5-alkyl-6-benzyl-2-(2-oxo-2-phenylethylsulfanyl)pyrimidin-4(3H)-ones, a series of anti-HIV-1 agents of the dihydro-alkoxy-benzyl-oxopyrimidine family with peculiar structure - Activity relationship profile

Article abstract of DOI:10.1021/jm800340w

A series of dihydro-alkylthio-benzyl-oxopyrimidines (S-DABOs) bearing a 2-aryl-2-oxoethylsulfanyl chain at pyrimidine C2, an alkyl group at C5, and a 2,6-dichloro-, 2-chloro-6-fluoro-, and 2,6-difluoro-benzyl substitution at C6 (oxophenethyl-S-DABOs, 6-8)

Full text of DOI:10.1021/jm800340w

J. Med. Chem. 2008, 51, 4641–4652
4641
5-Alkyl-6-benzyl-2-(2-oxo-2-phenylethylsulfanyl)pyrimidin-4(3H)-ones, a Series of Anti-HIV-1
Agents of the Dihydro-alkoxy-benzyl-oxopyrimidine Family with Peculiar Structure-Activity
Relationship Profile
Maxim B. Nawrozkij,†,‡ Dante Rotili,§,‡ Domenico Tarantino,§ Giorgia Botta,§,| Alexandre S. Eremiychuk,† Ira Musmuca,§
Rino Ragno,§ Alberta Samuele, Samantha Zanoli, Mercedes Armand-Ugo´n,# Imma Clotet-Codina,# Ivan A. Novakov,†
Boris S. Orlinson,† Giovanni Maga, Jose´ A. Este´,# Marino Artico,§ and Antonello Mai*,§
Volgograd State Technical UniVersity, Pr. Lenina, 28, 400131 Volgograd, Russia, Dipartimento di Studi Farmaceutici, Sapienza UniVersita` di
Roma, P. le A. Moro 5, 00185 Roma, Italy, Istituto di Genetica Molecolare IGM-CNR, Via Abbiategrasso 207, 27100 PaVia, Italy, and
RetroVirology Laboratory IrsiCaixa, Hospital UniVersitari Germans Trias i Pujol, UniVersitat Auto`noma de Barcelona, 08916 Badalona, Spain
ReceiVed March 26, 2008
A series of dihydro-alkylthio-benzyl-oxopyrimidines (S-DABOs) bearing a 2-aryl-2-oxoethylsulfanyl chain
at pyrimidine C2, an alkyl group at C5, and a 2,6-dichloro-, 2-chloro-6-fluoro-, and 2,6-difluoro-benzyl
substitution at C6 (oxophenethyl-S-DABOs, 6-8) is here described. The new compounds showed low
micromolar to low nanomolar (in one case subnanomolar) inhibitory activity against wt HIV-1. Against
clinically relevant HIV-1 mutants (K103N, Y181C, and Y188L) as well as in enzyme (wt and K103N,
Y181I, and L100I mutated RTs) assays, compounds carrying an ethyl/iso-propyl group at C5 and a 2,6-
dichloro-/2-chloro-6-fluoro-benzyl moiety at C6 were the most potent derivatives, also characterized by
low fold resistance ratio. Interestingly, the structure-activity relationship (SAR) data drawn from this DABO
series are more related to HEPT than to DABO derivatives. These findings were at least in part rationalized
by the description of a fair superimposition between the 6-8 and TNK-651 (a HEPT analogue) binding
modes in both WT and Y181C RTs.
Introduction
Dihydro-alkoxy-benzyl-oxopyrimidines (DABOs)1,2 are a
family of non-nucleoside reverse transcriptase (RT) inhibitors
(NNRTIs)3–5 endowed with micromolar to subnanomolar activ-
ity against human immunodeficiency virus type 1 (HIV-1). We
disclosed the first series of these inhibitors in 1992 by describing
the synthesis and antiviral activity of a number of pyrimidin-
4(3H)-ones bearing an alkoxy chain at the C2 position and a
benzyl/3-methylbenzyl/3,5-dimethylbenzyl group at the C6
position of the pyrimidine ring (DABOsa, Figure 1).6–9 The
replacement of the C2-alkoxy with a C2-alkylthio chain gave
an increase of the antiviral potency of the derivatives, which
showed submicromolar activity against wild type (wt) HIV-1
(S-DABOs, DATNOs; Figure 1).10–12 In our hands, the intro-
duction of a C6-naphthylmethyl moiety instead of the C6-benzyl
* To whom correspondence should be addressed. Phone: +396-4991-
3392. Fax:+396-491491. E-mail:antonello.mai@uniroma1.it.
†
Volgograd State Technical University.
MBN and DR contributed equally to this work.
Sapienza Universita` di Roma.
Present address: Dipartimento Farmaco Chimico Tecnologico, Univer-
‡
Figure 1. The DABO family.
one did not furnish a significant improvement of activity.12
§
|
Differently, the substitution of the C6-benzyl ring with halogens
(chloro, fluoro) at the 2- and 2,6-positions afforded highly potent
sita` degli Studi di Siena, via A. Moro, 53100 Siena, Italy.
Istituto di Genetica Molecolare IGM-CNR.
Universitat Auto`noma de Barcelona.
#
compounds (F2-S-DABOs, Figure 1), active at nanomolar
concentration against wt HIV-1.13–15 Conformational restriction
applied to F2-S-DABOs further improved the anti-HIV-1 activity
of the derivatives, leading to compounds active at low nanomolar
level against the wild type virus and also active against the
HIV-1 Y181C mutant.16–18 By replacing the C2-alkylthio with
a C2-alkylamino moiety, two further series (F2-NH-DABOs and
F2-N,N-DABOs, Figure 1) of highly potent, broad spectrum
NNRTI compounds belonging to the DABO family have been
obtained.19–22
a Abbreviation: AIDS, acquired immunodeficiency syndrome; BHAP,
bis(heteroaryl)piperazine; CC50, compound concentration toxic for 50% of
cells; DABOs, dihydro-alkoxy-benzyl-oxopyrimidines; DATNOs, dihydro-
alkylthio-naphthylmethyl-oxopyrimidines; EC50, effective concentration able
to protect 50% of cells from the HIV-1 induced cytopathogenicity; EFV,
efavirenz; F2-N,N-DABOs, 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-dif-
luorophenylalkyl)pyrimidin-4(3H)ones; HEPT, 1-[(2-hydroxyethoxy)m-
ethyl]-6-(phenylthio)thymine; HIV, human immunodeficiency virus; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NH-DABOs,
dihydro-alkylamino-benzyl-oxopyrimidines; NNBS, non-nucleoside binding
site; NNRTIs, non-nucleoside reverse transcriptase inhibitors; NVP, nevi-
rapine; PDB, protein data bank; RCSB, research collaboratory for structural
bioinformatics; RT, reverse transcriptase; SAR, structure-activity relation-
ship; S-DABOs, dihydro-alkylthio-benzyl-oxopyrimidines; WT, wild type.
In summary, we can draw the following structure-activity
relationships (SAR) (Figure 2) about the DABO family to obtain
10.1021/jm800340w CCC: $40.75
2008 American Chemical Society
Published on Web 07/17/2008

4642 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Nawrozkij et al.
Figure 2. SAR graphical summary of DABOs.
the highest HIV-1 inhibitory activity: (i) The pyrimidin-4(3H)-
one ring must carry a 2,6-difluoro-benzyl/-1-phenylethyl sub-
stituent at the C6 position, and a properly sized alkoxy, alkylthio,
or alkylamino chain at the C2 position. (ii) The HNCdO
fragment at N3/C4 position of the pyrimidine must be un-
changed. (iii) With the exception of the methylthiomethyl-S-
DABOs,14 the C5 position of the pyrimidine has to be substituted
with a methyl group (thymine derivatives): the uracil, 5-ethy-
luracil, and 5-iso-propyluracil derivatives are typically less
potent than the corresponding thymines. These SAR are quite
different from those of the DABO-related HEPT derivatives,
in which, to display the highest anti-HIV-1 activity, the
pyrimidine ring must be substituted with a 3,5-dimethylbenzyl
moiety at C6 and a iso-propyl group at C5.23–28
As a rule, to insert the C2-alkoxy, -alkylthio, or -alkylamino
chain in the DABO compounds, we linked either linear,
branched, or cyclic alkyl moieties with a growing number of
carbon units at the heteroatom at the C2 position of the
pyrimidine ring. The highest inhibitory activity was registered
with the insertion of iso-propyl, n-propyl, sec-butyl, n-butyl,
or cyclopentyl chains. Examples of C2-aryl(alkyl)thio chains
in the S-DABO series were performed by us (compounds 1a-p,
Figure 3), but the obtained compounds showed lower anti-HIV
activity when compared with the corresponding C2-alkylthio
counterparts (1q,r) (Table 1),29 thus confirming the negative
trend highlighted by Mugnaini et al. with some S-aryl-S-
DABOs.30
different substitution patterns at position 2, 5, and 6 of the
pyrimidine ring.31 The obtained new lead compound 4, having
a 4-methoxybenzylthio moiety at C2, a methyl group at C5,
and a 1-(2,6-difluorophenylethyl) portion at C6, displayed
picomolar activity against wt HIV-1 and submicromolar activi-
ties against some clinically relevant HIV-1 mutants.31 In the
same paper, four compounds having a phenacyl moiety at the
sulfur atom at C2 of the pyrimidine ring have been reported,
but they were endowed with low (if any) antiviral activity both
against wt HIV-1 and mutant strains.31 Moreover, a series of
5-alkyl-2-(2-aryl-2-oxoethylsulfanyl)-6-(1-naphthylmethyl)py-
rimidin-4(3H)-ones was reported by He et al. as unique NNRTIs
of the S-DABO series.32,33 In these compounds, the highest
activity (30 nM against wt HIV-1) has been obtained with the
introduction of a 2-(4-methoxyphenyl)-2-oxoethylsulfanyl chain
at the C2 position, and a iso-propyl group at the C5 position of
the pyrimidine ring (5, Figure 3). However, limited data on the
effects of such compounds against HIV mutant strains are
available, and no data have been reported about RT inhibition.
From these findings, with the aim to acquire new SAR
information and to explore the effect of the insertion of a C2-
oxo-phenethylsulfanyl moiety in our S-DABOs, on the activity
against wt HIV-1 and a panel of clinically relevant HIV-1
mutants, we prepared a further series of S-DABOs (oxophen-
ethyl-S-DABOs 6-8, Figure 3), characterized by the insertion
of a 2-oxo-2-[(substituted)phenyl]ethylsulfanyl chain at C2, a
2,6-dichloro-, 2-chloro-6-fluoro-, or 2,6-difluorobenzyl group
at C6, and hydrogen, methyl, ethyl, iso-propyl, or sec-butyl
substituent at C5.
In 2005, Manetti et al. reported that S-DABOs showing a
4-methoxybenzyl-/4-methoxyphenylethyl-thio substitution at C2,
a methyl group at C5, and a 2,6-dichlorobenzyl moiety at C6
(2 and 3, Figure 3) displayed nanomolar to subnanomolar
activity against both wt HIV-1 and the clinical isolate IRLL98
and micromolar/submicromolar activity against the HIV-1
K103N and Y188L mutant strains.30 Very recently, the same
group optimized their molecules by studying the effects of
Chemistry. The ethyl arylacetylacetates 9a-l, key intermedi-
ates for the synthesis of the title DABO derivatives, were
prepared through two alternative routes. The 2-H- and 2-methyl-
ꢀ-oxoesters 9a,13b,13e,f,j,13 were obtained by reaction of the
appropriate acylimidazolide with potassium ethyl malonate and
2-methylmalonate in the presence of triethylamine and magne-

Anti-HIV-1 Agents of the DABO Family
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4643
Figure 3. Arylalkyl-S-DABOs (1-4), 5-iso-propyl-2-[2-(4-methoxyphenyl)-2-oxoethylsulfanyl]-6-(1-naphthylmethyl)pyrimidin-4(3H)-one (ox-
ophenethyl-DATNO, 5), and oxophenethyl-S-DABOs (6-8).
Table 1. Cytotoxicity and Anti-HIV-1 Activity of
4(1H)-ones 10a-l, which were alkylated with the proper
Arylalkyl-F2-S-DABOs 1a-pa
ω-bromoacetophenones in the presence of sodium methoxide
compd
R
R2
CC50,b µM EC50,c µM
SId
in methanol (route d, for 6 and 7) or, alternatively, of potassium
carbonate in N,N-dimethylformamide (route e, for 8) to give
the title compounds 6-8 (Scheme 1).
Chemical and physical data for compounds 6-10 are listed
in Table 2.
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
Ph
PhCH2
2,6-F2-PhCH2
PhCH2CH2
PhCH2CH2CH2
CH3COCH2
PhCOCH2
4-Cl-PhCOCH2
4-NO2-PhCOCH2
>76e
>73
>66
>70
32
>81
>67
>61
22
>70
>37
>65
>65
>70
>70
>70
>84
>77
1.4
0.1
1.0
0.3
0.2
3.1
1.0
9.8
8.4
0.13
1.2
0.4
14.0
10.1
5.9
0.06
0.017
0.006
>54
>730
>66
>233
160
H
H
H
H
H
H
H
H
>26
>67
>6
Results and Discussion
Cytotoxicity and Wild Type HIV-1 Inhibiting Activity. The
oxophenethyl-S-DABOs 6–8 were tested in MT-4 cells to
evaluate their cytotoxicity and their capability to inhibit the HIV-
induced cytopathic effect (HIV-1 strain: NL4-3) by 50%.
Nevirapine (NVP) and efavirenz (EFZ) were also tested as
reference drugs (Table 3).
The majority of the tested compounds were not cytotoxic up
to 65 µM. Some compounds showed >11/>14 µM values of
CC50 (compound concentration toxic for 50% of cells) because
higher concentration could not be achieved for the precipitation
of the compounds in the culture medium.
When tested against wt HIV-1, the uracils 6a-c and 7a-c
displayed micromolar/submicromolar activity. With the insertion
of an alkyl substituent at the C5 position of the pyrimidine, the
inhibitory activity increased according to the order iso-propyl
(i-Pr)/sec-butyl (s-Bu) > Et > Me. Differently from other
DABO series, in which the optimal substituent at the C5 position
was the methyl group7,9,10,15,17,20,22,32 in oxophenethyl-S-DA-
BOs, the thymine derivatives (6d-f, 7d,f, 8a-c) showed anti-
HIV-1 activity in the high nanomolar range (200 to 15 nM),
whereas the 5-ethyluracil analogues (6g,h, 7g-i, 8d-f) were
more potent in inhibiting wt HIV-1 (EC50, concentration needed
to inhibit 50% HIV-induced cytopathic effect, values ranging
from 20 to 5 nM). Exceptions to this rule were the 6-(2-choro-
6-fluorobenzyl)-5-methylpyrimidin-4(3H)-one 7e, which was
more potent than the other thymines being active at 2 nM, and
the 6-(2,6-dichlorobenzyl)-5-ethyl-2-[2-(4-methoxyphenyl)-2-
oxoethylsulfanyl] derivative 6i, which inhibited HIV-1 at
>3
Me PhCH2
>538
>31
>162
>5
Me 4-NO2-PhCH2
Me 2,4-Me2-PhCH2
Me 3,5-Me2-PhCH2
Me 2-pyridyl-CH2
Me 3-pyridyl-CH2
Me 4-pyridyl-CH2
>7
>12
>1167
4941
12833
H
iso-propyl
Me iso-propyl
a Values are means ( SD determined from at least two experiments.
The alkylthio-substituted F2-S-DABOs 1q,r have been added for a direct
comparison. 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. e Higher concentra-
tions could not be tested due to precipitation of compounds in the culture
medium.
sium chloride, according to the previously reported simple and
high-yielding method (Scheme 1, route a).13 The 2-ethyl-, 2-iso-
propyl-, and 2-sec-butyl-substituted ꢀ-oxoesters 9c,d,g-i,k,15l15
were obtained by a modified Blaise procedure involving the
reaction between the proper arylacetonitriles and ethyl 2-bro-
mobutanoate, 2-bromo-3-methylbutanoate, or 2-bromo-3-me-
thylpentanoate35 in the presence of freshly prepared zinc
turnings, followed by acidic hydrolysis of the enamine inter-
mediate (Scheme 1, route b). Further condensation of the
ꢀ-oxoesters 9a-l with thiourea in alkaline medium afforded
the 5-alkyl-6-(2,6-dihalobenzyl)-2-thioxo-2,3-dihydropyrimidin-

4644 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Nawrozkij et al.
Scheme 1a
a (a) (1) KOOCCHRCOOEt, MgCl2, Et3N, CH3CN, reflux; (2) 4 N HCl; (b) (1) RCH(Br)COOEt, Zn, THF, reflux; (2) 13% HCl, rt; (c) NH2CSNH2,
EtOK, EtOH, reflux; (d) ω-bromoacetophenone, MeONa, MeOH, rt; (e) ω-bromoacetophenone, K2CO3, DMF, rt.
micromolar level. The 6-benzyl-5-iso-propyl-2-(2-oxo-2-phe-
nylethylsulfanyl)pyrimidin-4(3H)-ones 6j, 7j-l, and 8g-i as
well as the 5-sec-butyl analogue 7n were active at single-digit
nanomolar concentration, and 6l displayed anti-HIV-1 activity
at subnanomolar level (EC50 ) 0.4 nM).
The effect of halogen substitution of the C6-benzyl group in
this series of compounds was different from other series of
DABOs reported by us13–15,17,20,22 and others.30–32 In the
compounds described herein, with some exceptions, the 5-ethyl/
5-iso-propyl-2,6-dichlorobenzyl derivatives 6g-l were more
potent than the corresponding 2-chloro-6-fluoro- and 2,6-
difluoro-analogues 7g-l and 8d-i in inhibiting wt HIV-1.
In oxophenethyl-S-DABOs, we introduced at C2 a 2-phenyl-,
2-(4-fluorophenyl)-, or 2-(4-methoxyphenyl)-2-oxoethylthio chain.
We chose these substituents as they gave the highest anti-HIV
activity to the 5-ethyl/5-iso-propyl-DATNO analogues reported
by He et al.33,34 Nevertheless, in our 2,6-Cl2-, 2-Cl-6-F-, and
2,6-F2-oxophenethyl-S-DABOs, the substituent at the para-
position of the phenethyl moiety did not influence the inhibitory
activity of the compounds, or only marginally. In some cases,
the 4-methoxy (see for example 6l, 7l, and 8f) and in others the
4-H and/or 4-F group (6g,h, 7e,h, and 8a,b) seem to be
preferred.
[EC50sK103N (µM)/fold resistance: 5.1/1275 (6j) and 3.29/658
(7j); EC50sY181C (µM)/fold resistance: 0.11/27 (6j) and 0.21/
42 (7j); EC50sY188L (µM)/fold resistance: 5.5/1375 (6j) and 7.7/
1540 (7j)]. The 6-(2,6-difluorobenzyl)-5-ethyl- and -5-iso-
propyl counterparts 8d,g showed good anti-HIV-1 activity
against the K103N and Y181C mutants [EC50sK103N(µM)/fold
resistance: 1.3/108 (7d), and 4.46/2230 (7g); EC50sY181C(µM)/
fold resistance: 0.2/17 (8d) and 0.24/120 (8g)] but were inactive
against the Y188L mutant strain. The compound 6l, active at
subnanomolar level against wt HIV-1, was totally inefficient in
inhibiting the K103N and Y188L virus strains and retained its
activity only against Y181C but with a large loss of potency.
The insertion of a 4-fluoro or a 4-methoxy substituent at the
C2-oxophenethyl moiety gave in all cases a drastic reduction
joined to a high fold-resistance ratio, or a total loss of activity
of the compounds against the tested HIV-1 mutants.
Enzyme Inhibiting Activity: Effect on HIV-1 RT Wild
type and NNRTI-resistant Mutants. The oxophenethyl-S-
DABOs 6-8 were tested against recombinant wt HIV-1 RT as
well as against a panel of recombinant RTs carrying known
NNRTI-resistance mutations (K103N, Y181I, and L100I) (Table
5). As observed in in-cell tests, the SAR data drawn from our
previously reported DABOs are not applicable to the oxophen-
ethyl compounds. In the wt RT assay, the highest activity of
the derivatives is associated with a iso-propyl > ethyl > H >
methyl substitution at C5, and with the introduction of a
2-chloro-6-fluoro- > 2,6-dichloro- > 2,6-difluorobenzyl moiety
at C6. As in cellular assays, also in enzyme tests the differently
substituted chains at C2 showed only weak modulation of the
inhibitory activity. The most potent wt RT inhibitory action was
displayed by 7l, which showed ID50 (inhibitory dose 50,
compound dose required to inhibit the HIV-1 rRT activity by
50%) ) 0.7 nM. Optimal, single-digit nanomolar activities were
registered by the 6-(2-chloro-6-fluorobenzyl) derivatives 7a, 7g,
7i, 7j, and 7n as well as by the 2,6-difluorobenzyl analogue 8g.
Nevertheless, when tested against the mutated RTs, such
compounds retained in part their activities but together with
high fold resistance ratio (7a, 7j, 7n, and 8g), or were active
against only one mutated RT (7g and 7i). Differently, the 6-(2,6-
dichlorobenzyl)-5-ethyl-2-(2-phenyl-2-oxoethylsulfanyl)pyrimi-
din-4(3H)-ones 6g-i, which showed ID50 values against wt RT
ranging from 71 to 23 nM, displayed low micromolar to
Inhibitory Activity Against Clinically Relevant HIV-1
Mutant Strains. The oxophenethyl-S-DABOs 6-8 were also
tested against a panel of clinically relevant HIV-1 mutants
(K103N, Y181C, and Y188L) (Table 4). Among the three
different substitutions studied in this work (at C5 position, R;
at the C6-benzyl moiety, R1; at the phenyl ring of the
C2-oxophenethylsulfanyl chain, R2), the insertion of a ethyl
group at C5, a 2,6-dichloro- or 2-chloro-6-fluoro-benzyl portion
at C6, and a unsubstituted 2-oxo-2-phenylethylsulfanyl chain
at C2 seem to be suitable to obtain compounds highly active
against the HIV-1 mutants. Indeed, high, wide-spectrum inhibi-
tory activity was registered by the 6-(2,6-dichlorobenzyl)- and
6-(2-chloro-6-fluorobenzyl)-5-ethyl-2-(2-oxo-2-phenylethylsul-
fanyl)pyrimidin-4(3H)-ones 6g and 7g [EC50sK103N (µM)/fold
resistance: 1.2/171 (6g) and 1.2/100 (7g); EC50sY181C: 0.046/7
(6g) and 0.39/32 (7g); EC50sY188L: 5.0/714 (6g) and 9.38/782
(7g)]. The corresponding 5-iso-propyl analogues 6j and 7j
showed low micromolar to submicromolar inhibition of the
tested HIV-1 mutants but with higher fold resistance ratio

Anti-HIV-1 Agents of the DABO Family
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4645
Table 2. Physical and Chemical Data for Compounds 6-10
compd
R
R1
R2
mp (°C)
recryst solventa
synth methodb
% yield
formulac
6a
6br
6c
6d
6e
6f
6g
6h
6i
H
H
H
Me
Me
Me
Et
Et
Et
i-Pr
i-Pr
i-Pr
H
H
H
Me
Me
Me
Et
Et
Et
i-Pr
i-Pr
i-Pr
s-Bu
s-Bu
Me
Me
Me
Et
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2,6-Cl2
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
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
2,6-Cl2
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2,6-Cl2
2,6-Cl2
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F
H
F
OMe
H
F
OMe
H
F
OMe
H
F
OMe
H
F
OMe
H
F
OMe
H
F
OMe
H
F
OMe
F
OMe
H
F
OMe
H
F
OMe
H
F
OMe
204–205
207–209
219–220.5
205–206
220.5–221.5
239.5–240
200.5–201.5
225.5–227
234–235
210.5–211.5
229–230
219–211
201–204.5
200–201
229–230.5
191.5–194.5
203.5–204.5
236.5–238
189–190.5
210–211
222.5–225.5
190.5–196
223–224
190–195
208.5–209.5
196.5–197.5
215–218
219–221
222–224
194–196
206–208
228–230
221–223
234–237
187–191
oil
A
A
A
A
B
A
A
A
B
B
B
B
A
A
A
B
A
A
B
A
A
B
B
B
B
A
C
D
D
D
D
D
D
D
D
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
e
e
e
e
e
e
e
e
e
b
b
a
a
b
b
b
c
c
c
c
c
c
c
87
86
75
76
56
72
70
79
59
65
54
63
86
82
78
59
70
73
56
79
69
51
48
64
61
83
84
80
76
78
77
72
70
73
67
65
62
92
78
72
93
76
81
57
78
84
31
32
21
C19H14Cl2N2O2S
C19H13Cl2FN2O2S
C20H16Cl2N2O3S
C20H16Cl2N2O2S
C20H15Cl2FN2O2S
C21H18Cl2N2O3S
C21H18Cl2N2O2S
C21H17Cl2FN2O2S
C22H20Cl2N2O3S
C22H20Cl2N2O2S
C22H19Cl2FN2O2S
C23H22Cl2N2O3S
C19H14ClFN2O2S
C19H13ClF2N2O2S
C20H16ClFN2O3S
C20H16ClFN2O2S
C20H15ClF2N2O2S
C21H18ClFN2O3S
C21H18ClFN2O2S
C21H17ClF2N2O2S
C22H20ClFN2O3S
C22H20ClFN2O2S
C22H19ClF2N2O2S
C23H22ClFN2O3S
C23H21ClF2N2O2S
C24H24ClFN2O3S
C20H16F2N2O2S
C20H15F3N2O2S
C21H18F2N2O3S
C21H18F2N2O2S
C21H17F3N2O2S
C22H20F2N2O3S
C22H20F2N2O2S
C22H19F3N2O2S
C23H22F2N2O3S
C14H16Cl2O3
6j
6k
6l
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
8a
8b
8c
8d
8e
8f
8g
8h
8i
9c
9d
9e
9f
9g
9h
9i
10c
10d
10e
10f
10g
10h
10i
Et
Et
i-Pr
i-Pr
i-Pr
Et
i-Pr
H
Me
Et
i-Pr
s-Bu
Et
i-Pr
H
Me
Et
i-Pr
s-Bu
oil
48–50
oil
oil
oil
oil
270–271
262–264
268–270.5
243–244 dec
228–230
240–242
217–219
C15H18Cl2O3
C12H12ClFO3
C13H14ClFO3
C14H16ClFO3
C15H18ClFO3
C16H20ClFO3
C13H12Cl2N2OS
C14H14Cl2N2OS
C11H8ClFN2OS
C12H10ClFN2OS
C13H12ClFN2OS
C14H14ClFN2OS
C15H16ClFN2OS
E
F
F
G
F
F
F
H
a A: ethanol/N,N-dimethylformamide; B: 2-propanol/ N,N-dimethylformamide; C: methanol; D: acetonitrile/tetrahydrofuran; E: n-hexane; F: ethanol; G:
glacial acetic acid; H: acetonitrile. b See Scheme 1. c Analytic results were within ( 0.40% of the theoretical values.
submicromolar activities against K103N, Y181I, and L100I
mutated RTs, without large loss of activity [ID50sK103N (µM)/
fold resistance: 1.88/26 (6g), 1.79/43 (6h), 0.48/21 (6i);
ID50sY181I (µM)/fold resistance: 0.33/5 (6g), 0.24/6 (6h), 0.14/6
(6i); ID50sL100I (µM)/fold resistance: 3.54/50 (6g), 1.82/43 (6h),
2.42/105 (6i)].
Molecular Modeling and Binding Mode Studies. In previ-
ous molecular modeling studies, we described the putative
binding modes of F2-S-DABOs and F2-NH-DABOs,19,20 high-
lighting the differences with the experimental bound conforma-
tion of TNK-651 (Figure 4), a potent HEPT analogue,36 in both
WT and Y181C HIV-1 RTs. In the oxophenethyl-S-DABOs
6-8 reported here, we noted a peculiar SAR profile of activity
more related to HEPT derivatives than to DABOs. Thus, we
compared the binding modes of 6-8 with those of TNK-651
in WT (PDB37 entry code 1RT2),36 Y181C (PDB entry code
1JLA),38 and L100I (PDB entry code 1S1V)39 RTs by the means
of Autodock 4.0.1.40 About TNK-651, the analysis of the
experimental binding modes revealed that substantially the
overall binding mode is not influenced by the Y181C or L100I
mutation (Figure S1 in Supporting Information). Furthermore,
in the three RT isoforms (1RT2, 1JLA, and 1S1V) it is possible
to identify at least five important binding regions (Figure 5):
(1) a C5-iso-propyl binding pocket (red colored area in Figure
5), formed by the side-chain of Val179 and the backbone atoms
of Tyr181, Val189, and Gly190, (2) a π-rich hydrophobic region
[side-chains of Leu100 (Ile100), Tyr181 (Cys181), Tyr188,
Phe227, Trp229, and Leu234], hosting the TNK-651 C6-benzyl
moiety (blue colored area in Figure 5), (3) a short hydrophobic
channel [one entrance of the non nucleoside binding site
(NNBS), green-colored area in Figure 5], in which the -CH2-O-
CH2- part of the TNK-651 N1-benzyloxymethyl chain makes
interaction mainly with the terminal part of the Val106 and
Tyr318 side chains, (4) the end of the NNBS entrance channel
(purple-colored area in Figure 5), in which the side chain of
Pro236 interacts with the benzyloxy portion of TNK-651, and
(5) a H-bonding hydrophilic region (cyan-colored area in Figure
5), in which the backbone amido oxygen of Lys101 makes an

4646 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Nawrozkij et al.
Table 3. Cytotoxicity and Anti-HIV-1 Activity (wt HIV-1 Strain:
NL4-3) of 6-8a
Table 4. Anti-HIV-1 Activity of 6-8 against Clinically Relevant HIV-1
Mutant Strainsa
compd
R
R1
R2 CC50,b µM EC50,c µM selectivity indexd
EC50,b µM (fold resistance)c
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
H
H
2,6-Cl2
2,6-Cl2
2,6-Cl2 OMe
H
F
>12e
>59
>11
>60
>57
>56
>52
>55
>54
>56
>54
>14
>13
>61
>12
>12
>59
>12
>12
>57
>11
>12
>56
>11
>54
40
1.78
0.78
1.22
>7
>76
compd
R
R1
R2
K103N
>12
>59
OMe >11
Y181C
Y188L
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
H
H
2,6-Cl2
2,6-Cl2
2,6-Cl2
H
F
>12
>12
>9
>59
>59
Me 2,6-Cl2
Me 2,6-Cl2
Me 2,6-Cl2 OMe
Et
Et
Et
i-Pr 2,6-Cl2
i-Pr 2,6-Cl2
i-Pr 2,6-Cl2 OMe
H
H
H
Me 2-Cl-6-F H
Me 2-Cl-6-F F
Me 2-Cl-6-F OMe
Et
Et
Et
i-Pr 2-Cl-6-F H
i-Pr 2-Cl-6-F F
i-Pr 2-Cl-6-F OMe
s-Bu 2-Cl-6-F F
s-Bu 2-Cl-6-F OMe
Me 2,6-F2
Me 2,6-F2
Me 2,6-F2
H
F
0.038
0.046
0.067
0.007
0.007
3.3
0.004
0.064
0.0004
1.26
>1579
>1239
>836
>7429
>7857
>16
>11
>11
Me 2,6-Cl2
Me 2,6-Cl2
Me 2,6-Cl2
Et
Et
Et
i-Pr 2,6-Cl2
i-Pr 2,6-Cl2
i-Pr 2,6-Cl2
H
H
H
Me 2-Cl-6-F
Me 2-Cl-6-F
Me 2-Cl-6-F OMe >12
Et
Et
Et
i-Pr 2-Cl-6-F
i-Pr 2-Cl-6-F
i-Pr 2-Cl-6-F OMe >11
s-Bu 2-Cl-6-F >54
s-Bu 2-Cl-6-F OMe >40
Me 2,6-F2
Me 2,6-F2
Me 2,6-F2
Et
Et
Et
H
F
3.79 (100)
>57
0.48 (13)
>57
8.82 (232)
>57
2,6-Cl2
2,6-Cl2
2,6-Cl2 OMe
H
F
OMe >56
H
F
OMe >54
H
F
OMe >14
H
F
1.2 (18)
0.046 (7)
0.49 (70)
24.1 (7)
0.11 (27)
>54
0.23 (575)
>13
13.9 (48)
>12
1.68 (37)
>56
2,6-Cl2
2,6-Cl2
2,6-Cl2
1.2 (171)
4.7 (671)
5.0 (714)
36.3 (5186)
>54
6j
6k
6l
H
F
>14000
>844
6j
6k
6l
5.1 (1275)
>54
5.5 (1375)
>54
>35000
>10
>14
7a
7b
7c
7d
7e
7f
7g
7h
7i
2-Cl-6-F H
2-Cl-6-F F
2-Cl-6-F OMe
7a
7b
7c
7d
7e
7f
7g
7h
7i
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F OMe >12
>13
>61
>13
0.29
1.55
>210
>61
>8
>12
0.045
0.002
0.2
0.012
0.005
0.02
>267
H
F
9.48 (211)
8.72 (4360) 0.28 (140)
>12
>29500
>60
>59
1.57 (8)
>12
2-Cl-6-F H
2-Cl-6-F F
2-Cl-6-F OMe
>1000
>11400
>550
>2400
>14000
>11000
>3177
>6667
4333
>2480
>1250
4667
>3158
>6444
>30000
>11600
16500
4571
2-Cl-6-F
2-Cl-6-F
2-Cl-6-F OMe 4.16 (208)
H
F
1.2 (100)
4.14 (828)
0.39 (32)
1.93 (386)
0.84 (42)
0.21 (42)
0.56 (140)
0.16 (160)
0.86 (51)
>40
0.52 (35)
0.35 (14)
0.65 (13)
0.2 (17)
9.38 (782)
>57
>11
7j
7k
7l
0.005
0.004
0.001
0.017
0.006
0.015
0.025
0.048
0.012
0.019
0.009
0.002
0.005
0.002
0.007
<0.00014
0.000025
0.030
0.13
7j
7k
7l
H
F
3.29 (658)
>56
7.7 (1540)
>55
>11
7m
7n
8a
8b
8c
8d
8e
8f
7m
7n
8a
8b
8c
8d
8e
8f
F
>54
>40
H
F
19 (1267)
>62
>65
H
F
OMe
H
F
OMe
H
F
65
>62
>62
>60
56
>60
>58
>60
>58
33
OMe >60
H
F
OMe >58
H
F
OMe 17.5 (8750) 0.067 (33)
4.7 (671) ND
0.87 (6214) ND
>60
2,6-F2
2,6-F2
2,6-F2
1.3 (108)
5.14 (270)
>56
Et
Et
Et
2,6-F2
2,6-F2
2,6-F2
0.72 (38)
0.25 (28)
>60
>58
8g
8h
8i
i-Pr 2,6-F2
i-Pr 2,6-F2
i-Pr 2,6-F2
4.46 (2230) 0.24 (120)
>58 0.18 (36)
>60
8g
8h
8i
i-Pr 2,6-F2
i-Pr 2,6-F2
i-Pr 2,6-F2
>58
14.7 (7350)
>32
OMe
2d
2f
32
3d
6.9 (49286)
3f
>57
>12.4
203
>7
>3
>407143
>496000
6766
4e
0.55 (22000) 0.30 (12000) 0.95 (38000)
4.96 (38)
0.39 (56)
4g
NVP
EFZ
>7
0.044 (6)
>7
1.57 (224)
5h
NVP
EFZ
>54
>429
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 wt NL4-3 strain strain (Table 3). d Ref 31. e Ref 32.
0.007
a Values are means ( SD determined from at least three 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. e Higher concentrations
could not be tested due to precipitation of compounds in the culture medium.
f Ref 31. g Ref 32. h Ref 33.
NNBS space (part left of Figure S3 in Supporting Information),
with some loss of ligand stabilizing interactions. On the other
hand, in L100I, there is a new methyl group (of the Ile100 sec-
butyl moiety) that in some cases clashes against the S-DABO
pyrimidine ring, thus not allowing for the oxophenethyl-S-
DABO compounds to keep their WT or Y181C binding modes
(right part of of Figure S3 of the Supporting Information).
Interestingly, the docked conformations of almost the all C5-
ethyl substituted compounds (6g, 6h, 6i, 7g, 7h, 7i, 8d, and 8e)
revealed a limited influence of the L100I mutation, and this is,
at least in part, in agreement with their activity profile (see Table
5).
Somehow, the bound conformations of 6g, 6h, 6i, 7g, 7h, 7i,
8d, and 8e place the plane of the pyrimidine ring at a positive
contributing Lennard-Jones interaction with the Ile100 sec-butyl
methyl (Figure S4 in Supporting Information) and retain the
WT or Y181C binding mode.
The introduction of the 2-oxo-2-phenylethyl group is defi-
nitely one important feature of the oxophenethyl-S-DABOs. The
inspection of their docked conformations in either the WT or
Y181C RTs revealed that the carbonyl oxygen makes an
hydrogen bond interaction with Lys103 amide NH, which could
explain the decreased anti-HIV potency against the clinically
relevant K103N mutant strain as the Lys103 f Asn103 mutation
hydrogen bond with most of the RT cocrystallized NNRTIs (not-
shown). The inspection of the Autodock-proposed binding
modes of the oxophenethyl-S-DABOs 6-8 revealed a fair
superimposition to those of TNK-651 both in WT and Y181C
RTs, showing an overall common binding conformation (Figure
6 and Figure S2 in Supporting Information). It is noteworthy
to underline that, in the WT RT structure, all the 6-8 derivatives
share the same binding mode (part A of Figure S2 of the
Supporting Information) whereas in the Y181C RT, although
they are still highly superimposable (part B of Figure S2 of the
Supporting Information), the various compounds display some
influence of the Tyr181 f Cys181 mutation, so that the
structure-based aligned conformations are slightly less over-
lapped. The mutation Leu100 f Ile100 seems to be detrimental
for the conservation of such a binding (part C of Figure 6 and
part C of Figure S2 of the Supporting Information). Indeed,
the 6-8 bound conformations into the L100I RT (part C of
Figure S2 of the Supporting Information) are somehow disor-
dered, and there is not any common binding mode. It seems
likely that the Leu f Ile mutation on one hand led to a wider

Anti-HIV-1 Agents of the DABO Family
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4647
Table 5. Inhibitory Activity of 6-8 against HIV-1 RT Wild Type and
the influence of the Y181C mutation, as it represents a further
anchor point that fill the loss introduced by the Tyr181 f
Cys181 mutation, similarly to that previously reported for the
NH-DABOs.19,20 As a additional proof of the importance of
the introduced feature, docking experiments were also carried
out on the reported F2-S-DABO MC92213 (Figure 4) in
comparison with 6-8. The docked conformation of MC922
clearly show that the position of the pyrimidine ring is somehow
slightly shifted toward the top of the red-colored region in
respect to the average position of the 6-8 as well as to the
TNK-651 pyrimidine (Figure 7). Thus, the C5-methyl of MC922
is neither overlapped with the C5-substituent of 6-8 nor with
the C5-iso-propyl group of TNK-651 (red-colored region). This
is in agreement with the herein reported SAR profile, in which
an increase of bulkiness at the pyrimidine C5 position led to
more active oxophenethyl-S-DABO derivatives, differently from
that observed with the F2-S-DABOs. Furthermore, a direct
comparison to the TNK-651 and delavirdine (a well-known
NNRTI approved for therapy, Figure 4, PDB entry code
1KLM)41 bound conformations (WT RT structures) highlighted
that the oxophenethyl-S-DABOs showed a sort of mixed TNK-
651/delavirdine structural profile (Figure S5 in Supporting
Information). This last observation could be useful to design
novel series of NNRTIs through the building of a sort of chimera
between HEPT, DABO, and bis(heteroaryl)piperazine (BHAP)
compounds (Figure S6 in Supporting Information).
NNRTI-Resistant Mutantsa
ID50,b µM (fold resistance)c
compd
R
R1
R2
WT
0.012 3.7 (308)
0.21 9.7 (46)
K103N
Y181I
L100I
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
H
H
2,6-Cl2
2,6-Cl2
H
F
0.26 (22)
9.7 (46)
11.0 (917)
26.1 (124)
>40
7.8 (34)
10.7 (56)
>40
3.54 (50)
1.82 (43)
2.42 (105)
2,6-Cl2 OMe 0.054 37.5 (694) 0.11 (2)
Me 2,6-Cl2
Me 2,6-Cl2
Me 2,6-Cl2 OMe 0.094 >40
Et
Et
Et
H
F
0.23
0.19
12.0 (53)
>40
1.38 (6)
0.8 (4)
0.46 (5)
0.33 (5)
0.24 (6)
0.14 (6)
2,6-Cl2
2,6-Cl2
H
F
0.071 1.88 (26)
0.042 1.79 (43)
2,6-Cl2 OMe 0.023 0.48 (21)
6j
6k
6l
i-Pr 2,6-Cl2
i-Pr 2,6-Cl2
i-Pr 2,6-Cl2 OMe 0.014 >40
H
F
0.031 27.7 (894) 16.8 (542) 10.1 (326)
0.020 NDd
30.6 (1530) ND
17.1 (1221) >40
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
H
H
2-Cl-6-F H
2-Cl-6-F F
2-Cl-6-F OMe 0.014 3.4 (243)
0.002 2 (1000)
0.93 20.7 (22)
0.4 (200)
1.5 (750)
9.2 (10)
>40
>40
0.5 (36)
3.3 (275)
8.8 (49)
25.0 (1389)
9.7 (1213)
5.6 (170)
2.9 (414)
1.3 (1300)
ND
Me 2-Cl-6-F H
Me 2-Cl-6-F F
Me 2-Cl-6-F OMe 0.018 2.7 (150)
0.012 23.0 (1917) 1.2 (100)
0.18
5.35 (30)
0.7 (4)
1.2 (67)
>40
0.38 (12)
>40
Et
Et
Et
2-Cl-6-F H
2-Cl-6-F F
2-Cl-6-F OMe 0.007 >40
0.008 >40
0.033 1.64 (50)
7j
7k
7l
i-Pr 2-Cl-6-F H
i-Pr 2-Cl-6-F F
i-Pr 2-Cl-6-F OMe 0.0007 >40
s-Bu 2-Cl-6-F F 0.024 >40
s-Bu 2-Cl-6-F OMe 0.007 0.59 (84)
Me 2,6-F2
Me 2,6-F2
Me 2,6-F2
0.001 4.5 (4500) >40
0.024 40 (1667)
>40
>40
>40
>40
32.7 (1363)
7m
7n
8a
8b
8c
8d
8e
8f
7.9 (1129) 27.4 (3914)
H
F
0.33
0.41
10.4 (32)
12.0 (29)
22.7 (87)
1.68 (5)
1.71 (4)
1.17 (4)
0.82 (19)
0.51 (8)
12.7 (38)
39.2 (96)
>40
5.4 (126)
6.9 (113)
>40
4.8 (600)
5.7 (70)
>40
OMe 0.26
H
F
OMe 0.031 3.41 (110) 0.58 (19)
H
F
Conclusion
Et
Et
Et
2,6-F2
2,6-F2
2,6-F2
0.043 3.7 (86)
0.061 3.65 (60)
In this paper, a further series of S-DABOs, the oxophenethyl-
S-DABOs 6-8 have been reported as NNRTIs active against
wt HIV-1 (NL4-3 strain) as well as a panel of clinically relevant
HIV-1 mutant strains (K103N, Y181C, and Y188L). Despite
the fact that they belong to the DABO family, in both cellular
and enzyme assays, the oxophenethyl-S-DABO compounds
showed SAR data deeply different from those typical of the
DABO family. In particular, to obtain the highest wide spectrum
anti-HIV-1 activity, it seems to be important to insert an ethyl/
iso-propyl group at the C5 position of the pyrimidine ring, and
a 2,6-dichlorobenzyl/2-chloro-6-fluorobenzyl moiety at the C6
position. The 6-(2,6-dichlorobenzyl)-5-ethyl-2-(2-oxo-2-phe-
nylethylsulfanyl)pyrimidin-4(3H)-one 6g was one of the com-
pounds endowed with the highest broad spectrum HIV-1
inhibitory activity, with EC50 ) 7 nM against the wt virus, EC50
) 46 nM against the Y181C variant, EC50s ) 1.2 and 5.0 µM
against the K103N and Y188L strains, respectively. In com-
parison with 2-4, 6g, which was equally or less active against
the wt virus but displayed slightly lower (vs 3 and 4) or higher
(vs 2) potency against the K103N strain, was 7-fold more potent
than 4 against Y181C and showed against the Y188L mutant
an intermediate inhibition value between those of 2/3 and 4.
When compared to 5, 6g was 4-fold more efficient in inhibiting
wt HIV-1. In respect to NVP and EFZ, 6g was clearly more
potent than NVP and showed similar activities as EFZ against
wt HIV-1 and the Y181C variant, whereas it was 3-fold less
active against the K103N and Y188L mutant strains.
8g
8h
8i
i-Pr 2,6-F2
i-Pr 2,6-F2
i-Pr 2,6-F2
0.008 1.58 (198) >40
0.082 5.82 (71) >40
OMe 0.026 37.3 (1435) >40
2e
0.004 102 (25500) 150 (37500) ND
3e
0.026 3.2 (123)
1.5 (58)
ND
4f
0.003 45 (15000) >20
ND
9 (22)
ND
NVP
EFZ
0.4
7 (17)
35 (87)
0.08 (3)
0.03
3 (100)
a Values are means ( SD determined from at least three experiments.
b Inhibitory dose 50, compound dose required to inhibit the HIV-1 rRT
activity by 50%. c Fold resistance: ratio of ID50mut/ID50wt values. d ND,
not determined. e Ref 31. f Ref 32.
Enzyme data are in agreement with the cellular ones. When
screened against the wt RT and a panel of mutated RTs (K103N,
Y181I, and L100I), 6g together with its 4-fluoro- and 4-meth-
oxy-analogues 6h,i showed the highest inhibitory activities
joined with the lowest fold resistance ratio. In comparison with
2-4 and EFZ, compounds 6g-i showed similar (vs 3, 4, and
EFZ) or one magnitude order lower (vs 2) potency against wt
RT, were 6- to 54-fold more efficient than 2-4 and EFZ against
the K103N mutated RT and displayed lower potency that EFZ
and higher potency than 2-4 against the Y181I RT.
Figure 4. Structures of the NNRTIs used for molecular modeling
studies.
involve movement of the whole 103 residue and therefore ligand
interactions with this amino acid are likely diminished. At the
same time, the new hydrogen bonding interaction seems to face

4648 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Nawrozkij et al.
Figure 5. Experimental bound TNK-651. The WT RT is shown in gray wire. The five RT/TNK-651 interaction regions are also displayed.
Figure 6. Derivative 6h (pale-purple-colored carbon atoms) docked into WT (A), Y181C (B), and L100I (C) reverse transcriptases. For comparison
purposes, the experimental bound conformation of TNK-651 (cyan-colored carbon atoms) is shown in stick representation. The five RT/TNK-651
interaction regions are also displayed for comparison.
Figure 7. Derivative 6h (pale purple) docked into WT (A), Y181C (B) and L100I (C) reverse transcriptases. For comparison purposes, the docked
conformation of MC922 (yellow), an earlier F2-S-DABO, is shown in stick representation. The five RT/TNK-651 interaction regions are also
displayed for comparison.
Molecular modeling and binding mode studies on both wt
and mutated (Y181C and L100I) RTs could, at least in part,
explain the peculiar, HEPT-like SAR observed with the ox-
ophenethyl-S-DABOs. In previous molecular modeling studies,
we highlighted the differences between the binding modes of
F2-S-DABOs/F2-NH-DABOs and the experimental bound con-
formation of the HEPT derivative TNK-651 in both WT and
Y181C RTs. With the oxophenethyl-S-DABOs, a fair super-
imposition between their putative binding modes and those of
TNK-651 in WT and Y181C RTs has been observed. Moreover,
some differences in the binding modes of 6-8 and the F2-S-
DABO MC922 in both the enzyme structures have been found.
Experimental Section
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. 1H NMR and 13C
NMR spectra were recorded at 400 MHz on a Bruker AC 400 or
at 300 MHz on a Varian Mercury 300 BB spectrometer; chemical
shifts are reported in δ (ppm) units relative to the internal reference
tetramethylsilane (Me4Si) or hexamethyldisiloxane [(Me3Si)2O]. All

Anti-HIV-1 Agents of the DABO Family
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4649
1
compounds were routinely checked by TLC and H NMR. Mass
spectra were obtained on a Agilent MS/5975 mass spectrometer.
TLC was performed on aluminum-backed silica gel plates [Merck
DC, Alufolien Kieselgel 60 F254 or AlUGRAM Nano-SIL G/UV254
(MACHEREY-NAGEL GmbH & Co. KG)] with spots visualized
by UV light. All 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 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 chemicals
were purchased from Aldrich Chimica, Milan (Italy), or from
Lancaster Synthesis GmbH, Milan (Italy), and were of the highest
purity. The specific examples presented below illustrate general
synthetic methods. As a rule, samples prepared for physical (Table
2) and biological studies (Tables 1, 3–5) were dried in high vacuum
over P2O5 for 20 h at temperatures ranging from 25 to 110 °C,
depending on the sample melting point.
General Procedure (Scheme 1, Route a) for the Preparation
of Ethyl 4-(2,6-Dihalophenyl)-3-oxobutanoates (9a,e,j) and Ethyl
4-(2,6-Dihalophenyl)-2-methyl-3-oxobutanoates (9b,f) (Example:
Ethyl 4-(2-Chloro-6-fluorophenyl)-3-oxobutanoate (9e)). Triethy-
lamine (13.4 mL, 96 mmol) and magnesium chloride (7.14 g, 75
mmol) were added to a stirred suspension of potassium malonate
monoethyl ester (10.7 g, 63 mmol) in dry acetonitrile (200 mL),
and stirring was continued at room temperature for 2 h. Then, a
solution of the 2-chloro-6-fluorophenylacetic imidazolide in the
same solvent (50 mL), prepared 15 min before by reaction between
2-chloro-6-fluorophenylacetic acid (5.66 g, 30 mmol) and N,N′-
carbonyldiimidazole (5.84 g, 36 mmol) in acetonitrile (50 mL), was
added. The reaction mixture was stirred overnight at room tem-
perature and then was heated at reflux for 2 h. After the mixture
was cooled, 13% HCl (400 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 (200
mL). The aqueous layer was extracted with ethyl acetate (3 × 150
mL), and the organic phases were collected, washed with a sodium
hydrogen carbonate saturated solution (3 × 150 mL) and brine (3
× 150 mL), dried, and concentrated to give the pure desired product
as a solid, which was directly used in the following step. 1H NMR
(CDCl3): δ 1.19 (t, 3H, OCH2CH3), 3.35 (s, 2H, COCH2CO), 4.13
(m, 2H, OCH2CH3), 4.18 (s, 2H, Ar-CH2), 7.02 (m, 1H, C4-Ar-
H), 7.25 (m, 2H, C3,5-Ar-H). Anal. C, H, Cl, F.
General Procedure (Scheme 1, Route b) for the Preparation
of Ethyl 2-Alkyl-4-(2,6-dihalophenyl)-3-oxobutanoates (9c,d,g-i,k,l)
(Example: Ethyl 4-(2-Chloro-6-fluorophenyl)-3-oxo-2-iso-propy-
lbutanoate (9h)). A few drops of ethyl 2-bromo-3-methylbutanoate
were added to a vigorously stirred, boiling mixture of freshly
prepared zinc turnings (50 g, 0.765 g-atom) and a trace of
mercury(II) chloride in absolute THF (150 mL). After the appear-
ance of the greenish color (5-7 min), the solution of 2-(2-chloro-
6-fluorophenyl)acetonitrile (19.2 g, 113 mmol) in absolute THF
(170 mL) was added. The remaining ethyl 2-bromo-3-methylbu-
tanoate (118.5 g, 567 mmol) was added dropwise to the resulting
refluxing mixture over a period of 2 h. After all the bromoester
was added, the mixture was stirred and refluxed for 30 min more,
the organic layer was decanted, and the unreacted zinc (14.1 g)
was washed with several portions of anhydrous THF. The combined
THF solutions were evaporated at slightly reduced pressure, thus
living the viscous residue, which was taken up in toluene (500 mL)
and quenched with 13% HCl (240 mL). The resulting mixture was
stirred for 2.5 h, and the organic solution was washed with water,
dried over MgSO4, filtered through a silica gel pad, and evaporated
under reduced pressure. The remaining oil was fractionated in vacuo
on a Vigreux column to give the title product 9h as a pale-yellow
oil. H1 NMR (CDCl3): δ 0.87 (t, 6H, J ) 6.84 Hz, CH(CH3)2),
1.14 (m, 3H, OCH2CH3), 2.38 (m, 1H, CH(CH3)2), 3.28 (dd, 1H,
J1 ) 1.71 Hz, J2 ) 7.69 Hz, COCHCO), 3.94 (d, 2H, J ) 10.26
Hz, ArCH2), 4.07 (q, 2H, J1 ) 7.69 Hz, J2 ) 6.84 Hz, OCH2CH3),
6.84 (m, 1H, C4-Ar-H), 7.05 (m, 2H, C3,5-Ar-H). Anal. C, H, Cl,
F.
General Procedure (Scheme 1, Route c) for the Preparation of
5-Alkyl-6-(2,6-dihalobenzyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-
ones (10c-i) (Example: 6-(2-chloro-6-fluorobenzyl)-5-iso-propyl-
2-thioxo-2,3-dihydropyrimidin-4(1H)-one (10h)). Absolute ethanol
(110 mL) was placed in a 500 mL round-bottom flask, and
potassium (3.7 g, 94.9 mg-atom) was added to the above in small
pieces. Then, thiourea (3.1 g, 40.7 mmol) and ethyl 4-(2-chloro-
6-fluorophenyl)-3-oxo-2-iso-propylbutanoate (9h) (5.6 g, 15.6
mmol) were subsequently added. The resulting mixture was refluxed
for 90 h on oil bath and the excess solvent was distilled at normal
pressure. The residue was dissolved in hot water (400 mL), and
the resulting solution was boiled with 0.5 g of charcoal (30 min),
cooled to room temperature, and filtered on a Buchner funnel. The
filtrate was extracted with diethyl ether (3 × 25 mL), acidified to
pH 4-5 with glacial acetic acid, and the resulting precipitate was
filtered off, washed with water (50 mL) and diethyl ether (3 × 15
mL), and air-dried. The dry product was dissolved in boiling ethanol
(50 mL), treated with 0.5 g of charcoal and filtered. The filtrate
was concentrated at normal pressure, then the solution was chilled
to room temperature and the separated crystals were filtered off
1
and washed with a little cold ethanol. H NMR (DMSO-d6): 0.81
(d, J ) 6.72 Hz, 6H, CH(CH3)2), 2.35-2.44 (m, 1H, CH(CH3)2),
3.99 (s, 2H, CH2Ar), 7.15-7.22 (m, 1H, C4-Ar-H), 7.30-7.37
(m, 2H, C3,5-Ar-H), 12.24 (s, 1H, NH), 12.27 (s, 1H, NH). Anal.
C, H, Cl, F, N, S.
General Procedure (Scheme 1, Route d) for the Preparation
of 5-Alkyl-6-(2,6-dihalobenzyl)-2-(2-aryl-2-oxoethylsulfanyl)pyri-
midin-4(3H)-ones (6, 7) (Example: 6-(2,6-dichlorobenzyl)-2-(2-oxo-
2-phenylethylsulfanyl)pyrimidin-4(3H)-one (6a)). 6-(2,6-Dichlo-
robenzyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one13 (0.5 g, 1.74
mmol) was added in one portion to a magnetically stirred 1 M
methanolic solution of sodium methoxide (0.19 g, 3.48 mmol), and
stirring was continued until a clear solution was obtained. 2-Bromo-
1-phenylethanone (0.36 g, 1.83 mmol) was added to this solution,
and a few minutes later, a bulky, white precipitate was deposited.
The mixture was quenched with 1 N acetic acid (to pH ) 4) and
filtered. The filter cake was washed with water, sucked dry, and
recrystallized from ethanol/N,N-dimethylformamide (2:1) mixture.
1H NMR (DMSO-d6): δ 3.88 (s, 2H, ArCH2), 4.64 (s, 2H, SCH2),
5.71 (s, 1H, C5-H), 7.15 (m, 3H, C3-5-Ar-H), 7.53 (m, 2H,
C3,5-Ph-H), 7.65 (t, 1H, J ) 7.33 Hz, C4-Ph-H), 7.88 (d, 2H, J
) 7.33 Hz, C2,6-Ph-H). 13C NMR (DMSO-d6): δ 24.9 (CH2), 33.5
(SCH2), 114.2 (C-5), 126.9 (2C, dichlorophenyl ring), 128.3 (1C,
dichlorophenyl ring), 128.6 (2C, Ph), 128.8 (2C, Ph), 132.9 (1C,
Ph), 135.9 (2C, dichlorophenyl ring), 137.5 (1C, Ph), 138.6 (1C,
dichlorophenyl ring), 153.4 (C-6), 162.2 (C-2), 167.9 (C-4), 194.3
(CdO). MS (EI) m/z 404 (M+). Anal. C, H, Cl, F, N, S.
General Procedure (Scheme 1, Route e) for the Preparation of
5-Alkyl-6-(2,6-difluorobenzyl)-2-(2-aryl-2-oxoethylsulfanyl)pyrimi-
din-4(3H)-ones (8) (Example: 6-(2,6-Difluorobenzyl)-5-ethyl-2-(2-
oxo-2-phenylethylsulfanyl)pyrimidin-4(3H)-one (8d)). Anhydrous
potassium carbonate (0.13 g, 0.97 mmol) and 2-bromo-1-phenyle-
thanone (0.19 g, 0.97 mmol) were added in succession to a
suspension of 6-(2,6-difluorobenzyl)-5-ethyl-2-thioxo-2,3-dihydro-
pyrimidin-4(1H)-one15 (0.25 g, 0.88 mmol) in dry N,N-dimethyl-
formamide (1.0 mL). After stirring for 5 h at room temperature,
the mixture was quenched with water (10 mL) and filtered. The
obtained solid residue was crystallized to furnish pure 8d. 1H NMR
(DMSO-d6): δ 0.98 (t, 3H, CH2CH3), 2.43 (q, 2H, CH2CH3), 3.77
(s, 2H, C6-CH2), 4.47 (s, 2H, SCH2CO), 6.64 (m, 2H, C3,5-Ar-
H), 6.99 (m, 1H, C4-Ar-H), 7.51-7.77 (m, 5H, Ph-H), 12.65 (s
1H, NH). 13C NMR (DMSO-d6): δ 10.9 (CH3), 15.4 (CH2), 17.8
(CH2), 33.7 (SCH2), 111.5 (2C, difluorophenyl ring), 111.8 (1C,
difluorophenyl ring), 128.3 (C-5), 128.7 (2C, Ph), 128.9 (2C, Ph),
129.1 (1C, difluorophenyl ring), 132.9 (1C, Ph), 137.4 (1C, Ph),
145.1 (C-6), 159.9 (C-2), 163.5 (C-4), 164.7 (2C, difluorophenyl
ring), 193.9 (CdO). MS (EI) m/z 400 (M+). Anal. C, H, F, N, S.

4650 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Nawrozkij et al.
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.42–44 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
postinfection, a tetrazolium-based colorimetric method (MTT
method) was used to evaluate the number of viable cells. The HIV-1
K103N, Y181C, or Y188L mutant were received from the Medical
Research Council Centralised Facility for AIDS Reagents, Her-
fordshire, UK.
Anti-HIV Reverse Transcriptase Assays. RNA-dependent
DNA polymerase activity was assayed as described45 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 8% final concentration of DMSO.
employed to generate the docking input files and to analyze the
docking results, the same procedure as described in the manual
were followed. All the nonpolar hydrogens and the water molecules
were removed. The AMBER 8.0 atomic charges were saved for
either the proteins or the ligands, and the prepare_receptor4.py and
the prepare_ligand4.py ADT scripts were used to transform in the
united atom paradigm. A grid box size of 58 × 76 × 66 points
with a spacing of 0.375 Å between the grid points was implemented
and extended more than 6 Å out of the NNBS. The grid was
centered on the mass center of the experimental bound TNK-651
coordinates. For all the inhibitors, the single bonds including the
amide bonds were treated as active torsional bonds. One hundred
docked structures, i.e., 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 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 for the Autodock 3 version,56 the
actual one (Autodock 4) proved to reproduce with lower rmsd
values the experimental structures of TNK-651 (0.53, 0.59, and
0.72 rmsd values for 1RT2, 1JLA and 1S1V, respectively). The
same preparation and docking protocols were applied also for the
delavirdine/RT complex (PDB entry code 1KLM), which was used
for comparison purposed. The structures of the newly synthesized
S-DABOs were drawn by the mean of the jchempaint (http://
jchempaint.sourceforge.net/) software,57 converted into three-
dimensional structure using the CDK libraries,58,59 and subsequently
optimized with the sander module of the AMBER8 suite. The
needed parameters to run the sander minimization were calculated
using the antechamber module similarly as reported above. The
docking results for each of the three enzyme isoforms (WT, L100I,
and Y181C) were then clustered using a rmsd tolerance value of
2.0. In each cluster, the best cluster conformation was coincident
with the best docked.56
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 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.45 All 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 (ID50) determination, an interval of inhibitor
concentrations between 0.2 ID50 and 5 ID50 was used in the
inhibition assays. ID50 values were determined with computer-aided
curve fitting of the experimental data to a fully noncompetitive
model. Curve fitting was performed with the program GraphPad
Prism 3.0.
Acknowledgment. This work was partially supported by the
EU Contract LSHB-CT-2003-503480-TRIoH (to G.M. and
J.A.E.) and the Spanish MEC project BFU2006-00966 and FIS
PI060624 (J.A.E.). I. Clotet-Codina holds a FI scholarship from
Generalitat de Catalunya. S. Zanoli is recipient of a Fondazione
Buzzati-Traverso Fellowship.
Molecular Modeling. RT Structure Preparation. The WT
(1RT2), Y181C (1JLA) and L100I (1S1V) X-ray crystal structures
of RTs complexed with TNK-651 were retrieved from the Protein
Data Bank.46–48 All the complexes were superimposed each other
arbitrarily using the WT RT as reference complex. The superim-
position of the RT complexes was performed by the means of the
program ProFit version 2.249 using the implemented McLachlan
algorithm.50 All residue’s backbone atoms comprised in a 20 Å
core from TNK-651 were selected with the Chimera program51 (182
residues) and used as 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 complexes superimpositions. Then, using the AMBER 8.0
suite,52 the complexes were minimized to alleviate steric contacts
that usually arise due to the random way in which hydrogens are
added to the heavy atoms. Hydrogens were added to the receptors
with the tLeap module. Protonation states were assumed to be those
most common at pH 7, i.e., 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 neutralization. The solvated complex was then refined by
minimization using the SANDER module of AMBER. The
parameters for the cocrystallized NNRTIs 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.
Supporting Information Available: Chemical and physical data
of compounds 1a-p. Elemental analyses of compounds 1, 6-8.
1H NMR, 13C NMR, and MS (EI) data for compounds 6-8. Figures
S1-S6 (Molecular Modeling Section). This material is available
free of charge via the Internet at http://pubs.acs.org.
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