1H and 13C NMR analysis of 2-acetamido-3-mercapto-3- methyl-N-aryl-butanamides and 2-acetamido-3-methyl-3-nitrososulfanyl-N-aryl- butanamide derivatives

Article abstract of DOI:10.1002/mrc.3944

The complete assignment of the ¹H and ¹³C NMR spectra of various 2-acetamido-3-mercapto-3-methyl-N-aryl-butanamides and 2-acetamide-3-methyl-3-nitrososulfanyl-N-aryl-butanamides with p-methoxy, o-chloro and m-chloro substituents is r

Full text of DOI:10.1002/mrc.3944

MRC Letters
Received: 6 October 2012
Revised: 9 February 2013
Accepted: 11 February 2013
Published online in Wiley Online Library: 11 March 2013
(wileyonlinelibrary.com) DOI 10.1002/mrc.3944
13
1H and C NMR analysis of 2-acetamido-3-
mercapto-3-methyl-N-aryl-butanamides and
2-acetamido-3-methyl-3-nitrososulfanyl-
N-aryl-butanamide derivatives
Rafael Germano Santana,a Derisvaldo Rosa Paiva,b
Roberto da Silva Gomesc and Adriana Karla C. A. Reisb*
The complete assignment of the 1H and 13C NMR spectra of various 2-acetamido-3-mercapto-3-methyl-N-aryl-butanamides
and 2-acetamide-3-methyl-3-nitrososulfanyl-N-aryl-butanamides with p-methoxy, o-chloro and m-chloro substituents is
reported. Copyright © 2013 John Wiley & Sons, Ltd.
1
Keywords: NMR; H NMR; 13C NMR; N-aryl-butanamides; S-nitrosothiol
anhydride (1 : 3) at room temperature.[11] This compound was
utilized to produce the 2-acetamide-3-methyl-3-mercapto-N-
Introduction
It is well-established that nitric oxide (NO), a gaseous free radical,
regulates vascular tone and participates in cellular signaling
events.[1] S-nitrosothiols have been implicated as major trans-
ducers of NO bioactivity, acting as both potent vasodilators and
inhibitors of platelet aggregation. However, the mechanisms by
which these reactive nitrogen species affect cellular functions
are not fully established.[2–4]
aryl-butanamides through a reaction with aniline and substituted
anilines (o-Cl, m-Cl, and p-MeO) in chloroform.
The S-nitrosothiol derivatives were produced by reacting
2-acetamide-3-methyl-3-mercapto-N-aryl-butanamides in acetone
and tert-butyl-nitrite (1: 2).[9]
Covalent attachment of NO to protein or to low molecular
weight thiols (S-nitrosylation) is generally used in cells as a
reversible signal.[1] Through S-nitrosylation, S-nitrosothiols modify
a number of proteins, including protein kinases, phosphatases,
and transcription factors.[5,6]
1
1
O
O
6
6
2
2
7
7
5
5
NH
NH
3
4
3
4
8
HS
ONS
9
9
O
NH 10
O
NH 10
A number of theoretical and experimental studies involving the
formation of S-nitrosothiols are described in the literature.[7,8] The
majority of these studies involved the structural characterization
of the compound and its relationship with the biological properties.
S-nitroso-N-acetyl-penicillamine is a widely characterized
nitrosothiol[9] whose functionality and unique aqueous stability
suggest that penicillamine derivatives are good substrates for
the synthesis of novel and stable S-nitrosothiols.[10] These com-
pounds must be fairly water soluble to facilitate biological testing.
In this context, S-nitrosopenicillamine derivatives are worthy of
synthesis and would include simple a-amino derivatives.
Therefore, the aim of this work was to prepare a series of
2-acetamido-3-methyl-3-mercapto-N-aryl-butanamides (1–4)
and S-nitrosothiol derivatives (5–8) (Scheme 1) and to characterize
them through their 1H NMR and 13C NMR spectra.
11
11
13
15
13
12
14
12
14
15
16
16
Y
Y
17
17
1
2
3
4
Y = H
5
6
7
8
Y = H
Y = o-Cl
Y = m-Cl
Y = o-Cl
Y = m-Cl
Y= p-OMe
Y = p-OMe
Scheme 1. Structures and numbering of the compounds studied (1–8).
*
Correspondence to: Adriana K. C. A. Reis, Departamento de Ciências Exatas e
da Terra, Universidade Federal de São Paulo (UNIFESP), 09972-270 Diadema,
São Paulo, Brazil. E-mail: adriana.amorim@unifesp.br
a Departamento de Bioquímica, Universidade Federal de São Paulo (UNIFESP),
São Paulo, São Paulo, Brazil
Experimental
b Departamento de Ciências Exatas e da Terra, Universidade Federal de São
Paulo (UNIFESP), Rua Prof. Artur Riedel, 275, Jardim Eldorado, 09972-270
Diadema, São Paulo, Brazil
Compounds
Initially, 3-acetamido-4,4-dimethylthietan-2-one was obtained
from the reaction of (Æ) penicillamine in dry pyridine and acetic
c
Universidade Federal do Mato Grosso do Sul (UFMS), Campo Grande, Mato
Grosso do Sul, Brazil
Magn. Reson. Chem. 2013, 51, 316–319
Copyright © 2013 John Wiley & Sons, Ltd.

1
Assignment of the H and 13C NMR spectra of 3-mercapto-N-aryl-butanamides and S-nitrosothiol derivatives
Table 1. Physical and elemental analysis data for the 2-acetamide-3-methyl-3-mercapto-N-aryl-butanamides (1–4) and S-nitrosothiol derivatives
(5–8)
Analysis (%)
Compound
Melting point (ꢀC/mmHg)
Molecular formula
13H18N2O2S
Formula weight
266.4
C
H
N
1
194–198
C
Calc.
Found
Calc.
58.62
58.53
51.91
51.85
51.91
51.84
56.73
56.43
52.86
53.81
47.34
47.93
47.34
48.23
51.68
52.86
6.81
6.83
5.70
5.76
5.70
5.82
6.80
6.88
5.80
6.09
4.89
5.01
4.89
5.11
5.89
6.23
10.52
10.30
9.31
2
3
4
5
6
7
8
176–177
210–211
192–193
255–256
218.5–220
255–256
237–238
C
C
13H17 C1N2O2S
13H17 C1N2O2S
300.8
300.8
296.4
295.4
329.8
329.8
325.4
Found
Calc.
9.11
9.31
Found
Calc.
8.98
C
14H20N2O3S
13H17N3O3S
9.45
Found
Calc.
8.70
C
14.23
13.57
12.74
12.15
12.74
11.77
12.91
11.33
Found
Calc.
C
C
13H16 C1N3O3S
13H16 C1N3O3S
Found
Calc.
Found
Calc.
C
14H19 N3O4S
Found
All compounds were obtained at a 60–97% yield. Elemental
analyses were carried out on a Perkin-Elmer 2400 CHN (Central
Analítica - USP -SP - Brazil) standard analyzer instrument (Table 1).
was performed with 1k  512 data point matrices zero-filled to 2k
1k, with 40 transients per t1 increment. Spectral widths of 30248
and 5000 Hz were used for 13C and 1H, respectively. Proton-
detected heteronuclear correlations were measured using a
gradient in HMBC that was optimized for 8 Hz long-range coupling.
The 1H and 13C chemical shifts are given on the d scale (ppm)
and were referenced to internal tetra-methyl-silane; coupling
constants J are reported in hertz (Hz). The abbreviations s, d,
and m were used for singlet, doublet, and multiplet, respectively.
Spectra
1H NMR and 13C NMR spectra were recorded at room temperature on
a Varian Inova 300 spectrometer (Central Analítica - USP -SP - Brazil)
(10% in DMSO-d6 solutions at 298 K) operating at 299.956 and
75.418 MHz. Data processing was carried out on a Solaris workstation.
The 1H NMR parameters were as follows: spectral width,
4507.6 Hz; data points, 16k, zero-filled to 32k; acquisition time,
3.78 s; and digital resolution, 0.28 Hz. The 13C NMR parameters
were as follows: spectral width, 8 kHz; data points, 32k, zero-filled
to 64k; acquisition time, 1.92 s; and digital resolution, 0.25 Hz,
with a delay of 2.05 s between transients.
Results and Discussion
The 1H and 13C NMR data from compounds 1–8 are summarized
in Tables 2 and 3. Initially, the spectra of compounds 1 and 5
(Y = H) were fully assigned, and the signals of the remaining
compounds were assigned by analogy.
The greater electronegativity of the S–NO group has a
deshielding effect that is readily identified by downfield shifts
in the resonance of the b-proton (H-4) and a-carbon (C-5) atoms
attached to the S–NO group in S-nitrosothiols (5–8).
1
The HMBC experiments for the structural assignment of the H
and 13C signals (using standard 2D-NMR pulse sequences on BRUKER
software) were performed on a Bruker Avance DRX 500 spectrom-
eter (Central Analítica - USP -SP - Brazil) equipped with a 2.5 mm di-
rect probe with a field z-gradient. The two-dimensional experiment
Table 2. 1H NMR chemical shifts (ppm), constant coupling (Hz), and multiplicities for 2-acetamide-3-methyl-3-mercapto-N-aryl-butanamides (1–4)
and S-nitrosothiol derivatives (5–8)
Comp. H1
H3
H4
H6*
H7*
H8
H10
H12/H13
H14
H15
H16
H17
1
2
3
4
5
6
7
8
1.92 s 8.15 d (3JH3,H4 = 9.6 Hz) 4.65 d (3JH4,H3 = 9.6 Hz) 1.39 s 1.42 s 2.74 s 10.22 s 7.23 m 6.50 m 6.50 m 7.06 m
—
2.11 s 8.19 d (3JH3,H4 = 9.6 Hz) 4.75 d (3JH4,H3 = 9.6 Hz) 1.37 s 1.62 s 2.65 s
1.95 s 8.17 d (3JH3,H4 = 9.3 Hz) 4.69 d (3JH4,H3 = 9.3 Hz) 1.39 s 1.41 s 2.79 s 10.42 s 7.48 m 7.34 m
1.95 s 8.18 d (3JH3,H4 = 9.3 Hz) 4.70 d (3JH4,H3 = 9.3 Hz) 1.39 s 1.41 s 2.79 s 10.42 s 7.43 m 7.13 m 7.13 m
9.78 s 7.23 m 7.07 m 7.25 m 6.74 m 6.74 m
7.13 m 7.13 m
—
—
3.35 s
2.04 s 8.58 d (3JH3,H4 = 9.6 Hz) 5.46 d (3JH4,H3 = 9.6 Hz) 2.04 s 2.00 s
2.11 s 8.54 d (3JH3,H4 = 9.6 Hz) 5.59 d (3JH4,H3 = 9.6 Hz) 2.04 s 1.89 s
2.04 s 8.60 d (3JH3,H4 = 9.6 Hz) 5.42 d (3JH4,H3 = 9.6 Hz) 1.99 s 1.89 s
2.04 s 8.55 d (3JH3,H4 = 9.9 Hz) 5.40 d (3JH4,H3 = 9.9 Hz) 1.99 s 1.89 s
—
—
—
—
10.50 s 7.60 m 7.32 m 7.32 m 7.08 m
—
10.20 s 7.53 m 7.35 m 7.53 m 7.24 m 7.24 m
10.70 s 7.42 m 7.26 m 7.15 m 7.15 m
10.36 s 7.52 m 7.50 m 7.50 m 3.72 s
—
—
* The assignments can be interchanged.
Magn. Reson. Chem. 2013, 51, 316–319
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

R. G. Santana et al.
Table 3. 13C NMR chemical shifts (ppm) for the 2-acetamide-3-methyl-3-mercapto-N-aryl-butanamides (1–4) and S-nitrosothiol derivatives (5–8)
Comp.
C1
C2
C4
C5
C6*
C7*
C9
C11
C12
C13
C14
C15
C16
C17
1
2
3
4
5
6
7
8
23.79
23.20
22.29
23.40
22.17
22.19
22.18
23.07
171.87
170.47
169.50
170.63
169.46
169.43
169.54
170.30
61.80
61.08
61.43
60.65
59.60
59.59
59.33
55.96
46.54
45.75
45.89
46.28
59.58
59.02
59.72
60.44
29.04
28.68
28.48
28.70
24.77
24.83
24.69
25.67
31.61
31.13
30.97
31.01
26.49
26.72
26.39
27.38
168.59 140.86 120.12 120.12 131.06 131.06 125.92
168.32 134.01 122.60 129.31 125.45 127.47 124.05
168.57 139.80 117.74 118.77 130.35 132.95 123.20
167.89 130.53 121.81 121.81 114.09 114.09 156.60
167.19 138.18 123.78 123.78 128.68 128.68 119.51
167.58 134.07 126.96 129.55 127.05 127.35 127.53
167.65 139.57 117.91 118.93 130.45 132.98 123.55
167.50 132.18 121.93 121.93 114.70 114.70 156.39
—
—
—
55.47
—
—
—
60.53
* The assignments may be interchanged.
Figure 1. 1H 13C correlation spectrum of 1 (HMBC). The horizontal axis shows 1D 1H spectrum; the vertical axis shows 13C projection.
Figure 2. 1H 13C correlation spectrum of 5 (HMBC). The horizontal axis shows 1D 1H spectrum; the vertical axis shows 13C projection.
wileyonlinelibrary.com/journal/mrc
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2013, 51, 316–319

1
Assignment of the H and 13C NMR spectra of 3-mercapto-N-aryl-butanamides and S-nitrosothiol derivatives
For compounds 1–8, the singlet signals at d = 2.0 ppm in media
correspond to H-1 of the acetamido group (Table 2).
Doublets at d = 8.1 ppm for compounds 1–4 and d = 8.6 ppm
for compounds 5–8 correspond to H-3 of the amidic nitrogen
of the acetamido group and arise because of the coupling
constants of d = 9.4 and 9.6 Hz for compounds 1–4 and 5–8,
respectively. In compounds 1–4, the doublet signals at d = 4.7
ppm and the coupling constant of d = 9.4 Hz refer to H-4 attached
to the chirality center.
Acknowledgements
The authors thank Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP – 2010/51784-3) for their financial support of
this research and Conselho Nacional de Desenvolvimento e
Tecnológico (CNPq) for fellowships (to R.G.S and D.R.P). The
authors also graciously acknowledge Prof. Paulo Roberto Olivato
for providing laboratory facilities.
References
In S-nitrosothiols 5–8, this signal is d = 5.5, and the coupling
constant is d = 9.6 Hz. The singlet in d = 2.7 ppm refers to the
H-8 of the thiol group for compounds 1–4.
[1] D. T. Hess, A. Matsumoto, S.-O. Kim, H. E. Marshall, J. Stamler. Nat.
Rev. Mol. Cell Biol. 2005, 6, 150–166.
The high-frequency multiplet signals at d = 6.5 to 7.8 ppm
were assigned to the aromatic protons. Empirically calculated
chemical shifts are in close agreement with the experimental
values.[12,13]
[2] D. D. Thomas, D. Jourd’heuil. Antioxid. Redox Signal. 2012, 17(7), 934–936.
[3] M.-F. Garcia-Saura, F. Saijo, N. S. Bryan, S. Bauer, J. Rodriguez,
M. Feelisch. Antioxid, Redox Signal. 2012, 17(7), 937–950.
[4] R. Priora, A. Margaritis, S. Frosali, L. Coppo, D. Summa, D. Di
Giuseppe, C. Aldinucci, G. Pessina, A. Di Stefano, P. Di Simplicio.
Pharmacol. Res. 2011, 64(3), 289–297.
The 13C NMR chemical shifts for 1–8 are presented in Table 3.
The remaining quaternary carbons were assigned mainly on
the basis of the HMBC spectra from 1 and 5 (Figs. 1 and 2).
All of the 13C NMR chemical shifts from the remaining
compounds were determined on the basis of their similarity
to compounds 1–8.
[5] H. P. Monteiro, J. Gruia-Gray, T. M. Peranovich, L. C. de Oliveira,
A. Stern. Free Rad. Biol. Med. 2000, 28, 174–182.
[6] M. W. Foster, T. J. McMahon, J. S. Stamler. Trends Mol. Med. 2003, 9,
160–168.
[7] N. L. Chan, P. H. Rogers, A. Arnone. Biochemistry 1998, 37(47),
16459–16464.
[8] M. D. Bartberger, K. N. Houk, S. C. Powell, J. D. Mannion, K. Y. Lo,
J. S. Stamler, E. J. Toone. J. Am. Chem. Soc. 2000, 122, 5889–5894.
[9] S. C. Askew, A. R. Butler, F. W. Flitney, G. D. Kemp, I. L. Megson. Bioorg.
Med. Chem. 1995, 3, 1–9.
[10] H. A. Moynihan, S. M. Roberts. J. Chem. Soc. Perkin Trans. 1994, (1),
797–805.
[11] L. Soulére, J. C. Sturm, L. J. Nuúnez-Vergara, P. Hoffmann, J. Périe.
Tetrahedron 2001, 57, 7173–7180.
[12] H. Gunther, NMR Spectroscopy. Basic Principles, Concepts and
Applications in Chemistry, 2nd edn, John Wiley, Chichester, 1995.
[13] R. J. Abraham, J. J. Byrne, L. Griffiths. Magn. Reson. Chem. 2008, 46, 667.
The two 13C signals at d = 168 ppm and d = 170 ppm were
attributed to carbonyl carbons C-2 and C-9 based on the
correlations observed between atoms H-3 and H-10, respectively,
in the HMBC spectrum.
Carbon C-5 presents chemical shifts at d = 46 ppm for 1–4 and
d = 60 ppm for 5–8, considering that both signals present
correlations with H-4. The chirality center C-4 presents chemical
shifts at d = 61 ppm for 1–4 and d = 59 ppm for 5–8, confirming
the correlations observed with H-3.
Magn. Reson. Chem. 2013, 51, 316–319
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc