Synthesis, characterization and antioxidant activity of angiotensin converting enzyme inhibitors

Article abstract of DOI:10.1039/c0ob00823k

Angiotensin converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to angiotensin II (Ang II). ACE also cleaves the terminal dipeptide of vasodilating hormone bradykinin (a nonapeptide) to inactivate this hormone. Therefore, inhibition of ACE is generally used as one of the methods for the treatment of hypertension. 'Oxidative stress' is another disease state caused by an imbalance in the production of oxidants and antioxidants. A number of studies suggest that hypertension and oxidative stress are interdependent. Therefore, ACE inhibitors having antioxidant property are considered beneficial for the treatment of hypertension. As selenium compounds are known to exhibit better antioxidant behavior than their sulfur analogues, we have synthesized a number of selenium analogues of captopril, an ACE inhibitor used as an antihypertensive drug. The selenium analogues of captopril not only inhibit ACE activity but also effectively scavenge peroxynitrite, a strong oxidant found in vivo. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0ob00823k

Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Volume 9 | Number 5 | 7 March 2011 | Pages 1257–1668
ISSN 1477-0520
FULL PAPER
Mugesh et al.
Synthesis, characterization and
antioxidant activity of angiotensin
converting enzyme inhibitors
1477-0520(2011)9:5;1-D

Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 1356
www.rsc.org/obc
PAPER
Synthesis, characterization and antioxidant activity of angiotensin converting
enzyme inhibitors†
Bhaskar J. Bhuyan and Govindasamy Mugesh*
Received 4th October 2010, Accepted 4th November 2010
DOI: 10.1039/c0ob00823k
Angiotensin converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to angiotensin
II (Ang II). ACE also cleaves the terminal dipeptide of vasodilating hormone bradykinin (a
nonapeptide) to inactivate this hormone. Therefore, inhibition of ACE is generally used as one of the
methods for the treatment of hypertension. ‘Oxidative stress’ is another disease state caused by an
imbalance in the production of oxidants and antioxidants. A number of studies suggest that
hypertension and oxidative stress are interdependent. Therefore, ACE inhibitors having antioxidant
property are considered beneficial for the treatment of hypertension. As selenium compounds are
known to exhibit better antioxidant behavior than their sulfur analogues, we have synthesized a number
of selenium analogues of captopril, an ACE inhibitor used as an antihypertensive drug. The selenium
analogues of captopril not only inhibit ACE activity but also effectively scavenge peroxynitrite, a strong
oxidant found in vivo.
important in the treatment of hypertension. Captopril (1),5 the first
Introduction
pharmaceutical compound designed to inhibit a protease, prevents
Angiotensin II (Ang II), the peptide-based blood pressure regulat-
the conversion of Ang I to Ang II by inhibiting ACE. After the
ing hormone in the renin-angiotensin system (RAS)1 is produced
synthesis of captopril, several ACE inhibitory antihypertensive
drugs such as enalaprilat (2),6 zofenoprilat (3),7 fosinoprilat (4)8
etc. have been reported. It should be noted that most of these drugs
contain a proline residue, which appears to be important for the
ACE inhibition (Fig. 2).
in vivo from the decapeptide angiotensin I (Ang I) by cleavage
of the C-terminal dipeptide His-Leu. The conversion of Ang I to
Ang II is catalyzed by angiotensin converting enzyme (ACE), a
metalloprotease having a zinc(II) ion at the active site (Fig. 1).2
As the overproduction of Ang II leads to hypertension, ACE
inhibitors are commonly used to treat cardiovascular diseases
such as high blood pressure, heart failure, coronary artery disease
and kidney failure.1–3 Furthermore, ACE is also responsible for
the elevation of blood pressure by cleaving the terminal dipeptide
(Phe-Arg) of vasodilator hormone bradykinin (nonapeptide) to its
inactive form (bradykinin 1-7).4 Therefore, inhibition of ACE is
Fig. 2 Proline-based ACE inhibitors: captopril (1), enalaprilat (2),
zofenoprilat (3) and fosinoprilat (4).
Fig. 1 ACE-catalyzed conversion of Ang I to Ang II and bradykinin
to bradykinin (1-7). Elevation of Ang II and depletion of bradykinin
concentrations result in the elevation of blood pressure.
Recent evidence suggests that hypertension and oxidative stress
are related to each other and that hypertension plays an important
role in the pathogenesis of oxidative stress and inflammation.9
It has been shown that hypertension induces oxidative stress by
activating NADPH oxidase, leading to the overproduction of
Department of Inorganic and Physical Chemistry, Indian Institute of Science,
Bangalore 560 012, INDIA. E-mail: mugesh@ipc.iisc.ernet.in; Fax: +91-80-
2360 1552/2360 0683
† Electronic supplementary information (ESI) available: X-ray crystal
structures and HPLC data. CCDC reference numbers 775133, 797125,
797126. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c0ob00823k
∑
superoxide anion (O2 -). These reactive superoxide anions interact
with nitric oxide (∑NO) to generate peroxynitrite (ONOO-),10
a highly reactive intermediate known to nitrate protein tyro-
sine residues and cause cellular damage.11 Furthermore, Ang
II-induced endothelial dysfunction has been associated with
1356 | Org. Biomol. Chem., 2011, 9, 1356–1365
This journal is
The Royal Society of Chemistry 2011
©

increased production of peroxynitrite (PN).12 Therefore, antihy-
pertensive drugs having antioxidant activity may play an impor-
tant role in the treatment of hypertension. It is known that cap-
topril (1) and zofenoprilat (3) have significant antioxidant activity
that may contribute to their cardio- and endothelium-protective
effects.13 As selenium compounds are generally better antioxidants
than their sulfur analogues,14 it was thought worthwhile to
synthesize the selenium analogues of captopril and a few cysteine-
and selenocysteine-containing dipeptides (Fig. 3) and study their
ACE inhibition properties. In this paper, we report that the
selenium analogues of captopril and Cys-Pro and Sec-Pro peptides
(5–11) not only inhibit ACE activity but also effectively scavenge
peroxynitrite.
Scheme 1 Synthesis of compounds 5 and 6. a) p-Methoxy benzyl
diselenide, NaBH4, anhyd. DMF, 36 h; b) I2, 1 : 1 MeOH–water, 15 min;
c) MeOH, NaBH4,10 min; Cy = Cyclohexyl.
Fig. 3 Selenium analogues of captopril and related derivatives (5–11).
over the (S,R)-diastereomer. The non-centrosymmetric space
group (P1) and Flack parameter16 (0.21) confirm the diastere-
omeric purity of the crystals. It should be noted that the bromine
atom in compound 13 is disordered over two positions. The crystal
structure of compound 15 indicates that there was no racemization
during the conversion of 14 to 15. Compound 15 crystallized in a
P21 space group and all four chiral centers in this compound are
present in the S-configuration. The absolute structure parameter
for this compound (0.036) indicates that the crystals are optically
pure (crystallographic details are given in the ESI†).
Compounds 7 and 8 were synthesized by following a different
synthetic route (Scheme 2). In this method, the selenium moiety
was introduced to bromo-propionic acid or bromo-acetic acid
as PMB-protected selenolate to produce compounds 19 and 20,
respectively, in reasonably good yield. In addition to spectro-
scopic analysis, compound 20 was characterized by single crystal
X-ray studies (ESI†). After coupling of the L-Pro moiety, the
PMB-protecting group at the selenium center was removed by
iodine from compounds 21 and 22 to obtain the corresponding
diselenides (23 and 24). Hydrolysis of the ester group by LiOH
followed by reduction with NaBH4 produced the selenols 7 and
8. The stability of 7 and 8 was found to be identical to that of
Se-captopril.
Synthesis
In contrast to the synthesis of captopril (1),5 the synthesis of
the selenium analogues is complicated due to a facile oxidation
of the selenol to the corresponding diselenide. Therefore, we
approached the synthesis of the diselenide 15 starting from the
L-proline-based compound 12 (Scheme 1). Compound 12 can
be obtained in pure form by coupling L-proline with (R,S)-3-
bromo-2-methyl-propionic acid. Due to the unavailability of the
optically pure form of (R,S)-3-bromo-2-methyl-propionic acid,
the resolution of the diastereomers is the crucial step in the
synthetic procedure. The diastereomeric mixture of compound 12
canbe resolvedby preferentialcrystallization oftheir dicyclo-hexyl
amine (DCHA) salts (13 an◦d 16).15 When the mixture was treated
with DCHA and kept at 4 C for 12 h, one of the diastereomers
(compound 13) was crystallized preferentially, leaving the other
diastereomer (compound 16) in the solution. Removal of DCHA
from compounds 13 and 16 by treatment with 1 M KHSO4
solution followed by reactions with p-methoxy benzyl selenolate
(PMB-selenolate) afforded the PMB-protected compounds 14 and
17, respectively. The diastereomerically pure diselenides 15 and 18
were obtained by removal of the PMB-group with iodine. The
selenols 5 and 6 required for the inhibition studies were prepared
by reducing the corresponding diselenides with NaBH4.
Captopril (1) and Se-captopril (5) are structurally similar
to D-Cys-L-Pro and D-Sec-L-Pro dipeptides, respectively. Harris
et al. have shown that the replacement of the methyl group in
captopril by an amino group decreases the inhibition activity
of the parent compound.17 The D-Sec-L-Pro dipeptide (11) re-
sembles Se-captopril (5), whereas, L-Sec-L-Pro (9) resembles the
Compounds 13 and 15 were characterized by single crystal
X-ray diffraction studies. The crystal structure of compound 13
shows that there is a hydrogen bond between the carboxylic group
of proline residue and the amino group of DCHA. This also
indicates that the (S,S)-diastereomer has crystallized preferentially
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1356–1365 | 1357
©

Scheme 2 Synthesis of compounds 7 and 8.
R,S-diastereomer of Se-captopril (6). To understand the effect
34. Removal of the Boc group by treatment with TFA afforded the
Sec-Pro dipeptides. The selenols 9 and 11 could be conveniently
prepared by reducing the diselenides 35 and 36, respectively, by
NaBH4. The Cys-Pro dipeptide (10) was synthesized following a
similar procedure (Scheme 4).
of the methyl group in captopril and its selenium analogues,
we have synthesized Cys-Pro and Sec-Pro dipeptides (9–11) and
studied their ACE inhibition activities. Dipeptides containing Sec-
Pro and Cys-Pro were synthesized by following solution phase
peptide synthetic routes.18 Due to the presence of reactive thiol
and selenol moieties, the protection of the side chain is important
in the synthesis of Cys- and Sec-containing dipeptides.19 Sec-Pro-
containing dipeptides were synthesized by following the procedure
given in Scheme 3.
Scheme 4 Synthesis of Cys-Pro dipeptide (compound 10).
Inhibition of angiotensin converting enzyme
Scheme 3 Synthesis of Sec-Pro dipeptides (compounds 9 and 11).
The ACE inhibition activities of captopril and its selenium
analogues (5–11) were studied by a HPLC method (Scheme 5).
Ang I was used as the substrate for the enzyme and a decrease
in the formation of Ang II with an increase in the concentration
of inhibitors was followed at 215 nm wavelength. The peak area
corresponding to Ang II was obtained for the initial 5–10% of the
reaction and, wherever possible, the IC50 values (concentration of
inhibitors required to inhibit 50% of the enzyme activity) were
determined. The reaction mixture was incubated at 37 ◦C for
To understand the importance of chirality in dipeptides, we
have used enantiomerically pure D- or L-serine. The tosylated
compounds 27 and 28 were obtained from the corresponding Boc-
protected serines. The PMB-Se moiety was then introduced to ob-
tain enantiomerically pure 29 and 30. DCC-mediated coupling of
L-Pro-OMe to the PMB-protected Sec afforded the corresponding
dipeptides 31 and 32. The cleavage of the PMB group under mild
conditions using iodine led to the formation of diselenides 33 and
1358 | Org. Biomol. Chem., 2011, 9, 1356–1365
This journal is
The Royal Society of Chemistry 2011
©

indicating that the presence of the selenol moiety is important for
ACE inhibition.
To understand the nature of ACE inhibition by the captopril
analogues, we have carried out further kinetic experiments at
different concentrations of inhibitors (5 and 6) by varying
the concentrations of the substrate (Ang I) for each inhibitor
concentration. The initial rates for the hydrolysis of Ang I by
ACE in the presence of inhibitors were obtained by following the
formation of Ang II. For each concentration of the inhibitor, a
linear line was obtained when the reciprocal of the initial rate was
plotted against the reciprocal of the concentration of Ang I. These
linear lines (Lineweaver–Burk plots) for various concentrations of
inhibitors intersect the ordinate with almost identical Vmax values
(Fig. 5), suggesting that inhibition of ACE activity by compounds
5 and 6 is competitive with respect to the substrate. It is known
that the inhibition of ACE by captopril is competitive with respect
to ACE substrates.5 Therefore, the binding of compounds 5 and 6
to the active site of ACE is similar to that of captopril.
Scheme 5 Inhibition of ACE-catalyzed conversion of Ang I to Ang II by
the selenium analogues of captopril.
30 min prior to analysis. The selenols (5–8) were freshly prepared
by reducing the corresponding diselenides by NaBH4 prior to use
and were kept under N2 atmosphere during the assay. The IC50
values obtained for the inhibition of ACE-catalyzed conversion
of Ang I to Ang II by the captopril analogues are summarized
in Fig. 4. In contrast to the thiol and selenols, the IC50 values
could not be determined for the disulfide and diselenides up to a
concentration of 50 mM.
Fig. 5 Lineweaver–Burk plots obtained for (a) compound 5 and (b)
compound 6 at various concentrations of Ang I and fixed concentration
of the inhibitor (for more details, see the ESI†).
Fig. 4 IC50 values for ACE inhibition by 1, 5–11. a At this concentration,
only 30% of the enzyme activity was inhibited. b Only 40% inhibition was
observed. Assay conditions: the reaction was carried out in HEPES–HCl
buffer (50 mM, pH 8.3) at 37 ◦C with a final concentration of 50 mM Ang
I, 60mM NaCl and 2 milliunits of ACE in 400 mL reaction mixture.
The crystal structure of the ACE-captopril complex reveals that
the proline residue of captopril interacts with the S2¢ subsite of
the enzyme active site (Fig. 6).20 This interaction is favored by two
histidine residues at the enzyme active site. It has been shown that
the thiol group of captopril interacts with the zinc(II) ion to form
a zinc-thiolate complex. As compound 5 is structurally similar to
that of captopril, we propose the formation of a zinc-selenolate
complex. This is further supported by the observations that the
PMB-protected compounds 14 and 17 and the diselenides 15 and
18 do not inhibit the enzyme activity. Although the formation
of zinc-selenolate may be more favored than the formation of a
It is clear from Fig. 4 that selenol 5 is an excellent inhibitor of
ACE although the IC50 value (36.4 1.5 nM) is almost two times
higher than that of captopril (1, 18.1 1.0 nM). Interestingly, the
diastereomer 6 was found to be much less active than 5. The IC50
value obtained for 6 (7500 500 nM) is almost 200 times higher
than that of 5. This indicates that the correct stereochemistry
at the side chain is important for an efficient inhibition. The
IC50 value obtained for compound 7 (5300 440 nM) indicates
that the absence of the methyl group significantly reduces the
inhibitory activity. However, this compound is moderately more
active than compound 6. The distance between the proline moiety
and the selenol group appears to be important for the inhibition
as compound 8, having only one methylene group, exhibited
very poor inhibition. At 30 mM concentration, this compound
inhibited only 30% of the enzyme activity. Similar to the captopril
analogues, the ACE inhibition activities by the dipeptides depends
on the chirality. However, it was observed that the replacement
of the methyl group of Se-captopril by an amino group in
dipeptide 11 dramatically reduces the ACE inhibition potency.
This compound inhibited only 40% of the enzyme activity up to
50 mM concentration. In contrast, the other diastereomer (L-Sec-
L-Pro-OMe, compound 9) exhibited very good activity (IC50: 342
33 nM). In the case of dipeptides, sulfur analogue (compound 10,
IC50: 6480 640 nM) showed about 20 times less activity than
the selenium analogue. In contrast to the selenols and thiols, the
diselenides and disulfides did not show any noticeable inhibition,
Fig.
6
Possible binding of captopril (1), Se-captopril (5) and
L-Sec-L-Pro-OMe (9) to the enzyme active site.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1356–1365 | 1359
©

zinc-thiolate complex,21 the partial oxidation of the selenol to the
corresponding diselenide is probably responsible for the relatively
higher IC50 value for Se-captopril as compared to that of captopril.
The poor inhibition by compound 6 indicates that the binding of
captopril and its analogues to the ACE active site is stereospecific.
Interestingly, the higher activity of compound 7 as compared to 6
indicates that the side chain lacking any substituent is probably a
better inhibitor than the one having incorrect stereochemistry. As
the distance between the selenol group and proline moiety plays an
important role in the inhibitor binding, shortening of this distance
by removing one methylene group may reduce the binding ability.
In agreement with this, compound 8, having one –CH2– group,
shows very weak inhibition. In the case of dipeptides, replacement
of the non-polar methyl group by the polar amino group in the
same stereochemistry hinders the binding of compound 11 to the
enzyme active site. However, when the orientation of the amino
group is reversed, compounds 9 and 10 can probably interact
with the polar amino acid residues present at the active site. The
higher activity of the Sec-containing peptide as compared to the
Cys-containing peptide may be attributed to the stabilization of
the reactive selenol of compound 9 by the electron-withdrawing
nature of the amino functional group to form a strongly bound
zinc(II)-selenolate complex.
Fig. 7 Peroxynitrite-scavenging activity of various captopril analogues.
Assay conditions: reactions were carried out in sodium phosphate buffer
(100 mM, pH 7.5) at 22 ◦C with a final concentration of 20 mM Ang II
and 300 mM PN.
is, however, not surprising since the selenium compounds exhibit
their inhibitory effects mainly by scavenging the peroxynitrite.
In addition to the inhibition of Ang II nitration, the disulfides
and diselenides effectively protect against PN-mediated nitration
of bovine serum albumin (BSA). At 60 mM concentration of
these compounds, ~40–60% inhibition of tyrosine nitration was
observed (Fig. 8). Similar to the effect of these compounds on the
nitration of Ang II, the diselenides were found to be more effective
than the corresponding disulfide derivatives (41 and 42). Although
compounds 25 and 26 were found to be more active than the other
disulfides and diselenides in the Ang II assay, these compounds
were slightly less potent than the captopril analogues 15 and 18 in
the inhibition of the nitration of BSA.
Inhibition of PN-mediated nitration reactions
To evaluate the antioxidant activity of the sulfur and selenium
compounds, we have studied the effect of these compounds on
the peroxynitrite-mediated nitration22 of Ang II. It has been
demonstrated that nitration of the tyrosine residue in Ang II
completely inhibits its vasoconstrictive properties, which may
correlate with the endothelial dysfunction observed at the early
stage of Ang II action.12c,23 As the formation of 3,5-dinitro-
Ang II was also observed in the reaction, only the initial 5–
10% of the conversion was followed, for which the mononitro
compound was produced as the major product (Scheme 6). In
order to compare the PN-scavenging activity of the diselenides
with that of a disulfide, we have synthesized captopril-disulfide
(compound 42) from captopril using K3[Fe(CN)6] as oxidizing
agent.24 The IC50 values obtained for the inhibition of PN-
mediated nitration of Ang II are summarized in Fig. 7. Interest-
ingly, all the selenium compounds strongly inhibited the nitration
reaction. The inhibitory activity of the selenium analogue of
captopril (5) was found to be almost 10 times higher than that
of captopril (1). The Sec-containing dipeptides were observed to
be as potent inhibitors of PN-mediated nitration reactions as
the other selenium-containing compounds. The PN-scavenging
ability of Cys-containing peptides was lower than that of the Sec-
containing dipeptides. In contrast to the ACE inhibition activities,
the PN-scavenging activity does not depend on the chirality, but
it depends on the presence of a sulfur or selenium center. This
Fig. 8 Immunoblots for the inhibition of PN-mediated nitration of BSA.
100 mM of BSA was incubated with 60 mM of inhibitor and 1.2 mM of PN
for 30 min at 20 ◦C and then subjected to gel electrophoresis.
Conclusions
In conclusion, we provided the first experimental evidence that the
selenium analogues of captopril not only inhibit angiotensin con-
verting enzyme but also effectively protect against peroxynitrite-
mediated nitration of tyrosyl residues in peptides and proteins.
The inhibition of PN-mediated nitration of Ang II is particularly
important as such nitration has been implicated in cardiovas-
cular diseases. This study reveals that the ACE inhibition is
Scheme 6 Peroxynitrite-mediated nitration of Angiotensin II to mono-ni-
tro angiotensin II.
1360 | Org. Biomol. Chem., 2011, 9, 1356–1365
This journal is
The Royal Society of Chemistry 2011
©

highly stereospecific to the inhibitor conformation, whereas, PN-
scavengening activity depends on the presence of the selenium
or sulfur center. Selenium-containing compounds are better
scavengers of peroxynitrite. Although the selenols are oxidized
to the corresponding diselenides in air, biological thiols such as
glutathione (GSH) can prevent the oxidation.
34.4, 41.8, 47.9, 59.6, 174.3, 175.3; HRMS (ESI mode) calcd. for
C9H14BrNO3 [M+Na]+ 264.0235, found 264.0229.
Synthesis of 16. This compound was obtained from the filtrate
from the above reaction. The excess DCHA was removed by
acidification. Yield 0.41 g (40%). 1H NMR (CDCl3) d (ppm): 1.24–
1.28 (d, 3H), 2.06–2.10 (m, 3H), 2.35–2.36 (m, 1H), 3.07–3.11 (m,
1H), 3.37–3.41 (m, 1H), 3.60–3.68 (m, 3H), 4.15–4.67(dd, 1H);
13C NMR (CDCl3) d (ppm): 17.4, 25.3, 29.1, 34.7, 41.3, 48.0, 59.2,
174.1, 176.0; HRMS (ESI mode) calcd. for C9H14BrNO3 [M+Na]+
264.0235, found 264.0245.
Experimental section
General procedure. Angiotensin converting enzyme (ACE)
and captopril (1) were purchased from Sigma-Aldrich Chemical
Co. All experiments involving selenols and thiols were carried out
under dry and oxygen free nitrogen using standard Schlenk tech-
niques. Column chromatography was performed on glass columns
loaded with silica gel or on an automated flash chromatography
system (Biotage) using pre-loaded silica cartridges. 1H (400 MHz),
13C (100.56 MHz), and 77Se (76.29 MHz) NMR spectra were
obtained on a Bruker 400 MHz NMR spectrometer. Chemical
shifts are cited with respect to SiMe4 as internal (1H and 13C),
and Me2Se as external (77Se) standards. Mass spectral studies
were carried out on a Q-TOF micro mass spectrometer or on
a Bruker Daltonics 6000 plus mass spectrometer with ESI-MS
mode analysis. The purities of the compounds were greater than
95% as determined by reversed-phase HPLC.
Synthesis of 14. To a solution of 4-methoxy benzyl diselenide
(0.24 g, 0.6 mmol) in dry DMF, NaBH4 was added in small
portions (~0.09 g, 2.4 mmol). The mixture was stirred for 15 min
to obtain the corresponding selenol. A solution of 13 (0.26 g,
1.0 mmol in 15 mL ethanol) in 10 mL DMF was added to the
selenol over 15 min at 0 ◦C. The reaction mixture was stirred
for 36 h at room temperature under nitrogen atmosphere. To
this, 100 mL of 1 M KHSO4 was added and the compound
was extracted with ethyl acetate. The organic layer was washed
three times with brine and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure and the compound
was purified by flash chromatography (hexane–ethyl acetate; 1 : 2)
to afford a pale yellow liquid. Yield 0.21 g (53%). 1H NMR
(CDCl3) d (ppm): 1.15–1.17 (d, 3H), 1.97–2.00 (m, 4H), 2.49–
2.54 (m, 2H), 2.83–2.89 (m, 1H), 3.38–3.44 (m, 1H), 3.76–3.79
(m, 6H), 4.61–4.63 (dd, 1H), 6.82–6.84 (dd, 2H), 7.20–7.22 (dd,
2H); 13C NMR (CDCl3) d (ppm): 18.7, 25.2, 27.2, 28.1, 28.6,
40.1, 47.9, 55.8, 59.9, 114.4, 130.4, 131.9, 158.9, 174.5, 176.7; 77Se
NMR (CDCl3, Me2Se) d (ppm): 247; HRMS (ESI mode) calcd.
for C17H23NO4Se [M+Na]+ 408.0690, found 408.0693.
Synthesis of compound 12. To a solution of 3-bromo-2-methyl
propionic acid (0.75 g, 4.5 mmol) in benzene (20 mL), SOCl2
(1 mL, 12.5 mmol) was added dropwise at 0 ◦C. To this mixture,
DMF (0.034 mL, 0.4 mmol) was added as catalyst. The reaction
mixture was allowed to attain the room temperature and the
stirring was continued for 3 h. Solvent and excess SOCl2 were
evaporated under reduced pressure to yield the acyl chloride,
which was dissolved in chloroform and cooled to 0 ◦C. L-proline
(0.78 g, 6.7 mmol) was added to the above solution, followed by
the addition of Et3N (0.9 mL, 6.5 mmol). The reaction mixture was
allowed to attain the room temperature and stirred for 10 h. The
mixture was acidified by adding 1 M aqueous KHSO4 solution and
the compound was extracted with chloroform. The organic layer
was washed two times with brine. The solvent was evaporated and
the compound was purified by column chromatography (hexane–
ethyl acetate; 1 : 1). Yield 1.0 g (84%). 1H NMR (CDCl3) d (ppm):
1.24–1.29 (d, 3H), 2.05–2.11 (m, 3H), 2.34–2.35 (m, 1H), 3.05–3.10
(m, 1H), 3.31–3.35 (m, 1H), 3.60–3.68 (m, 3H), 4.15–4.67(dd, 1H);
13C NMR (CDCl3) d (ppm): 17.3, 25.2, 29.1, 34.4, 41.8, 47.9, 59.6,
174.3, 175.3; HRMS (ESI mode) calcd. for C9H14BrNO3 [M+Na]+
264.0235, found 264.0240.
Synthesis of 17. Compound 17 was synthesized following a
similar procedure to that given for compound 14. Yield 0.22 g
1
(55%). H NMR (CDCl3) d (ppm): 1.14–1.16 (d, 3H), 1.99–2.05
(m, 3H), 2.51–2.55 (m, 1H), 2.68–2.75 (m, 1H), 2.82–2.87 (m, 1H),
3.34–3.36 (m, 1H), 3.45–3.48 (m, 1H), 3.76–3.79 (m, 6H), 4.54–
4.56 (d, 1H), 6.81–6.84 (dd, 2H), 7.21–7.23 (d, 2H); 13C NMR
(CDCl3) d (ppm): 18.2, 25.1, 27.3, 28.2, 40.3, 48.1, 55.7, 60.5, 114.4,
130.5, 131.8, 158.9, 173.5, 177.5; 77Se NMR (CDCl3, Me2Se) d
(ppm): 245; HRMS (ESI mode) calcd. for C17H23NO4Se [M+Na]+
408.0690, found 408.0702.
Synthesis of 15. To a 10 mL methanolic solution of 14 (0.20 g,
0.5 mmol), iodine (0.15 g, 0.6 mmol) was added followed by 10 mL
of water. The reaction mixture was stirred for 15 min. To this,
0.5 mL of hydrazine hydrate was added to decompose the excess
iodine. Methanol was removed under reduced pressure and 20 mL
of 1 M KHSO4 solution was added. The compound was extracted
three times with ethyl acetate and purified by flash chromatography
(hexane–ethyl acetate; 1 : 3). Yield 0.13 g (95%). 1H NMR (CDCl3)
d (ppm): 1.25–1.27 (d, 6H), 2.02–2.10 (m, 6H), 2.46–2.50 (m, 2H),
3.08–3.12 (m, 2H), 3.33–3.37 (m, 2H), 3.60–3.62 (m, 2H), 3.65–
3.69 (m, 4H), 4.67–4.70 (dd, 2H); 13C NMR (CDCl3) d (ppm): 18.3,
25.3, 28.8, 32.67, 39.9, 48.0, 59.8, 174.9, 175.9; 77Se NMR (CDCl3,
Me2Se) d (ppm): 305; HRMS (ESI mode) calcd. for C18H28N2O6Se2
[M+Na]+ 551.0175, found 551.0173.
Synthesis of 13. Compound 12 (1.01 g, 3.7 mmol) was
dissolved in 10 mL chloroform and dicyclohexyl amine (DCHA)
(0.71 g, 3.8 mmol) was added. To this, 10 mL of acetonitrile
was added and the mixture was kept at 4 ◦C for 12 h. After
this period, dicyclohexyl amine salt of compound 13 crystallizes
out. The solvent was removed and the crystals were washed with
acetonitrile. The crystals were dissolved in chloroform and washed
several times with a saturated solution of KHSO4. The organic
layer was dried with anhydrous Na2SO4 and the solvent was
1
removed under reduced pressure. Yield 0.45 g (45%). H NMR
(CDCl3) d (ppm): 1.24–1.29 (d, 3H), 2.05–2.11 (m, 3H), 2.34–2.35
(m, 1H), 3.05–3.10 (m, 1H), 3.31–3.35 (m, 1H), 3.60–3.68 (m, 3H),
4.15–4.67 (d, 1H); 13C NMR (CDCl3) d (ppm): 17.3, 25.2, 29.1,
Synthesis of 18. Compound 18 was synthesized following a
similar procedure to that given for compound 15 using compound
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1356–1365 | 1361
©

17 as the starting material. Yield 0.13 g (95%). 1H NMR (CDCl3)
d (ppm): 1.22–1.24 (d, 6H), 1.96–1.38 (m, 6H), 2.90–3.02 (m,
6H), 3.21–3.23 (m, 2H), 3.53–3.56 (m, 2H), 3.76–3.79 (m, 2H),
4.49–4.52 (dd, 2H); 13C NMR (CDCl3) d (ppm): 17.6, 25.2, 28.9,
30.1, 39.8, 48.0, 60.1, 174.4, 176.1; 77Se NMR (CDCl3, Me2Se) d
(ppm): 306; HRMS (ESI mode) calcd. for C18H28N2O6Se2 [M+H]+
529.0356, found 529.0341.
was extracted with ethyl acetate. The organic layer was dried
over anhydrous Na2SO4 and the compound was purified by flash
chromatography (hexane–ethyl acetate; 1 : 1). Yield 0.10 g (88%).
1H NMR (CDCl3) d (ppm): 1.91–2.02 (m, 8H), 2.15–2.16 (d, 2H),
3.64–3.67 (m, 2H), 3.84 (s, 2H) 4.40–4.44 (dd, 2H); 13C NMR
(CDCl3) d (ppm): 23.2, 25.7, 28.1, 48.7, 55.0, 171.7, 174.8; 77Se
NMR (CDCl3, Me2Se) d (ppm): 335; HRMS (ESI mode) calcd.
for C13H18N2O6Se2 [M+Na]+ 494.9549, found 494.9621.
Synthesis of 19. To a solution of 4-methoxy benzyl diselenide
(0.93 g, 2.3 mmol) in ethanol under N2 atmosphere, NaBH4 was
added in small portions (~0.37 g, 10.0 mmol). The mixture was
stirred for 1 h to obtain the corresponding selenol. To this, 15 mL
ethanolic solution of 3-bromo acetic acid (0.83 g, 6.0 mmol) was
added over a period of 15 min at 0 ◦C. The reaction mixture was
stirred for 12 h at room temperature under nitrogen atmosphere.
The solvent was evaporated under reduced pressure and acidified
with 1 M KHSO4 solution. The compound was extracted from the
aqueous mixture with CHCl3 and washed three times with brine.
The organic layer was dried over anhydrous Na2SO4. Removal
of solvent afforded a pale yellow liquid. Pure compound was
obtained by column chromatography (hexane–ethyl acetate; 2 : 1).
Yield 1.21 g (77%). 1H NMR (CDCl3) d (ppm): 3.04 (s, 2H), 3.80 (s,
3H), 3.97 (s, 2H), 6.84–6.87 (d, 2H), 7.27–7.29 (d, 2H); 13C NMR
(CDCl3) d (ppm): 21.5, 28.1, 55.3 114.0, 129.9, 130.4, 158.7, 178.2;
77Se NMR (CDCl3, Me2Se) d (ppm): 312; HRMS (ESI mode)
calcd. for C10H12O3Se [M+Na]+ 282.9849, found 282.9857.
Synthesis of 20. This compound was synthesized by following
a similar procedureto that given for compound 19. Yield 72%. 1H
NMR (CDCl3) d (ppm): 2.68 (s, 4H), 3.80 (s, 5H), 6.82–6.84 (d,
2H), 7.20–7.22 (d, 2H); 13C NMR (CDCl3) d (ppm): 17.2, 27.2,
35.5, 55.6, 114.3, 131.1, 158.8, 178.0; 77Se NMR (CDCl3, Me2Se)
d (ppm): 270; HRMS (ESI mode) calcd. for C11H14O3Se [M+Na]+
297.0006, found 296.9972.
Synthesis of 22. Compound 22 was synthesized by following
a similar procedure to that given for compound 21. Yield 0.27 g
(53%); 1H NMR (CDCl3) d (ppm): 1.94–2.04 (m, 3H), 2.13–2.16
(m, 2H), 2.53–2.63 (m, 2H), 2.76–2.80 (m, 1H), 3.38–3.41 (m,
1H), 3.51–3.54 (m, 1H), 3.70 (s, 3H), 3.81 (s, 5H), 4.46–4.48 (dd,
1H), 6.80–6.82 (d, 2H), 7.19–7.21 (d, 2H); 13C NMR (CDCl3) d
(ppm): 16.5, 21.4, 24.3, 28.1, 34.6, 45.8, 51.5, 54.2, 57.5, 112.8,
128.8, 129.9, 157.3, 169.6, 171.4; 77Se NMR (CDCl3, Me2Se) d
(ppm): 267; HRMS (ESI mode) calcd. for C17H23NO4Se [M+H]+
386.0871, found 385.9808.
Synthesis of 21. To a solution of 19 (0.35 g, 1.3 mmol) in 50 mL
chloroform, dicyclohexyl carbodiimide (DCC) (0.29 g, 1.4 mmol)
and 1-hydroxy benzotriazole (HOBt) (0.21 g, 1.4 mmol) were
Synthesis of 24. Compound 24 was synthesized by following a
similar procedure to that given for compound 15 using compound
22 as starting material. Yield 95%. H NMR (CDCl3) d (ppm):
1
◦
added at 0 C successively and the resulting mixture was stirred
1.97–2.02 (m, 4H), 2.15–2.19 (m, 2H), 2.81–2.86 (m, 4H), 3.07–
3.16 (m, 2H), 3.34–3.79 (m, 18H), 4.37–4.48 (dd, 2H); 13C
NMR (CDCl3) d (ppm): 23.6, 24.7, 29.2, 36.1, 47.0, 52.3, 58.7,
170.4, 172.8; 77Se NMR (CDCl3, Me2Se) d (ppm): 325; HRMS
(ESI mode) calcd. for C18H28N2O6Se2 [M+Na]+ 551.0175, found
551.0163.
for 20 min. To this, L-proline-OMe (0.19 g, 1.5 mmol) was added.
The reaction mixture was allowed to attain room temperature in
1 h and the stirring was continued for a further 10 h. The solvent
was evaporated and the residue was dissolved in ethyl acetate. The
organic layer was washed three times with 1 M solution of KHSO4
and brine. The organic layer was dried over anhydrous Na2SO4.
Removal of solvent afforded a pale yellow liquid. The product
was purified by flash chromatography (hexane–ethyl acetate; 1 : 1).
Yield 0.37 g (66%). 1H NMR (CDCl3) d (ppm): 1.95–2.09 (m, 3H),
2.14–2.22 (m, 1H), 3.13 (s, 2H), 3.46–3.52 (m, 1H), 3.60–3.65 (m,
1H), 3.73 (s, 2H), 3.77 (s, 3H), 3.86–3.95 (q, 2H), 6.80–6.82 (d,
2H), 7.29–7.31 (d, 2H); 13C NMR (CDCl3) d (ppm): 22.9, 24.9,
27.0, 29.4, 47.5, 52.3, 55.3, 59.0, 113.9, 130.4, 130.7, 158.5, 167.0,
172.8; 77Se NMR (CDCl3, Me2Se) d (ppm): 275; ESI-MS: m/z
calcd. for C16H21NO4Se [M+H]+ 372.0714, found 371.9364.
Synthesis of 26. This compound was synthesized by following
a similar procedure to that given for 25 using compound 24 as
the starting material. Yield 76%. 1H NMR (CDCl3) d (ppm):
1.97–2.02 (m 4H), 2.15–2.19 (m, 2H), 2.81–2.86 (m, 4H), 3.07–
3.16 (m, 2H), 3.34–3.79 (m, 12H), 4.37–4.48 (dd, 2H); 13C NMR
(CDCl3) d (ppm): 23.6, 24.7, 29.3, 36.1, 47.0, 52.3, 170.4, 172.8;
77Se NMR (CDCl3, Me2Se) d (ppm): 324; HRMS (ESI mode)
calcd. for C16H24N2O6Se2 [M+Na]+ 522.9862, found 522.9901.
Synthesis of 29. In a two-necked 250 mL round-bottom flask,
p-methoxy benzyl diselenide (3.02 g, 7.5 mmol) and NaBH4
(1.10 g, 30.0 mmol) were mixed under nitrogen atmosphere. To this,
100 mL of dry DMF was added at 0 ◦C and the reaction mixture
was stirred for 10 min. 29 (2.50 g, 7.0 mmol) was dissolved in 25 mL
dry DMF and was dropwise added to the reaction mixture. The
reaction mixture was allowed to attain 25 ◦C and the stirring was
continued for 36 h. After this time, 200 mL 1 M KHSO4 was added
to the reaction mixture and the compound was extracted with
ethyl acetate. The ethyl acetate layer was washed three times with
brine. Solvent was evaporated and the compound was purified by
column chromatography (hexane–ethyl acetate; 1 : 1). Yield 1.49 g
(55%); 1H NMR (CDCl3) d (ppm): 1.43 (s, 9H), 2.61–2.69 (m, 2H),
3.76–3.77 (d, 5H), 4.32–4.35 (m, 1H), 6.78–6.79 (d, 2H), 7.19–7.20
Synthesis of 23. Compound 23 was synthesized following a
similar procedure to that given for compound 15 using compound
21 as the starting material. Yield 90%. 1H NMR (CDCl3) d (ppm):
1.91–2.02 (m, 8H), 2.14–2.16 (m, 2H), 3.55–3.58 (m, 2H), 3.65
(s, 6H), 3.84 (s, 4H), 4.40–4.44 (dd, 2H); 13C NMR (CDCl3) d
(ppm): 23.2, 25.3, 27.8, 28.8, 48.4, 55.8, 60.2, 170.7, 174.1; 77Se
NMR (CDCl3, Me2Se) d (ppm): 335; ESI-MS: m/z calcd. for
C16H24N2O6Se2 [M+Na]+ 501.0043, found 500.8780.
Synthesis of 25. 0.12 g (0.3 mmol) of compound 23 was
dissolved in 10 mL ethanol and 20 mg (0.5 mmol) of LiOH·H2O
was added to it followed by 10 mL of water. The reaction mixture
was stirred for 3 h. Ethanol was evaporated under reduced pressure
and the aqueous layer was acidified with KHSO4. The compound
1362 | Org. Biomol. Chem., 2011, 9, 1356–1365
This journal is
The Royal Society of Chemistry 2011
©

(d, 2H); 13C NMR (CDCl3) d (ppm): 22.1, 28.7, 35.7, 54.0, 61.3,
81.6, 128.3, 130.3, 132.6, 145.8, 155.3, 171.8; 77Se NMR (CDCl3) d
(ppm): 213; HRMS (ESI mode) calcd. for C16H23NO5Se [M+Na]+
412.0639, found 412.0653.
172.8; 77Se NMR (CDCl3) d (ppm) 223; ESI-MS: m/z calcd. for
C22H32N2O6Se [M+Na]+ 523.1323, found 523.1168.
Synthesis of 34. Compound 34 was synthesized following a
1
similar method to that given for compound 15. Yield 95%. H
NMR (CDCl3) d (ppm): 1.36 (s, 18H), 1.94–1.96 (m, 6H), 2.15–
2.17 (m, 2H), 3.09–3.13 (m, 2H), 3.22–3.25 (m, 2H), 3.49 (s, 6H),
3.70–3.73 (m, 4H), 4.35–4.37 (d, 2H), 4.70–4.73 (d, 2H); 13C NMR
(CDCl3) d (ppm): 25.1, 28.7, 29.5, 32.8, 47.6, 52.6, 52.9, 59.5,
80.3, 155.4, 169.7, 172.6; 77Se NMR (CDCl3) d (ppm) 303; HRMS
(ESI mode) calcd. for C28H46N4O10Se2 [M+Na]+ 781.1442, found
781.1450.
Synthesis of 31. To a solution of 29 (0.80 g, 2.0 mmol) in
120 mL chloroform, DCC (0.62 g, 3.0 mmol) and HOBt (0.46 g,
3.0 mmol) were added at 0 ◦C and stirred for 20 min. L-Pro-OMe
(0.39 g, 3 mmol) was added to this and the mixture was stirred
at 25 ◦C for 10 h. The precipitate was filtered and the filtrate
was washed three times each with 1 M KHSO4 solution, 1 M
Na2CO3 solution and brine. The solvent was evaporated and the
compound was purified by column chromatography (hexane–ethyl
acetate; 1 : 2). Yield 0.68 g (65%); 1H NMR (CDCl3) d (ppm): 1.44
(s, 9H), 1.96–2.04 (m, 3H), 2.18–2.21 (m, 1H), 2.65–2.70 (m, 1H),
2.81–2.86 (m, 1H), 3.51–3.59 (m, 2H), 3.70 (s, 3H), 3.78 (s, 3H),
3.81 (s, 2H), 4.48–4.51 (dd, 1H), 4.60–4.64 (m, 1H), 6.81–6.83 (d,
2H), 7.26–7.28 (d, 2H); 13C NMR (CDCl3) d (ppm): 25.2, 25.7,
27.7, 28.8, 29.5, 47.5, 52.3, 52.7, 55.7, 59.4, 80.3, 114.4, 130.6,
131.4, 155.7, 158.9, 170.5, 172.7; 77Se NMR (CDCl3) d (ppm):
222; ESI-MS: m/z calcd. for C22H32N2O6Se [M+Na]+ 523.1323,
found 523.0989.
Synthesis of 36. This compound was synthesized following a
similar method to that given for compound 35 by using compound
34 as the starting material. Yield 83%. 1H NMR (CDCl3) d
(ppm): 1.92–2.04 (m, 4H), 2.15–2.21 (m, 2H), 2.63–2.67 (m, 2H),
2.76–2.81 (m, 2H), 3.51–3.53 (m, 2H), 3.71–3.72 (d, 4H), 3.76 (s,
6H), 4.41–4.44 (dd, 2H), 4.64–4.66 (m, 2H); 13C NMR (CDCl3)
d (ppm): 25.7, 27.6, 29.5, 47.6, 52.7, 55.7, 59.4, 158.8, 172.7;
77Se NMR (CDCl3) d (ppm) 295; HRMS (ESI mode) calcd. for
C18H30N4O6Se2 [M+Na]+ 581.0393, found 581.0350.
Synthesis of 38. 1.35 g (3.0 mmol) Boc-L-cystine was dissolved
in 75 mL ethanol. To it NaBH4 (0.37 g, 10.0 mmol) was added. The
mixture was stirred for 15 min under nitrogen atmosphere. To it an
ethanolic solution of p-methoxy benzyl chloride (1 mL, 7.5 mmol)
was added dropwise. The reaction mixture was further stirred for
6 h. The ethanol was evaporated under reduced pressure and the
compound was dissolved in chloroform. The organic layer was
washed 2 times with KHSO4 solution. The organic layer was dried
over anhydrous Na2SO4 and compound was purified by column
chromatography (hexane–ethyl acetate; 1 : 1). Yield 1.48 g (70%);
1H NMR (CDCl3) d (ppm): 1.45 (s, 9H), 2.83–2.89 (m, 2H), 3.69
(s, 2H), 3.79 (s, 3 H), 4.50–4.51 (d, 1H), 6.82–6.85 (d, 2H), 7.20–
7.23 (d, 2H); 22.2, 29.6, 34.9, 54.5, 60.1, 80.4, 129.0, 131.1, 132.9,
146.8, 154.9, 171.8; ESI-MS: m/z calcd. for C16H23NO5S [M+Na]+
364.1195, found 363.8573.
Synthesis of 33. This compound was synthesized by following
a similar procedure to that reported for compound 15 by using
compound 31. Yield 0.18 g (95%); H NMR (CDCl3) d (ppm):
1.38 (s, 18H), 1.97–1.99 (m, 6H), 2.15–2.17 (m, 2H), 3.10–3.14 (m,
2H), 3.25–3.28 (m, 2H), 3.67 (s, 6H), 3.70–3.73 (m, 4H), 4.46–
4.47 (d, 2H), 4.71–4.72 (d, 2H); 13C NMR (CDCl3) d (ppm):
25.2, 28.7, 29.3, 32.5, 47.5, 52.7, 52.9, 59.3, 80.3, 155.7, 170.2,
172.5; 77Se NMR (CDCl3) d (ppm): 309; ESI-MS: m/z calcd. for
C28H46N4O10Se2 [M+Na]+ 781.1442, found 781.2930.
1
Synthesis of 35. Compound 33 (0.38 g, 0.5 mmol) was taken
in a round-bottom flask and 2 mL of trifluoroacetic acid (TFA)
was added. The flask was sealed and stirred for 3 h. TFA was
evaporated under reduced pressure to obtain the Boc free dipeptide
35. Yield 0.26 g (93%); 1H NMR (CDCl3) d (ppm): 1.94–2.00 (m,
6H), 2.15–2.19 (m, 2H), 2.63–2.68 (m, 2H), 2.79–2.84 (m, 2H),
3.73–3.76 (dd, 4H), 3.80 (s, 6H), 4.46–4.49 (dd, 2H), 4.58–4.60
(m, 2H); 13C NMR (CDCl3) d (ppm): 25.6, 27.7, 29.4, 47.4, 52.7,
55.6, 59.3, 158.8, 172.6; 77Se NMR (CDCl3) d (ppm): 298; HRMS
(ESI mode) calcd. for C18H30N4O6Se2 [M+Na]+ 581.0393, found
581.0291.
Synthesis of 39. This compound was synthesized following a
similar procedure to that given for compound 31 using the PMB-
1
protected L-Cys derivative (38). Yield 52%. H NMR (CDCl3) d
(ppm): 1.35 (s, 9H), 1.84–1.86 (m, 3H), 2.05–2.08 (m, 1H), 2.49–
2.52 (m, 1H), 2.68–2.73 (m, 1H), 3.39–3.42 (m, 2H), 3.57 (s, 3H),
3.65 (s, 2H), 3.67 (s, 3H), 4.37–4.40 (dd, 1H), 4.47–4.51 (m, 1H),
6.72–6.75 (d, 2H), 7.16–7.19 (d, 2H); 13C NMR (CDCl3) d (ppm):
25.2, 28.7, 29.4, 33.7, 36.1, 47.4, 51.9, 52.6, 55.6, 59.3, 80.2, 114.3,
130.3, 130.6, 155.8, 159.1, 170.4, 172.6; ESI-MS: m/z calcd. for
C22H32N2O6S [M+Na]+ 475.1879, found 475.0789.
Synthesis of 30. This compound was synthesized following a
1
method similar to the synthesis of compound 29. Yield 63%. H
NMR (CDCl3) d (ppm): 1.45 (s, 9H), 2.93–2.94 (d, 2H), 4.62–
4.64 (dd, 1H), 6.81–6.84 (dd, 2H), 7.19–7.22 (dd, 2H); 13C NMR
(CDCl3) d (ppm): 14.7, 28.8, 48.0, 55.7, 59.8, 80.5, 114.4, 130.6,
131.4, 155.7, 158.9, 171.2, 174.6; 77Se NMR (CDCl3) d (ppm) 223.
Synthesis of 40. This compound was synthesized from com-
pound 39 by using a similar method to that given for compound
15. Yield 90%. 1H NMR (CDCl3) d (ppm): 1.38 (s, 18H), 1.98–2.00
(m, 6H), 2.17–2.19 (m, 2H), 2.85–2.87 (m, 2H), 3.04–3.06 (m, 2H),
3.67 (s, 6H), 3.70–3.76 (m, 4H), 4.48–4.50 (m, 2H), 4.74–4.76 (d,
2H); 13C NMR (CDCl3) d (ppm): 25.3, 28.7, 29.4, 41.4, 47.6, 51.8,
52.7, 59.4, 80.4, 155.93, 170.4, 172.6; HRMS (ESI mode) calcd.
for C28H46N4O10S2 [M+Na]+ 685.2553, found 685.2550.
Synthesis of 32. This compound was synthesized following a
1
similar method to that given for compound 31. Yield 61%. H
NMR (CDCl3) d (ppm): 1.42 (s, 9H), 1.89–1.99 (m, 3H), 2.16–
2.19 (m, 1H), 2.61–2.66 (m, 1H), 2.75–2.80 (m, 1H), 3.49–3.52
(m, 2H), 3.69 (s, 3H), 3.74 (s, 2H), 3.75 (s, 3H), 4.39–4.42 (dd,
1H), 4.63–4.65 (m, 1H), 6.77–6.80 (d, 2H), 7.20–7.22 (d, 2H);
13C NMR (CDCl3) d (ppm): 25.1, 25.8, 27.7, 28.8, 29.6, 47.6,
52.3, 52.8, 55.7, 59.4, 80.3, 114.3, 130.6, 131.4, 155.5, 158.9, 170.3,
Synthesis of 41. This compound was synthesized from com-
pound 40 by using a similar method to that given for compound
1
35. Yield 86%. H NMR (CDCl3) d (ppm): 1.93–1.96 (m, 4H),
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1356–1365 | 1363
©

2.15–2.18 (m, 2H), 2.56–2.61 (m, 2H), 2.78–2.83 (m, 2H), 3.49–
3.52 (m, 2H), 3.66–3.67 (d, 4H), 3.76 (s, 6H), 4.46–4.49 (dd, 2H),
4.57–4.59 (m, 2H); 13C NMR (CDCl3) d (ppm): 24.9, 33.5, 35.8,
47.1, 52.3, 55.3, 59.0, 158.8, 170.1; HRMS (ESI mode) calcd. for
C18H30N4O6S2 [M+Na]+ 485.1504, found 485.1537.
and with increasing concentration of the inhibitor added to the
assay mixture. The reaction mixture was incubated for 5 min before
injection. The formation of nitro-Ang II was monitored at the
wavelength of 215 nm. The inhibition plots were obtained by using
Origin 6.1 software utilizing sigmoidal curve fitting and these plots
were used for the calculation of IC50 values.
Synthesis of peroxynitrite (PN). Peroxynitrite was synthesized
by following the literature method with minor modifications.25
A
Inhibition of nitration of BSA. For bovine serum albumin
(BSA), the nitration was performed by the addition of PN
(1.2 mM) to BSA (0.1 mM) in 0.5 M phosphate buffer of pH 7.0 at
20 ◦C. After the addition of PN, the final pH was maintained below
7.5. The reaction mixture was incubated for 30 min at 20 ◦C. The
reactions of BSA with PN were performed in the presence of
various inhibitors at 60 mM final concentration. Upon performing
the reactions, the mixture was denatured by boiling at 100 ◦C
for 5 min in the presence of sample loading dye and subjected to
polyacrylamide gel electrophoresis and western blot analyses.
solution of 30% (~8.8 M) H2O2 (5.7 mL) was diluted to 50 mL
◦
with water, chilled to about 4 C in an ice/water mixture, added
to 30 mL of 5 N NaOH and 5 mL of 0.04 M DTPA in 0.05 N
NaOH with gentle mixing, and then diluted to a total volume
of 100 mL. The concentration of H2O2 in the final solution was
0.5 M, with the pH ranging from 12.5 to 13.0. The buffered
H2O2 was stirred vigorously with an equimolar amount of isoamyl
nitrite (6.7 mL, 50 mmol) for 3–4 h at room temperature. The
reaction was monitored by withdrawing aliquots at intervals of
15 or 30 min, and assaying for peroxynitrite at 302 nm by UV-
Vis spectrophotometer. When the yield of peroxynitrite reached a
maximum, the aqueous phase was washed with 3 ¥ 100 mL volume
of dichloromethane, chloroform and hexane in a separatory
funnel to remove the contaminating isoamyl alcohol and isoamyl
nitrite. The unreacted H2O2 was removed by passing the aqueous
phase through a column filled with 25 g of granular MnO2. The
concentration of the stock solution of peroxynitrite was measured
after 500 times dilution with 0.1 N NaOH solution and then
assaying for peroxynitrite at 302 nm (e = 1670 M-1cm-1) by UV-Vis
spectrophotometric method.
Electrophoretic analysis. Gel was prepared with 10% polyacry-
lamide with 6% stacking gel. The gel was run in the running buffer
of pH 8.3 with glycine and SDS. After separating the proteins,
the gel was analyzed by western blotting. The proteins were
transferred to a PVDF membrane and the non-specific binding
sites were blocked by 5% non-fat skimmed milk in PBST (blocking
solution) for 1 h. Then the membrane was probed with rabbit
polyclonal primary antibody against nitro-tyrosine (1 : 20000
dilutions) in blocking solution for 1 h followed by incubation
with horseradish peroxidase-conjugated donkey polyclonal anti-
rabbit IgG (1 : 20000 dilutions) for another 1 h. The probed
membrane was then washed three times with blocking solution
with 0.1% Tween 20 and the immunoreactive protein was detected
by luminol-enhanced chemiluminiscence (ECL, Amersham).
ACE assay. The assay was performed in 400 mL sample vials
and an autosampler was used for sample injection. Ang I and
AngII were analyzed by reverse-phase HPLC method (Princeton
C18 column, 4.6 ¥ 150 mm, 5 mm) with isocratic elution of 50 : 50
MeOH–0.1% TFA in water. In the ACE inhibition assay, we
employed a mixture of 50 mM Ang I, 60 mM sodium chloride and
2 milliunits of ACE in 50 mM HEPES-HCl buffer at pH 8.3 with
various concentrations of the inhibitors. The reaction mixture was
incubated at 37 ◦C for 30 min prior to injection. Selenols and thiols
were freshly prepared by reducing the diselenides and disulfides by
NaBH4 prior to use and were kept under N2 atmosphere during the
assay. The decrease in the formation of Ang II with an increase
in the concentration of inhibitor was monitored at 215 nm and
the % inhibition was calculated by comparing the peak areas.
The inhibition plots were obtained by using Origin 6.1 software
utilizing sigmoidal curve fitting and these plots were used for the
calculation of IC50 values.
Acknowledgements
This study was supported by the Department of Science and
Technology (DST), New Delhi. GM acknowledges the DST for
the award of Ramanna and Swarnajayanti fellowships and BJB
thanks the Council of Scientific and Industrial Research, New
Delhi and Indian Institute of Science Bangalore for a research
fellowship.
Notes and References
1 (a) L. T. Skeggs, F. E. Doven, M. Levins, K. Lentz and J. R. Kahn, inThe
Renin-Angiotensin System, ed. J. A. Johnson, R. R. Anderson. Plenum
Press, New York 1980, pp 1–27; (b) T. Matsusaka and I. Ichikawa,
Annu. Rev. Physiol., 1997, 59, 395–412.
2 M. R. W. Ehlers, E. A. Fox, D. J. Strydom and J. F. Riordan, Proc.
Natl. Acad. Sci. U. S. A., 1989, 86, 7741–7745; M. R. W. Ehlers and
J. F. Riordan, Biochemistry, 1991, 30, 7118–7126; R. Natesh, S. L. U.
Schwager, E. D. Sturrock and K. R. Acharya, Nature, 2003, 421, 551–
554; E. D. Sturrock, R. Natesh, J. M. V. Rooyen and K. R. Acharya,
Cell. Mol. Life Sci., 2004, 61, 2677–2686.
3 L. Hansson, L. H. Lindholm, L. Niskanen, J. Lanke, T. Hedner, A.
Niklason, K. Luomanma¨ki, B. Dahlo¨f, U. de Faire, C. Mo¨rlin, B. E.
Karlberg, P. O. Wester and J.-E. Bjo¨rck, Lancet, 1999, 353, 611–616;
H. R. Corradi, I. Chitapi, B. T. Sewell, D. Georgiadis, V. Dive, E. D.
Sturrock and K. R. Acharya, Biochemistry, 2007, 46, 5473–5478.
4 (a) E. G. Erdo¨s and H. Y. T. Yang, Life Sci., 1967, 6, 569–574; (b) H. Y. T.
Yang and E. G. Erdo¨s, Nature, 1967, 215, 1402–1403; (c) H. Y. T. Yang,
E. G. Erdo¨s and Y. J. Levin, Pharmacol. Exp. Ther., 1971, 177, 291–300.
Nitration of angiotensin II. High performance liquid chro-
matography (HPLC) experiments were carried out on a Waters-
Alliance system (Milford, MA) consisting of a 2695 separation
module, a 2996 photodiode-array detector and a fraction collector.
The assays were performed in 1.8 mL sample vials and a built-
in autosampler was used for sample injection. The Alliance
HPLC System was controlled with EMPOWER software (Waters
Corporation, Milford, MA). The nitration assay of Ang II was
analyzed by reverse phase HPLC method (Princeton C18 column,
4.6 ¥ 150 mm, 5 mm) with isocratic elution of 45 : 55 MeOH–
0.1% TFA in water. In the PN-mediated nitration of Ang II, we
employed a mixture containing Ang II (20 mM) and peroxynitrite
(300 mM) in sodium phosphate buffer (100 mM) of pH 7.5, without
1364 | Org. Biomol. Chem., 2011, 9, 1356–1365
This journal is
The Royal Society of Chemistry 2011
©

5 M. A. Ondetti, B. Rubin and D. W. Cushman, Science, 1977, 196, 441–
444; D. W. Cushman, H. S. Cheung, E. F. Sabo and M. A. Ondetti,
Biochemistry, 1977, 16, 5484–5491.
6 A. A. Patchett, E. Harris, E. W. Tristram, M. J. Wyvratt, M. T. Wu,
D. Taub, E. R. Peterson, T. J. Ikeler, J. tenBroeke, L. G. Payne, D. L.
Ondeyka, E. D. Thorsett, W. J. Greenlee, N. S. Lohr, R. D. Hoffsommer,
H. Joshua, W. V. Ruyle, J. W. Rothrock, S. D. Aster, A. L. Maycock,
F. M. Robinson, R. Hirschmann, C. S. Sweet, E. H. Ulm, D. M. Gross,
T. C. Vassil and C. A. Stone, Nature, 1980, 288, 280–283.
7 J. Krapcho, C. Turk, D. W. Cushman, J. R. Powell, J. M. DeForrest,
E. R. Spitzmiller, D. S. Karanewsky, M. Duggan, G. Rovnsak, J.
Schwartz, S. Natarajan, J. D. Godfrey, D. E. Ryono, R. Neubeck, K. S.
Atwa and E. W. Petrillo, Jr, J. Med. Chem., 1988, 31, 1148–1160.
8 E. W. Petrillo, D. W. Cushman, M. E. Duggan, J. E. Karanewsky, M. A.
Ondetti, B. O’ Reilly, G. C. Rovnyak, J. Schwartz, E. R. Spitzmiller
and N.-Y. Wang, in Peptides: Structure and Function. Proceedings of
the Eighth American Peptide Symposium; ed. V. J. Hurby, D. H. Rich,
Pierce Chemical Co. Rockford, 1983 p 541.
Wattanapitayakul, S.-F. Piao, D. G. Hoyt and J. A. Bauer, Biochem.
Pharmacol., 2003, 65, 1189–1197.
13 T. Mak, A. M. Freedman, B. F. Dickens and W. Weglicki, Biochem.
Pharmacol., 1990, 40, 2169–2175; X. Liu, R. M. Engelman, J. A.
Rousou, G. A. Cordis and D. K. Das, Cardiovasc. Drugs Ther., 1992,
6, 437–443.
14 G. Mugesh, W.-W. du Mont and H. Sies, Chem. Rev., 2001, 101, 2125–
2179; S. Kumar, L. Engman, L. Valgimigli, R. Amorati, M. G. Fumo
and F. Pedulli, J. Org. Chem., 2007, 72, 6046–6055.
15 D. H. Nam, C. S. Lee and D. D. Y. Ryu, J. Pharm. Sci., 1984, 73,
1843–1844.
16 H. D. Flack, Acta Crystallogr., Sect. A: Found. Crystallogr., 1983, 39,
876–881.
17 R. B. Harris, J. T. Ohlsson and I. B. Wilson, Arch. Biochem. Biophys.,
1981, 206, 105–112.
18 M. Mutter and E. Bayer, in The Peptides: Analysis, Synthesis and
Biology, ed. J. Gross and J. Meienhofer, Academic Press, New York
1980, vol. 2, 285–382.
9 R. W. Alexander, Hypertension, 1995, 25, 155–161; C. Kunsch and
19 M. Muttenthaler and P. F. Alewood, J. Pept. Sci., 2008, 14, 1223–1239.
20 H. R. Evans, E. R. Sturrock and K. R. Acharya, Biochemistry, 2004,
43, 8718–8724.
21 R. G. Pearson, Coord. Chem. Rev., 1990, 100, 403–425; M. H. Salter
Jr., J. H. Reibenspies, S. B. Jones and R. D. Hancock, Inorg. Chem.,
2005, 44, 2791–2797.
22 S. A. B. Greenacre and H. Ischiropoloulos, Free Radical Res., 2001, 34,
541–581; K. P. Bhabak and G. Mugesh, Inorg. Chim. Acta, 2010, 363,
2812.
23 S. K. Wattanapitayakul, D. M. Weinstein, B. J. Holycross and J. A.
Bauer, FASEB J, 2000, 14, 271–278.
R. M. Medford, Circ. Res., 1999, 85, 753–766.
10 K. K. Griendling, C. A. Minieri, J. D. Ollerenshaw and R. W. Alexander,
Circ. Res., 1994, 74, 1141–1148.
11 L. A. MacMillan-Crow, J. P. Crow, J. D. Kerby, J. S. Beckman and
J. A. Thompson, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 11853–
11858; S. N. Savvides, M. Scheiwein, C. C. Bohme, G. E. Arteel, P. A.
Karplus, K. Becker and R. H. Schirmer, J. Biol. Chem., 2002, 277, 2779–
2784; K. P. Bhabak and G. Mugesh, Chem.–Eur. J., 2010, 16, 1175–
1185.
12 S. Rajagopalan, S. Kurz, T. Mu¨nzel, M. Tarpey, B. A. Freeman, K. K.
Griendling and D. G. Harrison, J. Clin. Invest., 1996, 97, 1916–1923; G.
Kojda and D. Harrison, Cardiovasc. Res., 1999, 43, 562–571; V. Turko
and F. Murad, Pharmacol. Rev., 2002, 54, 619–634; M. J. Mihm, S. K.
24 S. L. Wang, M. Wang and Q. M. Li, Chin. Chem. Lett., 2009, 20, 88–91.
25 (a) R. M. Uppu and W. A. Pryor, Anal. Biochem., 1996, 236, 242–249;
(b) R. M. Uppu, Anal. Biochem., 2006, 354, 165–168.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 1356–1365 | 1365
©