Novel propanamides as fatty acid amide hydrolase inhibitors

European Journal of Medicinal Chemistry 136 (20 1 7 ) 523e542

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Novel propanamides as fatty acid amide hydrolase inhibitors

,

Alessandro Deplano ^{a 1}, Carmine Marco Morgillo ^{c 1}, Monica Demurtas ^a,

b

€

Emmelie Bjorklund , Mariateresa Cipriano , Mona Svensson , Sanaz Hashemian ,

Giovanni Smaldone ^d, Emilia Pedone ^e, F. Javier Luque ^f, Maria G. Cabiddu ^g,

Ettore Novellino ^c, Christopher J. Fowler ^b, Bruno Catalanotti ^{c *}, Valentina Onnis ^a

,

^aDepartment of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale

72, Cagliari I-09124, Italy

^bDepartment of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden

c

ꢀ

Department of Pharmacy, Universita Degli Studi di Napoli Federico II, Napoli, Italy

^dIRCCS SDN, Via Emanuele Gianturco 113, 80143 Napoli, Italy

^eInstitute of Biostructures and Bioimaging, CNR, Naples, Italy

f

ꢁ

ꢀ

ꢁ

ꢀ

ꢁ

Departament de Nutricio, Ciencies de l'Alimentacio i Gastronomia and Institut de Biomedicina (IBUB), Facultat de Farmacia i Ciencies de l'Alimentacio,

Universitat de Barcelona, Santa Coloma de Gramenet, Spain

^gDepartment of Chemical and Geological Sciences, University of Cagliari, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the

hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH in-

hibition represents a promising strategy to activate the cannabinoid system, since it does not result in the

psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here

we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(tri-

ﬂuoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed

potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack

activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the deﬁ-

nition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat

and mouse FAAH on selected compounds of the series demonstrated that slight modiﬁcations of the

chemical structure could inﬂuence the binding mode and give rise to competitive (TPA1) or non-

competitive (TPA14) inhibition modes.

Received 3 March 2017

Received in revised form

2 May 2017

Accepted 11 May 2017

Available online 12 May 2017

Keywords:

FAAH inhibitors

Heteroaryl propanamides

Fatty acid amide hydrolase

Endocannabinoids

Anandamide

1. Introduction

unusual catalytic triad Ser²¹⁷-Lys¹⁴²-Ser²⁴¹

.

Blockade of FAAH activity is a suitable strategy to modulate the

endocannabinoid system compared to exogenous cannabinoid re-

ceptor agonists, whose therapeutic proﬁle is heavily limited by CB₁-

mediated psychotropic and peripheral side effects such as impair-

ment in cognition, motor coordination, and psychoses. It is now

well established that the endocannabinoid system is involved in

the regulation of mood, and in animal models of anxiety, and that

FAAH inhibitors show a potentially useful proﬁle [1]. Additionally,

FAAH inhibitors may be useful in disorders ranging from cannabi-

noid dependence [2] to preventing or reducing the inﬂammatory

Anandamide (AEA), 2-acylethanolamines and 2-arachidonoyl

glycerol are signaling lipids belonging to the class of endocanna-

binoids, endogenous agonists of the cannabinoid receptor type-1

(CB₁) and type-2 (CB₂). The cannabinoid system regulates many

physiological functions, both in the central and peripheral nervous

systems and in peripheral organs. In order to maintain the normal

functionality of nerve systems, many fatty acid amides, including

AEA, are degraded by fatty acid amide hydrolase (FAAH), a member

of the serine hydrolase enzyme family, characterized by the

process associated with Ab deposition in Alzheimer's Disease [3]

and in the treatment of comorbidity between psychological disor-

ders and cardiac disease [4]. The principal classes of covalent and

non-covalent inhibitors as well as the FAAH inhibitors entered into

* Corresponding author.

E-mail address: brucatal@unina.it (B. Catalanotti).

1

These authors equally contribute to the present paper.

http://dx.doi.org/10.1016/j.ejmech.2017.05.033

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A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

clinical studies have been recently reviewed [5]. Clinical studies so

far undertaken indicate that FAAH inhibitors are generally well

tolerated [6,7]. A serious brain injury was reported in a phase I

study with the FAAH inhibitor BIA 10-2474, but this appears to be

an off-target effect of the drug, not correlated to FAAH inhibition

[8]. Indeed, a computer-based proteome-docking approach sug-

gested that BIA 10-2474 could interact with several targets in a

manner likely to cause brain haemorrhaging [9].

reﬂuxing acetonitrile (MeCN) solution to yield the enaminone 4 in

good yields. Upon reacting with an excess of N,N-dimethylforma-

mide dimethyl acetal (DMF-DMA) in reﬂuxing toluene, the enam-

inone 4 was converted into 1,1,1,triﬂuorohexadienone 5 in 88%

yield. The treatment of intermediate 5 with ammonium acetate in

DMF at 100 ^ꢀC produced pyridine ring closure to ester 6 in 75%

yield. Hydrolysis of compound 6 in hydroalcoholic sodium hy-

droxide solution afforded acid 7.

We previously reported that some NSAIDs as ibuprofen display

modest FAAH inhibitory activity. The conversion of the carboxylic

group of ibuprofen into heterocyclic amide namely 2-(4-

Finally, treatment of acid 7 with the appropriate amine in the

presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-

drochloride (EDC) and 1-hydroxybenzotriazole (HOBt) in MeCN

solution gave amides TPA1-16, 18-21 in moderate to good yields.

In order to study the structure-activity relationship of TPA

compounds and to discover the better modiﬁcation to improve the

activity as FAAH inhibitors, we modiﬁed all moieties of the

molecule.

isobutylphenyl)-N-(3-methylpyridin-2-yl)propanamide

(Ibu-

AM5) that showed high FAAH inhibitory activity, while retaining

COX inhibitory properties of the parent ibuprofen [10e12].

Encouraged by these ﬁnding, we have been prompted to investigate

further new Ibu-AM5 analogs as potential FAAH inhibitors. Since

superimposition of Ibu-AM5 with FAAH co-crystallized inhibitors

indicated that the isobutyl chain of Ibu-AM5 overlap aromatic

hydrophilic moieties, as a rational development we designed and

synthesized a new series of compounds where the isobutyl group is

replaced by a 2-(triﬂuoromethyl)pyridin-4-ylamino group (Fig. 1).

In the present study we report the discovery, synthesis, pharma-

cological and biochemical characterization of these compounds, as

well as their putative binding mode of the lead and ﬁnally the ef-

forts to identify key structural features of this novel series of N-

(heteroaryl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)amino)phenyl)

propanamides as FAAH inhibitors. With the aim to deﬁne the

critical requirements for FAAH inhibitory activity we have explored

the effects of structural modiﬁcation in the heteroaryl group, in the

linker between the heteroaryl group and the phenylcarbonyl moi-

ety, the effect of replacement of the methyl on the C-2 with smaller

and larger substituents, and ﬁnally the effect of introducing

different polar rings in the (triﬂuoromethyl)pyridine region.

First we focused our attention upon the 3-methylpyridin-2-yl

moiety. To evaluate the effect of the presence of the methyl group

and of its position on the pyridine ring upon the FAAH inhibitory

properties. Speciﬁcally, the 2-aminopyridine, 3-aminopyridine and

4-aminopyridine analogs TPA4-6 and the 5-methyl TPA2 and 4-

methyl TPA3 analogs were prepared as reported in Scheme 1. To

evaluate the effect of pyridine replacement by other rings, the

phenyl analog TPA7, the pyrimidine (TPA8), pyrazine (TPA9),

quinoline (TPA10) and arylpiperazino analogues (TPA18e21) were

also prepared (Scheme 1). Finally, the 3-methylpyrido group was

replaced with halogens and triﬂuoromethyl group (TPA11e14).

To investigate the importance for FAAH inhibition of the dis-

tance between the carbonyl group and the pyridine ring, the acid 7

was condensed with 2-picolylamine and 3-picolylamine to obtain

amides TPA15 and TPA16 respectively (Scheme 1). Further linker

extension was accomplished through introduction of a second

amide moiety to give TPA17. As indicated in Scheme 2, TPA17 was

prepared by condensation between 7 and ethyl glycinate hydro-

chloride using the EDC method. The obtained ester 8 was hydro-

lyzed under basic conditions to obtain compound 9. This acid was

ﬁnally condensed with the 2-amino-3-methylpiridine using the

EDC method to obtain the amide TPA17.

2. Results and discussion

2.1. Chemistry

To examine the importance of the substituent on C-

a to the

The synthesis of the target amides was undertaken as outlined

in Schemes 1-6. The ﬁrst step of the synthetic approach to the new

series of TPA amides was based on a previously described proced-

ure that allows the construction of the triﬂuoromethylpyridine ring

linked by an amino nitrogen to an aromatic ring [13]. This synthetic

approach was modiﬁed and successfully applied for the preparation

of 2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)amino)phenyl)propanoic

acid (7) starting from the easily available 2-(4-nitrophenyl)propi-

onic acid (1), which was ﬁrst converted into its methyl ester and

then submitted to reduction of the nitro group. The resulting

carbonyl group ﬁrstly, a slight modiﬁcation of the procedure used

to afford acid 7 was employed. Thus, the unsubstituted derivative

TPA22 was prepared starting from the methyl ester of the 2-(4-

aminophenyl)acetic acid (10). The obtained acid 14 was subse-

quently condensed with 2-amino-3-methylpyridine using EDC

method (Scheme 3).

To introduce a second methyl or a cycloalkyl group on the C-a 2-

(4-nitrophenyl)acetic acid was converted into its methyl ester de-

rivative 15, which was treated with iodomethane in DMF solution

in the presence of sodium hydride to obtain intermediate 16.The

nitro group of 16 was reduced into its corresponding amine (17).

Compound 17 was used as starting material for the construction of

the triﬂuoromethylpyridine ring through the reaction sequence

above described and then the acid 21 was converted into TPA23 by

EDC method (Scheme 4).

methyl 2-(4-aminophenyl)propanoate

2 reacted with 1,1,1-

triﬂuoro-4-methoxypent-3-en-2-one (3) in 1:1.5 molar ratio in

TPA24e27 were prepared in an analogous manner starting from

the nitro derivative 15 and the appropriate di-halogen derivative,

namely 1,2-dibromoethane, 1,3-diiodopropane, 1,4-diiodobutane,

1,5-diiodopentane respectively that were reacted modifying a

described method [14] (Scheme 5).

Finally, we investigated the importance for FAAH inhibition of

the triﬂuoromethylpyridine ring. To this purpose, the ester 2 was

hydrolyzed to the acid 46. That is subsequently treated with 4-

chloro-8-triﬂuoromethylquinoline

and

4-chloro-7-

triﬂuoromethylquinoline to obtain the corresponding acids 47

and 48, which are condensed with 2-amino-3-methylpyridine by

Fig. 1. Design of the representative member TPA1 from Ibu-AM5.

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

525

Scheme 1. Reagents and conditions: (i) MeOH, HCl 37%, reﬂux, 4 h; (ii) Fe, HCl 37%, MeOH, H₂O, reﬂux overnight; (iii) MeCN, reﬂux, 2 h; (iv) DMF-DMA, PhMe, reﬂux, 1 h; (v)

NH₄OAc, DMF, reﬂux, 1.5 h; (vi) EtOH, 5 N aq. NaOH, r.t., 24 h; (vii) R-NH₂, EDC, HOBt, MeCN, r.t., 18e36 h.

Scheme 2. Reagents and conditions: (i) Ethyl glicinate hydrochloride, EDC, HOBt, TEA, MeCN, r.t., 4 h; (ii) EtOH, 5 N aq. NaOH, r.t., 24 h; (iii) EDC, HOBt, MeCN, r.t., 36 h.

EDC method to obtain TPA28 and TPA29, respectively (Scheme 6).

enzyme via an irreversible mechanism, upon prolonged pre-

incubation the potency should become greater; a constant IC₅₀

,

conversely, supports a reversible mechanism. For a fully reversible

compound, the percentage of control activity seen in the green bars

(i.e. following the dilution) should be higher than in the orange bars

(the remaining activity seen at the undiluted concentrations) but

equal to the white bars (the free concentrations after the dilution).

TPA1 showed no time-dependent increase in potency, consistent

with a reversible mechanism of action (Fig. 2A). This was conﬁrmed

in dilution experiments (Fig. 2B). The kinetics of inhibition was

investigated for both rat and mouse FAAH. For the rat, the data was

better ﬁtted by a model assuming a competitive mode of inhibition

than by a mixed model inhibition (Fig. 2C), whereas in the mouse,

the mixed model inhibition ﬁtted the data best (Fig. 2D). The lower

potency in the mouse compared to the rat has also been seen for

2.2. FAAH inhibitory activity

The racemate of compounds TPA1-29, along with the reference

compound Ibu-AM5, were evaluated for their ability to inhibit

FAAH. The inhibition assays were performed using 0.5 m

M [³H]AEA

as substrate and rat brain homogenates as enzyme source. In all

cases reported here, concentration-response curves were con-

structed from values from at least 3 experiments using at least ﬁve

concentrations over the appropriate range (in half log units, i.e. 0.3,

1, 3, 10, 30, 100 mM). The results of these primary assays are showed

in Tables 1, and 3-5. The replacement of the Ibu-AM5 isobutyl chain

with a triﬂuoromethylamino moiety to give TPA1 led to similar

inhibitory activity (IC₅₀values of 0.52 and 0.59 mM, respectively).

Ibu-AM5

and

2-(2-ﬂuoro-(1,1'-biphenyl)-4-yl)-N-(3-

methylpyridin-2-yl)propanamide (Flu-AM1) [12].

2.2.1. Mode of inhibition of FAAH by TPA1

To explore the inhibition mechanism, a preincubation time-

course study was performed on TPA1. If a compound inhibits the

526

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

Scheme 3. Reagents and conditions: (i) MeCN, reﬂux, 2 h; (ii) DMF-DMA, PhMe, reﬂux, 1 h; (iii) NH₄OAc, DMF, reﬂux, 1.5 h; (iv) EtOH, 5 N aq. NaOH, r.t., 24 h; (v) EDC, HOBt, MeCN,

r.t., 36 h.

Scheme 4. Reagents and conditions: (i) MeI, NaH, DMF, 0 ^ꢀC to r.t.; (ii) SnCl₂2H₂O, EtOH, 85 ^ꢀC, 4 h; (iii) MeCN, reﬂux, 2 h; (iv) DMF-DMA, PhMe, reﬂux, 1 h; (v) NH₄OAc, DMF,

reﬂux, 1.5 h; (vi) EtOH, 5 N aq. NaOH, r.t., 24 h; (vii) EDC, HOBt, MeCN, r.t., 36 h.

2.2.2. Binding mode of TPA1

enantiomer for computational studies on TPA1. Docking was per-

formed with Autodock 4.2 targeting all the binding sites reported

for known competitive inhibitors (i.e., the membrane access

channel, the acyl chain binding channel, the catalytic site and the

cytosolic port). The results suggest that TPA1 preferentially binds

the acyl chain binding (ABP) channel in proximity to the catalytic

triad. Four arrangements that differ in the orientation and

Taking into account the competitive inhibition mechanism of

TPA1 for rat FAAH, we studied its binding mode by combining

docking and molecular dynamics (MD) simulations using the pro-

tocol reported previously [12]. Given that (S)-Ibu-AM5 is 10-fold

more potent FAAH inhibitor than (R)-Ibu-AM5, while (S)-Flu-

AM1 and (R)-Flu-AM1 are almost equipotent, we used the (S)-

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

527

Scheme 5. Reagents and conditions: (i) DMF, NaH, di-haloalkane, 0 ^ꢀC to r.t.; (ii) AcOEt, SnCl₂2H₂O, 75 ^ꢀC, 4 h; (iii) MeCN, reﬂux, 2 h; (iv) DMF-DMA, PhMe, reﬂux, 1 h; (v) NH₄OAc,

DMF, reﬂux, 1.5 h; (vi) EtOH, 5 N aq. NaOH, r.t., 24 h; (vii) EDC, HOBt, MeCN, r.t., 36 h.

Scheme 6. Reagents and conditions: (i) EtOH, 5 N aq. NaOH, r.t., 24 h; (ii) EtOH, appropriate 4-chlorotriﬂuoromethylquinoline, reﬂux, 24 h; (iii) EDC, HOBt, MeCN, r.t., 36 h.

positioning of the ligand along the ABP channel were found

(Table 2; Supplementary data Fig. S1), which have the amide

moiety pointing towards the cytosolic port (A1-mode), the catalytic

triad (A2-mode), the membrane access channel (B1-mode), or the

Figs. S2eA), and showed convergence toward a common binding

mode in the two monomers (Figs. S1eB). On the contrary, A2, B1

and B2 binding modes underwent larger rearrangements, showing

less stability and a lower degree of convergence. (RMSD ꢂ 2.5 Å;

Supplementary data Fig. S3).

membrane interacting helices

a18-a19 (B2-mode). Even though

binding modes A1 and B1 are the most populated clusters,

encompassing 50% and 30% of the docked poses, the score of the

four poses differ by less than 0.7 kcal/mol, which impedes to rule

out any of the four arrangements. Therefore, we examined the

structural stability of the four poses by running MD simulations

(100 ns) using the dimeric form of the enzyme with the ligand

loaded in the two monomers, thus allowing to enhance the sta-

tistics of our analysis.

The stability of the binding mode along the trajectory was

examined by monitoring the ligand RMSD proﬁle, and the

convergence of the binding in the monomers was evaluated by

comparing the main clusters. The A1-binding mode was very stable

along the trajectories, (RMSD ꢁ 1.5 Å; Supplementary data

To analyze further the results of the MD reﬁnement, the binding

free energies were estimated using Molecular Mechanics General-

ized Born Surface Area (MM/GBSA) [15], and Solvated Interaction

Energy (SIE) [16], which relies on speciﬁc parameterization of the

PBSA method. The differences in binding afﬁnity consistently sup-

ported the larger stability of the A1-binding mode. Nevertheless,

taking into account that very similar compounds, such as Flu-AM1

and Ibu-AM5, showed binding modes comparable to the B2-mode

[12], we further assessed the interaction energy between TPA1 and

the protein using hybrid quantum mechanical molecular me-

chanics (QM/MM) calculations for each representative binding

mode, which indicated that the A1 binding mode has the most

favored interaction energy (Table 2; Supplementary data Table S1).

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A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

Table 1

In the A1-mode (Fig. 3) TPA1 binds prevalently through hy-

FAAH Inhibitory activity of compounds TPA1-14.

drophobic interactions with the apolar gorge of ABP, supported by

few hydrogen bonds. In particular, the methyl-pyridine moiety is in

the proximity of the catalytic triad, tightly packed against Met¹⁹¹

,

Leu¹⁹², Ile²³⁸, Val²⁷⁰and Leu²⁷⁸. The carbonyl oxygen of the amide

group is near the anionic hole formed by the main chains of resi-

dues Ile²³⁸, Gly²³⁹, Gly²⁴⁰and Ser²⁴¹, while the NH group forms a

stable hydrogen bond with the backbone of Met¹⁹¹. The interactions

of the amide group and of the methyl-pyridine ring required the

adoption of a speciﬁc conformation, which is characterized by a

relative torsion of 48^ꢀ. This arrangement seems to be a key requisite

for FAAH inhibition of this class of compounds. Further apolar in-

teractions of the aromatic groups with lipophilic residues along the

channel, including Trp⁵³¹, Phe⁴³²and Thr⁴⁸⁸and a water-bridged H-

bond of aromatic and the backbone of Leu¹⁹²contribute to stabilize

the binding.

Compound

R

Max inhibition (%) IC₅₀

(

m

M) pIC₅₀s.e.m^a

6.23 0.03

TPA1

100

0.59

TPA2

TPA3

TPA4

TPA5

TPA6

TPA7

68

4

11

4.95 0.08

5.40 0.01

4.92 0.04

5.19 0.04

4.28 0.08

6.13 0.02

Finally, we also compared the TPA1 A1-binding mode with

those experimentally determined for other inhibitors. TPA1 adopts

a binding conformation that matches well the structure of methyl

arachidonyl ﬂuorophosphonate (MAFP; PDB ID: 1MT5), although

this latter compound binds even deeper in the MAC (Supplemen-

tary data Figs. S4eA). A similar behavior is observed from the su-

100

93

4.0

12

3

2

perposition with three compounds of the series of

a-

ketoheterocycle covalent FAAH inhibitors [17e20]. (Supplementary

data Fig. S4-B-D). In particular, the carbonyl moiety of the two

compounds ﬁlls the same position, pointing towards the anionic

hole, while the nitrogen on the pyridine ring of TPA1 overlaps the

nitrogen in the oxazole moiety (Supplementary data Figs. S4eD).

Albeit the A1-binding mode strictly resembles that of substrates, or

86

6.4

52

100

covalent inhibitors such as a-ketoheterocycles, the absence of time-

dependency clearly showed that TPA1 is not hydrolyzed by FAAH

(Fig. 2A), since such hydrolysis would have resulted in a decreasing

potency with increasing preincubation time. In order to verify if

conditions for a nucleophilic attack of Ser217 to the TPA1 amide

group subsisted during the MD, we analyzed the geometrical fea-

tures of the catalytic triad, by monitoring key generic descriptors as

proposed by Palermo et al. [21]. Results demonstrated that during

the whole trajectory key geometrical descriptors do not assume

values suitable for a catalytic attack of the TPA1 amide bond

(Supplementary data Fig. S5).

0.74

TPA8

TPA9

TPA10

100

75

0.99

2.0

6.00 0.03

5.70 0.04

5.37 0.15

The involvement of Thr⁴⁸⁸in the A1-binding mode suggests that

TPA1 binding would be affected by mutating this residue in the

wild-type FAAH. This was investigated in experiments where HeLa

cells transfected expressing wild-type FAAH or FAAH^T488Awere

used (Supplementary data Table S2). For comparative purposes,

(R)-Flu-AM1 was also investigated, since previous data indicated

that the potency of this compound was indeed lower towards the

FAAH^T488Amutant [12]. Even though the results might suggest a

reduction in the inhibitory potency in the mutated enzyme, vari-

ation between transfection batches in the sensitivity to inhibition

by TPA1 and by (R)-Flu-AM1 suggest caution in interpretation of

the data (see Supplementary data).

7

4.3

TPA11

100

0.13

6.87 0.03

TPA12

TPA13

100

0.10

0.33

6.99 0.09

6.48 0.02

2.2.3. SAR of TPA1 analogs

With the aim to explore the SAR of this new series of reversible

FAAH inhibitors, we designed a number of derivatives to gain

insight into the role of different moieties of the lead TPA1. In

particular we explored the effect of modiﬁcations of the 3-

TPA14

100

0.058

7.24 0.02

methylpyridin-2-yl moiety, the methyl group in C-

a to the

carbonyl group, and the 2-(triﬂuoromethyl)pyridine ring.

Effect of the distance between the arylpropanoic acid core

and pyridine ring. The effect of the length of the linker between

the arylpropanoic acid core and pyridine ring was determined

(Table 3). Insertion of a methylene unit (TPA15 and TPA16) signif-

icantly reduced the inhibitory activity as compared to TPA1, as the

a

pI₅₀values (i.e.,-log10(IC₅₀) values) are indicated because the analysis method

returns theseSE values, which are then antilogged to give the IC₅₀values for the

inhibitable component (max inhibition) of the FAAH activity.

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

529

Table 2

Docking and MD reﬁning results and analysis. In cases of non-convergent binding modes in the two monomers, data are reported for the best cluster in the monomer A (a0) and

B (b0). MMGBSA, SIE and QM/MM free energy values are in kcal/mol.

DOCKING

mode

MD analysis^b

cluster

Pop%

AD score

MMGBSA^a

Pop%

D

G

D

SIE

G

DG

MMGBSA

QM/MM^c

A1

A2

50

3

ꢃ9.1

ꢃ8.4

- 59.5

- 49.6

0

97.8

96.2

38.6

66.7

75.1

94.5

48.7

- 58.5

- 48.0

- 48.6

- 45.8

- 48.2

- 42.4

- 47.6

- 10.2

- 9.3

- 9.4

- 8.9

- 9.1

- 8.6

- 9.3

- 75.0

- 57.2

- 64.7

- 53.1

- 52.5

- 63.3

- 59.8

a0

b0

a0

b0

a0

b0

B1

B2

30

9

ꢃ9.0

ꢃ8.7

- 49.2

- 50.7

a

Estimated for the docked pose of the ligand.

b

c

Values determined for a set of 50 snapshots taken on 0.1 ns along the MD trajectory.

The solvation energy term ( Gsol) was calculated by MM/GBSA.

D

Table 3

FAAH Inhibitory activity of compounds TPA15-21.

Compound

R

Max inhibition (%)

100

IC₅₀

23

(m

M)

pIC₅₀s.e.m^a

4.63 0.01

TPA15

TPA16

TPA17

100

32

4.50 0.02

5.62 0.01

2.4

TPA18

TPA19

100

27

17

4.57 0.14

4.76 0.15

65

8

TPA20

TPA21

24 2% inhibition at 100

24 3% inhibition at 100

m

M

a

pI₅₀values (i.e.,-log10(IC₅₀) values) are indicated because the analysis method returns theseSE values, which are then antilogged to give the IC₅₀values for the inhibitable

component (max inhibition) of the FAAH activity.

IC₅₀values increase by about 40-fold. Likewise, introduction of a

piperazine ring had a similar effect (TPA18 and TPA19) or even

eliminated the inhibitory activity (TPA20 and TPA21). In contrast,

insertion of an additional amide unit (TPA17) still reduced the

inhibitory potency, but only by a factor of 4 with respect to TPA1.

Taken together these results indicate an ortho-substituted aromatic

amide as essential for FAAH inhibitory activity.

Furthermore, the data suggest that the linker present in TPA1 is

likely to be optimal for the activity. These results are consistent

with the proposed A1-binding mode, as the methyl pyridine moiety

is well packed in the pocket deﬁned by residues Met¹⁹¹, Leu¹⁹²

,

Ile²³⁸, Val²⁷⁰and Leu²⁷⁸, while the amide bond is involved in polar

interactions with the anionic hole and the backbone of Met¹⁹¹

Accordingly, docking of TPA15 in the FAAH showed that the

.

530

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

Table 4

presence of the 3-methyl substituent served to enhance activity by

a factor of about 20 (TPA4 IC₅₀12.0 M versus 0.59 M for TPA1).

FAAH Inhibitory activity of compounds TPA22-27.

m

Similarly, shifting the methyl group into position 5 (TPA2) or po-

sition 4 (TPA3) reduced the activity by 20- and 6-fold, respectively.

This suggests a key role of the methyl group on the conformational

behavior of TPA1, determining the relative positioning of the

methyl-pyridine ring with respect to the amide bond, besides the

speciﬁc apolar interactions formed by the methyl group with side

chains of Val²⁷⁰and Leu¹⁹². To analyze further the effect of the

methyl group, thermodynamic integration (TI) calculations [23]

were used to calculate the differences in binding free energy be-

tween TPA1 and TPA4. TI is best used in situations where small

changes in structure correlate with relatively substantial changes in

the binding afﬁnity. Therefore, TI was used to elucidate the role of

substitution of the methyl group on the pyridine ring in TPA1 with

the hydrogen group in TPA4. The calculation yielded a free energy

difference of 1.0 kcal/mol, which would indicate a 5.7-fold higher

afﬁnity of TPA1 versus TPA4, in good agreement with the experi-

mental results.

Compound

X

Max inhibition (%)

IC₅₀

(m

M)

pIC₅₀s.e.m^a

TPA22

TPA23

TPA24

CH₂

C(CH₃)₂

100

48

1.8

14

4.31 0.06

5.74 0.06

4.87 0.08

TPA25

TPA26

100

9.1

60

5.04 0.05

4.22 0.13

Effect of modiﬁcation of the pyridinamino moiety. The

importance of the 2-pyridine ring was explored by comparing the

inhibitory activity of TPA4 with its analogues bearing different

heterocyclic rings (Table 1). Substitution of the 2-pyridine moiety

by a 3-pyridine (TPA5) resulted in about 2-fold increased activity

TPA27

30 3% inhibition at100

m

M

a

pI₅₀values (i.e.,-log10(IC₅₀) values) are indicated because the analysis method

returns theseSE values, which are then antilogged to give the IC₅₀values for the

inhibitable component (max inhibition) of the FAAH activity.

(TPA5, IC₅₀6.4 mM versus 12.0 mM of TPA4). Conversely the intro-

duction of a 4-pyridine ring led to the poor FAAH inhibitor TPA6. On

the other hand, replacement of the 2-pyridine ring of TPA4 with

pyrimidine (TPA8) improved the inhibitory potency (IC₅₀of

Table 5

FAAH Inhibitory activity of compounds TPA28 and TPA29.

0.99

mM), while the change by pyrazine (TPA9) and quinoline

(TPA10) led to more modest improvements. Finally, the replace-

ment of pyridine nitrogen of TPA1 with a carbon atom to give the

phenyl analog TPA7 (IC₅₀0.74 mM) produced no signiﬁcant differ-

ence in inhibitory activity. This reinforces the relevance of the 3-

methyl group for FAAH inhibition. Overall, these results are in

agreement with the A1 binding mode, where the pyridine ring is

embedded in a hydrophobic environment.

Effect of the modiﬁcation of the 3-methyl group on the

pyridine ring. Taking into account that the best inhibitory activity

was found for compounds bearing a 3-methyl substituent and the

lipophilic nature of the binding pocket, the effect of replacing the

methyl with other lipophilic groups was explored (Table 1). The

replacement of the methyl group with a halogen atom and a tri-

ﬂuoromethyl group increased inhibitory activity as compared to

TPA1; the triﬂuoromethyl substituted (TPA13) IC₅₀value was

Compound

Y

Max inhibition (%)

100

IC₅₀

27

(m

M)

pIC₅₀s.e.m^a

4.56 0.04

TPA28

TPA29

100

3.1

5.51 0.03

0.33

mM, while those of bromine (TPA11) and iodine (TPA12) de-

rivatives were 0.13

m

M and 0.10 M respectively. The activity

m

remarkably increased in the chlorine analog (TPA14). This com-

pound showed a tenfold enhanced activity as compared to TPA1

(TPA14 IC₅₀0.058

mM versus 0.59 mM of TPA1).

a

Effect of replacing the methyl group on C-

group. Removal of the methyl group (TPA22) is detrimental for the

inhibitory activity (Table 4).

a

to carbonyl

pI₅₀values (i.e.,-log10(IC₅₀) values) are indicated because the analysis method

returns theseSE values, which are then antilogged to give the IC₅₀values for the

inhibitable component (max inhibition) of the FAAH activity.

However, insertion of a second methyl group on the same car-

bon atom (TPA23) slightly reduced the inhibitory activity with

introduction of a methylene forced the methyl pyridine moiety

partially outside the hydrophobic pocket toward the water ﬁlled

cytosolic port (Supplementary data Fig. S6). Therefore, modiﬁca-

tions altering the distance between the aromatic ring and the

carbonyl should result in loss of activity by altering the proper

positioning of this moiety. Moreover, this is consistent with the

previous work that profenamides endowed with three-atom linker

showed high FAAH inhibitory activity [11].

respect to TPA1 (IC₅₀value 1.8

mM). Taken together these results

indicated that the presence of a lipophilic group on C-

a

to the

carbonyl group is necessary for the activity and that the relative

position of methyl and aromatic ring is not a key requisite. A

comparison of substituent effects revealed that the introduction of

3, 4, 5, and 6 atom rings on C-a led to compounds TPA24, TPA25,

TPA26 and TPA 27 showing a progressive reduction in inhibitory

activity correlated to ring size increase.

Effect of replacing 2-(triﬂuoromethyl)pyridine ring. The 8-

triﬂuoromethylquinoline (TPA28) and 7-triﬂuoromethylquinoline

(TPA29) analogs showed respectively 45- and 5- fold reduction in

Role of the 3-methyl group on the pyridine ring. The impor-

tance of the 3-methyl group on pyridine ring was explored by

comparing the inhibitory activity of TPA1 and TPA4 (Table 1).The

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

531

Fig. 2. Mode of inhibition of FAAH by TPA1. Panel A shows the time-dependency for rat. In Panel B, rat homogenates (at 20-fold normal strength) were preincubated with either

vehicle, 0.4, 0.8 or 1.2 M TPA1 for 60 min. Aliquots were then diluted 20-fold and assayed for FAAH activity. These are shown as 0.4 / 0.02, 0.8 / 0.0.04 and 1.2 / 0.0.06.

Concomitantly, TPA1 was added to vehicle-preincubated aliquots to give concentrations of 0.02, 0.04 and 0.06 M (representing free concentrations after a 20- fold dilution), 0.4, 0.8

and 1.2 M ﬁnal concentrations. The panel shows the data as % of corresponding control. Panel C shows the kinetics of the inhibition of rat FAAH. The data was better ﬁtted by a

m

competitive mode of inhibition (K_ivalue ¼ 0.39 0.03

better ﬁtted by a mixed model inhibition (K_i¼ 6.3 1.2

inhibition. All data are means SEM, n ¼ 3e4.

m

M) than by a mixed-type inhibition. Panel D shows the corresponding curves for inhibition of mouse FAAH. The data was

M; /∞; for a non-competitive inhibitor, ¼ 1]) than by a competitive mode of

a

¼ 4.2 1.6 [for a competitive inhibitor,

a

inhibitory activity as compared to TPA1 (Table 5). These results

indicate that the presence of a larger aromatic system is detri-

mental for the activity, and that the presence of the 8-

triﬂuoromethyl group in TPA28, which resembles the 2-

triﬂuoromethyl group of TPA1, is relevant for the activity.

2.2.5. Mode of inhibition of FAAH by TPA14

Compound TPA14 showed no time-dependent improvement of

potency, indicating its reversible mechanism of action (Fig. 5A).

This was conﬁrmed by dilution experiments (Fig. 5B). The kinetics

of inhibition were also investigated on rat and mouse FAAH. In the

rat, TPA14 inhibition of [³H]AEA hydrolysis was better ﬁtted by a

non-competitive inhibition mode than by a mixed one, whereas in

the mouse, TPA14 showed

a mixed-type inhibition of the

hydrolysis.

2.2.4. COX inhibitory activity

Among the novel compounds, TPA1 and the most potent TPA14

were chosen for further investigation. Given the ability of Ibu-AM5

and Flu-AM1 to inhibit COX [11,23], we investigated the ability of

TPA1 and TPA14 to inhibit COX-1 and COX-2 (Fig. 4). Two substrates

were investigated, arachidonic acid (AA, for both COX isoforms) and

2-acylglycerol (2-AG, for COX-2; COX-1 does not catalyze the

cyclooxygenation of this compound). Flurbiprofen was used as

positive control. In contrast to Ibu-AM5 and Flu-AM1, TPA1

showed a very weak COX-1 inhibition and did not inhibit COX-2

cyclooxygenation of either AA or 2-AG. A similar pattern of inhi-

bition was seen with TPA14 on COX-2, although this compound at a

2.2.6. Cross competition experiments

To investigate if TPA1 and TPA14 act in a mutually exclusive

manner or in a cooperative manner, the two compounds were

assayed together. The FAAH activity in the presence of different

concentrations of both compounds was assessed and the data

plotted per se in the left columns of Fig. 6 and as 1/v vs. the con-

centration of one of the compounds in the right columns. When

compounds act as mutually exclusive inhibitors, the lines in the

right columns should be parallel [25].

This is the case for TPA1 and TPA14 in both rat and mouse,

suggesting that the compounds behave as mutually exclusive in-

hibitors. These data indicate that TPA1 and TPA14 act as FAAH in-

hibitors with different inhibition modes, suggesting that these

very high concentration (100 mM) produced a partial inhibition of

COX-1. Thus, the structural modiﬁcations described in the present

paper result in a loss of activity towards COX isoforms.

532

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

Fig. 3. A1-mode of TPA1 reﬁned after 100 ns of MD. A) Hydrogen bond of TPA1 (cyan, in sticks) in A1-mode. B) Apolar interactions in the binding pocket of the FAAH. The ligand

TPA1 is represented in cyan by van der Waals spheres. C) LIGPLOTþ [22] interaction diagram. TPA1 (purple) and protein side chains (orange) are shown in ball-and-stick repre-

sentation. Hydrogen bonds are shown as green dotted lines, spiked arcs represent apolar interactions. (For interpretation of the references to colour in this ﬁgure legend, the reader

is referred to the web version of this article.)

structurally closely related compounds may involve distinct bind-

ing modes with the enzyme, presumably through either partial

overlapping of the binding sites or conformational reshaping of the

binding site.

In order to analyze the molecular basis of these subtle effects,

we used TI calculations, to carry out the alchemical mutation from

methyl (TPA1) to bromine (TPA11), triﬂuoromethyl (TPA13) or

chlorine (TPA14). TI calculations showed that only the tri-

ﬂuoromethyl substitution increased the afﬁnity in the A1-mode

with a triﬂuoromethylamino moiety led to a novel class of FAAH

inhibitors. The N-(heteroaryl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-

yl)amino)phenyl)propanamides were found to act as reversible

inhibitors. The 2-amino-3-methylpyridine amide (TPA1) retained

the FAAH inhibitory potency of Ibu-AM5 and behaved essentially as

competitive inhibitor in the rat enzyme. Docking and MD studies

indicated the acyl chain binding (ABP) channel in proximity to the

catalytic triad is the preferential TPA1 binding site. Structure-

activity relationship studies indicated that the FAAH inhibitory

activity was enhanced by the presence of 3-substituted pyr-

idinamide moiety. The 2-amino-3-chloropyridine amide (TPA14)

was the most potent analogue in this series and behaved essentially

as non-competitive inhibitor in the rat.

(

DDG ¼ ꢃ0.83 kcal/mol), while both bromine and chlorine sub-

stitutions resulted in a slight detrimental effect on the binding free

energy (0.2 and 0.3 kcal/mol, respectively). These latter results

were in contrast with the observed activities of TPA11 and TPA14

(0.13 mM and 0.058 mM, respectively), suggesting that for haloge-

nated derivatives, the binding is different (albeit mutually exclu-

sive) from the A1-mode described for TPA1. The simplest

explanation is that there are two binding modes for the com-

pounds, one competitive and another non-competitive, and that

the compounds show different afﬁnities for the two sites. The

different potencies and different inhibition kinetics determined in

rat and mouse FAAH (Figs. 2 and 5) suggest that the modestly

different structures of these enzymes (Supplementary data Figs. S7

and S8) are sufﬁcient to alter the relative afﬁnities (and hence mode

of inhibition) of the two sites towards the compounds. Moreover,

we found a similar behavior for IbuAM5 and FluAM1, lower po-

tencies in mouse FAAH and different inhibition modes. Further

experiments are required to clarify the molecular mechanism of

inhibition.

4. Experimental section

4.1. Chemistry

4.1.1. General methods

Commercially available solvents and reagents were used

without further puriﬁcation unless otherwise stated. Tetrahydro-

furan (THF) was freshly distilled from sodium/benzophenone. Re-

actions requiring anhydrous conditions were performed in oven-

dried glassware under argon atmosphere. ¹H NMR spectra were

recorded on a Varian Inova 500 spectrometer. The chemical shifts

(d) are reported in part per million downﬁeld from tetramethylsi-

lane (TMS), which was used as internal standard, and the spectra

were recorded in DMSO-d₆. Splitting patterns are designated as

singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m).

Infrared spectra were recorded on a Bruker Vector 22 spectrometer.

The main bands are given in cm^ꢃ1. Melting points (m.p.) were

determined on a Stuart Scientiﬁc Melting point SMP1 apparatus

3. Conclusions

In the present study by replacement of Ibu-AM5 isobutyl chain

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

533

Fig. 4. Effects of A, TPA1 and B, TPA14 upon the cyclooxygenation of 10

tension following addition of enzyme in the presence of the compounds. The concentrations, in

ﬂurbiprofen, used as a positive control). The enzyme isoform and substrate used is given above each panel. COX-1 does not metabolise 2-AG.

m

M arachidonic acid or 2-AG by COX-1 and -2. Shown are means SEM, n ¼ 3e4 for the changes in oxygen

m

M, of the compounds are shown on the right of each panel (F30 refers to 30

mM

and are uncorrected. The purity of tested compounds was deter-

mined by combustion elemental analyses conducted by the

Microanalytical Laboratory of the Chemistry Department of the

University of Ferrara with a Yanagimoto MT-5 CHN recorder

elemental analyzer and all values were within 0.4% of the calculated

values, which indicates >95% purity of the tested compounds.

Analytical thin layer chromatography was performed using

0.25 mm silica gel 60-F plates. Flash chromatography was per-

formed using 200e400 mesh silica gel (Merk KGaA). Unless

otherwise stated, yields refer to chromatography and spectro-

scopically pure materials.

was dissolved in CH₂Cl₂(40 mL) and extracted with a saturated

NaHCO₃aqueous solution (3 ꢄ 35 mL), dried over anhydrous

Na₂SO₄and evaporated under vacuum to give the methyl-2-(4-

aminophenyl)propanoate (2) as orange oil. Yield 75%.¹H NMR

(DMSO-d₆)

d

1.45 (d, J ¼ 7.0 Hz, 3H), 3.60 (s, 3H), 3.75 (s, 2H), 3.80

(m, 1H), 6.65 (d, J ¼ 7.0 Hz, 2H), 7.05 (d, J ¼ 7.0 Hz, 2H). IR (Film)

3458, 3374, 2979, 2951, 1729, 1626 cm^ꢃ1. Anal. Calcd. for C₁₀H₁₃NO₂

(179.21): C, 67.02; H, 7.31; N, 7.82. Found: C, 67.08; H, 7.32; N, 7.85.

4.1.3. Methyl (E)-2-(4-((5,5,5-triﬂuoro-4-oxopent-2-en-2-yl)

amino)phenyl)propanoate (4)

A mixture of methyl ester 2 (1.79 g, 10 mmol), and enol ether 3

(2.52 g, 15 mmol) in anhydrous MeCN (10 mL) was reﬂuxed for 2 h.

After cooling, the formed precipitate was collected by ﬁltration,

washed with di-isopropyl ether (iPr₂O), dried, and used without

further puriﬁcation. Yield 78%. Mp 43e45 ^ꢀC. ¹H NMR (DMSO-d₆)

4.1.2. Methyl 2-(4-aminophenyl)propanoate (2)

A solution of 2-(4-nitrophenyl)propanoic acid (1) (5 g, 26 mmol)

in methanol (MeOH) (25 mL) was treated at room temperature with

37% aqueous hydrochloric acid (HCl) (1 mL) and then reﬂuxed for

4 h. The solvent was removed under vacuum and crude methyl

ester was used for the further step. Iron powder (14 g, 256 mmol)

was suspended in a mixture of MeCN (50 mL) and water (4 mL); the

mixture was treated with 37% HCl (0.1 mL), than reﬂuxed for 1 h.

After cooling at room temperature a solution of crude methyl ester

in MeOH (5 mL) was added dropwise in 30 min and the resulting

solution was reﬂuxed overnight. The hot suspension was ﬁltered on

a celite pad and the ﬁltrate evaporated to afford an orange oil that

d

1.45 (d, J ¼ 5.0 Hz, 3H), 2.24 (s, 3H), 3.70 (s, 3H), 3.98 (m, 1H), 5.74

(s, 1H), 6.94 (d, J ¼ 8.4 Hz, 2H), 7.73 (d, J ¼ 8.4 Hz, 2H), 12.48 (s, 1H).

IR (Nujol) 3376, 2981, 2936, 1729 cm^ꢃ1. Anal. Calcd. for C₁₅H₁₆F₃NO₃

(315.29): C, 57.14; H, 5.12; N, 4.44. Found: C, 57.18; H, 5.13; N, 4.46.

4.1.4. Methyl 2-(4-(((1E,3E)-1-(dimethylamino)-6,6,6-triﬂuoro-5-

oxohexa-1,3-dien-3-yl)amino)phenyl)propanoate (5)

A mixture of 4 (1.58 g, 5 mmol), and DMF-DMA (1.79 g,15 mmol)

534

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

Fig. 5. Mode of inhibition of FAAH by TPA14. Panel A shows the time-dependency for rat. In Panel B, rat homogenates (at 20-fold normal strength) were preincubated with either

vehicle, 1.6, 3.2 or 4.8 M TPA14 for 60 min. Aliquots were then diluted 20-fold and assayed for FAAH activity. These are shown as 1.6 / 0.08 etc. Concomitantly, TPA14 was added to

vehicle-preincubated aliquots to give concentrations of 0.08, 0.16 and 0.24 M (representing free concentrations after a 20- fold dilution), 1.6, 3.2 and 4.8 M ﬁnal concentrations.

The panel shows the data as % of corresponding control. Panel C shows the kinetics of the inhibition of rat FAAH. The data was better ﬁtted by a non-competitive mode of inhibition

(K_ivalue ¼ 0.11 0.0017 M) than by a mixed-type inhibition. Panel D shows the corresponding curves for inhibition of mouse FAAH. The data was better ﬁtted by a mixed model

inhibition (K_i¼ 3.0 0.65 M; 3.2 1.3) than by a competitive mode of inhibition. All data are means SEM, n ¼ 3.

m

a

in anhydrous toluene (20 mL) was reﬂuxed for 1 h, then was

allowed to reach the room temperature and stirred for additional

24 h. The mixture was carefully concentrated in vacuum to give 5.

under vacuum, ice was added to the residue and then acidiﬁed with

aqueous 20% HCl solution until pH 3-4. The formed precipitate was

ﬁltered, washed with water and re-crystallized with EtOH to obtain

the title compound. Yield 69%. Mp 37e39 ^ꢀC. ¹H NMR (DMSO-d₆)

Yield 88%. Oil. ¹H NMR (DMSO-d₆)

d

1.41 (d, J ¼ 6.5 Hz, 3H), 2.75 (s,

3H), 3.10 (s, 3H), 3.60 (s, 3H), 3.85 (m, 1H), 4.98 (d, J ¼ 10.5 Hz, 1H),

5.74 (s, 1H), 7.25 (d, J ¼ 7.5 Hz, 2H), 7.33 (d, J ¼ 7.5 Hz, 2H), 7.91 (d,

J ¼ 10.5 Hz, 1H), 12.76 (s, 1H). IR (Film) 1722, 1671 cm^ꢃ1. Anal. Calcd.

for C₁₈H₂₁F₃N₂O₃(370.37): C, 58.37; H, 5.72; N, 7.56. Found: C,

58.42; H, 5.73; N, 7.53.

d

1.37 (d, J ¼ 7.0 Hz, 3H), 3.67 (q, J ¼ 7.0 Hz, 1H), 7.08 (m, 1H), 7.20

(m, 3H), 7.30 (m, 2H), 8.30 (m, 1H), 9.22 (s, 1H), 12.28 (s, 1H). IR

(Nujol) 3336, 3296, 2485, 1683, 1603 cm^ꢃ1. Anal. Calcd. for

C₁₅H₁₃F₃N₂O₂(320.27): C, 58.07; H, 4.22; N, 9.03. Found: C, 58.12;

H, 4.21; N, 9.06.

4.1.5. Methyl 2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)amino)phenyl)

propanoate (6)

4.1.7. General procedure for the synthesis of amides TPA1-29

A solution of acids 7, 9, 14, 21, 42-45 (1 mmol), EDC (0.19 g,

1.1 mmol) and HOBt (0.13 g, 1 mmol) in anhydrous MeCN (10 mL)

was stirred at r.t. for 30 min, then the appropriate amine (1 mmol)

was added. The mixture was stirred at r.t. for 12 h in the case of

aliphatic amines and 36 h in the case of heteroaromatic and aro-

matic amines. After the solvent was removed under vacuum. The

residue was dissolved in ethyl acetate (AcOEt) (20 mL) and washed

sequentially with brine (2 ꢄ 5 mL), 10% citric acid (2 ꢄ 5 mL),

saturated NaHCO₃aqueous solution (2 ꢄ 5 mL) and water

(2 ꢄ 5 mL). The organic layer was dried over anhydrous Na₂SO₄and

evaporated under vacuum to give the title amides.

To a solution of 5 (0.74 g, 2 mmol) in dry DMF (5 mL), ammo-

nium acetate (0.31 g, 4 mmol) was added and the mixture was

gently reﬂuxed for 1.5 h. The mixture was carefully concentrated in

vacuum, and then ice-water (15 mL) was added. The formed solid

was ﬁltered off, washed with water, air-dried, and then recrystal-

lized with iPr₂O to give 6. Yield 75%. Mp 36e39 ^ꢀC. ¹H NMR (DMSO-

d₆)

d

1.57 (d, J ¼ 6.5 Hz, 3H), 3.92 (m, 1H), 3.98 (s, 3H), 6.50 (m, 2H),

6.75 (m, 2H), 7.02 (m, 2H), 8.48 (m, 1H), 9.69 (s, 1H). IR (Nujol) 3346,

3312, 1737, 1670 cm^ꢃ1. Anal. Calcd. for C₁₆H₁₅F₃N₂O₂(324.30): C,

59.26; H, 4.66; N, 8.64. Found: C, 59.33; H, 4.65; N, 8.67.

4.1.6. 2-(4-((2-(Triﬂuoromethyl)pyridin-4-yl)amino)phenyl)

propanoic acid (7)

To a solution of the ester 6 (0.65 g; 2 mmol) in EtOH (20 mL) 5 N

solution of NaOH (4 mL) and water (4 mL) were added. The

resulting mixture was stirred at r.t. for 24 h. After removing EtOH

4.1.7.1. N-(3-Methylpyridin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-

4-yl)amino)phenyl)propanamide (TPA1). Obtained following the

general procedure by the condensation between 7 and 2-amino-3-

methylpyridine. Yield 79%. M p 109e110 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.15 (d, J ¼ 6.2 Hz, 3H), 1.50 (s, 3H), 3.79 (q, J ¼ 6.2 Hz, 1H),

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

535

Fig. 6. Multiple-inhibitor experiments for the inhibition of A, B rat brain and C, D mouse brain [³H]AEA (0.5

untransformed data and are means SEM, N ¼ 6. The right panels show the Dixon plots of the mean data.

mM) hydrolysis using TPA1 and TPA14. The left panels show the

7.18e7.53 (m, 8H), 8.40 (m, 2H), 9.28 (s, 1H), 10.26 (s, 1H). ¹³C NMR

(DMSO-d₆) 25.3, 26.1, 52.2, 110.0, 113.5, 117.9, 129.1, 136.1, 136.2,

144.4, 145.8, 155.5, 155.8, 157.7, 159.8, 183.4. IR (Nujol) 1674, 1620,

1603 cm^ꢃ1. Anal. Calcd. for C₂₁H₁₉F₃N₄O (400.40): C, 62.99; H, 4.78;

N, 13.99. Found: C, 62.94; H, 4.79; N, 14.02.

4.1.7.4. N-(pyridin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)

amino)phenyl)propanamide (TPA4). Obtained following the general

procedure by the condensation between 7 and 2-aminopyridine.

d

Yield 30%. M p 133e135 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.14 (m, 3H), 4.22

(m, 1H), 7.18e7.84 (m, 10H), 8.39 (m, 1H), 9.34 (s, 1H). ¹³C NMR

(DMSO-d₆) 25.3, 52.2, 109.8, 113.7, 115.8, 117.6, 122.3, 130.0, 136.0,

d

136.2, 145.6, 155.8, 157.9, 159.5, 182.9. IR (Nujol) 3298, 2924, 1668,

1602 cm^ꢃ1. Anal. Calcd. for C₂₀H₁₇F₃N₄O (386.37): C, 62.17; H, 4.43;

N, 14.50. Found: C, 62.22; H, 4.42; N, 14.53.

4.1.7.2. N-(5-methylpyridin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-

4-yl)amino)phenyl)propanamide (TPA2). Obtained following the

general procedure by the condensation between 7 and 2-amino-5-

methylpyridine. Yield 25%. M p 148e150 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.46 (m, 3H), 2.64 (s, 3H), 3.90 (m, 1H), 7.19e7.69 (m, 9H), 8.39 (m,

4.1.7.5. N-(pyridin-3-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)

amino)phenyl)propanamide (TPA5). Obtained following the general

procedure by the condensation between 7 and 3-aminopyridine.

1H), 9.39 (s, 1H). ¹³C NMR (DMSO-d₆)

d

25.3, 27.4, 52.2, 110.0, 113.5,

117.9, 129.1, 136.2, 137.0, 145.9, 153.2, 155.5, 157.7, 158.3, 159.7, 183.2.

IR (Nujol) 1666, 1601 cm^ꢃ1. Anal. Calcd. for C₂₃H₂₂F₃N₅O₂(457.45):

C, 60.39; H, 4.85; N, 15.31. Found: C, 60.33; H, 4.84; N, 15.35.

Yield 44%. M p 156e158 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.44 (m, 3H), 3.99

d 25.3, 52.2, 110.0,

(m, 1H), 7.13e8.33 (m, 11H). ¹³C NMR (DMSO-d₆)

113.6, 117.7, 121.4, 130.0, 135.9, 136.2, 140.9, 141.4, 144.0, 145.7, 158.0,

159.3, 182.7. IR (Nujol) 3274, 2955, 1695, 1603 cm^ꢃ1. Anal. Calcd. for

4.1.7.3. N-(4-Methylpyridin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-

4-yl)amino)phenyl)propanamide (TPA3). Obtained following the

general procedure by the condensation between 7 and 2-amino-4-

C₂₀H₁₇F₃N₄O (386.37): C, 62.17; H, 4.43; N, 14.50. Found: C, 62.12; H,

4.41; N, 14.48.

methylpyridine. Yield 31%. M p 53e55 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.47

(m, 3H), 2.65 (s, 3H), 3.94 (m, 1H), 7.06e7.56 (m, 9H), 8.40 (m, 1H),

9.40 (s, 1H). ¹³C NMR (DMSO-d₆)

25.3, 27.3, 52.1, 110.0, 113.6, 117.9,

4.1.7.6. N-(pyridin-4-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)

amino)phenyl)propanamide (TPA6). Obtained following the general

procedure by the condensation between 7 and 4-aminopyridine.

d

120.3, 136.1, 144.4, 146.0, 155.7, 155.8, 156.2, 158.1, 159.7, 179.8. IR

(Nujol) 2923, 1681, 1601 cm^ꢃ1. Anal. Calcd. for C₂₃H₂₂F₃N₅O₂

(400.40): C, 62.99; H, 4.78; N, 13.99. Found: C, 63.06; H, 4.79; N,

13.95.

Yield 22%. M p 128e130 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.15 (d, J ¼ 5.5 Hz,

3H), 3.61 (q, J ¼ 5.5 Hz, 1H), 7.06 (m, 2H), 7.19 (m, 4H), 7.31 (m, 2H),

7.57 (m, 1H), 8.28 (m, 2H), 9.24 (s, 1H), 10.54 (s, 1H). ¹³C NMR

536

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

(DMSO-d₆)

d

25.3, 52.3, 109.8, 112.3, 113.1, 118.0, 130.0, 134.2, 136.1,

iodopyridine. Yield 33%. mp 210e211 ^ꢀC. ¹H NMR (DMSO-d₆)

d 1.44

141.3, 114.9, 152.4, 155.6, 158.1, 159.1, 181.9. IR (Nujol) 1602,

1515 cm^ꢃ1. Anal. Calcd. for C₂₀H₁₇F₃N₄O (386.37): C, 62.17; H, 4.43;

N, 14.50. Found: C, 62.12; H, 4.44; N, 14.48.

(d, J ¼ 7.0 Hz, 3H), 3.88 (q, J ¼ 7.5 Hz, 1H), 7.05 (m, 2H), 7.18 (m, 1H),

7.19 (d, J ¼ 8.0 Hz, 2H), 7.41 (d, J ¼ 8.5 Hz, 2H), 8.28 (m, 2H), 8.41 (m,

1H), 9.19 (s, 1H), 10.23 (s, 1H). ¹³C NMR (DMSO-d₆)

d 26.2, 52.7, 90.3,

116.0,129.4,136.1,136.5,145.4,145.8,147.2,153.9,155.8,158.1,160.8,

181.3. IR (Nujol) 1667, 1602, 1531 cm^ꢃ1

Anal. Calcd. for

₂₀H₁₆F₃IN₄O (512.27): C, 46.89; H, 3.15; N, 10.94. Found: C, 48.94;

H, 3.16; N, 10.97.

4.1.7.7. N-(o-tolyl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)amino)

phenyl)propanamide (TPA7). Obtained following the general pro-

cedure by the condensation between 7 and 2-methylaniline. Yield

.

C

60%. M p 212e214 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.03 (d, J ¼ 6.0 Hz, 3H),

2.08 (s, 3H), 3.92 (q, J ¼ 7.0 Hz, 1H), 7.07 (m, 2H), 7.13e7.23 (m, 5H),

7.32 (m, 1H), 7.44 (m, 2H), 8.29 (m, 1H), 9.22 (s, 1H), 9.38 (s, 1H). ¹³

NMR (DMSO-d₆) 19.8, 25.3, 52.2, 109.9, 112.0, 117.9, 126.7, 129.8,

4.1.7.13. N-(3-(triﬂuoromethyl)pyridin-2-yl)-2-(4-((2-(tri-

ﬂuoromethyl)pyridin-4-yl)amino)phenyl)propanamide (TPA13).

Obtained following the general procedure by the condensation

C

d

130.4, 136.2, 139.8, 145.8, 155.5, 157.6, 159.7, 180.9. IR (Nujol) 1667,

1604 cm^ꢃ1. Anal. Calcd. for C₂₂H₂₀F₃N₃O (399.40): C, 66.16; H, 5.05;

N, 10.52. Found: C, 66.22; H, 5.03; N, 10.55.

between 7 and 2-amino-3-(triﬂuoromethyl)pyridine. Yield 59%. Mp

188e190 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.42 (d, J ¼ 7.0 Hz, 3H), 3.91 (q,

J ¼ 7.0 Hz, 1H), 7.05 (m, 1H), 7.17 (s, 1H), 7.20 (d, J ¼ 6.5 Hz, 2H), 7.39

(d, J ¼ 7.0 Hz, 2H), 7.53 (m, 1H), 8.20 (m, 1H), 8.28 (m, 1H), 8.72 (m,

4.1.7.8. N-(pyrimidin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)

amino)phenyl)propanamide (TPA8). Obtained following the general

procedure by the condensation between 7 and 2-aminopyrimidine.

1H), 9.20 (s, 1H), 10.28 (s, 1H). ¹³C NMR (DMSO-d₆)

d 26.2, 52.7,

116.8,117.1,120.3,129.2,136.3,136.5,142.9,145.3,145.9,147.2,155.8,

158.0, 160.9, 179.9. IR (Nujol) 1671, 1603 cm^ꢃ1. Anal. Calcd. for

Yield 31%. mp 98e100 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.40 (d, J ¼ 7.0 Hz,

C₂₁H₁₆F ₆N₄O (454.37): C, 55.51; H, 3.55; N, 12.33. Found: C, 55.56;

H, 3.56; N, 12.36.

3H), 4.10 (q, J ¼ 7.0 Hz, 1H), 7.17 (m, 1H), 7.20 (m, 4H), 7.40 (m, 2H),

8.28 (m, 1H), 8.64 (m, 2H), 9.21 (s, 1H), 10.71 (s, 1H). ¹³C NMR

(DMSO-d₆)

d

25.2, 52.2, 109.7, 117.8, 125.9, 127.6, 136.0, 136.2, 144.6,

4.1.7.14. N-(3-Chloropyridin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-

4-yl)amino)phenyl)propanamide (TPA14). Obtained following the

general procedure by the condensation between 7 and 2-amino-3-

chloropyridine. Yield 78%. Mp 148e150 ^ꢀC. ¹H NMR (DMSO-d₆)

146.2, 154.2, 157.7, 158.7, 160.3, 183.0. IR (Nujol) 1701, 1602 cm^ꢃ1

.

Anal. Calcd. for C₁₉H₁₆F₃N₅O (387.36): C, 58.91; H, 4.16; N, 18.08.

Found: C, 58.96; H, 4.15; N, 18.05.

d

1.44 (d, J ¼ 5.5 Hz, 3H), 3.93 (q, J ¼ 5.5 Hz, 1H), 7.07 (m, 1H), 7.21

(m, 3H), 7.32 (m, 1H), 7.41 (m, 2H), 7.96 (m, 1H), 8.29 (m, 1H), 8.38

(m, 1H), 9.22 (s, 1H), 10.32 (s, 1H). ¹³C NMR (DMSO-d₆)

26.2, 52.8,

4.1.7.9. N-(pyrazin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)

amino)phenyl)propanamide (TPA9). Obtained following the general

procedure by the condensation between 7 and 2-aminopyrazine.

d

113.6, 117.8, 129.3, 136.2, 136.4, 145.2, 145.4, 146.0, 154.0, 155.8,

Yield 46%. mp 82e83 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.44 (d, J ¼ 7.0 Hz,

3H), 4.06 (q, J ¼ 7.0 Hz, 1H), 7.07 (m, 1H), 7.21 (m, 3H), 7.32 (m, 1H),

7.43 (m, 2H), 8.32 (m, 2H), 9.22 (s, 1H), 9.34 (s, 1H), 10.95 (s, 1H). ¹³

NMR (DMSO-d₆) 25.3, 52.2, 110.0, 117.9, 129.0, 136.0, 137.5, 144.1,

145.8, 150.5, 155.4, 157.7, 159.9, 183.5. IR (Nujol) 1701, 1602 cm^ꢃ1

156.7, 160.5, 180.3. IR (Nujol) 1670, 1603 cm^ꢃ1. Anal. Calcd. for

C₂₀H₁₆ClF₃N₄O (420.81): C, 57.08; H, 3.83; N, 13.31. Found: C, 57.12;

C

H, 3.84; N, 13.34.

d

.

4.1.7.15. N-(pyridin-2-ylmethyl)-2-(4-((2-(triﬂuoromethyl)pyridin-

4-yl)amino)phenyl)propanamide (TPA15). Obtained following the

Anal. Calcd. for C₁₉H₁₆F₃N₅O (387.36): C, 58.91; H, 4.16; N, 18.08.

Found: C, 58.87; H, 4.17; N, 18.11.

general procedure by the condensation between

7

and 2-

1.49

picolylamine. Yield 71%. Mp 73e75 ^ꢀC. ¹H NMR (DMSO-d₆)

d

4.1.7.10. N-(quinolin-8-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)

amino)phenyl)propanamide (TPA10). Obtained following the gen-

(m, 3H), 3.29 (m, 1H), 4.47 (s, 2H), 7.19e7.49 (m, 7H), 7.83 (m, 1H),

8.41 (m, 1H), 8.59 (m, 1H), 8.76 (m, 1H), 9.39 (s, 1H). ¹³C NMR

eral procedure by the condensation between

aminoquinoline. Yield 69%. mp 185e187 ^ꢀC. ¹H NMR (DMSO-d₆)

1.50 (d, J ¼ 7.0 Hz, 3H), 3.57 (d, J ¼ 7.0 Hz, 1H), 7.15 (d, J ¼ 8.0 Hz,

7

and 8-

(DMSO-d₆) d 25.2, 52.8, 54.0, 109.8, 113.5, 117.9, 120.3, 127.2, 129.0,

135.8, 136.0, 145.8, 146.2, 155.5, 157.7, 158.8, 159.6, 177.7. IR (Nujol)

2924, 1600, 1532 cm^ꢃ1. Anal. Calcd. for C₂₁H₁₉F₃N₄O (400.40): C,

62.99; H, 4.78; N, 13.99. Found: C, 62.93; H, 4.80; N, 13.95.

d

4H), 7.57 (m, 5H), 8.26 (m, 1H), 8.37 (d, J ¼ 8.5 Hz, 1H), 8.63 (d,

J ¼ 8.0 Hz, 1H), 8.85 (s, 1H), 9.21 (s, 1H). ¹³C NMR (DMSO-d₆)

d 25.3,

52.2, 115.2, 117.8, 118.2, 123.4, 128.0, 129.2, 130.1, 136.1, 136.3, 137.2,

138.4, 144.4, 145.8, 150.2, 155.5, 157.6, 159.9, 183.3. IR (Nujol) 1657,

1600, 1523 cm^ꢃ1. Anal. Calcd. for C₂₄H₁₉F₃N₄O (436.43): C, 66.05; H,

4.39; N, 12.84. Found: C, 66.10; H, 4.38; N, 12.81.

4.1.7.16. N-(pyridin-3-ylmethyl)-2-(4-((2-(triﬂuoromethyl)pyridin-

4-yl)amino)phenyl)propanamide (TPA16). Obtained following the

general procedure by the condensation between

picolylamine. Yield 58%. Mp 143e145 ^ꢀC. ¹H NMR (DMSO-d₆)

1.14 (m, 3H), 2.64 (s, 2H), 4.39 (m, 1H), 7.16e7.66 (m, 8H),

8.40e8.71 (m, 3H), 9.36 (s, 1H). ¹³C NMR (DMSO-d₆)

25.2, 52.7,

53.7, 109.9, 113.5, 117.9, 127.2, 130.2, 135.9, 136.1, 145.8, 146.1, 148.2,

155.4, 155.5, 157.5, 159.6, 177.8. IR (Nujol) 2906, 1657, 1601 cm^ꢃ1

7 and 3-

d

4.1.7.11. N-(3-bromopyridin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-

4-yl)amino)phenyl)propanamide (TPA11). Obtained following the

general procedure by the condensation between 7 and 2-amino-3-

bromopyridine. Yield 77%. mp 199e201 ^ꢀC. ¹H NMR (DMSO-d₆)

d

.

Anal. Calcd. for C₂₁H₁₉F₃N₄O (400.40): C, 62.99; H, 4.78; N, 13.99.

Found: C, 62.93; H, 4.77; N, 13.95.

d

1.43 (d, J ¼ 6.5 Hz, 3H), 3.88 (q, J ¼ 6.5 Hz,1H), 7.05 (m, 1H), 7.18 (d,

J ¼ 8.5 Hz, 2H), 7.22 (m, 2H), 7.41 (d, J ¼ 8.0 Hz, 2H), 8.10 (m, 1H),

8.28 (m, 1H), 8.41 (m, 1H), 9.19 (s, 1H), 10.26 (s, 1H). ¹³C NMR

4.1.7.17. N-(2-((3-Methylpyridin-2-yl)amino)-2-oxoethyl)-2-(4-((2-

(triﬂuoromethyl)pyridin-4-yl)amino)phenyl)propanamide (TPA17).

Obtained following the general procedure by the condensation

(DMSO-d₆)

d 26.1, 52.8, 113.3, 115.8, 129.4, 136.0, 136.5, 145.2, 145.6,

146.3, 153.9, 155.8, 158.1, 160.3, 180.1. IR (Nujol) 1670, 1603, 1532,

1462, 1377 cm^ꢃ1. Anal. Calcd. for C₂₀H₁₆BrF ₃N₄O (465.27): C, 51.63;

H, 3.47; N, 12.04. Found: C, 51.58; H, 3.46; N, 12.07.

between

228e230 ^ꢀC. ¹H NMR (DMSO-d₆)

3H), 7.19 (m, 2H), 7.30 (m, 2H), 7.46 (m, 2H), 7.53 (m, 2H), 8.36 (m,

1H), 8.53 (m, 1H). ¹³C NMR (DMSO-d₆)

25.3, 26.2, 49.8, 52.2, 109.8,

9

and 2-amino-3-methylpyridine. Yield 76%. mp

d

1.38 (m, 3H), 2.62 (s, 3H), 3.98 (m

4.1.7.12. N-(3-iodopyridin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyridin-4-

yl)amino)phenyl)propanamide (TPA12). Obtained following the

general procedure by the condensation between 7 and 2-amino-3-

d

113.3, 118.0, 129.1, 136.0, 136.1, 144.6, 145.8, 155.4, 155.7, 157.7, 159.9,

182.8. IR (Nujol) 1658, 1602 cm^ꢃ1. Anal. Calcd. for C₂₃H₂₂F₃N₅O₂

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

537

(457.45): C, 60.39; H, 4.85; N, 15.31. Found: C, 60.35; H, 4.60; N,

N, 14.50. Found: C, 62.22; H, 4.44; N, 11.53.

15.35.

4.1.7.23. 2-Methyl-N-(3-methylpyridin-2-yl)-2-(4-((2-(tri-

ﬂuoromethyl)pyridin-4-yl)amino)phenyl)propanamide (TPA23).

4.1.7.18. 1-(4-(3-Chlorophenyl)piperazin-1-yl)-2-(4-((2-(tri-

ﬂuoromethyl)pyridin-4-yl)amino)phenyl)propan-1-one

Obtained following the general procedure by the condensation

between and 1-(3-chlorophenyl)piperazine. Yield 67%. mp

78e80 ^ꢀC. ¹H NMR (DMSO-d₆)

(TPA18).

Obtained following the general procedure by the condensation

between acid 21 and 2-amino-3-methylpyridine. Yield 61%. Mp

7

219e221 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.60 (s, 6H), 2.06 (s, 3H), 7.09 (s,

1H), 7.23 (m, 4H), 7.46 (s, 2H), 7.64 (s, 1H), 8.24 (s, 1H), 8.30 (s, 1H),

9.24 (s, 1H). ¹³C NMR (DMSO-d₆)

26.1, 28.1, 56.3, 110.0, 113.5, 117.9,

d

1.44 (d, J ¼ 7.5 Hz, 3H), 2.66 (m,1H),

2.99 (m, 1H), 3.18 (m, 2H), 3.59 (m, 4H), 4.16 (q, J ¼ 7.0 Hz, 1H), 6.78

d

(m, 1H), 6.83 (m, 1H), 6.89 (m, 1H), 7.03 (m, 1H), 7.18 (m, 4H), 7.31

129.1, 136.1, 136.2, 144.4, 145.8, 155.5, 155.8, 157.7, 159.8, 187.2. IR

(Nujol) 3290, 1661, 1605 cm^ꢃ1. Anal. Calcd. for C₂₂H₂₁F₃N₄O

(414.42): C, 63.76; H, 5.11; N, 13.52. Found: C, 63.71; H, 5.16; N,

13.57.

(m, 2H), 8.26 (m, 1H), 9.18 (s, 1H). ¹³C NMR (DMSO-d₆)

d 25.6, 48.2,

50.8, 54.5, 110.1, 112.9, 114.1, 117.8, 123.0, 129.0, 132.8, 136.7, 136.4,

144.4, 145.8, 151.3, 155.4, 157.5, 159.8, 183.6. IR (Nujol) 1596,

1519 cm^ꢃ1. Anal. Calcd. for C₂₅H₂₄ClF₃N₄O (488.93): C, 61.41; H,

4.95; N, 11.46. Found: C, 61.47; H, 4.94; N, 11.50.

4.1.7.24. N-(3-Methylpyridin-2-yl)-1-(4-((2-(triﬂuoromethyl)pyr-

idin-4-yl)amino)phenyl)cyclopropane-1-carboxamide

(TPA24).

4.1.7.19. 1-(4-(4-Chlorophenyl)piperazin-1-yl)-2-(4-((2-(tri-

Obtained following the general procedure by the condensation

ﬂuoromethyl)pyridin-4-yl)amino)phenyl)propan-1-one

Obtained following the general procedure by the condensation

between and 1-(4-chlorophenyl)piperazine. Yield 82%. Mp

83e85 ^ꢀC. ¹H NMR (DMSO-d₆)

(TPA19).

between acid 42 and 2-amino-3-methylpyridine. Yield 78%. Mp

209e211 ^ꢀC. ¹H NMR (DMSO-d₆)

d 1.20 (m, 2H), 1.46 (m, 2H), 2.10 (s,

7

3H), 7.18 (m, 1H), 7.21 (m, 1H), 7.24 (m, 3H), 7.48 (m, 2H), 7.63 (m,

d

1.32 (d, J ¼ 6.5 Hz, 3H), 2.66 (m,1H),

1H), 8.19 (s, 1H), 8.30 (m, 1H), 8.79 (s, 1H), 9.28 (s, 1H). ¹³C NMR

2.95 (m, 1H), 3.11 (m, 2H), 3.61 (m, 4H), 4.17 (q, J ¼ 6.5 Hz, 1H), 6.89

(d, J ¼ 8.0 Hz, 2H), 7.03 (m, 1H), 7.19 (m, 5H), 7.30 (d, J ¼ 8.0 Hz, 2H),

(DMSO-d₆) d 19.2, 26.2, 30.9, 110.0, 113.5, 117.9, 121.9, 128.2, 134.7,

136.1, 144.4, 145.8, 153.7, 155.8, 157.7, 159.8, 182.3. IR (Nujol) 3401,

3244, 2924, 1688, 1599 cm^ꢃ1. Anal. Calcd. for C₂₂H₁₉F₃N₄O (412.40):

C, 64.07; H, 4.64; N, 13.59. Found: C, 64.01; H, 4.63; N, 13.55.

8.27 (m, 1H), 9.18 (s, 1H). ¹³C NMR (DMSO-d₆)

d 25.6, 48.2, 50.8,

54.5, 110.1, 117.8, 118.2, 130.3, 129.0, 136.4, 144.4, 145.8, 151.3, 155.4,

157.5, 159.8, 183.5. IR (Nujol) 1645, 1600 cm^ꢃ1. Anal. Calcd. for

C

₂₅H₂₄ClF₃N₄O (488.93): C, 61.41; H, 4.95; N, 11.46. Found: C, 61.35;

4.1.7.25. N-(3-Methylpyridin-2-yl)-1-(4-((2-(triﬂuoromethyl)pyr-

H, 4.93; N, 11.49.

idin-4-yl)amino)phenyl)cyclobutane-1-carboxamide

(TPA25).

Obtained following the general procedure by the condensation

4.1.7.20. 1-(4-(2,3-Dimethylphenyl)piperazin-1-yl)-2-(4-((2-(tri-

between acid 43 and 2-amino-3-methylpyridine. Yield 21%. Mp.

ﬂuoromethyl)pyridin-4-yl)amino)phenyl)propan-1-one

(TPA20).

184e186 ^ꢀC. ¹H NMR (DMSO-d₆)

d 1.83 (m, 2H), 1.98 (m, 4H), 2.88 (s,

Obtained following the general procedure by the condensation

3H), 7.06 (m, 1H), 7.19 (m, 2H), 7.22 (m, 2H), 7.47 (m, 2H), 7.61 (m,

between 7 and 1-(2,3-dimethylphenyl)piperazine. Yield 62%. Mp

1H), 8.21 (s, 1H), 8.30 (m, 1H), 9.22 (s, 1H), 9.70 (s, 1H). ¹³C NMR

84e86 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.33 (d, J ¼ 6.0 Hz, 3H), 2.14 (s, 3H),

(DMSO-d₆) d 17.9, 26.2, 33.8, 54.3, 110.0, 113.5, 117.9, 122.7, 130.2,

2.19 (s, 3H), 2.31 (m, 1H), 2.65 (m, 2H), 2.73 (m, 1H), 3.55 (m, 4H),

4.16 (q, J ¼ 6.0 Hz,1H), 6.74 (d, J ¼ 7.5 Hz,1H), 6.86 (d, J ¼ 7.0 Hz,1H),

6.98 (m, 1H), 7.05 (m, 1H), 7.19 (m, 3H), 7.31 (d, J ¼ 8.0 Hz, 2H), 8.28

136.1, 142.5, 145.8, 153.7, 155.8, 157.7, 159.8, 182.5. IR (Nujol) 3583,

2925, 1652, 1606, 1536 cm^ꢃ1. Anal. Calcd. for C₂₃H₂₁F₃N₄O (426.43):

C, 64.78; H, 4.96; N, 13.14. Found: C, 64.72; H, 4.95; N, 13.17.

(m, 1H), 9.20 (s, 1H). ¹³C NMR (DMSO-d₆)

d 20.2, 25.6, 28.7, 48.2,

50.8, 54.5, 109.8, 110.1, 118.2, 121.3, 122.4, 128.5, 129.0, 136.1, 139.0,

144.4, 145.8, 150.8, 155.4, 157.5, 159.8, 183.2. IR (Nujol) 3281, 2925,

1600 cm^ꢃ1. Anal. Calcd. for C₂₇H₂₉F₃N₄O (482.54): C, 67.20; H, 6.06;

N, 11.61. Found: C, 67.14; H, 6.04; N, 11.65.

4.1.7.26. N-(3-Methylpyridin-2-yl)-1-(4-((2-(triﬂuoromethyl)pyr-

idin-4-yl)amino)phenyl)cyclopentane-1-carboxamide

(TPA26).

Obtained following the general procedure by the condensation

between acid 44 and 2-amino-3-methylpyridine. Yield 27%. Mp

220 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.69 (m, 4H), 1.92 (m, 4H), 2.72 (s, 3H),

7.05 (m, 1H), 7.20 (m, 4H), 7.46 (m, 2H), 7.58 (m, 1H), 8.19 (s, 1H),

8.27 (m, 1H), 9.22 (s, 1H), 9.51 (s, 1H). ¹³C NMR (DMSO-d₆)

25.3,

4.1.7.21. 1-(4-(o-Tolyl)piperazin-1-yl)-2-(4-((2-(triﬂuoromethyl)pyr-

idin-4-yl)amino)phenyl)propan-1-one (TPA21). Obtained following

the general procedure by the condensation between 7 and 1-(2-

methylphenyl)piperazine. Yield 68%. Mp 79e81 ^ꢀC. ¹H NMR

d

26.2, 41.3, 54.2, 109.8, 113.5, 117.8, 122.6, 130.2, 136.0, 142.4, 145.7,

154.0, 155.8, 157.5, 159.7, 181.6. IR (Nujol) 3583, 3201, 2926, 1650,

1607 cm^ꢃ1. Anal. Calcd. for C₂₄H₂₃F₃N₄O (440.46): C, 65.44; H, 5.26;

N, 12.72. Found: C, 65.38; H, 5.24; N, 12.76.

(DMSO-d₆)

d

1.33 (d, J ¼ 6.5 Hz, 3H), 2.22 (s, 3H), 2.36 (m, 1H), 2.69

(m, 2H), 2.78 (m, 1H), 3.64 (m, 4H), 4.16 (q, J ¼ 6.5 Hz, 1H), 6.87 (m,

1H), 6.94 (m, 1H), 7.15 (m, 5H), 7.31 (m, 1H), 7.31 (m, 2H), 8.28 (m,

1H), 9.21 (s, 1H). ¹³C NMR (DMSO-d₆)

d

20.3, 25.6, 48.2, 50.8, 54.5,

4.1.7.27. N-(3-Methylpyridin-2-yl)-1-(4-((2-(triﬂuoromethyl)pyr-

110.1, 118.2, 120.7, 124.9, 128.6, 129.1, 13.4, 135.7, 136.2, 144.4, 145.8,

153.5, 155.5, 157.5, 159.7, 183.2. IR (Nujol) 3280, 1601, 1518,

1462 cm^ꢃ1. Anal. Calcd. for C₂₆H₂₇F₃N₄O (468.51): C, 66.65; H, 5.81;

N, 11.96. Found: C, 66.72; H, 5.82; N, 11.92.

idin-4-yl)amino)phenyl)cyclohexane-1-carboxamide

Obtained following the general procedure by the condensation

(TPA27).

between acid 45 and 2-amino-3-methylpyridine. Yield 55%. Mp.

>250 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.53 (m, 1H), 1.68 (m, 1H), 1.70e1.75

(m, 6H), 1.97 (s, 3H), 2.59 (m, 2H), 7.07e7.24 (m, 5H), 7.49 (m, 2H),

7.62 (m, 1H), 8.20 (s, 1H), 8.27 (m, 1H), 9.45 (s, 1H), 9.47 (s, 1H). ¹³

NMR (DMSO-d₆) 21.3, 26.2, 27.3, 48.6, 109.9, 113.5, 117.9, 122.5,

4.1.7.22. N-(3-Methylpyridin-2-yl)-2-(4-((2-(triﬂuoromethyl)pyr-

idin-4-yl)amino)phenyl)acetamide (TPA22). Obtained following the

general procedure by the condensation between acid 14 and 2-

amino-3-methylpyridine. Yield 78%. mp 138e140 ^ꢀC. ¹H NMR

C

d

130.2, 136.0, 142.3, 145.7, 154.0, 155.8, 157.5, 159.7, 182.0. IR (Nujol)

3583, 3288, 2925, 1645, 1606 cm^ꢃ1. Anal. Calcd. for C₂₅H₂₅F₃N₄O

(454.48): C, 66.07; H, 5.54; N, 12.33. Found: C, 66.13; H, 5.55; N,

12.30.

(DMSO-d₆)

(m, 1H), 8.28 (m, 1H), 9.25 (s, 1H), 10.20 (s, 1H). ¹³C NMR (DMSO-d₆)

25.8, 53.9, 110.0, 113.5, 117.9, 129.1, 136.1, 136.2, 144.4, 145.8, 155.5,

d 2.08 (s, 3H), 3.67 (s, 2H), 7.05 (m,1H), 7.16 (m, 7H), 7.37

d

155.8, 157.7, 159.8, 179.9. IR (Nujol) 1602, 1518, 1462, 1377, 1180,

4.1.7.28. N-(3-Methylpyridin-2-yl)-2-(4-((8-(triﬂuoromethyl)quino-

lin-4-yl)amino)phenyl)propanamide (TPA28). Obtained following

1135 cm^ꢃ1. Anal. Calcd. for C₂₀H₁₇F₃N₄O (368.37): C, 62.17; H, 4.43;

538

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

the general procedure by the condensation between acid 47 and 2-

amino-3-methylpyridine. Yield 78%. Mp 230e232 ^ꢀC. ¹H NMR

Found: C, 55.87; H, 4.67; N, 4.67.

(DMSO-d₆)

d

1.40 (d, J ¼ 6.5 Hz, 3H), 2.02 (s, 3H), 3.97 (q, J ¼ 6.5 Hz,

4.1.11. Methyl 2-(4-(((1E,3E)-1-(dimethylamino)-6,6,6-triﬂuoro-5-

oxohexa-1,3-dien-3-yl)amino)phenyl)acetate (12)

1H), 6.95 (m, 1H), 7.18 (m, 1H), 7.34 (m, 4H), 7.46 (m, 1H), 7.62 (m,

2H), 8.10 (m, 1H), 8.53 (m, 1H), 8.70 (m, 1H), 9.20 (s, 1H), 10.16 (s,

Prepared using the same procedure used for the synthesis of the

1H), 12.31 (s, 1H). ¹³C NMR (DMSO-d₆)

d 25.3, 26.1, 52.2, 110.0, 115.1,

compound 5, starting from ester 11. Yield 95%. Mp 105e106 ^ꢀC (n-

119.1, 124.0, 124.3,128.9,129.1, 129.6, 136.1,136.2,144.4, 153.5,155.1,

hexane). ¹H NMR (DMSO-d₆)

d 2.77 (s, 3H), 3.28 (s, 3H), 3.92 (s, 3H),

155.8, 157.9, 183.2. IR (Nujol) 1663, 1583 cm^ꢃ1. Anal. Calcd. for

4.03 (s, 2H), 5.02 (d, J ¼ 12.8 Hz, 1H), 5.96 (s, 1H), 7.49 (m, 2H), 8.03

C

₂₅H₂₁F₃N₄O (450.45): C, 66.66; H, 4.70; N, 12.44. Found: C, 66.60;

(m, 3H), 12.86 (s, 1H). IR (Nujol) 1724, 1661 cm^ꢃ1. Anal. Calcd. for

H, 4.71; N, 12.47.

C₁₇H₁₉F₃N₂O₃(356.34): C, 57.30; H, 5.37; N, 7.86. Found: C, 57.34; H,

5.36; N, 7.82.

4.1.7.29. N-(3-Methylpyridin-2-yl)-2-(4-((7-(triﬂuoromethyl)quino-

lin-4-yl)amino)phenyl)propanamide (TPA29). Obtained following

the general procedure by the condensation between acid 48 and 2-

amino-3-methylpyridine. Yield 83%. Mp 112e114 ^ꢀC. ¹H NMR

4.1.12. Methyl 2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)amino)

phenyl)acetate (13)

Prepared using the same procedure used for the synthesis of

(DMSO-d₆)

d

1.45 (d, J ¼ 6.5 Hz, 3H), 2.01 (s, 3H), 3.96 (q, J ¼ 6.5 Hz,

compound 6, starting from ester 12. Yield 90%. Mp 48e50 ^ꢀC (n-

1H), 6.98 (m, 1H),7.35 (m, 4H), 7.47 (m, 1H), 7.80 (m, 2H), 8.56 (m,

hexane).¹H NMR (DMSO-d₆)

d 3.58 (s, 2H), 3.93 (s, 3H), 6.93 (m, 1H),

1H), 8.63 (m, 2H) 9.19 (m, 1H), 10.17 (s, 1H). ¹³C NMR (DMSO-d₆)

7.18 (m, 2H), 7.59 (m, 2H), 7.72 (m, 1H), 8.48 (m, 1H), 9.70 (s, 1H). IR

(Nujol) 3362, 3205, 1717 cm^ꢃ1. Anal. Calcd. for C₁₅H₁₃F₃N₂O₂

(310.27): C, 58.07; H, 4.22; N, 9.03. Found: C, 58.03; H, 4.23; N, 9.06.

d

25.3, 26.1, 52.2, 110.0, 116.8, 121.9, 122.8, 123.1, 124.3, 129.1, 130.2,

133.5, 136.1, 136.2, 144.4, 150.2, 153.5, 154.3, 155.8, 183.2. IR (Nujol)

1671, 1587 cm^ꢃ1. Anal. Calcd. for C₂₅H₂₁F₃N₄O (450.45): C, 66.66; H,

4.70; N, 12.44. Found: C, 66.71; H, 4.72; N, 12.40.

4.1.13. 2-(4-((2-(Triﬂuoromethyl)pyridin-4-yl)amino)phenyl)acetic

acid (14)

4.1.8. Ethyl 2-(2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)amino)

phenyl)propanamido)acetate (8)

Prepared using the same procedure used for the synthesis of 7,

starting from ester 13. Yield 84%. Mp 219e220 ^ꢀC$¹H NMR (DMSO-

The acid 7 (0.62 g, 2 mmol), EDC (0.39 g, 2.2 mmol) and HOBt

(0.27 g, 2 mmol) were dissolved in MeCN (10 mL). The mixture was

stirred at r.t. for 30 min, then TEA (0.4 mL, 4 mmol) and ethyl gli-

cinate hydrochloride (0.56 g, 4 mmol) were added. The mixture was

stirred at r.t. for 4 h. After the solvent was removed under vacuum.

The residue was dissolved in AcOEt (20 mL) and washed sequen-

tially with brine (2 ꢄ 5 mL), 10% citric acid (2 ꢄ 5 mL), saturated

NaHCO₃aqueous solution (2 ꢄ 5 mL) and water (2 ꢄ 5 mL). The

organic layer was dried over anhydrous Na₂SO₄and evaporated

under vacuum. The residue was treated with iPr₂O; the precipitate

was then ﬁltrated and puriﬁed by recrystallization from 2-propanol

d₆)

d

3.67 (s, 2H), 7.19 (m, 1H), 7.31 (m, 3H), 7.40 (d, J ¼ 8.4 Hz, 2H),

8.40 (d, J ¼ 5.8 Hz, 1H), 9.33 (s, 1H). IR (Nujol) 3325, 2495, 1898,

1693 cm^ꢃ1. Anal. Calcd. for C₁₄H₁₁F₃N₂O₂(296.24): C, 56.76; H, 3.74;

N, 9.46. Found: C, 56.71; H, 3.73; N, 9.48.

4.1.14. Methyl 2-methyl-2-(4-nitrophenyl)propanoate (16)

Methyl ester 15 (1.56 g, 8 mmol) was dissolved in anhydrous

DMF (20 mL). The solution was cooled to 0 ^ꢀC, then NaH (0.63 g,

16 mmol, 60% dispersion in oil) was added slowly and cautiously.

The resulting mixture was allowed to warm to r.t. and then stirred

for additional 20 min. The solution was cooled again to 0 ^ꢀC.

Iodomethane (2 mL, 32 mmol) was added dropwise, and the

resulting solution was allowed to stir at 0 ^ꢀC. for about 30 min. The

solution was warmed to r.t. and stirred for additional 1 h. The so-

lution was cooled to 0 ^ꢀC and quenched with water, maintaining

0 ^ꢀC throughout the quenching process. The obtained mixture was

extracted with dichloromethane (CH₂Cl₂) (2 ꢄ 15 mL). The com-

bined organic layers were washed with water (2 ꢄ 5 mL) and brine

(2 ꢄ 5 mL); dried over anhydrous Na₂SO₄. After removing the

Na₂SO₄by ﬁltration, the ﬁltrate was concentrated under vacuum.

The residue was puriﬁed by column chromatography (silica gel, 8:1

petroleum ether: AcOEt) and gave the title compound as a yellow

solid material. Yield 80%. Mp. 33e35 ^ꢀC. lit. 45e46 ^ꢀC [26]. ¹H NMR

(2-PrOH). Yield 80%. Mp 90e91 ^ꢀC. ¹H NMR (DMSO-d₆)

d 1.27 (t,

J ¼ 7.2 Hz, 3H), 1.39 (d, J ¼ 6.8 Hz, 3H), 3.79 (m, 3H), 4.15 (q,

J ¼ 7.2 Hz, 2H), 7.07 (m, 1H), 7.20 (m, 3H), 7.36 (m, 2H), 8.30 (m, 2H),

10.02 (s, 1H),. IR (Nujol) 1724,1636, 1605, 1592 cm^ꢃ1. Anal. Calcd. for

C

₁₉H₂₀F₃N₃O₃(395.38): C, 57.72; H, 5.10; N, 10.63. Found: C, 57.77;

H, 5.11; N, 10.60.

4.1.9. (2-(4-((2-(Triﬂuoromethyl)pyridin-4-yl)amino)phenyl)

propanoyl)glycine (9)

The ester 8 (0.40 g, 1 mmol) was dissolved in EtOH (10 mL). To

this solution a 5 N aqueous NaOH (2 mL) and water (2 mL) were

added. The resulting mixture was stirred at r.t. for 24 h. After

removing EtOH under vacuum, ice and then aqueous 20% HCl so-

lution until pH 3-4 were added. The formed precipitate was ﬁl-

trated, washed with water and re-crystallized with EtOH. Yield 94%.

(DMSO-d₆)

d

1.69 (s, 6H), 3.74 (s, 3H), 7.45 (d, J ¼ 8.5 Hz, 2H), 8.13 (d,

J ¼ 8.5 Hz, 2H). Anal. Calcd. for C₁₁H₁₃NO₄(223.22): C, 59.19; H,

5.87; N, 6.27. Found: C, 59.24; H, 5.86; N, 6.30.

Mp 229e231 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.33 (d, J ¼ 7.5, 3H), 3.72 (m,

3H), 7.04 (m, 1H), 7.17 (m, 3H), 7.34 (m, 2H), 8.28 (m, 2H), 9.19 (s,

1H), 12.46 (s, 1H). IR (Nujol) 3350, 3281, 2854, 2522, 1900, 1707,

1657,1606 cm^ꢃ1. Anal. Calcd. for C₁₇H₁₆F₃N₃O₃(367.32): C, 55.59; H,

4.39; N, 11.44. Found: C, 55.54; H, 4.38; N, 11.47.

4.1.15. Methyl 2-(4-aminophenyl)-2-methylpropanoate (17)

To a solution of ester 16 (1.34 g, 6 mmol) in EtOH (15 mL) tin (II)

chloride dihydrate (5.5 g, 24 mmol) was added. The resulting so-

lution was stirred at 85 ^ꢀC for 4 h. After cooling, the pH was

adjusted to 10 with 40% aqueous NaOH. The mixture was extracted

with AcOEt (3 ꢄ 5 mL). The combined organic layers were washed

with water (2 x 5 mL) and brine (2 ꢄ 5 mL) and dried over Na₂SO₄.

After removing of the Na₂SO₄the ﬁltrate was concentrated under

vacuum to obtain the title compound as yellow oil. Yield 87%. ¹H

4.1.10. Methyl (E)-2-(4-((5,5,5-triﬂuoro-4-oxopent-2-en-2-yl)

amino)phenyl)acetate (11)

Was prepared using the same procedure used for the synthesis

of the compound 4, starting from methyl 2-(4-aminophenyl)ace-

tate 10. Yield 96%. Mp 164e165 ^ꢀC. ¹H NMR (DMSO-d₆)

d

2.24 (s,

NMR (DMSO-d₆) d 0.63 (s, 3H), 0.91 (s, 3H), 3.54 (s, 3H), 4.96 (s, 2H),

3H), 3.54 (s, 2H), 3.98 (s, 3H), 5.82 (s, 1H), 7.34 (d, J ¼ 7.6 Hz, 2H),

6.49 (d, J ¼ 8.5 Hz, 2H), 6.91 (d, J ¼ 8.5 Hz, 2H). IR (Film) 3435, 3349,

2956, 1723, 1626 cm^ꢃ1. Anal. Calcd. for C₁₁H₁₅NO₂(193.24): C,

68.37; H, 7.82; N, 7.25. Found: C, 68.31; H, 7.85; N, 7.23.

7.81 (d, J ¼ 7.6 Hz, 2H), 12.52 (s, 1H). IR (Nujol) 3260, 1717 cm^ꢃ1

.

Anal. Calcd. for C₁₄H₁₄F₃NO₃(301.26): C, 55.82; H, 4.68; N, 4.65.

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

539

4.1.16. Methyl (E)-2-methyl-2-(4-((5,5,5-triﬂuoro-4-oxopent-2-en-

2-yl)amino)phenyl)propanoate (18)

added with 5 N solution of NaOH (4 mL) and water (4 mL). The

resulting mixture was stirred at r.t. for 20 h. After removing EtOH

under vacuum ice was added and then aqueous 20% HCl solution

until pH 3-4. The formed precipitate was ﬁltered and washed with

water and dried.

Prepared using the same procedure used for the synthesis of the

compound 4, starting from ester 17. Yield 87%.Oil. ¹H NMR (DMSO-

d₆)

d 1.53 (s, 6H), 2.27 (s, 3H), 3.77 (s, 3H), 5.77 (s, 1H), 7.01 (d,

J ¼ 8.5 Hz, 2H), 7.77 (d, J ¼ 8.5 Hz, 2H), 12.58 (s, 1H). IR (Film) 2925,

1721, 1571 cm^ꢃ1. Anal. Calcd. for C₁₆H₁₈F₃NO₃(329.31): C, 58.35; H,

5.51; N, 4.25. Found: C, 58.30; H, 5.52; N, 4.27.

4.1.20.1. 1-(4-((2-(Triﬂuoromethyl)pyridin-4-yl)amino)phenyl)cyclo-

propane-1-carboxylic acid (42). Prepared using the general pro-

cedure starting from dienaminone 34. Yield 59%. Mp 208e210 ^ꢀC.

4.1.17. 2-Methyl-2-(4-((2-(triﬂuoromethyl)pyridin-4-yl)amino)

phenyl)propanoic acid (21)

¹H NMR (DMSO-d₆)

d 1.13 (m, 2H), 1.45 (m, 2H), 7.10 (m, 1H), 7.16

(m, 2H), 7.23 (m, 1H), 7.33 (m, 2H), 8.29 (s, 1H), 9.34 (s, 1H). IR

(Nujol) 3285, 2924, 1677, 1602 cm^ꢃ1. Anal. Calcd. for C₁₆H₁₃F₃N₂O₂

(322.28): C, 59.63; H, 4.07; N, 8.69. Found: C, 59.68; H, 4.06; N, 8.71.

A mixture of 18 (1.65 g, 5 mmol) and DMF-DMA (1.79 g,

15 mmol) in anhydrous toluene (20 mL) was reﬂuxed for 1 h, then

allowed to reach the room temperature and stirred for additional

24 h. The mixture was carefully concentrated in vacuum. Without

isolate the dienaminone intermediate (19) the residue was dis-

solved in dry DMF (5 mL), ammonium acetate (0.31 g, 4 mmol) was

added and the mixture was gently heated for 1.5 h. The mixture was

carefully concentrated in vacuum, and then ice-water (15 mL) was

added. The formed solid was ﬁltered off and washed with water.

The obtained triﬂuoromethylpyridine ester (20) was dissolved in

EtOH (20 mL), 5 N solution of NaOH (4 mL) and water (4 mL) were

added. The resulting mixture was stirred at r.t. for 24 h. After

removing EtOH under vacuum, the residue was ice added and the

resulting mixture acidiﬁed with aqueous 20% HCl solution until pH

3-4. The formed precipitate was ﬁltered and washed with water.

4.1.20.2. 1-(4-((2-(Triﬂuoromethyl)pyridin-4-yl)amino)phenyl)cyclo-

butane-1-carboxylic acid (43). Prepared using the general proced-

ure starting from dienaminone 35. Yield 78%. Mp 184e186 ^ꢀC. ¹H

NMR (DMSO-d₆)

d 1.75 (m, 1H), 1.96 (m, 1H), 2.38 (m, 2H), 2.70 (m,

2H), 7.08 (m,1H), 7.21 (m, 3H), 7.30 (m, 2H), 8.28 (s,1H), 9.26 (s,1H).

IR (Nujol) 3319, 2855, 1671, 1622, 1606 cm^ꢃ1. Anal. Calcd. for

C₁₇H₁₅F₃N₂O₂(336.31): C, 60.71; H, 4.50; N, 8.33. Found: C, 60.77;

H, 4.48; N, 8.37.

4.1.20.3. 1-(4-((2-(Triﬂuoromethyl)pyridin-4-yl)amino)phenyl)cyclo-

pentane-1-carboxylic acid (44). Prepared using the general pro-

cedure starting from dienaminone 36. Yield 86%.Mp > 250 ^ꢀC. ¹H

Yield 42%. Mp 173e175 ^ꢀC. ¹H NMR (DMSO-d₆)

d

1.48 (s, 6H), 7.02

NMR (DMSO-d₆) d 1.65 (m, 4H), 1.79 (m, 4H), 7.07 (m, 1H), 7.18 (m,

(m, 1H), 7.17 (m, 3H), 7.52 (m, 2H), 8.28 (m, 1H), 9.25 (s, 1H). IR

3H), 7.34 (m, 2H), 8.27 (s, 1H), 9.27 (s, 1H), 12.24 (s, 1H). IR (Nujol)

3301, 2921, 1691, 1618, 1601 cm^ꢃ1. Anal. Calcd. for C₁₈H₁₇F₃N₂O₂

(350.33): C, 61.71; H, 4.89; N, 8.00. Found: C, 61.65; H, 4.88; N, 8.04.

(Nujol) 3285, 2924, 1690, 1623, 1602 cm^ꢃ1. Anal. Calcd. for

C

₁₆H₁₅F₃N₂O₂(324.30): C, 59.26; H, 4.66; N, 8.64. Found: C, 59.19;

H, 4.64; N, 8.67.

4.1.20.4. 1-(4-((2-(Triﬂuoromethyl)pyridin-4-yl)amino)phenyl)cyclo-

hexane-1-carboxylic acid (45). Prepared using the general proced-

ure starting from dienaminone 37. Yield 88%. Mp > 250 ^ꢀC. ¹H NMR

4.1.18. General procedure for the synthesis of esters 22-25

Compound 15 (2.50 g, 12.8 mmol) was dissolved in anhydrous

DMF (20 mL). The solution was cooled to 0 ^ꢀC. NaH (1.02 g,

25.6 mmol, 60% dispersion in oil) was added slowly and cautiously.

The resulting mixture was allowed to warm to r.t. and was stirred

for additional 20 min. The solution was cooled again to 0 ^ꢀC The

appropriate dihaloalkane (25.6 mmol) was added dropwise, and

the resulting solution was allowed to stir at 0 ^ꢀC for 30 min. The

solution was warmed to r.t. and stirred for additional 1 h. The so-

lution was cooled to 0 ^ꢀC and quenched with water, maintaining

0 ^ꢀC throughout the quenching process. The obtained mixture was

extracted with CH₂Cl₂(2 ꢄ 20 mL). The combined organic layers

were washed with water and brine and dried over Na₂SO₄. This was

removed by ﬁltration and the ﬁltrate concentrated under vacuum

to give the crude esters 22-25 (see supplementary data for physical

and spectral data).

(DMSO-d₆)

d 1.22 (m, 1H), 1.43 (m, 1H), 1.59 (m, 6H), 2.64 (m, 2H),

7.07 (m, 1H), 7.18 (m, 3H), 7.39 (m, 2H), 8.27 (s, 1H), 9.28 (s, 1H). IR

(Nujol) 3305, 1687, 1622, 1603, 1523, 1459, 1364, 1320, 1229,

1178 cm^ꢃ1. Anal. Calcd. for C₁₉H₁₉F₃N₂O₂(364.36): C, 62.63; H, 5.26;

N, 7.69. Found: C, 62.60; H, 5.30; N, 7.72.

4.1.20.5. 2-(4-Aminophenyl)propanoic acid (46). To a solution of the

ester 2 (1.79 g, 10 mmol) in EtOH (50 mL) 5 N solution of NaOH

(20 mL) and water (20 mL) were added. The resulting mixture was

stirred at r.t. for 24 h. After removing EtOH under vacuum to the

resulting solution ice was added and then 20% aqueous HCl solution

until pH 3-4. The formed precipitate was ﬁltered, washed with

water, dried and re-crystallized from EtOH. Yield 97%. Mp

123e125 ^ꢀC. ¹H NMR (DMSO-d₆)

J ¼ 7.0 Hz, 1H), 6.48 (d, J ¼ 8.5 Hz, 2H), 6.90 (d, J ¼ 8.5 Hz, 2H). IR

(Nujol) 2854, 2610, 2188,1632,1591 cm^ꢃ1. Anal. Calcd. for C₉H₁₁NO₂

(165.19): C, 65.44; H, 6.71; N, 8.48. Found: C, 65.48; H, 6.68; N, 8.52.

d

1.26 (d, J ¼ 7.0 Hz, 3H), 3.43 (q,

4.1.19. General procedure for the synthesis of esters 26-29

To a solution of ester 22-25 (9.78 mmol) in AcOEt (230 mL)

stannous chloride hydrate (110 g, 48.9 mmol was added). The re-

action mixture was stirred at 75 ^ꢀC for 4 h. Reaction mixture was

cooled at r.t and then diluted with AcOEt (100 mL) and 25% aqueous

ammonia solution until pH 10 was added. The organic layer was

separated out, dried over Na₂SO₄and the solvent evaporated under

vacuum to obtain the desired compound 26-29 (see supplementary

data for physical and spectral data).

4.1.21. General procedure for the synthesis of acids 47-48

A solution of acid 46 (0.34 g, 2 mmol) in EtOH (40 mL) was

added with the appropriate 4-chloroquinoline (2 mmol) and

reﬂuxed for 24 h. Then the solvent was removed under vacuum and

the formed solid was ﬁltered, treated with iPr₂O, ﬁltered and then

dried.

4.1.20. General procedure for the synthesis of acids 42e45

4.1.21.1. 2-(4-((8-(Triﬂuoromethyl)quinolin-4-yl)amino)phenyl)prop-

To a solution of dienaminone 34-37 (2 mmol) in dry DMF (5 mL),

ammonium acetate (0.308 g, 4 mmol) was added and the mixture

was gently reﬂuxed for 1.5 h. The mixture was carefully concen-

trated in vacuum, and then ice-water (15 mL) was added. The

formed solid was dissolved in EtOH (20 mL), this solution was

anoic

triﬂuoromethylquinoline. Yield 89%. Mp 158e160 ^ꢀC. ¹H NMR

(DMSO-d₆) 1.42 (m, 3H), 4.09 (m, 1H), 6.93 (m, 1H), 7.45 (m, 4H),

acid

(47). Obtained

starting

from

4-chloro-8-

d

7.87 (m, 1H), 8.38 (m, 1H), 8.46 (m, 1H), 9.08 (m, 1H). IR (Nujol)

2926,1726, 1619,1589 cm^ꢃ1. Anal. Calcd. for C₁₉H₁₅F₃N₂O₂(360.33):

540

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

C, 63.33; H, 4.20; N, 7.77. Found: C, 63.38; H, 4.19; N, 7.80.

mixed-type or non-competitive inhibition in the GraphPad Prism

programme, and the best ﬁt determined using Akaike's Informative

Criteria. Using the methodology reported here, carprofen has been

4.1.21.2. 2-(4-((7-(Triﬂuoromethyl)quinolin-4-yl)amino)phenyl)

propanoic acid (48). Obtained starting from 4-chloro-7-

triﬂuoromethylquinoline. Yield 87%. Mp 163e165 ^ꢀC. ¹H NMR

shown to inhibit rat brain AEA (0.5

value of 32 M [24]. Arachidonoylserotonin inhibited 2

hydrolysis with an IC₅₀value of 1.2 M [29]. URB597 is an irre-

versible inhibitor, and thus its potency will be dependent upon the

preincubation time used. In our hands, the compound inhibits 2

mM) hydrolysis with an IC₅₀

m

mM AEA

(DMSO-d₆)

7.49 (m, 2H), 8.12 (m, 1H), 8.45 (m, 1H), 8.60 (m, 1H), 9.03 (m, 1H),

11.16, (s, 1H), 12.40 (s, 1H, OH). IR (Nujol) 2960, 1720, 1598 cm^ꢃ1

d

1.41 (m, 3H), 3.80 (m, 1H), 6.90 (m, 1H), 7.44 (m, 2H),

m

.

mM

Anal. Calcd. for C₁₉H₁₅F₃N₂O₂(360.33): C, 63.33; H, 4.20; N, 7.77.

Found: C, 63.30; H, 4.23; N, 7.74.

AEA hydrolysis following a 10 min preincubation period with IC₅₀

values of 64 and 18 nM at assay pH values of 6 and 8, respectively

[30].These values are consistent with the literature using other

assays [31e33].

4.2. Biological assays

4.2.1. Preparation of rat, mouse brain and HeLa transfected cells

homogenates

4.2.3. COX assays

The assay of Meade et al. [34] with minor modiﬁcations [35] was

Brains (minus cerebella) from adult Wistar or Sprague-Dawley

rats (killed by decapitation) and from male B6CBAF1/J mice (kil-

led by cervical dislocation), stored at ꢃ80 ^ꢀC, were thawed, weighed

and homogenized in cold buffer (20 mM HEPES, 1 mM MgCl₂pH

7.0). Homogenates were centrifuged (35,000 g at 4 ^ꢀC for 20 min)

before the pellet was resuspended in cold homogenization buffer.

Centrifugation and resuspension was repeated twice. The suspen-

sion was incubated at 37 ^ꢀC for 15 min to degrade any endogenous

substrate able to interfere with the FAAH assay. After centrifugation

(35,000 g at 4 ^ꢀC for 20 min), the pellet was resuspended in cold

buffer (50 mM Tris-HCl, 1 mM EDTA, 3 mM MgCl₂, pH 7.4). The

protein concentration was determined according to reported

method [27] after which the samples were frozen in aliquots

at ꢃ80 ^ꢀC. The homogenates from HeLa cells transfected with FAAH

wt and FAAH T488A were performed as previously reported [12].

used. Brieﬂy, buffer containing 1

EDTA, substrate (arachidonic acid or 2-AG, 10

m

M haematin, 2 mM phenol, 5 mM

mM ﬁnal concentra-

tion) and 0.1 M TriseHCl, pH 7.4 at room temperature was added to

an oxygen electrode chamber with an integral stirring unit (Oxy-

graph System, Hansatech Instruments, King'sLynn, U.K.). The ﬁnal

assay volume was 2 mL, and the oxygen electrode was calibrated

with respect to air pressure and r.t. After addition of 20 mL test

compound dissolved in EtOH, a baseline was established over a

period of 5 min. Reactions were started by addition of COX (ovine

COX-1 or human recombinant COX-2, as appropriate, 200U per

assay), and the oxygen consumption was measured in 10 s blocks.

Data are presented as the change in oxygen consumption (

the point of addition of the enzyme.

mM) from

4.3. Molecular docking

4.2.2. Assay of [³H]AEA hydrolysis in the homogenates

Rigid docking calculations were performed using the software

Autodock 4.2 [36]. The 3D structures of compounds were built with

Pymol 1.74. Docking was performed on a single monomer of rat

FAAH (PDB code 3QK5) [37], after crystallized ligand and water

removal. Lys¹⁴²was considered in the basic form according to the

proposed catalytic mechanism [38]. Rigid docking in the competi-

tive binding site was performed using a cubic docking box of 60³

grid points, centered on the monomer A of rat FAAH active site. The

rotatable bonds of the compound were automatically detected by

Autodock default settings. For each docking run,100 iterations were

performed using default parameters of Lamarckian genetic algo-

rithm (GALS). The results were clustered on the basis of RMSD

criterion (ꢁ4 Å). The energetically favored poses of the best clus-

ters, according to Autodock 4.2 score, were loaded in the two

monomers of the dimeric form of rat FAAH and each system were

used for further analysis.

The assays were performed with minor modiﬁcations of our

previously described method [28]. Brieﬂy, homogenates in assay

buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) were mixed with test

TPA dissolved in ethanol or DMSO on ice. Hydrolysis reactions were

initiated by addition of substrate ([³H]AEA in 10 mM Tris-HCl, 1 mM

EDTA, pH 7.4 containing 10 mg/mL fatty acid-free bovine serum,

assay concentration 0.5

tion at 37 ^ꢀC. For the brain homogenates, hydrolysis at 37 ^ꢀC was

allowed to proceed for 10 min (assay volume was 200 L, with 1

and 0.3 g/assay protein concentrations of the rat and mouse ho-

mM unless otherwise stated) and incuba-

m

mogenates, respectively). For the transfected cells, a 2 h incubation

was used. For the kinetic experiments, the AEA: fatty acid-free

bovine serum albumin ratio was kept constant at 1: ~4.5 (

Following incubation, reactions were stopped by addition of 400

activated charcoal (80 L 0.5 M HCl)

L activated charcoal þ 320

and placement of samples on ice before centrifugation at 2500 rpm

for 10 min. Aliquots (200 L) of the supernatant were analyzed for

mM).

mL

m

4.4. Molecular dynamics

tritium content by liquid scintillation spectroscopy with quench

correction. Sample sizes were chosen on the basis of previous data

investigating FAAH inhibitory properties of compounds. IC₅₀values

were calculated by ﬁtting the data (as % of control) to the algorithm

log (inhibitor) vs. response e Variable slope (four parameters) built

into the GraphPad prism programme (versions 5-7 for the Macin-

tosh; GraphPad Software Inc., San Diego, CA, USA). The equation

used is Y¼Bottom þ (Top-Bottom)/(1 þ 10^((LogIC50-X)*Hill-

Slope)). Two constraints were used: one where the top (uninhib-

ited) value was set to 100 and the bottom (maximum inhibition)

value was set to 0 and one where only the top value was con-

strained. The curve ﬁts were then compared using Akaike's Infor-

mative Criteria. When the best ﬁt had a bottom value > 0, this is

shown in the results as “maximum inhibition”, where the value

given is 100- the calculated bottom value. For the kinetic experi-

ments, the data was analysed using algorithms for competitive,

MD simulations were performed to reﬁne best docking results

with Amber15 [39]. Each complex was immersed in a pre-

equilibrated octahedral box of TIP3P water molecules, and the

system was neutralized. The ﬁnal system contained about 80000

atoms. All simulations were performed with the Amber 99SBildn

force ﬁeld [40] for the protein and the gaff force ﬁeld [39] for the

ligands. The charge distribution of the inhibitors was reﬁned using

RESP charges [41] ﬁtted to the B3LYP/6-31G(d) electrostatic po-

tential obtained with Gaussian09 [42]. For each complex the ge-

ometry was minimized using convergence criterion for the energy

gradient set to 0.01 kcal/mol$Å²in three steps, which involve: i)

hydrogen atoms in the system (5000 steps of steepest descent and

10000 steps of conjugate gradient), ii) hydrogen atoms, water

molecules and counterions (2000 steps of steepest descent and

18000 steps of conjugate gradient), iii) ﬁnally the whole system

A. Deplano et al. / European Journal of Medicinal Chemistry 136 (2017) 523e542

541

(2000 steps of steepest descent and 18000 steps of conjugate

1

Z

gradient). Thermalization of the system was performed in four

steps of 60 ps, increasing the temperature from 50 to 298 K.

Concomitantly, the atoms that deﬁne the protein backbone were

restrained during thermalization using a variable restraining force.

Thus, a force constant of 30 kcal/mol$Å²was used in the ﬁrst stage

of the thermalization and was subsequently decreased by in-

crements of 5 kcal/mol$Å²in the next stages. Then, an additional

step of 250 ps was performed in order to equilibrate the system

density at constant pressure (1 bar) and temperature (298 K).

Finally, an extended trajectory was run using a time step of 2 fs.

SHAKE was used for those bonds containing hydrogen atoms in

conjunction with periodic boundary conditions at constant pres-

sure and temperature, particle mesh Ewald for the treatment of

long range electrostatic interactions, and a cutoff of 9 Å for non-

bonded interactions. The structural analysis was performed using

in-house software and standard codes of AmberTools 15.

vVð

l

Þ

DG ¼

dl

0

4.8. Homology building

The amino acid sequence of mouse FAAH was retrieved from the

Universal Protein Resource database (http://www.uniprot.org

accession ID O08914). The 3D structure of the target protein was

modeled using SWISSMODEL [46]. The X-ray determined structure

of rat FAAH 3QKV was used as template (covered sequence 100%;

sequence identity of 91.6%). The global model quality estimation

yielded a score of 0.92.

Authors will release the atomic coordinates upon publication.

Acknowledgments

4.5. Free energy calculations

This work was supported by grants from the Regione Autonoma

della Sardegna (Project L.R. 7/2007, no. 2012_CRP-59473) to VO; the

Swedish Research Council (Grant no. 12158, medicine) and the

Research Funds of the Medical Faculty, Umeå University to CJF; the

Generalitat de Catalunya (2014SGR1189), ICREA Academia, and

Consorci de Serveis Universitaris de Catalunya to FJL. CMM and BC

Analysis of the MD trajectory was carried out by calculating

Molecular Mechanics Generalized Born Surface Area (MM/GBSA)

[15], and Solvated Interaction Energy (SIE) [16], on a 0.1 ns interval

at the end of the trajectories. We used open source AmberTools

package (MMPBSA.py) [43] to perform binding free energy

calculations.

acknowledge the CINECA (ISCRA

C projects ID HP10C34LJ7,

HP10CPFIRC and HP10CSJ2H8) for the access to high performance

computing resources.

4.6. QM/MM calculations

Appendix B. Supplementary data

The contribution due to the formation of the ligand-protein

complex in the gas phase was determined by QM/MM calcula-

tions. To this end, the ligand was treated at the QM level and all the

residues within 15 Å of the ligand were treated classically.

Accordingly, the electrostatic term accounts for the QM interaction

(determined at the B3LYP/6-311 þ G(d,p) level) of the ligand with

the set of point charges of the residues included in the MM region.

QM/MM calculations were performed with Gaussian09 [42] for a

set of 50 snapshots taken on a 0.1 ns interval along the last 5 ns of

the trajectories, averaging the electrostatic and van der Waals

components of the individual snapshots. The van der Waals term

was determined using the 6-12 expression as implemented in

AMBER. The contribution due to the solvation of the complexes

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2017.05.033.

These data include additional ﬁgures and tables illustrating: i)

computational data, ii) alignment and superimposition of mouse

and rat FAAH, iii) general synthetic procedure, physical and spectral

data for compounds 22-37, and 42-45, iv) FAAH inhibitory activity

in HeLa cell homogenates expressing wild type and mutant FAAH v)

Molecular Formula Strings Spreadsheet.

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Article Doi

Novel propanamides as fatty acid amide hydrolase inhibitors

DOI: 10.1016/j.ejmech.2017.05.033

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Full text of DOI:10.1016/j.ejmech.2017.05.033

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