Exploring the fatty acid amide hydrolase and cyclooxygenase inhibitory properties of novel amide derivatives of ibuprofen

Article abstract of DOI:10.1080/14756366.2020.1743283

Inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents such as sulindac and indomethacin in experimental animals, suggesting that a dual-action FAAH-cyclooxygenase (COX) inhib

Full text of DOI:10.1080/14756366.2020.1743283

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Exploring the fatty acid amide hydrolase and
cyclooxygenase inhibitory properties of novel
amide derivatives of ibuprofen
Alessandro Deplano, Jessica Karlsson, Mona Svensson, Federica Moraca,
Bruno Catalanotti, Christopher J. Fowler & Valentina Onnis
To cite this article: Alessandro Deplano, Jessica Karlsson, Mona Svensson, Federica
Moraca, Bruno Catalanotti, Christopher J. Fowler & Valentina Onnis (2020) Exploring the fatty
acid amide hydrolase and cyclooxygenase inhibitory properties of novel amide derivatives
of ibuprofen, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 815-823, DOI:
10.1080/14756366.2020.1743283
To link to this article: https://doi.org/10.1080/14756366.2020.1743283
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 23 Mar 2020.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 815–823
https://doi.org/10.1080/14756366.2020.1743283
RESEARCH PAPER
Exploring the fatty acid amide hydrolase and cyclooxygenase inhibitory properties
of novel amide derivatives of ibuprofen
a
, Jessica Karlssonb† , Mona Svenssonb†, Federica Moracac , Bruno Catalanottic
,
ꢀ
Alessandro Deplano
Christopher J. Fowlerb†
and Valentina Onnisa
aUnit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, Department of Life and Environmental Sciences, University of Cagliari,
b
c
Cagliari, Italy; Department of Integrative Medical Biology, Umeå University, Umeå, Sweden; Department of Pharmacy, University of Napoli
Federico II, Napoli, Italy
ABSTRACT
ARTICLE HISTORY
Received 23 January 2020
Revised 6 March 2020
Accepted 9 March 2020
Inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-
steroidal anti-inflammatory agents such as sulindac and indomethacin in experimental animals, suggesting
that a dual-action FAAH-cyclooxygenase (COX) inhibitor could have useful therapeutic properties. Here,
we have investigated 12 novel amide analogues of ibuprofen as potential dual-action FAAH/COX inhibi-
tors. N-(3-Bromopyridin-2-yl)ꢁ2-(4-isobutylphenyl)propanamide (Ibu-AM68) was found to inhibit the
hydrolysis of [3H]anandamide by rat brain homogenates by a reversible, mixed-type mechanism of inhib-
ition with a Ki value of 0.26 mM and an a value of 4.9. At a concentration of 10 mM, the compound did not
inhibit the cyclooxygenation of arachidonic acid by either ovine COX-1 or human recombinant COX-2.
However, this concentration of Ibu-AM68 greatly reduced the ability of the COX-2 to catalyse the cycloox-
ygenation of the endocannabinoid 2-arachidonoylglycerol. It is concluded that Ibu-AM68 is a dual-acting
FAAH/substrate-selective COX inhibitor.
KEYWORDS
Ibuprofen amides; FAAH
inhibition; fatty acid amide
hydrolase; endocannabinoid;
cyclooxygenase
Introduction
30-(aminocarbonyl)ꢁ6-hydroxy[1,10-biphenyl] ꢁ 3-yl ester) and with
indomethacin as NSAID4. A second endocannabinoid, 2-arachido-
noylglycerol (2-AG) is primarily hydrolysed by monoacylglycerol
lipase, and inhibition of that enzyme also reduces the ulcerogenic
The non-steroidal anti-inflammatory agents (NSAIDs) such as ibu-
profen, naproxen and diclofenac have widespread usage around
the world, but their use is hampered by the incidence of severe
gastrointestinal side effects. The elderly have a high consumption
of NSAIDs, and this has resulted in a high incidence of NSAID-
related hospitalisations and deaths1. There is thus much to be
gained by the discovery and development of compounds that are
as efficacious as the NSAIDs, but which lack these deleterious
gastrointestinal effects.
potency of diclofenac5,6
.
Taken together, the studies above suggest that a compound
with dual-action effects towards both cyclooxygenase (COX, the
primary target of NSAIDs) and FAAH (or monoacylglycerol lipase)
may be a potentially useful anti-inflammatory agent lacking the
problematic gastrointestinal unwanted effects associated with
NSAIDs. In 2015, the Piomelli group reported the synthesis and
pharmacological properties of ARN2508 (( )ꢁ2-[3-fluoro-4-[3-(hex-
ylcarbamoyloxy)phenyl]phenyl]propanoic acid), a compound com-
bining the structural elements of URB597 and the NSAID
flurbiprofen7,8. The compound inhibited FAAH, COX-1 and COX-2
with IC50 values of 31, 12 and 420 nM, respectively, and produced
anti-inflammatory effects in vivo without causing gastric irritation7.
The carbamate group in the molecule was required for (presum-
ably irreversible) FAAH inhibition, but not for COX-inhibition5.
Similar to the profens9, the compound shows substrate-selective
inhibition of COX-2, being more potent when 2-AG is used as sub-
strate than when arachidonic acid (AA) is used10.
In a key study from 2009, Naidu, Lichtman and colleagues2
reported that the ulcerogenic potency of the NSAID diclofenac
was lower in mice lacking the enzyme fatty acid amide hydrolase
(FAAH) than in the corresponding wild-type mice. A similar result
was found in wild-type mice pre-treated with the irreversible
FAAH inhibitor URB597 ((30-(aminocarbonyl)[1,10-biphenyl] ꢁ 3-yl)-
cyclohexylcarbamate). Further, URB597 and diclofenac acted syner-
gistically in reducing acetic acid-induced visceral nociception2.
FAAH catalyses the hydrolysis of the endogenous cannabinoid
(endocannabinoid) ligand anandamide (AEA, arachidonoylethano-
lamide)3 and the effects of FAAH inhibition upon diclofenac-
induced ulceration were not seen in mice lacking cannabinoid-1
receptors2. The ability of FAAH inhibition to reduce the ulcero-
genic potency of NSAIDs has also been seen with a peripherally-
An alternative approach has been to design compounds based
on ibuprofen, which has modest FAAH-inhibitory activity11, and to
optimise the FAAH-inhibitory properties while retaining the COX-
restricted FAAH inhibitor, URB937 (N-cyclohexyl-carbamic acid, inhibitory properties of the parent compound. The first such
CONTACT Valentina Onnis
vonnis@unica.it
Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, Department of Life and Environmental
Sciences, University of Cagliari, University Campus, S.P. nꢂ 8, Km 0.700, I-09042 Monserrato, Italy
ꢀ
Pharmacelera, Placa Pau Vila, 1, Sector 1, Edificio Palau de Mar, Barcelona, 08039, Spainpresent address
†At the time of the work presented here, the address was the Department of Pharmacology and Clinical Neuroscience, Umeå University. Since 1 Jan 2020, the
Pharmacology Unit is now part of the Department of Integrative Medical Biology at Umeå University
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

816
A. DEPLANO ET AL.
1
products reported showed H NMR spectra in agreement with the
assigned structures. The purity of the tested compounds was
determined by combustion elemental analyses conducted by the
Microanalytical Laboratory of the Department of Chemical and
Pharmaceutical Sciences of the University of Ferrara with a MT-5
CHN recorder elemental analyser (Yanagimoto, Kyoto, Japan) and
the values found were within 0.4% of theoretical values. Ibuprofen
amides Ibu-AM38-73 were synthesised according to Schemes 1
and 2.
Methyl 2–(4-isobutylphenyl)acetate (2)
Figure 1. Structure of Ibu-AM5 and TPA-14.
A solution of Ibufenac 1 (1.92 g, 10 mmol) in CH3OH (10 ml) was
added at room temperature (r.t.) with 37% HCl (0.5 ml) and
refluxed for 4 h. The solvent was removed under vacuum and the
crude methyl ester was used without purification in the further
step. Yield 85%. Oil. 1H NMR (DMSO-d6) d 0.98 (d, J ¼ 7.0 Hz, 6H,
CH3), 1.93 (m, 1H, CH), 2.42 (d, J ¼ 7.0 Hz, 2H, CH2), 3.65 (s, 2H,
CH2), 3.78 (s, 3H, CH3), 7.15 (m, 2H, Ar), 7.19 (m, Hz, 2 H, Ar).
Elemental analysis: calculated for C13H18O2 (206.29)% C 75.69; H
8.80; found % C 75.75; H 8.77. Physical and spectral data were in
accordance with literature values18.
compound, a heterocyclic amide ibuprofen analoge, Ibu-AM5
(2–(4-isobutylphenyl)-N-(3-methylpyridin-2-yl)propenamide, Figure
1) had been shown previously by one of us in 2003 to have anal-
gesic activity with respect to acetic acid-induced visceral nocicep-
tion in the mouse, without appreciable ulcerogenic potency12,
and successively further described in 2007 for its FAAH inhibitory
activity13. Further studies by us have shown that the compound
inhibits FAAH in a mixed-type manner in sub-micromolar concen-
trations (i.e. 2-3 orders of magnitude more potent than ibuprofen
itself) while retaining the substrate-selective inhibition of COX-2
seen with ibuprofen14,15
.
General procedure for the synthesis of esters 3 and 4
While Ibu-AM5 is a potentially useful compound, it would be
useful to explore its structure to determine whether more potent
FAAH/COX dual inhibitors can be identified. SAR studies so far
reported by us have14,16,17, however, been unsuccessful in that
the most potent FAAH-inhibitory compound so far described,
N-(3-chloropyridin-2-yl)ꢁ2–(4-((2-(trifluoromethyl)pyridin-4-yl)amino)
phenyl)propenamide, TPA-14 (N-(3-chloropyridin-2-yl)ꢁ2–(4-((2-
(trifluoromethyl)pyridin- 4-yl)amino)phenyl)propenamide, Figure 1),
had a similar potency to that of Ibu-AM5, but lacked COX-inhibi-
tory potency16. In the present study, we report the identification
of a novel Ibu-AM5 analogue that is more potent than Ibu-AM5
but which retains its substrate-selective inhibition of COX-2.
Lithium bis-(trimethylsilyl)amide (4.00 g, 24 mmol) was added to a
solution of ester 2 (2.00 g, 9.7 mmol) in dry THF (40 ml) under
argon at ꢁ78 ꢂC, the mixture was stirred at this temperature for
45 min. Then methyl iodide (3.40 g, 24 mmol) or 1,2-dibromo-
ethane (4.51 g, 12 mmol) was added dropwise to the stirred solu-
tion for an additional 1 h. The mixture was poured in water and
the desired product was extracted with diethyl ether (2 ꢃ 30 ml).
The solvent was dried over Na2SO4, then it was evaporated under
reduced pressure. The crude residue was purified via silica gel
(200-400 mesh silica gel Merk KGaA) chromatography using pet-
roleum ether 40–60 ꢂC and AcOEt 20:1.
Experimental
Methyl 2–(4-isobutylphenyl)-2-methylpropanoate (3)
Yield 80%. Oil. 1H NMR (DMSO-d6) d 0.95 (d, J ¼ 7.0 Hz, 6H, CH3),
1.63 (s, 6H, CH3), 1.82 (m, 1H, CH), 2.42 (d, J ¼ 7.0 Hz, 2H, CH2),
3.66 (s, 3H, CH3), 7.07 (d, J ¼ 7.5 Hz, 2H, Ar), 7.25 (d, J ¼ 7.5 Hz, 2 H,
Ar). Elemental analysis: calculated for C15H22O2 (234.16)% C 76.88;
H 9.46; found % C 76.92; H 9.44. Physical and spectral data were
in accordance with literature values19.
Materials
Anandamide [ethanolamine-1-3H] ([3H]AEA, specific activity 2.22
TBq mmolꢁ1
) was purchased from American Radiolabeled
Chemicals, Inc (St. Louis, MO). Non-radioactive AEA, ovine COX-1
(cat. no. 60100), human recombinant COX-2 (cat. no. 60122), ara-
chidonic acid (AA) and 2-AG were purchased from the Cayman
Chemical Co. (Ann Arbour, MI, USA). All commercially available sol-
vents and reagents were used without further purification and
were purchased from Sigma-Aldrich (Milan, Italy).
Methyl 1-(4-isobutylphenyl)cyclopropane-1-carboxylate (4)
Yield 60%. Oil. 1H NMR (DMSO-d6) d 0.91 (d, J ¼ 7.0 Hz, 6H, CH3),
1.25 (m, 2H, CH2), 1.58 (m, 2H, CH2), 1.84 (m, 1H, CH), 2.43 (d,
J ¼ 7.0 Hz, 2H, CH2), 3.69 (s, 3H, CH3), 7.15 (d, J ¼ 7.5 Hz, 2 H, Ar),
7.23 (d, J ¼ 7.5 Hz, 2 H, Ar). Elemental analysis: calculated for
C15H20O2 (232.32)% C 77.55; H 8.68; found % C 77.62; H 8.72.
Physical and spectral data were in accordance with litera-
ture values19.
Chemistry
NMR spectra were recorded on an Inova 500 spectrometer (Varian,
Palo Alto, CA). The chemical shifts (d) are reported in part per mil-
lion downfield from tetramethylsilane (TMS), which was used as
internal standard, and the spectra were recorded in hexadeuterio-
dimethylsulphoxide (DMSO-d6). Infra-red spectra were recorded on
a Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol
mulls. The main bands are given in cmꢁ1. Positive-ion electrospray
General procedure for the synthesis of acids 5 and 6
ionisation (ESI) mass spectra were recorded on a double-focusing To a solution of ester 3 or 4 (1 mmol) in EtOH (10 ml) 5 N solution
MAT 95 instrument (Finnigan, Waltham, MA) with BE geometry. of NaOH (2 ml) and water (2 ml) were added. The resulting mixture
Melting points (mp) were determined on a SMP1 Melting Point was stirred at r.t. for 24 h. After removing EtOH under vacuum to
apparatus (Stuart Scientific, Stone, UK) and are uncorrected. All the resulting solution ice was added and then acidified with

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
817
Scheme 1. Synthetic pathway for Ibu-AM72 and 73. Reagents and conditions: (i) HCl 37%, MeOH, reflux 4 h; (ii) Lithium bis-(trimethylsilyl)amide, THF, ꢁ78 ꢂC, 45 min,
then MeI or 1,2-dibromoethane, 1 h; (iii) 5 N NaOH, H2O, EtOH, r.t., 24 h; (iv) EDC, HOBt, MeCN, r.t. 36 h.
Scheme 2. Synthetic pathway for Ibuprofen aryl- and pyridyl-amides. Reagents and conditions i) EDC, HOBt, MeCN, r.t. 36h.
aqueous 20% HCl solution until pH 3–4. The formed precipitate sequentially with brine (2 ꢃ 5 ml), 10% citric acid (2 ꢃ 5 ml), satu-
was filtrated, washed with water and re-crystallized from
n-hexane.
rated NaHCO3 aqueous solution (2 ꢃ 5 ml) and water (2 ꢃ 5 ml).
The organic layer was dried over anhydrous Na2SO4 and evapo-
rated under vacuum to give the title amides.
2–(4-Isobutylphenyl)-2-methylpropanoic acid (5)
Obtained following the general procedure starting by ester 4.
Yield 90%. m.p. 70-72 ꢂC. 1H NMR (DMSO-d6) d 0.90 (d, J ¼ 7.0 Hz,
6H, CH3), 1.64 (s, 6H, CH3), 1.90 (m, 1H, CH), 2.55 (d, J ¼ 7.0 Hz, 2H,
CH2), 7.09 (d, J ¼ 7.5 Hz, 2H, Ar), 7.33 (d, J ¼ 7.5 Hz, 2 H, Ar).
Elemental analysis: calculated for C14H20O (220.15)% C 76.33; H
9.15; found % C 76.33; H 9.03. Physical and spectral data were in
accordance with literature values19.
2–(4-Isobutylphenyl)-N-(o-tolyl)propanamide (Ibu-AM38)
Obtained following the general procedure by the condensation
between ibuprofen and 2-methylaniline. Yield 95%. Oil. 1H NMR
(DMSO-d6) d 0.85 (d, J ¼ 7.0 Hz, 6H, CH3), 1.41 (d, J ¼ 7.0 Hz, 3H,
CH3), 1.83 (q, J ¼ 7.0 Hz 1H, CH), 2.04 (s, 3H, CH3), 2.42 (q,
J ¼ 7.0 Hz, 2H, CH2), 3.88 (q, J ¼ 7.0 Hz, 1H, CH), 7.04–7.10 (m, 6H,
Ar), 7.31 (m, 2H, Ar), 9.30 (s, 1H, NH). IR (Film) 3265, 2955, 2929,
1659, 1528 cmꢁ1. Elemental analysis: calculated for C20H25NO
(295.43)% C 81.31; H 8.53; N 4.74; found % C 81.39; H 8.56; N 4.72.
1–(4-Isobutylphenyl)cyclopropane-1-carboxylic acid (6)
Obtained following the general procedure starting by ester 5.
Yield 90%. 74-76 ꢂC. 1H NMR (DMSO-d6) d 0.87 (d, J ¼ 7.0 Hz, 6H,
CH3), 1.08 (m, 2H, CH2), 1.41 (m, 2H, CH2), 1.80 (m, 1H, CH), 2.42
(d, J ¼ 7.0 Hz, 2H, CH2), 7.06 (d, J ¼ 7.5 Hz, 2 H, Ar), 7.21 (d,
J ¼ 7.5 Hz, 2 H, Ar). Elemental analysis: calculated for C14H18O2
(218.30)% C 77.03; H 8.31; found % C 77.10; H 8.27. Physical and
spectral data were in accordance with literature values19.
N-(3-Chloropyridin-2-yl)-2-(4-isobutylphenyl)propanamide
(Ibu-AM58)
Obtained following the general procedure by the condensation
between ibuprofen and 2-amino-3-chloropyridine. Yield 39%. Oil.
Yield 39%. Oil. 1H NMR (DMSO-d6) d 0.85 (d, J ¼ 6.5 Hz, 6H, CH3),
1.34 (d, J ¼ 7.0 Hz, 3H, CH3), 1.41 (d, J ¼ 7.0 Hz, 2H, CH2), 1.82 (m,
J ¼ 7.0 Hz, 1H, CH), 3.88 (m, 1H, CH), 7.09–8.37 (m, 7H, Ar), 10.25 (s,
1H, NH). 13 C NMR (DMSO-d6) d 21.6, 25.4 (2 C), 32.7, 47.3, 116.9,
120.2, 130.1 (2 C), 132.0 (2 C), 142. 6, 143.2, 147.8, 150.1, 160.2,
178.7.IR (Film) 1660, 1512 cmꢁ1. Elemental analysis: calculated for
C18H21ClN2O (316.83)% C 68.24; H 6.68; N 8.84; found % C 68.30;
General procedure for the synthesis of amides Ibu-AM38-73
The solution of the appropriate acid 2, 5 or 6 (1 mmol), 1-(3-
dimethylaminopropyl)ꢁ3-ethylcarbodiimide hydrochloride (EDC)
(0.19 g, 1.1 mmol) and 1-hydroxybenzotriazole (HOBt) (0.13 g,
1 mmol) in anhydrous MeCN (10 ml) was stirred at r.t., after 30 min
the opportune amine (1 mmol) was added and the mixture was
stirred at r.t. for 36 h. After the solvent was removed under vac-
uum. The residue was dissolved in AcOEt (20 ml) and washed H 6.65; N 8.81.

818
A. DEPLANO ET AL.
N-(2-Hydroxyphenyl)-2-(4-isobutylphenyl)propanamide
(Ibu-AM59)
C21H27NO2 (325.45)% C 77.50; H 8.36; N 4.30; found % C 77.56; H
8.39; N 4.28.
Obtained following the general procedure by the condensation
between ibuprofen and 2-hydroxyaniline. Yield 52%. m.p.
123 ꢁ 125 ꢂC. 1H NMR (DMSO-d6) d 0.85 (d, J ¼ 7.0 Hz, 6H, CH3),
1.40 (d, J ¼ 6.5 Hz, 3H, CH3), 1.82 (hept, J ¼ 6.5-7.0 Hz 1H, CH), 2.40
(q, J ¼ 6.5 Hz, 2H, CH2), 4.00 (q, J ¼ 6.5 Hz, 1H, CH), 6.72 (m, 1H, Ar),
6.82 (m, 1H, Ar), 6.91 (m, 1H, Ar), 7.10 (m, 2H, Ar), 7.30 (m, 2H, Ar),
7.79 (m, 1H, Ar), 9.12 (s, 1H, OH), 9.73 (s, 1H, NH). IR (Nujol) 3359,
3091, 2733, 1654, 1592 cmꢁ1. Elemental analysis: calculated for
C19H23NO2 (297.17)% C 76.74; H 7.80; N 4.71; found % C 76.68; H
7.83; N 4.73.
N-(3-Bromopyridin-2-yl)-2-(4-isobutylphenyl)propenamide
(Ibu-AM68)
Obtained following the general procedure by the condensation
between ibuprofen and 2-amino-3-bromopyridine. Yield 67%. Oil.
1H NMR (DMSO-d6) d 0.85 (d, J ¼ 6.0 Hz, 6H, CH3), 1.34 (d,
J ¼ 7.5 Hz, 3H, CH3), 1.42 (d, J ¼ 7.0 Hz, 2H, CH2), 1.81 (m, 1H, CH),
3.86 (q, J ¼ 7.0 Hz, 1H, CH), 7.1–8.41 (m, 7H, Ar), 10.22 (s, 1H, NH).
13 C NMR (DMSO-d6) d 21.7, 25.3 (2 C), 32.7, 47.4, 106.3, 116.7,
130.2 (2 C), 132.1 (2 C), 141.7, 142.6, 143.2, 150.1, 159.4, 178.7. IR
(Film) 3240, 1703, 1580 cmꢁ1. Elemental analysis: calculated for
C18H21BrN2O (360.08)% C 59.84; H 5.86; N 7.75; found % C 59.90;
H 5.84; N 7.79.
2-(4-Isobutylphenyl)-N-(3-(trifluoromethyl)pyridin-2-yl)
propanamide (Ibu-AM60)
Obtained following the general procedure by the condensation
between ibuprofen and 2-amino-3-(trifluoromethyl)pyridine. Yield
N-(3-Iodopyridin-2-yl)-2-(4-isobutylphenyl)propanamide
(Ibu-AM69)
1
59%. m.p. 112–114 ꢂC. H NMR (DMSO-d6) d 0.85 (d, J ¼ 6.0 Hz, 6H,
CH3), 1.39 (d, J ¼ 7.0 Hz, 3H, CH3), 1.81 (q, J ¼ 6.5 Hz, 1H, CH), 2.42
(d, J ¼ 7.0 Hz, 2H, CH2), 3.87 (q, J ¼ 7.0 Hz, 1H, CH), 7.10–8.71 (m,
Obtained following the general procedure by the condensation
between ibuprofen and 2-amino-3-iodopyridine. Yield 53%. Oil. H
1
7H, Ar), 10.25 (s, 1H, NH). IR (Nujol) 3253, 1670, 1583 cmꢁ1
.
NMR (DMSO-d6) d 0.85 (d, J ¼ 7.0 Hz, 6H, CH3), 1.42 (d, J ¼ 7.0 Hz,
3H, CH3), 1.80 (hept, J ¼ 7.0 Hz, 1H, CH), 2.41 (m, 2H, CH2), 3.84 (q,
J ¼ 7.0 Hz, 1H, CH), 7.02–8.40 (m, 7H Ar), 10.18 (s, 1H, NH). IR (Film)
Elemental analysis: calculated for C19H21F3N2O (350.16)% C 65.13;
H 6.04; N 8.00; found % C 65.08; H 6.06; N 7.96.
3233, 1675, 1574 cmꢁ1
.
Elemental analysis: calculated for
C18H21IN2O (408.28)% C 52.95; H 5.18; N 6.86; found % C 53.01; H
5.16; N 6.90.
N-(4-Hydroxyphenyl)-2-(4-isobutylphenyl)propanamide
(Ibu-AM65)
Obtained following the general procedure by the condensation
between ibuprofen and 4-hydroxyaniline. Yield 47%. m.p.
N-(3-Hydroxypyridin-2-yl)-2-(4-isobutylphenyl)propenamide
(Ibu-AM70)
1
113–115 ꢂC. H NMR (DMSO-d6) d 0.85 (d, J ¼ 7.0 Hz, 6H, CH3), 1.37
(d, J ¼ 7.0 Hz, 3H, CH3), 1.80 (q, J ¼ 7.0 Hz, 1H, CH), 2.40 (d,
J ¼ 7.0 Hz, 2H, CH2), 3.72 (q, J ¼ 7.0 Hz, 1H, CH), 6.66 (d, J ¼ 8.5 Hz,
2H Ar), 7.01 (d, J ¼ 8.0 Hz, 2H, Ar), 7.28 (d, J ¼ 8.0 Hz, 2H, Ar), 7.35
(d, J ¼ 8.5 Hz, 2H, Ar), 9.14 (s, 1H, OH), 9.77 (s, 1H, NH). IR (Nujol)
3299, 1653, 1609, 1538 cmꢁ1. Elemental analysis: calculated for
C19H23NO2 (297.17)% C 76.74; H 7.80; N 4.71; found % C 76.80; H
7.77; N 4.73.
Obtained following the general procedure by the condensation
between ibuprofen and 2-amino-3-hydroxypyridine. Yield 38%. Oil.
1H NMR (DMSO-d6) d 0.85 (d, J ¼ 7.0 Hz, 6H, CH3), 1.42 (d,
J ¼ 7.5 Hz, 3H, CH3), 1.79 (hept, J ¼ 7.0 Hz, 1H, CH), 2.40 (d,
J ¼ 7.0 Hz, 2H, CH2), 3.62 (q, J ¼ 7.0 Hz, 1H, CH), 7.09–7.88 (m, 7H,
Ar), 10.24 (s, 1H, NH), 10.75 (s, 1H, OH). IR (Film) 1662, 1512 cmꢁ1
.
Elemental analysis: calculated for C18H22N2O2 (298.39)% C 72.46; H
7.43; N 9.39; found % C 72.54; H 7.45; N 9.42.
N-(4-Hydroxy-2-methylphenyl)-2-(4-isobutylphenyl)propanamide
(Ibu-AM66)
2-(4-Isobutylphenyl)-2-methyl-N-(3-methylpyridin-2-yl)
propanamide (Ibu-AM72)
Obtained following the general procedure by the condensation
between ibuprofen and 4-hydroxy-2-methylaniline. Yield 63%. m.p.
Obtained following the general procedure by the condensation
1
133–135 ꢂC. H NMR (DMSO-d6) d 0.83 (d, J ¼ 7.0 Hz, 6H, CH3), 1.35
between 2-(4-isobutylphenyl)ꢁ2-methylpropanoic acid (5) and 2-
1
(d, J ¼ 7.0 Hz, 3H, CH3), 1.77 (hept, J ¼ 7.0 Hz, 1H, CH), 1.90 (s, 3H,
CH3), 2.40 (d, J ¼ 7.0 Hz, 2H, CH2), 3.75 (q, J ¼ 7.0 Hz, 1H, CH),
6.48–7.28 (m, 7H, Ar), 9.09 (s, 1H, OH). IR (Nujol) 3398, 3292, 1656,
1610 cmꢁ1. Elemental analysis: calculated for C20H25NO2 (311.43)%
C 77.14; H 8.09; N 4.50; found % C 77.06; H 8.11; N 4.52.
amino-3-methylpyridine. Yield 70%. Oil. H NMR (DMSO-d6) d 0.86
(d, J ¼ 7.0 Hz, 6H, CH3), 1.57 (s, 6H, CH3), 2.02 (m, 1H, CH), 2.03 (s,
3H, CH3), 2.44 (d, J ¼ 7.0 Hz, 2H, CH2), 7.12–7.20 (m, 3H, Ar),
7.30–7.35 (m, 2H, Ar), 7.62 (m, 1H, Ar), 8.22 (m, 1H, Ar), 9.27 (s, 1H,
NH). IR (Film) 3167, 2956, 1686, 1583 cmꢁ1. Elemental analysis: cal-
culated for C20H26N2O (310.44)% C 77.38; H 8.44; N 9.02; found %
C 77.31; H 8.47; N 9.06.
2-(4-Isobutylphenyl)-N-(4-methoxy-2-methylphenyl)propanamide
(Ibu-AM67)
3-Methylpyridin-2-yl 1–(4-isobutylphenyl)cyclopropane-1-
carboxylate (Ibu-AM73)
Obtained following the general procedure by the condensation
between ibuprofen and 4-methoxy-2-methylaniline. Yield 49%.
1
m.p. 100–102 ꢂC. H NMR (DMSO-d6) d 0.85 (d, J ¼ 7.0 Hz, 6H, CH3),
Obtained following the general procedure by the condensation
between 1–(4-isobutylphenyl)cyclopropane-1-carboxylic acid (6)
and 2-amino-3-methylpyridine.Yield 73%. Oil.1H NMR (DMSO-d6) d
0.87 (d, J ¼ 7.0 Hz, 6H, CH3), 1.10 (m, 2H, CH2), 1.44 (m, 2H, CH2),
1.40 (d, J ¼ 7.0 Hz, 3H, CH3), 1.81 (hept, J ¼ 7.0 Hz, 1H, CH), 1.99 (s,
3H, CH3), 2.42 (d, J ¼ 7.0 Hz, 2H, CH2), 3.70 (s, 3H, CH3), 3.81 (d,
J ¼ 7.0 Hz, 1H, CH), 6.69-7.31 (m, 7H, Ar), 9.21 (s, 1H, NH). IR (Nujol)
3298, 1655, 1613 cmꢁ1
.
Elemental analysis: calculated for 1.82 (m, 1H, CH), 2.03 (s, 3H, CH3), 2.42 (d, J ¼ 7.0 Hz, 2H, CH2),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
819
7.07–7.25 (m, 4H, Ar), 7.37 (m, 1H, Ar), 7.62 (m, 1H, Ar), 8.19 (m, 11.1223. The protonation state of Ibu-AM68 at pH 7.4 in water has
1H, Ar), 8.59 (s, 1H, NH). IR (Film) 3397, 1687, 1582 cmꢁ1
Elemental analysis: calculated for C20H24N2O (308.43)% C 77.89; H
7.84; N 9.08; found % C 77.82; H 7.87; N 9.04.
.
been calculated using the Epik module27. Finally, Ibu-AM68 was
then minimised using a protocol already adopted for Ibu-AM5:17
OPLS 2005 force field using the Polak-Ribiere Conjugate Gradient
(PRCG)28 algorithm and 2500 iteration steps.
Pharmacology
Docking of Ibu-AM68 in FAAH
FAAH assay
The molecular docking of Ibu-AM68 was performed only on the
monomer A of the rat FAAH (rFAAH) receptor. Docking procedure
was carried out with the Glide software package29, using the
Standard Precision (SP) algorithm of the GlideScore function30,31
and the OPLS 2005 force field32. A grid box of 29 ꢃ 29 ꢃ 29 Å cen-
tred on the ligand binding cavity was created. A total amount of
200 poses was generated and the conformational sampling of the
ligand was enhanced by two times, as reported by the default set-
ting of Glide. Docking conformations of Ibu-AM68 were then clus-
terised based on their RMSD cut-off of 2 Å. Globally, ten clusters
were obtained and, among them, only the conformation included
in the most populated cluster owing both the Glide Emodel and
GlideScore lowest-energy value was considered (Figure 4). Such
conformation was, finally, submitted to a further minimisation
protocol using the OPLS 2005 force field32, 20,000 minimisation
steps and the Polak-Ribiere Conjugate Gradient (PRCG)
algorithm28.
Frozen (ꢁ80 ꢂC) brains (minus cerebella) from adult Wistar or
Sprague-Dawley rats were thawed and homogenised in 20 mM
HEPES, 1 mM MgCl2, pH 7.0. The homogenates were then centri-
fuged at ꢄ35000 x g for 20 min at 4 ꢂC followed by washing (by
recentrifugation and by resuspension in the buffer) twice before
incubation at 37 ꢂC for 15 min in order to hydrolyse all endogen-
ous FAAH substrates. After a further centrifugation, pellets were
resuspended in 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM
EDTA and 3 mM MgCl2, and frozen at ꢁ80 ꢂC in aliquots until
used for the assay. For the FAAH assay20, test compounds, homo-
genates and [3H]AEA (diluted with non-radioactive AEA to give a
substrate concentration of 0.5 lM) in 10 mM Tris- HCl, 1 mM EDTA,
pH 7.4, containing 1% w/v fatty acid-free bovine serum albumin
were incubated for 10 min at 37 ꢂC. Activated charcoal in 0.5 M
HCl was added to adsorb the unmetabolized [3H]AEA and the
samples were mixed and left at r.t. for ꢄ30 min. Following centri-
fugation at 2000 g for 10 min, aliquots of the supernatants, con-
taining the [3H]ethanolamine produced by hydrolysis of [3H]AEA,
were analysed for tritium content by liquid scintillation spectros-
copy with quench correction. Blank values were obtained by the
use of buffer rather than homogenate.
Results and discussion
The potency of Ibu-AM5 towards AEA hydrolysis has been meas-
ured by different groups with different assay methodologies,
FAAH preparations (rat brain, mouse brain, recombinant human
FAAH) and substrate concentrations (0.5–2 mM)13–15,33,34. The IC50
value for Ibu-AM5 from different studies in our laboratory using
the same assay as here (of importance given the mixed-type
nature of its inhibition of FAAH) ranges from 0.52 ꢁ 1.2 mM14,15
and we therefore have used the most potent value for compara-
tive purposes, since the aim is to identify more
potent compounds.
Data were expressed as % of vehicle (ethanol) control and ana-
lysed using the algorithm log(inhibitor) vs. response – variable
slope (four parameters) built into the GraphPad Prism computer
programme v8.3 for the Macintosh (GraphPad Software Inc., San
Diego, CA). The programme reports 95% confidence limits (profile
likelihood) for the IC50 values and these presented in the results.
COX-1 and 2 assay
The assay was performed essentially according to the method of
Meade et al21. An oxygen electrode chamber with integral stirring
(Oxygraph System, Hansatech Instruments, King ꢀs Lynn, U.K.) was
calibrated daily to ambient temperature and air pressure. The
assay buffer contained 0.1 M Tris-HCl buffer pH 7.4, 1 lM haem-
atin, 2 mM phenol, 5 mM EDTA, 10 lM substrate (AA or 2-AG) in a
final assay volume was 2 ml. After addition of test compound, a
baseline was established for 5 min before initiation of reaction by
addition of 200 units ovine COX-1 or human recombinant COX-2.
The change in oxygen consumption as a measurement of enzyme
activity was monitored for approximately 5 min.
FAAH inhibition
Three series of Ibu-AM5 analogues were synthesised according to
Schemes 1 and 2 and tested towards rat brain FAAH-catalysed
hydrolysis of AEA. The first series of two compounds was moti-
vated by the finding that for Ibu-AM5 removal of the methyl
group at the C-2 carbon atom (“Ibufenac-AM1”) reduced the
potency roughly 60-fold15. In order to explore whether or not the
methyl group at that position was optimal, two compounds were
synthesised, with a dimethyl (Ibu-AM72) and cyclopropyl (Ibu-
AM73) groups instead of the methyl group at the C-2 carbon
atom. The amides were obtained starting from Ibufenac (1) that
was converted into its methyl ester 2 and then alkylated at C-2
position to give intermediates 3 and 4 that were hydrolysed to
the corresponding acid 5 and 6. These last were coupled with 2-
amino-3-methylpyridine by EDC method to afford the target
amides Ibu-AM72 and Ibu-AM73. The compounds inhibited FAAH
with IC50 values of 1.0 and 4.1 mM for Ibu-AM72 and Ibu-AM73,
respectively (Figure 2A, Table 1). Although this is a very limited
series, it would suggest that there is little to be gained by adjust-
ing the methyl group at the C-2 carbon atom, and so we moved
on to the amido moiety of Ibu-AM5 3-substituent on the pyridine
Computational studies
FAAH receptor and ligand preparation
The crystal structure of the rat fatty acid amide hydrolase (rFAAH)
(PDB ID: 3QK5) was downloaded from the Protein Data Bank web-
site. Both monomers A and B were treated with the Protein
Preparation Wizard22 tool implemented in Maestro ver. 11.1223, in
order to add all the hydrogen atoms and assign the correct bond
orders. Subsequently, both the co-crystallized ligands and water
molecules were removed. Residue Lys142 was considered in its
deprotonated form, according to the proposed catalytic mechan-
ism of FAAH24–26. The 3 D structure of Ibu-AM68 was built using
the Graphical User Graphical User Interface (GUI) of Maestro ver. ring of Ibu-AM5.

820
A. DEPLANO ET AL.
Figure 2. Inhibition of [3H]AEA hydrolysis by analogues of Ibu-AM5. (A–C) show concentration-response curves for the inhibition by the compounds shown of the
hydrolysis of 0.5 mM [3H]AEA by rat brain homogenates. In (D), the homogenates were preincubated with Ibu-AM68 for the times shown prior to addition of 0.5 mM
[3H]AEA. In e, rat homogenates (at 20-fold normal strength) were preincubated with either vehicle, 0.3, 0.6 or 0.9 mM Ibu-AM68 for 60 min. Aliquots were then diluted
20-fold and assayed for FAAH activity with 0.5 mM [3H]AEA. These are shown as 0.3 ! 0.015, 0.6 ! 0.03 and 0.9 ! 0.045 in the figure. Concomitantly, Ibu-AM68
was added to vehicle-preincubated aliquots to give concentrations of 0.015, 0.03 and 0.045 mM (representing free concentrations after a 20-fold dilution), 0.3, 0.6 and
0.9 mM final concentrations. (F) shows the kinetics of the inhibition of rat FAAH by Ibu-AM68. The data was better fitted by a mixed-type inhibition mode of inhibition
(Ki value of 0.26 mM and an a value of 4.9) than by a competitive mode of inhibition. In (A–D), data are means SEM (when not enclosed by the symbols), N ¼ 3,
except for the data for Ibu-AM69 in Figure 1(B) where N ¼ 3-7. In (D), data are means SEM, N ¼ 4.
In our initial study12, we reported that the potency of the pyri-
dinamides of ibuprofen towards FAAH is related to the presence
of both methyl and pyridine nitrogen in ortho positions to the
amide nitrogen as in Ibu-AM5, the methyl absence or its moving
in a position different from ortho to the amide nitrogen results in
activity decrease. On this basis to evaluate the influence on the
activity of the pyridine nitrogen, we changed pyridine ring with a
Table 1. IC50 values for the inhibition by novel Ibu-AM compounds of the
hydrolysis of 0.5 mM [3H]AEA by rat brain homogenates.
Compound
IC50 (mM)
95% confidence limits of the IC50
Ibu-AM72
Ibu-AM73
Ibu-AM38
Ibu-AM59
Ibu-AM65
1.0
4.1
2.0
8.5
0.86–1.2
3.2–5.3
1.3–3.0
6.2–12
2.3–6.5
3.8
phenyl. With this purpose, we prepared the 2-methylphenyl ana- Ibu-AM66
0.35
7.9
0.29–0.42
5.9–11
Ibu-AM67
logue of Ibu-AM5. The replacement of pyridine ring with a phenyl
Ibu-AM58
as in Ibu-AM38 produced activity decrease (IC50 2.0 lM). After fur-
Ibu-AM68
0.91
0.083
0.12
0.36
2.7
0.72–1.1
0.038–0.15
0.078–0.19
0.32–0.41
1.1–6.4
ther evaluation a group able to establish hydrogen bond with the
Ibu-AM69
enzyme was inserted on the amide phenyl ring. With this purpose,
the 2-hydroxy (Ibu-AM59) and the 4-hydroxy (Ibu-AM65) deriva-
tives were designed. As indicated by their IC50 values, the pres-
ence of the hydroxy group in 4-position caused an increase in
activity (3.8 mM for Ibu-AM65 vs 8.5 mM for Ibu-AM59), although
these compounds were among the least potent in the series. Next
step was the integration of this activity enhancement with the
presence of a 2-methyl group, with this purpose the compound
Ibu-AM66 was prepared by condensation of ibuprofen with 2-
methyl-4-hydroxyaniline. Ibu-AM66 showed very good activity
with an IC50 value of 0.35 lM. With the aim to study, the influence
on the inhibitory activity of hydrogen bond donor or acceptor
group Ibu-AM66 was modified by replacement of the 4-hydroxy
by a methoxy group to afford Ibu-AM67 (IC50 4.6 lM). The result
was decrease in activity, indicating that a hydrogen bond donor is
better than an acceptor. Thereafter, we focussed on alternative
Ibu-AM60
Ibu-AM70
in Figure 2(B) and summarised in Table 1. The observed potencies
of the substituents were -I (Ibu-AM69) and -Br (Ibu-AM68) > -CF3
(Ibu-AM60) > -Cl (Ibu-AM58) > -OH (Ibu-AM70). The 95% confi-
dence intervals for the mean IC50 values for Ibu-AM69
(0.078–0.19 mM) and Ibu-AM68 (0.038–0.15) overlap, so we regard
the two compounds as equipotent but more potent than
Ibu-AM5.
The inhibition of FAAH by Ibu-AM68 was investigated in more
detail. Preincubation of Ibu-AM68 with the homogenates for up
to 60 min prior to addition of substrate did not increase the
observed inhibition, indicating that there is no time-dependence
of the inhibition (Figure 2(D)). For a fully reversible inhibitor, pre-
substituents to the 3-methyl group such us halogens, trifluoro- incubation for 60 min with a concentration “x” of compound fol-
methyl and hydroxy groups. The data for these amides are shown lowed by a 20-fold dilution prior to addition of substrate should

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
821
Figure 3. The influence of Ibu-AM68 upon the cyclooxygenation of 10mM arachidonic acid (AA) and 2-arachidonoylglycerol (2-AG) by COX-1 and COX-2. Shown are
means SEM, N ¼ 3 in each graph for the changes in oxygen tension following addition of enzyme in the presence of Ibu-AM68. The concentrations of Ibu-AM68, in
mM, are shown on the right of each panel. The enzyme isoform and substrate used is given above each panel. Note that there are two different panels for COX-1 and
AA and for COX-2 and AA. This was because the experiments were performed on different occasions with different batches of the enzyme. COX-1 does not metabolise
2-AG.
produce the same observed inhibition as seen with a concentra- acting as functional heterodimers, whereby the binding of the R-
tion of “x/20” of the compound added together with the sub- profen to one site acts allosterically to block 2-AG but not AA
strate, and this was found to be the case for Ibu-AM68 (Figure cyclooxygenation. It is possible that such a mechanism can explain
2(E)). Finally, kinetic experiments indicated a simple mixed-model the actions of Ibu-AM5 and Ibu-AM68.
inhibition of FAAH with a Ki value of 0.26 mM and an a value (the
ratio of the Kiintercept: Kislope values; for pure competitive inhibition,
FAAH docking on Ibu-AM68
a! 1) of 4.9 (Figure 2(F)). Thus, Ibu-AM68 is a reversible mixed
inhibitor of rat brain FAAH with a greater potency than Ibu-AM5.
Molecular docking calculations on (S)-Ibu-AM68 were performed
with the Glide software29–31 in the crystal structure of rat FAAH
(PDB ID: 3QK5)37. The software Glide was chosen since it showed
Inhibition of COX isoenzymes by Ibu-AM68
to be able to well reproduce the binding poses of (R)- and (S)-
The ability of Ibu-AM68 to inhibit the cyclooxygenation of AA Ibu-AM5 resulting by molecular dynamics and free energy calcula-
and 2-AG by COX-1 and COX-2 was investigated (Figure 3). In our tions 0.17 The results were clustered and successively ranked
hands under the assay conditions used, 30 mM ibuprofen itself pro- according to the Glide Emodel and the Glide Score. The best pose
duces approximately 50% inhibition of the cyclooxygenation of showed the isobutyl moiety pointing to the catalytic triad and the
AA by COX-1 with at best minor inhibition of COX-2 at this con- pyridine moiety entering the membrane access channel (MAC)
centration. However, ꢅ10 and 30 mM ibuprofen produce a marked channel (Figure 4). In particular, the substituted pyridine ring
inhibition of 2-AG and AEA cyclooxygenation by COX-2 (neither established hydrophobic contacts with Leu404, Ile407, a T-shaped
endocannabinoid is a substrate for COX-1)14,35. Ibu-AM5 also p-p interaction with Trp531 and a H-bond interaction with the
shows substrate selective inhibition, reducing the rate of cyclooxy- hydroxyl group of Thr488. Moreover, polar contacts between the
genation to about half at concentrations of 50 mM (COX-1, AA as bromine atom and the backbone hydrogens of residues Asp403
substrate) and 3 mM (COX-2, AEA as substrate) whilst 100 mM Ibu- and Leu404 were observed. An additional H-bond was established
AM5 is without effect upon COX-2 with AA as substrate.14 At a between the NH group of the ligand with the carbonyl of the
concentration of 10 mM, a modest inhibition of AA cyclooxygena- Gly485. The Ibu-AM68 hydrophobic isobutyl-phenyl moiety
tion by Ibu-AM68 was seen with COX-1 whereas the cyclooxyge- resulted embedded in a hydrophobic region in the Acyl Chain
nation of 2-AG by COX-2 was almost completely inhibited. Higher Binding channel (ACB), and established hydrophobic contacts with
concentrations of Ibu-AM68 (50 and 100 mM) produced a com- residues Leu192 Phe244, Leu380, Thr488 and Ile491. The compari-
plete inhibition of COX-1 but did not inhibit AA cyclooxygenation son with the binding mode of Ibu-AM5 showed high similarity in
by COX-2. This suggests that the substrate-selective inhibition of the isobutyl-phenyl moiety, but a different conformation of the
COX-2 reported first for the R-profens by Marnett and colleagues35 pyridine ring with respect to the amide moiety. This different con-
is also seen with Ibu-AM68. The mechanism of this inhibition has formational behaviour maybe be likely due to different dipole
not been investigated, but Marnett et al.9,36 have suggested that alignment, being the slightly negative bromine atom better
it may be related to COX-2 (which has a homodimeric structure) aligned with the NH group of the amide bond, while the methyl

822
A. DEPLANO ET AL.
Figure 4. (A) 3 D structure of monomers A and B of rFAAH. The rectangular box indicates the Ibu-AM68 ligand binding cavity. (B) Focus on the binding mode of Ibu-
AM68 within the ACB channel. Polar contacts engaged by bromine atom with Leu404 and Asp403 are depicted as magenta dashed lines, while hydrogen bond inter-
actions with Gly485 and Thr488 are shown as dashed black lines. (C) Overlap between the binding mode of Ibu-68AM (tan stick) and Ibu-AM5 (light cyan stick), high-
lighting the different orientation of the substituted pyridine ring.
substituent preferred an orientation orthogonal to the car-
bonyl group.
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