-alkylamino chain at the
pyrimidine-C2, in addition to the highly favorable 2,6-difluo-
robenzyl substitution at the C6 position of the pyrimidine ring.
Compounds bearing all such chemical features were more potent
than the F2-S-DABO and F2-NH-DABO prototypes 2a and 3a
and than NVP and EFV used as reference drugs. The introduc-
tion of a cyclic amine (pyrrolidine, piperidine, morpholine,
thiomorpholine, etc.) at the C2 position of the pyrimidine ring
instead of the N-methyl-N-alkylamino moiety led to derivatives
active at the nanomolar (against HIV-1 wt) or submicromolar
(HIV-1 mutant strains) level, they being more potent than NVP
and active at the same range of 2a, 3a, and EFV. Enzyme
inhibitory data fully agree with cellular evaluation. Binding
mode analyses performed on F2-NH-DABO 3a and F2-N,N-
DABO 7h suggest that the higher adaptability showed by 7h
in comparison with 3a, by changing conformation (wiggle) and
reorienting and repositioning (jiggle) in response to changes in
the HIV-1 RT NNBS may almost in part explain its higher
inhibitory effect against the mutated RTs.
General Procedure for the Preparation of 6-[(2,6-Difluo-
rophenyl)alkyl]-3,4-dihydro-2-dimethylamino-5-(un)substitut-
edpyrimidin-4(3H)-ones (4a, 5a, 6a, 6c, and 7a-e). Example:
6-[1-(2,6-Difluorophenyl)ethyl]-3,4-dihydro-2-dimethylamino-5-
methylpyrimidin-4(3H)-one (7a). Sodium metal (0.68 g, 29.75
mmol) was dissolved in 100 mL of absolute ethanol, and 1,1-
dimethylguanidine sulfate (2.70 g, 9.92 mmol) and ethyl 4-(2,6-
difluorophenyl)-2-methyl-3-oxopentanoate26 (2.68 g, 9.92 mmol)
were added to the clear solution. The mixture was heated at reflux
for 5 h. After cooling, the solvent was evaporated in a vacuum at
40-50 °C until dry, and the residue was dissolved in a small amount
of water (20 mL), neutralized with 1 N HCl, and extracted with
ethyl acetate (3 × 25 mL). The organic extracts were washed with
brine (3 × 50 mL), dried, and evaporated in a vacuum, and the
residue was purified by chromatography on silica gel eluting with
1
ethyl acetate/chloroform 1:1. H NMR (DMSO-d6): δ 1.65-1.66
(d, 3H, ArCHCH3), 1.92 (s, 3H, C5-CH3), 3.11 (s, 6H, NCH3),
4.53-4.58 (q, 1H, ArCHCH3), 6.77-6.85 (m, 2H, C3,5-Ar-H),
7.12-7.20 (m, 1H, C4-Ar-H). Anal. C, H, F, N.
General Procedure for the Preparation of 2-N,N-Disubsti-
tuted- (or cyclic) -amino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-
dihydro-5-(un)substitutedpyrimidin-4(3H)-ones (4e-q, 5e-u,
and 7i-o). Example: 6-[1-(2,6-Difluorophenyl)ethyl]-3,4-dihy-
dro-5-methyl-2-(morpholin-4-yl)pyrimidin-4(3H)-one (7n). A
mixture of 6-(2,6-difluorophenylmethyl)-3,4-dihydro-2-methylthio-
5-methylpyrimidin-4(3H)-one24 (0.3 g, 1.01 mmol) and morpholine
(5 mL) was heated in a sealed tube at 170 °C for 8 h. After cooling,
the crude residue was dissolved in ethyl acetate (60 mL) and water
(60 mL). The aqueous phase was extracted with ethyl acetate (2 ×
50 mL). The organic extracts were washed with brine (3 × 50 mL),
dried, evaporated under reduced pressure, and purified by column
chromatography (silica gel, ethyl acetate/chloroform 1:1). 1H NMR
Experimental Section
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. 1H NMR spectra were
recorded at 400 MHz on a Bruker AC 400 spectrometer; chemical

5422 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Mai et al.
(DMSO-d6): δ 1.51-1.55 (d, 3H, ArCHCH3), 1.80 (s, 3H,
C5-CH3), 3.55-65 (m, 8H, morpholine ring), 4.50-4.53 (q, 1H,
ArCHCH3), 6.95-7.05 (m, 2H, C3,5-Ar-H), 7.31-7.41 (m, 1H,
C4-Ar-H). Anal. C, H, F, N.
(d, 3H, CHCH3), 4.11 (q, 1H, CHCH3), 6.87 (m, 3H, C3-5-Ar-H),
7.21 (m, 2H, C2-4-Ar-H). Anal. C, H, F.
Biology. Anti-HIV Activity in Lymphoid Cells. Biological
activity of the compounds was tested in the lymphoid MT-4 cell
line (received from the NIH AIDS Reagent Program) against the
wt HIV-1 NL4-3 strain and three different HIV-1 strains, as
described before.52 Briefly, MT-4 cells were infected with the
appropriate HIV-1 strain (or mock-infected to determine cytotox-
icity) in the presence of different drug concentrations. At day five
post-infection, a tetrazolium-based colorimetric method (MTT
method) was used to evaluate the number of viable cells. The
IRLL98 HIV-1 strain contains the following mutations in the RT
coding sequence:53 M41L, D67N, Y181C, M184V, R211K, T215Y
(conferring resistance to NRTIs) and mutations K101Q, Y181C,
G190A (conferring resistance to NNRTIs). The HIV-1 strains
containing the multi-NNRTI mutation, K103N, or the Y188L
mutant were received from the Medical Research Council Central-
ized Facility for AIDS Reagents, Herfordshire, UK.
In the same conditions, the reaction of pyrrolidine with 6-(2,6-
difluorophenylmethyl)-3,4-dihydro-2-methylthiopyrimidin-4(3H)-
one26 and 6-(2,6-difluorophenylmethyl)-3,4-dihydro-2-methylthio-
5-methylpyrimidin-4(3H)-one26 afforded a mixture of two compounds
(4g,h and 5i,j, respectively), that were separated by column
chromatography (silica gel, ethyl acetate/chloroform 3:1). Ex-
ample: 6-(2,6-Difluorophenylmethyl)-5-methyl-2-(pyrrolidin-1-yl)-
pyrimidin-4(3H)-one (5i): 1H NMR (CDCl3): δ 1.90 (m, 4H,
NCH2CH2CH2), 2.04 (s, 3H, C5-CH3), 3.35 (m, 4H, CH2NCH2),
4.14 (s, 2H, C6-CH2), 6.84 (m, 2H, C3,5-Ar-H), 7.12 (m, 1H,
C4-Ar-H), 10.74 (s, 1H, NH). Anal. C, H, F, N.
6-[2-Fluoro-6-(pyrrolidin-1-yl)-phenylmethyl]-5-methyl-2-(pyr-
rolidin-1-yl)pyrimidin-4(3H)-one (5j): 1H NMR (CDCl3): δ 1.87
(m, 8H, 2 × NCH2CH2CH2), 2.03 (s, 3H, C5-CH3), 3.10-3.38
(m, 8H, 2 × CH2NCH2), 4.12 (s, 2H, C6-CH2), 6.67 (m, 2H,
Anti-HIV Reverse Transcriptase Assays. RNA-dependent
DNA polymerase activity was assayed as described54 in the presence
of 0.5 µg of poly(rA)/oligo(dT)10:1 (0.3 µM 3′-OH ends), 10 µM
[3H]-dTTP (1 Ci/mmol), and 2-4 nM RT in the presence of an
8% final concentration of DMSO.
C
3,5-Ar-H), 7.05 (m, 1H, C4-Ar-H), 10.96 (s, 1H, NH). Anal.
C, H, F, N.
General Procedure for the Preparation of 2-N,N-dialkyl-
amino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-(un)substi-
tutedpyrimidin-4(3H)-ones (4b-d, 5b-d, 6b, and 7f-h). Ex-
ample: 6-[1-(2,6-Difluorophenyl)methyl]-3,4-dihydro-2-(N-ethyl-
N-methylamino)-5-methylpyrimidin-4(3H)-one (5b). N-Ethyl-N-
methylammonium acetate salt was prepared by slowly the addition
of 2.1 mL (0.036 mol) of glacial acetic acid to 3.1 mL (0.036 mol)
of N-ethyl-N-methylamine in an ice bath. Afterward, 6-(2,6-
difluorophenylmethyl)-3,4-dihydro-5-methyl-2-methylthiopyrimi-
din-4(3H)-one26 (0.53 g, 1.8 mmol) was added, and the mixture
was fused for 5 h at 160 °C. After cooling, the residue was
partitioned between water (60 mL) and ethyl acetate (60 mL), and
the aqueous phase was extracted with ethyl acetate (2 × 50 mL).
The combined organic extracts were washed with brine (3 × 50
mL) and dried. Evaporation of the solvent furnished a crude residue,
which was purified by column chromatography (silica gel, ethyl
acetate/chloroform 2:1). 1H NMR (CDCl3): δ 0.98 (t, 3H,
NCH2CH3), 2.04 (s, 3H, C5-CH3), 2.96 (s, 3H, NCH3), 3.37 (q,
2H, NCH2CH3), 3.87 (s, 2H, C6-CH2), 6.85 (m, 2H, C3,5-Ar-H),
7.15 (m, 1H, C4-Ar-H), 11.26 (s, 1H, NH). Anal. C, H, F, N.
Synthesis of 6-(2,6-Difluorophenylmethyl)-2-(1H-pyrrol-1-yl)
pyrimidin-4(3H)-one (4r). 2,5-Dimethoxytetrahydrofuran (1.96 g,
14.8 mmol) was added to a solution of 2-amino-6-(2,6-difluoro-
phenylmethyl)pyrimidin-4(3H)-one 1128 (3.00 g, 13.5 mmol) in
glacial acetic acid (50 mL), and the resulting mixture was stirred
at reflux for 5 min. After the completion of the reaction, the mixture
was poured into crushed ice and stored at 0-5 °C overnight. The
resulting solid was then filtered, washed with water, dried, and
Reagents. [3H]-dTTP (40 Ci/mmol) was from Amersham and
unlabeled dNTP’s from Boehringer. Whatman was the supplier of
the GF/C filters. All of the other reagents were of analytical grade
and purchased from Merck or Fluka. The homopolymer poly(rA)
(Pharmacia) was mixed at weight ratios in nucleotides of 10:1, to
the oligomer oligo(dT)12-18 (Pharmacia) in 20 mM Tris-HCl (pH
8.0), containing 20 mM KCl and 1 mM EDTA, heated at 65 °C
for 5 min and then slowly cooled at room temperature.
Proteins. Recombinant proteins expression and purification was
as described.54 All of the enzymes were purified to >95% purity.
RT Inhibition Assays. Time-dependent incorporation of radio-
active nucleotides into poly(rA)/oligo(dT)10:1 at different nucleotide
substrate concentrations was monitored by removing 25 µL-aliquots
at 2-min time intervals. Initial velocities of the reaction were then
plotted against the corresponding substrate concentrations. For
inhibition constant (Ki) determination, an interval of inhibitor
concentrations between 0.2 and 5 Ki was used in the inhibition
assays. Data were then plotted according to Lineweaver-Burke
and Dixon.
Molecular Modeling Studies. RT Structures Preparation.
Sixty-five publicly available X-ray crystal structures of reverse
transcriptase complexed with a NNRTI were retrieved from the
Protein Data Bank55-57 (Supporting Information). Of the 65
complexes, 45 belonged to the wild-type enzyme (because the long
distance from the NNBS, the tetra mutant 1RT3, and the C280S
mutated forms were considered as wild-type RT) and 20 belonged
to RT mutated forms. All of the complexes were superimposed
onto each other arbitrarily using nevirapine/RT (1VRT) as a
reference complex. The superimposition of the RT complexes was
performed by the means of the program ProFit version 2.2,58 using
the implemented McLachlan algorithm.59 All of the residue’s
backbone atoms comprised in a 20 Å core from nevirapine (1VRT)
were selected with the Chimera program60 (182 residues) and used
as a reference for the fitting, the same number of residues (Trp88A-
Tyr115A, Ser156A-Leu210A, Trp212A, Leu214A-Ile244A, Lys263A,
Asn265A-Tyr271A, Glu312A-Ile326A, Tyr339A-Thr351A, Ile375A,
Thr377A-Lys385A, Gln23B, Pro25B-Glu28B, Ile31B-Lys32B,
Val35B, Thr131B-Arg143B) was used for all of the complexes’
superimpositions.
Then, using the AMBER 8.0 suite, the complexes were mini-
mized to alleviate steric contacts, which usually arise due to the
random way in which hydrogen atoms are added to the heavy atoms.
Hydrogen atoms were added to the receptors with the tLeap module.
Protonation states were assumed to be those most common at pH
7, that is, lysines, arginines, histidines, aspartates, and glutamates
were considered in the ionized form. Each complex was solvated
(SOLVATEOCT command) with water molecules (TIP3 model)
in a box extending 10 Å, and counterions were added to neutraliza-
1
purified by recrystallization from acetonitrile. H NMR (CDCl3-
CD3OD): δ 4.01 (s, 2H, C6-CH2), 6.08 (d, 2H, C3,4-pyrrole-H),
6.29 (s, 1H, C5-H), 6.93 (m, 2H, C3,5-Ar-H), 7.28 (m, 1H,
C4-Ar-H), 7.62 (d, 2H, C2,5-pyrrole-H). Anal. C, H, F, N.
Synthesis of 2-(2-Fluorophenyl)propionic Acid (10b). A di-
ethylamine (2.5 mL, 24.2 mmol) solution in dry THF (5 mL) was
added dropwise at -78 °C to a buthyllithium (19.4 mL of 2.5 M
solution in n-hexane, 48.4 mmol) solution in dry THF (10 mL),
and the resulting mixture was stirred for 0.5 h at 0 °C. A solution
of 2-fluorophenylacetic acid (3.39 g, 22.0 mmol) in anhydrous THF
(5 mL) was then added dropwise at -78 °C to the mixture. After
0.5 h of stirring at 0 °C, a solution of methyl iodide (3.43 g, 1.5
mL, 24.2 mmol) in dry THF (5 mL) was added at -78 °C. The
mixture was stirred and gradually warmed from -78 °C to room
temperature over 1 h and then poured into water (150 mL) and
extracted with ethyl acetate (3 × 50 mL). The aqueous layer was
acidified with concentrated hydrochloric acid at 0 °C and then
extracted with ethyl acetate (4 × 50 mL). The combined organic
layers were washed with brine to neutral pH and dried with
anhydrous sodium sulfate. Evaporation of the solvent gave the crude
acidic fraction, which was purified by column chromatography
(silica gel, ethyl acetate/chloroform 1:5). 1H NMR (CDCl3): δ 1.50

2-Aminopyrimidin-4(3H)-ones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5423
(9) Balzarini, J. Current Status of the Non-Nucleoside Reverse Tran-
scriptase Inhibitors of Human Immunodeficiency Virus Type 1. Curr.
Top. Med. Chem. 2004, 4, 921-944.
tion. The solvated complex was then refined by minimization using
the SANDER module of AMBER. The parameters for the cocrys-
tallized NNRTI were calculated using the antechamber module of
AMBER and the atomic charges were calculated using the AM1
Hamiltonian. Once the minimizations were complete, the ligands
(NNRTIs) and the receptors (RTs) were extracted into separate files
(locks and keys) to be used for the subsequent docking set up.
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(NNRTIs): Past, Present and Future. Chem. BiodiVersity 2004, 1,
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M.; Lange, J. M. TMC125 Exerts Similar Initial Antiviral Potency
as a Five-Drug, Triple Class Antiretroviral Regimen. AIDS 2003,
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Activities of Isotrimethoprim and Some Related Derivatives. Eur. J.
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Sbardella, G.; Massa, S.; Artico, M. 3,4-Dihydro-2-alkoxy-6-benzyl-
oxopyrimidines (DABOs): Development of a Potent Class of Non-
Nucleoside Reverse Transcriptase Inhibitors. Recent Res. DeVel.
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Tramontano, E;, La Colla, P. 3,4-Dihydro-2-alkoxy-6-benzyl-4-
oxopyrimidines (DABOs): A New Class of Specific Inhibitors of
Human Immunodeficiency Virus Type 1. AntiViral Chem. Chemother.
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HIV-1 Activity of 3,4-Dihydro-2-alkoxy-6-benzyl-4-oxopyrimidines
(DABOs), New Non-Nucleoside Reverse Transcriptase Inhibitors.
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A. G.; Scano, P.; La Colla, P. Synthesis and Antiviral Activity of
New 3,4-Dihydro-2-alkoxy-6-benzyl-4-oxopyrimidines (DABOs),
Specific Inhibitors of Human Immunodeficiency Virus Type-1.
AntiViral Chem. Chemother. 1995, 6, 1-8.
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E.; Scano, P.; La Colla, P. Synthesis and Anti-HIV-1 Activity of
Thio Analogues of Dihydroalkoxybenzyloxopyrimidines. J. Med.
Chem. 1995, 38, 3258-3263.
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P. 6-(3-Methylbenzyl)-2-(2-methylpropyl)thio-4(3H)-pyrimidinone
(DABO 622). Acta Crystallogr. 1995, C52, 2115-2117.
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A. G.; De Montis, A.; Scintu, F.; Putzolu, M.; La Colla, P. Dihydro-
(alkylthio)(naphthylmethyl) Oxopyrimidines: Novel Non-Nucleoside
Reverse Trasncriptase Inhibitors of the S-DABO Series. J. Med.
Chem. 1997, 40, 1447-1454.
Docking Set-Up. Autodock 3.0.544 was used for all of the
docking calculations. The AutodockTools package version 1.4.4
was employed to generate the docking input files and to analyze
the docking results, the same procedure as described in the manual
was followed. All of the nonpolar hydrogen atoms and the water
molecules were removed. The Kollmann charges were loaded for
the proteins, whereas the Gasteiger charges were used for the
ligands. A grid box size of 61 × 81 × 61 points with a spacing of
0.375 Å between the grid points was implemented and covered
more than 8 Å of the NNBS. The grid was centered on the mass
center of the experimental bound nevirapine (1VRT) coordinates
taken as a NNRTI representative. For all of the inhibitors, the single
bonds including the amide bonds were treated as active torsional
bonds. One hundred docked structures, that is, 100 runs, were
generated by using genetic algorithm searches. A default protocol
was applied, with an initial population of 50 randomly placed
individuals, a maximum number of 2.5 × 105 energy evaluations,
and a maximum number of 2.7 × 104 generations. A mutation rate
of 0.02 and a crossover rate of 0.8 were used. Results differing by
less than 2.0 Å in a positional root-mean-square deviation (rmsd)
were clustered together and represented by the result with the most-
favorable free energy of binding. As previously reported, Autodock
proved to reproduce the experimental structure of the NNRTIs
with low rmsd values. The representative F2-NH-DABO (3a) and
F2-N,N-DABO (7h) structures were built by means of the stand-
alone version of the program PRODRG61 in conjunction with
GROMACS62 and then cross-docked in the 66 RTs using the above
settings. The cross-docking results for each of the seven enzyme
isoforms (WT, L100I, V106A, K103N, Y181C, Y188C, and
Y188L) and were then reclustered using a rmsd tolerance value of
2.0. In each cluster, the best cluster41 conformation coincided with
the best docked conformation,41 therefore only seven conformations
for both 3a and 7h were used for the binding mode analyses.
Acknowledgment. This work was partially supported by the
EU Contract LSHB-CT-2003-503480-TRIoH (to GM and JAE),
and the Spanish MEC project BFU2006-00966 and FIS PI060624
(JAE). I. Clotet-Codina holds a FI scholarship from Generalitat
de Catalunya.
(22) Mai, A.; Artico, M.; Sbardella, G.; Massa, S.; Novellino, E.; Greco,
G.; Loi, A. G.; Tramontano, E.; Marongiu, M. E.; La Colla, P.
5-Alkyl-2-(alkylthio)-6-(2,6-dihalophenylmethyl)-3,4-dihydropyrimi-
din-4(3H)-ones: Novel Potent and Selective Dihydro-alkoxy-benzyl-
oxopyrimidine Derivatives. J. Med. Chem. 1999, 42, 619-627.
(23) Sbardella, G.; Mai, A.; Artico, M.; Massa, S.; Marceddu, T.; Vargiu,
L.; Marongiu, M. E.; La Colla, P. Does the 2-Methylthiomethyl
Substituent Really Confer High Anti-HIV-1 Activity to S-DABOs?
Med. Chem. Res. 2000, 10, 30-39.
Supporting Information Available: Molecular modeling de-
tails; elemental analyses of compounds 4-7. This material is
available free of charge via the Internet at http://pubs.acs.org.
(24) Sbardella, G.; Mai, A.; Artico, M.; Chimenti, P.; Massa, S.; Loddo,
R.; Marongiu, M. E.; La Colla, P.; Pani, A. Structure-Activity
Relationship. Studies on New DABOs: Effect of Substitutions at
Pyrimidine C-5 and C-6 Positions on Anti-HIV-1 Activity. AntiViral
Chem. Chemother. 2001, 12, 37-50.
(25) Pani, A.; Musiu, C.; Loi, A. G.; Mai, A.; Loddo, R.; La Colla, P.;
Marongiu, M. E. DABOs as Candidates to Prevent Mucosal HIV
Transmission. AntiViral Chem. Chemother. 2001, 12 (Suppl. 1), 51-
59.
(26) Mai, A.; Sbardella, G.; Artico, M.; Ragno, R.; Massa, S.; Novellino,
E.; Greco, G.; Lavecchia, A.; Musiu, C.; La Colla, M.; Murgioni,
C.; La Colla, P.; Loddo, R. Structure-Based Design, Synthesis, and
Biological Evaluation of Conformationally Restricted Novel 2-Alkyl-
thio-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones
as Non-Nucleoside Inhibitors of HIV-1 Reverse Transcriptase. J. Med.
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(27) Ragno, R.; Mai, A.; Sbardella, S.; Artico, M.; Massa, S.; Musiu, C.;
Mura, M.; Marceddu, T.; Cadeddu, A.; La Colla, P. Computer-Aided
Design, Synthesis, and Anti-HIV-1 Activity In Vitro of 2-Alkyl-
amino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-
4(3H)-ones as Novel Potent Non-Nucleoside Reverse Transcriptase
Inhibitors, Also Active Against the Y181C Variant. J. Med. Chem.
2004, 47, 928-934.
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