Octahydropyrrolo[3,4-c]pyrrole: A diamine scaffold for construction of either α4β2 or α7-selective nicotinic acetylcholine receptor (nAChR) ligands. Substitutions that switch subtype selectivity

Article abstract of DOI:10.1021/jm900249k

A series of 5-(pyridine-3-yl)octahydropyrrolo[3,4-c]pyrroles have been prepared that exhibit high affinity to α4β2 and/or α7 nicotinic acetylcholine receptors (nAChRs). Simple substitution patterns have been identified that allow construction of ligands that are highly selective for either nAChR subtype. The effects of substitution on subtype selectivity provide some insight into the differences in the ligand binding domains of the α4β2 and R7 receptors, especially in regions removed from the cation binding pocket.

Full text of DOI:10.1021/jm900249k

4126 J. Med. Chem. 2009, 52, 4126–4141
DOI: 10.1021/jm900249k
Octahydropyrrolo[3,4-c]pyrrole: A Diamine Scaffold for Construction of Either r4β2 or r7-Selective
Nicotinic Acetylcholine Receptor (nAChR) Ligands. Substitutions that Switch Subtype Selectivity
William H. Bunnelle,*,† Karin R. Tietje,† Jennifer M. Frost,† Dan Peters,‡ Jianguo Ji,† Tao Li,† Marc J. C. Scanio,† Lei Shi,†
David J. Anderson,† Tino Dyhring,‡ Jens H. Grønlien,† Hilde Ween,† Kirsten Thorin-Hagene,† and Michael D. Meyer†
Neuroscience Research, Abbott Laboratories, Department R47W, Building AP9A, Abbott Park, Illinois 60064-6117, and NeuroSearch A/S, 93
Pederstrupvej, DK-2750 Ballerup, Denmark
Received February 27, 2009
A series of 5-(pyridine-3-yl)octahydropyrrolo[3,4-c]pyrroles have been prepared that exhibit high
affinity to R4β2 and/or R7 nicotinic acetylcholine receptors (nAChRs). Simple substitution patterns
have been identified that allow construction of ligands that are highly selective for either nAChR
subtype. The effects of substitution on subtype selectivity provide some insight into the differences in the
ligand binding domains of the R4β2 and R7 receptors, especially in regions removed from the cation
binding pocket.
Introduction
memory, and information processing,10-14 including gating
deficits that are common in schizophrenia,15,16 while periph-
eral R7 nAChRs have been shown to play a role in inflamma-
tion response.17,18 Central R4β2 nAChRs have been identified
as key mediators of the analgesic effects of nicotinic agoni-
sts19-22 but are also linked to learning, memory, and atten-
tional effects.23-28 A numberof selectiveR4β2 andR7 nAChR
ligands, including AZD-3480 (1),29,30 ABT-089 (2),31,32
MEM-3454,33,34 PHA-543613 (3),35 and GTS-21 (4),36,37
among others (Figure 1), have been advanced to human
clinical trials for treatment of cognitive disorders.
Nicotinic acetylcholine receptors have become prominent
targets for drug discovery efforts aimed at treating a wide
range of CNSa disorders, including cognitive dysfunction
(attention-deficit hyperactivity disorder, cognitive deficits in
schizophrenia), neurodegenerative conditions (Alzheimer’s
disease, Parkinson’s disease), substance abuse, and pain,
among others.1-4 Consequently, the nAChR pharmacology
offers great potential for the development of new drugs in
areas of significant unmet medical need. A plethora of
nAChR subtypes exist in the brain, and much current effort
is devoted to understanding the structure, distribution, and
function of each.5,6 An early and still useful categorization
divides neuronal nAChRs into two major families, namely
those with high (nanomolar) affinity to (S)-nicotine and a
distinct population that is labeled selectively by the snake
venom component R-bungarotoxin. The former group con-
sists mainly of receptors constructed from heteropentameric
combinations of R4 and β2 subunits, probably with stoichi-
ometry (R4)2(β2)3, while the latter is represented mainly by a
homopentamer of the R7 subunit.6 The R4β2 and R7 subtypes
are differentially distributed in the CNS, but both are ex-
pressed in brain regions (cortex, hippocampus) that are im-
portant for cognitive processes, where their main function
appears to be to modulate the release of neurotransmitters
(including acetylcholine, glutamate, GABA, dopamine, and
serotonin) that are important for a wide range of CNS
functions.7-9 The R7 nAChR is a rapid-opening, rapidly
desensitizing ion channel that is implicated in learning,
Structural features of the nAChR agonist binding site were
originally inferred from site-specific mutagenesis and photo-
affinity labeling studies38 and nicely confirmed by the three-
dimensional structure of the acetylcholine binding protein
(AChBP) isolated from snail glia.39 A key feature is the
convergence of several tyrosine and tryptophan side chains
to form a pocket that receives the protonated or quaternized
amine that is an essential element of the nicotinic agonist
pharmacophore.40,41 A π-cation interaction has been estab-
lished as a substantial contributor to binding of acetylcho-
line42 but appears less important for nicotine.43,44 Hydro-
gen-bonding interactions have been proposed to account
for charge compensation of protonated amines within that
site.45-47 General features of the cation binding site are well-
conserved across nAChR subtypes, including R4β2 and R7,
and do not provide much basis for designing subtype-selective
ligands. On the other hand, homology models based on
AChBP structures have revealed differences in the ligand
binding site outside of the cation binding domain,48,49 and it
has been suggested that the R7 binding site is more lipophilic
and presents a less negative electrostatic surface than that for
the R4β2 subtype.50 Although it has not been very difficult to
develop ligands that exhibit high selectivity for either R4β2 or
R7 subtype over the other, to our knowledge, there have been
no systematic studies of structure and substitution effects that
determine this selectivity. We have identified a series of
N-pyridinyldiamines that exhibit high selectivity for either
R4β2 or R7 nAChR subtype depending upon simple changes
*To whom correspondence should be addressed. Phone: (847) 938-
9765. Fax: (847) 937-9195. E-mail: William.h.bunnelle@abbott.com.
Address: Abbott Laboratories, R4DE, AP9A, 100 Abbott Park Road,
Abbott Park, IL 60064.
† Abbott Laboratories.
‡ NeuroSearch A/S.
a Abbreviations: CNS, central nervous system; nAChR, nicotinic
acetylcholine receptor; AChBP, acetylcholine binding protein; GABA,
gamma aminobutyric acid; FLIPR, fluorometric light-imaging plate
reader; POETs, parallel oocyte elctrophysiology test station.
r
pubs.acs.org/jmc
Published on Web 06/24/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4127
Chemistry
in substitution patterns. In addition to providing a general
scaffold for preparation of ligands for either subtype, the SAR
trends have the potential to clarify pharmacophore models of
the R4β2 and R7 binding sites.
The Boc-protected diamine 5 was prepared (Scheme 1) by
a four-step process involving azomethine ylide cycloaddi-
tion51 to maleimide, reduction of the imide, and exchange of
protecting groups. N-Arylation was accomplished either by
base-mediated nucleophilc aromatic substitution (for acti-
vated heteroaryl halides) or through Pd-catalyzed amina-
tion of less reactive aryl and heteroaryl halides.52-54 Thus, a
variety of 3-bromopyridines reacted smoothly with 5 under
Buchwald-Hartwig conditions to provide the pyridinyl
amines Boc-6 to Boc-17 (Scheme 2). Removal of the Boc
group with acid provided the amines 6-17. The liberated
secondary amine could be further substituted by reductive
alkylation. Alternatively, Boc removal and N-methylation
could be carried out in a single step under Eschweiler-
Clarke conditions.
Other substituents were accessible by straightforward ma-
nipulation of the pyridine coupling products. Hydrogenolysis
of the benzyl ether Boc-15 provided the hydroxylpyridine 18
after deprotection. Monoamination of 3,5-dibromopyridine
and 5-bromo-2-chloropyridine provided Boc-11 and Boc-8,
respectively, which could be elaborated by Suzuki-Miyaura
coupling to the arylated analogues 19-22.
Figure 1. Selective nAChR ligands advanced to human clinical
trials.
Scheme 1. Preparation of the Diaminea
Other dihalogenated aromatics were also efficiently mono-
aminated (Scheme 3). Thus, symmetrical dihalides 25a and
26a proceeded to the corresponding aminoarylhalides Boc-25
and Boc-26. Amination of 2,5-dibromopyridine (24a) reacted,
as previously reported,54 with substitution at C(2) to provide
Boc-24. The regioisomeric Boc-10 was easily prepared by
electrophilic bromination of Boc-6. All of these halogenated
derivatives were useful substrates for coupling with arylboro-
nic acids to provide biaryl derivatives 27-46.
a Reagents and conditions: (a)CF3CO2H, CH2C12, 5 °C (83%); (b)
LiAIH4, THF, 70 °C; (c) Boc2O, CH2Cl2, 20 °C; (d) H2 (60 psi), Pd
(OH)2, CH3OH, 50 °C (75%).
Scheme 2a
a Reagents and Conditions: (a) Pd2dba3, rac-BINAP, NaOt-Bu, toluene, 85 °C; (b) H+, 0-20 °C; (c) HCHO/H2O, HCO2H, 100 °C, (d) H2, Pd(OH)2,
50 °C; (e) PhB(OH)2, Pd(0), Na2CO3(aq)/toluene or Cs2CO3/dioxane, 80 °C.

4128 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Bunnelle et al.
Scheme 3a
a Reagents and conditions: (a) Pd2dba3, rac-BINAP, NaOt-Bu, toluene, 85 °C; (b) PhB(OH)2, Pd(0), Na2CO3(aq)/toluene or Cs2CO3/dioxane, 80 °C;
(c) H+, 0-20 °C; (d) HCHO, NaBH(OAc)3; (e) N-bromosuccinimide, CH3CN, 0-20 °C.
Scheme 4a
were normalized to the average of those to 10 mM ACh,
applied to the same oocyte before and after the test com-
pound.
Results and Discussion
Binding affinities for native R4β2 and R7 receptors in rat
brain are collected in Table 1. It should be noted that typical
“nonselective” (acharacterization that refers to activityacross
heteromeric receptor subtypes) nAChR ligands like (-)-nico-
tine and epibatidine actually exhibit significantly higher affi-
nity for the R4β2 nAChR compared to the R7 subtype: for
these examples approximately 200-fold and 60-fold differ-
ences, respectively. The parent compound of the series, 6,
a Reagents and conditions: (a) iPr2NEt, DMSO, 105 °C; (b) H+, 0-
20 °C; (c) HCHO, HCO2H, 100 °C.
exhibits potent (subnanomolar) affinity for the R4β2 subtype
and approximately 400-fold weaker R7 activity.
The SAR trends for 6 follow the patterns established for
other pyridine-based nAChR ligands.60-67 In particular, only
the 3-pyridinyl attachment affords compounds with potent
binding affinity, while the 2- and 4-pyridinyl analogues (47
and 48, respectively) are substantially weaker at both R4β2
and R7 subtypes. Also typical of most pyridinyl diamines,64,68
N-methylation (7, 9) causes a very sharp (ca. 200-fold)
attenuation of R4β2 binding, which in these instances is
mirrored by a similar decrease in R7 affinity. The N,N-
dimethylated quaternary ammonium salt 23 shows a further
drop in potency at the R4β2 receptor, but enhanced R7 affinity
relativeto9, so that 23is essentially nonselective for bindingto
these major brain subtypes. The effect of sequential N-methy-
lation on the R4β2 activity of other diamines has been
studied;68 quaternization generally improved potency relative
to the tertiary amine. In this context, 23 resembles the
analogous homopiperazine system where the lower activity
of the quaternary dimethyl ammonium derivative was attrib-
uted to a slight change in ligand orientation in the cation
binding domain that pivoted the 6-substituent of the hetero-
aryl group, resulting in an unfavorable interaction with a
leucine residue at the remote edge of the ligand binding site.69
Because the R7 nAChR already accommodates larger substit-
uents in this position than does the R4β2 subtype (see below),
it seems reasonable that quaternization might have a differ-
ential effect on R7 vs R4β2 binding.
Direct coupling of 5 to 3-chloro-6-phenylpyridazine, ac-
complished with base in DMSO, provided more convenient
access to 31 and 32 (Scheme 4). Similar conditions sufficed for
amination of 2-chloropyridine to form 47, but 4-bromopyr-
idine reacted very sluggishly, and Pd catalysis was required for
synthesis of 48.
Biological Assays
The affinities of these ligands for native R4β2 and R7
nAChR subtypes in rat brain membranes were measured by
radioligand binding using previously reported methods.55,56
Briefly, displacement of [3H]-cytisine was taken to represent
binding to the R4β2 subtype, while the R7 affinities were
determined by displacement of the R7-selective agonist [3H]-
A-585539.56
Selected compounds were also evaluated for agonist activity
at the R4β2 and R7 receptor subtypes. For the heteromeric
nAChR, functional activity was determined from calcium influx
detected by fluorometric light-imaging plate reader (FLIPR)
methodology, according to the reported methods,57,58 employ-
ing an HEK-293 cell line expressing recombinant R4β2
nAChR. Experiments were run in 96-well plate format, and
responses were normalized to that of 100 μM (-)-nicotine on
the same plate.
The rapid desensitization of the R7 nAChR precludes use of
FLIPR methodology for this subtype, so agonist activity was
measured in oocytes expressing the human R7 nAChR using
the automated electrophysiological robotic system parallel
oocyte electrophysiology test (POET station).59 Responses
The effects of pyridine substituents on R4β2 activity have
been extensively investigated, with remarkable consistency
across a wide range of structure types.60-67 In general, only
small (e.g., halogen) substitutents are tolerated at the pyridine

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4129
Table 1. Binding Affinities at R4β2 (Cytisine Binding) and R7 (A-585539 Binding) in Rat Brain
structure
cytisine binding
Ki (nM) SEM range
0.94
A-585539 binding
Ki (nM) SEM range
176
2.7
selectivity
example
NR
R5
R6
(R4β2:R7)
(-)-nicotine
epibatidine
(0.46-1.9)
(0.035-0.063)
(0.11-0.14)
(23-29)
(120-260)
(2.5-3.0)
(43-62)
190
57
0.047
0.12
6
7
H
H
H
H
H
H
Br
H
51
1590
420
61
CH3
H
H
26
0.20
38
(1550-1630)
(90-360)
8
Cl
Cl
Br
H
(0.14-0.28)
(34-42)
180
900
9
CH3
H
>10000
290
66
>260
1000
1100
2900
41000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
47
48
0.28
(0.21-0.39)
(43-85)
(200-420)
(58-76)
H
0.060
0.081
0.035
0.041
0.058
0.051
H
OCH3
OCH2CH3
H
(0.073-0.089)
(0.031-0.040)
(0.036-0.047)
(0.038-0.089)
(0.045-0.058)
(44-52)
240
1440
(190-320)
(870-2400)
H
H
H
OCH2CH2CH3
OCH2C6H5
H
>10000
>10000
>10000
>10000
>10000
4560
>240000
H
H
>170000
>200000
>210
H
OCH(CH3)2
morpholin-4-yl
H
H
H
47
H
OH
C6H5
C6H5
H
H
0.75
0.16
(0.66-0.85)
(0.13-0.20)
(43-61)
>13000
28000
>190
H
H
(3200-6500)
CH3
H
H
51
>10000
C6H5
C6H5
Cl
H
96
28000
210
(80-114)
8.4
9.6
340
(6.2-11.4)
(9.1-10.2)
(280-400)
0.09
0.0003
1.6
CH3
(CH3)2
H
H
(22000-35000)
(190-240)
(250 280)
H
H
260
>10,000
>10000
>10000
>38
H
H
Table 2. Effect of Aminoaryl Group on nAChR Affinity
structure
cytisine binding
A-585539 binding
selectivity
example
R
Y
Z
Ki (nM)
(SEM range)
Ki (nM)
(SEM range)
(R7:R4β2)
21
22
27
28
29
30
31
32
33
34
H
CH
CH
N
N
96
28000
(80-114)
(22000-35000)
8.4
9.6
(6.2-11.4)
(9.1-10.2)
(27-35)
(11-28)
(26-30)
(8.8-16.1)
(17.1-18.0)
(9.0-12.9)
(180-620)
(5.0-7.1)
11
2900
CH3
H
N
CH
CH
CH
CH
N
>100000
>100000
>10000
>100000
5320
30
17
>3300
>6000
>350
>8300
300
CH3
H
N
CH
CH
N
28
12
CH3
H
(2800-10000)
17.5
10.8
330
5.98
CH3
Et
N
N
>100000
>100000
>10000
>9200
>300
>1600
N
N
(CH3)2
N
N
6-position, while the 5-position accommodates a much broad-
er range of groups. Not surprisingly, the data in Table 1
reinforce this trend for effects on R4β2 affinity. Substituents at
the pyridine 5-position ranging from Br (11), straight and
branched chain alkyl ethers (12-16), and phenyl (19) main-
tain or even improve the affinity for the R4β2 nAChR. In
contrast, the morpholino group (17) has a detrimental effect
on nAChR affinity. The 5-hydroxyl substitution results in a
6-fold drop in binding potency, but 18 remains a subnano-
molar compound. Likewise, while halogen (8, 10) substitution
atthe 6-positioniswell-tolerated, a 6-phenylgroup (21) results
in a nearly 800-fold loss of affinity for the R4β2 nAChR.
Toourknowledge, the effect of pyridine substitutionsonR7
binding affinity has not been studied systematically. Studies of
epibatidine SAR indicate that analogues with small groups at
the pyridine 5-position retain R7 activity,65,66 but phenyl
substitution is not tolerated.67 The results in Table 1 extend
those findings to the N-pyridinyl diamine scaffold and illus-
trate that pyridine substitutions have nearly opposite effects
on R7 binding as compared to R4β2 activity. For example,

4130 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Table 3. Effects of Biaryl Substituents on nAChR Binding Affinities
Bunnelle et al.
structure
Y
cytisine binding
A-585539 binding
selectivity
example
R
(R7:R4β2)
SEM range
Ki (nM)
11.6
4.42
6.7
44
SEM range
(R7:R4β2)
35
36
37
38
39
40
41
42
43
44
45
46
N
4-methyl
3-methyl
3-methyl
3-phenyl
>100000
>100000
750
(7.4-18.1)
(3.7-5.3)
(6.0-7.2)
(40-48)
>8600
>22000
112
N
CH
CH
N
(590-930)
9800
(8400-11500)
220
3,5-dimethyl
2-methyl
4-methoxy
4-hydroxy
3-methoxy
3-hydroxy
3-nitro
>100000
>10000
>100000
>10000
>100000
21000
>10000
690
22
N
(620-770)
(15-32)
>14
>4500
>1800
>2000
2660
N
N
5.3
(5.0-5.7)
(4.5-5.5)
(5.2-12)
(25-35)
CH
N
5.0
7.9
(18000-25000)
(22000-29000)
CH
2600
>100000
30
0.24
87
>400000
(0.21-0.28)
Scheme 5
nearly all substitutions at the pyridine 5-position that are
compatible withhighR4β2 affinity attenuate R7 binding. Only
5-Br (11) and 5-OMe (12) retain submicromolar R7 affinity,
and the potency drops rapidly as the size of the alkoxy group
increases. The 5-phenyl substituent (19) causes a 90-fold loss
of binding affinity relative to the parent 6. On the other hand,
6-phenyl substitution (21) results in a 6-fold enhancement of
potency (compared to 6) at the R7 nAChR. Coupled with the
decreased affinity for R4β2, 21 achieved 11-fold selectivity for
binding to the R7 vs R4β2 nAChR. Somewhat surprisingly in
light of the effect of N-methylation on binding for 7 and 9,
N-methylation of the 6-phenyl analogue (as in 22) does not
change R7 affinity significantly, although it does cause the
expected loss of R4β2 potency. The combination of N-methy-
lation and 6-phenyl substitution, then, results in a shift of
selectivity from a compound that has 400-fold selectivity for
R4β2 (6) to 22, with ∼2900-fold selectivity for R7 binding.
High selectivity for R4β2 binding, on the other hand, is
favored by a pyrrolidine NH and substitutions on the pyridine
5-position. Nearly 3000-fold selectivity is achieved already
with a 5-methoxy group (12) and larger substituents (13-16)
drive the affinity ratio still higher.
Further SAR studies reveal that R7 binding affinity is much
less sensitive to the nature of the N-aryl ring than is R4β2
binding. While a shift in the aza-ring position from 6 to 47
results in a 2000-fold loss of potency at R4β2, the correspond-
ing 5-phenyl-2-pyridinyl R7 ligand (28) is nearly as potent as
the 6-phenyl-3-pyridinyl isomer 22. Infact, the pyridine canbe
replaced with a phenyl ring (30) without loss of R7 affinity.
These, and the pyridazine analogue 32, actually show en-
hanced R7 vs R4β2 selectivity compared to 22 due to replace-
ment of the 3-pyridinyl pharmacophore that is critical for
potent R4β2 binding. The high R7 affinity of the biphenyl
analogues 29 and 30 is consistent with the suggestion that the
R7 ligand binding domain is more lipophilic than that for
R4β250 and further suggests that hydrogen bonding to the
N-aryl group is not a prerequisite for strong binding. Never-
theless, the more hydrophilic pyridine and pyridazine linking
rings are certainly well-tolerated. We have demonstrated that
32, in particular, is an exceptionally useful compound for
evaluating R7 nAChR activity in vitro and in vivo and have
shown that it is potent and efficacious across several animal
models of learning, memory, and attention.70,71
Similar to 21 and 22, the R7 activities of the biaryl diamines
27-32 are barely influencedby methylation of the basic amine
group. In fact, the N-methyl derivatives 28, 30, and 32 exhibit
slightly greater R7 binding potency than the corresponding
des-methyl analogues 27, 29, and 31. Homologation to an
N-ethyl group, on the other hand, causes a 30-fold loss of
binding potency (33 vs 32), while the N,N-dimethyl quatern-
ary salt (34) retains high affinity for the R7 nAChR.
The terminal phenyl ring of the biaryl system accommo-
dates further substitution. A para- (35) or meta-methyl group
(36, 37) does not change R7 affinity, and even a meta-phenyl
substituent (38) is tolerated with only a small decrease in
potency. On the other hand, substitution at both meta posi-
tions (39) completely eliminates R7 binding, suggesting that
one edge of the terminal phenyl ring already makes close
contact with receptor protein in the R7 binding pocket. Like-
wise, even a single ortho-substituent (40) causes a sharp drop
in in potency. In this case, it seems likely that a coplanar
geometry of the biaryl system is preferred for R7 binding and
the ortho-methyl prevents that arrangement. This explanation
is consistent with the potent R7 affinity reported for the
structurally related, but conformationally constrained analo-
gue 47 (Scheme 5).72
Electron donating (41-44) groups appear to be slightly
preferredto electron-withdrawing (45) groups, but the effect is
not dramatic. The notion that a para-situated hydrogen bond
donor (42 vs 41) seemed to enhance R7 binding affinity was
inspiration for replacement of the terminal phenyl by an

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4131
Table 4. Agonist Activity for Selected Compounds at Recombinant hR4β2 and hR7 nAChRs Expressed in Vitro
R4β2 FLIPR
R7 POETs
EC50 (μM) (SEM range)
example
EC50 (μM) (SEM range)
max
max
(-) nicotinea
6.6 (5.9-7.4)
0.040 (0.034-0.046)
0.37 (0.27-0.51)
>100
101 ( 3
146 ( 16
118 ( 10
<10
91 (78-104)
4.47 (2.42-8.27)
>100
74 ( 3
129 ( 25
37b
6
13
19
31
32
46
>100
<10
>100
>100
>100
<10
<10
1.15 (0.62-2.15)
4.26 (2.64-6.61)
0.32 (0.14-0.66)
29 ( 4
52 ( 5
87 ( 11
<10
a Nicotine data published previously.62,73 b Maximum response at 100 μM.
indolyl group, resulting in the most potent R7 ligand (46)
identified in this series.
which also disfavors R4β2 binding. N-Methylation improves
R7 further, mainly by reducing affinity at the R4β2 nAChR.
These simple structure changes on a common scaffold should
help to illuminate the differences in the ligand binding
domains for R4β2 and R7 nAChRs, particularly in the regions
extending away from the conserved cation binding pocket.
Having established structural modifications that can be
manipulated to design ligands with high selectivity for binding
to either R4β2 or R7 nAChRs, it remained to evaluate the
functional activity of selective ligands. Binding experiments
reflect interaction of the ligand with a desensitized, inactive
state of the receptor and do not provide information about
channel-activating (agonist) activity that is considered to be
important for selective nAChR ligands that have advanced to
clinical trials.29,32,34,35 Agonist data for selected compounds is
presented in Table 4. It can be seen that 6 is a full agonist at
both R4β2 and R7 nAChRs and significantly more potent
than (-)-nicotine. Comparison of agonist potencies across
R4β2 and R7 subtypes must be done with caution because the
assays are run in different systems, but 6 is clearly more
selective toward activating the R4β2 nAChR than is nicotine.
More to the point, the 5-ethoxy derivative 13, which exhibits
high selectivity for R4β2 in the binding assay, is also a potent
full agonist at this receptor but only partially activates the R7
nAChR at high concentrations. It can be noted that the size of
the 5-substituent does eventually affect the agonist activity of
these ligands; the 5-phenyl analogue 19, also a highly selective
R4β2 ligand, fails to activate the receptor in functional assays.
It is likewise inactive at the R7 subtype.
In contrast, the 6-aryl analogues like 31 and 32 are fairly
potent partial agonists of the R7 nAChR and highly selective
for that subtype because they do not activate the R4β2
receptor. It is interesting to note that 31 appears to be a little
more potent as an agonist than is 32, opposite to their relative
binding affinities, but 32 exhibits higher agonist efficacy than
31. The partial efficacy (30-50% of the maximal response
achieved by the native transmitter acetylcholine) observed in
the electrophysiological assay does not appear to be a limita-
tion for the in vivo pharmacological effects; the R7 partial
agonists MEM-3454 and GTS-2136,37 have been shown to
enhance cognitive performance in human clinical trials, and
we have reported that 32 achieves full efficacy in a variety of
preclinical animal models of cognition, memory, and atten-
tion.70,71 Nevertheless, the data for 46 demonstrates that it is
possible to identify ligands in this series with nearly full
agonist efficacy while maintaining the R7-selective profile.
Experimental Section
General. Unless otherwise specified, all reagents and solvents
were obtained from commercial suppliers and were used without
further purification. Flash chromatography was performed
using silica gel (230-400 mesh) from EM Science or prepacked
columns supplied by Analogix, Isco, or Silicycle. Proton NMR
spectra were recorded at 300 MHz in the solvent indicated, and
chemical shifts are listed in ppm downfield of internal tetra-
methylsilane. Mass spectra were obtained in chemical ionization
mode, and only parent ions are listed. Elemental analyses were
performed by Robertson Microlit Laboratories (Madison, NJ)
or QTI Inc. (Whitehouse, NJ). Compounds for biological test-
ing were typically prepared as water-soluble salts in g95%
purity, in accord with results from combustion analysis.
Preparation of t-Butyl cis-Hexahydropyrrolo[3,4-c]pyrrole-
2(1H)-carboxylate (5). Step 1. cis-5-Benzyltetrahydropyrrolo[3,
4-c]pyrrole-1,3-dione. A solution of N-benzyl-N-methoxymethyl-
N-(trimethylsilyl)methylamine51 (76.68 g, 0.29 mol) in dichloro-
methane (130 mL) was added to an ice-cooled mixture of mal-
eimide (25.53 g, 0.26 mol) and trifluoroacetic acid (2.2 mL, 0.028
mol)indichloromethane (350 mL) over 40 minsothat the reaction
temperature remained between 0 and 5 °C. The resulting bright-
yellow solution was allowed to warm gradually and stirred at
room temperature for 27 h. The mixture was washed with
saturated NaHCO3(aq) (80 mL), and the organic phase was dried
(MgSO4) and concentrated under vacuum. The residual oil was
stirred with 10% EtOAc-heptane (300 mL) for 15 h, and the
resulting precipitate was isolated by filtration, washed with 10%
EtOAc-heptane (150 mL) and dried under vacuum at
50 °C to provide a white solid (54.25 g), contaminated by vari-
able amounts of the N-(N-benzyl-N-methylaminomethyl) Man-
nich base of the title compound. The crude product was
stirred with methanol (500 mL) and 50% aqueous H2NOH
(4.2 mL) at room temperature for 20 h. This mixture was
concentrated under vacuum, and the residue was taken up in
EtOAc (500 mL) and filtered to remove some insoluble material.
The filtrate was concentrated under vacuum to leave the title
compound as an off-white solid (49.9 g, 83% yield), sufficiently
pure to use in the next step. An analytical sample was obtained by
crystallization from EtOAc. 1H NMR (CD3OD) δ 2.32-2.43 (m,
2 H), 3.18 (d, J = 9.8 Hz, 2 H), 3.20-3.26 (m, 2 H), 3.59
(s, 2 H), 7.13-7.35 ppm (m, 5 H). MS m/z 231 (MH)+. Anal.
(C13H14N2O2) C, H, N.
Summary
We have identified an N-aryldiamine scaffold that can be
manipulated to produce ligands that are highly selective for
binding to either the R4β2 or R7 nAChRs. Beginning with 6,
incorporation of substituents at the pyridine 5-position leads
to highly selective R4β2 ligands, mainly by strong attenuation
of binding to the R7 subtype. On the other hand, potent R7
binding is favored by an aryl group at the pyridine 6-position,
Step 2. t-Butyl cis-5-Benzylhexahydropyrrolo[3,4-c]pyrrole-
2(1H)-carboxylate. A three-necked, 2 L flask with mechanical
stirrer, thermometer, and addition funnel was charged with
LiAlH4 (16.9 g, 0.445 mol) and THF (200 mL). The slurry
was cooled in ice under a nitrogen atmosphere as a solution of

4132 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Bunnelle et al.
cis-5-benzyltetrahydropyrrolo[3,4-c]pyrrole-1,3-dione (49.0 g,
0.202 mol) in THF (250 mL) was added dropwise over 20 min
such that the temperature remained below 14 °C. After the
addition was complete, the reaction mixture was stirred for
40 min with ice cooling and then heated at reflux for 3 h. The
mixture was again cooled in ice and quenched by gradual,
successive addition of water (17 mL), 15% NaOH(aq) (17 mL),
and water (51 mL). The resulting slurry was filtered and the cake
washed well with EtOAc (2 ꢀ 400 mL). The combined filtrate
and wash was concentrated under vacuum to provide a colorless
oil (43.5 g), which was dissolved in dichloromethane (200 mL).
The solution was stirred with ice cooling as di-t-butyl dicarbo-
nate (46.3 g, 0.212 mol) was added gradually (gas evolution),
and the resulting solution was allowed to warm to room
temperature and stirred for 1 h. Aspartic acid (5.3 g, 0.04 mol)
was added, and the mixture was stirred for 30 min and
then transferred to a separatory funnel and washed with 1 N
NaOH(aq) (100 mL). The organic layer was dried (Na2SO4) and
concentrated to provide the titled compound as a colorless oil,
and then cooled to room temperature and poured onto ice-
water (100 g). The mixture was extracted with ethyl ether (2 ꢀ
50 mL), and the combined extract was washed with satd brine
(40 mL), dried (Na2SO4), and concentrated. The residual oil
(a mixture of starting dibromide and product, with some DMF)
was purified by flash chromatography (silica, hexanes-EtOAc
1
80:20) to provide the title compound (1.15 g, 25% yield). H
NMR (CDCl3) δ 1.05 (t, J = 7.4 Hz, 3 H), 1.83 (tq, J = 7.0, 6.8
Hz, 2 H), 3.96 (t, J = 6.4 Hz, 2 H), 7.36 (dd, J = 2.6, 1.8 Hz,
1 H), 8.23 (d, J = 2.6 Hz, 1 H), 8.27 ppm (d, J = 1.8 Hz, 1 H).
MS m/z 216/218 (MH)+.
3-Bromo-5-isopropyloxypyridine. Solid K2CO3 (1.3 g, 9.44
mmol) was added to a solution of 5-bromopyridin-3-ol
(705 mg, 4.05 mmol) and 2-iodopropane (1.70 g, 1.00 mmol)
in DMF (10 mL). The mixture was purged with a nitrogen
stream for 15 min and then heated under nitrogen at 80 °C for
16 h. The reaction was cooled to room temperature, diluted with
water (100 mL), and extracted with hexanes (2 ꢀ 50 mL). The
combined extract was washed with water (50 mL), satd brine
(40 mL), dried (Na2SO4), and concentrated to a pale-yellow oil.
Bulb-to-bulb distillation (115-120 °C air bath/21 Torr) pro-
vided the title compound as a colorless oil (0.78 g, 88% yield). 1H
NMR (CDCl3)δ 1.36 (d, J = 6.1 Hz, 6 H), 4.56 (heptet, J =
6.0 Hz, 1 H), 7.35 (dd, J = 2.4, 1.7 Hz, 1 H), 8.20 (d, J = 2.4 Hz,
1 H), 8.25 ppm (d, J = 1.7 Hz, 1 H). MS m/z 216/218 (MH)+.
4-(5-Bromopyridin-3-yl) Morpholine. Morpholine (0.33 g,
3.83 mmol), 3,5-dibromopyridine (1.09 g, 4.60 mmol), race-
mic-2,20-bis(diphenylphosphino)-1,10-binaphthyl (rac-BINAP,
0.14 g, 0.23 mmol), tris(dibenzylideneacetone)dipalladium(0)
(Pd2(dba)3, 70 mg, 0.08 mmol), and sodium t-butoxide (0.55 g,
5.75 mmol) were combined with toluene (25 mL). The suspen-
sion was evacuated and purged with nitrogen. The mixture was
heated at 115 °C under nitrogen for 18 h. The residue was filtered
through a pad of silica, followed by an EtOAc (150 mL) rinse.
The filtrate was concentrated under vacuum, and the residue
was purified by flash chromatography (hexanes-EtOAc, 50:50)
1
sufficiently pure for use in the next step (64.73 g, 108%). H
NMR (CD3OD) δ 1.45 (s, 9 H), 2.34 (dd, J = 9.3, 3.9 Hz, 2 H),
2.69-2.78 (m, 2 H), 2.78-2.88 (m, 2 H), 3.20-3.30 (m, 2 H),
3.39-3.50 (m, 2 H), 3.59 (s, 2 H), 7.15-7.39 ppm (m, 5 H). MS
m/z 303 (MH)+.
Step 3. t-Butyl cis-Hexahydropyrrolo[3,4-c]pyrrole-2(1H)-
carboxylate (5). The crude t-butyl cis-5-benzylhexahydropyrro-
lo[3,4-c]pyrrole-2(1H)-carboxylate (64.70 g, 0.212 mol) was
dissolved in methanol (250 mL). Pearlman’s catalyst (Pd-
(OH)2, 20% on C, 6.5 g) was added, and the mixture was shaken
at 50 °C under hydrogen (60 psi) for 4 h. The mixture was cooled
to room temperature, filtered under nitrogen to remove the
catalyst, and the filtrate was concentrated under vacuum to
leave a pale oil (46.55 g). This was dissolved in ether (150 mL),
applied to a column of silica gel (300 g), and eluted with ether
(1 L). The column was then eluted with methanol (2 L), and the
methanol fraction was concentrated under vacuum. The residue
was taken up in EtOAc (500 mL), filtered to remove insoluble
material, and concentrated under vacuum to provide the title
compound as an off-white solid (33.84 g, 75% yield over two
1
to provide the title compound (0.66 g, 71% yield). H NMR
(CD3OD) δ 3.20-3.26 (m, 4 H), 3.77-3.87 (m, 4 H), 7.50-7.60
(m, 1 H), 8.03 (d, J = 1.7 Hz, 1 H), 8.20 ppm (d, J = 2.4 Hz, 1 H).
MS m/z 243/245 (MH)+.
1
steps), sufficiently pure for use in subsequent operations. H
NMR (CD3OD) δ 1.45 (s, 9 H), 2.65 (dd, J = 11.5, 4.4 Hz, 2 H),
2.75-2.89 (m, 2 H), 3.01-3.11 (m, 2 H), 3.20 (dd, J = 11.5, 3.7
Hz, 2 H), 3.47-3.58 ppm (m, 2 H).
General Pyridine-Amine Coupling Procedure (A). t-Butyl cis-
5-(3-Pyridinyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate
(Boc-6). A solution of 5 (0.52 g, 2.45 mmol) in toluene (30 mL)
was dried by azeotropic distillation under N2 (1 atm) to a volume
of ∼20 mL. The solution was cooled to 35 °C and tris(dibenzy-
lideneacetone)dipalladium(0) (Pd2(dba)3) (45 mg, 0.049 mmol)
and racemic-2,20-bis(diphenylphosphino)-1,10-binaphthyl (rac-
BINAP, 75 mg, 0.12 mmol)wereadded. Themixturewas warmed
to 85 °C for 10 min and then cooled to 35 °C. Sodium t-butoxide
(371 mg, 3.86 mmol) and 3-bromopyridine (426 mg, 2.70 mmol)
were added, and the mixture was warmed to 85 °C under N2 for
2 h. The mixture was cooled to 30 °C and filtered through
diatomaceous earth with an EtOAc (100 mL) rinse. The filtrate
was concentrated under reduced pressure to provide a red oil,
which was purified by flash chromatography (CH2Cl2-CH3OH,
16:1) to provide the title compound as a pale-yellow solid (0.62 g,
87% yield). 1H NMR (CDCl3) δ 1.46 (s, 9 H), 2.96-3.12 (m, 2 H),
3.24(dd, J = 9.5, 4.1 Hz, 2 H), 3.34 (m, 2 H), 3.55 (dd, J = 9.5, 7.5
Hz, 2 H), 3.60-3.72 (m, 2 H), 6.81 (dd, J = 8.3, 1.9 Hz, 1 H), 7.13
(dd, J = 8.3, 4.6 Hz, 1 H), 7.89-8.07 ppm (m, 2 H). MS m/z
290 (MH)+.
General Procedure for Boc Deprotection with HCl. cis-2-(3-
Pyridinyl)octahydropyrrolo[3,4-c]pyrrole Dihydrochloride (6).
HCl/dioxane (4M, 2 mL, 8 mmol) was added to a solution of
Boc-6 (310 mg, 1.07 mmol) in ethanol (5 mL), and the solution
was stirred at 20 °C for 4 h. The solution was concentrated under
reduced pressure, and the residual solid was crystallized from
ethanol:EtOAc (1:5) to provide the title compound as a white
solid (203 mg, 72% yield). mp 250-252 °C (dec). 1H NMR
(CD3OD) δ 3.32 (m, 4H), 3.57 (dd, J = 10.7, 3.0 Hz, 2H), 3.65
3-Bromo-5-ethoxypyridine. Freshly cleaned sodium metal
(1.46 g, 63.3 mmol) was added gradually to ethanol (50 mL)
and stirred until the metal had reacted completely to give a clear
solution (1 h). The solution was concentrated under vacuum,
and the residue was suspended in dry toluene (20 mL) and
concentrated to a white solid (toluene treatment repeated a
second time). The solid was taken up in dry DMF (60 mL),
and 3,5-dibromopyridine (10.0 g, 42.2 mmol) was added. The
mixture was heated at 70 °C for 2.5 h, then cooled to room
temperature and poured into ice water (250 g). The mixture was
extracted with ethyl ether (2 ꢀ 100 mL), and the combined
extract was washed successively with water (50 mL) and satu-
rated brine (50 mL), dried (Na2SO4), and concentrated. The
residual brown oil was purified by flash chromatography (silica,
hexanes-EtOAc 80:20) to provide the title compound as a pale
oil (5.83 g, 69% yield). 1H NMR (CDCl3) δ1.43 (t, J = 7.0 Hz, 3
H), 4.07 (q, J = 7.2 Hz, 2 H), 7.34 (dd, J = 2.5, 1.7 Hz, 1 H), 8.22
(d, J = 2.5 Hz, 1 H), 8.26 ppm (d, J = 1.7 Hz, 1 H). MS m/z 202/
216 (MH)+.
3-Bromo-5-propyloxypyridine. Freshly cleaned sodium metal
(0.73 g, 32 mmol) was stirred with 1-propanol (20 mL) until the
metal had reacted completely to give a clear solution (3 h). The
solution was concentrated under vacuum, and the residue was
suspended in dry toluene (20 mL) and concentrated to a white
solid (toluene treatment repeated a second time). The solid was
taken up in dry DMF (30 mL), and 3,5-dibromopyridine (5.0 g,
21 mmol) was added. The mixture was heated at 70 °C for 4 h

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4133
(m, 4H), 7.78 (m, 1H), 7.83 (dd, J = 8.2, 5.1 Hz, 1H), 8.09
(d, J = 5.1 Hz, 1H), 8.12 ppm (d, J = 2.2 Hz, 1H). MS m/z 190
cis-5-(6-Bromo-pyridin-3-yl)-hexahydro-pyrrolo[3,4-c]pyrrole
Trifluoroacetate (10). Trifluoroacetic acid (3 mL) was added to a
solution of Boc-10 (100 mg, 0.27 mmol) in CH2Cl2 (4 mL), and
the solution was stirred at room temperature for 30 min and then
concentrated under vacuum. The residue was combined with
toluene (3 mL) and concentrated under vacuum. This process
was repeated once more to ensure removal of excess acid. The
residual oil was taken up in EtOAc (2 mL), and ethyl ether
(3 mL) was added gradually. The resulting suspension was
stirred at room temperature for 16 h. The precipitate was
isolated by filtration and dried under vacuum to provide the
(MH)+. Anal. (C11H15N3 2HCl) C, H, N.
3
General Procedure for Boc Deprotection with Concomitant
N-Methylation. cis-2-Methyl-5-(3-pyridinyl)octahydropyrro-
lo[3,4-c]pyrrole Dihydrochloride (7). A mixture of Boc-6
(200 mg, 0.69 mmol) in 88% formic acid (1.8 mL) and 37%
formalin (3.5 mL) was warmed to 95 °C for 3 h. The solution
was concentrated under reduced pressure, and the resulting
pale-yellow solid was taken up in 20% aq KOH (5 mL) and
extracted into ethyl acetate (2 ꢀ 20 mL). The organic phases
were combined, dried (MgSO4), and concentrated under
reduced pressure. The residue was dissolved in EtOH
(10 mL), treated with HCl/dioxane (4M, 2 mL), and concen-
trated under reduced pressure to leave an oily solid, which was
crystallized from EtOH:ethyl acetate (1:20) to provide the
title compound as an off-white, hygroscopic solid: mp 207-
1
title salt (89 mg, 86% yield). H NMR (CD3OD) δ 3.18-3.28
(m, 4 H), 3.32-3.41 (m, 2 H), 3.46 (dd, J = 9.8, 2.2 Hz, 2 H),
3.54-3.65 (m, 2 H), 7.08 (dd, J = 8.8, 3.1 Hz, 1 H), 7.39 (d, J =
8.5 Hz, 1 H), 7.78 ppm (d, J = 3.4 Hz, 1 H). MS m/z 268/270
(MH)+. Anal. (C11H14N3Br CF3CO2H 0.5H2O) C, H, N.
3
3
t-Butyl cis-5-(5-Bromopyridin-3-yl)hexahydro-pyrrolo[3,4-c]-
pyrrole-2-carboxylate (Boc-11). Prepared from 5 (212 mg, 1.0
mmol was coupled with 3,5-dibromopyridine (255 mg, 1.08
mmol) according to the procedure A as described for Boc-6.
The crude product was purified by chromatography on silica
gel, eluting with hexanes-EtOAc (1:1) to provide the title
compound as a light-yellow solid (256 mg, 70% yield). 1H
NMR (CDCl3) δ 1.46 (s, 9 H), 2.97-3.08 (m, 2 H), 3.23 (dd,
J = 9.7, 3.8 Hz, 2 H), 3.26-3.40 (m, 2 H), 3.55 (dd, J = 9.1, 7.5
Hz, 2 H), 3.60-3.74 (m, 2 H), 6.95 (br s, 1 H), 7.88 (br s, 1 H),
8.00 ppm (br s, 1 H). MS m/z 368/370 (MH)+.
1
209 °C. H NMR (CD3OD) δ 2.93-2.98 (2 s, endo and exo
CH3, 3 H) 3.00-3.10 (m, 1 H) 3.32-3.39 (m, 1 H) 3.39-3.55
(m, 3 H) 3.58-3.65 (m, 2 H) 3.65-3.75 (m, 2 H) 3.94-4.07
(m, 1 H) 7.76-7.88 (m, 2 H) 8.05-8.20 ppm (m, 2 H). MS m/z
190 (MH)+. Anal. (C12H17N3 2HCl 0.5H2O) C, H, N.
3
3
t-Butyl cis-5-(6-Chloro-3-pyridinyl)hexahydropyrrolo[3,4-c]-
pyrrole-2(1H)-carboxylate (Boc-8). Diamine 5 (1.70 g, 8 mmol)
and 5-bromo-2-chloropyridine (2.11 g, 8.8 mmol) were coupled
according to procedure A (described for Boc-6) to provide the
title compound as a yellow solid (1.18 g, 46% yield). 1H NMR
(CDCl3) δ 1.46 (s, 9 H), 2.98-3.12 (m, 2 H), 3.20 (dd, J = 9.5, 4.0
Hz, 2 H), 3.24-3.41 (m, 2 H), 3.52 (dd, J = 9.3, 7.3 Hz, 2 H),
3.58-3.72 (m, 2 H), 6.79 (dd, J = 8.7, 3.2Hz, 1 H),7.12 (d, J = 8.7
Hz, 1 H), 7.68 ppm (d, J = 3.2 Hz, 1 H). MS m/z 324/326 (MH)+.
cis-2-(6-Chloro-3-pyridinyl)octahydropyrrolo[3,4-c]pyrrole Hy-
drochloride (8). A solution of HCl in 1,4-dioxane (4M, 2 mL,
8 mmol) was added to Boc-8 (360 mg, 1.11 mmol) in dichlor-
omethane (20 mL), and the mixture was stirred at room tempera-
ture for 2 h and then concentrated under vacuum. The residual
yellow solid was crystallized from ethanol:EtOAc (2:1) after
carbon treatment to provide the title compound as a white solid
(198 mg, 69% yield): mp 230-236 °C (dec). 1H NMR (CD3OD) δ
3.28 (m, 4 H), 3.49 (m, 4 H), 3.62 (m, 2 H), 7.45 (dd,
J = 8.8, 3.1 Hz, 1 H), 7.52 (d, J = 9 Hz, 1 H), 7.90 ppm (d, J =
cis-2-(5-Bromopyridin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole
Trifluoroacetate (11). A solution of the product of Boc-11
(30 mg, 0.082 mmol) in CH2Cl2 (1 mL) was cooled in ice and
treated with trifluoroacetic acid (500 μL). The pale-yellow
solution was stirred at 0 - 5 °C for 1 h and then concentrated
under vacuum. The residue was triturated with ether (3 mL),
and the solvent was decanted. After a second trituration with
10% methanol-ether (3 mL), the resulting pale-yellow solid
was isolated by filtration and dried under vacuum at 50 °C to
provide the title compound (25 mg, 75% yield). 1H NMR
(CD3OD) δ 3.20-3.30 (m, 4 H), 3.38-3.51 (m, 4 H), 3.55-3.65
(m, 2 H), 7.33 (t, J = 2.2 Hz, 1 H), 7.98 ppm (m, 2 H). MSm/z 268/
270 (MH)+. Anal. (C11H14N3Br CF3CO2H 0.5H2O) C, H, N.
3
3
cis-2-(5-Methoxy-3-pyridinyl)octahydropyrrolo[3,4-c]pyrrole-
Dihydrochloride (12). Prepared from 5 (300 mg, 1.4 mmol) and 3-
bromo-5-methoxypyridine (290 mg, 1.5 mmol) according to
procedure A described for Boc-6, followed by deprotection with
HCl in dioxane and EtOAc to provide the title compound as a
white crystalline solid (150 mg, 37% yield). 1H NMR (CD3OD)
δ 3.32 (m, 4 H), 3.58 (dd, J = 10.7, 3.1 Hz, 2 H), 3.63 (m, 4 H),
4.00 (s, 3 H), 7.21 (t, J = 2.2 Hz, 1 H), 7.78 (d, J = 2.4 Hz, 1 H),
7.85 ppm (d, J = 2.0 Hz, 1 H). MS m/z 220 (MH)+. Anal.
3 Hz, 1 H). MS m/z 224/226 (MH)+. Anal. (C11H14N3Cl HCl)
3
C, H, N.
cis-2-(6-Chloro-3-pyridinyl)-5-methyloctahydropyrrolo[3,4-c]-
pyrrole (9). Aqueous 37% formalin (3.5 mL) was added to a
mixture of Boc-8 (207 mg, 0.64 mmol) and 88% formic acid (1.8
mL), and the solution was heated at 95 °C for 2 h. The solution
was concentrated under reduced pressure, and the resulting solid
was purified by chromatography (CH2Cl2:CH3OH:Et2NH,
94:5:1) to provide the title compound (135 mg, 88% yield). 1H
NMR (CDCl3) δ 2.36 (s, 3 H) 2.53 (dd, J = 9.5, 3.2 Hz, 2 H)
2.65-2.75 (m, 2 H) 2.94-3.08 (m, 2 H) 3.19 (dd, J = 9.3, 3.0 Hz,
2 H) 3.35-3.48 (m, 2 H) 6.88 (dd, J = 8.7, 3.2 Hz, 1 H) 7.11
(d, J = 8.7 Hz, 1 H) 7.75 ppm (d, J = 2.8 Hz, 1 H). MS m/z 238/
240 (MH)+. Anal. (C12H16N3Cl) C, H, N.
(C12H17N3O 2HCl) C, H, N.
3
cis-2-(5-Ethoxy-3-pyridinyl)octahydropyrrolo[3,4-c]pyrrole- Di-
hydrochloride (13). Prepared from 5 (600 mg, 2.8 mmol) and 3-
bromo-5-ethoxypyridine (625 mg, 3.1 mmol) by procedure A as
described for Boc-6, followed by deprotection and salt formation
with HCl in refluxing ethyl acetate-EtOH (3:1, 20 mL). After
cooling, the mixture was filtered to provide the title compound as a
white crystalline solid (435 mg, 51% yield): mp 226-227 °C. 1H
NMR (CD3OD) δ 1.57 (t, J = 7.0 Hz, 3 H), 3.30 (m, 4 H), 3.55
(dd, J = 10.6, 2.9 Hz, 2 H), 3.61 (m, 4 H), 4.25 (q, J = 7.0 Hz, 2 H),
7.19 (dd, J = 2.4, 1.7 Hz, 1 H), 7.78 (d, J = 2.4 Hz, 1 H), 7.84 ppm
t-Butyl cis-5-(6-Bromo-pyridin-3-yl)-hexahydro-pyrrolo[3,4-
c]pyrrole-2-carboxylate (Boc-10). A solution of N-bromosucci-
nimide(0.62 g, 3.46 mmol) inCH3CN(5mL) was addeddropwise
over 15 min to a solution of Boc-6 (1.0 g, 3.46 mmol) in CH3CN
(40 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min
and then warmed to room temperature. The reaction was diluted
with H2O (10 mL), and the layers were separated. The aqueous
layer was extracted with CH2Cl2 (2 ꢀ 5 mL), and the combined
organic phase was washed with brine (5 mL), dried over Na2SO4,
and concentrated under reduced pressure to give 1.24 g (3.38
(d, J = 1.7 Hz, 1 H). MS m/z 234 (MH)+. Anal. (C13H19N3O 2
3
HCl 0.5H2O) C, H, N.
3
cis-3-(5-Propyloxy-3-pyridinyl)-3,7-diazabicyclo[3.3.0]octane
Semifumarate (14). Prepared from 5 (300 mg, 1.4 mmol) and 3-
bromo-5-(1-propyloxy)pyridine (333 mg, 1.5 mmol) according
to procedure A as described for Boc-6, followed by deprotection
with HCl in refluxing ethyl acetate-EtOH (4:1, 10 mL). After
3 h, the solution was concentrated and the residue was purified
by flash chromatography (CH2Cl2-EtOH-NH4OH, 96:3:0.5)
to provide the free base of the title compound (70 mg). This was
1
mmol, 98% yield) of the title compound. H NMR (CDCl3) δ
1.46 (s, 9 H), 2.95-3.08 (m, 2 H), 3.20 (dd, J = 9.8, 4.1 Hz, 2 H),
3.23-3.40 (m, 2 H), 3.52 (dd, J = 9.9, 7.5 Hz, 2 H), 3.60-3.73 (m,
2 H), 6.71 (dd, J = 8.8, 3.1 Hz, 1 H), 7.25 (d, J = 8.8 Hz, 1 H), 7.69
ppm (d, J = 3.4 Hz, 1 H). MS m/z 368/370 (MH)+.

4134 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Bunnelle et al.
dissolved in ethyl acetate (8 mL) and methanol (1 mL), and a
solution of fumaric acid (34 mg) in methanol (1.2 mL) was added
dropwise. The solution was diluted with diethyl ether (5 mL) and
scratched to induce crystallization. The mixture was filtered and
the solid dried under vacuum to provide the title compound (50
mg, 17% yield). 1H NMR (CD3OD) δ 1.06 (t, J = 7.5 Hz, 3 H),
1.81 (qt, J = 7.5, 6.4 Hz, 2 H), 3.20 (m, 4 H), 3.40 (m, 4 H), 3.54
(m, 2 H), 3.99 (t, J = 6.4 Hz, 2 H), 6.65 (s, 1 H), 6.68 (t, J = 2.4
Hz, 1 H), 7.62 ppm (m, 2 H). MS m/z 248 (MH)+. Anal.
yield). 1H NMR (CD3OD) δ 3.05 (m, 2 H), 3.20 (dd, J = 9.8, 4.1
Hz, 2 H), 3.28 (m, 2 H), 3.51 (dd, J = 9.8, 7.5 Hz, 2 H), 3.64 (m, 2
H), 6.42 (t, J = 2.4 Hz, 1 H), 7.40 (d, J = 2.4 Hz, 1 H), 7.43 ppm
(d, J = 2.4 Hz, 1 H).
cis-2-(5-Hydroxy-3-pyridinyl)octahydropyrrolo[3,4-c]pyrrole-
Dihydrochloride (18). A solution of Boc-18 (150 mg, 0.49 mmol)
in EtOAc-CH3OH (1:2, 15 mL) was treated with HCl/1,4-
dioxane (4M, 1 mL, 4 mmol). The mixture was stirred at room
temperature for 16 h and then cooled in ice to complete
precipitation. The mixture was filtered to provide the title
compound as a white solid (136 mg, 99% yield). 1H NMR
(CD3OD) δ 3.30 (m, 4 H), 3.51 (dd, J = 10.3, 2.4 Hz, 2 H), 3.60
(m, 4 H), 7.06 (dd, J = 2.2, 1.8 Hz, 1 H), 7.62 (d, J = 1.8 Hz,
1 H), 7.69 ppm (d, J = 2.2 Hz, 1 H). MS m/z 206 (MH)+. Anal.
(C11H15N3O 2HCl 0.5H2O) C, H, N.
(C14H21N3O 0.5 C4H4O4) C, H, N.
3
t-Butyl cis-5-[5-(Benzyloxy)-3-pyridinyl]hexahydropyrrolo-
[3,4-c]pyrrole-2(1H)-carboxylate (Boc-15). Prepared from 5
(1.0 g, 4.7 mmol) and 3-(benzyloxy)-5-bromopyridine (1.37 g,
5.2 mmol) according to coupling procedure A described for Boc-
6 to provide the title compound as a solid (1.5 g, 81% yield). 1H
NMR (CD3OD) δ 1.45 (s, 9 H), 2.96-3.11 (m, 2 H), 3.21 (dd,
J = 9.9, 3.6 Hz, 2 H), 3.24-3.31 (m, 2 H), 3.52 (dd, J = 9.9, 7.1
Hz, 2 H), 3.64 (dd, J = 9.5, 7.1 Hz, 2 H), 5.13 (s, 2 H), 6.61 (t, J =
2.2 Hz, 1 H), 7.26-7.46 (m, 5 H), 7.51 (d, J = 2.4 Hz, 1 H), 7.61
ppm (d, J = 2.0 Hz, 1 H). MS m/z 396 (MH)+.
3
3
t-Butyl cis-5-(5-Phenylpyridin-3-yl)hexahydro-pyrrolo[3,4-c]-
pyrrole-2-carboxylate (Boc-19). A mixture of Boc-11 (158 mg,
0.43 mmol), phenylboronic acid (63 mg, 0.52 mmol) and tetrakis
(triphenylphosphine)palladium (13 mg, 0.011 mmol) was com-
bined with ethanol (6 mL) and 5% Na2CO3(aq) (1 mL). The
mixture was evacuated and purged with nitrogen (3 cycles)
and stirred under nitrogen at 85 °C for 1 h. The mixture was
cooled to 30 °C, diluted with water (25 mL), and extracted with
ethyl acetate (2 ꢀ 25 mL). The combined extract was concentrated
under vacuum, and the residue was purified by chromatography
on silica gel, eluting with 1:1 hexanes-EtOAc to provide the title
compound as a white foam (142 mg, 91% yield). 1H NMR
(CDCl3) δ 1.45 (s, 9 H), 3.06 (m, 2 H), 3.32 (dd, J = 9.7, 3.9 Hz,
2 H), 3.37 (br m, 2 H), 3.66 (m, 4 H), 7.03 (br s, 1 H), 7.46 (m, 3 H),
7.58 (m, 2 H), 7.95 (br s, 1 H), 8.20 ppm (br s, 1 H). MS m/z 366
(MH)+.
cis- 2-(5-(Benzyloxy)pyridin-3-yl)octahydropyrrolo[3,4-c]pyr-
role (15). Trifluoroacetic acid (2 mL) was added to an ice-cooled
solution of Boc-15 (363 mg, 0.92 mmol) in CH2Cl2 (5 mL). The
yellow solution was stirred with ice cooling for 1 h, then at room
temperature for 16 h. The solution was concentrated under
vacuum, and the residue was purified by chromatography
(CH2Cl2-EtOH-NH4OH, 90:10:1) to provide an off-white
solid, which was triturated with ethyl ether (10 mL) and dried
1
to provide the title compound (172 mg, 63% yield). H NMR
(CD3OD) δ 2.81 (dd, J = 11.5, 3.6 Hz, 2 H), 3.19 (m, 2 H), 3.18
(dd, J = 11.5, 7.1 Hz, 2 H), 3.25 (dd, J = 9.9, 2.4 Hz, 2 H), 3.39
(dd, J = 9.5, 7.5 Hz, 2 H), 5.14 (s, 2 H), 6.69 (t, J = 2.2 Hz, 1 H),
7.29-7.47 (m, 5 H), 7.59 (d, J = 2.4 Hz, 1 H), 7.64 ppm (d, J = 2.0
cis-2-(5-Phenylpyridin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole Bis-
trifluoroacetate (19). A solution of Boc-19 (42 mg, 0.115 mmol) in
CH2Cl2 (1.5 mL) was cooled in ice and treated with trifluoroacetic
acid (800 μL). The pale-yellow solution was stirred at 0 °C for 1 h
and then concentrated under vacuum. The residue was taken up in
ether (5 mL) and concentrated under vacuum, then triturated with
10% methanol-ether. The resulting white precipitate was filtered
and dried under vacuum at 50 °C to provide the title compound (45
mg, 80% yield). 1H NMR (CD3OD) δ 3.32 (m, 4 H), 3.63 (m, 6 H),
7.54 (m, 3 H), 7.73 (d, J = 1.4 Hz, 1 H), 7.76 (m, 2 H), 8.07 (d, J =
2.7 Hz, 1 H), 8.34 ppm (d, J = 1.4 Hz, 1 H). MS m/z 266 (MH)+.
Hz, 1 H). MS m/z296 (MH)+.Anal.(C18H21N3O 0.1H2O) C, H, N.
3
cis-3-(5-Isopropyloxy-3-pyridinyl)-3,7-diazabicyclo[3.3.0]oc-
tane Semi(fumarate) (16). Prepared in 63% yield from 5
(0.73 mmol) and 3-bromo-5-isopropyloxypyridine according
to the procedures described for 15. 1H NMR (CD3OD) δ 1.32
(d, J = 6.1 Hz, 6 H), 3.11-3.25 (m, 4 H), 3.33-3.40 (m, 2 H),
3.44 (dd, J = 9.8, 2.4 Hz, 2 H), 3.47-3.61 (m, 2 H), 4.65
(heptet, J = 6.0 Hz, 1 H), 6.65 (s, 1 H), 6.66 (t, J = 2.4 Hz, 1 H),
7.60 ppm (d, J = 2.4 Hz, 2 H). MS m/z 148 (MH)+. Anal.
(C14H21N3O 0.5C4H4O4) C, H, N.
Anal. (C17H19N3 2CF3CO2H) C, H, N.
3
3
cis-4-(5-(Hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridin-3-
yl)morpholine Sesqui-fumarate (17). Prepared from 5 (138 mg,
0.65 mmol) and 4-(5-bromopyridin-3-yl)morpholine (190 mg,
0.78 mmol) according to coupling procedure A as described for
Boc-6, followed by deprotection with trifluoroacetic acid in
CH2Cl2. The crude product was taken up with 1.0 M Na2-
CO3(aq) (100 mL) and extracted with CHCl3-iPrOH (4:1, 2 ꢀ
50 mL). The combined extract was dried (Na2SO4) and con-
centrated under vacuum. The residue was dissolved in a mini-
mal amount of methanol (ca. 1 mL), and a solution of fumaric
acid (50 mg, 0.43 mmol) in ether-methanol (10:1, 5.0 mL) was
added dropwise with stirring over 5 min. After 1 h, the
precipitate was collected by filtration, rinsed with ether, and
dried to afford the title compound (83 mg, 25% yield). 1H
NMR (CD3OD) δ 3.17-3.22 (m, 4 H), 3.23-3.39 (m, 6 H),
3.46-3.78 (m, 4 H), 3.80-3.86 (m, 4 H), 6.65 (t, J = 2.2 Hz,
1 H), 6.70 (s, 3.4 H; C4H4O4), 7.54 (d, J = 2.4 Hz, 1 H), 7.68
ppm (d, J = 2.4 Hz, 1 H). MS m/z 275 (MH)+. Anal.
cis-2-(5-Phenylpyridin-3-yl)-5-methyl-octahydro-pyrrolo[3,4-c]-
pyrrole Sesqui-fumarate (20). A solution of Boc-19 (100 mg, 0.274
mmol)and37%formalin(66 mg, 0.814 mmol)in88% formic acid
(1 mL) was warmed to 100 °C for 100 min. The mixture was
cooled to 30 °C and concentrated under vacuum. The residue was
taken up in 20% K2CO3(aq) (4 mL) and 25% NaOH(aq) (2 mL),
and the resulting mixture was extracted with CH2Cl2 (2 ꢀ 8 mL).
The combined extract was dried over K2CO3 and concentrated
under vacuum. The residue was dissolved in 10% methanol-
ether (3 mL) and treated with a solution of fumaric acid (33 mg,
0.284 mmol) in 10% methanol-ether (10 mL). The mixture was
scratched until an even white suspension resulted, and the solid
was isolated by filtration and dried under vacuum at 50 °C to
provide the title compound (59 mg, 46% yield). 1H NMR
(CD3OD) δ 2.91 (s, 3 H), (3.33 (m, 6 H), 3.64 (m, 4 H), 6.70
(s, 3 H, 1.5 fumaric acid), 7.48 (dd, J = 2.7, 2.7 Hz, 1 H), 7.41 (t,
J = 7.4 Hz, 1 H), 7.49 (t, J = 7.3 Hz, 2 H), 7.64 (d, J = 7.4 Hz, 2
H), 8.05 (d, J = 2.7 Hz, 1 H), 8.20 ppm (d, J = 1.7 Hz, 1 H). MS
m/z 280 (MH)+. Anal. (C18H21N3 1.5C4H4O4 0.2H2O) C, H, N.
t-Butyl cis-5-(6-Phenyl-pyridin-3-yl)-octahydro-pyrrolo[3,4-c]-
pyrrole-2-carboxylate (Boc-21). A pressure tube was charged with
Boc-8 (200 mg, 0.62 mmol), phenylboronic acid (238 mg, 1.24
mmol), Pd2dba3 (20 mg, 0.022 mmol), Cs2CO3 (611 mg, 1.86
mmol), and p-dioxane (15 mL). Solid 1,3-bis(2,6-di(isopropyl)
phenyl)imidazolium chloride (26 mg, 0.061 mmol) was added, and
the tube was closed under a nitrogen purge. The mixture was
heated at 85 °C for 13 h, and the mixture was cooled to room
(C15H22N4O 1.7C4H4O4) C, H, N.
3
3
3
t-Butyl cis-5-(5-Hydroxy-3-pyridinyl)hexahydropyrrolo[3,4-
c]-pyrrole-2(1H)-carboxylate (Boc-18). A solution of Boc-15
(1.3 g, 3.8 mmol) in 2-propanol (100 mL) was shaken with
10% Pd/C (0.65 g) under hydrogen (4 atm) at ambient tempera-
ture for 18 h. The catalyst was removed by filtration, and the
filtrate was concentrated under reduced pressure. The residue
was triturated with a minimum amount of EtOAc (ca. 2 mL) and
filtered to provide the title compound as a solid (0.76 g, 66%

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4135
temperature and filtered. The filtrate was concentrated under
vacuum, and the residue was purified by flash chromato-
graphy (hexanes-EtOAc, 50:50) to provide the title com-
t-Butyl cis-5-(4-Bromo-phenyl)-hexahydro-pyrrolo[3,4-c]pyr-
role-2-carboxylate (Boc-25). Prepared from 5 (0.75 g, 3.53
mmol) and 1,4-dibromobenzene (25a, 0.83 g, 3.53 mmol) ac-
cording to coupling procedure A as described for Boc-6, except
the heating period was extended to 24 h at 100 °C. The crude
product was purified by flash chromatography (hexanes-
EtOAc, 70:30) to provide the title compound (0.65 g, 1.8 mmol,
1
pound (200 mg, 88% yield). H NMR (CDCl3) δ 1.46 (s, 9 H),
2.99-3.09 (m, 2 H), 3.29 (dd, J = 9.8, 3.7 Hz, 2 H), 3.30-3.44
(m, 2 H), 3.55-3.74 (m, 4 H), 6.89 (dd, J = 8.6, 2.9 Hz, 1 H),
7.27-7.34 (m, 1 H), 7.38-7.46 (m, 2 H), 7.60 (d, J = 8.8 Hz, 1 H),
7.87-7.94 (m, 2 H), 8.07 ppm (d, J = 2.7 Hz, 1 H). MS m/z
366 (MH)+.
1
50% yield). H NMR (CDCl3) δ 1.45 (s, 9 H), 2.94-3.05 (m,
2 H), 3.17 (dd, J = 9.7, 3.9 Hz, 2 H), 3.20-3.42 (m, 2 H), 3.50
(dd, J = 8.8, 7.4 Hz, 2 H), 3.64 (dd, J = 11.2, 7.5 Hz, 2 H), 6.43
(d, J = 8.8 Hz, 2 H), 7.30 ppm (d, J = 8.8 Hz, 2 H). MS m/z 367/
369 (MH)+.
cis-2-(6-Phenyl-pyridin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole
Bis-trifluoroacetate (21). Trifluoroacetic acid (1 mL) was added
to a solution of Boc-21 (72 mg, 0.20 mmol) in CH2Cl2 (2 mL),
and the solution was stirred at room temperature for 1 h and
then concentrated under vacuum. Toluene (5 mL) was added
to the residue, and the mixture concentrated under vacuum.
This was repeated once more to remove excess acid. The residue
was dissolved in EtOAc (1.5 mL), and ethyl ether (3 mL) was
added gradually. The resulting suspension was stirred for 5 h
and then cooled in ice. The precipitate was isolated by filtration
and dried under vacuum to provide the title salt (72 mg, 74%
yield). 1H NMR (CD3OD) δ 3.32 (m, 4 H), 3.62 (m, 6 H), 7.57
(m, 3 H), 7.68 (dd, J = 9.2, 2.7 Hz, 1 H), 7.81 (m, 2 H), 8.01 ppm
t-Butyl cis-5-(6-Chloro-pyridazin-3-yl)-hexahydro-pyrrolo[3,
4-c]pyrrole-2-carboxylate (Boc-26). A mixture of 5 (5.0 g, 23.6
mmol), 3,6-dichloropyridazine (26a, 4.70 g, 30.6 mmol), Pd2-
(dba)3, (944 mg, 1.03 mmol), 1,3-bis(2,6-di(isopropyl)phenyl)-
imidazolium chloride (1.23 g, 2.90 mmol), and Cs2CO3 (23.2 g,
70.8 mmol) in p-dioxane (60 mL) was heated at 85 °C with stirring
under nitrogen for 18 h. The reaction was cooled to room
temperature, filtered, and concentrated under vacuum. The
resulting solid was triturated with hexanes-EtOAc (50:10, 100
mL) and filtered to provide the title compound as an off-white
solid (3.40 g). The filtrate was concentrated, and the residue was
purified by flash chromatography (hexanes-EtOAc, 50:50-
0:100) to provide additional product (total 4.44 g, 58% yield).
1H NMR (CDCl3) δ 1.46 (s, 9 H), 3.05 (s, 2 H), 3.21-3.57 (m,
4 H), 3.58-3.83 (m, 4 H), 6.61 (d, J = 9.5 Hz, 1 H), 7.19 ppm
(d, J = 9.5 Hz, 1 H). MS m/z 325/327 (MH)+.
(m, 2 H). MS m/z 266 (MH)+. Anal. (C17H19N3 2CF3CO2H)
3
C, H, N.
cis-2-Methyl-5-(6-phenyl-pyridin-3-yl)-octahydro-pyrrolo[3,4-
c]pyrrole L-Tartrate (22). Trifluoroacetic acid (2 mL) was added
to a solution of Boc-21 (200 mg, 0.55 mmol) in CH2Cl2 (4 mL),
and the solution was stirred at room temperature for 1 h and
then concentrated under vacuum. The residue was taken up in
20% K2CO3(aq) (4 mL) and 15% NaOH(aq) (4 mL) and extracted
with CH2Cl2 (6 ꢀ 8 mL). The combined extract was dried
(Na2SO4) and concentrated to leave the free base (122 mg). This
was dissolved in CH3CN (5 mL), and formalin (37%, 4 mL) was
added, followed by NaBH(OAc)3 (134 mg, 0.60 mmol). The
mixture was stirred at room temperature for 12 h and concen-
trated under vacuum. The residue was extracted with CH2Cl2
(4 ꢀ 5 mL), the combined organic phase was concentrated under
vacuum, and the residue was purified by chromatography
(CH2Cl2-CH3OH-NH4OH, 95:5:0.7) to provide the N-methy-
lated free base (46 mg). This was dissolved in warm EtOAc
(4 mL) and a solution of L-tartaric acid (49 mg, 0.33 mmol,
2 equiv) in CH3OH (1.5 mL) was added dropwise. The resulting
suspension was stirred at room temperature for 1 h, then filtered
and the solid dried under vacuum to provide the title compound
(80 mg, 25% yield). 1H NMR (CD3OD) δ 2.87 (s, 3 H), 3.30 (m,
6 H), 3.60 (m, 4 H), 4.40 (s, 2 H), 7.29 (dd, J = 8.8, 2.7 Hz, 1 H),
7.35 (m, 1 H), 7.44 (m, 2 H), 7.71 (d, J = 8.5 Hz, 1 H), 7.83 (m,
2 H), 8.12 ppm (d, J = 3.1 Hz, 1 H). MS m/z 280 (MH)+. Anal.
(C18H21N3 C4H6O6 0.1H2O) C, H, N.
t-Butyl cis-5-(5-Phenyl-pyridin-2-yl)-hexahydro-pyrrolo[3,4-c]-
pyrrole-2-carboxylate (Boc-27). A mixture of Boc-24 (0.25 g,
0.68 mmol), phenylboronic acid (0.26 g, 1.36 mmol), Pd2(dba)3
(40 mg, 0.044 mmol), 1,3-bis(2,6-di(isopropyl)phenyl)imidazo-
lium chloride (52 mg, 0.12 mmol), and Cs2CO3 (0.67 g, 1.9 mmol)
in 15 mL dioxane was heated at 85 °C for 16 h. The mixture
was cooled to room temperature and concentrated under vacuum.
The residue was purified by chromatography (hexanes-EtOAc,
1
50:50) to provide the title compound (0.20 g, 80% yield). H
NMR (CDCl3)δ1.46 (s, 9 H), 2.96-3.10(m, 2H), 3.21-3.55 (m, 4
H), 3.58-3.83 (m, 4 H), 6.44 (d, J =8.9Hz, 1 H), 7.30 (dt, J =7.0,
1.2 Hz, 1 H), 7.38-7.45 (m, 2 H), 7.47-7.55 (m, 2 H), 7.71 (d, J =
9.0 Hz, 1 H), 8.42 ppm (dd, J = 2.4, 0.7 Hz, 1 H). MS m/z 3
66 (MH)+.
cis-2-(5-Phenyl-pyridin-2-yl)-octahydro-pyrrolo[3,4-c]pyrrole-
Bis-trifluoroacetate (27). Deprotection and salt formation from
Boc-27 (0.20 g, 0.55 mmol) was accomplished with trifluoroa-
cetic acid according to the procedure described for 15 to provide
the title compound (233 mg, 84% yield). 1H NMR (CD3OD) δ
3.33 (m, 2 H), 3.40 (m, 2 H), 3.70 (m, 4 H), 3.92 (m, 2 H), 7.08
(d, J = 9.5 Hz, 1 H), 7.52 (m, 3 H), 7.65 (m, 2 H), 8.18 (br d, J =
2.4 Hz, 1 H), 8.25 ppm (dd, J = 9.5, 2.4 Hz, 1 H). MS m/z 266
3
3
cis-5-(6-Chloropyridin-3-yl)-2,2-dimethyloctahydropyrrolo[3,4-c]-
pyrrol-2-ium Iodide (23). Methyl iodide (0.06 mL, 130 mg, 0.93
mmol) was added at room temperature to a solution of 9 (110 mg,
0.46 mmol) in CH2Cl2 (5 mL). A precipitate formed within 2 min,
and the suspension was stirred for 1 h, and then filtered. The solid
was washed with ethyl ether (3 mL) and dried under vacuum to
provide the title compound (137 mg, 78% yield). 1H NMR
(CD3OD) δ 3.07-3.16 (m, 2 H), 3.17 (s, 3 H), 3.27 (s, 3 H),
3.29-3.36 (m, 2 H), 3.38-3.51 (m, 2 H), 3.54-3.72 (m, 2 H),
3.87-4.05 (m, 2 H), 7.16-7.37 (m, 2 H), 7.88 ppm (br s, 1 H). MS
m/z 252 (M)+. Anal. (C13H19N3ClI) C, H, N.
(MH)+. Anal. (C17H19N3 2CF3CO2H) C, H, N.
3
cis-2-Methyl-5-(5-phenyl-pyridin-2-yl)-octahydro-pyrrolo[3,4-
c]pyrrole 4-Toluenesulfonate (28). NaBH(OAc)3 (0.21 g, 0.57
mmol) was added to a solution of 27 (0.20 g, 0.40 mmol) in 37%
formalin (7 mL), and the resulting mixture was stirred at room
temperature for 8 h and then concentrated under vacuum. The
residue was taken up in CH2Cl2 (3 mL) and purified by flash
chromatography (CH2Cl2-CH3OH-NH4OH, 90:10:1) to pro-
vide the methylated free base (74 mg, 66% yield). This was
combined with p-toluenesulfonic acid (53 mg, 0.28 mmol) in
warm EtOAc (10 mL) and the resulting precipitate was filtered
and dried to provide the title compound (85 mg, 69% yield). 1H
NMR (CD3OD) δ 2.35 (s, 3 H), 2.96 (s, 3 H), 3.22 (m, 2 H), 3.43
(m, 3 H), 3.66 (m, 4 H), 3.98 (m, 1 H), 6.82 (d, J = 9.2 Hz, 1 H),
7.22 (m, 2 H), 7.34 (tt, J = 7.1, 2.0 Hz, 1 H), 7.44 (m, 2 H), 7.56
(m, 2 H), 7.68 (m, 2 H), 7.92 (dd, J = 8.8, 2.4 Hz, 1 H), 8.32 ppm
t-Butyl cis-5-(5-Bromo-pyridin-2-yl)-hexahydro-pyrrolo[3,4-c]-
pyrrole-2-carboxylate (Boc-24). Prepared from 5 (7.032 g, 33.2
mmol) and 2,5-dibromopyridine (24a, 22.03 g, 92.9 mmol) accord-
ing to coupling procedure A as described for Boc-6. The crude
product was purified by column chromatography (hexanes-
EtOAc, 95:5-40:60) to provide the title compound as an off-
white solid (7.71 g, 62% yield). 1H NMR (CDCl3) δ 1.45 (s, 9 H)
2.94-3.14 (m, 2 H) 3.17-3.38 (m, 4 H) 3.51-3.76 (m,
4 H) 6.26 (d, J = 8.9 Hz, 1 H) 7.50 (dd, J = 9.0, 2.5 Hz, 1 H)
8.16 ppm (d, J = 2.4 Hz, 1 H). MS m/z 368/370 (MH)+.
(d, J = 2.0 Hz, 1 H). MS m/z 280 (MH)+. Anal. (C18H21N3
3
C7H8O3S 0.6H2O) C, H, N.
t-Butyl cis-2-Biphenyl-4-yl-octahydro-pyrrolo[3,4-c]pyrrole-2-
carboxylate (Boc-29). Aqueous Na2CO3 (2 M, 3 mL) was added
3

4136 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Bunnelle et al.
to a mixture of Boc-25 (250 mg, 0.68 mmol), Pd2(dba)3 (25 mg,
0.027 mmol), 1,3-bis(2,6-di-i-propylphenyl)imidazolium chlor-
ide (29 mg, 0.068 mmol) and phenylboronic acid (262 mg,
1.37 mmol) in toluene (25 mL). The mixture was evacuated
and purged with nitrogen (3 cycles) and stirred under nitrogen at
85 °C for 60 h. The mixture was cooled to room temperature,
filtered, and concentrated under vacuum. The residue was
purified by flash chromatography (hexanes-EtOAc 70:30) to
provide the title compound (160 mg, 64% yield). 1H NMR
(CDCl3) δ 1.46 (s, 9 H), 2.93-3.08 (m, 2 H), 3.19-3.45 (m, 4 H),
3.51-3.72 (m, 4 H), 6.63 (d, J = 8.5 Hz, 2 H), 7.21-7.30
(m, 1 H), 7.39 (t, J = 7.6 Hz, 2 H), 7.50 (d, J = 8.8 Hz, 2 H),
7.54 ppm (d, J = 7.5 Hz, 2 H). MS m/z 365 (MH)+.
(d, J = 9.5 Hz, 1 H), 7.53 (m, 3 H), 7.96 (m, 2 H), 8.17 ppm
(d, J = 9.8 Hz, 1 H). MS m/z 267 (MH)+. Anal. (C16H18N4
2CF3CO2H 0.2H2O) C, H, N.
3
3
cis-2-(6-Phenyl-pyridazin-3-yl)-5-methyloctahydro-pyrrolo[3,4-c]-
pyrrole (32). Aqueous formalin (37%, 1.1 mL, 14.7 mmol) was
added to a solution of Boc-31 (4.91 g, 13.40 mmol) in 88% formic
acid (25 mL). The dark mixture was heated at 100 °C for 1 h and
then cooled to room temperature and concentrated under vacuum.
The dark-amber residue was combined with 20% Na2CO3(aq) (25
mL), and the mixture was extracted with CH2Cl2 (2 ꢀ 50 mL). The
combined organic extract was washed with brine (25 mL) and
concentrated under vacuum. The residue was recrystallized from
EtOAc (50 mL) to provide a slightly off-white, crystalline solid (2.00
1
cis-2-Biphenyl-4-yl-octahydro-pyrrolo[3,4-c]pyrrole Trifluor-
oacetate (29). Trifluoroacetic acid (4 mL) was added dropwise
over 5 min to a solution of Boc-29 (160 mg, 0.44 mmol) in
CH2Cl2 (5 mL). The solution was stirred at room temperature
for 2 h and then concentrated under vacuum. Toluene (3 mL)
was added to the residue and the mixture was concentrated
under vacuum to remove excess acid (repeated once more). The
residue was triturated with a mixture of EtOAc (2 mL) and ethyl
ether (2 mL). The resulting suspension was filtered and the solid
dried under vacuum to provide the title salt (144 mg, 83%
yield). 1H NMR (CD3OD) δ 3.29 (m, 6 H), 3.52 (d, J =
8.8 Hz, 2 H), 3.61 (m, 2 H), 6.83 (m, 2 H), 7.23 (m, 1 H), 7.37
(m, 2 H), 7.52 ppm (m, 4 H). MS m/z 265 (MH)+. Anal.
(C18H20N2 CF3CO2H 0.2H2O) C, H, N.
g, 53%). H NMR (CD3OD) δ 2.35 (s, 3 H), 2.53 (dd, J = 9.5,
4.0 Hz, 2 H), 2.84 (dd, J = 9.5, 7.1 Hz, 2 H), 3.02-3.17 (m, 2 H),
3.56 (dd, J = 10.9, 3.0 Hz, 2 H), 3.71 (dd, J = 11.1, 8.0 Hz, 2 H),
7.08 (d, J = 9.5 Hz, 1 H), 7.37-7.51 (m, 3 H), 7.84 (d, J = 9.5 Hz, 1
H), 7.89-7.95 ppm (m, 2 H). MS m/z 281 (MH)+. Anal.(C17-
H20N4) C, H, N.
cis-2-Ethyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]-
pyrrole Bis-p-toluenesulfonate (33). NaBH(OAc)3 (112 mg, 0.53
mmol) was combined with 31 (200 mg, 0.40 mmol) and acetalde-
hyde (5 mL), and the mixture was stirred at room temperature
for 16 h. The reaction mixture was concentrated under vacuum,
and the residue was taken up in CH2Cl2 (15 mL) and washed
successively with satd NaHCO3(aq) (2 ꢀ 5 mL) and brine (5 mL)
and then dried (Na2SO4) and concentrated under vacuum. The
residue was purified by chromatography (CH2Cl2-CH3OH-
NH4OH, 90:10:1) to provide the free base (31 mg). This was
dissolved in EtOAc (3 mL) and a solution of p-toluenesulfonic
acid monohydrate (42 mg, 2 equiv) In CH3OH (1 mL) was added
dropwise with stirring. The resulting precipitate was isolated by
filtration and dried under vacuum to provide the title salt (38 mg,
15% yield). 1H NMR (CD3OD) δ 1.37 (q, J = 7.7 Hz, 3 H), 2.30 (s,
6 H), 3.41 (m, 6 H), 3.94 (m, 6 H), 7.18 (d, J = 7.8 Hz, 4 H),
7.58 (m, 3 H), 7.65 (d, J = 8.1 Hz, 4 H), 7.74 (dd, J = 28.1, 10.2 Hz,
1 H), 7.96 (d, J = 3.7 Hz, 2 H), 8.32 ppm (dd, J = 27.0, 10.0 Hz,
1 H). MS m/z 295 (MH)+. Anal. (C18H22N4 2C7H8O3S 0.2H2O)
3
3
cis-2-Biphenyl-4-yl-5-methyl-octahydro-pyrrolo[3,4-c]pyrrole
Dihydrochloride (30). Solid NaBH(OAc)3 (59 mg, 0.28 mmol)
was added to a solution of 29 (105 mg, 0.21 mmol) in 37%
formalin (3 mL). The mixture was stirred at room temperature
for 2 h and then quenched by addition of satd NaHCO3 (2 mL).
The mixture was extracted with CH2Cl2 (4 ꢀ 3 mL), and the
combined extract was concentrated under vacuum. The residue
was purified by flash chromatography (silica, CH2Cl2-
CH3OH-NH4OH, 90:10:1) to provide the crude N-methylated
free base (88 mg), contaminated with paraformaldehyde. This
material was taken up in EtOAc (3 mL) and filtered to remove
some insoluble matter. HCl/dioxane (4 M, 0.7 mL) was added
to the filtrate, and the resulting suspension was stirred at room
temperature for 2 h. The precipitate was collected by filtration
and dried under vacuum to provide the title salt (40 mg, 54%
yield). 1H NMR (CD3OD) δ 2.92 (s, 3 H), 3.21 (m, 2 H), 3.40
(m, 4 H), 3.64 (m, 3 H), 3.98 (m, 1 H), 6.96 (m, 2 H), 7.25 (t, J =
7.8 Hz, 1 H), 7.38 (t, J = 7.6 Hz, 2 H), 7.54 ppm (m, 4 H). MS m/
3
3
C, H, N.
cis-5-(6-Phenylpyridazin-3-yl)-2,2-dimethyloctahydropyrrolo-
[3,4-c]pyrrol-2-ium Iodide (34). Iodomethane (0.2 mL) was
added to a solution of 32 (39 mg, 0.14 mmol) in CH2Cl2 (3
mL). The mixture was stirred at room temperature for 12 h, then
concentrated under vacuum. The residue was triturated with
ether (5 mL) and filtered to provide the title salt as an off-white
solid (52 mg, 88% yield). 1H NMR (D2O) δ 3.09 (s, 3 H), 3.17
(s, 3 H), 3.25-3.35 (m, 2 H), 3.45 (m, 4 H), 3.77 (d, J = 9.8 Hz, 2
H), 3.93 (br dd, J = 11.0, 4.2 Hz, 2 H), 7.17 (d, J = 9.5 Hz, 1 H),
7.43-7.53 (m, 3 H), 7.75-7.80 (m, 2 H), 7.82 ppm (d, J =
9.5 Hz, 1 H). MS m/z 295 (M)+. Anal. (C18H23N4I) C, H, N.
cis-2-(6-Chloro-pyridazin-3-yl)-5-methyl-octahydro-pyrrolo-[3,4-c]-
pyrrole. A solution of Boc-26 (1.0 g, 3.1 mmol) in 37% formalin (10
mL) and 88% formic acid (20 mL) was heated at 100 °Cfor2.5h.The
mixture was cooled to room temperature and concentrated under
vacuum. The residual oil was purified by flash chromatography
(CH2Cl2-CH3OH-NH4OH, 90:10:1) to provide the title compound
(0.43 g, 58% yield). 1H NMR (CD3OD) δ 2.41 (s, 3 H) 2.62 (dd, J =
10.0, 3.9 Hz, 2 H) 2.89 (dd, J = 10.3, 7.3 Hz, 2 H) 3.05-3.16 (m, 2 H)
3.50 (dd, J = 11.2, 3.1 Hz, 2 H) 3.65 (dd, J = 11.5, 8.1 Hz, 2 H)
7.04 (d, J = 9.5 Hz, 1 H) 7.41 ppm (d, J = 9.5 Hz, 1 H). MS m/z 239
(MH)+.
cis-2-Methyl-5-(6-(4-methylphenyl)pyridazin-3-yl)-octahydro-
pyrrolo[3,4-c]pyrrole Dihydrochloride (35). A mixture of cis-2-(6-
chloro-pyridazin-3-yl)-5-methyl-octahydro-pyrrolo[3,4-c]pyr-
role (0.23 g, 9.5 mmol), p-tolylboronic acid (0.17 g, 1.2 mmol),
Cs2CO3 (0.93 g, 2.8 mmol), 1,3-bis(2,6-di-i-propylphenyl)imi-
dazolium chloride (52 mg, 0.12 mmol), and Pd2(dba)3 (40 mg,
4.4 mmol) was charged to a glass pressure tube with 1,4-dioxane
(15 mL). The mixture was stirred at 85 °C for 72 h then cooled to
ambient temperature. The mixture was filtered, concentrated
z 279 (MH)+. Anal. (C19H22N2 2HCl) C, H, N.
3
t-Butyl cis-5-(6-Phenyl-pyridazin-3-yl)-hexahydro-pyrrolo-
[3,4-c]pyrrole-2-carboxylate (Boc-31). Solid 3-chloro-6-phenyl-
pyridazine (2.82 g, 14.79 mmol) and 5 (3.14 g, 14.79 mmol) were
charged to a 100 mL flask with stir bar and condenser. N,N-
Diisopropylethylamine (9 mL, 51.5 mmol) and DMSO (9 mL)
were added, and the slurry was heated at 105 °C under nitrogen
for 49 h. The dark-amber solution was cooled to 40 -50 °C,
diluted with water (50 mL), and stirred vigorously for 1 h. The
precipitate was isolated by filtration, washed well with water,
and sucked dry on the filter. The solid was triturated with ether
(2 ꢀ 50 mL) to remove unreacted 3-chloro-6-phenylpyridazine,
and filtered to provide the title compound as a beige solid (4.91
1
g, 91% yield). H NMR (CD3OD) δ 1.46 (s, 9 H), 3.03-3.17
(m, 2 H), 3.29-3.37 (m, 2 H), 3.50 (dd, J = 10.7, 4.0 Hz, 2 H),
3.61-3.74 (m, 2 H), 3.82 (dd, J = 11.1, 7.1 Hz, 2 H), 7.04 (d, J =
9.5 Hz, 1 H), 7.35-7.43 (m, 1 H), 7.47 (t, J = 7.3 Hz, 2 H), 7.85
(d, J = 9.5 Hz, 1 H), 7.92 ppm (d, J = 7.1 Hz, 2 H). MS m/z 367
(MH)+.
cis-2-(6-Phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole
Bis-trifluoroacetate (31). Deprotection of Boc-31 (0.16 g, 0.44
mmol) with trifluoroacetic acid according to the procedure
described for 19 provided the title salt as an off-white solid
1
(0.30 mmol, 69% yield). H NMR (CD3OD) δ 3.36 (m, 4 H),
3.65 (m, 2 H), 3.75 (dd, J = 11.5, 3.1 Hz, 2 H), 3.89 (m, 2 H), 7.46

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4137
under reduced pressure, and the residue was purified by flash
chromatography (CH2Cl2-CH3OH-NH4OH, 90:10:1) to pro-
vide the free base of the title product (0.13 g, 37% yield). This
was dissolved in 10% CH3OH-diethyl ether (5 mL) and 4 N
HCl/dioxane (1 mL) was added. The suspension was stirred for
1 h, and the precipitate was collected by filtration, washed with
ether, and dried to provide the title salt (0.11 g, 64% yield). 1H
NMR (CD3OD) δ 2.43 (s, 3 H), 2.97 (s, 3 H), 3.17 (m, 2 H), 3.54
(m, 2 H), 3.93 (m, 6 H), 7.40 (d, J = 7.8 Hz, 2 H), 7.76 (m, 1 H),
7.88 (m, 2 H), 8.38 ppm (d, J = 9.5 Hz, 1 H). MS m/z 295
(MH)+. Anal. (C18H22N4 2HCl 1.5H2O) C, H, N.
for 36. The free base (34 mg) was dissolved in EtOAc (2 mL) and a
solution of p-toluenesulfonic acid (19 mg, 0.096 mmol) in 10%
CH3OH-EtOAc (1 mL) was added with stirring. After 30 min,
the precipitate was isolated by filtration and dried to provide the
title salt(35 mg, 68% yieldfor the salt). 1H NMR (CD3OD) δ 2.34
(s, 3 H), 2.96 (s, 3 H), 3.35 (m, 6 H), 3.62 (m, 4 H), 7.21 (m, 2 H),
7.36 (m, 2 H), 7.46 (m, 2 H), 7.53 (m, 1 H), 7.63 (ddd, J = 7.8, 1.7,
1.0 Hz, 1 H), 7.69 (m, 4 H), 7.81 (ddd, J = 7.8, 1.7, 1.0 Hz, 1 H)
7.82 (m, 1 H), 8.10 (dd, J = 2.0, 1.4 Hz, 1 H), 8.15 ppm (br d, J =
2.7 Hz, 1 H). MS m/z 356 (MH)+. Anal. (C24H25N3 C7H8O3S
3
3
0.5H2O) C, H, N.
3
3
cis-2-Methyl-5-(6-(3-methylphenyl)pyridazin-3-yl)-octahydro-
pyrrolo[3,4-c]pyrrole Dihydrochloride (36). Step 1. Procedure
for Anhydrous Miyaura-Suzuki Coupling. To a solution of
Boc-26 (1.45 g, 4.47 mmol) in 50 mL p-dioxane was added m-
tolylboronic acid (0.79 g, 5.82 mmol), Pd2(dba)3 (0.24 g, 0.26
mmol), 1,3-bis(2,6-di-i-propylphenyl)imidazolium chloride
(0.312 g, 0.73 mmol), and Cs2CO3 (4.4 g, 13.4 mmol). This
mixture was warmed to 85 °C and stirred for 20 h. The reaction
was then cooled to room temperature and concentrated under
vacuum. Diethyl ether (25 mL) and hexanes (25 mL) were added
to the crude material, and the resulting solid was filtered to
provide the title compound (1.28 g, 75% yield). 1H NMR
(CDCl3) δ1.46 (s, 9 H), 2.43 (s, 3 H), 2.99-3.12 (m, 2 H),
3.21-3.45 (m, 2 H), 3.46-3.65 (m, 2 H), 3.63-3.74 (m, 2 H),
3.75-3.91 (m, 2 H), 6.70 (d, J = 9.5 Hz, 1 H), 7.21 (d, J =
7.5 Hz, 1 H), 7.35 (t, J = 7.6 Hz, 1 H), 7.64 (d, J = 9.5 Hz, 1 H),
7.75 (d, J = 7.8 Hz, 1 H), 7.86 ppm (s, 1 H). MS m/z 381 (MH)+.
Step 2. Deprotection and N-Methylation. Trifluoroacetic acid
(10 mL) was added to a solution of the product from Step 1 (1.18
g, 3.1 mmol) in CH2Cl2 (20 mL), and the solution was stirred at
room temperature for 90 min and then concentrated under
vacuum. Toluene (15 mL) was added to the residue, and the
mixture concentrated under vacuum;this was repeated once
more to remove excess acid. The residue was taken up in EtOAc
(15 mL), and the amine salt was precipitated by dropwise
addition of diethyl ether (8 mL). The solid was filtered and dried
(1.5 g). This material was dissolved in 37% formalin (25 mL) and
NaBH(OAc)3 (0.82 g, 3.84 mmol) was added. The mixture was
stirred for 18 h at room temperature and then concentrated
under vacuum. The residue was diluted with satd NaHCO3
(20 mL) and extracted with CH2Cl2 (5 ꢀ 10 mL). The combined
extract was washed with brine (10 mL) and concentrated under
vacuum. The residue was dissolved in 10% ethanol-EtOAc
(15 mL) and a solution of 4 N HCl/dioxane (4 mL) was added.
The solution was stirred at room temperature as ethyl ether (8
mL) was added gradually to produce a precipitate. The mixture
was filtered and the collected solid was washed with ether and
dried to provide the title compound (1.0 g, 81% yield). 1H NMR
(CD3OD) δ 2.45 (s, 3 H), 2.96 and 3.02 (endo/exo NHMe, s,
3 H), 3.20 (m, 1 H), 3.48 (m, 2 H), 3.61 (m, 1 H), 3.79 (m, 1 H),
3.98 (m, 5 H), 7.44 (m, 2 H), 7.81 (m, 3 H), 8.41 and 8.44 ppm
(endo/exo salt, d, J = 9.8 Hz, 1 H). MS m/z 295 (MH)+. Anal.
(C18H22N4 2HCl 2H2O) C, H, N.
cis-2-[6-(3,5-(Dimethylphenyl)pyridazin-3-yl]-5-methyl-octa-
hydro-pyrrolo[3,4-c]pyrrole Dihydrochloride (39). Prepared in
51% yield (73% coupling, 70% methylation and salt forma-
tion) from Boc-26 and 3,5-dimethylphenylboronic acid accord-
ing to the procedures described for 36. 1H NMR (CD3OD)
δ 2.41 (s, 6 H), 2.97 (s, 3 H), 3.17 (m, 1 H), 3.52 (m, 3 H), 3.91
(m, 6 H), 7.25 (s, 1 H), 7.58 (s, 2 H), 7.78 (t, J = 9.2 Hz, 1 H),
8.39 ppm (m, 1 H). MS m/z 309 (MH)+. Anal. (C19H24N4
2
3
HCl 1.7H2O) C, H, N.
3
cis-2-Methyl-5-(6-(2-methylphenyl)pyridazin-3-yl)-octahydro-
pyrrolo[3,4-c]pyrrole Bis-L-tartrate (40). Step 1: Biphasic
Miyaura-Suzuki Coupling. Toluene (10 mL) was added to a
mixture of Boc-26 (150 mg, 0.46 mmol), o-tolylboronic acid (126
mg, 0.93 mmol), Pd2(dba)3 (20 mg, 0.021 mmol), 1,3-bis(2,6-di
(isopropyl)phenyl)imidazolium chloride (26 mg, 0.062 mmol).
Aqueous Na2CO3 (2M, 1 mL) was added and the mixture was
stirred at 85 °C for 16 h and then cooled to room temperature.
The mixture was diluted with EtOAc (40 mL) and washed
successively with water (15 mL) and brine (15 mL). The organic
phase was dried (Na2SO4) and concentrated under vacuum. The
residue was purified by flash chromatography (hexanes-
EtOAc, 20:80) to provide the title compound as a white solid
(167 mg, 95% yield). 1H NMR (CDCl3) δ 1.46 (s, 9 H), 2.42 (s,
3 H), 3.02-3.11 (m, 2 H), 3.28-3.46 (m, 2 H), 3.46-3.63 (m,
2 H), 3.63-3.75 (m, 2 H), 3.78-3.91 (m, 2 H), 6.69 (d, J = 9.5
Hz, 1 H), 7.26-7.31 (m, 3 H), 7.34 (d, J = 9.5 Hz, 1 H), 7.38-
7.43 ppm (m, 1 H). MS m/z 381 (MH)+.
This material was deprotected and N-methylated (50% yield)
according to the methods described for 36, and the free base was
converted to the L-tartrate salt as described for 37 to provide the
title salt. 1H NMR (CD3OD) δ 2.31 (s, 3 H), 2.94 (s, 3 H), 3.26
(m, 2 H), 3.40 (m, 2 H), 3.67 (m, 4 H), 3.80 (m, 2 H), 4.45 (s, 4 H),
7.17 (d, J = 9.5 Hz, 1 H), 7.32 (m, 4 H), 7.56 ppm (d, J = 9.5 Hz,
1 H). MS m/z 295 (MH)+. Anal. (C18H22N4 2C4H6O6) C, H, N.
3
cis-2-[6-(4-Methoxy-phenyl)-pyridazin-3-yl]-5-methyl-octahy-
dro-pyrrolo[3,4-c]pyrrole p-Toluenesulfonate (41). Step 1. t-Bu-
tyl cis-5-[6-(4-Methoxyphenyl)pyridazin-3-yl]-octahydro-pyrrolo-
[3,4-c]pyrrole-2-carboxylate (41a). Aqueous Na2CO3 (2 M, 2
mL) was added to a mixture of Boc-26 (200 mg, 0.62 mmol), 4-
(methoxy)phenylboronic acid (187 mg, 1.23 mmol), Pd2(dba)3 (23
mg, 0.025 mmol), and 1,3-bis(2,6-di(isopropyl)phenyl)imidazo-
lium chloride (26 mg, 0.062 mmol) in toluene (20 mL). The mixture
was evacuated and purged with nitrogen (3 cycles)and then heated
under nitrogen at 85 °C for 60 h. The reaction was cooled to room
temperature, filtered through a pad of diatomaceous earth, and
concentrated under vacuum. The residue was purified by flash
chromatography (silica, eluted with hexanes-EtOAc 20:80) to
provide the title compound as a beige solid (180 mg, 74% yield).
1H NMR (CD3OD) δ 1.46 (s, 9 H), 3.05-3.17 (m, 2 H), 3.31-3.36
(m, 2 H), 3.48 (dd, J = 10.9, 3.7 Hz, 2 H), 3.67 (dd, J = 9.3, 7.3Hz,
2 H), 3.81 (dd, J = 10.9, 7.5 Hz, 2 H), 3.84 (s, 3 H), 7.01 (d, J = 9.5
Hz, 1 H), 7.03 (d, J = 8.6 Hz, 2 H), 7.79 (d, J = 9.5 Hz, 1 H), 7.86
ppm (d, J = 8.8 Hz, 2 H). MS m/z 397 (MH)+.
3
3
cis-2-Methyl-5-(6-m-tolyl-pyridin-3-yl)-octahydro-pyrrolo[3,4-c]-
pyrrole Bis-L-tartrate (37). Prepared in 39% overall yield from
Boc-8 (200 mg, 0.62 mmol) and m-tolylboronic acid (96 mg, 0.68
mmol) according to the anhydrous Miyaura-Suzuki coupling and
N-methylation procedures described for 36. The free base (45 mg)
was combined with L-tartaric acid (46 mg) in methanol (1 mL) and
EtOAc (2 mL) and the precipitate was isolated to provide the title
salt. 1H NMR (CD3OD) δ 1.24 (m, 2 H), 2.41 (s, 3 H), 2.93 (s, 3 H),
3.31 (m, 4 H), 3.62 (d, J = 8.8 Hz, 2 H), 3.69 (m, 2 H), 4.45 (s, 4 H),
7.18 (d, J = 7.5 Hz, 1 H), 7.31 (m, 2 H), 7.61 (d, J = 7.8 Hz, 1 H),
7.66 (s, 1 H), 7.70 (d, J = 8.8 Hz, 1 H), 8.11 (d, J = 2.7 Hz, 1 H).
Step 2. cis-2-[6-(4-Methoxyphenyl)pyridazin-3-yl]-octahydro-
pyrrolo[3,4-c]pyrrole Bis-trifluoroacetate (41b). Trifluoroacetic
acid (2 mL) was added to a stirring solution of 41a (180 mg,
0.45 mmol) in CH2Cl2 (5 mL). After 1 h, the solution was
concentrated under vacuum, and the residue was stirred with
toluene (3 mL) and concentrated under vacuum to remove
MS m/z 294 (MH)+. Anal. (C19H23N3 2.3C4H6O6) C, H, N.
3
cis-2-(6-Biphenyl-3-yl-pyridin-3-yl)-5-methyl-octahydro-pyrro-
lo-[3,4-c]pyrrole p-Toluenesulfonate (38). Prepared in 34% yield
from Boc-10 and 3-biphenylboronic acid according to the
coupling, deprotection and N-methylation procedures described

4138 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Bunnelle et al.
excess acid. The residue was taken up in EtOAc (2 mL) to give a
solution from which the salt soon crystallized. The solid was
collected by filtration and dried to provide the title compound
(215 mg, 90% yield). 1H NMR (CD3OD) δ 3.32-3.46 (m, 4 H),
3.66 (dd, J = 11.5, 7.1 Hz, 2 H), 3.74 (dd, J = 11.7, 3.2 Hz, 2 H),
3.93 (dd, J = 11.9, 7.4 Hz, 2 H), 6.89-6.99 (m, 2 H), 7.65 (d, J =
9.8 Hz, 1 H), 7.80-7.85 (m, 2 H), 8.29 ppm (d, J = 9.8 Hz, 1 H).
MS m/z 283 (MH)+.
Step 3. cis-2-[6-(4-Methoxyphenyl)pyridazin-3-yl]-5-methyl-
octahydro-pyrrolo[3,4-c]pyrrole p-Toluenesulfonate (41). A solu-
tion of 41b (215 mg, 0.41 mmol) in 37% formalin (5 mL) was
stirred at room temperature as NaBH(OAc)3 (113 mg, 0.53
mmol) was added. After 14 h, the reaction was quenched by
addition of satd NaHCO3(aq) (2 mL). The mixture was extracted
with CH2Cl2 (4 ꢀ 4 mL) and the combined extract was dried
(MgSO4) and concentrated under vacuum. The residue was
purified by flash chromatography (CH2Cl2-CH3OH-
NH4OH, 90:10:1) to provide the free base of the title compound
(100 mg). This was dissolved in EtOAc (3 mL) and EtOH
cis-3-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyri-
dazin-3-yl]-phenol dihydrochloride (44). Prepared from Boc-26
and 3-(methoxy)phenylboronic acid using the biphasic
Miuaura-Suzuki coupling method described for 40 (83% yield)
and further processed according to the methods described for 42
to provide the free base of the title compound. This (16 mg) was
dissolved in 10% CH3OH in EtOAc (1 mL), and excess 4 N HCl/
dioxane (0.2 mL) was added. The resulting precipitate was
isolated via filtration to give 20 mg of the title compound
(20 mg, overall 42% yield). 1H NMR (CD3OD) δ 2.96 (s,
3 H), 3.31 (m, 4 H), 3.61 (m, 6 H), 6.86 (ddd, J = 7.9, 2.5,
1.2 Hz, 1 H), 7.16 (d, J = 9.5 Hz, 1 H), 7.29 (t, J = 7.8 Hz, 1 H),
7.37 (m, 2 H), 7.86 ppm (d, J = 9.5 Hz, 1 H). MS m/z 297
(MH)+. Anal. (C17H20N4O 2.5HCl 0.75H2O) C, H, N.
3
3
cis-2-Methyl-5-[6-(3-nitrophenyl)-pyridin-3-yl]-octahydro-py-
rrolo[3,4-c]pyrrole p-toluenesulfonate (45). Prepared from Boc-8
and 3-nitrophenylboronic acid by the anhydrous Miyaura-
Suzuki coupling procedure as described for 36 (20% yield),
followed by deprotection and N-methylation as described for b
(82% yield). The free base (28 mg, 0.86 mmol) was dissolved in
EtOAc (1 mL), and MeOH (3 drops) was added. A solution of
p-toluenesulfonic acid monohydrate (17 mg, 0.86 mmol) in
warm EtOAc (1 mL) was added, and the resulting solution
was stirred at room temperature as a precipitate formed slowly.
After 16 h, the solid was isolated by filtration and dried to
provide the title salt (33 mg, 73% yield). 1H NMR (CD3OD) δ
2.35 (s, 3 H), 2.94 (s, 3 H), 3.26 (m, 3 H), 3.43 (m, 3 H), 3.68 (m, 3
H), 3.99 (m, 1 H), 7.22 (m, 2 H), 7.33 (m, 1 H), 7.69 (m, 3 H), 7.88
(d, J = 8.8 Hz, 1 H), 8.21 (m, 2 H), 8.28 (m, 1 H), 8.77 ppm (dd,
(0.3 mL) and pTsOH H2O (64 mg, 1 equiv) was added with
3
warming to dissolve. The solution was cooled to room tempera-
ture and ether (1 mL) was added gradually. The resulting
suspension was stirred at room temperature for 14 h and then
filtered and the solid dried to provide the title compound
1
(141 mg, 64% yield). H NMR (CD3OD) δ 2.35 (s, 3 H), 2.95
(s, 3 H), 3.37 (m, 4 H), 3.65 (m, 4 H), 3.78 (m, 2 H), 3.85 (s, 3 H),
7.04 (m, 2 H), 7.15 (d, J = 9.5 Hz, 1 H), 7.21 (d, J = 7.8 Hz, 2 H),
7.69 (m, 2 H), 7.86 ppm (m, 3 H). MS m/z 311 (MH)+. Anal.
(C18H22N4O C7H8O3S) C, H, N.
3
cis-4-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyri-
dazin-3-yl]-phenol Bis-p-toluenesulfonate (42). Step 1. cis-4-
[6-(Hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-phe-
nol Hydrobromide (42a). A solution of BBr3 (1 M in heptane,
7.3 mL, 7.3 mmol) was added dropwise under nitrogen to a cold
(-70 °C) solution of 41a (725 mg, 1.83 mmol) in CH2Cl2
(70 mL). The reaction mixture was kept at -70 °C for 30 min
after the addition and then allowed to warm gradually to room
temperature and stirred for 14 h. The mixture was cooled to
-20 °C and quenched by dropwise addition of CH3OH (20 mL)
and allowed to warm to room temperature for 1 h. The suspension
was filtered and the solid dried to provide the crude title salt
(728 mg,>100% yield) used directly in the next reaction. 1H NMR
(CD3OD) δ 3.35-3.50 (m, 4 H), 3.61-3.73 (m, 2 H), 3.81 (dd, J=
11.4, 2.5 Hz, 2 H), 3.98 (dd, J=11.5, 7.1 Hz, 2 H), 6.97 (d, J=8.8
Hz, 2 H), 7.76 (d, J=9.8 Hz, 1 H), 7.85 (d, J=8.8 Hz, 2 H), 8.37
ppm (d, J=9.8 Hz, 1 H). MS m/z 283 (MH)+.
J = 1.9, 1.9 Hz, 1 H). MS m/z 325 (MH)+. Anal. (C18H20N4O2
3
C7H8O3S H2O) C, H, N.
3
cis-5-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyri-
dazin-3-yl]-1H-indole Bis-p-toluenesulfonate (46). Prepared from
Boc-26 and indol-5-ylboronic acid according to the procedures
described for 36. 1H NMR (C5D5N) δ 2.17 (s, 6 H) 3.01 (s, 3 H)
3.26-3.39 (m, 4 H) 3.52 (dd, J = 10.7, 6.8 Hz, 2 H) 3.78-3.92
(m, J = 9.8 Hz, 4 H) 6.80-6.85 (m, 1 H) 6.94 (d, J = 9.2 Hz, 1 H)
7.18 (d, J = 8.0 Hz, 4 H), 7.60 (t, J = 2.8 Hz, 1 H) 7.79 (d, J =
9.0 Hz, 1 H) 7.91 (d, J = 9.0 Hz, 1 H) 8.31 (d, J = 9.2, 1.5 Hz,
1 H) 8.33 (d, J = 8.0 Hz, 4 H) 8.63 (m, 1 H) 12.30 ppm (s, 1 H).
MS m/z 320 (MH)+. Anal. C19H21N5 2C7H8O3S H2O: C, H, N.
3
3
t-Butyl cis-5-(Pyridin-2-yl)-octahydro-pyrrolo[3,4-c]pyrrole-
2-carboxylate (Boc-47). 2-Chloropyridine (120 mg, 1.06 mmol)
and Na2CO3 (95.8 mg, 0.90 mmol) were added to a solution of 5
(109.7 mg, 0.52 mmol) in DMSO (1.5 mL). The mixture was
heated at 130 °C for 18 h and then cooled to room temperature
and diluted with methanol (1 mL). The solution was purified by
Step 2. cis-4-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-
yl)-pyridazin-3-yl]-phenol Bis-p-toluenesulfonate (42). The crude
salt 42a (91 mg, 0.23 mmol) was carried through reductive
methylation according to the procedure described for 36 to
provide the N-methylated free base (62 mg, 91% yield). This
was dissolved in 10% ethanol in EtOAc (3 mL) and p-toluene-
sulfonic acid (83 mg, 0.44 mmol) in 1 mL 10% ethanol in EtOAc
was added. The resulting solids were recrystallized from ethanol
˚
preparative HPLC (Phenomenex Luna C8(2) 5 μm 100 A AXIA
column (30 mm ꢀ 75 mm), eluted with acetonitrile/0.1% aqu-
eous trifluoroacetic acid, 10:90-90:10) to provide the title
compound as a white solid (69.8 mg, 47% yield). 1H NMR
(CDCl3) δ 1.45 (s, 9 H), 2.92-3.06 (m, 2 H), 3.18-3.51 (m, 4 H),
3.59-3.75 (m, 4 H), 6.35 (d, J = 8.7 Hz, 1 H), 6.56 (dd, J = 6.5,
5.4 Hz, 1 H), 7.45 (ddd, J = 8.5, 6.9, 2.0 Hz, 1 H), 8.16 ppm (ddd,
J = 5.2, 2.0, 0.8 Hz, 1 H). MS m/z 290 (MH)+.
1
to provide the title compound (34 mg, 23% yield). H NMR
(CD3OD) δ 2.31 (s, 6 H), 3.00 (s, 3 H), 3.20 (m, 1 H), 3.47 (m,
3 H), 3.89 (m, 6 H), 6.97 (d, J = 8.8 Hz, 2 H), 7.19 (d, J = 8.1 Hz,
4 H), 7.65 (d, J = 8.1 Hz, 4 H), 7.67 (m, 1 H), 7.81 (d, J = 8.5 Hz,
2 H), 8.24 ppm (m, 1 H). MS m/z 297 (MH)+. Anal.
cis-2-(Pyridin-2-yl)-octahydro-pyrrolo[3,4-c]pyrrole Bis-tri-
fluoroacetate (47). Trifluoroacetic acid (1 mL) was added to
a solution of Boc-47 (64.8 mg, 0.22 mmol) in CH2Cl2 (5 mL),
and the solution was stirred at 20 °C for 1 h. The solution was
concentrated under vacuum, and the residue was triturated
with 10% CH3OH-ethyl ether to produce an even suspension.
The solid was filtered, washed with ethyl ether, and dried under
vacuum to provide the title compound as a light-yellow solid
(80 mg, 86% yield). 1H NMR (CD3OD) δ 3.32-3.47 (m, 4 H),
3.59-3.72 (m, 4 H), 3.80-3.92 (m, 2 H), 6.98 (ddd, J = 7.1, 6.1,
0.8 Hz, 1 H), 7.07 (dt, J = 8.8, 1.0 Hz, 1 H), 7.95-8.03 ppm
(C17H20N4O 2C7H8O3S) C, H, N.
3
cis-2-[6-(3-Methoxy-phenyl)-pyridin-3-yl]-5-methyl-octahydro-
pyrrolo[3,4-c]pyrrole Bis-L-tartrate (43). Prepared from Boc-8 and
3-(methoxy)phenylboronic acid according to the anhydrous cou-
pling procedure described for 36 (78% yield), followed by
deprotection, reductive methylation, and salt formation as de-
scribed for 37 to provide the title salt. 1H NMR (CD3OD)
δ 2.93 (s, 3 H), 3.30 (m, 6 H), 3.61 (m, 2 H), 3.70 (m, 2 H), 3.85
(m, 3 H), 4.44 (m, 4 H), 6.92 (m, 1 H), 7.32 (m, 4 H), 7.71 (d, J =
8.8 Hz, 1 H), 8.12 ppm (d, J = 3.1 Hz, 1 H). MS m/z 310 (MH)+.
Anal. (C19H23N3O 2C4H6O6 2H2O) C, H, N.
(m, 2 H). MS m/z 190 (MH)+. Anal. (C11H15N3 2CF3CO2H)
3
C, H, N.
t-Butyl cis-5-(Pyridin-4-yl)-octahydro-pyrrolo[3,4-c]pyrrole-
2-carboxylate (Boc-48). A 100 mL flask with stir bar and
3
3

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4139
condenser was charged with 5 (234 mg, 1.10 mmol) and
4-bromopyridine hydrochloride (225 mg, 1.157 mmol). Toluene
(15 mL) was added, followed by triethylamine (6 mL,
43.0 mmol). A solution of Pd2(dba)3 (25 mg) and rac-BINAP
(21 mg) in toluene (2 mL) was added, followed by NaOtBu
(250 mg). The mixture was warmed at reflux under nitrogen for
8 h and then cooled to room temperature and concentrated
under vacuum. The residue was purified by flash chromatogra-
phy (silica, CH2Cl2-MeOH-NH4OH (95:5:0-90:10:1) to pro-
vide the adduct as an orange-brown solid (202 mg, 63% yield).
1H NMR (CD3OD) δ 1.35-1.57 (m, 9 H), 2.97-3.13 (m, J =
1.7 Hz, 2 H), 3.20-3.29 (m, 4 H), 3.50-3.74 (m, 4 H), 6.53
(d, J = 6.4 Hz, 2 H), 8.05 ppm (d, J = 6.4 Hz, 2 H). MS m/z
290 (MH)+.
was determined using a Packard TopCount Microplate β coun-
ter. IC50 values were determined by nonlinear regression in
Microsoft Excel or Assay Explorer. Ki values were calculated
from the IC50s using the Cheng-Prusoff equation, where Ki =
IC50/1 + [Ligand]/KD].
Functional Assays. Ca2+ Signaling in FLIPR. An HEK-293
cell line expressing the recombinant human R4β2 subunit com-
bination was used to measure functional agonist activity by
measuring intracellular calcium changes using the fluorometric
imaging plate reader (GFLIPR; Molecular Devices, Sunnyvale,
CA). Cells were plated at densities of 25000-50000 cells/well in
Dulbecco’s modified Eagle’s medium (Gibco) supplemented
with 10% fetal bovine serum (Gibco) in 96-well clear bottom
black-walled plates precoated with poly-D-lysine and allowed to
incubate for 24-48 h at 37 °C in 5% CO2 in a humidified
environment. After the media was aspirated off, the cells were
incubated in the dark at room temperature for ∼0.75-1 h with
2-4 μM Fluo-4 AM calcium indicator dye (Molecular Probes,
Eugene, OR) dissolved in 0.1-0.2% v/v of DMSO in Ringer
buffer (in mM: 140 NMDG, 5 KCl, 1 MgCl2, 10 HEPES, and 10
CaCl2, pH 7.4). Cells were placed in the FLIPR, and 50 μL of 3ꢀ
stock concentrations of test compounds or buffer prepared in
the same Ringer buffer was added. Raw fluorescence data were
corrected by subtracting fluorescence values from wells that
received buffer-only additions. Peak fluorescent values were
exported to Microsoft Excel, corrected for background signal,
and expressed as a percentage of the peak response for the
positive control of 100 μM nicotine. Dose-response data for
half-log concentration increments were fitted using a single
sigmoidal function in GraphPad Prism for determination of
EC50 and maximum response. Data are expressed as means (
SEM for at least n = 6 (two replicates across three different
plates).
cis-2-(Pyridin-4-yl)-octahydro-pyrrolo[3,4-c]pyrrole Bis-tri-
fluoroacetate (48). Trifluoroacetic acid (1 mL) was added over
5 min to an ice-cooled solution of Boc-48 (200 mg, 0.691 mmol)
in CH2Cl2 (5 mL). The cold solution was allowed to warm
gradually to room temperature and stirred overnight. The
orange solution was concentrated under vacuum, and the
residue was suspended in ether (15 mL) and concentrated
(repeat twice) to remove residual trifluoroacetic acid. Finally,
the residue was stirred with 10% MeOH-ether (15 mL) for 2 h.
The tan solid was collected by filtration, washed with 10%
MeOH-ether (2 ꢀ 4 mL), and dried under vacuum to provide
1
the title compound (243 mg, 84% yield). H NMR (CD3OD)
δ 3.31-3.46 (m, 4 H), 3.57-3.72 (m, 4 H), 3.82-3.95 (m, J =
7.5 Hz, 2 H), 6.91 (d, J = 7.8 Hz, 2 H), 8.17 ppm (d, J = 7.8 Hz,
2 H). MS m/z 190 (MH)+. Anal. (C11H15N3 2C2HO2F3)
3
C, H, N.
Experimental [3H]-Cytisine Binding. Binding conditions were
modified from the procedures described.74 Membrane enriched
fractions from rat brain minus cerebellum (ABS Inc., Wilming-
ton, DE) were slowly thawed at 4 °C, washed, and resuspended
in 30 volumes of BSS-Tris buffer (120 mM NaCl/5 mM KCl/2
mM CaCl2/2 mM MgCl2/50 mM Tris-Cl, pH 7.4, 4 °C). Samples
containing 100-200 μg of protein and 0.75 nM [3H]-cytisine
(30 Ci/mmol; Perkin-Elmer/NEN Life Science Products,
Boston, MA) were incubated in a final volume of 500 μL for
75 min at 4 °C. Seven log-dilution concentrations of each
compound were tested in duplicate. Nonspecific binding was
determined in the presence of 10 μM (-)-nicotine. Bound
radioactivity was isolated by vacuum filtration onto prewetted
glass fiber filter plates (Millipore, Bedford, MA) using a 96-well
filtration apparatus (Packard Instruments, Meriden, CT) and
were then rapidly rinsed with 2 mL of ice-cold BSS buffer (120
mM NaCl/5 mM KCl/2 mM CaCl2/2 mM MgCl2). Packard
MicroScint-20 scintillation cocktail (40 μL) was added to each
well and radioactivity determined using a Packard TopCount
instrument. The IC50 values were determined by nonlinear
regression in Microsoft Excel software. Ki values were calcu-
lated from the IC50s using the Cheng-Prusoff equation, where
Ki = IC50/1 + [Ligand]/KD].
Electrophysiological Measurements. Functional evaluation at
human R7 nAChR was performed using recombinant receptors
expressed in Xenopus laevis oocytes. Responses were measured
at room temperature using the two-electrode voltage clamp
(-60 mV) in the presence of 0.5 μM atropine to block endogen-
ous muscarinic receptors. Assays were sonducted in the POETs
apparatus58,59 using a robotic pipettor. Responses were mea-
sured as the peak (maximal) inward current relative to the
baseline holding current and were normalized to the maximal
response to acetylcholine (10 mM) determined in the each
oocyte before and after application of each test compound.
Dose-response data for half-log concentration increments were
fitted using a single sigmoidal function in GraphPad Prism for
determination of EC50 and maximum response. Data are ex-
pressed as means ( SEM.
Acknowledgment. We thank Eric Stoner and Alan Christesen
of Abbott Global Pharmaceutical Process Research for help in
preparing the intermediate 5.
[3H]-A-585539 Binding. [3H]-(S,S)-2,2-dimethyl-5-(6-phe-
nyl-pyridazin-3-yl)-5-aza-2-azonia-bicyclo[2.2.1]heptane iodide
([3H]-A-585539), binding to the R7 nAChR subtype was deter-
mined using membrane enriched fractions from rat brain minus
cerebellum or human cortex (ABS Inc., Wilmington, DE) as
previously described.56 Pellets were thawed at 4 °C, washed, and
resuspended with a Polytron at a setting of 7 in 30 volumes of
BSS-Tris buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM
MgCl2, and 50 mM Tris-Cl, pH 7.4, 4 °C). Seven log-dilution
concentrations of test compounds containing 100-200 μg of
protein and 0.5 nM [3H]A-585539 (62.8 Ci/mmol; R46 V,
Abbott Laboratories) were incubated in a final volume of
500 μL for 75 min at 4 °C in duplicate. Nonspecific binding
was determined in the presence of 10 μM methyllycaconitine.
Bound radioactivity was collected on Millipore MultiScreen
harvest plates FB presoaked with 0.3% PEI using a Packard cell
harvester, washed with 2.5 mL ice-cold buffer, and radioactivity
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