Discovery of 3-{5-[(6-amino-1H-pyrazolo[3,4-b]pyridine-3-yl)methoxy]-2- chlorophenoxy}-5-chlorobenzonitrile (MK-4965): A potent, orally bioavailable HIV-1 non-nucleoside reverse transcriptase inhibitor with improved potency against key mutant viruses

Article abstract of DOI:10.1021/jm800856c

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been shown to be a key component of highly active antiretroviral therapy (HAART). The use of NNRTIs has become part of standard combination antiviral therapies producing clinical outcomes with

Full text of DOI:10.1021/jm800856c

J. Med. Chem. 2008, 51, 6503–6511
6503
Discovery of 3-{5-[(6-Amino-1H-pyrazolo[3,4-b]pyridine-3-yl)methoxy]-
2-chlorophenoxy}-5-chlorobenzonitrile (MK-4965): A Potent, Orally Bioavailable HIV-1
Non-Nucleoside Reverse Transcriptase Inhibitor with Improved Potency against Key Mutant
Viruses
Thomas J. Tucker,*,† John T. Sisko,† Robert M. Tynebor,† Theresa M. Williams,† Peter J. Felock,§ Jessica A. Flynn,‡
Ming-Tain Lai,§ Yuexia Liang,# Georgia McGaughey,† Meiquing Liu,§ Mike Miller,§ Gregory Moyer,‡ Vandna Munshi,§
Rebecca Perlow-Poehnelt,† Sridhar Prasad,† John C. Reid,† Rosa Sanchez,# Maricel Torrent,† Joseph P. Vacca,† Bang-Lin Wan,†
and Youwei Yan†
Departments of Medicinal Chemistry and Structural Biology, AntiViral Research, Vaccines and Biologics Research, and Drug Metabolism,
Merck Research Laboratories, WP14-3, 770 Sumneytown Pike, P.O. Box 4, West Point, PennsylVania 19486-0004
ReceiVed July 11, 2008
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been shown to be a key component of highly
active antiretroviral therapy (HAART). The use of NNRTIs has become part of standard combination antiviral
therapies producing clinical outcomes with efficacy comparable to other antiviral regimens. There is, however,
a critical issue with the emergence of clinical resistance, and a need has arisen for novel NNRTIs with a
broad spectrum of activity against key HIV-1 RT mutations. Using a combination of traditional medicinal
chemistry/SAR analyses, crystallography, and molecular modeling, we have designed and synthesized a
series of novel, highly potent NNRTIs that possess broad spectrum antiviral activity and good pharmacokinetic
profiles. Further refinement of key compounds in this series to optimize physical properties and
pharmacokinetics has resulted in the identification of 8e (MK-4965), which has high levels of potency against
wild-type and key mutant viruses, excellent oral bioavailability and overall pharmacokinetics, and a clean
ancillary profile.
Introduction
Non-nucleoside reverse transcriptase inhibitors (NNRTIsa) are
a key component of highly active antiretroviral therapy (HAART).
NNRTIs target an allosteric binding pocket on the reverse
transcriptase enzyme, and their binding is noncompetitive with
respect to dNTPs and template/primer.1 There are currently three
commercially available first generation NNRTIs: efavirenz,
nevirapine, and delavirdine (Figure 1).2 Despite the demonstrated
clinical efficacy of these compounds, the emergence of clinical
resistance has become a key issue and a major cause of treatment
failure. Recently, the second generation NNRTI etravirine
(TMC-125, Figure 1) has been approved by the FDA and has
demonstrated activity toward a number of clinically observed
mutations.3 However, the need remains for alternative second
generation NNRTIs that have a broad spectrum of activity versus
mutant viruses and a high genetic barrier to the selection of
new resistant strains. Since compliance is also a key issue in
Figure 1. Currently marketed NNRTIs.
anti-HIV therapy, the need also exists for compounds that are
safe, are easy to dose orally once a day, and have low potential
for drug-drug interactions. Our efforts have focused on the
design and synthesis of novel NNRTIs with potent and broad
spectrum mutant activity that are also safe and have pharma-
cokinetics suitable for once a day oral dosing.
* To whom correspondence should be addressed. Phone: 1-215-652-5275.
Fax: 1-215-652-3971. E-mail: tom_tucker@merck.com.
Figure 2
†
Department of Medicinal Chemistry and Structural Biology.
Department of Antiviral Research.
Department of Vaccines and Biologics Research.
Department of Drug Metabolism.
§
In a previous manuscript, we described our early efforts
toward the design and synthesis of novel second generation
NNRTIs and presented a potent and novel diaryl ether/indazole
template 1 (Figure 2).4 Compound 1 possesses potent antiviral
activity versus WT and a variety of clinically relevant mutant
viruses.4 Unfortunately, 1 has low solubility and low oral
‡
#
a Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhibitor;
HAART, highly active antiretroviral therapy; WT, wild type; CIC95, cell
inhibitory concentration that blocks 95% of viral replication; FBS, fetal
bovine serum; NHS, normal human serum; MOI, multiple of infection.
10.1021/jm800856c CCC: $40.75
2008 American Chemical Society
Published on Web 10/01/2008

6504 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Tucker et al.
good yield. The methyl group in the 4-position was homologated
to an ethyl group via directed metalation and treatment with
iodomethane to give 15. Removal of the Boc protecting group
with TFA and subsequent diazotization/cyclization of the amino
intermediate 16 yielded the 3-methylpyrazolopyridine derivative
17. Compound 17 was converted to the desired bromide 6c via
a two-step protection/bromination as described above.
The 6-amino-7-aza derivative 8e was synthesized using a
strategy similar to that detailed above (Scheme 3). Ortho directed
metalation of 2,6-difluoropyridine (19) and subsequent quench-
ing with the requisite Weinreb amide gave the ketone 20 in
modest yield. Numerous attempts to modify the conditions of
this metalation failed to provide increased yields. The intermedi-
ate ketone 20 was treated with hydrazine in the presence of
titanium isopropoxide to give the hydrazone intermediate, and
the hydrazone was refluxed in ethanol to effect efficient
cyclization to the 3-methyl-6-fluoropyrazolopyridine intermedi-
ate 21. In a manner similar to that described previously for the
des-fluoro analogues, 21 was Boc protected and brominated to
give the bromide intermediate 23. In a procedure similar to that
described above (Scheme 1), alkylation of the phenol 5 with
bromide 23 provided 24 in good yield. Displacement of the
fluorine of 24 with PMB amine also removed the N1-Boc
protecting group to provide the protected intermediate 25.
Heating 25 in neat TFA provided the desired target compound
8e in good yield.
Figure 3. X-ray crystal structure of 1 bound in WT NNRTI binding
site (from ref 4). View is looking inward along the enzyme-solvent
interface under P236 (magenta), showing the solvent exposed lower
surface of the indazole C ring.
bioavailability that precluded its further development. Further
efforts in this series have focused on preparing more soluble
versions of 1 that we hypothesized would have better overall
physical properties and improved pharmacokinetic profiles after
oral dosing. Crystallographic studies with 14 showed that the
lower edge of the indazole C ring lies under the P236 residue
along the enzyme/solvent interface (Figure 3), and as such, we
hypothesized that this region should be amenable to function-
alization with polar, solubilizing substituents. We proposed to
prepare a number of analogues of 1 in which the phenyl ring of
the indazole moiety was replaced with a pyridine ring, with the
goal of finding a close derivative that showed enhanced
solubility and oral bioavailability while retaining the desirable
overall biological profile seen with 1. In this manuscript, we
describe the design, synthesis, and biological profile of this series
of pyridine analogues that has led to the discovery of novel
NNRTIs suitable for clinical development.
Results and Discussion
Table 1 details the in vitro biological data for efavirenz, 1,
and 8a-e. Compounds were evaluated for intrinsic enzyme
inhibitory potency versus WT RT (wild type HIV-1 reverse
transcriptase), as well as the K103N and Y181C mutant RTs.8
Compounds were also evaluated for antiviral potency (CIC95)
against WT and the key mutant viruses in the presence of 10%
FBS (fetal bovine serum; WT and mutants) as well as 50% NHS
(normal human serum; WT) to evaluate the effects of protein
binding.9 In general, inhibitory activity and antiviral activity
improved as the nitrogen was moved around the aromatic ring
from the 4-position (8a) to the 7-position (8d) in accord with
our earlier hypothesis. The 7-aza analogue 8d exhibited highly
potent enzyme inhibitory activity versus WT and the key
mutants and also showed excellent antiviral potency against the
same panel. Pharmacokinetic studies with 8d (Table 2) showed
that the compound possessed promising pharmacokinetics after
both iv and po dosing in several species, and as such, 8d became
a compound of high interest in our program. Crystallographic
studies with 8d10 (Figure 4) demonstrate that the compound
binds to the K103N mutant-NNRTI binding pocket in a manner
analogous to that reported previously for 1 in the WT binding
site. All of the key interactions of the western A ring and central
B ring with the Y181C-Y188C-W229 lipophilic binding
pocket are maintained as seen previously with the structure of
1 bound to the WT enzyme. The Y181 side chain is rotated by
90° as seen in previous crystal structures that we have reported
in this series.4 The two nitrogens of the pyrazolo moiety
maintain two key hydrogen bonding interactions with the
backbone carbonyl oxygen and NH of the K103 residue, and it
is likely that this key interaction is critical for the high levels
of potency observed versus the K103N mutant. The pyrazol-
opyridine heterocycle makes a stacking interaction with Y318
and also appears to make lipophilic interactions with the side
chains of F227, P236, and V106. The bottom edge of this pyridyl
ring lies along the enzyme-solvent interface as anticipated, and
the nitrogen atom appears to be solvent exposed.
Chemistry
Target compounds were prepared by the generic method
shown in Scheme 1. SNAr reaction of phenol 2 and aryl fluoride
3 provided the protected diaryl ether intermediate 4 in good
yield. Removal of the methyl protecting group with boron
tribromide progressed in excellent yield to give the key phenol
intermediate 5. The target compounds were then prepared in a
two-step sequence via alkylation of the phenol 5 with the 1-Boc-
3-bromomethylpyrazolopyridines 6a-d, followed by removal
of the Boc protecting group with TFA to give the target
compounds 8a-d. The critical bromo intermediates 6a5 and 6d5
were prepared via literature methods; however, bromides 6b
and 6c were not known in the literature and were prepared by
modification of related literature methodologies6,7 as detailed
in Scheme 2. Ortho directed metalation of 4-chloropyridine 9
and reaction with the Weinreb amide provided the ketone
intermediate 10. Heating 10 with hydrazine in the presence ot
titanium isopropoxide effected ring closure to the 3-methylpyra-
zolopyridine 11. Compound 11 was protected selectively in the
1-position using standard conditions, and the protected inter-
mediate 12 was brominated with NBS to provide the desired
bromide 6b in moderate yield. Bromide 6c was synthesized in
a similar manner, starting from 3-amino-4-methylpyridine 13.
Protection of the amino group using sodium hexamethyldisila-
zane and Boc anhydride gave the protected intermediate 14 in

A Chlorobenzonitrile as NNRTI
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6505
Scheme 1a
a Reagents: (a) K2CO3, NMP, 120 °C (75%); (b) BBr3, CH2Cl2 (95%); (c) NaH or Cs2CO3, DMF; (d) TFA (10-80% for two steps).
Scheme 2. Synthesis of Bromides 6b and 6ca
a Reagents: (a) LDA, N-methoxy-N-methylacetamide, THF, -78 °C (34%); (b) Ti(iOPr)4, hydrazine hydrate, ethylene glycol, 120 °C (47%); (c) (Boc)2O,
DMAP, TEA, acetonitrile (67%); (d) NBS, benzoyl peroxide, CCl4 (20%); (e) Na(TMS)2, (Boc)2O, THF (70%); (f) t-BuLi, CH3I, THF (98%); (g) TFA
(69%); (h) NaNO2, 50% H2SO4 (51%); (i) (Boc)2O, DMAP, TEA, acetonitrile (59%); (j) NBS, benzoyl peroxide, CCl4, reflux (36%).
Further evaluation of 8d versus a wider panel of clinically
relevant mutant viruses11 (Table 3) showed that the compound
possessed broad activity versus most of the mutants tested. Only
the Y188L mutation provided notable decreases in potency; this
is presumably due to the direct interaction between the phenyl
ring of 8d and the aryl ring of the Y188 residue. When the
residue is mutated to a leucine, much of the interaction between
the phenyl ring and the side chain is lost because of the
decreased size of the side chain as well as the loss of the
π-stacking interaction. Y188L is a rare mutation, occurring in
only 0.6% of clinical isolates.12 The Y188L mutation requires
a two-base change in its codon, indicating that progression to
this mutation occurs in a stepwise manner via several intermedi-
ate mutations.12 While the Y188L virus itself exhibits good
fitness, the intermediate mutation Y188H is a much less fit virus,
and the Y188F intermediate mutation appears to be slow
growing.12 In-house cell culture selection experiments with later
members of this class have failed to select for the Y188L
mutation under a variety of conditions (details of the data for
the final development candidate will be presented later in this
manuscript). While these studies suggest that the Y188L
mutation may not be a serious issue for this series of compounds,
it is also possible that secondary mutations could confer added
fitness to one of the Y188L intermediates given the preexisting
pool of viral mutations in a treatment experienced population.
Therefore, only actual clinical experience with a compound of
this class will provide a suitable answer to this question.
On the basis of its excellent overall profile, 8d became a
development candidate and was designated as MK-1107.8 Full
ancillary evaluation of 8d showed the compound to be devoid
of significant off-target activities, and the compound was also
negative in preliminary genotoxicity determinations. Unfortu-
nately, as 8d was being studied in depth to support its further
development, difficulty was encountered producing a formula-
tion that could provide the high exposures necessary to support
po dosing in these safety studies. Further in vivo studies showed
that the compound was orally bioavailable only when dosed as
a solution, and this became an issue given the low solubility of
the compound especially as higher doses/exposures were needed.
While 8d did have improved solubility versus 1 (solubility of

6506 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Tucker et al.
Scheme 3a
a Reagents: (a) LDA, N-methoxy-N-methylacetamide, THF, -78 °C (25%); (b) Ti(iOPr)4, hydrazine hydrate, EtOH, reflux (95%); (c) (Boc)2O, DMAP,
TEA, acetonitrile (64%); (d) NBS, benzoyl peroxide, CCl4, reflux (64%); (e) Cs2CO3, 5, NMP (86%); (f) PMBNH2, NMP, 95 °C (31%); (g) TFA, 60 °C
(30%).
Table 1. Enzyme Inhibition and Antiviral Activity for Compounds 1 and 8a-e
inhibition of RT polymerase, IC50 (nM)a
WT K103N Y181C
1.0 18.9 7.2
antiviral activity in cell culture, CIC95 (nM)b
compd
W
X
Y
Z
WT (10% FBS) K103N (10% FBS) Y181C (10% FBS) WT (50% NHS)
efavirenz
4.6
245
87
42
1
CH CH CH
CH 1.4 ( 0.3
1.1 ( 0.3
2.6 ( 0.4
22.6 ( 1.2
33 ( 1.9
102 ( 1.8
114 ( 1.7
8a
8b
8c
8d
8e
N
CH CH
CH 3.6
40
740
307
2767
ND
922
CH
CH CH
N
CH
N
CH 1.2
CH 0.8
3.8
1.3
0.5 ( 0.2
0.5 ( 0.2
6.2
3.9
0.2 ( 0.1
0.4 ( 0.2
169
10.8
10.3 ( 1.1
8.4 ( 1.0
922
34.2
14.6 ( 1.1
11.4 ( 1.1
278
31
22 ( 1.2
19 ( 1.0
307
34
34 ( 1.3
34 ( 1.1
CH CH CH
CH CH C-NH2
N
N
0.4 ( 0.2
0.2 ( 0.1
a Compounds were evaluated in a standard SPA assay. For compounds 8a-c, n ) 2 and the values are the geometric mean of two determinations; all
individual values are within 25% of the mean. For compound 1 where n > 3 and compounds 8d and 8e where n > 50, standard deviations are included.
Assay protocols are detailed in ref 8. b CIC95 (cell culture inhibitory concentration) is defined as the concentration at which the spread of virus is inhibited
by >95% in MT-4 human T-lymphoid cells maintained in RPMI 1640 medium containing either 10% FBS or 50% NHS. Details of the assay protocols are
provided in ref 9. ND ) not determined. For compounds 8a-c, n ) 2 and the values are the geometric mean of two determinations; all individual values
are within 25% of the mean. For compound 1 where n > 3 and 8d and 8e where n > 50, standard deviations are included. No cytotoxicity was observed
for any of the compounds up to the upper limit of the assay (8.3 µM).
8d at pH 7 is 0.1 µg/mL, while the solubility of 1 is so low that
it cannot be determined), it appeared that further enhancements
in solubility would be necessary to provide a compound whose
exposure was not limited by solubility. As a result, development
of the compound was halted, and we were forced to reevaluate
our efforts in an attempt to further enhance the solubility and
physical properties of this series. Once again, we sought to make
minor changes to the structure of 8d in an effort to retain the
aforementioned excellent biological profile of the compound.
Using 8d as a starting point, we hypothesized that by adding
an amino group in the 6-position of the pyridine ring, we could
further enhance the basicity and possibly increase the solubility
(especially at acidic pH) of this series of compounds while also
maintaining the excellent overall profile of previous compounds
in this series. A pyridyl nitrogen was reasonably well tolerated
at the 6-position in a previous analogue in this series (Table 1,
8c), suggesting that the introduction of an amino group at this
position might also be well tolerated. Molecular modeling
studies13 suggested that this compound would be accommodated
by the active site in a similar manner to 1 and 8d, with the
6-amino group lying in the solvent exposed region and not
making any detrimental interactions with the enzyme. Com-
pound 8e was synthesized (Scheme 3) and was shown to possess
enzyme inhibitory activity and antiviral activity similar to that
observed with 8d (Table 1). 8e was shown to be several orders
of magnitude more soluble than 8d especially at acidic pH
(solubility of 8d is <0.1 µg/mL at pH 2; solubility of 8e is 10
µg/mL at pH 2), and these data encouraged further evaluation
of the compound. Pharmacokinetic evaluation of 8e (Table 2)
demonstrated that this compound possessed a similar profile to
8d in several species. More importantly, additional in vivo
studies with 8e showed that the compound was fully orally
bioavailable in a dose proportional manner when dosed as either
a suspension or a solution. Evaluation of 8e (Table 3) against
a wider panel of clinically relevant mutations confirmed that
the compound retained excellent antiviral activity versus most

A Chlorobenzonitrile as NNRTI
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6507
Table 2. Pharmacokinetic Parameters for 8d and 8e in Rat, Dog, and
Rhesus
Table 3. Antiviral Potency of 8d and 8e versus Various Clinically
Isolated Mutant Viruses (NL4-3 Isolate)a
8d
8e
IC50 (nM)
rata
dogb
rhesusc
ratd
doge
rhesusf
mutation
8d
1.5
1.8
6.8
1.1
2.5
4.1
8e
1.35
1.5
7.6
1.8
4.4
5.6
T1/2 (h)
4.1
28
9.5
5.3
58
11.8
8.9
1.6
2.6
17
8.2
19
0.3
4.4
3
3.5
9
22.7
2
3.9
5.8
31.3
1.7
2.1
15.8
0.3
1.9
1
WT
Cl ((mL/min)/kg)
AUCpo (µM h)
Vd (L/kg)
G190A
G190S
L100I
K103N
Y181C
Y188L
V106A
V179E
P236L
F227L
K101E/G190A
K103N/G190A
K103N/P225H
K103N/V179E
K103N/Y181C
Y181C/G190A
K103N/Y181C/G190A
F (%)
52
47
a Average of three Sprague-Dawley rats dosed at 10 mpk po (Imwitor/
Tween) and 2 mpk iv (DMSO). All values are within 25% of the mean.
b Average of two beagle dogs dosed at 2 mpk po (Imwitor/Tween) and
0.25 mpk iv (DMSO). c Average of two rhesus macaques dosed at 10 mpk
po (Imwitor/Tween) and 1 mpk iv (DMSO). d Average of three Sprague-
Dawley rats dosed at 10 mpk po (0.5% methocel) and 3 mpk iv (DMSO).
All values are within 25% of the mean. e Average of three beagle dogs
dosed at 10 mpk po (0.5% methocel) and 2 mpk iv (DMSO). All values
are within 25% of the mean. f Average of three rhesus macaques dosed at
10 mpk po (0.5% methocel) and 2 mpk iv (DMSO). All values are within
25% of the mean.
900
>1000
ND
ND
ND
ND
4.8
4.0
4.8
146.7
5
4.2
0.7
10.1
9.6
1.2
9.6
84
14.8
98.3
6.0
34.1
17.1
44.7
a Compounds were analyzed in a Monogram Bioscience Phenoscreen
assay in the presence of 40% NHS. The IC50 is defined as the concentration
of compound in cell culture required to block 50% of viral replication. For
all compounds, n ) 2 and the value is geometric mean. All individual values
are within (30% of the mean. Details are provided in ref 11.
Figure 4. X-ray crystal structure of 8d (gray) bound in the K103N
mutant NNRTI binding site (3.1 angstrom resolution). Key active site
residues are labeled in black. Details of the crystallographic studies
are provided in ref 10.
of the mutants that were evaluated. Once again, only the Y188L
mutant showed notable decreases in antiviral potency, as
observed with previous compounds in this series. Cell culture
selection experiments with this compound have shown that the
Y188L mutation does not appear to arise after treatment with
the inhibitor. Under low MOI (multiple of infection) conditions
((1-5) × CIC95), small amounts of Y181C, P236L, and V106A
are observed. Under higher MOI conditions at a 200 nM
concentration, the V106A mutation is selected after 36 days.
Under the highest MOI conditions (1000 nM), no mutations
are observed after multiple months of treatment. Crystallographic
studies14 with 8e were performed in the WT and Y181C mutant
enzymes. Figure 5 shows the crystal structure of 8e bound in
the WT active site. The WT crystal structure is essentially
identical to the previous structures we have reported in this
series, with all previously noted interactions with the active site
maintained. Figure 6 shows the crystal structure of 8e in the
Y181C mutant binding site. The binding mode in the Y181C
mutant is strikingly similar to that seen in the WT enzyme. The
binding of the compound is completely uninfluenced by the
mutation of the 181 residue, as the inhibitor does not make any
direct interaction with this residue. In total, all crystal structures
that we have reported for this series demonstrate remarkably
similar binding modes for the inhibitors in the WT and mutant
active sites, with only very subtle differences in the central aryl
ether conformation and slight movements of the compounds’
relative positions in the site noted. It has been proposed in the
Figure 5. X-ray crystal structure of 8e (gray) bound in the WT NNRTI
binding site (2.6 angstrom resolution). Key active site residues are
labeled in black. Details of the crystallographic studies are provided
in ref 14.
literature that the ability of NNRTIs to be somewhat confor-
mationally flexible is of critical importance for their broader
spectrum antiviral activity.15 Our results are consistent with this
hypothesis; however, the differences in binding modes between
the wild-type and mutant enzymes are more subtle. It is likely
that the ability of compounds such as 8e to make direct
interactions with the backbone of residue 103 and the ability to
avoid direct interaction with Y181 are critical factors in
determining the mutant profile of the compounds.
Further preclinical evaluation of 8e showed the compound
to be free of major ancillary activities, including no effects on
QTc, heart rate, or blood pressure in a conscious cardiovascular
dog model up to 55 µM plasma levels.16 8e demonstrates
balanced metabolism in human microsomes via two pathways:
oxidation by CYP3A4 on the methylene linker between the
central ring and the pyrazolopyridine ring and glucuronidation
at N1 of the pyrazolopyridine.16 In accord with its improved
solubility, the compound did not exhibit any difficulties with
formulation and had sufficient exposures in safety assessment
studies. On the basis of available comparative in-house data,
we believe that the overall pharmacokinetic profile of 8e

6508 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Tucker et al.
(17.00 mmol) of 1 M boron tribromide (Aldrich) added as a steady
stream. The resulting solution was stirred in the cold for 30 min
and allowed to warm to room temperature overnight. The reaction
was quenched by adding excess water, and the resulting mixture
was extracted with 2 × 50 mL portions of ether. The combined
ethereal extracts were washed with 25 mL of brine and dried over
anhydrous MgSO4. The mixture was filtered and the filtrate
concentrated in vacuo to give 1.55 g (98%) of the desired product
as an off-white solid. The material was determined to be of adequate
purity (HPLC purity of 96% at 215 nm) to be used without further
1
purification. H NMR δ (ppm) (CDCl3): 4.94 (1H, s), 6.62 (1H,
m), 6.73 (1H, m), 7.05 (1H, m), 7.16 (1H, m), 7.35 (1H, s), 7.37
(1H, d). MS (ES+) m/z: [M + H] ) 280.
tert-Butyl3-{[4-Chloro-3-(3-chloro-5-cyanophenoxy)phenoxy]me-
thyl}-1H-pyrazolo[4,3-b]pyridine-1-carboxylate (7a). To a solution
of 83 mg (0.30 mmol) of 5 in 1 mL of anhydrous DMF was added
22 mg (0.33 mmol) of 60% NaH dispersion in oil (Aldrich). The
mixture was stirred for 30 min before 93 mg (0.30 mmol) of 6a
was added as a solution in 2 mL of anhydrous DMF. The resulting
mixture was stirred for 1 h and was diluted with 10 mL of water
and 10 mL of EtOAc. The mixture was extracted with 2 × 20 mL
of EtOAc, and the combined organic layers were washed with 4 ×
10 mL of water and 2 × 10 mL of brine, dried (anhydrous MgSO4),
and concentrated in vacuo to give 160 mg of crude product as a
brown oil. The crude product was determined to be 80-90% pure
by LC/MS analysis and was used in the next reaction without further
purification or characterization.
tert-Butyl3-{[4-Chloro-3-(3-chloro-5-cyanophenoxy)phenoxy]me-
thyl}-1H-pyrazolo[4,3-c]pyridine-1-carboxylate (7b). A mixture of
40 mg (0.14 mmol) of 5, 45 mg (0.14 mmol) of 6b, and 141 mg
(0.43 mmol) of Cs2CO3 in 3 mL of anhydrous DMF was stirred at
ambient temperature overnight or until complete by LC/MS
analysis. The mixture was concentrated in vacuo to give 15 mg of
crude 7a as a brown oil. The crude material (80-90% purity by
LC/MS analysis) was used as recovered in the next step without
further purification.
Figure 6. X-ray crystal structure of 8e (copper) bound in the Y181C
mutant NNRTI binding site (2.9 angstrom resolution). Key active site
residues are labeled in black. Details of the crystallographic studies
are provided in ref 14.
supports development of the compound as a once a day oral
agent. As such, the compound was moved forward as a clinical
development candidate, labeled as MK-4965.8 8e was shown
to have a low potential for drug-drug interactions in preclinical
assessments and was clean in genotoxicity assessments, and no
major issues were observed in safety assessment studies.16 On
the basis of the results of these studies, 8e was advanced into
phase I clinical trials. Further details of the early clinical studies
with 8e, as well as the pharmacokinetics, formulation, process
development, and more extensive SAR studies in this series,
will be presented in subsequent publications from our laboratories.
Experimental Section
Silica gel flash chromatography was performed on an Isco
Combi-Flash or a Biotage unit using prepacked Isco or Biotage
silica cartridges. Reversed phase chromatographic separations were
performed on a Gilson unit using Phenomenex Gemini C18 columns.
1H NMR spectra were routinely obtained on a 400 MHz Varian
FT NMR spectrometer and are referenced versus TMS. NMR
solvents used were CDCl3, CD3OD, or DMSO-d6 as indicated.
Chemical shifts are reported in δ units relative to TMS. HPLC was
performed on a Waters Alliance 2695/Micromass ZQ LC/MS
system, using a YMC Pro C18 5 µm/120 Å, 3 mm × 50 mm column
and a 95:5 to 5:95 A/B gradient (A ) 92% water/8% acetonitrile/
0.05% TFA; B ) 100% acetonitrile with 0.0425% TFA) over 4 or
6 min at a flow rate of 2 mL/min, with UV (215 or 254 nm) and
mass spec detection. Aldrich Sure-Seal solvents were used when
anhydrous solvents were necessary.
tert-Butyl3-{[4-Chloro-3-(3-chloro-5-cyanophenoxy)phenoxy]me-
thyl}-1H-pyrazolo[3,4-c]pyridine-1-carboxylate (7c). In a manner
identical to that described above for the preparation of 7b, from
41 mg (0.15 mmol) of 5, 46 mg (0.15 mmol) of 6c, and 144 mg
(0.43 mmol) of Cs2CO3 was obtained 90 mg of crude 7c as a brown
oil. The crude material (80-90% purity by LC/MS analysis) was
used as recovered in the next step without further purification.
tert-Butyl3-{[4-Chloro-3-(3-chloro-5-cyanophenoxy)phenoxy]me-
thyl}-1H-pyrazolo[3,4-b]pyridine-1-carboxylate (7d). In a manner
identical to that described above for the preparation of 7a, from 80
mg (0.29 mmol) of 5, 91 mg (0.29 mmol) of 6d, and 12 mg (0.30
mmol) of 60% NaH dispersion was obtained 150 mg of crude 7d
as a brown oil. The crude material (80-90% purity by LC/MS
analysis) was used as recovered in the next step without further
purification.
3-Chloro-5-[2-chloro-5-(1H-pyrazolo[4,3-b]pyridine-3-yl-meth-
oxy)phenoxy]benzonitrile (8a). A solution of 150 mg of crude 7a
was dissolved in 4 mL of TFA and stirred for 30 min. The crude
mixture was concentrated in vacuo and was purified using silica
gel chromatography (10-60% EtOAc/hexane) to afford 82 mg
(67% for two steps) of the title compound as an amorphous white
solid. 1H NMR δ (ppm) (CDCl3): 5.58 (2 H, s), 6.93 (1 H, d, J )
3 Hz), 6.97-7.01 (2 H, m), 7.16 (1 H, t, J ) 2 Hz), 7.31-7.37 (2
H, m), 7.42 (1 H, dd, J ) 9, 5 Hz), 7.95 (1 H, dd, J ) 9, 1 Hz),
8.66-8.68 (1 H, m). HRMS (ES+) m/z: [M + H] theoretical )
411.0410, obsd ) 411.0391. LC purity, >99%; tR ) 2.39 min at
215 nm.
3-Chloro-5-(2-chloro-5-methoxyphenoxy)benzonitrile (4). A mix-
ture of 1.00 g (6.31 mmol) of phenol 2, 1.28 g (8.20 mmol) of
3-fluoro-5-chlorobenzonitrile 3, and 2.62 g (18.92 mmol) of
anhydrous potassium carbonate in 4 mL of anhydrous 1-methyl-
2-pyrrolidinone (NMP) was stirred at 120 °C in a nitrogen
atmosphere for 4 h, then stirred at room temperature overnight.
The mixture was filtered, and the solids were washed with 25 mL
of ethyl acetate. The filtrate was washed with 10 mL of 1 N HCl,
10 mL of 1 N NaOH, and 10 mL of brine. The organic layer was
dried over anhydrous MgSO4 and was filtered, and the filtrate was
concentrated to an orange oil. The crude oil was purified by flash
chromatography over silica gel with 1:1 hexanes/chloroform to give
1
1.75 g (94%) of the desired product as a white solid. H NMR δ
(ppm) (CDCl3): 3.81 (3H, s), 6.44 (1H, m), 6.62 (1H, m), 6.78
(1H, m), 7.14 (1H, m), 7.22 (1H, m), 7.37 (1H, m). MS (ES+)
m/z: [M + H] ) 295.
3-Chloro-5-(2-chloro-5-hydroxyphenoxy)benzonitrile (5). A so-
lution of 1.65 g (5.61 mmol) of 4 was dissolved in 15 mL of
methylene chloride, and the resulting solution cooled to -15 °C
under a nitrogen atmosphere. The solution was treated with 17 mL
3-Chloro-5-[2-chloro-5-(1H-pyrazolo[4,3-c]pyridine-3-yl-meth-
oxy)phenoxy]benzonitrile (8b). In a manner identical to that
described above for the preparation of 8a, from 15 mg of crude 7b
was obtained 6 mg (10% for two steps) of the desired product as
a white amorphous solid. H NMR δ (ppm) (CD3OD): 5.56 (2 H,
s), 6.99 (1 H, d, J ) 2.87 Hz), 7.06-7.12 (2 H, m), 7.15 (1 H, t,
J ) 2 Hz), 7.45-7.52 (2 H, m), 7.56 (1 H, d, J ) 6. Hz), 8.33 (1
1

A Chlorobenzonitrile as NNRTI
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6509
H, d, J ) 6 Hz), 9.15 (1 H, s). LC purity, >99%; tR ) 2.40 min at
215 nm. HRMS (ES+) m/z: [M + H] theoretical ) 411.0410, obsd
) 411.0426.
dried (anhydrous MgSO4), filtered, and concentrated in vacuo to
provide the crude product. The crude product was purified by silica
gel chromatography (40-100% EtOAc/hexane gradient) to afford
1
0.71 g (67%) of the title compound as an oily off-white solid. H
3-Chloro-5-[2-chloro-5-(1H-pyrazolo[3,4-c]pyridine-3-ylmethoxy)-
phenoxy]benzonitrile (8c). In a manner identical to that described
above for the preparation of 8a, from 90 mg of crude 7c was
obtained 48 mg (78% for two steps) of the desired product as a
white amorphous solid. 1H NMR δ (ppm) (CD3OD): 5.55 (2 H, s),
6.99 (1 H, d, J ) 2.90 Hz), 7.06 (1 H, dd, J ) 8.91, 3.01 Hz), 7.11
(1 H, dd, J ) 6.15, 2.32 Hz), 7.15 (1 H, t, J ) 2.16 Hz), 7.47 (1
H, d, J ) 8.89 Hz), 7.52 (1 H, t, J ) 1.72 Hz), 7.88 (1 H, dd, J )
5.72, 1.40 Hz), 8.23 (1 H, d, J ) 5.71 Hz), 9.00 (1 H, d, J ) 1.40
Hz). HRMS (ES+) m/z: [M + H] theoretical ) 411.0410, observed
) 411.0422. LC purity, >99%; tR ) 2.48 min at 254 nm.
3-Chloro-5-[2-chloro-5-(1H-pyrazolo[3,4-b]pyridine-3-ylmethoxy)-
phenoxy]benzonitrile (8d). In a manner identical to that described
above for the preparation of 8a, from 150 mg of crude 7d was
obtained crude 8d as a brown oil. The oil was purified by reversed
phase preparative HPLC (Gilson) to give 100 mg (68% for 2 steps)
of the TFA salt of the desired product as a fluffy white amorphous
NMR δ (ppm) (CDCl3): 1.75 (9H, s), 2.68 (3H, 2), 7.95 (1H, d, J
) 6 Hz), 8.62 (1H, d, J ) 6 Hz), 9.02 (1H, s). MS (ES+) m/z: [M
+ H - Boc] ) 134. LC purity, >99%; tR ) 1.05 min at 254 nm.
tert-Butyl 3-(Bromomethyl)-1H-pyrazolo[4,3-c]pyridine-1-car-
boxylate (6b). A stirred solution of 500 mg (2.14 mmol) of 12 in
20 mL of carbon tetrachloride was treated with 51 mg (0.21 mmol)
of benzoyl peroxide and 400 mg (2.25 mmol) of N-bromosuccin-
imide. The resulting mixture was heated to reflux for 2 h and was
cooled to room temperature and concentrated in vacuo. The crude
mixture was purified by silica gel chromatography (0-50% ethyl
acetate/hexane gradient) to provide 65 mg (20%) of the desired
product as an off-white solid. LC purity, >99%; tR ) 2.01 min at
1
254 nM. H NMR δ (ppm) (CDCl3): 1.75 (9H, s), 4.80 (3H, s),
7.98 (1H, d, J ) 6 Hz), 8.65 (1H, d, J ) 6 Hz 1H), 9.22 (1H, s).
MS (ES+) m/z: [M + H - Boc] ) 212.3. LC purity, >99%; tR )
2.01 min at 254 nm.
1
powder after lyophilization. H NMR δ (ppm) (DMSO-d6): 5.42
tert-Butyl (4-Methylpyridin-3-yl)carbamate (14). A solution of
5.00 g (108.15 mmol) of 13 in 100 mL of anhydrous THF was
treated dropwise with 92.40 mL (92.40 mmol) of 1.0 M NaHMDS
in THF (Aldrich). A solution of 9.58 g (43.90 mmol) of di-tert-
butyl dicarbonate in 30 mL of anhydrous THF was added via a
cannula, and the mixture was stirred for 2.5 h at room temperature.
The reaction was quenched with excess water and was extracted
with 3 × 40 mL of EtOAc. The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concentrated
in vacuo to provide the crude product as an oil. The mixture was
purified by silica gel chromatography (20-60% EtOAc/hexane
gradient) to provide 5.95 g (70%) of the desired product as a brown
(2H, s), 7.08 (1H, dd, J ) 2 Hz, 9 Hz), 7.12 (1H, d, J ) 3 Hz),
7.21 (1H, q), 7.37 (1H, m), 7.46 (1H, m), 7.55 (1H, d, J ) 11 Hz),
7.80 (1H,m), 8.30 (1H, dd, J ) 1 Hz, 8 Hz), 8.54 (1H, dd, J ) 1
Hz, 5 Hz). LC purity, >99%; tR ) 2.54 min at 215 nm. HRMS
(ES+) m/z: [M + H] theoretical ) 411.0410, observed ) 411.0416.
1-(4-Chloropyridin-3-yl)ethanone (10). A solution of 4.80 mL
(33.80 mmol) of diisopropylamine in 20 mL of anhydrous THF
cooled to - 78 °C in a nitrogen atmosphere was treated with 21
mL (33.6 mmol) of 1.6 M n-BuLi in hexanes (Aldrich). The
resulting mixture was warmed to 0 °C for 30 min, recooled to
-78C, and was treated dropwise with a solution of 3.20 g (28.30
mmol) of 9 in 10 mL of THF. The mixture was stirred for 2 h and
was treated with 3.00 mL (28.30 mmol) of N-methoxy-N-methy-
lacetamide. The resulting mixture was warmed to room temperature
overnight, was poured into water (100 mL), and was extracted with
3 × 50 mL of ethyl acetate. The combined organic layers were
washed with brine, dried (MgSO4), filtered, and concentrated in
vacuo to provide the crude product. The mixture was purified by
silica gel chromatography (0-100% EtOAc/hexane gradient) to
afford 1.5 g (34%) of the title compound as an oil. 1H NMR δ
(ppm) (CDCl3): 2.64 (3H, s), 7.38 (1H, d), 8.78 (1H, d), 8.80 (1H,
s). MS (ES+) m/z: [M + H] ) 156.3. LC purity, >99%; tR ) 1.04
min at 254 nm.
1
oil/solid. H NMR δ (ppm) (CDCl3): 1.52 (9H, s), 2.25 (3H, s),
6.18 (1H, bs), 7.18 (1H, d, J ) 5 Hz), 8.24 (1H, s, J ) 5 Hz, 1H),
8.85 (1H, s). MS (ES+) m/z: [M + H - Boc] ) 153.4. LC purity,
>99%; tR ) 1.09 min at 215 nm (6 min gradient).
tert-Butyl (4-Ethylpyridin-3-yl)carbamate (15). A solution of
4.00 g (19.20 mmol) of 14 in 80 mL of anhydrous THF was cooled
to -78 °C under a nitrogen atmosphere and was treated dropwise
with 28.20 mL (48.00 mmol) of 1.7 M t-BuLi in pentane (Aldrich).
The resulting mixture was stirred at -78 °C for 1 h, was warmed
to -20 °C for 45 min, and was recooled to -30 °C. The mixture
was treated with a solution 4.08 g (28.80 mmol) of iodomethane
in 20 mL of THF (20 mL) added via a cannula. The resulting
mixture was maintained between -20 and -30 °C for 45 min. The
reaction was quenched with brine, warmed to room temperature,
and was extracted with 3 × 40 mL of EtOAc. The combined organic
layers were washed with brine, dried (anhydrous MgSO4), filtered,
and concentrated in vacuo to provide the crude product as an oil.
The crude mixture was purified by silica gel chromatography
(10-100% EtOAc/hexanes gradient) to provide 4.15 g (98%) of
the desired product as an oil. 1H NMR δ (ppm) (CDCl3): 1.30 (3H,
t), 1.54 (9H, s), 2.60 (2H, q), 6.10 (1H, bs), 7.12 (1H, d, J ) 8
Hz), 8.32 (1H, d, J ) 8 Hz), 8.88 (1H, s). MS (ES+) m/z: [M +
H-Boc] ) 167.4. LC purity, >99%; tR ) 1.33 min at 215 nm (6
min gradient).
3-Methyl-1H-pyrazolo[4,3-c]pyridine (11). A solution of 1.50 g
(9.60 mmol) of 10 in 15 mL of CH2Cl2 was treated with 5.45 g
(19.20 mmol) of titanium isopropoxide at room temperature. The
resulting mixture was stirred for 30 min and cooled to 0 °C, and
0.96 g (19.20 mmol) of hydrazine hydrate was added. The ice bath
was removed and the mixture stirred for 2 h before water (5 mL)
was added. The resulting mixture was stirred vigorously for 30 min.
The thick precipitate was filtered off and washed with CH2Cl2 (100
mL), and the filtrate was concentrated in vacuo. The crude residue
was dissolved in 15 mL of ethylene glycol, and the solution was
heated at 120 °C for 18 h. The reaction mixture was diluted with
an excess of water and was extracted with 100 mL of ethyl acetate.
The ethyl acetate extract was washed with 50 mL of brine, dried
(anhydrous MgSO4), filtered, and concentrated in vacuo to give
0.60 g (47%) of the crude product as an oil. The material was used
as obtained from the reaction without further purification. 1H NMR
δ (ppm) (CDCl3): 2.65 (s, 3H), 7.35 (d, J ) 5.8 Hz, 1H), 8.43 (d,
J ) 5.8 Hz, 1H), 9.08 (s, 1H), 10.10 (bs, 1H). MS (ES+) m/z: [M
+ H] ) 134.0. LC purity, >99%; tR ) 0.37 min at 254 nm.
tert-Butyl 3-Methyl-1H-pyrazolo[4,3-c]pyridine-1-carboxylate
(12). A suspension of 0.60 g (4.50 mmol) of 11 in 20 mL of
acetonitrile was treated with 0.55 g (4.53 mmol) of N,N-dimethy-
laminopyridine, 0.66 mL (4.75 mmol) of triethylamine, and 1.18 g
(5.43 mmol) of di-tert-butyl dicarbonate. The resulting mixture was
stirred at room temperature for 1 h and was diluted with an equal
volume of brine and extracted with 3 × 25 mL of ethyl acetate.
The combined organic extract was washed with 25 mL of brine,
4-Ethylpyridin-3-amine (16). A solution of 4.15 g (18.82 mmol)
of 15 in 20 mL of TFA was stirred at room temperature for 30
min. The reaction mixture was concentrated in vacuo and the crude
oil purified by silica gel chromatography (0-40% MeOH/CH2Cl2
gradient) to give 3.03 g (69%) of the TFA salt of the desired
1
product. H NMR δ (ppm) (CD3OD): 1.32 (3H, t), 2.70 (2H, q),
7.60 (1H, d, J ) 8 Hz), 7.92 (1H, d, J ) 8 Hz), 7.96 (1H, s). MS
(ES+) m/z: [M + H ] ) 123.3. LC purity, >99%; tR ) 0.42 min
at 215 nm.
3-Methyl-1H-pyrazolo[3,4-c]pyridine (17). A solution of 550 mg
(4.50 mmol) of 16 in H2O/H2SO4 (1:1 volume) cooled to 0 °C was
treated with a solution of 341 mg (4.96 mmol) of NaNO2 in 1.4
mL of water. The resulting mixture was stirred at 0 °C for 0.5 h,
and the solution was cannulated into 20 mL of 20% aqueous sodium
acetate solution. The resulting mixture was stirred for 1.25 h at 0

6510 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Tucker et al.
°C and was extracted with 2 × 25 mL of EtOAc. The combined
organic layers were washed with brine, dried (MgSO4), filtered,
and concentrated in vacuo to give the crude product. The crude
product was purified by silica gel chromatography (0-100%
MeOH/CH2Cl2 gradient) to give 304 mg (51%) of the desired
product as a tan oil/solid. 1H NMR δ (ppm) (CDCl3): 2.62 (3H, s),
7.60 (1H, d, J ) 5 Hz), 8.32 (1H, d, J ) 5 Hz), 8.98 (1H, s). MS
(ES+) m/z: [M + H ] ) 134.3. LC purity, >93%; tR ) 0.38 min
at 254 nm.
The mixture was stirred at room temperature for 30 min, was
quenched with brine, and was extracted with 2 × 150 mL of EtOAc.
The combined organic extracts were washed with 2 × 100 mL of
water and 2 × 100 mL of brine, dried (anhydrous MgSO4), and
filtered, and the filtrate was concentrated in vacuo. The crude oil
was purified by silica gel chromatography (25% EtOAc/hexane)
to provide the 25 g (64%) of the desired compound as a clear oil/
1
solid. H NMR δ (ppm) (CDCl3): 1.74 (9H, s), 2.60 (3H, s), 6.95
(1H, d, J ) 8 Hz), 8.08 (1H, t, J ) 8 Hz). HRMS (ES+) m/z: [M
+ H] theoretical ) 252.1143, obsd ) 252.1141. LC purity, >99%;
tR ) 1.96 min at 254 nm.
tert-Butyl 3-Methyl-1H-pyrazolo[3,4-c]pyridine-1-carboxylate
(18). In a manner identical to that described above for the
preparation of 12, from 95 mg (0.71 mmol) of 17, 104 µL (0.75
mmol) of TEA, 87 mg (0.71 mmol) of DMAP, and 185 mg (0.85
mmol) of di-tert-butyl dicarbonate (185 mg, 0.85 mmol) was
tert-Butyl 3-(Bromomethyl)-6-fluoro-1H-pyrazolo[3,4-b]pyridine-
1-carboxylate (23). A solution of 25.00 g (99.00 mmol) of 22 in
500 mL of carbon tetrachloride was treated with 19.48 g (109.00
mmol) of NBS and 2.41 g (9.95 mmol) of benzoyl peroxide. The
resulting mixture was heated at reflux for 6 h. The mixture was
cooled to room temperature and filtered, and the filtrate was
concentrated in vacuo to yield the crude product as an oil. The
mixture was purified by silica gel chromatography (10-25%
EtOAc/hexane gradient) to give 21.00 g (64%) of the desired
product as an off-white/pale-yellow solid that was stored in the
freezer. 1H NMR δ (ppm) (CDCl3): 1.75 (9H, s), 4.75 (2H, s), 7.00
(1H, d, J ) 8 Hz), 8.30 (1H, t, J ) 8 Hz). HRMS (ES+) m/z: [M
+ Na] theoretical ) 352.0067, obsd ) 352.0079. LC purity, >99%;
tR ) 1.94 min at 254 nm.
1
obtained 97 mg (59%) of the desired product. H NMR δ (ppm)
(CD3OD): 1.75 (9H, s), 2.64 (3H, s), 7.85 (1H, d, J ) 5 Hz), 8.48
(1H, d, J ) 5 Hz), 9.40 (1H, s). MS (ES+) m/z: [M + H - Boc]
) 134.3. LC purity, >99%; tR ) 1.30 min at 215 nm.
tert-Butyl 3-(Bromomethyl)-1H-pyrazolo[3,4-c]pyridine-1-car-
boxylate (6c). In a manner identical to that described above for the
preparation of compound 12, from 97 mg (0.42 mmol) of 18, 7
mg (0.44 mmol) of NBS, and 10 mg (0.04 mmol) of benzoyl
peroxide was obtained 47 mg (36%) of the desired product as an
off-white solid. 1H NMR δ (ppm) (CDCl3): 1.77 (9H, s), 4.80 (2H,
2), 7.75 (1H, dd, J ) 1, 5 Hz), 8.55 (1 h, d, J ) 5 Hz), 9.50 (1H,
s). MS (ES+) m/z: [M + H - Boc] ) 212.2. LC purity, >99%; tR
) 1.77 min at 254 nm.
1-(2,6-Difluoropyridin-3-yl)ethanone (20). A solution of 32.70
g (323.00 mmol) of diisopropylamine in 550 mL of anhydrous THF
was cooled to -78 °C under a nitrogen atmosphere and was treated
with 200.00 mL (318.00 mmol) of 1.6 M n-BuLi in hexanes
(Aldrich). The resulting solution naturally warmed to -20 °C and
was maintained at -20 °C for 1.5 h. The solution was recooled to
-78 °C, and a solution of 31.00 g (269.00 mmol) of 19 in 100 mL
of anhydrous THF was added dropwise. The reaction was continued
with stirring for 2 h at -78 °C and was treated with a solution of
28.60 mL (269.00 mmol) of N-methoxy-N-methylacetamide in 20
mL of THF added via a cannula. The resulting solution was warmed
to ambient temperature overnight and was quenched with excess
brine solution. The mixture was extracted with 3 × 100 mL of
ethyl acetate, and the combined organic layers were washed with
100 mL of water and 2 × 100 mL of brine, dried (anhydrous
MgSO4), filtered, and concentrated in vacuo. The crude oil was
purified via silica gel chromatography (0-40% EtOAc/hexane
gradient) to afford 10.70 g (25%) of the desired product a clear,
pale-yellow oil. 1H NMR δ (ppm) (CDCl3): 2.68 (3H, s), 6.95 (1H,
d, J ) 6 Hz), 8.50 (1H, m). MS (ES+)m/z: [M + H] ) 158.0. LC
purity, >99%; tR ) 1.40 min at 254 nm.
6-Fluoro-3-methyl-1H-pyrazolo[3,4-b]pyridine (21). A solution
of 29.00 g (185.00 mmol) of 20 in 450 mL of CH2Cl2 was treated
with 108.00 mL (369.00 mmol) of titanium(IV) isopropoxide at
room temperature. The resulting mixture was stirred for 15 min,
and 17.96 mL (369.00 mmol) of hydrazine hydrate was added.
Stirring continued for an additional 1.5 h. Water (40 mL) was added,
and the resulting thick mixture was stirred vigorously for 20 min.
The reaction mixture was filtered, and the solids were washed with
100 mL of CH2Cl2. The filtrate was concentrated in vacuo to provide
the crude hydrazone intermediate as an oil. The crude hydrazone
was dissolved in 150 mL of ethanol, and the solution was heated
at 80 °C for 24 h. The mixture was cooled and concentrated in
vacuo to provide 26.50 g (95%) of crude product as an oil. The
crude product was used in the next reaction without further
purification. 1H NMR δ (ppm) (CDCl3): 2.56 (3H, s), 6.78 (1H, d,
J ) 8 Hz), 8.08 (1H, t, J ) 8 Hz). MS (ES+) m/z: [M + H] )
152.0. LC purity, 85%; tR ) 1.19 min at 254 nm.
3-Chloro-5-{2-chloro-5-[(6-fluoro-1H-pyrazolo[3,4-b]pyridin-3-
yl)methoxy]phenoxy}benzonitrile (24). A solution of 2.97 g (10.68
mmol) of 5 in 15 mL of 1-methyl-2-pyrrolidinone was treated with
6.58 g (20.30 mmol) of Cs2CO3. The mixture was stirred for 20
min, and a solution of 3.34 g (10.15 mmol) of 23 in 20 mL of
1-methyl-2-pyrrolidinone was added. The resulting mixture was
stirred for 1.5 h and was poured into 2.5 L of ice-water. The
mixture was extracted with 3 × 150 mL EtOAc, and the combined
organic layers were washed with 4 × 100 mL of water and 2 ×
100 mL of brine. The organic layer was dried (MgSO4) and filtered,
and the filtrate was concentrated in vacuo to give the crude product
as a brown oil. The crude product was purified by silica gel
chromatography (10-60% EtOAc/hexanes gradient) to afford
1
4.60 g (86%) of the desired product as an oil/solid. H NMR δ
(ppm) (CDCl3): 1.73 (9H, s), 5.42 (2H, s), 6.80 (1H, m), 6.93-6.98
(2H, m), 7.01 (1H, m), 7.13 (1H, m), 7.36 (1H, m), 7.42 (1H, d, J
) 8 Hz), 8.28 (1H, t, J ) 8 Hz). HRMS (ES+) m/z: [M + H]
theoretical ) 546.1106, obsd ) 546.1128. LC purity, >99%; tR )
3.73 min at 254 nm.
3-Chloro-5-[2-chloro-5-({6-[(4-methoxybenzyl)amino]-1H-pyra-
zolo[3,4-b]pyridin-3-yl}methoxy)phenoxy]benzonitrile (25). A solu-
tion of 200 mg (0.30 mmol) of 24 in 2.50 mL of 1-methyl-2-
pyrrolidinone was treated with 410 mg (3.00 mmol) of
p-methoxybenzylamine, and the resulting solution was heated at
95 °C for 18 h. The mixture was cooled to room temperature and
was diluted with 5 mL of 1/1 brine/H2O solution. The resulting
mixture was extracted with 3 × 25 mL of ethyl acetate, and the
combined extracts were washed with 4 × 25 mL of water and 2 ×
25 mL of brine. The extract was dried (anhydrous MgSO4) and
filtered, and the filtrate was concentrated in vacuo to give the crude
product as a brown oil. The crude material was purified by silica
gel chromatography (20-100% EtOAc/hexane gradient) to provide
1
51 mg (31%) of the desired product as an oil. H NMR δ (ppm)
(CDCl3): 3.80 (3H, s), 4.58 (2H, s), 4.98 (1H, m), 5.30 (2H, s),
6.30 (1H, d,, J ) 8.4 Hz), 6.82 (1H, m), 6.85 (2H, d, J ) 8.4 Hz),
6.92 (1H, d, J ) 8.4 Hz), 7.00 (1H, s), 7.10 (1H, s), 7.25-7.38
(4H, m), 7.78 (1H, d, J ) 8.4 Hz). MS (ES+)m/z: [M + H] )
546.3. LC purity, >99%; tR ) 2.51 min at 254 nm.
3-{5-[(6-Amino-1H-pyrazolo[3,4-b]pyridin-3-yl)methoxy]-2-chlo-
rophenoxy}-5-chlorobenzonitrile (8e). A solution of 51 mg (0.09
mmol) of 25 in 5 mL of TFA was heated at 60 °C for 7 h. The
reaction mixture was concentrated in vacuo and purified using
reverse phase chromatography (Gilson) to afford 15 mg (30%) of
the TFA salt of the desired compound as a fluffy white amorphous
tert-Butyl 6-Fluoro-3-methyl-1H-pyrazolo[3,4-b]pyridine-1-car-
boxylate (22). A solution of 23.65 g (156.00 mmol) of 21 in 400
mL of acetonitrile was treated with 19.12 g (156.00 mmol) of
DMAP and 22.90 mL (164.00 mmol) of TEA. The resulting solution
was stirred for 5 min before a solution of 43.60 mL (188.00 mmol)
of di-tert-butyl dicarbonate in 100 mL of acetonitrile was added.
1
solid after lyophilization. H NMR δ (ppm) (CD3OD): 5.40 (2H,
s), 6.58 (1H, d, J ) 9 Hz), 6.93 (1H, d, J ) 3 Hz), 7.04 (1H, dd,

A Chlorobenzonitrile as NNRTI
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6511
(11) The compound was evaluated in a Monogram Bioscience PhenoSense
assay versus a panel of clinically derived RT mutants in the presence
of 40% normal human serum. Values are the average of two de-
terminations. Assays were performed by Monogram Bioscience, South
San Francisco, CA. Details of the assay protocols are available at http://
www.monogramhiv.com/assays/hcp/phenoHIVTechnology.aspx.
(12) Dykes, C.; Demeter, L. M. Y188H, Which Is a Potential Intermediate
between Wild-Type (WT) and Y188L, Has Markedly Reduced
Replication Efficiency. Abstracts of Papers, 14th Conference on
Retroviruses and Opportunistic Infections, Los Angeles, CA, Feb 25-
28, 2007; Poster 633.
J ) 3, 9 Hz), 7.11 (1H, m), 7.15 (1H, m), 7.46 (1H, d, J ) 9 Hz),
7.52 (1H,m), 8.10 (1H, d, J ) 9 Hz). HRMS (ES+) m/z: [M + H]
theoretical ) 426.0519, obsd ) 426.0519. LC purity, >99%; tR )
1.93 min at 254 nm.
Acknowledgment. The authors thank Dr. Charles Ross and
Joan Murphy for high resolution mass spectral data. We also
thank Dr. Sandor Varga for NMR support.
(13) Compound 8e was placed in the NNRTI binding pocket based on
prior knowledge of related compounds in NNRTI crystal structures.
Approximately 30 conformations of each compound was generated
using two algorithms: a distance geometry (ref (a)) and a knowledge
based (ref (b)) method. All conformations were then subjected to
energy minimization using the distance dependent dielectric
constant of 2 and applying the MMFFs force field (ref (c)). All
figures were created in PyMol (ref (d)). Modeling discussion
references: (a) Crippen, C. M.; Havel, T. F. In Distance Geometry
and Molecular Conformation; Bawden, E., Ed.; Research Studies
Press: Wiley, NY, 1988. (b) Kuszewski, J.; Nilges, M.; Brunger, A. T.
Sampling and efficiency of metric matrix distance geometry: a novel
partial metrization algorithm. J. Biomol. NMR 1992, 2, 33–56. (c)
Feuston, B. P.; Miller, M. D.; Culberson, J. C.; Nachbar, R. B.;
Kearsley, S. L. Comparison of knowledge-based and distance geometry
approaches for generation of molecular conformations. J. Chem. Inf.
Comput. Sci. 2001, 41, 754–763. (d) Halgren, T. A. Merck molecular
force field. I. Basis, form, scope, parameterization, and performance
of MMFF94. J. Comput. Chem. 1996, 17, 490, 520, 553, and 616. (e)
Halgren, T. A.; Nachbar, R. B. Merck molecular force field. IV.
Conformational energies and geometries for MMFF94. J. Comput.
Chem. 1996, 17, 587–615. (f) PyMol, version 99, distributed by
DeLano Scientific LLC, was utilized to generate all images.
(14) The X-ray diffraction data of the wild type RT and inhibitor 8e
complex crystal (Figure 5) was collected at 2.6 Å resolution with
Rsym of 0.057 and 100% completeness. The structure was refined
to an R-factor of 0.187. The structure has been deposited in the
PDB; PDB code is 3DRP. The X-ray diffraction data of the Y181C
RT and inhibitor 8e complex crystal (Figure 6) was collected at
2.9 Å resolution with Rsym of 0.067 and 100% completeness. The
structure was refined to an R-factor of 0.183. The structure has
been deposited in the PDB; PDB code is 3DRR.
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(10) The X-ray diffraction data of the K103N RT and inhibitor 8d complex
crystal (Figure 4) were collected at 3.15 Å resolution with Rsym of
0.085 and 100% completeness. The structure was refined to an R-factor
of 0.184. The structure has been deposited in the PDB; PDB code is
3DRS.
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(16) Unpublished data, Merck Research Laboratories.
JM800856C