Biaryl ethers as novel non-nucleoside reverse transcriptase inhibitors with improved potency against key mutant viruses

Article abstract of DOI:10.1021/jm901230r

Biaryl ethers were recently reported as potent NNRTIs. Herein we disclose a detailed SAR study that led to the biaryl ether 6. This compound possessed excellent potency against WT RT and key clinically observed RT mutants and had an excellent pharmacokinetic profile in rats, dogs, and rhesus macaques. The compound also exhibited a clean safety profile in preclinical safety studies.

Full text of DOI:10.1021/jm901230r

J. Med. Chem. 2009, 52, 7163–7169 7163
DOI: 10.1021/jm901230r
Biaryl Ethers as Novel Non-nucleoside Reverse Transcriptase Inhibitors with Improved Potency
against Key Mutant Viruses
Dai-Shi Su,*,† John J. Lim,† Elizabeth Tinney,† Bang-Lin Wan,† Mary Beth Young,† Kenneth D. Anderson,† Deanne Rudd,†
Vandna Munshi,‡ Carolyn Bahnck,‡ Peter J. Felock,‡ Meiquing Lu,‡ Ming-Tain Lai,‡ Sinoeun Touch,§ Gregory Moyer,§
Daniel J DiStefano,§ Jessica A. Flynn,§ Yuexia Liang, Rosa Sanchez, Rebecca Perlow-Poehnelt,† Mike Miller,‡ Joe P. Vacca,†
Theresa M. Williams,† and Neville J. Anthony†
†Department of Medicinal Chemistry and Structural Biology, ‡Antiviral Research, §Vaccines and Biologics Research and Drug Metabolism,
Merck Research Laboratory, P.O. Box 4, West Point, Pennsylvania 19486
Received August 18, 2009
Biaryl ethers were recently reported as potent NNRTIs. Herein we disclose a detailed SAR study that led
to the biaryl ether 6. This compound possessed excellent potency against WT RT and key clinically
observed RT mutants and had an excellent pharmacokinetic profile in rats, dogs, and rhesus macaques.
The compound also exhibited a clean safety profile in preclinical safety studies.
Introduction
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)a
are key components of the most current HIV therapy, highly
active antiretroviral therapy (HAART), a combination of
nucleoside and non-nucleoside reverse transcriptase inhibitors
(NRTIs, NNRTIs) and protease inhibitors (PIs). HAART can
be very effective at delaying disease progression. However, due
to the propensity of HIV to rapidly mutate, the efficacy and
durability of HAART can be compromised. The most fre-
Figure 1. Lead compounds 1 and 2.
quent HIV RT mutations observed in patients failing therapy
with first generation NNRTIs are K103N and Y181C. There-
fore, new agents with better activity profiles against mutant
HIV-1 RT are needed. Recently, biaryl ethers were reported to
be potent NNRTIs (Figure 1).1-3 In this manuscript, we report
in a two-step sequence via displacement of the fluorine atom
the detailed structural-activity relationship (SAR) study of
this series of compounds that led to a compound which
other compounds in Table 1 were synthesized in an analogous
exhibited excellent potency against wild type (WT) reverse
previously reported 1-Boc-3-bromomethyl-6-fluoro-pyrazolo-
pyridine2 delivered 1d. The final compound (6) was prepared
with 4-methoxybenzylamine and simultaneous removal of the
4-methoxybenzyl and Boc protecting groups with TFA. The
fashion.
The isoxazolopyridines 12 to 20 in Table 2 were synthesized
according tothe routedepicted inScheme2. Deprotonationof
transcriptase (RT) and key clinically observed RT mutants
and had a favorable pharmacokinetic profile.
2,6-difluoropyridine by the action of LDA followed by {[t-
butyl(dimethyl)silyl]oxy}acetaldehyde addition afforded the
Chemistry
Compounds described in this paper were prepared accord-
ing to the route depicted in Scheme 1. Treatment of the
commercially available 1-fluoro-3,5-dimethyoxybenzene with
BBr3 followed by chlorination of the resulting intermediate
with NCS provided a mixture of two regioisomers1a and 1b in
a ratio of about 2:1. The SNAr reaction of 1a with 3-chloro-
5-fluorobenzonitrile and demethylation by BBr3 yielded the
key intermediate 1c. Alkylation of phenol (1c) with the
desired alcohol 1e. Swern oxidation and treatment of the
resulting ketone with hydroxyl amine and Ti(iOPr)4 formed
the oxime derivative 1f. Thermal cyclization yielded a mixture
of desired cyclized product (1g), the desilylated cyclized
product (1h), and the desilylated starting material. Conver-
sion of the resulting alcohol (1h) to mesylate provided the
compound 1i. Coupling 3-chloro-5-(2-chloro-5-hydroxyphe-
noxy)benzonitrile2 with mesylate 1i under basic conditions
afforded the mesylate 1m in 71% yield, presumably via an in
situ displacement of fluorine by the newly generated anionic
mesylate group. The compounds (13-20) in Table 2 were then
prepared by the displacement of mesylate with a variety of
amines.
*To whom correspondence should be addressed. Phone: 610-917-
5879. Fax: 610-917-6151. E-mail: Dai-Shi.X.Su@gsk.com. Present ad-
dress: GlaxoSmithKline, 1250 South Collegeville Road, UP1205,
Collegeville, PA 19426.
a Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhi-
bitors; HAART, highly active antiretroviral therapy; PI, protease
inhibitors; SAR, structural-activity relationship; WT, wild type, RT,
reverse transcriptase; FBS, fetal bovine serum; NHS, normal human
serum; PK, pharmacokinetic; ECG, electrocardiogram.
The synthesis of the intermediates for the preparation of
compound 21 is shown in Scheme 3. Treatment of fluoride 1g
with sodium cyanide afforded the cyano intermediate 1n.
r
2009 American Chemical Society
Published on Web 11/02/2009
pubs.acs.org/jmc

7164 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Scheme 1. Synthesis of Compound 6a
Su et al.
a (a) BBr3, CH2Cl2, -15 to 23 °C, 67%; (b) NCS, DCE, 84 °C, ∼2:1 ratio, 36% for 1a; (c) 3-chloro-5-fluorobenzonitrile, Cs2CO3, NMP, 120 °C, 88%;
(d) BBr3, CH2Cl2, 0-23 °C, 35%; (e) 1-Boc-3-bromomethyl-6-fluoro-pyrazolopyridine, Cs2CO3, NMP, 23 °C, 85%; (f) (1) PMB-amine, NMP, 95 °C, (2)
TFA, 65 °C, 4% for two steps.
Table 1. B-Ring SAR Results
RT-Pola
Spread (nM)b
K103Nc
compd
R1
R2
R3
X
WT/K103N/Y181C(nM)
10% FBS/50%NHS (WT)
Y181Cc
K103N/Y181Cc
1
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
Cl
Cl
H
F
H
0.1/0.2/0.2
0.2/0.4/0.4
2.7/4.6/3.2
0.4/1.9/0.9
0.5/0.9/0.6
0.1/0.4/0.3
5.2/0.8/13
120/>910/270
02/3.3/1.3
47/350/120
17/140/100
10/52
5/26
15
11
22
27
31
31
31
14
31
53
101
277
278
93
2
H
Cl
CH3
F
NH2
H
3
18/103
11/103
34
103
4
H
5
H
16/59
8/69
11/103
11
18
6
F
NH2
H
62
93
7
F
11
8
H
F
NH2
NH2
H
2778/>8333
<3.8/103
3767/>8300
>8300/>8300
8333
309
9
H
F
10
11
H
H
H
>8300
>8300
H
Cl
H
a Ki, compounds were evaluated in a standard ECL assay. The values are the geometric mean of at least two determinations. All individual values are
within 25% of the mean. 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% fetal bovine serum (FBS) or 50% normal human serum
(NHS). The antiviral activity of compound against wild-type ABI R8 virus was measured. The values are the geometric mean of at least two
determinations. All individual values are within 25% of the mean. c In the presence of 10%FBS.
The cyano group in 1n was then converted to imidazoline by a
two-step sequence: formation of the ethyl imidate and cycliza-
tion with ethylene diamine. Treatment of the resulting alcohol
with thionyl chloride afforded the intermediate 1p. Com-
pound 21 was prepared in a manner analogous to the route
described in Scheme 2.
species (Figure 1). The SAR study results suggested that the
chlorine atom at the R1 position of B-ring is critical for the
potency and mutation profile of compound (Table 1).1 There-
fore, multiple substitutions on the B-ring with chloride anchor-
ing the R1 position were investigated. Several potent analogues
were produced. At the R2 position, a chlorine atom, a methyl
group, and a fluorine atom were well tolerated (3, 4, and5). The
chlorine and fluorine combination was the most promising (5).
Compound 5maintained high antiviral activity against WT RT
and its key mutants in both enzyme (RT_Pol)4 and cell-based
Biological Results and Discussion
The previously reported compounds 1 and 2 showed excel-
lent potencies and had good oral bioavailability in preclinical

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7165
Table 2. C-Ring SAR Results
a Ki, compounds were evaluated in a standard ECL assay. The values are the geometric mean of at least two determinations. All individual values are
within 25% of the mean. b CIC95 (cell culture inhibitory concentration) is defined as the concentration at which the spread of the virus is inhibited by
>95% in MT-4 human T-lymphoid cells maintained in RPMI 1640 medium containing either 10% fetal bovine serum (FBS) or 50% normal human
serum (NHS). The antiviral activity of compound against wild-type ABI R8 virus was measured. The values are the geometric mean of at least two
determinations. All individual values are within 25% of the mean. c In the presence of 10%FBS.
Scheme 2. Synthesis of Compound 13-20a
a Reagents and conditions: (a) LDA, then aldehyde, 77%; (b) (COCl)2, DMSO, TEA, DCM, 94%; (c) NH2OH, Ti(iPrO)4, 66%; (d) ethylene glycol,
150 °C, 5 h, 17% (1h), 42-60% (1g); (e) MsCl, TEA, 73%; (f) tBuOK, 1i, THF, 78%; (g) amine, Cs2CO3, DMF, 60 °C.
Scheme 3. Synthesis of the Intermediates for Compound 21a
a Reagents and conditions: (a) NaCN, DMSO, 88%; (b) (i) HCl (g), EtOH, (ii) ethylene diamine, EtOH, 88%, (iii) SOCl2.
assays (Spread assay).5 Introduction of an amino group on the
pyrazolo pyridine C-ring was reported to affect both the
physiochemical properties and the potency of compounds 1
and 2.2 Incorporating the amino group in 5 led to compound 6.
Gratifyingly, compound 6 exhibited excellent intrinsic enzyme
inhibitory potency versus WT and mutant RTs and also had

7166 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Su et al.
excellent antiviral activity in cell-based assays against the
same panel of mutants. Relocation of the chlorine atom
from R1 to R3 position (7) was also well tolerated. We
were encouraged that 7 had equivalent potency compared
with 5 in our assays. However, introduction of the amino
group in the C-ring unexpectedly caused a significant
loss of activity (8). Monohalogenation at the R2 and R3
positions of the B-ring led to a loss of potency in the Spread
assay (9-11).
Co-crystallization of compound 2 in the allosteric WT RT
binding site suggested that the NH group and the imido
nitrogen atom in the pyrazole motif make key interactions
with the carbonyl oxygen and NH of the backbone K103
residue, respectively.2 To our surprise, the isoxazolopyridine
analogue 12, in which the NH of pyrazolopyridine was
replaced with an oxygen atom, exhibited nanomolar activities
in RT_Pol assay against WT and K103N and Y181C mutants
and potency decreased only 2-fold in the WT Spread assay
compared with that of 1 (Table 2). The amino analogues
(13 and 14) showed similar intrinsic potency to that of 2 and 6,
respectively, and were about 2-fold less potent in the cell-
based assay. Although the substituted amino analogues (15
and 16) showed nanomolar antiviral activities in the enzyme
assay, they generally were less potent than the correspond-
ing pyrazolopyridines in the Spread assay. The 6-amino
group in the C-ring of pyrazolopyridine (2) is accommo-
dated within the solvent channel lying under Pro-236.2 We
speculated that introduction of a polar and basic group at
the 6-position of the isoxazolopyridine C-ring would de-
crease the lipophilicity of compounds and hence reduce the
fold-shift in the Spread assay. Therefore a structurally
diverse array of basic heterocycles were installed at the
6-position of the C-ring. Nearly all the compounds pre-
pared (17-21) exhibited potency less than 5 nM against the
WT and the key mutant RTs in the enzyme assay. However,
these compounds proved less potent in the cell-based assay
compared to the unsubstituted amino-compounds (13 and
14). A higher shift between sera was also observed for the
most of analogues, indicating the influence of protein
binding. Of the heterocycles, 20, containing imidazole at
the 6-position, had the best overall profile. While com-
pound 20 displayed subnanomolar potency against the WT
RT and the key mutants in the enzyme assay, it was
significantly less potent in the cell-based assay.
Figure 2. Antiviral potency vs clinically isolated RT mutants of
compounds 1, 2, and 6.
Table 3. Pharmacokinetics of Compounds 2 and 6a
2
6
ratb
dogc
rhesusd
ratb
dogc
rhesusd
CL
Vd
9
2
5.8
1.7
31
16
1.9
0.3
2.1
1
10
4
2.3
6
15
3.5
18
3.6
1.7
4.2
13
AUCpo
t1/2
23
3.5
52
3.9
47
4.6
49
7
63
F
a CL: mL/min/kg; Vd: L/kg; AUCpo: μM h; t1/2: hours; F: % oral
3
bioavailability. b Sprague-Dawley rats (n = 3); po: 10 mg/kg, iv: 2 mg/
kg. c Beagle dogs (n = 3) for dog singles; po: 1 mg/kg, iv: 0.5 mg/kg.
d Rhesus macaques (n = 3); po: 5 mg/kg; iv: 1 mg/kg. Interanimal
variability was less than 20%.
volume distribution, and half-life in three preclinical species
(rat, dog, and monkey), showing a similar profile to that of
compound 2 but with higher Vd, leading to longer t1/2 across
species. (Table 3).
Compound 6 is not a potent competitive inhibitor of the
major cytochrome P450 isoforms (CYP3A4 IC50: 4.4 μM;
CYP2C9 10.3 μM; CYP2D6 47.4 μM) nor did it show time-
dependent inhibition of CYP3A4 in human liver microsomes.
The binding affinity of 6 in a human PXR-dependent reporter
gene assay showed an inductive response value of 51% of that
of the positive control (10 μM rifampcin) at a concentration of
3.3 μM.
Further preclinical evaluation of compound 6 showed that it
is devoid of ancillary activities, including effects on mean arterial
pressure, heart rate, or electrocardiogram (ECG) in a conscious
cardiovascular dog study with peak plasma exposure of 25.9 μM
at 10 mg/kg (cumulative dosing).8 Compound 6 was negative in
preliminary genotoxicity determinations.9 There are no signifi-
cant findings for 6in a seven-day rat study in which high levels of
exposure were observed (21.78 μM h and 180.3 μM h over a
Compounds 3 and 6 showed promising pharmacokinetic
profiles in rats following iv administration (3: Cl, 5.5 mL/min/
kg; Vd, 1.8 L/kg; AUCiv, 3.5 μM h; t1/2, 9.4 h; 6 (vide infra):
3
Cl, 10.1 mL/min/kg; Vd: 3.5 L/kg; AUCiv, 7.4 μM h, t1/2
,
3
4.6 h). However, other compounds in this series generally
exhibited high clearance in rats following iv administration.6
In a related in vitro study in hepatocytes, an analogue
structurally similar to 6 underwent predominantly O-deal-
kylationwithloss of the heterocyclicC-ring toform a phenolic
metabolite (M1) along with glucuronidation of parent mole-
cule and M1. The major metabolic pathways in microsomes
were O-dealkylation to form M1 and subsequent further
oxidation of M1.
3
3
period of 24 h at 10 and 100 mg/kg, respectively).10
Conclusion
In summary, compound 6 was identified in an effort to
modify the previously reported compound 2. Compound 6
possessed excellent antiviral activity against WT RT and key
clinically observed RT mutants with a favorable pharmaco-
kinetic profiles. The compound also exhibited a clean safety
profile in preclinical safety studies. While compound 6 had an
excellent overall profile for a second generation NNRTI,
overall, it was very similar to 2. Further study is required to
determine if compound 6 has developmental potential.
On the basis of its overall profile (potency against WT and
mutants and PK), compound 6 was selected for further
evaluation. Examination of compound 6 against a broader
panel of clinically relevant mutant HIV-1viruses showed that
the compound possessed excellent antiviral activity against
these mutants (Figure 2).7 In terms of pharmacokinetics,
compound 6 hadexcellent bioavailability, low clearance, good

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7167
Experimental Procedures
(1-20% EtOAc/hexanes) of the crude material gave 3-chloro-
5-(2-chloro-3-fluoro-5-hydroxyphenoxy)benzonitrile (1c) (3.2 g,
35%). 1H NMR (400 MHz, CDCl3, ppm): δ 6.40 (dd, 1H), 6.63
(dd, 1H), 7.09 (dd, 1H), 7.19 (t, 1H), 7.38 (t, 1H). LCMS (ES) m/z
298.2 (M)þ, 300.2 (M þ 1)þ.
General Procedures. 1H and 13C (300, 400, or 600 MHz) NMR
spectra were recorded on a Varian VXR 300, Unity Inova 400, or
Unity Inova 600 spectrometer. The chemical shifts are reported in
δ (ppm) using the δ 0.00 signal of Me4Si as an internal standard.
LC/MS data were obtained on a Waters 2690 separations module
and Micromass ZMD. High resolution MS (HRMS) data were
obtained on a Bruker 3T or 7T FTICR MS with either electro-
spray ionization or APCI. HPLC spectra were recorded on a
Hewlett-Packard 1100 with a YMC-Pack Pro C-18 column or
Atlantis dC18 column with a 5-95% CH3CN/H2O (with 0.05%
TFA) gradient at 215 nm. The purity of compound is g95%.
2-Chloro-3-fluoro-5-methoxyphenol (1a). Under nitrogen at-
mosphere, 1-fluoro-3,5-dimethoxybenzene (25 g, 160 mmol) was
diluted in CH2Cl2 (200 mL, 0.8M) and then cooled to -15 °C.
BBr3 (176 mL, 176 mmol, 1 M in CH2Cl2) was slowly added to the
reaction mixture. The reaction mixture was stirred at -15 °C for
one and a half hours at room temperature for 10 min. The reaction
mixture was then cooled to 0 °C and slowly quenched with water
(150 mL). The aqueous layer was then extracted with methylene
chloride (3 ꢀ 100 mL). The organic extracts were dried over
sodium sulfate, filtered, and concentrated in vacuo. Silica gel
chromatography (1%-30% EtOAc/hexanes) of the crude residue
gave 3-fluoro-5-methoxyphenol (15 g, 67%). 1H NMR (400 MHz,
CDCl3, ppm): δ 3.76 (s, 3H), 4.93 (s, 1H), 6.19 (m, 3H).
Under a nitrogen atmosphere, 3-fluoro-5-methoxyphenol
(15 g, 106 mmol) was diluted in DCE (150 mL, 0.7 M). To this
solution NCS (15.5 g, 116 mmol) was added, and the reaction
mixture was heated to reflux for 4 h. The reaction was then
cooled to room temperature and quenched with water (100 mL).
The aqueous layer was extracted with methylene chloride (3 ꢀ
50 mL). The organic extracts were dried over sodium sulfate,
filtered, and concentrated in vacuo. Silica gel chromatography
(1-30% EtOAc/hexanes) of the crude material gave 2-chloro-3-
fluoro-5-methoxyphenol (1a) (6.7 g, 36%), 1H NMR (400 MHz,
CDCl3, ppm): δ 3.76 (s, 3H), 5.59 (s, 1H), 6.33 (d, 1H), 6.36 (d,
1H), 6.40 (dd, 1H). ESMS, M þ Hþ found 177.2, and 4-chloro-
3-fluoro-5-methoxyphenol (1b) (2.8 g, 15%). 1H NMR (400
MHz, CDCl3, ppm): δ 3.87 (s, 3H), 4.93 (s, 1H), 6.28 (dd, 1H),
6.29 (d, 1H), 6.31 (d, 1H). ESMS, M þ Hþ found 177.2. LCMS
(ES) m/z 177.2 (M)þ, 179.2 (M þ 1)þ. The starting material,
3-fluoro-5-methoxyphenol was also recovered (4.3 g).
3-Chloro-5-(2-chloro-3-fluoro-5-hydroxyphenoxy)benzonitrile
(1c). Under a nitrogen atmosphere, 2-chloro-3-fluoro-5-meth-
oxyphenol (1a) (6.7 g, 37.9 mmol) was diluted in NMP (40 mL,
0.95 M). To this solution Cs2CO3 (24.73 g, 76 mmol) was added,
and the reaction was allowed to stir at room temperature for
5 min. Then 3-chloro-5-fluorobenzonitrile (11.81 g, 76 mmol)
was added to the reaction and it was then heated to 120 °C. After
2 h, the reaction was cooled to room temperature and then
diluted with EtOAc (40 mL). It was partitioned with water
(20 mL) and then extracted with EtOAc (3 ꢀ 30 mL). The organic
extracts were then washed with water (3 ꢀ 20 mL) and brine (1 ꢀ
20 mL), dried over sodium sulfate, filtered, and concentrated in
vacuo. Silica gel chromatography (1-15% EtOAc/hexanes) of
the crude residue gave 3-chloro-5-(2-chloro-3-fluoro-5-methoxy-
phenoxy)benzonitrile(10.4g, 88%). 1H NMR (400 MHz, CDCl3,
ppm): δ 3.80 (s, 3H), 6.47 (t, 1H), 6.69 (dd, 1H), 7.07 (t, 1H), 7.19
(t, 1H), 7.36 (s, 1H).
Under nitrogen atmosphere, 3-chloro-5-(2-chloro-3-fluoro-
5-methoxyphenoxy)benzonitrile (9.6 g, 30.8 mmol) was diluted
in CH2Cl2 (60 mL, 0.5M) and then cooled to 0 °C. To this
solution BBr3 (61.5 mL, 61.5 mmol, 1 M in CH2Cl2) was slowly
added to the reaction. The reaction mixture was allowed to
slowly warm to room temperature and stir for 12 h. It was then
cooled to 0 °C and slowly quenched with water (60 mL). The
aqueous layer was then extracted with methylene chloride (3 ꢀ
30 mL). The organic extracts were dried over sodium sulfate,
filtered, and concentrated in vacuo. Silica gel chromatography
3-{5-[(6-Amino-1H-pyrazolo[3,4-b]pyridin-3-yl)methoxy]-2-
chloro-3-fluorophenoxy}-5-chlorobenzonitrile (6). Under a nitro-
gen atmosphere, 3-chloro-5-(2-chloro-3-fluoro-5-hydroxyphe-
noxy)benzonitrile (1c) (150 mg, 0.503 mmol) was dissolved in
NMP (4 mL, 0.1 M). Cs2CO3 (164 mg, 0.503 mmol) was added,
and the reaction was allowed to stir at room temperature
for 15 min. Then, 1-Boc-3-bromomethyl-6-fluoro-pyrazolopyri-
dine (166 mg, 0.503 mmol, prepared by the method described in
ref 2) was added to the reaction. The reaction was stirred at room
temperature for 2 h. It was diluted with EtOAc (4 mL), parti-
tioned with water (3 mL), and then extracted with EtOAc (3 ꢀ
4 mL). The organic extracts were then washed with water (3 ꢀ
4 mL) and brine (1 ꢀ 4 mL), dried over sodium sulfate, filtered, and
concentrated in vacuo. Silica gel chromatography (1-20% EtOAc/
hexanes) of the crude gave intermediate 1d (230 mg, 85%). 1HNMR
(400 MHz, CDCl3, ppm): δ 1.73 (s, 9H), 5.39 (s, 2H), 6.59 (m, 1H),
6.84(dd, 1H), 6.98(dd, 1H),7.06(m,1H),7.14(t,1H),7.38(m, 1H),
8.24 (t, 1H). ESMS, M þ Hþ found 446.9 (M-100, loss of Boc).
Under a nitrogen atmosphere, intermediate 1d (230 mg,
0.420 mmol) was dissolved in NMP (4 mL, 0.1 M). 4-Methoxy-
benzylamine (288 mg, 2.101 mmol) was added and the reaction
mixture was heated to 95 °C for 2 h. It was then diluted
with EtOAc (4 mL), partitioned with water (3 mL), and then
extracted with EtOAc (3 ꢀ 4 mL). The organic extracts were
then washed with water (3 ꢀ 4 mL) and brine (1 ꢀ 4 mL), dried
over sodium sulfate, filtered, and concentrated in vacuo. The
crude reaction material was then diluted with TFA (3 mL) and
heated to 65 °C for 2 h. The reaction mixture was concentrated
and purified directly on a reverse phase HPLC system (5-95%
AcCN/H2O with 0.05% TFA) to give 6 (8 mg, 4%). 1H
NMR (400 MHz, CDCl3, ppm): δ 4.72 (b, 2H), 5.30 (s, 2H),
6.41 (d, 1H), 6.61 (s, 1H), 6.81 (dd, 1H), 7.04 (s, 1H), 7.17 (m,
1H), 7.38 (s, 1H), 7.81 (d, 1H), 9.90 (b, 1H). ESMS, M þ Hþ
found 444.0.
3-Chloro-5-[2,3-dichloro-5-(1H-pyrazolo[3,4-b]pyridin-3-ylmeth-
oxy)phenoxy] Benzonitrile (3). 1H NMR (400 MHz, CDCl3, ppm):
δ5.43 (s, 2H), 6.76 (d, 1H), 7.04 (s, 1H), 7.15 (m, 2H), 7.22 (dd, 1H),
7.38 (s, 1H), 8.19 (d, 1H), 8.61 (s, 1H), 10.6 (b, 1H). High resolution
MS: m/z found 445.0014 (M þ 1), calculated 445.0021 (M þ 1).
3-Chloro-5-[2-chloro-3-methyl-5-(1H-pyrazolo[3,4-b]pyridin-
3-ylmethoxy)phenoxy] Benzonitrile (4). 1H NMR (400 MHz,
CDCl3, ppm): δ 2.39 (s, 3H), 5.43 (s, 2H), 6.69 (d, 1H), 6.89 (d,
1H), 6.99 (m, 1H), 7.13 (t, 1H), 7.22 (dd, 1H), 7.32 (t, 1H), 8.21
(d, 1H), 8.60 (s, 1H). High resolution MS: m/z found 425.0565
(M þ 1), calculated 425.0567 (M þ 1).
3-Chloro-5-[2-chloro-3-fluoro-5-(1H-pyrazolo[3,4-b]pyridin-
3-ylmethoxy) henoxy] Benzonitrile (5). 1H NMR (400 MHz,
CDCl3, ppm): δ 5.49 (s, 2H), 6.62 (t, 1H), 6.83 (dd, 1H), 7.03 (s,
1H), 7.16 (t, 1H), 7.39 (s, 1H), 7.44 (dd, 1H), 8.56 (m, 2H).
ESMS, M þ Hþ found 429.6.
3-Chloro-5-[4-chloro-3-fluoro-5-(1H-pyrazolo[3,4-b]pyridin-
1
3-ylmethoxy)phenoxy] Benzonitrile (7). H NMR (400 MHz,
CDCl3, ppm): δ 5.43 (s, 2H), 6.63 (t, 1H), 6.84 (dd, 1H), 7.07 (t,
1H), 7.17 (t, 1H), 7.23 (dd, 1H), 7.38 (t, 1H), 8.20 (dd, 1H). 8.60
(d, 1H). ESMS, M þ Hþ found 429.0.
3-{3-[(6-Amino-1H-pyrazolo[3,4-b]pyridin-3-yl)methoxy]-4-
chloro-5-fluorophenoxy}-5-chlorobenzonitrile (8). 1H NMR
(400 MHz, CD3OD, ppm): δ 5.41 (s, 2H), 6.47 (d, 1H), 6.65
(dd, 1H), 6.87 (t, 1H), 7.34 (m, 2H), 7.61 (t, 1H), 7.92 (d, 1H).
ESMS, M þ Hþ found 444.1.
3-{3-[(6-Amino-1H-pyrazolo[3,4-b]pyridin-3-yl)methoxy]-5-
fluorophenoxy}-5-chlorobenzonitrile (9). 1H NMR (400 MHz,
CD3OD, ppm): δ 5.44 (s, 2H), 6.55 (dt, 1H), 6.63 (s, 1H), 6.66
(d, 1H), 6.78 (dt, 1H), 7.32 (s, 1H), 7.35 (t, 1H), 7.59 (s, 1H),
8.22 (d, 1H). ESMS, M þ Hþ found 410.0.

7168 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
3-Chloro-5-[4-fluoro-3-(1H-pyrazolo[3,4-b]pyridin-3-ylmeth-
oxy)phenoxy] Benzonitrile (10). H NMR (400 MHz, CDCl3,
Su et al.
(500 mg, 2.97 mmol) in 10 mL of dichloromethane was added
TEA (0.415 mL, 2.97 mmol) and cooled to 0 °C, and then
methane sulfonyl chloride was added (0.232 mL, 2.97 mmol)
and stirred for 15 min at 0 °C. The mixture was quenched with
5 mL of water and diluted with dichloromethane. The organic layer
was washed once each with water and brine, dried over sodium
sulfate, filtered, and concentrated in vacuo. Flash chromatography
of the crude residue (10-60% EtOAc/hexanes) afforded 533 g
(73%) 1i as a yellow oil. ESMS, M þ Hþ found 247.2.
1
ppm): δ 5.53 (s, 2H), 6.60 (m, 1H), 6.93 (m, 1H), 7.06 (s, 1H),
7.11 (m, 2H), 7.24 (t, 1H), 7.31 (s, 1H), 8.32 (d, 1H), 8.61 (s, 1H),
11.20 (b, 1H). ESMS, M þ Hþ found 395.1.
3-Chloro-5-[4-chloro-3-(1H-pyrazolo[3,4-b]pyridin-3-ylmeth-
oxy)phenoxy] Benzonitrile (11). 1H NMR (400 MHz, CD3OD,
ppm): δ 5.54 (s, 2H), 6.66 (dd, 1H), 7.06 (d, 1H), 7.24 (m, 3H),
7.42 (d, 1H), 7.53 (t, 1H), 8.40 (dd, 1H), 8.52 (dd, 1H). ESMS,
M þ Hþ found 411.5.
3-{[4-Chloro-3-(3-chloro-5-cyanophenoxy)phenoxy]methyl}-
isoxazolo[5,4-b]pyridine-6-yl Methanesulfonate (1m). To a so-
lution of 3-chloro-5-(2-chloro-5-hydroxyphenoxy)benzonitrile
(600 mg, 2.14 mmol, prepared by the method described in ref 2)
in 10 mL of THF was added t-BuOK (1 M solution in THF, 2.14
mmol), stirred for 10 min at room temperature, 1i (527 mg, 2.14
mmol) dissolved in 5 mL of THF was added and stirred at room
temperature for 90 min. The reaction mixture was then quenched
with 5 mL of water and diluted with EtOAc. The organic layer
was washed once each with water and brine, dried over sodium
sulfate, filtered, and concentrated in vacuo. Flash chromato-
graphy of the crude residue (100% CHCl3) gave 850 mg (78%)
of 1m as a white solid. (ESMS, M þ Hþ found 506.0).
2-{[t-Butyl(dimethyl)silyl]oxy}-1-(2,6-difluoropyridin-3-yl)-
ethanol (1e). 2,6-Difluoro pyridine (3.3 g, 28.7 mmol) was
dissolved in 20 mL of THF and cooled to -78 °C. To this
solution was added LDA (mono THF complex in hexanes,
1.5M, 28.7 mmol) dropwise, and the resulting mixture was
stirred for 2 h at -78 °C. In a separate flask {[t-butyl-
(dimethyl)silyl]oxy}acetaldehyde (5 g, 28.7 mmol) was dis-
solved in 20 mL of THF and cooled to -78 °C. The lithiated
pyridine solution was then transferred into the aldehdyde
solution via cannula over 15 min. The reaction mixture was
stirred an additional 15 min at -78 °C and then warmed to
room temperature for 1 h. The reaction mixture was quenched
with 50 mL of water and diluted with EtOAc. The combined
organic layer was washed once each with water and brine,
dried over sodium sulfate, filtered, and concentrated in vacuo.
Flash chromatography of the crude residue (0-20% EtOAc/
hexanes) gave 6.4 g (77%) of 1e as a yellow oil. 1H NMR (400
MHz, CDCl3, ppm): δ 0.07 (m, 6H), 0.942 (s, 9H), 3.05 (t, 1H),
3.53 (t, 1H), 3.92 (dd, 1H), 5.01 (d, 1H), 6.88 (d, 1H), 8.11 (q,
1H). ESMS, M þ Hþ found 290.2.
3-Chloro-5-(2-chloro-5-{[6-(methylamino)isoxazolo[5,4-b]-
pyridine-3-yl)methoxy}phenoxy) Benzonitrile (15). To a solution
of 3-{[4-chloro-3-(3-chloro-5-cyanophenoxy)phenoxy]methyl}-
isoxazolo[5,4-b]pyridine-6-yl methanesulfonate (95 mg, 0.221
mmol) in 1 mL of DMF was added Cs2CO3 (71.9 mg, 0.221
mmol) and methyl amine (0.22 L mL, 1.0 M solution in THF).
The resulting reaction mixture was heated for 12 h at 60 °C,
cooled to room temperature, and diluted with EtOAc. The
organic layer was washed with water (3 ꢀ 5 mL) and brine (1 ꢀ
5 mL), dried over sodium sulfate, filtered, and concentrated in
vacuo. The crude residue was purified via reverse phase chromato-
graphy (5-95% AcCN/water/0.05% TFA) to afford 17 mg
(15%) of the title compound. 1H NMR (400 MHz, CDCl3, ppm):
δ 3.05 (d, 3H), 5.05(b, 1H), 5.33 (s, 2H), 6.37 (d, 1H), 6.77 (d, 1H),
6.92 (dd, 1H), 6.96 (t, 1H), 7.13 (t, 1H), 7.34 (t, 1H), 7.40 (d, 1H),
7.71 (d, 1H). High resolution MS: m/z found 441.0497 (M þ 1),
calculated 441.0516 (M þ 1).
2-{[t-Butyl(dimethyl)silyl]oxy}-1-(2,6-difluoropyridin-3-yl)-
N-hydroxyethanimine (1f). DMSO (0.123 mL, 1.73 mmol) was
added dropwise to a solution of oxalyl chloride (0.302 mL, 3.46
mmol) in 10 mL of dichloromethane at -78 °C and then stirred
for 30 min. 2-{[t-Butyl(dimethyl)silyl]oxy}-1-(2,6-difluoropyri-
din-3-yl)ethanol (500 mg, 1.728 mmol) was added to the cooled
solution, and the mixture was stirred for 15 min. TEA (1.2 mL,
8.64 mmol) was added and then warmed to 0 °C for 30 min. The
reaction mixture was diluted with EtOAc. The organic layer was
washed once each with water and brine, dried over sodium
sulfate, filtered, and concentrated in vacuo. Flash chromatogra-
phy of the crude residue (5-15% EtOAc/hexanes) gave 470 mg
(94%) of 2-{[t-butyl(dimethyl)silyl]oxy}-1-(2,6-difluoropyridin-
3-yl)ethanone as a yellow oil. 1H NMR (400 MHz, CDCl3, ppm):
δ 0.126 (s, 6H), 0.930 (s, 9H), 4.82 (s, 2H), 6.97 (d, 1H), 8.52 (q,
1H). ESMS, M þ Hþ found 287.2.
3-Chloro-5-[2-chloro-5-(isoxazolo[5,4-b]pyridine-3-yl)methoxy)-
phenoxy] Benzonitrile (12). 1H NMR (400 MHz, CDCl3, ppm): δ
5.47 (s, 2H), 6.80 (d, 1H), 6.94 (dd, 1H), 7.00 (t, 1H), 7.13 (t, 1H),
7.35 (m, 2H), 7.43 (d, 1H), 8.22 (d, 1H), 8.67 (d, 1H). High
resolution MS: m/z found 412.0234 (M þ 1), calculated 412.0250
(M þ 1).
3-{5-[(6-Aminoisoxazolo[5,4-b]pyridin-3-yl]-2-chlorophenoxy}-
5-chlorobenzonitrile (13). 1H NMR (400 MHz, CDCl3, ppm): δ
5.1 (b, 2H), 5.35 (s, 2H), 6.48 (d, 1H), 6.78 (d, 1H), 6.93 (dd, 1H),
6.96 (t, 1H), 7.14 (t, 1H), 7.35 (s, 1H), 7.40 (d, 1H), 7.81 (d, 1H).
High resolution MS: m/z found 427.0341 (M þ 1), calculated
427.0346 (M þ 1).
To a solution of 2-{[t-butyl(dimethyl)silyl]oxy}-1-(2,6-di-
fluoropyridin-3-yl)ethanone (6.8 g, 23..66 mmol) in 200 mL of
dichloromethane was added titanium isopropoxide (13.87 mL,
47.3 mmol) followed by hydroxyl amine hydrochloride (3.13 g,
47.3 mmol). The reaction mixture was stirred at room tempera-
ture for 1 h and then quenched with 10 mL of water. The
reaction was allowed to stir overnight and then filtered and
concentrated in vacuo. Flash chromatography of the crude
residue (5-20% EtOAc/hexanes) gave 4.7 g (66%) of 1f. ESMS,
M þ Hþ found 303.2.
3-{5-[(6-Aminoisoxazolo[5,4-b]pyridin-3-yl]-2-chloro-3-fluoro-
phenoxy}-5-chlorobenzonitrile (14). 1H NMR (400 MHz, CDCl3,
ppm): δ 5.34 (s, 2H), 6.50 (d, 1H), 6.58 (t, 1H), 6.81 (dd, 1H), 7.01
(s, 1H), 7.17 (t, 1H), 7.39 (t, 1H), 7.82 (d, 1H). High resolution
MS: m/z found 445.0271 (M þ 1), calculated 445.0265 (M þ 1).
3-Chloro-5-(2-chloro-5-{[6-(dimethylamino)isoxazolo[5,4-b]-
pyridine-3-yl)methoxy}phenoxy) Benzonitrile (16). 1H NMR
(400 MHz, CDCl3, ppm): δ 3.20 (s, 6H), 5.33 (s, 2H), 6.52 (d,
1H), 6.78 (d, 1H), 6.92 (dd, 1H), 6.98 (t, 1H), 7.12 (t, 1H), 7.35
(t, 1H), 7.39 (d, 1H), 7.76 (d, 1H). High resolution MS: m/z
found 455.0660 (M þ 1), calculated 455.0672 (M þ 1).
3-Chloro-5-{2-chloro-5-[(6-piperdin-1-yl-isoxazolo[5,4-b]pyridine-
3-yl)methoxy]phenoxy} Benzonitrile (17). 1H NMR (400 MHz,
CDCl3, ppm): δ 1.66 (m, 4H), 1.71 (m, 2H), 3.71 (t, 4H), 5.32
(s, 2H), 6.62 (d, 1H), 6.77 (d, 1H), 6.92 (dd, 1H), 6.99 (t, 1H), 7.12
(t, 1H), 7.34 (t, 1H), 7.39 (d, 1H), 7.73 (d, 1H). High resolution MS:
m/z found 495.0967 (M þ 1), calculated 495.0985 (M þ 1).
3-Chloro-5-{2-chloro-5-[(6-morpholin-4-yl-isoxazolo[5,4-b]-
pyridine-3-yl)methoxy]phenoxy} Benzonitrile (18). 1H NMR
(6-Fluoroisoxazolo[5,4-b]pyridine-3-yl)methanol (1h) and
3-({[t-Butyl(dimethyl)silyl]oxy}methyl)-6-fluoroisoxazolo[5,4-b]-
pyridine (1g). 2-{[t-Butyl(dimethyl)silyl]oxy}-1-(2,6-difluoropyri-
din-3-yl)-N-hydroxyethanimine (3 g, 9.92 mmol) was dissolved in
100 mL of ethylene glycol and heated to 150 °C for 5 h. The mixture
was cooled to room temperature and diluted with EtOAc. The
organic layer was washed once each with water and brine, dried over
sodium sulfate, filtered, and concentrated in vacuo. Flash chroma-
tography of the crude residue (1-4% MeOH/CH2Cl2) afforded
480 mg (17%) of 1h, 1 g (60%) of 1g, and 550 mg (30%) of desilyla-
ted starting material. For 1h, 1H NMR (400 MHz, CDCl3, ppm): δ
5.1 (s, 2H), 7.00 (d, 1H), 8.34 (t, 1H). ESMS, M þ Hþ found 169.2.
(6-Fluoroisoxazolo[5,4-b]pyridine-3-yl)methanesulfonate (1i).
To a solution of (6-fluoroisoxazolo[5,4-b]pyridine-3-yl)methanol

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7169
(400 MHz, CDCl3, ppm): δ 3.71 (t, 4H), 3.81 (t, 4H), 5.34 (s,
2H), 6.63 (d, 1H), 6.77 (d, 1H), 6.92 (dd, 1H), 6.97 (t, 1H), 7.13
(t, 1H), 7.34 (t, 1H), 7.40 (d, 1H), 7.81 (d, 1H). High resolution
MS: m/z found 497.0748 (M þ 1), calculated 497.0778 (M þ 1).
3-Chloro-5-{2-chloro-5-[(6-piperazin-1-yl-isoxazolo[5,4-b]pyridine-
3-yl)methoxy]phenoxy} Benzonitrile (19). 1H NMR (400 MHz,
CDCl3, ppm): δ 2.98 (t, 4H), 3.71 (t, 4H), 5.33 (s, 2H), 6.63 (d,
1H), 6.77 (d, 1H), 6.91 (dd, 1H), 6.99 (t, 1H), 7.13 (t, 1H), 7.34 (t,
1H), 7.39 (d, 1H), 7.78 (d, 1H). High resolution MS: m/z found
496.0946 (M þ 1), calculated 496.0938 (M þ 1).
Supporting Information Available: PhenoScreen assay data
from Monogram Bioscience and rat PK of selected compounds
(iv administration). This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) Tucker, T. J.; Sisko, J. T.; Tynebor, R. M.; Williams, T. M.; Felock,
P. J.; Flynn, J. A.; Lai, M.-T.; Liang, Y.; McGaughey, G.; Liu, M.;
Miller, M.; Moyer, G.; Munshi, V.; Perlow-Poehnell, R.; Prasad, S.;
Reid, J. C.; Sanchez, R.; Torrent, M.; Vacca, J. P.; Wan, B.-L.; Yan,
Y. Discovery of 3-{5-[(6-Amino-1H-pyrazolo[3,4-b]pyridine-3-yl)-
3-Chloro-5-(2-chloro-5-{[6-(1H-imidazol-1-yl)isoxazolo[5,4-b]-
pyridine-3-yl)methoxy]phenoxy} Benzonitrile (20). 1H NMR (400
MHz, CDCl3, ppm): δ 5.47 (s, 2H), 6.80 (d, 1H), 6.93 (dd, 1H),
7.00 (t, 1H), 7.14 (t, 1H), 7.25 (s, 1H), 7.36 (t, 1H), 7.45 (dd, 1H),
7.76 (s, 1H), 8.31 (d, 1H), 8.47 (s, 1H). High resolution MS: m/z
found 478.0456 (M þ 1), calculated 478.0468 (M þ 1).
methoxy]-2-chlorophenoxy}-5-chlorobenzonitrile (MK-4965):
A
Potent, Orally Bioavailable HIV-1 Non-Nucleoside Reverse Tran-
scriptase Inhibitor with Improved Potency against Key Mutant
Viruses. J. Med. Chem. 2008, 51, 6503–6511.
(2) Tucker, T. J.; Saggar, S.; Sisko, J. T.; Tynebor, R. M.; Williams,
T. M.; Felock, P. J.; Flynn, J. A.; Lai, M.-T.;Liang, Y.;McGaughey,
G.; Liu, M.; Miller, M.; Moyer, G.; Munshi, V.; Perlow-Poehnell,
R.; Prasad, S.; Sanchez, R.; Torrent, M.; Vacca, J. P.; Wan, B.-L.;
Yan, Y. The Design and Synthesis of Diaryl Ether Second Genera-
tion HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitor with
Enhanced Potency versus Key Clinical Mutations. Bioorg. Med.
Chem. Lett. 2008, 18, 2959–2966.
(3) (a) Sweeney, Z. K.; Harris, S. F.; Arora, N.; Javanbakht, H.; Li, Y.;
Fretland, J.; Davidson, J. P.; Biledeau, J. R.; Gleason, S. K.;
Hirschfeld, D.; Kennedy-Smith, J. J.; Mirazadegan, T.; Roetz, R.;
Smith, M.; Sperry, S.; Suh, J. M.; Wu, J.; Tsing, S.; Villasenor, A. G.;
Paul, A.; Su, G.; Heilek, G.; Hang, J. Q.; Zhou, A. S.; Jernelius, J. A.;
Zhang, F.-J.; Klumpp, K. Design of Annulated Pyrazoles as In-
hibitors of HIV-1 Reverse Transcriptase. J. Med. Chem. 2008, 51
(23), 7449–7458. (b) Elworthy, T. R.; Dunn, J. P.; Hogg, J. H.; Lam, G.;
Saito, Y. D.; Silva, T. M. A. C.; Stefanidis, D.; Woroniecki, W.;
Zhornisky, E.; Zhou, A. S.; Klumpp, K. Orally bioavailable prodrugs
of a BCS class 2 molecule, an inhibitor of HIV-1 reverse transcriptase.
Bioorg. Med. Chem. Lett. 2008, 18, 6344–6347.
(4) The polymerase activity of reverse transcriptase was measured
using a 500nt heteropolymeric RNA template and a biotinylated
DNA primer. BrdU incorporation by purified recombinant RT
(wild-type, K103N, or Y181C) was detected using a ruthenylated
anti-BrdU antibody and a Bioveris M384 chemiluminescence
detection instrument. Data represent mean and standard devia-
tions of g2 experiments. Efavirenz was tested in this assay as a
control (Ki (nM): 0.4/8.5/0.3 (WT/K103N/Y181C)).
(5) The antiviral potency of compounds against wild-type (H9IIIB)
virus or H9IIIB with K103N or Y181C mutations was measured in a
multiple-cycle replication assay in MT2 cells. Cells were infected
overnight (moi ≈ 0.01) in the absence of compound, washed, and
cultured for 3 days in varying compound concentrations. Viral
replicationwasassessedbymeasuringasp24inculturesupernatants,
and the CIC95 is the lowest concentration of compound inhibiting
replication by g95%. Mutants K103N, Y181C, and K103N/Y181C
are prepared by the Advanced Biotechnology, Inc. Also see Vacca,
J. P.; Dorsey, B. D.; Schleif, W. A.; Levin, R. B.; McDaniel, S. L.;
Darke, P. L.; Zygay, J.; Quintero, J. C.; Blahy, O. M.; Roth, E.;
Sardana, V. V.; Schlabach, A. J.; Graham, P. I.; Condra, J. H.;
Gotlib, L.; Holloway, M. K.; Lin, J.; Chen, I.; Vastag, K.; Ostovic,
D.; Anderson, P. S.; Emini, E. A.; Huff, J. R. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 4096. Efavirenz was tested in this assay as a control
(CIC95 (nM): 4.3/38 (WT, 10% FBS/50% NHS), 232 (K103N, 10%
FBS), 8.0 (Y181C, 10% FBS), and 272 (K103N/Y181C, 10% FBS)).
(6) See Supporting Information for detailed rat PK of selected com-
pounds (iv administration).
3-Chloro-5-(2-chloro-5-{[6-(4,5-dihydro-1H-imidazol-2-yl)iso-
xazolo[5,4-b]pyridine-3-yl)methoxy}phenoxy] Benzonitrile (21).
1H NMR (400 MHz, CDCl3, ppm): δ 3.70-4.10 (b, 4H), 5.48
(s, 2H), 6.80 (d, 1H), 6.92 (dd, 1H), 7.00 (t, 1H), 7.13 (t, 1H), 7.35
(t, 1H), 7.45 (dd, 1H), 8.30 (s, 2H). High resolution MS: m/z
found 480.0623 (M þ 1), calculated 480.0625 (M þ 1).
3-({[t-Butyl(dimethyl)silyl]oxy}methyl)isoxazolo[5,4-b]pyridine-
6-carbonitrile (1n). To a solution of 3-({[t-butyl(dimethyl)silyl]-
oxy}methyl)-6-fluoroisoxazolo[5,4-b]pyridine (470 mg, 1.66 mmol)
in 2 mL of DMSO was added NaCN (82 mg, 1.664 mmol) and the
reaction mixture was allowed to stir for 12 h at 90 °C. The reaction
was then cooled to room temperature and diluted with EtOAc. The
organic layer was washed with water (3 ꢀ 10 mL) and brine (1 ꢀ
10 mL), dried over sodium sulfate, filtered, and concentrated in
vacuo. Flash chromatography of the crude residue (10-85%
EtOAc/hexanes) gave 425 mg (88%) of 1n. 1H NMR (400 MHz,
CDCl3, ppm): δ 0.15 (s, 6H), 0.92 (s, 9H), 5.11 (s, 2H), 7.72 (d, 1H),
8.40 (d, 1H). ESMS, M þ Hþ found 290.2.
3-(Chloromethyl)-6-(4,5-dihydro-1H-imidazol-2-yl)isoxazolo-
[5,4-b]pyridine (1p). To a solution of 3-({[t-butyl(dimethyl)-
silyl]oxy}methyl)isoxazolo[5,4-b]pyridine-6-carbonitrile (270 mg,
0.933 mmol) in 5 mL of EtOH at 0 °C was bubble HCl (g) for
2 min. The ice water bath was removed, and the reaction
mixture was allowed to warm to room temperature for 4 h.
The residual HCl gas was purged with N2, and the reaction
mixture was concentrated in vacuo. The crude residue was
redissolved in 5 mL of EtOH to which was added ethylene
diamine (0.077 mL, 0.993 mmol). The reaction was allowed to
stir overnight at room temperature and concentrated in vacuo.
Flash chromatography of the crude residue (0-8% MeOH/
dichloromethane/0.1% TEA) gave 425 mg (88%) of [6-(4,5-
dihydro-1H-imidazol-2-yl)isoxazolo[5,4-b]pyridin-3-yl]methanol
as a clear oil. 1H NMR (400 MHz, DMSO, ppm): δ 3.78 (s, 4H),
4.91 (d, 2H), 5.92 (t, 1H), 8.21 (d, 1H), 8.58 (d, 1H). ESMS,
M þ Hþ found 219.3.
To a solution of [6-(4,5-dihydro-1H-imidazol-2-yl)isoxazolo-
[5,4-b]pyridin-3-yl]methanol (56 mg, 0.257 mmol) in 2 mL
of dichloromethane was added thionyl chloride (37.5 uL,
0.513 mmol) and the reaction was allowed to stir at room
temperature for 30 min. The reaction was then concentrated in
vacuo to afford 61 mg of crude material as the HCl salt, which
was used for the next reaction without further purification. 1H
NMR (400 MHz, DMSO, ppm): δ 4.09 (s, 4H), 5.31 (s, 2H), 8.37
(d, 1H), 8.92 (d, 1H), 8.58 (d, 1H), 9.5 (b, 1H), 11.15 (s, 2H)
ESMS, M þ Hþ found 237.2.
(7) Assays performed by Monogram Bioscience, San Francisco, CA.
Values are the average of two determinations. Detailed assay
protocols are available at: http://www.monogramhiv.com/assays/
hcp/phenolHIVTechnology.aspx.
(8) iv dose, 1, 3, and 10 mg/kg (cumulative); vehicle: 100% DMSO,
5 mL/30 min; 3 anesthetized, vagotomized, and ventilated dogs.
(9) Compound 6 was tested over a concentration range of 7.11-4000.00
μg/mL with and without S-9, in a forward mutation assay in Salmonella
typhimurium strain FU100. Compound 6 did not produce any sig-
nificant increases in mutants compared with the mean historical control
levels in either the presence or absence of S-9 metabolic activation and is
thus considered negative in the Exploratory 5-Fluorouracil Microbial
Forward Mutation Assay developed at Merck.
Acknowledgment. We are pleased to acknowledge the
efforts of Thomas J. Tucker, John T. Sisko, Robert M.
Tynebor, Dr. C. W. Ross, Dr. S. M. Pitzenberger, S. L. Varga,
and J. S. Murphy.
(10) Exploratory 7-day tolerability study, po dose, vehicle: 10% poly-
sorbate and 80/90% water.