Design, synthesis, and evaluation of trifluoromethyl ketones as inhibitors of SARS-CoV 3CL protease

Article abstract of DOI:10.1016/j.bmc.2008.02.040

A series of trifluoromethyl ketones as SARS-CoV 3CL protease inhibitors was developed. The inhibitors were synthesized in four steps from commercially available compounds. Three different amino acids were explored in the P1-position and in the P2-P4 positions varying amino acids and long alkyl chain were incorporated. All inhibitors were evaluated in an in vitro assay using purified enzyme and fluorogenic substrate peptide. One of the inhibitors showed a time-dependent inhibition, with a K_i value of 0.3 μM after 4 h incubation.

Full text of DOI:10.1016/j.bmc.2008.02.040

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4652–4660
Design, synthesis, and evaluation of trifluoromethyl ketones
as inhibitors of SARS-CoV 3CL protease
Yi-Ming Shao,a,b,c Wen-Bin Yang,a Tun-Hsun Kuo,b Keng-Chang Tsai,a
Chun-Hung Lin,a,b,c An-Suei Yang,a Po-Huang Lianga,b,c and Chi-Huey Wonga,b,c,d,
*
aGenomics Research Center, Academia Sinica, No. 128, Section 2, Academia Road, Taipei 11529, Taiwan
bInstitute of Biological Chemistry, Academia Sinica, No.128, Section 2, Academia Road, Taipei 11529, Taiwan
cInstitute of Biochemical Sciences, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan
dDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 23 November 2007; revised 10 February 2008; accepted 11 February 2008
Available online 15 February 2008
Abstract—A series of trifluoromethyl ketones as SARS-CoV 3CL protease inhibitors was developed. The inhibitors were synthe-
sized in four steps from commercially available compounds. Three different amino acids were explored in the P1-position and in
the P2–P4 positions varying amino acids and long alkyl chain were incorporated. All inhibitors were evaluated in an in vitro assay
using purified enzyme and fluorogenic substrate peptide. One of the inhibitors showed a time-dependent inhibition, with a Ki value
of 0.3 lM after 4 h incubation.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
virus main protease (Cys-144 and His-41) and human
coronavirus 229E main protease (Cys-144 and His-
41).8 In addition, it cleaves the replicase polyprotein at
no less than 11 conserved sites with canonical Leu-
Gln#(Ser, Ala, Gly) sequences.9 Taken together, this
information provides good understanding to the design
of potent inhibitors.
Severe acute respiratory syndrome-associated coronavi-
rus (SARS-CoV), identified to be the causative agent of
this life-threatening epidemic,1–5 leads to a respiratory
disease with the symptoms including cough, high fever,
chills, rigor, myalgia, headache, dizziness, and progres-
sive radiographic changes of the chest and lymphopenia.
The spread of this contagious disease in 2003 infected
more than 8000 people with a high mortality. In total,
there were 774 deaths reported around the world. Dur-
ing the life cycle of SARS-CoV, 3CL protease cleaves
the polyprotein into individual polypeptides to provide
all the essential proteins for viral replication and tran-
scription.6,7 This enzyme is thus recognized as a primary
target for the therapeutic intervention.
To date, a number of 3CL protease inhibitors have been
prepared, including C2-symmetric diols,10 bifunctional
aryl boronic acids,11 keto-glutamine analogs,12 isatin
derivatives,13 a,b-unsaturated esters,14 anilide,15 and
benzotriazole.16 Here, we report the synthesis of trifluo-
romethyl ketones as inhibitors against SARS-CoV 3CL
protease, and provide kinetic analysis and computer
modeling to address the issue of covalent binding.
In contrast to the common serine proteases containing a
Ser-His-Asp catalytic triad, SARS-CoV 3CL protease
has a Cys-His catalytic dyad (Cys-145 and His-41),
which is similar to porcine transmissible gastroenteritis
Trifluoromethyl ketones (TFMKs) are well known as
the inhibitors of serine17 and cysteine18 proteases. Owing
to the high electronegativity of fluorine, the carbonyl
carbon of TFMK is a highly active electrophile. It is
generally believed that hemiketal or hemithioketal is
formed by the nucleophilic attack of the hydroxyl or
thiol group at the active site when TFMKs are employed
as the inhibitors against serine or cysteine proteases,
respectively. Previous studies19 indicated that TFMKs
demonstrate a competitive slow, tight-binding inhibition
Keywords: SARS; Protease inhibitor; Trifluoromethyl ketone; Time-
dependent inhibition.
*
Corresponding author. Fax: +886 2 2789 8771; e-mail addresses:
chwong@gate.sinica.edu.tw; wong@scripps.edu
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.02.040

Y.-M. Shao et al. / Bioorg. Med. Chem. 16 (2008) 4652–4660
4653
against human leukocyte elastase. Recently, Zhang
et al.20a described N,N-dimethyl glutaminyl fluorometh-
yl ketones as 3CL protease inhibitors. One of these com-
pounds was found to have low toxicity in mice, and
another one was found to have an EC50 value of
2.5 lM based on the cytopathic effect (CPE) inhibition
assay. However, the in vitro inhibition has not been
characterized in detail. Sydnes et al.20b also reported
the synthesis of glutamic acid and glutamine peptides
with a CF3-ketone unit as 3CL protease inhibitors.
DIEA (or Et3N) to afford 4a–g. Final oxidation using
Dess–Martin reagent generated the desired trifluoro-
methyl ketones 5a–h.
TFMKs 5a–h were evaluated to interfere with SARS-
CoV 3CL protease activity according to the reported
procedure21 (Table 1). The activity of 5a, 5b, 5f, 5g,
and 5h, having benzyl group as the side chain at the
P1 site, supports the idea that the P2–P4 sites still have
a significant contribution to the binding affinity though
they are far from the active site. The best inhibitor 5h,
containing the same residues as the reported substrate
sequence at the P2, P3, and P4 sites, displayed a compet-
2. Results and discussion
In order for the synthetic simplicity, we assumed that
the benzyl group as the P1 site can mimic the Gln resi-
due of the substrate. Scheme 1 shows the four-step syn-
thesis of various N-protected trifluoromethyl ketones.
The preparation of nitro alcohols 3 was carried out by
C–C bond formation between nitroalkanes 2 and trifluo-
roacetaldehyde ethyl hemiacetal under the basic condi-
tion of catalytic potassium carbonate. The choice of
nitroalkanes defines the P1 group of the final inhibitor.
For instance, 1-nitro-2-phenylethane 2a introduces a
benzyl group at the P1 site. Subsequent reduction to
amine alcohols was performed either by PtO2- or Raney
nickel-catalyzed hydrogenation. The use of PtO2 was
avoided in the reduction of 3a because undesired satura-
tion of the phenyl ring was observed. At this stage, the
trifluoroamine alcohols were coupled with N-protected
amino acids or long-chain acids by using HBTU and
Table 1. Inhibition of trifluoromethyl ketones against SARS-CoV
3CL protease
R
CF3
X-HN
O
No.
R
X
IC50 (lM)
5a
5b
5c
5d
5e
5f
Bn
Bn
Me
H
Cbz-Leu
Cbz-Phe
15
20
40
40
50
50
>50
10
Boc-Leu
Boc-cGlu(OtBu)-Ala
cGlu-Ala
CH3(CH2)8CO-Leu
CH3(CH2)7CO-Leu
Cbz-Ala-Val-Leu
H
Bn
Bn
Bn
5g
5h
R
R
R
a
b
CF3
OH
O2N
O2N
Br
1 R = Bn
2a R = Bn
2b R = Me
2c R = H
3a R = Bn (45%)
3b R = Me (77%)
3c R = H (90%)
4a R = Bn, X = Cbz-Leu (70%)
4b R = Bn, X = Cbz-Phe (66%)
4c R = Me, X = Boc-Leu (78%)
R
c or d,
then e
CF3
OH
X-HN
4d R = H, X = Boc-γGlu(OtBu)-Ala (54%)
4e R = Bn, X = CH3(CH2)8CO-Leu (58%)
4f R = Bn, X = CH3(CH2)7CO-Leu (95%)
4g R = Bn, X = Cbz-Ala-Val-Leu (73%)
5a R = Bn, X = Cbz-Leu (86%)
5b R = Bn, X = Cbz-Phe (87%)
5c R = Me, X = Boc-Leu (86%)
R
f
CF3
5d R = H, X = Boc-γGlu(O
5e R = H, X = γGlu-Ala
tBu)-Ala (14%)
g
X-HN
O
5f R = Bn, X = CH3(CH2)8CO-Leu (85%)
5g R = Bn, X = CH3(CH2)7CO-Leu (70%)
5h R = Bn, X = Cbz-Ala-Val-Leu (67%)
Scheme 1. Synthesis of trifluoromethyl ketones 5a–5h. Reagents and conditions: (a) NaNO2 (1.3 equiv), DMF, ꢀ78 ! 23 ꢁC, 15 h, 68%; (b)
trifluoroacetaldehyde ethyl hemiacetal (1.27 equiv), K2CO3 (cat.), neat, 50–60 ꢁC, 3 h, then 23 ꢁC, 25.5 h, 45–90%; (c) H2 (1 atm), cat. PtO2Æ·H2O
(79–84% Pt), MeOH/CHCl3 (16:1), 23 ꢁC, 43 h; (d) H2 (1 atm), Ra-Ni, H2O, EtOH, 23 ꢁC, 14 h; (e) N-protected amino acids or long-chain acids,
HBTU, DIEA (or Et3N), DMF, 23 ꢁC, 36 h, 54–95%; (f) Dess-Martin reagent (3 equiv), TFA (3 equiv), CH2Cl2, 22 ꢁC, 3 h, 14–87%; (g) TFA, 40.5 h.
DMF = N, N-dimethylformamide; HBTU = (1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; DIEA = diisopropylethyl-
amine; TFA = trifluoroacetic acid.

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Y.-M. Shao et al. / Bioorg. Med. Chem. 16 (2008) 4652–4660
Figure 1. Lineweaver-Burk plots of compound 5h incubated with 3CL protease for 4 h. The enzyme activities were measured using 8–40 lM
fluorogenic substrate in the absence (Ç) or presence of 1 · IC50 (j) and 2· IC50 (m) inhibitor. The pattern of these plots displayed competitive
inhibition.
itive inhibition against 3CL protease (Fig. 1). Moreover,
in consistence with the previous reports of cathepsin B
and human leukocyte elastase,18b,19 prolonged incuba-
tion of 3CL protease with 5h exhibited a time-dependent
decrease in enzyme activity as a function of the inhibitor
concentration. The inhibitor was found to produce pro-
gressive tightening of inhibition, as shown by a 30-fold
decrease in the Ki value (from 8.8 to 0.3 lM) in 4 hr
(Table 2 and Fig. 2). As indicated by the NMR studies,
the trifluoromethyl ketone moiety exists as an equilib-
rium mixture of ketone and hydrate forms. The time-
dependent tightening of inhibition is likely due to the
slow formation of a covalent adduct through the nucle-
ophilic attack of the thiol group on the carbonyl carbon.
2
1.5
1
0 uM
2 uM
4 uM
7 uM
10 uM
0.5
0
0
20
40
60
80
100
120
140
Time (min)
Compound 5h and 3CL protease complex have been
crystallized in our laboratory, but the X-ray crystallog-
raphy experiments were nevertheless unsuccessful in
structural refinement due to fragmented electron density
maps. Alternatively, computational molecular modeling
was used to construct a model for the acyl–enzyme com-
plex. On the basis of the crystal structure of 3CL prote-
ase with a chloromethyl ketone (CMK) inhibitor, the
analog of trifluoromethyl ketone, determined by Yang
et al.,7 we first constructed the models for the four pos-
sible stereoisomers of the covalent adducts between the
protein and compound 5h. All the models were con-
strained with a covalent link between the thiol group
of Cys-145 and compound 5h, in consistent with the
analog experimental complex structure by Yang et al.7
In comparison with the analog experimental structure,
Figure 2. The progress curves in the presence of 2–10 lM inhibitor for
reactions initiated by adding enzyme (final concentration of 0.005 lM)
into a mixture of substrate (6 lM) and inhibitor 5h. Over the entire
120 min time window, the uninhibited enzyme displayed a linear
progress curve, whereas the inhibited enzyme with a different concen-
tration of inhibitor showed a time-dependent reduction of activity.
only the (S,S,S,S) isomer of compound 5h with the R
configuration of carbonyl carbon adjacent to CF3 group
agreed with the binding mode of the CMK inhibitor, in
particular all four amino acid side chains of compound
5h fitted into the bind pockets of the 3CL protease
active site. All the other three stereoisomers were ruled
out because all these molecules were unable to bind to
the active site under the covalent constraint. The com-
putational model of the (S,S,S,S) isomer is different
from the binding mode of the CMK-3CL protease com-
plex structure in that the P1, P2, P4 side chains in com-
pound 5h occupied S2, S1, and S4 sites, respectively, in
3CL protease (Fig. 3). The binding mode discrepancies
were expected consequences due to the difference be-
tween the amino acid side chains of the two inhibitor
analogs. The proposed detailed covalent attacking
mechanism was shown in Figure 4.
Table 2. Time-dependent inhibition of 5h against SARS-CoV 3CL
protease
Incubation time
IC50 (lM)
Ki (lM)
10 min
30 min
1 h
10
7
8.76 1.61
2.69 0.47
1.30 0.19
0.73 0.07
0.29 0.09
4
2 h
4 h
2
0.8

Y.-M. Shao et al. / Bioorg. Med. Chem. 16 (2008) 4652–4660
4655
ber septa or three-way T taps under a positive pressure
of argon or nitrogen. Air- and moisture-sensitive liquids
and solutions were transferred via syringe. Organic solu-
tions were concentrated by rotary evaporation at 23–
80 ꢁC (water-bath temperature). Column chromatogra-
˚
phy was performed employing Merck silica gel (60 A
pore size, 70–230 mesh ASTM). Analytical thin-layer
chromatography (TLC) was performed using glass
˚
plates pre-coated with Merck silica gel (60 A pore size)
impregnated with a fluorescent indicator (254 nm).
TLC plates were visualized by I2 vapors, UV lamp,
phosphomolybdic acid solution in ethanol, or 0.5% nin-
hydrin in ethanol followed by brief heating on a hot
plate. Commercial solvents and reagents were used as re-
ceived without further purification. They were pur-
chased from Aldrich, ACROS, BACHEM, or other
commercial sources. Compounds are characterized by
nuclear magnetic resonance spectroscopy and high reso-
lution mass spectroscopy. Proton nuclear magnetic res-
onance (1H NMR) spectra and carbon nuclear
magnetic resonance (13C NMR) spectra were recorded
with Bruker Avance 600 (600 MHz/ 150 MHz), Bruker
DRX 500 (500 MHz/ 125 MHz), and Bruker Avance
400 (400 MHz/100 MHz) NMR spectrometers. Chemi-
cal shifts for protons are reported in parts per million
(ppm; d scale) and are referenced to residual protium
in the NMR solvents (CHCl3: d 7.26, D2HCOD: d
3.31, C2D5HSO: d 2.50, C2D5HCO: d 2.05). Chemical
shifts for carbon are reported in parts per million
(ppm; d scale) and are referenced to the carbon reso-
nances of the solvent (CDCl3: d 77.23, CD3OD: d
49.15, DMSO-d6: d 39.50, acetone-d6: d 29.84). Data
are represented as follows: chemical shift, multiplicity
(s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad), coupling constant in Hz, integration, and
assignment. High resolution mass spectra were obtained
using Bruker Daltonics BioTOF III.
Figure 3. The model of compound 5h and SARS-CoV 3CL protease.
The hydrogen bondings are shown in the green and blue (oxyanion
hole) dotted lines, and the hydrophobic interactions are shown in
yellow dotted lines. The thiol group on Cys-145 forms a covalent
bonding to compound 5h.
3. Conclusion
The substrate-based design and synthesis of trifluoro-
methyl ketones as SARS-CoV 3CL protease inhibitors
have been reported. The most potent inhibitor 5h, which
possesses the same moiety as the substrate on P1-P4 site,
supported the covalent binding. Also, the time-depen-
dent inhibition displayed by inhibitor 5h advanced our
understanding of the interactions between the cysteine
protease and the electrophilic compound, thereby fur-
thering the discovery of cysteine protease inhibitors.
4.2. SARS-CoV 3CL protease inhibition assay
As described,21,22 the inhibitory effects of each com-
pound on the enzymatic activities of 3CL protease were
evaluated using purified enzyme and fluorogenic sub-
strate peptide. The kinetic measurements were per-
formed in 20 mM Bis–Tris (pH 7.0) at 25 ꢁC. The
initial velocities of the inhibited reactions of 50 nM
4. Experimental
4.1. General methods
All reactions with air- and moisture-sensitive materials
were performed in oven-dried glassware fitted with rub-
His41
ImH
His41
Im
Cys145
S
His41
Im
Cys145
S
H
H
Ph
Ph
Ph
O
Cys145
S
CF3
HO OH
CF3
N
X-HN
F3C
X-HN
X-HN
O
H
O
5h
H
O
N
Gly143
Figure 4. Proposed mechanism of inhibition of 3CL protease with compound 5h.

4656
Y.-M. Shao et al. / Bioorg. Med. Chem. 16 (2008) 4652–4660
3CL protease and 6 lM fluorogenic substrate were plot-
ted against the different inhibitor concentrations to ob-
tain the IC50 by fitting with Eq. 1. Ki measurement
was performed at two fixed inhibitor concentrations of
1 · IC50 and 2· IC50. Substrate concentrations ranged
from 8 to 40 lM in a reaction mixture containing
50 nM 3CL protease. Lineweaver–Burk plots of kinetic
data were fitted with the computer program KinetAsyst
II (IntelliKinetics, State College, PA) by nonlinear
regression to obtain the Ki values of competitive inhibi-
tors using Eq. 2
4.3.3. 3-Nitro-1,1,1-trifluorobutan-2-ol (3b). Compound
3b was prepared in a similar way to compound 3a, ex-
cept nitroethane was used here in place of 2-nitrophenyl
1
ethane (77% yield). Rf = 0.47 (hexanes/EtOAc 2:1); H
NMR (400 MHz, CDCl3): d = 4.87–4.39 (m, 2H;
O2NCH + CHOHCF3), 3.68–3.47 (br s, 1H; OH), 1.69
(d, J = 6.9 Hz, 3H; CH3); 13C NMR (100 MHz, CDCl3):
d = 123.5 (q, J = 280.5 Hz), 82.1, 71.1, 14.4; HRMS
(ESI): calcd for C4H5F3NO3 [MꢀH]ꢀ: 172.0222, found:
172.0237.
4.3.4. 3-Nitro-1,1,1-trifluoropropan-2-ol (3c). Compound
3c was prepared in a similar way to compound 3a, ex-
cept nitromethane was used here in place of 2-nitro-
phenyl ethane (90% yield). Rf = 0.72 (hexanes/EtOAc
AðIÞ ¼ Að0Þ ꢁ f1 ꢀ ½½Iꢂ=ð½Iꢂ þ IC50Þꢂg
1=V ¼ Km=V mðþ½Iꢂ=KiÞ1=½Sꢂ þ 1=V m
ð1Þ
ð2Þ
1
1:1); H NMR (400 MHz, CDCl3): d = 4.83 (br s, 1H;
In Eq. 1, A(I) is the enzyme activity with inhibitor con-
centration [I], A(0) is the enzyme activity without inhib-
itor, and [I] is the inhibitor concentration. In Eq. 2, Km
is the Michaelis constant of the substrate, Vm is the max-
imal velocity, Ki is the inhibition constant, and [I] and
[S] represent the inhibitor and substrate concentrations
in the reaction mixture, respectively.
CHOHCF3), 4.67 (dd, J = 14.2, 2.6 Hz, 1H; O2NCHH0),
4.58 (dd, J = 14.0, 9.5 Hz, 1H; O2NCHH0), 4.13 (br s,
1H; OH); 13C NMR (100 MHz, CDCl3): d 123.2 (q,
J = 280.3 Hz; CF3), 74.3 (CNO2), 67.6 (q, J = 32.8 Hz;
CH(OH)CF3).
4.3.5. 3-[N-(N-tert-Butoxycarbonyl-L-Leu)]-1,1,1-trifluo-
robutan-2-ol (4a). To a stirred Raney-nickel (aqueous
suspension) solution was added compound 3a
(472.8 mg, 1.90 mmol) in EtOH (8 mL), and the mixture
was hydrogenated under H2 bubbling at 23 ꢁC for 14 h.
The catalyst was filtered over Celite, and ethanol and
water were evaporated under reduced pressure to afford
the amine as a white solid (396.7 mg, 95%). Rf = 0.13
(hexanes/EtOAc 2:1); 1H NMR (500 MHz, CD3OD):
d = 7.33–7.21 (m, 5H; ArH), 3.91–3.68 (m, 1H;
CHOHCF3), 3.30–3.20 (m, 1H; H2NCH), 3.11–2.56
(dd, J = 13.4, 7.8 Hz, 2H; CH2Ph); 13C NMR
(125 MHz, CD3OD): d = 139.6, 130.5, 129.8, 127.8,
73.5, 53.5, 40.1; HRMS (ESI): calcd for C10H11F3NO
[MꢀH]ꢀ: 218.0793, found: 218.0855.
4.3. Synthesis of compounds 2–5
4.3.1. 1-Nitro-2-phenylethane (2a). In a 25 mL round-
bottom flask fitted with a stirrer were placed sodium ni-
trite (156 mg, 2.26 mmol) and anhydrous DMF (10 mL).
The clear solution was cooled to ꢀ78 ꢁC and stirred un-
der N2 (in the absence of light) for 10 min, after which
(2-bromoethyl)benzene (238 lL, 1.74 mmol) was added.
The mixture was stirred for 15 h, during which the tem-
perature was gradually returned to 23 ꢁC. DMF was re-
moved under reduced pressure, and the residue was
extracted with EtOAc. The organic layers were washed
with H2O, dried over Na2SO4, filtered, and concentrated
under reduced pressure to afford compound 2a as a yel-
1
low oil (178.9 mg, 68%). H NMR (400 MHz, CDCl3):
d = 7.39–7.23 (m, 5H; ArH), 4.62 (t, J = 7.34 Hz, 2H;
O2NCH2), 3.33 (t, J = 7.35 Hz, 2H; O2NCH2CH2); 13C
NMR (100 MHz, CDCl3): d = 135.6, 128.8, 128.4,
127.2, 76.1, 33.2.
To a stirred solution of the above amine (385.9 mg,
1.76 mmol) and Cbz–Leu–OH (492 mg, 1.76 mmol) in
dry DMF (15 mL) were added HBTU (1720 mg,
4.40 mmol) and Et3N (1227 lL, 8.80 mmol). The reaction
mixture was stirred under N2 at 23 ꢁC for 36 h. DMF was
evaporated under reduced pressure, and the resulting
brown oil was diluted with CH2Cl2 and washed with
1 N HCl. The water layer was separated and extracted
with CH2Cl2 for three times. The organic layers were com-
bined and washed with H2O for two times, dried over
Na2SO4, filtered, and concentrated under reduced pres-
sure. The residue was then purified by SiO2 column chro-
matography (20% ! 25% EtOAc–hexanes) to give 4a as a
yellow solid (574 mg, 70%). Rf = 0.26 (hexanes/EtOAc
4.3.2. 3-Nitro-4-phenyl-1,1,1-trifluorobutan-2-ol (3a). To
compound 2a (4161 mg, 27.5 mmol) at 23 ꢁC were
added trifluoroacetaldehyde ethyl hemiacetal (90%,
4519 lL, 35 mmol) and K2CO3 (255 mg, 1.84 mmol).
The mixture was stirred at 50–60 ꢁC for 3 h, and then
at 23 ꢁC for 25.5 h. 1 N HCl (20 mL) and Et2O
(20 mL) were added and the water layer was separated.
After extraction with Et2O (twice), the combined organ-
ic layers were washed with H2O, dried over Na2SO4, fil-
tered, and concentrated. The residue was purified by
1
2:1); H NMR (500 MHz, CD3OD): d = 7.34–7.18 (m,
10H; ArH), 5.14–5.05 (m, 2H; PhCH2O), 4.42–4.31 (m,
silica
gel
chromatography
(9% ! 20% ! 100%
EtOAc–hexanes) to give 3a as a yellow oil (3117 mg,
45%). Rf = 0.44 (hexanes/EtOAc 2:1); 1H NMR
(500 MHz, CDCl3): d = 7.41–7.20 (m, 5H; ArH), 5.02–
4.96 (m, 1H; O2NCH), 4.67–4.27 (m, 1H; CHOHCF3),
3.90–3.65 (br s, 1H; OH), 3.44–3.35 (m, 2H; CH2Ph);
13C NMR (125 MHz, CDCl3): d = 134.4, 129.1, 128.8,
128.1, 123.3 (q, J = 281 Hz), 87.2, 70.4 (q, J = 32 Hz),
36.3; HRMS (ESI): calcd for C10H9F3NO3 [MꢀH]ꢀ:
248.0535, found: 248.0555.
1H; CH(OH)CF3), 4.14–3.89 (m, 2H; 2· CHa), 3.12–
2.77 (m, 2H; CH2b(Phe)), 1.63–1.00 (m, 3H; CH2b(Leu)
+
CHc(Leu)), 0.91–0.76 (m, 6H; 2· CH3d(Leu)); 13C NMR
(125 MHz, CD3OD): d = 174.8, 158.3, 139.1, 138.5,
130.6, 130.4, 129.6, 129.5, 129.4, 129.3, 129.0, 128.9,
128.8, 128.7, 127.7, 127.5, 127.4, 55.0, 51.3, 42.0, 37.6,
25.7, 23.3, 21.9; 19F NMR (376 MHz, CDCl3):
d = ꢀ77.88, ꢀ77.90, ꢀ77.97, ꢀ77.99; HRMS (ESI): calcd
for C24H28F3N2O4 [MꢀH]ꢀ: 465.2001, found: 465.2044.

Y.-M. Shao et al. / Bioorg. Med. Chem. 16 (2008) 4652–4660
4657
4.3.6. 3-[N-(N-Benzyloxycarbonyl-L-Leu)]-4-phenyl-1,1,1-
trifluorobutan-2-ol (4b). Compound 4b was prepared in a
similar way to compound 4a, except Cbz–Phe–OH was
used here in place of Cbz–Leu–OH. Compound 4b
was isolated as a white solid (1240.7 mg, 66%).
Rf = 0.21 (hexanes/EtOAc 7:3); 1H NMR (400 MHz,
CD3OD): d = 7.33–7.06 (m, 15H; ArH), 5.05–4.94 (m,
2H; PhCH2O), 4.48–3.93 (m, 3H; 2· CHa +
CHOHCF3), 3.15–2.50 (m, 4H; 2 · CH2b(Phe)); 13C
NMR (100 MHz, CD3OD): d = 173.7, 158.3, 139.3–
138.3 (C(Ar)), 130.7–127.4 (CH(Ar) + CF3), 72.3–69.5
(CHOHCF3), 67.6, 57.8, 51.4, 39.1, 36.1.
1H; NH), 7.20 (br s, 1H; NH), 5.48 (br s, 1H; NH),
4.51 (br s; CHOHCF3), 4.09–3.36 (br, 4H; 4 · CHa),
2.35–1.93 (br, 4H; CH2b(Glu) + CH2c(Glu)), 1.46–1.43
(2s, 21H; 2· OC(CH3)3 + CH3b(Ala)); 13C NMR
(150 MHz, CDCl3): d = 173.9, 172.7, 171.5, 155.9,
124.4 (q, J = 280.5 Hz; CF3), 82.4, 80.2, 69.1 (q,
J = 30 Hz; CHOHCF3), 53.5, 49.3, 49.1, 39.6, 31.9,
28.4, 27.8, 17.7.
4.3.9. 3-{N-[N-CH3(CH2)8(C@O)-L-Leu]}-4-phenyl-1,1,1-
trifluorobutan-2-ol (4e). To a stirred solution of com-
pound 4a (107.4 mg, 0.23 mmol) in MeOH (10 mL) was
added Pd(OH)2 (20% Pd, 81 mg), and the whole mixture
was stirred under H2. When tlc analysis indicated that
the starting material has reacted completely, the catalyst
was removed by filtration through Celite. Solvent was
evaporated, and the crude amine (78.5 mg) was used with-
out further purification. Subsequent coupling was per-
formed using decanoic acid (40.7 mg, 0.24 mmol),
HBTU (98.7 mg, 0.26 mmol), and DIEA (117 lL,
0.71 mmol) by the routine procedure. After SiO2 column
chromatography (10% ! 15% ! 25% EtOAc–hexanes),
compound 4e was obtained as a white solid (65.2 mg,
58%). Rf = 0.45 (hexanes/EtOAc 2:1); 1H NMR
(400 MHz, CDCl3): d = 7.23–7.09 (m, 5H; ArH), 7.60–
7.30, 7.00–5.30 (m, 2H; 2· NH), 4.60–3.80 (m, 3H; 2·
CHa + CHOHCF3), 3.00–2.83 (m, 2H; CH2b(Phe)), 2.13–
2.03 (m, 2H; CH2b(Leu)), 1.54–1.09 (br m, 16H; CH3
(CH2)8C(@O)NH), 0.86–0.73 (m, 7H; CHc(Leu) + 2·
CH3d(Leu)), 0.65 (d, J = 5.9 Hz, 3H; CH3(CH2)8
C(@O)NH); 13C NMR (100 MHz, CDCl3): d = 174.3,
172.3, 137.1, 129.2, 128.5, 126.7, 124.6 (q, J = 281.4 Hz),
70.3 (q, J = 30.1 Hz), 51.8, 50.4, 40.8, 36.3, 31.8, 29.4,
29.3, 29.2, 25.7, 24.5, 22.6, 22.3, 22.1, 14.0; HRMS
(ESI): calcd for C26H42F3N2O3 [M + H]+: 487.3148,
found: 487.3161.
4.3.7. 3-[N-(N-Benzyloxycarbonyl-L-Phe)]-4-phenyl-1,1,1-
trifluorobutan-2-ol (4c). Compound 3b was hydrogenated
under H2 using PtO2 as catalyst and MeOH/CHCl3
(16:1) as solvent. After the work-up, the corresponding
amine hydrochloride salt was obtained in a satisfactory
1
yield (3482.8 mg, 84%). H NMR (400 MHz, DMSO-
d6): d = 8.38 (br s, 3H; NH3), 4.29 (m, 1H; CHOHCF3),
3.35 (m, 1H; H3NCH), 1.21 (d, J = 6.6 Hz, 3H; CH3);
13C NMR (100 MHz, DMSO-d6): d = 124.9 (q,
J = 281.9 Hz), 68.9 (q, J = 29.7 Hz), 46.8, 13.7; HRMS
(ESI): calcd for C4H8ClF3NO [MꢀH]ꢀ: 178.0247,
found: 178.0268. The subsequent coupling reaction is
similar to that for compound 4a, except Boc–Leu–OH
was used here in place of Cbz–Leu–OH. Compound 4c
was isolated as
a white solid (855.7 mg, 78%).
Rf = 0.53 (hexanes/EtOAc 1:1); 1H NMR (400 MHz,
CDCl3): d = 7.06 (d, J = 6.7 Hz, 1H; NH), 6.06 (br s,
1H; OH), 5.22 (d, J = 7.9 Hz, 1H; NH), 4.25–3.92 (m,
3H; 2· CHa + CHOHCF3), 1.57 (m, 2H; CH2b(Leu)),
1.41 (s, 10H; OC(CH3)3 + CHc(Leu)), 1.31 (d,
J = 6.5 Hz, 3H; CH3b(Ala)), 0.90 (s, 6H; 2 · CH3d(Leu));
13C NMR (100 MHz, CD3OD): d = 172.8, 156.2, 124.5
(q, J = 281.4 Hz), 80.6, 72.1 (q, J = 29.9 Hz), 53.1,
45.1, 40.7, 28.2, 24.5, 22.5, 22.1, 17.5; HRMS (ESI):
calcd for C15H27F3N2NaO4 [M + Na]+: 379.1821,
found: 379.1865.
4.3.10. 3-{N-[N-CH3(CH2)7(C@O)-L-Leu]}-4-phenyl-1,1,1-
trifluorobutan-2-ol (4f). Compound 4f was prepared in a
similar way to compound 4e, except nonanoic anhydride
was used here in place of decanoic acid (95% yield).
Rf = 0.42 (hexanes/EtOAc 2:1); 1H NMR (400 MHz,
CDCl3): d = 7.60–6.10 (m, 7H; ArH+2· NH), 5.10–3.15
(m, 3H; 2· CHa + CHOHCF3), 3.15–2.80 (m, 2H;
CH2b(Phe)), 2.25–2.05 (m, 2H; CH2b(Leu)), 1.70–0.65 (m,
24H; CH3(CH2)7C(@O)NH + CHc(Leu) + 2 · CH3d(Leu));
13C NMR (100 MHz, CDCl3): d = 178.1, 173.4, 137.0,
129.1, 128.7, 126.9, 71.8, 51.8, 51.5, 40.6, 36.4, 34.0, 31.8,
29.2, 25.6, 24.8, 22.6, 15.0; HRMS (ESI): calcd for
C25H39F3N2NaO3 [M+Na]+: 495.2810, found: 495.2797.
4.3.8. 3-{N-[N-tert-Butoxycarbonyl-L-cGlu(OtBu)-L-Ala]}-
1,1,1-trifluoropropan-2-ol (4d). Compound 3c was
hydrogenated under H2 using Ra-Ni as catalyst to give
the corresponding amine, which was used directly for cou-
pling to Cbz-Ala-OSu to produce dipeptide adduct.
Rf = 0.19 (hexanes/EtOAc 1:1); 1H NMR (400 MHz,
CDCl3): d = 7.32 (m, 5H; ArH), 7.05 (br s, 1H; NH),
5.70 (br d, J = 6.3 Hz, 1H; NH), 5.06 (AB, J = 12.1 Hz,
m
ab = 25.0 Hz, 2H; PhCH2O), 4.89 (br s, 1H; OH), 4.23
(t, J = 6.6 Hz, 1H; CHOHCF3), 4.04 (br s, 1H; CHa),
3.68 (br s, 1H; CHa), 3.28 (m, 1H; CHa), 1.34 (d,
J = 7.0 Hz, 3H; CH3b(Ala));13C NMR (100 MHz, CDCl3):
d = 174.8, 156.4, 135.7, 128.3, 128.0, 127.9, 124.3 (q,
1J(C,F) = 280.4 Hz; CF3), 68.7 (q, 2J(C,F) = 15.1 Hz;
CH(OH)CF3), 67.2, 50.8, 39.4, 17.8; HRMS (ESI): calcd
for C14H17F3N2NaO4 [M + Na]+: 357.1038, found:
357.1001.
4.3.11. 3-[N-(N-Benzyloxycarbonyl-L-Ala-L-Val-L-Leu)]-
4-phenyl-1,1,1-trifluorobutan-2-ol (4g). Compound 4g was
prepared in a similar way to compound 4e, except Cbz–
Ala–Val–OH was used here in place of decanoic acid
1
(73% yield). Rf = 0.41 (hexanes/EtOAc 1:1); H NMR
(400 MHz, CD3OD): d = 7.34–7.14 (m, 10H; ArH),
5.13–4.92 (m, 2H; OCH2Ph), 4.47–3.83 (m, 5H;
4 · CHa + CHOHCF3), 3.15–2.74 (m, 2H; CH2b(Phe)),
2.06 (m, 1H; CHb(Val)), 1.50–0.75 (m, 18H; 5 · CH3
+CH2b(Leu) + CH c(Leu)); 13C NMR (100 MHz, CD3OD):
d = 175.8, 174.0, 173.2, 158.5, 139.0, 138.1, 130.5, 129.6,
129.5, 129.1, 128.9, 127.9, 67.8, 60.3, 53.1, 51.9, 41.9,
The Cbz group of the above dipeptide adduct was
deprotected followed by amino acid coupling using
Boc–Glu–OtBu as acid. Compound 4d was isolated as
1
a white solid (1238.7 mg, 54%). Rf = 0.48 (EtOAc); H
NMR (600 MHz, CDCl3): d = 7.56 (d, J = 22.0 Hz,

4658
Y.-M. Shao et al. / Bioorg. Med. Chem. 16 (2008) 4652–4660
39.0, 36.4, 32.1, 31.5, 25.8, 23.6, 22.0, 20.0, 18.9, 18.2;
HRMS (ESI): calcd for C32H43F3N4NaO6 [M + Na]+:
659.3032, found: 659.3000.
0.95–0.85 (m, 6H; 2· CH3d(Leu)); 13C NMR (100 MHz,
CDCl3): d = 175.4, 156.1, 123.2 (q, J = 287.0 Hz), 94.6
(q, J = 30.5 Hz), 80.9, 53.2, 50.9, 40.6, 28.2, 24.6, 22.8,
21.8, 14.5; HRMS (ESI): calcd for C15H24F3N2O4
[M ꢀ H]ꢀ: 353.1688, found: 353.1705.
4.3.12. 3-[N-(N-Benzyloxycarbonyl-L-Leu)]-4-phenyl-1,1,1-
trifluorobutan-2-one (5a). To a solution of 4a (57.6 mg,
0.12 mmol) in dry CH2Cl2 (5 mL) was added the
Dess–Martin reagent (15 wt% soln. in CH2Cl2, 769 lL,
0.37 mmol). TFA (28 lL, 0.37 mmol) was added and
then the reaction mixture was stirred at 22 ꢁC for 3 h.
The reaction was concentrated under reduced pressure
and the remaining residue was treated with a mixture
of EtOAc and saturated aqueous solutions of NaHCO3.
The water layer was extracted with EtOAc, washed with
brine, dried (Na2SO4), filtered, and concentrated under
vacuum. Purification (twice) by SiO2 column chroma-
tography (1st: 25% EtOAc–hexanes; 2nd: 10% EtOAc–
CH2Cl2) afforded 5a as a white solid (49.6 mg, 86%).
Rf = 0.17 (CH2Cl2/EtOAc 5:1); 1H NMR (400 MHz,
CDCl3): d = 7.35–7.13 (m, 10H; ArH), 6.68–6.45 (m,
1H; NH), 5.14–4.88 (m, 3H; PhCH2O + NH), 4.29–
3.93 (m, 2H; 2· CHa), 3.31–3.23 (m, 1H; CHbH0b(Phe)),
3.02–2.87 (m, 1H; CHbH0b(Phe)), 1.60–1.36 (m, 3H;
CH2b(Leu) + CHc(Leu)), 0.90–0.75 (m, 6H; 2· CH3d(Leu));
13C NMR (100 MHz, CDCl3): d = 189.7 (m;
(C=O)CF3), 171.9, 156.3, 144.0, 135.7, 134.1, 129.2,
129.0, 128.9, 128.8, 128.6, 128.4, 128.1, 127.7, 126.9,
67.4, 54.9, 53.0, 40.4, 36.0, 24.6, 22.7, 21.8; 19F NMR
(376 MHz, CDCl3): d = ꢀ 77.0, ꢀ77.1 (ketones),
ꢀ82.8, ꢀ 83.1 (hydrates); HRMS (ESI): calcd for
C24H28F3N2O4 [M + H]+: 465.2001, found: 465.2001.
Notes: In this and many other TFMK related com-
pounds described, the NMR data are rather complex
due to the presence of diastereomers and ketone/hydrate
mixtures, which are not routinely separated.
4.3.15. 3-{N-[N-tert-Butoxycarbonyl-L-cGlu(OtBu)-L-
Ala]}-1,1,1-trifluoropropan-2-one (5d). Compound 5d
was prepared in a similar way to compound 5a, except
the starting material used was 4d (14% yield). Rf = 0.55
(EtOAc); 1H NMR (400 MHz, acetone-d6): d = 7.86 (m,
1H; NH), 7.60 (d, J = 6.3 Hz, 1H; NH), 6.52 (br d,
J = 30.2 Hz, 1H; OH), 6.26 (d, J = 7.9 Hz, 1H; NH),
4.50–4.41 (m, 1H; CHa), 4.04–3.99 (m, 1H; CHa), 3.57
(d, J = 5.8 Hz, 2H; CH2C(OH)2CF3), 3.10 (br s, 1H;
OH), 2.38–1.85 (m, 4H; CH2b(Glu)+ CH2c(Glu)), 1.44 (s,
9H; OC(CH3)3), 1.41 (s, 9H; OC(CH3)3), 1.34 (d,
J = 7.1 Hz, 3H; CH3b(Ala)); 13C NMR (100 MHz, ace-
tone-d6): d = 177.5, 173.8, 173.1, 157.3, 125.2 (q,
J = 285.7 Hz; CF3), 94.7 (q, J = 30.5 Hz; C(OH)2CF3),
82.3, 80.0, 55.6, 50.6, 45.7, 33.2, 31.4, 29.3, 28.9, 18.6;
HRMS
(ESI):
calcd
for
C20H34F3N3NaO8
[M+H2O+Na]+: 524.2196, found: 524.2222.
4.3.16. 3-(N-L-cGlu-L-Ala)-1,1,1-trifluoropropan-2-one
(5e). Compound 5d (7.2 mg) was dissolved in TFA
(5 mL) and stirred for 40.5 h. TFA was evaporated un-
der reduced pressure to give 5e (TFA salt) as a white so-
lid. 1H NMR (400 MHz, CD3OD): d = 4.30 (m, 1H;
CHa), 3.97 (m, 1H; CHa), 3.62 (m, 1H; CHa), 3.44 (m,
1H; CHa), 2.48 (t, J = 7.3 Hz, 2H; CH2c(Glu)), 2.20–
2.06 (m, 2H; CH2b(Glu)), 1.28 (d, J = 7.1 Hz, 3H;
CH3b(Ala)); 13C NMR (100 MHz, CD3OD): d = 176.8,
174.3, 171.6, 95.9, 53.7, 50.7, 42.4, 32.4, 27.2, 18.0; 19F
NMR (376 MHz, CD3OD): d = ꢀ 74.7, ꢀ75.0, ꢀ80.3,
ꢀ80.6; HRMS (ESI): calcd for C11H19F3N3O6
[M + H2O + H]+: 346.1226, found: 346.1066.
4.3.13. 3-[N-(N-Benzyloxycarbonyl-L-Phe)]-4-phenyl-1,1,1-
trifluorobutan-2-one (5b). Compound 5b was prepared in
a similar way to compound 5a, except the starting mate-
rial used was 4b (87% yield). Rf = 0.56 (hexanes/EtOAc
1:1; Notes: This compound has very similar mobility
to the precursor alcohol 4b, but stains very differently
4.3.17. 3-{N-[N-CH3(CH2)8(C@O)-L-Leu]}-4-phenyl-1,1,
1-trifluorobutan-2-one (5f). Compound 5f was prepared in
a similar way to compound 5a, except the starting mate-
rial used was 4e (85% yield). Rf = 0.41 (hexanes/EtOAc
2:1); 1H NMR (400 MHz, CDCl3): d = 7.33–7.14 (m,
5H; ArH), 6.38–5.27 (m, 2H; 2· NH), 5.10–4.13 (m,
2H; 2· CHa), 3.51–2.70 (m, 2H; CH2b(Phe)), 2.18–2.07
(m, 2H; CH2b(Leu)), 1.97–0.69 (m, 26H; CH3(CH2)8
C(@O)NH + CHc(Leu) + 2· CH3d(Leu)); 13C NMR
(100 MHz, CDCl3): d = 189.4 (q, J = 34.4 Hz), 174.8,
173.7, 136.1, 129.1, 128.5, 126.6, 123.2 (q, J =
287.5 Hz), 94.5 (q, J = 30.9 Hz), 55.3, 51.6, 40.4, 36.4,
34.2, 33.3, 31.8, 29.4, 29.3, 29.2, 29.1, 25.6, 24.6, 22.6,
22.1, 14.1; HRMS (ESI): calcd for C26H41F3N2NaO4
[M + H2O + Na]+: 525.2916, found: 525.2906.
1
with phosphomolybdic acid on silica TLC plates.); H
NMR (400 MHz, acetone-d6): d = 7.27–7.07 (m, 15H;
ArH), 6.61–6.40 (m, 2H; 2· NH), 4.98–4.92 (m, 2H;
PhCH2O), 4.43–4.34 (m, 2H; 2· CHa), 3.29–2.50 (m,
4H; 2· CH2b(Phe)); 13C NMR (100 MHz, acetone-d6):
d = 190.9–189.7 (C(=O)CF3), 175.1–171.7, 157.3–155.9,
139.5–137.1 (C(Ar)), 130.5–127.3 (CH(Ar)), 116.8 (q,
J = 291.6 Hz; CF3), 98.0–94.8 (q, J = 29.8 Hz;
C(OH)2CF3), 67.0, 57.0, 50.9, 38.7, 35.3; 19F NMR
(376 MHz, acetone-d6): d = ꢀ 71.5, ꢀ72.3 (ketones),
ꢀ76.8, ꢀ77.1 (hydrates); HRMS (ESI): calcd for
C27H26F3N2O4 [M + H]+: 499.1845, found: 499.1890.
4.3.18. 3-{N-[N-CH3(CH2)7(C@O)-L-Leu]}-4-phenyl-1,1,
1-trifluorobutan-2-one (5g). Compound 5g was prepared
in a similar way to compound 5a, except the starting
material used was 4f (70% yield). Rf = 0.26 (hexanes/
EtOAc 2:1); 1H NMR (400 MHz, CDCl3): d = 7.34–
7.14 (m, 5H; ArH), 7.10–5.65 (m, 2H; 2· NH), 5.36–
4.95 (m, 1H; CHa), 4.48–4.11 (m, 1H; CHa), 3.30–2.79
(m, 2H; CH2b(Phe)), 2.20–2.07 (m, 2H; CH2b(Leu)),
4.3.14. 3-[N-(N-tert-Butoxycarbonyl)-L-Leu]-1,1,1-triflu-
orobutan-2-one (5c). Compound 5c was prepared in a
similar way to compound 5a, except the starting mate-
rial used was 4c (86% yield). Rf = 0.24 (hexanes/EtOAc
2:1); 1H NMR (400 MHz, CDCl3): d = 7.30–7.07 (m,
1H; NH), 5.23–4.88 (m, 1H; NH), 4.25–4.00 (m, 2H;
2· CHa), 1.66–1.49 (m, 3H; CH2b(Leu)+ CHc(Leu)), 1.42
(s, 9H; OC(CH3)3), 1.34 (d, J = 6.9 Hz, 3H; CH3b(Ala)),
1.74–0.71 (m, 24H; CH3(CH2)7C(@O)NH + CHc(Leu)
+

Y.-M. Shao et al. / Bioorg. Med. Chem. 16 (2008) 4652–4660
4659
2· CH3d(Leu)); 13C NMR (100 MHz, CDCl3): d = 189.6,
References and notes
174.6, 172.1, 136.0, 129.1, 128.5, 126.7, 117.0, 94.6, 55.7,
51.5, 40.4, 36.5, 31.8, 29.7, 29.2, 29.1, 25.6, 24.6, 22.6,
14.0; HRMS (ESI): calcd for C25H39F3N2NaO4
[M + H2O + Na]+: 511.2760, found: 511.2763.
1. Ksiazek, T. G.; Erdman, D.; Goldsmith, C. S.; Zaki, S. R.;
Peret, T.; Emery, S.; Tong, S.; Urbani, C.; Comer, J. A.;
Lim, W.; Rollin, P. E.; Dowell, S. F.; Ling, A.-E.;
Humphrey, C. D.; Shieh, W.-J.; Guarner, J.; Paddock,
C. D.; Rota, P.; Fields, B.; DeRisi, J.; Yang, J.-Y.; Cox,
N.; Hughes, J. M.; LeDuc, J. W.; Bellini, W. J.; Anderson,
L. J.the SARS working group N. Engl. J. Med. 2003, 348,
1953–1966.
2. Peiris, J. S. M.; Lai, S.-T.; Poon, L. L.-M.; Guan, Y.;
Yam, L. Y.-C.; Lim, W.; Nicholls, J.; Yee, W. K.-S.; Yan,
W. W.; Cheung, M.-T.; Cheng, V. C.-C.; Chan, K.-H.;
Tsang, D. N.-C.; Yung, R. W.-H.; Ng, T. K.; Yuen,
K.-Y.members of the SARS study group Lancet
2003, 361, 1319–1325.
3. Rota, P. A.; Oberste, M. S.; Monroe, S. S.; Nix, W. A.;
Campagnoli, R.; Icenogle, J. P.; Pen˜aranda, S.; Bankamp,
B.; Maher, K.; Chen, M.-H.; Tong, S.; Tamin, A.; Lowe,
L.; Frace, M.; DeRisi, J. L.; Chen, Q.; Wang, D.; Erdman,
D. D.; Peret, T. C. T.; Burns, C.; Ksiazek, T. G.; Rollin, P.
E.; Sanchez, A.; Liffick, S.; Holloway, B.; Limor, J.;
McCaustland, K.; Olsen-Rasmussen, M.; Fouchier, R.;
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4.3.19. 3-{N-[N-Benzyloxycarbonyl-L-Ala-L-Val-L-Leu]}-
4-phenyl-1,1,1-trifluorobutan-2-one (5h). Compound 5h
was prepared in a similar way to compound 5a, except
the starting material used was 4g (67% yield).
Rf = 0.32 (hexanes/EtOAc 1:1); 1H NMR (400 MHz,
CD3OD): d = 7.34–7.12 (m, 10H; ArH), 5.09 (m, 2H;
OCH2Ph), 4.61 (br s, 2H; C(OH)2CF3), 4.57–4.00 (m,
4H; 4 · CHa), 3.27–3.04 (m, 1H; CHbH0b(Phe)), 2.80–
2.68 (m, 1H; CHbH0b(Phe)), 2.02 (m, 1H; CHb(Val)),
1.38–0.70 (m, 18H; 5 · CH3+ CH2b(Leu) + CHc(Leu));
13C NMR (100 MHz, CD3OD): d = 175.9, 174.5,
173.9, 158.5, 139.3, 138.1, 130.7, 129.6, 129.5, 129.2,
129.0, 127.5, 97.3 (q, J = 29.0 Hz), 67.9, 60.2, 55.6,
53.0, 52.2, 41.6, 35.4, 31.9, 25.7, 23.6, 21.9, 19.8, 18.7,
18.3; HRMS (ESI): calcd for C32H43F3N4NaO7
[M + H2O+Na]+: 675.2982, found: 675.3005.
4.4. Computer modeling
4. Marra, M. A.; Jones, S. J. M.; Astell, C. R.; Holt, R. A.;
Brooks-Wilson, A.; Butterfield, Y. S. N.; Khattra, J.;
Asano, J. K.; Barber, S. A.; Chan, S. Y.; Cloutier, A.;
Coughlin, S. M.; Freeman, D.; Girn, N.; Griffith, O. L.;
Leach, S. R.; Mayo, M.; McDonald, H.; Montgomery, S.
B.; Pandoh, P. K.; Petrescu, A. S.; Robertson, A. G.;
Schein, J. E.; Siddiqui, A.; Smailus, D. E.; Stott, J. M.;
Yang, G. S.; Plummer, F.; Andonov, A.; Artsob, H.;
Bastien, N.; Bernard, K.; Booth, T. F.; Bowness, D.;
Czub, M.; Drebot, M.; Fernando, L.; Flick, R.; Garbutt,
M.; Gray, M.; Grolla, A.; Jones, S.; Feldmann, H.;
Meyers, A.; Kabani, A.; Li, Y.; Normand, S.; Stroher, U.;
Tipples, G. A.; Tyler, S.; Vogrig, R.; Ward, D.; Watson,
B.; Brunham, R. C.; Krajden, M.; Petric, M.; Skowronski,
D. M.; Upton, C.; Roper, R. L. Science 2003, 300, 1399–
1404.
The crystal structure of SARS-CoV 3CL protease in
complex with a substrate-analog inhibitor (coded
1uk4) was obtained from The Protein Data Bank
(PDB; http://www.rcsb.org/pdb/). We constructed four
stereomeric compound 5h complex as hemithioketal
(DISCOVERY STUDIO 1.7) to determine which iso-
mer can form the protein-inhibitor adduct. GOLD
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formational flexibility. The atoms of the enzyme and
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charges25 with SYBYL 7.3 program.26 To distinguish
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complex, the carbonyl carbon adjacent to the CF3
group of compound 5h was constrained to form a cova-
lent bonding with the sulfur atom of Cys-145. Initial
1000 independent genetic algorithm running cycles
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Acknowledgments
This work is supported by National Science Council,
Taiwan and Genomics Research Center, Academia Sini-
ca. The SYBYL computation was conducted at the Na-
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