5-Alkyl-2-alkylamino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H) -ones, a new series of potent, broad-spectrum non-nucleoside reverse transcriptase inhibitors belonging to the DABO family

Article abstract of DOI:10.1016/j.bmc.2005.01.005

2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin- 4(3H)-ones (F₂-NH-DABOs) 4, 5 belonging to the dihydro-alkoxy-benzyl- oxopyrimidine (DABO) family and bearing different alkyl- and arylamino side chains at the C₂-position of the pyrimidine ring were designed as active against wild type (wt) human immunodeficiency virus type 1 (HIV-1) and some relevant HIV-1 mutants. Biological evaluation indicated the importance of the further anchor point of compounds 4, 5 into the non-nucleoside binding site (NNBS): newly synthesized compounds were highly active against both wild type and the Y181C HIV-1 strains. In anti-wt HIV-1 assay the potency of amino derivatives did not depend on the size or shape of the C₂-amino side chain, but it associated with the presence of one or two methyl groups (one at the pyrimidine C₅-position and the other at the benzylic carbon), being thymine, α-methyluracil or α-methylthymine derivatives almost equally active in reducing wt HIV-1-induced cytopathogenicity in MT-4 cells. Against the Y181C mutant strain, 2,6-difluorobenzyl-α-methylthymine derivatives 4d, 5h′-n′ showed the highest potency and selectivity among tested compounds, both a properly sized C₂-NH side chain and the presence of two methyl groups (at C₅ and benzylic positions) being crucial for high antiviral action.

Full text of DOI:10.1016/j.bmc.2005.01.005

Bioorganic & Medicinal Chemistry 13 (2005) 2065–2077
5-Alkyl-2-alkylamino-6-(2,6-difluorophenylalkyl)-3,4-
dihydropyrimidin-4(3H)-ones, a new series of potent,
broad-spectrum non-nucleoside reverse transcriptase
inhibitors belonging to the DABO family
Antonello Mai,a,* Marino Artico,a Rino Ragno,b Gianluca Sbardella,c Silvio Massa,d
Chiara Musiu,e Massimo Mura,e Flavia Marturana,e Alessandra Cadeddu,e
Giovanni Magaf and Paolo La Collag,*
a
`
Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Universita degli Studi di Roma ‘La Sapienza’,
P. le A. Moro 5, I-00185 Roma, Italy
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita degli Studi di Roma ‘La Sapienza’,
b
`
P. le A. Moro 5, I-00185 Roma, Italy
`
c
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Salerno, via Ponte Don Melillo, I-84084 Fisciano (SA), Italy
d
`
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, via A. Moro, I-53100 Siena, Italy
e
`
Cooperative Laboratories Idenix, Universita degli Studi di Cagliari, Cittadella Universitaria SS 554,
I-09042 Monserrato (Cagliari), Italy
fIstituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, I-27100 Pavia, Italy
g
`
Dipartimento di Scienze e Tecnologie Biomediche, Universita degli Studi di Cagliari,
Cittadella Universitaria SS 554, I-09042 ‘Monserrato (Cagliari), Italy
Received 21 September 2004; revised 30 December 2004; accepted 7 January 2005
Abstract—2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F2-NH-DABOs) 4, 5 belonging
to the dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family and bearing different alkyl- and arylamino side chains at the C2-posi-
tion of the pyrimidine ring were designed as active against wild type (wt) human immunodeficiency virus type 1 (HIV-1) and some
relevant HIV-1 mutants. Biological evaluation indicated the importance of the further anchor point of compounds 4, 5 into the non-
nucleoside binding site (NNBS): newly synthesized compounds were highly active against both wild type and the Y181C HIV-1
strains. In anti-wt HIV-1 assay the potency of amino derivatives did not depend on the size or shape of the C2-amino side chain,
but it associated with the presence of one or two methyl groups (one at the pyrimidine C5-position and the other at the benzylic
carbon), being thymine, a-methyluracil or a-methylthymine derivatives almost equally active in reducing wt HIV-1-induced cyto-
pathogenicity in MT-4 cells. Against the Y181C mutant strain, 2,6-difluorobenzyl-a-methylthymine derivatives 4d, 5h0–n0 showed
the highest potency and selectivity among tested compounds, both a properly sized C2–NH side chain and the presence of two
methyl groups (at C5 and benzylic positions) being crucial for high antiviral action.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
deficiency virus type 1 (HIV-1) infections when used
in combination with other anti-HIV agents such as
nucleoside analogs (NRTIs) and protease inhibitors
(PIs).1–4
Non-nucleoside reverse transcriptase inhibitors (NNR-
TIs) are useful drugs for the treatment of human immuno-
First generation NNRTIs include more than 30 classes
of compounds. Among them, nevirapine5,6 (Viramune,
Boehringer Ingelheim) was the first product approved
in 1996 as anti-AIDS drug by the US Food and Drug
Administration (FDA), followed in 1997 by delavirdine
Keywords: HIV-1; Reverse transcriptase; Dihydro-alkoxy-benzyl-oxo-
pyrimidines; HIV-1 mutant strains; Docking.
*
Corresponding authors. Tel.: +39 649913392; fax: +39 6491491
(A.M.); tel.: +39 706754148; fax: +39 706754210 (P.L.C.); e-mail
addresses: antonello.mai@uniroma1.it; placolla@unica.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.005

2066
A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
mesylate7,8 (Rescriptor, Pharmacia & Upjohn) (Fig. 1).
These inhibitors of HIV-1 reverse transcriptase (RT)
show low toxicity and favorable pharmacokinetic
properties, but they suffer from the rapid selection for
resistant viral populations both in cell cultures and
patients.6,9–11 Mutations commonly selected for NNR-
TIs occur at 98–108, 179–190, and 230–236 amino acid
positions, with Y181C, K103N or both being the most
clinically relevant. Therefore, in the NNRTI field the ef-
forts are now focused on the development of new com-
pounds endowed with higher anti-HIV-1 activity and a
resistance profile different from that of known drugs
(second generation NNRTIs).
more potent and selective compounds.19–28 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-alk-
oxy (DABOs, 1) or C2-alkylthio (S-DABOs, 2) side
chain is a structural determinant for the antiviral activ-
ity of these derivatives, with the length and size of the C2
side chain having only modulator effects on potency.19–22
Moreover, the NHCO fragment at N3/C4-positions of
pyrimidine ring, stabilized by a hydrogen bond between
the 3-NH function of S-DABOs and the carbonyl oxy-
gen of Lys101, cannot be modified without abolishing
the antiviral activity of DABO derivatives,24 and the
2,6-difluoro substitution at the C6 phenylmethyl moiety
of S-DABOs produces favorable p-stacking interactions
with the Tyr188 side chain and leads to compounds
active in the nanomolar range (difluoro-thio-DABOs,
F2-S-DABOs 3, i.e., 3a,b).25 Finally, the presence in
the S-DABO skeleton of two methyl groups, the former
at the pyrimidine C5-position and the latter on the meth-
ylene bridge between pyrimidine and phenyl rings (benz-
ylic position), furnished new conformationally
restricted F2-S-DABOs 3 (i.e., 3c,d) exhibiting potent
activity in the low nanomolar range against wild-type
HIV-1 and at submicromolar concentrations against
the Y181C mutant strain of HIV-1 (Fig. 1).27 2-Cyclo-
pentylthio-6-[1-(2,6-difluorophenyl)ethyl]-3,4-dihydro-
5-methylpyrimidin-4(3H)-one 3d turned out the most
potent and selective among the S-DABOs reported to
Efavirenz (Sustiva, DuPont)12 has been approved by
FDA in 1998 for the therapy of HIV-1 infections in
combination with other anti-HIV drugs, and the imida-
zole derivative capravirine (AG1549, Pfizer)13 is under
Phase III clinical trials. Clinical development of emivi-
rine, a HEPT derivative formerly known as MKC-
442,14,15 has been halted by Triangle Pharmaceuticals
in January 2002 when a comparative study showed
emivirine to be less potent than other antiretrovirals
(Fig. 1).
Dihydro-alkoxy-benzyl-oxopyrimidines (DABOs) were
reported in 1992 as a novel NNRTI class.16–18 Since
then, a number of oxopyrimidines were synthesized
and tested as anti-HIV-1 agents with the aim to obtain
Figure 1. Structures of known non-nucleoside reverse transcriptase inhibitors.

A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
2067
date, with EC50 against wt HIV-1 = 6 nM, and EC50
against the Y181C HIV-1 mutant = 200 nM.
On the basis of these findings, we made computational
studies and structure-based design of a novel series of
DABOs (compounds 4a–d, difluoro-amino-DABOs,
F2-NH-DABOs), characterized by the replacement of
the C2-cyclopentylthio moiety of 3d with a C2-cyclopent-
ylamino side chain, beside the hydrogen or methyl sub-
stitutions at the C5 and/or at the benzylic carbon, and
the highly favorable 2,6-difluorophenylmethyl moiety
at C6-position of the pyrimidine ring (Fig. 1).28 The
aim of such modification was to introduce structural
features in the DABO skeleton able to give more inter-
action points with the HIV-1 RT non-nucleoside bind-
ing site (NNBS), that is, through a hydrogen bond
formed between the novel C2–NH function and the en-
zyme backbone, necessary for a tighter binding with RT.
Figure 2. New designed amino-DABO derivatives.
2. Chemistry
As 4a–d were highly active in both anti-RT and
anti-HIV biological assays, and 2-cyclopentylamino-6-
[1-(2,6-difluorophenyl)ethyl]-3,4-dihydro-5-methylpyr-
imidin-4(3H)-one 4d showed an EC50 against Y181C
HIV-1 mutant variant = 160 nM with the fold resistance
value (5.3) 2-fold lower than that of its 2-cyclopentylthio
counterpart 3d (13),28 we prepared a large series of 2-
alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-
5-alkylpyrimidin-4(3H)-one derivatives 5a–n0, bearing
differently sized and shaped alkyl- and arylamino side
chains at the C2-position of the pyrimidine ring, to be
tested as anti-HIV-1 agents (Fig. 2). The inhibitory ef-
fects against wild type HIV-1 rRT and a panel of mutant
RTs (K103N, L100I, V106A, V179D, Y181I, and
Y188L) were determined on 3d and 4d, as representative
members of F2-S- and F2-NH-DABOs, respectively, and
the activity of selected 4, 5 compounds were assessed
against Y181C HIV-1 mutant strain in comparison with
those of F2-S-DABOs 3a–d.
Most of difluoro-amino-DABOs 5a–n0 were obtained
from 5-alkyl-6-(2,6-difluorophenylalkyl)-3,4-dihydro-2-
methylthiopyrimidin-4(3H)-ones25,27 by nucleophilic
displacement of the C2-methylthio group of the pyrim-
idine ring with the appropriate primary amine. Two dif-
ferent procedures have been used to achieve methylthio
displacement, depending on the amine boiling points.
With highly boiling amines (bp >60 ꢁC) the reaction
was carried out in a sealed tube heating the starting
material in the presence of an excess of the amine at
the temperature of 130 ꢁC (anilines) or 170 ꢁC (alkyl-
and arylalkylamines) (Scheme 1). With amine having a
boiling point of <60 ꢁC, the reaction was performed in
oil bath at 180 ꢁC by mixing the 2-methylthioderivative
with the amine as acetate salt (Scheme 1). 2-Methyl-
amino derivatives 5a,l,a0,h0 and their by-products
2-amino-3-methylpyrimidin-4(3H)-ones 6a–c, 2-amino-
pyrimidin-4(3H)-one 7, and 2-cyanoamino- and
2-nitroaminopyrimidin-4(3H)-one derivatives 8 and 9
have been prepared through condensation of the appro-
priate ethyl 2,6-difluorophenylacetylacetates with
methylguanidine, guanidine, cyanoguanidine, or nitro-
guanidine in alkaline medium (Scheme 2). Acylation of
7 with propionyl chloride under standard conditions
furnished the 2-propionamide-4(3H)-pyrimidinone 10
(Scheme 2).
In parallel with the synthetic effort, we performed dock-
ing analysis of 3d and 4d into the NNBS of both wt and
Y181C HIV-1 RT, in comparison with TNK-651,15,29
a
HEPT analog, which was co-crystallized in wt as well as
Y181C HIV RT enzymes.
Difluoro-amino-DABOs 6–10 bearing at C2–NH-posi-
tion a hydrogen (6, 7) or chemical functions different
from alkyl or aryl chains (8–10) (Fig. 2) were also pre-
pared and tested to gain further SAR information.
Chemical and physical data for derivatives 5–10 are
reported in Table 1.
Scheme 1. Reagents and conditions: (a) X–NH2, 130–170 ꢁC, sealed tube (compounds 5f–k,o–z,d0–g0,k0–n0); (b) CH3COONH3–X, 180 ꢁC, oil bath
(compounds 5b–e,m,n,b0,c0,i0,j0). R = R1 = H, Me; X = Et, n-Pr, i-Pr, cyclopropyl, n-Bu, s-Bu, methoxyethyl, methylthioethyl, cyclohexyl,
(substituted)phenyl, phenylmethyl, 1-naphthyl.

2068
A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
Scheme 2. Reagents and conditions: (a) EtONa, EtOH, reflux; (b) propionyl chloride, Et3N, THF, rt. R = R1 = H, Me; X = H, Me, CN, NO2,
COCH2CH3.
3. Results and discussion
In spite of this small difference in potency among com-
pounds carrying different alkyl/arylamino chains, the
introduction of a C5-methyl group in the uracil base fur-
nished more potent and selective derivatives (compare
5l–s with the corresponding C5–H analogs). Less evi-
dently, this rule was confirmed by the EC50 values shown
by the 6-[1-(2,6-difluorophenyl)ethyl] analogs 5a0–g0, and
5h0–n0, in which the thymine compounds resulted 2–3
times more active than the uracil counterparts.
3.1. Cytotoxicity, anti-HIV-1 and anti-RT activities,
and SAR studies
Title compounds 5a–n0 are characterized by the presence
of differently shaped alkylamino or arylamino side
chains at C2-position of the pyrimidine, and they can
be divided into four groups: (i) 6-(2,6-difluorophenyl-
methyl)uracils 5a–k, (ii) 6-(2,6-difluorophenylmethyl)-
thymines 5l–z, and (iii) and (iv) 6-[1-(2,6-difluorophen-
yl)ethyl] analogs of both the uracil and thymine series
5a0–g0 and 5h0–n0, respectively.
The introduction of a methyl substituent in the benzylic
carbon of F2-NH-DABOs in the uracil series (group
(iii)) gave compounds showing EC50 values from 3 to
14 times lower than the corresponding values of unsub-
stituted derivatives (compare 5a0–g0 with their benzylic-
unsubstituted analogs). Differently, in the thymine series
(group (iv)) this substitution did not influence the antivi-
ral activity, with the exception of the 2-methylamino
compound 5h0, which resulted 13 times more active than
the unsubstituted analog 5l. Therefore, while in previ-
ously reported F2-S-DABOs the methylation of the benz-
ylic carbon improved anti-HIV-1 inhibitory activity
together with selectivity, in F2-NH-DABOs the double
C5 and benzylic methyl substitution did not produce
increase of antiviral activity.
All designed compounds were evaluated for cytotoxicity
and anti-HIV-1 activity in MT-4 cells, in comparison
with nevirapine and emivirine used as reference drugs
(Table 2).
Most of compounds 5a–z were not cytotoxic for MT-4
cells at doses as high as 200 lM, and only six of them
(5j,k,q,r,t,z) showed CC50 values at concentrations lower
than 100 lM. Introduction of a methyl group at the benz-
ylic position of F2-NH-DABOs (compounds 5a0–n0)
led to more cytotoxic derivatives, both in the uracil
and thymine related series, with CC50 values ranging
from 44 to 115 lM. Exceptions to this rule were 5h0,j0,
and n0, which resulted less cytotoxic (CC50 values
P200 lM).
Comparing F2-NH-DABOs with the reference drugs,
most of DABO derivatives sharing C5 and/or benzylic
methyl substituents resulted 8- to 10-fold more potent
than nevirapine and endowed with the same potency
of MKC-442. Moreover, two compounds (5i0 and 5j0)
were 3- to 6-fold more potent and three (5m,o, and 5b)
were more selective than MKC-442.
In previous DABO series19–22,24–27 the maximum anti-
HIV-1 potency correlated with the size of the C2-alkyl-
thio side chain rather than with the nature of the base
(uracil or thymine), whereas in the F2-NH-DABOs the
antiviral activity resulted slightly or not dependent on
the size of the C2-alkyl/arylamino substituent. In fact,
compounds bearing 2-alkyl/arylamino chains with dif-
ferent length and size showed very similar EC50 values.
Exceptions to this rule are the C2-methylamino and -eth-
ylamino derivatives 5a,b,l,a0, which having a small C2
substituent showed a decrease of anti-HIV-1 activity,
and the cyclopropylamino derivative 5e, 8- and 29-fold
less potent than the n-propylamino and i-propylamino
counterparts 5c and 5d, respectively. The 2-methoxyeth-
ylamino compound 5h resulted 8 times less potent than
the corresponding 2-n-butylamino analog 5f, showing
that the introduction of lipophilic side chain was pre-
ferred at C2-position.
Representative F2-NH-DABOs were tested in enzyme
assays against highly purified HIV-1 rRT using
poly(rC)–oligo(dG)12–18 as template primer, and an
excellent correlation between the IC50 (enzyme inhibit-
ing activity) and EC50 (in vitro antiproliferative effect)
values was found (Table 3). Moreover, the F2-S-DABO
and the F2-NH-DABO prototypes 3d and 4d were
assayed against a panel of rRTs containing mutations
(K103N, L100I, V106A, V179D, Y181I, and Y188L)
known to confer resistance to NNRTIs (Table 4).
Despite 4d being less potent than 3d against wt RT, data
reported in Table 4 clearly show for 4d a better
fold-resistance profile against mutant RTs than that
for 3d.

A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
Table 1. Physical and chemical data of compounds 5–10
2069
Compound
R
R1
X
Mp (ꢁC)
Recryst solvent
Synth scheme (method)a % Yield Formulab
5a
5b
5c
5d
5e
5f
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
Methyl
Ethyl
202–203
138–140
136–137
150–151
183–184
130–131
140–141
120–121
Acetonitrile
n-Hexane/cyclohexane 1 (b)
2
58
60
44
52
58
65
57
68
59
60
68
52
46
65
70
51
55
67
57
55
48
58
64
59
47
56
49
69
55
58
62
52
48
55
58
67
52
44
59
54
18
12
16
78
86
79
85
88
75
C12H11F2N3O
C13H13F2N3O
C14H15F2N3O
C14H15F2N3O
H
H
n-Propyl
iso-Propyl
Cyclopropyl
n-Butyl
Cyclohexane
Diethyl ether
Benzene/cyclohexane
n-Hexane
1 (b)
1 (b)
1 (b)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
2
H
H
C
C
14H13F2N3O
15H17F2N3O
H
5g
5h
5i
H
sec-Butyl
Methoxyethyl
Diethyl ether
Acetonitrile
C15H17F2N3O
14H15F2N3O2
C14H15F2N3OS
17H19F2N3O
C17H13F2N3O
13H13F2N3O
H
C
H
Methylthioethyl 128.5–129 Acetonitrile
5j
H
Cyclohexyl
Phenyl
Methyl
143–144
223–224
210–211
156–157
165–166
192–193
Oil
Diethyl ether
Acetonitrile
Acetonitrile
Acetonitrile
n-Hexane
C
5k
5l
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
C
5m
5n
5o
5p
5q
5r
n-Propyl
iso-Propyl
n-Butyl
1 (b)
1 (b)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
1 (a)
2
C15H17F2N3O
C15H17F2N3O
C16H19F2N3O
Acetonitrile
sec-Butyl
C16H19F2N3O
C15H17F2N3OS
C18H21F2N3O
Methylthioethyl 150–151
Acetonitrile
n-Hexane
Cyclohexyl
Phenyl
150–151
226–227
214–215
272–273
231–232
245–246
235–236
>280
5s
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
Acetonitrile
Cyclohexane
C
18H15F2N3O
C18H13F4N3O
18H14ClF2N3O
C18H14ClF2N3O
18H14ClF2N3O
5t
2,6-F2-phenyl
2-Cl-phenyl
3-Cl-phenyl
4-Cl-phenyl
4-Me-phenyl
1-Naphthyl
Phenylmethyl
5u
5v
5w
5x
5y
5z
5a0
5b0
5c0
5d0
5e0
5f0
5g0
5h0
5i0
5j0
5k0
5l0
5m0
5n0
6a
6b
6c
7
C
C
C19H17F2N3O
C22H17F2N3O
C19H17F2N3O
C13H13F2N3O
C15H17F2N3O
C15H17F2N3O
C16H19F2N3O
C16H19F2N3O
C15H17F2N3OS
C18H21F2N3O
C14H15F2N3O
C16H19F2N3O
C16H19F2N3O
C17H21F2N3O
C16H19F2N3OS
C19H23F2N3O
C19H17F2N3O
C12H11F2N3O
182–183
145–146
Oil
Me Methyl
Me n-Propyl
Me iso-Propyl
Me n-Butyl
Me sec-Butyl
H
1 (b)
1 (b)
1 (a)
1 (a)
1 (a)
1 (a)
2
H
Oil
Oil
H
H
Oil
H
Me Methylthioethyl 113–114
Me Cyclohexyl
Diethyl ether
H
Oil
193–194
Oil
Me Me Methyl
Me Me n-Propyl
Me Me iso-Propyl
Me Me n-Butyl
Benzene/cyclohexane
1 (b)
1 (b)
1 (a)
1 (a)
1 (a)
1 (a)
2
157–157.5 Diethyl ether
Oil
Me Me Methylthioethyl 106–107
Me Me Cyclohexyl
Me Me Phenyl
Diethyl ether
Oil
210–211
215–216
197–198
197–198
Acetonitrile
Acetonitrile
Acetonitrile
Benzene
H
H
H
Me
2
C13H13F2N3O
Me Me
2
C14H15F2N3O
C11H9F2N3O
C12H8F2N4O
H
H
H
H
H
H
H
H
CN
276.5–277 Ethanol
2
8a
8b
9a
9b
10
H
255–256
276–277
212–214
197–198
Ethanol/water
2
Me
H
CN
NO2
Ethanol/water
Ethanol/water
Ethanol/water
2
C13H10F2N4O
2
C11H8F2N3O3
C12H10F2N3O3
C14H13F2N3O2
Me
H
NO2
COCH2CH3
2
163–163.5 Benzene
2
a See Schemes 1 and 2.
b All compounds were analyzed for C, H, N, S, F. Analytical results were within 0.4% of the theoretical values.
Finally, selected F2-NH-DABOs 4, 5 were tested in cell
culture using MT-4 cells and both wild-type virus and
virus containing a mutation in residue Tyr181 to Cys,
known to be crucial to confer NNRTI resistance in trea-
ted patients, in comparison with compounds 3a–d (F2-S-
DABOs), nevirapine, and efavirenz (Table 5).
DABO series, similarly to that observed with F2-S-DA-
BOs,27 the contemporary presence of two methyl groups
both in C5 and in benzylic position of the pyrimidine ring
strongly contributed to maintain the antiviral potency
against the Y181C strain in the low submicromolar range.
Particularly, 4d and 5k0 showed EC50 values against
Y181C mutant only 4–5 times higher than those showed
against wild-type HIV-1. The C2-methylamino derivative
5h0, belonging to the same series of 4d and 5k0 (group (iv)),
showed a Y181C/wt HIV-1 EC50 ratio of 650, showing
that beside the double-methyl substitution a bulky C2 side
All tested DABO derivatives (with the exception of 5s) re-
tained their inhibitory activity against the HIV-1 Y181C
mutant strain, being their EC50 values included in the
low micromolar/submicromolar range. In the amino-

2070
A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
Table 2. Cytotoxicity and anti-HIV-1 activity of compounds 5–10a
b
c
Compound
R
R1
X
CC50 (lM)
EC50 (lM)
SId
>133
5a
5b
H
H
Methyl
Ethyl
n-Propyl
>200e
>200
200
1.5
0.8
H
H
>250
1818
5c
5d
H
H
0.11
0.4
H
H
iso-Propyl
Cyclopropyl
n-Butyl
>200
>200
100
>500
>63
1000
5e
H
H
3.17
0.1
5f
5g
H
H
H
H
sec-Butyl
>200
>200
>100
66
0.13
0.80
0.3
>1543
>250
>333
476
5h
5i
H
H
Methoxyethyl
Methylthioethyl
Cyclohexyl
Phenyl
H
H
5j
5k
H
H
0.14
0.29
0.4
H
H
60
135
207
335
5l
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Methyl
n-Propyl
5m
5n
H
>200
190
0.02
0.03
0.02
0.06
0.016
0.03
0.06
0.14
0.13
0.2
>10,000
6333
H
iso-Propyl
n-Butyl
sec-Butyl
5o
5p
H
>200
200
82
>10,000
3333
5125
H
5q
5r
H
Methylthioethyl
Cyclohexyl
Phenyl
H
30
1000
>3333
396
5s
H
>200
55.5
>200
>200
>200
>200
>200
50
5t
5u
H
2,6-F2-phenyl
2-Cl-phenyl
3-Cl-phenyl
4-Cl-phenyl
4-Me-phenyl
1-Naphthyl
Phenylmethyl
Methyl
H
>1538
>1000
>2000
>1111
5v
H
5w
5x
H
0.1
0.18
H
5y
H
>200
5z
H
0.50
0.11
0.02
0.03
0.03
0.04
0.013
0.022
0.03
0.006
0.005
0.01
0.14
0.026
0.024
55
100
964
5a0
5b0
5c0
5d0
5e0
5f0
5g0
5h0
5i0
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
106
103
H
n-Propyl
5150
3830
1730
2150
3385
2545
6666
11,666
40,000
8300
>714
2231
>8333
>3
H
iso-Propyl
n-Butyl
sec-Butyl
115
52
H
H
86
44
H
Methylthioethyl
Cyclohexyl
Methyl
H
56
200
Me
Me
Me
Me
Me
Me
Me
H
n-Propyl
iso-Propyl
n-Butyl
70
200
5j0
5k0
5l0
5m0
5n0
6a
83
>100
58
Methylthioethyl
Cyclohexyl
Phenyl
>200
>200
>200
93
6b
6c
H
Me
Me
H
2.4
>93
>83
Me
H
7
8a
H
CN
>200
>200
>200
>200
>200
>200
>200
>200
76
108
>200
>200
>200
>200
>200
0.09
0.02
0.03
0.02
0.03
0.3
>1.8
H
H
8b
9a
Me
H
H
CN
NO2
H
9b
Me
H
H
NO2
10
H
COCH2CH3
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
4af
4bf
4cf
4df
Emivirine
Nevirapine
H
H
>2222
>10,000
3500
Me
H
H
Me
Me
Me
90
200
4500
6666
>200
>666
a Data represent mean values of at least two separate experiments.
b Compound dose required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.
c Compound dose required to achieve 50% protection of MT-4 cells from HIV-1-induced cytopathogenicity, as determined by the MTT method.
d Selectivity index, CC50/EC50 ratio.
e Higher concentrations could not be achieved for crystallization of compounds in the culture medium.
f Ref. 28.
chain (at least four carbon atoms) is requested to obtain a
high anti-Y181C HIV-1 activity.
Compared with the thio-DABO 3d, the amino-DABOs
4d and 5k0 showed a lower fold resistance of antiviral

A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
Table 3. Activity of selected F2-NH-DABOs 4, 5 against HIV-1 rRTa
2071
3.2. Binding mode investigation of F2-NH-DABOs in
wild-type and Y181C mutant forms of HIV-1 RT
b
Compound
R
R1
X
IC50 (lM)
5a
4ac
H
H
Methyl
2.5
In a previous study28 we found that the possible role of
S ! NH replacement in the F2-S-DABO series was the
introduction of a new anchor point for the binding of
F2-NH-DABOs into the NNBS of the HIV-1 RT.
Docking studies revealed that a hydrogen bond is
formed between the C2-NH group and the carbonyl
H
H
Cyclopentyl
Cyclopentyl
Phenyl
0.07
0.03
0.15
0.04
0.035
0.04
4bc
Me
Me
H
H
5s
4cc
4dc
Emivirine
H
Me
Me
Cyclopentyl
Cyclopentyl
Me
˚
a Data represent mean values of at least two separate experiments.
b Compound dose required to inhibit the HIV-1 rRT activity by 50%.
c Ref. 28.
oxygen of Lys101 (N–O distance = 2.779 A). The F2-
NH-DABO/RTwt hydrogen bonding pattern resembles
that of the DNA base pairs, and such interactions seem
to stick the ligand in such a way that in part diminish
the consequence of the double methyl substitution at
C5 and benzylic carbon positions of the pyrimidine,
which was crucial for the anti-HIV-1 activity of F2-S-
DABOs (compare EC50 values of 3a–d25,27 and 4a–d28
against wild type HIV-1). Moreover, an improvement
of the anti-HIV activity profile against the Y181C
mutant strain was observed for the F2-NH-DABOs
4a–d.28
Table 4. Activity of the F2-S-DABO 3d and the F2-NH-DABO 4d
against a panel of mutant rRTs
HIV-1 RT
Ki Values (lM) (n)a
3d
4d
WT
0.005
0.03 (6)
0.3 (60)
0.03
K103N
L100I
V106A
V179D
Y181I
Y188L
0.08 (3)
0.5 (17)
0.5 (17)
0.3 (10)
1.7 (57)
1.1 (37)
In view of the above observations, docking studies were
performed in parallel on the wild type (RTwt) and the
Y181C mutant (RTY181C) form of the HIV-1 RT. For
these studies, 4d was used as representative member of
F2-NH-DABOs and docked into the NNBS of either
RTwt or RTY181C. For comparison purposes, the same
docking procedure was carried out on the conforma-
tionally constrained F2-S-DABO 3d and also on TNK-
651, a potent HEPT analog, to assess the reliability of
the docking procedure.
0.5 (100)
0.3 (60)
0.9 (180)
1.4 (280)
a (n): Fold resistance.
Table 5. Activity of selected DABOs against Y181C HIV-1 mutant
strain: comparison between F2-S-DABO (3) and F2-NH-DABO (4 and
5) activities
The coordinates of TNK-651 co-crystallized in either the
RTwt (PDB entry code 1rt2)15 or RTY181C (PDB entry
code 1jla)29 were used to define the NNBSwt and
a
Compound
R
R1
X
EC50 (lM)
(Y181C/
WTIIIB
b
)
WTIIIB Y181C
NNBSY181C
.
3a
3b
H
H
H
Cyclopentyl 0.3
Cyclopentyl 0.04
5.3
1.2
(18)
(30)
(50)
(13)
(10)
(42)
(25)
(5.3)
(70)
(38)
Me
H
First-ranking docked conformations for 4d, 3d and the
experimental binding conformations of TNK-651 are re-
ported in Figure 3.
3c
Me Cyclopentyl 0.05
2.5
0.2
3dc
4a
Me Me Cyclopentyl 0.015
H
H
H
Cyclopentyl 0.2
Cyclopentyl 0.02
2.1
0.8
4b
4c
4dc
Me
H
Me Cyclopentyl 0.02
0.5
It can be seen that the two binding modes, in the
NNBSwt and NNBSY181C, for 4d as proposed by AUTO-
DOCK are almost indistinguishable to each other. In
either the cases, 4d binding conformations are character-
ized by a Ôpropeller-likeÕ disposition of thymine and 2,6-
difluorophenyl rings, with s1 and s2 values25,27 of ꢀ30.7ꢁ
and 78.3ꢁ.
Me Me Cyclopentyl 0.03
0.16
0.7
5o
5p
Me
Me
Me
H
H
H
H
n-Butyl
sec-Butyl
Phenyl
0.01
0.06
0.05
0.03
0.04
0.02
0.01
0.02
0.04
0.37
0.004
2.3
5s
>100
0.6
1.4
5d0
Me n-Butyl
Me sec-Butyl
(20)
(35)
(650)
(20)
(29)
(4.5)
(>81)
(6)
5e0
H
5h0
Me Me Methyl
Me Me n-Propyl
Me Me iso-Propyl
Me Me n-Butyl
13
0.2
5i0
5j0
0.5
0.2
Experimental conformations of TNK-651 bound into
NNBSwt/NNBSY181C differ only for the disposition of
the ligand C5 iso-propyl groups, that were found rotated
each other of about 180ꢁ.15,29
5k0
Nevirapine
Efavirenz
>30
0.025
a Compound dose required to achieve 50% protection of MT-4 cells for
wild-type and Y181C HIV-1-induced cytopathogenicity, as deter-
A different scenario is observed for the F2-S-DABO 3d,
for which the two RTwt and RTY181C docked conforma-
tions are not exactly superimposable. In fact, while the
two molecules are conformationally almost identical,
the 3d RTY181C bound conformation is slightly rotated
in a way that the ligand difluorophenyl moiety seems
to approach to Tyr188, thus trying to replace the miss-
ing interactions with the mutated Y181C.
mined by the MTT method.
b Fold resistance.
c Ref. 28.
activity from wild type to Y181C HIV-1 strains
(3d = 10; 4d = 5.3; 5k0 = 4.5).

2072
A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
Figure 3. (A): Superimposition of the docked conformations of 4d in the RTwt (red) and RTY181C (blue). (B): Superimposition of the docked
conformations of 3d in the RTwt (green) and RTY181C (cyan). (C): Superimposition of the experimental TNK-651 conformations in the RTwt (white)
and RTY181C (yellow).
The DNA-like hydrogen bonding profile of 4d bound
into NNBSwt28 is still maintained in the RTY181C/4d com-
plex. A deeper inspection of the RTY181C/4d complex re-
veals a binding mode of the inhibitor in which three
potential hydrogen bonds can be observed: (i) a hydro-
gen bond donated by the 3-NH function of the pyrimi-
dine to the carbonyl oxygen of Lys101 (N–O
At the same time, inspection of the RTY181C/3d complex
reveals that the F2-S-DABO inhibitor seats on
the Pro236 hairpin, its C5 methyl group pointing
towards the above described hydrophobic pocket. Less
important interactions are made between the two
benzylic/C5 methyl groups of 3d and the Cys181
˚
side chain (CH3-benz–b-CH2-CYS181 distance = 5.45 A;
˚
˚
distance = 3.702 A), (ii) a hydrogen bond accepted by
the C-4 carbonyl oxygen of the pyrimidine from the ami-
CH3-C5–b-CH2-CYS181 distance = 4.31 A), and only
appreciable favorable interactions are made by the 3d
difluorophenyl group with Tyr188.
˚
dic NH of Lys101 (O–N distance = 2.769 A), and (iii) a
hydrogen bond donated by the C-2 NH group to the car-
˚
bonyl oxygen of Lys101 (N–O distance = 2.770 A). This
hydrogen bond pattern, compared to that of 3d, arranges
4d so as to lift up into the RT pocket (Fig. 4). In such
way, the 4d benzylic methyl group exactly lies in the
same spatial position of the C5 iso-propyl of TNK-651,
making favorable steric interactions within a hydro-
phobic pocket formed by the b-methylene of Cys181
4. Conclusion
2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihy-
dro-5-alkylpyrimidin-4(3H)-one derivatives 4, 5 belong-
ing to the DABO family and bearing different alkyl-
and arylamino side chains at the C2-position of the
pyrimidine ring were designed as active against wild type
and some relevant mutants of HIV-1. Molecular model-
ing studies strongly suggested the synthesis of such com-
pounds as they can make an additional hydrogen bond
˚
(CH3-benz–b-CH2-CYS181 distance = 3.49 A) and other
portion of Val106, Val179, and Tyr188 residues. Finally,
the 4d difluorophenyl moiety is still in good position to
make favorable p–p stacking interaction with Tyr188.
Figure 4. Stereoview of all the six structures in the NNBSwt (white lines) and in NNBSY181C (yellow lines). The ligands are color coded as in Figure 3.

A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
2073
between the NH group at C2-position and the carbonyl
oxygen of Lys101, with a tighter binding into the enzyme
NNBS. Biological evaluation of F2-NH-DABOs indi-
cated the importance of the further anchor point of com-
pounds 4, 5 into the NNBS: newly synthesized
compounds were highly active against both wild type
and the Y181C HIV-1 strains. In anti-wt HIV-1 assay,
the potency of derivatives did not depend on the size or
shape of the C2-amino side chain, but it associated with
the presence of one or two methyl groups, one at C5-po-
sition and the other at the benzylic carbon of the pyrim-
idine. Differently from that observed with F2-S-DABOs,
against wt HIV-1 in the amino-DABO series the double
methyl substitution at C5 and benzylic positions is not
necessary for a significant improvement of antiviral
activity: thymine and a-methyluracil or a-methylthymine
derivatives are almost equally active in reducing wt HIV-
1-induced cytopathogenicity in MT-4 cells. Conversely,
against the Y181C mutant strain the 2,6-difluorobenz-
yl-a-methylthymine derivatives 4d, 5h0–n0 were en-
dowed with higher potency and selectivity, in this series
both a properly sized C2–NH side chain and the presence
of two methyl groups (at C5 and benzylic positions) being
crucial for high antiviral action. Docking studies per-
formed on wt (RTwt) and Y181C mutant (RTY181C) form
of HIV-1 RT using 3d and 4d as F2-S-DABO and F2-
NH-DABO representative members, respectively,
showed that the further anchor point (hydrogen bond
with C2–NH function) of amino-DABOs, lacking in
the thio-analogs, produced a tighter binding of the ligand
with the enzyme, so that a mutation at 181 residue (Tyr
to Cys) is less effective in reducing the anti-HIV-1 activity
(4d resistance ratio = 5.3; 3d resistance ratio = 13).
5.1.1. General procedure for the preparation of 2-alkyl-
amino-6-[(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkyl-
pyrimidin-4(3H)-ones 5f–k,o–z,d0–g0,k0–n0. Example: 6-
(2,6-difluorophenylmethyl)-3,4-dihydro-2-(2-methoxyeth-
yl)aminopyrimidin-4(3H)-one (5h). A mixture of 6-(2,6-
difluorophenylmethyl)-3,4-dihydro-2-methylthiopyrim-
idin-4(3H)-one25 (0.5 g, 1.8 mmol) and 2-methoxyethyl-
amine (5 mL) was heated in a sealed tube at 170 ꢁC
for 8 h. After cooling, the crude residue was dissolved
in ethyl acetate (50 mL), washed with water (50 mL),
0.5 N HCl (50 mL), and brine (50 mL), and then was
evaporated under reduced pressure and purified by col-
1
umn chromatography (silica gel/chloroform). H NMR
(CDCl3): d 3.27 (s, 3H, OCH3), 3.44–3.46 (d, 4H,
OCH2 and NCH2 overlapped), 3.78 (s, 2H, C6–CH2),
5.45 (s, 1H, C5–H), 6.51 (br s, 1H, C2–NH exchanged
with D2O), 6.84–6.88 (m, 2H, C3,5 Ar–H), 7.13–7.17
(m, 1H, C4 Ar–H), 11.75 (br s, 1H, CONH exchanged
with D2O). Anal. C, H, N, F.
5.1.2. General procedure for the preparation of 2-alkyl-
amino-6-[(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyr-
imidin-4(3H)-ones 5b–e,m,n,b0,c0,i0,j0. Example: 6-(2,6-
difluorophenylmethyl)-3,4-dihydro-5-methyl-2-n-propyl-
amino pyrimidin-4(3H)-one (5m). n-Propylammonium-
acetate was prepared by slowly adding 2.1 mL (0.036
mol) of glacial acetic acid to 2.9 mL (0.036 mol) of n-pro-
pyl-amine in an ice bath. 6-(2,6-Difluorophenylmethyl)-
3,4-dihydro-5-methyl-2-methylthiopyrimidin-4(3H)-one25
(0.53 g, 1.8 mmol) was added and fused for 5 h at
160 ꢁC. After cooling, the residue was partitioned be-
tween 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 puri-
fied by column chromatography (silica gel/chloroform).
1H NMR (CDCl3): d 0.73–0.80 (t, 3H, NHCH2-
CH2CH3), 1.32–1.43 (m, 2H, NHCH2CH2CH3), 2.01
(s, 3H, C5–CH3), 3.05–3.08 (m, 2H, NHCH2CH2CH3),
3.86 (s, 2H, C6–CH2), 6.26 (br s, 1H, NHCH2CH2CH3
exchanged with D2O), 6.80–6.87 (m, 2H, C3,5 Ar–H),
7.12–7.20 (m, 1H, C4 Ar–H), 11.43 (br s, 1H, CONH
exchanged with D2O). Anal. C, H, N, F.
5. Experimental
5.1. Chemistry
Melting points were determined on a Buchi 530 melting
¨
point apparatus and are uncorrected. Infrared (IR) spec-
tra (KBr) were recorded on a Perkin–Elmer 297 instru-
ment. H NMR spectra were recorded at 200 MHz on
1
a Bruker AC 200 spectrometer; chemical shifts are re-
ported in d (ppm) units relative to the internal reference
tetramethylsilane (Me4Si). All compounds were rou-
5.1.3. General procedure for the preparation of 2-amino-
6-[(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimi-
din-4(3H)-ones 5a,l,a0,h0,6–9. Example: 6-[1-(2,6-difluoro-
phenyl)ethyl]-3,4-dihydro-2-methylaminopyrimidin-4(3H)-
one (5a0) and 2-amino-6-[1-(2,6-difluorophenyl)ethyl]-
3,4-dihydro-3-methylpyrimidin-4(3H)-one (6b). Sodium
metal (0.58 g, 25.2 mmol) was dissolved in 50 mL of
absolute ethanol, and 1-methylguanidine hydrochloride
(1.66 g, 15.1 mmol) and ethyl 4-(2,6-difluorophenyl)-3-
oxopentanoate27 (2.56 g, 10.1 mmol) were added to the
clear solution. The mixture was heated at reflux for
5 h. After cooling, the solvent was distilled in vacuo at
40–50 ꢁC until dry and the residue was dissolved in a
little water (20 mL), made acid with 2 N HCl, and
extracted with ethyl acetate (3 · 25 mL). The organic
extracts were washed with brine (3 · 50 mL), dried,
and evaporated in vacuo, and the residue was purified
by chromatography on silica gel eluting with ethyl
1
tinely checked by TLC and H NMR. TLC was per-
formed on aluminum-backed silica gel plates (Merck
DC-Alufolien Kieselgel 60 F254) 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 extrac-
tions 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.
The specific examples presented below illustrate general
synthetic methods A and B. As a rule, samples prepared
for physical (Table 1) and biological studies (Tables 2–5)
were dried in high vacuum over P2O5 for 20 h at temper-
atures ranging from 25 to 110 ꢁC, depending on the sam-
ple melting point.

2074
A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
acetate–chloroform 3:1. The early eluates deprived of
the solvent furnished 6b as a pure solid; with further
elution, pure 5a0 was isolated. Compound 6b: 1H
NMR (DMSO-d6): d 1.48–1.51 (d, 3H, ArCHCH3),
3.21 (s, 3H, NCH3), 4.06–4.13 (q, 1H, ArCHCH3),
5.51 (s, 2H, C5–H), 6.99–7.07 (m, 4H, C3,5 Ar–H
and NH2), 7.28–7.36 (m, 1H, C4 Ar–H). Anal. C, H,
F, N.
5.2.4. Anti-HIV assays. Activity of the compounds
against HIV-1 and HIV-2 multiplication in acutely in-
fected cells was based on the inhibition of virus-induced
in MT-4 and C8166 cells, respectively. Briefly, 50 lL of
culture medium containing 1 · 104 cells were added to
each well of flat-bottom microtiter trays containing
50 lL of culture medium with or without various con-
centrations of the test compounds. Then 20 lL of an
HIV suspension containing 100 (HIV-1) or 1000 (HIV-
2) CCID50 (50% cell culture infective dose) were added.
After a 4 days incubation (5 days for HIV-2) at 37 ꢁC,
the number of viable cells was determined by the
3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) method.30 Cytotoxicity of the compounds
was evaluated in parallel with their antiviral activity. It
was based on the viability of mock-infected cells, as
monitored by the MTT method.
1
Compound 5a0: H NMR (DMSO-d6): d 1.40–1.55 (d,
3H, ArCHCH3), 2.66–2.68 (d, 3H, NHCH3), 4.10–4.14
(q, 1H, ArCHCH3), 5.36 (s, 2H, C5–H), 6.35 (br s,
1H, NHCH3 exchanged with D2O), 7.03–7.09 (m, 2H,
C3,5 Ar–H), 7.32–7.36 (m, 1H, C4 Ar–H), 10.71 (br s,
1H, NHCO exchanged with D2O). Anal. C, H, F, N.
5.1.4. 6-(2,6-Difluorophenylmethyl)-3,4-dihydro-2-propion-
ylaminopyrimidin-4(3H)-one (10). To a cooled (0 ꢁC)
solution of 7 (1.26 mmol, 0.3 g) in dry THF (10 mL), tri-
ethylamine (1.45 mmol, 0.21 mL) and propionyl chlo-
ride (1.39 mmol, 0.13 mL) were added in succession.
After stirring for 30 min, the mixture was evaporated
under reduced pressure and the residue was partitioned
between water (50 mL) and ethyl acetate (50 mL). The
organic phase was separated, and the aqueous one was
extracted with ethyl acetate (2 · 30 mL). The combined
organic solution was washed with brine (2 · 50 mL),
dried, and evaporated under reduced pressure. The
obtained solid residue was crystallized to furnish
pure 10. 1H NMR (CDCl3): d 1.15–1.24 (t, 3H,
COCH2CH3), 2.45–2.56 (q, 2H, COCH2CH3), 3.82 (s,
2H, C6–CH2), 5.87 (s, 1H, C5–H), 6.84–6.92 (m, 2H,
C3,5 Ar–H), 7.15–7.27 (m, 1H, C4 Ar–H). Anal. C, H,
N, F.
5.2.5. RT assays. Assays were performed as previously
described.31 Briefly, purified rRT was assayed for its
RNA-dependent polymerase-associated activity in a 50 lL
volume containing: 50 mM Tris–HCl (pH 7.8), 80 mM
KCl, 6 mM MgCl2, 1 mM DTT, 0.1 mgmLꢀ1 BSA,
0.5 OD260 unit mLꢀ1 template:primer [poly(rC)–oligo-
(dG)12–18] and 10 mM [3H]dGTP (1 Ci mmolꢀ1). After
incubation for 30 min at 37 ꢁC, the samples were spotted
on glass fiber filters (Whatman GF/A), and the acid-
insoluble radioactivity was determined.
5.3. Computational studies
All molecular modeling calculations and manipulations
were performed using the software packages MACRO-
MODEL 7.1,32 AUTODOCK 3.0.5,33 running on Silicon
Graphics O2 R10000, IBM compatible Intel Pentium
IV 1.4 GHz and AMD Athlon 1.9 GHz workstations.
For any minimization the all-atom Amber force field34
was adopted as implemented in the MACROMODEL
package.
5.2. Antiviral assay procedures
5.2.1. Compounds. Compounds were solubilized in
DMSO at 200 mM and then diluted into culture
medium.
The geometry of the RTwt NNBS was taken from the
structure of HIV-1 RT/TNK-651 complex filed in the
Brookhaven Protein Data Bank35 (entry code 1rt2).
5.2.2. Cells and viruses. MT-4, C8166, H9/IIIB, and
CEM cells were grown at 37 ꢁC in a 5% CO2 atmosphere
in RPMI 1640 medium, supplemented with 10% fetal
calf serum (FCS), 100 IU/mL penicillin G and 100 lg/
mL streptomycin. Cell cultures were checked periodi-
cally for the absence of mycoplasma contamination with
a MycoTect Kit (Gibco). Human immunodeficiency
viruses type-1 (HIV-1, IIIB strain) and type-2 (HIV-2
ROD strain, kindly provided by Dr. L. Montagnier,
Paris) were obtained from supernatants of persistently
infected H9/IIIB and CEM cells, respectively. HIV-1
and HIV-2 stock solutions had titers of 4.5 · 106 and
1.4 · 105 50% cell culture infectious dose (CCID50)/
mL, respectively.
˚
All the residues within 20 A from any ligandÕs atom
(TNK-651) were used to define the NNBS. Analogously
it was done to define the RTY18C NNBS using the
structure of HIV-1 RTY181C/TNK-651 (PDB entry code
1jla).
The representative compound 4d was modeled in its
R configuration as previously reported.36 Compound
4d was chosen as F2-NH-DABO representative mem-
ber for the following reasons: (i) it is one of the most ac-
tive derivative in both enzymatic and cell based assays;
(ii) it has a cyclopentyl moiety attached to the NH–C2
that reduce the number of conformations, and also
no further chiral center is introduced by the cyclo-
pentyl; (iii) it is active against the Y181C mutant strain
of HIV.
5.2.3. HIV titration. Titration of HIV was performed in
C8166 cells by the standard limiting dilution method
(dilution 1:2, four replica wells per dilution) in 96-well
plates. The infectious virus titer was determined by light
microscope scoring of cytopathogenicity after 4 days of
incubation and the virus titers were expressed as
CCID50/mL.
The starting conformation for docking studies was ob-
tained using molecular dynamics with simulated anneal-
ing as implemented in MACROMODEL version 7.1 and

A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
2075
conducted as following: 4d was energy minimized to a
low gradient. The nonbonded cut-off distances were set
˚
to 20 A for both Van der Waals and electrostatic inter-
Acknowledgements
Authors thank Italian Ministero della Sanita—Istituto
`
Superiore di Sanita—IV Programma nazionale di ric-
`
actions. An initial random velocity to all atoms corre-
sponding to 300 K was applied. Three subsequent
molecular dynamic runs were then performed. The first
was carried out for 10 ps with a 1.5 fs time step at a con-
stant temperature of 300 K for equilibration purposes.
The next molecular dynamic was carried out for 20 ps,
during which the system is coupled to a 150 deg thermal
bath with a time constant of 5 ps. The time constant
represents approximately the half life for equilibration
with the bath; consequently the second molecular
dynamic command caused the molecule to slowly cool
to approximately 150 K. The third and last dynamic
cooled the molecule to 50 K over 20 ps. A final energy
minimization was then carried out for 250 iterations
using conjugate gradient. The minimizations and the
molecular dynamics were in all cases performed in aque-
ous solution. The atom charges automatically assigned
by the batchmin module were retained on 4d for the
docking calculations.
erca sullÕ AIDS 2001 (Grants nos. 40D.08 and 9403-
59) for financial aid. Acknowledgements are due to
Italian MURST (40% funds) and Regione Autonoma
`
Sardegna (Assessorato Sanita) for partial support.
References and notes
1. Artico, M. Nonnucleoside anti-HIV-1 reverse transcrip-
tase inhibitors (NNRTIs): a chemical survey from lead
compounds to selected drugs for clinical trials. Farmaco
1995, 51, 305.
2. De Clercq, E. New perspectives for the treatment of HIV
infections. Collect. Czech. Chem. Commun. 1998, 63, 449.
3. De Clercq, E. The role of non-nucleoside reverse trans-
criptase inhibitors (NNRTIs) in the therapy of HIV-1
infection. Antiviral Res. 1998, 38, 153.
4. Pedersen, O. S.; Pedersen, E. B. Nonnucleoside reverse
transcriptase inhibitors: the NNRTI boom. Antiviral
Chem. Chemother. 1999, 10, 285.
5. Merluzzi, V. J.; Hargrave, K. D.; Labadia, M.; Grozinger,
K.; Skoog, M.; Wu, J. C.; Shih, C.-K.; Eckner, R. J.;
Koup, R. A.; Sullivan, J. L. Inhibition of HIV-1 replica-
tion by a nonnucleoside reverse transcriptase inhibitor.
Science 1990, 250, 1411.
6. Cywin, C. L.; Klunder, J. M.; Hoermann, M.; Brickwood,
J. R.; David, E.; Grob, P. M.; Schwartz, R.; Pauletti, D.;
Barringer, K. J.; Shih, C.-K.; Sorge, C. L.; Erickson, D.
A.; Joseph, D. P.; Hattox, S. E. Novel nonnucleoside
inhibitors of HIV-1 reverse transcriptase. 8. 8-Aryloxy-
methyl- and 8-arylthiomethyldipyridodiazepinones. J.
Med. Chem. 1998, 41, 2972.
28,37–45
For the docking procedure the program AUTODOCK
was used to explore the binding conformation of 4d. For
˚
the docking a grid spacing of 0.375 A and 60 · 80 · 60
number of points was used. The grid was centered on
the mass center of the experimental bound TNK-651 coor-
dinates. The GA-LS method was adopted using the default
settings. Amber united atom charges were assigned to the
protein using the program ADT (AUTODOCK TOOLS).46
AUTODOCK generated 100 possible binding conformations
for 4d (see Table 5).
7. Romero, D. L.; Morge, R. A.; Biles, C.; Berrios-Pena, N.;
May, P. D.; Palmer, J. R.; Johnson, P. D.; Smith, H. W.;
Busso, M.; Tan, C.-K.; Voorman, R. L.; Reusser, F.;
Althaus, I. W.; Downey, K. M.; So, A. G.; Resnick, L.;
Tarpley, W. G.; Aristoff, P. A. Discovery, synthesis, and
bioactivity of bis(heteroaryl)piperazines. 1. A novel class
of non-nucleoside HIV-1 reverse transcriptase inhibitors.
J. Med. Chem. 1994, 37, 999.
8. Genin, M. J.; Toni, J. P.; May, P. D.; Kopta, L. A.; Yagi,
Y.; Olmsted, R. A.; Friis, J. M.; Voorman, R. L.; Adams,
W. J.; Thomas, R. C.; Romero, D. L. Synthesis and
structure–activity relationships of the (alkylamino)piper-
idine-containing BHAP class of non-nucleoside reverse
transcriptase inhibitors: effect of 3-alkylpyridine ring
substitution. J. Med. Chem. 1999, 42, 4140.
To validate the use of the AUTODOCK program, the
docking studies were performed on the reference com-
pound TNK-651 and for comparison purposes also on
the previously reported F2-S-DABO 3d. AUTODOCK suc-
cessfully reproduced the experimental binding confor-
mations of the reference drug TNK-651 with
acceptable root mean square deviation (RMSD) of atom
coordinates in either the RTwt (RMSD = 1.01) or
RTY181C (RMSD = 1.08) NNBS.
The results of the AUTODOCK runs that were clustered
˚
using an RMSD tolerance of 0.5 A are summarized in
Table 6.
9. Nunberg, J. H.; Schleif, W. A.; Boots, E. J.; OÕBrien, J. A.;
Quintero, J. C.; Hoffman, J. M.; Emini, E. A.; Goldman,
M. E. Viral resistance to human immunodeficiency virus
type 1-specific pyridinone reverse transcriptase inhibitors.
J. Virol. 1991, 65, 4887.
Table 6. Autodock results
10. Miller, V.; de Bethune, M.-P.; Kober, A.; Sturmer, M.;
Hertogs, K.; Pauwels, R.; Stoffels, P.; Staszewski, S.
Patterns of resistance and cross-resistance to human
immunodeficiency virus type 1 reverse transcriptase inhibi-
tors in patients treated with the non-nucleoside reverse
transcriptase inhibitor loviride. Antimicrob. Agents Chemo-
ther. 1998, 42, 3123.
11. Campiani, G.; Ramunno, A.; Maga, G.; Nacci, V.;
Fattorusso, C.; Catalanotti, B.; Morelli, E.; Novellino,
E. Non-nucleoside HIV-1 reverse transcriptase (RT)
inhibitors: past, present, and future perspectives. Curr.
Pharm. Design 2002, 8, 615.
Complex
No of
No of multi-
member
No of con-
formations
in the first
ranked
distinct
conforma-
tional
conforma-
tional clusters
clusters
cluster
3d/RTwt
3d/RTY181C
4d/RTwt
5
6
2
4
6
8
8
8
58
68
54
58
8
17
13
75
69
4d/RTY181C
TNK-651/RTwt
TNK-651/RTY181C
14

2076
A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
12. Young, S. D.; Britcher, S. F.; Tran, L. O.; Payne, L. S.;
Lumma, W. C.; Lyle, T. A.; Huff, J. R.; Anderson, P. S.;
Olsen, D. B.; Carroll, S. S.; Pettibone, D. J.; OÕBrien, J. A.;
Ball, R. G.; Balani, S. K.; Lin, J. H.; Chen, I.-W.; Schleif,
W. A.; Sardana, V. V.; Long, W. J.; Byrnes, V. W.; Emini,
E. A. L-743,726 (DMP-266): a novel, highly potent non-
nucleoside inhibitor of the human immunodeficiency virus
type 1 reverse transcriptase. Antimicrob. Agents Chemo-
ther. 1995, 39, 2602.
Colla, P. Dihydro(alkylthio)(naphthylmethyl) oxopyrimi-
dines: novel non-nucleoside reverse transcriptase inhibi-
tors of the S-DABO series. J. Med. Chem. 1997, 40, 1447.
25. 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-dihalophen-
ylmethyl)-3,4-dihydropyrimidin-4(3H)-ones: novel potent
and selective dihydro-alkoxy-benzyl-oxopyrimidine deriv-
atives. J. Med. Chem. 1999, 42, 619.
13. Fujiwara, T.; Sato, A.; El-Farrash, M.; Miki, S.; Abe, K.;
Isaka, Y.; Kodama, M.; Wu, Y.; Chen, B. L.; Harada, H.;
Sugimoto, H.; Hatanaka, M.; Hinuma, Y. S-1153 inhibits
replication of known drug-resistant strains of human
immunodeficiency virus type 1. Antimicrob. Agents Chemo-
ther. 1998, 42, 1340.
14. Baba, M.; Shigeta, S.; Yuasa, S.; Takashima, H.; Sekiya,
K.; Ubasawa, M.; Tanaka, H.; Miyasaka, T.; Walker, R.
T.; De Clercq, E. Preclinical evaluation of MKC-442. A
highly potent and specific inhibitor of human immunode-
ficiency virus type 1 in vitro. Antimicrob. Agents Chemo-
ther. 1994, 38, 688.
26. Artico, M.; Mai, A.; Sbardella, G.; 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.
27. 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 evalua-
tion of conformationally restricted novel 2-alkylthio-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. Chem. 2001, 44, 2544.
15. Hopkins, A. L.; Ren, J.; Esnouf, R. M.; Willcox, B. E.;
Jones, E. Y.; Ross, C.; Miyasaka, T.; Walker, R. T.;
Tanaka, H.; Stammers, D. K.; Stuart, D. I. Complexes of
HIV-1 reverse transcriptase with inhibitors of the HEPT
series reveal conformational changes relevant to the design
of potent non-nucleoside inhibitors. J. Med. Chem. 1996,
39, 1589.
16. Artico, M. Selected non-nucleoside reverse transcriptase
inhibitors (NNRTIs): the DABOs family. Drug Fut. 2002,
27, 159.
17. Marongiu, M. E.; Pani, A.; Musiu, C.; La Colla, P.; Mai,
A.; 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. Develop. Med. Chem. 2002, 1, 65.
18. Marongiu, M. E.; Pani, A.; Artico, M.; Massa, S.; Mai,
A.; La Colla, P. Selective inhibition of HIV-1 replication
by a novel series of 2-substituted 6-benzyl-pyrimidines.
VIII International Conference on AIDS. 1992, Amster-
dam, July 19–24, PoA 2315.
19. Artico, M.; Massa, S.; Mai, A.; Marongiu, M. E.; Piras,
G.; 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. 1993, 4, 361.
20. Tramontano, E.; Marongiu, M. E.; De Montis, A.; Loi, A.
G.; Artico, M.; Massa, S.; Mai, A.; La Colla, P.
Characterization of the anti-HIV-1 activity of 3,4-dihy-
dro-2-alkoxy-6-benzyl-4-oxopyrimidines (DABOs), new
non-nucleoside reverse transcriptase inhibitors. Microbio-
logica 1994, 17, 269.
21. Massa, S.; Mai, A.; Artico, M.; Sbardella, G.; Tramont-
ano, E.; Loi, 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. Chemo-
ther. 1995, 6, 1.
28. 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-alkylamino-6-[1-(2,6-difluor-
ophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones as
novel potent non-nucleoside reverse transcriptase inhibi-
tors, also active against the Y181C variant. J. Med. Chem.
2004, 47, 928.
29. Ren, J.; Nichols, C.; Bird, L.; Chamberlain, P.; Weaver,
K.; Short, S.; Stuart, D. I.; Stammers, D. K. Structural
mechanisms of drug resistance for mutations at codons
181 and 188 in HIV-1 reverse transcriptase and the
improved resilience of second generation non-nucleoside
inhibitors. J. Mol. Biol. 2001, 312, 795.
30. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols,
D.; Herdewijn, P.; Desmyster, J.; De Clercq, E. Rapid and
automated tetrazolium-based assay for the detection of
anti-HIV compounds. J. Virol. Methods 1988, 20, 309.
31. Tramontano, E.; Cheng, Y.-C. HIV-1 reverse transcrip-
tase inhibition by a dipyridodiazepinone derivative: BI-
RG-587. Biochem. Pharmacol. 1992, 43, 1371.
32. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.;
Liskamp, R.; Lipton, M.; Caufield, C.; Chang, G.;
Hendrickson, T.; Still, W. C. MacroModel—An inte-
grated software system for modeling organic and bioor-
ganic molecules using molecular mechanics. J. Comput.
Chem. 1990, 11, 440.
33. Goodsell, D. S.; Morris, G. M.; Olson, A. J. Automated
docking of flexible ligands: applications of AutoDock. J.
Mol. Recognit. 1995, 9, 1.
34. Pearlman, D. A.; Case, D. A.; Caldwell, J. W.; Ross, W. S.;
Cheatham, T. E. I.; Debolt, S.; Ferguson, D. M.; Seibel, G.
L.; Kollman, P. A. AMBER, a package of computer
programs for applying molecular mechanics, normal-mode
analysis, molecular dynamics and free energy calculations
to simulate the structural and energetic properties of
molecules. Comput. Phys. Commun. 1995, 91, 1.
22. Mai, A.; Artico, M.; Sbardella, G.; Massa, S.; Loi, A. G.;
Tramontano, E.; Scano, P.; La Colla, P. Synthesis and
anti-HIV-1 activity of thio analogs of dihydro-
alkoxybenzyloxopyrimidines. J. Med. Chem. 1995, 38,
3258.
23. Ettorre, A.; Mai, A.; Artico, M.; Massa, S.; De Montis,
A.; La Colla, P. 6-(3-Methylbenzyl)-2-(2-methylprop-
yl)thio-4(3H)-pyrimidinone (DABO 622). Acta Crystal-
logr. 1995, C52, 2115.
35. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.;
Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E.
The protein data bank. Nucleic Acids Res. 2000, 28,
235.
36. Quaglia, M.; Mai, A.; Sbardella, G.; Artico, M.; Ragno,
R.; Massa, S.; del Piano, D.; Setzu, G.; Doratiotto, S.;
Cotichini, V. Chiral resolution and molecular modeling
investigation of rac-2-cyclopentylthio-6-[1-(2,6-difluoro-
phenyl)ethyl]-3,4-dihydro-5-methylpyrimidin-4(3H)-one (MC-
1047), a potent anti-HIV-1 reverse transcriptase agent of
the DABO class. Chirality 2001, 13, 75.
24. Mai, A.; Artico, M.; Sbardella, G.; Quartarone, S.; Massa,
S.; Loi, A. G.; De Montis, A.; Scintu, F.; Putzolu, M.; La

A. Mai et al. / Bioorg. Med. Chem. 13 (2005) 2065–2077
2077
37. Chen, H. F.; Yao, X. J.; Li, Q.; Yuan, S. G.; Panaye, A.;
Doucet, J. P.; Fan, B. T. Comparative study of non
nucleoside inhibitors with HIV-1 reverse transcriptase
based on 3D-QSAR and docking. SAR QSAR Environ.
Res. 2003, 14, 455.
38. Zhigang, Z.; Madura, J. Use of dock and PB in
reproducing the binding mode and predicting the bind-
ing-free energies of non-nucleoside inhibitors to HIV-1
reverse transcriptase. Presented at the 224th ACS National
Meeting, Boston, MA, August 18–22, 2002, COMP-170.
39. Di Santo, R.; Costi, R.; Artico, M.; Ragno, R.; Massa, S.;
La Colla, M.; Loddo, R.; La Colla, P.; Pani, A.
Arylthiopyridylmethylisopropylpyrrole carbinols, novel
NNRTIs endowed with potent anti-HIV-1 activity. Med.
Chem. Res. 2002, 11, 153.
41. Sotriffer, C. A.; Ni, H.; McCammon, J. A. HIV-1
integrase inhibitor interactions at the active site: predic-
tion of binding modes unaffected by crystal packing. J.
Am. Chem. Soc. 2000, 122, 6136.
42. Hetenyi, C.; Maran, U.; Karelson, M. A comprehensive
docking study on the selectivity of binding of aromatic
compounds to proteins. J. Chem. Inf. Comput. Sci. 2003,
43, 1576.
43. Anderson, A. C. The process of structure-based drug
design. Chem. Biol. 2003, 10, 787.
44. Campbell, S. J.; Gold, N. D.; Jackson, R. M.; Westhead,
D. R. Ligand binding: functional site location, similarity
and docking. Curr. Opin. Struct. Biol. 2003, 13, 389.
45. Hu, X.; Balaz, S.; Shelver, W. H. A practical approach to
docking of zinc metalloproteinase inhibitors. J. Mol.
Graph. Model 2004, 22, 293.
46. Michel, F. S. Python: A programming language for
software integration and development. J. Mol. Graph.
Mod. 1999, 17, 57.
40. Schames, J. R.; Henchman, R. H.; Siegel, J. S.; Sotriffer,
C. A.; Ni, H.; McCammon, J. A. Discovery of a novel
binding trench in HIV integrase. J. Med. Chem. 2004, 47,
1879.