The design, synthesis and evaluation of novel HIV-1 protease inhibitors with high potency against PI-resistant viral strains

Article abstract of DOI:10.1016/S0960-894X(03)00474-8

Replacement of the pyridylmethyl moiety in indinavir with a pyridyl oxazole yielded HIV-1 protease inhibitors (PI) with greatly improved potency against PI-resistant HIV-1 strains. A meta-methoxy group on the pyridyl ring and a gem-dimethyl methyl linkage afforded compound 10 with notable in vitro antiviral activity against HIV-1 viral strains with reduced susceptibility to the clinically available PIs. Compound 10 also demonstrated favorable in vivo pharmacokinetics in animal models.

Full text of DOI:10.1016/S0960-894X(03)00474-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2573–2576
The Design, Synthesis and Evaluation of Novel HIV-1 Protease
Inhibitors with High Potency Against PI-Resistant Viral Strains
Fengqi Zhang,a,* Kevin T. Chapman,a William A. Schleif,b David B. Olsen,c
Mark Stahlhut,c Carrie A. Rutkowski,c Lawrence C. Kuo,d Lixia Jin,e
Jiunn H. Lin,e Emilio A. Eminib and James R. Tataa
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
Received 10 February 2003; accepted 23 April 2003
Abstract—Replacement of the pyridylmethyl moiety in indinavir with a pyridyl oxazole yielded HIV-1 protease inhibitors (PI) with
greatly improved potency against PI-resistant HIV-1 strains. A meta-methoxy group on the pyridyl ring and a gem-dimethyl methyl
linkage afforded compound 10 with notable in vitro antiviral activity against HIV-1 viral strains with reduced susceptibility to the
clinically available PIs. Compound 10 also demonstrated favorable in vivo pharmacokinetics in animal models.
# 2003 Elsevier Ltd. All rights reserved.
The introduction of HIV-1 protease inhibitors (PIs) into
the clinic in 1996 was a great advance in the treatment
of AIDS.1 However, there is an increasing incidence of
viral resistance to these agents.2 Therefore, there exists a
need forPIs that possess increased potency against the
wild-type virus as well as against viral strains with
reduced PI susceptibility.
indinavir.4 The aldehydes used forthe final erductive
amination were prepared by formylation of the corres-
ponding pyridyl furans. After much experimentation, a
unique synthetic strategy for the incorporation of a
gem-dimethyl linkage between the piperazine and the
oxazole was developed. Scheme 1 illustrates this suc-
cessful strategy for the preparation of compound 10.
The bis-protected piperazine I was first deprotected with
TFA.3 Silver triflate induced substitution with 2-bromo-
2-methylpropionic acid afforded amino acid II. In a
separate route, 3,5-dibromopyridine was converted into
3-bromo-5-methoxypyridine with sodium methoxide.5
Treatment of III with isopropylmagnesium chloride
followed by addition to the Weinreb amide of BOC-
glycine provided aminoketone V. PyBOP mediated
coupling of II and V gave ketoamide VI, which was
treated with fuming sulfuric acid to afford oxazole VII
with concomitant deprotection of the piperazine. Epox-
ide opening of VIII6 with VII, followed by the final
deprotection under acid conditions gave the desired
product 10.
Recently, we have reported modifications of the indin-
avir( 1) scaffold that have afforded compounds with
increased potency against resistant HIV-1 viral strains,
as exemplified by compound 2 (Fig. 1).3 Although
compound 2 has desirable antiviral activity and a good
pharmacokinetic profile, it is a potent inhibitor of CYP
isomers 3A4, 2D6 and 2C9, a property that would dra-
matically complicate its clinical use due to the potential
for serious drug–drug interactions. This work details
our efforts to modify this P450 inhibition profile while
maintaining antiviral potency and bioavailability. These
efforts have resulted in compound 10.
The synthesis of compounds 4, 5 and 6 (Table 1), which
lack the gem-dimethyl substituent, was similarto that of
The compounds, including indinavir, were tested for
theirability to inhibit the protease enzyme (IC 50) and to
inhibit the spread of viral infection in MT4 human
T-lymphoid cells infected eitherby wild-type HIV-1
*Corresponding author. Fax: +1-732-594-8080; e-mail: abe_zhang@
merck.com
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00474-8

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F. Zhang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2573–2576
Figure 1. Modifications of indinavirthat lead to compound 10.
Table 1. Enzyme inhibition and antiviral activity of HIV protease inhibitors
Compd
P3 moiety
IC50 (nM)
Viral speed CIC95 (nM)
K-60C
NL4-3
4X Virus
V-18C
Q-60C
Indinavir0.60
50
400
>1000
>1000
>1000
3
0.05
0.13
0.14
0.10
0.04
0.04
0.02
0.02
ꢁ8
ꢁ8
31
15
15
4
23
27
31
62
—
125
250
125
62
125
125
250
15
5
125
125
31
6
23
62
7
ꢁ8
ꢁ8
ꢁ8
ꢁ8
ꢁ8
ꢁ8
ꢁ8
ꢁ8
8
31
125
31
31
9
16
ꢁ8
31
10
15
15
Scheme 1. (a) TFA, CH2Cl2; (b) 2-bromo-2-methylpropionic acid, silver trifluoromethane sulfonate, TEA, THF; (c) NaOCH3, MeOH, DMF, 90 ꢀC,
6 h; (d) N-(tert-butoxycarbonyl)glycine N0-methoxy-N0-methylamide, isopropylmagnesium chloride, THF; (e) HCl, MeOH; (f) PyBop, HOAt,
DIEA, DMF; (g) fuming sulfuric acid, 55 ꢀC; (h) EtOH, reflux; (i) MeOH, HCl.

F. Zhang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2573–2576
Table 3. Pharmacokinetics of HIV protease inhibitors in dog
2575
(strain NL4-3) or by various PI-resistant strains.7,8a The
results, presented in Table 1, are given as the concentration
of compound that inhibits at least 95% of infectious viral
spread in the cell culture relative to the untreated virus
control culture (CIC95). Each of the PI-resistant strains
(4X, K-60C, V-18C, Q-60C) represent engineered viral
constructs that express varying patterns of multiple PI
resistance as described in our previous reports.3,8
Compd
Dose PO/IV
(mpk)
Cmax
(mM)
T1/2
(min)
CLp
(mL/min/kg)
F
(%)
8
9
10
10/2
10/2
10/2
3.8
1.0
4.3
94
92
61
21
19
18
10
8
30
Table 4. Pharmacokinetics of compound 10 dosed in rat and rhesus
As indicated in Table 1, indinavirexpressed no activity
against all of the resistant viral strains whereas the other
tested compounds expressed varying degrees and pat-
terns of activity. When the furan moiety in compound 3
was replaced with the oxazole (compound 4), a significant
loss in potency was observed. Some of this potency was
regained in the gem-dimethyl analogues (compare 4 with
7 and 5 with 8). A further increase in potency was realized
with the addition of a meta substituent to the pyridyl
ring. Chloro or methoxy substituents at this position
(compounds 9 and 10) provided a substantial increase in
activity against all of the viral variants.
Species
Dose
(mpk)
Cmax
(mM)
T1/2
(min)
AUC
(mM-h)
CLp
(mL/min/kg)
F
(%)
Rhesus
Rat
10/2
10/5
20
50
160
0.10
0.04
0.37
3.06
6.88
73
26
0.12
0.03
0.38
4.02
33
104
2.2
1.5
9.2
40.5
47.2
15.67
Table 5. Incubation of compound 10 in livermicrosomes CL int (mL/
min/kg)
Rat
87
Dog
67
Rhesus
120
Human
13
Previous studies have shown that CYP3A4 is the pri-
mary P450 enzyme that is responsible for the metabo-
lism of indinavir.9 Furthermore, a correlation between
inhibition of 3A4 and metabolic stability has been
demonstrated for indinavir related analogues.10
Although the inhibition of 3A4 could result in drug–
drug interactions, clinical experience with both indinavir
and ritonavir has shown this to be manageable.11 In
fact, treatment strategies have been devised to take
advantage of this property; thus the inhibition of 3A4
without significant inhibition of either2D6 or2C9 is
considered acceptable.12
10–160 mg/kg range. Both Cmax and AUC values
increased in the rat in a greater than dose-proportional
manner(see Table 4), suggesting clearance via a satur-
able first pass metabolic pathway. Further evidence of
this was obtained by comparing the steady-state drug
concentrations in the systemic circulation during portal
vein verses femoral vein infusion. The hepatic extraction
ratio dropped from 90% at low dose (0.6 mg min) to
38% at high dose (12 mg.min).
As expected, the CYP inhibition profile for the pyridyl–
oxazole series (Table 2) parallels the pyridyl–furan ser-
ies.3 Once again, the meta-pyridine provided the best
selectivity forCYP3A4 over2D6 and 2C9. The ternd
toward better metabolic stability (shown in Table 2 as
CLint) with increasing 3A4 inhibition also holds here,
with the most potent inhibitor, compound 10, also being
the most metabolically stable.
In the rhesus, compound 10 displayed a high clearance
with poorbioavailability ( Table 4), a result that is con-
sistent with its poormetabolic stability in hresus liver
microsomes.
The differences in in vivo clearance rate of compound 10
among species was further investigated by incubation of
the compound in livermicorsomes. As can be seen in
Table 5, compound 10 is most stable in human micro-
somes and least stable in rhesus microsomes.
The pharmacokinetic profiles of compounds 8, 9, and 10
were evaluated in dogs dosed both IV and PO.13 Com-
pound 10 displayed reasonable PK parameters in dogs
(Table 3).
Assuming that compound 10 would be cleared primarily
by hepatic metabolism in humans, as it is in rat and most
likely in dog and rhesus, it would be expected to display
non-linearpharmacokinetics in the clinic. Accordingly,
at highercompound plasma concentartions, its clea-r
ance would be saturated enabling the maintenance of
clinically relevant drug levels. This property, along with
the compound’s stability in human microsomes and its
excellent antiviral potency, make the compound poten-
Follow up studies were performed on compound 10 in
both rat and rhesus. In the rat, compound 10 displayed
non-linear pharmacokinetics with oral doses in the
Table 2. In vitro metabolism assays of HIV protease inhibitors
a
Compd
CLint
(mL/min/kg)
CYP3A4 IC50 CYP2D6 IC50 CYP2C9 IC50
(mM)
14
(mM)
(mM)
tially suitable forfurtherinvestigation.
Indinavir50
7
8
9
10
0.15
>30.00
22.2
10.5
4.17
8.7
>30.00
28
33
21
30
500
27
35
13
5.0
0.11
0.04
0.04
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