Syn hesis and Pha macological Cha ac e iza ion o a No el
Cannabinoid Recep o 1 An agonis
Ike Bengoe xea de Tena, Go ka Pe ei a-Cas elo, Jona an Ma ínez-Ga deazabal,
Ma a Mo eno-Rod íguez, I án Manuel, Claudio Ma ínez, Belén Vaz, Ja ie González-Rica e,
Rosana Al a ez, Angel To es-Mozas, F ancesca Pecca i, Gonzalo Jiménez-Osés,
Angel Rod íguez de Le a, and Ra ael Rod íguez-Pue as*
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ABSTRACT: The endocannabinoid (eCB) sys em egula es se e al
b ain unc ions and is implica ed in nume ous condi ions a ec ing he
b ain. Thus, he pha macological blockade o cannabinoid ecep o s has a
he apeu ic po en ial bu p oduces se e e psychia ic side e ec s. Hence,
new cannabinoid compounds wi h di e en pha macological p o iles a e
needed o po en ially minimize his oxici y. The objec i e o his s udy,
ea u ing o iginal chemical insigh s, pha macological analysis, and obus
compu a ional me hods, was o syn hesize and cha ac e ize a se ies o
no el an agonis s/in e se agonis s o cannabinoid ecep o s. To do so,
we i s syn hesized and hen sc eened 11 no el compounds o a ini y o cannabinoid ecep o s. A e ha , we cha ac e ized in
dep h he pha macological p o ile o he mos p omising one, UVI3502, which showed a ini y o wo [3H]CP55,940 binding si es
(IC50Hi 0.026 ±0.43 nM and IC50Lo 772 ±49.40 nM, R2= 0.59) in he a co ex. Binding assays pe o med in memb anes
o e exp essing cannabinoid ecep o s 1 and 2 (CB1and CB2) con i med mode a e a ini y o bo h ecep o sub ypes, abou 10- old
highe o he i s one, indica ing limi ed ecep o sub ype speci ici y. In key b ain a eas om he oden b ain, which ha e a much
highe CB1 ecep o densi y han CB2, he a ini y o UVI3502 was u he s udied wi h neu oana omical speci ici y by
au o adiog aphy. Func ional [35S]GTPγS assays demons a ed ha UVI3502 beha ed as an an agonis o CB1 ecep o s, blocking
he s imula ion e oked by he po en cannabinoid ecep o agonis CP55,940. The in silico cha ac e iza ion o he binding o he
CB1 ecep o h ough molecula docking and molecula dynamics sugges s ha his ac i i y is explained by he plana and igid
s uc u e o UVI3502, which is op imal o in e ac ions wi h he inac i e s a e o he ecep o . Hence, we syn hesized and
cha ac e ized UVI3502 as a no el an agonis o CB1, making i a new pha macological ool o he s udy o he eCB sys em and o
blocking cannabinoid ecep o s in he cen al ne ous sys em.
■INTRODUCTION
The endocannabinoid (eCB) sys em is essen ial o p ese ing
ene gy balance and me abolism
1
bu is also implica ed in
modula ing cogni i e unc ions, including lea ning and memo-
y.
2
This sys em in ol es wo iden i ied G p o ein-coupled
ecep o s (GPCRs), cannabinoid ecep o s o ype-1 and -2, o
CB1and CB2, espec i ely. Thei exp ession di e s signi ican ly,
wi h CB1exhibi ing high le els o exp ession in he cen al
ne ous sys em (CNS), including in b ain a eas associa ed wi h
he psychoac i e e ec s o Δ-9- e ahyd ocannabinol (Δ9-
THC); he basal ganglia, he ce ebellum, po ions o he
hippocampus, and he co ical egions.
3
In con as , he a eas
wi h he highes le els o CB2 ecep o s a e p ima ily he
immune sys em and he spleen.
4,5
As one o he mos ele an neu omodula o y ne wo ks o he
CNS, he eCB sys em egula es impo an physiological
p ocesses, such as neu ode elopmen , synap ic plas ici y, and
adap i e esponses,
2
all o which a ec cogni ion. Hence, despi e
he well-known dele e ious e ec s o cannabinoids on memo y,
6
he e is a g owing in e es in de eloping new cannabinoid
compounds o he ea men o neu ological and neu o-
degene a i e diseases.
7,8
In ac , a ious componen s o he
eCB sys em unde go al e a ions in pos -mo em human samples
om Alzheime ’s disease (AD) pa ien s,
9−11
and animal model
s udies sugges ha he pha macological manipula ion o he
eCB sys em can in luence he his opa hological and biochemical
ma ke s associa ed wi h his disease.
12−14
In addi ion o AD, he
po en ial o cannabinoid ea men s in animal models o o he
neu odegene a i e and also neu ode elopmen al diso de s,
Recei ed: Decembe 17, 2024
Re ised: Feb ua y 6, 2025
Accep ed: Feb ua y 7, 2025
Published: June 2, 2025
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including Pa kinson’s (PD) and Hun ing on’s diseases (HD), o
Williams−Beu en synd ome, has also been desc ibed.
15−18
While many o hese ea men s in ol e he ac i a ion o he
eCB sys em, ei he h ough di ec ac ion on cannabinoid
ecep o s o h ough egula ion o he syn hesis and deg ada ion
enzymes o endocannabinoids, he pha macological blockade o
cannabinoid ecep o s also exe s posi i e e ec s depending on
he con ex . Fo ins ance, in wo mouse models o Down’s
synd ome (DS), ea men s wi h he gold-s anda d in e se
agonis o CB1 ecep o s, SR141716A o imonaban ,
19
es o ed
key cogni i e pheno ypes a ec ed by he pa hology.
20
Simila ly,
in ano he neu ode elopmen al condi ion, agile X synd ome,
CB1blockade es o ed cogni ion and no malized he mo phol-
ogy o dend i ic spines, while blocking CB2only no malized
anxie y le els.
21,22
In a e y di e en con ex , mice ea ed wi h
he well-known musca inic an agonis scopolamine, known o
p oduce ansien choline gic hypo unc ion and cogni i e
de ici s, co ea men wi h MK-7128, a CB1 ecep o in e se
agonis , imp o ed pe o mance in di e en beha io al asks and
his was achie ed a mode a e le els o CB1occupancy in he
b ain.
23
The pha macological inhibi ion o cannabinoid ecep o s,
pa icula ly CB1, o e s he po en ial o amelio a e a wide a ay o
cogni i e de ici s a ising om a ious causes. Howe e , he mos
used compound in hese s udies, imonaban , is a e y high-
a ini y in e se agonis which p oduced se e e psychia ic side
e ec s, including anxious and dep essi e diso de s, when i was
adminis e ed o pa ien s su e ing om obesi y.
24,25
Thus, he e
is a need o de elop new compounds wi h a simila
pha macological p o ile ha can po en ially a oid such ad e se
e ec s.
Thus, ou objec i e was o pe o m he chemical syn hesis and
pha macological cha ac e iza ion in he oden b ain using in
i o and in silico me hods o a numbe o no el an agonis s/
in e se agonis s o cannabinoid ecep o s. We desc ibe ha one
o hese compounds, UVI3502, is a no el an agonis o CB1
ecep o s, which blocks he s imula ion p oduced by CP55,940,
a po en cannabinoid agonis , in some o he mos ele an b ain
a eas ha con ol lea ning and memo y p ocesses.
■RESULTS AND DISCUSSION
Conside ing he he apeu ic po en ial o he eCB sys em
ega ding a ious condi ions a ec ing he b ain, he e is a
need o de elop no el compounds a ge ing cannabinoid
ecep o s bo h as new esea ch ools and as po en ial ea men s
o hese diso de s. In his s udy, we ha e pe o med he
syn hesis and pha macological sc eening o a se ies o no el
compounds o a ini y o cannabinoid ecep o s, ollowed by a
pha macological p o iling o he mos p omising compound,
using a ange o in i o and in silico me hods. Hence, his wo k
encompasses he comp ehensi e p ocess o d ug disco e y o a
no el compound, de ailing he design, chemical syn hesis, and in
i o and in silico pha macodynamical cha ac e iza ion.
Chemical Syn hesis o he No el Compounds. As a
ollow-up o p e iously desc ibed s udies ega ding he
palladium-ca alyzed he e ocycliza ion/oxida i e Heck coupling
cascade as a syn he ic app oach o used he e ocycles, we ha e
explo ed he easibili y o pe o ming he eac ion sequence in a
one-po ashion. The me ge o Sonogashi a he e ocycliza ion
and oxida i e Heck p ocesses in he same s ep using he same
ca alys gene a es a new se ies o dihyd oindolo[3,2-d]
benzazepine-6(5H)-ones s a ing om simple p o ec ed o ho-
iodoanilines and acyl o ho-alkynylanilines (see Scheme 1).
In he syn he ic design, we ha e also conside ed he be e
solubili y o he indole de i a i es p o ec ed as ca bama es,
which a e easie o pu i y and c ys allize han he co esponding
ee indoles wi h he same subs i u ion pa e n, while he
ca bama e g oup does no p e en cycliza ion h ough he
ni ogen in he nucleopallada ion s ep.
26
The syn hesis o he p ecu so s included wo s aigh o wa d
eac ions, as shown in Scheme 2, namely, he p e iously
desc ibed p ocedu es o 2-haloa ylca bama es 2
26,27
and he
condensa ion o he 2-e hynylanilines 3wi h he co esponding
acid chlo ide o he acyl de i a i es 4, bo h p oceeding in good
yields.
26,28,29
Based on he p e iously epo ed condi ions o he o ma ion
o used indoles in a one-po sequence,
26
he cons uc ion o he
a ge skele on included: (a) ea men o he co esponding
o ho-iodoaniline 2(1 equi ) and alkyne 4(2 equi ) wi h
PdCl2(PPh3)2(5 mol %) as he ca alys and CuI (20 mol %),
Ph3P (5 mol %), and E 3N (2 equi ) as he addi i es in N,N-
dime hyl o mamide (DMF) unde an a gon a mosphe e a 50
°C o 0.5 h and (b) opening he lask o ai and hea ing o 50 °C
o 17−22 h. Indoles 5a− we e ob ained in 47−75% yields
depending upon he subs i u ion pa e n, which can be
conside ed as a e y e icien p o ocol gi en he inc ease in
s uc u al complexi y esul ing om h ee consecu i e syn he ic
s eps.
S aigh o wa d syn he ic modi ica ions (dep o ec ion and
hyd olysis o he es e ) allowed con e sion o he ca bama e/
es e o 5a in o 6a and 5 in o 6 (see Scheme 2). Hyd ogena ion
o he conjuga ed es e 5a upon ca alysis o Pd(OH)2in e hyl
ace a e and dep o ec ion o he ca bama e wi h TBAF a o ded
7a in a combined 73% yield, which was al e na i ely hyd olyzed
o 8o con e ed in o he used iazole 9in an o e all 87% yield
upon combined ea men wi h Lawesson’s eagen a 60 °C,
Scheme 1. Syn he ic App oach o he Cons uc ion o he dihyd oindolo[3,2-d] Benzazepine-6(5H)-One Skele on, including he
In a- and In e molecula Ve sion
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ollowed by hyd azine hyd a e and ie hyl o hoace a e in THF
a 80 °C (see Scheme 2).
Wi h he syn he ic app oach used, we ha e u he inc eased
he e iciency o he in amolecula oxida i e Heck cascade
eac ion,
26
by inco po a ing a Sonogashi a c oss-coupling p io
o he he e ocycliza ion−Heck, as desc ibed o o he he e o-
cycles. This s a egy led o he o ma ion and N-cycliza ion o
o ho-alkynylaniline in e media es s a ing om app op ia ely
p o ec ed o ho-iodoanilines and e minal alkynylanilines. The
sequence o chemical ans o ma ions was pe o med in he
same eac ion lask while addi ional eagen s and ca alys s we e
added a di e en ime in e als,
30
ano he example o he “one-
po ” mul icomponen eac ion (MCR).
31
This combina ion o
consecu i e Sonogashi a, nucleopallada ion, and oxida i e Heck
couplings con enien ly allowed he p epa a ion o a new se ies
o 7,12-dihyd oindolo[3,2-d]benzazepine-6(5H)-ones. The
skele on o he indolobenzazepinones was u he modi ied by
inco po a ion o addi ional subs i uen s o by i s con e sion in o
he used [1,2,4] iazoloazepines in an e icien manne .
Sc eening o he Newly Syn hesized Compounds o
Cannabinoid Recep o A ini y. Following he syn hesis, we
e alua ed he pha macodynamic pa ame e s o he no el
compounds using adioligand a ini y assays wi h [3H]CP55,940
pe o med in memb ane homogena es pu i ied om he a
co ical issue, which na u ally con ain CB1and CB2 ecep o s.
5
Ra co ical issue was used o hese assays, gi en he
a o emen ioned ele ance o he eCB sys em in neu ological
condi ions.
The IC50 alues o each compound ob ained in he
compe i ion cu es a e summa ized in Table S1. A compa ison
o i s was pe o med o e e y cu e, and he s a is ically
p e e ed model (one si e s wo si es) was chosen in each case.
Ou o he 11 compounds analyzed, one o hem, UVI3502,
showed a ini y o cannabinoid ecep o s. UVI3502 showed a
ela i ely high a ini y wi h wo binding si es in he [3H]-
CP55,940 compe i ion cu e (IC50Hi 0.026 ±0.43 nM and
IC50Lo 772 ±49.40 nM, R2= 0.59; see Figu e 1 and Table S1).
Gi en ha he assay was pe o med using a i ia ed agonis ,
[3H]CP55,940, he wo binding si es obse ed a e expec ed o
co espond o di e en ecep o s, mos likely CB1o CB2, a he
han o wo di e en a ini y s a es (e.g., high and low) o he
same ecep o , which can only be obse ed by pe o ming he
compe i ion wi h a i ia ed an agonis .
32
The o al inhibi ion o
[3H]CP55,940 binding exe ed by UVI3502 was abou 58%,
wi h app oxima ely 18% co esponding o he high-a ini y
binding si e and he emaining 40% co esponding o he low-
a ini y one. None o he o he compounds showed signi ican
a ini y o cannabinoid ecep o s (i.e., inhibi ion o [3H]-
CP55,940 binding). Consequen ly, UVI3502 was selec ed as he
Scheme 2
Reagen s and Reac ion Condi ions:
a
ClCO2E , K2CO3, Ace one, 25 °C (2a−e, 74-81%).
b
E hyl Fuma oyl Chlo ide, NaHCO3, Dioxane, 0 o 25
°C, 23 h (4a−c, 66-90%).
c
(i) PdCl2(PPh3)2(5 mol %), CuI (20 mol %), PPh3(5 mol %), E 3N, DMF, 60 °C, 12 h and (ii) Ai , 100 °C, 15 h (5a−
, 47-75%).
d
LiOH H2O, THF, 50 °C, 12 h (6a, 86%; 8, 79%).
e
TBAF, THF, 80 °C, 21 h (6 , 71%).
(i) H2, Pd(OH)2, E OAc, 25 °C and (ii) 1M
TBAF in THF, 80 °C, 21 h (7a, 73%).
g
(i) Lawesson’s Reagen , THF, 60 °C, 3 h; (ii) H2NNH2·H2O, THF, 20 °C, and (iii) CH3C(OE )3, THF,
80 °C, 1 h (9, 87%).
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bes candida e compound o subsequen pha macodynamic
cha ac e iza ion using bo h in i o and in silico echniques.
Sc eening o he A ini y o UVI3502 o Cannabinoid
Recep o Sub ypes. The pha macological p o iling o
UVI3502 as a no el cannabinoid ligand was comple ed by
pe o ming inhibi ion cu es o [3H]CP55,940 s a ange o
concen a ions o UVI3502 in memb ane homogena es om
CHO cells o e exp essing human CB1and CB2 ecep o s. As a
u he con ol, inhibi ion cu es we e also pe o med in he a
spleen issue, due o he high exp ession o CB2and e y low
le els o CB1 ecep o s in his o gan.
33
UVI3502 showed a ini y o CB1 ecep o s and a single
binding si e in CB1o e exp essing cells (IC50 4641 ±1595 nM,
R2= 0.55; see Figu e 2A), ollowing he same compa ison o i s
pe o med (one si e s wo si es). The o al inhibi ion o
[3H]CP55,940 binding exe ed by UVI3502 in his issue was
app oxima ely 38%. These esul s indica e ha UVI3502
pa ially binds he CB1 ecep o . UVI3502 also showed
app oxima ely 10- old lowe a ini y o he CB2 ecep o ,
Figu e 1. Compe i ion cu es o [3H]CP55,940 s inc easing concen a ions anging om 10−12 M o 10−4M o he 11 no el compounds in
memb ane homogena es om he Sp ague−Dawley a b ain co ex (n= 5). Ou o he 11 compounds, one o hem, UVI3502 (cen e up), inhibi ed
[3H]CP55,940 binding wi h wo di e en binding si es, likely co esponding o wo di e en ecep o sub ypes. Cu es ep esen he mean o n= 2
independen expe imen s wi h a ange o 4−5 echnical eplica es o e e y compound excep UVI3502, in which he cu e ep esen s he mean o n= 5
independen expe imen s wi h 11 echnical eplica es.
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inhibi ing [3H]CP55,940 binding in CB2o e exp essing cells
and in spleen memb ane homogena es in concen a ions a he
low mic omola ange (CB2o e exp essing cells: IC50 16200 ±
130.67 nM, R2= 0.83; see Figu e 2B; spleen memb ane
homogena es: IC50 10230 ±17.83 nM, R2= 0.62; see Figu e
2C). The maximum inhibi ion achie ed was app oxima ely 83%
and 61%, espec i ely, indica ing ha UVI3502 pa ially
displaces [3H]CP55,940 binding in cells o e exp essing he
CB2 ecep o wi h low a ini y. Toge he , he esul s ob ained in
[3H]CP55,940 binding assays pe o med in he a co ical
issue as well as in CB1and CB2o e exp essing memb ane
homogena es indica e a limi ed ecep o sub ype speci ici y o
UVI3502 o CB1, and he possibili y ha he high-a ini y
binding si e obse ed in Figu e 1 could co espond o a hi d,
non-CB1/-CB2, ecep o .
34
Cha ac e iza ion o he Binding o UVI3502 o he CB1
Recep o wi h Neu oana omical Speci ici y in he
Roden B ain. The pha macological p o ile o UVI3502 was
u he cha ac e ized in he oden b ain wi h neu oana omical
speci ici y by pe o ming [3H]CP55,940 au o adiog aphic
assays in b ain slices om wo mos used animal models in
esea ch, nai e Sp ague−Dawley a s and Swiss mice (see Figu e
3), ocusing on b ain egions associa ed wi h lea ning and
memo y p ocesses.
[3H]CP55,940 au o adiog aphy was pe o med in he
p esence o bo h adioligand and UVI3502, as well as in he
p esence o he adioligand and SR141716A, a known in e se
agonis o CB1 ecep o s, in o de o ha e a e e ence o he
amoun o [3H]CP55,940 binding inhibi ed by he no el
compound. UVI3502 was able o pa ially inhibi [3H]CP55,940
binding in all a eas ha we e analyzed in bo h a and mouse
b ain slices (see Table S2). Gi en he much highe densi y o
CB1o e CB2 ecep o s in he b ain,
35,36
hese esul s con i m
ha UVI3502 binds CB1 ecep o s and indica e ha i inhibi s a
ac ion o he [3H]CP55,940 binding inhibi ed by he ull
in e se agonis SR141716A.
[35S]GTPγS Func ional Assay o Cha ac e ize he
Ac i i y o UVI3502 a he CB1Recep o . To s udy he
ac i i y o UVI3502 a CB1 ecep o s, [35S]GTPγS unc ional
assays in memb ane homogena es om he a co ical issue
and in CHO cells o e exp essing he human CB1 ecep o we e
pe o med. In hese assays, UVI3502 did no s imula e he
coupling o CB1 ecep o s o Gi/o p o eins in any o he issues
used, and no signi ican educ ions in baseline le els o Gi/o
p o ein coupling we e obse ed, especially in CB1o e -
exp essing cells. These esul s sugges ha UVI3502 ac s as an
an agonis o CB1 ecep o s (see Figu e S1).
Cha ac e iza ion o he Ac i i y o UVI3502 wi h
Neu oana omical Speci ici y in he Roden B ain. A
subsequen unc ional au o adiog aphic assay o de e mine he
ac i i y o UVI3502 in key b ain a eas o he oden b ain was
hen conduc ed. The assay was pe o med by incuba ing
[35S]GTPγS wi h CP55,940 (10 μM) alone, as a known agonis
o CB1 ecep o s, and consecu i e slices wi h UVI3502 (10 μM)
alone and in he p esence o bo h CP55,940 (10 μM) and
UVI3502 (10 μM) (see Figu e 4).
Figu e 2. Inhibi ion cu es o [3H]CP55,940 s inc easing concen a ions anging om 10−12 M o 10−4M o UVI3502 in (a) CB1o e exp essing
memb anes, (b) CB2o e exp essing memb anes, and (c) Sp ague−Dawley a spleen memb anes (n= 5). No e ha he cu e shows a single binding
si e wi h CB1o e exp essing memb anes, indica ing ha UVI3502 binds CB1 ecep o s (IC50 4641 ±1595 nM, R2= 0.55). No e ha UVI3502 also
shows low a ini y o CB2 ecep o s bo h in CB2o e exp essing memb anes (IC50 16200 ±130.67 nM, R2= 0.83) and in CB2-en iched spleen issue
(IC50 10230 ±17.83 nM, R2= 0.62). Each cu e ep esen s he mean o n= 3−4 independen expe imen s wi h a ange o 6−10 echnical eplica es.
Figu e 3. Rep esen a i e au o adiog ams o a (n= 5) and mouse (n= 5) b ain sagi al sec ions showing [3H]CP55,940 binding alone, [3H]CP55,940
binding in he p esence o UVI3502, and [3H]CP55,940 binding in he p esence o SR141716A. [3H] mic oscales used as s anda ds in Ci/g .e. No e
he pa ial inhibi ion o [3H]CP55,940 binding by UVI3502 in mos o he b ain a eas exp essing CB1 ecep o s. Scale ba = 0.5 cm.
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Resul s om Sp ague−Dawley a and Swiss mouse b ain
slices indica e ha UVI3502 ac s as an an agonis , as he
s imula ion e oked by he agonis CP55,940 was supp essed in
all o he analyzed a eas (see Table S3). In spi e o he ac ha
UVI3502 only inhibi s a ac ion o [3H]CP55,940 binding (see
Figu e 3 and Table S2), i ac s as a po en an agonis in
supp essing CP55,940-e oked G-p o ein coupling (see Figu e 4
and Table S3), and his highligh s i s po en ial as a esea ch ool
o he s udy o he eCB sys em in he b ain.
In e es ingly, and unlike wha was obse ed in he
[35S]GTPγS unc ional assays in a co ex memb ane
homogena es and in CHO cells o e exp essing he human
CB1 ecep o , [35S]GTPγS binding in he p esence o UVI3502
was lowe han baseline le els o [35S]GTPγS binding in he
amygdala, he co ex, he hippocampus, he nucleus basalis
magnocellula is (NBM), he s ia um, and he g ay ma e o he
ce ebellum (see Table S3). These esul s could indica e an
in e se-agonis -like ac i i y o UVI3502 in hese a eas. In
unc ional au o adiog aphy, he in e p e a ion o basal
[35S]GTPγS binding in he p esence o no d ug, and hus o
he concep o “in e se agonis ”, emains a subjec o
con o e sy. While nume ous epo s sugges ha di e en
ecep o s can be cons i u i ely ac i e in he absence o any
ligand,
37−39
o he epo s sugges oles o endogenous ligands in
so-called basal ac i i y, such as he o ma ion o adenosine
du ing incuba ion
40
o he p esence o endogenous lysophos-
pha idic acid (LPA) ac i a ing LPA1 ecep o s.
41
The seemingly
con adic o y da a in [35S]GTPγS assays pe o med in
memb ane homogena es s b ain au o adiog aphy migh de i e
om he di e en p o ocols used in bo h cases and could be
a ec ed by he highe p esence o endogenous ligands in he
b ain issue.
Figu e 4. Rep esen a i e au o adiog ams o a (n= 5) and mouse (n= 5) b ain sagi al sec ions ha show [35S]GTPγS basal binding as well as
[35S]GTPγS binding in he p esence o CP55,940 (10 μM) alone, in he p esence o UVI3502 (10 μM) alone and in he p esence o bo h CP55,940
and UVI3502 (bo h a 10 μM). No e ha UVI3502 inhibi s he s imula ion e oked by CP55,940 in mos b ain a eas, indica ing ha UVI3502 beha es
as a CB1an agonis . [14C] mic oscales used as s anda ds in Ci/g .e. Scale ba = 0.5 cm.
Figu e 5. (a) C ys al s uc u e o he human cannabinoid ecep o CB1in complex wi h in e se agonis AM-6538 (in o ange s icks; e minal ni a e
g oup is no shown due o he absence o elec on densi y), PDB 5TGZ. (b) Docking pose o UVI3502 (in g een s icks) on he human cannabinoid
ecep o CB1( ecep o s uc u e aken om PDB 5TGZ). (c) Space- illing iew o he same docking pose wi h indica ion o he main egions in ol ed
in UVI3502 binding. (d) O e lay o 5 snapsho s sampled wi h an e en s ide o 20 ns om a molecula dynamics simula ion o he UVI3502:CB1
complex. Key esidues o an agonis /in e se agonis binding a e shown as yellow s icks.
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The ac i i y o UVI3502 as an an agonis is no ewo hy, gi en
ha i sha es some s uc u al esemblance o he amino-
alkylindole WIN55,212−2, a po en cannabinoid ecep o
agonis .
42
To explain his coun e in ui i e obse a ion, molec-
ula docking and classical molecula dynamics we e pe o med,
modeling he binding o UVI3502 o he human CB1 ecep o .
Modeling o UVI3502 Binding o CB1.Binding o
UVI3502 o he human CB1 ecep o was modeled h ough a
combina ion o molecula docking and classical molecula
dynamics. UVI3502 and ela ed de i a i es we e docked on a
h ee-dimensional model o CB1gene a ed om i s c ys allo-
g aphic s uc u e in complex wi h he known an agonis /in e se
agonis AM-6538 (see Figu e 5A).
43
Figu e 5B shows he bes
sco ing docking pose (sco e = 35.5); like AM-6538, UVI3502 is
deeply bu ied in he binding pocke o CB1, oughly occupying
he same egion, and is engaged in hyd ophobic con ac s wi h
Phe170, Phe174, Phe268, T p356, and Phe379. The binding
pose is d i en by a igh shape complemen a i y (see Figu e 5C);
he main di e ences obse ed be ween he wo ligands a e a
educed occupa ion o he long channel (lined by T p279 and
Me 363) o UVI3502 owing o he size o he ca bama e moie y
and inc eased in e ac ions wi h he uppe pa o he pocke
(Phe268 and Phe379) h ough he icyclic co e and me hoxy
g oup. The wo ligands i he gap and side pocke egions o a
simila ex en . Excep o UVI3501 ea u ing a la ge es e g oup
a he indole ing, all o he analogues show a binding pose simila
o UVI3502 wi h he same a angemen o he a oma ic co e
inside he binding si e. Consequen ly, he lexible ca bama e,
ca boxyl, o es e moie ies ex end in o he long channel, and he
ca boxylic o es e g oups occupy he gap egion (see Figu e S2).
Compounds equipped wi h he igid icyclic hyd ophobic co e
( s e acyclic ones) and an es e g oup ( s a ca boxyla e) show
he bes binding p ope ies o he CB1 ecep o . A 100 ns
classical molecula dynamics simula ion o he UVI3502:CB1
complex was pe o med by using he docking pose as he s a ing
geome y o e alua e he pe sis ence o key binding con ac s.
Figu e 5D shows an o e lay o i e ames sampled wi h an e en
s ide om he molecula dynamics simula ion. Al hough some
lexibili y is obse ed, he posi ioning and o ien a ion o
UVI3502 in he binding pocke emain cons an , as well as he
packing o he su ounding hyd ophobic esidues, co obo a ing
he docking pose as a plausible ep esen a ion o he binding
in e ac ion.
The an agonis ac i i y o UVI3502 can be en a i ely
explained in e ms o he shape complemen a i y wi h he
binding pocke in he ac i e and inac i e s a es o he ecep o
(Figu e 6). Indeed, UVI3502 is p edic ed o bind in a simila
a angemen o p e iously epo ed an agonis /in e se agonis
AM-653843, ex ending in o he side pocke h ough i s e ical
axis and joining he long channel and gap egion along i s
Figu e 6. Side iew (le ) and op iew ( igh ) o agonis s and an agonis s/in e se agonis s bound o human CB1(AM-11542, PDB 5XRA; AM-6538,
PDB 5TGZ) and CB2(WIN55,212−2, PDB 6PT0) ecep o s om c ys allog aphic s uc u es and docking pose o UVI3502. The binding ca i y is
shown as anspa en su aces. Agonis s a e shown in yellow ( op) and an agonis s/in e se agonis s in o ange (bo om).
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longi udinal axis. The la e is a cha ac e is ic ea u e sha ed by
o he known in e se agonis s (e.g., AM-251, imonaban )
44
and
is key o op imal in e ac ions wi h he ecep o in he inac i e
s a e (see Figu e 6), in which i ma ches he oughly T-shaped
binding ca i y. Fo UVI3502, his is enabled by he qui e plana
and igid amewo k ex ending om he ca bama e o he
exocyclic double bond. Con e sely, known agonis s, such as
AM-1154243 and WIN55,212−2, bind wi h an angula shape
o en eme ging om lexible alkyl and a oma ic side chains (see
Figu e 6), which in u n p o ide op imal con ac s wi h a
ma ching oughly V-shaped ca i y in he less longi udinally
ex ended ac i e s a e. Owing o i s igid amewo k, we
hypo hesize ha UVI3502 canno adop such an angula
shape, p o iding a molecula basis o i s ac i i y and p o iding
insigh s in o he s uc u al nuances o CB1ac i a ion.
■CONCLUSIONS
In summa y, we de e mined he an agonis p ope ies o he
no el compound UVI3502 mainly o CB1 ecep o s, wi h a 10-
old lowe a ini y o CB2, using bo h in i o and in silico
app oaches. Via unc ional au o adiog aphy, we de e mined ha
UVI3502 e icien ly blocks he coupling o CB1 o Gi/o p o eins
elici ed by a po en cannabinoid agonis in key b ain a eas
con olling lea ning and memo y in he b ain issue om wo o
he mos used expe imen al models, a s and mice. By using
molecula docking and dynamics, we could explain his ac i i y
by he plana and igid s uc u es o UVI3502, which op imally
in e ac wi h he inac i e s a e o he ecep o . While his s udy
o e s impo an insigh s in o he pha macological p ope ies o
UVI3502, i mus be acknowledged ha he in i o and in silico
me hodologies, while obus , may no ully eplica e in i o
condi ions. The adminis a ion o UVI3502 o oden s on i s
own, as well as by coadminis a ion o his compound along wi h
a po en cannabinoid ecep o agonis , like CP55,940, as
pe o med in i o, would o e aluable in o ma ion ega ding
he po en ial o his compound in he apy. I would be o
pa icula in e es o analyze UVI3502 in e ms o i s e ec o
he egula ion o me abolism, as well as ega ding po en ial
psychia ic side e ec s, which ha e been obse ed o o he
in e se agonis s o CB1 ecep o s, mos no ably imonaban .
45
I
would also be ele an o analyze he physicochemical p ope ies
and in i o abso p ion, dis ibu ion, me abolism, and exc e ion
(ADME) p ope ies o he compound, such as me abolic
s abili y, in es inal abso p ion, binding o plasma p o eins, o
blood−b ain ba ie pe mea ion. Howe e , hese ho ough
analyses a e beyond he scope o he p esen s udy and should be
in es iga ed in he u u e. Ne e heless, he p esen esul s open
he doo o he use o his newly syn hesized compound as a
new esea ch ool o he s udy o he eCB sys em and,
po en ially, o he in i o inhibi ion o cannabinoid ecep o s in
he CNS.
■METHODS
Reagen s, D ugs, and Chemicals. All necessa y com-
pounds o he di e en p ocedu es we e o he highes
comme cially a ailable quali y o he pu pose o ou s udies.
Fo he syn hesis o he no el compounds, chemical eagen s o
he highes pu i y a ailable we e pu chased om Sigma-Ald ich
and used as ecei ed excep when indica ed.
[3H]CP55,940 (149 Ci/mmol) and [35S]GTPγS (1250 Ci/
mmol) we e acqui ed om Re i y (Wal ham, MA, USA). The
[3H]-mic oscales and [14C]-mic oscales used as s anda ds in he
au o adiog aphic expe imen s we e pu chased om ARC
(Ame ican Radiolabeled Chemicals, S . Louis, MO, USA).
The β- adia ion sensi i e ilms, Kodak Biomax MR, bo ine
se um albumin (BSA), DL-di hio h ei ol (DTT), guanosine 5′-
diphospha e (GDP), guanosine 5′-O-3- hio iphospha e
(GTPγS), ke amine, and xylazine we e all acqui ed om
Sigma-Ald ich (S Louis, MO, USA).
5-(4-Chlo ophenyl)-1-(2,4-dichlo ophenyl)-4-me hyl-N-1-
pipe idinyl-1H-py azole-3-ca boxamide hyd ochlo ide
(SR141716A) and (11R)-2-Me hyl-11-[(mo pholin-4-yl)-
me hyl]-3-(naph halene-1-ca bonyl)-9-oxa-1-aza icyclo-
[6.3.1.04,12]dodeca-2,4(12),5,7- e aene (WIN55,212−2)
we e acqui ed om Toc is (B is ol, UK). (−)-cis-3-[2-
Hyd oxy-4-(1,1-dime hylhep yl)phenyl]- ans-4-(3-hyd oxy-
p opyl) cyclohexanol (CP55,940) was acqui ed om Sigma-
Ald ich (S Louis, MO, USA). [(1S,2S,5S)-2-[2,6-Dime hoxy-4-
(2-me hyloc an-2-yl)phenyl]-7,7-dime hyl-4-bicyclo[3.1.1]-
hep -3-enyl]me hanol (HU308) was acqui ed om Me ck
(Da ms ad , Ge many).
Chemical Syn hesis o he No el Compounds. We ha e
p e iously desc ibed he palladium-ca alyzed he e ocycliza ion/
oxida i e Heck coupling cascade as asyn he ic app oach o used
he e ocycles, including 3-alkenyl-subs i u ed benzo u ans, in-
doles, 1H-isoch omen-1-imines, e ahyd odibenzo u ans, and
e ahyd obenzo[c]ch omen-6-imines
46−50
and ex ended he
p ocedu e o he syn hesis o analogues wi h he 7,12-
dihyd oindolo[3,2-d]benzazepine-6(5H)-one skele on. The
syn he ic p o ocol allowed he egioselec i e cons uc ion o
he co e indole and benzazepinone he e ocycles o polycyclic
compounds, also known as alkylidenepaullones, which we e
u he cha ac e ized as ac i a o s o he epigene ic enzyme
NAD+-dependen class o his one deace ylases (si uins, Si 1)
in biochemical assays.
26
The comple e in o ma ion ega ding
he syn hesis o each o he no el compounds is desc ibed in
de ail in he Suppo ing In o ma ion.
Animals, Tissues and Cells. Animal su e ing was
minimized o he maximum ex en , and he lowes possible
numbe o animals was used. All p ocedu es using all animal
species we e pe o med in acco dance wi h he Guide o he
Ca e and Use o Labo a o y Animals as adop ed and
p omulga ed by he U.S. Na ional Ins i u es o Heal h, wi h
he Eu opean animal esea ch laws (Di ec i e 2010/63/EU)
and he Spanish Na ional p o ocols, and we e app o ed by he
Local E hical Commi ee o Animal Resea ch o he Uni e si y
o he Basque Coun y (CEEA-UPV/EHU 2024/23). All
animals used in his s udy we e p o ided by he gene al acili ies
o he Uni e si y o he Basque Coun y (UPV/EHU).
Sp ague−Dawley Ra s. Male Sp ague−Dawley a s, wi h a
weigh o abou 200−300 g, we e housed in g oups o 3−4 pe
cage, wi h a cons an empe a u e o app oxima ely 22 °C, in a
oom wi h con olled humidi y (65%) and wi h a ligh /da k
cycle o 12:12 h. Animals had access o ood and wa e ad
libi um. Spleens and b ains om Sp ague−Dawley (n= 5) a s
we e used o p epa e memb ane homogena es o adioligand
a ini y assays. B ains om Sp ague−Dawley a s (n= 5) we e
also used o au o adiog aphic s udies.
Swiss Mice. Male Swiss mice, wi h a weigh o abou 20−30 g,
we e housed in a single cage wi h a cons an empe a u e o
app oxima ely 22 °C in a oom wi h con olled humidi y (65%)
and wi h a ligh /da k cycle o 12:12 h. Animals had access o
ood and wa e ad libi um. B ains om con ol Swiss mice (n=
5) we e used o au o adiog aphic s udies.
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Memb ane Homogena es om CHO Cells O e exp essing
CB1and CB2Recep o s. Memb ane homogena es om CHO
cells o e exp essing human CB1and CB2 ecep o s, as well as
ma ched wild- ype cells, we e used o es newly syn hesized
compounds and we e acqui ed om Sigma-Ald ich (S Louis,
MO, USA).
P epa a ion o Memb ane Homogena es. Sp ague−
Dawley a s (n= 5) we e anes he ized and sac i iced by
decapi a ion. Spleens and b ains we e hen quickly emo ed by
dissec ion a 4 °C, and in he case o he b ain issue, he co ex
was dissec ed o he p epa a ion o he memb ane homoge-
na es. Fo his p ocedu e, spleen and co ex samples we e
homogenized using a Te lon-glass g inde (15 up-and-down
s okes a 800 pm) in 30 olumes o homogeniza ion bu e (1
mM EGTA, 3 mM MgCl2, 50 mM T is−HCl; pH 7.4)
supplemen ed wi h 0.25 mM suc ose, a 4 °C. The ob ained
homogena es we e cen i uged o 5 min a 1500 pm. Pelle s
we e emo ed, and supe na an s we e cen i uged again o 15
min a 14,000 pm. Fo washing, he ob ained pelle s we e
esuspended in a bu e and cen i uged again, and he
supe na an was emo ed. The esul ing aliquo s we e s o ed
a −80 °C un il use.
Radioligand Binding Assays. [3H]CP55,940 Binding
Assays. To sc een he a ini y o he newly syn hesized
compounds o cannabinoid ecep o s, hese we e used in
concen a ions anging om 10−12 o 10−4M and incuba ed
wi h a p o ein concen a ion o 0.1 mg/mL o a co ex
homogena es (2 h, 37 °C) wi h agi a ion. The incuba ion was
pe o med wi h 0.5 nM o [3H]CP55,940. To de ine nonspeci ic
binding, 10−4M o SR141716A was added o he incuba ion. To
s op he eac ion, an ice-cold wash bu e (50 mM T is−HCl
and 0.5% BSA, pH 7.4) was added. Then, he memb anes we e
e ained by acuum il a ion o a Wha man GF/C glass
mic o ibe il e (Sigma-Ald ich, S . Louis, MO, USA) and he
ee adioligand was disca ded. Fil e s wi h he bound
adioligand we e ans e ed o ials con aining 5 mL o Ul ima
Gold cock ail (Re i y, Bos on, MA, USA) and measu ed wi h a
Packa d T i-Ca b 2200CA liquid scin illa ion coun e (Re i y,
Bos on, MA, USA).
A e ha , he compound wi h he bes a ini y was es ed in
cell memb ane homogena es o e exp essing human CB1and
CB2 ecep o s. A concen a ion o 0.02 mg/mL o comme cial
WT (as con ol) and CB1and CB2o e exp essing CHO cells
we e used, and he same p o ocol desc ibed abo e was ollowed.
Ra spleen homogena es we e also used a a concen a ion o 0.1
mg/mL as a u he cha ac e iza ion o binding o he CB2
ecep o , gi en he high exp ession o his ecep o in his issue
and he p ac ical lack o CB1in i .
33
To de ine nonspeci ic
binding, WIN55,212−2 (CB1/CB2agonis ) o SR141716A
(speci ic CB1in e se agonis ) was added o he incuba ion,
depending on he issue used o each assay.
[3H]CP55,940 Recep o Au o adiog aphy. Fo he pe o m-
ance o cannabinoid ecep o au o adiog aphy using [3H]-
CP55,940, esh ozen sec ions om b ain samples om wild-
ype Sp ague−Dawley a s (n= 5) and Swiss mice (n= 5) we e
used o es he newly syn hesized compound, which had shown
he bes a ini y o cannabinoid ecep o s.
All b ain sec ions we e ai -d ied o 30 min and la e
imme sed in Coplin ja s o p eincuba ion in a bu e con aining
50 mM T is−HCl and 1% o BSA (pH 7.4) o 30 min a oom
empe a u e. The objec i e o his p eincuba ion was o emo e
endogenous ligands. Two issue slices we e la e incuba ed in he
p esence o he [3H]CP55,940 adioligand (3 nM) o 2 h a 37
°C and, in wo consecu i e slices, he incuba ion was pe o med
also in he p esence o he a ge compound (10 μM) and in he
p esence o he known CB1in e se agonis SR141716A (10
μM). Following incuba ion, issue slices we e washed wi h an
ice-cold p eincuba ion bu e , dipped in dis illed wa e , and
d ied o e nigh . To gene a e au o adiog ams, d y sec ions we e
placed in he me ically closed casse es and exposed o β-
adia ion-sensi i e ilms o 21 days a 4 °C. To calib a e he
op ical densi ies o mol/mg issue equi alen , [3H]-mic oscales
we e exposed o he ilms. To quan i y he calib a ed ilms a e
scanning, Fiji so wa e (Be hesda, MA, USA) was used.
[35S]GTPγS Func ional Binding Assays. The compound wi h
he bes a ini y was also es ed using unc ional [35S]GTPγS
binding assays o cha ac e ize i s ac i i y as an agonis o
an agonis /in e se agonis . A p o ein concen a ion o 0.1 mg/
mL o a co ex homogena es and a p o ein concen a ion o
0.02 mg/mL o comme cial CB1o e exp essing CHO cells we e
used o hese assays, suspended in a eac ion bu e (T is−HCl
50 mM, EGTA 1 mM, MgCl23 mM, NaCl 100 mM, 0.5% BSA;
pH 7.4). The a ge compound was used in concen a ions
anging om 10−11 o 10−4M and incuba ed o 2 h a 37 °C wi h
agi a ion in he p esence o 0.5 nM [35S]GTPγS and 50 μM
GDP. Basal coupling o [35S]GTPγS o Gi/o p o eins was
de e mined by incuba ing he memb ane aliquo s wi h he
adioligand in he absence o he a ge compound. To de ine
nonspeci ic binding, 10 μM o unlabeled GTPγS was added o
he incuba ion. A e he incuba ion, he same p ocedu e
de ailed o he [3H]CP55,940 binding assay was ollowed.
Func ional [35S]GTPγS Au o adiog aphy. To pe o m unc-
ional au o adiog aphy
51
o cannabinoid ecep o s, esh ozen
sec ions om b ain samples om wild- ype Sp ague−Dawley
a s (n= 5) and Swiss mice (n= 5) we e used o es he newly
syn hesized compound which had shown he bes a ini y o
cannabinoid ecep o s.
All b ain sec ions we e ai -d ied o 30 min and hen
imme sed in Coplin ja s o p eincuba ion (4 imes, 15 min each
ime) in an HEPES-based bu e (50 mM HEPES, 100 mM
NaCl, 3 mM MgCl2, 0.2 mM EGTA, 0.5% BSA; pH 7.4) a 30
°C. The objec i e o his p eincuba ion was he emo al o
endogenous ligands. Slices we e hen incuba ed o 2 h a 30 °C
in he same bu e supplemen ed wi h 2 mM GDP, 1 mM DTT,
and 0.04 nM [35S]GTPγS and he a ge compound (10 μM)
alone, as well as he a ge compound and CP55,940 (10 μM)
oge he . Basal [35S]GTPγS binding was de ined in he absence
o he agonis s in wo consecu i e slices. To de ine nonspeci ic
binding, 10 μM o unlabeled GTPγS was added o he
incuba ion in ano he sec ion. Following incuba ion, slices
we e wice washed in an ice-cold 50 mM HEPES bu e (pH
7.4), d ied, and exposed o 48 h o β- adia ion sensi i e ilm
wi h a se o [14C] s anda ds calib a ed o [35S]. Calib a ed ilms
we e scanned and quan i ied using Fiji so wa e (Fiji, Be hesda,
MA, USA). The signal co esponding o nonspeci ic binding was
p e iously sub ac ed om he basal and agonis -s imula ed
binding. Then, he da a was exp essed as he pe cen age o
s imula ion o e basal ollowing he o mula: ([35S]GTPγS
agonis -s imula ed binding) ×100/([35S]GTPγS basal bind-
ing)-100.
Molecula Docking Simula ions. Molecula docking
calcula ions we e pe o med using GOLD (CCDC Disco e y
2020) and he ChemSco e i ness unc ion.
52,53
The s uc u e o
human cannabinoid ecep o CB1was aken om PDB 5TGZ.
43
The ecep o was p epa ed o docking using UCSF Chime a o
add hyd ogen a oms.
54
The docking ca i y was cen e ed on he
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