Aza vinyl sulfones: Synthesis and evaluation as antiplasmodial agents

Article abstract of DOI:10.1016/j.bmc.2011.10.018

A series of novel aza vinyl sulfones were designed, synthesized in good yields and evaluated as antiplasmodial agents. Tested compounds did not show activity against papain or the Plasmodium falciparum cysteine protease falcipain-2. However, a number of t

Full text of DOI:10.1016/j.bmc.2011.10.018

Bioorganic & Medicinal Chemistry 19 (2011) 7635–7642
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aza vinyl sulfones: Synthesis and evaluation as antiplasmodial agents
Paulo M.C. Glória a, Jiri Gut b, Lídia M. Gonçalves a, Philip J. Rosenthal b, Rui Moreira a,
Maria M.M. Santos a,
⇑
a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
b Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94143, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel aza vinyl sulfones were designed, synthesized in good yields and evaluated as antiplas-
modial agents. Tested compounds did not show activity against papain or the Plasmodium falciparum cys-
teine protease falcipain-2. However, a number of the new compounds effectively inhibited the in vitro
development of P. falciparum. Compounds containing a squaramide group were the most active, with
Received 11 July 2011
Revised 6 October 2011
Accepted 7 October 2011
Available online 14 October 2011
IC50 values between 0.95 and 4.5
l
M, suggesting that these are potential lead compounds for the devel-
opment of new antimalarial agents.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Aza vinyl sulfone
Squaramate
Cysteine protease inhibitor
Antiplasmodial activity
Malaria
1. Introduction
P1
R2
N
Malaria is one of the most important infectious diseases in the
world. The most severe form of malaria in humans is caused by
Plasmodium falciparum. Although several antimalarial drugs are
known, resistance to available drugs is widespread and increasing.1
As a consequence, there is an urgent need to find new antimalarial
agents. A promising target for antimalarial drug design is falcipain-
2, a cysteine protease from P. falciparum, that has received consid-
erable attention due to its key role in the life cycle of the parasite.2
Falcipain-2 belongs to the papain family or CA clan, which is
one of the largest subfamilies of cysteine proteases. Several pepti-
dyl vinyl sulfone cysteine protease inhibitors have been described
as potent irreversible falcipain-2 inhibitors.3–5 These compounds
act as Michael acceptors of the catalytic cysteine residue.6 How-
ever, peptidyl inhibitors generally exhibit low bioavailability be-
cause of their susceptibility to degradation by other proteases. In
biologically active peptide analogs, the replacement of the alpha
carbon by a nitrogen atom to give azapeptides has led to enhanced
activity and selectivity as well as improved properties, such as pro-
longed duration of action and metabolic stability.7
O
R4HN
SO2R1
N
H
R3
P2
O
1
Figure 1. Chemical structure of aza vinyl sulfones 1.
we report the synthesis of novel antiplasmodial agents 1 based on
the aza vinyl sulfone scaffold (Fig. 1). These compounds were de-
signed in order to explore the impact of CH/N exchange in the P1
position of dipeptide vinyl sulfones on enzyme–inhibitor interac-
tion and on antiplasmodial activity.
2. Results and discussion
The required methyl and phenyl vinyl sulfones acids were syn-
thesized from propiolic acid and the corresponding thiol sodium
salts, followed by oxidation of the trans-isomer with excess of
mCPBA.15 The next stage was the design of selective falcipain-2
inhibitors, by incorporating an appropriate recognition moiety to
the double bond. We started by studying the reaction of Boc pro-
tected hydrazine 2a with methyl and phenyl vinyl sulfones acids.
Compounds 3a–b were obtained in good yields (62% and 71%
Several cysteine protease inhibitors containing azapeptide scaf-
folds have already been reported,8–11 but this strategy has not, to
our knowledge, been applied to the P1 position of peptidyl vinyl
sulfones. As part of our ongoing interest in the design and synthe-
sis of Michael acceptors as cysteine protease inhibitors,12–14 herein
⇑
Corresponding author. Tel.: +351 217946400; fax: +351 217946470.
E-mail address: mariasantos@ff.ul.pt (M.M.M. Santos).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.018

7636
P. M. C. Glória et al. / Bioorg. Med. Chem. 19 (2011) 7635–7642
HO2C
SO2R1
O
H
N
1)TFA, CH2Cl2
H
N
SO2R1
CDI
SO2R1
BocHN
BocHN NH2
BocHN
N
H
THF/DMF (1:1)
2) BocLeuOH, CDI
THF/DMF (1:1)
O
O
2a
3a R1 = Me
1a, R1 = Me
3b R1 =Ph
1b, R1 =Ph
Scheme 1. Synthesis of aza vinyl sulfones 1a–b.
HO2C
SO2R1
R2
O
R2
NH
O
R2
N
1)TFA, CH2Cl2
CDI
BocHN
BocHN
SO2R1
BocHN NH
N
H
N
H
2) BocLeuOH, CDI
THF/DMF (1:1)
THF/DMF (1:1)
O
4a R2 = CH2Ph
4b R2 = CH2CH2Ph
2
2
2b
1c
R = CH2Ph
R = CH2Ph, R1 = Me
2c R2 = CH2CH2Ph
1d R2 = CH2Ph, R1 =Ph
2
1
1e
R = CH2CH2Ph, R = Me
1f R2 = CH2CH2Ph, R1 =Ph
Scheme 2. Synthesis of aza vinyl sulfones 1c–f.
yields). Deprotection of the Boc group, followed by coupling with
Leu led to aza vinyl sulfones 1a–b containing Gly in P1 (Scheme 1).
The aza vinyl sulfones containing Phe and homoPhe in P1 were
synthesized starting from the corresponding Boc protected hydra-
zines 2b–c. Deprotection of the Boc group with TFA, followed by
coupling with BocLeuOH led to azadipeptides 4a–b. Finally, cou-
pling with the acid derivative of the corresponding vinyl sulfones
led to aza vinyl sulfones 1c–f in 62–78% yields (Scheme 2).
Compounds 1a–f were then screened against the parasite cys-
teine protease falcipain-2 and against the chloroquine-resistant
W2 strain of P. falciparum (Table 1). Compounds 1a–f were devoid
of enzyme inhibitory activity. However, most of the compounds
demonstrated modest activity against cultured P. falciparum. Prob-
ably, falcipain-2 inhibition is not the primary mode by which
derivatives 1a–f exert antiplasmodial activities. The most active
compound in this series, 1f, contained aza-homoPhe in P1 and a
phenyl vinyl sulfone, and thus we decided to prepare a new series
of compounds containing these features and Cbz-protected amino
acids in P2. Leu, Phe and Gly residues were selected in order to
modulate the interaction with P2 (Scheme 3).16 Two compounds
were also synthesized with a Mu-protected Leu at P2 and an aza-
Gly or aza-homoPhe in P1 (Scheme 4).
led to azadipeptides 6a–c. Reduction of the double bond with
NaBH3CN in the presence of sulfonic acid led to azadipeptides
7a–c.16 Finally, coupling with the acid derivative of the corre-
sponding vinyl sulfones led to aza vinyl sulfones 1g–i in 73–86%
yields (Scheme 3). Despite the fact that compounds 1g–i belong
to the same class of compounds 1a–f, a different synthetic pathway
was adopted in order to avoid the formation of the dialkylated sub-
product of 2b–c, observed during the preparation of 1c–f.
Compounds 1j and 1l were obtained from aza vinyl sulfones 1b
and 1f in 72% and 87% yields, respectively, by deprotection of the
Boc group with TFA, followed by reaction with 4-morpholinecar-
bonyl chloride (Scheme 4).
All compounds were evaluated for activity against papain, the
prototype for clan CA cysteine proteases. As observed before for fal-
cipain-2, our compounds were not active against papain (Table 2).
Compounds 1g–l were also evaluated against cultured P. falciparum.
Inspection of the data in Table 2, allows the following observations
to be made:
1. For the Cbz-protected compounds containing an aza-homoPhe
in P1 and a phenyl vinyl sulfone, that is, vinyl sulfones 1g–i,
the presence of a Cbz-protected Phe in P2 (i.e., vinyl sulfone
1i) improved inhibitory activity against cultured parasites when
compared to the compound with Boc-protected Leu in P2, that
is, 1f. The presence of a Leu or Gly in P2 resulted in lost of anti-
parasitic activity vinyl sulfones 1g and 1h).
For the Cbz series, the recognition moiety was obtained starting
with different N-Cbz amino acids (Leu, Gly and Phe) and Boc pro-
tected hydrazine 2a in the presence of CDI and NEt3. Deprotection
of the Boc group with TFA, followed by reaction with PhCH2CHO
Table 1
Antiplasmodial activity for compounds 1a–f
O
R2
N
SO2R1
BocHN
N
H
O
R1
R2
Falcipain-2 IC50
(lM)
P. falciparum W2 IC50 (lM)
a
Compds
1a
1b
1c
1d
1e
1f
Me
Ph
Me
Ph
Me
Ph
H
H
ND
ND
>50
>50
>50
>50
0.11 0.025
34.2 4.2
33.5 1.4
21.6 1.8
>50
46.6 1.6
15.4 0.4
1.94 0.004
CH2Ph
CH2Ph
CH2CH2Ph
CH2CH2Ph
E-64
a
ND = not done.

P. M. C. Glória et al. / Bioorg. Med. Chem. 19 (2011) 7635–7642
7637
Ph
O
O
1) TFA, CH2Cl2
CbzAAOH, CDI
CbzHN
N
BocHN NH2
CbzHN
NHBoc
N
H
2) PhCH2CHO,
NEt3, MeOH
N
H
NEt3, THF/DMF (1:1)
R3
R3
2a
6a R3 = CH2CHMe2
6b R3 = CH2Ph
6c R3 = H
5a R3 = CH2CHMe2
5b R3 = CH2Ph
5c R3 = H
Ph
Ph
HO2C
SO2Ph
CbzHN
O
O
NaBH3CN, THF
pTsOH
CDI
CbzHN
NH
N
N
SO2Ph
6a-c
N
H
THF/DMF (1:1)
H
R3
R3
O
3
3
7a
7b
7c
R = CH2CHMe2
1g
1h
1i
R = CH2CHMe2
3
3
R = CH2Ph
R = H
3
R3 = CH2Ph
R = H
Scheme 3. Synthesis of aza vinyl sulfones 1g–i.
R1
N
O
R1
N
O
1) TFA, CH2Cl2
MuHN
SO2Ph
BocHN
SO2Ph
N
H
N
H
2) MuCOCl,
NEt3, CH2Cl2
O
O
1j R1 = H
1l R1 = CH2CH2Ph
1
1b
R = H
1f R1 = CH2CH2Ph
Scheme 4. Synthesis of aza vinyl sulfones 1j–l.
Table 2
Papain inhibition and antiplasmodial activity for compounds 1g–l
O
R1
N
R3HN
SO2Ph
N
H
R2
O
Compds
R1
R2
R3
Papain IC50
(lM)
P. falciparum W2 IC50 (lM)
1g
1h
1i
1j
1l
CH2CH2Ph
CH2CH2Ph
CH2CH2Ph
H
CH2CHMe2
H
CH2Ph
CH2CHMe2
CH2CHMe2
Cbz
Cbz
Cbz
Mu
Mu
>50
>50
>50
>50
>50
>10
>10
9.0 0.9
>10
3.4 0.04
1.94 0.004
CH2CH2Ph
E-64
0.002 0.0001
2. For compounds containing an aza-Gly in P1 and Leu in P2 the
presence of a Mu protective group resulted in lost of activity
against cultured parasites (i.e., vinyl sulfones 1j, versus their
Boc-protected counterpart 1b). The opposite result was
observed for inhibitors containing an aza-homoPhe in P1 and
Leu in P2. In this case a Mu protective group (i.e., aza vinyl sul-
fone 1l) increased the activity ꢀ5-fold compared to that of its
Boc-protected counterpart 1f.
Compounds8a–iwerescreenedagainsttheparasitecysteinepro-
tease falcipain-2 and against the cultured P. falciparum (Table 3).
Most of the compounds had low micromolarantiplasmodialactivity,
with IC50 values ranging from 0.95 to 4.5 lM. Except for the aza-
homoPhe derivatives 8h and 8i, the presence of a methoxy or a but-
oxy group in the squaric moiety did not markedly change activity
against P. falciparum (compounds 8b and 8e vs compounds 8c and
8f, respectively). Forthephenylvinylsulfones,theorderofinhibitory
activity against P. falciparum depended on the nature of the P1 resi-
due and varied in the order aza-Phe > aza-Gly > aza-homoPhe. In
the case of methyl vinyl sulfones, inhibitory activity was similar for
aza-Gly (8a) and aza-homoPhe (8g), but in the presence of aza-Phe
(8d) activity against P. falciparum was lost. As observed before, all
compounds were devoid of enzyme inhibitory activity.
To further study aza vinyl sulfones as antiplasmodial agents we
prepared compounds 8a–i, in which the aza vinyl sulfone scaffold
is coupled to a squarate moiety. In fact, since the first synthesis of
squaric acid a number of derivatives have been reported in the
fields of bioorganic and medicinal chemistry.17–20 Very recently,
a few compounds containing a squarate moiety were described
in the GSK database as antimalarials.21
3. Conclusion
The synthesis of derivatives 8a–i involved the deprotection of
the Boc group of aza vinyl sulfones 1a–f, followed by reaction with
dimethoxy or dibutoxy squaric acid in 61–69% yields (Scheme 5).
Dipeptide aza vinyl sulfones incorporating a recognition moiety
for Falcipain-2 inhibition were inactive for cysteine proteases of

7638
P. M. C. Glória et al. / Bioorg. Med. Chem. 19 (2011) 7635–7642
R2
N
O
R2
N
O
H
N
R3O
O
1) TFA, CH2Cl2
SO2R1
BocHN
SO2R1
N
H
N
H
2)
O
O
O
O
O
R3O
OR3
1a-f
8a R3 = Me, R2 = H, R1 = Me
3
2
1
8b
R = Me, R = H, R = Ph
8c R3 = Bu, R2 = H, R1 = Ph
3
2
1
8d
R = Me, R = CH2Ph, R = Me
8e R3 = Me, R2 = CH2Ph, R1 = Ph
3
2
1
8f
R = Bu, R = CH2Ph, R = Ph
8g R3 = Me, R2 = CH2CH2Ph, R1 = Me
3
2
1
8h
R = Me, R = CH2CH2Ph, R = Ph
8i R3 = Bu, R2 = CH2CH2Ph, R1 = Ph
Scheme 5. Synthesis of aza vinyl sulfones squaramates 8a–i.
Table 3
Antiplasmodial activity for compounds 8a–i
O
R2
N
H
N
R3O
O
SO2R1
N
H
O
O
Compds
R1
R2
R3
Falcipain-2 IC50
(lM)
P. falciparum W2 IC50 (lM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
Me
Ph
Ph
Me
Ph
Ph
Me
Ph
Ph
H
H
H
CH2Ph
CH2Ph
Me
Me
Bu
Me
Me
Bu
Me
Me
Bu
>50
>50
>50
>50
>50
>50
>50
>50
2.3 0.1
3.5 0.02
2.6 0.5
>10
0.95 0.06
1.2 0.1
2.5 0.5
>10
CH2Ph
CH2CH2Ph
CH2CH2Ph
CH2CH2Ph
>50
0.11 0.025
4.5 0.2
1.94 0.004
E-64
clan CA but displayed antiplasmodial activity against a chloro-
quine-resistant strain of P. falciparum, with IC50 values ranging
from 13 to 47 lM. In fact, the exchange of CH/N in the P1 position
led to much poorer antiplasmodial activity compared to classical
peptidyl vinyl sulfone cysteine protease inhibitors previously
reported.
performed on Merck Silica Gel (200–400 mesh). 1H and 13C NMR
spectra were recorded on a Bruker 400 Ultra-Shield (400 MHz).
1H and 13C chemical shifts are expressed in d (ppm) referenced to
the solvent used and the proton coupling constants (J) in hertz.
4.1.1. General preparation of vinyl sulfones 3a–b
Antiplasmodial activity was significantly improved when Mu
was used as protective group or a squaric moiety was coupled to
the dipeptide Leu-aza vinyl sulfones. The most active compounds,
squaric-Leu-azaPhe-VSPh, 8e and 8f, presented IC50 values of 0.95
To a solution of the suitable vinyl sulfone acid (1 equiv) in a
mixture of THF/DMF (1:1) was added CDI (1 equiv) and the mix-
ture stirred at rt for 30 min. After this time, the t-butyl carbazate
2a (1 equiv) was added. The mixture was stirred overnight at rt.
The reaction mixture was then diluted with water and extracted
with AcOEt and the organic layers combined, dried with anhydrous
Na2SO4, filtered and concentrated to dryness. The obtained residue
was purified by column chromatography (Hexane/AcOEt 1:1)
affording the corresponding product as a white solid.
and 1.2 lM, suggesting that these are potential lead compounds for
the development of new antimalarial agents. Since the squaric
moiety is a known zinc binding group, it is possible that these com-
pounds target the heme polymerization.22 Further studies are
underway to identify the molecular targets responsible for the
activity of the most potent compounds.
4.1.2. General preparation of Boc Leu aza-Gly vinyl sulfones 1a–b
To a solution of the suitable Boc aza vinyl sulfone 3 (1 equiv) in
DCM was added TFA (4 equiv). After 2 h the solvent was removed
under reduced pressure affording quantitatively a slightly yellow
oil.
4. Experimental
4.1. Chemistry
To a solution of BocLeuOH (1 equiv) in a mixture of THF/DMF
(1:1) was added CDI (1 equiv) and the mixture stirred at rt for
30 min. After this time, the deprotected aza vinyl sulfone (1 equiv)
was added followed by NEt3 (2 equiv). The mixture was stirred
overnight at rt. The reaction mixture was then diluted with water
and extracted with AcOEt and the organic layers combined, dried
with anhydrous Na2SO4, filtered and concentrated to dryness.
The obtained residue was purified by column chromatography
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. Melting points
were determined using a Kofler camera Bock monoscope M and
are uncorrected. The infrared spectra were collected on a Nicolet
Impact 400 FTIR infrared spectrophotometer. High resolution mass
spectra (HMRS) were performed in Unidade de Espectrometria de
Masas, Santiago de Compostela. Merck Silica Gel 60 F254 plates
were used as analytical TLC; flash column chromatography was

P. M. C. Glória et al. / Bioorg. Med. Chem. 19 (2011) 7635–7642
7639
(Hexane/AcOEt 3:2) affording the corresponding product as a
white solid.
(2CH3). HRMS-ESI-TOF: m/z calcd C27H35N3O6SNa (M++Na)
552.2144, found 552.2140.
4.1.2.1. BocLeuazaGlyCOVSMe 1a.
Obtained in 64% yield. Mp
4.1.3.3. BocLeuazahomoPheCOVSMe 1e.
Obtained in 73%
88–89 °C. IR (cmÀ1) 3442 (NH), 2929, 2858, 1743 (C@O), 1718
(C@O), 1637 (C@O), 1461, 1404, 1303, 1144. 1H NMR (400 MHz,
CDCl3) d 7.84 (d, J = 16 Hz, 1H), 7.19 (d, J = 16 Hz, 1H), 6.87 (d,
J = 8 Hz, 2H), 4.52 (t, J = 8 Hz, 1H), 3.85 (s, 1H), 2.93 (s, 3H), 2.18
(m, 1H), 1.72 (t, J = 8 Hz, 1H), 1.45 (sl, 10H), 1.01 (d, J = 8 Hz, 6H).
13C NMR (100 MHz, CDCl3) d 173.66 (C@O), 168.56 (C@O), 159.31
(C@O), 146.86 (CHSO2CH3), 131.71 (CH@CHSO2CH3), 80.65
(C(CH3)3), 50.58 (CH), 40.43 (CH2), 37.99 (CH@CHSO2CH3), 28.41
(C(CH3)3), 25.38 (CH), 23.14 (2CH3). HRMS-ESI-TOF: m/z calcd
C15H27N3O6SNa (M++Na) 400.1518, found 400.1509.
yield. Mp 103–104 °C. IR (cmÀ1) 3340 (NH), 3244 (NH), 3051,
2969, 2914, 1722 (C@O), 1647 (C@O), 1538, 1416, 1265. 1H NMR
(400 MHz, CDCl3) d 8.16 (d, J = 16 Hz, 1H), 7.29 (m, 3H), 7.17 (m,
3H), 6.57 (s, 1H), 4.88 (sl, 1H), 3.84 (s, 1H), 3.45 (t, J = 8 Hz, 1H),
3.33 (t, J = 8 Hz, 1H), 2.93 (s, 3H), 2.82 (t, J = 8 Hz, 2H), 1.86 (m,
1H), 1.60 (dd, J = 8 Hz, J = 4 Hz, 1H), 1.54 (dd, J = 8 Hz, J = 4 Hz,
1H), 1.43 (s, 9H), 1.00 (d, J = 4 Hz, 6H). 13C NMR (100 MHz, CDCl3)
d 170.87 (C@O), 161.64 (C@O), 158.31 (C@O), 147.17 (CHSO2CH3),
138.54 (Cq), 133.52 (CH@CHSO2CH3), 129.20 (CHAr), 129.09 (CHAr),
126.37 (CHAr), 80.65 (C(CH3)3), 51.09 (CH), 49.77 (CH2), 40.43
(CH2), 37.99 (SO2CH3), 30.94 (CH2), 28.41 (C(CH3)3), 25.38 (CH),
23.14 (2CH3). HRMS-ESI-TOF: m/z calcd C23H35N3O6SNa (M++Na)
504.2144, found 504.2137.
4.1.2.2. BocLeuazaGlyCOVSPh 1b.
Obtained in 73% yield. Mp
91–92 °C. IR (cmÀ1) 3358 (NH), 3051, 2969, 2914, 1715 (C@O),
1654 (C@O), 1525, 1456, 1368, 1231, 1150. 1H NMR (400 MHz,
CDCl3) d 7.95 (t, J = 4 Hz, 2H), 7.64–7.58 (m, 4H), 7.17 (s, 1H),
7.04 (d, J = 16 Hz, 1H), 6.81 (s, 1H), 4.62 (t, J = 4 Hz, 1H), 4.29 (s,
1H), 1.64 (m, 2H), 1.46 (sl, 10H), 1.01 (d, J = 4 Hz, 6H). 13C NMR
(100 MHz, CDCl3) d 173.66 (C@O), 168.56 (C@O), 158.31 (C@O),
140.93 (Cq), 137.57 (CHSO2Ph), 133.05 (CHAr), 130.28 (CH@CH-
SO2Ph), 129.10 (CHAr), 128.17 (CHAr), 80.65 (C(CH3)3), 50.58 (CH),
40.43 (CH2), 28.41 (C(CH3)3), 25.38 (CH), 23.14 (2CH3). HRMS-
4.1.3.4. BocLeuazahomoPheCOVSPh 1f.
Obtained in 78%
yield. Mp 112–113 °C. IR (cmÀ1) 3409 (NH), 3326 (NH), 3051,
2969, 2928, 1715 (C@O), 1668 (C@O), 1511, 1272, 1150. 1H NMR
(400 MHz, CDCl3) d 8.02 (s, 1H), 7.96 (m, 2H), 7.59 (m, 3H), 7.25
(m, 5H), 7.14 (d, J = 16 Hz, 2H), 6.69 (s, 1H), 4.77 (t, J = 8 Hz, 1H),
4.36 (s, 1H), 3.56 (t, J = 8 Hz, 1H), 3.47 (t, J = 8 Hz, 1H), 2.89 (m,
2H), 1.59 (m, 2H), 1.50 (m, 1H), 1.43 (s, 9H), 0.89 (d, J = 4 Hz,
6H). 13C NMR (100 MHz, CDCl3) d 170.87 (C@O), 161.64 (C@O),
158.31 (C@O), 140.93 (Cq), 139.58 (CHSO2Ph), 138.54 (CHAr),
133.0 (CHAr), 130.14 (CH@CHSO2Ph), 129.20 (CHAr), 129.09 (CHAr),
128.17 (CHAr), 126.37 (CHAr), 80.65 (C(CH3)3), 51.09 (CH), 49.77
(CH2), 40.44 (CH2), 30.95 (CH2), 28.41 (C(CH3)3), 25.39 (CH),
23.14 (2CH3). HRMS-ESI-TOF: m/z calcd C28H37N3O6SNa (M++Na)
566.2301, found 566.2297.
ESI-TOF: m/z calcd
C
20H29N3O6SNa (M++Na) 462.1675, found
462.1668.
4.1.3. General preparation of N-Boc azahomoPhe and azaPhe
vinyl sulfones 1c–f
To a solution of the suitable vinyl sulfone acid (1 equiv) in a
mixture of THF/DMF (1:1) was added CDI (1 equiv) and the mix-
ture stirred at rt for 30 min. After this time, the suitable azadipep-
tide (1 equiv) was added. The mixture was stirred overnight at rt.
The reaction mixture was then diluted with water and extracted
with AcOEt and the organic layers combined, dried with anhydrous
Na2SO4, filtered and concentrated to dryness. The obtained residue
was purified by column chromatography (Hexane/AcOEt 3:2)
affording the corresponding product as a white solid.
4.1.4. General preparation of Cbz aza vinyl sulfones 1g–i
To a solution of the suitable vinyl sulfone acid (1 equiv) in a
mixture of THF/DMF (1:1) was added CDI (1 equiv) and the mix-
ture stirred at rt for 30 min. After this time, the suitable azapeptide
(1 equiv)16 was added. The mixture was stirred overnight at rt. The
reaction mixture was then diluted with water and extracted with
AcOEt and the organic layers combined, dried with anhydrous
Na2SO4, filtered and concentrated to dryness. The obtained residue
was purified by column chromatography (Hexane/AcOEt 3:2)
affording the corresponding product as a white solid.
4.1.3.1. BocLeuazaPheCOVSMe 1c.
Obtained in 62% yield. Mp
98–99 °C. IR (cmÀ1) 3409 (NH), 3340 (NH), 3065, 2969, 2914, 1722
(C@O), 1675 (C@O), 1518, 1368, 1265. 1H NMR (400 MHz, CDCl3) d
7.65 (d, J = 16 Hz, 1H), 7.40 (d, J = 16 Hz, 1H), 7.25 (m, 5H), 6.87 (s,
1H), 5.06 (s, 1H), 4.89 (t, J = 8 Hz, 1H), 4.32 (s, 2H), 2.95 (s, 3H), 1.76
(m, 1H), 1.53 (m, 11H), 1.03 (d, J = 4 Hz, 6H). 13C NMR (100 MHz,
CDCl3) d 170.87 (C@O), 165.32 (C@O), 158.31 (C@O), 147.17
(CHSO2CH3), 136.21 (Cq), 133.52 (CH@CHSO2CH3), 128.77 (CHAr),
128.43 (CHAr), 127.60 (CHAr), 80.65 (C(CH3)3), 53.18 (CH2), 51.09
(CH), 40.43 (CH2), 37.99 (SO2CH3), 28.41 (C(CH3)3), 25.38 (CH),
23.14 (2CH3). HRMS-ESI-TOF: m/z calcd C22H33N3O6SNa (M++Na)
490.1988, found 490.1969.
4.1.4.1. CbzLeuazahomoPheCOVSPh 1g.
Obtained in 73%
yield. Mp 115–116 °C. IR (cmÀ1) 3299 (NH), 3051, 2969, 1715
(C@O), 1647 (C@O), 1531, 1450, 1361, 1225, 1156. 1H NMR
(400 MHz, CDCl3) d 7.96 (d, J = 8 Hz, 2H), 7.75 (d, J = 16 Hz, 1H),
7.61 (m, 3H), 7.29 (m, 8H), 7.17 (m, 3H), 6.49 (s, 1H), 5.38 (s,
2H), 4.87 (t, J = 4 Hz, 1H), 3.61 (s, 1H), 3.49 (t, J = 4 Hz, 1H), 3.14
(t, J = 4 Hz, 1H), 2.75 (t, J = 4 Hz, 2H), 1.70 (m, 2H), 1.57 (t,
J = 4 Hz, 1H), 1.05 (d, J = 4 Hz, 6H). 13C NMR (100 MHz, CDCl3) d
170.87 (C@O), 161.64 (C@O), 159.04 (C@O), 140.93 (Cq), 139.58
(CHSO2Ph), 138.54 (CHAr), 136.99 (CHAr), 133.05 (CHAr), 130.14
(CH@CHSO2Ph), 129.20 (CHAr), 129.09 (CHAr), 128.33 (CHAr),
128.20 (CHAr), 128.18 (CHAr), 126.37 (CHAr), 67.05 (CH2), 51.09
(CH), 4977 (CH2), 40.43 (CH2), 30.94 (CH2), 25.38 (CH), 23.14
(2CH3). HRMS-ESI-TOF: m/z calcd C31H35N3O6SNa (M++Na)
600.2144, found 600.2137.
4.1.3.2. BocLeuazaPheCOVSPh 1d.
Obtained in 71% yield. Mp
101–102 °C. IR (cmÀ1) 3299 (NH), 3051 (NH), 2969, 2955, 1715
(C@O), 1647 (C@O), 1531, 1225, 1156. 1H NMR (400 MHz, CDCl3)
d 8.33 (s, 1H), 7.86 (m, 2H), 7.57 (m, 3H), 7.42 (m, 2H), 7.31 (m,
2H), 7.23 (m, 1H), 6.96 (d, J = 16 Hz, 1H), 6.50 (d, J = 16 Hz, 1H),
4.49 (t, J = 8 Hz, 1H), 4.16 (s, 2H), 3.06 (s, 1H), 1.50 (m, 12H), 1.36
(t, J = 8 Hz, 1H), 1.01 (d, J = 8 Hz, 6H). 13C NMR (100 MHz, CDCl3)
d 170.87 (C@O), 165.32 (C@O), 158.31 (C@O), 140.93 (CHAr),
139.58 (CHSO2Ph), 136.21 (CHAr), 133.05 (CHAr), 130.05 (CHAr),
130.14 (CH@CHSO2Ph), 129.10 (CHAr), 128.77 (CHAr), 128.43
(CHAr), 128.17 (Cq), 127.60 (Cq), 80.65 (C(CH3)3), 53.18 (CH2),
51.09 (CH), 40.43 (CH2), 28.41 (C(CH3)3), 25.38 (CH), 23.14
4.1.4.2. CbzGlyazahomoPheCOVSPh 1h.
Obtained in 86%
yield. Mp 114–115 °C. IR (cmÀ1) 3292 (NH), 3285 (NH), 2969,
1722 (C@O), 1647 (C@O), 1518, 1450, 1375, 1238, 1150. 1H NMR
(400 MHz, CDCl3) d 9.08 (s, 1H), 7.91 (m, 2H), 7.58 (m, 3H), 7.24
(m, 11H), 6.46 (d, J = 16 Hz, 1H), 5.39 (s, 2H), 4.13 (s, 1H), 3.97 (s,
1H), 3.84 (s, 1H), 3.48 (t, J = 8 Hz, 1H), 3.20 (t, J = 4 Hz, 1H), 2.86

7640
P. M. C. Glória et al. / Bioorg. Med. Chem. 19 (2011) 7635–7642
(t, J = 8 Hz, 1H). 13C NMR (100 MHz, CDCl3) d 171.16 (C@O), 161.64
(C@O), 157.43 (C@O), 140.75 (Cq), 139.58 (CHSO2Ph), 138.64
(CHAr), 137.08 (CHAr), 133.04 (CHAr), 130.14 (CH@CHSO2Ph),
129.19 (CHAr), 129.08 (CHAr), 129.06 (CHAr), 128.31 (CHAr),
128.16 (CHAr), 128.15 (CHAr), 126.36 (CHAr), 66.68 (CH2), 49.77
(CH2), 43.68 (CH2), 31.42 (CH2). HRMS-ESI-TOF: m/z calcd
slightly yellow oil. The residue was dissolved in methanol and
NEt3 (2 equiv) was added followed by the addition of the suitable
squaric acid derivative (1 equiv) under N2. The reaction mixture
was stirred overnight at rt. The solvent was then removed under
reduced pressure and the obtained residue was purified by column
chromatography (Hexane/AcOEt 3:2) affording the corresponding
product as a white solid.
C
27H27N3O6SNa (M++Na) 544.1518, found 544.1510.
4.1.4.3. CbzPheazahomoPheCOVSPh 1i. Obtained in 77%
4.1.6.1. SQMeLeuazaGlyCOVSMe 8a.
Obtained in 61% yield.
yield. Mp 126–127 °C. IR (cmÀ1) 3305 (NH), 2971, 1727 (C@O),
1657 (C@O), 1523, 1242, 1153, 1031. 1H NMR (400 MHz, CDCl3) d
7.94 (m, 3H), 7.52 (m, 3H), 7.29 (m, 15H), 7.10 (d, J = 16 Hz, 1H),
6.70 (s, 1H), 5.39 (s, 2H), 5.14 (sl, 1H), 3.71 (s, 1H), 3.43 (t,
J = 4 Hz, 1H), 3.36 (t, J = 4 Hz, 1H), 3.23 (dd, J = 12 Hz, J = 4 Hz,
1H), 2.84 (t, J = 8 Hz, 2H). 13C NMR (100 MHz, CDCl3) d 168.38
(C@O), 161.64 (C@O), 159.04 (C@O), 140.93 (Cq), 139.58
(CHSO2Ph), 138.54 (CHAr), 137.59 (CHAr), 136.99 (CHAr), 133.05
(CHAr), 130.14 (CH@CHSO2Ph), 129.42 (CHAr), 129.20 (CHAr),
129.10 (CHAr), 129.07 (CHAr) 128.33 (CHAr), 128.20 (CHAr), 128.18
(CHAr), 127.17 (CHAr), 126.37 (CHAr), 67.05 (CH2), 56.18 (CH),
49.77 (CH2), 38.45 (CH2), 30.94 (CH2). HRMS-ESI-TOF: m/z calcd
Mp 126–127 °C. IR (cmÀ1) 3436 (NH), 3051, 2955, 1715 (C@O),
1661 (C@O), 1525, 1272, 1156, 1040. 1H NMR (400 MHz, CDCl3) d
7.81 (d, J = 16 Hz, 1H), 7.41 (s, 1H), 7.22 (m, 2H), 5.07 (t, J = 8 Hz,
1H), 4.97 (s, 1H), 3.93 (s, 3H), 2.95 (s, 3H), 1.60 (t, J = 8 Hz, 1H),
1.46 (m, 2H), 1.01 (sl, 6H). 13C NMR (100 MHz, CDCl3) d 191.89
(C@O), 179.35 and 178.94 (Cq and C@O), 173.66 (C@O), 168.56
(C@O), 161.68 (Cq), 146.86 (CHSO2CH3), 131.71 (CH@CHSO2CH3),
61.45 (OCH3), 57.78 (CH), 40.43 (CH2), 37.99 (SO2CH3), 25.38
(CH), 23.14 (2CH3). HRMS-ESI-TOF: m/z calcd C15H21N3O7SNa
(M++Na) 410.0998, found 410.0988.
4.1.6.2. SQMeLeuazaGlyCOVSPh 8b.
Obtained in 67% yield.
C
34H33N3O6SNa (M++Na) 634.1988, found 634.1973.
Mp 147–148 °C. IR (cmÀ1) 3354 (NH), 3051, 2969, 2914, 1722
(C@O), 1654 (C@O), 1525, 1456, 1381, 1313, 1136, 1040. 1H NMR
(400 MHz, CDCl3) d 7.93 (d, J = 16 Hz, 1H), 7.87 (m, 2H), 7.58 (m,
3H), 7.25 (s, 1H), 6.88 (d, J = 16 Hz, 1H), 6.10 (s, 1H), 4.67 (t,
J = 8 Hz, 1H), 3.91 (s, 3H), 3.00 (s, 1H), 1.59 (m, 3H), 1.03 (sl, 6H).
13C NMR (100 MHz, CDCl3) d 191.95 (C@O), 179.41 and 179.00
(C@O and Cq), 173.71 (C@O), 168.61 (C@O), 161.73 (Cq), 140.99
(CHAr), 137.62 (CHSO2Ph), 133.11 (Cq), 130.34 (CH@CHSO2Ph),
129.15 (CHAr), 128.23 (CHAr), 61.51 (OCH3), 57.84 (CH), 40.49
(CH2), 25.44 (CH), 23.19 (2CH3). HRMS-ESI-TOF: m/z calcd
4.1.5. General preparation of Mu aza vinyl sulfones 1j–l
To a solution of the suitable Boc azadipeptide vinylsulfone 1
(1 equiv) in DCM was added TFA (4 equiv). After 2 h the solvent
was removed under reduced pressure affording quantitatively a
slightly yellow oil. The residue was dissolved in DCM and NEt3
(2 equiv) was added followed by 4-morpholinecarbonyl chloride
(1 equiv) under N2. The reaction mixture was stirred at rt for 2 h.
The solvent was then removed under reduced pressure and the ob-
tained residue was purified by column chromatography (Hexane/
AcOEt 3:2) affording the corresponding product as a white solid.
C
20H23N3O7SNa (M++Na) 472.1154, found 472.1147.
4.1.6.3. SQBuLeuazaGlyCOVSPh 8c. Obtained in 66% yield.
4.1.5.1. MuLeuGlyCOVSPh 1j.
Obtained in 72% yield. Mp 98–
Mp 132–133 °C. IR (cmÀ1) 3340 (NH), 3051 (NH), 2969, 2914,
1709 (C@O), 1668 (C@O), 1518, 1470, 1245, 1081. 1H NMR
(400 MHz, CDCl3) d 7.95 (m, 3H), 7.62 (m, 3H), 7.23 (s, 1H), 6.90
(d, J = 16 Hz, 1H), 6.17 (s, 1H), 4.70 (t, J = 4 Hz, 1H), 4.42 (t,
J = 8 Hz, 2H), 3.34 (s, 1H), 1.78 (m, 2H), 1.66 (m, 3H), 1.53 (m,
2H), 1.05 (m, 9H). 13C NMR (100 MHz, CDCl3) d 191.89 (C@O),
181.91 (Cq), 180.01 (C@O), 173.66 (C@O), 168.56 (C@O), 164.00
(Cq), 140.93 (CHAr), 137.57 (CHSO2Ph), 133.05 (CHAr), 130.28
(CH@CHSO2Ph), 129.10 (CHAr), 128.17 (CHAr), 73.09 (OCH2), 57.78
(CH), 40.43 (CH2), 31.14 (CH2), 25.38 (CH), 23.14 (2CH3), 19.94
(CH2), 14.01 (CH3). HRMS-ESI-TOF: m/z calcd C23H29N3O7SNa
(M++Na) 514.1624, found 514.1615.
99 °C. IR (cmÀ1) 3312 (NH), 2941, 1661 (C@O), 1593, 1456, 1381,
1197. 1H NMR (400 MHz, CDCl3) d 8.26 (s, 1H), 7.93 (m, 2H), 7.59
(m, 5H), 6.99 (d, J = 16 Hz, 1H), 4.76 (t, J = 4 Hz, 1H), 4.03 (s, 1H),
3.90 (t, J = 4 Hz, 2H), 3.79 (t, J = 4 Hz, 4H), 3.57 (t, J = 4 Hz, 2H),
1.87 (m, 2H), 1.53 (t, J = 4 Hz, 1H), 1.08 (d, J = 8 Hz, 6H). 13C NMR
(100 MHz, CDCl3) d 173.66 (C@O), 168.56 (C@O), 157.25 (C@O),
140.93 (Cq), 137.57 (CHSO2Ph), 133.05 (CHAr), 130.28 (CH@CH-
SO2Ph), 129.10 (CHAr), 128.17 (CHAr), 67.21 (2CH2), 51.33 (CH),
46.36 (2CH2), 40.43 (CH2), 25.38 (CH), 23.14 (2CH3). HRMS-ESI-
TOF: m/z calcd C20H28N4O6SNa (M++Na) 475.1627, found 475.1619.
4.1.5.2. MuLeuhomoPheCOVSPh 1l.
Obtained in 87% yield.
Mp 93–94 °C. IR (cmÀ1) 3370 (NH), 3051, 2969, 2914, 1722
(C@O), 1642 (C@O), 1518, 1450, 1368, 1313, 1259, 1143. 1H NMR
(400 MHz, CDCl3) d 7.94 (t, J = 4 Hz, 2H), 7.58 (m, 3H), 7.49 (d,
J = 16 Hz, 1H), 7.31 (m, 5H), 6.73 (d, J = 16 Hz, 1H), 4.56 (sl, 1H),
3.88 (s, 1H), 3.81 (m, 6H), 3.57 (sl, 1H), 3.52 (sl, 2H), 3.26 (t,
J = 4 Hz, 1H), 2.93 (t, J = 4 Hz, 2H). 13C NMR (100 MHz, CDCl3) d
170.87 (C@O), 161.64 (C@O), 157.71 (C@O), 140.75 (Cq), 139.58
(CHSO2Ph), 138.64 (Cq), 133.04 (Cq), 130.14 (CH@CHSO2Ph),
129.19 (CHAr), 129.08 (CHAr), 129.06 (CHAr), 128.14 (CHAr),
126.36 (CHAr), 66.10 (2CH2), 51.98 (CH), 49.77 (CH2), 46.58
(2CH2), 40.44 (CH2), 31.42 (CH2), 25.39 (CH), 23.14 (2CH3).
4.1.6.4. SQMeLeuazaPheCOVSMe 8d.
Obtained in 68% yield.
Mp 148–149 °C. IR (cmÀ1) 3340 (NH), 3244 (NH), 3051, 2969,
1722 (C@O), 1647 (C@O), 1538, 1265, 1150, 1088, 1034. 1H NMR
(400 MHz, CDCl3) d 7.24 (m, 7H), 6.48 (s, 1H), 5.02 (t, J = 4 Hz,
1H), 4.33 (s, 1H), 4.16 (s, 1H), 3.85 (s, 3H), 3.46 (s, 1H), 2.91 (s,
3H), 1.90 (m, 1H), 1.69 (m, 1H), 1.54 (m, 1H), 1.09 (m, 6H). 13C
NMR (100 MHz, CDCl3) d 191.89 (C@O), 179.35 and 178.94 (Cq
and C@O), 170.87 (C@O), 165.33 (C@O), 161.68 (Cq), 147.17
(CHSO2CH3), 136.21 (CHAr), 133.52 (CH@CHSO2CH3), 128.77 (CHAr),
128.44 (CHAr), 127.61 (CHAr), 61.46 (OCH3), 58.31 (CH), 53.18
(CH2), 40.44 (CH2), 37.99 (SO2CH3), 25.39 (CH), 23.14 (2CH3).
HRMS-ESI-TOF: m/z calcd
C
28H36N4O6SNa (M++Na) 579.2253,
HRMS-ESI-TOF: m/z calcd C
22H27N3O7SNa (M++Na) 500.1467,
found 579.2245.
found 500.1456.
4.1.6. General procedure for the preparation of squaric aza vinyl
sulfones derivatives 8a–i
To a solution of the suitable Boc azapeptide vinylsulfone 1
(1 equiv) in DCM was added TFA (4 equiv). After 2 h the solvent
was removed under reduced pressure affording quantitatively
4.1.6.5. SQMeLeuazaPheCOVSPh 8e.
Obtained in 67% yield.
Mp 143–144 °C. IR (cmÀ1) 3392 (NH), 3218 (NH), 3070, 1703
(C@O), 1644 (C@O), 1600, 1466, 1311, 1192, 1125. 1H NMR
(400 MHz, CDCl3) d 7.84 (m, 3H), 7.54 (m, 1H), 7.47 (m, 2H), 7.18
(m, 5H), 7.05 (d, J = 16 Hz, 1H), 6.22 (s, 1H), 5.00 (t, J = 8 Hz, 1H),

P. M. C. Glória et al. / Bioorg. Med. Chem. 19 (2011) 7635–7642
7641
4.39 (s, 1H), 3.94 (s, 1H), 3.89 (s, 3H), 3.55 (s, 1H), 1.74 (t, J = 8 Hz,
1H), 1.65 (t, J = 8 Hz, 1H), 1.52 (m, 1H), 1.01 (sl, 6H). 13C NMR
(100 MHz, CDCl3) d 191.89 (C@O), 179.35 and 178.94 (Cq and
C@O), 170.87 (C@O), 165.32 (C@O), 161.68 (Cq), 140.93 (Cq),
139.58 (CHSO2Ph), 136.21 (CHAr), 133.05 (CHAr), 130.14 (CH@CH-
SO2Ph), 129.10 (CHAr), 128.77 (CHAr), 128.43 (CHAr), 128.17 (CHAr),
127.60 (CHAr), 61.45 (OCH3), 58.31 (CH), 53.18 (CH2), 40.43 (CH2),
25.38 (CH), 23.14 (2CH3). HRMS-ESI-TOF: m/z calcd C27H29N3O7S-
Na (M++Na) 562.1624, found 562.1617.
(CHAr), 125.37 (CHAr), 72.09 (CH2), 57.31 (CH), 48.77 (CH2), 39.43
(CH2), 30.14 (CH2), 29.94 (CH2), 24.38 (CH), 22.14 (2CH3), 18.94
(CH2), 13.01 (CH3). HRMS-ESI-TOF: m/z calcd C31H37N3O7SNa
(M++Na) 618.2250, found 618.2243.
4.2. Pharmacology
4.2.1. Falcipain-2 assays
Falcipain-2 was assayed at 25 °C using 25 lM Z-Leu-Arg-AMC
as substrate in 100 mM sodium acetate pH 6.0, 5 mM DTT, 0.75%
DMSO. One microliter of serial dilutions of each compound dis-
4.1.6.6. SQBuLeuazaPheCOVSPh 8f.
Obtained in 63% yield.
Mp 138–139 °C. IR (cmÀ1) 3177 (NH), 2945, 2765, 1797 (C@O),
1644 (C@O), 1574, 1421, 1300, 1146, 1044. 1H NMR (400 MHz,
CDCl3) d 7.85 (d, J = 8 Hz, 2H), 7.79 (d, J = 16 Hz, 1H), 7.55 (m,
3H), 7.30 (m, 5H), 6.86 (s, 1H), 6.61 (d, J = 16 Hz, 1H), 5.19 (t,
J = 4 Hz, 1H), 4.46 (m, 3H), 4.33 (s, 1H), 3.26 (s, 1H), 1.81 (m, 5H),
1.52 (m, 2H), 1.07 (sl, 9H). 13C NMR (100 MHz, CDCl3) d 191.89
(C@O), 181.91 (Cq), 180.01 (C@O), 170.87 (C@O), 165.33 (C@O),
164.01 (Cq), 140.94 (Cq), 139.59 (CHSO2Ph), 136.21 (CHAr), 133.05
(CHAr), 130.14 (CH@CHSO2Ph), 129.10 (CHAr), 128.77 (CHAr),
128.44 (CHAr), 128.18 (CHAr), 127.61 (CHAr), 73.09 (OCH2), 58.31
(CH), 53.18 (CH2), 40.44 (CH2), 31.15 (CH2), 25.39 (CH), 23.14
(2CH3), 19.95 (CH2), 14.02 (CH3). HRMS-ESI-TOF: m/z calcd
solved in DMSO (highest concentration was 50
eight steps resulting in the lowest concentration used 0.65 nM)
was diluted into 100 L of assay buffer. Enzyme (1 nM) was added,
the mixture was incubated for 10 min, the reaction was initiated
by addition of 50 L of assay buffer containing the substrate, and
lM diluted 1:5 in
l
l
fluorescence was read immediately in a Fluoroskan Ascent micro-
plate spectrofluorometer. IC50 values were determined by plotting
percentage inhibition relative to controls without inhibitor using
GraphPad Prism 4 (GraphPad Software).
4.2.2. In vitro antiplasmodial activity in human red blood cells
Human red blood cells infected with ꢀ1% parasitemia of ring
stage P. falciparum synchronized with 5% sorbitol were incubated
with tested compounds in 96 well plates at 37 °C for 48 h in
RPMI-1640 medium, supplemented with 25 mM HEPES pH 7.4,
10% heat inactivated human serum (or 0.5% Albumax, 2% human
C
30H35N3O7SNa (M++Na) 604.2093, found 604.2087.
4.1.6.7. SQMeLeuazahomoPheCOVSMe 8g. Obtained in 69%
yield. Mp 138–139 °C. IR (cmÀ1) 3409 (NH), 3299 (NH), 3051,
2969, 1722 (C@O), 1647 (C@O), 1511, 1368, 1265, 1156, 1034. 1H
NMR (400 MHz, CDCl3) d 7.25 (m, 6H), 7.07 (d, J = 16 Hz, 1H),
6.47 (s, 1H), 4.87 (t, J = 4 Hz, 1H), 3.76 (s, 3H), 3.53 (t, J = 4 Hz,
1H), 3.37 (t, J = 4 Hz, 1H), 3.30 (s, 1H), 2.93 (s, 3H), 2.85 (t,
J = 4 Hz, 2H), 1.87 (m, 1H), 1.66 (m, 1H), 1.53 (m, 1H), 1.09 (d,
J = 4 Hz, 6H). 13C NMR (100 MHz, CDCl3) d 191.89 (C@O), 179.35
(Cq), 178.94 (C@O), 170.87 (C@O), 161.68 and 161.64 (C@O and
Cq), 147.17 (CHSO2CH3), 138.54 (CHAr), 133.52 (CH@CHSO2CH3),
129.20 (CHAr), 129.09 (CHAr), 126.37 (CHAr), 61.45 (OCH3), 58.31
(CH), 49.77 (CH2), 40.43 (CH2), 37.99 (SO2CH3), 30.94 (CH2),
25.38 (CH), 23.14 (2CH3). HRMS-ESI-TOF: m/z calcd C23H29N3O7S-
Na (M++Na) 514.1624, found 514.1617.
serum), and 100 lM hypoxanthine under an atmosphere of 3%
O2, 5% CO2, 91% N2. After 48 h the cells were fixed in 2% HCHO in
PBS, transferred into PBS with 100 mM NH4Cl, 0.1% Triton X-100,
1 nM YOYO-1, and then analyzed in a flow cytometer (FACSort,
Beckton Dickinson; EX 488 nm, EM 520 nm). IC50s were calculated
using GraphPad PRISM software.
4.2.3. Papain assays
Assays were carried out in 200
7, 4, 5 mM DTT) containing 20 L of papain activated in assay buffer
at 5 g/mL, and 5 L of each concentration of tested inhibitors.
Reactions were initiated by the addition of 30 M fluorogenic sub-
lL assay buffer (10 mM PBS, pH
l
l
l
l
strate (Z-Leu-Leu-Arg-AMC, from Bachem, Germany) and activity
was monitored (excitation 355 nm; emission 460 nm) for 30 min,
at 37 °C on a Fluorescence Microplate Reader Tecan infinite M200
(Tecan, Switzerland). The Km of this substrate of papain was previ-
4.1.6.8. SQMeLeuazahomoPheCOVSPh 8h.
Obtained in 63%
yield. Mp 134–135 °C. IR (cmÀ1) 3299 (NH), 3051 (NH), 2983,
1709 (C@O), 1681 (C@O), 1647 (C@O), 1525, 1422, 1265, 1129,
1040. 1H NMR (400 MHz, CDCl3) d 7.89 (d, J = 8 Hz, 2H), 7.55 (m,
3H), 7.36 (d, J = 16 Hz, 1H), 7.20 (m, 5H), 7.03 (d, J = 16 Hz, 1H),
6.26 (s, 1H), 4.92 (t, J = 8 Hz, 1H), 3.78 (s, 3H), 3.60 (t, J = 8 Hz,
1H), 3.39 (t, J = 8 Hz, 1H), 3.31 (s, 1H), 2.82 (t, J = 8 Hz, 2H), 1.86
(m, 1H), 1.70 (m, 1H), 1.67 (m, 1H), 1.06 (d, J = 4 Hz, 6H). 13C NMR
(100 MHz, CDCl3) d 191.89 (C@O), 179.35 (Cq), 178.94 (C@O),
170.87 (C@O), 161.68 (C@O), 161.64 (CHAr), 148.14 (CHSO2Ph),
140.93 (CHAr), 139.58 (CHAr), 138.54 (CHAr), 133.05 (CHAr), 130.14
(CH@CHSO2Ph), 129.20 (CHAr), 129.10 (CHAr), 128.17 (CHAr),
126.37 (CHAr), 61.45 (OCH3), 58.31 (CH), 49.77 (CH2), 40.43 (CH2),
30.94 (CH2), 25.38 (CH), 23.14 (2CH3). HRMS-ESI-TOF: m/z calcd
ously determined to be 0.4 lM (data not shown). For all assays, sat-
urated substrate concentrations were used in order to obtain linear
fluorescence curves. Inhibitors stock solutions were prepared in
DMSO, and serial dilutions were made in DMSO. Controls were per-
formed using enzyme alone, substrate alone, enzyme with DMSO
and a positive control (trans-epoxysucciny-L-leucyl-amido(4-gua-
nidino)butane E64, Calbiochem, Germany). The IC50 values were
determined by non-linear regression analysis based on the log of
inhibitors concentrations versus the percentage of activity using
GraphPad PRISM software. Assays were performed in triplicate.23
C
28H31N3O7SNa (M++Na) 576.1780, found 576.1768.
Acknowledgments
4.1.6.9. SQBuLeuazahomoPheCOVSPh 8i. Obtained in 65%
yield. Mp 142–143 °C. IR (cmÀ1) 3409 (NH), 3065 (NH), 2969,
2914, 1722 (C@O), 1675 (C@O), 1518, 1368, 1313, 1265, 1143,
1040. 1H NMR (400 MHz, CDCl3) d 7.90 (m, 3H), 7.59 (m, 3H),
7.21 (m, 6H), 6.60 (s, 1H), 5.17 (t, J = 4 Hz, 1H), 4.40 (t, J = 8 Hz,
2H), 4.23 (s, 1H), 3.65 (m, 2H), 2.86 (t, J = 8 Hz, 2H), 1.63 (m, 7H),
1.02 (sl, 9H). 13C NMR (100 MHz, CDCl3) d 191.89 (C@O), 180.91
(Cq), 179.01 (C@O), 169.87 (C@O), 163.00 (Cq), 160.64 (C@O),
139.93 (CHAr), 138.58 (CHSO2Ph), 137.54 (CHAr), 132.05 (CHAr),
129.14 (CH@CHSO2Ph), 128.20 (CHAr), 128.10 (CHAr), 127.17
This work was supported by Fundação para a Ciência e a Tecn-
ologia (Portugal) through postdoctoral fellowship to P.M.C.G. P.J.R.
is a Distinguished Clinical Scientist of the Doris Duke Charitable
Foundation.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.10.018.

7642
P. M. C. Glória et al. / Bioorg. Med. Chem. 19 (2011) 7635–7642
13. Glória, P. M. C.; Coutinho, I.; Gonçalves, L. M.; Baptista, C.; Soares, J.; Newton, A.
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