A Palladium-Catalyzed Carbonylation Approach to Eight-Membered Lactam Derivatives with Antitumor Activity

Article abstract of DOI:10.1002/chem.201504443

The reactivity of 2-(2-alkynylphenoxy)anilines under PdI₂/KI-catalyzed oxidative carbonylation conditions has been studied. Although a different reaction pathway could have been operating, N-palladation followed by CO insertion was the favored

Full text of DOI:10.1002/chem.201504443

DOI: 10.1002/chem.201504443
Full Paper
&
Reaction Mechanisms
A Palladium-Catalyzed Carbonylation Approach to Eight-
Membered Lactam Derivatives with Antitumor Activity
Raffaella Mancuso,*[a] Dnyaneshwar S. Raut,[a] Nadia Marino,[b] Giorgio De Luca,[c]
Cinzia Giordano,[d] Stefania Catalano,[d] Ines Barone,[d] Sebastiano Andò,[d] and
Bartolo Gabriele*[a]
Abstract: The reactivity of 2-(2-alkynylphenoxy)anilines
under PdI2/KI-catalyzed oxidative carbonylation conditions
has been studied. Although a different reaction pathway
could have been operating, N-palladation followed by CO in-
sertion was the favored pathway with all substrates tested,
including those containing an internal or terminal triple
bond. This led to the formation of a carbamoylpalladium
species, the fate of which, as predicted by theoretical calcu-
lations, strongly depended on the nature of the substituent
on the triple bond. In particular, 8-endo-dig cyclization pref-
erentially occurred when the triple bond was terminal, lead-
ing to the formation of carbonylated z-lactam derivatives,
the structures of which have been confirmed by XRD analy-
sis. These novel medium-sized heterocyclic compounds
showed antitumor activity against both estrogen receptor-
positive (MCF-7) and triple negative (MDA-MB-231) breast
cancer cell lines. In particular, z-lactam 3j’ may represent
a novel and promising antitumor agent because biological
tests clearly demonstrate that this compound significantly
reduces cell viability and motility in both MCF-7 and MDA-
MB-231 breast cancer cell lines, without affecting normal
breast epithelial cell viability.
Introduction
tively large ring) factors, which tend to raise the cyclization ac-
tivation energy.[4]
Medium-sized ring-based scaffolds (7–11-membered, carbo-
and heterocyclic) are molecular frameworks of particular inter-
est, owing to their biological activity[1] and occurrence in many
important natural products.[2] Despite their significance, rela-
tively few efficient synthetic methods for their preparation
through cyclization of acyclic precursors are known so far,[3]
compared with the quite abundant annulation protocols for
the preparation of five- and six-membered rings. This is funda-
mentally due to a combination of unfavorable enthalpic (trans-
annular interactions) and entropic (difficulty in closing a rela-
Catalytic cyclocarbonylation reactions are currently widely
recognized as one of the most important and powerful syn-
thetic tools for the direct preparation of cyclic carbonyl com-
pounds from acyclic precursors.[5,6] Although numerous exam-
ples are known in the literature for the formation of five-, six-,
and even four-membered rings by cyclocarbonylation ap-
proaches, very few examples have been reported so far for the
direct synthesis of medium-sized rings.[5b,6i] Considering our ex-
perience in oxidative carbonylation chemistry,[5d,7] in particular,
the use of the PdI2/KI catalytic system we proposed some
years ago for alkyne carbonylation,[8] herein we have studied
the reactivity of 2-(2-alkynylphenoxy)anilines under PdI2/KI-cat-
alyzed oxidative carbonylation conditions, with the aim of ob-
taining novel carbonylated medium-sized rings of biological in-
terest.
[a] Dr. R. Mancuso, Dr. D. S. Raut, Prof. Dr. B. Gabriele
Laboratory of Industrial and Synthetic Organic Chemistry (LISOC)
Department of Chemistry and Chemical Technologies
University of Calabria, Via Pietro Bucci, 12/C
87036 Arcavacata di Rende (CS) (Italy)
E-mail: raffaella.mancuso@unical.it
bartolo.gabriele@unical.it
Results and Discussion
[b] Dr. N. Marino
Department of Chemistry and Chemical Technologies
University of Calabria, Via Pietro Bucci, 14/C
87036 Arcavacata di Rende (CS) (Italy)
In principle, different reaction pathways could be followed
when 2-(2-alkynylphenoxy)anilines 1 are allowed to react in
the presence of the PdI2/KI catalytic system (leading in situ to
K2PdI4) under oxidative carbonylation conditions (Scheme 1).
The first possibility (path a) could correspond to N-palladation
of 1, with the formation of complex I, stabilized by triple-bond
coordination, followed by CO insertion to give the carbamoyl-
palladium intermediate II. The latter species could then evolve
either by direct attack from an external alcohol ROH, to afford
acyclic carbamates 2 (path b), or through intramolecular syn 8-
[c] Dr. G. De Luca
Institute on Membrane Technology, National Research Council
Via Pietro Bucci, 17/C, 87036 Arcavacata di Rende (CS) (Italy)
[d] Dr. C. Giordano, Prof. Dr. S. Catalano, Dr. I. Barone, Prof. Dr. S. Andò
Health Center and Department of Pharmacy
and Health and Nutritional Science
University of Calabria, 87036 Arcavacata di Rende (CS) (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201504443.
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Scheme 1. Possible divergent pathways in the PdI2/KI-catalyzed oxidative carbonylation of 2-(2-alkynylphenoxy)anilines 1.
exo-dig triple-bond insertion to yield vinylpalladium intermedi-
ate III. Alkoxycarbonylation of the latter would eventually lead
to the eight-membered lactam derivative 3 (path c). In both
cases, the obtained Pd0 species would be reoxidized back to
[PdI4]2À according to the mechanism we demonstrated several
years ago,[8] which involved oxidation of hydrogen iodide (also
ensuing from the carbonylation process) to I3À, followed by ox-
idative addition of the latter to Pd0 in the presence of iodide li-
gands. However, another possible reaction pathway could start
with anti-intramolecular attack of the nucleophilic amino
group to the triple bond coordinated to the metal center
(path d; reactivity that we have observed in several other car-
bonylative heterocyclization reactions),[5d] followed by alkoxy-
carbonylation of the ensuing vinylpalladium complexes IV and/
or V, finally leading to carbonylated heterocycles 4 and/or 5.
Clearly, from a conceptual and synthetic point of view, the
most interesting process would correspond to the carbonyla-
tive z-lactamization route (path a followed by path c), leading
to eight-membered lactams 3, also considering the importance
of the z-lactam core, which is extensively found in natural and
biologically relevant compounds.[9]
To predict which pathway, of those shown in Scheme 1, was
likely to be followed, starting from substrates 1, we carried out
theoretical calculations. Thus, a comparative analysis of the en-
ergies and geometries of the intermediates shown in
Scheme 1 was carried out.[10] First, optimization of the geome-
tries of the isoelectronic reaction intermediates III, VI, and VII
(with R1 =CH3, chosen as a simple model system for an internal
triple bond) was carried out. All frequencies of these intermedi-
ates were positive, which indicated that they were minima in
the respective reaction pathways. The energy difference be-
tween intermediates III and VI was +5.02 kcalmolÀ1, whereas
that between III and VII was À2.01 kcalmolÀ1. These values in-
dicate that paths a (leading to III) and d (leading to VI and/or
VII) are equally probable. With reference to path d, intermedi-
ates IV and V (precursors of VI and VII, respectively) were also
stable minima on the potential energy surface (PES). However,
going towards the reactants, discrimination between paths a
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and d could, in principle, still be at work, and could be related
to different possible coordination modes of substrate 1 to the
metal center. To prove this assumption, three structures (A–C)
were built, representing the starting geometries for optimiza-
tion (Figure 1). In particular, structure A differs from those of B
and C in the palladium orientation with respect to the triple
bond: in structure A, the metal center is close to the amino
group, whereas in B and C palladium is coordinated from the
opposite position with respect to the ÀNH2 group. In turn,
structure B differs from that of C in the ÀNH2 orientation with
respect to the triple bond, in particular, in B, NH···p (orbitals)
hydrogen bonding is present.[11]
Figure 2. Optimized geometries for structures A and B, labeled A’ (a) and B’
(b, c), respectively (pink: Pd; violet: I; red: O, gray: C; white: H).
position. The displacement of the PdI3···p moiety from its initial
geometry is caused by a marked rotation of the Ph-O-Ph dihe-
dral angle, as shown in Figure 2c. Key distances in B’, as those
given for A’, are 5.4 Š (N···Pd bond) and 4.5 Š (N···C bond), re-
spectively (Figure 2b). However, the formation of a NÀC bond,
leading to intermediate IV, would imply an unfavorable move-
ment of the ÀNH2 group toward the position shown in the
nonoptimized structure B (Figure 1b), with an inverse rotation
of Ph-O-Ph dihedral angle. With regard to starting geometry C,
shown in Figure 1c, a marked rotation of the dihedral angle
Ph-O-Ph was always obtained after optimization; again, this ro-
tation arranged the phenyl rings perpendicularly, with the
ÀNH2 farther away from the triple bond with respect to the
starting geometry.
Figure 1. Possible coordination modes of substrate 1 (R1 =Me) to the metal
center: starting geometries A (a), B (b), and C (c) used for geometry optimi-
zation (pink: Pd; violet: I; red: O, gray: C; white: H).
All optimized structures obtained from these initial geome-
tries showed positive frequencies, which demonstrated that
they were minima. The optimized geometries for structures A
and B, labeled A’ and B’, are shown in Figure 2.
Therefore, this computational analysis suggests that path a,
via intermediate I, should be favored with respect to path d,
via intermediates IV/V (Scheme 1), and, as a result, products 2
and/or 3 (ensuing from path a) are predicted to be formed
preferentially over products 4 and/or 5 (derived from path d).
It is important to emphasize that the geometry of intermediate
I was also optimized, and its existence as a stable minimum on
the PES was confirmed; the optimized structure is shown in
Figure 3.
These structures appear to be stabilized by an intramolecu-
lar hydrogen bond (NH···I, 2.9 Š), involving an iodine atom and
a hydrogen atom on the amino group (Figure 2a and b). The
energy difference between B’ and A’ was only À3.43 kcal
molÀ1, which suggested that two minima were in equilibrium.
Comparing A’ (Figure 2a) with the non-optimized structure A
(Figure 1a), it can be seen that the catalyst orientation does
not change significantly from the initial (A) to the optimized
(A’) structure. In A’, the N···Pd and N···C distances (in which C is
the carbon of the triple bond closest to nitrogen) were 4.4 and
4.9 Š, respectively. This suggests that A’ should preferentially
evolve through the formation of a NÀPd bond, leading to in-
termediate I, rather than through that of a NÀC bond, which
would lead to intermediate IV. On the other hand, comparing
the optimized structure B’ (Figure 2b) with the initial structure
B (Figure 1b), it can be observed that the NH···p hydrogen
bond in the initial geometry B is broken down after geometry
optimization, in favor of a more stable NH···I noncovalent
bond, with simultaneous rotation of the catalyst from its initial
According to path a (Scheme 1), CO insertion in the NÀPd
bond of intermediate I would lead to carbamoylpalladium in-
termediate II, which, in turn, could follow either path b (from
external nucleophilic displacement by ROH, to give acyclic car-
bamates 2) or path c (from intramolecular triple bond insertion
into the CÀPd bond, to give intermediate III, followed by
alkoxycarbonylation, eventually leading to z-lactams 3). The
latter pathway was expected to be less favored in the case of
an internal triple bond (R1 ¼ H), as usually observed in other
[PdI4]2À-catalyzed heterocyclocarbonylation processes,[5d] for
steric reasons. Thus, to predict which product could be formed
preferentially between 2 (ensuing from path b) or 3 (ensuing
from path c), depending on the nature of the triple bond (in-
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namic point of view, the difference in energy between inter-
mediates III and II yields information on the relative stability of
these structures in equilibrium between them. The energy gap
between intermediates G and E (R1 =H) was 8.24 kcalmolÀ1
larger than the corresponding energy difference between F
and D (R1 =CH3). This means that the stability of intermediate
III with respect to II is greater when R1 =H; thus confirming
that the formation of III is more favored when R1 =H.
Thus, the results obtained by the theoretical calculations can
be summarized as follows: 1) Both paths a and d, leading to in-
termediates II and VI and/or VII, respectively, can, in principle,
be followed. 2) Path a is expected to be more favored with re-
spect to path d. 3) Path c, derived from path a and leading to
the desired z-lactam derivative 3 through the formation of car-
bamoylpalladium complex II, is more likely to be followed
when R1 =H, otherwise path b, leading to acyclic carbamate 2,
is more favored. This may be ascribed to the steric effect exert-
ed by the R1 substituent on the triple bond, which hinders its
insertion into the carbamoylpalladium bond when R1 ¼ H.
To verify these hypotheses, we began to investigate the re-
activity of a substrate containing an internal triple bond, such
as 2-[2-(2-phenylethynyl)phenoxy]aniline (1a; R1 =Ph). This
substrate was allowed to react with CO, O2, and MeOH (R=
Me), which was also used as the solvent (substrate concentra-
tion=0.05 mmol of 1 per mL of MeOH), at 1008C and under
40 atm (at 258C) of a 4:1 mixture of CO/air, in the presence of
catalytic amounts of PdI2 (2 mol%) in conjunction with KI (KI/
PdI2 molar ratio=10). After 24 h, carbamate 2a was isolated in
74% yield [Eq. (1)]. Similar results were obtained with other
substrates, containing p-bromophenyl, alkenyl, or alkyl sub-
stituents on the triple bond; the corresponding acyclic carba-
mates were formed in 50–62% yield after 24–48 h [Eq. (1)]. The
structure of 2b (R1 =p-Br-C6H4) was confirmed by XRD analysis
(see the Supporting Information for details), and unidentified
heavy products (chromatographically immobile materials) ac-
counted for the remaining part of the converted substrates.
Figure 3. Optimized geometry for intermediate I (R1 =CH3).
Figure 4. Optimized geometries for intermediate II with R1 =Me (D) and H
(E).
ternal or external), the geometries of intermediates II with
both R1 =CH3 (D) and R1 =H (E) were optimized (Figure 4).
As seen from Figure 4, in D (R1 =Me) the C···C distance be-
tween the carbon of the carbonyl and the carbon of the triple
bond is 5.1 Š, and the H2CH···I distance is 3.1 Š, whereas in E
(R1 =H) both the C···C and H···I distances are appreciably short-
er (4.6 and 2.9 Š, respectively). This finding indeed suggests
that the cyclocarbonylation pathway (path c) may be favored
with an external triple bond (R1 =H), whereas, in the case of an
internal triple bond (R1 =Me), this route may be significantly
more difficult, since the key distances are systematically longer
owing to steric hindrance exerted by the R1 substituent. To fur-
ther confirm this supposition, the geometries of intermediates
III with both R1 =CH3 (F) and H (G) were also optimized
(Figure 5). For both F and G, the Hessian showed positive ei-
genvalues. Once again, they were minima on the PES, and
therefore, they were both possible. The energy differences be-
tween structures F and G and intermediates D and E, respec-
tively, were then calculated to support the hypothesis that the
formation of III was favored when R1 =H. From a thermody-
Thus, the results obtained with substrates containing an in-
ternal triple bond showed that, in agreement with theoretical
predictions, path a was preferentially followed with respect to
path d, and then path b was favored over path c, eventually
leading to acyclic carbamates 2. On the other hand, as seen
above, theoretical calculations also predicted that intermediate
II (from path a) could follow path c when the triple bond was
terminal (R1 =H). Accordingly, we next turned our attention to
the reactivity of substrates containing a terminal triple bond.
The first experiments were carried out with 2-(2-ethynylphe-
Figure 5. Optimized geometries for intermediate III with R1 =Me (F) and H
(G).
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noxy)aniline (1e), which was allowed to react under
conditions similar to those employed previously for
substrates 1a–d. Gratifyingly, after 15 h, z-lactam 3e
was isolated in 63% yield (Table 1, entry 1); thus
confirming the validity of our hypotheses. This result
was noteworthy when considering the possible
competitive pathways that could have been at work
(Scheme 1), the importance of realizing a novel z-
lactamization process by a direct carbonylative ap-
proach, and the potential importance of the product
obtained for various applications. The reaction was
repeated in EtOH and a slightly higher yield of the
corresponding ethyl ester 3e’ was obtained (71%,
Table 1, entry 2). The structure of 3e’ was confirmed
by XRD analysis (see the Supporting Information for
details), and the Z stereochemistry around the
double bond was in perfect agreement with the
mechanism shown in Scheme 1, path c. The next ex-
periments, carried out with differently substituted
substrates, confirmed the formation of the corre-
sponding eight-membered lactams 3 f–i and 3 f’–k’
in satisfactory yields in all cases (53–75%; Table 1,
entries 3–12).
Table 1. Synthesis of alkyl (Z)-(6-oxo-5,6-dihydro-12-oxa-5-azadibenzo[a,d]cycloocten-
7-ylidene)acetates 3e–i and 3e’–k’ by PdI2/KI-catalyzed oxidative cyclocarbonylation–
alkoxycarbonylation of 2-(2-ethynylphenoxy)anilines 1e–k.[a]
Entry
1
R
t
[h]
3
Yield
[%][b]
1
Me 15
63
71
72
70
62
75
73
69
72
63
2
3
1e
Et
24
Me 15
Considering the bioactivity reported in the litera-
ture for eight-membered lactam systems, we tested
the newly synthesized z-lactams for their biological
activity as antitumor agents against different breast
cancer cell lines. To this aim, we evaluated the ef-
fects of increasing concentrations (1, 10, 25, 50, 75,
and 100 mm) of z-lactams for 96 h on cell viability in
estrogen receptor (ER)-positive MCF-7 and ER-nega-
tive, progesterone receptor (PR)-negative, and
human epidermal growth factor receptor 2 (HER2)-
negative MDA-MB-231 human breast cancer cells by
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assays. Although almost all
tested compounds induced a decrease in breast
cancer cells viability (data not shown), z-lactam 3j’
was the most active in reducing anchorage-depen-
dent growth of breast cancer cells (Figure 6). In par-
ticular, as shown in Figure 6A, treatment for 96 h
with 3j’ induced a significant decrease in cell viabili-
ty in MCF-7 (Figure 6A, top) and in MDA-MB-231
cells (Figure 6A, middle).
4
1 f
Et
24
5
Me 15
6
1g
Et
24
7
Me 15
8
1h
Et
24
The half-maximal inhibitory concentration (IC50)
values of z-lactam 3j’ for the cell lines tested are
shown in Table 2. Notably, treatment with z-lactam
3j’ at doses of IC50 values calculated in our breast
cancer models did not elicit any noticeable effects in
MCF-10A nonmalignant breast epithelial cell viability
(Figure 6A, bottom). The ability of 3j’ to inhibit cell
growth was also evaluated by using anchorage-inde-
pendent growth assays (soft-agar assay), which
better reflected in vivo three-dimensional tumor
growth. MCF-7 and MDA-MB-231 cells were plated
in soft agar and then treated with z-lactam 3j’
(50 mm) for 14 days. At the end of treatment, colo-
9
Me 15
10
1i
Et
24
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in view of the biological relevance of these com-
pounds. Biological tests showed that the newly syn-
thesized z-lactam 3j’ exerted antiproliferative effects
on different breast cancer cell lines, without affecting
normal breast epithelial cell viability. Other studies
are currently ongoing to elucidate its biochemical
mechanism of action, and will be reported in due
course.
Table 1. (Continued)
Entry
11
1
R
t
[h]
3
Yield
[%][b]
Experimental Section
Et
24
24
74
General
1
Melting points are uncorrected. H and 13C NMR spectra
were recorded on a Bruker DPX Avance 300 spectrome-
ter at 258C in CDCl3 at 300 or 500 MHz, and 75 or
126 MHz, respectively, with Me4Si as an internal standard.
12
Et
53
19F NMR spectra were recorded on
a Bruker DPX
Avance 500 spectrometer at 258C in CDCl3 or [D6]DMSO
at 471 MHz with CFCl3 as an internal standard. Chemical
shifts (d) and coupling constants (J) are given in ppm
and Hz, respectively. IR spectra were recorded on
a JASCO FTIR 4200 spectrometer. GC-MS spectra were re-
corded on a Shimadzu QP-2010 GC-MS apparatus at an
ionization voltage of 70 eV. Microanalyses were carried
out with a Thermo-Fischer Elemental Analyzer Flash
2000 instrument. All reactions were analyzed by TLC on
silica gel 60 F254 or on neutral alumina (Merck) and by gas liquid
chromatography (GLC) with a gas chromatograph equipped with
capillary columns with polymethylsilicone and 5% phenylsilicone
as the stationary phase. Column chromatography was performed
on silica gel 60. Evaporation refers to the removal of solvent under
reduced pressure.
[a] All reactions were carried out at 1008C in ROH as the solvent (0.05 mmol of 1 per
mL of solvent) under 40 atm of a 4:1 mixture of CO/air (at 258C) in the presence of
PdI2 (2 mol%) in conjunction with KI (20 mol%). [b] Yield of product isolated based on
starting material 1.
Table 2. IC50 values of 3j’ for MDA-MB 231 and MCF-7 cells on anchor-
age-dependent growth.
Cell lines
IC50 [mm]
95% confidence interval
MDA-MB 231
MCF-7
27.34
21.29
20.37–36.69
11.96–37.89
Preparation of substrates 1
2-(2-Alkynylphenoxy)anilines were prepared by coupling between
the corresponding 2-(2-iodophenoxy)anilines[12] and a suitable ter-
minal alkyne, followed (in the case of the reaction with trimethyl-
silylacetylene) by deprotection, as described below. All other mate-
rials were commercially available and were used without further
purification.
nies of >50 mm in diameter were counted. As shown in Fig-
ure 6B, treatment with 3j’ significantly inhibited anchorage-in-
dependent growth of both MCF-7 and MDA-MB-231 cells. We
then examined the ability of 3j’ to inhibit cell movement in
wound-healing scratch assays (Figure 6C). During 24 h of ob-
servation, MCF-7 and MDA-MB-231 cells moved in either direc-
tion to close the gap, although treatment with 3j’ strongly re-
duced breast cancer cell movement. Taken together, these
data clearly indicate that the new z-lactam derivative 3j’ is
able to inhibit breast cancer cell proliferation and motility,
without affecting normal breast epithelial cell viability.
General procedure for the preparation of 1a–d and 2-(2-tri-
methylsilanylethynylphenoxy)anilines
Under nitrogen and with stirring, anhydrous Et3N (330 mg,
3.3 mmol), [PdCl2(PPh3)2] (12.8 mg, 3.2”10À2 mmol), CuI (30.4 mg,
0.16 mmol), and the 1-alkyne [2.4 mmol; phenylacetylene, 245 mg;
p-bromophenylacetylene,
434 mg;
1-ethynylcyclohex-1-ene,
255 mg; tert-butylacetylene, 200 mg; trimethylsilylacetylene,
235 mg] were added in this order to a solution of the 2-(2-iodo-
phenoxy)aniline [1.6 mmol; 2-(2-iodophenoxy)aniline, 498 mg; 2-(2-
iodophenoxy)-5-methylaniline, 520 mg; 2-(2-iodophenoxy)-5-me-
thoxyaniline, 546 mg; 1-[3-amino-4-(2-iodophenoxy)phenyl]etha-
none, 565 mg; methyl 3-amino-4-(2-iodophenoxy)benzoate,
591 mg; 2-(2-iodophenoxy)-5-cyanoaniline, 538 mg; 2-(2-iodophe-
noxy)-5-triluoromethylaniline, 607 mg] in anhydrous THF (5 mL).
The resulting mixture was allowed to stir at room temperature for
8 h (for p-bromophenylacetylene, tert-butylacetylene, and trime-
thylsilylacetylene) or at 408C for 15 h (for 1-ethynylcyclohex-1-ene).
After cooling to room temperature, a saturated aqueous solution
of NH4Cl was added (10 mL), and the mixture was extracted with
Conclusion
We found that readily available 2-(2-ethynylphenoxy)anilines 1,
containing a terminal triple bond (R1 =H), could be directly
converted into a novel class of medium-sized heterocyclic de-
rivatives 3, through a new PdI2/KI-catalyzed carbonylative z-lac-
tamization–alkoxycarbonylation process. In agreement with
theoretical calculations, the process started with N-palladation
of 1, followed by CO insertion, intramolecular triple bond inser-
tion, and alkoxycarbonylation. The formation of z-lactams 3
from simple building blocks (1, CO, ROH, and O2) in a multi-
component manner represents a significant achievement, also
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Figure 6. The z-lactam derivative 3j’ inhibits breast cancer cell growth. A) MTT assays in MCF-7, MDA-MB-231, and MCF-10A cells treated with vehicle (À) or
increasing concentrations (1, 10, 25, 50, 75, and 100 mm) of 3j’ for 96 h. Cell proliferation is expressed as fold change versus control (vehicle-treated cells). The
values represent the meansÆstandard deviation (SD) of three different experiments, each performed with triplicate samples. B) Soft-agar growth assay in
MCF-7 and MDA-MB-231 cells plated in 0.35% agarose and treated with z-lactam 3j’ (50 mm). After 14 days of growth, colonies of >50 mm diameter were
counted. Data are the mean colony numberÆSD of three plates and representative of two independent experiments. C) Cells were subjected to wound-heal-
ing scratch assays with images captured at 0 and 24 h after incubation with vehicle (À) or 3j’ (50 mm) by using phase-contrast microscopy. Small squares,
time=0. *P<0.05 and **P<0.001 compared with vehicle-treated cells.
AcOEt (3”15 mL). The collected organic layers were washed with
brine (40 mL) and dried over Na2SO4. After filtration and evapora-
tion of the solvent, the residue was purified by column chromatog-
raphy on silica gel as the stationary phase with 7:3 hexane/AcOEt
as the eluent to give pure 1a–d and 2-(2-trimethylsilanylethynyl-
phenoxy)anilines. 2-(2-Phenylethynylphenoxy)aniline (1a), yellow
solid, 365 mg, 80%; 2-[2-(4-bromophenylethynyl)phenoxy]aniline
(1b), yellow solid, 466 mg, 80%; 2-(2-cyclohex-1-eynylethynylphe-
noxy)aniline (1c), yellow oil, 375 mg, 81%; 2-[2-(3,3-dimethylbut-1-
ynylphenoxy)aniline (1d), yellow solid, 344 mg, 81%; 2-(2-trime-
thylsilanylethynylphenoxy)aniline, gray solid, 342 mg, 76%; 5-
methyl-2-(2-trimethylsilanylethynylphenoxy)aniline, yellow solid,
340 mg, 72%; 5-methoxy-2-(2-trimethylsilanylethynylphenoxy)ani-
line, brown oil, 419 mg, 84%; 1-[3-amino-4-(2-trimethylsilanylethy-
nylphenoxy)phenyl]ethanone, brown solid, 440 mg, 85%; methyl 3-
amino-4-(2-trimethylsilanylethynylphenoxy)benzoate, yellow solid,
478 mg, 88%; 3-amino-4-(2-trimethylsilanylethynylphenoxy)benzo-
nitrile, white solid, 358 mg, 88%; 5-trifluoromethyl-2-(2-trimethylsi-
lanylethynylphenoxy)aniline, yellow solid, 458 mg, 82%.
Deprotection of 2-[2-(trimethylsilylethynyl)phenoxy]anilines
to give 1e–k
With stirring and under nitrogen, K2CO3 (622 mg, 4.5 mmol) was
added to a solution of the 2-[2-(trimethylsilylethynyl)phenoxy]ani-
line obtained as described above [1.5 mmol; 2-(2-trimethylsilanyle-
thynylphenoxy)aniline, 422 mg; 5-methyl-2-(2-trimethylsilanylethy-
nylphenoxy)aniline, 443 mg; 5-methoxy-2-(2-trimethylsilanylethy-
nylphenoxy)aniline, 467 mg; 1-[3-amino-4-(2- trimethylsilanylethy-
nylphenoxy)phenyl]ethanone, 485 mg; methyl 3-amino-4-(2-trime-
thylsilanylethynylphenoxy)benzoate,
509 mg;
3-amino-4-(2-
trimethylsilanylethynylphenoxy)benzonitrile, 460 mg; 5-trifluoro-
methyl-2-(2-trimethylsilanylethynylphenoxy)aniline, 524 mg] in an-
hydrous methanol (5 mL). The resulting mixture was allowed to stir
at room temperature for 3 h, and then it was filtered through
Celite to remove K2CO3, followed by washing with MeOH (50 mL).
The resulting filtrate was evaporated to dryness to give the crude
product, which was dissolved in CH2Cl2 (40 mL) and washed with
brine (50 mL) and water (50 mL). After drying over Na2SO4 and
Chem. Eur. J. 2016, 22, 3053 – 3064
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evaporation of the solvent, the residue was purified by column
chromatography on silica gel by using hexane/AcOEt from 95:5 to
7:3 as the eluent to give pure 1e–k. 2-(2-Ethynylphenoxy)aniline
(1e), white solid, 245 mg, 78%; 5-methyl-2-(2-ethynylphenoxy)ani-
line (1 f), yellow oil, 255 mg, 76%; 5-methoxy-2-(2-ethynylphenoxy)
aniline (1g), yellow solid, 302 mg, 84%; 1-[3-amino-4-(2-ethynyl-
phenoxy)phenyl]ethanone (1h), yellow solid, 305 mg, 81%; methyl
3-amino-4-(2-ethynylphenoxy)benzoate (1i), yellow solid, 337 mg,
84%; 3-amino-4-(2-ethynylphenoxy)benzonitrile (1j), white solid,
257 mg, 72%; 2-(2-ethynylphenoxy)-5-trifluoromethylaniline (1k),
yellow oil, 362 mg, 87%.
calcd (%) for C18H19NO (265.35): C 81.47, H 7.22, N 5.28; found: C
81.51, H 7.20, N 5.29.
Compound 1e: Colorless solid, m.p. 64–658C; IR (KBr): n˜ =3464 (w,
br), 3377 (m, br), 3281 (m, br), 2107 (vw), 1620 (m), 1500 (s), 1482
(s), 1443 (m), 1270 (m), 1231 (s), 1192 (m), 886 (w), 748 cmÀ1 (s);
1H NMR (300 MHz, CDCl3): d=7.55–7.48 (m, 1H; aromatic), 7.26–
7.16 (m, 1H; aromatic), 7.04–6.94 (m, 2H; aromatic), 6.88 (distorted
d, J=8.1 Hz, 1H; aromatic), 6.83–6.66 (m, 3H; aromatic), 3.76 (brs,
2H; NH2), 3.29 ppm (s, 1H; ꢀCH); 13C NMR (75 MHz, CDCl3): d=
158.6, 142.7, 138.6, 134.1, 130.2, 125.3, 122.5, 120.5, 118.7. 116.6,
116.0, 112.8, 81.9, 79.4 ppm; GC-MS: m/z (%): 209 (100) [M+], 183
(32), 180 (77), 168 (57), 152 (19), 139 (5), 108 (9), 89 (9), 80 (39), 65
(16); elemental analysis calcd (%) for C14H11NO (209.24): C 80.36, H
5.30, N 6.69, found: C 80.40, H 5.29, N 6.67.
Compound 1a: Yellow solid, m.p. 119–1208C; IR (KBr): n˜ =3473 (m,
br), 3384 (m, br), 3057 (m), 2215 (vw), 1619 (m), 1500 (s), 1447 (m),
1266 (m), 1221 (s), 1188 (w), 1099 (m), 889 (w), 753 (s), 691 cmÀ1
(m); 1H NMR (300 MHz, CDCl3): d=7.63–7.39 (m, 3H; aromatic),
7.39–7.17 (m, 4H; aromatic), 7.12–6.64 (m, 6H; aromatic), 3.87 ppm
(brs, 2H; NH2); 13C NMR (75 MHz, CDCl3): d=157.8, 143.6, 138.5,
133.6, 131.7, 129.7, 128.3, 124.8, 123.4, 122.9, 119.7, 118.6, 117.2,
116.4, 114.7, 94.3, 85.2 ppm; GC-MS: m/z (%): 285 (100) [M+], 268
(29), 256 (29), 244 (66), 230 (5), 215 (12), 183 (78), 176 (14), 165
(21), 150 (9), 127 (12), 80 (20); elemental analysis calcd (%) for
C20H15NO (285.34): C 84.19, H 5.30, N 4.91; found: C 84.21, H 5.29,
N 4.92.
Compound 1 f: Yellow oil; IR (film): n˜ =3466 (w, br), 3377 (w, br),
3280 (m, br), 2107 (vw), 1621 (m), 1510 (m), 1481 (s), 1444 (m),
1305 (w), 1232 (s), 1200 (m), 862 (w), 754 cmÀ1 (m); 1H NMR
(300 MHz, CDCl3): d=7.51 (dd, J=7.7, 1.8 Hz, 1H; aromatic), 7.24–
7.17 (m, 1H; aromatic), 6.97 (td, J=7.6, 1.1 Hz, 1H; aromatic), 6.80
(distorted d, J=8.1 Hz, 1H; aromatic), 6.74 (dd, J=8.4, 0.8 Hz, 1H;
aromatic), 6.64–6.59 (m, 1H; aromatic), 6.55–6.50 (m, 1H; aromatic),
3.72 (brs, 2H; NH2), 3.18 (s, 1H; ꢀCH), 2.38 ppm (s, 3H; Me);
13C NMR (75 MHz, CDCl3): d=159.3, 141.1, 138.6, 135.1, 134.3,
130.2, 122.4, 120.5, 119.4, 117.4, 116.2, 113.2, 81.7, 79.7, 20.9 ppm;
GC-MS: m/z (%): 223 (100) [M+], 208 (11), 194 (24), 183 (34), 182
(23), 168 (17), 152 (8), 122 (11), 94 (20), 77 (18); elemental analysis
calcd (%) for C15H13NO (223.27): C 80.69, H 5.87, N 6.27; found: C
80.71, H 5.86, N 6.26.
Compound 1b: Yellow solid, m.p. 67–688C; IR (KBr): n˜ =3466 (m,
br), 3379 (m, br), 2218 (vw), 1619 (m), 1500 (s), 1478 (m), 1446 (w),
1264 (w), 1221 (s), 1187 (w), 1069 (w), 1010 (m), 823 (m), 747 cmÀ1
(s); 1H NMR (300 MHz, CDCl3): d=7.58–7.51 (m, 1H; aromatic),
7.47–7.38 (m, 2H; aromatic), 7.34–7.26 (m, 2H; aromatic), 7.26–7.21
(m, 1H; aromatic), 7.10–7.02 (m, 1H; aromatic), 7.02–6.93 (m, 1H;
aromatic), 6.91–6.82 (m, 3H; aromatic), 6.75–6.67 (m, 1H; aromatic),
3.76 ppm (brs, 2H; NH2); 13C NMR (75 MHz, CDCl3): d=157.7, 143.5,
138.3, 133.5, 133.1, 131.5, 130.0, 124.8, 122.9, 122.5, 122.3, 119.6,
118.7, 117.2, 116.4, 114.3, 93.2, 86.3 ppm; GC-MS: m/z (%): 365 (51)
[M+2+], 363 (51) [M+], 324 (36), 322 (36), 283 (65), 268 (18), 254
(27), 239 (10), 215 (9), 183 (100), 176 (18), 163 (15), 150 (12), 142
(30), 127 (22), 113 (8), 80 (20); elemental analysis calcd (%) for
C20H14BrNO (364.24): C 65.95, H 3.87, Br 21.94, N 3.85; found: C
65.99, H 3.86, Br 21.96, N 3.86.
Compound 1g: Yellow solid; m.p. 36–388C; IR (KBr): n˜ =3468 (w,
br), 3377 (w, br), 3278 (m, br), 2104 (vw), 1623 (m), 1509 (s), 1481
(m), 1443 (w), 1231 (s), 1208 (s), 1029 (w), 754 cmÀ1 (w); 1H NMR
(300 MHz, CDCl3): d=7.49 (dd, J=7.6, 1.7 Hz, 1H; aromatic), 7.23–
7.15 (m, 1H; aromatic), 6.95 (td, J=7.5, 1.1 Hz, 1H; aromatic), 6.85
(distorted d, J=8.5 Hz, 1H; aromatic), 6.72 (dd, J=8.5, 1.1 Hz, 1H;
aromatic), 6.35 (distorted d, J=2.9 Hz, 1H; aromatic), 6.26 (dd, J=
8.8, 2.9 Hz, 1H; aromatic), 3.74 (s, 5H; OMe+NH2), 3.31 ppm (s,
1H; ꢀCH); 13C NMR (75 MHz, CDCl3): d=159.7, 157.9, 140.0, 137.2,
134.3, 130.2, 122.2, 121.8, 115.7, 113.0, 104.0, 102.7, 81.8, 79.8,
55.7 ppm; GC-MS: m/z (%): 239 [M+] (100), 224 (61), 196 (41), 183
(32), 167 (16), 152 (6), 138 (44), 110 (20), 95 (14); elemental analysis
calcd (%) for C15H13NO2 (239.27): C 75.30, H 5.48, N 5.85; found: C
75.35, H 5.47, N 5.84.
Compound 1c: Yellow oil; IR (film): n˜ =3468 (m, br), 3381 (m, br),
2930 (m), 2204 (w), 1619 (m), 1501 (s), 1483 (s), 1446 (m), 1267 (w),
1
1218 (s), 748 (s) cmÀ1; H NMR (300 MHz, CDCl3): d=7.45 (dd, J=
7.6, 1.6 Hz, 1H; aromatic), 7.22–7.14 (m, 1H; aromatic), 7.05–6.92
(m, 2H; aromatic), 6.89–6.78 (m, 3H; aromatic), 6.75–6.66 (m, 1H;
aromatic), 6.19–6.11 (m, 1H; =CH), 3.86 (brs, 2H; NH2), 2.22–2.06
(m, 4H; cyclohexenyl ring), 1.70–1.52 ppm (m, 4H; cyclohexenyl
ring); 13C NMR (75 MHz, CDCl3): d=157.5, 143.6, 139.8, 138.5, 135.3,
133.4, 129.1, 124.7, 122.8, 119.8, 118.6, 117.0, 116.4, 96.2, 82.4, 29.1,
25.8, 22.4, 21.5 ppm; GC-MS: m/z (%): 289 (100) [M+], 272 (14), 260
(47), 246 (95), 233 (27), 220 (27), 205 (16), 183 (78), 165 (19), 152
(16), 139 (8), 115 (14), 102 (7), 80 (20), 65 (12); elemental analysis
calcd (%) for C20H19NO (289.37): C 83.01, H 6.62, N 4.84; found: C
83.04, H 6.60, N 4.83.
Compound 1h: Pale yellow solid; m.p. 92–938C; IR (KBr): n˜ =3466
(w, br), 3365 (m, br), 3281 (m, br), 2104 (vw), 1674 (m), 1619 (m),
1589 (m), 1507 (w), 1482 (m), 1442 (m), 1359 (w), 1302 (m), 1226
1
(s), 1195 (m), 755 cmÀ1 (w); H NMR (300 MHz, CDCl3): d=7.56 (d,
J=7.7 Hz, 1H; aromatic), 7.47–7.40 (m, 1H; aromatic), 7.36–7.23
(m, 2H; aromatic), 7.12 (t, J=7.6 Hz, 1H; aromatic), 6.92 (d, J=
8.2 Hz, 1H; aromatic), 6.74 (d, J=8.4 Hz, 1H; aromatic), 4.04 (brs,
2H; NH2), 3.22 (s, 1H; ꢀCH), 2.53 ppm (s, 3H; Me); 13C NMR
(75 MHz, CDCl3): d=196.8, 157.5, 148.2, 138.3, 134.6, 134.2, 130.4,
124.0, 119.6, 118.9, 117.6, 115.9, 115.0, 82.3, 79.0, 26.2 ppm; GC-MS:
m/z (%): 251 (100) [M+], 236 (44), 208 (40), 195 (12), 181 (27), 180
(27), 168 (9), 152 (24), 118 (4), 77 (7); elemental analysis calcd (%)
for C16H13NO2 (251.28): C 76.48, H 5.21, N 5.57; found: C 76.53, H
5.20, N 5.55.
Compound 1d: Pale yellow solid, m.p. 75–768C; IR (KBr): n˜ =3384
(w, br), 3348 (m, br), 2969 (m), 2231 (vw), 1620 (s), 1485 (m), 1265
1
(m), 745 cmÀ1 (vs); H NMR (300 MHz, CDCl3): d=7.42 (dd, J=7.8,
1.6 Hz, 1H; aromatic), 7.24–7.16 (m, 1H; aromatic), 7.07–6.99 (m,
1H; aromatic), 6.98–6.88 (m, 2H; aromatic), 6.83–6.74 (m, 2H; aro-
matic), 6.72–6.64 (m, 1H; aromatic), 3.90 (brs, 2H; NH2), 1.22 ppm
(s, 9H; tBu); 13C NMR (75 MHz, CDCl3): d=157.2, 144.1, 138.0, 133.4,
128.8, 124.1, 123.0, 118.5, 118.2, 117.9, 116.1, 115.8, 103.8, 74.6,
30.8, 28.0 ppm; GC-MS: m/z (%): 265 (100), 250 (98), 234 (37), 220
(62), 209 (48), 208 (69), 183 (55), 180 (27), 157 (14), 141 (13), 128
(15), 115 (22), 102 (8), 91 (11), 80 (17), 65 (15); elemental analysis
Compound 1i: Yellow solid; m.p. 63–648C; IR (KBr): n˜ =3435 (w,
br), 3349 (w, br), 3281 (w, br), 3071 (w), 2106 (vw), 1725 (s), 1621
(m), 1592 (m), 1572 (m), 1509 (m), 1486 (m), 1445 (s), 1302 (s), 1260
(s), 1117 (m), 995 (w), 915 (w), 752 cmÀ1 (m); H NMR (300 MHz,
CDCl3): d=7.55 (distorted dd, J=7.6, 1.4 Hz, 1H; aromatic), 7.52–
7.47 (m, 1H; aromatic), 7.41–7.34 (m, 1H; aromatic), 7.34–7.25 (m,
1H; aromatic), 7.10 (td, J=7.6, 1.0 Hz, 1H; aromatic), 6.90, (dd, J=
Chem. Eur. J. 2016, 22, 3053 – 3064
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8.3, 0.6 Hz, 1H; aromatic), 6.75 (d, J=8.3 Hz, 1H; aromatic), 3.87 (s,
5H; OMe+NH2), 3.24 ppm (s, 1H; ꢀCH); 13C NMR (75 MHz, CDCl3):
d=166.9, 157.2, 147.5, 137.9, 134.4, 130.4, 126.1, 123.8, 120.3,
118.3, 117.7, 117.2, 114.3, 82.3, 78.8, 52.0 ppm; GC-MS: m/z (%): 267
(100) [M+], 241 (9), 236 (28), 235 (19), 226 (13), 208 (36), 195 (21),
183 (21), 180 (24), 168 (13), 152 (17), 138 (4), 118 (6), 104 (7), 90
(11), 75 (8); elemental analysis calcd (%) for C16H13NO3 (267.28): C
71.90, H 4.90, N 5.24; found: C 71.94, H 4.89, N 5.23.
with CO (32 atm) and air (up to 40 atm). After being stirred at
1008C for the required time (15 h for the reaction of 1e–i with
MeOH; 24 h for the reactions of 1e–k with EtOH), the autoclave
was cooled, degassed, and opened. The solvent was evaporated
and the product was purified by column chromatography on silica
gel to give pure 3e–i (R=Me) for 6:4 hexane/AcOEt as the eluent
or 3e’–3k’ (R=Et) for 65:35 hexane/AcOEt as the eluent: 3e,
yellow solid, 65 mg, 63%; 3e’, white solid, 77 mg, 71%; 3 f, yellow
solid, 78 mg, 72%; 3 f’, yellow solid, 79 mg, 70%; 3g, yellow solid,
71 mg, 62%; 3g’, gray solid, 89 mg, 75%; 3h, white solid, 86 mg,
73%; 3h’, yellow oil, 85 mg, 69%; 3i, white solid, 89 mg, 72%; 3i’,
white solid, 81 mg, 63%; 3j’, 87 mg, 74%; 3k’, colorless solid,
70 mg, 53%.
Compound 1j: Pale yellow solid; m.p. 104–1058C; IR (KBr): n˜ =
3468 (w, br), 3360 (w, br), 3283 (w, br), 2225 (m), 1619 (m), 1507 (s),
1402 (m), 1305 (w), 1235 (s), 863 (m), 756 cmÀ1 (w); 1H NMR
(300 MHz, CDCl3): d=7.56 (dd, J=7.7, 1.6 Hz, 1H; aromatic), 7.38–
7.30 (m, 1H; aromatic), 7.15 (td, J=7.6, 1.1 Hz, 1H; aromatic), 7.03
(distorted d, J=1.8 Hz, 1H; aromatic), 6.98–6.89 (m, 2H; aromatic),
6.67 (distorted d, J=8.4 Hz, 1H; aromatic), 4.20 (brs, 2H; NH2),
3.21 ppm (s, 1H; ꢀCH); 13C NMR (75 MHz, CDCl3): d=156.8, 147.9,
138.9, 134.7, 130.5, 124.6, 122.7, 119.4, 119.0, 118.7, 117.8, 115.3,
107.7, 82.7, 78.7 ppm; GC-MS: m/z (%): 234 (100) [M+], 205 (52),
193 (27), 183 (26), 168 (6), 151 (10), 105 (17), 89 (12), 75 (16); ele-
mental analysis calcd (%) for C15H10N2O (234.25): C 76.91, H 4.30, N
11.96; found: C 76.96, H 4.28, N 11.95.
Compound 2a: Yield: 90 mg (74% from 1a); yellow oil; IR (film):
n˜ =3427 (m, br), 2220 (vw), 1739 (s), 1610 (m), 1529 (s), 1454 (s),
1326 (w), 1253 (s), 1065 (m), 753 cmÀ1 (s); 1H NMR (300 MHz,
CDCl3): d=8.26–8.16 (m, 1H; NH), 7.59 (dd, J=7.7, 1.4 Hz, 1H; aro-
matic), 7.39–7.22 (m, 7H; aromatic), 7.22–7.07 (m, 2H; aromatic),
7.07–6.91 (m, 2H; aromatic), 6.80 (distorted dd, J=8.1, 1.3 Hz, 1H;
aromatic), 3.73 ppm (s, 3H; CO2Me); 13C NMR (75 MHz, CDCl3): d=
156.5, 153.9, 145.6, 133.8, 131.6, 129.8, 129.4, 128.4, 128.3, 124.3,
123.9, 122.9, 119.5, 119.2, 117.0, 116.1, 94.9, 84.4, 52.3 ppm; GC-MS:
m/z (%): 343 (66) [M+], 311 (20), 310 (22), 283 (29), 282 (27), 268
(59), 254 (30), 244 (100), 241 (31), 226 (7), 209 (11), 176 (13), 165
(16), 150 (10), 127 (8), 91 (3), 77 (8); elemental analysis calcd (%) for
C22H17NO3 (343.38): C 76.95, H 4.99, N 4.08; found: C 76.98, H 4.98,
N 4.06.
Compound 1k: Yellow oil; IR (film): n˜ =3478 (w, br), 3389 (w, br),
3299 (w, br), 2109 (vw), 1625 (m), 1515 (w), 1484 (m), 1446 (m),
1339 (s), 1229 (s), 1196 (m), 1165 (m), 1119 (m), 862 (w), 755 cmÀ1
(w); H NMR (300 MHz, CDCl3): d=7.55 (distorted dd, J=7.5 Hz, 1.1,
1H; aromatic), 7.34–7.24 (m, 1H; aromatic), 7.09 (td, J=7.5, 1.1 Hz,
1H; aromatic), 7.05–6.98 (m, 1H; aromatic), 6.95–6.84 (m, 2H; aro-
matic), 6.80 (distorted d, J=8.2, 1H; aromatic), 4.08 (brs, 2H; NH2),
3.25 ppm (s, 1H; ꢀCH); 13C NMR (75 MHz, CDCl3): d=157.2, 146.0,
138.4, 134.4, 130.5, 126.6 (q, J=32.4 Hz), 124.2 (q, J=271.9 Hz),
123.9, 118.4, 118.0, 115.4 (q, J=4.2 Hz), 114.1, 112.8 (q, J=3.7 Hz),
82.4, 78.9 ppm; 19F NMR (471 MHz, CDCl3): d=À62.6 ppm (s, 3F;
CF3); GC-MS: m/z (%): 277 (100) [M+], 248 (25), 236 (28), 208 (14),
180 (21), 148 (11), 128 (3), 101 (7), 75 (9); elemental analysis calcd
(%) for C15H10F3NO (277.24): C 64.98, H 3.64, F 20.56, N 5.05; found:
C 65.03, H 3.63, F 20.57, N 5.03.
Compound 2b: Yield: 86 mg (58% from 1b); yellow solid; m.p.
58–608C; IR (KBr): n˜ =3459 (w, br), 2175 (vw), 1739 (s), 1608 (w),
1524 (s), 1474 (m), 1452 (m), 1323 (w), 1227 (s), 1208 (s), 1060 (m),
1
820 (m), 756 cmÀ1 (m); H NMR (300 MHz, CDCl3): d=8.20 (brd, J=
7.6 Hz, 1H; NH), 7.57 (dd, J=7.7, 1.4 Hz, 1H; aromatic), 7.48–7.38
(m, 3H; aromatic), 7.33 (td, J=8.2, 1.7 Hz, 1H; aromatic), 7.22–7.15
(m, 3H; aromatic), 7.14–7.07 (m, 1H; aromatic), 7.01 (distorted d,
J=8.2 Hz, 1H; aromatic), 6.95 (td, J=8.1, 1.5 Hz, 1H; aromatic),
6.78 (distorted dd, J=8.2, 1.2 Hz, 1H; aromatic), 3.74 ppm (s, 3H;
CO2Me); 13C NMR (75 MHz, CDCl3): d=156.4, 153.9, 145.5, 133.7,
133.0, 131.6, 130.1, 129.3, 124.3, 123.9, 123.0, 122.7, 121.8, 119.6,
119.2, 116.9, 115.6, 93.8, 85.5, 52.3 ppm; GC-MS: m/z (%) 423 (100)
[M+2+], 421 (98) [M+], 391 (22), 389 (20), 348 (32), 346 (35), 324
(97), 322 (99), 310 (29), 254 (65), 241 (64), 209 (18), 176 (27), 150
(20), 126 (16), 113 (12); elemental analysis calcd (%) for C22H16BrNO3
(422.27): C 62.57, H 3.82, Br 18.92, N 3.32; found: C 62.61, H 3.83,
Br 18.94, N 3.31.
General procedure for the oxidative carbonylation of 1a–g
to give 2a–d [Eq. (1)]
A 35 mL stainless-steel autoclave was charged in the presence of
air with PdI2 (2.5 mg, 6.94”10À3 mmol), KI (11.5 mg, 6.93”
10À2 mmol), and a solution of 1 [0.35 mmol: 1a (100 mg), 1b
(127 mg), 1c (101 mg), 1d (93 mg)] in MeOH (7.0 mL). The auto-
clave was sealed and, while the mixture was stirred, the autoclave
was pressurized with CO (32 atm) and air (up to 40 atm). After
being stirred at 1008C for the required time (24 h for 1a–c; 48 h
for 1d), the autoclave was cooled, degassed, and opened. The sol-
vent was evaporated and the products were purified by column
chromatography on silica gel to give pure 2a–d (eluent: 7:3
hexane/AcOEt for 2a and 2b; 8:2 hexane/AcOEt for 2c and 2d):
2a, yellow oil, 90 mg, 74%; 2b, yellow solid, 86 mg, 58%; 2c,
yellow oil, 61 mg, 50%; 2d, yellow oil, 70 mg, 62%.
Compound 2c: Yield: 61 mg (50% from 1c); yellow oil; IR (film):
n˜ =3433 (w, br), 3366 (w, br), 2929 (m), 2858 (w), 2208 (vw), 1740
(s), 1609 (w), 1529 (s), 1479 (m), 1445 (s), 1227 (s), 1065 (w),
1
751 cmÀ1 (m); H NMR (300 MHz, CDCl3): d=8.22–8.11 (m, 1H; NH),
7.47 (distorted dd, J=7.7, 1.7 Hz, 1H; aromatic), 7.30–7.22 (m, 2H;
aromatic), 7.15–7.03 (m, 2H; aromatic), 7.00–6.88 (m, 2H; aromatic),
6.74 (distorted dd, J=8.2, 1.2 Hz, 1H; aromatic), 6.09–6.01 (m, 1H;
=CH), 3.78 (s, 3H; CO2Me), 2.16–1.96 (m, 4H; cyclohexenyl ring),
1.67–1.49 ppm (m, 4H; cyclohexenyl ring); 13C NMR (75 MHz,
CDCl3): d=156.1, 153.9, 145.7, 135.7, 133.6, 129.3, 129.2, 124.2,
123.6, 122.8, 120.5, 119.6, 119.0, 117.3, 116.8, 96.9, 81.7, 52.3, 28.8,
25.8, 22.3, 21.5 ppm; GC-MS: m/z (%): 347 (100) [M+], 319 (64), 315
(23), 304 (62), 286 (54), 278 (26), 272 (89), 260 (45), 245 (36), 231
(24), 218 (10), 208 (50), 194 (17), 183 (27), 165 (36), 152 (23), 139
(12), 115 (15); elemental analysis calcd (%) for C22H21NO3 (347.41): C
76.06, H 6.09, N 4.03; found: C 76.12, H 6.08, N 4.04.
General procedure for the oxidative carbonylation of 1e–k
to give 3e–i and 3e’–k’ (Table 1)
A 35 mL stainless-steel autoclave was charged in the presence of
air with PdI2 (2.5 mg, 6.94”10À3 mmol), KI (11.5 mg, 6.93”
10À2 mmol), and a solution of 1 [0.35 mmol; 1e (73 mg), 1 f
(78 mg), 1g (84 mg), 1h (88 mg), 1i (94 mg), 1j (82 mg), 1k
(97 mg)] in ROH (R=Me or Et; 7 mL). The autoclave was sealed
and, while the mixture was stirred, the autoclave was pressurized
Compound 2d: Yield: 70 mg (62% from 1d); yellow oil; IR (film):
n˜ =3435 (m, br), 2969 (m), 2931 (m), 2867 (w), 2242 (w), 1739 (s),
1611 (m), 1539 (m), 1480 (w), 1456 (m), 1328 (w), 1219 (m), 1113
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1
(w), 1065 (m), 954 (w), 755 cmÀ1 (s); H NMR (300 MHz, CDCl3): d=
Compound 3g: Yield: 71 mg (62% from 1g; Table 1, entry 5);
yellow solid; m.p. 243–2458C; IR (KBr): n˜ =3429 (m, br), 2922 (w),
1709 (s), 1677 (s), 1607 (m), 1495 (w), 1476 (w), 1373 (w), 1258 (w),
1200 (s), 1161 (m), 1030 (m), 756 cmÀ1 (m); 1H NMR (300 MHz,
CDCl3): d=7.47–7.28 (m, 4H; NH+3H aromatic), 7.20–7.11 (m, 1H;
aromatic), 7.05–6.98 (m, 1H; aromatic), 6.81–6.72 (m, 2H; aromatic),
5.81 (s, 1H; =CH), 3.76 (s, 3H; CO2Me), 3.74 ppm (s, 3H; OMe at C-
3); 13C NMR (75 MHz, CDCl3): d=168.7, 164.4, 157.8, 157.3, 150.1,
146.4, 132.7, 131.7, 130.5, 127.0, 124.9, 123.7, 121.2, 119.8, 115.2,
111.1, 56.0, 51.6 ppm; GCMS: m/z (%): 325 (68) [M+], 308 (4), 297
(11), 294 (41), 278 (20), 266 (100), 265 (71), 250 (41), 238 (20), 222
(31), 210 (10), 195 (16), 180 (6), 167 (23), 139 (13), 115 (4), 101 (9),
89 (7); elemental analysis calcd (%) for C18H15NO5 (325.32): C 66.46,
H 4.65, N 4.31; found: C 66.49, H 4.64, N 4.30.
8.16 (brd, J=9.1 Hz, 1H; NH), 7.45 (distorted dd, J=7.7, 1.4 Hz,
2H; aromatic), 7.31–7.23 (m, 1H; aromatic), 7.16–6.98 (m, 3H; aro-
matic), 6.91 (td, J=8.1, 1.5 Hz, 1H; aromatic), 6.65 (distorted dd,
J=8.1, 1.2 Hz, 1H; aromatic), 3.79 (s, 3H; CO2Me), 1.12 ppm (s, 9H;
tBu); 13C NMR (75 MHz, CDCl3): d=155.8, 154.0, 146.1, 133.8, 128.9,
124.5, 123.2, 122.7, 120.3, 119.0, 115.9, 104.7, 89.1, 73.9, 52.3, 30.6,
28.0 ppm; GC-MS: m/z (%): 323 (97) [M+], 308 (33), 291 (9), 276
(100), 266 (31), 248 (46), 234 (43), 233 (33), 219 (14), 209 (13), 178
(5), 165 (4), 152 (5), 141 (10), 128 (8), 115 (10); elemental analysis
calcd (%) for C20H21NO3 (323.39): C 74.28, H 6.55, N 4.33; found: C
74.33, H 6.53, N 4.32.
Compound 3e: Yield: 65 mg (63% from 1e; Table 1, entry 1);
yellow solid; m.p. 223–2258C; IR (KBr): n˜ =3468 (w, br), 3378 (m,
br), 2952 (w), 1723 (s), 1622 (m), 1503 (m), 1483 (m), 1435 (m),
1354 (w), 1306 (w), 1268 (m), 1216 (s), 755 cmÀ1 (m); H NMR
(300 MHz, CDCl3): d=7.89 (brs, 1H; NH), 7.47–7.05 (m, 8H; aromat-
ic), 5.79 (s, 1H; =CH), 3.74 ppm (s, 3H; CO2Me); 13C NMR (75 MHz,
CDCl3): d=168.9, 164.3, 157.0, 152.4, 149.9, 131.9, 131.7, 130.5,
129.2, 126.7, 126.4, 126.3, 125.1, 123.2, 121.3, 120.0, 51.6 ppm; GC-
MS: m/z (%): 295 (59) [M+], 278 (12), 263 (97), 236 (97), 235 (100),
207 (29), 190 (14), 180 (36), 165 (28), 152 (35), 139 (6), 104 (10), 89
(17), 75 (13); elemental analysis calcd (%) for C17H13NO4 (295.29): C
69.15, H 4.44, N 4.74; found: C 69.19, H 4.43, N 4.73.
Compound 3g’: Yield: 89 mg (75% from 1g; Table 1, entry 6); off-
white solid; m.p. 219–2218C; IR (KBr): n˜ =3294 (m, br), 2927 (w),
1712 (s), 1676 (s), 1609 (m), 1502 (m), 1477 (m), 1390 (m), 1270 (m),
1208 (s), 1180 (s), 1032 (m), 760 cmÀ1 (m); 1H NMR (300 MHz,
CDCl3): d=7.91 (brs, 1H; NH), 7.46–7.28 (m, 3H; aromatic), 7.20–
7.11 (m, 1H; aromatic), 7.0 (distorted d, J=8.8 Hz, 1H; aromatic),
6.83–6.72 (m, 2H; aromatic), 5.80 (s, 1H; =CH), 4.21 (q, J=7.1 Hz,
2H; CH2CH3), 3.76 (s, 3H; OMe), 1.29 ppm (t, J=7.1 Hz, 3H;
CH2CH3); 13C NMR (75 MHz, CDCl3): d=168.8, 164.0, 157.8, 157.3,
149.7, 146.3, 132.8, 131.5, 130.5, 127.1, 124.9, 123.6, 121.1, 120.3,
115.1, 111.1, 60.8, 56.0, 14.2 ppm; GC-MS: m/z (%): 339 (52) [M+],
311 (8), 294 (31), 293 (41), 278 (17), 266 (100), 265 (68), 250 (37),
238 (22), 222 (23), 207 (13), 195 (15), 178 (7), 167 (23), 152 (8), 139
(11), 115 (4), 101 (9), 89 (7); elemental analysis calcd (%) for
C19H17NO5 (339.34): C 67.25, H 5.05, N 4.13; found: C 67.30, H 5.03,
N 4.12.
Compound 3e’: Yield: 77 mg (71% from 1e; Table 1, entry 2);
white solid; m.p. 231–2338C. IR (KBr): n˜ =3291 (w, br), 2926 (w),
1716 (s), 1674 (m), 1500 (w), 1470 (m), 1446 (m), 1384 (m), 1290
(w), 1260 (w), 1217 (m), 1190 (m), 1107 (w), 1032 (w), 755 cmÀ1 (s);
1H NMR (300 MHz, CDCl3): d=7.89 (brs, 1H; NH), 7.47–7.09 (m, 8H;
aromatic), 5.79 (s, 1H; =CH), 4.20 (q, J=7.1 Hz, 2H; CH2CH3),
1.29 ppm (t, J=7.1 Hz, 3H; CH2CH3); 13C NMR (75 MHz, CDCl3): d=
169.2, 163.8, 156.6, 151.9, 149.3, 131.9, 131.7, 130.4, 129.2, 126.7,
126.3, 126.0, 125.0, 123.1, 121.2, 120.3, 60.8, 14.1 ppm; GC-MS: m/z
(%): 309 (37) [M+], 281 (6), 264 (33), 263 (73), 236 (100), 235 (75),
219 (6), 207 (21), 190 (8), 180 (26), 165 (18), 152 (21), 89 (7); ele-
mental analysis calcd (%) for C18H15NO4 (309.32): C 69.89, H 4.89, N
4.53; found: C 69.93, H 4.88, N 4.54.
Compound 3h: Yield: 86 mg (73% from 1h; Table 1, entry 7); col-
orless solid; m.p. 265–2678C; IR (KBr): n˜ =3425 (w, br), 2952 (w),
1721 (s), 1673 (s), 1598 (w), 1448 (w), 1389 (w), 1283 (m), 1199 (s),
1
854 (w), 768 cmÀ1 (w); H NMR (300 MHz, [D6]DMSO): d=10.4 (brs,
1H; NH), 7.93–7.83 (m, 1H; aromatic), 7.65 (brs, 1H; aromatic),
7.61–7.21 (m, 5H; aromatic), 5.89 (s, 1H; =CH), 3.65 (s, 3H;
CO2Me), 2.55 ppm (s, 3H; MeCO); 13C NMR (75 MHz, [D6]DMSO):
d=196.4, 167.6, 163.6, 155.3, 154.0, 149.3, 134.7, 133.2, 132.0,
130.3, 128.9, 126.4, 125.4, 124.5, 123.2, 121.1, 119.2, 51.5, 26.6 ppm;
GC-MS: m/z (%): 337 (47) [M+], 305 (48), 279 (100), 278 (65), 262
(41), 261 (31), 249 (12), 235 (22), 222 (15), 206 (21), 191 (7), 178
(14), 166 (10), 151 (18), 139 (15), 89 (10); elemental analysis calcd
(%) for C19H15NO5 (337.33): C 67.65, H 4.48, N 4.15; found: C 67.69,
H 4.46, N 4.15.
Compound 3 f: Yield: 78 mg (72% from 1 f; Table 1, entry 3); pale
yellow solid; m.p. 205–2078C; IR (KBr): n˜ =3307 (br), 1721 (s), 1673
(s), 1478 (w), 1448 (w), 1385 (m), 1211 (m), 1173 (m), 757 cmÀ1 (s);
1H NMR (300 MHz, CDCl3): d=7.87 (brs, 1H; NH), 7.47–7.28 (m, 3H;
aromatic), 7.21–6.93 (m, 4H; aromatic), 5.80 (s, 1H; =CH), 3.75 (s,
3H; CO2Me), 2.30 ppm (s, 3H; Me at C-3); 13C NMR (75 MHz, CDCl3):
d=169.0, 164.4, 157.1, 150.2, 150.0, 136.5, 131.8, 131.7, 130.5,
129.8, 127.1, 126.5, 125.0, 122.8, 121.3, 119.9, 51.6, 20.8 ppm; GC-
MS: m/z (%): 309 (94) [M+], 277 (100), 250 (99), 249 (95), 235 (15),
221 (39), 207 (93), 193 (54), 179 (31), 165 (15), 147 (21), 135 (28), 89
(25), 73 (46); elemental analysis calcd (%) for C18H15NO4 (309.32): C
69.89, H 4.89, N 4.53; found: C 69.92, H 4.90, N 4.52.
Compound 3h’: Yield: 85 mg (69% from 1h, Table 1, entry 8); col-
orless solid; m.p. 271–273; IR (KBr): n˜ =3342 (w, br), 2982 (w), 1723
(s), 1683 (m), 1596 (w), 1432 (w), 1263 (m), 1207 (s), 1027 (w),
1
759 cmÀ1 (w); H NMR (300 MHz, [D6]DMSO): d=10.4 (brs, 1H; NH),
7.89 (distorted dd, J=8.5, 2.1 Hz, 1H; aromatic), 7.83 (d, J=2.1 Hz,
1H; aromatic), 7.60–7.23 (m, 5H; aromatic), 5.85 (s, 1H; =CH), 4.12
(q, J=7.0 Hz, 2H; CH2CH3), 2.55 (s, 3H; MeCO), 1.21 ppm (t, J=
7.0 Hz, 3H; CH2CH3); 13C NMR (75 MHz, [D6]DMSO): d=196.3, 167.6,
163.2, 155.3, 154.0, 148.9, 134.7, 133.3, 131.9, 130.3, 129.0, 126.4,
125.4, 124.5, 123.2, 121.0, 119.6, 60.2, 26.6, 13.9 ppm; GC-MS: m/z
(%): 351 (39) [M+], 323 (3), 306 (7), 278 (45), 262 (25), 236 (100),
207 (11), 192 (6), 180 (8), 165 (30), 152 (11); elemental analysis
calcd (%) for C20H17NO5 (351.35): C 68.37, H 4.88, N 3.99; found: C
68.40, H 4.88, N 4.00.
Compound 3 f’: Yield: 79 mg (70% from 1 f; Table 1, entry 4);
yellow solid; m.p. 220–2228C; IR (KBr): n˜ =3396 (m, br), 1712 (s),
1674 (s), 1501 (w) 1477 (w), 1385 (m), 1305 (w), 1275 (w), 1209 (m),
1180 (m), 1037 (w), 758 cmÀ1 (m); 1H NMR (300 MHz, CDCl3): d=
8.32 (brs, 1H; NH), 7.47–7.29 (m, 3H; aromatic), 7.20–6.95 (m, 4H;
aromatic), 5.80 (s, 1H; =CH), 4.22 (q, J=7.1 Hz, 2H; CH2CH3), 2.29
(s, 3H; Me at C-3), 1.30 ppm (t, J=7.1 Hz, 3H; CH2CH3); 13C NMR
(75 MHz, CDCl3): d=169.0, 163.9, 157.1, 150.2, 149.7, 136.4, 131.9,
131.6, 130.5, 129.7, 127.2, 126.5, 124.9, 122.8, 121.3, 120.4, 60.8,
20.8, 14.2 ppm; GC-MS: m/z (%): 323 (39) [M+], 295 (5), 278 (33),
277 (66), 250 (100), 249 (79), 232 (7), 221 (17), 207 (14), 193 (18),
179 (15), 165 (11), 152 (9), 139 (3), 128 (4), 101 (6), 89 (9), 77 (8); el-
emental analysis calcd (%) for C19H17NO4 (323.34): C 70.58, H 5.30,
N 4.33; found: C 70.61, H 5.29, N 4.31.
Compound 3i: Yield: 89 mg (72% from 1i; Table 1, entry 9); color-
less solid; m.p. 244–2468C; IR (KBr): n˜ =3512 (m, br), 1721 (s), 1675
1
(m), 1398 (m), 1280 (w), 1201 cmÀ1 (w); H NMR (300 MHz, CDCl3):
d=8.02–7.82 (m, 4H; NH+3H aromatic), 7.52–7.30 (m, 2H; aro-
matic), 7.25–7.15 (m, 2H; aromatic), 5.83 (s, 1H; =CH), 3.91 (s, 3H;
Chem. Eur. J. 2016, 22, 3053 – 3064
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CO2Me), 3.77 ppm (s, 3H; CO2Me); 13C NMR (75 MHz, CDCl3): d=
169.0, 165.6, 164.1, 156.0, 155.1, 149.0, 132.1, 132.0, 130.5, 130.4,
128.4, 127.3, 126.6, 125.5, 123.3, 121.3, 120.4, 52.4, 52.0 ppm; GC-
MS: m/z (%): 353 (80) [M+], 322 (40), 321 (74), 294 (100), 293 (58),
262 (41), 235 (20), 206 (26), 178 (19), 151 (11), 131 (11), 89 (11); ele-
mental analysis calcd (%) for C19H15NO6 (353.33): C 64.59, H 4.28, N
3.96; found: C 64.63, H 4.27, N 3.97.
core potential (LANL2DZ ECP)[13] was used instead for the palladi-
um atom. The convergence threshold was set to 10À6 a.u. for the
self-consistent field procedure and to 10À5 a.u. for the electron
density root-mean-square. All structures were fully optimized by
using analytical gradients with approximate Hessian updates. The
optimization convergences, based on the maximum and root-
mean-square gradient thresholds, were set equal to 10À6 a.u.,
whereas the maximum and root-mean-square of the Cartesian dis-
placement vectors were set to 6”10À6 and 4”10À6 a.u., respective-
ly. An accurate characterization of the structures on the PES re-
quired very high convergence criteria for Hessian evaluation. Thus,
in the Hessian calculations, the energy convergence threshold was
set to 10À8 a.u. for the self-consistent field procedure and 10À7 a.u.
for the electron density root-mean-square.
Compound 3i’: Yield: 81 mg (63% from 1i; Table 1, entry 10);
white solid; m.p. 220–2228C; IR (KBr): n˜ =3461 (m, br), 1720 (s),
1682 (m), 1400 (m), 1283 (m), 1178 (m), 1100 (w), 759 cmÀ1 (w);
1H NMR (300 MHz, [D6]DMSO): d=10.43 (brs, 1H; NH), 7.85 (dist
dd, J=8.5, 2.2 Hz, 1H; aromatic), 7.70 (d, J=2.2 Hz, 1H; aromatic),
7.61–7.23 (m, 5H; aromatic), 5.86 (s, 1H; =CH), 4.12 (q, J=7.1 Hz,
2H; CH2CH3), 3.84 (s, 3H; CO2Me), 1.21 ppm (t, J=7.1 Hz, 3H;
CH2CH3); 13C NMR (75 MHz, DMSO): d=167.6, 164.9, 163.2, 155.2,
154.0, 148.9, 133.3, 131.9, 130.3, 129.3, 127.5, 126.3, 125.9, 125.5,
123.5, 121.0, 119.7, 60.3, 52.3, 13.8 ppm; GC-MS: m/z (%): 367 (55)
[M+], 339 (7), 322 (35), 321 (61), 294 (100), 293 (49), 262 (28), 235
(15), 108 (17), 178 (15), 152 (8), 131 (4), 89 (7); elemental analysis
calcd (%) for C20H17NO6 (367.35): C 65.39, H 4.66, N 3.81; found: C
65.42, H 4.64, N 3.83.
Cell cultures
Human breast cancer epithelial cell line MCF-7 (ER-positive) was
cultured in Dulbecco‘s modified Eagle‘s medium (DMEM) contain-
ing 5% fetal bovine serum (FBS), 1% l-glutamine, 1% Eagle’s non-
essential amino acids, and 1 mgmLÀ1 penicillin–streptomycin at
378C with 5% CO2 in air. Triple-negative human breast cancer cell
line MDA-MB-231 (ER-, PR-, HER2-negative) was cultured in
DMEM:F12 containing 5% FBS. Human normal breast epithelial cell
line MCF-10A was grown in DMEM-F12 medium containing 5%
horse serum. Before each experiment, cells were grown in phenol
red free medium, containing 5% charcoal-stripped FBS for 2 days
and treated as described.
Compound 3j’: Yield: 87 mg (74% from 1j; Table 1, entry 11); col-
orless solid; m.p. 249–2518C; IR (KBr): n˜ =3396 (m, br), 2233 (vw),
1712 (s), 1670 (m), 1634 (w), 1477 (w), 1385 (m), 1198 (m), 1026
(m), 767 cmÀ1 (w); 1H NMR (300 MHz, [D6]DMSO): d=10.45 (brs,
1H; NH), 7.78 (distorted dd, J=8.4, 2.1 Hz, 1H; aromatic), 7.63–7.40
(m, 5H; aromatic), 7.33–7.25 (m, 1H; aromatic), 5.89 (s, 1H; =CH),
4.15 (q, J=7.1 Hz, 2H; CH2CH3), 1.23 ppm (t, J=7.1 Hz, 3H;
CH2CH3); 13C NMR (75 MHz, [D6]DMSO): d=167.5, 163.2, 155.2,
154.1, 148.3, 134.6, 132.5, 131.9, 130.1, 129.1, 126.6, 125.6, 124.6,
121.1, 120.4, 117.4, 109.2, 60.4, 13.9 ppm; GC-MS: m/z (%): 334 (16)
[M+], 288 (68), 261 (100), 260 (63), 247 (10), 232 (22), 216 (11), 205
(29), 190 (22), 177 (19), 164 (5), 151 (20), 116 (6), 101 (18), 89 (24),
75 (27); elemental analysis calcd (%) for C19H14N2O4 (334.33): C
68.26, H 4.22, N 8.38; found: C 68.31, H 4.21, N 8.39.
Cell viability assays
Cell viability was determined with the MTT (Sigma, Milan, Italy)
assay. MCF-7, MDA-MB-231, and MCF-10A cells (2”104 cellsmLÀ1
)
were grown in 24-well plates and exposed to treatments as indi-
cated for 96 h, in phenol red free minimum essential medium
(MEM) containing 5% charcoal-stripped FBS. The MTT assay was
performed as follows: 100 mL MTT stock solution in PBS
(2 mgmLÀ1) was added to each well and incubated at 378C for 2 h,
followed by media removal, and solubilization in DMSO (500 mL).
After shaking the plates for 15 min, the absorbance in each well,
including the blanks at l=570 nm, was measured by using a Beck-
man Coulter spectrophotometer. Data are representative of three
independent experiments, each performed in triplicate. Data were
analyzed for statistical significance by using a two-tailed Student’s
t-test, performed by using the Graph Pad Prism 4 program (Graph-
Pad Software, Inc., San Diego, CA). SD values are shown. A mini-
mum of three experiments, containing eight different doses of
z-lactam 3j’ in triplicate, was combined for IC50 calculations. The
absorbance readings were used to determine the IC50 values by
using the GraphPad Prism 4 program. Briefly, values were log-trans-
formed, then normalized, and nonlinear regression analysis was
used to generate a sigmoidal dose-response curve to calculate IC50
values for each cell line.
Compound 3k’: Yield: 70 mg (53% from 1k; Table 1, entry 12); col-
orless solid; m.p. 218–2208C; IR (KBr): n˜ =3103 (m, br), 1718 (s),
1673 (m), 1504 (m), 1478 (m), 1449 (m), 1391 (m), 1339 (s), 1198
(m), 1167 (m), 1126 (m), 882 (w), 856 (w), 767 cmÀ1 (w); 1H NMR
(300 MHz, [D6]DMSO): d=10.48 (brs, 1H; NH), 7.71–7.63 (m, 1H; ar-
omatic), 7.61–7.39 (m, 5H; aromatic), 7.33–7.24 (m, 1H; aromatic),
5.89 (s, 1H; =CH), 4.13 (q, J=7.1 Hz, 2H; CH2CH3), 1.20 ppm (t, J=
7.1 Hz, 3H; CH2CH3); 13C NMR (75 MHz, [D6]DMSO): d=167.6, 163.2,
155.1, 153.2, 148.7, 134.0, 132.0, 130.3, 126.8 (q, J=32.6 Hz), 126.3,
125.6, 125.5 (q, J=1.5 Hz), 124.3, 123.4 (q, J=272.2 Hz), 121.1,
120.0, 60.3, 13.8 ppm; 19F NMR (471 MHz, CDCl3): d=À60.6 ppm (s,
3F; CF3); GC-MS: m/z (%): 377 (45) [M+], 349 (5), 332 (28), 331 (58),
305 (17), 304 (100), 276 (20), 275 (22), 248 (19), 234 (5), 208 (8), 207
(12), 178 (13), 152 (9), 128 (5), 101 (11), 89 (12), 75 (18); elemental
analysis calcd (%) for C19H14F3NO4 (377.31): C 60.48, H 3.74, F 15.11,
N 3.71; found: C 68.45, H 3.72, F 15.13, N 3.73.
Theoretical calculations
Soft-agar anchorage-independent growth assays
Quantum calculations were carried out by using the NWChem
package,[13] within the framework of DFT. Becke–Lee–Yang–Parr16
(B3LYP) hybrid potentials and energy functionals were used to take
into account the exchange and correlation contributions. The Cou-
lomb and exchange-correlation potentials were numerically inte-
grated on an adaptive grid with fine accuracy. All calculations were
performed by using linear combinations of Gaussian-type orbitals:
triple-z basis set with polarization function (6-311G*) for C, N, O,
and H atoms and 3-21G* basis set for iodine. Relativistic effective
Cells (104 per well) were plated in 0.35% agarose (4 mL) with 5%
charcoal-stripped FBS in phenol red free media, with a 0.7% agar-
ose base in 6-well plates. Two days after plating, media containing
vehicle or z-lactam 3j’ were added to the top layer and replaced
every two days. After 14 days, MTT (300 mL) was added to each
well and allowed to incubate at 378C for 4 h. Plates were then
placed at 48C overnight and colonies of >50 mm in diameter from
triplicate assays were counted. Data are the mean colony number
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of three plates and representative of two independent experi-
ments, analyzed for statistical significance by using a two-tailed
Student’s t-test, performed by using the Graph Pad Prism 4 pro-
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Wound-healing scratch assays
Motility was assessed by wound-healing scratch assays. Briefly, cell
monolayers were scraped and treated as indicated. Wound closure
was monitored over 24 h; cells were fixed and stained with Coo-
massie brilliant blue. Pictures were taken at 10” magnification by
means of phase-contrast microscopy and were representative of
three independent experiments.
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Keywords: antitumor agents · carbonylation · heterocycles ·
palladium · reaction mechanisms
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Published online on January 28, 2016
Chem. Eur. J. 2016, 22, 3053 – 3064
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