Two distinct classes of novel pyrazolinecarboxamides as potent cannabinoid CB1 receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2010.07.056

The synthesis and SAR of 3-alkyl-4-aryl-4,5-dihydropyrazole-1-carboxamides 1-23 and 1-alkyl-5-aryl-4,5-dihydropyrazole-3-carboxamides 24-27 as two novel cannabinoid CB₁ receptor agonist classes were described. The target compounds elicited high

Full text of DOI:10.1016/j.bmcl.2010.07.056

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4992–4998
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Two distinct classes of novel pyrazolinecarboxamides as potent cannabinoid
CB1 receptor agonists
*
Jos H. M. Lange , Amos Attali, Martina A. W. van der Neut, Henri C. Wals, Arie Mulder, Hicham Zilaout,
Ate Duursma, Hans H. M. van Aken, Bernard J. van Vliet
Abbott Healthcare Products B.V., Chemical Design & Synthesis Unit, C.J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR of 3-alkyl-4-aryl-4,5-dihydropyrazole-1-carboxamides 1–23 and 1-alkyl-5-aryl-
4,5-dihydropyrazole-3-carboxamides 24–27 as two novel cannabinoid CB1 receptor agonist classes were
described. The target compounds elicited high affinities to the CB1 as well as the CB2 receptor and were
found to act as CB1 receptor agonists. The key compound 19 elicited potent CB1 agonistic and CB2 inverse
agonistic properties in vitro and showed in vivo activity in a rodent model for multiple sclerosis after oral
administration.
Received 28 June 2010
Revised 13 July 2010
Accepted 14 July 2010
Available online 17 July 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Cannabinoid
CB1 receptor
Agonist
EAE assay
Multiple sclerosis
SAR
Pyrazoline
CNS/plasma ratio
Cannabinoids constitute an area of intensive research since the
endocannabinoid system plays an important role in many physiolog-
ical processes.1,2 Cannabinoid CB1 receptor agonists have good pros-
pects for the treatment of various disorders such as (neuropathic)
pain, inflammation, multiple sclerosis, glaucoma, gastrointestinal
disorders, and chemotherapy-induced nausea and vomiting.3,4 The
cannabinoid receptor has a high density in several brain areas and
is also found in peripheral tissues including the eye, gastrointestinal
tract, liver, pancreas, prostate, testis, adipose tissue, and heart. Both
CB1 and CB2 receptor belong to the class A, G-protein-coupled recep-
tor (GPCR) superfamily.
pattern.11,12 Since many CB1 receptor agonists contain a flexible al-
kyl chain (cf. Fig. 1) it was anticipated that replacement of one of
the vicinal aryl groups by a flexible alkyl chain in our 3,4-diarylpy-
razoline antagonist chemotype might lead to a functional switch
from CB1 antagonism to CB1 receptor agonism.
These considerations resulted in the design of the target com-
pounds 1–27. The synthesis of the target compounds 1–21 is
depicted in Scheme 1.
Ketones 28–32 are either commercially available or easily
accessible, for example, via the reaction of the appropriate
Weinreb amide with a Grignard reagent ArCH2MgCl in THF at
0 °C, followed by acidification in 4 N HCl. The enones 33–37 were
obtained from the corresponding ketones 28–32, respectively,
using a Mannich reaction/elimination sequence6 and further cyclo-
condensed in the presence of hydrazineꢀH2O into the pyrazolines
38–42. Subsequent isocyanate addition at room temperature deliv-
ered the target compounds 1–13 (Route 1). Alternatively, the pyr-
azoline 38 was converted in situ to the corresponding carbonyl
chloride 43 by treatment with diphosgene at ambient temperature.
Subsequent treatment with the appropriate amines R1–NH2 in the
presence of Hünig’s base furnished the target compounds 14–18
(Route 2). Preparative HPLC separation of the endo-[1R,2S,4R]-bor-
nyl derivative 9 produced the pure diastereomers (ꢁ)-19 and (+)-
20, respectively. The 1-naphthoyl substituted pyrazoline 21 was
prepared from 38 in moderate chemical yield.
Both naturally occurring CB1 receptor agonists (e.g., the endo-
cannabinoid anandamide and the herbal
D
9-tetrahydrocannabinol
(dronabinol)) and synthetic cannabinoids (e.g., CP-55,940, WIN
55,212-2, and nabilone) have been disclosed5 (Fig. 1).
Previous research efforts concentrated on diarylpyrazoline
derivatives as CB1/CB2 subtype selective CB1 receptor antago-
nists/inverse agonists.6–10 However, pyrazoline-based CB1 receptor
agonists have hitherto not been reported. This prompted the intro-
duction of the pyrazoline heterocyclic template in CB1 receptor
agonist drug design. It has been reported that the CB1 receptor
pharmacophore incorporates
a
vicinal diaryl substitution
* Corresponding author. Tel.: +31 (0) 294 479731; fax +31 (0) 294 477138.
E-mail address: jos.lange@abbott.com (J.H.M. Lange).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.056

J. H. M. Lange et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4992–4998
4993
OH
O
O
O
OH
OH
OH
OH
H
H
N
H
H
H
O
N
O
O
O
N
OH
CP 55,940
Anandamide
Δ9-THC (dronabinol)
Nabilone
WIN 55,212-2
Figure 1. Representative naturally occurring and synthetic CB1 receptor agonists.
g
R
R
R
R
R
Ar
Ar
Ar
Cl
Ar
Ar
d
c
b
a
N
N
N
O
N
N
H
Route 2
N
N
N
O
O
O
NHR1
O
28-32
33-37
38-42
43
1-18
f
e
21
9
(-)-19 + (+)-20
Route 1
Compound
R
Ar
Route
1
R1
Compound
R
Ar
Route
1
R1
10
H
C6H5
1
CH3 C6H5
11
H
3-F-C6H4
1
CH3
CH3
2
3
4
5
6
7
8
9
C6H5
C6H5
1
1
1
1
1
1
1
1
F
H
12
13
14
H
H
2-F-C6H4
3-thienyl
1
1
2
F
H
CH3 C6H5
CH3 C6H5
CH3 C6H5
CH3
CH3
C6H5
C6H5
15
16
17
18
2
2
2
2
C6H5
CH3
CH3 C6H5
CH3 C6H5
CH3 C6H5
CH3
C6H5
CH3
C6H5
F3C
Scheme 1. Reagents and conditions: (a) CH2O (35% aq), HOAc, piperidine, MeOH, 55 °C, 60 h (92%); (b) H2NNH2ꢀH2O, EtOH, N2, reflux, 4 h; (c) triphosgene (0.33 mol equiv),
DIPEA, CH2Cl2, 30 °C, 1 h; (d) R1–NH2, DIPEA, CH2Cl2, 30 °C, 18 h; (e) R1–N@C@O, Et3N (cat.), benzene, rt, 16 h (60–70%); (f) Chiral preparative HPLC separation, stationary
phase: ChiralpakÒ AD 20
lm, mobile phase: acetone/methanol (95:5 (v/v), flow rate 2 ml/min; (g) 1-naphthoyl chloride, toluene, rt, 16 h (53%).
The target compounds 22 and 23 had to be prepared by an alter-
native route since the Mannich reaction/elimination sequence as
outlined above (Scheme 1)—but with the enolisable acetone in-
stead of formaldehyde—was unsuccessful. This alternative route
is depicted in Scheme 2.
Addition of the isopropyl group to the ketone 29 under basic
conditions13 provided 44 in a moderate yield. Subsequent radical
bromination of 44 with N-bromosuccinimide, followed by an elim-
ination of HBr at elevated temperature in dimethylformamide fur-
nished the desired enone 45 which was converted in the presence

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J. H. M. Lange et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4992–4998
Compound
R1
b, c
a
d
e
N
O
O
O
22
23
N
N
N
H
29
44
45
O
R1
N
H
46
22-23
Scheme 2. Reagents and conditions: (a) 2-iodopropane, NaOCH3, N2, reflux, 1 h (40%); (b) NBS, dibenzoyl peroxide (cat.), CCl4, reflux, 6 h, (ꢂ100%); (c) LiCl, DMF, N2, 130 °C,
90 min (46%); (d) H2NNH2ꢀH2O, EtOH, N2, reflux, 4 h; (e) R1–N@C@O, Et3N (cat.), toluene, rt, 16 h (55–58%).
H
O
b, c
a
d
e
N
NH
N
N
N
N
N
N
CO2Et
CO2H
CO2Et
48
CO2Et
O
N
H
47
50
49
24
Scheme 3. Reagents and conditions: (a) n-pentylhydrazine, EtOH, 80 °C, 16 h (93%); (b) NCS, EtOAc, N2, 60 °C, 1 h; (c) styrene, NaHCO3, H2O, 70 °C, 16 h (22%); (d) LiOH, THF,
H2O, 70 °C, 1 h, followed by acidification (Et2O, 1 N HCl) (74%); (e) 2-adamantamineꢀHCl, DIPEA, CIP, CH2Cl2, N2, rt, 16 h (67%).
of H2NNH2ꢀH2O to the pyrazoline 46 as outlined above for 38–42.
Reaction with the appropriate isocyanates smoothly yielded the
target compounds 22 and 23.
chidonic acid release functional assay,6 using the same recombinant
cell line. CB1 agonist stimulation leads to activation of PLA2 followed
by release of [3H]arachidonic acid into the medium.
Besides the 3-alkyl-4-aryl-4,5-dihydropyrazole-1-carboxamides
1–23 we were also interested in the structurally related—but dis-
tinct—1-alkyl-5-aryl-4,5-dihydropyrazole-3-carboxamides 24–27.
The synthesis of 24 is depicted in Scheme 3.
The commercially available oxoacetic acid ester 47 was reacted
with n-pentylhydrazine14 to give 48. Subsequent chlorination with
N-chlorosuccinimide, followed by cyclization with styrene10 affor-
ded the ester 49 which was saponified into the corresponding acid
50. Target compound 24 was obtained by amidating 50 with
2-adamantamineꢀHCl in the presence of the coupling reagent CIP
in 67% yield.
The synthesis of the target compounds 25–27 is shown in
Scheme 4. Amidation of the carboxylic acids15 51 and 52 in the
presence of the coupling reagent HBTU led to the amides 53–55,
respectively. Treatment of 53–55 with n-pentylhydrazine in etha-
nol produced the target compounds 25–27 in moderate yields.
The pharmacological results of the reference cannabinoid recep-
tor agonists WIN 55,212-2,16,17 CP-55,940,16,17 nabilone,18,19 and the
target compounds 1–27 are given in Table 1. They were evaluated
in vitro at the human CB1 and CB2 receptor, stably expressed into
Chinese hamster ovary (CHO) cells,6 utilizing radioligand binding
studies (displacement of the specific binding of [3H]-CP-55,940).
CB1 receptor agonism was measured using a CP-55,940 induced ara-
The compounds 1–27 elicited moderate to high CB1 receptor
affinities. In the 3-alkyl-4-aryl-4,5-dihydropyrazole-1-carboxam-
ides series (target compounds 1–23), the compounds bearing a
bulky and apolar carboxamide N-substituent—such as 2, 7, 19,
and 22—showed single digit nanomolar CB1 receptor affinities
comparable to the values obtained for nabilone. In accordance with
this SAR trend, the alternative 1-alkyl-5-aryl-4,5-dihydropyrazo-
line series (target compounds 24–27) contained three of such high
CB1 receptor binders, viz. the 2-adamantyl substituted 24 and the
endo-bornyl substituted 26 and 27.
The compounds 1–27 also elicited moderate to high CB2 recep-
tor affinities. Again, the target compounds bearing a bulky and
apolar carboxamide N-substituent—such as 2, 4, 9, 19, 23, 24,
and 27—showed single digit nanomolar CB2 receptor affinities
comparable to the values obtained for nabilone. It should be noted
that the degree of observed CB1/CB2 receptor subtype selectivity of
the target compounds 1–27 is modest, their values ranging from
0.21 to 5.4. These CB1/CB2 selectivity values are in the same range
as those obtained for CP-55,940 and nabilone.
In general, the target compounds were found to behave as CB1
receptor agonists. The target compounds 9, 19, and 24–27 showed
pA2 values ranging from 8.1 to 9.2 which are comparable to the ob-
served values of nabilone and CP-55,940, respectively.
Compound
R
R1
R
R
N
N
R
F
25
H
a
b
O
O
O
O
26
27
H
F
O
OH
NHR1
NHR1
51-52
53-55
25-27
Scheme 4. Reagents and conditions: (a) R1–NH2, DIPEA, O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU), CH2Cl2, rt, 16 h (60–70%);
(b) n-pentylhydrazine, HOAc, EtOH, N2, 60 °C, 8 h (40–50%).

J. H. M. Lange et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4992–4998
4995
Table 1
Pharmacological in vitro results of the reference compounds WIN 55,212-2, CP-55,940, nabilone, and the target compounds 1–27
Compound
Ki (CB1),a (nM)
pEC50 (CB1),b
Ki (CB2),c (nM)
CB1/CB2 selectivityd
WIN 55,212-2
CP-55,940
Nabilone
1
2
3
4
5
6
94.5 18
1.36 0.18
5.1 1.6
68 32
6.4 2.1
15.6 11.5
16.1 2.1
15.3 2.9
51 13
7.5 0.4
8.9 0.1
8.4 0.1
6.4e
9.1 3.4
0.62 0.09
17.6g
14.9 5.9
6.3 3.4
13.3 3.3
8.0 2.3
53 16
260 60
36.6 8.9
17.6f
7.4 3.3
35.6 19.7
74 11
0.1
0.46
3.5
0.22
1.0
0.85
0.50
3.5
5.1
4.9
5.4
0.26
2.5
1.6
2.8
3.2
1.5
1.3
2.2
0.68
0.21
0.64
0.77
0.63
1.3
0.21
0.77
1.0
7.6e
5.7e
5.7 0.4
n.d.f
6.4e
7
8
9
7.4 0.5
5.8e
3.26 1.27
28.5 14.1
14.2 3.0
44.9 16.4
32.0 8.1
19.3 15.5
52 19
15.3 8.7
11.9 4.9
33.1 15.9
70 44
8.4e
9.2 0.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
8.4e
7.0 0.1
6.9 0.2
7.4 0.2
n.d.f
89 19
62 17
79g
n.d.f
19.8 6.8
26.6 6.6
22.5 4.7
15.0 5.0
5.3 0.8
36.0 3.7
103g
11.9 3.1
3.4 0.4
7.1 1.5
121 23
20.1 9.2
9.1 3.1
n.d.f
n.d.f
n.d.f
8.3 3.4
8.4 0.4
6.8 0.3
7.5 0.5
8.1e
7.4 0.3
8.1 0.1
8.4 0.4
8.3 0.2
8.4 0.2
46.8 5.7
164 118
9.0 3.7
16.1 2.1
9.2 2.6
118 68
4.7 1.9
6.1 1.8
4.3
1.5
a
Displacement of specific CP-55,940 binding in CHO cells stably transfected with human CB1 receptor, expressed as Ki SEM (nM). The values represent the mean result
based on at least three independent experiments, unless otherwise noted.
b
[3H]Arachidonic acid release in CHO cells expressed as pEC50 SEM values. The values represent the mean result based on at least three independent experiments, unless
otherwise noted.
c
Displacement of specific CP-55,940 binding in CHO cells stably transfected with human CB2 receptor, expressed as Ki SEM (nM). The values represent the mean result
based on three independent experiments, unless otherwise noted.
d
CB1/CB2 selectivity values are provided as single values instead of their ranges based on the underlying CB1 and CB2 receptor affinity SEM values.
Result of single measurement.
n.d., not determined.
Result of duplicate measurement.
e
f
g
It is interesting to note that 19 showed significantly higher CB1
and CB2 receptor affinities and a higher CB1 agonistic potency than
its diastereomeric counterpart 20, indicating that these chiral
ligands bind to some extent stereoselectively to the CB1 and CB2
receptor.
with the reported results of other CB1 receptor agonists (data not
shown).
More interestingly, it was decided to test the effect of our CB1
receptor agonist 19 on the disease course of acute EAE by oral
administration in the male Lewis rat starting the day after the
appearance of the first paralysis symptoms in the entire cohort
(day 11). The results are depicted in Figure 2. Compound 19
(20 mg/kg po, once daily) significantly (p < 0.0001) reduced the
EAE induced motor deficits by reducing the intensity of the paral-
ysis (AUC and average score).
At the end of the EAE experiment, defined as the day all animals
had completely recovered, plasma and brains were sampled at sev-
eral time points (0.5, 1, 3, 7, 14, and 24 h) after the last administra-
tion of 19 to determine the exposure levels and CNS/plasma ratio
after repeated administration ( Fig. 3). These data revealed that
the levels of 19 in CNS as well as plasma returned to the basis with-
in 24 h and thereby corroborate the absence of accumulation of 19
during the course of the EAE experiment. In addition, these data
showed a significant CNS exposure of 19. It is interesting to note
that the levels of 19 at the end of the experiment were comparable
to the values obtained during day 1 (data not shown) which is an
indication that the degree of its metabolic breakdown remained
unaffected during the course of the EAE experiment.
Reference compounds such as anandamide,
D
9-tetrahydrocan-
nabinol, WIN 55,212-2, CP-55,940, HU210, O-2545, and nabilone
have been reported to act as agonists at both the CB1 receptor
and the CB2 receptor. Intriguingly, compound 19 elicited a potent
antagonistic effect (dose-dependent antagonism of the selective
CB2 receptor agonist JWH133) on human CB2 receptor mediated
adenylate cyclase activity in vitro (pA2 = 9.2), which nicely corre-
sponded to the obtained value (pA2 = 9.4) in our human CB2 cAMP
accumulation assay.20,21 Furthermore, our CB2 cAMP accumulation
assay revealed significant inverse agonistic properties of 19
(pEC50 = 8.5 0.3) in the absence of JWH133 at the constitutively
active20 CB2 receptor. It can be concluded that 19 has a distinct
cannabinoid in vitro profile as compared to the CB1/2 agonistic ref-
erence compounds mentioned hereinabove.
The Experimental Autoallergic Encephalomyelitis (EAE) mod-
el22 is regarded as a key pharmacological model for multiple scle-
rosis.23–25 It has been reported26 in animal studies that the
cannabinoid system is neuroprotective27 during EAE. In such re-
ported experiments the administration of the CB1 receptor agonist
was started from day one of the EAE experiment. By using such a
dosing regime it was observed that 19 by oral administration in
the male Lewis rat reduced the EAE induced motor deficits in line
In general, cannabinoid CB1 receptor agonists are known as
highly lipophilic compounds. Despite the high calculated log D
value of 19 (5.7 at pH 7.4), both its relatively low molecular
weight and its low polar surface area (calculated PSA = 45 Å2)

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J. H. M. Lange et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4992–4998
Figure 2. In vivo results of 19 in the EAE model.
1200
1000
800
600
400
200
0
900
800
700
600
500
400
300
200
100
0
Plasma levels
CNS levels
0
0.5
1
3
7
14
24
Post Drug Time (hr)
Figure 3. Plasma and CNS levels at different time points after the last administration of 19 in the EAE experiment.
3. Pertwee, R. G. Br. J. Pharmacol. 2009, 156, 397.
were anticipated to favorably contribute to its CNS penetra-
bility.
4. Ware, M. A.; Daeninck, P.; Maida, V. Ther. Clin. Risk Manag. 2008, 4, 99.
5. Thakur, G. A.; Tichkule, R.; Bajaj, S.; Makriyannis, A. Expert Opin. Ther. Patents
2009, 19, 1647.
6. Lange, J. H. M.; Coolen, H. K. A. C.; van Stuivenberg, H. H.; Dijksman, J. A. R.;
Herremans, A. H. J.; Ronken, E.; Keizer, H. G.; Tipker, K.; McCreary, A. C.;
Veerman, W.; Wals, H. C.; Stork, B.; Verveer, P. C.; den Hartog, A. P.; de Jong, N.
M. J.; Adolfs, T. J. P.; Hoogendoorn, J.; Kruse, C. G. J. Med. Chem. 2004, 47, 627.
7. Lange, J. H. M.; van Stuivenberg, H. H.; Veerman, W.; Wals, H. C.; Stork, B.;
Coolen, H. K. A. C.; McCreary, A. C.; Adolfs, T. J. P.; Kruse, C. G. Bioorg. Med. Chem.
Lett. 2005, 15, 4794.
In conclusion, two structurally related pyrazoline classes28 were
presented as a novel CB1 receptor agonist chemotype. The target
compounds 1–27 elicited high affinities to the CB1 and CB2 recep-
tor and were in general found to act as CB1 receptor agonists. The
key compound 19 showed oral in vivo activity in a rodent model
for multiple sclerosis (EAE), even if treatment was started a day
after the first symptom was observed.
8. Srivastava, B. K.; Joharapurkar, A.; Raval, S.; Patel, J. Z.; Soni, R.; Raval, P.; Gite,
A.; Goswami, A.; Sadhwani, N.; Gandhi, N.; Patel, H.; Mishra, B.; Solanki, M.;
Pandey, B.; Jain, M. R.; Patel, P. R. J. Med. Chem. 2007, 50, 5951.
9. Lange, J. H. M.; Kruse, C. G. Chem. Rec. 2008, 8, 156.
Acknowledgments
10. Lange, J. H. M.; van der Neut, M. A. W.; den Hartog, A. P.; Wals, H. C.;
Hoogendoorn, J.; van Stuivenberg, H. H.; van Vliet, B. J.; Kruse, C. G. Bioorg. Med.
Chem. Lett. 2010, 20, 1752.
Chris Kruse is gratefully acknowledged for his helpful sugges-
tions, Pieter Spaans for analytical support, Harry Hiemstra for sta-
tistical support, Petra den Ridder-Lubbers and Sandra de Fluiter-
Mittelmeijer for pharmacology support, Jessica Dijksman for bio-
analytical support, and Syncom B.V. (The Netherlands) for syn-
thetic support.
11. Reggio, P. H. Curr. Pharm. Des. 2003, 9, 1607.
12. Lange, J. H. M.; Kruse, C. G. Drug Discovery Today 2005, 10, 693.
13. Myers, T. C.; Pratt, R. J.; Morgan, R. L.; O’Donnell, J.; Jensen, E. V. J. Am. Chem.
Soc. 1955, 77, 5655.
14. Stroh, H.-H.; Scharnow, H.-G. Chem. Ber. 1965, 98, 1588.
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separated and successively washed with an aqueous Na2S2O3 solution and
water. The organic layer was dried over Na2SO4, filtered and concentrated. The
residue was further purified using Sepacore equipment: (petroleum ether/
Et2O = 19:1 (v/v)) to give 44 (3.52 g) as a colorless oil; 1H NMR (400 MHz,
CDCl3) d 0.66 (d, J = 7, 3H), 0.81 (t, J = 7, 3H), 0.96 (d, J = 7, 3H), 1.10–1.24 (m,
2H), 1.36–1.54 (m, 2H), 2.29–2.47 (m, 3H), 3.30 (d, J = 10 Hz, 1H), 7.20–7.33 (m,
5H). To a magnetically stirred solution of 44 (4.31 g, 0.02 mol) in CCl4 (40 ml)
was added a catalytic amount of dibenzoyl peroxide and NBS (6.56 g). The
resulting mixture was heated for 6 h at reflux temperature. The obtained
mixture was allowed to attain rt. The formed precipitate was removed by
filtration. The filtrate was concentrated to give crude 2-methyl-3-bromo-3-
24. Owens, T.; Sriram, S. Neurol. Clin. 1995, 13, 51.
25. Steinman, L.; Zamvil, S. S. Ann. Neurol. 2006, 60, 12.
26. Pryce, G.; Ahmed, Z.; Hankey, D. J. R.; Jackson, S. J.; Croxford, J. L.; Pocock, J. M.;
Ledent, C.; Petzold, A.; Thompson, A. J.; Giovannoni, G.; Cuzner, M. L.; Baker, D.
Brain 2003, 126, 2191.
27. De Lago, E.; Gómez-Ruiz, M.; Moreno-Martet, M.; Fernández-Ruiz, J. Expert Rev.
Clin. Pharmacol. 2009, 2, 645.
phenyloctan-4-one as a dark-colored oil (6.77 g) which was dissolved in
anhydrous DMF (35 ml) under a N2 atmosphere. LiCl (3.2 g, 0.075 mol) was
added and the resulting mixture was heated at 130 °C for 90 min. The resulting
mixture was allowed to attain rt and was subsequently poured into water and
extracted with Et2O. The organic layer was separated and washed with water
(four portions). The organic layer was dried over Na2SO4, filtered and
concentrated. The obtained residue was purified using Sepacore equipment:
(petroleum ether/Et2O = 98:2 (v/v)) to give 45 (1.96 g, 46% yield) as a pale
yellow oil; 1H NMR (400 MHz, CDCl3) d 0.81 (d, J = 7, 3H), 1.13–1.24 (m, 2H),
1.43–1.52 (m, 2H), 1.66 (s, 3H), 2.00 (s, 3H), 2.25 (t, J = 7, 2H), 7.15 (br d, J = 8,
28. Yields refer to isolated pure products unless otherwise noted and were not
maximized. Coupling constants (J) are expressed in Hz. Flash chromatography
was performed using silica gel 60 (0.040–0.063 mm, Merck). Sepacore
chromatographic separations were carried out using Supelco equipment,
VersaFLASH™ columns, VersaPak™ silica cartridges, Büchi UV monitor C-
630, Büchi pump module C-605, Büchi fraction collector C-660, and Büchi
pump manager C-615. Selected data for target compounds 3, 13, 19–22, 24, 25,
and 27, the protocols for the in vitro cannabinoid-CB2 receptor antagonism
assay and the acute EAE assay. Synthesis of compound 3: To a magnetically
stirred solution of hexanoic acid methoxymethylamide (12.2 g, 77 mmol) at
0 °C in THF was slowly added BnMgCl (20% in THF, 90 ml, 116 mmol) and the
resulting mixture was reacted for 2 h. The reaction mixture was poured in
excess HCl (4 N) and extracted with tert-butyl-methyl ether (MTBE).
Concentration, followed by flash chromatographic purification (heptane/
EtOAc = 40:1 (v/v)) gave 28 (11.6 g, 79% yield) as an oil; 1H NMR (300 MHz,
CDCl6) d 0.86 (t, J = 7, 3H), 1.20–1.27 (m, 4H), 1.52–1.60 (m, 2H), 2.40–2.46 (m,
2H), 3.68 (s, 2H), 7.18–7.33 (m, 5H). To a magnetically stirred solution of 28
(11.6 g, 61 mmol) in MeOH (100 ml) was added piperidine (1 ml) and HOAc
(1 ml), followed by formalin (20 ml (35% aq.), 226 mmol) and the resulting
mixture was stirred at 55 °C for 60 h. The reaction mixture was cooled to rt,
concentrated and taken up in a mixture of MTBE and water. The organic layer
was collected, dried over Na2SO4, filtered and concentrated to give 33 (11.4 g,
92% yield) as an oil. 1H NMR (400 MHz, CDCl3) d 0.80 (t, J = 7, 3H), 1.18–1.30
(m, 4H), 1.54–1.63 (m, 2H), 2.65 (t, J = 7, 2H), 5.80 (s, 1H), 6.02 (s, 1H), 7.20–
7.32 (m, 5H). To a magnetically stirred solution of 33 (5 g, 24.7 mmol) in EtOH
(30 ml) was added hydrazineꢀH2O (2.46 ml, 50.7 mmol) and the resulting
solution was heated at reflux temperature for 4 h. The resulting solution was
allowed to attain rt, concentrated and taken up in a mixture of MTBE and
water. The organic layer was collected, dried over Na2SO4, filtered and
concentrated to give crude 38 (4.8 g) as an impure oil which was used
immediately in the subsequent step. Compound 38 (2.2 g, 10.3 mmol) was
dissolved in benzene (25 ml) and treated with cis-myrtanylisocyanate (2.12 g,
11.8 mmol)—which was prepared from (ꢁ)-cis-myrtanylamine (CAS 38235-68-
6) and diphosgene in CH2Cl2 at 0 °C—and five drops of Et3N and the resulting
solution was stirred at rt for 16 h. The solution was concentrated, followed by
flash chromatographic purification (heptane/EtOAc = 6:1 (v/v)) to give 3 as an
oil. 1H NMR (400 MHz, CDCl3) d 0.85–0.95 (m, 4H), 1.06 (s, 3H), 1.19–1.31 (m,
7H), 1.38–1.60 (m, 3H), 1.82–2.41 (m, 9H), 3.22–3.40 (m, 2H), 3.83–3.90 (m,
1H), 4.12 (dd, J = 12 and 7, 1H), 4.18–4.26 (m, 1H), 5.92–5.96 (m, 1H), 7.15 (br
d, J = 8, 2H), 7.25–7.37 (m, 3H). Compound 10: 1H NMR (400 MHz, CDCl3) d 0.86
(t, J = 7, 3H), 1.21–1.33 (m, 2H), 1.38–1.54 (m, 2H), 1.75 (s, 3H), 1.77 (s, 3H),
2.04–2.22 (m, 2H), 3.82 (dd, J = 9.7 and 5.6, 1H), 4.07–4.20 (m, 2H), 6.38 (br s,
1H), 7.13–7.36 (m, 8H), 7.48 (br d, J = 8, 2H). Compound 13: 1H NMR (400 MHz,
CDCl3) d 0.85 (t, J = 7, 3H), 1.21–1.57 (m, 4H), 1.74 (s, 3H), 1.77 (s, 3H), 2.05–
2.25 (m, 2H), 3.79 (dd, J = 11 and 7, 1H), 4.08–4.13 (m, 1H), 4.28 (dd, J = 11 and
7, 1H) 6.36 (br s, 1H), 6.91 (dd, J = 6 and 2, 1H), 7.06–7.08 (m, 1H), 7.19–7.24
(m, 1H), 7.30–7.37 (m, 3H), 7.45–7.49 (m, 2H); HRMS exact mass calcd for
2H), 7.20–7.39 (m, 3H). To
a magnetically stirred solution of 45 (1.96 g,
9.07 mmol) in abs EtOH (15 ml) was added hydrazineꢀhydrate (0.88 ml,
18.14 mmol) and the resulting solution was heated at reflux temperature for
4 h under a N2 atmosphere. The resulting solution was allowed to attain rt,
concentrated and taken up in a mixture of MTBE and water. The organic layer
was collected, dried over MgSO4, filtered and concentrated to give crude 46
(2.06 g) as an oil. 1H NMR (400 MHz, CDCl3) d 0.80–0.89 (m, 6H), 1.23–1.37 (m,
5H), 1.42–1.54 (m, 2H), 2.06–2.35 (m, 2H), 3.52 (s, 1H), 4.90 (br s, 1H), 7.07 (br
d, J = 8 Hz, 2H), 7.19–7.38 (m, 3H). Crude 46 (1.03 g, ꢂ4.48 mmol) was
dissolved in toluene (10 ml) and treated with 1-methyl-1-phenyl-
ethylisocyanate (0.72 g, 4.48 mmol) and two drops of Et3N and the resulting
solution was stirred at rt for 16 h. The solution was concentrated and purified
using Sepacore equipment: (petroleum ether/diethyl ether = 85:15 (v/v)) to
give 22 as a colorless oil (0.97 g, 55% yield). 1H NMR (400 MHz, CDCl3) d 0.88 (t,
J = 7, 3H), 1.08 (s, 3H), 1.26–1.39 (m, 2H), 1.44 (s, 3H), 1.45–1.56 (m, 2H), 1.70
(s, 3H), 1.76 (s, 3H), 2.09–2.18 (m, 1H), 2.25–2.35 (m, 1H), 3.70 (s, 1H), 6.59 (br
s, 1H), 7.03 (br d, J = 8 Hz, 2H), 7.20 (br t, J = 8 Hz, 1H), 7.26–7.36 (m, 5H), 7.44
(br d, J = 8 Hz, 2H); HRMS exact mass calcd for C25H34N3O m/z 392.2702 [MH]+,
found 392.2711. Synthesis of compound 24: To a magnetically stirred solution of
47 (35.08 ml, 177 mmol; 50% solution in toluene) in EtOH (450 ml) was added
n-pentylhydrazine (21.7 g, 212 mmol) and the resulting solution was heated at
80 °C for 16 h. The obtained mixture was allowed to attain rt and concentrated.
The resulting residue was taken up in EtOAc and water. The organic layer was
separated and subsequently dried over MgSO4, filtered and concentrated to
give 48 (32.2 g, 93% yield) as a purple colored oil. 1H NMR (400 MHz, CDCl3) d
0.87–0.94 (m, 3H), 1.25–1.42 (m, 7H), 1.55–1.68 (m, 2H), 3.17–3.23 (m, 1H),
3.35–3.45 (m, 1H), 4.28 (q, J = 7, 2H), 6.51 (br s, 1H), 6.72 (s, 1H). To a
magnetically stirred solution of 48 (35.16 g, 179 mmol) in EtOAc (450 ml) was
added NCS (26.34 g, 197 mmol) and the resulting mixture was heated at 60 °C
for 1 h in
a N2 atmosphere. To the reaction mixture was added styrene
(41.1 ml, 359 mmol) and KHCO3 (89.8 g, 897 mmol) and water (8 ml). The
resulting mixture was heated at 70 °C for 16 h. The resulting mixture was
allowed to attain rt, concentrated and the resulting residue was
chromatographically separated using Sepacore equipment (CH2Cl2/
MeOH = 98:2 v/v) to give 49 (12.1 g, 22% yield) as an oil. 1H NMR (400 MHz,
CDCl3) d 0.83 (t, J = 7, 3H), 1.13–1.28 (m, 4H), 1.35 (t, J = 7, 3H), 1.53–1.67 (m,
2H), 2.89 (dd, J = 16 and 13, 1H), 3.01–3.09 (m, 1H), 3.14–3.22 (m, 1H), 3.41
(dd, J = 16 and 12, 1H), 4.31 (double (diastereotopic) quartet, J = 7, 2H), 4.63
(dd, J = 13 and 12, 1H), 7.27–7.39 (m, 5H). To a magnetically stirred solution of
49 (11.76 g, 38.74 mmol) in THF (100 ml) and water (100 ml) was added LiOH
(1.86 g, 77.5 mmol) and the resulting mixture was heated at 70 °C for 1 h. The
reaction mixture was allowed to attain rt and Et2O (200 ml) and concentrated
HCl (7 ml) were added. The organic layer was separated, washed three times
with water and with brine and subsequently dried over Na2SO4, filtered and
concentrated to give 50 (7.9 g, 74% yield) as an oil. 1H NMR (400 MHz, CDCl3) d
0.84 (t, J = 7, 3H), 1.15–1.28 (m, 4H), 1.53–1.65 (m, 2H), 2.92 (dd, J = 17 and 13,
1H), 3.02–3.11 (m, 1H), 3.18–3.27 (m, 1H), 3.44 (dd, J = 17 and 13, 1H), 4.75 t,
J = 13, 1H), 7.31–7.41 (m, 5H), 7.42–9.00 (br s, 1H). To a magnetically stirred
solution of 50 (0.70 g, 2.55 mmol) in CH2Cl2 (40 ml) was successively added 2-
adamantanamine.HCl (480 mg, 2.55 mmol), DIPEA (1.78 ml, 10.22 mmol) and
2-chloro-1,3-dimethylimidazolinium hexafluorophosphate (CIP) (853 mg,
C
21H28N3OS m/z 370.1953 [MH]+, found 370.1963. Compound 19: ([
a]D) ꢁ85 (c
1.55 g/100 ml, MeOH). 1H NMR (400 MHz, CDCl3) d 0.80–0.94 (m, 10H), 0.97 (s,
3H), 1.20–1.69 (m, 10H), 1.74–1.83 (m, 1H), 2.00–2.22 (m, 2H), 2.33–2.45 (m,
1H), 3.83–3.89 (m, 1H), 4.09–4.27 (m, 3H), 6.02 (br d, J = 10, 1H), 7.16 (br d,
J = 8, 2H), 7.27–7.37 (m, 3H). 13C NMR (100 MHz, CDCl3) d 13.73, 13.93, 18.73,
20.00, 22.31, 25.75, 28.03, 28.26, 28.46, 31.36, 38.12, 44.99, 48.00, 49.37, 53.34,
53.62, 54.41, 127.56, 127.68, 129.06, 139.71, 155.78, 158.83; HRMS exact mass
calcd for C25H38N3O m/z 396.3015 [MH]+, found 396.3028. Compound 20: ([
a]D)
+124 (c 1.3 g/100 ml, MeOH). 1H NMR (400 MHz, CDCl3) d 0.80–0.92 (m, 10H),
0.97 (s, 3H), 1.20–1.69 (m, 10H), 1.74–1.83 (m, 1H), 2.00–2.22 (m, 2H), 2.33–
2.45 (m, 1H), 3.83–3.89 (m, 1H), 4.09–4.27 (m, 3H), 6.02 (br d, J = 10, 1H), 7.16
(br d, J = 8, 2H), 7.27–7.37 (m, 3H). 13C NMR (100 MHz, CDCl3) d 13.74, 13.93,
18.74, 20.00, 22.32, 25.76, 28.05, 28.27, 28.45, 31.35, 38.20, 44.97, 47.99, 49.29,
53.30, 53.58, 54.42, 127.54, 127.64, 129.05, 139.67, 155.87, 158.88. Compound
21: 1H NMR (400 MHz, CDCl3) d 0.80–0.90 (m, 3H), 1.02–1.40 (m, 6H), 1.92–
2.11 (m, 2H), 4.21–4.30 (m, 2H), 4.57–4.65 (m, 1H), 7.20 (d, J = 8, 2H), 7.29–
7.55 (m, 6H), 7.66 (d, J = 8, 1H), 7.84–7.94 (m, 2H), 8.03 (br d, J = 8, 1H); HRMS
exact mass calcd for C25H27N2O m/z 371.2123 [MH]+, found 371.2142. Synthesis
of compound 22: To an ice-cold magnetically stirred mixture of 29 (7.04 g,
0.04 mol) and NaOCH3 (4.32 g, 0.08 mol) was added dropwise 2-iodopropane
(15 ml) in a N2 atmosphere. The resulting mixture was heated for 1 h at reflux
temperature. The obtained mixture was allowed to attain rt and concentrated.
The resulting residue was taken up in Et2O and water. The Et2O layer was
3.07 mol) and the resulting mixture was reacted at rt for 16 h in
a N2
atmosphere. The reaction mixture was successively washed twice with water,
twice with aqueous citric acid (0.5 M), twice with NaHCO3 (5% aqueous
solution) and brine, and subsequently dried over Na2SO4, filtered and
concentrated to give crude 24 (1.26 g) as an orange oil. Further
chromatographic purification using Sepacore equipment (petroleum ether/
Et2O = 85:15 (v/v)) gave 24 (750 mg, 67% yield) as an oil. 1H NMR (400 MHz,
CDCl3) d 0.85 (t, J = 7, 3H), 1.21–1.30 (m, 4H), 1.55–1.65 (m, 2H), 1.65–1.70 (m,
2H), 1.76 (br s, 2H), 1.75–1.92 (m, 8H), 1.97–2.01 (m, 2H), 2.82 (dd, J = 17 and
14, 1H), 2.92–2.97 (m, 2H), 3.42 (dd, J = 17 and 11, 1H), 4.09–4.14 (m, 1H), 4.40
(dd, J = 14 and 11, 1H), 6.99–7.07 (m, 1H), 7.28–7.38 (m, 5H); HRMS exact mass
calcd for C25H36N3O m/z 394.2858 [MH]+, found 394.2880. Compound 25: 1H
NMR (400 MHz, CDCl3) d 0.85 (t, J = 7, 3H), 1.20–1.31 (m, 4H), 1.54–1.67 (m,
2H), 1.74 (s, 3H), 1.75 (s, 3H), 2.77 (dd, J = 17 and 14, 1H), 2.90–2.97 (m, 2H),

4998
J. H. M. Lange et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4992–4998
of test compounds were determined as inhibition of 0.1
3.35 (dd, J = 17 and 11, 1H), 4.38 (dd, J = 14 and 11, 1H), 6.94 (br s, 1H), 6.98–
l
M JWH133-
7.04 (m, 2H), 7.27–7.43 (m, 7H); HRMS exact mass calcd for C24H31FN3O m/z
396.2451 [MH]+, found 396.2451. Synthesis of compound 27: To a magnetically
stirred solution of 55 in EtOH (50 ml) was successively added AcOH (660 ml,
11.58 mmol) and n-pentylhydrazine (1.45 ml, 9.65 mmol) and the resulting
mixture was reacted in a N2 atmosphere at 60 °C for 8 h in an oil bath. The
reaction mixture was allowed to attain rt and concentrated. The residue was
dissolved in CH2Cl2, washed with water and subsequently dried over MgSO4,
filtered and concentrated. Further chromatographic purification using
Sepacore equipment (petroleum ether/EtOAc = 95:5 (v/v)) gave 27 (940 mg,
46% yield) as an oil. 1H NMR (400 MHz, CDCl3) d 0.83–0.94 (m, 10H), 1.20–1.85
(m, 14H), 2.32–2.42 (m, 1H), 2.74–2.85 (m, 1H), 2.91–3.02 (m, 2H), 3.43–3.54
(m, 1H), 4.26–4.36 (m, 1H), 4.69–4.80 (m, 1H), 6.63–6.70 (m, 1H), 7.02–7.09
(m, 1H), 7.12–7.19 (m, 1H), 7.25–7.31 (m, 1H), 7.46–7.54 (m, 1H); HRMS exact
mass calcd for C25H37FN3O m/z 414.2921 [MH]+, found 414.2891. Compound
55: 1H NMR (400 MHz, CDCl3) d 0.84–1.00 (m, 10H), 1.21–1.30 (m, 1H), 1.38–
1.48 (m, 1H), 1.53–1.62 (m, 1H), 1.70–1.87 (m, 2H), 2.34–2.44 (m, 1H), 4.21–
4.30 (m, 1H), 7.10–7.25 (m, 3H), 7.38–7.45 (m, 1H), 7.70–7.75 (m, 1H), 7.85 (d,
J = 16, 1H), 8.12 (d, J = 16, 1H). In vitro cannabinoid-CB2 receptor antagonism.
Functional activity of 19 at the CB2 receptor was assessed using a forskolin-
stimulated cAMP accumulation assay in Chinese ovarian hamster (CHO) K1
cells expressing human CB2 receptor. CHO cells were grown in a CHO-S-SFM-II
culture medium, supplemented with 10% heat-inactivated fetal calf serum,
decreased [3H]cAMP formation. After aspiration, the reaction was stopped
with 1 ml trichloroacetic acid (5% w/v). The [3H]ATP and [3H]cAMP formed in
the cellular extract were assayed as follows: a volume of 0.8 ml of the extract
was passed over Dowex (50WX-4200-400 mesh) and aluminum oxide
columns, eluted with water and 0.1 M imidazole (pH 7.5). Eluates were
mixed with 7 ml Ultima-Flo [AP] and the b-radioactivity was counted with a
liquid scintillation counter. The conversion of [3H]ATP into [3H]cAMP was
expressed as the ratio in percentage radioactivity in the cAMP fraction as
compared to the combined radioactivity in both cAMP and ATP fractions, and
basal activity was subtracted to correct for spontaneous activity. Compounds
were studied in a concentration range of 10ꢁ10–10ꢁ6 M. pEC50 and pA2 values
were calculated according to Cheng–Prusoff equation. Two independent
experiments were performed in triplicate. Induction of Acute EAE in the Lewis
rat. Male Lewis rats (Harlan Laboratories B.V., the Netherlands) kept under
normal housing conditions with water and food ad libitum and weighing
between 175 and 225 g at the start of the experiment, were inoculated on day
0 as previously described.22 Briefly, a 100
50 L Complete Freund Adjuvant H37 RA (CFA, Difco Laboratories, Detroit, MI),
500 L Mycobacterium tuberculosis type H37RA (Difco) and 20 g guinea pig
lL saline based emulsion containing
l
l
l
myelin basic protein (MBP) was injected subcutaneously in the pad of left hind
paw of isoflurane anaesthetized animals. Animals were group housed per 3 and
cages were randomized across treatments. Disease symptoms and weights of
all animals were recorded daily. The following scores for motor dysfunctions
were used: 0, healthy animal with normal curling reflex at the tail; 1, paralysis
of the tip of the tail; 2, loss of muscle tone at the base of the tail; 3, low posture
of hind limbs; 4, instability at hips; 5, partial hind limb paralysis; 6, complete
hind limb paralysis; 7, paralysis include midriff; 8, quadriplegia; 9, moribund;
10, death due to EAE. All experimental procedures were approved by Abbott’s
Institutional Animal Care and Use Committee.
2 mM glutamine, 400 lg/ml Hygromycine B and 500 lg/ml G418 at 37 °C in
93% air/5% CO2. For incubation with test compounds, confluent cultures grown
in 24-well plates were used. Each condition or substance was routinely tested
in quadruplicate. Cells were loaded with 1 mCi [3H]adenine in 0.5 ml medium
per well. After 2 h, cultures were washed with 0.5 ml PBS containing 1 mM
IBMX and incubated for 20 min with 0.5 ml PBS containing 1 mM IBMX and
3 ꢃ 10ꢁ7 M forskolin with or without the test compound. Antagonistic effects