Fast synthesis of amides from ethyl salicylate under microwave radiation in a solvent-free system

Article abstract of DOI:10.1039/c7ra11434f

In this study, amide bond formation, one of the most important reactions in organic chemistry, it was evaluated using ethyl salicylate and ten different primary amines. Under the optimized experimental conditions, i.e. 60 °C, hexane, phenylboronic acid-PBA (15 mol%), boric acid-BA (15 mol%) or without catalyst-WC, using a hot-plate for 24 h, amides were obtained in excellent isolated yields (WC, 77-94% to S-Aa-Ad; PBA, 11-94% to S-Ae-Aj; and BA, 28-90% to S-Ae-Aj). The reaction employing CAL-B also permitted a moderate conversion for the production of amides S-Ae-Aj (3-42%). However, in our efforts to reduce the amide synthesis time (24 h), the reactions were performed in the presence of microwave-MW radiation using a free-solvent system [60 °C, PBA (15 mol%) or WC], which reduced the time of the reaction by 32-fold (45 min) and afforded nine amides (S-Aa-Ah and S-Aj) in 80-99% isolated yield and S-Ai in 23% yield. A cytotoxicity assay demonstrated that the amide S-Ag was capable of inhibiting four human tumor cell lines (MCF-7, HCT116, HepG2, and HL-60) with an IC₅₀ ranging from 8.68 to 17.57 μg mL^-1. In this study, MW radiation provided an attractive way for faster reactions, improved yields, and cleaner reactions, as well as the synthesis of amide S-Ag with cytotoxic activity.

Full text of DOI:10.1039/c7ra11434f

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Fast synthesis of amides from ethyl salicylate under
microwave radiation in a solvent-free system†
Cite this: RSC Adv., 2017, 7, 56566
a
Rafaely N. Lima,
Valdenizia R. Silva,b Luciano de S. Santos,b Daniel P. Bezerra,b
b
a
*
Milena B. P. Soares and Andre L. M. Porto
´
In this study, amide bond formation, one of the most important reactions in organic chemistry, it was
evaluated using ethyl salicylate and ten different primary amines. Under the optimized experimental
conditions, i.e. 60 ꢀC, hexane, phenylboronic acid-PBA (15 mol%), boric acid-BA (15 mol%) or without
catalyst-WC, using a hot-plate for 24 h, amides were obtained in excellent isolated yields (WC, 77–94%
to S-Aa-Ad; PBA, 11–94% to S-Ae-Aj; and BA, 28–90% to S-Ae-Aj). The reaction employing CAL-B also
permitted a moderate conversion for the production of amides S-Ae-Aj (3–42%). However, in our efforts
to reduce the amide synthesis time (24 h), the reactions were performed in the presence of microwave-
MW radiation using a free-solvent system [60 ꢀC, PBA (15 mol%) or WC], which reduced the time of the
reaction by 32-fold (45 min) and afforded nine amides (S-Aa-Ah and S-Aj) in 80–99% isolated yield and
S-Ai in 23% yield. A cytotoxicity assay demonstrated that the amide S-Ag was capable of inhibiting four
human tumor cell lines (MCF-7, HCT116, HepG2, and HL-60) with an IC50 ranging from 8.68 to 17.57 mg
mLꢁ1. In this study, MW radiation provided an attractive way for faster reactions, improved yields, and
cleaner reactions, as well as the synthesis of amide S-Ag with cytotoxic activity.
Received 16th October 2017
Accepted 28th November 2017
DOI: 10.1039/c7ra11434f
rsc.li/rsc-advances
have demonstrated that aspirin plus dipyridamole and indi-
vidual ASA present antithrombotic activity aer ischemic
stroke.8,9 Salicylanilides display activity against tuberculosis and
cytotoxicity to multi-drug resistant bacteria.10 SA conjugated to
hydroquinone (SAH) displayed better activity for the treatment
of melasma through chemical peeling as compared to its iso-
lated compounds, and SAH had higher lipophilicity and skin
absorption.11
Introduction
Salicylic acid (SA) is a phenolic compound and member of the b-
hydroxy acid family, which is widely found in many plants to
induce a disease resistance response.1 It has also been widely
used as a key ingredient in many skin-care products for the
treatment of acne, wrinkles, warts, exfoliation, and aging;2–4
however, the use of SA in these formulations has been limited
due to strong irritation of skin caused by it.5
To minimize or improve the activity of SA, novel methodol-
ogies or analogues compounds have been developed or
synthesized. For example, due to its low solubility in water,
microcapsules containing SA have been developed, which show
controlled release and improved antibacterial activity against
Staphylococcus aureus, Staphylococcus epidermidis, and Propio-
nibacterium acnes.5
Fatty amide derivatives of SA also show a high affinity for
stratum corneum and present safer topical application since
they do not metabolize and enter into systemic circulation,
accumulating in the uppermost layer of skin.12,13 Lipophilic
hydroxy acid derivatives of SA reduced the number of non-
inammatory lesions of comedonal acne by up to 55.6% as
compared to salicylic acid, which reduced it by 48.5%;14 SA
amides also present antioxidant activity and HIV-1 integrase
inhibition,15,16 same as observed with 3-keto SA chalcones,
exhibiting HIV-1 integrase inhibition.17
Therefore, SA and its analogues (amides, esters, fatty amides,
and fatty acid) present a large number of biological activities
and have been largely studied in academic, medicinal, and
pharmaceutical areas. Thus, it is important to synthesize
amides from SA for applications in biotechnology, agriculture,
and medicine according to Green Chemistry principles using
facile procedures and eco-friendly methodologies with lower
amounts of waste, solvent, and time. Microwave irradiation has
become a very usual technique in organic synthesis, improving
products yields and decreasing reaction times; moreover, when
Aspirin (acetylsalicylic acid, ASA), an ester product obtained
from SA, is a drug used worldwide due to its analgesic, anti-
thermal, and anti-inammatory activities.6,7 Investigations
a
´
´
ˆ
´
´
˜
Laboratorio de Quımica Organica e Biocatalise, Instituto de Quımica de Sao Carlos,
˜
˜
´
Universidade de Sao Paulo, Av. Joao Dagnone, 1100, Ed. Quımica Ambiental, Santa
˜
Angelina, 13563-120, Sao Carlos, SP, Brazil. E-mail: almporto@iqsc.usp.br; Fax:
+55 16 33739952; Tel: +55 16 33738103
b
˜
˜
Fundaçao Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz, R. Waldemar Falcao,
121, Candeal, 40296-710, Salvador – BA, Brazil
† Electronic supplementary information (ESI) available: NMR spectroscopic
spectra, GC-MS analysis, mass spectra and infrared analysis. See DOI:
10.1039/c7ra11434f
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it is used under solvent-free conditions, it results in cleaner
reactions.18–20
To optimize the production of amide S-Aa, the reaction was
carried out at 45 ꢀC using 3 equivalents of amine Aa (0.90 mmol)
Herein, we report a facile, rapid, high yielding, and eco- while decreasing the amount of solvent to 1 mL of hexane. The
friendly solvent-free synthesis of amide derivatives from ethyl conversion obtained in this assay was 74% in the presence of
salicylate under microwave radiation in the absence or presence CAL-B (50 mg) and 75% without CAL-B. Similar results were
of lipase obtained from Candida antarctica (CAL-B) and phe- obtained aer 48 h when the same conditions were used: the
nylboronic acid or boric acid as catalysts. The amides synthe- amide S-Aa was obtained with 87% conversion in the presence
sized were also submitted to an in vitro cytotoxicity assay using of CAL-B and with 79% conversion without CAL-B. Again, it was
four human tumor cells lines (breast adenocarcinoma, colon observed that CAL-B had a modest effect on the reaction yield.
carcinoma, hepatocellular carcinoma, and promyelocytic
leukemia).
In accordance with the previous experiments, the difference
in conversion aer 24 h or 48 h in the presence or absence of
CAL-B was not signicant when 3 equivalents of amine (0.90
mmol) and 1 mL of hexane were used. However, optimization of
the reaction conditions was signicant since it was not neces-
sary to add the enzyme to achieve a high yield of amide S-Aa at
Results and discussion
Aminolysis reactions of ethyl salicylate using Candida
ꢀ
45 C aer 48 h using 3 equiv. of amine and 1 mL of hexane
(79%).
antarctica lipase and phenylboronic acid
In addition, the aminolysis reaction of ES (0.3 mmol) and
amine Aa (0.90 mmol) in hexane (1 mL) without CAL-B at 60 ꢀC
produced a 93% conversion of S-Aa aer 24 h (Table 1). 2-
Hydroxy-N-(2-hydroxyethyl)benzamide S-Aa was isolated (92%
yield) and characterized using GC-MS, IR, and NMR analysis.
The optimized reaction conditions were used to realize the
reaction between ethyl salicylate ES (0.30 mmol) and nine
different amines Ab-Aj (0.90 mmol, 60 ꢀC, 1 mL of hexane,
450 rpm, and 24 h). High isolated yields were obtained for 2-
hydroxy-N-(3-hydroxypropyl)benzamide S-Ab (94%), 2-hydroxy-
N-(5-hydroxypentyl)benzamide S-Ac (84%), and 2-hydroxy-N-(2-
hydroxypropyl)benzamide S-Ad (77%), as reported in Table 2.
Interestingly, it was observed that for the amides N-butyl-2-
hydroxybenzamide S-Ae, 2-hydroxy-N-pentylbenzamide S-Af, N-
heptyl-2-hydroxybenzamide S-Ag, N-allyl-2-hydroxybenzamide
S-Ai, and N-benzyl-2-hydroxybenzamide S-Aj, the aminolysis
To investigate the synthetic reactions of amides, ethyl salicylate
ES (50 mg, 0.30 mmol) and 2-aminoethan-1-ol Aa (0.30 mmol)
have been used during the reaction optimization process, and
the optimized conditions are as follows: initial temperature at
ꢀ
45 C, 3 mL of hexane as a solvent, and immobilized commer-
cial CAL-B (50 mg). In this assay, it was observed that CAL-B did
not catalyze the aminolysis reaction aer 24 h. Upon increasing
the amount of amine Aa to 3 equivalents (0.90 mmol), amide S-
Aa was produced in 59% yield aer 24 h. However, a similar
yield (57%) without the use of the enzyme was obtained. These
reactions showed that the use of CAL-B did not efficiently
catalyze the reaction aer 24 h. However, upon prolonging the
reaction time to 48 h, 79% conversion was obtained in the
presence of CAL-B, whereas 62% conversion was obtained
without CAL-B. In this case, the enzyme showed a moderate
catalytic effect in the reaction.
Table 1 Optimization of the aminolysis reaction conditions using ethyl salicylate ES and 2-aminoethan-1-ol Aa in the presence and absence of
CAL-Ba
24 h
48 h
c (%)
c (%)
c (%)
c (%)
T (ꢀC) Hexane (mL) Ratio ES : Aa amide S-Aa with CAL-B amide S-Aa without CAL-B amide S-Aa with CAL-B amide S-Aa with CAL-B
45
3
3
1
1
3
1
(1 : 1)
(1 : 3)
(1 : 1)
(1 : 3)
(1 : 3)
(1 : 3)
0
57
26
75
0
—
62
41
79
93
—
79
53
87
94
59
29
74
68
94
60
59
93 (92)b
a
Performed in duplicate. b Isolated yield. c ¼ conversion determined by the GC-FID analysis.
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Table 2 The aminolysis reactions using ethyl salicylate ES and amines
Ab-Ad in the absence of CAL-B
functionalized amines (Ae-Aj), boric acid (BA, H3BO3), as
a cheaper and non-toxic catalyst, was used with the aim to
reduce the reaction cost.
Upon carrying out the reaction using BA under the same
reaction conditions (60 ꢀC, 15 mol%, 450 rpm, 1 mL of hexane,
and 24 h) used with phenylboronic acid, it was observed that by
increasing the amine carbon chain (Ae < Af < Ag < Ah), inferior
yields were obtained as compared to the case of PBA (S-Ae, 53%;
S-Af, 77%; S-Ag, 86%; and S-Ah, 90%).
The observed effect was attributed to the lower solubility of
BA in hexane as compared to that of PBA, which reduced the
catalytic effect of BA and consequently the production of
amides. On the other hand, both catalysts have the same effect
on the increased yields, which were observed in the order of S-
Ae < S-Af < S-Ag < S-Ah (BA and PBA).
PBA can also infer some p electronic repulsion or interaction
with the amines Ai and Aj, which have a similar p-bond
component causing a reduction in the amide conversion (S-Ai,
11% and S-Aj, 63%) as compared to BA (S-Ai, 28% and S-Aj, 77%)
with the yields being greater than those obtained with PBA.
Thus, both catalysts allowed high isolated yields for the
synthesis of several amide derivatives of ethyl salicylate ES.
Fig. 1 shows an adapted proposal21–23 for the catalytic cycle
using phenylboronic acid as the catalyst to perform the ami-
nolysis reactions between ethyl salicylate ES and the amines
Ae-Aj providing the amides S-Ae-Aj, respectively. The catalytic
cycle starts with the formation of a cyclic intermediate
between PBA and ethyl salicylate ES. This intermediate acti-
vates the carbonyl group of ES via hydrogen bonding; this
facilitates amine attack, consequent formation of the amide,
and regeneration of PBA.
Amines
Amide products
c (%) Yielda (%)
93
85
94
84
83b
77
Isolated yield. b Diastereoisomer mixture. c ¼ conversion determined
a
by GC-FID analysis.
As PBA showed good catalytic effect for the production of
amides from mono-functionalized amines, it was also used for
reactions did not occur without the use of CAL-B as a catalyst bi-functionalized amines, Aa-Ad, in the aminolysis reactions
with the exception of amide N-dodecyl-2-hydroxybenzamide S- with ES. The results showed that PBA was not able to catalyze
Ah, which was formed with only 6% conversion.
the reactions for the production of S-Aa (93%), S-Ab (94%), S-Ac
The reaction conversions (3–42%) observed in the prepara- (84%), and S-Ad (77%) since the results obtained were very
tion of amides S-Ae to S-Aj using CAL-B showed the moderate similar to those achieved in the absence of the catalyst aer 24 h
catalytic effect of the lipase (Table 3).
of reaction. These results can be explained through the intra-
To obtain the amides S-Ae-Aj in better yields, phenylboronic molecular interaction observed with amino-alcohols between
acid (PBA) was used as a catalyst. The reaction to produce S-Ae the N–H and O–H functional groups that forms a cyclic
was carried out using 5 mol%, 10 mol%, and 15 mol% PBA for conformation, which does not occur with mono-functionalized
24 h. It was observed that 15% PBA was able to catalyze the amines. These interactions can cause steric hindrance and
production of amide S-Ae with 95% conversion (determined by electronic effects that make it difficult for the catalyst to react
GC-FID), and a 79% isolated yield was obtained via ash and accelerate the reaction.24
column chromatography using silica gel. Lower yields of the
amide S-Ae were obtained using 10 mol% PBA (yield ¼ 70%) and
Aminolysis reactions of ethyl salicylate ES and amines Aa-Aj
under microwave radiation
5 mol% PBA (yield ¼ 60%) (Scheme 1). Following the procedure
described, the amides S-Ae-Aj were analyzed by GC-FID and
isolated, and the yields were obtained (Table 3).
To decrease the reaction time obtained previously using a hot
In the presence of PBA, ethyl salicylate, ES, was substantially plate at 24 hours, the aminolysis reaction was conducted under
converted to its respective amides in high isolated yields aer microwave radiation to accelerate the reaction. As hexane is the
24 h (S-Af, 86%; S–Ag, 91%; and S-Ah, 90%). However, the best solvent for the aminolysis reactions with ethyl salicylate ES,
amides S-Ai and S-Aj were obtained in only 11% and 63% yields, as reported in the previous studies and according to the litera-
respectively (Table 3).
ture,25 it does not present a good effect when applied under
As phenylboronic acid was observed to be a good catalyst in MW; thus, the aminolysis reactions in this study have been
the aminolysis reactions using ethyl salicylate ES and mono- carried out using a solvent-free system.
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Table 3 The aminolysis reactions of ethyl salicylate ES and mono-functionalized amines Ae-Aj in the presence of phenylboronic acid (PBA) and
boric acid (BA)
Amines
Amide products
c (%) CAL-B
c (%) PBA
Yielda (%) PBA
Yielda (%) BA
3
95
79
53
20
40
96
97
86
91
77
86
42
15
12
97
43
82
90
11
63
90
28
77
a
Isolated yield. c ¼ conversion determined by GC-FID analysis.
The reaction under MW using ethyl salicylate ES and 2-
According to the high performance of the aminolysis reac-
aminoethan-2-ol Aa (3 equiv.) in a solvent-free system at 60 ꢀC was tion under MW, the amides S-Aa-Aj were synthesized using this
followed by thin layer chromatography-CCD, and aer 45 min, the methodology. All products were obtained in excellent yield (80–
ES was totally consumed; this decreased the reaction time deter- 99%), except for amide S-Ai (23%). The low yield of the amide S-
mined earlier by conventional heating in hexane (24 h) by 32-fold. Ai can be explained by the low vapor pressure and boiling point
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results were slightly higher than those obtained upon heating
on a hot-plate (Table 3). The exception was S-Ai (23%) although
its yield was double of that obtained with conventional heating
on a hot plate (11%).
Finally, due to the good yields obtained in the aminolysis
reaction using the optimized procedure with 3 equivalents of
amine under MW radiation, the reaction was investigated using
1 equivalent of amine Aa. In this case, the reaction to produce S-
Scheme 1 The synthesis of amide S-Ae via the aminolysis reaction of
n-butylamine and ES in the presence of PBA as a catalyst.
ꢀ
Aa was then scaled up to 1 mmol and carried out at 100 C for
45 min and 2 h. Under these conditions, S-Aa was obtained in
75% and 80% yields. Therefore, it was concluded that even
ꢀ
upon increasing the temperature (100 C) and prolonging the
reaction time to 2 h, the yield did not increase (only 5%); this
conrmed the need to use an excess amount of amine to achieve
a better reaction performance and yield.
MW irradiation has been used for the aminolysis reactions
without the use of an organic solvent, and good isolated yields
have been obtained; however, these methodologies still employ
tosyl chloride as the condensing agent26 or high temperatures
(150 C),18,27 which results in a less eco-friendly methodology.
ꢀ
Others previous studies have described the synthesis of the
amide S-Aa from methyl salicylate without the use of an organic
solvent at high temperatures (210–215 ꢀC).28 A similar proce-
dure was also used to produce the amides S-Aa, S-Ab, and S-Ac at
170 ꢀC.29,30 An one-pot system using 2-chlorobenzoyl chloride
and dichloromethane (step-one, 5 h) and KOH, CuI, 1,10-phe-
nanthroline, and water at 100 ꢀC (step-two, 10 h) also produced
the amide S-Aa.31
Fig. 1 The proposed catalytic cycle of the aminolysis reaction of ethyl
salicylate ES with different amines catalyzed by PBA.
Amide S-Ae was previously obtained using a solution of
phenol in toluene, BCl3, and n-butylisocyanate aer 12 h of
reaction.32 The amide S-Af was synthetized using N-butyl-o-
iodobenzamide, LiOH, CuBr, and 1,10-phenanthroline in
DMSO at 100 ꢀC aer 12 h.33 The amide S-Aj was synthetized
using salicyloyl chloride in dichloromethane at 0 ꢀC and TEA
(t.a., overnight).34
(53 ꢀC) of the amine Ai. When submitted to a system at 60 ꢀC, it
can be evaporated into the atmosphere even if the reaction has
been performed using a reux apparatus.
As observed in Table 4, several amides were obtained in high
isolated yields (from 80 to 99%) under MW radiation, and the
Greener conditions were applied to produce the amide S-Ag
using salicylic acid and 10% CAL-B at 75 ꢀC (18 h) and amide S-
Ah using ethyl salicylate and 5 g molꢁ1 of CAL-B without the use
of a solvent at 65 ꢀC (20 h).12,33 The amide S-Ai was obtained
from salicylic acid using silica gel 60 (230–400 mesh) under
microwave radiation (200 W, 130 ꢀC, and 20 min).36 As observed,
Table 4 The aminolysis reactions of ethyl salicylate ES and several
amines Aa-Aj under microwave radiation
ꢀ
most of these reactions required temperatures between 100 C
and 215 ꢀC, time in the range from 12 to 20 h, complex catalysts,
and chloride or iodine reagents. Milder, greener, and faster
reactions were obtained using CAL-B (65–75 ꢀC, 18–20 h)12,33 and
MW irradiation (200 W, 130 ꢀC, 20 min).36 These conditions are
superior (temperature or time) when compared with those of
the procedure reported by us in this study (17 W, 60 ꢀC,
15 mol% PBA or BA, without a solvent, and 45 min).
Amide products
Yielda (%)
2-Hydroxy-N-(2-hydroxyethyl)benzamide (S-Aa)
2-Hydroxy-N-(3-hydroxypropyl)benzamide (S-Ab)
2-Hydroxy-N-(5-hydroxypentyl)benzamide (S-Ac)
2-Hydroxy-N-(2-hydroxypropyl)benzamide (S-Ad)
N-Butyl-2-hydroxybenzamide (S-Ae)
2-Hydroxy-N-pentylbenzamide (S-Af)
N-Heptyl-2-hydroxybenzamide (S-Ag)
N-Dodecyl-2-hydroxybenzamide (S-Ah)
N-Allyl-2-hydroxybenzamide (S-Ai)
N-Benzyl-2-hydroxybenzamide (S-Aj)
99b
91b
87b
92b
80c
94c
97c
97c
23c
86c
Antitumor activity
Table 5 shows the IC50 data obtained for the amide series S-Aa-
Aj synthesized from ethyl salicylate. As shown in Table 5, only
amide S-Ag exhibited good cytotoxic activity for the four-tumor
cell lines (MCF-7, HCT116, HepG2, and HL-60) investigated
herein. The best IC50 observed was obtained for MCF-7 cells
a
Isolated yield. b Without catalyst. c 15 mol% PBA.
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Table 5 The IC50 values obtained for cytotoxic activity in human
tumor cell lines versus non-tumor cell lines after 72 h of exposurea
aminopropan-1-ol Ab (99%), 5-aminopentan-1-ol Ac (95%), DL-1-
aminopropan-2-ol Ad ($90%), butan-1-amine Ae (99%), pentan-
1-amine Af ($99%), heptan-1-amine Ag (97%), dodecan-1-
amine Ah (98%), prop-2-en-1-amine Ai (99%), phenylmethan-
amine Aj ($99%), ethyl salicylate (99%), and phenylboronic
acid (95%) and boric acid (99.5%) were purchased from Sigma
Aldrich. Hexane was purchased from Synth, ethyl acetate
(EtOAc) was obtained from NEON, and silica gel C18 on TLC
(Thin Layer Chromatography) was obtained from ALUGRAM®
XTra SIL G/UV245. All reagents were used without further
purication.
Human tumor cell lines
MCF7
HCT116
HepG2
HL-60
Amides
(mg mLꢁ1
)
(mg mLꢁ1
)
(mg mLꢁ1
)
(mg mLꢁ1
)
S-Aa
S-Ab
S-Ac
S-Ad
S-Ae
S-Af
S-Ag
S-Ah
S-Ai
>25
>25
>25
ND
>25
>25
8.68
>25
>25
ND
>25
>25
>25
>25
>25
>25
17.57
>25
>25
>25
0.11
>25
>25
>25
>25
>25
>25
15.56
>25
>25
>25
0.07
>25
>25
>25
>25
>25
>25
16.30
>25
>25
>25
0.05
GC-FID analysis of the aminolysis reactions
The analyses of the aminolysis reactions were performed using
a gas chromatograph (GC2010, Shimadzu Corporation, Japan)
equipped with an AOC-20i (Shimadzu) automatic injection
sampler and ame ionization detector (FID). The ionization
process was carried out using nitrogen gas (30 mL minꢁ1) and
the ame formed from hydrogen 5.0 (40 mL minꢁ1) and
synthetic air (400 mL minꢁ1). The separations were performed
on a fused silica capillary column (DB-5MS 5%-phenyl–95%-
dimethylpolysiloxane 30 m ꢂ 0.25 mm internal diameter,
0.25 mm lm thickness) in a stream of nitrogen 5.0 of 0.95
S-Aj
DOX
1.78
a
MCF7 (human breast adenocarcinoma), HCT116 (human colon
carcinoma), HepG2 (human hepatocellular carcinoma), and HL-60
(human promyelocytic leukemia). Doxorubicin (DOX) was used as
a positive control. ND ¼ not determined.
(8.68 mg mLꢁ1). The amide S-Ag was also able to inhibit HepG2,
HL-60, and HCT116 cells with the IC50 values of 15.56 mg mLꢁ1
,
mL minꢁ1. The injector temperature was 230 C, and 1.0 mL of
ꢀ
16.30 mg mLꢁ1, and 17.57 mg mLꢁ1, respectively. The other
amides presented IC50 values above 25 mg mLꢁ1 and thus were
not considered to have a promising cytotoxic activity.37,38
the sample was used. The oven temperature setting started from
100 ꢀC and increased at 7 ꢀC minꢁ1 until 300 ꢀC, and this
temperature was maintained for 5 min. The total time of the
analysis was 33.57 min.
Conclusion
Biocatalytic synthesis of the amide S-Aa
Aer employing three different catalysts for the synthesis of ten
amides S-Aa-Aj from ethyl salicylate ES, it was observed that
CAL-B showed a moderate effect for catalyzing the aminolysis
reactions (3–42%), especially for the mono-functionalized
amines Ae-Aj using a hot-plate and hexane as a solvent. The
reactions with phenylboronic acid (63–91%; S-Ae-Ah, S-Aj) and
boric acid (53–90%; S-Ae-Ah, S-Aj) as catalysts resulted in high
isolated yields, except for S-Ai (11% and 23%, respectively). The
use of microwave radiation in a solvent-free system allowed the
preparation of amides in higher yields (80–99%; S-Aa-Ah, S-Aj)
than those obtained using a hot plate (63–94%; S-Aa-Ah, S-Aj),
and a 32-fold reduction in the reaction time as compared to the
case of conventional heating (24 h) was observed. Via the results
obtained, it was possible to verify the great technological
potential of microwave radiation to improve the reaction time
in a solvent-free system and the capacity of the catalysts based
on boron to improve the synthesis of amides, e.g. amide S-Ag
that presented cytotoxic activity in the presence of four tumor
cell lines.
In a 5 mL vial, ethyl salicylate ES (50 mg, 0.30 mmol), 2-
aminoethan-1-ol Aa (0.30 or 0.90 mmol), Candida antarctica
lipase (50 mg), and hexane (1 or 3 mL) were added. The vial was
kept under magnetic stirring (450 rpm) on a hot-plate (IKA®/
RCT basic) at 45 ꢀC or 60 ꢀC. The samples (200 mL) were ob-
tained, diluted with hexane in a 5 mL volumetric ask, and
analyzed by GC-FID aer 24 and 48 hours. The yield was
calculated based on an analytical curve (R2 ¼ 0.9952) obtained
by the GC-FID analysis of a stock solution (670 ppm, 33.5 mg/50
mL) and dilutions (570, 470, 370, 170, 70, and 28.7 ppm) (ESI†).
Reactions without CAL-B were also performed and analyzed by
GC-FID.
Synthesis of the amides S-Aa-Ad
In a 5 mL vial, ethyl salicylate ES (50 mg, 0.30 mmol), amine Aa-
Ad (0.90 mmol), and hexane (1 mL) were added. The reaction
mixtures were kept under magnetic stirring (450 rpm) on a hot-
plate (IKA®/RCT basic) at 60 ꢀC for 24 h. To minimize the
solvent cost, the amides were not subjected to an extraction
process. Aer the reaction, the amides were isolated using
preparative thin layer chromatography. For the amides S-Aa-Ad,
90% EtOAc and 10% hexane were used as the eluent for the
Experimental
Material and methods
Lipase obtained from Candida antarctica (CAL-B), expressed in isolation and purication of the products.
Aspergillus niger and immobilized on acrylic resin, deuterated
2-Hydroxy-N-(2-hydroxyethyl)benzamide (S-Aa).28–31 Molec-
chloroform (CDCl3-99.8% with 0.5% TMS), deuterated meth- ular formula: C9H11NO3; MM: 181.19 g molꢁ1; yield: 99%, beige
anol (CD3OD, 99.9%), 2-aminoethan-2-ol Aa ($99%), 3- solid; mp 102–103 ꢀC; IR (silicon plate) n (cmꢁ1): 3397, 3298,
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1
2934, 1634, 1589, 1549; H NMR (400 MHz, CD3OD) d (ppm): 6.89 (m, 2H), 7.35 (qd, 1H, J ¼ 1.71 and 7.33 Hz), 7.79 (dd, 1H, J
3.52 (t, 2H, J ¼ 5.86 Hz), 3.71 (t, 2H, J ¼ 5.87 Hz), 6.89 (m, 2H), ¼ 1.71 and 7.83 Hz); 13C NMR (126 MHz, CD3OD) d (ppm): 14.1,
7.37 (qd, 1H, J ¼ 1.71 Hz and 7.33 Hz), 7.78 (dd, 1H, J ¼ 1.23 Hz 21.2, 32.6, 40.2, 117.0, 118.4, 120.0, 128.7, 134.6, 161.2, 170.9;
and 7.83 Hz); 13C NMR (126 MHz, CD3OD) d (ppm): 43.0, 61.5, MS (70 eV), m/z (%): M+c 193 (24), 121 (100), 120 (25), 65 (17).
117.1, 118.3, 120.1, 129.1, 134.7, 161.0, 171.0; MS (70 eV), m/z
(%): M+c 181 (27), 163 (23), 121 (100), 120 (20).
2-Hydroxy-N-pentylbenzamide (S-Af).33 Molecular formula:
C
12H17NO2; MM: 207.27 g molꢁ1; yield: 94%, beige liquid; IR
2-Hydroxy-N-(3-hydroxypropyl)benzamide (S-Ab).29,30 Molec- (silicon plate) n (cmꢁ1): 3375, 3065, 2959, 2932, 2864, 1641,
ular formula: C10H13NO3; MM: 195.22 g molꢁ1; yield: 91%, beige 1595, 1545; 1H NMR (400 MHz, CD3OD) d (ppm): 0.93 (t, 3H, J ¼
liquid; IR (silicon plate) n (cmꢁ1): 3362, 3084, 2934, 2882, 1638, 6.85 Hz), 1.37 (m, 4H), 1.63 (quintet, 2H, J ¼ 7.33 Hz), 3.37 (t,
1597, 1597; 1H NMR (400 MHz, CD3OD) d (ppm): 1.83 (quintet, 2H, J ¼ 7.09 Hz), 6.88 (m, 2H), 7.36 (qd, 1H, J ¼ 1.71 and 7.34
2H, J ¼ 6.36 Hz), 3.49 (t, 2H, J ¼ 6.85 Hz), 3.65 (t, 2H, J ¼ 6.11 Hz), 7.74 (dd, 1H, J ¼ 1.22 and 7.83 Hz); 13C NMR (126 MHz,
Hz), 6.87 (m, 2H), 7.36 (qd, 1H, J ¼ 1.71 and 7.34 Hz), 7.73 (dd, CD3OD) d (ppm): 14.4, 23.4, 30.1, 30.3, 40.5, 117.0, 118.4, 120.0,
1H, J ¼ 1.22 and 7.82 Hz); 13C NMR (126 MHz, CD3OD) d (ppm): 128.7, 134.6, 161.2, 171.9; MS (70 eV), m/z (%): M+c 207 (30), 151
33.2, 37.7, 60.6, 117.0, 118.4, 120.0, 128.7, 134.7, 161.1, 171.0; (18), 150 (21), 121 (100), 120 (22).
MS (70 eV), m/z (%): M+c 195 (19), 151 (17), 150 (20), 121 (100),
N-Heptyl-2-hydroxybenzamide (S-Ag).35 Molecular formula:
120 (20).
C
14H21NO2; MM: 235.33 g molꢁ1; yield: 97%, amorphous white
2-Hydroxy-N-(5-hydroxypentyl)benzamide (S–Ac).29,30 Molec- solid; IR (silicon plate) n (cmꢁ1): 3375, 3065, 2955, 2928, 2857,
ular formula: C12H17NO3; MM: 223.27 g molꢁ1; yield: 87%, 1639, 1595, 1545; H NMR (400 MHz, CD3OD) d (ppm): 0.90 (t,
1
amorphous beige solid; IR (silicon plate) n (cmꢁ1): 3358, 3080, 3H, J ¼ 6.85 Hz), 1.35 (m, 8H), 1.61 (quintet, 2H, J ¼ 7.09 Hz),
2938, 2864, 1638, 1597, 1549. 1H NMR (400 MHz, CD3OD) 3.37 (t, 2H, J ¼ 7.33 Hz), 6.88 (m, 2H), 7.35 (qd, 1H, J ¼ 1.47 and
d (ppm): 1.45 (m, 2H), 1.62 (m, 4H), 3.39 (t, 2H, J ¼ 7.34 Hz), 3.57 7.09 Hz), 7.74 (dd, 1H, J ¼ 1.47 and 7.82 Hz); 13C NMR (126
(t, 2H, J ¼ 6.61 Hz), 6.88 (m, 2H), 7.36 (qd, 1H, J ¼ 1.71 and 7.34 MHz, CD3OD) d (ppm): 14.4, 23.7, 28.1, 30.1, 30.4, 32.9, 40.5,
Hz), 7.74 (dd, 1H, J ¼ 1.22 and 7.83 Hz); 13C NMR (126 MHz, 117.1, 118.4, 120.0, 128.7, 134.6, 161.2, 170.8; MS (70 eV), m/z
CD3OD) d (ppm): 24.4, 30.3, 33.3, 40.5, 62.8, 117.0, 118.4, 120.0, (%): M+c 235 (29), 151 (24), 150 (24), 121 (100), 120 (20).
128.7, 134.6, 161.2, 170.9; MS (70 eV), m/z (%): M+c 223 (17), 150
(20), 121 (100), 120 (20).
N-Dodecyl-2-hydroxybenzamide (S-Ah).39 Molecular formula:
C
19H31NO2; MM: 305.46 g molꢁ1; yield: 97%, white amorphous
2-Hydroxy-N-(2-hydroxypropyl)benzamide (S-Ad). Molecular solid; mp 66–68 ꢀC; IR (silicon plate) n (cmꢁ1): 3406, 3059, 2953,
formula: C10H13NO3; MM: 195.22 g molꢁ1; yield: 92%, beige 2916, 2851, 1643, 1584, 1541; 1H NMR (400 MHz, CDCl3)
liquid; IR (silicon plate) n (cmꢁ1): 3364, 3084, 2974, 2930, 2880, d (ppm): 0.87 (t, 3H, J ¼ 6.85 Hz), 1.30 (m, 18H), 1.63 (quintet,
1639, 1597, 1549; 1H NMR (400 MHz, CD3OD) d (ppm): 1.20 (d, 2H, J ¼ 7.09 Hz), 3.43 (td, 2H, J ¼ 5.63 and 7.10 Hz), 6.84 (qd, 1H,
3H, J ¼ 6.36 Hz), 3.32 (dd, 1H, J ¼ 7.09 and 13.70 Hz), 3.46 (dd, J ¼ 0.97 and 7.09 Hz), 6.98 (dd, 1H, J ¼ 0.98 and 8.32 Hz), 7.36
1H, J ¼ 4.64 and 13.69 Hz), 3.96 (m, 1H), 6.89 (m, 2H), 7.35 (qd, (m, 2H); 13C NMR (126 MHz, CDCl3) d (ppm): 14.1, 22.7, 26.9,
1H, J ¼ 1.72 and 7.34 Hz), 7.80 (dd, 1H, J ¼ 1.22 and 7.83 Hz); 29.3, 29.4, 29.4, 29.5, 29.5, 29.6, 31.9, 39.7, 114.4, 118.5, 118.6,
13C NMR (126 MHz, CD3OD) d (ppm): 21.0, 47.8, 67.4, 117.3, 125.2, 134.0, 161.5, 169.9; MS (70 eV), m/z (%): M+c 305 (26), 164
118.3, 120.1, 129.3, 134.7, 160.8, 170.8; MS (70 eV), m/z (%): M+c (17), 151 (42), 121 (100), 120 (17).
195 (20), 151 (26), 150 (31), 121 (100), 65 (20).
N-Allyl-2-hydroxybenzamide (S-Ai).34 Molecular formula:
C
10H11NO2; MM: 177.20 g molꢁ1; yield: 23%, colorless liquid; IR
(silicon plate) n (cmꢁ1): 3364, 3078, 2926, 2855, 1639, 1597,
1543; 1H NMR (400 MHz, CD3OD) d (ppm): 4.01 (dt, 2H, J ¼ 1.47
and 5.38 Hz), 5.14 (dq, 1H, J ¼ 1.47 and 10.51 Hz), 5.23 (dq, 1H, J
Synthesis of the amides S-Ae-Aj using phenylboronic acid and
boric acid as catalysts
In a 5 mL ask, ethyl salicylate ES (50 mg, 0.30 mmol), butan-1- ¼ 1.71 and 17.11 Hz), 5.94 (qt, 1H, J ¼ 5.38 and 10.76 Hz), 6.89
amine Ae (0.90 mmol), hexane (1 mL), and the organic catalyst (m, 2H), 7.35 (qd 1H, J ¼ 1.47 and 7.34 Hz), 7.79 (dd, 1H, J ¼ 1.71
phenylboronic acid (PBA, 5 mol%, 10 mol% and 15 mol%) or and 7.82 Hz); 13C NMR (126 MHz, CD3OD) d (ppm): 42.7, 116.2,
boric acid (BA, 15 mol%) were added, and the reaction was 117.0, 118.4, 120.1, 128.9, 134.7, 135.4, 161.1, 170.7; MS (70 eV),
conducted at 60 C for 24 h. Aer the purication process, the m/z (%): M+c 177 (36), 121 (100), 120 (33), 93 (20), 92 (20), 57 (47),
ꢀ
best aminolysis reaction condition was applied to 6 different 56 (31), 65 (22).
amines (Ae-Aj). Aer the reaction, the amides were isolated
using thin layer chromatography. For the amides S-Ai and S-Aj,
N-Benzyl-2-hydroxybenzamide (S-Aj).36 Molecular formula:
C
14H13NO2; MM: 227.26 g molꢁ1; yield: 86%, white solid; mp
a mixture of 90% hexane and 10% EtOAc was used as the eluent, 128–129 C; IR (silicon plate) n (cmꢁ1): IR (nmax): cmꢁ1 3356,
ꢀ
1
and for S-Ae-Ah, a mixture of 80% hexane and 20% EtOAc was 3026, 2938, 1639, 1589; H NMR (400 MHz, CD3OD) d (ppm):
used as the eluent. The control reactions did not detect 4.58 (s, 2H), 6.89 (qd, 2H, J ¼ 0.74 and 7.34 Hz), 7.24 (tt, 1H, J
production of the amides S-Ae-Aj.
¼ 1.47 and 6.36 Hz), 7.35 (m, 5H), 7.79 (dd, 1H J ¼ 1.47 and
N-Butyl-2-hydroxybenzamide (S-Ae).32 Molecular formula: 7.83 Hz); 13C NMR (126 MHz, CD3OD) d (ppm): 44.0, 117.0,
C
11H15NO2; MM: 193.25 g molꢁ1; yield: 80%, beige liquid; IR 118.4, 120.1, 128.2, 128.5, 128.9, 129.5, 134.8, 140.0, 161.2,
(silicon plate) n (cmꢁ1): 3375, 3067, 2961, 2934, 2870, 1641, 170.9; MS (70 eV), m/z (%): 91 (100), M+c 227 (60), 121 (23), 106
1595, 1545; 1H NMR (400 MHz, CD3OD) d (ppm): 0.96 (t, 3H, J ¼ (21), 65 (17).
7.58 Hz), 1.40 (m, 2H), 1.60 (m, 2H), 3.37 (t, 2H, J ¼ 7.34 Hz),
56572 | RSC Adv., 2017, 7, 56566–56574
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salt, Alamar blue is reduced in proliferating cells. Its oxidized
form is blue (non-uorescent/non-viable cell), and the reduced
form is pink (uorescent/viable cell). The reduction of Alamar
blue reects cell proliferation. It was initially used to indicate
cell growth and/or viability in monitoring lymphocyte prolifer-
ation,41 and currently has several applications. Initially, the cells
were plated in 96-well plates (100 mL per well of a solution of 0.3
ꢂ 106 cells per mL for cells in suspension and 0.7 ꢂ 105 cells
per mL for adhered cells). Aer 24 hours of incubation, amides
S-Aa-Aj (previously dissolved in DMSO) were added to the
human tumor cells lines and incubated at 37 ꢀC for 72 h.
Doxorubicin was used as a positive control. The negative control
received the same amount of DMSO. Then, four hours (twenty-
four hours for PBMC) before the end of the incubation period,
20 mL of a stock solution (0.312 mg mLꢁ1) of Alamar blue
(resazurin) was added to each well. The absorbance was
measured at the wavelength of 570 nm (reduced) and 595 nm
(oxidized) using a plate reader.41 The percentage of inhibition
was calculated, the percentage of inhibition ꢂ log of the
concentration was calculated, and its IC50 was determined from
the non-linear regression using the Prisma version 5.0 program
(GraphPad Soware).
Synthesis of the amides S-Aa to S-Ad under microwave
radiation without the use of a catalyst and using
phenylboronic acid for the amides S-Ae to S-Aj
In a 5 mL round-bottomed ask, ES (50 mg, 0.30 mmol) and the
amines Aa-Ad (0.90 mmol) were added. For the S-Ae-Aj
synthesis, ES, the amines Ae to Aj, and 15 mol% of PBA were
used. For the production of the amides S-Aa-Ad, the use of
a catalyst was not necessary. The reaction was carried out
without the addition of an organic solvent and subjected to
microwave radiation (17 W, 60 ꢀC, 45 min). To avoid the use of
excess amine, the reactions were also carried out using ES and
ꢀ
the amine Aa in equivalent proportions (1 mmol) at 100 C for
45 min and 2 h.
Gas chromatography-mass spectrometry (GC-MS)
The reactions analyses were conducted using a gas chromato-
graph (GC-MS-QP2010 Ultra Shimadzu Corporation, Japan)
equipped with an auto-sampler injection AOC-20i (Shimadzu).
An electron capture detector was used with the electronic
impact of 70 eV, a scanning speed of 1.666, and a scan interval
of 0.30 fragments per s; the fragments were detected from 40 to
500 Da. The separations were performed using a fused silica
capillary column (DB-5MS 5% phenyl–95%–dimethylpolysilox-
ane, 30 m ꢂ 0.25 mm internal diameter, and 0.25 mm lm
thickness) in a stream of helium 5.0 at 0.95 mL minꢁ1. The
injector temperature, oven temperature, rate increase, amount
of sample, and total time analysis were the same as presented in
the GC-FID analysis (see the ESI†).
Cells
Tumor cells, MCF-7 (human breast adenocarcinoma), HCT116
(human colon carcinoma), HepG2 (human hepatocellular
carcinoma), and HL-60 (human promyelocytic leukemia), were
obtained from the American Type Culture Collection (ATCC)42
and used in the assays. The tumor cells were cultured in cell
culture bottles (75 cm3, 250 mL volume) in media comprising
RPMI 1640 and supplemented with 10% fetal bovine serum.
The tumor cells were maintained in incubators under a 5% CO2
atmosphere at 37 ꢀC. Cellular growth was monitored daily using
an inversion microscope. The medium was changed whenever
the cell growth reached the necessary conuence for nutrient
renewal. For maintenance of the adhered cells, trypsin (0.25%)
was used for the cells to escape from the walls of the bottles. Cell
cultures showed microplasma negatives, as judged by place-
ment with Hoechst (Mycoplasma Stain Kit, Cat. MYC1, Sigma-
Aldrich, St. Louis, MO, USA).
Fourier transform infrared (FTIR) analysis
FTIR spectra were obtained using a Shimadzu IRAffinity-1
spectrometer. The analyses were performed using a thin lm
formed by the application of diluted samples (CHCl3) on silicon
plates. The transmittance was expressed in cmꢁ1 for the bands
observed between 4000 and 400 cmꢁ1
.
Nuclear magnetic resonance (NMR)
NMR spectra were obtained using an Agilent apparatus oper-
ated at 500 MHz for 1H and 126 MHz for 13C. The samples were
solubilized in CDCl3 or CD3OD, and the chemical shis were
expressed in ppm relative to an internal standard, TMS.
Coupling constants (J) are reported in hertz (Hz). The analyses
were conducted using 15–20 mg of isolated amides. The
acquisition time for 1H analysis was 4.0894 s at a sweep width of
8012.82 Hz, and for 13C analysis, the acquisition time was
1.0486 s at a sweep width of 31 250.00 Hz.
Conflicts of interest
There are no conicts of interest to declare.
Cytotoxicity assay
Acknowledgements
The samples (S-Aa-Aj) were diluted in sterile and pure DMSO
ꢁ1
˜
(5 mg mL ) and tested at concentrations ranging from 0.19 to RNL is thankful for the fellowship provided by the Coordenaçao
25 mg mLꢁ1. To evaluate the cytotoxicity of the amides S-Aa-Aj, de Aperfeiçoamento de Pessoal de Nıvel Superior (CAPES).
´
an Alamar blue test was performed aer 72 h of exposure with ALMP thanks the Conselho Nacional de Desenvolvimento
´ ´
Cientıco e Tecnologico (CNPq/Proc. 301987/2013-0) and
the test substances.
Alamar blue, identied as resazurin,40 is a uorescent/ Fundaçao de Amparo a Pesquisa do Estado de Sao Paulo
colorimetric indicator with redox properties. As a tetrazolium (FAPESP/Proc. 2014/18257-0) for nancial support.
˜
`
˜
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Paper
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56574 | RSC Adv., 2017, 7, 56566–56574
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