Synthesis, structure-activity relationship, and evaluation of SR141716 analogues: Development of central cannabinoid receptor ligands with lower lipophilicity

Article abstract of DOI:10.1021/jm020157x

Exploration of the central CB₁ cannabinoid receptors using positron emission tomography (PET) will allow for an understanding of the pharmacological and physiological role played by these receptors in the CNS. Current tracers are highly lipophi

Full text of DOI:10.1021/jm020157x

642
J . Med. Chem. 2003, 46, 642-645
Syn th esis, Str u ctu r e-Activity Rela tion sh ip , a n d Eva lu a tion of SR141716
An a logu es: Develop m en t of Cen tr a l Ca n n a bin oid Recep tor Liga n d s w ith Low er
Lip op h ilicity
Reeti Katoch-Rouse,† Olga A. Pavlova,† Tara Caulder,‡ Alexander F. Hoffman,‡ Alexey G. Mukhin,† and
Andrew G. Horti*,†
Neuroimaging Research Branch and Cellular Neurophysiology Section, National Institute on Drug Abuse, Intramural Research
Program, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, Maryland 21224
Received April 15, 2002
Exploration of the central CB1 cannabinoid receptors using positron emission tomography (PET)
will allow for an understanding of the pharmacological and physiological role played by these
receptors in the CNS. Current tracers are highly lipophilic compounds that exhibit very high
nonspecific to specific binding ratios and as a result are inapt for use in humans. We have
synthesized a series of less lipophilic analogues of SR141716 to serve as potential radioligands.
Binding affinities of the series and a functional electrophysiological assay of three of our
compounds have been presented.
In tr od u ction
with thionyl chloride, followed by acylation with the
desired amine. The formation of 4′-hydroxy analogue 7b
was carried out through 6b, prepared by demethylation
of the corresponding 4′-methoxy carboxylic acid 6a
(Scheme 1). Mitsunobu coupling of 7b with (() N-t-BOC-
prolinol followed by deprotection with TFA gave 7k .
Lipophilicity is an important parameter in developing
in vivo imaging agents1 for positron emission tomogra-
phy (PET) and single photon emission computed tomog-
raphy (SPECT). The existing CB1 radiotracers,2-6 mainly
analogues of the selective CB1 antagonist SR141716,
exhibit high nonspecific binding and modest affinity,
which limits their application in imaging. This paper
describes our effort to develop novel SR141716 ana-
logues, as potential PET tracers, that display lower
lipophilicity than existing CB1 radiotracers while still
maintaining high affinity. The limiting factor for the
design of our target compounds was the necessity to
introduce either a fluoro/bromo substituent, which
would to be suitable for radiolabeling with positron-
emitting halogens (18F, 76Br), or a methyl-containing
substituent that could be radiolabeled with [11C]methyl
iodide. Additionally, since antagonists serve as superior
radioligands than agonists for in vivo use because they
do not impose the potential psychoactive effects, func-
tional assays were carried out on a few of the compounds
to confirm their antagonistic property.
The depicted method in Scheme 1 proved unsuccessful
for the preparation of 4-bromopyrazole derivatives and
led us to explore an alternate pathway (Scheme 2) which
was an improvement over previously reported proce-
dures.8 To insert a fluoro group in position 4, the
synthesis was carried out from 2-fluoroacetophenone 11
which was synthesized from 10 via a modification of an
earlier procedure.9 The desired fluorinated ketone 11
was then converted to the ethyl ester 5i (Scheme 3,
pathway I). To ensure that 1,5-diarylpyrazole derivative
was obtained via pathway I, as opposed to the undesir-
able 1,3-diaryl product, an alternate synthesis of the
fluoro ester 5i was carried out using pathway II by
fluorination of 8 with Selectfluor. To our knowledge, this
is the first reported method for inserting a fluoro group
in the C-4 position of pyrazoles using an N-F reagent.
Such a method is more practical for a conventional
chemistry lab than the fluorination of pyrazole deriva-
tives with F2.10
Resu lts a n d Discu ssion
Ch em istr y. The general strategy for preparation of
SR141716 analogues with modified substituents on the
phenyl rings is presented in Scheme 1. Some of the
intermediate compounds were prepared according to
known methods.7 Condensation of the diketone ester
lithium enolate obtained from the requisite propiophe-
none 4, with substituted phenylhydrazine hydrochlo-
ride, followed by refluxing in acetic acid resulted in the
formation of the core pyrazole structure 5. Conversion
of the ethyl ester to the desired hydrazide 7 was
accomplished via basic hydrolysis to give the corre-
sponding carboxylic acid 6 and subsequent treatment
Log Doct Deter m in a tion . Octanol-water partition
coefficient at pH 7.4 (ElogDoct) was determined utilizing
the rp-HPLC protocol11 as well as using software
(clogD).12
In Vitr o Bin d in g Assa y. The binding affinities of
all compounds were determined by using a competition
assay with rat cerebellum homogenate against [125 I]-
AM251.13
St r u ct u r e-Act ivit y R ela t ion sh ip s (SAR ). The
first stage of exploring the SAR of pyrazole analogues
was to modify the R1 substituent in the C-5 benzene
ring (Table 1). Substitution of p-chloro with a p-methoxy
group (7a ) resulted in lower lipophilicity and binding
affinity compared to SR141716 (3). The methylenedioxy
group in 7c also decreased lipophilicity and resulted in
* Corresponding author. Tel.: +1-410-550-2916. Fax: +1-410-550-
2724; E-mail: ahorti@intra.nida.nih.gov.
† Neuroimaging Research Branch.
‡ Cellular Neurophysiology Section.
10.1021/jm020157x CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/18/2003

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 643
Sch em e 1a
a
Reagents: (a) LiHMDS, Et2O, (CO2Et)2, -78 f 0 °C; (b) For 5a ,b,g,n and 8: 2,4-dichlorophenylhydrazine hydrochloride; 5d :
2-chlorophenylhydrazine hydrochloride; 5e: 2-methoxyphenylhydrazine hydrochloride; 5f: 2-fluorophenylhydrazine hydrochloride, EtOH,
rt; (c) AcOH, reflux; (d) KOH, MeOH, reflux; (e) SOCl2, PhCH3, reflux; (f) For 7a -g,k : 1-aminopiperidine; 7l: 2-(aminoethyl)-1-
ethylpyrrolidine; 7m ,n : 1-aminohomopiperidine, Et3N, CH2Cl2; (g) HBr, gl. CH3COOH, reflux; (h) Br2, CH2Cl2, -5 °C; (i) (()N-t-BOC-
prolinol, DEAD, Ph3P, THF; (j) CF3COOH, CH2Cl2, rt.
Ta ble 1.
compd
name
AM251
AM281
SR141716
R1
R2
R3
Cl
R4
R5
Ki ( SEM (nM)
clogD
ElogDoct
1
2
3
I
I
Cl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
Br
F
Br
CH3
CH3
CH3
Br
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
Cl
Cl
H
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
piperidinyl
0.6 ( 0.04
4.5 ( 0.1
1.8 ( 0.2
4.1 ( 0.1
104 ( 10
7.1 ( 0.2
8.0 ( 0.1
440 ( 24
37.8 ( 1.7
9.0 ( 1
1.4 ( 0.2
18.2 ( 1.4
6.2 ( 1
390 ( 55
380 ( 15
7.0 ( 0.3
5.2 ( 0.5
5.25
3.71
4.81
4.06
3.46
4.11
3.45
2.86
2.91
4.35
4.94
3.43
3.58
1.23
1.61
4.62
4.75
5.46
-
Cl
Cl
Cl
Cl
Cl
Cl
OCH3
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
5.36
4.78
4.20
4.69
3.72
3.89
4.03
5.14
5.38
4.01
4.09
2.09
3.15
5.34
5.17
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
NIDA-41020
NIDA-41057
NIDA-41075
NIDA-41087
NIDA-41093
NIDA-41127
NIDA-41119
NIDA-41109
NIDA-42033
NIDA-42055
NIDA-42071
NIDA-42087
NIDA-42077
NIDA-42093
OCH3
OH
-OCH2O-
OCH3
OCH3
OCH3
Cl
Cl
OCH3
OCH3
prolinolyl
OCH3
OCH3
OCH3
H
Cl
Cl
Cl
Cl
2-aminoethyl-1-ethyl pyrrolidinyl
homopiperidinyl
homopiperidinyl
slightly decreased binding. Incorporation of a p-hydroxy
(7b) or a pyrrolidinyl moiety (7k ) in the para-position
showed desirably low lipophilicity, but the compounds
exhibited low binding affinity. Remarkably, the binding
affinities of compounds of R1-subseries (1, 3, 7a , 7b, 7k )
display a linear correlation with clogD values according
to eq 1. Correlation of the binding affinities (pKi ) -log
Ki) of another SR141716 R1-subseries7 with the calcu-
lated12 clogD values gave a similar linear trend line
following eq 2.16
pKi ) 0.6987 × clogD + 5.3167 (R2 ) 0.862, n ) 6)
(1)
pKi ) 0.4939 × clogD + 5.3621 (R2 ) 0.663, n ) 6)
(2)

644 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Brief Articles
Sch em e 2a
Sch em e 3a,b
a
Reagents: (a) Selectfluor, MeOH, reflux; (b) LiHMDS, Et2O,
(CO2Et)2, -78 °C f 0 °C; (c) 2-chlorophenylhydrazine hydrochlo-
ride, EtOH, rt; (d) AcOH, reflux; (e) Selectfluor, CH3CN, reflux;
(f) KOH, MeOH, reflux; (g) SOCl2, PhCH3, reflux; (h) 1-aminopi-
peridine, Et3N, CH2Cl2. bCompound 11 has been synthesized
previously18 but was prepared according to our protocol for this
publication. All analytical data correlated with that found in the
literature.
to conclude that within the synthesized series, more
lipophilic R2 substituents (Br > CH3 > F > H) enhance
binding affinity. These observations are consistent with
other studies,14 indicating that replacement of a methyl
group with more lipophilic bromine and iodine leads to
better binding. Modification of the hydrazide portion of
the lead molecule was also carried out. Substitution with
a pyrrolidinyl group (7l) and homopiperidinyl group(7m
and 7n ), did not exhibit improved affinities. This dis-
crepancy may be attributed to the specific role of p-OMe
group in the C-5 phenyl ring.
F u n ction a l Assa y. To assess the functional proper-
ties of these compounds, we performed extracellular
electrophysiological recordings of field potentials in the
nucleus accumbens of rat brain slices, where CB ago-
nists have been previously shown to exhibit glutamate
release.15 Pretreatment of the slices with 7a , 7c, or 7d
blocked the ability of the CB agonist WIN 55,212-2 to
reduce the field potentials, but had no effect on the
baseline (pretreatment) synaptic responses (data not
shown). When these compounds were applied subse-
quently to WIN 55,212-2, the inhibitory effect of the
agonist was rapidly and completely reversed.16 This
reversal was due to antagonism, rather than “wash-out”
of WIN 55,212-2, since previous data have shown that
this lipophilic agonist does not typically wash out of
brain slices.15
a
Reagents: (a) Br2, CH2Cl2, -5 °C; (b) NBS, CH3CN, rt; (c)
KOH, MeOH, reflux; (d) SOCl2, PhCH3, reflux; (e) 1-aminopiperi-
dine, Et3N, CH2Cl2.
This finding suggests the possibility that analogues
of SR141716 with more hydrophilic R1 substituents than
chlorine are likely to display reduced binding affinity.
It is also in agreement with the previously proposed
pharmacophoric alignment of C-5 benzene ring in
SR141716 with the hydrophobic pentyl side-chain of ∆9-
THC.19
The lower lipophilicity of 7a , combined with its
similar binding to the 3, led us to investigate substitu-
tions on the N-1 aryl ring of the pyrazole ring (R3 and
R4) while keeping the methoxy group intact. Elimination
of the R4 p-chloro group resulted in a lower binding
analogue 7d . However, its lower clogD value led us to
explore different substituents at the ortho-position (R3).
The replacement of the o-chloro group with an o-fluoro
7f and o-methoxy 7e produced low affinity analogues.
Comparison of the R3 subseries of compounds 7d , 7e,
and 7f demonstrates the extreme sensitivity of R3
substituent toward receptor recognition.
Substitution of the C-4 methyl of the pyrazole ring
with a bromo substituent (7h ) led to a more lipophilic
compound possessing enhanced binding affinity. How-
ever, replacement of the C-4 methyl in SR141716 with
hydrogen yielded a less lipophilic compound 7g, with
lower binding affinity. The presence of a fluoro group
in the position C-4 lowered the affinity and lipophilicity
(compare 7i with 7d and 7j). Thus, it seems reasonable
Con clu sion
We have synthesized a series of SR141716 (3) ana-
logues as potential ligands for PET studies of cannab-
inoid receptors targeting high affinity ligands with low
lipophilicity. Some of these compounds possess lower
lipophilicity values and comparable binding affinity to

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 645
(6) Katoch-Rouse, R.; Horti, A. G. Synthesis of N-(piperidin-1-yl)-
5-(4-methoxyphenyl)-1-(2-chlorophenyl)-4-[18F]fluoro-1H-pyrazole-
3-carboxamide by nucleophilic [18F]fluorination: a PET radiotrac-
er for studying CB1 cannabinoid receptors. J . Labeled Cpd.
Radiopharm. 2003, 46, 93-98.
(7) Lan, R.; Liu, Q.; Fan, P.; Lin, S.; Fernando, S. R.; McCallion,
D.; Pertwee, R.; Makriyannis, A. Structure-activity relation-
ships of pyrazole derivatives as cannabinoid receptor antago-
nists. J . Med. Chem. 1999, 42, 769-776.
(8) Penning, T. D.; et al. Synthesis and biological evaluation of the
1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identi-
fication of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-
1-yl]benzenesulfonamide (SC-58635, celecoxib). J . Med. Chem.
1997, 40, 1347-1365.
3. A facile method for the synthesis of C-4 fluoro
analogues of 3 has been developed. Moreover, using a
functional electrophysiological assay, we have demon-
strated that several compounds of the series (7a , 7c,
and 7d ) possess antagonistic properties and are able
to rapidly and completely reverse the effects of WIN
55,221-2.
Currently, 7a ,17 the methoxy analogue of 3, has been
selected as a potential target for the development of
corresponding 11CH3-labeled derivatives.
(9) Stavber, S.; J ereb, M.; Zupan, M. Solvent directing immediate
fluorination of aromatic ketones using 1-fluoro-4-hydroxy-1,4-
diazoniabicyclo[2.2.2] octane bis(tetrafluoroborate). Chem. Com-
mun. 2000, 1323-1324.
Ack n ow led gm en t. The authors would like to thank
Dr. Amina Woods for providing mass spectroscopy data.
The research was supported by Intramural Research
Program of the National Institute on Drug Abuse, NIH.
(10) Storer, R.; et al. The synthesis and antiviral activity of 4-fluoro-
1-â-D-ribofuranosyl-1H-pyrazole-3-carboxamide. Nucleosides Nu-
cleotides 1999, 18, 203-216.
Su p p or tin g In for m a tion Ava ila ble: Experimental data.
Correlation of binding affinity versus lipophilicity (Figure 1).
Functional reversal of CB agonist mediated inhibition of
potentials in brain slices by SR141716 analogues (Figure 2).
This material is available free of charge via the Internet at
http://pubs.acs.org.
(11) Lombardo, F.; Shalaeva, M. Y.; Tupper, K. A.; Gao, F. Elog Doct
:
a tool for lipophilicity determination in drug discovery. 2. Basic
and neutral compounds. J . Med. Chem. 2001, 44, 2490-2497.
(12) ACD/Log D Suite, v.4.5. Advanced Chemistry Development:
Toronto, Ontario, Canada.
(13) Gatley, S. J .; Lan, R.; Pyatt, B.; Gifford, A. N.; Volkow, N. D.;
Makriyannis, A. Binding of nonclassical cannabinoid CP 55,-
940, and the diarylpyrazole AM251 to rodent brain cannabinoid
receptors. Life Sci. 1997, 61, PL 191-197.
(14) Wiley, J . L.; J efferson, R. G.; Grier, M. C.; Mahadevan, A.;
Razdan, R. K.; Martin, B. R. Novel pyrazole cannabinoids:
insights into CB1 receptor recognition and activation. J . Phar-
macol. Exp. Ther. 2001, 296, 1013-1022.
Refer en ces
(1) Kessler, R. M.; Ansari, M. S.; de Paulis, T.; Schmidt, D. E.;
Clanton, J . A.; Smith, H. E.; Manning, R. G.; Gillespie, D.; Ebert,
M. H. High affinity dopamine D2 receptor radioligands. 1.
Regional rat brain distribution of iodinated benzamides. J . Nucl.
Med. 1991, 32, 1593-1600.
(2) Mathews, W. B.; Ravert, H. T.; Musachio, J . L.; Frank, R. A.;
Rinaldi-Carmona, M.; Barth, F.; Dannals, R. F. Synthesis of [18F]
SR144385: a selective radioligand for positron emission tomo-
graphic studies of brain cannabinoid receptors. J . Labeled Cpd.
Radiopharm. 1999, 42, 589-596.
(3) Mathews, W. B.; Scheffel, U.; Finley, P.; Ravert, H. T.; Frank,
R. A.; Rinaldi-Carmona, M.; Barth, F.; Dannals, R. F. Biodis-
tribution of [18F] SR144385 and [18F]SR147963: selective radio-
ligands for positron emission tomographic studies of brain
cannabinoid receptors. Nuc. Med. Biol. 2000, 27, 757-762.
(4) Lan, R.; Gatley, S. J .; Makriyannis, A. Preparation of iodine-
123 labeled AM251: a potential SPECT radioligand for the brain
cannabinoid CB1 receptors. J . Labeled Cpd. Radiopharm. 1996,
38, 875-881.
(5) 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 radio-
ligand that binds to cannabinoid CB1 receptors in vivo. J .
Neurochem. 1998, 70, 417-423.
(15) Robbe, D.; Alonso, G.; Duchamp, F.; Bockaert, J .; Manzoni, O.
J . Localization and mechanisms of action of cannabinoid recep-
tors at the glutamatergic synapses of the mouse nucleus accum-
bens. J . Neurosci. 2001, 21, 109-116.
(16) See Supporting Information.
(17) Caulder, T.; Katoch-Rouse, R.; Horti, A. G.; Bell, J . A.; Hoffman,
A. F. Comparison of cannabinoid-mediated inhibition of excita-
tory transmission in the core and shell of the nucleus accumbens.
Presented at the Society for Neuroscience Meeting, San Diego,
CA, Nov 10-15, 2001.
(18) Bridge, C. F.; O’Hagan, D. The synthesis of R-fluoroketones by
1,4-additions of mono-fluorinated enamines to Michael acceptors.
J . Fluorine Chem. 1997, 82, 21-24.
(19) Thomas, B. F.; Gilliam, A. F.; Burch, D. F.; Roche, M. J .;
Seltzman, H. H. Comparative receptor binding analyses of
cannabinoid agonists and antagonists. J . Pharmacol. Exp. Ther.
1998, 285, 285-292.
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