Potent multitarget FAAH-COX inhibitors: Design and structure-activity relationship studies

Article abstract of DOI:10.1016/j.ejmech.2015.12.036

Non-steroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological effects by inhibiting cyclooxygenase (COX)-1 and COX-2. Though widely prescribed for pain and inflammation, these agents have limited utility in chronic diseases due to serious mechanism-based adverse events such as gastrointestinal damage. Concomitant blockade of fatty acid amide hydrolase (FAAH) enhances the therapeutic effects of the NSAIDs while attenuating their propensity to cause gastrointestinal injury. This favorable interaction is attributed to the accumulation of protective FAAH substrates, such as the endocannabinoid anandamide, and suggests that agents simultaneously targeting COX and FAAH might provide an innovative strategy to combat pain and inflammation with reduced side effects. Here, we describe the rational design and structure-active relationship (SAR) properties of the first class of potent multitarget FAAH-COX inhibitors. A focused SAR exploration around the prototype 10r (ARN2508) led to the identification of achiral (18b) as well as racemic (29a-c and 29e) analogs. Absolute configurational assignment and pharmacological evaluation of single enantiomers of 10r are also presented. (S)-(+)-10r is the first highly potent and selective chiral inhibitor of FAAH-COX with marked in vivo activity, and represents a promising lead to discover novel analgesics and anti-inflammatory drugs.

Full text of DOI:10.1016/j.ejmech.2015.12.036

Accepted Manuscript
Potent multi-target FAAH-COX inhibitors: Design and structure-activity relationship
studies
Marco Migliore, Damien Habrant, Oscar Sasso, Clara Albani, Sine Mandrup Bertozzi,
Andrea Armirotti, Daniele Piomelli, Rita Scarpelli
PII:
S0223-5234(15)30422-0
10.1016/j.ejmech.2015.12.036
EJMECH 8276
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 October 2015
Revised Date: 9 December 2015
Accepted Date: 19 December 2015
Please cite this article as: M. Migliore, D. Habrant, O. Sasso, C. Albani, S.M. Bertozzi, A. Armirotti, D.
Piomelli, R. Scarpelli, Potent multi-target FAAH-COX inhibitors: Design and structure-activity relationship
studies, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2015.12.036.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Potent Multi-target FAAH-COX Inhibitors: Design
and Structure-Activity Relationship Studies
a
a, 1
a
a, 2
a
Marco Migliore, Damien Habrant, Oscar Sasso, Clara Albani, Sine Mandrup Bertozzi,
a
a,b*
a*
Andrea Armirotti, Daniele Piomelli and Rita Scarpelli
a
Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Via Morego 30,
1
6163 Genova, Italy
b
Departments of Anatomy and Neurobiology, Pharmacology and Biological Chemistry,
University of California, Irvine, USA, 92697-4621
Present addresses:
1
In-Cell-Art, 21 rue de la Noue Bras de Fer, 44200 Nantes, France.
2
Covagen AG, Wagistrasse 25, 8952, Zürich Schlieren, Switzerland.
*
Corresponding authors:
piomelli@uci.edu; phone: +1(949) 824-9179
rita.scarpelli@iit.it; phone: +39 010 71781233
1

ACCEPTED MANUSCRIPT
ABSTRACT
Non-steroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological effects by
inhibiting cyclooxygenase (COX)-1 and COX-2. Though widely prescribed for pain and
inflammation, these agents have limited utility in chronic diseases due to serious mechanism-
based adverse events such as gastrointestinal damage. Concomitant blockade of fatty acid amide
hydrolase (FAAH) enhances the therapeutic effects of the NSAIDs while attenuating their
propensity to cause gastrointestinal injury. This favorable interaction is attributed to the
accumulation of protective FAAH substrates, such as the endocannabinoid anandamide, and
suggests that agents simultaneously targeting COX and FAAH might provide an innovative
strategy to combat pain and inflammation with reduced side effects. Here, we describe the
rational design and structure-active relationship (SAR) properties of the first class of potent
multi-target FAAH-COX inhibitors. A focused SAR exploration around the prototype 10r
(
ARN2508) led to the identification of achiral (18b) as well as racemic (29a-c and 29e) analogs.
Absolute configurational assignment and pharmacological evaluation of single enantiomers of
0r are also presented. (S)-(+)-10r is the first highly potent and selective chiral inhibitor of
1
FAAH-COX with marked in vivo activity, and represents a promising lead to discover novel
analgesics and anti-inflammatory drugs.
KEYWORDS. FAAH, COX, hybrid scaffold, multitarget inhibitors, structure-activity
relationship, inflammation.
2

ACCEPTED MANUSCRIPT
1
.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely utilized to treat pain and
inflammation, [1] but their chronic use is hindered by a variety of potentially serious adverse
events that include gastrointestinal (GI) mucosal lesions, bleeding and perforations. [2-5]
Conventional NSAIDs inhibit the two isoforms of cyclooxygenase (COX), COX-1 and COX-2,
which catalyze the first committed steps in the biosynthetic pathway that converts arachidonic
acid (AA) into inflammatory prostanoids such as prostaglandin E
2 2 2
(PGE ) and thromboxane A
(
TXA ). [6] The dual role of COX-1-derived PGE as inflammation promoter and mucosal tissue
2
2
protectant explains, at least in part, why NSAIDs cause damage to the GI tract. [7-10] Efforts to
overcome this problem have led to the development of selective COX-2 inhibitors, which
combine a high level of anti-inflammatory efficacy with a reduced propensity to cause injury to
the GI mucosa. [6] Nevertheless, the use of COX-2 inhibitors has been linked to a distinctive set
of adverse cardiovascular effects. [11, 12] Thus, the need for safe and effective drugs that can be
used in the treatment of chronic inflammatory disorders remains urgent.
A promising approach to meet this need is offered by targeting with a single agent more than
one component of the inflammatory cascade. [13-15] Agents designed to achieve this objective
include nitric oxide (NO) donors-NSAIDs, [16, 17] COX-2 inhibitors-NO-donors, [18, 19]
hydrogen sulfide (H S) donors-NSAIDs, [20-22] as well as compounds that block distinct
2
enzymes of the AA pathway, such as COX/lipoxygenase [23, 24] and COX-2/soluble epoxy
hydrolase (sEH). [25] Another potential multi-target strategy to treat inflammation is the
concomitant inhibition of COX and fatty acid amide hydrolase (FAAH) [26], [27-33] a serine
hydrolase that deactivates a family of analgesic and anti-inflammatory lipid amides that are
produced by host-defense cells and other cells in the body. [34, 35] These lipid mediators include
3

ACCEPTED MANUSCRIPT
the endocannabinoid anandamide (arachidonoylethanolamide) – which engages cannabinoid-1
(
CB ) and CB receptors to suppress neutrophil migration [36] and prevent immune-cell
1 2
recruitment [37, 38] – as well as the endogenous peroxisome proliferator-activate receptor-α
PPAR-α) agonists, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). [39-41] In
(
addition to opposing pain and inflammation, these FAAH substrates are also protective of the GI
mucosa. [42, 43] Indeed, studies in animal pain models have shown that co-administration of
FAAH and COX inhibitors results in a synergistic potentiation of analgesia along with reduced
gastric damage. [44-46]
In several chronic inflammatory conditions, including inflammatory bowel disease (IBD),
FAAH [47-49] and COX-2 [50] are expressed at abnormally high levels. This simultaneous up-
regulation may help establish a pathological state that exacerbates inflammation by amplifying
inflammatory COX-dependent signals at the expense of defensive FAAH-regulated mediators.
This hypothesis predicts that drugs targeting both COX and FAAH should have substantial anti-
inflammatory efficacy combined with reduced GI toxicity. In a recent study, we provided
support to this hypothesis using a multi-target modulator based on the hybrid scaffold 1 (Figure
1
). [51] This scaffold merges key pharmacophores of two known classes of FAAH and COX
inhibitors – O-aryl carbamates [52-58] such as [3-(3-carbamoylphenyl)phenyl] N-
cyclohexylcarbamate (URB597, 2) [54, 57], and 2-aryl propionic acids [6] such as flurbiprofen,
3
1
a [59-61] – which share a biphenyl core as a common structural motif (A and B rings, Figure
). Moreover, structure-activity relationship (SAR) studies of these scaffolds supported the
hypothesis of additional elements of structural overlapping, such as the oxygenated substituents
at the 3’-position of the A phenyl ring, corresponding to the carbamate functionality of 2 [53, 54,
5
4
6] and the ether moieties of 3b or 3c, [61] respectively (Figure 1).

ACCEPTED MANUSCRIPT
Figure 1. Rational design of a ‘hybrid scaffold’ for FAAH and COX inhibition.
This SAR work led to the identification of compound 10r ((±)-2-[3-fluoro-4-[3-
(hexylcarbamoyloxy)phenyl]phenyl]propanoic acid, ARN2508) [51] as a potent in vivo active
inhibitor of intracellular FAAH and COX activities, which exerts profound anti-inflammatory
effects in mouse models of IBD without causing COX-dependent gastric toxicity. [51] In the
present study, (a) we outline the in-depth SAR investigations that led to the discovery of
compound 10r [51]; (b) we report an expansion of this SAR work, which culminated in the
identification of several new and potent multitarget inhibitors (18b, 29a-c and 29e); and, finally
(c) we describe the absolute configurational assignment and pharmacological properties of single
enantiomers of 10r, identifying (S)-(+)-10r as the first chiral inhibitor of FAAH-COX with
marked in vivo activity.
5

ACCEPTED MANUSCRIPT
2
.
Results and discussion
2
.1 Chemistry
Compounds 10a-t were synthetized from the corresponding phenol 8 through a carbamoylation
reaction, using commercially available isocyanates, followed by the hydrolysis of the methyl
esters 9a-t, under acidic conditions (Scheme 1).
b
c
4
5
, R = H
, R = CH
6
7
a
3
d
f
e
1
0a, R = c-hexyl
1
0b, R = c-pentyl
1
0c, R = c-butyl
1
0d, R = c-propyl
9a-t
8
(9r, R = n-hexyl)
g
1
1
0e, R = c-hexyl-CH
0f, R = c-hexyl-(CH
2 2
)
2
1
1
0g, R = iso-propyl
0h, R = iso-butyl
1
1
1
1
1
0i, R = Ph
,
R = Ph-CH
2
11
0j
0k, R = Ph-(CH
0l, R = Ph-(CH
0m, R = Ph-(CH
2 2
)
2 3
)
2
)
4
h
1
1
1
1
1
1
1
0n, R = ethyl
0o, R = n-propyl
0p, R = n-butyl
0q, R = n-pentyl
0r, R = n-hexyl
0s, R = n-heptyl
0t, R = n-octyl
( )
5
12
Scheme 1. Synthesis of compounds 10a-t and 12. Reagents and conditions: (a) MeOH, conc.
SO , rt, 15 h, 93%; (b) HCO NH , 10% Pd/C, MeOH, rt, 3 h, 94%; (c) NaNO , 3M HCl, 0 °C,
0 min, then NaI, 60 °C, 2 h, 55%; (d) (3-hydroxyphenyl)boronic acid, Pd(OAc) , K CO ,
H
2
4
2
4
2
3
2
2
3
EGME/H O, rt, 15 h, 84%; (e) RNCO, DMAP, MeCN, rt, 15 h, 38-99%; (f) 6M HCl, THF, rt, 2
2
d, 26-73%; (g) ZrCl , NaBH , THF, rt, 2 h, 96%; (h) n-hexyl-NCO, DMAP, MeCN, rt, 15 h,
4
4
7
3%.
6

ACCEPTED MANUSCRIPT
The intermediate 8 was prepared in four steps, starting from the acid 4, obtained as previously
described. [62] Compound 4 was converted to the corresponding methyl ester 5, under standard
acidic conditions, to afford, after catalytic hydrogenation with ammonium formate in the
presence of Pd/C, the resulting aniline 6. Compound 6 was then transformed into the
corresponding diazonium salt, that was reacted in situ with NaI to obtain the phenyl iodide 7 in
good yield, which was converted, under ligand less Suzuki cross coupling conditions, [63] to the
biphenyl derivatives 8 and 13a-c in excellent yield (Schemes 1-3).
3
-Hydroxypropyl derivative 12 was synthesized by reduction of the methyl ester 8 to the
alcohol 11 (Scheme 1). Although lithium aluminum hydride succeeded in reducing the ester 8, a
significant des-fluorinated side product was observed and separation of the two compounds was
troublesome. Therefore, a milder reducing agent, such as zirconium borohydride generated in
situ, was used to afford a clean conversion of 8 to 11, [64] which was then converted to 12 under
standard carbamoylation reaction conditions (Scheme 1).
Carbamates 15a-b and urea 15c were prepared from the corresponding phenols 13a-b and
aniline 13c, respectively, through a carbamoylation reaction using n-hexyl-isocyanate, followed
by acidic hydrolysis of the methyl esters 14a-c (Scheme 2 and 3). The reverse carbamate 15d
was prepared upon activation of the aniline 13c with triphosgene, and, then, reaction with n-
hexanol, followed by acidic hydrolysis of the methyl ester 14d (Scheme 3).
7

ACCEPTED MANUSCRIPT
OCH3
O
I
F
7
a
d
OCH3
OCH3
O H
2'
O
O
4
'
F
F
HO
13a
13b
b
e
( )
5
HN
OCH3
OCH3
O
O
O
O
O
( )
5
F
F
N
H
O
14a
14b
c
f
( )
5
HN
OH
OH
O
O
O
O
O
( )
5
F
F
N
H
O
15a
15b
Scheme 2. Synthesis of compounds 15a-b. Reagents and conditions: (a) (2-
hydroxyphenyl)boronic acid, Pd(OAc) , K CO , EGME/H O, rt, 15 h, 84%; (b) n-hexyl-NCO,
2
2
3
2
DMAP, MeCN, rt, 15 h, 88%; (c) 6M HCl, THF, rt, 2 d, 90%; (d) (4-hydroxyphenyl)boronic
acid, Pd(OAc) , K CO , EGME/H O, rt, 15 h, 59%; (e) n-hexyl-NCO, DMAP, MeCN, rt, 15 h,
2
2
3
2
7
2%; (f) 6M HCl, THF, rt, 2 d, 46%.
8

ACCEPTED MANUSCRIPT
3'
a
7
13c
b
d
(
)
5
( )
5
14c
14d
c
e
(
)
5
( )5
15c
15d
Scheme 3. Synthesis of compounds 15c-d. Reagents and conditions: (a) (3-aminophenyl)boronic
acid, Pd(OAc) , K CO , EGME/H O, rt, 15 h, 91%; (b) n-hexyl-NCO, DMAP, MeCN, rt, 15 h,
2
2
3
2
6
5%; (c) 6M HCl, THF, rt, 2 d, 38%; (d) triphosgene, toluene, reflux, 15 h, then n-hexanol, rt, 15
h, 82%; (e) 6M HCl, THF, rt, 2 d, 59%.
Compounds 18a and 21a-b were synthetized by reacting the phenyl iodides 16b and 19a-b,
with (3-hydroxyphenyl)boronic acid under Suzuki cross coupling conditions, followed by
carbamoylation reaction of phenols 17 and 20a-b under standard conditions (Scheme 4 and 5).
Compounds 18a and 21b were then transformed into the corresponding acids 18b and 21c
under standard acidic hydrolysis (Scheme 4 and 5).
9

ACCEPTED MANUSCRIPT
b
c
( )
5
1
6a, R = H
17
18a, R = Me
18b, R = H
a
d
1
6b, R = Me
Scheme 4. Synthesis of compound 18b. Reagents and conditions: (a) MeOH, conc. H SO , rt, 15
2
4
h, quant.; (b) (3-hydroxyphenyl)boronic acid, Pd(OAc)
2 2 3 2
, K CO , EGME/H O, rt, 15 h, 71%; (c)
n-hexyl-NCO, DMAP, MeCN, rt, 15 h, 64%; (d) 6M HCl, THF, rt, 2 d, 62%.
a
b
( )
5
1
1
9a, R = CH
9b, R = CO
3
20a, R = CH
20b, R = CO
3
21a, R = CH
21b, R = CO
2
1c, R = CO H
3
2
Me
2
Me
2
Me
c
2
Scheme 5. Synthesis of compound 21a and 21c. Reagents and conditions: (a) (3-
hydroxyphenyl)boronic acid, Pd(OAc) , K CO , EGME/H O, rt, 15 h, 86-92%; (c) n-hexyl-
2
2
3
2
NCO, DMAP, MeCN, rt, 15 h, 86%-quant.; (d) 6M HCl, THF, rt, 2 d, 34%.
Compounds 29a-g were synthesized following the synthetic sequence described in Scheme 6.
p-Nitrofluorobenzenes 22a-d were reacted with diethylmethylmalonate followed by
decarboxylation to the corresponding acids 23a-d. 23a-d and the commercially available 23e
were converted into methyl esters 24a-e in acidic MeOH. In addition, the phenolic intermediate
2
4d was directly converted into the corresponding O-Bn protected 24f, under standard reaction
conditions.
Reduction of the nitro group was carried out using iron in presence of HCl for compounds 24a
and 24f, and ammonium formate in the presence of Pd/C for compounds 24b-c and 24e.
Compound 25f was obtained from 25e by standard nitration reaction. Diazotation/Sandmayer
reaction of the anilines 25a-f gave the iodides 26a-f, which were converted to carbamates 28a-f
via Suzuki and carbamoylation reactions. Compounds 28a-f were then transformed into the
1
0

ACCEPTED MANUSCRIPT
corresponding acids 29a-f under standard acidic hydrolysis. Finally, the aniline 29g was obtained
from the nitrophenyl 29f, under standard hydrogenation conditions.
a
d (or e)
g
2
2
2
2
2a, X = Cl
2b, X = CH
2c, X = CF
2d, X = OH
23a, X = Cl, R = H
23b, X = CH , R = H
23c, X = CF , R = H
23d, X = OH, R = H
3e, X = H, R = H
25a, X= Cl
25b, X= CH
26a, X= Cl
26b, X= CH
3
3
3
3
3
3
25c, X = CF
25d, X= OBn
25e, X= H
3
26c, X = CF
26d, X= OBn
26e, X= H
3
2
f
2
5f, X= NO
2
26f, X= NO
2
b
2
2
2
2
4a, X = Cl, R = CH
4b, X = CH , R = CH
4c, X = CF , R = CH
4d, X = OH, R = CH
24e, X = H, R = CH
4f, X = OBn, R = CH
3
3
3
h
3
3
3
c
3
2
3
j
i
( )
5
( )
5
2
9a, X = Cl
28a, X = Cl
28b, X = CH
28c, X= CF
28d, X = OBn
28e, X = H
27a, X= Cl
27b, X= CH
27c, X = CF
3
27d, X= OBn
27e, X= H
27f, X= NO
2
2
9b, X = CH
9c, X = CF
9d, X = OH
9e, X = H
9f, X = NO
9g, X = NH
3
3
3
3
2
2
3
2
2
2
28f, X = NO
2
2
k
2
Scheme 6. Synthesis of compounds 29a-g. Reagents and conditions: (a) diethyl methylmalonate,
NaOH, DMF, rt, 15 h; then AcOH, H SO , H O 110 °C, 24 h, 48-87%; (b) MeOH, conc. H SO ,
2
4
2
2
4
rt, overnight, 81-98%; (c) BnBr, K CO , acetone, 60 °C, 15 h, 63%; (d) Fe, HCl, MeOH, 65 °C,
2
3
2
2
3
h, 64-94%, for 24a and 24f; (e) HCO
4e; (f) Ac O, HNO 0 °C, 2 h then H SO
0 min, then NaI, 60 °C, 2 h, 55-72%; (h) (3-hydroxyphenyl)boronic acid, Pd(OAc)
O, rt, 15 h, 59-84%; (i) n-hexyl-NCO, DMAP, MeCN, rt, 15 h, 89%-quant.; (j) 2M
HCl, dioxane, 80 °C, 15 h, 73-95%; (k) cyclohexene, 10% Pd/C 80 °C, 2 h then 2M HCl, 55%.
2
NH
4
, 10% Pd/C, MeOH, rt, 3 h, quant., for 24b-c, and
, 3M HCl, 0 °C,
3
, K CO ,
2
3
2
4
, MeOH, 65 °C, 2 h, 98%; (g) NaNO
2
2
2
EGME/H
2
1
1

ACCEPTED MANUSCRIPT
2
2
.2. SAR exploration of the first class of potent multi-target FAAH-COX inhibitors.
.2.1. Rational drug design: merging strategy and identification of hit 10a.
We started our SAR exploration with compound 10a, [51] which was designed by merging
essential pharmacophores of the FAAH inhibitor, URB597, 2, and those of the NSAID,
flurbiprofen, 3a (Figure 1). The inhibitory potencies of 2, 3a and 10a against rat brain FAAH,
ovine testis COX-1 and human recombinant COX-2 are reported in Table 1.
Compound 10a inhibited FAAH and COX activities with relatively weak potencies (IC50
values, in µM: FAAH = 8.2; COX-1 = 7.9; COX-2 > 100). Nevertheless, these initial results
encouraged us because 10a was one of the most potent FAAH/COX-1 inhibitors previously
reported. [27, 28, 30, 32, 33, 65]
We started, therefore, an SAR exploration around 10a with the objective of identifying
chemical and structural determinants that might improve potency on the three targets in a
balanced manner.
2
.2.2. Study of the effect of the nature of R group: cycloalkanes, small-branched alkanes and
phenyls.
We prepared a series of analogs bearing cycloalkyl groups with different ring size at the N-
terminal of the carbamate functionality (Table 1).
1
2

ACCEPTED MANUSCRIPT
Table 1. SAR exploration on the nature of R group: cycloalkanes, small-branched alkanes and
phenyls.
a,b
a,b
a,b
FAAH
IC50(µM)±SD
COX-1
COX-2
Compound
R
IC50 (µM)±SD
IC50 (µM)±SD
2
,
-
-
0
.0017±0.001
100
>100
>100
URB597
a,
flurbiprofen
0a
3
>
0.15±0.018
7.9±2.1
1.06±0.53
>100
1
c-hexyl
c-pentyl
8.2±2.4
4.8±3.2
48.7±9.0
>100
1
0b
0c
0d
0e
0f
0g
0h
4.4±2.0
0.72±0.02
5.4±2.9
> 100
>100
1
c-butyl
1
c-propyl
c-hexyl-CH
74.3±6.1
>100
1
2
0.36±0.06
0.60±0.04
0.15±0.03
3.9±2.1
1
c-hexyl-(CH
iso-propyl
iso-butyl
Ph
2
)
2
0.018±0.007
>100
10.8±2.2
>100
1
1
4.1±2.1
8.2±2.1
>100
1
0i
41.2±3.4
4.18±2.8
0.17±0.07
0.09±0.01
0.027±0.010
0.27±0.07
1.3±0.6
2.7±0.3
>100
1
0j
Ph-CH
2
1
0k
0l
0m
Ph-(CH
Ph-(CH
Ph-(CH
2
)
2
3
4
6.3±2.2
>100
1
2
2
)
)
0.58±0.09
3.7±2.8
6.2±0.3
>100
1
a
b
Values are reported as mean values of ≥3 experiments performed; IC50 values were not
determined for compounds showing less than 50% inhibition at concentrations of 100 µM for
FAAH and COXs.
1
3

ACCEPTED MANUSCRIPT
We observed that, while the potency against FAAH was retained with the c-pentyl analog 10b
IC =4.8 µM), a 10-fold loss in potency occurred with the c-butyl derivative 10c (IC =48.7
(
5
0
50
µM) and complete loss of activity (IC50>100 µM) with the c-propyl derivative 10d. With regard
to COX activity, while the c-pentyl analog 10b showed a comparable potency against COX-1
(
IC =4.4 µM), the c-butyl analog 10c was 10-fold more potent than compound 10a (IC =0.72
50 50
µM). Conversely, the c-propyl analog 10d displayed an IC50 value similar to compounds 10a and
0b against COX-1 (=5.4 µM) and was indeed the only compound in this series that showed
modest activity against COX-2 (IC =74.3 µM).
1
5
0
The N-terminal region of the carbamate functionality in 10a may engage in beneficial
interactions with the acyl chain-binding domain of FAAH [26], [56, 57] as well as the
hydrophobic channels present in COX-1 and COX-2. [6] [61] To capture such interactions, we
prepared a series of analogs bearing lipophilic aliphatic and aromatic N-terminal substituents
with diverse steric properties (Table 1).
The insertion of a methylene group adjacent to the c-hexyl ring of 10a - compound 10e- led to
a significant increase of potency toward FAAH (23-fold) and COX-1 (10-fold), but no COX-2
inhibition (IC >100 µM). A further homologation, compound 10f, showed a 400-fold increase
5
0
in potency toward FAAH and a 50-fold increase in potency toward COX-1, compared to 10a.
Interestingly, 10f also inhibited COX-2 with an IC of 10.8 µM.
50
Next, we investigated the effects of small and branched alkyl groups, the iso-propyl 10g and
the iso-butyl 10h - as truncated analogs of 10a and 10e, respectively. These modifications were
detrimental for FAAH and COX inhibitory activities compared to 10a and 10e, respectively.
While the replacement of the c-hexyl ring with a phenyl group (10i) was not tolerated by
FAAH, in analogy to previous reports on the class of O-aryl carbamates, [56, 57] this
1
4

ACCEPTED MANUSCRIPT
modification led to a gain in inhibitory activity toward COX-1 and COX-2. The insertion of a
methylene group adjacent to the phenyl ring of 10i - compound 10j- caused a 10-fold increase in
potency toward FAAH, compared to 10i, but had almost no impact on COX-1 activity and
dramatic loss on COX-2. Homologation (10k-m) resulted in a progressive enhancement of the
inhibitory potency toward FAAH, but this trend was more erratic for COX-1 and COX-2:
compound 10l was most active analog with IC50 = 0.58 µM and 6.2 µM against COX-1 and
COX-2, respectively.
These findings might reflect differences in the depth of lipophilic pockets of FAAH and COX
enzymes. [6, 26]
2
.2.3. Study of the effect of the nature of the R group: linear alkanes. Identification of 10r
(ARN2508).
Since the (CH ) homologation at the N-terminal site appeared to be critical for the modulation
2
n
of the biological activities at both targets, we prepared a series of carbamates bearing linear alkyl
groups (alkyl = (CH (CH ) ) with n= 1 to 7) at N- terminal region (Table 2).
3
2 n
In analogy to the reported SAR results on the class of O-aryl carbamates, [56] potency toward
FAAH increased with increased length of the (CH ) chain (n = 1-7). A different trend was
2
n
observed for COX-1 and COX-2, where insertion of short (CH
compounds (10n-o) that were weak COX-1 inhibitors and had no activity against COX-2. On the
other hand, insertion of n = 3-5 (CH chains (10p-r) increased the inhibitory potencies for
COX-1 and COX-2 from sub-micromolar to nano-molar IC , whereas insertion of n = 6-7
2 n
) chains (n = 1-2) led to
2 n
)
5
0
(
CH ) chains (10s-t) was detrimental.
2 n
1
5

ACCEPTED MANUSCRIPT
2 n
These results are in agreement with those above reported in the homologation of the Ph(CH )
chain series (n = 1-4, compounds 10i-m, Table 1).
Table 2. SAR exploration on the nature of the R group: linear alkanes
a,b
a,b
a,b
FAAH
COX-1
COX-2
Compound
R
IC (µM)±SD IC (µM)±SD
IC (µM)±SD
5
0
50
50
1
0n
0o
ethyl
>100
>100
2.1±0.9
>100
>100
1
n-propyl
n-butyl
n-pentyl
1.65±0.06
0.26±0.07
1
1
0p
0q
7.0±1.8
>100
0.57±0.15
0.020±0.009
0.16±0.02
1
0r,
n-hexyl
0.031±0.002
0.012±0.002
0.43±0.02
ARN2508
1
0s
0t
n-heptyl
n-octyl
0.011±0.003
0.003±0.002
0.37±0.10
0.99±0.07
0.32±0.005
1
28.8±8.4
a
b
Values are reported as mean values of ≥3 experiments performed; IC50 values were not
determined for compounds showing less than 50% inhibition at concentrations of 100 µM for
FAAH and COXs.
From this SAR exploration, we identified 10r (ARN2508), [51] which bears a n-hexyl chain at
the N-terminal site, as a potent multi-target inhibitor of FAAH, COX-1 and COX-2 (IC : FAAH
5
0
=
31 nM; COX-1 = 12 nM; COX-2 = 430 nM) (Table 2). In addition to its high balanced
potency, the highest reported thus far, [27, 28, 30, 32, 33, 65] we found that 10r displays no off-
1
6

ACCEPTED MANUSCRIPT
target activities on a panel of >90 biologically relevant targets, and effectively engages its
intended targets after oral administration in mice. [51]
These results encouraged us to initiate a more focused SAR exploration to define the effect of
additional chemical and structural modifications in various regions of 10r scaffold.
2
.2.4. Focused SAR exploration around 10r (ARN2508) and identification of 18b, 29a-c, e and
(S)-(+)-10r.
In particular, we focused our interest on the role and position of carbamate group in the A
phenyl ring (Table 3 and Table 4), as well as the role of the propionic acid functionality and the
fluorine atom in the B phenyl ring (Table 5 and Table 6).
2
.2.4.1. Role and position of carbamate group in the A phenyl ring.
We first investigated the effect of the position of the carbamate group in the A phenyl ring,
which indeed appeared to play an important role in the inhibition of both FAAH and COX
Table 3). In agreement with the rational design of our hybrid scaffold 1 (Figure 1), the C(2’)-
(
derivative 15a (ortho derivative) showed a 70-fold decrease in potency toward FAAH, a 60-fold
decrease in potency toward COX-1, and a complete loss of activity toward COX-2, when
compared to the C(3’)-isomer 10r (meta derivative) (Table 3).
1
7

ACCEPTED MANUSCRIPT
Table 3. Effect of the position of the carbamate functionality on the A phenyl ring.
2'
A
4'
a,b
a,b
a,b
FAAH
COX-1
COX-2
Compound position
IC50 (µM) ±SD IC50 (µM) ±SD IC50 (µM) ±SD
1
5a
C(2’)
C(4’)
2.2±0.6
0.72±0.04
>100
>100
>100
1
5b
0.068±0.012
a
b
Values are reported as mean values of ≥3 experiments performed; IC50 values were not
determined for compounds showing less than 50% inhibition at concentrations of 100 µM for
FAAH and COXs.
On the other hand, the C(4’)-derivative 15b (para derivative) exhibited a slight loss of potency
toward FAAH compared to 10r, but both COX inhibitions were completely suppressed (Table
3
). These results support the hypothesis that the bent shape of the O-biphenyl moieties, which is
known to better fit the FAAH enzyme surface, [53] is also important in the recognition by COX-
and COX-2, possibly through a better superimposition to the conformations adopted by the
1
fatty acyl chain of the natural substrate/product (the first two cis-double bonds of AA) when
bound to COX-1 [66] and COX-2 [67].
Next, we replaced the carbamate moiety with alternative functional groups, such as urea (15c)
[51] and reversed carbamate (15d) [51] (Table 4).
As expected from the rational design of our class of multitarget inhibitors, 15c and 15d showed
a significant decrease in potency toward FAAH, whilst retaining COX-1 and COX-2 inhibitory
activities compared to 10r.
1
8

ACCEPTED MANUSCRIPT
Table 4. Carbamate replacement: urea and reversed carbamate derivatives
a
a
a
FAAH
IC (µM) ±SD
COX-1
COX-2
Compound
Y
X
IC (µM) ±SD
IC (µM) ±SD
5
0
50
50
1
5c
NH NH
NH
88.4±2.3
14.9±1.6
0.014±0.003
0.03±0.01
0.56±0.12
0.17±0.01
1
5d
O
a
Values are reported as mean values of ≥3 experiments performed.
These results support the hypothesis that the mechanism of action of this class of compounds is
similar to the one reported for the O-aryl carbamates (acylation of FAAH Ser 241) [57] and that
COX inhibition does not rely on any irreversible binding mode at the expense of the carbamate
group of 10r. Reported dialysis experiments on 10r are in agreement with this mechanistic
speculation. [51]
2
.2.4.2. Role of the propionic acid functionality in the B phenyl ring.
We then turned our attention to the role of the propionic acid in the B phenyl ring (Table 5).
Replacing the propionic acid group of 10r with several substituents had only a minor impact on
the potency toward FAAH, compared to the effect observed on COX activities. In fact, methyl
ester 9r retained FAAH inhibitory activity, compared to 10r, but completely lost activity toward
both COX-1 and COX-2. Replacement of the carboxylic acid of 10r with the corresponding
1
9

ACCEPTED MANUSCRIPT
primary alcohol 12 resulted in a 10-fold improvement in potency toward FAAH (IC50=3 nM), a
1
00-fold loss in potency toward COX-1 (IC =1.1 µM) and in a complete loss of activity toward
50
COX-2.
Table 5. SAR exploration on the R group: role of the propionic acid functionality on the B
phenyl ring
B
a,b
a,b
a,b
FAAH
COX-1
COX-2
Compound
R
IC (µM) ±SD IC (µM) ±SD IC (µM) ±SD
5
0
50
50
9
r
CH(CH
3
)CO
2
CH
3
0.052±0.010
0.003±0.002
0.063±0.010
0.026±0.09
0.085±0.006
>100
>100
>100
1
2
CH(CH
3
)CH
2
OH
1.1±0.3
2.1±0.1
>100
1
8b
1a
1c
CH
2
CO
2
H
0.24±0.04
>100
2
CH
3
2
CO
2
H
>100
>100
a
b
Values are reported as mean values of ≥3 experiments performed; IC50 values were not
determined for compounds showing less than 50% inhibition at concentrations of 100 µM for
FAAH and COXs.
On the other hand, the removal of the α-methyl group, as in the achiral des-methylated
derivative 18b, caused a 2-fold decrease of the potency toward FAAH, compared to 10r (IC50
3 nM and 31 nM, respectively), and a 180-fold reduction of potency toward COX-1 (IC50 = 2.1
µM and 12 nM, respectively). The activity against COX-2 was slightly improved (IC = 0.24
=
6
5
0
µM and 0.43 µM respectively). The methyl analog 21a [51] was active against FAAH in the
2
0

ACCEPTED MANUSCRIPT
same potency range of 10r (IC50 = 26 nM and 31 nM, respectively), while a completely loss of
activity against COX enzymes was observed. A similar result was obtained with the carboxylic
analog 21c, which also showed a 3-fold reduction in potency toward FAAH, compared to 10r
(
IC = 85 nM and 31 nM, respectively).
50
We conclude that FAAH tolerates substituents with different steric and electronic properties at
the 4-position of the B phenyl ring, while COX-1 and COX-2 display a stringent requirement for
a propionic or acetic acid groups in the same position.
2
.2.4.3. Role of the fluorine atom in the B phenyl ring.
To complete the SAR exploration of the B phenyl ring, we evaluated the effect of substituents
with different electronic and steric properties, alternative to the fluorine atom (Table 6).
Substituting the fluorine with chlorine was tolerated: indeed, 29a was virtually equipotent
against FAAH and COX-1, and marginally less potent on COX-2, compared to 10r. The same
trend was observed with the methyl derivative 29b, which was slightly more potent than 10r
against FAAH and equally potent on COX-1, but less active against COX-2. The CF
9c showed a 6-fold and 2-fold increase in potency toward FAAH and COX-2, respectively, and
was as potent as 10r on COX-1.
3
derivative
2
Removal of the fluorine atom (29e) resulted in a 10-fold increase in potency toward FAAH,
compared to 10r, and a slight decrease in activity for COX-1 and COX-2.
Compounds 29d and 29g, which bear -OH or -NH groups, respectively, inhibited FAAH with
2
potencies similar to that of 10r, whereas a clear loss in potency for both COX-1 and COX-2 was
2
observed. On the other hand, the NO derivative 29f had higher potency toward FAAH but loss
2
1

ACCEPTED MANUSCRIPT
lower potency toward both COX-1 and COX-2. We interpret these results to suggest that the
electronic and steric properties of the substituents in the 3-position of the B phenyl ring affect
FAAH recognition only slightly, whereas these same substituents influence COX-1 and COX-2
more markedly, with lipophilic groups being better tolerated than polar or H-bond donator
groups.
Table 6. SAR exploration on the role of the X substituent on the B phenyl ring
B
3
a
a
a
FAAH
IC (µM)±SD
COX-1
50
COX-2
50
Compound
9a
9b
9c
9d
9e
9f
9g
X
Cl
IC (µM)±SD
IC (µM)±SD
5
0
2
0.023±0.008
0.010±0.001
0.005± 0.001
0.035±0.010
0.003±0.001
0.009±0.002
0.049±0.023
0.009±0.001
0.011±0.001
0.01±0.003
0.65±0.07
0.73±0.21
1.40±0.31
0.2±0.08
2
CH
3
2
CF
3
2
OH
H
13.0 ±2.1
0.69±0.02
0.930±0.15
12.1±0.6
2
0.054±0.011
0.13±0.03
2
NO
2
2
NH
2
0.22±0.09
a
Values are reported as mean values of ≥3 experiments performed.
2
2

ACCEPTED MANUSCRIPT
2
.2.4.4. Stereochemical and pharmacological studies of 10r enantiomers.
Finally, we subjected the best studied member of this class of inhibitors, the racemic
compound 10r, [51] to chiral HPLC separation and tested each of its enantiomers – (-)-10r (first
eluted) and (+)-10r (second eluted) – for the ability to inhibit FAAH, COX-1 and COX-2 (Table
7
). FAAH showed no preference for either enantiomer, with each being more active than the
racemate 10r. By contrast, in analogy to prior studies on different classes of FAAH/COX
inhibitors, [30, 33] substantial differences were observed on COX-1 and COX-2. Compound (+)-
1
1
0r was highly potent on both COX-1 (IC50=0.29 nM) and COX-2 (IC50=50 nM), whereas (-)-
0r was weakly active on either target.
Table 7. Evaluation of the enantiomers of 10r
a
a
a
r-FAAH
COX-1
COX-2
Compound
IC50(µM) ±SD IC50 (µM) ±SD IC50 (µM) ±SD
(
±)-10r
0.031±0.002
0.0099±0.002
0.012±0.002
4.0±1.3
0.43±0.02
22.8±8.7
b
(-)-10r
c
(+)-10r
0.0094±0.0003 0.00029±0.00004
0.050±0.012
a
b
Values are reported as mean values of ≥3 experiments performed; (R)- configurated
c
enantiomer of 10r (see Supporting Information for details); (S)-configurated enantiomer of 10r
(see Supporting Information for details).
We completed our exploration on the two enantiomers of 10r by assigning their absolute
stereo-configurations. As reported in Supporting Information (Scheme S1), a stereochemical
correlation study allowed us unambiguously to assign the absolute stereochemistry of (-)-10r and
(+)-10r to the (R)- and (S)- configurations, respectively. These results are in agreement with
2
3

ACCEPTED MANUSCRIPT
earlier reports showing that the (S)-enantiomer is responsible for the COX-inhibiting activity of
aryl-propionic acid derivatives such as flurbiprofen. [29, 31, 33, 59]
2
.2.4.5. In vivo experiments on (S)-(+)-10r.
Finally, pharmacological experiments indicate that compound (S)-(+)-10r strongly engages its
intended molecular targets in live mice. Intravenous administration of (S)-(+)-10r (1 mg/kg)
lowered the concentrations of two COX products in circulation, prostacyclin and TXA , as
assessed surveying the stable metabolites, 6-keto-PGF1 and TXB (Figure 2A and 2B).
2
α
2
Moreover, (S)-(+)-10r increased plasma levels of the FAAH substrate, OEA (Figure 2C). In
addition, (S)-(+)-10r demonstrated no off-target activities on a panel of >90 biologically relevant
receptors, enzymes [including N-acylethanolamine amide hydrolase (NAAA), which is the
primary enzyme involved in the deactivation of PEA and OEA in innate immune cells] and ion
channels (Table S1). Further pharmacological studies on the (R)- and (S)- series of this class of
inhibitors will be reported in due course.
2
4

ACCEPTED MANUSCRIPT
Figure 2. Plasma levels of COX metabolites and FAAH substrate after intravenous
administration of (S)-(+)-10r (1 mg/kg): 6-keto-PGF (A) TXA (B) and OEA (C). Results are
1
α
2
expressed as mean ± s.e.m. of 6 independent determinations. *P<0.05, **P<0.01 and
**P<0.001 compared to vehicle mice, two-tailed Student’s t test.
*
3
.
Conclusions
The present study outlines key SAR properties of a novel class of dual inhibitors of
intracellular FAAH and COX activities, which are based on the hybrid scaffold 1. Several
chemical variations of this scaffold were considered, which involved the carbamate moiety at the
3
’-position of the A phenyl ring, the R groups, and the propionic acid moiety and fluorine atom
in the B phenyl ring. Introduction of different alkyl and aromatic groups in the N-terminal region
of the carbamate functionality improved inhibitory potency toward both FAAH and COX. A
more focused exploration around the potent, selective and orally available racemic inhibitor 10r
[51] led to the identification of novel potent analogs, 29a-c, and e. Because of the problems
associated with the development of racemic compounds, we extended our studies and identified
two additional molecules, the achiral compound 18b and the enantiomer (S)-(+)-10r, which also
display high inhibitory potency for FAAH/COX-1/COX-2.
2
5

ACCEPTED MANUSCRIPT
The in vivo activity of (S)-(+)-10r suggests that this agent may be used to probe the therapeutic
utility of simultaneous FAAH-COX inhibition, especially in pathologies in which these enzymes
are abnormally expressed.
4
.
Experimental part
4
.1. Synthesis.
Solvents and reagents were obtained from commercial suppliers and were used without further
purification. URB597 was prepared following a reported procedure. [54] Flurbiprofen was
purchased from Sigma-Aldrich (Milan, Italy). Melting points were determined on a Büchi M-560
capillary melting point apparatus and are uncorrected. Automated column chromatography
®
purifications were done using a Teledyne ISCO apparatus (CombiFlash Rf) with pre-packed
silica gel columns of different sizes (from 4 g until 120 g). Mixtures of increasing polarity of Cy
and EtOAc or DCM and MeOH were used as eluents. Preparative TLC analyses were performed
1
13
using Macherey-Nagel pre-coated 0.05 mm TLC plates (SIL G-50 UV ). H and C-NMR
2
54
1
experiments were run on a Bruker Avance III 400 system (400.13 MHz for H, and 100.62 MHz
1
3
19
for C), equipped with a BBI probe and Z-gradient coil. F-NMR experiments were run on a
1
9
Bruker Avance III 600 system (546.6 MHz for F), equipped with a 5 mm CryoProbe QCI
1
19
13 15
H/ F– C/ N–D quadruple resonance and a Z-gradient coil. Spectra were acquired at 300 K,
using deuterated dimethylsulfoxide (DMSO-d ) or deuterated chloroform (CDCl ) as solvents.
Chemical shifts for H and C spectra were recorded in parts per million using the residual non-
6
3
1
13
1
13
deuterated solvent as the internal standard (for DMSO-d : 2.50 ppm, H; 39.52 ppm, C; for
6
2
6

ACCEPTED MANUSCRIPT
1
13
CDCl
3
: 7.26 ppm, H and 77.16 ppm, C). Data are reported as follows: chemical shift (ppm),
multiplicity (indicated as: bs, broad signal; s, singlet; d, doublet; t, triplet; q, quartet; p, quintet,
sx, sextet; m, multiplet and combinations thereof), coupling constants (J) in Hertz (Hz) and
integrated intensity. UPLC/MS analyses were run on a Waters ACQUITY UPLC/MS system
consisting of a SQD (Single Quadropole Detector) Mass Spectrometer equipped with an
Electrospray Ionization interface and a Photodiode Array Detector. PDA range was 210-400 nm.
Analyses were performed on an ACQUITY UPLC BEH C18 column (50x2.1 mmID, particle
size 1.7 µm) with a VanGuard BEH C18 pre-column (5x2.1 mmID, particle size 1.7 µm). Mobile
4 2 4
phase was either 10 mM NH OAc in H O at pH 5 adjusted with AcOH (A) and 10 mM NH OAc
in MeCN-H O (95:5) at pH 5 (B). Electrospray ionization in positive and negative mode was
2
applied. Analyses were performed with method A or B. Method A for compounds 10a-t, 15a-d,
1
8b, 21c and 29b-g: Gradient: 5 to 95% B over 3 min. Flow rate 0.5 mL/min. Temperature 40
C. Method B for compounds 9r, 12, 21a and 29a: Gradient: 50 to 100% B over 3 min. Flow rate
.5 mL/min. Temperature 40 °C. Purifications by preparative HPLC/MS were run on a Waters
°
0
Autopurification system consisting of a 3100 Single Quadropole Mass Spectrometer equipped
with an Electrospray Ionization interface and a 2998 Photodiode Array Detector. HPLC system
included a 2747 Sample Manager, 2545 Binary Gradient Module, System Fluidic Organizer and
5
15 HPLC Pump. PDA range was 210-400 nm. Purifications were performed on a XBridgeTM
Prep C18 OBD column (100x 19 mmID, particle size 5 µm) with a XBridgeTM Prep C18 (10x
1
5
9 mmID, particle size 5 µm) Guard Cartridge. Mobile phase was 10 mM NH OAc in H O at pH
4 2
adjusted with AcOH (A) and 10 mM NH OAc in MeCN-H O (95:5) at pH 5 (B). Electrospray
4
2
ionization in positive and negative mode was used. Analyses by chiral HPLC were run on a
Waters Alliance HPLC instrument consisting of an e2695 Separation Module and a 2998
2
7

ACCEPTED MANUSCRIPT
Photodiode Array Detector. PDA range was 210-400 nm. Analyses were performed isocratic on
a Daicel ChiralPak AD column (250x4.6 mmID, particle size 10 µm). Mobile phase was 0.1 %
TFA Heptane/2-Propanol (75:25). Separations of 10r by preparative chiral HPLC were run on a
Waters Alliance HPLC instrument consisting of a 1525 Binary HPLC Pump, Waters Fraction
Collector III and a 2998 Photodiode Array Detector. UV detection was at 240 nm. Purifications
were performed isocratic on a Daicel ChiralPak AD column (250 x 10mmID, particle size 10
µm). Mobile phase was 0.1 % TFA Heptane/2-Propanol (75: 25). Optical rotations were
measured on a Rudolf Research Analytical Autopol II Automatic polarimeter using a sodium
3
lamp (589 nm) as the light source; concentrations expressed in g/100 mL using CHCl as a
solvent and a 1 dm cell. Accurate mass measurement was performed on a Synapt G2
Quadrupole-ToF Instrument (Waters, USA), equipped with an ESI ion source; compounds were
diluted to 50 ꢀM in H2O/MeCN and analyzed. Leucine Enkephalin (2 ng/mL) was used as lock
mass reference compound for spectra calibration. All final compounds displayed ≥ 95% purity as
determined by NMR and UPLC/MS analysis.
All the analytical data of intermediate compounds are reported in Supporting Material.
4
.1.1. (±)-2-(3-fluoro-4-nitro-phenyl)propanoic acid (4). Compound 4 was obtained as brown
clear oil (4.50 g, 81 %), according to the procedure reported in the literature starting from 2,4-
difluoronitrobenzene (4.77 g, 30 mmol). [62]
4
.1.2. (±)-Methyl 2-(4-nitro-3-fluoro-phenyl)propanoate (5). To a solution of 4 (4.50 g, 21.11
mmol) in MeOH (40 mL), concentrated H SO (0.1 mL) was added and the resulting solution
2
4
2
8