2-(Alkyl/Aryl)amino-6-benzylpyrimidin-4(3 H)-ones as inhibitors of wild-type and mutant HIV-1: Enantioselectivity studies

Article abstract of DOI:10.1021/jm201308v

The single enantiomers of two pyrimidine-based HIV-1 non-nucleoside reverse transcriptase inhibitors, 1 (MC1501) and 2 (MC2082), were tested in both cellular and enzyme assays. In general, the R forms were more potent than their S counterparts and racemat

Full text of DOI:10.1021/jm201308v

Brief Article
pubs.acs.org/jmc
2-(Alkyl/Aryl)Amino-6-Benzylpyrimidin-4(3H)-ones as Inhibitors of
Wild-Type and Mutant HIV-1: Enantioselectivity Studies
Dante Rotili,†,# Alberta Samuele,‡,# Domenico Tarantino,† Rino Ragno,† Ira Musmuca,† Flavio Ballante,†
Giorgia Botta,† Ludovica Morera,† Marco Pierini,† Roberto Cirilli,§ Maxim B. Nawrozkij,∥
Emmanuel Gonzalez,⊥ Bonaventura Clotet,⊥ Marino Artico,† Jose A. Este,*,⊥ Giovanni Maga,*,‡
́
́
and Antonello Mai*,†
†Istituto PasteurFondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Universita
̀
degli Studi di Roma
“La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy
‡Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy
§Dipartimento del Farmaco, Istituto Superiore di Sanita,
̀
Viale Regina Elena 299, 00161 Rome, Italy
∥Volgograd State Technical University, prospekt Lenina, 28, 400131 Volgograd, Russia
⊥Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Auton
Badalona, Spain
̀
oma de Barcelona, 08916
S
* Supporting Information
ABSTRACT: The single enantiomers of two pyrimidine-based HIV-1 non-nucleoside reverse transcriptase inhibitors, 1
(MC1501) and 2 (MC2082), were tested in both cellular and enzyme assays. In general, the R forms were more potent than
their S counterparts and racemates and (R)-2 was more efficient than (R)-1 and the reference compounds, with some exceptions.
Interestingly, (R)-2 displayed a faster binding to K103N RT with respect to WT RT, while (R)-1 showed the opposite behavior.
Chart 1. Pyrimidine-Based NNRTIs That Are the Object of
the Present Study
INTRODUCTION
■
Since 1992, our research team has discovered excellent dihydro-
alkyloxy-benzyl-oxopyrimidine (DABO) classes of non-nucleo-
side reverse transcriptase (RT) inhibitors (NNRTIs) such as
F2-S-DABOs,1 F2-NH-DABOs,2 and F2-N,N-DABOs.3 Such
compounds had inhibitory potencies in the (sub)nanomolar
range against wild-type (WT) HIV-1 without significant
cytotoxicity at higher concentrations, and with potencies in
the submicromolar range against clinically relevant mutant
strains. The F2-N,N-DABO derivative 1 (MC1501),3 charac-
terized by a N-methyl-N-propyl side chain at the C-2
pyrimidine ring position and by a double pyrimidine C-5/C-6
benzylic position methyl substitution, with its subnanomolar
inhibitory potency against WT HIV-1 and the Y181C mutant
strain, can be considered one of the most promising DABO
compounds reported so far (Chart 1).
Diarylpyrimidine (DAPY) derivatives are one of the most
successful classes of pyrimidine-based NNRTIs, as demon-
strated by the recent approval of etravirine and rilpivirine for
clinical use.4,5 SAR studies on the DAPY class had also shown
how the para-cyanoaniline substitution at the C-2 pyrimidine
ring position is crucial for a strong inhibitory activity. Recently,
combining the 2,6-difluorobenzyl or 1-(2,6-difluorophenyl)-
ethyl group at the C-6 position characteristic of DABOs with
the para-cyanoaniline group at the C-2 of the pyrimidine ring
typical of DAPYs, and inserting at the C-4 pyrimidine ring
position groups found in either DABOs (−OH) or DAPYs
(−Cl, −H, −NH2), a new series of highly potent anti-HIV-1
agents called DAPY-DABO hybrids has been developed.6
Among those, the derivative 2 (MC2082) (Chart 1) was the
most potent due to its (sub)nanomolar activity against WT and
clinically relevant mutant HIV-1 strains, such as K103N and
Y188L.6
Both the pyrimidine-based NNRTIs 1 and 2 contain a
stereogenic center at the C-6 benzylic position and, therefore,
exist as racemic mixtures. Because of the high potency of these
two lead compounds, we decided to perform a systematic
investigation on their potential enantioselective anti-HIV-1
activity. Here we describe, through a multidisciplinary
approach, the enantioseparation, the absolute configuration
assignment, and the enzymatic and cellular evaluation of the
Received: October 11, 2011
Published: March 19, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
HIV-1 inhibitory activity of the single enantiomers in
comparison with the corresponding racemic mixtures of both
compounds. The application of a comparative binding energy
(ComBinE) model built to quantitate the influence of single
point HIV-RT mutations to modeled bound conformations of
both 1 and 2 was in good agreement with the experimental
findings and gave insights on the absolute configuration
assignment of both 1 and 2 separated enantiomers.
Chemistry. The racemic mixture of 2 was prepared as
previously described.6 The racemic mixture of 1 was prepared
starting from the β-oxoester 31 and using, differently from
previously reported,3 a route characterized by only two steps
and by a final nucleophilic displacement under mild conditions
and with high yield. After condensation of the β-oxoester 3 with
nitroguanidine, the resulting 6-[1-(2,6-difluorophenyl)-ethyl]-
5-methyl-2-nitroamino-3H-pyrimidin-4-one 4 was heated in a
sealed tube of a Parr high pressure reactor in the presence of an
excess of N-methyl-N-propylamine to give directly the racemic
1 (Scheme 1).
(+)-1 and (S)-(+)-2, first-eluted enantiomers on the Chiralpak
AD CSP) and the (R) configuration assigned to the
levorotatory counterparts ((R)-(−)-1 and (R)-(−)-2, second-
eluted enantiomers).
The racemic compounds 1 and 2 and their enantiomers were
tested in MT-4 cells to evaluate their cytotoxicity and their
capability to inhibit by 50% the HIV-induced cytopathic effect
(HIV-1 strain: NL4−3). The compounds were also tested
against a panel of clinically relevant HIV-1 mutant strains
(K103N, Y181C, and Y188L). Nevirapine (NVP), efavirenz
(EFV), and dapivirine (TMC120) were also tested as reference
drugs (Table 1).
The enantiomer (R)-1 was much more potent (ranging from
about 2800- to 11000-fold) than the other enantiomer (S)-1 in
inhibiting WT HIV-1 and the three tested mutant strains. (R)-1
was slightly better, both in terms of activity and of selectivity,
than the corresponding racemic mixture. The enantiomer (R)-2
inhibited WT and mutant HIV-1 strains from 900- to 10000-
fold more efficiently than the other enantiomer and was slightly
more potent than the racemic mixture. With the only exception
of the mutant Y181C, (R)-2 was always more efficient than
(R)-1 and TMC120 in the inhibition of both WT and mutant
(K103N and Y188L) HIV-1 strains (Table 1). Moreover, (R)-2
was 10-fold less toxic than TMC120, yielding an highly
improved selectivity index (SI(R)‑2 = 300000; SITMC120 = 5000).
We evaluated the RNA-dependent DNA polymerase activity
of WT RT and those enzymes carrying the most common
NNRTI-resistance mutations (K103N, L100I, Y181I, V106A,
Y188L) in the presence of increasing concentrations of the 1
and 2 enantiomers (Table 2). In the inhibition assays, the
enantiomers (R)-1 and (R)-2 showed higher potency than their
corresponding S isomers toward RT WT and mutants.
Moreover, (R)-2 showed the lowest ID50 values with respect
to the majority of the mutants evaluated, such as K103N,
V106A, and Y188L, whereas its efficacy appeared slightly lesser
than that observed for (R)-1 in the case of L100I and Y181I. As
illustrated in Table 2, in all cases both racemic compounds
presented intermediate levels of potency compared to the
corresponding pairs of enantiomers.
a
Scheme 1
a
(i) EtONa, nitroguanidine, dry EtOH, reflux; (ii) N-methyl-N-
propylamine, 120 °C, Parr high pressure reactor.
RESULTS AND DISCUSSION
■
The direct resolution of 1 and 2 was achieved by HPLC on the
coated-type Chiralpak AD chiral stationary phase (CSP) using
pure ethanol in mixture with a small percentage (0.1%) of
diethylamine (DEA) as eluent (see Supporting Information).
Efforts to obtain the absolute configuration of 1 and 2 by X-ray
crystallography were not successful, therefore stereochemical
information on the four chiral compounds were obtained by
analyzing their chiroptical properties. The (S) configuration
was assigned to the dextrorotatory enantiomers of 1 and 2 ((S)-
Steady-state kinetic assays were performed for the two most
potent isomers, (R)-1 and (R)-2, to assess the inhibition
mechanism against RT WT and RT carrying K103N mutation.
The enzyme activity was measured in the presence of fixed
Table 1. Cytotoxicity and Anti-HIV-1 Activity against WT (NL4-3) and Clinically Relevant HIV-1 Mutant Strains of Racemic
a
Compounds 1 and 2 and their Enantiomers
b
c
EC50, nM (fold resistance)
d
e
compd
NL4−3
K103N
Y181C
Y188L
CC50, nM
selectivity index
1
1.9 0.2
15000 50
1.3 0.1
16.5 0.5 (9)
>78000
17 1 (9)
>78000
49 4 (26)
>78000
>78000
>41000
>5.2
(S)-1
(R)-1
2
>78000
11 0.7 (8)
35 3 (60)
4500 10 (4)
0.8 0.01 (8)
1.2 0.2 (2)
6100 20 (47)
340 4 (47)
13 1 (10)
40 2 (70)
>38000
28 2 (22)
27 3 (40)
6500 (6)
75000 50
40000 70
38000 50
31000 50
3000 30
7500 20
58000
70000
40
0.6 0.1
(S)-2
(R)-2
TMC120
NVP
EFV
1000 10
0.1 0.02
0.6 0.07
31 2 (300)
1.2 0.2 (2)
>7500
7.1 0.5 (70)
330 10 (500)
>7500
300000
5000
58
130
1
7.3 0.5
10 1 (1.4)
1600 5 (220)
3200
8
440
a
b
Values are means SD determined from at least three experiments. Effective concentration 50, concentration needed to inhibit 50% HIV-induced
c
cytopathic effect, evaluated with the MTT method in MT-4 cells. Fold resistance: ratio of EC50 value against drug-resistant strain and EC50 of the
WT NL4−3 strain. Cytotoxic concentration 50, concentration to induce 50% death of noninfected cells, evaluated with the MTT method in MT-4
cells. Selectivity index, CC50/EC50.
d
e
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Journal of Medicinal Chemistry
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Table 2. Comparison of Inhibition Potencies, Expressed as ID50 (nM), between the Racemic Compounds 1 and 2 and their
a
Enantiomers, Tested against RT Wild-Type (WT) and K103N, L100I, Y181I, V106A, Y188L RT Mutants
b
c
ID50, nM (fold resistance)
compd
WT
30
K103N
L100I
Y181I
V106A
Y188L
1
3
174 4 (6)
>20000
177 9 (6)
350 8 (0.4)
96 1 (50)
280 3 (20)
>15000
1260 8 (40)
>20000
19 1 (0.6)
120 8 (0.1)
1430 20 (50)
>20000
(S)-1
(R)-1
2
975
2
5
0.2
2
96 9 (48)
22 3 (1.6)
240 9 (1.3)
450 9 (200)
1700 10 (120)
>20000
3
0.2 (1.5)
195 5 (100)
150 6 (11)
500 8 (3)
d
14
180
8
ND
(S)-2
(R)-2
TMC120
NVP
EFV
8
64 4 (0.3)
1
8
1 (1)
150 9 (20)
90 1 (13)
ND
750 8 (90)
>1000
0.3 0.02 (0.04)
3
0.2 (0.4)
7
1
100 10 (14)
7000 100 (20)
3000 50 (100)
ND
ND
ND
380 3 (50)
400 10
30
35000 100 (87)
80 3 (3)
ND
ND
3
ND
a
b
Values are means SD determined from at least three experiments. Inhibitory dose 50, compound dose required to inhibit the HIV-1 rRT activity
c
d
by 50%. Fold resistance: ratio of ID50mut/ID50WT values. ND: not determined.
concentrations of the compounds and variable concentrations
of one of the two substrates (either nucleic acid or nucleotide),
while the other was maintained at saturating doses. The kinetic
parameters Vmax and Km were derived by fitting the
experimental data to the appropriate rate equations (Table S1
in Supporting Information). For both compounds, either
varying the nucleic acid (NA) or the nucleotide (dNTP)
concentrations, the Vmax values of RT WT or K103N decreased
as the inhibitor concentrations increased while the Km values
did not change. Thus, the mechanism of action of (R)-1 and
(R)-2 against RT WT and K103N was fully noncompetitive.
The (R)-2 showed Ki values lower than those of (R)-1. Binding
experiments were subsequently carried out, with RT WT and
K103N to evaluate the association (kon) and the dissociation
rate (koff) values for each inhibitor with respect to the different
catalytic forms of the enzyme (free, binary complex, ternary
complex) along the reaction pathway (Table S2 in Supporting
Information). The kon rate values were used to calculate the
relative association index (RAI = kon WT/kon mut). RAI values
were >1 for (R)-1 toward the RT binary and ternary complexes,
while they scored <1 for (R)-2 (toward the free RT and binary
complex). This indicated a different interaction mechanism of
the two compounds with RT: (R)-1 experienced a slower
binding to the mutated form with respect to RT WT, while
(R)-2, surprisingly, associated faster to the mutant RT (Figure
1A). Such unexpected behavior could be related to the presence
of the cyano group into the (R)-2 structure and its ability to
disrupt the K103−Y188 hydrogen bond in the apo form of the
mutated enzyme.7 koff values were used to derive the relative
dissociation index (RDI = koff mut/koff WT). RDI values were,
for both inhibitors, >1 toward the free enzyme and <1 in the
case of the binary and ternary complexes, indicating that the
binding of the compounds to the K103N RT was more stable
than with WT RT once the enzymes were bound to the
substrates (Figure 1B).
Figure 1. (A) Relative association index (RAI) derived from the
association rates (kon) of the inhibitors (R)-1 and (R)-2 to RT K103N
and RT WT, expressed as ratio kon WT/kon mut. (B) Relative
dissociation index (RDI) derived from the dissociation rates (koff) of
the inhibitors from RT K103N and RT WT, expressed as ratio koff
mut/koff WT. free, free enzyme; bin, binary complex; ter, ternary
complex. Error bars indicate the standard deviations ( SD).
Binding of substrates to RT is known to stabilize specific
conformations of the enzyme, thus narrowing its range of
inherent flexibility. According to the studies mentioned above,
this implies that a given mutation will exert its effects on a
different set of possible enzyme conformations, depending on
whether the enzyme is unliganded, bound to nucleic acid, or in
the ternary complex conformation. Our data suggest that such
substrate-induced conformational changes are capable of
influencing the ability of a certain mutation to affect the
different kinetic steps of NNRTI interaction.
Biosensor-based analysis of binding of different NNRTIs to
HIV-1 RT either WT or carrying drug-resistance mutants8,9
supported the notion that the inherent flexibility of HIV-1 RT
might explain the existence of high-affinity and low-affinity
forms of RT with respect to NNRTI binding. Moreover, those
studies revealed that the resistance induced by some mutations
(such as K103N) was due to effects on both the association
(kon) and dissociation (koff) rates of NNRTIs in an inhibitor-
specific manner. In particular, the K103N mutation was found
to stabilize the low-affinity conformation of the enzyme,
thereby increasing both the kon and koff rates of some NNRTIs.
A structure-base study by means of ComBinE10-like protocol
was undertaken (see Supporting Information) to further
support the experimental evidence on the absolute config-
uration of the most- and the least-active 2 enantiomers and to
provide a quantitative assessment of the mutation influence on
the activity potency. The statistically most robust ComBinE
model CM4, (previously developed without consideration of
these compounds, Ballante, F., et al. J. Comput.-Aided Mol. Des.,
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Journal of Medicinal Chemistry
Brief Article
submitted for publication) was used to verify the mutation role
on the activity profile of (R)- and (S)-1 and of (R)- and (S)-2.
It could be argued that the use of static unrefined complexes
cannot account for the dynamic aspect of ligand binding,
nevertheless the CM4 models is able to highlight some aspects
that would be otherwise ignored to interpret the experimental
data. Application of the CM4 model to the inhibitors
successfully reproduced the experimental eudismic ratio of
(R) and (S) enantiomers as well as the increase of potency of 2
compared to 1. In particular, among the residues around the
RT NNBP a main role can be quantitatively attributed to
Lys101 that was not considered in the RT mutated list. Other
important residues for the binding strength were the highly
conserved Trp229 and Tyr188 residues. The latter (Tyr188 in
the WT isoform) serves as a steric anchor point to fulfill the
concomitant lack of interactions that occurs in other mutation
points such as in the case of V106A (Figure 2).
effect of K103N RT mutation on the (R)-1 and (R)-2 activities
was further characterized. A ComBinE model based on 14
complexes between RT and EFV and NVP without any
knowledge of the herein reported compounds, reproduced with
acceptable errors of prediction quantitatively the differences
between different pyrimidinone derivatives and their enan-
tiomers and was used to gain insight into the role of RT
mutations on the inhibitors’ biological activities.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. H NMR spectra were
■
1
recorded at 400 MHz on a Bruker AC 400 spectrometer; chemical
shifts are reported in δ units relative to the internal reference
tetramethylsilane (Me4Si). All of the compounds were routinely
1
checked by TLC and H NMR. TLC was performed on aluminum
backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254) with
spots visualized by UV light. All of the solvents were reagent grade
and, when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions involved the
use of a rotary evaporator operating at a reduced pressure of ca. 20
Torr. Organic solutions were dried over anhydrous sodium sulfate.
Elemental analysis has been used to determine purity of the described
compounds, that is >95%. 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.
Preparation of 6-[1-(2,6-Difluorophenyl)-ethyl]-5-methyl-2-
nitroamino-3H-pyrimidin-4-one (4). See Supporting Information.
Preparation of 6-[1-(2,6-Difluorophenyl)-ethyl]-5-methyl-2-
(methyl-n-propyl-amino)-3H-pyrimidin-4-one (1). A mixture of
6-[1-(2,6-difluorophenyl)-ethyl]-5-methyl-2-nitroamino-3H-pyrimi-
din-4-one 4 (100 mg, 0.322 mmol) and N-methyl-N-propylamine
(707.2 mg, 9.66 mmol, 0.99 mL) was heated in a sealed tube of a Parr
apparatus at 120 °C for 5 h. After cooling, the crude residue was
dissolved in ethyl acetate (15 mL) and water (15 mL). The aqueous
phase was extracted with ethyl acetate (3 × 15 mL). The organic
extracts were washed with brine (1 × 20 mL), dried, evaporated under
reduced pressure, and purified by column chromatography (silica gel,
ethyl acetate/chloroform 1:2) to give the desired product 1 as a white
powder (75.1 mg, 72.6%); mp 128−130 °C (dichloromethane/diethyl
Figure 2. Comparison between (R)-2 (green carbon atoms) docked
into WT (yellow) and V106A mutated (gray) RT. The only residue
that appreciably moves is Tyr188. The mutated Ala106 is displayed in
magenta.
1
ether). H NMR (CDCl3) δ 10.70 (s, 1H, NH), 7.13 (m, 1H, H
benzene ring), 6.81 (m, 2H, H benzene ring), 4.54 (q, 1H, CHCH3),
3.50 (m, 1H, NCHHCH2CH3), 3.37 (m, 1H, NCHHCH2CH3), 3.06
(s, 3H, NCH3), 1.93 (s, 3H, CH3), 1.64 (d, 3H, CHCH3), 1.54 (m,
2H, NCH2CH2CH3), 0.88 (s, 3H, NCH2CH2CH3). Anal. C, H, N, F:
% Calcd C, 63.54; H, 6.59; N, 13.08; F, 11.82, Percent found C, 63.22;
H, 6.48; N, 13.25; F, 12.01.
Enantioseparation and Chiroptical Characterization. See
Supporting Information.
Molecular Modeling: ComBinE and Docking Calculations.
See Supporting Information.
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.11,12 For a brief description, see Supporting Information.
Anti-HIV Reverse Transcriptase Assays. RNA-dependent DNA
polymerase activity was assayed as described13 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. For a brief description, see Supporting
Information.
CONCLUSION
■
Among the most potent pyrimidine derivatives described by us
as anti-HIV-1 agents, the N,N-DABO 1 and the DABO−DAPY
hybrid 2, both carrying a stereogenic center at the C6-benzylic
position and previously prepared and tested as racemates, were
resolved and characterized for their enantioselective activities in
HIV-1-infected cells as well as by enzyme assays. In both
cellular and enzyme assays, the R enantiomers of the two
compounds were significantly more potent than the S
counterparts, the racemates having an intermediate behavior
compared to the corresponding single enantiomers. (R)-2 was
typically more efficient than (R)-1 as well as than the reference
compounds TMC120, NVP, and EFV in inhibiting the
cytopathic effect of HIV-1 strains in MT-4 cells with the
exception of the Y181C mutant, against which TMC120 and
EFV showed the highest effects. However, (R)-2 was one
magnitude order less toxic than TMC120 and EFV, thus
reaching a very high selectivity index. In enzyme assays, (R)-2
displayed the highest inhibitory activities against RT WT,
K103N, V106A, and Y188L, while against L100I and Y181I
(joined in this case to EFV) (R)-1 was more efficient. The
ASSOCIATED CONTENT
■
S
* Supporting Information
Details on enantioseparation and absolute configuration
assignment, biochemistry and molecular modeling studies,
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Journal of Medicinal Chemistry
Brief Article
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and experimental section. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For J.A.E.: phone, +34-934656374; fax, +34-934653968; E-
mail, jaeste@irsicaixa.es. For G.M.: phone, +39 0382 546354;
fax, +39 0382 422286; E-mail: maga@igm.cnr.it. For A.M.:
phone, +39 06 49913392; fax, +39 06 49693268; E-mail,
antonello.mai@uniroma1.it.
Author Contributions
#Equal contribution.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partially supported by MICINN (BFU2009-
06958 and SAF2010-21617-C02 to J.A.E. and B.C.) and by the
Italian National Program for AIDS Research Grant 40H26 (to
G.M.).
ABBREVIATIONS USED
■
(13) Maga, G.; Amacker, M.; Ruel, N.; Hubscher, U.; Spadari, S.
Resistance to nevirapine of HIV-1 reverse transcriptase mutants: loss
of stabilizing interactions and thermodynamic or steric barriers are
induced by different single amino acid substitutions. J. Mol. Biol. 1997,
274, 738−747.
CC50, compound concentration toxic for 50% of cells; CSP,
chiral stationary phase; DABOs, dihydro-alkoxy-benzyl-oxopyr-
imidines; DAPYs, diarylpyrimidines; DEA, diethylamine; 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-difluoro-
phenylalkyl) pyrimidin-4(3H)ones; HIV, human immunodefi-
ciency virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide; NH-DABOs, dihydro-alkylamino-benzyl-
oxopyrimidines; NNBS, non-nucleoside binding site; NNRTIs,
non-nucleoside reverse transcriptase inhibitors; NVP, nevir-
apine; RT, reverse transcriptase; SAR, structure−activity
relationship; S-DABOs, dihydro-alkylthio-benzyl-oxopyrimi-
dines; WT, wild-type
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