From a Designer Drug to the Discovery of Selective Cannabinoid Type 2 Receptor Agonists with Favorable Pharmacokinetic Profiles for the Treatment of Systemic Sclerosis

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From a Designer Drug to the Discovery of Selective Cannabinoid

Type 2 Receptor Agonists with Favorable Pharmacokinetic Proﬁles

for the Treatment of Systemic Sclerosis

Bei-Er Jiang,^⊥Xingwu Jiang,^⊥Qiansen Zhang,^⊥Qiuwen Liang, Zi-Liang Qiu, Xiang-Bai Sun,

Jun-Jie Yang, Si Chen, Chunyang Yi, Xiaolei Chai, Mingyao Liu, Li-Fang Yu,* Weiqiang Lu,*

and Han-Kun Zhang *

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ABSTRACT: Synthetic cannabinoids, as exempliﬁed by SDB-001

(1), bind to both CB1 and CB2 receptors and exert

cannabimimetic eﬀects similar to (−)-trans-Δ⁹-tetrahydrocannabi-

nol, the main psychoactive component present in the cannabis

plant. As CB1 receptor ligands were found to have severe adverse

psychiatric eﬀects, increased attention was turned to exploiting the

potential therapeutic value of the CB2 receptor. In our eﬀorts to

discover novel and selective CB2 receptor agonists, 1 was selected

as a starting point for hit molecule identiﬁcation and a class of 1H-

pyrazole-3-carboxamide derivatives were thus designed, synthesized, and biologically evaluated. Systematic structure−activity

relationship investigations resulted in the identiﬁcation of the most promising compound 66 as a selective CB2 receptor agonist with

favorable pharmacokinetic proﬁles. Especially, 66 treatment signiﬁcantly attenuated dermal inﬂammation and ﬁbrosis in a

bleomycin-induced mouse model of systemic sclerosis, supporting that CB2 receptor agonists might serve as potential therapeutics

for treating systemic sclerosis.

a selective CB1 receptor blocker originally approved in Europe

as an antiobesity agent in 2006, was removed from the market

due to its severe adverse psychiatric eﬀects such as anxiety,

depression, and suicidal thoughts.⁹⁻¹¹In contrast, the CB2

receptor is abundant in the peripheral immune system and has

much lower expression in the CNS compared to the CB1

receptor.¹⁶It was found to be heavily implicated in a host of

pathophysiological conditions including chronic and neuro-

pathic pain,^12,13neurodegenerative disorders,^14,15cancers,¹⁶

osteoporosis,¹⁷and inﬂammation-associated pathologies.^18,19

Thus, the development of novel ligands targeting CB2 receptor

with high potency and selectivity may hold promise to fulﬁll

multiple unmet clinical needs while avoiding the psychotropic

side eﬀects caused by interaction with the CB1 receptor. To

date, there are several cannabinoid drugs such as Nabiximols

(approved in the United Kingdom), Nabilone (approved in the

United States), Cannabidiol (approved in the United States),

and Dronabinol (approved in the United States) that have been

INTRODUCTION

■

Cannabis sativa has a widespread use for recreational, medical,

and religious purposes in many cultures for thousands of years

and is the unique source of more than 60 phytocannabinoids.¹

Among these biologically active substances, (−)-trans-Δ⁹-

tetrahydrocannabinol (Δ⁹-THC, 2) is the principal psychoactive

component in C. sativa and the most studied one (Figure 1). It

was originally isolated and elucidated by Mechoulam and his co-

workers in 1964, which resulted in the further identiﬁcation of

the endocannabinoid system (ECS).²⁻⁴The ECS mainly

consists of two types of cannabinoid receptors (CB1 and

CB2) and a series of endogenous ligands such as anandamide

and 2-arachidonoylglycerol, as well as some metabolic enzymes

responsible for the biosynthesis and degradation of endocanna-

binoids.⁵

CB1 and CB2 receptors are both class A rhodopsin-like G-

protein-coupled receptors (GPCRs), sharing 44% overall

homology and more than 60% homology in the transmembrane

domain that encompasses the ligand-binding pocket.⁶The CB1

receptor is widely expressed in the central nervous system

(CNS), having a high level particularly in the brain and spinal

cord.^7,8It is a potential target for treating obesity, metabolic

syndrome, and several neurodegenerative diseases. However, it

is also believed that the stimulation of the CB1 receptor is

responsible for several undesirable side eﬀects. Rimonabant (3),

Received: June 15, 2020

Published: December 31, 2020

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Figure 1. Representative cannabinoid receptor ligands disclosed in the literature.

approved for clinical use, all of which are derived from naturally

occurring active constituents (2 and 4) of C. sativa. On the other

hand, as shown in Figure 1, a number of synthetic cannabinoids

with a preference for the CB2 receptor are under clinical

evaluation mostly in the area of pain management or

inﬂammation-related diseases, such as GW842166X²⁰(5),

LY2828360²¹(6), and S-777469²²(7). Among these investiga-

tional drugs, ajulemic acid (JBT-101, 8), an orally active

synthetic CB2 agonist, is the most promising one developed by

Corbus Pharmaceuticals and has entered phase 3 clinical studies

for treating dermatomyositis and systemic sclerosis (SSc).²³

SSc is a complex autoimmune disease that is characterized by

vasculopathy, inﬂammation, and autoimmunity, which leads to

progressive ﬁbrosis in the skin and multiple internal organs.^24,25

It has a worldwide distribution and severely impacts patient

quality of life with high morbidity and mortality. SSc is

heterogeneous in its clinical presentation, and common

treatments such as immunomodulatory drugs and glucocorti-

coids have limited therapeutic eﬀects and poor prognosis.²⁶At

present, safe and eﬀective therapeutics that can alleviate or

reverse the progression of SSc are urgently needed.²⁷Recently,

mounting studies have demonstrated that the ECS plays key

roles in modulating ﬁbrosis, inﬂammation, and vasodilatation, all

of which are dysregulated in SSc.²⁸It was found that CB2

receptor knockout mice (CB2^‑/‑mice) showed increased

susceptibility to bleomycin (BLM)-induced dermal ﬁbrosis

compared to wild-type mice. In addition, activation of CB2

receptor can exert antiﬁbrotic eﬀects in experimental models of

SSc by reducing leukocyte inﬁltration into skin lesions and

preventing tissue damage in pathologic ﬁbrosis.²⁹In terms of

CB2 receptor ligands, WIN55212−2 (9), a nonselective

cannabinoid receptor agonist, dramatically reduced the dermal

ﬁbrosis and the synthesis of extracellular matrix components in a

mouse model of BLM-induced SSc.³⁰Compound 8 received

orphan drug designation from the FDA as a Fast Track

Development Program for the treatment of chronic inﬂamma-

tion and SSc, and is currently in a phase 3 clinical trial.^23,31

Taken together, the evidence suggests that the CB2 receptor

might be a potential molecular target for treating SSc.

SDB-001 (1) is an indole-based synthetic cannabinoid and

was ﬁrst identiﬁed as a designer drug in the illegal market by

Japanese researchers from the National Institute of Health

Sciences in 2012. It exhibits full agonist activity at both CB1

(EC₅₀= 34 nM, E_max= 98%) and CB2 (EC₅₀= 29 nM, E_max

=

91%) receptors, and has potent cannabimimetic eﬀects in rats.³²

Although its in vivo pharmacological activity was similar to that

of partial agonist 2, compound 1 may cause more severe

psychiatric complications due to its full agonism and the longer

duration of its pharmacologic eﬀects on the CB1 receptor.^33,34

In addition, as the primary psychoactive components in K2/

Spice products, derivatives of compound 1 are diﬃcult to detect

in standardized drug testing due to their structural diversity.³⁵

Since then, considerable research eﬀorts have been devoted to

unraveling the structure−activity relationships (SARs) of

compound 1 and its structural analogues for better under-

standing the pharmacological mechanism underlying the

distinct toxic eﬀects of synthetic cannabinoids compared to

compound 1.³⁶⁻⁴¹In most of these studies, the central indole

ring of compound 1 was retained, and the SAR studies mainly

focused on the substituents at the 1-indole position and in the

carboxamide group at the 3-indole position. Recently, a number

of studies have indicated that compound 1 and its derivatives

were rapidly metabolized when incubated with human

hepatocytes or human liver microsomes in vitro.^42,43Therefore,

it is not surprising that all of the in vivo eﬃcacy studies disclosed

in the above-mentioned articles were carried out through

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Figure 2. Overview of the design of 1H-pyrazole-3-carboxamide derivatives reported in this work.

a

Scheme 1. Synthesis of Compounds 17−40, 45−52, and 54−68

a

Reagents and conditions: (a) potassium tert-butoxide (t-BuOK), diethyl oxalate, anhydrous tetrahydrofuran (THF), room temperature (rt), 5 h;

(b) NH₂NH₂·H₂O, acetic acid (AcOH), anhydrous THF, reﬂux, 3 h, 52−88%; (c) R⁴Br, anhydrous THF, reﬂux, overnight; (d) LiOH·H₂O,

CH₃OH:H₂O:THF = 2:1:2, rt, overnight, 87−98%; (e) R¹NH₂, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuorophosphate

(HATU), N,N-diisopropylethylamine (DIPEA), CH₂Cl₂, rt, 4 h, 65−80%; (f) R⁴NHNH₂·HCl, AcOH, anhydrous THF, reﬂux, 3 h, 38−76%.

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a

Scheme 2. Synthesis of Compounds 41−44

a

Reagents and conditions: (a) N-bromosuccinimide (NBS), dibenzoyl peroxide, chloroform (CHCl₃), reﬂux, overnight, 52%; (b) N-

chlorosuccinimide (NCS), dibenzoyl peroxide, CHCl₃, reﬂux, overnight, 58%; (c) Cu(NO₃)₂, triﬂuoroacetic anhydride (TFAA), CHCl₃, rt,

overnight, 62%; (d) Zn, 3N HCl, CH₃OH, ice bath, 1 h, 43%.

intraperitoneal injection, suggesting that the pharmacokinetic

(PK) proﬁles of these amidoalkylindoles were not optimal and

still had some room for improvement.

regioisomers 15a (29%) and 16a (33%), which were separated

by ﬂash chromatography and characterized by heteronuclear

multiple-bond coherence (HMBC) experiments (see the

Supporting Information). In addition, it should be noted that

the 1,3-regioisomer 16a showed a lower R_fvalue compared to its

1,5-counterpart (15a) when monitored via thin-layer chroma-

tography (TLC) analysis. This pattern was also applicable to all

pyrazoles described in the present work. Upon ester hydrolysis,

compounds 15a−o and 16a−o were coupled with various

amines using 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetrame-

thyluronium hexaﬂuoro (HATU) to furnish the desired

products 17−40 and 45−52. Alternatively, 13a was cyclized

with diﬀerent arylhydrazines to produce pyrazole-3-carbox-

amide intermediates 53a−o as major products,^48,49which were

further transformed to ﬁnal compounds 54−68 by employing

the same strategy as described for compound 26. In an eﬀort to

further investigate the SAR of the 4-position of pyrazole ring in

compound 26, various substituents were introduced in this

position, as outlined in Scheme 2. In the presence of the radical

initiator dibenzoyl peroxide, compound 26 was treated with

NBS or NCS in CHCl₃to aﬀord compounds 41 and 42,

respectively. Compound 43 was obtained via nitration of

compound 26, and further reduction of the nitro group to the

amino group in compound 43 provided the desired product 44.

In Vitro Functional Studies. In vitro functional proﬁles of

compounds 17−52 and 54−68 against CB1 and CB2 receptors

were evaluated in calcium mobilization assay through co-

expressing Gα16 and human CB1 and CB2 receptors in CHO

cells.⁵⁰All of the ﬁnal compounds were initially screened for

their agonism activities at a concentration of 10 μM.

Compounds exhibiting over 50% activation were next evaluated

for their EC₅₀values. CP55940 (11) and AM630 (12) were

tested concurrently as nonselective cannabinoid receptor

agonist and selective CB2 receptor antagonist, respectively.

Maximal eﬃcacy (E_max) was determined as maximal eﬀect

relative to compound 11. We ﬁrst explored the SARs between

the 1,3- and 1,5-regioisomers starting with R¹substituents.

Pyrazole-5-carboxamide analogues 17−20 were much less active

than their counterparts 23−26 at both CB1 and CB2 receptors,

suggesting that the 1,3-regioisomer pyrazole was preferred to

maintain agonist potency at cannabinoid receptors (Table 1).

Compound 23, acting as a partial agonist, exhibited moderate

activity at both CB1 and CB2 receptors (EC_50,CB1= 270.6 nM,

EC_50,CB2= 194.2 nM). Replacing the adamantane in compound

In our eﬀorts to discover novel and selective CB2 receptor

agonists, a series of 1H-pyrazole-3-carboxamide derivatives were

designed to overcome the metabolic liability of N-alkyl indole-

based synthetic cannabinoids while maintaining the major

pharmacophoric elements of compound 1 (Figure 2). Pyrazole-

3-carboxamide fragment is well known as a privileged skeleton in

medicinal chemistry and is found in many naturally occurring or

synthetic substances possessing a variety of biological functions.

The methods of synthesizing 1H-pyrazole-3-carboxamide

derivatives are ﬂexible and reliable, which allow the rapid

preparation of diverse analogues based on this moiety and

facilitate the structural optimization process. Besides, it is

noteworthy that the 1H-pyrazole-3-carboxamide scaﬀold has

been applied in the development of CB1 receptor antagonists/

inverse agonists and in PET tracers for clinical use such as 2 and

[

¹²³I]-AM281⁴⁴⁻⁴⁷(10), indicating that the discovery of

selective CB2 receptor agonists based on 1H-pyrazole-3-

carboxamide scaﬀold may be saturated with challenges and

opportunities. Therefore, structural modiﬁcation needs to be

cautiously implemented to achieve this goal. In this work, the

target compounds were initially obtained as pyrazole-3-

carboxamide (1,3-regioisomer) and pyrazole-5-carboxamide

(1,5-regioisomer) analogues, as exempliﬁed by compounds 17

and 23, to investigate which regioisomer was preferred to

maintain the potency against CB2 receptor. Further structure

optimization along with a series of pharmacological evaluations

led to the identiﬁcation of compound 66, which is a potent and

selective CB2 receptor agonist with high eﬃcacy. In addition,

compound 66 signiﬁcantly attenuated dermal ﬁbrosis in a BLM-

induced mouse model of SSc, further supporting a tight interplay

between the cannabinoid system and SSc.

RESULTS AND DISCUSSION

■

Chemistry. The syntheses of pyrazole analogues 17−52 and

54−68 were accomplished as depicted in Schemes 1 and 2. The

ketones 13a−g were treated with diethyl oxalate in the presence

of t-BuOK by the Claisen condensation to aﬀord the

corresponding γ-diketo esters. These intermediates were further

cyclized with hydrazine monohydrate to produce 14a−g. The

N-alkylation of 14a with 1-bromopentane led to a mixture of

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a

Table 1. Agonist Activity of Compounds 17−34 against the Human CB1 and CB2 Receptors in Calcium Mobilization Assays

a

See the Experimental Section. Data represent the mean standard error of the mean (SEM) values of at least three independent experiments.

b

The EC₅₀values were determined by calcium mobilization assay and calculated with GraphPad Prism 7 software. Compound 11 served as a

c

positive control with EC₅₀values of 42.0 and 35.0 nM against the CB1 and CB2 receptors, respectively. ND: not determined; for compounds with

EC₅₀> 10⁵nM, their maximal eﬀects were not determined.

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ad

Table 2. Agonist Activity of Compounds 35−44 against the Human CB1 and CB2 Receptors in Calcium Mobilization Assays

a

b

See the Experimental Section. Data represent the mean SEM values of at least three independent experiments. The EC₅₀values were

determined by calcium mobilization assay and calculated with GraphPad Prism 7 software. Compound 11 served as a positive control with EC₅₀

c

values of 42.0 and 35.0 nM against the CB1 and CB2 receptors, respectively. ND: not determined; for compounds with EC₅₀> 10⁵nM, maximal

d

eﬀects were not further determined. All of the ﬁnal compounds were tested for their antagonist activity at the CB2 receptor, and only compound

40 behaved as a selective antagonist at the CB2 receptor (IC₅₀= 87.1 nM). Cells were preincubated with tested compounds at diﬀerent

concentrations for 15 min, followed by the addition of compound 11 (100 nM).

23 with an aromatic group such as 4-chlorophenyl (27) or 4-

chlorobenzyl (28) resulted in a further loss of activity, with the

exception of pyridine analogue 30 which exhibited a slightly

better functional activity than compound 23 at both receptors.

However, compound 29 with two methyl groups in the α-

position of the 4-Cl-phenyl was about 2- to 3-fold more potent

compared to compound 23 at both CB1 and CB2 receptors.

Unexpectedly, introduction of a hydroxyl group into the

adamantane of compound 23 resulted in a selective CB₁

receptor agonist 24 (EC₅₀= 4.0 nM). In consideration of

monohydroxylation of the adamantyl moiety being one of the

primary metabolic pathways for 1 and its analogues,⁴³it is likely

that these monohydroxylated metabolites also behave as potent

CB1 receptor agonists. On the other hand, the alkyl-substituted

compounds 25, 26, and 31 displayed a signiﬁcant increase of

potency in comparison to compound 23 at both receptors. In

particular, the terminal alcohol 26 acted as a high eﬃcacy agonist

with low nanomolar activities at both receptors and showed a

ﬁvefold preference for CB2 receptor (EC_50,CB1= 17.4 nM,

EC_50,CB2= 2.9 nM). In addition, although introducing D/L

amino acid methyl esters such as leucine or valine (32−34) was

beneﬁcial for functional potency at both receptors, compound

33 was readily degraded in human liver microsomal stability

assay (Table 4).

As compound 26 was the most promising hit obtained so far,

our subsequent SAR studies focused on the R²and R³positions

in the pyrazole ring (Table 2). Replacing the tert-butyl in

compound 26 with a triﬂuoromethyl (35), isopropyl (36), or

neopentyl (37) group resulted in varying decreases of potency at

CB2, while the eﬃcacy and selectivity of these analogues

declined as well. In addition, the cycloalkyl substitutions (38 and

39) yield a total loss of agonistic activity against both receptors.

Interestingly, a 2,4-diﬂuorophenyl substitution resulted in a

selective CB2 receptor antagonist 40 (IC₅₀= 87.1 nM). Next,

pyrazole ring 4-position substituents were investigated. As

shown in Table 2, introduction of various substituents

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Table 3. Agonist Activity of Compounds 45−52 and 54−68 against the Human CB1 and CB2 Receptors in Calcium Mobilization

a

Assays

a

See the Experimental Section. Data represent the mean

SEM values of at least three independent experiments. The EC₅₀values were

determined by calcium mobilization assay and calculated with GraphPad Prism 7 software. Compound 11 served as a positive control with EC₅₀

b

values of 42.0 and 35.0 nM against the CB1 and CB2 receptors, respectively. ND: not determined; for compounds with EC₅₀> 10⁵nM, their

maximal eﬀects were not determined.

(compounds 41−44) in this position was well tolerated and the

agonist activity of these derivatives at the CB2 receptor

remained at a low nanomolar level. With the exception of

nitro analogues 43, these compounds behaved as high-eﬃcacy

agonists toward the CB2 receptor and exhibited improved

selectivity over the CB1 receptor compared to parent compound

26.

Finally, in an attempt to further improve CB2 receptor

selectivity and PK properties, the R⁴substituent of compound

26 was interrogated. As shown in Table 3, shortening or

extending the pentyl side chain in the N-1 position of compound

26 with one carbon atom (46 and 47) was tolerated for CB2

agonist activity, while the two carbon shorter homologue 45

showed a distinct reduction in CB2 receptor agonist activity and

was completely inactive at the CB1 receptor. In comparison to

their parent compounds, terminal ﬂuorinated analogues (48, 49,

and 50) were generally less active and selective at the CB2

receptor. Replacement of the pentyl substituent in compound

26 with a cyclopropylmethyl (51) or 2-methoxyethyl (52)

group led to a signiﬁcant drop of agonistic activity at both

receptors.

phenyl ring in the N-1 position, was found to be totally inactive

toward both CB1 and CB2 receptors. However, further

structural optimization demonstrated that various substituents

attached to the benzene ring of compound 54 had a considerable

eﬀect on its agonist potency against the CB2 receptor. For

instance, the 2,4-diﬂuroro substitution yielded a selective CB2

receptor agonist 61 with an EC₅₀value of 1725 nM, and mono-

substitutions at the para-position resulted in compounds 55−57

that were 2- to 8-fold more active than compound 61, while

compounds 58−60 completely lost their cannabinoid receptor

activities. In the ﬁeld of medicinal chemistry, the substitution of

a CH group with a nitrogen atom in an aromatic system is a

common strategy to improve the biochemical potency and

physicochemical proﬁles of targeted molecules.⁵¹In this study,

incorporation of a nitrogen atom at the 2-position of the phenyl

ring (62) was beneﬁcial for CB2 receptor activity compared to

parent compound 55, while its 3-pyridine analogue 63 and

pyrimidine analogue 68 were inactive against both cannabinoid

receptors. Introduction of substituents at the 4-position of

phenyl in compound 62 greatly improved the agonist activity

toward the CB2 receptor (compounds 64−66); in particular,

the triﬂuoromethyl analogue 66 exhibited low nanomolar CB2

agonist potency and more than 100-fold selectivity over CB1

receptor (EC_50,CB1= 1839 nM, EC_50,CB2= 15.2 nM). Meanwhile,

it behaved as a high-eﬃcacy CB2 receptor agonist and a weak

agonist at the CB1 receptor (E_max= 30%), and its CB2 agonist

Since N-dealkylation was one of the main metabolic pathways

for compound 1 and its derivatives,⁴³a series of aryl substituents

were introduced into the N-1 position of compound 26 for

improving the metabolic stability of these pyrazole-3-carbox-

amide ligands. Unfortunately, compound 54, possessing a

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activity could be eﬀectively blocked by a selective CB2

antagonist AM630 (Figure S1). Further modiﬁcation aﬀorded

compound 67 with an extra chlorine atom at the 6-position of

pyridine ring, which was entirely ineﬀective at both receptors.

To further explore the interactions between 66 and 40 with

CB2 receptor and characterize the diﬀerent binding modes, we

conducted molecular docking studies for 66 on active CB2

(PDB code: 6PT0)⁵²and 40 on inactive CB2 (PDB code:

5ZTY).⁵³The docking poses of 66 and 40 are quite similar in the

orthostatic binding pocket along with the key interacting

residues (Figure 3). The −OH moiety of both compounds can

Table 4. Human Liver Microsome Stability Data for

Compounds 26, 33, 40, 44, and 66

a

b

c

t_1/2

CL_int(mic)

CL_int(liver)

remaining

compd.

(min) (μL/min/mg) (mL/min/kg) (T = 60 min)

26

33

40

44

66

22.2

11.8

21.4

13.3

112.6

18.8

10.8

62.4

117.0

64.6

104.4

12.3

73.6

128.9

189.1

56.1

105.3

58.2

94.0

11.1

66.3

116.0

170.2

14.9%

2.9%

14.1%

4.6%

64.0%

10.7%

2.0%

testosterone

diclofenac

propafenone

7.3

0.4%

a

b

t

_1/2, half-life. CL_int(mic), intrinsic clearance, 0.693/half-life/mg

c

microsome protein per mL. CL_int(liver)= CL_int(mic)× microsomal

protein/liver weight (mg/g) × liver weight/body weight (g/kg).

Table 5, compound 66 demonstrated an acceptable half-life (t_1/2

= 130.8 min) and favorable drug exposure (AUC_0‑∞= 1672.9

ng·h/mL), as well as excellent oral bioavailability (F = 79.5%)

when dosed orally. In general, compound 66 exhibited a

favorable PK proﬁle and was suitable for further animal eﬃcacy

studies.

In Vitro Pharmacological Characterization of Com-

pound 66. Previous studies have reported that CB2 receptor

activation typically suppresses the formation of intracellular

cyclic adenosine monophosphate (cAMP) via inhibiting

adenylyl cyclase activity.⁵⁴Compound 66 was chosen to be

evaluated in an HTRF-based cAMP assay using CHO cells

expressing CB2 (Figure 4A,B). Compound 66 signiﬁcantly

inhibited cAMP production (EC₅₀= 10.7 nM), which was more

potent than that of positive control 11 (EC₅₀= 16.3 nM) and in

line with the results obtained from the calcium mobilization

assay (66, EC_50,CB2= 15.2 nM; 11, EC_50,CB2= 35.0 nM).

Extracellular signal-regulated kinase (ERK) is a key cellular

eﬀector of GPCR activation.⁵⁴We then detected ERK activation

in CHO cells transiently transfected with CB2 expression or

empty vector. As shown in Figure 4C,D, compound 66 induced

ERK phosphorylation in CHO cells co-expressing CB2, but not

in negative control with empty vector. The eﬀects of compound

66 were completely inhibited by a speciﬁc CB2 receptor

antagonist AM630. Activation of cannabinoid receptors is

capable of stimulating G-protein-independent β-arrestin signal-

ing pathway. To evaluate whether 66 was able to aﬀect the CB1-

or CB2-dependent β-arrestin2 traﬃcking, a typical Tango β-

arrestin recruitment assay was used to assess GPCR activation.⁵⁵

As shown in Figure S2, positive control 11 exhibited high

potency against both CB1 and CB2 receptors(EC_50,CB1= 0.3

nM, EC_50,CB2= 9.8 nM). Compound 66 was not active in CB1-

TANGO assay (EC₅₀> 10 μM). On the contrary, it dose-

dependently induced β-arrestin2 traﬃcking in HEK293-CB2

cells (EC₅₀= 561.6 nM), suggesting that compound 66 was

biased toward G-protein signaling at the CB2 receptor (calcium

mobilization assay: EC₅₀= 15.2 nM). Moreover, compound 66

was evaluated in a series of endocannabinoid-related GPCRs

(GPR119, GPR132, and prostaglandin EP2 and EP4 receptors),

and it exhibited neither agonist nor antagonist activity against

these receptors at 10 μM (data not shown). Taken together,

these ﬁndings suggested that compound 66 activated a range of

downstream signaling pathways speciﬁcally through CB2

receptors.

Figure 3. Comparison of docking results of agonist 66 on active CB2

receptor (PDB code: 6PT0) and antagonist 40 on inactive CB2

receptor (PDB code: 5ZTY). (Left) Superposition of docking poses of

agonist 66 (yellow) on activated CB2 (orange) and antagonist 40

(magenta) on inactive CB2 receptor (sky blue). (Right) Detailed

interactions of the 66 (top) or 40 (bottom) on CB2 receptor.

form a hydrogen bond with residue S90^2.60, the pyridine moiety

from compound 66 and benzene moiety from compound 40 are

predicted to form strong π−π interactions with F183^ECL2and

hydrophobic interactions with W194^5.43and V261^6.51. More-

over, F87^2.57, F91^2.61, I110^3.29, F117^3.36, F281^7.35, and S285^7.39are

important for recognition of the CB2 agonist 66, and F83^ECL2

,

V113^3.32, T114^3.33, and L191^5.40via hydrophobic interactions

with compound 40. The most diﬀerent feature is that the agonist

66 does not extend suﬃciently deep to constrain the

conformation of W258^6.48with a distance of 5.5 Å, whereas

the alkyl chain of compound 40 approaches closer to W258^6.48to

form potentially hydrophobic interactions (Figure 3).

In Vitro Metabolic Stability and In Vivo PK Study. To

assess the metabolic stability of synthesized 1H-pyrazole-3-

carboxamide derivatives, selected compounds were initially

tested in the human liver microsome assay in vitro. As shown in

Table 4, compound 66 exhibited a much better metabolic

stability proﬁle than other four compounds, with a half-time of

nearly 2 h and at least 64% of the parent compound remained

upon 1 h incubation at a concentration of 10 μM. Apparently,

introduction of a pyridyl in the N-1 position of pyrazole notably

improved metabolic stability. However, compounds bearing an

amino acid methyl ester group or an amino group in the 4-

position of the pyrazole were less stable than the parent

compound (33 vs 26, 44 vs 26).

Next, the preliminary in vivo PK proﬁles of 66 were further

characterized in vivo. Compound 66 was intravenously or orally

administered at 1 or 5 mg/kg (Balb/c mice). As depicted in

Moreover, it has been reported previously that repeated

stimulation of a GPCR with its agonist leads to receptor

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Table 5. Preliminary PK Parameters of Compound 66 In Vivo

route

dose (mg/kg)

C

_max(ng/mL)

AUC_0‑∞(ng·h/mL)

Vd (L/kg)

CL (L/h/kg)

t_1/2(min)

F (%)

iv

po

1

5

959.1

603.0

422.0

1672.9

4.5

9.2

2.4

3.1

79.2

130.8

79.5

Figure 4. In vitro functional activity of compound 66. (A, B) Compounds 11 and 66 inhibited cAMP formation that is stimulated by forskolin in the

CB2-expressing CHO cells as determined by HTRF assay. Data represent the mean SEM values of at least three independent experiments. (C, D)

Cells were subjected to indicated treatments, and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation was detected by Western

blotting. EGF: epidermal growth factor.

Figure 5. Compound 66 induced desensitization of CB2 receptor. CHO-CB2-Gα16 cells were initially stimulated with 10 μM compound 11,

compound 66, or DMSO (0.1%), and the intracellular calcium response was detected (left). The cells were washed with HBSS buﬀer upon incubation

for 10 min and restimulated (right, second arrow) with compound 11 (10 μM). Representative intracellular calcium signals were present with at least

three independent measurements. (B) Quantitative analysis, the signiﬁcance of the diﬀerence was assessed via unpaired two-tailed Student’s t-test. ***,

p < 0.001.

desensitization, which is a protective mechanism to restrict

constant GPCR activation.^30,56To determine whether com-

pound 66 was capable of inducing CB2 desensitization, CHO-

CB2-Gα16 cells were ﬁrst stimulated with tested compounds

(10 μM) or dimethyl sulfoxide (DMSO, 0.1%). As shown in

Figure 5, compounds 11 and 66 both caused a signiﬁcant

increase in intracellular calcium concentration. The cells were

washed with HBSS buﬀer upon 10 min incubation and then

restimulated with 10 μM compound 11. The cells pretreated

with 0.1% DMSO responded suﬃciently to the stimulation of

compound 11, while the cells pretreated with compounds 11 or

66 displayed a distinct decline in calcium response following the

second stimulus.

Antiﬁbrotic Eﬀects of Compound 66 in a Mouse Model

of SSc. Encouraged by the favorable pharmacological properties

in vitro and PK proﬁle in vivo, compound 66 was next selected for

further in vivo characterization in a BLM-induced mouse model

of SSc. Upon subcutaneous injections of BLM for 2 weeks,

dermal ﬁbrosis was induced in the back skin of the Balb/c mice.

Compound 9 was used as a positive control for the animal study

due to its well-documented in vivo antiﬁbrotic activity.⁵⁰The

mice were subsequently treated with compound 66 (1 or 5 mg/

kg/day) or 9 (1 mg/kg/day) for another 4 weeks. Skin ﬁbrosis

was characterized by determining the dermal thickness and

collagen deposition. As shown in Figure 6A,B, BLM treatment

resulted in a remarkable increase in dermal thickness and dermal

collagen deposition (sirius red positive area) compared to the

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Figure 6. Compound 66 alleviated BLM-induced dermal ﬁbrosis and inﬂammation in mice. (A) H&E, sirius red, and α-SMA immunohistochemistry

staining of skin sections with scale bar of 200 μm. (B) Dermal thickness data were measured at the end of the experiment (shown as mean standard

deviation (SD), n = 8). (C) Body weight was measured twice a week. The signiﬁcance of the diﬀerence between mean values was evaluated using one-

way analysis of variance (ANOVA) followed by Tukeyʼs multiple comparison test. ***, P < 0.001.

control group. In compound 66-treated groups, BLM-induced

dermal pathological symptoms were signiﬁcantly attenuated and

the skin thickness in the high-dose group was reduced nearly to

the control level. Meanwhile, immunohistochemical analysis

results demonstrated that compound 66 could also dramatically

decrease the expression of α-smooth muscle actin (α-SMA), one

of the key cellular mediators of ﬁbrosis in SSc. In addition, no

signiﬁcant body weight loss was observed in mouse cohorts,

suggesting that compound 66 was well tolerated at the tested

dosages (Figure 6C). Moreover, it was found that BLM

signiﬁcantly increased the mRNA expression of a series of

proinﬂammatory and ﬁbrotic mediators such as Col1α, α-SMA,

TNF-α, and TGF-β in skin lesions, which was markedly

suppressed after 66 treatment (Figure 7) in a dose-dependent

manner. Collectively, these data demonstrated that compound

66 could eﬀectively attenuate both dermal inﬂammation and

ﬁbrosis in a BLM-induced mouse model of SSc.

agonist. In addition, compound 66 exhibited good metabolic

stability and favorable PK properties both in vitro and in vivo that

warranted its subsequent animal eﬃcacy study. In a BLM-

induced mouse model of SSc, subcutaneous injections of

compound 66 at 1 or 5 mg/kg for 4 weeks signiﬁcantly relieved

BLM-induced dermal thickness and collagen deposition, as well

as suppressed the mRNA expression of a series of pro-

inﬂammatory and ﬁbrotic mediators such as Col1α, α-SMA,

TNF-α, and TGF-β in skin lesions. Together, this evidence

suggested that compound 66 eﬀectively delayed ﬁbrosis

progress and is expected to be a potential preclinical candidate

for the treatment of SSc.

EXPERIMENTAL SECTION

■

Chemistry. General. All anhydrous solvents, starting materials, and

reagents were purchased from commercial sources and used without

any further puriﬁcation. Reactions in this work were run under a N₂

atmosphere. All reactions were monitored using TLC on SiO₂and

visualized with UV light. NMR spectra were recorded on Bruker 400,

500, or 600 MHz instruments. The values of chemical shifts were shown

in δ (ppm) using DMSO-d₆(2.50 and 39.51 ppm) or CDCl₃(7.26 and

77.00 ppm) as the standard. Mass spectra (MS) were obtained on

GCMS-QP2010 in electrospray ionization (ESI) mode, while high-

resolution mass spectra (HRMS) were obtained using a Bruker Micro

TOF-Q II liquid chromatography-mass spectrometry (LC-MS)

instrument in ESI mode. Melting points (mp) were determined on

an SGWX-4/4A/4B capillary apparatus. Compound purities were

detected using high-pressure liquid chromatography (Agilent Tech-

nologies 1200 Series) with an ACE Excel 5 C18 column (5 μm, 4.6 mm

CONCLUSIONS

■

In summary, we prepared a series of novel 1H-pyrazole-3-

carboxamide derivatives to develop selective CB2 agonists that

lacked CB1 associated psychiatric side eﬀects. Initial screening

of compounds 17−34 revealed that the 1,3-regioisomer

pyrazoles were preferred to maintain agonist activity at

cannabinoid receptors. Further structural modiﬁcations to

improve the potency, selectivity, and PK proﬁles of designed

compounds resulted in identifying the most promising

compound 66 as a highly potent and selective CB2 receptor

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Figure 7. mRNA expression levels of Col1α (A), α-SMA (B), TNF-α (C), and TGF-β (D) in skin lesions. Data are shown as mean SD, n = 8. The

signiﬁcance between diﬀerent groups was assessed using one-way ANOVA followed by Bonferroniʼs multiple comparison test. *, P < 0.05, **, P < 0.01,

***, P < 0.001.

× 250 mm) at 40 °C with a ﬂowrate of 1.5 mL/min. The detection was

done at 254 or 280 nm on a G1314B detector. Gradient A, 20−90%

CH₃OH in water over 20 min; gradient B, 20−40% CH₃OH (0−5

min), 40−90% CH₃OH (5−7 min), 90% CH₃OH (7−15 min), 90−

20% CH₃OH (15−20 min). All of the ﬁnal biologically tested

compounds had a conﬁrmed purity of ≤95%.

General Synthetic Procedure of N-alkyl-Substituted Pyr-

azoles. Ethyl 5-(tert-Butyl)-1H-pyrazole-3-carboxylate (14a). To a

solution of 3,3-dimethyl-2-butanone (1.0 g, 10.0 mmol) in anhydrous

THF (250 mL) was added t-BuOK (1.46 mg, 13.0 mmol) with ice

cooling. After stirring for 10 min, diethyl oxalate (1.36 mL, 10.0 mmol)

was added and the reaction mixture was reﬂuxed for 5 h. Then,

hydrazine hydrate (550 mg, 11.0 mmol) was added dropwise to the

mixture, followed by the addition of AcOH (1.5 mL). The mixture was

stirred at 60 °C for 3 h. Then, the reaction was concentrated under

vacuum and the crude mixture was extracted with EtOAc. The

combined organic layers were washed with brine, dried over anhydrous

Na₂SO₄, and concentrated. The resulted residue was puriﬁed by ﬂash

chromatography on silica gel to give the title compound 14a (1.54 g,

78.3%). ¹H NMR (500 MHz, DMSO-d₆) δ 13.21 (s, 1H), 6.46 (s, 1H),

4.31−4.19 (m, 2H), 1.28 (m, 12H).

Ethyl 3-(tert-Butyl)-1-pentyl-1H-pyrazole-5-carboxylate (15a)

and Ethyl 5-(tert-Butyl)-1-pentyl-1H-pyrazole-3-carboxylate (16a).

To a solution of 14a (564 mg, 2.88 mmol) in anhydrous THF (30 mL)

was added t-BuOK (337 mg, 3.45 mmol) with ice cooling. Then, 1-

bromopentane (0.43 mL, 3.45 mmol) was slowly added and the mixture

was reﬂuxed overnight. The reaction was cooled to room temperature

(rt) and concentrated under vacuum. The crude mixture was extracted

with EtOAc, and the combined organic layers were washed with brine,

dried over anhydrous Na₂SO₄, and concentrated. The residue was

puriﬁed by ﬂash chromatography on silica gel to give the isomer

compound ethyl 3-(tert-butyl)-1-pentyl-1H-pyrazole-5-carboxylate

(406 mg, 29%) and compound ethyl 5-(tert-butyl)-1-pentyl-1H-

pyrazole-3-carboxylate (220 mg, 33%). Ethyl 3-(tert-butyl)-1-pentyl-

1H-pyrazole-5-carboxylate (15a): ¹H NMR (500 MHz, CDCl₃) δ 6.55

(s, 1H), 4.45−4.33 (m, 2H), 4.25−4.15 (m, 2H), 1.96 (m, 2H), 1.43−

1.32 (m, 16H), 0.93 (t, J = 6.1 Hz, 3H). Ethyl 5-(tert-butyl)-1-pentyl-

1H-pyrazole-3-carboxylate (16a): ¹H NMR (500 MHz, CDCl₃) δ 6.69

(s, 1H), 4.50 (t, J = 7.4 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 1.82 (m, 2H),

1.40 (t, J = 7.1 Hz, 3H), 1.37−1.28 (m, 13H), 0.90 (t, J = 6.6 Hz, 3H).

N-(1-Adamantyl)-3-(tert-butyl)-1-pentyl-1H-pyrazole-5-carboxa-

mide (17). A solution of 15 (78 mg, 0.29 mmol) and LiOH·H₂O (74

mg, 1.76 mmol) in 10 mL of MeOH:H₂O:THF = 2:1:2 was stirred at rt

for 1 h. The reaction was then concentrated under vacuum and acidiﬁed

to pH 4−5 with 1 M aqueous HCl. The mixture was extracted with

EtOAc, and the combined organic layers were washed with brine, dried

over anhydrous Na₂SO₄, and concentrated to aﬀord the crude

compound 3-(tert-butyl)-1-pentyl-1H-pyrazole-5-carboxylic acid. To

a solution of 3-(tert-butyl)-1-pentyl-1H-pyrazole-5- carboxylic acid (72

mg, 0.30 mmol) and 1-adamantylamine in CH₂Cl₂(5 mL) was added

HATU (137 mg, 0.36 mmol), followed by DIPEA (0.1 mL, 0.60

mmol). The mixture was stirred at rt for 5 h. Then, the reaction was

concentrated under vacuum and the crude mixture was extracted with

EtOAc. The combined organic layers were washed with brine, dried

over anhydrous Na₂SO₄, and concentrated. The residue was puriﬁed by

ﬂash chromatography on silica gel to give the title compound 17 (62

1

mg, 68.8%) as a white solid. H NMR (500 MHz, CDCl₃) δ 6.19 (s,

1H), 5.62 (s, 1H), 4.43 (t, J = 7.4 Hz, 2H), 2.12−2.09 (m, 9H), 1.80−

1.76 (m, 2H), 1.75−1.68 (m, 6H), 1.29−1.25 (m, 13H), 0.87 (t, J = 6.9

Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.5, 152.8, 145.6, 103.6,

51.6, 51.5, 41.7 (3C), 36.5 (3C), 31.4, 30.5, 30.1 (3C), 29.5, 29.0 (3C),

22.4, 14.0. HRMS (ESI): calcd for C₂₃H₃₇N₃NaO [M + Na]⁺,

394.2834; found 394.2801. mp: 136−138 °C. HPLC purity = 97.9%; t_R

= 11.6 min.

3-(tert-Butyl)-N-(3-hydroxyadamantan-1-yl)-1-pentyl-1H-pyra-

zole-5-carboxamide (18). The title compound was prepared by the

same procedure described for 17 except using 3-amino-1-adamantanol

1

instead of 1-adamantylamine as a white solid. H NMR (500 MHz,

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CDCl₃) δ 6.20 (s, 1H), 5.69 (s, 1H), 4.44−4.41 (m, 2H), 2.32 (s, 2H),

2.01 (s, 2H), 2.00 (s, 4H), 1.74−1.72 (m, 4H), 1.64−1.54 (m, 4H),

1.29−1.24 (m, 13H), 0.89 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 159.9, 158.8, 136.5, 103.9, 67.8, 54.6, 50.5, 49.3, 44.7,

35.3, 32.1, 30.9 (3C), 30.5 (2C), 30.4, 28.6 (2C), 22.1 (2C), 14.4 (2C).

HRMS (ESI): calcd for C₂₃H₃₇N₃NaO₂[M + Na]⁺, 410.2783; found

410.2758. mp: 143−145 °C. HPLC purity = 99.9%; t_R= 11.5 min.

N,3-Di-tert-butyl-1-pentyl-1H -pyrazole-5-carboxamide (19). The

title compound was prepared by the same procedure described for 17

except using tert-butylamine instead of 1-adamantylamine as a white

solid. ¹H NMR (500 MHz, CDCl₃) δ 6.19 (s, 1H), 5.75 (s, 1H), 4.45−

4.42 (m, 2H), 1.82−1.76 (m, 2H), 1.44 (s, 9H), 1.31−1.25 (m, 13H),

0.87 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 160.2,

158.8, 136.6, 103.8, 51.4, 50.4, 32.1, 30.9 (3C), 30.4, 29.0 (3C), 28.6,

22.1, 14.3. HRMS (ESI): calcd for C₁₇H₃₂N₃O [M + H]⁺, 294.2545;

found 294.2546. mp: 118−120 °C. HPLC purity = 95.1%; t_R= 8.9 min.

3-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-pentyl-1H-

pyrazole-5-carboxamide (20). The title compound was prepared by

the same procedure described for 17 except using 2-amino-2-methyl-1-

propanol instead of 1-adamantylamine as a white solid. ¹H NMR (500

MHz, CDCl₃) δ 6.24 (s, 1H), 6.00 (s, 1H), 4.44−4.41 (m, 2H), 3.67 (s,

2H), 1.81−1.75 (m, 2H), 1.39 (s, 6H), 1.33−1.23 (m, 13H), 0.88 (t, J =

7.2 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 161.0, 159.9, 135.3, 102.6,

70.5, 56.5, 51.2, 32.0, 30.5, 30.5 (3C), 28.7, 24.5 (2C), 22.3, 14.0.

HRMS (ESI): calcd for C₁₇H₃₂N₃O₂[M + H]⁺, 310.2495; found

310.2496. mp: 136−138 °C. HPLC purity = 96.1%; t_R= 8.6 min.

3-(tert-Butyl)-1-pentyl-N-(4-chlorophenyl)-1H-pyrazole-5-car-

boxamide (21). The title compound was prepared by the same

procedure described for 17 except using 4-chloroaniline instead of 1-

adamantylamine as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 8.71

(s, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 6.63 (s, 1H),

4.25−4.13 (m, 2H), 2.02−1.93 (m, 2H), 1.47−1.37 (m, 13H), 0.96 (t, J

= 6.9 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 160.2, 153.5, 144.4,

136.7, 128.9 (2C), 128.62, 120.8 (2C), 104.2, 51.7, 31.5, 30.4, 30.1

(3C), 29.0, 22.4, 14.0. HRMS (ESI): calcd for C₁₉H₂₇ClN₃O [M + H]⁺,

348.1843; found 348.1836. mp: 136−138 °C. HPLC purity = 96.3%; t_R

= 9.7 min.

14.0. HRMS (ESI): calcd for C₂₃H₃₇N₃NaO [M + Na]⁺, 394.2834;

found 394.2803. mp: 56−58 °C. HPLC purity = 95.4%; t_R= 5.2 min.

5-(tert-Butyl)-N-(3-hydroxyadamantan-1-yl)-1-pentyl-1H-pyra-

zole-3-carboxamide (24). The title compound was prepared by the

same procedure described for 23 except using 3-amino-1-adamantanol

1

instead of 1-adamantylamine as a white solid. H NMR (500 MHz,

CDCl₃) δ 6.69 (s, 1H), 6.49 (s, 1H), 4.13−4.10 (m, 2H), 2.33−2.27

(m, 2H), 2.16−2.11 (m, 2H), 1.97−1.90 (m, 2H), 1.74 (m, 4H), 1.61

(m, 6H), 1.49−1.31 (m, 13H), 0.93 (t, J = 7.0 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 161.5, 152.9, 145.3, 103.7, 69.3, 54.0, 51.5, 49.2, 44.2

(2C), 40.3 (2C), 35.0, 31.4, 30.7 (2C), 30.5, 30.1 (3C), 29.0, 22.4, 14.0.

HRMS (ESI): calcd for C₂₃H₃₇N₃NaO₂[M + Na]⁺, 410.2783; found

410.2750. mp: 93−95 °C. HPLC purity = 97.3%; t_R= 10.5 min.

N,5-Di-tert-butyl-1-pentyl-1H-pyrazole-3-carboxamide (25). The

title compound was prepared by the same procedure described for 23

except using tert-butylamine instead of 1-adamantylamine as a white

solid. ¹H NMR (500 MHz, CDCl₃) δ 6.72 (s, 1H), 6.50 (s, 1H), 4.13−

4.10 (m, 2H), 1.92−1.90 (m, 2H), 1.45 (s, 9H), 1.38−1.37 (m, 4H),

1.36 (s, 9H), 0.93 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

161.8, 152.8, 145.5, 103.6, 51.5, 50.9, 31.4, 30.5, 30.1 (3C), 29.0, 28.9

(3C), 22.4, 14.0. HRMS (ESI): calcd for C₁₇H₃₁N₃NaO [M + Na]⁺,

316.2365; found 316.2350. mp: 90−93 °C. HPLC purity = 97.6%; t_R=

10.3 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-pentyl-1H-

pyrazole-3-carboxamide (26). The title compound was prepared by

the same procedure described for 23 except using 2-amino-2-methyl-1-

propanol instead of 1-adamantylamine as a white solid. ¹H NMR (500

MHz, CDCl₃) δ 6.91 (s, 1H), 6.51 (s, 1H), 4.14−4.11 (m, 2H), 3.67 (s,

2H), 1.94−1.88 (m, 2H), 1.38 (s, 6H), 1.37−1.34 (m, 11H), 1.27−1.23

(m, 2H), 0.93 (t, J = 6.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

163.2, 153.2, 144.3, 103.9, 71.0, 56.1, 51.6, 31.4, 30.5, 30.0 (3C), 29.0,

24.9 (2C), 22.4, 14.0. HRMS (ESI): calcd for C₁₇H₃₁N₃NaO₂[M +

Na]⁺, 332.2314; found 332.2297. mp: 92−95 °C. HPLC purity =

98.5%; t_R= 6.0 min.

5-(tert-Butyl)-1-pentyl-N-(4-chlorophenyl)-1H-pyrazole-3-car-

boxamide (27). The title compound was prepared by the same

procedure described for 23 except using 4-chloroaniline instead of 1-

adamantylamine as a colorless oil. ¹H NMR (600 MHz, CDCl₃) δ 8.71

(s, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 6.63 (s, 1H),

4.25−4.13 (m, 2H), 2.02−1.93 (m, 2H), 1.47−1.37 (m, 13H), 0.96 (t, J

= 6.9 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 160.2, 153.5, 144.4,

136.8, 128.9 (2C), 128.6, 120.8 (2C), 104.2, 51.7, 31.5, 30.4, 30.1 (3C),

29.0, 22.4, 14.0. HRMS (ESI): calcd for C₁₉H₂₆ClN₃NaO [M + Na]⁺,

370.1662; found 370.1682. HPLC purity = 98.8%; t_R= 10.5 min.

5-(tert-Butyl)-1-pentyl-N-(4-chlorobenzyl)-1H-pyrazole-3-car-

boxamide (28). The title compound was prepared by the same

procedure described for 23 except using 4-chlorobenzylamine instead

of 1-adamantylamine as a white solid. ¹H NMR (500 MHz, CDCl₃) δ

7.28−7.26 (m, 4H), 7.17 (s, 1H), 6.56 (s, 1H), 4.57 (d, J = 6.2 Hz, 2H),

4.12−4.11 (m, 2H), 1.91−1.86 (m, 2H), 1.37 (s, 9H), 1.36−1.33 (m,

4H), 0.91 (t, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 162.0,

152.6, 144.7, 139.5, 131.6, 129.6 (2C), 128.5 (2C), 103.8, 51.4, 41.7,

31.4, 30.6, 30.2 (3C), 28.8, 22.4, 14.3. HRMS (ESI): calcd for

C₂₀H₂₈ClN₃NaO [M + Na]⁺, 384.1819; found 384.1808. mp: 136−138

°C. HPLC purity = 98.6%; t_R= 9.7 min.

3-(tert-Butyl)-1-pentyl-N-(4-chlorobenzyl)-1H-pyrazole-5-car-

boxamide (22). The title compound was prepared by the same

procedure described for 17 except using 4-chlorobenzylamine instead

of 1-adamantylamine as a white solid. ¹H NMR (500 MHz, DMSO-d₆)

δ 8.96−8.90 (m, 1H), 7.38 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H),

6.73 (s, 1H), 4.45−4.34 (m, 4H), 1.72−1.59 (m, 2H), 1.27−1.21 (m,

11H), 1.18−1.12 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H). ¹³C NMR (126

MHz, DMSO-d₆) δ 160.0, 159.2, 138.9, 135.2, 131.8, 129.6 (2C), 128.7

(2C), 103.8, 50.7, 41.9, 32.1, 30.9 (3C), 30.5, 28.6, 22.1, 14.3. HRMS

(ESI): calcd for C₂₀H₂₉ClN₃O [M + H]⁺, 362.1999; found 362.2001.

mp: 136−138 °C. HPLC purity = 98.6%; t_R= 9.2 min.

N-(1-Adamantyl)-5-(tert-butyl)-1-pentyl-1H-pyrazole-3-carboxa-

mide (23). A solution of 16a (52 mg, 0.19 mmol) and LiOH·H₂O (48

mg, 1.1 mmol) in 10 mL of MeOH:THF:H₂O = 2:2:1 was stirred at rt

for 1 h. Then, the reaction was concentrated under vacuum and

acidiﬁed to pH 4−5 with 1 M aqueous HCl. The mixture was extracted

with EtOAc, and the combined organic layers were washed with brine,

dried over anhydrous Na₂SO₄, and concentrated to aﬀord the crude

compound 5-(tert-butyl)-1-pentyl-1H-pyrazole-3-carboxylic acid. To a

solution of 5-(tert-butyl)-1-pentyl-1H-pyrazole-3- carboxylic acid (43

mg, 0.18 mmol) and 1-adamantylamine in CH₂Cl₂(10 mL) was added

HATU (82 mg, 0.22 mmol), followed by DIPEA (0.1 mL, 0.36 mmol).

The mixture was stirred at rt for 5 h. Then, the reaction was

concentrated under vacuum and the crude mixture was extracted with

EtOAc. The combined organic layers were washed with brine, dried

over anhydrous Na₂SO₄, and concentrated. The residue was puriﬁed by

ﬂash chromatography on silica gel to give the title compound 23 (40

5-(tert-Butyl)-1-pentyl-N-(2-phenylpropan-2-yl)-1H-pyrazole-3-

carboxamide (29). The title compound was prepared by the same

procedure described for 23 except using 2-phenylpropan-2-amine

1

instead of 1-adamantylamine as a white solid. H NMR (500 MHz,

CDCl₃) δ 7.47−7.45 (m, 2H), 7.34−7.31 (m, 2H), 7.23−7.18 (m, 2H),

6.50 (s, 1H), 4.15−4.12 (m, 2H), 1.95−1.92 (m, 2H), 1.80 (s, 6H),

1.42−1.38 (m, 4H), 1.35 (s, 9H), 0.94 (t, J = 7.0 Hz, 3H). ¹³C NMR

(151 MHz, CDCl₃) δ 161.6, 152.8, 147.3, 145.3, 128.3 (2C), 126.5,

124.9 (2C), 103.8, 55.7, 51.5, 31.4, 30.5, 30.1 (3C), 29.4 (2C), 29.0,

22.4, 14.0. HRMS (ESI): calcd for C₂₂H₃₄N₃O [M + H]⁺, 356.2702;

found 356.2703. mp: 138−140 °C. HPLC purity = 99.1%; t_R= 10.2

min.

1

mg, 67.5%) as a white solid. H NMR (500 MHz, CDCl₃) δ 6.61 (s,

1H), 6.48 (s, 1H), 4.25−4.00 (m, 2H), 2.16−2.04 (m, 9H), 1.95−1.85

(m, 2H), 1.72−1.68 (m, 6H), 1.42−1.31 (m, 13H), 0.93 (t, J = 6.9,

3H). ¹³C NMR (151 MHz, CDCl₃) δ 161.6, 152.7, 145.7, 103.5, 51.5,

51.5, 41.7 (3C), 36.5 (3C), 31.4, 30.4, 30.1 (3C), 29.5 (3C), 29.0, 22.4,

5-(tert-Butyl)-1-pentyl-N-(pyridin-4-yl)-1H-pyrazole-3-carboxa-

mide (30). The title compound was prepared by the same procedure

described for 23 except using 4-aminopyridine instead of 1-adamantyl-

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amine as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 8.83 (s, 1H),

8.50 (d, J = 6.2 Hz, 2H), 7.63 (d, J = 6.3 Hz, 2H), 6.62 (s, 1H), 4.19−

4.13 (m, 2H), 1.98−1.91 (m, 2H), 1.41−1.36 (m, 13H), 0.93 (t, J = 6.9

Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 160.7, 153.8, 150.5 (2C),

145.2, 143.8, 113.5 (2C), 104.4, 51.8, 31.5, 30.5, 30.0 (3C), 29.0, 22.4,

14.0. HRMS (ESI): calcd for C₁₈H₂₇N₄O [M + H]⁺, 315.2185; found

315.2154. HPLC purity = 97.9%; t_R= 9.5 min.

5-(tert-Butyl)-N-cyclohexyl-1-pentyl-1H-pyrazole-3-carboxamide

(31). The title compound was prepared by the same procedure

described for 23 except using cyclohexane instead of 1-adamantylamine

as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 6.70 (d, J = 7.8 Hz,

1H), 6.52 (s, 1H), 4.13−4.10 (m, 2H), 3.95−3.85 (m, 1H), 1.98−1.91

(m, 2H), 1.90−1.88 (m, 2H), 1.74−1.72 (m, 2H), 1.54−1.33 (m,

15H), 1.26−1.14 (m, 4H), 0.93 (t, J = 6.8 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 161.4, 152.7, 144.9, 103.8, 51.5, 47.7, 33.3 (2C), 31.4,

30.5, 30.1 (3C), 29.0, 25.7, 25.0, 22.4, 14.0, 1.0. HRMS (ESI): calcd for

C₁₉H₃₃N₃NaO [M + Na]⁺, 342.2521; found 342.2537. HPLC purity =

98.2%; t_R= 7.6 min.

71.0, 56.1, 49.3, 30.2, 28.8, 25.4, 24.9 (2C), 22.8 (2C), 22.3, 13.9.

HRMS (ESI): calcd for C₁₆H₂₉N₃NaO₂[M + Na]⁺, 318.2157; found

318.2134. mp: 37−38 °C. HPLC purity = 97.4%; t_R= 7.3 min.

N-(1-Hydroxy-2-methylpropan-2-yl)-5-neopentyl-1-pentyl-1H-

pyrazole-3-carboxamide (37). The title compound was prepared by

the same procedure described for 26 except using 4,4-dimethyl-2-

1

pentanone instead of 3,3-dimethyl-2-butanone as a white solid. H

NMR (500 MHz, CDCl₃) δ 6.96 (s, 1H), 6.54 (s, 1H), 4.03−3.99 (m,

2H), 3.68 (s, 2H), 2.51 (s, 2H), 1.86−1.79 (m, 2H), 1.39 (s, 6H),

1.38−1.33 (m, 2H), 1.32−1.27 (m, 2H), 0.95 (s, 9H), 0.91 (t, J = 7.2

Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.2, 144.8, 142.5, 106.8,

70.9, 56.1, 49.6, 39.0, 32.1, 20.0, 29.4 (3C), 28.8, 24.9 (2C), 22.3, 13.9.

HRMS (ESI): calcd for C₁₈H₃₃N₃NaO₂[M + Na]⁺, 346.2470; found

346.2442. mp: 46−48 °C. HPLC purity = 98.9%; t_R= 4.2 min.

5-Cyclopropyl-l-N-(1-hydroxy-2-methylpropan-2-yl)-1-pentyl-

1H-pyrazole-3-carboxamide (38). The title compound was prepared

by the same procedure described for 26 except using cyclopropylmethyl

1

ketone instead of 3,3-dimethyl-2-butanone as a light yellow solid. H

NMR (500 MHz, CDCl₃) δ 6.91 (s, 1H), 6.32 (s, 1H), 4.16−4.13 (m,

2H), 3.66 (s, 2H), 1.90−1.83 (m, 2H), 1.73−1.68 (m, 1H), 1.38 (s,

6H), 1.36−1.28 (m, 4H), 1.03−0.96 (m, 2H), 0.92 (t, J = 7.1 Hz, 3H),

0.72−0.54 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.1, 147.0,

144.8, 103.0, 71.0, 56.2, 49.7, 29.8, 28.8, 25.0 (2C), 22.3, 13.9, 7.3 (2C),

6.1. HRMS (ESI): calcd for C₁₆H₂₇N₃NaO₂[M + Na]⁺, 316.2001;

found 316.1989. mp: 45−48 °C. HPLC purity = 98.9%; t_R= 4.2 min.

5-Cyclohexyl-N-(1-hydroxy-2-methylpropan-2-yl)-1-pentyl-1H-

pyrazole-3-carboxamide (39). The title compound was prepared by

the same procedure described for 26 except using 1-cyclohexyletha-

Methyl (R)-2-(5-(tert-Butyl)-1-pentyl-1H-pyrazole-3-carboxami-

do)-3,3-dimethyl-butanoate (32). The title compound was prepared

by the same procedure described for 23 except using methyl (R)-2-

amino-3,3-dimethylbutanoate instead of 1-adamantylamine as a

1

colorless oil. H NMR (500 MHz, CDCl₃) δ 7.38 (d, J = 9.6 Hz,

1H), 6.52 (s, 1H), 4.63 (d, J = 9.7 Hz, 1H), 4.14 (t, J = 7.8 Hz, 2H), 3.73

(s, 3H), 1.99−1.90 (m, 2H), 1.42−1.37 (m, 4H), 1.36 (s, 9H), 1.04 (s,

9H), 0.93 (t, J = 6.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.1,

162.0, 152.9, 143.8, 104.1, 59.6, 51.7, 51.6, 35.0, 31.4, 30.4, 30.1 (3C),

29.0, 26.6 (3C), 22.4, 14.0. HRMS (ESI): calcd for C₂₀H₃₅N₃NaO₃[M

+ Na]⁺, 388.2576; found 388.2551. HPLC purity = 96.1%; t_R= 9.2 min.

Methyl (5-(tert-Butyl)-1-pentyl-1H-pyrazole-3-carbonyl)-^D-vali-

nate (33). The title compound was prepared by the same procedure

described for 23 except using methyl D-valinate instead of 1-

1

none instead of 3,3-dimethyl-2-butanone as a white solid. H NMR

(500 MHz, CDCl₃) δ 6.92 (s, 1H), 6.51 (s, 1H), 4.00−3.98 (m, 2H),

3.67 (s, 2H), 2.51 (t, J = 9.0 Hz, 1H), 1.85−1.82 (m, 6H), 1.38−1.30

(m, 16H), 0.92 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

163.1, 150.4, 145.0, 103.1, 71.1, 56.2, 49.3, 35.3, 33.2 (2C), 30.2, 28.8,

26.3 (2C), 25.7, 25.0 (2C), 22.3, 13.93. HRMS (ESI): calcd for

C₁₉H₃₃N₃NaO₂[M + Na]⁺, 358.2470; found 358.2469. mp: 84−86 °C.

HPLC purity = 97.7%; t_R= 6.0 min.

5-(2,4-Diﬂuorophenyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-

pentyl-1H-pyrazole-3-carboxamide (40). The title compound was

prepared by the same procedure described for 26 except using 2′,4′-

diﬂuoroacetophenone instead of 3,3-dimethyl-2-butanone as a white

solid. ¹H NMR (500 MHz, CDCl₃) δ 7.31−7.28 (m, 1H), 6.99−6.94

(m, 3H), 6.77 (s, 1H), 3.96 (t, J = 7.4 Hz, 2H), 3.71 (s, 2H), 1.80−1.74

(m, 2H), 1.42 (s, 6H), 1.18−1.12 (m, 2H), 1.19−1.11 (m, 2H), 0.83 (t,

J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.6, 145.8, 138.1,

132.6, 114.2, 112.0, 108.1, 104.7, 70.8, 56.2, 50.3, 29.6, 28.5, 24.9 (2C),

24.8 (2C), 22.0, 13.8. HRMS (ESI): calcd for C₁₉H₂₅F₂N₃NaO₂[M +

Na]⁺, 388.1813; found 388.1786. mp: 85−88 °C. HPLC purity =

96.2%; t_R= 6.0 min.

5-(tert-Butyl)-4-chloro-N-(1-hydroxy-2-methylpropan-2-yl)-1-

pentyl-1H-pyrazole-3-carboxamide (41). To a solution of 26 (1.878

g, 5.7 mmol) and NCS (837 mg, 6.3 mmol) in CHCl₃(20 mL) was

slowly added dibenzoyl peroxide (138 mg, 0.57 mmol). The mixture

was reﬂuxed overnight. Then, the reaction was concentrated under

vacuum and the crude mixture was extracted with EtOAc. The

combined organic layers were washed with brine, dried over anhydrous

Na₂SO₄, and concentrated. The residue was puriﬁed by ﬂash

chromatography on silica gel to give the title compound 41 (1.190 g,

69.4%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 6.88 (s, 1H),

4.23−4.20 (m, 2H), 3.68 (s, 2H), 1.90−1.75 (m, 2H), 1.51 (s, 9H),

1.40−1.30 (m, 10H), 0.92 (t, J=7.0, 3H). ¹³C NMR (101 MHz, CDCl₃)

δ 162.0, 146.3, 140.2, 108.0, 70.7, 56.3, 53.7, 33.7, 30.8, 30.2 (3C), 28.7,

24.9 (2C), 22.3, 14.0. HRMS (ESI): calcd for C₁₇H₃₀ClN₃NaO₂[M +

Na]⁺, 366.1924; found 366.1941. mp: 82−84 °C. HPLC purity =

95.3%; t_R= 9.3 min.

1

adamantylamine as a colorless oil. H NMR (500 MHz, CDCl₃) δ

7.28 (s, 1H), 6.52 (s, 1H), 4.77−4.57 (m, 1H), 4.14 (t, J = 7.8 Hz, 2H),

3.75 (s, 3H), 2.28−2.20 (m, 1H), 1.98−1.85 (m, 2H), 1.43−1.31 (m,

13H), 1.03−0.96 (m, 6H), 0.93 (t, J = 6.8 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ 172.6, 162.3, 152.8, 143.9, 103.9, 56.7, 52.1, 51.6, 31.6,

31.4, 30.4, 30.1 (3C), 29.0, 22.4, 19.1, 18.0, 14.0. HRMS (ESI): calcd

for C₁₉H₃₃N₃NaO₃[M + Na]⁺, 374.2420; found 374.2395. HPLC

purity = 98.0%; t_R= 8.7 min.

Methyl (5-(tert-Butyl)-1-pentyl-1H-pyrazole-3-carbonyl)-L-vali-

nate (34). The title compound was prepared by the same procedure

described for 23 except using methyl L-valinate instead of 1-

1

adamantylamine as a colorless oil. H NMR (500 MHz, CDCl₃) δ

7.28 (d, J = 9.3 Hz, 1H), 6.53 (s, 1H), 4.71 (dd, J = 9.2, 5.3 Hz, 1H),

4.14 (t, J = 7.9 Hz, 2H), 3.75 (s, 3H), 2.33−2.16 (m, 1H), 1.99−1.60

(m, 2H), 1.42−1.38 (m, 4H), 1.36 (s, 9H), 0.99 (dd, J = 10.4, 6.9 Hz,

6H), 0.94 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.6.

162.3, 152.9, 143.9, 104.0, 56.8, 52.0, 51.6, 31.5, 31.4, 30.4, 30.1 (3C),

29.0, 22.4, 19.1, 18.0, 14.0. HRMS (ESI): calcd for C₁₉H₃₃N₃NaO₃[M

+ Na]⁺, 374.2420; found 374.2389. HPLC purity = 96.2%; t_R= 10.5

min.

N-(1-Hydroxy-2-methylpropan-2-yl)-1-pentyl-5-(triﬂuorometh-

yl)-1H-pyrazole-3-carboxamide (35). The title compound was

prepared by the same procedure described for 26 except using 1,1,1-

triﬂuoroacetone instead of 3,3-dimethyl-2-butanone as a colorless oil.

¹H NMR (500 MHz, CDCl₃) δ 7.07 (s, 1H), 6.88 (s, 1H), 4.18 (t, J =

7.5 Hz, 2H), 3.69 (s, 2H), 1.92−1.87 (m, 2H), 1.40 (s, 6H), 1.38−1.30

(m, 4H), 0.91 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ

161.3, 145.6, 137.0−129.9, 119.6, 108.1, 70.5, 56.3, 51.9, 29.8, 28.5,

24.8, 22.1, 13.8, 1.0. HRMS (ESI): calcd for C₁₄H₂₂F₃N₃NaO₂[M +

Na]⁺, 344.1562; found 344.1578. HPLC purity = 97.8%; t_R= 5.0 min.

N-(1-Hydroxy-2-methylpropan-2-yl)-5-isopropyl-1-pentyl-1H-

pyrazole-3-carboxamide (36). The title compound was prepared by

the same procedure described for 26 except using 3-methyl-2-butanone

4-Bromo-5-(tert-butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-

pentyl-1H-pyrazole-3-carboxamide (42). The title compound was

prepared by the same procedure described for 41 except using NBS

instead of NCS as a yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 6.90 (s,

1H), 4.29−4.21 (m, 2H), 3.69 (s, 2H), 1.89−1.80 (m, 2H), 1.53−1.47

(m, 9H), 1.43−1.30 (m, 10H), 0.93 (t, J = 7.0 Hz, 3H). ¹³C NMR (101

1

instead of 3,3-dimethyl-2-butanone as a light yellow solid. H NMR

(500 MHz, CDCl₃) δ 6.93 (s, 1H), 6.54 (s, 1H), 5.27 (s, 1H), 4.00 (t, J

= 7.5 Hz, 2H), 3.67 (s, 2H), 2.96−2.86 (m, 1H), 1.88−1.80 (m, 2H),

1.38 (s, 6H), 1.37−1.28 (m, 4H), 1.25 (d, J = 6.8 Hz, 6H), 0.92 (t, J =

7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.2, 151.3, 145.1, 102.6,

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MHz, CDCl₃) δ 162.2, 147.4, 141.5, 91.8, 70.7, 56.2, 54.1, 34.0, 31.0,

30.5 (3C), 28.7, 24.9 (2C), 22.3, 14.0. HRMS (ESI): calcd for

C₁₇H₃₀BrN₃NaO₂[M + Na]⁺, 410.1419; found 410.1401. mp: 87−88

°C. HPLC purity = 97.4%; t_R= 8.7 min.

prepared by the same procedure described for 26 except using 1-

bromo-3-ﬂuoropropane instead of 1-bromopentane as a white solid. ¹H

NMR (500 MHz, CDCl₃) δ 6.93 (s, 1H), 6.55 (s, 1H), 4.61 (t, J = 5.5

Hz, 1H), 4.51 (t, J = 5.5 Hz, 1H), 4.37−4.32 (m, 2H), 3.67 (s, 2H),

2.42−2.37 (m, 1H), 2.35−2.30 (m, 1H), 1.38 (s, 15H). ¹³C NMR (101

MHz, CDCl₃) δ 162.9, 153.9, 144.8, 104.1, 81.9−80.2, 70.9, 56.1, 47.6,

31.4, 31.3, 29.9 (3C), 24.9 (2C). HRMS (ESI): calcd for

C₁₅H₂₆FN₃NaO₂[M + Na]⁺, 322.1907; found 322.1893. mp: 81−83

°C. HPLC purity = 99.7%; t_R= 2.9 min.

5-(tert-Butyl)-1-(4-ﬂuorobutyl)-N-(1-hydroxy-2-methylpropan-2-

yl)-1H-pyrazole-3-carboxamide (49). The title compound was

prepared by the same procedure described for 26 except using 1-

bromo-4-ﬂuorobutane instead of 1-bromopentane as a white solid. ¹H

NMR (500 MHz, CDCl₃) δ 6.90 (s, 1H), 6.53 (s, 1H), 4.56 (t, J = 5.8

Hz, 1H), 4.47 (t, J = 5.8 Hz, 1H), 4.22−4.19 (m, 2H), 3.67 (s, 2H),

2.10−2.05 (m, 2H), 1.86−1.78 (m, 2H), 1.38 (s, 6H), 1.37 (s, 9H). ¹³C

NMR (101 MHz, CDCl₃) δ 163.1, 153.4, 144.5, 104.0, 84.5−82.8, 71.0,

56.2, 51.0, 31.4, 30.0 (3C), 27.7, 26.9, 25.0 (2C). HRMS (ESI): calcd

for C₁₆H₂₈FN₃NaO₂[M + Na]⁺, 336.2063; found 336.2034. mp: 97−

98 °C. HPLC purity = 99.1%; t_R= 3.1 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-4-nitro-1-pen-

tyl-1H-pyrazole-3-carboxamide (43). To a solution of 26 (279 mg, 0.9

mmol) in CHCl₃(10 mL) was added copper dinitrate (253 mg, 1.3

mmol). Then, TFAA (1.3 mL, 9.0 mmol) was gradually added. The

mixture was stirred at rt overnight. Then, the reaction was concentrated

under vacuum and the crude mixture was extracted with EtOAc. The

combined organic layers were washed with brine, dried over anhydrous

Na₂SO₄, and concentrated. The residue was puriﬁed by ﬂash

chromatography on silica gel to give the title compound 43 (225 g,

70.5%) as a yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 6.69 (s, 1H),

4.17−4.13 (m, 2H), 3.67 (s, 2H), 1.99−1.85 (m, 2H), 1.60−1.25 (m,

19H), 0.95 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.6,

143.6, 136.3, 132.1, 70.1, 56.4, 52.9, 33.3, 30.2, 28.8 (3C), 28.8 (2C),

24.8, 22.3, 13.9. HRMS (ESI): calcd for C₁₇H₃₀N₄NaO₄[M + Na]⁺,

377.2165; found 377.2179. mp: 92−95 °C. HPLC purity = 97.5%; t_R=

5.2 min.

5-(tert-Butyl)-1-(5-ﬂuoropentyl)-N-(1-hydroxy-2-methylpropan-

2-yl)-1H-pyrazole-3-carboxamide (50). The title compound was

prepared by the same procedure described for 26 except using 1-

bromo-5-ﬂuoropentane instead of 1-bromopentane as a yellow solid.

¹H NMR (500 MHz, DMSO-d₆) δ 7.06 (s, 1H), 6.31 (s, 1H), 5.08 (s,

1H), 4.49 (t, J = 6.0 Hz, 1H), 4.41 (t, J = 6.0 Hz, 1H), 4.17−4.14 (m,

2H), 3.40 (d, J = 5.6 Hz, 2H), 1.88−1.82 (m, 2H), 1.74−1.66 (m, 2H),

1.48−1.42 (m, 2H), 1.33 (s, 9H), 1.29 (s, 6H). ¹³C NMR (101 MHz,

CDCl₃) δ 162.7, 153.7, 144.0, 104.4, 84.5−82.9, 70.8, 56.3, 51.4, 39.0,

31.5, 30.0 (3C), 30.0, 24.8 (2C), 22.6. HRMS (ESI): calcd for

C₁₇H₃₀FN₃NaO₂[M + Na]⁺, 350.2220; found 350.2235. mp: 161−163

°C. HPLC purity = 97.1%; t_R= 9.5 min.

5-(tert-Butyl)-1-(cyclopropylmethyl)-N-(1-hydroxy-2-methylpro-

pan-2-yl)-1H-pyrazole-3-carboxamide (51). The title compound was

prepared by the same procedure described for 26 except using

(bromomethyl)cyclopropane instead of 1-bromopentane as a white

solid. ¹H NMR (400 MHz, CDCl₃) δ 6.94 (s, 1H), 6.54 (s, 1H), 4.05

(d, J = 6.8 Hz, 2H), 3.67 (s, 2H), 1.38 (s, 6H), 1.37 (s, 9H), 1.29−1.27

(m, 1H), 0.66−0.58 (m, 2H), 0.48−0.40 (m, 2H). ¹³C NMR (101

MHz, CDCl₃) δ 163.2, 153.2, 144.4, 104.0, 71.0, 56.1, 55.7, 31.5, 30.2

(3C), 25.0 (2C), 11.6, 4.1 (2C). HRMS (ESI): calcd for

C₁₆H₂₇N₃NaO₂[M + Na]⁺, 316.2001. found 316.1985. HPLC purity

= 95.6%; t_R= 9.5 min.

4-Amino-5-(tert-butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-

pentyl-1H-pyrazole-3-carboxamide (44). To a solution of 43 (100

mg, 0.3 mmol) in MeOH (10 mL) and 3N HCl (10 mL) was slowly

added zinc powder (1.961 g, 30.0 mmol) with ice cooling. The mixture

was stirred at rt for 1 h. The resulting mixture was ﬁltered through a pad

of diatomaceous earth and washed with MeOH. The ﬁltrate was

concentrated under vacuum, and the crude mixture was extracted with

EtOAc. The combined organic layers were washed with brine, dried

over anhydrous Na₂SO₄, and concentrated. The residue was puriﬁed by

ﬂash chromatography on silica gel to give the title compound 44 (41

mg, 42.8%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 6.80 (s,

1H), 4.12−3.99 (m, 2H), 3.65 (s, 2H), 1.94−1.78 (m, 2H), 1.45 (s,

9H), 1.41−1.31 (m, 10H), 0.92 (t, J = 6.9 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 165.3, 134.8, 131.6, 128.6, 71.1, 56.0, 52.4, 32.9, 30.5,

30.4 (3C), 28.9, 25.0 (2C), 22.4, 14.0. HRMS (ESI): calcd for

C₁₇H₃₂N₄NaO₂[M + Na]⁺, 347.2423; found 347.2404. HPLC purity =

97.8%; t_R= 5.3 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-propyl-1H-

pyrazole-3-carboxamide (45). The title compound was prepared by

the same procedure described for 26 except using 1-bromopropane

1

instead of 1-bromopentane as a white solid. H NMR (500 MHz,

CDCl₃) δ 6.92 (s, 1H), 6.52 (s, 1H), 4.11−4.08 (m, 2H), 3.67 (s, 2H),

1.98−1.91 (m, 2H), 1.38 (s, 6H), 1.36 (s, 9H), 0.99 (t, J = 7.4 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ 163.2, 153.3, 144.3, 103.9, 71.0, 56.2,

53.1, 31.4, 30.1 (3C), 25.0 (2C), 24.0, 11.2. HRMS (ESI): calcd for

C₁₅H₂₇N₃NaO₂[M + Na]⁺, 304.2001; found 304.1987. mp: 58−60 °C.

HPLC purity = 96.9%; t_R= 9.5 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-(2-methox-

yethyl)-1H-pyrazole-3-carboxamide (52). The title compound was

prepared by the same procedure described for 26 except using 1-

1

bromo-2-methoxyethane instead of 1-bromopentane. H NMR (500

MHz, CDCl₃) δ 6.84 (s, 1H), 6.38 (s, 1H), 4.54 (t, J = 5.5 Hz, 2H), 3.78

(t, J = 4.9 Hz, 2H), 3.69 (s, 2H), 3.33 (s, 3H), 1.37 (s, 6H), 1.29 (s, 9H).

¹³C NMR (126 MHz, CDCl₃) δ 163.0, 154.1, 144.8, 104.1, 71.0, 59.2,

56.1, 50.8, 38.6, 31.4, 30.0 (3C), 24.9 (2C). HRMS (ESI): calcd for

C₁₅H₂₇N₃NaO₃[M + Na]⁺, 320.1950; found 320.1959. mp: 64−66 °C.

HPLC purity = 96.5%; t_R= 9.2 min.

5-(tert-Butyl)-1-butyl-N-(1-hydroxy-2-methylpropan-2-yl)-1H-

pyrazole-3-carboxamide (46). The title compound was prepared by

the same procedure described for 26 except using 1-bromobutane

1

instead of 1-bromopentane as a white solid. H NMR (500 MHz,

CDCl₃) δ 6.91 (s, 1H), 6.52 (s, 1H), 4.15−4.12 (m, 2H), 3.67 (s, 2H),

1.92−1.88 (m, 2H), 1.46−1.41 (m, 2H), 1.38 (s, 6H), 1.37 (s, 9H),

0.99 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.2, 153.2,

144.4, 103.9, 71.0, 56.1, 51.4, 32.8, 31.4 (3C), 30.1 (2C), 25.0, 20.1,

13.8. HRMS (ESI): calcd for C₁₆H₂₉N₃NaO₂[M + Na]⁺, 318.2157;

found 318.2135. mp: 81−83 °C. HPLC purity = 97.2%; t_R= 9.3 min.

5-(tert-Butyl)-1-hexyl-N-(1-hydroxy-2-methylpropan-2-yl)-1H-

pyrazole-3-carboxamide (47). The title compound was prepared by

the same procedure described for 26 except using 1-bromohexane

General Synthetic Procedure of N-Aryl-Substituted Pyra-

zoles. Ethyl 5-(tert-Butyl)-1-phenyl-1H-pyrazole-3-carboxylate

(53a). To a solution of 3,3-dimethyl-2-butanone (1002 mg, 10.0

mmol) in anhydrous THF (250 mL) was added t-BuOK (1459 mg,

13.0 mmol) with ice cooling. After stirring for 10 min, diethyl oxalate

(1.36 mL, 10.0 mmol) was added. The reaction mixture was reﬂuxed for

5 h. Then, phenylhydrazine hydrochloride (1591 mg, 11.0 mmol) was

added dropwise to the mixture, followed by the addition of acetic acid

(1.5 mL). The mixture was stirred at 60 °C for 3 h. Then, the reaction

was concentrated under vacuum and the crude mixture was extracted

with EtOAc. The combined organic layers were washed with brine,

dried over anhydrous Na₂SO₄, and concentrated. The residue was

puriﬁed by ﬂash chromatography on silica gel to give the title

compound 53a (2.350 g, 86.2%). ¹H NMR (400 MHz, CDCl₃) δ 7.48−

7.35 (m, 5H), 6.74 (s, 1H), 4.39 (d, J = 6.8 Hz, 2H), 1.37 (t, J = 6.6 Hz,

3H), 1.18 (s, 9H). LRMS (ESI): calcd for C₁₆H₂₀N₂O₂, 272.15; found

272.

1

instead of 1-bromopentane as a white solid. H NMR (500 MHz,

CDCl₃) δ 6.95 (s, 1H), 6.52 (s, 1H), 4.15−4.12 (m, 2H), 3.67 (s, 2H),

1.94−1.90 (m, 2H), 1.43−1.39 (s, 8H), 1.36 (s, 9H), 1.34−1.33 (m,

4H), 0.91 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.1,

153.2, 144.3, 104.0, 71.1, 56.2, 51.6, 31.4, 31.4, 30.7, 30.1 (3C), 26.5,

25.0 (2C), 22.5, 14.0. HRMS (ESI): calcd for C₁₈H₃₃N₃NaO₂[M +

Na]⁺, 346.2470; found 346.2466. mp: 88−90 °C. HPLC purity =

96.0%; t_R= 10.3 min.

5-(tert-Butyl)-1-(3-ﬂuoropropyl)-N-(1-hydroxy-2-methylpropan-

2-yl)-1H-pyrazole-3-carboxamide (48). The title compound was

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5-(tert-Butyl)-1-phenyl-N-(1-hydroxy-2-methylpropan-2-yl)-1H-

pyrazole-3-carboxamide (54). A solution of 53a (254 mg, 1.1 mmol)

and LiOH·H₂O (240 mg, 5.7 mmol) in 10 mL of MeOH:THF:H₂O =

2:2:1 was stirred at rt for 1 h. Then, the reaction was concentrated under

vacuum and acidiﬁed to pH 4−5 with 1 M aqueous HCl. The mixture

was extracted with EtOAc, and the combined organic layers were

washed with brine, dried over anhydrous Na₂SO₄, and concentrated to

aﬀord the crude compound 5-(tert-butyl)-1-phenyl-1H-pyrazole-3-

carboxylic acid. To a solution of 5-(tert-butyl)-1-phenyl-1H-pyrazole-3-

carboxylic acid (215 mg, 1.1 mmol) and 2-amino-2-methylpropan-1-ol

(0.11 mL, 1.1 mmol) in CH₂Cl₂(10 mL) was added HATU (521 mg,

1.4 mmol), followed by DIPEA (0.40 mL, 2.3 mmol). The mixture was

stirred at rt for 5 h. Then, the reaction was concentrated under vacuum

and the crude mixture was extracted with EtOAc. The combined

organic layers were washed with brine, dried over anhydrous Na₂SO₄,

and concentrated. The residue was puriﬁed by ﬂash chromatography on

silica gel to give the title compound 54 (253 mg, 72.8%) as a white solid.

¹H NMR (500 MHz, CDCl₃) δ 7.55−7.49 (m, 3H), 7.39 (d, J = 7.1 Hz,

prepared by the same procedure described for 54 except using (4-

methoxyphenyl)hydrazine hydrochloride instead of phenylhydrazine

hydrochloride as a yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 7.31−

7.30 (m, 2H), 7.03−6.97 (m, 2H), 6.92 (s, 1H), 6.71 (s, 1H), 3.88 (s,

3H), 3.67 (s, 2H), 1.35 (s, 6H), 1.18 (s, 9H). ¹³C NMR (151 MHz,

CDCl₃) δ 163.0, 160.4, 155.7, 145.3, 134.2, 129.6 (2C), 113.9 (2C),

104.6, 70.8, 56.3, 55.6, 32.1, 30.6 (3C), 24.9 (2C). HRMS (ESI): calcd

for C₁₉H₂₈N₃O₃[M + H]⁺, 346.2131; found 346.2156. mp: 207−210

°C. HPLC purity = 97.7%; t_R= 8.4 min.

5-(tert-Butyl)-1-(4-(triﬂuoromethoxy)phenyl)-N-(1-hydroxy-2-

methylpropan-2-yl)-1H-pyrazole-3-carboxamide (60). The title

compound was prepared by the same procedure described for 54

except using 4-((triﬂuoromethoxy)phenyl)hydrazine hydrochloride

1

instead of phenylhydrazine hydrochloride as a yellow solid. H NMR

(500 MHz, CDCl₃) δ 7.43−7.37 (m, 2H), 7.34−7.32 (m, 2H), 6.87 (s,

1H), 6.74 (s, 1H), 5.03 (s, 1H), 3.68 (s, 2H), 1.36 (s, 6H), 1.19 (s, 9H).

¹³C NMR (151 MHz, CDCl₃) δ 162.7, 155.8, 149.9, 145.9, 139.9, 130.2

(2C), 121.1 (2C), 105.0 (2C), 70.8, 56.4, 32.2, 30.6(3C), 24.9 (2C).

HRMS (ESI): calcd for C₁₉H₂₅F₃N₃O₃[M + H]⁺, 400.1848; found

400.1874. mp: 196−198 °C. HPLC purity = 98.1%; t_R= 8.6 min.

5-(tert-Butyl)-1-(2,4-diﬂuorophenyl)-N-(1-hydroxy-2-methylpro-

pan-2-yl)-1H-pyrazole-3-carboxamide (61). The title compound was

prepared by the same procedure described for 54 except using 4-(2,4-

diﬂuorophenyl)hydrazine hydrochloride instead of phenylhydrazine

hydrochloride as a yellow solid. ¹H NMR (600 MHz, CDCl₃) δ 7.43−

7.39 (m, 1H), 7.04−6.97 (m, 2H), 6.86 (s, 1H), 6.70 (s, 1H), 4.96 (s,

1H), 3.66 (s, 2H), 1.35 (s, 6H), 1.18 (s, 9H). ¹³C NMR (151 MHz,

CDCl₃) δ 162.6, 156.5, 146.7, 131.7, 131.6, 111.6, 111.5, 105.3, 105.1,

105.0, 70.8, 56.3, 32.0, 30.0 (3C), 24.9 (2C). HRMS (ESI): calcd for

C₁₈H₂₄F₂N₃O₂[M + H]⁺, 352.1837; found 352.1838. mp: 247−249

°C. HPLC purity = 98.1%; t_R= 8.3 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-(pyridin-2-

yl)-1H-pyrazole-3-carboxamide (62). The title compound was

prepared by the same procedure described for 54 except using 2-

hydrazinylpyridine hydrochloride instead of phenylhydrazine hydro-

chloride as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.59 (s, 1H),

7.95−7.88 (m, 1H), 7.52−7.41 (m, 2H), 6.96 (s, 1H), 6.76 (s, 1H),

3.66 (s, 2H), 1.35 (s, 6H), 1.25 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ

162.8, 155.9, 154.0, 148.3, 146.2, 138.6, 124.4, 122.1, 105.5, 70.8, 56.3,

32.5, 30.5 (3C), 24.9 (2C). HRMS (ESI): calcd for C₁₇H₂₄N₄NaO₂[M

+ Na]⁺, 339.1797; found 339.1765. mp: 138−140 °C. HPLC purity =

98.9%; t_R= 9.2 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-(pyridin-3-

yl)-1H-pyrazole-3-carboxamide (63). The title compound was

prepared by the same procedure described for 54 except using 3-

hydrazinylpyridine hydrochloride instead of phenylhydrazine hydro-

chloride as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.78 (s, 1H),

8.68 (s, 1H), 7.77 (d, J = 7.3 Hz, 1H), 7.51 (d, J = 6.6 Hz, 1H), 6.88 (s,

1H), 6.77 (s, 1H), 3.68 (s, 2H), 1.36 (s, 6H), 1.19 (s, 9H). ¹³C NMR

(101 MHz, CDCl₃) δ 162.6, 156.4, 150.3, 148.9, 146.4, 138.4, 136.3,

123.7, 105.2, 70.7, 56.3, 32.2, 30.7 (3C), 24.8 (2C). HRMS (ESI): calcd

for C₁₇H₂₄N₄NaO₂[M + Na]⁺, 339.1797; found 339.1765. mp: 108−

110 °C. HPLC purity = 97.8%; t_R= 9.0 min.

5-(tert-Butyl)-1-(5-ﬂuoropyridin-2-yl)-N-(1-hydroxy-2-methylpro-

pan-2-yl)-1H-pyrazole-3-carboxamide (64). The title compound was

prepared by the same procedure described for 54 except using 5-ﬂuoro-

2-hydrazinylpyridine hydrochloride instead of phenylhydrazine hydro-

chloride as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.42 (s, 1H),

7.64−7.59 (m, 1H), 7.53−7.48 (m, 1H), 6.93 (s, 1H), 6.76 (s, 1H),

3.67 (s, 2H), 1.36 (s, 6H), 1.25 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ

162.7, 160.4, 158.3, 156.1, 146.3, 136.2, 125.5, 123.4, 105.6, 70.8, 56.3,

32.5, 30.4 (3C), 24.9 (2C). HRMS (ESI): calcd for C₁₇H₂₃FN₄NaO₂

[M + Na]⁺, 357.1703; found 357.1670. mp: 117−119 °C. HPLC purity

= 99.2%; t_R= 3.3 min.

2H), 6.97 (s, 1H), 6.74 (s, 1H), 3.67 (s, 2H), 1.35 (s, 6H), 1.18 (s, 9H).

¹³C NMR (126 MHz, CDCl₃) δ 163.0, 155.6, 145.4, 141.5, 129.8, 128.9

(2C), 128.5 (2C), 104.7, 70.9, 56.3, 32.2, 30.6 (3C), 24.9 (2C). HRMS

(ESI): calcd for C₁₈H₂₅N₃NaO₂[M + Na]⁺, 338.1844; found 338.1833.

mp: 180−182 °C. HPLC purity = 98.1%; t_R= 3.9 min.

5-(tert-Butyl)-1-(p-tolyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1H-

pyrazole-3-carboxamide (55). The title compound was prepared by

the same procedure described for 54 except using 4-methylphenylhy-

drazine hydrochloride instead of phenylhydrazine hydrochloride as a

1

light yellow solid. H NMR (500 MHz, DMSO-d₆) δ 7.35−7.31 (m,

4H), 7.09 (s, 1H), 6.55 (s, 1H), 5.00 (t, J = 5.6 Hz, 1H), 3.38 (d, J = 5.6

Hz, 2H), 2.41 (s, 3H), 1.29 (s, 6H), 1.13 (s, 9H). ¹³C NMR (151 MHz,

CDCl₃) δ 163.1, 155.6, 145.4, 139.9, 139.0, 129.4 (2C), 128.23 (2C),

104.6, 70.9, 56.3, 32.2, 30.6 (3C), 24.9 (2C), 21.3. HRMS (ESI): calcd

for C₁₉H₂₈N₃O₂[M + H]⁺, 330.2182; found 330.2180. mp: 180−182

°C. HPLC purity = 96.0%; t_R= 8.5 min.

5-(tert-Butyl)-1-(4-ﬂuorophenyl)-N-(1-hydroxy-2-methylpropan-

2-yl)-1H-pyrazole-3-carboxamide (56). The title compound was

prepared by the same procedure described for 54 except using (4-

ﬂuorophenyl)hydrazine hydrochloride instead of phenylhydrazine

hydrochloride as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 7.37−

7.34 (m, 2H), 7.21−7.15 (m, 2H), 6.88 (s, 1H), 6.72 (s, 1H), 5.08 (s,

1H), 3.67 (s, 2H), 1.35 (s, 6H), 1.18 (s, 9H). ¹³C NMR (151 MHz,

CDCl₃) δ 163.8, 162.8, 162.2, 155.8, 145.7, 137.6, 130.4, 130.4, 115.9,

115.8, 104.9, 70.8, 56.3, 32.2, 30.6 (3C), 24.9 (2C). HRMS (ESI): calcd

for C₁₈H₂₅FN₃O₂[M + H]⁺, 334.1931; found 334.1927. mp: 181−184

°C. HPLC purity = 99.2%; t_R= 8.4 min.

5-(tert-Butyl)-1-(4-chlorophenyl)-N-(1-hydroxy-2-methylpropan-

2-yl)-1H-pyrazole-3-carboxamide (57). The title compound was

prepared by the same procedure described for 54 except using 4-

chlorophenylhydrazine hydrochloride instead of phenylhydrazine

1

hydrochloride as a yellow solid. H NMR (500 MHz, CDCl₃) δ 7.47

(d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.6 Hz, 2H), 6.88 (s, 1H), 6.73 (s, 1H),

5.05 (s, 1H), 3.67 (s, 2H), 1.35 (s, 6H), 1.18 (s, 9H). ¹³C NMR (151

MHz, CDCl₃) δ 162.8, 155.8, 145.8, 140.1, 135.8, 129.9 (2C), 129.1

(2C), 105.0, 70.8, 56.3, 32.2, 30.6 (3C), 24.9 (2C). HRMS (ESI): calcd

for C₁₈H₂₅ClN₃O₂[M + H]⁺, 350.1635; found 350.1634. mp: 245−248

°C. HPLC purity = 95.2%; t_R= 8.6 min.

5-(tert-Butyl)-1-(4-(triﬂuoromethyl)phenyl)-N-(1-hydroxy-2-

methylpropan-2-yl)-1H-pyrazole-3-carboxamide (58). The title

compound was prepared by the same procedure described for 54

except using 4-(triﬂuoromethyl)phenylhydrazine hydrochloride in-

1

stead of phenylhydrazine hydrochloride as a yellow solid. H NMR

(500 MHz, DMSO-d₆) δ 7.93 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 8.2 Hz,

2H), 7.15 (s, 1H), 6.62 (s, 1H), 5.05 (t, J = 5.5 Hz, 1H), 3.37 (d, J = 5.5

Hz, 2H), 1.29 (s, 6H), 1.14 (s, 9H). ¹³C NMR (151 MHz, CDCl₃) δ

162.6, 155.9, 146.1, 144.6, 132.1, 131.9, 129.1 (2C), 126.1, 126.1,

105.2, 70.8, 56.3, 32.2, 30.7 (3C), 24.9 (2C). HRMS (ESI): calcd for

C₁₉H₂₅F₃N₃O₂[M + H]⁺, 384.1899; found 384.1900. mp: 247−249

°C. HPLC purity = 97.1%; t_R= 8.5 min.

5-(tert-Butyl)-1-(5-chloropyridin-2-yl)-N-(1-hydroxy-2-methyl-

propan-2-yl)-1H-pyrazole-3-carboxamide (65). The title compound

was prepared by the same procedure described for 54 except using 5-

chloro-2-hydrazinylpyridine hydrochloride instead of phenylhydrazine

hydrochloride as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.40

(s, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.96 (s, 1H), 7.72 (d, J = 8.7 Hz, 1H),

5-(tert-Butyl)-1-(4-methoxyphenyl)-N-(1-hydroxy-2-methylpro-

pan-2-yl)-1H-pyrazole-3-carboxamide (59). The title compound was

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6.65 (s, 1H), 4.71 (t, J = 6.0 Hz, 1H), 3.44 (d, J = 6.0 Hz, 2H), 1.30 (s,

9H), 1.25 (s, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 163.3, 161.4, 150.5,

145.2, 139.4, 138.9, 129.8, 117.8, 108.8, 69.1, 56.7, 32.4, 30.1 (3C), 24.5

(2C). HRMS (ESI): calcd for C₁₇H₂₃ClN₄NaO₂[M + Na]⁺, 373.1407;

found 373.1404. mp: 156−158 °C. HPLC purity = 96.1%; t_R= 4.5 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-(5-

(triﬂuoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide (66).

The title compound was prepared by the same procedure described

for 54 except using 2-hydrazinyl-5-(triﬂuoromethyl)pyridine hydro-

chloride instead of phenylhydrazine hydrochloride as a white solid. ¹H

NMR (400 MHz, CDCl₃) δ 8.84 (s, 1H), 8.13 (d, J = 8.3 Hz, 1H), 7.74

(d, J = 8.4 Hz, 1H), 6.92 (s, 1H), 6.81 (s, 1H), 4.93 (s, 1H), 3.68 (s,

2H), 1.37 (s, 6H), 1.33 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 162.4,

156.6, 147.1, 145.1, 136.0, 126.6, 124.5, 121.7, 121.0, 106.7, 70.6, 56.3,

32.8, 30.4 (3C), 24.8 (2C). HRMS (ESI): calcd for C₁₈H₂₃F₃N₄NaO₂

[M + Na]⁺, 407.1671; found 407.1640. mp: 136−138 °C. HPLC purity

= 97.6%; t_R= 5.9 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-(3-chloro-5-

(triﬂuoromethyl)pyridin-2-yl)-1H-pyrazole-3-carboxamide (67).

The title compound was prepared by the same procedure described

for 54 except using 2-hydrazinyl-3- chloro -5-(triﬂuoromethyl)pyridine

hydrochloride instead of phenylhydrazine hydrochloride as a white

solid. ¹H NMR (500 MHz, CDCl₃) δ 8.80 (s, 1H), 8.19 (s, 1H), 6.87 (s,

1H), 6.79 (s, 1H), 3.68 (s, 2H), 1.35 (s, 6H), 1.21 (s, 9H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 160.8, 155.7, 153.4, 148.4, 144.8, 138.7, 130.8,

123.8, 121.7, 104.9, 68.4, 54.6, 32.2, 30.1 (3C), 23.6 (2C). HRMS

(ESI): calcd for C₁₈H₂₃ClF₃N₄O₂[M + H]⁺, 419.1462; found

419.1460. mp: 199−201 °C. HPLC purity = 97.8%; t_R= 8.5 min.

5-(tert-Butyl)-N-(1-hydroxy-2-methylpropan-2-yl)-1-(pyrimidin-

2-yl)-1H-pyrazole-3-carboxamide (68). The title compound was

prepared by the same procedure described for 54 except using 2-

hydrazinylpyrimidine hydrochloride instead of phenylhydrazine hydro-

chloride as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.95−8.88 (m,

2H), 7.50−7.44 (m, 1H), 7.04 (s, 1H), 6.79 (s, 1H), 3.66 (s, 2H), 1.36

(s, 6H), 1.29 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 161.1, 160.0

(2C), 158.8, 155.2, 147.3, 122.8, 104.9, 68.5, 54.6, 32.4, 30.5 (3C), 23.7

(2C). HRMS (ESI): calcd for C₁₆H₂₄N₅O₂[M + H]⁺, 318.1930; found

318.1927. mp: 111−113 °C. HPLC purity = 97.4%; t_R= 7.9 min.

Calcium Mobilization Assay. Calcium mobilization assays were

performed as previously described,⁵⁷and the EC₅₀or IC₅₀values were

calculated using GraphPad Prism 7 software (GraphPad).

cAMP HTRF Assay. cAMP HTRF assays were performed using a

Cisbio HTRF dynamic 2 cAMP kit (Gif-sur-Yvette, France) according

to the manufacturer’s instructions.⁵⁰HEK293-CB2 cells were

resuspended in HBSS buﬀer (containing 10 mM HEPES, 500 μM 3-

isobutyl-1-methylxanthine) with a ﬁnal cell density of 1 × 10⁶cells/mL.

Cell suspension (5 μL, 5000 cells/well) loaded into shallow 384-well

plates was mixed with 5 μL of cAMP assay buﬀer containing the

indicated concentrations of compounds and forskolin (3 μM) for 15

min at rt. Then, detection buﬀer consisting of cAMP-d2 (5 μL) and

anti-cAMP-cryptate (5 μL) was added to terminate the reaction. Upon

2 h incubation at rt away from light, HTRF signals were recorded using

a Cytation 5 Imaging Reader (BioTek, Winooski, Vermont).

β-Arrestin Recruitment Assay. HEK293 cells were transfected

with CB1/CB2-tTA fusion expression vector, a β-arrestin2-TEV fusion

gene, and a tTA-dependent luciferase reporter using Lipofectamine

2000. Then, the transfected cells were seeded into 96-well white clear-

bottom cell culture plates with a density of 25 000 cells per well. After

24 h, 20 μL of designed drug solutions was prepared and added to each

well. Next day, luciferase activities were determined by adding Bright-

Glo solution (Promega) and detected with a Cytation 5 Imaging Reader

(BioTek, Winooski, Vermont). GraphPad Prism was used for data

analysis.

Signaling Technology), and anti-GAPDH (1:10000; Sigma-Aldrich)

were used.

Human Liver Microsome Assay. Human liver microsome assay

was conducted by Wuxi AppTec Co. Ltd. The microsomes (Cat No.

452117) (0.5 mg/mL) were preincubated in 100 mM potassium

phosphate buﬀer (pH = 7.4) with 1 μM tested compounds, 1 μM

NADPH, and 10 mM MgCl₂for 5 min at 37 °C. The reactions were

initiated by the addition of 1 mM NADPH. After 0, 5, 10, 30, and 60

min incubations at 37 °C, the mixtures were quenched by adding cold

CH₃CN with internal standard. Then, the samples were centrifuged at

4000 rpm for 20 min under 4 °C. Supernatants were used for analysis

using a developed LC-MS/MS method.

PK Study. 18 BALB/c female mice, weighing between 20 and 24 g,

were randomly divided into two groups. Compound 66 was

intravenously or orally injected at 1 or 5 mg/kg. Plasma samples were

collected at the following time points: 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24

h upon administration (n = 3/group). Biological data collection and

analysis of 66 were performed by a developed LC-MS/MS method. The

PK parameters were then calculated using a nonatrioventricular model

(Phoenix WinNonlin 7.0, Pharsight).

BLM-Induced SSc Model. The BLM-induced SSc model was

developed as previously described.^28,50Brieﬂy, female Balb/c mice (6−

8 weeks old) were obtained from the Experimental Animal Centre of

the East China Normal University (ECNU) and fed in a sterile

environment at 20 °C with a 12 h light/dark cycle. All of the animal

experiments were approved by the Animal Experiments Committee of

the ECNU(AR2013/06002). BLM in phosphate-buﬀered saline (PBS,

100 μL, 1 mg/mL) was subcutaneously injected in the shaved upper

back of the mice once daily for 2 weeks. The mice were next randomly

divided into ﬁve groups including negative controls (PBS only), BLM

plus vehicle (BLM + PBS), BLM plus 9 (BLM + 9), and BLM plus 66

(BLM + 66, 1 or 5 mg/kg). Compounds 66 and 9 were dissolved in PBS

with 0.3% Tween 80 and 0.1% DMSO, and subcutaneously injected in

the shaved upper back once daily for 4 weeks. Finally, the mice were

euthanized using CO₂asphyxiation and the treated skin was extracted

for further histological analysis.

RNA Isolation and Quantitative Real-Time PCR Assay. The

total RNA of the mouse skin was isolated using RNAiso Plus (TaKaRa

Biotechnology, Dalian, China). Reverse-transcription PCR was next

performed using PrimeScript RT Master Mix (TaKaRa Biotechnology,

Dalian, China). Quantitative real-time PCR (QPCR) analysis was

performed using Applied Biosystems QuantStudio 3 Real-Time PCR

Systems (Applied Biosystems, Rotkreuz, Switzerland). QPCR primers

are listed in Table S1.

Docking Stimulations. Molecular docking studies between

compound 40, 66, and CB2 receptor were performed using

59

̈

Schrodinger Glide software. The X-ray crystal structure of human

CB2 co-crystallized with antagonist AM10257 (PDB code: 5ZTY)⁵³

and Cryo-EM structure of human CB2-G_iprotein in complex with

agonist WIN 55,212−2 (PDB code: 6PT0)⁵²were downloaded from

the Protein Data Bank. The co-crystallized ligands were used to help

deﬁne the active sites for docking studies. LigPrep was used to prepare

high-quality,⁶⁰all-atom three-dimensional (3D) structures of com-

pounds 40 and 66. Receptor grids were generated using Receptor Grid

Generation in the Glide application of Maestro, and ligands were

docked to the CB2 receptor using Glide version 5.8 docking protocol.

The docking conformations with the lowest binding energy were

selected for further analysis.

Statistical Data Analysis. Data were shown as mean

SD.

Datasets consisting of more than two groups were assessed using one-

way ANOVA followed by Tukeyʼs multiple comparison test. Statistical

comparisons between two groups were analyzed using a two-tailed

Student’s t-test. Dose−response curve ﬁtting and all of the statistical

analyses were performed using GraphPad Prism 7 software (Graph-

Pad). Statistical signiﬁcance was deﬁned as *P < 0.05, **P < 0.01, and

***P < 0.001.

ERK1/2 Phosphorylation Assay. Cells were starved with serum-

free medium for 2 h and treated with indicated compounds for 15 min.

Then, the treated cells were washed and harvested in RIPA lysis buﬀer

with protease and phosphatase inhibitors (Roche; San Francisco, CA).

Western blotting was conducted as previously described.^28,58The

primary anti-ERK1/2 antibody (1:1000; Cell Signaling Technology),

anti-phospho-ERK1/2 antibody (Thr202/Tyr204) (1:1000; Cell

400

https://dx.doi.org/10.1021/acs.jmedchem.0c01023

J. Med. Chem. 2021, 64, 385−403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Si Chen − Drug Discovery Unit, Shanghai Key Laboratory of

Regulatory Biology, Institute of Biomedical Sciences and School

of Life Sciences, East China Normal University, Shanghai

200241, P. R. China

Chunyang Yi − Drug Discovery Unit, Shanghai Key Laboratory

of Regulatory Biology, Institute of Biomedical Sciences and

School of Life Sciences, East China Normal University,

Shanghai 200241, P. R. China

Xiaolei Chai − Drug Discovery Unit, Shanghai Key Laboratory

of Regulatory Biology, Institute of Biomedical Sciences and

School of Life Sciences, East China Normal University,

Shanghai 200241, P. R. China

Mingyao Liu − Drug Discovery Unit, Shanghai Key Laboratory

of Regulatory Biology, Institute of Biomedical Sciences and

School of Life Sciences, East China Normal University,

Shanghai 200241, P. R. China; orcid.org/0000-0001-

7339-5048

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01023.

■

sı

CB2 receptor antagonist AM630 inhibit calcium mobi-

lization induced by 66; primer sequences of QPCR; and

characterization of key intermediates by 2D HMBC and

¹H NMR experiments (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Li-Fang Yu − Shanghai Engineering Research Center of

Molecular Therapeutics and New Drug Development, School of

Chemistry and Molecular Engineering, East China Normal

University, Shanghai 200062, P. R. China; orcid.org/

0000-0002-3250-6773; Email: lfyu@sat.ecnu.edu.cn

Weiqiang Lu − Drug Discovery Unit, Shanghai Key Laboratory

of Regulatory Biology, Institute of Biomedical Sciences and

School of Life Sciences, East China Normal University,

Shanghai 200241, P. R. China; orcid.org/0000-0003-

1349-6597; Email: wqlu@bio.ecnu.edu.cn

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01023

Author Contributions

^⊥B.-E.J., X.J., and Q.Z. contributed equally to this work.

Notes

Han-Kun Zhang − Drug Discovery Unit, Shanghai Key

Laboratory of Regulatory Biology, Institute of Biomedical

Sciences and School of Life Sciences, East China Normal

University, Shanghai 200241, P. R. China; orcid.org/

0000-0002-5008-0825; Email: hkzhang@bio.ecnu.edu.cn

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors thank the National Key R&D Program of China

(2018YFA0507001), the National Natural Science Foundation

of China (82073706, 21977032, 81830083, and 81972828), the

Shanghai Committee of Science and Technology

(18431900100, 18431900500, and 19ZR1473500), the In-

novation Program of Shanghai Municipal Education Commis-

sion (2017−01−07−00−05-E00011), the Shenzhen Municipal

Government of China (KQTD2017081060226082), and the

ECNU Multifunctional Platform for Innovation (011). In

addition, they thank Dr. Stefan Siwko for his assistance in

proofreading the manuscript.

Authors

Bei-Er Jiang − Drug Discovery Unit, Shanghai Key Laboratory

of Regulatory Biology, Institute of Biomedical Sciences and

School of Life Sciences, East China Normal University,

Shanghai 200241, P. R. China; Navy Medical Research

Institute, Second Military Medical University, Shanghai

200433, P. R. China

Xingwu Jiang − Drug Discovery Unit, Shanghai Key Laboratory

of Regulatory Biology, Institute of Biomedical Sciences and

School of Life Sciences, East China Normal University,

Shanghai 200241, P. R. China; Department of Materials

Science, Fudan University, Shanghai 200433, P. R. China

Qiansen Zhang − Drug Discovery Unit, Shanghai Key

Laboratory of Regulatory Biology, Institute of Biomedical

Sciences and School of Life Sciences, East China Normal

University, Shanghai 200241, P. R. China; orcid.org/

0000-0002-8608-3067

Qiuwen Liang − Drug Discovery Unit, Shanghai Key

Laboratory of Regulatory Biology, Institute of Biomedical

Sciences and School of Life Sciences, East China Normal

University, Shanghai 200241, P. R. China

Zi-Liang Qiu − Drug Discovery Unit, Shanghai Key Laboratory

of Regulatory Biology, Institute of Biomedical Sciences and

School of Life Sciences, East China Normal University,

Shanghai 200241, P. R. China

ABBREVIATIONS

■

Δ⁹-THC, (−)-trans-Δ⁹-tetrahydrocannabinol; ECS, endocan-

nabinoid system; SSc, systemic sclerosis; BLM, bleomycin; SAR,

structure−activity relationship; PK, pharmacokinetic; t-BuOK,

potassium tert-butoxide; rt, room temperature; AcOH, acetic

acid; HATU, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetrame-

thyluronium hexaﬂuoro; DIPEA, N,N-diisopropylethylamine;

NBS, N,N-diisopropylethylamine; NCS, N-chlorosuccinimide;

TFAA, triﬂuoroacetic anhydride; cAMP, cyclic adenosine

monophosphate; ERK, extracellular signal-regulated kinase;

EGF, epidermal growth factor; α-SMA, α-smooth muscle actin;

mp, melting point

REFERENCES

■

(1) Pertwee, R. G. Cannabinoid pharmacology: the first 66 years. Br. J.

Pharmacol. 2006, 147, S163−S171.

Xiang-Bai Sun − Drug Discovery Unit, Shanghai Key

Laboratory of Regulatory Biology, Institute of Biomedical

Sciences and School of Life Sciences, East China Normal

University, Shanghai 200241, P. R. China

Jun-Jie Yang − Drug Discovery Unit, Shanghai Key Laboratory

of Regulatory Biology, Institute of Biomedical Sciences and

School of Life Sciences, East China Normal University,

Shanghai 200241, P. R. China; orcid.org/0000-0003-

3997-1851

(2) Pertwee, R. G. The pharmacology of cannabinoid receptors and

their ligands: an overview. Int. J. Obes. 2006, 30, S13−S18.

(3) Gaoni, Y.; Mechoulam, R. Isolation, structure, and partial

synthesis of an active constituent of hashish. J. Am. Chem. Soc. 1964,

86, 1646−1647.

(4) De Petrocellis, L.; Di Marzo, V. An introduction to the

endocannabinoid system: from the early to the latest concepts. Best

Pract. Res., Clin. Endocrinol. Metab. 2009, 23, 1−15.

401

https://dx.doi.org/10.1021/acs.jmedchem.0c01023

J. Med. Chem. 2021, 64, 385−403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(5) Brindisi, M.; Maramai, S.; Gemma, S.; Brogi, S.; Grillo, A.;

Mannelli, L. D. C.; Gabellieri, E.; Lamponi, S.; Saponara, S.; Gorelli, B.;

et al. Development and pharmacological characterization of selective

blockers of 2-arachidonoyl glycerol degradation with efficacy in rodent

models of multiple sclerosis and pain. J. Med. Chem. 2016, 59, 2612−

2632.

(6) Pertwee, R. G.; Howlett, A. C.; Abood, M. E.; Alexander, S. P. H.;

Marzo, V. D.; Elphick, M. R.; Greasley, P. J.; Hansen, H. S.; Kunos, G.;

Mackie, K.; et al. International union of basic and clinical

pharmacology. LXXIX. Cannabinoid receptors and their ligands:

Beyond CB1 and CB2. Pharmacol. Rev. 2010, 62, 588−631.

(7) Panagis, G.; Mackey, B.; Vlachou, S. Cannabinoid regulation of

brain reward processing with an emphasis on the role of CB1 receptors:

a step back into the future. Front. Psychiatry 2014, 5, 92.

(23) https://www.corbuspharma.com/ (accessed Sep 25, 2019).

(24) Beyer, C.; Schett, G.; Distler, O.; Distler, J. H. W. Animal models

of systemic sclerosis: Prospects and limitations. Arthritis Rheum. 2010,

62, 2831−2844.

(25) Varga, J.; Abraham, D. Systemic sclerosis: a prototypic

multisystem fibrotic disorder. J. Clin. Invest. 2007, 117, 557−567.

(26) Yazawa, N.; Fujimoto, M.; Tamaki, K. Recent advances on

pathogenesis and therapies in systemic sclerosis. Clin. Rev. Allergy.

Immunol. 2007, 33, 107−112.

(27) Denton, C. P. Advances in pathogenesis and treatment of

systemic sclerosis. Clin. Med. 2015, 15, s58−s63.

(28) Gonzalez, E. G.; Selvi, E.; Balistreri, E.; Akhmetshina, A.;

Palumbo, K.; Lorenzini, S.; Lazzerini, P. E.; Montilli, C.; Capecchi, P.

L.; Lucattelli, M.; Baldi, C.; Gianchecchi, E.; Galeazzi, M.; Pasini, F. L.;

Distler, J. H. Synthetic cannabinoid ajulemic acid exerts potent

antifibrotic effects in experimental models of systemic sclerosis. Ann.

Rheum. Dis. 2012, 71, 1545−1551.

(29) Akhmetshina, A.; Dees, C.; Busch, N.; Beer, J.; Sarter, K.;

Zwerina, J.; Zimmer, A.; Distler, O.; Schett, G.; Distler, J. H. The

cannabinoid receptor CB2 exerts antifibrotic effects in experimental

dermal fibrosis. Arthritis Rheum. 2009, 60, 1129−1136.

(30) Balistreri, E.; Garcia-Gonzalez, E.; Selvi, E.; Akhmetshina, A.;

Palumbo, K.; Lorenzini, S.; Maggio, R.; Lucattelli, M.; Galeazzi, M.;

Distler, J. W. The cannabinoid WIN55, 212-2 abrogates dermal fibrosis

in scleroderma bleomycin model. Ann. Rheum. Dis. 2011, 70, 695−699.

(31) https://www.americanpharmaceuticalreview.com/1315-News/

175177-FDA-Designates-Corbus-Pharmaceuticals-Resunab-an-

Orphan-Drug/ (accessed Sep 30, 2019).

(32) Banister, S. D.; Wilkinson, S. M.; Longworth, M.; Stuart, J.;

Apetz, N.; English, K.; Brooker, L.; Goebel, C.; Hibbs, D. E.; Glass, M.;

et al. The synthesis and pharmacological evaluation of adamantane-

derived indoles: cannabimimetic drugs of abuse. ACS Chem. Neurosci.

2013, 4, 1081−1092.

(33) Martinotti, G.; Santacroce, R.; Papanti, D.; Elgharably, Y.;

Prilutskaya, M.; Corazza, O. Synthetic cannabinoids: psychopharma-

cology, clinical aspects, psychotic onset. CNS Neurol. Disord.: Drug

Targets 2017, 16, 567−575.

(34) Tai, S.; Fantegrossi, W. E. Pharmacological and toxicological

eﬀects of synthetic cannabinoids and their metabolites. In Neuro-

pharmacology of New Psychoactive Substances (NPS); Current Topics in

Behavioral Neurosciences; Springer: Cham, 2016; Vol. 32, pp 249−

262.

(35) Basavarajappa, B. S.; Subbanna, S. Potential mechanisms

underlying the deleterious effects of synthetic cannabinoids found in

spice/K2 products. Brain Sci. 2019, 9, 14.

(36) Banister, S. D.; Stuart, J.; Kevin, R. C.; Edington, A.; Longworth,

M.; Wilkinson, S. M.; Beinat, C.; Buchanan, A. S.; Hibbs, D. E.; Glass,

M.; Connor, M.; McGregor, I. S.; Kassiou, M. Effects of bioisosteric

fluorine in synthetic cannabinoid designer drugs JWH-018, AM-2201,

UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-135. ACS Chem.

Neurosci. 2015, 6, 1445−1458.

(37) Banister, S. D.; Moir, M.; Stuart, J.; Kevin, R. C.; Wood, K. E.;

Longworth, M.; Wilkinson, S. M.; Beinat, C.; Buchanan, A. S.; Glass,

M.; Connor, M.; McGregor, I. S.; Kassiou, M. Pharmacology of indole

and indazole synthetic cannabinoid designer drugs AB-FUBINACA,

ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-

ADB-PINACA, ADBICA, and 5F-ADBICA. ACS Chem. Neurosci.

2015, 6, 1546−1559.

(38) Banister, S. D.; Longworth, M.; Kevin, R.; Sachdev, S.; Santiago,

M.; Stuart, J.; Mack, J. B.; Glass, M.; McGregor, I. S.; Connor, M.;

Kassiou, M. Pharmacology of valinate and tert-leucinate synthetic

cannabinoids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA,

MDMB-FUBINACA, MDMB-CHMICA, and their analogues. ACS

Chem. Neurosci. 2016, 7, 1241−1254.

(8) Farquhar-Smith, W. P.; Egertova, M.; Bradbury, E. J.; McMahon,

S. B.; Rice, A. S.; Elphick, M. R. Cannabinoid CB(1) receptor

expression in rat spinal cord. Mol. Cell. Neurosci. 2000, 15, 510−521.

(9) Blasio, A.; Iemolo, A.; Sabino, V.; Petrosino, S.; Steardo, L.; Rice,

K. C.; Orlando, P.; Iannotti, F. A.; Di Marzo, V.; Zorrilla, E. P.; Cottone,

P. Rimonabant precipitates anxiety in rats withdrawn from palatable

food: role of the central amygdala. Neuropsychopharmacology 2013, 38,

2498−2507.

(10) Jones, D. End of the line for cannabinoid receptor 1 as an anti-

obesity target? Nat. Rev. Drug Discovery 2008, 7, 961−962.

́

(11) Despres, J.-P.; Golay, A.; Sjostrom, L. Rimonabant in Obesity-

Lipids Study, G. Effects of rimonabant on metabolic risk factors in

overweight patients with dyslipidemia. N. Engl. J. Med. 2005, 353,

2121−2134.

(12) Hollinshead, S. P.; Tidwell, M. W.; Palmer, J.; Guidetti, R.;

Sanderson, A.; Johnson, M. P.; Chambers, M. G.; Oskins, J.; Stratford,

R.; Astles, P. C. Selective cannabinoid receptor type 2 (CB2) agonists:

optimization of a series of purines leading to the identification of a

clinical candidate for the treatment of osteoarthritic pain. J. Med. Chem.

2013, 56, 5722−5733.

(13) Cheng, Y.; Hitchcock, S. A. Targeting cannabinoid agonists for

inflammatory and neuropathic pain. Expert Opin. Invest. Drugs 2007, 16,

951−965.

́

(14) Benito, C.; Romero, J. P.; Tolon, R. M.; Clemente, D.; Docagne,

F.; Hillard, C. J.; Guaza, C.; Romero, J. Cannabinoid CB1 and CB2

receptors and fatty acid amide hydrolase are specific markers of plaque

cell subtypes in human multiple sclerosis. J. Neurosci. 2007, 27, 2396−

2402.

(15) Cassano, T.; Calcagnini, S.; Pace, L.; De Marco, F.; Romano, A.;

Gaetani, S. Cannabinoid receptor 2 signaling in neurodegenerative

disorders: from pathogenesis to a promising therapeutic target. Front.

Neurosci. 2017, 11, 30.

(16) Sarfaraz, S.; Afaq, F.; Adhami, V. M.; Mukhtar, H. Cannabinoid

receptor as a novel target for the treatment of prostate cancer. Cancer

Res. 2005, 65, 1635−1641.

(17) Idris, A. I. Cannabinoid receptors as target for treatment of

osteoporosis: a tale of two therapies. Curr. Neuropharmacol. 2010, 8,

243−253.

(18) McKallip, R. J.; Lombard, C.; Fisher, M.; Martin, B. R.; Ryu, S.;

Grant, S.; Nagarkatti, P. S.; Nagarkatti, M. Targeting CB2 cannabinoid

receptors as a novel therapy to treat malignant lymphoblastic disease.

Blood 2002, 100, 627−634.

(19) Thakur, G. A.; Tichkule, R.; Bajaj, S.; Makriyannis, A. Latest

advances in cannabinoid receptor agonists. Expert Opin. Ther. Pat.

2009, 19, 1647−1673.

(20) Ostenfeld, T.; Price, J.; Albanese, M.; Bullman, J.; Guillard, F.;

Meyer, I.; Leeson, R.; Costantin, C.; Ziviani, L.; Nocini, P. F.; Milleri, S.

A randomized, controlled study to investigate the analgesic efficacy of

single doses of the cannabinoid receptor-2 agonist GW842166,

ibuprofen or placebo in patients with acute pain following third

molar tooth extraction. Clin. J. Pain 2011, 27, 668−676.

(21) https://clinicaltrials.gov/ct2/show/NCT01319929 (accessed

Sep 20, 2019).

(39) Longworth, M.; Connor, M.; Banister, S. D.; Kassiou, M.

Synthesis and pharmacological profiling of the metabolites of synthetic

cannabinoid drugs APICA, STS-135, ADB-PINACA, and 5F-ADB-

PINACA. ACS Chem. Neurosci. 2017, 8, 1673−1680.

(22) https://clinicaltrials.gov/ct2/show/NCT00697710 (accessed

Sep 20, 2019).

402

https://dx.doi.org/10.1021/acs.jmedchem.0c01023

J. Med. Chem. 2021, 64, 385−403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(40) Shi, Y.; Duan, Y. H.; Ji, Y. Y.; Wang, Z. L.; Wu, Y. R.;

Gunosewoyo, H.; Xie, X. Y.; Chen, J. Z.; Yang, F.; Li, J.; Tang, J.; Xie, X.;

Yu, L. F. Amidoalkylindoles as potent and selective cannabinoid type 2

receptor agonists with in vivo efficacy in a mouse model of multiple

sclerosis. J. Med. Chem. 2017, 60, 7067−7083.

(41) Moir, M.; Lane, S.; Lai, F.; Connor, M.; Hibbs, D. E.; Kassiou, M.

Strategies to develop selective CB2 receptor agonists from indole

carboxamide synthetic cannabinoids. Eur. J. Med. Chem. 2019, 180,

291−309.

(42) Kevin, R. C.; Lefever, T. W.; Snyder, R. W.; Patel, P. R.; Fennell,

T. R.; Wiley, J. L.; McGregor, I. S.; Thomas, B. F. In vitro and in vivo

pharmacokinetics and metabolism of synthetic cannabinoids CUMYL-

PICA and 5F-CUMYL-PICA. Forensic Toxicol. 2017, 35, 333−347.

(43) Sobolevsky, T.; Prasolov, I.; Rodchenkov, G. Study on the phase I

metabolism of novel synthetic cannabinoids, APICA and its fluorinated

analogue. Drug Test. Anal. 2015, 7, 131−142.

(44) Gatley, S. J.; Lan, R.; Volkow, N. D.; Pappas, N.; King, P.; Wong,

C. T.; Gifford, A. N.; Pyatt, B.; Dewey, S. L.; Makriyannis, A. Imaging

the brain marijuana receptor: development of a radioligand that binds

to cannabinoid CB1 receptors in vivo. J. Neurochem. 1998, 70, 417−

423.

androgens on the periphery and their involvement in the pathogenesis

of common diseases (Review). Int. J. Mol. Med. 2016, 38, 1642−1651.

(55) Wetter, J. A.; Revankar, C.; Hanson, B. J. Utilization of the Tango

beta-arrestin recruitment technology for cell-based EDG receptor assay

development and interrogation. J. Biomol. Screening 2009, 14, 1134−

1141.

(56) Rajagopal, S.; Shenoy, S. K. GPCR desensitization: Acute and

prolonged phases. Cell Signalling 2018, 41, 9−16.

(57) Jiang, X. W.; Jiang, B. E.; Liu, H.; Liu, Z. T.; Hu, L. L.; Liu, M.; Lu,

W.; Zhang, H. K. Design, synthesis, and biological evaluations of

phenylpropiolic acid derivatives as novel GPR40 agonists. Eur. J. Med.

Chem. 2018, 158, 123−133.

(58) Jiang, X.; Lu, W.; Shen, X.; Wang, Q.; Lv, J.; Liu, M.; Cheng, F.;

Zhao, Z.; Pang, X. Repurposing sertraline sensitizes non-small cell lung

cancer cells to erlotinib by inducing autophagy. JCI Insight 2018, 3,

No. e98921.

(59) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic,

J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.;

Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: a new approach for rapid,

accurate docking and scoring. 1. Method and assessment of docking

accuracy. J. Med. Chem. 2004, 47, 1739−1749.

(60) Greenwood, J. R.; Calkins, D.; Sullivan, A. P.; Shelley, J. C.

Towards the comprehensive, rapid, and accurate prediction of the

favorable tautomeric states of drug-like molecules in aqueous solution.

J. Comput.-Aided Mol. Des. 2010, 24, 591−604.

(45) Francisco, M. E.; Seltzman, H. H.; Gilliam, A. F.; Mitchell, R. A.;

Rider, S. L.; Pertwee, R. G.; Stevenson, L. A.; Thomas, B. F. Synthesis

and structure-activity relationships of amide and hydrazide analogues of

the cannabinoid CB(1) receptor antagonist N-(piperidinyl)- 5-(4-

chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-car-

boxamide (SR141716). J. Med. Chem. 2002, 45, 2708−2719.

(46) Szabo, G.; Varga, B.; Payer-Lengyel, D.; Szemzo, A.; Erdelyi, P.;

Vukics, K.; Szikra, J.; Hegyi, E.; Vastag, M.; Kiss, B.; Laszy, J.; Gyertyan,

I.; Fischer, J. Chemical and biological investigation of cyclopropyl

containing diaryl-pyrazole-3-carboxamides as novel and potent

cannabinoid type 1 receptor antagonists. J. Med. Chem. 2009, 52,

4329−4337.

(47) Donohue, S. R.; Dannals, R. F.; Halldin, C.; Pike, V. W. N-(4-

cyanotetrahydro-2H-pyran-4-yl) and N-(1-cyanocyclohexyl) deriva-

tives of 1,5-diarylpyrazole-3-carboxamides showing high affinity for 18

kDa translocator protein and/or cannabinoid receptors. J. Med. Chem.

2011, 54, 2961−2970.

(48) Hwang, S. H.; Wagner, K. M.; Morisseau, C.; Liu, J. Y.; Dong, H.;

Wecksler, A. T.; Hammock, B. D. Synthesis and structure-activity

relationship studies of urea-containing pyrazoles as dual inhibitors of

cyclooxygenase-2 and soluble epoxide hydrolase. J. Med. Chem. 2011,

54, 3037−3050.

(49) Tyagarajan, S.; Chakravarty, P. K.; Zhou, B.; Taylor, B.; Fisher,

M. H.; Wyvratt, M. J.; Lyons, K.; Klatt, T.; Li, X.; Kumar, S.; Williams,

B.; Felix, J.; Priest, B. T.; Brochu, R. M.; Warren, V.; Smith, M.; Garcia,

M.; Kaczorowski, G. J.; Martin, W. J.; Abbadie, C.; McGowan, E.;

Jochnowitz, N.; Parsons, W. H. Substituted biaryl pyrazoles as sodium

channel blockers. Bioorg. Med. Chem. Lett. 2010, 20, 5480−5483.

(50) Jiang, X.; Chen, S.; Zhang, Q.; Yi, C.; He, J.; Ye, X.; Liu, M.; Lu,

W. Celastrol is a novel selective agonist of cannabinoid receptor 2 with

anti-inflammatory and anti-fibrotic activity in a mouse model of

systemic sclerosis. Phytomedicine 2020, 67, 153160.

(51) Pennington, L. D.; Moustakas, D. T. The Necessary nitrogen

atom: a versatile high-impact design element for multiparameter

optimization. J. Med. Chem. 2017, 60, 3552−3579.

(52) Xing, C.; Zhuang, Y.; Xu, T. H.; Feng, Z.; Zhou, X. E.; Chen, M.;

Wang, L.; Meng, X.; Xue, Y.; Wang, J.; Liu, H.; McGuire, T. F.; Zhao,

G.; Melcher, K.; Zhang, C.; Xu, H. E.; Xie, X. Q. Cryo-EM structure of

the human cannabinoid receptor CB2-Gi signaling complex. Cell 2020,

180, 645−654.

(53) Li, X.; Hua, T.; Vemuri, K.; Ho, J. H.; Wu, Y.; Wu, L.; Popov, P.;

Benchama, O.; Zvonok, N.; Locke, K.; Qu, L.; Han, G. W.; Iyer, M. R.;

Cinar, R.; Coffey, N. J.; Wang, J.; Wu, M.; Katritch, V.; Zhao, S.; Kunos,

G.; Bohn, L. M.; Makriyannis, A.; Stevens, R. C.; Liu, Z. J. Crystal

structure of the human cannabinoid receptor CB2. Cell 2019, 176,

459−467.

̌

(54) Dobovisek, L.; Hojnik, M.; Ferk, P. Overlapping molecular

pathways between cannabinoid receptors type 1 and 2 and estrogens/

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https://dx.doi.org/10.1021/acs.jmedchem.0c01023

J. Med. Chem. 2021, 64, 385−403

Article Doi

From a Designer Drug to the Discovery of Selective Cannabinoid Type 2 Receptor Agonists with Favorable Pharmacokinetic Profiles for the Treatment of Systemic Sclerosis

DOI: 10.1021/acs.jmedchem.0c01023

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Article abstract of DOI:10.1021/acs.jmedchem.0c01023

Full text of DOI:10.1021/acs.jmedchem.0c01023

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