VT-NMR and cytotoxic evaluation of some new ortho-(alkylchalcogen)acetanilides

Article abstract of DOI:10.1016/j.tet.2014.09.055

Several novel atropisomeric N-alkyl-N-[(2-alkylchalcogen)phenyl)]acetamides have been synthesized and fully characterized by ¹H and ¹³C NMR, FTIR, and HRMS (FAB). The barriers of the restricted rotation about the N-aryl bond between the two atropisomeric forms were measured by accurate lineshape simulation of variable temperature NMR (VT-NMR) spectra obtained in DMSO-d₆ solution and ranged from 17.0 to 20.5 kcal/mol. The relationship between the structure of the different acetanilide moieties and both coalescence temperature and energy of rotation are herein discussed. Taking in mind the acetamide structural resemblances with nimesulide related compounds known by their anticancer activity, the in vitro cytotoxicity of 20 representative acetanilides, against human breast (MCF-7) and prostate (LNCaP) cancer cell lines as well as normal human dermal fibroblasts (NHDF) was also preliminary evaluated. Interestingly a selective antiproliferative activity was observed for cancerous cells with prominence to LNCaP within the most potent O- and/or N-benzylic and -hexyl acetanilides.

Full text of DOI:10.1016/j.tet.2014.09.055

Tetrahedron 70 (2014) 8930e8937
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
VT-NMR and cytotoxic evaluation of some new
ortho-(alkylchalcogen)acetanilides
,
,c
Susana S. Ramos a c, Sandra S. Almeida b, Paula M. Leite b, Renato E.F. Boto b
,
Samuel Silvestre b, Paulo Almeida b
UMTP-UBI, Unit of Textile and Paper Materials, University of Beira Interior, Rua Marques d’Avila e Bolama, 6201-001 Covilha, Portugal
CICS-UBIdHealth Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, Portugal
Department of Chemistry, University of Beira Interior, Rua Marques d’Avila e Bolama, 6201-001 Covilha, Portugal
,c,
*
a
ꢀ
^
~
b
c
~
ꢀ
^
~
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2014
Received in revised form 15 September 2014
Accepted 17 September 2014
Available online 28 September 2014
Several novel atropisomeric N-alkyl-N-[(2-alkylchalcogen)phenyl)]acetamides have been synthesized
and fully characterized by 1H and 13C NMR, FTIR, and HRMS (FAB). The barriers of the restricted rotation
about the N-aryl bond between the two atropisomeric forms were measured by accurate lineshape
simulation of variable temperature NMR (VT-NMR) spectra obtained in DMSO-d6 solution and ranged
from 17.0 to 20.5 kcal/mol. The relationship between the structure of the different acetanilide moieties
and both coalescence temperature and energy of rotation are herein discussed. Taking in mind the
acetamide structural resemblances with nimesulide related compounds known by their anticancer ac-
tivity, the in vitro cytotoxicity of 20 representative acetanilides, against human breast (MCF-7) and
prostate (LNCaP) cancer cell lines as well as normal human dermal fibroblasts (NHDF) was also pre-
liminary evaluated. Interestingly a selective antiproliferative activity was observed for cancerous cells
with prominence to LNCaP within the most potent O- and/or N-benzylic and -hexyl acetanilides.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Atropisomerism
Axial chirality
Cytotoxic evaluation
Dynamic VT-NMR
Nimesulide analogues
ortho-(Alkylchalcogen)acetanilides
1. Introduction
After the pioneering work by Curran et al. on atropisomeric
anilides,1 the potential use of non-biaryl atropisomers in medicinal
chemistry has driven the interest and research around their syn-
thesis and enantioselective synthesis, the stereoselectivity of their
reactions as well as their use as chiral auxiliaries and chiral
ligands.2
Recently, as part of our efforts to definitely clarify the identity
of the products resulting from the hydroxylation of 2-
methylbenzoazolium iodides, we described the synthesis of some
representativeortho-(alkylchalcogen)-N-ethylacetanilides 3 (X¼O, S
or Se, Scheme 1).3 The acetanilides 3 were obtained as atropisomers,
in moderate to good yields, after an alkylation step used to trap the
unstable sulfur and selenium open (as well as the more stable oxy-
gen analogues) N-ethyl-(2-thiol/selenol/hydroxyl)acetanilide 2b,
resulting from the cleavage of the 2-hydroxybenzoazole moiety of
the initially formed intermediate 2a.
Scheme 1. Reagents and conditions: (a) NEt3, 96% ethanol, reflux; (b) ethanol, NaOH,
RI or RBr, reflux.
affording, respectively, enantiomeric and diasteroisomeric
forms.2q,3,4 This restricted rotation around the amide NeCO bond
originates endo and exo isomers (benzene ring cis and trans to
carbonyl oxygen), which are easily differentiated by 1H NMR. In
general, N-substituted anilides exist predominantly as the exo iso-
mer.2q,3e5 The atropisomeric forms resulting from the circum-
scribed rotation about the amide N-aryl bond are easily recognized
in solution by the signals of the two magnetically nonequivalent
geminal N-CH2 protons clearly displayed in the 1H NMR spectra.
The dynamic process of the E diastereoisomer of five representative
acetanilides 3 was thus investigated by variable temperature NMR
(VT-NMR) spectroscopy and the corresponding rotation barriers
N-Alkyl-(2-alkylchalcogen)acetanilides 3 have restricted rota-
tions around their N-aryl bond as well as amide NeCO bond,
* Corresponding author. Tel.: þ351 275329174; fax: þ351 275319056; e-mail
addresses: paulo.almeida@ubi.pt, pjsa@ubi.pt (P. Almeida).
http://dx.doi.org/10.1016/j.tet.2014.09.055
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

S.S. Ramos et al. / Tetrahedron 70 (2014) 8930e8937
8931
(
D
Gz) at 298 K and the coalescence temperatures (Tc) were de-
All N-alkyl-(2-alkylchalcogen)acetanilides
3
were prepared
termined, ever since is less than 413 K.3
from the appropriate 3-alkyl-2-methylbenzazol-3-ium iodides 1
(or bromides), in moderate to good yields, following the same
procedure already described3 (Scheme 1, Table 1). In our hands,
none O-alkylated products of 2a were observed, which is in
agreement with the more expectable nucleophilic anionic forms of
thiophenol, selenophenol, and phenol moieties present in acetan-
ilides 2b in relation to the alkoxy form present in 2a.
Following our preliminary results,3 herein we describe a full set
of 23 ortho-substituted acetanilides 3aeh (X¼O), 3ieu (X¼S), and
3u,w (X¼Se), 18 of them are, to the best of our knowledge, novel
(3b, deh, j, leu, w). In this serial, the nature of the chalcogen atom
(X) and the nature of the alkyl group linked to both amide group
(R1) or chalcogen atom (R2) were varied (Scheme 1, Table 1).
Hence, the full characterization by 1H and 13C NMR, FTIR, and
HRMS (FAB) of these atropisomeric N-alkyl-N-[((2-alkylchalcogen)
All the new acetanilides 3 were structurally characterized by 1H
and 13C NMR, FTIR, and HRMS (FAB).
phenyl)]acetamides 3aej,lew are here presented. The
D
Gz between
As expected, the 1H NMR analysis reveals four stereoisomeric
forms, as already stressed in our previous work.3 The Ra and Sa exo
forms were always predominant with an E/Z ratio ꢀ95:5, as usual
for N-substituted anilides.3e5 As example, both axially enantio-
meric forms Sa-endo and Sa-exo, are present in Fig. 1.
the two atropisomeric forms of acetanilides 3aew, with emphasis
for alkyloxy- 3aeh and alkylthioanilides 3ieu, have been de-
termined by accurate lineshape simulation of VT-NMR spectra ob-
tained in DMSO-d6 solution and ranged from 17.0 to 20.5 kcal/mol.
The relationship between the structure of the different moieties of
acetanilides and both Tc and
D
Gz for all acetanilides is herein dis-
cussed. The cytotoxicity against normal human dermal fibroblasts
(NHDF) and human breast (MCF-7) and prostate (LNCaP) cancer cell
lines of several representative acetanilides 3aed,gem,qew and
anilines 4a,b additionally prepared was also evaluated and is here
also presented and discussed.
Table 1
Reaction times, overall yields, DG
z and Tc for N-alkyl-(2-alkylchalcogen)acetanilides
3
Compd 3
X
R1
R2
Reaction times Yielda
DG
z (ꢃ0.2 kcal/mol) Tc (K)
Fig. 1. Sa-endo and Sa-exo axially enantiomeric forms of acetanilides 3.
(%)
Step (a) Step
(min)
(b) (h)
Since the exo diastereoisomer is almost the exclusive form, the
ab
b
cb
d
e
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
S
S
S
S
S
S
Et
Et
Et
Et
Pr
Pr
Et
Pr
60
45
Hex 60
45
45
Hex 45
3
4
6
6
3
6
5
3
3
4
6
6
3
3
6
3
6
3
5
5
6
4
5
79
70
68
66
71
65
71
64
87
77
75
78
82
81
69
84
72
83
65
62
59
85
74
18.1
17.9
17.1
17.2
18.1
17.9
17.4
17.0
18.3
18.2
17.8
18.0
18.1
18.2
18.1
18.0
17.5
17.5
17.9
18.1
18.2
20.5
17.8
388
391
393
393
395
398
401
D
Gz between the two axially enantiomeric forms (Sa and Ra) in
solution have been determined by accurate lineshape simulation of
VT-NMR spectra using gNMR modeling software (Cherwell Scien-
tific). These lineshape simulations were based on the experimental
spectra obtained for each compound, namely on the chemical shifts
and coupling constants of both two geminal N-CH2 protons mag-
netically nonequivalent and on the distance between both signals.
1H NMR representative pattern signals of one of the N-CH2 geminal
proton of acetanilides are presented in Fig. 2.
Bn
Et
f
g
Hex Pr
60
60
30
30
h
ib
j
Bn
Et
Et
Et
Et
Pr
Pr
Pr
Hex Et
Hex Pr
Bn
Bn
Et
Et
Pr
402
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
kb
l
Hex 30
Bn
Me
Et
30
30
30
m
n
o
q
p
r
s
t
u
vb
w
Hex 30
45
45
30
45
45
Et
Bn
MeBn Et
MeBn MeBn 60
Se Et
Se Et
Et
30
Hex 45
Fig. 2. 1H NMR representative pattern signals (400 MHz; DMSO-d6) of one of the
geminal N-CH2 proton: (a) R1¼Et; (b) R1¼Pr or Hex; and (c) R1¼Bn.
a
Isolated overall yield.
Previously described.3
>413 K.
b
c
Lineshape simulation of the spectra using gNMR modeling
software gave a rate constant for interconversion (k) at a range of
temperatures (T in K), which was converted into a barrier to rota-
tion at a given temperature using the Eyring equation.6
2. Results and discussion
In our previous described work,3 the nature of both alkyl groups
were restricted to N-ethylacetanilides (3, R1¼Et) and to ortho-
(ethylchalcogen)- and ortho-(hexylchalcogen)acetanilides (3, X¼O,
S, Se; R2¼Et, Hex).
D
Gz ¼ 4:569 ꢁ 10ꢂ3Tð10:319 þ log T=kÞ
As a representative example, both experimental and simulated
1H NMR N-CH2 signals of the N-ethyl-N-(2-propoxyphenyl)acet-
amide 3b, from 298 to 391 K (k variation from 1 to 225 sꢂ1) are
presented in Fig. 3.
Thus, in order to determine a more accurate relationship be-
tween the structure of the different moietiesof acetanilidesand both
Tc and
D
Gz, a setof18 newacetanilides was synthesizedin addition to
The Eyring plot of ln (k/T) versus 1/T gives a straight line from
which, using the equation y¼mxþc, the slope (m) gives the enthalpy
the 5 previously described. In this series, the nature of the chalcogen
atom as well as the N-acetamide and chalcogen substituents were
varied [X¼O, S or Se; R1 and/or R2¼methyl, ethyl, propyl, hexyl,
benzyl (Bn), and/or 2-methylbenzyl (MeBn) (Scheme 1)].
of rotation (
tion (
equation for free energy (
D
Hz) and the y-intercept (c) gives the entropy of rota-
D
Sz) as described above. Inserting theses values into Gibbs’
D
Gz¼
D
HzꢂT
D
Sz) gives an estimation of the

8932
S.S. Ramos et al. / Tetrahedron 70 (2014) 8930e8937
Fig. 3. Experimental (left) 1H NMR spectra of N-CH2 protons of 3b in DMSO-d6 as a function of temperature and lineshape simulation (right) for the same compound using gNMR
software with the rate constants for the interconversion between the two rotamers.
D
Gz at room temperature (taken to be 25 ꢄC). Eyring plots ln (k/T)
However, Tc was not achieved for ortho-(alkylthio)- 3ieu and or-
tho-(alkylseleno)acetanilides 3v,w and therefore rate constants
determined above this temperature could not be measured. The
estimated
pounds 3aew) are in accordance with the values disclosed in the
versus 1/T for alkyloxyacetanilide 3b and alkylthioacetanilide 3j
were also present as representative examples (Fig. 4) as well as the
activation parameters determined from these plots (Table 2).
D
Gz ranging from 17.0 to 20.5 kcal/mol (Table 1, com-
literature for anilides possessing
a single bulky ortho-sub-
stituent.1,2p,q,7 The Tc from 388 to 402 K herein presented are re-
stricted for the ortho-(alkyloxy)acetanilides 3aeh, ever since the
lowest temperatures at which the two rotamers of remaining ac-
etanilides 3iew merge were determined to be above the maximum
spectrometer’s working temperature (413 K).
Despite the fact that acetanilides 3 show the same N-CH2
geminal protons splitting patterns within the same alkyl group
linked to the chalcogen atom serial (Fig. 2), they differ in the region
where these signals are displayed [
d
3.33e5.01 ppm (X¼O);
d
2.97e5.42 ppm (X¼S);
d
3.08e3.95 ppm (X¼Se)]. Another sig-
nificant difference lies on the chemical shift difference (DdAB) be-
tween the two geminal N-CH2 protons, with the values being higher
as the size of the chalcogen atom increases from oxygen to sulfur
Fig. 4. Representative Eyring plots of ln (k/T) versus 1/T for acetanilides 3b and 3j.
[DdAB from 0.21 to 0.55 (X¼O); DdAB from 0.82 to 1.51 (X¼S)]. This
observation is in agreement with the DdAB between nonequivalent
methylene protons observed for other acetanilides already de-
scribed, where DdAB also depends on the size of the ortho-sub-
stituent of the aniline moiety.4a,d However, when the size of the
chalcogen increases from sulfur to selenium atom, the shift differ-
ences remain almost the same within the two alkylselenoacetani-
lides 3v,w compared to their thio-congeners 3i,k.
Table 2
Representative acetanilides 3b and 3j activation parameters determined from plots
of ln (K/T) versus 1/T using Eyring equation
m
D
Hz (J)
c
D
S
z (J)
D
Gz (J)
3b
3j
ꢂ6943.1
ꢂ6214.0
57,728.2
51,666.1
16.847
13.926
ꢂ57.5
ꢂ81.8
74,871.8
76,054.8
Another direct consequence of the size of the chalcogen is the Tc
of the acetanilides. As a matter of this fact, while the Tc of ortho-
(alkyloxy)acetanilides 3aeh was determined to be below 413 K, for
ortho-(alkylthio) 3ieu and (alkylseleno)acetanilides 3v,w we are
not able to reach the coalescence due to the limitation of the
In our case, the linear regression analysis performed for each
compound 3aew was obtained from a set of 7 to 12 data points,
with correlation coefficients between 0.9873 and 0.9990, showing
consistently a very high degree of linearity for the data obtained.

S.S. Ramos et al. / Tetrahedron 70 (2014) 8930e8937
8933
spectrometer’s working temperature, as above mentioned. In ad-
dition, within the set of ortho-(alkyloxy)acetanilides 3aeh, the Tc
raises with the increase of the size of both R1 and R2 n-alkyl groups
(Hex>Prop>Et).
acetanilides (Table 2, entries 3e6, 9, 10, 12e16, 18) revealed to be
more toxic and more selective (frequently up to 2-folder) to both
cancer cell lines than the corresponding methyl, ethyl, and propyl
congeners.
The previously described relationship between the rotation
barrier values with both alkyl R1 and R2 size groups based just on
These results prompt us to suspect that the lipophilicity of the
compounds could be related to their antiproliferative activity. Thus,
by using the software ChemDraw 9.0, the log P of all compounds
considered in this assay was calculated (Table 3) and the relation-
ship between these two variables was evaluated. From this analysis,
only a modest association between the lipophilicity and cytotoxic
effects was found for LNCaP cells (r2¼0.5379) and for MCF-7 cells
(r2¼0.3933) and no association was evidenced for NHDF cells
(r2¼0.0032). Moreover, a careful analysis within X2N2, X6N2, and
X3N6 series [Table 3, entries 1, 3, 5, 7, 9, 12, 17, and 18; Xn and Nn
mean the number of carbons (2¼ethyl, 3¼propyl, and 6¼hexyl)
linked to the chalcogen (X¼O, S or Se) or nitrogen, respectively]
reveals a generally higher cytotoxicity for the ortho-(alkyloxy)ac-
etanilides (X¼O) and a lower one for the ortho-(alkylseleno)acet-
anilides (X¼Se).
five different acetanilides,3 are herein reconfirmed. Specifically, Gz
D
values slightly increase with the size of the chalcogen (Se>S>O).
Nevertheless, and in contrary to our expectations, the increase in
the rotational barrier in function of the alkyl group size (Hex>-
Prop>Et) within each chalcogen serial renders lower
accordance with our previous report.3 This statement is reinforced
by the lower
Gz value observed when the hexyl or benzylic (benzyl
D
Gz values, in
D
and 2-methylbenzyl) group is introduced in R1 or R2 position.
Taking into account the structural similarly between the acet-
anilides herein described and nimesulide related compounds as
anticancer agents,8 we decided to evaluate the in vitro anti-
proliferative effect against normal human dermal fibroblasts
(NHDF) and against the human hormone-dependent breast (MCF-
7), and prostate (LNCaP) cancer cell lines, for several representative
ortho-(alkyloxy)acetanilides 3aed,g,h, ortho-(alkylthio)acetani-
lides 3iem,qeu, and ortho-(alkylseleno)acetanilides 3v,w. In order
to evaluate the carbonyl group role in the cytotoxicity of these
acetanilides, two representative ortho-(alkylchalcogen)-N,N-dia-
lkylanilines 4a,b were additionally prepared as previously de-
scribed3 and their in vitro antiproliferative activity evaluated and
compared to their ortho-(alkylthio)acetanilides 3i,m congeners.
The evaluation of the relative cell viability of MCF-7, NHDF, and
LNCaP cells was performed through the MTT assay, following
a previously described procedure.9 Briefly, cells were seeded in 48-
The ortho-(alkylthio)anilines 4a,b (Table 3, entries 19 and 20)
revealed to be less active and less selective than their ortho-
(alkylthio)acetanilides 3i,m congeners (Table 3, entries 7 and 11),
pointing to the importance of the carbonyl amide group for the
antiproliferative activity and to their needless reduction into
a methylene group.
The highest antiproliferative activity and superior selectivity
against both cancerous cells (almost 6-fold and 2-fold higher for
MCF-7 and LNCaP, respectively, when compared to NHDF cells)
observed for the 2-methylbenzyl disubstituted thioacetanilide
3u (Table 3, entry 16) should be emphasized. In fact, this com-
pound revealed to have a higher selectivity to breast cancer cells
(13.71% proliferation vs control) in comparison to both prostate
(39.03%) and normal skin fibroblasts (79.02%). This important
result is now being explored in order to clarify the potential
interest of this compound as a future antitumor agent. Moreover,
when comparing acetanilides 3u and 3s (Table 3, entries 16 and
14) it is clear that the introduction of an ortho-methyl group
increases the antiproliferative activity of the benzylic moiety as
well as in their selectivity for MCF-7 cells in special. A similar
effect can also be observed for compounds 3t and 3r, and again
the ortho-methylated (3t) revealed a higher antiproliferative
effect that the non-methylated derivative (3r) (Table 3, entries
15 and 13).
well plates and treated with a 30 mM concentration of the different
compounds during approximately 48 h, with untreated cells serv-
ing as negative control and H2O2 (1 mM) as positive control and at
the end of incubation the MTT test was effected. The results of these
experiments are presented in Table 3.
Table 3
Relative cell viability of MCF-7, NHDF, and LNCaP cells incubated with compounds
3aed,gem,qew and 4a,b at 30 mM, determined by the MTT assay after 48 h of ex-
position. Mean valuesꢃSD (% of the negative control) were obtained from five ex-
perimental determinations and data are expressed as a percentage of cell viability in
comparison with the respective negative control
Entry Compound (30
m
M) MCF-7
LNCaP
NHDF
log Pa
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3g
3h
3i
3j
3k
3l
3m
3q
3r
3s
3t
3u
93.45ꢃ3.38 68.32ꢃ10.63 85.59ꢃ10.74 1.73
As the highest antiproliferative effects in cancer cell lines were
observed with compound 3u, we also performed doseeresponse
studies with this and with compound 3q, aiming to obtain their
half-medium inhibitory concentration (IC50) values. For this, cells
were treated with six different concentrations (0.01, 0.1, 1, 10, 50,
92.54ꢃ3.64 74.29ꢃ10.14 83.97ꢃ6.84
2.21
3.47
3.12
3.95
55.53ꢃ4.85 38.46ꢃ2.62
88.84ꢃ2.70 52.97ꢃ4.41
51.52ꢃ9.41 45.86ꢃ2.01
78.34ꢃ7.75
75.77ꢃ2.96
84.34ꢃ5.20
87.59ꢃ3.58 65.75ꢃ10.13 91.07ꢃ12.63 3.12
99.50ꢃ5.98 75.99ꢃ9.89
89.14ꢃ7.94 59.74ꢃ4.03
67.65ꢃ5.63 53.19ꢃ6.17
95.33ꢃ7.97 51.71ꢃ5.02
95.34ꢃ6.77 65.78ꢃ2.66
47.58ꢃ6.46 59.65ꢃ9.99
89.37ꢃ5.62 62.44ꢃ3.19
79.81ꢃ5.21 52.63ꢃ6.08
77.00ꢃ7.68 41.85ꢃ1.75
13.71ꢃ4.71 39.03ꢃ2.59
98.23ꢃ5.71 86.20ꢃ8.98
82.63ꢃ6.97 59.35ꢃ1.60
99.72ꢃ3.40 83.65ꢃ6.86
82.65ꢃ3.13
76.19ꢃ6.36
83.48ꢃ8.95
79.17ꢃ7.78
81.49ꢃ4.77
82.99ꢃ8.86
80.05ꢃ6.03
2.29
2.78
4.03
3.69
1.96
4.52
3.69
and 100 mM) of the compounds, again during nearly 48 h, before the
9
MTT test and the doseeresponse curves and IC50 values were
established by sigmoid fitting. This study not only confirmed the
relevant cytotoxic effects of these compounds in these cells but also
revealed that the anilide 3u has an interesting IC50 value in LNCaP
cells. In fact, considering a 95% confidence interval, the IC50 values
10
11
12
13
14
15
16
17
18
19
20
21
96.68ꢃ12.68 5.08
75.21ꢃ6.90 4.18
for compounds 3u and 3q in LNCaP cells were 17.24
m
M (r2¼0.8632)
79.02ꢃ10.39 6.06
and 61.64
m
M (r2¼0.8723), and in MCF-7 cells were 40.93
mM
3v
3w
4a
4b
95.45ꢃ8.18
98.90ꢃ7.91
93.00ꢃ6.44
1.88
3.47
3.77
3.43
d
(r2¼0.9422; 95% CI) and 41.84
m
M (r2¼0.9407), respectively. As
these compounds presented relatively low cytotoxicity against
NHDF cells we decided not to determine the IC50 values with these
non-cancerous cells.
97.19ꢃ2.32 76.91ꢃ16.91 88.27ꢃ7.15
H2O2 (1 mM)
0.17ꢃ0.32 11.22ꢃ1.67
1.51ꢃ2.56
a
Calculated lipophilicity, using the software ChemDraw 9.0 (CambridgeSoft).
Although more studies are needed to further explore the po-
tential interest of this acetanilide family as antiproliferative agents,
particularly those based in hexyl or substituted benzylic (as acet-
anilide 3u) groups, the results herein presented, can be significantly
important for the future development of more selective and pow-
erful structures.
Considering the data presented in Table 3 it is clear that, despite
a low to moderate antiproliferative activity, generally these com-
pounds seems to be more toxic to LNCaP cells than to MCF-7 and
NHDF cells. Moreover, the benzylic 3d,h,l,reu and hexyl 3c,g,k,q,w

8934
S.S. Ramos et al. / Tetrahedron 70 (2014) 8930e8937
3. Conclusions
internal standard. Coupling constants (J values) are reported in
hertz (Hz) and splitting multiplicities are described as s¼singlet;
d¼doublet; t¼triplet; q¼quartet; or combinations of the above; or
m¼multiplet. All new compounds were determined to be >95%
pure by 1H NMR. ESI-TOF mass spectrometry was performed on
a microTOF (focus) mass spectrometer (Bruker Daltonics, Bremen,
Germany). Ions were generated using an Apollo II (ESI) source.
Ionization was achieved by electrospray, using a voltage of 4500 V
applied to the needle, and a counter voltage between 100 and 150 V
applied to the capillary. Samples were prepared by adding a spray
solution of 70:30 (v/v) acetonitrile/water with 0.1% of formic acid
into a solution of the sample in CH2Cl2 at a v/v ratio of 1 to 5% to
give the best signal-to-noise ratio. Data acquisition was performed
using the microTOFControl software version 2.1, and data pro-
cessing was performed using the DataAnalysis software, version 3.4
both from Bruker Daltonics.
In summary, we prepared a series of atropisomeric ortho-
(alkylchalcogen)-N-alkylacetanilides 3 from quaternary ammo-
nium salts 1 in overall yields of 59e87%.
The dynamics of the N-aryl bond rotation of the major E
diastereoisomers of these acetanilides have been evaluated by
VT-NMR techniques and computed by gNMR calculations. While
the magnitude of the energetic barrier to the interconversion
between atropisomers is just slightly dependent on the chalcogen
and on the nature of both alkyl groups linked to N-aryl or to the
chalcogen atom, the Tc appears to be much more structurally
dependent.
The evaluation of the in vitro antiproliferative effects of a range
of representative acetanilides against normal human dermal fi-
broblasts (NHDF) and MCF-7 and LNCaP cancer cell lines was also
performed. Curiously, this study revealed not only a general se-
lective cytotoxicity against tumor cells versus normal cells, espe-
cially pronounced for prostate cancer cells, but also that benzylic
and hexyl acetanilides were the most powerful compounds. The
best result was observed for the methylphenyl disubstituted acet-
anilide (compound 3v), which is now being studied as a potential
future lead compound as anti-breast cancer agent. In order to en-
large structureeactivity relationship data in this context, the de-
velopment of new hexylic and/or benzylic related acetanilides is
also now object of our interest.
4.2. Synthesis and structural characterization
The acetanilides 3aew and anilines 4a,b were prepared as al-
ready described.3 The characterization of both novel or not yet fully
characterized acetanilides 3aeg,kew and aniline 4a described
herein is presented.
4.2.1. N-(2-Ethoxyphenyl)-N-ethylacetamide 3a. IR
2980, 2931, 1682, 1651 (C]O), 1596, 1500, 1455, 1403, 1264, 1231,
1043, 924, 732, 702. 1H NMR (400 MHz, DMSO-d6):
(
nmax/cmꢂ1):
Additionally, this new synthetic way to prepare ortho-(alkyl-
chalcogen)-N-alkylacetanilides 3 appears to be a promising alter-
native to develop new compounds resembling nimesulide,
especially in cases where the benzothiazole starting material pos-
sesses a nitro group or other electrowithdrawing group derived
from in the 6-position. Besides, other steps as acetanilide hydrolyze
followed by aniline derivatization could be considered.
d¼7.34 (dt, 1H,
J¼6.0, 1.7 Hz), 7.22 (dd, 1H, J¼7.5, 1.7 Hz), 7.13 (dd, 1H, J¼7.2, 1.1 Hz),
6.99 (dt, 1H, J¼6.2, 1.3 Hz), 4.02e4.15 (m, 2H), 3.65 (dq, 1H, J¼13.7,
7.0 Hz), 3.43 (dq, 1H, J¼13.7, 7.0 Hz), 1.64 (s, 3H), 1.30 (t, 3H,
J¼7.0 Hz), 0.96 (t, 3H, J¼7.0 Hz) ppm. 13C NMR (100 MHz, DMSO-
d6):
d
¼169.6, 154.7, 131.4, 130.3, 129.8, 121.3, 113.7, 64.0, 42.6, 22.4,
15.1, 13.3 ppm. HRMS (ESI-TOF) m/z 208.13391 (208.13375 calcd for
Finally, it is our belief that our search for more interesting re-
lated compounds will forward us to acetanilides with a second
C
12H18NO2, [MþHþ]).
bulky ortho-substituted group and therefore presenting a
D
Gz suf-
4.2.2. N-Ethyl-N-(2-propoxyphenyl)acetamide 3b. IR (nmax/cmꢂ1):
2968, 2934, 2877, 1655 (C]O), 1596, 1499, 1454, 1395, 1286, 1265,
ficiently high to allow isolation of stereochemically stable atro-
pisomers by this new simple, effective, and regiospecific way herein
depicted. Thus, an energy of rotation high enough to allow the
separation and isolation of both atropisomers of each acetanilide
will open to us the opportunity to study their biological interest,
such as the cytotoxicity against various human cancer cell lines,
both as racemic or as their isolated atropisomeric forms.
1231, 1043, 978, 750. 1H NMR (400 MHz, DMSO-d6):
d
¼7.34 (dt, 1H,
J¼7.8, 1.7 Hz), 7.22 (dd, 1H, J¼7.6, 1.7 Hz), 7.13 (dd, 1H, J¼8.3, 1.0 Hz),
6.99 (dt, 1H, J¼7.5, 1.2 Hz), 3.98 (t, 2H, J¼5.9 Hz), 3.65 (dq, 1H,
J¼13.9, 7.0 Hz), 3.43 (dq, 1H, J¼13.9, 7.0 Hz), 1.70 (sext, J¼6.9, 2H),
1.63 (s, 3H), 0.96 (dt, 3H, J¼7.4, 1.6 Hz) ppm. 13C NMR (100 MHz,
DMSO-d6):
d
¼169.7,155.0,131.4, 130.2,129.8,121.3,113.6, 69.7, 42.8,
22.7, 22.4, 13.4, 11.0 ppm. HRMS (ESI-TOF) m/z 222.14940
(222.14940 calcd for C13H20NO2, [MþHþ]).
4. Experimental
4.1. General considerations
4.2.3. N-Ethyl-N-[2-(hexyloxy)phenyl]acetamide 3c. IR (nmax/cmꢂ1):
2960, 2931, 2871, 1657 (C]O), 1595, 1499, 1455, 1397, 1283, 1264,
Reagents and solvents were purchased from standard sources
and purified and/or dried whenever necessary using standard
procedures prior to use. TLC analysis was performed routinely us-
ing 0.20 mm Al-backed silica-gel plates (Macherey-Nagel 60 F254).
Compounds were visualized using UV light (254 nm). Attenuated
total reflectance (ATR) IR spectra were collected on a Thermo-
scientific Nicolet iS10: smart iTR, equipped with a diamond ATR
crystal. For ATR data acquisition, a drop of the sample oil was
placed onto the crystal and the spectrum was recorded. An air
spectrum was used as a reference in absorbance calculations. The
sample spectra were collected at room temperature in the
4000e400 cmꢂ1 range by averaging 32 scans at a spectral resolu-
tion of 2 cmꢂ1. NMR spectra were acquired on a Bruker Avance
400 MHz spectrometer (1H NMR at 400 MHz and 13C NMR at
100 MHz) and were processed with the software TOPSPIN 3.1
(Bruker, Fitchburg, WI, USA). Deuterated dimethylsulfoxide
(DMSO-d6) was used as solvent. Chemical shifts are reported in
1231, 1013, 935, 750. 1H NMR (400 MHz, DMSO-d6):
d¼7.34 (dt, 1H,
J¼7.8, 1.8 Hz), 7.21 (dd, 1H, J¼7.8, 1.8 Hz), 7.13 (dd, 1H, J¼8.3, 1.0 Hz),
6.99 (dt, 1H, J¼7.6, 1.3 Hz), 4.00 (t, 2H, J¼6.3 Hz), 3.64 (dq, 1H,
J¼13.8, 6.9 Hz), 3.43 (dq, 1H, J¼13.8, 6.9 Hz), 1.64e1.72 (m, 2H), 1.62
(s, 3H), 1.36e1.43 (m, 2H), 1.26e1.32 (m, 4H), 0.95 (t, 3H, J¼7.1 Hz),
0.86 (t, 3H, J¼7.1 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
¼169.5,
d
154.9, 131.3, 130.2, 129.8, 121.2, 113.5, 68.0, 42.6, 31.3, 29.0, 25.5,
22.5, 22.2, 14.3, 13.3 ppm. HRMS (ESI-TOF) m/z 264.19524
(264.19635 calcd for C16H26NO2, [MþHþ]).
4.2.4. N-[2-(Benzyloxy)phenyl]-N-ethylacetamide
3d. IR
(nmax/
cmꢂ1): 2972, 2932, 2872, 1652 (C]O), 1595, 1499, 1401, 1397, 1282,
1264, 1222, 1075, 950, 750, 735. 1H NMR (400 MHz, DMSO-d6):
d
¼7.41e7.44 (m, 3H), 7.35e7.40 (m, 2H), 7.30e7.34 (m, 1H),
7.24e7.27 (m, 2H), 7.03 (dt, 1H, J¼7.5, 1.5 Hz), 5.14e5.21 (m, 2H),
3.72 (dq, 1H, J¼13.4, 7.0 Hz), 3.41 (dq, 1H, J¼13.4, 7.0 Hz), 1.65 (s,
3H), 0.96 (t, 3H, J¼7.2 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
parts per million (
d
) relative to TMS or deuterated solvent as an
d¼169.7, 154.6, 137.4, 131.6, 130.5, 129.9, 129.0, 129.0, 128.4, 127.8,

S.S. Ramos et al. / Tetrahedron 70 (2014) 8930e8937
8935
127.8, 121.7, 114.2, 70.0, 42.7, 22.4, 13.4 ppm. HRMS (ESI-TOF) m/z
270.14819 (270.14940 calcd for C17H20NO2, [MþHþ]).
7.0 Hz), 3.05 (dq, 1H, J¼13.1, 6.9 Hz), 2.88 (t, 2H, J¼7.3 Hz), 1.59 (s,
3H), 1.50e1.58 (m, 2H), 0.95 (t, 3H, J¼7.6 Hz), 0.91 (t, 3H,
J¼7.6 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
¼170.4, 139.2, 137.1,
d
4.2.5. N-(2-Ethoxyphenyl)-N-propylacetamide 3e. IR (nmax/cmꢂ1):
2966, 2933, 2875, 1652 (C]O), 1596, 1499, 1455, 1398, 1281, 1249,
1221, 1122, 1043, 923, 751, 734. 1H NMR (400 MHz, DMSO-d6):
130.0,129.5,126.6,126.0, 42.2, 32.4, 22.2, 21.9,13.5,13.1 ppm. HRMS
(ESI-TOF) m/z 238.12530 (238.12656 calcd for C13H20NOS, [MþHþ]).
d
¼7.34 (dt,1H, J¼7.8,1.6 Hz), 7.22 (dd,1H, J¼7.8,1.6 Hz), 7.13 (dd,1H,
4.2.11. N-Ethyl-N-[2-(hexylthio)phenyl]acetamide
3k. Previously
J¼8.0, 1.2 Hz), 6.99 (dt, 1H, J¼7.6, 1.2 Hz), 4.05e4.13 (m, 2H), 3.58
(ddd, 1H, J¼13.2, 8.8, 6.7 Hz), 3.55 (ddd, 1H, J¼13.2, 8.6, 6.8 Hz), 1.64
(s, 3H), 1.34e1.42 (m, 2H), 1.30 (t, 3H, J¼7.1 Hz), 0.81 (t, 3H,
described.3
4.2.12. N-[2-(Benzylthio)phenyl]-N-ethylacetamide 3l. IR
(nmax/
J¼7.6 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
d
¼169.9,154.7,131.6,
cmꢂ1): 2974, 2922, 2849, 1663 (C]O), 1579, 1494, 1466, 1455, 1379,
130.2, 129.7, 121.2, 113.6, 64.2, 49.6, 22.2, 21.1, 15.1, 11.8 ppm. HRMS
1289, 1265, 1063, 1028, 994, 919, 762, 735. 1H NMR (400 MHz,
(ESI-TOF) m/z 222.14959 (222.14940 calcd for C13H20NO2, [MþHþ]).
DMSO-d6):
d
¼7.54e7.56 (m, 1H), 7.36e7.42 (m, 3H), 7.29e7.33 (m,
2H), 7.21e7.27 (m, 3H), 4.29 (s, 2H), 3.91 (dq, 1H, J¼13.4, 7.2 Hz),
4.2.6. N-[2-(Hexyloxy)phenyl]-N-propylacetamide
3f. IR
(nmax/
3.04 (dq, 1H, J¼14.0, 7.2 Hz), 1.56 (s, 3H), 0.97 (t, 3H, J¼7.1 Hz) ppm.
cmꢂ1): 2956, 2930, 2872, 1657 (C]O), 1595, 1499, 1455, 1395, 1280,
13C NMR (100 MHz, DMSO-d6):
d¼169.2, 139.6, 137.3, 137.1, 130.2,
1248, 1221, 1123, 1044, 1014, 937, 750. 1H NMR (400 MHz, DMSO-
129.3, 129.3, 129.2, 128.9, 128.9, 127.7, 127.5, 126.4, 45.4, 35.2, 22.4,
d6):
d
¼7.33 (dt, 1H, J¼7.8, 1.8 Hz), 7.21 (dd, 1H, J¼7.6, 1.8 Hz), 7.12
13.4 ppm. HRMS (ESI-TOF) m/z 286.12683 (286.12656 calcd for
C
(dd, 1H, J¼8.3, 1.2 Hz), 6.98 (dt, 1H, J¼7.5, 1.2 Hz), 4.00 (t, 2H,
J¼6.1 Hz), 3.58 (ddd, 1H, J¼13.2, 8.4, 6.4 Hz), 3.33 (ddd, 1H, J¼13.2,
8.8, 6.1 Hz), 1.64e1.72 (m, 2H), 1.63 (s, 3H), 1.33e1.43 (m, 4H),
1.26e1.31 (m, 4H), 0.86 (t, 3H, J¼6.8 Hz), 0.80 (t, 3H, J¼7.5 Hz) ppm.
17H20NOS, [MþHþ]).
4.2.13. N-[2-(Methylthio)phenyl]-N-propylacetamide 3m. IR (nmax
/
cmꢂ1): 2961, 2930, 2872, 1661 (C]O), 1579, 1470, 1374, 1265, 1124,
13C NMR (100 MHz, DMSO-d6):
d¼169.9, 154.9, 131.7, 130.1, 129.8,
1068, 1042, 953, 731, 702. 1H NMR (400 MHz, DMSO-d6):
121.2, 113.5, 68.0, 49.5, 31.2, 29.0, 25.5, 22.5, 22.2, 21.1, 14.3,
d
¼7.40e7.44 (m, 1H), 7.35 (d, 1H, J¼7.8 Hz), 7.23e7.24 (m, 2H), 3.88
11.7 ppm. HRMS (ESI-TOF) m/z 278.21234 (278.21200 calcd for
(ddd, 1H, J¼13.2, 9.2, 6.7 Hz), 2.98 (ddd, 1H, J¼13.6, 9.2, 5.9 Hz), 2.47
C
17H28NO2, [MþHþ]).
(s, 3H),1.64 (s, 3H),1.34e1.51 (m, 2H), 0.82 (t, 3H, J¼7.4 Hz) ppm. 13
C
NMR (100 MHz, DMSO-d6):
d¼169.5, 139.3, 138.5, 129.9, 129.4,
4.2.7. N-Hexyl-N-(2-propoxyphenyl)acetamide 3g. IR (nmax/cmꢂ1):
2958, 2928, 2872, 2858, 1656 (C]O), 1596, 1499, 1454, 1395, 1274,
1257,1235, 1122,1044, 978, 750, 727. 1H NMR (400 MHz, DMSO-d6):
125.6, 125.5, 48.5, 13.9, 11.7 ppm. HRMS (ESI-TOF) m/z 224.11042
(224.11091 calcd for C12H18NOS, [MþHþ]).
d
¼7.33 (dt,1H, J¼7.9,1.8 Hz), 7.21 (dd,1H, J¼7.6,1.8 Hz), 7.12 (dd,1H,
4.2.14. N-[2-(Ethylthio)phenyl]-N-propylacetamide 3n. IR
(nmax/
J¼8.2, 1.0 Hz), 6.99 (dt, 1H, J¼7.5, 1.0 Hz), 3.97 (t, 2H, J¼6.2 Hz), 3.60
(ddd, 1H, J¼13.2, 8.0, 6.7 Hz), 3.39 (ddd, 1H, J¼13.2, 8.0, 6.6 Hz), 1.70
(sext, 2H, J¼7.0 Hz), 1.63 (s, 3H), 1.29e1.39 (m, 2H), 1.15e1.25 (m,
6H), 0.96 (t, 3H, J¼7.6 Hz), 0.82 (t, 3H, J¼7.0 Hz) ppm. 13C NMR
cmꢂ1): 2965, 2930, 2873, 1656 (C]O), 1583, 1470, 1392, 1295, 1232,
1149,1096,1065,1034, 973, 765, 733. 1H NMR (400 MHz, DMSO-d6):
d
¼7.37e7.42 (m, 2H), 7.20e7.26 (m, 2H), 3.88 (ddd, 1H, J¼13.2, 8.8,
7.2 Hz), 3.01 (q, 2H, J¼7.4 Hz), 2.97 (ddd, 1H, J¼12.7, 9.1, 6.4 Hz), 1.63
(s, 3H), 1.39e1.50 (m, 2H), 1.27 (t, 3H, J¼7.3 Hz), 0.82 (t, 3H,
(100 MHz, DMSO-d6):
d¼169.8, 154.8, 131.6, 130.0, 129.7, 121.2,
113.5, 69.6, 47.8, 31.6, 27.8, 26.5, 22.5, 22.5, 22.2, 14.3, 10.9 ppm.
HRMS (ESI-TOF) m/z 278.21166 (278.21200 calcd for C17H28NO2,
[MþHþ]).
J¼7.7 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
¼170.9, 139.4,
d
136.7, 130.0, 129.6, 126.6, 126.1, 49.0, 24.7, 22.3, 21.0, 14.0, 11.6 ppm.
HRMS (ESI-TOF) m/z 238.12549 (238.12656 calcd for C13H20NOS,
[MþHþ]).
4.2.8. N-benzyl-N-(2-ethoxyphenyl)acetamide 3h. IR (nmax/cmꢂ1):
2984, 2930, 1738, 1652 (C]O), 1500, 1396, 1264, 1217, 1044, 733,
4.2.15. N-[2-(Hexylthio)phenyl]-N-propylacetamide 3o. IR
(nmax/
701. 1H NMR (400 MHz, DMSO-d6):
d¼7.20e7.35 (m, 4H), 7.17 (d,
cmꢂ1): 2958, 2928, 2857, 1660 (C]O), 1583, 1469, 1437, 1392, 1295,
2H, J¼7.4 Hz), 7.07 (d, 1H, J¼8.0 Hz), 7.02 (dd, 1H, J¼8.0, 1.4 Hz), 6.88
(t, 1H, J¼7.6 Hz), 5.01 (d, 1H, J¼15.7 Hz), 4.46 (d, 1H, J¼14.9 Hz),
3.88e4.10 (m, 2H), 1.74 (s, 3H), 1.27 (t, 3H, J¼7.4 Hz) ppm. 13C NMR
1255, 1232, 1149, 1096, 1064, 1036, 733. 1H NMR (400 MHz, DMSO-
d6):
d
¼7.36e7.38 (m, 2H), 7.20e7.25 (m, 2H), 3.87 (ddd, 1H, J¼13.2,
8.8, 7.8 Hz), 2.99 (t, 2H, J¼7.5 Hz), 2.94 (ddd, 1H, J¼13.2, 8.8, 6.2 Hz),
1.63 (s, 3H),1.56e1.62 (m, 2H),1.37e1.50 (m, 4H),1.24e1.28 (m, 4H),
0.86 (t, 3H, J¼7.0 Hz), 0.82 (t, 3H, J¼7.7 Hz) ppm. 13C NMR (100 MHz,
(100 MHz, DMSO-d6):
d¼170.5, 154.4, 138.2, 131.3, 130.0, 129.8,
128.6, 128.6, 128.5, 128.5, 127.4, 121.0, 113.7, 64.0, 51.4, 22.2,
15.0 ppm. HRMS (ESI-TOF) m/z 270.14944 (270.14940 calcd for
C
DMSO-d6):
d¼169.4, 139.8, 137.3, 130.2, 129.3, 126.6, 125.9, 48.7,
17H20NO2, [MþHþ]).
31.2, 30.5, 28.5, 28.4, 22.5, 22.4, 21.2,14.3,11.7 ppm. HRMS (ESI-TOF)
m/z 294.18792 (294.18916 calcd for C17H28NOS, [MþHþ]).
4.2.9. N-Ethyl-N-[2-(ethylthio)phenyl]acetamide 3i. IR (nmax/cmꢂ1):
2972, 2930, 2872, 1655 (C]O), 1582, 1567, 1470, 1441, 1392, 1296,
1263, 1245, 1144, 1102, 1080, 1063, 1035, 994, 918, 759, 734. 1H NMR
4.2.16. N-[2-(ethylthio)phenyl]-N-hexylacetamide
3p. IR
(nmax/
cmꢂ1): 2955, 2927, 2857, 1660 (C]O), 1583, 1528, 1469, 1456, 1392,
(400 MHz, DMSO-d6):
d
¼7.38e7.40 (m, 2H), 7.21e7.23 (m, 2H), 3.94
1374, 1309, 1241, 1171, 1156, 1126, 1096, 1065, 1034, 1015, 758, 733.
(dq, 1H, J¼14.0, 7.1 Hz), 3.08 (dq, 1H, J¼14.0, 7.1 Hz), 2.98 (q, 2H,
J¼7.0 Hz), 1.62 (s, 3H), 1.25 (t, 3H, J¼6.9 Hz), 0.99 (t, 3H,
1H NMR (400 MHz, DMSO-d6):
d
¼7.37e7.42 (m, 2H), 7.21e7.26 (m,
2H), 3.91 (ddd, 1H, J¼13.6, 9.6, 6.5 Hz), 3.01 (q, 2H, J¼7.5 Hz),
2.94e3.02 (m, 1H, under q at 3.01 ppm), 1.63 (s, 3H), 1.36e1.47 (m,
2H), 1.27 (t, 3H, J¼7.2 Hz), 1.17e1.25 (m, 6H), 0.83 (t, 3H,
J¼6.9 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
¼169.3, 139.5,
d
137.3, 130.2, 129.3, 126.5, 125.9, 41.9, 24.7, 22.5, 14.0, 13.3 ppm.
HRMS (ESI-TOF) m/z 224.11063 (224.11091 calcd for C12H18NOS,
[MþHþ]).
J¼6.8 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
¼170.8, 139.4,
d
136.8, 129.9, 129.5, 126.6, 126.1, 47.3, 31.3, 27.6, 26.3, 24.7, 22.3, 22.3,
14.2, 14.0 ppm. HRMS (ESI-TOF) m/z 280.17253 (280.17351 calcd for
4.2.10. N-Ethyl-N-[2-(propylthio)phenyl]acetamide 3j. IR
(nmax
/
C
16H26NOS, [MþHþ]).
cmꢂ1): 2965, 2932, 2873, 1660 (C]O), 1583, 1470, 1441, 1393, 1298,
1268, 1145, 1063, 1036, 761, 735. 1H NMR (400 MHz, DMSO-d6):
4.2.17. N-Hexyl-N-[2-(propylthio)phenyl]acetamide 3q. IR
(nmax/
d¼7.32e7.37 (m, 2H), 7.13e7.21 (m, 2H), 3.87 (dq, 1H, J¼13.6,
cmꢂ1): 2959, 2927, 2857, 1660 (C]O), 1583, 1528, 1470, 1456, 1434,

8936
S.S. Ramos et al. / Tetrahedron 70 (2014) 8930e8937
1393, 1300, 1260, 1241, 1171, 1157, 1126, 1095, 1065, 1015, 795, 745,
729. 1H NMR (400 MHz, DMSO-d6):
J¼7.8 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
¼169.2,141.4,133.0,
d
d
¼7.35e7.42 (m, 2H), 7.18e7.26
130.4, 129.7, 129.5, 127.1, 42.1, 22.8, 18.7, 15.2, 13.5 ppm. HRMS (ESI-
(m, 2H), 3.90 (ddd, 1H, J¼13.2, 9.2, 6.7 Hz), 3.02 (ddt, 1H, J¼13.3,
9.06, 5.0 Hz), 2.98 (t, 2H, J¼7.1 Hz), 1.64 (s, 3H), 1.58e1.67 (m, 2H),
1.36e1.47 (m, 2H), 1.19e1.26 (m, 6H), 0.99 (t, 3H, J¼7.1 Hz), 0.83 (t,
TOF) m/z 272.05426 (272.05536 calcd for C12H18NOSe, [MþHþ]).
4.2.23. N-Ethyl-N-[2-(hexylseleno)phenyl]acetamide 3w. IR
(nmax/
3H, J¼6.8 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
d
¼169.4, 139.9,
cmꢂ1): 2957, 2928, 2855, 1656 (C]O), 1579, 1467, 1441, 1390, 1375,
137.3, 130.3,129.3,126.6,125.9, 47.0, 32.4, 31.5, 27.8, 26.6, 22.5, 22.4,
22.0, 14.4, 13.7 ppm. HRMS (ESI-TOF) m/z 294.18892 (294.18916
calcd for C17H28NOS, [MþHþ]).
1296, 1265, 1144, 1099, 1051, 1030, 993, 917, 759, 729. 1H NMR
(400 MHz, DMSO-d6):
d
¼7.50 (dd, 1H, J¼7.8, 1.3 Hz), 7.36 (dt, 1H,
J¼7.5, 1.6 Hz), 7.29 (dt, 1H, J¼7.2, 1.4 Hz), 7.24 (dd, 1H, J¼7.6, 1.6 Hz),
3.95 (dq, 1H, J¼13.7, 7.0 Hz), 3.08 (dq, 1H, J¼13.7, 7.0 Hz), 3.00 (t, 2H,
J¼7.5 Hz), 1.65e1.70 (m, 2H), 1.64 (s, 3H), 1.29e1.41 (m, 2H),
1.24e1.28 (m, 4H), 1.02 (t, 3H, J¼7.2 Hz), 0.85 (t, 3H, J¼6.8 Hz) ppm.
4.2.18. N-Benzyl-N-[2-(ethylthio)phenyl]acetamide 3r. IR
(nmax/
cmꢂ1): 2969, 2926, 2869, 1653 (C]O), 1582, 1467, 1434, 1388, 1280,
1249, 1209, 1064, 1030, 970, 774, 759, 700. 1H NMR (400 MHz,
13C NMR (100 MHz, DMSO-d6):
d¼169.1, 141.3, 133.2, 130.3, 129.7,
DMSO-d6):
d
¼7.41 (dd, 1H, J¼8.0, 1.4 Hz), 7.34 (dt, 1H, J¼7.6, 1.6 Hz),
129.5, 127.0, 42.1, 31.1, 29.5, 29.4, 25.1, 22.7, 22.4, 14.3, 13.4 ppm.
HRMS (ESI-TOF) m/z 328.11670 (328.11796 calcd for C16H26NOSe,
[MþHþ]).
7.22e7.30 (m, 3H), 7.17e7.20 (m, 2H), 7.06 (dt, 1H, J¼7.5, 1.6 Hz),
6.80 (dd, 1H, J¼7.9, 1.4 Hz), 5.42 (d, 1H, J¼14.2 Hz), 3.97 (d, 1H,
J¼14.2 Hz), 3.02 (dq, 2H, J¼7.3, 3.0 Hz), 1.72 (s, 3H), 1.28 (t, 3H,
J¼7.2 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
d
¼170.0,139.4,137.9,
4.2.24. N,N-Diethyl-2-(ethylthio)aniline 4a. IR (nmax/cmꢂ1): 2977,
137.0, 130.2, 129.4, 128.9, 128.9, 128.7, 128.7, 127.7, 126.7, 125.7, 50.3,
24.7, 22.4, 14.1 ppm. HRMS (ESI-TOF) m/z 286.12667 (286.12656
calcd for C17H20NOS, [MþHþ]).
2929, 1584, 1471, 1391, 1303, 1265, 1067, 1050, 730, 702. 1H NMR
(400 MHz, DMSO-d6):
d
¼7.15e7.18 (m, 1H), 7.04e7.12 (m, 3H), 2.96
(q, 4H, J¼7.1 Hz), 2.85 (q, 2H, J¼7.3 Hz), 1.26 (t, 3H, J¼7.3 Hz), 0.92 (t,
6H, J¼7.1 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
¼147.8, 136.5,
d
4.2.19. N-Benzyl-N-[2-(benzylthio)phenyl]acetamide 3s. IR
(
nmax
/
125.7, 124.9, 123.1, 47.1, 24.4, 14.1, 12.7 ppm. HRMS (ESI-TOF) m/z
210.13053 (210.13165 calcd for C12H20NS, [MþHþ]).
cmꢂ1): 3028, 2926, 1655 (C]O), 1582, 1494, 1469, 1454, 1435, 1386,
1357, 1284, 1210, 1064, 1029, 971, 776, 755, 729, 698. 1H NMR
(400 MHz, DMSO-d6):
d
¼7.56 (dd, 1H, J¼8.1, 1.6 Hz), 7.40e7.43 (m,
4.2.25. N-Ethyl-2-(methylthio)-N-propylaniline 4b. IR (nmax/cmꢂ1):
2959, 2920, 2871, 2810, 1578, 1470, 1438, 1379, 1268, 1225, 1171,
1126, 1084, 1068, 1042, 752, 731. 1H NMR (400 MHz, DMSO-d6):
2H), 7.29e7.35 (m, 3H), 7.20e7.28 (m, 4H), 7.10e7.13 (m, 2H), 7.06
(dt, 1H, J¼7.5, 1.2 Hz), 6.77 (dd, 1H, J¼7.7, 1.2 Hz), 5.37 (d, 1H,
J¼14.7 Hz), 4.30 (s, 2H), 3.86 (d, 1H, J¼13.9 Hz), 1.64 (s, 3H) ppm. 13
C
d
¼7.10e7.12 (m, 2H), 7.07e7.09 (m, 2H), 2.93 (q, 2H, J¼7.1 Hz), 2.87
NMR (100 MHz, DMSO-d6):
d
¼170.0, 139.7, 137.8, 137.7, 136.7, 130.2,
(t, 2H, J¼7.2 Hz), 2.31 (s, 3H), 1.34 (sext, 2H, J¼7.3 Hz), 0.93 (t, 3H,
129.4, 129.3, 129.0, 128.9, 128.7, 128.0, 127.7, 127.6, 126.2, 50.5, 35.4,
J¼7.1 Hz), 0.82 (t, 3H, J¼7.4 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
22.4 ppm. HRMS (ESI-TOF) m/z 348.14149 (348.114221 calcd for
d
¼147.7, 138.0, 125.1, 124.5, 124.2, 123.0, 54.5, 48.0, 20.6, 13.9, 12.5,
C
22H22NOS, [MþHþ]).
12.1 ppm. HRMS (ESI-TOF) m/z 210.13139 (210.13165 calcd for
C
12H20NS, [MþHþ]).
4.2.20. N-[(2-Ethylthio)phenyl]-N-(2-methylbenzyl)acetamide 3t. IR
nmax/cmꢂ1): 2970, 2929, 2872, 1658 (C]O), 1584, 1471, 1440, 1386,
1379, 1359, 1301, 1271, 1248, 1049, 1040, 969, 764, 738. 1H NMR
(400 MHz, DMSO-d6):
(
4.3. VT-NMR spectroscopy
d
¼7.40 (dd, 1H, J¼8.0, 1.3 Hz), 7.32 (dt, 1H,
VT-NMR experiments were run on a Bruker Avance 400 MHz
spectrometer equipped with a variable temperature probe. Tem-
perature calibrations were performed before the experiments by
means of a thermocouple with an uncertainly not exceeding ꢃ2 K.
The conditions were kept as equal as possible with all subsequent
work. To selectively irradiate the desired signal, a 50 Hz wide
shaped pulse was calculated with a refocusing-SNOB shape10 and
a pulse width of 37 ms.
J¼7.6, 1.4 Hz), 7.09e7.14 (m, 2H), 7.04e7.08 (m, 2H), 7.02 (dd, 1H,
J¼7.5, 1.4 Hz), 6.74 (dd, 1H, J¼7.8, 1.3 Hz), 5.34 (d, 1H, J¼14.7 Hz),
4.18 (d, 1H, J¼14.7 Hz), 3.02 (q, 2H, J¼7.3 Hz), 2.06 (s, 3H), 1.71 (s,
3H), 1.27 (t, 3H, J¼7.3 Hz) ppm. 13C NMR (100 MHz, DMSO-d6):
d
¼169.7, 139.1, 137.0, 136.8, 135.5, 130.4, 130.2, 129.8, 129.4, 127.8,
126.7, 126.2, 125.7, 47.2, 24.8, 22.4, 19.0, 14.2 ppm. HRMS (ESI-TOF)
m/z 300.14192 (300.14221 calcd for C18H22NOS, [MþHþ]).
4.2.21. N-(2-Methylphenyl)-N-[2-(2-methylbenzylthio)phenyl]acet-
amide 3u. IR (nmax/cmꢂ1): 3051, 3021, 2979, 2927, 1655 (C]O),
1584, 1493, 1471, 1391, 1265, 1050, 1037, 971, 730, 702. 1H NMR
4.4. In vitro cell viability studies using MCF-7, NHDF, and
LNCaP cells
(400 MHz, DMSO-d6):
d
¼7.59 (dd, 1H, J¼8.0, 1.0 Hz), 7.34 (dt, 1H,
The cells used in this study were human breast (MCF-7) and
prostate (LNCaP) cancer cells as well as normal human dermal fi-
broblasts (NHDF) (all acquired to ATCCdAmerican Type Culture
Collection).
Unless otherwise stated, chemicals (analytical grade), assay re-
agents, culture media, and supplements are all from SigmaeAldrich.
The studied compounds were dissolved in dimethylsulfoxide
(DMSO) and the final solvent concentration in the MTT experiment
was 0.3%. This concentration of DMSO has no significant effect on
cell viability (data not shown).
J¼7.7, 1.4 Hz), 7.28 (d, 1H, J¼7.5 Hz), 7.17e7.22 (m, 2H), 7.01e7.14 (m,
5H), 6.94 (d, 1H, J¼7.3 Hz), 6.73 (dd, 1H, J¼7.8, 1.2 Hz), 5.30 (d, 1H,
J¼14.7 Hz), 4.24e4.30 (m, 2H), 4.05 (d, 1H, J¼14.7 Hz), 2.40 (s, 3H),
2.03 (s, 3H), 1.64 (s, 3H) ppm. 13C NMR (100 MHz, DMSO-d6):
d
¼169.6, 139.5, 137.2, 136.7, 135.4, 134.7, 130.9, 130.4, 130.2, 130.1,
129.8, 129.3, 128.3, 128.1, 127.8, 126.4, 126.3, 126.1, 55.4, 49.1, 34.2,
22.3, 19.0 ppm. HRMS (ESI-TOF) m/z 376.17314 (376.17351 calcd for
C
24H26NOS, [MþHþ]).
4.2.22. N-Ethyl-N-[2-(ethylseleno)phenyl]acetamide 3v. IR
(nmax/
cmꢂ1): 2972, 2930, 2872, 1655 (C]O), 1582, 1470, 1441, 1392, 1376,
4.4.1. Culture of cells. Cells were routinely maintained in 75 or
150 cm2 T-flasks at 37 ꢄC in a humidified atmosphere containing 5%
CO2. Human dermal fibroblasts were cultured in RPMI medium
supplemented with 10% fetal bovine serum (FBS), HEPES (0.01 M),
1297, 1263, 1144, 1161, 1102, 1063, 1035, 994, 760, 734. 1H NMR
(400 MHz, DMSO-d6):
d
¼7.50 (dd, 1H, J¼7.9, 1.2 Hz), 7.37 (dt, 1H,
J¼7.3, 1.8 Hz), 7.30 (dt, 1H, J¼7.9, 1.2 Hz), 7.25 (dd, 1H, J¼7.6, 1.8 Hz),
3.94 (dq, 1H, J¼14.0, 7.0 Hz), 3.08 (dq, 1H, J¼14.0, 7.0 Hz), 3.00 (q,
2H, J¼7.5 Hz), 1.64 (s, 3H), 1.40 (t, 3H, J¼7.8 Hz), 1.02 (t, 3H,
L-glutamine (0.02 M), and sodium pyruvate (0.001 M) and 1%
antibiotic/antimycotic (10,000 units/ml penicillin, 10 mg/ml

S.S. Ramos et al. / Tetrahedron 70 (2014) 8930e8937
8937
Jørgensen, K. A. Angew. Chem., Int. Ed. 2006, 45, 1147; (c) Ishichi, Y.; Ikeura, Y.;
Natsugari, H. Tetrahedron 2004, 60, 4481; (d) Albert, J. S.; Ohnmacht, C.;
Bernstein, P. R.; Rumsey, W. L.; Aharony, D.; Masek, B. B.; Dembofsky, B. T.;
Koether, G. M.; Potts, W.; Evenden, J. L. Tetrahedron 2004, 60, 4337; (e) Curran,
D. P.; Chen, C. H.-T.; Geib, S. J.; Lapierre, A. J. B. Tetrahedron 2004, 60, 4413; (f)
Clayden, J.; McCarthy, C.; Helliwell, M. Chem. Commun. 1999, 2059; (g) Curran,
D. P.; Geib, S. J.; DeMello, N. C. Tetrahedron 1999, 55, 5681; (h) Curran, D. P.; Liu,
W.; Chen, C. H.-T. J. Am. Chem. Soc. 1999, 121, 11012; (i) Avalos, M.; Babiano, R.;
streptomycin and 25 mg/ml amphotericin B). Dubelco’s Modified
Eagle’s Medium high glucose supplemented with 10% fetal bovine
serum and 1% antibiotic/antimycotic was used to culture MCF-7
cells. LNCaP cells were cultured in RPMI medium supplemented
with 10% FBS and 1% antibiotic (10,000 units/ml penicillin and
10 mg/ml streptomycin). The cells used in the experiments were in
passages 5e6 (NHDF), 36e37 (MCF-7), and 23e24 (LNCaP).
ꢀ
Cintas, P.; Higes, F. J.; Jimenez, J. L.; Palacios, J. C.; Silvero, G. Tetrahedron:
Asymmetry 1999, 10, 4071; (j) Fujita, M.; Kitagawa, O.; Izawa, H.; Dobashi, A.;
Fukaya, H.; Taguchi, T. Tetrahedron Lett. 1999, 40, 1949; (k) Clayden, J. Synlett
1998, 810; (l) Ikeura, Y.; Ishichi, Y.; Tanaka, T.; Fujishima, A.; Murabayashi, M.;
Kawada, M.; Ishimaru, T.; Kamo, I.; Doi, T.; Natsugari, H. J. Med. Chem. 1998, 41,
4232; (m) Ikeura, Y.; Ishimaru, T.; Doi, T.; Kawada, M.; Fujishima, A.; Natsugari,
H. Chem. Commun. 1998, 2141; (n) Kitagawa, O.; Izawa, H.; Sato, K.; Dobashi, A.;
Taguchi, T. J. Org. Chem. 1998, 63, 2634; (o) Ahmed, A.; Bragg, R. A.; Clayden, J.;
Lai, L. W.; McCarthy, C.; Pink, J. H.; Westlund, N.; Yasin, S. A. Tetrahedron 1998,
54, 13277; (p) Clayden, J. Angew. Chem., Int. Ed. 1997, 36, 949; (q) Curran, D. P.;
Hale, G. R.; Geib, S. J.; Balog, A.; Cass, Q. B.; Degani, A. L. G.; Hernandez, M. Z.;
Freitas, L. C. G. Tetrahedron: Asymmetry 1997, 8, 3955; (r) Clayden, J.; Westlund,
N.; Wilson, F. X. Tetrahedron Lett. 1996, 37, 5577; (s) Thayumanavan, S.; Beak, P.;
Curran, D. P. Tetrahedron Lett. 1996, 37, 2899; (t) Hughes, A. D.; Price, D. A.;
Shishkin, O.; Simpkins, N. S. Tetrahedron Lett. 1996, 37, 7607; (u) Bowles, P. J.;
Clayden, J. M.; Tomkinson, M. Tetrahedron Lett. 1995, 36, 9219.
4.4.2. MTT assay. Cell viability was evaluated by quantifying the
extent of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) according to a previously de-
scribed procedure.9 Briefly, cells were seeded in 48-well plates
(0.5ꢁ104 cells/well) in the culture medium and after 48 h they were
treated with the different compounds at 30 mM for 48 h, with un-
treated cells serving as negative control and H2O2 (1 mM) as pos-
itive control. For the doseeresponse studies, the concentrations
evaluated were 0.01, 0.1, 1, 10, 50, and 100 mM. At the end of in-
cubation the media in wells were removed and replaced with fresh
media and MTT solution (5 mg/ml in phosphate buffer saline) and
incubated at 37 ꢄC for 4 h. Thereafter, media-containing MTT were
removed and formazan crystals were dissolved with DMSO and
absorbance was recorded in an Anthus 2020 microplate reader at
570 nm. The extent of cell death was expressed as the percentage of
cell viability in comparison with control cells.
3. Ramos, S. S.; Reis, L. V.; Boto, R. E. F.; Santos, P. F.; Almeida, P. Tetrahedron Lett.
2013, 54, 5441.
4. Representative examples: (a) Siddall, T. H., III. J. Org. Chem. 1966, 31, 3719; (b)
Siddall, T. H., III; Steward, W. E. J. Org. Chem. 1969, 34, 2927; (c) Steward, W. E.;
Siddall, T. H., III. Chem. Rev.1970, 70, 517; (d) Staskun, B. J. Org. Chem.1981, 46,1643.
5. Yamasaki, R.; Tanatani, A.; Azumaya, I.; Saito, S.; Yamaguchi, K.; Kagechika, H.
Org. Lett. 2003, 5, 1265.
6. Eyring, H. Chem. Rev. 1935, 17, 65.
7. Representative examples: (a) Shvo, Y.; Taylor, E. C.; Mislow, K.; Raban, M. J. Am.
Chem. Soc.1967, 89, 4910; (b) Siddall, T. H., III; Steward, W. E. J. Phys. Chem.1969, 73,
40; (c) Kondo, O. K.; Iida, T.; Fujita, H.; Suzuki, T.; Yamaguchi, K.; Murakami, Y.
Tetrahedron 2000, 56, 8883; (d) Ates, A.; Curran, D. P. J. Am. Chem. Soc. 2001, 123,
5130; (e) Clayden, J., Ed. (Tetrahedron Symposium-in-Print on Axially Chiral Am-
ides)Tetrahedron 2004, 60, 4325e4558; (f) Kitagawa, O.; Yoshikawa, M.; Tanabe, H.;
Morita, T.; Takahashi, M.; Dobashi, Y.; Taguchi, T. J. Am. Chem. Soc. 2006, 128, 12923.
8. (a) Kankanala, K.; Reddy, V. R.; Devi, Y. P.; Mangamoori, L. N.; Mukkanti, K.; Pal,
S. J. Chem. 2013, 1e8 http://dx.doi.org/10.1155/2013/816769 Hindawi Publishing
Corporation, http://www.hindawi.com/journals/jchem; (b) Mareddy, J.; Nalla-
pati, S. B.; Anireddy, J.; Devi, Y. P.; Mangamoori, L. N.; Kapavarapu, R.; Pal, S.
Bioorg. Med. Chem. Lett. 2013, 23, 6721; (c) Zhong, B.; Cai, X. H.; Chennamaneni,
S.; Yi, X.; Liu, L. L.; Pink, J. J.; Dowlati, A.; Xu, Y.; Zhou, A. M.; Su, B. Eur. J. Med.
Chem. 2012, 47, 432; (d) Zhong, B.; Lama, R.; Smith, K. M.; Xu, Y.; Su, B. Bioorg.
Med. Chem. Lett. 2011, 21, 5324; (e) Su, B.; Chen, S. A. Bioorg. Med. Chem. Lett.
2009, 19, 6733; (f) Su, B.; Darby, M. V.; Brueggerneier, R. W. J. Comb. Chem. 2008,
10, 475; (g) Su, B.; Diaz-Cruz, E. S.; Landini, S.; Brueggemeier, R. W. J. Med. Chem.
2006, 49, 1413; (h) Su, B.; Landini, S.; Davis, D. D.; Brueggemeier, R. W. J. Med.
Chem. 2007, 50, 1635; (i) Su, B.; Tian, R.; Darby, M. V.; Brueggemeier, R. W. J.
Med. Chem. 2008, 51, 1126.
4.4.3. Statistics. The experiments were performed in quintuplicate
and the results of the cell proliferation were expressed as aver-
ageꢃstandard deviation (SD). These calculations as well as the
quantification of the potential association between the lipophilicity
(log P) and cytotoxic effects were performed using the program
Microsoft Excel 2010. The half-medium inhibitory concentration
(IC50) values were calculated from the obtained doseeresponse
curve by sigmoid fitting.
Acknowledgements
This work was financed by FCT (Project PTDC/QUI-QUI/100896/
2008) and COMPETE (Project Pest-C/SAU/UI0709/2011).
References and notes
~
9. (a) Cruz, C.; Cairrao, E.; Silvestre, S.; Breitenfeld, L.; Almeida, P.; Queiroz, J. A.
PLoS One 2011, 6, e27078; (b) Cytotoxicity In Culture of Animal Cells: A Manual of
Basic Technique; Freshney, R. I., Ed.; John Wiley & Sons: Hoboken, NJ, 2005;
Chapter 22, pp 359e373.
1. Curran, D. P.; Qi, H.; Geib, S. J.; DeMello, N. C. J. Am. Chem. Soc. 1994, 116, 3131.
2. (a) Brandes, S.; Niess, B.; Bella, M.; Prieto, A.; Overgaard, J.; Jørgensen, K. A.
Chem.dEur. J. 2006, 12, 6039; (b) Brandes, S.; Bella, M.; Kjærsgaard, A.;
ꢁ
10. Kupce, E.; Boyd, J.; Campbell, I. D. J. Magn. Reson., Ser. B 1995, 106, 300.