De novo parallel design, synthesis and evaluation of inhibitors against the reverse transcriptase of human immunodeficiency virus type-1 and drug-resistant variants

Article abstract of DOI:10.1021/jm0613121

We used molecular modeling to design de novo broad-range inhibitors against wild type and drug-resistant variants of the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1). First, we screened for small fragments that would interact with each one of four RT structures (one wild type and three mutants). Then, these fragments were linked to build a scaffold molecule. Out of 27 different compounds that were synthesized, four inhibited the DNA polymerase activity of RT with IC₅₀ values below 10 μM. Compound 5f inhibited RT with an IC₅₀ value of about 3.5 μM, while inhibiting drug-resistant RT variants more efficiently than the clinically used drug, nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b: 2′,3′-e-]-[1,4]diazepin-6-one). 5f also inhibited the RT ribonuclease H activity with an IC₅₀ of 20 μM and therefore, unlike nevirapine, targets both RT activities. Accordingly, 5f can serve as lead for developing novel inhibitors against RT that may be used to suppress HIV-1 growth.

Full text of DOI:10.1021/jm0613121

2370
J. Med. Chem. 2007, 50, 2370-2384
De NoWo Parallel Design, Synthesis and Evaluation of Inhibitors against the Reverse
Transcriptase of Human Immunodeficiency Virus Type-1 and Drug-Resistant Variants
Alon Herschhorn,‡,§ Lena Lerman,# Michal Weitman,# Iris Oz Gleenberg,‡ Abraham Nudelman,*,# and Amnon Hizi*,‡,|
Department of Cell and DeVelopmental Biology, Sackler School of Medicine, Tel AViV UniVersity, Tel AViV 69978, and Chemistry Department,
Bar Ilan UniVersity, Ramat Gan 52900, Israel
ReceiVed NoVember 12, 2006
We used molecular modeling to design de noVo broad-range inhibitors against wild type and drug-resistant
variants of the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1). First, we screened
for small fragments that would interact with each one of four RT structures (one wild type and three mutants).
Then, these fragments were linked to build a scaffold molecule. Out of 27 different compounds that were
synthesized, four inhibited the DNA polymerase activity of RT with IC50 values below 10 µM. Compound
5f inhibited RT with an IC50 value of about 3.5 µM, while inhibiting drug-resistant RT variants more efficiently
than the clinically used drug, nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b:2′,3′-e]-
[1,4]diazepin-6-one). 5f also inhibited the RT ribonuclease H activity with an IC50 of 20 µM and therefore,
unlike nevirapine, targets both RT activities. Accordingly, 5f can serve as lead for developing novel inhibitors
against RT that may be used to suppress HIV-1 growth.
Introduction
proximity to the DNA polymerase active site of the enzyme;3
most of them are highly specific against HIV-1 RT with minimal
effects on the closely related HIV-2 RT.4 Both classes of
inhibitors are currently used in the therapy against HIV-1 as
part of the highly active anti-retroviral therapy, which simul-
taneously targets the RT, the viral protease, and most recently
the entry step of the virus.5
The reverse transcriptase (RTa) of HIV-1 is an essential
enzyme in the retroviral life cycle. After penetrating into the
target cell, RT copies the viral plus sense and single-stranded
genomic RNA into double-stranded DNA. This process is
catalyzed solely by RT and depends on two fundamental
enzymatic activities: that of the DNA polymerase (which copies
both RNA and DNA into DNA) and that of the RNase H, which
concomitantly cleaves the viral RNA strand in the RNA/DNA
heteroduplex. The resulting double-stranded DNA is transported
into the nucleus as part of a preintegration complex and is
subsequently incorporated into the DNA of the cell by the viral
enzyme integrase.1 Since the identification, two decades ago,
of HIV-1 as the cause for AIDS, a massive search for molecules
that block RT activities has been conducted worldwide. These
efforts led to the identification of two classes of anti-RT
compounds: nucleoside/nucleotide RT inhibitors (NRTIs) and
non-nucleoside RT inhibitors (NNRTIs).2 NRTIs are competitive
inhibitors that mimic normal nucleotides but lack the 3′-OH.
When incorporated into the nascent DNA by RT, NRTIs prevent
additional incorporation of nucleotides and, hence, terminate
chain elongation. The NNRTIs are a variety of noncompetitive
inhibitors that bind specifically to a hydrophobic pocket in
A significant obstacle for the use of NNRTIs is their very
high specificity that reduces their efficacy against mutated
variants of the virus.6 Given that the protein targets for therapy,
especially the RT and protease, are quite flexible and can tolerate
mutations and still remain active, resistance develops rapidly
during treatment even when a combination of drugs is used.7
Consequently, intense efforts have been directed in recent years
to find broad spectrum NNRTIs that inhibit both wild type and
drug-resistant variants of RT. This intensive search led to the
discovery of several highly efficient inhibitors, including 4-[[6-
amino-5-bromo-2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-
3,5-dimethylbenzonitrile (TMC-125), N-[4-(aminosulfonyl)-2-
methylphenyl]-2-[4-chloro-2-(3-chloro-5-cyanobenzoyl)-
phenoxy]acetamide (GW678248), and 5-bromo-N-(4-chloro-5-
isopropyl-3-methyl-1,3-thiazol-2(3H)-ylidene)-2-hydroxy-
benzenesulfonamide (YM-215389).8-10 However, identifying
such inhibitors by screening random libraries and optimizing
them by systematic chemical modifications are highly time and
resources consuming. Therefore, faster and more efficient
strategies that facilitate and shorten the discovery process would
be extremely beneficial.
Molecular modeling is one approach that could be used to
narrow down a library containing an extraordinarily high number
of random molecules into a smaller list of the potentially
effective inhibitors. Two major tools that have been used
intensively in this arena are virtual screening and de noVo drug
design, both of them based either on the known crystal structure
of the specific target enzyme or on known inhibitors against
the enzyme. Virtual screening is usually performed by docking
each member of a large available chemical database into the
enzyme’s active site. This leads, in most cases, to a selective
list enriched in active compounds. The top scored molecules
of such screens can be then easily obtained and tested for
* To whom correspondence should be addressed. A. Hizi, Tel: 972-
36409974. Fax: 972-36407432. Email: ahizy@post.tau.ac.il.; A. Nudelman,
Tel: 972-35318314. Fax: 972-35351250. Email: nudelman@mail.biu.ac.il.
‡ Tel Aviv University.
# Bar Ilan University.
§ This work was performed in partial fulfillment of the requirements for
a Ph.D. degree of A. Herschhorn, Sackler Faculty of Medicine, Tel Aviv
University, Israel.
| The Gregorio and Dora Shapira Chair for the Research of Malignancies.
a Abbreviations: RT, reverse transcriptase; HIV, human immunodefi-
ciency virus; RNase H, ribonuclease H; AIDS, acquired immunodeficiency
syndrome; NRTIs, nucleoside/nucleotide reverse transcriptase inhibitors;
NNRTIs, non-nucleoside reverse transcriptase inhibitors; PETT, pheneth-
ylthiazolylthiourea; DABO, dihydroalkoxybenzyloxopyrimidine; XTT, 2,3-
bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetra-
zolium hydroxide; NMR, nuclear magnetic resonance; CFF, consistent
forcefield; RMS, root mean square; PLP, piecewise linear potential; CI,
chemical ionization; DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic
acid; TBE, tris borate ethylenediamine tetraacetic acid; dTTP, deoxythy-
midine triphosphate.
10.1021/jm0613121 CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/26/2007

NoVel Broad-Range RT Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2371
Table 1. Ludi Scores of the Fragments with the Highest Affinity to the
NNRTI Binding Pocket of All the Crystal Structures of RT Tested
Table 2. Docking and Scoring of the Scaffold and Control Molecules
into the Various RT Structuresa
crystal
structure
scoring
efavirenz
ref_10087_NCI
functionb (positive control) scaffold (negative control)
Wild type Ligscore1
PLP2
3.56
97.31
3.53
101.85
4.16
3.97
98.32
3.85
89.93
4.23
2.4
64.3
2.6
57.6
2.7
T181C
Ligscore1
PLP2
K103N
Ligscore1
PLP2
96.69
3.02
97.66
4.28
71.8
2.5
L100I +
K103N
Ligscore1
PLP2
Ligscore1
PLP2
81.27
3.65
92.99
97.98
4.13
94.89
79.1
2.56
68.19
average
a The values reported are the scores of the best conformation for each
molecule. b The docked molecules were scored using Ligscore1 and PLP2
scoring functions in the ligandfit module of cerius 2. The positive value of
PLP2 is reported and in both functions the highest score represent the
strongest binding.
four structures were linked together to construct a final
compound. The resulting molecule was used as a scaffold for
the synthesis of various chemically related molecules that were
subsequently tested in Vitro for inhibiting the activities of HIV-1
RT.
Results
De NoWo Design of Non-nucleoside RT Inhibitors. To
construct a wide range of NNRTIs against both wild type and
drug-resistant HIV-1 RT variants, we used four structures of
the RT: wild type, mutant K103N, double mutant K103N +
L100I, and mutant T181C. The NNRTI binding site was defined
in all four structures, and each one was screened separately
against a library of 1000 small fragments using the Ludi module
in the Cerius 2 software.17 Ludi generates a map of interaction
sites within the NNRTI binding site based on favorable
orientations for H-bonds and hydrophobic contacts.18,19 The
module then fits different fragments from a library of small
molecules (5-30 atoms) onto these sites and scores each
fragment according to the bonds that it can make with the RT.
Screening against the four RT structures resulted in four separate
lists of fragments. In each list, every fragment was scored
according to its affinity to the specific NNRTI binding site in
the specific structure. The results were filtered to retain only
those fragments that appeared in all RT structures, and the scores
of these fragments were averaged over all structures. Nine
molecules were identified in the search, and their location within
the hydrophobic pocket of the NNRTI was verified (Table 1).
From this group, the phenyl and pyrrole rings were chosen and
were colocalized within the NNRTI binding site in the Ludi
module. Using the same module, we conducted a second search
to connect the fragments. This search suggested to link the two
fragments through a phenyl group and to add an amide group
to the pyrrole. In addition, the pyrrole was replaced by an
imidazole to simplify the synthesis of such molecules (scaffold,
see Table 2).
inhibitory activity against the enzyme.11 The de noVo design of
new molecules is done by first identifying the functional groups
that bind the active site of the target protein and then linking
them into the final inhibitor molecules. The desired molecules
are then synthesized and tested for their enzymatic inhibitory
effects.12
Since the discovery of NNRTIs, crystal structures of wild
type and drug-resistant variants of HIV-1 RT have been used
extensively in the design of novel NNRTIs. Mao et al. used a
flexible binding site based on several structures to design a few
PETT and DABO derivatives with improved profiles against
drug resistance mutants of HIV-1 RT.13 Moreover, Vinkers et
al. have recently designed de noVo several NNRTIs, using
advanced software that includes a synthesis route for each
generated molecule.14
Here, we present a novel approach for de noVo design of
NNRTIs that inhibit wild type as well as drug-resistant variants
of HIV-1 RT. We have used four different crystal structures of
RT, one wild type and three mutated enzymes, which are
resistant to several of the commonly used NNRTIs.15,16 On the
basis of these structures, we searched for small fragments of
synthetic molecules that could bind separately to each variant
of the RT enzyme. The fragments that interacted with each RT
structure were compared and only those that interacted with all

2372 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Scheme 1. Synthesis of the Different Molecules in the Study
Herschhorn et al.
To ensure that the novel scaffold molecule can bind the RT
in an energetically favorable mode, the molecule was energy
minimized and then docked into the NNRTI binding site of all
four structures, using the module ligand fit of Cerius 2.20 We
performed the same docking with two reference molecules.
Efavirenz, a clinically used NNRTI drug, which was originally
cocrystallized with RT, was used as a positive control and a
random molecule from the NCI database (ref_10087_NCI) was
used as negative control (Table 2). The top ranked structures
of the docking process were scored with the Ligscore1 and PLP2
scoring functions. These functions showed the best selectivity
with negative and positive training sets of molecules against
RT in a preliminary analysis (out of the nine scoring functions
in ligand fit). The scores from the docking of the scaffold
molecule against all structures were averaged and compared to
the positive and negative controls (Table 2). As judged from
the scoring results, the final molecule exhibited scores compa-
rable to those of efavirenz and fitted the RT structures much
better then the negative control molecule, indicating that the
new scaffold molecule was a reasonable candidate for further
evaluations.
We then synthesized and tested three groups of derivatives
of the scaffold molecule. Group I included the phenyl ring
attached to an amido substituted imidazole/thiazole (compounds
8a-10f, Scheme 1). In group II, the amido-substituted hetero-
cyclic ring was replaced with a 2-oxopropanamide fragment
(compounds 6a-g, Scheme 1), and in group III it was replaced
with a 2-oxopropanenitrile/2-oxobutanenitrile fragment (com-
pounds 5a-h, Scheme 1). These replacements were intended
to introduce functional groups that were more flexible than those
of the scaffold molecule due to the additional rotatable bonds
of the molecules. The resulting molecules would presumably
have better ability to accommodate themselves in the binding
pocket of either the wild type or the mutant RTs. In all three

NoVel Broad-Range RT Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2373
Table 3. The Effects of the Different Molecules Synthesized on RT
groups, the phenyl ring was further substituted with either
another phenyl group or halogen atoms. In addition, one member
of the third group was substituted with a phosphate group.
Enzymatic Activities and on the Viability of Jurkat Cellsa
DNA
c
polymerase,b
RNase H,
CC50
Chemical Synthesis. Compounds 1, 3a-d, 3f, and 5g are
commercially available and served as primary building blocks
for the synthesis. Compound 3e was prepared by a Suzuki
reaction between phenylboronic acid and 3a.
compd
5a
5b
5c
5d
5e
5f
5h
6a
6b
6d
6e
8b
8c
8d
8e
8g
9a
9b
9c
9d
9e
9g
10a
10c
10d
10e
10g
nevirapine
IC50 [µM]
IC50 [µM]
[µM]
.312
4.1 ( 0.5
.312
ND
28 ( 2
ND
98 ( 6
84 ( 5
20 ( 1
112 ( 2
.312
ND
ND
ND
.312
41 ( 9
ND
ND
ND
ND
.312
ND
.312
249 ( 45
.312
ND
ND
77 ( 13
ND
ND
.312
ND
251 ( 18
233 ( 19
247 ( 28
.312
.312
ND
ND
ND
.312
213 ( 3
ND
ND
ND
ND
.312
ND
.312
244 ( 8
.312
ND
ND
.312
ND
9.8 ( 2.4
33.4 ( 9.6
3.5 ( 0.8
64.3 ( 16.9
29.6 ( 5.7
.312
The synthesis of the various heterocyclic compounds started
with the conversion of the carboxylic acids 3a-f to acyl
chlorides 4a-f. Alkylation of the latter with cyanoacetic acid
in the presence of n-BuLi gave compounds 5a-f in about 60%
isolated yield. The phosphorylated analogue 5h was obtained
from 2-bromo-3′-hydroxyacetophenone (1) in a two-step syn-
thesis involving phosphorylation and replacement of bromide
by potassium cyanide. Hydration of the â-ketonitriles 5a-g in
the presence of concentrated H2SO4 gave the amides 6a-g. This
step was problematic for compound 5e, since under these
conditions the external electron rich phenyl ring was converted
into an arylsulfonic acid.21 To overcome this problem, the
synthetic sequence was changed, with the introduction of the
second phenyl ring following the construction of the amide
moiety. On the basis of this strategy, compound 6a was reacted
in a Suzuki reaction with phenylboronic acid to give 6e.
R-Bromination of amides 6a-g provided 7a-g, which were
used as intermediate building blocks for a variety of heterocyclic
rings. The construction of the imidazole moiety suffered from
extremely low yields, and numerous attempts were made to
improve it. Finally, the imidazoles 8a-g were synthesized by
heating of 7a-g in formamide to which a few drops of
concentrated H2SO4 had been added. The aminothiazole deriva-
tives 9a-g were prepared in good yields by reaction of 7a-g
with thiourea. Deamination of the latter gave the thiazoles 10a-
g.
.312
.312
79.8 ( 18.8
9.9 ( 1.4
.312
.312
300 ( 84
125.4 ( 14.2
54.4 ( 8.9
118.2 ( 19.9
44.1 ( 10.8
59.2 ( 7.6
80.7 ( 17.6
.312
87.5 ( 11.2
16.3 ( 4.6
.312
283.4 ( 28.6
1.7 ( 0.2
ND
ND
ND
ND
a IC50 ) concentration of inhibitor at which the specified RT activity
was reduced by 50% compared to the inhibitor-free control. b DNA
polymerase activity was assayed with poly(rA)n•oligo(dT)12-18 substrate,
as described under the Experimental Section. c CC50 ) concentration of
inhibitor at which the cell viability was reduced by 50% compared to the
inhibitor-free control.
RT Enzymatic Activity. A total of 27 synthesized molecules
were tested against the DNA polymerase activity of HIV-1 RT.
The IC50 values, compound concentrations inhibiting 50% of
the initial activity of RT, were calculated for each compound
from dose-response curves (Table 3). Fourteen compounds
inhibited the DNA polymerase activity with apparent IC50 values
below 100 µM, and four showed IC50 values below 10 µM
(Table 3). In group I, out of sixteen molecules, eight displayed
IC50 values below 100 µM. Compounds 10d and 8c were the
most effective ones in this group with IC50 values of about 16
and 9.9 µM, respectively. In compound 10d, the distance
between the two phenyl groups was increased by inserting a
carbonyl group between them, and in 8c, Br and Cl atoms were
added to the middle phenyl group. In both cases, the inhibitory
effect was significantly improved compared to analogous
molecules lacking these functional groups (10e and 8g). In group
II, only compound 6a showed a significant inhibitory effect,
suggesting a preferred interaction of the iodine in the context
of this structure with specific groups in the RT enzyme. In the
substances of group III, four out of the six molecules were
effective. In compound 5d, as in 10d, increasing the distance
between the phenyl groups by an insertion of a carbonyl group
improved the inhibition relative to molecule 5e. Addition of a
chloride atom in compound 5b resulted in a potent inhibitor,
whereas addition of an iodide atom at the same position
(compound 5a) abolished the activity against RT. Compound
5f, which possesses a 2-oxobutanitrile substituent is an analogue
of 5a, where the 2-oxopropanitrile found in 5a is separated from
the aromatic ring by an additional methylene group. Interest-
ingly, this extension of the molecule was beneficial and 5f
showed the most effective inhibition with an IC50 value of
approximately 3.5 µM. For these studies, nevirapine, a clinically
used RT inhibitor was used as reference, its IC50 value under
these conditions was 1.7 µM.
All thirteen compounds with apparent IC50 values below 85
µM for inhibition of the HIV-1 RT-associated DNA polymerase
activity were further tested for inhibition of the RNase H activity
of the enzyme. Out of the tested compounds, five did not inhibit
this activity even at concentrations as high as 312 µM, while
the other eight molecules showed different degrees of inhibition
(Table 3). Compound 6a inhibited only the DNA polymerase
activity and had no effect on the RNase H activity. The
inhibition of RT by compound 5d was more specific to the DNA
polymerase activity compared to the inhibition of the RNase H
activity, with IC50 values of about 9.8 µM and 98 µM,
respectively. In contrast, compounds 8c, 5b, and 5f showed
broader inhibition and were effective against both activities of
RT. Compound 5f displayed the lowest IC50 value of 20 µM
against the RNase H activity and was the most effective against
both activities (Table 3).
Two of the most effective RT inhibitors, compounds 8c and
5f, were further evaluated against the DNA polymerase activity
of two drug-resistant mutants of HIV-1 RT. T181C, a single
mutant RT that carries a cysteine residue instead of a tyrosine
at position 181 and is highly resistant to nevirapine;16 L100I +
K103N, a double mutant RT that carries an isoleucine instead
of a leucine at position 100 and an asparagine instead of a lysine
at position 103 and is resistant to efavirenz, another clinically
used drug.15 The single mutation, K103N, was not tested, since
this mutation is already included in the double mutant variant
that shows higher resistance to efavirenz than K103N alone.
Compound 8c inhibited the wild-type RT as well as the T181C

2374 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Herschhorn et al.
mutants compared to the wild type RT. Compound 5f was even
more effective than 8c, as it inhibited all three RT variants with
IC50 values below 10 µM and only an ∼2.7 fold difference in
inhibition of the mutant RT, relative to the wild type RT.
Compound 5f was substantially more effective than nevirapine,
which inhibited the T181C and the L100I+K103N RT mutants
only at IC50 values of 100 and 77 µM, respectively, i.e., the
mutants had a 45-59 fold increase in resistance to nevirapine
(Figure 2). All analyses of the measured data were well fitted
to the four-parameter logistic equation with high correlation
coefficients (higher than 0.98).
The mode of inhibition of RT-associated DNA polymerase
activity by 5f, the most efficient inhibitor, was further inves-
tigated in a DNA polymerase primer-extension assay. In this
assay, the RT is allowed to extend a 5′-end-labeled 15-mer
primer that was annealed to a φX174 single-stranded DNA. The
reaction products are then analyzed by urea-PAGE, and as was
shown by us previously,22 the length of the DNA products is
usually up to about 500nt in length, with several strong pausing
sites (Figure 3). The experiments were divided into two sets:
in the first one HIV-1 RT was initially preincubated with the
inhibitor followed by the addition of the labeled template-primer;
in the second, the RT was first preincubated with the template-
Figure 1. Outline of the design process of new inhibitors against HIV-1
RT.
and L100I-K103N mutants with IC50 values of about 9.9, 26,
and 20 µM, respectively (Figure 2). This means that 8c was
only about two times less effective in the inhibition of the
Figure 2. Inhibition of wild type and drug-resistant mutant RTs by compounds 8c and 5f. Wild type RT (left column), single mutant T181C RT
(middle column), and double mutant RT L100I-K103N (right column) were assayed for DNA polymerase activity in the present of various
concentrations of three compounds: 8c (top raw), 5f (middle raw), and nevirapine (bottom raw). The dose-response curves were fitted to a four-
parameter logistic equation, and the IC50 parameter as well as standard errors and correlation coefficients are reported.

NoVel Broad-Range RT Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2375
with the template-primer prior to the addition of compound 5f
(lane 5 compared to lane 3). On the other hand, the order of
adding the reaction components had little effect on the inhibition
obtained by nevirapine (lane 6 compared to lane 4).
Compounds 8c and 5f were also tested in a gel mobility shift
assay for their effects on the formation of RT-DNA complex.
In this assay, the DNA is 5′-end labeled with [32P]ATP and
incubated with RT. The amount of the complexed DNA-RT
(as well as the free DNA) that formed can be monitored after
separating the reaction products by electrophoresis on nonde-
naturating polyacrylamide gels. As shown in Figure 4, none of
the compounds, including the NNRTI inhibitor nevirapine,
prevented the binding of RT to the DNA. This conclusion is
drawn from the fact that in the case of the two novel molecules
the amount of the bound DNA was similar to that detected when
RT was incubated with the DNA without any inhibitor. In
contrast to compounds 8c and 5f, 1,4-benzenediol, 2-[[(1R,2R,5S,-
8aS)-1,2,3,5,6,7,8,8a-octahydro-1,2,5-trimethyl-5-[2-[(1R,2S,-
4aR)-1,2,3,4,4a,5,6,7-octahydro-1,2,5,5-tetramethyl-1-naphthalenyl]-
ethyl]-1-naphthalenyl]methyl], bis(hydrogen sulfate) (toxiusol),
a natural product that was shown by us to be a potent RT
inhibitor,23 prevented the binding of the enzyme to DNA and
was used as a positive control.
The thirteen most effective molecules (Table 3) were also
tested for their cytotoxic effects on Jurkat cells, using the XTT
assay that measures cell death (see Experimental Section). In
this assay, none of the tested molecules showed any substantial
effect on the viability of the cells. Seven of them were not toxic
at the tested concentrations, while the other six showed mod-
erate toxicity, with CC50 values in the range of 213-251 µM
(Table 3).
Figure 3. DNA primer extension by HIV-1 RT in the presence of
compound 5f and nevirapine. In all conducted reactions HIV-1 RT was
preincubated on ice for 30 min. In lanes 2-4 RT was preincubated
with an inhibitor followed by the addition of the template-primer
(T/P), and in lanes 5-6 RT was preincubated with T/P followed by
addition of the inhibitor. The reactions were initiated with the addition
of the relevant substrates and were then further incubated at 37 °C for
30 min. M: DNA molecular size marker. A nonphosphorylated
φX174am3 double-stranded DNA, Hinf I cut (from Promega) was 5′-
end labeled with [γ-32P] ATP. Lane 1: T/P only, lane 2: RT without
inhibitors, lane 3: RT in the presence of compound 5f, lane 4: RT in
the presence of nevirapine, lane 5: RT in the presence of compound
5f, after preincubation with the T/P for 5 min on ice, lane 6: RT in the
presence of nevirapine, after preincubation with the T/P for 5 min on
ice. All reactions included 2% DMSO.
Discussion
On the basis of the known various crystal structures of HIV-1
RT, we designed in silico a molecule that is capable of binding
to both wild type and drug-resistant mutant RTs. The molecule
was used as a scaffold for the synthesis of various compounds,
and 27 of them were tested for their ability to inhibit the DNA
polymerase activity of RT. More than 50% of the tested
compounds displayed a significant capacity to inhibit the RT
activity with apparent IC50 values below 100 µM, out of which
29% demonstrated IC50 values below 10 µM. This undoubtedly
shows that the collection of novel molecules was enriched for
their capacity to inhibit RT. Moreover, this hit rate of active
molecules is significantly higher than the proportion expected
from a high throughput screening of a random list of compounds.
primer followed by the addition of the inhibitor. The primer
elongation, obtained in the presence of either 5f or nevirapine,
was strongly impaired when RT was first incubated with the
inhibitors (lanes 3 and 4, respectively, compared to lane 2). This
inhibition was mostly abolished when the RT was preincubated
Figure 4. The effect of compounds 8c, 5f, and nevirapine on the formation of RT-DNA complex. The PAGE mobility shift assay was performed
as described in the Experimental Section. The lanes in the autoradiogram are of the following reactions: lane 1, control with no RT present; lane
2, binding in the absence of inhibitors; lanes 3-8, binding in the presence of compounds 8c, 5f, and nevirapine at the indicated concentrations.
Lane 9, positive control, of binding in the presence of toxiusol.

2376 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Herschhorn et al.
The parallel screen of small fragments against various
structures of RT successfully led to several effective inhibitors.
This novel method had several advantages. First, it enabled
filtering molecules that could interact with the wild type and
mutant variants. Second, it took into account subtle movements
of residues in the different structures that might represent a better
description of the binding site in the RT conformation. Third,
several cycles of docking into different structures was presum-
ably more reliable than only a single docking into a specific
RT structure. Therefore, it was expected to increase the
proportion of true binders and to decrease the proportion of false
positive binders.
the RT, since the catalytic domains for the DNA polymerase
and RNase H are located in different domains of RT (although
there are tight interactions between these two domains, and many
mutations affect both RT activities).28
Both activities of RT were inhibited most effectively by
compound 5f, which was only slightly cytotoxic to Jurkat cells
with a CC50 of 247 µM (Table 3). This compound showed an
improved profile against drug-resistant mutants compared to
nevirapine. Compound 5f inhibited the DNA polymerase activity
of wild type RT slightly less effectively than nevirapine (by
2-folds). More importantly, 5f inhibited more effectively than
nevirapine the T181C mutant and the L100I-K103N double
mutant RTs by 10- and 8-fold, respectively (Figure 2).
The computational approach presented in this study could
serve as an alternative or complementary tool to other fragment-
based methods, such as NMR or functional assay screenings at
high compound concentrations, both of which have gained
increasing popularity during recent years.24-27 While application
of the computational approach is purely theoretic, it can be easily
used to screen a huge amount of compounds, sampling a wide
range of the chemical space encompassed by all the possible
molecules. Yet, any potential compound has to be tested in Vitro
for confirming its inhibition or binding effects on the target
protein. On the other hand, screening fragments by NMR or
functional assays represent experimental techniques intended
to isolate specific fragments interacting with a target protein.
NMR has low false positive hits and high sensitivity and gives
some information on the binding mode (but no confirmation
for actual inhibitory activity), while only functional assays can
validate the inhibitory nature of the compounds.24 Nevertheless,
both techniques are limited by the number of the substances
that can be tested, and the solubility of the fragments that have
to be tested at high concentrations. Evidently, computational
methods can complement any of these experimental methods
by preselecting potential fragments for the experimental screen
or by aiding the optimization and linkage of the fragments
isolated.
Interestingly, the additional methylene in compound 5f
compared to 5a (by replacing the 2-oxopropanenitrile with
2-oxobutanitrile) had a drastic effect on its ability to inhibit RT.
While 5a did not inhibit RT at all, the additional methylene
rendered compound 5f to be a highly potent inhibitor against
RT. This might be due to steric hindrance of 5a inside the
hydrophobic pocket within the RT or to greater flexibility or
accessibility of 5f.
DNA-primer extension experiments showed that the prior
binding of the template-primer to RT before interaction of RT
with 5f mostly abrogates the inhibition of RT (see Figure 3,
lane 5 compared to lane 3). In addition, compound 5f did not
prevent the formation of DNA-RT complexes, as evident from
the gel shift assay (Figure 4). In all, it is likely that 5f did not
alter the binding of RT to DNA, but formation of the RT-
DNA complex interfered substantially with the interaction of
the inhibitor with RT. This pattern of inhibition could be
explained if the conformational changes of the RT, induced by
binding of the template-primer to RT, could obstruct inhibitor
binding. Alternatively, binding of the DNA to RT might partially
obscure free access of the inhibitor to its binding site. In light
of this behavior as well as the effective inhibition of the drug-
resistant RT variants, it seems that 5f inhibits RT by a different
mechanism from that of nevirapine.
Docking of compounds 5f and 8c into the HIV-1 RT structure
(Figure 5) showed that they both fit well into the NNRTI binding
pocket. In both molecules the phenyl group is positioned in the
vicinity of Trp 229, Tyr 188, and Tyr 181 residues of RT
(located in the p66 subunit), while other functional groups of
the compounds are extended to interact with other residues in
the hydrophobic pocket of RT. According to this model, 8c
formed H-bonds with both Lys 101 and His 235 residues of
the p66 subunit of RT (Figure 5a). One bond was formed
between the backbone carbonyl of Lys 101 and the hydrogen
of the NH group in the imidazole ring, and a second bond was
formed between the backbone carbonyl of His 235 and the
hydrogen of the amide group of 8c. This model is supported
by the experimentally observed interaction of NNRTI inhibitors3
with Lys 101. Compound 5f formed one H-bond between the
ketone group of the compound and the hydroxyl group of the
side chain of Tyr 318, located in the p66 subunit of RT (Figure
5b). Since 5f formed fewer H-bonds with RT than 8c but
inhibited the enzyme more efficiently, it may have a more
favorite hydrophobic interaction with RT than 8c.
Incorporation of halogens in the inhibitor has been shown in
various examples to notably improve the inhibition of RT.
Efavirenz, which is the most potent inhibitor currently used
nowadays in clinical treatment, has a chlorine and a trifluo-
romethyl group. Masuda et al. demonstrated that the effective-
ness of several RT inhibitors was significantly improved upon
substitution by halogens and NO2.9 Therefore, it is not surprising
that addition of halogens in compounds 8c and 6a led to a
dramatic increase in the efficacy of the inhibition. This effect
is probably due to the overall combination of the constituents
and the molecular orientation of the functional groups in the
molecule rather than to the presence of the halogens since in
the iodine-containing 10a no inhibitory effect was detected.
Interestingly, the same improved inhibitory effect was observed
after the addition of a chlorine in compound 5b and a benzoyl
group in compound 5d; both molecules contain 2-oxopropaneni-
trile groups and showed a marked inhibition of RT compared
to the other molecules with the same 2-oxopropanitrile group.
The most efficient inhibitors were further tested for their
capacity to inhibit the RNase H activity of RT, and all of them
showed higher IC50 values than the values measured for
inhibition of the DNA polymerase activity. This difference was
mostly prominent with molecules, such as 6a and 5d. Other
compounds, such as 8c, 5b, and 5f, inhibited both RT-associated
activities with about 4- to 7-fold reduced inhibition efficacy of
the RNase H activity compared to the DNA polymerase activity
(Table 3). These results suggest that these broad range inhibitors
might have various modes of binding leading to inhibition of
Of the molecules studied, 8c, 5d, 5b, and 5f were the most
effective against HIV-1 RT enzymatic activities, and all four
of them have drug-like properties (Table 4). Their molecular
weight ranges from 179.6 to 300.5, and they have between two
and four H-bond acceptor atoms and between zero and three
H-bond donor atoms. The LogP values of the molecules range
from 1.6 to 2.4, and they have no more than four rotatable bonds.

NoVel Broad-Range RT Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2377
Figure 5. Docking compounds 8c and 5f into the NNRTI binding site of the wild type RT. 8c (A) and 5f (B) were docked into the structure of
RT found in PDB entry 1fkp using surflex software. The top scored conformation of each molecule is shown. Amino acids in the pocket are
specified in three-letter codes and different colors, Glu 138 is part of the p51 subunit, and all the other amino acids are part of the p66 subunit. The
two compounds are depicted in CPK (Corey, Pauling, and Kultin) colors and are displayed as a stick model.
Table 4. Molecular Properties of the Most Efficient Inhibitors of HIV-1
RT
and it suggested linking the two groups through another phenyl
group. The resulting molecule was subjected to another link mode
(single links) search, in which an amide group was added to the
pyrrole ring.
mol
H-bond
H-bond
rotatable
bonds
compd weight donors acceptors miLogPa TPSAb
Docking of molecules to the NNRTI binding site of all RT
structures was performed with the module ligand fit within Cerius
2 module.20 The docking site was generated with the site search
function, based on the bond inhibitor efavirenz (before its deletion
from the structure), with the opening size parameter set to 5 Å and
the resolution parameter to 0.5 Å. The molecule was energy
minimized using the CFF1.02 field force and then docked into the
NNRTI binding site using the following parameters: flexible fit,
number of trials ) 150000, maximal number of conformations )
4, search step for torsions w/polar hydrogen ) 5 degrees, saved
only diverse conformers (rms thresholds ) 1.5 Å), energy
minimized ligands in the protein and PLPv.1 energy grid. The final
docking poses were scored with Ligscoere1 and PLP2 scoring
functions (negative values of PLP2 function were replaced with
positive ones for consistency).
5b
5d
5f
179.6
249.3
285.1
300.5
0
0
0
3
2
3
2
4
1.572
2.349
1.802
2.072
40.9
57.9
40.9
71.8
2
4
3
2
8c
a Molinspiration calculated LogP. b Topological polar surface area [Å2].
In addition, the polar surface area of all of them is less than 60
Å2 which is a good indication for bioavailability.29 None of the
four molecules violates any of the Lipinski rule of 5,30 and
therefore they represent attractive compounds for further
research. These substances are currently under further develop-
ment and will be tested in the future for their capacity to inhibit
HIV in a cell-based assay.
Docking of compounds 8c and 5f into RT was performed with
the Surflex software.32 The original inhibitor in structure 1fkp was
used to generate a protomol, an idealized binding site ligand, using
the defaults setting of the program. For each compound, the charge
was calculated, and the structure was energy minimized and then
docked into 1fkp structure using the generated protomol. Surflex
docks the molecules by fragmentation each one and fitting each
conformation of each fragment to the protomol to yield poses that
maximize molecular similarity to the protomol. The docked
conformation with the highest score, for each molecule, was selected
and analyzed visually with Discovery studio visualizer 1.6 (Accelrys
software inc.).
Experimental Section
Molecular Modeling. The RT structures 1fkp, 1fko, and 1jkh
were downloaded from the Research Collaboratory for Structural
Bioinformatics site (http://www.rcsb.org/pdb) as a complex with
efavirenz and were saved as pdb files. 1fkp is the structure of a
wild type RT with a resolution of 2.5 Å, 1fko carries the mutation
K103N and has a resolution of 2.9 Å and 1jkh carries the mutation
T181C and has a resolution of 2.5 Å. All structures were modified
with the Cerius 2 software as follows: first the inhibitor and water
molecules were removed. The structures were then inspected for
the correct double bonds, hydrogen atoms were added to each
structure, and only these atoms were energy minimized with the
CFF 1.02 force field. The double mutant K103N + L100I is not
found in the protein database, and its structure was generated by
replacing leucine 100 in the 1fko structure with isoleucine and then
minimizing the energy of the amino acid, using the CFF 1.02 force
field.
A scaffold inhibitor molecule was designed by using the Ludi
module of the Cerius 2 software (Accelrys inc. http://www.accel-
rys.com). The center of the NNRTI binding site in each structure
was defined based on the amino acid residues that form the binding
site, and a de noVo search with a radius of 10 Å was initiated with
a library of 1000 fragments. The bond rotation parameter was set
to one at a time to allow flexibility to the search, and the search
type parameter was set to best. Fragments that interacted with the
binding site were scored with the energy_estimate_3 scoring
function31 (that includes aromatic-aromatic interactions in addition
to the other terms) and were averaged over all structures. From the
top scored fragments the phenyl and trimethylpyrrole fragments
were repositioned in the binding site of all structures, and a second
search in a link mode (double links) was initiated. This search looks
for fragments that could link the phenyl to the trimethylpyrrole,
The molecular properties of the best inhibitors were calculated
with the molinspiration on-line calculator (http://www.molinspira-
tion.com/cgi-bin/properties).
1
Chemistry. General. H and 13C NMR spectra were obtained
on 200, 300, and 600 MHz Bruker AC-200, AM-300, and DMX-
600 spectrometers. Chemical shifts are expressed in ppm downfield
from Me4Si used as internal standard. The values are given in δ
scale. Mass spectra were obtained on a Varian Mat 731 spectrom-
eter. HRMS were obtained on a VG AutoSpec E spectrometer. The
reaction progress was monitored by TLC on silica gel (Merck, Art.
5554) or alumina. (Riedel-de Haen, Art. 37349). Flash chroma-
tography was carried out on silica gel (Merck, Art. 9385).
Commercially available compounds were used without further
purification.
3-(2-Bromoacetyl)phenyl Diethyl Phosphate (2).33 Diethyl
chlorophosphate (1.39 mmol, 0.2 mL) and triethylamine (1.16
mmol, 0.16 mL) were added in sequence to a stirred solution of 1
(1.16 mmol 0.25 g) in anhydrous CH2Cl2 (25 mL). The obtained
solution was stirred at room temperature for 3 h followed by
addition of distilled water. The organic phase was separated, washed
with brine, and dried over MgSO4. The filtrate was evaporated to

2378 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Herschhorn et al.
give brown colored oil. Upon addition of ether, a brown solid was
obtained and removed. The yellow ethereal solution was evaporated,
and the residue was purified by column chromatography (CH2Cl2/
1.21 Hz, H-C4), 7.7-7.48 (m, 6H, Ph, H-C5). 13C NMR (300 MHz,
CDCl3) δ 168.44 (CO), 142.25 (Ph), 139.19 (C3), 133.93 (C4),
133.82 (C1), 130.18 (C2), 129.86 (C6), 129.49 (C5), 129.147 (Ph),
128.31 (Ph), 127.18 (Ph).
1
EtOAc ) 20:1) to provide 2 as a colorless oil in 43% yield. H
2-(3-Iodophenyl)acetyl Chloride (4f).41 Compound 3f (1 mmol)
and oxalyl chloride (2 mmol) were dissolved in anhydrous CH2Cl2
to which 1 drop of DMF was added. The final solution was stirred
at room temperature for 2 h and then concentrated under vacuum
to give 4f as a yellow solid which was used without further
NMR (300 MHz, acetone-d6) δ 7.92-7.84 (m, 2H, H-C4, H-C6),
7.62 (t, 1H, J ) 7.2 Hz, H-C5), 7.56-7.53 (m, 1H, H-C2), 5.05 (s,
2H, CH2Br), 4.23 (AB sys of qd, 1H, J ) 10; 8; 7 Hz, CH2Me),
1.33 (td, 6H, J ) 7.2; 1.2 Hz, CH2CH3). 13C NMR (300 MHz,
acetone-d6, where applicable Jcp, in Hz, are given in parentheses)
δ 191.1 (CO), 152.15 (C3, d, J ) 6 Hz), 137.05 (C1), 131.2 (C5),
126.1 (C4, d, J ) 4.5 Hz), 125.9 (C6), 120.4 (C2, d, J ) 4.5 Hz),
65.2 (CH2Me, d, J ) 6 Hz), 47.5 (CH2Br), 16.4 (CH2CH3, d, J )
6.5 Hz). 31P NMR (81.01 MHz, acetone-d6) δ -5.85 (broad quint,
J ) 8 Hz). MS (CI+) m/z: 257.057 ([M - CH2Br], 100), 352.999
(MH+, 30). HRMS: calcd for C12H17O5BrP (MH+, CI+) 352.9977
found 352.9986.
1
purification. H NMR (300 MHz, CDCl3) δ 7.67 (d, 1H, J ) 7.7
Hz, H-C4), 7.63 (s, 1H, H-C2), 7.23 (d, 1H, J ) 7.7 Hz, H-C6),
7.09 (t, 1H, J ) 7.7 Hz, H-C5), 4.08 (s, 2H, CH2CO). 13C NMR
(300 MHz, CDCl3) δ 171.61 (CO), 138.4, 137.3 (C2 and C4),
133.56 (C1), 130.8, 129.0 (C5 and C6), 94.95 (C1), 52.32 (CH2-
CO).
General Procedure for the Synthesis of Cyanoacetonitriles
5a-f.42 To a cooled (-78 °C) solution of cyanoacetic acid (15
mmol) in anhydrous THF (40 mL) was added n-BuLi (2.5 M, 30
mmol, 12 mL) dropwise, and the resulting mixture was stirred for
15 min followed by dropwise addition of an aroyl chloride (7.5
mmol). The mixture was stirred at -78 °C for 1 h and then for an
additional 1 h at room temperature, poured into ice-water, and
washed with ether, and the organic phase was removed. The
aqueous phase was acidified with 1 N HCl and washed twice with
ether, the combined organic phase was washed with saturated
NaHCO3 and brine, dried with MgSO4, filtered, and evaporated to
provide the desired compound.
3-Phenyl-benzoic Acid (3e).34 To a solution of NaOH (0.048
mol), 3-iodobenzoic acid 3a (0.024 mol), and phenylboronic acid
(0.026 mol) in H2O (100 mL) was added Pd(OAc)2 (5% mol), and
the colorless mixture was stirred at room temperature for 2 h, while
at the end it turned to a black colored solution. The solution was
acidified with 1N HCl and extracted with EtOAc. The organic phase
was washed with brine, dried over MgSO4, and filtered through a
celite bed. The colorless filtrate was evaporated to give 3e as a
white solid in quantitative yield; mp 155-157 °C [lit.34 112-114
1
°C]. H NMR (300 MHz, DMSO-d6) δ 8.2-8.15 (t, 1H, J ) 1.9
Hz, H-C2), 7.96-7.9 (m, 2H, H-C4, H-C6), 7.71-7.68 (m, 2H,
Ph), 7.62-7.57 (t, 1H, J ) 7.8 Hz, H-C5), 7.51-7.46 (m, 2H,
Ph), 7.42-7.37 (m, 1H, Ph). 13C NMR (300 MHz, DMSO-d6) δ
167.66 (CO), 142.18 (Ph), 140.82 (C3), 132.19 (C4), 130.0 (C2),
129.87 (C6), 129.68 (C1), 129.31 (C5), 128.77 (Ph), 128.65 (Ph),
127.79 (Ph). MS (CI+) m/z: 198.064 (M+, 100). HRMS: calcd
for C13H10O2 (M+, CI+) 198.0681 found 198.0637. Anal. calcd
for (C13H10O2‚0.3H2O): C 76.68, H 5.25; Found: C 76.99, H 5.02.
General Procedure for the Synthesis of Aroyl Chlorides.35
The known aroyl halides 4a-e were prepared by treatment of the
corresponding acids with an excess of SOCl2 at reflux over night.
The excess SOCl2 was removed, and the residual aroyl halides,
obtained in quantitative yield, were isolated as oils or crystals. The
products were used without further purification.
3-(3-Iodophenyl)-3-oxopropanenitrile (5a).43 Compound 5a
was obtained as a yellow solid from 4a in 60% yield; mp 105-
106 °C. 1H NMR (300 MHz, CDCl3) δ 8.24-8.23 (t, 1H, J ) 1.5
Hz, H-C2), 8-7.96 (ddd, 1H, J ) 7.8; 1.8; 1.2 Hz, H-C6), 7.88-
7.85 (ddd, 1H, J ) 7.8; 1.8; 1.2 Hz, H-C4), 7.29-7.24 (t, 1H, J )
7.8 Hz, H-C5), 4.06 (s, 2H, CH2). 13C NMR (300 MHz, CDCl3) δ
185.8 (CO), 144.44 (C4), 137.27 (C2), 135.82 (C1), 130.72 (C6),
127.49 (C5), 113.27 (CN), 94.73 (C3), 29.37 (CH2). MS (CI+)
m/z: 230.93 ([M - CH2CN], 100), 270.947 (M+, 30). HRMS: calc.
for C9H6NOI (M+, CI+) 270.9494 found 270.9472. Anal. calcd
for (C9H6INO): C 39.88, N 5.17, H 2.23; Found: C 39.75, N 5.21,
H 2.19.
3-(3-Chlorophenyl)-3-oxopropanenitrile (5b).44 Compound 5b
was obtained as a white solid from 4b in 68% yield; mp 73 °C. 1H
NMR (300 MHz, CDCl3) δ 7.9-7.88 (td, 1H, J ) 2.1; 0.3 Hz,
H-C2), 7.81-7.77 (ddd, 1H, J ) 7.8; 1.5; 0.9 Hz, H-C6), 7.65-
7.61 (ddd, 1H, J ) 7.8; 2.1; 0.9 Hz, H-C4), 7.5-7.43 (td, 1H, J )
7.8; 0.6 Hz, H-C5), 4.09 (s, 2H, CH2). 13C NMR (600 MHz, CDCl3)
δ 186.06 (CO), 135.66 (C1), 135.59 (C3), 134.67 (C4), 130.47 (C2),
128.46 (C6), 126.5 (C5), 113.32 (CN), 29.54 (CH2). MS (CI+)
m/z: 138.98 ([M - CH2CN], 100), 180.02 (MH+, 10). HRMS:
calc. for C9H7NOCl (M+, CI+) 180.0216 found 180.0199. Anal.
calcd for (C9H6ClNO‚0.1H2O): C 59.59, N 7.72, H 3.44; Found:
C 59.45, N 7.93, H 3.38.
3-Iodobenzoyl Chloride (4a).36 Compound 4a was obtained as
1
a colorless oil from 3-iodobenzoic acid 3a. H NMR (300 MHz,
CDCl3) δ 8.46-8.45 (td, 1H, J ) 1.8; 0.6 Hz, H-C2), 8.13-8.1
(ddd, 1H, J ) 7.8; 1.8; 1.79 Hz, H-C6), 8.05-8.02 (ddd, 1H, J )
7.8; 1.8; 1.79 Hz, H-C4), 7.32-7.27 (td, 1H, J ) 7.8; 0.6 Hz,
H-C5). 13C NMR (300 MHz, CDCl3) δ 167.03 (CO), 144.09 (C4),
139.84 (C2), 134.92 (C1), 130.55 (C6), 130.51 (C5), 94.2 (C3).
3-Chlorobenzoyl Chloride (4b).37 Compound 4b was obtained
as a colorless oil from compound 3b. 1H NMR (200 MHz, acetone-
d6) δ 8.08-8.02 (m, 2H, H-C2, H-C6), 7.84-7.78 (ddd, 1H, J )
8.2; 1.8; 1.3 Hz, H-C4), 7.69-7.6 (td, 1H, J ) 8.2; 0.72 Hz, H-C5).
13C NMR (200 MHz, acetone-d6) δ 167.5 (CO), 136.36 (C4), 135.8
(C1, C3), 131.82 (C2), 131.32 (C5), 130.47 (C6).
3-(3-Bromo-4-chlorophenyl)-3-oxopropanenitrile (5c). Com-
pound 5c was obtained as a white solid from 4c in 58% yield; mp
3-Bromo-4-chlorobenzoyl Chloride (4c).38 Compound 4c was
obtained as a white solid from compound 3c. 1H NMR (200 MHz,
acetone-d6) δ 8.34-8.33 (d, 1H, J ) 2.18 Hz, H-C2), 8.13-8 (dd,
1H, J ) 8.5; 2.18 Hz, H-C6), 7.84-7.8 (d, 1H, J ) 8.5 Hz, H-C5).
13C NMR (300 MHz, acetone-d6) δ 166.65 (CO), 142.7 (C1), 136.57
(C2), 133.81 (C4), 132.15 (C6), 132.12 (C5), 123.63 (C3).
3-Benzoyl-benzoyl Chloride (4d).39 Compound 4d was obtained
as a white solid from compound 3d. 1H NMR (300 MHz, acetone-
d6) δ 8.47-8.45 (m, 1H, H-C2), 8.41-8.36 (m, 1H, H-C6), 8.2-
8.16 (m, 1H, H-C4), 7.87-7.8 (m, 3H, Ph, H-C5), 7.71-7.68 (m,
1H, Ph), 7.61-7.56 (m, 2H, Ph). 13C NMR (300 MHz, acetone-
d6) δ 195 (CO), 167 (COCl), 139 (C3), 137 (Ph), 136.39 (C4),
134.42 (C6), 133.2 (C1), 133.08 (Ph), 132.01 (C2), 129.86 (Ph),
129.75 (C5), 128.67 (Ph).
1
95-98 °C. H NMR (300 MHz, CDCl3) δ 8.4-8.13 (d, 1H, J )
2.1 Hz, H-C2), 7.79-7.76 (dd, 1H, J ) 8.4; 2.1 Hz, H-C6), 7.6-
7.57 (d, 1H, J ) 8.4 Hz, H-C5), 4.1 (s, 2H, CH2). 13C NMR (300
MHz, CDCl3) δ 184.97 (CO), 141.68 (C1), 133.66 (C4, C2), 131.1
(C6), 127.99 (C5), 123.84 (C3), 113.02 (CN), 29.41 (CH2).
HRMS: calcd for C9H5NOBrCl (M+, CI+) 258.9214 found
258.9217. Anal. calcd for (C9H5BrClNO‚0.2C4H10O): C 43.06, N
5.12, H 2.58; Found: C 43.24, N 5.39, H 2.17.
3-Benzoyl-3-oxopropanenitrile (5d). Compound 5d was ob-
tained as a yellow solid in 54% yield from 4d; mp 125-128 °C.
1H NMR (300 MHz, acetone-d6) δ 8.37-8.36 (td, 1H, J ) 1.8;
0.3 Hz, H-C2), 8.31-8.27 (ddd, 1H, J ) 7.8; 1.8; 1.2 Hz, H-C6),
8.09-8.06 (ddd, 1H, J ) 7.8; 1.8; 1.2 Hz, H-C4), 7.84-7.67 (m,
4H, Ph, H-C5), 7.61-7.55 (m, 2H, Ph), 4.69 (s, 2H, CH2). 13C
NMR (300 MHz, acetone-d6) δ 194.72 (CO), 188.47 (COCH2CN),
138.32 (C3), 136.93 (Ph), 134.77 (C4), 133.84 (C1), 132.94 (C6),
131.82 (C2), 129.84 (Ph), 129.26 (Ph), 128.26 (C5), 114.51 (CN),
29.49 (CH2). MS (CI+) m/z: 209.064 ([M - CH2CN]+, 100),
3-Phenylbenzoyl Chloride (4e).40 Compound 4e was obtained
as colorless oil from compound 3e. 1H NMR (300 MHz, CDCl3) δ
8.4-8.38 (t, 1H, J ) 1.62 Hz, H-C2), 8.19-8.14 (ddd, 1H, J )
7.74; 1.62; 1.21 Hz, H-C6), 7.97-7.93 (ddd, 1H, J ) 7.74; 1.62;

NoVel Broad-Range RT Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2379
249.072 (M+, 40). HRMS: calcd for C16H11NO2 (M+, CI+)
249.0790 found 249.0768.
7.69 (dt, 1H, J ) 6.9; 2.1 Hz, H-C6(enol)), 7.67-7.63 (ddd, 1H,
J ) 8.1; 2.1; 1.2 Hz, H-C4(ket)), 7.58-7.53 (t, 1H, J ) 7.8 Hz,
H-C5(ket)), 7.49-7.44 (m, 2H, H-C4(enol), H-C5(enol)), 7.12 (bs,
1H, NH(ket/enol)), 6.68-6.52 (bs, 1H, NH(ket/enol)), 5.9 (s, 1H,
CH(enol)), 3.96 (s, 2H, CH2(ket)). 13C NMR (600 MHz, acetone-
d6) δ 194.12 (CO(ket)), 175.47 (CON(ket)), 169.02 (CON(enol)),
139.52 (C1(ket)), 137.55 (C1(enol)), 135.12 (C3(ket)), 134.99 (C3-
(enol)), 133.77 (C4(ket)), 131.26 (C2(ket)), 131.18 (C4(enol)),
131.13 (C2(enol)), 129.1 (C6(ket)), 127.94 (C5(ket)), 126.2 (C6-
(enol)), 124.78 (C5(enol)), 89.73 (CH(enol)), 47.42 (CH2(ket)). MS
(CI+) m/z: 197.027; 198.03 (M+, 100; 85). HRMS: calcd for C9H8-
NO2Cl (M+, CI+) 197.0244 found 197.0266.
3-Phenyl-3-oxopropanenitrile (5e). Compound 5e was obtained
1
as a white solid in 15% yield from 4e; mp 85-87 °C. H NMR
(300 MHz, CDCl3) δ 8.13-8.12 (t, 1H, J ) 1.8 Hz, H-C2), 7.88-
7.83 (dd, 2H, J ) 7.9; 1.8 Hz, H-C6, H-C4), 7.6-7.57 (m, 2H,
Ph), 7.51-7.41 (m, 4H, Ph, H-C5), 4.1 (s, 2H, CH2). 13C NMR
(300 MHz, CDCl3) δ 187.25 (CO), 142.58 (Ph), 133.48 (C4),
132.45 (C3), 129.93 (C2), 129.21 (C6), 129.06 (C5), 128.35 (Ph),
127.97 (C1), 127.3 (Ph), 127.23 (Ph), 113.89 (CN), 29.65 (CH2).
MS (CI+) m/z: 181.066 ([M - CH2CN]+, 100), 221.086 (M+,
55). HRMS: calcd for C15H11NO (M+, CI+) 221.0841 found
221.0857.
3-(3-Bromo-4-chlorophenyl)-3-oxopropanamide (6c). Com-
pound 6c was obtained as a mixture of ketone and enol forms (1:
1.7) as a white solid in 78% yield from compound 5c; mp 90-92
4-(3-Iodophenyl)-3-oxobutanenitrile (5f). Compound 5f was
obtained as a cream colored solid in as a 78% yield from 4e; mp
96-98 °C. 1H NMR (300 MHz, acetone-d6) δ 7.65 (m, 2H, H-C2,
H-C4), 7.28 (d, 1H, J ) 7.8 Hz, H-C6), 7.14 (t, 1H, J ) 7.8 Hz,
H-C5), 4.02, 3.98 (s, s, 4H, Ph-CH2, CH2CN). 13C NMR (300 MHz,
acetone-d6) δ 196.8 (CO), 139.6 (C2), 137.1 (C1), 136.9 (C3),
130.27 (C6 and C5), 115.2 (CN), 48.0 (Ph-CH2), 29.6 (CH2CN).
MS (CI+) m/z: 216.937 ([M - COCH2CN]+, 65), 284.966 (M+,
25). HRMS: calcd for C10H8NOI (M+, CI+) 284.9651 found
284.9659.
1
°C. H NMR (300 MHz, acetone-d6) δ 8.32 (m, 1H, H-C2(ket))
8.05 (d, 1H, J ) 1.8 Hz, H-C2(enol)), 8.03-8 (m, 1H, H-C6 (ket)),
7.8-7.7 (m, 2H, H-C5(ket), H-C6(enol)), 7.65-7.63 (d, 1H, J )
8.4 Hz, H-C5(enol)), 77.11 (bs, 1H, NH(ket/enol)), 6.67 (bs, 1H,
NH(ket/enol)), 5.9 (s, 1H, CH(enol)), 3.96 (s, 2H, CH2(ket)). 13C
NMR (300 MHz, acetone-d6) δ 193.27 (CO(ket)), 175.26 (CON-
(ket)), 168.64 (CON(enol)), 137.78 (C1(ket)), 136.62 (C1(enol)),
135.91 (C4(ket)), 134.64 (C4(enol), C2(ket)), 131.59 (C5(ket)),
131.47 (C2(enol), C5(enol)), 129.82 (C6(ket)), 126.78 (C6(enol)),
123.04 (C3(ket)), 122.92 (C3(enol)), 90.01 (CH(enol)), 47.33 (CH2-
(ket)). MS (CI+) m/z: 276.934 (M+, 100). HRMS: calcd for C9H7-
NO2ClBr (M+, CI+) 276.9328 found 276.9343.
3-(2-Cyanoacetyl)phenyl Diethyl Phosphate (5h).45 A solution
of KCN (54 mg, 0.83 mmol) in distilled water (a few drops) was
added in one portion to a solution of 2 (117 mg, 0.33 mmol) in
95% EtOH (4 mL). The mixture became yellow and was stirred at
room temperature for 5 h. The reaction course was followed by
TLC, and an additional amount of KCN was added if needed. To
the mixture were then added CH2Cl2 and water and acidified with
glacial acetic acid (pH ) 5-6). The organic phase was separated,
washed with brine dried over MgSO4, and evaporated. Final
purification by column chromatography (eluent CH2Cl2/EtOAc )
10:1) gave 5h as a colorless oil in 40% yield. 1H NMR (300 MHz,
acetone-d6) δ 7.86 (m, 1H, H-C6), 7.83 (m, 1H, H-C2), 7.62 (t,
1H, J ) 8.5 Hz, H-C5), 7.59 (m, 1H, H-C4), 4.62 (s, 2H, CH2Br),
4.23 (AB sys of qd, 1H, J ) 10; 8; 7 Hz, CH2Me), 1.32 (td, 6H,
J ) 7; 1 Hz, CH2CH3). 13C NMR (300 MHz, acetone-d6, where
applicable Jcp, in Hz, are given in parentheses) δ 188.9 (CO), 152.3
(C3), 137.5 (C1), 131.3 (C5), 126.6 (C4, d, J ) 4.5 Hz), 125.9
(C6), 120.4 (C2, d, J ) 5 Hz), 115.33 (CN), 65.3 (CH2Me, d, J )
6 Hz), 30.3 (CH2Br), 16.3 (CH2CH3, d, J ) 6.5 Hz). 31P NMR
(81.01 MHz, acetone-d6) δ -5.85 (br quint, J ) 8 Hz). MS (CI+)
m/z: 257 ([M - CH2CN]+, 41), 297 (M+, 8), 298 (MH+, 11).
HRMS: calcd for C13H17NO5P (MH+, CI+) 298.0844 found
298.0869.
General Procedure for the Synthesis of â-Ketoamides 6a-
g.46 A cyanoacetonitrile 5a-g (1 mmol) was dissolved in concen-
trated H2SO4 (5 mL) and the solution was stirred at room
temperature for 3-5 h and was then poured into ice water, basified
with NH4OH and extracted with EtOAc. The organic phase was
washed with brine, dried with MgSO4, filtered and evaporated to
provide the desired amide. Some of the compounds were obtained
only in a keto form and others as enol-keto mixtures. When the
stirring period was reduced most of the products were obtained in
the keto form.
3-(3-Benzoylphenyl)-3-oxopropanamide (6d). Compound 6d
was obtained as a mixture of ketone and enol forms (2:1) as a white
solid in 75% yield from compound 5d. 1H NMR (300 MHz, CDCl3)
δ 8.37 (m, 1H, H-C2(ket)), 8.23-8.18 (d, 1H, J ) 7.5 Hz, H-C4-
(ket)), 8.15 (m, 1H, H-C2(enol)), 8.05-8.03 (d, 1H, J ) 7.5 Hz,
H-C6(ket)), 8-7.98 (d, 1H, J ) 7.5 Hz, H-C4(enol)), 7.86-7.83
(d, 1H, J ) 7.5 Hz, H-C6(enol)), 7.8-7.77 (m, 4H, Ph(enol/ket)),
7.66-7.59 (m, 4H, H-C5(enol/ket), Ph(enol/ket), 7.54-7.48 (m,
4H, Ph(enol/ket)), 7.05 (bs, 1H, NH(ket)), 5.85 (bs, 1H, NH(ket)),
5.6 (s, 1H, CH(enol)), 5.51 (bs, 2H, NH(enol)), 4 (s, 2H, CH2-
(ket)). 13C NMR (300 MHz, CDCl3) δ 195.37 (CO(ket/enol)),
194.65 (CO(ket)), 171 (CON(ket)), 167.74 (CON(enol)), 138.46
(C3(ket)), 136.84 (C3(enol)), 136.24 (Ph(ket)), 135.58 (Ph(enol)),
134.97 (C4(ket)), 134.48 (C1(ket)), 134.16 (C1(enol)), 133.12 (C4-
(enol)), 132.96 (C6(ket)), 132.72 (C6(enol)), 132.16 (C2(enol)),
131.96 (C2(ket)), 131.75 (C5(enol)), 129.99 (Ph(ket/enol)), 129.61
(C5(ket)), 129.36 (Ph(enol)), 129.33 (C-OH(enol)), 129.07 (Ph-
(ket)), 128.53 (Ph(ket/enol)), 87 (CH(enol)), 45.24 (CH2(ket)). MS
(ES+) m/z: 268 (MH+, 60), 290 (MNa+, 50). HRMS: calcd for
C16H13NO3 (M+, CI+) 267.0895 found 267.0900.
3-Oxo-3-(3-phenylphenyl)propanamide (6e).48 A mixture of
amide 6a (0.34 mmol), phenylboronic acid (0.38 mmol), 2 M
aqueous solution of Na2CO3 (0.13 g), and Pd(OAc)2 (3.88 mg) in
EtOH 95% (10 mL) was refluxed over night and then cooled and
filtered through a celite bed. The filtrate was evaporated, and the
obtained crude mixture was extracted with EtOAc and NaHCO3.
The organic phase was washed with brine, dried over MgSO4 and
evaporated to dryness to give 6e in quantitative yield; mp 95 °C.
1H NMR (600 MHz, CDCl3) δ 8.18 (s, 1H, H-C2), 7.93-7.92 (d,
1H, J ) 7.8 Hz, H-C6), 7.8-7.79 (d, 1H, J ) 7.8 Hz, H-C4), 7.58-
7.56 (m, 2H, Ph), 7.46-7.42 (m, 3H, Ph), 7.38-7.35 (t, 1H, J )
7.8 Hz, H-C5), 7.3-7.2 (bs, 1H, NH), 6.3-6.2 (bs, 1H, NH), 4.01
(s, 2H, CH2). 13C NMR (600 MHz, CDCl3) δ 195.36 (CO), 168.72
(CON), 141.96 (C1), 139.68 (Ph), 136.33 (C3), 132.62 (C4), 129.52
(C2), 128.92 (Ph), 127.93 (C5), 127.32 (C6), 127.1 (Ph), 45.19
(CH2). MS (CI+) m/z: 105.035 ([M - CH2CONH2], 100), 239.091
(M+, 25). HRMS: calcd for C15H13NO2 (M+, CI+) 239.0946 found
239.0907.
3-(3-Iodophenyl)-3-oxopropanamide (6a). Compound 6a was
obtained as a yellow solid in 80% yield from compound 5a; mp
1
90-95 °C. H NMR (300 MHz, CDCl3) δ 8.35-8.34 (m, 1H,
H-C2), 8.02-7.93 (m, 2H, H-C6, H-C4), 7.29-7.24 (t, 1H, J )
8.5 Hz, H-C5), 7.1-7 (bs, 1H, NH), 6.1-5.8 (bs, 1H, NH), 3.97
(s, 2H, CH2). 13C NMR (300 MHz, CDCl3) δ 194.12 (CO), 167.79
(CON), 142.83 (C4), 137.4 (C2), 137.28 (C1), 130.52 (C6), 127.4
(C5), 94.7 (C3), 44.91 (CH2). MS (CI+) m/z: 164.038 ([M - I],
100), 230.914 ([M - CH2CONH2], 30), 289.972 (MH+, 40).
HRMS: calcd for C9H9NO2I (MH+, CI+) 289.9678 found 289.9718.
3-(3-Chlorophenyl)-3-oxopropanamide (6b).47 Compound 6b,
present as a mixture of ketone and enol forms (1:1.9), was obtained
as a yellow solid in 80% yield from 5b; mp 125-130 °C [lit.47
135-137 °C]. 1H NMR (300 MHz, acetone-d6) δ 8-7.93 (m, 2H,
H-C2(ket), H-C6 (ket)), 7.76-7.74 (m, 1H, H-C2(enol)), 7.72-
3-Oxo-3-phenylpropanamide (6g).49 Compound 6g was ob-
tained from compound 5g as a white solid in 75% yield. 1H NMR
(300 MHz, CDCl3) δ 8-7.98 (d, 2H, J ) 7.5 Hz, H-C2), 7.64-
7.46 (m, 3H, H-C3, H-C4), 3.99 (s, 2H, CH2). 13C NMR (300 MHz,

2380 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Herschhorn et al.
CDCl3) δ 195.56 (CO), 168.87 (CON), 136.16 (C1), 134.02 (C4),
128.82 (C2), 128.54 (C3), 45.36 (CH2). MS (ES+) m/z: 186
(MNa+, 20).
Hz, H-C4), 7.54-7.49 (t, 2H, J ) 7.5 Hz, H-C3), 7-6.9 (bs, 1H,
NH), 6.34-6.36 (bs, 1H, NH), 5.59 (s, 1H, CHBr). 13C NMR (200
MHz, CDCl3) δ 190.7 (CO), 166.9 (CON), 134.51 (C4), 133.72
(C1), 129.27 (C2), 128.98 (C3), 43.27 (CH). MS (ES+) m/z:
242.244 (MH+, 10), 264.266 (MNa+, 100).
General Procedure for Bredereck Synthesis of Imidazoles
8a-g.52 An R-bromoketone compound 7a-g (2.61 mmol) was
dissolved in formamide (4 mL) followed by addition of 3 drops of
concentrated H2SO4. The solution was refluxed at 150 °C for 2 h
and was quenched with 1 N Na2CO3. Chloroform was added, and
the organic phase was separated, dried over MgSO4 and evaporated.
The crude mixture was further purified by column chromatography
(eluent hexane/EtOAc 3:1) to give 8a-g.
4-(3-Iodophenyl)-1H-imidazole-5-carboxamide (8a). Com-
pound 8a was obtained as a yellow solid in 5% yield from 7a. 1H
NMR (300 MHz, CDCl3) δ 8.57-8.56 (t, 1H, J ) 1.5 Hz, H-C2),
8.26-8.22 (ddd, 1H, J ) 7.8; 1.5; 1.4 Hz, H-C6), 7.9 (s, 1H, CH),
7.75-7.7 (ddd, 1H, J ) 7.8; 1.5; 1.4 Hz, H-C4), 7.18-7.15 (t,
1H, J ) 7.8 Hz, H-C5), 6.35-6.26 (bs, 3H, NH). 13C NMR (200
MHz, CDCl3) δ 159.22 (CON), 150.07 (CH), 141.09 (Ph-C-N),
138.67 (C4), 138.05 (C2), 130.06 (C6), 129.4 (C1), 128.7 (C5),
128.47 (C-CON), 94.05 (C3). MS (CI+) m/z: 312.968; 313.979;
314.99 (MH+, 40; 100; 20). HRMS: calcd for C10H9N3OI (MH+,
CI+) 313.979 found 313.9793.
4-(3-Chlorophenyl)-1H-imidazole-5-carboxamide (8b). Com-
pound 8b was obtained as a white solid in 20% yield; mp 160 °C.
1H NMR (600 MHz, acetone-d6) δ 8.54 (t, 1H, J ) 1.8; 0.6 Hz,
H-C2), 8.36-8.34 (dt, 1H, J ) 7.8; 1.8 Hz, H-C4), 8.34 (s, 1H,
CH), 7.47-7.46 (t, 1H, J ) 7.8 Hz, H-C5), 7.44-7.42 (ddd, 1H,
J ) 7.8; 1.8; 1.2 Hz, H-C6). 13C NMR (300 MHz, CDCl3) δ 158.88
(CON), 149.87 (CH), 142.64 (Ph-C-N), 138.82 (C1), 134.29 (C3),
131.45 (C-CON), 129.76 (C5), 129.57 (C4), 129.25 (C2), 127.51
(C6). MS (CI+) m/z: 222.04; 223.04; 224.04 (MH+, 100; 56; 37).
HRMS: calcd for C10H9N3OCl (MH+, CI+) 222.0434 found
222.0403.
General Procedure for Synthesis of r-Brominated 7a-g.50
A mixture of a ketone (0.65 mmol) and CuBr2 (0.65 mmol) in
anhydrous EtOAc was heated to reflux. The reaction was monitored
by TLC, and an additional amount of CuBr2 was added if needed.
The reaction was stopped at the point when no starting material
spot was observed, and the mixture was filtered through a short
silica column and washed with EtOAc. The filtrate was evaporated
and if needed followed by further purification by column chroma-
tography.
2-Bromo-3-(3-iodophenyl)-3-oxopropanamide (7a). Compound
7a was obtained as a yellow solid in 80% yield from 6a; mp 93-
1
95 °C. H NMR (300 MHz, CDCl3) δ 8.24-8.23 (t, 1H, J ) 1.8
Hz, H-C2), 7.97-7.94 (m, 2H, H-C6, H-C4), 7.27-7.22 (t, 1H, J
) 8.1 Hz, H-C5), 6.9-6.8 (bs, 1H, NH), 6.25-6.15 (bs, 1H, NH),
5.51 (s, 1H, CH). 13C NMR (300 MHz, CDCl3) δ 189.28 (CO),
166.41 (CON), 143.19 (C1), 138.06 (C2), 135.38 (C4), 130.5 (C6),
128.32 (C5), 94.52 (C3), 43.21 (CH). MS (CI+) m/z: 230.932 ([M
- CHBrCONH2], 100), 368.866 (M+, 10). HRMS: calcd for C9H7-
NO2I (M+, CI+) 368.8684 found 368.8661.
2-Bromo-3-(3-chlorophenyl)-3-oxopropanamide (7b). Com-
pound 7b was obtained as a yellow solid in 81% yield from 6b;
1
mp 110-115 °C. H NMR (300 MHz, CDCl3) δ 7.99-7.98 (t,
1H, J ) 1.8 Hz, H-C2), 7.9-7.87 (m, 1H, H-C4), 7.62-7.58 (ddd,
1H, J ) 8.1; 2.1; 0.9 Hz, H-C6), 7.48-7.43 (t, 1H, J ) 7.8 Hz,
H-C5), 6.83 (bs, 1H, NH), 6.17 (bs, 1H, NH), 5.52 (s, 1H, CH).
13C NMR (300 MHz, CDCl3) δ 189.46 (CO), 166.39 (CON), 135.46
(C1), 135.25 (C3), 134.46 (C4), 130.25 (C5), 129.24 (C2), 127.3
(C6), 43.37 (CH). MS (CI+) m/z: 275.939; 277.939 (MH+, 84;
100). HRMS: calcd for C9H8NO2ClBr (MH+, CI+) 277.9406 found
277.9387.
2-Bromo-3-(3-bromo-4-chlorophenyl)-3-oxopropanamide (7c).
Compound 7c was obtained as a white solid in 80% yield from 6c.
1H NMR (300 MHz, CDCl3) δ 8.23-8.22 (d, 1H, J ) 2.1 Hz,
H-C2), 7.88-7.84 (dd, 1H, J ) 8.4; 2.1 Hz, H-C6), 7.57-7.54 (d,
1H, J ) 8.4 Hz, H-C5), 6.9 (bs, 1H, NH), 6.24 (bs, 1H, NH), 5.49
(s, 1H, CH). 13C NMR (300 MHz, acetone-d6) δ 188.54 (CO),
166.91 (CON), 148.1 (C1), 135.01 (C2, C4), 131.87 (C5), 130.23
(C6), 123.33 (C3), 43.35 (CH).
4-(3-Bromo-4-chlorophenyl)-1H-imidazole-5-carboxamide (8c).
Compound 8c was obtained as a white solid in 5% yield from
compound 7c. 1H NMR (300 MHz, acetone-d6) δ 8.91-8.9 (d, 1H,
J ) 2.1 Hz, H-C2), 8.47-8.43 (dd, 1H, J ) 8.7; 2.1 Hz, H-C6),
8.38 (s, 1H, CH), 7.67-7.65 (d, 1H, J ) 8.7 Hz, H-C5), 7.58 (bs,
1H, NH), 7.15 (bs, 1H, NH). 13C NMR (300 MHz, acetone-d6) δ
159.5 (CON), 151.82 (CH), 141.5 (Ph-C-N), 137 (C4), 134.99 (C2),
131.65 (C1), 130.9 (C5), 130.53 (C-CON), 130.38 (C6), 122.26
(C3).
2-Bromo-3-(3-benzoylphenyl)-3-oxopropanamide (7d). Com-
pound 7d was obtained as a white solid in 80% yield from 6d; mp
1
125-126 °C. H NMR (300 MHz, CDCl3) δ 8.39-8.38 (t, 1H, J
) 1.28 Hz, H-C2), 8.23-8.21 (dt, 1H, J ) 7.8; 1.28 Hz, H-C4),
8.04-8.01 (dt, 1H, J ) 7.58; 1.28 Hz, H-C6), 7.79-7.76 (m, 2H,
Ph), 7.64-7.58 (m, 2H, Ph), 7.51-7.28 (m, H-C5, Ph), 7-6.8 (bs,
1H, NH), 6.15-6 (bs, 1H, NH), 5.6 (s, 1H, CH). 13C NMR (300
MHz, CDCl3) δ 195.4 (CO), 189.77 (CO), 167.08 (CON), 138.34
(C3), 136.67 (Ph), 135.49 (C4), 133.7 (C1), 133.08 (C6), 132.85
(C2), 130.7 (C5), 130.17 (Ph), 129.29 (Ph), 128.67 (Ph), 43.89
4-(3-Benzoylphenyl)-1H-imidazole-5-carboxamide (8d). Com-
pound 8d was obtained as a white solid in 7% yield from compound
7d. 1H NMR (600 MHz, CDCl3) δ 8.65-8.64 (t, 1H, J ) 1.2 Hz,
H-C2), 8.51-8.49 (dt, 1H, J ) 7.8; 1.2 Hz, H-C4), 7.94 (s, 1H,
CH), 7.87-7.86 (m, 3H, H-C6, Ph), 7.6-7.59 (m, 2H, H-C5, Ph),
7.52-7.5 (m, 2H, Ph), 6.37 (bs, 1H, NH), 6.04 (bs, 1H, NH). 13C
NMR (600 MHz, CDCl3) δ 195.34 (CO), 159.02 (CON), 149.98
(CH), 143.06 (Ph-C-N), 137.64 (Ph-q), 137.29 (C3), 133.2 (Ph),
132.57 (C6), 131.07 (C2), 130.9 (C4), 130.22 (Ph), 130.02 (C5),
129.99 (C1), 129.41 (C-CONH2), 128.32 (Ph). MS (ES+) m/z: 293
(MH+, 70), 315 (MNa+, 100). HRMS: calcd for C17H14N3O2 (MH+,
CI+) 292.1086 found 292.1078.
(CH). MS (ES+) m/z: 346; 348 (MH+, 100; 90). HRMS: calcd
81
for C16H1379BrNO3 (MH+, CI+) 346.0079 found 346.0051; C16H13
-
BrNO3 (MH+, CI+) 346.9980 found 346.9998.
2-Bromo-3-(3-phenylphenyl)-3-oxopropanamide (7e). Com-
pound 7e was obtained from 6e and was purified by column
chromatography (eluent hexane/EtOAc ) 2:1) to give 7e as a yellow
4-(3-Phenylphenyl)-1H-imidazole-5-carboxamide (8e). Com-
pound 8e was obtained from 7e by general procedure 5. The crude
mixture was purified by column chromatography (eluent hexane/
EtOAc ) 1:1) to give the desired compound as a yellow solid in
1
solid in 80% yield. H NMR (300 MHz, CDCl3) δ 8.23-8.22 (t,
1H, J ) 1.8 Hz, H-C2), 8-7.96 (ddd, 1H, J ) 7.8; 1.8; 1.2 Hz,
H-C6), 7.87-7.83 (ddd, 1H, J ) 7.8; 1.8; 1.2 Hz, H-C4), 7.62-
7.55 (m, 3H, H-C5, Ph), 7.5-7.36 (m, 3H, Ph), 6.9 (bs, 1H, NH),
6.2 (bs, 1H, NH), 5.65 (s, 1H, CHBr). 13C NMR (300 MHz, CDCl3)
δ 190.82 (CO), 167.05 (CON), 142.36 (C1), 139.74 (Ph), 134.21
(C3), 133.34 (C4), 129.58 (C2), 129.16 (Ph), 128.22 (C5), 128.13
(C6), 128.06 (Ph), 127.33 (Ph), 43.49 (CHBr). MS (CI+) m/z:
105.044 ([M - CH2CONH2], 80), 239.096 ([M - Br], 20), 317.004
(M+, 5).
1
15.3% yield. H NMR (200 MHz, CDCl3) δ 8.52-8.5 (td, 1H, J
) 1.93; 0.64 Hz, H-C2), 8.21-8.16 (ddd, 1H, J ) 7.5; 1.93; 1.12
Hz, H-C4), 7.94 (s, 1H, CH), 7.7-7.3 (m, 7H, H-C5, H-C6, Ph),
6.34-6 (bs, 3H, NH, NH2). 13C NMR (300 MHz, CDCl3, ppm δ):
159.44 (CON), 150.13 (CH), 141.32 (Ph-C-N), 140.86 (Ph), 130.82
(C1), 130.33 (C3), 129.22 (C-CONH2), 128.94 (Ph), 128.53 (C5),
128.31 (C4), 128.24 (C6), 127.57 (C2), 127.34 (Ph).
2-Bromo-3-oxo-3-phenylpropanamide (7 g).51 Compound 7g
was obtained as a yellow solid in 75% yield by bromination of 6g;
mp 105-108 °C [lit.51 124-125 °C]. 1H NMR (300 MHz, CDCl3)
δ 8.03-8.01 (d, 2H, J ) 7.5 Hz, H-C2), 7.67-7.62 (t, 1H, J ) 7.5
4-Phenyl-1H-imidazole-5-carboxamide (8g). Compound 8g was
1
obtained as a yellow solid in 80% yield from compound 7g. H
NMR (300 MHz, CDCl3) δ 8.19-8.15 (m, 2H, H-C2), 7.91 (s,
1H, CH), 7.46-7.37 (m, 3H, H-C3, H-C4), 6.49-6.36 (bs, 1H,

NoVel Broad-Range RT Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2381
NH), 6.36-6.29 (bs, 1H, NH). 13C NMR (200 MHz, CDCl3) δ 159.5
(CON), 149.96 (CH), 143.68 (Ph-C-N), 131.9 (C1), 129.6 (C3),
129.21 (C4), 128.24 (C2), 124.87 (C-CONH2). MS (ES+) m/z: 171
([M - NH2], 40), 189 (MDH+, 100), 211 (MDNa+, 40).
General Procedure for the Synthesis of Aminothiazoles 9a-
g.53 Τhiourea (0.265 mmol) was dissolved in 95% EtOH (10 mL)
followed by addition of an R-bromoketone (0.265 mmol). The
reaction mixture was stirred at room temperature and evaluated with
TLC (hexane/EtOAc ) 2:1). The reaction was stopped after 4 h
when no starting material spot was detected. Evaporation of the
reaction mixture to dryness and recrystallization from MeOH/Et2O
provided the desired product.
General Procedure for the Synthesis of Thiazoles 10a-g.55
To an aminothiazole derivative 9a-g (0.5 mmol) dissolved in 40%
H2SO4 (6 mL) at 0 °C was added NaNO2 (0.9 mmol) in H2O (0.3
mL), and the resulting yellow mixture was stirred at this temperature
for 30 min. The mixture was then added to a suspension of Ca-
(H2PO4)2 (0.5 mmol) in water (2.15 mL), and the final mixture
was stirred at 0 °C for additional 1 h. The obtained suspension
was filtered, the residue washed with CHCl3, and the filtrate further
extracted with CHCl3. The organic phase washed with brine, dried
over MgSO4, and evaporated. The crude residue was purified by
column chromatography (eluent hexane/EtOAc ) 2:1) to provide
compounds 10a-g.
4-(3-Iodophenyl)thiazole-5-carboxamide (10a). Compound 10a
was obtained from 9a as a yellow solid in 15.8% yield; mp 137-
2-Amino-4-(3-iodophenyl)thiazole-5-carboxamide Hydrobro-
mide (9a). Compound 9a was obtained as a yellow solid in 72%
1
1
140 °C. H NMR (300 MHz, CDCl3) δ 8.9 (s, 1H, CH), 8.06-
yield from 7a; mp 248-250 °C. H NMR (200 MHz, CD3OD) δ
8.05 (t, 1H, J ) 1.5 Hz, H-C2), 7.84-7.8 (ddd, 1H, J ) 7.8; 1.5
Hz, H-C4), 7.67-7.63 (ddd, 1H, J ) 7.8; 1.5 Hz, H-C6), 7.26-
7.2 (t, 1H, J ) 7.8 Hz, H-C5), 5.99-5.77 (bs, 2H, NH). 13C NMR
(300 MHz, CDCl3) δ 163.26 (CON), 155.35 (CH), 139.2 (C1),
138.8 (C4), 138.23 (C2), 135.42 (Ph-C-N), 130.66 (C5), 129.36
(C-CONH2), 128.62 (C6), 94.83 (C3). C10H7N2IO3S (M+, CI+)
329.9324 found 329.9315.
8.03-8.01 (td, 1H, J ) 1.68; 0.38 Hz, H-C2), 7.99-7.93 (ddd,
1H, J ) 7.9; 1.68; 1 Hz, H-C6), 7.67-7.62 (ddd, 1H, J ) 7.9;
1.68; 1 Hz, H-C4), 7.37-7.3 (td, 1H, J ) 7.9; 0.38 Hz, H-C5). 13
C
NMR (300 MHz, CD3OD) δ 170.79 (C-NH2), 162.95 (CON),
141.37 (C4), 140.18 (Ph-C-N), 139.1 (C2), 131.95 (C6), 130.69
(C1), 129.85 (C5), 117.54 (C-CONH2), 95.18 (C3). MS (CI+)
m/z: 345.951 (MH+, 35). HRMS: calcd for C10H9N3OSI (MH+,
CI+) 345.9511 found 345.9508.
4-(3-Chlorophenyl)thiazole-5-carboxamide (10b). Compound
10b was obtained from 9b as a yellow solid in 12% yield; mp 183-
2-Amino-4-(3-chlorophenyl)thiazole-5-carboxamide Hydro-
bromide (9b). Compound 9b was obtained as a yellow solid in
72% yield from 7b; mp 138-140 °C. 1H NMR (300 MHz,
CD3OD) δ 7.71-7.7 (m, 1H, H-C2), 7.66-7.56 (m, 3H, H-C4,
H-C5, H-C6). 13C NMR (200 MHz, CD3OD) δ 170.6 (C-NH2),
162.83 (CON), 140.09 (Ph-C-N), 136.17 (C3), 136.14 (C1), 132.41
(C5), 131.96 (C4), 130.44 (C2), 121.99 (C6), 117.77 (C-CONH2).
2-Amino-4-(3-bromo-4-chlorophenyl)thiazole-5-carboxam-
ide Hydrobromide (9c). Compound 9c was obtained as a white
solid in 72% yield from 7c; mp 245-247 °C. 1H NMR (300 MHz,
CD3OD) δ 8 (d, 1H, J ) 2.1 Hz, H-C2), 7.73-7.7 (d, 1H, J ) 8.4
Hz, H-C5), 7.65-7.59 (dd, 1H, J ) 8.4; 2.1 Hz, H-C6). 13C NMR
(200 MHz, CD3OD) δ 170.73 (C-NH2), 162.83 (CON), 139.47
(Ph-C-N), 138.38 (C4), 135.75 (C2), 132.11 (C5), 131.03 (C6),
130.6 (C1), 129.02 (C3), 122.81 (C-CONH2). MS (CI+) m/z:
332.915 (M+, 100). HRMS: calcd for C10H7N3OSClBr (M+, CI+)
332.9161 found 332.9153.
1
185 °C. H NMR (300 MHz, CD3OD) δ 9.1 (s, 1H, CH), 7.84-
7.82 (m, 1H, H-C2), 7.75-7.72 (ddd, 1H, J ) 6.3; 2.7; 1.8 Hz,
H-C4), 7.47-7.43 (m, 2H, H-C5, H-C6). 13C NMR (300 MHz,
CD3OD) δ 165.26 (CON), 155.13 (CH), 153.9 (Ph-C-N), 136.84
(C3), 134.98 (C1), 130.92 (C5), 129.85 (C4), 129.73 (C2), 129.53
(C-CONH2), 128.36 (C6). MS (CI+) m/z: 237.997; 238.986;
239.996 (M+, 90; 60; 36). HRMS: calcd for C10H7N2OSCl (M+,
CI+) 237.9968 found 237.9968.
4-(3-Bromo-4-chlorophenyl)thiazole-5-carboxamide (10c). Com-
pound 10c was obtained from 9c as a white solid in 13% yield;
1
mp 154-156 °C. H NMR (300 MHz, acetone-d6) δ 9.09 (s, 1H,
CH), 8.2-8.19(d, 1H, J ) 2.1 Hz, H-C2), 7.87-7.84 (dd, 1H, J )
8.4; 2.1 Hz, H-C6), 7.66-7.63 (d, 1H, J ) 8.4 Hz, H-C5), 7.35
(bs, 1H, NH), 7.16 (bs, 1H, NH). 13C NMR (300 MHz, acetone-d6)
δ 163.64 (CON), 154.57 (CH), 135.64 (Ph-C-N), 134.99 (C2),
134.82 (C4), 131.6 (C1), 131.1 (C5), 130.46 (C6), 129.25 (C3),
122.41 (C-CONH2). MS (CI+) m/z: 316.903 (M+, 100), 317.904
(MH+, 90). HRMS: calcd for C10H6N3OS35Cl79Br (MH+, CI+)
298.9461 found 298.9438.
2-Amino-4-(3-benzoylphenyl)thiazole-5-carboxamide Hydro-
bromide (9d). Compound 9d was obtained as a highly hygroscopic,
yellow solid in 72% yield from 7d. 1H NMR (300 MHz, acetone-
d6) δ 8.06-8.05 (t, 1H, J ) 1.8 Hz, H-C2), 7.99-7.96 (ddd, 1H,
J ) 7.8; 1.8; 1.5 Hz, H-C6), 7.87-7.8 (m, 3H, H-C4, H-C5, Ph),
7.7-7.55 (m, 4H, Ph). 13C NMR (300 MHz, CD3OD) δ 196.98
(CO), 170.86 (C-NH2), 162.99 (CON), 141.26 (Ph-C-N), 139.67
(Ph), 138.09 (C3), 134.27 (Ph), 133.35 (C6), 131.85 (C2), 131.15
(Ph), 130.58 (C4), 130.47 (C1), 129.75 (Ph, C5), 129.26
(C-CONH2). MS (CI+) m/z: 324.076 (MH+, 100). HRMS: calcd
for C17H14N3O2S (MH+, CI+) 324.0807 found 324.0759.
2-Amino-4-(3-phenylphenyl)thiazole-5-carboxamide Hydro-
bromide (9e). Compound 9e was obtained as a yellow solid in
55% yield from 7e; mp 223-227 °C. 1H NMR (300 MHz,
CD3OD) δ 7.9-7.85 (m, 2H, H-C2, H-C4), 7.74-7.59 (m, 4H,
H-C5, H-C6, Ph), 7.5-7.45 (m, 2H, Ph), 7.39-7.36 (m, 1H, Ph).
13C NMR (300 MHz, CD3OD) δ 170.75 (C-NH2), 163.15 (CON),
143.59 (Ph-C-N), 141.41 (Ph), 140.83 (C1), 132.58 (C3), 131.07
(C5), 130.95 (C4), 130.13 (Ph), 129.83 (C-CONH2), 129.17 (C6),
129.14 (Ph), 128.96 (C2), 128.12 (Ph). MS (CI+) m/z: 295.078
(M+, 10). HRMS: calcd for C16H13N3OS (M+, CI+) 295.0779
found 295.0779.
4-(3-Benzoylphenyl)thiazole-5-carboxamide (10d). Compound
10d was obtained from 9d as a yellow solid in 12% yield; mp 180-
1
183 °C. H NMR (300 MHz, CDCl3) δ 8.91 (s, 1H, CH), 8.11-
8.1 (t, 1H, J ) 1.8 Hz, H-C2), 7.94-7.89 (m, 2H, H-C4, H-C6),
7.83-7.8 (m, 2H, Ph), 7.65-7.58 (m, 2H, H-C5, Ph), 7.52-7.46
(m, 2H, Ph), 5.84-5.82 (bs, 2H, NH). 13C NMR (300 MHz, CDCl3)
δ 195.87 (CO), 162.54 (CON), 155.07 (CH), 154.32 (Ph-C-N),
138.46 (Ph), 136.92 (C3), 133.73 (C1), 133.12 (C6), 132.87 (C2),
131.04 (C4), 130.82 (C5), 130.08 (Ph), 129.08 (Ph), 128.49 (C-
CONH2), 128.48 (Ph). MS (CI+) m/z: 231.004 ([M - Ph], 60),
308.063 (M+, 100). HRMS: calcd for C17H12N2O2S (M+, CI+)
308.0619 found 308.0634.
4-(3-Phenylphenyl)thiazole-5-carboxamide (10e). Compound
10e was obtained from 9e as a yellow solid in 12% yield. 1H NMR
(300 MHz, CDCl3) δ 8.92 (s, 1H, CH), 7.91-7.89 (td, 1H, J )
1.8; 0.6 Hz, H-C2), 7.74-7.71 (dt, 1H, J ) 7.5; 1.5 Hz, H-C4),
7.64-7.55 (m, 4H, H-C5, H-C6, Ph), 7.5-7.35 (m, 3H, Ph), 5.79-
5.72 (bs, 2H, NH). 13C NMR (300 MHz, CDCl3) δ 164.25 (CON),
155.33 (CH), 129.64 (C5), 128.98 (Ph), 128.54 (C4), 128.23 (Ph),
128.17 (C6), 127.92 (C2), 127.19 (Ph). MS (CI+) m/z: 264.094
([M - NH2]+, 40), 280.071 (M+, 100). HRMS: calcd for C16H12N2-
OS (M+, CI+) 280.067 found 280.0713.
2-Amino-4-phenylthiazole-5-carboxamide Hydrobromide (9g).54
Compound 9g was obtained as a yellow solid in 59% yield from
1
7g; mp 225-230 °C. H NMR (200 MHz, DMSO-d6) δ 7.59-
7.56 (m, 2H, H-C2), 7.52-7.49 (m, 3H, H-C3, H-C4), 7.17-7.0
(bs, 1H, NH), 5.27-5.11 (bs, 3H, NH). 13C NMR (200 MHz,
DMSO-d6) δ 167.92 (C-NH2), 161.2 (CON), 143.25 (Ph-C-N),
129.94 (C1), 129.85 (C4), 129.17 (C3), 128.48 (C2), 114.9
(C-CONH2). MS (CI+) m/z: 219.045 (M+, 100). HRMS: calcd
for C10H9N3OS (M+, CI+) 219.0433 found 219.0452.
4-Phenylthiazole-5-carboxamide (10g). Compound 10g was
1
obtained from 9g as a yellow solid in 16% yield; mp 150 °C. H
NMR (300 MHz, CDCl3) δ 8.91 (s, 1H, CH), 7.66-7.63 (m, 2H,
H-C2), 7.54-7.49 (m, 2H, H-C3, H-C4), 6.46 (bs, 1H, NH), 5.8
(bs, 1H, NH). 13C NMR (300 MHz, CDCl3) δ 163.42 (CON), 155.49
(CH, Ph-C-N), 133.57 (C1), 130.01 (C3), 129.75 (C-CONH2),

2382 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Herschhorn et al.
129.51 (C4), 129.31 (C2). MS (CI+) m/z: 204.038 (M+, 100),
205.045 (MH+, 90). HRMS: calcd for C10H9N2OS (MH+, CI+)
205.0436 found 205.0453.
mM KCl, at final pH 8.0. These reactions were further incubated
for 10 min at 32 °C and were stopped by the addition of 2 µL of
loading buffer (1% bromophenol blue, 30% glycerol). The reaction
mixtures underwent electrophoresis through 6.5% polyacrylamide
nondenatured gel in TBE buffer (89 mM Tris base, 89 mM borate,
and 2 mM EDTA, pH 8.3) at 4 °C under 15 V/cm for about 2.5 h.
The dried gels were subjected to autoradiography at -80 °C.
Biology. Expression and Purification of RTs. Wild-type p66/
p51 HIV-1 RT, derived from BH-10 clone of HIV-1, was expressed
in bacteria. This enzyme, which has six histidines tag at the
C-terminus of the p66 subunit, was purified on Ni2+ nitrilotriacetic
acid agarose (Ni-NTA) column followed by cation exchange
chromatography, as was previously described.56 The plasmids
encoding for the single mutant T181C and double mutant L100I +
K103N were a kind gift from Dr. S. Hughes of NCI, NIH in
Frederick, MD. The mutant T181C RT was expressed and purified
in a similar manner on Ni-NTA column. The double mutant L100I-
K103N was expressed as a homodimer enzyme with two identical
66 kDa subunits; it was expressed in DH5R E. coli and purified in
a similar manner as above.
DNA Polymerase Assay. The DNA polymerase activity of RT
was assayed by measuring the incorporation of [3H]dTTP into
poly(rA)n‚oligo(dT)12-18 template-primer as described previously.57
All inhibitors were serially diluted (5-fold or 3-fold for assays with
compounds 8c and 5f) in DMSO, and 1 µL of each concentration
(or 1 µL of DMSO only as a control) was added to 79 µL of 31.25
mM Tris-HCl pH 7.5, 50 mM KCl, and 8 mM MgCl2. A 10 µL
amount of purified HIV-1 RT (14 ng) was added to each tube, and
the solutions were incubated on ice for 10 min. The enzymatic
reactions were initiated by the addition of 10 µL of the substrate
mix [50 µg/mL poly(rA)n‚oligo(dT)12-18, 50 µM dTTP, 150 µCi/
mL [3H]dTTP] followed by incubation for 30 min at 37 °C. The
reaction was stopped by adding 50 µg/mL herring sperm carrier
DNA and 40 mM sodium pyrophosphate followed by precipitation
with ice-cold 10% (w/v) trichloroacetic acid. The precipitates were
collected on Whatman GF/C fiberglass filters, and the filters were
washed with 5% cold trichloroacetic acid and then with 50% cold
ethanol. The dried filters were put in a scintillation fluid and counted
in a â scintillation counter.
The reported measurements are the average of at least two
experiments for compounds with IC50 values higher than 10 µM
and at least three experiments for compounds with IC50 values lower
than 10 µM. The dose-response curves were nonlinearly fitted to
the four-parameter logistic equation58 using Origin software.
RNase H Assay. The RNase H activity of RT was assayed with
fluorescence resonance energy transfer technology as described
previously.59 Briefly, RT was incubated with a substrate of hybrid
of fluorescein-RNA and DNA-4-[[4′-(dimethylamino)phenyl]azo]-
benzoic acid and with (or without) a specific inhibitor. Hydrolysis
of the substrate and the resulting emitting fluorescence was
measured with a fluorescence spectrometer after 30 min of
incubation at 37 °C. To improve the signal-to-noise ratio, the assay
was slightly modified to contain 6 nM RT and 1 mM DTT. All
measurements were done in triplicates in a 96-well plate, and IC50
values were interpolated from the dose-response curves.
PAGE Mobility Shift Assay. Complex formation between [32P]-
5′ end labeled DNA oligonucleotide and HIV-1 RT was detected
by the electrophoretic retardation of the DNA as a result of its
association with RT, as previously described in detail.60 HIV-1 RT
(1.5 pmol) was preincubated with or without compounds 8c, 5f,
and nevirapine, each at a final concentration of 250 and 625 µM,
in addition to 50 µM toxiusol that served as a positive control.23
After 10 min at 4 °C, the binding reactions were initiated by the
addition of 0.12 pmol of the [32P]-end labeled double-stranded DNA
substrate. This substrate was a duplex of two 54 nt-long synthetic
oligonucleotide. The first one with the sequence:
DNA-Primer Extension Reactions. Single-stranded circular
φX174am3 DNA that served as a template was primed with a
synthetic 15-mer oligonucleotide (5′-AAAGCGAGGGTATCC-3′),
which hybridizes at positions 588-602 of the template-DNA.22 The
primer, prelabeled at its 5′-end with [γ-32P] ATP using T4
polynucleotide kinase, was annealed to a 2-fold molar excess of
unlabeled template DNA. The reactions were performed in 6.6 mM
Tris-HCl, pH 8.0, 4 mM DTT, 6 mM MgCl2, 24 µg/mL bovine
serum albumin, 33 mM NaCl, 4 nM template-primer, and all four
dNTPs (each at a final concentration of 20 µM) and 1.6 nM of
purified HIV-1 RT. The reactions, each at a final volume of 12.5
µL, were incubated at 37 °C for 30 min and were stopped by adding
12.5 µL of formamide loading buffer (90% formamide, 10 mM
EDTA, 1 mg/mL bromophenol blue, 1 mg/mL xylene cyanole).
The samples were heat-denatured, quickly cooled on ice, and loaded
onto 6 M urea 12% polyacrylamide denaturing gels, followed by
electrophoresis (urea-PAGE) as previously described.22 The gels
were dried and subjected to autoradiography at -80 °C or at room
temperature to obtain a close to linear exposure.
Cytotoxic Assay. Cytotoxic assay was performed in 96-well plate
with XTT substrate as previously described.61 Absorbance was
recorded at 450 nm and the reference wavelength was recorded at
620 nm.
Acknowledgment. The authors thank Dr. S. Loya and Dr.
R. Shultz for helpful comments on the manuscript, Dr. S. H.
Hughes from NCI for sending us the plasmids that express the
mutant HIV-1 RTs studied here, and to Dr. M. A. Parniak from
University of Pittsburgh, School of Medicine, for the substrate
for the RNase H assay. We also thank Dr. A. N. Jain for kindly
supplying the suflex software, Accelrys inc. for access to Cerius
2 software and Dr. H. Senderowitz for constructive guidance
to the molecular modeling studies. The generous support for
this work by the “Marcus Center for Pharmaceutical and
Medicinal Chemistry” at Bar Ilan University is gratefully
acknowledged.
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