Synthesis and activity of novel HIV protease inhibitors with improved potency against multiple PI-resistant viral strains.

Article abstract of DOI:10.1016/S0960-894X(02)00425-0

Substitution of the t-butylcarboxamide substituent in analogues of the HIV protease inhibitor (PI) Indinavir with a trifluoroethylamide moiety confers greater potency against both the wild-type (NL4-3) virus and PI-resistant HIV. The trifluoroethyl substi

Full text of DOI:10.1016/S0960-894X(02)00425-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2423–2426
Synthesis and Activity of Novel HIV Protease Inhibitors with
Improved Potency Against Multiple PI-Resistant Viral Strains
Joseph L. Duffy,a,* Nancy J. Kevin,a Brian A. Kirk,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 26 March 2002; accepted 17 May 2002
Abstract—Substitution ofthe t-butylcarboxamide substituent in analogues ofthe HIV protease inhibitor (PI) Indinavir with a tri-
fluoroethylamide moiety confers greater potency against both the wild-type (NL4-3) virus and PI-resistant HIV. The trifluoroethyl
substituent also affords a slower clearance rate in vivo (dogs); however, this may be due to more potent inhibition ofat least two
P450 isoforms. # 2002 Elsevier Science Ltd. All rights reserved.
The advent ofchemotherapeutic agents to suppress
human immunodeficiency virus (HIV-1) infection has
proven to be beneficial in the treatment ofacquired
immune deficiency syndrome (AIDS). Several HIV pro-
tease inhibitors (PIs), including Indinavir (Fig. 1), have
been approved for use in combination with other thera-
pies for the treatment of HIV infection.1 However, the
clinical emergence ofdrug-resistant variants ofthe HIV
virus underscores the need for more potent and bio-
available protease inhibitors to achieve sustained viral
suppression in vivo. Furthermore, the alarming increase
in the number ofviral strains that are resistant to mul-
tiple protease inhibitors highlights the necessity for
compounds that are active against a wide variety of
HIV mutations.2
carboxamide moiety in Indinavir analogues affords
compounds with further increased potency against PI-
resistant virus, as well as improvements in pharmaco-
kinetic properties.
The synthesis ofthe carboxamide analogues was
designed so as to differentiate the amide position as late
as possible in the synthetic sequence (Scheme 1). The
bis-protected piperazine-2-(S)-carboxylic acid I4 was
first protected as the benzyl ester under standard condi-
tions. Deprotection ofthe distal nitrogen, ofllowed by
copper mediated alkylation, afforded intermediate II.5
Cyclization ofthis intermediate with 4-hydroxy-3,5-
diiodopyridine with the Castro–Stevens reaction affor-
ded the pyridylfuran III.6 Protecting group manipulation,
followed by hydrogenolysis, afforded the free acid IV.
Amide coupling with a variety ofamine nucleophiles
under standard conditions (EDC and HOAT) afforded
the desired amides V. Deprotection ofthe piperazine,
followed by nucleophilic epoxide opening with inter-
mediate VII7 and final deprotection under acid condi-
tions, gave the desired products VIII.
Recently, we reported that modification ofthe amino-
indanol moiety and the pyridylmethyl substituent on Indi-
navir afforded compounds, such as compound 1 (Fig. 1),
with both improved bioavailability and increased potency
against PI-resistant viral strains.3 In this communication
we report that modification ofthe piperazine tert-butyl-
The biological activity ofthe compounds synthesized is
shown in Table 1. The compounds were tested for the
ability to inhibit the protease enzyme (IC50) and to
*Corresponding author. Fax: +1-732-594-5350; e-mail: joseph_duffy
@merck.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00425-0

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J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2423–2426
by testing a set ofcompounds against PI cross-resistant
viral strains (Table 2). The viral constructs employed
were engineered from clinical viral isolates of patients
infected with multiply PI-resistant HIV, and the geno-
type and phenotype ofthese isolates have been reported
elsewhere.11 While Indinavir remains largely inactive
against the resistant viral strains, the substitution ofthe
trifluoroethylamide moiety (14, Table 2) affords a
limited increase in potency against the 4X virus and
the V-18 and Q-60 viral constructs. Similarly, while
compound 1 represents a substantial improvement in
potency over Indinavir, the trifluoroethyl analogue (11)
affords a further 2-fold increase in potency for each
viral construct.
The pharmacokinetic impact ofthe trifluoroethylamide
substituent was next investigated by dosing compound 11
in dogs, both orally (5 mg/kg) and iv (2 mg/kg) (Table
3).12 Remarkably high plasma levels ofcompound 11 were
observed after oral dosing, with a maximum concentra-
tion of17.0 mM. In fact, the trifluoroethylamide sub-
stituent appears to confer substantially greater plasma
exposure than the analogous t-butylamide compound
(compound 1), as measured by the A.U.C. In addition,
the overall clearance (CLp) ofthe trifluoroethylamide
analogue was less than a third the rate ofthe corre-
sponding t-butylamide. Both compounds compared
quite favorably with the pharmacokinetic profile of
Indinavir in dogs.12
Figure 1. Aminoindanol and pyridylmethyl replacements that afford
improved potency and bioavailability.
inhibit the spread ofviral inefction in MT4 human
T-lymphoid cells using NL4-3 virus (CIC95).8 In addi-
tion, the compounds were tested using the 4X mutant
virus model system, as an initial indication oftheir
potential potency against PI-resistant HIV.9 The results
indicate that the t-butylamide substituent (1, Table 1) is
highly optimized for potency in this series, and all
modifications with aliphatic amides (2–7) resulted in a
loss ofactivity. Furthermore, the arylamides and
arylalkylamides (8–10) were all substantially less potent
than the t-butylamide in this series ofcompounds. The
fluoroalkyl compounds (11–13) exhibited variable effects
on potency depending on their size, with the optimum
substituent being the trifluoroethylamide (11).10 This
substituent afforded increased potency in both the
enzyme inhibition assay and the inhibition ofviral
spread assay. In addition, a 4-fold increase in potency
over the t-butylamide substituted compound was
observed against the 4X mutant virus.
The substantially lower in vivo clearance observed for
compound 11 versus 1 in dogs was further probed by
comparative studies in a series ofin vitro metabolic assays.
In human liver microsomes the trifluoroethylamide sub-
stituent conferred slightly greater metabolic stability to
the compound than the corresponding t-butylamide, as
measured by the intrinsic clearance (CLint, Table 4).
However, both ofthe investigated compounds exhibited
greater in vitro metabolic stability than Indinavir.13 The
metabolic stability ofIndinavir may be attributed to the
competitive inhibition ofthe cyctochrome P450 isoform
3A4 (CYP3A4), which is the isoform primarily respon-
sible for metabolism of this compound in vivo.14 While
The impact ofthe trifluoroethylamide substituent on the
potency ofIndinavir analogues was further investigated
Scheme 1. (a) TFA, CH2Cl2; (b) HCꢀC(CH3)2Cl, CuCl, Cu(0), Et3N; (c) 4-hydroxy-3,5-diiodopyridine, Cu2O, pyridine, 115 ꢁC; (d) Pd2(dba)3,
DPPB, thiosalicylic acid, THF; (e) di-tert-butyldicarbonate, DMAP, CH2Cl2; (f ) H, Pd/C, Et3N, MeOH; (g) RNH2, EDC, HOAT, Et3N, CH2Cl2;
2
(h) TFA, CH2Cl2; (i) VII, iPrOH, 85 ꢁC; (j) HCl (g), MeOH.

J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2423–2426
2425
Table 1. Influence ofcarboxamide substituents on in vitro potency ofHIV-protease inhibitors ofstructure VIII
Compd
R
IC50 (nM)
0.08
CIC95 (nM) NL4-3
CIC95 (nM) 4X
31
1
ꢂ8.0
2
1.16
0.61
0.40
0.60
0.50
0.37
13.8
2.19
0.65
0.07
0.14
1.60
31
31
500
62
3
4
15
125
5
62
250
6
46
125
7
ꢂ8.0
250
125
94
31
8
>1000
250
9
10
11
12
13
125
ꢂ8.0
ꢂ8.0
62
ꢂ8.0
62
500
Table 2. Potency (CIC95) (nM) against PI-resistant viral isolate constructs in the viral spread assay
Compd
Scaffold
Amide substituent
NL4-3
4X Virus
Viral constructs
K-60C
V-18C
Q-60C
Indinavir
14
R=C(CH3)3
R=CH2CF3
50
62
400
125
>1000
>1000
>1000
500
>1000
62
1
11
R=C(CH3)3
R=CH2CF3
9.2
ꢂ8.0
16
ꢂ8.0
500
250
125
62
125
62

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J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2423–2426
Table 3. Pharmacokinetics ofHIV-protease inhibitors in dogs po (5 mg/kg) and iv (2 mg/kg)
Compd
R
Cmax (mM)
t1=2 (min)
A.U.C. (mM h)
CLp (mL/min/kg)
% F
1
11
C(CH3)3
CH2CF3
8.8
17.0
62
52
7.4
18.8
14.2
4.01
81
65
Table 4. In vitro metabolic assays ofinvestigational HIV-protease inhibitors
a
Compd
CLint (mL/min/kg)
CYP3A4 IC50 (mM)
CYP2D6 IC50 (mM)
Indinavirb
1
11
0.15
0.31
0.03
>30.0
2.43
0.52
34 (64)
22 (50)
aMeasured in human liver microsomes.
bClearance values for Indinavir measured in tandem experiments are given in parentheses.
6. Castro, C. E.; Gaughan, E. J.; Owsley, D. C. J. Org. Chem.
1966, 31, 4071.
the t-butylamide analogue (1) is a potent inhibitor of
CYP3A4 in its own right (IC50=0.31 mM), the corre-
sponding trifluoroethylamide substituent confers over a
10-fold increase in potency against this metabolic
enzyme. This same increase in potency was observed in
7. The epoxide VII may be synthesized in direct analogy to
the aminoindanol derivative: Maligres, P. E.; Upadhyay, V.;
Rossen, K.; Cianciosi, S. J.; Purick, R. M.; Eng, K. K.;
Reamer, R. A.; Askin, D.; Reider, P. J. Tetrahedron Lett.
1995, 36, 2195.
8. Vacca, J. P.; Dorsey, B. D.; Schleif, W. A.; Levin, R. B.;
McDaniel, S. L.; Darke, P. L.; Zugay, 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.-W.; Vastag, K.; Ostovic, D.; Anderson, P. S.;
Emini, E. A.; Huff, J. R. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 4096.
the related metabolic enzyme CYP2D6. Indinavir is
15
a very weak inhibitor ofthis P450 isoofrm,
while
the t-butylamide (1) exhibits micromolar inhibition
potency, and the analogous trifluoroethylamide affords
almost a further 5-fold increase in potency (0.52 mM).
Although the P450 inhibitory properties ofcompound
11 may afford slower clearance ofthis compound in
vivo, they also greatly increase the likelihood ofcom-
plicating drug–drug interactions ifthis compound were
employed as part ofa clinical regimen.
9. Schock, H. B.; Garsky, V. M.; Kuo, L. C. J. Biol. Chem.
1996, 271, 31957.
10. Data for compound 11. 1H NMR (CDCl3, 400MHz) 9.42 (t,
J=4.8 Hz, 1H), 8.90 (s, 1H), 8.52 (d, J=6.0 Hz, 1H), 7.37 (d,
J=5.6 Hz, 1H), 7.30 (m, 5H), 7.11 (t, J=8.4 Hz, 1H), 7.06 (d,
J=7.6 Hz, 1H), 6.78 (m, 2H), 6.67 (s, 1H), 5.91 (d, J=8.4 Hz,
1H), 5.15 (dd, J=4.0 Hz, 1H), 4.27 (m, 1H), 4.06 (d, J=10.4 Hz,
1H), 4.00 (dd, J=4.8 Hz, J=11.6 Hz, 1H), 3.76 (m, 3H), 3.46 (s,
1H), 3.37 (s, 1H), 3.11 (d, J=11.6 Hz, 1H), 2.85 (m, 4H), 2.70
(m, 4H), 2.44 (m, 2H), 2.10 (d, J=5.2 Hz, 1H), 1.90 (t, J=11.2
Hz, 1H), 1.57 (s, 8H); HPLC-MS (ES) 724.6 (M+1).
11. (a) Olsen, D. B.; Stahlhut, M. W.; Rutkowski, C. A.;
Schock, H. B.; vanOlden, A. L.; Kuo, L. C. J. Biol. Chem.
1999, 274, 23699. (b) Condra, J. H.; Holder, D. J.; Schleif,
W. A.; Blahy, O. M.; Danovich, R. M.; Gabyelski, L. J.;
Graham, D. J.; Laird, D.; Quintero, J. C.; Rhodes, A.; Rob-
bins, H. L.; Roth, E.; Shivaprakash, M.; Yang, T.; Chodake-
witz, J. A.; Deutsch, P. J.; Leavitt, R. Y.; Massari, F. E.;
Mellors, J. W.; Squires, K. E.; Steigbigel, R. T.; Teppler, H.;
Emini, E. A. J. Virol. 1996, 70, 8270. The most highly cross-
resistant viral isolates reported in ref11b were chosen for this
investigation without regard to their amino acid substitution
pattern.
12. The use ofanimals was done under the purview ofan
Institutional Animal Care and Use Committee, and all applic-
able regulations and laws pertaining to the use oflaboratory
animals were followed. For experimental methods for both in
vitro and in 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.
13. Chiba, M.; Hensleigh, M.; Lin, J. H. Biochem. Pharmacol.
1997, 53, 1187.
Modification ofthe piperazine carboxamide substituent
on Indinavir and a related series ofcompounds can sig-
nificantly impact the inhibitory potency ofthe compounds
against the HIV protease enzyme. The trifluoro-
ethylamide substituent in compound 11 (Table 1) imparts
greater potency in halting the viral spread ofboth the
wild-type virus and a number ofPI-resistant variants of
HIV. This compound also exhibited more favorable phar-
macokinetic properties in vivo as compared to the analo-
gous t-butyalmide; however, this may be due to the
striking increase in inhibitory potency ofat least two
important P450 isoforms, CYP3A4 and CYP2D6.
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