Synthesis of glutamic acid and glutamine peptides possessing a trifluoromethyl ketone group as SARS-CoV 3CL protease inhibitors

Article abstract of DOI:10.1016/j.tet.2006.06.052

Trifluoromethyl-β-amino alcohol 11 [(4S)-tert-butyl 4-amino-6,6,6-trifluoro-5-hydroxyhexanoate] was synthesized in five steps starting from Cbz-l-Glu-OH 5 where the key step involved the introduction of the trifluoromethyl (CF₃) group to oxazolidinone 7, resulting in the formation of silyl ether 8 [(4S,5S)-benzyl 4-(2-(tert-butoxycarbonyl)ethyl)-5-(trifluoromethyl)-5-(trimethylsilyloxy)oxazolidine-3-carboxylate]. Compound 11 was then converted into four tri- and tetra-glutamic acid and glutamine peptides (1-4) possessing a CF₃-ketone group that exhibited inhibitory activity against severe acute respiratory syndrome coronavirus protease (SARS-CoV 3CL^pro).

Full text of DOI:10.1016/j.tet.2006.06.052

Tetrahedron 62 (2006) 8601–8609
Synthesis of glutamic acid and glutamine peptides possessing
a trifluoromethyl ketone group as SARS-CoV 3CL
protease inhibitors
a
a
b
Magne O. Sydnes,a Yoshio Hayashi,a, Vinay K. Sharma, Takashi Hamada, Usman Bacha,
*
Jennifer Barrila,b Ernesto Freireb and Yoshiaki Kisoa,
*
aDepartment of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, 21st Century COE Program,
Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
bDepartment of Biology, Johns Hopkins University, Baltimore, MD, USA
Received 9 May 2006; revised 13 June 2006; accepted 14 June 2006
Available online 14 July 2006
Abstract—Trifluoromethyl-b-amino alcohol 11 [(4S)-tert-butyl 4-amino-6,6,6-trifluoro-5-hydroxyhexanoate] was synthesized in five steps
starting from Cbz-L-Glu-OH 5 where the key step involved the introduction of the trifluoromethyl (CF3) group to oxazolidinone 7, resulting in
the formation of silyl ether 8 [(4S,5S)-benzyl 4-(2-(tert-butoxycarbonyl)ethyl)-5-(trifluoromethyl)-5-(trimethylsilyloxy)oxazolidine-3-car-
boxylate]. Compound 11 was then converted into four tri- and tetra-glutamic acid and glutamine peptides (1–4) possessing a CF3-ketone group
that exhibited inhibitory activity against severe acute respiratory syndrome coronavirus protease (SARS-CoV 3CLpro).
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
the carbonyl prone to nucleophilic substitution by water, the
active site Ser hydroxyl or Cys thiol group present in serine
or cysteine proteases. Nucleophilic attack by the active site
thiol in SARS-CoV 3CLpro would convert the CF3-ketone
A to the tetrahedral adduct B (Scheme 1), which is believed
to mimic the substrate–enzyme intermediate formed during
substrate peptide-bond hydrolysis. Since adduct B is rela-
tively stable, compound Awould behave as a protease inhib-
itor,8 suggesting that compounds containing a CF3-ketone
moiety may play an important role as 3CLpro inhibitors.
CF3-ketone A also forms a relatively stable hydrate adduct
C upon reacting with water. A unique and conservative
recognition of the substrate’s Gln residue at the P1 site has
been identified in the CoV cysteine protease family.9 There-
fore, a Gln-derived CF3-ketone residue would contribute to
the activity of SARS-CoV 3CLpro inhibitors. Based on these
considerations, a new synthetic method for forming Gln and
Glu derivatives possessing a CF3-ketone moiety was devel-
oped and this strategy was used in the synthesis of four
peptides (compounds 1–4).
In May 2003, two groups reported that a novel coronavirus
(CoV) was the causative agent of severe acute respiratory
syndrome (SARS).1,2 CoVencodes a chymotrypsin-like pro-
tease (3CLpro) that plays a pivotal role in the replication of
the virus.3 3CLpro is functionally analogous to the main
picornavirus protease 3Cpro and both are cysteine proteases
with a catalytic dyad (Cys-145 and His-41) in the active
site, with Cys as the nucleophile and His as the general
base.4,5 Although a global SARS crisis was avoided in
2003 and the infection was contained, it is still a matter of
necessity to find compounds that can inhibit SARS-CoV in
case that the disease might re-emerge.
Compounds containing a trifluoromethyl ketone (CF3-
ketone) moiety form an important group of biologically
useful fluorinated molecules6 that can be used as protease in-
hibitors, as first described by Abeles et al.7 The CF3 group
next to the carbonyl group thermodynamically stabilizes
the hemi-ketal form relative to the ketone form, thus making
H2O
Enz-SH
O
Enz-S OH
CF3
HO OH
CF3
Keywords: Trifluoromethyl ketone; Protease inhibitors; Severe acute respi-
ratory syndrome coronavirus protease (SARS-CoV 3CLpro).
R
R
CF3
R
* Corresponding authors. Tel.: +81 75 595 4636; fax: +81 75 595 4787
(Y.H.); tel.: +81 75 595 4635; fax: +81 75 591 9900 (Y.K.); e-mail
addresses: yhayashi@mb.kyoto-phu.ac.jp; kiso@mb.kyoto-phu.ac.jp
B
A
C
Scheme 1. Trifluoromethyl ketone adducts.
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.06.052

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M. O. Sydnes et al. / Tetrahedron 62 (2006) 8601–8609
2. Results and discussion
target compound 10 as a ca. 4.5:1 mixture of diastereomers,
as determined by 1H and 13C NMR analyses, in 69% yield.
Among the different synthetic routes tried, treating a metha-
nol solution of silyl ether 8 with NaBH4 seems to be an effi-
cient route to synthesize alcohol 10. Under these conditions,
we obtained the desired compound 10 in 68% yield (Scheme
2). Finally, the protecting group within substrate 10 could be
easily cleaved off by hydrogenation over Pd/C (10%) afford-
ing alcohol 11 in quantitative yield.
2.1. Synthesis of trifluoromethyl-b-amino alcohol 11
The target compounds were envisioned being synthesized in
two parts, viz, the peptide part and b-amino alcohol 11 con-
taining the CF3 unit. These two parts would then be coupled
together and further elaborated to the desired target com-
pounds. The synthesis of the key compound 11 started
with oxazolidinone acid 6 prepared from Cbz-L-Glu-OH
(5) under conditions described by Moore et al.10 The result-
ing acid 6 was then converted to tert-butyl ester 7 (45%) that
was expediently converted to silyl ether 8 (92% yield)
(Scheme 2), which was isolated as a single diastereomer as
determined by 1H and 13C NMR analyses, by utilizing a lit-
erature method.11,12 The depicted stereochemistry for com-
pound 8 is based on literature precedence for a very similar
compound in which the addition of the CF3 anion is anti to
the side chain.11 Product 8 was then readily desilylated
upon treatment with tetrabutylammonium fluoride (TBAF)
giving alcohol 9 in 77% yield.
2.2. Synthesis of glutamic acid and glutamine peptides
with a CF3-ketone unit
With compound 11 prepared, focus could now shift toward
the synthesis of the acid component coupling partners,
namely peptides 12, 14, and 15. Protected dipeptides 12
and 15 could be prepared following literature procedures14,15
while tripeptide 14 could be prepared from dipeptide 12 as
outlined in Scheme 3. The Cbz group within compound 12
could be removed using standard hydrogenation conditions,
thus giving dipeptide 13 that was used directly in the next
step. Coupling compound 13 with Cbz-L-Ala-OSu16 af-
forded dipeptide 14 in 67% yield over the two steps.
t
CO2R
O
CO2 Bu
CO2H
CO2H
a
c
Cbz
b
H
N
H
N
Cbz
h
CF3
OSi(CH3)3
Cbz
N
N
Cbz
CO2H
CO2H
a
N
H
N
H
H2N
O
O
O
O
5
6 R = H, quant.
8 92%
d or e
t
12
13
t
7 R = Bu, 45%
b
f, g
Ph
t
t
CO2 Bu
CO2 Bu
CO2 Bu
H
N
O
H
N
H
N
Cbz
CO2H
g
i
CO2H
N
Cbz
N
H
H
Cbz
CF3
OH
CF3
OH
Cbz
CF3
OH
O
N
H2N
N
O
H
15
O
14 67% from 12
11 quant.
10 50% from 7
9 condition d, 77%
68% from 8
condition e, quant.
Scheme 3. Synthesis of tripeptide 14 from dipeptide 12. Reagents and
conditions: (a) H2, Pd/C (10%), MeOH/water/AcOH (9.5:5:1), rt, 2 h; (b)
Cbz-L-Ala-OSu,16 Et3N, DMF, 0 ꢀC–rt, 16 h.
69% from 9
Scheme 2. Synthesis of b-amino alcohol 11. Reagents and conditions: (a)
paraformaldehyde, p-TsOH$H2O, toluene, reflux, 1.67 h; (b) t-BuOH,
EDC$HCl, DMAP, Et3N, THF, rt, 16 h; (c) CsF, CF3Si(CH3)3, THF, amb.
temp, sonication, 2 h; (d) TBAF, THF, 0 ꢀC–rt, 0.5 h; (e) MeOH/water
(9:1), rt, 3 h; (f) CsF, CF3Si(CH3)3, THF, sonication, amb. temp, 2 h then
water, sonication, amb. temp, 0.5 h; (g) NaBH4, MeOH, rt, 21 h; (h)
NaBH4, MeOH, rt, 16 h; (i) H2, Pd/C (10%), MeOH, rt, 16 h.
Coupling of peptide 12 with b-amino alcohol 11 gave the ex-
pected amide (Scheme 4) that was used directly in the next
step affording ketone 16. Peptides 14 and 15 were subjected
to the exactly same reaction sequences giving ketones 17
and 18.
We observed that compound 8 was partly converted to the
desilylated product 9 when exposed to air. The cause of
the partial protio-desilylation might be due to the mois-
ture-sensitive nature of compound 8. In the patent literature,
there is one report of desilylation occurring upon stirring
similar compounds in methanol,13 most likely caused by
water present in the methanol. For our substrate, we found
that this method only proceeded when the reaction was car-
ried out on a small scale (20 mg or less). However, by adding
water to the methanol [methanol/water (9:1 v/v)], substrate 8
could be fully converted to compound 9 after 3 h stirring at
room temperature (Scheme 2).
t
CO2 Bu
CO2H
c
a, b
11
R
CF3
O
R
CF3
O
N
H
N
H
16, R = Cbz-L-Val-L-Leu
17, R = Cbz-L-Phe-L-Ala
18, R = Cbz-L-Ala-L-Val-L-Leu
1, R = Cbz-L-Val-L-Leu
19, R = Cbz-L-Phe-L-Ala
20, R = Cbz-L-Ala-L-Val-L-Leu
d or e
2, R = Cbz-L-Val-L-Leu,
8% from 12
CONH2
3, R = Cbz-L-Phe-L-Ala,
5% from 15
4, R = Cbz-L-Ala-L-Val-L-Leu,
4% from 14, Method A
R
CF3
N
H
12% from 14, Method B
Compound 7 was also converted to the corresponding alco-
hol 9 (72% yield) in a one-pot reaction by adding small
amount of water to the reaction mixture of intermediate 8
followed by sonication for an additional half hour. Finally,
the desired alcohol 10 was obtained by treating compound
9 with NaBH4 in methanol at room temperature. This gave
O
Scheme 4. The final steps toward the target compounds. Reagents and con-
ditions: (a) peptide (12, 14 or 15), HOBt, EDC$HCl, DMF, 0 ꢀC–rt, 21 h; (b)
Dess–Martin periodinane, CH2Cl2, rt, 19 h; (c) TFA, CH2Cl2, rt, 16 h; (d)
HOBt, EDC$HCl, ammonia solution (28% aq solution), DMF, 16 h (Method
A); (e) Boc2O, NH4HCO3, pyridine, 1,4-dioxane, rt, 23 h (Method B).

M. O. Sydnes et al. / Tetrahedron 62 (2006) 8601–8609
8603
Treating compound 16 with trifluoroacetic acid (TFA) re-
sulted in clean removal of the tert-butyl group forming tri-
peptide 1. Examination of the 13C NMR spectrum did
reveal that inhibitor 1 exists predominantly as the hydrate
form in CDCl3 (containing one drop of DMSO-d6). 19F
NMR analysis of the same sample not only showed that
the hydrate form was the dominant tautomer in the sample
but that the two other possible tautomeric forms of tripeptide
1 were also present in small amount.17 The equilibrium be-
tween the different tautomeric forms of this compound
might shift depending on solvent. Due to the small amount
of compound available, it was decided to study this in
more detail by using a simpler model compound (vide infra).
keto and hydrate forms as evident from 19F and 13C NMR
analyses (Scheme 5). Attempts to convert compound 21 to
the free acid 22 only resulted in the formation of decompo-
sition products.
The lack of stability for our desired model compound forced
us to use a slightly more complex acid for these studies.
Compound 25 was synthesized in a three-step process, as
outlined in Scheme 5, by first coupling Cbz-L-Ala-OH
with amine 11. This gave the desired alcohol 23, which
was directly oxidized to ketone 24 (52% yield over the
two steps). From the 13C and 19F NMR analyses of this
ketone, it became evident that the ketone exists as a ca.
7:3 mixture of the hydrate and keto forms. The rather unsta-
ble ketone 24 was then deprotected giving dipeptide 25 in al-
most quantitative yield in ca. 90% purity as determined by
HPLC analysis. The 13C NMR spectrum suggested that com-
pound 25 exists mostly as the cyclic hemiacetal in CDCl3
(resonance shifts from >170 to 75 ppm). This was also the
case when the 13C NMR spectrum was obtained for the
same sample in CD3OD.
Compounds 17 and 18 were subjected to the exactly same re-
action conditions as ester 16 affording peptides 19 and 20.
The remaining crude tripeptide 1 and peptides 19 and 20
were subjected directly to the coupling conditions outlined
in Scheme 4 (Method A), thus giving products 2–4 in 8, 5,
and 4% yield over the four steps, respectively, after HPLC
purification. The low chemical yield for the target com-
pounds is a result of the last reaction sequence that seems
to be very inefficient giving rise to many side products. In
an effort to improve the yield for the last step, compound 4
was prepared by a mixed anhydride strategy using a slightly
modified literature procedure (Method B).18 By such means,
we were able to improve the overall yield of inhibitor 4, from
peptide 14, from 4 to 12%.
NMR studies of model compounds 21, 24, and 25 in the pre-
dominant keto, hydrate, and hemiacetal forms, respectively,
supported our assignment of compound 1 as existing mainly
in the hydrate form in CDCl3. This evidence was derived
from the 19F and 13C NMR spectra of ketones 21 and 24
that were both present as a mixture of the keto and hydrate
forms.23 The work with the model compound also suggests
that the form these acids appear in solution is highly solvent-
and concentration-dependent.
19F NMR studies of the three glutamine peptides showed that
compounds 2 and 4 only existed in the cyclic form while tri-
peptide 3 was a ca. 3.3:1 mixture of the cyclic and keto forms
in CDCl3.19 Recently, similar observations were reported for
glutamine fluoromethyl ketones by Cai et al.20 Previously,
there have also been reports that glutaminal compounds
mostly exist as the hemiaminal in organic solvent.21,22
2.4. Inhibitory activity of synthesized compounds
The inhibitory activity of the target compounds against
SARS-CoV 3CLpro was tested using a fluorescence-based
peptide cleavage assay (Table 1).23 We originally thought
that the glutamine peptides (compounds 2–4) would be the
more potent inhibitors in these assays. However, the gluta-
mate-possessing inhibitor 1 was the most potent of the group.
The conformation that these compounds exist in during their
interaction with the active site of SARS-CoV 3CLpro is be-
lieved to contribute to binding affinity. Cai and co-workers
found that their Gln fluoromethyl ketones exhibited low ac-
tivity in their assays, a fact which they explained by referring
to that their inhibitors predominantly exist in the cyclic form
as evident from NMR studies.20 Indeed, the cyclic form is
2.3. Synthesis of model Glu-CF3 compounds
As previously noted, target peptide 1 was predominantly
present in the hydrate form in CDCl3. However, as alluded
to in the previous section, this might differ depending on
the solvent used for the NMR studies. Therefore, we decided
to synthesize acid 22, which is a much simpler molecule than
the real system but, nevertheless, thought to be a good model
for this study. To this end, alcohol 10 was converted to
ketone 21 in 81% yield and as a ca. 2:1 mixture of the
t
t
t
t
t
CO2 Bu
CO2 Bu
CO2 Bu
CO2 Bu
CO2 Bu
O
O
O
H
N
H
N
H
N
c
a
a
CF3
OH
CF3
O
CF3
Cbz
CF3
Cbz
Cbz
CF3
Cbz
N
H
Cbz
N
H
10
11
Cbz
N
H
N
H
N
H
HO OH
HO OH
O
21 81%
b
23
24 52% from amine 11
b
CO2H
CO2H
CO2H
CF3
O
R
O
N
CF3
R
CF3
R
N
H
N
H
N
H
HO CF3
H
HO OH
O
O
R = Cbz-L-Ala
22
25 99%
Scheme 5. Attempted synthesis of model compound 22 and synthesis of model compound 25. Reagents and conditions: (a) Dess–Martin periodinane, CH2Cl2,
rt, 16 h; (b) TFA, CH2Cl2, rt, 16 h; (c) HOBt, EDC$HCl, Cbz-L-Ala-OH, DMF, rt, 21 h.

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M. O. Sydnes et al. / Tetrahedron 62 (2006) 8601–8609
Table 1. Inhibitory activity of peptides against the SARS-CoV 3CLpro
HPLC. Solvents used for HPLC were of HPLC grade and all
other chemicals were of analytical grade or better.
Compound
Structure
Ki (mM)
1
2
3
4
Cbz-Val-Leu-Glu-CF3
Cbz-Val-Leu-Gln-CF3
Cbz-Phe-Ala-Gln-CF3
Cbz-Ala-Val-Leu-Gln-CF3
116.1ꢃ13.6
>1000
4.1.1. (S)-3-[3-(Benzyloxycarbonyl)-5-oxooxazolidin-4-
yl]propanoic acid 6.10 This compound was synthesized
according to the procedure in Ref. 10. [a]2D6 +80.5 (c 3.9,
MeOH) {lit.24 [a]2D5 +73 (c 2.35, MeOH)}.
844.4ꢃ120.3
134.5ꢃ31.6
not expected to interact effectively with the active site of
SARS-CoV.20 The Gln compounds synthesized in our study
were also found to be mainly in the cyclic form which may
explain the low biological activity for these compounds.
However, the Glu inhibitor 1 was found to mainly exist in
the hydrate form, which is a form that most likely will inter-
act more effectively with the active site.
4.1.2. (S)-tert-Butyl-3-[3-benzyloxycarbonyl-5-oxooxazo-
lidin-4-yl]propanoate 7. DMAP (428 mg, 3.50 mmol) and
EDC$HCl (1.83 g, 9.56 mmol) were added to a stirred
solution of oxazolidinone acid 6 (2.26 g, 7.78 mmol) and
t-BuOH (2.2 mL, 23.0 mmol) in THF (80 mL) at room
temperature. The reaction mixture was then stirred for
5 min before triethylamine (1.1 mL, 7.89 mmol) was added
dropwise. The reaction mixture was then stirred for 16 h
before being diluted with EtOAc (100 mL) and washed
with citric acid (2ꢂ50 mL of a 5% aq solution), NaHCO3
(2ꢂ50 mL of a 5% aq solution) and brine (2ꢂ50 mL) before
being dried (Na2SO4). Filtration and concentration under re-
duced pressure gave a light-yellow oil, which was subjected
to flash chromatography (silica, hexane/hexane/EtOAc
9:1/4:1 gradient eluent). Concentration of the relevant
fractions (Rf 0.3 in hexane/EtOAc 4:1) gave the title com-
pound 725 (1.22 g, 45%) as a clear, colorless oil: [a]2D5
+63.2 (c 3.86, EtOH) {lit.25 [a]D22 +27.9 (c 1.58, EtOH)};
1H NMR (300 MHz, CDCl3) d 7.41–7.30 (m, 5H), 5.54
(br s, 1H), 5.22 (d, J¼4.8 Hz, 1H), 5.19 (s, 2H), 4.37
(t, J¼5.3 Hz, 1H), 2.37–2.11 (m, 4H), 1.43 (s, 9H); MS
(ESI+) m/z 372 (M++Na, 100%).
3. Conclusion
A simple five-step procedure for the synthesis of b-amino
alcohol 11 containing a CF3 group was developed. This alco-
hol was further elaborated into four tri- and tetra-Glu and
Gln peptides. Compounds 1 and 4 were found to be moderate
SARS-CoV 3CLpro inhibitors. Current work is focused on
the co-crystallization of compounds 1 and 4 with SARS-
CoV 3CLpro in an attempt to elucidate their mode of action.
4. Experimental
4.1. General procedures
Melting points were measured on a Yanagimoto micro hot-
stage apparatus and are uncorrected. Proton (1H) and carbon
(13C) NMR spectra were recorded on either a JEOL JNM-
AL300 spectrometer operating at 300 MHz for proton and
75 MHz for carbon, or a Varian UNITY INOVA 400NB
spectrometer operating at 400 MHz for proton and
101 MHz for carbon. Chemical shifts were recorded as
d values in parts per million (ppm) downfield from tetra-
methylsilane (TMS). Fluorine (19F) NMR spectra were re-
corded on a Varian UNITY INOVA 400 spectrometer
operating at 376 MHz for fluorine. Fluorine NMR spectra
were referenced externally to C6F6 at 0.00 ppm. Low-resolu-
tion mass spectra (ESI) were recorded on a Finnigan SSQ-
7000 spectrophotometer. Low- and high-resolution mass
spectra (FAB) were recorded on a JEOL JMS-SX102A spec-
trometer equipped with JMA-DA7000 data system. Low-
and high-resolution mass spectra (CI) were recorded on
a JEOL JMS-GCmate. Optical rotations were measured
with a Horiba High-speed Accurate Polarimeter SEPA-300
at the sodium-D line (589 nm) at the concentrations (c,
g 100 mLꢁ1). The measurements were carried out between
22 and 28 ꢀC in a cell with path length (l) of 0.5 dm. Specific
rotations [a]D are given in 10ꢁ1 deg cm2 gꢁ1. Preparative
HPLC was carried out on a C18 reverse phase column
(20ꢂ250 mm; YMC Pack ODS SH343-5) with a binary sol-
vent system (a linear gradient of CH3CN and aq TFA (0.1%)
at a flow rate of 5.0 mL minꢁ1), detected at 230 nm. Analyt-
ical HPLC was performed using a C18 reverse phase column
(4.6ꢂ150 mm; YMC Pack ODS AM302) with a binary sol-
vent system (a linear gradient of CH3CN and aq TFA (0.1%)
at a flow rate of 0.9 mL minꢁ1), detected at 230 nm. The tR
given for the target compounds are obtained from analytical
4.1.3. (4S,5S)-Benzyl 4-[2-(tert-butoxycarbonyl)ethyl]-
5-trifluoromethyl-5-(trimethylsilyloxy)oxazolidine-3-
carboxylate 8. Cesium fluoride (87.8 mg, 0.58 mmol) and
(trifluoromethyl)trimethylsilane (0.73 mL, 4.94 mmol)
were added to a solution of oxazolidinone 7 (986.0 mg,
3.98 mmol) in dry THF (20 mL) maintained under an argon
atmosphere. The reaction mixture was then sonicated for 2 h
at ambient temperature before being diluted with EtOAc
(40 mL). The resulting solution was washed with water
(1ꢂ20 mL) and brine (1ꢂ20 mL) before being dried
(MgSO4). Filtration and concentration under reduced pres-
sure gave the title compound 8 (1.80 g, 92%) as a clear,
1
yellow oil, which was >95% pure (as judged by H NMR
analysis): [a]D28 +37.8 (c 1.26, CHCl3); 1H NMR
(400 MHz, CDCl3) d 7.36–7.27 (m, 5H), 5.41–5.29 (m,
1H), 5.14 (s, 2H), 4.83 (br s, 1H), 4.37 (br s, 1H), 2.32
(app. br s, 2H), 1.94 (6, J¼7.0 Hz, 1H), 1.79 (app. br s,
1H), 1.41 (s, 9H), 0.20 (s, 9H); 13C NMR (101 MHz,
CDCl3) d 172.0, 153.9, 135.7, 128.5, 128.2, 127.9, 122.2
(q, JC–F¼287.3 Hz), 102.2 (br), 80.3, 77.8, 67.8, 59.1,
31.7, 28.0, 23.9, 1.0; MS (CI+) m/z 492 (M++H, 1%), 436
(10), 401 (4), 392 (6), 334 (7),107 (7), 91 (100), 57 (47);
HRMS (CI+): calcd for C22H33NO6F3Si (M++H)
492.2029, found 492.2030.
4.1.4. (4S,5R)-Benzyl 4-[2-(tert-butoxycarbonyl)ethyl]-5-
trifluoromethyl-5-hydroxyoxazolidine-3-carboxylate 9.
Method A: TBAF (0.11 mL of a 1 M solution in THF,
0.11 mmol) was added dropwise to a stirred solution of com-
pound 8 (44.5 mg, 0.091 mmol) in THF (2.0 mL) at 0 ꢀC.
The reaction mixture was then allowed to heat to room tem-
perature and stirred for 0.5 h before being diluted with

M. O. Sydnes et al. / Tetrahedron 62 (2006) 8601–8609
8605
EtOAc (15 mL). The organic phase was washed with water
(2ꢂ10 mL) and brine (1ꢂ10 mL) before being dried
(MgSO4). Filtration and concentration under reduced pres-
sure gave a yellow oil, which was subjected to flash chroma-
tography (silica, hexane/EtOAc 4:1 eluent). Evaporation of
the relevant fractions (Rf 0.2) gave the title alcohol 9
(29.3 mg, 77%) as a clear, yellow oil: [a]2D6 +41.0 (c 0.97,
CHCl3); 1H NMR (400 MHz, CDCl3) d 7.40–7.30 (m,
5H), 5.34 (br s, 1H), 5.17 (s, 2H), 4.88 (d, J¼4.8 Hz, 1H),
4.40 (t, J¼6.7 Hz, 1H), 2.40 (t, J¼7.1 Hz, 2H), 2.14–1.97
(m, 2H), 1.43 (s, 9H); 13C NMR (101 MHz, CDCl3)
d 173.5, 153.9, 135.6, 128.6, 128.4, 128.0, 122.4 (q,
JC–F¼286.1 Hz), 101.1 (q, JC–F¼33.2 Hz), 81.4, 77.9,
67.9, 58.3, 31.2, 28.0, 22.8; MS (CI+) m/z 420 (M++H,
2%), 364 (15), 91 (100); HRMS (CI+): calcd for
C19H25NO6F3 (M++H) 420.1634, found 420.1633. Method
B: A solution of silyl ether 8 (129.3 mg, 0.26 mmol) in
MeOH (4.5 mL) and water (0.5 mL) was stirred at room
temperature for 3 h. The reaction mixture was then concen-
trated under reduced pressure to give the title alcohol 9
(110.2 mg, quant.), which was identical, in all respects,
with the material obtained by Method A. The product was
>95% pure (as judged by 1H NMR analysis).
23.7; MS (CI+) m/z 392 (M++H, 3%), 336 (27), 292 (12),
91 (100); HRMS (CI+): calcd for C18H25NO5F3 (M++H)
392.1684, found 392.1689.
4.1.7. Synthesis of compound 10 from compound 8.
Sodium borohydride (78.8 mg, 2.08 mmol) was added to
a stirred solution of silyl ether 8 (89.0 mg, 0.18 mmol) in
MeOH (5.0 mL) at room temperature. The reaction mixture
was then stirred at room temperature for 16 h before being
quenched by addition of water (5.0 mL). The water phase
was extracted with EtOAc (3ꢂ15 mL) and the combined or-
ganic fractions were dried (MgSO4). Filtration and concen-
tration under reduced pressure gave a light-yellow oil, which
was subjected to flash chromatography (silica, hexane/
EtOAc 3:1 eluent). Concentration of the relevant fractions
(Rf 0.2) gave the title alcohol 10 (48.3 mg, 68%) as a viscous
colorless oil and as a ca. 4.5:1 mixture of diastereomers (as
judged by 1H and 13C NMR analyses). The material obtained
via this method was identical, in all respects, with the mate-
rial obtained via the reduction of alcohol 9.
4.1.8. (4S)-tert-Butyl 4-amino-6,6,6-trifluoro-5-hydroxy-
hexanoate 11. Amine 10 (204.3 mg, 0.52 mmol) and Pd/C
(10%) (21.0 mg) were stirred vigorously for 16 h in MeOH
(6.0 mL) under an atmosphere of H2. The reaction mixture
was then diluted with MeOH (10 mL) and filtered through
a plug of CeliteÒ and washed afterwards with MeOH
(3ꢂ10 mL). Concentration of the filtrate under reduced pres-
sure gave the title amine 11 (134.3 mg, quant.) as a white
solid: mp 93–95 ꢀC and as a ca. 4.5:1 mixture of diastereo-
4.1.5. One-pot synthesis of compound 9 from compound
7. Method C: Cesium fluoride (37.9 mg, 0.25 mmol) and
(trifluoromethyl)trimethylsilane (0.31 mL, 2.02 mmol)
were added to a solution of oxazolidinone 7 (427.0 mg,
1.72 mmol) in dry THF (9.0 mL) maintained under an argon
atmosphere. The reaction mixture was then sonicated for 2 h
at ambient temperature before water (0.30 mL) was added
and the reaction mixture was sonicated for an additional
0.5 h. The reaction mixture was then diluted with EtOAc
(30 mL) and washed with water (1ꢂ10 mL) and brine
(1ꢂ10 mL) before being dried (MgSO4). Filtration and con-
centration under reduced pressure gave the title alcohol 9
(520.8 mg, 72%), which was identical, in all respects, with
the material obtained via the stepwise method.
1
1
mers (as judged by H and 13C NMR analyses): H NMR
(400 MHz, CDCl3) d 5.50–4.20 (br s, 3H), 4.10–3.85 (m,
1H), 3.54 (app. br s, 0.18H), 3.39 (app. br s, 0.82H), 2.55–
2.29 (m, 2H), 2.17–1.85 (m, 2H), 1.45 (s, 9H); 13C NMR
(101 MHz, CDCl3) d 172.7, 172.2, 125.3 (q, JC–F
¼
284.0 Hz), 125.2 (q, JC–F¼283.4 Hz), 81.0, 80.8, 71.4 (q,
JC–F¼28.6 Hz), 70.2 (q, JC–F¼29.4 Hz), 51.6, 48.3, 32.3,
32.0, 29.7, 28.0 (2), 27.9 (9); MS (CI+) m/z 258 (M++H,
12%), 242 (8), 202 (36), 102 (100); HRMS (CI+): calcd
for C10H19NO3F3 (M++H) 258.1317, found 258.1319.
Anal. Calcd for C10H18F3NO3$0.5H2O: C, 45.11; H, 7.19;
N, 5.26. Found: C, 45.27; H, 6.79; N, 5.44.
4.1.6. (4S)-tert-Butyl 4-(benzyloxycarbonyl)amino-6,6,6-
trifluoro-5-hydroxyhexanoate 10. Sodium borohydride
(0.76 g, 20.09 mmol) was added to a stirred solution of alco-
hol 9 (1.09 g, 2.60 mmol) in THF (70 mL) under an atmo-
sphere of argon. The resulting reaction mixture was stirred
for 23 h before being quenched by addition of water
(10 mL). The water phase was then extracted with EtOAc
(3ꢂ30 mL) and the combined organic fractions were dried
(MgSO4). Filtration and concentration under reduced pres-
sure gave a light-yellow oil, which was subjected to flash
chromatography (silica, hexane/EtOAc 3:1 eluent). Concen-
tration of the relevant fractions (Rf 0.2) gave the title alcohol
10 (702.1 mg, 69%) as a clear, viscous, colorless oil and as
4.1.9. N-Benzyloxycarbonyl-L-valyl-L-leucine 12.14 This
compound was synthesized according to the procedure in
Ref. 14: mp 134–136 ꢀC (lit.4 mp 135–137 ꢀC); [a]2D5
ꢁ20.5 (c 0.47, CH2Cl2) {lit.4 [a]D20 ꢁ24.0 (c 0.49,
CH2Cl2)}. Anal. Calcd for C19H28N2O5: C, 62.62; H, 7.74;
N, 7.69. Found: C, 62.82; H, 7.94; N, 7.87.
4.1.10. N-Benzyloxycarbonyl-L-alanyl-L-valyl-L-leucine
14. Protected dipeptide 12 (1.00 g, 2.74 mmol) and Pd/C
(10%) (100.0 mg) were stirred vigorously in a mixture of
MeOH (9.5 mL), water (5.0 mL), and acetic acid (1.0 mL)
under an atmosphere of H2 for 2 h. The reaction mixture
was then filtered through a plug of CeliteÒ and washed after-
wards with MeOH (3ꢂ10 mL). Concentration under reduced
pressure gave peptide 1326 (500.0 mg), which was used
directly in the next step without further purification. A solu-
tion of N-hydroxysuccinimide ester of Cbz-L-Ala-OH16
(694.0 mg, 2.17 mmol) in DMF (5.0 mL) was added drop-
wise to a solution of amine 13 (500.0 mg) and triethylamine
(0.606 mL, 4.34 mmol) in DMF (10 mL) maintained at 0 ꢀC
1
a ca. 4.5:1 mixture of diastereomers (as judged by H and
1
13C NMR analyses): H NMR (400 MHz, CDCl3) d 7.33–
7.23 (m, 5H), 5.58 (d, J¼9.3 Hz, 0.17H), 5.54 (d,
J¼9.3 Hz, 0.83H), 5.10 (br s, 0.83H), 5.05 (s, 1.67H), 4.87
(br s, 0.17H), 4.62 (s, 0.33H), 4.09–3.82 (m, 2H), 2.29 (t,
J¼7.4 Hz, 2H), 1.97–1.74 (m, 2H), 1.41 (s, 7.4H), 1.40 (s,
1.6H); 13C NMR (101 MHz, CDCl3) d 173.1, 173.0, 156.5
(9), 156.5 (5), 136.0, 135.9, 128.4, 128.3, 128.1, 128.0,
127.9, 127.7, 127.5, 126.9, 124.4 (q, JC–F¼283.2 Hz), 81.0
(4), 81.0 (1), 71.9 (q, JC–F¼29.4 Hz), 70.6 (q, JC–F
¼
30.1 Hz), 67.0, 66.9, 51.2, 49.6, 31.8, 31.6, 27.8, 27.2,

8606
M. O. Sydnes et al. / Tetrahedron 62 (2006) 8601–8609
over the course of 30 min. The reaction mixture was then al-
lowed to heat to room temperature and stirred for 16 h before
being concentrated under reduced pressure. The resulting
substrate was dissolved in EtOAc (40 mL) and washed
with citric acid (2ꢂ20 mL of a 5% aq solution) and brine
(1ꢂ20 mL) before being dried (Na2SO4). Filtration and con-
centration under reduced pressure gave a light-yellow solid,
which was recrystallized from hexane/EtOAc to give the title
protected peptide 1427 (800.0 mg, 67% over the two steps) as
a white solid: mp 194–195 ꢀC; [a]D25 ꢁ52.4 (c 0.71, MeOH);
1H NMR (300 MHz, CDCl3) d 7.38–7.30 (m, 5H), 6.99 (br d,
J¼7.3 Hz, 1H), 6.65 (d, J¼7.3 Hz, 1H), 5.47 (br d, J¼6.6 Hz,
1H), 5.11 (s, 2H), 4.56–4.49 (m, 1H), 4.26 (app. t, J¼7.9 Hz,
2H), 3.49–3.40 (m, 1H), 2.17–1.54 (m, 3H), 1.3 (d,
J¼7.0 Hz, 3H), 0.95–0.88 (m, 12H) (one signal due to OH
in COOH could not be discerned); 13C NMR (75 MHz,
CDCl3+one drop of DMSO-d6) d 174.0, 172.3, 170.5,
155.6, 136.1, 128.1, 127.7, 66.3, 58.0, 50.4, 40.8, 33.6,
30.5, 22.4, 21.5, 18.9, 17.6 (one signal obscured or overlap-
ping); MS (FAB+) m/z 436 (M+H, 7%), 305 (8), 222 (7), 91
(100); HRMS (FAB+): calcd for C22H40N3O6 (M++H)
436.2448, found 236.2451. Anal. Calcd for C22H33N3O6:
C, 60.67; H, 7.64; N, 9.65. Found: C, 60.89; H, 7.91; N, 9.88.
solution of the relevant compound from the previous step
in CH2Cl2 (4.0 mL) at 0 ꢀC. The reaction mixture was
then allowed to heat to room temperature and stirred for
16 h before being concentrated under reduced pressure.
The crude product was used directly in the next step without
further purification except for a small amount of the crude
peptide 1, which was purified at this stage in order to provide
a sample for biological assaying.
4.1.13. (S)-4-[N-(Benzyloxycarbonyl)-L-valyl-L-leucyl]-
amino-6,6,6-trifluoro-5-oxohexanoic acid 1. Part of the
resulting yellow oil was subjected to preparative HPLC
purification in order to provide a sample for biological test-
ing. Concentration of the relevant fractions (tR 23.7 min)
gave the title compound 1 (5.9 mg) as a white solid and as
a ca. 6:1 mixture of the hydrate and keto forms (as judged
by 19F NMR analysis) and the hydrate form existed as
a ca. 1:1 mixture of rotamers (as judged by 13C NMR anal-
ysis). Trace amounts of the cyclic form of this compound
19
could also be seen by F NMR: mp 172–173 ꢀC; [a]D22
1
ꢁ15.6 (c 0.28, MeOH); H NMR (400 MHz, CDCl3+one
drop of DMSO-d6) d 7.40–7.20 (m, 6H), 6.00 (dd, J¼8.3
and 24.3 Hz, 1H), 5.1 (app. dd, J¼12.1 and 16.5 Hz, 2H),
4.48–4.44 (m, 1H), 4.22–4.13 (m, 1H), 4.01 (q, J¼8.1 Hz,
1H), 3.80–2.70 (br s, 2H), 2.35 (app. s, 2H), 2.23–1.88 (m,
3H), 1.72–1.47 (m, 3H), 0.96–0.88 (m, 12H); 13C NMR
(101 MHz, CDCl3+one drop of DMSO-d6) d 175.9, 175.8,
174.4, 173.8, 172.0, 156.9, 156.7, 136.0, 128.4, 128.2,
128.1, 94.0 (q, JC–F¼29.8 Hz), 67.2, 67.0, 60.9, 60.8, 53.8,
53.6, 52.0, 51.9, 30.7, 30.6, 30.4, 24.5 (4), 24.4 (7), 23.1,
22.9, 22.8, 21.5, 21.4, 19.1, 17.7 (signal due to CF3 group
carbon could not be discerned); 19F NMR (376 MHz,
CDCl3+one drop of DMSO-d6) d ꢁ74.8 (cyclic), ꢁ76.4
(keto), ꢁ76.5 (keto), ꢁ81.8 (hydrate), ꢁ81.9 (hydrate),
ꢁ82.1 (hydrate), ꢁ82.2 (hydrate). (The appearance of four
signals for the hydrate form of this compound in 19F NMR
is probably due to partial racemization over time at the a
position of this compound. The extent of racemization at
the time 19F NMR was measured was less than 10%.28)
MS (FAB+) m/z 546 (M++H, 5%), 502 (1), 412 (1), 347
(4), 91 (100); HRMS (FAB+): calcd for C25H35N3O7F3
(M++H) 546.2427, found 546.2421. Anal. Calcd for
C25H34F3N3O7$1/4CF3COOH$H2O: C, 51.73; H, 6.17; N,
7.10. Found: C, 51.79; H, 6.34; N, 7.13.
4.1.11. N-Benzyloxycarbonyl-L-phenylalanyl-L-alanine
15.15 This compound was synthesized according to the pro-
cedure in Ref. 15: mp 157–159 ꢀC (lit.15 mp 157–158 ꢀC);
[a]D24 ꢁ8.1 (c 0.65, EtOH) {lit.15 [a]2D5 ꢁ9.5 (c 1.0, EtOH)}.
4.1.12. General procedure for the synthesis of (S)-4-[N-
(benzyloxycarbonyl)-L-valyl-L-leucyl]amino-6,6,6-tri-
fluoro-5-oxohexanoic acid 1, (S)-4-[N-(benzyloxycar-
bonyl)-L-phenylalanyl-L-alanyl]amino-6,6,6-trifluoro-5-
oxohexanoic acid 19, and (S)-4-[N-(benzyloxycarbonyl)-
L-alanyl-L-valyl-L-leucyl]amino-6,6,6-trifluoro-5-oxo-
hexanoic acid 20. Coupling: HOBt (59.6 mg, 0.39 mmol)
and EDC$HCl (80.4 mg, 0.42 mmol) were added to a stirred
solution of the relevant protected peptide (0.39 mmol) in
DMF (6.0 mL) at 0 ꢀC. The reaction mixture was then
stirred for 15 min before amine 11 (100.0 mg, 0.39 mmol)
dissolved in DMF (6.0 mL) was added dropwise. The reac-
tion mixture was then allowed to heat to room temperature
and stirred for 21 h before DMF was removed under reduced
pressure. The resulting residue was diluted with EtOAc
(30 mL) and washed with citric acid (2ꢂ10 mL of a 5%
aq solution), NaHCO3 (2ꢂ10 mL of a 5% aq solution),
and brine (2ꢂ10 mL) before being dried (Na2SO4). Filtra-
tion and concentration under reduced pressure gave the de-
sired compound in quantitative yield. The crude product,
which contained small amounts of impurities, was used in
the next step without further purification. All products had
satisfactory low-resolution mass spectra. Oxidation: Dess–
Martin periodinane (439 mg, 1.04 mmol) was added to
a stirred solution of the relevant peptide from the previous
step in CH2Cl2 (15.0 mL) at 0 ꢀC. The resulting reaction
mixture was then allowed to heat to room temperature and
stirred for 19 h before being filtered through a plug of
CeliteÒ and washed afterwards with EtOAc (3ꢂ15 mL).
Concentration under reduced pressure gave the desired
compound as a yellow oil. The material was used in the
next step without further purification. All products had
satisfactory low-resolution mass spectra. Deprotection:
TFA (0.115 mL, 1.55 mmol) was added dropwise to a stirred
4.1.14. (S)-4-[N-(Benzyloxycarbonyl)-L-phenylalanyl-L-
alanyl]amino-6,6,6-trifluoro-5-oxohexanoic acid 19. MS
(ESIꢁ) m/z 550 (MꢁH, 64%), 442 (100).
4.1.15. (S)-4-[N-(Benzyloxycarbonyl)-L-alanyl-L-valyl-L-
leucyl]amino-6,6,6-trifluoro-5-oxohexanoic acid 20. MS
(ESIꢁ) m/z 615 (MꢁH, 100%), 507 (60).
4.1.16. General procedure for the synthesis of (S)-4-[N-
(benzyloxycarbonyl)-L-valyl-L-leucyl]amino-6,6,6-tri-
fluoro-5-oxohexanamide 2, (S)-4-[N-(benzyloxycar-
bonyl)-L-phenylalanyl-L-alanyl]amino-6,6,6-trifluoro-5-
oxohexanamide 3, and (S)-4-[N-(benzyloxycarbonyl)-L-
alanyl-L-valyl-L-leucyl]amino-6,6,6-trifluoro-5-oxohexa-
namide 4. Method A: HOBt (25.0 mg, 0.16 mmol) and
EDC$HCl (31.0 mg, 0.16 mmol) were added to a stirred
solution of the relevant peptide from the previous step in
DMF (7.0 mL) at 0 ꢀC. The resulting reaction mixture

M. O. Sydnes et al. / Tetrahedron 62 (2006) 8601–8609
8607
was then stirred for 15 min before ammonia solution
(31.0 mL of a 28% aq solution) was added dropwise.
The reaction mixture was then allowed to heat to room
temperature and stirred for 16 h before DMF was removed
under reduced pressure. The residue thus obtained was
then dissolved in EtOAc (20 mL) and washed with citric
4.1.19. (S)-4-[N-(Benzyloxycarbonyl)-L-alanyl-L-valyl-L-
leucyl]amino-6,6,6-trifluoro-5-oxohexanamide 4. Con-
centration of the relevant fractions (tR 25.0 min) gave the
title compound 4 (8.5 mg, 4% from acid 14) as a white solid
and as a ca. 1:1 mixture of rotamers (as judged by 13C and
19F NMR analyses): mp 140–141 ꢀC; [a]2D7 ꢁ4.5 (c 0.11,
CHCl3); 1H NMR (400 MHz, CDCl3) d 7.38–7.36 (m,
3H), 7.33–7.30 (m, 2H), 7.13 (app. br d, J¼8.4 Hz, 0.8H),
7.06–6.96 (m, 1.2H), 6.62 (app. br d, J¼4.4 Hz, 0.8H),
6.49 (app. br d, J¼4.4 Hz, 0.2H), 6.20 (s, 0.8H), 6.10 (s,
0.2H), 5.28 (br s, 0.8H), 5.24 (br s, 0.2H), 5.13 (d,
J¼4.0 Hz, 2H), 4.69 (dt, J¼3.6 Hz, 0.2H), 4.64–4.56 (m,
0.2H), 4.54 (dt, J¼3.6 Hz, 0.8H), 4.50–4.43 (m, 0.8H),
4.16–4.01 (m, 2H), 3.70–3.30 (br s, 0.2H), 2.64–2.14 (m,
4H), 1.95–1.90 (m, 1H), 1.87–1.76 (m, 1H), 1.45 (d,
J¼7.1 Hz, 3H), 0.96 (d, J¼6.6 Hz, 6H), 0.91–0.86 (m, 6H)
(the signal for three protons were obscured by the signal
for residual water in the sample); 1H NMR (400 MHz,
CDCl3+one drop of DMSO-d6) d 7.38–7.31 (m, 5H), 7.07–
6.99 (m, 3H), 6.81 (app. br s, 1H), 6.42–6.23 (m, 2H),
5.11 (s, 2H), 4.55–4.39 (m, 2H), 4.19–4.08 (m, 2H), 3.70–
3.30 (br s, 1H), 2.56–2.41 (m, 2H), 1.94–1.86 (m, 1H),
1.77–1.53 (m, 4H), 1.39 (d, J¼7.1 Hz, 3H), 0.98–0.89 (m,
12H); 19F NMR (376 MHz, CDCl3+one drop of DMSO-
d6) d ꢁ83.2 (cyclic), ꢁ83.3 (cyclic); MS (ESI+) m/z 654
(M++K, 35%), 638 (M++Na, 100), 616 (M++H, 68);
HRMS (FAB+): calcd for C28H41N5O7F3Na (M++Na)
638.2778, found 638.2783.
acid (2ꢂ10 mL of
a
5% aq solution), NaHCO3
(2ꢂ10 mL of a 5% aq solution), and brine (2ꢂ10 mL)
before being dried (Na2SO4). Filtration and concentration
under reduced pressure gave the crude product, which
was purified by preparative HPLC. Concentration of the
relevant fractions gave the desired compounds in the
yields stated below.
4.1.17. (S)-4-[N-(Benzyloxycarbonyl)-L-valyl-L-leucyl]-
amino-6,6,6-trifluoro-5-oxohexanamide 2. Concentration
of the relevant fractions (tR 24.2 min) gave the title com-
pound 2 (16.9 mg, 8% from acid 12) as a white solid and
as a ca. 1:1 mixture of rotamers (as judged by 1H, 13C, and
19F NMR analyses): mp 112–113 ꢀC; [a]2D6 +32.0 (c 0.07,
CHCl3); 1H NMR (400 MHz, CDCl3) d 7.45 (br d,
J¼8.1 Hz, 0.5H), 7.39–7.31 (m, 5H), 7.08 (app. br d,
J¼8.1 Hz, 0.5H), 6.83 (br s, 0.5H), 6.52 (br s, 0.5H), 6.48–
6.41 (m, 1H), 5.38 (app. br d, J¼5.7 Hz, 0.5H), 5.30 (app.
br d, J¼5.7 Hz, 0.5H), 5.12 (s, 2H), 4.66 (app. br s, 0.5H),
4.47 (app. br s, 1H), 4.38 (app. br d, J¼6.6 Hz, 0.5H), 3.93
(app. br s, 1H), 2.66–2.43 (m, 2H), 2.22–1.45 (m, 11H),
0.97–0.88 (m, 12H); 13C NMR (101 MHz, CDCl3)
d 172.5, 172.1, 172.0, 171.8 (4), 171.8 (0), 171.7 (6),
157.0, 135.6, 128.7, 128.6, 128.5, 128.2, 128.1, 67.8, 67.6,
61.3, 61.2, 52.3, 47.0, 40.0, 39.6 (4), 39.6 (0), 30.3, 24.8,
24.7, 22.8, 22.7, 21.6, 19.2, 19.1, 17.9, 17.8 (signal due to
CF3 group carbon and signal due to the carbon adjacent
to the CF3 group could not be discerned); 19F NMR
(376 MHz, CDCl3) d ꢁ82.7 (cyclic), ꢁ83.3 (cyclic);
MS (ESI+) m/z 567 (M++Na, 100%), 545 (M++H, 5);
HRMS (FAB+): calcd for C25H36N4O6F3 (M++H) 545.2587,
found 545.2591. Anal. Calcd for C25H35F3N4O6$1/
4CF3COOH$1/4H2O: C, 53.03; H, 6.24; N, 9.70. Found:
C, 53.26; H, 6.35; N, 9.79.
4.1.20. (S)-4-[N-(Benzyloxycarbonyl)-L-alanyl-L-valyl-L-
leucyl]amino-6,6,6-trifluoro-5-oxohexanamide 4. Method
B: Pyridine (0.133 mL, 1.64 mmol) was added dropwise to
a stirred solution of peptide 20 (39.1 mg) and di-tert-butyl
dicarbonate (23.6 mg, 0.18 mmol) in 1,4-dioxane (13 mL)
under an argon atmosphere at room temperature. Ammo-
nium bicarbonate (324 mg, 4.10 mmol) was then added to
the resulting solution and the reaction mixture was stirred
at room temperature for 23 h before being diluted with
EtOAc (20 mL). The organic phase was washed with citric
acid (1ꢂ10 mL of a 5% aq solution), NaHCO3 (1ꢂ10 mL
of a 5% aq solution), and brine (1ꢂ10 mL) before being
dried (Na2SO4). Filtration and concentration under reduced
pressure gave a light-yellow solid, which was purified by
preparative HPLC. Concentration of the relevant fractions
(tR 25.4 min) gave the title compound 4 (8.4 mg, 12%
from peptide 14), which was identical, in all respects, with
the material obtained via Method A.
4.1.18. (S)-4-[N-(Benzyloxycarbonyl)-L-phenylalanyl-L-
alanyl]amino-6,6,6-trifluoro-5-oxohexanamide 3. Con-
centration of the relevant fractions (tR 21.9 min) gave the
title compound 3 (8.0 mg, 5% from acid 15) as an off-white
solid and as a ca. 1:1 mixture of rotamers (as judged by
13C and 19F NMR analyses) and as a ca. 3.3:1 mixture of
the cyclic and keto forms (as judged by 19F NMR):
mp 108–111 ꢀC; [a]D27 ꢁ6.8 (c 0.24, CHCl3); 1H NMR
(400 MHz, CDCl3) d 7.38–7.07 (m, 14.5H), 5.47 (br s,
0.5H), 5.08–4.94 (m, 2H), 4.64–4.22 (m, 3H), 3.19–2.85
(m, 2H), 2.44 (app. br s, 2H), 2.11–1.77 (m, 2H), 1.28
(app. br s, 3H); 13C NMR (101 MHz, CDCl3) d 172.3 (3),
172.2 (8), 172.0 (2), 171.9 (7), 156.7, 156.6, 156.5, 135.7,
129.1, 128.9, 128.8, 128.6, 128.5, 128.3, 128.2, 128.0,
127.4, 67.6, 67.4, 56.5, 56.3, 49.2, 46.5, 45.8, 37.9, 37.8,
37.6, 29.6, 28.8, 22.8, 17.8, 17.1 (signal due to CF3 group
carbon and signal due to the carbon adjacent to the CF3
group could not be discerned); 19F NMR (376 MHz,
CDCl3) d ꢁ76.2 (keto), ꢁ82.9 (cyclic), ꢁ83.2 (cyclic);
MS (FAB+) m/z 573 (M++Na, 7%), 551 (M++H, 5);
HRMS (FAB+): calcd for C26H30N4O6F3 (M++H)
551.2117, found 551.2114.
4.1.21. (S)-tert-Butyl 4-(benzyloxycarbonyl)amino-6,6,6-
trifluoro-5-oxohexanoate 21. Dess–Martin periodinane
(220.0 mg, 0.52 mmol) was added to a stirred solution of al-
cohol 10 (91.8 mg, 0.24 mmol) in CH2Cl2 (5.0 mL) at room
temperature. The reaction mixture was then stirred for 16 h
before being filtered through a plug of CeliteÒ and washed
afterwards with EtOAc (3ꢂ5 mL). The filtrate was concen-
trated under reduced pressure to give a light-yellow crude
product, which was purified by flash chromatography (silica,
hexane/EtOAc/triethylamine 50:49.6:0.4). Concentration of
the relevant fractions (Rf 0.56 in hexane/EtOAc 1:1) gave the
title compound 21 (74.4 mg, 81%) as a clear oil and as a ca.
2:1 mixture of the keto and hydrated forms (as judged by 19F
1
and 13C NMR analyses): [a]2D5 +3.6 (c 0.73, MeOH); H
NMR (300 MHz, CDCl3) d 7.38–7.31 (m, 5H), 5.57 (br d,

8608
M. O. Sydnes et al. / Tetrahedron 62 (2006) 8601–8609
J¼7.7 Hz, 0.5H), 5.46 (br d, J¼7.7 Hz, 0.5H), 5.11 (s, 2H),
4.88–4.78 (m, 0.5H), 3.96–3.86 (m, 0.5H), 2.42–2.09 (m,
3H), 1.97–1.86 (m, 1H), 1.43 (s, 4.5H), 1.42 (s, 4.5H); 13C
NMR (101 MHz, CDCl3) d 190.3 (q, JC–F¼34.7 Hz),
173.5, 171.8, 157.9, 155.8, 135.7, 128.6, 128.5 (0), 128.4
(8), 128.3, 128.2, 128.1, 128.0, 127.7, 127.6 (4), 127.5 (8),
4.1.23. 4-[N-(Benzyloxycarbonyl)-L-alanyl]amino-6,6,6-
trifluoro-5-oxohexanoic acid 25. TFA (0.10 mL,
1.30 mmol) was added dropwise to a solution of ester 24
(40.0 mg, 0.087 mmol) in CH2Cl2 (5 mL) at room tempera-
ture. The reaction mixture was then stirred at room temper-
ature for 24 h before being concentrated under reduced
pressure to give the title compound 25 (34.9 mg, crude yield
99%) as a yellow oil and ca. 90% pure (as judged by HPLC
analysis) and as a ca. 6:2:1 mixture of cyclic, keto, and hy-
drate forms (as judged by 19F NMR analysis) and the cyclic
form existed as a ca. 1:1 mixture of rotamers (as judged by
127.0, 123.1 (q, JC–F¼288.6 Hz), 115.5 (q, JC–F
¼
292.3 Hz), 94.2 (q, JC–F¼30.5 Hz), 81.5, 81.3, 67.5, 67.4,
55.5, 55.0, 31.8, 30.9, 27.9, 25.3, 23.2; 19F NMR
(376 MHz, CDCl3) d ꢁ76.5 (keto), ꢁ82.3 (hydrate); MS
(ESIꢁ) m/z 388 (MꢁH, 70%), 280 (100).
1
19F NMR analysis): [a]D24 +10.5 (c 0.77, CHCl3); H NMR
4.1.22. (S)-tert-Butyl 4-[N-(benzyloxycarbonyl)-L-ala-
nyl]amino-6,6,6-trifluoro-5-oxohexanoate 24. HOBt
(31.8 mg, 0.21 mmol) and EDC$HCl (43.6 mg, 0.23 mmol)
were added to a stirred solution of Cbz-L-Ala-OH
(44.1 mg, 0.198 mmol) in DMF (3.0 mL) at 0 ꢀC. The reac-
tion mixture was then stirred for 15 min before amine 11
(49.0 mg, 0.19 mmol) dissolved in DMF (2.0 mL) was added
dropwise. The reaction mixture was then allowed to heat to
room temperature and stirred for 21 h before DMF was re-
moved under reduced pressure. The residue thus obtained
was dissolved in EtOAc (20 mL) and washed with citric
acid (2ꢂ10 mL of a 5% aq solution), NaHCO3 (2ꢂ10 mL
of a 5% aq solution), and brine (2ꢂ10 mL) before being dried
(Na2SO4). Filtration and concentration under reduced pres-
sure gave the title compound 23 (66.8 mg) as a clear, yellow
oil. The material was used directly in the next step without
further purification: MS (FAB+) m/z 485 (M++Na, 5%),
463 (M++H, 10), 407 (32), 363 (18), 91 (100); HRMS
(FAB+): calcd for C21H30N2O6F3 463.2056, found 463.2061.
(400 MHz, CD3OD) d 7.38–7.25 (m, 5H), 5.08 (s, 2H),
4.18 (q, J¼7.3 Hz, 1H), 3.04–2.86 (m, 1H), 2.58 (t,
J¼6.5 Hz, 1H), 1.38 (d, J¼7.3 Hz, 3H), 1.46–1.23 (m, 2H)
(signal for one proton was obscured by the signal for meth-
anol); 13C NMR (101 MHz, CDCl3) d 177.5, 176.9, 155.9,
136.0, 128.5, 128.2, 128.1, 122.4 (q, JC–F¼283.1 Hz), 75.0
(q, JC–F¼31.7 Hz), 67.1, 49.5, 34.0, 29.7, 27.7, 18.3; 13C
NMR (101 MHz, CD3OD) d 176.5, 176.0, 158.4, 138.2,
129.4, 129.0, 128.8, 75.8 (q, JC–F¼30.1 Hz), 67.5, 50.8,
35.3, 28.2, 17.9 (signal due to CF3 group carbon could not
be discerned and one signal was obscured or overlapping);
19F NMR (376 MHz, CDCl3) d ꢁ74.6 (2) (cyclic), ꢁ74.6
(4) (cyclic), ꢁ76.2 (keto), ꢁ82.2 (1) (hydrate), ꢁ82.2 (3)
(hydrate), ꢁ82.3 (0) (hydrate), ꢁ82.3 (2) (hydrate). (The ap-
pearance of four signals for the hydrate form of this com-
pound in 19F NMR is probably due to partial racemization
over time at the a position of this compound. The extent
of racemization at the time 19F NMR was measured was
less than 5%.28) MS (ESIꢁ) m/z 403 (MꢁH, 46%), 222
(69), 199 (100).
Dess–Martin periodinane (138.8 mg, 0.33 mmol) was added
to a stirred solution of alcohol 23 (66.8 mg) in CH2Cl2
(3.0 mL) at room temperature. The reaction mixture was
then stirred for 16 h before being diluted with EtOAc
(10 mL) and filtered through a plug of CeliteÒ and washed
with EtOAc (3ꢂ10 mL). Concentration under reduced pres-
sure gave a light-yellow oil, which was subjected to flash
chromatography (silica, hexane/EtOAc/Et3N 50:49.8:0.2
eluent). Concentration of the relevant fractions (Rf 0.2 in hex-
ane/EtOAc 1:1) gave the title compound 24 (45.7 mg, 52%
overthetwosteps) asa clear, colorlessoiland asa ca. 7:3mix-
4.2. Enzyme inhibitory assay
The inhibitory assay was performed using a com-
mercially available fluorogenic substrate Dabcyl-
KTSAVLQSGFRKME-Edans (Genesis Biotech, Taiwan)
corresponding to the N-terminal autocleavage site of
SARS 3CLpro 29
The change in fluorescence intensity was
.
monitored in a Cary Eclipse fluorescence spectrophotometer
(Varian) with 355 and 538 nm for excitation and emission
wavelengths, respectively. Kinetic measurements were per-
formed at 25 ꢀC in buffer containing 10 mM sodium phos-
phate (pH 7.4), 10 mM sodium chloride, 1 mM EDTA, and
1 mM TCEP. The inhibition constant, Ki, was determined
by measuring the apparent kinetic parameters at a constant
substrate concentration with varying inhibitor concentra-
tions (0–1 mM). The protease (final concentration of
1 mM) was incubated with inhibitor for 10 min at room tem-
perature and the reaction was initiated by adding the sub-
strate (a volume corresponding to a final concentration of
5 mM in the reaction mixture). The dependence of activity
on the inhibitor concentration was analyzed in a manner
similar to what was reported earlier.30 Briefly, the kinetic
parameters were determined by global nonlinear regression
analysis to the equation.
ture of the hydrate and keto forms (as judged by 1H and 19
F
NMR analyses) and both tautomers exist as a ca. 1:1 mixture
of rotamers (as judged by 1H, 13C, and 19F NMR analyses):
1
[a]2D4 ꢁ9.1 (c 2.02, CHCl3); H NMR (400 MHz, CDCl3)
d 7.56–7.27 (m, 5H), 7.14 (app. br d, J¼6.6 Hz, 0.3H), 7.03
(app. d, J¼8.4 Hz, 0.3H), 6.08–5.42 (br m, 1.7H), 5.12–
5.05 (m, 2H), 4.90 (br s, 0.3H), 4.38–4.06 (m, 2H), 2.42–
2.11 (m, 3H), 1.98–1.84 (m, 1H), 1.44 (s, 4H), 1.42 (4) (s,
4H), 1.42 (1) (s, 1H), 1.39–1.34 (m, 3H); 13C NMR
(101 MHz, CDCl3) d 189.6 (q, JC–F¼34.7 Hz), 189.5 (q,
JC–F¼34.7 Hz), 175.0, 174.8, 173.6, 173.2, 172.8, 172.3,
172.1, 156.3, 156.2, 156.0, 136.0, 135.9, 135.8, 128.5,
128.3, 128.2 (0), 128.1 (6), 128.0 (7), 128.0 (3), 128.0 (1),
123.1 (q, JC–F¼288.8 Hz), 115.5 (q, JC–F¼292.6 Hz), 94.3
(q, JC–F¼30.9 Hz), 94.2 (q, JC–F¼30.5 Hz), 81.7, 81.6,
81.3, 81.2, 67.3, 67.1, 53.9, 53.8, 53.7, 50.9, 50.7, 50.1,
31.8, 30.9 (2), 30.9 (0), 29.7, 27.9, 24.8, 23.4, 23.2, 18.4,
18.1; 19F NMR (376 MHz, CDCl3) d ꢁ76.5 (keto), ꢁ82.0
(hydrate), ꢁ82.1 (hydrate) (one signal obscured or overlap-
ping); MS (ESIꢁ) m/z 459 (MꢁH, 38%), 351 (100).
vI=v0 ¼ Vmax½Sꢄ=f½Sꢄ þ Kmð1 þ ½Iꢄ=KiÞg
where vI and v0 are the rate of substrate cleavage in the pres-
ence and absence of inhibitor, respectively. Vmax is the

M. O. Sydnes et al. / Tetrahedron 62 (2006) 8601–8609
8609
9. Gao, F.; Ou, H.-Y.; Chen, L.-L.; Zheng, W.-X.; Zhang, C.-T.
FEBS Lett. 2003, 553, 451–456.
maximal rate, [S] is the substrate concentration, [I] is the in-
hibitor concentration, and Km is the Michaelis constant.31
10. Luesch, H.; Hoffmann, D.; Hevel, J. M.; Becker, J. E.;
Golakoti, T.; Moore, R. E. J. Org. Chem. 2003, 68, 83–91.
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Schofield, C. J. Tetrahedron Lett. 1995, 36, 7761–7764.
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B.; Bahr, J.; Wagner, K.; Fischer, P. Synthesis 1987, 236–241.
15. Katritzky, A. R.; Suzuki, K.; Singh, S. K. Synthesis 2004,
2645–2652.
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Acknowledgements
This research was supported by various grants from MEXT
(Ministry of Education, Culture, Sports, Science, and Tech-
nology, Japan: the ‘Academic Frontier’ Project for Private
Universities: matching fund subsidy from MEXT, the 21st
Century Center of Excellence Program ‘Development of
Drug Discovery Frontier Integrated from Tradition to Pro-
teome’ and Grant-in-Aid for Scientific Research on Priority
Areas 17035085) and the National Institutes of Health (GM
57144, USA). We thank Dr. J.-T. Nguyen for valuable help
during manuscript preparation.
Supplementary data
NMR spectra for all new compounds 1, 2, 3, 4, 8, 9, 10, 11,
21, 24, and 25, and HPLC chromatograms of compounds 1,
2, 3, and 4. Supplementary data associated with this article
can be found in the online version, at doi:10.1016/
j.tet.2006.06.052.
18. Brimble, M. A.; Trotter, N. S.; Harris, W. R.; Sieg, F. Bioorg.
Med. Chem. 2005, 13, 519–532.
19. We found that 19F NMR analysis was the best method to deter-
mine the structure of these compounds in solution in cases
where the amount of available material for NMR studies was
scarce.
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