Pyrrolobenzoxazepinone derivatives as non-nucleoside HIV-1 RT inhibitors: Further structure-activity relationship studies and identification of more potent broad-spectrum HIV-1 RT inhibitors with antiviral activity

Article abstract of DOI:10.1021/jm990150o

Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose prototype is (±)-6-ethyl-6-phenylpyrrolo[2,1- d][1,5]benzoxazepin-7(6H)-one (6

Full text of DOI:10.1021/jm990150o

4462
J . Med. Chem. 1999, 42, 4462-4470
P yr r oloben zoxa zep in on e Der iva tives a s Non -Nu cleosid e HIV-1 RT In h ibitor s:
F u r th er Str u ctu r e-Activity Rela tion sh ip Stu d ies a n d Id en tifica tion of Mor e
P oten t Br oa d -Sp ectr u m HIV-1 RT In h ibitor s w ith An tivir a l Activity
Giuseppe Campiani,*,§ Elena Morelli,| Monica Fabbrini,| Vito Nacci,| Giovanni Greco, Ettore Novellino,
Anna Ramunno, Giovanni Maga,‡ Silvio Spadari,‡ Giuseppe Caliendo, Alberto Bergamini,† Emanuela Faggioli,†
Ilaria Uccella,† Francesca Bolacchi,† Stefano Marini,X Massimiliano Coletta,X Angelo Nacca,∇ and Silvio Caccia∇
Dipartimento di Scienze Farmaceutiche, Facolta’ di Farmacia, Universita’ degli Studi di Salerno, via Ponte don Melillo,
84084 Fisciano (SA), Italy, Dipartimento Farmaco Chimico Tecnologico, Facolta’ di Farmacia, Universita’ degli Studi di Siena,
via Aldo Moro, 53100 Siena, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, Universita’ di Napoli “Federico II”,
via D. Montesano 49, 80131 Napoli, Italy, Dipartimento di Sanita’ Pubblica e Biologia Cellulare (DSP&BC) and Dipartimento
di Medicina Sperimentale e Scienze Biochimiche (DMSSB), Universita’ degli Studi di Roma “Tor Vergata”,
via Tor Vergata 135, 00133 Roma, Italy, Istituto di Genetica Biochimica ed Evoluzionistica (IGBE) - CNR,
via Abbiategrasso 207, 27100 Pavia, Italy, and Istituto di Ricerche Farmacologiche “Mario Negri”, via Eritrea 62,
20157 Milano, Italy
Received March 30, 1999
Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency
virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose
prototype is (()-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-7(6H)-one (6). Docking studies
based on the three-dimensional structure of RT prompted the synthesis and biological evaluation
of novel derivatives and analogues of 6 featuring a meta-substituted phenyl or a 2-thienyl
ring at C-6 and a pyridine system in place of the fused-benzene ring to yield pyrrolopyridoox-
azepinones (PPOs). Compared with the lead 6 and nevirapine, several of the synthesized
compounds (PBOs 13a -d and PPOs 13i-k ) displayed higher inhibitory activity against wild-
type RT and clinically relevant mutant RTs containing the single amino acid substitutions
L100I, K103N, V106A, Y181I, and Y188L. The most potent inhibitors were further evaluated
for in vitro antiviral activity on lymphocytes and monocyte-macrophages, for cytotoxicity on a
panel of cell lines, and for potential synergistic antiviral activity with AZT. Pharmacokinetic
studies performed on 13b, 13c, and 13i showed that these compounds achieve high concentra-
tions in the brain. The results of the biological and pharmacokinetic experiments suggest a
potential clinical utility of analogues such as 13b-d , 13i, and 13j, in combination with
nucleoside RT inhibitors, against strains of HIV-1 bearing those mutations that confer resistance
to known NNRTI.
In tr od u ction
nucleoside inhibitors (NRTIs) that interact competi-
tively with the catalytic site of the RT and the non-
nucleoside inhibitors (NNRTIs) that inhibit the enzyme
by an allosteric interaction with a site adjacent to the
NRTI binding site. A competitive inhibitor (NRTI)
currently employed in the treatment of acquired im-
mune deficiency syndrome (AIDS) is AZT (1), but its
therapeutic usefulness is limited by poor brain penetra-
tion, emergence of viral resistance, toxicity, and low
specificity.6-8 NNRTIs have been found to bypass some
of the disadvantages inherent to nucleoside inhibitors,
but despite high specificity, their therapeutic effective-
ness is limited by the onset of skin rashes and the rapid
development of resistance. Representative compounds
of the NNRTI class are nevirapine (2),9 TIBO com-
pounds (3),10 thioureas such as trovirdine (4),11 and
pyridone derivatives (5).12 Although these compounds
are structurally unrelated, they interact with the allo-
steric site of RT by a similar three-dimensional ar-
rangement.13 We have recently described a new class
of NNRTIs based on a pyrrolobenzoxazepinone (PBO)
structure (6)14 (Chart 1). The most potent compound was
(()-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-
7(6H)-one (6) which had significant antiviral efficacy but
Reverse transcriptase (RT) is a key multifunctional
enzyme in the life cycle of the type-1 human immuno-
deficiency virus (HIV-1). The enzyme shows both RNA-
and DNA-dependent polymerase activities and is re-
sponsible 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 Although several targets for therapeutic
intervention have been identified in the life cycle of HIV-
1, the lack of RT activity in the eukaryotic cells has
made RT one of the most attractive targets for the
development of anti-HIV-1 agents.2-5 Its inhibition is
considered one of the most practicable approaches to
prevent the spread of infection. The RT inhibitors
known to date can be divided into two main classes:
* To whom correspondence should be addressed. Tel: +39-089-
962809. Fax: +39-089-962828. E-mail: campiani@unisa.it.
§ Universita’ degli Studi di Salerno.
| Universita’ degli Studi di Siena.
Universita’ degli Studi di Napoli “Federico II”.
‡ IGBE - CNR.
† DSP&BC, Universita’ degli Studi di Roma “Tor Vergata”.
X DMSSB, Universita’ degli Studi di Roma “Tor Vergata”.
∇ Istituto di Ricerche Farmacologiche “Mario Negri”.
10.1021/jm990150o CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/05/1999

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4463
Ch a r t 1
Sch em e 1
Ch a r t 2. Planned Modifications to Our Lead and the
Newly Designed Antiviral Agents
displayed by selected compounds and preliminary phar-
macokinetic studies.
Ch em istr y
The pyrrolo[2,1-d][1,5]benzoxazepinones (PBOs) and
pyrrolo[1,2-d]pyrido[3,2-b][1,5]oxazepinones
(PPOs)
13a -k were synthesized as shown in Scheme 1. Start-
ing from the 1-(2-hydroxyphenyl)pyrrole 8, previously
described by Artico,15 and from 1-(3-hydroxy-2-pyridyl)-
pyrrole 9, obtained starting from 2-amino-3-hydroxy-
pyridine 7, the esters 10a -i were prepared by O-alky-
lation with the appropriate ethyl R-bromo esters (see
refs 14 and 16a-c). Saponification of the ethyl ester
group (NaOH, H2O)11a-i followed by intramolecular
cyclization using phosphorus pentachloride gave ketones
12a -j. Treatment of the corresponding enolates with
alkyl halides finally yielded the desired oxazepinones
13a -k . The corresponding O-alkylated compounds were
obtained as byproducts (10-25% yield).
limited spectrum of activity. To improve the pharma-
cological properties of this lead, and to further explore
the potential of PBOs as anti-HIV agents, additional
compounds in this series were designed by docking
studies based on the structure of wild-type RT (Chart
2). The compounds were then tested as inhibitors of
RTwt and mutant RTs as well as for inhibition of HIV-1
replication in T4 lymphocytes and monocyte-macro-
phages. This strategy led to the identification of anti-
HIV-1 agents with greater inhibitory potency and
broader spectrum compared with 6 or nevirapine. We
also report synergistic antiretroviral activity with AZT
Com p u ter -Aid ed Design
The discovery of PBOs as a new class of NNRTIs
prompted molecular modeling studies aimed at super-

4464 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Brief Articles
Ta ble 1. Inhibition of HIV-1wt and HIV-1 Mutant RT
Enzymes, Containing the Single Amino Acid Substitutions
L100I, K103N, V106A, Y181I, and Y188L
6 through computer intensive methods would have still
left unsolved the question of which could be the pre-
ferred configuration for each designed virtual compound.
Thus, both NNBS/R-6 and NNBS/S-6 complexes were
analyzed as though the binding modes of the enanti-
omers of 6 had the same probability to occur. Inspection
of the NNBS/S-6 complex revealed room within the
NNBS that could be filled by a small-size substituent
inserted at the meta-position of the phenyl ring. Such
a substituent was expected to occupy the NNBS pocket
which accommodates the methyl group of nevirapine.
Interestingly, a meta-substituent should be tolerated
also on the pendant phenyl of the R-enantiomer because
it would fit into the NNBS locus hosting the chlorine of
9-Cl-TIBO18 and the proxymal part of the CH2OCH2-
CH2OH side chain of HEPT.19 Accordingly, the m-tolyl
and m-methoxyphenyl derivatives 13b and 13c were
proposed for synthesis. To validate our model, we
predicted that the p-tolyl derivative 13e would result
less active than its isomer 13c because (i) Tyr181 and
the Trp229 aromatic rings restrict the space accessible
by the p-methyl in the S-enantiomer and (ii) the
p-methyl in the R-enantiomer would collide with the
carbonyl atoms of His235 and Pro236. Since the Tyr181
side chain appeared in a suitable orientation for a
π-stacking interaction with the phenyl of S-6, electron-
withdrawing groups introduced on this ring as well as
its replacement with a more electron-deficient het-
eroaromatic ring (i.e. a thiophene20) were modifications
expected to promote a putative charge-transfer interac-
tions between the rings. Therefore, we planned the
synthesis of 13a , 13d , and 13f.
Ki (µM)a polyrA(dT)12-18
compd
WT
L100I K103N V106A Y181I Y188L
6
0.19
0.10
0.06
0.07
0.03
0.97
1.5
0.75
0.16
0.011
0.1
0.07
NT
1.7
NT
NT
7.7
3.9
0.4
>10
4
>10
4
0.97
NT
6.8
NT
NT
4
0.43
1.2
36
>10
1.5
>10
1.9
13a
13b
13c
13d
13e
13f
13g
13h
13i
0.07
0.022
1.1
0.4
NT
2.7
NT
NT
0.3
0.045
0.03
7
0.01
0.25
0.11
NT
3.0
NT
NT
0.07
0.09
0.4
0.3
NT
>10
NT
NT
1.5
0.475
1.5
18
NA
NA
0.022 0.04
0.093 0.09
0.021 0.044
13j
13k
nevirapine 0.4
9
10
a
Inhibition of HIV-1 RT activities. All the data listed were
compared to the corresponding test results for nevirapine, per-
formed at the same time. Each value is the mean of at least three
experiments. NT, not tested; NA, not active, inhibition e10% at
10 µM final drug.
imposing the lead compound 6 on nevirapine as it is
found in its crystallographic complex with RT.17 It was
felt that a pharmacophore-consistent alignment of these
two compounds would facilitate the derivation of a
model of the RT/6 complex and subsequent structure-
based design of novel inhibitors. We have recently
proposed14 that 6 binds in a conformation featuring the
pendant phenyl ring in an equatorial disposition. Since
both enantiomers of 6 (assayed as racemic mixture)
were superimposable on nevirapine in a “butterfly-like”
arrangement (Figure 1) we could not single out the
preferred configuration of this compound. The pharma-
cophore-based conformations of S-6 and R-6 were each
in turn placed into the non-nucleoside binding site
(NNBS) extracted from the crystal structure of RT/
nevirapine complex17 to yield the corresponding com-
plexes of NNBS/S-6 and NNBS/R-6 shown in Figure 2.
The following features of the two docking models are
worthy to be outlined. The pendant phenyl (fused-
pyrrole) ring of S-6 (R-6) is embedded within a hy-
drophic pocket made up by the side chains of Pro95,
Tyr181, Tyr188, Trp229, Leu100, and Leu234. The
pyrrole (phenyl) moiety is surrounded by backbone
atoms of His235, Pro236, Asp237, Lys101, and Lys102
and by side chains of Leu100, Lys103, and Val106. The
ethyl group projects toward Val179 and Gly190 back-
bone. No intermolecular hydrogen bonds involve the
oxygens of the ligand. The fused-benzene ring of both
enantiomers extends to the NNBS entrance delimited
by Glu138, Val179, Lys101, and Lys103 side chains. To
roughly estimate the degree of steric complementarity
between the docked ligand and the protein, we calcu-
lated the volume wherein the two partners of the
complex overlap (OVOL). The higher OVOL the lower
is the enzyme/inhibitor shape complementarity. This
volume is linked to steric conflicts that can be poten-
tially relieved by molecular dynamics and geometry
optimization. The OVOLs associated with the NNBS/
S-6 and NNBS/R-6 complexes (red contours in Figure
2) were not sufficiently dissimilar (13 and 4 Å3, respec-
tively) so as to conclude that only one complex (enan-
tiomer) had biological relevance. Indeed, predicting
differences in potency between the two enantiomers of
Further lead optimizations attempts assumed that
the fused-benzene ring is not a pharmacophore element
and that it may slightly worsen the steric match of both
R- and S-enantiomers with the NNBS. Particularly, the
OVOL calculated on the NNBS/S-6 complex is es-
sentially related to overlap of the fused-benzene ring
C-1 and C-2 hydrogens with the Lys101 carbonyl and
the Lys103 side chain atoms. Most of the OVOL associ-
ated with the NNBS/R-6 complex is due to steric
conflicts between the fused-benzene ring C-1 hydrogen
and the Tyr181 side chain. The possibility to remove
the fused-benzene was discarded owing to exceeding
synthetic difficulties. A more feasible modification to
reduce steric bulk at position 1 was the isosteric
replacement of the fused-benzene 1-CH with a nitrogen,
to give the PPO 13i. The pendant phenyl ring of 13i
was replaced by a 2-crotyl or a 2-isopentenyl to yield
compound 13g or 13h , respectively. The likelihood for
these 6-alkenyl derivatives to elicit inhibitory activity
was strongly supported by the finding that in the PBO
series changing the phenyl to a 2-isopentenyl produced
an inhibitor as active as the lead 6.14 Moreover, X-ray
crystallographic experiments have shown that the 2-iso-
pentenyl chain in 8-Cl-TIBO21 and 9-Cl-TIBO18 fills the
same locus of the enzyme hosting the nevirapine A ring.
Compounds 13j and 13k were prepared shortly after
the biological assays on 13c, 13d , and 13i and showed
improved activities over the lead 6. It was hoped, in fact,
that combining structural features beneficial to activity,
i.e., a m-tolyl or a 2-thienyl at position 6 with 1-CH/N
replacement, would synergistically increase the inhibi-
tory potency.

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4465
F igu r e 1. Overlay of the phenyl equatorial conformation of S-6 (left) and R-6 (right) on the RT-bound conformation of nevirapine.17
The pendant phenyl and the fused-pyrrole rings of S-6 match the pyridine A and C rings of nevirapine, respectively. The fitting
of the aromatic rings is inverted for R-6. In both alignments the ethyl group is superimposed on the cyclopropyl ring of nevirapine.
Compound 6 and nevirapine are depicted in light and dark gray, respectively.
F igu r e 2. NNBS/S-6 (left) and NNBS/R-6 (right) complexes obtained by replacing nevirapine with the pharmacophore-based
conformations of S-6 and R-6. Only a subset of residues of the NNBS is displayed for sake of clarity. Red mesh contours delimit
the overlap volume (OVOL) obtained by intersecting the ligand and the protein volumes.
Resu lts a n d Discu ssion
as active as 6 in our previous work.14 It is worth
outlining that insertion of a m-methyl on the phenyl
ring (so as to mimic the methyl group in nevirapine)
improves activity in the PBO series (compare 13c vs 6)
but is irrelevant in the PPO series (compare 13k vs 13i).
Taken together, these data suggest that PBOs can bind
as S-enantiomers (Figure 1 on the left). As stated, 13j
and 13k were designed by combining features favorable
for activity, i.e. the m-tolyl in 13c, the 2-thienyl in 13d ,
and the fused-pyrido ring in 13i. Although more potent
than the lead 6, these two “hybrids” did not however
exhibit synergistically increased efficacies resulting
from additive effects of structural modifications. If we
admit that the active configuration/binding mode of
PBOs and PPOs is dictated by the overall stereoelec-
tronic properties of each ligand, we can also explain why
13j and 13k were not so active as expected. In fact, if
the nevirapine A ring is mimicked by the aryl rings of
13c and 13d (binding as S-enantiomers) as well as by
the fused pyridine ring of 13i (binding as R-enantiomer),
none of the enantiomers of 13j and 13k can obviously
P r im a r y Tests. An tien zym a tic Assa ys. All the new
compounds described were tested in vitro against RTwt
and several mutant RTs containing the single amino
acid substitutions L100I, K103N, V106A, Y181I, and
Y188L, known to confer NNRTI resistance in treated
patients. The results are summarized in Table 2 as Ki
values. Most of the compounds showed improved efficacy
on RTwt with respect to the lead 6. Particularly, the
gain of potency obtained through 13a -d ,i-k varied
from 2- to 9-fold. The p-tolyl derivative 13e was found
14-fold less active than the m-tolyl isomer 13c in
accordance with our theoretical model. Contrary to our
expectations, 13f-h were devoid of appreciable activity.
Inactivity of the 2-crotyl and 2-isopentenyl derivatives
13g and 13h suggests that their 2-alkenyl chains are
not oriented similarly to the 2-isopentenyl group of
TIBO inhibitors within the NNBS,18,21 thus supporting
the hypothesis that active PPOs bind preferentially as
R-enantiomers (similarly to R-6 on the right in Figure
1). Conversely, the fused-benzene isoster of 13h resulted

4466 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Brief Articles
Ta ble 2. Inhibition of HIV-1 Infection in CEM-SS Cells,a Inhibition of HIV-IIIB Infection in C8166 Cells, and Cytotoxicity on NSO
Murine Cell Line, Daudi Human (DH) Cell Line, 3T3 Fibroblasts (3T3F) Murine Cell Line, and Normal Human Lymphocytes (HL)
CEM-SS cells
C8166 cells
TC50 (mM)e
compd
IC50 (µM)b
EC50 (µM)c
SId
IC50 (µM)b
EC50 (µM)c
SId
NSOf
DHg
3T3Fh
HLi
6j
4.9
2.0
0.2
119
11
NT
3.9
>2.0
>2.0
>1
0.47
0.072
0.04
0.35
0.115
NT
0.069
0.15
0.027
0.0019
11
28
5
338
95
NT
56
>13
>75
526
10
13.8
7.0
0.8
1.1
12.5
12.7
212
122
NT
0.024
0.72
0.23
>1
0.026
0.72
0.28
>1
0.033
0.71
0.21
>1
0.032
0.72
0.2
>1
NT
13a
13b
13c
13d
13f
13i
13j
13k
AZT
0.033
0.425
NT
0.65
0.054
1.2
52
NT
17.1
6.0
20.5
7.2
NT
NT
NT
26
NT
NT
NT
NT
111
16.6
26.7
>250
0.014
0.026
0.41
0.013
0.029
0.38
0.01
0.035
0.40
0.013
0.030
0.40
0.27
0.4
>100
a
Testing was performed by the National Cancer Institute’s Developmental Therapeutics Program, AIDS Antiviral Screening Program.
All the data listed were compared to the corresponding test results for AZT which served as the treated control, performed at the same
b
time. The IC50 value is the test drug concentration which results in a 50% survival of uninfected untreated control CEM-SS cells (e.g.
cytotoxicity of the test drug) or macrophages cells (C8166). c The EC50 value is the test drug concentration which produces a 50% survival
d
of HIV-1-infected cells relative to uninfected untreated controls (e.g. in vitro anti-HIV-1 activity). SI, selectivity index (IC50/EC50). e Blank
was subtracted from OD measured at 570 nm. All data reported are measured OD × 1000. Standard deviations are not reported but
g
never exceded 5% of the mean value. f OD measured in control row was 0.125 ( 0.015. OD measured in control row was 0.129 ( 0.018.
h
i
j
OD measured in control row was 0.131 ( 0.014. OD measured in control row was 0.114 ( 0.01. Reference 14. NT, not tested.
Ta ble 3. EC50 and Combination Index (CI) for the Anti-HIV-1 Compounds 13c and 13i in Association with AZT, Calculated Using
Both the Mutually Nonexclusive and the Mutually Exclusive Assumptionsa
EC50 (nM)
CIb
of compounds
alone
of compounds
with AZT
of AZT
with compounds
mutually exclusive
assumption
mutually nonexclusive
assumption
compd
none
13c
13i
400
3.25
3.17
425
54
11
17
0.040
0.302
0.040
0.303
a
b
C8166 cells were infected with HIV-IIIB. CI > 1, CI ) 1, and CI < 1 indicate antagonistic, additive, and synergistic activities,
respectively. The CI values were calculated at 50% antiviral activity using both the mutually nonexclusive and the mutually exclusive
forms of the equation.
position both favorable aromatic moieties into the same
NNBS region. With respect to our lead 6, the PBOs
13a -c and the PPO 13i were found much more active
against L100I, K103N, and V106A mutants. Of particu-
lar interest was the submicromolar potency elicited by
the 6-(2-thienyl) derivatives 13d and 13j against all
the considered mutant RTs. Mager has proposed that
conformational flexibility and overall size of the inhibi-
tor are the main determinants of binding affinity to
mutant forms of RT.22 Molecular mechanics calculations
performed in our laboratories revealed that the 2-thienyl
ring at the 6-position in compound 13d is more free to
rotate about the C-6-C-1′ bond compared with the
corresponding phenyl ring in 6 (unpublished observa-
tions). The remarkable performance of 13d and 13j
might depend on a greater capability to adapt their
conformation to changes in the shape of the NNBS
resulting from a single mutation.
Secon d a r y Tests. A subset of compounds with
promising antienzymatic profile in primary screening
was further evaluated for in vitro antiviral activity on
lymphocytes and monocyte-macrophages (Table 2), for
cytotoxicity on a panel of cell lines (Table 2), and for
potential synergistic antiviral activity with AZT (Table
3).
1. Cell Cu ltu r e Assa ys. On the CEM-SS cell line
(Table 2), 13c resulted practically equipotent, whereas
the remaining selected compounds (13a -d ,i-k ) turned
out to be more active compared with the lead 6 (13b
and 13k by 12- and 17-fold, respectively). The better
antiviral properties of the newly synthesized compounds
were confirmed on C8166 cells infected with HIV-III
Ba-L (Table 2), except for 13a , whose activity was found
slightly lower than that of 6.23 Remarkably, 13b and
13i showed 24- and 14-fold improvement of potency over
6. Testing of 13c and 13i on monocyte-macrophages
characterized these compounds as promising antiviral
agents in terms of efficacy (EC50 values of 42 and 43
nM, respectively) and low cytotoxicity (SI values of 826
and >46, respectively).
2. MTT Assa ys. To confirm the low cytotoxicity data
shown in cell culture assays, compounds 13a -c,i-k
were further screened in a MTT assay by using NSO
murine, Daudi human, and 3T3 fibroblast murine cell
lines and normal human lymphocytes (Table 2). In these
tests, the title compounds showed low toxicity, with the
exception of 13i and 13j whose toxicity is in the
micromolar range and is comparable with that exhibited
by the lead 6. Compound 16c stands out for its ex-
tremely low cytotoxicity (TC50 values > 1 mM).
3. Syn er gistic An tivir a l Activity w ith AZT. The
recent therapeutic developments of AIDS converge to
favor multidrug regimens which have shown to improve
the chances of success. A further improvement in
multidrug therapy would be to administer combinations
of drugs able to interact synergistically. This so-called
“second level” of combinations consists of drugs acting
at different sites within the same viral protein.24 Ac-
cording to this approach, we decided to investigate the
synergistic antiviral activity of our NNRTIs, with AZT
(NRTI). Two of the most active compounds (13c, 13i)
showed synergistic anti-HIV-1 activity (CI <1) when
tested in combination with AZT, whether the mutually
exclusive or nonexclusive assumption formulas were
used (Table 3).

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4467
was removed in vacuo, and the residue was extracted with
EtOAc. The organic layers were washed with 5% NaHCO3
solution and brine, dried, and evaporated. The crude product
was purified by silica gel column chromatography using
dichloromethane/ethyl acetate (8:2) as eluant. Fractions con-
taining the product were combined, dried, and recrystallized
by ethyl ether to give analytically pure 9 (4.7 g) as white
P h a r m a cok in etic Stu d ies. Initial pharmacokinetic
studies were done on compounds 13b and 13c. Only
trace amounts of the unchanged compounds were de-
tectable in whole brain (not in blood or plasma) within
60 min of oral dosing to mice (10-20 mg/kg, suspended
in 10% Tween 80 in water). This may be referred to an
incomplete absorption and/or an extensive first-pass
effect as a result of the water insolubility of these
compounds and their high lipophilicity. The log P values
calculated for 13b and 13c were 4.95 and 5.53, respec-
tively (see Experimental Section). After subcutaneous
injection (20 mg/kg) the two derivatives were measur-
able in whole brain (and trace amounts of 13b were also
detectable in plasma), but the concentrations were
relatively low and variable, particularly for the more
lipophilic 13c. The mean brain maximum concentrations
(observed 30 min after dosing) approximated 0.5 nmol/g
for 13c and 1.2 nmol/g for 13b, with coefficients of
variation approaching respectively 90% and 50%.25
Pharmacokinetic studies were successively extended
to 13i. This PPO derivative is less lipophilic (calculated
log P value 4.05) than 13b, as also indicated by the
different elution characteristics (see Experimental Sec-
tion). When given to mice orally (20 mg/kg), as a solution
in 10% Tween 80-5% ethanol in HCl, 10-3 M, it rapidly
reached the systemic circulation, although mean Cmax
(0.6 nmol/mL, 15 min after dosing) and AUCt were only
about 30% of those after the same dose subcutaneously.
This compound also showed good brain penetration,
with brain Cmax (8.0 ( 1.7 nmol/g, 15 min after subcu-
taneous dosing) and AUC (542 nmol/g min) about 4
times the corresponding plasma values (2.1 nmol/mL
and 141 nmol/mL min). Its elimination t1/2 was calcu-
lated from the 0.5-3 h time points of the subcutaneous
plasma and brain profiles and was about 65 min in both
tissues.26
1
prisms: IR (KBr) 2928 cm-1; H NMR (CD3OD) δ 6.08 (m, 2
H), 6.96 (dd, 1 H, J ) 7.9, 4.6 Hz), 7.21 (d, 1 H, J ) 7.9 Hz),
7.42 (m, 2 H), 7.75 (d, 1H, J ) 4.6 Hz). Anal. (C9H8N2O) C, H,
N.
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
10a -i. This procedure is illustrated for the preparation of (()-
R-[[2-(1H-pyrrol-1-yl)phenyl]oxy](m-fluorophenyl)acetic acid
ethyl ester (10a ). Sodium hydride (347 mg, 14.4 mmol) was
added to a solution of 1-(2-hydroxyphenyl)pyrrole (2.1 g, 13.1
mmol) in anhydrous THF at room temperature. The reaction
mixture was stirred for 1 h at room temperature, and then a
solution of ethyl R-bromo-m-fluorophenylacetate (3.0 g, 13.1
mmol) in anhydrous THF (20 mL) was added dropwise. After
16 h at room temperature, the solvent was removed in vacuo,
and the residue was taken up in dichloromethane. The organic
layer was washed with brine, dried, and concentrated. The
residue was purified by flash chromatography (dichloromethane/
hexanes, 2:1) to give 10a as a colorless oil: IR (neat) 1748 cm-1
;
1H NMR (CDCl3) δ 7.21 (m, 10 H), 6.42 (m, 2 H), 5.58 (s, 1 H),
4.21 (q, 2 H, J ) 7.6 Hz), 1.22 (t, 3 H, J ) 7.7 Hz). Anal.
(C20H18FNO3) C, H, N.
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
11a -i. This procedure is illustrated for the preparation of (()-
R-[[2-(1H-pyrrol-1-yl)phenyl]oxy](m-fluorophenyl)acetic acid
(11a ). The ester 10a (1.0 g, 29 mmol) was dissolved in 10 mL
of EtOH and 5% aqueous NaOH was added. The reaction
mixture was stirred at room temperature for 17 h, concen-
trated, and acidified with 1 N HCl. The aqueous phase was
extracted with EtOAc, and the organic layer was washed with
brine, dried, and concentrated. The residue was recrystallized
to give the acid 11a as colorless prisms: IR (Nujol) 1745 cm-1
;
1H NMR (CDCl3) δ 9.31 (br, s, 1 H), 7.17 (m, 10 H), 6.34 (m, 2
H), 5.49 (s, 1 H). Anal. (C18H14FNO3) C,H,N.
Gen er a l P r oced u r e for P r ep a r a tion of Oxa zep in on es
12a -d ,f-j. This procedures is illustrated for the preparation
of (()-6-(m-fluorophenyl)pyrrolo[2,1-d][1,5]benzoxazepin-7(6H)-
one (12a ). Phosphorus pentachloride (0.55 g, 2.3 mmol) was
added to a solution of acid 11a (0.64 g, 2.06 mmol) within 20
min. The reaction mixture was stirred at room temperature
for 5 h and then was poured into crushed ice, basified with
10% NaOH solution, and extracted with chloroform. The
organic layers were washed with brine, dried, and evaporated.
The residue was purified by flash chromatography (dichlo-
romethane) and recrystallized (hexanes). The title compound
Con clu sion s
With the goal to develop more potent and broad-
spectrum analogues within the series of oxazepinone RT
inhibitors, we investigated several structural features
for conferring optimum enzyme inhibition. Focusing
directly on a small number of structural alterations,
dictated by docking studies, we have been able to
identify analogues of 6 with a greatly improved phar-
macological profile, in terms of efficacy, broad spectrum,
and low cytotoxicity. The antiviral activity and the
synergistic antiviral properties with AZT suggest a
potential clinical utility of analogues such as 13b-d ,
13i, and 13j, in combination with NRTIs, against strains
of HIV-1 bearing those mutations that confer resistance
to the known NNRTIs.
was obtained as colorless prisms: IR (Nujol) 1741 cm-1 1H
;
NMR (CDCl3) δ 7.20 (m, 10 H), 6.49 (m, 1 H), 5.47 (s, 1 H).
Anal. (C18H12FNO2) C,H,N.
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
13a -k . This procedure is illustrated for the preparation of (()-
6-ethyl-6-(m-fluorophenyl)pyrrolo[2,1-d][1,5]benzoxazepin-7(6H)-
one (13a ). A solution of 12a (0.42 g, 1.43 mmol) in anhydrous
THF (2.6 mL) was added to a suspension of potassium hydride
(183 mg, 1.6 mmol, 35% in oil). The reaction mixture was
stirred for 1.5 h at room temperature, and then ethyl iodide
(231 mL, 2.9 mmol) was added. After an additional 15 h at
room temperature the solvent was removed, and the residue
was extracted with dichloromethane. The organic layers were
washed with brine, dried, and evaporated. The residue was
purified by flash chromatography (chloroform and hexanes,
1/1) and recrystallized to give 13a as colorless prisms: IR
(Nujol) 1697 cm-1; 1H NMR (CDCl3) δ 7.08 (m, 10 H), 6.44 (m,
Exp er im en ta l Section
For general experimental information, see ref 14. The
R-bromo esters used for the synthesis of compounds 10a -i
were synthesized by halogenation with N-bromosuccinimide
of the corresponding arylacetic acid ethyl esters, by using a
synthetic procedure described in refs 14 and 16, except for
R-bromobutyric acid ethyl ester which was purchased from
Aldrich.
1-(3-Hyd r oxy-2-p yr id yl)p yr r ole (9). To a solution of
2-amino-3-hydroxypyridine (7) (7.5 g, 68.1 mmol) in 250 mL
of glacial acetic acid, heated at 95-100 °C, was added 2,5-
dimethoxytetrahydrofuran (9.0 g, 68.1 mmol) in glacial acetic
acid (20 mL). The mixture was stirred for 30 min, the solvent
1 H), 2.34 (m, 2 H), 1.00 (t, 3 H, J ) 7.5 Hz). Anal. (C20H16
FNO2) C,H,N.
-
Molecu la r Mod elin g. Molecular modeling studies were
performed using the SYBYL program27 running on a Silicon
Graphics Iris Indigo XS24 workstation. The crystal structure
of the RT/nevirapine complex solved at 2.2 Å resolution by Ren

4468 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Brief Articles
et al.17 was retrieved from the Brookhaven Protein Data
Bank28 (entry code 1VRT). Nevirapine and the NNBS, this
latter consisting of a set of amino acids located within 10 Å
from any non-hydrogen atom of the bound inhibitor, were
extracted from the complex. Water molecules were deleted, and
hydrogens were added to the unfilled valences of the amino
acids. Nevirapine was removed, and the pharmacophore-based
conformations of S-6 and R-6 were each in turn placed into
the NNBS to yield the corresponding complexes of NNBS/S-6
and NNBS/R-6. Modeling of 6 and its alignment on nevirapine
are described in a previous study.14 Manipulation and display
of molecular volumes were realized with the MVOLUME
command of SYBYL. Semiempirical quantum-mechanics cal-
culations of benzene and thiophene LUMO energies were
performed using the AM129 and PM330 Hamiltonians avail-
able in the MOPAC program (version 6.0, QCPE, Indiana
University, Bloomington, IN) under default settings. The
calculated n-octanol/water log P values for compounds 13b,
13c, and 13i were obtained running the ClogP program
(version 2.0, Biobyte Corp., Claremont, CA).
HIV-1 RT RNA-Dep en d en t DNA P olym er a se Activity
Assa y. RNA-dependent DNA polymerase activity assay was
assayed as follows: a final volume of 25 µM contained reaction
buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.2 mg/mL BSA,
4% glycerol), 10 mM MgCl2, 0.5 µg of poly(rA):oligo(dT)10:1 (0.3
µM 3′-OH ends), 10 µM [3H]dTTP (1 Ci/mmol), and 2-4 nM
RT. Reactions were incubated at 37 °C for the indicated time;
20-µL aliquots were then spotted on glass fiber filters GF/C
which were immediately immersed in 5% ice-cold TCA. Filters
were washed twice in 5% ice-cold TCA and once in ethanol for
5 min and dried, and acid-precipitable radioactivity was
quantitated by scintillation counting.31
In h ibition Assa y. Reactions were performed under the
conditions described for the HIV-1 RT RNA-dependent DNA
polymerase activity assay. Incorporation of radioactive dTTP
into poly(rA):oligo(dT) was monitored in the presence of
increasing amounts of the inhibitors to be tested. Data were
then plotted according to Lineweaver-Burke and Dixon. For
Ki determinations an interval of inhibitor concentrations
between 0.2 Ki and 5 Ki was used. Experiments have been done
in triplicate. Experimental errors ((SD) were e10%.
In Vitr o An ti-HIV Assa ys. Cell Cu ltu r e Assa y. 1. CEM
Cell Lin e. The ability of the test compounds to protect HIV-
1-infected T4 lymphocytes (CEM cells) from cell death was
determined following the reported procedure.32 All compounds
were compared with a positive (AZT-treated) control performed
at the same time under identical conditions.
2. Oth er Cells. Mature macrophages were obtained by
incubating in 48-well plates (Costar, Cambridge, MA) 106
PBMC/mL of complete medium (containing Roswell Park
Memorial Institute (RPMI)-1640, penicillin 100 units/mL,
streptomycin 100 mg/mL, L-glutamine 0.3 mg/mL, and 20%
heat-inactivated fetal calf serum). After 7 days of incubation
in 5% CO2 at 37 °C, nonadherent cells (lymphocytes) were
removed by extensive washing. Using this method, the yield,
after removal of the nonadherent cells, was 105 macrophages/
well. Further details of this procedure are described else-
where.33 Macrophages obtained with this method are >95%
pure as detected by nonspecific esterase activity. C8166 is a
CD4+ T-cell line containing an HTLV-I genome of which only
the tat gene is expressed.34
3. Vir u s. A laboratory, lymphocyte-tropic strain of HIV-1
(HIV-1-IIIB) was used to infect C8166. A laboratory monocyte-
tropic strain of HIV-1 (HTLV-III Ba-L, also called HIV Ba-L)
was used to infect macrophages. Details of the characteristics
of the laboratory strain are reported elsewhere.35,36 The sources
of the HIV-1 laboratory lymphocyte-tropic and monocyte-tropic
strains were respectively the supernatants of H9/IIIB (H9)
cultures (H9 is a CD4+ T-cell line persistently infected with
HIV-1)35 and the supernatants of infected macrophages cul-
tures. Titration to determine the infectivity was performed in
human macrophages as previously described. The titer of the
virus stocks, expressed as 50% tissue culture infectious dose
(TCID50), was determined as previously described.37
4. An tivir a l Agen t. Zidovudine (AZT) was purchased from
Sigma Chimica.
5. Toxicity. Chemicals toxicity in C8166 was evaluated with
a procedure involving a colorimetric assay (MTT assay) that
monitors the ability of viable, but not dead, cells to reduce 3-(4-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
to a blue formazan product, which can be measured spectro-
photometrically. Details of this methods are described else-
where.38,39
6. Assa y of An tivir a l Activity. For evaluation of antiviral
activity in acutely infected C8166 cultures, the cells were
distributed into 15-mL polyethylene tubes at a concentration
of 90 × 103 cells/mL and exposed to 100 TCID50 of HIV in the
presence or absence of antiviral compounds. After 2 h incuba-
tion in 5% CO2 at 37 °C the cells were washed twice in PBS
and inoculated in each well of a 96-well plate (Falcon 3042,
Falcon, Basel, Switzerland) at a concentration of 35 × 103 cells/
200 µL of complete medium. The cells were subsequently
cultured in the presence of the same concentrations of drugs
as before for 5 days. The antiviral activity of the compounds
was assessed by quantifying the HIV-induced cytopathogenic-
ity by the MTT assay. The percentage of protection from the
cytopathic effect achieved by each compound in HIV-infected
cells measured by the MTT assay was calculated by the
following formula: (ODT)HIV - (ODC)HIV/(ODC)mock - (ODC)HIV
,
expressed in %, whereby (ODT)HIV is the optical density
measured with a given concentration of compound in HIV-
infected cells, (ODC)HIV is the optical density measured for the
control untreated HIV infected cells, and (ODC)mock is the
optical density measured for the control untreated mock
infected cells. The optical density at 490/650 nm was measured
using a plate reader. The assay to evaluate anti-HIV drug
efficacy in acutely infected mature macrophages has been
previously described.33 The antiviral activity of the compounds
was assessed by measuring HIV-p24 antigen production in the
supernatants of infected cultures as previously described35 by
using a commercially available HIV-antigen kit.
7. Im m u n oflu or escen ce Vir u s Bin d in g Assa y. Calcula-
tion of the 50% effective dose (ED50) and 50% inhibitory dose
(ID50) was done from pooled values in the effective dynamic
range of the antiviral and toxicity assays (5-95%) using the
median effect equation as previously described.40
Toxicity Tests. 1. Cell Lin es. All cell lines were obtained
from ATCC. The cells were cultured in RPMI-1640 supple-
mented with 5% FCS, 0.1 mM glutamine, 1% penicillin, and
streptomycin. Cells were grown in Nunc clone plastic bottles
(TedNunc, Roskilde, Denmark) and split twice weekly at
different cell densities according to standard procedure. 3T3
cells were grown as monolayer and split by using trypsin.
Peripheral blood mononuclear cells (MNC) were separated
from heparinized whole blood obtained from a healthy donor
on a Fycoll-Hypaque gradient as previously described.41 MNC
thus obtained were washed twice with RPMI-1640 supple-
mented with 10% FCS, glutamine, and antibiotics, suspended
at 200 000 viable cells/mL in medium containing, as mitogen,
5 µg/mL PHA (Sigma), and used in toxicity tests.
2. Ch em ica ls. MTT was purchased from Aldrich. It was
dissolved at a concentration of 5 mg/mL in sterile PBS at room
temperature, and the solution was further sterilized by filtra-
tion and stored at 4 °C in a dark bottle. SDS was obtained
from Sigma. Lysis buffer was prepared as follows: 20% w/v of
SDS was dissolved at 37 °C in a solution of 50% of each DMF
and demineralized water; pH was adjusted to 4.7 by adding
2.5% of an 80% acetic acid and 2.5% 1 N HCl solution.
3. Toxicity Exp er im en ts. Cells were plated at different
concentrations on flat-bottom 96-well microplates (0.1 mL/
well). Lymphocytes were plated out at 20 000 cells/well. 3T3
cells (murine fibroblast line) were plated at 10 000 cells/well.
NSO cells (plasmocytoma murine cell line) were plated out at
3 000 cells/well, and Daudi cells (human lymphoblastoid cell
line) were plated at 300 cells/well. 12 h after plating, different
concentrations of each compound were added to each well.
After 48 h, MTT assay was performed to analyze cytotoxicity
of the different compounds. Some experiments were performed

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4469
(8) Richman, D. D. Resistance of Clinical Isolates of Human
Immunodeficiency Virus to Antiretroviral Agents. Antimicrob.
Agents Chemother. 1993, 37, 1207-1221.
by using confluent cells: compounds were added to 3T3
monolayer 3 days after plating. Tests were then run as
described above.
(9) (a) Hargrave, K. D.; Proudfoot, J . R.; Grozinger, K. G.; Cullen,
E.; Kapadia, S. R.; Patel, U. R.; Fuchs, V. U.; Mauldin, S. C.;
Vitous, J .; Behnke, M. L.; Klunder, J . M.; Pal, K.; Skiles, J . W.;
McNeil, D. W.; Rose, J . M.; Chow, G. C.; Skoog, M. T.; Wu, J .
C.; Schmidt, G.; Engel, W. W.; Eberlein, W. G.; Saboe, T. D.;
Campbell, S. J .; Rosenthal, A. S.; Adams, J . Novel Non-
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cyclic Pyridobenzo- and Dipyridodiazepinones. J . Med. Chem.
1991, 34, 2231-2241 and references therein. (b) Cohen, K. A.;
Hopkins, J .; Ingraham, R. H.; Pargellis, G.; Wu, J . C.; Palladino,
D. E. H.; Kinkade, P.; Warren, T. C.; Rogers, S.; Adams, J .;
Farina, P. R.; Grob, P. M. Characterization of the Binding Site
4. MTT/F or m a za n Extr a ction P r oced u r e. 20 µL of the
5 mg/mL stock solution of MTT was added to each well; after
2 h of incubation at 37 °C, 100 µL of the extraction buffer was
added. After an overnight incubation at 37 °C, the optical
densities at 570 nm were measured using a Titer-Tech 96-
well multiscanner, employing the extraction buffer as the
blank.
Syn er gy Ca lcu la tion s. The multiple drug effect analysis
of Chou and Talalay42 was used to calculate combined drug
effects.
of Nevirapine (BI-RG-587),
a Non-Nucleoside Inhibitor of
Dr u g Ad m in istr a tion a n d P la sm a a n d Br a in Sa m -
p lin g. Male CD1 mice weighing about 25 g (Charles River,
Italy) were used. Procedures involving animals and their care
were conducted in conformity with the institutional guidelines
that are in compliance with national laws (D.L. n. 116, G.U.,
Suppl. 40, Feb 18, 1992; Circolare No. 8, G.U., 1994) and
international laws and policies (EEC Council Directive 86/609,
OJ L 358,1, Dec 12, 1987; Guide for the Care and Use of
Laboratory Animals, U.S. National Research Council, 1996).
Mice received the test compounds orally (suspended in 10%
Tween 80 in water) and subcutaneously (10% Tween 80 in
water or 10% Tween 80-5% ethanol in 10-3 M hydrochloric
acid) at doses of 10-20 mg/kg and were killed by decapitation
under deep anesthesia at various times after dosing. Blood
samples were collected in heparinized tubes and centrifuged
and the plasma was stored at -20 °C. Brains were rapidly
removed, blotted with paper to remove excess surface blood,
and stored at -20 °C until analysis.
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Ack n ow led gm en t. We are grateful to the Istituto
Superiore di Sanita’, Italy, for financial support (Pro-
getto Patologia, Clinica e Terapia dell′ AIDS, Grant 30A/
0/44). This work has also been supported by the CNR
Target Project on Biotechnology (to S.S.) and by the ISS-
AIDS Fellowship (to G.M.). We also thank Claudia
Fracasso for expert technical assistance. Giuseppe Cali-
endo thanks Assessorato Ricerca Scientifica - Regione
Campania, Italy (POP 5.4.2).
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails relative to synthesis and physical and chemical data of
compounds 10-13, synergy calculations, and pharmacokinetic
studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
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4470 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Brief Articles
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