Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further SAR studies and identification of a novel orally bioavailable hydrazine-based antiviral agent

Article abstract of DOI:10.1021/jm0010365

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Full text of DOI:10.1021/jm0010365

J . Med. Chem. 2001, 44, 305-315
305
Articles
Qu in oxa lin yleth ylp yr id ylth iou r ea s (QXP Ts) a s P oten t Non -Nu cleosid e HIV-1
Rever se Tr a n scr ip ta se (RT) In h ibitor s. F u r th er SAR Stu d ies a n d Id en tifica tion
of a Novel Or a lly Bioa va ila ble Hyd r a zin e-Ba sed An tivir a l Agen t
Giuseppe Campiani,*,§ Francesca Aiello,| Monica Fabbrini,| Elena Morelli,∇ Anna Ramunno,§ Silvia Armaroli,|
Vito Nacci,| Antonio Garofalo, Giovanni Greco,∇ Ettore Novellino,∇ Giovanni Maga,‡ Silvio Spadari,‡
Alberto Bergamini,† Laura Ventura,† Barbara Bongiovanni,† Marcella Capozzi,† Francesca Bolacchi,†
Stefano Marini,) Massimiliano Coletta,) Giovanna Guiso,# and Silvio Caccia#
Dipartimento di Scienze Farmaceutiche, Facolta’ di Farmacia, Universita’ degli Studi di Salerno, via Ponte Don Melillo 11C,
84084 Fisciano (Salerno), Italy, Dipartimento Farmaco Chimico Tecnologico, Universita’ degli Studi di Siena, via Aldo Moro,
53100 Siena, Italy, Universita’ della Calabria, 87036 Arcavacata, Cosenza, Italy, Dipartimento di Chimica Farmaceutica e
Tossicologica, Universita’ di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italy, Istituto di Genetica Biochimica ed
Evoluzionistica (IGBE) - CNR, via Abbiategrasso 207, 27100 Pavia, Italy, Dipartimento di Sanita’ Pubblica e Biologia
Cellulare (DSP&BC), Universita’ degli Studi di Roma “Tor Vergata”, via Tor Vergata 135, 00133 Roma, Italy, Dipartimento di
Medicina Sperimentale e Scienze Biochimiche (DMS&SB), Universita’ degli Studi di Roma Tor Vergata, via Tor Vergata 135,
00133 Roma, Italy, and Istituto di Ricerche Farmacologiche “Mario Negri”, via Eritrea 62, 20157 Milano, Italy
Received J uly 20, 2000
Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency
virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose
prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT
prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-
HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and
significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents
and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity
relationships are discussed in terms of the possible interaction with the RT binding site.
Although the new QXPTs analogues show potent antiviral activity, none of the compounds
tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral
agents, which in general have very low oral bioavailability. Through an integrated effort
involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we
investigated the structural dependence of the poor bioavailability and rapid clearance within
the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine
linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic
properties in terms of antiviral activity and oral bioavailability.
In tr od u ction
site, adjacent to the NRTI (nucleoside reverse tran-
scriptase inhibitor) binding site, by a similar three-
dimensional arrangement. HEPT and TIBO derivatives
(2)3 were the first compounds described in this class,
followed by nevirapine (3)4 and efavirenz (4), both of
which have been approved for clinical use for the
therapy of acquired immunodeficiency syndrome (AIDS).
Recently the PETT class of RT inhibitors has been
described. The representative compound of this series
is trovirdine (5), a potent RT inhibitor that, however,
has low oral bioavailability.5-7a,b In the course of our
research aimed at identifying novel antiviral agents,8
we discovered that members of a series of quinoxalin-
ylethylthioureas inhibited the replication of HIV-1 in
different cell lines.9 First, we identified the tricyclic
quinoxaline system as a critical structural requirement
for optimal interaction with the NNRTI binding site,
and 6-FQXTP (6), a potent broad-spectrum second-
generation NNRTI but characterized by unsatisfactory
oral bioavailability, was selected as the lead compound.9
Among the antiviral agents so far proposed for the
treatment of human immunodeficiency virus type 1
(HIV-1) infection,1 the non-nucleoside reverse tran-
scriptase inhibitors (NNRTI) represent an intriguing
class of therapeutic agents. Although NNRTIs are
highly specific and less toxic than nucleoside inhibitors
(Chart 1, zidovudine (AZT), 1),2 their therapeutic ef-
fectiveness is limited by relatively rapid emergence of
drug-resistant HIV-1 strains and by the onset of skin
rashes. NNRTIs (Chart 1) include structurally unrelated
subclasses of compounds that bind a common allosteric
* To whom correspondence should be addressed. Tel: 0039-089-
964381. Fax: 0039-089-962828. E-mail: campiani@unisa.it.
§ Universita’ degli Studi di Salerno.
| Universita’ degli Studi di Siena.
Universita’ della Calabria.
∇ Universita’ degli Studi di Napoli “Federico II”.
‡ IGBE - CNR Pavia.
† DSP&BC Universita’ degli Studi di Roma “Tor Vergata”.
) DMS&SB Universita’ degli Studi di Roma “Tor Vergata”.
#
Istituto di Ricerche Farmacologiche “Mario Negri”.
10.1021/jm0010365 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/03/2001

306 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Campiani et al.
Ch a r t 1
Sch em e 1
Ch a r t 2. Planned Modifications to 6-FQXPT and the
Newly Designed Antiviral Agents
To discover analogues with greater potency and better
pharmacokinetic properties, we extended our structure-
activity relationship (SAR) studies to assess the effect
of a variety of substituents and heterocyclic systems (7)
in place of the quinoxalinyl scaffold of 6-FQXPT (Chart
2). Potent broad spectum RT inhibitors with high
antiviral efficacy were identified (12 and 15). Despite
these promising results, studies performed to evaluate
the pharmacokinetic properties of the new compounds
were disappointing. None of the tested compounds
showed an improved pharmacokinetic profile over the
known thioureidic antiviral agents in terms of oral
bioavailability. Thus, starting from 6, the objective of
the present study was also the development of QXPT-
related antiviral agents with better oral bioavailability
to bypass the pharmacokinetic disadvantages inherent
to the thioureas. Exploiting synthesis, molecular model-
ing, and biological evaluation, our SAR studies (Chart
2) led to the identification of the quinoxaline derivative
8, representative lead of a new class of RT inhibitors,
offering satisfactory oral bioavailability and higher
antiviral activity than that of nevirapine. Furthermore,
this novel antiviral agent readly crosses the blood-brain
barrier and has potent synergistic activity with AZT.
Herein we report the synthesis and biological evaluation
of a series of heterocyclic analogues of 6-FQXPT (12a -
g, 15a -c) and the development of 8. Pharmacokinetic
and synergistic studies for the most active analogues
were also done.
Ch em istr y
Syntheses were accomplished as schematized in
Schemes 1 and 2. The desired products 12a -g and
15a -c were prepared starting from the corresponding
key amino intermediates 11a -g and 14a -c following
a similar synthetic procedure using 2-(5-bromopyridyl)-
isothiocyanate, which is in turn prepared from 2-amino-
5-bromopyridine and 1,1′-thiocarbonyldiimidazole or
thiophosgene.9 The azido intermediates 10a -g and

QXPTs as Potent HIV-1 RT Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 307
Ta ble 1. Inhibition of HIV-1 Wild-Type RT and HIV-1 Mutant
HIV-1 RT Enzymes Containing the Single Amino Acid
Sch em e 2
Substitutions L100I, V179D (for 15b,c), K103N, V106A, Y181I,
and Y188L by Compounds 8, 12a -g, 15a -c, and 18a ,b, and 19
HIV-1 RT Ki (µM)a
poly rA (dT)12-18
compd
WT L100I V179D K103N V106A Y181I Y188L
8
0.19 0.3
0.029 0.7
0.04 0.8
0.022 0.1
0.02 0.16
0.009 0.095
0.25 1.0
0.015 0.1
0.058 0.15
0.053 0.12
0.22 0.5
2.6
NA
0.65
0.4
0.8
2.5
1.0
0.9
1.6
0.24
0.4
1.4
1.8
1.2
0.2
0.45
0.9
0.13
5.0
0.4
0.7
0.1
1.5
12a
12b
12c
12d
12e
12f
0.04
0.06
0.03
0.02
0.05
0.35
0.33
0.029 0.3
0.12
0.15
NA
0.15
0.25
0.24
0.021
0.35
0.21
0.45
12g
15a
15b
15c
18a
18b
19
0.52
0.07
0.8
0.09
0.66
1.9
4.0
QXPTb
0.058 0.105
0.1
0.16
0.16
7
0.112 4.0
0.015 0.2
0.04
10
2.5
0.2
0.3
6-FQXPTb 0.009 0.17
efavirenz 0.03 0.12
0.15
36
nevirapine 0.4
9
18
a
Inhibition of HIV-1 RT activities. All data listed were com-
pared to the corresponding test results for nevirapine, performed
at the same time.The Ki is stated as the mean of at least three
13a -g were obtained starting from the lactams 9a -g
by direct alkylation with tosylethyl azide, while 13h
(R ) 6-F, A ) CH, X ) N, Y ) C, and Z ) CH) and 13i
(R ) 7,8-diMe, A ) CH, X ) N, Y ) C, and Z ) CH)
were previously synthesized.9 The lactams 9a -g were
prepared according to previously described proce-
dures.10-14 Usually the O-alkylated products were ob-
tained in 10-25% yield. After examination of several
reduction methods, we found that treatment of azides
10a -g and 13d ,h ,i with 1,3-propanedithiol-triethyl-
amine in methanol15 provided the corresponding amines
11a -g and 14a -c. Last, by treatment of 11a -g and
14a -c with 2-(5-bromopyridyl)isothiocyanate in the
presence of triethylamine we obtained the thioureas
12a -g and 15a -c. The syntheses of compounds 8 and
18a ,b are outlined in Scheme 2. Lactam 1610 was
transformed to the hydrazine derivatives 17 by means
of phosphorus oxychloride and hydrazine. Treatment of
17 with triphosgene and 2-amino-5-bromopyridine pro-
vided 18a , while treatment of 17 with 5-bromopyridyl-
2-isothiocyanate (in turn obtained from 2-amino-5-
bromopyridine and 1,1′-thiocarbonyldiimidazole) fur-
nished 18b. Moreover, reaction of 17 with pyrazinecar-
boxylic acid or pyridine-2-carboxylic acid, in the pres-
ence of triphenylphosphine and Aldrithiol-2,16 gave 8
and 19, respectively, in good overall yield.
b
experiments. From ref 9. NA ) not active.
Y188L, a mutation of RT that appears in patients
treated with both nevirapine and AZT, was also in-
cluded.
The first question addressed was whether, by expand-
ing the SAR studies of the QXPT class of antiviral
agents, the antiviral activity, the broadening of the
spectrum of antienzymatic activity, and, possibly, the
pharmacokinetic properties could be improved over the
known thioureas. SAR studies were conducted by ap-
propriately varying the substituents on the benzo-fused
ring and by introducing different heteroaryl systems in
place of the pyrroloquinoxaline of the lead 6 (6-FQXPT).
As illustrated in Table 1 for compounds 12a -g and
15a -c, the potency of inhibition of RTwt and mutated
RTs is strictly dependent on the nature of the substit-
uents on the aromatic system. By comparing the inhibi-
tory activity (Kis) of compounds 12a -c to that of
6-FQXPT (6), it clearly appears that the electron-
withdrawing fluorine is preferred at C-6 (6-FQXPT), this
being 4-fold more potent against RT wt than its 7-, 9-,
and 7,9-fluorinated analogues (12a -c). While against
the V106A mutant 12a -c show a comparable activity
to that of 6-FQXPT (6), the 7,9-difluoro analogue 12c
proved to be inactive against the K103N mutant,
maintaining significant potency vs Y188L mutant. The
analogue 12d (7-ClQXPT) is slightly less potent than 6
(6-FQXPT) vs RT wt, but it is about 4-fold more active
than 12a (7-FQXPT) against L100I, V106A, K103N, and
Y181I. In general, the halogenated analogues show
greatly improved antienzymatic properties over their
unsubstituted counterpart QXPT.9 Furthermore, within
this series, the antienzymatic potency and the broaden-
ing of spectrum of activity are also influenced by the
nature of the heterocyclic skeleton (12e-g vs 6-FQXPT).
By comparing the Kis (Table 1) of unsubstituted QXPT
and 6-FQXPT for inhibition of RTwt, L100I, K103N, and
V106A with those of 12f (ImQXPT), the key role played
by the pyrrole ring with respect to the imidazole
Biologica l Resu lts
En zym a tic Assa ys. In Vitr o SAR Stu d ies. The
newly described ethylpyridylthioureas 12a -g and 15a -
c, and the analogue 8, were tested in an in vitro HIV-1
RTwt assay to evaluate their potential as anti-HIV
drugs. The results are summarized in Table 1 as Ki
values. In addition, in order to evaluate the potential
of these antienzymatic derivatives as novel broad-
spectrum antiviral agents, the whole set of active
compounds was also tested on a panel of RT mutants
including L100I, Y181I, V106A, one of the most com-
monly identified mutations associated with dipyridodi-
azepinones, and K103N, a mutant associated with
resistance to pyridones and nevirapine as well. The

308 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Campiani et al.
(unsubstituted QXPT9 vs 12f) is evident. On the con-
trary, the pyrazole ring in place of the pyrrole (12g vs
unsubstituted QXPT9) greatly improves the inhibitory
activity against Y181I and Y188L mutants (12g, Ki )
0.52 and 0.40 µM, respectively). We also investigated
the analogue 12e (PyrPZPT) in which the benzo-fused
ring of 6 is replaced by a pyridine system. In this case
12e shows increased anti-Y181I/Y188L activity, main-
taining high potency against RTwt. Taking into account
the inhibitory potency against RTwt and the other
mutants, especially vs Y188L, it appears that 12e
(PyrPZPT) represents one of the most potent second-
generation RT inhibitors.
The O-alkylated regioisomers 15a -c were further
investigated and included in Table 1. Compounds 15a
and 15b proved to be potent broad-spectrum RT inhibi-
tors, being as active as their N-alkylated isomers (6 and
12d ). In particular, 15b demostrated higher potency vs
Y181I and Y188L than its N-alkylated isomer 12d .
When subjected to preliminary pharmacokinetic stud-
ies, the most representative thioureas proved to be not
bioavailable after oral administration, probably by rapid
metabolic inactivation and clearance (first-pass hepatic
elimination) (see Preliminary Pharmacokinetic Studies
section). Since this unfavorable behavior was shown by
thioureas characterized by different heteroaryl systems,
the subsequent question addressed was whether through
an alteration of the linker between the heteroaryl and
the 5-bromopyridine groups an antiviral agent with
higher hydrophilicity and, of consequence, higher oral
bioavailability, maintaining the 3D pharmacophoric
arrangement for an optimal interaction with the NNRTI
binding site, could be developed. Starting from QXPTs,
by replacing the ethylthiourea moiety with a semicar-
bazide or a thiosemicarbazide group, two inactive
compounds were obtained (18a ,b). Furthermore, short-
ening the linker with hydrazine, and maintaining the
distal pyridine ring, compound 19 was obtained, char-
acterized by a low antienzymatic activity. Conversely,
replacing the pyridinoyl with a pyrazinoyl group, we
identified the new reverse transcriptase inhibitor 8. As
shown in Table 1, although less potent than thioureas
(8 vs unsubstituted QXPT9 and 6), compound 8 displays
antienzymatic activity against RT wt and mutants
higher than that of nevirapine, and it represents a great
improvement in oral bioavailability over known ethyl-
thioureas (see Preliminary Pharmacokinetic Studies
section). Replacement of the pyrazine nucleus of 8 with
pyridine (19) and substituted pyridines (data not shown),
leading to inactive compounds, demostrated the critical
role played by the distal nitrogen of the pyrazine ring.
F igu r e 1. Model of the RT/8 complex. Dotted lines highlight
intramolecular hydrogen bonds. To improve clarity, only
residues within 4 Å from the inhibitor are displayed and
Glu138 and Pro236 have been excluded.
F igu r e 2. Overlay of the putative RT-bound conformations
of 8 (yellow) and 15a (red) extracted together with Lys101 from
the RT/8 and RT/15a complexes.
the backbone NH group of Lys101. This interaction
rationalizes the loss of inhibitory activity resulting from
replacement of the pyrazine N4 with a CH fragment to
yield the pyridine derivative (19). An additional inter-
molecular hydrogen bond is hypothesized to occur
between the hydrazide CONH and the Val179 carbonyl
oxygen. The pyrroloquinoxaline and pyrazine rings are
arranged in a butterfly-like arrangement adopted by
most NNRTIs.20 The tricyclic system is hosted in cavity
made up by the aromatic side chains of Tyr181, Tyr188,
and Trp229.
Figure 2 shows a superposition of the putative RT-
bound geometries of 8 and 15a 7b extracted together with
Lys101 from the corresponding complexes. The pyrrolo-
quinoxaline systems dock similarly although they are
not coincident atom by atom. None of the pyrazine
nitrogens of 8 match the pyridine nitrogen of 15a . Each
of the thioureidic NH groups of 15a makes a hydrogen
bond: one with the pyridine nitrogen (intramolecular)
and the other one with the Lys101 carbonyl oxygen
Computational studies were performed to develop
docking models of 8 and 15a into the HIV-1 NNRTI
binding site which could guide further lead optimization
efforts. The coordinates of the RT/nevirapine complex
solved by Ren et al.17 were employed to model the target
protein. All calculations were performed using molecular
mechanics (Tripos force field18), conformational analysis,
docking, and graphics routines available within the
SYBYL program.19 Details of these procedures are
described in the Experimental Section, whereas results
are summarized below.
According to our model of RT/8 complex (see Figure
1), the pyrazine N4 atom receives a hydrogen bond from

QXPTs as Potent HIV-1 RT Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 309
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)
b
b
compd
12a
12b
12c
12d
12e
12f
15a
IC50 (µM)
EC50c (µM)
SId
IC50 (µM)
EC50c (µM)
SId
NSOe
DHe
3T3Fe
HLe
>200
>2
0.063
0.0019
0.085
0.0018
0.0052
0.013
>3170
>110
>23
>1080
>380
>150
>690
15
21
0.032
0.2
468
105
0.02
0.52
NT
0.97
0.7
0.68
>1
>1
0.019
0.51
NT
0.96
0.69
0.68
>1
>1
0.86
0.8
1.0
0.02
0.5
NT
0.98
0.69
0.69
>1
>1
0.88
0.8
0.02
0.51
NT
0.97
0.68
0.67
>1
>1
0.87
>2
>2
7
11
88
0.061
0.124
0.32
0.020
0.025
0.4
123
89
275
6250
3720
40
>2
>2
>2
0.0029
125
93
15b
8
18
17
0.87
0.8
1.0
QXPTf
6-FQXPTf
8-ClTIBO
AZT
0.38
45
0.8
0.96
>0.02
>1
0.00047
0.0019
>43
200
50
>100
0.025
0.030
0.4
7990
1670
>250
1.0
526
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 compound concentration which results in a 50% survival of uninfected untreated control CEM-SS and
C8166 cells (e.g., cytotoxicity of the test drug). c The EC50 value is the test compound concentration which produces a 50% survival of HIV
d
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 O.D. measured at 570 nm. All data reported in table are measured O.D. × 1000. Standard deviations are not reported
in the table but never exceded 5% of the mean value. O.D.s measured in the control row were as follows: NSO 0.125 ( 0.015;
DH 0.129 ( 0.018; 3T3F 0.131 ( 0.014; HL 0.114 ( 0.01. f From ref 9.
(intermolecular). Differences in the binding modes of 8
and 15a imply different lead optimization strategies in
the hydrazide- and thiourea-based series.
Ta ble 3. Inhibition of HIV-1-p24 Antigen Production in
Monocyte-Macrophagesa by Compounds 8 and 12a
compd
IC50 (µM)b
EC50 (µM)c
SId
125
Secon d a r y Tests. A selected set of active compounds
in enzymatic assays was evaluated for its ability to
inhibit the HIV-1 wt cytopathicity on T4 cells (CEM-
SS), to inhibit syncytia formation on C8166 cells infected
with wild-type HIV-1IIIB virus. Furthermore, compounds
12a and 8, representative of the two series of anti-
enzymatic agents, were further tested on monocyte-
macrophages infected with HTLV-III Ba-L (a laboratory
adapted monocytotropic strain) in order to confirm their
potential as antiviral agents. MTT assays were also
conducted as a measure of the cytotoxic effect of the new
compounds on four different cell lines, and synergistic
antiviral activity with AZT has been calculated for
representative antiviral agents.
8
25
49
17
0.2
0.004
0.002
12a (7-FQXPT)
12200
8800
6-FQXPTe
a
Antiviral activity was determined after 14 days after infection.
For comparison, HIV-1-p24 antigen production in control cells was
22.789 pg/mL. All the data listed were compared to the corre-
sponding test results for AZT which served as the treated control,
b
performed at the same time. The IC50 value is the test compound
concentration which results in
a 50% survival of uninfected
untreated control macrophages cells (e.g., cytotoxicity of the test
drug). c The EC50 value is the test drug concentration which
produces a 50% reduction of HIV-1-p24 antigen production in
infected cells relative to uninfected untreated controls (e.g., in vitro
d
anti-HIV-1 activity). SI ) selectivity index (IC50/EC50). e From
ref 9.
Cell Cu ltu r e Assa ys a n d MTT Assa ys. The results
are presented in Table 2. For compounds bearing the
quinoxaline system (12a -d ), potent antiviral activity
in CEM-SS and C8166 cells was found, comparable to
that of 6, although 12b, in C8166 cells, proved to be less
active than the counterparts 12a and 12d . Replacement
of the benzo-fused ring with a pyridine led to an
analogue with great antiviral efficacy (12e vs QXPT),
while replacement of the pyrrole ring of unsubstituted
QXPT with an imidazole (12f) maintained antiviral
activity (C8166 cells). The O-alkylated regioisomers of
12 (15a ,b) were also tested on CEM-SS and C8166 cells.
By comparing the data for 6 and 15a in Table 2 (CEM-
SS and C8166), an apparent discrepancy arose. In fact,
15a shows low antiviral efficacy on C8166 cells while
its EC50 on CEM-SS cells is equal to 2 nM. In C8166
cells, the new hydrazine derivative 8 showed a lower
antiviral efficacy with respect to those of QXPTs and
related compounds, although it was comparable to that
of AZT tested in the same experimental conditions.
Furthermore, two representative derivatives (8 and 12a )
were also studied on monocyte-macrophages infected by
HTLV-III Ba-L (Table 3). HIV-1 infection of monocyte-
macrophages is an important event in the pathogenesis
of AIDS. Monocyte-macrophages differ from T cells for
a number of aspects which could possibly affect the anti-
HIV activity of some antiviral agents. In our case, 12a
proved to be extremely potent in this test, while 8
confirmed its significant efficacy, in the submicromolar
range. Taking into account the data shown in assays
using different infected cells, 12a -e,g appear to be
among the most potent second-generation anti-HIV
agents, while the quinoxaline derivative 8, although less
potent than QXPTs, is a representative lead of a new
class of antiviral agents.
The low cytotoxicity was also confirmed by screening
compounds in a MTT assay (3-(4,5-dimethylthiazol-2-
yl)2,5-diphenyltetrazolium bromide) by using NSO mu-
rine, Daudi human, 3T3 fibroblast murine cell lines, and
normal human limphocytes (Table 2). Most of the tested
compounds show weak cytotoxicity.
Syn er gistic An tivir a l Activity w ith AZT (a n d
DDI). Resistance is a major concern in the chemo-
therapy of AIDS, and the recent therapeutic strategies
converge to favor multidrug regimens which are more
safe and effective against the HIV-1 infection, with
simultaneous reduction on drug dosages, toxicity, and
resistance. A further improvement in multidrug therapy
would be to administer combinations of drugs able to
interact synergistically. One of the clinically preferred

310 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Campiani et al.
Ta ble 4. EC50 and Combination Index for Different Anti-HIV-1 Compounds in Association with AZT (and DDI for 8), Calculated
Using Both the Mutually Nonexclusive and the Mutually Exclusive Assumptiona
EC50 (nM)
CIb
mutually nonexclusive
assumption
of compound
alone
of compound
of AZT (DDI)c
with compound
mutually exclusive
assumption
compound
none
with AZT (DDI)c
400 (1300)
8
450
66
124
588
25
15 (140)
57
50
460
2
98 (0.84)
0.640 (1.18)
0.700
0.470
1.070
0.091
0.570 (0.97)
12a (7-FQXPT)
12e (PyrPZPT)
12f (ImQXPT)
6-FQXPTd
85
15
66
3
0.600
0.450
0.940
0.090
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
form of the equation. c EC50 and the combination index for 8 in association with DDI are shown in brackets. From ref 9.
d
combinations among those drugs acts on different
receptor sites within the same viral protein.21 Accord-
ingly, to evaluate the potential of the new series of
inhibitors in a multidrug approach, we investigated the
synergistic antiviral activity of 8, 12a , 12e, and 12f in
combination with the nucleoside analogue AZT (and
DDI for 8) (Table 4). The compounds tested proved to
be able to potentiate the antiviral activity of AZT, and
AZT was able to potentiate their activity. The extent of
the synergistic activity was quantified according to Chou
and Talalay by the calculation of the combination index
(CI). Compounds 12a ,e showed a synergistic anti-HIV-1
activity (CI < 1) when tested in combination with AZT,
notwithstanding the mutually exclusive or nonexclusive
assumption formulas that were used (Table 4). On the
contrary, 12f showed an additive antiviral activity
(CI ) 1). It is noteworthy that the new antiviral agent
F igu r e 3. Plasma and brain concentration-time curves of
8 showed a strong synergistic anti-HIV-1 activity in
combination with AZT. Synergistic activity was less
evident when 8 was tested in combination with DDI.
Data shown in Table 4 confirm these new reverse
transcriptase inhibitors to be potentially useful in
combination therapies.
P r elim in a r y P h a r m a cok in etic Stu d ies.22-26a,b In
previous studies we showed that the ethylpyridylthio-
ureidic derivative 6 (6-FQXPT)9 rapidly reaches the
systemic circulation and enters the brain after subcu-
taneous dosing in mice. However, its plasma and brain
concentrations were low and variable and rapidly fell
below the limit of quantitation, possibly because of rapid
and extensive biotransformation. Findings were similar
with its 9-fluoro-substituted analogue 12b (data not
shown). After oral dosing (20 mg/kg), compounds 6 and
12b were undetectable in mouse plasma within the
limits of sensitivity of the analytical procedure (about
0.1 nmol/ml, using 0.2 mL of plasma).
In the same experimental conditions (20 mg/kg orally,
suspended in PEG400:Tween 80, 80:20, v/v) the 7,9-
difluoro-substituted analogue 12c showed low oral bio-
availability too. The same was achieved with the more
lipophilic O-alkylated regioisomer 15a (i.e., plasma
concentrations were less than 0.1 nmol/mL within 2 h
of oral dosing). Possibly the water insolubility and high
lipophilicity of these ethylpyridylthioureidic derivatives
prevent their efficient absorption and/or lead to exten-
sive presystemic biotransformation.
compound 8 after subcutaneous and oral administration in
mice. Each point represents the mean plasma (solid line) and
brain concentrations (n ) 3) of subcutaneously (closed circles)
and orally (open circles) administered 8.
hydrazide derivative was expected to possess a higher
solubility and a lower lipophilicity compared with the
thioureidic analogues of type 12 and 15 as suggested
by the following calculated n-octanol/water logP values
of 8, 12c, and 15a : 1.1, 3.7, and 4.9, respectively.26b
Figure 3 shows the plasma concentration-time curves
of compound 8 after subcutaneous and oral dosing in
mice. Subcutaneous absorption was relatively slow, as
indicated by the plasma tmax which averaged 120 min
after a 20 mg/kg dose. The mean Cmax was 12.9 ( 3.1
nmol/ml (n ) 3). Under the assumption of complete
absorption of 8 from the subcutaneous injection site into
the systemic circulation, the mean total body Cl aver-
aged 22.5 mL/min/kg. In conjunction with a relatively
high Vd of 3 L/kg, this resulted in a mean elimination
t1/2 of 1.5 h, although this could only be calculated with
limited accuracy because of the short sampling interval.
Absorption of 8 was faster after oral dosing, with a
mean plasma Cmax (6.5 ( 1.7 nmol/ml) at 30 min. Mean
plasma concentrations of the compound declined from
the peak with elimination t1/2 of 1.4 h, similar to that
after subcutaneous injection. The relative bioavailabil-
ity, calculated from the AUC of the compound after an
oral (21.7 nmol/ml/h) and subcutaneous (48.7 nmol/ml/
h) dose of 20 mg/kg, was about 45%.
Synthetic and pharmacokinetic efforts were then
focused on the development and characterization of
novel NNRTIs: by modifications of the structure of
QXPTs, the new lead 8 was identified (see Table 1). This
Since the brain is a site of infection and viral replica-
tion for HIV-1, effective brain entry is a desirable
property of new NNRTIs. It was therefore of interest
preliminarily to examine the passage of 8 across the

QXPTs as Potent HIV-1 RT Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 311
and 4-(2-azidoethoxy)-7-fluoropyrrolo[1,2-a]quinoxaline (13a ).
To a suspension of sodium hydride (69.7 mg, 2.81 mmol) in
anhydrous DMF (10 mL) was added the pirroloquinoxaline 9a
(0.6 g, 2.81 mmol) in one portion.The reaction mixture was
stirred at room temperature for 2 h, and then tosylethyl azide
(667 mg, 2.81 mmol) was added. After 24 h of stirring at 80
°C the solvent was removed under vacuum, and the residue
was dissolved in ethyl acetate. The organic phase was washed
with brine, dried, and concentrated. The residue was purified
by flash chromatography (ethyl acetate and hexanes, 1:3, as
eluant) to afford 13a and 10a as white solids (compound 13a
eluted first). After recrytstallization (ethyl acetate and hex-
anes, 1:3) the N-ethylazide 10a was obtained as colorless
prisms. 10a : (59%) mp 133-134 °C; IR (Nujol) 2104, 1653,
blood-brain barrier and the relationship between brain
and plasma concentrations. Reflecting the time profiles
in the systemic circulation, 8 achieved mean brain Cmax
at approximately the same time as in plasma after
either subcutaneous (3.0 ( 1.4 nmol/g, at 120 min) or
oral dosing (0.9 ( 0.1 nmol/g, at 30 min). After the peak,
mean brain concentrations disappeared almost in paral-
lel with plasma concentrations, with similar elimination
t1/2 looking at the whole brain concentrations (Figure
2). Mean brain concentrations remained consistently
15-25% of the plasma concentrations within 4 h of
dosing, yielding a mean brain-to-plasma distribution
ratio (plasma/brain AUC) of about 0.2 regardless of the
route of administration.
1
1611 cm-1; H NMR (CDCl3) δ 3.72 (m, 2 H), 4.41 (m, 2 H),
6.66 (m, 1 H), 6.95 (m, 1 H), 7.15 (m, 1 H), 7.25 (m, 1 H), 7.66
(m, 2 H). Anal. Calcd for C13H10FN5O: C, 57.56; H, 3.72; N,
25.82. Found: C, 57.57; H, 3.69; N, 25.65.
Con clu sion s
13a : (20%) colorless prisms, mp 66-67 °C; IR (CHCl3) 2109
cm-1; 1H NMR (CDCl3) δ 3.70 (m, 2 H), 4.78 (m, 2 H), 6.76 (m,
1 H), 6.94 (m, 1 H), 7.08 (m, 1 H), 7.38 (m, 1 H), 7.68 (m, 1 H),
7.78 (m, 1 H). Anal. Calcd for C13H10FN5O: C, H, N.
In summary, we have prepared a number of highly
potent antiviral agents with an ethylthioureidic back-
bone, structurally related to 6-FQXPT. Within the series
of QXPTs, the SAR studies led to the identification of
12e, a potent second-generation antiviral agent, in
which the benzo-fused ring of the earlier-described
QXPTs is replaced by a pyridine ring. Furthermore, in
the course of this work we have also investigated the
structural dependence of the poor bioavailability and
rapid clearance of QXPT compounds, in order to identify
antivirals with better oral bioavailability over the
known thioureas. Alterations of the molecular structure
of our QXPTs 6 and 12 enabled us to develop the
quinoxaline derivative 8, a prototype antiviral agent of
a new class of RT inhibitors. Its unique structural
feature is the presence of a hydrazine group linking the
heterocyclic system to the aroyl moiety. Compound 8
represents a new generation inhibitor of HIV-1 replica-
tion with potent synergistic antiviral activity with AZT.
Preliminary pharmacokinetic studies in the mouse
suggest that the QXPT antiviral agents, like other
thioureidic antivirals, offer low oral bioavailability and/
or rapid clearance. In contrast, the new quinoxaline
derivative 8 has much better pharmacokinetic proper-
ties, being rapidly absorbed from the gastrointestinal
tract, with mean Cmax of about 7 µM after a 20 mg/kg
dose. Although oral bioavailability is about half that
after subcutaneous dosing, it remains to be seen whether
this is due to an extensive first-pass metabolism or to
incomplete absorption from the gastrointestinal tract.
Compound 8 also has an intermediate-low Cl compared
to liver blood flow and its Vd exceeds the total body
water volume of the mouse, suggesting extensive uptake
into tissue. Although its brain-to-plasma distribution
ratio averaged 0.2 after a single dose, it remains to be
seen whether it is similar across species because of
differences in blood flow, brain size, protein binding, and
other mechanisms such as active transport that govern
the extent of brain uptake.
Gen er a l P r oced u r e for P r ep a r a tion of th e Am in oeth yl
Der iva tives 11a -g a n d 14a -c. This procedure is illustrated
for the preparation of 5-(2-aminoethyl)-7-fluoropyrrolo[1,2-a]-
quinoxalin-4(5H)-one (11a ).
The azide 10a (112 mg, 0.4 mmol) was suspended in dry,
freshly distilled methanol (4 mL). The flask was purged with
argon, and propane-1,3-dithiol (120 µL, 1.2 mmol) and anhy-
drous triethylamine (166 µL, 1.2 mmol) were added. The
solution was stirred at room temperature for 24 h, the solvent
was then removed under vacuum, and the residue was purified
by flash chromatography (elution with methanol and triethyl-
amine, 9:1) to obtain 11a as an amorfous solid (89%). The
amine was used in the next step without further purification.
1
IR (Nujol) 1635 cm-1; H NMR (CDCl3) δ 1.5 (br s, 2 H), 3.05
(m, 2 H), 4.32 (m, 2 H), 6.63 (m, 1 H), 6.95 (m, 1 H), 7.10 (m,
1 H), 7.20 (m, 1 H), 7.55-7.65 (m, 2 H).
Gen er a l P r oced u r e for P r ep a r a tion of QXP T Rela ted
Com p ou n d s 12a -g a n d 15a -c. This procedure is illustrated
for the preparation of N-[2-(7-fluoro-4-oxopyrrolo[1,2-a]quin-
oxalin-5-yl)ethyl]-N′-[2-(5-bromopyridyl)]thiourea (11a ). To a
solution of aminoethylquinoxalinone 11a (127 mg, 0.5 mmol)
in anhydrous THF (2 mL) was added 2-(5-bromopyridyl)-
isothiocyanate (107 mg, 0.5 mmol), and the reaction mixture
was stirred at room temperature for 1 h. The resulting
precipitate was collected by filtration through a sintered glass
filter and washed with another portion of THF. Recrystalli-
zation from THF gave 12a (211 mg) as colorless prisms
(91%): mp 256-257 °C. IR (Nujol) 3420, 1653 cm-1; 1H NMR
(DMSO-d6) δ 3.95 (m, 2 H), 4.48 (m, 2 H), 6.70 (m, 1 H), 7.04
(m, 3 H), 7.83 (dd, 1 H, J ) 11.15, 2.3 Hz), 7.94 (dd, 1 H, J )
8.85, 2.1 Hz), 8.14 (m, 3 H), 10.74 (b s, 1 H), 11.35 (br s, 1 H).
Anal. Calcd for C19H15BrFN5OS: C, H, N.
N-(P yr r olo[1,2-a ]qu in oxa lin -4-yl)h yd r a zin e (17). A so-
lution of 16 (1.04 g, 5.6 mmol) and a catalytic amount of N,N-
dimethylaniline in phosphorus oxychloride (16.9 mL, 184
mmol) was refluxed under argon for 5 h. After cooling, the
excess phosphorus oxychloride was distilled off, and the
residue was partitioned between dichloromethane and sodium
hydrogencarbonate solution. The organic solution was washed
with brine, dried, and concentrated. The residue was purified
by column chromatography (dichloromethane/EtOAc, 9:1) and
recrystallized from hexanes to give the 4-chloro intermediate
(0.67 g, 59%) as colorless prisms: mp 262-264 °C. This chloro
derivative was dissolved in methanol (15 mL), and the solution
was cooled at 0 °C. Then a solution of hydrazine monohydrate
(140 µL) in methanol (3 mL) was slowly added. After the
mixture was stirred for 2 h at room temperature, the solid was
filtered off and the solution was concentrated to give 17 as a
white solid which was recrystallized from methanol (590 mg,
Exp er im en ta l Section
For general experimental information, see ref 11. Lactams
9a -g used for the synthesis of N-alkylated and O-alkylated
compounds were synthesized by using a procedure described
in ref 10, while azides 13h ,i were previously reported in ref 9.
Gen er a l P r oced u r e for P r ep a r a tion of N-Alk yla ted
Qu in oxa lin on es 10a -g a n d of O-Alk yla ted Qu in oxa lin e
13a . This procedure is illustrated for the preparation of 5-(2-
azidoethyl)-7-fluoropyrrolo[1,2-a]quinoxalin-4(5H)-one (10a )
86%): mp 159-160 ° C; IR (Nujol) 3300 cm-1
;
1H NMR
(DMSO-d6) δ 4.53 (br s, 2 H), 6.68 (m, 1 H), 6.89 (m, 1 H),

312 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Campiani et al.
7.30 (m. 2 H), 7.51 (d, 1 H, J ) 7.6 Hz), 8.05 (d, 1 H, J ) 7.6
Hz), 8,20 (m, 1 H), 8.71 (br s, 1 H). Anal. Calcd for C11H10N4:
C, H, N.
standard distances and valence angles of the Tripos force field.
By manually modifying the torsion angles about the CAr-N
and N-N rotatable bonds of 8, we found a butterfly-like
orientation of this ligand superimposable on nevirapine about
the aromatic systems (corresponding to the butterfly wings):
the pyrroloquinoxaline B ring and the pyrazine ring of 8 were
matched with the pyridine A and B rings of nevirapine,
respectively. The fitting points were, more specifically, pseudo-
atoms placed at 1 Å along the normals to the plane of each
aromatic ring passing through the ring centroid.
4-(5-Br om op yr id in -2-yl)-1-(p yr r olo[1,2-a ]qu in oxa lin -4-
yl)sem ica r ba zid e (18a ). To a solution of triphosgene (54 mg,
0.85 mmol) in anhydrous dichloromethane (1 mL) was added
a solution of 2-amino-5-bromopyridine (86 mg, 0.5 mmol) and
diisopropylethylamine (192 µL, 1.1 mmol) in anhydrous dichlo-
romethane (1 mL). After the mixture was stirred for 5 min at
room temperature, a solution of 17 (100 mg, 0.5 mmol) in
anhydrous dichloromethane (1 mL) was added. The resulting
solution was stirred at room temperature overnight. The
solvent was evaporated, and the residue was taken up in
EtOAc. The organic layer was washed with brine, dried, and
concentrated. The residue was purified by chromatography
(dichlorometane and 10% methanol and 1% ammonium hy-
droxide) to give a yellow solid which was recrystallized (EtOAc)
to give 73 mg (41%) of 18a as yellow prisms: mp 114-115 °
The so-obtained pharmacophore-based conformation of 8
was then placed into the NNRTI binding cleft extracted from
the crystal structure of the RT/nevirapine complex. The
binding site comprised 71 amino acids located within
a
distance of 10 Å from any non-hydrogen atom of the bound
inhibitor. Water molecules were deleted. Hydrogens were
added to the unfilled valences of the amino acids, and the Lys,
Asp, and Glu side chains were modeled in their ionized forms.
The trial RT/8 complex was first geometry-optimized by
keeping fixed the coordinates of the protein. The resulting
(highly distorted) geometry of the ligand was energy-mini-
mized alone while retaining the conformation of the spacer
through appropriate constraints applied to torsional angles.
The optimized output geometry of the ligand was then placed
into the NNRTI binding site through an overlay on the
corresponding input geometry about non-hydrogen atoms.
Finally, the complex was fully energy-minimized by keeping
fixed the protein backbone until an attractive energy of 25 kcal/
mol was reached. This protocol did not produce significant
changes in the starting structure.
1
C. IR (Nujol) 3410, 1653 cm-1; H NMR (DMSO-d6) δ 6.06 (s,
1 H), 6.45 (d, 1 H, J ) 6.6 Hz), 7.22 (m, 4 H), 7.59 (m, 1 H),
8.02 (m, 3 H), 9.98 (br s, 1 H), 10.11 (br s, 1 H), 11.64 (br s, 1
H). Anal. Calcd for C17H13BrN6O: C, H, N.
4-(5-Br om op yr id in -2-yl)-1-(p yr r olo[1,2-a ]qu in oxa lin -4-
yl)th iosem ica r ba zid e (18b). Starting from 17, the title
compound was obtained following the synthetic procedure
described in ref 9. Compound 18b was obtained as yellow
prisms (41%): mp 236-237 ° C. IR (Nujol) 3410, 1620 cm-1
;
1H NMR (DMSO-d6) δ 6.80-7.59 (m, 5 H), 7.80-8.70 (m, 4
H), 9.97 (br s, 1 H), 10.62 (br s, 1 H), 10.91 (br s, 1 H). Anal.
Calcd for C17H13BrN6S: C, H, N.
A model of the RT/15a complex was obtained by following
a computational procedure similar to the one described above.
To check whether the RT-bound conformations of 8 and 15a
were energetically feasible, they were fully geometry optimized
in vacuo. The resulting nearest local minimum conformers
revealed to be more stable by 1.3 and, respectively, 3.1 kcal/
mol, meaning that the input geometries were not affected by
steric conflicts.
HIV-1 RT RNA-d ep en d en t DNA P olym er a se Activity
Assa y. In h ibition 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. A total of 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
N-(P yr a zin eca r bon yl)-N′-(p yr r olo[1,2-a ]qu in oxa lin -4-
yl)h yd r a zin e (8). To a magnetically stirred suspension of
pyrazinecarboxylic acid (31 mg, 0.25 mmol) in anhydrous
dichloromethane (1 mL) were added, portionwise within 1 h,
triphenylphosphine (132 mg, 0.5 mmol) and 2,2′-dipyridyl
disulfide (Aldrithiol-2) (111 mg, 0.5 mmol), and the disappear-
ance of the starting material was monitored by TLC (EtOAc).
Then a solution of 17 (50 mg, 0.25 mmol) in anhydrous
dichlometane (3 mL) was added, and the reaction mixture was
stirred overnight at room temperature. The solvent was
removed, and the residue was taken up in a mixture of EtOAc
and 5% hydrochloric acid. The organic phase was washed with
1 N sodium hydroxide and brine, dried, and concentrated. The
residue was chromatographed (dichlorometane and 10% metha-
nol and 1% ammonium hydroxide) to give a yellow solid which
was recrystallized (EtOAc) to give 45 mg of 8 as yellow
prisms: mp 216-218 ° C; IR (Nujol) 3420, 1653 cm-1; 1H NMR
(DMSO-d6) δ 6.83 (s, 1 H), 7.20-7.30 (m, 3 H), 7.44 (m, 1 H),
8.13 (m, 1 H), 8.34 (s, 1 H), 8.87 (s, 1 H), 8.98 (s, 1 H), 9.25 (s,
1 H), 9.65 (br s, 1 H), 10.96 (br s, 1 H). Anal. Calcd for
C
16H12N6O: C, H, N.
N-(P yr a zin eca r bon yl)-N′-(p yr r olo[1,2-a ]qu in oxa lin -4-
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 0.2Ki and 5Ki was
used. Experiments have been done in triplicate. Experimental
errors ((SD) were e 10%.
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,
yl)h yd r a zin e (19). Following a synthetic procedure similar
to that described for 18a , using pyridine-2-carboxylic acid, the
title compound was obtained in 68% yield as colorless prisms:
mp 148-149 °C; IR (Nujol) 3420, 1635 cm-1; 1H NMR (CDCl3)
δ 6.86 (m, 1 H), 7.10-7.25 (m, 3 H), 7.40-7.50 (m, 2 H), 7.83
(m, 1 H), 8.19 (m, 1 H), 8.46-8.74 (m, 2 H), 9.03-9.35 (m, 2
H), 10.16 (br s, 1 H). Anal. Calcd for C17H13N5O: C, H, N.
Molecu la r Mod elin g. Molecular modeling studies were
performed using the SYBYL software package19 running on a
Silicon Graphics Iris Indigo R10000 workstation. The crystal
structure of the RT/nevirapine complex solved at 2.2 Å
resolution by Ren et al. was retrieved from the Brookhaven
Protein Data Bank27 (entry code 1VRT).
Intramolecular and intermolecular energies were calculated
using the molecular mechanics Tripos force field including the
electrostatic contribution. Atom centered partial charges were
calculated according to the Gasteiger-Hu¨ckel method.28,29
Geometry optimizations were realized with the SYBYL/MAXI-
MIN2 minimizer by applying the BFGS algorithm30 and
setting a root-mean-square gradient of the forces acting on
each atom at 0.1 kcal/mol Å as a convergence criterion.
Models of compounds 8 and 15a were built according to

QXPTs as Potent HIV-1 RT Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 313
after removal of the nonadherent cells, was 105 macrophages
per well. Test was conducted as described elsewhere.33 Mac-
rophages 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
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.
3. Vir u s. A laboratory lymphocyte-tropic strain of HIV-1
(HIV-1-IIIB) was used to infect C8166, while macrophages
were infected with a laboratory monocyte-tropic strain of
HIV-1 (HTLV-III-Ba-L, also called HIV-Ba-L).35,36 Titration to
determine the infectivity was performed in human macro-
phages as previously described. The titer of the virus stocks,
expressed as 50% tissue culture infectious dose (TCID50), was
determined as previously described.37
Dr u g An a lysis.8b Plasma and brain concentrations of the
test compounds were determined by high-performance liquid
chromatography (HPLC). An internal standard (IS) was used
in each assay. Brain concentrations of 8 were not corrected
for the compound contributed by residual blood because, in
terms of concentrations, this amounted only to 5-10% of the
brain concentrations.
4. An tivir a l Agen t. Zidovudine (AZT) was purchased from
Sigma Chimica.
5. Toxicity. Chemicals toxicity in C8166 was evalueted with
a procedure involving a colorimetric assay (MTT assay) that
monitors the ability of viable, but not dead, cells to reduce
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) to a blue formazan product, which can be measured
spectrophotometrically.38,39
6. Assa y of An tivir a l Activity. Antiviral activity of the
tested compounds in acutely infected C8166 cultures was
performed following an already described procedure.9 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.
Ack n ow led gm en t. We are grateful to the Istituto
Superiore di Sanita’, Roma, Italy, for financial support
(Progetto Patogenesi, Immunita’ e Vaccino, Grant No.
40B.69) and to Universita’ di Siena (PAR 99). Further-
more, this work has been partially supported by the ISS-
Programma Nazionale di Ricerca sull’ AIDS (Contract
30C.70) and by the CNR Target Project on Biotechnol-
ogy to S.S. Authors thank Dr. Massimo Pregnolato
(Dipartimento di Chimica Farmaceutica, Universita’ di
Pavia) for a gift of efavirenz.
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 11-15, to synergy calculations, and to drug
administration and plasma and brain sampling/drug analysis,
and a table with elemental analyses of compounds 8, 12a -g,
15a -c, 18a ,b and 19. This material is available free of charge
via the Internet at http://pubs.acs.org.
7. Im m u n oflu or escen ce Vir u s Bin d in g Assa y. Calcula-
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(ID50) was performed. The ED50 and ID50 values were calcu-
lated 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
Refer en ces
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 a monolayer and were split by using
tripsin. Perypheral blood mononuclear cells (MNC) were
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healthy donor on a Fycoll-Hypaque gradient as previously
described.41 MNC thus obtained were washed twice with RPMI
1640 supplemented 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 (3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide) 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
filtration 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
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line) were plated at 300 cells/well. Twelve hours 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 by using confluent cells: compounds were
added to the 3T3 monolayer 3 days after plating. Tests were
then run as described above.
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