Effect of alkyl group on transnitrosation of N-nitrosothiazolidine thiocarboxamides

Article abstract of DOI:10.1016/j.bmc.2015.09.008

S-Nitrosoglutathione (GSNO) relaxes vascular smooth muscles, prevents platelet aggregation, and acts as a potential in vivo nitric oxide donor. 3-Nitroso-1,3-thiazolidine-4-thiocarboxamide (1), a N-nitrosothio-proline analogue, exhibited a high GSNO formation activity. In this study, two compounds (2 and 3) based on compound 1 were newly synthesized by introducing either one or two methyl groups onto a nitrogen atom on the thioamide substituent in 1. The pseudo-first-order rate constants (k_obs) for the GSNO formation for the reaction between the compound and glutathione followed the order 1>2=3. Thus, the introduction of a methyl group(s) onto the thioamide group led to a decrease in the transnitrosation activity. On the basis of density functional theoretical calculations, the transnitrosation for the N-nitrosothiazolidine thiocarboxamides was proposed to proceed via a bridged intermediate pathway. Specifically, the protonated compound 1 forms a bridged structure between the nitrogen atom in the nitroso group and two sulfur atoms - one in the ring and the other in the substituent. The bridged intermediate gives rise to a second intermediate in which the nitroso group is bonded to the sulfur atom in the thioamide group. Finally, the nitroso group is transferred to GSH to form GSNO.

Full text of DOI:10.1016/j.bmc.2015.09.008

Bioorganic & Medicinal Chemistry 23 (2015) 6733–6739
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effect of alkyl group on transnitrosation of N-nitrosothiazolidine
thiocarboxamides
a
b
c
Keiko Inami a,b, , Yuta Ono , Sonoe Kondo , Ikuo Nakanishi , Kei Ohkubo d,e, Shunichi Fukuzumi d,e,f
,
⇑
Masataka Mochizuki a,b
a Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki 2641, Noda-shi, Chiba 278-8510, Japan
b Kyoritsu University of Pharmacy, Tokyo, Japan
c Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of Radiological
Sciences (NIRS), Inage-ku, Chiba 263-8555, Japan
d Department of Material and Life Science, Graduate School of Engineering, Osaka University, ALCA and SENTAN, Japan Science and Technology Agency (JST), Suita, Osaka
565-0871, Japan
e Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, Republic of Korea
f Faculty of Science and Engineering, Meijo University, ALCA and SENTAN, Japan Science and Technology Agency (JST), Nagoya, Aichi 468-0073, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2015
Revised 24 August 2015
Accepted 2 September 2015
Available online 5 September 2015
S-Nitrosoglutathione (GSNO) relaxes vascular smooth muscles, prevents platelet aggregation, and acts as
a potential in vivo nitric oxide donor. 3-Nitroso-1,3-thiazolidine-4-thiocarboxamide (1), a N-nitrosothio-
proline analogue, exhibited a high GSNO formation activity. In this study, two compounds (2 and 3) based
on compound 1 were newly synthesized by introducing either one or two methyl groups onto a nitrogen
atom on the thioamide substituent in 1. The pseudo-first-order rate constants (kobs) for the GSNO forma-
tion for the reaction between the compound and glutathione followed the order 1 > 2;3. Thus, the intro-
duction of a methyl group(s) onto the thioamide group led to a decrease in the transnitrosation activity.
On the basis of density functional theoretical calculations, the transnitrosation for the N-nitrosothiazo-
lidine thiocarboxamides was proposed to proceed via a bridged intermediate pathway. Specifically, the
protonated compound 1 forms a bridged structure between the nitrogen atom in the nitroso group
and two sulfur atoms—one in the ring and the other in the substituent. The bridged intermediate gives
rise to a second intermediate in which the nitroso group is bonded to the sulfur atom in the thioamide
group. Finally, the nitroso group is transferred to GSH to form GSNO.
Keywords:
S-Nitrosoglutathione
Transnitrosation
N-Nitrosothioproline
1,3-Thiazolidine
Thiocarboxamide
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
stability in aqueous solutions, and it readily degrades upon
exposure to light.10
Nitric oxide (NO) is important for many biological events,
specifically in signalling pathways, immune responses, and vasodi-
lation.1 S-Nitrosothiols (RSNOs), such as S-nitrosoglutathione
(GSNO), S-nitrosocysteine, and S-nitrosoalbumin, represent a cir-
culating endogenous reservoir of NO and are potential donors of
NO.2–4 RSNOs have anti-platelet properties, a theoretical role in
treating asthma, and potential for treating infectious diseases.
Thus, RSNOs have been studied as NO donors in therapeutic
drugs.5–9
N-Nitrosamines have been reported to transfer a nitrosonium
ion (NO+) to other amines to form the corresponding N-nitroso
derivatives.12–15 Hence, as NO donors in the transnitrosation,
N-nitrosamines can transnitrosate to a sulfur atom in proteins to
form S-nitrosothiols. Ohwada et al. demonstrated that some
bicyclic nitrosamines can generate GSNO without releasing NO.16
We have previously focused on the GSNO formation via the
transnitrosation of monocyclic N-nitrosamines. Although many
N-nitrosodialkylamines are potent carcinogens, some N-nitrosamines,
such as N-nitrosoproline and N-nitrosothioproline, are known to be
non-mutagenic and non-carcinogenic.17,18
GSNO is one of the most abundant S-nitrosothiols present in the
body. It plays an important role in many important physiological
functions of NO.10 Specifically, GSNO has been tested as
a
We have recently reported that the non-carcinogenic
therapeutic agent for clinical use.5,11 However, GSNO has limited
N-nitrosothioproline analogues transfer a nitroso group to
glutathione (GSH) to form GSNO under acidic conditions.19 The
reaction rate of GSNO formation significantly increases when a
thioamide group instead of a carboxyl group is introduced onto
⇑
Corresponding author. Tel./fax: +81 4 7121 4436.
E-mail address: inami@rs.noda.tus.ac.jp (K. Inami).
http://dx.doi.org/10.1016/j.bmc.2015.09.008
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 6733–6739
Figure 1. Structure of the alicyclic N-nitrosamines synthesized in this study.
the N-nitrosothioproline. N-Nitrosothioproline thiocarboxamide is
a 3-nitroso-1,3-thiazolidine-4-thiocarboxamide (1) with a sulfur
atom in its substituent and in its ring. To increase the rate of GSNO
formation,
a series of N-nitrosothiazolidine thiocarboxamides
(1–3) were synthesized (Fig. 1). The transnitrosation mechanism
for N-nitrosothiazolidine thiocarboxamide 1 was proposed on the
basis of the computational calculations.
Figure 2. Formation of GSNO from the reaction of N-nitrosamines (1–3) with GSH.
The reaction conditions were as follows: N-nitrosamine 1 (N), 2 (j), or 3 (d),
0.45 mM; GSH, 5.0 mM; pH 1.5; 37 °C.
2. Results
2.1. Chemistry
To increase the nucleophilicity of the sulfur atom in the thioa-
mide group in 1, one or two electron-donating methyl groups were
introduced to the nitrogen atom on the thioamide group. We
designed a N-methylthioamide-substituted compound (2) and a
N,N-dimethyl-thioamide-substituted compound (3).
Table 1
Reaction rate constant (kobs) and GSNO yield in transnitrosation
GSNOc N-Nitrosamine
GSNO
yieldc
(%)
b
N-
kobs
Nitrosamine
consumptionc
(ꢁ10ꢀ7/s)
(mM)
(mM)
1a
2a
3a
1301
464
405
0.43
0.38
0.38
0.45
0.45
0.45
95
85
84
The procedures for synthesizing the compounds (2 and 3) are
shown in Scheme 1. Methyl 1,3-thiazolidine-4-carboxylate was
prepared via a reaction between thioproline and thionyl chloride
in methanol. The methyl 1,3-thiazolidine-4-carboxylate solid was
directly added to the methylamine-containing aqueous solution
to obtain N-methyl-1,3-thiazolidine-4-carboxamide. The prepara-
tion of N,N-dimethyl-1,3-thiazolidine-4-carboxamide was per-
formed in the same manner using dimethylamine. The nitrogen
atom in the ring was protected with a tert-butoxycarboxyl (Boc)
group. Under ultrasonication, carboxamides in the Boc compounds
were directly thiolated with diphosphorous pentasulfide to pro-
vide the thioamides.20 The Boc group was then removed under
acidic conditions and subsequently nitrosated with sodium nitrite
to afford the desired compound.
a
The reaction conditions are as follows: N-nitrosamine, 0.45 mM; GSH, 5.0 mM;
pH 1.5; 37 °C; in the dark.
b
The kobs for GSNO formation was calculated from the slope in ln{[GSNO1]/
([GSNO1] ꢀ [GSNO])} versus time, where GSNO and GSNO1 refer to the GSNO
concentration at the time and the final concentration (0.45 mM), respectively. The
kobs were determined by the method of the least-squares.
c
Data were acquired after 24 h.
relationship between the GSNO concentration and the reaction
time (Fig. 2). The transnitrosation results are summarised in
Table 1. The kobs values followed the order 1 > 2;3 (Table 1).
The GSNO yields after 24 h of reaction time were calculated by
dividing the [GSNO formation] by the [N-nitrosamine consump-
tion] (Table 1). The GSNO yields were 95% for 1, 85% for 2, and
84% for 3. These results indicate that the transnitrosation to GSH
proceeded selectively without decomposition of the compound.
Compound
1 was synthesized according to the method
described in the literature19 by thiolating 1,3-thiazolidine-4-car-
boxamide using ultrasonication. However, the product yield of
1,3-thiazolidine-4-thio-carboxamide was very low (26%). Then, 1
was prepared after the Boc group was introduced using the same
method used for 2 and 3 (Scheme 1).
2.3. The pH-rate profile for GSNO formation
The logarithms of kobs for GSNO formation in the reaction of 1
with GSH were linearly correlated with the oxonium-ion concentra-
tion. The pH-rate (logkobs) profile gave a slope of ꢀ1.32 0.43
(Fig. 3). Thus, the transnitrosation proceeded via a specific acid
catalysis and the protonated 1 was involved in the rate-determining
step.21
2.2. Transnitrosation activity evaluation based on GSNO
formation
The transnitrosation activity of the compounds was evaluated
in terms of their capacity to form GSNO. The pseudo-first-order
rate constants (kobs) were determined using the slope of the linear
Scheme 1. Synthesis of N-nitrosothiazolidine thiocarboxamides.

K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 6733–6739
6735
Figure 4. DFT-optimised structure of the protonated 1b (B3LYP/6-311G(d) level).
Figure 3. The pH-rate profile for the formation of GSNO (kobs) in 1 and GSH. The
reaction conditions were as follows: N-nitrosamine, 0.45 mM; GSH, 5.0 mM; pH 1.4
(d), 1.7 (N), 2.0 (j), 2.2 (s), 2.8 (4), or 3.3 (h); 37 °C.
Interestingly, the optimised structure of the protonated 1 (1b)
was a bridged form between the nitrogen atom in the nitroso
group and two sulfur atoms—one in the ring and the other on
the substituent (Fig. 4).
A transition state (TS) is expected to be similar to the bridged
intermediate. The transition state is proposed to include a nitroso
group bonded with three intramolecular atoms (two sulfur atoms
and the nitrogen atom that attaches to the nitroso group). The TS
was validated by an intrinsic reaction coordinate (IRC) calculation.
The activation energy in gas phase has been determined and con-
firmed by the IRC calculations. The energy diagrams including TS
energies for the main reaction course are given in Figure 5.
The stability of the intermediates c and d is compared, and the
energies of the intermediate c is lower than that of d (Fig. 5). The
data indicates that the path A for 1 is preferred.
2.4.
Theoretical
calculations
of
N-nitrosothiazolidine
thiocarboxamides
Density functional theoretical (DFT) calculations were per-
formed to find out the thermodynamically most favored pathway
of the transnitrosation mechanism and to gain insight into some
key intermediate or transition states involved in the reaction. The
geometries of the species (1) in the transnitrosation reaction were
optimised in the computational calculationsusing the double hybrid
density functional B3LYP and the basis set 6-311G(d), as imple-
mented in Gaussian09. The single-point energy was subsequently
calculated.22 The free-energy difference (
DG) was obtained by sub-
3. Discussion
tracting the single-point energy of a former form from that of follow-
ing intermediate in the stepwise reaction.
Because GSNO is a potential therapeutic agent,5,10 we focused
on the GSNO formation from N-nitrosamines by transnitrosation.
Non-mutagenic N-nitrosothioproline was used as the basic struc-
ture for developing new transnitrosating N-nitrosamines.19 Com-
pounds 2 and 3, which were introduced with one or two methyl
groups, respectively, on the thioamide nitrogen in 1, were newly
synthesized, and N-nitrosothiazolidine thiocarboxamides could
transnitrosate to GSH to form GSNO. Contrary to the expectation
that the introduction of the electron-donating methyl group(s) into
the thioamide increases their nucleophilicity and is followed by an
increase in the kobs for the GSNO formation, compounds 2 and 3
decreased the kobs for the GSNO formation.
To elucidate the transnitrosation mechanism for 1, the effect of
pH on kobs was investigated and a computational study was per-
formed. The pH-rate profile with a slope of ꢀ1.32 0.43 indicated
that protonated form of the N-nitrosamine was appeared in the
rate-determining step.21 The pH-dependency of GSNO formation
was consistent with the formation step of the bridged 1b in TS as
shown in Figure 5.
In previous studies, N-nitrosoproline acts as an NO donor in the
presence of thiourea, whereas 1 containing sulfur atoms both in
the ring and as a substituent exhibited high activity for GSNO for-
mation without thiourea.19 The data indicate that the intramolec-
ular sulfur atoms in the N-nitrosamines were involved in the
transnitrosation, and we then proposed a presumable transnitrosa-
tion pathway for N-nitrosamine 1 (Scheme 2).
Protonation of nitrogen atom attaching the nitroso group and
protonation of oxygen atom are present at an equilibrium process.
The initial protonation site is the nitrogen in the N-nitrosothiazo-
lidine ring which was determined by the comparison of the ener-
gies of protonated isomers calculated by the DFT. The energy of
the protonation at the N-nitrosothiazolidine ring is more stable
than that of protonation at the oxygen atom of NO group
(+9.6 kcal molꢀ1) and amine moiety of thiocarboxamide group
(+19.3 kcal molꢀ1). The nitroso group in N-protonated 1 is then
transferred to a sulfur atom of one of the thioamide groups or to
the sulfur atom of the ring. The two routes are presented for paths
A and B, which lead to c and d, respectively. Then, the intermediate
transfers the nitroso group to GSH to form GSNO.
In the computational studies of the protonated 1, the optimised
structure was a bridged form between the nitrogen atom in a
Scheme 2. Proposed intermediates in the transnitrosation mechanism for N-nitrosothiazolidine thiocarboxamide 1 under acidic conditions.

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K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 6733–6739
Figure 5. Potential energies of transnitrosation between N-nitrosamine 1 and GSH at the B3LYP/6-311(g) level.
Scheme 3. Transnitrosation mechanism for N-nitrosothiazolidine thiocarboxamide 1 under acidic conditions.
nitroso group and two sulfur atoms, one in the ring and the other
in the substituent (Fig. 4). The formation of the bridged structure is
strongly supported, because the GSNO formation of the N-ni-
trosothiazolidine thiocarboxamides is significantly enhanced by
introducing two sulfur atoms into the molecule. A few reports indi-
cates that the SAN(@O)AS structure is formed by transnitrosation
with S-nitrosothiol and another thiol.23–25 The reported data were
similar to our data in that an intramolecular SAN(@O)AS bridged
intermediate was formed. Although the bridged structure is highly
distorted, it is a bicyclic structure with a five-membered ring and a
six-membered ring.
and finally raises the GSNO formation. We considered a reason to
decrease the kobs in 2 and 3, the bridged form and/or the S
(thioamide)ANO form was unstable in 2 and 3. The instability in
2 and 3 structures is due to the steric hindrance between the ring
and the methyl group. Thus, we assumed that the electronic effect
of the methyl group in their intermediates increased the stability,
whereas the steric hindrance between the methyl group and the
ring led a decrease the stability of their intermediates of 2 and 3.
N-Nitrosamine 1 has the highest GSNO yield (95%) even though
the intermediate 1b was stable. Therefore, we thought that a rate-
determining step for 2 and 3 was different from that for 1 and a
formation step of S(thioamide)ANO structure was the rate-deter-
mining step. In the reaction mixture, compounds 2 and 3 could
exist in their bridged forms, resulting in lower GSNO yields (85%
for 2 and 84% for 3).
According to the computations, the intermediate c for path A or
d for path B was assumed to exist, and path A was preferred
(Fig. 5). The computational data was in good agreement with reso-
nance stability of C in path A.
Unexpectedly, the kobs for GSNO formation of 2 and 3 were
lower than that of 1. Introduction of methyl group into thioamide
group caused decrease the GSNO formation. Generally, the intro-
duction of a methyl group onto a thioamide group enhances the
partial N@N double-bond character via the electron-donating abil-
ity of the methyl group, increases the stability of intermediates,
Our experimental and theoretical evidence clearly confirmed
the mechanism in Scheme 3 for the transnitrosation of the
N-nitrosamine 1.
In summary, the transnitrosation step was characterised. It
begins with the protonation of the N-nitrosamine and is followed
by the formation of a stable intermediate. The transition state is

K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 6733–6739
6737
similar to the bridged form. The transition state is confirmed to
include a nitroso group bonded with three intramolecular atoms
(two sulfur atoms and the nitrogen atom that attaches to the
nitroso group). The bridged intermediate then gives rise to a sec-
ond intermediate in which the nitroso group is bonded to a sulfur
atom in the thioamide group. Finally, the nitroso group is trans-
ferred to GSH to form GSNO.
1,3-thiazolidine-4-carboxylate hydrochloride in an ice bath and
stirred at room temperature until the starting material disappeared
in the TLC chromatograms. The reaction mixture was extracted with
CHCl3, dried over anhydrous sodium sulfate, filtered, and concen-
trated under reduced pressure. The crude product was purified on
a silica-gel column to afford the desired product as a single
compound.
N-Methyl-1,3-thiazolidine-4-carboxamide; 1H NMR (400 MHz,
CDCl3) d 4.51 (d, J = 1.2, 4.1 Hz, 1H, S-CH2-N), 4.07 (dq, J = 1.5,
11.2 Hz, 1H, CONHCH3), 4.02 (d, J = 5.6 Hz, 1H, S-CH2-N), 3.96 (dd,
J = 1.7, 6.6 Hz, 1H, N-CH-CO), 3.31 (dd, J = 1.7, 12.0 Hz, 1H, CH-CH2-S),
3.19 (ddd, J = 1.7, 1.9, 6.6 Hz), 2.85 (d, J = 1.2 Hz, 3H, NHCH3).
4. Conclusion
GSNO is a potential therapeutic agent; however, its poor sta-
bility is problematic. Therefore, we focused on a series of N-ni-
trosothiazolidine thiocarboxamides for GSNO formation. Three
newly synthesized N-nitrosothiazolidine thiocarboxamides (1–3)
exhibited transnitrosation activity under acidic conditions. The
activity of the GSNO formation for 1 was higher than that for 2
and 3. In the transnitrosation mechanism, a protonated structure
is formed as a transition state. An intramolecular SAN(@O)AS
bridge structure was characterised as an intermediate based on
the result of DFT calculations. Thus, we propose that the transni-
trosation activity of N-nitrosothiazolidine thiocarboxamides can
enhance by introduction of a sulfur-containing substituent, which
has an electron-donating effect and less steric hindrance. The
studied compounds are therapeutic candidates for GSNO-related
diseases.
N,N-Dimethyl-1,3-thiazolidine-4-carboxamide;
1H
NMR
(400 MHz, CDCl3) d 4.46 (d, J = 9.5 Hz, 1H, S-CH2-N), 4.11 (d,
J = 9.5 Hz, 1H, S-CH2-N), 3.89 (dd, J = 6.6, 9.1 Hz, 1H, N-CH-CO),
3.20 (dd, J = 6.7, 10.0 Hz, 1H, CH-CH2-S), 3.11 (s, 3H, N-CH3), 3.01
(s, 3H, N-CH3), 2.68 (t, J = 10.0 Hz, 1H, CH-CH2-S).
5.3.2. Preparation of 3-tert-butoxycarbonyl-1,3-thiazolidine-4-
carboxamides
A solution of di-tert-butyl dicarbonate in acetone was added to
a solution of the corresponding 1,3-thiazolidine-4-carboxamides in
acetone under nitrogen atmosphere. The reaction mixture was stir-
red at room temperature until the starting material disappeared in
the TLC chromatograms. After the reaction mixture was concen-
trated under reduced pressure, the crude product was purified on
a silica-gel column to afford the single desired product.
5. Experimental
5.1. Chemicals
3-tert-Butoxycarbonyl-1,3-thiazolidine-4-carboxamide;
1H
NMR (400 MHz, methanol-d4) d 5.45 (br, 1H, N-CH-CO), 4.63 (br,
1H, S-CH2-N), 4.44 (br, 1H, S-CH2-N), 3.48 (br, 1H, C-CH2-S), 3.13
(br, 1H, C-CH2-S), 1.46 (s, 9H, tert-butyl).
3-tert-Butoxycarbonyl-N-methyl-1,3-thiazolidine-4-carboxam-
ide; 1H-NMR (400 MHz, CDCl3) d 4.65 (d, br, J = 9.5 Hz, 2H, N-CH-
CO, S-CH2-N), 4.35 (br, 1H, S-CH2-N), 3.42 (br, 1H, C-CH2-S), 3.20
(br, 1H, C-CH2-S), 2.85 (d, J = 4.6 Hz, 3H, N-CH3), 1.48 (s, 9H, tert-
butyl).
3-tert-Butoxycarbonyl-N,N-dimethyl-1,3-thiazolidine-4-carbox-
amide; 1H NMR (400 MHz, methanol-d4) d 5.15 (br, 1H, N-CH-CO),
4.89 (br, 1H, S-CH2-N), 4.75 (br, 1H, S-CH2-N), 4.52 (d, J = 8.8 Hz,
1H, C-CH2-S), 3.31 (dd, J = 3.7, 11.2 Hz, 1H, C-CH2-S), 3.12 (s, 3H,
N-CH3), 2.99 (s, 3H, N-CH3), 1.51 (s, 9H, tert-butyl).
L-Thioproline was obtained from Tokyo Kasei Kogyo Co., Ltd
(Tokyo, Japan). Diphosphorous pentasulfide was purchased from
Sigma–Aldrich Co., Inc. (St. Louis, MO, USA). Diethylenetriamine-
N,N,N0,N00,N00-pentaacetic acid (DTPA) was acquired from Dojindo
Laboratories (Kumamoto, Japan). Unless otherwise noted, chemi-
cals were purchased from Wako Pure Chemical Industries (Osaka,
Japan) at the highest available purity and were used without
further purification.
5.2. General procedure
The reaction progress was monitored using thin-layer chro-
matography (TLC) on silica gel 60 F254 (0.25 mm, Merck) and alu-
minium oxide 150 F254 neutral plates (Merck). Column
chromatography was performed using silica gel 60 (0.01–
0.063 mm, Merck) or aluminium oxide 90 (0.063–0.200 mm, Mer-
ck) active neutral solid phases. Melting points were determined
using a Yanaco micro-melting-point apparatus without correction.
HPLC was performed using a Shimadzu LC system [SPD-20A UV
5.3.3. Preparation of 3-tert-butoxycarbonyl-1,3-thiazolidine-4-
thiocarboxamides
Diphosphorous pentasulfide (1 equiv) was added to a solution
of the corresponding 3-tert-butoxycarbonyl-1,3-thiazolidine-4-
carboxamide in THF.20 The reaction mixture was ultrasonicated
below 25 °C until the starting material disappeared in the TLC
chromatograms. After the addition of acetone, the reaction mixture
was filtered and concentrated under reduced pressure. The crude
product was purified on silica-gel columns to afford the single
desired product.
3-tert-Butoxycarbonyl-1,3-thiazolidine-4-thiocarboxamide;
white needle crystals (chloroform); yield 47%; mp 174.0–
176.0 °C (decomp.); 1H NMR (400 MHz, CDCl3) d 7.76 (br, 1H,
CSNH2), 5.03 (t, J = 5.5 Hz, 1H, N-CH-CS), 4.68 (d, J = 9.5 Hz, 1H,
S-CH2-N), 4.48 (br, 1H, S-CH2-N), 3.45 (br, 2H, C-CH2-S), 1.48 (s,
9H, tert-butyl); 13C NMR (100 MHz, CDCl3) d 206.41 (C@S),
154.01 (C@O), 82.36 (C(CH3)3), 69.41 (N-CH-CS), 50.32 (S-CH2-N),
37.94 (C-CH2-S), 28.20 (C(CH3)3); HRMS (EI) 248.0650 (calcd for
C9H16N2O2S2 248.0653).
spectrometric detector, Shiseido Capcell Pak column (5 lm,
250 ꢁ 4.6 mm)]. The NMR spectra were recorded with a JEOL
JNM-LA400 spectrometer (Tokyo, Japan). The chemical shifts are
expressed in ppm and were shifted downfield from TMS. The
high-resolution mass spectra were collected using a JEOL JMS-
SX102A mass spectrometer. GSNO was synthesised using the Hart
method [kmax (H2O): 335 nm (
e = 918) (lit. kmax: 336 nm (e
= 922)].26
5.3. General procedure for alicyclic N-nitrosamines
5.3.1. Preparation of N-alkyl-1,3-thiazolidine-4-carboxamide by
alkylamines and 1,3-thiazolidine-4-carboxamide
3-tert-Butoxycarbonyl-N-methyl-1,3-thiazolidine-4-thiocar-
boxamide; white needle crystals (chloroform and hexane); yield
81%; mp 149.0–152.0 °C; 1H NMR (400 MHz, CDCl3) d 8.06 (br,
1H, NH), 5.07 (t, J = 5.3 Hz, 1H, N-CH-CS), 4.65 (d, J = 9.8 Hz, 1H,
S-CH2-N), 4.45 (d, J = 8.8 Hz, 1H, S-CH2-N), 3.44 (br, 2H, C-CH2-S),
Methyl 1,3-thiazolidine-4-carboxylate hydrochloride and
1,3-thiazolidine-4-carboxamide were prepared according to
a
previously reported procedure.19 An excess of the corresponding
N-alkylamine aqueous solution was added dropwise to methyl

6738
K. Inami et al. / Bioorg. Med. Chem. 23 (2015) 6733–6739
3.22 (d, J = 4.9 Hz, 3H, N-CH3), 1.46 (s, 9H, tert-butyl); 13C NMR
(100 MHz, CDCl3) d 201.59 (C@S), 154.09 (C@O), 82.22 (C(CH3)3),
70.16 (N-CH-CS), 50.46 (S-CH2-N), 37.73 (C-CH2-S), 32.66 (NCH3),
28.20 (C(CH3)3); HRMS (EI) 262.0813 (calcd for C10H18N2O2S2
262.0810).
3-tert-Butoxycarbonyl-N,N-dimethyl-1,3-thiazolidine-4-thio-
carboxamide; white needle crystals (chloroform and hexane); yield
49%; mp 56.5–58.5 °C; 1H NMR (400 MHz, CDCl3) d 5.32 (t,
J = 6.8 Hz, 1H, N-CH-CS), 5.13 (br, 1H, N-CH-CS), 4.85 (br, 1H,
S-CH2-N), 4.74 (br, 2H, S-CH2-N), 3.51 (s, 6H, N-CH3), 3.45 (s, 3H,
N-CH3), 3.37 (d, J = 7.6 Hz, 1H, C-CH2-S), 3.35 (d, J = 7.6 Hz, 1H,
C-CH2-S), 3.23 (br, 2H, C-CH2-S), 1.47 (s, 9H, tert-butyl), 1.40 (s,
9H, tert-butyl); 13C NMR (100 MHz, CDCl3) d 203.64, 202.67
(C@S), 153.17, 152.41 (C@O), 80.95 (C(CH3)3), 64.00, 50.59
(N-CH-CS), 50.20, 45.21 (S-CH2-N), 41.62, 41.39 (NCH3), 37.34,
36.18 (C-CH2-S), 28.36 (C(CH3)3); HRMS (EI) 276.0964 (calcd for
C11H20N2O2S2 276.0966).
5.4. Reaction of 1–3 with GSH
The GSH (50 mM) and DTPA (24 lM) were dissolved in 0.1 M
sodium phosphate buffer (pH 7.4). The N-nitroso compound
(4.5 mM) was dissolved in acetonitrile. Aliquots of GSH (300 L,
5.0 mM) and DTPA (150 L, 1.2 M) were mixed, and the pH of
l
l
l
the resulting mixture was adjusted to 1.5 with 0.1 M HCl for a total
volume of 2.7 mL. The reaction was initiated by adding a solution
of the compound (300 lL, 0.45 mM) at 37 °C. The aliquots were
collected at specified intervals, and the GSNO yield was deter-
mined using HPLC with a Shiseido UG80 (5
column with methanol/0.05% trifluoroacetic acid (5:95) as the
eluent at 1.0 mL/min at 335 nm.
l
m, 150 ꢁ 4.6 mm2)
5.5. Kinetic analysis of the transnitrosation reactions
The reaction rate constants were determined by measuring the
GSNO formation at 37 °C. The pseudo-first-order rates (kobs) were
determined using graphical analysis of the initial linear portion
of the curve obtained from a plot of ln{[GSNO ]/([GSNO ] ꢀ
5.3.4. Preparation of 3-nitroso-1,3-thiazolidine-4-
thiocarboxamides (1, 2, 3)
1
1
The corresponding 3-tert-butoxycarbonyl-1,3-thiazolidine-4-
thiocarboxamide was dissolved in methanol and was acidified to
pH 3 by the addition of 1 M HCl. The reaction mixture was stirred at
60 °C until the starting material disappeared in the TLC chro-
matograms. After the solution cooled to room temperature, sodium
nitrite (1 equiv) was added and the resulting mixture was stirred
for 1 h until the deprotected compound disappeared. The reaction
mixture was extracted with CH2Cl2, and the combined organic layers
were washed with water, dried over anhydrous sodium sulfate, fil-
tered, and concentrated under reduced pressure. The crude product
was purified on silica-gel columns to afford the single desired
compound.
[GSNO])} versus t. The theoretical value for GSNO1 was the initial
concentration of the N-nitroso compound. All rate values were
calculated using the least-squares method, and each experiment
was conducted in three separate trials.
The quantity of N-nitroso that remained was quantified simul-
taneously with the quantity of GSNO formed. The yield of S-nitro-
sation from N-nitrosamines after the 24 h reaction time was
calculated by dividing the percentage of GSNO that was formed
by the percentage of the N-nitroso compound.
5.6. pH-rate profile
1; white needle crystals (chloroform); yield 13%; mp 121.5–
122.0 °C; 1H NMR (400 MHz, DMSO-d6) d 10.07 (br, 1H, E-CSNH2),
9.79 (br, 1H, Z-CSNH2), 9.47 (br, 1H, E-CSNH2), 9.39 (br, 1H,
Z-CSNH2), 5.88 (dd, J = 3.7, 7.3 Hz, 1H, E-N-CH-CS), 5.86 (d,
J = 11.0 Hz, 1H, Z-S-CH2-N), 5.26 (d, J = 10.3 Hz, 1H, Z-S-CH2-N),
4.99 (t, J = 7.9 Hz, 1H, Z-N-CH-CS), 4.87 (d, J = 12.2 Hz, 1H,
E-S-CH2-N), 4.56 (d, J = 12.2 Hz, 1H, E-S-CH2-N), 3.63 (dd, 1H,
E-CH-CH2-S), 3.58 (dd, J = 7.9, 12.2 Hz, 1H, Z-CH-CH2-S), 3.33 (dd,
1H, E-CH-CH2-S), 3.20 (dd, J = 7.9, 11.6 Hz, 1H, Z-CH-CH2-S).
2; white needle crystals (chloroform and hexane); yield 7%; mp
149.0–149.5 °C; 1H NMR (400 MHz, CDCl3) d 6.00 (dd, J = 1.7,
4.9 Hz, 1H, E-N-CH-CS), 5.75 (d, J = 10.0 Hz, 1H, Z-S-CH2-N), 5.22
(t, J = 3.7 Hz, 1H, Z-N-CH-CS), 5.19 (s, 1H, E-S-CH2-N), 4.52 (d,
J = 12.2 Hz, 1H, E-S-CH2-N), 4.02 (m, 1H, E-CH-CH2-S), 4.00 (m,
1H, Z-CH-CH2-S), 3.54 (dd, J = 6.8, 6.6 Hz, 1H, E-C-CH2-S), 3.42
(dd, J = 7.8, 7.8 Hz, 1H, Z-C-CH2-S), 3.25 (d, J = 4.9 Hz, 3H, E-N-
CH3), 3.16 (d, J = 4.9 Hz, Z-N-CH3); 13C NMR (100 MHz, CDCl3) d
202.4 (E-CSNH2), 200.6 (Z-CSNH2), 70.7 (E-N-CH-CS), 66.2 (Z-N-
CH-CS), 53.5 (Z-S-CH2-N), 47.8 (E-S-CH2-N), 35.2 (E-S-CH2-C),
34.8 (Z-S-CH2-C); HRMS (EI) 191.0188 (calcd for C5H9N3OS2
191.2745).
The GSH and DTPA solutions were adjusted to the desired pH.
The reaction mixtures were prepared and analysed as previously
described.
5.7. Theoretical calculations
DFT calculations were performed using Gaussian 09.55. The
calculations were performed on a 32-processor QuantumCube at
the B3LYP/6-311+G(d) level of theory by applying the polarizable
continuum model (PCM) using the integral equation formalism
variant (IEF-PCM).22
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.09.008.
References and notes
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3; a tan-yellow crystals (chloroform and hexane); yield 62%; mp
97.0–97.5 °C; 1H NMR (400 MHz, CDCl3) d 6.03 (t, J = 7.1 Hz, 1H, Z-
S-CH2-N), 5.78 (d, J = 4.9 Hz, 1H, E-S-CH2-N), 5.48 (d, J = 1.0 Hz, 1H,
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