Peptide-based inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase): Model compounds towards small molecule inhibitors

Article abstract of DOI:10.1016/S0968-0896(03)00190-1

From L-α-aminobutyric acid (Abu) a set of electrophilic and non-electrophilic replacements for the P1 cysteine of substrate and product inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase) serine protease have been synthesised and co

Full text of DOI:10.1016/S0968-0896(03)00190-1

Bioorganic & Medicinal Chemistry 11 (2003) 2955–2963
Peptide-Based Inhibitors of Hepatitis C Virus Full-Length NS3
(Protease-Helicase/NTPase): Model Compounds Towards Small
Molecule Inhibitors
Karin Oscarsson,a Anton Poliakov,b Stefan Oscarson,a U. Helena Danielson,b
Anders Hallbergc and Bertil Samuelssona,d,
*
aDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-10691 Stockholm, Sweden
bDepartment of Biochemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
cDepartment of Organic Pharmacutical Chemistry, BMC, Uppsala University, Box 574, S-751 23 Uppsala, Sweden
dMedivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden
Received 24 September 2002; accepted 21 March 2003
Abstract—From l-a-aminobutyric acid (Abu) a set of electrophilic and non-electrophilic replacements for the P1 cysteine of sub-
strate and product inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase) serine protease have been synthesised
and coupled to a model pentapeptide furnishing a set of hexapeptide inhibitors. Promising inhibitory activities with Ki values of
0.18 mM (11b, P1 electrophilic a,b-unsaturated ketone), 0.46 mM (12e, P1 electrophilic alkyl ketone) and 0.98 mM (10e, P1 non-
electrophilic alkenyl alcohol as diastereomeric mixture). The reference hexapeptide product inhibitor had a Ki value of 1.54 mM (14,
P1 Abu–OH). The electrophilic inhibitors exhibit increased potency as compared with the corresponding product inhibitor, and
notably also the non-electrophilic P1 alkenyl alcohol 10e. This represents the first example of non-electrophilic inhibitors that are
not P1 amides or product inhibitors.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
One of the most studied molecular targets for antiviral
therapy against HCV is the NS3 serine protease.8,9 The
NS3 is a multifunctional protein which contains a serine
protease domain and a RNA helicase domain.10 The
protease cofactor NS4A, which is a relatively small
protein, is absolutely required for enhanced serine pro-
tease activity.9,11,12 The NS3 serine protease is essential
in the viral lifecycle and therefore presents a prime tar-
get for development of new hepatitis C drugs. For inhi-
Hepatitis C virus (HCV) was identified 1989 as the
major etiological agent of non-A, non-B hepatitis.1 An
estimated 175 million people worldwide are affected
from HCV infection, which becomes chronic in about
50% of cases leading to secondary diseases such as liver
cirrhosis and hepatocellular carcinoma.2ꢀ5 The pre-
valence of chronic hepatitis C is of such magnitude that
patients with the disease constitute the highest propor-
tion of candidates for liver transplantation.5 Current
therapies involve treatment with interferon-a (IFN-a)
either alone or in combination with ribavirin. These
treatment regimes although effective in selective patients
show overall disappointing efficacy as many patients do
not respond to therapy or tolerate therapy poorly
underscoring the need for improved therapeutic
options.6,7
bitor
design,
both
X-ray
crystal
structure
information,13ꢀ15 consensus cleavage sites of the HCV
polyprotein and cleavage kinetics of different peptide
substrates have been used to develop a first generation
of inhibitors.6,9,16 From analysis of the substrate bind-
ing site as revealed by X-ray crystal structure, it has
been shown that the binding site of the NS3 protease is
remarkably shallow and solvent exposed making small
molecule inhibitor design a challenge. NS3 protease
inhibitors in general fall into two structural classes
characterised by either an electrophilic carbonyl P1
group, acting as classical serine-trap or warhead for the
catalytic serine of the NS3 protease active site,9,17ꢀ24 or
*Corresponding author. Tel.: +46-8-608-3104; fax: +46-8-608-3199;
e-mail: bertil.samuelsson@medivir.se
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00190-1

2956
K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 2955–2963
by having a P1 carboxylic acid characterised as product
based inhibitors.9,17,18,25ꢀ29 Recently, we suggested,
supported by modelling, that the helicase domain parti-
cipates in the binding of the inhibitors to the native full-
A
series of inhibitors of the full-length protease with Ki in
nanomolar range were identified.30,31
example, 11b (Ki=0.18 mM) showing over 8-fold
increase in potency over the corresponding product
inhibitor 14, and interestingly the non-electrophilic
alcohol 10e (diastereomeric mixture, Ki=0.98 mM)
being substantially more potent than the corresponding
product inhibitor 14.
length NS3 protein (protease-helicase/NTPase).30,31
We have been intrigued by the possibility to use potent
serine-trap based inhibitors as a starting point for opti-
misation of small-molecule drug-like non-electrophilic
inhibitors targeting the non-truncated protease. As part
of this program we have also discovered P1 alkenyl
alcohols as a potentially new class of non-electrophilic
inhibitors. In this report we disclose our initial findings
from the optimisation of a new P1 serine-trap and new
P1 alkenyl alcohols based on Abu (a-aminobutyric acid)
which is incorporated into a model P1-P6 spanning
peptide, Ac-Asp-d-Glu-Leu-Ile-Cha-P1. Ingallinella et
al. recently reported on the potent hexapeptide Ac-Asp-
d-Glu-Leu-Ile-Cha-Cys-OH (Cha, b-cyclohexyl-Ala)
which inhibits NS3 proteases with an IC50=0.015 mM.28
We have for comparison synthesised the corresponding
P1 Abu peptide (14) which in our hand exhibited a Ki
value of 1.54 mM. Furthermore, Ingallinella et al.
reported that Ac-Asp-Glu-Met-Glu-Glu-Cys-OH and
the corresponding P1 Abu showed an inhibitory IC50
potency of 1 and 5.8 mM, respectively.28 These data
consistently show that a substantial reduction of inhibi-
tion potency can be expected from replacing P1 Cys
with Abu.18 However, cysteine is generally not con-
sidered as a tractable P1 for drug discovery purposes,
being chemically reactive and unstable, so for this
model study we have chosen to use the chemically stable
Abu as a starting point for P1 modifications. Numerous
other stable and potent P1 Cys replacements have been
disclosed that further underscores the rationale behind
this methodology. 2-Amino-4,4-difluoro-butyric acid
(difluoroAbu) was recently developed and successfully
incorporated into potent tripeptide a-ketoacid inhibi-
tors.21 Moreover, 1-aminocyclopropane-1-carboxylic
acid has been introduced as a chirally stable and potent
P1 amino acid.25,26,29 This line of work was further
explored resulting in 1-amino-vinylcyclopropane-1-car-
boxylic acid as P1 amino acid, which yielded potent
product based inhibitors.32,33 There are, thus, several
currently available chemically stable drugable P1 sub-
stituents, which can be used in place of the model P1
Abu used in this study to further optimise this set of
inhibitors. Several other serine traps, for example
boronic acids,34 a-diketones,20 a-ketoamides,20,23 and
a-ketoesters,20 have previously been disclosed to show
activity against HCV NS3 protease.
Chemistry and Structure–Activity Relationship
The target inhibitors (10a–e, 11a–e, and 12a–e) were
synthesised starting from Boc protected l-a-aminobu-
tyric acid (Boc-Abu-OH, 1), which was transformed to
phosphonate 3, and reacted further in a Horner–Wads-
worth–Emmons reaction to give the trans-enones. The
ketones moiety of the trans-enones were subsequently
reduced to the corresponding alcohols, the Boc groups
cleaved off and the resulting amines coupled to the
pentapeptide 6 giving 10a–e after deprotection. Oxida-
tion of the alcohols delivered the ketones 11a–e after
deprotection. The saturated ketones 12a–e were pre-
pared by chemo-selective hydrogenation of the C–C
double bonds followed by deprotection.
The Boc protected amino acid 1 was converted to the
methyl ester 2 in 96% yield, using methyl iodide and
potassium carbonate in DMF (Scheme 1) and reacted
further with 6 equiv of the lithium salt of dimethyl
methylphosphonate at ꢀ78 ꢁC in THF, according to
Chakravarty et al.,36 to yield the phosphonate 3 in 92%
yield.
The trans-enones 4a–e were obtained in good to mod-
erate yields (24–90%) using Horner–Wadsworth–
Emmons conditions, that is by reacting the phospho-
nate 3 with the appropriate aldehyde in dry ethanol
using 1 equiv of potassium carbonate as base.37 Initially,
this reaction was accompanied by racemisation, and all
attempts to suppress racemisation at the a-carbon of
the trans-enones failed. Numerous different bases
were explored, for example NaH in THF,38 DIPEA
In this report, we present the synthesis of novel series of
a,b-unsaturated alcohols (10a–e), a,b-unsaturated
ketones (11a–e) and saturated ketones (12a–e) starting
from the unnatural amino acid Abu. These three struc-
turally related P1 inhibitor classes (10a–e, 11a–e and
12a–e) were assessed in an in vitro assay system com-
prising the native bifunctional full-length NS3 protein
(protease-helicase/NTPase).30,31,35 Several hexapeptides
were identified exhibiting promising Ki values, for
Scheme 1. Synthesis of key intermediates 5a–e.

K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 2955–2963
2957
(N,N-ethyldiisopropylamine), Et3N, DBU or diisopro-
pylamine in CH3CN with or without addition of LiCl/
LiBr,39ꢀ41 without success. However, with potassium
carbonate as a base, the racemisation of the condensa-
tion products could be minimised. 5-Methylhexanal,
used in the formation of condensation product 4e, was
synthesised by oxidation of 5-methyl-1-hexanol
employing pyridinium dichromate (PDC) in CH2Cl2.42
Cleavage of the Boc group on compounds 4a–e was
unsuccessful and therefore the ketones were reduced
using sodium borohydride to produce the alcohols 5a–e
in 74–96% yield as diastereomeric mixtures. Synthesis
of the pentapeptide, Ac-Asp(OtBu)-d-Glu(OtBu)-Leu-
Ile-Cha-OH (6), was performed using a Fmoc solid
TM43
phase methodology on SASRIN
acid 6.44,45
yielding the pure
Boc deprotection was then performed by stirring 5a–e
with 1 M TMSOTf and 1.5 M 2,6-lutidine in CH2Cl2 to
furnish the corresponding free amines (Scheme 2).22
Attempts to cleave off the Boc group using the standard
conditions, for example TFA and HCl, resulted in
decomposition of the alcohols 5a–e. Subsequent coupling
Scheme 2. Synthesis of target compounds 10–12a–e.
Table 1. Percent inhibition at 10 and 1 mM and inhibition constant against full-length NS3 (protease-helicase/NTPase) protease
R
Compd
% inhib at
10 mM
% inhib at
1 mM
Ki (mM)
R
Compd
% inhib at
10 mM
% inhib at
1 mM
Ki (mM)
14
60
8
17
nm
nm
nm
nm
nm
58
1.54ꢂ0.16
nm
11c
11d
11e
95
50
0.32ꢂ0.03
0.20ꢂ0.01
ꢂ0.02
10a
10b
10c
10d
10e
11a
11b
96
54
7
nm
95
420.39
47
7
2.30ꢂ0.21
nm
12a 5
12b
12c
802.928
81
ꢂ0.1
30
nm
40
0.88ꢂ0.05
nm
64
96
100
0.98ꢂ0.09
0.21ꢂ0.03
0.18ꢂ0.01
64
12d
12e
80
0.65ꢂ0.04
0.46ꢂ0.07
64
91
31
nm, not measured.

2958
K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 2955–2963
of the corresponding amines with the acid 6 was accom-
plished using benzotriazol-1-yloxytris(pyrrolidino)pho-
sphonium hexafluorophosphate (PyBOP) and DIPEA in
CH2Cl2 furnishing 7a (66%), 7b (87%), 7c (50%), 7d
(52%) and 7e (64%) as diastereomeric mixtures.46
hydrolysis of a depsipeptide substrate, Ac-DED(E-
dans)EEAbuc[COO]ASK(Dabcyl)-NH2 (AnaSpec, San
Jose, USA), was measured spectrofluorometrically in
the presence of a peptide cofactor, KKGSVVIV-
GRIVLSGK (Ake Engstrom, Department of Medical
Biochemistry and Microbiology, Uppsala University,
Sweden).49 The enzyme (1 nM) was incubated in 50 mM
HEPES, pH 7.5, 10 mM DTT, 40% glycerol, 0.1%
n-octyl-b-d-glucoside, with 25 mM cofactor and inhi-
bitor at 30 ꢁC for 10 min, whereupon the reaction was
initiated by addition of 0.5 mM substrate. Inhibitors
were dissolved in DMSO, sonicated for 30 s and vor-
texed. The solutions were stored at ꢀ20 ꢁC between
measurements. The final concentration of DMSO in the
assay sample was adjusted to 3.3%. The rate of hydro-
lysis was corrected for inner filter effects according to
published procedures.50 Ki values were estimated by
non-linear regression analysis (GraFit, Erithacus Soft-
ware, Staines, UK), using a model for competitive inhi-
bition and a fixed value for Km (0.15 mM). A minimum
of two replicates was performed for all measurements.
Oxidation of compound 7a–e to the ketones 8a (69%), 8b
(53%), 8c (73%), 8d (78%) and 8e (96%) was performed
using Dess–Martin periodinane reagent in CH2Cl2.47
The unsaturated ketones 8a–e were hydrogenated over
palladium on carbon in EtOAc delivering the saturated
ketones 9a–e in quantitative yields. Finally, the t-butyl
esters were cleaved in compounds 7a–e, 8a–e and 9a–e,
using TFA in CH2Cl2 containing Et3SiH as scavenger,
to afford the target compounds 10a–e (as diastereomeric
mixtures), 11a–e and 12a–e in quantitative yields (no
racemisations were observed).48
The reference hexapeptide 14, Ac-Asp-d-Glu-Leu-Ile-
Cha-Abu-OH was synthesised by coupling of t-butyl
l-a-aminobutyrate (H-Abu-OtBu) with pentapeptide 6
using PyBOP and DIPEA in CH2Cl2 yielding the pro-
tected hexapeptide Ac-Asp(OtBu)-d-Glu(OtBu)-Leu-
Ile-Cha-Abu-OtBu 13 in 52% yield. The t-butyl esters
were then cleaved off, as above, furnishing unprotected
14 in quantitative yield.
General
All glassware was dried over an open flame before use in
connection with an inert atmosphere. Concentrations
were performed under reduced pressure at <40 ꢁC (bath
temperature). Thin layer chromatography was per-
formed using silica gel 60 F-254 plates with detection by
UV and charring with ninhydrine or KMnO4. Silica gel
(0.040–0.063 mm) was used for column chromato-
graphy. Me4Si (0.0 ppm) was used as an internal stan-
From examination of the three classes of inhibitors in
Table 1, the a,b-unsaturated alcohol, 10e (diastereo-
meric mixture) shows promising inhibitory activity with
a 1.6-fold increased activity as compared with the refer-
ence product inhibitor 14. The most potent group of
inhibitors are the a,b-unsaturated ketones, 11a–e, all
showing promising Ki values in the range 0.18–0.39 mM,
with potency selectivity of 4–8.5 over the product inhibitor
14. The potency of the a,b-unsatureted reduced ketones
appears especially promising in view of the activity of the
alkyl ketones 12d and 12e with Ki=0.65 and 0.46mM,
respectively, and anticipating reversible kinetics.
1
dard in H NMR and Me4Si or CDCl3 (77.0 ppm) were
used in 13C NMR. MALDI-TOF spectra were recorded
on a Bruker Biflex III using 20,40,60-trihydroxy-aceto-
phenone monohydrate (THAP) as matrix. Unless stated
otherwise, all materials were obtained from commercial
suppliers and used without further purification. The
preparative RP-HPLC was performed on a Wares-
Micromass: 600 controller and ZMD mass on a Xterra
MS C8 (5 mm, 19ꢃ100 mm), using an acetonitrile/H2O
gradient with 10 mM NH4HCO3, using MS detection
(positive ionisation with ES). The analytical RP-HPLC
was performed on a Waters-Micromass: 2790 Waters
pump, 996 DAD Waters, and Micromass ZMD mass
on a Phenomex C18 Synergi Max Rp-80A, (4 m,
50ꢃ4.6 mm), using an acetonitrile/H2O gradient with
10 mM NH4HCO3, using UV (DAD and 214 and
254 nm) and MS detection (positive ionisation with ES).
Conclusion
Potent inhibitors of the HCV NS3 protease have been
obtained from a limited set of hexapeptides containing a
P1 modified Abu showing Ki values in the 0.1–2.0 mM
range. Preliminary SAR has been obtained, and simple
P1 alkenyl alcohols and alkyl ketones appear to hold
much promise for further optimisations towards small
molecule inhibitors. Interestingly the non-electrophilic
alkenyl alcohol 10e have superior potency over the cor-
responding product based inhibitor 14, thus providing a
novel entry of non-electrophilic non-product based
inhibitors.
General method for the preparation of 5a–e. Oven dried
K2CO3 (268 mg, 1.94 mmol) was added to a stirred
solution of phosphonate 336 (600 mg, 1.94 mmol) and
the appropriate aldehyde (2mmol) in absolute EtOH
(15 mL). After 16 h the reaction mixture was filtered and
the filtrate was neutralised by glacial acetic acid. The
solvent was removed and the residue was portioned
between EtOAc and saturated aqueous NaHCO3. The
aqueous phase was extracted with EtOAc and the com-
bined organic phases were dried over Na2SO4 and con-
centrated. The crude product was purified by silica gel
column chromatography (solvent system A; toluene;
toluene–EtOAc 20:1, solvent system B; toluene–EtOAc
Experimental
Inhibition assay
The inhibition of full-length hepatitis C NS3 was mea-
sured essentially as described elsewhere.35 Briefly, the

K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 2955–2963
2959
3:1) providing the ketones 4a–e. NaBH4 (1 equiv) was
added at 0 ꢁC in small portions over 10 min, to a stirred
solution of 4a–e (1 equiv) in MeOH. After 2.5 h at 0 ꢁC,
the solution was neutralised with glacial acetic acid, the
solvent was removed and the residue was partitioned
between EtOAc and saturated aqueous NaHCO3. The
aqueous phase was extracted with EtOAc and the com-
bined organic phases were dried over Na2SO4 and con-
centrated. The crude product was purified by silica gel
column chromatography (solvent system C; toluene;
toluene–EtOAc 6:1, solvent system D; toluene–EtOAc
1:1) providing the alcohol 5a–e as a diasteromeric mix-
ture.
according to general procedure, vide supra (solvent sys-
1
tem D). H NMR (300 MHz, CDCl3) d 7.70, 7.68 (d,
1H), 7.17, 7.19 (d, 1H), 6.90 (d, 1H), 6.55, 6.60 (d, 1H),
4.9, 4.8 (d, 1H), 4.38 (m, 1H). 3.8–3.4 (m, 2H), 1.68–1.33
(m, 11H), 0.94 (t, 3H), 13C NMR (75 MHz, CDCl3) d
166.6, 157.3, 143.3, 135.7, 124.4, 118.6, 80.1, 74.6, 73.0,
57.4, 56.6, 28.5, 23.6, 11.1, 11.0. Anal. calcd for
(C14H22N2O3S): C, 56.4; H, 7.4; N, 9.4. Found: C, 56.3;
H, 7.5; N, 9.1.
(5S)-5-[N-(tert-Butyloxycarbonyl)amino]-4-hydroxy-2-
hepten 5d. The title compound was prepared in a two-
step synthesis according to general procedure, vide
supra. The intermediate 4d was prepared in 74% yield
(335 mg, 1.4 mmol) according to general procedure, vide
supra (solvent system A). 13C NMR (75 MHz, CDCl3) d
198.1, 155.6, 144.8, 128.6, 79.6, 58.3, 28.4, 25.5, 18.5,
9.2. The title compound was prepared in 79% yield
(222 mg, 0.94 mmol) according to general procedure,
vide supra (solvent system C). 1H NMR (300 MHz,
CDCl3) d 5.70 (m, 1H), 5.44 (m, 1H), 4.74, 4.63 (bd,
1H), 4.06, 3.98 (m, 1H), 3.52–3.38 (m, 2H), 3.13 (bs,
1H), 1.66 (d, 3H), 1.61–1.19 (m, 11H), 0.90 (t, 3H) 13C
NMR (75 MHz, CDCl3) d 157.2, 129.9, 128.7, 128.5,
79.7, 75.4, 74.4, 57.3, 56.8, 28.6, 23.5, 18.0, 18.0, 10.9,
10.8. Anal. calcd for (C12H23NO3): C, 62.9; H, 10.11; N,
6.1. Found: C, 62.7; H, 10.1; N, 5.9.
(4S)-4-[N-(tert-Butyloxycarbonyl)amino]-3-hydroxy-1-
benzyl-1-hexen 5a. The title compound was prepared in
a two-step synthesis according to general procedure,
vide supra. The intermediate 4a was prepared in 90%
yield (507 mg, 1.8 mmol) according to general proce-
dure, vide supra (solvent system A). 13C NMR
(75 MHz, CDCl3) d 197.8, 155.3, 143.8, 134.0, 130.5,
128.7, 128.2, 122.5, 79.5, 58.8, 28.3, 25.3, 9.20. The title
compound was prepared in 96% yield (289 mg,
1.0 mmol) according to general procedure, vide supra
(solvent system C). 1H NMR (300 MHz, CDCl3) d 7.44–
7.26 (m, 4H), 6.64, 6.16 (d, 1H), 6.26–6.16 (m, 1H), 4.82,
4.7, (d, 1H), 4.37, 4.28 (bs, 1H), 3.73–3.46 (m, 1H), 3.47
(bs, 1H), 1.72–1.31 (m, 2H), 1.40, 1.43 (s, 9H), 0.96 (t,
3H) 13C NMR (75 MHz, CDCl3) d 157.3, 136.9, 131.5,
131.9, 128.8, 128.7, 128.5, 127.9, 126.8, 126.7, 80.0, 75.5,
74.5, 57.6, 57.0, 28.6, 23.6, 11.1, 11.0. Anal. calcd for
(C17H25NO3): C, 70.1; H, 8.7; N, 4.8. Found: C, 69.7;
H, 8.6; N, 4.8.
(3S)-3-[N-(tert-Butyloxycarbonyl)amino]-4-hydroxy-10-
methyl-5-undecaen 5e. The title compound was pre-
pared in a two-step synthesis according to general pro-
cedure, vide supra. The intermediate 4e was prepared in
24% yield (140 mg, 0.47 mmol) according to general
procedure, vide supra (solvent system A). 13C NMR
(75 MHz, CDCl3) d 198.3, 155.7, 149.9, 127.1, 79.6,
58.4, 38.6, 33.1, 28.5, 28.0, 26.0, 25.8, 22.7, 9.3. The title
compound was prepared in 74% yield (104 mg,
0.35 mmol) according to general procedure, vide supra
(solvent system C). 1H NMR (300 MHz, CDCl3) d 5.64–
5.55 (m, 1H), 5.38–5.30 (m, 1H), 5.00–4.80 (m, 1H),
3.98–3.90 (m, 1H), 3.48–3.30 (m, 3H), 1.95–1.90 (m,
2H), 1.34 (s, 9H), 1.52–1.13 (m, 4H), 0.83 (t, 3H), 0.77
(s, 3H), 0.75 (s, 3H), 13C NMR (75 MHz, CDCl3) d
157.2, 133.8, 133.5, 139.8, 128.6, 79.6, 75.0, 74.0, 57.3,
56.8, 39.0, 38.6, 32.7, 28.4, 27.9, 27.1, 23.2, 22.6, 10.8,
10.6. Anal. calcd for (C17H33NO3): C, 68.2; H, 11.1; N,
4.7. Found: C, 68.4; H, 11.1; N, 4.4.
(4S)-4-[N-(tert-Butyloxycarbonyl)amino]-3-hydroxy-1-
(4-methoxybenzyl)-1-hexen 5b. The title compound was
prepared in a two-step synthesis according to general
procedure, vide supra. The intermediate 4b was pre-
pared in 82% yield (590 mg, 1.6 mmol) according to
general procedure, vide supra (solvent system A). 13C
NMR (75 MHz, CDCl3) d 198.2, 162.1, 155.7, 144.2,
130.5, 127.2, 120.7, 114.7, 79.7, 59.0, 55.6, 28.6, 25.9,
9.4. The title compound was prepared in 79% yield
(283 mg, 0.88 mmol) according to general procedure,
vide supra (solvent system C). 1H NMR (300 MHz,
CDCl3) d 7.34–7.28 (m, 2H), 6.86–6.81 (m, 2H), 6.55,
6.57 (d, 1H), 6.11–6.00 (m, 1H), 4.62, 4.72 (bd, 1H),
4.33, 4.24 (m, 1H), 3.8 (s, 3H), 3.76–3.53 (m, 2H), 3.13
(bd, 1H), 1.67–1.31 (m 11H). 0.96 (t, 3H), 13C NMR
(75 MHz, CDCl3) d 159.5, 157.3, 131.5, 129.7, 128.0,
127.9, 126.1, 114.2, 114.2, 80.0, 75.7, 74.8, 57.3, 55.5,
28.6, 23.7, 11.1, 10.9. Anal. calcd for (C18H27NO4): C,
67.3; H, 8.5; N, 4.4. Found: C, 67.3; H, 8.5; N, 4.3.
Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha-OH 6. The
peptide was synthesised using standard Fmoc solid-
phase peptide synthesis techniques. Fmoc-Cha-OH
TM
(5 eqiuv.) was loaded to the SASRIN (1 equiv) using
2,6-dichlorobenzoyl chloride (5 equiv) and pyridine
(5.5 equiv) in DMF with a loading time of 16 h. The
resin was washed with MeOH, DMF, and DCM.
Removal of the Fmoc group was achieved by reaction
with 20% piperidine in DMF for 10+10 min. Peptide
elongation was performed using Fmoc-AA-OH
(2equiv), DIC (2equiv) and HOBt (2 equiv) in DCM.
The peptide was cleaved from the resin using 3% TFA
in DCM and Et3SiH as scavenger, producing 6. 13C
NMR (75 MHz, CDCl3) d 175.2, 174.4, 167.9, 172.6,
172.0, 171.9, 171.7, 171.5, 57.4, 53.0, 52.1, 49.9, 49.9,
(4S)-4-[N-(tert-Butyloxycarbonyl)amino]-3-hydroxy-1-
(thiazol-2-yl)-1-hexen 5c. The title compound was pre-
pared in a two-step synthesis according to general pro-
cedure, vide supra. The intermediate 4c was prepared in
75% yield (500 mg, 1.7 mmol) according to general pro-
cedure, vide supra (solvent system B). 13C NMR
(75 MHz, CDCl3) d 197.9, 163.6, 155.4, 145.3, 134.8,
126.3, 122.3, 80.0, 59.7, 28.5, 25.2, 9.5. The title com-
pound was prepared in 76% yield (382mg, 1.3 mmol)

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K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 2955–2963
40.0, 38.7, 36.4, 35.2, 33.7, 33.2, 31.9, 29.9, 26.0, 25.8,
25.6, 25.5, 24.3, 24.3, 22.4, 21.7, 20.6, 14.6, 10.0.
36.3, 34.1, 33.8, 31.8, 31.7, 28.0, 26.5, 26.4, 26.0, 25.4,
24.8, 22.8, 22.5, 22.5, 21.8, 18.4, 17.8, 16.9, 15.4, 12.6,
11.2, 11.2, 10.7; MS (MALDI-TOF) m/z mass calcd for:
C47H82N6O11, 906.60 found: 945.58 (MK+) 929.63
(MNa+) 907.65 (MH+).
General method for the preparation of 7a–e
Compound 5a–e was added to a solution of TMSOTf
(1 M, 1 mL) and 2,6-lutidine (1.5 M, 1 mL) in CH2Cl2.
After 6 h of stirring the mixture was evaporated. PyBOP
(1 equiv), the pentapeptide 6 (1 equiv) and DIPEA
(2equiv) was added to a stirred solution of the residue
in CH2Cl2. After 16 h the reaction mixture was washed
with aqueous 5% KHSO4 (2ꢃ) and aqueous 5%
NaHCO3 (2ꢃ), dried over Na2SO4 and concentrated.
The residue was purified by silica gel column chroma-
tography (CH2Cl2; CH2Cl2–MeOH 40:1) to give the
target compound 7a–e.
(3S)-3-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-4-hydroxy-10-methyl-5-undecaen 7e. The title
compound was prepared in 64% yield (200 mg,
0.20 mmol) according to general procedure, vide supra.
13C NMR (75 MHz, CDCl3) d 174.3, 173.7, 172.6,
172.3, 172.1, 172.1, 170.4, 170.3, 132.7, 128.7, 82.0, 81.0,
74.9, 59.5, 56.7, 54.9, 54.0, 53.4, 53.4, 51.8, 50.9, 43.0,
40.1, 39.1, 38.7, 36.6, 36.3, 34.2, 33.8, 32.7, 31.9, 31.8,
28.0, 26.5, 25.4, 26.1, 25.4, 25.3, 24.9, 22.8, 22.6, 22.6,
21.6, 18.5, 17.0, 15.4, 12.7, 11.3, 10.8; MS (MALDI-
TOF) m/z mass calcd for: C52H92N6O11, 976.68 found:
1015.64 (MK+) 999.68 (MNa+).
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-3-hydroxy-1-benzyl-1-hexen 7a. The title com-
pound was prepared in 66% yield (161 mg, 0.17 mmol)
according to general procedure, vide supra. 13C NMR
(75 MHz, CDCl3) d 173.9, 173.4, 172.7, 172.1, 172.0,
171.9, 170.2, 136.8, 131.2, 121.2, 128.7, 128.4, 128.4, 127.4,
126.4, 81.9, 81.0, 74.7, 59.0, 56.3, 53.4, 53.1, 53.0, 51.5,
40.1, 39.9, 36.4, 36.3, 33.9, 33.5, 31.7, 31.6, 29.6, 27.8, 26.3,
26.1, 25.8, 25.0, 24.7, 22.7, 22.3, 22.0, 21.3, 15.2, 11.0, 10.5;
MS (MALDI-TOF) m/z mass calcd for: C52H84N6O11,
968.62found: 1007.58 (MK +) 998.58 (MNa+).
General method for the preparation of 8a–e
Dess–Martin reagent (1 equiv) was added to a stirred
solution of the alcohol 7a–e in CH2Cl2 and the mixture
was stirred for 16 h at room temperature. The reaction
mixture was diluted with CH2Cl2 and washed with
Na2S2O3 (7 equiv) in aqueous saturated NaHCO3 (2ꢃ)
and water (1ꢃ). The combined aqueous phases were
washed with CH2Cl2 (2ꢃ) and the combined organic
phases were dried over Na2SO4 and concentrated. The
residue was purified by silica gel column chromatography
(CH2Cl2; CH2Cl2–MeOH 40:1) and on preparative RP-
HPLC producing the target compound 8a–e.
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-3-hydroxy-1-(4-methoxybenzyl)-1-hexen 7b. The
title compound was prepared in 87% yield (217 mg,
0.22 mmol) according to general procedure, vide supra.
13C NMR (75 MHz, CDCl3) d 173.9, 173.5, 172.6,
172.1, 172.1, 172.0, 170.0, 169.8, 159.1, 140.9, 129.7,
127.6, 126.6, 113.7, 113.7, 74.9, 58.9, 56.2, 54.9, 54.6,
53.3, 53.0, 50.7, 42.6, 40.0, 36.3, 36.3, 33.8, 33.5, 31.5,
27.6, 26.0, 25.7, 25.0, 24.6, 22.5, 22.0, 21.0, 17.9, 16.5,
15.0, 12.2, 10.8, 10.3; MS (MALDI-TOF) m/z mass
calcd for: C53H86N6O12, 998.63 found: 1037.60 (MK+)
1021.61 (MNa+).
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-1-benzyl-1-hexen-3-one 8a. The title compound
was prepared in 69% yield (70 mg, 0.07mmol) according
to general procedure, vide supra. 13C NMR (75 MHz,
CDCl3) d 198.2 , 172 .9, 172 .3, 172 .2 , 172 .1, 171.8, 170.2 ,
144.8, 134.5, 131.07, 129.1, 128.8, 122.4, 81.9, 81.1, 59.6,
58.6, 58.5, 53.1, 51.5, 51.4, 50.6, 40.7, 39.4, 36.7, 34.1, 33.7,
32.5, 31.9, 28.0, 26.5, 26.3, 26.2, 26.1, 25.0, 24.9, 24.6, 22.9,
22.5, 21.5, 15.5, 11.0, 9.7; MS (MALDI-TOF) m/z mass
calcd for: C52H82N6O11, 966.60 found: 1005.65 (MK+)
989.68 (MNa+); RP-LC MS (EI) found: 966.57 (MH+).
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-3-hydroxy-1-(thiazol-2-yl)-1-hexen 7c. The title
compound was prepared in 50% yield (164 mg,
0.17 mmol) according to general procedure, vide supra.
13C NMR (75 MHz, CDCl3) d 173.7, 172.6, 172.5,
172.3, 172.2, 172.1, 172.0, 170.2, 170.1, 167.4, 142.4,
142.3, 136.7, 123.5, 118.6, 118.5, 81.9, 80.9, 73.5, 59.6,
55.9, 55.8, 55.7, 53.9, 53.8, 53.2, 51.7, 51.6, 50.7, 42.8,
36.4, 34.0, 31.6, 31.5, 27.8, 26.3, 26.3, 26.2, 26.1, 25.8,
25.2, 24.7, 22.5, 22.3, 21.3, 18.2, 16.7, 15.2, 12.4, 11.1,
11.0,10.5, 10.4; MS (MALDI-TOF) m/z mass calcd for:
C49H81N7O11S, 975.57 found: 1014.45 (MK+) 998.49
(MNa+) 976.51 (MH+).
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-1-(4-methoxybenzyl)-1-hexen-3-one 8b. The title
compound was prepared in 53% yield (60 mg,
0.06 mmol) according to general procedure, vide supra.
13C NMR (75 MHz, CDCl3) d 198.3, 173.6, 173.5,
172.9, 172.3, 172.3, 172.1, 172.0, 170.2, 162.2, 144.8,
130.7, 127.2, 120.1, 114.6, 81.8, 81.18, 58.6, 58.5, 58.4,
55.4, 53.2, 51.4, 50.7, 40.7, 39.4, 36.7, 34.1, 33.7, 32.4,
32.9, 29.7, 27.9, 26.5, 25.3, 26.2, 26.1, 25.0, 24.9, 24.7,
24.7, 22.9, 22.4, 21.4, 15.4, 11.0, 9.7; MS (MALDI-
TOF) m/z mass calcd for: C53H84N6O12, 996.61 found:
1035.63 (MK+) 1019.54 (MNa+) RP-LC MS (EI)
found: 997.64 (MH+).
(5S)-5-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-4-hydroxy-2-hepten 7d. The title compound was
prepared in 52% yield (140 mg, 0.15 mmol) according to
general procedure, vide supra. 13C NMR (75 MHz,
CDCl3) d 174.2, 173.6, 172.6, 172.3, 172.2, 172.2, 172.1,
170.3, 130.2, 127.8, 81.9, 80.9, 74.9, 59.4, 56.5, 54.8,
53.9, 53.4, 51.7, 50.9, 46.4, 46.3, 42.9, 40.1, 39.1, 36.5,
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-1-(thiazole-2-yl)-1-hexen-3-one 8c. The title com-
pound was prepared in 73% yield (70 mg, 0.0.7 mmol)
according to general procedure, vide supra. 13C NMR

K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 2955–2963
2961
(75 MHz, CDCl3) d 197.5, 173.7, 173.3, 172.9, 172.3,
172.2, 172.1, 172.0, 170.2, 144.7, 134.6, 126.7, 122.6,
81.8.81.0, 59.3, 59.2, 58.6, 53.1, 51.4, 50.7, 40.6, 39.4,
36.7, 34.1, 33.7, 32.6, 31.8, 31.7, 29.7, 27.9, 26.5, 26.3,
26.1, 26.1, 25.0, 24.9, 24.0, 22.9, 22.3, 21.3, 15.4, 10.9,
9.7; MS (MALDI-TOF) m/z mass calcd for:
C49H97N7O11S, 973.56 found: 1012.50 (MK+) 996.54
(MNa+) 974.57 (MH+); RP-LC MS (EI) found: 974.59
(MH+).
172.8, 172.4, 172.4, 172.3, 172.2, 170.2, 158.1, 133.2,
129.4, 113.9, 81.7, 80.9, 60.3, 58.7, 55.0, 53.3, 53.2, 51.3,
50.9, 41.0, 40.4, 39.1, 36.6, 36.5, 34.1, 33.7, 32.3, 31.7,
28.5, 27.8, 26.4, 26.3, 26.1, 25.9, 25.1, 24.9, 23.6, 22.7,
22.1, 21.2, 15.3, 10.8, 9.8; MS (MALDI-TOF) m/z mass
calcd for: C53H86N6O12, 998.63 found: 1037.65 (MK+)
1021.68 (MNa+).
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-1-(thiazole-2-yl)-3-hexenone 9c. The title com-
pound was prepared in 100% yield (23 mg, 0.024 mmol)
according to general procedure, vide supra. 13C NMR
(75 MHz, CDCl3) d 207.9, 173.9, 173.6, 172.8, 172.4,
172.4, 172.4, 172.3, 170.2, 141.6, 119.0, 81.7, 80.9, 60.3,
58.3, 53.3, 53.2, 51.3, 50.9, 40.3, 39.1, 38.5, 36.6, 36.5,
34.1, 33.7, 32.1, 31.7, 27.8, 26.6, 26.4, 26.3, 26.1, 25.9,
25.2, 24.9, 23.6, 22.7, 22.1, 21.16, 15.3, 10.8, 9.8; MS
(MALDI-TOF) m/z mass calcd for: C49H81N7O11S,
975.57 found: 1014.62(MK +) 998.67 (MNa+) 976.67
(MH+).
(5S)-5-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-2-hepten-4-one 8d. The title compound was pre-
pared in 78% yield (78 mg, 0.09 mmol) according to
general procedure, vide supra. 13C NMR (75 MHz,
CDCl3) d 198.1, 198.0, 173.4, 172.9, 172.8, 172.3, 172.2,
172.0, 171.8, 145.5, 128.3, 81.8, 81.1, 58.9, 58.3, 58.1,
57.8, 53.3, 53.2, 53.1, 53.0, 52.7, 52.3, 50.6, 50.3, 40.1,
39.0, 36.8, 36.7, 35.9, 34.2, 34.1, 33.9, 33.7, 32.5, 31.8,
31.7, 29.8, 28.0, 26.5, 26.3, 26.1, 26.0, 25.2, 25.0, 24.9,
24.6, 24.3, 23.0, 22.9, 22.4, 21.4, 21.2, 18.4, 18.4, 16.9,
15.4, 15.3, 13.6, 10.9, 10.8, 10.0, 9.5; MS (MALDI-
TOF) m/z mass calcd for: C47H80N6O11, 904.59 found:
943.65 (MK+) 927.66 (MNa+) 905.67 (MH+); RP-LC
MS (EI) found: 905.60 (MH+).
(5S)-5-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-4-heptenone 9d. The title compound was pre-
pared in 100% yield (25 mg, 0.0.28 mmol) according to
general procedure, vide supra. 13C NMR (75 MHz,
CDCl3) d 210.4, 210.2, 173.9, 173.8, 173.2, 172.9, 172.8,
172.7, 172.4, 172.1, 172.0, 1720, 170.2, 170.1, 81.9, 81.7,
81.1, 60.2, 60.1, 59.1, 58.7, 53.4, 53.3, 53.2, 51.3, 51.2,
50.8, 41.4, 41.2, 40.6, 40.4, 39.1, 39.0, 36.8, 36.7, 36.6,
35.7, 34.2, 34.2, 33.9, 33.7, 32.2, 31.8, 27.9, 26.5, 26.4,
26.2, 26.1, 25.9, 25.3, 25.1, 25.9, 23.8, 23.6, 23.0, 22.8,
22.3,.21.3, 21.1, 16.9, 16.9, 15.4, 15.3, 13.5, 11.0, 10.2,
9.9; MS (MALDI-TOF) m/z mass calcd for:
C47H82N6O11, 906.60 found: 945.65 (MK+) 929.69
(MNa+)
(3S)-3-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-10-methyl-5-undecaen-4-one 8e. The title com-
pound was prepared in 96% yield (95 mg, 0.10 mmol)
according to general procedure, vide supra. 13C NMR
(75 MHz, CDCl3) d 198.1, 173.4, 172.9, 172.7, 172.3,
172.2, 172.0, 171.8, 170.2, 150.4, 126.6, 81.8, 81.0, 58.3,
57.8, 53.1, 52.9, 51.3, 50.6, 40.7, 39.6, 39.4, 38.9, 38.6,
36.8, 36.7, 34.1, 33.7, 33.1, 32.5, 31.8, 28.9, 26.5, 26.3,
26.2, 26.1, 25.9, 24.9, 24.7, 22.9, 22.5, 22.4, 22.4, 22.4,
21.4, 15.4, 10.9, 9,5; MS (MALDI-TOF) m/z mass calcd
for: C52H90N6O11, 974.67 found: 1015.64 (MK+) 999.68
(MNa+); RP-LC MS (EI) found: 975.70 (MH+).
(3S)-3-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-10-methyl-4-undecaenone 9e. The title com-
pound was prepared in 100% yield (24 mg, 0.025 mmol)
according to general procedure, vide supra. 13C NMR
(75 MHz, CDCl3) d 210.3, 173.8, 173.3, 172.9, 172.4,
172.2, 170.2, 81.8, 81.0, 60.1, 58.7, 53.3, 53.2, 51.3, 50.8,
40.4, 39.3, 39.2, 38.9, 36.7, 36.6, 34.1, 33.8, 32.2, 31.8,
29.5, 28.0, 27.9, 27.3, 26.5, 26.4, 26.1, 25.9, 25.1, 25.9,
23.8, 23.5, 22.9, 22.8, 22.4, 22.2, 21.3, 15.3, 10.9, 9.9; MS
(MALDI-TOF) m/z mass calcd for: C52H92N6O11,
976.66 found: 1015.70 (MK+) 999.72(MNa +).
General method for the preparation of 9a–e
A catalytic amount of Pd/C (10%) was added to 8a–e in
MeOH. Hydrogen was added at atmospheric pressure
to the system and the reaction mixture was stirred for
1 h. The suspension was then filtrated through Celite
and the solvent was removed to give 9a–e.
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-1-benzyl-3-hexenone 9a. The title compound was
prepared in 100% yield (33 mg, 0.034 mmol) according
to general procedure, vide supra. 13C NMR (75 MHz,
CDCl3) d 209.2, 173.9, 173.5, 172.8, 172.4, 172.4, 172.3,
172.2, 170.2, 141.0, 128.4, 128.4, 126.1, 81.9, 81.0, 60.3,
58.7, 53.3, 53.2, 51.3, 50.9, 40.8, 40.4, 39.1, 36.6, 36.6,
34.1, 33.7, 32.2, 31.7, 29.4, 27.8, 26.4, 26.3, 26.1, 25.9,
25.1, 24.9, 23.6, 22.7, 22.1, 21.2, 15.3, 10.9, 9.8; MS
(MALDI-TOF) m/z mass calcd for: C52H84N6O11,
968.62found: 1007.58 (MK +) 998.58 (MNa+).
General method for the preparation of 10a–e, 11a–e and
12a–e
Compound 7a–e, 8a–e or 9a–e was added to a stirred
solution of TFA (30 equiv) and Et3SiH (6 equiv) in
CH2Cl2 (100 equiv). After 1 h, toluene (3 mL) was added
and the mixture was concentrated to give the free acid
10a–e, 11a–e and 12a–e. The peptides were analysed by
MALDI-TOF and by analytical RP-HPLC. All the
measured weight were within +0.13 to +0.19 mass unit
of the calculated weight (M+H+, M+Na+, M+K+),
of the compounds.
(4S)-4-[N-(Ac-Asp(OtBu)-D-Glu(OtBu)-Leu-Ile-Cha)-
amino]-1-(4-methoxybenzyl)-3-hexenone 9b. The title
compound was prepared in 100% yield (36 mg,
0.036 mmol) according to general procedure, vide supra.
13C NMR (75 MHz, CDCl3) d 209.3, 1173.8, 173.4,
Ac-Asp-D-Glu-Leu-Ile-Cha-Abu-OH 14. H-N-Abu-OtBu
ꢃ HCl (23 mg, 0.14 mmol) was added to a mixture of

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K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 2955–2963
PyBOP (65 mg, 0.13 mmol), 6 (100 mg, 0.13 mmol) and
DIPEA (47 mL, 0.27 mmol) in CH2Cl2. After 16 h stir-
ring at rt the reaction mixture was washed with aqueous
5% KHSO4 (2ꢃ) and aqueous 5% NaHCO3 (2ꢃ), dried
over Na2SO4 and concentrated. The residue was pur-
ified by silica gel column chromatography (CH2Cl2;
CH2Cl2–MeOH 40:1) to give Ac-Asp(OtBu)-d-
Glu(OtBu)-Leu-Ile-Cha-Abu-OtBu 13 in 52% yield
(68 mg, 0.08 mmol). 13C NMR (75 MHz, CDCl3) d
173.4, 172.9, 172.4, 172.3, 171.9, 171.6, 171.5, 170.9,
169.7, 81.4, 81.2, 80.5, 57.8, 54.0, 52.6, 52.4, 50.6, 50.3,
40.1, 38.9, 36.2, 33.7, 33.6, 33.3, 31.9, 31.2, 27.3, 27.2,
26.0, 25.8, 25.7, 25.6, 24.6, 24.4, 24.4, 22.3, 21.7, 20.7,
14.8, 10.2, 9.1. Compound 13 (25 mg, 0.03 mmol) was
added to a stirred solution of TFA (85 mL, 1.14 mmol)
and Et3SiH (29 mL, 0.18 mmol) in CH2Cl2 (185 mL,
2.9 mmol). After 1 h, toluene (3 mL) was added and the
solvent was removed to give the target compound 14 in
quantitative yield (23 mg, 0.03 mmol). The peptide was
analysed by MALDI-TOF m/z mass calcd for:
C36H60N6O12, 768.43 found: 807.43 (MK+) 791.46
(MNa+) 769.46 (MH+) and by analytical RP-HPLC:
791.45 (MNa+) 769.48 (MH+).
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Acknowledgements
We gratefully acknowledge Medivir AB, Huddinge,
Sweden for financial support.
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