Synthesis and evaluation of chloromethyl sulfoxides as a new class of selective irreversible cysteine protease inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.07.045

The synthesis and biological evaluation of a new class of selective irreversible cysteine protease inhibitors is described. A set of amino acid based chloromethyl sulfoxides was prepared and they were found to inhibit irreversibly the cysteine protease papain. They were selective for cysteine proteases since no inhibition was found for the serine protease chymotrypsin.

Full text of DOI:10.1016/j.bmc.2007.07.045

Bioorganic & Medicinal Chemistry 15 (2007) 6985–6993
Synthesis and evaluation of chloromethyl sulfoxides as a new
class of selective irreversible cysteine protease inhibitors
Arwin J. Brouwer, Anton Bunschoten and Rob M. J. Liskamp*
Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
PO Box 80082, NL-3508 TB Utrecht, The Netherlands
Received 23 May 2007; revised 18 July 2007; accepted 25 July 2007
Available online 22 August 2007
Abstract—The synthesis and biological evaluation of a new class of selective irreversible cysteine protease inhibitors is described. A
set of amino acid based chloromethyl sulfoxides was prepared and they were found to inhibit irreversibly the cysteine protease
papain. They were selective for cysteine proteases since no inhibition was found for the serine protease chymotrypsin.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Cysteine proteases are important targets in medicinal
chemistry, as they have been implicated in different dis-
eases.1 Therefore, there is a continuous demand for new
and more selective cysteine protease inhibitors. Inhibi-
Figure 1.
tors are also needed as activity-based probes for track-
ing protease activities in cells, tissues and whole
moiety, (Fig. 1) are known to react irreversibly with cys-
teine proteases, but also with serine proteases.4
animals.2 Many types of reversible and irreversible
inhibitors have been described.3,4 The more selective
It is expected that by lowering the reactivity of the
inhibitors of cysteine proteases, that is, inhibitors with
chloromethyl ketone moiety the selectivity can be
a strong preference for cysteine versus serine proteases,
improved. We were wondering whether replacement
are typically irreversible inhibitors. Among the irrevers-
of the carbonyl by a sulfoxide would reduce the reac-
ible inhibitors are diazomethyl ketones (Fig. 1), epoxides
tivity sufficiently to uncover new selective irreversible
(E64), Michael acceptors and chloromethyl ketones
cysteine protease inhibitors. This replacement would
require a chloromethyl sulfoxide moiety (Fig. 1), which
(Fig. 1).1,3,4 All these inhibitors contain an electrophilic
group which can react with the thiol function present in
as far as we know has not been described in the liter-
the active site of a cysteine protease. The reactivity of
ature for incorporation in protease inhibitors. Amino
this electrophilic group greatly determines the selectivity
acid based chloromethyl sulfoxides are known com-
and reaction rate of formation of the covalent inhibitor–
pounds and have been used for example in the synthe-
enzyme complex. With respect to this, peptidyl chlorom-
sis of mono oxodithioacetal containing natural
ethanes containing the electrophilic chloromethyl ketone
products and derivatives, that is, sparsomycin and
c-glutamyl marasmin.5a–d In this synthesis the chloro-
methyl sulfoxide moiety was used for reaction with
sodium methylmercaptide5a and with other thiolates.5c
Abbreviations: ESI-MS, electrospray ionization mass spectrometry;
Boc, tert-butyloxycarbonyl; BOP, (1H-benzotriazol-1-yloxy)tris(dime-
thylamino)phosphonium hexafluorophosphate; DBU, 1,8-diazabicy-
In the literature there is one procedure described5e in
which a few alcohols were reacted with chloromethyl
clo[5.4.0]undec-7-ene; DTT, dithiothreitol; DiPEA, N,N-diisopropyl-
methyl sulfoxide. However, these reactions were slow
ethylamine; TFA, trifluoroacetic acid; TMS, trimethylsilyl.
Keywords: Chloromethyl sulfoxide; Cysteine protease; Irreversible
inhibitor; Papain; Amino acid.
(18 h) and needed elevated temperatures (50 ꢁC). If
chloromethyl sulfoxides indeed are capable of inhibit-
ing cysteine proteases, they might become a new class
of selective irreversible inhibitors of cysteine proteases.
*
Corresponding author. Tel.: +31 30 253 7396/7307; fax: +31 30 253
6655.; e-mail: R.M.J.Liskamp@uu.nl
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.045

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A. J. Brouwer et al. / Bioorg. Med. Chem. 15 (2007) 6985–6993
In addition, by altering the amino part of the chloro-
methyl sulfoxide containing inhibitor, their specificity
and binding affinity might be tuned towards inhibition
of specific enzymes.
2. Results and discussion
2.1. Chemistry
It was decided to start with the most simple and easier
accessible chloromethyl sulfoxide derived from glycine.
To this end 2-(Boc-amino)ethyl bromide (1)6 was
reacted with in situ generated cesium thioacetate to give
thioacetate 2 in high yield (Scheme 1).
The thioacetate (2) was oxidized to the sulfinyl chloride
(3) by treatment with sulfuryl chloride and acetic anhy-
dride.7 The crude sulfinyl chloride (3) was directly used
in a reaction with diazomethane in dichloromethane to
yield the chloromethyl sulfoxide (4a).5a–d,8 Due to prob-
lems with the commercial availability of the starting
material (diazogen) of diazomethane, it was decided to
perform the reaction with TMS-diazomethane (Scheme
1), which is safe and not explosive and can be stored
for longer periods.9a TMS-diazomethane has been used
in the literature for different types of reactions,9 but
not for the synthesis of chloromethyl sulfoxides. The
purity and yield (37%) of 4a obtained with TMS-dia-
zomethane were almost equal to the reaction with dia-
zomethane, and comparable to the yield reported in
the literature.5d
Scheme 2. Synthesis of chloromethyl sulfoxides 4b–e from Boc-
protected amino acids.
Table 1. Yields (%) for the synthesis of chloromethyl sulfoxides 4b–e
from Boc-protected amino acids
Amino acid
6
7
8
4
Overall yield
Boc-Ala-OH (b)
Boc-Val-OH (c)
Boc-Leu-OH (d)
Boc-Phe-OH (e)
89
95
96
64
90
69
75
83
82
94
85
96
19
20
20
27
12
12
12
14
and the relatively large by-product formation caused
tedious purifications. In order to decrease reaction
time and by-product formation it was decided to per-
form the reaction in a microwave oven (Scheme 2).
Initial experiments, which were carried out in a
domestic microwave oven using a low power setting,
were already successful. After 4 cycles of only 15 s
TLC showed completion of the reaction and by-prod-
uct was absent. Thioacetates 8b–e could now be pre-
pared in high yields (82–96%) (Table 1) and—if
necessary—easily purified by silica gel column chroma-
tography. For comparison thioacetate 8e was also pre-
pared using a dedicated microwave reactor. After
irradiation for only 20 s at 140 ꢁC thioacetate 8e was
isolated in a comparable yield (88%). Next, thioace-
tates (8b–e) were oxidized to sulfinyl chlorides 9b–e,
analogous to the preparation of 3. The crude almost
pure sulfinyl chlorides (9b–e) were used directly in
the reaction with TMS-diazomethane in dichlorometh-
ane to afford the chloromethyl sulfoxides (4b–e), albeit
in low yields (19–27%) (Table 1), but still comparable
or better than the yields reported in the literature.9c,d
After preparation of the glycine derived chloromethyl
sulfoxide, the next step was the preparation of chloro-
methyl sulfoxides derived from amino acids with differ-
ent side chains. To this end Boc-protected amino acids
(Ala, Val, Leu, Phe) (5b–e) were used as the starting
materials which were reduced to their corresponding
alcohols (6b–e) (Scheme 2) using the method described
by Rodriguez et al.10 by reduction (NaBH4) of the
in situ prepared mixed anhydride.
Alcohols 6b–e were obtained in high yields and were
converted to mesylates 7b–e also in good to high
yields (after crystallization, Table 1). Introduction of
the thioacetate group, was performed using the proce-
dure used for 2 (Scheme 1), by reaction with in situ
generated cesium thioacetate in DMF at rt overnight.
Unfortunately the yields were low, variable (5–50%)
Before evaluation of chloromethyl sulfoxides 4a–e in
biological assays, we were interested in obtaining an
indication of their reactivity towards different nucleo-
philes (Scheme 3).
When chloromethyl sulfoxide 4a was treated with
sodium ethoxide in ethanol no product was found after
stirring for 40 h, only starting material was visible on
TLC. Treatment with benzylamine in ethanol with
Scheme 1. Synthesis of the glycine derived chloromethyl sulfoxide.

A. J. Brouwer et al. / Bioorg. Med. Chem. 15 (2007) 6985–6993
6987
serine protease. In addition, it is expected that no reac-
tion will take place with the abundantly present lysine
amino groups.
For biological evaluation a (fluorescently) labeled chlo-
romethyl sulfoxide was needed. It was decided to attach
a dansyl-group to the N-terminus of chloromethyl sulf-
oxide 4a. Since the dansyl-group is relatively bulky, a
glycine spacer was introduced. Thus, the Boc-group in
chloromethyl sulfoxide 4a was removed by TFA, fol-
lowed by coupling of dansylglycine 13 and labeled chlo-
romethyl sulfoxide 14 was obtained in 68% yield
(Scheme 4).
Scheme 3. Reactions of chloromethyl sulfoxides 4a with different
nucleophiles.
2.2. Biological evaluation
Papain was chosen as a target enzyme for evaluation of
the chloromethyl sulfoxides. Papain is the most widely
studied member of the cysteine protease class of
enzymes. First, papain (6.86 lM) was activated with
DTT in a phosphatebuffer (0.05 M, pH = 6.5) and
subsequently incubated with chloromethyl sulfoxide 14
(10 lM) for 1 h at room temperature. After centrifuga-
tion of the solution through a filter with a cut off of
5 kDa, the fluorescence of the filtrate was measured. A
significant difference in the fluorescence was found
between the chloromethyl sulfoxide containing sample
and a control sample without papain which had received
the same treatment. The difference in fluorescence
indicated that the chloromethyl sulfoxide (14) was
capable of binding of ca. 40% of the papain which was
a very promising result. However it was also possible
that chloromethyl sulfoxide 14 binds reversibly to
papain. To this end the cut off filter was extensively
washed with the phosphate buffer. Since hardly any
fluorescence was measured in the concentrated filtrate,
it appeared that chloromethyl sulfoxide 14 was bound
irreversibly to papain.
Scheme 4. Synthesis of dansyl-labeled chloromethyl sulfoxide 14.
DBU gave similar results, even when the reaction was
stirred at 50 ꢁC. As expected product formation was
found after reaction with the sodium thiolate of benzyl
mercaptan in ethanol. After stirring for 16 h at rt more
than half of chloromethyl sulfoxide 4a was consumed.
After a total of 80 h, and addition of more reagent,
mono oxodithioacetal 12 was obtained in 85% yield
after purification. These simple test-reactions clearly
showed the preference of a chloromethyl sulfoxide (4a)
for a sulfur nucleophile and as a consequence, its poten-
tial to react selectively with the cysteine thiol of a cys-
teine protease, as opposed to the serine-hydroxyl of a
The next step was to evaluate the chloromethyl sulfox-
ides in an activity-based enzyme assay with papain using
Bz-L-Arg-pNA as a substrate. Although a relatively high
0.20
0.19
0.18
0.17
0.16
0.15
0.14
0.13
0.12
0.11
0.10
5
0
10
15
20
25
30
Minutes
Figure 2. Progress curve of a papain enzyme assay for comparison of the inhibitory activity of chloromethyl sulfoxides 4a–e and 14 (j, control; m,
4a; h, 4b; , 4c; n, 14; Ç, 4d; +, 4e). [E] = 800 nM, [S] = 1.0 mM, [I] = 500 lM.
*

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A. J. Brouwer et al. / Bioorg. Med. Chem. 15 (2007) 6985–6993
concentration of 500 lM was required for sufficient inhi-
bition, we were enraptured that our chloromethyl sulf-
oxides could indeed decrease the activity of papain.
Next, chloromethyl sulfoxides 4a–e were tested for their
inhibitory activity. No large differences were expected
since the S1 subsite of papain is not very selective.
Although it is reported that this site shows some prefer-
ence for Arg and Lys side chains,11 most amino acid side
chains are accepted. However, when chloromethyl sulf-
oxides 4a–e were compared in this assay, some remark-
able selectivity’s were found (Fig. 2). Glycine derived
chloromethyl sulfoxide 4a showed no inhibition while
dansyl-labeled chloromethyl sulfoxide 14 (Scheme 4)
was clearly one of the best inhibitors.
thesis of 14 (Scheme 4). Unfortunately, chloromethyl
sulfoxide 15 displayed poor inhibition, possibly indicat-
ing that the phenylalanine side chain cannot be properly
positioned to bind in the S2 subsite. Based on the found
affinities, together with the known preference for the pa-
pain subsites, it is assumed that the side chains of chlo-
romethyl sulfoxides 4b–e bind to the S2 subsite instead
of the S1 subsite of papain.
Molecular modeling corroborated this assumption. The
three dimensional structure of the diazomethyl ketone
inhibitor Z-LFG-CH2-papain covalent complex has
been determined by X-ray crystallography.14 This inhib-
itor is suitable for comparison with chloromethyl sulfox-
ide inhibitor 4e since it contains a phenylalanine residue
which binds to the S2 subsite of papain (Fig. 3).
Phenylalanine derived chloromethyl sulfoxide 4e
showed the highest inhibition. High inhibition was also
found for valine derived chloromethyl sulfoxide 4c.
Since the S1 subsite of papain does not accept the side
chain of valine,12,13 the isopropyl side chain of this chlo-
romethyl sulfoxide (4c) is presumably binding in the S2
subsite of papain instead of the S1 subsite. In addition, it
is known from the literature that the S2 subsite of pa-
pain has a preference for the side chains of phenylala-
nine and valine residues.13 Since dansyl containing
chloromethyl sulfoxide 14 was one of the best inhibitors
it is likely that its aromatic part also binds to the S2 sub-
site. Therefore, we surmised that chloromethyl sulfoxide
15, containing an N-terminal phenylalanine residue,
would display inhibitory behavior similarly to 14. Chlo-
romethyl sulfoxide 15 was obtained in high yield (89%,
Fig. 5) by the same procedure as was used for the syn-
Using the program Yasara Structure, molecular model-
ing of the 4e-papain complex clearly showed that the
side chain of chloromethyl sulfoxide 4e binds to the
same (S2) pocket of papain (A) as the diazoketone
inhibitor (B). Although only the S(Ca)R(S@O) diaste-
reomer covalently bound to papain (Fig. 3a) is shown
here, both diastereoisomers of 4e were found to be able
to bind in a similar way to the active site of papain.
105
100
95
90
85
80
75
1.0
1.5
2.0
2.5
3.0
[I]-1 x 103 L/mol
Figure 4. The dependence of j upon the concentration of inhibitor 4e,
plotted as reciprocals in accordance to Kitz and Wilson kinetics.15
0.6
0.45
0.3
0.15
0
-200
-100
0
100
200
300
400
500
[I] (µM)
Figure 5. A Dixon plot of inhibition of papain by chloromethyl
sulfoxide 4e ([S] (mM): j, 1.0; m, 1.2; Ç, 1.4; h, 1.6; n, 1.8; Æ, 2.0).
Figure 3. Comparison of covalent bound 4e-papain (a) with a crystal
structure of Z-LFG-CH2-papain14 (b).

A. J. Brouwer et al. / Bioorg. Med. Chem. 15 (2007) 6985–6993
6989
Table 2. Inhibitor constants of chloromethyl sulfoxides 4a–e, 14 and
15 (R-W[CH2S(O)]–CH2Cl)a
The best Ki (142 lM) was obtained for phenylalanine
derived chloromethyl sulfoxide 4e, which was compara-
ble to the Ki determined by the time-dependent assay
(179 lM). Although this Ki is modest, we think that this
result is encouraging and that affinity for the active site
can be improved by modification of the N-terminus, for
example by introduction of (an) amino acid residue(s).
In addition, it is possible that one of both diastereoiso-
mers, which so far could not be separated, has a higher
affinity and therefore better Ki. Lineweaver–Burk plots
indicated that the mode of inhibition by the chloro-
methyl sulfoxides was competitive for the substrate.
Chloromethyl sulfoxide
R
Ki (lM)
4a
15
4b
4c
4d
14
4e
Boc-Gly
No inhibition
3671 558
1426 149
543 21
Boc-Phe-Gly
Boc-Ala
Boc-Val
Boc-Leu
Ds-Gly-Gly
Boc-Phe
485 51
471 78
142 5b
a Ki values were determined using Dixon plots.16
b Determination using Kitz and Wilson kinetics15 gave Ki = 179 lM.
To determine whether the chloromethyl sulfoxides were
selective towards cysteine proteases, an enzyme assay
was performed with the serine protease chymotrypsin,
which clearly showed that none of the chloromethyl
sulfoxides were capable of inhibition of this protease.
3. Conclusions
A series of amino acid based chloromethyl sulfoxides
and N-terminal modified chloromethyl sulfoxides have
been successfully synthesized in moderate to high yields.
The synthesis of the intermediate thioacetates was opti-
mized using the microwave leading to high yields and
very short reaction times. Instead of the highly toxic
and explosive diazomethane, the stable and safe TMS-
diazomethane was found to be equally suitable in the
synthesis of the chloromethyl sulfoxides from the corre-
sponding sulfinyl chlorides.
Figure 6. Structures of chloromethyl sulfoxides 4a–e, 14 and 15.
For determination of the inhibition constants and
inactivation rates, the chloromethyl sulfoxides were
subjected to time-dependent assays according to Kitz
and Wilson.15 In the first assays no significant irrevers-
ible binding was observed after 90 min, indicating that
the chloromethyl sulfoxides were not bound covalently
to the active site and thus were binding as reversible
inhibitors. Fortunately, after 4 h the binding was found
to be indeed irreversible. As expected the rate constant
for the conversion of the reversible complex to the irre-
The described chloromethyl sulfoxides were capable of
irreversible inhibition of the cysteine protease papain.
In addition, the affinities were found to be dependent
on the side chain of the inhibitor, which points at the
possibilities for tuning the selectivity. The chloromethyl
sulfoxides were also found to be selective cysteine prote-
ase inhibitors, since no inhibition of the serine protease
chymotrypsin was observed. Although relatively modest
affinities and rate constants were found, we believe that
these chloromethyl sulfoxides might become a new
important class of irreversible cysteine protease inhibi-
tors of which the affinity can be further improved by
using different amino acid side chains and/or by extend-
ing the N terminus. Currently, we are preparing small li-
braries of N-terminal functionalized amino acid based
chloromethyl sulfoxides for finding inhibitors with high-
er affinities for biologically more relevant cysteine prote-
ases. Under present investigation is also the influence of
the backbone length on the rate constant.
versibly inhibited enzyme (k3) was low (1.5 · 10À5 sÀ1
)
(Fig. 4).
The low reaction rate reflects to insufficient reactivity of
the thiol in the active site of papain towards the chloro-
methyl sulfoxide moiety and/or to low reactivity of the
chloromethyl sulfoxide. Another explanation is that
the chloromethyl sulfoxide moiety of the reversibly
bound inhibitor is not properly positioned with respect
to the active site thiol, thus making formation of a cova-
lent bond less likely.
Since all prepared chloromethyl sulfoxides showed no
significant irreversible binding in the first hour it was
decided to determine the inhibitor constants Ki using
Dixon plots (Fig. 5), which normally are used for revers-
ible inhibition.16
4. Experimental
Boc-protected amino acids were purchased from
Advanced Chemtech Europe Ltd (France). Thioacetic
acid was distilled and used within 4 weeks. Peptide grade
solvents for synthesis were purchased from Biosolve
(The Netherlands) and were stored on molecular sieves
The Ki values found (Table 2) for all prepared chloro-
methyl sulfoxides (Fig. 6) were in agreement with the
results from the comparison assay (Fig. 1).
˚
(4 A). Reactions were carried out at ambient tempera-
ture unless stated otherwise. TLC analysis was per-

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A. J. Brouwer et al. / Bioorg. Med. Chem. 15 (2007) 6985–6993
formed on Merck pre-coated silicagel 60 F-254
(0.25 mm) plates. Spots were visualized with UV light,
ninhydrin or Cl2-TDM.17 Solvents were evaporated un-
der reduced pressure at 40 ꢁC. Column chromatography
was performed on ICN silicagel 60 (32–63 lm). Melting
and DMF was evaporated in vacuo. The residue was
dissolved in EtOAc, washed with 5% NaHCO3, water,
1 N KHSO4, water and brine. Drying (Na2SO4) and
concentration in vacuo afforded the crude thioacetate
which was purified by column chromatography. All thi-
oacetates were obtained as pale orange oils, which crys-
tallized either directly or overnight. Spectroscopic data
for 8b, c, and e were consistent with the literature.5c,d
points were measured on a Buchi Schmelzpunktbestim-
¨
mungsapparat (according to Dr. Tottoli) and are uncor-
rected. Electrospray mass spectra were recorded on a
Shimadzu LCMS-QP-8000 spectrometer, or a Finnigan
LCQ Deca XP MAX spectrometer. Elemental analyses
were carried out at Kolbe Mikroanalytisches Laborato-
4.4.1. Alanine derived thioacetate 8b. The scale of the
reaction was 15.0 mmol and 8b was obtained (2.87 g,
82%) after column chromatography (hexanes/CH2Cl2/
EtOAc, 15:4:1).
rium (Mulheim an der Ruhr, Germany). Microwave as-
¨
sisted reactions were carried out in either a Biotage
Initiator or in a SMC (E70-TFA, 650 W) domestic
1
microwave oven. H NMR (300 MHz) and 13C NMR
4.4.2. Valine derived thioacetate 8c. The scale of the reac-
tion was 15.0 mmol and 8c was obtained (3.67 g, 94%)
after column chromatography (hexanes/CH2Cl2/EtOAc,
10:4:1).
(75 MHz) spectra were recorded on a Varian G-300
spectrometer. Chemical shifts are reported in ppm rela-
tive to TMS (0 ppm) or DMSO (2.50 ppm) for the H
1
NMR and to CDCl3 (77 ppm) or DMSO (39.5 ppm)
for the 13C NMR spectra as internal standards. 13C
NMR spectra were recorded using the attached proton
test (APT) pulse sequence. HSQC spectra of 12 and 14
and a HMBC spectrum of 12 were recorded on a Varian
Inova-500 spectrometer.
4.4.3. Leucine derived thioacetate 8d. The scale of the
reaction was 15.0 mmol and 8d was obtained (3.51 g,
85%) after column chromatography (hexanes/CH2Cl2/
EtOAc, 5:4:1).
Rf = 0.51 (hexanes/CH2Cl2/EtOAc, 5:4:1). Mp = 51 ꢁC.
4.1. Alcohols 6b–d: general procedure
1H NMR (300 MHz, CDCl3): d = 0.91 (2d, J = 1.9 Hz,
6H, CH(CH3)2), 1.32 (m, 2H, CH2CH(CH3)2), 1.43 (s,
9H, C(CH3)3), 1.66 (m, 1H, CH2CH(CH3)2), 2.35 (s,
3H, SC(O)CH3), 2.96 (dd, Jgem = 13.7 Hz, Jvic = 6.9 Hz,
1H, NCHCHa), 3.13 (dd, Jgem = 13.7 Hz, Jvic = 4.7 Hz,
1H, NCHCHb), 3.83 (m, 1H, NCH), 4.47 (d,
J = 8.8 Hz, 1H, NH).
Alcohols 6b–d were synthesized according to Rodriquez
et al.10 The described method afforded the alcohols in
high yields (Table 1).
4.2. Mesylates 7b–e: general procedure
Mesylates 7b–e were prepared following an earlier de-
scribed protocol.18 All products were recrystallized
from EtOAc/hexanes and obtained as white fluffy crys-
tals. All spectroscopic data were consistent with the
literature.19
13C NMR (75 MHz, CDCl3): d = 21.9, 22.8
(CH2CH(CH3)2),
24.6
(CH2CH(CH3)2),
28.1
(C(CH3)3), 30.3 (SC(O)CH3), 34.2 (CH2CH(CH3)2),
43.2 (SCH2), 48.3 (NCH), 78.8 (C(CH3)3), 155.2
(C(O)C(CH3)3), 195.3 (SC(O)CH3).
4.3. Glycine derived thioacetate 2
ESI-MS: m/z = 176.50 [M-Boc+H]+, 220.33 [M-t-
Thioacetic acid (3.8 mL, 53.8 mmol) and cesium carbon-
ate (9.45 g, 29 mmol) were dissolved in DMF and this
solution was added to Boc-(2-bromoethyl)amine6
(10.0 g, 44.8 mmol). The flask was wrapped in alumi-
num foil and stirred overnight at rt. After evaporation
of the DMF, ethylacetate (500 mL) was added. The or-
ganic layer was washed with NaHCO3(2.5%), water,
brine and dried (Na2SO4). After concentration and
coevaporation with chloroform, the product was ob-
tained as a brownish oil (9.43 g, 96%). Spectroscopic
data were consistent with the literature.20
Bu+H]+, 276.33 [M+H]+, 298.50 [M+Na]+.
4.4.4. Phenylalanine derived thioacetate 8e. The scale of
the reaction was 15.0 mmol (domestic microwave oven)
and 8e was obtained and used without further purifica-
tion (4.45 g, 96%). With a dedicated microwave reactor
the scale was 2.0 mmol and 8e was obtained (545 mg,
88%) after column chromatography (hexanes/CH2Cl2/
EtOAc, 5:4:1).
4.5. Chloromethyl sulfoxides 4a–e: general procedure
4.4. Thioacetates 8b–e: general procedure
A solution of the thioacetate (8a–e) (12.3 mmol) in dry
dichloromethane (25 mL) was cooled under a nitrogen
atmosphere to À20 ꢁC. Acetic anhydride (1.16 mL,
12.3 mmol) was added all at once and sulfuryl chloride
(1.99 mL, 24.6 mmol) was added dropwise. After stir-
ring for 1 h at approximately À10 ꢁC, the reaction was
warmed up to rt. The mixture was concentrated in vacuo
and the residue was dissolved in dry dichloromethane
(15 mL). TMS-diazomethane (2 M in hexanes)
(12.3 mL, 24.6 mmol) was cooled under a nitrogen
A solution of thioacetic acid (1.12 mL, 15.8 mmol) and
cesium carbonate (2.69 g, 8.25 mmol) in DMF (60 mL)
was added to the mesylate (7b–e, 15.0 mmol) in a pres-
sure vessel. The closed vessel was subjected to micro-
wave irradiation in either a domestic microwave oven
(four or five times 15 s at the defrost setting) or in a ded-
icated microwave reactor (in 40 s from rt to 140 ꢁC, 20 s
at 140 ꢁC). The formed white precipitate was filtered off

A. J. Brouwer et al. / Bioorg. Med. Chem. 15 (2007) 6985–6993
6991
atmosphere to À20 ꢁC and the dissolved residue was
4.6. Mono oxodithioacetal 12
added to the cooled TMS-diazomethane dropwise. After
2 h stirring, the excess TMS-diazomethane was decom-
posed by addition of a few drops of acetic acid until
no more nitrogen gas evolved. Concentration in vacuo
followed by column chromatography afforded the chlo-
romethyl sulfoxides (4a–e). All chloromethyl sulfoxides
were obtained as white solids. Spectroscopic data for
4a–c, e were consistent with the literature.5c,d
To a solution of chloromethyl sulfoxide 4a (73 mg,
0.30 mmol) in dry methanol was added benzyl mercap-
tan (39 lL, 0.33 mmol) and a solution of sodium meth-
oxide in methanol (76 lL, 30% w/w). After stirring
overnight (20 h) at rt the conversion was 50% according
to TLC. Additional portions of benzyl mercaptan and
sodium methoxide solution were added and after 2 h
the conversion was already 80%. After additional stir-
ring for ca, 2.5 days, the reaction mixture was concen-
trated in vacuo. Column chromatography (DCM/
EtOAc, 1:1) afforded mono oxodithioacetal 12 as a
white solid (84 mg, 85%).
4.5.1. Glycine derived chloromethyl sulfoxide 4a. The
scale of the reaction was 9.0 mmol and 4a was obtained
(790 mg, 37%) after column chromatography (EtOAc/
CH2Cl2, 1:1). Glycine derived chloromethyl sulfoxide
4a was also prepared using freshly prepared diazome-
thane. The same procedure was followed and similar
yields were found.
Rf = 0.43 (EtOAc). Mp = 87 ꢁC.
1H NMR (300 MHz, CDCl3): d = 1.45 (s, 9H, C(CH3)3),
2.86 (dt, Jgem = 13.2 Hz, Jvic = 5.5 Hz, 1H, NCH2CHa
S(O)), 3.13 (dt, Jgem = 13.2 Hz, Jvic = 6.6 Hz, 1H,
NCH2CHbS(O)), 3.67 (m, 4H, NCH2, SCH2Ph), 3.92
(s, 2H, S(O)CH2S), 5.13 (bs, 1H, NH), 7.34 (m, 5H,
Ar–CH).
4.5.2. Alanine derived chloromethyl sulfoxide 4b. The
scale of the reaction was 12.3 mmol and 4b was obtained
(596 mg, 19%) after column chromatography (EtOAc/
CH2Cl2, 3:7).
4.5.3. Valine derived chloromethyl sulfoxide 4c. The scale
of the reaction was 14.0 mmol and 4c was obtained
(787 mg, 20%) after column chromatography (EtOAc/
CH2Cl2, 25:75).
13C NMR (75 MHz, CDCl3): d = 28.3 (C(CH3)3), 35.2
(NCH2), 37.0 (SCH2Ph), 50.9 (4 lines) (S(O)CH2S),
50.7 (2 lines) (NCH2CH2S(O)), 79.8 (C(CH3)3), 129.2,
127.3, 128.8, 136.3, 136.4 (ArAC), 155.8 (C@O (Boc).
ESI-MS: m/z = 351.95 [M+Na]+.
4.5.4. Leucine derived chloromethyl sulfoxide 4d. The
scale of the reaction was 10.3 mmol and 4d was obtained
(610 mg, 20%) after column chromatography (EtOAc/
CH2Cl2 gradient from 5:95 to 20:80).
Anal. Calcd for C15H23NO3S2: C, 54.68; H, 7.04; N,
4.25; S, 19.46. Found: C, 54.78; H, 6.98; N, 4.20; S,
19.40.
Rf = 0.53 (EtOAc). Mp = 139 ꢁC.
4.7. Ds-Gly-GlyW[CH2S(O)]-CH2Cl (14)
1H NMR (300 MHz, CDCl3): d = 0.93, 0.95 (2d,
J = 4.4 Hz, 6H, CH(CH3)2), 1.47, 1.71 (2m, 3H,
CH2CH(CH3)2), 1.43 (s, 9H, C(CH3)3), 2.88 (dd,
Jgem = 13.5 Hz, Jvic = 8.9 Hz, 0.5H, NCHCH2), 3.00
(dd, Jgem = 13.2 Hz, Jvic = 3.7 Hz, 0.5H, NCHCH2),
3.14 (dd, Jgem = 13.2 Hz, Jvic = 7.7 Hz, 0.5H,
NCHCH2), 3.31 (dd, Jgem = 13.5 Hz, Jvic = 3.8 Hz,
0.5H, NCHCH2), 3.92, 4.10 (2m, 1H, NCH), 4.40,
4.51 (2d, J = 11.0 Hz, 1H, S(O)CH2Cl), 4.64, 4.74 (2d,
1H, S(O)CH2Cl), 4.70, 5.03 (s d, 1H, NH).
Chloromethyl sulfoxide 4a (483 mg, 2.00 mmol) was
Boc-deprotected by treatment with TFA/DCM
(10 mL, 1:1) for 5 min. After evaporation, the residue
was dissolved in dichloromethane (10 mL). A solution
of dansyl-glycine21 (678 mg, 2.20 mmol) in dichloro-
methane (150 mL) was added to the Boc-deprotected
chloromethyl sulfoxide solution. BOP (973 mg,
2.20 mmol) and DiPEA (660 lL, 4.00 mmol) were
added, and the reaction was stirred overnight at rt
with the flask covered with aluminum foil. If necessary
more DiPEA was added to maintain a basic pH. After
stirring for an additional hour followed by evapora-
tion in vacuo, the residue was dissolved in EtOAc
and washed with 1 N KHSO4, water and brine. The
organic layer was dried (Na2SO4) and concentrated
in vacuo. Column chromatography (MeOH/EtOAc,
5:95) and trituration using MeOH, afforded 14 as a
yellow/greenish solid (585 mg, 68%).
13C NMR (75 MHz, CDCl3): d = 21.9, 22.6
(CH2CH(CH3)2), 24.7, 24.9 (CH2CH(CH3)2), 28.2
(C(CH3)3), 43.2, 44.0 (CH2CH(CH3)2), 45.0, 45.2
(NCH), 55.4, 56.7 (NCHCH2), 56.7, 56.9 (S(O)CH2Cl),
79.9 (C(CH3)3), 155.4 (C(O)C(CH3)3).
ESI-MS: m/z = 198.63, 200.88 [M-Boc+H]+, 242.39,
244.40 [M-t-Bu+H]+, 298.37, 300.31 [M+H]+, 320.49,
322.43 [M+Na]+: due to chlorine isotopes.
Rf = 0.43 (10% MeOH/EtOAc). Mp = 87 ꢁC.
Anal. Calcd for C12H24ClNO3S: C, 48.39; H, 8.12; N,
4.70. Found: C, 48.46; H, 8.03; N, 4.72.
1H NMR (300 MHz, DMSO-d6) d, 2.83 (s, 6H,
N(CH3)3), 2.85–2.93 (m, 2H, NCH2CH2), 3.30–3.39
(m, 2H, NCH2CH2), 3.44 (s, 2H, NCH2C(O)), 4.77 (d,
J = 11.3 Hz, 1H, S(O)–CHa–Cl), 4.96 (d, J = 11.3 Hz,
1H, S(O)–CHb–Cl), 7.26, 7.60, 8.11, 8.29, 8.46 (m, 6H,
Ds-Ar–CH).
4.5.5. Phenylalanine derived chloromethyl sulfoxide 4e.
The scale of the reaction was 14.3 mmol and 4e was ob-
tained (1.29 g, 27%) after column chromatography
(EtOAc/CH2Cl2, 25:75).

6992
A. J. Brouwer et al. / Bioorg. Med. Chem. 15 (2007) 6985–6993
13C NMR (75 MHz, DMSO-d6) d: 32.7 (NCH2CH2),
45.3 (N(CH3)2), 49.2 (NCH2CH2), 58.3 (S(O)–CH2–
Cl), 115.4, 119.4, 123.8, 128.1, 128.4, 129.7 (Ar–CH),
129.3, 136.0, 151.5 (Ar–C), 168.4 (C(O)).
used without further purification. Bz-L-Arg-pNA and
Bz-L-Tyr-pNA were purchased from Bachem (Switzer-
land). Kinetic enzyme assays were done in a lQuant
Biotek plate reader for 1 h at rt at 405 nm. All assays
were done in triplicate or quadruplicate.
The signals of NCH2C(O) were found in the proton
spectra but were absent in the carbon spectra. From a
HSQC-measurement the (NCH2C(O))-signal was deter-
mined at 45.3 ppm.
4.10.2. Papain assays. A colorimetric assay in 96-well
plates was used. In this assay a phosphate buffer
(0.10 M, pH 6.5) containing EDTA (1.5 mM) and
DTT (2.0 mM) was used, and Bz-L-Arg-pNA as a sub-
strate. The optimal papain and substrate concentrations
were found to be 800 nM and 1.0 mM. The chloro-
methyl sulfoxide was first pre-incubated for 1 h with
the activated papain before addition of the substrate.
ESI-MS: m/z = 432.25, 434.25 [M+Na]+: due to chlorine
isotopes.
4.8. Boc-Phe-GlyW[CH2S(O)]-CH2Cl (15)
Chloromethyl sulfoxide 15 was synthesized according to
the preparation of 14 on a scale of 0.40 mmol. The prod-
uct was isolated after workup by crystallization (EtOAc/
hexanes) as a white solid (143 mg, 89%).
Papain was dissolved in buffer without DTT (8.16 lM)
and after shaking for 5 min the solution was centrifuged.
The substrate Bz-L-Arg-pNA was first dissolved in
DMSO and then diluted twice (50.0 mM) with buffer
without DTT. The chloromethyl sulfoxides were dis-
solved in DMSO and diluted twice (25.0 mM) with buf-
fer without DTT. In a typical assay to each well was
added inhibitor solution (4.0 lL), buffer (172.0 lL)
and enzyme solution (20.0 lL). For the controls a
DMSO/buffer without DTT solution (1:1) was used in-
stead of the inhibitor solution. After 1 h pre-inhibition
a sample (98.0 lL) was taken from each well and added
to wells containing substrate solution (2.0 lL), and sub-
sequently the liberation p-nitroaniline from Bz-L-Arg-
pNA was measured at 405 nm during 1 h. Final concen-
trations in the wells were: enzyme: 800 nM; substrate:
1.0 mM; inhibitor: 500 lM.
Rf = 0.30 (EtOAc). Mp = 125 ꢁC.
1H NMR (300 MHz, CDCl3) d, 1.40 (s, 9H, C(CH3)3),
2.84–3.11 (m, 4H, CH2Ph, NCH2CH2), 3.59–3.78 (m,
2H, NCH2), 4.32 (m, 1H, NCH), 4.39 (dd,
Jgem = 11.3 Hz, Jvic = 5.0 Hz, 1H, S(O)–CHa–Cl), 4.47
(dd, Jgem = 11.3 Hz, Jvic = 4.1 Hz, 1H, S(O)–CHb–Cl),
5.07 (bd, 1H, BocNH), 6.77 (bs, 1H, CH2NHC@O),
7.17–7.33 (m, 5H, Ar–CH).
13C NMR (75 MHz, CDCl3) d: 28.2 (C(CH3)3), 33.2,
33.4 (NCH2), 38.6 (CH2Ph), 49.2 (S(O)CH2CH2), 55.7
(NCH), 56.5 (CH2Cl), 80.1 (C(CH3)3), 126.9, 128.6,
129.3, 136.6 (ArAC), 155.3 (C@O (Boc)), 172.2
(NHCHC@O).
For the assays for determination of the Ki’s with Dixon
plots15 a similar procedure was used. Stock solutions of
the substrate and inhibitors were prepared using only
DMSO and with different concentrations. Substrate
concentrations were 200, 180, 160, 140, 120 and
100 mM, and inhibitor concentrations were 100, 50
and 25 mM in the stock solutions (for 4e: 50, 25 and
12.5 mM). To each well was added inhibitor solution
(2.0 lL), substrate solution (2.0 lL), buffer (176.0 lL)
and enzyme solution (20.0 lL). For the controls DMSO
was added instead of inhibitor solution. The liberation
of p-nitroaniline was directly measured for 1 h. Final
concentrations in the wells were: enzyme: 800 nM; sub-
strate: 1.0–2.0 mM; inhibitor: 0.25, 0.50 or 1.00 mM (for
4e: 0.125, 0.25 or 0.50 mM).
ESI-MS: m/z = 411.53, 413.29 [M+Na]+: due to chlorine
isotopes.
4.9. Fluorescence based binding test with papain
An aqueous solution of papain (7.2 lM, 500 lL) was buf-
fered with 25 lL sodium phosphate buffer (1.0 M) con-
taining EDTA (30 mM) and DTT (40 mM) yielding a
final 50 mM sodium phosphate buffer at a pH of 6.5 con-
taining 1.5 mM EDTA and 2 mM DTT. To this mixture
and the reference mixture without Papain, 2.2 lL of
inhibitor 14 (2.38 mM in DMSO) was added, yielding a
concentration of 10 lM. After incubation for 1 h at RT,
450 lL of each sample was subjected to ultra filtration
with a filter with a cut off of 5000 Da in a centrifuge at
10,000 rpm. This treatment was continued with the same
buffer without Papain or probe until the volume of the fil-
trate was 1.5 mL, the filtrate of each sample was measured
with an excitation wavelength of 321 nm and an emission
wavelength of 564 nm to monitor the decrease of 14.
For the time-dependent assays15 the stock solution of
the substrate was 100 mM in DMSO, and the stock
solutions of the inhibitor were 40, 55, 70, 85 and
100 mM. To vials containing inhibitor solution (10 lL)
or DMSO (10 lL, controls) was added buffer (880 lL)
and enzyme solution (100 lL). The rate of irreversible
inhibition was followed by withdrawing samples
(99 lL) at different time intervals. These samples were
added to wells containing substrate solution (1.0 lL)
and the remaining enzyme activity was measured.
4.10. Enzyme assays
4.10.1. General remarks for kinetic experiments. Buffer
solutions were prepared using distilled water. Stock as-
say solutions were centrifuged before use. Papain and
chymotrypsin were purchased from Sigma and were
4.10.3. Chymotrypsin assay. The following stock solu-
tions were prepared: buffer: sodium phosphate
(50 mM, pH 7.0); enzyme: chymotrypsin was dissolved

A. J. Brouwer et al. / Bioorg. Med. Chem. 15 (2007) 6985–6993
6993
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in buffer (1.1 lM); substrate: Bz-L-Tyr-pNA was dis-
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sulfoxides were dissolved in DMSO (20.0 mM). To each
well was added inhibitor solution (10.0 lL) and enzyme
solution (180.0 lL). For the controls DMSO was used
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a sample (95.0 lL) was taken from each well and added
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enzyme: 1.0 lM; substrate: 0.25 mM; inhibitor: 1.0 mM.
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´
Arevalo, M. A.; Lazaro, E.; Ballesta, J. P. G.; Lelieveld,
Molecular modeling was performed using the program
Yasara Structure (version 6.6.20; www.yasara.org). In
the crystal structure of papain covalently bound to the
diazomethyl ketone inhibitor (Z-LFG-CH2-N2) was
used (Pdb-file 1KHQ),14 the bound diazomethyl ketone
inhibitor was replaced by chloromethyl sulfoxide 4e fol-
lowed by minimization without changing the atomic
coordinates of the enzyme. The following settings were
used: Yamber2 force field; PME electrostatics with a
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˚
cutoff at 7.8 A; simulated annealing; the simulation cell
˚
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The static minimization was done from 300 K until the
temperature dropped below 10 K. The minimized com-
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5.0. The enzyme surface was colored according to elec-
trostatic surface potential: red is negatively charged;
blue is positively charged.
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Acknowledgments
We thank Dr. J. Kemmink and Dr. J.H. Ippel for
recording the 500 MHz NMR spectra, Dr. D.T.S.
Rijkers and Mr. D.J. van Zoelen for their helpful discus-
sions about enzyme kinetics, Mr. H.B. Albada and
Dr. E.E. Moret for their assistance with the molecular
modeling, and finally Ms. A.C. Dechesne for measuring
some of the mass spectra.
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Synthesis 2000, 1579–1584.
References and notes
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