Thiirancarboxamides as inhibitors of papain

Article abstract of DOI:10.1002/ardp.200300820

Derivatives of the thiirancarboxylic acid building-block containing a peptide bond were synthesised and screened against the model cysteine protease papain. The most active of the series showed a second-order rate constant of inactivation comparable to that of the parent compound. The insertion of a peptide moiety seems to compensate the lack of a free carboxylate interacting with the histidinium ion at the enzyme's active site.

Full text of DOI:10.1002/ardp.200300820

90
Bruno and Schirmeister
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 90−95
Gemma Bruno,
Tanja Schirmeister
Thiirancarboxamides as Inhibitors of Papain
Derivatives of the thiirancarboxylic acid building-block containing a peptide bond
were synthesised and screened against the model cysteine protease papain.
The most active of the series showed a second-order rate constant of inacti-
vation comparable to that of the parent compound. The insertion of a peptide
moiety seems to compensate the lack of a free carboxylate interacting with the
histidinium ion at the enzyme’s active site.
Department of Chemistry and
Pharmacy,
Institute of Pharmacy and
Food Chemistry
Keywords: Thiirane; Episulfide; Cysteine protease; Inhibitor; Papain
Received: May 30, 2003; Accepted: August 29, 2003 [FP820]
DOI 10.1002/ardp.200300820
Introduction
Replacement of the oxirane ring with an aziridine gave
rise to new classes of peptidic inhibitors: the ring nitro-
gen offered a third position for derivatisation and,
therefore, the possibility to influence activity and selec-
tivity of those inhibitors.
Cysteine proteases belong to a heterogeneous family
of peptidases almost ubiquitous in nature [1]. Their key
role in the life cycle of certain viruses [2Ϫ4], bacteria
[
5], and protozoa [6] offers a starting point for the de-
Thiirane is structurally related to oxirane and aziridine,
being susceptible to ring-opening by nucleophiles as
sign of cysteine protease inhibitors as therapeutic ag-
ents.
Lysosomal and cytoplasmatic cysteine proteases in
mammals (cathepsins and calpains) fulfil specific
house-keeping functions. An abnormal expression or
an imbalance between proteases and endogenous in-
hibitors seems to be associated with a number of dis-
eases such as malignancy of certain tumours, e.g. in
prostate carcinoma [7], progression of rheumatoid ar-
thritis [8], destruction of muscle fiber in sarcoid myo-
pathy [9], neurodegenerative processes as in Alzhei-
mer’s disease [10] and Huntington’s disease [11], isch-
emic neuronal death [12], calcium-triggered cell death
[
13], and atrial fibrillation [14].
Therefore, the urgency of new therapeutically active
compounds is reflected by the strong efforts done
lately in the development of more potent cysteine pro-
tease inhibitors. Three-membered heterocycles rank
among the most promising irreversible cysteine pro-
tease inhibitors, due to their sensitivity against the thi-
olate at the active site of the enzyme.
Since the discovery of the naturally occuring irrevers-
ible inhibitor E-64, epoxysuccinyl peptides have rep-
resented a fruitful field for the development of potent
cysteine protease inhibitors: Loxistatin reached phase
III in the clinical trial, but was stopped later because of
side-effects [15].
Correspondence: Tanja Schirmeister, Department of
Chemistry and Pharmacy, Institute of Pharmacy and Food
Chemistry, Am Hubland, D-97074 Wuerzburg, Germany.
Phone: +49 931 888-5440, Fax: +49 931 888-5494; e-mail:
schirmei@pharmazie.uni-wuerzburg.de
Figure 1. Thiirane and epoxide derivatives as cysteine
protease inhibitors.
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2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 90−95
Thiirancarboxamides as Inhibitors 91
well. However, it shows a slower reaction rate of ring
opening under either acidic or basic conditions, when
compared with oxirane and aziridine [16-18], due to
less ring strain and to lower electronegativity of the
sulphur.
A direct comparison with an epoxide analogue is not
possible, since no oxirane-2-carboxylate was tested
so far against cysteine proteases; however, methyl-
and benzyl esters of the homologue (S,S)-2-benzyl-
3,4-epoxybutanoic acid (BEBA) (2a, 2b, Figure 1) irre-
Ϫ1 Ϫ1
versibly inactivate papain (k
18 M
s
and 49
2
nd
As reported previously [19], (S)-thiirancarboxylic acid
Ϫ1 Ϫ1
M
s
, respectively) [21] and epoxyketone derivates
(
1a, Figure 1) irreversibly inactivates the cysteine pro-
tease papain. (S)-methyl- and ethyl thiirancarboxylate
1b, 1c) showed a non time-dependent inhibition. A
with a peptidic chain (3a, 3b) were reported to be po-
tent irreversible inhibitors of cruzain [22].
(
strong ionic interaction between the inhibitor and the
imidazolium ion at the active site justified the import-
ance of the free carboxylic acid for good inhibition.
When the reaction rate of the (S)-aziridine-2-car-
boxylic acid is compared with that of its amide, the lack
of ionic interactions affecting the inactivation rates of
papain is evident [20].
Indeed, the impact of an amide bond on the affinity
and the alkylating capability of the thiirane-2-car-
boxylic acid is of interest.
Mechanistic studies on the inactivation of papain by
epoxysuccinyl inhibitors suggested that a free car-
boxylate in those structures may not be an absolute
Scheme 1. Syntheses of thiirancarboxamides 8aϪd.
Scheme 2. Reaction of thiirancarboxylic acid with benzyl amine/DCC.
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92
Bruno and Schirmeister
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 90−95
requirement for a good enzyme-inhibitor interaction.
The alkylation constant values (ki) of amide, ester, and
ketone (4b, 4c, 4d, Figure 1) derivates of EP-475
(
(
4a) are comparable to that of the parent compound
4a), indicating the need for at least a carbonyl group
for hydrogen bonding to the enzyme [23].
In order to explore how the inhibitory activity of the
thiirancarboxylic acid building block can vary by substi-
tuting the free acid with a secondary amide group, we
synthesised a series of thiirancarboxamides with size-
increasing side chains. In this paper, we discuss the
inhibition constants of these compounds found for the
inhibition of the model enzyme papain.
Results and discussion
Figure 2. Continuous assays with papain and deriva-
tive 8d. Progress curves of the hydrolysis of the sub-
strate L-BAPA (absorption versus time) at increasing
inhibitor concentrations: ᭺ without inhibitor; ᭹ [I] =
(
L)-(R,R)-Cystine was protected with tBOC following a
known procedure [24] and then coupled with the ap-
propriate L-amino acid by the symmetric anhydride ap-
proach with dicyclohexylcarbodiimide/N-hydroxy-
benzotriazole (Scheme 1).
0.037 mM; ᮀ [I] = 0.124 mM; ᭿ [I] = 0.249 mM; ᭝ [I] =
.747 mM; ᭡ [I] = 0.871 mM.
0
This step ran with pretty good yields between 60 and
82%. Alternatively, the (S,S)-isoleucine derivative was
synthesised by the asymmetric anhydride approach
with isobutylchloroformate. After cleavage of the tBOC
group with trifluoroacetic acid, the synthesis of the epi-
sulfides was carried out in the presence of nitrous
acid, running with inversion of configuration, according
to a mechanism proposed by Owen and Leone [25].
The end product (8aϪd) was isolated in good overall
yield. Carrying out the reaction at lower pH values led
to dithiane (9) via dimerisation [26]. Attempts, to obtain
8
aϪd by peptide coupling of (S)-thiirancarboxylic acid
(
1a) with the appropriate amino acid esters using dif-
ferent standard coupling procedures (DCC; EEDQ;
DPPA; pentafluorophenyl ester of the thiirancarboxylic
acid; acid chloride of the thiirancarboxylic acid) failed.
The reaction of thiirancarboxylic acid with DCC and
benzylamine as model amine to test several coupling
methods led to the formation of benzyl acrylamide (10)
Figure 3. Inactivation of papain with 8d. Plot of
pseudo first-order inactivation rates kobs versus inhibi-
tor concentration.
(
Scheme 2).
The second-order rates of inactivation (k2nd) shown in
Table 1 were determined for the inhibition of papain by
the inhibitors 8aϪd; the method was described pre-
viously [19, 27].
This kind of interaction is known to be important for
the inhibitory activity of epoxysuccinyl peptides and
probably for the thiirancarboxylic acid (1a).
Thiirancarboxamides (8aϪd) irreversibly inactivate
papain (Figures 2, 3). The second-order rate constant
for inhibition of papain by the most potent of the series
shows the same order of magnitude as the parent
compound 1a (Table 1). These products completely
lack any negatively charged moiety that could interact
with the histidinium ion at the enzyme’s active site.
The insertion of a peptidic moiety, however, positively
influences the affinity of the inhibitor for the active site
of the enzyme. This suggests that an electrostatic in-
teraction of the thiirane carboxylate moiety is not an
absolute requirement if another part of the molecule
can stabilize the enzyme-inhibitor interaction.
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2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 90−95
Thiirancarboxamides as Inhibitors 93
Table 1. Second-order rate constants determined for the inhibition of papain by thiirancarboxamides 8aϪda.
R
X
AA
ki (s؊1)
K (mM)
i
k2nd (M؊1 min؊1
)
8
8
8
8
1
a
b
c
d
H
Me
i-Pr
sec-But
Et
Gly
Ala
Val
Ile
0.0008
1.93
24
33
156
234
222
b
b
Me
Me
Me
0.0012
0.0009
0.47
0.2
1
9
a
a
b
Assays were conducted in 0.05 M phosphate buffer, pH 6.5, 5 mM EDTA and 5 mM cysteine, at 30°C.
Measurements were limited to the linear range, with [I] Ӷ K . Therefore, only the second-order rate constant could
i
be obtained.
(
R,R)-{2-tert-Butoxycarbonylamino-3-[2-tert-butoxy-
The lipophilicity of the molecules grows with the size
of the side-chain. A correspondance between the im-
provement of the inhibitory activity and the size of the
amino acid residue is observed. This can be explained
with a better fitting of the molecule into papain’s active
site by positioning the hydrophobic side-chain in the
S2 subsite which is known to accept hydrophobic resi-
dues like Leu and Ile, respectively. This higher affinity
carbonylamino-2-(ethoxycarbonylmethylcarbamoyl)-ethyl-
disulfanyl]-propionylamino}-acetic acid ethyl ester (6a)
To a stirred solution of (R,R)-bis-BOC-cystine 5 (0.55 g, 1.25
mmol) in dry dichloromethane, 1-hydroxybenzotriazole (0.337
g, 2.5 mmol) and a solution of N,N-dicyclohexylcarbodiimide
(
0.515 g, 2.5 mmol) in dichloromethane were added. A solu-
tion of glycine ethyl ester, freshly prepared by dropwise ad-
dition of triethylamine (1.095 g, 25 mmol) to an ice-cold and
stirred solution of glycine ethyl ester hydrochloride (0.349 g,
is reflected by the decreasing K -values of the com-
i
3
2
.5 mmol) in dry DMF (15 cm ), was then added. The mixture
pounds while the k -values remain approximately con-
i
was stirred overnight, filtered off, and the residue was
stant. According to these results we propose a binding
mode in which the Ile side chain of inhibitor 8d is lo-
cated in the S2 subsite of papain consisting of the am-
ino acids Trp69, Tyr67, Phe207, Pro68, Ala160,
Val133, and Val157 (papain numbering) [1].
3
washed with dichloromethane (15 cm ). The organic phases
were evaporated, the residue dissolved in ethyl acetate,
washed with 2 M hydrochloric acid (2 ϫ 25 cm ), brine (2 ϫ
1
The crude peptide was purified by column chromatography
on silica gel. Eluent: ethyl acetate/cyclohexane 3/2. Yield:
3
3
5 cm ), dried with magnesium sulphate, and evaporated.
60%.
(
S,R,S,R)-2-{2-tert-Butoxycarbonylamino-3-[2-tert-butoxy-
Acknowledgments
carbonylamino-2-(1-ethoxycarbonylethylcarbamoyl)-ethyl-
Financial support by the DFG (Deutsche Forschungs-
gemeinschaft) and FCI (Fonds der Chemischen Indus-
trie) is gratefully acknowledged.
disulfanyl]-propionylamino}-propionic acid methyl ester (6b)
The reaction was performed as for (6a) using the amino acid
(S)-Ala. Eluent: ethyl acetate/cyclohexane 1/2. Yield: 63%.
(
S,R,S,R)-2-{2-tert-Butoxycarbonylamino-3-[2-tert-butoxy-
carbonylamino-2-(1-ethoxycarbonyl-2-methylpropionyl-
carbamoyl)-ethyldisulfanyl]-propionylamino}-3-methyl-
butyric acid methyl ester (6c)
Experimental
General methods
NMR spectra were recorded on a Bruker Avance-400 instru-
ment (Bruker, Rheinstetten, Germany). J values are given in
Hz. Mass spectra were recorded on a Finnigan MAT 8200
The reaction was performed as for (6a) using the amino acid
(
S)-Val. Eluent: ethylacetate. Yield: 82%.
(
Finnigan, San Jose, CA, USA) under electron impact (EI) or
(S,S,R,S,S,R)-2-{2-tert-Butoxycarbonylamino-3-[2-tert-
butoxycarbonylamino-2-(1-ethoxycarbonyl-2-methyl-
butylcarbamoyl)-ethyldisulfanyl]-propionylamino}-3-methyl-
pentanoic acid methyl ester (6d)
chemical ionisation (CI) conditions. IR spectra were recorded
on a Perkin-Elmer 841 instrument (Perkin-Elmer, Ueber-
lingen, Germany). All melting points were determined on a
Buechi type 530 (Buechi, Flawil, Switzerland) and the values
are uncorrected. Optical rotations were determined using a
Perkin-Elmer type 241.
To a stirred and cooled (Ϫ15°C) solution of (R,R)-bis-BOC-
3
cystine 5 (2.0 g, 4.54 mmol) in dry tetrahydrofuran (20 cm )
N-methylmorpholine (1.049 mL, 9.45 mmol) and then isobu-
3
Column chromatography was performed with Merck silica gel
tylchloroformate (1.297 cm , 10 mmol) were added succes-
6
0 (0.063Ϫ0.200). Analytical TLC was performed on alumina
sively. After 5 min a solution of (S,S)-isoleucine methyl ester
hydrochloride (1.645 g, 9.09 mmol) was added. The mixture
was stirred for 5 min at Ϫ15°C and then allowed to stand 20
hours at room temperature (rt). The solvent was evaporated
under vacuum. The residue was taken up in ethyl acetate and
extracted with water. The organic phase was washed suc-
sheets coated with silica gel 60 F254 (Merck, Darmstadt,
Germany). Anhydrous reactions were conducted under an at-
mosphere of dry nitrogen. Anhydrous solvents were prepared
following literature standard procedures. Other solvents were
of reagent grade. All other reagents were used as received.
Solvents were removed by rotary evaporation.
3
cessively with 20 cm of a saturated sodium bicarbonate solu-
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94
Bruno and Schirmeister
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 90−95
tion, 20 cm of water, 20 cm3 of 1[TH]M hydrochloric acid,
3
(S,S,S)-3-Methyl-2-(thiirancarbonylamino)-pentanoic
methyl ester (8d)
acid
3
and 20 cm of water. The solution was dried with magnesium
sulphate and then concentrated under vacuum. The peptide
was purified by chromatography on silica gel. Eluent: ethyl
acetate. The product was recrystallized from ethyl acetate
The reaction was performed as for (8a). Yield: 14% as
colourless viscous oil. Eluent: ethyl acetate/cyclohexane 1/1.
δ
(
H
(400 MHz; CDCl
1H, m); 1.91 (1H, m); 2.64 (2H, dd, JAX 6.8, JAB 16.9); 3.37
(1H, dd, JAX 6.8, JBX 5.3); 3.71 (3H, s), 4.51 (1H, dd, J 4.8,
J 8.8); 6.63 (1H, br d); δ (CDCl ): 11.9; 15.8; 24.7; 25.5;
2.6; 38.0; 52.5; 57.0; 169.1; 172.3; νmax/cm (FT-IR): 3302
m), 3082 (w), 2963 (m), 1740 (s), 1656 (s), 1459-1436 (m),
3 4
; Me Si) 0.91 (6H, m); 1.15 (1H, m); 1.40
(
315 mg). Yield: 10%.
General procedure for the cleavage of the BOC group
C
3
To a vigorously stirred ice cold solution of 6aϪd (0.43 mmol)
Ϫ1
3
(
3
3
in 5 cm dichloromethane, 5 cm trifluoracetic acid were ad-
ded dropwise. The solution was stirred for 15 min at 0°C
and then 5 min at rt. The solvent was evaporated and the
products (7aϪd) were used for the next step without further
purification.
20
1
D 3
532 (s), 1205 (s); [α] +0.6 (c 4 in CHCl ); m/z (EI) 231.1
+
(
M , 1.9%); 172.1 (9.0); 102 (1.77).
Abbreviations
(
S)-Thiirancarbonylamino acetic acid ethyl ester (8a)
AA amino acid, BEBA (S,S)-2-benzyl-3,4-epoxybutanoic
acid, L-BAPA L-benzoyl arginyl p-nitroanilide, DCC dicyclo-
hexylcarbodiimide, DPPA diphenyl phosphorazidate; EEDQ
1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline; HOBt N-
hydroxybenzotriazole, NMM N-methyl morpholine, TEA tri-
ethylamine, E-64 (S,S)-1-[N-[[(L-3-trans-carboxyoxiran-2-yl)-
carbonyl]-L-leucyl]amino]-4-guanidinobutane, EDTA ethylen-
edinitrilotetraacetic acid.
To a suspension of 7a (1.070 g, 1.678 mmol) in 9 mL of 0.2
M hydrochloric acid, sodium nitrite (463 mg, 6.714 mmol) was
added. The mixture was stirred for one hour at rt. Then, the
3
solution was extracted with chloroform (2 ϫ 10 cm ), the or-
ganic phase dried with magnesium sulphate, and evaporated
under vacuum at 30°C. The crude product was purified twice
by column chromatography on silica gel to give 190 mg
(
1
60%) of a white solid. Eluents: ethyl acetate/petroleum ether
/1 and ethyl acetate/petroleum ether 1/2.
References
δ
H
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D
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7
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2
0
mp 65Ϫ68°C from ethyl acetate/cyclohexane; [α]
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D
Ϫ1.0 (c
+
in CHCl
3
) m/z (EI) 189.0 (M , 3.9%); 130 (21.5); 103 (15.6);
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7 3
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6
4
1
H
(400 MHz; CDCl ; Me Si) 0.9 (6H, dd, JAX 6.8, JMX 21.8);
.17 (1H, m, JAX 6.8, JAX 4.8, JMX 21.8); 2.64 (2H, dd, JAX
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3
4
[
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C 3
(CDCl ): 18.0,
Ϫ1
(
FT-IR): 3293 (m), 3078 (w), 1746 (s), 1649 (s), 1554 (s),
373 (w), 1199 (s); [α]
2
0
1
D 3
+2.8 (c 2 in CHCl ); m/z (EI) 217.1
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(
M , 6.71%); 184 (12.8); 158 (54.0); HRMS 217.078 (217.077
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 90−95
Thiirancarboxamides as Inhibitors 95
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