HIV protease inhibitors with picomolar potency against PI-resistant HIV-1 by extension of the P3 substituent

Article abstract of DOI:10.1016/S0960-894X(03)00475-X

A biaryl pyridylfuran P₃ substituent on the hydroxyethylene isostere scaffold affords HIV protease inhibitors (PI's) with picomolar (IC₅₀) potency against the protease enzymes from PI-resistant HIV-1 strains. Inclusion of a gem-dimethyl substituent afforded compound 3 with 100% oral bioavailability (dogs) and more than double the t_1/2 of indinavir. Inhibition of multiple P450 isoforms is dependent on the regiochemistry of the pyridyl nitrogen in these compounds.

Full text of DOI:10.1016/S0960-894X(03)00475-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2569–2572
HIV Protease Inhibitors with Picomolar Potency Against
PI-Resistant HIV-1 by Extension of the P3 Substituent
Joseph L. Duffy,a,* Thomas A. Rano,a Nancy J. Kevin,a Kevin T. Chapman,a
William A. Schleif,b DavidB. Olsen, Mark Stahlhut,c Carrie A. Rutkowski,c
Lawrence C. Kuo,d Lixia Jin,e Jiunn H. Lin,e Emilio A. Eminib andJames R. Tata
c
a
aDepartment of Basic Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Virus and Cell Biology, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Structural Biology, Merck Research Laboratories, West Point, PA 19486, USA
eDepartment of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
Received10 February 2003; accepted23 April 2003
Abstract—A biaryl pyridylfuran P3 substituent on the hydroxyethylene isostere scaffold affords HIV protease inhibitors (PI’s) with
picomolar (IC50) potency against the protease enzymes from PI-resistant HIV-1 strains. Inclusion of a gem-dimethyl substituent
afforded compound 3 with 100% oral bioavailability (dogs) and more than double the t1/2 of indinavir. Inhibition of multiple P450
isoforms is dependent on the regiochemistry of the pyridyl nitrogen in these compounds.
# 2003 Elsevier Ltd. All rights reserved.
The use of HIV protease inhibitors (PI’s) in anti-
retroviral therapy has demonstrated remarkable success
over the past six years by decreasing the mortality rate
wild-type virus, but greatly improved potency against a
panel of clinically isolatedPI-resistant strains of HIV-1.
1
associatedwith HIV-1 infection.
However, as the
For those compounds from Figure 1 where R=H, the
synthesis may be accomplishedby direct analogy to the
syntheses of indinavir (4, 5, and 6 from Table 1).6 For
compound 3, which contains the gem-dimethyl moiety,
an alternate strategy has been employedas illustrated
in Scheme 1. The biaryl substituent I was assembled
by Stille coupling of 3-bromopyridine with the
2-stanylfuran. Acylation of this intermediate with
N-methoxy-N-methylacetamide afforded the ketone II.
The piperazine intermediate III was then condensed
with this ketone in neat TMSCN,7 resulting in the ami-
nonitrile intermediate IV. Deprotection of the proximal
nitrogen on this intermediate, followed by addition of
methylmagnesium bromide, yielded the gem-dimethyl
piperazine fragment V. Addition of this fragment to the
lactone VI8 afforded the cyclic intermediate VII. The
lactone was openedunder basic conditions, then treated
with excess silylating agent, followedby selective
hydrolysis of the silyl ester under neutral conditions,
duration of this treatment is extended an alarming
increase has been observedin the occurrence of viral
resistance to currently approvedtherapies. To address
2
this problem the next generation of PI’s must meet sev-
eral criteria. They must achieve more effective suppres-
sion of the wild-type virus through increased potency
3
andhigher plasma levels. They must be broadly active
against PI-resistant variants of HIV-1. Finally, they
must have outstanding patient tolerability as a compo-
nent of a multi-drug regimen. Many protease inhibitors
are currently in development to address these issues.4
In order to meet these criteria we have been engaged in a
program to further optimize the hydroxyethylene iso-
stere class of PI’s such as indinavir, which has resulted in
compound 2 (Fig. 1).5 We now report that extension of
the P3 pyridylfuran moiety in 2 to a biaryl system affords
compounds with similar in vitro potency against the
which gave the acidintermeidate
VIII. This inter-
mediate was then coupled with the aminochrominol.9
Final removal of the silyl protecting group afforded the
fully elaboratedmolecule 3.
*Corresponding author. Fax: +1-732-594-5350; e-mail: joseph_duffy@
merck.com
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00475-X

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J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2569–2572
are presentedin Table 1. The activity (IC50) of the
compounds against this panel of HIV-1 proteases is
presentedin Table 2. The compounds were also tested
for their ability to inhibit 95% of the spreadof viral
infection in MT4 human T-lymphoidcells (CIC 95) using
viral constructs derived from the strains described
above, andthese values are also listedin Table 2.11 It
must be notedthat the most highly cross-resistant viral
phenotypes we have identified10a were chosen for opti-
mization of the leadclass without regardto the amino
acidsubstitution pattern of their protease enzyme.
As is evident from Table 2, the resistant viral strains
K-60C, V-18C, andQ-60C have lost almost all sensitiv-
ity to indinavir in the viral spread assay. Compound 2,5a
with substitutions for the aminoindanol, carboxamide,
andpyridylmethyl moieties shows improvedinhibitory
potency against the enzymes from wild-type and resis-
tant strains of HIV-1, andthis translates directly into
improvedpotency in the viral spreadassays as well.
Extension of the P3 pyridylfuran substituent to the
biaryl system in 3 afforded additional gains in potency,
with 3 being at or near the potency limit of detection of
the spreadassay for each of the resistant strains tested.
We further foundthat the exclusion of the gem-dime-
thyl substituent afforded similar or slightly improved
potency in the enzyme inhibition assays, achieving
Figure 1. Extension of the P3 pyridylfuran substituent to a biaryl
moiety where R=H or Me, andX, Y, or Z=N.
The compounds synthesized were tested for the ability
to inhibit HIV-1 protease. The enzymes employedwere
derived from both the wild-type (NL4-3) virus, and a
series of clinical viral isolates from patients infectedwith
multiply PI-resistant HIV-1 (K-60, Q-60, andV-18).
The genotype andphenotype of these isolates has been
reported,10 andthe protease sequences for these strains
Table 1. Sequence mutations from wild-type HIV-1 protease for resistant viral strains
NL4-3a Leu-10 Met-36 Asn-37 Arg-41 Met-46 Ile-54 Arg-57 Gln-58 Ile-62 Leu-63 Ile-64 Ala-71 Gly-73 Val-77 Val-82 Ile-84 Leu-90 Ile-93
K-60
Q-60
V18
Ile
Ile
Ile
Ile
Ile
Ile
Val
Val
Glu
Pro
Pro
Pro
Val
Ile
Ile
Phe
Ala
Val
Val
Met
Met
Met
Leu
Leu
Leu
Ser
Asp
Lys
Val
Val
Val
Ile
Lys
Ser
aFor the complete sequence of the NL4-3 virus, see ref 10c.
Scheme 1. (a) Pd(PPh3)4, DIEA, DMF, 100 ꢀC; (b) BuLi, N-Methoxy-N-methylacetamide, THF, À78 ꢀC; (c) TMSCN (neat), 60 ꢀC; (d) Thiosalicilic
acid, Pd2(dba)3, DPPB, THF; (e) MeMgCl, DME, À20 ꢀC; (f) DIEA, iPrOH; (g) 1.0 N aq LiOH, DME, 0 ꢀC; (h) TBSOTf, DIEA, CH2Cl2, 0 ꢀC; (i)
30% H2O in THF; (j) HBTU, HOBT, DIEA, NMP; (k) TBAF, THF, 45 ꢀC.

J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2569–2572
2571
Table 2. Enzyme inhibitory concentrations (IC50) andviral spreadinhibitory concentrations (CIC 95) for HIV protease inhibitors
CompdP
substituent
HIV-1 Protease inhibition IC50 (nM)
Viral SpreadCIC 95 (nM)a
3
NL4-3
0.60
K-60C
61.2
V-18C
43.6
Q-60C
20.1
NL4-3
50
K-60C
V-18C
Q-60C
1
2
Indinavir
>1000
>1000
>1000
0.07
5.8
5.2
1.6
ꢂ8
ꢂ8
250
62
12
62
0.05
0.3
1.2
0.1
ꢂ8
ꢂ8
3
4
5
0.08
0.03
0.6
0.3
1.3
0.7
0.8
ꢂ8
ꢂ8
15
15
31
15
31
n.d.
ꢂ8
6
0.05
0.5
0.7
0.4
ꢂ8
31
15
15
aThe ꢂand ꢃvalues denote the lower and upper concentrations tested in our assays.
picomolar inhibition against all enzymes for compound
6. The activity of 3–6 were comparable in the viral
spreadassays, andthe regiochemistry of the pyridyl
nitrogen appearedto have little effect on antiviral
potency.
pound. The 3-pyridyl isomer 5 exhibiteda further dra-
matic decrease in A.U.C. The corresponding 2-pyridyl
isomer 6 was similarly poor, even at twice the oral dose
(10 mpk). Thus the bioavailability of this series of com-
pounds is critically dependent on the location of the
pyridyl nitrogen.
The pharmacokinetic properties of 3–6 were examined
by dosing the compounds both orally and IV (Table
3).12 Compound 3 exhibitedextraoridnary bioavail-
ability when dosed at 5 mpk PO in dogs (ꢁ100%). A
follow-up experiment was performedin rhesus maca-
ques that confirmedthe favorable PK profile of this
compound. Removal of the gem-dimethyl moiety as in 4
causeda substantial decrease in the A.U.C. of the com-
The source of SAR surrounding the bioavailability of
these compounds is apparent from the in vitro metabo-
lism assays in Table 4. The bioavailability of the com-
pounds when dosed orally in dogs (Table 3) is inversely
proportional to the stability of the compounds when
incubatedin human liver microsomes, as measuredby
their intrinsic clearance (CLint, Table 4). Thus, com-
Table 3. Pharmacokinetics of HIV protease inhibitors dosed in vivo
CompdSpecies
Dose PO/IV (mpk)
Cmax (
mM)
11.4
7.3
5.3
0.2
0.9
4.4
t1/2 (min)
A.U.C. (mM*h)
CLp (mL/min/kg)
%F
Indinavir
Dog
Dog
Dog
Dog
Dog
10/2
5/2
5/2
5/2
10/2
10/2
28
70
60
41
50
91
12.5
11.3
2.6
0.1
0.4
16.0
22.5
24.5
25.2
33.5
20.4
72
100
55
2
4
90
3
4
5
6
3
Rhesus
9.9
Table 4. In vitro metabolism assays for HIV protease inhibitors
a
int
CompdCL
(mL/min/kg)
CYP3A4 IC50 (mM)
CYP2D6 IC50 (mM)
CYP2C9 IC50 (mM)
Indinavir
50
19
133
233
655
0.15
0.01
0.32
0.44
5.20
>30.00
0.36
0.53
6.40
29.60
>30.00
0.41
5.50
>30.00
>30.00
3
4
5
6
aDeterminedin human liver microsomes.

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J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2569–2572
pound 3 was remarkably stable in this assay, while 5
and 6 were rapidly metabolized.
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Schleif, W. A.; Olsen, D. B.; Stahlhut, M.; Rutkowski, C. A.;
Kuo, L. C.; Jin, L.; Lin, J. H.; Emini, E. A.; Tata, J. R.
Bioorg. Med. Chem. Lett. 2002, 12, 2423. (b) Cheng, Y. C.;
Zhang, F.; Rano, T. A.; Lu, Z.; Schleif, W. A.; Gabrylski, L.;
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McDaniel, S. L.; Darke, P. L.; Zugay, J.; Quintero, J. C.;
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12. The use of animals was done under the purview of an
Institutional Animal Care andUse Committee, andall
applicable regulations andlaws pertaining to the use of
laboratory animals were followed. For experimental methods
for both in vitro andin vivo pharmacokinetic investigations,
see: Lin, J. H.; Chiba, M.; Balani, S. K.; Chen, I.-W.; Kwei,
G. Y.-S.; Vastag, K. J.; Nishime, J. A. Drug Metab. Disp.
1996, 24, 1111.
It is equally apparent from Table 4 that the microsome
stability of these compounds mirrors their potency as
inhibitors of the P450 isoform CYP3A4. In previous
investigations we have establishedthat CYP3A4 is the
isoform primarily responsible for the metabolism of indi-
13
navir, andthat indinavir is an inhibitor of this isoform.
Compound 3 is 10-foldmore potent than indinavir as an
inhibitor of CYP3A4, but is also a potent inhibitor of the
relatedisoforms 2D6 and2C9. It shouldbe noted, how-
ever, that 3 is not a comprehensive P450 inhibitor, as it is
essentially inactive against the isoforms 2C19, 1A2, and
2A6 (IC50 >50 mM). The des-methyl derivative 4 remains
a sub-micromolar inhibitor of 3A4 and2D6, but is a
micromolar inhibitor of 2C9. The corresponding 3-pyr-
idyl isomer 5 loses all activity as an inhibitor of 2C9, and
was only a sub-micromolar inhibitor of 3A4. Finally, the
2-pyridyl isomer 6 hadlost substantial activity as an
inhibitor of all of the isoforms. Thus it may be possible to
achieve greater selectivity among the P450 isoforms by
modification of the pyridy moiety.
Compound 3 shows high potency against PI resistant
HIV-1 strains, andoutstanding bioavailability in two
species.14 Therefore it wouldlikely meet the first two
criteria for a secondgeneration protease inhibitor.
However it wouldnot meet our thirdcriteria, which is
tolerability as a component of a multi-drug anti-
retroviral regimen. The profile of P450 isoform inhibi-
tion observedwith 3 increases the likelihoodof drug-
drug interactions with other medications metabolized
by these P450 isoforms, andthis preclueddfurther
development of this molecule. It is also apparent that
the P450 isoform inhibition profile is exquisitely sensi-
tive to the regiochemistry of the pyridyl substituent,
indicating that further modification of the P3 region of
the molecule may afforda more optimal PK profile.
References and Notes
13. Chiba, M.; Hensleigh, M.; Nishime, J. A.; Balani, S. K.;
Lin, J. H. Drug Metab. Disp. 1996, 24, 307.
1. Kempf, D. J.; Molla, A.; Hsu, A. In Antiretroviral Therapy;
De Clercq, E. Ed.; ASM: Washington D. C., 2001; pp 147–
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2. Romano, L.; Venturi, G.; Giomi, S.; Pippi, L.; Valensin,
P. E.; Zazzi, M. J. Med. Virol. 2002, 66, 143.
3. Dorsey, B. D.; McDoough, C.; McDaniel, S. L.; Levin,
R. B.; Newton, C. L.; Hoffman, J. M.; Darke, P. L.; Zugay-
Murphy, J. A.; Emini, E. A.; Schleif, W. A.; Olsen, D. B.;
Stahlhut, M. W.; Rutkowski, C. A.; Kuo, L. C.; Lin, J. H.;
Chen, I.-W.; Michelson, S. R.; Holloway, M. K.; Huff, J. R.;
Vacca, J. P. J. Med. Chem. 2000, 43, 3386.
14. Data for compound 3: 1H NMR (CDCl3, 400 MHz) d 9.29
(t, J=6.4 Hz, 1H), 8.62 (dd, J=1.6 Hz, J=4.8 Hz, 1H), 7.47
(dd, J=1.6 Hz, J=4.4 Hz, 1H), 7.29 (m, 5H), 7.10 (t, J=8.8
Hz, 1H), 7.06 (d, J=7.6 Hz, 1H), 6.34 (d, J=3.2 Hz, 1H), 5.95
(d, J=8.0 Hz, 1H), 5.16 (dd, J=4.4 Hz, 1H), 4.13 (m, 1H),
4.07 (d, J=12.4 Hz, 1H), 4.00 (dd, J=5.2 Hz, J=12.0 Hz,
1H), 3.81 (m, 1H), 3.75 (t, J=10.0 Hz, 1H), 3.65 (m, 1H), 3.35
(s, 1H), 3.10 (d, J=11.6 Hz, 1H), 2.86 (m, 4H), 2.64 (m, 2H),
2.39 (m, 2H), 1.89 (t, J=10.8 Hz, 1H), 1.65 (t, 1H), 1.56 (s,
3H), 1.53 (s, 3H); HPLC-MS (ES) 750.4 (M+1).