Synthesis, computational evaluation and pharmacological assessment of acetylsalicylic esters as anti-inflammatory agents

Article abstract of DOI:10.1007/s00044-018-02284-3

A convenient approach to the synthesis of alkyl esters of aspirin (ASA-OR) has been developed. The synthesis of ASA-OR has been realized in two steps: (1) direct esterification of salicylic acid with alcohols in the presence of dicyclohexylcarbodiimide to give alkyl salicylates (SAL-OR); (2) acetylation of SAL-OR with acetyl chloride to yield ASA-OR. Molecular mechanics simulations, performed to calculate the kinetic radii of several ASA-OR, indicated that the pentyl and hexyl acetylsalicylates possess the best properties to cross cell membranes. The in vitro biological tests demonstrate their anti-inflammatory activity, superimposable to that of aspirin. The results of our study suggest that ASA-OR may be used as anti-inflammatory drugs for topical application.

Full text of DOI:10.1007/s00044-018-02284-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-02284-3
ORIGINAL RESEARCH
Synthesis, computational evaluation and pharmacological
assessment of acetylsalicylic esters as anti-inflammatory agents
Raffaella Mancuso1 Nadia Ferlazzo2 Giorgio De Luca3 Roberta Amuso1 Antonio Palumbo Piccionello4
●
●
●
●
●
Salvatore V. Giofrè2 Michele Navarra
Bartolo Gabriele1
2
●
●
Received: 10 August 2018 / Accepted: 24 December 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A convenient approach to the synthesis of alkyl esters of aspirin (ASA-OR) has been developed. The synthesis of ASA-OR
has been realized in two steps: (1) direct esterification of salicylic acid with alcohols in the presence of
dicyclohexylcarbodiimide to give alkyl salicylates (SAL-OR); (2) acetylation of SAL-OR with acetyl chloride to yield
ASA-OR. Molecular mechanics simulations, performed to calculate the kinetic radii of several ASA-OR, indicated that the
pentyl and hexyl acetylsalicylates possess the best properties to cross cell membranes. The in vitro biological tests
demonstrate their anti-inflammatory activity, superimposable to that of aspirin. The results of our study suggest that ASA-
OR may be used as anti-inflammatory drugs for topical application.
●
●
●
●
Keywords Acetylsalicylic acid Acetylsalicylic esters Anti-inflammatory activity Aspirin Computational study
Introduction
516 moiety of COX-2 (Loll et al. 1995), being 170 times
more effective in inhibiting the former (Vane et al. 1998).
The blockage of these enzymes leads to a decrease of the
production of prostaglandins and thromboxane-A2,
responsible for its anti-inflammatory and anti-platelet
effects, respectively. Despite its significant activity against
different conditions, inflammatory and cardiovascular dis-
eases, fever and pain, ASA also presents several important
collateral effects, mainly on the gastrointestinal apparatus
(Lavie et al. 2017). In order to reduce them, transdermal
delivery offers an alternative route for administering ASA
that bypasses the gut and may be a more convenient, safer
and non-invasive mean for its delivery especially in case of
long-term use. For the topic treatment of pain or inflam-
matory diseases, the use of a formulation based on a slow
and low-dose transdermal delivery of ASA would be highly
desirable (Shamsher et al. 2010). To avoid its rapid
hydrolysis to salicylic acid (SAL) during skin absorbtion, it
has been suggested to use more stable aspirin derivatives,
such as salicylic esters ASA-OR (R = alkyl), able to effi-
ciently cross the skin and then release the more hydrophilic
ASA subcutaneously by hydrolysis (McMahon et al. 1988).
Some alkyl esters of ASA were previously prepared by
esterification of acetylsalicyloyl chloride with the corre-
sponding alcohols, yet in low to modest yields (7–22%)
(Gerber et al. 2006). In this work, we have synthesized four
salicylic esters ASA-OR 1–4, with R = pentyl (1), hexyl (2),
Aspirin (acetylsalicylic acid, ASA) is one of the most
known and used non-steroidal anti-inflammatory drugs
(Desborough and Keeling 2017). It acts by irreversibly
inhibiting both cyclooxygenase (COX) enzymes. ASA
acetylates the serine 530 moiety of COX-1, and the serine
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-018-02284-3) contains supplementary
material, which is available to authorized users.
* Michele Navarra
mnavarra@unime.it
1
Laboratory of Industrial and Synthetic Organic Chemistry
(LISOC), Department of Chemistry and Chemical Technologies,
University of Calabria, via Pietro Bucci 12/C, Arcavacata di Rende
(CS), Rende 87036, Italy
2
Department of Chemical, Biological, Pharmaceutical and
Environmental Sciences, University of Messina, viale SS
Annunziata, Messina 98168, Italy
3
Institute on Membrane Technology, National Research Council
(ITM-CNR), Via Pietro Bucci 17/C, Arcavacata di Rende (CS),
Rende 87036, Italy
4
Department of Biological, Chemical and Pharmaceutical Science
and Technology-STEBICEF, University of Palermo, viale delle
Scienze Ed.17, Palermo 90128, Italy

Medicinal Chemistry Research
decyl (3), and tetradecyl (4), by a new and convenient
procedure, based on two synthetic steps: (1) direct ester-
ification of SAL with aliphatic alcohols (ROH), in the
presence of dicyclohexylcarbodiimide (DCC), to give alkyl
salicylates (SAL-OR); (2) acetylation of SAL-OR with
acetyl chloride to yield ASA-OR. We have also performed
molecular mechanics simulations to evaluate whether these
agents are able to cross cellular membranes and assessed
their anti-inflammatory activity in vitro.
with AcOEt, and the solution was first acidified with 1 M
HCl and then neutralized with saturated NaHCO3. The
organic layer was washed with water (3 × 50 mL) and then
dried over Na2SO4. After filtration and evaporation of the
solvent, the crude product was purified by column chro-
matography on silica gel, using hexane-AcOEt 99:1 as
eluent.
Pentyl salicylate Yield: 300 mg, starting from 500 mg of
SAL (40%). Yellow oil. IR (film): v = 3183 (w, br), 1676
(s), 1327 (m), 1302 (m), 1214 (m), 1158 (m), 757 (m), 701
1
Materials and methods
Synthesis of alkyl acetylsalicylates (ASA-OR) 1–4
General
(w); H NMR (300 MHz, CDCl3): δ (ppm) = 10.87 (s, 1H,
OH), 7.85 (dd, J = 8.0, 1.7, 1H, aromatic), 7.49–7.40 (m,
1H, aromatic), 6.94 (d, J = 8.4, 1H, aromatic), 6.92–6.88
(m, 1H, aromatic), 4.34 (t, J = 6.7, 2H, OCH2), 1.87–1.72
(m, 2H, OCH2CH2CH2), 1.51–1.33 (m, 4H, CH2CH2CH3),
0.94 (t, J = 7.1, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ
(ppm) = 170.3, 161.7, 135.6, 129.9, 119.1, 117.6, 112.6,
65.5, 28.3, 28.1, 22.3, 14.0; GC-MS: m/z = 208 (21) [M+],
138 (31), 120 (100), 92 (13), 65 (8). HRMS-ESI [(M-H)‒]:
m/z calcd for (C12H15O3)‒: 207.1027; found, 207.1035.
Anhydrous THF was purchased from Sigma Aldrich
(Sigma-Aldrich Italia s.r.l., Milano, Italy). All other solvents
and chemicals were reagent grade and used without further
purification (Sigma-Aldrich). Starting material, salicylic
acid, was commercially available (Sigma-Aldrich). All
reactions were analyzed by TLC on silica gel 60F254
(Merck) and by GLC by using a Shimadzu GC-2010 gas
chromatograph and capillary columns with poly-
methylsilicone + 5% phenylsilicone (HP-5) as the sta-
tionary phase. Evaporation refers to the removal of solvent
Hexyl salicylate Yield: 325 mg, starting from 500 mg of
SAL (40%). Yellow oil. IR (film): v = 3184 (m, br), 1676
(s), 1586 (w), 1302 (m), 1214 (m), 1091 (m), 757 (m), 701
1
(w); H NMR (300 MHz, CDCl3): δ (ppm) = 10.87 (s, 1H,
OH), 7.83 (dd, J = 8.0, 1.7, 1H, aromatic), 7.47–7.37
(m, 1H, aromatic), 6.96 (dd, J = 8.5, 1.7, 1H, aromatic),
6.90–6.81 (m, 1H, aromatic), 4.32 (t, J = 6.7, 2H, OCH2),
1.76 (q, J = 6.8, 2H, OCH2CH2CH2), 1.51–1.21 [m, 6H,
(CH2)3CH3], 0.99–0.81 (m, 3H, CH3); 13C NMR (75 MHz,
CDCl3): δ (ppm) = 170.3, 161.7, 135.5, 129.9, 119.1,
117.6, 112.7, 65.5, 31.5, 28.6, 25.7, 22.6, 14.0; GC-MS: m/
z = 222 (10) [M+], 138 (47), 120 (100), 92 (15). The
spectroscopic properties agreed with those reported
(Chighine et al. 2009).
1
under reduced pressure. Melting points are uncorrected. H
NMR and 13C NMR spectra were recorded at 25 °C with a
Bruker DPX Avance 300 Spectrometer in CDCl3 solutions
at 300 and 75 MHz, respectively, with Me4Si as internal
standard. Chemical shifts (δ) and coupling constants (J) are
given in ppm and in Hz, respectively. IR spectra were taken
with a JASCO FT-IR 4200 spectrometer. Mass spectra were
obtained by using a Shimadzu QP-2010 GC-MS apparatus
at 70 eV ionization voltage and by electrospray ionization
mass spectrometry (ESI-MS) by using an Agilent 6540
UHD accurate-mass Q-TOF spectrometer equipped with a
Dual AJS ESI source working in positive or negative mode.
Decyl salicylate Yield: 458 mg, starting from 500 mg of
SAL (45%). Yellow oil. IR (film), v: 3190 (m, br), 1675 (s),
1302 (m), 1198 (m), 1158 (m), 757 (m), 701 (w); 1H NMR
(300 MHz, CDCl3): δ (ppm) = 10.87 (s, 1H, OH), 7.83 (dd,
J = 8.0, 1.5, 1H, aromatic), 7.48–7.38 (m, 1H, aromatic),
6.96 (d, J = 8.4, 1H, aromatic), 6.90–6.82 (m, 1H, aro-
matic), 4.33 (t, J = 6.7, 2H, OCH2), 1.85–1.69 (m, 2H,
OCH2CH2CH2), 1.52–1.15 [m, 14H, (CH2)7CH3], 0.94–
0.83 (m, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ (ppm) =
170.3, 161.7, 135.5, 129.9, 119.1, 117.6, 112.7, 65.5, 31.9,
29.58, 29.56, 29.35, 29.28, 28.6, 26.0, 22.7, 14.1; GC-MS:
m/z = 278 (31) [M+], 138 (89), 120 (100), 92 (11). HRMS-
ESI [(M-H)‒]: m/z calcd for (C17H25O3)‒: 277.1809; found,
277.1793. The spectroscopic properties agreed with those
reported (Kalbasi et al. 2010).
Esterification of salicylic acid (SAL) to give alkyl salicylates
(SAL-OR)
To a solution of SAL (500 mg, 3.62 mmol) in anhydrous
THF (200 mL), maintained at 0 °C and under nitrogen, was
added the alcohol ROH (5.43 mmol; 1.5 equiv) (R = pentyl:
480 mg; hexyl: 555 mg; decyl: 860 mg, tetradecyl: 1.17 g).
To the resulting mixture was added, dropwise and under
nitrogen, a solution of DCC (1.57 g, 7.61 mmol) in anhy-
drous THF (200 mL). After stirring at room temperature for
20 h, the mixture was concentrated under vacuum and fil-
tered to remove the excess DCC. The solid was washed

Medicinal Chemistry Research
Tetradecyl salicylate Yield: 489 mg, starting from 500 mg
of SAL (40%). Yellow solid, m.p. 37–38 °C. IR (KBr): v =
3125 (w, br), 1673 (s), 1470 (m), 1303 (m), 1215 (m), 1157
(m), 762, 668 (w); 1H NMR (300 MHz, CDCl3): δ (ppm) =
10.87 (s, 1H, OH), 7.84 (d, J = 7.8, 1H, aromatic), 7.50–
7.36 (m, 1H, aromatic), 6.97 (d, J = 8.4, 1H, aromatic),
6.92–6.80 (m, 1H, aromatic), 4.33 (t, J = 6.6, 2H, OCH2),
1.86–1.70 (m, 2H, OCH2CH2CH2), 1.50–1.05 [m, 22H,
(CH2)11CH3], 0.94–0.79 (m, 3H, CH3); 13C NMR (75 MHz,
CDCl3): δ (ppm) = 170.3, 161.7, 135.5, 129.9, 119.1,
117.6, 112.7, 65.5, 31.9, 29.72, 29.71, 29.69, 29.67, 29.61,
29.54, 29.39, 29.26, 28.6, 26.0, 22.7, 14.1; GC-MS: m/z =
334 (37) [M+], 138 (100), 120 (80), 92 (8). HRMS-ESI
[(M-H)‒]: m/z calcd for (C21H33O3)‒: 333.2435; found,
333.2445.
1.81–1.62 (m, 2H, OCH2CH2CH2), 1.52–1.20 [m, 6H,
(CH2)3CH3], 0.98–0.80 (m, 3H, CH3); 13C NMR (75
MHz, CDCl3): δ (ppm) = 169.7, 164.6, 150.7, 133.7,
131.7, 126.0, 123.8, 123.5, 65.3, 31.5, 28.6, 25.6, 22.5,
21.0, 14.0; HRMS-ESI [(M+Na)+]: m/z calcd for
(C15H20NaO4)+: 287.1254; found, 287.1234.
Decyl acetylsalicylate (3) Yield: 208 mg, starting from 400
mg of decyl salicylate (45%). Yellow solid, m.p. 41–42 °C.
IR (KBr): v = 1776 (s), 1720 (s),1606 (m), 1373 (m), 1256
(s), 1200 (m), 1081 (m); 1H NMR (300 MHz, CDCl3):
δ (ppm) = 8.02 (dd, J = 7.8, 1.6, 1H, aromatic), 7.59–7.50
(m, 1H, aromatic), 7.35–7.27 (m, 1H, aromatic), 7.10 (dd,
J = 8.0, 1.1, 1H, aromatic), 4.26 (t, J = 6.7, 2H, OCH2),
2.35 (s, 3H, COMe), 1.82–1.66 (m, 2H, OCH2CH2CH2),
1.47–1.18 [m, 14H, (CH2)7CH3], 0.94–0.83 (m, 3H, CH3);
13C NMR (75 MHz, CDCl3): δ (ppm) = 169.7, 164.6,
150.7, 133.7, 131.7, 126.0, 123.8, 123.5, 65.3, 31.9, 29.54,
29.52, 29.30, 29.28, 28.6, 26.0, 22.7, 21.0, 14.1; HRMS-
ESI [(M+Na)+]: m/z calcd for (C19H28NaO4)+: 343.1880;
found, 343.1857.
Acetylation of alkyl salicylates to give alkyl acetylsalicylates
ASA-OR 1–4
To a solution of the alkyl salicylate (1.44 mmol) (R =
pentyl: 300 mg; hexyl: 320 mg; decyl: 400 mg, tetradecyl:
482 mg) in anhydrous CH2Cl2 (13 mL), was added, at room
temperature and under nitrogen, Et3N (365 mg, 3.6 mmol)
followed by AcCl (290 mg, 3.7 mmol). After stirring at
40 °C for 20 h, satd NH4Cl was added (5 mL). The resulting
mixture was extracted at room temperature with CH2Cl2
(3 × 15 mL), and the collected organic layers dried over
Na2SO4. After filtration and evaporation of the solvent, the
crude product was purified by column chromatography on
silica gel, using hexane-AcOEt99:1 as eluent.
Tetradecyl acetylsalicylate (4) Yield: 300 mg, starting
from 482 mg of tetradecyl salicylate (55%). Yellow solid,
m.p. 39–40 °C. IR (KBr): v = 1766 (s), 1720 (s), 1255 (s),
1201 (m), 1084 (m); 1H NMR (300 MHz, CDCl3):
δ (ppm) = 8.06–7.97 (m, 1H, aromatic), 7.61–7.49 (m, 1H,
aromatic), 7.36–7.25 (m, 1H, aromatic), 7.13–7.06 (m,
1H, aromatic), 4.26 (t, J = 6.8, 2H, OCH2), 2.36 (s, 3H,
COMe), 1.87–1.61 (m, 2H, OCH2CH2CH2), 1.58–1.02 [m,
22H, (CH2)11CH3], 1.01–0.79 (m, 3H, CH3); 13C NMR (75
MHz, CDCl3): δ (ppm) = 169.7, 164.5, 150.7, 133.7,
131.7, 126.0, 123.8, 123.5, 65.3, 31.9, 29.69, 29.66, 29.60,
29.5, 29.4, 29.3, 28.6, 26.0, 22.7, 21.0, 14.1; HRMS-ESI
[(M+Na)+]: m/z calcd for (C23H36NaO4)+: 399.2506;
found, 399.2479.
Pentyl acetylsalicylate (1) Yield: 163 mg, starting from
300 mg of pentyl salicylate (45%). Yellow oil. IR (film): v
= 1778 (s), 1723 (s), 1294 (m), 1196 (s), 1160 (s), 1083
(m); 1H NMR (300 MHz, CDCl3): δ (ppm) = 8.03 (dd, J =
7.8, 1.7, 1H, aromatic), 7.60–7.50 (m, 1H, aromatic), 7.36–
7.27 (m, 1H, aromatic), 7.10 (dd, J = 8.0, 0.7, 1H, aro-
matic), 4.27 (t, J = 6.8, 2H, OCH2), 2.35 (s, 3H, COMe),
1.82–1.66 (m, 2H, OCH2CH2CH2), 1.46–1.31 (m, 4H,
CH2CH2CH3), 0.97–0.86 (m, 3H, CH3); 13C NMR (75
MHz, CDCl3): δ (ppm) = 169.7, 164.5, 150.6, 133.7, 131.7,
126.0, 123.8, 123.5, 65.3, 28.3, 28.1, 22.3, 21.0, 13.9;
HRMS-ESI [(M+Na)+]: m/z calcd for (C14H18NaO4)+:
273.1077; found, 273.1080.
Computational approach
The ASA-OR conformational analysis was performed by
Molecular Mechanics using the universal force field and
Avogadro code (Hanwell et al. 2012), which is an
application of the Open Babel platform (O’Boyle et al.
2011). A search method converging to the low energy
conformers, by weighing torsion angles as function of the
total energy, was employed. For each acetylsalicylic
esters, a sample of 30 × 104 conformers was analyzed and
the weighed rotor search was applied to find the more
stable conformations. Finally, the most stable fifty
structures were used for the calculation of the kinetic
radii, after a last geometry optimization using a conjugate
gradient algorithm.
Hexyl acetylsalicylate (2) Yield: 252 mg, starting from
320 mg of hexyl salicylate (66%). Yellow oil. IR (film): v
= 1772 (s), 1723 (s), 1294 (m), 1196 (s), 1083 (m), 915
(m); 1H NMR (300 MHz, CDCl3): δ (ppm) = 8.02 (d, J =
7.8, 1H, aromatic), 7.61–7.49 (m, 1H, aromatic), 7.36–
7.24 (m, 1H, aromatic), 7.09 (d, J = 8.1, 1H, aromatic),
4.26 (t, J = 6.7, 2H, OCH2), 2.35 (s, 3H, COMe),

Medicinal Chemistry Research
Cell culture and cytotoxicity study
as 1. All the experiments were carried out in triplicate and
repeated three times (Romeo et al. 2014a).
Cytotoxic studies
Real-time PCR
Experiments were carried out on the human leukemia
monocytic cell line THP-1, originally obtained from ATCC
(Rockville, MD, USA). The cultures were grown according
to the literature (Currò et al. 2016). Stock solutions of each
tested compound (ASA, 1, 2, and 3) were prepared in
dimethylsulfoxide (DMSO) at 100 mM concentration and
stored in aliquot at −20 °C. Compound 4 was insoluble in
DMSO, thus not allowing us to test its biological activity.
Products were diluted in culture media to the desired con-
centration just prior the use. The same DMSO concentra-
tions used to dissolve the molecules served as vehicle
controls. First, in order to assess the potential cytotoxicity of
molecules used in this study, preliminary experiments were
carried out on THP-1 cells using both 3-(4,5-dimethylthia-
zole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
(Navarra et al. 2015) and lactate dehydrogenase (LDH) test
(Romeo et al. 2014a). The former measures the mitochon-
drial activity of living cells, providing indirect information
on cell viability. The latter determines the release of the
cytosolic enzyme lactate dehydrogenase into the culture
medium when the cell plasma membrane is damaged (dead
cells). In both tests, THP-1 cells were incubated in 96-well
culture plates at a density of 5 × 103 cells/well with fresh
red-phenol free medium containing increasing concentra-
tions of molecules (ASA, 1, 2, and 3) at 37 °C for 24 h.
Then, for MTT assay, the plates were centrifuged, the
supernatants were replaced with 100 μL of media containing
0.5 mg/mL of MTT (Sigma-Aldrich, Milan, Italy), and the
plates were returned in the incubator for 4 h. After a cen-
trifugation, the supernatants were removed and the crystals
of formazan (MTT metabolic product) were solubilized
with 100 μL HCl/isopropanol 0.1 N lysis buffer. Absor-
bance was determined at 570 nm with reference at 690 nm,
using a microplate reader (iMark™ microplate reader).
Results are expressed as percentages of MTT reduction
respect to control cultures. All experiments were performed
in eight replicates and repeated three times (Romeo et al.
2014b).
The experimental model used to evaluate the anti-
inflammatory activity of the molecules 1, 2, and 3 con-
sisted in THP-1 cells stimulated with lipopolysaccharide
(LPS; 500 ng/ml) for 3 h or 6 h (Risitano et al. 2014). Cells
(1 × 106) were seeded into six/wells plates and the next day
were treated or not with 100 µM ASA, 1, 2, or 3, prior to add
LPS (30 min). Extraction of total cellular RNA and cDNA
synthesis were performed as described (Ferlazzo et al. 2016)
Briefly, after RNA isolation with TRIzol reagent (Invitrogen
Life Technologies, Milan, Italy), RNA (2 µg) was reverse
transcribed with High Capacity cDNA Archive kit (Applied
Biosystems, Applera Corp., Milan, Italy) according to the
manufacturer’s instructions. Then, mRNA levels of IL-1β,
TNF-α, cyclooxygenase-1 and 2 (COX-1 and COX-2), and
inducible nitricoxide synthase (iNOS) were analyzed by
real-time PCR. β-Actin mRNA was used as endogenous
control. Quantitative PCR reactions were set up in triplicate
in a 96-well plate and were carried out in 20 µL reactions
containing 1xSYBR® Select Master Mix (Applied Biosys-
tems), 0.2 µM of each primer (they are listed in Table 1) and
25 ng RNA converted into cDNA. The analyses were per-
formed in a 7500 real-time PCR System (Applied Biosys-
tems) and the data were collected and analyzed using the
2-ΔΔCT relative quantification method.
Results and discussion
Synthesis of acetylsalicylic esters (ASA-OR) 1–4
As said in the Introduction, some alkyl acetylsalicylates
ASA-OR (R = Me, Et, Pr, i-Pr, Bu, 1-methylpropyl, t-Bu,
pentyl, 1-methylbutyl, and 1-ethylpropyl) were previously
Table 1 Primers used for real-time PCR
Gene product
COX1
Primers sequence
Forward: 5ʹ -CTTGTGCTCCTCTTGATT- 3ʹ
Reverse: 5ʹ -CACTCCACCATTCTGTCT- 3ʹ
Forward: 5ʹ -GCCTGGTCTGATGATGTA - 3ʹ
Reverse: 5ʹ -TCTGGAACAACTGCTCAT - 3ʹ
Forward: 5ʹ -GGATGACAACCGATACCA - 3ʹ
Reverse: 5ʹ -GAAGGCAATGGACTCAGA - 3ʹ
Forward: 5ʹ -GCTTATTACAGTGGCAATGA - 3ʹ
Reverse: 5ʹ -TAGTGGTGGTCGGAGATT - 3ʹ
Forward:5ʹ -GTGAGGAGGACGAACATC - 3ʹ
Reverse: 5ʹ -GAGCCAGAAGAGGTTGAG - 3ʹ
Forward: 5ʹ -TTGTTACAGGAAGTCCCTTGCC - 3ʹ
Reverse: 5ʹ -ATGCTATCACCTCCCCTGTGTG - 3ʹ
In LDH assay, we used a commercial LDH kit (CytoTox
96® Non-Radioactive Cytotoxicity Assay, Promega),
according to the manufacturer’s protocol. Briefly, plates
were centrifuged at 400 × g for 5 min, and 100 μL of
supernatant from each well was transferred to correspond-
ing wells on a new plate. Then, freshly prepared 100 μL
LDH reaction solution was added to each well, and the plate
placed on an orbital shaker for 30 min at room temperature.
Absorbance at 490 nm was quantified spectro-
photometrically. LDH levels are extrapolated as the values
detected in control cells, which are arbitrarily expressed
COX2
iNOS
IL1-β
TNF-α
β-Actin

Medicinal Chemistry Research
Fig. 1 Synthesis of acetylsalicylic esters (ASA-OR) 1–4 by direct esterification of salicylic acid (SAL) to give alkyl salicylates (SAL-OR) followed
by acetylation
Table 2 Minimum (rinf kic) and maximum (rsup kic) kinetic radii of five
ASA-OR (in brackets the corresponding % of isomers)
prepared in 7–22% yield by esterification of acetylsalicyloyl
chloride with the corresponding alcohols (Gerber et al.
2006). We present here an alternative and convenient
approach to alkyl acetylsalicylates (with R = pentyl, hexyl,
decyl, and tetradecyl, in particular), based on the direct
esterification of salicylic acid with alcohols ROH in the
presence of DCC followed by acetylation with acetyl
chloride, according to the scheme shown in Fig. 1.
As can be seen from the Scheme, fair isolated yields of
the final alkyl acetylsalicylates 1–4 (18–26% over the two
steps) were obtained under particularly mild conditions and
starting from readily available substrates.
Acetylsalicylic
esters (ASA-OR)
rinf
and %
isomer
(Å) rsup
(Å) % Isomer
with 5 Å <
Log
Ko/w
kic
kic
a
and %
isomer
rkic < 6 Å
R = butyl
5 (10)
5 (5)
8 (5)
9 (1)
9 (5)
9 (5)
9 (10)
63
61
15
33
50
1.8
1.9
R = pentyl
R = 2-metylpentyl
R = hexyl
6 (15)
6 (33)
6 (50)
2.4
R = 2-metylhexyl
aValues for acetates ROAc (Abraham et al. 1994)
Computational analysis of acetylsalicylic esters
kinetic radii
Therefore, the diffusion of ASA-OR exponentially
decreases as their kinetic radius increases, and increses as
the partition constants rise (Mitragotri 2003). According to
Eq. (1), the kinetic radius of the most stable ASA-OR
conformers provides information on the diffusion of mole-
cules having similar Ko/w. Different empirical formulas are
available in the literature to calculate the kinetic radii as
function of the molecular weights. Clearly, each ASA-OR
shows a huge number of conformers, so these empirical
formulas are not directly applicable in our case. For this
reason, in this work, the rkic of the acetylsalicylic esters
were calculated using a previously validated algorithm
(Buekenhoudt et al. 2013), which takes into account the
elevated number of conformers. In particular, the kinetic
radii of the 50 more stable conformers, selected from a
sample of 30 × 104 conformers were calculated. From this
calculation, it was possible to determine the minimum and
Four permeation pathways can be considered to describe the
passage of organic compounds across the cute: (a) diffusion
through the double lipid layers of the cellular membrane (due
to the presence of free volumes); (b) solute transport (ensuing
from lateral diffusion along lipid layers; for molecules with
Mw > 400 Da and partition constant Ko/w > 1); (c) diffusion
through pores (for small molecules with Ko/w < 0.01; and (d)
diffusion through hair follicles and sweat glands (for com-
pounds with Mw > 10,000 and Ko/w < 0.01).
In the case of small and hydrophobic solutes, with Mw <
400 Da and Ko/w > 1, the permeation is mainly controlled by
the diffusion through the free volumes of the double lipid
layers. The ASA-OR, theoretically investigated in this work
(R = butyl, pentyl, 2-methylpentyl, hexyl, 2-methylhexyl)
possess molecular masses < 400 Da and the partition coef-
ficients for acetates ROAc are all >1. Thus, the diffusion
through the double lipid layers should be the predominant
permeation mechanism with respect to the others. The dif-
fusion coefficient can be evaluated as function of the solutes
partition constant, Ko/w and kinetic radius, rkic, by means of
the following model equation (Mitragotri 2003):
maximum kinetic radii, that are, rinf
and rsup kic, respec-
kic
tively, and the percentage of ASA-OR conformers having
rkic between 5 Å and 6 Å; the results are shown in Table 2.
All the stable conformers of the investigated ASA-OR
show rkic higher than 5 Å. The range rinf kic – rsup kic remains
substantially constant as the number of the carbon atoms in
the chain of the alkyl chain increases. This means that the
0;7
o=w
À
Á
À
Á
diffusion of the ASA-OR is mainly controlled by K
.
0;7
o=w
À6
Kpfv rkic; Ko=w ¼ 5; 6 Â 10
K
exp À0; 46rk2ic
ð1Þ
From Table 2, it can be seen that logKo/w increases with the

Medicinal Chemistry Research
Fig. 2 Effects of ASA, 1, 2, and
3 on cell proliferation and LDH
release. THP-1 cells were
exposed to increased
concentration (µM) of
compounds for 24 h.
a Proliferation rate assessed by
MTT assay. b Cytotoxic effect
assessed in terms of LDH
release after 24 h of exposure.
Each value is the mean S.E.M.
of three experiments performed
eight times (MTT) or in
A
140
120
100
80
ASA
1
2
3
**
**
**
**
**
**
60
40
20
triplicate (LDH) and repeated
three different times. **p < 0.01
vs. ctrl, ***p < 0.001 vs. ctrl
0
B
3,0
2,5
2,0
1,5
1,0
0,5
0,0
ASA
1
2
3
***
***
***
***
***
**
length of the alkyl chain, therefore acetylsalicylic esters
with longer R chains should permeate more easily as long as
the (rinf kic – rsup kic) range remains constant. Furthermore,
the distribution of the kinetic radius in this range is another
important feature to consider. In fact, comparing the kinetic
radii of the ASA-OR with R = pentyl and hexyl, the 56% of
the conformers with R = pentyl shows rkic equal to 6 Å,
while only 33% of the conformers with R = hexyl displays
an equal kinetic radius. This means that the number of the
ASA-OR conformers with R = pentyl, which possess a rkic
equal to 6 Å, is greater than the number of the ASA-OR
conformers with R = hexyl. Since the logKo/w of these
ASA-OR are similar, the derivative with R = pentyl should
show a slightly greater diffusion compared to the derivative
with R = hexyl derivative. On the other hand, the ASA-OR
with R = butyl should show a similar permeability with
respect to the ester R = pentyl. The 50% of the more stable
conformers of ASA-OR with R = 2-metylhexyl shows a
kinetic radius around 6 Å; as a result, they should show a
permeability similar to the derivative with R = pentyl.
Moreover, the ASA-OR with R = 2-metylpentyl has only
the 15% of its conformers with rkic equal to 6 Å, which
means that this ASA-OR should permeate the double lipid
layers less effectively than ASA-OR with R = pentyl.
Biological experiments on acetylsalicylic esters
(ASA-OR) 1–3
In order to find the concentration of the molecules 1, 2, and
3 that did not induce cytotoxicity, preliminary experiments
were carried out using both MTT and LDH tests. Exposure
of THP-1 monocytes to different concentrations of tested
compounds (in a range 2.5–100 µM) for 24 h did not show
any significant cytotoxic effects (Fig. 2), while the 250 and
500 µM concentrations produced a significant reduction of
cell viability (Fig. 2a), accompanied by cell death (Fig. 2b).
Therefore, we chose the 100 µM concentration to detect the
anti-inflammatory activity of the molecules 1, 2, and 3.
Moreover, based on published data (Risitano et al. 2014),
we used LPS at the concentration of 500 ng/mL that has
been shown to trigger the release of pro-inflammatory
mediators in THP-1 cells within 3 h.
Data of the real-time PCR analyses showed that stimulation
of THP-1 monocytes with 500 ng/mL LPS for 3 h produced a
dramatic increase of IL-1β, TNF-α and COX-2 mRNA levels,
without affecting those of both of COX-1 and iNOS. In par-
ticular, in LPS-treated cells, the levels of IL-1β mRNA were
more than 1000-fold higher than those found in untreated cells
(Fig. 3), as well as those of TNF-α and COX-2 were almost

Medicinal Chemistry Research
Fig. 3 Effects of ASA and molecules 1, 2, and 3 on cytokine gene
expression in THP-1 cells stimulated with LPS for 3 h. THP-1 cells
were treated with 100 µM of ASA, 1, 2, or 3 before exposure to 500
ng/mL of LPS for 3 h. Results from real-time PCR are expressed as
relative fold change detected in treated cells compared to the untreated
culture, after normalization against β-actin used as endogenous con-
trol. Columns and bars represent means SEM from triplicate
experiments. °°°p < 0.001 versus control cells; ***p < 0.001 versus
LPS-treated cells (ANOVA followed by Student-Newman Keuls
multiple comparisons test)
Fig. 4 Real-time PCR of cytokine gene expression at 6 h following
LPS exposure. The cells were treated with ASA, 1, 2 or 3 compound
100 µM prior to be exposed to 500 ng/mL of LPS for 6 h. The
experimental results are expressed as relative fold change detected in
treated cells compared to the untreated culture, after normalization to
β-actin used as endogenous control. Columns and bars represent
means SEM of three independent experiments. °°°p < 0.001 and °°p
< 0.01 versus control cells; *p < 0.05, **p < 0.01, ***p < 0.001 versus
LPS-treated cells (ANOVA followed by Student-Newman Keuls
multiple comparisons test)

Medicinal Chemistry Research
150-fold and about 28-fold higher, respectively, than those in
control cells. These effects were decreased in presence of ASA
or the 1, 2, and 3 molecules. Specifically, the increase of LPS-
induced IL-1β mRNA levels were significantly reduced by
100 µM ASA, 1, 2 or 3 compounds up to 35%, 65%, 55% and
20%, respectively. Similarly, the pre-treatment with ASA, 1, 2,
or 3 molecules significantly diminished the rise of TNF-α
mRNA caused by LPS by 50%, 30%, 25% and 30%,
respectively, as well as reduced the COX-2 up-regulation by
35%, 43%, 35% and 30% (Fig. 3). After LPS stimulation for
6 h, the mRNA levels of IL-1β, TNF-α and COX-2 showed
similar behavior as those observed after 3 h exposure, either in
presence or absence of tested molecules (Fig. 4). It is note-
worthy that, in the cells exposed to LPS, the mRNA levels of
COX-1 and iNOS were more than doubled with respect to
untreated cells (Fig. 4). The pre-incubation of THP-1 cells
with ASA produced a 30% and 35% reduction of LPS-
induced COX-1 and iNOS mRNA levels, respectively, like-
wise the molecules 1, 2, and 3 that decreased COX-1 and
iNOS levels by 20–25% and 25–30%, respectively (Fig. 4).
ceramic nanofiltration and ultrafiltration membranes. J Membr Sci
439:36–47
Chighine A, Crosignani S, Arnal MC, Bradley M, Linclau B (2009)
Microwave-assisted ester formation using O-alkylisoureas: a
convenient method for the synthesis of esters with inversion of
configuration. J Org Chem 74:4753–4762
Currò M, Risitano R, Ferlazzo N, Cirmi S, Gangemi C, Caccamo D,
Ientile R, Navarra M (2016) Citrus bergamia juice extract
attenuates β-amyloid-induced pro-inflammatory activation of
THP-1 cells through MAPK and AP-1 Pathways. Sci Rep 6:20809
Desborough JR, Keeling DM (2017) The aspirin story – from willow
to wonder drug. Br J Haematol 177:674–683
Ferlazzo N, Cirmi S, Russo M, Ursino MR, Trapasso E, Lombardo
GE, Gangemi S, Calapai G, Navarra M (2016) NF-κB mediates
the antiproliferative and proapoptotic effects of bergamot juice in
HepG2 cells. Life Sci 146:81–91
Gerber M, Breytenbach JC, Hadgraft J, du Plessis J (2006) Synthesis
and transdermal properties of acetylsalicylic acid and selected
esters. Int J Pharm 310:31–36
Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E,
Hutchison GRJ (2012) Avogadro: an advanced semantic chemical
editor, visualization, and analysis platform. J Cheminform 4:17
Kalbasi RJ, Massah AR, Barkhordari Z (2010) An efficient and green
approach for the esterification of aromatic acids with various
alcohols over H3PO4/TiO2-ZrO2. Bull Korean Chem Soc
31:2361–2367
Lavie CJ, Howden CW, Scheiman J, Tursi J (2017) Upper gastro-
intestinal toxicity associated with long-term aspirin therapy:
consequences and prevention. Curr Probl Cardiol 42:146–163
Loll PJ, Picot D, Garavito RM (1995) The structural basis of aspirin
inferred from the crystal structure of inactivated prostaglandin
H2 synthase. Nat Struct Biol 2:637–643
McMahon GP, O’Connor SJ, Fitzgerald DJ, Roy SL, Kelly MT (1988)
Determination of aspirin and salicylic acid in transdermal perfu-
sates. J Chromatogr B 707:322–327
Mitragotri S (2003) Modeling skin permeability to hydrophilic and
hydrophobic solutes based on four permeation pathways. J
Control Release 86:69–92
Navarra M, Ferlazzo N, Cirmi S, Trapasso E, Bramanti P, Lombardo
GE, Minciullo PL, Calapai G, Gangemi S (2015) Effects of ber-
gamot essential oil and its extractive fractions on SH-SY5Y human
neuroblastoma cell growth. J Pharm Pharmacol 67:1042–1053
O’Boyle NM, Banck M, James CA, Morley C, Vandermeersch T,
Hutchison GR (2011) Open Babel: an open chemical toolbox. J
Chemin- 3:33
Conclusion
In conclusion, we have developed a convenient new method
for the synthesis of ASA-OR (R = alkyl) as potential can-
didates for the transdermal application of ASA. The newly
synthesized alkyl acetylsalicylates 1, (R = pentyl), 2 (R =
hexyl), and 3 (R = decyl) showed anti-inflammatory prop-
erties in vitro superimposable to those of ASA. Molecular
mechanics simulations, performed to calculate the kinetic
radii of several ASA-OR, indicated that the pentyl and
hexyl acetylsalicylates possess the best properties to cross
cell membranes. Our study reinforces the finding that alkyl
esters of aspirin may be used as anti-inflammatory drugs for
topical application.
Risitano R, Currò M, Cirmi S, Ferlazzo N, Campiglia P, Caccamo D,
Ientile R, Navarra M (2014) Flavonoid fraction of Bergamot juice
reduces LPS-induced inflammatory response through SIRT1-
mediated NF-κB inhibition in THP-1 monocytes. PLoS ONE 9:
e107431
Romeo R, Giofrè SV, Carnovale C, Chiacchio MA, Campisi A,
Mancuso R, Cirmi S, Navarra M (2014a) Synthesis and biolo-
gical activity of triazole-appended N,O-nucleosides. Eur J Org
Chem 25:5422–5447
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Romeo R, Navarra M, Giofrè SV, Carnovale C, Cirmi S, Lanza G,
Chiacchio MA (2014b) Synthesis and biological activity of new
arenediyne-linked isoxazolidines. Bioorg Med Chem 22:3379–
3385
Shamsher AA, Charoo NA, Kohli K, Pillai K, Rahman Z (2010) Effect
of transdermally delivered aspirin on blood coagulation para-
meters. Am J Biomed Sci 2:129–141
References
Abraham MH, Chadha HS, Whiting GS, Mitchell RC (1994) Hydro-
gen bonding. 32. An analysis of water-octanol and water-alkane
partitioning and the delta log P parameter of seiler. J Pharm Sci
83:1085–1100
Buekenhoudt A, Bisignano F, De Luca G, Vendezande P, Wouters M,
Verhulst K (2013) Unravelling the solvent flux behaviour of
Vane JR, Bakhle YS, Botting RM (1998) Cyclooxygenases 1 and 2.
Ann Rev Pharmacol Toxicol 38:97–120