Design, synthesis and biological evaluation of second-generation benzoylpiperidine derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors

European Journal of Medicinal Chemistry 209 (2021) 1 12857

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European Journal of Medicinal Chemistry

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

Research paper

Design, synthesis and biological evaluation of second-generation

benzoylpiperidine derivatives as reversible monoacylglycerol lipase

(MAGL) inhibitors

,

h

Carlotta Granchi ^a, Giulia Bononi ^a, Rebecca Ferrisi ^a, Eleonora Gori ^a, Giulia Mantini ^g

,

Sandra Glasmacher ^b, Giulio Poli ^a, Stefano Palazzolo ^c, Isabella Caligiuri ^c,

Flavio Rizzolio ^c^d, Vincenzo Canzonieri ^{c e}, Tiziana Perin ^c, Jürg Gertsch ^b, Andrea Sodi ^f,

,

, *

Elisa Giovannetti ^g^h, Marco Macchia ^a, Filippo Minutolo ^a, Tiziano Tuccinardi ^a

,

Andrea Chicca ^b

^aDepartment of Pharmacy, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy

^bInstitute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, CH-3012, Bern, Switzerland

^cPathology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, 33081, Aviano, Italy

^dDepartment of Molecular Sciences and Nanosystems, Ca’ Foscari University, 30123, Venezia, Italy

e

ꢀ

Department of Medical, Surgical and Health Sciences, Universita Degli Studi di Trieste, Strada di Fiume 447, Trieste, Italy

^fDepartment of Neurosciences, Psychology, Drug Research and Child Health Eye Clinic, University of Florence, AOU Careggi, 50139, Florence, Italy

^gDepartment of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, DeBoelelaan 1117, 1081HV, Amsterdam, the Netherlands

^hCancer Pharmacology Lab, Fondazione Pisana per La Scienza, Via Giovannini 13, 56017, San Giuliano Terme, Pisa, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

An interesting enzyme of the endocannabinoid system is monoacylglycerol lipase (MAGL). This enzyme,

which metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG), has attracted great interest due

to its involvement in several physiological and pathological processes, such as cancer progression.

Experimental evidences highlighted some drawbacks associated with the use of irreversible MAGL in-

hibitors in vivo, therefore the research ﬁeld concerning reversible inhibitors is rapidly growing. In the

present manuscript, the class of benzoylpiperidine-based MAGL inhibitors was further expanded and

optimized. Enzymatic assays identiﬁed some compounds in the low nanomolar range and steered mo-

lecular dynamics simulations predicted the dissociation itinerary of one of the best compounds from the

enzyme, conﬁrming the observed structure-activity relationship. Biological evaluation, including assays

in intact U937 cells and competitive activity-based protein proﬁling experiments in mouse brain

membranes, conﬁrmed the selectivity of the selected compounds for MAGL versus other components of

the endocannabinoid system. An antiproliferative ability in a panel of cancer cell lines highlighted their

potential as potential anticancer agents. Future studies on the potential use of these compounds in the

clinical setting are also supported by the inhibition of cell growth observed both in cancer organoids

derived from high grade serous ovarian cancer patients and in pancreatic ductal adenocarcinoma pri-

mary cells, which showed genetic and histological features very similar to the primary tumors.

Received 31 August 2020

Received in revised form

16 September 2020

Accepted 17 September 2020

Keywords:

Monoacylglycerol lipase inhibitors

MAGL

Benzoylpiperidine derivatives

1. Introduction

Abbreviations: MAGL, monoacylglycerol lipase; GPCRs, G protein-coupled re-

ceptors; ECS, endocannabinoid system; eCBs, endocannabinoids; AEA, anandamide;

The endocannabinoid system (ECS) is composed of two seven-

transmembrane protein-coupled receptors (GPCRs) named

2-AG, 2-arachidonoylglycerol; FAAH, fatty acid amide hydrolase; ABHD6,

a/b hy-

G

drolase-6; ABHD12, hydrolase-12; MD, molecular dynamics; 2-OG, 2-oleoyl

a/b

cannabinoid receptors type-1 and type-2 (CB1R, CB2R), a family of

lipophilic molecules that binds to CB receptors called endocanna-

binoids (eCBs) and several biosynthetic and degrading enzymes

involved in the production and metabolism of eCBs. CB1R is one of

glycerol; 4-NPA, 4-nitrophenylacetate; 4-NP, 4-nitrophenol; GAFF, General Amber

force ﬁeld; SMD, steered molecular dynamics; ABPP, activity-based protein

proﬁling; PDTXs, primary patient-derived tumor xenografts.

* Corresponding author.

E-mail address: tiziano.tuccinardi@unipi.it (T. Tuccinardi).

https://doi.org/10.1016/j.ejmech.2020.1 12857

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

the most abundant GPCRs in mammalian brain and it is highly

expressed in neuronal cells, where it regulates neurotransmitter

release. CB2R is prevalently expressed in immune cells, where it

controls the activation state during inﬂammation. Anandamide

(AEA) and 2-arachidonoylglycerol (2-AG) are the most important

eCBs and are biosynthesized on-demand from phospholipid pre-

cursors in the inner leaﬂet of the plasma membrane and released

into the extracellular environment [1]. After activating CB re-

ceptors, eCBs are transported into the cytoplasm via facilitated

diffusion mediated by a putative eCB membrane transporter. [2,3]

Intracellular eCBs degradation is mediated by fatty acid amide hy-

drolase (FAAH) for AEA, and by monoacylglycerol lipase (MAGL)

enzyme, and usually permanently block its catalytic activity; 2)

reversible inhibitors, which interact with MAGL for a limited time,

and then the activity of the protein is restored. Irreversible in-

hibitors are usually highly potent, with inhibition activities in the

low nanomolar range; however, they manifested some drawbacks

when tested in in vivo studies. Fig. 1 shows three representative

irreversible MAGL inhibitors, i.e. derivative

1 (JZL184, 4-

nitrophenyl-4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperi-

dine-1-carboxylate, Fig. 1) [8], 2 (CAY10499, benzyl(4-(5-methoxy-

2-oxo-1,3,4-oxadiazol-3(2H)-yl)-2-methylphenyl)carbamate, Fig .1)

[9], and 3 (ABX-1431, 1,1,1,3,3,3-Hexaﬂuoropropan-2-yl-4-[[2-(pyr-

rolidin-1-yl)-4-(triﬂuoromethyl)phenyl]methyl]piperazine-1-

carboxylate) [10]. In detail, genetic deletion and irreversible MAGL

inhibition determine the loss of CB1R-mediated biological effects

and induce cross-tolerance to exogenous CB1R agonists [1 1e15].

These effects are provoked by prolonged pharmacological blockage

or genetic inactivation of MAGL, which leads to an excessive in-

crease of 2-AG concentration (5-10-times over basal levels), which

triggers CB1R desensitization. Moreover, MAGL-deﬁcient mice are

characterized by an impaired CB1R-dependent synaptic plasticity

and physical dependence [1 1]. These aspects may represent a

serious limitation for the therapeutic use of most irreversible MAGL

inhibitors, especially for chronic treatments. In the last years, MAGL

inhibitors endowed with a reversible mode of action gained more

interest in the scientiﬁc community as potential alternative strat-

egy to modulate MAGL. Initially, only natural compounds, such as

the terpenoid Euphol 4 (Fig. 1), a potent MAGL inhibitor with an

and a/b hydrolase-6 and -12 (ABHD6 and ABHD12) for 2-AG. MAGL

is the main hydrolytic enzyme for 2-AG and it is responsible for

approximately 85% of 2-AG hydrolysis in the brain, with a minor

contribution of ABHD6 and ABHD12. Modulation of eCB levels

represents a promising pharmacological strategy to activate the

ECS without the typical side effects associated with direct CB1R

agonists [4]. Several studies indicate the therapeutic potential of

selective eCB reuptake inhibitors, as well as of selective FAAH and

MAGL inhibitors in different animal disease models of inﬂamma-

tion, pain, anxiety and other neuroinﬂammatory diseases [3,5,6].

In the last decades, many academic research groups and phar-

maceutical industries have focused their research on the discovery

of new MAGL inhibitors, and some of the most representative in-

hibitors are reported in Fig. 1 [6,7]. MAGL inhibitors reported in the

literature can be classiﬁed on the basis of their mechanism of ac-

tion: 1) irreversible inhibitors, which bind covalently to the

IC₅₀value in the nanomolar range [16], and the triterpenoid

b-

Fig. 1. Structures of some of the most representative MAGL inhibitors.

2

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

amyrin 5 (Fig. 1) [1 7], less potent than Euphol, were identiﬁed as

reversible MAGL inhibitors. However, they are not selective for

MAGL: Euphol was active on other targets, affecting the cell cycle of

chemical class. The structural optimization was focused mainly on

the phenolic ring (in blue, Fig. 2) as well as on the benzoyl moiety

(in red, Fig. 2). All the newly synthesized compounds were

designed keeping in mind that the meta-hydroxyl group on the

amidic phenolic ring should not be changed, since it proved to be

fundamental for the inhibition activity of the enzyme, establishing

a strategic hydrogen bond network with two active site residues,

E53 and H272 [23]. As the ﬁrst modiﬁcation we added a second

ﬂuorine atom on the phenolic ring of 11, maintaining the other part

of the molecule ﬁxed, thus obtaining compound 12 (Fig. 3). This

choice was determined by considering that the presence of a

ﬂuorine atom in para to the phenolic hydroxyl group (as in com-

pound 11, IC₅₀¼ 80 nM) and in para position to the amide carbonyl

group (the analogue compound of 11 bearing the ﬂuorine atom in

this position showed an IC₅₀value of 110 nM) [24], proved to be

fundamental for the improvement of the enzyme inhibition po-

tency. As the second modiﬁcation, the ﬂuorine atom of compound

11 was substituted by other groups. Considering that modeling

studies suggested that this part of the molecule is located in a small

polar pocket of the enzyme, normally hosting the glycerol portion

of the substrate 2-AG, the groups suitable to be introduced in this

position should not be too bulky, to maintain the same binding

disposition of the parent molecule [23]. The groups were chosen

among those characterized by electron-withdrawing properties,

such as triﬂuoromethyl and nitro groups, as in compounds 13a and

13b (Fig. 3) or electron-donating properties, such as amine and

methyl groups, as in compounds 13c and 13d (Fig. 3), to determine

if a variation of the electronic density on the amidic part could

inﬂuence the activity of the enzyme. The last modiﬁcation con-

cerning the phenolic ring was the introduction of hydroxy-pyridine

rings, in which the relative position between the pyridine nitrogen

and the hydroxyl group is diversiﬁed, thus originating compounds

14e17 (Fig. 3). In the second part of our structural optimization, the

benzoyl moiety of the reference compound 11 was varied, although

the modiﬁcations concerning this part of the scaffold were intro-

duced maintaining the phenolic ring of the amide moiety, but

without the ﬂuorine atom of compound 11. This choice was guided

by the easy accessibility and the low cost of the 3-methoxybenzoic

acid, used for the synthesis of the compounds bearing the 3-

hydroxy-phenyl substitution pattern, compared to the 2-ﬂuoro-5-

cancer cells [18], whereas

b-amyrin inhibited ABHDs too [1 7].

ꢁ

Hernandez-Torres et al. discovered the ﬁrst synthetic and selective

MAGL

inhibitor,

benzo[d]

[1,3]dioxol-5-ylmethyl

6-

phenylhexanoate 6 (Fig. 1): this nanomolar inhibitor was able to

slow down the clinical progression and to decrease the symptoms

of multiple sclerosis in an experimental autoimmune encephalo-

myelitis mouse model, without inducing unwanted CB1-mediated

side effects [19]. Tabrizi et al. disclosed the 1,5-diphenylpyrazole-

3-carboxamide 7 (Fig. 1): this reversible MAGL inhibitor mitigated

the neuropathic hypersensitivity induced in vivo by oxaliplatin [20].

A series of long-chain salicylketoxime derivatives was developed as

MAGL inhibitors and, among them, 8 (Fig. 1) was the most active

compound of this series. Compound 8 proved to be selective for

MAGL over other targets of the ECS and to exert antiproliferative

activity in a series of cancer cells [21]. Very recently, Takeda Phar-

maceuticals identiﬁed a series of piperazinyl pyrrolidin-2-ones,

exempliﬁed by compound 9 (Fig. 1) [22], which showed high po-

tency in vitro (IC₅₀value in the subnanomolar range). Some de-

rivatives of this class showed promising efﬁcacy in vivo, decreasing

arachidonic acid level and increasing 2-AG level in mouse brain.

Compound 10 (Fig. 1) belongs to the class of the benzoylpiperidine-

based MAGL inhibitors [23e25], developed by our group since

2014. This compound derives from an optimization process of this

scaffold, possessing a phenolic amidic moiety and a 4-isopropyl-

benzoyl ring, and these portions are both necessary to reach a

nanomolar afﬁnity for MAGL.

2. Results and discussion

2.1. Design

In the last years, a series of benzoylpiperidine-based compounds

was identiﬁed and developed as reversible and selective MAGL

inhibitors, reaching inhibition values in the nanomolar range. In the

present manuscript, the most potent inhibitor so far discovered

within this series of compounds (11, Fig. 2) [24] was further opti-

mized, to ﬁnd more potent MAGL inhibitors belonging to this

Fig. 2. Design of new benzoylpiperidine derivatives. Previously published reference compound 11 was schematically represented as located in the MAGL binding site. The planned

modiﬁcations of the benzoyl part and of the amidic moiety are represented in red and blue, respectively. (For interpretation of the references to color in this ﬁgure legend, the reader

is referred to the Web version of this article.)

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C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

Fig. 3. New series of benzoylpiperidine derivatives. Upper panel (A): newly synthesized compounds with different phenolic moieties (12, 13a-d, 14e17). Lower panel (B): newly

synthesized compounds bearing new substituents in the benzoyl portion (18, 19, 20a-i).

methoxy benzoic acid necessary for the synthesis of compound 11.

The isopropyl group of compound 11 demonstrated to be beneﬁcial

for the inhibition potency, however, we considered the possibility

to introduce bulkier and lipophilic moieties in this position, since

modeling studies revealed that the benzoyl part extends in the

wide lipophilic channel pointing outwards, in which the long un-

saturated chain of 2-AG accommodates. A ﬁrst modiﬁcation

involved the replacement of the isopropyl group by some cycloalkyl

rings, such as cyclohexyl or cyclopropyl rings (compounds 18 and

19, Fig. 3). Moreover, we considered that the previously obtained

analogue of 11, where the isopropyl group was replaced by a second

phenyl ring, showed a moderately good activity (IC₅₀value of

460 nM) [23], therefore we hypothesized that a more ﬂexible

portion could be better accommodated into the lipophilic channel

of the protein. Therefore, the second phenyl ring was connected to

the benzoyl moiety by means of a linker, which could be oxygen or

sulphur atoms (compounds 20b and 20c, respectively, Fig. 3), NH,

sulfone, sulfoxide or methylene groups (compounds 20a, 20d, 20e

and 20f, respectively, Fig. 3). In addition, in some cases the triazole

ring was used as the spacer, alone or in combination with a short

alkyl chain (1 or 3 carbon atoms), thus obtaining compounds 20g-i

(Fig. 3). Finally, the inhibition potencies shown by the ﬁrst two

groups of new MAGL inhibitors (panel A and panel B of Fig. 3)

prompted us to investigate the combinations of chemical modiﬁ-

cations on both sides of the benzoylpiperidine scaffold (compounds

21a,b and 22a,b, Fig. 4). In particular, the newly optimized com-

pounds possess a ﬂuorine atom in para to the phenolic hydroxyl

group (compounds 21a,b), as in compound 11, or the simultaneous

presence of two ﬂuorine atoms in para and in ortho positions to the

phenolic OH, as in compound 12, together with the presence of a

sulphur atom or a methylene group as linkers between the benzoyl

ring and the second phenyl, as in compounds 20c and 20f.

2.2. Chemistry

The synthesis of compounds 12, 13a and 13b started from an

amide condensation between amine 23 [24] and the corresponding

commercially available substituted benzoic acids, which are 2,4-

diﬂuoro-5-methoxybenzoic

acid

24,

5-methoxy-2-(tri-

ﬂuoromethyl)benzoic acid 26 and 5-methoxy-2-nitrobenzoic acid

28, as shown in Scheme 1. The amide formation was performed in

the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-

triazolo[4,5-b]pyridinium 3-oxide hexaﬂuorophosphate (HATU) as

the condensing agent, DIPEA as the base and dry N,N-dime-

thylformamide as the solvent, as previously reported [24]. Finally,

the methoxy groups of these intermediates were deprotected by

boron tribromide in dichloromethane to yield the ﬁnal hydroxy-

substituted compounds.

For the preparation of amino- and methyl-substituted de-

rivatives 13c and 13d, the corresponding methoxylated benzoic

acids bearing the proper moiety in position para to the methoxy

group were not commercially available, therefore they were pre-

pared starting from appropriate precursors. In order to obtain

compound 32, 5-methoxy-2-nitrobenzoic acid 28 was converted to

methyl ester 30, to facilitate the isolation and puriﬁcation proced-

ures of the subsequent intermediates. Nitro group of compound 30

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European Journal of Medicinal Chemistry 209 (2021) 112857

Fig. 4. New series of benzoylpiperidine derivatives, deriving from the combinations of compounds 11 and 20c, f (compounds 21a,b) and of compounds 12 and 20c,f (compounds

22a,b).

Scheme 1. Synthesis of compounds 12, 13a and 13b. Reagents and conditions: (a) HATU, DIPEA, dry DMF, RT, 2e3 h [59e84%]; (b) 1 M BBr₃, dry CH₂Cl₂, ꢀ10 to 0 ^ꢁC, then RT, 1.5e4 h

[6e60%].

was reduced to an amine by using stannous chloride dihydrate in a

mixture of chloroform and methanol as the solvent. Then, the

methyl ester of compound 31 was hydrolyzed under basic condi-

tions to obtain 2-amino-5-methoxybenzoic acid 32 (Scheme 2). For

the synthesis of compound 36, commercially available 5-hydroxy-

2-methylbenzoic acid 34 was subjected to a reaction with methyl

iodide by using potassium carbonate in DMF and in these

conditions both the phenolic and the carboxylic acid were meth-

ylated. The methyl ester of intermediate 35 was hydrolyzed to

generate the free carboxylic group of 5-methoxy-2-methylbenzoic

acid 36 (Scheme 2). At this point, the previously adopted reaction

conditions were followed, which consisted in the amide conden-

sation with amine 23 and the subsequent BBr₃-promoted depro-

tection of intermediates 33 and 27 to obtain ﬁnal compounds 13c

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European Journal of Medicinal Chemistry 209 (2021) 112857

Scheme 2. Synthesis of compounds 13c and 13d. Reagents and conditions: (a) HATU, DIPEA, dry DMF, RT, 1.5e2 h [54e70%]; (b) 1 M BBr₃, dry CH₂Cl₂, ꢀ10 to 0 ^ꢁC, then RT, 1e6.5 h

[48e59%]; (c) MeI, K₂CO₃, DMF, RT, 4e5 h [85e91%]; (d) SnCl^.₂2H₂O, CHCl₃/MeOH, 65 ^ꢁC, 1 h [85%]; (e) aq. 2 N LiOH, THF/MeOH 1:1 v/v, RT, overnight [77e99%].

and 13d, respectively (Scheme 2).

Compounds 18 and 19 bearing cycloalkyl rings, such as cyclo-

propyl or cyclohexyl groups, on the benzoyl ring were prepared

starting from the same precursor, acetylated isonipecotic acid 42

[24]. The synthetic pathway is reported in Scheme 4: common in-

termediate 42 was reﬂuxed with thionyl chloride in dichloroethane

to convert it to the corresponding acyl chloride and then reacted in

a Friedel-Crafts reaction with phenylcyclohexane in the presence of

aluminum chloride in dichloroethane to obtain compound 47, or

with bromobenzene under the same conditions to give compound

43. For the introduction of the cyclopropyl ring, a palladium

The synthesis of hydroxy-pyridine derivatives 14e17 was per-

formed in one step (Scheme 3), since in these cases the use of

commercially available hydroxy-substituted pyridine-carboxylic

acids was preferred to their methoxylated counterparts. Therefore,

amine 23 was condensed with 4-hydroxypyridine-2-carboxylic

acid 38, 5-hydroxynicotinic acid 39, 2-hydroxypyridine-4-

carboxylic acid 40 or 6-hydroxypyridine-2-carboxylic acid 41 to

obtain ﬁnal compounds 14, 15, 16 or 17, respectively, in moderate

yields (Scheme 3).

Scheme 3. Synthesis of compounds 14e17. Reagents and conditions: (a) HATU, DIPEA, dry DMF, RT, 3e4 h [50e57%].

6

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European Journal of Medicinal Chemistry 209 (2021) 112857

Scheme 4. Synthesis of compounds 18e19. Reagents and conditions: (a) i. SOCl₂, dry 1,2-DCE, 60 ^ꢁC, 4 h; ii. bromobenzene (for 43) or phenylcyclohexane (for 47), AlCl₃, dry 1,2-DCE,

90 ^ꢁC, overnight [40e64%]; (b) cyclopropylboronic acid, Pd(OAc)₂, Cy₃P 20% toluene, K₃PO₄, toluene, 100 ^ꢁC, 24 h [92%]; (c) NaOH 1 N, EtOH, 90 ^ꢁC, overnight [81e86%]; (d) 3-

methoxybenzoic acid, HATU, DIPEA, dry DMF, RT, 3 h [68e75%]; (e) 1 M BBr₃, dry CH₂Cl₂, ꢀ10 to 0 ^ꢁC, then RT, 2 h [28e59%].

catalyzed cross-coupling reaction with cyclopropylboronic acid,

using the catalytic system comprised of palladium acetate, together

with tricyclohexylphosphine as the catalyst ligand and potassium

phosphate as the base, allowed the insertion of the desired group,

thus giving compound 44. After the different functionalization of

the benzoyl ring, hydrolysis under aqueous basic conditions and

heating removed the acetyl group of the piperidine nitrogen, thus

obtaining amines 45 and 48, which were then submitted to amidic

condensation with 3-methoxybenzoic acid to obtain amides 46 and

49. The ﬁnal demethylation step afforded compounds 18 and 19.

Compounds 20a and 20b, bearing a benzoyl moiety with two

phenyl rings connected through a nitrogen (20a) or an oxygen atom

(20b), were prepared starting from the previously described

bromo-substituted intermediate 43 (Scheme 5). Two different

palladium-catalyzed cross-coupling reactions under suitable reac-

tion conditions gave the desired compounds (Scheme 5): a) with

aniline, using tris(dibenzylideneacetone)dipalladium as the cata-

lyst, XPhos as the ligand and potassium tert-butoxide as the base, in

toluene, for compound 55 or b) with phenol, using palladium ac-

etate as the catalyst, tetramethyl di-tert-butylXPhos as the ligand

and potassium phosphate as the base, in toluene, for compound 56.

Compounds 55 and 56 were then subjected to the same sequence of

reactions, consisting in basic aqueous hydrolysis of the N-acetyl

group of the piperidine, amidic condensation with 3-

methoxybenozic acid and, ﬁnally, deprotection of the methoxy

group to produce phenolic derivatives 20a-b. Compounds 20f, 21b

and 22b have in common the methylene group as the linker be-

tween the two phenyl rings, and they were prepared following a

common synthetic strategy (Scheme 5). N-Acetylated isonipecotic

acid 42 was transformed to the corresponding acyl chloride and

then reacted in a Friedel-Crafts reaction with diphenylmethane in

the presence of aluminum chloride in dichloroethane. Hydrolysis of

the N-acetyl group furnished free amine 51, which was reacted with

the proper benzoic acid: 3-methoxybenzoic acid to obtain amide

52, 2-ﬂuoro-5-methoxybenzoic acid to obtain amide 53 or 2,4-

diﬂuoro-5-methoxybenzoic acid for amide 54. The ﬁnal step,

common for all these intermediates, is the deprotection of the

methoxy group promoted by boron tribromide (Scheme 5).

Scheme 6 displays the synthetic pathways for the sulphur-based

compounds 20c,d,e, 21a and 22a, starting from the previously

described bromo-substituted derivative 43. Compound 43 was

subjected to a cross-coupling reaction with thiophenol, using

tris(dibenzylideneacetone)dipalladium as the catalyst, XanthPhos

as the ligand and potassium carbonate as the base, in toluene, to

obtain aryl sulﬁde derivative 61. This intermediate was subjected to

the same sequence of reactions, that are the hydrolysis of N-ace-

tylpiperidine group (compound 62), amide formation with the

proper unsubstituted or ﬂuoro-substituted benzoic acids (com-

pounds 63e65) and deprotection of the methoxy group, thus ﬁnally

obtaining compounds 20c, 21a and 22a. Alternatively, intermediate

61 was oxidized by using oxone® in a mixture water/1,4-dioxane to

obtain the corresponding sulfone 66. Similarly, compound 66 fol-

lowed the usual synthetic sequence to get compound 20d, which is

the sulfone analogue of derivative 20c. We also synthesized the

sulfoxide analogue of 20c, compound 20e: in this case compound

20c was oxidized in the presence of a mild oxidizing system

composed of hydrogen peroxide and 1,3,5-triazo-2,4,6-

triphosphorine-2,2,4,4,6,6-tetrachloride (TAPC) in acetic acid as

the solvent.

Some of the newly synthesized compounds, such as derivatives

20g-i, contain 1,2,3-triazole portions in their structures. The tri-

azole ring acts as a linker between the benzoyl phenyl ring and the

second phenyl ring. In some cases, a short linear carbon chain (1

or 3 carbon chain, in compounds 20h or 20i, respectively) was also

added between the triazole and the peripheral phenyl ring

(Scheme 7). These compounds were prepared starting from

compound 43, which is a common intermediate for the synthesis

of other compounds shown in the previously described schemes,

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European Journal of Medicinal Chemistry 209 (2021) 112857

Scheme 5. Synthesis of compounds 20a,b,f, 21b and 22b. Reagents and conditions: (a) i. SOCl₂, dry 1,2-DCE, 60 ^ꢁC, 4 h; ii. bromobenzene (for 43) or diphenylmethane (for 50), AlCl₃,

dry 1,2-DCE, 90 ^ꢁC, overnight [49e64%]; (b) if X ¼ NH (55): aniline, Pd₂(dba)₃, XPhos, KOtBu, toluene, 100 ^ꢁC, 24 h [99%], if X ¼ O (56): phenol, Pd(OAc)₂, Me₄tBuXPhos, K₃PO₄,

toluene, 100 ^ꢁC, 24 h [84%]; (c) NaOH 1 N, EtOH, 90 ^ꢁC, overnight [91e99%]; (d) 3-methoxybenzoic acid (for 52, 59 and 60), or 2-ﬂuoro-5-methoxybenzoic acid (for 53) or 2,4-

diﬂuoro-5-methoxybenzoic acid 24 (for 54), HATU, DIPEA, dry DMF, RT, 3e4 h [65e85%]; (e) 1 M BBr₃, dry CH₂Cl₂, ꢀ10 to 0 ^ꢁC, then RT, 1e5 h [25e90%].

Scheme 6. Synthesis of compounds 20c-e, 21a and 22a. Reagents and conditions: (a) thiophenol, Pd₂(dba)₃, XantPhos, K₂CO₃, toluene, 120 ^ꢁC, 24 h [97%]; (b) NaOH 1 N, EtOH, 90 ^ꢁC,

overnight [81e99%]; (c) 3-methoxybenzoic acid (for 63 and 68), or 2-ﬂuoro-5-methoxybenzoic acid (for 64) or 2,4-diﬂuoro-5-methoxybenzoic acid 24 (for 65), HATU, DIPEA, dry

DMF, RT, 3 h [49e85%]; (d) 1 M BBr₃, dry CH₂Cl₂, ꢀ10 to 0 ^ꢁC, then RT, 1e3 h [45e78%]; (e) oxone®, H₂O/1,4-dioxane, 0 ^ꢁC, then RT, overnight [99%]; (f) 35% H₂O₂, TAPC, AcOH, RT,

48 h [78%].

which was subjected to a copper-catalyzed Sonogashira reaction

with alkyne 2-methyl-3-butyn-2-ol (MEBYNOL), in the presence

of palladium acetate, triphenylphosphine, with triethylamine as

the base and DMF as the solvent [26]. After 4 h the desired

compound 69 was obtained, then removal of the 2-hydroxypropyl

group was achieved under basic conditions, reacting 69 with po-

tassium phosphate and potassium hydroxide in reﬂuxing toluene

for 24 h, thus obtaining compound 70 bearing the terminal alkyne

8

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

Scheme 7. Synthesis of compounds 20g-i. Reagents and conditions: (a) MEBYNOL, Pd(OAc)₂, PPh₃, K₂CO₃, CuI, Et₃N, dry DMF, 90 ^ꢁC, 4 h [99%]; (b) K₃PO₄, KOH, toluene, 115 ^ꢁC, 24 h

[82%]; (c) for 71: NaNO₂, NaN₃, HCl aq, H₂O, 0 ^ꢁC for 1 h, then RT, overnight [70%]; (d) for 72, 73: imidazole-1-sulfonyl azide hydrochloride, K₂CO₃, CuSO₄^.5H₂O, MeOH, RT, 2.5 h

[61e77%]; (e) CuSO^.₄5H₂O, sodium ascorbate, H₂O/tBuOH 1:1 v/v, 80 ^ꢁC, overnight [66e97%]; (f) NaOH 1 N, EtOH, 90 ^ꢁC, overnight [69e88%]; (g) 3-methoxybenzoic acid, HATU,

DIPEA, dry DMF, RT, 3 h [87e99%]; (h) 1 M BBr₃, dry CH₂Cl₂, ꢀ10 to 0 ^ꢁC, then RT, 1e3.5 h [8e61%].

moiety. At this point, compound 70 was subjected to a Cu(I)-

catalyzed 1,3-dipolar cycloaddition also known as “Huisgen

cycloaddition” or “click-reaction” [27] leading to the formation of

triazole-substituted intermediates 77e79. Azides 74e76 were

prepared from the corresponding amines, following two different

procedures, according to the type of amine, either aliphatic or

aromatic: 1) aniline 71 was converted to the corresponding azide

74 by using sodium nitrite and sodium azide in aqueous hydro-

chloridric acid (condition c, Scheme 7); 2) differently, benzyl-

amine 72 and 3-phenyl-1-propylamine 73 were subjected to a

2.3. Enzymatic assays

The herein reported compounds were evaluated for their inhi-

bition activity on human MAGL by using 4-nitrophenylacetate as

the substrate [25], comparing the inhibition data with those of

some reference compounds, which are the previously published

benzoylpiperidine derivatives (1-(3-hydroxybenzoyl)piperidin-4-

yl)(4-isopropylphenyl)methanone

(10)

and

(1-(2-ﬂuoro-5-

hydroxybenzoyl)piperidin-4-yl)(4-isopropylphenyl)methanone

(11) and the irreversible MAGL inhibitor CAY10499 (2). In Table 1,

the activity results related to the modiﬁcation of the phenolic

fragment of compound 11 are reported. It is evident that the

addition of a second ﬂuorine atom in para position to the amide

moiety led to an evident increase of the inhibition potency. In fact,

compound 12 was about 2.6-fold more active than mono-ﬂuoro

substituted derivative 11. All the other modiﬁcations of the

phenolic portion, such as the substitution with different groups as

well as the presence of a pyridine ring, were detrimental for the

inhibition activity, since compounds 13a-d and 14e17 showed

higher IC₅₀values that fall in the micromolar range, at best.

Once the best substituted phenolic fragment for an optimal

interaction with human MAGL was identiﬁed, we evaluated the

modiﬁcations of the benzoyl moiety, considering the larger di-

mensions of the binding site cavity in which this fragment is sup-

posed to be located. As reported above, in order to speed up the

synthesis of the new compounds and reduce the costs, we

preferred to maintain in ﬁrst instance the simple phenolic ring of

compound 10 at this stage of the optimization process. As shown in

Table 2, among the two cycloalkyl rings introduced in the para

position to the benzoyl ring, the cyclohexyl group of compound 18

was preferred compared to the smaller cyclopropyl ring of com-

pound 19, reaching a good IC₅₀value, slightly lower than that of

reference compound 10 (IC₅₀value of 129 nM compared to

142 nM). In the group of compounds in which the benzoyl ring and

a terminal phenyl ring are connected by different moieties, the

most promising results were achieved by the introduction of a

sulphur atom (compound 20c) or a methylene moiety (compound

20f). The IC₅₀values of 20c and 20f (74 and 78 nM, respectively)

reaction with

a suitable diazotransfer reagent, imidazole-1-

sulfonyl azide hydrochloride, which was prepared as previously

reported [28], in a copper-catalyzed reaction in the presence of

potassium carbonate in methanol, thus obtaining azides 75 and 76

(condition d, Scheme 7). After the formation of the triazole ring,

compounds 77e79 followed the usual sequence of reactions to

obtain compounds 20g-i.

Analysis of ¹H and ¹³C NMR spectra of the newly synthesized

benzoylpiperidine derivatives revealed that in two cases, in

particular in compounds 13a and 13d bearing bulky groups in

ortho position to the amidic carbonyl group (triﬂuoromethyl for

13a and methyl group for 13d), it was possible to identify two

rotational conformers generated by the hindered rotation around

the CeC bond between the amidic phenyl ring and the carbonyl

group nearby, as previously observed by us for similar compounds

[23]. The splitting of NMR signals was not observed for other

compounds bearing nitro (13b) or amino group (13c) in the same

position, since the presence of the ortho-NH₂moiety allowed the

formation of an intramolecular H-bond between the hydrogen

atom of the amino group and the amide carbonyl oxygen atom,

thus inducing stabilization of only one conformer. Similarly, the

ortho-NO₂substituted derivative 13b was visible as a single

conformer by NMR analysis, because the repulsion between the

partially negatively charged oxygen of the amide carbonyl group

and the negatively charged oxygen of the NO₂group induces a

block of rotation, stabilizing only the conformer in which these

two portions are distant.

9

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

Table 1

In vitro inhibitory activity on MAGL (IC₅₀, nM)^aof derivatives 12, 13a-d, 14e17.

a

Enzymatic values are the mean of three or more independent experiments, performed in duplicate.

were about two-fold lower than that of compound 10. Unfortu-

nately, when the spacer is an NH group (20a) or an oxygen atom

(20b), the inhibition activities decreased (IC₅₀¼ 758 and 258 nM,

respectively). The inhibition potency was negatively affected by the

presence of more polar groups, such as a sulfoxide or a sulphone

spacer between the two aromatic portions (20e and 20d). Likewise,

the triazole moiety present in compounds 20g-i led to high IC₅₀

values in the micromolar range.

The results shown in Tables 1 and 2 demonstrated that the best

inhibition activity of MAGL was obtained when: a) the phenolic

moiety is substituted with one or two ﬂuorine atoms in appropriate

positions, and b) on the opposite side of the benzoylpiperidine

scaffold, the benzoyl ring and the peripheral phenyl ring are spaced

by means of a sulphur atom or a methylene moiety. Therefore, these

features were simultaneously combined to obtain compounds

21a,b and 22a,b, and their inhibitory activities were tested on

MAGL. As shown in Table 3, mono-ﬂuoro-substituted compounds

21a and 21b exhibited IC₅₀values 2.7-fold or 1.6-fold lower,

respectively, than their isopropyl parent compound 11 (Table 1),

conﬁrming that the diphenylmethane and diphenyl sulphide moi-

eties are optimal lipophilic moieties that nicely ﬁt in the open

channel of the MAGL active site. Diﬂuoro-substituted compound

22b showed similar potency to that of compound 21a, whereas the

best result was obtained with compound 22a, in which the diﬂuoro

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European Journal of Medicinal Chemistry 209 (2021) 112857

Table 2

Table 3

In vitro inhibitory activity on MAGL (IC₅₀and K_i, nM)^aof derivatives 21a,b, 22a,b.

In vitro inhibitory activity on MAGL (IC₅₀, nM)^aof derivatives 18, 19, 20a-i.

Compounds

R

X

IC₅₀(nM)

K_i(nM)

Compounds

R

IC₅₀(nM)

21a

21b

22a

22b

H

F

S

CH₂

S

30

49

18

36

2

3

1

27

37

11

30

3

1

2

10

142

8

F

CH₂

a

Enzymatic values are the mean of three or more independent experiments,

performed in duplicate.

18

129

4

substitution was combined with the diphenyl sulphide moiety,

showing an IC₅₀value of 18 nM, thus reaching the highest potency

of this series of MAGL inhibitors.

19

368 16

758 88

With the aim to conﬁrm the reversible mechanism of inhibition,

already proved for other members of this chemical class [23,24], the

four derivatives 21a,b, 22a,b were subjected to preincubation and

dilution assays. With regards to the preincubation assay, a higher

potency after longer incubation times should be produced by an

irreversible inhibition, whereas for a reversible inhibitor a constant

inhibition potency over all the different incubation times should be

observed. The four compounds were thus preincubated with the

enzyme for 0, 30 and 60 min before adding the substrate to start the

enzymatic reaction. As shown in Fig. 5A, the test suggests a

reversible binding mode for all compounds, as they showed very

similar activities at all the three different incubation times. As a

second test, we studied the effect of dilution on the inhibitory ac-

tivity of these four compounds. In this test, the potency of an

irreversible mechanism of inhibition should not decrease after

dilution, whereas for a reversible inhibitor, the potency should be

strongly reduced after dilution. Therefore, the inhibition produced

by incubation with a 50 nM concentration of compounds 21a,b and

22a,b was measured after a 40x dilution and compared to the po-

tency observed by a 2000 nM and a 50 nM concentration of the

same compounds. As shown in Fig. 5B, for all compounds, the in-

hibition produced at a concentration of 50 nM was similar to that

obtained after a 40x dilution and was considerably lower than that

produced by the same compound at a concentration of 2000 nM,

thus supporting a reversible mechanism of inhibition.

20a

20b

20c

20d

278 12

74

6

3400 400

4300 500

20e

20f

78

6

20g

1000 100

4400 400

The inhibition mode of the four best compounds was then

evaluated by measuring MichaeliseMenten kinetics at various in-

hibitor concentrations. The datasets were plotted as substrate

concentration versus enzyme activity and analyzed by applying the

mixed-model inhibition ﬁt of GraphPad Prism 5.0, which includes

competitive, uncompetitive, and noncompetitive inhibition terms.

20h

20i

The model evaluates the V_max, K_mand the

indicative of the inhibition mechanism. When

a

parameter, which is

corresponds to

3300 300

a

one, the inhibitor does not alter the binding of the substrate to the

enzyme, and the mixed-model can be considered as a noncom-

petitive inhibition. When

interaction prevents the substrate binding and the mixed-model

corresponds to competitive inhibition. Finally, when is a very

small value, the binding of the inhibitor increases the binding of the

substrate and the mixed model corresponds to an uncompetitive

a is a very large value, the inhibitor

CAY10499 (2)

130 13

a

Enzymatic values are the mean of three or more independent experiments,

performed in duplicate.

model. Kinetic studies indicate for 21a,b and 22a,b an

a value

greater than 10 000, thus supporting a competitive behavior for

these compounds (Fig. S1) and ss shown in Table 3, the K_ivalues

measured for 21a,b and 22a,b ranged from 11 to 37 nM.

11

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

Fig. 5. Analysis of the mechanism of MAGL inhibition of compounds 21a,b and 22a,b. A) IC₅₀(nM) values at different preincubation times with MAGL (0 min, 30 min and 60 min). B)

Dilution assay: the ﬁrst two columns indicate the inhibition percentage of the compounds at a concentration of 2000 nM and 50 nM. The third column indicates the inhibition

percentage of the compounds after dilution (ﬁnal concentration ¼ 50 nM).

2.4. Molecular modeling studies

reference ligand for molecular modeling studies. A thorough

docking procedure performed with AUTODOCK 4.2, aimed at pre-

dicting its potential binding mode, was initially performed (See

Experimental Section for details). The docking calculations

Compound 22a, which is one of the most promising MAGL in-

hibitors among the synthesized derivatives, was used as the

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C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

conﬁrmed the general binding disposition of this series of ligands

that guided the structure-based optimization of this class of com-

pounds [23]. In fact, the phenol moiety of compound 22a was

located into the terminal portion of MAGL binding site, showing the

key H-bond network with H272 and E53. As expected, the diphenyl

sulphide moiety was placed into the lipophilic channel at the

entrance of the catalytic site, while the central carbonyl group of

the ligand formed H-bonds interactions with A51 and M123 within

the oxyanion hole (Fig. S2). The binding mode generated by docking

for compound 22a was then reﬁned and further studied by running

molecular dynamics (MD) simulation analysis, followed by ligand-

protein binding energy evaluations. The predicted 22a-MAGL

complex was subjected to an MD protocol consisting in 250 ns of

simulation, which conﬁrmed the reliability of both the disposition

of the ligand into MAGL binding site (with an average RMSD of 1.9 Å

during the MD simulation) and the ligand-protein interactions

predicted by docking (Table S1). Fig. 6 shows the minimized

average structure of 22a within the MAGL catalytic site generated

from the last 200 ns of MD simulation. The ligand slightly adjusted

its binding conformation during the MD simulation, especially at

the level of the terminal phenyl ring of the diphenyl sulphide

moiety, which showed to be highly mobile and able to establish

multiple hydrophobic interactions with the residues located at the

entrance of the binding pocket, such as A151, A156, F159, L205,

L213, and L241. The central phenyl ring of the ligand is sandwiched

between A51 and L213 from one side and L241 from the other,

forming lipophilic interactions with these residues, while the

adjacent carbonyl group establishes stable H-bonds with the

backbone nitrogen of both A51 and M123, which were maintained

for most of the MD simulation (Table S1). The diﬂuorophenolic

maintained for 77% of the simulation, could well compensate the

reduced strength of the H-bond with H272.

With the aim to evaluate the reliability of the binding mode

predicted for compound 22a from a quantitative point of view, the

last 200 ns of MD simulation of the 22a-MAGL complex were

employed to perform ligand-protein binding energy evaluations,

using the MM-GBSA method. Notably, the binding afﬁnity esti-

mated for the complex (ꢀ71.9 kcal/mol) was found to outperform

of 7 kcal/mol the afﬁnity previously predicted for the parent

compound 11 (ꢀ64.9 kcal/mol) [24]. This result is consistent with

the higher inhibitory activity of compound 22a with respect to 11,

and could thus conﬁrm the reliability of the binding mode and the

ligand-protein interactions predicted for 22a.

The 22a-MAGL complex was then subjected to steered molec-

ular dynamics (SMD) simulations aimed at predicting the potential

dissociation process of this class of inhibitors from their complex

with the enzyme. The simulation of the putative ligand unbinding

process was motivated by the attempt of obtaining a deeper insight

of the inhibition mechanism of this class of compounds in terms of

ligand-protein interactions, analyzing the importance of the key

structural moieties of these ligands from a different point of view

and also providing a ﬁrst hint about the ligand-binding process. In

view of this last purpose, we aimed at simulating the exit of the

ligand through the lipophilic channel at the entrance of the MAGL

catalytic site, which should constitute the access gate to MAGL

substrates and inhibitors. In particular, three different SMD simu-

lations were performed following three different unbinding path-

ways (P1-3), each deﬁned based on the

a carbon of a speciﬁc

protein residue: S48, M123 and A126, respectively (Fig. S3A). In

each simulation, the distance between the terminal phenyl ring of

moiety of 22a showed the

p-p

stacking with Y194 and the H-bond

the ligand and the residue a carbon was progressively increased by

network with E53 and H272, already predicted for reference

compound 11 and other phenolic derivatives of this series [24].

However, while the charged H-bond with E53 showed to be ﬁrmly

maintained during the whole simulation, the H-bond with H272

was found to be less stable, being maintained for about 64% of the

MD simulation. Nevertheless, a further H-bond, that was not

initially predicted by docking, was observed between the ﬂuorine

atom in ortho position to the hydroxyl group of the ligand and the

guanidine moiety of R57; this additional interaction, which was

a total 30 Å, to allow the dissociation of the ligand from the protein

through the desired direction (see Experimental section for details).

Importantly, in each SMD the ligand was moved at a constant ve-

locity of 0.1 Å/ns, which required to perform 300 ns of simulation

for each unbinding pathway, for a total of 0.9

ms of SMD. This

approach allowed us to consider the simulated ligand dissociation

process as reversible, and the work associated with it as the free

energy variation of the process [29,30]. As shown in Fig. S3B, the

unbinding pathway P2 demonstrated to be the most energetically

Fig. 6. Minimized average structure of compound 22a within the MAGL catalytic site (PDB code 3PE6).

13

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

favored one since its corresponding total free energy (48.6 kcal/

mol) was found to be signiﬁcantly lower than those associated with

P1 (70.8 kcal/mol) and P3 (64.7 kcal/mol). Therefore, P2 was

considered as a likely unbinding pathway of compound 22a and

was thus further analyzed from both the quantitative and qualita-

tive point of view. The MM-GBSA method was used to perform a

per-frame ligand-protein binding energy evaluation, to monitor the

free energy of binding during the simulation. In parallel, the SMD

trajectory showing the whole ligand unbinding process was

inspected in relation to the binding energy proﬁle, to correlate the

change in ligand disposition with the change in the corresponding

binding free energy. Fig. 7 shows different snapshots of the SMD

simulation that illustrate the evolution of the 22a-MAGL complex

during the different stages of the ligand unbinding process.

Right after the ﬁrst few nanoseconds of simulation, the ligand

loses some of the interactions with the protein, such as the H-bond

with R57 and even the H-bond with M123. Nevertheless, similarly

to what observed in the predicted binding mode of the parent

compound 11 [24], the loss of the key interaction with the oxyanion

hole is compensated by the formation of a stable H-bond with the

hydroxyl group of S181. Therefore, for about the ﬁrst 55 ns of

simulation, the ligand globally maintains its binding mode (Fig. 7A),

30 ns of simulation (between 155 and 185 ns of SMD). In this time-

lapse, the binding free energy of the complex ﬂuctuates steadily

around ꢀ26 kcal/mol. The ligand then moves completely out of the

binding pocket; after about 200 ns from the beginning of the

simulation, 22a only interacts with the residue at the entrance of

the lipophilic channel from the outside region of the protein

(Fig. 7E), with a binding energy around ꢀ10 kcal/mol. After that, the

ligand moves away from MAGL catalytic site, forming only transient

and very weak interactions (with average

D

G_bindaround ꢀ6 kcal/

mol) with the protein surface residues until about 245 ns of SMD. A

complete detachment of compound 22a from MAGL is then

observed (Fig. 7F), consistently with the total drop of the binding

free energy, which remains to zero until the end of the simulation,

except for few frames in which the ligand forms transient occa-

sional interactions with the protein surface. For a deeper analysis of

the ligand unbinding process, we focused our attention on the

simulation interval ranging from about 100 to 185 ns, which was

found to be associated with a quite stable DG of binding (Fig. S4). By

carefully inspecting the SMD trajectory in this time-lapse, we

identiﬁed two different ligand conformations that are stably

maintained for a considerable time. In particular, when compound

22a starts crossing the entrance of the MAGL catalytic site, it as-

sumes for about 18 ns (110e127 ns of SMD) a binding pose in which

it interacts predominantly with H269 (and secondarily with S122)

through its OH group. The phenolic ring also shows van der Waals

contacts with A51, Y194 and L184, while the piperidine core and the

diphenyl sulphide moiety of 22a form extensive hydrophobic in-

teractions with F159, P178, I179, L241 and L205, placed at the

entrance of the lipophilic channel (Fig. S5A). Subsequently, as

mentioned above, the ligand reaches a stable conformation that is

maintained for a quite long time-lapse (between 155 and 185 ns of

SMD) with respect to the whole unbinding process. Although a

major portion of the ligand is already outside MAGL binding pocket,

the compound is anchored to the protein through two H-bonds

with the backbone nitrogen of D178 and L205. Additionally, the

hydroxyl group of 22a forms a water bridged interaction with S122

(Fig. S5B). Taken together, these results highlight the key role of the

ligand phenolic moiety, which is not only necessary for achieving a

high MAGL inhibitory activity, but may also be important for the

ligand recognition and binding process. Indeed, the interaction

which is only slightly weakened, since the

DG of binding is still

around ꢀ60 kcal/mol (Fig. S4). Subsequently, the central carbonyl

group of 22a is slowly pulled away from the oxyanion hole, thus

losing the H-bond with A51 (Fig. 7B). Moreover, the H-bond be-

tween the phenolic OH group of the ligand and H272 is also lost in

this stage (between 55 and 85 ns of SMD), in which the binding

energy continuously decreases up to about ꢀ40 kcal/mol. However,

both the H-bond with E53 and S181 are still maintained. During the

following 40 ns, the diphenyl sulphide moiety of the ligand moves

outside the entrance of the lipophilic channel, while its phenol ring

is dragged out the polar pocket of MAGL binding site (Fig. 7C), with

the consequent disruption of the H-bond with E53 and the further

drop of the

D

G of binding to about ꢀ30 kcal/mol. During this stage,

even the loss of the H-bond with S181 is eventually observed,

although this interaction is maintained for almost the ﬁrst 110 ns of

SMD. After about 130 ns from the beginning of the SMD, compound

22a further slides out MAGL catalytic site, reaching a sort of inter-

mediate, half-unbound state (Fig. 7D) that is maintained for about

Fig. 7. Evolution of the 22a-MAGL complex during the SMD simulation. Six different SMD snapshots, obtained after A) 25, B) 75, C) 120, D) 170, E) 200 and F) 250 ns from the

beginning of the simulation, are shown.

14

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

with E53 is maintained for the ﬁrst 30% of the SMD; when this is

disrupted, multiple additional H-bonds established by the ligand

OH group were observed during the dissociation process. For the

same reason, the two carbonyl groups of 22a could also play a role

in the ﬁrst stages of ligand binding to MAGL. Finally, the in-

teractions observed with D178 and L205 highlight two potential

pharmacophore interactions that might be exploited in the design

of more potent MAGL inhibitors.

selectivity over other relevant enzymes in the ECS. Compounds 21a

and 21b showed high selectivity also towards other serine hydro-

lases up to 30

exhibited

m

M (Fig. S6, Table S2), while compound 22a and 22b

a

partial inhibition of two non-identiﬁed bands

(approximately 50 and 55 KDa) at 10 and 30 mM (Fig. S6, Table S2).

2.8. Cell viability assays

2.5. Selectivity

The four compounds 21a,b and 22a,b were selected for further

in vitro experiments to evaluate their anticancer potency against a

series of cancer cells. Compound CAY10499 (2) was used as the

reference compound. Due to the key role that MAGL plays in the

tumor progression of breast, colon and ovarian cancers, ﬁve tumor

cell lines were chosen: human breast MDA-MB-231, colorectal

HCT116 and ovarian CAOV3, OVCAR3 and SKOV3 cancer cells

(Table 6) [34e36]. With the exception of OVCAR3, all compounds

produced an appreciable inhibition of cell viability in tested cancer

cell lines. With respect to the covalent reference inhibitor 2, com-

pounds 21a,b and 22a,b showed more potent cytotoxic activities on

HCT116, MDA-MB-231, CAOV3 and SKOV3, whereas the prolifera-

tion of the OVCAR3 tumor cells was most signiﬁcantly affected only

by compound 21b.

Compounds 21a,b and 22a,b were proﬁled for their selectivity

towards the other components of the ECS. As shown in Table 4, at

the concentration of 10

bound to CB1R and CB2R or inhibited ABHD6 and ABHD12; only

compound 21a showed a weak inhibition of ABHD6 (40% at 10 M).

Importantly, the four MAGL inhibitors showed high selectivity over

mM none of the compounds signiﬁcantly

m

FAAH, with no signiﬁcant inhibition at 10 mM (Table 4).

2.6. Cell-based assays of MAGL inhibition

In order to conﬁrm the MAGL inhibition in a more physiological

system, 21a,b and 22a,b were tested in intact U937 cells as previ-

ously described [24]. As shown in Fig. 8, all four compounds

showed an IC₅₀value in the nanomolar range; in particular, com-

pound 22a was the most potent inhibitor with an IC₅₀value of

568 nM.

2.9. Human cancer organoids

Beyond the cancer cell lines, human cancer models include the

use of primary patient-derived tumor xenografts (PDTXs) that can

mimic the biological characteristics of the tumor better than in vitro

culture models. However, limitations of PDTXs include the use of

animals and limited engraftment efﬁciencies for subsets of patient

tumors. Moreover, the approach is expensive, time-consuming,

resource-consuming, and PDTXs may undergo mouse-speciﬁc tu-

mor evolution [37]. In this scenario, cancer organoids represent an

emerging approach for creating patient-derived in vitro cancer

models that closely recapitulate the pathophysiological features of

natural tumorigenesis and metastasis [38]. Cancer-derived orga-

noids are three-dimensional tissue-resembling cellular clusters

derived from tumor-speciﬁc stem cells that mimic the in vivo tumor

characteristics, as well as tumor cell heterogeneity. Organoids have

been demonstrated to closely recapitulate the drug response in

clinical setting and could be utilized to measure the efﬁcacy and

toxicity of small molecules [39e42]. Furthermore, the generation of

cancer organoids is inexpensive, ease to use, and can be accom-

plished in 1e2 months.

2.7. Activity-based protein proﬁling experiments

With the aim to assess the selectivity of the four MAGL in-

hibitors in a broader context of serine hydrolase family, we per-

formed competitive activity-based protein proﬁling (ABPP)

experiments for compounds 21a,b and 22a,b using mouse brain

membrane preparations. ABPP is a functional proteomic technol-

ogy, which exploits chemical probes that react with mechanisti-

cally related classes of enzymes [31]. TAMRA-ﬂuorophosphonate

(TAMRA-FP) is used to visualize serine hydrolases, which include

the major eCB degrading enzymes [32]. An important advantage of

ABPP relative to other approaches is that it can detect changes in

the activity of very low-abundance enzymes in highly complex

samples and can simultaneously assess the potency and selectivity

of an inhibitor towards the entire family of serine hydrolases in a

speciﬁc tissue.

As shown in Fig. 9, at the concentration of 10

mM the four

compounds signiﬁcantly inhibited the two bands associated to

MAGL (doublet band 3 and 4 as MAGL migrates as double band in

the ABPP) [33]. In agreement with the data obtained in intact cells,

compounds 21a, 21b, 22a and 22b showed a concentration-

dependent inhibition of MAGL (Fig. S6, band 3 and 4) without

affecting FAAH (Fig. S6, band 1), ABHD6 (Fig. S6, band 5) and

On these bases, the abilities of the four compounds 21a,b and

22a,b to inhibit the growth of cancer cells prompted us to inves-

tigate their activities directly on cancer organoids derived from

human tumor tissues. In our study, the high grade serous ovarian

cancer (HGSOC) was selected, since it is one of the most aggressive

cancer types with a lethality/incidence ratio of more than 60% [43].

Cancer organoids derived from three HGSOC patients were treated

ABHD12 (Fig. S6, band 2) up to 30 mM, indicating a high MAGL

Table 4

with 100 mM of 21a,b, 22a,b and carboplatin as the positive control.

Pharmacological characterization of compounds 21a,b, 22a,b towards the other

components of the ECS. Data represent IC₅₀values ( M, mean SD) and the % of

receptor binding/enzyme inhibition at the concentration of 10 M (in brackets).

Carboplatin turned out to be the most effective agent with a range

of values of inhibition from 50% to 67%. Compound 22b was the

least effective inhibitor with a percentage of inhibition ranging

from 18% to 35%. Compounds 21a,b and 22a inhibited the growth of

cancer organoids in the range of 42%e63%, thus moving near the

values shown by carboplatin (Fig. 10). Considering the targeting

nature of MAGL inhibitors, the results obtained for compounds

21a,b and 22a are comparable to that of carboplatin and therefore

open up to an in-depth analysis by considering a higher number of

patients.

m

IC₅₀values (

CB1

m

M, mean SD)

CB2

Compound

FAAH

ABHD6

ABHD12

21a

21b

22a

22b

>10 (<10%) >10 (<10%) >10 (<10%) >10 (40%) >10 (26%)

>10 (<10%) >10 (<10%) >10 (<10%) >10 (21%) >10 (20%)

>10 (<10%) >10 (<10%) >10 (22%)

>10 (<10%) >10 (<10%) >10 (21%)

>10 (18%) >10 (27%)

>10 (10%) >10 (28%)

15

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

Fig. 8. Concentration-dependent inhibition of 2-OG hydrolysis in intact U937 cells for compounds 21a,b and 22a,b. Cells were pre-incubated for 30 min with the compounds and

then with 2-OG/[³H]2-OG (ﬁnal concentration 10 M) for additional 15 min at 37 ^ꢁC. JZL184 1 (1

M) was used as a positive control for full inhibition of MAGL. Data represent

mean SD of two independent experiments in triplicate. IC₅₀values are expressed as mean (95% CI). Data were normalized (residual 2-OG hydrolysis of 30%).

m

2.10. PDAC cells viability assay

pancreatic neoplasm, the later disease diagnosis and the low

effectiveness of available chemotherapeutic agents are the main

causes of PDAC poor prognosis [46]. Thus, developing new and

more effective drugs to treat PDAC represents an urgent and unmet

medical need.

The use of preclinical models which do not take into account the

complex molecular and histopathologic features of PDAC may be

one of the reasons of the limited effects shown by most of the

anticancer agents developed so far in clinical trials. The use of

patient-derived primary cell cultures which are ex vivo cell pop-

ulations recovered directly from fresh surgically resected tissue

samples has the advantage of preserving the most important

original tumor features. Therefore, the primary patient-derived

PDAC-3 cell culture was chosen as cellular model in the present

Compounds 21a,b and 22a,b were also subjected to

Sulforhodamine-B (SRB) assay in order to evaluate their anti-

proliferative activity on a pancreatic ductal adenocarcinoma (PDAC)

model: PDAC-3 primary cell culture. Gemcitabine was used as

reference compound because it is a drug of choice in the treatment

of PDAC, but chemoresistance to this drug is common and should

prompt the development of new therapeutic approaches. PDAC is

an extremely lethal cancer with a 5-year survival rate of approxi-

mately 10% in the USA [44], and because of its increasing incidence

and mortality rates it is expected to become the second leading

cause of cancer-related deaths before 2030 [45]. Despite the recent

improvements in the molecular knowledge about this type of

16

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

Fig. 9. ABPP ﬂuorescent screen of mouse brain membrane preparations (4 mg/mL) with TAMRA-Fluorophosphonate serine hydrolase probe (TAMRA-FP). The proteome was

preincubated for 25 min with either DMSO, URB597 (FAAH inhibitor, 4 M), JZL184 (MAGL inhibitor, 1 M), WWL70 (ABHD6 inhibitor, 10 M), THL (ABHD6 and 12 inhibitor, 20 M),

DO264 (ABHD12 inhibitor, 5 M) or compounds 21a,b and 22a,b at 10

M followed by 5 min labeling with TAMRA-FP (125 nM ﬁnal concentration). Band 1 ¼ FAAH, band

m

2 ¼ ABDH12, band 3 and 4 ¼ doublet of MAGL, band 5 ¼ ABHD6.

Table 6

Cell growth inhibitory activities (IC₅₀values) of CAY10499 (2) and compounds 21a, 21b, 22a and 22b.

IC₅₀M, mean SD)

(

m

Compound

HCT116

38

MDA-MB-231

CAOV3

OVCAR3

53

>100

20.7

>100

SKOV3

33

CAY10499 (2)

4

83

43

25

63

65

5

3

5

4

95

34

70

42

44

4

2

5

4

2

21a

21b

22a

22b

5.1 0.8

4.6 0.7

17

15

2

1

8

1

11

12

2

1

5.3 0.9

Fig. 10. Growth inhibition of cancer-derived human organoids derived from three HGSOC patients. The activity of compounds 21a,b and 22a,b was compared to carboplatin

standard chemotherapeutic drug. The % of inhibition is shown at 100 M dilution.

m

17

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

study, considering that its genetic, metastatic and histopathological

characteristics are comparable to the originator tumor [47]. These

cells were cultured in vitro only for a few passages (less than 20) to

maintain the genetic hallmarks of the primary tumor and before

entering into senescence. Moreover, PDAC-3 cell culture was cho-

sen as cellular model since showed an over-expression of MAGL

respect to the normal cell line HPDE (fold change ¼ 2.7). Com-

pounds 21a,b and 22a,b were tested on PDAC-3 primary cell culture

at 8 different concentrations (from 156.25 to 20 000 nM) showing a

remarkable antiproliferative activity, with IC₅₀values in the range

sheets that were visualized under a UV lamp. Evaporation was

performed in vacuo (rotating evaporator). Sodium sulfate was al-

ways used as the drying agent. Proton (¹H) and carbon (¹³C) NMR

spectra were obtained with a Bruker Avance III 400 MHz spec-

trometer using the indicated deuterated solvents. Chemical shifts

are given in parts per million (ppm) (d relative to residual solvent

peak for ¹H and ¹³C). ¹H NMR spectra are reported in this order:

multiplicity and number of protons. Standard abbreviation indi-

cating the multiplicity were used as follows:

s

¼

singlet,

d ¼ doublet, dd ¼ doublet of doublets, ddd ¼ doublet of doublet of

of 9.28e14.6 mM (Table 7).

doublets, t ¼ triplet, tt ¼ triplet of triplets, dt ¼ doublet of triplets,

td

¼

triplet of doublets, quint

¼

quintet, sept

¼

septet,

m ¼ multiplet, bm ¼ broad multiplet and bs ¼ broad singlet. HPLC

analysis was used to determine purity: all target compounds (i.e.,

assessed in biological assays) were ꢂ95% pure by HPLC, as

3. Conclusions

In the present work, the class of benzoylpiperidine-based MAGL

inhibitors was expanded and optimized, leading to the identiﬁca-

tion of compounds 21a,b and 22a,b, which are characterized by a

potent MAGL inhibition activity, with K_ivalues ranging from 11 to

37 nM, a reversible mode of action, as well as a high selectivity for

MAGL vs. CB1R, CB2R, FAAH, ABHD6 and ABHD12 (IC₅₀

conﬁrmed via UV detection (

l

¼ 254 nm). Analytical reversed-

phase HPLC was conducted using a Kinetex EVO C18 column

(5

m, 150 ꢃ 4.6 mm, Phenomenex, Inc.); eluent A, water; eluent B,

m

CH₃CN; after 5 min at 25% B, a gradient was formed from 25% to 75%

of B in 5 min and held at 75% of B for 10 min; ﬂow rate was 1 mL/

min. HPLC analyses were performed at 254 nm. Elemental analysis

was used to further characterize the ﬁnal compounds; analytical

results are within 0.4% of the theoretical value. Yields refer to

isolated and puriﬁed products derived from non-optimized pro-

cedures. Compound 2 was purchased from Cayman Chemical and

compounds 10, 11, 23 and 42 were synthesized as previously re-

ported [24].

value > 10 mM in all cases). These four derivatives were also tested

in intact human monocytic cell line U937, where they inhibited 2-

OG hydrolysis, and showed antiproliferative activities in human

breast MDA-MB-231, colorectal HCT116, ovarian CAOV3 and SKOV3

cancer cells at micromolar concentrations. ABPP gels conﬁrmed a

broad selectivity towards other serine hydrolases for compound

21a,b, while compound 22a,b showed potential off-target inhibi-

tion of two non-identiﬁed bands at 50e55 KDa. The potential

applicability to the clinical setting was supported by the results

obtained in additional preclinical models that better mimic the

molecular and histopathological complexity of the tumors, such as

primary PDAC cells and cancer organoids derived from high grade

serous ovarian cancer patients. These models have the advantage of

maintaining the original genotype and phenotype of the tumors

and represent an important tool for the experimental testing of

anticancer agents, that could directly counsel individualized de-

cisions in future clinical trials. Taken together, these results suggest

that these derivatives are among the most active and selective

reversible MAGL inhibitors reported in the literature so far and may

therefore open up new perspectives for studying the therapeutic

potential of reversible MAGL inhibition. Finally, the applicability in

clinical setting was demonstrated in cancer organoids derived from

high grade serous ovarian cancer patients.

4.1.1. General procedure for the synthesis of amide derivatives 25,

27, 29, 33, 37, 14e17, 46, 49, 52e54, 59, 60, 63e65, 68, 83-85

HATU (1.05 equiv) was added to a solution of the appropriate

benzoic acid (1 equiv) in dry DMF (2.1 mL), then DIPEA (4 equiv)

was added dropwise. The resulting mixture was stirred at room

temperature for 30 min and then the proper amine (100 mg, 1

equiv) was added and left under stirring at room temperature until

consumption of starting material (TLC). After this time, the residue

was diluted with water and extracted with EtOAc. The organic layer

was repeatedly washed with brine, dried over Na₂SO₄and the

solvent was removed under reduced pressure. The residue was

puriﬁed with a ﬂash column chromatography (silica gel, an

appropriate mixture of n-hexane or petroleum ether/ethyl acetate

or CHCl₃/MeOH) and pure fractions containing the desired com-

pound were evaporated to dryness affording the amides.

4. Experimental section

4.1.2. General procedure for the synthesis of O-deprotected

benzoylpiperidines 12, 13a-d, 18, 19, 20a-d, 20f-i, 21a,b, 22a,b

A solution of pure O-methylated amides (0.23 mmol) in anhy-

drous CH₂Cl₂(2.7 mL) was cooled to ꢀ10 ^ꢁC and treated dropwise

with a 1.0 M solution of BBr₃in CH₂Cl₂(0.73 mL) under argon. The

mixture was left under stirring at the same temperature for 5 min

and then at 0 ^ꢁC for 1 h and ﬁnally at RT until starting material was

consumed (TLC). The mixture was then diluted with water and

extracted with ethyl acetate. The organic phase was washed with

4.1. Synthesis. General procedures and materials

All solvents and chemicals were used as purchased without

further puriﬁcation. Chromatographic separations were performed

on silica gel columns by ﬂash chromatography (Kieselgel 40,

0.040e0.063 mm; Merck). Reactions were followed by thin layer

chromatography (TLC) on Merck aluminum silica gel (60 F254)

Table 7

Cell growth inhibitory activities (IC₅₀values) of Gemcitabine and compounds 21a,b and 22a,b.

IC₅₀M) SEM

(

m

Compound

PDAC-3

Gemcitabine

0.00554 0.00044

9.28 0.91

12.5 0.1

14.6 1.3

12.0 0.6

21a

21b

22a

22b

18

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

brine, dried and concentrated. The crude product was puriﬁed by

ﬂash chromatography over silica gel. Elution with n-hexane/EtOAc

or CHCl₃/MeOH mixtures afforded the desired compounds.

in vacuo. The residue was puriﬁed by silica gel chromatography

using appropriate n-hexane or petroleum ether/EtOAc mixtures.

4.1.8. Procedure for the synthesis of compound 44

4.1.3. Procedure for the synthesis of sulfoxide derivative 20e

To the mixture of sulﬁde 20c (27 mg, 1 equiv) in acetic acid

(4 mL), TAPC (0.22 equiv) and 30% H₂O₂(2.2 equiv) were added at

room temperature with continuous stirring overnight. The progress

of the reaction was monitored by TLC and TAPC (0.22 equiv) and

30% H₂O₂(2.2 equiv) were added to consume the starting material.

After the completion of the reaction, H₂O was added to the reaction

mixture and the residue was then extracted with EtOAc and the

combined extracts were dried (Na₂SO₄). The ﬁltrate was evaporated

and the corresponding sulfoxide was obtained after puriﬁcation by

ﬂash chromatography.

Substituted bromobenzene 43 (96 mg, 0.31 mmol, 1 equiv),

cyclopropylboronic acid (1.3 equiv), and K₃PO₄(3.5 equiv) were

added to a sealed vial under argon atmosphere. Toluene (1.4 mL),

20 wt % solution of P(Cy)₃in toluene (0.1 equiv, 0.05 mL) and

Pd(OAc)₂(0.05 equiv) were added to the reaction mixture. The

reaction mixture was heated to 100 ^ꢁC and stirred for 24 h. After

removing from heat, the reaction mixture was diluted with EtOAc,

ﬁltered through Celite, and concentrated. The crude product was

puriﬁed by ﬂash column chromatography to obtain the desired

compound.

4.1.9. General procedure for the synthesis of piperidine derivatives

45, 48, 51, 57, 58, 62, 67, 80-82

4.1.4. General procedure for the synthesis of methyl 3-

methoxybenzoates 30 and 35

To a solution of N-acetylated intermediates 44, 47, 50, 55, 56, 61,

66, 77e79 (400 mg) in 17 mL of EtOH,1 N aqueous solution of NaOH

was added (17 mL). The reaction mixture was heated at 90 ^ꢁC

overnight. The solution was cooled to room temperature, then

concentrated under reduced pressure, diluted with water and

extracted with EtOAc. The organic layer was washed with brine,

dried over Na₂SO₄and concentrated to dryness to obtain the pure

desired compounds that were used in the next step without further

puriﬁcation.

A solution of commercially available 5-methoxy-2-nitrobenzoic

acid 28 or 5-hydroxy-2-methylbenzoic acid 34 (500 mg, 1 equiv) in

10 mL of DMF was treated with anhydrous K₂CO₃(2.2 equiv) and

iodomethane (3 equiv) and the reaction mixture was stirred at

room temperature for 4e5 h. The mixture was diluted with water

and extracted with ethyl acetate. The organic extract was washed

with brine, dried over Na₂SO₄and the organic solvent was removed

under vacuum on a rotary evaporator. The crude product afforded

the pure desired compounds 30 and 35, that were used in the next

step without further puriﬁcation.

4.1.10. Procedure for the synthesis of compound 55

A solution of Pd₂dba₃(0.02 equiv), XPhos (0.08 equivl), potas-

sium tert-butoxide (1.4 equiv), commercially available aniline (1.2

equiv) and intermediate 43 (100 mg, 1 equiv) in toluene (0.6 mL)

was stirred at 100 ^ꢁC under inert atmosphere in a sealed vial for

24 h. The reaction mixture was allowed to cool to room tempera-

ture, then ﬁltered through a small pad of Celite, washed with ethyl

acetate and concentrated under vacuum. The obtained crude res-

idue was puriﬁed by ﬂash column chromatography (eluent mixture

n-hexane/EtOAc) to give intermediate 55.

4.1.5. Procedure for the synthesis of compound 31

Nitro-substituted derivative 30 (240 mg,1.14 mmol,1 equiv) was

dissolved in 1:1 v/v CHCl₃/MeOH mixture (6.2 mL), then

SnCl₂$2H₂O (8 equiv) was added and the mixture was heated at

65 ^ꢁC for 1 h. Upon consumption of starting material, the mixture

was concentrated, diluted with water, neutralized with 1 N aqueous

NaOH, extracted with EtOAc and the organic phase was washed

with brine. The combined organic phase was dried over anhydrous

sodium sulfate, ﬁltered and evaporated, affording a crude residue

which was used in the next step without further puriﬁcation.

4.1.11. Procedure for the synthesis of compound 56

A sealed vial was charged with Me₄tBuXPhos (0.03 equiv),

Pd(OAc)₂(0.02 equiv), K₃PO₄(2 equiv), intermediate 43 (100 mg, 1

equiv), commercially available phenol (1.2 equiv) and toluene

(0.6 mL) under a positive pressure of argon. The resulting mixture

was heated at 100 ^ꢁC for 24 h. Then the mixture was allowed to cool

to room temperature and then ﬁltered through a small pad of

Celite, washed several times with ethyl acetate and the ﬁltrate was

concentrated under reduced pressure. The crude product was pu-

riﬁed by ﬂash chromatography on silica gel (n-hexane/EtOAc

mixture) to afford compound 56.

4.1.6. General procedure for the synthesis of 3-methoxybenzoic

acids 32 and 36

Methyl esters 31 or 35 (190 mg, 1 equiv) were dissolved in a 1:1

v/v mixture of THF/methanol (10.6 mL) and treated with 3.2 mL of

2 N aqueous solution of LiOH. The reaction was stirred until con-

sumption of starting material, then the solvents were evaporated,

and the residue was treated with 1 N aqueous HCl and extracted

with EtOAc. The organic phase was dried and evaporated to afford

the pure desired carboxylic acid derivatives.

4.1.7. General procedure for the synthesis of compounds 43, 47, 50

To a cooled suspension of 1-acetylpiperidine-4-carboxylic acid

42 (500 mg,1 equiv) in 2.7 mL of anhydrous 1,2-dichloroethane was

slowly added SOCl₂(2.3 equiv). The mixture was stirred at 60 ^ꢁC for

4 h (the mixture turned from white to orange) and then evaporated

under vacuum. Under argon atmosphere, the residue acyl chloride

was dissolved in 2.2 mL of anhydrous 1,2-dichloroethane, then the

reddish solution was cooled at 0 ^ꢁC and AlCl₃(2 equiv) was slowly

added. Finally, a solution of the aromatic reagent (1 equiv; bro-

mobenzene for 43, phenylcyclohexane for 47, diphenylmethane for

50) in anhydrous 1,2-dichloroethane (1.3 mL) was added dropwise.

The mixture was stirred at 90 ^ꢁC overnight. The solution was cooled

to room temperature and poured into ice. The water layer was

extracted with EtOAc, the combined organic phase was washed

with brine, dried over anhydrous sodium sulfate and concentrated

4.1.12. Procedure for the synthesis of compound 61

A sealed vial was charged with K₂CO₃(1 equiv), compound 43

(200 mg, 1 equiv), Pd₂(dba)₃(0.1 equiv), Xantphos (0.11 equiv),

toluene (9.7 mL), and commercially available thiophenol (1.25

equiv). After purging with argon, the resulting mixture was then

heated at 120 ^ꢁC for 24 h. After cooling to room temperature, the

mixture was diluted with EtOAc and water, the organic phase was

washed with brine, dried over Na₂SO₄and concentrated under

reduced pressure. The crude product was then puriﬁed by silica gel

chromatography to afford the expected product.

4.1.13. Procedure for the synthesis of sulfone derivative 66

An aqueous solution (1.1 mL) of Oxone® (50% w/v, 3 equiv) was

added dropwise to a stirred solution of the sulﬁde derivative 61

(100 mg, 1 equiv) in 1,4-dioxane (3.0 mL) at 0 ^ꢁC, and the reaction

19

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

was allowed to proceed with stirring at RT for 12 h. The reaction

mixture was diluted with water, extracted with EtOAc, the organic

phase was washed successively with water and brine, and dried

over anhydrous sodium sulfate. After ﬁltration, the solvent from the

organic fraction was evaporated to give the desired crude product

which was used in the next reaction without any further

puriﬁcation.

4.1.17.1. (1-(2,4-Diﬂuoro-5-hydroxybenzoyl)piperidin-4-yl)(4-

isopropylphenyl)methanone (12). White solid; 60% yield from 25,

eluent n-hexane/EtOAc 6:4.¹H NMR (DMSO‑d₆)

d (ppm): 1.22 (d, 6H,

J ¼ 6.9 Hz), 1.38e1.54 (bm, 2H), 1.70e1.79 (bm, 1H), 1.84e1.93 (bm,

1H), 2.91e3.04 (m, 2H), 3.17e3.28 (m, 1H), 3.44e3.54 (m, 1H),

3.68e3.79 (m, 1H), 4.43e4.53 (m, 1H), 6.89 (dd, 1H, J ¼ 9.5, 6.6 Hz),

7.29 (dd, 1H, J ¼ 11.1, 9.3 Hz), 7.41 (d, 2H, J ¼ 8.2 Hz). 7.93 (d, 2H,

J ¼ 8.4 Hz), 10.13 (exchangeable bs, 1H). ¹³C NMR (DMSO‑d₆)

4.1.14. Procedure for the synthesis of compound 69

d (ppm): 23.46 (2C), 28.27, 28.62, 33.47, 40.74, 42.09, 45.96, 105.00

A solution of Pd(OAc)₂(0.05 equiv) and triphenylphosphine

(0.25 equiv) in dry DMF (0.2 mL) was stirred at RT under argon for

10 min. After that period, aryl-bromide 43 (100 mg,1 equiv), copper

iodide (0.05 equiv), 2-methyl-3-butyn-2-ol (1.2 equiv) and Et₃N

(1.0 mL) were sequentially added. The mixture was stirred at 90 ^ꢁC

for 4 h. After being cooled to RT, the mixture was diluted with water

and extracted several times with EtOAc. The combined organic

phase was washed with brine, dried over anhydrous sodium sulfate

and concentrated. The crude product was puriﬁed by ﬂash chro-

matography to yield the product.

(dd, J_CF¼ 27.4, 23.0 Hz),116.00 (t, J_CF¼ 4.5 Hz),120.07 (dd, J_CF¼ 19.7,

4.2 Hz), 126.78 (2C), 128.51 (2C), 133.31, 141.85 (dd, J_CF¼ 12.3,

2.9 Hz), 149.05 (dd, J_CF¼ 104.0, 11.4 Hz), 151.46 (dd, J_CF¼ 112.0,

11.4 Hz), 154.19, 163.13, 201.30. HPLC analysis: retention

time ¼ 12.486 min; peak area, 97% (254 nm). Elemental analysis for

C

₂₂H₂₃F₂NO₃, calculated: % C, 68.20; % H, 5.98; % N, 3.62; found: % C,

68.02; % H, 6.30; % N, 3.35.

4.1.17.2. (1-(5-Hydroxy-2-(triﬂuoromethyl)benzoyl)piperidin-4-

yl)(4-isopropylphenyl)methanone (13a). Light yellow solid; 6% yield

from 27, eluent n-hexane/EtOAc 6:4.¹H NMR (DMSO‑d₆; asterisk

4.1.15. Procedure for the synthesis of compound 70

denotes isomer peaks)

d

(ppm): 1.22 (d, 6H, J ¼ 6.9 Hz), 1.34e1.58

Compound 69 (130 mg, 1 equiv) was suspended in toluene

(16.5 mL), then potassium hydroxide (1 equiv) and potassium

phosphate (1 equiv) were added and the mixture was reﬂuxed at

115 ^ꢁC for 24 h. The reaction mixture was cooled to RT, ﬁltered

through a small pad of Celite and washed several times with ethyl

acetate. Evaporation under vacuum of the ﬁltrate afforded com-

pound 70.

(bm, 2H), 1.62e1.77 (m, 1H), 1.83e1.95 (bm, 1H), 2.90e3.29 (m, 4H),

3.64e3.80 (bm, 1H), 4.45e4.55 (m, 1H), 6.70* (d, 1H, J ¼ 2.2 Hz),

6.75 (d, 1H, J ¼ 2.4 Hz), 6.91e6.99 (m, 1H), 7.41 (d, 2H, J ¼ 7.8 Hz),

7.59 (d, 1H, J ¼ 8.8 Hz), 7.90e7.98 (m, 2H), 10.59 (exchangeable s,

1H). ¹³C NMR (DMSO‑d₆; asterisk denotes isomer peaks)

d (ppm):

23.40 (2C), 23.42* (2C), 27.64*, 27.89, 28.09, 28.22*, 33.42, 40.17,

40.31*, 41.90*, 42.08, 45.86*, 45.92, 113.33, 113.52*, 115.51*,115.74,

124.23 (q, J_CF¼ 281.7 Hz), 126.74 (2C), 128.46 (2C), 128.57, 133.24,

136.88, 154.13*, 154.16, 160.52*, 160.63, 165.71*, 165.86, 201.27.

HPLC analysis: retention time ¼ 12.898; peak area, 96% (254 nm).

Elemental analysis for C₂₃H₂₄F₃NO₃, calculated: % C, 65.86; % H,

5.77; % N, 3.34; found: % C, 65.50; % H, 5.92; % N, 3.71.

4.1.16. General procedure for the synthesis of azides 74-76

To a solution of aniline 71 (500 mg, 5.37 mmol, 1 equiv) in

aqueous 4 N HCl (7.8 mL) cooled to ꢀ5 ^ꢁC was added dropwise a

solution of sodium nitrite (1.2 equiv) in water (1.4 mL) and the

mixture was stirred at the same temperature for 30 min. Then,

another solution of sodium azide (1.2 equiv) in water (1.7 mL) was

added dropwise, keeping the temperature below 0 ^ꢁC. Stirring was

continued for 1 h at 0 ^ꢁC and overnight at room temperature. The

reaction mixture was then extracted with EtOAc and the combined

organic phase was washed with a saturated solution of NaHCO₃and

brine, then dried and concentrated under vacuum, to obtain pure

74 without any further puriﬁcation. In the case of aliphatic azides

75 and 76, imidazole-1-sulfonyl azide hydrochloride (0.86 g,

4.9 mmol, 1.2 equiv) [28] was added to the commercially available

amine (benzylamine 72 or 3-phenyl-1-propylamine 73, 1 equiv),

K₂CO₃(1.7 equiv) and CuSO₄^.5H₂O (0.01 equiv) in MeOH (20.6 mL)

and the mixture was stirred at room temperature for 2.5 h. The

mixture was concentrated, diluted with H₂O, acidiﬁed with conc.

HCl and extracted with EtOAc. The combined organic layers were

dried, ﬁltered and concentrated. The crude product was puriﬁed by

ﬂash chromatography to give the desired azides.

4.1.17.3. (1-(5-Hydroxy-2-nitrobenzoyl)piperidin-4-yl)(4-

isopropylphenyl)methanone (13b). Light yellow solid; 22% yield

from 29, eluent EtOAc/MeOH 95:5.¹H NMR (DMSO‑d₆)

d (ppm): 1.22

(d, 6H, J ¼ 6.9 Hz), 1.41e1.61 (bm, 2H), 1.61e1.75 (bm, 1H), 1.84e1.97

(bm, 1H), 2.91e3.07 (m, 2H), 3.07e3.18 (bm, 1H), 3.35e3.47 (m, 1H),

3.65e3.80 (m, 1H), 4.40e4.57 (m, 1H), 6.68e6.78 (m, 1H), 6.95 (dd,

1H, J ¼ 9.1, 2.6 Hz), 7.41 (d, 2H, J ¼ 8.2 Hz), 7.88e8.00 (m, 2H), 8.12

(d, 1H, J ¼ 8.3 Hz), 11.29 (exchangeable bs, 1H).¹³C NMR (DMSO‑d₆)

d

(ppm): 23.45 (2C), 28.09, 33.46, 40.40, 113.66, 116.16, 126.45,

126.77 (2C), 127.69, 128.50 (2C), 129.90, 133.31, 135.73, 149.20,

154.20, 163.46.

HPLC analysis: retention time ¼ 12.279 min; peak area, 98%

(254 nm). Elemental analysis for C₂₂H₂₄N₂O₅, calculated: % C, 66.65;

% H, 6.10; % N, 7.07; found: % C, 66.99; % H, 6.12; % N, 7.01.

4.1.17.4. (1-(2-Amino-5-hydroxybenzoyl)piperidin-4-yl)(4-

isopropylphenyl)methanone (13c). Light yellow solid; 59% yield

4.1.17. General procedure for the synthesis of triazole-substituted

derivatives 77-79

from 33, eluent n-hexane/EtOAc 3:7.¹H NMR (DMSO‑d₆)

d (ppm):

1.22 (d, 6H, J ¼ 6.9 Hz), 1.45e1.58 (m, 2H), 1.74e1.85 (m, 2H), 2.97

(sept, 1H, J ¼ 6.7 Hz), 3.02e3.14 (m, 2H), 3.70 (tt, 1H, J ¼ 11.4,

3.6 Hz), 3.95e4.10 (bm, 2H), 4.48 (s, 2H), 6.43 (t, 1H, J ¼ 1.6 Hz), 6.57

(d, 2H, J ¼ 1.6 Hz), 7.41 (d, 2H, J ¼ 8.2 Hz), 7.93 (d, 2H, J ¼ 8.4 Hz),

Alkyne 70 (287 mg, 1 equiv) and azides 74, 75 or 76 (1 equiv)

were suspended in a 1:1 mixture of water and tert-butyl alcohol

(4.5 mL). A freshly prepared sodium ascorbate aqueous solution (0.1

equiv, 1.1 mL of water) was added, followed by copper (II) sulfate

pentahydrate aqueous solution (0.01 equiv, 0.04 mL of water). The

heterogeneous mixture was stirred vigorously at 80 ^ꢁC in a sealed

vial until consumption of the starting materials. The reaction

mixture was cooled, diluted with water, extracted with EtOAc. The

combined organic extracts were washed with brine, dried with

anhydrous Na₂SO₄, ﬁltered and concentrated. The crude product

was puriﬁed by ﬂash column chromatography to obtain the title

triazole-substituted compounds.

8.62 (exchangeable s, 1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 23.44 (2C),

28.52, 33.44, 42.36, 113.27, 117.08, 117.33, 120.93, 126.74 (2C), 128.46

(2C), 133.37, 137.81, 147.87, 154.09, 168.42, 201.45. HPLC analysis:

retention time ¼ 11.174 min; peak area, 95% (254 nm). Elemental

analysis for C₂₂H₂₆N₂O₃, calculated: % C, 72.11; % H, 7.15; % N, 7.64;

found: % C, 71.84; % H, 7.29; % N, 7.31.

4.1.17.5. (1-(5-Hydroxy-2-methylbenzoyl)piperidin-4-yl)(4-

isopropylphenyl)methanone (13d). White solid; 48% yield from 37,

20

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

eluent n-hexane/EtOAc 55:45.¹H NMR (DMSO‑d₆; asterisk denotes

(bm, 2H), 1.70e1.95 (bm, 2H), 2.90e3.07 (m, 2H), 3.14e3.30 (bm,

1H), 3.53e3.68 (bm, 1H), 3.69e3.80 (m, 1H), 4.32e4.50 (bm, 1H),

6.35e6.52 (bm, 2H), 7.41 (d, 2H, J ¼ 8.2 Hz), 7.46e7.57 (bm,1H), 7.94

(d, 2H, J ¼ 8.3 Hz), 11.65 (exchangeable bs, 1H). ¹³C NMR (DMSO‑d₆)

isomer peaks)

d

(ppm): 1.22 (d, 6H, J ¼ 6.9 Hz), 1.34e1.55 (bm, 2H),

1.64e1.77 (bm, 1H), 1.85e1.94 (bm, 1H), 2.07 (s, 3H), 2.10* (s, 3H),

2.90e3.05 (bm, 1H), 2.97 (sept, 1H, J ¼ 7.0 Hz), 3.08e3.20 (m, 1H),

3.35e3.45 (bm, 1H), 3.65e3.77 (bm, 1H), 4.48e4.59 (bm, 1H), 6.51*

(bs, 1H), 6.54 (bs, 1H), 6.68 (dd, 1H, J ¼ 8.2, 2.6 Hz), 7.04 (d, 1H,

J ¼ 8.2 Hz), 7.41 (d, 2H, J ¼ 8.2 Hz), 7.93 (d, 2H, J ¼ 8.4 Hz), 9.39

d

(ppm): 23.48 (2C), 27.92, 28.40, 33.49, 40.81, 42.15, 46.04, 126.80

(2C), 128.53 (2C), 133.32, 140.43, 154.20 (2C), 162.31, 163.45, 201.34.

HPLC analysis: retention time ¼ 10.682 min; peak area, 99%

(254 nm). Elemental analysis for C₂₁H₂₄N₂O₃, calculated: % C, 71.57;

% H, 6.86; % N, 7.95; found: % C, 71.88; % H, 6.89; % N, 8.04.

(exchangeable s, 1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 17.49, 23.43

(2C), 28.39, 28.82, 33.44, 42.21, 45.30, 45.81, 112.01, 115.48, 123.17,

126.76 (2C), 128.47 (2C), 131.13, 133.27, 137.32, 154.15, 155.13,

168.34, 201.34. HPLC analysis: retention time ¼ 12.215 min; peak

area, 98% (254 nm). Elemental analysis for C₂₃H₂₇NO₃, calculated: %

C, 75.59; % H, 7.45; % N, 3.83; found: % C, 75.68; % H, 7.40; % N, 3.69.

4.1.17.10. (4-(4-Cyclohexylbenzoyl)piperidin-1-yl)(3-hydroxyphenyl)

methanone (18). Light yellow solid; 28% yield from 49, eluent n-

hexane/EtOAc 6:4.¹H NMR (DMSO‑d₆)

d (ppm): 1.30e1.55 (bm, 5H),

1.66e1.94 (bm, 8H), 2.53e2.64 (bm, 1H), 2.86e3.27 (bm, 3H),

3.56e3.80 (bm, 2H), 4.36e4.53 (bm, 1H), 6.71e6.75 (m, 1H), 6.77

(dt, 1H, J ¼ 7.7, 1.2 Hz), 6.81 (ddd, 1H, J ¼ 8.2, 2.5, 0.9 Hz), 7.22 (t, 1H,

J ¼ 7.8 Hz), 7.38 (d, 2H, J ¼ 8.3 Hz), 7.93 (d, 2H, J ¼ 8.4 Hz), 9.67

4.1.17.6. (1-(4-Hydroxypicolinoyl)piperidin-4-yl)(4-isopropylphenyl)

methanone (14). White solid; 56% yield from 23 and 4-

hydroxypyridine-2-carboxylic acid 38, eluent CHCl₃/MeOH

95:5.¹H NMR (DMSO‑d₆)

d

(ppm): 1.22 (d, 6H, J ¼ 6.9 Hz), 1.43e1.60

(exchangeable s, 1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 25.46, 26.18

(bm, 2H), 1.67e1.77 (bm, 1H), 1.83e1.93 (bm, 1H), 2.91e3.05 (m,

2H), 3.14e3.26 (bm, 1H), 3.66e3.80 (bm, 2H), 4.41e4.52 (bm, 1H),

6.70e6.94 (bm, 2H), 7.41 (d, 2H, J ¼ 8.3 Hz), 7.94 (d, 2H, J ¼ 8.4 Hz),

8.16e8.30 (bm, 1H), 10.94 (exchangeable bs, 1H). ¹³C NMR (acetone-

(2C), 28.50 (2C), 33.51 (2C), 42.24, 43.76, 46.43 (2C), 113.36, 116.27,

117.04, 127.15 (2C), 128.48 (2C), 129.58, 133.31, 137.49, 153.26,

157.25, 168.90, 201.37. HPLC analysis: retention time ¼ 13.220 min;

peak area, 96% (254 nm). Elemental analysis for C₂₅H₂₉NO₃,

calculated: % C, 76.70; % H, 7.47; % N, 3.58; found: % C, 77.05; % H,

7.80; % N, 3.86.

d₆)

d (ppm): 23.93 (2C), 34.88, 42.23 (2C), 43.68, 47.25 (2C), 112.72,

114.11, 127.62 (2C), 129.45 (2C), 129.64, 131.96, 134.81, 147.92,

155.25, 166.92, 201.83. HPLC analysis: retention time ¼ 9.940 min;

peak area, 99% (254 nm). Elemental analysis for C₂₁H₂₄N₂O₃,

calculated: % C, 71.57; % H, 6.86; % N, 7.95; found: % C, 71.60; % H,

6.93; % N, 8.01.

4.1.17.11. (4-(4-Cyclopropylbenzoyl)piperidin-1-yl)(3-

hydroxyphenyl)methanone (19). White solid; 59% yield from 46,

eluent n-hexane/EtOAc 5:5.¹H NMR (DMSO‑d₆)

d (ppm): 0.74e0.80

(m, 2H), 1.01e1.09 (m, 2H), 1.41e1.55 (m, 2H), 1.63e1.94 (bm, 2H),

1.96e2.05 (m, 1H), 2.87e3.05 (bm, 1H), 3.07e3.25 (bm, 1H),

3.58e3.77 (bm, 2H), 4.40e4.52 (bm, 1H), 6.72e6.75 (m, 1H), 6.76

(dt, 1H, J ¼ 7.6, 1.2 Hz), 6.81 (ddd, 1H, J ¼ 8.1, 2.4, 0.9 Hz), 7.19e7.26

(m, 3H), 7.88 (d, 2H, J ¼ 8.4 Hz), 9.67 (exchangeable s, 1H). ¹³C NMR

4.1.17.7. (1-(5-Hydroxynicotinoyl)piperidin-4-yl)(4-isopropylphenyl)

methanone (15). Light yellow solid; 57% yield from 23 and 5-

hydroxynicotinic acid 39, eluent CHCl₃/MeOH 95:5.¹H NMR

(DMSO‑d₆)

d

(ppm): 1.22 (d, 6H, J ¼ 6.9 Hz), 1.45e1.58 (m, 2H),

1.68e1.96 (bm, 2H), 2.93e3.09 (bm, 1H), 2.97 (sept, 1H, J ¼ 6.9 Hz),

3.18e3.37 (m, 1H), 3.55e3.68 (bm, 1H), 3.73 (tt, 1H, J ¼ 11.3, 3.5 Hz),

4.39e4.54 (bm, 1H), 7.14 (dd, 1H, J ¼ 2.7, 1.8 Hz), 7.41 (d, 2H,

J ¼ 8.2 Hz), 7.94 (d, 2H, J ¼ 8.4 Hz), 8.04 (d, 1H, J ¼ 1.7 Hz), 8.18 (d,

1H, J ¼ 2.8 Hz), 10.25 (exchangeable bs, 1H). ¹³C NMR (DMSO‑d₆)

(DMSO‑d₆) d (ppm): 10.49 (2C), 15.33, 28.49 (2C), 42.17, 46.44 (2C),

113.35, 116.27, 117.04, 125.49 (2C), 128.41 (2C), 129.58, 132.64,

137.48, 150.19, 157.24, 168.90, 201.16. HPLC analysis: retention

time ¼ 11.559 min; peak area, 99% (254 nm). Elemental analysis for

C

₂₂H₂₃NO₃, calculated: % C, 75.62; % H, 6.63; % N, 4.01; found: % C,

d

(ppm): 23.44 (2C), 28.22, 28.59, 33.46, 40.99, 42.15, 46.51, 120.10,

75.83; % H, 6.59; % N, 4.00.

126.76 (2C), 128.50 (2C), 132.50, 133.30, 137.81, 138.83, 153.32,

154.14, 166.51, 201.28. HPLC analysis: retention time ¼ 11.049 min;

peak area, 98% (254 nm). Elemental analysis for C₂₁H₂₄N₂O₃,

calculated: % C, 71.57; % H, 6.86; % N, 7.95; found: % C, 71.66; % H,

7.02; % N, 8.14.

4.1.17.12. (1-(3-Hydroxybenzoyl)piperidin-4-yl)(4-(phenylamino)

phenyl)methanone (20a). Yellow solid; 25% yield from 59, eluent

CHCl₃/MeOH 98:2.¹H NMR (DMSO‑d₆)

d (ppm): 1.40e1.90 (m, 5H),

2.83e3.06 (bm, 1H), 3.55e3.72 (m, 2H), 4.37e4.55 (bm, 1H),

6.72e6.79 (m, 2H), 6.81 (ddd, 1H, J ¼ 8.2, 2.4, 0.9 Hz), 7.00 (t, 1H,

J ¼ 7.3 Hz), 7.07 (d, 2H, J ¼ 8.9 Hz), 7.17e7.21 (m, 2H), 7.23 (t, 1H,

J ¼ 7.8 Hz), 7.29e7.36 (m, 2H), 7.89 (d, 2H, J ¼ 8.9 Hz), 8.85

(exchangeable s,1H), 9.67 (exchangeable s,1H). ¹³C NMR (DMSO‑d₆)

4.1.17.8. (1-(2-Hydroxyisonicotinoyl)piperidin-4-yl)(4-

isopropylphenyl)methanone (16). Amber solid; 50% yield from 23

and 2-hydroxypyridine-4-carboxylic acid 40, eluent CHCl₃/MeOH

95:5.¹H NMR (DMSO‑d₆)

d

(ppm): 1.22 (d, 6H, J ¼ 6.9 Hz), 1.42e1.55

d (ppm): 28.81, 41.08, 41.72, 46.56, 113.37, 113.96 (2C), 116.29,117.07,

(m, 2H), 1.71e1.81 (bm, 1H), 1.82e1.92 (bm, 1H), 2.90e3.03 (m, 2H),

3.14e3.29 (m, 1H), 3.56e3.66 (m, 1H), 3.72 (tt, 1H, J ¼ 11.0, 3.1 Hz),

4.36e4.45 (m, 1H), 6.12 (dd, 1H, J ¼ 6.6, 1.6 Hz), 6.23 (d, 1H,

J ¼ 0.8 Hz), 7.41 (d, 2H, J ¼ 8.2 Hz), 7.44 (d, 1H, J ¼ 6.8 Hz), 7.93 (d,

2H, J ¼ 8.3 Hz), 11.74 (exchangeable bs, 1H). ¹³C NMR (DMSO‑d₆)

119.52 (3C), 122.07, 125.84, 129.36 (2C), 129.62, 130.54, 137.54,

141.14, 148.72, 157.29, 168.93, 199.27. HPLC analysis: retention

time ¼ 11.606 min; peak area, 96% (254 nm). Elemental analysis for

C

₂₅H₂₄N₂O₃, calculated: % C, 74.98; % H, 6.04; % N, 7.00; found: % C,

74.70; % H, 5.87; % N, 7.12.

d

(ppm): 23.46 (2C), 28.07, 28.55, 33.47, 40.50, 42.12, 45.99, 102.88,

116.45, 126.77 (2C), 128.51 (2C), 133.29, 136.50, 148.57, 154.14,

161.99, 166.25, 201.24. HPLC analysis: retention time ¼ 10.364 min;

peak area, 97% (254 nm). Elemental analysis for C₂₁H₂₄N₂O₃,

calculated: % C, 71.57; % H, 6.86; % N, 7.95; found: % C, 71.45; % H,

6.99; % N, 7.90.

4.1.17.13. (1-(3-Hydroxybenzoyl)piperidin-4-yl)(4-phenoxyphenyl)

methanone (20b). White solid; 78% yield from 60, n-hexane/EtOAc

5:5.¹H NMR (DMSO‑d₆)

d (ppm): 1.43e1.55 (m, 2H), 1.67e1.94 (bm,

2H), 2.89e3.26 (bm, 2H), 3.59e3.77 (m, 2H), 4.39e4.53 (bm, 1H),

6.72e6.75 (m, 1H), 6.77 (dt, 1H, J ¼ 7.4, 1.2 Hz), 6.82 (ddd, 1H, J ¼ 8.1,

2.4, 0.9 Hz), 7.06 (AA’XX’, 2H, J_AX¼ 8.9 Hz, J_AA’/XX’¼ 2.4 Hz),

7.11e7.16 (m, 2H), 7.19e7.28 (m, 2H), 7.42e7.50 (m, 2H), 8.04

(AA’XX’, 2H, J_AX¼ 8.9 Hz, J_AA’/XX’¼ 2.4 Hz), 9.67 (exchangeable s,

4.1.17.9. (1-(6-Hydroxypicolinoyl)piperidin-4-yl)(4-isopropylphenyl)

methanone (17). White solid; 53% yield from 23 and 6-

hydroxypyridine-2-carboxylic acid 41, eluent CHCl₃/MeOH

1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 28.47 (2C), 40.94, 42.20, 46.42,

98:2.¹H NMR (DMSO‑d₆)

d

(ppm): 1.22 (d, 6H, J ¼ 6.9 Hz), 1.45e1.68

113.38,116.29,117.07,117.30 (2C),120.01 (2C),124.80,129.60,130.16,

21

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

130.35 (2C), 130.87 (2C), 137.49, 154.95, 157.27, 161.31, 168.91,

200.37. HPLC analysis: retention time ¼ 12.218 min; peak area, 95%

(254 nm). Elemental analysis for C₂₅H₂₃NO₄, calculated: % C, 74.79;

% H, 5.77; % N, 3.49; found: % C, 74.89; % H, 5.86; % N, 3.70.

4.1.17.18. (1-(3-Hydroxybenzoyl)piperidin-4-yl)(4-(1-phenyl-1H-

1,2,3-triazol-4-yl)phenyl)methanone (20g). White solid; 61% yield

from 83, CHCl₃/MeOH 97:3.¹H NMR (DMSO‑d₆)

d (ppm): 1.46e1.60

(m, 2H), 1.75e1.98 (bm, 2H), 2.92e3.35 (bm, 2H), 3.62e3.76 (bm,

1H), 3.81 (tt,1H, J ¼ 11.2, 3.4 Hz), 4.42e4.55 (bm,1H), 6.75e6.85 (m,

3H), 7.23 (t,1H, J ¼ 7.8 Hz), 7.54 (t, 1H, J ¼ 7.3 Hz), 7.62e7.69 (m, 2H),

7.95e8.00 (m, 2H), 8.08e8.18 (m, 4H), 9.48 (s, 1H), 9.67

4.1.17.14. (1-(3-Hydroxybenzoyl)piperidin-4-yl)(4-(phenylthio)

phenyl)methanone (20c). White solid; 65% yield from 63, n-hexane/

(exchangeable s, 1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 28.46 (2C),

EtOAc 6:4.¹H NMR (DMSO‑d₆)

d (ppm): 1.40e1.53 (m, 2H),

42.38, 113.37, 116.28, 117.05, 120.12 (2C), 120.93, 125.48 (2C), 128.90,

129.17 (2C), 129.57, 129.95 (2C), 134.66, 134.83, 136.50, 137.48,

146.26, 157.24, 168.92, 201.27. HPLC analysis: retention

time ¼ 11.670 min; peak area, 98% (254 nm). Elemental analysis for

1.65e1.92 (bm, 2H), 2.86e3.24 (bm, 2H), 3.58e3.73 (m, 2H),

4.37e4.52 (bm, 1H), 6.71e6.74 (m, 1H), 6.76 (dt, 1H, J ¼ 7.5, 1.3 Hz),

6.81 (ddd, 1H, J ¼ 8.1, 2.4, 1.0 Hz), 7.22 (t, 1H, J ¼ 7.8 Hz), 7.26

(AA’XX’, 2H, J_AX¼ 8.7 Hz, J_AA’/XX’¼ 2.0 Hz), 7.46e7.55 (m, 5H), 7.94

(AA’XX’, 2H, J_AX¼ 8.7 Hz, J_AA’/XX’¼ 2.0 Hz), 9.66 (exchangeable s,

C

₂₇H₂₄N₄O₃, calculated: % C, 71.67; % H, 5.35; % N, 12.38; found: % C,

72.04; % H, 5.51; % N, 12.60.

1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 28.37 (2C), 40.97, 42.32, 46.32,

113.37, 116.29, 117.06, 126.68, 127.44, 128.42, 129.13, 129.23, 129.60,

130.06 (2C), 131.19, 132.94, 133.56 (2C), 137.47, 143.68, 157.26,

168.91, 200.86. HPLC analysis: retention time ¼ 12.659 min; peak

area, 95% (254 nm). Elemental analysis for C₂₅H₂₃NO₃S, calculated:

% C, 71.92; % H, 5.55; % N, 3.35; found: % C, 72.20; % H, 5.40; % N,

2.99.

4.1.17.19. (4-(4-(1-Benzyl-1H-1,2,3-triazol-4-yl)benzoyl)piperidin-1-

yl)(3-hydroxyphenyl)methanone (20h). Light yellow solid; 8% yield

from 84, CHCl₃/MeOH 98:2.¹H NMR (DMSO‑d₆)

d (ppm): 1.45e1.60

(m, 2H), 1.70e1.98 (bm, 2H), 2.90e3.24 (bm, 2H), 3.55e3.83 (bm,

2H), 4.35e4.60 (bm, 1H), 5.67 (s, 2H), 6.73e6.80 (m, 2H), 6.81 (ddd,

1H, J ¼ 8.2, 2.3, 0.9 Hz), 7.23 (t, 1H, J ¼ 7.8 Hz), 7.32e7.43 (m, 5H),

8.01 (d, 2H, J ¼ 8.6 Hz), 8.08 (d, 2H, J ¼ 8.5 Hz), 8.80 (s, 1H), 9.67

4.1.17.15. (1-(3-Hdroxybenzoyl)piperidin-4-yl)(4-(phenylsulfonyl)

(exchangeable bs, 1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 28.71 (2C),

phenyl)methanone (20d). Off-white solid; 75% yield from 68, n-

38.20, 42.31, 53.11, 79.13, 113.34, 116.26, 117.03, 122.83, 125.28,

127.90 (5C), 128.19, 128.80, 129.07, 129.55, 134.50, 135.06, 135.80,

137.46, 145.60, 157.23, 168.89, 201.21. HPLC analysis: retention

time ¼ 11.351 min; peak area, 99% (254 nm). Elemental analysis for

hexane/EtOAc 3:7.¹H NMR (DMSO‑d₆)

d (ppm): 1.39e1.52 (m, 2H),

1.72e1.90 (bm, 2H), 2.89e3.22 (bm, 2H), 3.58e3.70 (bm, 1H), 3.74

(tt, 1H, J ¼ 11.0, 3.5 Hz), 4.34e4.50 (bm, 1H), 6.71-6-73 (m, 1H), 6.76

(dt, 1H, J ¼ 7.4, 1.2 Hz), 6.81 (ddd, 1H, J ¼ 8.1, 2.4, 1.0 Hz), 7.21 (t, 1H,

J ¼ 7.8 Hz), 7.62e7.68 (m, 2H), 7.73 (tt, 1H, J ¼ 7.4, 1.6 Hz), 7.98e8.03

(m, 2H), 8.10 (d, 2H, J ¼ 8.7 Hz), 8.17 (d, 2H, J ¼ 8.7 Hz), 9.64

C

₂₈H₂₆N₄O₃, calculated: % C, 72.09; % H, 5.62; % N, 12.01; found: % C,

72.10; % H, 5.68; % N, 11.83.

(exchangeable s, 1H). ¹³C NMR (DMSO‑d₆)

d

(ppm): 28.07, 42.88,

4.1.17.20. (1-(3-Hydroxybenzoyl)piperidin-4-yl)(4-(1-(3-

113.36, 116.28, 117.02, 127.57 (2C), 127.85 (2C), 129.48 (2C), 129.54,

129.88 (2C), 134.10, 137.41, 139.30, 140.33, 144.58, 157.23, 168.90,

201.28. HPLC analysis: retention time ¼ 11.233 min; peak area, 99%

(254 nm). Elemental analysis for C₂₅H₂₃NO₅S, calculated: % C,

66.80; % H, 5.16; % N, 3.12; found: % C, 66.87; % H, 5.15; % N, 3.01.

phenylpropyl)-1H-1,2,3-triazol-4-yl)phenyl)methanone

White solid; 12% yield from 85, CHCl₃/MeOH 98:2.¹H NMR

(DMSO‑d₆) (ppm): 1.43e1.60 (m, 2H), 1.70e1.98 (bm, 2H), 2.20

(20i).

d

(quint, 2H, J ¼ 7.2 Hz), 2.62 (t, 2H, J ¼ 7.8 Hz), 2.90e3.25 (bm, 2H),

3.54e3.85 (bm, 2H), 4.39e4.53 (bm, 1H), 4.44 (t, 2H, J ¼ 7.2 Hz),

6.73e6.85 (m, 3H), 7.16e7.26 (m, 4H), 7.27e7.33 (m, 2H), 8.01 (d,

2H, J ¼ 8.6 Hz), 8.10 (d, 2H, J ¼ 8.5 Hz), 8.79 (s, 1H), 9.67

4.1.17.16. (1-(3-Hydroxybenzoyl)piperidin-4-yl)(4-(phenylsulﬁnyl)

phenyl)methanone (20e). Light yellow solid; 78% yield from 20c, n-

(exchangeable bs, 1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 28.43 (2C),

31.18, 31.87, 40.14, 42.34, 46.53, 49.19, 113.37, 116.28, 117.06, 122.60,

125.24, 126.00, 128.33, 128.38 (5C), 129.10, 129.58, 134.44, 135.30,

137.48, 140.69, 145.31, 157.25, 168.92, 201.23. HPLC analysis:

retention time ¼ 11.959 min; peak area, 97% (254 nm). Elemental

analysis for C₃₀H₃₀N₄O₃, calculated: % C, 72.85; % H, 6.11; % N, 11.33;

found: % C, 72.95; % H, 6.44; % N, 11.20.

hexane/EtOAc 2:8.¹H NMR (DMSO‑d₆)

d (ppm): 1.38e1.52 (m, 2H),

1.63e1.92 (bm, 2H), 2.84e3.25 (bm, 2H), 3.54e3.80 (m, 2H),

4.31e4.53 (bm, 1H), 6.69e6.84 (m, 3H), 7.22 (t, 1H, J ¼ 7.8 Hz),

7.48e7.59 (m, 3H), 7.72e7.80 (m, 2H), 7.88 (d, 2H, J ¼ 8.4 Hz), 8.13

(d, 2H, J ¼ 8.3 Hz), 9.67 (exchangeable bs, 1H). ¹³C NMR (DMSO‑d₆)

d

(ppm): 28.22 (2C), 42.65, 46.40, 113.36, 116.28, 117.05, 124.28 (2C),

124.32 (2C), 129.30 (2C), 129.57, 129.68 (2C), 131.45, 137.35, 137.43,

145.31, 150.77, 157.24, 168.89, 201.33. HPLC analysis: retention

time ¼ 10.290 min; peak area, 99% (254 nm). Elemental analysis for

4.1.17.21. (1-(4-Fluoro-3-hydroxybenzoyl)piperidin-4-yl)(4-(phenyl-

thio)phenyl)methanone (21a). White solid; 78% yield from 64, n-

hexane/EtOAc 5:5.¹H NMR (DMSO‑d₆)

d (ppm): 1.37e1.53 (m, 2H),

C

₂₅H₂₃NO₄S, calculated: % C, 69.26; % H, 5.35; % N, 3.23; found: % C,

1.68e1.78 (bm, 1H), 1.83e1.92 (bm, 1H), 2.90e3.11 (m, 1H),

3.12e3.26 (bm, 1H), 3.43e3.51 (m, 1H), 3.63e3.73 (m, 1H),

4.45e4.53 (bm, 1H), 6.63e6.69 (m, 1H), 6.77e6.83 (m, 1H), 7.08 (t,

1H, J ¼ 9.1 Hz), 7.26 (AA’XX’, 2H, J_AX¼ 8.7 Hz, J_AA’/XX’¼ 1.9 Hz),

7.46e7.55 (m, 5H), 7.93 (AA’XX’, 2H, J_AX¼ 8.7 Hz, J_AA’/XX’¼ 2.0 Hz),

69.34; % H, 5.26; % N, 2.88.

4.1.17.17. (4-(4-Benzylbenzoyl)piperidin-1-yl)(3-hydroxyphenyl)

methanone (20f). White solid; 79% yield from 52, eluent n-hexane/

EtOAc 5:5.¹H NMR (DMSO‑d₆)

d

(ppm): 1.40e1.53 (m, 2H),

9.65 (exchangeable s, 1H). ¹³C NMR (DMSO‑d₆)

d (ppm): 28.23,

1.67e1.91 (bm, 2H), 2.89e3.24 (bm, 2H), 3.60e3.76 (m, 2H), 4.02 (s,

2H), 4.37e4.52 (bm, 1H), 6.71e6.74 (m, 1H), 6.76 (dt, 1H, J ¼ 7.8,

1.2 Hz), 6.81 (ddd, 1H, J ¼ 8.2, 2.5, 1.0 Hz), 7.16e7.33 (m, 6H), 7.39 (d,

28.53, 40.57, 42.16, 45.90, 113.96, 116.42 (d, J_CF¼ 23.2 Hz), 117.20 (d,

J_CF¼ 7.9 Hz), 124.66 (d, J_CF¼ 20.2 Hz), 127.43 (2C), 129.13, 129.23

(2C), 130.06 (2C), 131.18, 132.90, 133.55 (2C), 143.71, 150.71 (d,

J_CF¼ 235.2 Hz), 153.74 (d, J_CF¼ 1.5 Hz), 163.78, 200.77. HPLC anal-

ysis: retention time ¼ 12.853 min; peak area, 96% (254 nm).

Elemental analysis for C₂₅H₂₂FNO₃S, calculated: % C, 68.95; % H,

5.09; % N, 3.22; found: % C, 68.85; % H, 5.13; % N, 3.39.

2H, J ¼ 8.4 Hz), 7.93 (d, 2H, J ¼ 8.4 Hz), 9.66 (exchangeable s,1H). ¹³

C

NMR (DMSO‑d₆)

d (ppm): 28.41 (2C), 40.89, 42.28, 46.29 (2C),

113.34, 116.25, 117.02, 126.16, 128.50 (2C), 128.55 (2C), 128.75 (2C),

129.09 (2C), 129.55, 133.39, 137.46, 140.40, 147.02, 157.23, 168.88,

201.39. HPLC analysis: retention time ¼ 12.325 min; peak area, 98%

(254 nm). Elemental analysis for C₂₆H₂₅NO₃, calculated: % C, 78.17;

% H, 6.31; % N, 3.51; found: % C, 77.81; % H, 6.15; % N, 3.28.

4.1.17.22. (4-(4-Benzylbenzoyl)piperidin-1-yl)(2-ﬂuoro-5-

hydroxyphenyl)methanone (21b). White solid; 90% yield from 53,

22

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

eluent n-hexane/EtOAc 6:4.¹H NMR (DMSO‑d₆)

d

(ppm): 1.39e1.52

(m, 2H).

(bm, 2H), 1.69e1.77 (m, 1H), 1.84e1.91 (bm, 1H), 2.93e3.02 (bm,

1H), 3.15e3.26 (bm, 1H), 3.43e3.51 (bm, 1H), 3.67e3.76 (bm, 1H),

4.02 (s, 2H), 4.44e4.54 (m, 1H), 6.59e6.69 (m, 1H), 6.77e6.83 (m,

1H), 7.07 (t, 1H, J ¼ 9.0 Hz), 7.20 (tt, 1H, J ¼ 7.1, 1.6 Hz), 7.23e7.32 (m,

4H), 7.39 (d, 2H, J ¼ 8.4 Hz), 7.93 (d, 2H, J ¼ 8.4 Hz), 9.66

4.1.17.27. (4-(4-Isopropylbenzoyl)piperidin-1-yl)(5-methoxy-2-

nitrophenyl)methanone (29). 59% yield from 23 and 5-methoxy-2-

nitrobenzoic acid 28.¹H NMR (CDCl₃; asterisk denotes isomer

peaks)

d

(ppm): 1.27 (d, 6H, J ¼ 6.9 Hz), 1.69e1.88 (bm, 3H),

(exchangeable s, 1H). ¹³C NMR (DMSO‑d₆)

d

(ppm): 28.22, 28.55,

2.06e2.15 (bm, 1H), 2.99 (sept, 1H, J ¼ 6.9 Hz), 3.09e3.25 (bm, 2H),

3.43e3.60 (bm, 2H), 3.92 (s, 3H), 4.58e4.75 (m, 1H), 6.78e6.82* (m,

1H), 6.83 (d, 1H, J ¼ 2.6 Hz), 6.98 (dd, 1H, J ¼ 9.3, 2.7 Hz), 7.29e7.32*

(m, 1H), 7.34 (d, 2H, J ¼ 8.1 Hz), 7.83e7.86* (m, 1H), 7.88 (d, 2H,

J ¼ 8.4 Hz), 8.21 (d, 1H, J ¼ 9.2 Hz).

40.55, 40.89, 42.12, 45.85, 113.92 (d, J_CF¼ 3.9 Hz), 116.38 (d,

J_CF¼ 23.1 Hz),117.17 (d, J_CF¼ 7.7 Hz),124.66 (d, J_CF¼ 20.1 Hz),126.15,

128.49 (2C), 128.55 (2C), 128.74 (2C), 129.09 (2C), 133.35, 140.39,

147.04, 150.69 (d, J_CF¼ 235.2 Hz), 153.72, 163.76, 201.30. HPLC

analysis: retention time ¼ 12.537 min; peak area, 97% (254 nm).

Elemental analysis for C₂₆H₂₄FNO₃, calculated: % C, 74.80; % H, 5.79;

% N, 3.36; found: % C, 74.99; % H, 5.48; % N, 3.03.

4.1.17.28. Methyl 5-methoxy-2-nitrobenzoate (30). 91% yield from

5-methoxy-2-nitrobenzoic acid 28.¹H NMR (CDCl₃)

d (ppm): 3.92 (s,

3H), 3.94 (s, 3H), 7.02 (dd, 1H, J ¼ 9.0, 2.8 Hz), 7.05 (d, 1H,

4.1.17.23. (1-(2,4-Diﬂuoro-5-hydroxybenzoyl)piperidin-4-yl)(4-(phe-

nylthio)phenyl)methanone (22a). White solid; 45% yield from 65, n-

J ¼ 2.6 Hz)), 8.04 (d, 1H, J ¼ 9.0 Hz).

hexane/EtOAc 5:5.¹H NMR (DMSO‑d₆)

d

(ppm): 1.37e1.52 (m, 2H),

4.1.17.29. Methyl 2-amino-5-methoxybenzoate (31). 85% yield from

1.68e1.79 (bm, 1H), 1.82e1.93 (bm, 1H), 2.91e3.02 (m, 1H),

3.13e3.25 (bm, 1H), 3.43e3.53 (bm, 1H), 3.63e3.74 (m, 1H),

4.42e4.52 (m, 1H), 6.88 (dd, 1H, J ¼ 9.5, 6.9 Hz), 7.23e7.33 (m, 3H),

7.44e7.58 (m, 5H), 7.93 (d, 2H, J ¼ 8.6 Hz), 10.14 (exchangeable bs,

30.¹H NMR (CDCl₃)

d (ppm): 3.77 (s, 3H), 3.88 (s, 3H), 6.68 (d, 1H,

J ¼ 8.9 Hz), 6.96 (dd, 1H, J ¼ 8.9, 3.0 Hz), 7.36 (d, 1H, J ¼ 3.0 Hz).

4.1.17.30. 2-Amino-5-methoxybenzoic acid (32). 99% yield from

1H). ¹³C NMR (DMSO‑d₆)

d

(ppm): 28.18, 28.52, 40.69, 42.11, 45.92,

31.¹H NMR (DMSO‑d₆)

6.94 (dd, 1H, J ¼ 9.0, 3.1 Hz), 7.18 (d, 1H, J ¼ 3.1 Hz), 8.35 (bs, 2H).

d

(ppm): 3.65 (s, 3H), 6.70 (d, 1H, J ¼ 9.0 Hz),

104.98 (dd, J_CF¼ 27.4, 23.0 Hz), 116.00 (t, J_CF¼ 4.9 Hz), 120.00 (dd,

J_CF¼ 19.5, 4.1 Hz), 127.41 (2C), 129.09, 129.20 (2C), 130.02 (2C),

131.17, 132.89, 133.52 (2C), 141.89 (d, J_CF¼ 15.1 Hz), 143.68, 149.01

(dd, J_CF¼ 109.0, 11.3 Hz), 151.42 (dd, J_CF¼ 117.2, 11.3 Hz), 163.10,

200.74. HPLC analysis: retention time ¼ 13.036 min; peak area, 95%

(254 nm). Elemental analysis for C₂₅H₂₁F₂NO₃S, calculated: % C,

66.21; % H, 4.67; % N, 3.09; found: % C, 65.84; % H, 4.96; % N, 2.80.

4.1.17.31. (1-(2-Amino-5-methoxybenzoyl)piperidin-4-yl)(4-

isopropylphenyl)methanone (33). 54% yield from 23 and 32.¹H NMR

(CDCl₃)

d

(ppm): 1.27 (d, 6H, J ¼ 6.9 Hz), 1.76e1.98 (bm, 4H),

2.50e2.86 (bm, 3H), 2.97 (sept, 1H, J ¼ 6.9 Hz), 3.06e3.20 (m, 2H),

3.53 (tt, 1H, J ¼ 10.7, 4.0 Hz), 3.74 (s, 3H), 4.15e4.50 (bm, 1H), 6.69

(d, 1H, J ¼ 2.7 Hz), 6.75 (d, 1H, J ¼ 8.8 Hz), 6.80 (dd, 1H, J ¼ 8.7,

2.7 Hz), 7.33 (d, 2H, J ¼ 8.2 Hz), 7.88 (d, 2H, J ¼ 8.3 Hz).

4.1.17.24. (4-(4-Benzylbenzoyl)piperidin-1-yl)(2,4-diﬂuoro-5-

hydroxyphenyl)methanone (22b). White solid; 78% yield from 54,

eluent n-hexane/EtOAc 5:5.¹H NMR (DMSO‑d₆)

d

(ppm): 1.38e1.52

4.1.17.32. Methyl 5-methoxy-2-methylbenzoate (35). 85% yield from

(bm, 2H), 1.69e1.78 (bm, 1H), 1.83e1.92 (bm, 1H), 2.92e3.03 (m,

1H), 3.14e3.27 (bm, 1H), 3.43e3.53 (bm, 1H), 3.67e3.77 (bm, 1H),

4.02 (s, 2H), 4.42e4.52 (m, 1H), 6.89 (dd, 1H, J ¼ 9.5, 6.7 Hz), 7.20 (tt,

1H, J ¼ 7.0, 1.8 Hz), 7.22e7.34 (m, 5H), 7.39 (d, 2H, J ¼ 8.4 Hz), 7.93

(d, 2H, J ¼ 8.4 Hz), 10.14 (exchangeable s, 1H). ¹³C NMR (DMSO‑d₆)

5-hydroxy-2-methylbenzoic acid 34.¹H NMR (CDCl₃)

d (ppm): 2.52

(s, 3H), 3.82 (s, 3H), 3.89 (s, 3H), 6.96 (dd,1H, J ¼ 8.4, 2.8 Hz), 7.15 (d,

1H, J ¼ 8.4 Hz), 7.44 (d, 1H, J ¼ 2.9 Hz).

4.1.17.33. 5-Methoxy-2-methylbenzoic acid (36). 77% yield from

d

(ppm): 28.22, 28.58, 40.71, 40.90, 42.11, 45.93, 105.00 (dd,

35.¹H NMR (acetone-d₆)

d (ppm): 2.50 (s, 3H), 3.82 (s, 3H), 7.03 (dd,

J_CF¼ 27.4, 23.0 Hz), 115.96 (t, J_CF¼ 4.6 Hz), 120.03 (dd, J_CF¼ 19.6,

4.0 Hz), 126.17, 128.51 (2C), 128.57 (2C), 128.75 (2C), 129.10 (2C),

133.33, 140.41, 141.84 (dd, J_CF¼ 12.4, 2.9 Hz), 147.08, 149.02 (dd,

J_CF¼ 104.3, 11.2 Hz), 151.99 (dd, J_CF¼ 111.3, 11.2 Hz), 163.10, 201.30.

HPLC analysis: retention time ¼ 12.712 min; peak area, 98%

(254 nm). Elemental analysis for C₂₆H₂₃F₂NO₃, calculated: % C,

71.71; % H, 5.32; % N, 3.22; found: % C, 72.10; % H, 5.16; % N, 3.24.

1H, J ¼ 8.4, 2.8 Hz), 7.21 (d, 1H, J ¼ 8.5 Hz), 7.48 (d, 1H, J ¼ 2.9 Hz),

11.14 (bs, 1H).

4.1.17.34. (4-(4-Isopropylbenzoyl)piperidin-1-yl)(5-methoxy-2-

methylphenyl)methanone (37). 70% yield from amine 23 and 36.¹H

NMR (CDCl₃; asterisk denotes isomer peaks)

d (ppm): 1.27 (d, 6H,

J ¼ 6.9 Hz), 1.68e1.86 (bm, 3H), 1.98e2.09 (bm, 1H), 2.22 (s, 3H),

2.26* (s, 3H), 2.97 (sept, 1H, J ¼ 6.9 Hz), 3.02e3.17 (bm, 2H),

3.45e3.56 (bm, 1H), 3.56e3.66 (m, 1H), 3.78 (s, 3H), 4.67e4.82 (bm,

1H), 6.67e6.77 (m, 1H), 6.82 (dd, 1H, J ¼ 8.5, 2.7 Hz), 7.08e7.16 (m,

1H), 7.33 (d, 2H, J ¼ 8.2 Hz), 7.88 (d, 2H, J ¼ 8.3 Hz).

4.1.17.25. (1-(2,4-Diﬂuoro-5-methoxybenzoyl)piperidin-4-yl)(4-

isopropylphenyl)methanone (25). 84% yield from 23 to 2,4-diﬂuoro-

5-methoxybenzoic acid 24.¹H NMR (CDCl₃)

d (ppm): 1.27 (d, 6H,

J ¼ 6.9 Hz), 1.75e1.88 (bm, 3H), 1.98e2.06 (bm, 1H), 2.97 (sept, 1H,

J ¼ 7.0 Hz), 3.03e3.14 (bm, 1H), 3.16e3.31 (bm, 1H), 3.48e3.59 (bm,

1H), 3.65e3.75 (bm, 1H), 3.89 (s, 3H), 4.64e4.73 (bm, 1H), 6.88 (dd,

1H, J ¼ 10.7, 8.8 Hz), 6.99 (dd, 1H, J ¼ 9.1, 6.1 Hz), 7.33 (d, 2H,

J ¼ 8.2 Hz), 7.88 (d, 2H, J ¼ 8.4 Hz).

4.1.17.35. 1-(4-(4-Bromobenzoyl)piperidin-1-yl)ethanone

(43).

(ppm):

64% yield from 42 and bromobenzene. ¹H NMR (CDCl₃)

d

1.60e1.70 (m, 1H), 1.74e1.96 (m, 3H), 2.11 (s, 3H), 2.77e2.88 (m,

1H), 3.17e3.27 (m, 1H), 3.43 (tt, 1H, J ¼ 10.8, 4.0 Hz), 3.86e3.95 (m,

1H), 4.53e4.62 (m, 1H), 7.63 (AA’XX’, 2H, J_AX

J_AA’XX’¼ 2.1 Hz), 7.80 (AA’XX’, 2H, J_AX¼ 8.6 Hz, J_AA’XX’¼ 2.1 Hz).

¼

8.7 Hz,

4.1.17.26. (4-(4-Isopropylbenzoyl)piperidin-1-yl)(5-methoxy-2-(tri-

ﬂuoromethyl)phenyl)methanone (27). 75% yield from 23 and 5-

methoxy-2-(triﬂuoromethyl)benzoic acid 26.¹H NMR (CDCl₃;

4.1.17.36. 1-(4-(4-Cyclopropylbenzoyl)piperidin-1-yl)ethanone (44).

asterisk denotes isomer peaks)

d

(ppm): 1.27 (d, 6H, J ¼ 6.9 Hz),

92% yield from 43.¹H NMR (CDCl₃)

d (ppm): 0.75e0.81 (m, 2H),

1.66e1.87 (bm, 3H), 1.98e2.10 (bm, 1H), 2.90e3.05 (bm, 1H),

3.05e3.18 (bm, 2H), 3.43e3.58 (bm, 2H), 3.86* (s, 3H), 3.87 (s, 3H),

4.65e4.74 (bm, 1H), 6.79* (d, 1H, J ¼ 2.2 Hz), 6.84 (d, 1H, J ¼ 2.4 Hz),

6.95e7.01 (m, 1H), 7.29e7.36 (m, 2H), 7.58e7.64 (m, 1H), 7.83e7.91

1.03e1.11 (m, 2H), 1.57e1.91 (bm, 4H), 1.91e1.99 (m, 1H), 2.11 (s,

3H), 2.74e2.92 (bm, 1H), 3.11e3.29 (bm, 1H), 3.41e3.51 (m, 1H),

3.80e3.98 (bm,1H), 4.47e4.64 (bm,1H), 7.13 (d, 2H, J ¼ 8.3 Hz), 7.83

(d, 2H, J ¼ 8.4 Hz).

23

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European Journal of Medicinal Chemistry 209 (2021) 112857

4.1.17.37. (4-Cyclopropylphenyl)(piperidin-4-yl)methanone

(45).

7.88 (d, 2H, J ¼ 8.3 Hz).

86% yield from 44.¹H NMR (CDCl₃)

d (ppm): 0.75e0.81 (m, 2H),

1.03e1.10 (m, 2H), 1.67e1.80 (m, 2H), 1.83e1.92 (m, 2H), 1.92e1.99

(m, 1H), 2.44 (bs, 1H), 2.81 (td, 2H, J ¼ 12.1, 2.6 Hz), 3.22 (dt, 2H,

J ¼ 12.8, 3.6 Hz), 3.39 (tt, 1H, J ¼ 10.9, 3.8 Hz), 7.12 (AA’XX’, 2H,

J_AX¼ 8.3 Hz, J_AA’XX’¼ 1.9 Hz), 7.83 (AA’XX’, 2H, J_AX¼ 8.4 Hz, J_AA’XX

4.1.17.46. (4-(4-Benzylbenzoyl)piperidin-1-yl)(2,4-diﬂuoro-5-

methoxyphenyl)methanone (54). 73% yield from 51 to 2,4-diﬂuoro-

5-methoxybenzoic acid 24.¹H NMR (CDCl₃)

d (ppm): 1.75e1.87 (bm,

3H), 1.97e2.06 (bm, 1H), 3.01e3.13 (m, 1H), 3.14e3.31 (m, 1H),

3.45e3.57 (bm, 1H), 3.64e3.74 (m, 1H), 3.89 (s, 3H), 4.04 (s, 2H),

4.63e4.73 (m, 1H), 6.88 (dd, 1H, J ¼ 10.7, 8.7 Hz), 6.99 (dd, 1H,

J ¼ 9.1, 6.1 Hz), 7.15e7.20 (m, 2H), 7.23 (tt, 1H, J ¼ 7.3, 1.8 Hz),

7.27e7.34 (m, 4H), 7.87 (d, 2H, J ¼ 8.4 Hz).

¼ 1.9 Hz).

’

4.1.17.38. (4-(4-Cyclopropylbenzoyl)piperidin-1-yl)(3-

methoxyphenyl)methanone (46). 75% yield from 45 and 3-

methoxybenzoic acid. ¹H NMR (CDCl₃)

d (ppm): 0.75e0.81 (m,

2H), 1.04-1-11 (m, 2H), 1.72e1.87 (bm, 3H), 1.90e2.01 (m, 2H),

2.95e3.23 (bm, 2H), 3.45e3.57 (bm, 1H), 3.74e3.97 (bm, 1H), 3.82

(s, 3H), 4.56e4.78 (bm, 1H), 6.92e6.99 (m, 3H), 7.14 (d, 2H,

J ¼ 8.3 Hz), 7.31 (dd, 1H, J ¼ 9.0, 7.5 Hz), 7.84 (AA’XX’, 2H,

J_AX¼ 8.4 Hz, J_AA’/XX’¼ 1.8 Hz).

4.1.17.47. 1-(4-(4-(Phenylamino)benzoyl)piperidin-1-yl)ethanone

(55). 99% yield from 43 and aniline. ¹H NMR (CDCl₃)

d (ppm):

1.64e1.93 (bm, 4H), 2.13 (s, 3H), 2.77e2.93 (bm,1H), 3.12e3.29 (bm,

1H), 3.39e3.49 (m, 1H), 3.84e3.99 (bm, 1H), 4.49e4.64 (bm, 1H),

7.01 (AA’XX’, 2H, J_AX¼ 8.8 Hz, J_AA’/XX’¼ 2.3 Hz), 7.10 (tt, 1H, J ¼ 7.4,

1.1 Hz), 7.16e7.22 (m, 2H), 7.32e7.39 (m, 2H), 7.87 (AA’XX’, 2H,

J_AX¼ 8.8 Hz, J_AA’/XX’¼ 2.3 Hz).

4.1.17.39. 1-(4-(4-Cyclohexylbenzoyl)piperidin-1-yl)ethanone (47).

40% yield from 42 and phenylcyclohexane. ¹H NMR (CDCl₃)

d

(ppm): 1.34e1.50 (bm, 4H), 1.56e1.98 (bm, 10H), 2.13 (s, 3H),

4.1.17.48. 1-(4-(4-Phenoxybenzoyl)piperidin-1-yl)ethanone

(56).

2.47e2.64 (m, 1H), 2.72e3.03 (bm, 1H), 3.07e3.33 (bm, 1H),

3.43e3.54 (m, 1H), 3.77e4.02 (bm, 1H), 4.45e4.65 (bm, 1H), 7.31 (d,

2H, J ¼ 8.2 Hz), 7.87 (AA’XX’, 2H, J_AX¼ 8.4 Hz, J_AA’XX’¼ 1.8 Hz).

84% yield from 43 and phenol. ¹H NMR (CDCl₃)

d

(ppm): 1.59e1.95

(bm, 4H), 2.12 (s, 3H), 2.77e2.90 (bm, 1H), 3.15e3.28 (bm, 1H), 3.45

(tt, 1H, J ¼ 10.4, 3.9 Hz), 3.85e3.97 (bm, 1H), 4.50e4.63 (bm, 1H),

7.02 (AA’XX’, 2H, J_AX¼ 8.9 Hz, J_AA’/XX’¼ 2.4 Hz), 7.05e7.10 (m, 2H),

7.21 (tt, 1H, J ¼ 7.4, 1.1 Hz), 7.37e7.44 (m, 2H), 7.93 (AA’XX’, 2H,

J_AX¼ 8.9 Hz, J_AA’/XX’¼ 2.4 Hz).

4.1.17.40. (4-Cyclohexylphenyl)(piperidin-4-yl)methanone

(48).

81% yield from 47.¹H NMR (CDCl₃)

d

(ppm): 1.33e1.50 (bm, 4H),

1.65e1.95 (bm, 10H), 2.24 (bs, 1H), 2.51e2.61 (bm, 1H), 2.80 (td, 2H,

J ¼ 12.1, 2.1 Hz), 3.15e3.27 (m, 2H), 3.40 (tt,1H, J ¼ 10.9, 3.7 Hz), 7.29

(d, 2H, J ¼ 8.2 Hz), 7.87 (d, 2H, J ¼ 8.4 Hz).

4.1.17.49. (4-(Phenylamino)phenyl)(piperidin-4-yl)methanone (57).

99% yield from 55.¹H NMR (CDCl₃)

d (ppm): 1.63e1.90 (m, 4H), 2.77

(td, 2H, J ¼ 12.1, 2.6 Hz), 3.14e3.24 (m, 2H), 3.34 (tt, 1H, J ¼ 11.3,

4.1.17.41. (4-(4-Cyclohexylbenzoyl)piperidin-1-yl)(3-methoxyphenyl)

3.6 Hz), 6.10 (exchangeable s, 1H), 7.00 (AA’XX’, 2H, J_AX¼ 8.8 Hz, J_AA’/

methanone (49). 68% yield from 48 and 3-methoxybenzoic acid. ¹H

¼ 2.3 Hz), 7.08 (tt,1H, J ¼ 7.4,1.1 Hz), 7.15e7.21 (m, 2H), 7.31e7.38

XX’

NMR (CDCl₃)

d

(ppm): 1.43e1.50 (bm, 5H), 1.70e2.03 (bm, 9H),

(m, 2H), 7.87 (AA’XX’, 2H, J_AX¼ 8.8 Hz, J_AA’/XX’¼ 2.3 Hz).

2.51e2.62 (bm, 1H), 2.98e3.20 (bm, 2H), 3.47e3.58 (bm, 1H),

3.77e4.00 (bm, 1H), 3.82 (s, 3H), 4.57e4.80 (bm, 1H), 6.91e7.00 (m,

3H), 7.27e7.34 (m, 3H), 7.87 (d, 2H, J ¼ 8.4 Hz).

4.1.17.50. (4-Phenoxyphenyl)(piperidin-4-yl)methanone

(58).

93% yield from 56.¹H NMR (CDCl₃)

d

(ppm): 1.61e1.74 (m, 2H),

1.75e1.92 (m, 2H), 2.76 (td, 2H, J ¼ 12.3, 2.7 Hz), 3.19 (dt, 2H,

J ¼ 12.5, 3.4 Hz), 3.35 (tt, 1H, J ¼ 11.4, 3.6 Hz), 7.00 (AA’XX’, 2H,

J_AX¼ 8.9 Hz, J_AA’/XX’¼ 2.4 Hz), 7.04e7.10 (m, 2H), 7.20 (tt, 1H, J ¼ 7.4,

1.1 Hz), 7.36e7.43 (m, 2H), 7.93 (AA’XX’, 2H, J_AX¼ 8.9 Hz, J_AA’/

4.1.17.42. 1-(4-(4-Benzylbenzoyl)piperidin-1-yl)ethanone

(50).

49% yield from 42 and diphenylmethane. ¹H NMR (CDCl₃)

d

(ppm):

1.68e1.80 (bm, 2H),1.85e1.93 (bm, 2H), 2.13 (s, 3H), 2.77e3.28 (bm,

2H), 3.42e3.51 (m, 1H), 3.83e4.00 (bm, 1H), 4.04 (s, 2H), 4.42e4.62

(bm, 1H), 7.16e7.20 (m, 2H), 7.23 (tt, 1H, J ¼ 7.3, 2.2 Hz), 7.27e7.33

(m, 4H), 7.86e7.89 (m, 2H).

¼ 2.4 Hz).

XX’

4.1.17.51. (1-(3-Methoxybenzoyl)piperidin-4-yl)(4-(phenylamino)

phenyl)methanone (59). 65% yield from 57 and 3-methoxybenzoic

4.1.17.43. (4-Benzylphenyl)(piperidin-4-yl)methanone

(51). 91%

acid. ¹H NMR (CDCl₃)

d (ppm): 1.72e2.07 (bm, 4H), 2.94e3.22

yield from 50.¹H NMR (CDCl₃)

d

(ppm): 1.66e1.78 (m, 2H),

(bm, 2H), 3.43e3.52 (bm, 1H), 3.78e3.95 (bm, 1H), 3.82 (s, 3H),

4.60e4.80 (bm, 1H), 6.92e7.03 (m, 5H), 7.10 (t, 1H, J ¼ 7.4 Hz),

7.16e7.22 (m, 2H), 7.27e7.39 (m, 3H), 7.87 (d, 2H, J ¼ 8.8 Hz).

1.82e1.91 (m, 2H), 2.80 (td, 2H, J ¼ 12.2, 2.7 Hz), 3.21 (dt, 2H,

J ¼ 12.7, 3.6 Hz), 3.38 (tt, 1H, J ¼ 10.8, 3.7 Hz), 4.03 (s, 2H), 7.15e7.20

(m, 2H), 7.20e7.26 (m, 1H), 7.26e7.33 (m, 4H), 7.86 (AA’XX’, 2H,

J_AX¼ 8.3 Hz, J_AA’/XX’¼ 1.8 Hz).

4.1.17.52. (1-(3-Methoxybenzoyl)piperidin-4-yl)(4-phenoxyphenyl)

methanone (60). 85% yield from 58 and 3-methoxybenzoic acid. ¹H

4.1.17.44. (4-(4-Benzylbenzoyl)piperidin-1-yl)(3-methoxyphenyl)

NMR (CDCl₃) d (ppm): 1.73e2.10 (m, 4H), 2.96e3.21 (bm, 2H),

methanone (52). 80% yield from 51 and 3-methoxybenzoic acid. ¹H

3.45e3.55 (m, 1H), 3.80e3.95 (bm, 1H), 3.82 (s, 3H), 4.63e4.76 (bm,

1H), 6.92e6.99 (m, 3H), 7.02 (AA’XX’, 2H, J_AX¼ 8.9 Hz, J_AA’/

NMR (CDCl₃)

d (ppm): 1.72e2.06 (bm, 4H), 3.00e3.17 (bm, 2H),

3.46e3.55 (m, 1H), 3.78e3.92 (bm, 1H), 3.82 (s, 3H), 4.04 (s, 2H),

4.61e4.74 (bm, 1H), 6.92e6.98 (m, 3H), 7.15e7.20 (m, 2H), 7.22 (tt,

1H, J ¼ 7.4, 2.2 Hz), 7.27e7.34 (m, 5H), 7.87 (d, 2H, J ¼ 8.2 Hz).

¼ 2.4 Hz), 7.05e7.10 (m, 2H), 7.21 (tt, 1H, J ¼ 7.4, 1.1 Hz), 7.31 (dd,

XX’

1H, J ¼ 9.0, 7.5 Hz), 7.37e7.44 (m, 2H), 7.94 (AA’XX’, 2H, J_AX¼ 8.9 Hz,

J_AA’/XX’¼ 2.4 Hz).

4.1.17.45. (4-(4-Benzylbenzoyl)piperidin-1-yl)(2-ﬂuoro-5-

methoxyphenyl)methanone (53). 77% yield from 51 and 2-ﬂuoro-5-

4.1.17.53. 1-(4-(4-(phenylthio)benzoyl)piperidin-1-yl)ethanone (61).

97% yield from 43 and thiophenol. ¹H NMR (CDCl₃)

d (ppm):

methoxybenzoic acid. ¹H NMR (CDCl₃)

d

(ppm): 1.74e1.86 (bm, 3H),

1.63e1.69 (m, 1H), 1.73e1.93 (m, 3H), 2.11 (s, 3H), 2.75e2.85 (m,

1H), 3.15e3.25 (m, 1H), 3.41 (tt, 1H, J ¼ 10.6, 4.1 Hz), 3.85e3.94 (m,

1H), 4.51e4.60 (m, 1H), 7.21 (AA’XX’, 2H, J_AX¼ 8.7 Hz, J_AA’/

1.97e2.04 (m, 1H), 3.02e3.12 (m, 1H), 3.313e3.30 (bm, 1H),

3.44e3.54 (bm, 1H), 3.65e3.74 (m, 1H), 3.80 (s, 3H), 4.04 (s, 2H),

4.64e4.74 (m, 1H), 6.85e6.92 (m, 2H), 7.00 (t, 1H, J ¼ 8.6 Hz),

7.15e7.20 (m, 2H), 7.23 (tt, 1H, J ¼ 7.3, 2.2 Hz), 7.27e7.33 (m, 4H),

¼ 2.0 Hz), 7.39e7.43 (m, 3H), 7.48e7.53 (m, 2H), 7.80 (AA’XX’,

XX’

2H, J_AX¼ 8.7 Hz, J_AA’/XX’¼ 2.0 Hz).

24

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

4.1.17.54. (4-(phenylthio)phenyl)(piperidin-4-yl)methanone

(62).

J_AX¼ 8.6 Hz, J_AA’/XX’¼ 1.8 Hz).

4.1.17.63. Azidobenzene (74). 70% yield from 71.¹H NMR (CDCl₃)

99% yield from 61.¹H NMR (CDCl₃)

d (ppm): 1.57e1.87 (m, 4H),

2.67e2.80 (m, 2H), 2.95e3.22 (m, 2H), 3.29 (tt, 1H, J ¼ 11.4, 3.8 Hz),

7.21 (AA’XX’, 2H, J_AX¼ 8.6 Hz, J_AA’/XX’¼ 2.0 Hz), 7.37e7.43 (m, 3H),

7.47e7.53 (m, 2H), 7.81 (AA’XX’, 2H, J_AX¼ 8.6 Hz, J_AA’/XX’¼ 2.0 Hz).

d

(ppm): 7.01e7.06 (m, 2H), 7.14 (tt,1H, J ¼ 7.4,1.1 Hz), 7.32e7.39 (m,

2H).

4.1.17.55. (1-(3-Methoxybenzoyl)piperidin-4-yl)(4-(phenylthio)

4.1.17.64. (Azidomethyl)benzene (75). 61% yield from 72.¹H NMR

phenyl)methanone (63). 73% yield from 62 and 3-methoxybenzoic

(CDCl₃)

d (ppm): 4.34 (s, 2H), 7.30e7.42 (m, 5H).

acid. ¹H NMR (CDCl₃)

d (ppm): 1.72e2.04 (m, 4H), 2.95e3.20 (bm,

2H), 3.39e3.52 (m, 1H), 3.76e3.96 (bm, 1H), 3.82 (s, 3H), 4.58e4.76

(bm, 1H), 6.92e6.98 (m, 2H), 7.22 (AA’XX’, 2H, J_AX¼ 8.6 Hz, J_AA’/

4.1.17.65. (3-Azidopropyl)benzene (76). 77% yield from 73.¹H NMR

(CDCl₃)

3.29 (t, 2H, J ¼ 6.8 Hz), 7.17e7.24 (m, 3H), 7.27e7.33 (m, 2H).

d

(ppm): 1.92 (quint, 2H, J ¼ 6.7 Hz), 2.71 (t, 2H, J ¼ 7.6 Hz),

¼ 2.0 Hz), 7.30 (dd, 1H, J ¼ 8.9, 7.5 Hz), 7.38e7.44 (m, 4H),

XX’

7.48e7.54 (m, 2H), 7.78e7.84 (m, 2H).

4.1.17.66. 1-(4-(4-(1-Phenyl-1H-1,2,3-triazol-4-yl)benzoyl)piperidin-

4.1.17.56. (1-(2-Fluoro-5-methoxybenzoyl)piperidin-4-yl)(4-(phenyl-

1-yl)ethanone (77). 79% yield from 70 and 74.¹H NMR (CDCl₃)

thio)phenyl)methanone (64). 85% yield from 62 and 2-ﬂuoro-5-

d (ppm): 1.73e2.00 (bm, 4H), 2.14 (s, 3H), 2.81e2.96 (bm, 1H),

methoxybenzoic acid. ¹H NMR (CDCl₃)

d

(ppm): 1.72e1.86 (m,

3.17e3.32 (bm, 1H), 3.54 (tt, 1H, J ¼ 10.6, 4.0 Hz), 3.86e4.00 (bm,

1H), 4.51e4.67 (bm, 1H), 7.49 (tt, 1H, J ¼ 7.5, 1.2 Hz), 7.54e7.61 (m,

2H), 7.78e7.83 (m, 2H), 8.05 (s, 4H), 8.30 (s, 1H).

3H), 1.95e2.03 (m, 1H), 2.98e3.10 (m, 1H), 3.11e3.27 (bm, 1H),

3.41e3.50 (bm, 1H), 3.65e3.74 (m, 1H), 3.79 (s, 3H), 4.65e4.73 (m,

1H), 6.84e6.91 (m, 2H), 7.00 (t, 1H, J ¼ 8.7 Hz), 7.21 (AA’XX’, 2H,

J_AX¼ 8.5 Hz, J_AA’/XX’¼ 2.0 Hz), 7.38e7.44 (m, 3H), 7.48e7.53 (m, 2H),

7.81 (AA’XX’, 2H, J_AX¼ 8.6 Hz, J_AA’/XX’¼ 2.0 Hz).

4.1.17.67. 1-(4-(4-(1-Benzyl-1H-1,2,3-triazol-4-yl)benzoyl)piperidin-

1-yl)ethanone (78). 66% yield from 70 and 75.¹H NMR (CDCl₃)

d

(ppm): 1.63e1.72 (bm, 1H), 1.77e1.96 (bm, 3H), 2.11 (s, 3H),

4.1.17.57. (1-(2,4-Diﬂuoro-5-methoxybenzoyl)piperidin-4-yl)(4-

(phenylthio)phenyl)methanone (65). 77% yield from 62 to 2,4-

2.80e2.90 (m, 1H), 3.18e3.29 (m, 1H), 3.50 (tt, 1H, J ¼ 10.7, 4.1 Hz),

3.86e3.96 (m, 1H), 4.52e4.62 (m, 1H), 5.60 (s, 2H), 7.31e7.35 (m,

2H), 7.37e7.44 (m, 3H), 7.75 (s, 1H), 7.92 (d, 2H, J ¼ 8.6 Hz), 7.98 (d,

2H, J ¼ 8.6 Hz).

diﬂuoro-5-methoxybenzoic acid 24.¹H NMR (CDCl₃)

d (ppm):

1.72e1.86 (m, 3H), 1.95e2.04 (bm, 1H), 2.99e3.12 (bm, 1H),

3.12e3.30 (bm, 1H), 3.41e3.52 (bm, 1H), 3.64e3.74 (m, 1H), 3.89 (s,

3H), 4.62e4.73 (m, 1H), 6.88 (dd, 1H, J ¼ 10.5, 8.7 Hz), 6.95e7.02 (m,

1H), 7.21 (d, 2H, J ¼ 8.4 Hz), 7.38e7.44 (m, 3H), 7.47e7.54 (m, 2H),

7.81 (d, 2H, J ¼ 8.5 Hz).

4.1.17.68. 1-(4-(4-(1-(3-Phenylpropyl)-1H-1,2,3-triazol-4-yl)benzoyl)

piperidin-1-yl)ethanone (79). 97% yield from 70 and 76.¹H NMR

(CDCl₃)

d (ppm): 1.61e1.73 (bm, 1H), 1.77e1.99 (bm, 3H), 2.12 (s,

3H), 2.32 (quint, 2H, J ¼ 7.3 Hz), 2,.71 (t, 2H, J ¼ 7.4 Hz), 2.81e2.90

(bm,1H), 3.20e3.30 (m,1H), 3.52 (tt,1H, J ¼ 10.6, 3.9 Hz), 3.89e3.96

(m,1H), 4.43 (t, 2H, J ¼ 7.1 Hz), 4.57e4.64 (m,1H), 7.17e7.25 (m, 3H),

7.28e7.35 (m, 2H), 7.81 (s, 1H), 7.95 (d, 2H, J ¼ 8.6 Hz), 8.01 (d, 2H,

J ¼ 8.6 Hz).

4.1.17.58. 1-(4-(4-(Phenylsulfonyl)benzoyl)piperidin-1-yl)ethanone

(66). 99% yield from 61.¹H NMR (CDCl₃)

d (ppm): 1.57e1.92 (bm,

4H), 2.11 (s, 3H), 2.78e2.89 (bm, 1H), 3.15e3.28 (bm, 1H), 3.44 (tt,

1H, J ¼ 10.7, 4.0 Hz), 3.83e3.96 (bm, 1H), 4.48e4.60 (bm, 1H),

7.51e7.56 (m, 2H), 7.61 (tt, 1H, J ¼ 7.3, 1.6 Hz), 7.94e7.99 (m, 2H),

8.00e8.07 (m, 4H).

4.1.17.69. (4-(1-Phenyl-1H-1,2,3-triazol-4-yl)phenyl)(piperidin-4-yl)

methanone (80). 82% yield from 77.¹H NMR (DMSO‑d₆)

d (ppm):

4.1.17.59. (4-(Phenylsulfonyl)phenyl)(piperidin-4-yl)methanone (67).

1.59e1.73 (m, 2H), 1.80e1.93 (m, 2H), 2.85e2.96 (m, 2H), 3.14e3.26

(m, 2H), 3.65e3.77 (bm, 1H), 7.51e7.58 (m, 1H), 7.62e7.71 (m, 2H),

7.93e8.02 (m, 2H), 8.13 (s, 4H), 9.50 (s, 1H).

81% yield from 66.¹H NMR (CDCl₃)

d (ppm): 1.60e1.73 (m, 4H),

1.77e1.85 (m, 2H), 2.75 (td, 1H, J ¼ 12.4, 2.4 Hz), 3.17 (dt, 1H,

J ¼ 12.5, 3.2 Hz), 3.33 (tt,1H, J ¼ 11.4, 3.6 Hz), 7.50e7.56 (m, 2H), 7.60

(tt, 1H, J ¼ 7.4, 1.9 Hz), 7.93e7.99 (m, 2H), 7.99e8.06 (m, 4H).

4.1.17.70. (4-(1-Benzyl-1H-1,2,3-triazol-4-yl)phenyl)(piperidin-4-yl)

methanone (81). 88% yield from 78.¹H NMR (CDCl₃)

d (ppm): 1.70

4.1.17.60. (1-(3-Methoxybenzoyl)piperidin-4-yl)(4-(phenylsulfonyl)

(td, 2H, J ¼ 11.7, 3.6 Hz), 1.81e1.89 (m, 2H), 2.77 (td, 2H, J ¼ 12.4,

2.7 Hz), 3.19 (dt, 2H, J ¼ 9.6, 3.3 Hz), 3.39 (tt, 1H, J ¼ 11.3, 3.6 Hz),

5.60 (s, 2H), 7.31e7.35 (m, 2H), 7.37e7.44 (m, 3H), 7.42 (s, 1H), 7.90

(d, 2H, J ¼ 8.4 Hz), 7.98 (d, 2H, J ¼ 8.5 Hz).

phenyl)methanone (68). 49% yield from 67 and 3-methoxybenzoic

acid. ¹H NMR (CDCl₃)

d (ppm): 1.70e2.01 (m, 4H), 2.89e3.19 (bm,

2H), 3.44e3.54 (m, 1H), 3.76e3.96 (bm, 1H), 3.82 (s, 3H), 4.56e4.74

(bm, 1H), 6.91e6.97 (m, 3H), 7.30 (t, 1H, J ¼ 8.1 Hz), 7.50e7.57 (m,

2H), 7.61 (tt, 1H, J ¼ 7.5, 2.3 Hz), 7.94e7.99 (m, 2H), 8.00e8.08 (m,

4H).

4.1.17.71. (4-(1-(3-Phenylpropyl)-1H-1,2,3-triazol-4-yl)phenyl)(-

piperidin-4-yl)methanone (82). 69% yield from 79.¹H NMR (CDCl₃)

d

(ppm): 1.63e1.76 (m, 2H), 1.82e1.91 (m, 2H), 2.32 (quint, 2H,

4.1.17.61. 1-(4-(4-(3-Hydroxy-3-methylbut-1-yn-1-yl)benzoyl)piper-

J ¼ 7.3 Hz), 2.71 (t, 2H, J ¼ 7.4 Hz), 2.79 (td, 2H, J ¼ 12.4, 2.4 Hz),

3.16e3.24 (m, 2H), 3.42 (tt, 1H, J ¼ 11.0, 3.6 Hz), 4.42 (t, 2H,

J ¼ 7.1 Hz), 7.17e7.25 (m, 3H), 7.28e7.34 (m, 2H), 7.80 (s,1H), 7.93 (d,

2H, J ¼ 8.6 Hz), 8.01 (d, 2H, J ¼ 8.6 Hz).

idin-1-yl)ethanone (69). 99% yield from 43.¹H NMR (CDCl₃)

d

(ppm): 1.63 (s, 6H), 1.75e1.95 (bm, 4H), 2.11 (s, 3H), 2.78e2.88 (m,

1H), 3.17e3.27 (m, 1H), 3.46 (tt, 1H, J ¼ 10.9, 3.9 Hz), 3.86e3.95 (m,

1H), 4.53e4.63 (m, 1H), 7.51 (d, 2H, J ¼ 8.6 Hz),7.88 (d, 2H,

J ¼ 8.6 Hz).

4.1.17.72. (1-(3-Methoxybenzoyl)piperidin-4-yl)(4-(1-phenyl-1H-

1,2,3-triazol-4-yl)phenyl)methanone (83). 98% yield from 80 and 3-

4.1.17.62. 1-(4-(4-Ethynylbenzoyl)piperidin-1-yl)ethanone

(70).

methoxybenzoic acid. ¹H NMR (CDCl₃)

d (ppm): 1.78e2.13 (m, 4H),

82% yield from 69.¹H NMR (CDCl₃)

d

(ppm): 1.62e1.95 (bm, 4H),

3.03e3.24 (bm, 2H), 3.54e3.64 (bm, 1H), 3.81e3.98 (bm, 1H), 3.83

(s, 3H), 4.63e4.80 (bm, 1H), 6.93e7.01 (m, 3H), 7.28e7.35 (m, 1H),

7.46e7.52 (m,1H), 7.54e7.61 (m, 2H), 7.81 (d, 2H, J ¼ 8.0 Hz), 8.05 (s,

4H), 8.30 (s, 1H).

2.12 (s, 3H), 2.77e2.91 (bm, 1H), 3.14e3.30 (bm, 1H), 3.26 (s, 1H),

3.47 (tt, 1H, J ¼ 10.8, 3.9 Hz), 3.84e3.97 (bm, 1H), 4.51e4.63 (bm,

1H), 7.59 (AA’XX’, 2H, J_AX¼ 8.4 Hz, J_AA’/XX’¼ 1.7 Hz), 7.89 (AA’XX’, 2H,

25

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

4.1.17.73. (4-(4-(1-Benzyl-1H-1,2,3-triazol-4-yl)benzoyl)piperidin-1-

described above. The minimized complexes were used as input

structures for the MD simulations, which were run using Particle

Mesh Ewald (PME) electrostatics, a cutoff of 10 Å for the non-

bonded interactions and periodic boundary conditions. SHAKE al-

gorithm was used to constrain all bonds involving hydrogen atoms

and a time step of 2.0 fs was thus used for the simulation. Initially,

an MD step of 0.5 ns in which the temperature of the system was

raised from 0 to 300 K was performed using constant-volume pe-

riodic boundary conditions. An equilibration step of constant

pressure periodic boundary MD was run for 3 ns, keeping the

temperature of the system at the constant value of 300 K with

Langevin thermostat. Finally, a further MD step with constant

yl)(3-methoxyphenyl)methanone (84). 87% yield from 81 and 3-

methoxybenzoic acid. ¹H NMR (CDCl₃)

d (ppm): 1.73e2.10 (bm,

4H), 3.00e3.24 (bm, 2H), 3.50e3.60 (bm, 1H), 3.82 (s, 3H),

3.84e3.99 (bm, 1H), 4.57e4.79 (bm, 1H), 5.60 (s, 2H), 6.92e6.99 (m,

3H), 7.28e7.35 (m, 3H), 7.37e7.44 (m, 3H), 7.76 (s, 1H), 7.92 (d, 2H,

J ¼ 8.6 Hz), 7.98 (d, 2H, J ¼ 8.6 Hz).

4.1.17.74. (1-(3-Methoxybenzoyl)piperidin-4-yl)(4-(1-(3-

phenylpropyl)-1H-1,2,3-triazol-4-yl)phenyl)methanone

99% yield from 82 and 3-methoxybenzoic acid. ¹H NMR (CDCl₃)

(ppm): 1.76e1.89 (bm, 3H), 1.92e2.10 (bm, 1H), 2.33 (quint, 2H,

(85).

d

J ¼ 7.3 Hz), 2.71 (t, 2H, J ¼ 7.5 Hz), 3.00e3.23 (bm, 2H), 3.52e3.62

(bm, 1H), 3.83 (s, 3H), 3.84e3.95 (bm, 1H), 4.43 (t, 2H, J ¼ 7.1 Hz),

4.62e4.80 (bm, 1H), 6.93e7.00 (m, 3H), 7.17e7.25 (m, 3H), 7.28-7-

34 (m, 3H), 7.81 (s, 1H), 7.95 (d, 2H, J ¼ 8.6 Hz), 8.02 (d, 2H,

J ¼ 8.6 Hz).

pressure conditions was run for 246.5 ns. All a carbons of the

protein were subjected to a harmonic potential of 10 kcal/molꢄÅ²

during the three MD steps, for a total of 250 ns of simulation. The

ﬁnal structure of 22a-MAGL complex corresponded to the average

of the last 200.0 ns of MD minimized by the CG method until a

convergence of 0.05 kcal/molꢄÅ². The average structure was ob-

tained using the Cpptraj program [56] implemented in AMBER 16.

4.2. Docking calculations

The X-ray structure of MAGL (PDB code 3PE6) [48] was down-

loaded from the Protein Data Bank [49]. Hydrogen atoms were

added to the ligand-protein complex, which was then minimized

using Amber16 software [50] and ff14SB force ﬁeld at 300 K. The

complex was placed at the center of a rectangular parallelepiped

box and solvated with a 20 Å water cap, generated using TIP3P

explicit solvent model. Sodium ions were then added to neutralize

the system, which was then energy minimized using a two-step

protocol. In the ﬁrst step, only the minimization of the solvent

was performed, since a position restraint of 500 kcal/molꢄÅ²was

applied on all solute atoms. In the second step, 5000 cycles of

steepest descent followed by conjugate gradient (CG) were used to

minimize the whole system, until a convergence of 0.05 kcal/

Åꢄmol. The ligand was built with Maestro [51] and then subjected

to energy minimization performed with Macromodel [52] until a

convergence value of 0.05 kcal/Åꢄmol, by employing the CG algo-

rithm, MMFFs force ﬁeld and a distance-dependent dielectric

constant of 1.0. Docking calculations were performed using

AUTODOCK 4.0 software [53]. Autodock Tools was used to auto-

matically identify the torsion angles in the ligand, add the solvent

model and determine ligand and protein atomic charges. Gasteiger

and Kollmann charges were assigned for ligand and protein,

respectively. A grid box of 82, 40, and 30 points in the x, y, and z

directions centered on the bound inhibitor ZYH was used as the

docking site, in which the energetic maps were calculated using a

grid spacing of 0.375 Å and a distance-dependent function of the

dielectric constant. The ligand was docked performing 200 runs of

Autodock search with the Lamarckian Genetic Algorithm, following

a robust protocol [54,55]. For each docking run, 10 000 000 steps of

energy evaluations were performed, the number of individuals in

the initial population was set to 500 and a maximum of 10 000 000

generations were simulated. An RMS cut-off 2.0 Å was used for pose

clustering.

4.4. SMD simulations

Steered molecular dynamics (SMD) simulations were performed

using AMBER16 [50]. The ﬁnal MD frame obtained from the

simulation of 22a-MAGL complex was used as the starting structure

for the SMD simulations, which were thus performed at the same

constant-pressure periodic boundary conditions and at the con-

stant temperature of 300 K. However, no position restraint was

applied on the protein

a carbons during the simulations. Three

different SMD simulations using three different initial pulling di-

rections were performed. Each pulling direction was imposed by

selecting a couple of atoms, one belonging to the protein and one

belonging to the ligand, which set the initial distance to be

stretched during the SMD: a) the

of the ligand; b) the carbon of M123 and the C47 atom of the

ligand; c) the carbon of A126 and the C47 atom of the ligand. In all

a carbon of S48 and the C47 atom

a

simulation, the ligand was pulled out from the protein binding site

by increasing the initial distance between the selected couple of

atoms by 30 Å, through the application of a spring constant of

5 kcal/molꢄÅ². Each SMD simulation was performed at the constant

velocity of 0.1 Å/ns by setting the simulation length to 300 ns; this

allowed us to consider the simulated ligand unbinding process as

reversible, and the pulling work associated to the process as the

exact free energy [29,30].

4.5. Binding energy evaluations

The total and per-frame ligand-protein binding afﬁnity of the

22a-MAGL complex were calculated with AMBER 16 using the MM-

GBSA method. The trajectories corresponding to the last 200 ns of

the classic MD simulation and the full trajectory of the SMD

simulation (300 ns) were used for the evaluation, which was thus

performed on a total of 200 and 300 MD frames (1 per ns),

respectively. MOLSURF program and the MM-PBSA module of

AMBER 16 were used to calculate nonpolar and polar energies,

respectively, while van der Waals, electrostatic and internal con-

tributions were estimated with SANDER module [57,58]. The li-

gand’s entropy was not taken into account in the calculation.

4.3. MD simulations

Molecular dynamics (MD) simulations were performed using

AMBER, version 16 [50], using the ff14SB force ﬁeld. General Amber

force ﬁeld (GAFF) parameters were used for the ligand, whose

partial charges were assigned using the Antechamber suite of

AMBER 16, based on the AM1-BCC method. The complex was

placed at the center of a rectangular parallelepiped box and sol-

vated with a 20 Å water cap, generated using TIP3P explicit solvent

model. Sodium ions were then added to neutralize the system,

which was then energy minimized following the same procedure

4.6. MAGL inhibition assay

Human recombinant MAGL, 2 and 4-nitrophenylacetate (4-NPA)

substrate were purchased from Cayman Chemical. The IC₅₀values

were generated in 96-well microtiter plates. The MAGL reaction

was carried out at room temperature, at a ﬁnal volume of 200 mL in

26

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

10 mM Tris buffer, pH 7.2, containing 1 mM EDTA and BSA 0.1 mg/

37 ^ꢁC. The reaction was stopped by the addition of 400

mL of ice-

mL. A total of 150

containing the suitable amount of compound. The reaction was

initiated by the addition of 40 L of MAGL (11 ng/well) in such a way

m

L of 4-NPA 133.3

m

M was added to 10

m

L of DMSO

cold CHCl₃:MeOH (1:1) and samples were vortexed and centri-

fuged (16 000ꢃg, 10 min, 4 ^ꢁC). Aliquots (200

mL) of the aqueous

m

phase were assayed for tritium content by liquid scintillation

spectroscopy. Blank values were recovered from tubes containing

no enzyme, whereas basal 2-OG hydrolysis occurring in non-

transfected HEK293 cells was subtracted. For FAAH and MAGL ac-

that the assay was linear over 30 min. After the reaction had pro-

ceeded for 30 min, absorbance values were then measured by using

a Victor X3 Microplates Reader (PerkinElmer®) at 405 nm.⁵³Two

reactions were also run: one reaction containing no compounds

and the second one containing neither compound nor MAGL. IC₅₀

values were derived from experimental data using the Sigmoidal

doseꢀresponse ﬁtting of GraphPad Prism software. Final values

were obtained from duplicates of three independent experiments.

To remove possible false-positive results, for each compound con-

centration a blank analysis was carried out, and the ﬁnal absor-

bance results were obtained detracting the absorbance produced

by the presence of all the components except MAGL in the same

conditions. In the enzyme kinetics experiments, compounds 21a,b,

22a,b were tested in the presence of scalar concentrations of 4-NPA.

They were added in scalar amounts (concentration

tivity assay 1.0 ꢃ 10⁶of intact U937 cells were suspended in 400

mL

of assay buffer (Tris-HCl 10 mM, EDTA 1 mM plus fatty acid-free

0.1% BSA, pH 8) in plastic tubes and incubated with different con-

centrations of the screening compounds at 37 ^ꢁC (co-incubation).

Then, 10

[1,2,3-³H]2-OG or 1

1-³H]AEA were added and cells were incubated for 15 min at 37 ^ꢁC

with shaking. The reaction was stopped by the addition of 800 L of

mM of nonradioactive (2-OG) and a small tracer (0.5 nM) of

mM of AEA containing 2 nM of [ethanolamine-

m

a methanol chloroform ice-cold mixture 1:1 (v/v) and, after

vigorous vortexing, aqueous and organic phases were separated by

centrifugation at 10 000ꢃg, for 10 min, at 4 ^ꢁC. Aliquots (400

mL) of

both the aqueous and organic phases were transferred in scintil-

lation tubes and mixed with 3 mL of Ultima Gold scintillation liquid.

The radioactivity associated with the [³H]glycerol or [³H]ethanol-

amine formation for the aqueous phase was measured for tritium

content by liquid scintillation spectroscopy. Compounds were

tested in three independent experiments, each performed in

triplicates.

range ¼ 1e0.125

for 22a) to a reaction mixture containing scalar concentrations of 4-

NPA (15e1400 M). Finally, MAGL solution was added (11 ng/well).

mM for compounds 21a,b, 22b and 0.5e0.0625 mM

m

The MAGL activity was measured by recording the increase in 4-

nitrophenol absorbance using the Victor X3 Microplates Reader

(PerkinElmer®). The experimental data were analyzed by non-

linear regression analysis with GraphPad Prism software, using a

second order polynomial regression analysis, and by applying the

mixed-model inhibition ﬁt.

4.10. CB1 and CB2 binding assay

Binding assay to cannabinoid receptor 1 and 2 (CB1 and CB2)

were performed as previously described.⁶Brieﬂy, clean membranes

expressing hCB1 or hCB2 were resuspended in binding buffer

(50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl₂, 0.5% fatty acid-free

bovine serum albumin (BSA), pH 7.4) and incubated with vehicle

or compounds and 0.5 nM of [³H]CP55,940 for 90 min at 30 ^ꢁC.

Non-speciﬁc binding was determined in the presence of 10 mM of

WIN55,512. After incubation, membranes were ﬁltered through a

pre-soaked 96-well microplate bonded with GF/B ﬁlters under

4.7. MAGL preincubation assay

The MAGL reaction was conducted in the same conditions re-

ported above. A total of 150

10 L of DMSO containing the appropriate amount of compound.

After 0 min, 30 min, and 60 min of incubation time the reaction was

initiated by the addition of 40 L of 4-NPA 500 M. The enzyme

mL of MAGL (11 ng/well) was added to

m

activity was then measured according to the procedure described

above. Final values were obtained from triplicates of two inde-

pendent experiments.

vacuum and washed twelve times with 150 mL of ice-cold binding

buffer. The radioactivity was measured and the results expressed as

[³H]CP55,940 binding. Compounds were tested, at a screening

4.8. MAGL dilution assay

concentration of 10 mM, in two independent experiments, each

performed in triplicates.

The enzyme (880 ng in 75

bated during 60 min at room temperature with 5

21a,b, 22a,b (concentration of 2 M in the mixture) dissolved in

m

L of Tris buffer, pH 7.2) was incu-

mL of compounds

4.11. Competitive activity-based protein proﬁling (ABPP)

m

DMSO. The MAGL-inhibitor mixture was then diluted 40-fold with

the buffer. After 15 min of incubation, the reaction was initiated on

a 160 mL aliquot by the addition of 40 mL of 4-NPA 500 mM and the

enzyme activity was measured according to the procedure

described above. Final values were obtained from triplicates of two

independent experiments.

Activity-based protein proﬁling (ABPP) experiments were per-

formed using mouse brain membrane preparations at a ﬁnal con-

centration of 4 mg/mL in PBS. Sample preparation was performed

as previously described.⁶Samples (19.5

m

L) were preincubated with

either DMSO (vehicle control), URB597 (4 M), JZL184 (1 M),

WWL70 (10 M), THL (20 M), DO264 (5 M) or compound 21a,

m

21b, 22a, 22b (different concentrations) for 25 min at 25 ^ꢁC under

shaking and then added with TAMRA-FP probe (125 nM ﬁnal

concentration) and incubated for 5 min at 25 ^ꢁC under shaking.

URB597, JZL184, WWL70 and THL/DO264 were used as positive

controls for FAAH, MAGL, ABHD6 and ABHD12 inhibition, respec-

4.9. Enzyme activity assays

Enzyme activity assays were performed as previously described

using cell homogenates HEK-293 cells stably transfected with

ABHD6 and ABHD12 (ABHD6 and ABHD12), and intact U937 cells

(FAAH and MAGL) [24]. Brieﬂy, hABHD6 and hABHD12 activity as-

says were performed using cell homogenates from hABHD6 and

hABHD12 stably transfected HEK293 cells. Compounds at the

tively. The reaction was stopped by adding 10 mL of 3x Laemmli

buffer and the samples kept for 3 min at room temperature, boiled

for 10 min at 90 ^ꢁC, cooled down to room temperature and

centrifuged at 10⁰000 g for 1 min. The samples were loaded on an

11% SDS-PAGE gel and resolved by electrophoresis at 120 V for

180 min. The gel was scanned with a Typhoon FLA 9500 using

TAMRA settings at the excitation wavelength of 542 nm and

emission light wavelength 568 nm. After coomassie staining and

destaining, the gels were scanned in Cy5 settings.

screening concentration of 10

cell homogenate for 30 min at 37 ^ꢁC in assay buffer (Tris 1 mM,

EDTA 10 mM plus fatty acid-free 0.1% BSA, pH 7.6). WWL70 10

or THL 20 M were used as positive controls, while DMSO as vehicle

control. Then, 10 M of 2-OG was added and incubated for 5 min at

mM were pre-incubated with 40 mg of

m

M

m

27

C. Granchi, G. Bononi, R. Ferrisi et al.

European Journal of Medicinal Chemistry 209 (2021) 112857

4.12. Cell viability assay

(TRIS buffer), and the optical density (OD) was measured at

wavelengths of 490 nm and 540 nm. Cell growth inhibition was

calculated as the percentage of drug treated cells versus vehicle-

treated cells (“control”) OD (corrected for OD before drug addic-

tion, “day-0”). The SRB assays were carried out in triplicate and

repeated at least three times. The data were evaluated using

GraphPad Prism 8.2.1 (Intuitive Software for Science, San Diego, CA,

USA).

Human breast MDA-MB-231, colorectal HCT116 and ovarian

CAOV3, OVCAR3 and SKOV3 cancer cell lines (from ATCC) were

maintained at 37 ^ꢁC in a humidiﬁed atmosphere containing 5% CO₂

according to the supplier. Cells (5 ꢃ 10²) were plated in 96-well

culture plates. The day after seeding, vehicle or compounds were

added at different concentrations to the medium. Compounds were

added to the cell culture at a concentration ranging from 200 to

0.02 mM. Cell viability was measured after 96 h according to the

4.15. RNA-sequencing data analysis

supplier (CellTiter-Glo® luminescence assay, Promega G7571) with

a Tecan M1000 PRO instrument. IC₅₀values were calculated from

logistical dose-response curves and performed in triplicates. Error

bars are standard deviations.

Sample preparation of primary PDAC cells and normal cells

HPDE was performed as described previously [60] and sequenced

using 100 bp SE with Illumina Platform. Raw data were pre-

processed for quality ﬁltering and adapter trimming using FASTX

Toolkit (version 0.7) and subsequently mapped to the Human

genome (GRCh38) using STAR alignment tool (version 2.5.3a) [61].

We obtained ~90% of reads mapped to the Human Genome per

sample. Gene counts were normalized to FPKM through CuffLinks

algorithm [62] and plots were generated in R Studio with R version

3.5.0.

4.13. Human cancer organoids

Patient-derived organoids (PDO) were obtained from totally

anonymized surgical specimens. However, biobank informed con-

sent for research purposes was available to collect the samples.

Tissues were processed and cultured using the protocol described

by Stappenbeck [59]. Summarizing, a piece of tissue was dissected

and incubated with 2 mg/mL of collagenase for 30’. After the

enzymatic digestion, the tissue was transferred into a clean 15 mL

tube with 10 mL of washing medium and centrifugated at 0.5ꢃg.

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Supernatant was discarded, the pellet was resuspended in 10

Cultrex® BME (Trevigen, MD, US) and plated in a 24 mw. After

solidiﬁcation of the matrix, 500 L of medium derived from Hill

mL of

m

et al. [39], was added to each well. Cell viability was done utilizing

the CellTiter-Glo® luminescence assay as previously described.

Acknowledgments

We are grateful to the University of Pisa (Progetti di Ricerca di

Ateneo, prog. PRA-2018-18), MIUR (PRIN 2017, project 2017SA5837)

and the Italian Ministry of Health e Ricerca Finalizzata 2016 - NET-

2016-02363765 for funding.

4.14. PDAC cells viability assay

The newly synthesized benzoylpiperidines 21a,b and 22a,b

were dissolved in DMSO. RPMI medium was from Gibco (Gai-

thersburg, MD, USA), sterile ﬁltered Newborn Calf Serum (NBCS)

was from Biowest, Pen e Strep (10.000 U Penicillin/mL, 10.000

Streptomycin/mL) was from Lonza (Verviers, Belgium). All other

chemicals were from Sigma (Zwijndrecht, theNetherlands). The

HPDE immortalized pancreatic ductal cell line was kindly supplied

by Dr. Tsao, Ontario, Canada) and cultured in supplemented KGM

medium (Lonza). PDAC-3 cells were isolated from a patient at Pisa

University Hospital as described previously [47]. The cells were

cultured in RPMI-1640 (Roswell Park Memorial Institute 1640)

supplemented with 10% sterile ﬁltered NBCS and 1% penicillin/

streptomycin. The cells were kept in a humidiﬁed atmosphere of 5%

CO2 and 95% air at 37 ^ꢁC and harvested with TrypsineEDTA solu-

tion (Sigma, Zwijndrecht, theNetherlands). The in vitro anti-

proliferative activity of the compounds 21a,b and 22a,b was

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.1 12857.

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Design, synthesis and biological evaluation of second-generation benzoylpiperidine derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors

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