Potent 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-based macrocyclic inhibitors of hepatitis C virus NS3 protease

Article abstract of DOI:10.1021/jm050520a

The NS3 protease of hepatitis C virus (HCV) has emerged as one of the best characterized targets for next-generation HCV therapy. The tetrapeptide 1 and pentapeptide 2 are α-ketoamide-type HCV serine protease inhibitors with modest potency. We envisioned

Full text of DOI:10.1021/jm050520a

J. Med. Chem. 2006, 49, 567-574
567
Potent 7-Hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid-Based Macrocyclic Inhibitors
of Hepatitis C Virus NS3 Protease
Kevin X. Chen,* F. George Njoroge, John Pichardo, Andrew Prongay, Nancy Butkiewicz, Nanhua Yao, Vincent Madison, and
Viyyoor Girijavallabhan
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-3-3545, Kenilworth, New Jersey 07033
ReceiVed June 3, 2005
The NS3 protease of hepatitis C virus (HCV) has emerged as one of the best characterized targets for
next-generation HCV therapy. The tetrapeptide 1 and pentapeptide 2 are R-ketoamide-type HCV serine
protease inhibitors with modest potency. We envisioned that the 1,2,3,4-tetrahydroisoquinoline-3-
carboxylamide (Tic) moiety could be cyclized to the P3 capping group. The resulting macrocycle could
enhance the binding through its extra contact with the Ala156 methyl group. Macrocyclization could also
provide a less peptidic HCV inhibitor. Synthesis started from dipeptide 5, which was obtained via a coupling
of two amino acid derivatives. The N-terminal was capped as hept-6-enoylamide to give 6. Hydroboration
of the double bond afforded alcohol 7, the precursor to the macrocycle 8. The macrocyclization was achieved
under Mitsunobu conditions (PPh3, ADDP). The macrocyclic acid 9 was then combined with appropriate
right-hand fragments 12, 14, or 16, which was prepared from common intermediate 11. Finally, oxidation
of R-hydroxyamide provided target molecule R-ketoamides 17, 18, and 21. The C-terminal esters were then
elaborated to carboxylic acids 19 and 20, and amides 20 and 23. The inhibitors 17-23 were tested in HCV
NS3 protease continuous assay. Tripeptide 17 was more potent than the larger acyclic tetrapeptide 1. The
tetrapeptides 18-20 were as active as 17. Most significantly, the pentapeptides (21-23) were much better
inhibitors (Ki* ) 0.015-0.26 µM). The carboxylic acid (22) and amide (23) were 57-80 times more potent
than the acyclic analogue 2. The X-ray crystal structure of compound 23 bound to the protease revealed
that the macrocycle adopted a donutlike conformation and had close contact with the Ala156 methyl group.
The ketone carbonyl formed a reversible covalent bond with Ser139. The n-propyl of P1 novaline and the
aromatic ring of P2′ phenylglycine formed a C-shaped clamp around the Lys136 side chain.
Introduction
The hepatitis C virus (HCV) is the principal etiologic agent
of chronic hepatitis C infection, which could lead to cirrhosis,
hepatocellular carcinoma, and, ultimately, liver failure. It has
infected more than 170 million people worldwide and is
emerging as a global health problem.1 Current standard therapy
involves pegylated R-interferon administered subcutaneously,
either alone or in combination with the broad-spectrum antiviral
agent ribavirin. These therapies have limited efficacy and
frequently are accompanied by serious side effects.2 Given the
prevalence of HCV infections, a more effective, convenient,
and well-tolerated small molecule drug is highly desirable.
Responsible for processing the nonstructural (NS) portion of
the polyprotein in hepatitis C virus, the virally encoded NS3
protease is located in the N-terminal portion of the NS3 protein.
Studies have confirmed that NS3 protease belongs to the trypsin
or chymotrypsin super family of serine protease.3 It is unique
Figure 1. Tetra- and pentapeptide HCV protease inhibitors.
in that it requires a cofactor protein NS4A for efficient
processing. The NS3 protease is essential for viral replication,4
and it has been a prime target for drug discovery.5 However,
the fact that the binding pockets of the NS3 protease are shallow,
solvent-exposed, and relatively featureless has made it a
challenging endeavor. Although a number of potent inhibitors
have been reported by various research laboratories around the
world,6 none has gone beyond phase II clinical trial.
the substrates,7 intensive research has been focused on various
derivatives of these peptide inhibitors. Our early efforts led to
moderately potent tetrapeptide inhibitor 1 and pentapeptide
inhibitor 2, with binding affinity (Ki*) of 15 and 1.2 µM,
respectively8 (Figure 1). Both compounds have tetrahydroiso-
quinoline-3-carboxylamide (Tic) as the P2 residue. The chal-
lenge was to enhance the potency while the peptidic nature of
these inhibitors was decreased. Peptides are known to be easily
cleaved by peptidases and thus suffer from low bioavailability
and poor pharmacological profiles.9 One promising approach
in depeptization is macrocyclization.10 Macrocycles are con-
formationally preorganized for enzyme binding and are not
Since the discovery of competitive inhibitors of the HCV
NS3/NS4 protease from the hexapeptide cleavage products of
* To whom correspondence should be addressed. Tel: (908) 740-7556.
Fax: (908) 740-7152. E-mail: kevin.chen@spcorp.com.
10.1021/jm050520a CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/20/2005

568 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Chen et al.
Scheme 1a
Scheme 2a
a Reaction conditions: (a) glyoxylic acid monohydrate, Et3N, MeOH, 0
°C, then rt, 99%; (b) H2, Pd-C, AcOH, rt, 66%; (c) (Boc)2O, dioxane/
H2O, rt, 89%; (d) NH4Cl, HOOBt, EDC, NMM, DMF -20 °C, 31%; (e) 4
M HCl, dioxane, rt, quantitative; (f) glycine benzyl ester hydrochloride,
HOOBt, EDC, NMM, DMF/CH2Cl2, -20 °C, 95%; (g) 4 M HCl, dioxane,
rt, quantitative; (h) H2, Pd-C, EtOH, rt, 98%; (i) phenylglycine tert-butyl
ester hydrochloride, HOOBt, EDC, NMM, DMF/CH2Cl2, -20 °C, 80%;
(j) 2 M HCl in EtOAc/dioxane (1:1), rt, quantitative.
a Reaction conditions: (a) HOOBt, EDC, NMM, DMF/CH2Cl2, -20 °C,
97%; (b) 4 M HCl, 1,4-dioxane; then 1-hept-6-enoic acid, HOOBt, EDC,
NMM, DMF/CH2Cl2, -20 °C, 31%; (c) BH3‚THF, THF, 0 °C, 93%; (d)
PPh3, ADDP, CH2Cl2, rt, 66%; (e) LiOH, MeOH/THF/H2O, rt, 98%.
easily recognized by peptidases. As a result, they could provide
better potency and stability.11 Using X-ray crystal structure data
of the enzyme, computer modeling of compounds 1 and 2 at
the enzyme active site indicated that the P2 moiety was close
to the P3 Boc capping group. We envisioned that macrocy-
clization from the P2 benzene ring to the P3 capping group
would not only provide additional binding but also effectively
depeptize the P2-P3 moiety. On the basis of molecular
modeling results, the optimal ring size was estimated to be 17.
The macrocycle formation could be accomplished through an
aryl-alkyl ether linkage created by a Mitsunobu reaction.12
Herein, we report the discovery of highly potent Tic-based
macrocyclic HCV NS3 protease inhibitors.
Preparation of the right-hand fragments of the inhibitors
started with synthesis of R-hydroxy-â-amino acid derivative 11
(Scheme 2). Thus, in the presence of triethylamine, 1-nitrobutane
reacted with glyoxylic acid to afford R-hydroxy-â-nitrohexanoic
acid 10. Low-pressure hydrogenation reduced the nitro group
to an amino group, which was protected as a Boc derivative.
The acid 11 was converted to primary amide, which, after
removal of Boc-protecting group, gave desired intermediate 12.
Similarly, coupling of 11 with glycine benzyl ester hydrochloride
provided dipeptide 13, which was then converted to intermediate
14. On the other hand, hydrogenation of benzyl ester 13
provided acid 15. Standard coupling of 15 with phenylglycine
tert-butyl ester furnished a tripeptide, whereupon Boc-depro-
tection, gave the desired intermediate 16.
Synthesis of Inhibitors
With synthesis of both left-hand and right-hand subunits
accomplished, time came for the assembly of the final targets.
Thus, macrocyclic acid 9 was coupled to amine 12 under
standard conditions (Scheme 3). The resulting R-hydroxyamide
was oxidized to tripeptide R-ketoamide 17 via a modified
Moffatt oxidation13 (DMSO, EDC, Cl2CHCO2H). The coupling
between 9 and dipeptide amine 14 provided an R-hydroxyamide
intermediate, which, upon Dess-Martin periodinane oxidation,14
afforded tetrapeptide R-ketoamide 18. The benzyl ester 18 was
converted to its corresponding carboxylic acid 19 through
catalytic hydrogenation. Amide bond formation from 19 fur-
nished the benzyl amide derivative 20. Similarly, coupling of
compounds 9 and 16 and subsequent oxidation gave pentapep-
tide R-ketoamide 21. When treated with trifluoroacetic acid, tert-
butyl ester 21 was converted to carboxylic acid 22. Finally,
dimethyl amide derivative 23 was obtained from acid 22 through
amide formation.
Synthesis of the macrocyclic left-hand portion of the inhibitors
is outlined in Scheme 1. Amino acids Boc-cyclohexylglycine
(3) and 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid (Tic) methyl ester hydrochloride (4) were coupled to give
dipeptide 5 under standard conditions (HOOBt, EDC, NMM).
After removal of the Boc protecting group, the resulting amine
hydrochloride was coupled to 1-hept-6-enoic acid to afford
compound 6. Hydroboration of the terminal alkene provided
primary alcohol 7. The key step, macrocyclization of this phenol
alcohol, was accomplished under Mitsunobu reaction12 condi-
tions using triphenylphosphine and 1,1′-(azodicarbonyl) dipip-
eridine (ADDP) in dichloromethane at room temperature.
Macrocyclic ester 8 was obtained in good yield (66%). To
complete the synthesis of the left-hand section of the target
molecule, the methyl ester was hydrolyzed to carboxylic acid
9.

Tic-Based Inhibitors of HCV NS3 Protease
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 569
Scheme 3a
Figure 2. X-ray crystal structure of compound 23 bound to HCV NS3
protease.
(0.26 µM). More encouraging was that the Ki* values of
carboxylic acid 22 and dimethyl carboxamide 23 were 15 and
21 nM, respectively.
Comparing the results of new macrocyclic inhibitors to that
of acyclic analogues, it was clear that macrocyclization signifi-
cantly enhanced potency. Tripeptide 17 was a smaller molecule
than acyclic tetrapeptide 1, yet it was a more effective inhibitor.
Compounds 18-20 were similar in size to 1, but the potency
was improved 5-9-fold. More dramatic enhancement was
observed from cyclization of acyclic pentapeptide 2 to macro-
cyclic compounds 22 and 23. The improvement in Ki* was 50-
80-fold.
Compound 23 was soaked with crystalline HCV NS3/4A
protease protein, and an X-ray crystal structure of the inhibitor
bound to the enzyme was obtained. The reversible covalent bond
between the hydroxyl of the enzyme active site serine (Ser139)
and the ketone carbonyl of the inhibitor was confirmed. The
most interesting feature, however, was that the macrocycle
encircled the methyl group of Ala156. This donutlike conforma-
tion provided additional interaction between the inhibitor and
the protease. The n-propyl side chain of P1 norvaline extended
deep inside the S1 pocket. On the prime side, the phenyl ring
of the P2′ residue extended over the far side of residue Lys136
in such a way that the P1 propyl group and P2′ phenyl group
formed a C-shaped clamp around the lysine side chain. There
were also several hydrogen-bonding interactions between the
inhibitor amide chain and the protease backbone. To examine
the pharmacokinetic properties of these macrocyclic inhibitors,
compound 23 was also selected for rat PK studies. It has good
AUC in rats (5.5 µM h) when the compounds were administered
subcutaneously. However, oral dosing gave a low AUC in rats
(0.27 µM‚h), which led a low bioavailability (<10%). The
results were not surprising, considering the fact that the
backbone of the molecule is still mostly peptidic in nature, even
though macrocyclization reduced some peptide characteristics
at the P2-P3 moiety. Further depeptization and designing of
smaller potent inhibitors are in progress.
a Reaction conditions: (a) HOOBt, EDC, NMM, DMF/CH2Cl2, -20 °C,
92%; (b) EDC, Cl2CHCO2H, DMSO/toluene, rt, 33%; (c) same as step a,
66%; (d) Dess-Martin periodinane, CH2Cl2, rt, 87%; (e) H2, Pd-C, EtOH/
MeOH, rt, 98%; (f) benzylamine, HOOBt, EDC, NMM, DMF/CH2Cl2, -20
°C, 53%; (g) same as step a, 80%; (h) same as step d, 88%; (i) CF3CO2H,
CH2Cl2, rt, quantitative; (j) Me2NH‚HCl, HOOBt, EDC, NMM, DMF/
CH2Cl2, -20 °C, 56%.
Table 1. Inhibitory Activity against HCV NS3 Protease
compd
Ki* (µM)
compd
Ki* (µM)
1
2
17
18
19
15
20
21
22
23
2.0
1.2
2.1
1.7
3.0
0.26
0.015
0.021
Results and Discussions
The macrocyclic R-ketoamides (17-23) were examined in a
continuous spectrophotometric assay15 for inhibitory activity
against the NS4A-tethered single chain NS3 serine protease.16
The assay was based on the proteolysis of chromogenic ester
substrates and was suitable for continuous monitoring of HCV
NS3 protease activity. The inhibition constants (Ki*)17 were
determined and are listed in Table 1. Due to the acidic nature
of the P1 R-proton, all the compounds were tested as a mixture
of two diastereomers. When pure P1 isomers were used,
extensive racemization was observed under assay conditions.
The small molecule tripeptide 17 was modestly potent (Ki* )
2.1 µM). The tetrapeptide inhibitors (18-20) had similar binding
affinity (Ki* ) 1.7-3.0 µM). Additional amino acid (P1′
glycine) did not provide much enhancement to potency.
However, when a second amino acid residue (P2′ phenylglycine)
was incorporated, the resulting pentapeptide inhibitors were
significantly more active against HCV protease. The Ki* of the
tert-butyl ester derivative (21) was in the sub-micromolar range
Conclusion
A short synthesis of a 17-membered macrocycle 8 has been
accomplished. The macrocyclization of the phenol alcohols was
achieved through a Mitsunobu protocol (PPh3, ADDP). The
macrocyclic acid 9 was coupled to mono-, di-, and tripeptide
right-hand intermediates 12, 14, and 16. The resulting R-hy-
droxyamides were oxidized to the final target R-ketoamides.
The inhibition constants (Ki*) from HCV protease continuous
assay demonstrated that these macrocyclic inhibitors were much

570 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Chen et al.
the preparation of compound 5. Flash chromatography (5-30%
EtOAc-CH2Cl2) of the crude product afforded 6 (2.30 g, 31%,
two steps) as a white solid. Two rotamers were observed (ca., 4:1
more potent (5-80-fold) than acyclic analogues of comparable
molecular weight. The pentapeptide acid 22 and dimethyl amide
23 were the most potent inhibitors, with Ki* values (15 and 21
nM, respectively) approaching single digit nanomolar range. The
X-ray structure of compound 23 bound to the enzyme revealed
that the macrocycle formed a donutlike shape around the methyl
group of Ala156. It also confirmed the formation of a covalent
bond between the ketone carbonyl and Ser139 hydroxyl. A
C-shaped clamp around the Lys136 side chain formed by P1
norvaline and P2′ phenylglycine was also observed.
1
ratio) according to H NMR spectra. The two sets of peaks only
partially collapsed when the NMR sample was heated to 90 °C.
They were, however, proved to be exchangeable protons in a 2D
NOESY experiment: 1H (500 MHz, DMSO-d6) δ 9.37 & 9.31 (s,
1 H), 8.12 & 8.09 (d, J ) 8.8 & 9.4 Hz, 1 H), 6.99 & 6.97 (d, J
) 8.2 & 8.2 Hz, 1 H), 6.64-6.54 (m, 2 H), 5.79-5.39 (m, 1 H),
5.05-4.89 (m, 4 H), 4.71 & 4.62 (t, J ) 8.8 & 9.2 Hz, 1 H), 4.51
& 4.23 (d, J ) 15.6 & 17.7 Hz, 1 H), 3.54 & 3.46 (s, 3 H), 3.12-
2.90 (m, 2 H), 2.16-1.93 (m, 4 H), 1.75-1.59 (m, 5 H), 1.50-
0.97 (m, 10 H); 13C NMR (125 MHz, DMSO-d6) δ 171.9, 171.6,
171.5, 171.3, 171.1, 170.7, 156.0, 155.9, 138.5, 134.0, 132.8, 129.1,
128.6, 122.5, 121.3, 114.6, 114.5, 114.1, 114.0, 112.4, 54.2, 52.9,
52.5, 52.2, 52.1, 51.9, 45.0, 43.1, 34.48, 34.46, 32.8, 32.7, 30.6,
29.4, 29.0, 28.6, 28.0, 27.9, 27.7, 27.6, 25.84, 25.78, 25.52, 25.46,
25.39, 25.35, 24.7; HRMS calcd for C26H37N2O5 (M + H)+
457.2702, found 457.2712.
Experimental Section
General Methods. Chemical reagents and solvents, including
anhydrous THF, dichloromethane, and DMF, were purchased from
Aldrich or other commercial sources and were used without further
purification. Reactions that were moisture sensitive or using
anhydrous solvents were performed under either a nitrogen or an
argon atmosphere. Analytical thin-layer chromatography (TLC) was
performed on precoated silica gel plates obtained from Analtech.
Visualization was accomplished with UV light or by staining with
basic KMnO4 solution, ethanolic H2SO4, or Vaughn’s reagent.
Compounds were purified by flash chromatography either on a glass
column using Merck silica gel 60 (230-400 mesh) or on an ISCO
RediSep disposable silica gel column. NMR spectra were recorded
at 300, 400, or 500 MHz for 1H NMR and at 75, 100, or 125 MHz
for 13C NMR, on a Bruker or Varian spectrometer with CDCl3 or
d6-DMSO as solvent. The chemical shifts are given in ppm,
referenced to the internal TMS or deuterated solvent signal.
2-(2-tert-Butoxycarbonylamino-2-cyclohexylacetyl)-7-hydroxy-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid Methyl Ester
(5). To a solution of N-Boc-cyclohexylglycine (3) (3.50 g, 12.7
mmol), 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
(Tic) methyl ester hydrochloride (4) (3.03 g, 12.4 mmol), 3-hy-
droxy-1,2,3-benzotriazin-4(3H)-one (HOOBt) (2.15 g, 13.2 mmol),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochlo-
ride (2.85 g, 14.9 mmol) in anhydrous DMF (100 mL), and CH2-
Cl2 (100 mL) at -20 °C was added 4-methylmorpholine (NMM)
(4.10 mL, 37.3 mmol). After being stirred at -20 °C for 30 min,
the reaction mixture was kept in a freezer overnight (18 h). It was
then warmed to room temperature. EtOAc (300 mL), brine (100
mL), and 5% H3PO4 (100 mL) were added and the layers were
separated. The organic solution was washed consecutively with 5%
H3PO4 (200 mL), saturated aqueous sodium bicarbonate solution
(2 × 200 mL), water (200 mL), and brine (200 mL); dried with
magnesium sulfate; filtered; and concentrated under reduced
pressure to afford dipeptide 5 (5.35 g, 97% yield) as a pale yellow
solid. An analytical sample was obtained through flash chroma-
tography (2-5% MeOH/CH2Cl2): 1H NMR (500 MHz, CDCl3) δ
6.87 (d, J ) 7.9 Hz, 1 H), 6.59 (d, J ) 2.2 Hz, 1 H), 6.56 (dd, J
) 8.0, 2.5 Hz, 1 H), 5.49 (d, J ) 9.2 Hz, 1 H), 5.42 (dd, J ) 6.0,
4.0 Hz, 1 H), 4.76 (d, J ) 5.5 Hz, 1 H), 4.67 (dd, J ) 9.2, 5.4 Hz,
1 H), 4.55 (d, J ) 5.5 Hz, 1 H), 3.60 (s, 3 H), 3.10 (dd, J ) 15.7,
4.0 Hz, 1 H), 2.89 (dd, J ) 15.7, 5.8 Hz, 1 H), 1.90-1.67 (m, 4
H), 1.44 (s, 9 H), 1.38-0.76 (m, 7 H); 13C NMR (125 MHz, CDCl3)
δ 171.9, 171.2, 156.3, 155.3, 132.7, 129.1, 123.0, 114.1, 112.9,
80.3, 55.1, 52.3, 51.9, 45.4, 41.0, 29.7, 29.6, 28.4, 28.3, 27.3, 26.2,
26.1, 26.0; HRMS calcd for C24H35N2O6 (M + H)+ 447.2492, found
447.2495.
2-[2-Cyclohexyl-2-(7-hydroxyheptanoylamino)acetyl]-7-hy-
droxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid Methyl
Ester (7). To the solution of 6 (2.20 g, 4.82 mmol) in anhydrous
THF (100 mL) under nitrogen at 0 °C was added borane-THF
solution (20 mL, 1.0 M, 20 mmol) cautiously. The mixture was
stirred at 0 °C under nitrogen for 1 h 40 min. Then, ethanol (10
mL) and pH 7 buffer (15 mL) were added, followed by 30%
aqueous hydrogen peroxide solution (15 mL). After 20 min, the
mixture was warmed to room temperature and stirred for 2 h. EtOAc
(400 mL) and brine (200 mL) were added and the two layers were
separated. Aqueous solution was extracted with EtOAc (2 × 150
mL). The combined organic solution was dried with magnesium
sulfate, filtrated, and concentrated in vacuo. Flash chromatography
(3-5% MeOH-CH2Cl2) afforded 7 (2.18 g, 4.47 mmol, 93%) as
a white solid. Similar to the NMR spectra of compound 6, two
1
rotamers were observed (ca., 4:1 ratio) in both H and 13C NMR
spectra at room temperature. The two sets of peaks only partially
collapsed in 1H NMR at 90 °C. They were, however, proved to be
exchangeable protons in a 2D NOESY experiment. One set of peaks
were observed in 13C NMR at 90 °C: 1H NMR (500 MHz, DMSO-
d6) δ 9.37 & 9.31 (s, 1 H), 8.09 & 8.07 (d, J ) 8.9 & 10.4 Hz, 1
H), 6.99 & 6.97 (d, J ) 8.2 & 8.2 Hz, 1 H), 6.63-6.54 (m, 2 H),
5.04-5.02 (d, J ) 5.4 & 5.4 Hz, 1 H), 4.70 & 4.61 (t, J ) 8.5 &
9.2 Hz, 1 H), 4.51 & 4.23 (d, J ) 15.4 & 17.9 Hz, 1 H), 4.33 (t,
J ) 5.2 Hz, 1 H), 3.54 & 3.46 (s, 3 H), 3.36 (s, 1 H), 3.35 & 3.32
(t, J ) 2.6 & 5.9 Hz, 2 H), 3.15-2.90 (m, 2 H), 2.14-1.99 (m, 2
H), 1.74-1.57 (m, 5 H), 1.49-1.31 (m, 4 H), 1.27-0.84 (m, 10
H); 13C NMR (125 MHz, DMSO-d6, 90 °C) δ 172.1, 171.4, 171.0,
156.0, 134.0, 128.5, 122.6, 114.3, 112.6, 60.8, 53.0, 52.3, 51.6,
45.1, 35.0, 32.3, 29.5, 28.9, 28.4, 28.0, 25.8, 25.5, 25.4, 25.2, 25.1;
HRMS calcd for C26H39N2O6 (M + H)+ 475.2812, found 475.2808.
3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo[11.5.3.016,20]-
henicosa-13(21),14,16(20)-triene-18-carboxylic Acid Methyl Es-
ter (8). A solution of phenol alcohol 7 (2.08 g, 4.38 mmol) and
1,1′-(azodicarbonyl)dipiperidine (ADDP) (3.00 g, 11.9 mmol) in
anhydrous CH2Cl2 was bubbled with argon through a frit-glass
bubbler for 20 min. To this solution at 0 °C was added triphen-
ylphosphine (3.45 g, 13.2 mmol). After stirring at 0 °C for 20 min,
the solution was warmed to room temperature and stirred overnight
(18 h) under argon. TLC indicated the presence of starting material.
A second batch of ADDP (3.00 g, 11.9 mmol) and triphenylphos-
pine (3.45 g, 13.2 mmol) was added, and the mixture was stirred
under argon for 2 days and 16 h. The reaction was concentrated
under reduced pressure. The residue was purified by flash chro-
matography (1-2% MeOH in CH2Cl2) to afford the macrocycle 8
(1.31 g, 66% yield): 1H NMR (500 MHz, CDCl3) δ 7.08 (d, J )
8.2 Hz, 1 H), 6.93 (d, J ) 2.5 Hz, 1 H), 6.79 (dd, J ) 8.2, 2.5 Hz,
1 H), 5.92 (d, J ) 10.0 Hz, 1 H), 5.22 (d, J ) 14.5 Hz, 1 H), 5.02
(t, J ) 10.3 Hz, 1 H), 4.52 (dd, J ) 11.9, 6.3 Hz, 1 H), 4.36-4.31
(m, 1 H), 4.31 (d, J ) 14.5 Hz, 1 H), 4.14-4.07 (m, 1 H), 3.77 (s,
3 H), 3.20 (dd, J ) 14.9, 6.3 Hz, 1 H), 2.88 (dd, J ) 14.4, 11.7
Hz, 1 H), 2.28 (ddd, J ) 14.9, 7.4, 3.1 Hz, 1 H), 2.01-1.95 (m, 1
2-(2-Cyclohexyl-2-hept-6-enoylaminoacetyl)-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic Acid Methyl Ester (6). The
dipeptide 5 (7.20 g, 16.1 mmol) was dissolved in 4 M HCl (100
mL, 400 mmol), and the resulting solution was stirred at room
temperature. The progress of the reaction was monitored by TLC.
After 4 h, the solution was concentrated and the residue was left
under vacuum overnight to give product amine hydrochloride as
an off-white solid: HRMS calcd for C19H27N2O4 (M + H)+
347.1971, found 347.1965. The desired product 6 was prepared
from the amine hydrochloride and 1-hept-6-enoic acid (2.90 g, 22.6
mmol) under similar coupling conditions as described above for

Tic-Based Inhibitors of HCV NS3 Protease
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 571
H), 1.87-1.62 (m, 10 H), 1.42-0.98 (m, 9 H); 13C NMR (125
MHz, CDCl3) δ 172.3, 171.8, 171.5, 158.0, 136.8, 128.0, 126.1,
116.5, 112.7, 66.9, 54.9, 52.8, 52.3, 46.2, 40.2, 35.1, 30.2, 29.5,
28.6, 27.5, 27.4, 26.4, 25.61, 25.57, 24.4, 24.0; HRMS calcd for
C26H37N2O5 (M + H)+ 457.2707, found 457.2702.
27.7, 19.4, 19.3, 13.2, 13.1; HRMS calcd for C11H22NO5 248.1498
(M + H)+, found 248.1510.
3-Amino-2-hydroxyhexanoic Acid Amide Hydrochloride (12).
Carboxylic acid 11 (1.50 g, 6.07 mmol) was converted to the
primary amide (0.462 g, 31% yield) under standard coupling
conditions as described above for the preparation of 5, except that
excess ammonium chloride (5 equivalent) was used as amine and
DMF was the only solvent used in the reaction: HRMS calcd for
C11H23N2O4 (M + H)+ 247.1658, found 247.1652. This product
was treated with 4 M hydrochloric acid in dioxane at room
temperature for 3 h. It was concentrated under reduced pressure to
give an pale yellow solid (quantitative yield) as a mixture of four
diastereomers, which was used in subsequent reactions without
further purification: HRMS calcd for C6H15N2O2 (M + H)+
147.1134, found 147.1139.
(3-tert-Butoxycarbonylamino-2-hydroxyhexanoylamino)ace-
tic Acid Benzyl Ester (13). The coupling of carboxylic acid 11
(3.00 g, 12.0 mmol) and glycine benzyl ester hydrochloride (2.56
g, 13.0 mmol) was carried out in a manner similar to that described
above for the preparation of 5. The crude product was purified by
flash chromatography (25-50% acetone/hexanes) to afford the
dipeptide product (4.50 g, 95%) as a mixture of four diastereo-
mers: 1H NMR (500 MHz, DMSO-d6) δ 8.21 & 8.18 (t, 1 H, J )
6.5 Hz), 7.40-7.33 (m, 5 H), 6.39 & 5.94 (d, J ) 9.0, 9.5 Hz, 1
H), 5.89 &5.76 (d, J ) 6, 5.5 Hz, 1 H), 5.14 (bs, 2 H), 3.99-3.70
(m, 4 H), 1.39 &1.36 (s, 9 H), 1.48-1.07 (m, 4 H), 0.85 & 0.77 (t,
J ) 7.0, 6.5 Hz, 3 H); 13C NMR (125 MHz, DMSO-d6), δ, 173.9,
173.2, 170.5, 170.4, 156.1, 136.8, 129.3, 128.9, 128.8, 128.7, 78.5,
78.4, 74.5, 73.1, 66.6, 53.5, 41.4, 30.5, 29.1, 29.0, 19.8, 19.7, 14.7,
14.6; LRMS m/z MH+ ) 395; HRMS calcd for C20H31N2O6
395.2182 (M + H)+, found 395.2199.
3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo[11.5.3.016,20]-
henicosa-13(21),14,16(20)-triene-18-carboxylic Acid (9). A solu-
tion of lithium hydroxide (0.21 g, 8.75 mmol) in water (30 mL)
was added to a solution of methyl ester 8 (1.30 g, 2.85 mmol) in
THF (30 mL) and methanol (30 mL) at 0 °C. The mixture was
allowed to warm to room temperature along with the ice bath in 4
h. The progress of the reaction was monitored by TLC. The mixture
was concentrated under reduced pressure to one-third of its original
volume. EtOAc (50 mL) and water (30 mL) were added and the
two layers were separated. The aqueous solution was washed with
CH2Cl2 (50 mL), after which it was acidified to pH 1 using 2 M
aqueous hydrochloric acid. EtOAc was then added (100 mL) and
the aqueous solution was saturated with solid sodium chloride. After
separation of the layers, the aqueous layer was extracted with EtOAc
(2 × 60 mL). Organic solutions were combined, dried with
magnesium sulfate, filtered, and concentrated in vacuo to afford 9
(1.23 g, 98% yield) as an off-white solid, which was used without
further purification: 1H NMR (500 MHz, DMSO-d6) δ 12.64 (bs,
1 H), 7.99 (d, J ) 9.9 Hz, 1 H), 7.16 (d, J ) 8.2 Hz, 1 H), 6.80
(dd, J ) 8.2, 2.6 Hz, 1 H), 6.75 (d, J ) 2.2 Hz, 1 H), 5.10 (d, J )
14.8 Hz, 1 H), 4.74 (t, J ) 10.0 Hz, 1 H), 4.32 (dd, J ) 11.3, 6.4
Hz, 1 H), 4.25 (d, J ) 14.4 Hz, 1 H), 4.22-4.18 (m, 2 H), 4.07-
4.03 (m, 1 H), 3.16 (dd, J ) 14.8, 6.3 Hz, 1 H), 2.84 (dd, J )
14.4, 11.3 Hz, 1 H), 2.0.05-2.04 (m, 1H), 1.83-1.77 (m, 1 H),
1.69-1.47 (m, 8 H), 1.35-0.82 (m, 11 H); 13C NMR (125 MHz,
DMSO-d6) δ 172.5, 171.7, 169.9, 157.0, 137.0, 128.2, 126.6, 115.7,
112.5, 66.4, 54.2, 52.2, 44.8, 33.6, 30.3, 29.4, 29.1, 27.9, 27.3, 27.0,
26.0, 25.3, 25.2, 24.3, 23.8; HRMS calcd for C25H35N2O5 (M +
H)+ 443.2546, found 443.2558.
3-tert-Butoxycarbonylamino-2-hydroxyhexanoic Acid (11). To
a stirred solution of 1-nitrobutane (16.5 g, 0.160 mol) and glyoxylic
acid monohydrate (28.1 g, 0.305 mol) in MeOH (120 mL) at 0 °C
was added triethylamine (93.0 mL, 0.667 mol) dropwise over 2 h.
The solution was warmed to room temperature, stirred overnight,
and concentrated to give an oil. The oil was dissolved in water
(100 mL), and the solution was acidified to pH 1 with 10% aqueous
HCl solution and extracted with EtOAc (3 × 200 mL). The
combined organic solution was washed with brine, dried over Na2-
SO4, filtered, and concentrated to dryness to give the 3-nitro-2-
hydroxyhexanoic acid (28.1 g, 99% yield). To a stirred solution of
the nitro acid (240 g, 1.35 mol) in acetic acid (1.25 L) was added
10% Pd/C (37 g). The resulting solution was hydrogenated at 59
psi for 20 h. The acetic acid was then evaporated and the residue
was azeotroped three times with toluene. It was then triturated with
MeOH and ether. The solid that formed was separated by filtration
and azeotroped twice with toluene to give the amino acid product
as an off-white solid (131 g, 66% yield). To a stirred solution of
this amino acid (2.0 g, 13.6 mmol) in dioxane (10 mL) and H2O (5
mL) at 0 °C was added 1 N aqueous NaOH solution (14.0 mL,
14.0 mmol). After stirring for 10 min, di-tert-butyl dicarbonate (3.10
g, 14.0 mmol) was added and the mixture was stirred for 15 min
at 0 °C. It was then warmed to room temperature and stirred for an
additional 45 min before it was placed in a refrigerator overnight.
The reaction was then concentrated to dryness. The residue was
dissolved in EtOAc (100 mL). Ice (∼50 g), KHSO4 (3.36 g), and
H2O (32 mL) were added, and the mixture was stirred for 5 min.
The layers were then separated, the aqueous layer was extracted
twice with EtOAc (2 × 50 mL), and the combined organic solution
was washed with water and brine, dried (Na2SO4), filtered, and
concentrated to dryness to yield the product 11 (3.0 g, 89% yield)
as a mixture of four diastereomers: 1H NMR (500 MHz, CD3-
OD,) δ, 4.19 & 4.15 (d, J ) 4, 2.5 Hz, 1 H), 3.98-3.95 & 3.91-
3.90 (m, 1 H), 1.59-1.35 (m, 10 H), 1.46 & 1.43 (s, 9 H), 0.98 &
0.94 (t, 3 H, J ) 7.0 Hz).13C NMR (125 MHz, CD3OD) δ, 175.2,
174.8, 157.1, 156.9, 79.1, 73.3, 72.1, 53.2, 42.9, 34.1, 31.1, 27.8,
(3-Amino-2-hydroxyhexanoylamino)acetic Acid Benzyl Ester
Hydrochloride (14). Compound 12 was treated with 4 M hydro-
chloric acid in dioxane at room temperature for 3 h. It was
concentrated under reduced pressure to give an off-white solid as
a mixture of four diastereomers (quantitative yield), which was used
in subsequent reactions without further purification: HRMS calcd
for C15H23N2O4 295.1658 (M + H)+, found 295.1654.
(3-tert-Butoxycarbonylamino-2-hydroxyhexanoylamino)ace-
tic Acid (15). To a solution of the benzyl ester 13 (4.50 g, 11.4
mmol) in ethanol (50 mL) was added 5% Pd-C (1.0 g). The
mixture was stirred vigorously under a hydrogen atmosphere at
room temperature for 3 h before it was filtered through a Celite
pad. The filter cake was washed with EtOAc (2 × 30 mL). The
solution was concentrated under reduced pressure to afford the
product 15 (3.40 g, 98% yield) as a mixture of four diastereomers:
HRMS calcd for C13H25N2O6 (M + H)+ 305.1713, found 305.1699.
[2-(3-Amino-2-hydroxyhexanoylamino)acetylamino]phenyl-
acetic Acid tert-butyl Ester Hydrochloride (16). The coupling
of carboxylic acid 15 (2.00 g, 6.57 mmol) and phenyl glycine tert-
butyl ester hydrochloride (1.76 g, 6.57 mmol) was carried out in a
manner similar to that described above for the preparation of 5.
The crude product was purified by flash chromatography (1:1
EtOAc/hexanes) to afford a tripeptide product (2.60 g, 80%) as a
mixture of four diastereomers: 1H NMR (500 MHz, DMSO-d6) δ
8.64 & 8.58 (d, J ) 7.0 Hz, 1 H), 7.91 &7.87 (m, 1 H), 7.42-7.32
(m, 5 H), 6.40 & 5.99 (dd, J ) 4.5 & 5.00 Hz, 1 H), 5.77-5.73
(m, 1 H), 5.30-5.28 (m, 1 H), 3.97-3.70 (m, 4 H), 1.51-1.08
(m, 22 H), 0.86 & 0.79 (t, J ) 6.0 Hz, 3 H); 13C NMR (125 MHz,
DMSO-d6) δ 173.4, 172.8, 170.3, 169.4, 169.3, 155.7, 137.5, 129.5,
129.0, 128.3, 128.2, 82.2, 82.1, 78.4, 74.6, 73.2, 57.7, 53.4, 42.3,
33.7, 29.1, 29.0, 28.3, 19.7, 14.7, 14.6; HRMS calcd for C25H40N3O7
494.2866 (M + H)+, found 494.2863. A solution of this tert-butyl
ester (2.6 g, 5.30 mmol) in 2 M HCl in EtOAc/dioxane (1:1) was
stirred at room temperature for 15 min. The reaction mixture was
concentrated in vacuo to yield 16 (quantitative yield) as a pale
yellow solid which was used in subsequent reactions without further
purification: HRMS calcd for C20H32N3O5 394.2342 (M + H)+,
found 394.2336.
3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo [11.5.3.016,20]-
henicosa-13(21),14,16(20)-triene-18-carboxylic Acid (1-Ami-

572 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Chen et al.
nooxalylbutyl)amide (17). Coupling of acid 9 (59 mg, 0.133 mmol)
and amine hydrochloride 12 (34 mg, 0.187 mmol) was carried out
in a manner similar to that described above for the preparation of
5. The intermediate product was obtained as a mixture of diaster-
eomers (70 mg, 92% yield). To the solution of this product and
EDC (235 mg, 1.23 mmol) in DMSO/toluene (1:1, 10 mL) at 0 °C
was added dichloroacetic acid (0.055 mL, 0.667 mmol) dropwise.
The mixture was stirred at 0 °C for 15 min and at room temperature
for 2 h. EtOAc (100 mL) was added and the mixture was washed
with 5% H3PO4 (2 × 60 mL), saturated aqueous sodium bicarbonate
solution (2 × 60 mL), and brine (60 mL). It was then dried with
magnesium sulfate, filtered, and concentrated in vacuo. Flash
chromatography (3-6% MeOH/CH2Cl2) afforded 16 (25 mg, 33%
yield) as a mixture of diastereomers. The ratio of the two
diastereomers was variable and dependent on the reaction condi-
tions: 1H NMR (500 MHz, DMSO-d6) δ 8.21 & 8.08 (d, J ) 7.3
& 7.3 Hz, 1 H), 8.00 (s, 1 H), 7.91 (t, J ) 9.6 Hz, 1 H), 7.87 (d,
J ) 10.4 Hz, 1 H), 7.13 (t, J ) 8.2 Hz, 1 H), 6.82-6.79 (m, 1 H),
6.74 (dd, J ) 11.0, 2.4 Hz, 1 H), 5.09-4.98 (m, 1 H), 4.74 (ABq,
J ) 9.8 Hz, 1 H), 4.38 & 4.36 (dd, J ) 8.9, 6.5 & 9.5, 6.5 Hz, 1
H), 4.30 & 4.21 (d, J ) 14.7 & 14.7 Hz, 1 H), 4.21-4.18 (m, 1
H), 4.06-4.03 (m, 1 H), 3.14-3.09 (m, 1 H), 2.75 (ddd, J ) 15.5,
11.2, 5.7 Hz, 1 H), 2.06 (t, J ) 5.7 Hz, 2 H), 1.80-1.45 (m, 12
H), 1.43-0.82 (m, 14 H); 13C NMR (125 MHz, DMSO-d6) δ 197.6,
197.5, 171.72, 171.69, 171.1, 171.0, 170.2, 169.8, 163.1, 163.0,
157.04, 156.96, 137.4, 137.2, 128.0, 127.8, 127.0, 126.8, 115.7,
115.6, 112.6, 112.5, 66.7, 66.6, 54.9, 54.7, 53.2, 53.1, 52.5, 52.4,
45.4, 45.3, 33.5, 33.4, 31.74, 31.65, 30.0, 29.9, 29.6, 29.4, 28.1,
28.0, 27.2, 27.1, 27.0, 26.9, 26.0, 25.34, 25.28, 24.1, 23.9, 23.8,
23.7, 18.63, 18.55, 13.5, 13.4; HRMS calcd for C31H45N4O6 (M +
H)+ 569.3339, found 569.3336.
{3-[(3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo[11.5.3.016,20]-
henicosa-13(21),14,16(20)-triene-18-carbonyl)amino]-2-oxohexa-
noylamino}acetic Acid Benzyl Ester (18). Coupling of carboxylic
acid 9 (150 mg, 0.339 mmol) and amine hydrochloride 14 (155
mg, 0.468 mmol) was carried out in a manner similar to that
described above for the preparation of compound 5. After purifica-
tion by flash chromatography (2-5% MeOH/CH2Cl2), the inter-
mediate product (160 mg, 66% yield) was obtained as a mixture
of diastereomers. To the solution of this product (150 mg, 0.209
mmol) in anhydrous CH2Cl2 (80 mL) at room temperature was
added Dess-Martin periodinane (0.220 g, 0.519 mmol). The
mixture was stirred for 4 h. Saturated aqueous sodium bicarbonate
and sodium thiosulfate solutions (40 mL each) were added. After
stirring for 10 min, the layers were separated. The aqueous solution
was extracted with CH2Cl2 (2 × 60 mL). The combined organic
solution was dried with magnesium sulfate, filtered, and concen-
trated in vacuo. Flash chromatography (1-5% MeOH/CH2Cl2)
afforded 18 (130 mg, 87% yield) as a mixture of diastereomers.
The ratio of the two diastereomers was variable and dependent on
the reaction conditions: 1H NMR (500 MHz, DMSO-d6) δ 9.08 (t,
J ) 6.0 Hz, 1 H), 8.27 & 8.14 (d, J ) 6.8 & 7.1 Hz, 1 H), 7.92 (t,
J ) 9.5 Hz, 1 H), 7.39-7.32 (m, 5 H), 7.13 (dd, J ) 10.6, 8.4 Hz,
1 H), 6.80 (ddd, J ) 8.4, 6.0, 2.4 Hz, 1 H), 6.74 (dd, J ) 10.2, 2.4
Hz, 1 H), 5.17-5.10 (m, 2 H), 5.09-5.00 (m, 2 H), 4.74 (dd, J )
18.7, 10.0 Hz, 1 H), 4.40-4.35 (m, 1 H), 4.33-4.18 (m, 2 H),
4.06-4.00 (m, 1 H), 3.99-3.93 (m, 1H), 3.11 (dd, J ) 14.8, 6.3
Hz, 1 H), 2.78-2.71 (m, 1 H), 2.07-2.05 (m, 2 H), 1.77-1.46
(m, 10 H), 1.42-0.84 (m, 16 H); 13C NMR (125 MHz, DMSO-d6)
δ 196.5, 196.4, 171.74, 171.69, 171.2, 171.1, 170.2, 169.8, 168.7,
161.2, 161.1, 157.1, 157.0, 137.4, 137.3, 135.7, 128.3, 128.03,
127.99, 127.92, 127.86, 127.83, 127.0, 126.8, 115.7, 115.6, 112.54,
112.50, 66.7, 66.6, 66.0, 55.0, 54.7, 53.4, 53.3, 52.6, 51.8, 45.3,
40.6, 33.5, 33.4, 31.6, 31.46, 31.45, 31.2, 30.0, 29.9, 29.6, 29.5,
28.1, 28.0, 27.2, 27.1, 27.0, 26.9, 26.01, 26.02, 25.34, 25.29, 24.1,
23.9, 23.8, 23.7, 18.6, 18.5, 13.5, 13.4; HRMS calcd for C40H52N4O8
(M + H)+ 717.3863, found 717.3859.
was added 5% Pd-C (0.50 g). The mixture was stirred vigorously
under hydrogen at room temperature for 3 h before it was filtered
through a 1 in. Celite pad. The filter cake was washed with EtOAc
(2 × 30 mL). The solution was concentrated under reduced pressure
to afford the product 19 (103 mg, 98% yield) as a mixture of
diastereomers: 1H NMR (500 MHz, DMSO-d6) δ 12.7 (bs, 1 H),
8.88 (t, J ) 6.0 Hz, 1 H), 8.26 & 8.13 (d, J ) 7.0 & 7.5 Hz, 1 H),
7.92 (t, J ) 9.4 Hz, 1 H), 7.14 (dd, J ) 12.0, 8.2 Hz, 1 H), 6.80
(td, J ) 7.2, 2.4 Hz, 1 H), 6.74 (dd, J ) 9.5, 2.4 Hz, 1 H), 5.09-
5.01 (m, 2 H), 4.74 (ABq, J ) 9.7 Hz, 1 H), 4.39-4.34 (m, 1 H),
4.33-4.18 (m, 2 H), 3.76 (d, J ) 6.0 Hz, 2 H), 3.11 (dd, J ) 15.0,
6.3 Hz, 1 H), 2.75 (ddd, J ) 15.0, 11.2, 7.0 Hz, 1 H), 2.07-2.05
(m, 2 H), 1.79-1.01 (m, 21 H), 0.94-0.79 (m, 5 H); 13C NMR
(125 MHz, DMSO-d6) δ 196.8, 196.7, 171.74, 171.69, 171.2, 171.1,
170.2, 169.8, 161.0, 160.9, 157.1, 157.0, 137.4, 137.3, 128.0, 127.9,
126.8, 126.5, 115.6, 112.5, 66.7, 66.6, 56.2, 55.0, 54.7, 53.4, 53.3,
52.6, 45.3, 40.5, 33.5, 33.4, 31.6, 31.5, 30.0, 29.9, 29.6, 29.4, 28.1,
28.0, 27.2, 27.1, 27.0, 26.9, 26.3, 26.0, 25.34, 25.29, 24.1, 23.8,
23.7, 18.7, 18.6, 13.5, 13.4; LRMS m/z (M + H)+ ) 627.3.
3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo[11.5.3.016,20]-
henicosa-13(21),14,16(20)-triene-18-carboxylic Acid [1-(Benzyl-
carbamoylmethyl)aminooxalyl]butyl)amide (20). The coupling
of carboxylic acid 19 (90 mg, 0.144 mmol) and benzylamine (0.020
mL, 0.183 mmol) was carried out in a manner similar to that
described above for the preparation of compound 5. The product
was purified by flash chromatography (2-5% MeOH/CH2Cl2) to
afford 20 (55 mg, 53% yield) as a mixture of diastereomers: 1H
NMR (500 MHz, DMSO-d6) δ 8.83-8.79 (m, 1 H), 8.44 (dd, J )
10.3, 5.5 Hz, 1 H), 8.27 & 8.13 (d, J ) 7.6 & 6.6 Hz, 1 H), 7.93
(t, J ) 10.2 Hz, 1 H), 7.34-7.23 (m, 5 H), 7.14 (t, J ) 8.7 Hz, 1
H), 6.83-6.80 (m, 1 H), 6.76-6.74 (m, 1 H), 5.11-5.03 (m, 2 H),
4.75 (ABq, J ) 10.5 Hz, 1 H), 4.41-4.36 (m, 1 H), 4.33-4.19
(m, 4 H), 4.07-4.04 (m, 1 H), 3.82 (d, J ) 6.1 Hz, 1 H), 3.12 (td,
J ) 14.4, 6.2 Hz, 1 H), 2.75 (t, J ) 13.1 Hz, 1 H), 2.08 (s, 2 H),
1.77-1.25 (m, 18 H), 1.14-0.87 (8 H); 13C NMR (125 MHz,
DMSO-d6) δ 196.4, 196.3, 171.73, 171.69, 171.2, 171.0, 170.2,
170.0, 167.7, 160.8, 160.7, 157.1, 157.0, 139.1, 137.4, 137.3, 128.1,
128.0, 127.8, 127.1, 127.0, 126.8, 126.7, 115.7, 115.5, 112.54,
112.52, 66.7, 66.6, 55.0, 54.7, 53.3, 53.2, 45.4, 45.3, 42.0, 41.9,
41.8, 33.5, 33.4, 31.88, 31.85, 29.97, 29.93, 29.6, 29.4, 28.1, 28.0,
27.2, 27.1, 27.0, 26.9, 26.0, 25.33, 25.29, 24.1, 23.91, 23.90, 23.8,
23.7, 18.6, 18.5, 13.5, 13.4; HRMS calcd for C40H54N5O7 (M +
H)+ 716.4023, found 716.4021.
(2-{3-[(3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo[11.
5.3.016,20]henicosa-13(21),14,16(20)-triene-18-carbonyl)amino]-
2-oxohexanoylamino}acetylamino)phenylacetic Acid tert-Butyl
Ester (21). The coupling of carboxylic acid 9 (390 mg, 0.881 mmol)
and amine hydrochloride 16 (390 mg, 0.907 mmol) was carried
out in a manner similar to that described above for the preparation
of 5. The intermediate product (580 mg, 80% yield) was obtained
as a mixture of diastereomers. To the solution of this product (0.360
g, 0.440 mmol) in anhydrous CH2Cl2 (80 mL) at room temperature
was added Dess-Martin reagent (0.467 g, 1.10 mmol). The mixture
was stirred for 4 h. Saturated aqueous sodium bicarbonate and
sodium thiosulfate solutions (50 mL each) were added. After stirring
for 10 min, the layers were separated. The aqueous solution was
extracted with CH2Cl2 (2 × 80 mL). The organic solutions were
combined, dried with magnesium sulfate, filtered, and concentrated
in vacuo. Flash chromatography (2-5% MeOH/CH2Cl2) afforded
21 (0.316 g, 88% yield) as a mixture of diastereomers. The ratio
of the two diastereomers was variable and dependent on the reaction
conditions: 1H NMR (500 MHz, DMSO-d6) δ 8.74 (ABq, J ) 5.9
Hz, 1 H), 8.70 (t, J ) 8.0 Hz, 1 H), 8.25 & 8.11 (d, J ) 6.9 & 8.0
Hz, 1 H), 7.92 (t, J ) 10.0 Hz, 1 H), 7.40-7.33 (m, 5 H), 7.13
(dd, J ) 10.4, 8.1 Hz, 1 H), 6.81 (ddd, J ) 7.8, 5.2, 2.6 Hz, 1 H),
6.74 (dd, J ) 9.0, 2.3 Hz, 1 H), 5.26 (t, J ) 7.4 Hz, 1 H), 5.09-
5.01 (m, 2 H), 4.74 (ABq, J ) 10.0 Hz, 1 H), 4.36 (td, J ) 11.3,
5.6 Hz, 1 H), 4.31 & 4.22 (d, J ) 14.5 & 14.9 Hz, 1 H), 4.21-
4.19 (m, 1 H), 4.06-4.02 (m, 1 H), 3.92-3.80 (m, 2 H), 3.11 (ddd,
J ) 14.8, 6.3, 2.74 (ddd, J ) 14.8, 11.2, 8.0 Hz, 1 H), 2.07-2.05
(m, 2 H), 1.77-1.42 (m, 12 H), 1.34 & 1.33 (s, 9 H), 1.41-1.23
{3-[(3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo [11.5.3.
016,20]henicosa-13(21),14,16(20)-triene-18-carbonyl)amino]-2-
oxohexanoylamino}acetic Acid (19). To a solution of the benzyl
ester 18 (120 mg, 0.167 mmol) in ethanol/methanol (2:1, 45 mL)

Tic-Based Inhibitors of HCV NS3 Protease
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 573
References
(m, 4 H), 1.21-0.91 (m, 5 H), 0.89-0.82 (m, 5 H); 13C NMR
(125 MHz, DMSO-d6) δ 197.6, 197.5, 172.7, 172.6, 172.1, 172.0,
171.3, 171.1, 170.8, 170.3, 168.49, 168.47, 161.8, 161.7, 158.0,
157.9, 138.4, 138.3, 137.4, 132.91, 132.89, 132.4, 132.3, 129.7,
129.6, 129.5, 129.0, 128.8, 128.3, 128.0, 127.8, 116.6, 116.5,
113.49, 113.47, 82.2, 82.1, 67.6, 67.5, 57.8, 57.7, 56.0, 55.7, 54.3,
54.2, 46.3, 42.31, 42.27, 34.48, 34.46, 34.3, 32.65, 32.60, 32.1,
30.954, 30.947, 30.9, 30.52, 30.51, 30.4, 29.04, 28.99, 28.3, 28.2,
28.1, 28.0, 27.9, 27.0, 26.3, 26.2, 25.1, 24.9, 24.7, 24.6, 19.6, 19.5,
14.43, 14.35; HRMS calcd for C45H62N5O9 (M + H)+ 816.4548,
found 816.4554.
(1) (a) Cohen, J. The Scientific Challenge of Hepatitis C. Science 1999,
285, 26-30. (b) Alter, M. J.; Kruszon-Moran, D.; Nainan, O. V.;
McQuillan, G. M.; Gao, F.; Moyer, L. A.; Kaslow, R. A.; Margolis,
H. S. The Prevalence of Hepatitis C Virus Infection in The United
States, 1988 Through 1994. N. Engl. J. Med. 1999, 341, 556-562.
(c) Cuthbert, J. A. Hepatitis C: Progress and Problems. Clin.
Microbiol. ReV. 1994, 7, 505-532.
(2) (a) Neumann, A. U.; Lam, N. P.; Dahari, H.; Gretch, D. R.; Wiley,
T. E.; Layden, T. J.; Perelson, A. S. Hepatitis C Virus Dynamics in
vivo and the Antiviral Efficacy of Interferon-R Therapy. Science
1998, 282, 103-107. (b) Rosen, H. R.; Gretch, D. R. Hepatitis C
Virus: Current Understanding and Prospects for Future Therapies.
Mol. Med. Today 1999, 5, 393-399. (c) Di Bisceglie, A. M.;
McHutchison, J.; Rice, C. M. New therapeutic strategies for hepatitis
C. Hepatology 2002, 35, 224-231.
(3) (a) Kwong, A. D.; Kim, J. L.; Rao, G.; Lipovsek, D.; Raybuck, S.
A. Hepatitis C Virus NS3/4A Protease. AntiViral Res. 1998, 40, 1-18.
(b) Yan, Y.; Li, Y.; Munshi, S.; Sardana, V.; Cole, J. L.; Sardana,
M.; Steinkuehler, C.; Tomei, L.; De Francesco, R.; Kuo, L. C.; Chen,
Z. Complex of NS3 protease and NS4A Peptide of BK Strain
Hepatitis C Virus: A 2.2 Å Resolution Structure in a Hexagonal
Crystal Form. Protein Sci. 1998, 7, 837-847.
(4) Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C. M.
Hepatitis C Virus-Encoded Enzymatic Activities and Conserved RNA
Elements in the 3′ Nontranslated Region Are Essential for Virus
Replication In Vivo. J. Virol. 2000, 74, 2046-2051.
(2-{3-[(3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo[11.
5.3.016,20]henicosa-13(21),14,16(20)-triene-18-carbonyl)amino]-
2-oxohexanoylamino}acetylamino)phenylacetic Acid (22). A
solution of tert-butyl ester 21 (210 mg, 0.257 mmol) in trifluoro-
acetic acid (15 mL) and CH2Cl2 (15 mL) was stirred at room
temperature for 4 h. The mixture was concentrated in vacuo. The
residue was redissolved in 1:1 MeOH/CH2Cl2 (10 mL) and
concentrated to dryness to afford product 22 (198 mg, quantitative)
as a mixture of diastereomers: 1H NMR (500 MHz, DMSO-d6) δ
8.74 (t, J ) 6.1 Hz, 1 H), 8.63 (d, J ) 6.9 Hz, 1 H), 8.25 & 8.11
(d, J ) 7.0 & 7.6 Hz, 1 H), 7.91 (t, J ) 9.4 Hz, 1 H), 7.38-7.33
(m, 5 H), 7.31-7.28 (m, 1 H), 7.13 (t, J ) 9.1 Hz, 1 H), 6.80
(ddd, J ) 7.5, 5.0, 2.5 Hz, 1 H), 6.74 (dd, J ) 8.2, 2.2 Hz, 1 H),
5.25 (t, J ) 7.5 Hz, 1 H), 5.08-5.01 (m, 2 H), 4.74 (ABq, J )
10.2 Hz, 1 H), 4.36 (td, J ) 11.4, 6.3 Hz, 1 H), 4.31 & 4.22 (d, J
) 14.8 & 14.5 Hz, 1 H), 4.20-4.18 (m, 1 H), 4.06-4.03 (m, 1 H),
3.86 (ddd, J ) 20.3, 16.5, 6.3 Hz, 2 H), 3.11 (ddd, J ) 14.8, 6.2,
4.1 Hz, 1 H), 2.74 (ddd, J ) 14.8, 11.2, 8.3 Hz, 1 H), 2.06 (t, J )
6.0 Hz, 2 H), 1.77-1.45 (m, 12 H), 1.44-1.21 (m, 4 H), 1.19-
1.03 (m, 5 H), 0.91-0.80 (m, 5 H); 13C NMR (125 MHz, DMSO-
d6) δ 196.7, 196.5, 171.73, 171.68, 171.5, 171.2, 171.1, 170.2,
169.8, 167.1, 160.8, 160.7, 157.1, 157.0, 137.4, 137.3, 128.3,
128.24, 128.18, 128.0, 127.8, 127.6, 127.5, 127.30, 127.27, 127.1,
126.8, 115.7, 115.6, 112.53, 112.52, 66.7, 66.6, 56.5, 55.0, 54.7,
53.4, 53.2, 52.4, 48.5, 45.4, 45.3, 41.5, 33.5, 33.4, 31.7, 31.6, 30.0,
29.9, 29.6, 29.4, 28.1, 28.0, 27.2, 27.1, 27.0, 26.9, 26.0, 25.34,
25.27, 24.1, 23.9, 23.8, 23,7, 18.6, 18.5, 13.5, 13.4; HRMS calcd
for C41H54N5O9 (M + H)+ 760.3922, found 760.3943.
3-Cyclohexyl-2,5-dioxo-12-oxa-1,4-diazatricyclo[11.5.3.016,20]-
henicosa-13(21),14,16(20)-triene-18-carboxylic Acid [1-(Dimeth-
ylcarbamoylphenylmethyl)carbamoyl]methyl)aminooxalyl)butyl)-
amide (23). The coupling of carboxylic acid 22 (50 mg, 0.0658
mmol) and dimethylamine hydrochloride (10 mg, 0.123 mmol) was
carried out in a manner similar to that described above for the
preparation of 5. The product was purified by flash chromatography
(2-5% MeOH/CH2Cl2) to afford 23 (29 mg, 56% yield) as a
mixture of diastereomers. The ratio of the two diastereomers was
variable and dependent on the reaction conditions: 1H NMR (500
MHz, DMSO-d6) δ 8.73 (ABq, J ) 6.0 Hz, 1 H), 8.57-8.54 (m,
1 H), 8.24 & 8.09 (d, J ) 7.3 & 7.5 Hz, 1 H), 7.91 (t, J ) 9.4 Hz,
1 H), 7.39-7.28 (m, 6 H), 7.13 (t, J ) 8.8 Hz, 1 H), 6.82-6.73
(m, 2 H), 5.81 & 5.78 (d, J ) 7.8 & 7.8 Hz, 1 H), 5.09-5.01 (m,
2 H), 4.77 (dd, J ) 21.1, 10.0 Hz, 1 H), 4.36 (td, J ) 11.3, 6.0 Hz,
1 H), 4.28-4.18 (m, 2 H), 4.10-4.02 (m, 1 H), 3.87-3.76 (m, 2
H), 3.17-3.09 (m, 1 H), 2.91 & 2.89 (s, 3 H), 2.84 & 2.83 (s, 3
H), 2.80-2.71 (m, 1 H), 2.07-2.05 (m, 2 H), 1.77-1.24 (m, 16
H), 1.19-1.05 (m, 5 H), 0.89-0.77 (m, 5 H); 13C NMR (125 MHz,
DMSO-d6) δ 196.7, 196.5, 171.72, 171.67, 171.14, 170.07, 170.2,
169.8, 169.15, 169.12, 166.9, 166.8, 160.9, 160.7, 157.1, 156.9,
137.41, 137.38, 137.3, 128.5, 127.83, 127.77, 127.70, 127.65, 127.1,
126.8, 115.6, 115.5, 112.54, 112.51, 66.7, 66.6, 55.0, 54.8, 54.70,
54.68, 53.3, 53.2, 52.94, 52.92, 45.4, 45.3, 41.51, 41.47, 36.48,
36.46, 35.26, 35.24, 33.5, 33.3, 31.7, 31.6, 30.0, 29.9, 29.6, 29.4,
28.1, 28.0, 27.2, 27.1, 27.0, 26.9, 26.02, 25.98, 25.33, 25.28, 24.1,
23.9, 23.8, 23.7, 18.6, 18.5, 13.5, 13.4; HRMS calcd for C43H59N6O8
(M + H)+ 787.4394, found 787.4404.
(5) For recent reviews, see: (a) Tan, S.-L.; He, Y.; Huang, Y.; Gale,
M., Jr. Strategies for Hepatitis C Therapeutic Intervention: Now and
Next. Curr. Opin. Pharmacol. 2004, 4, 465-470. (b) Zhang, X.
Inhibitors of Hepatitis CsA Review of the Current Patent Literature.
Idrugs 2002, 5, 154-158. (c) Dymock, B. W. Emerging Therapies
for Hepatitis C Virus Infection. Expert Opin. Emerging Drugs 2001,
6, 13-42. (d) Dymock, B. W.; Jones, P. S.; Wilson, F. X. Novel
Approaches to the Treatment of Hepatitis C Virus. AntiViral. Chem.
Chemother. 2000, 11, 79-96.
(6) For recent representative reports on HCV NS3 protease inhibitors,
see: (a) Malancona, S.; Colarrusso, S.; Ontoria, J. M.; Marchetti,
A.; Poma, M.; Stansfield, I.; Laufer, R.; Marco, A. D.; Taliani, M.;
Verdirame, M.; Gonzalez-paz, O.; Matassa, V. G.; Narjes, F. SAR
and Pharmacokinetics Studies on Phenethylamide Inhibitors of the
Hepatitis C Virus. Bioorg. Med. Chem. Lett. 2004, 14, 4575-4579.
(b) Llinas-Brunet, M.; Bailey, M. D.; Ghiro, E.; Gorys, V.; Halmos,
T.; Poirier, M.; Rancourt, J.; Goudreau, N. A Systematic Approach
to the Optimization of Substrate-Based Inhibitors of the Hepatitis C
Virus NS3 Protease: Discovery of Potent and Specific Tripeptide
Inhibitors. J. Med. Chem. 2004, 47, 6584-6594. (c) Tsantrizos, Y.
S.; Bolger, G.; Bonneau, P.; Cameron, D. R.; Gordreau, N.; Kukolj,
G.; LaPlante, S. R.; Llinas-Brunet, M.; Nar, H.; Lamarre, D.
Macrocyclic Inhibitors of the NS3 Protease as Potential Therapeutic
Agents of Hepatitis C Virus Infection. Angew. Chem., Int. Ed. 2003,
42, 1355-1360. (d) Zhang, R.; Durkin, J. P.; Windsor, W. T.
Azapeptides as Inhibitors of the Hepatitis C Virus NS3 Serine
Protease. Bioorg. Med. Chem. Lett. 2002, 12, 1005-1008. (e)
Ingallinella, P.; Fattori, D.; Altamura, S.; Steinkuhler, C.; Koch, U.;
Cicero, D.; Bazzo, R.; Cortese, R.; Bianchi, E.; Pessi, A. Prime Site
Binding Inhibitors of a Serine Protease: NS3/4A of Hepatitis C Virus.
Biochemistry 2002, 41, 5483-5492. (f) Beevers, R.; Carr, M. G.;
Jones, P. S.; Jordan, S.; Kay, P. B.; Lazell, R. C.; Raynham, T. M.
Solution and Solid-Phase Synthesis of Potent Inhibitors of Hepatitis
C Virus NS3 Protease. Bioorg. Med. Chem. Lett. 2002, 12, 641-
643.
(7) (a) Llinas-Brunet, M.; Bailey, M. D.; Fazal, G.; Goulet, S.; Halmos,
T.; LePlante, S.; Maurice, R.; Poirier, M.; Poupart, M.-A.; Thibeault,
D.; Wernic, D.; Lamarre, D. Peptide-Based Inhibitors of the Hepatitis
C Virus Serine Protease. Bioorg. Med. Chem. Lett. 1998, 8, 1713-
1718. (b) Steinkuhler, C.; Biasiol, G.; Brunetti, M.; Urbani, A.; Koch,
U.; Cortese, R.; Pessi, A.; De Francesco, R. Product Inhibition of
the Hepatitis C Virus NS3 Protease. Biochemistry 1998, 37, 8899-
8905.
(8) Schering-Plough Research Institute unpublished results.
(9) West, M. L.; Fairlie, D. P. Targeting HIV-1 Protease: A Test of
Drug-Design Methodologies. Trends Pharm. Sci. 1995, 16, 67-75.
(10) (a) Tyndall, J. D.; Fairlie, D. P. Macrocycles Mimic the Extended
Peptide Conformation Recognized by Aspartic, Serine, Cysteine and
Metallo Proteases. Curr. Med. Chem. 2001, 8, 893-907. (b) Fairlie,
D. P.; West, M. L.; Wong, A. K. Towards Protein Surface Mimetics.
Curr. Med. Chem. 1998, 5, 29-62.

574 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Chen et al.
(11) (a) Reid, B. L.; Fairlie, D. P. In Amino Acid Mimetics and
peptidomimetics; Abell, A., Ed.; JAI Press Inc.: London, 1997; Vol.
1, p 77. (b) R. P.; Fairlie, D. P. Macrocyclic Peptidomimetics:
Potential for Drug Development. Curr. Opin. Drug DiscoVery DeV.
1998, 1, 208-217.
(12) (a) Hughes, D. L. The Mitsunobu Reaction. Org. Reactions 1992,
42, 335. (b) Mitsunobu, O. The Use of Diethyl Azodicarboxylate
and Triphenylphosphine in Synthesis and Transformation of Natural
Products. Synthesis 1981, 1-28.
(13) (a) Tidwell, T. T. Oxidation of Alcohols to Carbonyl Compounds
via Alkoxysulfonium Ylides: The Moffatt, Swern and Related
Oxidations. Org. React. 1990, 39, 297. (b) Epstein, W. W.; Sweat,
F. W. Dimethyl Sulfoxide Oxidations. Chem. ReV. 1967, 67, 247-
260.
(14) Dess, D. B.; Martin, J. C. A Useful 12-I-5 Triacetoxyperiodinane
(the Dess-Martin Periodinane) for the Selective Oxidation of Primary
or Secondary Alcohols and a Variety of Related 12-I-5 Species. J.
Am. Chem. Soc. 1991, 113, 7277-7287.
(15) Zhang, R.; Beyer, B. M.; Durkin, J.; Ingram, R.; Njoroge, F. G.;
Windsor, W. T.; Malcolm, B. A. A Continuous Spectrophotometric
Assay for the Hepatitis C Virus Serine Protease. Anal. Biochem. 1999,
270, 268-275.
(16) Taremi, S. S.; Beyer, B.; Maher, M.; Yao, N.; Prosise, W.; Weber,
P. C.; Malcolm, B. A. Construction, Expression, and Characterization
of a Novel Fully Activated Recombinant Single-Chain Hepatitis C
Virus Protease. Protein Sci. 1998, 7, 2143-1249.
(17) For a definition of Ki* and discussions, see: Morrison, J. F.; Walsh,
C. T. In AdVances in Enzymology; Meister, A., Ed.; 1988; Vol. 61,
pp 201-301.
JM050520A