Biaryl ethers as potent allosteric inhibitors of reverse transcriptase and its key mutant viruses: Aryl substituted pyrazole as a surrogate for the pyrazolopyridine motif

Article abstract of DOI:10.1016/j.bmcl.2010.06.083

Biaryl ethers were recently reported as potent NNRTIs. Herein, we disclose a detailed effort to modify the previously reported compound 1. We have designed and synthesized a series of novel pyrazole derivatives as a surrogate for pyrazolopyridine motif th

Full text of DOI:10.1016/j.bmcl.2010.06.083

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4328–4332
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biaryl ethers as potent allosteric inhibitors of reverse transcriptase and its
key mutant viruses: Aryl substituted pyrazole as a surrogate for the
pyrazolopyridine motif
a
a
a
a
a
Dai-Shi Su a, , John J. Lim , Elizabeth Tinney , Thomas J. Tucker , Sandeep Saggar , John T. Sisko ,
Bang-Lin Wan a, Mary Beth Young a, Kenneth D. Anderson a, Deanne Rudd a, Vandna Munshi b,
Carolyn Bahnck b, Peter J. Felock b, Meiquing Lu b, Ming-Tain Lai b, Sinoeun Touch c, Gregory Moyer c,
Daniel J. DiStefano c, Jessica A. Flynn c, Yuexia Liang d, Rosa Sanchez d, Rebecca Perlow-Poehnelt a,
Mike Miller b, Joe P. Vacca a, Theresa M. Williams a, Neville J. Anthony a
*
a Department of Medicinal Chemistry and Structural Biology, Merck Research Laboratory, PO Box 4, West Point, PA 19486, United States
b Antiviral Research, Merck Research Laboratory, PO Box 4, West Point, PA 19486, United States
c Vaccines and Biologics Research, Merck Research Laboratory, PO Box 4, West Point, PA 19486, United States
d Drug Metabolism, Merck Research Laboratory, PO Box 4, West Point, PA 19486, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Biaryl ethers were recently reported as potent NNRTIs. Herein, we disclose a detailed effort to modify
the previously reported compound 1. We have designed and synthesized a series of novel pyrazole
derivatives as a surrogate for pyrazolopyridine motif that were potent inhibitors of HIV-1 RT with
nanomolar intrinsic activity on the WT and key mutant enzymes and potent antiviral activity in
infected cells.
Received 21 May 2010
Revised 10 June 2010
Accepted 14 June 2010
Available online 17 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Non-nucleoside reverse transcriptase
inhibitors (NNRTI)
Structural–activity relationship (SAR)
Mutant virus
Aryl substituted pyrazoles
Surrogate for pyrazolopyridine
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are key
components of current HIV therapy, HAART (Highly Active Antiret-
roviral Therapy), a combination of nucleoside and non-nucleoside
reverse transcriptase inhibitors (NRTIs, NNRTIs), and protease inhib-
itors (PIs). HAART can be very effective at delaying disease progres-
sion. However, due to the propensity of HIV to rapidly mutate, the
efficacy and durability of HAART can be compromised. The most fre-
quent HIV RT mutations observed in patients failing therapy with
Cl
CN
B
Cl
CN
A
H
N
A
Ar
Y
N
X
N
O
O
O
O
C
z
NH2
B
Cl
Cl
compound 1 (MK-4965)
Scheme 1. Lead Compounds 1 and replacement of pyrazolopyridine C-ring.
* Correspondingauthoratpresentaddress:1250S. CollegevilleRd., UP1205,GlaxoSmithKline, Collegeville, PA19426,UnitedStates. Tel.:+16109175879;fax:+16109176151.
E-mail address: Dai-Shi.X.Su@gsk.com (D.-S. Su).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.083

D.-S. Su et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4328–4332
4329
Table 1
SAR results
Cl
CN
R
Het
O
O
Cl
RT-Pola
Compound
Het
R
Spreadb (nM)
WT/K103N/Y181C (nM)
10% FBS/50% NHS (WT)
K103Nc
Y181Cc
2
3
—
39/179/140
922/8300
222/924
8300
—
NH
NH
N
N
0.9/2.4/12.5
924
—
833
4
460/>340/>3000
8300/>8300
—
NH
O
N
N
O
N
5
6
7
2.6/5.5/20.4
31/115/763
1.6/4.6/49.5
926/8333
>8300/>8300
168/307
2778
>8333
922
833
—
N
S
178
NH
O
8
0.3/10/1.5
97/922
922
833
NH
NH
N
N
N
N
9
10
11
12
0.7/4.2/3*
3.2/9.6/8.9
1.1/5.2/6.7
0.7/4.2/4.6
32/307
97/307
32/307
25/102
102
307
307
102
278
>833
278
278
N
NH
NH
N
N
N
N
NH
NH
NH
N
N
N
+
13
14
5.3/21/46
44/307
34/922
307
307
500
—
N
O
0.7/3.6/12*
CN
Cl
N
15
16
0.4/0.5/0.6*
0.5/0.7/0.5*
922/922
97/307
2767
102
—
NH
NH
NH
N
N
Cl
Cl
278
N
H
N
17
0.3/0.7/0.6*
2778/>8333
8333
—
N
N
N N
a
Compounds were evaluated in a standard SPA assay.
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
b
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.
*
Compounds were evaluated in a standard ECL assay.

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D.-S. Su et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4328–4332
first generation NNRTIs are K103N and Y181C. Therefore, new
agents with better activity profiles against mutant HIV-1 RT are
needed.
The current study was initiated with the previously reported
biaryl ethers lead compound 1 (Scheme 1).1–4 In this Letter, we re-
port the SAR study results of a structural alternative, substituted
pyrazole, as a replacement of pyrazolopyridine (the C-ring), that
exhibited excellent potency against wild-type (WT) reverse trans-
criptase (RT) and key clinically observed RT mutants.
The previous reported compound 1 showed excellent potencies
and had good oral bioavailablity in preclinical species (Scheme 1).2
In an effort to increase the structural diversity of our compounds,
we focused on the replacement of the pyrazolopyridine C-ring.
We speculated that the pyrazolopyridine moiety (a.k.a. fused aryl
pyrazole) could be replaced with aryl substituted pyrazole. To test
this hypothesis, the truncated analogue (2) was synthesized
(Table 1). Gratifyingly, compound 2 maintained potency against
WT RT and its key mutants in the enzyme assay (RT_Pol).5 How-
ever, its activity in the cell based assay (Spread assay) was signifi-
cantly reduced.6 We were encouraged that introduction of a phenyl
group at the 5-position of the pyrazole (compound 3) improved the
potency in both enzyme and cell based assays and decreased the
sera shift between 10% FBS and 50% NHS. The isomeric 4-phenyl
pyrazole analogue (compound 4) was inactive. It is known that
the isoxazolopyridine can be an effective isostere for the pyrazolo-
pyridine.1 Replacement of pyrazole with isoxazoles led to com-
pounds 5 and 6. The 5-phenyl isoxazole analogue (5) exhibited
good intrinsic potencies in the enzyme assay, but it lost potency
in the Spread assay. The isomeric 3-phenyl isoxazole (6) was less
potent than 5. Replacement of benzene with thiophene, furan,
and methylated pyrazole produced compounds 7–9 that had im-
proved potency, especially in cell based assay, compared with 3.
While a significant shift between the sera was frequently observed,
compound 9 showed excellent activities in both enzyme and the
cell based assays against WT and key mutant enzymes. The amino
group and the pyrido nitrogen atom in pyrazolopyridine were re-
ported to be critical for both the physiochemical properties and
the potency of compound 1.2 We speculated that introduction of
a pyridine moiety would decrease the lipophilicity of compounds
and hence reduce the effects of protein binding in the Spread assay.
Therefore, a pyridine moiety was installed. All three pyridine ana-
logues (10–12) showed potent intrinsic activity and improved po-
tency in the Spread assay versus benzene analogue (3). Of the three
pyridine analogues prepared, the 4-pyridyl derivative (12) had the
best overall profile. It showed excellent intrinsic antiviral activity
against WT, K103N, and Y181C mutants. Compound 12 also exhib-
ited excellent activity in the cell base assay against the same panel
of mutants with small shift in activity between 10% FBS and 50%
NHS. Oxidation of 12 led to the N-oxide 13 was accompanied by
a significant loss of activity, especially against the mutants.
Replacement of pyridine with 4-cyanobenzene led to analogue
14 that was less potent. Incorporation of a chloride atom in the
pyrazole ring yielded subnanomolar compounds (15–17) against
WT and mutant enzymes in the RT_Pol assay, but they were signif-
icantly less potent in the cell based assay, indicating the influence
of protein binding.
Table 2
SAR results
Cl
CN
Het
R
O
O
The other structural diverse ring systems that were also inves-
tigated are shown in Table 2. Nearly all the compounds prepared
(18–25) exhibited good potency (630 nM) against the WT RTs in
the enzyme assay. Compounds 22, 24, and 25 showed the potency
around or less than 10 nM against WT and K103N mutant viruses.
However, these compounds proved much less active in the cell
based assay, presumably due to their high protein binding. The re-
lated other ring systems, such as, thioazole, imidazole, triazole, and
the other oxazoles, showed lower activity (data not shown).
Compound 3 showed promising pharmacokinetic profiles, low
clearance, good volume distribution, and long half life in both rats
and dogs following iv coadministration (rat: Cl: 7.3 mL/min/kg, Vd:
Cl
Compound Het
R
RT-Pola
WT/
K103N/
Y181C
(nM)
Spreadb (nM)
10% FBS/50%
NHS (WT)
N
N
N
68/946/
>10,000
18
19
20
—
N
N
N
N
15/56/240
29/69/120
—
O
N
N
>8300/>8300
5.2 L/kg, AUCiv: 2.6
0.5 L/kg, AUCiv: 4.4
l
M h, t1/2: 10.4 h; dog: Cl: 2.2 mL/min/kg, Vd:
O
l
M h, t1/2: 9.5 h) (Table 3). Replacement of ben-
N
zene ring with thiophene (7), 4-pyridine (12), and 4-N-oxide pyri-
dine (13) led to higher clearance in rat. However, the 2-pyridine
analogue (10) showed improved clearance in rat and longer t1/2
21
22
23
24
25
2.1/30/170
5.2/9.3/85
>8300/>8300
926/926
O
OMe
OMe
Cl
O
N
N
N
N
N
31/130/
3100
>8300/>8300
2778/830
O
Table 3
Pharmacokinetics of selected compoundsa
O
4.9/11/91
Compound #
Rat
Dog
N
Cl
AUC
Cl
7.3
22.9
3.9
44.7
30.5
17.9
50.5
Vd
5.2
2.9
1.3
3.2
12.8
4.9
t1/2
AUC
Cl
2.2
2.5
4.5
—
—
Vd
t1/2
9.5
3.5
13.3
—
—
—
1.5
O
3
7
2.6
0.9
5.0
0.4
0.6
1.0
0.4
10.4
4.5
5.5
1.2
5.4
4.4
4.0
1.1
—
—
—
0.5
0.4
1.6
—
—
—
2.1/4.4/170 926/277
N
Cl
10
12
13
15
16
The values are the geometric mean of at least two determinations. All individual
values are within 25% of the mean.
a
Compounds were evaluated in a standard SPA assay.
CIC95 (cell culture inhibitory concentration) is defined as the concentration at
10.2
1.5
—
48.2
b
3.9
0.2
3.7
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.
a
iv cassette administration (co-administration with other analogues). AUC:
M h, CL: mL/min/Kg, Vd: L/kg, t1/2: hours. Vehicle: DMSO. Sprague–Dawley rats,
0.5 mg/Kg. Beagle dog: 0.25 mg/Kg. Interanimal variability was less than 20%.
l

D.-S. Su et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4328–4332
4331
gram Bioscience.7 It showed good antiviral activities against a
broader panel of clinically relevant mutant HIV-1 viruses (Table 4).
These results further suggest that the aryl substituted pyazole is
an effective surrogate for the indazole/pyrazolopyridine moieties.
Compounds described in this letter were prepared straightfor-
wardly according to the method reported previously.1,2 The key
intermediate, halogenated methyl pyrazoles/oxazoles, were synthe-
sized according to the route depicted in Scheme 2. Either radical bro-
mination of substituted methyl pyrazoles/oxazoles with NBS in the
presence of benzoyl peroxide or reduction of corresponding pyra-
zole ester with LAH following by treatment with thionyl chloride
afforded the desired halogenated methyl pyrazoles/oxazoles (the
intermediates for 3–9). The final product (7) was then prepared by
the coupling of the bromomethyl pyrazole with the 3-chloro-5-
(2-chloro-5-hydroxyphenoxy)benzonitrile under basic conditions.
The other analogues were prepared in an analogous manner. The
pyrazole ester required for analogue 12 was prepared from 1-(4-
pyridinyl)ethanone. Addition of the enolate to diethyl oxalate fol-
lowing by the cyclization with hydrazine delivered the desired ester.
The chlorinated pyrazoles were synthesized from the corresponding
pyrazole alcohol with NCS under basic conditions (the intermediate
for 16). The ester intermediate for the tetrazole analogue (17) was
prepared from the corresponding cyano derivative by the action of
sodium azide in the presence of zinc bromide.
Table 4
Antiviral potency of compounds 12 against clinically isolated mutant viruses (NL4-3
isolates)a
Mutantion
Compound 12
b
IC50
(
lM)
IC50 fold shift
WT
L100I
K103N
Y181C
Y188L
G190A
G190S
K103N/V179E
K103N/Y181C
K103N/G190A
Y181C/G190A
K103N/P225H
K101E/G190A
K101E/Y181C/G190A
K103N/Y181C/G190A
V106A/G190A/F227L
0.0045
0.0219
0.0197
0.0362
>1.000
0.0223
0.1607
0.0902
>1.000
0.1196
0.1309
0.0324
0.1181
0.3315
>1.000
>1.000
1
4.9
4.4
8.0
>222
5.0
35.7
20.0
>222
26.6
29.1
7.2
26.2
73.7
>222
>222
a
Phenoscreen assay, monogram bioscience, in presence of 40% NHS. For details,
see Ref. 7.
b
The IC50 is defined as the concentration of compound in cell culture required to
inhibit 50% of viral replication.
In summary, the pyrazole 12 was identified in an effort to mod-
ify the previously reported compound 1. We have designed and
synthesized a series of novel pyrazole derivatives as a surrogate
for pyrazolopyridine motif that were potent inhibitors of HIV-1
RT with nanomolar intrinsic activity on the WT and key mutant
enzymes and potent antiviral activity in infected cells. This com-
in dog. Introduction of chlorine atom at the pyrazole ring increased
clearance significantly in both rat and dog.
To assess the antiviral activities of compounds in this series
against a panel of clinically relevant mutants, the pyrazole com-
pound 12 was selected for a PhenoScreen assay performed by Mono-
Cl
CN
N
Cl
S
O
OH
a
Cl
S
S
N
Br
O
O
N
H
N
N
H
b
d
N
NBoc
Cl
7
O
c
O
O
8
OH
Cl
O
OEt
N
N
H
N
NH
N
N
H
O
O
N
O
EtO
d
N
f
N
OEt
12
OEt
O
Cl
OH
N
H
N
N
H
N
e
N
N
N
H
g
h
N
Cl
Cl
N
O
d
N
Cl
16
OH
OEt
Cl
N
H
N
N
N
N
N
H
H
N
N
NC
NC
j
N
Cl
i
O
O
N
Cl
O
17
H
OEt
OEt
OEt
N
N
N
N
H
H
N
N
H
Scheme 2. Reagents and conditions: (a) (1) Boc2O, DMAP, TEA, CH3CN, 84%; (2) NBS, benzoyl peroxide, CCl4, reflux, 66%; (b) (1) 3-chloro-5-(2-chloro-5-hydroxyphen-
oxy)benzonitrile, Cs2CO3, NMP; (2) HCl, EtOAc, 0°C to rt, 38%; (c) LAH, THF, 86%; (d) SOCl2, neat, >95%; (e) (1) NaOEt, THF, then diethyl oxalate; (2) hydrazineÁHCl, water, 36%
for two steps; (f) LAH, THF, 68%; (g) NCS, NaOH, THF, 59%; (h) LAH, THF, 37%; (i) NCS, NaOH, THF, 81%; (j) NaN3, ZnBr2, IPA/water, 90 °C, 94%.

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D.-S. Su et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4328–4332
Villasenor, A. G.; Paul, A.; Su, G.; Heilek, G.; Hang, J. Q.; Zhou, A. S.; Jernelius, J. A.;
Zhang, F.-J.; Klumpp, K. J. Med. Chem. 2008, 51, 7449; (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. Bioorg. Med. Chem. Lett. 2008, 18, 6344.
5. Experimental protocols for the HIV-1 RT polymerase SPA assay are described in
the following patent application: Saggar, S. A.; Sisko, J. T.; Tucker, T. J.; Tynebor,
R. M.; Su, D.-S.; Anthony, N. J. US Patent Appl. US 2007/021442A1, 2007.
Efavirenz was tested in this assay as a control (Ki (nM): 0.4/8.5/0.3 (WT/K103N/
Y181C)).
pound possesses broad activity against a number of clinically sig-
nificant mutant viruses.
Acknowledgments
We are pleased to acknowledge the efforts of Drs. C. W. Ross, S.
M. Pitzenberger, Mr. S. L. Varga, and Mrs. J. S. Murphy.
6. 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 replication was assessed by measuring as p24 in culture
supernatants, and the CIC95 is the lowest concentration of compound inhibiting
replication by P95%. 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)).
References and notes
1. Su, D.-S.; Lim, J. J.; Tinney, E.; Wan, B.-L.; Young, M. B.; Anderson, K. D.; Rudd, D.;
Munshi, V.; Bahnck, C.; Felock, P. J.; Lu, M.; Lai, M.-T.; Touch, S.; Moyer, G.;
DiStefano, D. J.; Flynn, J. A.; Liang, Y.; Sanchez, R.; Perlow-Poehnelt, R.; Miller,
M.; Vacca, J. P.; Williams, T. M.; Anthony, N. J. J. Med. Chem. 2009, 52, 7163.
2. 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. J. Med. Chem. 2008, 51, 6503.
3. 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. Bioorg. Med. Chem. Lett. 2008, 18, 2959.
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.
4. (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.;