Design of annulated pyrazoles as inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1021/jm800527x

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of HIV. These regimens are extremely effective in suppressing virus replication. Structure-based o

Full text of DOI:10.1021/jm800527x

J. Med. Chem. 2008, 51, 7449–7458
7449
Design of Annulated Pyrazoles as Inhibitors of HIV-1 Reverse Transcriptase|
Zachary K. Sweeney,*,† Seth F. Harris,*,‡ Nidhi Arora,† Hassan Javanbakht,§ Yu Li, Jennifer Fretland,# James P. Davidson,[
J. Roland Billedeau,† Shelley K. Gleason,† Donald Hirschfeld,† Joshua J. Kennedy-Smith,† Taraneh Mirzadegan,† Ralf Roetz,†
Mark Smith,† Sarah Sperry,† Judy M. Suh,† Jeffrey Wu,† Stan Tsing,‡ Armando G. Villasen˜or,‡ Amber Paul,§ Guoping Su,§
Gabrielle Heilek,§ Julie Q. Hang, Amy S. Zhou,# Jesper A. Jernelius,[ Fang-Jie Zhang,[ and Klaus Klumpp
Departments of Medicinal Chemistry, DiscoVery Sciences and Technologies, Viral Disease Biology, Viral Disease Biochemistry, Non-Clinical
Safety, and Chemical Synthesis, Roche Palo Alto LLC, 3431 HillView AVenue, Palo Alto, California 94304
ReceiVed May 7, 2008
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred
combination antiretroviral therapies used for the treatment of HIV. These regimens are extremely effective
in suppressing virus replication. Structure-based optimization of diaryl ether inhibitors led to the discovery
of a new series of pyrazolo[3,4-c]pyridazine NNRTIs that bind the reverse transcriptase enzyme of human
immunodeficiency virus-1 (HIV-RT) in an expanded volume relative to most other inhibitors in this class.
The binding mode maintains the ꢀ13 and ꢀ14 strands bearing Pro236 in a position similar to that in the
unliganded reverse transcriptase structure, and the distribution of interactions creates the opportunity for
substantial resilience to single point mutations. Several pyrazolopyridazine NNRTIs were found to be highly
effective against wild-type and NNRTI-resistant viral strains in cell culture.
Chart 1. NNRTIs Efavirenz, Etravirine, and Trovirdine,
Highlighting the Aniline Motif
Introduction
Non-nucleoside reverse transcriptase inhibitors bind to an
induced hydrophobic pocket adjacent to the polymerase active
site of HIV-reverse transcriptase and prevent the progression
of DNA synthesis from the viral RNA template.1 Clinical studies
have demonstrated that therapy with a combination of NNRTIsa
and nucleoside or nucleotide reverse transcriptase inhibitors is
able to provide a sustained reduction of viral replication in
treatment-naive patients.2 Nevertheless, a single nucleotide
mutation in the HIV-genome can produce a virus that is highly
resistant to NNRTIs, and treatment failure is often accompanied
by the emergence of resistant viruses.3 Many research groups
have therefore attempted to discover new NNRTIs that inhibit
commonly observed NNRTI-associated resistant viruses and that
have a higher genetic barrier to resistance.4-6
Binding of NNRTIs to HIV-RT is thought to restrict the
motion of key residues in the polymerase active site and prevent
incorporation of nucleotides into the DNA chain during reverse
transcription.7 The reverse transcriptase enzyme is very flexible,
and lead optimization using structure-based drug design and
computational analysis of ligand affinity is complicated by the
dynamic nature of the induced ligand binding site.8-10 Relative
to the apo enzyme, NNRTI-bound reverse transcriptases show
large conformational adjustments in backbone positions and side
chain rotamers, particularly of Tyr181, Tyr183, and Tyr188,
that create the NNRTI pocket (Figure 1).11,12 This induced
conformation also maintains a remarkable degree of plasticity,
as the geometry and volume of the final protein pocket are
sensitive to the shape and properties of the bound ligand.
All NNRTIs form direct or water-mediated hydrogen bonds
with the protein backbone of lysine residue Lys101 or Lys103.13
Inhibitors that maintain hydrogen bond interactions with Lys101
include efavirenz,14 etravirine,15 and trovirdine16 (Chart 1). In
structures of most smaller K101-binding inhibitors, strands ꢀ13
and ꢀ14 (residues 232-241) are displaced relative to their
position in the apo structure. Pro236, which is located on the
intervening turn, translates approximately 3 Å.14 This movement
allows for the formation of a direct hydrogen bond between
the carbonyl of Pro236 and the Lys103 amide NH and provides
favorable hydrophobic interactions between the aniline portion
of the inhibitors and hydrophobic elements of the underlying
shifted ꢀ sheet (Figure 1b).
Another class of NNRTIs contains a hydrogen bond acceptor
that directly interacts with the Lys103 backbone NH. In addition
to delavirdine, this group includes many “next-generation”
inhibitors that maintain potency against the commonly observed
Lys103Asn mutant virus.4,6 Structural analysis of these com-
pounds bound to HIV-RT shows that the ꢀ13-ꢀ14 turn region
is in a position similar to that found in the unliganded structure
(Figure 1c). The inhibitors that interact with the backbone of
Lys103 fill the space adjacent to the loop with functionality
capable of maintaining substantial hydrophobic contacts with
P236 and F227. For example, the investigational drug candidates
31 (GW678248)17 and 32 (RDEA806)18 contain an aromatic
|
PDB codes for cocrystals of compounds 6 and 10 with HIV-RT are
3DYA and 3E01, respectively.
* To whom correspondence should be addressed. For Z.K.S.: phone, 650-
855-6376; fax, 650-852-1311; e-mail, zachary.sweeney@roche.com. For
S.F.H.:
phone,
650-855-5403;
fax,
650-852-1528;
e-mail,
seth.harris@roche.com.
†
Department of Medicinal Chemistry.
‡
§
Department of Discovery Sciences and Technologies.
Department of Viral Disease Biology.
Department of Viral Disease Biochemistry.
Department of Non-Clinical Safety.
#
[
Department of Chemical Synthesis.
a Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhibitor;
HIV-RT, human immunodeficiency virus-reverse transcriptase.
10.1021/jm800527x CCC: $40.75
2008 American Chemical Society
Published on Web 11/14/2008

7450 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Sweeney et al.
Figure 1. (a) NNRTI binding pocket in apo enzyme structure (1HMV). (b) Structure of efavirenz in the NNRTI binding pocket (1FK9). Reorganization
of the protein creates volume to accommodate the NNRTI. Smaller compounds such as this one result in an inward motion of P236 that hydrogen-
bonds to the backbone of K103. (c) Structure of delavirdine in NNRTI binding pocket (1KLM). The larger compound stabilizes Pro236 and the
flanking ꢀ strands in a more open conformation.
Chart 2. NNRTIs Delavirdine, 31, and 32, Highlighting the
Scheme 1. Preparation of Ester Intermediates 15a
Portions of the Inhibitor That Contact Lys103 and Pro236
a (a) NaO-t-Bu, 2,3,4-trifluoronitrobenzene, THF, 0 °C; (b) (i) NaH, tert-
butyl ethylmalonate, NMP; (ii) 12, NMP, 0 °C to room temperature; (c)
TFA, dichloroethane, 75 °C (d) (i) Fe, NH4Cl, EtOH, H2O, 80 °C; (ii)
NaNO2, aqueous hydrogen bromide, CuBr, LiBr, CH3CN, 30 °C; (e) LiOH,
CH3CN, H2O, room temperature; (f) (BOC)2O, DMAP, t-BuOH, EtOAc,
40 °C.
amide functionality in which the amide portion of the inhibitor
interacts with the Lys103 NH while the substituted phenyl group
makes extensive hydrophobic contacts (Chart 2).
discovery of annulated pyrazole NNRTIs that maintain excellent
activity against wild-type and NNRTI-resistant viruses.20 Struc-
tural analysis reveals a distribution of significant interactions
between the most potent inhibitors and HIV-RT. These contacts
allow this class of compounds to inhibit a broad range of
NNRTI-resistant viruses.
In earlier studies of diaryl ether NNRTIs, we had discovered
that triazolinones related to structure A are potent inhibitors of
viral replication (Figure 2).19 Structural analysis revealed that
the triazolinone ring simultaneously forms hydrogen bonds with
the carbonyl and amide NH functionality of Lys103. Modeling
of the triazolinones A and similar amide-based inhibitors B
indicated that the amides made more extensive contact with the
ꢀ13-ꢀ14 region. This observation led to the design of annulated
pyrazole NNRTIs C, which should make hydrogen bonding
interactions similar to those of the triazolinone while more
effectively engaging in hydrophobic interactions with the distal
portions of the ꢀ12-ꢀ14 sheet. In this paper, we describe the
Chemistry
The synthesis of the diaryl ether NNRTIs commenced with
a highly regioselective reaction between phenol and 2,3,4-
trifluoronitrobenzene as described in Scheme 1. A malonate
addition/decarboxylation sequence allowed for the installation
of the acetic acid ester group in good yield. Reduction of the
nitro functionality and a Sandmeyer reaction with the resulting
aniline gave ester 14. The ethyl ester could be converted to the
corresponding tert-butyl ester 15 using standard procedures.
The synthetic procedure used for the preparation of annulated
pyrazole NNRTIs starting from 14 is exemplified for compound
4 in Scheme 2. In the key transformation, a Claisen condensation
between an acylimidazole and the sodium enolate of ester 14
or 15 provided ꢀ-ketoesters that could be decarboxylated and
treated with hydrazine to provide the annulated pyrazole
inhibitors. This route proved to be generally useful for the
preparation of pyrazoles that contained different functionality
on the annulated phenyl ring. The acid utilized for the
preparation of pyrazolo[3,4-c]pyridazines 6 and 8-10 was
prepared in four steps starting from commercially available
3-carboxy-2,6-dichloropyridazine (Scheme 3). Similarly, pyra-
zolopyridone 3 was prepared employing acid 24 (synthesis
Figure 2. Design of annulated pyrazole NNRTIs.

Annulated Pyrazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7451
Scheme 2. General Synthesis of Annulated Pyrazole Analoguesa
a (a) 16, 1,1′-carbonyldiimidazole, DMF, 50 °C; then 14 or 15, NaH, -10 °C.; (b) for R ) Et, H2O, DMSO, 150 °C; for R ) t-Bu, TFA, CH2Cl2, room
temperature; (c) hydrazine, EtOH, 50 °C.
Table 1. Structure and Activity of Annulated Pyrazole Compounds 1-7
Scheme 3. Preparation of Pyridazine Acid for the Synthesis of
Inhibitors 6 and 8-10a
a (a) Trimethylsilyldiazomethane, MeOH, CH2Cl2, 0 °C; (b) 2,4-
difluorophenol, NaH, THF, 0-50 °C; (c) palladium on carbon, ammonium
formate, MeOH, room temperature to 60 °C; then LiOH, THF, MeOH,
H2O, room temperature.
IC50, nMa
EC50, nMb
compd
X7
X6
X5 X4 WT 103N/181C WT 103N/181C
1
2
3
4
5
6
7
CCN CH
CNH2 CH
CdO NCH3 CH CH
CH CH 24
CH CH 24
15
22
12
ntc
9
5
8
4
1
2
1
25<
53
84
97
12
18
Scheme 4. Preparation of Pyridone Acid 24a
8
8
5
3
3
N
CH
N
CH
N
N
CH CH
CH CH
CH CH
3
14
4
N
CH
CH
N
>100
a Inhibition of RNA-dependent DNA polymerase activity using the wild
type or Lys103Asp/Tyr181Cys polymerase. b Inhibition of virus-induced
cell death in MT-4 cells. c Not tested.
a (a) Pd carbon, xylenes, 160 °C; (b) CH3I, K2CO3, DMF, 50 °C; (c)
LiOH, THF, room temperature.
Results and Discussion
Scheme 5. Synthesis of Ester Intermediate 30a
The inhibitors 1-7 were evaluated in enzymatic and cellular
assays using standard procedures, and the results are displayed
in Table 1. Inhibition of HIV polymerase activity was initially
assessed using purified recombinant wild-type or Lys103NAsn/
Tyr181Cys HIV polymerase and a poly(rA)/oligo(dT)16 template-
primer. The ability of the NNRTIs to reduce HIV replication
in cell culture was determined in MT-4 cells using wild-type
virus and clinically relevant mutant strains. Further details are
provided in the Experimental Section.
In order to test the hypothesis that annulated pyrazole
compounds would be potent inhibitors of polymerase activity,
compound 1 was initially prepared (Table 1). Modeling had
suggested that the nitrile substituent of the indazole ring would
emerge from the binding pocket through an opening that
separates Lys104 and Pro225, and we were encouraged to find
that this compound did exhibit strong inhibition of polymerase
activity in the enzyme assay. Furthermore, 1 displayed good
potency in the cellular antiviral assay. Replacement of the nitrile
of the indazole ring with an amino group (2) or conversion of
the indazole phenyl ring to a methylated pyridone (3) did not
significantly alter activity against the wild-type or mutant
viruses. Pyrazolopyridine 4, which contains a single nitrogen
atom in the 7-position of the phenyl ring, had excellent activity
in the cellular assay. This potency was maintained for regioi-
somer 5. Additional activity was gained for the pyrazolo[3,4-
c]pyridazine 6, which significantly inhibited virus replication
at subnanomolar concentrations in the cellular assay. Isomeric
a (a) K2CO3, DMF, room temperature; (b) Fe, NH4Cl, H2O, EtOH, 100
°C; (c) 2-tert-butoxyethylzinc chloride, bis(tri-tert-butylphosphine)palladium,
dioxane, room temperature; (d) t-BuONO, CuBr2, LiBr, CH3CN, 60 °C.
described in Scheme 4). The ester intermediate used for the
synthesis of compound 10 was prepared in four steps as
described in Scheme 5. A nucleophilic aromatic substitution
reaction between phenol 25 and fluorobenzene 26 provided the
diaryl ether. Acetic acid ester 29 was prepared by reduction of
the nitro group with iron, followed by a palladium-catalyzed
reaction with 2-tert-butoxyethylzinc chloride. A Sandmeyer
reaction was then used to install the desired halogen.

7452 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Chart 3. Additional Pyrazolo[3,4-c]pyridazine Inhibitors
Sweeney et al.
replication of the isolates. Compound 8 and etravirine were able
to effectively inhibit a similar proportion of viruses in this panel
(effective inhibition defined as (IC50 mutant)/(IC50 wild-type)
< 10). Importantly, compound 8 maintained potency against
all viruses that contained the common K103 and Y181 muta-
tions. A substantial (>10-fold) reduction in potency was
observed only for isolates that contain the Y188L/G190A and
A98/V108/G190/F227 combination mutations.
Pharmacokinetic profiling revealed that 8 is also stable in
vivo (Table 4). This inhibitor had a moderate volume of
distribution, and acceptable half-lives were observed in several
preclinical species.
The structures of compounds 6 and 10 bound to wild-type
HIV-RT were solved in order to further understand the binding
mode of the inhibitors to HIV-RT (Figures 3 and 4). As
designed, the compounds induce the expanded NNRTI pocket
where Pro236 and the flanking ends of strands ꢀ13 and ꢀ14
are in a position similar to that found in the apo structure. This
configuration is stabilized by the pyrazolopyridazine moiety.
The pyrazole makes hydrogen bonds to both the backbone amide
and carbonyl of Lys103. The pyridazine extension parallels the
face of Pro236 of the ꢀ13-ꢀ14 hairpin, and its amphipathic
edges are well suited to the site: the hydrophobic edge faces
the pocket interior, packing with Phe227 and Leu234, while
the polar nitrogen atoms of the six-membered ring are exposed
to solvent through one of the few openings of the highly buried
cavity (Figure 5).
The diaryl ether system of both 6 and 10 partly overlaps the
“butterfly wing” binding region of the early NNRTIs,21 but the
two inhibitors maintain markedly different angles through the
ether linkage. Both 6 and 10 sit closer to strands ꢀ6 and ꢀ10
than most NNRTIs, which allows Tyr181 to remain in the
general position it occupies in apo or DNA-bound RT
structures.22,23 Concomitantly, Tyr188 rotates slightly to form
aromatic stacking interactions with the terminal phenyl ring of
the inhibitors. This portion of the molecule is also braced against
the conserved Trp229 residue, and a distortion of strand ꢀ12
and the primer grip region similar to that observed in first-
generation NNRTIs is achieved. The large bromine on the core
ring of 6 fills the volume between Val106, Val179, Tyr181,
and Tyr188, with closest approach (3.5 Å) to the backbone
carbonyl of Tyr188, suggesting a favorable halogen-carbonyl
interaction as seen in other recent examples.24
The primary difference between compounds 6 and 10 is the
relative disposition of the substituents about the central phenyl
ring (Figure 6). Compound 10 also extends more broadly across
the binding pocket, reaching slightly closer to both Trp229 at
the back of the pocket and Lys103 at the opposite side. While
the amino acids on each end of the pocket shift outward slightly
to accommodate the ligand, the tightness of fit and the lower
potency of 10 compared to 6 suggests that the organization of
the inhibitor may extend beyond the optimal size for the wild
type pocket. The central phenyl ring of 10 is pivoted such that
the bromine atom does not interact significantly with the Tyr188
carbonyl group and has fewer contacts in this hydrophobic
subpocket. The central phenyl ring of 10 is also removed from
the carbonyl group of K101 relative to the more potent inhibitor
compounds in which the nitrogen atom in the 6-position was
moved to other positions (e.g., 7) generally had inferior potency
in the cellular assay, particularly in tests with the Lys103Asn/
Tyr181Cys mutant enzyme.
The pyrazolopyridazine heterocycle found in inhibitor 6 was
used to prepare a small family of NNRTIs (Chart 3). Compounds
8 and 9 were also found to maintain good potency against the
wild-type and mutant viruses. Inhibitor 10, which was prepared
in order to test the requirement for a meta disposition of the
methylene and ether linkages about the central phenyl ring, had
reasonable activity against the wild-type virus but did not inhibit
the Lys103Asn/Tyr181Cys mutant at tested concentrations.
Further investigation revealed that 10 also did not maintain good
potency against the Lys103Asn or Tyr181Cys single mutant
viruses (IC50(wt) ) 30 nM, IC50(Lys103Asn) ) 120 nM,
IC50(Tyr181Cys) ) 800 nM).
Compound 8, which had an excellent preliminary antiviral
profile and moderate molecular weight (MW ) 415 Da), was
selected for further profiling. Tests with a large panel of NNRTI-
resistant mutant viruses revealed that single nucleotide mutations
of the wild-type virus do not significantly reduce the potency
of this compound (Table 2). In particular, 8 strongly inhibits
replication of the prevalent 103Asn, 181Cys, and 190Ala
mutants. For comparison, data obtained with efavirenz are also
shown. Inhibitor 8 was more potent than efavirenz against
several NNRTI resistant mutants and the wild-type virus. The
relative potencies of efavirenz and 8 in this assay against the
Lys103Asn mutant were particularly striking.
The potential of compound 8 to prevent the replication of
NNRTI-resistant viruses that contain several mutations of the
viral genome was assessed in testing against a panel of NNRTI-
resistant clinical isolates (Table 3). These viruses were selected
(PhenoScreen, Monogram Biosciences) on the basis of their
reduced susceptibility to efavirenz, and the diversity of viral
isolates was chosen to represent the majority of NNRTI
resistance patterns that have been described in clinical practice.
Etravirine, which has shown excellent potency against NNRTI-
resistant viruses,15 was also tested for its ability to inhibit the
Table 2. Potency of 8 and Efavirenz against WT-HIV and Selected NNRTI-Resistant Mutant Virusesa
W
T
WTb
40%
compd
190A
190S
100 I 101E 225H 103N 106A 108I 179D 227 L 236 L 138K 181I 188H 181C
8
EFV
1
2
5
18
1
6
<1
>100
<1
7
2
12
<1
1
<1
66
2
6
1
3
1
8
1
1
1
<1
2
2
1
2
<1
4
1
3
a EC50 (nM) for inhibition of virus-induced cell death in MT-4 cells. b Tests performed in the presence of 40% human serum.

Annulated Pyrazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7453
Table 3. IC50 for 8 and Etravirine against NNRTI-Resistant Clinical
Isolatesa
virus
8
Etr
NNRTI-associated mutations
CNDO
1
2
1
2
2
28
6
100
10
2
G190, Y188
A98, K101, Y181, G190
L100, K103
6
12
14
16
17
20
21
29
31
32
34
36
43
47
1
3
3
5
4
3
2
7
7
5
2
6
6
1
1
14
7
17
3
73
9
5
3
L100, K103
A98, K103, V108, M230
K103, P225
A98, K103, Y181
K103, Y181
K103, V179, Y181
K103, Y181
A98, V179, Y181
V106, V179, Y181, G190
K101, V106, Y181
K103, V106, Y181
K103, Y181, P225
A98, V108, G190, F227
Figure 4. Structure of compound 10 in the NNRTI binding pocket
(3E01). The pocket is formed similarly to compound 6, though the
para disposition around the core forces slight setbacks of W229 and
K103.
13
1
24
a IC50 (nM) in Monogram Biosciences PhenoScreen single-cycle antiviral
assay. Etr ) etravirine.
Table 4. Selected Pharmacokinetic Parameters for Compound 8a
species
Cl ((mL/min)/kg)
Vss (L/kg)
T1/2 (h)
rat
dog
monkey
2.9
4.6
1.3
0.91
0.99
1.21
4.2
5.6
12.2
a Clearance, volume, and half-life observed following intravenous
administration of 0.5 mg/kg dose in 30% DMSO/55% PEG-400/15% water.
Figure 5. View of the pyrazolo[3,4-c]pyridazine ring in the 6-RT
complex. On the pyrazole moiety, the carbon atoms face the interior
hydrophobic environment while the nitrogen atoms are relatively
exposed to solvent via a small opening in the enclosed binding pocket.
between the pyrazolopyridazine compounds and mutable resi-
dues such as Val106, Tyr188, and Phe227 does not significantly
impact the inhibition potency. In addition, the architecture of 6
includes malleable linkages that allow for conformational and
positional adjustment.26-28 As a consequence of the soft energy
potentials around those connections, binding energy is lost as
the compound adapts to the mutant protein’s changed surface.
Finally, specific polar interactions target invariant backbone
atoms rather than mutable side chain groups. These motifs are
again spread across the binding pocket and include traditional
hydrogen bond interactions with the pyrazolopyridazine het-
erocycle, CH-hydrogen bond interactions of the central phenyl
ring, and halogen-carbonyl interactions of the bromine atom.
Some of these interactions are not observed in the structure of
the enzyme complex with inhibitor 10, and their absence may
contribute to the relatively poor potency of this inhibitor.
Figure 3. Structure of compound 6 in the NNRTI binding pocket
(3DYA). The diaryl ether fills the core hydrophobic pocket. The
pyrazolo[3,4-c]pyridazine forms hydrogen bonds to the backbone of
K103 and stabilizes Pro236 in an apo-like position, creating an expanded
pocket volume relative to efavirenz.
6. Many NNRTIs maintain a strong hydrogen bond donor-
acceptor relationship with the carbonyl group of Lys101, and
it is possible that the CH-hydrogen bond through which 6
interacts with this moiety contributes to the activity of the
inhibitor (Figure 3).25
As has been noted previously, NNRTI-resistant HIV-reverse
transcriptases generally have an expanded pocket volume
relative to the wild type enzyme.12 That is, the majority of
NNRTI resistance-pathways involve the substitution of residues
that make hydrophobic interactions with the enzyme inhibitor.
As a consequence of the larger size of compound 6 and the
expanded binding pocket that it induces in the reverse tran-
scriptase enzyme, the interactions of this inhibitor are spread
across a larger area of the protein than first generation NNRTIs.
All portions of the molecule are making significant interactions
with the enzyme, and no individual position appears to provide
a majority of the interaction energy. This conclusion is supported
by the observation that perturbation of the significant interactions
Conclusions
Structure-based design of novel inhibitors that interact with
the K103 backbone of HIV-reverse transcriptase has led to the
discovery of NNRTIs that contain an unusual 1H-pyrazolo[3,4-
c]pyridazine heterocycle. This heterocycle stabilizes a large
binding pocket with many structural features less changed from
the apoprotein conformation than seen in early NNRTIs. The
optimized fit to this expanded pocket provides compounds that
potently inhibit the replication of wild-type and NNRTI-resistant

7454 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Sweeney et al.
Hz, 1H), 7.06 (m, 1H), 4.21 (q, J ) 7.1 Hz, 2H), 3.80 (d, J ) 1.5
Hz, 2H), 1.28 (t, J ) 7.1 Hz, 3H).
[4-Bromo-3-(3-chloro-5-cyanophenoxy)-2-fluorophenyl]acetic Acid
Ethyl Ester (14). A 250 mL round-bottom flask was charged with
13 (8.0 g, 21.12 mmol) and absolute EtOH. To the reaction vessel
was added NH4Cl (2.26 g, 42.2 mmol), water (30 mL), and iron
(1.17 g, 21.1 mmol). The mixture was stirred and heated to 80 °C
for 4 h. When 13 was consumed, the heterogeneous mixture was
filtered and the filter cake was washed with EtOAc. The aqueous
filtrate was extracted with EtOAc and washed with water and brine.
The combined EtOAc extracts were dried over Na2SO4 and filtered.
The solvent was removed in vacuo to afford a pale oil which was
purified by column chromatography, eluting with EtOAc/hexane
(15:85) to afford the aniline (6.0 g, 87%). The aniline (20.0 g, 57.3
mmol), CuBr (24.7 g, 172 mmol), and LiBr (7.45 g, 86.0 mmol)
were placed in CH3CN (400 mL) and heated to 30 °C. A 48%
aqueous HBr solution (13.0 mL, 115 mmol) was added, which
resulted in a slight exotherm. Sodium nitrite (11.5 mL of a 40%
aqueous solution, 86 mmol) was slowly added over 30 min, during
which time the starting material was consumed and the solution
became homogeneous. The solution was stirred for an additional
hour, solid NaHSO3 (6.0 g) and SiO2 (40 g) were added, and the
mixture was cooled to room temperature. The acetonitrile solvent
was removed by distillation, toluene (400 mL) was added, and the
solution was filtered. The toluene was removed by distillation, and
the remaining solids were crystallized from isopropyl alcohol and
Figure 6. Compound 6 (3DYA) and compound 10 (3EO1) in the
NNRTI binding pocket.
viruses. Pharmacokinetic studies revealed that inhibitor 8 has
properties suitable for use as an NNRTI that could provide
improved treatment durability for HIV-infected patients. These
compounds and related diaryl ether NNRTIs will be the subject
of future reports.
Experimental Section
1
water to provide 14 (20.2 g, 85%). H NMR (DMSO-d6): δ 7.82
NMR spectra were recorded on a Bruker 300 MHz or Bruker
400 MHz spectrometer. Elemental analyses were performed at
Roche Palo Alto. All purchased starting materials were used without
further purification. Solvents used in reaction mixtures were
anhydrous and stored under nitrogen. All reactions were performed
in oven-dried flasks under a nitrogen atmosphere unless otherwise
noted.
3-Chloro-5-(2,3-difluoro-6-nitrophenoxy)benzonitrile (12). A 250
mL round-bottom flask was charged with (6.0 g, 39.0 mmol) and
anhydrous THF (100 mL), and the solution was cooled to 0 °C. To
the cooled solution was added sodium tert-butoxide (46.9 g, 45.0
mmol), and the resulting solution stirred for 1 h. 2,3,4-Trifluoroni-
trobenzene (6.9 g, 39.0 mmol) was added dropwise while maintain-
ing the mixture at 0 °C until the phenol was completely consumed.
The mixture was quenched by addition of 10% aqueous HCl, and
the resulting mixture was stirred for an additional hour. The mixture
was extracted with EtOAc and washed with water and brine. The
EtOAc was dried over Na2SO4 and filtered. The solvent was
removed in vacuo to yield a yellow oil which was purified by
column chromatography on silica gel with EtOAc/hexanes to afford
ether 12 (10 g, 82%). 1H NMR (DMSO-d6): δ 8.20 (m, 1H), 7.85
(m, 1H), 7.75-7.70 (m, 3H).
[3-(3-Chloro-5-cyanophenoxy)-2-fluoro-4-nitrophenyl]acetic Acid
Ethyl Ester (13). A solution of tert-butyl ethyl malonate (10.3 g,
54.8 mmol) in anhydrous NMP (200 mL) was cooled to 0 °C and
stirred under a nitrogen atmosphere. To this solution was added
NaH (40% in mineral oil, 1.84 g, 76.7 mmol). The mixture was
allowed to stir at 0 °C for 1 h. The aryl ether 12(15.00 g, 49.80
mmol) was then added to the reaction vessel and stirred under
nitrogen at room temperature until the reaction was complete. The
mixture was quenched by addition of aqueous 10% HCl at room
temperature, extracted with EtOAc, and washed with water and
brine. The EtOAc was dried over Na2SO4 and filtered, and the
solvent was removed in vacuo to afford a light-yellow oil. This oil
(24.0 g, 50.2 mmol) was dissolved in dichloroethane (300 mL) and
TFA (6.29 g, 55.2 mmol) and heated to 75 °C for 24 h. The mixture
was cooled to room temperature, and solvent and excess TFA were
removed in vacuo. The crude oil was dissolved in CH2Cl2 and
cooled to 0 °C, and aqueous NaHCO3 was added. The mixture was
extracted with CH2Cl2 and washed with water and brine. The
organic layer was dried over Na2SO4 and filtered, and the solvent
was removed in vacuo to afford a yellow oil. The crude oil was
purified by column chromatography on silica gel, eluting with
EtOAc/hexanes (1:9) to afford 13 (15.0 g, 80%). 1H NMR (CDCl3):
δ 7.89 (dd, J ) 8.5 Hz, 1.9 Hz, 1H), 7.41 (m, 2H), 7.20 (t, J ) 2.2
(t, J ) 1.5 Hz, 1H), 7.51-7.47 (m, 2H), 7.43-7.40 (m, 2H), 4.11
(q, J ) 7.1 Hz, 2H), 3.84 (s, 2H), 1.29 (t, J ) 7.1 Hz, 3H).
[4-Bromo-3-(3-chloro-5-cyanophenoxy)-2-fluorophenyl]acetic Acid
tert-Butyl Ester (15). A solution of 14 (15 g, 0.036 mol) in
acetonitrile (75 mL) was heated to 50 °C. An aqueous solution of
lithium hydroxide (1.6 g in 10 mL water, 1.1 equiv) was added
over 2.5 h with stirring. The mixture was stirred overnight at 50
°C, diluted with toluene (100 mL), and cooled to 5 °C. After aging
the mixture for 1 h at 5 °C, the mixture was filtered, washed with
cold acetonitrile, and dried in a vacuum oven to provide the lithium
salt of [4-bromo-3-(3-chloro-5-cyanophenoxy)-2-fluorophenyl]acetic
acid (12.9 g, 92%). This salt was suspended in MTBE (20 mL/g)
and washed three times with 10% sulfuric acid in water. The organic
layer was washed with brine, decolorizing carbon (75 mg/g, Darco
KB) and silica gel (75 mg per gram) were added, and the mixture
was stirred at ambient temperature for 2 h. The mixture was filtered
and concentrated under reduced pressure to provide the acid as an
oil that solidified on standing. The acid (12.7 g, 0.033 mol, 1 equiv),
tert-BuOH (2.45 g, 5 equiv), Boc2O (7.20 g, 1.05 equiv), and
DMAP (120 mg, 0.03 equiv) were dissolved in EtOAc (33 mL).
The mixture was warmed to 40 °C for 3 h and cooled to room
temperature, and an equal volume of water was added, followed
by H2SO4 (4 equiv). The aqueous layer was separated and discarded,
and the organic layer was washed with water. The organic layer
was washed with brine, concentrated to about 20% of original
volume, and diluted with hexanes back to the original volume.
Decolorizing carbon (75 mg/g, Darco G60) and silica gel (75 mg/
g) were added, and the mixture stirred at ambient temperature for
2 h. The mixture was filtered and concentrated under reduced
pressure to give the ester as an oil that solidified on standing (12.4
1
g, 85%). H NMR (CDCl3): δ 7.44 (m, 1H), 7.34 (m, 1H), 7.15
(m, 2H), 7.04 (m, 1H), 3.61 (s, 1H), 1.44 (s, 9H).
General Procedure for Preparation of 1-10 from Ester 14.
Step 1. Synthesis of 3-(6-Bromo-3-{2-[2-(2,4-difluorophenoxy)py-
ridin-3-yl]-2-oxoethyl}-2-fluorophenoxy)-5-chlorobenzonitrile (18).
CDI (1.57 g, 9.7 mmol) was added to a solution of acid 17 (1.45
g, 9.2 mmol) in dry DMF (30 mL). The solution was heated to 50
°C for 1 h and then cooled to -10 °C. A solution of ester 14 (4.00
g, 9.7 mmol) in DMF (15 mL) was added, followed by a 60%
dispersion of NaH (1.35 g, 31 mmol). The orange solution was
warmed slowly to room temperature, stirred for 30 min, and added
to a saturated aqueous solution of ammonium chloride. The aqueous
layer was extracted with EtOAc. The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concentrated

Annulated Pyrazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7455
in vacuo to provide an orange oil. This oil was dissolved in DMSO
(20 mL) and brine (1 mL), and the solution was placed in a oil
bath that had been heated to 150 °C. After 10 min, the mixture
was poured into water (500 mL) and the aqueous mixture was
extracted with EtOAc. The combined organic layers were washed
with brine, dried over MgSO4, filtered, and concentrated. The
residue was purified by column chromatography on silica gel,
eluting with a EtOAc/hexanes gradient (25-100% EtOAc) to
6.33 (d, J ) 7.3 Hz, 1H), 4.36 (q, J ) 7.1 Hz, 2H), 4.01 (s, 3H),
3.57 (s, 3H), 1.38 (t, J ) 7.1 Hz, 3H).
3-Methoxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylic Acid
(24). Ethyl ester 23 (0.75 g, 3.6 mmol) was dissolved in THF (35
mL). To this solution was added a solution of LiOH ·H2O (0.18 g,
4.2 mmol) in H2O (12 mL). After being stirred for 18 h, the mixture
was quenched with 10% HCl to pH 1 and extracted with EtOAc.
The organic layers were washed with brine, dried over MgSO4,
filtered, and concentrated to provide acid 24 (0.55 g, 85%). 1H NMR
(CDCl3): δ 7.17 (d, J ) 7.1 Hz, 1H), 6.73 (d, J ) 7.1 Hz, 1H),
4.28 (s, 3H), 3.60 (s, 3H).
1
provide 18 (3.32 g, 73%) as a white solid. H NMR (CDCl3): δ
8.54 (dd, J ) 4.7 Hz, 1.5 Hz, 1H), 7.88 (dd, J ) 8.2 Hz, 1.6 Hz,
1H), 7.49 (dd, J ) 8.2 Hz, 1.8 Hz, 1H), 7.37 (m, 2H), 7.16 (m,
2H), 6.99 (m, 1H), 4.39 (s, 1H).
3-[6-Bromo-2-fluoro-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrazo-
lo[3,4-c]pyridin-3-ylmethyl)phenoxy]-5-chlorobenzonitrile (3). 1,1′-
Carbonyldiimidazole (0.53 g, 3.6 mmol) was added in one portion
to a solution of acid 24 (0.55 g, 3.25 mmol) in DMF (5.0 mL).
After being heated for 30 min at 50 °C, the solution was cooled to
0 °C and 15 (1.43 g, 3.6 mmol) in DMF (10 mL) was introduced.
NaH (0.40 g of a 60% dispersion, 12.2 mmol) was then slowly
added in one portion, and the reaction mixture was allowed to warm
to room temperature over 2 h. The mixture was then cooled to 0
°C, quenched with saturated NH4Cl, diluted with water, and
extracted with EtOAc. The organic layers were washed with brine,
dried over MgSO4, filtered, and concentrated. The residue obtained
was dissolved in CH2Cl2 (21 mL) and treated with TFA (7 mL)
for 18 h. Upon concentration, silica gel chromatography using a
MeOH/CH2Cl2 gradient (1-5% MeOH) provided the desired ketone
(0.90 g, 60%). Hydrazine (0.28 mL, 9 mmol) was slowly added to
a solution of this ketone (0.76 g, 1.5 mmol) in 1,4-dioxane (15
mL) at room temperature. The reaction was heated to 65 °C for
18 h, concentrated, and purified by column chromatography on silica
gel using a EtOAc/hexanes gradient (33 to 100% EtOAc) to provide
3 (0.19 g, 26%). 1H NMR (DMSO-d6): δ 13.83 (s, 1H, NH), 7.80
(m, 1H), 7.58 (dd, J ) 8.5 Hz, 1.7 Hz, 1H), 7.45 (dd, J ) 2.45 Hz,
1.3 Hz, 1H), 7.38 (m, 1H), 7.25 (m, 1H), 7.18 (d, J ) 7.2 Hz, 1H),
6.37 (d, J ) 7.2 Hz, 1H), 4.24 (s, 2H). MS (ESI): 487 (M + H).
Anal. Calcd for C21H13BrClFN4O2 ·0.1H2O: C, 51.52; H, 2.72; N,
11.45. Found: C, 51.25, H, 2.63; N, 11.30.
Step 2: 3-[6-Bromo-2-fluoro-3-(1H-pyrazolo[3,4-b]pyridin-3-
ylmethyl)phenoxy]-5-chlorobenzonitrile (4). Hydrazine (0.44 mL,
2 equiv) was added to a solution of the ketone 18 (3.32 g, 6.9 mmol)
in a mixture of dioxane (20 mL) and EtOH (3 mL), and the solution
was slowly heated to 50 °C. After 2 h, the volatile materials were
removed, and the solid was triturated with a 1:1 mixture of EtOAc
and hexanes. The remaining solids were dissolved in 25% MeOH/
CH2Cl2 and washed with water, saturated aqueous NaHCO3
solution, and brine. The volatile materials were removed in vacuo
to provide the desired product 4 (1.90 g, 59%). 1H NMR (DMSO-
d6): δ 13.43 (s, 1H), 8.50 (dd, J ) 4.5 Hz, 1.5 Hz, 1H), 8.10 (dd,
J ) 8.0 Hz, 1.6 Hz, 1H), 7.80 (m, 1H), 7.59 (dd, J ) 8.4 Hz, 1.8
Hz, 1H), 7.48 (dd, J ) 2.4 Hz, 1.3 Hz, 1H), 7.40 (t, J ) 2.2 Hz,
1H), 7.26 (m, 1H), 7.15 (dd, J ) 8.1 Hz, 4.5 Hz, 1H), 4.37 (s,
2H). MS (ESI): 457 (M
+
H). Anal. Calcd for
C20H11BrClFN4O·0.4H2O: C, 51.67; H, 2.56; N, 12.05. Found: C,
51.67; H, 2.55; N, 12.42.
3-[4-Bromo-3-(3-chloro-5-cyanophenoxy)-2-fluorobenzyl]-1H-in-
dazole-7-carbonitrile (1). 1 was prepared as described in the general
procedure from 3-cyano-2-fluorophenylacetic acid and 14 (24%
overall yield). 1H NMR (DMSO-d6): δ 13.81 (s, 1H, NH), 8.06 (d,
J ) 8.3 Hz, 1H), 7.93 (d, J ) 8.3 Hz, 1H), 7.80 (m, 1H), 7.58 (dd,
J ) 8.3 Hz, 1.7 Hz, 1H), 7.48 (dd, J ) 2.5, 1.2 Hz, 1H), 7.40 (t,
J ) 2.1 Hz, 1H), 7.25 (m, 2H), 4.41 (s, 1H). MS (ESI): 484 (M +
H). Anal. Calcd for C22H11BrClFN4O·0.25H2O: C, 54.34; H, 2.38;
N: 11.53. Found: C, 54.71; H, 2.31; N, 11.18.
3-[6-Bromo-2-fluoro-3-(1H-pyrazolo[3,4-c]pyridin-3-ylmeth-
yl)phenoxy]-5-chlorobenzonitrile (5). 5 was prepared as described
in the general procedure from 3-fluoroisonicotinic acid and ester
14 (10% overall yield). 1H NMR (DMSO-d6): δ 13.40 (s, 1H, NH),
9.00 (d, J ) 1.3 Hz, 1H), 8.20 (d, J ) 5.7 Hz, 1H), 7.81 (m, 1H),
7.57 (m, 2H), 7.50 (dd, J ) 2.4 Hz, 1.3 Hz, 1H), 7.40 (t, J ) 2.0
Hz, 1H), 4.41 (s, 2H). MS (ESI): 457 (M + H). Anal. Calcd for
C20H11BrClFN4O·0.35H2O: C, 51.77; H, 2.54; N, 12.08. Found:
C, 51.78; H, 2.25; N, 11.93.
3-[3-(7-Amino-1H-indazol-3-ylmethyl)-6-bromo-2-fluorophenoxy]-
5-chlorobenzonitrile (2). As described in the general procedure,
3-[6-bromo-2-fluoro-3-(7-nitro-1H-indazol-3-ylmethyl)phenoxy]-5-
chlorobenzonitrile was prepared in 15% overall yield from 2-chloro-
3-nitrobenzoic acid and 14. This material (0.070 g, 0.013 mmol)
was dissolved in EtOH (0.4 mL) and H2O (0.1 mL). Iron powder
(0.032 g, 0.058 mmol) and NH4Cl (0.031, 0.058 mmol) were added.
The solution was heated to 90 °C for 30 min, cooled to room
temperature, filtered, and concentrated in vacuo to provide the
amine. The resulting product was purified by column chromatog-
raphy on silica gel, eluting with an EtOAc/hexane gradient
6-Chloro-3-(2,4-difluorophenoxy)pyridazine-4-carboxylic Acid
Methyl Ester (20). A solution of (trimethylsilyl)diazomethane (2.0
M in hexane) was slowly added to a 0 °C solution of 3,6-dichloro-
4-carboxypyridazine (7.5 g, 39 mmol) in CH2Cl2 (30 mL) and
MeOH (10 mL) until a persistent yellow color was observed. After
addition was complete, the solvents were removed in vacuo. The
crude product was purified by column chromatography on silica
gel, eluting with an EtOAc/hexane gradient (10-25% EtOAc) to
afford 3.89 g (86%) of the methyl ester as a brown oil that solidified
on standing. Sodium hydride (1.53 g, 38.27 mmol) was suspended
in dry THF (70 mL) under a N2 atmosphere and cooled, and 2,4-
difluorophenol (3.31 mL, 34.94 mmol) was added dropwise via
syringe. After the addition was complete, the mixture was stirred
for 15 min. Then the cooling bath was removed for 30 min, and
finally the solution was again cooled to 0 °C. A solution of the
methyl ester (6.89 g, 33.3 mmol) in dry THF (20 mL) was added
through a cannula. The resulting mixture was stirred at room
temperature overnight and then heated to 50 °C for 3 h. The mixture
was cooled to room temperature, and saturated aqueous NH4Cl (40
mL) was added followed by water (60 mL). The mixture was
extracted with EtOAc, dried (MgSO4), filtered, and evaporated. The
crude product was purified by SiO2 chromatography, eluting with
an EtOAc/hexane gradient (10-20% EtOAc) to afford 8.15 g (82%)
of 20 as a light-yellow oil. 1H NMR (CDCl3): δ 7.94 (s, 1H), 7.27
(m, 1H), 6.95 (m, 1H), 4.02 (s, 3H). MS (ESI): 301 (M + H).
1
(33-70% EtOAc) to afford 2 (0.068 g, 91%). H NMR (DMSO-
d6): δ 12.36 (s, 1H, NH), 7.80 (m, 1H), 7.54 (dd, J ) 8.5, 1.7 Hz,
1H), 7.47 (dd, J ) 2.3, 1.2 Hz, 1H), 7.39 (t, J ) 2.2 Hz, 1H), 7.2
(m, 1H), 6.77 (m, 2H), 6.45 (dd, J ) 6.7 Hz, 1.4 Hz, 1H), 5.28 (s,
2H, NH2), 4.26 (s, 2H). MS (ESI): 471 (M + H). Anal. Calcd for
C21H13BrClFN4O: C, 53.47; H, 2.78; N, 11.88. Found: C, 53.51;
H, 2.63; N, 11.65.
3-Methoxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylic Acid
Ethyl Ester (23). Palladium on carbon (1.2 g of a 10 weight-percent
dispersion) was added to ester 22 (5.30 g, 28.6 mmol) that was
suspended in xylenes (100 mL). This mixture was then heated to
160 °C for 20 h. Upon cooling, the mixture was diluted with 1:1
MeOH/CHCl3 (200 mL) and filtered. The solution obtained was
concentrated in vacuo. This material was redissolved in DMF (100
mL), and to the mixture was added K2CO3 (20.3 g, 143 mmol)
and iodomethane (7.2 mL, 114 mmol). After being stirred for 3 h
at 50 °C, the mixture was filtered, quenched with 10% HCl (30
mL), diluted with water, and extracted with EtOAc. The organic
layers were washed with brine, dried over MgSO4, filtered, and
concentrated. Column chromatography on silica gel, eluting with
a EtOAc/hexanes gradient (50-100% EtOAc), provided the ethyl
ester 23 (2.14 g, 35%). 1H NMR (CDCl3): δ 7.07 (d, J ) 7.3, 1H),

7456 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Sweeney et al.
3-(2,4-Difluorophenoxy)pyridazine-4-carboxylic Acid (21). To a
solution of 20 (8.15 g, 127.1 mmol) in MeOH (40 mL) was added
ammonium formate (8.55 g, 1.1 equiv) followed by 10% palladium
on carbon (0.5 g). The mixture was heated to 50 °C for 20 min
and then to 60 °C for 35 min. The mixture was cooled to room
temperature and filtered. The volatile solvents were evaporated, and
the residual material was partitioned between CH2Cl2 (80 mL) and
H2O. The CH2Cl2 layer was separated, and the aqueous layer was
extracted twice with CH2Cl2 (80 mL). The combined extracts were
dried (MgSO4), filtered, and evaporated. The crude product was
purified by SiO2 chromatography, eluting with an EtOAc/hexane
gradient (10-50% EtOAc) to afford 5.5 g (76%) of the monochlo-
ride intermediate as a viscous yellow oil. To a solution of this
intermediate (5.0 g, 19.0 mmol) in THF (40 mL) and MeOH (10
mL) was added an aqueous solution of LiOH (21.6 mL, 1.0 M
solution). The mixture was stirred for 15 min and concentrated,
and the residue was diluted with H2O (25 mL) and THF (20 mL)
and then adjusted to pH 2-3 with 10% HCl. The resulting solid
was collected by filtration and washed with water (50 mL) and
EtOAc (30 mL) to obtain 4.08 g (86%) of 21 as a white powder.
1H NMR (CDCl3): δ 9.21 (d, J ) 4.7 Hz, 1H), 8.08 (d, J ) 4.7
Hz, 1H), 7.51 (m, 2H), 7.17 (m, 1H). MS (ESI): 253 (M + H).
3-[6-Bromo-2-fluoro-3-(1H-pyrazolo[3,4-c]pyridazin-3-ylmeth-
yl)phenoxy]-5-chlorobenzonitrile (6). 6 was prepared as described
in the general procedure from 14 and 21. 1H NMR (DMSO-d6): δ
14.25 (s, 1H, NH), 9.08 (d, J ) 5.5 Hz, 1H), 8.03 (d, J ) 5.5 Hz,
1H), 7.80 (m, 1H), 7.60 (dd, J ) 8.5 Hz, 1.7 Hz, 1H), 7.49 (dd, J
) 2.3 Hz, 1.2 Hz, 1H), 7.40 (t, J ) 2.2 Hz, 1H), 7.33 (t, J ) 8.1
Hz, 1H), 4.45 (s, 2H). MS (ESI): 458 (M + H). Anal. Calcd for
C19H10BrClFN5O: C, 49.75; H, 2.20; N, 15.27. Found: C, 49.81;
H, 2.16; N, 15.17.
Anal. Calcd for C20H10BrFN6O: C, 53.47; H, 2.24; N, 18.71. Found:
C, 53.55; H, 2.27; N, 18.65.
3-(4-Bromo-2-nitrophenoxy)-5-methylbenzonitrile (27). Potas-
sium carbonate (8.30 g, 60 mmol) was added to a stirred solution
of 3-hydroxy-5-methylbenzonitrile 25 (4.0 g, 30 mmol) and
4-bromo-1-fluoro-2-nitrobenzene 26 (3.7 mL, 30 mmol) in DMF
(300 mL). The resulting mixture was stirred at room temperature
for 48 h. The crude reaction mixture was concentrated under
reduced pressure to remove excess DMF. The residue was diluted
with EtOAc, washed with water, aqueous 1 M HCl solution,
saturated aqueous LiCl solution and dried over MgSO4. Filtration
and evaporation of the volatile materials in vacuo provided 27 as
a light-brown solid (9.40 g, 94%). 1H NMR (CDCl3): δ 8.14 (d, J
) 2.4 Hz, 1H), 7.71 (dd, J ) 8.8, 2.4 Hz), 7.26s7.28 (m, 1H),
7.04s7.07 (m, 2H), 6.98 (d, 1H, J ) 8.8 Hz), 2.39 (s, 3H).
3-(2-Amino-4-bromophenoxy)-5-methylbenzonitrile (28). Iron
powder (6.3 g, 112 mmol), NH4Cl (6.03 g, 112 mmol), and water
(180 mL) were added to a solution of 27 (49.4 g, 28.2 mmol) in
EtOH (180 mL). The mixture was heated to 100 °C for 4 h, filtered,
and evaporated to dryness under reduced pressure. The residue was
diluted with EtOAc, washed with saturated aqueous NaHCO3
solution, brine, and dried over MgSO4. Filtration and evaporation
of the volatile materials in vacuo provided 28 as an off-white solid
(1.50 g, 66%). 1H NMR (CDCl3): δ 7.14-7.16 (m, 1H), 6.96-7.00
(m, 3H), 6.85 (dd, J ) 8.5, 2.2 Hz, 1H), 6.72 (d, J ) 8.5 Hz, 1H),
2.35 (s, 1H).
[3-Amino-4-(3-cyano-5-methylphenoxy)phenyl]acetic Acid tert-
Butyl Ester (29). A solution of 2-tert-butoxyethylzinc chloride
(74.80 mL, 37.40 mmol) in THF was added to a solution of 28
(6.30 g, 20.8 mmol) and bis(tri-tert-butylphosphine)palladium (1.60
g, 3.11 mmol) in 1,4-dioxane (200 mL). After being stirred for
20 h, the reaction mixture was filtered and evaporated to dryness
under reduced pressure. Column chromatography on silica gel using
an EtOAc/hexanes gradient (20-50% EtOAc) provided the desired
product 29 as an oil that slowly crystallized on standing (2.30 g,
3-[6-Bromo-2-fluoro-3-(1H-pyrazolo[3,4-b]pyrazin-3-ylmeth-
yl)phenoxy]-5-chlorobenzonitrile (7). 1-1′-Carbonyldiimidazole (0.56
g, 3.46 mmol) was added in one portion to a solution of
3-chloropyrazine-2-carboxylic acid (0.50 g, 3.15 mmol) in DMF
(5.3 mL). After being heated for 30 min at 50 °C, the solution was
cooled to 0 °C and a solution of 15 (1.50 g, 3.46 mmol) in DMF
(11 mL) was introduced. Sodium hydride (0.43 g, 60% dispersion
in mineral oil, 9.45 mmol) was then slowly added, and the reaction
mixture was allowed to warm to room temperature over 2 h. The
mixture was then cooled to 0 °C, quenched with saturated NH4Cl,
diluted with water, and extracted with EtOAc. The organic layers
were washed with brine, dried over magnesium sulfate, filtered,
and concentrated. The residue was redissolved in CH2Cl2 (21 mL)
and treated with TFA (7 mL) for 18 h. Concentration of the volatile
materials in vacuo and silica gel chromatography using a EtOAc/
hexanes gradient (5-35% EtOAc) provided 1.0 g of the desired
ketone. This material was dissolved in 1,4-dioxane (21 mL), and
hydrazine (0.20 mL) was slowly added. The mixture was heated
to 60 °C for 1 h, cooled to room temperature, concentrated, and
1
33%). H NMR (CDCl3): δ 7.11s7.14 (m, 1H), 6.98s7.01 (m,
2H), 6.81 (d, J ) 8.15 Hz, 1H), 6.77 (d, J ) 2.05 Hz, 1H), 6.64
(dd, J ) 8.2, 2.1 Hz, 1H), 3.74 (s, 2H), 3.45 (s, 2H), 2.33 (s, 3H),
1.46 (s, 9H).
[3-Bromo-4-(3-cyano-5-methylphenoxy)phenyl]acetic Acid tert-
Butyl Ester (30). tert-Butyl nitrite solution (1.07 mL, 8.15 mmol)
was added to a mixture of CuBr2 (1.51 g, 6.79 mmol) and LiBr
(1.76 g, 6.79 mmol) in MeCN (90 mL) at 60 °C. After 10 min, a
solution of 29 (2.30 g, 6.79 mmol) in MeCN (20 mL) was added.
After being stirred at 60 °C for 24 h, the reaction mixture was
quenched with aqueous 5% HBr solution (100 mL). The organics
were extracted with EtOAc, washed with brine, dried over MgSO4,
filtered, and evaporated to dryness in vacuo. Column chromatog-
raphy on silica gel using an EtOAc/hexanes gradient (10-50%
EtOAc) provided the desired product 30 (0.70 g 25%) as an oil.
1H NMR (CDCl3): δ 7.58 (d, J ) 2.0 Hz, 1H), 7.22s7.27 (m,
1H), 7.15s7.18 (m, 1H), 6.93s7.05 (m, 3H), 3.53 (s, 2H), 2.35
(s, 3H), 1.47 (s, 9H).
3-[2-Bromo-4-(1H-pyrazolo[3,4-c]pyridazin-3-ylmethyl)phenoxy]-
5-methylbenzonitrile (10). Carbonyldiimidazole (0.297 g, 1.83
mmol) was added to a solution of 3-(2,4-difluorophenoxy)py-
ridazine-4-carboxylic acid (0.462 g, 1.83 mmol) in DMF (8 mL).
The contents of the flask were heated at 50 °C under an atmosphere
of argon. After 50 min, the reaction mixture was cooled to room
temperature and cannulated into a solution of 15 (0.67 g, 1.67 mmol)
in DMF (8 mL), followed by the addition of NaH (0.128 g, 5.34
mmol). After 30 min the reaction was quenched by the addition of
aqueous 1 M HCl solution. The organics were extracted with
EtOAc, washed with brine, dried over MgSO4, filtered, and
evaporated to dryness under reduced pressure. The residue was
passed through a silica gel plug to provide the intermediate ester
as a colorless oil (0.200 g). The ester was dissolved in toluene (1.2
mL) and treated with p-toluenesulfonic acid (0.015 g, 0.08 mmol).
The reaction mixture was heated at 150 °C for 4 h and then cooled
to room temperature. The crude reaction mixture was quenched by
the addition of saturated aqueous NaHCO3 solution. The organics
1
triturated with Et2O to give 7 (0.51 g, 32%). H NMR (DMSO-
d6): δ 13.87 (s, 1H, NH), 8.58 (m, 2H), 7.80 (m, 1H), 7.55 (d, J )
8.6 Hz, 1H), 7.47 (s, 1H), 7.33 (t, J ) 7.8 Hz, 1H), 4.43 (s, 2H).
MS (APCI, negative): 456 (M
C19H10BrClFN5O: C, 49.75; H, 2.20; N, 15.27. Found: C, 49.84;
H, 2.23; N, 15.14.
- H). Anal. Calcd for
3-Chloro-5-[6-chloro-2-fluoro-3-(1H-pyrazolo[3,4-c]pyridazin-
3-ylmethyl)phenoxy]benzonitrile (8). 8 was prepared from 4-chloro-
3-(3-chloro-5-cyanophenoxy)-2-fluorophenyl]acetic acid ethyl ester
and 21 using the general procedure. 1H NMR (DMSO-d6): δ 14.29
(s, 1H, NH), 9.09 (d, J ) 5.6 Hz, 1H), 8.04 (d, 1H, J ) 5.6 Hz),
7.81 (m, 1H), 7.53 (dd, J ) 2.3 Hz, 1.2 Hz, 1H), 7.50-7.35 (m,
3H), 4.47 (s, 2H). MS (ESI): 414 (M + H). Anal. Calcd for
C19H10Cl2FN5O: C, 55.09; H, 2.43; N: 16.91. Found: C, 54.99; H,
2.31; N: 16.69.
5-[6-Bromo-2-fluoro-3-(1H-pyrazolo[3,4-c]pyridazin-3-ylmeth-
yl)phenoxy]isophthalonitrile (9). 9 was prepared from 4-bromo-3-
(3,5-dicyanophenoxy)-2-fluorophenyl]acetic acid ethyl ester and 21
using the general procedure. 1H NMR (MeOH-d3): δ 9.04 (d, J )
5.5 Hz, 1H), 8.03 (d, J ) 5.5 Hz, 1H), 7.91 (m, 1H), 7.60 (m, 2H),
7.53 (m, 1H), 7.29 (m, 1H), 4.49 (s, 2H). MS (ESI): 449 (M + H).

Annulated Pyrazoles as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7457
were extracted with EtOAc, washed with brine, dried over MgSO4,
filtered, and evaporated to dryness under reduced pressure. Column
chromatography on silica gel using an EtOAc/hexanes gradient
(0-40% EtOAc) provided the intermediate ketone as a brown oil
Test compounds were prepared as 2 mM solutions in dimethyl
sulfoxide (DMSO). Four replicate, serial 2-fold dilutions in GM10
were then prepared and 50 µL amounts placed in 96-well plates
over a final nanomolar concentration range of 625-1.22. Fifty
microliters GM10 and 3.5 × 104 infected cells were then added to
each well. Control cultures containing no cells (blank), uninfected
cells (100% viability, four replicates), and infected cells without
compound (total virus-mediated cell death, four replicates) were
also prepared. The cultures were then incubated at 37 °C in a
humidified atmosphere of 5% CO2 in air for 5 days.
A fresh solution of 5 mg/mL MTT was prepared in 0.01 M
phosphate buffered saline, pH 7.2, and 20 µL added to each culture.
The cultures were further incubated as before for 2 h. They were
then mixed by pipetting up and down 170 µL of Triton X-100 in
acidified isopropanol (10% v/v Triton X-100 in 1:250 mixture of
concentrated HCl in isopropanol). When the formazan deposit was
fully solubilized by further mixing, the absorbance (OD) of the
cultures was measured at 540 and 690 nm wavelength (690 nm
readings were used as blanks for artifacts between wells). The
percent protection for each treated culture was then calculated from
the equation
1
(0.081 g, 9% over two steps). H NMR (CDCl3): δ 9.17 (d, J )
4.75 Hz, 1H), 7.82 (d, J ) 4.75 Hz, 1H), 6.94-7.06 and 7.17-7.36
(m, 9H), 4.46 (s, 2H), 2.36 (s, 3H). A mixture containing this ketone
(0.081 g, 0.15 mmol), p-toluenesulfonic acid (0.057 g, 0.30 mmol),
and hydrazine monohydrate (17 µL, 0.30mmol) in propan-2-ol was
placed under an atmosphere of nitrogen and heated at 80 °C for
14 h. Once cooled, the reaction mixture was treated with 20%
aqueous NaCO3 solution. The organics were extracted with EtOAc,
washed with brine, dried over MgSO4, filtered, and evaporated in
vacuo. Chromatography over silica gel using an EtOAc/hexanes
gradient (70-100%) provided 10 as a light-yellow oil (0.019 g,
30%). 1H NMR (DMSO-d6): δ 14.3 (s, 1H, NH), 9.09 (d, J ) 5.6
Hz, 1H), 8.14 (d, J ) 5.6 Hz, 1H), 7.78 (d, J ) 2.14 Hz, 1H), 7.42
(m, 1H), 7.38 (dd, J ) 8.4 Hz, 2.0 Hz, 1H), 7.20s7.05 (m, 3H),
4.41 (s, 2H), 2.34 (s, 3H). MS (ESI): 420 (M + H). HRMS (ESI)
calcd for C20H15BrN5O (M + H): 420.0460. Found: 420.0462.
HIV-1 Reverse Transcriptase Assay. RNA-dependent DNA
polymerase activity was measured using a biotinylated primer
oligonucleotide and tritiated dNTP substrate. Newly synthesized
DNA was quantified by capturing the biotinylated primer molecules
on streptavidin coated scintillation proximity assay (SPA) beads
(Amersham). The sequences of the polymerase assay substrate were
as follows: 18nt DNA primer, 5′-biotin/GTC CCT GTT CGG GCG
CCA-3′; 47nt RNA template, 5′-GGG UCU CUC UGG UUA GAC
CAC UCU AGC AGU GGC GCC CGA ACA GGG AC-3′. The
biotinylated DNA primer was obtained from the Integrated DNA
Technologies Inc., and the RNA template was synthesized by
Dharmacon. The DNA polymerase assay (final volume 50 µL)
contained 32 nM biotinylated DNA primer, 64 nM RNA substrate,
dGTP, dCTP, dTTP (each at 5 µM), 103 nM [3H]dATP (specific
activity ) 29 µCi/mmol) in 45 mM Tris-HCl, pH 8.0, 45 mM NaCl,
2.7 mM Mg(CH3COO)2, 0.045% Triton X-100 w/v, 0.9 mM EDTA.
The mixtures contained 5 µL of serial compound dilutions in 100%
DMSO for IC50 determination, and the final concentrations of
DMSO were 10%. Reactions were initiated by the addition of 30
µL of the HIV room temperature enzyme (final concentrations of
1-3 nM). Protein concentrations were adjusted to provide linear
product formation for at least 30 min of incubation. After incubation
at 30 °C for 30 min, the reaction was quenched by addition of 50
µL of 200 mM EDTA (pH 8.0) and 2 mg/mL SA-PVT SPA beads
(Amersham, RPNQ0009, reconstituted in 20 mM Tris-HCl, pH 8.0,
100 mM EDTA, and 1% BSA). The beads were left to settle
overnight, and the SPA signals were counted in a 96-well top
counter-NXT (Packard). IC50 values were obtained by sigmoidal
regression analysis using GraphPad.
Antiviral Assay. Anti-HIV antiviral activity was assessed using
an adaptation of the method of Pauwels et al.29 The method is based
on the ability of compounds to protect HIV-infected T lympho-
blastoid cells (MT4 cells) from cell death mediated by the infection.
The end point of the assay was calculated as the concentration of
compound at which the cell viability of the culture was preserved
by 50% (“50% inhibitory concentration”, IC50). The cell viability
of a culture was determined by the uptake of soluble, yellow 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and
its reduction to a purple insoluble formazan salt. After solubilization,
spectrophotometric methods were employed to measure the amount
of formazan product.
MT4 cells were prepared to be in logarithmic-phase growth and
a total of 2 × 106 cells infected with the HXB2-strain of HIV at a
multiplicity of 0.0001 infectious units of virus per cell in a total
volume of between 200 and 500 µL. The cells were incubated with
virus for 1 h at 37 °C before removal of virus. The cells are then
washed in 0.01 M phosphate buffered saline, pH 7.2, before being
resuspended in culture medium for incubation in culture with serial
dilutions of test compound. The culture medium used was RPMI
1640 without phenol red, supplemented with penicillin, strepto-
mycin, L-glutamine, and 10% fetal calf serum (GM10).
% protection ) {[(OD drug treated cultures) -
(OD untreated virus control cultures)] ⁄
[(OD uninfected cultures) -
(OD untreated virus control cultures)]} × 100%
The IC50 can be obtained from graph plots of percent protection
versus log10 of the drug concentration.
Antiviral Assay Using Clinical Isolates. Viruses were selected
from a panel of clinical isolates available at Monogram Biosciences
based on their reduced susceptibility to efavirenz. Compounds were
evaluated in the PhenoScreen single cycle antiviral assay at
Monogram Biosciences as described previously.29b
Crystallization and Structure Determination. Amino acids
1-561 of HIV-1 reverse transcriptase were overexpressed in E.
coli. Supernatant from disrupted and centrifuged cell pellet was
passed over Q Sepharose, heparin HP, C3 HIC, SP Sepharose, and
Superdex 200 SEC columns, concentrated to ∼12 mg/mL, and
stored in 20 mM Tris, pH 7.8, 200 mM NaCl, 1 mM EDTA, 2
mM DTT at -80 °C. An aliquot of thus purified p66/p51
heterodimer reverse transcriptase was mixed with 0.4 mM 6 or 10.
Crystals were grown by sitting drop vapor diffusion over a reservoir
of 1.15 M sodium malonate, 50 mM potassium phosphate, pH 7.2,
5% ethylene glycol. Crystals were exchanged into a cryoprotectant
of 2 M sodium malonate, 50 mM potassium phosphate, pH 7.5,
5% glycerol, 0.5 M sodium ascorbate including 0.1 mM compound.
Crystals were frozen in liquid nitrogen and data collected at the
Advanced Light Source beamline 5.0.2 and Stanford Synchrotron
Radiation Laboratory beamline 9.2. Data were reduced with
HKL2000 and phased using coordinates from an internal structure
(i.e., rigid-body refinement in Refmac rather than a full molecular
replacement search). Model inspection, building, and refinement
were done using PyMOL,30 COOT,31 and Refmac.32,33 Statistics
and further details are included in the Supporting Information.
Acknowledgment. The authors thank a reviewer for cor-
rections and suggestions that substantially improved the quality
of this manuscript.
Supporting Information Available: Additional crystallographic
information. This material is available free of charge via the Internet
at http://pubs.acs.org.
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