"Photo-Rimonabant": Synthesis and Biological Evaluation of Novel Photoswitchable Molecules Derived from Rimonabant Lead to a Highly Selective and Nanomolar " Cis-On" CB1R Antagonist

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Research Article

“Photo-Rimonabant”: Synthesis and Biological Evaluation of Novel

Photoswitchable Molecules Derived from Rimonabant Lead to a

Highly Selective and Nanomolar “Cis-On” CB₁R Antagonist

Diego A. Rodr í guez-Soacha, Julia Fender, Yesid A. Ram í rez, Juan Antonio Collado, Eduardo Mun

Rangan Maitra, Christoph Sotri ﬀ er, Kristina Lorenz, and Michael Decker *

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oz,

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ABSTRACT: Human cannabinoid receptor type 1 (hCB₁R) plays important roles in the regulation of appetite and development of

addictive behaviors. Herein, we describe the design, synthesis, photocharacterization, molecular docking, and in vitro characterization

of “photo-rimonabant”, i.e., azo-derivatives of the selective hCB₁R antagonist SR1411716A (rimonabant). By applying azo-extension

strategies, we yielded compound 16a, which shows marked aﬃnity for CB₁R (K_{i (cis form)}= 29 nM), whose potency increases by

illumination with ultraviolet light (CB₁R K_itrans/cis ratio = 15.3). Through radioligand binding, calcium mobilization, and cell

luminescence assays, we established that 16a is highly selective for hCB₁R over hCB₂R. These selective antagonists can be valuable

molecular tools for optical modulation of CBRs and better understanding of disorders associated with the endocannabinoid system.

KEYWORDS: Photopharmacology, photochromic molecules, cannabinoid receptor, G-protein-coupled receptor, azo-extension,

azologization

between the receptor and multiple pathologies in the tissues in

which the receptor is present.^3,4,8−11By study of the

association between hCBs and appetite increase, hCB₁R was

found to be an important target for treatment of metabolic

disorders such as obesity, anorexia, and cachexia. By activation

of CB₁R in speciﬁc areas of the brain, such as nucleus

accumbens and hippocampus, an increase in the desire to eat

palatable food is produced, which is enhanced by a possible

relationship between the cannabinoid system and the

modulation of metabolic hormones in the blood that stimulate

lipogenesis and fat accumulation.¹²⁻¹⁴

INTRODUCTION

■

The isolation and elucidation of Δ⁹-tetrahydrocannabinol (Δ⁹-

THC) as one of the main active components of marijuana¹

gave way to the subsequent discovery of the receptors involved

in the endocannabinoid system. To date, two canonical

receptors have been described as part of this system.^2,3

These G-protein-coupled receptors (GPCRs) are named as

human cannabinoid receptor type 1 (hCB₁R) and type 2

(hCB₂R). The main diﬀerences between them lie in the

characteristics of their transduction system, given by diﬀer-

ences between their primary structures.²The distribution of

both receptors is diverse. The density of hCB₂R is

predominant in lung and testicles, as well as peripheral tissues

with immune function, such as tonsils, spleen, thymus, mast

cells, leukocytes, and macrophages.^2,4,5Expression of hCB₁R is

noted in the central and peripheral nervous system and in

peripheral tissues including liver, gastrointestinal tract, and

some tissues of the musculoskeletal, cardiovascular, and

reproductive systems.^3,6,7This wide distribution of hCB₁R

has generated several studies to understand the relationship

Through the discovery and development of selective ligands

for hCB₁R, a novel orally active and selective hCB₁R antagonist

Received: February 13, 2021

Accepted: March 29, 2021

Published: April 15, 2021

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was reported in 1994.¹⁵This compound, known as

SR141716A or rimonabant, was marketed as an anorectic

medicine for the treatment of obesity and metabolic disorders.

However, despite its pronounced therapeutical eﬀects during

the clinical research phases and application, it was withdrawn

from the market in 2008 due to adverse events related to

increased anxiety and depression.¹⁶Even being withdrawn

from the market, the rimonabant scaﬀold remains a highly

interesting pharmacological structure and entity. Recently

published studies suggest that rimonabant-derived molecules

are eﬀective in in vitro assays against tuberculosis-causing

pathogens.¹⁷Similarly, the role of cannabinoids in light-

induced retinal degeneration has been studied, where hCB₁R

antagonists such as rimonabant are shown to be eﬀective in

protecting against photoreceptor cell death in murine

models.¹⁸Additionally, hCB₁R antagonists were found to

have interesting pharmacological properties for the treatment

of addiction to drugs of abuse. Studies suggest that rimonabant

acts dually to generate anorectic eﬀects, increasing satiety and

decreasing neural response to positive stimuli in areas of the

brain involved with reward, thereby inhibiting motivation for

eating palatable food.^19,20This mechanism is complemented

by the relationship between hCB₁R and memory-related

plasticity, suggesting that hCB₁R antagonists have a positive

eﬀect on relapse prevention in patients with addiction to

abused drugs by modifying the impact on reward-related

memories.^21,22

These pharmacological advances highlight the need to

further investigate the speciﬁc roles of the hCB₁R in diﬀerent

brain regions in more detail by novel selective molecular

probes. Molecular tool compounds are necessary in which an

optimal response in the speciﬁc tissue can be obtained with the

possibility to control this action also in time (spatiotemporal

control). In the pharmaceutical ﬁeld, numerous approaches

have been followed to control drug activation in the body

precisely at its site of action. These applications are based on

the use of an external stimulus such as heat, electricity,

magnetic ﬁeld, ultrasound, among others, to release the active

drug in a speciﬁc tissue.²³Although these advances allow the

release in precise doses and speciﬁc times, nevertheless most of

these are irreversible, preventing the subsequent inactivation of

the molecule by nonmetabolic routes once it has been released.

A strategy used to generate reversibility in the activation of a

molecule is “photopharmacology”, which is based on the

design and synthesis of molecules whose activation can be

modulated by the application of light irradiation.²⁴When these

molecules are irradiated, their molecular structure changes,

which generates a change in their pharmacological activity.^23,24

The use of photoswitchable molecules such as stilbenes,

azobenzenes, or diarylethenes, and their coupling to speciﬁc

molecular ligands, has allowed creation of systems that can be

modiﬁed with high spatiotemporal precision.²⁴This allows the

study of the type of interaction between ligands and their

receptors, as well as interaction between ligands and other

biological processes after the activation/inactivation of the

target molecules. All these possibilities generate useful tools for

the study of complex pathologies where diﬀerent physiological

processes are simultaneously involved.²⁵Applying the

principles of photopharmacology for GPCRs, several research

groups have successfully synthesized new photoligands, which

act irreversibly or reversibly over their target protein (opioid

receptors, adrenoreceptors, glutamate receptors, acetylcholine

receptors, among others), thus expanding the toolbox for

understanding the mechanisms of action and physiological

responses at diﬀerent levels in the organism.²⁶⁻²⁹

The use of photopharmacology in the design of molecules

involved in the function of hCBR is of great interest because of

the multiple biological processes related to this receptor. The

synthesis of molecules based on the Δ⁹-THC pharmacophore

and the coupling of an azobenzene moiety have been reported,

with which it was possible to yield two photoswitchable hCB₁R

agonists, achieving control over hCB₁R and its associated

signaling pathways through isomerization of compounds.³⁰

Although this most interesting type of molecule allows

activation of the receptor with high spatiotemporal resolution,

the selectivity over other targets in the endocannabinoid

system is an important element to consider. Δ⁹-THC binds

with similar aﬃnity to both hCB₁and hCB₂in radioligand

displacement assays.³¹Cannabinoids such as Δ⁹-THC have

been found to bind to a large extent to several targets, such as

potassium channels and glycine receptors, among others.³²

This lack of selectivity leads, in vivo, to greater unwanted side

eﬀects, which is why ligands with a high degree of speciﬁcity

for their biological targets are preferred. In a previous work by

our research group, it was possible to obtain hCB₂R partial

agonist photochromic compounds with high selectivity over

hCB₁R.³³These compounds were based on abenzimidazole

structure and showed a higher aﬃnity for its cis photoisomer

and with variations in its biological parameters depending on

the substitution pattern introduced in the molecule. Very

recently, the synthesis of hybrid compounds by combining the

structural characteristics of the cannabinoid agonists HU-308

and AM841 was described and ligands based on HU-308

proved to be highly selective CB₂R ligands with the potential

to be chemically coupled to ﬂuorescent and photoswitchable

units.³⁴Subsequent work by the Carreira and Frank groups led

to a set of highly remarkable CB₂R ligands that enabled real-

time control of calcium release allowing monitoring of CB₂R

signaling, which shows the remarkable potential of selective

photoswitchable ligands as molecular tool compounds.^34,35

Herein, we describe the design, synthesis, photochemical

characterization, and aﬃnity and eﬃcacy studies of “photo-

rimonabant” as new photoswitchable molecules based on the

structure of the hCB₁R antagonist. Using molecular docking

and in vitro biological assays, we demonstrate that compound

16a has a very high aﬃnity in the nanomolar range, which is

even higher in its cis isoform. Additionally, this molecule is

highly selective for hCB₁R over hCB₂R, becoming the ﬁrst

molecule with these characteristics reported to date.

RESULTS AND DISCUSSION

■

Chemistry. Compounds were developed based on

published rimonabant’s structure−activity relationship (SAR)

studies.³⁶⁻³⁸We deﬁne that the most reasonable positions to

incorporate the photoswitch are atoms 3 and 5 of the central

pyrazole ring (Chart 1). For target compounds 9a−e and

14a,b, the azologization strategy was applied, by which the

photoswitch is introduced inside the scaﬀold of the

pharmacophoric structure, in such a way that the azoarene

replaces in shape and size analogs coming from the original

molecule.^39,40For this case, the pyrazole ring of rimonabant

was used, to which a diazene unit (NN) was attached in

positions 3 or 5 for the introduction of the second aromatic

ring. For target compounds 16a−d, the azo-extension strategy

was applied, whereby a phenylazo group is added to the

pharmacophore in a position expected to generate changes in

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Chart 1. Design of Rimonabant Photoderivatives “Photo-

Rimonabant” by Azologization (Blue) and Azo-Extension

(Orange) Strategies

Scheme 1. Synthesis of Analogs of Rimonabant with the

Introduction of Photoswitch in Position 5

a

ligand−receptor interaction when photoisomerization is

carried out.^39,41For this project, we considered position 3 as

optimal, extending the molecule through introduction of

azobenzene in the position of the piperidin-1-ylcarbamoyl

moiety.

a

Reagents and conditions: (a) CH₃CH₂CN, 18-crown-6, t-BuOK,

For the choice of the photoswitchable moiety to introduce,

we considered that incorporation of 1,2-dithienylethenes leads

to photoswitchable ligands with high thermal stability and

quantum yields, e.g., enabling their application in kinetic

studies.^25,42For the present study, however, we decided to take

azoarenes as the photoswitches of choice. The main advantage

of azoarenes in this case is their comparatively small molecular

size. For the introduction of the photoswitch in the diﬀerent

positions of the pyrazole ring, it is necessary to get similar

proportions to the benzene ring (position 5) or to the

piperidine ring (position 3) of the parent molecule

rimonabant. With the introduction of an azo group attached

to an aromatic ring, we could generate similar-sized structures.

Another consideration is the thermal stability of the cis-isomer.

By introducing diﬀerent substituents on the aromatic rings, it is

possible to obtain a wide variety of molecules with thermal

half-lives from seconds to several days.⁴³Finally, the ease of

synthesis was also considered, since azoarenes are highly stable

molecules under acidic or basic conditions, so the possibilities

for chemical synthesis are broad.^44,45

For the introduction of the azoarene into position 5 of the

pyrazole ring (Scheme 1), the synthesis started from the

reaction between diethyl oxalate (1) and propionitrile in the

presence of potassium tert-butoxide and 18-crown-6, yielding

compound 2. Subsequently, reﬂux of 2 in EtOH was carried

out together with 2,4-dichlorophenyl hydrazine, generating the

N-substituted pyrazole 3. Hydrolysis of 3 with lithium

hydroxide at room temperature yielded the carboxylic acid 4.

In a ﬁrst approach, compound 4 was reacted with (COCl)₂

and subsequent reaction with N-aminopiperidine in the

presence of HBTU and TEA, yielding the respective

carboxamide 5a. However, formation of byproduct 5b with

physicochemical characteristics like 5a did not allow the

continuation of the synthetic route due to problems related to

complex puriﬁcation. As an alternative, it was decided to carry

out the incorporation of the azoarene prior to formation of the

carboxamide. For this purpose, the respective substituted

anilines 6a−e were reacted with MPS for partial oxidation

yielding nitroso compounds 7a−e. Subsequently, compound 4

was coupled in position 5 of the central ring by reﬂuxing with

7a−e in 40% aqueous solution of NaOH and pyridine,

obtaining substituted azoarenes 8a−e. Finally, after activation

THF, 60 °C, 30 min, 81%; (b) 2,4-dichlorophenylhydrazine

hydrochloride, EtOH, reﬂux, 2 h, 25%; (c) LiOH, THF, H₂O, rt,

overnight, quant; (d) MPS, DCM/H₂O, rt, overnight; (e) 40%

aqueous NaOH/pyridine, 80 °C, 2 h, 20−25%; (f) (COCl)₂, DMF,

DCM, rt, 1 h; (g) N-aminopiperidine, HBTU, TEA, DCM, rt, 4 h,

45−61%.

with (COCl)₂and subsequent amide formation with N-

aminopiperidine, compounds 9a−e were obtained.

For generation of compounds with azoarene moieties in

position 3, azologization (compounds 14a,b) and azoextension

(compound 16a) strategies were used in order to enlarge the

structural diversity for photoswitchable CB₁R ligands. Syn-

thesis started from commercially available rimonabant

carboxylic acid 10 (Scheme 2). As a ﬁrst step, the activation

with (COCl)₂and subsequent reaction with ammonia at room

temperature were performed, yielding primary amide 11.

Subsequently, a Hofmann rearrangement was carried out with

NBS and metallic sodium in MeOH to generate the secondary

amide 12. Reﬂuxing with NaOH and MeOH was carried out,

yielding primary amine 13 by carbamate hydrolysis. For the

azo-coupling in position 3 of the pyrazole ring, reﬂux with 40%

of aqueous NaOH and pyridine solution in the presence of

nitroso compounds 7a,b was performed obtaining target

compounds 14a,b. In parallel, compound 10 and 4-amino-

benzylamine were treated with TEA and ECF to generate

amide 15. For the formation of the respective azobenzene,

coupling was carried out using nitrosobenzene through a

Baeyer−Mills reaction under acidic conditions, yielding 16a.

By studying the crystal structure of hCB₁R, it has been

established that residues Phe170, Phe174, and Met384 are

important binding points for the activity of antagonist ligands,

especially for hydrophobic interactions with the piperidin-1-

ylcarbamoyl moiety.⁴⁶This is conﬁrmed by SAR studies that

suggest that introduction of an additional aromatic ring at

position 5 generates an increase in the activity of the

antagonists, possibly due to a greater interaction between

this second ring with the hydrophobic residues of the

receptor.³⁸In the present work, an additional synthetic route

was designed to obtain analogs of compound 16a (Scheme 3).

The aim was to evaluate the inﬂuence of aromatic substitution

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azobenzenes, carrying out the reaction of the amines 18b,c

with nitrosobenzene, or 1,3-dihydroisobenzofuran-5-amine

with nitroso-compound 19d, yielding photoligands 20b−d.

Subsequent deprotection of the aliphatic amines in acidic

medium generated compounds 21b−d. Finally, the coupling of

the respective amines with 10 was carried out in the presence

of ECF and TEA, yielding ﬁnal compounds 16b−d. All

synthesized target compounds showed stability in aqueous

media and organic solvents during synthesis, puriﬁcation, and

biological assays.

Scheme 2. Synthesis of Analogs of Rimonabant with the

Introduction of Photoswitch in Position 3

a

Photophysicochemical Properties. To conﬁrm photo-

isomeration and to rule out possible photofatigue (loss of

photochromic behavior after multiple isomerization cycles) of

the new analogs, the photostationary states were evaluated by

UV/vis spectroscopy. Through the use of light at diﬀerent

wavelengths, it was established that all the tested compounds

show their highest photoconversion to their cis forms by UV

irradiation (λ = 366 nm) and to their trans photoisomers by

blue light (λ = 454 nm) (Figure 1). These diﬀerences are

a

Reagents and conditions: (a) (COCl)₂, DMF, DCM, rt, 1 h; (b)

25% NH₄OH, DCM, rt, 30 min, quant; (c) Na, NBS, MeOH, reﬂux, 2

h, 51%; (d) NaOH, MeOH, 70 °C, 12 h, quant; (e) 40% aqueous

NaOH/pyridine, 80 °C, 2 h, 30−34%; (f) ECF, TEA, 4-amino-

benzylamine, DMF, rt, 0 °C, 3 h, rt, overnight, 88%; (g)

nitrosobenzene, acetic acid/TFA/toluol, rt, overnight, 41%.

a

Scheme 3. Synthesis of Analogs of 16a

Figure 1. Representation of the photoisomerization of 16a and its UV

absorbance spectra by irradiation with λ = 454 nm blue light (blue,

solid line) and λ = 366 nm UV light (purple, dotted line).

a

Reagents and conditions: (a) TEA, Boc anhydride, DCM, 0 °C, 2 h,

quant; (b) nitrosobenzene, acetic acid, rt, overnight, 58−68%; (c)

MPS, DCM/H₂O, rt, overnight; (d) 1,3-dihydroisobenzofuran-5-

amine, acetic acid, rt, overnight, 62%; (e) TFA, DCM, rt, overnight,

70−96%; (f) rimonabant carboxylic acid, ECF, TEA, DMF, rt, 0 °C, 3

h, rt, overnight, 88−95%.

explained by π → π* and n → π* transitions, typical of this

type of photoswitchable compounds.⁴⁷Compounds 9a−e have

maximum absorption value of ∼350 nm for the trans

photoisomer and ∼450 nm for the cis form. Compounds

14a,b and 16a−d show slightly lower values, being close to 325

and 430 nm for the trans and cis photoisomers, respectively. By

alternately irradiating with UV and blue light, it was shown that

all compounds can easily be photoconverted between their cis

and trans states (t < 3 min). This process can be repeated for

many cycles without noticeable photofatigue (Figure 2).

Most of the compounds show long thermal stability (>2 h,

37 °C); the only exception is compound 9c, which presented a

reversal in the photoconversion of 45% after being stored in

the dark for 2 h. This instability can be explained by the push−

pull system eﬀect observed for substituted photocompounds.⁴⁸

This eﬀect is due to the introduction of electron-withdrawing

groups in the para position of the azoarene, giving a

considerable decrease in the thermal cis → trans relaxation

time.⁴⁹Furthermore, the photostationary distribution of the

compounds was quantiﬁed by HPLC. All the compounds are

of azobenzene on the biological activity of the photoligand, in a

similar way as previously published for hCB₂.³³Our hypothesis

was that structural changes between the diﬀerently substituted

azobenzene isomers could generate a diﬀerent interaction

behavior with the hydrophobic pocket of the receptor.

Additionally, through the synthesis of analog 16d, we evaluated

whether an oxygen atom incorporated in the distal position of

azobenzene leads to signiﬁcant changes in pharmacological

activity.

The synthesis started using amino-substituted aminobenzyl-

amines in the meta (17b), ortho (17c), and para (17d)

positions, which were selectively protected by coupling with

Boc on the aliphatic amine, obtaining compounds 18b−d.

Subsequently, the aromatic amine 18d was partially oxidized

using MPS, yielding nitroso-compound 19d. Baeyer−Mills

reaction in acidic medium was conducted for generation of

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Table 1. Aﬃnity Values at rCB₁and hCB₂Determined by

Radioligand Binding Studies

CB₁

trans/cis

ratio

CB₂(K_iSD) or

CB₁(K_iSD)

[³H]CP55950 displ at

a

b

photoisomer

[nM]

10 μM

rimonabant

MN-I-79

45 18

<10%

38 7.8 nM

14%

51%

61%

81%

55%

62%

45%

68%

46%

65%

21%

<10%

60%

31%

<10%

14%

9a

cis

461 337

458 239

716 26

602 85

358 62

401 33

384 40

294 66

679 160

410 103

249 155

538 66

195 63

521 200

29 2.8

444 116

311 196

377 243

142 28

224 148

221 109

602 416

1.0

0.83

1.11

0.76

0.60

2.16

2.74

15.31

1.23

1.58

2.72

trans

cis

9b

trans

cis

9c

trans

cis

9d

trans

cis

9e

trans

cis

trans

cis

trans

cis

trans

cis

trans

cis

trans

cis

Figure 2. Photochemical characterization of 16a: (a) HPLC

chromatogram and the changes in their respective stationary states

induced by light irradiation; (b) switching cycles without noticeable

photofatigue; (c) thermal stability in a period of >3 h (buﬀer pH 7.4;

37 °C).

14a

14b

16a

16b

16c

16d

distributed mainly toward their trans isomer (70−84%), which

is thermodynamically more stable. By use of UV light at λ =

366 nm, the ratio shifts in favor of its cis isomer. For

chlorinated compounds 9b, 9c, and 14b, their cis forms are

given with a maximum of approximately 50%, while for

nonchlorinated compounds their cis photoisomer can be

obtained almost quantitatively.

Radioligand Binding Studies. As all the tested

compounds showed diﬀerent extents of photoisomerization

upon irradiation in physicochemical characterization, i.e., they

are photoswitchable, it was decided to test their aﬃnity at CB₁

and CB₂to evaluate whether these diﬀerences indicate a

change in aﬃnity or selectivity for the receptors. The

radioligand binding assay was used to assess whether the cis

photoisomers had a higher or lower aﬃnity for the receptor

(“switching-on”/“switching-oﬀ”). In contrast to optical in vitro

assays, radioligand binding studies can be performed under

light exclusion and therefore eﬀectively avoid false-negative (or

false-low) diﬀerences between the in vitro activity of the

diﬀerent photoisomers due to back-isomerization upon

measurement.^27,28

14%

46%

<10%

32%

trans

a

Performed on CB₁membranes prepared from rat brain homogenate.

Values are mean values from at least two independent experiments,

each performed in triplicate. Performed on CB₂membranes

harvested from stably transfected hCB₂-HEK cells.

b

Regarding the eﬀect of aromatic substitution of the

photoswitch, we analyzed the binding of the azo-extended

compounds with para (16a), meta (16b), and ortho (16c)

substitution. As a result, the substitution in para position had

the highest aﬃnity toward CB₁. The substitution in ortho

position showed moderate activity on its cis form (K_i(CB₁) =

142 nM), and the substitution in meta showed weaker aﬃnity.

Concerning the selectivity parameter, photocompounds 16a

and 14a showed strong selectivity for CB₁over CB₂in the

radioligand displacement assay at 10 μM.

All the tested compounds have a notable aﬃnity for CB₁,

showing values in the 2 or 3 digits nanomolar range (Table 1).

The 9a−e family of compounds, despite having a good aﬃnity

for CB₁, showed no change in their binding values (K_ivalue)

when irradiated with λ = 366 nm UV light or λ= 454 nm blue

light. Compounds with photoswitch incorporation at position

3 of the pyrazole ring showed diﬀerences in the aﬃnity of their

respective photoisomers. Compounds 14a and 14b, obtained

by the azologization strategy, showed a slight change in aﬃnity

when irradiated with λ= 366 nm UV light (trans/cis ratio =

2.16 for 14a; 2.74 for 14b). In contrast, 16a, obtained using

the azo-extension strategy, showed the highest diﬀerence

regarding this key pharmacological parameter. The aﬃnity

(ratio trans/cis) is more than 15-fold higher for CB₁in its cis

photoisomer (K_i(CB₁) = 29 nM) compared to the trans iso-

form (K_i(CB₁) = 444 nM) (Figure 3a). Additionally, its cis

form showed even stronger aﬃnity for CB₁than the reference

compound rimonabant (K_i(CB₁) = 45 nM).

Cell Luminescence Assay. To further study the possible

activation mechanism by which the tested compounds bind to

hCB₁R, we studied the eﬀect over HEK293-CB₁and HEK293-

CB₂cells, transfected with the reporter plasmid pCRE-luc. In

the luciferase assay with HEK-293-CB₂-CRELuc cells, no

activity was observed for the tested molecules and hCB₂R.

None of the compounds could inhibit forskolin-induced

luciferase activity (agonist mode assay) (Supporting Informa-

tion, Figures S14 and S15). Similarly, no compound was able

to recover the activity in cells previously treated with the

agonist CP55940 (antagonist mode assay).

Regarding the luciferase assay with HEK-293-CB₁-CRELuc

cells, the compounds showed antagonistic behavior at hCB₁R.

Compounds tested at doses of >1 μM antagonized the activity

of the agonist CP55940 (1 μM) (Figure 3b). These results

support the selectivity of compounds such as 16a, which for

the radioligand binding assay showed high aﬃnity at hCB₁R

but no aﬃnity at hCB₂R.

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of 16a is predicted to occupy the same side pocket as the 4-

substituted phenyl ring of AM6538, thereby addressing

Phe102, Phe268, and Trp356 through aryl−aryl interactions,

as well as Leu193, Val196, Leu359 through hydrophobic

interactions.

Moreover, the proximal phenyl ring of the photoswitchable

group of 16a occupies a similar space as the piperidin-1-

ylcarbamoyl moiety of AM6538, forming interactions with the

hydrophobic residues Ile105, Phe170, and Phe174.

The shared structural features between 16a and AM6538, a

rimonabant derivative, lead to similar interaction networks

between the ligands and the receptor, as suggested by our

docking experiments. This may account for the experimentally

observed CB₁R antagonistic activity of both 16a photoisomers.

Finally, our docking experiments suggest that the distal phenyl

ring of the photoswitchable group of 16a occupies a

hydrophobic pocket deﬁned by Ile105, Ile119, and Phe381

in the cis conformation, while the nitrogen atoms on the

double bond address Ser123 through hydrogen bonds (Figure

4d). In contrast, the distal phenyl ring of the photoswitchable

group is pushed toward the solvent in the trans conformation,

exiting the globular core of the receptor (Figure 4e). We

hypothesize that the energy penalty of the binding mode

resulting from this conformational change on the ligand may

contribute to the ∼15-fold aﬃnity decrease of the trans

conformation of 16a, as compared to the cis conformation.

To explain the potential reasons for selectivity of 16a for

CB₁over CB₂, the superimposed structures of antagonist-

bound CB₁(AM6538) and CB₂(AM10257) reveal that, in

contrast to CB₂, CB₁antagonist pushes α-helices I and II

outward at the extracellular part of the receptor (Supporting

Information Figure S13a, denoted by arrows). These helices

adopt a more compact conformation in the antagonist-bound

(AM10257) CB₂structure. The compact binding pocket of

CB₂would cause the CB₁-bound superimposed pose of 16a to

clash with V36, F87, and F91 located at α-helices I and II in

CB₂(Supporting Information Figure S13b). Additionally, S123

(which is predicted to form hydrogen bonds with 16a in CB₁)

is replaced by C40 at the equivalent position in CB₂, which

may aﬀect hydrogen bond formation and promote CB₁

selectivity. These observations are congruent with the

incompatibility of the CB₂binding pocket geometry with the

extended conformation of rimonabant, as it has been suggested

in the literature.⁵⁶

Figure 3. Pharmacological evaluation of compound 16a. (a)

Radioligand binding data of cis (purple, dotted line) and trans

(blue, solid line) photoisomers. Symbols represent mean values

SEM (n = 9). (b) Cell luminescence assay, hCB₁R receptor

antagonism: results of HEK-293-CB₁-CRELuc cells pretreated with

16a-cis and stimulated with the agonist CP55940 (1 μM). Results are

shown as the mean SD (n = 6), p < 0.05; **p < 0.01; ***p < 0.001;

****p < 0.0001. versus negative control.

hCB₁Calcium Mobilization Assay for Antagonism.

Antagonism of 16a was evaluated by measuring hCB₁-activated

Gα_q16-coupled calcium mobilization in CHO-K₁cells over-

expressing hCB₁, using a ﬂuorescent dye as described in

previous studies.^50,51Compound 16a (tested as a mixture of

isomers due to interference between the dyes and the external

light sources used for isomerization)²⁷was found to be capable

of moving the concentration response curve of the CB₁

reference agonist CP55940 rightwards, thus conﬁrming the

activity 16a as a human CB₁antagonist (see section “Methods”

below). The apparent antagonist dissociation equilibrium

constant (K_e) of 16a was determined (∼85.3 nM),

corroborating the data obtained by radioligand binding assay

and cell luminescence assay.

Molecular Docking. To gain insights into the molecular

framework of 16a binding to the CB₁R, we optimized a

docking protocol using the coordinates of the AM6538-CB₁

complex (PDB code 5TGZ). By testing of all scoring functions

available within GOLD,^52,53the ASP⁵⁴scoring function was

found to consistently reproduce the experimental pose of

AM6538. Rescoring with the DSX_CSD⁵⁵scoring function

yielded a top-ranked pose that diﬀered only by 0.45 Å from the

experimentally observed binding mode of AM6538 (Figure 4).

The 4-methylpyrazole core of the molecules forms hydro-

phobic interactions with the side chains of Phe170 and

Phe379, while the 2,4-dichlorophenyl arm shows an edge-face

aryl−aryl interaction with Phe170 and a hydrophobic

interaction with Val196. The 4-substituted chlorophenyl ring

CONCLUSIONS

■

Through the application of the azo-extension and azologization

approaches, we were able to successfully synthesize molecules

derived from rimonabant, which have a photoswitchable

moiety in position 3 or 5 of the central pyrazole ring. These

compounds can be easily switched between their cis and trans

photoforms by short-time irradiation with light. This isomer-

ization can be repeated for many cycles without showing

photofatigue.

By pharmacological characterization, it was found that

photorimonabant molecules have a high aﬃnity for CB₁in the

nanomolar range. The incorporation of the photoswitch at

position 3 of the pyrazole generated molecules with higher

aﬃnity diﬀerences between their photoisomers (14a,b; 16a−

d) with respect to their modiﬁed homologues in position 5

(9a−e). In turn, by use of the azo-extension approach, the

aﬃnity diﬀerence between the cis and trans forms was further

increased. By synthesis of compound 16a (“photo-rimona-

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Figure 4. (a) Pose reproduction of AM6538 (0.45 Å RMSD). (b) Top scored docking pose for 16a-cis (1.30 Å partial RMSD) and (c) 16a-trans

(0.59 Å partial RMSD). (d) Detail of the interaction network of the photoswitchable group of 16a-cis within CB₁, as predicted by docking.

Hydrogen bonds are denoted by black dashed lines. (e) Comparison of the predicted binding poses of the two photoisomers of 16a. The

photoswitchable group in the trans conformation is pushed toward the solvent.

with a Bruker AV-400 NMR instrument (Bruker, Karlsruhe,

Germany) in deuterated solvents, and chemical shifts were expressed

bant”), a >15-fold higher aﬃnity was evidenced in its cis form

(K_i(CB₁) = 29 nM), compared to the trans isomer (K_i(CB₁) =

444 nM). Likewise, it was found that 16a conserves its activity

as an antagonist (K_e∼ 85.3 nM), with high selectivity for CB₁

over CB₂. These excellent pharmacological properties are

explained by the interaction between 16a and diﬀerent

aromatic residues of CB₁. Our predictions indicate that

residues Ile105, Ile119, and Phe381 are of special importance

for the marked aﬃnity of 16a-cis, mainly due to hydrophobic

interactions. However, when switched to the trans photo-

isomer, conformational changes in the distal azobenzene ring

generate increased exposure toward the solvent that leads to a

decrease in aﬃnity for the receptor.

in ppm (DMSO, H, 2.50 ppm, ¹³C, 39.52 ppm; CDCl₃, H, 7.26

ppm, ¹³C, 77.16 ppm; MeOD, ¹H, 4.87 ppm, ¹³C, 49.0 ppm; acetone-

d₆¹H, 2.05 ppm, ¹³C, 203.6 ppm). To monitor the purity of the

products by HPLC, a Shimadzu kit, equipped with a DGU-20A3R

degassing unit, a LC20AB liquid chromatograph, and an SPD-20A

UV/vis detector, was used. By use of an LCMS 2020, mass spectra

were obtained. The stationary phase was a Synergi 4 μm fusion-RP

(150 mm × 4.6 mm) column, and a MeOH/water gradient with 0.1%

formic acid was used as the mobile phase (parameters, A, water; B,

MeOH, V(B)/(V(A) + V(B)) = from 5% to 90% over 10 min, V(B)/

(V(A) + V(B)) = 90% for 5 min, V(B)/(V(A) + V(B)) = from 90%

to 5% over 3 min. The method was performed with a ﬂow rate of 1.0

mL/min and scan range of 60−1000 m/z. The new compounds were

accepted with a purity of ≥95%. UV/vis spectra and experiments were

made on a Varian Cary 50 Bio UV/vis spectrophotometer using

Hellma (type 100-QS) cuvettes (10 mm light path). Target

compounds used for biological evaluation were tested only if the

purity was ≥95%.

1

Since photo-rimonabant shows “cis-on” activation and holds

all in vitro characteristics of a GPCR molecular tool compound

in terms of aﬃnity and selectivity in various assays, we

currently use photorimonabant in several advanced pharmaco-

logical applications.

General Procedure I for the Partial Oxidation of Anilines

(7b−e, 19d). The respective aniline (1 equiv) was dissolved in DCM.

Then, an aqueous solution of potassium peroxymonosulfate (MPS) (2

equiv) was added to it (water/DCM, 1:1). The reaction mixture was

vigorously stirred at room temperature overnight. After reaction, the

mixture was diluted with DCM and washed with water. The

combined organic phases were dried over Na₂SO₄, and the solvent

was partially removed in vacuo; the brown-green crude resulting

solution was used immediately for the next reaction step without

puriﬁcation.

General Procedure II for Azo-Coupling under Alkaline

Conditions (8a−e). Crude DCM solution of nitroso compound (4

equiv) was added to the respective amine (1 equiv) dissolved in

pyridine and 40% aq NaOH (1:1). The mixture was stirred at 80 °C

for 4 h. The reaction mixture was cooled, quenched with water,

METHODS

■

Chemistry. General Methods. Both solvents and commonly used

reagents were purchased directly from commercial suppliers and used

without further puriﬁcation. Tetrahydrofuran (THF) was distilled

from sodium/benzophenone under an argon atmosphere. The

monitoring of the reactions was performed with thin layer

chromatography (TLC) on silica gel 60 on alumina foils with

ﬂuorescent indicator, and spots were detected with UV light (254

nm). Melting points were determined with a Stuart melting point

SMP3 apparatus (Bibby Sterilin Ltd., Staﬀordshire, U.K.). Preparative

TLC puriﬁcation (prep-TLC) was done by preparing glass surface

chromatographic plates (20 cm × 20 cm) with silica gel 60GF254

(Merck). For column chromatography, silica gel 60, 230−400 mesh

(Merck) was used. Nuclear magnetic resonance spectra were recorded

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acidiﬁed to pH 2−5, and extracted with ethyl acetate and water (3×).

The combined organic layers were dried over Na₂SO₄, and the solvent

was removed under reduced pressure.

reduced pressure. The residue was puriﬁed with column chromatog-

raphy (petroleum ether/ethyl acetate = 3/1) to yield to yield the title

compound (3), as a yellow solid (197 mg, 25%). Mp 180−183 °C. ¹H

NMR (400 MHz, DMSO): δ = 7.86 (d, J = 2.2 Hz, 1H), 7.58 (dd, J =

8.5, 2.3 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 5.20 (s, 2H), 4.21 (q, J =

7.1 Hz, 2H), 2.05 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H) ppm. ¹³C NMR

(101 MHz, CDCl₃): δ = 162.9 (1C), 144.0 (1C), 142.3 (1C), 136.2

(1C), 134.2 (1C), 132.8 (1C), 130.9 (1C), 130.0 (1C), 128.1 (1C),

102.6 (1C), 60.6 (1C), 14.3 (1C), 8.1 (1C) ppm. LC: t_R= 9.57 and

11.92 min, purity = 84.42%. MS: [M + H]⁺calcd for

[C₁₃H₁₃Cl₂N₃O₂]⁺= 314.05, found 314.05.

5-Amino-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-

carboxylic Acid (4). Lithium hydroxide monohydrate (66 mg, 1.54

mmol, 2 equiv) was added to a solution of 3 (242 mg, 0.77 mmol, 1

equiv) in THF/H₂O (2:1). The reaction was stirred at room

temperature for 72 h. The mixture was acidiﬁed with HCl to pH 2−5

and extracted with ethyl acetate. The combined organic layers were

washed with water, dried over Na₂SO₄and the solvent was

concentrated under reduced pressure to yield the title compound

(4) as yellow solid (216 mg, quant). Mp 198 °C (dec). ¹H NMR (400

MHz, DMSO): δ = 7.86 (d, J = 2.2 Hz, 1H), 7.58 (dd, J = 8.5, 2.3 Hz,

1H), 7.50 (d, J = 8.5 Hz, 1H), 5.14 (s, 2H), 2.05 (s, 3H) ppm. ¹³C

NMR (101 MHz, acetone-d₆): δ = 163.9 (1C), 149.1 (1C), 136.7

(1C), 135.9 (1C), 133.5 (1C), 131.7 (1C), 130.5 (1C), 129.0 (1C),

128.8 (1C), 106.3 (1C), 9.2 (1C) ppm. LC: t_R= 8.47 min, purity =

98.53%. MS: [M + H]⁺calcd for [C₁₁H₉Cl₂N₃O₂]⁺= 286.01, found

286.00.

2,3-Dichloro-1-nitrosobenzene (7b). The reaction was carried

out according to general procedure I, using 2,3-dichloroaniline (227

mg, 1.4 mmol) and MPS (0.86 g, 2.8 mmol). The crude product was

immediately used without further puriﬁcation for the next reaction

step.

2,4-Dichloro-1-nitrosobenzene (7c). The reaction was carried

out according to general procedure I, using 2,4-dicholoroaniline (243

mg, 1.5 mmol) and MPS (0.92 g, 3 mmol). The crude product was

immediately used without further puriﬁcation for the next reaction

step.

1-Methyl-2-nitrosobenzene (7d). The reaction was carried out

according to general procedure I, using o-toluidine (255 μL, 2.37

mmol) and MPS (1.46 g, 4.74 mmol). The crude product was

immediately used without further puriﬁcation for the next reaction

step.

1-Methyl-3-nitrosobenzene (7e). The reaction was carried out

according to general procedure I, using m-toluidine (150 μL, 1.42

mmol) and MPS (0.87 g, 2.84 mmol). The crude product was

immediately used without further puriﬁcation for the next reaction

step.

General Procedure III for Amidation in Position 3 of

Pyrazole (9a−e). To a stirred solution of the respective carboxylic

acid (1 equiv) in DCM were added 3 drops of DMF. The reaction

mixture was cooled to 0 °C and then, (COCl)₂(1.7 equiv) was added

dropwise. The reaction mixture was stirred for 1 h to room

temperature. Excess of (COCl)₂was removed under reduced

pressure. In another ﬂask were introduced N-aminopiperidine (1.5

equiv) and TEA (1 equiv) in DCM, and the mixture was cooled to 0

°C. Subsequently, a solution of the intermediate acyl chloride in

DCM was added dropwise at the same temperature. The reaction

mixture was stirred at 0 °C for 15 min and for 15 min more at room

temperature. The mixture was diluted with DCM, washed with

NaHCO₃and brine, dried over Na₂SO₄, and concentrated under

reduced pressure.

General Procedure IV for Selective Boc-Protection of

Aminometylanilines (18b−d). A solution of the respective diamine

(1 equiv) and TEA (2 equiv) in DCM was cooled to 0 °C. Then, a

solution of di-tert-butyl decarbonate (1 equiv) in DCM was added

dropwise to the mixture. The reaction was stirred at 0 °C for a further

2 h. After completing the reaction, the mixture was washed with sat.

NaHCO₃(aq). The organic layers were dried over Na₂SO₄, and the

solvent was removed under reduced pressure.

General Procedure V for Azo-Coupling under Acidic

Conditions (20b−d). The respective amine (1 equiv) and the

nitroso compound (1.1 equiv) were dissolved in acetic acid. The

mixture was stirred at room temperature overnight. After reaction, the

solvent was removed in vacuo. The raw material was dissolved in

DCM and washed with sat. NaHCO₃(aq). The organic layers were

dried over Na₂SO₄, and the solvent was removed under reduced

pressure.

General Procedure VI for Boc-Deprotection of Amino-

metylanilines (21b−d). The Boc-protected amine was dissolved in

a mixture DCM/TFA (9:1) and stirred at room temperature

overnight. After reaction, the mixture was neutralized with

NaHCO₃(aq) and washed with brine.

General Procedure VII for Incorporation of Azobenzene by

Azo-Extension Strategy (16b−d). Rimonabant carboxylic acid (1

equiv) and TEA (2.1 equiv) were dissolved in anhydrous DMF. Then,

ECF (2.05 equiv) was added dropwise and the reaction was

maintained under stirring at 0 °C for 2 h. To the reaction mixture,

the respective azobenzene (2.4 equiv) was added, and the reaction

was stirred at 0 °C for 1 h and subsequently at room temperature

overnight. After the reaction was completed, the reaction was poured

into ice−water. The precipitate was ﬁltered and washed with water

and petroleum ether.

Potassium (Z)-3-Cyano-1-ethoxy-1-oxobut-2-en-2-olate (2).

Diethyl oxalate (1.5 mL, 10.94 mmol, 1 equiv) was added to a

solution of 18-crown-6 (280 mg, 1.1 mmol, 0.1 equiv) and sodium

tert-butoxide (1.22 g, 10.94 mmol, 1 equiv) in anhydrous THF at 0

°C. The mixture was heated at 60 °C while anhydrous propionitrile

(0.82 mL, 10.94 mmol, 1 equiv) was added dropwise. The reaction

mixture was stirred at 60 °C for 1 h. After cooling, the solid was

ﬁltered and washed with anhydrous diethyl ether to yield the title

compound (2) as yellow powder (1.71 g, 81%). Mp 114 °C (dec). ¹H

NMR (400 MHz, DMSO): δ = 4.07−3.92 (m, 3H), 1.51 (s, 2H),

1.23−1.13 (m, 3H) ppm. ¹³C NMR (101 MHz, DMSO): δ = 167.9

(1C), 162.7 (1C), 128.5 (1C), 63.6 (1C), 58.9 (1C), 14.5 (1C), 12.5

(1C) ppm. LC: t_R= 6.52 min, purity = 99.15%. MS: [M − K]⁻calcd

for [C₇H₈KNO₃− K]⁻= 154.05, found 154.05.

(E)-1-(2,4-Dichlorophenyl)-4-methyl-5-(phenyldiazenyl)-1H-

pyrazole-3-carboxylic Acid (8a). The reaction was carried out

according to general procedure II, using nitrosobenzene (117 mg,

1.09 mmol) and 4 (155 mg, 0.55 mmol). The residue was puriﬁed by

column chromatography (dichloromethane/methanol/formic acid =

98/2/0.05) to yield the title compound (8a) as an orange solid. (38.5

1

mg, 25%). Mp 248 °C. H NMR (400 MHz, DMSO) δ 7.95 (d, J =

2.2 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.67 (dd, J = 8.5, 2.3 Hz, 1H),

7.61−7.51 (m, 5H), 2.61 (s, 3H) ppm. ¹³C NMR (101 MHz,

DMSO): δ = 163.2 (1C), 152.2 (1C), 148.9 (1C), 135.6 (1C), 135.5

(1C), 134.8 (1C), 132.1 (1C), 131.7 (1C), 130.8 (1C), 129.3 (2C),

129.1 (1C), 128.0 (1C), 121.9 (2C), 113.4 (1C), 9.4 (1C) ppm. LC:

t_R(min) = 10.69 (cis) + 11.11 (trans), purity = 76.8%. MS: [M + H]⁺

calcd for [C₁₇H₁₂Cl₂N₄O₂]⁺= 375.04, found 375.05.

(E)-1-(2,4-Dichlorophenyl)-5-((2,3-dichlorophenyl)-

diazenyl)-4-methyl-1H-pyrazole-3-carboxylic Acid (8b). The

reaction was carried out according to general procedure II, using 7b

solution (1.4 mmol, 4 equiv) and 4 (100 mg, 0.35 mmol, 1 equiv)

The residue was puriﬁed by column chromatography (petroleum

ether/ethyl acetate/formic acid = 1/1/0.05) to achieve the title

compound (8b) as an orange solid (31.1 mg, 20%). Mp 253 °C (dec).

¹H NMR (400 MHz, DMSO): δ = 13.35 (s, 1H), 7.97 (d, J = 2.2 Hz,

Ethyl-5-amino-1-(2,4-dichlorophenyl)-4-methyl-1H-pyra-

zole-3-carboxylate (3). 2,4-Dichlorophenylhydrazine hydrochloride

(718 mg, 3.36 mmol, 1.3 equiv) was added to a suspension of 2 (500

mg, 2.58 mmol, 1 equiv) in EtOH. The reaction mixture was reﬂuxed

for 2 h. After cooling, water was added and the precipitate was

collected, washed with water, and dried. Then, the compound was

dissolved in ethyl acetate and the organic layer was washed with

water, dried over Na₂SO₄and the solvent was concentrated under

1H), 7.83 (dd, J = 7.9, 1.5 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.69

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(dd, J = 8.5, 2.3 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.41 (dd, J = 8.2,

1.5 Hz, 1H), 2.66 (s, J = 8.1 Hz, 3H) ppm. ¹³C NMR (101 MHz,

DMSO): δ = 163.0 (1C), 149.7 (1C), 149.3 (1C), 143.4 (1C), 135.9

(1C), 135.13 (1C), 133.3 (1C), 133.2 (1C), 132.7 (1C), 131.8 (1C),

130.8 (1C), 129.7 (1C), 128.8 (1C), 128.4 (1C), 116.8 (1C), 115.1

(1C), 9.7 (1C) ppm. LC: t_R(min) = 10.80 (cis) +11.79 (trans), purity

= 99.0%. MS: [M + H]⁺calcd for [C₁₇H₁₀Cl₄N₄O₂]⁺= 442.96,

444.96, found 442.95, 444.95.

(1C). LC: t_R(min) = 10.73 (cis) + 11.62 (trans), purity = 99.1%. MS:

m/z [M + H]⁺calcd for (C₂₂H₂₂Cl₂N₆O)⁺= 457.13, found 457.10.

1-(2,4-Dichlorophenyl)-4-methyl-5-[(E)-2-(2,3-

dichlorophenyl)diazen-1-yl]-N-(piperidin-1-yl)-1H-pyrazole-3-

carboxamide (9b). The reaction was carried out according to

general procedure III, using 8b (38 mg, 0.09 mmol, 1 equiv),

(COCl)₂(12.5 μL, 0.15 mmol, 1.7 equiv), N-aminopiperidine (16.3

μL, 0.14 mmol, 1.5 equiv), and TEA (12.5 μL, 0.09 mmol, 1 equiv).

The residue was puriﬁed by column chromatography (petroleum

ether/ethyl acetate = 2/1) to achieve the title compound as an orange

solid (27.5 mg, 0.052 mmol, 61%). Mp 164 °C. ¹H NMR (400 MHz,

CDCl₃): δ = 7.65 (s, 1H), 7.56 (dt, J = 3.4, 1.7 Hz, 1H), 7.54−7.48

(m, 1H), 7.45−7.39 (m, 2H), 7.37−7.30 (m, 1H), 7.19 (t, J = 8.0 Hz,

1H), 2.94−2.80 (m, 4H), 2.76 (s, 3H), 1.79−1.72 (m, 4H), 1.49−

1.36 (m, 2H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 159.1 (1C),

150.2 (1C), 150.0 (1C), 144.4 (1C), 135.9 (1C), 135.8 (1C), 134.4

(1C), 134.2 (1C), 132.7 (1C), 132.5 (1C), 129.9 (1C), 129.8 (1C),

127.6 (1C), 127.0 (1C), 116.4 (1C), 114.5 (1C), 56.9 (2C), 25.2

(2C), 23.1 (1C), 9.6 (1C) ppm. LC: t_R(min) = 11.12 (cis) + 12.39

(trans), purity = 99.2%. MS: m/z [M + H]⁺calcd for

(C₂₂H₂₀Cl₄N₆O)⁺= 525.05, 527.05, found 525.00, 527.00.

1-(2,4-Dichlorophenyl)-5-[(E)-2-(2,4-dichlorophenyl)diazen-

1-yl]-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide

(9c). The reaction was carried out according to general procedure III,

using 8c (40 mg, 0.09 mmol, 1 equiv), (COCl)₂(12.5 μL, 0.15 mmol,

1.7 equiv), N-aminopiperidine (16.3 μL, 0.14 mmol, 1.5 equiv), and

TEA (12.5 μL, 0.09 mmol, 1 equiv). The residue was puriﬁed by

column chromatography (petroleum ether/ethyl acetate = 4/1) to

achieve the target compound as an orange solid (25 mg, 52%). Mp

144−147 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.64 (s, 1H), 7.58−

7.55 (m, 1H), 7.52 (d, J = 2.1 Hz, 1H), 7.44−7.37 (m, 3H), 7.23 (dd,

J = 8.8, 2.2 Hz, 1H), 2.91−2.81 (m, 4H), 2.75 (s, 3H), 1.81−1.72 (m,

4H), 1.49−1.39 (m, 2H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ =

159.8 (1C), 150.8 (1C), 147.9 (1C), 145.0 (1C), 138.6 (1C), 137.6

(1C), 136.6 (1C), 136.4 (1C), 133.3 (1C), 131.0 (1C), 130.6 (1C),

130.5 (1C), 128.3 (1C), 128.2 (1C), 117.7 (1C), 116.6 (1C), 57.6

(2C), 25.9 (2C), 23.8 (1C), 10.3 (1C) ppm. LC: t_R(min) = 10.94

(cis) + 12.56 (trans), purity = 96.2%. MS: m/z [M + H]⁺calcd for

(C₂₂H₂₀Cl₄N₆O)⁺= 525.05, 527.05, found 525.05, 527.05.

(E)-1-(2,4-Dichlorophenyl)-5-((2,4-dichlorophenyl)-

diazenyl)-4-methyl-1H-pyrazole-3-carboxylic Acid (8c). The

reaction was carried out according to general procedure II, using 7c

solution (1.4 mmol, 4 equiv) and 4 (100 mg, 0.35 mmol, 1 equiv).

The residue was puriﬁed by column chromatography (petroleum

ether/ethyl acetate/formic acid = 2/1/0.05) to achieve the title

1

compound (8c) as an orange solid. (37.5 mg, 24%). Mp 242 °C. H

NMR (400 MHz, DMSO): δ = 7.93 (t, J = 2.6 Hz, 1H), 7.86 (d, J =

2.2 Hz, 1H), 7.75 (dd, J = 8.5, 2.4 Hz, 1H), 7.68−7.63 (m, 1H), 7.53

(dd, J = 8.8, 2.2 Hz, 1H), 7.43 (d, J = 8.8 Hz, 1H), 2.62 (s, 3H) ppm.

¹³C NMR (101 MHz, DMSO): δ = 163.0 (1C), 149.4 (1C), 146.9

(1C), 143.4 (1C), 137.5 (1C), 136.0 (1C), 135.8 (1C), 135.1 (1C),

131.8 (1C), 130.9 (1C), 130.4 (1C), 129.7 (1C), 128.7 (1C), 128.4

(1C), 117.6 (1C), 116.5 (1C), 9.8 (1C) ppm. LC: t_R(min) = 9.77

(cis) + 10.86 (trans), purity = 80.5%. MS: [M + H]⁺calcd for

[C₁₇H₁₀Cl₄N₄O₂]⁺= 442.96, 444.96, found 442.95, 444.95.

(E)-1-(2,4-Dichlorophenyl)-4-methyl-5-(o-tolyldiazenyl)-1H-

pyrazole-3-carboxylic Acid (8d). The reaction was carried out

according to general procedure II, using 7d solution (2.4 mmol, 6

equiv) and 4 (113 mg, 0.4 mmol, 1 equiv). The residue was puriﬁed

by column chromatography (petroleum ether/ethyl acetate/formic

acid = 2/1/0.1) to achieve the title compound (8d) as an orange

solid. (38.9 mg, 25%). Mp 225 °C. ¹H NMR (400 MHz, DMSO): δ =

7.94 (d, J = 2.3 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.66 (dd, J = 8.5,

2.3 Hz, 1H), 7.46−7.31 (m, 3H), 7.26 (ddd J = 8.3, 6.7, 1.7 Hz, 1H)

2.61 (s, 3H), 2.29 (s, 3H) ppm. ¹³C NMR (101 MHz, DMSO): δ =

163.6 (1C), 150.8 (1C), 149.9 (1C), 143.6 (1C), 139.9 (1C), 136.8

(1C), 135.4 (1C), 132.8 (1C), 132.3 (1C), 132.0 (1C), 131.2 (1C),

129.9 (1C), 128.8 (1C), 127.2 (1C), 115.9 (1C), 114.5 (1C), 17.1

(1C), 10.1 (1C) ppm. LC: t_R(min) = 10.26 (cis) + 11.19 (trans),

purity = 74.1%. MS: [M + H]⁺calcd for [C₁₈H₁₄Cl₂N₄O₂]⁺= 389.06,

found 389.05.

(E)-1-(2,4-Dichlorophenyl)-4-methyl-5-(m-tolyldiazenyl)-

1H-pyrazole-3-carboxylic Acid (8e). The reaction was carried out

according to general procedure II, using 7e solution (1.27 mmol, 6

equiv) and 4 (60 mg, 0.21 mmol, 1 equiv). The residue was puriﬁed

by column chromatography (petroleum ether/ethyl acetate/formic

acid = 2/1/0.1) to achieve the title compound (8e) as an orange

solid. (16.0 mg, 20%). Mp 237 °C (dec). ¹H NMR (400 MHz,

DMSO): δ = 7.85 (d, J = 2.2 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.59−

7.55 (m, 1H), 7.34 (d J = 7.2 Hz, 1H), 7.30 (d J = 7.6 Hz, 1H), 7.28−

7.19 (m, 2H), 2.50 (s, 3H), 2.26 (s, 3H) ppm. ¹³C NMR (101 MHz,

DMSO): δ = 163.5 (1C), 152.5 (1C), 149.7 (1C), 144.6 (1C), 139.4

(1C), 136.2 (1C), 135.3 (1C), 133.2 (1C), 132.3 (1C), 131.2 (1C),

129.7 (1C), 128.5 (1C), 123.7 (1C), 118.8 (1C), 114.0 (1C), 111.5

(1C), 21.0 (1C), 9.9 (1C) ppm. LC: t_R(min) = 10.77 (cis) + 11.75

(trans), purity = 82.5%. MS: [M + H]⁺calcd for [C₁₈H₁₄Cl₂N₄O₂]⁺=

389.06, found 389.05.

1-(2, 4-Dichlorophenyl)-4-methyl-5-[(1E)-2-(2-

methylphenyl)diazen-1-yl]-N-(piperidin-1-yl)pyrazole-3-car-

boxamide (9d). The reaction was carried out according to general

procedure III, using 8d (44 mg, 0.11 mmol, 1 equiv), (COCl)₂(16

μL, 0.19 mmol, 1.7 equiv), N-aminopiperidine (18 μL, 0.17 mmol, 1.5

equiv), and TEA (15 μL, 0.11 mmol, 1 equiv). The residue was

puriﬁed by column chromatography (petroleum ether/ethyl acetate =

2/1) to achieve the target compound as an orange solid (23 mg,

1

45%). Mp 172−174 °C. H NMR (400 MHz, DMSO): δ = 9.26 (s,

1H), 7.95 (d, J = 2.2 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.68 (dd J =

8.5, 2.3 Hz, 1H), 7.42 (dt J = 14.1, 6.6 Hz, 2H), 7.35 (d, J = 7.2 Hz,

1H), 7.28 (t, J = 7.4 Hz, 1H), 2.84−2.77 (m, 4H), 2.60 (s, 3H), 2.32

(s, 3H), 1.59 (dd J = 10.6, 5.5 Hz, 4H), 1.25 (s, 2H). ¹³C NMR (101

MHz, CDCl₃): δ = 160.5 (1C), 159.8 (1C), 151.2 (1C), 144.6 (1C),

139.5 (1C), 135.9 (1C), 133.2 (1C), 132.1 (1C), 131.6 (1C), 130.3

(1C), 130.2 (1C), 128.9 (1C), 127.9 (1C), 126.6 (1C), 114.7 (1C),

114.6 (1C), 57.3 (2C), 25.6 (2C), 23.5 (1C), 17.5 (1C), 9.9 (1C).

LC: t_R(min)= 10.47 (cis) + 11.25 (trans), purity = 98.0%. MS: m/z

[M + H]⁺calcd for (C₂₃H₂₄Cl₂N₆O)⁺= 471.15, found 471.15.

1-(2, 4-Dichlorophenyl)-4-methyl-5-[(1E)-2-(3-

methylphenyl)diazen-1-yl]-N-(piperidin-1-yl)-1H-pyrazole-3-

carboxamide (9e). The reaction was carried out according to

general procedure III, using 8e (24 mg, 0.062 mmol, 1 equiv),

(COCl)₂(8 μL, 0.086 mmol, 1.4 equiv), N-aminopiperidine (11 μL,

0.092 mmol, 1.5 equiv), and TEA (9 μL, 0.062 mmol, 1 equiv). The

residue was puriﬁed by prep-TLC (petroleum ether/ethyl acetate =

4/1) to achieve the ﬁnal compound as an orange solid (16 mg, 55%).

Mp 134 °C. ¹H NMR (400 MHz, CDCl₃): δ = 7.67 (s, 1H), 7.58 (d, J

= 2.0 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.45−7.38 (m, 3H), 7.32 (t, J

= 7.6 Hz, 1H), 7.29−7.27 (m, J = 6.4 Hz, 1H), 2.95−2.83 (m, 4H),

2.73 (s, 3H), 2.41 (s, 3H), 1.85−1.71 (m, 4H), 1.51−1.40 (m, 2H).

¹³C NMR (101 MHz, CDCl₃): δ = 159.8 (1C), 153.1 (1C), 150.2

1-(2,4-Dichlorophenyl)-4-methyl-5-[(E)-2-phenyldiazen-1-

yl]-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (9a). The

reaction was carried out according to general procedure III, using

8a (250 mg, 0.55 mmol, 1 equiv), (COCl)₂(0.08 mL, 0.94 mmol, 1.7

equiv), N-aminopiperidine (0.1 mL, 0.83 mmol, 1.5 equiv), and TEA

(0.08 mL, 0.55 mmol, 1 equiv). The residue was puriﬁed by prep-

TLC (DCM/MeOH = 99/1) to achieve the title compound (9a) as

1

an orange solid (155 mg, 61%). Mp 141 °C. H NMR (400 MHz,

CDCl₃) δ 7.68−7.61 (m, 3H), 7.59−7.55 (m, 1H), 7.46−7.39 (m,

5H), 2.86 (dd, J = 15.7, 10.6 Hz, 4H), 2.73 (s, 3H), 1.80−1.71 (m,

4H), 1.48−1.38 (m, 2H). ¹³C NMR (101 MHz, CDCl₃): δ = 159.7

(1C), 152.9 (1C), 150.1 (1C), 144.6 (1C), 136.3 (1C), 135.9 (1C),

133.2 (1C), 131.9 (1C), 130.3 (1C), 130.0 (1C), 129.3 (2C), 127.7

(1C), 122.8 (2C), 114.0 (1C), 57.23 (2C), 25.6 (2C), 23.5 (1C), 9.8

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(1C), 144.6 (1C), 139.2 (1C), 136.4 (1C), 135.9 (1C), 133.2 (1C),

132.8 (1C), 130.4 (1C), 130.0 (1C), 129.1 (1C), 127.8 (1C), 124.0

(1C), 119.4 (1C), 114.1 (1C), 57.2 (2C), 25.6 (2C), 23.5 (1C), 21.4

(1C), 9.8 (1C). LC: t_R(min) = 10.74 (cis) + 11.73 (trans), purity =

98.4%. MS: m/z [M + H]⁺calcd for (C₂₃H₂₄Cl₂N₆O)⁺= 471.15,

found 471.10.

The organic layer was dried over Na₂SO₄and the solvent removed

under reduced pressure. The residue was puriﬁed by prep-TLC

(petroleum ether/ethyl acetate = 20/1) to achieve the target

1

compound as an orange solid (15 mg, 30%). Mp 160 °C. H NMR

(400 MHz, CDCl₃): δ = 8.03−7.97 (m, 2H), 7.55−7.44 (m, 3H),

7.42 (dd, J = 5.3, 3.1 Hz, 2H), 7.36−7.28 (m, 3H), 7.18−7.11 (m,

2H), 2.39 (d, J = 5.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ =

160.9 (1C), 153.2 (1C), 143.3 (1C), 136.2 (1C), 136.0 (1C), 135.1

(1C), 133.2 (1C), 131.4 (1C), 131.0 (2C), 130.9 (1C), 130.3 (1C),

129.2 (2C), 129.1 (2C), 128.0 (1C), 127.4 (1C), 123.1 (2C), 110.9

(1C), 9.9 (1C). LC: t_R(min) = 11.06 (cis) + 12.07 (trans), purity =

98.5%. MS: m/z [M + H]⁺calcd for (C₂₂H₁₅Cl₃N₄)⁺= 441.04, found

440.95.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-

pyrazole-3-carboxamide (11). Four drops of DMF were added to

a stirred solution of rimonabant carboxylic acid (100 mg, 0.262 mmol,

1 equiv) in DCM (4 mL). The reaction mixture was cooled to 0 °C,

and then, (COCl)₂(40 μL, 0.445 mmol, 1.7 equiv) was added

dropwise. The reaction mixture was stirred for 1 h at room

temperature. Excess of (COCl)₂was removed under reduced

pressure. Then, the intermediate acyl chloride was diluted in DCM,

cooled to 0 °C, and introduced ammonia solution 25% dropwise (500

μL). The reaction mixture was stirred at 0 °C for 15 min and for 15

min more at room temperature. The mixture was quenched with

NH₄Cl saturated solution and washed with water and DCM. The

organic layer was dried over Na₂SO₄and concentrated under reduced

pressure to yield the title compound (11) as a white solid without

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-3-[(E)-2-(2,3-

dichlorophenyl)diazen-1-yl]-4-methyl-1H-pyrazole (14b). This

compound was synthesized analogously as 14a, using 7b (4 equiv)

and 13 (100 mg, 0.26 mmol, 1 equiv). The residue was puriﬁed by

prep-TLC (petroleum ether/ethyl acetate = 15/1) to achieve the ﬁnal

1

compounds as an orange solid (46 mg, 34%). Mp 154 °C. H NMR

(400 MHz, CDCl₃): δ= 8.06 (d, J = 8.1, 1.0 Hz, 1H), 7.88 (d, J = 7.9,

1.1 Hz, 1H), 7.75 (d, J = 2.2 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.63

(td, J = 5.2, 2.8 Hz, 3H), 7.59−7.53 (m, 1H), 7.43 (t, J = 7.5 Hz, 2H),

2.68 (d, J = 16.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 161.5

(1C), 150.8 (1C), 143.6 (1C), 136.2 (1C), 136.1 (1C), 135.3 (1C),

134.4 (1C), 133.0 (1C), 132.4 (1C), 131.1 (2C), 130.7 (1C), 130.4

(1C), 129.2 (1C), 129.1 (2C), 128.0 (1C), 127.5 (1C), 127.1 (1C),

115.4 (1C), 109.0 (1C), 10.8 (1C). LC: t_R(min) = 11.86 (cis) +

14.12 (trans), purity = 97.3%. MS: m/z [M + H]⁺calcd for

(C₂₂H₁₃Cl₅N₄)⁺= 508.97, 510.96, found 508.90, 510.95.

1

further puriﬁcation (109.3 mg, quant.). Mp 185−187 °C. H NMR

(400 MHz, DMSO): δ = 7.75 (dd, J = 13.7, 5.4 Hz, 2H), 7.57 (dd, J =

8.3, 2.2 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.28 (s, 1H), 7.23 (d, J =

8.4 Hz, 2H), 2.24 (s, 3H) ppm. ¹³C NMR (101 MHz, DMSO): δ =

164.0 (1C), 144.8 (1C), 142.5 (1C), 135.8 (1C), 135.0 (1C), 133.7

(1C), 132.1 (1C), 131.9 (1C), 131.2 (2C), 129.6 (1C), 128.7 (2C),

128.3 (1C), 127.3 (1C), 116.5 (1C), 9.20 (1C) ppm. LC: t_R= 10.98;

purity = 97.5%. MS: [M + H]⁺calcd for [C₁₇H₁₂Cl₃N₃O]⁺= 380.01,

found 380.00.

Methyl N-[5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-

methyl-1H-pyrazol-3-yl]carbamate (12). Metallic sodium (3.25

g, 140.4 mmol, 30 equiv) was dissolved in methanol. Then,

compound 11 (1.8 g, 4.68 mmol, 1 equiv) and NBS (1.26 g, 7.02

mmol, 1.5 equiv) were added to the previously solution. The reaction

was carried out under reﬂux for 1.5 h, and then the solvent was

concentrated under reduced pressure. The crude was dissolved in

ethyl acetate, washed with water and brine, and dried over Na₂SO₄.

The residue was puriﬁed by ﬂash chromatography (petroleum ether/

ethyl acetate = 3:1) to achieve the title compound (12), as a white

solid (0.989 g, 51%). Mp 176−178 °C. ¹H NMR (400 MHz,

DMSO): δ = 9.42 (s, 1H), 7.73 (d, J = 2.1 Hz, 1H), 7.60−7.48 (m,

2H), 7.44 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 3.65 (s, 3H),

1.94 (s, 3H) ppm.¹³C NMR (101 MHz, DMSO): δ = 154.8 (1C),

146.7 (1C), 141.0 (1C), 136.1 (1C), 134.3 (1C), 133.4 (1C), 132.0

(1C), 131.8 (1C), 130.8 (2C), 129.6 (1C), 128.7 (2C), 128.3 (1C),

127.9 (1C), 110.0 (1C), 51.9 (1C), 8.31 (1C) ppm. LC: t_R= 10.72

min, purity = 90.8%. MS: [M + 2]⁺calcd for [C₁₈H₁₄Cl₃N₃O₂]⁺=

410.02, 412.02, found 410.00, 412.00.

N-(4-Aminobenzyl)-5-(4-chlorophenyl)-1-(2,4-dichloro-

phenyl)-4-methyl-1H-pyrazole-3-carboxamide (15). Rimona-

bant carboxylic acid (200 mg, 0.524 mmol, 1 equiv) and TEA

(0.15 mL, 1.10 mmol, 2.1 equiv) were dissolved in anhydrous DMF.

Then, ECF (0.11 mL, 1.07 mmol, 2.05 equiv) was added dropwise

and the reaction was maintained under stirring at 0 °C for 2 h. To the

reaction mixture, 4-aminobenzylamine (0.14 mL, 1.26 mmol, 2.4

equiv) was added, and the reaction was stirred at 0 °C for an hour and

subsequently at room temperature overnight. After the reaction was

completed, the reaction was poured into ice−water. The precipitate

was ﬁltered and washed with water and petroleum ether to aﬀord the

title compound (15) as a white solid (225 mg, 88%). Mp 102 °C. ¹H

NMR (400 MHz, CDCl₃): δ = 7.44−7.36 (m, 1H), 7.31−7.24 (m,

4H), 7.15 (d, J = 8.3 Hz, 3H), 7.06 (t, J = 8.2 Hz, 2H), 6.64 (d, J = 8.4

Hz, 2H), 4.49 (d, J = 5.8 Hz, 2H), 4.11−3.08 (m, 2H), 2.41 (d, J =

9.7 Hz, 3H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 162.5 (1C),

145.9 (1C), 145.1 (1C), 143.1 (1C), 136.1 (1C), 136.0 (1C), 135.0

(1C), 133.1 (1C), 130.9 (2C), 130.6 (1C), 130.4 (1C), 129.5 (2C),

129.0 (2C), 128.4 (1C), 128.0 (1C), 127.4 (1C), 117.9 (1C), 115.3

(2C), 42.8 (1C), 9.6 (1C) ppm. LC: t_R= 10.00 min, purity = 93.8%.

MS: [M + H]⁺calcd for [C₂₄H₁₉Cl₃N₄O]⁺= 485.07, found 485.05.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-((4-

[(1E)-2-phenyldiazen-1-yl]phenyl)methyl)-1H-pyrazole-3-car-

boxamide (16a). Nitrosobenzene (48 mg, 0.45 mmol, 2 equiv) was

added to a solution of 15 (110 mg, 0.226 mmol, 1 equiv) in a mixture

of acetic acid/toluene/TFA (6:6:1). Then, the reaction was stirred at

room temperature overnight. After reaction, the mixture was diluted

with ethyl acetate and washed with water. The organic layers were

separated, dried over Na₂SO₄, and concentrated under reduced

pressure. The residue was puriﬁed by prep-TLC (petroleum ether/

ethyl acetate = 4/1) to achieve the title compound (53 mg, 41%) as

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-

pyrazol-3-amine (13). Compound 12 (30 mg, 0.073 mmol, 1

equiv) was dissolved in methanol. Then to the mixture was added

NaOH (30 mg, 6.5 mmol, 90 equiv). The reaction was heated to 70

°C for 12 h. After completion of the reaction, the solvent was

concentrated under reduced pressure. The crude was dissolved in

ethyl acetate, washed with water and brine, and dried with Na₂SO₄to

yield the title compound (13) as a white solid without further

1

puriﬁcation (26 mg, quant). Mp 140−142 °C. H NMR (400 MHz,

DMSO): δ = 7.66 (d, J = 2.3 Hz, 1H), 7.49−7.35 (m, 4H), 7.17−7.09

(m, 2H), 4.90 (s, 2H), 1.91 (s, 3H) ppm.¹³C NMR (101 MHz,

DMSO): δ = 155.8 (1C), 140.4 (1C), 137.1 (1C), 133.1 (1C), 132.7

(1C), 132.1 (1C), 131.7 (1C), 130.5 (2C), 129.5 (1C), 129.0 (1C),

128.5 (2C), 128.0 (1C), 102.3 (1C), 7.66 (1C) ppm. LC: t_R= 10.86

1

an orange solid. Mp 93 °C. H NMR (400 MHz, CDCl₃): δ = 7.70

(ddd, J = 8.3, 4.1, 1.7 Hz, 3H), 7.34−7.27 (m, 4H), 7.21 (d, J = 1.8

Hz, 1H), 7.16 (t, J = 6.1 Hz, 1H), 7.12−7.02 (m, 5H), 6.90−6.84 (m,

2H), 6.63 (ddd, J = 6.5, 4.3, 1.6 Hz, 1H), 4.43 (t, J = 55.8, 6.2 Hz,

2H), 2.20 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ = 163.1 (1C),

153.1 (1C), 152.5 (1C), 145.2 (1C), 143.6 (1C), 142.0 (1C), 136.5

(1C), 136.4 (1C), 135.4 (1C), 133.4 (1C), 131.4 (1C), 131.3 (2C),

130.9 (1C), 130.8 (1C), 129.5 (2C), 129.4 (2C), 129.0 (2C), 128.4

(1C), 127.6 (1C), 123.6 (2C), 123.3 (2C), 118.4 (1C), 43.1 (1C),

9.9 (1C). LC: t_R(min) = 11.12 (cis) + 12.23 (trans), purity = 99.9%.

min, purity = 91.3%. MS: [M + H]⁺calcd for [C₁₆H₁₂Cl₃N₃]⁺

352.02, found 352.00.

=

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-[(E)-

2-phenyldiazen-1-yl]-1H-pyrazole (14a). Nitrosobenzene (24

mg, 0.11 mmol, 2 equiv) was added to a solution of 13 (40 mg,

0.11 mmol, 1 equiv) in pyridine/NaOH 40% (1:1). The mixture was

stirred at 80 °C for 2 h. Then, the reaction was cooled, quenched with

water, acidiﬁed to pH 2−5, and extracted with ethyl acetate and water.

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MS: m/z [M + H]⁺calcd for (C₃₀H₂₂Cl₃N₅O)⁺= 574.09, found

574.05.

7.75 (s, 1H), 7.40 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H), 5.14

(s, 4H), 5.08 (s, 1H), 4.37 (d, J = 5.2 Hz, 2H), 1.47 (s, 9H) ppm. ¹³

NMR (101 MHz, CDCl₃): δ = 155.7 (1C), 152.4 (1C), 151.6 (1C),

141.9 (1C), 140.1 (1C), 127.8 (1C), 123.6 (2C), 122.9 (2C), 121.2

(2C), 113.9 (1C), 79.4 (1C), 73.1 (2C), 44.1 (1C), 28.2 (3C) ppm.

LC: t_R(min) = 9.10 (cis) + 10.38 (trans), purity = 87.7%. MS: [M +

H]⁺calcd for [C₂₀H₂₃N₃O₃]⁺= 354.18, found 354.13.

C

tert-Butyl (3-Aminobenzyl)carbamate (18b). The reaction was

carried out according to general procedure IV using 3-aminobenzyl-

amine (1 g, 8.19 mmol), TEA (2.27 mL, 16.4 mmol), and di-tert-butyl

decarbonate (1.79, 8.19 mmol). The product was used for the next

step without further puriﬁcation. The title compound (18b) was

1

obtained as a white solid (1.80 g, quant). Mp 50−54 °C. H NMR

(E)-(3-(Phenyldiazenyl)phenyl)methanamine (21b). The re-

action was carried out according to general procedure VI, using 20b

(80 mg, 0.26 mmol) dissolved in TFA (500 μL) and DCM (4500 μL)

to aﬀord the title product (21b) as an orange oil. No further

(400 MHz, CDCl₃): δ = 7.11 (t, J = 7.7 Hz, 1H), 6.63 (d, J = 7.5 Hz,

1H), 6.56 (dd, J = 11.0, 3.1 Hz, 2H), 4.83 (s, 1H), 4.20 (d, J = 5.6 Hz,

2H), 3.64 (s, 2H), 1.45 (s, 9H) ppm. ¹³C NMR (101 MHz, CDCl₃):

δ = 156.2 (1C), 146.6 (1C), 140.3 (1C), 129.7 (1C), 117.7 (1C),

114.1 (1C), 79.3 (1C), 44.9 (1C), 28.5 (1C), 28.5 (3C) ppm.

tert-Butyl (2-Aminobenzyl)carbamate (18c). The reaction was

carried out according to general procedure IV using 3-aminobenzyl-

amine (1 g, 8.1gmmol), TEA (2.27 mL, 16.4 mmol), and di-tert-butyl

decarbonate (1.79, 8.19 mmol). The product was used for the next

step without further puriﬁcation. The title compound (18c) was

1

puriﬁcation was performed (57 mg, quant.). H NMR (400 MHz,

CDCl₃): δ = 7.85−7.80 (m, 2H), 7.68 (s, 1H), 7.64 (d, J = 7.7 Hz,

1H), 7.43−7.39 (m, 5H), 3.74 (s, 2H), 1.41 (s, 2H) ppm. ¹³C NMR

(101 MHz, CDCl₃): δ = 154.0 (1C), 153.6 (1C), 142.1 (1C), 130.0

(1C), 129.2 (1C), 129.0 (2C), 128.8 (1C), 121.7 (2C), 121.0 (1C),

118.9 (1C), 45.3 (1C) ppm. LC: t_R(min) = 5.24 (cis) + 7.26 (trans),

purity = 81.6%. MS: [M + H]⁺calcd for [C₁₃H₁₃N₃]⁺= 212.12, found

212.20.

1

obtained as a white solid (1.74 g, 96%). Mp 85−88 °C. H NMR

(400 MHz, CDCl₃): δ = 7.12 (td, J = 7.7, 1.5 Hz, 1H), 7.13 (d, J = 6.9

Hz, 1H), 6.89 (t, J = 7.4 Hz, 2H), 4.91 (s, 1H), 4.36−4.16 (m, 4H),

1.45 (s, 9H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 157.7 (1C),

146.2(1C), 132.6 (1C), 129.0 (1C), 124.2 (1C), 119.0 (1C), 116.7

(1C), 79.7 (1C), 42.3 (1C), 26.7 (3C) ppm.

(E)-(2-(Phenyldiazenyl)phenyl)methanamine (21c). The re-

action was carried out according to general procedure VI, using 20c

(80 mg, 0.26 mmol) dissolved in TFA (500 μL) and DCM (4500 μL)

to aﬀord the title product (21c) as an orange oil. No further

1

puriﬁcation was performed (55 mg, quant). H NMR (400 MHz,

tert-Butyl (4-Aminobenzyl)carbamate (18d). The reaction was

carried out according to general procedure IV using 4-aminobenzyl-

amine (1 g, 8.19 mmol), TEA (2.27 mL, 16.4 mmol), and di-tert-butyl

decarbonate (1.79, 8.19 mmol). The product was used for the next

step without further puriﬁcation. The title (18d) compound was

obtained as a white solid (1.86 g, quant). Mp 107−110 °C. ¹H NMR

(400 MHz, CDCl₃): δ = 7.07 (d, J = 8.2 Hz, 2H), 6.64 (d, J = 8.3 Hz,

2H), 4.71 (s, 1H), 4.18 (d, J = 5.1 Hz, 2H), 3.62 (s, J = 55.5 Hz, 2H),

1.45 (s, 9H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 156.0 (1C),

145.8 (1C), 138.7 (1C), 129.0 (2C), 115.3 (2C), 79.38 (1C), 44.51

(1C), 28.57 (3C) ppm.

tert-Butyl (4-Nitrosobenzyl)carbamate (19d). The reaction

was carried out according to general procedure I, using 18d (200 mg,

0.89 mmol) and MPS (0.55 g, 1.8 mmol). The crude product was

immediately used without further puriﬁcation for the next reaction

step.

CDCl₃): δ = 7.99 (d, J = 7.3 Hz, 2H), 7.80 (d, J = 7.9 Hz, 1H), 7.60−

7.49 (m, 5H), 7.42−7.39 (m, 1H), 3.72 (s, 2H), 1.52 (s, 2H) ppm.

¹³C NMR (101 MHz, CDCl₃): δ = 152.8 (1C), 150.0 (1C), 142.6

(1C), 131.5 (1C), 131.2 (2C), 129.2 (1C), 129.1 (2C), 127.7 (1C),

123.0 (1C), 115.9 (1C), 43.5 (1C) ppm. LC: t_R(min) = 5.86 (cis) +

7.21 (trans), purity = 84.3%. MS: [M + H]⁺calcd for [C₁₃H₁₃N₃]⁺=

212.12, found 212.20.

(E)-(4-((1,3-Dihydroisobenzofuran-5-yl)diazenyl)phenyl)-

methanamine (21d). The reaction was carried out according to

general procedure VI, using 20d (170 mg, 0.67 mmol) dissolved in

TFA (500 μL) and DCM (4500 μL). The crude material was puriﬁed

by column chromatography (DCM/MeOH/NH₃= 10/1/0.1) to

1

aﬀord the title product (21d) as an orange oil. (119 mg, 70%). H

NMR (400 MHz, DMSO): δ = 7.97−7.90 (m, J = 12.6, 5.0 Hz, 3H),

7.88 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.0 Hz, 1H), 5.16

(s, 4H), 4.02 (s, 2H) ppm. ¹³C NMR (101 MHz, DMSO): δ = 153.8

(1C), 153.0 (1C), 146.1 (1C), 144.6 (1C), 142.7 (1C), 130.7 (2C),

125.5 (1C), 124.5 (2C), 124.0 (1C), 116.1 (1C), 74.4 (1C), 74.3

(1C), 46.0 (1C) ppm. LC: t_R(min) = 5.49 (cis) + 7.18 (trans), purity

= 91.3%. MS: [M + H]⁺calcd for [C₁₅H₁₅N₃O]⁺= 254.13, found

254.05.

tert-Butyl (E)-(3-(Phenyldiazenyl)benzyl)carbamate (20b).

The reaction was carried out according to general procedure V,

using 18b (100 mg, 0.45 mmol) and nitrosobenzene (53 mg, 0,49

mmol). The crude product was puriﬁed by column chromatography

using DCM to aﬀord the title product (20b) as an orange oil. (94 mg,

1

68%). H NMR (400 MHz, CDCl₃): δ = 8.00−7.95 (m, 2H), 7.90−

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-((3-

[(1E)-2-phenyldiazen-1-yl]phenyl)methyl)pyrazole-3-carboxa-

mide (16b). The reaction was carried out according to general

procedure VII, using rimonabant carboxylic acid (58 mg, 0.15 mmol,

1 equiv), TEA (43 μL, 0.31 mmol, 2.1 equiv), ECF (29 μL, 0.3 mmol,

2.05 equiv), and 21b (74 mg, 0.35 mmol, 2.4 equiv). The product was

puriﬁed by prep-TLC (petroleum ether/ethyl acetate = 4/1) to aﬀord

7.87 (m, 2H), 7.68−7.70 (m, 4H), 7.64−7.38 (m, 1H), 5.29−5.13

(m, 1H), 4.63 (d, J = 5.1 Hz, 2H), 1.74 (s, 9H) ppm. ¹³C NMR (101

MHz, CDCl₃): δ = 159.3 (1C), 154.0 (1C), 155.7 (1C), 143.1 (1C),

134.4 (2C), 132.6 (1C), 131.5 (1C), 131.3 (1C), 130.6 (2C), 125.0

(1C), 123.2 (1C), 81.5 (1C), 47.3 (1C), 30.3 (3C) ppm.

tert-Butyl (E)-(2-(Phenyldiazenyl)benzyl)carbamate (20c).

The reaction was carried out according to general procedure V,

using 18c (100 mg, 0.45 mmol) and nitrosobenzene (53 mg, 0,49

mmol). The crude product was puriﬁed by column chromatography

using DCM to aﬀord the title product (20c) as an orange oil. (80 mg,

1

the target compound as an orange oil (82 mg, 95%). H NMR (400

MHz, DMSO): δ = 7.87 (dd, J = 8.0, 1.6 Hz, 3H), 7.84−7.72 (m,

3H), 7.64−7.51 (m, 6H), 7.50−7.41 (m, 2H), 7.28−7.04 (m, 3H),

4.54 (d, J = 6.2 Hz, 2H), 2.25 (s, 3H). ¹³C NMR (101 MHz, DMSO):

δ = 162.2(1C), 151.9(1C), 144.6(1C), 142.6(1C), 141.5(1C),

135.8(1C), 135.1(1C), 133.8(1C), 132.2(1C), 132.0 (1C),

131.5(1C), 131.3(2C), 130.7(1C), 129.6(1C), 129.5(2C),

129.3(1C), 128.8(1C), 128.7(1C), 128.3(1C), 127.2(1C),

122.5(2C), 121.4(1C), 121.2(1C), 119.7(1C), 116.5(1C),

41.7(1C), 9.1 (1C). LC: t_R(min) = 10.99 (cis) + 11.92 (trans),

purity = 98.6%. MS: m/z [M + H]⁺calcd for (C₃₀H₂₂Cl₃N₅O₂)⁺=

574.09, found 574.10.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-((2-

[(1E)-2-phenyldiazen-1-yl]phenyl)methyl)-1H-pyrazole-3-car-

boxamide (16c). The reaction was carried out according to general

procedure VII, using rimonabant carboxylic acid (50 mg, 0.13 mmol,

1 equiv), TEA (36 μL, 0.26 mmol, 2.1 equiv), ECF (25 μL, 0.26

mmol, 2.05 equiv), and 21c (63 mg, 0.3 mmol, 2.4 equiv). The crude

1

58%). H NMR (400 MHz, CDCl₃): δ = 7.87 (d, J = 7.2 Hz, 2H),

7.69 (d, J = 7.9 Hz, 1H), 7.57−7.50 (m, 4H), 7.46−7.41 (m, 1H),

7.40−7.36 (m, 1H), 5.08 (s, 1H), 4.85 (d, J = 5.0 Hz, 2H), 1.47 (s,

9H) ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 156.0 (1C), 152.9

(1C), 150.1 (1C), 138.11 (2C), 131.5 (1C), 131.4 (1C), 129.8 (2C),

129.3 (1C), 128.3 (1C), 123.2 (1C), 116.0 (1C), 79.5 (1C), 41.2

(1C), 28.6 (3C)ppm.

tert-Butyl (E)-(4-((1,3-Dihydroisobenzofuran-5-yl)diazenyl)-

benzyl)carbamate (20d). The reaction was carried out according to

general procedure V, using (1,3-dihydroisobenzofuran-5-yl)-

methanamine (110 mg, 0.81 mmol) and crude 19d (0,89 mmol).

The crude product was puriﬁed by column chromatography using

DCM to aﬀord the title product (20d) as an orange oil. (177 mg,

1

62%). H NMR (400 MHz, CDCl₃): δ = 7.85 (t, J = 6.1 Hz, 3H),

1642

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ACS Chem. Neurosci. 2021, 12, 1632−1647

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

product was puriﬁed by column chromatography (petroleum ether/

ethyl acetate = 4/1) to aﬀord the target compound as an orange oil

(69 mg, 92%). Mp 73 °C. ¹H NMR (400 MHz, DMSO): δ = 7.96 (q,

J = 6.5, 3.3 Hz, 2H), 7.82−7.70 (m, 2H), 7.68−7.50 (m, 6H), 7.50−

7.34 (m, 3H), 7.34−7.11 (m, 3H), 7.05−6.94 (m, 1H), 4.90 (d, J =

101.7, 6.0 Hz, 2H), 2.27 (s, 3H). ¹³C NMR (101 MHz, DMSO): δ =

162.0(1C), 152.2(1C), 149.0(1C), 144.6(1C), 142.6(1C),

138.5(1C), 135.7(1C), 135.0(1C), 133.8(1C), 132.1(1C),

131.9(1C), 131.5(1C), 131.2(2C), 129.6(1C), 129.4(2C),

128.8(2C), 128.7(1C), 128.3(1C), 127.6(1C), 127.2(1C),

122.8(2C), 119.9(1C), 116.4(1C), 115.0(1C), 38.1(1C), 9.1(1C).

LC: t_R(min) = 11.09 (cis) + 12.52 (trans), purity = 98.9%. MS: m/z

[M + H]⁺calcd for (C₃₀H₂₂Cl₃N₅O₂)⁺= 574.09, found 574.10.

5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-((4-[(1E)-2-

(1,3-dihydro-2-benzofuran-5-yl)diazen-1-yl]phenyl)methyl)-4-

methyl-1H-pyrazole-3-carboxamide (16d). The reaction was

carried out according to general procedure VII, using rimonabant

carboxylic acid (20 mg, 0.05 mmol, 1 equiv), TEA (15 μL, 0.10 mmol,

2.1 equiv), ECF (10 μL, 0.10 mmol, 2.05 equiv), and 21d (33 mg,

0.05 mmol, 1 equiv). The crude product was puriﬁed by prep-TLC

(petroleum ether/ethyl acetate = 4/1) to aﬀord the target compound

reproduction performance was assessed by RMSD between docked

poses and the experimental binding pose of AM6538 (PDB code

5TGZ). Geometry optimization of AM6538 was performed using the

MMFF94x^65,66force ﬁeld (gradient convergence criterion: RMS

0.001·kcal·mol⁻¹·Å⁻¹). The atoms on the nitrate group of AM6538

were not considered as they were not visible in the electron density.

The GOLD program^52,53(version 5.4.1) was used to dock the ligand

under default settings. The knowledge-based scoring function ASP⁵⁴

was found to robustly model experimentally observed intermolecular

interactions and to yield the best crystal pose reproduction among all

of GOLD’s scoring functions. The 50 best ASP poses were rescored

using the knowledge-based scoring function DSX⁵⁵(version 0.89),

with CSD and PDB potentials (version 05/11). The cis and trans

isomers of compound 16a were manually prepared in MOE’s Builder

tool and subsequently prepared and evaluated under the same

conditions as AM6538. All ﬁgures representing structures and

superimpositions were prepared using PyMOL (The PyMOL

̈

Molecular Graphics System, version 1.8.0.6, Schrodinger, LLC).

Biological Assays. Cell Luminescence Assay. The assay was

established in a similar way as previously described.⁶⁷HEK-293 cells

stably expressing hCB₁R or hCB₂R were cultured in DMEM

supplemented with FBS 10%, ^L-glutamine 2 mM, and penicillin/

streptomycin 1%. All cells were maintained at 37 °C and 5% CO₂in a

humidiﬁed atmosphere. The CRE luciferase reporter contains the

ﬁreﬂy luciferase gene under the control of multimerized cAMP

response element (CRE) located upstream of a minimal promoter.

The cells were treated with the compounds (previously isomerized to

their cis or trans states with light of 366 or 454 nm, respectively) for 5

h.

1

as an orange oil (27 mg, 88%). Mp 102 °C. H NMR (400 MHz,

CDCl₃): δ = 7.77 (dd, J = 12.9, 4.9 Hz, 3H), 7.67 (s, J = 11.8 Hz,

1H), 7.42 (d, J = 8.3 Hz, 2H), 7.36−7.25 (m, 3H), 7.23−7.13 (m,

4H), 7.03−6.93 (m, 2H), 5.13−4.85 (m, 4H), 4.61 (d, J = 6.1 Hz,

2H), 2.31 (d, J = 10.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃): δ =

162.8 (1C), 152.8 (1C), 152.0 (1C), 144.9 (1C), 143.3 (1C), 142.3

(1C), 141.79 (1C), 140.5 (1C), 136.15 (1C), 136.0 (1C), 135.1

(1C), 133.1 (1C), 130.9 (2C), 130.6 (1C), 130.5 (1C), 129.1 (2C),

128.7 (2C), 128.0 (1C), 127.3 (1C), 124.0 (1C), 123.3 (2C), 121.6

(1C), 118.1 (1C), 114.3 (1C), 73.5 (2C), 42.8 (1C), 9.6 (1C). LC: t_R

(min) = 10.80 (cis) + 11.56 (trans), purity = 99.1%. MS: m/z [M +

H]⁺calcd for (C₃₂H₂₄Cl₃N₅O₂)⁺= 616.10, found 616.15.

For hCB₁R agonistic activity, HEK-293-CB₁-CRELuc cells were

stimulated with CP55940 1 μM as a positive control. For hCB₁R

antagonistic activity, HEK-293-CB₁-CRELuc cells were pretreated

with the test compounds for 30 min and then stimulated with

CP55940 1 μM for 5 h. As a positive control, the cells were stimulated

with rimonabant 1 μM. For hCB₂R agonistic assay, HEK-293-CB₂-

CRELuc cells were pretreated with the test compounds for 30 min

and then stimulated with forskolin 10 μM for 5 h. As a positive

control the cells were stimulated with CP55940 1 μM. For hCB₂R

antagonistic assay, HEK-293-CB₂-CRELuc cells were pretreated with

test compounds for 30 min and then stimulated with forskolin 10 μM

+ CP55940 1 μM for 5 h. As positive controls, the cells were

stimulated with SR144528 10 μM and AM630 10 μM.

After stimulation and incubation, the cells were dissolved in a

solution containing tris-phosphate 25 μmol/L (pH = 7.8); MgCl₂, 8

μmol/L; dithiothreitol, 1 μmol/L; Triton X-100, 1%; and glycerol,

7%. The luciferase activity was measured on lysed cells with the

luciferase assay kit (Promega) according to the manufacturer’s

protocol in a Tristar²LB942 multimode reader machine (Berthold

Technologies). To avoid isomerization reversion, all the experiments

were carried out in the dark. Statistical analyses were performed using

one-way ANOVA followed by Dunnett’s multiple comparison post-

tests using GraphPad Prism 6 for Windows (version 6.01, September

21, 2012).

Rimonabant Hydrochloride. Five drops of DMF were added to

a stirred solution of rimonabant carboxylic acid (1 g, 2.62 mmol, 1

equiv) in DCM (10 mL). The reaction mixture was cooled to 0 °C,

and then (COCl)₂(0.37 mL, 4.45 mmol, 1.7 equiv) was added

dropwise. The reaction mixture was stirred for 1 h at room

temperature. Excess of (COCl)₂was removed under reduced

pressure. In another ﬂask were introduced N-aminopiperidine (0.43

mL, 3.93 mmol, 1.5 equiv) and TEA (0.36 mL, 2.62 mmol, 1 equiv)

in DCM, and the mixture was cooled to 0 °C. Subsequently, to the

mixture was added dropwise a solution of the intermediate acyl

chloride in DCM at the same temperature. The reaction mixture was

stirred at 0 °C for 15 min and for 15 min more at room temperature.

The mixture was diluted with DCM, washed with NaHCO₃and brine,

dried over Na₂SO₄, and concentrated under reduced pressure,

yielding rimonabant without further puriﬁcation as a pale-yellow

solid (1.2 g, quant). The product was treated with a solution of HCl

in 2-propanol. The precipitate was collected and washed with

anhydrous diethyl ether to obtain the respective rimonabant

1

hydrochloride salt as a beige powder. Mp 225−227 °C (dec). H

NMR (400 MHz, CDCl₃): δ = 7.62 (s, 1H), 7.42 (d, J = 1.9 Hz, 1H),

7.32−7.27 (m, 4H), 7.05 (d, J = 8.3 Hz, 2H), 2.94−2.80 (m, 4H),

2.36 (s, 3H), 1.75 (dt, J = 11.1, 5.6 Hz, 4H), 1.41 (t, J = 14.5 Hz, 2H)

ppm. ¹³C NMR (101 MHz, CDCl₃): δ = 160.4 (1C), 144.9 (1C),

143.4 (1C), 136.4 (1C), 135.4 (1C), 133.5 (1C), 132.9 (1C), 131.3

(2C), 131.0 (1C), 130.8 (1C), 129.4 (2C), 128.4 (1C), 127.7 (1C),

Radioligand Binding Assay. For the experiments, hCB₂-HEK

cells were a kind gift from AbbVie Laboratories (Chicago, U.S.). Cells

were grown in Dulbecco’s modiﬁed Eagle’s medium containing high

glucose supplemented with 8% fetal calve serum and 25 μg/mL

zeocin in a 37 °C incubator in the presence of 5% CO₂. The

respective hCB₂R membranes were prepared as described in the

literature.³³The rCB₁R membrane homogenate was prepared from

brains of adult female rats.^15,68The tissues were obtained from an

alternate project of the Institute of Pharmacology and Toxicology at

118.7 (1C), 57.6 (2C), 25.9 (2C), 23.8 (1C), 9.8 (1C) ppm. LC: t_R=

+

10.68 min, purity = 99.9%. MS: [M]⁺calcd for [C₂₂H₂₁Cl₃N₄O]

463.09, found 463.05.

=

Molecular Docking. The 2D structures of the compounds were

drawn with ChemDraw (version 18.0, PerkinElmer) and Marvin-

Sketch for Windows (version 19.10.0, ChemAxon Ltd.). Subse-

quently, the input ﬁles for the molecular docking studies were

generated. The crystal structure of the CB₁R (PDB code 5TGZ) was

prepared for docking using MOE (version 2016.08).⁵⁷Removal of

ligands and water molecules was followed by automatic assignment of

tautomer and protonation states, using MOE’s protein structure

preparation pipeline⁵⁸⁻⁶³and Protonate3D⁶⁴at pH 7.5. Pose

the University of Wurzburg and frozen at −80 °C. The respective

rCB₁R membranes were freshly prepared according to the protocol

described by Catani and Gasperi for preparation of membrane

homogenates.⁶⁹

̈

Saturation and competition assays were done according to the

protocol previously established in M. Decker’s research group.³³For

the determination of K_Dvalue of the membrane samples, saturation

assays were done with 8 concentrations of [³H]CP55940 (Hartmann

1643

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ACS Chem. Neurosci. 2021, 12, 1632−1647

ACS Chemical Neuroscience

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Research Article

Analytic GmbH) ranging from 0.088 nM to 4.4 nM. Reactions were

started by adding membrane (25 μg/well for rCB₁or 8 μg/well for

hCB₂) of a 96 well Multiscreen ﬁlter plate (Millipore) containing the

radioligand in assay buﬀer (50 mM Tris-HCl, pH = 7.4; 5 mM

MgCl₂·6H₂O; 2.5 mM EDTA; 2 mg/mL BSA). After 3 h incubation

at room temperature, the reaction was stopped by vacuum ﬁltration

and each well was washed 4 times with cold binding buﬀer (50 mM

Tris-HCl, pH = 7.4; 5 mM MgCl₂·6H₂O; 2.5 mM EDTA). The ﬁlter

plate was dried at 45 °C. Lately, 20 μL of IRGA Safe plus-scintillation

cocktail (PerkinElmer) was added to each well. The activity was

counted in a Micro Beta Trilux counter (Wallac). Competition assays

were done with 5−10 concentrations (0.1 nM to 0.4 mM) of target

compound (previously isomerized to their cis or trans states with light

of 366 or 454 nm, respectively) and 0.6 nM [³H]CP55940. The

positive controls for the assays over CB₁and CB₂were respectively

the selective ligands rimonabant and MN-I-79 (both synthesized in

house).

Authors

Diego A. Rodríguez-Soacha − Pharmazeutische und

Medizinische Chemie, Institut fur Pharmazie und

Lebensmittelchemie, Julius-Maximilians-Universität

Wurzburg, D-97074 Wurzburg, Germany

Julia Fender − Institut fur Pharmakologie und Toxikologie,

Julius-Maximilians-Universität Wurzburg, D-97078

Wurzburg, Germany

Yesid A. Ramírez − Pharmazeutische und Medizinische

Chemie, Institut fur Pharmazie und Lebensmittelchemie,

Julius-Maximilians-Universität Wurzburg, D-97074

Wurzburg, Germany; Departmento de Ciencias

̈

Farmacéuticas, Facultad de Ciencias Naturales, Universidad

Icesi, 760031 Cali, Valle del Cauca, Colombia

Juan Antonio Collado − Instituto Maimónides de

Investigación Biomédica de Córdoba, Departamento de

Biología Celular, Fisiología e Inmunología, Universidad de

Córdoba, Hospital Universitario Reina Sofía, 14004

Córdoba, Spain

The stock solutions of all tested compounds (4 mM) were

prepared by dissolving in DMSO. The dilution series of all stock

solutions were prepared diluting with binding buﬀer.

Statistical analyses and sigmoidal dose−response curve ﬁttings were

performed with GraphPad Prism 6 for Windows (version 6.01,

September 21, 2012).

Eduardo Munoz − Instituto Maimónides de Investigación

̃

Biomédica de Córdoba, Departamento de Biología Celular,

Fisiología e Inmunología, Universidad de Córdoba, Hospital

Universitario Reina Sofía, 14004 Córdoba, Spain

Rangan Maitra − Discovery Science and Technology, RTI

International, Research Triangle Park, North Carolina

27709-2194, United States; orcid.org/0000-0001-6663-

6800

K_ivalues were determined according to the Cheng−Prusoﬀ

equation:

EC₅₀

K_i=

[L*]

1 +

K_D

with [L*] as radioligand concentration. The K_ivalue was calculated

from at least two individual experiments. K_Dvalues were measured in

at least two individual experiments for each new batch of prepared

membrane.

hCB₁Calcium Mobilization Assay. By use of a functional

ﬂuorescent hCB₁activated Gα_q16-coupled intracellular calcium

mobilization assay in CHO-K1 cells, the antagonist eﬀect of the

target compound was characterized pharmacologically as described

previously in the literature and apparent aﬃnity (K_e) values were

determined.^50,51Calcium ﬂux was monitored in a 96-well format using

the ﬂuorescent calcium-5 dye in an automated plate reader (FLIPR

Tetra, Molecular Devices). The antagonist activity of 16a was

measured by its ability to shift the concentration−response curve of

the reference CB₁agonist CP55940 rightward using the equation

Christoph Sotriﬀer − Pharmazeutische und Medizinische

̈

Chemie, Institut fur Pharmazie und Lebensmittelchemie,

Julius-Maximilians-Universität Wu

Wurzburg, Germany

Kristina Lorenz − Institut fu

Julius-Maximilians-Universität Wu

Wurzburg, Germany; Leibniz-Institut fu

̈

rzburg, D-97074

̈

r Pharmakologie und Toxikologie,

rzburg, D-97078

r Analytische

̈

WissenschaftenISAS e.V., 44139 Dortmund, Germany

Complete contact information is available at:

https://pubs.acs.org/10.1021/acschemneuro.1c00086

Author Contributions

K_e= [ligand]/[DR − 1]

M.D. was responsible for the supervision and development of

the whole project. D.A.R.-S. performed the chemical synthesis,

testing of the physicochemical properties of the target

compounds, cell luminesce assay, and radioligand binding

assays. The cell luminesce assay was supervised by J.A.C. and

E.M. The computational docking studies were performed by

Y.A.R. with supervision of C.S. The radioligand binding assays

were made in collaboration with J.F. and K.L. The calcium

mobilization assay was performed by R.M. All authors have

given approval to the ﬁnal version of the manuscript.

where DR is the EC₅₀ratio of CP55940 in the presence or absence of

a test agent. Statistical analysis was performed using GraphPad Prism

6 for Windows (version 6.01, September 21, 2012).

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acschemneuro.1c00086.

■

sı

HPLC chromatogram of target compounds, molecular

docking and scoring (16a), potential selectivity mech-

anism of 16a for CB₁over CB₂, and hCBRs cell

luminescence assays for agonism (16a) (PDF)

Funding

Financial support from the German Research Foundation

(Deutsche Forschungsgemeinschaft under Grant DFG

DE1546/10-1) and the European Cooperation in Science

and Technology under the COST Action CA15135 (MuTa-

LiG), as well as a Ph.D. scholarship for D.A.R.-S. by the

German Academic Exchange Service (Deutscher Akademisch-

er Austauschdienst, DAAD) is gratefully acknowledged. A

Ph.D. position was awarded to J.F. by the Elite Network of

Bavaria within the International Doctoral Program “Receptor

Dynamics”. R.M. is supported by Grants AA023256 (NIAAA),

DK124615 (NIDDK), and DA052495 (NIDA) from NIH.

AUTHOR INFORMATION

Corresponding Author

■

Michael Decker − Pharmazeutische und Medizinische

Chemie, Institut fur Pharmazie und Lebensmittelchemie,

̈

Julius-Maximilians-Universität Wu

Wurzburg, Germany; orcid.org/0000-0002-6773-6245;

̈

rzburg, D-97074

̈

Phone: +49 931 3189676; Email: michael.decker@uni-

wuerzburg.de

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pubs.acs.org/chemneuro

Research Article

to appetite and metabolism: a randomized, double-blind, placebo-

controlled, human laboratory study. Transl. Psychiatry 10 (1), 71.

(15) Rinaldi-Carmona, M., Barth, F., Heaulme, M., Shire, D.,

Calandra, B., Congy, C., Martinez, S., Maruani, J., Neliat, G., Caput,

D., Ferrara, P., Soubrie, P., Breliere, J. C., and Le Fur, G. (1994)

SR141716A, a potent and selective antagonist of the brain

cannabinoid receptor. FEBS Lett. 350 (2−3), 240−244.

(16) Moreira, F. A., and Crippa, J. A. (2009) The psychiatric side-

effects of rimonabant. Rev. Bras. Psiquiatr. 31 (2), 145−53.

(17) Gajbhiye, J. M., More, N. A., Patil, M. D., Ummanni, R.,

Kotapalli, S. S., Yogeeswari, P., Sriram, D., and Masand, V. H. (2015)

Discovery of Rimonabant and its potential analogues as anti-TB drug

candidates. Med. Chem. Res. 24 (7), 2960−2971.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

We thank Jessica Pfeiﬀer for her support in the isolation of the

rat brains used to perform radioligand binding assays.

■

ABBREVIATIONS

■

hCB₁R, human cannabinoid receptor 1; hCB₂R, human

cannabinoid receptor 2; rCB₁R, rat cannabinoid receptor 1;

DCM, dichloromethane; DMF, dimethylformamide; ECF,

Ethyl chloroformate; MPS, potassium peroxymonosulfate.;

NBS, N-bromosuccinimide; TEA, triethylamine; TFA, tri-

ﬂuoroacetic acid; THF, tetrahydrofuran HBTU (2-(1H-

benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuorophos-

phate; TLC, thin layer chromatography

(18) Imamura, T., Tsuruma, K., Inoue, Y., Otsuka, T., Ohno, Y.,

Ogami, S., Yamane, S., Shimazawa, M., and Hara, H. (2017)

Rimonabant, a selective cannabinoid 1 receptor antagonist, protects

against light-induced retinal degeneration in vitro and in vivo. Eur. J.

Pharmacol. 803, 78−83.

(19) Horder, J., Harmer, C. J., Cowen, P. J., and McCabe, C. (2010)

Reduced neural response to reward following 7 days treatment with

the cannabinoid CB1 antagonist rimonabant in healthy volunteers.

Int. J. Neuropsychopharmacol. 13 (8), 1103−1113.

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"Photo-Rimonabant": Synthesis and Biological Evaluation of Novel Photoswitchable Molecules Derived from Rimonabant Lead to a Highly Selective and Nanomolar " Cis-On" CB1R Antagonist

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