Hepatitis C virus NS3 protease inhibitors comprising a novel aromatic P1 moiety

Article abstract of DOI:10.1016/j.bmc.2007.12.041

Inhibition of the hepatitis C virus (HCV) NS3 protease has emerged as an attractive approach to defeat the global hepatitis C epidemic. In this work, we present the synthesis and biochemical evaluation of HCV NS3 protease inhibitors comprising a non-natural aromatic P₁ moiety. A series of inhibitors with aminobenzoyl sulfonamides displaying submicromolar potencies in the full-length NS3 protease assay was prepared through a microwave-irradiated, palladium-catalyzed, amidocarbonylation protocol.

Full text of DOI:10.1016/j.bmc.2007.12.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2955–2967
Hepatitis C virus NS3 protease inhibitors comprising
a novel aromatic P1 moiety
a
a
a
b
a
˚
Robert Ro¨nn, Anna Lampa, Shane D. Peterson, Thomas Gossas, Eva Akerblom,
b
a
a,
*
´
U. Helena Danielson, Anders Karlen and Anja Sandstro¨m
aDepartment of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University,
BMC, Box 574, SE-751 23 Uppsala, Sweden
bDepartment of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
Received 18 October 2007; revised 14 December 2007; accepted 20 December 2007
Available online 27 December 2007
Abstract—Inhibition of the hepatitis C virus (HCV) NS3 protease has emerged as an attractive approach to defeat the global hep-
atitis C epidemic. In this work, we present the synthesis and biochemical evaluation of HCV NS3 protease inhibitors comprising a
non-natural aromatic P1 moiety. A series of inhibitors with aminobenzoyl sulfonamides displaying submicromolar potencies in the
full-length NS3 protease assay was prepared through a microwave-irradiated, palladium-catalyzed, amidocarbonylation protocol.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
protease, in complex with its co-factor NS4A, is a key
actor in the processing of the HCV polyprotein.4 More-
over, it has been suggested that the NS3 protease inter-
feres with cellular mechanisms involved in the host
immune response to an HCV infection.5 Thus, inhibi-
tion of the protease is an attractive antiviral approach
that will block viral replication as well as potentially re-
store the host immune response.
Hepatitis C is a global epidemic with an estimated prev-
alence of 2%. The disease is caused by the hepatitis C
virus (HCV) which is the major cause of chronic liver
disease and liver transplantations in the developed
world.1 The current standard treatment with a combina-
tion of pegylated interferon and ribavirin has a sus-
tained virological response rate of only ꢀ55%.2
Moreover, this therapy is associated with high costs
and severe adverse effects. Clearly, more efficient and
better-tolerated therapies against HCV are highly
sought.
The NS3 protease was early recognized as a potential
drug target, possibly as a result of the successful use of
HIV-1 protease inhibitors, and initial substrate-based
structure–activity relationship (SAR) studies identified
hexapeptides as potent NS3 protease inhibitors.6,7 To-
day, approximately one decade after the first reports
on NS3 protease inhibitors, a huge arsenal of preclinical
shorter peptide-based inhibitors has emerged.8–10 Addi-
tionally, in the last couple of years we have witnessed the
first NS3 protease inhibitors to enter clinical trials:
BILN 2061 (ciluprevir), VX-950 (telaprevir) and SCH
503034 (Fig. 1).11 These three inhibitors have shown
powerful antiviral effects in HCV-infected patients and
both telaprevir and SCH 503034 are currently being
evaluated in phase II trials whereas the clinical evalua-
tion of ciluprevir has been stopped due to cardiac toxic-
ity in animals.12–15 ITMN-191, a novel inhibitor
developed by InterMune, is structurally related to
ciluprevir, but one interesting modification is that the
cyclopropyl acyl sulfonamide group has replaced the
The HCV genome consists of a single-stranded, positive
sense RNA molecule of approximately 9600 nucleotides.
Translation of the viral RNA generates a polyprotein
that is proteolytically cleaved into 10 viral proteins of
different function: the structural proteins C, E1 and
E2, p7, and the non-structural proteins NS2, NS3,
NS4A, NS4B, NS5A and NS5B.3
The NS3 protein is a bifunctional enzyme with a heli-
case/NTPase domain and a protease domain. The NS3
Keywords: HCV; NS3; Protease inhibitor; Carbonylation; Acyl
sulfonamide; Palladium.
*
Corresponding author. Tel.: +46 18 471 4957; fax: +46 18 471
4474; e-mail: Anja.Sandstrom@orgfarm.uu.se
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.041

2956
R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
O
H
N
N
N
S
H
N
N
O
O
O
NH
O
O
NH
N
HN
O
O
H
N
O
O
N
O
N
OH
VX-950 (telaprevir)
Ki = 39 nM; EC50 = 310 nM
O
O
N
H
BILN 2061 (ciluprevir)
Ki = 0.66 nM; EC50 = 3 nM
F
N
O
O
H
N
N
O
O
O
O
O
O
H
N
O
N
S
O
NH
O
N
NH2
NH
H
O
O
N
H
O
SCH 503034
Ki* = 14 nM; EC50 = 200 nM
ITMN-191
IC50 < 0.25 nM; EC50 = 2.1 nM
Figure 1. Chemical structures and inhibition data of BILN 2061,12 VX-950,17 SCH 50303418 and ITMN-191.19,20
C-terminal carboxylic acid (Fig. 1). Clinical evaluation
of ITMN-191 has recently been initiated.16
benzoyl sulfonamide moiety were found to be the most
promising. A series of compounds was generated by
the use of a palladium-catalyzed amidocarbonylation
protocol providing NS3 protease inhibitors displaying
a diverse SAR and with submicromolar potencies.
One major concern regarding future HCV therapy in
general and thus with NS3 protease inhibitors is the
development of viral resistance. HCV strains resistant
to ciluprevir, telaprevir and SCH 503034 have been ob-
served in cell studies and more recently in patients trea-
ted with telaprevir.21–24 Consequently, the development
of NS3 protease inhibitors comprising novel structural
motifs is highly sought in order to combat HCV.
2. Results
2.1. Chemistry
Synthesis of compound 1, herein used as a starting point
for further synthesis, has previously been reported.26
Compounds 2–4, comprising the aminobenzoic acid
moiety, were prepared through amide bond formation
between carboxylic acid 1 and the aminobenzoic
acid ester using N-[(dimethylamino)-1H-1,2,3-triazolo-
[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide (HATU) as the activating
agent in the presence of N,N-diisopropylethylamine
(DIEA) at 45 ꢁC in CH2Cl2. Subsequent ester hydrolysis
provided the desired carboxylic acids 2–4 (Scheme 1).
Compound 5, comprising the 2-aminophenylacetic acid
moiety, was prepared according to the same procedure
as compounds 2–4 (Scheme 1).
The vast majority of NS3 protease inhibitors developed
so far consist of a peptide backbone comprising a-amino
acids.8–10 Although ciluprevir, telaprevir and SCH
503034 are evaluated as oral drugs, the use of peptides
as drugs is often associated with poor oral bioavailabil-
ity and rapid metabolic degradation.25
We were interested in examining whether the a-amino
acid in P1 could be substituted with a non-natural aro-
matic moiety. Such a replacement would not only make
more rigid and less peptide-like inhibitors, but also en-
able us to explore the vital C-terminal end of the inhib-
itors—interacting with the catalytic machinery of the
protease—and the S1 pocket in a new way. Further-
more, the aryl moiety allows the use of a more diverse
range of synthetic methods in analogue preparation.
Carboxylic acid 1 was also used in the preparation of
compounds 6–8, 18 and 19, comprising a bromoaniline
moiety (Scheme 2). Amide bond formation between
1 and 2-, 3- or 4-bromoaniline was enabled by the use
of HATU as described above, providing the aryl
bromides 6–8 in good yields. The coupling between 1
and 2-bromo-6-methylaniline and 2-bromo-5-(trifluoro-
methyl)aniline required the use of a different activating
agent. We found that the use of POCl3 in pyridine as
Herein, we present the synthesis and biochemical evalu-
ation of NS3 protease inhibitors comprising a non-nat-
ural aromatic P1 moiety. Aminobenzoic acids and
aminobenzoyl sulfonamides, aimed at interacting with
the active site of the protease, were investigated as po-
tential P1 motifs and compounds containing the amino-

R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
2957
O
R
R
O
P
OAc
OAc
Pd
Pd
N
N
P
R
R
O
O
R =
o-tolyl
a,b
Figure 2. Chemical structure of Herrmann’s palladacycle.
N
N
NH
OH
O
BocHN
BocHN
O
O
O
O
OH
n=0,1
nation of Herrmann’s palladacycle (Fig. 2)29 and the Fu
salt, [(t-Bu)3PH]BF4,30 as well as Mo(CO)6 as a conve-
nient carbon monoxide source.31
1
2, n=0, ortho (35%)
3, n=0, meta (65%)
4, n=0, para (59%)
5, n=1, ortho (72%)
2.2. Biochemical evaluation
Scheme 1. Reagents and conditions: (a) 2-aminobenzoic acid methyl
ester, 3-aminobenzoic acid ethyl ester, 4-aminobenzoic acid methyl
ester or 2-aminophenylacetic acid methyl ester, HATU, DIEA,
CH2Cl2, 45 ꢁC; (b) LiOH, THF, MeOH, H2O, rt.
The compounds 1–17, 20 and 21 were biochemically
evaluated in a protease activity assay for the full-length
NS3 protein (Ki values) and the results are summarized
in Tables 1 and 2.32
reported by Rijkers and co-workers27 was very conve-
nient and furnished the aryl bromides 18 and 19 in good
yields.
Carboxylic acid 1 lacking the aromatic P1 moiety is a
very weak inhibitor of the NS3 protease with a Ki value
of 66 lM (Table 1). Compounds 2–4 comprising the
aminobenzoic acid moiety display inhibitory potencies
of 5.0, 6.8 and 12 lM for the ortho, meta and para-com-
pound, respectively. Introduction of a carbon spacer be-
tween the aromatic moiety and the carboxylic acid
group as in compound 5 was not beneficial for the po-
tency and increased the Ki value to 17 lM. The aryl bro-
mide precursors 6–8 displayed Ki-values of 3.9, 1.4 and
1.8 lM for the ortho-, meta- and para-bromide, respec-
tively. Replacement of the aromatic carboxylic acid
The aryl bromides 6–8, 18 and 19 served as precursors
for the synthesis of compounds 9–17, 20 and 21 compris-
ing the acyl sulfonamide functionality (Scheme 2). A
palladium-catalyzed amidocarbonylation protocol using
microwave irradiation recently presented by our group
yielded the desired acyl sulfonamide containing com-
pounds 9–17, 20 and 21 in reasonably good yields.28
This method employs the thermostable catalytic combi-
O
O
9, ortho, R=phenyl (35%)
10, meta, R=phenyl (44%)
11, para, R=phenyl (35%)
N
N
12, ortho, R=methyl (50%)
O
O
13, ortho, R=cyclopropyl (62%)
14, ortho, R=benzyl (56%)
15, ortho, R=4-methoxyphenyl (43%)
16, ortho, R=4-(trifluoromethyl)phenyl (42%)
17, ortho, R=2-thienyl (37%)
O
S
R
N
N
O
b
O
NH
Br
NH
N
H
BocHN
BocHN
O
O
O
O
6, ortho (77%)
7, meta (63%)
8, para (82%)
a
c
1
O
O
N
N
O
O
O
b
O
S
NH
O
N
N
Br
NH
NH
BocHN
BocHN
O
O
O
O
R1
R1
R2
R2
18, R1=CH3, R2=H (76%)
19, R1=H, R2=CF3 (88%)
20, R1=CH3, R2=H (45%)
21, R1=H, R2=CF3 (35%)
Scheme 2. Reagents and conditions: (a) 2-, 3- or 4-bromoaniline, HATU, DIEA, CH2Cl2, 45 ꢁC; (b) Herrmann’s palladacycle, [(tBu)3PH]BF4,
RSO2NH2, Mo(CO)6, DBU, 1,4-dioxane, microwave irradiation at 140 ꢁC for 15 min; (c) 2-bromo-6-methylaniline or 2-bromo-5-(trifluoro-
methyl)aniline, POCl3, pyridine, À15 ꢁC to rt.

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R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
Table 1.
Table 2.
O
O
N
N
O
O
O
R3
O
S
NH
O
N
N
NH
NH
R
BocHN
O
O
R1
BocHN
O
O
R2
Compound
R
Position
Ki SD (lM)a
Compound
R1
R2
R3
Ki SD (lM)a
1b
2
3
4
5
6
7
8
9
—
—
66 30
5.0 1.5
6.8 2.5
–COOH
–COOH
–COOH
ortho
meta
para
ortho
ortho
meta
para
ortho
meta
para
9
12
13
–H
–H
–H
–H
–H
–H
0.83 0.11
5.9 1.1
2.0 0.3
12
17
5
5
–CH2COOH
–Br
–Br
–Br
3.9 0.6
1.4 0.3
1.8 0.3
–CONHSO2Ph
–CONHSO2Ph
–CONHSO2Ph
0.83 0.11
0.78 0.15
1.1 0.2
10
11
14
15
–H
–H
–H
–H
0.61 0.09
1.2 0.2
SD, standard deviation.
a Determined in a full-length NS3 protein inhibition assay.
b See Scheme 1 for structure.
O
group in compounds 2–4 with the corresponding phenyl
acyl sulfonamide in compounds 9–11 was successful and
the inhibitory potencies improved between 6- and 11-
fold, rendering inhibitors with submicromolar potencies.
CF3
16
17
20
21
–H
–H
–H
–H
0.31 0.05
0.92 0.15
3.2 0.4
The ortho acyl sulfonamide compounds were further ex-
plored as HCV NS3 protease inhibitors by variation of
the sulfonamide substituent (Table 2). The SAR study
included alkyl groups (12–13), a benzyl group (14) and
several aryl groups (15–17). The methyl and cyclopropyl
aliphatic substituents used in compounds 12 and 13,
respectively, produced inhibitors with potencies weaker
than the initial phenyl-substituted inhibitor 9. Inhibitor
14, comprising the benzylic substituent, was slightly
more potent than inhibitor 9, displaying a Ki value of
0.61 lM. The 4-methoxyphenyl-based inhibitor 15 was
–H
S
–CH3
–H
–CF3
0.35 0.05
SD, standard deviation.
a Determined in a full-length NS3 protein inhibition assay.
slightly less potent than
9 but the 4-(trifluoro-
methyl)phenyl group rendered inhibitor 16 with im-
proved potency compared to 9 and a Ki value of
0.31 lM. The heteroaromatic thiophene-based inhibitor
17 (Ki = 0.92 lM) was equipotent to 9.
3. Discussion
One common structural feature for the vast majority of
NS3 protease inhibitors is the presence of a peptide
backbone comprising a-amino acids (Fig. 1). In an effort
to develop less peptide-like inhibitors, we decided to
investigate the replacement of the normal a-amino acid
with a non-natural aromatic P1 moiety. Although a
large structural modification like this was expected to
produce less efficient inhibitors as compared to those
comprising well-optimized a-amino acids in P1, at least
initially, we looked forward to finding a new starting
point in a forthcoming drug discovery process. More-
over, this structural modification would enable synthetic
methods not possible on non-aromatic P1 groups.
Furthermore, we explored the effect of an additional sub-
stituent on the aromatic P1 moiety, as in compounds 20
and 21 (Table 2). A methyl substituent ortho to the aniline
nitrogen present in compound 20 resulted in an inhibitor
with a Ki value of 3.2 lM. In comparison to the unsubsti-
tuted analogue 9, the ortho methyl compound 20 is
approximately 4 times weaker in inhibitory potency. On
the other hand, the introduction of a trifluoromethyl sub-
stituent para to the acyl sulfonamide group was well toler-
ated and provided inhibitor 21 with a Ki value of 0.35 lM.
Compound 21 was the only inhibitor displaying effect in a
cell-based subgenomic HCV replicon assay with an EC50
of 5.2 lM (CC50 = 28 lM).33
We envisioned that properly substituted anilines could
work well as P1 moieties and that an acidic substituent

R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
2959
would be favorable for interactions with the active site
of the NS3 protease, as utilized in product-based NS3
protease inhibitors such as ciluprevir (Fig. 1). Conse-
quently, compounds 2–5 comprising a carboxylic acid
were synthesized but failed to produce highly potent
inhibitors. Apparently, the carboxylate group is not
capable of interacting with the oxyanion hole in an opti-
mal way as it does in inhibitors with P1 a-amino acids.34
pared (compounds 12–17, Table 2). The new inhibitors
displayed a diverse SAR, which was encouraging. The
most potent inhibitor 16 with
a
4-(trifluoro-
methyl)phenyl substituent (Ki = 0.31 lM) is 19 times
more potent than inhibitor 12 with a methyl substituent
(Ki = 5.9 lM) and 16 times more potent than the corre-
sponding carboxylic acid inhibitor 2 (Ki = 5.0 lM).
Interestingly, the cyclopropyl substituent in compound
13 did not function as well as was previously seen for
inhibitors with (1R,2S)-1-amino-2-vinyl-cyclopropane-
carboxylic acid in the P1 position.26,39 This emphasizes
that the acyl sulfonamide group in the inhibitors com-
prising an aromatic P1 has a different binding mode than
it does in inhibitors with a P1 a-amino acid.
We have previously used the acyl sulfonamide group as
a potent P1 C-terminal carboxylic acid replacement and
introduction of this group in the aromatic P1 moiety re-
sulted in submicromolar inhibitors (compounds 9–
11).35–37 Molecular modeling of compound 9 (Fig. 3)
suggests that the aromatic carbonyl is not positioned
in the oxyanion hole, which is also indicated by the
modest potency observed for the carboxylic acid 2. On
the other hand, the sulfone oxygens in 9 have the possi-
bility to hydrogen bond to the side chains of Gln41 and
Lys136 in the protease. These hydrogen bonds, in com-
bination with additional interactions between the prote-
ase and the sulfonamide substituent as well as
electrostatic interactions between Lys136 and the acyl
sulfonamide part, could explain the difference in potency
between compounds 2 and 9.
According to molecular modeling studies, it is possible
that the aromatic P1 moiety interacts with the aromatic
ring of Phe154, which forms the bottom of the S1 pocket
of the protease (Fig. 3). This is also indicated by the fact
that the bromoanilines 6–8, lacking the acidic function-
ality, are modest inhibitors of the NS3 protease and that
compound 1, lacking the aromatic P1 moiety, is a very
weak inhibitor (Table 1). This encouraged us to intro-
duce an additional substituent on the aromatic P1 moi-
ety that could interact more favorably with the S1
pocket. Hence, compound 20, with a methyl group ortho
to the aniline nitrogen, was prepared but resulted in an
inhibitor with four times lower potency compared to the
unsubstituted analogue 9. On the other hand, the trifluo-
romethyl group para to the acyl sulfonamide group in
compound 21 rendered an inhibitor displaying a Ki of
0.35 lM, almost three times more potent than the
unsubstituted inhibitor 9. Compound 21 was the only
inhibitor displaying a measurable inhibition in a cell-
based HCV replicon assay with an EC50 of 5.2 lM.
Although no significant difference in potency between
the ortho, meta and para compounds was observed, we
decided to further explore the ortho acyl sulfonamide
compound since this analogue bears closer resemblance
to an inhibitor with a P1 a-amino acid. A series of inhib-
itors with different sulfonamide substituents was pre-
4. Conclusions
Herein, we present the synthesis and biochemical evalu-
ation of the first account of HCV NS3 protease inhibi-
tors comprising a non-natural aromatic P1 moiety. The
aminobenzoyl sulfonamide fragment was identified as
a novel P1 structural motif. A microwave irradiated, pal-
ladium catalyzed, amidocarbonylation protocol enabled
the facile preparation of a series of compounds display-
ing submicromolar potencies in the full-length NS3 as-
say. Encouragingly, a diverse SAR was found both for
the sulfonamide substituent and for the substituent on
the aromatic P1 moiety. This indicates that the inhibitors
have two sites that can be utilized in a forthcoming opti-
mization process. Further studies are underway to fully
exploit this new P1 moiety.
Figure 3. Compound 9 docked in the NS3 protease binding site of the
1CU1 crystal structure.38 The enzyme is shown in a ribbon structure
with residues Gln41, Lys136 and Phe154 displayed as sticks with
carbon atoms in green and heteroatoms colored according to atom
type (oxygens in red and nitrogens in blue). The hydrogen bond
distances shown are between the sulfone oxygens of the inhibitor and
5. Experimental
5.1. Chemistry
˚
˚
the side chains of Lys136 (1.95 A) and Gln41 (2.47 and 2.67 A). The
aromatic P1 moiety of 9 is positioned above the aromatic ring of
Phe154. All essential ligand hydrogens as well as the hydrogens of the
protease thought to participate in hydrogen bonding with the acyl
sulfonamide group are displayed. Ligand atoms are colored according
to atom type (oxygens in red, nitrogens in blue and sulfur in yellow).
5.1.1. General methods. TLC was performed using alumi-
num sheets precoated with silica gel 60 F254 (0.2 mm,
Merck). Chromatographic spots were visualized using
UV-detection and/or ethanolic ninhydrin solution (2%)

2960
R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
followed by heating. Column chromatography was per-
formed using silica gel 60 (40–63 lm, Merck). Analytical
HPLC–MS was performed on a Gilson-Finnigan Ther-
moQuest AQA system equipped with a C18 (Onyx Mono-
lithic C18 (50 · 4.6 mm)) or a C4 (Hichrom ACE C4
(5 lm, 50 · 4.6 mm)) column using MeCN/H2O (0.05%
HCOOH) as the mobile phase with UV (254 nm) and
MS (ESI) detection. HPLC purity of the compounds
was determined using the C18 or the C4 column through
integration of the UV-trace at 254 nm. Preparative
HPLC–MS was performed on a Gilson-Finnigan Ther-
moQuest AQA system equipped with a C8 (Sorbax SB-
C8 (5 lm, 150 · 21.2 mm) column using MeCN/H2O
(0.05% HCOOH) as the mobile phase with UV (254 nm)
and MS (ESI) detection. Elemental analyses were per-
formed by Analytische Laboratorien, Lindlar, Germany
or by MikroKemi AB, Uppsala, Sweden. NMR spectra
were recorded on a Varian Mercury plus spectrometer
(1H at 399.8 MHz, 13C at 100.5 MHz) at ambient temper-
ature. Chemical shifts (d) are reported in ppm referenced
indirectly to TMS via the solvent signals (1H: CHCl3 d
7.26, CHD2OD d 3.31; 13C: CDCl3 d 77.16, CD3OD d
49.00). Microwave irradiated reactions were carried out
in a SmithSyntheSizerTM or in an InitiatorTM single-mode
microwave cavity producing controlled irradiation at
2450 MHz (Biotage AB, Uppsala, Sweden).
11.88 (br s, NH), 8.64 (d, J = 8.7 Hz, 1H), 7.96–7.94
(m, 2H), 7.89–7.86 (m, 2H), 7.50 (d, J = 2.0 Hz, 1H),
7.45 (ddd, J = 1.6, 7.6, 8.7 Hz, 1H), 7.40–7.38 (m, 3H),
7.01–6.96 (m, 2H), 6.80 (s, 1H), 5.30 (d, J = 9.8 Hz,
NH), 5.30–5.29 (m, 1H), 4.83 (dd, J = 7.9, 9.1 Hz,
1H), 4.59 (dm, J = 11.6 Hz, 1H), 4.39 (d, J = 9.8 Hz,
1H), 4.14 (dm, J = 11.6 Hz, 1H), 3.88 (s, 3H), 2.66
(ddm, J = 7.9, 13.8 Hz, 1H), 2.39 (ddd, J = 4.3, 9.1,
13.8 Hz, 1H), 1.37 (s, 9H), 1.09 (s, 9H). 13C NMR
(CDCl3): d (10:1 mixture of rotamers, major rotamer re-
ported) 172.2, 171.1, 169.5, 162.1, 160.9, 159.1, 156.2,
149.8, 141.4, 138.8, 134.1, 131.6, 129.8, 128.8, 128.1,
123.4, 122.8, 120.2, 119.0, 116.6, 115.2, 105.9, 98.7,
80.1, 76.7, 60.9, 59.1, 55.8, 54.5, 35.6, 35.0, 28.4, 26.6.
MS [M+H]+ 697.5. HPLC purity: C18 column 99%,
C4 column >99%. Anal. Calcd for C39H44N4O8Æ1/
2H2O: C, 66.37; H, 6.43; N, 7.94. Found: C, 66.58; H,
6.57; N, 8.06.
5.1.2.2. Compound 3. Prepared according to the gen-
eral procedure described above. Amide bond formation:
1 (40 mg, 0.069 mmol), 3-aminobenzoic acid ethyl ester
(17 mg, 0.10 mmol), HATU (32 mg, 0.084 mmol), DIEA
(45 lL, 0.26 mmol) and CH2Cl2 (2 mL). Reaction time:
3 h. Evaporation and purification by column chroma-
tography (EtOAc:i-hexane 1:2) gave the ester intermedi-
ate as a white solid (46 mg). Ester hydrolysis: ester
intermediate (25 mg, 0.034 mmol), LiOH (3.6 mg,
0.15 mmol), H2O (0.41 mL), THF (0.95 mL) and MeOH
(0.14 mL). Reaction time: overnight. Extraction: EtOAc
(3 · 5 mL). Purification by preparative HPLC–MS gave
5.1.2. General procedure for the synthesis of compounds
2–5. Amide bond formation: a mixture of 1, aminobenzo-
ic acid ester or 2-aminophenylacetic acid methyl ester,
HATU, DIEA and CH2Cl2 was stirred in a sealed reac-
tion tube under N2-atmosphere at 45 ꢁC for 3–26 h.
Aqueous workup and/or purification by column chro-
matography gave the corresponding ester intermediates
that were hydrolyzed in the next step without further
characterization. Ester hydrolysis: a solution of LiOH
in H2O was added to a solution of the ester intermediate
in THF and MeOH and the resulting mixture was stir-
red at room temperature. The reaction mixture was
thereafter neutralized with 1.0 M HCl (aq) and the or-
ganic solvents were evaporated. The remaining aqueous
phase was acidified to pH 4 using 1.0 M HCl (aq) and
extracted three times with EtOAc. The combined organ-
ic layer was washed with brine, dried (MgSO4), filtered
and evaporated. Purification by preparative HPLC–
MS or column chromatography gave the desired car-
boxylic acids 2–5.
1
compound 3 (17 mg, 65%) as a white solid. H NMR
(CDCl3): d (10:1 mixture of rotamers, major rotamer re-
ported) 9.46 (br s, NH), 8.11 (dm, J = 8.2 Hz, 1H), 8.08–
8.03 (m, 4H), 7.78 (dm, J = 7.8 Hz, 1H), 7.51–7.42 (m,
4H), 7.38 (dd, J = 7.8, 8.2 Hz, 1H), 7.05 (dd, J = 2.5,
9.1 Hz, 1H), 7.03 (s, 1H), 5.66 (d, J = 9.4 Hz, NH),
5.43 (m, 1H), 5.04 (t, J = 7.4 Hz, 1H), 4.56 (dm,
J = 11.6 Hz, 1H), 4.41 (d, J = 9.4 Hz, 1H), 4.09 (dd,
J = 4.9, 11.6 Hz, 1H), 3.93 (s, 3H), 3.01–2.95 (m, 1H),
2.61–2.55 (m, 1H), 1.42 (s, 9H), 1.02 (s, 9H). 13C
NMR (CDCl3): d (10:1 mixture of rotamers, major rot-
amer reported) 173.5, 169.4, 168.6, 161.6, 160.4, 159.3,
156.2, 140.1, 138.2, 131.0, 129.5, 129.3, 128.9, 127.8,
125.8, 124.5, 123.3, 121.4, 118.7, 115.2, 107.2, 104.9,
98.2, 80.1, 76.3, 59.9, 59.5, 55.7, 54.4, 35.5, 33.1, 28.4,
26.6. MS [M+H]+ 697.5. HPLC purity: C18 column
>99%, C4 column >99%. Anal. Calcd for
C39H44N4O8Æ1/2H2O: C, 66.37; H, 6.43; N, 7.94. Found:
C, 66.12; H, 6.54; N, 7.79.
5.1.2.1. Compound 2. Prepared according to the gen-
eral procedure described above. Amide bond formation:
1 (40 mg, 0.069 mmol), 2-aminobenzoic acid methyl es-
ter (16 mg, 0.11 mmol), HATU (32 mg, 0.084 mmol),
DIEA (45 lL, 0.26 mmol) and CH2Cl2 (2 mL). Reaction
time: 26 h. Evaporation and purification by column
chromatography (gradient elution, EtOAc:i-hexane
1:2–2:3) gave the ester intermediate as a white solid
(22 mg). Ester hydrolysis: ester intermediate (21 mg,
0.030 mmol), LiOH (2.8 mg, 0.12 mmol), H2O
(0.41 mL), THF (0.95 mL) and MeOH (0.14 mL). Reac-
tion time: overnight. Extraction: EtOAc (3 · 5 mL).
Purification by preparative HPLC–MS gave compound
5.1.2.3. Compound 4. Prepared according to the gen-
eral procedure described above. Amide bond formation:
1 (40 mg, 0.069 mmol), 4-aminobenzoic acid methyl es-
ter (16 mg, 0.11 mmol), HATU (32 mg, 0.084 mmol),
DIEA (45 lL, 0.26 mmol) and CH2Cl2 (2 mL). Reaction
time: 3.5 h. Evaporation and purification by column
chromatography (EtOAc:i-hexane 1:2) gave the ester
intermediate as a white solid (40 mg). Ester hydrolysis:
Ester intermediate (38 mg, 0.053 mmol), LiOH (5.0 mg,
0.21 mmol), H2O (0.41 mL), THF (0.95 mL) and MeOH
(0.14 mL). Reaction time: overnight. Extraction: EtOAc
(3 · 5 mL). Purification by preparative HPLC–MS gave
1
2 (16 mg, 35%) as a white solid. H NMR (CDCl3): d
(10:1 mixture of rotamers, major rotamer reported)

R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
2961
1
compound 4 (27 mg, 59%) as a white solid. H NMR
(CDCl3): d (10:1 mixture of rotamers, major rotamer re-
ported) 9.94 (br s, NH), 8.06–8.03 (m, 2H), 7.85–7.83
(m, 2H), 7.77 (d, J = 9.1 Hz, 1H), 7.53–7.46 (m, 5H),
7.33 (d, J = 2.3 Hz, 1H), 6.97 (s, 1H), 6.92 (dd, J = 2.3,
9.1 Hz, 1H), 5.39 (m, 1H), 5.31 (d, J = 9.5 Hz, NH),
5.10 (t, J = 7.7 Hz, 1H), 4.52–4.48 (m, 2H), 4.19–4.15
(m, 1H), 3.81 (s, 3H), 2.92–2.86 (m, 1H), 2.58–2.52 (m,
1H), 1.23 (s, 9H), 1.09 (s, 9H). 13C NMR (CDCl3): d
(10:1 mixture of rotamers, major rotamer reported)
173.1, 170.8, 169.1, 161.4, 160.2, 159.1, 155.6, 151.0,
142.7, 140.1, 131.1, 129.5, 128.8, 127.9, 125.1, 122.4,
118.8, 118.7, 114.7, 107.0, 97.9, 80.1, 76.1, 60.2, 59.1,
55.5, 54.1, 36.3, 33.8, 28.3, 26.6. MS [M+H]+ 697.6.
HPLC purity: C18 column >99%, C4 column >99%.
Anal. Calcd for C39H44N4O8Æ1/2H2O: C, 66.37; H,
6.43; N, 7.94. Found: C, 66.68; H, 6.42; N, 7.82.
5.1.3.1. Compound 6. Prepared according to the gen-
eral procedure described above using: 1 (150 mg,
0.260 mmol), 2-bromoaniline (42 lL, 0.385 mmol),
HATU (120 mg, 0.316 mmol), DIEA (167 lL,
0.958 mmol) and CH2Cl2 (5 mL). Reaction time: 28 h.
Repeated purification by column chromatography (gra-
dient elution, EtOAc:i-hexane 1:2–2:3) gave compound
1
6 (146 mg, 77%) as a white solid. H NMR (CD3OD):
d (10:1 mixture of rotamers, major rotamer reported)
8.09 (d, J = 9.2 Hz, 1H), 8.05–8.02 (m, 2H), 7.74 (dd,
J = 1.6, 8.1 Hz, 1H), 7.57 (dd, J = 1.5, 8.1 Hz, 1H),
7.54–7.47 (m, 3H), 7.37 (d, J = 2.6 Hz, 1H), 7.32–7.28
(m, 1H), 7.17 (s, 1H), 7.09–7.02 (m, 2H), 6.45 (d,
J = 8.9 Hz, NH), 5.45–5.44 (m, 1H), 4.95 (dd, J = 8.0,
9.1 Hz, 1H), 4.55 (dm, J = 11.9 Hz, 1H), 4.30–4.27 (m,
1H), 4.01 (dm, J = 11.9 Hz, 1H), 3.92 (s, 3H), 2.75
(ddm, J = 8.0, 14.0 Hz, 1H), 2.51 (ddd, J = 4.2, 9.1,
14.0 Hz, 1H), 1.30 (s, 9H), 1.04 (s, 9H). 13C NMR
(CDCl3): d (10:1 mixture of rotamers, major rotamer re-
ported) 172.9, 168.5, 161.6, 160.4, 159.4, 156.0, 151.7,
140.5, 136.1, 132.8, 129.5, 129.0, 128.4, 127.8, 125.8,
123.3, 122.4, 118.7, 115.3, 113.9, 107.7, 98.2, 80.1,
76.3, 60.0, 58.9, 55.8, 54.0, 35.8, 32.7, 28.5, 26.6. MS
[M+H]+ 731.2, 733.2. HPLC purity: C18 column
>99%, C4 column >99%. Anal. Calcd for
C38H43BrN4O6: C, 62.38; H, 5.92; N, 7.66. Found: C,
62.07; H, 6.11; N, 7.50.
5.1.2.4. Compound 5. Prepared according to the gen-
eral procedure described above. Amide bond formation:
1 (60 mg, 0.10 mmol), 2-aminophenylacetic acid methyl
ester40 (26 mg, 0.16 mmol), HATU (47 mg, 0.12 mmol),
DIEA (67 lL, 0.38 mmol) and CH2Cl2 (2 mL). Reaction
time: 15 h. The mixture was diluted with EtOAc (15 mL)
and washed with aqueous NaOAc buffer (pH 4,
2 · 8 mL), 5% aqueous NaHCO3 (8 mL) and brine
(8 mL). The organic layer was dried (MgSO4), filtered
and evaporated. Purification by column chromatogra-
phy (EtOAc:i-hexane 2:3) gave the ester intermediate
as a white solid (60 mg). Ester hydrolysis: ester interme-
diate (60 mg, 0.083 mmol), LiOH (30 mg, 1.3 mmol),
H2O (1.1 mL), THF (2.4 mL) and MeOH (0.3 mL).
Reaction time: 1 h. Extraction: EtOAc (2 · 9 mL). Puri-
fication by column chromatography (gradient elution:
CH2Cl2:MeOH:HCOOH 96:4:0.2–94:6:0.2) gave com-
pound 5 (53 mg, 72%) as a white solid. 1H NMR
(CD3OD): d (10:1 mixture of rotamers, major rotamer
reported) 8.15 (d, J = 9.2 Hz, 1H), 8.05–8.03 (m, 2H),
7.59–7.53 (m, 3H), 7.45 (dd, J = 1.5, 7.9 Hz, 1H), 7.39
(d, J = 2.5 Hz, 1H), 7.29–7.23 (m, 2H), 7.28 (s, 1H),
7.19–7.15 (m, 1H), 7.11 (dd, J = 2.5, 9.2 Hz, 1H), 6.51
(d, J = 8.9 Hz, NH), 5.57–5.56 (m, 1H), 4.84 (dd,
J = 7.4, 10.0 Hz, 1H), 4.60 (dm, J = 12.0 Hz, 1H),
4.27–4.25 (m, 1H), 4.07 (dd, J = 3.5, 12.0 Hz, 1H),
3.93 (s, 3H), 3.78 (d, J = 15.6 Hz, 1H), 3.58 (d,
J = 15.6 Hz, 1H), 2.81 (ddm, J = 7.4, 14.1 Hz, 1H),
2.51 (ddd, J = 4.2, 10.0 14.1 Hz, 1H), 1.28 (s, 9H), 1.04
(s, 9H). 13C NMR (CD3OD): d (10:1 mixture of rota-
mers, major rotamer reported) 176.0, 173.5, 172.5,
163.8, 163.2, 160.5, 158.0, 150.2, 139.5, 137.1, 131.9,
131.7, 131.3, 130.0, 129.2, 128.6, 127.5, 127.0, 124.8,
119.9, 116.4, 105.8, 100.5, 80.4, 78.9, 61.1, 60.7, 56.2,
55.3, 39.4, 36.1, 35.9, 28.6, 27.0. MS [M+H]+ 711.3.
HPLC purity: C18 column >99%, C4 column >99%.
Anal. Calcd for C40H46N4O8Æ1/2H2O: C, 66.74; H,
6.58; N, 7.78. Found: C, 67.05; H, 6.56; N, 7.38.
5.1.3.2. Compound 7. Prepared according to the gen-
1 (50 mg,
eral procedure described above using:
0.087 mmol), 3-bromoaniline (14 lL, 0.13 mmol),
HATU (41 mg, 0.11 mmol), DIEA (56 lL, 0.32 mmol)
and CH2Cl2 (2.5 mL). Reaction time: 3 h. Purification
by column chromatography (EtOAc:i-hexane 1:2) gave
1
compound 7 (40 mg, 63%) as a white solid. H NMR
(CD3OD): d (10:1 mixture of rotamers, major rotamer
reported) 8.08 (d, J = 9.1 Hz, 1H), 8.05–8.02 (m, 2H),
7.90–7.89 (m, 1H), 7.55–7.43 (m, 4H), 7.37 (d,
J = 2.5 Hz, 1H), 7.20–7.16 (m, 3H), 7.04 (dd, J = 2.5,
9.1 Hz, 1H), 6.50 (d, J = 8.9 Hz, NH), 5.50–5.49 (m,
1H), 4.77 (dd, J = 7.5, 10.0 Hz, 1H), 4.58 (dm,
J = 11.7 Hz, 1H), 4.30–4.27 (m, 1H), 4.07 (dm,
J = 11.7 Hz, 1H), 3.92 (s, 3H), 2.74 (ddm, J = 7.5,
14.0 Hz, 1H), 2.41 (ddd, J = 3.9, 10.0, 14.0 Hz, 1H),
1.28 (s, 9H), 1.06 (s, 9H). 13C NMR (CD3OD:CDCl3
1:1): d (10:1 mixture of rotamers, major rotamer re-
ported) 173.5, 168.0, 161.5, 160.3, 159.3, 155.8, 151.6,
140.4, 139.2, 130.4, 129.5, 129.0, 127.7, 127.5, 123.1,
122.9, 118.6, 118.3, 115.2, 107.6, 104.9, 98.1, 80.2,
76.0, 59.3, 59.0, 55.6, 54.0, 35.5, 31.9, 28.4, 26.6. MS
[M+H]+ 731.2, 733.2. HPLC purity: C18 column
>99%, C4 column >99%. Anal. Calcd for
C38H43BrN4O6: C, 62.38; H, 5.92; N, 7.66. Found: C,
62.22; H, 6.04; N, 7.57.
5.1.3.3. Compound 8. Prepared according to the gen-
eral procedure described above using:
1 (50 mg,
5.1.3. General procedure for the synthesis of compounds
6–8. A mixture of 1, bromoaniline, HATU, DIEA and
CH2Cl2 was stirred in a sealed reaction tube under N2-
atmosphere at 45 ꢁC for 3–28 h. Evaporation and purifi-
cation by column chromatography gave the desired
compounds 6–8.
0.087 mmol), 4-bromoaniline (22 mg, 0.13 mmol),
HATU (41 mg, 0.11 mmol), DIEA (56 lL, 0.32 mmol)
and CH2Cl2 (2.5 mL). Reaction time: 4 h. Purification
by column chromatography (EtOAc:i-hexane 3:7) gave
compound 8 (52 mg, 82%) as a white solid.1H NMR
(CD3OD): d (10:1 mixture of rotamers, major rotamer

2962
R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
reported) 8.08 (d, J = 9.1 Hz, 1H), 8.05–8.02 (m, 2H),
7.54–7.47 (m, 5H), 7.39–7.37 (m, 3H), 7.19 (s, 1H),
7.04 (dd, J = 2.5, 9.1 Hz, 1H), 6.48 (d, J = 8.9 Hz,
NH), 5.49–5.47 (m, 1H), 4.77 (dd, J = 7.4, 10.0 Hz,
1H), 4.58 (dm, J = 11.6 Hz, 1H), 4.29–4.27 (m, 1H),
4.05 (dm, J = 11.6 Hz, 1H), 3.92 (s, 3H), 2.73 (ddm,
J = 7.4, 14.0 Hz, 1H), 2.40 (ddd, J = 4.0, 10.0, 14.0 Hz,
1H), 1.28 (s, 9H), 1.05 (s, 9H). 13C NMR (CDCl3): d
(10:1 mixture of rotamers, major rotamer reported)
173.4, 168.1, 161.5, 160.2, 159.3, 155.8, 151.6, 140.4,
137.0, 132.1, 129.4, 128.9, 127.7, 123.1, 121.3, 118.6,
117.1, 115.1, 107.5, 98.1, 80.2, 76.0, 59.4, 59.0, 55.6,
54.0, 35.6, 32.0, 28.4, 26.5. MS [M+H]+ 731.2, 733.2.
HPLC purity: C18 column >99%, C4 column >99%.
Anal. Calcd for C38H43BrN4O6Æ1/2H2O: C, 61.62; H,
5.99; N, 7.56. Found: C, 61.83; H, 6.12; N, 7.54.
5.1.4.2. Compound 10. Prepared according to the gen-
eral procedure described above using: Herrmann’s palla-
dacycle (1.5 mg, 0.0016 mmol), [(tBu)3PH]BF4 (0.94 mg,
0.0032 mmol), 7 (24 mg, 0.033 mmol), benzenesulfon-
amide (15 mg, 0.095 mmol), Mo(CO)6 (8.6 mg,
0.033 mmol), 1,4-dioxane (0.5 mL) and DBU (15 lL,
0.10 mmol). Purification by column chromatography
(CH2Cl2:MeOH 96:4) gave compound 10 (12 mg, 44%)
1
as a white solid. H NMR (CD3OD): d (10:1 mixture
of rotamers, major rotamer reported) 8.18 (d,
J = 9.2 Hz, 1H), 8.07–8.03 (m, 4H), 8.00–7.99 (m, 1H),
7.74 (ddd, J = 0.9, 2.2, 8.1 Hz, 1H), 7.63–7.51 (m, 7H),
7.42 (d, J = 2.4 Hz, 1H), 7.36–7.32 (m, 2H), 7.14 (dd,
J = 2.4, 9.2 Hz, 1H), 6.51 (d, J = 8.9 Hz, NH), 5.64–
5.62 (m, 1H), 4.81 (dd, J = 7.2, 10.0 Hz, 1H), 4.64
(dm, J = 12.0 Hz, 1H), 4.27–4.25 (m, 1H), 4.10 (dm,
J = 12.0 Hz, 1H), 3.97 (s, 3H), 2.82 (ddm, J = 7.2,
13.9 Hz, 1H), 2.46 (ddd, J = 4.0, 10.0, 13.9 Hz, 1H),
1.27 (s, 9H), 1.05 (s, 9H). 13C NMR (CD3OD): d (10:1
mixture of rotamers, major rotamer reported) 173.5,
171.9, 169.5, 163.9, 163.4, 160.7, 158.1, 150.3, 142.3,
139.9, 139.5, 136.0, 134.1, 131.3, 130.0, 129.8, 129.2,
129.0, 125.1 (two signals), 124.9, 120.9, 119.9, 116.5,
105.8, 100.6, 80.5, 79.0, 61.3, 60.9, 56.2, 55.4, 36.2,
35.9, 28.7, 28.6, 27.0. MS [M+H]+ 836.3. HPLC purity:
C18 column >99%, C4 column >99%. Anal. Calcd for
C45H49N5O9SÆ1/2H2O: C, 63.96; H, 5.96; N, 8.29.
Found: C, 63.64; H, 6.13; N, 8.08.
5.1.4. General procedure for the synthesis of compounds
9–17, 20 and 21. A microwave process vial was charged
with Herrmann’s palladacycle (trans-di(l-acetato)bis[o-
(di-o-tolylphosphino)benzyl]dipalladium(II)), [(t-Bu)3-
PH]BF4, aryl bromide 6, 7, 8, 18 or 19, the appropriate
sulfonamide and Mo(CO)6. 1,4-Dioxane followed by
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) was added
and the process vial was thereafter sealed and rigor-
ously stirred before it was exposed to microwave irra-
diation for 15 min at 140 ꢁC. The process vial was
cooled to room temperature before it was diluted with
CH2Cl2 and filtered through a plug of cotton. Purifi-
cation by column chromatography and/or preparative
HPLC–MS gave the desired compounds 9–17, 20
and 21.
5.1.4.3. Compound 11. Prepared according to the gen-
eral procedure described above using: Herrmann’s palla-
dacycle (2.7 mg, 0.0029 mmol), [(tBu)3PH]BF4 (1.5 mg,
0.0052 mmol), 8 (38 mg, 0.052 mmol), benzenesulfon-
amide (29 mg, 0.18 mmol), Mo(CO)6 (14 mg,
0.053 mmol), 1,4-dioxane (0.6 mL) and DBU (23 lL,
0.15 mmol). Purification by column chromatography
(CH2Cl2:MeOH 96:4) gave compound 11 (15 mg, 35%)
5.1.4.1. Compound 9. Prepared according to the gen-
eral procedure described above using: Herrmann’s palla-
dacycle (2.5 mg, 0.0027 mmol), [(t-Bu)3PH]BF4 (1.6 mg,
0.0055 mmol), 6 (40 mg, 0.055 mmol), benzenesulfon-
amide (45 mg, 0.29 mmol), Mo(CO)6 (16 mg,
0.061 mmol), 1,4-dioxane (0.3 mL) and DBU (24 lL,
0.16 mmol). Purification by column chromatography
(gradient elution, CH2Cl2:MeOH 97:3–95:5) followed
by preparative HPLC–MS gave compound 9 (16 mg,
1
as a white solid. H NMR (CD3OD): d (10:1 mixture
of rotamers, major rotamer reported) 8.17 (d,
J = 9.2 Hz, 1H), 8.07–8.04 (m, 4H), 7.81–7.79 (m, 2H),
7.67–7.60 (m, 3H), 7.57–7.52 (m, 5H), 7.42 (d, J = 2.5
Hz, 1H), 7.33 (s, 1H), 7.12 (dd, J = 2.5, 9.2 Hz, 1H),
6.49 (d, J = 8.9 Hz, NH), 5.63–5.61 (m, 1H), 4.81 (dd,
J = 7.3, 9.9 Hz, 1H), 4.64 (dm, J = 12.1 Hz, 1H), 4.27–
4.25 (m, 1H), 4.10 (dm, J = 12.1 Hz, 1H), 3.96 (s, 3H),
2.80 (ddm, J = 7.3, 14.0 Hz, 1H), 2.45 (ddd, J = 4.0,
9.9, 14.0 Hz, 1H), 1.27 (s, 9H), 1.05 (s, 9H). 13C NMR
(CD3OD): d (10:1 mixture of rotamers, major rotamer
reported) 173.5, 172.0, 168.6, 163.8, 163.1, 160.9,
158.0, 150.7, 144.1, 142.2, 134.2, 131.2, 130.5, 130.0,
129.8, 129.6, 129.2, 129.1, 124.8, 120.1 (two signals),
119.8, 116.5, 106.1, 100.5, 80.5, 78.8, 61.4, 60.8, 56.2,
55.4, 36.1, 35.9, 28.6, 27.0. MS [M+H]+ 836.3. HPLC
purity: C18 column >99%, C4 column >99%. Anal.
Calcd for C45H49N5O9SÆH2O: C, 63.29; H, 6.02; N,
8.20. Found: C, 62.98; H, 5.98; N, 7.95.
1
35%) as a white solid. H NMR (CD3OD): d (10:1 mix-
ture of rotamers, major rotamer reported) 8.33 (d,
J = 9.2 Hz, 1H), 8.22 (dd, J = 1.0, 8.3 Hz, 1H), 8.07–
8.04 (m, 2H), 7.96–7.94 (m, 2H), 7.90 (dd, J = 1.6,
8.0 Hz, 1H), 7.64–7.58 (m, 3H), 7.48 (s, 1H), 7.44–7.35
(m, 5H), 7.23 (dd, J = 2.5, 9.2 Hz, 1H), 7.07–7.02 (m,
1H), 5.71–5.69 (m, 1H), 4.75 (dd, J = 7.4, 10.1 Hz,
1H), 4.66 (dm, J = 12.0 Hz, 1H), 4.23 (s, 1H), 4.18
(dm, J = 12.0 Hz, 1H), 3.97 (s, 3H), 2.93 (ddm,
J = 7.4, 14.0 Hz, 1H), 2.46 (ddd, J = 4.0, 10.0, 14.0 Hz,
1H), 1.25 (s, 9H), 1.03 (s, 9H). 13C NMR
(CD3OD:CDCl3 1:1): d (10:1 mixture of rotamers, major
rotamer reported) 172.7, 171.7, 170.3, 164.6, 164.3,
158.8, 157.1, 146.6, 143.2, 139.4, 135.9, 132.7, 132.3,
131.9, 130.9, 129.8, 129.7, 128.9 (two signals), 125.1,
123.9, 123.7, 121.4, 120.4, 115.6, 102.8, 100.7, 80.2,
79.3, 61.6, 59.8, 56.3, 54.6, 35.6, 35.5, 28.5, 26.7. MS
[M+H]+ 836.2. HPLC purity: C18 column >99%, C4
column >99%. Anal. Calcd for C45H49N5O9SÆH2O: C,
63.29; H, 6.02; N, 8.20. Found: C, 63.40; H, 6.10; N,
8.01.
5.1.4.4. Compound 12. Prepared according to the gen-
eral procedure described above using: Herrmann’s palla-
dacycle (2.6 mg, 0.0028 mmol), [(tBu)3PH]BF4 (1.6 mg,
0.0055 mmol), 6 (40 mg, 0.055 mmol), methanesulfon-
amide (26 mg, 0.27 mmol), Mo(CO)6 (14 mg,
0.053 mmol), 1,4-dioxane (0.3 mL) and DBU (25 lL,

R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
2963
0.17 mmol). Purification by column chromatography
(gradient elution, CH2Cl2:MeOH 97:3 to CH2Cl2:
MeOH:HCOOH 96.9:3:0.1) gave compound 12
MS gave compound 14 (26 mg, 56%) as a white solid.
1H NMR (CD3OD): d (10:1 mixture of rotamers, major
rotamer reported) 8.31 (d, J = 9.2 Hz, 1H), 8.22 (dm,
J = 8.0 Hz, 1H), 8.08–8.05 (m, 2H), 7.91 (dm,
J = 7.9 Hz, 1H), 7.64–7.62 (m, 3H), 7.43–7.38 (m, 3H),
7.31–7.28 (m, 2H), 7.24 (dd, J = 2.5, 9.2 Hz, 1H),
7.22–7.18 (m, 3H), 7.10–7.06 (m, 1H), 6.48 (d,
J = 8.8 Hz, NH), 5.60–5.58 (m, 1H), 4.68–4.58 (m,
4H), 4.23–4.18 (m, 2H), 3.99 (s, 3H), 2.71 (ddm,
J = 7.5, 14.1 Hz, 1H), 2.32–2.25 (m, 1H), 1.23 (s, 9H),
1.03 (s, 9H). 13C NMR (CD3OD): d (10:1 mixture of
rotamers, major rotamer reported) 173.5, 173.0, 171.1,
166.0, 165.3, 159.1, 157.9, 146.2, 139.9, 135.9, 133.0,
132.7, 132.3, 131.9, 131.3, 130.4, 129.7, 129.4, 129.1,
126.1, 125.4, 124.2, 122.0, 121.0, 116.2, 102.5, 101.7,
80.4, 80.3, 62.2, 60.8, 58.8, 56.6, 55.0, 36.0, 35.8, 28.5,
27.0. MS [M + H]+ 850.2. HPLC purity: C18 column
>99%, C4 column >99%. Anal. Calcd for C46H51N5O9-
SÆH2O: C, 63.65; H, 6.15; N, 8.07. Found: C, 63.44; H,
6.15; N, 7.90.
1
(21 mg, 50%) as a white solid. H NMR (CD3OD): d
(10:1 mixture of rotamers, major rotamer reported)
8.31 (d, J = 9.2 Hz, 1H), 8.20 (dd, J = 1.0, 8.4 Hz, 1H),
8.08–8.05 (m, 2H), 7.93 (dd, J = 1.6, 7.9 Hz, 1H),
7.65–7.62 (m, 3H), 7.47 (s, 1H), 7.44–7.42 (m, 2H),
7.25 (dd, J = 2.5, 9.2 Hz, 1H), 7.13–7.09 (m, 1H), 6.51
(d, J = 8.9 Hz, NH), 5.71–5.70 (m, 1H), 4.80 (dd,
J = 7.5, 10.2 Hz, 1H), 4.67 (dm, J = 12.3 Hz, 1H),
4.25–4.22 (m, 2H), 4.00 (s, 3H), 3.17 (s, 3H), 2.96
(ddm, J = 7.5, 14.3 Hz, 1H), 2.58 (ddd, J = 4.0, 10.2,
14.3 Hz, 1H), 1.23 (s, 9H), 1.04 (s, 9H). 13C NMR
(CD3OD): d (10:1 mixture of rotamers, major rotamer
reported) 173.6, 172.8, 171.4, 165.8, 165.2, 159.4,
158.1, 146.8, 139.8, 136.5, 133.2, 132.6, 131.3, 130.4,
129.6, 126.0, 125.6, 124.4, 122.3, 121.0, 116.4, 103.0,
101.6, 80.4, 80.3, 62.3, 60.9, 56.6, 55.2, 41.2, 36.2, 35.9,
28.6, 27.0. MS [M+H]+ 774.3. HPLC purity: C18 col-
umn >99%, C4 column >99%. Anal. Calcd for
C40H47N5O9SÆ1/2H2O: C, 61.36; H, 6.18; N, 8.95.
Found: C, 61.22; H, 6.41; N, 8.70.
5.1.4.7. Compound 15. Prepared according to the gen-
eral procedure described above using: Herrmann’s palla-
dacycle (2.2 mg, 0.0023 mmol), [(tBu)3PH]BF4 (1.4 mg,
0.0048 mmol), 6 (35 mg, 0.048 mmol), 4-methoxyben-
zenesulfonamide (45 mg, 0.24 mmol), Mo(CO)6 (13 mg,
0.049 mmol), 1,4-dioxane (0.3 mL) and DBU (21 lL,
0.14 mmol). Filtration of the crude product through a
short plug of silica gel (CH2Cl2:MeOH 95:5), purifica-
tion by column chromatography (gradient elution,
CH2Cl2:MeOH 97:3–95:5) followed by preparative
HPLC–MS gave compound 15 (18 mg, 43%) as a white
5.1.4.5. Compound 13. Prepared according to the gen-
eral procedure described above using: Herrmann’s palla-
dacycle (2.9 mg, 0.0031 mmol), [(tBu)3PH]BF4 (1.6 mg,
0.0055 mmol), 6 (40 mg, 0.055 mmol), cyclopropylsulf-
onamide (33 mg, 0.27 mmol), Mo(CO)6 (15 mg,
0.057 mmol), 1,4-dioxane (0.3 mL) and DBU (25 lL,
0.17 mmol). Purification by column chromatography
(gradient elution, CH2Cl2:MeOH 98:2–95:5) followed
by preparative HPLC–MS gave compound 13 (27 mg,
1
solid. H NMR (CD3OD): d (10:1 mixture of rotamers,
1
62%) as a white solid. H NMR (CD3OD): d (10:1 mix-
major rotamer reported) 8.27 (d, J = 9.3 Hz, 1H), 8.19
(dm, J = 8.2 Hz, 1H), 8.06–8.04 (m, 2H), 7.90–7.86 (m,
3H), 7.59–7.54 (m, 3H), 7.44 (s, 1H), 7.40–7.36 (m,
1H), 7.39 (d, J = 2.6 Hz, 1H), 7.19 (dd, J = 2.6, 9.3 Hz,
1H), 7.06 (ddm, J = 7.5, 7.7 Hz, 1H), 6.92–6.89 (m,
2H), 6.55 (d, J = 8.6 Hz, NH), 5.66–5.64 (m, 1H), 4.75
(dd, J = 7.7, 10.1 Hz, 1H), 4.64 (dm, J = 12.1 Hz, 1H),
4.23–4.21 (m, 1H), 4.15 (ddm, J = 3.2, 12.1 Hz, 1H),
3.95 (s, 3H), 3.74 (s, 3H), 2.90 (ddm, J = 7.7, 14.0 Hz,
1H), 2.45 (ddd, J = 4.1, 10.1, 14.0 Hz, 1H), 1.24 (s,
9H), 1.00 (s, 9H). 13C NMR (CD3OD): d (10:1 mixture
of rotamers, major rotamer reported) 172.7, 170.5,
170.4, 163.9, 163.4, 159.3, 157.0, 147.7, 139.3, 137.0,
133.7, 133.2, 131.5, 131.2, 130.6, 130.4, 129.7, 128.8,
124.8, 123.9, 123.1, 121.8, 120.1, 115.6, 114.2, 103.7,
100.4, 80.3, 78.7, 61.4, 59.7, 56.2, 55.9, 54.5, 35.6, 35.4,
28.5, 26.7. MS [M+H]+ 866.3. HPLC purity: C18 col-
umn >99%, C4 column >99%. Anal. Calcd for
C46H51N5O10SÆH2O: C, 62.50; H, 6.04; N, 7.92. Found:
C, 62.50; H, 6.00; N, 7.75.
ture of rotamers, major rotamer reported) 8.26 (d,
J = 9.2 Hz, 1H), 8.22 (dd, J = 1.0, 8.2 Hz, 1H), 8.07–
8.05 (m, 2H), 7.89 (dd, J = 1.6, 7.9 Hz, 1H), 7.62–7.59
(m, 3H), 7.42–7.40 (m, 3H), 7.20 (dd, J = 2.4, 9.2 Hz,
1H), 7.12–7.08 (m, 1H), 6.51 (d, J = 8.9 Hz, NH),
5.68–5.67 (m, 1H), 4.77 (dd, J = 7.6, 10.1 Hz, 1H),
4.64 (dm, J = 12.1 Hz, 1H), 4.25–4.23 (m, 1H), 4.20
(dm, J = 12.1 Hz, 1H), 3.98 (s, 3H), 3.05–2.98 (m, 1H),
2.92 (ddm, J = 7.6, 14.0 Hz, 1H), 2.55 (ddd, J = 4.1,
10.1, 14.0 Hz, 1H), 1.24 (s, 9H), 1.12–1.08 (m, 2H),
1.04 (s, 9H), 0.95–0.90 (m, 2H). 13C NMR (CD3OD):
d (10:1 mixture of rotamers, major rotamer reported)
173.5, 172.2, 171.2, 165.2, 164.9, 159.4, 158.0, 147.3,
139.8, 136.9, 133.2, 132.3, 131.1, 130.3, 129.6, 125.7,
125.2, 124.4, 122.2, 120.7, 116.3, 103.4, 101.4, 80.3,
80.0, 62.2, 60.8, 56.5, 55.1, 36.2, 35.8, 31.5, 28.6, 27.0,
5.9. MS [M + H]+ 800.3. HPLC purity: C18 column
>99%, C4 column >99%. Anal. Calcd for C42H49N5O9-
SÆH2O: C, 61.67; H, 6.28; N, 8.56. Found: C, 61.49; H,
6.35; N, 8.40.
5.1.4.8. Compound 16. Prepared according to the gen-
eral procedure described above using: Herrmann’s palla-
dacycle (2.2 mg, 0.0023 mmol), [(tBu)3PH]BF4 (1.4 mg,
5.1.4.6. Compound 14. Prepared according to the gen-
eral procedure described above using: Herrmann’s
palladacycle (2.6 mg, 0.0028 mmol), [(tBu)3PH]BF4
(1.6 mg, 0.0055 mmol), 6 (40 mg, 0.055 mmol), a-tolu-
enesulfonamide (47 mg, 0.27 mmol), Mo(CO)6 (14 mg,
0.053 mmol), 1,4-dioxane (0.3 mL) and DBU (25 lL,
0.17 mmol). Purification by column chromatography
(CH2Cl2:MeOH 95:5) followed by preparative HPLC–
0.0048 mmol),
methyl)benzenesulfonamide
6
(35 mg, 0.048 mmol), 4-(trifluoro-
(54 mg, 0.24 mmol),
Mo(CO)6 (13 mg, 0.049 mmol), 1,4-dioxane (0.3 mL)
and DBU (21 lL, 0.14 mmol). The crude product was
diluted in a total of 15 mL CH2Cl2 and thereafter
washed with aqueous NaOAc buffer (pH 4, 3 · 10 mL)

2964
R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
and brine (10 mL). The organic layer was dried
(MgSO4), filtered and evaporated. Purification by col-
umn chromatography (EtOAc:i-hexane:MeOH 48:48:4)
palladacycle (2.2 mg, 0.0023 mmol), [(tBu)3PH]BF4
(1.4 mg, 0.0048 mmol), 18 (35 mg, 0.047 mmol), ben-
zenesulfonamide (37 mg, 0.24 mmol), Mo(CO)6 (13 mg,
0.049 mmol), 1,4-dioxane (0.3 mL) and DBU (21 lL,
0.14 mmol). The crude product was diluted in a total
of 15 mL CH2Cl2 and thereafter washed with aqueous
NaOAc buffer (pH 4, 3 · 10 mL) and brine (10 mL).
The organic layer was dried (MgSO4), filtered and evap-
orated. Repeated purification by column chromatogra-
phy (EtOAc:i-hexane:MeOH 48:48:4 followed by
CH2Cl2:MeOH 97:3) gave compound 20 (18 mg, 45%)
1
gave compound 16 (18 mg, 42%) as a white solid. H
NMR (CD3OD): d (10:1 mixture of rotamers, major rot-
amer reported) 8.38 (ddm, J = 1.1, 8.4 Hz, 1H), 8.17 (d,
J = 9.2 Hz, 1H), 8.13–8.11 (m, 2H), 8.08–8.02 (m, 3H),
7.72–7.69 (m, 2H), 7.55–7.47 (m, 3H), 7.41–7.35 (m,
2H), 7.26 (s, 1H), 7.09 (dd, J = 2.6, 9.2 Hz, 1H), 7.05–
7.00 (m, 1H), 6.48 (d, J = 9.0 Hz, NH), 5.57–5.55 (m,
1H), 4.78 (dd, J = 7.8, 9.9 Hz, 1H), 4.60 (dm,
J = 11.9 Hz, 1H), 4.29–4.23 (m, 2H), 3.94 (s, 3H), 2.90
(ddm, J = 7.8, 14.1 Hz, 1H), 2.55 (ddd, J = 3.8, 9.9,
14.1 Hz, 1H), 1.22 (s, 9H), 0.99 (s, 9H). 13C NMR
(CD3OD): d (10:1 mixture of rotamers, major rotamer
reported) 174.4, 173.6, 171.5, 163.4, 162.5, 161.2,
157.9, 151.6, 149.1, 140.9, 140.7, 133.7 (q,
2JCF = 32 Hz), 132.9, 132.1, 130.7, 129.8, 129.2, 128.9,
1
as a white solid. H NMR (CD3OD): d (9:1 mixture of
rotamers, major rotamer reported) 8.14 (d, J = 9.2 Hz,
1H), 8.06–8.04 (m, 2H), 7.91–7.88 (m, 2H), 7.58–7.50
(m, 4H), 7.40 (d, J = 2.5 Hz, 1H), 7.39–7.34 (m, 3H),
7.22 (dm, J = 7.6 Hz, 1H), 7.12 (s, 1H), 7.07 (dd,
J = 2.5, 9.2 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 5.41–5.39
(m, 1H), 4.76 (t, J = 8.1 Hz, 1H), 4.47 (dm,
J = 12.0 Hz, 1H), 4.36 (s, 1H), 4.15 (dm, J = 12.0 Hz,
1H), 3.96 (s, 3H), 2.38–2.24 (m, 2H), 2.07 (s, 3H), 1.30
(s, 9H), 1.00 (s, 9H). 13C NMR (CD3OD): d (9:1 mixture
of rotamers, major rotamer reported) 175.4, 173.7,
171.8, 163.3, 162.3, 161.2, 157.8, 152.0, 144.3, 141.2,
137.2, 135.1, 134.6, 134.2, 133.0, 130.7, 129.9, 129.6,
129.2, 128.7, 127.7, 127.2, 124.8, 119.5, 116.4, 107.2,
100.2, 80.8, 78.1, 61.0, 60.5, 56.0, 55.4, 36.5, 36.0, 28.6,
27.0, 18.8. MS [M + H]+ 850.2. HPLC purity: C18 col-
umn 98%, C4 column 98%. Anal. Calcd for
C46H51N5O9SÆ2H2O: C, 62.36; H, 6.26; N, 7.90. Found:
C, 62.22; H, 6.10; N, 7.34.
3
1
126.5 (q, JCF = 4 Hz), 125.2, 125.2 (q, JCF = 271 Hz),
124.8, 123.8, 121.4, 119.5, 116.5, 106.8, 100.3, 80.5,
78.5, 62.8, 60.5, 56.0, 55.2, 36.4, 36.0, 28.5, 26.9. MS
[M+H]+ 904.2. HPLC purity: C18 column >99%, C4
column >99%. Anal. Calcd for C46H48F3N5O9SÆ3H2O:
C, 57.67; H, 5.68; N, 7.31. Found: C, 57.02; H, 5.30;
N, 6.91.
5.1.4.9. Compound 17. Prepared according to the gen-
eral procedure described above using: Herrmann’s palla-
dacycle (2.6 mg, 0.0028 mmol), [(tBu)3PH]BF4 (1.6 mg,
0.0055 mmol), 6 (40 mg, 0.055 mmol), 2-thiophenesulf-
onamide (45 mg, 0.28 mmol), Mo(CO)6 (14 mg,
0.053 mmol), 1,4-dioxane (0.3 mL) and DBU (25 lL,
0.17 mmol). The crude product was diluted in a total of
15 mL CH2Cl2 and thereafter washed with aqueous NaO-
Ac buffer (pH 4, 3 · 10 mL) and brine (10 mL). The or-
ganic layer was dried (MgSO4), filtered and evaporated.
Repeated purification by column chromatography (gra-
5.1.4.11. Compound 21. Prepared according to the
general procedure described above using: Herrmann’s
palladacycle (2.2 mg, 0.0023 mmol), [(tBu)3PH]BF4
(1.4 mg, 0.0048 mmol), 19 (38 mg, 0.048 mmol), ben-
zenesulfonamide (37 mg, 0.24 mmol), Mo(CO)6 (13 mg,
0.049 mmol), 1,4-dioxane (0.3 mL) and DBU (21 lL,
0.14 mmol). The crude product was diluted in a total
of 15 mL CH2Cl2 and thereafter washed with aqueous
NaOAc buffer (pH 4, 3 · 10 mL) and brine (10 mL).
The organic layer was dried (MgSO4), filtered and evap-
orated. Purification by column chromatography (EtOA-
c:i-hexane:MeOH 48:48:4) followed by preparative
HPLC–MS gave compound 21 (15 mg, 35%) as a white
solid. 1H NMR (CD3OD:CDCl3 19:1): d (9:1 mixture of
rotamers, major rotamer reported) 8.62 (m, 1H), 8.41 (d,
J = 9.3 Hz, 1H), 8.07–8.04 (m, 3H), 7.96–7.93 (m, 2H),
7.69–7.62 (m, 3H), 7.61 (s, 1H), 7.50–7.44 (m, 2H),
7.43–7.38 (m, 2H), 7.31 (dd, J = 2.4, 9.3 Hz, 1H),
7.31–7.28 (m, 1H), 5.82–5.81 (m, 1H), 4.79 (dd,
J = 7.4, 10.4 Hz, 1H), 4.73 (dm, J = 12.3 Hz, 1H), 4.22
(s, 1H), 4.22 (dm, J = 12.3 Hz, 1H), 4.01 (s, 3H), 3.04
(ddm, J = 7.4, 14.2 Hz, 1H), 2.53 (ddd, J = 3.8, 10.4,
14.2 Hz, 1H), 1.26 (s, 9H), 1.05 (s, 9H). 13C NMR
(CD3OD:CDCl3 19:1): d (9:1 mixture of rotamers, major
rotamer reported) 173.6, 171.0, 170.8, 167.5, 166.2,
158.2, 158.0, 144.1, 143.9, 140.5, 134.1 (q,
2JCF = 32 Hz), 133.7, 133.5, 132.7, 132.2, 130.6, 129.8,
dient
47.5:47.5:5 followed by CH2Cl2:MeOH 97:3–96:4) gave
elution,
EtOAc:i-hexane:MeOH
48:48:4–
1
compound 17 (17 mg, 37%) as a white solid. H NMR
(CD3OD): d (10:1 mixture of rotamers, major rotamer re-
ported) 8.36 (dd, J = 1.3, 8.4 Hz, 1H), 8.16 (d, J = 9.2 Hz,
1H), 8.09 (dd, J = 1.6, 7.9 Hz, 1H), 8.07–8.03 (m, 2H),
7.63 (dd, J = 1.4, 3.7 Hz, 1H), 7.55–7.47 (m, 3H), 7.40–
7.36 (m, 2H), 7.37 (d, J = 2.5 Hz, 1H), 7.21 (s, 1H), 7.09
(dd, J = 2.5, 9.2 Hz, 1H), 7.02 (ddd, J = 1.3, 7.4, 7.9 Hz,
1H), 6.87 (dd, J = 3.7, 4.9 Hz, 1H), 5.51–5.49 (m, 1H),
4.73 (dd, J = 8.0, 9.8 Hz, 1H), 4.58 (dm, J = 11.7 Hz,
1H), 4.29 (s, 1H), 4.25 (dm, J = 11.7 Hz, 1H), 3.93 (s,
3H), 2.81 (ddm, J = 8.0, 14.1 Hz, 1H), 2.51 (ddd,
J = 4.1, 9.8, 14.1 Hz, 1H), 1.25 (s, 9H), 0.98 (s, 9H). 13C
NMR (CD3OD): d (10:1 mixture of rotamers, major rot-
amer reported) 174.4, 173.5, 171.5, 163.3, 162.3, 161.3,
157.9, 151.9, 146.3, 141.1, 140.5, 133.0, 132.2, 131.7,
131.2, 130.7, 129.8, 129.2, 127.5, 125.3, 124.8, 123.9,
121.5, 119.4, 116.5, 107.0, 100.2, 80.5, 78.4, 62.7, 60.5,
56.0, 55.3, 36.3, 36.1, 28.6, 26.9. MS [M+H]+ 842.2.
HPLC purity: C18 column >99%, C4 column >99%.
Anal. Calcd for C43H47N5O9S2Æ3H2O: C, 57.64; H, 5.96;
N, 7.82. Found: C, 57.99; H, 5.19; N, 7.47.
1
129.3, 128.4, 128.3, 126.6, 125.0 (q, JCF = 272 Hz),
3
3
121.7, 120.1 (q, JCF = 4 Hz), 118.3 (q, JCF = 4 Hz),
116.2, 102.4, 100.6, 81.2, 80.3, 62.1, 60.8, 56.8, 55.0,
36.1, 35.7, 28.6, 27.0. MS [M+H]+ 904.2. HPLC purity:
C18 column 98%, C4 column >99%. Anal. Calcd for
5.1.4.10. Compound 20. Prepared according to the
general procedure described above using: Herrmann’s

R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
2965
3
C46H48F3N5O9SÆ11/2H2O: C, 59.34; H, 5.52; N, 7.52.
Found: C, 59.61; H, 5.48; N, 6.97.
1JCF = 271 Hz), 124.4, 124.1 (q, JCF = 4 Hz), 123.3 (q,
3JCF = 4 Hz), 121.9, 119.3, 116.5, 107.4, 99.9, 80.5,
78.1, 60.8, 60.7, 56.0, 55.3, 36.0, 35.6, 28.6, 26.9. MS
[M+H]+ 799.2, 801.2. HPLC purity: C18 column
>99%, C4 column >99%. Anal. Calcd for
C39H42BrF3N4O6: C, 58.58; H, 5.29; N, 7.01. Found:
C, 59.14; H, 5.57; N, 6.42.
5.1.5. General procedure for the synthesis of compounds
18 and 19. A mixture of 1, pyridine and substituted 2-
bromoaniline was cooled to À15 ꢁC whereafter POCl3
was added dropwise over approximately 1 min. The
resulting mixture was stirred at À15 ꢁC for 45 min
and thereafter at room temperature for 1 h. The mix-
ture was cooled to À15 ꢁC and another portion of
POCl3 was added. The mixture was stirred at À15
ꢁC for 30 min and then allowed to reach room tem-
perature. H2O was added and the mixture was there-
after extracted repeatedly with EtOAc. The
combined organic layer was washed with brine, dried
(MgSO4), filtered and evaporated. Purification by col-
umn chromatography (EtOAc:i-hexane 2:3) gave the
desired compounds 18 and 19.
5.2. Enzyme inhibition
The protease activity of the full-length HCV NS3 pro-
tein (protease-helicase/NTPase) was measured using a
FRET-assay as previously described.32,41 In short, 1
nM enzyme was incubated for 10 minutes at 30 ꢁC in
50 mM HEPES, pH 7.5, 10 mM DTT, 40 % glycerol,
0.1 % n-octyl-b-D-glucoside, 3.3 % DMSO with 25 lM
of the peptide cofactor 2K-NS4A (KKGSVVIVGRIV
LSGK) and inhibitor. The reaction was started by the
addition of 0.5 lM substrate (Ac-DED(Edans)EEAbuw-
[COO]ASK(Dabcyl)-NH2) obtained from AnaSpec Inc.
(San Jose, USA). The non-linear regression analysis
was made using Grafit 5.0.8 (Erithacus Software
Limited).
5.1.5.1. Compound 18. Prepared according to the gen-
1 (45 mg,
eral procedure described above using:
0.078 mmol), pyridine (0.25 mL), 2-bromo-6-methylani-
line (10 lL, 0.079 mmol) and POCl3 (8 lL + 4 lL, 0.088
mmol + 0.044 mmol). Compound 18 (44 mg, 76%) was
1
obtained as a white solid. H NMR (CD3OD): d 8.12
5.3. Computational methodology
(d, J = 9.2 Hz, 1H), 8.07–8.05 (m, 2H), 7.56–7.47 (m,
3H), 7.43 (dm, J = 7.8 Hz, 1H), 7.38 (d, J = 2.5 Hz,
1H), 7.23 (s, 1H), 7.21 (dm, J = 7.8 Hz, 1H), 7.08 (t,
J = 7.8 Hz, 1H), 7.06 (dd, J = 2.5, 9.2 Hz, 1H), 6.39 (d,
J = 8.9 Hz, NH), 5.52–5.50 (m, 1H), 4.93 (dd, J = 7.6,
9.8 Hz, 1H), 4.58 (dm, J = 11.7 Hz, 1H), 4.27–4.25 (m,
1H), 4.04 (dm, J = 11.7 Hz, 1H), 3.93 (s, 3H), 2.83
(ddm, J = 7.6, 14.0 Hz, 1H), 2.51 (ddd, J = 4.1, 9.8,
14.0 Hz, 1H), 2.32 (s, 3H), 1.30 (s, 9H), 1.04 (s, 9H).
13C NMR (CD3OD): d 173.4, 172.6, 163.1, 162.0,
161.3, 157.9, 152.2, 141.3, 140.5, 135.4, 131.4, 130.8,
130.6, 129.9, 129.8, 129.0, 124.4, 123.9, 119.3, 116.5,
107.3, 100.0, 80.5, 78.2, 60.7, 60.4, 56.0, 55.3, 36.2,
36.0, 28.6, 26.9, 19.3. MS [M + H]+ 745.2, 747.2. HPLC
purity: C18 column >99%, C4 column >99%. Anal.
Calcd for C39H45BrN4O6: C, 62.82; H, 6.08; N, 7.51.
Found: C, 62.98; H, 6.32; N, 6.88.
The FLO+ docking suite42 was used for all flexible
docking calculations. FLO+ was used partially due to
the fact that it allows for protein flexibility, which is a
more accurate representation of protein-ligand interac-
tions.43 The active site used for docking was developed
using the NS3 protease/helicase crystal structure (PDB
code 1CU1). Following constrained minimization, the
active site was formed by extracting all residues within
˚
9 A of the last eleven residues of the C-terminus. This
active site has been used previously36,37,39 and greater
detail regarding its derivation has been published
previously.36
The docking protocol used herein relied on conforma-
tional analysis (limited Monte Carlo perturbation) fol-
lowed by simulated annealing. To retain the
conformation obtained using Monte Carlo, an energy
2
˚
5.1.5.2. Compound 19. Prepared according to the gen-
penalty of 20 kJ/(molA ) was applied in the simulated
annealing step when the similarity distance between
two sequential conformations differed by more than
eral procedure described above using:
0.078 mmol), pyridine (0.25 mL), 2-bromo-5-(trifluoro-
1
(45 mg,
˚
0.2 A. The FLO+ software suite allows control over
methyl)aniline (11 lL, 0.077 mmol) and POCl3 (8 lL +
4 lL, 0.088 mmol
+
0.044 mmol). Compound 19
the flexibility of particular residues in the active site
and Arg155 and Lys136 were allowed full conforma-
tional freedom without energy penalty. However, move-
1
(55 mg, 88%) was obtained as a white solid. H NMR
(CD3OD): d (10:1 mixture of rotamers, major rotamer
reported) 8.19 (d, J = 2.3 Hz, 1H), 8.10 (d, J = 9.1 Hz,
1H), 8.05–8.03 (m, 2H), 7.77 (d, J = 8.4 Hz, 1H), 7.54–
7.47 (m, 3H), 7.38 (d, J = 2.4 Hz, 1H), 7.35 (dd,
J = 2.3, 8.4 Hz, 1H), 7.19 (s, 1H), 7.04 (dd, J = 2.4, 9.1
Hz, 1H), 6.50 (d, J = 9.0 Hz, NH), 5.49–5.47 (m, 1H),
4.97 (dd, J = 7.7, 9.5 Hz, 1H), 4.58 (dm, J = 11.8 Hz,
1H), 4.30–4.27 (m, 1H), 4.01 (ddm, J = 3.5, 11.8 Hz,
1H), 3.92 (s, 3H), 2.77 (ddm, J = 7.7, 14.0 Hz, 1H),
2.53 (ddd, J = 4.2, 9.5, 14.0 Hz, 1H), 1.30 (s, 9H), 1.04
(s, 9H). 13C NMR (CD3OD): d (10:1 mixture of rota-
mers, major rotamer reported) 173.6, 172.3, 163.1,
161.9, 161.2, 157.9, 152.2, 141.3, 138.1, 134.9, 131.3 (q,
2JCF = 33 Hz), 130.6, 129.8, 129.0, 125.1 (q,
˚
ment of all other active site residues by more than 0.2 A
2
was penalized by 20 kJ/(molA ). Docking in the NS3
˚
protease/helicase is complicated by the shallow and fea-
tureless nature of the protein. Therefore, it was neces-
sary to add several zero-order bonds to hold the
ligand in the general vicinity of the catalytic region in
the active site.
Acknowledgments
We gratefully acknowledge support from The Knut and
Alice Wallenberg Foundation.

2966
R. Ro¨nn et al. / Bioorg. Med. Chem. 16 (2008) 2955–2967
Lemieux, C.; Pieti Opie, L.; Sullivan, F.; Neitzel, N.;
Supplementary data
Hingorani, G.P.; Otten, J.; Brandhuber, B.; Vigers, G.;
Josey, J.A.; Blatt, L.M. Preclinical Characteristics of
ITMN 191, an Orally Active Inhibitor of the HCV NS3/
4A Protease Nominated for Preclinical Development.
Digestive Disease Week, May 20-25, Los Angeles 2006,
Poster #T1793.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.12.041.
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Bernat, B.A.; Brandhuber, B.J.; Andrews, S.W.; Josey,
J.A.; Blatt, L.M. Structure-Based Design of Novel Iso-
indoline Inhibitors of HCV NS3/4A Protease and Binding
Mode Analysis of ITMN-191 by X-ray Crystallography.
Digestive Disease Week, May 20-25, Los Angeles 2006,
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