Design, synthesis, biological properties, and molecular modeling investigations of novel tacrine derivatives with a combination of acetylcholinesterase inhibition and cannabinoid CB1 receptor antagonism

Article abstract of DOI:10.1021/jm901614b

Pyrazolines 7-10 were designed as novel CB₁ receptor antagonists, which exhibited improved turbidimetric aqueous solubilities. On the basis of their extended CB₁ antagonist pharmacophore, hybrid molecules exhibiting cannabinoid CB₁ receptor antagonistic as well as acetylcholinesterase (AChE) inhibiting activities were designed. The target compounds 12, 13, 20, and 21 are based on 1 (tacrine) as the AChE inhibitor (AChEI) pharmacophore and two different CB₁ antagonistic pharmacophores. The imidazole-based 20 showed high CB₁ receptor affinity (48 nM) in combination with high CB₁/CB₂ receptor subtype selectivity (>20-fold) and elicited equipotent AChE inhibitory activity as 1. Molecular modeling studies revealed the presence of a binding pocket in the AChE enzyme which nicely accommodates the CB₁ pharmacophores of the target compounds 12, 13, 20, and 21. 2010 American Chemical Society.

Full text of DOI:10.1021/jm901614b

1338 J. Med. Chem. 2010, 53, 1338–1346
DOI: 10.1021/jm901614b
Design, Synthesis, Biological Properties, and Molecular Modeling Investigations of Novel Tacrine
Derivatives with a Combination of Acetylcholinesterase Inhibition and Cannabinoid CB1 Receptor
Antagonism
Jos H. M. Lange,* Hein K. A. C. Coolen, Martina A. W. van der Neut, Alice J. M. Borst, Bob Stork, Peter C. Verveer, and
Chris G. Kruse
Solvay Pharmaceuticals, Research Laboratories, C. J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands
Received November 3, 2009
Pyrazolines 7-10 were designed as novel CB1 receptor antagonists, which exhibited improved
turbidimetric aqueous solubilities. On the basis of their extended CB1 antagonist pharmacophore,
hybrid molecules exhibiting cannabinoid CB1 receptor antagonistic as well as acetylcholinesterase
(AChE) inhibiting activities were designed. The target compounds 12, 13, 20, and 21 are based on 1
(tacrine) as the AChE inhibitor (AChEI) pharmacophore and two different CB1 antagonistic pharma-
cophores. The imidazole-based 20 showed high CB1 receptor affinity (48 nM) in combination with high
CB1/CB2 receptor subtype selectivity (>20-fold) and elicited equipotent AChE inhibitory activity as 1.
Molecular modeling studies revealed the presence of a binding pocket in the AChE enzyme which nicely
accommodates the CB1 pharmacophores of the target compounds 12, 13, 20, and 21.
Introduction
place preference and blocked the induction and persistence of
cocaine-evoked hyperlocomotion. AChEIs can thus be ap-
proached as novel and potential therapeutic agents for drug
addiction. Cannabinoid CB1 receptor antagonists have also
been associated14 with the treatment of drug addiction and
addictive behavior.
The multifactorial and complex ethiology of AD has hitherto
prompted several so-called “single entity-multitarget ligand”
approaches,15 such as combined AChEI-histamine H3 receptor
antagonists,16 dual binding site AChEIs,17 NO-donor-AChEI
hybrids,18 tacrine-dihydropyridine hybrids,19 tacrine-
melatonin hybrids,20 dual inhibitors of monoamine oxidase
and AChE,21 dual AChE/serotonin transporter inhibitors,22
tacrine-ferulic acid hybrids,23 and benzofuran-based hybrid
compounds.24
Alzheimer’s disease (ADa) is a neurodegenerative disorder
whose prevalence is increasing together with the life expec-
tancy throughout the world. Cholinergic-enhancing drugs are
themain currenttherapy.1 The AChEI 1 was the first synthetic
drug approved by the U.S. Food and Drug Administration
for the treatment of AD (Figure 1). The tricyclic 1 is a
reversible inhibitor that reduced the inactivation of acetylcho-
line (ACh).2
AChEIs3 have shown efficacy not only in AD4 but also in
other cognitive disorders such as dementia with Lewy bodies,5
Parkinson’s disease,6 vascular dementia,7 traumatic brain
injury,8 and cognitive impairment in multiple sclerosis.9
Cognitive disorders constitute also a potential therapeutic
area for cannabinoid CB1 receptor antagonists,10 since CB1
receptor antagonists were shown to increase ACh release in
certain brain areas including the cortical region and hippo-
campus.11 On the basis of the observations that CB1 receptor
antagonists as well as AChEIs improved performance in a
variety of animal memory models, Wise et al. found12 that
combined administration of subthreshold doses of the CB1
receptor antagonist rimonabant and the AChEI donezepil,
which had no discernible effects on performance when given
alone, enhanced memory.
Design
Molecular design which was based on our previous work on
pyrazoline-based CB1 receptor antagonists25,26 such as 2
revealed that substitution of its amidine N-methyl group by
sterically more demanding substituents would lead to com-
pounds with retained CB1 receptor antagonistic properties.
First, this basic hypothesis is validated by the design of the
target compounds 5-10 which contain either a polar group or
an ionizable nitrogen atom in their amidine N substituent in
order to produce novel CB1 receptor antagonists with im-
proved aqueous solubilities.
It was also found13 that AChEIs acting on the brain
suppressed both cocaine- and morphine-induced conditioned
*To whom correspondence should be addressed. Telephone: þ31
(0)294 479731. Fax: þ31 (0)294 477138. E-mail: jos.lange@solvay.com.
a Abbrevations: ACh, acetylcholine; AChE, acetylcholinesterase;
AChEI, acetylcholinesterase inhibitor; AD, Alzheimer’s disease; CHO,
Chinese hamster ovary; DMAP, 4-dimethylaminopyridine; EDC, 1-(3-
methylaminopropyl)-3-ethylcarbodiimide; GPCR, G-protein-coupled
receptor; HEK, human embryonic kidney; HOAt, 1-hydroxy-7-azaben-
zotriazole; HRMS, high resolution mass spectrometry; NO, nitric oxide;
PAS, peripheral anionic site; PDB, Protein Data Bank; SEM, standard
error of the mean.
Second, the impact of both AChE inhibition and CB1
receptor antagonism on cognitive deficits and drug addiction
prompted the design of dual acting AChEIs/CB1 receptor
antagonists. Although the general concept of designing dual
acting compounds based on 1 is not new as has been outlined
above, our approach to dual acting compounds by combining
the CB1 antagonist pharmacophore with 1 is unprecedented
and is of great importance because of the reported beneficial
r
pubs.acs.org/jmc
Published on Web 01/04/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1339
Figure 1. Chemical structures of the AChEI 1, CB1 receptor antagonists 2 and 3, and Esteve’s dual acting CB1 receptor antagonist-μ-opioid
receptor modulator 4.
roles of CB1 receptor antagonists as well as AChEIs in
cognitive impairment.
Results and Discussion
The CB1 receptor has a highly lipophilic binding site, and
consequently CB1 receptor agonists as well as antagonists
generally are lipophilic entities which in general exhibit low
aqueous solubilities. A poor water solubility may lead to
practical difficulties in pharmacological testing and may
necessitate the use of solubilizing agents and complex com-
pound formulations. The compounds 5-10 were designed
with the goal to produce CB1 receptor antagonists with
improved aqueous solubilities, due to the presence of either
a polar group (5 and 6) or an ionizable nitrogen atom in their
carboxamidine side chain (7-10).
Compounds 5-10 were synthesized in reasonable yields,
ranging from 39% to 65%, by chlorinating the key inter-
mediate25 11 with PCl5 in chlorobenzene, followed by treat-
ment with various amines (Scheme 1). Compound 7 was
subsequently converted to the corresponding hydrochloric
acid salt by treatment with 1 M HCl in ethanol.
The pharmacological results of the target compounds 5-10
and the reference compound 2 are given in Table 1. The CB1
receptor binding data of 5-10 corroborated our hypothesis
that the replacement of the amidine N-methyl group by larger
and more polar groups leads to retained CB1 receptor affi-
nities. Compound 7 with the N,N-dimethylaminopropyl tail
showed the highest CB1 receptor affinity (3.1 nM) in this
series. In general, the compounds 5-10 elicited high CB1/CB2
receptor selectivities, which is in line with the results from the
reference compound 2. It is interesting to note that the
compounds 7-10, which all have an ionizable nitrogen atom
incorporated in their side chain, have approximately 10-fold
lower CB1 antagonistic potencies compared with the com-
pounds 2 and 5, 6 which are devoid of the presence of such an
ionizable nitrogen atom. According to expectation, the com-
pounds 7-10 exhibited improved aqueous solubilities as
compared to 2 and 5, 6, in particular under acidic conditions
(Table 1).
In our design strategy, two molecular entities (interacting
with high selectivity either to the CB1 receptor or to the AChE
enzyme) were applied as the starting points, thereby combin-
ing their key structural elements to incorporate activity at
both targets into a single molecule. The 3,4-diarylpyrazoline25
2 and 1,2-diarylimidazole27,28 3 were selected as suitable CB1
antagonistic structural elements29 for this study (Figure 1),
whereas 1 was preferred as a compact AChEI moiety. It was
envisioned that connecting the CB1 antagonistic units 2 and 3,
respectively, to 1 by an alkylene spacer of suitable length
would result in dual acting AChEIs/CB1 receptor antagonists.
The choice of 2 and 3 enabled us to investigate the effect of the
difference in sprouting of the tail connecting the tacrine
moiety.
In the course of our investigations, researchers from Esteve
patented30 compound 4 and some derivatives with different
alkylene spacer lengths as dual acting CB1 receptor antag-
onists-μ-opioid receptor modulators. Compound 4 was
claimed by Esteve to have moderate CB1 receptor affinity
(Ki = 701 nM) and μ-opioidreceptor affinity (Ki = 6543nM).
It is interesting to note that 4 showed in vivo activities after
intraperitoneal administration in CB1 receptor as well as
opioid receptor mediated models despite these observed
moderate receptor affinities and its high molecular weight of
737. The reported31 synergistic effect between CB1 and
μ-opioid receptor modulation may have a positive impact
on the observed in vivo activities of 4.
It was also envisioned that the tacrine moiety would be
amenable for derivatization with preservation of AChE in-
hibiting ability, based on the reported structure of tacrine
derivative 17 with the AChE enzyme32 (Protein Data Bank
(PDB) code 1UT6). The alkylene chain of 17 points toward
the outer surface into an unoccupied region. The orientation
of the tacrine skeleton is the same33 as that in the structure of
AChE with 1 based on X-ray diffraction data (PDB code
1ACJ). These observations corroborated our design hypothe-
sis toward dual AChEI/CB1 receptor antagonists.
Our AChEI/CB1 receptor antagonist combination design
considerations led to the synthesis of the series based on 2,
targeting compounds 12 and 13 according to Scheme 2.

1340 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Lange et al.
Scheme 1a
a Reagents and conditions: (a) PCl5, chlorobenzene, reflux, 1 h; (b) RH, CH2Cl2, room temp, 16 h.
Table 1. In Vitro Pharmacological Results of Compounds 2 and 5-10
compd
Ki(CB1),a nM
pA2(CB1),b
Ki(CB2),c nM
aqueous solubility,d μM, (pH 2)
aqueous solubility,e μM, (pH 7.4)
2
25 ( 7
8.7 ( 0.3
9.0 ( 0.1
8.6 ( 0.2
7.8 ( 0.1
7.8 ( 0.1
7.6 ( 0.2
7.9 ( 0.2
>1000
>1000
571 ( 215
>1000
>1000
>1000
>1000
4
4
2
4
5
43 ( 11
31 ( 17
3.1 ( 0.9
19 ( 7
9.6 ( 1.4
27 ( 9
6
<7
>100
65
<3
38
11
4
7
8
9
10
65
38
11
a Displacement of specific CP-55,940 binding in CHO cells stably transfected with human CB1 receptor, expressed25 as Ki ( SEM values (nM).
b [3H]Arachidonic acid release in CHO cells expressed25 as pA2 ( SEM values. c Displacement of specific CP-55,940 binding in CHO cells stably
transfected with human CB2 receptor, expressed25 as Ki ( SEM values (nM). The values represent the mean result based on at least three independent
experiments. d Turbidimetric aqueous solubility, pH 2. Estimated precipitation concentration expressed as μM. e Turbidimetric aqueous solubility,
pH 7.4. Estimated precipitation concentration expressed as μM.
The chlorination reaction to convert 11 into its chloroimidoyl
derivative 14 was carried out under milder reaction conditions
(using a phosphorus oxychloride (POCl3)/4-dimethylamino-
pyridine (DMAP) combination) compared to the synthesis of
5-10. Subsequent reaction with the tacrine-alkylene spacer
properties, but compound 13 with a longer spacer length elicited
a higher pIC50 value.
The imidazoles 20 and 21 showed also AChE inhibiting
properties but in a reverse order. Compound 20 with the
shorter spacer length is the more potent compound in this
series, having AChE inhibitory activities comparable to those
of the AChEI reference compound 1.
In order to explain these observations, molecular modeling
studies were performed on both the CB1 receptor and the
AChE enzyme. Several CB1 receptor homology models have
been reported in the literature.25,36
In the majority of these models the loop regions spanning
the helices of the G-protein-coupled receptor (GPCR) have
been omitted. We have rebuilt our model using the rigid
template of the recently reported37 human β2-adrenergic
structure and taking the extracellular loop 2 into account,
including the cysteine bridge which is presumed to be present
in the lipids GPCR cluster. After docking of 2, all the essential
features of the binding as reported earlier25 were maintained
including the key interaction between Lys192 and the sulfon-
amide moiety of 2 and the multiple π-π stacking interactions
of its pyrazoline aryl rings witha seriesof aromatic residueson
the helices 5 and 6. The only difference with our previous
model is a different orientation of the p-Cl-phenyl ring
attached to the SO2 group of 2, which now points downward
between the helices 3 and 6. The resulting conformation of 2
derivatives34 15 and 16 in the presence of Hunig’s base
€
furnished the target compounds 12 and 13 in 36% and 35%
yield, respectively.
The synthesis of the 3-based series, aiming at 20 and 21,
started with the hydrolysis of the ethyl ester precursor27,28 18.
Hydrolysis with LiOH in a water/THF mixture led to the
corresponding carboxylic acid 19 in 84% yield. Subsequent
amidation with the tacrine building blocks 15 and 16 in the
presence of suitable coupling reagents led to the formation of
the target compounds 20 and 21 in 53% and 62% yield,
respectively (Scheme 3).
The pharmacological results of the target compounds 12,
13, 20, and 21, the reference compounds 1-3, and the known
CB1 receptor antagonist rimonabant35 are given in Table 2.
According to our expections, 1 was found inactive as CB1
receptor ligand but active in the AChEI assay. In addition,
both rimonabant and 3 are potent CB1 receptor antagonists
without significant AChE inhibiting properties. The target
compounds 12, 13, 20, 21 all showed significant CB1 receptor
affinities and in general acted as CB1 receptor antagonists.
The pyrazoline derivative 12 showed moderate AChE inhibiting

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1341
Scheme 2a
a
€
Reagents and conditions: (a) POCl3, DMAP, CH2Cl2, reflux, 5 h; (b) 15 or 16, 0 °C; (c) Hunig’s base, CH2Cl2, reflux, 72 h (35-36%).
Scheme 3a
a Reagents and conditions: (a) LiOH, THF/H2O, 70 °C, 16 h (84%); (b) concentrated HCl; (c) 15 or 16, 1-hydroxy-7-azabenzotriazole (HOAt), 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide HCl (EDC), CH2Cl2, room temp, 72 h (53-62%).
3
Table 2. In Vitro Pharmacological Results of Rimonabant, Compounds 1-3, 12-13, 20, 21
d
compd
Ki(CB1),a nM
pA2(CB1)b
Ki(CB2),c nM
AChE inhibition (pIC50)
rimonabant HCl
3
25 ( 15 (11.5)35
>1000
8.6 ( 0.1
nde
1580 ( 150 (1640)35
>1000
>1000
4.6 ( 0.2
6.6 ( 0.2
nde
tacrine (1)
2
25 ( 7
14 ( 5
42 ( 13
54 ( 14
48 ( 27
88 ( 30
8.7 ( 0.3
9.8 ( 0.2
8.1 ( 0.3
7.4 ( 0.2
nde
3
>1000
>1000
<4.5
12
13
20
21
5.6 ( 0.4
6.0 ( 0.3
6.5 ( 0.3
5.9 ( 0.3
>1000
>1000
>1000
8.2f
a Displacement of specific CP-55,940 binding in CHO cells stably transfected with human CB1 receptor, expressed as Ki ( SEM values (nM).
b [3H]Arachidonic acid release in CHO cells expressed as pA2 ( SEM values. c Displacement of specific CP-55,940 binding in CHO cells stably transfected
with human CB2 receptor, expressed as Ki ( SEM values (nM). The values represent the mean result based on at least three independent experiments,
unless indicated otherwise. d AChEI; human recombinant (HEK-293 cells); photometric detection; Cerep. e nd = not determined. f Result based on two
independent experiments (8.2 and 8.2, respectively).
has a higher resemblance to our earlier reported X-ray
structure of the 4S-enantiomer of 2.25
Inthe loopregion, positionedjustabove the CB1 antagonist
binding pocket, an additional pocket was recognized that can
accommodate 1. The docking result of 12 in the homology
model of the CB1 receptor is shown in Figure 2.
The protonated quinolinic nitrogen of 1 (ACD/pKa =
9.2)38 can interact with the Asp266 residue which also forms
a salt bridgewithLys192. The aromatic ring of the tacrinepart
is postulated to stack between Phe177, Phe174, and His178.
Its condensed cyclohexyl ring is enclosed by a small lipophilic
pocket limited by Leu193 and Phe268. It is noted that the CB1
pharmacophore part of 12 is positioned almost exactly as in
the case of 2.
The negative impact of linking a tacrine unit to the CB1
antagonist pharmacophore on the binding and activity of
12, 13, 20, and 21 is small, since they all still show CB1
receptor affinity values in the nanomolar range. The re-
duction in CB1 receptor affinities of the tacrine derivatives
in the imidazole-based series (20 and 21) seems to be
somewhat more pronounced (about 3- to 4-fold) than in
the pyrazoline-based series (12 and 13) (∼2-fold). This
might be rationalized by the fact that in the former series
one of the pharmacophoric features (the piperidinyl ring) is
replaced by the linker while in the latter series the corre-
sponding feature (the p-Cl-phenyl moiety), which is known
to be important for CB1 receptor interaction, is main-
tained.

1342 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Lange et al.
Figure 4. Docking result of the 3,4-diarylpyrazoline-based CB1
pharmacophore in the structure of AChE.
Figure 2. Docking result of 12 in the homology model of the CB1
receptor. Helices 5 and 6 are not displayed for clarity.
Figure 5. Structure-based alignment of 12 and 20 showing the
effect of the different sprouting of the tail from their CB1 antagonist
part.
Figure 3. Docking result of 20 (shown in purple) in the structure of
AChE. The lipophilic residues of the PAS accommodating the
diaryl moiety of the CB1 antagonistic part are given in orange.
Some regions of the protein are not displayed for clarity.
tacrine moiety has a negative impact on the AChE inhibiting
properties in the pyrazoline (2-based) series (compound 12),
whereas in the imidazole (3-based) series compound 20 is
equipotent compared to 1.
The docking of 20 in AChE is depicted in Figure 3. The
tacrine unit is found at the bottom of the active site gorge at
exactly the same position of unlinked 1, stacking between
Trp84 and Phe330 as well as bound by a hydrogen bond with
the backbone carbonyl group of His440. At the outer surface,
at the so-called peripheral anionic site (PAS), there is a
constitution of residues that accommodate the pyrazoline-
based CB1 pharmacophore surprisingly well (Figure 4).
The aromatic cluster Trp279, Phe288, Phe290, Phe330, and
Phe331 can bind the aromatic rings of the CB1 antagonists by
multiple π-π stacking interactions similar to the aromatic
residues on helices 5 and 6 of the GPCR. The H-bonding to
the sulfonamide of the 2 series and the corresponding amide of
the 3 series can be realized by Gln74. The linking tail runs
through the gorge following the trajectory of ACh on its way
to the catalytic site.
The distance betweenthe anilinicnitrogen atom of1 and the
attachment nitrogen of docked 2 in the PAS is approximately
˚
7.8 A, while the same distance in the case of 3 is only 5.0 A
˚
(Figure 5).
The latter can be bridged well by a C4 chain (6.3 A) which
˚
results in 20, while in the former case the C4 chain connection,
resulting in 12, will prevent an optimal fit into the AChE
structure. This further illustrates that the different modes of
attachment of the tacrine-spacer unit to the CB1 antagonist
pharmacophore in compounds 12 and 20, respectively, have
an impact on the optimal spacer lengths.29
Finally, two additional points are of interest. First, the
AChEI 1 and some of its metabolites have been reported39 to
elicit toxic effects. In this regard our compounds 12, 13, 20,
and 21 might be expected to elicit some of the adverse effects
According to the results in Table 2, the connection of the
CB1 antagonist part via the n = 4 alkylene spacer to the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1343
of 1. However, it is realized that N,N0-bis(1,2,3,4-tetrahydro-
acridin-9-yl)heptane-1,7-diamine (bis(7)-tacrine) demon-
strated40 a minor toxicity in comparison with 1 and accord-
ingly the tacrine heterodimers 12, 13, 20, and 21 would be
anticipated to be considerably less toxic than 1. Second,
although the more general term “antagonist” is used in this
article, it is with the understanding that the majority of the
reported cannabinoid CB1 receptor antagonists29,41 (includ-
ing compounds such as rimonabant, 2, and 3) behave as
inverse agonists42 at the constitutively active43 CB1 GPCR.
Therefore, it can be anticipated that the compounds 5-10, 12,
13, 20, and 21 will also act as inverse agonists at the CB1
receptor.
27.42, 27.45, 50.9, 54.8, 57.4, 70.5, 127.3, 127.4, 128.1, 128.4,
128.6, 128.7, 129.0, 129.5, 136.6, 137.4, 139.1, 143.3, 152.2,
157.5. HRMS (C26H27Cl2N4O3S) [M þ H]þ: found m/z
545.1188, calcd 545.1181. Anal. (C26H26Cl2N4O3S) C, H, N.
[3-(4-Chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyrazol-1-yl]-
[4-(1,1-dioxothiomorpholinyl)methylene]-4-chlorobenzenesul-
fonamide (6). Compound 6 was obtained from 11 and
thiomorpholine 1,1-dioxide according to the procedure des-
cribed for 7. 1H NMR (400 MHz, CDCl3) δ 3.28-3.46 (m,
4H), 3.80 (dd, J = 11 and 5 Hz, 1H), 4.18-4.24 (m, 4H), 4.39 (t,
J = 11 Hz, 1H), 4.62 (dd, J = 11 and 5 Hz, 1H), 7.07-7.12 (m,
2H), 7.26-7.36 (m, 5H), 7.39 (d, J = 9 Hz, 2H), 7.48 (d, J = 9
Hz, 2H), 7.82 (d, J = 9 Hz, 2H). HRMS (C26H25Cl2N4O4S2)
[M þ H]þ: found m/z 591.0707, calcd 591.0694. Anal. (C26H24-
Cl2N4O4S2) C, H, N.
3-(4-Chlorophenyl)-N0-((4-chlorophenyl)sulfonyl)-N-[3-(dimethy-
lamino)propyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxami-
Conclusion
Novel hybrid molecules 12, 13, 20, 21 exhibiting AChE
inhibiting activities as well as significant cannabinoid CB1
receptor antagonistic properties were discovered that are
based on 1 as the AChEI pharmacophore and known canna-
binoid CB1 antagonistic counterparts. The imidazole-based
20 showed significant CB1 receptor affinity (48 nM) in
combination with high CB1/CB2 receptor subtype selectivity
(>20-fold) and elicited equipotent AChE inhibitory activity
compared with 1.
dine HCl (7). A magnetically stirred mixture of 1125 (2.37 g, 5.0
3
mmol) and PCl5 (1.05 g, 5.0 mmol) in chlorobenzene (50 mL)
was heated at reflux temperature for 1 h. After thorough
concentration, the formed 13 was suspended in CH2Cl2 and
reacted with excess cold 3-(dimethylamino)propylamine (2.55 g,
25 mmol). After being stirred at room temperature for 16 h, the
mixture was washed with water, dried over MgSO4, filtered, and
concentrated in vacuo. Flash chromatographic purification
(eluant ethyl acetate/methanol = 1/2 (v/v)) gave pure 3-(4-
chlorophenyl)-N0-[(4-chlorophenyl)sulfonyl]-N-[3-(dimethylamino)-
propyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine 7 as its
free base: 1H NMR (400 MHz, CDCl3) δ1.77 (quint, J = 6 Hz, 2H),
2.29 (s, 6H), 2.48 (t, J = 6.2 Hz, 2H), 3.70 (q, J = 5.6 Hz, 2H), 4.10
(dd, J = 11.3 and 5.2 Hz, 1H), 4.53 (t, J = 11.4 Hz, 1H), 4.61-4.68
(m, 1H), 7.10-7.14 (m, 2H), 7.22-7.33 (m, 5H), 7.36 (d, J= 8.8Hz,
2H), 7.52 (d, J = 8.8 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 8.62 (br s,
1H). Subsequent treatment with 1 M HCl/EtOH (5 mL) and
trituration with diethyl ether gave the corresponding hydrochloric
acid salt as a white solid (1.83 g, 62% yield). Mp: 236-237 °C. 1H
NMR (400 MHz, CDCl3) δ 1.65 (br s, ∼1H), 2.26 (quint, J = 7 Hz,
2H), 2.80 (s, 6H), 3.05-3.17 (m, 2H), 3.74-3.82 (m, 2H), 4.11 (dd,
J= 11 and 5 Hz, 1H), 4.56 (t, J= 11 Hz, 1H), 4.71 (dd, J=11and5
Hz, 1H), 7.14 (br d, J = 8 Hz, 2H), 7.21-7.35 (m, 5H), 7.39 (br d,
J = 8 Hz, 2H), 7.61-7.67 (m, 3H), 7.85 (br d, J = 8 Hz, 2H). 13C
NMR (100 MHz, CDCl3) δ 25.0, 40.6, 43.0, 51.3, 55.1, 57.5, 127.2.
127.4, 128.1, 128.2, 128.8, 129.0, 129.1, 129.5, 136.7, 137.4, 139.0,
143.7, 151.4, 158.0. HRMS (C27H30Cl2N5O2S) [M þ H]þ: found m/z
Experimental Section
Chemistry. 1H and 13C NMR spectra were recorded on a
Varian UN400 instrument (400 MHz) using CDCl3 as solvent
with tetramethylsilane as an internal standard. Chemical shifts
are given in parts per million (ppm) (δ scale) downfield from
tetramethylsilane. Coupling constants (J) are expressed in hertz
(Hz). Thin-layer chromatography was performed on Merck
precoated 60 F254 plates, and spots were visualized with UV
light. Flash chromatography was performed using silica gel 60
(0.040-0.063 mm, Merck). Column chromatography was per-
formed using silica gel 60 (0.063-0.200 mm, Merck). Melting
€
points were recorded on a Buchi B-545 melting point apparatus
and are uncorrected. Mass spectra were recorded on a Micro-
mass QTOF-2 instrument with MassLynx application software
for acquisition and reconstruction of the data. Exact mass
measurement by HRMS was done of the quasimolecular ion
[M þ H]þ. Elemental analyses were performed on a Vario EL
elemental analyzer by Solvay Pharmaceuticals. Yields refer to
isolated pure products and were not maximized. The purity of
the target compounds 3, 5-10, 12, 13, 20, and 21 was established
as g95%, based on combustion analysis data.
558.1513, calcd 558.1497. Anal. (C26H30Cl2N5O2S HCl) C, H, N.
3
3-(4-Chlorophenyl)-N0-((4-chlorophenyl)sulfonyl)-N-[4-(methyl)-
piperidin-4-yl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxami-
dine (8). Compound 8 was obtained from 11 and 1-methylpiper-
idin-4-ylamine according to the procedure described for 7. Mp:
202-203 °C. 1H NMR (400 MHz, CDCl3) δ 1.58-1.74 (m, 3 H),
1.97-2.17 (m, 4H), 2.31 (s, 3H), 2.74-2.84 (m, 2H), 3.98-4.22 (m,
2H), 4.49-4.71 (m, 2H), 7.11 (d, J = 6.6 Hz, 2H), 7.23-7.35 (m,
5H), 7.39(d, J= 8.6 Hz, 2H), 7.49 (d, J=8.6Hz, 2H), 7.84(d, J=
8.6 Hz, 2H). HRMS (C28H30Cl2N5O2S) [M þ H]þ: found m/z
570.1497, calcd 570.1497. Anal. (C28H29Cl2N5O2S) C, H, N.
[3-(4-Chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyrazol-1-yl]-
{[2-(dimethylamino)ethylpiperazin-4-yl]methylene}-4-chloroben-
1-(4-Chlorophenyl)-2-(2-chlorophenyl)-5-ethyl-N-(piperidin-1-yl)-
1H-imidazole-4-carboxamide 2/3H2O (3). Compound 3 was ob-
3
tained from 19 according to reported procedures.27,28 Mp:
207-208 °C. 1H NMR (400 MHz, CDCl3) δ 1.08 (t, J = 7.5 Hz,
3H), 1.39-1.48 (m, 2H), 1.69-1.81 (m, 5H), 2.86 (br s, 3H), 2.94 (q,
J = 7.5 Hz, 2H), 7.09 (d, J = 8.7 Hz, 2H), 7.20-7.38 (m, 6H), 7.94
(s, 1H). 13C NMR (100 MHz, CDCl3) δ 13.9, 17.7, 23.4, 25.5, 57.3,
126.7, 129.0, 129.4, 129.7, 129.8, 130.0, 131.0, 132.5, 133.8, 134.5,
135.1, 140.3, 143.2, 160.5. HRMS (C23H25Cl2N4O) [M þ H]þ:
found m/z443.1413, calcd 443.1405. Anal. (C23H24Cl2N4O) C, H, N.
3-(4-Chlorophenyl)-N0-((4-chlorophenyl)sulfonyl)-N-(2-hydroxy-
2-methylpropyl)-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxami-
dine (5). Compound 5 was obtained from 11 and 1-amino-2-
methylpropan-2-ol according to the procedure described for 7.
Mp: 154-155 °C. 1H NMR (400 MHz, CDCl3) δ 1.30 (s, 3H),
1.31 (s, 3H), 1.63 (br s, 1H), 2.30 (br s, 1H), 3.63-3.71 (m, 2H),
4.07 (dd, J = 11.6 and 5.5 Hz, 1H), 4.52 (t, J = 11.6 Hz, 1H),
4.66 (dd, J = 11.6 and 5.5 Hz, 1H), 7.12 (d, J = 6.6 Hz, 2H),
7.23-7.34 (m, 5H), 7.37 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.8 Hz,
2H), 7.86 (d, J = 8.6 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ
zenesulfonamide 1/4H2O (9). Compound 9 was obtained from
3
11 and dimethyl(2-piperazin-1-ylethyl)amine according to
the procedure described for 7. Mp: 164-165 °C. 1H NMR
(400 MHz, CDCl3) δ 2.30 (s, 6H), 2.50 (t, J = 6.5 Hz, 2H),
2.57-2.62 (m, 2H), 2.64-2.78 (m, 4H), 3.72-3.89 (m, 5H), 4.42
(t, J = 11.2 Hz, 1H), 4.52-4.58 (m, 1H), 7.11 (br d, J = 8 Hz,
2H), 7.23-7.34 (m, 5H), 7.36 (d, J = 8.6 Hz, 2H), 7.49 (d, J =
8.8 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 45.8, 50.0, 50.1, 53.4, 56.4, 56.8, 58.2, 127.1,
127.3, 128.0, 128.5, 128.70, 128.74, 129.0, 129.5, 136.4, 137.1,
139.1, 144.1, 153.0, 157.2. HRMS (C30H35Cl2N6O2S) [M þ H]þ:
found m/z 613.1932, calcd 613.1919. Anal. (C30H34Cl2N6O2S)
C, H, N.

1344 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Lange et al.
3-(4-Chlorophenyl)-N0-((4-chlorophenyl)sulfonyl)-N-[4-(pyrrolidin-
1-yl)butyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (10).
Compound 10 was obtained from 11 and 4-(pyrrolidin-1-yl)-
butylamine according to the procedure described for 7. Mp:
126-127 °C. 1H NMR (400 MHz, CDCl3) δ 1.56-1.65 (m, 2H),
1.65-1.75 (m, 3H), 1.75-1.81 (m, 4H), 2.44-2.54 (m, 6H),
3.59-3.67 (m, 2H), 4.12 (dd, J= 11.3 and 4.7 Hz, 1H), 4.55 (t,
J= 11.5 Hz, 1H), 4.61 -4.67 (m, 1H), 7.10-7.14 (m, 2H), 7.23-7.34
(m, 5H), 7.38 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H), 7.85 (d,
J = 8.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 23.4, 26.2, 28.2,
44.9, 50.6, 54.2, 55.9, 57.7, 127.3, 127.4, 128.0, 128.5, 128.6, 128.7,
129.0, 129.5, 136.5, 137.3, 139.1, 143.3, 151.9, 157.4. HRMS
(C30H34Cl2N5O2S) [M þ H]þ: found m/z 598.1824, calcd 598.1810.
Anal. (C30H33Cl2N5O2S) C, H, N.
J = 7, 2H), 3.70 (br s, 1H), 7.12 (dt, J = 8 and 2, 2H), 7.22-7.28
(m, 1H), 7.29-7.38 (m, 5H).
N-[4-(1,2,3,4-Tetrahydroacridin-9-ylamino)butyl]-2-(2-chloro-
phenyl)-1-(4-chlorophenyl)-5-ethyl-1H-imidazole-4-carboxa-
mide H2O (20). Compound 20 was obtained from 15 and 19
3
according to the procedure described for 21. Mp: 179-180 °C.
1H NMR (400 MHz, CDCl3) δ 1.06 (t, J = 7 Hz, 3H), 1.40-1.52
(m, 6H), 1.55-1.68 (m, 2H), 1.80-2.02 (m, 6H), 2.60 (t, J=6 Hz,
2H), 2.95 (q, J=7 Hz, 2H), 3.35 (t, J = 6 Hz, 2H), 3.42 (q, J = 7
Hz, 2H), 3.91 (br s, 2H), 5.60 (br s, 1H), 7.09 (d, J=8 Hz, 2H),
7.20-7.36 (m, 6H), 7.45 (t, J = 7 Hz, 1H), 7.70 (t, J = 7 Hz, 2H),
8.17 (d, J = 8 Hz, 1H), 8.60 (br d, J = 7 Hz, 1H). HRMS
(C35H36Cl2N5O) [M þ H]þ: found m/z 612.2300, calcd 612.2297.
Anal. (C35H35Cl2N5O) C, H, N.
4-Chloro-N-{[3-(4-chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyra-
zolyl][4-(1,2,3,4-tetrahydroacridin-9-ylamino)butylamino]methy-
lene}benzenesulfonamide 2H2O 1.5MeOH (12). Compound
N-[7-(1,2,3,4-Tetrahydroacridin-9-ylamino)heptyl]-2-(2-chloro-
phenyl)-1-(4-chlorophenyl)-5-ethyl-1H-imidazole-4-carboxami-
de 1.5H2O 2MeOH (21). To a magnetically stirred solution of
3
3
3
3 0
N-[90-(10,20,3 ,40-tetrahydroacridinyl)]-1,7-diaminoheptane 16
(0.837 g, 3.11 mmol) in dichloromethane (40 mL) was succes-
sively added 19 (0.75 g, 2.08 mmol), HOAt (0.34 g, 2.5 mmol),
and EDC (0.48 g, 2.5 mmol). The resulting mixture was stirred at
room temperature for 18 h and successively washed with water
(2 ꢀ 50 mL) and brine (50 mL). The organic layer was succes-
sively dried over Na2SO4, filtered, and concentrated in vacuo.
The obtained crude product was purified by flash chromato-
graphy (gradient, dichloromethane/ethanol=99/1 f dichloro-
methane/ methanol = 90/10 (v/v)) to give pure 21 (780 mg, 62%
yield). Mp: 110-111 °C. 1H NMR (400 MHz, CDCl3) δ 1.07 (t,
J = 7 Hz, 3H), 1.67-1.85 (m, 4H), 1.88-1.99 (m, 4H), 2.68-2.78
(m,2H), 2.95(q, J= 7 Hz, 2H), 3.06-3.15 (m, 2H), 3.45-3.63 (m,
4H), 7.10 (br d, J=8 Hz, 2H), 7.20-7.41 (m, 9H), 7.52-7.62
(m, 1H), 7.92-8.05 (m, 2H). HRMS (C38H42Cl2N5O) [M þ
H]þ: found m/z 654.2773, calcd 654.2766. Anal. (C38H41Cl2-
N5O) C, H, N.
12 was obtained from 11 and 15 according to the procedure
described for 13. Mp: 125-126 °C. 1H NMR (400 MHz,
CDCl3) δ 1.80-2.01 (m, 8H), 2.60-2.70 (m, 2H), 3.25-3.34
(m, 2H), 3.77-3.86 (m, 2H), 3.95-4.06 (m, 3H), 4.43 (t, J = 12
Hz, 1H), 4.67 (dd, J=12 and 5 Hz, 1H), 6.30 (br s, 1H), 7.10 (br
d, J = 8 Hz, 2H), 7.15-7.31 (m, 6H), 7.36-7.44 (m, 3H), 7.52
(d, J = 8 Hz, 2H), 7.65 (t, J = 8 Hz, 1H), 7.83 (d, J = 8 Hz,
2H), 8.20 (d, J = 8 Hz, 1H), 8.52 (d, J = 8 Hz, 1H). 13C NMR
(100 MHz, CDCl3) δ 20.6, 22.0, 24.1, 27.1, 27.9, 28.6, 43.9,
47.2, 50.8, 57.3, 111.3, 115.9, 120.8, 124.3, 125.1, 127.1, 127.3,
128.1, 128.3, 128.76, 128.80, 129.0, 129.5, 132.1, 136.6, 137.5,
138.8, 139.0, 143.3, 151.4, 151.6, 155.6, 157.7. HRMS
(C39H39Cl2N6O2S) [M þ H]þ: found m/z 725.2255, calcd
725.2232. Anal. (C39H38Cl2N6O2S) C, H, N.
4-Chloro-N-{[3-(4-chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyra-
zolyl]-[7-(1,2,3,4-tetrahydroacridin-9-ylamino)heptylamino]-
methylene}benzenesulfonamide 3/4H2O (13). To a magnetically
3
stirred solution of 1125 (1.5 g, 3.16 mmol) in dichloromethane
were successively added (30 mL) DMAP (1.707 g, 13.9 mmol)
and POCl3 (0.59 g, 3.85 mmol), and the resulting magnetically
stirred mixture was heated at reflux temperature for 5 h. The
resulting mixture was allowed to attain room temperature and
concentrated in vacuo to give crude 14, which was dissolved in
dichloromethane (30 mL) and reacted at 0 °C with N-[90-
(10,20,30,40-tetrahydroacridinyl)]-1,7-diaminoheptane 16 (1.48 g,
4.75 mmol). After addition of N,N-diisopropylethylamine
(DIPEA) (1.02 g, 7.9 mmol) the reaction mixture was heated at
reflux temperature for 72 h. The resulting mixture was allowed to
attain room temperature and was washed with water and brine
successively, dried over Na2SO4, filtered, and concentrated. The
obtained crude product was purified by flash chromatography
(gradient, dichloromethane f dichloromethane/methanol = 95/5
(v/v)) to give pure 13 (0.85 g, 35% yield). Mp: 90-91 °C. 1H NMR
(400 MHz, CDCl3) δ 1.20-1.73 (m, 10H), 1.86-1.96 (m, 4H),
2.66-2.74 (m, 2H), 3.05-3.12 (m, 2H), 3.47-3.56 (m, 2H),
3.58-3.67 (m, 2H), 4.07-4.14 (m, 1H), 4.52 (t, J=12 Hz, 1H),
4.58-4.66 (m, 1H), 7.11 (br d, J=8 Hz, 2H), 7.21-7.40 (m, 8H),
7.49 (br d, J= 8 Hz, 2H), 7.56 (br t, J= 8 Hz, 1H, 7.83 (br d, J =8
Hz, 2H), 7.93-8.00 (m, 2H). HRMS (C42H45Cl2N6O2S) [M þ
H]þ: found m/z 767.2734, calcd 767.2702. Anal. (C42H44Cl2N6-
O2S) C, H, N.
2-(2-Chlorophenyl)-1-(4-chlorophenyl)-5-ethyl-1H-imidazole-
4-carboxylic Acid (19). To a magnetically stirred solution of 1827
(5.80 g, 0.0149 mol) in tetrahydrofuran (40 mL) was added a
solutionof LiOH(0.715 g) inwater (40mL). The resulting mixture
was heated at 70 °C for 16 h. The resulting mixture was allowed to
attain room temperature and subsequently treated with concen-
trated hydrochloric acid (3.5 mL). The tetrahydrofuran was
evaporated in vacuo, and the resulting mixture was stirred over-
night. The formed precipitate was collected by filtration and
washed with petroleum ether (40-60) to give 19 (4.52 g, 84%
yield). 1H NMR (400 MHz, CDCl3): δ 1.09 (t, J = 7, 3H), 2.90 (q,
Turbidimetric Aqueous Solubility Assay. Four dilutions of the
test compound (10 mM in DMSO) were prepared in DMSO (3,
1, 0.3, and 0.1 mM). Each test compound concentration was
then further diluted 1 in 100 in buffer (typically 0.01 M phos-
phate buffered saline, pH 7.4, or a buffer at pH 2) so that the
final DMSO concentration was 1% and the final test compound
concentrations were 1, 3, 10, 30, and 100 μM. The experiment
was performed at 37 °C, and seven replicate wells were desig-
nated per concentration. Following the addition of the DMSO
dilution to the buffer, the plates were incubated for 2 h at 37 °C
before the absorbance was measured at 620 nm. The solubility
was estimated from the concentration of test compound that
produced an increase in absorbance above vehicle control (i.e.,
1 % DMSO in buffer). Nicardipine and pyrene were included as
control compounds. The solubility of nicardipine was pH
dependent, whereas the solubility of pyrene was pH indepen-
dent. Results were given as a calculated midrange value, based
on measured lower and upper bound values.
Inhibition of AChE in Human HEK-293 Cells.44 Test com-
pounds were dissolved in DMSO (10 mM) and diluted to test
concentrations in assay buffer. Testing was performed at a 3 log
concentration range around a predetermined IC50 for the re-
spective assay: e.g., 10, 1, 0.1, and 0.01 μM for IC50 of 0.3 μM
and 300, 30, 3, and 0.3 nM for IC50 of 10 nM. All determinations
were performed as duplicates. The highest concentration tested
for primes was 10 μM. Following incubation of test compound
with an AChE enzyme preparation (human recombinant ex-
pressed in HEK-293 cells) and the substrate acetylthiocholine
(50 μM) for 30 min at 37 °C, the thio conjugate product was
determined by photometry. Results were expressed as percentage of
total product formed at each concentration of compound tested
(duplicates). From the concentration-production inhibition
curves, IC50 values were determined by nonlinear regression ana-
lysis using Hill equation curve fitting. Results were expressed as
pIC50 values. Compounds with no significant affinity at concentra-
tions of 10 μM and higher were considered inactive: pIC50 < 5.0.

Article
Molecular Modeling. Modeling studies were performed using
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1345
(12) Wise, L. E.; Iredale, P. A.; Stokes, R. J.; Lichtman, A. H.
Combination of rimonabant and donezepil prolongs spatial mem-
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€
€
Schrodinger software (Maestro, version 8.5; Schrodinger LLC:
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model was built with Prime using the X-ray structure of the
β2-adrenergic receptor (PDB code 2RH1) as rigid template. The
extracellular loop 2 was modeled with the loop prediction
module. The loop was modified in order to make the Cys bridge
between C257 and C264 possible via a stepwise decreasing
distance constraint minimization and after building the bridge,
a full minimization of the loop. The tacrine moiety was docked
in the described region manually without any further modifica-
tions.
ꢀ
(14) (a) Cohen, C.; Perrault, G.; Voltz, C; Steinberg, R.; Soubrie, P.
SR141716, a central cannabinoid (CB1) receptor antagonist, blocks
the motivational and dopamine releasing effects of nicotine in rats.
Behav. Pharmacol. 2002, 13, 451–463. (b) Solinas, M.; Paulilio, L. V.;
Antoniou, K.; Pappas, L. A.; Goldberg, S. R. The cannabinoid CB1
antagonist N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-
4-methylpyrazole-3-carboxamide (SR-141716A) differentially alters
the reinforcing effects of heroin under continuous reinforcement, fixed
ratio, and progressive ratio schedules of drug self-administration in rats.
J. Pharmacol. Exp. Ther. 2003, 306, 93–102.
The X-ray structure of AChE was processed with the Protein
Preparation Wizard in Maestro. The CB1 receptor binding site
was disclosed by adjusting the rotameric state of two lipophilic
residues (Phe331 gþf g-, Phe330 trans f g-). The CB1 receptor
antagonist moieties of 12, 13, 20, and 21 were docked manually
into this site with the tacrine part kept in the same position as
that in its X-ray structure with AChE (PDB code 1ACJ).
(15) (a) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.;
Tumiatti, V.; Recanatini, M.; Melchiorre, C. Multi-target-directed
ligands to combat neurodegenerative diseases. J. Med. Chem. 2008,
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target therapeutics: when the whole is greater than the sum of the parts.
Drug Discovery Today 2007, 12, 34–42. (c) Morphy, R.; Rankovic, Z.
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Acknowledgment. Hugo Morren is gratefully acknow-
ledged for supply of the high resolution mass spectrometry
(HRMS) data. Peter Pfaffenbach is gratefully acknowledged
for supply of the combustion analysis data. Pieter Spaans is
acknowledged for supply of the turbidimetric aqueous solu-
bility data. Mercachem B.V. (The Netherlands) is acknow-
ledged for the synthesis of compounds 12, 13, 20, and 21.
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T. W.; Carruthers, N. I. Lead identification of acetylcholinesterase
inhibitors-histamine H3 receptor antagonists from molecular mod-
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