Specific targeting of highly conserved residues in the HIV-1 reverse transcriptase primer grip region. 2. Stereoselective interaction to overcome the effects of drug resistant mutations

Article abstract of DOI:10.1021/jm801395v

Starting from the prototypic compound 4, we describe new, potent, and broad-spectrum pyrrolobenzo(py-rido)oxazepinones antivirals. A biochemical and enzymological investigation was performed for defining their mechanism of inhibition at either recombinant

Full text of DOI:10.1021/jm801395v

1224
J. Med. Chem. 2009, 52, 1224–1228
Specific Targeting of Highly Conserved Residues in the HIV-1 Reverse Transcriptase Primer
Grip Region. 2. Stereoselective Interaction to Overcome the Effects of Drug Resistant Mutations
Stefania Butini,†,‡ Margherita Brindisi,†,‡ Sandro Cosconati,§,† Luciana Marinelli,§,† Giuseppe Borrelli,†,‡
,†
Salvatore Sanna Coccone,†,‡ Anna Ramunno,†,‡ Giuseppe Campiani,†,‡,* Ettore Novellino,§,† Samantha Zanoli,
,†
Alberta Samuele, Gianluca Giorgi,| Alberto Bergamini,# Michela Di Mattia,3 Silvana Lalli,3 Bruno Galletti,3
,†
Sandra Gemma,†,‡ and Giovanni Maga
European Research Centre for Drug DiscoVery and DeVelopment (NatSynDrugs), UniVersita` di Siena, Italy, Dipartimento Farmaco Chimico
Tecnologico (DFCT), UniVersita` di Siena, Via Aldo Moro 2, 53100 Siena, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica
(DCF&T), UniVersita` di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy, Istituto di Genetica Molecolare (IGM)sCNR, Via
Abbiategrasso 207, 27100 PaVia, Italy, Dipartimento di Chimica (DC), UniVersita` di Siena, Via Aldo Moro 2, 53100 Siena, Italy, Dipartimento
di Sanita` Pubblica e Biologia Cellulare, UniVersita` degli Studi di Roma “Tor Vergata”, Via Tor Vergata 135, 00133 Roma, Italy, Sigma-Tau
Industrie Farmaceutiche Riunite spa, Via Pontina Km 30, 400, 00040 Pomezia, Italy
ReceiVed NoVember 4, 2008
Starting from the prototypic compound 4, we describe new, potent, and broad-spectrum pyrrolobenzo(py-
rido)oxazepinones antivirals. A biochemical and enzymological investigation was performed for defining
their mechanism of inhibition at either recombinant HIV-1 RT wild type and non-nucleoside reverse
transcriptase inhibitors (NNRTIs)-resistant mutants. For the novel compounds (S)-(+)-5 and (S)-(-)-7, a
clear-cut stereoselective mechanism of enzyme inhibition was found. Molecular modeling studies were
performed for revealing the underpinnings of this behavior.
Chart 1. Reference and Title Compounds
Introduction
Reverse transcriptase (RT) is a key multifunctional enzyme
in the life cycle of the human immunodeficiency virus type-1
(HIV-1), the causative agent of AIDS. The enzyme shows both
RNA- and DNA-dependent polymerase activities and is respon-
sible for conversion of the single-stranded RNA retroviral
genome into the double-stranded proviral DNA, which is
successively integrated into the host cell chromosome.1 RT
inhibitors are classified in nucleoside inhibitors (NRTIs) and
non-nucleoside inhibitors (NNRTIs). The NRTIs are chain-
terminators and interact competitively with the catalytic site of
the RT, whereas NNRTIs interact with an allosteric site adjacent
to the catalytic site of RT. Consequently, they are highly
susceptible to the development of viral resistance because
mutations in RT result in the preservation of the enzymatic
function while impairing inhibitor binding. NNRTIs are impor-
tant components of highly active antiretroviral therapy (HAART)
strategies, together with NRTIs and protease inhibitors (PI).2
NNRTIs can be grouped into two main classes: the so-called
first generation NNRTIs (e.g., nevirapine, 1, Chart 1), which
are very susceptible to single-point resistance mutations within
RT, and the second generation NNRTIs (e.g., efavirenz, 2, Chart
1). These latter are characterized by higher potency, a broader
spectrum of activity against HIV-1 variants, and more favorable
pharmacokinetic properties. However, a rapid development of
drug-resistant strains was observed upon introduction of both
first and second generation drugs in therapy. Because most
NNRTIs share a common binding site (with the exception of
TSAO compounds and congeners),3 one or two mutations
selected by a particular drug are able to confer cross resistance
to the entire class.4 Although NNRTIs are all considered by
definition noncompetitive inhibitors of RT, recent studies4,5 have
shown a peculiar mechanism of action for some of the second
generation NNRTIs. Investigation of the kinetics of efavirenz
binding to RT, by determination of equilibrium dissociation
constants and microscopic rates of binding and dissociation,
evidenced that this ligand preferentially interacted with RT
complexed with both the DNA and deossinucleotide triphosphate
(dNTP) substrates. It displayed lower affinity for the binary RT:
DNA complex and negligible affinity for the free enzyme.
Recently, we described the development of a new class of
NNRTIs6 whose prototype was the pyrrolobenzoxazepinone
(PBO) 3a (Chart 1). For this compound, a significant antiviral
efficacy was combined to a limited spectrum of activity.7 Later
on, a thorough exploitation of structure-activity relationship
(SAR) trends, aimed at the discovery of more potent and broad-
spectrum NNRTIs allowed us the identification of a novel class
* To whom correspondence should be addressed. Phone: 0039-0577-
234172. Fax: 0039-0577-234333. E-mail:campiani@unisi.it.
†
European Research Centre for Drug Discovery and Development
(NatSynDrugs).
‡
DFCTsUniversita` degli Studi di Siena.
DCF&TsUniversita` degli Studi di Napoli “Federico II”.
DCsUniversita` degli Studi di Siena.
§
|
IGMsIstituto di Genetica Molecolare, Pavia.
DSP&BCsUniversita` di Roma Tor Vergata.
Sigma-Tau Industrie Farmaceutiche Riunite spa.
#
3
10.1021/jm801395v CCC: $40.75
2009 American Chemical Society
Published on Web 01/26/2009

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1225
Table 1. Inhibition (Ki, µM) of HIV-1 wt and HIV-1 Mutant RT Enzymes, Containing the Single Amino Acid Substitutions L100I, K103N, Y181I,
V106A, V179D, and Y188La
Ki (µM)
compd
wt
L100I
K103N
Y181I
V106A
NTb
V179D
Y188L
NTb
(()-5
0.1 ( 0.01
0.25 ( 0.02
0.11 ( 0.01
0.4 ( 0.03
0.25 ( 0.02
0.3 ( 0.02
1.5 ( 0.2
0.8 ( 0.1
7 ( 1
<10
0.3 ( 0.02
3.6 ( 0.4
2 ( 0.1
2 ( 0.1
0.1 ( 0.01
(()-6
NTb
<10
1.8 ( 0.2
<10
(()-7
0.8 ( 0.1
9 ( 1
0.12 ( 0.01
<10
NTb
NTb
nevirapine
efavirenz
36 ( 4
10 ( 1
0.04 ( 0.006
18 ( 2
0.38 ( 0.04
0.03 ( 0.005
0.16 ( 0.02
0.15 ( 0.01
a All the data listed were compared to the corresponding test results for nevirapine, performed at the same time. Each value is the mean of at least three
experiments ( SD. b NT, not tested.
Figure 1. (A) Residual activity of HIV-1 RT tested with PBOs and PPOs (10 µM). RTwt is represented in white bars, K103N in light gray bars,
L100I in dark gray bars, and V179D in black. (B-D) Decrease of Kiapp (µM) of (S)-(+)-5 (circles) and (S)-(-)-7 (triangles) vs K103N (B), L100I
(C), and V179D (D). HIV-1 RT was tested on poly (rA)/oligo dT in presence of the different derivatives under the conditions described in the
Supporting Information in the presence of different fixed concentrations of TTP (1.5, 2.5, 10, and 20 µM). The Kiapp for different inhibitors at each
substrate concentrations were calculated from eq 1 as described in the Supporting Information. The variation of the Kiapp values as a function of the
TTP concentrations was analyzed according to eq 2 (in the Supporting Information) to calculate the true Kiter values. (E) Variation of Ki values for
the ternary complex. Each inhibitor was tested vs RTwt (white), K103N (light gray), and V179D (black).
of inhibitors typified by the pyrrolopyridooxazepinone (PPO)
3b (Chart 1). These analogues were potent antiviral agents
selectively targeting the HIV-1 RT ternary complex, and
potentially useful in combination with NNRTIs selectively
binding the free enzyme or the binary complex.8 Recently, we
further improved the pharmacological profile of PBOs by
synthesizing a series of C6-modified derivatives of 3a capable
of specifically targeting the highly conserved amino acid
residues within the ꢀ12-ꢀ13 hairpin (F227, W229, M230). The
resulting C6-extended analogues (typified by 4, Chart 1) were
identified as novel NNRTIs characterized by high antiviral
efficacy and improved mutant resilience.9 Herein we describe
the chemical and biological characterization of PBOs and novel
PPOs. Antiviral activity and drug resistance profiles of racemates
((()-5,9 (()-6, and (()-7, Chart 1) are compared to those of
the pure enantiomers ((S)-(+)-5, (R)-(-)-5, (S)-(-)-7, and (R)-
(+)-7) vs RT wild type and its mutant forms (K103N, L100I,
V179D, Tables 1-2, Table 1 of the Supporting Information
(SI), and Figures 1-3). A stereoselective interaction of the pure
enantiomers with the HIV-1 RT, which also correlates with
cellular assays, was demonstrated and rationalized through a
molecular modeling study.
Biological Results and Discussion
In enzymatic assays, compounds (()-5-7 demonstrated
good inhibitory properties against RT wild type and its mutant
forms (L100I, K103N, Y181I, V106A, V179D, and Y188L,
Table 1). In general, compounds (()-5 and (()-7 were more
potent than (()-6. Therefore they were selected for further
biological studies, their enantiomers were separated by HPLC
techniques, and the absolute configuration was determined
by X-ray analysis (Figure 1 SI and Figure 2 SI).
When the residual activity of wild type and mutant forms of
HIV-1 RT was tested in the presence of (()-5, (()-7, and their

1226 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Brief Articles
Figure 2. (A) Values of fold resistance derived from the ratio Kitermutant/Kiterwt. (B) Values of the kofftermutant/koffterwt representing the fold
increase in rate of dissociation. (C) Values of the konterwt/kontermutant representing the fold decrease in rate of association.
Figure 3. Docked structures of (S)-(+)-5 (A), and (S)-(-)-7 (B), (S)-(-)-7 in comparison with the experimentally found structure of TMC278 (C)
in the 3D crystal structure of HIV-1 RT (PDB code 2ZD1). Hydrogens are omitted for sake of clarity. (S)-(+)-5 and (S)-(-)-7 are displayed with
white sticks, TMC278 is displayed with magenta sticks, and key interacting residues are shown in blue or orange if belonging to the primer grip
region. The figure was created using Chimera software.11
enzyme (Kifree) values were almost equal to those for the binary
Table 2. Inhibition of HIV-IIIB Replication and Cytotoxicity in C8166
Cells
complex [E:DNA] (Kibin), whereas the Kiter values were signifi-
cell. assaysa
cantly lower. These data clearly indicate a higher affinity of
d
compd
(()-5
(R)-(-)-5
(S)-(+)-5
(()-7
(R)-(+)-7
(S)-(-)-7
8-ClTIBO
AZT
EC50 (µM)b
TC50 (µM)c
TI50
(S)-(+)-5 and (S)-(-)-7 for the catalytically competent ternary
complex of HIV-1 RT. In particular, we calculated the Kifree
/
0.1500
4.7000
0.0440
0.0690
1.9400
0.0500
0.0300
0.0019
0.0027
40.300
60.880
106.21
12.600
0.0188
111.39
50.10
<1
268.66
12.950
2413.0
Kiter (Table 1 SI and Figure 1E) in order to estimate the
magnitude of the decrease of Ki values for the ternary complex
with respect to the free enzyme for the mutant forms. It resulted
that (()-5 showed the highest selectivity toward the K103N
(Kifree/Kiter ) 50.0) enzyme, whereas (()-7 and (S)-(-)-7 showed
the highest selectivity for the L100I mutant (Kifree/Kiter ) 20.0).
The selectivity of (()-5 for V179D (Kifree/Kiter ) 23.3) was
similar to (S)-(+)-5 (Kifree/Kiter ) 26.0). We also compared the
Kiter mutant/Kiter wt for (()-5, (()-7, (S)-(+)-5, and (S)-(-)-7
(Figure 2A, in the form of bar plots) to determine the relative-
fold resistance. When compared to the wild type RT, all the
compounds showed very low resistance values (Kitermutant/
Kiterwt e 3) with the exception of V179D mutant toward (S)-
(-)-7 (-fold resistance values of 16.7). An inhibitor could show
a lower affinity for its target enzyme by either a decreased
association rate (kon), or an increased dissociation rate (koff), or
both. To elucidate the mechanism responsible for the observed
resistance toward our compounds, equilibrium binding experi-
ments were performed (as described in Supporting Information)
with the aim of determining the association (kon) and dissociation
(koff) rates for all the inhibitors to the different RT forms (free
enzyme, binary complex, and the catalytically competent ternary
complex) along the enzyme’s reaction pathway, with HIV-1 RT
wild type and L100I, K103N, and V179D mutants. The
calculated kon and koff values (Table 1 SI) for the binary and
ternary complexes were then compared to each other and to
182.60
<103.19
<2227.2
1670.0
<526.32
<370.37
efavirenz
<1
a All the data listed were compared to the corresponding test results for
AZT, which served as the treated control, and 8-ClTIBO performed at the
same time. b The EC50 value is the test drug concentration that produces a
50% survival of HIV-1 infected cells relative to uninfected untreated controls
(e.g., in vitro anti-HIV-1 activity). c TC50 value is the test drug concentration
that results in a 50% death of uninfected untreated control C8166 cells
(e.g., cytotoxicity of the test drug). d TI50 therapeutic index (TC50/EC50).
(R)- and (S)- enantiomers (Figure 1A), (S)-(+)-5, (S)-(-)-7, and
(()-5 significantly inhibited HIV-1 RT, both in its wild type
and mutant forms, whereas the enantiomers (R)-(-)-5 and (R)-
(+)-7 were almost inactive. These results indicate a strictly
stereoselective mode of inhibition. (S)-(+)-5 and (S)-(-)-7
showed an increase in potency (reduction of the apparent
inhibition constants Kiapp values), which was dependent on the
thymidine triphosphate (TTP) concentrations (Figure 1B-D).
From these data, it was possible to calculate the true dissociation
constant (Kiter) for binding of each inhibitor to the ternary
complex between the enzyme and its substrates nucleic acid
and dNTP. The derived values are listed in Table 1 SI. For all
the inhibitors tested, the apparent binding constant for the free

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1227
the corresponding values for the free enzyme. The values of
experimental data (SARs and site-directed mutagenesis). In our
previous paper,9a SAR analysis demonstrated that the PBO
scaffold itself and the pendant phenyl at the C6 position are
both involved in the interaction with the enzyme. Moreover,
the elongation of the pendant branch with a phenyloxymethyl
moiety was demonstrated to be crucial for activity. On the basis
of these experimental data, we discarded all the solutions in
which either the PBO scaffold or the pendant moiety at C6 did
not take direct contact with the enzyme binding site. In regards
to the (S)-enantiomers, this criterion allowed retention of only
the solutions calculated for the pseudoequatorial (S)-(+)-5, and
(S)-(-)-7 in complex with RT structure coded 2ZD1.10 This
latter is the enzyme conformation in which the inspected ligands
can better accommodate themselves. Accordingly, docking on
this 3D binding site structure produced the lowest ∆GAD4 values
(see Table 2 SI) with respect to the other docking experiments.
A close analysis of the computational results for the pseu-
doequatorial (S)-(+)-5 revealed a binding motif with the benzo-
fused ring of (S)-(+)-5 in proximity of the L100 side chain,
engaged in a T-shaped interaction with Y318, while the pyrrole
ring establishes favorable van der Waals contacts with V179
and V106 (Figure 3A). Consequently, the ethyl at C6 was in
close contact with L100, while the pendant phenyl ring was
embedded in the aromatic cleft delimited by Y181, Y188, F227,
and W229 residues establishing multiple charge-transfer interac-
tions. This region is flanked on one side by the ꢀ9-ꢀ10 hairpin
and on the other by the so-called primer grip region (ꢀ12-ꢀ13
hairpin), which is known to be directly involved in the catalytic
polymerase activity of HIV-1 RT.9a The phenyloxymethyl
branch is in close proximity with the primer grip region and
engages a T-shaped interaction with Y183 side chain, which is
reinforced by the electron-withdrawing meta-chlorine atom. This
latter residue was demonstrated to be crucial for the enzyme
polymerase activity being part of the YMDD motif in the
ꢀ9-ꢀ10 hairpin.10 In regard to the C6-extended PPO analogue
7, the enantiomers were separated and (S)-(-)-7 was found as
the active enantiomer. Docking studies highlighted binding
interactions for (S)-(-)-7 similar to (S)-(+)-5 (Figure 3B).
Interestingly, when comparing the predicted poses for both (S)-
(+)-5 and (S)-(-)-7 with the experimental one found for
TMC278 cocrystallized with HIV-1 RT in 2ZD1, an impressive
resemblance was found despite the chemical diversity (Figure
3C). It is worth noting that the above-described binding poses
were not detected in the other used X-ray structures due to a
different conformation of the ꢀ9-ꢀ10 hairpin that occludes the
aromatic tunnel formed by the primer grip and the hairpin itself
(see Figure 3 SI). If compared with the results obtained for the
(S)-enantiomers, a diverse behavior was observed for the (R)-
ones. In fact, regardless the enzyme conformation, the pheny-
loxymethyl branch did not allocate itself in the NNRTIs binding
site and is rather solvent exposed. Moreover, in the 2ZD1
conformation, the lowest energy pose of both (R)-(-)-5 and
(R)-(+)-7 does not allow any interaction with the residues in
both the primer grip region and the ꢀ12-ꢀ13 hairpin (Figure 4
of Supporting Information). Visual inspection of the crystal
structure of HIV-1 RT in complex with AZTMP-terminated
DNA reveals that the aforementioned primer grip region is
placed underneath the DNA. However, this portion in the
predicted (S)-PBO/enzyme and (S)-PPO/enzyme complexes
sterically hampers the interaction with the DNA (Figure 5 SI).
Therefore, it is likely that (S)-(+)-5 and (S)-(-)-7 exert their
inhibitory activity against HIV-1 RT by stabilizing a specific
conformation of the primer grip region and by preventing its
ter
koff mutant/koffterwt, indicating the -fold increase in the dis-
sociation constant (koff), are shown in Figure 2B. The values of
the konterwt/kon mutant, measuring the -fold decrease in the
ter
association constant (kon) for the different RT mutants, are shown
in Figure 2C. As can be seen, (()-7 and (S)-(-)-7 showed no
significant variations in their koff rates in the case of the mutant
K103N, whereas the L100I and V179D mutations caused an
increase in the koff rates of either (()-7 or (S)-(-)-7, respectively.
Compounds (()-5 and (S)-(+)-5 showed an increase in their
dissociation rates for all the mutants tested (Figure 2B).
Remarkably, all the compounds (with the only exception of
(()-7 toward K103N) showed increased association rates to the
mutants with respect to the wild type RT (Figure 2C), as
indicated by the -fold increase in kon values <1. Thus, the
relatively low level of resistance of these inhibitors (Figure 2A)
can be explained by faster dissociation rates from the mutants
(Figure 2B), which are in turn compensated by higher associa-
tion rates (Figure 2C).
Cellular tests were performed in order to evaluate ability of
inhibiting syncytia formation on C8166 cells infected with wild-
type HIV-1IIIB virus (Table 2). These tests were in line with
enzymatic assays, and confirmed better activity of the S
enantiomers. (S)-(+)-5 and (S)-(-)-7 showed a very potent
antiviral efficacy in C8166 cells, similar to that of 8-ClTIBO.
They also exhibited extremely favorable therapeutic indexes,
being endowed with low toxicity for C8166 noninfected cells
(TC50 between 106.2 and 11.4 µM).
Molecular Modeling Studies
The binding interactions established by our PBOs with wild
type HIV-1 RT were probed in previous studies6 and led to the
rational design of new derivatives (typified by 4), characterized
by an extended substitution at the C6-pendant phenyl ring.9b
Such a modification allowed a specific targeting of highly
conserved residues (e. g., W229), thus resulting in a lower
sensitivity to resistance mutations. In the same studies, the (S)-
enantiomer of these compounds was predicted to be the active
one.9b Moreover, the isosteric switch from the PBO scaffold to
the PPO tricyclic system resulted in new derivatives still
endowed with inhibitory activity against HIV-1 RT and some
of its mutants.8
Herein, we confirm our previous assumptions for the C6-
extended PBO analogue 5 by resolving the racemate into the
pure enantiomers, and we present two new PPO C6-extended
analogues, 6 and 7, of which only the (S)-enantiomer was
demonstrated to be active. In the present study, the reasons for
the stereoselective binding of 5 and 7 in the HIV-1 RT allosteric
binding site were investigated using a well-known ligand-receptor
docking approach (Autodock program (vs 4.0)). The three-
dimensional (3D) structure of wild type HIV-1 RT has been
reported in complex with several inhibitors and, so far, more
than 50 structures have been deposited on the Protein Data Bank
(PDB). To take into account the intrinsic plasticity displayed
by RT in the interaction with different NNRTIs, we decided to
use the nine highest resolution X-ray complexes (see Supporting
Information for details) to dock both the pseudoaxial and
pseudoequatorial pendant phenyl ring conformations of the
aforementioned ligands. A first analysis of the docking results
was made by taking into account the binding free energies
(∆G AD4) associated to the best (lowest energy) predicted
ligand-enzyme complexes for the nine X-ray structures (Table
2 SI). Then the lowest energy conformations of each calculated
ligand-enzyme complex was visually inspected and related to

1228 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Brief Articles
7, enantiomers crystallization, X-ray crystallography, biological
assays, and molecular modeling. This material is available free of
charge via the Internet at http://pubs.acs.org.
relocation. Consequently, the inactivity of the (R)-isomers may
be due to the lack of interaction with the primer grip region of
RT.
Conclusions
References
(1) De Clercq, E. HIV Inhibitors targeted at the reverse transcriptase. AIDS
Res. Hum. RetroVirus 1992, 8, 119–134.
We identified (S)-(+)-5 and (S)-(-)-7 as stereoselective
inhibitors of HIV-1 RT, and specific drug resistance RT
mutations (K103N, L100I and V179D) were demonstrated to
differentially affect enzyme sensitivity to the active compounds
and their racemic forms. Moreover, a selective binding for the
catalytically competent ternary complex of HIV-1 RT was also
demonstrated for these ligands. Cellular assays substantially
confirmed activity of the compounds and are in line with
stereoselectivity of interaction with HIV-1 RT. Molecular
modeling studies provided an explanation of this behavior at
the molecular level.
(2) Kauffmann, G. R.; Cooper, D. A. Antiretroviral therapy of HIV-1
infection: Established treatment strategies and new therapeutic options.
Curr. Opin. Microbiol. 2000, 3, 508–514.
(3) Tomassi, C.; Van Nhien, A. N.; Marco-Contelles, J.; Balzarini, J.;
Pannecouque, C.; De Clercq, E.; Soriano, E.; Postela, D. Synthesis,
anti-HIV-1 activity, and modeling studies of N-3 Boc TSAO com-
pound. Bioorg. Med. Chem. Lett. 2008, 18, 2277–2281.
(4) Campiani, G.; Ramunno, A.; Maga, G.; Nacci, V.; Fattorusso, C.;
Catalanotti, B.; Morelli, E.; Novellino, E. Non-nucleoside HIV-1
reverse transcriptase (RT) inhibitors: past, present, and future perspec-
tives. Curr. Pharm. Des. 2002, 8, 615–657.
(5) Maga, G.; Ubiali, D.; Salvetti, R.; Pregnolato, M.; Spadari, S. Selective
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Chemother. 2000, 44, 1186–1194.
(6) Campiani, G.; Morelli, E.; Fabbrini, M.; Nacci, V.; Greco, G.;
Novellino, E.; Ramunno, A.; Maga, G.; Spadari, S.; Caliendo, G.;
Bergamini, A.; Faggioli, E.; Uccella, I.; Bolacchi, F.; Marini, S.;
Coletta, M.; Nacci, A.; Caccia, S. Pyrrolobenzoxazepinone derivatives
as non-nucleoside HIV-1 RT inhibitors: Further structure-activity
relationship studies and identification of more potent broad-spectrum
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(7) Locatelli, G. A.; Campiani, G.; Cancio, R.; Morelli, E.; Ramunno,
A.; Gemma, S.; Hubscher, U.; Spadari, S.; Maga, G. Effects of drug
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(8) Maga, G.; Ramunno, A.; Nacci, V.; Locatelli, G. A.; Spadari, S.;
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Experimental Procedures
The synthesis of compounds (()-6 and (()-7 was performed
according to Scheme 1 as reported in SI. We describe herein the
synthesis of the final compounds (()-6 and (()-7.
(()-6-Ethyl-6-[4-(phenyloxymethyl)phenyl]-6,7-dihydropyr-
rolo[1,2-d]pyrido[3,2-b][1,4]oxazepin-7-one ((()-6). To a solution
of phenol (22 mg, 0.23 mmol) in dry tetrahydrofuran (2.0 mL),
sodium tert-butoxide (30 mg, 0.31 mmol) was added, and the
resulting mixture was heated to 40 °C. After 1 h, a solution of
bromide 9 (60 mg, 0.15 mmol) in dry terahydrofuran (2.0 mL) was
slowly added, and the resulting mixture was stirred overnight. The
solvent was removed in vacuo, and the residue was taken up with
dichloromethane. The organic phase was washed with brine, dried,
and concentrated. The residue was purified by means of flash
chromatography (dichloromethane/methanol 19:1) to give (()-6 as
a white amorphous solid (73% yield): 1H NMR (CDCl3) δ 1.08 (t,
3H, J ) 7.4 Hz), 2.42 (q, 2H, J ) 7.5 Hz), 4.92 (s, 2H), 6.47 (m,
1H), 6.75-6.79 (m, 1H), 6.89-6.96 (m, 2H), 7.00-7.05 (m, 1H),
7.13-7.42 (m, 8H), 7.49 (m, 1H), 8.08 (m, 1H). Anal. (C26H22N2O3)
C, H, N.
(9) (a) Fattorusso, C.; Gemma, S.; Butini, S.; Huleatt, P.; Catalanotti, B.;
Persico, M.; De Angelis, M.; Fiorini, I.; Nacci, V.; Ramunno, A.;
Rodriguez, M.; Greco, G.; Novellino, E.; Bergamini, A.; Marini, S.;
Coletta, M.; Maga, G.; Spadari, S.; Campiani, G. Specific targeting
highly conserved residues in the HIV-1 reverse transcriptase primer
grip region. Design, synthesis, and biological evaluation of novel,
potent, and broad spectrum NNRTIs with antiviral activity. J. Med.
Chem. 2005, 48, 7153–7165. (b) Zanoli, S.; Gemma, S.; Butini, S.;
Brindisi, M.; Joshi, B. P.; Campiani, G.; Fattorusso, C.; Persico, M.;
Crespan, E.; Cancio, R.; Spadari, S.; Hu¨bscher, U.; Maga, G. Selective
targeting of the HIV-1 reverse transcriptase catalytic complex through
interaction with the “primer grip” region by pyrrolobenzoxazepinone
non-nucleoside inhibitors correlates with increased selectivity towards
drug-resistant mutants. Biochem. Pharmacol. 2008, 76, 156–168.
(10) Das, K.; Bauman, J. D.; Clark, A. D., Jr.; Frenkel, Y. V.; Lewi, P. J.;
Shatkin, A. J.; Hughes, S. H.; Arnold, E. High-resolution structures
of HIV-1 reverse transcriptase/TMC278 complexes: Strategic flexibility
explains potency against resistance mutations. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 1466–1471.
(()-6-Ethyl-6-[4-[(3-chlorophenyloxy)methyl]phenyl]-6,7-di-
hydropyrrolo[1,2-d]pyrido[3,2-b][1,4]oxazepin-7-one ((()-7).
(()-7 was obtained following the procedure described for (()-6.
The residue was purified by flash chromatography (dichloromethane/
methanol 19:1) to give (()-7 as a white amorphous solid (69%
yield): 1H NMR (CDCl3) δ 1.09 (t, 3H, J ) 7.5 Hz), 2.43 (m, 2H),
4.93 (s, 2H), 6.48 (m, 1H), 6.75-6.79 (m, 1H), 6.89-6.94 (m,
2H), 7.00-7.05 (m, 1H), 7.13-7.39 (m, 7H), 7.49 (m, 1H), 8.09
(m, 1H); 13C NMR (75 MHz, CDCl3) δ 8.9, 33.7, 69.7, 94.4, 112.0,
113.5 115.5, 121.5, 122.3, 123.4, 126.8, 127.3, 128.1, 130.5, 132.9,
134.3, 135.1, 136.9, 137.5, 141.9, 143.9, 144.7, 159.5, 189.4; ESI-
MS m/z 445 (M + H)+, 466 (100) (M + Na)+. Anal.
(C26H21ClN2O3) C, H, N.
Acknowledgment. We thank the European Research Centre
for Drug Discovery and Development for financial support. S.Z.
is the recipient of a Buzzati-Traverso Fellowship.
(11) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF ChimerasA
Visualization system for exploratory research and analysis. J. Comput.
Chem. 2004, 25, 1605–1612.
Supporting Information Available: Elemental analyses for final
compounds, Table for enzymatic assays, chemistry, experimental
procedures for intermediates, HPLC separation of (()-5 and (()-
JM801395V