Synthesis and biological evaluation of reversible inhibitors of IdeS, a bacterial cysteine protease and virulence determinant

Article abstract of DOI:10.1016/j.bmc.2009.03.026

Analogues of the irreversible protease inhibitors TPCK and TLCK have been synthesized and tested as inhibitors of the bacterial cysteine protease IdeS excreted by Streptococcus pyogenes. Eight compounds were identified as inhibitors of IdeS in an in vitro

Full text of DOI:10.1016/j.bmc.2009.03.026

Bioorganic & Medicinal Chemistry 17 (2009) 3463–3470
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of reversible inhibitors of IdeS,
a bacterial cysteine protease and virulence determinant
Kristina Berggren a,b, Björn Johansson c, Tomas Fex b, Jan Kihlberg d, Lars Björck c, Kristina Luthman a,
*
a Department of Chemistry, Medicinal Chemistry, University of Gothenburg, SE-412 96 Göteborg, Sweden
b Lead Generation, AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden
c Department of Clinical Science, Division of Infection Medicine, BMC, B14, SE-221 84 Lund, Sweden
d Medicinal Chemistry, AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogues of the irreversible protease inhibitors TPCK and TLCK have been synthesized and tested as
inhibitors of the bacterial cysteine protease IdeS excreted by Streptococcus pyogenes. Eight compounds
were identified as inhibitors of IdeS in an in vitro assay. The most potent compounds contained an alde-
hyde function, thus acting as efficient reversible inhibitors, nitrile and azide derivatives showed moderate
activity.
Received 31 October 2008
Revised 23 January 2009
Accepted 13 March 2009
Available online 19 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Cysteine proteases
Reversible inhibitors
IdeS
Streptococcus pyogenes
1. Introduction
been found, which is explained by the requirement for a specific
protein–protein interaction between IdeS and IgG before cleavage
Streptococcus pyogenes, a Gram-positive bacterium, is the caus-
ative agent for common and relatively mild human diseases such
as strep throat (pharyngitis) and skin infections (impetigo, erysip-
elas), but it is also responsible for the life-threatening conditions
streptococcal toxic shock syndrome and necrotizing fasciitis. In
addition, S. pyogenes infections give rise to clinically important
sequelae, and acute rheumatic fever following a throat or skin
infection is the most common cause of heart disease in children
world-wide. Several S. pyogenes virulence factors have been de-
scribed and among these is a secreted cysteine protease called IdeS
or Mac-1.1–3 IdeS (IgG-degrading enzyme of S. pyogenes) is a 35 kDa
enzyme that cleaves IgG by hydrolyzing the peptide bond between
two glycine residues in the hinge region of IgG, generating one
F(ab0)2 fragment and two 1/2 Fc fragments. IgG antibodies repre-
sent the dominating class of antibodies in human blood, and they
play an important role in immunity by recognizing invading micro-
organisms and promoting their phagocytosis and killing. The cleav-
age of IgG antibodies bound to the surface of S. pyogenes by IdeS,
therefore represents a sophisticated mechanism by which the
bacterium overcomes IgG attack.4 Moreover, a recent study has
demonstrated that the Fc fragments generated by IdeS cleavage
of IgG also interfere with human defences.5 IdeS has a unique
degree of specificity and apart from IgG no other substrate has
can occur.2,6 In many autoimmune conditions (rheumatoid arthri-
tis, systemic lupus, myasthenia gravis, immune thrombocytopenic
purpura (ITP), etc.) IgG autoantibodies binding to human mole-
cules contribute to the disease development, and they also cause
acute transplant rejection. Given this background and the unique
specificity of IdeS for IgG, the effect of IdeS in animal models of
autoimmune diseases has been investigated, and the enzyme was
found to prevent the development of rheumatoid arthritis in mice
and to cure mice from lethal ITP.7,8 So far IdeS has not been used to
treat autoimmune disease in humans, but if and when this hap-
pens, there could be a need for compounds that block and control
the activity of the enzyme. Moreover, the fact that IdeS protects S.
pyogenes against IgG attack, suggests that blocking the activity of
IdeS during S. pyogenes infection could be a new way of treating
these sometimes hyper-acute and life-threatening infections. The
present investigation was therefore undertaken to synthesize and
identify inhibitors of IdeS.
As mentioned above, IdeS is a cysteine protease for which IgG
is the only known substrate.2 IdeS is inhibited by well known
protease inhibitors such as iodoacetate, Z-LVG-CHN2, tosyl-
L-
phenylalanine chloromethyl ketone (TPCK) and tosyl- -lysine
L
chloromethyl ketone (TLCK), but not by E-64, another common
cysteine protease inhibitor (Fig. 1).2,9 These electrophilic inhibi-
tors have been used to characterize IdeS and bind covalently
via the catalytically active cysteine, acting as irreversible
inhibitors.10,11
* Corresponding author. Tel.: +46 31 7722894; fax: +46 31 7723480.
E-mail address: luthman@chem.gu.se (K. Luthman).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.026

3464
K. Berggren et al. / Bioorg. Med. Chem. 17 (2009) 3463–3470
O
O
O
O
H
N
H
N
Cl
O
S
A
R
B
R
N
H
CHN2
S
O
O
Enzyme
A
S-
Enzyme
Z-LVG-CHN 2
O
B
O
H
N
H
N
S
O-
S+
O
O
O
O
O-
Cl
Cl
Cl
R
R
S
Enzyme
Enzyme
NH2
1
2
TPCK,
NH
TLCK,
O
Figure 3. Nucleophilic attack by cycteine can occur at two positions in the
irreversible -chloro ketone inhibitor giving the covalent-enzyme-inhibitor com-
plex via either route A or route B.
a
O
H
N
HN
H2N
N
attacked by the cysteine is discussed (Fig. 3).11 Attack can occur
OH
H
O
either at the
a-position forming a complex through route A or at
O
the carbonyl carbon forming the same complex via the intermedi-
ates in route B. The inhibitor–enzyme thioether complex has been
crystallized, but the cyclic sulfonium intermediate has not been
detected.16
E-64
Figure 1. Irreversible inhibitors of cysteine proteases.
We have synthesized analogues of TLCK and TPCK with varia-
tions in the side chain and in the warhead, and tested their ability
to act as inhibitors of IdeS in an in vitro assay. The analogues were
designed to provide indications whether reactivity of the warhead
alone is responsible for the inhibitory activity, or if side chain inter-
actions with the active site also contribute to the inhibition.
Inhibitors containing aldehyde or nitrile functionalities can re-
act reversibly with the cysteine residue forming covalent com-
plexes which differ in geometry (Fig. 2).12
Up to now there are only a few known non-covalently bound
inhibitors for cysteine proteases. For example, development of
inhibitors to cathepsin S and K have been reported in the litera-
ture.13,14 These inhibitors are non-peptidic and are based on either
a pyrimidine scaffold or a piperidine–pyrazole fused system. They
are not chemically reactive and depend instead on other strong
interactions with the active site. Such unreactive inhibitors are
prone to be more selective, less toxic and therefore more useful
as drugs.
TPCK and TLCK derived from the tosyl-substituted amino acids
phenylalanine and lysine (Fig. 1), have shown inhibitory activity
against IdeS (Mac-1).9 They are also reported to have different po-
tency and binding modes towards papain, another cysteine prote-
ase.15 These differences imply that the side chain is of importance
for the recognition and interaction between the inhibitor and the
active site of the enzyme. Since the two side chains are different
in structure and also have different physicochemical properties,
further variation of the substitution pattern in this position could
lead to new, more selective and potent inhibitors. Improved inter-
action via the side chain would make the reactive warhead less
important and possibly new inhibitors binding reversibly or non-
covalently could be developed.
2. Results and discussion
2.1. Design
The design of novel potential IdeS inhibitors was based on the
structures of TLCK and TPCK. The structures of the warhead X
and the side chain R were varied (Fig. 4). If the warhead is less reac-
tive, the impact of the side chain should increase. Instead of the a-
chloro ketone moiety other electrophilic functional groups such as
aldehyde and nitrile were introduced. In addition, a tosyloxy func-
tionality was used as warhead to investigate if it could act as a
leaving group in a reaction with the active site cysteine. The war-
head was also exchanged for alcohol and azide functionalities. Fur-
thermore, the side chain structures were varied. The benzyl and 4-
aminobutyl groups corresponding to TPCK and TLCK, and also the
less flexible and sterically demanding isopropyl group, were used.
To analyse if the amino group in TLCK contributes to the interac-
tion, the unfunctionalized n-butyl group was also used as side
chain. We aimed for all possible combinations of warheads and
side chains, and after synthesis 23 different test compounds were
obtained.
TPCK and TLCK act via the highly reactive
a-chloro ketone war-
head. In the literature, the position where the
a-chloro ketone is
O
O
H
S
S-
R
R
H
N
H
N
Enzyme
Enzyme
S
X
Enzyme
O
O
R
N-
S
X = OH, OTs, N3, CN, CHO
R
R
S-
R = PhCH2-, CH3(CH2)3-,
(CH3)2CH-, H2N(CH2)4-,
BocNH(CH2)4-
Enzyme
Figure 2. The aldehyde and nitrile functionalities are attacked by the cysteine
residue to form covalent inhibitor–enzyme complexes.
Figure 4. Compounds synthesized as potential inhibitors of IdeS.

K. Berggren et al. / Bioorg. Med. Chem. 17 (2009) 3463–3470
3465
2.2. Synthesis
O
CbzNH(CH2)4
OH
The synthetic route to compounds 4a–8c in which the side
chain R is either benzyl, n-butyl or i-propyl is shown in Scheme 1.
NH2
10
Initially, the amino alcohol derivatives of
L-phenylalanine, L-
norleucine and -valine 3a–c were reacted with tosylchloride and
L
triethylamine in dichloromethane to form N-tosylated alcohols
(4a–c) in good yields (86–95%) (Scheme 1). Attempts to convert
the alcohols to the corresponding azides (6a–c) using Mitsunobu
conditions resulted in aziridine formation (9a–c) (Scheme 2).
However, addition of water to the reaction mixture facilitated
aziridine ring opening affording 6a in low yield (31%) from 9a. Try-
ing to compete with the intramolecular ring formation by using an
excess of NaN3 (3 or 10 equiv), still gave the aziridine as the only
product (35–47%). Aziridine formation has been reported earlier,
either when treating amino alcohols under Mitsunobu conditions
or the corresponding mesylate or tosylate using Et3N, KOH or
K2CO3 as the base.17,18
Instead, ditosylated intermediates (5a–c) were synthesized
using two equivalents of tosylchloride in reactions with the amino
alcohols (3a–c) to afford products in low to moderate yields (43–
63%). Pyridine was used as base and no aziridine formation was de-
tected. This is in agreement with the literature which states that
aziridine formation had been favoured if triethylamine had been
i, ii
O
N
S
OH
O
O
O
NH
S
11
Scheme 3. Reagents and conditions: (i) LiAlH4, THF; (ii) TsCl, Et3N, DMAP, DCM.
0 °C?rt, o.n.
used.19 Using azide and cyanide ion as nucleophiles to expel the
O-tosyl group, gave the corresponding azides (6a–c) and nitriles
(7a–c) in moderate or excellent yields, respectively, and with high
purities when DMSO was used as the solvent. Using KCN in DMF
did not result in any conversion of the starting material, due to
the low solubility of KCN in DMF. The nitriles 7a–c were reduced
by DIBAL in toluene at ꢀ65 °C followed by hydrolysis of the formed
imines to give the corresponding aldehydes 8a–c in low to moder-
ate yields (22–60%).20 All 15 compounds (4a–8c) synthesized by
this highly convergent route were tested for inhibitory activity
against IdeS.
R
R
i
OH
OH
TsNH
4a
NH2
3a
3b
3c
R = benzyl
R = n-butyl
R = i-propyl
91%
4b 95%
4c
86%
ii
For the amino butyl derivatives 14–21 the synthetic route de-
scribed above had to be modified. The amino alcohol derivative
of e-protected lysine is not commercially available. Using the expe-
rience from synthesis of compounds 4–8 we initially aimed for the
ditosylated derivative 5 (R = CbzNH(CH2)4). However, reduction of
R
R
N
OTs
iii
N+
TsNH
6a
N-
TsNH
the carboxylic acid group of
e-Cbz-protected lysine (10) using
5a
63%
65%
6b 76%
6c
LiAlH4 followed by tosylation gave instead the N-methylated deriv-
5b 43%
ative 11 as the sole product (Scheme 3).21
5c
44%
77%
Therefore, the protecting groups were changed and reduction of
e-Boc-protected lysine methyl ester (12) using NaBH4 was per-
iv
formed. This resulted in a low yield of the corresponding alcohol
(3, R = BocNH(CH2)4) due to difficulties monitoring the reaction
and the work-up of the polar product. As shown in Scheme 4, the
route was instead started by N-tosylation of 12 to obtain 13 in
excellent yield (95%). Now the reduction could be monitored by
TLC and afforded a high yield (94%) of alcohol 14. The alcohol
was O-tosylated in a rather low yield (37%) as pyridine had to be
used as base to avoid aziridine formation. The ditosylated product
(15) was further reacted with either sodium azide or sodium cya-
nide in DMSO to give compounds 16 and 17, respectively, in high
yields (97% and 93%, respectively). The nitrile (17) was converted
to the corresponding aldehyde (18) in the same manner as de-
scribed above, again in a low yield (26%). Finally, the Boc-protected
alcohol (14), azide (16) and nitrile (17) derivatives were deprotec-
ted by TFA in dichloromethane to give 19–21 in the yields 78%, 80%
and 93%, respectively. Unfortunately, the unprotected aldehyde
derivative could not be isolated with a satisfying purity.
R
R
H
v
N
TsNH
7a 87%
TsNH
O
8a 44%
7b
8b
98%
7c 94%
22%
8c 60%
Scheme 1. Reagents and conditions: (i) TsCl, Et3N, DMAP, DCM 0 °C?rt, o.n.; (ii)
TsCl, pyridine, DCM 0 °C?rt, 6 h; (iii) NaN3, DMSO, 35 °C, 6 h or o.n.; (iv) NaCN,
DMSO, 35 °C, 6 h or o.n.; (v) DIBAL in toluene, ꢀ65 °C, 50 to 90 min.
R
i
ii
4a
4b
4c
6a 31%
N
Ts
9a 51%
9b
47%
9c 35%
2.3. Inhibitory activity of the synthesized compounds
IgG is composed of two heavy (56 kDa each) and two light
chains (25 kDa each) that are held together by disulfide bonds.
Scheme 2. Reagents and conditions: (i) NaN3, PPh3, DIAD, THF, 0 °C?rt, o.n.; (ii)
Water, rt, o.n.

3466
K. Berggren et al. / Bioorg. Med. Chem. 17 (2009) 3463–3470
kDa
250
130
95
O
BocNH(CH2)4
A
O
O
O
BocNH(CH2)4
NH
O
72
O
S
i
NH2
55
12
36
28
13 95%
IdeS 4b 7b 4c 7c 4a 6a 7a
1
2
6b
17
IgG
ii
B
BocNH(CH2)4
O
RNH(CH2)4
OTs
OH
O
O S
NH
NH
O
S
iii
8b 6c
8c
8a
19 20 21 IdeS IgG
15
37%
iv
14 R= Boc 94%
19
vi
R= H 78%
C
RNH(CH2)4
O
X
NH
16
R= Boc, X= N3 97%
O
S
vi
vi
20 R= H, X= N3 80%
17 R= Boc, X= CN 93%
21 R= H, X= CN 93%
v
14
16 17 18 5b
5c
5a
15 E-64
18
R= Boc, X= CHO 26%
Figure 5. A–C Cleavage of IgG by IdeS in the presence of test compounds 4a–8c, 14–
21 and controls. The positive controls (1 and 2) are shown in 5A and the negative
control (E-64) is shown in 5C.
Scheme 4. Reagents and conditions: (i) TsCl, Et3N, DCM, 0 °C?rt, o.n.; (ii) NaBH4,
THF/ethanol, 0 °C?rt, o.n.; (iii) TsCl, pyridine, DMAP, DCM, 0 °C?rt, o.n.; (iv) NaN3,
DMSO, rt, o.n. or NaCN, DMSO, 35 °C, o.n.; (v) DIBAL in toluene, THF, ꢀ65 °C, 45 min;
(vi) TFA in DCM, rt, 60–90 min.
warhead were shown to efficiently inhibit the hydrolyzing activity
of IdeS. In fact, all aldehydes tested (8a–c, 18), substituted by ben-
zyl, n-butyl, i-propyl and 4-tert-butyloxycarbonylaminobutyl
showed as strong inhibitory activity as the positive controls TPCK
(1) and TLCK (2). Apparently, the different side chains did not seem
to contribute to the binding as the same degree of inhibition was
observed for all derivatives.
Only one nitrile derivative (7b), substituted with an n-butyl
group, showed some inhibition of IdeS (Fig. 5A). This implies that
among these test compounds the n-butyl substituted derivative
is interacting strongest with the active site of IdeS.
Three of the tested azide derivatives (6a–c) were shown to be
moderate inhibitors of IdeS. Some of the first azides reported to
be potent inhibitors of various cysteine proteases were recently
shown to decompose into aldehydes when exposed to light.22,23
These aldehydes were suggested to be the active species. Therefore
the stability of our azide derivatives was tested and they were
found to be stable in the solid state as no impurities of the corre-
sponding aldehyde could be detected by 1H NMR spectroscopy.
The azides were also found to be stable in buffer solutions (pH
6.9 and 7.4) for 48 h, as determined by analytical HPLC (see the
Experimental section 4.9.)
Both covalent and non-covalent interactions between azides
and nucleophiles have been proposed but their mode of action as
inhibitors has so far not been studied in detail. Nucleophilic attack
of the cysteine residue as well as electrostatic interaction via the
dipolar resonance structures of the azide functionality have been
suggested.24 Furthermore, thiols such as dithiothreitol, mercap-
Thus, when run on SDS–PAGE under reducing conditions, IgG gives
rise to two bands of approximately 25 and 56 kDa (see Fig. 5B).
IdeS with a molecular weight of 35 kDa (see Fig. 5B), cleaves IgG
in the heavy chain generating two heavy chain fragments of 25
and 31 kDa, respectively. Therefore, incubation of IgG with IdeS
followed by separation on SDS–PAGE under reducing conditions,
will generate a new 31 kDa band from the heavy chain (the other
25 kDa peptide from the heavy chain is hidden in the light chain
band), and the SDS–PAGE analysis is based on the appearance of
this 31 kDa band. As mentioned above, IdeS is uniquely specific
for IgG. This is explained by the fact that the enzyme has to bind
to a site in Fc before cleavage can occur in the hinge region of
the heavy chain. The requirement for this initial protein–protein
interaction has made it impossible to develop a quantitative and
practical screening assay for the proteolytic activity of IdeS, which
is based on the hydrolysis of synthetic and natural peptides cover-
ing the cleavage site in the hinge.5 However, to identify inhibitors
of IdeS the qualitative SDS–PAGE analysis used in this work, was
found to be sufficient.
Purified IdeS was preincubated with the test compounds 4a–8c,
14–21, or 1, 2 and E-64 as positive and negative controls, respec-
tively. Following the addition of polyclonal human IgG and incuba-
tion, the samples were separated by SDS–PAGE, and the results are
shown in Figure 5A–C (for experimental details, see Experimental
Section 4.10).
In the in vitro assay, eight out of the 23 compounds tested were
able to inhibit IdeS. Compounds containing an aldehyde function as

K. Berggren et al. / Bioorg. Med. Chem. 17 (2009) 3463–3470
3467
toethanol and even cysteine, are reported to reduce azides to
4.2. General synthetic procedure for monotosylation
amines via a covalent intermediate, which could explain a revers-
ible inhibition of IdeS.25,26
A solution of tosyl chloride (1.05 equiv) in dichloromethane
(6 mL) was added dropwise over 90 min to a mixture of the amino
alcohol (4 mmol), DMAP (0.1 equiv) and Et3N (2 equiv) in dichloro-
methane (10 mL) at 0 °C. The reaction mixture was stirred over
night at room temperature, and then diluted with dichlorometh-
ane. The solution was washed with water (3ꢁ) and brine (1ꢁ),
dried (MgSO4) and filtered. The solvent was removed in vacuo.
Purification by flash chromatography (dichloromethane/methanol
95:5) gave the product.
Compounds with alcohol (4a–c, 14, 19) and O-tosyl (5a–c, 15)
groups as warheads were not able to inhibit IdeS, irrespectively
of the side chains used. Neither the lysine-derived azides (16, 20)
were active. The aziridine derivatives with n-butyl (9b) and i-pro-
pyl (9c) groups as side chains were also tested (results not shown),
but did not result in any inhibition, even though aziridines have
been reported to act as inhibitors for other cysteine proteases.27
Reasons for the inactivity of O-tosyl and aziridine derivatives could
either be low reactivity, steric hindrance or unfavourable spatial
orientation of the ligand in the active site of IdeS.
4.2.1. (S)-3-Phenyl-2-tosylamino-1-propanol (4a)
White solid; Yield 91%; Mp 66–69 °C (lit.28 73–74 °C); ½a D20
ꢂ
3. Conclusion
ꢀ47.5 (c 1.93, EtOH) (lit.28 ꢀ38.8); 1H NMR d 7.57 (d, J = 8.1 Hz,
2H, H20), 7.23–7.16 (m, 5H, H30, H200, H400), 6.99–6.94 (m, 2H,
H300), 4.67 (d, J = 5.5 Hz, 1H, NH), 3.68–3.49 (m, 2H, CH2OH),
3.49–3.39 (m, 1H, CH), 2.78 (dd, J = 7.0, 13.9 Hz, 1H, PhCHA), 2.68
(dd, J = 7.3, 13.9 Hz, 1H, PhCHB), 2.42 (s, 3H, CH3), 2.04–1.96 (m,
1H, OH); 13C NMR data were in agreement with those reported.28
To the best of our knowledge, no compounds have been pub-
lished with the specific aim to develop potential inhibitors of IdeS
as previously reported inhibitors of IdeS have been used only for
characterizing purposes.2,6,9 In the present study we have used a
highly convergent synthetic route to compounds which were
tested for their ability to inhibit IdeS. The results showed that
reversible binding of warheads, such as the aldehyde, can be suffi-
cient to efficiently inhibit IdeS. In addition, nitrile and azide deriv-
atives showed moderate inhibitory activity. For these inhibitors
the binding of the warhead is still of major importance, but irre-
versible binding is not necessary. Finally, interactions between
IdeS and other substituents than the warhead of the inhibitor were
found to be of importance for the inhibition. The identification of
reversible inhibitors of IdeS could be an important step towards
a potential treatment of acute and severe S. pyogenes infections.
IR (KBr)
m .
3445, 3159, 1316, 1157 cmꢀ1
4.2.2. (S)-2-Tosylamino-1-hexanol (4b)
White solid, Yield 95%, Mp 86–88 °C (lit.29 81–83 °C); ½a D20
ꢂ
,
1H
and 13C NMR data were in agreement of those reported.29
4.2.3. (S)-3-Methyl-2-tosylamino-1-butanol (4c)
White solid; Yield 86%; Data were in agreement of those
reported.30
4.3. General synthetic procedure for ditosylation
4. Experimental
A solution of tosyl chloride (2.1 equiv) in dichloromethane
(5 mL) was added dropwise to a mixture of the amino alcohol
(6 mmol) and pyridine (2.1 equiv) in dichloromethane (7 mL) at
0 °C. The reaction mixture was stirred for 6 h at room temperature,
and then diluted with dichloromethane. The solution was washed
with water (3ꢁ) and brine (1ꢁ), dried (MgSO4) and filtered. The
solvent was removed in vacuo. Purification by flash chromatogra-
phy (hexane/ethyl acetate 3:1) gave the product.
4.1. General methods
Melting points were determined with a Büchi B-545 apparatus
and are uncorrected. Optical rotations were measured with a Perkin
Elmer 341 LC Polarimeter at 20 °C. 1H and 13C NMR spectra were re-
corded on a JEOL Eclipse400 spectrometer at 400 MHz and 100 MHz,
respectively, in CDCl3 if nothing else is stated. Chemical shifts are re-
ported in ppm with the solvent residual peak as internal standard
(CHCl3 dH 7.26, dC 77.00, CH3OH dH 3.30, dC 49.00). Infrared spectra
were recorded on a Perkin Elmer 16 PC FTIR spectrometer. Only
the major peaks are listed. HRMS-analyses were run at BioAnSer,
Gothenburg, Sweden. Analytical TLC was performed on Merck Silica
Gel, grade 60 F254 and the spots were visualized by UV light
(254 nm). Flash chromatography was performed on Merck Silica
Gel 60. The hexane used was a mixture of isomers. Analytical HPLC
wasperformedon a Waters2690system(PhotodiodeArrayDetector
at 254 nm and flow rate 1.5 mL/min) equipped with a Genesis Light-
4.3.1. (S)-3-Phenyl-2-tosylamino-propyl-1-toluenesulfonate
(5a)
White solid; Yield 63%; Mp 100–102 °C (lit.31 97–98 °C); ½a D20
ꢂ
ꢀ51.2 (c 1.0, MeOH) (lit.31 ꢀ57.4); 1H NMR d 7.76 (d, J = 8.4 Hz,
2H, H20), 7.52 (d, J = 8.4 Hz, 2H, H2), 7.36 (d, J = 8.4 Hz, 2H, H3),
7.22–7.09 (m, 5H, H30, H200, H400), 6.92–6.83 (m, 2H, H300), 4.61 (d,
J = 8.1 Hz, 1H, NH), 4.16–3.81 (m, 2H, CH2O), 3.61–3.52 (m, 1H,
CH), 2.82 (dd, J = 7.3, 13.9 Hz, 1H, PhCHA), 2.67 (dd, J = 7.0,
13.9 Hz, 1H, PhCHB), 2.47 (s, 3H, PhCH3), 2.41 (s, 3H, PhCH3); 13C
NMR d 145.2, 143.4, 136.7, 135.6, 132.2, 130.0, 129.7, 129.1,
ning C8 4 lm, 50 mm, ID 4.6 mm column. Starting materials, re-
128.7, 128.0, 126.9, 70.1, 53.5, 37.5, 21.7, 21.5; IR (KBr)
m 3447
agents, polyclonal IgG and molecular mass markers were
purchased from Sigma–Aldrich and were used as such.
For the assignment of 1H NMR signals, the numbering used is
shown in the structure below.
(broad), 3291, 1364, 1174 cmꢀ1
.
4.3.2. (S)-2-Tosylamino-hexyl-1-toluenesulfonate (5b)
White solid; Yield 43%; Mp 95–98 °C; ½a D20
ꢀ44.4 (c 1.0, CHCl3);
ꢂ
1H NMR d 7.73 (d, J = 8.4 Hz, 2H, H20), 7.69 (d, J = 8.0 Hz, 2H, H2),
7.35 (d, J = 8.4 Hz, 2H, H30), 7.30–7.23 (m, 2H, H3), 4.56 (d,
J = 8.4 Hz, 1H, NH), 3.98–3.80 (m, 2H, CH2O), 3.40–3.30 (m, 1H,
CH), 2.46 (s, 3H, PhCH3), 2.42 (s, 3H, PhCH3), 1.53–1.30 (m, 2H,
CH2CH2CH), 1.16–0.90 (m, 4H, CH3(CH2)2), 0.74 (t, J = 7.0 Hz, 3H,
CH3CH2); 13C NMR d 145.1, 143.6, 137.4, 132.2, 129.9, 129.7,
127.9, 127.0, 71.1, 52.3, 31.2, 27.2, 22.0, 21.6, 21.5, 13.7; IR (KBr)
3´´
2´´
1´´
O
O
1
2
4´´
S
3
O
4
O
O
NH
S
1´
2´
3´
3276, 2949, 1600, 1438, 1357, 1174 cmꢀ1; HRMS (FT-ICR-MS)
Calcd for C20H27NO5S2 [M+H]+ 426.1402, found 426.1390.
4´
m

3468
K. Berggren et al. / Bioorg. Med. Chem. 17 (2009) 3463–3470
4.3.3. (S)-3-Methyl-2-tosylamino-butyl-1-toluenesulfonate (5c)
White solid; Yield 44%; Data were in agreement of those
reported.31,32
mixture was stirred at ꢀ65 °C for 50–90 min. The excess of reagent
was quenched by acetone (2 mL) at ꢀ65 °C. The mixture was syr-
inged into an aqueous HCl solution (5%) (3 mL) at 0 °C. The phases
were separated and the organic phase was washed twice with
brine, dried (MgSO4) and filtered. The solvents were removed in
vacuo at room temperature. Purification by flash chromatography
(hexane/ethyl acetate 1:1) gave the product.
4.4. General synthetic procedure for azide derivatives 6a–c
The ditosylated amino alcohol (0.3 mmol) was dissolved in
DMSO (3 mL). Sodium azide (2 equiv) was added and the reaction
mixture was heated to 35 °C over night. The mixture was diluted
with water (5 mL) and extracted with ethyl acetate (3 ꢁ 5 mL).
The combined organic phases were washed with brine, dried
(MgSO4) and filtered. The solvent was removed in vacuo to give
the product.
4.6.1. (S)-4-Phenyl-3-tosylamino-1-butanal (8a)
Pale yellow oil; Yield 44%; ½a D20
ꢂ
ꢀ31.8 (c 0.6, CH2Cl2) (lit.36
½ ꢂ
a 2D3
ꢀ18); IR, 1H and 13C NMR data were in agreement with those
reported.36
4.6.2. (S)-3-Tosylamino-1-heptanal (8b)
4.4.1. (S)-1-Azido-3-phenyl-2-tosylamino-propane (6a)
Transparent oil; Yield 22%; ½a D20
ꢂ
+26.7 (c 1.0 , CHCl3) (lit.37
Pale yellow oil; Yield 65%; ½a D20
ꢂ
ꢀ29.4 (c 1.0, CHCl3); IR, 1H and
+24.8); IR, 1H and 13C NMR data were in agreement with those
reported.37
13C NMR data were in agreement with those reported.33
4.4.2. (S)-1-Azido-2-tosylamino-hexane (6b)
4.6.3. (S)-4-Methyl-3-tosylamino-1-pentanal (8c)
Transparent oil; Yield 76%; ½a D20
ꢂ
ꢀ38.8 (c 1.0, CHCl3); 1H NMR
Pale yellow oil; Yield 60%; ½a D20
ꢂ
+34.6 (c 1.0, CHCl3); 1H NMR d
and IR data were in agreement with those reported.34 13C NMR d
9.58 (s, 1H, CHO), 7.73 (d, J = 8.4 Hz, 2H, H20), 7.29 (d, J = 8.4 Hz,
2H, H30), 4.76 (d, J = 8.8 Hz, 1H, NH), 3.51–3.39 (m, 1H, CHNH),
2.64–2.48 (m, 2H, CH2CHO), 2.42 (s, 3H, PhCH3), 1.86–1.76 (m,
1H, (CH3)2CH), 0.80 (d, J = 7.0 Hz, 3H, CH3ACH), 0.78 (d, J = 7.0 Hz,
3H, CH3BCH); 13C NMR d 200.6, 143.5, 137.6, 129.7, 127.1, 54.6,
143.6, 137.7, 129.7, 127.0, 54.8, 53.2, 32.2, 27.4, 22.1, 21.5, 13.7.
4.4.3. (S)-1-Azido-3-methyl-2-tosylamino-butane (6c)
White solid; Yield 77%; Mp 78–80 °C; ½a D20
ꢀ57.1 (c 1.0, CHCl3);
ꢂ
1H NMR d 7.76 (d, J = 8.1 Hz, 2H, H20), 7.31 (d, J = 8.4 Hz, 2H, H30),
4.59 (d, J = 7.7 Hz, 1H, NH), 3.37 (dd, J = 4.0, 12.4 Hz, 1H, CHAN3),
3.25 (dd, J = 5.1, 12.4 Hz, 1H, CHBN3), 3.13–3.05 (m, 1H, CHNH),
2.43 (s, 3H, PhCH3), 1.86–1.73 (m, 1H, (CH3)2CH), 0.82 (d,
J = 7.0 Hz, 6H, (CH3)2CH); 13C NMR d 143.6, 137.7, 129.7, 127.0,
45.9, 31.7, 21.5, 18.7, 18.2; IR (KBr)
m 3284, 2964, 1721, 1325,
1157 cmꢀ1 HRMS (FT-ICR-MS) Calcd for C13H19NO3S [M+H]+
;
270.1158, found 270.1159.
4.7. Synthetic procedures to lysine derivatives
58.4, 52.7, 29.7, 21.5, 19.0, 18.1; IR (KBr)
m 3261, 2962, 2094,
1461, 1314, 1162 cmꢀ1; HRMS (FT-ICR-MS) Calcd for C12H18N4O2S
[M+H]+ 283.1223, found 283.1223.
4.7.1. (S)-Methyl 6-tert-butoxycarbonylamino-2-tosylamino
hexanoate (13)
A solution of tosyl chloride (2.12 g, 11.1 mmol) in dichloro-
methane (30 mL) was added dropwise over 40 min to a mixture
of H-Lys(Boc)-OMe ꢃ HCl (3.0 g; 10.1 mmol) and Et3N (3.08 g,
30.4 mmol) in dichloromethane (45 mL) at 0 °C. The reaction mix-
ture was stirred over night at room temperature, and then diluted
with dichloromethane. The solution was washed with water
(2 ꢁ 25 mL) and brine (2 ꢁ 25 mL), dried (MgSO4) and filtered.
The solvent was removed in vacuo. Purification by flash chroma-
tography (hexane/ethyl acetate 5:1) gave 13 as a white solid
4.5. General synthetic procedure for nitrile derivatives 7a–c
The different nitrile derivatives were synthesized as described
for the azides using sodium cyanide (2 equiv) instead of sodium
azide.
4.5.1. (S)-4-Phenyl-3-tosylamino-butane-1-nitrile (7a)
White solid; Yield 87%; Mp 90–92 °C (lit.33 102–103 °C); ½a 2D0
ꢂ
ꢀ43.0 (c 1.0, CHCl3); IR, 1H and 13C NMR data were in agreement
(3.98 g, 95%). Mp 85–86 °C; ½a D20
ꢂ
+21.9 (c 0.6, CHCl3); 1H NMR d
with those reported.33
7.70 (d, J = 8.1 Hz, 2H, H20), 7.28 (d, J = 8.1 Hz, 2H, H30), 5.21 (d,
J = 9.2 Hz, 1H, NH), 4.54 (br s, 1H, CONH), 3.92–3.83 (m, 1H, CH),
3.47 (s, 3H, OCH3), 3.13–2.97 (m, 2H, NHCH2), 2.41 (s, 3H, PhCH3),
4.5.2. (S)-3-Tosylamino-heptane-1-nitrile (7b)
White solid; Yield 98%; Mp 83–86 °C (lit.34 73–74 °C); ½a D20
ꢂ
1.78–1.56 (m, 2H, CH2CH), 1.49–1.22 (m, 13H, (CH3)3; CH2CH2); 13
C
ꢀ57.9 (c 1.1, CHCl3); IR, 1H and 13C NMR data were in agreement
NMR d 172.1, 155.9, 143.6, 136.6, 129.6, 127.2, 79.1, 55.4, 52.4,
40.0, 32.7, 29.2, 28.3, 22.0, 21.5; IR (KBr) 3383, 2930, 1737,
1692, 1531, 1343, 1167 cmꢀ1
with those reported.34
m
.
4.5.3. (S)-4-Methyl-3-tosylamino-pentane-1-nitrile (7c)
White solid; Yield 94%; Mp 91–95 °C; ½a D20
ꢂ
ꢀ77.6 (c 1.0, CHCl3);
4.7.2. (S)-6-tert-Butoxycarbonylamino-2-tosylamino-1-hexanol
(14)
1H NMR d 7.77 (d, J = 8.4 Hz, 2H, H20), 7.33 (d, J = 8.4 Hz, 2H, H30),
4.95 (d, J = 8.4 Hz, 1H, NH), 3.33–3.17 (m, 1H, CHNH), 2.59 (d,
J = 5.1 Hz, 2H, CH2CN), 2.44 (s, 3H, PhCH3), 1.92 (oct, 1H, J = 6.6,
Hz, (CH3)2CH), 0.83 (d, J = 7.0 Hz, 3H, CH3ACH), 0.81 (d, J = 7.3 Hz,
3H, CH3BCH); 13C NMR d 143.9, 137.0, 129.8, 127.1, 116.9, 55.5,
A solution of 13 (1.8 g, 4.3 mmol) in THF/ethanol (2:3) (30 mL)
was added to a slurry of NaBH4 (0.49 g, 13.0 mmol) in THF (4 mL)
at 0 °C. More NaBH4 (0.1 g, 2.6 mmol) was added after 6 h and
the reaction mixture was stirred at room temperature over night.
The reaction was quenched by careful addition of water (3 mL)
while stirring. A white precipitate was removed by filtration and
the filtrate was concentrated. The residue was redissolved in
dichloromethane (100 mL). Aqueous NaOH (25%) (1 mL) and water
(25 mL) were added and the water phase was extracted twice with
dichloromethane (20 mL). The combined organic phases were
washed twice with brine, dried (MgSO4) and filtered. The solvent
was removed in vacuo to yield 14 as a white solid (1.58 g, 94%).
30.9, 22.6, 21.5, 19.1, 17.5; IR (KBr)
m 3247, 2961, 2251 w, 1451,
1328, 1154 cmꢀ1
; HRMS (FT-ICR-MS) Calcd for C13H18N2O2S
[M+H]+ 267.1162, found 267.1164.
4.6. General synthetic procedure for aldehyde derivatives 8a–c35
The nitrile (0.64 mmol) was dissolved in dry THF (3 mL) under
nitrogen atmosphere at ꢀ65 °C. A solution of DIBAL in toluene
(1 M, 5 equiv) was added in portions over 15 min and the reaction
Mp 159–161 °C; ½a D20
ꢂ
ꢀ5.0 (c 1.0, MeOH); 1H NMR d 7.76 (d,

K. Berggren et al. / Bioorg. Med. Chem. 17 (2009) 3463–3470
3469
J = 8.1 Hz, 2H, H20), 7.29 (d, J = 8.1 Hz, 2H, H30), 4.90 (d, J = 7.3 Hz,
1H, SO2NH), 4.50 (br s, 1H, CONH), 3.52–3.38 (m, 2H, NHCH2),
3.26–3.15 (m, 1H, CH), 3.15–3.02 (m, 1H, CHAOH), 3.02–2.91 (m,
1H, CHBOH), 2.42 (s, 3H, PhCH3), 1.47–1.04 (m, 6H, (CH2)3CH),
1.43 (s, 9H, (CH3)3CO); 13C NMR d 156.4, 143.5, 137.8, 129.7,
1696, 1517, 1452, 1329, 1159 cmꢀ1; HRMS (FT-ICR-MS) Calcd for
C19H29N3O4S [M+H]+ 396.1951, found 396.1954.
4.7.6. (S)-7-tert-Butoxycarbonylamino-3-tosylamino-1-
heptanal (18)
127.1, 79.4, 64.0, 55.3, 39.3, 30.8, 29.8, 28.4, 22.0, 21.5; IR (KBr)
m
A solution of 17 (0.3 g, 0.79 mmol) in dry THF (4 mL) under
nitrogen atmosphere was cooled to ꢀ65 °C. A solution of DIBAL
in toluene (1 M) (4 mL, 3.95 mmol) was added in portions over
15 min and the reaction mixture was stirred at ꢀ65 °C for an addi-
tional 30 min. Excess of reagent was quenched by addition of ace-
tone (1.5 mL) and the mixture was syringed into an aqueous HCl
solution (5%) (4 mL) at 0 °C. The phases were separated and the or-
ganic phase was washed twice with brine, dried (MgSO4) and fil-
tered. The solvent was removed in vacuo at room temperature.
Purification by flash chromatography (hexane/ethyl acetate 1:1)
3530, 3283, 2933, 1681, 1530, 1162 cmꢀ1; HRMS (FT-ICR-MS)
Calcd for C18H30N2O5S [M+H]+ 387.1947, found 387.1944.
4.7.3. (S)-6-tert-Butoxycarbonylamino-2-tosylamino-hexyl-1-
toluenesulfonate (15)
A solution of tosyl chloride (0.81 g, 7.12 mmol) in dichloro-
methane (10 mL) was added dropwise to a mixture of 14 (1.5 g,
3.8 mmol), pyridine (0.92 g, 19.4 mmol) and DMAP (47 mg,
0.65 mmol) in dichloromethane (30 mL) at 0 °C. The reaction mix-
ture was stirred over night at room temperature, and then diluted
with dichloromethane. The solution was washed with water and
brine, dried (MgSO4) and filtered. The solvent was removed in va-
cuo. Purification by flash chromatography (hexane/ethyl acetate
gave 18 as a transparent oil (80 mg, 26%). ½a D20
ꢀ10.6 (c 1.0, CHCl3);
ꢂ
1H NMR d 9.62 (s, 1H, CHO), 7.74 (d, J = 8.1 Hz, 2H, H20), 7.30 (d,
J = 8.1 Hz, 2H, H30), 5.08 (br s, 1H, SO2NH), 4.47 (br s, 1H, CONH),
3.63–3.51 (m, 1H, CH), 3.05–2.90 (m, 2H, NHCH2), 2.70–2.52 (m,
2H, CH2CHO), 2.43 (s, 3H, PhCH3), 1.44 (s, 9H, (CH3)3CO), 1.38–
1.02 (m, 6H, (CH2)3CH); 13C NMR d 200.7, 156.2, 143.7, 137.8,
129.8, 127.2, 49.4, 48.7, 39.9, 34.3, 29.5, 28.5, 22.7, 21.6; IR (neat)
2:1) gave 15 as a transparent oil (0.78 g, 37%). ½a D20
ꢀ31.4 (c 1.0,
ꢂ
1
CHCl3); H NMR d 7.72 (d, J = 8.4 Hz, 2H, H20), 7.69 (d, J = 8.1 Hz,
2H, H2), 7.34 (d, J = 8.4 Hz, 2H, H30), 7.27–7.24 (m, 2H, H3), 5.08
(d, J = 7.7 Hz, 1H, SO2NH), 4.50 (br s, 1H, CONH), 3.96 (dd, J = 3.3,
9.9 Hz, 1H, CHAO), 3.80 (dd, J = 5.1, 9.9 Hz, 1H, CHBO), 3.39–3.27
(m, 1H, CH), 2.99–2.88 (m, 2H, NHCH2), 2.45 (s, 3H, PhCH3), 2.42
(s, 3H, PhCH3), 1.43 (s, 9H, (CH3)3CO), 1.34–0.94 (m, 6H, (CH2)3CH);
13C NMR d 156.2, 145.2, 143.6, 137.3, 132.2, 130.0, 129.7, 128.0,
m
3279, 2931, 1691, 1519, 1327, 1161 cmꢀ1; HRMS (FT-ICR-MS)
Calcd for C19H30N2O5S [M+H]+ 399.1947, found 399.1947.
4.8. Synthetic procedures for Boc-deprotection
127.1, 79.4, 71.1, 52.2, 30.8, 29.5, 28.4, 21.9, 21.7, 21.5; IR (KBr)
m
4.8.1. (S)-6-Amino-2-tosylamino-1-hexanol TFA (19)
*
3290, 2870, 1692, 1359, 1167 cmꢀ1; HRMS (FT-ICR-MS) Calcd for
C25H36N2O7S2 [M+H]+ 541.2035, found 541.2025.
TFA (0.2 mL, 2.7 mmol) was added to a solution of 14 (80 mg,
0.19 mmol) in dichloromethane (3 mL). The mixture was stirred
at room temperature for 1 h. Excess of reagent and solvent were re-
4.7.4. (S)-1-Azido-6-tert-butoxycarbonylamino-2-tosylamino-
hexane (16)
moved in vacuo to give 19 as a transparent oil (60 mg, 78%). ½a D20
ꢂ
+9.0 (c 1.1, MeOH); 1H NMR (CD3OD) d 7.74 (dd, J = 8.1, 8.4 Hz,
2H, H20), 7.36 (d, J = 8.1 Hz, 2H, H30), 4.23–4.10 (m, 1H, CH),
3.35–3.14 (m, 2H, NH3CH2), 2.86–2.75 (m, 2H, CH2OH), 2.42 (s,
3H, CH3), 1.66–1.20 (m, 6H, (CH2)3CH); 13C NMR (CD3OD) d
144.6, 140.2, 130.68, 128.0, 65.0, 56.2, 40.5, 32.0, 28.2, 23.5, 21.4;
Sodium azide (55 mg, 0.85 mmol) was added to a solution of
15 (230 mg, 0.42 mmol) in DMSO (5 mL) and the reaction mixture
was stirred at room temperature over night. The mixture was di-
luted with water (12 mL) and extracted with ethyl acetate
(3 ꢁ 7 mL). The combined organic phases were washed with brine
(4 ꢁ 7 mL), dried (MgSO4) and filtered. The solvent was removed
in vacuo to give 16 as a white solid (0.17 g, 97%). Mp 74–77 °C;
IR (neat)
m ;
3258 (broad), 2921, 1683, 1430, 1319, 1156 cmꢀ1
HRMS (FT-ICR-MS) Calcd for C13H22N2O3S [M+H]+ 287.1423, found
287.1411.
½
a 2D0
ꢂ
ꢀ28.8 (c 0.22, CHCl3); 1H NMR d 7.76 (d, J = 8.1 Hz, 2H,
H20), 7.31 (d, J = 7.7 Hz, 2H, H30), 4.83 (br s, 1H, SO2NH), 4.49
(br s, 1H, CONH), 3.36–3.22 (m, 3H, CH, CH2N3), 3.05–2.95 (m,
2H, NHCH2), 2.44 (s, 3H, PhCH3), 1.44 (s, 9H, (CH3)3CO), 1.40–
1.06 (m, 6H, (CH2)3CH); 13C NMR d 156.1, 143.6, 137.7, 129.7,
127.0, 79.3, 54.8, 53.0, 39.8, 31.9, 29.6, 28.4, 22.3, 21.5; IR (KBr)
4.8.2. (S)-6-Azido-5-tosylamino-hexylamine TFA (20)
*
TFA (0.2 mL, 2.7 mmol) was added to a solution of 16 (150 mg,
0.36 mmol) in dichloromethane (3 mL). The mixture was stirred at
room temperature for 90 min. Excess of reagent and solvent were
removed in vacuo to give 20 as a pale yellow oil (124 mg, 80%).
m
3368, 3286, 2938, 2094, 1681, 1527, 1316, 1159 cmꢀ1; HRMS
½
a 2D0
ꢂ
ꢀ5.0 (c 1.0, MeOH); 1H NMR (CD3OD) d 7.76 (d, J = 8.4 Hz,
(FT-ICR-MS) Calcd for C18H29N5O4S [M+H]+ 412.2011, found
412.2010.
2H, H20), 7.38 (d, J = 8.8 Hz, 2H, H30), 3.36–3-27 (m, 1H, CH), 3.19
(dd, J = 5.1, 12.4 Hz, 1H, CHAN3), 3.14 (dd, J = 5.1, 12.4 Hz, 1H,
CHBN3), 2.82 (t, J = 7.7 Hz, 2H, NH3CH2), 2.43 (s, 3H, CH3), 1.62–
1.22 (m, 6H, (CH2)3CH); 13C NMR (CD3OD) d 144.7, 140.1, 130.8,
4.7.5. (S)-7-tert-Butoxycarbonylamino-3-tosylamino-heptane-
1-nitrile (17)
128.0, 56.0, 54.3, 40.6, 33.2, 28.2, 23.5, 21.4; IR (neat)
m 3169
Sodium cyanide (91 mg, 1.85 mmol) was added to a solution of
15 (0.5 g, 0.93 mmol) in DMSO (13 mL) and the reaction mixture
was heated to 35 °C over night. The mixture was diluted with
water (20 mL) and extracted with ethyl acetate (3 ꢁ 15 mL). The
combined organic phases were washed with brine (3 ꢁ 10 mL),
dried (MgSO4) and filtered. The solvent was removed in vacuo to
give 17 as a transparent oil (0.34 g, 93%). No further purification
(broad), 2930, 2105, 1679, 1526, 1433, 1319, 1152 cmꢀ1; HRMS
(FT-ICR-MS) Calcd for C13H21N5O2S [M+H]+ 312.1488, found
312.1480.
4.8.3. (S)-7-Amino-3-tosylamino-heptane-1-nitrile TFA (21)
*
TFA (0.2 mL, 2.7 mmol) was added to a solution of 17 (130 mg,
0.33 mmol) in dichloromethane (3 mL). The mixture was stirred at
room temperature for 1 h. Excess of reagent and solvent were re-
moved in vacuo to give 21 as a pale yellow oil (130 mg, 93%).
was needed. ½a 2D0
ꢂ
ꢀ39.0 (c 0.5, CHCl3); 1H NMR d 7.76 (d,
J = 8.4 Hz, 2H, H20), 7.33 (d, J = 8.4 Hz, 2H, H30), 5.22 (d, J = 7.0 Hz,
1H, SO2NH), 4.51 (br s, 1H, CONH), 3.53–3.33 (m, 1H, CH), 3.10–
2.93 (m, 2H, NHCH2), 2.69–2.53 (m, 2H, CH2CN), 2.44 (s, 3H,
PhCH3), 1.44 (s, 9H, (CH3)3CO), 1.71–1.02 (m, 6H, (CH2)3CH); 13C
NMR d 156.4, 143.9, 137.0, 129.8, 127.0, 116.9, 79.5, 49.9, 39.4,
½
a 2D0
ꢂ
ꢀ14.0 (c 1.0, MeOH); 1H NMR (CD3OD) d 7.76 (d, J = 8.1 Hz,
2H, H20), 7.38 (d, J = 8.4 Hz, 2H, H30), 3.50–3.40 (m, 1H, CH), 2.78
(t, J = 7.3 Hz, 2H, NH3CH2), 2.40–2.61 (m, 2H, CH2CN), 2.42 (s, 3H,
CH3), 1.64–1.45 (m, 4H, CH2CH2), 1.39–1.14 (m, 2H, CH2CH); 13C
NMR (CD3OD) d 145.0, 139.8, 130.9, 128.0, 118.3, 51.1, 40.4, 34.4,
32.8, 29.4, 28.4, 24.8, 21.9, 21.5; IR (neat)
m 3280, 2924, 2251,

3470
K. Berggren et al. / Bioorg. Med. Chem. 17 (2009) 3463–3470
3. Lei, B. F.; DeLeo, F. R.; Hoe, N. P.; Graham, M. R.; Mackie, S. M.; Cole, R. L.; Liu, M.
27.9, 25.2, 23.4, 21.4; IR (neat)
m
3173 (broad), 2920, 2254, 1674,
Y.; Hill, H. R.; Low, D. E.; Federle, M. J.; Scott, J. R.; Musser, J. M. Nat. Med. 2001,
7, 1298.
4. von Pawel-Rammingen, U.; Johansson, B. P.; Tapper, H.; Björck, L. Nat. Med.
2002, 8, 1043.
1531, 1428, 1326, 1154 cmꢀ1
; HRMS (FT-ICR-MS) Calcd for
C14H21N3O2S [M+H]+ 296.1426, found 296.1414.
5. Söderberg, J. J.; von Pawel-Rammingen, U. Mol. Immunol. 2008, 45, 3347.
6. Vincents, B.; von Pawel-Rammingen, U.; Björck, L.; Abrahamson, M.
Biochemistry 2004, 43, 15540.
4.9. Azide stability test
7. Nandakumar, K. S.; Johansson, B. P.; Björck, L.; Holmdahl, R. Arthritis Rheum.
2007, 56, 3253.
8. Johansson, B. P.; Shannon, O.; Björck, L. PLoS ONE 2008, 3, e1692.
9. Agniswamy, J.; Lei, B. F.; Musser, J. M.; Sun, P. D. J. Biol. Chem. 2004, 279, 52789.
10. For review of inhibitors of cysteine proteases, see: Otto, H.-H.; Schirmeister, T.
Chem. Rev. 1997, 97, 133.
Azide 6c (10 mg) was dissolved in acetonitrile (0.5 mL) and
either phosphate buffer (0.5 mL) (pH 7.4) or ammonium acetate
buffer (pH 6.9) and samples were exposed to ambient laboratory
light. Aliquots (0.1 mL) were taken out at t = 0, 2, 4, 6, 8, 24, 48 h,
analyzed twice directly or diluted with ammonium acetate buf-
fer/acetonitrile (1:1) (0.9 mL) and analyzed in duplicate on analyt-
ical HPLC.
11. For review of irreversible inhibitors, see: Powers, J. C.; Asgian, J. L.; Ekici, O. D.;
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4.10. IdeS inhibition assay
IdeS was produced in E. coli and purified as previously de-
scribed.2 IdeS (2
lL; 1 mg/mL) diluted in phosphate buffered saline
(PBS) (pH 7.4) was incubated with the test compounds dissolved in
18. Berry, M. B.; Craig, D. Synlett 1992, 41.
DMSO (10 mg/mL; 100ꢁ excess) for 30 min at room temperature in
19. Osborn, H. M. I.; Sweeney, J. Tetrahedron: Asymmetry 1997, 8, 1693.
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the dark. Human polyclonal IgG (5 lL, 1 mg/mL) in PBS was added,
1
21. Spectral data of compound 11 H NMR d 7.78 (d, J = 8.1 Hz, 2H, H20), 7.63 (d,
followed by incubation at 37 °C for 1 h. The samples were boiled
with an equal volume of SDS–PAGE sample buffer containing 4%
SDS and 5% 2-mercaptoethanol, and subjected to SDS–PAGE (4–
20% Precise Gels from Pierce). Gels were stained with Coomassie
Blue.
J = 8.4 Hz, 2H, H200), 7.39–7.27 (m, 4H, H30, H300), 5.17 (d, J = 7.7 Hz, 1H, NH),
3.52–3.42 (m, 2H, CH2OH), 3.21–3.19 (m, 1H, CH), 2.91–2.83 (m, 2H, CH2N),
2.64 (s, 3H, CH3N), 2.42 (s, 6H, 2ꢁ CH3), 1.59–1.09 (m, 6H, (CH2)3CH); MS
[MꢀH]ꢀ 453.3.
22. Le, G. T.; Abbenante, G.; Madala, P. K.; Hoang, H. N.; Fairlie, D. P. J. Am. Chem.
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We thank Ingbritt Gustavsson for help with the enzyme inhibi-
tion tests. We also thank the Swedish Research Council and Astra-
Zeneca R&D Mölndal for the graduent student fellowship for KB. LB
is supported by the Swedish Research Council (project 7480), the
Swedish Government Funds for Clinical Research (ALF), the Foun-
dations of Greta and Johan Kock, Alfred Österlund, and Torsten
and Ragnar Söderberg, and Hansa Medical AB.
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