Exploration of acyl sulfonamides as carboxylic acid replacements in protease inhibitors of the hepatitis C virus full-length NS3

Article abstract of DOI:10.1016/j.bmc.2005.08.045

The hepatitis C virus (HCV) NS3 protease has emerged as a promising anti-HCV drug target. Herein, we present an investigation of NS3 inhibitors comprising the acyl sulfonamide functionality. A series of tetra- and tripeptide based acyl sulfonamide inhibit

Full text of DOI:10.1016/j.bmc.2005.08.045

Bioorganic & Medicinal Chemistry 14 (2006) 544–559
Exploration of acyl sulfonamides as carboxylic acid replacements
in protease inhibitors of the hepatitis C virus full-length NS3
a
a
b
a
˚
Robert Ro¨nn, Yogesh A. Sabnis, Thomas Gossas, Eva Akerblom,
U. Helena Danielson,b Anders Hallberga and Anja Johanssona,*
aDepartment of Medicinal Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
bDepartment of Biochemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
Received 13 May 2005; revised 12 August 2005; accepted 19 August 2005
Available online 5 October 2005
Abstract—The hepatitis C virus (HCV) NS3 protease has emerged as a promising anti-HCV drug target. Herein, we present an
investigation of NS3 inhibitors comprising the acyl sulfonamide functionality. A series of tetra- and tripeptide based acyl sulfon-
amide inhibitors and their structure–activity relationships from both enzymatic and cell-based in vitro assays are presented. In sum-
mary, the acidity of the acyl sulfonamide functionality, the character of the P1 side chain, and the acyl sulfonamide substituent were
found to be important for the inhibitory potencies.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
structures and guided the design of less peptide-like
inhibitors ultimately leading to the discovery of BILN
2061, a cyclic inhibitor with a C-terminal carboxylate
group and with a powerful anti-HCV activity in
human.5 The C-terminal carboxylate seems crucial for
both potency and selectivity of the product-based
HCV NS3 protease inhibitors.8,9
The hepatitis C virus (HCV) is a major health problem
with an estimated 170 million people infected world-
wide. Approximately 80% of the infected individuals
develop a chronic infection that might progress into cir-
rhosis and eventually liver cancer.1 Unfortunately, low
efficacy and severe side effects have hampered the
success of the present therapy regime and the need of
improved antiviral agents is critical.2
We recently found that the C-terminal carboxylate could
successfully be replaced by carboxylic acid bioisosteres
such as the tetrazole and the phenyl acyl sulfonamide
in hexa- and pentapeptide inhibitors (Fig. 1).10,11 The
acyl sulfonamide inhibitors displayed an impressive in-
crease in the potency as compared to the corresponding
carboxylic acids. Furthermore, the acyl sulfonamide
inhibitors exhibited an exceptional specificity for the
NS3 protease.12 Campbell and co-workers at Bristol-
Myers Squibb have recently devoted substantial efforts
in developing acyl sulfonamide based NS3 protease
inhibitors.13–18
The virally encoded non-structural protein 3 (NS3) is
a bifunctional protein with both serine protease and
helicase/NTPase activities that is crucial for the deliv-
ery of essential viral components.3 Studies have vali-
dated the role of the protease in the replication of
the virus and an NS3 protease inhibitor has recently
been proven to be effective in reducing the viral load
in humans.4,5
`
Steinkuhler et al. and Llinas-Brunet et al. observed that
¨
the HCV NS3 protease is subjected to N-terminal prod-
uct inhibition and potent hexapeptides derived from the
P6-P1 residues of the natural substrates were identi-
fied.6,7 These hexapeptides subsequently served as lead
We felt encouraged to further explore the potential of
the acyl sulfonamide in HCV NS3 protease inhibitors.
Herein, we report a more detailed investigation of the
promising acyl sulfonamide functionality in tetra- and
tripeptide based inhibitors with both enzymatic and
cell-based inhibition data. Systematic alterations of the
acyl sulfonamide group were conducted and the influ-
ence of the P1 side chain was assessed. Notably, we
Keywords: HCV; NS3; Acyl sulfonamide; Protease inhibitor.
*
Corresponding author. Tel.: +46 18 471 4957; fax +46 18 471 4474;
e-mail: Anja.Johansson@orgfarm.uu.se
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.045

R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
545
Table 1. Inhibition constants of tetrapeptide inhibitors of HCV NS3
A
O
O
H
N
H
N
HO
R
N
H
N
H
O
O
O
HO
O
Figure 1. Impact of replacing the C-terminal carboxylic acid for a
tetrazole or a phenyl acyl sulfonamide.11
B
O
O
H
N
H
N
HO
R
N
H
N
H
demonstrate that not only the acidity of the acyl sulfon-
amide is important for inhibitory potency but also sug-
gest that the two sulfonyl oxygens per se are involved in
interactions with the enzyme.
O
O
O
Peptide
structure
Compound
R
Ki SDa (lM)
O
O
A
1
46
12
3
1
OH
2. Chemistry
O
O
The inhibitors included in the SAR study (Tables 1–3)
required preparation via different routes. The tetrapep-
tide inhibitors 1 and 4 with a C-terminal carboxylic acid
were prepared using the standard Fmoc/t-Bu SPPS
methodology.19 The acyl sulfonamide containing tetra-
peptides 2, 3, 5, and 6 were prepared by an inverse SPPS
method where tri-tert-butoxysilyl/fluorenylmethyl ester
protected amino acids were used (Table 1).20
S
2
N
H
O
O
O
O
O
3b
8.4 0.8
S
N
H
B
4
28
3
OH
O
The tripeptide acyl sulfonamide inhibitors encompass-
ing a P1 norvaline (Nva) side chain were synthesized
by a convergent route and provided compounds with
C-terminal carboxylic acid, alkyl/aryl acyl sulfonamide,
benzylamide, phenyl b-ketosulfone, and N-methylated
phenyl acyl sulfonamide. Thus, the P1-P10 building
block (Scheme 1) and the key intermediate 1513 compris-
ing the P3-P2 residues were prepared for ultimate
attachment via amide bond formation to suppress epi-
merization at the Nva a-carbon (Scheme 2). Synthesis
of 15 has been previously described by others.13
O
O
S
5
6
9.7 0.8
5.3 1.0
N
H
O
O
S
N
H
SD, standard deviation.
a Evaluated in an enzymatic assay comprising the full-length NS3
protein.
b 60:40 mixture of diastereomers at Nva.
The P1-P10 building blocks were prepared as described
in Scheme 1. The amide 7 was synthesized in solution
using O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) as coupling reagent and
benzylamine; a reaction that delivered 90% yield. After
Boc deprotection, the desired hydrochloride 8 was ob-
tained quantitatively. The b-ketosulfone 10 was success-
fully prepared in 85% yield from Boc-L-Nva-OMe using
two equiv of methyl phenyl sulfone and four equiv of n-
butyl lithium.21,22 The subsequent Boc deprotection
gave 11 in quantitative yield. Partial racemization oc-
curred during synthesis of 11 (approximately 20%
racemization according to HPLC). However, at room
temperature, this racemization was considerably sup-
pressed (Method A in Section 6, <5% racemization
according to HPLC). The N-methylated acyl sulfon-
amide 12f was prepared from 12c in 81% yield using
methyl iodide and Cs2CO3 at 65 ꢁC. Boc deprotection
of the acyl sulfonamide P1-P10 building blocks gave
the desired hydrochlorides 13a–f in 93–100% yields.
The hydrochlorides 13b and d were converted to 14b
and d in quantitative yields using propylene oxide.25
1
according to H NMR of the final inhibitor 24). The
acyl sulfonamide building blocks 12a–e were prepared
in 36–82% yields from Boc-L-Nva and the correspond-
ing sulfonamides using 1,1-carbonyldiimidazole (CDI)
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).11,23,24
Analysis of the final inhibitors revealed that partial race-
mization had occurred under these conditions. Initially,
the reaction was heated, which resulted in a significant
degree of racemization (Method B in Section 6, ꢀ10%
The Nva containing tripeptide inhibitors were finally pre-
pared as described in Scheme 2. The methyl ester 16 was
synthesized from 1513 and commercially available HClÆ
L-Nva-OMe, which after ester hydrolysis using LiOH
gave the carboxylic acid inhibitor 17 in 87% yield (2
steps). The inhibitors 18–25 were prepared by coupling
of 1513 with the corresponding P1-P10 building blocks

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R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
Table 2. Inhibition constants and EC50 values of tripeptide inhibitors
of HCV NS3: exploring the acyl sulfonamide functionality
separated on silica gel and the major diastereomer in each
case was subjected to biochemical evaluation (except for
24 that was tested as an 80:20 mixture of diastereomers).
The N-methylated acyl sulfonamide 25 is susceptible to
nucleophilic attack at the acyl sulfonamide carbonyl car-
bon and is thus moderately stable in solvents such as
water or methanol.26 Fortunately, we found it to be com-
pletely stable for 2 h in the buffer system used in the enzy-
matic assay, which facilitated biochemical evaluation.
O
N
O
N
O
O
NH
R
NH
O
O
The inhibitors containing the 1-amino-cyclopropane-
carboxylic acid (ACCA) or the (1R, 2S)-1-amino-2-vi-
nyl-cyclopropanecarboxylic acid (vinyl-ACCA), with
no risk of epimerization, were prepared in a linear fash-
ion (Scheme 2). The ester protected P1 amino acid was
coupled to 1513 using HBTU to give 2613 and 27 in
84% and 76% yields, respectively. Ester deprotection
with LiOH gave carboxylic acid inhibitors 2813 and
2927 in good yields. The acyl sulfonamide inhibitors
30–32 were prepared in moderate yields (75%, 60%,
and 63%, respectively) from the corresponding acids uti-
lizing preactivation with HATU and N,N-diisopropyl-
ethylamine (DIEA) followed by addition of the
sulfonamide, 4-dimethylaminopyridine (DMAP), and
DBU.28
Compound
R
Ki SDa (lM) EC50b(lM)
O
17
0.76 0.10
0.77 0.05
0.58 0.07
>10
>10
>10
OH
O
O
O
18
19
S
N
H
O
O
O
S
N
H
O
O
O
S
N
20
21
0.30 0.04
0.18 0.01
>10
H
3. Biochemical evaluation
The Ki-values for inhibitors 1–6, 17–25, and 28–32 were
determined in an in vitro assay comprising the full-
length NS3 protein and the central part of NS4A as
cofactor (Tables 1–3).29,30 EC50-values for compounds
17–24 and 28–32 were determined in an in vitro HCV rep-
licon assay comprising human hepatic Huh-7 cells trans-
fected with subgenomic HCV RNA (Tables 2 and 3).31
O
O
S
O
O
4.6
N
H
O
O
S
N
H
22
23
0.42 0.10
5.2 1.0
2.7 0.6
3.7 0.4
>10
>10
>10
n.d.
O
4. Results and discussion
N
H
After having introduced the acyl sulfonamide function-
ality in hexa- and pentapeptide inhibitors10,11, we felt
prompted to address the issue whether this group could
serve as a proper carboxylic acid replacement also on
shorter scaffolds. Two different tetrapeptide sequences
originating from our tetrapeptide library and an opti-
mized tripeptide scaffold developed by the researchers
at Boehringer Ingelheim were chosen.30,32 The isopro-
pyl and the phenyl acyl sulfonamides were evaluated
on the two tetrapeptide sequences (Table 1), whereas
a more detailed investigation of acyl sulfonamide sub-
stituents (Me, iPr, Ph, Bn, and phenethyl) was per-
formed on the tripeptide scaffold (Table 2). Also in
these two series the acyl sulfonamide group rendered
inhibitors with improved potencies compared to the
corresponding carboxylic acids, although the methyl
acyl sulfonamide 18 was found to be equipotent to
the carboxylic acid 17. The response to the bioisosteric
replacement is most pronounced when the acyl sulfon-
amides have substituents containing an aromatic ring
system (20–22). Molecular modeling suggested three
putative binding modes of the acyl sulfonamide substi-
tuent. The preference for aromatic groups could be
O
O
S
O
O
24c
O
O
S
25d
N
SD, standard deviation.; n.d., not determined.
a Evaluated in an enzymatic assay comprising the full-length NS3
protein.
b Evaluated in
determinations.
a HCV Replicon assay. Mean value of two
c 80:20 mixture of diastereomers at Nva.
d Did not bind irreversible to the enzyme.
using HBTU or O-(7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU). In
case of diastereomers (see above), they were successfully

R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
547
Table 3. Inhibition constants and EC50 values of tripeptide inhibitors of HCV NS3: influence from the P1 side chain
O
N
O
N
O
O
NH
P1
R
NH
O
O
Ki SDa (lM)
EC50 (lM)
b
Compound
P1
R
O
17
0.76 0.10
>10
OH
O
O
S
O
N
H
20
28
30
29
0.30 0.04
0.32 0.03
>10
>10
3.2
O
OH
O
O
O
S
N
H
0.055 0.007
0.030 0.006
O
1.1
(S)
OH
O
O
O
O
S
N
31
0.00076 0.00026
0.018 0.003
0.040
0.28
H
O
O
S
32
N
H
SD, standard deviation.
a Evaluated in an enzymatic assay comprising the full-length NS3 protein.
b Evaluated in a HCV Replicon assay. Mean value of two determinations.
explained by favorable pi-stacking interactions between
the phenyl ring and the side chain of Q41, as suggested
by molecular modeling of 20 (Fig. 2B), and which is in
line with the X-ray crystal structure of an a-ketoamide
inhibitor encompassing a phenyl ring in a position sim-
ilar to that reported by Liu et al.33 However, the C-ter-
minal phenyl group could also be seen docked in two
other orientations. One in which it stacks over the ali-
phatic side chain of K136 as previously proposed for
amide inhibitors by Colarusso et al.,34 and the other
where the C-terminal phenyl lies in the S10 site between
the backbones of K136, G137 on one side and Q41, S42
on the other.
additional carbon spacer did not improve the potency
(22, Ki = 0.42 lM).
To find out whether the acidity or the presence of the
sulfonyl oxygens was important for efficient inhibitory
potency of the acyl sulfonamides, three non-acidic ana-
logs of 20 were synthesized, the amide 23, the b-keto-
sulfone 24, and the N-methylated acyl sulfonamide 25
(Table 2). The amide 23 is 17-fold less potent than
the corresponding acyl sulfonamide 20, which empha-
sizes the importance of the sulfone group in the acyl
sulfonamides for efficient inhibition. The b-ketosulfone
24 and the N-methylated acyl sulfonamide 25 both dis-
played approximately 10 times lower potency as com-
pared to 20, suggesting that the acidity of the acyl
sulfonamides is important for efficient enzyme inhibi-
The benzyl substituent (21, Ki = 0.18 lM) was found to
be optimal in the tripeptide series. Elongation with one

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R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
Scheme 1. Reagents: (a) benzylamine, HBTU, DIEA, DMF; (b) HCl in EtOAc or 1,4-dioxane; (c) MeI, Cs2CO3, DMF; (d) MeSO2Ph, n-BuLi, THF;
(e) H2NSO2R, CDI, DBU, THF; (f) propylene oxide, EtOH or EtOH/MeOH.
Scheme 2. Reagents: (a) HClÆL-Nva-OMe, HBTU, DIEA, DMF; (b) LiOH, THF, MeOH, H2O; (c) 13a, 14b, 13c, 14d, 13e, 8, 11 or 13f, HBTU or
HATU, DIEA, DMF; (d) HClÆACCA-OMe or HClÆvinyl-ACCA-OEt, HBTU, DIEA, DMF; (e) H2NSO2R00, HATU, DIEA, DMAP, DBU, DMF.
tion. However, 24 and 25 are still slightly more potent
than 23, indicating that also the sulfonyl oxygens are
involved in interactions with the enzyme. According
to docking studies of 20 it seems likely that one of
the sulfonyl oxygens is H-bonded to the eNHs of
Q41 and the other to the backbone NH of G137
(Fig. 2B). Attempts were made to model the acyl sul-
fonamides constraining the two sulfonyl oxygens in
the oxyanion hole as reported for a-ketoacids by Di
Marco et al.35 However, the compounds have a high
internal strain in this pose indicating a non-favorable
binding mode.
Rancourt et al. have recently reported the bright design
of the optimized P1 amino acid vinyl-ACCA, which
yielded inhibitors more potent than the ones containing

R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
549
Figure 2. (A) Connolly surface representation of the full-length NS3 protein with docked inhibitor 20 occupying the active site. The helicase domain
is depicted in blue, the protease domain in green, and the catalytic triad in red (H57, D81, and S139). (B) Possible H-bonding interactions between the
acyl sulfonamide functionality of inhibitor 20 and the full-length NS3 protein. The P1 carbonyl is seen lying in the oxyanion hole (NHs of G137 and
S139), the sulfonyl oxygens bind to the oxyanion hole, and the eNHs of Q41 and the phenyl ring pi-stacks with the side chain of Q41. H-bond
˚
distances: 2.0–2.7 A. Atoms are colored according to atom type (oxygen in red, nitrogen in blue, and sulfur in yellow). Only essential hydrogens are
displayed for clarity.
the native cysteine in tetrapeptides with a C-terminal
carboxylate.36 On the contrary, Orvieto et al. observed
that when using a scaffold similar to the one used herein,
the P1 vinyl-ACCA was detrimental to the potencies of
their amide-based inhibitors.37 In addition, we have seen
that the potencies of our hexa- and pentapeptide acyl
sulfonamide inhibitors were dependent on the P1 side
chain.11 Consequently, we decided to explore the out-
come from incorporation of vinyl-ACCA and its unsub-
stituted analog ACCA in the tripeptide acyl sulfonamide
inhibitors for comparison to the linear Nva-analogs (Ta-
ble 3). These two modifications render inhibitors closely
related to the ones disclosed in Bristol-Myers Squibbꢀs
patent application.13 Thus, for inhibitors with a C-ter-
minal carboxylate the vinyl-ACCA (29)27 gave a 25-fold
increase in potency as compared to the Nva-based inhib-
itor (17) and a 11-fold increase in potency as compared
to the ACCA-based inhibitor (28)13. However, we were
surprised to see the effect of exchanging the C-terminal
carboxylic acid for the phenyl acyl sulfonamide in inhib-
itors comprising the P1 vinyl-ACCA (2927 vs. 31). This
alteration gave a 39-fold increase in the potency, as com-
pared to a 2.5-fold and a sixfold increase for the same
substitution in the Nva- (17 vs. 20) and the ACCA-
based (2813 vs. 30) inhibitors, respectively. In contrast
to the observations made by Orvieto et al. on neutral
amide inhibitors,37 the P1 vinyl-ACCA seems to be opti-
mal for acyl sulfonamide based inhibitors. Modeling
studies suggest that the vinyl-ACCA lies more inside
the S1 pocket than Nva or ACCA thereby packing its
inhibitors more tightly with the enzyme.
The acyl sulfonamide NS3 protease inhibitors are not
only more potent than the corresponding carboxylic acids
in the enzymatic assay but also in the cell-based replicon
assay, as seen from the EC50 values (Tables 2 and 3).
The benzyl acyl sulfonamide 21 was the only Nva contain-
ing inhibitor showing detectable potency (EC50
=
4.6 lM). The most potent inhibitor 31, which has a Ki
of 0.76 nM, also displays a low EC50 of 40 nM. Compared
to the inhibitors containing a C-terminal carboxylic
acid, the acyl sulfonamide inhibitors can be structurally
modified to enhance the cellular potency.
5. Conclusion
We have performed an investigation of acyl sulfona-
mides as replacements to the C-terminal carboxylic acid
found in product-based protease inhibitors of the HCV
full-length NS3. The tetra- and tripeptide acyl sulfon-
amide inhibitors synthesized were shown to exhibit
inhibitory potencies higher than those of the corre-
sponding carboxylic acids in both enzymatic and cell-
based assays. Results herein demonstrate that the acidi-
ty of the acyl sulfonamide functionality is important for
inhibitory potency and that the sulfonyl oxygens of the
acyl sulfonamide and the acyl sulfonamide substituent
contribute to the inhibitory potencies. Moreover, it
has been demonstrated that the positive outcome of
the acyl sulfonamide inhibitors is highly dependent on
the character of the P1 side chain. Taken together, this
study emphasizes the potential of acyl sulfonamides as
carboxylic acid replacements in product based NS3
protease inhibitors.
Surprisingly, no gain in potency was observed upon
combining the effects of the vinyl-ACCA and the
benzyl substituent of 21 that was the most potent
inhibitor in the Nva series. In fact, the benzyl acyl
sulfonamide 32 turned out to be 24 times less potent
than the corresponding phenyl substituted inhibitor
31. This finding might be explained by a different
binding mode of the vinyl-ACCA inhibitors
compared to the Nva-based inhibitors as discussed
above.
6. Experimental
6.1. Chemistry
Reagents and solvents were obtained commercially and
used without further purification. Thin layer chroma-
tography (TLC) was performed using aluminum sheets

550
R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
precoated with silica gel 60 F254 (0.2 mm, E. Merck).
Chromatographic spots were visualized using UV-de-
tection and/or spraying with an ethanolic ninhydrin
solution (2%) followed by heating. Column chromatog-
raphy was performed using silica gel 60 (40–63 lm, E.
Merck). Analytical RP-HPLC-MS was performed on
a Gilson-Finnigan ThermoQuest AQA system equipped
4.23–4.19 (m, 1H), 4.07 (d, J = 6.2 Hz, 1H), 3.59–3.51
(m, 1H), 3.45–3.39 (m, 1H), 3.20–3.14 (m, 1H), 2.61–
2.51 (m, 4H), 2.20–2.14 (m, 2H), 1.90–1.55 (m, 9H),
1.46–1.30 (m, 3H), 1.28 (d, J = 6.9 Hz, 3H), 1.28 (d,
J = 6.9 Hz, 3H), 1.23–1.05 (m, 5H), 0.91 (dd, J = 7.3,
7.4 Hz, 3H). HPLC purity (C18 column: 96%, C4 column:
100%). HRMS (M+H+) calcd for C38H54N5O11S:
788.3541; found: 788.3575. Amino acid analysis Chg
0.99, Glu 1.02, Nva 0.99.
with
a
C18 (Chromolith Performance RP-18e
(4.6 · 100 mm)) or a C4 (Hichrom ACE C4 (5 lm,
4.6 · 50 mm)) column using a MeCN/H2O (0.05%
HCOOH) gradient with UV (214 and 254 nm) and
MS (ESI, 10 eV) detection. HPLC-purity of the tetra-
peptides was determined at 214 nm. Preparative RP-
HPLC-MS was performed on a Gilson-Finnigan Ther-
moQuest AQA system equipped with a C8 (Zorbax SB-
C8 (5 lm, 21.2 · 150 mm)) column using a MeCN/H2O
(0.05% HCOOH) or MeCN/H2O (50 mM NH4OAc)
gradient with UV (214 and 254 nm) and MS (ESI,
10 eV) detection. Preparative RP-HPLC was performed
on a system equipped with a C18 (Vydac C18
218TP1022) column using a MeCN/H2O (0.1% TFA)
or a MeCN/H2O (25 mM NH4OAc) gradient with UV
detection. Elemental analyses were performed by Ana-
lytische Laboratorien, Lindlar, Germany. Amino acid
analyses were performed at the Department of Bio-
chemistry, Uppsala University, Sweden, on 72 h hydro-
lyzates with an LKB 4151 alpha plus analyzer using
ninhydrin detection. Exact molecular masses were
determined on a Micromass Q-Tof2 mass spectrometer
equipped with an electrospray ion source. NMR spectra
were recorded on a JEOL JNM EX270 spectrometer
(1H at 270.2 MHz, 13C at 67.8 MHz), a Varian Mercury
plus spectrometer (1H at 300.0 MHz, 13C at 75.5 MHz),
a Varian Mercury plus spectrometer (1H at 399.8 MHz)
or on a JEOL JNM EX400 spectrometer (1H at
399.8 MHz, 13C at 100.5 MHz) at ambient temperature.
Chemical shifts (d) are reported in ppm referenced indi-
rectly to TMS via the solvent signals (1H: CHCl3 d 7.26,
CHD2OD d 3.31, DMSO-d5 d 2.50; 13C: CDCl3 d 77.16,
CD3OD d 49.00, DMSO-d6 d 39.50).
6.1.4. Suc-Chg-Glu-2-Nal-Nva-NHSO2 Ph (3). 1H NMR
(CD3OD, 400 MHz) d (60:40 mixture of diastereomers
at Nva) 7.97–7.93 (m, 2H), 7.80–7.72 (m, 3H), 7.68–7.67
(m, 1H), 7.58–7.47 (m, 3H), 7.43–7.34 (m, 3H), 4.71–
4.65 (m, 1H), 4.28–4.22 (m, 2H), 4.11 (d, J = 6.3 Hz,
0.6H), 4.04 (d, J = 6.5 Hz, 0.4H), 3.37–3.35 (m, 1H),
3.12–3.07 (m, 1H), 2.66–2.53 (m, 4H), 2.28–2.16 (m,
2H), 2.00–1.92 (m, 1H), 1.90–1.81 (m, 1H), 1.75–1.58
(m, 6H), 1.55–1.50 (m, 1H), 1.27–1.00 (m, 7H), 0.97–
0.88 (m, 1H), 0.84 (t, J = 7.4 Hz, 1.8H), 0.59
(t, J = 7.3 Hz, 1.2H). HPLC purity (C18 column: 96%,
C4 column: 100%). HRMS (M+H+) calcd for
C41H52N5O11S: 822.3384; found: 822.3347. Amino acid
analysis Chg 1.00, Glu 1.02, Nva 0.98.
6.1.5. Suc-Chg-Ile-Cha-Nva-OH (4). 1H NMR (CD3OD,
400 MHz) d 4.46 (dd, J = 5.8, 9.8 Hz, 1H), 4.29–4.25 (m,
1H), 4.22–4.17 (m, 2H), 2.58–2.50 (m, 4H), 1.88–1.52 (m,
16H), 1.42–1.10 (m, 15H), 0.92 (d, J = 6.8 Hz, 3H), 0.92 (t,
J = 7.2 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H). HPLC purity
(C18 column: 100%, C4 column: 97%). HRMS (M+H+)
calcd for C32H55N4O8: 623.4020; found: 623.4012. Amino
acid analysis Chg 0.98, Ile 1.00, Cha 1.01, Nva 1.00.
6.1.6. Suc-Chg-Ile-Cha-Nva-NHSO2iPr (5). 1H NMR
(CD3OD, 400 MHz) d 4.42 (dd, J = 5.5, 9.8 Hz, 1H),
4.24 (dd, J = 5.3, 8.8 Hz, 1H), 4.14 (d, J = 8.0 Hz, 1H),
4.11 (d, J = 7.0 Hz, 1H), 3.72–3.63 (m, 1H), 2.61–2.48
(m, 4H), 1.84–1.91 (m, 1H), 1.80–1.58 (m, 17H), 1.37
(d, J = 6.8 Hz, 3H), 1.36 (d, J = 6.8 Hz, 3H), 1.32–1.10
(m, 13H), 0.94 (t, J = 7.4 Hz, 3H), 0.91 (d, J = 7.0 Hz,
3H), 0.90 (dd, J = 7.2, 7.4 Hz, 3H). HPLC purity (C18
column: 95%, C4 column: 99%). HRMS (M+H+) calcd
for C35H62N5O9S: 728.4268; found: 728.4260. Amino
acid analysis Chg 1.01, Ile 1.00, Cha 0.99, Nva 1.00.
6.1.1. General procedure for the preparation of com-
pounds 1–6. Prepared according to the procedures de-
scribed by Johansson et al.11 Purification by
preparative RP-HPLC-MS or preparative RP-HPLC
gave the desired tetrapeptides 1–6.
1
6.1.7. Suc-Chg-Ile-Cha-Nva-NHSO2 Ph (6). H NMR
6.1.2. Suc-Chg-Glu-2-Nal-Nva-OH (1). 1H NMR
(CD3OD, 400 MHz) d 7.84–7.72 (m, 4H), 7.44–7.37
(m, 3H), 4.74 (dd, J = 4.6, 9.0 Hz, 1H), 4.30–4.25 (m,
2H), 4.07 (d, J = 6.6 Hz, 1H), 3.44–3.39 (m, 1H), 3.18–
3.13 (m, 1H), 2.61–2.50 (m, 4H), 2.25–2.15 (m, 2H),
2.01–1.92 (m, 1H), 1.90–1.75 (m, 2H), 1.75–1.58 (m,
5H), 1.55–1.51 (m, 1H), 1.42–1.29 (m, 3H), 1.22–1.00
(m, 5H), 0.90 (t, J = 7.3 Hz, 3H). HPLC purity (C18 col-
umn: 97%, C4 column: 98%). HRMS (M+H+) calcd for
C35H47N4O10: 683.3292; found: 683.3301. Amino acid
analysis Chg 0.99, Glu 1.00, Nva 1.01.
(CD3OD, 400 MHz) d 7.97–7.93 (m, 2H), 7.59–7.55
(m, 1H), 7.52–7.48 (m, 2H), 4.41–4.33 (m, 1H), 4.23–
4.18 (m, 1H), 4.16–4.13 (m, 2H), 2.63–2.52 (m, 4H),
1.92–1.51 (m, 17H), 1.36–1.08 (m, 14H), 0.88
(t, J = 7.3 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H), 0.85 (t,
J = 7.2 Hz, 3H). HPLC purity (C18 column: 96%, C4
column: 98%). HRMS (M+H+) calcd for C38H60N5O9S:
762.4112; found: 762.4120. Amino acid analysis Chg
0.99, Ile 0.96, Cha 1.03, Nva 1.01.
6.1.8. tert-Butyl [(1S)-1-benzylcarbamoyl]butylcarbamate
(7). To a solution of Boc-L-Nva (0.420 g, 1.93 mmol) in
CH2Cl2 (50 mL) were added benzylamine (0.32 mL,
2.9 mmol), HBTU (0.880 g, 2.32 mmol), and DIEA
(1.35 mL, 7.73 mmol). The suspension was stirred at
room temperature for 2 h and thereafter washed with
6.1.3. Suc-Chg-Glu-2-Nal-Nva-NHSO2iPr (2). 1H NMR
(CD3OD, 400 MHz) d 7.85–7.82 (m, 1H), 7.79–7.76 (m,
2H), 7.73–7.72 (m, 1H), 7.45–7.33 (m, 3H), 4.73 (dd,
J = 4.6, 9.1 Hz, 1H), 4.25 (dd, J = 4.7, 8.5 Hz, 1H),

R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
551
5% aqueous citric acid (2 · 25 mL), 5% aqueous NaH-
CO3 (2 · 25 mL), 5% aqueous citric acid (25 mL), and
brine (30 mL). The organic layer was dried (MgSO4), fil-
tered, and evaporated. Purification by column chroma-
tography (EtOAc/i-hexane 1:1) gave 7 as a white solid
(0.531 g, 90%). 1H NMR (CDCl3, 400 MHz) d 7.32–
7.23 (m, 5H), 6.67 (m, 1H), 5.10 (br d, J = 7.4 Hz,
1H), 4.44 (dd, J = 5.8, 15.1 Hz, 1H), 4.39 (dd, J = 5.8,
15.1 Hz, 1H), 4.14–4.08 (m, 1H), 1.86–1.80 (m, 1H),
1.63–1.53 (m, 1H), 1.43–1.30 (m, 2H), 1.40 (s, 9H),
0.92 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3, 100 MHz)
d 172.4, 155.9, 138.1, 128.8, 127.7, 127.6, 80.2, 54.7,
43.5, 34.6, 28.4, 19.1, 13.9. MS (M+H+) 307.1. Anal.
Calcd for C17H26N2O3: C, 66.64; H, 8.55; N, 9.14;
found: C, 66.59; H, 8.59; N, 9.14.
mixture was stirred at room temperature for 7 h before
the addition of another portion of 5 M HCl in EtOAc
(0.50 mL, 2.0 mmol). The mixture was left for 3 days be-
fore evaporation, which gave 11 as a white solid
(0.029 g, 100%). 1H NMR (DMSO-d6, 400 MHz) d
8.44 (m, 3H), 7.96–7.94 (m, 2H), 7.79–7.56 (m, 1H),
7.70–7.66 (m, 2H), 5.12 (s, 2H), 4.16–4.13 (m, 1H),
1.91–1.82 (m, 1H), 1.73–1.64 (m, 1H), 1.36–1.23 (m,
1H), 1.20–1.07 (m, 1H), 0.83 (t, J = 7.2 Hz, 3H). 13C
NMR (DMSO-d6, 100 MHz) d 195.9, 139.4, 134.2,
129.3, 128.1, 61.7, 58.6, 29.8, 17.4, 13.5. MS (M+H+)
256.0. Anal. Calcd for C12H18ClNO3S: C, 49.39; H,
6.22; N, 4.80; found: C, 49.48; H, 6.20; N, 4.83.
6.1.12. General procedure for the preparation of com-
pounds 12a–e. Method A: A solution of Boc-L-Nva in
dry THF was added to a solution of CDI in dry THF
under N2 atmosphere. The mixture was stirred at room
temperature for 1 h before the addition of a solution of
sulfonamide in dry THF followed by a solution of DBU
in dry THF. The resulting mixture was then stirred at
room temperature for 6 h.
6.1.9. (2S)-2-Amino-pentanoic acid benzylamide hydro-
chloride (8). A 5 M solution of HCl in EtOAc (5.4 mL,
27 mmol) was added to a solution of 7 (0.417 g,
1.36 mmol) in EtOAc (3 mL) and the resulting mixture
was stirred at room temperature overnight. Coevapora-
tion with MeOH gave 8 as a white solid (0.33 g, 100%).
1H NMR (CD3OD, 400 MHz) d 7.36–7.24 (m, 5H), 4.45
(d, J = 14.7 Hz, 1H), 4.39 (d, J = 14.7 Hz, 1H), 3.88 (dd,
J = 6.6, 6.7 Hz, 1H), 1.90–1.75 (m, 2H), 1.46–1.36 (m,
2H), 0.97 (dd, J = 7.2, 7.4 Hz, 3H). 13C NMR (CD3OD,
100 MHz) d 170.1, 139.4, 129.6, 128.8, 128.5, 54.4, 44.3,
34.8, 19.2, 13.9. MS (M+H+) 207.1. Anal. Calcd for
Method B: A solution of Boc-L-Nva in dry THF was
added to a solution of CDI in dry THF under N2 atmo-
sphere. The mixture was stirred at room temperature for
1 h before the addition of a solution of sulfonamide in
dry THF followed by a solution of DBU in dry THF.
The mixture was stirred at room temperature for 1 h
and refluxed for 30 min. The resulting mixture was then
stirred at room temperature overnight.
1
C12H19ClN2O ꢁ H2O: C, 57.94; H, 7.97; N, 11.26;
3
found: C, 57.69; H, 7.88; N, 11.06.
6.1.10. tert-Butyl [1-(2-Benzenesulfonyl-acetyl)]butylcar-
bamate (10). A 1.4 M solution of n-BuLi in hexane
(2.0 mL, 2.8 mmol) was added dropwise over 30 min
to a solution of MeSO2Ph (0.203 mg, 1.30 mmol) in
dry THF (3 mL) at 0 ꢁC under N2 atmosphere. The mix-
ture was stirred at 0 ꢁC for 40 min and thereafter cooled
to ꢂ78 ꢁC. A solution of 9 (0.150 g, 0.649 mmol) in dry
THF (3 mL) was added dropwise over 20 min and the
resulting mixture was slowly allowed to reach ꢂ30 ꢁC
over 3 h and finally allowed to adopt room temperature
over 1 h. The mixture was quenched with saturated
aqueous NH4Cl (6 mL) and extracted with EtOAc
(3 · 7 mL). The organic layer was washed with brine
(20 mL), dried (MgSO4), filtered, and evaporated. Puri-
fication by column chromatography (EtOAc/i-hexane
1:3 followed by EtOAc/i-hexane 1:2) gave 10 as white
Methods A and B: The solvent was reduced to half by
evaporation, diluted with CH2Cl2 (ꢀ30 mL/mmol Boc-
L-Nva), and washed with 5% aqueous citric acid
(ꢀ20 mL/mmol Boc-L-Nva), H2O (ꢀ20 mL/mmol Boc-
L-Nva), and brine (ꢀ20 mL/mmol Boc-L-Nva). The
organic layer was dried (MgSO4), filtered, and evaporat-
ed. Purification by column chromatography or prepara-
tive RP-HPLC-MS gave the desired product.
6.1.13. tert-Butyl [(1S)-1-methanesulfonylaminocarbon-
yl]butylcarbamate (12a). Compound 12a was prepared
according to method A using Boc-L-Nva (0.800 g,
3.68 mmol), CDI (1.20 g, 7.37 mmol), methanesulfona-
mide
(0.527 g,
5.54 mmol),
DBU
(0.826 mL,
5.52 mmol), and dry THF (65 mL). Purification by col-
umn chromatography (EtOAc/i-hexane 2:1) gave 12a
as a white foam (0.866 g, 80%). 1H NMR (CDCl3,
400 MHz) d 9.70 (br s, 1H), 5.15 (br d, J = 6.9 Hz,
1H), 4.20–4.14 (m, 1H), 3.29 (s, 3H), 1.85–1.76
(m, 1H), 1.66–1.57 (m, 1H), 1.45 (s, 9H), 1.43–1.33 (m,
2H), 0.94 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3,
100 MHz) d 172.0, 156.4, 81.6, 55.0, 41.5, 33.3, 28.4,
18.9, 13.7. MS (M+H+) 295.1. Anal. Calcd for
C11H22N2O5S: C, 44.88; H, 7.53; N, 9.52; found: C,
45.03; H, 7.49; N, 9.47.
1
crystals (0.196 g, 85%). H NMR (CDCl3, 400 MHz) d
7.92–7.89 (m, 2H), 7.70–7.66 (m, 1H), 7.59–7.55 (m,
2H), 5.14 (br d, J = 8.4 Hz, 1H), 4.47 (d, J = 14.1 Hz,
1H), 4.26 (dt, J = 4.5, 8.4 Hz, 1H), 4.20 (d,
J = 14.1 Hz, 1H), 1.87–1.79 (m, 1H), 1.58–1.47 (m,
1H), 1.43 (s, 9H), 1.39–1.29 (m, 2H), 0.91 (t,
J = 7.3 Hz, 3H). 13C NMR (CDCl3, 100 MHz) d 198.4,
155.7, 139.0, 134.4, 129.4, 128.6, 80.6, 63.4, 60.4, 32.4,
28.4, 18.9, 13.8. MS (M+H+) 356.1. Anal. Calcd for
C17H25NO5S: C, 57.44; H, 7.09; N, 3.94; found: C,
57.34; H, 7.03; N, 4.09.
6.1.14. tert-Butyl {(1S)-1-[propane-2-sulfonylaminocar-
bonyl]}butylcarbamate (12b). Compound 12b was pre-
pared according to method B using Boc-L-Nva (0.869 g,
4.00 mmol), CDI (1.30 g, 8.00 mmol), isopropylsulfona-
mide (0.739 g, 6.00 mmol), prepared from isopropylsulfo-
6.1.11. 3-Amino-1-benzenesulfonyl-hexan-2-one hydro-
chloride (11). A 5 M solution of HCl in EtOAc
(0.50 mL, 2.0 mmol) was added to a solution of 10
(0.035 g, 0.099 mmol) in EtOAc (1.5 mL). The resulting

552
R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
nyl chloride,14 DBU (0.898 mL, 6.00 mmol) and dry THF
(73 mL). Purification by column chromatography
(EtOAc/i-hexane 2:1) gave 12b as a white foam (1.06 g,
overnight. The reaction was diluted with EtOAc (9 mL)
and washed with 5% aqueous NaHCO3 (4 · 5 mL), H2O
(5 mL), and brine (7 mL). The organic layer was dried
(MgSO4), filtered, and evaporated to give 12f as a yellow
1
82%). H NMR (CDCl3, 400 MHz) d 9.29 (br s, 1H),
1
5.11 (br d, J = 7.2 Hz, 1H), 4.17–4.12 (m, 1H), 3.79 (h,
J = 6.9 Hz, 1H), 1.87–1.74 (m, 1H), 1.68–1.54 (m, 1H),
1.44 (s, 9H), 1.41 (d, J = 6.9 Hz, 3H), 1.40 (d,
J = 6.9 Hz, 3H), 1.37–1.21 (m, 2H), 0.93 (t, J = 7.3 Hz,
3H). 13C NMR (CDCl3, 100 MHz) d 171.9, 156.3, 81.3,
55.2, 54.0, 33.3, 28.3, 18.9, 15.94, 15.90, 13.8. MS
(M+H+) 323.0. Anal. Calcd for C13H26N2O5S: C, 48.43;
H, 8.13; N, 8.69; found: C, 48.51; H, 8.16; N, 8.74.
oil (0.025 g, 81%). H NMR (CDCl3, 400 MHz) d 8.04–
8.02 (m, 2H), 7.65–7.61 (m, 1H), 7.56–7.52 (m, 2H), 5.17
(dt, J = 4, 9 Hz, 1H), 5.03 (d, J = 9 Hz, 1H), 3.26 (s,
3H), 1.82–1.74 (m, 1H), 1.55–1.34 (m, 3H), 1.39 (s, 9H),
0.92 (dd, J = 7.1, 7.2 Hz, 3H). 13C NMR (CDCl3,
100 MHz) d 174.7, 155.6, 138.5, 134.0, 129.3, 128.0,
80.0, 53.6, 36.0, 33.2, 28.4, 18.9, 13.8. MS (M+H+)
1
371.1. Anal. Calcd for C17H26N2O5S ꢁ H2O: C, 54.24;
H, 7.14; N, 7.44; found: C, 54.6; H, 7.0;2N, 7.0.
6.1.15. tert-Butyl[(1S)-1-benzenesulfonylaminocarbon-
yl]butylcarbamate (12c). Compound 12c was prepared
according to method A using Boc-L-Nva (0.602 g,
2.77 mmol), CDI (0.898 g, 5.54 mmol), benzenesulfona-
mide (0.654 g, 4.16 mmol), DBU (0.620 mL, 4.14 mmol),
and dry THF (50 mL). Purification by preparative RP-
HPLC-MS (MeCN/H2O (0.05% HCOOH)) gave 12c as
a white solid (0.554 g, 58%).11
6.1.19. N-[(2S)-2-Amino-pentanoyl]methanesulfonamide
hydrochloride (13a). A 5 M solution of HCl in EtOAc
(4.5 mL, 23 mmol) was added to a solution of 12a
(0.392 g, 1.33 mmol) in EtOAc (5.5 mL). The mixture
was stirred overnight and coevaporated with MeOH to
give 13a as a white solid (0.292 g, 95%). 1H NMR
(CD3OD, 400 MHz) d 3.95 (dd, J = 5.4, 7.2 Hz, 1H),
3.31 (s, 3H), 1.95–1.80 (m, 2H), 1.53–1.40 (m, 2H), 1.02
(t, J = 7.3 Hz, 3H). 13C NMR (CD3OD, 100 MHz) d
170.4, 54.8, 41.6, 34.0, 19.0, 13.9. MS (M + H+) 195.1.
Anal. Calcd for C6H15ClN2O3S: C, 31.24; H, 6.55; N,
12.14; found: C, 31.34; H, 6.54; N, 12.01.
6.1.16. tert-Butyl[(1S)-1-phenylmethanesulfonylaminocar-
bonyl]butylcarbamate (12d). Compound 12d was pre-
pared according to method B using Boc-L-Nva (0.869 g,
4.00 mmol), CDI (1.30 g, 8.00 mmol), a-toluenesulfon-
amide (1.03 g, 6.00 mmol), DBU (0.898 mL, 6.00 mmol),
and dry THF (73 mL). Purification by column chroma-
tography (EtOAc/i-hexane 1:1) gave 12d as a white foam
(0.858 g, 58%). 1H NMR (CDCl3, 400 MHz) d 9.18 (br s,
1H), 7.40–7.32 (m, 5H), 4.93 (br d, J = 7.1 Hz, 1H), 4.68–
4.60 (m, 2H), 4.11–4.06 (m, 1H), 1.82–1.72 (m, 1H), 1.59–
1.48 (m, 1H), 1.47–1.29 (m, 2H), 1.39 (s, 9H), 0.92 (t,
J = 7.3 Hz, 3H). 13C NMR (CDCl3, 67.9 MHz) d 172.2,
156.1, 130.9, 129.3, 129.0, 127.9, 81.3, 59.0, 54.8, 33.4,
28.3, 18.9, 13.7. MS (M+H+) 371.1. Anal. Calcd for
C17H26N2O5S: C, 55.12; H, 7.07; N, 7.56; found: C,
55.25; H, 7.06; N, 7.74.
6.1.20. N-[(2S)-2-Amino-pentanoyl]propane-2-sulfonamide
hydrochloride (13b). A 4 M solution of HCl in 1,4-dioxane
(5.5 mL, 22 mmol) was added to 12b (0.699 g, 2.17 mmol).
The mixture was stirred for 3 h and coevaporated with
1
MeOH to give 13b as a beige solid (0.559 g, 100%). H
NMR (CD3OD, 270 MHz) d 3.97 (dd, J = 5.6, 7.0 Hz,
1H), 3.72 (h, J = 6.9 Hz, 1H), 1.98–1.77 (m, 2H), 1.54–
1.36 (m, 2H), 1.40 (d, J = 6.9 Hz, 6H), 1.02 (dd, J = 7.2,
7.4 Hz, 3H). 13C NMR (CD3OD, 67.9 MHz) d 170.5,
55.1, 54.9, 34.1, 19.0, 16.5, 15.8, 13.9. MS (M+H+) 223.0.
1
Anal. Calcd for C8H19ClN2O3S ꢁ H2O: C, 35.88; H,
2
7.53; N, 10.46; found: C, 35.95; H, 7.39; N, 10.35.
6.1.17. tert-Butyl {(1S)-1-[2-phenyl-ethanesulfonylamino-
carbonyl]}butylcarbamate (12e). Compound 12e was pre-
pared according to method A using Boc-L-Nva (0.047 g,
0.22 mmol), CDI (0.070 g, 0.43 mmol), phenethylsulf-
onamide38 (0.060 g, 0.32 mmol) prepared from
phenethylsulfonyl chloride,14,39 DBU (0.049 mL,
0.33 mmol), and dry THF (4 mL). Purification by pre-
parative RP-HPLC-MS (MeCN/H2O (0.05% HCOOH))
gave 12e asa white solid(0.030 g, 36%). 1H NMR (CDCl3,
400 MHz) d 9.33 (br s, 1H), 7.32–7.20 (m, 5H), 4.97 (br d,
J = 6.9 Hz, 1H), 4.08–4.03 (m, 1H), 3.72–3.68 (m, 2H),
3.14–3.10 (m, 2H), 1.84–1.73 (m, 1H), 1.62–1.51 (m,
1H), 1.45 (s, 9H), 1.44–1.32 (m, 2H), 0.94 (t, J = 7.3 Hz,
3H). 13C NMR (CDCl3, 100 MHz) d 171.8, 156.4, 137.2,
129.0, 128.6, 127.2, 81.5, 55.0, 54.4, 33.1, 29.5, 28.3,
18.9, 13.8. MS (M+H+) 385.1. Anal. Calcd for
C18H28N2O5S: C, 56.23; H, 7.34; N, 7.29; found: C,
56.28; H, 7.50; N, 7.14.
6.1.21.
N-[(2S)-2-Amino-pentanoyl]benzenesulfonamide
hydrochloride (13c). A 5 M solution of HCl in EtOAc
(5.0 mL, 25 mmol) was added to a solution of 12c
(0.467 g, 1.31 mmol) in EtOAc (5 mL). The mixture was
stirred overnight and coevaporated with MeOH to give
13c as a white solid (0.371 g, 97%).11
6.1.22. N-[(2S)-2-Amino-pentanoyl]-C-phenyl-methanesulf-
onamide hydrochloride (13d). A 4 M solution of HCl in 1,4-
dioxane (8.2 mL, 33 mmol) was added to a solution of 12d
(0.622 g, 1.68 mmol) in 1,4-dioxane (5 mL) and MeOH
(3 mL). The resulting mixture was stirred overnight before
addition of another portion of 4 M HCl in 1,4-dioxane
(1.0 mL, 4.0 mmol). After an additional 4 h, the mixture
was coevaporated with MeOH to give 13d as a white solid
(0.504 g, 98%). 1H NMR (CD3OD, 270 MHz) d 7.45–7.37
(m, 5H), 4.74 (s, 2H), 3.86 (dd, J = 6.3, 6.4 Hz, 1H), 1.78–
1.70 (m, 2H), 1.47–1.31 (m, 2H), 0.96 (dd, J = 7.2, 7.3 Hz,
3H). 13C NMR (DMSO-d6/CD3OD 9:1, 67.9 MHz) d
169.7, 131.1, 129.0, 128.85, 128.83, 58.3, 52.9, 32.7, 17.7,
13.5. MS (M+H+) 271.0. Anal. Calcd for C12H19ClN2O3S:
C, 46.98; H, 6.24; N, 9.13; found: C, 47.01; H, 6.42; N, 9.16.
6.1.18. tert-Butyl {(1S)-1-[benzenesulfonyl-methyl-amino-
carbonyl]}butylcarbamate (12f). To a suspension of 12c
(0.030 g, 0.084 mmol) and Cs2CO3 (0.041 g, 0.13 mmol)
in dry DMF (3 mL) was added MeI (0.011 mL,
0.18 mmol). The resulting mixture was stirred at 65 ꢁC

R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
553
6.1.23. N-[(2S)-2-Amino-pentanoyl]-2-phenyl-ethanesulf-
onamide hydrochloride (13e). A 5 M solution of HCl in
EtOAc (1.0 mL, 5.0 mmol) was added to a solution of
12e (0.032 g, 0.083 mmol) in EtOAc (0.5 mL). The mix-
ture was stirred overnight and coevaporated with
6.1.28. Compound 16. To a solution of 15 (0.135 g,
0.240 mmol) in dry DMF (5 mL) were added HClÆ
L-NvaOMe (0.080 g, 0.479 mmol), HBTU (0.110 g,
0.290 mmol), and DIEA (167 ll, 0.958 mmol). The
resulting solution was stirred at room temperature for
2.5 h, diluted with EtOAc (25 mL) and washed with
5% aqueous NaHCO3 (10 mL), 35 mM aqueous NaH-
SO4 (10 mL), H2O (10 mL), and brine (10 mL). The
organic layer was dried (MgSO4), filtered, and evaporat-
ed. Purification by column chromatography (EtOAc/i-
hexane 1:1 followed by EtOAc/i-hexane 2:1) gave 16 as
a white solid (0.154 g, 95%). 1H NMR (CDCl3,
400 MHz) d 8.04–8.02 (m, 2H), 7.97 (d, J = 9.2 Hz,
1H), 7.50–7.40 (m, 4H), 7.28 (d, J = 7.9 Hz, 1H), 7.04
(dd, J = 2.6, 9.2 Hz, 1H), 6.99 (s, 1H), 5.38–5.34 (m,
2H), 4.81 (dd, J = 7.0, 7.7 Hz, 1H), 4.53 (dt, J = 5.3,
7.9 Hz, 1H), 4.37 (dm, J = 11.4 Hz, 1H), 4.25 (dd,
J = 7.2, 9.2 Hz, 1H), 4.00–3.96 (m, 1H), 3.91 (s, 3H),
3.68 (s, 3H), 2.79 (ddd, J = 5.3, 7.0, 13.9 Hz, 1H),
2.49–2.42 (m, 1H), 2.02–1.93 (m, 1H), 1.78–1.69 (m,
1H), 1.67–1.58 (m, 1H), 1.40–1.27 (m, 2H), 1.34 (s,
9H), 0.96 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H),
0.84 (dd, J = 7.2, 7.4 Hz, 3H). 13C NMR (CDCl3,
100 MHz) d 172.7, 172.6, 170.2, 161.7, 160.8, 158.7,
155.8, 150.3, 139.0, 129.7, 128.8, 127.8, 123.1, 118.7,
114.9, 106.5, 98.2, 79.7, 76.5, 58.6, 57.3, 55.6, 52.9,
52.29, 52.28, 34.3, 33.2, 31.4, 28.2, 19.2, 18.5, 17.8,
13.6. MS (M+H+) 677.3. Anal. Calcd for C37H48N4O8:
C, 65.66; H, 7.15; N, 8.28; found: C, 65.39; H, 7.13;
N, 8.12.
1
MeOH to give 13e as a white solid (0.025 g, 93%). H
NMR (CD3OD, 400 MHz) d 7.34–7.22 (m, 5H), 3.91
(dd, J = 5.3, 7.4 Hz, 1H), 3.80–3.68 (m, 2H), 3.18–3.06
(m, 2H), 1.89–1.74 (m, 2H), 1.50–1.41 (m, 2H), 1.01
(dd, J = 7.2, 7.4 Hz, 3H). 13C NMR (CD3OD,
100 MHz) d 170.4, 138.9, 129.9, 129.5, 128.0, 55.3,
54.8, 34.0, 30.4, 19.0, 13.9. MS (M+H+) 285.1. Anal.
Calcd for C13H21ClN2O3S: C, 48.67; H, 6.60; N, 8.73;
found: C, 48.48; H, 6.73; N, 8.63.
6.1.24. N-[(2S)-2-Amino-pentanoyl]-N-methyl-benzenesulf-
onamide hydrochloride (13f). A 5 M solution of HCl in
EtOAc (1.5 mL, 7.5 mmol) was added to a solution of
12f (0.021 g, 0.057 mmol) in EtOAc (0.5 mL). The mixture
was stirred for 7 h beforethe addition ofanotherportion of
5 M HCl in EtOAc (1.0 mL, 5.0 mmol) and finally left
overnight. Evaporation gave 13f as a white solid (0.016 g,
1
94%). H NMR (CDCl3, 300 MHz) d 8.72 (br s, 3H),
8.03–8.01 (m, 2H), 7.62–7.52 (m, 3H), 5.22–5.16 (m, 1H),
3.09 (s, 3H), 2.10–1.92 (m, 2H), 1.60–1.48 (m, 2H), 0.92 (t,
J = 7.0 Hz, 3H). 13C NMR (CDCl3, 75.5 MHz) d 170.8,
136.9, 134.3, 129.6, 128.1, 54.5, 33.9, 33.7, 18.3, 13.6. MS
(M+H+) 271.1. Anal. Calcd for C12H19ClN2O3S Æ H2O: C,
44.37; H, 6.52; N, 8.62; found: C, 44.4; H, 6.4; N, 8.1.
6.1.25. General procedure for the preparation of com-
pounds 14b and 14d. A solution of the deprotected acyl
sulfonamide building block in EtOH or EtOH/MeOH
was treated with propylene oxide and heated to 50 ꢁC
under N2 atmosphere in a closed reaction tube. Evapo-
ration after 5 h gave the desired product without further
purification.
6.1.29. Compound 17. A solution of LiOH (0.026 g,
1.1 mmol) in H2O (1.5 mL) was added to a solution of
16 (0.075 g, 0.11 mmol) in THF (4 mL) and MeOH
(0.5 mL). The resulting mixture was stirred at room tem-
perature for 6.5 h and neutralized with 1.0 M aqueous
HCl. The organic solvents were evaporated and the
remaining aqueous phase was acidified to pH 3 using
1.0 M HCl (aq) and extracted with EtOAc (3 · 15 mL).
The organic layer was washed with brine (20 mL), dried
(MgSO4), and filtered. Evaporation gave 17 as a white sol-
id (0.067 g, 92%). 1H NMR (CD3OD, 400 MHz) d 8.10 (d,
J = 9.2 Hz, 1H), 8.04–8.02 (m, 2H), 7.57–7.49 (m, 3H),
7.36 (d, J = 2.5 Hz, 1H), 7.21 (s, 1H), 7.09 (dd, J = 2.5,
9.2 Hz, 1H), 5.49–5.47 (m, 1H), 4.75 (dd, J = 7.6,
9.3 Hz, 1H), 4.59 (dm, J = 11.5 Hz, 1H), 4.39 (dd,
J = 4.9, 8.8 Hz, 1H), 4.12–4.02 (m, 2H), 3.92 (s, 3H),
2.72 (ddm, J = 7.6, 13.9 Hz, 1H), 2.42 (ddd, J = 4.4, 9.3,
13.9 Hz, 1H), 2.04–1.94 (m, 1H), 1.87–1.78 (m, 1H),
1.74–1.64 (m, 1H), 1.53–1.39 (m, 2H), 1.24 (s, 9H), 1.00
(d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H), 0.93 (dd,
J = 7.2, 7.4 Hz, 3H). 13C NMR (CD3OD, 100 MHz) d
176.0, 174.0, 173.4, 163.5, 162.8, 160.7, 157.9, 150.9,
140.1, 131.0, 130.0, 129.2, 124.7, 119.6, 116.4, 106.3,
100.3, 80.3, 78.5, 60.2, 59.4, 56.1, 54.5, 53.9, 36.0, 34.9,
31.8, 28.6, 20.0, 19.7, 19.0, 14.0. MS (M+H+) 663.4. Anal.
Calcd for C36H46N4O8: C, 65.24; H, 7.00; N, 8.45; found:
C, 65.01; H, 7.08; N, 8.30.
6.1.26. N-[(2S)-2-Amino-pentanoyl]propane-2-sulfonamide
(14b). Compound 13b (0.510 g, 1.97 mmol) was used
according to the general procedure with EtOH (17 mL)
and propylene oxide (1.54 mL, 22.0 mmol) to give 14b
(0.435 g, 99%) as a white solid. 1H NMR (CD3OD,
270 MHz) d 3.60 (dd, J = 5.4, 7.1 Hz, 1H), 3.55 (h,
J = 6.9 Hz, 1H), 1.96–1.71 (m, 2H), 1.55–1.39 (m, 2H),
1.30 (d, J = 6.9 Hz, 6H), 0.98 (dd, J = 7.2, 7.4 Hz, 3H).
13C NMR (CD3OD, 67.9 MHz) d 175.6, 56.8, 52.6,
34.9, 19.4, 16.9, 16.8, 14.1. MS (M+H+) 223.0. Anal.
Calcd for C8H18N2O3S: C, 43.22; H, 8.16; N, 12.60;
found: C, 43.30; H, 8.17; N, 12.46.
6.1.27. N-[(2S)-2-Amino-pentanoyl]-C-phenyl-methanesulf-
onamide (14d). Compound 13d (0.450 g, 1.47 mmol) was
used according to the general procedure with EtOH
(10 mL), MeOH (4 mL), and propylene oxide (1.15 mL,
16.4 mmol) to give 14d (0.383 g, 99%) as a white solid. 1H
NMR (CD3OD, 270 MHz) d 7.42–7.28 (m, 5H), 4.47 (s,
2H), 3.54 (dd, J = 5.3, 7.2 Hz, 1H), 1.87–1.62 (m, 2H),
1.48–1.29 (m, 2H), 0.94 (dd, J = 7.2, 7.3 Hz, 3H). 13C
NMR (CD3OD, 67.9 MHz) d 176.0, 132.7, 131.9, 129.2,
128.9, 58.6, 56.8, 34.9, 19.4, 14.1. MS (M+H+) 271.0. Anal.
CalcdforC12H18N2O3S:C,53.31;H,6.71;N,10.36;found:
C, 53.15; H, 6.80; N, 10.21.
6.1.30. Compound 18. To a solution of 15 (0.100 g,
0.177 mmol) and 13a (0.061 g, 0.27 mmol) in dry
DMF (3.8 mL) were added HBTU (0.081 g, 0.21 mmol)
and DIEA (0.124 mL, 0.710 mmol). The mixture was

554
R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
stirred for 3.5 h at room temperature, diluted with
EtOAc (25 mL), and washed with aqueous NaOAc
buffer (pH 4, 3 · 9 mL) and brine (13 mL). The organic
layer was dried (MgSO4), filtered, and evaporated.
Purification by column chromatography (CHCl3/
MeOH 96:4 followed by CHCl3/MeOH 95:5) gave 18
as a white solid (0.089 g, 68%). 1H NMR (CD3OD,
400 MHz) d 8.10 (d, J = 9.2 Hz, 1H), 8.05–8.02 (m,
2H), 7.58–7.49 (m, 3H), 7.34 (d, J = 2.5 Hz, 1H), 7.21
(s, 1H), 7.09 (dd, J = 2.5, 9.2 Hz, 1H), 5.47–5.45 (m,
1H), 4.74 (dd, J = 7.6, 9.3 Hz, 1H), 4.58 (dm,
J = 12.0 Hz, 1H), 4.29 (dd, J = 4.9, 8.9 Hz, 1H), 4.09–
4.01 (m, 2H), 3.92 (s, 3H), 3.16 (s, 3H), 2.71 (ddm,
J = 7.6, 13.9 Hz, 1H), 2.39 (ddd, J = 4.2, 9.3, 13.9 Hz,
1H), 1.98 (dh, J = 6.7, 8.7 Hz, 1H), 1.82–1.73 (m,
1H), 1.71–1.62 (m, 1H), 1.53–1.37 (m, 2H), 1.24 (s,
9H), 0.99 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz,
3H), 0.91 (dd, J = 7.2, 7.4 Hz, 3H). 13C NMR
(CD3OD, 100 MHz) d 175.4, 174.0, 173.6, 163.5,
162.8, 160.7, 157.8, 150.9, 140.1, 131.0, 129.9, 129.1,
124.7, 119.6, 116.3, 106.3, 100.3, 80.3, 78.5, 60.1,
59.4, 56.1, 55.6, 54.5, 41.2, 36.0, 34.9, 31.8, 28.6,
4, 4 · 7 mL) and brine (7 mL). The organic layer was
dried (MgSO4), filtered, and evaporated. Purification
by column chromatography (CHCl3/MeOH 96:4 fol-
lowed by CHCl3/MeOH 95:5) gave 20 as a white solid
1
(0.085 g, 79%). H NMR (CD3OD, 400 MHz) d 8.07
(d, J = 9.2 Hz, 1H), 8.04–8.02 (m, 2H), 7.96–7.94 (m,
2H), 7.58–7.51 (m, 4H), 7.48–7.44 (m, 2H), 7.34 (d,
J = 2.5 Hz, 1H), 7.19 (s, 1H), 7.08 (dd, J = 2.5, 9.2 Hz,
1H), 5.41–5.40 (m, 1H), 4.69 (dd, J = 7.9, 9.2 Hz, 1H),
4.53 (dm, J = 11.6 Hz, 1H), 4.28 (dd, J = 4.9, 8.5 Hz,
1H), 4.04 (d, J = 8.6 Hz, 1H), 4.00 (dd, J = 3.5,
11.6 Hz, 1H), 3.91 (s, 3H), 2.60 (ddm, J = 7.9, 13.9 Hz,
1H), 2.28 (ddd, J = 4.3, 9.2, 13.9 Hz, 1H), 1.96 (dh,
J = 6.7, 8.6 Hz, 1H), 1.70–1.61 (m, 1H), 1.58–1.49 (m,
1H), 1.34–1.20 (m, 2H), 1.23 (s, 9H), 0.96 (d,
J = 6.7 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.80 (dd,
J = 7.2, 7.4 Hz, 3H). 13C NMR (CD3OD, 100 MHz) d
174.9, 173.9, 173.3, 163.6, 163.0, 160.5, 157.8, 150.4,
142.0, 139.7, 134.0, 131.2, 130.0, 129.7, 129.2, 128.7,
124.8, 119.7, 116.3, 106.0, 100.4, 80.3, 78.6, 60.1, 59.4,
56.2, 55.6, 54.4, 35.9, 35.0, 31.8, 28.6, 19.69, 19.68,
19.0, 14.0. MS (M+H+) 802.4. Anal. Calcd for
C42H51N5O9S Æ H2O: C, 61.52; H, 6.52; N, 8.54; found:
C, 61.72; H, 6.28; N, 8.41.
19.9, 19.7, 19.0, 14.1. MS (M+H+) 740.3. Anal. Calcd
1
for C37H49N5O9S ꢁ H2O: C, 59.34; H, 6.73; N, 9.35;
found: C, 59.28; H2, 6.57; N, 9.16.
6.1.33. Compound 21. To a solution of 15 (0.080 g,
0.14 mmol) and 14d (0.058 g, 0.21 mmol) in dry DMF
(3 mL) were added HBTU (0.065 g, 0.17 mmol) and
DIEA (0.099 mL, 0.57 mmol). The mixture was stirred
for 3 h at room temperature, diluted with EtOAc
(20 mL), and washed with 5% aqueous NaHCO3
(7 mL), 35 mM aqueous NaHSO4 (7 mL), H2O (7 mL),
and brine (7 mL). The organic layer was dried (MgSO4),
filtered, and evaporated. Purification by column chro-
matography (CHCl3/MeOH 95:5) gave 21 as a white sol-
6.1.31. Compound 19. To a solution of 15 (0.080 g,
0.14 mmol) and 14b (0.048 g, 0.21 mmol) in dry DMF
(3 mL) were added HBTU (0.065 g, 0.17 mmol) and
DIEA (0.099 mL, 0.57 mmol). The mixture was stirred
for 3.5 h at room temperature, diluted with EtOAc
(20 mL), and washed with aqueous NaOAc buffer (pH
4, 3 · 7 mL) and brine (10 mL). The organic layer was
dried (MgSO4), filtered, and evaporated. Purification
by column chromatography (repeated columns,
EtOAc/i-hexane 2:1 and CHCl3/MeOH 96:4 followed
by CHCl3/MeOH 95:5) gave 19 as a white solid
1
id (0.052 g, 45%). H NMR (CD3OD, 400 MHz) d 8.13
(d, J = 9.2 Hz, 1H), 8.06–8.03 (m, 2H), 7.58–7.50 (m,
3H), 7.40–7.31 (m, 5H), 7.32 (d, J = 2.5 Hz, 1H), 7.24
(s, 1H), 7.10 (dd, J = 2.5, 9.2 Hz, 1H), 5.50–5.48 (m,
1H), 4.75 (dd, J = 7.6, 9.3 Hz, 1H), 4.63 (d,
J = 14.0 Hz, 1H), 4.62–4.58 (m, 1H), 4.57 (d,
J = 14.0 Hz, 1H), 4.28 (dd, J = 5.0, 8.7 Hz, 1H), 4.12–
4.04 (m, 2H), 3.93 (s, 3H), 2.73 (ddm, J = 7.6, 14.0 Hz,
1H), 2.40 (ddd, J = 4.3, 9.3, 14.0 Hz, 1H), 1.99 (dh,
J = 6.7, 8.8 Hz, 1H), 1.69–1.56 (m, 2H), 1.52–1.33 (m,
2H), 1.25 (s, 9H), 1.01 (d, J = 6.7 Hz, 3H), 0.97 (d,
J = 6.7 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR
(CD3OD, 100 MHz) d 175.0, 174.0, 173.5, 163.6,
162.9, 160.7, 157.9, 150.7, 140.0, 132.1, 131.1, 130.5,
130.0, 129.71, 129.66, 129.2, 124.8, 119.7, 116.4, 106.2,
100.4, 80.3, 78.6, 60.1, 59.5, 59.2, 56.2, 55.4, 54.5, 36.1,
35.0, 31.8, 28.6, 20.0, 19.7, 19.1, 14.0. MS (M+H+)
816.3. Anal. Calcd for C43H53N5O9S Æ H2O: C, 61.93;
H, 6.65; N, 8.40; found: C, 62.12; H, 6.36; N, 8.16.
1
(0.020 g, 18%). H NMR (CD3OD, 400 MHz) d 8.13
(d, J = 9.2 Hz, 1H), 8.07–8.04 (m, 2H), 7.58–7.50 (m,
3H), 7.39 (d, J = 2.5 Hz, 1H), 7.26 (s, 1H), 7.11 (dd,
J = 2.5, 9.2 Hz, 1H), 5.53–5.52 (m, 1H), 4.73 (dd,
J = 7.7, 9.3 Hz, 1H), 4.61 (dm, J = 11.7 Hz, 1H), 4.28
(dd, J = 5.0, 8.9 Hz, 1H), 4.08–4.04 (m, 2H), 3.95 (s,
3H), 3.64 (h, J = 6.9 Hz, 1H), 2.75 (ddm, J = 7.7,
13.9 Hz, 1H), 2.39 (ddd, J = 4.3, 9.3, 13.9 Hz, 1H),
1.99 (dh, J = 6.7, 8.7 Hz, 1H), 1.82–1.63 (m, 2H),
1.56–1.40 (m, 2H), 1.36 (d, J = 6.9 Hz, 3H), 1.35 (d,
J = 6.9 Hz, 3H), 1.25 (s, 9H), 1.00 (d, J = 6.7 Hz, 3H),
0.96 (d, J = 6.7 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H). 13C
NMR (CD3OD, 100 MHz) d 174.5, 174.1, 173.7,
163.4, 162.5, 161.2, 157.9, 151.6, 140.8, 130.8, 129.9,
129.1, 124.6, 119.5, 116.5, 106.8, 100.3, 80.3, 78.3,
60.1, 59.5, 56.1, 55.6, 54.6, 54.5, 36.1, 34.7, 31.7, 28.6,
20.0, 19.7, 19.0, 16.6, 15.8, 14.0. MS (M+H+) 768.3.
1
Anal. Calcd for C39H53N5O9S ꢁ H2O: C, 60.29; H,
7.01; N, 9.01; found: C, 60.48; H,27.07; N, 8.73.
6.1.34. Compound 22. To a solution of 15 (0.020 g,
0.036 mmol) and 13e (0.017 g, 0.053 mmol) in dry
DMF (0.6 mL) were added HBTU (0.016 g,
0.042 mmol) and DIEA (0.025 mL, 0.14 mmol). The
mixture was stirred for 3 h at room temperature, diluted
with EtOAc (10 mL) and washed with aqueous NaOAc
buffer (pH 4, 4 · 4 mL) and brine (6 mL). The organic
layer was dried (MgSO4), filtered, and evaporated. Puri-
6.1.32. Compound 20. To a solution of 15 (0.075 g,
0.13 mmol) and 13 c (0.059 g, 0.20 mmol) in dry DMF
(3 mL) were added HBTU (0.061 g, 0.16 mmol) and
DIEA (0.093 mL, 0.53 mmol). The mixture was stirred
for 3 h at room temperature, diluted with EtOAc
(20 mL), and washed with aqueous NaOAc buffer (pH

R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
555
fication by column chromatography (CHCl3/MeOH
97:3 followed by CHCl3/MeOH 96:4) gave 22 as a white
solid (0.024 g, 83%). H NMR (CD3OD, 400 MHz) d
dried (MgSO4), filtered, and evaporated. Purification
by column chromatography (EtOAc/i-hexane 1:1) gave
24 (80:20 mixture of diastereomers) as a white solid
1
1
8.13 (d, J = 9.2 Hz, 1H), 8.05–8.02 (m, 2H), 7.58–7.50
(m, 3H), 7.38 (d, J = 2.5 Hz, 1H), 7.26–7.13 (m, 6H),
7.11 (dd, J = 2.5, 9.2 Hz, 1H), 5.49–5.46 (m, 1H), 4.74
(dd, J = 7.7, 9.1 Hz, 1H), 4.59 (dm, J = 11.8 Hz, 1H),
4.24 (dd, J = 5.1, 8.8 Hz, 1H), 4.08–4.02 (m, 2H), 3.94
(s, 3H), 3.59 (ddd, J = 5.3, 11.5, 14.1 Hz, 1H), 3.49
(ddd, J = 5.6, 11.2, 14.1 Hz, 1H), 3.12 (ddd, J = 5.3,
11.2, 13.7 Hz, 1H), 3.02 (ddd, J = 5.6, 11.5, 13.7 Hz,
1H), 2.73 (ddm, J = 7.7, 14.0 Hz, 1H), 2.40 (ddd,
J = 4.4, 9.1, 14.0 Hz, 1H), 1.97 (dh, J = 6.7, 8.8 Hz,
1H), 1.80–1.62 (m, 2H), 1.56–1.40 (m, 2H), 1.25 (s,
9H), 0.98 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H),
0.94 (dd, J = 7.1, 7.4 Hz, 3H). 13C NMR (CD3OD,
100 MHz) d 175.8, 174.1, 173.5, 163.5, 162.7, 161.1,
158.0, 151.3, 140.6, 139.5, 130.9, 129.9, 129.7, 129.5,
129.2, 127.8, 124.6, 119.6, 116.4, 106.6, 100.4, 80.3,
78.3, 60.1, 59.5, 56.1, 55.9, 54.9, 54.5, 36.0, 34.7, 31.7,
30.8, 28.6, 20.1, 19.7, 19.1, 14.1. MS (M+H+) 830.3.
Anal. Calcd for C44H55N5O9S: C, 63.67; H, 6.68; N,
8.44; found: C, 63.46; H, 6.50; N, 8.38.
(0.030 g, 86%). H NMR (CDCl3, 400 MHz) d (major
diastereomer reported) 8.06–8.03 (m, 2H), 8.01 (d,
J = 9.2 Hz, 1H), 7.94–7.86 (m, 2H), 7.65–7.60 (m, 1H),
7.56–7.43 (m, 6H), 7.40 (br d, J = 7.3 Hz, 1H), 7.08
(dd, J = 2.5, 9.2 Hz, 1H), 7.00 (s, 1H), 5.38–5.35 (m,
1H), 5.33 (d, J = 9.4 Hz, 1H), 4.79 (dd, J = 7.5, 7.7 Hz,
1H), 4.50–4.42 (m, 2H), 4.45 (d, J = 14.2 Hz, 1H),
4.27–4.23 (m, 1H), 4.25 (d, J = 14.2 Hz, 1H), 4.00–3.97
(m, 1H), 3.95 (s, 3H), 2.75–2.68 (m, 1H), 2.61–2.55 (m,
1H), 2.02–1.92 (m, 1H), 1.88–1.79 (m, 1H), 1.64–1.48
(m, 1H), 1.37 (s, 9H), 1.35–1.25 (m, 2H), 0.94 (d,
J = 6.7 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.86 (dd,
J = 7.2, 7.4 Hz, 3H). 13C NMR (CDCl3, 100 MHz) d
(major diastereomer reported) 196.8, 172.9, 171.0,
161.6, 160.3, 159.3, 155.9, 151.3, 140.1, 139.1, 134.3,
129.5, 129.4, 128.9, 128.5, 127.7, 123.1, 118.6, 115.1,
107.3, 98.1, 80.0, 76.2, 63.8, 59.6, 58.9, 57.5, 55.7, 53.1,
33.4, 31.5, 31.4, 28.4, 19.4, 18.8, 17.9, 13.8. MS
(M+H+) 801.2. Anal. Calcd for C43H52N4O9S: C,
64.48; H, 6.54; N, 6.99; found: C, 64.73; H, 6.71; N,
7.07.
6.1.35. Compound 23. To a solution of 15 (0.050 g,
0.089 mmol) and 8 (0.033 g, 0.14 mmol) in dry DMF
(2 mL) were added HBTU (0.040 g, 0.11 mmol) and
DIEA (0.062 mL, 0.36 mmol). The mixture was stirred
for 3 h at room temperature, diluted with EtOAc
(13 mL), and washed with aqueous NaOAc buffer
(pH 4, 2 · 5 mL), 5% aqueous NaHCO3 (5 mL), H2O
(5 mL), and brine (5 mL). The organic layer was dried
(MgSO4), filtered, and evaporated. Purification by
preparative RP-HPLC-MS (MeCN/H2O (0.05%
6.1.37. Compound 25. To a solution of 15 (0.016 g,
0.028 mmol) and 13f (0.013 g, 0.042 mmol) in dry
DMF (1 mL) were added HBTU (0.013 g, 0.034 mmol)
and DIEA (0.020 mL, 0.12 mmol). The mixture was stir-
red for 3 h at room temperature, diluted with CH2Cl2
(15 mL), and washed with 5% aqueous NaHCO3
(3 · 7 mL), H2O (7 mL) and brine (8 mL). The organic
layer was dried (MgSO4), filtered, and evaporated. Puri-
fication by column chromatography (EtOAc/i-hexane
1:2 followed by EtOAc/i-hexane 2:1) gave 25 as a white
solid (0.014 g, 61%). 1H NMR (CDCl3, 400 MHz) d
8.06–8.01 (m, 4H), 8.00 (d, J = 9.2 Hz, 1H), 7.62–7.58
(m, 1H), 7.54–7.44 (m, 5H), 7.43 (d, J = 2.7 Hz, 1H),
7.12 (br d, J = 8.5 Hz, 1H), 7.08 (dd, J = 2.7, 9.2 Hz,
1H), 7.02 (s, 1H), 5.45 (dt, J = 3.8, 8.5 Hz, 1H), 5.38–
5.35 (m, 1H), 5.24 (d, J = 9.3 Hz, 1H), 4.76 (dd,
J = 6.9, 7.9 Hz, 1H), 4.38 (dm, J = 11.4 Hz, 1H), 4.29
(dd, J = 6.8, 9.3 Hz, 1H), 4.00 (dd, J = 4.7, 11.4 Hz,
1H), 3.95 (s, 3H), 3.22 (s, 3H), 2.76 (ddd, J = 5.1, 6.9,
13.9 Hz, 1H), 2.44 (ddd, J = 3.8, 7.9, 13.9 Hz, 1H),
2.01 (dh, J = 6.7, 6.8 Hz, 1H), 1.87–1.78 (m, 1H),
1.63–1.53 (m, 1H), 1.42–1.25 (m, 2H), 1.38 (s, 9H),
1.00 (d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H), 0.89
(t, J = 7.3 Hz, 3H). 13C NMR (CDCl3, 100 MHz) d
173.5, 172.9, 170.2, 161.5, 160.3, 159.4, 155.9, 151.6,
140.5, 138.3, 134.1, 129.44, 129.40, 128.9, 128.0, 127.7,
123.1, 118.6, 115.1, 107.6, 98.2, 79.9, 76.1, 58.7, 57.4,
55.7, 53.0, 52.9, 35.5, 33.2, 33.1, 31.5, 28.4, 19.5, 18.8,
17.9, 13.7. MS (M+H+) 816.2. Anal. Calcd for
C43H53N5O9S: C, 63.29; H, 6.55; N, 8.58; found: C,
63.11; H, 6.67; N, 8.42.
1
HCOOH)) gave 23 as a white solid (0.053 g, 79%). H
NMR (CD3OD, 400 MHz) d 8.08 (d, J = 9.2 Hz, 1H),
8.05–8.02 (m, 2H), 7.56–7.47 (m, 3H), 7.36 (d,
J = 2.5 Hz, 1H), 7.29–7.24 (m, 4H), 7.22–7.16 (m,
1H), 7.16 (s, 1H), 7.06 (dd, J = 2.5, 9.2 Hz, 1H),
5.45–5.43 (m, 1H), 4.71 (dd, J = 7.5, 9.6 Hz, 1H), 4.57
(dm, J = 11.8 Hz, 1H), 4.38 (d, J = 14.9 Hz, 1H), 4.33
(d, J = 14.9 Hz, 1H), 4.33 (dd, J = 5.5, 8.7 Hz, 1H),
4.06–4.01 (m, 2H), 3.92 (s, 3H), 2.68 (ddm, J = 7.5,
14.1 Hz, 1H), 2.31 (ddd, J = 4.3, 9.6, 14.1 Hz, 1H),
1.96 (dh, J = 6.7, 8.9 Hz, 1H), 1.81–1.64 (m, 2H),
1.50–1.34 (m, 2H), 1.27 (s, 9H), 0.97 (d, J = 6.7 Hz,
3H), 0.95 (d, J = 6.7 Hz, 3H), 0.91 (dd, J = 7.3,
7.4 Hz, 3H). 13C NMR (CD3OD, 100 MHz) d 174.3,
174.1, 173.6, 163.1, 161.9, 161.3, 157.9, 152.2, 141.4,
139.8, 130.5, 129.8, 129.5, 129.0, 128.5, 128.2, 124.4,
119.3, 116.4, 107.4, 100.1, 80.4, 78.0, 60.4, 59.7, 56.0,
55.0, 54.5, 44.0, 35.9, 35.1, 31.7, 28.6, 20.1, 19.7, 19.1,
14.0. MS (M+H+) 752.3. Anal. Calcd for
C43H53N5O7: C, 68.69; H, 7.10; N, 9.31; found: C,
68.46; H, 7.20; N, 9.20.
6.1.36. Compound 24. To a solution of 15 (0.025 g,
0.044 mmol) and 11 (0.019 g, 0.065 mmol) in dry DMF
(1 mL) were added HATU (0.020 g, 0.053 mmol) and
DIEA (0.031 mL, 0.18 mmol). The mixture was stirred
for 1 h at room temperature, diluted with EtOAc
(10 mL), and washed with aqueous NaOAc buffer (pH
4, 4 · 4 mL) and brine (7 mL). The organic layer was
6.1.38. Compound 26. To a solution of 15 (0.080 g,
0.14 mmol) and the hydrochloride salt of 1-amino-cyclo-
propanecarboxylic acid methyl ester (0.032 g,
0.21 mmol) in dry DMF (3 mL) were added HBTU
(0.064 g, 0.17 mmol) and DIEA (0.099 mL, 0.57 mmol).
The mixture was stirred for 2 h at room temperature,

556
R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
diluted with EtOAc (18 mL), and washed with aqueous
NaOAc buffer (pH 4, 2 · 7 mL), 5% aqueous NaHCO3
(7 mL), and brine (10 mL). The organic layer was dried
(MgSO4), filtered, and evaporated. Purification by col-
umn chromatography (EtOAc/i-hexane 2:1) gave 26 as
a white solid (0.079 g, 84%). 1H NMR (CD3OD,
400 MHz) d 8.06 (d, J = 9.2 Hz, 1H), 8.05–8.02 (m,
2H), 7.56–7.47 (m, 3H), 7.35 (d, J = 2.5 Hz, 1H), 7.19
(s, 1H), 7.05 (dd, J = 2.5, 9.2 Hz, 1H), 5.48–5.47 (m,
1H), 4.60 (dd, J = 7.6, 9.5 Hz, 1H), 4.58 (dm,
J = 11.5 Hz, 1H), 4.06–4.02 (m, 2H), 3.92 (s, 3H), 3.67
(s, 3H), 2.72 (ddm, J = 7.6, 14.0 Hz, 1H), 2.44 (ddd,
J = 4.2, 9.5, 14.0 Hz, 1H), 1.98 (dh, J = 6.7, 8.9 Hz,
1H), 1.57–1.53 (m, 1H), 1.44–1.39 (m, 1H), 1.24 (s,
9H), 1.23–1.13 (m, 2H), 1.00 (d, J = 6.7 Hz, 3H), 0.96
(d, J = 6.7 Hz, 3H). 13C NMR (CD3OD, 100 MHz) d
174.9, 174.2, 174.0, 163.1, 161.9, 161.3, 157.9, 152.2,
141.4, 130.5, 129.8, 129.0, 124.3, 119.3, 116.4, 107.3,
100.0, 80.3, 78.0, 60.3, 59.6, 56.0, 54.4, 52.9, 35.9, 34.3,
31.7, 28.6, 19.7, 19.1, 18.1, 17.3. MS (M+H+) 661.2.
1.0 M aqueous HCl. The organic solvents were evapo-
rated and the remaining aqueous phase was diluted with
H2O (10 mL), acidified to pH 3 using 1.0 M HCl (aq),
and extracted with EtOAc (3 · 35 mL). The organic
layer was washed with brine (40 mL), dried (MgSO4),
and filtered. Evaporation gave 28 as a white solid
1
(0.065 g, 96%). H NMR (CD3OD, 400 MHz) d 8.07
(d, J = 9.2 Hz, 1H), 8.04–8.02 (m, 2H), 7.57–7.48 (m,
3H), 7.35 (d, J = 2.5 Hz, 1H), 7.20 (s, 1H), 7.06 (dd,
J = 2.5, 9.2 Hz, 1H), 5.49–5.48 (m, 1H), 4.61 (dd,
J = 7.6, 9.5 Hz, 1H), 4.58 (dm, J = 11.9 Hz, 1H), 4.05–
4.01 (m, 2H), 3.92 (s, 3H), 2.74 (ddm, J = 7.6, 14.1 Hz,
1H), 2.49 (ddd, J = 4.4, 9.5, 14.1 Hz, 1H), 2.02–1.93
(m, 1H), 1.59–1.54 (m, 1H), 1.43–1.39 (m, 1H), 1.23 (s,
9H), 1.22–1.11 (m, 2H), 0.99 (d, J = 6.7 Hz, 3H), 0.96
(d, J = 6.7 Hz, 3H). 13C NMR (CD3OD, 100 MHz) d
175.9, 174.7, 174.0, 163.3, 162.3, 161.1, 157.9, 151.6,
140.8, 130.7, 129.8, 129.1, 124.5, 119.4, 116.4, 106.8,
100.2, 80.3, 78.3, 60.4, 59.7, 56.0, 54.4, 35.9, 34.2, 31.6,
28.6, 19.7, 19.1, 18.0, 17.2. MS (M+H+) 647.2. Anal.
1
Anal. Calcd for C36H44N4O8: C, 65.44; H, 6.71; N,
8.48; found: C, 65.20; H, 6.89; N, 8.30.
Calcd for C35H42N4O8 ꢁ H2O: C, 64.11; H, 6.61; N,
8.54; found: C, 63.93; H,26.95; N, 8.26.
6.1.39. Compound 27. To a solution of 15 (0.080 g,
0.14 mmol) and the hydrochloride salt of (1R, 2S)-1-
amino-2-vinyl-cyclopropanecarboxylic acid ethyl ester
(0.041 g, 0.21 mmol) in dry DMF (3 mL) were added
HBTU (0.064 g, 0.17 mmol) and DIEA (0.099 mL,
0.57 mmol). The mixture was stirred for 2 h at room
temperature, diluted with EtOAc (18 mL), and washed
with aqueous NaOAc buffer (pH 4, 2 · 7 mL), 5% aque-
ous NaHCO3 (7 mL), and brine (10 mL). The organic
layer was dried (MgSO4), filtered, and evaporated. Puri-
fication by column chromatography (EtOAc/i-hexane
6.1.41. Compound 29. A solution of LiOH (0.042 g,
1.8 mmol) in H2O (1.5 mL) was added to a solution of
27 (0.085 g, 0.12 mmol) in THF (4.7 mL) and MeOH
(0.7 mL). The resulting suspension was stirred at room
temperature overnight and thereafter neutralized with
1.0 M aqueous HCl. The organic solvents were evaporat-
ed and the remaining aqueous phase was acidified to pH 3
using 1.0 M HCl (aq) and extracted with EtOAc
(3 · 18 mL). The organic layer was washed with brine
(25 mL), dried (MgSO4), and filtered. Evaporation gave
1
29 as a white solid (0.069 g, 85%). H NMR (CD3OD,
1
2:3) gave 27 as a white solid (0.076 g, 76%). H NMR
400 MHz) d (7:1 mixture of rotamers, major rotamer
reported) 8.09 (d, J = 9.2 Hz, 1H), 8.05–8.03 (m, 2H),
7.57–7.48 (m, 3H), 7.38 (d, J = 2.5 Hz, 1H), 7.23 (s, 1H),
7.09 (dd, J = 2.5, 9.2 Hz, 1H), 5.86 (ddd, J = 9.3, 10.3,
17.1 Hz, 1H), 5.53–5.51 (m, 1H), 5.25 (dd, J = 2.0,
17.1 Hz, 1H), 5.07 (dd, J = 2.0, 10.3 Hz, 1H), 4.62 (dd,
J = 7.6, 9.6 Hz, 1H), 4.56 (dm, J = 11.9 Hz, 1H), 4.08–
4.01 (m, 2H), 3.94 (s, 3H), 2.72 (ddm, J = 7.6, 14.1 Hz,
1H), 2.47 (ddd, J = 4.5, 9.6, 14.1 Hz, 1H), 2.23–2.16 (m,
1H), 2.03–1.93 (m, 1H), 1.70 (dd, J = 5.0, 8.1 Hz, 1H),
1.44–1.41 (m, 1H), 1.24 (s, 9H), 0.98 (d, J = 6.7 Hz, 3H),
0.96 (d, J = 6.7 Hz, 3H). 13C NMR (CD3OD, 100 MHz)
d (7:1 mixture of rotamers, major rotamer reported)
174.4, 173.7, 173.6 (detected with HMBC), 163.3, 162.2,
161.2, 158.0, 151.9, 141.1, 136.0, 130.7, 129.8, 129.1,
124.4, 119.4, 117.4, 116.4, 107.1, 100.2, 80.5, 78.2, 60.7,
60.0, 56.0, 54.3, 41.1, 35.8, 34.8, 31.6, 28.6, 23.2, 19.7,
19.1. MS (M+H+) 673.2. Anal. Calcd for C37H44N4O8:
C,66.05;H,6.59;N,8.33;found:C,65.66;H,6.81;N,8.06.
(CD3OD, 400 MHz) d (5:1 mixture of rotamers, major
rotamer reported) 8.06 (d, J = 9.2 Hz, 1H), 8.05–8.01
(m, 2H), 7.56–7.48 (m, 3H), 7.36 (d, J = 2.5 Hz, 1H),
7.20 (s, 1H), 7.08 (dd, J = 2.5, 9.2 Hz, 1H), 5.76 (ddd,
J = 8.7, 10.3, 17.2 Hz, 1H), 5.50–5.49 (m, 1H), 5.27
(dd, J = 1.9, 17.2 Hz, 1H), 5.09 (dd, J = 1.9, 10.3 Hz,
1H), 4.63 (dd, J = 7.7, 9.4 Hz, 1H), 4.55 (dm,
J = 11.9 Hz, 1H), 4.14 (dq, J = 7.2, 10.6 Hz, 1H), 4.10
(dq, J = 7.2, 10.6 Hz, 1H), 4.06–4.00 (m, 2H), 3.92 (s,
3H), 2.70 (ddm, J = 7.7, 14.1 Hz, 1H), 2.41 (ddd,
J = 4.3, 9.4, 14.1 Hz, 1H), 2.26–2.20 (m, 1H), 1.98 (dh,
J = 6.7, 8.8 Hz, 1H), 1.72 (dd, J = 5.2, 8.2 Hz, 1H),
1.44–1.40 (m, 1H), 1.24 (s, 9H), 1.23 (t, J = 7.2 Hz,
3H), 0.98 (d, J = 6.7 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H).
13C NMR (CD3OD, 100 MHz) d (5:1 mixture of rota-
mers, major rotamer reported) 174.5, 173.9, 171.5,
163.3, 162.3, 160.9, 157.9, 151.5, 140.7, 135.1, 130.7,
129.8, 129.0, 124.4, 119.4, 118.1, 116.3, 106.9, 100.1,
80.4, 78.2, 62.4, 60.5, 59.9, 56.0, 54.3, 40.9, 35.9, 34.8,
31.5, 28.6, 23.2, 19.7, 19.1, 14.6. MS (M+H+) 701.3.
Anal. Calcd for C39H48N4O8: C, 66.84; H, 6.90; N,
7.99; found: C, 66.92; H, 6.55; N, 7.62.
6.1.42. Compound 30. A solution of 28 (0.030 g,
0.046 mmol), HATU (0.021 g, 0.055 mmol), and DIEA
(0.032 mL, 0.18 mmol) in dry DMF (1 mL) was stirred
for 1 h and 15 min before the addition ofa solution of ben-
zenesulfonamide (0.029 g, 0.18 mmol), DMAP (0.023 g,
0.19 mmol), and DBU (0.028 mL, 0.19 mmol) in dry
DMF (1 mL). The mixture was stirred at room tempera-
ture overnight, diluted with EtOAc (30 mL), and washed
with aqueous NaOAc buffer (pH 4, 2 · 10 mL), 5% aque-
6.1.40. Compound 28. A solution of LiOH (0.038 g,
1.6 mmol) in H2O (1 mL) was added to a solution of
26 (0.069 g, 0.10 mmol) in THF (3.5 mL) and MeOH
(1.5 mL). The resulting suspension was stirred at room
temperature overnight and thereafter neutralized with

R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
557
ous NaHCO3 (10 mL), and brine (15 mL). The organic
layer was dried (MgSO4), filtered, and evaporated. Purifi-
cation by preparative RP-HPLC-MS (MeCN/H2O
(0.05% HCOOH)) gave 30 as a white solid (0.027 g,
75%). 1H NMR (CD3OD, 400 MHz) d 8.08 (d,
J = 9.2 Hz, 1H), 8.05–8.03 (m, 2H), 7.99–7.97 (m, 2H),
7.65–7.61 (m, 1H), 7.56–7.48 (m, 5H), 7.37 (d,
J = 2.5 Hz, 1H), 7.23 (s, 1H), 7.10 (dd, J = 2.5, 9.2 Hz,
1H), 5.55–5.54 (m, 1H), 4.65 (dm, J = 11.8 Hz, 1H),
4.56 (dd, J = 6.6, 10.8 Hz, 1H), 4.12–4.04 (m, 2H), 3.94
(s, 3H), 2.62 (ddm, J = 6.6, 14.0 Hz, 1H), 2.36 (ddd,
J = 3.9, 10.8, 14.0 Hz, 1H), 2.19 (dh, J = 6.7, 9.3 Hz,
1H), 1.49–1.45 (m, 1H), 1.35–1.31 (m, 1H), 1.22 (s, 9H),
1.10–1.02 (m, 2H), 1.00 (d, J = 6.7 Hz, 3H), 0.98 (d,
J = 6.7 Hz, 3H). 13C NMR (CD3OD, 100 MHz) d 175.1,
174.4, 173.4, 163.4, 162.3, 161.0, 157.9, 151.4, 141.0,
140.6, 134.6, 130.8, 129.9 (two overlapping signals),
129.11, 129.05, 124.4, 119.5, 116.4, 106.8, 100.3, 80.3,
78.4, 61.1, 59.6, 59.5, 56.1, 54.5, 35.4, 31.5, 28.5, 19.9,
19.5, 19.1, 18.8. MS (M+H+) 786.3. Anal. Calcd for
C41H47N5O9S: C, 62.66; H, 6.03; N, 8.91; found: C,
62.47; H, 6.10; N, 8.83.
for 1.5 h before the addition of a solution of a-toluene-
sulfonamide (0.032 g, 0.18 mmol), DMAP (0.022 g,
0.18 mmol), and DBU (0.028 mL, 0.18 mmol) in dry
DMF (1 mL). The mixture was stirred overnight, diluted
with EtOAc (50 mL), and washed with aqueous NaOAc
buffer (pH 4, 2 · 15 mL), 5% aqueous NaHCO3
(15 mL), and brine (20 mL). The organic layer was dried
(MgSO4), filtered, and evaporated. Purification by pre-
parative RP-HPLC-MS (MeCN/H2O (0.05% HCOOH))
gave 32 as a white solid (0.022 g, 63%). 1H NMR
(CD3OD/CDCl3 2:1, 400 MHz) d 8.04 (d, J = 9.2 Hz,
1H), 8.00–7.98 (m, 2H), 7.55–7.47 (m, 3H), 7.38 (d,
J = 2.5 Hz, 1H), 7.37–7.30 (m, 5H), 7.14 (s, 1H), 7.07
(dd, J = 2.5, 9.2 Hz, 1H), 6.50 (d, J = 9.0 Hz, 1H), 5.87
(ddd, J = 8.9, 10.3, 17.1 Hz, 1H), 5.53–5.51 (m, 1H),
5.34 (dd, J = 1.7, 17.1 Hz, 1H), 5.20 (dd, J = 1.7,
10.3 Hz, 1H), 4.78 (d, J = 13.9 Hz, 1H), 4.54 (dm,
J = 11.9 Hz, 1H), 4.57 (dd, J = 7.0, 10.6 Hz, 1H), 4.46
(d, J = 13.9 Hz, 1H), 4.09 (dd, J = 3.6, 11.9 Hz, 1H),
4.04 (dd, J = 8.8, 9.0 Hz, 1H), 3.95 (s, 3H), 2.61 (ddm,
J = 7.0, 14.1 Hz, 1H), 2.36 (ddd, J = 4.2, 10.6, 14.1 Hz,
1H), 2.26–2.19 (m, 1H), 2.08 (dh, J = 6.7, 8.8 Hz, 1H),
1.97 (dd, J = 5.4, 8.3 Hz, 1H), 1.42 (dd, J = 5.4,
9.4 Hz, 1H), 1.26 (s, 9H), 0.93 (d, J = 6.7 Hz, 6H). 13C
NMR (CD3OD/CDCl3 2:1, 100 MHz) d 174.5, 173.9,
171.1, 162.8, 161.5, 160.9, 157.4, 151.5, 140.6, 133.9,
131.8, 130.4, 129.6, 129.5, 129.4, 128.8, 128.7, 123.9,
119.2, 118.8, 115.9, 106.8, 99.8, 80.2, 77.7, 60.5, 59.3,
58.8, 55.9, 54.1, 42.1, 35.7, 35.0, 31.2, 28.5, 24.4, 19.6,
18.7. MS (M+H+) 826.2. Anal. Calcd for C44H51N5O9S:
C, 63.98; H, 6.22; N, 8.48; found: C, 63.71; H, 6.45; N,
8.35.
6.1.43. Compound 31. A solution of 29 (0.040 g,
0.060 mmol), HATU (0.027 g, 0.071 mmol), and DIEA
(0.042 mL, 0.24 mmol) in dry DMF (5 mL) was stirred
for 1 h before the addition of a solution of benzenesulf-
onamide (0.038 g, 0.24 mmol), DMAP (0.030 g,
0.24 mmol), and DBU (0.036 mL, 0.24 mmol) in dry
DMF (1.5 mL). The mixture was stirred overnight,
diluted with EtOAc (60 mL), and washed with aqueous
NaOAc buffer (pH 4, 2 · 15 mL), 5% aqueous NaH-
CO3 (15 mL) and brine (20 mL). The organic layer
was dried (MgSO4), filtered, and evaporated. Purifica-
tion by preparative RP-HPLC-MS (MeCN/H2O
(0.05% HCOOH)) gave 31 as a white solid (0.029 g,
60%). 1H NMR (CD3OD, 400 MHz) d (3:1 mixture
of rotamers, major rotamer reported) 8.12 (d,
J = 9.2 Hz, 1H), 8.07–8.03 (m, 2H), 7.99–7.96 (m,
2H), 7.68–7.62 (m, 1H), 7.58–7.50 (m, 5H), 7.40 (d,
J = 2.5 Hz, 1H), 7.27 (s, 1H), 7.12 (dd, J = 2.5,
9.2 Hz, 1H), 5.59–5.56 (m, 1H), 5.52 (ddd, J = 8.9,
10.3, 17.1 Hz, 1H), 5.20 (dd, J = 1.8, 17.1 Hz, 1H),
4.96 (dd, J = 1.8, 10.3 Hz, 1H), 4.68 (dm,
J = 11.8 Hz, 1H), 4.56 (dd, J = 6.9, 10.4 Hz, 1H), 4.11
(dm, J = 11.8 Hz, 1H), 4.07–4.02 (m, 1H), 3.96 (s,
3H), 2.65 (ddm, J = 6.9, 14.4 Hz, 1H), 2.37 (ddd,
J = 3.9, 10.4, 14.4 Hz, 1H), 2.21–2.10 (m, 2H), 1.72
(dd, J = 5.2, 8.2 Hz, 1H), 1.33 (dd, J = 5.2, 9.4 Hz,
1H), 1.23 (s, 9H), 0.99 (d, J = 6.7 Hz, 3H), 0.98 (d,
J = 6.7 Hz, 3H). 13C NMR (CD3OD, 100 MHz) d
(3:1 mixture of rotamers, major rotamer reported)
174.9, 174.5, 170.4, 163.5, 162.4, 161.1, 158.0, 151.4,
140.9, 140.6, 134.6, 134.0, 130.9, 129.9, 129.8, 129.14,
129.14, 124.5, 119.6, 118.4, 116.4, 106.7, 100.4, 80.3,
78.4, 61.0, 59.6, 56.1, 54.6, 42.5, 35.8, 35.5, 31.5,
28.5, 24.0, 19.9, 19.2. MS (M+H+) 812.2. Anal. Calcd
for C43H49N5O9S Æ H2O: C, 62.23; H, 6.19; N, 8.44;
found: C, 62.33; H, 6.06; N, 8.42.
6.2. Enzyme Inhibition
The protease activity of the full-length HCV NS3 protein
(protease-helicase/NTPase) was measured using a FRET
assay as previously described.29,30 In short, 1 nM enzyme
was incubated for 10 min at 30 ꢁC in 50 mM Hepes, pH
7.5, 10 mM DTT, 40% glycerol, 0.1% n-octyl-b-D-gluco-
side, 3.3% DMSO with 25 lM of the peptide cofactor
2K-NS4A (KKGSVVIVGRIVLSGK) and inhibitor.
The reaction was started by the addition of 0.5 lM sub-
strate (Ac-DED(Edans)EEAbuw[COO]ASK(Dabcyl)-
NH2) obtained from AnaSpec Inc. (San Jose, USA).
Inhibitor 25, suspected to bind irreversibly to the enzyme,
was tested by incubation with the enzyme for several dif-
ferent periods of time, from 10 min to 1 h before addition
of substrate. The nonlinear regression analysis was made
using Grafit 5.0.8 (Erithacus software limited).
6.3. HCV replicon assay
Huh7 cells transfected with pFKI389luc/NS3-3ꢀ/ET, con-
stitutively expressing HCV replicons, were used for the
evaluation of the anti-HCV effect of our protease inhib-
itors in cell culture. The cells were maintained in Dul-
beccoꢀs modified Eagleꢀs media (DMEM) containing
10% FCS and 250 lg/ml G418 (geneticin). The day prior
to assay, cells were trypsinized and 15,000 cells were
plated in each well in a 96-well plate. The next day, serial
dilutions of test compounds in cell culture medium were
added to the cells and they were further incubated for
6.1.44. Compound 32. A solution of 29 (0.031 g,
0.046 mmol), HATU (0.021 g, 0.055 mmol), and DIEA
(0.032 mL, 0.18 mmol) in dry DMF (3 mL) was stirred

558
R. Ro¨nn et al. / Bioorg. Med. Chem. 14 (2006) 544–559
48 h. After three days, the medium was removed and the
cells were lysed with 50 ll lysis buffer. The luciferase
activity was measured using a Luciferase Assay kit
(Biothema, Haninge, Sweden) and a Microlumat Plus
(Berthold Technologies). IFN-aA was used as reference
inhibitor in each plate.
tein, we choose to dock the ligands by taking into
account the protein flexibility. FLO (also called QXP)
developed by McMartin et al. was employed to dock
the database of molecules into the active site of the cat-
alytic core of the enzyme.52 FLO search algorithms are
derived from the method of Monte Carlo perturbation
with energy minimization in Cartesian space. It uses
the modified version of the AMBER force field. Partial
charges were calculated using bond dipole moments.
Crucial amino acid residues in the binding pocket were
6.4. Computational methodology
(a) Ligand preparation. The ligands were constructed
and geometry optimized in SYBYL.40 All the ligands
˚
allowed to move freely up to 0.2 A. Movement larger
2
were assigned Gasteiger-Huckel charges.41,42 The con-
than 0.2 A was then penalized by 20.0 kJ/mol/A . The
inhibitor along with residue R155 had full conforma-
tional freedom.
˚
˚
¨
structed ligands were locally optimized as follows. The
first step in ligand optimization encompassed employing
the Powell Method43 using the conjugate gradient termi-
˚
nation criterion of 0.05 kcal/mol A for 1000 iterations.
(d) Docking protocol. The ligands were docked into the
active site by employing 2000 Monte Carlo perturba-
tion cycles and 10 unique binding poses for each ligand
were generated. Each of these poses was further sub-
jected to 20 steps of simulated annealing followed by
gradient minimization. Each perturbation cycle in-
volves 400 rapid Monte Carlo search steps followed
by energy minimization of the best conformer from
the set of 400. The conformations are randomly gener-
ated in such a way that each new conformation gener-
ated differs from the previous one by a similarity
˚
A distance dependent dielectric of 4 A was used for all
molecular mechanics (MM) calculations in order to take
into account the shielding in the protein. The ligands
were then further subjected to the BFGS minimization
method44–47 where they were optimized until a conjugate
˚
gradient of 0.001 kcal/mol A was reached.
(b) Preparation of protein coordinates and definition of
the active site. Reference protein coordinates used for
docking were taken from the X-ray structure of bifunc-
tional NS3 protein found in the Protein Databank under
˚
distance of 0.5 A and has an energy window of not
˚
more than 50.0 kJ/mol/A. The simulated annealing
the accession code 1CU1 resolved at 2.50 A.48 This 631-
˚
residue protein consists of the C-terminal helicase do-
main and the N-terminal protease domain covalently
linked to the NS4A cofactor. Although alternative rota-
meric states exist for a few side chains, we felt that
choosing the crystal coordinates was a reasonable choice
since the active site is defined by the C-terminal. Never-
theless, there exist a few structural discrepancies in
assigning the side chains of an X-ray structure resolved
implements the Beeman algorithm and rescales the
velocities to give an exact average kinetic temperature
at each step.53 Each of the complexes was submitted
to 3 fs of dynamics at 600 K following a 3000 fs equil-
ibration step. The maximum movement of an atom in
˚
any single time step was limited to 0.1 A. Hydrogen
vibrations were damped by assigning an atomic weight
of 10. This was followed by minimization of each saved
pose by the PRCG method.
˚
at more than 2.0 A. Therefore, the structure was subject-
ed to refinement by restrained minimization. The crys-
tallographic waters were removed and hydrogens were
added via an all-atom treatment with no atoms having
lone pairs. The refinement was done using the BatchMin
algorithm of Macromodel v.7.1,49 employing the AM-
BER*50 all-atom force field with a dielectric constant
of 78. The extended non-bonded cutoff distances for
van der Waals and Coulombic interactions were set to
Acknowledgments
We gratefully acknowledge support from the Swedish
Foundation for Strategic Research and the Swedish Re-
search Council. We also thank Karin Wiklund for assis-
tance in the synthetic work and Dr. Kristofer Olofsson
for linguistic help with the manuscript.
˚
˚
3.5 A and 6.0 A, respectively, while the hydrogen bond-
˚
ing cutoff distance was kept at 4.0 A. These cutoffs have
been shown to give optimal results in terms of speed and
accuracy.51 The protein was minimized with a total of
300 iterations employing the Polak-Ribiere Conjugate
Gradient (PRCG) method, and convergence was moni-
tored with the derivative convergence criterion using a
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