Design, synthesis, and in vitro evaluation of potential west nile virus protease inhibitors based on the 1-oxo-1,2,3,4-tetrahydroisoquinoline and 1-oxo-1,2-dihydroisoquinoline scaffolds

Article abstract of DOI:10.1021/cc100091h

The 1-oxo-1, 2, 3, 4-tetrahydroisoquinoline and 1-Oxo-1, 2-dihydroisoquinoline scaffolds were utilized in the design and solution phase synthesis of focused libraries of compounds for screening against West Nile Virus (WNV) protease. Exploratory studies have led to the identification of a WNV protease inhibitor (a 1-oxo-1, 2-dihydroisoquinoline-based derivative, 12j) which could potentially serve as a launching pad for a hit-to-lead optimization campaign. The identified hit was devoid of any inhibitory activity toward a panel of mammalian serine proteases.

Full text of DOI:10.1021/cc100091h

836
J. Comb. Chem. 2010, 12, 836–843
Design, Synthesis, and In Vitro Evaluation of Potential West Nile
Virus Protease Inhibitors Based on the 1-Oxo-1,2,3,4-
tetrahydroisoquinoline and 1-Oxo-1,2-dihydroisoquinoline Scaffolds
Dengfeng Dou,† Prasanth Viwanathan,‡ Yi Li, Guijia He,† Kevin R. Alliston,†
Gerald H. Lushington,§ Joshua D. Brown-Clay,‡ R. Padmanabhan,‡ and
William C. Groutas*,†
Department of Chemistry, Wichita State UniVersity, Wichita, Kansas 67260, Department of Microbiology
and Immunology, Georgetown UniVersity Medical Center, Washington, D.C. 20057, and Molecular
Graphics and Modeling Laboratory, The UniVersity of Kansas, Lawrence, Kansas 66045
ReceiVed May 29, 2010
The 1-oxo-1, 2, 3, 4-tetrahydroisoquinoline and 1-Oxo-1, 2-dihydroisoquinoline scaffolds were utilized in
the design and solution phase synthesis of focused libraries of compounds for screening against West Nile
Virus (WNV) protease. Exploratory studies have led to the identification of a WNV protease inhibitor (a
1-oxo-1, 2-dihydroisoquinoline-based derivative, 12j) which could potentially serve as a launching pad for
a hit-to-lead optimization campaign. The identified hit was devoid of any inhibitory activity toward a panel
of mammalian serine proteases.
Introduction
protease consists of a heterodimeric complex of NS2B and
NS3. The amino terminal part of NS3 that contains the serine
protease catalytic triad is sufficient for interaction with NS2B
and activation of the protease domain. The carboxy terminal
portion of NS3 contains ATPase, RNA helicase, and 5′ RNA
triphosphatase activities and performs other functions in the
virus life cycle such as viral replication and 5′ capping.7,8
The viral serine protease cofactor NS2B protein consists
of three hydrophobic regions flanking a conserved hydro-
philic domain of 45 amino acid residues. For the function
of the viral protease in infected host cells, the hydrophobic
regions of NS2B, as well as the hydrophilic domain are
required;7 however, the latter domain alone is sufficient for
the interaction with the NS3 protease domain and for in vitro
protease activity in cleaving the polyprotein substrate at
specific sites. These sites contain two basic amino acid
residues at the P1 and P2 positions,9 followed by a residue
with a short side chain (Ala, Ser, or Gly) at the P1′
position.6,10-12
Proteases account for ∼2% of the genes in humans and
infectious organisms and are involved in the regulation of a
multitude of essential physiological processes. A wide range
of human diseases are associated with the aberrant activity
of proteases.1,2 Furthermore, many proteases are essential
to the survival of bacterial, viral, parasitic, and fungal
pathogens.
West Nile virus (WNV), a member of the FlaViVirus genus
of the FlaViViridae family, has emerged as an important
mosquito-borne viral pathogen.3 No effective vaccines or
antiviral agents are currently available for the prevention or
treatment of WNV.4 WNV is a small, enveloped virus with
a single-stranded, positive sense 11-kb RNA genome, which
encodes a polyprotein precursor. Co- and post-translational
cleavage of the polyprotein produces three structural (C, prM,
and E) and seven nonstructural (NS1, NS2A, NS2B, NS3,
NS4A, NS4B, and NS5) proteins by a concerted action of a
host signal peptidase and the trypsin-like viral serine protease.
The viral protease encoded within NS3 in conjunction with
the NS2B cofactor cleaves at NS2A-NS2B, NS2B-NS3,
NS3-NS4A, and 4B-NS5 sites, as well as some internal sites
within C, NS3, and NS4A. Cleavage at these sites is essential
for viral replication. Thus, WNV NS2B/NS3 protease has
emerged as an important target for the design and develop-
ment of novel therapeutics against West Nile virus.5
Flavivirus NS2B is an endoplasmic membrane-associated
hydrophobic protein of 130 amino acid residues.6 Active
The substrate specificity of WNV protease has been probed
using peptidyl substrates and inhibitors4b,12,13 and X-ray
crystallography. The active site of WNV NS2B/NS3 pro-
tease, consisting of His51, Asp75, and Ser135, is located at
the interface of the N- and C-terminal lobes and prefers a
dibasic motif at P1-P2.14 X-ray crystal structures of WNV
NS2B-NS3pro complexed to aprotinin (Bowman pancreatic
trypsin inhibitor (BPTI)15 and Bz-Nle-Lys-Arg-Arg-CHO,16
respectively, have been reported. Inhibitors of WNV protease
containing a highly charged peptidyl or nonpeptidyl recogni-
tion component reflecting the substrate specificity of the
protease include peptidyl aldehydes,17 D-Arg-based pep-
tides,18 and guanidine-based compounds.19 Other low mo-
lecular weight inhibitors of the protease include pyrazoline20
* To whom correspondence should be addressed. Tel.: (316) 978 7374.
Fax: (316) 978 3431. E-mail: bill.groutas@wichita.edu.
† Wichita State University.
‡ Georgetown University.
§ The University of Kansas.
10.1021/cc100091h  2010 American Chemical Society
Published on Web 09/30/2010

West Nile Virus Protease Inhibitors
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6 837
Scheme 1a
Figure 1. (a) Design of transition state inhibitors (I) based on the
1-oxo-1, 2, 3, 4-tetrahydroisoquinoline scaffold; (b) Postulated
mechanism of action of (I).
a Reaction conditions: (a) Cl3COCOCl/dioxane/reflux; (b) AlCl3/DCM/
reflux; (c) LiBH4/THF; (d) (ClCO)2/DMSO/TEA.
and sultam thiourea21 derivatives and others.22 There is
currently a need for low molecular weight nonpeptidyl agents
that exhibit superior potency, selectivity and drug-like
characteristics. We report herein the results of preliminary
studies related to the use of 1-oxo-1,2,3,4-tetrahydroiso-
quinoline and 1-oxo-1,2-dihydroisoquinoline-based deriva-
tives as potential inhibitors of WNV protease.
Scheme 2a
Results and Discussion
Inhibitor Design. The biochemical rationale underlying
the use of the 1-oxo-1,2,3,4-tetrahydroisoquinoline scaffold
in the design of serine protease inhibitors rested on the
following considerations: (a) previous studies related to the
steric course and specificity of R-chymotrypsin-catalyzed
reactions have shown that D-1-oxo-1,2,3,4-tetrahydroiso-
quinoline 3-carboxymethyl ester (Figure 1a) behaves as a
relatively efficient substrate of the enzyme, indicating that
the heterocyclic scaffold is capable of binding productively
to the active site of the enzyme;23 (b) WNV NS2B-NS3pro
is a trypsin-like serine protease with a prototypical catalytic
triad and an active site that is located at the interface of the
N- and C-terminal lobes. We reasoned that an entity such
as (I) (Figure 1) that embodied a recognition element (1-
oxo-1,2,3,4-tetrahydroisoquinoline scaffold) and a suitably
positioned serine trap may function as a nonpeptidyl transi-
tion state inhibitor of the viral protease (Figure 1b).24 Of
particular interest were the R-ketoamide derivatives (structure
(I), Z ) CONHR), since judicious changes in the nature of
the R group could potentially lead to the exploitation of
additional favorable binding interactions.
Synthesis. We initially set out to prepare aldehyde 3 from
compound 2 (Scheme 1) since it would serve as a suitable
intermediate for synthesizing the desired inhibitors. Thus,
when compound 2 was treated with DIBAL,25 only the
starting material was recovered. An alternative method for
making 3, also unsuccessful, involved the treatment of 2 with
iodotrimethylsilane in methylene chloride to give the cor-
responding trimethylsilyl ether (RCOOSi(CH3)3) which was
then treated with DIBAL, as described in the literature.26
Lithium aluminum hydride reduction of ester 2 in the
presence of diethylamine27 failed to give 3 and gave instead
the corresponding alcohol. A second approach toward the
synthesis of 3 was pursued which involved reduction of 2
a Reaction conditions: (a) EDCI/DMF then ArNH2; (b) TFA or H2/Pd-C/
EtOAc; (c) Cl3COCOCl/dioxane/reflux; (d) AlCl3/CH2Cl2/reflux.
(LiBH4/THF) to give alcohol 4, followed by treatment with
pyridinium chlorochromate in methylene chloride. NMR
analysis of the complex reaction mixture showed the absence
of any aldehyde, despite complete consumption of the starting
alcohol. Swern oxidation28 of 4 yielded a white solid the
structure of which was established by X-ray crystallography
to be that of compound 5. It is evident that aldehyde 3 is
formed, which then dimerizes to 5 under the reaction
conditions. Further attempts to generate variants of I, such
as the direct formation of I with Z ) heterocycle,29 were
also unsuccessful and necessitated a change in strategy that
entailed the solution-phase generation of a library of
compounds based on the 1-oxo-1,2,3,4-tetrahydroisoquino-
line scaffold that could potentially function as reversible
competitive inhibitors of the enzyme. Thus, the synthesis of
compounds 7(a-f) proceeded uneventfully when aliphatic
amines were used (Scheme 2a); however, an alternative route
was utilized in the synthesis of compounds 7(g-j) derived
from aromatic amines (Scheme 2b). The unusually low yields
are probably the result of steric effects, the use of nonopti-
mized conditions, and loss of sample during the purification
of the compounds.
A fairly diverse set of amine inputs (Table 1) was utilized
for optimal space exploration. With the exception of com-
pound 7g (∼25% inhibition at 50 µM inhibitor concentra-
tion), the rest of the compounds were devoid of any inhibitory
activity against WNV protease, suggesting that these com-
pounds do not engage in multiple binding interactions with

838 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6
Table 1. Amine Inputs
Dou et al.
Scheme 3a
a Reaction conditions: (a) DDQ/1,4-dioxane/reflux; (b) NBS/dibenzoyl
peroxide/DCM; (c) 1 M LiOH/RT/1 h; (d) EDCI/DMF then RNH2.
Figure 2. (a) Predicted binding of energy-minimized compound
12j docked to the binding site of WNV protease. The enzyme
surface is colored as follows: red ) polar O, blue ) polar N, cyan
) donatable H, white ) mildly polar H/C, yellow ) nonpolar H/C/
S. (b) Conformer of compound 12j in the proteolytic active site of
WNV protease as predicted by molecular docking simulations. The
ligand is shown as CPK-colored sticks with white carbon atoms,
while a number of key interacting receptor residues are rendered
similarly but with green carbons. The overall structure of the protein
is depicted via secondary structure elements (purple helices, yellow
sheets/strands, and cyan coils).
the target enzyme. This series of compounds was inactive
against a representative panel of serine proteases, including
bovine R-chymotrypsin, human neutrophil elastase, cathepsin
G, and human skin chymase.30 Intriguingly, compounds 7f
and 7h were found to inhibit bovine trypsin (42% and 22%
inhibition, respectively, at a 50 µM inhibitor concentration).
The exploratory studies cited above suggested that the
nonplanar component in I may be inimical to binding, conse-
quently a focused library of compounds (10c, 10f, 12a-c, 12f,
12j) was generated based on the 1-oxo-1,2-dihydroisoquinoline
scaffold and its bromo variant using Scheme 3. Screening of
these libraries resulted in the identification of a hit (compound
12j) that was found to be a fair inhibitor of WNV protease
(IC50 ) 30 µM). A plausible mode of binding of energy-
minimized compound 12j to the active site of the enzyme is
illustrated in Figure 2a and b. The aromatic portion of the
bromisoquinoline moiety forms a π-stacking interaction with
Y161 and engages in end-on hydrophobic interactions with
Y150. The amide on the bromoisoquinoline group hydrogen
bonds with the hydroxyl side chain of the catalytic S135,
namely, the carbonyl oxygen on bromoisoquinoline serves as
a hydrogen acceptor from the S135 proton and the amide proton
serve as hydrogen bond donor to the S135 hydroxyl oxygen.
The amide proton on the linker between the bromoisoquinoline
and the (m-phenoxy)phenyl is predicted to hydrogen bond with
the backbone carbonyl of G151. The first aromatic ring in the
(m-phenoxy)phenyl group is solvent exposed and is not
predicted to participate in any significant favorable interactions.
However, the second aromatic ring interacts with the side chain
of V72, with the mildly hydrophobic ꢀ carbons of His51 and
D75, and has an end-on hydrophobic interaction with W50.
Although the aromatic portion of the bromoisoquinoline moiety
engages in π-stacking interactions and fits well within a very
small cavity, it might be possible to improve the affinity for
this cavity by noting that the end of the ring points toward highly
polar species, such as the D129 side chain and the Y130
backbone. Furthermore, the first aromatic ring in the (m-
phenoxy)phenyl component serves as a semi-rigid spacer of
ideal length for fostering the favorable interactions associated
with the bromoisoquinoline ring and on the second aromatic
ring in the (m-phenoxy)phenyl component. Consequently,
exploring other groups that maintain the same spacer distance
and orientation may provide additional opportunities for affinity
enhancement.
In summary, exploratory studies utilizing the 1-oxo-1,
2-dihydroisoquinoline scaffold have led to the identification
of an inhibitor of WNV protease suitable for further
exploration.
Experimental Section
General. The 1H NMR spectra were recorded on a Varian
XL-300 or XL-400 NMR spectrometer. A Gemini EM

West Nile Virus Protease Inhibitors
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6 839
microplate spectrofluorometer (Molecular Devices Corpora-
tion, Sunnyvale, CA) was used in the enzyme assays and
inhibition studies. Melting points were determined on a Mel-
Temp apparatus and are uncorrected. Reagents and solvents
were purchased from various chemical suppliers (Aldrich,
Acros Organics, TCI America, and Bachem). Silica gel
(230-450 mesh) used for flash chromatography was pur-
chased from Sorbent Technologies, Atlanta, GA. Thin layer
chromatography was performed using Analtech silica gel
plates. The TLC plates were visualized using iodine and/or
UV light. WNV protease was expressed in E. coli as
described for Dengue virus-2 protease12 and was purified as
previously described.14 Boc-Gly-Lys-Arg-AMC was pur-
chased from Bachem. The purity of all compounds was
established by TLC. HPLC analyses were performed on a
Varian Prostar 320 monitored at 280 nm using a C18 Luna2
250 × 4.6 mm column with an isocratic 45% acetonitrile
and 55% aqueous solution containing 5% methanol and 0.1%
formic acid.
Synthesis. (DL)Methyl 2-Isocyanato-3-phenylpropanoate
1. DL-Phenylalanine methyl ester hydrochloride (13.6 g; 63
mmol) was placed in a round-bottom flask and pumped under
high vacuum overnight. Dioxane (150 mL) was then added,
followed by trichloromethyl chloroformate (10.5 mL; 88.5
mmol). The resulting mixture was refluxed gently overnight.
The solvent was removed, and the residue was distilled under
reduced pressure to give the corresponding isocyanate 1 as
a colorless oil (12.9 g, 99% yield). 1H NMR (CDCl3): δ 3.03
(dd, J ) 13.7, 5.0 Hz, 1H), 3.16 (dd, J ) 13.7, 8.2 Hz, 1H),
3.81(s, 3H), 4.02-4.10 (m, 1H), 7.18-7.38 (m, 5H).
were dried over anhydrous sodium sulfate. Removal of the
solvent left compound 4 as a colorless oil (3.2 g, 90% yield).
1H NMR (CDCl3): δ 2.91 (d, J ) 7.0 Hz, 2H), 3.70 (dd, J
) 11.4, 4.7 Hz, 1H), 3.85 (dd, J ) 11.4, 7.7 Hz, 1H),
3.85-3.96 (m, 1H), 7.20-7.48 (m, 4H), 8.02-8.05 (d, J )
7.0 Hz, 1H). HRMS (ESI): calcd for C10H12NO2 [M + H]+
178.0868, found 178.0859; C10H11NO2Na [M + Na]+
200.0687, found 200.0699.
6,13-Dihydroxy-5,5a,6a,7,12,12a,13a,14-octahydrodi-
naphtho[2,3-a,d]-hexahydropyrazine-7,14-dione 5. A solu-
tion of 2 M oxalyl chloride in CH2Cl2 (2.75 mL, 5.5 mmol)
and 12 mL CH2Cl2 were placed in a 50 mL three-neck round-
bottom flask equipped with a thermometer, a CaCl2 drying
tube and two pressure-equalizing dropping funnels containing
DMSO (0.85 mL; 11 mmol) in 2.5 mL of CH2Cl2 and
compound 4 (0.88 g; 5 mmol) in 5 mL of CH2Cl2 and 1 mL
of DMSO, respectively. The DMSO solution was added to
the stirred oxalyl chloride solution at -50 to -60 °C. After
it was stirred for 2 min, the solution containing compound
4 was added over 5 min, and the mixture was stirred for an
additional 15 min. Triethylamine (3.5 mL; 25 mmol) was
added, and the reaction mixture was stirred for 5 min and
allowed to warm to room temperature for 3.5 h. Water (25
mL) was added, and the two layers were separated. The
aqueous layer was extracted with additional CH2Cl2 (25 mL),
and the combined organic extracts were washed with brine
(25 mL) and dried over anhydrous sodium sulfate. Removal
of the solvent left a crude product, which was purified by
flash chromatography (silica gel/ethyl acetate/hexanes) to
give compound 5 (150 mg; 8% yield) as a white solid, mp
1
154-156 °C. H NMR (DMSO-d6): δ 3.18 (dd, J ) 16.6,
(DL)Methyl 1-Oxo-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylate 2. To a solution of compound 1 (12.9 g; 63 mmol)
in dry methylene chloride (120 mL) was added AlCl3 (16.8
g; 126 mmol) in small portions, and the resulting mixture
was refluxed for 2 h. The reaction mixture was allowed to
cool to room temperature and then placed in an ice-water
bath. Water (100 mL) was slowly added, and the mixture
was stirred for 30 min. The two layers were separated and
the organic layer was dried over anhydrous sodium sulfate.
Removal of the solvent left a crude product which was
purified by flash chromatography (silica gel/ethyl acetate/
hexanes) to give compound 2 as a white solid (9.2 g; 71%
3.9 Hz, 2H), 3.44 (dd, J ) 16.6, 6.4 Hz, 2H), 3.91 (t, J )
7.1 Hz, 2H), 4.08 (s, 2H), 5.51 (d, J ) 7.6 Hz, 2H),
7.30-7.42 (m, 4H), 7.50-7.60 (t, J ) 6.3 Hz, 2H),
7.79-7.83 (d, J ) 6.7 Hz, 2H).
(DL)1-Oxo-1,2,3,4-tetrahydroisoquinoline-3-carboxyl-
ic acid 6. To a chilled solution of compound 2 (3.0 g, 14.7
mmol) in 30 mL of THF was added 15 mL of 1 M lithium
hydroxide, and the reaction mixture was stirred in an ice-
bath for 30 min. The solvent was removed on the rotary
evaporator, and the residue was treated with water (30 mL).
The solution was extracted with 50 mL of ethyl acetate. The
aqueous solution was acidified to pH 2, yielding a white
precipitate that was collected by suction filtration and air-
dried to yield a white solid (2.1 g, 75% yield), mp 224-226
1
yield), mp 98-100 °C. H NMR (CDCl3): δ 3.22 (dd, J )
15.7, 5.7 Hz, 1H), 3.34 (dd, J ) 15.7, 9.4 Hz, 1H), 3.80 (s,
3H), 4.40-4.46 (m, 1H), 6.65 (s, 1H), 7.23 (d, J ) 6.9 Hz,
1H), 7.38 (t, J ) 7.6 Hz, 1H), 7.46 (t, J ) 7.6 Hz, 1H), 8.08
(d, J ) 6.9 Hz, 1H). HRMS (ESI): calcd for C11H12NO3 [M
+ H]+ 206.0817, found 206.0811; C11H11NO3Na [M + Na]+
228.0637, found 228.0642.
1
°C. H NMR (CD3OD): δ 3.29 (dd, J ) 14.3, 5.9 Hz, 1H),
3.42 (dd, J ) 14.3, 5.9 Hz, 1H), 4.40 (t, J ) 5.6 Hz, 1H),
7.30-7.55 (m, 3H), 7.97 (d, J ) 7.8 Hz, 1H). HRMS (ESI):
calcd for C10H9NO3Na [M + Na]+ 214.0480, found 214.0465.
(DL)3-(Hydroxymethyl)-3,4-dihydroisoquinolin-1(2H)-
one 4. To a solution of compound 2 (4.1 g, 20.0 mmol) in
dry THF (20 mL) was added dropwise a solution of 2 M
LiBH4 in THF (10 mL; 20.0 mmol), followed by the
dropwise addition of 60 mL absolute ethanol. The reaction
was stirred at room temperature overnight. The reaction
mixture was cooled in an ice-bath, and the pH was adjusted
to 4 by adding 5% HCl. The solvent was removed and the
residue was treated with water (60 mL) and extracted with
ethyl acetate (3 × 50 mL). The combined organic extracts
General Procedure for Amide 7(a-f) Formation. A
solution of acid 6 (0.38 g; 2 mmol) in dry N,N-dimethyl-
formamide (5 mL) was treated with 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (0.38 g; 2 mmol),
followed by the appropriate amine (2 mmol). The reaction
mixture was stirred at room temperature overnight. The
solvent was removed in vacuo, and ethyl acetate (50 mL)
was added to the residue. The organic layer was washed with
5% aqueous HCl (2 × 10 mL), saturated aqueous NaHCO3
(2 × 10 mL), and brine (10 mL). The organic layer was

840 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6
Dou et al.
dried over anhydrous sodium sulfate; the solvent was
removed on the rotary evaporator, and the crude product was
purified using flash chromatography (silica gel/ethyl acetate/
hexanes) to give the corresponding amide 7a-f.
General Procedure for N-Boc/N-Cbz Phenylalanine
and Aromatic Amine Coupling Reactions. DL-N-Cbz-
phenylalanine (or L-N-Boc-phenylalanine) (5 mmol) was
dissolved in 10 mL ofDMF, and 1-[3-(dimethylamino)pro-
pyl]-3-ethylcarbodiimide hydrochloride (0.96 g; 5 mmol) was
added, followed by the appropriate aromatic amine (5.5
mmol). The resulting mixture was stirred at room temperature
overnight. Removal of the solvent on the rotary evaporator
left a residue that was dissolved in 50 mL ofethyl acetate.
The ethyl acetate solution was washed with 5% aqueous HCl
(3 × 25 mL), saturated aqueous NaHCO3 (3 × 25 mL), and
brine (25 mL). The organic layer was dried over anhydrous
sodium sulfate and the solvent was removed on the rotary
evaporator to give pure product 8.
(DL)N-(3-Methoxyphenethyl)-1-oxo-1,2,3,4-tetrahydroiso-
quinoline-3-carbox-amide 7a. White solid (10% yield), mp
1
124-126 °C. H NMR (CDCl3): δ 2.60-2.72 (m, 2H),
3.20-3.30 (dd, J ) 15.1, 4.4 Hz, 1H), 3.25-3.55 (m, 3H),
3.72 (s, 3H), 4.23 (q, J ) 3.2 Hz, 1H), 6.52-6.72 (m, 3H),
7.02-7.50 (m, 5H), 7.60 (d, J ) 2.9 Hz, 1H), 7.95 (d, J )
6.9 Hz, 1H). HRMS (ESI): calcd for C19H21N2O3 [M + H]+
325.1552, found 325.1547; C19H20N2O3Na [M + Na]+
347.1372, found 347.1364.
(DL)N-(2-Morpholinoethyl)-1-oxo-1,2,3,4-tetrahydroiso-
quinoline-3-carbox-amide 7b. White solid (26% yield), mp
(DL)tert-Butyl 1-Oxo-3-phenyl-1-(thiazol-2-ylamino)pro-
pan-2-ylcarbamate 8g. White solid (63% yield), mp 88-90
1
138-140 °C. H NMR (CD3OD): δ 2.28-2.52 (m, 6H),
1
°C. H NMR (CDCl3): δ 1.40 (s, 9H), 3.05-3.20 (m, 2H),
3.20-3.38 (m, 2H), 3.60 (t, J ) 5.0 Hz, 4H), 3.72(s, 2H),
4.26 (t, J ) 5.6 Hz, 1H), 7.27 (d, J ) 6.9 Hz, 1H), 7.35 (t,
J ) 7.5 Hz, 1H), 7.46 (t, J ) 7.6 Hz, 1H), 7.92 (d, J ) 6.9
Hz, 1H). HRMS (ESI): calcd for C16H22N3O3 [M + H]+
304.1661, found 304.1652.
4.64-4.78 (m, 1H), 5.18 (s, 1H), 7.00-7.28 (m, 6H), 7.54
(d, J ) 2.2 Hz, 1H), 11.20 (s, 1H). HRMS (ESI): calcd for
C17H22N3O3S [M + H]+ 348.1382, found 348.1368;
C17H21N3O3Na [M + Na]+ 370.1201, found 370.1190.
(DL)Benzyl 1-(Benzo[d][1,3]dioxol-5-ylamino)-1-oxo-3-
phenylpropan-2-yl-carbamate 8h. White solid (48% yield),
mp 145-147 °C. 1H NMR (CDCl3): δ 3.02-3.20 (m, 2H),
4.50 (q, J ) 7.8 Hz, 1H), 5.05 (s, 2H), 5.50 (s, 1H), 5.92 (s,
2H), 6.61 (dd, J ) 36.5, 7.2 Hz, 2H), 7.03 (s, 1H), 7.18-7.37
(m, 10H), 7.55 (s, 1H). HRMS (ESI): calcd for C24H23N2O5
[M + H]+ 419.1607, found 419.1594; C24H22N2O5Na [M +
Na]+ 441.1426, found 441.1412.
(DL)N-(Furan-2-ylmethyl)-1-oxo-1,2,3,4-tetrahydroiso-
quinoline-3-carbox-amide 7c. White solid (57% yield), mp
1
157-158 °C. H NMR (CDCl3): δ 3.30 (dd, J ) 15.6, 5.2
Hz, 1H), 3.43 (dd, J ) 15.6, 5.2 Hz, 1H), 4.26-4.33 (m,
1H), 4.32 (dd, J ) 14.3, 5.8 Hz, 1H), 4.46 (dd, J ) 14.3,
5.8 Hz, 1H), 6.01 (d, J ) 1.9 Hz, 1H), 6.22-6.24 (m, 1H),
6.55 (s, 2H), 7.20-7.55 (m, 4H), 7.99-8.02 (d, J ) 5.9 Hz,
1H). HRMS (ESI): calcd for C15H15N2O3 [M + H]+
271.1083, found 271.1068; C15H14N2O3Na [M + Na]+
293.0902, found 293.0894.
(DL)Benzyl 1-(4-Morpholinophenylamino)-1-oxo-3-phe-
nylpropan-2-yl-carbamate 8i. White solid (87% yield), mp
200-202 °C. 1H NMR (CDCl3): δ 3.05-3.22 (m, 6H), 3.85
(t, J ) 4.3 Hz, 4H), 4.51 (q, J ) 5.9 Hz, 1H), 5.08 (s, 2H),
5.48 (s, 1H), 6.80 (dd, J ) 9.4 Hz, 2H), 7.18-7.38 (m, 12H),
7.40 (s, 1H). HRMS (ESI): calcd for C27H30N3O4 [M + H]+
460.2236, found 460.2198; C27H29N3O4Na [M + Na]+
482.2056, found 482.2047.
(DL)N-(2,2-Diphenylethyl)-1-oxo-1,2,3,4-tetrahydroiso-
quinoline-3-carbox-amide 7d. White solid (23% yield), mp
1
166-168 °C. H NMR (CDCl3): δ 3.16 (dd, J ) 16.9, 5.8
Hz, 1H), 3.34 (dd, J ) 16.9, 6.8 Hz, 1H), 3.65-3.80 (m,
1H), 3.85-4.00 (m, 1H), 4.00-4.12 (m, 1H), 4.12-4.20 (m,
1H), 4.58 (s, 1H), 4.68 (s, 1H), 6.98-7.52 (m, 13H),
7.85-7.92 (d, J ) 9.4 Hz, 1H). HRMS (ESI): calcd for
C24H23N2O2 [M + H]+ 371.1760, found 371.1761;
C24H22N2O2Na [M + Na]+ 393.1579, found 393.1580.
(DL)N-Hexyl-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-
carboxamide 7e. White solid (24% yield), mp 99-101 °C.
1H NMR (CDCl3): δ 0.79 (t, J ) 7.3 Hz, 3H), 1.09-1.20
(m, 2H), 1.30-1.41(m, 2H), 3.18-3.23 (m, 2H), 3.31 (dd,
J ) 13.9, 5.0 Hz, 1H), 3.45 (dd, J ) 13.9, 5.0 Hz, 1H), 4.26
(q, J ) 3.8 Hz, 1H), 6.62 (s, 1H), 7.24 (d, J ) 8.4 Hz, 1H),
7.36 (t, J ) 7.8 Hz, 1H), 7.46 (t, J ) 7.8 Hz, 1H), 7.52 (s,
1H), 7.98 (d, J ) 8.3 Hz, 1H). HRMS (ESI): calcd for
C14H18N2O2Na [M + Na]+ 269.1266, found 269.1276;
C14H18N2O2K [M + K]+ 285.1005, found 285.1012.
(DL)Benzyl 1-Oxo-1-(3-phenoxyphenylamino)-3-phenyl-
propan-2-ylcarbamate 8j. White solid (100% yield), mp
1
132-134 °C. H NMR (CDCl3): δ 3.02-3.20 (m, 2H),
4.45-4.58 (m, 1H), 5.05 (s, 2H), 5.48 (s, 1H), 6.37-6.42
(m, 1H), 6.70-6.75 (m, 1H), 6.98-7.37 (m, 19H), 7.68 (s,
1H). HRMS (ESI) calcd: for C29H27N2O4 [M + H]+
467.1971, found 467.1963; C29H26N2O4Na [M + Na]+
489.1790, found 489.1777.
(DL)1-Oxo-N-(thiazol-2-yl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxamide 7g. Compound 8g (1.14 g; 3.28 mmol)
was dissolved in 15 mL of trifluoroacetic acid at room
temperature. The reaction mixture was stirred for 30 min,
and the trifluoroacetic acid was removed under vacuum.
Water (5 mL) was added, and the pH was adjusted to 9 using
a saturated solution of sodium carbonate. The free amine
was extracted with 50 mL of ethyl acetate, and the organic
layer was dried using anhydrous sodium sulfate. Removal
of the solvent left a colorless oil (0.60 g, 74% yield). The
oil was dissolved in 10 mL of dioxane, and trichloromethyl
chloroformate (0.4 mL, 3.4 mmol) was added. The resulting
mixture was stirred at room temperature overnight. Removal
of the solvent under vacuum left a residue which was
(DL)N-Benzyl-1-oxo-1,2,3,4-tetrahydroisoquinoline-3-
carboxamide 7f. White solid (110 mg; 20% yield), mp
1
165-167 °C. H NMR (CDCl3): δ 3.30 (dd, J ) 15.8, 6.2
Hz, 1H), 4.46 (dd, J ) 15.8, 6.2 Hz, 1H), 4.26 (t, J ) 3.4
Hz, 1H), 4.31 (dd, J ) 13.5, 5.1 Hz, 1H), 4.44 (dd, J )
13.5, 5.1 Hz, 1H), 6.82 (s, 1H), 6.89-7.54 (m, 9H), 7.91
(d, J ) 7.4 Hz, 1H). HRMS (ESI): calcd for C17H17N2O2
[M + H]+ 281.1290, found 281.1275; C17H16N2O2Na [M +
Na]+ 303.1109, found 303.1097.

West Nile Virus Protease Inhibitors
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6 841
dissolved in methylene chloride (20 mL). Aluminum chloride
(0.64 g, 4.8 mmol) was added, and the resulting mixture was
refluxed for 1 h. The reaction mixture was allowed to cool
to room temperature, and water (10 mL) was slowly added.
The mixture was stirred for 30 min and then transferred to
a separatory funnel. The organic layer was washed with water
(2 × 15 mL) and dried over anhydrous sodium sulfate.
Removal of the solvent gave a pure product 7g (50 mg; 6%
Methyl 1-Oxo-1,2-dihydroisoquinoline-3-carboxylate 9.
To a solution of compound 1-oxo-1,2,3,4-tetrahydroiso-
quinoline 3-carboxymethyl ester 2 (13.61 g; 66 mmol) in
200 mL of 1,4-dioxane was added 2,3-dichloro-5,6-dicy-
anobenzoquinone (DDQ) (16.0 g; 70.5 mmol) and the
mixture was refluxed overnight. The solvent was removed
in vacuo, and the residue was taken up in 200 mL of ethyl
acetate and washed with 5% NaOH (2 × 50 mL). The
organic layer was dried over anhydrous sodium sulfate and
the filtrate was evaporated, leaving a crude product which
was purified with flash chromatography (silica gel/ethyl
acetate/hexanes) to give compound 9 as a white solid (3.0
1
yield) as a gray solid, mp 202-204 °C. H NMR (CDCl3):
δ 2.96 (dd, J ) 13.2, 9.0 Hz, 1H), 3.45 (dd, J ) 12.0, 2.4
Hz, 1H), 4.40-4.48 (m, 1H), 5.58 (s, 1H), 7.20-7.38 (m,
6H), 7.77 (d, J ) 1.3 Hz, 1H). HRMS (ESI): calcd for
C13H12N3O2S [M + H]+ 274.0650, found 274.0659;
C13H11N3O2SNa [M + Na]+ 296.0470, found 296.0468.
1
g; 22% yield), mp 154-155 °C. H NMR (CDCl3): δ 3.92
(s, 3H), 7.52-7.68 (m, 4H), 8.37-8.41 (m, 1H), 9.12 (s,
1H). HRMS (ESI): calcd for C11H10NO3 [M + H]+ 204.0661,
found 204.0664; C11H9NO3Na [M + Na]+ 226.0480, found
226.0475.
Representative Procedure for the Synthesis of Com-
pounds 7h-j. (DL)N-(Benzo[d][1,3]dioxol-5-yl)-1-oxo-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide 7h. Com-
pound 8h (1.0 g, 2.39 mmol) was dissolved in 50 mL of
ethyl acetate; then 10% palladium/carbon (0.33 g) wetted
with 2 mL of ethyl acetate was added. The mixture was
placed on a Parr hydrogenator; 20 psi hydrogen gas was
applied, and the mixture was shaken for 3 h. The reaction
mixture was gravity filtered, and the filtrate was evaporated
to give a brown oil (0.5 g, 74% yield) that was dissolved in
8 mL of 1, 4-dioxane and treated with trichloromethyl
chloroformate (0.3 mL, 2.5 mmol). The resulting mixture
was stirred at room temperature overnight. The solvent was
removed under vacuum, and the residue was taken up in
methylene chloride (15 mL). Aluminum chloride (0.48 g,
3.6 mmol) was added, and the resulting mixture was refluxed
for 1 h and allowed to cool to room temperature. After water
(15 mL) was slowly added, the reaction mixture was stirred
for 30 min and then transferred to a separatory funnel. The
organic layer was washed with water (2 × 15 mL) and dried
over anhydrous sodium sulfate. Removal of the solvent left
a crude product which was purified by flash chromatography
(silica gel/ethyl acetate/hexanes) to give compound 7h (120
mg; 16% yield) as a white solid, mp 120-122 °C. 1H NMR
(CDCl3): δ 3.09 (dd, J ) 13.2, 3.6 Hz, 1H), 3.20 (dd, J )
13.2, 5.7 Hz, 1H), 4.35-4.40 (m, 1H), 5.95 (s, 2H),
6.50-6.59 (m, 2H), 6.81 (d, J ) 7.6 Hz, 1H), 6.88 (s, 1H),
7.20-7.35 (m, 5H). HRMS (ESI): calcd for C17H15N2O4 [M
+ H]+ 311.1032, found 311.1037; C17H14N2O4Na [M + Na]+
333.0851, found 333.0840.
Methyl 4-Bromo-1-oxo-1,2-dihydroisoquinoline-3-car-
boxylate 11. To a solution of compound 1-oxo-1,2,3,4-
tetrahydroisoquinoline 3-carboxymethyl ester 2 (3.56 g; 17.4
mmol) in 175 mL of methylene chloride were added
N-bromosuccinimide (NBS) (6.26 g; 35.2 mmol) and benzyl
peroxide (0.20 g; 0.8 mmol), and the reaction was refluxed
for 24 h. The reaction was cooled down to room temperature
and then washed with saturated NaHCO3 (3 × 80 mL) and
brine (80 mL). The organic layer was dried over anhydrous
sodium sulfate. The drying agent was filtered, and the filtrate
was concentrated. The crude product was precipitated in 30
mL ethyl acetate and filtered, yielding compound 11 as a
white solid (2.84 g; 57% yield), mp 186-187 °C. 1H NMR
(CDCl3): δ 4.05 (s, 3H), 7.68 (t, J ) 8.1 Hz, 1H), 7.85 (t, J
) 8.1 Hz, 1H), 8.27 (d, J ) 6.5 Hz, 1H), 8.46 (d, J ) 6.5
Hz, 1H), 9.45 (s, 1H). HRMS (ESI): calcd for C11H935BrNO3
[M + H]+ 281.9766, found 281.9767; C11H937BrNO3 [M +
H]+ 283.9745, found 283.9744; C11H835BrNO3Na [M + Na]+
303.9585, found 303.9584; C11H837BrNO3Na [M + Na]+
305.9565, found 305.9569.
Hydrolysis of Methyl Ester 9. To a solution of ester 9
(5 mmol) in 10 mL dioxane, was added 1 M lithium
hydroxide (10 mL), and the reaction mixture was stirred at
room temperature for 1 h. The solvent was removed on the
rotary evaporator, and the residue was treated with 20 mL
of water and acidified to pH 2, forming a precipitate. The
precipitate was collected by suction filtration and washed
with 20 mL of ethyl acetate to give the corresponding acid
(DL)N-(4-(1,3-Oxazinan-3-yl)phenyl)-1-oxo-1,2,3,4-tet-
rahydroisoquinoline-3-carboxamide 7i. White solid (5%
1
as a white solid (80% yield), mp 230-232 °C. H NMR
1
(DMSO-d6) δ 7.40 (s, 1H), 7.64 (t, J ) 7.3 Hz, 1H), 7.79 (t,
J ) 7.3 Hz, 1H), 7.85 (d, J ) 7.5 Hz, 1H), 8.22 (d, J ) 7.5
Hz, 1 H), 11.80 (s, 1H).
yield), mp 166-168 °C. H NMR (CDCl3): δ 3.01-3.35
(m, 6H), 3.85 (t, J ) 5.0 Hz, 4H), 4.37-4.41(m, 1H), 6.04
(s, 1H), 6.92 (d, J ) 9.4 Hz, 2H), 7.05 (d, J ) 9.4 Hz, 2H),
7.22-7.38 (m, 4H). HRMS (ESI): calcd for C20H22N3O3 [M
+ H]+ 352.1661, found 352.1648; C20H21N3O3Na [M + Na]+
374.1481, found 374.1467.
The acid derived from ester 11 was obtained the same
procedure as that above. White solid (64% yield), mp >260
1
°C. H NMR (DMSO-d6): δ 7.69 (t, J ) 6.9 Hz, 1H),
7.84-8.00 (m, 2H), 8.25 (d, J ) 5.3 Hz, 1H), 11.80 (s, 1H).
(DL)1-Oxo-N-(3-phenoxyphenyl)-1,2,3,4-tetrahydroiso-
quinoline-3-carboxamide 7j. White solid (7% yield), mp
93-95 °C. 1H NMR (CDCl3): δ 3.06 (dd, J ) 10.3, 5.1 Hz,
1H), 3.27 (dd, J ) 10.3, 2.5 H, 1H), 4.38-4.40 (m, 1H),
6.22 (s, 1H), 6.82-7.40 (m, 14H). HRMS (ESI): calcd for
C22H19N2O3 [M + H]+ 359.1396, found 359.1398;
C22H18N2O3Na [M + Na]+ 381.1215, found 381.1219.
General Coupling Reaction Procedure for Compounds
10c, 10f, 12a, 12c, 12f, 12i, and 12j. A solution of acid (2
mmol) derived from 9 or 11 in dry N,N-dimethylformamide
(5 mL) was treated with 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (EDCI) (0.38 g, 2 mmol),
followed by the appropriate amine (2 mmol). The reaction

842 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6
Dou et al.
mixture was stirred at room temperature overnight. The
solvent was removed under vacuum and the residue was
added ethyl acetate (30 mL). The organic layer was washed
with 5% aqueous HCl (3 × 10 mL), saturated aqueous
NaHCO3 (3 × 10 mL), and brine (10 mL). The organic layer
was dried over anhydrous sodium sulfate. The drying agent
was filtered and the solvent was removed. The crude pro-
duct was purified by flash chromatography (silica gel/ethyl
acetate/hexanes) to give compounds 10c, 10f, 12a, 12c, 12f,
12i, and 12j.
428.0625; C20H1937BrN3O3 [M + H]+ 430.0589, found 430.0592;
C20H1835BrN3O3Na [M + Na]+ 450.0429, found 450.0453;
C20H1837BrN3O3Na [M + Na]+ 452.0409, found 452.0429.
4-Bromo-1-oxo-N-(3-phenoxyphenyl)-1,2-dihydroiso-
quinoline-3-carboxamide 12j. White solid (33% yield), mp
1
216-218 °C. H NMR (DMSO-d6): δ 6.78 (d, J ) 7.4 Hz,
1H), 7.04 (d, J ) 7.4 Hz, 2H), 7.13 (d, J ) 7.0 Hz, 1H),
7.27-7.40 (m, 3H), 7.46-7.64 (m, 3H), 7.82 (t, J ) 7.4
Hz, 1H), 8.00 (d, J ) 7.5 Hz, 1H), 8.40 (d, J ) 7.5 Hz,
1H), 10.50 (s, 1H), 11.72 (s, 1H). HRMS (ESI): calcd for
C22H1635BrN2O3 [M + H]+ 435.0344, found 435.0340;
C22H1637BrN2O3 [M + H]+ 437.0324, found 437.0341;
C22H1535BrN2O3Na [M + Na]+ 457.0164, found 457.0155;
C22H1537BrN2O3Na [M + Na]+ 459.0143, found 459.0146.
WNV NS2B/NS3 Protease Assays.14 All assays were
performed in opaque 96-well plates. Reaction mixtures (100
µL/assay) contained 200 mM TRIS buffer, pH 9.5, 13.5 mM
NaCl, 30% glycerol, 0.025 µM enzyme, and 100 µM
fluorogenic peptide substrate (Boc-Gly-Lys-Arg-7-amino-4-
methyl coumarin). Enzyme incubations were at 25 °C for
30 min. The fluorescence of 7-amino-4-methyl coumarin
(AMC) released from the cleavage of the substrate was
monitored using excitation and emission wavelengths of 385
and 465 nm, respectively.
N-(Furan-2-ylmethyl)-1-oxo-1,2-dihydroisoquinoline-3-
carboxamide 10c. White solid (10% yield), mp 138-140
1
°C. H NMR (DMSO-d6): δ 4.48 (s, 2H), 6.34-6.36 (m,
1H), 6.60-6.62 (m, 1H), 7.35 (s, 1H), 7.57-7.80 (m, 4H),
8.22 (d, J ) 8.3 Hz, 1H). HRMS (ESI): calcd for C15H13N2O3
[M + H]+ 269.0926, found 269.0936; C15H12N2O3Na [M +
Na]+ 291.0746, found 291.0752.
N-Benzyl-1-oxo-1,2-dihydroisoquinoline-3-carboxam-
ide 10f. White solid (15% yield), mp 189-191 °C. 1H NMR
(CDCl3): δ 4.70 (d, J ) 3.5 Hz, 2H), 7.20-7.72 (m, 9H),
8.08 (d, J ) 7.7 Hz, 1H), 10.88 (s, 1H). HRMS (ESI): calcd
for C17H15N2O2 [M + H]+ 279.1134, found 279.1130;
C17H14N2O2Na [M + Na]+ 301.0953, found 301.0960.
4-Bromo-N-(3-methoxyphenethyl)-1-oxo-1,2-dihydroiso-
quinoline-3-carboxamide 12a. White solid (18% yield), mp
175-177 °C. 1H NMR (CDCl3): δ 2.97 (t, J ) 6.9 Hz, 2H),
3.82 (s, 3H), 3.80-3.86 (m, 2H), 6.80-6.89 (m, 3H),
7.23-7.30 (m, 1H), 7.64 (t, J ) 7.1 Hz, 1H), 7.67 (s, 1H),
7.79 (t, J ) 7.1 Hz, 1H), 8.03 (d, J ) 7.4 Hz, 1H), 8.45 (d,
J ) 7.4 Hz, 1H), 9.78 (s, 1H). HRMS (ESI): calcd for
C19H1835BrN2O3 [M + H]+ 401.0501, found 401.0502;
C19H1837BrN2O3 [M + H]+ 403.0480, found 403.0480;
C19H1735BrN2O3Na [M + Na]+ 423.0320, found 423.0320;
C19H1737BrN2O3Na [M + Na]+ 425.0300, found 425.0297.
4-Bromo-N-(furan-2-ylmethyl)-1-oxo-1,2-dihydroiso-
quinoline-3-carboxamide 12c. White solid (15% yield), mp
197-199 °C. 1H NMR (CDCl3): δ 4.70 (d, J ) 4.7 Hz, 2H),
6.37 (s, 2H), 7.41 (m, 1H), 7.64 (t, J ) 9.4 Hz, 1H), 7.80 (t,
J ) 9.4 Hz, 1H), 8.00 (s, 1H), 8.05 (d, J ) 9.9 Hz, 1H),
8.42 (d, J ) 9.9 Hz, 1H), 9.90 (s, 1H). HRMS (ESI): calcd
for C15H1235BrN2O3 [M + H]+ 347.0031, found 347.0029;
C15H1237BrN2O3 [M + H]+ 349.0011, found 348.9997;
C15H1135BrN2O3Na [M + Na]+ 368.9851, found 368.9850;
C15H1137BrN2O3Na [M + Na]+ 370.9830, found 370.9820.
N-Benzyl-4-bromo-1-oxo-1,2-dihydroisoquinoline-3-
carboxamide 12f. White solid (45% yield), mp 208-209 °C.
1H NMR (DMSO-d6): δ 4.48 (d, J ) 3.7 Hz, 2H), 7.23-8.27
(m, 9H), 9.33 (t, J ) 3.3 Hz, 1H), 12.10 (s, 1H). HRMS (ESI):
calcd for C17H1435BrN2O2 [M + H]+ 357.0239, found 357.0236;
C17H1437BrN2O2 [M + H]+ 359.0218, found 359.0235;
C17H1335BrN2O2Na [M + Na]+ 379.0058, found 379.0059;
C17H1337BrN2O2Na [M + Na]+ 381.0038, found 381.0041.
4-Bromo-N-(4-morpholinophenyl)-1-oxo-1,2-dihydroiso-
quinoline-3-carboxamide 12i. White solid (10% yield), mp
WNV NS2B/NS3 Protease Inhibition Assays.14 The
protease inhibitor assays contained 200 mM Tris-HCl buffer,
pH 9.5, 13.5 mM NaCl, 30% glycerol, 0.025 µM enzyme
(2.5 pmol), 100 µM fluorogenic peptide substrate, and 50
µM inhibitor. The inhibitors were dissolved in DMSO and
diluted in assay buffer. The DMSO concentration in the assay
mix was maintained at 1%, including in the no-inhibitor
control. The assay mixtures containing WNV protease with
an inhibitor (or without the inhibitor as a control) were
preincubated at room temperature for fifteen minutes. An
aliquot of the substrate (100 µM) was added, and the
incubation continued for an additional fifteen minutes.
Fluorescence values were obtained using excitation and
emission wavelengths of 385 and 465 nm, respectively. The
percent inhibition of protease activity was determined using
Microsoft Excel. For calculation of IC50 value, in vitro
protease assays were performed in triplicate as described
above in the presence of 10, 20, 30, 40, and 50 µM of
compound 12j or no-compound control. The % inhibition
(y-axis) was plotted against the concentration of the inhibitor
(x-axis) using GraphPad Prism 5.0 software.
Molecular Modeling. Molecular docking simulations were
performed via the AutoDock program.31 Compound 12j was
constructed in SYBYL 8.032 and structurally optimized to
default convergence thresholds using the Tripos Force Field33
and Gasteiger-Marsili partial atomic charges.34 The active
site model for WNV protease was prepared using the 2FP7
crystal structure.16 The structure was protonated in SYBYL,
stripped of all water molecules and bound ligands, and
electrostatically represented with Gasteiger-Marsili charges.
AutoDock simulations were performed using the Lamarckian
Genetic Algorithms (GA) subroutine at default settings for
GA population size, crossover rate, and mutation rate, and
starting with fully randomized ligand position, orientation
1
252-254 °C. H NMR (DMSO-d6): δ 3.05-3.15 (m, 4H),
3.70-3.80 (m, 4H), 6.96 (d, J ) 7.3 Hz, 2H), 7.56 (d, J )
7.3 Hz, 2H), 7.60-7.68 (m, 1H), 7.84-7.96 (m, 2H), 8.26
(d, J ) 6.9 Hz, 1H), 10.82 (s, 1H), 12.17 (s, 1H). HRMS
(ESI): calcd for C20H1935BrN3O3 [M + H]+ 428.0610, found

West Nile Virus Protease Inhibitors
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6 843
Liebscher, S.; Jensen, C. M.; Gan, C. H.; Gupta, P. K.; Xu,
W.-J.; Young, P. R.; Fairlie, D. P. J. Med. Chem. 2008, 51,
5714–5721.
and conformation. One hundred GA runs were performed
for the ligand-enzyme pair.
(18) Shiryaev, S. A.; Ratnikov, B. I.; Chakanov, A. V.; Sikora,
S.; Rozanov, D. V.; Godzik, A.; Wang, J.; Smith, J. W.;
Huang, Z.; Lindberg, I.; Samuel, M. S.; Diamond, M. S.;
Strongin, A. Y. Biochem. J. 2006, 393, 503–511.
(19) Ganesh, V. K.; Mueller, N.; Judge, K.; Luan, C.-H.; Pad-
manabhan, R.; Murthy, K. H. M. Bioorg. Med. Chem. 2005,
13, 257–264.
(20) Goodell, J. R.; Puig-Basagoiti, Forshey, B. M.; Shi, P.-Y.;
Ferguson, D. M. J. Med. Chem. 2006, 49, 2127–2137.
(21) Barklis, E.; Still, A.; Sabri, M. I.; Hirsch, A. J.; Nikolich-
Zugich, J.; Brien, J.; Dhenub, T. C.; Scholz, I.; Alfadhli, A.
Antimicrob. Agents Chemother. 2007, 51, 2642–2645.
(22) (a) Noueiry, A. O.; Olivo, P. D.; Slomenzyska, U.; Zhou, Y.;
Buscher, B.; Geiss, B.; Engle, M.; Roth, R. M.; Chung, K. M.;
Samuel, M.; Diamond, M. S. J. Virol. 2007, 81, 11992–12004.
(b) Ekonomiuk, D.; Su, X. C.; Ozawa, K.; Bodenreider, C.;
Lim, S. P.; Yin, Z.; Keller, T. H.; Beer, D.; Patel, V.; Otting,
G.; Caflisch, A.; Huang, D. PLoS Neglected Trop. Dis 2009,
3 (1), e-356.
Acknowledgment. This work was supported by the
National Institutes of Health (AI577045 and AI 070791).
References and Notes
(1) Abbenante, G.; Fairlie, D. P. Med. Chem. 2005, 1, 71–104.
(2) Maly, D. J.; Huang, L.; Ellman, J. A. ChemBiochem 2002, 3,
16–37.
(3) Burke, S. D.; Monath, T. P. Flaviviruses. In Field’s Virology,
4th ed.; Knipe, D. M., Howley, P. M., Eds.; Lippincott-Raven:
Philadelphia, PA, 2001.
(4) (a) Ray, D.; Shi, P. Y. Recent Pat. Anti-Infect. Drug DiscoVery
2006, 1, 45–55. (b) Chapell, K. J.; Stoermer, M. J.; Fairlie,
D. P.; Young, P. R. J. Biol. Chem. 2006, 281, 38448–38458.
(5) (a) Chappell, K. J.; Stoermer, M. J.; Fairlie, D. P.; Young,
P. R. Curr. Med. Chem. 2008, 15, 2771–2784. (b) Sampath,
A.; Padmanabhan, R. AntiViral Res. 2009, 81, 6–15. (c)
Steuber, H.; Hilgenfeld, R. Curr. Top. Med. Chem. 2010, 10,
323–345. (d) Diamond, M. S. AntiViral Res. 2009, 83, 214–
227.
(23) Hein, G. E.; Niemann, K. J. Am. Chem. Soc. 1962, 84, 4487–
(6) Clum, S.; Ebner, K. E.; Padmanabha, R. J. Biol. Chem. 1997,
4494.
272, 30715–30723.
(7) Lindenbach, B. D.; Rice. M. AdV. Virus Res. 2003, 59, 23–
61.
(24) Zhong, J.; Groutas, W. C. Curr. Top. Med. Chem. 2004, 4,
1203–1216.
(25) Zakharkin, L. I.; Khorlina, I. M. Tetrahedron Lett. 1962, 619–
(8) Padmanabhan, R.; Mueller, N.; Reichert, E.; Yon, C.; Tera-
moto, T.; Kono, Y.; Takhampunya, R.; Ubol, S.; Pattabiraman,
N.; Falgout, B.; Ganesh, V. K.; Murthy, K. NoVartis Found.
Symp. 2006, 277, 74–84.
(9) Nomenclature used is that of Schechter, I.; Berger, A.
Biochem. Biophys. Res. Commun. 1967, 27, 157-162, where
S1, S2, S3,...Sn and S′1, S2′, S3′, ...Sn′ correspond to the enzyme
subsites on either side of the scissile bond. Each subsite
accommodates a corresponding amino acid residue side chain
designated P1, P2, P3, ...Pn and P1′, P2′, P3′,...Pn′ of the substrate
or (inhibitor). S1 is the primary substrate specificity subsite,
and P1-P1′ is the scissile bond.
(10) Falgout, B.; Miller, R. H.; Lai, C.-J. J. Virol. 1993, 67, 2034–
2042.
(11) Chambers, T. J.; Nestorowicz, A.; Amberg, S. M.; Rice, C. M.
J. Virol 1993, 67, 6797–807.
(12) Yusof, R. S.; Clum, M.; Wetzel, H.; Murthy, M.; Padmanab-
han, R. J. Biol. Chem. 2000, 265, 9963–9.
(13) Chambers, T. J.; Hahn, C. S.; Galler, R.; Rice, C. M. Annu.
ReV. Microbiol. 1990, 44, 649–688.
(14) Mueller, N. H.; Yon, C.; Ganesh, V. K.; Padmanabhan, R.
Int. J. Biochem. Cell Biol. 2007, 39, 606–614.
(15) Aleshin, A. E.; Shiryaev, S. A.; Strongin, A. Y.; Liddington,
R. C. Protein Sci. 2007, 16, 795–806.
(16) Erbel, P.; Schiering, N.; D’Arcy, A.; Renatus, M.; Kroemer,
M.; Lim, S. P.; Yin, Z.; Keller, T. H.; Vasudevan, S. G.;
Hommel, U. Nat. Struct. Mol.Biol. 2006, 13, 372–373.
(17) (a) Knox, J. E.; Ma, N. L.; Yin, Z.; Patel, S. J.; Wang, W.-L.;
Chan, W.-L.; Rao, K. R. R.; Ngew, X.; Patel, V.; Beer, D.;
Lim, S. P.; Vasudevan, S. G.; Keller, T. H. J. Med. Chem.
2006, 49, 6585–6590. (b) Stoermer, M. J.; Chappell, K. J.;
620.
(26) Chandrasekhar, S.; Kumar, M. S.; Muralidhar, B. Tetrahedron
Lett. 1998, 909–910.
(27) Cha, J. S.; Kwon, S. S. J. Org. Chem. 1987, 52, 5487–5489.
(28) Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem. 1978,
43, 2480–2482.
(29) Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.; Fecik,
R. A.; Cheng, H.; Hwang, I.; Hedrick, M. P.; Leung, D.;
Acevedo, O.; Guimaraes, C. R. W.; Jorgensen, W. L.; Cravatt,
B. F. J. Med. Chem. 2005, 48, 1849–1856.
(30) (a) Huang, W.; Yamamoto, Y.; Li, Y.; Dou, D.; Alliston,
K. R.; Hanzlik, R. P.; Williams, T. D.; Groutas, W. C. J. Med.
Chem. 2008, 51, 2003–2008, and references cited therein. (b)
Li, Y.; Yang, Q.; Dou, D.; Alliston, K. R.; Groutas, W. C.
Bioorg. Med. Chem. 2008, 16, 692–698. (c) Lai, Z.; Gan, X.;
Wei, L.; Alliston, K. R.; Yu, H.; Li, Y. H.; Groutas, W. C.
Arch. Biochem. Biophys. 2004, 429, 191–197. (d) Zhong, J.;
Gan, X.; Alliston, K. R.; Lai, Z.; Yu, H.; Groutas, C. S.; Wong,
T.; Groutas, W. C. J.Comb. Chem. 2004, 6, 556–563. (e)
Kuang, R.; Epp, J. B.; Ruan, S.; Yu, H.; Huang, P.; He, S.;
Tu, J.; Schechter, N. M.; Turbov, J.; Froleich, C. J.; Groutas,
W. C. J. Am. Chem. Soc. 1999, 121, 8128–8129.
(31) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.;
Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem.
1998, 19, 1639–1662.
(32) SYBYL 8.0; The Tripos Associates: St. Louis, MO, 2008.
(33) Clark, M.; Cramer, R. D., III; Van Opdenbosch, N. J. Comput.
Chem. 1989, 10, 982–1012.
(34) Gasteiger, J.; Marsili, M. Tetrahedron Lett. 1978, 3181–3184.
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