Achiral oligoamines as versatile tool for the development of aspartic protease inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.08.012

Due to the important role that aspartic proteases play in many patho-physiological processes, they have intensively been targeted by modern drug development. However, up to now, only for two family members, renin and HIV protease, approved drugs are available. Inhibitor development, mostly guided by mimicking the natural peptide substrates, resulted in very potent inhibitors for several targets, but the pharmacokinetic properties of these compounds were often not optimal. Herein we report a novel approach for lead structure discovery of non-peptidic aspartic protease inhibitors using easily accessible achiral linear oligoamines as starting point. An initial library comprising 11 inhibitors was developed and screened against six selected aspartic proteases. Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was confirmed by determination of the crystal structure of two derivatives in complex with the HIV-1 protease, and represents a promising basis for the further inhibitor development.

Full text of DOI:10.1016/j.bmc.2008.08.012

Bioorganic & Medicinal Chemistry 16 (2008) 8574–8586
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Achiral oligoamines as versatile tool for the development of aspartic
protease inhibitors
Andreas Blum , Jark Böttcher , Benedikt Sammet , Torsten Luksch,
*
Andreas Heine, Gerhard Klebe, Wibke E. Diederich
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35032 Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to the important role that aspartic proteases play in many patho-physiological processes, they have
intensively been targeted by modern drug development. However, up to now, only for two family mem-
bers, renin and HIV protease, approved drugs are available. Inhibitor development, mostly guided by
mimicking the natural peptide substrates, resulted in very potent inhibitors for several targets, but the
pharmacokinetic properties of these compounds were often not optimal. Herein we report a novel
approach for lead structure discovery of non-peptidic aspartic protease inhibitors using easily accessible
achiral linear oligoamines as starting point. An initial library comprising 11 inhibitors was developed and
screened against six selected aspartic proteases. Several hits could be identified, among them selective as
well as rather promiscuous inhibitors. The design concept was confirmed by determination of the crystal
structure of two derivatives in complex with the HIV-1 protease, and represents a promising basis for the
further inhibitor development.
Received 3 June 2008
Revised 29 July 2008
Accepted 4 August 2008
Available online 7 August 2008
Keywords:
Oligoamines
Aspartic protease inhibitors
HIV-1 protease
Inhibitor design
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
(malaria)13,14 which all share a common folding motif: the N-ter-
minal and the C-terminal domain, each contributing one catalytic
Aspartic proteases belong to the class of endopeptidases and
have shown to play an important role in many physiological but
also patho-physiological processes. The active site comprises two
aspartic acid residues which activate a water molecule hence facil-
itating the nucleophilic attack at the scissile amide bond. The
cleavage of the substrate follows a general catalytic acid–base
mechanism in which one of the two aspartates is protonated, the
other deprotonated.1,2 The nucleophilic attack of the activated
water molecule leads to a tetrahedral gem-diol intermediate which
collapses under cleavage of the peptide bond (Scheme 1). In gen-
eral, 6–10 amino acids of the natural polypeptide substrates are
recognized by aspartic proteases, thus demanding an extended ac-
tive site. The standard nomenclature defines the substrate residues
as, for example, P3, P2, P1, P10, P20, and P30 and the corresponding
recognition pockets as, for example, S3, S2, S1, S10, S20, and S30 as
depicted in Scheme 1.3
(a)
P1'
O
P1'
P1
P1'
P1
P1
OH
+
NH
NH
OH
H2N
O
O
O
H
O
H
H
O
H
O
H
O
H
O
O
O
O
O
O
O
O
Asp
Asp
Asp
S1
Asp
Asp
Asp
S3
P3
S2'
(b)
O
P2'
O
O
P1
H
N
H
H
Two classes of aspartic proteases have received pronounced
attention as potential drug targets; the pepsin-like5 (family A1)
and the retroviral proteases (family A2).6–8 The family of the pep-
sin-like proteases includes, for example, renin (cardiovascular dis-
eases),9,10 BACE-1 (Alzheimer’s disease),11,12 and the plasmepsins
N
N
N
N
H
N
H
H
P1'
O
P3'
O
P2
O
S2
S1'
S3'
* Corresponding author. Tel.: +49 6421 28 25810; fax: +49 6421 28 26254.
E-mail address: wibke.diederich@staff.uni-marburg.de (W.E. Diederich).
These authors contributed equally to this work.
Scheme 1. (a) Schematic representation of the catalytic mechanism of aspartic
proteases. (b) Nomenclature of the protease’s subsites according to Berger and
Schechter,3 the scissile peptide bond is indicated by crossing lines.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.012

A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
8575
(Fig. 1a and c, PDB code: 5HVP).20 The HIV protease recognizes a
variety of natural substrates with a strong preference for those
bearing hydrophobic amino acids in P1 and P10 and variable amino
acids in farther positions (Table 1).21
Despite immense efforts in the development of clinically effec-
tive drugs targeting aspartic proteases, up to now, only for HIV pro-
tease22 and renin inhibitors have been approved for disease
therapy.23–25 The rational design of inhibitors has mostly been
guided by the structure of the natural peptide substrates thus
resulting in very potent inhibitors. However, the pharmacokinetic
properties of these peptidic compounds bearing secondary hydro-
xyl groups as transition state isosters are often not optimal, thus
hampering their clinical efficacy.26 The synthesis of these complex
and chiral inhibitors often turns out to be very challenging, and the
optimization of the sub-pocket-addressing moieties is very re-
source-intensive and time-consuming. Replacement of the second-
ary hydroxyl group in transition state mimetics by the nearly
isosteric amino functionality as well as by cyclic amidines has also
been successfully pursued.27–30 Additionally, reduced amide tran-
sition state isosters have been exploited recently as BACE-1 inhib-
itors.31,32 However, for a more efficient development of novel
aspartic protease inhibitors, an easily accessible and achiral core
structure would be preferable. Utilizing such scaffolds would facil-
itate the development of promising moieties addressing the prote-
ase’s sub-pockets. As suitable core structure for this approach we
selected secondary amines, which have already been successfully
utilized as anchoring groups for the development of aspartic prote-
ase inhibitors.
In the late 1990s, researchers at Roche discovered the piperi-
dine moiety as a novel privileged skeleton addressing the cata-
lytic dyad of aspartic proteases with its secondary nitrogen as
revealed by X-ray crystallography.33–35 Based on this discovery,
several projects for the development of non-peptidic amine-based
aspartic protease inhibitors have been pursued, which led to the
development of potent inhibitors for the aspartic proteases
plasmepsin (Plm) II and IV,36–39 BACE-1,40 and very recently for
HIV-1 protease.41 All inhibitors share in common a cyclic amino
functionality addressing the enzyme’s catalytic dyad directly or
mediated by a water molecule. However, numerous scaffolds
have been utilized. Depending on the nature of the scaffold and
the target enzyme studied, the specificity pockets are addressed
differently. In case of a pyrrolidine-based HIV-1 protease inhibi-
tor, the interaction of the endocyclic, secondary amino functional-
ity to the catalytic aspartic acid residues was studied by X-ray
crystallography and Poisson–Boltzmann calculations. The latter
study suggests the amine being in the protonated and the cata-
lytic dyad in the fully deprotonated state resulting in strong elec-
trostatic interactions.42
Figure 1. Crystal structures of the peptidomimetic inhibitor Pepstatin A, shown in
light blue, color-coded by atom type, in complex with the HIV-1 protease (a and c)
(PDB code: 5HVP) and pepsin (b and d) (PDB code: 1PSO). The proteins are
represented as cartoons and flap regions establishing interactions to the inhibitor
are highlighted in magenta. Hydrogen bonds are indicated by dashed lines. The
catalytic aspartates are color-coded by atom type in gray and selected flap amino
acid residues are color-coded by atom type in magenta. Figures 1–3 were created
using PyMol 0.99.4
aspartic acid residue, are connected via a b-sheet domain. The ac-
tive site is formed by the closure of a flexible loop region termed
‘flap’ which covers the active site and forms two hydrogen bonds
to the substrate carbonyl groups adjacent to the cleavage site
(Fig. 1b and d, PDB code: 1PSO).15 In the S1 and S10 pocket, prefer-
ably hydrophobic amino acids are recognized, whereas in the S2,
S3, S20, and S30 pockets the recognition is non-uniform (Table 1).
The retroviral aspartic proteases belong to the A2 family, among
which the HIV-1 protease was shown to be a valuable target for
treatment of HIV-infection.16–19 The HIV-1 protease is a C2-sym-
metric protein consisting of two identical subunits each contribut-
ing one catalytic aspartate. In contrast to family A1, the binding
pocket is formed by the closure of two flaps. A conserved water
molecule mediates the interactions of the peptide carbonyls adja-
cent to the cleavage site and the flap residues Ile50A and Ile50B
2. Results
Table 1
Cleavage sites in natural substrates of the selected aspartic proteases as annotated in
the MEROPS database7
To further exploit this promising anchoring group for the design
of novel, easily accessible inhibitors, linear oligoamines were se-
lected as novel core structure. This scaffold bears a central second-
ary amino function aimed to address the catalytic dyad. Via the
distal amino functionalities, the introduction of appropriate accep-
tor groups addressing the flap regions of the respective target en-
zyme is easily amenable. Concomitantly hydrophobic moieties
intended to address the S2 and S20 specificity pockets are intro-
duced. Via further alkylation of the distal nitrogen atoms, addi-
tional hydrophobic substituents can be implemented to address
the S1/S10 pockets, thereby avoiding the generation of any chiral
center. This strategy allows the quick and straightforward genera-
tion of achiral inhibitors following a highly flexible and short syn-
thetic route. To optimize the amine-acceptor distances with
respect to a certain target enzyme, oligoamines with varying chain
Protease
Substrate
P3
P2
P1
P10
P20
P30
Pepsin A
Plm II
Plm IV
Renin
—
—
R
R
F
—
M
M
H
K
F/L
F
F
L
M
F
L
L
L/V
D
—
S
S
V/I
A
—
F
F
H
Q
Hemoglobin
Hemoglobin
Angiotensinogen
a
a
-chain
-chain
BACE-1
Amyloid-precursor-protein
V
HIV-1 protease
Gag polyprotein
Q
R
T
A
G
F
L
E
K
N
V
I
N
N
N
N
T
I
Y
L
M
F
F
F
F
F
L
P
A
M
L
I
V
A
R
K
S
I
S
D
D
E
Q
H
Q
Q
I
L
Pol polyprotein
P
P
Y
F
V
L

8576
A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
F3C
HN
CF3
O
O
H2N
NH2
( )m
H2N
NH2
( )m
( )n
( )n
N
H
NH
( )m
N
( )n
N
BOC
BOC
2a-c
1a-c
3a-c
(n,m): a (1,1), b (1,2), c (2,2)
(n,m): a (1,1), b (1,2), c (2,2)
(n,m): a (1,1), b (1,2), c (2,2)
H
H
( )m
R
N
N
R
R
N
N
R
R
N
N
R
( )n
( )n
( )m
( )n
( )m
S
OO
N
S
OO
S
OO
N
S
OO
S
OO
N
H
S
OO
3a-c
BOC
BOC
4a-f
5a-f
6a-f
R = Ph: a (1,1), b (1,2), c (2,2)
R = -NO2Ph: d (1,1), e (1,2), f (2,2)
R = Ph: a (1,1), b (1,2), c (2,2)
R = -NO2Ph: d (1,1), e (1,2), f (2,2)
R = Ph: a (1,1), b (1,2), c (2,2)
R = -NH2Ph: d (1,1), e (1,2), f (2,2)
p
p
p
HN
NH
R
N
N
R
R
N
N
R
N
S
OO
N
S
OO
S
OO
N
H
S
OO
3a
BOC
BOC
8a R = Ph
9a R = Ph
7
8b R =
p-NO2Ph
9b R =
p-NH2Ph
HN
NH
( )m
Bn
N
N
Bn
Bn
N
N
Bn
( )n
( )n
( )m
( )n
( )m
N
N
N
H
3a-c
BOC
O
BOC
O
O
O
10a-c
a (1,1), b (1,2), c (2,2)
12a-c
a (1,1), b (1,2), c (2,2)
11a-c
a (1,1), b (1,2), c (2,2)
Scheme 2. Preparation of the oligoamines: for reaction conditions and yields refer to Section 5.
length between the central and each of the distal amino function-
alities can be employed.
ble via reductive amination of 3a with iso-butyraldehyde and
NaBH4 as reducing agent yielding 7, followed by condensation with
sulfonyl chlorides rendering 8a,b. The final inhibitors 9a,b were
obtained by reduction of the nitro functionality or by acidic cleav-
age of the Boc-protecting group as described above. Carboxamide
inhibitors 12 were synthesized following a similar synthetic se-
quence: the benzyl-substituted oligoamines 10a–c were obtained
by reductive amination of the amines 3a–c and benzaldehyde uti-
lizing Pd/BaSO4 as catalyst. Subsequent condensation with phenyl-
acetic acid chloride gave rise to the protected inhibitors 11a–c,
which were deprotected under acidic anhydrous conditions yield-
ing 12a–c.
For the design of an initial library, linkers with two and three
methylene groups separating the amino groups were selected. As
hydrophobic moieties intended to address the S1/S10 sub-pockets,
iso-butyl and benzyl groups resembling the side chains of leucine
and phenylalanine of substrate peptides were introduced. For
appropriate flap interactions, two types of acceptor functionalities
were investigated: on the one hand carboxamides present in the
natural substrate and on the other hand sulfonamides, which allow
additional rotational degrees of freedom. The introduction of resi-
dues addressing the S2/S20 pockets is easily achieved via further
substitution at the lateral nitrogen atoms. In case of the sulfona-
mides, the hydrophobic phenyl and the more polar p-amino phenyl
group were chosen. For carboxamides, phenyl acetic acid deriva-
tives were utilized offering additional flexibility between the rigid
amide functionality and the phenyl ring.
2.2. Kinetic characterization
To elucidate the potential of this new class of inhibitors, the
affinity toward selected aspartic proteases was determined in fluo-
rescence-based assays using commercially available substrates.
The results are listed in Table 2. The anthranyl-HIV protease sub-
2.1. Chemistry
strate was utilized in case of the HIV-1 protease (Km = 14.6
pepsin (Km = 13.3 M), and the plasmepsins II (Km = 63 M) and
IV (Km = 28 M). For renin and BACE-1, suitably labeled oligopep-
tides derived from their natural substrates with comparable affin-
ity were used (renin substrate Km = 3.3 M, BACE-1 substrate
Km = 7.9 M).
Except for BACE-1, for each of the investigated target enzymes
an inhibitor with an affinity in the single-digit micromolar range
could be identified (Table 2). In case of the HIV-1 protease, a strong
preference for inhibitors bearing ethylene linkers and a sulfonyl
lM),
Oligoamines 3a–c, Boc-protected at the pivotal, secondary ami-
no function, are accessible from commercially available precursors
1a–c by transient protection of the terminal amino groups as their
trifluoroacetamides 2a–c (Scheme 2).43 Condensation with sulfo-
nyl chlorides furnished the sulfonamides 4a–f which were further
alkylated with benzyl bromide giving rise to the protected inhibi-
tors 5a–f. The phenyl-substituted sulfonamides 6a–c were ob-
tained via deprotection of 5a–c with HCl in Et2O. The p-amino
phenyl-substituted sulfonamides 6d–f were synthesized by reduc-
tion of the corresponding nitro-compounds 5d–f with SnCl2ꢀ2H2O
in refluxing 32% HCl under concomitant cleavage of the Boc-pro-
tecting group. The iso-butyl-substituted inhibitors 9a,b are accessi-
l
l
l
l
l
moiety was observed, with affinities ranging from 10.0
up to 0.9 M for 9b. Considering the pepsin-like proteases, the tol-
erance of extended linkers is noteworthy. Particularly the plasmep-
lM for 9a
l

A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
8577
Table 2
Ki-Values of the inhibitors toward selected aspartic proteases in
l
M
P1
N ( )n
P1
P1
N ( )n
P1
( )mN
P2
S
P2
P2
P2
( )mN
S
OO
N
H
N
H
OO
O
O
6a-f, 9a-b
12a-c
n
m
P2
P1
HIV-1 pr.
3.8
Plm II
7.0
Plm IV
Renin
BACE-1
n.i.
Pepsin
5.7
6a
1
1
36
17
42
7.5
7.5
20
87
56
98
56
170
n.i.
6b
6c
1
2
1
1
2
1
1
1
1
2
2
2
1
2
2
1
1
1
2
2
14
18
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
5.7
n.i.
16
n.i.
150
n.i.
n.i.
64
15
n.i.
9.6
n.i.
n.i.
10
21
n.i.
3.9
n.i.
4.0
4.5
4.8
n.i.
n.i.
n.i.
6d
6e
4.2
22
NH2
NH2
NH2
6f
2.8
23
9a
n.i.
n.i.
n.i.
n.i.
n.i.
9b
12a
12b
12c
0.9
n.i.
n.i.
n.i.
6.8
91
NH2
63
120
n.i., IC50 > 250 lM.
sins II and IV reveal a remarkable tolerance toward the linker
2.3. Binding mode of 6d
length: inhibitor 6f exhibits the highest affinity toward Plm II
(2.8
IV (7.5
l
M) and the corresponding analogues 6d and 6e toward Plm
The binding mode is schematically represented in Scheme 3.
The pivotal secondary nitrogen of the inhibitor points toward
Asp25A forming polar contacts to the terminal oxygen atoms of
the side chain and to one sulfonyl oxygen atom of one of its sulfon-
amide functions. The N-benzyl moieties of the inhibitor address
the S1 and S10 pockets, whereas the p-amino-phenyl sulfonamide
groups occupy the S2 and S20 pockets. The p-amino substituents
form a similar hydrogen bond network in both pockets: hydrogen
bonds are observed to the main chain carbonyl oxygen atoms of
Asp30A and Asp30B and to the corresponding side chain carboxyl
oxygen atoms in each case mediated by a water molecule. One sul-
fonyl oxygen atom of each sulfonamide functionality establishes
hydrogen bonds to a tetrahedrally coordinated water molecule
mediating polar contacts to the main chain NHs of Ile50A and
Ile50B, which are located at the tips of the flaps. One of the sulfonyl
oxygen atoms remains unsatisfied showing no direct polar con-
tacts to the protein. The ligand is deeply embedded in the binding
pocket with a surface burial of 94%.
lM). An inhibition of renin could only be observed in case
of derivatives equipped with carbonyl groups as acceptor function-
alities. With compound 12a bearing the shortest linkers, the high-
est affinity (5.7
only accomplished with sulfonamide derivatives, 6d being the
most active compound (3.9 M) in the series. However, no prefer-
ence toward a certain chain length could be observed. In case of
BACE-1, the sulfonamides equipped with the longest linkers show
moderate inhibition with an affinity of 64 lM for compound 6f.
To elucidate whether the oligoamine derivatives indeed exhibit
the expected binding mode thus being in agreement with our ini-
tial design concept, the crystal structure in complex with one of the
target enzymes was determined. Since the crystallization of HIV-1
protease is well established in our lab, this enzyme was selected
for crystallization. The inhibitors 6a, 6d, and 9b exhibiting the
highest potency were selected for cocrystallization experiments.
However, crystals could only be obtained in case of the better sol-
uble compounds 6d and 9b. The X-ray structures of these com-
pounds in complex with HIV-1 protease were determined with a
resolution of 1.90 and 1.80 Å, respectively. The crystallographic
data and refinement statistics are listed in Table 3. Both complexes
adopt space group P21212 and the inhibitors are clearly visible in
the Fo ꢁ Fc density at a sigma level of 3. They could be refined as
one single conformer (Fig. 2).
lM) could be achieved. An inhibition of pepsin is
l
2.4. Binding mode of 9b
The crystal structure of 9b in complex with HIV-1 protease
exhibits a high similarity to the previously described structure.
Comparable polar interactions of the secondary amino group to
Asp25A are observed (Scheme 4). The sub-pocket occupancy

8578
A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
Table 3
Ile50A
Ile50B
X-ray data processing and refinement for the HIV-1 protease complexes of derivatives
6d and 9b
N
N
H
3.1
H
2.6
HOH
2.7
2.4
HOH
3.5
3.2
HOH
3.1
O
O
6d
9b
3.0
O
O
Resolution (Å)
Space group
Cell dimensions (Å)
a
b
c
50–1.80
P21212
30–1.90
P21212
O
O
NH2
O
H
N
H
N
O
S
N
H2N
S
N
3.1
O
S2'
3.0
O
57.4
85.7
46.5
1.83–1.80
49,890
56.0
85.9
46.5
S2
Asp30B
+
Asp30A
NH2
2.8
Highest resolution shell (Å)
No. of measured reflections
No. of independent reflections
Completenessa (%)
1.93–1.90
65,713
18,795
99.0 [100]
15.6 [2.6]
8.0 [49.0]
18.2; 20.9
23.9; 27.1
29.5; 27.1
33.1
S1'
S1
O
3.6
O
21,367
O
O
97.4 [97.6]
16.0 [2.0]
7.8 [44.4]
18.4; 20.8
23.0; 26.0
25.9; 22.4
35.1
Asp25A
Asp25B
I/ra
Rsym (%)
a
Scheme 4. Schematic representation of the binding mode of 9b. Hydrogen bonds
are indicated by dashed lines and distances are given in Å.
Rcryst (F > 4
Rfree (F > 4
r
Fo; Fo)
rFo; Fo)
Mean B-factor (Å2) protein (chain A; B)
Mean B-factor (Å2) ligand
Mean B-factor (Å2) water
Ramachandran plot
difference between both complexes is the deviating polar interac-
tion of the sulfonyl groups of the inhibitor to the flap. Although a
water molecule is involved in the mediation of polar contacts of
the sulfonyl oxygen atoms in both cases, a different coordination
is observed. In contrast to 6d, the water in the enzyme–inhibitor
complex of 9b is not tetrahedrally coordinated. It only forms
hydrogen bonds to two sulfonyl oxygen atoms and the main chain
NH of Ile50A. One of the sulfonyl oxygen atoms establishes an
intramolecular hydrogen bond to the pivotal oligoamine nitrogen
atom, as observed in case of 6d. In contrast to the previous struc-
ture, the second sulfonyl oxygen atom directly addresses Ile50
NH of the B chain, whereas in case of 6d, this oxygen atom remains
without any polar contacts. The overall burial of the inhibitor sums
up to 93%.
31.0
34.0
Most favored geometry (%)
Additionally allowed (%)
RMSD bonds (Å)
96.8
3.2
0.006
2.0
95.6
4.4
0.005
2.0
RMSD angles (°)
a
Values in brackets refer to the highest resolution shell.
3. Discussion
The biological data as well as the crystal structures of 6d and 9b
in complex with HIV-1 protease clearly reveal that our initial de-
sign concept has been successful. For all of the investigated aspar-
tic proteases inhibitors binding in the micromolar range could be
identified. Noteworthy, for most of these enzymes a preference to-
ward ethylene linkers is observed. The sulfonamide acceptor
groups allowing additional rotational degrees of freedom are fa-
vored compared to the carbonyl groups for all targets except for re-
nin. In the latter case the more rigid structure of N-alkylated
carboxamides is obviously preferred for inhibition.
Figure 2. Ligand geometries of 6d (a, color-coded by atom type in green) and 9b (b,
color-coded by atom type in yellow) in the cocrystal structures with the HIV-1
protease. The Fo ꢁ Fc omit maps for the ligands are displayed at a
r level of 3.0 as
blue mesh. The protein surface is schematically represented in gray and the
catalytic aspartates Asp25A and Asp25B are highlighted in red.
Ile50A
Ile50B
N
N
H
H
2.6
HOH
2.7
2.5
The binding modes of 6d and 9b in HIV-1 protease reveal how
these compounds fulfill the initial pharmacophore hypothesis. As
intended, the secondary amino group addresses the catalytic dyad,
and two of the sulfonamide oxygen atoms mimic the substrate’s
3.0
2.8
2.8
2.8
HOH
HOH
3.2
NH2
O
O
O
O
O
2.9
O
O
H
N
H
N
O
S
N
H2N
S
N
2.7
O
3.1
amide carbonyl groups. This is illustrated by the C -superposition
a
O
of the complex structures of 6d and 9b, respectively, with the crys-
tal structure of a substrate analogue oligopeptide in complex with
an inactive HIV-1 protease mutant (PDB code: 1KJH) (Fig. 3a and
b).21 In addition, the specificity pockets of the enzyme are occupied
analogously; however, the direction in which the S1/S10 pocket is
addressed deviates from the substrate’s one. The 10-fold lower
binding affinity of the inhibitor 6d compared to 9b can most likely
be attributed to this difference: the benzyl moieties of 6d are in our
case now slightly too large to occupy the S1/S10 pockets
appropriately.
S2'
S2
Asp30B
+
Asp30A
NH2
2.9
O
S1
Asp25B
O
3.8
S1'
O
O
Asp25A
Scheme 3. Schematic representation of the binding mode of 6d. Hydrogen bonds
are indicated by dashed lines and distances are given in Å.
resembles the binding situation of 6d: the iso-butyl moieties occu-
py the S1 and S10 pockets, and the p-amino-phenyl sulfonamide
groups are located in the S2 and S20 pockets. The amino substitu-
ents form a similar hydrogen bond network as already described
for 6d: hydrogen bonds to the main chain carbonyl oxygen of
Asp30A, Asp30B as well as water mediated to the corresponding
side chain carboxyl oxygens are formed. The most pronounced
The C -superposition of the crystal structures in complex with
a
the HIV-1 protease of the approved peptidomimetic inhibitor
Amprenavir (PDB code: 1HPV) and 9b, which bears the same P1
and P2 substituents, is shown in Figure 3c.44 Comparing the posi-
tions of the N-iso-butyl-N-p-amino-phenyl sulfonamide moieties
of the inhibitors which address the S1 and S2 pocket, only slight
differences can be observed. Although the inhibitors comprise

A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
8579
Figure 3. (a and b) C -superposition of the cocrystal structures of 6d (green, color-coded by atom type) and 9b (yellow, color-coded by atom type) with the structure of a
a
substrate analogue polypeptide (magenta, color-coded by atom type) in complex with an inactive Asp25Asn HIV protease mutant (PDB code: 1KJH). (c) Superposition of the
cocrystal structure of 9b (yellow, color-coded by atom type) with the cocrystal structure of the approved inhibitor Amprenavir (blue, color-coded by atom type, PDB code:
1HPV). The protein surface is schematically represented in gray and the catalytic aspartates Asp25A and Asp25B are highlighted in red.
different moieties, the occupation of the S10 and S20 pockets ap-
pears similar. However, due to their polymethylene linkers, the oli-
goamine derivatives are very flexible molecules and a high degree
of pre-organization prior to binding to the protein is required,
resulting in an entropic penalty thus lowering the overall affinity.
Additionally, the position of the flap-water molecule present in
the complexes of 6d and 9b is different in the oligoamine struc-
tures when compared to the substrate-like oligopeptide and
Amprenavir complexes. In case of 6d, a similar tetrahedral coordi-
nation of the water molecule is observed, but it is shifted about 1 Å
away from the catalytic dyad. A deviation from the usually ob-
served coordination is detected in case of the 9b complex leading
to a direct interaction of one sulfonyl oxygen atom with the flap.
These observations indicate that the flap interactions of the oligo-
amines 6d and 9b in case of HIV-1 might require some further
improvement. Taking all these facts into account, the lower affinity
by three orders of magnitude of the oligoamines compared to
Amprenavir (Ki = 0.6 nM) seems reasonable.
The observed peptide-like binding mode in case of the ethylene-
bearing inhibitors 6d and 9b in complex with HIV-1 protease sug-
gests a similar binding situation in case of the other pepsin-like
proteases. In this case, the acceptor functionalities of the inhibitors
mimic the carbonyl groups of the substrate adjacent to the cleav-
age site, resulting in the same subsite occupation as observed for
6d and 9b. However, in contrast to HIV protease, there is no clear
preference for the ethylene linkers. In pepsin-like proteases, amino
acids bearing flexible polar side chains are located in the flap re-
gion: Thr77 in case of pepsin (Fig. 1d), Ser76 and Thr77 in case
of renin, Thr72 and Gln73 in case of BACE-1, and Ser79 in case of
Plm II and Plm IV. In addition to the peptide recognition motifs,
these residues might also be addressed by the acceptor groups of
the inhibitors resulting in the preference for longer linkers. The
binding of inhibitors with propylene linkers results in a larger dis-
tance between the sub-pocket-addressing hydrophobic moieties
and could omit occupancy of the S1 or S10 pockets. This might ex-
plain the inhibition of BACE-1 only by the inhibitors 6c and 6f, be-
cause the S10 pocket of BACE-1 is featured to recognize an aspartic
acid side chain. Most likely, the studied oligoamine inhibitors are
not suitable to address this site.
enzymes, at least one single-digit micromolar inhibitor could be
identified. The crystal structures of two representatives in com-
plex with the HIV-1 protease proved the concept initially used
for their design. The central amino group addresses the catalytic
dyad, the acceptor groups establish polar interactions to the flap,
and the hydrophobic moieties occupy the sub-pockets of the en-
zyme. The binding mode exhibits high similarity to the binding
orientation of substrates as well as to that of peptidomimetic
inhibitors. Taking this into account, the generalization of this
binding situation to other aspartic proteases appears reasonable
thus providing a first insight into the observed structure–activity
relationships.
The straightforward synthesis allows the combinatorial intro-
duction of numerous different substituents, thus facilitating the
preparation of a plethora of putative aspartic protease inhibitors.
The derived SAR data from such libraries can easily be utilized
for the selection of promising moieties for selectively addressing
the specificity pockets of a target enzyme in order to optimize
inhibitors bearing other scaffolds. Hits from these oligoamine li-
braries can also be utilized as suitable starting point for further
optimization. Structural variation of the oligoamine chain based
on a certain inhibitor, at best guided by a crystal structure, can lead
to novel derived amine-based scaffolds. Increasing the rigidity of
the linker chain is the most promising approach, leading to a better
preorganization of the inhibitor and a reduction of the entropic
penalty upon binding hence most likely resulting in a gain of
affinity.
5. Experimental
5.1. Molecular biology
5.1.1. Enzyme assays
All enzyme assays were performed at room temperature on a
microplate reader (Safire2 ) using black 96-well microtiter plates
TM
purchased from Nunc. The assay volume was 200 ll, and inhibitors
and substrates were previously dissolved in dimethyl sulfoxide (fi-
nal concentration 4%). The hydrolysis of the substrates was re-
corded as increase in fluorescence intensity. IC50 values were
generated by nonlinear regression analysis from plots of vi/v0 ver-
sus inhibitor concentration, in which vi is the velocity in presence,
and v0 the velocity in the absence of an inhibitor. The kinetic con-
stants were determined by the method of Lineweaver and Burk,
and Ki values were consecutively calculated from the following
equation: Ki = IC50/[1 + (S/Km)]. The overall error of the assays is
estimated to be 40%.
4. Summary and conclusion
In this study, we present a novel rational strategy for the
development of aspartic protease inhibitors. This method is based
on achiral linear oligoamines, which are easily accessible via a
short and flexible synthetic route starting from commercially
available precursors. Within the initial compound series compris-
ing 11 inhibitors, several hits for the target enzymes could be
identified. Some of them reveal remarkable selectivity, others
show broad promiscuity. Noteworthy, for five out of six target
5.1.2. HIV-1 protease
Recombinant HIV-1 protease was expressed from Escherichia
coli and purified as previously described.45 The fluorogenic sub-

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A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
strate Abz-Thr-Ile-Nle-(p-NO2-Phe)-Gln-Arg-NH2 was purchased
from Bachem and exhibited a Km of 14.6 M. The assays were per-
formed in 100 mM MES, 300 mM KCl, 5 mM EDTA 1 mg/mL BSA,
5.2. Chemistry
l
5.2.1. General
pH 5.5, and a substrate concentration of 20
l
M (excitation wave-
Reported yields refer to the analytically pure product obtained
by column chromatography or recrystallization. All proton and car-
bon nuclear magnetic resonance spectra were recorded on a Jeol
Eclipse+ Spectrometer (1H NMR: 500 MHz, 13C NMR: 125 MHz)
using TMS as internal standard (0.00 ppm). 13C-spectra were refer-
enced to CDCl3 (77.0 ppm), DMSO-d6 (39.7 ppm), or CD3OD
(49.0 ppm). The values of chemical shifts (d) are given in ppm
and coupling constants (J) are given in Hz. Abbreviations:
br = broad, s = singulet, d = doublet, t = triplet, q = quartet,
sm = symmetric multiplet, m = multiplet. Mass spectra were ob-
tained from a double-focusing sectorfield Micromass VG-Autospec
spectrometer. Combustion analyses were determined on a Vario
Micro Cube by Elementar Analysen GmbH. Melting Points were
determined using a Leitz HM-Lux apparatus and are uncorrected.
Flash chromatography was performed with a Büchi Sepacore Flash
MPLC system using silica gel 60 (0.04–0.063 mm) purchased from
Macherey-Nagel and solvents as indicated. TLC was carried out
using 0.2 mm aluminium plates coated with silica gel 60 F254 by
Merck and visualized by UV-detection or ninhydrin staining. Sol-
vents and reagents that are commercially available were used
without further purification unless otherwise noted. All mois-
ture-sensitive reactions were carried out using oven-dried glass-
ware under a positive stream of argon.
length 337 nm, emission wavelength 410 nm).46
5.1.3. Plasmepsin II and plasmepsin IV
Recombinant plasmepsin II and plasmepsin IV were expressed
from Escherichia coli and purified following the protocol described
by Hill et al.47 The fluorogenic substrate Abz-Thr-Ile-Nle-(p-NO2-
Phe)-Gln-Arg-NH2 was purchased from Bachem (Km (Plm
II) = 63
in 100 mM NaAc, pH 4.5, 1 mg/mL BSA and a substrate concentra-
tion of 18 M (excitation wavelength 337 nm, emission wave-
lM and Km (Plm IV) = 28 lM). The assays were performed
l
length 410 nm).
5.1.4. Renin
The fluorogenic substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-
Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg and recombinant renin were
purchased from Sigma–Aldrich (Km = 3.3
lM). Assays were per-
formed in 50 mM Tris–HCl, 100 mM NaCl, 1 mM EDTA, pH 8.0,
and a substrate concentration of 5 lM (excitation wavelength
340 nm, emission wavelength 500 nm).48
5.1.5. b-Secretase
The fluorogenic substrate MOCAc-Ser-Glu-Val-Asn-Leu-Asp-
Ala-Glu-Phe-Arg-(2,4-dinitrophenyl)-Lys-Arg-Arg-NH2 and recom-
5.2.2. General procedure A for the preparation of N-Boc-protec-
binant b-secretase were purchased from Sigma (Km = 7.9
Assays were performed in 100 mM NaAc, pH 4.5, and a substrate
concentration of 5 M (excitation wavelength 320 nm, emission
lM).
ted bis-trifluoromethylacetamides of type 2
In accordance to a procedure of Hodacová et al.,43 ethyl trifluo-
ˇ
l
romethylacetate (15.6 g, 12.5 mL, 110 mmol) in CH2Cl2 (50 mL)
was added dropwise to a stirred solution of 50.0 mmol of the cor-
responding oligoamine 1 in CH2Cl2 (100 mL) at 0 °C, and the reac-
tion mixture was stirred for 30 min at 0 °C. After addition of CH2Cl2
(20 mL) to dissolve the resulting precipitate, the mixture was al-
lowed to warm to rt and stirred for additional 60 min. A mixture
of triethylamine (11.1 g, 15.3 mL, 110.0 mmol) and Boc2O (24.0 g,
110.0 mmol) in CH2Cl2 (50 mL) was then added dropwise to the
solution and stirring was continued for 5 h. The reaction mixture
was washed with a satd NaHCO3-solution (2ꢂ 80 mL) and water
(2ꢂ 80 mL), the organic layer separated, dried over K2CO3, and fil-
tered. After concentration of the organic layer to about one third of
its original volume, an equal volume of hexanes was added. The
mixture was kept in the freezer (ꢁ12 °C) rendering colorless crys-
tals, which were separated by filtration, washed with water fol-
lowed by hexanes, and finally dried under vacuum giving rise to
the respective N-Boc-protected bis-trifluoroacetamides 2a–c.
wavelength 405 nm).
5.1.6. Pepsin
Pepsin from porcine gastric mucosa was purchased from Sig-
ma–Aldrich, and the HIV protease substrate Abz-Thr-Ile-Nle-(p-
NO2-Phe)-Gln-Arg-NH2 was used for kinetic measurements
(Km = 13.3
3.5, and a substrate concentration of 20
337 nm, emission wavelength 410 nm).
lM). Assays were performed in 10 mM NaHCOO, pH
l
M (excitation wavelength
5.1.7. Crystallization of the HIV-1 protease inhibitor complexes
The HIV-1 protease inhibitor complexes were crystallized at
18 °C in 0.1 M Bis–Tris, pH 6.5, 2.5–3.0 M NaCl and a protein con-
centration of 7 mg/mL in the space group P21212 (crystal data, Ta-
ble 3). The crystals were obtained by cocrystallization of the
enzyme with inhibitor concentrations ranging from 20- to 100-fold
the Ki value. Crystals were further optimized using streak-seeding
techniques. For cryoprotection, the crystals were briefly soaked in
mother liquor containing 25% glycerol.
5.2.3. General procedure B for the preparation of N-Boc-protec-
ted oligoamines of type 3
According to a procedure of Hodacová et al.,43 an aqueous solu-
ˇ
5.1.8. Data collection, phasing, and refinement
tion of NaOH (5.00 g, 125.0 mmol, in 42 mL water) was added to a
solution of 10.0 mmol of the respective N-Boc-bis-trifluoroaceta-
mide 2 in MeOH (50 mL), and the reaction mixture stirred for 6 h
at rt. The major amount of methanol was removed under reduced
pressure and the remaining solution extracted with CH2Cl2 (3ꢂ
40 mL). The combined organic layers were dried over K2CO3, fil-
tered, evaporated, and finally dried in vacuum furnishing the
respective N-Boc-protected oligoamines 3a–c as colorless oils.
The data sets were collected on a Rigaku R-AXIS IV image plate
detector using CuK radiation from an in-house RU-H3R Rigaku
a
rotating anode. Data were processed and scaled with Denzo and
Scalepack as implemented in HKL2000.49 The structures were
determined by the molecular replacement method using Phaser,50
one monomer of the 1.50 Å HIV-1 protease in complex with a pyr-
rolidine-based inhibitor (PDB code: 2PQZ) was used as the search
model. The structure refinement was continued with SHELXL-
97,51 for each refinement step, at least 10 cycles of conjugate gra-
dient minimization were performed, with restraints on bond dis-
tances, angles, and B-values. Intermittent cycles of model
building were done with the program COOT.52 The coordinates
have been deposited in the PDB (http://www.rcsb.org/pdb/) with
access codes: 3BGC and 3BGB.
5.2.4. General procedure C for the preparation of N-Boc-protec-
ted bis-arylsulfonamides of type 4
To a stirred solution of the corresponding N-Boc-protected oli-
goamine 3 (5.0 mmol) in CH2Cl2 (17 mL), triethylamine (1.42 g,
1.95 mL, 14.0 mmol) was added, followed by slow addition of the
respective arylsulfonyl chloride (11.0 mmol) in CH2Cl2 (10 mL) at

A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
8581
0 °C. The reaction mixture was allowed to reach rt, stirred for addi-
tional 12 h, and quenched with H2O (42 mL). After addition of
MTBE (42 mL), the aqueous phase was separated and extracted
with MTBE (3ꢂ 17 mL). The combined organic layers were washed
with a satd NaHCO3-solution (1ꢂ 50 mL) and brine (1ꢂ 50 mL),
dried over MgSO4, filtered, and evaporated. The residual oil was
further purified by FC yielding the analytically pure N-Boc-pro-
tected bis-arylsulfonamides 4a–f.
allowing the reaction mixture to reach rt, stirring was continued
overnight. The reaction was quenched by addition of a satd NaH-
CO3-solution (10–20 mL) followed by CH2Cl2 (10–20 mL). The
aqueous layer was extracted with CH2Cl2 (2ꢂ 25–50 mL). The com-
bined organic phases were washed with brine (2ꢂ 30–60 mL),
dried over MgSO4, filtered, and evaporated. The resid was further
purified by MPLC rendering the respective derivatives 11a–c.
5.2.10. Bis-[2-(2,2,2-trifluoro-acetylamino)-ethyl]-carbamic acid
tert-butyl ester (2a)
5.2.5. General procedure D for benzylation of N-Boc-protected
bis-arylsulfonamides of type 4
Following the general procedure A, employment of oligoamine
1a (5.15 g, 50.0 mmol) yielded 14.12 g (71%) of 2a. The compound
exhibited identical physical and spectroscopic data to those de-
scribed earlier.43 Colorless crystals; mp: 125 °C; 1H NMR
(500 MHz, DMSO-d6) d = 1.36 (s, 9H), 3.30 (s, 8H), 9.41 (br s, 2H).
To a suspension of the respective N-Boc-protected bis-arylsulf-
onamide 4 (1.50 mmol) and anhydrous K2CO3 (4.50 mmol) in dry
CH3CN (12 mL), a catalytic amount of KI and benzyl bromide
(3.30 mmol) were added, and the reaction mixture was stirred
for 3 h under reflux. After cooling to rt, insoluble material was fil-
tered off, the remaining residue washed with MTBE, and the com-
bined filtrates concentrated in vacuum. The remaining oily residue
was purified by FC giving rise to the analytically pure bis-benzylat-
ed N-Boc-protected bis-arylsulfonamides 5a–f.
5.2.11. [2-(2,2,2-Trifluoro-acetylamino)-ethyl]-[3-(2,2,2-trifluoro-
acetylamino)-propyl]-carbamic acid tert-butyl ester (2b)
According to general procedure A, utilization of oligoamine 1b
(5.86 g, 50.0 mmol) gave rise to 17.66 g (86%) of 2b. Colorless crys-
tals; mp: 113 °C; 1H NMR (500 MHz, DMSO-d6) d = 1.38 (s, 9H),
1.69 (sm, 2H, J = 6.9), 3.16 (sm, 4H, J = 7.0), 3.26–3.30 (m, 4H),
9.39 (br s, 2H); 13C NMR (125 MHz, CDCl3, rotamers) d = 27.1,
27.7, 28.0, 37.2, 38.0, 44.3, 44.9, 45.1, 45.3, 78.9, 116.0 (q,
J = 287.9), 116.1 (q, J = 287.9), 154.8, 155.0, 156.4 (q, J = 36.5),
156.5 (q, J = 36.5); MS (ESI) m/z = 432 (100, [M+Na]+), 841 (25,
[2M+Na]+); HRMS (ESI) [M+NH4]+ Calcd 427.1780, found
427.1806; Anal. Calcd for C14H21F6N3O4: C, 41.08; H, 5.17; N,
10.27. Found: C, 41.20; H, 4.96; N, 10.20.
5.2.6. General procedure E for Boc-deprotection of N-Boc-protected
bis-arylsulfonamides of type 5
A solution of the respective N-Boc-protected bis-arylsulfona-
mide 5 (ꢃ0.5 mmol) in 2 M HCl in Et2O (8 mL) was stirred at rt un-
til TLC indicated completion of the reaction (12–36 h). The solution
was decanted from the resulting precipitate, which was thoroughly
washed with dry ether and finally dried in vacuum rendering the
N-Boc-deprotected
bis-arylsulfonamides
6a–c
as
their
hydrochlorides.
5.2.12. Bis-[3-(2,2,2-trifluoro-acetylamino)-propyl]-carbamic
acid tert-butyl ester (2c)
5.2.7. General procedure F for the reduction of nitro groups
and concomitant Boc-deprotection of N-Boc-protected bis-nitro-
arylsulfonamides 5d–f and 8b
Following general procedure A, employment of oligoamine 1c
(6.56 g, 50.0 mmol) furnished 15.28 g (72%) of 2c. Colorless crys-
tals; mp: 68 °C; 1H NMR (500 MHz, DMSO-d6) d = 1.38 (s, 9H),
1.68 (sm, 4H, J = 7.1), 3.11–3.18 (m, 8H); 13C NMR (125 MHz, CDCl3,
rotamers) d = 27.1, 27.9, 28.2, 36.1, 37.4, 43.0, 44.4, 81.0, 115.9 (q,
J = 286.9), 156.8, 157.3 (q, J = 37.0); MS (ESI) m/z = 324 (100, [M–
CO2–C4H8+H]), 441 (100, [M+NH4]+), 864 (9, [2M+NH4]+); HRMS
(ESI) Calcd 441.1937, found 441.1930; Anal. Calcd for
The corresponding Boc-protected nitroarylsulfonamide (0.13–
0.46 mmol) was suspended in 32% HCl (5–15 mL). After addition
of SnCl2ꢀ2H2O (8 equiv), the suspension was refluxed for 3.5 h
and subsequently allowed to reach rt. A 10 M NaOH-solution was
carefully added until pH 12 was obtained. The resulting mixture
was extracted with CH2Cl2 (3ꢂ 10 mL), and the combined organic
layers were washed with brine (2ꢂ 10 mL), dried over MgSO4, fil-
tered, and evaporated. The residual oil was purified by FC
(CH2Cl2/MeOH 95:5) and the product-containing fractions were
combined and concentrated. After treatment with 2 M HCl in
Et2O (8–20 mL), a precipitate was formed which was triturated
with Et2O and dried in vacuum yielding compounds 6d–f and 9b
as their hydrochlorides.
C
15H23F6N3O4ꢀH2O: C, 40.82; H, 5.71; N, 9.52. Found: C, 40.73; H,
5.56; N, 9.34.
5.2.13. Bis-(2-amino-ethyl)-carbamic acid tert-butyl ester (3a)
According to general procedure B, usage of bis-trifluoroaceta-
mide 2a (3.95 g, 10.0 mmol) yielded 1.89 g (93%) of 3a. The com-
pound exhibited identical physical and spectroscopic data to
those described earlier.43 Colorless oil; 1H NMR (500 MHz, CDCl3)
d = 1.23 (s, 4H), 1.43 (s, 9H), 2.81 (t, 4H, J = 6.9), 3.25 (br s, 4H).
5.2.8. General procedure G for the preparation of N-Boc-protected
bis-benzylamines of type 10 via reductive amination
To a solution of the corresponding N-Boc-protected oligoamine
of type 3 (6.1–9.8 mmol) in MeOH (100 mL), benzaldehyde (2
equiv) and 10% of Pd/BaSO4 were added. A permanent H2-atmo-
sphere was applied to the reaction flask and the mixture stirred
for 16 h. The catalyst was removed by filtration through a pad of
Celite, and the filtrate was concentrated in vacuum yielding the
corresponding N-Boc-protected bis-benzylamine derivatives
10a–c.
5.2.14. (2-Amino-ethyl)-(3-amino-propyl)-carbamic acid tert-
butyl ester (3b)
According to general procedure B, utilization of bis-trifluoroac-
etamide 2b (4.09 g, 10.0 mmol) gave rise to 1.71 g (79%) of 3b. Col-
orless oil; 1H NMR (500 MHz, DMSO-d6) d = 1.44 (s, 9H), 1.57 (br s,
4H), 1.65 (sm, 2H, J = 6.9), 2.69 (t, 2H, J = 6.6), 2.80 (t, 2H, J = 6.6),
3.21 (br s, 4H); 13C NMR (125 MHz, CDCl3, rotamers) d = 28.2,
31.4, 32.2, 38.8, 40.3, 40.5, 44.3, 44.8, 49.8, 79.3, 155.6; MS (EI)
m/z = 217 (1, [M]+), 188 (100, [MꢁC2H5]+), 175 (89, [MꢁC3H6]+),
132 (78), 131 (76), 127 (64), 119 (17); HRMS (ESI) Calcd
218.1869, found 218.1865.
5.2.9. General procedure H for the preparation of N-Boc-protected
bis-benzylated carboxamides of type 11 utilizing phenylacetic
acid chloride
To a stirred solution of the corresponding N-Boc-protected bis-
benzylated oligoamine of type 10 (0.68–1.5 mmol) in CH2Cl2 (5–
10 mL), phenylacetic acid chloride (2.2 equiv), triethylamine (2.5
equiv), and a catalytic amount of DMAP were added at 0 °C. After
5.2.15. Bis-(3-amino-propyl)-carbamic acid tert-butyl ester (3c)
Following general procedure A, employment of bis-trifluoroace-
tamide 2c (4.23 g, 10.0 mmol) gave rise to 2.22 g (96%) of 3c. Col-
orless oil; 1H NMR (500 MHz, CDCl3) d = 1.30 (s, 4H), 1.42 (s, 9H),

8582
A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
1.62 (sm, 4H, J = 6.9), 2.66 (t, 4H, J = 6.6), 3.23 (br s, 4H); 13C NMR
(125 MHz, CDCl3, rotamers) d = 28.0, 31.5, 32.1, 38.8, 39.0, 43.4,
43.9, 78.8, 155.3; MS (ESI) m/z = 232 (100, [M+H]+), 463 (80,
[2M+H]+); HRMS (ESI) Calcd 232.2025, found 232.2049; Anal. Calcd
for C11H25N3O2ꢀ0.5H2O: C, 54.97; H, 10.90; N, 17.48. Found: C,
54.59; H, 10.90; N, 17.08.
5.2.20. [2-(4-Nitro-benzenesulfonylamino)-ethyl]-[3-(4-nitro-
benzenesulfonylamino)-propyl]-carbamic acid tert-butyl ester
(4e)
Following the general procedure C, employment of 3b (0.71 g,
3.3 mmol) and 4-nitrobenzenesulfonyl chloride (1.61 g, 7.3 mmol)
yielded, after purification via FC (hexanes/EtOAc 3:2, then CH2Cl2/
MeOH 4:1), 1.41 g (73%) of bis-sulfonamide 4e. Colorless crystals;
mp: 81 °C; 1H NMR (500 MHz, DMSO-d6) d = 1.29 (s, 9H), 1.53 (br s,
2H), 2.72 (t, 2H, J = 7.3), 2.87 (t, 2H, J = 6.6), 3.05 (t, 2H, J = 7.1), 3.09
(t, 2H, J = 6.4), 8.01 (d, 4H, J = 8.9), 8.40 (d, 4H, J = 8.7); 13C NMR
(125 MHz, DMSO-d6, rotamers) d = 28.0, 28.6, 40.5, 40.9, 41.2,
44.7, 45.2, 46.6, 46.8, 79.0, 124.7, 128.1, 128.2, 146.1, 146.3,
149.7, 154.6; MS (ESI) m/z = 610 (100, [M+Na]+), 1197 (39,
[2M+Na]+); HRMS (ESI) Calcd 610.1254, found 610.1286; Anal.
Calcd for C22H29N5O10S2: C, 44.97; H, 4.97; N, 11.92; S, 10.91.
Found: C, 44.95; H, 4.85; N, 11.85; S, 10.56.
5.2.16. Bis-(2-benzenesulfonylamino-ethyl)-carbamic acid tert-
butyl ester (4a)
According to general procedure C, utilization of 3a (1.02 g,
5.0 mmol) and benzenesulfonyl chloride (1.42 mL, 11.0 mmol) fur-
nished, after FC (hexanes/EtOAc 1:1), 1.78 g (74%) of bis-sulfon-
amide 4a. Colorless crystals; mp: 58–62 °C; 1H NMR (500 MHz,
DMSO-d6) d = 1.32 (s, 9H), 2.80 (sm, 4H, J = 6.5), 3.12 (br s, 4H),
7.51–7.75 (m, 8H), 7.76–7.80 (m, 4H); 13C NMR (125 MHz,
DMSO-d6, rotamers) d = 28.2, 41.9, 42.2, 47.8, 48.5, 80.9, 126.6,
129.1, 132.6, 139.7, 156.1; MS (ESI) m/z = 506 (57, [M+Na]+), 989
(100, [2M+Na]+), 1472 (29, [3M+Na]+); HRMS (ESI) Calcd
484.1576, found 484.1553; Anal. Calcd for C21H29N3O6S2: C,
52.16; H, 6.04; N, 8.69. Found: C, 52.39; H, 5.89; N, 8.71.
5.2.21. Bis-[3-(4-nitro-benzenesulfonylamino)-propyl]-carbamic
acid tert-butyl ester (4f)
According to general procedure C, utilization of 3c (1.00 g,
4.3 mmol) and 4-nitrobenzenesulfonyl chloride (2.11 g, 9.5 mmol)
rendered 4f, which partly crystallized during work-up. The remain-
ing oily portion was purified by FC (hexanes/EtOAc 3:2, then
CH2Cl2/MeOH 3:1), overall 1.92 g (74%) of bis-sulfonamide 4f
was obtained. Yellow crystals; mp: 152 °C; 1H NMR (500 MHz,
DMSO-d6) d = 1.27 (s, 9H), 1.53 (sm, 4H, J = 6.7), 2.72 (t, 4H,
J = 7.1), 3.01 (t, 4H, J = 7.1), 3.30 (br s, 2H), 8.02 (d, 4H, J = 8.7),
8.40 (d, 4H, J = 8.7); 13C NMR (125 MHz, DMSO-d6) d = 28.0, 28.2,
40.2, 44.2, 78.7, 124.7, 128.2, 146.1, 149.7, 154.6; MS (ESI) m/
z = 602 (42, [M+H]+), 619 (100, [M+NH4]+), 624 (52, [M+Na]+);
HRMS (ESI) Calcd 624.1410, found 624.1403; Anal. Calcd for
5.2.17. (2-Benzenesulfonylamino-ethyl)-(3-benzenesulfonyl-
amino-propyl)-carbamic acid tert-butyl ester (4b)
Following general procedure C, employment of 4b (1.09 g,
5.0 mmol) and benzenesulfonyl chloride (1.42 mL, 11.0 mmol)
yielded, after purification via FC (hexanes/EtOAc 1:1), 1.60 g
(66%) of bis-sulfonamide 4b. Colorless crystals; mp: 55–60 °C; 1H
NMR (500 MHz, DMSO-d6) d = 1.29 (s, 9H), 1.46–1.54 (m, 2H),
2.65 (sm, 2H, J = 7.3), 2.79 (sm, 4H, J = 6.9), 3.01–3.10 (m, 4H),
7.40–7.61 (m, 8H), 7.77 (t, 4H, J = 6.8); 13C NMR (125 MHz,
DMSO-d6) d = 27.9, 28.2, 28.7, 39.8, 40.5, 41.7, 42.3, 44.1, 45.2,
46.9, 80.8, 126.6, 126.9, 129.0, 129.1, 132.4, 132.7, 139.7, 139.8,
156.1; MS (ESI) m/z = 515 (56, [M+H2O]+), 1012 (100,
[2M+H2O]+); HRMS (ESI) Calcd 498.1733, found 498.1748; Anal.
Calcd for C22H31N3O6S2: C, 53.10; H, 6.28; N, 8.44; S, 12.89. Found:
C, 52.75; H, 6.08; N, 8.26; S, 12.29.
C23H31N5O10S2: C, 45.91; H, 5.19; N, 11.64; S, 10.66. Found: C,
46.23; H, 5.37; N, 11.30; S, 10.50.
5.2.22. Bis-[2-(benzenesulfonyl-benzyl-amino)-ethyl]-carbamic
acid tert-butyl ester (5a)
According to general procedure D, utilization of bis-sulfon-
amide 4a (0.73 g, 1.5 mmol) furnished, after FC (hexanes/EtOAc
3:1), 0.47 g (47%) of bis-benzylated bis-sulfonamide 5a. Color-
less crystals; mp: 44 °C; 1H NMR (500 MHz, CDCl3) d = 1.35 (s,
9H), 2.95–3.03 (m, 4H), 3.05–3.14 (m, 4H), 4.33 (d, 4H,
J = 14.0), 7.10–7.29 (m, 10H), 7.49–7.53 (m, 6H), 7.80 (m, 4H);
13C NMR (125 MHz, CDCl3, rotamers) d = 28.3, 45.7, 45.9, 46.0,
46.8, 52.1, 52.7, 80.0, 127.1, 127.8, 128.0, 128.4, 128.6, 128.7,
129.1, 129.2, 132.5, 132.6, 135.9, 139.6, 140.0, 154.8; MS (ESI)
m/z = 686 (100, [M+Na]+), 1349 (49, [2M+Na]+); HRMS (ESI)
Calcd 664.2515, found 664.2508; Anal. Calcd for C35H41N3O6S2:
C, 63.32; H, 6.23; N, 6.33; S, 9.66. Found: C, 63.50; H, 6.11;
N, 6.22; S, 13.52.
5.2.18. Bis-(3-benzenesulfonylamino-propyl)-carbamic acid
tert-butyl ester (4c)
According to general procedure C, utilization of 3c (1.12 g,
5.0 mmol) and benzenesulfonyl chloride (1.42 mL, 11.0 mmol)
gave rise to 2.13 g (83%) of bis-sulfonamide 4c after FC (hexanes/
EtOAc 1:1). Colorless crystals; mp: 55–60 °C; 1H NMR (500 MHz,
CDCl3) d = 1.35 (s, 9H), 1.61 (br s, 4H), 2.88 (br s, 4H), 3.03–3.21
(m, 4H), 4.91 (s, 1H), 6.03 (s, 1H), 7.40–7.63 (m, 6H), 7.84 (d, 4H,
J = 7.1); 13C NMR (125 MHz, CDCl3, rotamers) d = 27.8, 28.2, 28.4,
39.8, 40.6, 43.1, 44.0, 80.3, 126.9, 129.0, 132.3, 132.6, 139.8,
140.2, 156.2; MS (ESI) m/z = 534 (85, [M+Na]+), 1045 (100,
[2M+Na]+), 1556 (20, [3M+Na]+); HRMS (ESI) Calcd 534.1708,
found 534.1674; Anal. Calcd for C23H33N3O6S2: C, 53.99; H, 6.50;
N, 8.21; S, 12.53. Found: C, 53.98; H, 6.46; N, 8.23; S, 12.45.
5.2.23. [2-(Benzenesulfonyl-benzyl-amino)-ethyl]-[3-(benzenesulfonyl-
benzyl-amino)-propyl]-carbamic acid tert-butyl ester (5b)
Following the general procedure D, employment of bis-sulfon-
amide 4b (1.39 g, 2.8 mmol) yielded, after purification via FC (hex-
anes/EtOAc 3:1), 1.25 g (66%) of bis-benzylated bis-sulfonamide
5b. Colorless crystals; mp: 46 °C; 1H NMR (500 MHz, CDCl3)
d = 1.30 (s, 9H), 1.36–1.39 (m, 2H), 2.60–3.22 (m, 8H), 4.21–4.33
(m, 4H), 7.21–7.35 (m, 10H), 7.45–7.64 (m, 6H), 7.81–7.88 (m,
4H); 13C NMR (125 MHz, CDCl3, rotamers) d = 27.3, 28.3, 44.6,
45.8, 46.1, 46.2, 52.1, 52.2, 53.2, 79.7, 127.06, 127.09, 127.8,
128.1, 128.3, 128.4, 128.6, 129.1, 132.5, 132.7, 136.0, 136.2,
139.3, 139.7, 155.0; MS (ESI) m/z = 700 (84, [M+Na]+), 1377 (100,
[2M+Na]+); HRMS (ESI) Calcd 700.2491, found 700.2522; Anal.
Calcd for C36H43N3O6S2: C, 63.79; H, 6.39; N, 6.20; S, 9.46. Found:
C, 63.70; H, 6.29; N, 6.12; S, 9.07.
5.2.19. Bis-[2-(4-nitro-benzenesulfonylamino)-ethyl]-carbamic
acid tert-butyl ester (4d)
According to general procedure C, utilization of 3a (1.02 g,
5.0 mmol)
and
4-nitrobenzenesulfonyl
chloride
(2.44 g,
11.0 mmol) furnished 1.71 g (60%) of bis-sulfonamide 4d after FC
(hexanes/EtOAc 3:2, then CH2Cl2/MeOH 4:1). Colorless crystals;
mp: 158 °C; 1H NMR (500 MHz, DMSO-d6) d = 1.33 (s, 9H), 2.89
(t, 4H, J = 6.6), 3.14 (t, 4H, J = 6.4), 8.02 (d, 4H, J = 8.7), 8.40 (d,
4H, J = 8.5); 13C NMR (125 MHz, DMSO-d6) d = 28.1, 40.8, 41.2,
47.5, 47.9, 79.2, 124.7, 128.1, 146.3, 149.7, 154.5; MS (ESI) m/
z = 596 (100, [M+Na]+), 612 (27, [M+K]+); HRMS (ESI) Calcd
596.1097, found 596.1118; Anal. Calcd for C21H27N5O10S2: C,
43.97; H, 4.74; N, 12.21; S, 11.18. Found: C, 44.32; H, 4.67; N,
12.13; S, 10.82.

A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
8583
5.2.24. Bis-[3-(benzenesulfonyl-benzyl-amino)-propyl]-carbamic
acid tert-butyl ester (5c)
5.2.28. Bis-(2-(phenylsulfonyl-N-benzyl-amino)-ethyl)-amine
hydrochloride (6a)
According to general procedure D, utilization of bis-sulfonamide
4c (0.73 g, 1.4 mmol) gave rise to 0.79 g (80%) of bis-benzylated
bis-sulfonamide 5c after FC (hexanes/EtOAc 3:1). Colorless crys-
tals; mp: 46 °C; 1H NMR (500 MHz, DMSO-d6) d = 1.24 (s, 9H),
1.15–1.32 (m, 4H), 2.65 (br s, 4H), 2.95 (t, 4H, J = 7.3), 4.30 (s,
4H), 7.21–7.45 (m, 10H), 7.64 (t, 4H, J = 7.3), 7.70 (t, 2H, J = 7.6),
7.84 (d, 4H, J = 7.3); 13C NMR (125 MHz, CDCl3, rotamers)
d = 27.2, 28.3, 43.9, 44.2, 46.2, 46.3, 52.4, 79.3, 126.8, 127.0,
127.9, 128.3, 128.6, 129.1, 132.5, 136.4, 139.6, 155.1; MS (ESI) m/
z = 714 (71, [M+Na]+), 1405 (100, [2M+Na]+); HRMS (ESI) Calcd
According to general procedure E, utilization of Boc-protected
bis-sulfonamide 5a (302 mg, 0.46 mmol) furnished 261 mg (96%)
of hydrochloride 6a. Colorless crystals; mp: 205 °C; 1H NMR
(500 MHz, DMSO-d6) d = 2.68 (br s, 4H), 3.29–3.33 (m, 4H), 4.34
(s, 4H), 7.25–7.32 (m, 10H), 7.70 (t, 4H, J = 7.6), 7.74 (t, 2H,
J = 7.5), 7.86 (d, 4H, J = 7.3), 8.93 (br s, 2H); 13C NMR (125 MHz,
DMSO-d6) d = 43.6, 44.9, 52.0, 126.9, 127.7, 128.1, 128.4, 129.5,
133.1, 136.1, 138.4; MS (ESI) m/z = 564 (100, [MꢁHCl+H]+), 586
(19, [MꢁHCl+Na]+), 1127 (26, [2Mꢁ2HCl+H]+), 1149 (19,
[2Mꢁ2HCl+Na]+); HRMS (ESI) Calcd 564.1991, found 564.1963;
Anal. Calcd for C30H34N3O4S2ClꢀH2O: C, 58.28; H, 5.87; N, 6.80; S,
10.37. Found: C, 58.28; H, 5.56; N, 6.84; S, 10.43.
714.2648, found 714.2680; Anal. Calcd for
C37H45N3O6S2: C,
64.23; H, 6.56; N, 6.07; S, 9.27. Found: C, 64.15; H, 6.51; N, 6.02;
S, 8.89.
5.2.29. (2-(Phenylsulfonyl-N-benzylamino)-ethyl)-(3-(phenyl-
sulfonyl-N-benzylamino)-propyl)-amine hydrochloride (6b)
Following the general procedure E, employment of Boc-pro-
tected bis-sulfonamide 5b (358 mg, 0.53 mmol) yielded 279 mg
(86%) of hydrochloride 6b. Colorless crystals; mp: 185 °C; 1H
NMR (500 MHz, DMSO-d6) d = 1.56 (sm, 2H, J = 7.6), 2.54 (s, 2H),
2.62 (s, 2H), 3.12 (t, 2H, J = 7.2), 3.32 (t, 2H, J = 7.3), 4.30 (s, 2H),
4.34 (s, 2H), 7.22–7.38 (m, 10H), 7.61–7.75 (m, 6H), 7.87 (t, 4H,
J = 7.1), 8.89 (br s, 2H); 13C NMR (125 MHz, DMSO-d6) d = 24.6,
43.8, 44.2, 45.0, 45.6, 51.6, 52.3, 127.0, 127.1, 127.7, 127.9, 128.2,
128.4, 128.6, 128.7, 129.6, 129.7, 133.1, 133.3, 136.4, 136.8,
138.5, 139.1; MS (ESI) m/z = 578 (100, [MꢁHCl+H]+), 1155 (19,
[2Mꢁ2HCl+H]+); HRMS (ESI) Calcd 578.2147, found 578.2148;
Anal. Calcd for C31H36N3O4S2Clꢀ0.5H2O: C, 59.74; H, 5.98; N, 6.74;
S, 10.29. Found: C, 60.14; H, 5.74; N, 6.79; S, 9.89.
5.2.25. Bis-{2-[benzyl-(4-nitro-benzenesulfonyl)-amino]-ethyl}-
carbamic acid tert-butyl ester (5d)
According to general procedure D, utilization of bis-nitrosulf-
onamide 4d (1.15 g, 2.0 mmol) furnished, after FC (hexanes/
EtOAc 3:2), 0.99 g (80%) of bis-benzylated bis-nitrosulfonamide
5d. Colorless crystals; mp: 167 °C; 1H NMR (500 MHz, CDCl3)
d = 1.34 (s, 9H), 3.03–3.11 (m, 4H), 3.17–3.21 (m, 2H), 3.22–
3.27 (m, 2H), 4.35 (s, 2H), 4.40 (s, 2H), 7.16–7.32 (m, 10H),
7.90 (d, 2H, J = 8.5), 7.94 (d, 2H, J = 8.5), 8.28 (d, 2H, J = 8.5),
8.33 (d, 2H, J = 8.5); 13C NMR (125 MHz, CDCl3, rotamers)
d = 28.3, 45.9, 46.2, 46.5, 47.1, 52.3, 52.8, 80.5, 124.3, 124.4,
128.3, 128.4, 128.6, 128.8, 128.9, 135.1, 145.4, 145.7, 149.9,
150.0, 154.8; MS (ESI) m/z = 776 (100, [M+Na]+), 1529 (37,
[2M+Na]+); HRMS (ESI) Calcd 776.2036, found 776.2079; Anal.
Calcd for C35H39N5O10S2: C, 55.76; H, 5.21; N, 9.29; S, 8.51.
Found: C, 55.56; H, 5.31; N, 8.96; S, 8.80.
5.2.30. Bis-(3-(phenylsulfonyl-N-benzylamino)-propyl)-amine
hydrochloride (6c)
According to general procedure E, utilization of Boc-protected
bis-sulfonamide 5c (321 mg, 0.46 mmol) gave rise to 260 mg
(89%) of hydrochloride 6c. Colorless crystals; mp: 175 °C; 1H
NMR (500 MHz, DMSO-d6) d = 1.59 (sm, 4H, J = 7.3), 2.50 (s,
4H), 3.14 (t, 4H, J = 7.3), 4.32 (s, 4H), 7.20–7.33 (m, 10H), 7.63
(t, 4H, J = 7.3), 7.71 (t, 2H, J = 7.3), 7.88 (d, 4H, J = 7.3), 8.74
(br s, 2H); 13C NMR (125 MHz, DMSO-d6) d = 24.5, 44.1, 45.7,
51.6, 127.0, 127.7, 128.2, 128.5, 129.5, 133.0, 136.8, 139.1; MS
(ESI) m/z = 592 (100, [MꢁHCl+H]+); HRMS (ESI) Calcd
592.2304, found 592.2311; Anal. Calcd for C32H38N3O4S2Cl: C,
61.18; H, 6.10; N, 6.69; S, 10.21. Found: C, 61.10; H, 5.99; N,
6.55; S, 9.67.
5.2.26. {2-[Benzyl-(4-nitro-benzenesulfonyl)-amino]-ethyl}-{3-
[benzyl-(4-nitro-benzenesulfonyl)-amino]-propyl}-carbamic
acid tert-butyl ester (5e)
Following the general procedure D, employment of bis-nitro-
sulfonamide 4e (0.93 g, 1.6 mmol) yielded, after purification via
FC (hexanes/EtOAc 3:2, then CH2Cl2), 0.89 g (73%) of bis-benzylat-
ed bis-nitrosulfonamide 5e. Colorless crystals; mp: 68 °C; 1H NMR
(500 MHz, DMSO-d6) d = 1.27 (s, 9H), 2.72 (br s, 2H), 2.81–2.90 (m,
2H), 2.97 (br s, 2H), 3.13 (br s, 2H), 4.35 (s, 2H), 4.37 (s, 2H), 7.16–
7.33 (m, 10H), 8.07–8.12 (m, 4H), 8.42 (d, 4H, J = 8.7); 13C NMR
(125 MHz, DMSO-d6) d = 26.6, 27.1, 28.0, 43.9, 44.6, 44.8, 45.4,
45.7, 45.9, 46.3, 51.5, 51.7, 52.2, 79.1, 124.9, 127.9, 128.0, 128.2,
128.6, 128.68, 128.72, 136.2, 136.4, 144.8, 144.9, 149.9, 154.3;
MS (ESI) m/z = 790 (100, [M+Na]+), 1557 (42, [2M+Na]+); HRMS
(ESI) Calcd 790.2193, found 790.2199; Anal. Calcd for
5.2.31. Bis-(2-(4-amino-phenylsulfonyl-N-benzylamino)-ethyl)-
amine hydrochloride (6d)
According to general procedure F, utilization of Boc-protected
bis-nitrosulfonamide 5d (100 mg, 0.13 mmol) furnished 60 mg
(64%) of bis-aminosulfonamide 6d as hydrochloride. Colorless
crystals; mp: 140 °C; 1H NMR (500 MHz, MeOH-d4) d = 2.67
(br s, 4H), 3.31 (br s, 4H), 4.30 (s, 4H), 7.05 (d, 4H, J = 8.0),
7.32 (s, 10H), 7.72 (d, 4H, J = 8.0); 13C NMR (125 MHz, DMSO-
d6) d = 43.9, 45.4, 52.4, 115.3, 125.6, 127.9, 128.3, 128.7,
129.2, 136.7, 150.3; MS (ESI) m/z = 594 (100, [MꢁHCl+H]+),
1187 (4, [2Mꢁ2HCl+H]+); HRMS (ESI) Calcd 594.2209, found
594.2237.
C36H41N5O10S2: C, 56.31; H, 5.38; N, 9.12; S, 8.35. Found: C,
56.13; H, 5.36; N, 8.98; S, 8.01.
5.2.27. Bis-{3-[benzyl-(4-nitro-benzenesulfonyl)-amino]-propyl}-
carbamic acid tert-butyl ester (5f)
According to general procedure D, utilization of bis-sulfonamide
4f (1.40 g, 2.3 mmol) gave rise to 0.99 g (55%) of bis-benzylated
bis-sulfonamide 5f after FC (hexanes/EtOAc 4:1, then CH2Cl2). Col-
orless crystals; mp: 113 °C; 1H NMR (500 MHz, DMSO-d6) d = 1.34
(s, 9H), 1.42–1.51 (m, 4H), 2.80 (t, 4H, J = 6.4), 3.10 (t, 4H, J = 7.3),
4.34 (s, 4H), 7.19–7.32 (m, 10H), 7.97 (d, 4H, J = 8.5), 8.35 (d, 4H,
J = 8.3); 13C NMR (125 MHz, CDCl3) d = 27.4, 28.3, 44.3, 46.4, 52.4,
79.7, 124.4, 128.2, 128.3, 128.8, 135.5, 145.5, 149.9, 155.2; MS
(ESI) m/z = 782 (13, [M+H]+), 804 (100, [M+Na]+), 820 (15,
[M+K]+); HRMS (ESI) Calcd 804.2349, found 804.2368; Anal. Calcd
for C37H43N5O10S2: C, 56.84; H, 5.54; N, 8.96; S, 8.20. Found: C,
56.84; H, 5.65; N, 8.81; S, 8.27.
5.2.32. (2-(4-Aminophenylsulfonyl-N-benzylamino)-ethyl)-(3-
(4-aminophenylsulfonyl-N-benzylamino)-propyl)-amine hydro-
chloride (6e)
Following the general procedure F, employment of Boc-pro-
tected bis-nitrosulfonamide 5e (200 mg, 0.26 mmol) yielded
65 mg (35%) of bis-aminosulfonamide 6e as hydrochloride. Color-
less crystals; mp: 115 °C; 1H NMR (500 MHz, MeOH-d4) d = 1.23–

8584
A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
1.31 (m, 2H), 2.01 (t, 2H, J = 7.1), 2.26 (t, 2H, J = 6.6), 2.95 (t, 2H,
J = 6.9), 3.06 (t, 2H, J = 6.6), 4.20 (s, 4H), 6.72 (d, 4H, J = 8.5), 7.21–
7.38 (m, 10H), 7.51–7.55 (m, 4H); 13C NMR (125 MHz, MeOH-d4)
d = 26.6, 46.4, 46.9, 47.4, 48.3, 54.7, 55.5, 119.4, 120.5, 129.5,
129.8, 130.2, 130.4, 130.5, 130.8, 131.0, 133.6, 137.5, 138.1,
145.7, 148.0; MS (ESI) m/z = 608 (100, [MꢁHCl+H]+); HRMS (ESI)
5.2.36. Bis-{2-[isobutyl-(4-nitro-benzenesulfonyl)-amino]-
ethyl}-carbamic acid tert-butyl ester (8b)
According to general procedure C, utilization of mono-Boc-pro-
tected oligoamine 7 (627 mg, 2.1 mmol) and 4-nitro benzenesulfo-
nyl chloride (1.03 g, 4.6 mmol) gave rise to 1.15 g (80%) of 8b after
FC (hexanes/EtOAc 4:1). Colorless crystals; mp: 125 °C; 1H NMR
(500 MHz, CDCl3) d = 0.88 (d, 6H, J = 6.4), 0.92 (d, 6H, J = 6.4), 1.45
(s, 9H), 1.84–1.94 (m, 2H), 2.94 (d, 2H, J = 7.3), 2.97 (d, 2H,
J = 7.6), 3.19–3.27 (m, 4H), 3.38–3.45 (m, 4H), 8.04 (d, 4H, J = 7.8),
8.38 (d, 4H, J = 8.5); 13C NMR (125 MHz, CDCl3) d = 19.7, 19.8,
26.9, 27.0, 28.3, 46.7, 47.0, 47.5, 57.1, 57.2, 80.5, 124.1, 124.4,
128.3, 128.4, 144.9, 145.1, 149.9, 154.8; MS (ESI) m/z = 708 (100,
[M+Na]+), 1393 (19, [2M+Na]+); HRMS (ESI) Calcd 708.2349, found
708.2371; Anal. Calcd for C29H43N5O10S2: C, 50.79; H, 6.32; N,
10.21; S, 9.35. Found: C, 50.81; H, 6.45; N, 10.17; S, 9.24.
Calcd 608.2365, found 608.2366; Anal. Calcd for
C31H38N5
O4S2Clꢀ3H2O: C, 53.32; H, 6.35; N, 10.03. Found: C, 53.09; H,
6.03; N, 10.06.
5.2.33. Bis-(3-(4-Aminophenylsulfonyl-N-benzylamino)-propyl)-
amine hydrochloride (6f)
According to general procedure F, utilization of Boc-protected
bis-nitrosulfonamide 5f (156 mg, 0.2 mmol) gave rise to 46 mg
(31%) of bis-aminosulfonamide 6f as hydrochloride. Yellow crys-
tals; mp: 103 °C; 1H NMR (500 MHz, MeOH-d4) d = 1.54–1.62 (m,
4H), 2.67 (t, 4H, J = 7.2), 3.20 (t, 4H, J = 6.4), 4.28 (s, 4H), 6.86 (d,
4H, J = 8.7), 7.33–7.40 (m, 10H), 7.65 (d, 4H, J = 8.7); 13C NMR
(125 MHz, DMSO-d6) d = 24.6, 44.2, 45.7, 51.8, 113.0, 123.5,
127.5, 128.2, 128.5, 129.1, 137.4, 153.1; MS (ESI) m/z = 622 (100,
[MꢁHCl+H]+); HRMS (ESI) Calcd 622.2522, found 622.2556; Anal.
Calcd for C32H40N5O4S2Clꢀ3H2O: C, 53.96; H, 6.51; N, 9.83. Found:
C, 53.89; H, 6.19; N, 9.56.
5.2.37. Bis-(2-(phenylsulfonyl-N-isobutyl-amino)-ethyl)-amine
hydrochloride (9a)
According to general procedure E, utilization of Boc-protected
bis-sulfonamide 8a (280 mg, 0.46 mmol) furnished, after purifica-
tion via FC (CH2Cl2/MeOH 98:2), 150 mg (62%) of hydrochloride
9a. Colorless crystals; mp: 45 °C; 1H NMR (500 MHz, CDCl3)
d = 0.89 (d, 12H, J = 6.6), 1.86 (sm, 2H, J = 6.9), 2.89 (d, 4H,
J = 7.6), 3.15 (t, 4H, J = 6.5), 3.38 (t, 4H, J = 6.6), 7.53 (t, 4H,
J = 7.8), 7.59 (t, 2H, J = 7.3), 7.86 (d, 4H, J = 8.7); 13C NMR
(125 MHz, CDCl3) d = 19.9, 27.1, 46.5, 47.7, 58.0, 127.4, 129.2,
132.8, 138.0; MS (ESI) m/z = 496 (100, [MꢁHCl+H]+), 518 (8,
[MꢁHCl+Na]+); HRMS (ESI) Calcd 496.2304, found 496.2323; Anal.
Calcd for C24H38N3O4S2Cl: C, 54.17; H, 7.20; N, 7.90. Found: C,
54.63; H, 7.27; N, 7.97.
5.2.34. Bis-(2-isobutylamino-ethyl)-carbamic acid tert-butyl
ester (7)
To a solution of 3a (0.85 g, 4.2 mmol) in dry MeOH (15 mL),
iso-butyric aldehyde (0.84 mL, 9.2 mmol) and triethyl orthofor-
mate (2.77 mL, 16.8 mmol) were added at rt. The reaction mix-
ture was stirred overnight, subsequently NaBH4 (0.95 g,
25.2 mmol) was added, and stirring was continued for 2 h.
The reaction mixture was quenched by careful addition of con-
cd HCl. The resulting precipitate was filtered off, washed with
MTBE, and discarded. The filtrate was made alkaline by addition
of a concd aqueous NaOH-solution and extracted with MTBE
(2ꢂ 50 mL). The combined organic phases were washed with
brine (2ꢂ 40 mL), dried over MgSO4, filtered, and evaporated.
The residual oil was purified by FC (CH2Cl2/MeOH 7:3 + 1%
NEt3) yielding 346 mg (24%) of mono-Boc-protected oligoamine
7. Colorless oil; 1H NMR (500 MHz, CDCl3) d = 0.88 (d, 12H,
J = 6.6), 1.44 (s, 9H), 1.64–1.75 (m, 4H), 2.40 (d, 4H, J = 6.9),
2.74 (t, 4H, J = 6.6), 3.31 (br s, 4H); 13C NMR (125 MHz, CDCl3)
d = 20.5, 28.26, 28.30, 47.6, 48.2, 57.7, 79.3, 155.7; MS (ESI) m/
z = 216 (100, [MꢁCO2ꢁC4H8+H]+), 316 (49, [M+H]+); HRMS (ESI)
5.2.38. Bis-(2-(4-amino-phenylsulfonyl-N-isobutylamino)-
ethyl)-amine hydrochloride (9b)
According to general procedure F, utilization of bis-nitrosulf-
onamide 8b (300 mg, 0.44 mmol) furnished 77 mg (28%) of 9b as
hydrochloride. Colorless crystals; mp: 123 °C; 1H NMR (500 MHz,
MeOH-d4) d = 0.94 (d, 12H, J = 6.4), 1.91 (sm, 2H, J = 6.6), 2.94 (d,
4H, J = 7.3), 3.33 (br s, 4H), 3.41 (t, 4H, J = 6.2), 7.12 (d, 4H,
J = 8.5), 7.78 (d, 4H, J = 8.7); 13C NMR (125 MHz, MeOH-d4)
d = 20.7, 28.6, 47.0, 49.4, 59.8, 119.5, 131.0, 131.3, 147.4; MS
(ESI) m/z = 526 (100, [MꢁHCl+H]+), 1051 (4, [2Mꢁ2HCl+H]+);
HRMS (ESI) Calcd 526.2522, found 526.2515; Anal. Calcd for
C
24H40N5O4S2Clꢀ2.5H2O: C, 47.47; H, 7.47; N, 11.53. Found: C,
47.45; H, 7.02; N, 11.27.
5.2.39. Bis-(2-benzylamino-ethyl)-carbamic acid tert-butyl
ester (10a)
Calcd 316.2964, found 316.2989; Anal. Calcd for
C17H37N3
O2ꢀ0.5H2O: C, 62.92; H, 11.80; N, 12.95. Found: C, 63.28; H,
According to general procedure G, utilization of mono-Boc-pro-
tected oligoamine 3a (1.34 g, 6.1 mmol) furnished 0.89 g (49%) of
mono-Boc-protected bis-benzylamine 10a after FC (CH2Cl2/MeOH,
95:5 then 9:1). Yellow oil; 1H NMR (500 MHz, CDCl3) d = 1.38 (s,
9H), 2.76 (br s, 4H), 3.33 (br s, 4H), 3.76 (s, 4H) 7.22–7.32 (m,
10H); 13C NMR (125 MHz, CDCl3) d = 28.3, 47.5, 47.7, 53.5, 79.5,
126.9, 127.9, 128.3, 140.0, 155.8; MS (ESI) m/z = 384 (100,
[M+H]+), 767 (34, [2M+H]+); HRMS (ESI) Calcd 384.2651, found
384.2624; Anal. Calcd for C23H33N3O2ꢀH2O: C, 68.80; H, 8.79; N,
10.46. Found: C, 68.80; H, 8.44; N, 10.22.
11.59; N, 13.21.
5.2.35. Bis-[2-(benzenesulfonyl-isobutyl-amino)-ethyl]-carbamic
acid tert-butyl ester (8a)
According to general procedure C, utilization of mono-Boc-
protected oligoamine 7 (423 mg, 1.3 mmol) and benzenesulfonyl
chloride (0.40 mL, 3.1 mmol) furnished, after FC (hexanes/EtOAc
4:1), 670 mg (83%) of 8a. Colorless crystals; mp: 111 °C; 1H
NMR (500 MHz, CDCl3) d = 0.89 (d, 6H, J = 6.6), 0.94 (d, 6H,
J = 6.6), 1.45 (s, 9H), 1.83–1.94 (m, 2H), 2.88 (d, 2H, J = 7.3),
2.90 (d, 2H, J = 7.3), 3.15 (t, 4H, J = 8.0), 3.35–3.43 (m, 4H),
7.49–7.60 (m, 6H), 7.85 (t, 4H, J = 8.6); 13C NMR (125 MHz,
CDCl3, rotamers) d = 19.9, 20.0, 27.0, 27.1, 28.4, 46.9, 47.1, 47.6,
57.2, 57.5, 80.2, 127.16, 127.21, 129.1, 132.4, 139.1, 139.3,
155.0; MS (ESI) m/z = 618 (100, [M+Na]+), 1213 (23, [2M+Na]+);
HRMS (ESI) Calcd 618.2648, found 618.2616; Anal. Calcd for
5.2.40. (2-Benzylamino-ethyl)-(3-benzylamino-propyl)-carbamic
acid tert-butyl ester (10b)
Following the general procedure G, employment of mono-Boc-
protected oligoamine 3b (2.10 g, 9.6 mmol) yielded 1.87 g (98%)
of mono-Boc-protected bis-benzylamine 10b. Brownish oil; 1H
NMR (500 MHz, CDCl3) d = 1.30–1.40 (m, 9H), 1.71 (sm, 2H,
J = 6.9), 2.60 (t, 2H, J = 6.9), 2.74–2.81 (m, 2H), 3.29 (br s, 4H),
3.76 (s, 2H), 3.79 (s, 2H), 7.20–7.37 (m, 10H); 13C NMR
C29H45N3O6S2: C, 58.46; H, 7.61; N, 7.05; S, 10.76. Found: C,
58.14; H, 7.59; N, 7.09; S, 10.90.

A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
8585
(125 MHz, CDCl3, rotamers) d = 28.3, 28.9, 45.0, 45.4, 46.3, 46.8,
47.4, 53.5, 53.8, 79.1, 126.58, 126.64, 127.76, 127.83, 128.07,
128.09, 140.2, 155.5; MS (ESI) m/z = 298 (100, [MꢁCO2ꢁC4H8+H]+),
398 (91, [M+H]+), 795 (12, [2M+H]+); HRMS (ESI) Calcd 398.2808,
found 398.2779; Anal. Calcd for C24H35N3O2: C, 72.51; H, 8.87; N,
10.57. Found: C, 71.96; H, 8.90; N, 10.80.
26.4, 27.3, 28.2, 40.8, 43.9, 44.2, 45.0, 48.1, 48.2, 51.25,
51.33, 79.1, 79.4, 79.8, 126.2, 126.6, 126.7, 127.1, 127.2,
127.4, 127.5, 127.9, 128.4, 128.5, 128.72, 128.77, 134.86,
134.94, 136.4, 136.5, 137.4, 154.9, 155.1, 155.2, 170.7, 171.2;
MS (ESI) m/z = 670 (100, [M+Na]+), 1316 (66, [2M+Na]+); HRMS
(ESI) Calcd 670.3621, found 670.3620; Anal. Calcd for
C
41H49N3O4ꢀ0.5H2O: C, 74.97; H, 7.67; N, 6.40. Found: C,
5.2.41. Bis-(3-benzylamino-propyl)-carbamic acid tert-butyl
ester (10c)
74.94; H, 7.59; N, 6.56.
According to general procedure G, utilization of mono-Boc-pro-
tected oligoamine 3c (2.26 g, 9.8 mmol) gave rise to 1.76 g (96%) of
mono-Boc-protected bis-benzylamine 10c. Brownish oil; 1H NMR
(500 MHz, CDCl3) d = 1.42 (s, 9H), 1.71 (sm, 4H, J = 6.9), 2.60 (t,
4H, J = 6.9), 3.20 (br s, 2H), 3.26 (br s, 2H), 3.77 (s, 4H), 7.20–7.35
(m, 10H); 13C NMR (125 MHz, CDCl3) d = 28.2, 29.0, 44.3, 44.8,
46.3, 46.5, 53.9, 79.0, 126.6, 127.9, 128.1, 140.3, 155.5; MS (ESI)
m/z = 312 (100, [MꢁCO2ꢁC4H8+H]+), 412 (95, [M+H]+), 823 (23,
[2M+H]+); HRMS (ESI) Calcd 412.2964, found 412.2967; Anal. Calcd
for C25H37N3O2: C, 72.95; H, 9.06; N, 10.21. Found: C, 72.27; H,
8.98; N, 9.90.
5.2.45. Bis-(2-(N-benzyl-phenylacetamido)-ethyl)-amine
hydrochloride (12a)
According to general procedure E, utilization of Boc-pro-
tected bis-carboxamide 11a (244 mg, 0.39 mmol) furnished
after FC (CH2Cl2/MeOH 98:2) 127 mg (58%) of hydrochloride
12a. Colorless crystals; mp: 38 °C; 1H NMR (500 MHz, CDCl3)
d = 2.49–3.09 (m, 4H), 3.49–3.69 (m, 4H), 3.78–3.84 (m, 4H),
4.57–4.68 (m, 4H), 7.00–7.38 (m, 20H); 13C NMR (125 MHz,
CDCl3) d = 40.4, 40.5, 40.7, 43.0, 43.4, 44.8, 45.7, 46.0, 46.2,
48.5, 48.7, 51.5, 51.8, 126.3, 126.5, 126.7, 127.3, 127.7, 127.9,
128.1, 128.3, 128.4, 128.6, 128.9, 129.1, 129.3, 129.6, 134.9,
135.0, 136.0, 136.1, 137.4, 171.5, 173.4, 174.0; MS (ESI) m/
z = 520 (100, [MꢁHCl+H]+), 1039 (11, [2Mꢁ2HCl+H]+); HRMS
(ESI) Calcd 520.2964, found 520.2995; Anal. Calcd for C34H38
N3O2ClꢀH2O: C, 71.12; H, 7.02; N, 7.32. Found: C, 71.51; H,
7.01; N, 7.51.
5.2.42. Bis-[2-(benzyl-phenylacetyl-amino)-ethyl]-carbamic
acid tert-butyl ester (11a)
According to general procedure H, utilization of bis-benzyl-
amine 10a (260 mg, 0.68 mmol) furnished 312 mg (74%) of bis-car-
boxamide 11a after FC (hexanes/EtOAc 7:3). Colorless resin; 1H
NMR (500 MHz, CDCl3) d = 1.39–1.47 (m, 9H), 3.05–3.52 (m, 8H),
3.64–3.81 (m, 8H), 4.50–4.61 (m, 4H), 7.05–7.13 (m, 2H), 7.15–
7.40 (m, 18H); 13C NMR (125 MHz, CDCl3, rotamers) d = 28.30,
28.33, 29.6, 40.2, 40.4, 40.6, 40.8, 44.8, 45.1, 45.3, 45.4, 45.7,
45.9, 48.1, 48.4, 51.3, 51.7, 52.3, 79.7, 80.2, 80.4, 81.1, 126.3,
126.6, 126.7, 126.8, 126.9, 127.0, 127.2, 127.3, 127.5, 127.6,
127.8, 127.9, 128.1, 128.5, 128.6, 128.7, 128.8, 128.9, 134.6,
134.8, 134.96, 135.04, 135.3, 136.2, 136.5, 137.2, 137.4, 137.5,
154.8, 155.0, 155.1, 155.4, 171.0, 171.4, 171.5, 171.7; MS (ESI) m/
z = 642 (100, [M+Na]+), 1261 (26, [2M+Na]+); HRMS (ESI) Calcd
642.3308, found 642.3322; Anal. Calcd for C39H45N3O4ꢀ0.5H2O: C,
74.49; H, 7.37; N, 6.68. Found: C, 74.76; H, 7.19; N, 6.28.
5.2.46. (2-(N-benzyl-phenylacetamido)-ethyl)-(3-(N-benzyl-
phenylacetamido)-propyl)-amine hydrochloride (12b)
Following general procedure E, employment of Boc-protected
bis-carboxamide 11b (359 mg, 0.39 mmol) yielded after column
chromatography (CH2Cl2/MeOH 95:5) 150 mg (67%) of hydrochlo-
ride 12b. Colorless crystals; mp: 51 °C; 1H NMR (500 MHz, CDCl3)
d = 1.82–2.01 (m, 2H), 2.45–3.25 (m, 4H), 3.43–3.88 (m, 8H),
4.55–4.73 (m, 4H), 7.02–7.43 (m, 20H); 13C NMR (125 MHz, CDCl3)
d = 24.3, 25.0, 25.4, 40.3, 40.4, 40.7, 42.6, 43.3, 45.1, 46.0, 47.6, 48.6,
51.3, 51.5, 51.7, 52.1, 126.3, 126.4, 126.54, 126.65, 126.72, 127.0,
127.2, 127.3, 127.7, 127.9, 128.0, 128.1, 128.3, 128.38, 128.42,
128.5, 128.7, 128.9, 129.0, 129.4, 129.5, 134.6, 134.8, 135.2,
135.5, 135.7, 136.0, 137.3, 171.0, 171.1, 171.4, 173.7, 173.8; MS
(ESI) m/z = 534 (100, [MꢁHCl+H]+), 1067 (7, [2Mꢁ2HCl+H]+);
HRMS (ESI) Calcd 534.3121, found 534.3099; Anal. Calcd for
5.2.43. [2-(Benzyl-phenylacetyl-amino)-ethyl]-[3-(benzyl-phenyl-
acetyl-amino)-propyl]-carbamic acid tert-butyl ester (11b)
Following the general procedure H, employment of bis-benzyl-
amine 10b (0.50 g, 1.3 mmol) yielded, after purification via FC
(hexanes/EtOAc 7:3), 0.68 g (77%) of bis-carboxamide 11b. Color-
less resin; 1H NMR (500 MHz, CDCl3) d = 1.35–1.40 (m, 9H), 1.61–
1.75 (m, 2H), 2.89–3.54 (m, 8H), 3.61–3.84 (m, 4H), 4.41–4.59
(m, 4H), 7.08–7.14 (m, 2H), 7.18–7.38 (m, 18H); 13C NMR
(125 MHz, CDCl3, rotamers) d = 26.1, 26.5, 26.8, 27.2, 27.6, 28.2,
40.4, 40.6, 40.7, 40.9, 43.7, 43.9, 44.1, 44.3, 44.9, 45.0, 45.6, 45.8,
46.4, 48.1, 48.5, 48.8, 51.3, 51.7, 52.0, 79.4, 79.7, 79.9, 126.20,
126.24, 126.6, 126.8, 127.2, 127.3, 127.4, 127.9, 128.2, 128.56,
128.64, 128.8, 129.2, 134.7, 134.9, 135.0, 136.3, 136.6, 137.5,
154.9, 155.1, 170.8, 171.16, 171.24, 171.5; MS (ESI) m/z = 656
(100, [M+Na]+); HRMS (ESI) Calcd 656.3464, found 656.3469; Anal.
Calcd for C40H47N3O4ꢀ0.5H2O: C, 74.74; H, 7.53; N, 6.54. Found: C,
74.44; H, 7.48; N, 6.28
C
35H40N3O2ClꢀH2O: C, 71.47; H, 7.20; N, 7.14. Found: C, 71.12; H,
7.12; N, 6.87.
5.2.47. Bis-(3-(N-benzyl-phenylacetamido)-propyl)-amine
hydrochloride (12c)
According to general procedure E, utilization of Boc-protected
bis-carboxamide 11c (320 mg, 0.49 mmol) furnished after FC
(CH2Cl2/MeOH 98:2, then 4:1) 181 mg (62%) of hydrochloride
12c. Colorless resin; 1H NMR (500 MHz, CDCl3) d = 1.95–2.15 (m,
4H), 2.49–2.81 (m, 4H), 3.29–3.51 (m, 4H), 3.70–3.80 (m, 4H),
4.56–4.61 (m, 4H), 7.09–7.43 (m, 20H), 9.55 (br s, 2H); 13C NMR
(125 MHz, CDCl3) d = 24.3, 24.4, 24.7, 40.3, 40.5, 40.9, 42.8, 43.1,
44.2, 45.0, 45.1, 45.4, 47.6, 51.3, 51.6, 126.3, 126.8, 127.0, 127.1,
127.3, 127.8, 128.0, 128.1, 128.5, 128.6, 128.7, 128.8,
128.97,129.04, 134.4, 134.6, 135.0, 135.4, 135.7, 137.2, 171.0,
173.1, 173.6; MS (ESI) m/z = 548 (100, [MꢁHCl+H]+); HRMS (ESI)
5.2.44. Bis-[3-(benzyl-phenylacetyl-amino)-propyl]-carbamic
acid tert-butyl ester (11c)
Calcd
548.3277,
found
548.3263;
Anal.
Calcd
for
C
36H42N3O2Clꢀ2H2O: C, 69.71; H, 7.48; N, 6.77. Found: C, 69.54;
According to general procedure H, utilization of bis-benzyl
amine 10c (615 mg, 1.5 mmol) gave rise to 693 mg (72%) of
bis-carboxamide 11c after FC (hexanes/EtOAc 3:2, then 1:1).
Colorless resin; 1H NMR (500 MHz, CDCl3) d = 1.32–1.36 (m,
9H), 1.46–1.78 (m, 4H), 2.65–3.35 (m, 8H), 3.69 (s, 2H), 3.75
(s, 2H), 4.49 (s, 2H), 4.59 (s, 2H), 7.03–7.13 (m, 2H), 7.15–
7.40 (m, 18H); 13C NMR (125 MHz, CDCl3, rotamers) d = 26.0,
H, 7.15; N, 6.71.
Acknowledgments
We thank the Fonds der Chemischen Industrie (FCI) and
the Deutsche Pharmazeutische Gesellschaft (DPhG) for funding.

8586
A. Blum et al. / Bioorg. Med. Chem. 16 (2008) 8574–8586
Folmer, R. H. A.; Geschwindner, S.; Koether, G.; Kolmodin, K.; Krumrine, J.;
References and notes
Mauger, R. C.; Murray, C. W.; Olsson, L. L.; Patel, S.; Spear, N.; Tian, G. J. Med.
Chem. 2007, 50, 5912.
31. Coburn, C. A.; Stachel, S. J.; Jones, K. G.; Steele, T. G.; Rush, D. M.; DiMuzio, J.;
Pietrak, B. L.; Lai, M.-T.; Huang, Q.; Lineberger, J.; Jin, L.; Munshi, S.; Katharine
Holloway, M.; Espeseth, A.; Simon, A.; Hazuda, D.; Graham, S. L.; Vacca, J. P.
Bioorg. Med. Chem. Lett. 2006, 16, 3635.
32. Stauffer, S. R.; Stanton, M. G.; Gregro, A. R.; Steinbeiser, M. A.; Shaffer, J. R.;
Nantermet, P. G.; Barrow, J. C.; Rittle, K. E.; Collusi, D.; Espeseth, A. S.; Lai, M.-T.;
Pietrak, B. L.; Holloway, M. K.; McGaughey, G. B.; Munshi, S. K.; Hochman, J. H.;
Simon, A. J.; Selnick, H. G.; Graham, S. L.; Vacca, J. P. Bioorg. Med. Chem. Lett.
2007, 17, 1788.
33. Oefner, C.; Binggeli, A.; Breu, V.; Bur, D.; Clozel, J.-P.; D’Arcy, A.; Dorn, A.;
Fischli, W.; Grüninger, F.; Güller, R.; Hirth, G.; Märki, H. P.; Mathews, S.; Müller,
M.; Ridley, R. G.; Stadler, H.; Vieira, E.; Wilhelm, M.; Winkler, F. K.; Wostl, W.
Chem. Biol. 1999, 6, 127.
34. Vieira, E.; Binggeli, A.; Breu, V.; Bur, D.; Fischli, W.; Güller, R.; Hirth, G.; Märki,
H. P.; Müller, M.; Oefner, C.; Scalone, M.; Stadler, H.; Wihelm, M.; Wostl, W.
Bioorg. Med. Chem. Lett. 1999, 9, 1397.
35. Güller, R.; Binggeli, A.; Breu, V.; Bur, D.; Fischli, W.; Hirth, G.; Jenny, C.; Kansy,
M.; Montavon, F.; Müller, M.; Oefner, C.; Stadler, H.; Vieira, E.; Wilhelm, M.;
Wostl, W.; Märki, H. P. Bioorg. Med. Chem. Lett. 1999, 9, 1403.
36. Prade, L.; Jones, A. F.; Boss, C.; Richard-Bildstein, S.; Meyer, S.; Binkert, C.; Bur,
D. J. Biol. Chem. 2005, 280, 23837.
37. Carcache, D. A.; Hörtner, S. R.; Bertogg, A.; Binkert, C.; Bur, D.; Märki, H. P.;
Dorn, A.; Diederich, F. ChemBioChem 2002, 3, 1137.
38. Hof, F.; Schütz, A.; Fäh, C.; Meyer, S.; Bur, D.; Liu, J.; Goldberg, D. E.; Diederich,
F. Angew. Chem., Int. Ed. 2006, 45, 2138.
1. Suguna, K.; Padlan, E. A.; Smith, C. W.; Carlson, W. D.; Davies, D. R. Proc. Natl.
Acad. Sci. U.S.A. 1987, 84, 7009.
2. Northrop, D. B. Acc. Chem. Res. 2001, 34, 790.
3. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157.
4. DeLano, W. L., DeLano Scientific, San Carlos, CA, 2002.
5. Dunn, B. M. Chem. Rev. 2002, 102, 4431.
6. Cooper, J. B. Curr. Drug Targets 2002, 3, 155.
7. Rawlings, N. D., Morton, F. R., Barrett, A. J., Nucleic Acids Res. 2006, 34, D270.
http://merops.sanger.ac.uk/.
8. Eder, J.; Hommel, U.; Cumin, F.; Martoglio, B.; Gerhartz, B. Curr. Pharm. Des.
2007, 13, 271.
9. Greenlee, W. J. Med. Res. Rev. 1990, 10, 173.
10. Tice, C. M.; Anthony, W.. In Annu. Rep. Med. Chem; Academic Press, 2006; Vol.
41, p 155.
11. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
12. Durham, T. B.; Shepherd, T. A. Curr. Opin. Drug Discov. Devel. 2006, 9, 776.
13. Silva, A. M.; Lee, A. Y.; Gulnik, S. V.; Majer, P.; Collins, J.; Bhat, T. N.; Collins, P. J.;
Cachau, R. E.; Luker, K. E.; Gluzman, I. Y.; Francis, S. E.; Oksman, A.; Goldberg, D.
E.; Erickson, J. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10034.
14. Ersmark, K.; Samuelsson, B.; Hallberg, A. Med. Res. Rev. 2006, 26, 626.
15. Fujinaga, M.; Chernaia, M. M.; Tarasova, N. I.; Mosimann, S. C.; James, M. N. G.
Protein Sci. 1995, 4, 960.
16. Wlodawer, A.; Erickson, J. W. Annu. Rev. Biochem. 1993, 62, 543.
17. Meadows, D. C.; Gervay-Hague, J. ChemMedChem 2006, 1, 16.
18. Randolph, J. T.; DeGoey, D. A. Curr. Top. Med. Chem. 2004, 4, 1079.
19. Chrusciel, R. A.; Strohbach, J. W. Curr. Top. Med. Chem. 2004, 4, 1097.
20. Fitzgerald, P. M.; McKeever, B. M.; VanMiddlesworth, J. F.; Springer, J. P.;
Heimbach, J. C.; Leu, C.; Herber, W. K.; Dixon, R. A.; Darke, P. L. J. Biol. Chem.
1990, 265, 14209.
21. Prabu-Jeyabalan, M.; Nalivaika, E.; Schiffer, C. A. Structure 2002, 10, 369.
22. Pomerantz, R. J.; Horn, D. L. Nat. Med. 2003, 9, 867.
23. Göschke, R.; Stutz, S.; Rasetti, V.; Cohen, N. C.; Rahuel, J.; Rigollier, P.; Baum, H.
P.; Forgiarini, P.; Schnell, C. R.; Wagner, T.; Gruetter, M. G.; Fuhrer, W.;
Schilling, W.; Cumin, F.; Wood, J. M.; Maibaum, J. J. Med. Chem. 2007, 50, 4818.
24. Cohen, N. C. Chem. Biol. Drug Des. 2007, 70, 557.
39. Boss, C.; Corminboeuf, O.; Grisostomi, C.; Meyer, S.; Jones, A. F.; Prade, L.;
Binkert, C.; Fischli, W.; Weller, T.; Bur, D. ChemMedChem 2006, 1, 1341.
40. John, V.; Beck, J. P.; Bienkowski, M. J.; Sinha, S.; Heinrikson, R. L. J. Med. Chem.
2003, 46, 4625.
41. Blum, A.; Böttcher, J.; Heine, A.; Klebe, G.; Diederich, W. E. J. Med. Chem. 2008,
51, 2078.
42. Czodrowski, P.; Sotriffer, C. A.; Klebe, G. J. Chem. Inf. Model. 2007, 47, 1590.
ˇ
´
ˇ
43. Košcová, S.; Budšínsky, M.; Hodacová, J. Collect. Czech. Chem. Commun. 2003, 68, 744.
44. Kim, E. E.; Baker, C. T.; Dwyer, M. D.; Murcko, M. A.; Rao, B. G.; Tung, R. D.;
Navia, M. A. J. Am. Chem. Soc. 1995, 117, 1181.
25. Jensen, C.; Herold, P.; Brunner, H. R. Nat. Rev. Drug Disc. 2008, 7, 399.
26. Thompson, L. A.; Tebben, A. J.. In Annu. Rep. Med. Chem.; Academic Press, 2001;
Vol. 36, p 247.
45. Taylor, A.; Brown, D. P.; Kadam, S.; Maus, M.; Kohlbrenner, W. E.; Weigl, D.;
Turon, M. C.; Katz, L. Appl. Microbiol. Biotechnol. 1992, 37, 205.
46. Toth, M. V.; Marshall, G. R. Int. J. Pept. Protein Res. 1990, 36, 544.
47. Hill, J.; Tyas, L.; Phylip, L. H.; Kay, J.; Dunn, B. M.; Berry, C. FEBS Lett. 1994, 352,
155.
48. Wang, G. T.; Chung, C. C.; Holzman, T. F.; Krafft, G. A. Anal. Biochem. 1993, 210,
351.
49. Otwinowski, Z.; Minor, W.. In Methods Enzymol; Carter, C. W., Jr., Ed.; Academic
Press, 1997; Vol. 276, p 307.
50. Storoni, L. C.; McCoy, A. J.; Read, R. J. Acta Crystallogr., Sect D: Biol. Crystallogr.
2004, 60, 432.
51. Sheldrick, G. M.; Schneider, T. R.. In Methods Enzymol; Charles, W. C. J., Robert,
M. S., Eds.; Academic Press, 1997; Vol. 277, p 319.
27. Arrowsmith, R. J.; Carter, K.; Dann, J. G.; Davies, D. E.; Harris, C. J.; Morton, J. A.;
Lister, P.; Robinson, J. A.; Williams, D. J. J. Chem. Soc., Chem. Commun. 1986, 755.
28. Yang, W.; Lu, W.; Lu, Y.; Zhong, M.; Sun, J.; Thomas, A. E.; Wilkinson, J. M.;
Fucini, R. V.; Lam, M.; Randal, M.; Shi, X. P.; Jacobs, J. W.; McDowell, R. S.;
Gordon, E. M.; Ballinger, M. D. J. Med. Chem. 2006, 49, 839.
29. Baxter, E. W.; Conway, K. A.; Kennis, L.; Bischoff, F.; Mercken, M. H.; DeWinter,
H. L.; Reynolds, C. H.; Tounge, B. A.; Luo, C.; Scott, M. K.; Huang, Y.; Braeken, M.;
Pieters, S. M. A.; Berthelot, D. J. C.; Masure, S.; Bruinzeel, W. D.; Jordan, A. D.;
Parker, M. H.; Boyd, R. E.; Qu, J.; Alexander, R. S.; Brenneman, D. E.; Reitz, A. B. J.
Med. Chem. 2007, 50, 4261.
30. Edwards, P. D.; Albert, J. S.; Sylvester, M.; Aharony, D.; Andisik, D.; Callaghan,
O.; Campbell, J. B.; Carr, R. A.; Chessari, G.; Congreve, M.; Frederickson, M.;
52. Emsley, P.; Cowtan, K. Acta Crystallogr., Sect D: Biol. Crystallogr. 2004, 60, 2126.