6-Chloropyridazin-3-yl derivatives active as nicotinic agents: Synthesis, binding, and modeling studies

Article abstract of DOI:10.1021/jm0208830

3,8-Diazabicyclo[3.2.1]octane (1), 2,5-diazabicyclo[2.2.1]heptane (2), piperazine (3), and homopiperazine (4) derivatives, substituted at one nitrogen atom with the 6-chloro-3-pyridazinyl group while the other nitrogen atom was either unsubstituted or mono- or dimethylated, were synthesized and tested for their affinity toward the neuronal nicotinic acetylcholine receptors (nAChRs). All of the compounds had K_i values in the nanomolar range. A molecular modeling study allowed location of their preferred conformations, the energies of which were recalculated in water with a continuum solvent model. Some of the compounds showed, in their populated conformations, only pharmacophoric distances longer than the values taken into consideration by the Sheridan model for nAChRs receptors. Thus, this SAR study gives support to the hypothesis that these longer distances are still compatible with affinity for α4β2 receptors in the nanomolar range.

Full text of DOI:10.1021/jm0208830

J . Med. Chem. 2002, 45, 4011-4017
4011
6-Ch lor op yr id a zin -3-yl Der iva tives Active a s Nicotin ic Agen ts: Syn th esis,
Bin d in g, a n d Mod elin g Stu d ies†
Lucio Toma,*,‡ Paolo Quadrelli,‡ William H. Bunnelle,§ David J . Anderson,§ Michael D. Meyer,§
Giorgio Cignarella,# Arianna Gelain,# and Daniela Barlocco*,#
Dipartimento di Chimica Organica, Universita` di Pavia, Via Taramelli 10, 27100 Pavia, Italy; Neurological and Urological
Diseases Research, D-47W, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064; and
Istituto di Chimica Farmaceutica e Tossicologica, Universita` di Milano, Viale Abruzzi 42, 20131 Milano, Italy
Received March 20, 2002
3,8-Diazabicyclo[3.2.1]octane (1), 2,5-diazabicyclo[2.2.1]heptane (2), piperazine (3), and ho-
mopiperazine (4) derivatives, substituted at one nitrogen atom with the 6-chloro-3-pyridazinyl
group while the other nitrogen atom was either unsubstituted or mono- or dimethylated, were
synthesized and tested for their affinity toward the neuronal nicotinic acetylcholine receptors
(nAChRs). All of the compounds had Ki values in the nanomolar range. A molecular modeling
study allowed location of their preferred conformations, the energies of which were recalculated
in water with a continuum solvent model. Some of the compounds showed, in their populated
conformations, only pharmacophoric distances longer than the values taken into consideration
by the Sheridan model for nAChRs receptors. Thus, this SAR study gives support to the
hypothesis that these longer distances are still compatible with affinity for R4â2 receptors in
the nanomolar range.
Ch a r t 1
In tr od u ction
The discovery of compounds that can safely treat both
acute and chronic pain without the side effects of drug
dependency is highly desirable in pain management. In
this respect, suggestions have been made that selective
neuronal nicotinic acetylcholine receptor (nAChR) ago-
nists may be useful. Nicotine itself has long been known
to have antinociceptive properties, but a variety of side
effects (mainly on the gastrointestinal tract and the
cardiovascular system) make it a poor therapeutic
choice.1-4 The discovery of epibatidine5-7 (Chart 1), a
potent analgesic and nAChR modulator, brought about
a renewed interest for compounds acting through
nAChRs. These receptors are widely distributed through
the central and peripheral nervous system. In particu-
lar, a major subtype in brain is composed of the R4â2
subunits combination, whereas ganglionic-type nAChRs
are thought to contain R3 in combination with â2 or â4
and possibly other R units. Because ganglionic-type
nAChRs are believed2 to at least partially mediate
several side effects of nicotinic agonists, the R4â2
subunits have become the target of our research. In a
previous paper8 we reported on the binding and anal-
gesic properties of 3-(6-chloro-3-pyridazinyl)-3,8-di-
azabicyclo[3.2.1]octane (1a , Chart 1), which we prepared
as a possible analogue of epibatidine. In the same
study,8 we performed molecular modeling investigations
at the semiempirical AM1 level on the protonated form
of 1a . Four minimum energy conformations were lo-
cated, among which the most stable one was found to
have a geometry similar to that of epibatidine. This
conformation of 1a appears to be stabilized by an
intramolecular hydrogen bond between the aromatic N2′
atom and one of the two hydrogen atoms present on the
1
positively charged N8. However, the H NMR spectrum
of 1a recorded in water was in disagreement with a
significant contribution of this conformation to the
overall population.8 To confirm, or to rule out, the
importance of such conformation stabilized by the
hydrogen bond, we decided to synthesize other models,
either bicyclic (2a ) or monocyclic (3a , 4a ) (Chart 1),
structurally related to the 3,8-diazabicyclo[3.2.1]octane
system; moreover, we prepared the mono- or dimethyl
derivatives 1b-4b and 1c-4c (Scheme 1) to set aside
the possibility of the hydrogen bond by replacement of
* Author to whom correspondence should be addressed L.T.: tele-
phone +39 0382 507843; fax +39 0382 507323; e-mail toma@
chifis.unipv.it; D.B.: telephone +39 02 50317515; fax +39 02
50317565; e-mail daniela.barlocco@unimi.it.
† Dedicated to Professor Paola Vita Finzi on the occasion of her 70th
birthday.
‡ Universita` di Pavia.
§ Abbott Laboratories.
#
Universita` di Milano.
10.1021/jm0208830 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/27/2002

4012 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Toma et al.
Sch em e 1a
a
(a) 3,6-Dichloropyridazine, NEt3, toluene, reflux. (b) 1, HCl, Et2O; 2, 6 N NaOH. (c) 1, HCOOH, 40% HCHO, 110 °C; 2, aqueous
NaHCO3. (d) CH3I, PMP, DMF, rt. (e) CH3I, Et2O, rt. (f) 3,6-Dichloropyridazine, 160 °C.
Ta ble 1. Radioligand Binding Properties of Compounds 1-4
one or both the ammonium hydrogen atoms with methyl
compd
Ki (nM)a
compd
Ki (nM)a
groups. We report here their synthesis together with
the binding affinity for the R4â2 nAChR subtype.
Moreover, modeling studies of all the compounds,
including also the influence of a continuum solvent
model, are reported.
1a
1b
1c
2a
2b
2c
4.1
39
2.3
8.8
65
3a
3b
3c
4a
4b
4c
32
14
0.44
1.5
220
120
4.3
Ch em istr y
a
[3H]Cytisine competition assay. Each experiment was per-
formed in triplicate. Ki values were from three experiments, which
agreed within 10%.
Compounds 1-4 were synthesized according to Scheme
1. The required diazaderivative either protected as a
Boc-derivative, as in the case of the diazabicyclo com-
pounds (5, 6),8-10 or unprotected (7, 8) was condensed
with 3,6-dichloropyridazine in refluxing toluene and in
the presence of triethylamine to give 1a -4a . In the case
of 1a and 2a the necessary deprotection step was easily
performed by treatment with a solution of hydrochloric
acid in diethyl ether. N-Methylation was then achieved
by treatment of 1a -4a with HCOOH/HCHO at 110 °C
for 2.5 h to afford 1b-4b. It should be noted that the
N-methyl derivatives 9-11 are commercial compounds.
Therefore, compounds 2b-4b could also be obtained by
treating them with 3,6-dichloropyridazine as described
for the corresponding nor-analogues. Finally, stirring
of 1b-4b with iodomethane in diethyl ether at room
temperature for 18 h led to the desired quaternary salts
1c-4c. However, in the case of 1c better yields were
obtained by repeated treatment of 1b with iodomethane
and 1,2,2,6,6-pentamethylpiperidine (PMP) in dimeth-
ylformamide at room temperature.11
In Vitr o Assa ys. Compounds were tested in binding
studies in [3H]cytisine competition assays. Rat cerebral
cortical membranes were used.12 Ki values are reported
in Table 1.
Mod elin g Stu d ies. Compounds 1a ,b-4a ,b, in their
protonated form, and 1c-4c, as cations, were submitted
to a modeling study through theoretical calculations
performed in two steps; first, the conformational space
was completely explored at the semiempirical AM1

6-Chloropyridazin-3-yl Derivatives Active as Nicotinic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4013
Ta ble 2. Relative Energy and Selected Geometrical Features of the B3LYP/6-31G(d) Calculated Minimum Energy Conformations of
Compounds 1-4a
Erel
E
rel(aq)
Erel
Erel(aq)
conformation
(kcal/mol)
(kcal/mol)
A-B (Å)
A-C (Å)
conformation
(kcal/mol)
(kcal/mol)
A-B (Å)
A-C (Å)
1a -A
1a -B
1a -C
1a -D
1b-A
1b-B
1b-C
1b-D
1b-E
1b-F
1b-G
1c-A
1c-B
1c-C
2a -A
2a -B
2a -C
2b-A
2b-B
2b-C
2b-D
2b-E
2b-F
2c-A
2c-B
2c-C
3a -A
3a -B
3a -C
3a -D
3b-A
3b-B
3b-C
3b-D
3b-E
3b-F
3b-G
3b-H
3c-A
3c-B
3c-C
3c-D
0.00
3.51
3.65
6.01
0.00
2.29
2.42
4.24
4.69
4.88
6.83
0.00
0.65
2.69
0.00
2.36
3.33
0.00
2.42
2.66
3.33
3.46
4.33
0.00
0.35
1.19
0.00
4.80
5.20
9.05
0.00
4.14
4.24
6.76
6.81
8.81
10.38
11.05
0.00
0.18
4.57
4.71
12.08
0.00
2.11
7.05
14.07
0.00
1.89
1.54
4.38
7.37
10.23
0.00
2.72
8.90
10.66
0.00
0.21
11.26
0.00
7.48
0.28
1.14
1.06
5.58
0.00
0.39
11.13
8.52
0.00
5.07
12.37
8.33
0.00
2.18
10.91
6.03
11.59
6.77
0.00
8.49
9.71
5.09
3.89
6.22
5.93
5.86
3.93
6.26
6.03
6.29
6.00
5.87
5.85
6.29
6.12
5.82
4.05
6.13
6.18
4.09
6.14
4.84
6.18
6.17
6.23
4.96
6.16
6.22
3.86
4.87
6.26
5.81
3.89
4.89
6.29
6.31
4.88
5.99
4.93
6.10
6.34
4.89
5.05
6.14
3.94
5.57
5.38
5.44
3.99
5.58
5.44
5.61
5.43
5.44
5.44
5.61
5.51
5.43
4.07
5.44
5.48
4.11
5.45
4.66
5.48
5.49
5.52
4.71
5.48
5.51
3.91
4.78
5.58
5.16
3.95
4.81
5.61
5.62
4.80
5.25
4.66
5.39
5.65
4.82
4.75
5.44
4a -A
4a -B
4a -C
4a -D
4a -E
4a -F
4a -G
4a -H
4a -I
0.00
0.33
1.37
7.79
8.77
10.42
10.54
10.89
11.13
11.48
12.61
0.00
0.16
0.98
6.50
7.76
8.83
8.92
9.23
9.48
9.61
9.62
9.88
9.97
10.05
10.22
10.65
11.24
11.27
11.67
12.55
13.72
0.00
0.76
0.76
1.03
1.85
2.47
2.71
3.02
3.66
6.40
7.08
6.88
0.00
2.12
2.09
2.61
5.83
4.04
4.57
0.26
7.83
8.51
8.15
0.00
2.06
4.97
2.30
2.44
3.60
2.95
3.18
5.90
4.87
4.19
7.86
7.14
4.23
0.18
2.89
5.98
2.54
0.00
3.33
1.92
1.45
6.08
5.13
2.28
0.89
6.50
3.96
0.32
3.85
3.85
3.83
5.46
5.16
5.81
5.39
5.05
5.55
5.93
6.53
3.89
3.87
3.86
5.48
5.20
4.89
5.51
5.80
5.19
5.43
5.50
5.08
5.95
5.58
4.77
5.15
5.43
6.55
6.06
5.37
6.58
5.53
4.99
5.20
5.56
4.87
5.25
5.45
6.21
5.07
5.45
6.61
3.98
4.00
3.89
5.17
4.87
5.17
4.88
4.74
5.27
5.04
5.63
4.02
4.04
3.93
5.20
4.91
4.69
5.22
5.16
4.90
4.92
5.27
4.77
5.06
5.30
4.65
4.95
4.92
5.65
5.45
4.85
5.67
5.25
4.79
4.94
5.33
4.75
5.05
4.94
5.60
4.77
4.92
5.70
4a -J
4a -K
4b-A
4b-B
4b-C
4b-D
4b-E
4b-F
4b-G
4b-H
4b-I
4b-J
4b-K
4b-L
4b-M
4b-N
4b-O
4b-P
4b-Q
4b-R
4b-S
4b-T
4b-U
4c-A
4c-B
4c-C
4c-D
4c-E
4c-F
4c-G
4c-H
4c-I
4c-J
4c-K
4.09
4.82
a
Boldface data represent conformations with Erel(aq) in a range of 3 kcal/mol above the global minimum. For compounds 4 only the
most significant conformations from the AM1 analysis have been reoptimized at the B3LYP/6-31G(d) level.
level;13 successively, all of the minimum energy confor-
mations thus located for 1-3 were reoptimized at the
higher B3LYP/6-31G(d) level.14,15 For 4, reoptimization
was limited to the most significant conformations found
with AM1. Moreover, the solvation energy was calcu-
lated with continuum solvent models, AM1-SM5.416 and
C-PCM,17 to take into account the strong influence of
water on the behavior of these compounds that bear a
positive charge under physiological conditions.
Table 2 reports the B3LYP calculated gas-phase and
water-solvated energies together with selected geo-
metrical features of the various conformations of the
compounds studied. Figure 1 reports the three-dimen-
sional plots of the global energy minimum conforma-
tions.
leads to an ∼10-fold decrease in affinity, whereas
addition of the second one restores it; indeed, quater-
narization slightly improves Ki with respect to the
parent nonmethylated compounds. Substantially dif-
ferent is the behavior of the series of compounds 3 and
4; in the piperazine series 3 double methylation to the
quaternary ammonium salt 3c improves Ki by 2 degrees
of magnitude, approaching the subnanomolar range;
conversely, in the homopiperazine series methiodide 4c
shows a 100-fold decrease in affinity with respect to 4a .
The monomethyl derivatives 3b and 4b in both series
stand close to the less active compound.
In binding assays, typical nicotinic ligands exhibit
diverse structure-activity patterns with respect to
N-substitution; whereas the affinity of S-nicotine is ∼20-
fold higher than that of its nor-analogue,18 N-methyla-
tion decreases by 2-fold the affinity of (-)-epibatidine19
and by several hundreds that of (+)-anatoxin.20 The
effects of the N-substitution have been discussed in a
recent review.21 The hypothesis has been made that it
may evoke indirect conformational effects in addition
to the exclusive steric effects usually taken into consid-
Discu ssion
The binding data reported in Table 1 indicate for all
of the compounds an affinity in the nanomolar range
as already shown for the model compound 1a . The
influence of methylation was similar in the series of
compounds 1 and 2: addition of the first methyl group

4014 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Toma et al.
F igu r e 1. Three-dimensional plots of the global energy minimum conformations of compounds 1-4 as optimized at the B3LYP/
6-31G(d) level and recalculated in water using the C-PCM model.
eration. N-Methyl derivatives 1b -4b were always
weaker ligands than their nonmethylated and/or dim-
ethylated analogues. This suggests that, in our case,
protonated tertiary amines are not able to profit from
the efficient binding modes of the protonated secondary
amines and quaternary ammonium compounds.22
The best compounds in the 1-4 series show affinity
for R4â2 nAChR in the same range as nicotine, and the
question arises whether they interact with the receptor
similarly to nicotine or epibatidine. Over the years, on
the basis of the structures of nicotine and other nicotinic
agonists, several pharmacophoric elements have been
proposed; the Beers and Reich model23 suggested a
distance of 5.9 Å between the center of positive charge
(N+) and a hydrogen acceptor (the lone pair of a pyridine
nitrogen atom or of a carbonyl oxygen). Subsequently,
Sheridan et al.24 proposed a model based on three
pharmacophoric elements, the cationic center N+ (A),
an electronegative atom capable of accepting a hydrogen
bond (B), and a dummy atom that defines the line along
which the hydrogen bond may form (C): the Sheridan
model indicates for a good affinity an A-B distance of
4.8 ( 0.3 Å, an A-C distance of 4.0 ( 0.3 Å, and a B-C
distance of 1.2 Å. Subsequently, a longer A-B distance
was proposed25 on the basis of the fact that epibatidine
presents minimum energy conformations with an A-B
distance of 5.5 Å. This finding was also supported by
the discovery of azetidinylmethoxypyridine A-85380,4
almost equipotent to epibatidine, which shows an even
longer A-B distance of 6.1 Å in its minimum energy
conformation. Actually, epibatidine possesses other low-
energy conformations with an A-B distance of 4.5 Å,
and also A-85380, being a rather flexible molecule,
presents conformers with A-B distances of <6.1 Å.
Moreover, when epibatidine is considered as a reference
compound, it should be noted that the barriers to
rotation of the pyridine ring are very low, 2.0-2.5 kcal/
mol as calculated by Campillo et al.26 at various levels
of calculations; none of its four energy minima can be
taken as a rigid model for R4â2 receptor agonists, and
it is only possible to say that the active conformation
has an A-B distance somewhere in the range of 4.5-
5.5 Å. The discovery3 of a series of derivatives of A-85380
variously substituted by a halogen atom on the pyridine
ring gave again support to the original Sheridan model
as only compounds able to present low energy confor-
mations with A-B of ∼4.4 Å possess affinity comparable
to that of A-85380. However, a comprehensive review21
on nicotinic agonists and antagonists has shown several
cases, in addition to those reported above, of good
affinity still compatible with pharmacophoric distances
higher than that of the Sheridan model.
The strong influence of the water solvation on the
energy of the various conformers in the case of charged
species should be noted. Table 3 shows as an example
how the energy values vary for the four conformations
of protonated epibatidine calculated with the B3LYP/
6-31G(d) method in gas phase and in water with a
continuum solvent model (see also Figure 2). Some
reranking of the conformations can be observed, which,
however, remain close in energy in a range of a few
kilocalories per mole.
As far as compounds 1-4 are concerned, the consid-
erations reported above for epibatidine should also apply

6-Chloropyridazin-3-yl Derivatives Active as Nicotinic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4015
Ta ble 3. Relative Energy and Selected Geometrical Features
of the B3LYP/6-31G(d) Calculated Minimum Energy
Conformations of Protonated Epibatidine
dihedral angle
C1-C2-
C3′-C2′
Erel
E
rel(aq) A-B A-C
(Å)
conformation
(deg)
(kcal/mol) (kcal/mol) (Å)
ep i-A
ep i-B
ep i-C
ep i-D
13
85
-90
-170
0.95
0.00
1.03
2.27
0.00
1.38
2.49
1.52
4.57 4.26
4.51 4.13
5.30 4.21
5.49 4.32
F igu r e 3. Superimposition of conformation 3c-A of compound
3c with the four energy minimum conformations of epibati-
dine.
with experimental NMR data. Conformers 1a -B-D
differ only in the orientation of the aryl ring and present
similar A-B (5.9-6.2 Å) and A-C (5.4-5.6 Å) distances.
The former distance appears in the upper limit for good
R4â2 agonists and in disagreement with the Sheridan
model; also, the latter distance does not conform to the
model as it is ∼1.5 Å longer.
Compound 1b presents seven energy minima; the first
one, 1b-A, corresponds to 1a -A, and the other six
correspond to the three conformers 1a-B-D each doubled
by the two possible orientations of the N-methyl group.
In compound 1c the possibility of a hydrogen bond is
precluded by the presence of two N-methyl groups;
hence, only the three conformers 1c-A-C, resembling
the 1a -B-D conformations of 1a , could be located. This
fact suggests that the conformations stabilized by a
hydrogen bond are not a necessary requisite for good
binding properties as the affinity of compound 1c is even
higher than that of 1a . For 1c the global minimum
conformation is the same in water and in the gas phase
and resembles the best “solvated” conformations of 1a
and 1b. This suggests that also for 1a and 1b the
“solvated” conformations should be more relevant for a
good affinity. In fact, although it is generally accepted
that ligands bind receptors after desolvation, probably
the bound conformation of compounds 1 resembles the
“solvated” conformations. On these bases, we were
induced to focus our discussion on the energy data in
water.
These considerations may be extended to compounds
2a -c and 3a -c, which also show, in their preferred
“solvated” conformations, A-B and A-C distances of
6.1-6.3 and 5.4-5.6 Å, respectively. It is worth pointing
out that 3c, the compound with the highest affinity in
our four series, presents a largely preferred conforma-
tion, 3c-A, with A-B and A-C distances significantly
high. However, a superimposition of this conformation
with the four conformations of epibatidine (Figure 3)
shows that the similarity of the molecules is greater
than was indicated by the analysis of the pharmacoph-
oric distances. This suggests that the three-point phar-
macophore is useful only for a preliminary comparison.
In fact, additional binding interactions may be present
and may derive from the two methyl groups that appear
able to superimpose to the ethylidenic bridge of epiba-
tidine in some of the pictures reported in Figure 3.
Finally, the great flexibility of the seven-membered
ring in 4a -c allows the existence of several conforma-
F igu r e 2. Three-dimensional plots of the energy minimum
conformations of epibatidine as optimized at the B3LYP/6-31G-
(d) level.
with an important difference: the presence of a second
nitrogen atom (N2′) in the heterocyclic ring limits the
rotational freedom; in fact, an interaction should exist
between this atom and the positively charged center.
This interaction could stabilize a particular orientation
of the aromatic ring as a consequence not only of the
possible existence of a hydrogen bond between N2′ and
one N-H hydrogen atom but also of the influence that
the solvent has on the strength of this bond. Moreover,
the orientation of the aromatic ring can be influenced
by the resonance with the lone pair of the nitrogen atom
to which it is connected, so that conformations that
make possible this conjugation should be preferred. The
results previously reported by us8 on compound 1a were
in some respect contradictory: whereas gas-phase AM1
calculations indicated as favored the conformation that
presents the intramolecular hydrogen bond N8-H‚‚‚N2′
and a boat conformation of the piperazine ring, vicinal
1
coupling constants data from the H NMR spectrum of
compound 1a registered in D2O support a chair confor-
mation of the piperazine ring. Reinvestigation of the
behavior of 1a at the B3LYP level confirmed conforma-
tion 1a -A, presenting the intramolecular hydrogen bond,
as the gas-phase global energy minimum. Three other
conformations, 1a -B-D, more compatible than 1a -A
1
with the H NMR data, were located; they all present a
chair conformation of the pyperazine ring, and two of
them, 1a -B and 1a -C, show the aryl ring correctly
oriented for a good conjugation with the N3 nitrogen
lone pair. When the energy of the four conformations
of 1a was recalculated in water with a continuum
solvent model,16,17 conformation 1a -A became a very
high energy conformer, less stable than the global
energy minimum 1a -B by >10 kcal/mol, in agreement

4016 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Toma et al.
Ta ble 4. Physicochemical Properties of Compounds 1-4
yield
(%)
compda
formulab
1H NMR (δ)
solvent
CDCl3
1a
46
C
10H13ClN4
1.80 (m, 4H + 1H, exch with D2O), 3.10 (m, 2H), 3.65 (m, 2H), 3.90 (m, 2H), 6.75 (d, 1H),
7.15 (d, 1H)
1b
1c
6
13
C
C
11H15ClN4
1.70 (m, 2H), 2.00 (m, 2H), 2.35 (s, 3H), 3.30 (m, 4H), 3.80 (d, 2H), 6.80 (d, 1H), 7.15 (d, 1H)
CDCl3
D2O
12H18ClIN4 2.20 (m, 2H), 2.45 (m, 2H), 3.20 (s, 3H), 3.35 (s, 3H), 3.80 (m, 2H), 4.10 (m, 4H), 7.35 (d, 1H),
7.55 (d, 1H)
2a
(1S,4S)
2b
(1S,4S)
2c
40
64
78
C9H11ClN4
2.10 (d, 1H), 2.20 (d, 1H), 3.2-3.3 (m, 2H+1H, exch with D2O), 3.70 (d, 1H), 3.80 (d, 1H),
CDCl3
4.60 (br s, 1H), 5.00 (br s, 1H), 7.30 (d, 1H), 7.70 (d, 1H)
C
C
10H13ClN4
1.90 (d, 1H), 2.05 (d, 1H), 2.45 (s, 3H), 2.90 (m, 2H), 3.40 (dd, 1H), 3.65 (d, 1H), 3.75 (bs, 1H), CDCl3
4.65 (bs, 1H), 6.70 (d, 1H), 7.15 (d, 1H)
11H16ClIN4 2.52 (br d, 1H), 2.73 (d, 1H), 3.23 (s, 3H), 3.35 (s, 3H), 3.81 (m, 3H), 4.14 (d, 1H),
D2O
(1S,4S)
3a
3b
3c
4a
4.6-5.1 (m, 2H, partly obscured by HOD peak), 7.18 (d, 1H), 7.58 (d, 1H)
21
44
40
42
C8H11ClN4
C9H13ClN4
C
1.70 (s, 1H, exch with D2O), 3.00 (t, 4H), 3.65 (t, 4H), 6.90 (d, 1H), 7.25 (d, 1H)
2.35 (s, 3H), 2.55 (t, 4H), 3.65 (t, 4H), 6.90 (d, 1H), 7.20 (d, 1H)
CDCl3
CDCl3
DMSO
CDCl3
10H16ClIN4 3.20 (s, 6H), 3.60 (t, 4H), 4.00 (t, 4H), 7.55 (d, 1H), 7.75 (d, 1H)
C9H13ClN4
1.90 (t, 2H), 2.20 (s, 1H, exch with D2O), 2.80 (t, 2H), 3.05 (t, 2H), 3.75 (m, 4H), 6.90 (d, 1H),
7.15 (d, 1H)
4b
4c
28
90
C
C
10H15ClN4
2.00 (m, 2H), 2.40 (s, 3H), 2.55 (t, 2H), 2.75 (t, 2H), 3.65 (t, 2H), 3.90 (t, 2H), 6.75 (d, 1H),
7.15 (d, 1H)
CDCl3
11H18ClIN4 2.35 (m, 2H), 3.24 (s, 6H), 3.62 (m, 2H), 3.67 (m, 2H), 3.72 (t, 2H), 4.19 (m, 2H), 7.26 (d, 1H),
7.52 (d, 1H)
CD3OD
a
With the exception of 3b (mp ) 115-116 °C) and the quaternary salts, which, however, decomposed before their melting points, all
b
of the compounds were oils. Elemental analyses for C, H, and N were within (0.4% of the calculated data.
400 mesh) was used for flash chromatography. All commercial
compounds were purchased from Aldrich Chemical Co.
Gen er a l Met h od for t h e Syn t h esis of Com p ou n d s
1a )4a . Method a. A solution of piperazine (7) or homopip-
erazine (8) (5 mmol), 3,6-dichloropyridazine (0.743 g, 5 mmol),
and triethylamine (TEA; 0.695 g, 5 mmol) in toluene (10 mL)
was refluxed under stirring overnight. The solvent was
evaporated under vacuum and the residue purified by flash
chromatography (CH2Cl2/CH3OH 75:25) to give the desired
compounds 3a and 4a , respectively. See Table 4 for data.
Method b. In the case of the diazabicyclo compounds, 8-tert-
butoxycarbonyl 3,8-diazabicyclo[3.2.1]octane (5)8 and the lower
homologue 2-tert-butoxycarbonyl-2,5-diazabicyclo[2.2.1]-hep-
tane (6)9,10 were reacted as reported for method a. Final
deprotection by treatment with a solution of hydrochloric acid
in diethyl ether gave the desired 1a and 2a as their hydro-
chlorides. The corresponding free base could be obtained by
treatment with 6 N NaOH and subsequent extraction with
Et2O. See Table 4 for data.
Gen er a l Met h od for t h e Syn t h esis of Com p ou n d s
1b)4b. A mixture of the appropriate 1a -4a (0.47 mmol), 99%
HCOOH (0.705 mL, 18.68 mmol), and 40% HCHO (0.034 mL,
0.47 mmol) was stirred at 110 °C for 2.5 h. After cooling, the
solvent was evaporated, and the residue was treated with a
solution of NaHCO3, extracted with CH2Cl2 (3 × 10 mL), and
dried over sodium sulfate. After evaporation of the solvent,
the product was purified by flash chromatography (CH2Cl2/
CH3OH 85:15). See Table 4 for data.
tions, and also the most stable “solvated” conformations
present a large distribution of the A-B (5.2-6.6 Å) and
A-C distances (4.9-5.7 Å).
Con clu sion s
Compounds 1-3 give support to the hypothesis21 that
A-B distances longer (∼6.0 Å) than in the Sheridan
model are still compatible with affinity for R4â2 recep-
tors in the nanomolar range. Our results put also
attention on the A-C distance that, contrarily to
epibatidine, in several of our compounds appears by
∼1.5 Å longer than in the Sheridan model. Compounds
4 appear to be more flexible and present larger ranges
of the A-B and A-C distances; however, it should be
noted that, if the conformations presenting the intramo-
lecular hydrogen bond are excluded, the A-C distance
remains longer than in the Sheridan model. The reason
only 3c, and not 1c, 2c, or 4c, shows a significant
improvement of the binding affinity with respect to the
nonmethylated and monomethylated analogue is not
evident from the analysis of the A-B and A-C dis-
tances; other factors, such as steric interactions, could
play a special role in the case of the quaternary
ammonium compounds. The three-dimensional plots of
1c-A, 2c-B, and 3c-A depicted in Figure 1 show a great
similarity among them. However, it should be noted that
1c and 2c, as well as 4c, have a molecular volume larger
than that of 3c; because all of the values found for their
volumes (V ) 283, 267, 257, and 275 Å3 for 1c, 2c, 3c,
and 4c, respectively) are greater than those of nicotine
(208 Å3) and epibatidine (234 Å3), it could be hypoth-
esized that the value found for 3c might represent the
maximum tolerance for the receptor.
Alternatively, compounds 2b-4b could be obtained by
heating the commercially available 9-11 with 3,6-dichloro-
pyridazine (molar ratio 1:1.5) at 160 °C for 70 min.
Gen er a l Met h od for t h e Syn t h esis of Com p ou n d s
1c)4c. Method a. Methyl iodide (100 µL, 1.4 mmol) was added
to a stirred solution of the required 1a -4a (0.10 mmol) in
diethyl ether (8 mL). A precipitate begins to form within 1 h,
and stirring was continued for 18 h at room temperature. The
solid was isolated by filtration, washed well with ether, and
dried under vacuum at 50 °C to provide the title compound as
an off-white powder. See Table 4 for data.
Method b. In the case of 1c an alternative procedure11 was
followed, which gave better results. Accordingly, to a solution
stirred at room temperature of 1a (80 mg, 0.356 mmol) in DMF
(3.5 mL) were added 1,2,2,6,6-pentamethylpiperidine (PMP;
0.128 mL, 0.712 mmol) and CH3I (0.044 mL, 0.712 mmol) every
24 h for four times; the last time the mixture was allowed to
stir for 72 h. The so-formed precipitate was filtered off by
suction and ethyl ether (10 mL) at 0 °C added to the filtrate
to give a red solid, which was constituted by a mixture of the
Exp er im en ta l Section
Melting points were determined on a Bu¨chi 510 capillary
melting point apparatus and are uncorrected. Analyses indi-
cated by the symbols were within (0.4 of the theoretical
values. 1H NMR spectra were recorded on a Bruker AC200
spectrometer; chemical shifts are reported as δ (parts per
million) relative to tetramethylsilane. TLC on silica gel plates
was used to check product purity. Silica gel 60 (Merck; 230-

6-Chloropyridazin-3-yl Derivatives Active as Nicotinic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4017
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desired product and PMP. The obtained solid was filtered by
suction and heated with acetone (15 mL) for a few minutes.
After filtration, the solid was further washed with methanol,
to give the desired compound in acceptable purity. See Table
4 for data.
Bin d in g Assa ys. 1. Mem br a n e P r ep a r a tion s. Rat cere-
bral cortical membranes were purchased from ABS Inc.
(Wilmington, DE). Prior to use, the frozen membrane pellets
were slowly thawed, washed, and resuspended in 30 volumes
of assay buffer (composition, mM: Tris HCl, 50; NaCl, 120;
KCl, 5; MgCl2, 1; and CaCl2, 2.5; pH 7.4 at 4 °C). The
homogenate was centrifuged at 45000g for 20 min at 4 °C and
the pellet resuspended in ice-cold buffer.
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approach17 implemented in the Gaussian 98 package. The A-B
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two nitrogen atoms, whereas the A-C distance was measured
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