Design, synthesis, and activity of a series of arylpyrid-3-ylmethanones as type i positive allosteric modulators of α7 nicotinic acetylcholine receptors

Article abstract of DOI:10.1021/jm400704g

A series of novel arylpyrid-3-ylmethanones (7a-aa) were designed as modulators of α7 nicotinic acetylcholine receptors (nAChRs). The methanones were found to be type I positive allosteric modulators (PAMs) of human α7 nAChRs expressed in Xenopus ooctyes. Structure-activity relationship (SAR) studies resulted in the identification of compound 7v as a potent and efficacious type I PAM with maximum modulation of a nicotine EC ₅ response of 1200% and EC₅₀ = 0.18 μM. Compound 7z was active in reversing the effect of scopolamine in the novel object recognition (NOR) paradigm with a minimum effective ip dose of 1.0 mg/kg (2.7 μmol/kg). This effect was blocked by the selective α7 nAChR antagonist methyllycaconitine (MLA). These compounds are potent type I positive allosteric modulators of α7 nAChRs that may have therapeutic value in restoring impaired sensory gating and cognitive deficits in schizophrenia and Alzheimer's disease.

Full text of DOI:10.1021/jm400704g

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Article
Design, Synthesis and Activity of a Series of Arylpyrid-3-ylmethanones as
Type I Positive Allosteric Modulators of #7 Nicotinic Acetylcholine Receptors
Derk J. Hogenkamp, Thomas A Ford-Hutchinson, Wen-Yen Li, Edward R Whittemore, Ryan F Yoshimura,
Minhtam B Tran, Timothy B C Johnstone, Gavin D. Bascom, Hannah Rollins, Lena Lu, and Kelvin W. Gee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400704g • Publication Date (Web): 04 Oct 2013
Downloaded from http://pubs.acs.org on October 5, 2013
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Design, Synthesis and Activity of a Series of Arylpyridꢀ3ꢀylmethanones as
Type I Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors
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Derk J. Hogenkamp* , Thomas A. FordꢀHutchinson , WenꢀYen Li , Edward R.
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Whittemore , Ryan F. Yoshimura , Minhtam B. Tran , Timothy B. C. Johnstone , Gavin D.
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Bascom , Hannah Rollins , Lena Lu and Kelvin W. Gee
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Med Surge 2, Room 372, Department of Pharmacology, College of Medicine, University of
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California, Irvine, Irvine, CA 92697. Current address: ClinDatrix, Inc. 6 Jenner St., Suite 200,
Irvine, CA 92618. Department of Chemistry, 4216 Natural Sciences 1, University of California,
Irvine, Irvine, CA 92697. Current address: Pharmatek Laboratories, Inc., 7330 Carroll Road,
Suite 200, San Diego, CA 92121.
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Abstract. A series of novel arylpyridꢀ3ꢀylmethanones (7aꢀaa) were designed as modulators of
α7 nicotinic acetylcholine receptors (nAChRs). The methanones were found to be Type I
positive allosteric modulators (PAMs) of human α7 nAChRs expressed in Xenopus ooctyes.
Structure activity relationship (SAR) studies resulted in the identification of compound 7v as a
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potent and efficacious Type I PAM with maximum modulation of a nicotine EC response of
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200% and EC = 0.18 ꢀM. Compound 7z was active in reversing the effect of scopolamine in
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the novel object recognition (NOR) paradigm with a minimum effective i.p. dose of 1.0 mg/kg
2.7 ꢀmol/kg). This effect was blocked by the selective α7 nAChR antagonist
(
methyllycaconitine (MLA). These compounds are potent Type I positive allosteric modulators of
α7 nAChRs that may have therapeutic value in restoring impaired sensory gating and cognitive
deficits in schizophrenia and Alzheimer’s disease.
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Introduction. The α7ꢀsubtype of nicotinic acetylcholine receptors (nAChR) has emerged as an
important target for the development of central nervous system (CNS) agents for the treatment of
a variety of disorders involving cognitive deficits and neurodegeneration, including
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schizophrenia, attention deficit hyperactivity disorder (ADHD) and Alzheimer’s disease. The
α7 nAChR is part of the Cysꢀloop family that includes γꢀaminobutyric acid (GABA ), serotonin
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(5ꢀHT ) and glycine receptors. The α7 receptor is a pentamer that forms an ion channel that
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binds acetylcholine (choline) as the endogenous ligand and enhances Ca conductance when
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open. Selective agonists and partial agonists have been described for the α7 nAChR as well as
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positive allosteric modulators (PAMs). PAMs may have advantages over compounds acting at
the orthosteric ligand binding site because they maintain the normal temporal and spatial pattern
of neurotransmission due to their lack of activity in the absence of an agonist. Clinical studies
with agonists have shown that targeting the α7 nAChR can be an effective method for improving
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cognitive deficits in schizophrenia. The fact that 80% of schizophrenics smoke also points to
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the importance of nAChRs in the disease. PAMs at nicotinic receptors have been further
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characterized as Type I and II, depending on their interactions with the receptor. Type I
modulators leave the fast native kinetics of the channel and its desensitization characteristics
unperturbed, while Type II compounds significantly retard channel kinetics and may reverse
desensitization. Structures of representative Type I and II PAMs are given in Figure 1.
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Compounds have also been characterized with properties in between the two extremes . Because
+
+
the α7 nAChR is highly Ca permeable, Type II modulators that reverse desensitization may
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have the potential to be cytotoxic, although this has not been observed in vivo. With some type
II modulators, the currents elicited from desensitized channels were drastically reduced at
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physiological temperature compared to rt. Despite these reduced currents, these Type II
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modulators have been found to be excitotoxic under more physiological conditions (e.g., 37 C
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Figure 1: Structures of Type I and Type II PAMs
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Type I PAMS
Cl
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CF3
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NH
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OH
Cl
AVL-3288
NS-1738
SB-206553
Type II PAMs
SO NH
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Cl
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MeO
HN
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Cl
O
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N
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H
H
OMe
SO NH
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2
PNU-120596
THQ
A-867744
We have described the activity of a series of enaminone esters and amides as potent
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allosteric modulators of GABA Rs. A small library of these compounds were screened for
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activity as PAMs of human α7 nAChRs expressed in Xenopus oocytes. Compounds were
identified with activity at both receptors and modifications to these hits resulted in the
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identification of novel compounds with selectivity for α7 nAChRs. The enaminone esters and
amides, exemplified by compounds 1a, b and c (Figure 2) emerged from these efforts, but were
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not optimized as potential drugs. For example, in vitro, the compounds were not selective for α7
nAChRs. Compound 1a was equally active at α7 nACh and GABA Rs, showing essentially
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equal efficacy at the nicotinic receptor and α β γ GAB Rs. The 4ꢀfluoro analog 1b was more
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efficacious at α7 receptors but still showed GABA R activity. In addition to this lack of
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selectivity, the H NMR spectra of both esters indicate that they are mixtures of Zꢀ and Eꢀ
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isomers. Based on the chemical shift for the aniline NH in the Zꢀisomers of 1a and b (Zꢀ1), a
hydrogenꢀbond (Hꢀbond) with the ester carbonyl oxygen atom is implicated (see Figure 2). The
major isomer, at least under NMR conditions in CDCl or DMSOꢀd , is Zꢀ1, presumably due to
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the stronger Hꢀbond with the ester in Zꢀ1 compared to the ketone in Eꢀ1. Unlike the
corresponding esters, enaminone amides like 1c that form two internal Hꢀbonds (one between the
amide NH and the ketone and a second between the amide oxygen atom and the aniline NH) are
1
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known to be single isomers. In vitro, 1c was similar in activity at α7 nAChRs to 1a with I/I
max
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tertꢀbutylbicyclophosphorothionate ([ S]TBPS, IC = 0.1 ꢀM). Furthermore the enaminone
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amides are alkylated anilines with the potential for the formation of toxic metabolites.
Assuming that the enaminone esters bind to the α7 nAChR as the major Zꢀisomer, and the
amides bind similarly, it was hoped that the Hꢀbonded array in 1c could be replaced with a
pyridine ring as in compound 2 (Figure 3). This tactic has been employed in a number of other
chemical series, where groups of Hꢀbonded atoms have been replaced with sixꢀmembered rings
1
3
and viceꢀversa. In many cases, the biological activity of the Hꢀbonded series has been
maintained or improved. Several criteria were felt to be important for the compounds to meet: (1)
They should be selective for α7 nAChRs over heteromeric nAChRs and GABA Rs. (2) The
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compounds should be Type I modulators of α7 nAChRs that preserve the native kinetics of the
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receptor and do not reverse desensitization. (3) The efficacy of the compounds at α7 nAChRs
should be maximized without decreasing their potency. This paper describes the synthesis and
biological activity of 2 as PAMs of α7 nAChRs.
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Figure 2. Structures of enaminone esters (1a and 1b), amide 1c and the Eꢀ and Zꢀisomers of 1a.
Figure 3. Design of (2ꢀalkylaminoꢀ5ꢀarylpyridineꢀ3ꢀyl)arylmethanones 2 from enaminone amide
c.
1
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Chemistry. The pyridines 2 were prepared by using one of three methods depending on the
substituents on the aryl group of the benzoyl moiety. Starting with arylacetic acids (Method A;
Scheme
1),
reaction
with
POCl /DMF
(Vilsmeier
reagent)
afforded
the
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aryl(dimethylamino)propenal 3, which was condensed with cyanoacetamide in the presence of
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sodium methoxide to give the pyridone 4.
Reaction of 4 with POCl followed by addition of
3
an amine afforded the 2ꢀaminoꢀ5ꢀarylꢀ3ꢀcyanopyridine 6. Addition of an arylmagnesium halide
and hydrolysis to the ketone then gave the products 7aꢀk.
Scheme 1. Method A: Synthesis of Arylpyridylmethanones Starting with Arylacetic Acids.
o
o
Reagents/Solvents: a) POCl /DMF/70 C, then aq. NaOH/100 C b) cyanoacetamide/NaOMe/MeOH c) POCl ,
3
3
reflux d) R NH /DMSO e) R PhMgX/THF reflux then aq. HCl, reflux f) MeNHPr/DMSO.
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2
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The intermediate aminocyanopyridines 6 were also prepared as shown in Scheme 2 (Method B),
starting with 2ꢀchloronicotinonitrile. Reaction with propylamine gave 8, followed by
bromination with Nꢀbromosuccinimide (NBS) afforded 5ꢀbromoꢀ2ꢀpropylaminoꢀ3ꢀ
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pyridinecarbonitrile 9. Reaction with arylboronic acids was carried out under Suzuki coupling
conditions. Conversion of 6 to the final product still necessitated the use of an arylmagnesium
halide. This final step was found to be low yielding and limited by the availability and stability
of the Grignard reagents. In order to avoid these issues and to allow for the preparation of a
wider variety of arylketones, a better method for introducing the benzoyl group was needed.
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Scheme 2. Method B: Conversion of 2ꢀchloronicotinonitrile to 7l, m, n and o.
Reagents/Solvents: a) Propylamine/DMSO b) NBS/DMF c) ArylB(OH) , toluene/EtOH/aq. Na CO /Pd(PPh ) d) 4ꢀ
2
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3
3 4
Fluorophenylmagnesium bromide/THF, reflux then aq. HCl, reflux
As shown in Scheme 3, use of 2ꢀfluoropyridine as starting material (Method C) improved the
versatility of the synthetic method, allowing for a more modular construction of the desired
product. With the wide variety of benzaldehydes available a larger variety of substitutions to the
benzoyl group were easily accessible.
Scheme 3. Method C: Use of 2ꢀfluoropyridine as starting material
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Reagents/Solvents: a) LDA/THF/R PhCHO, ꢀ78 C b) PCC/CH Cl c) R NH /DMSO or EtOH, rt d) NBS/DMF e)
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addition of a substituted benzaldehyde and oxidation with pyridinium chlorochromate (PCC)
gave the 3ꢀbenzoylꢀ2ꢀfluoropyridines 11. Addition of an amine R NH in DMSO or EtOH at rt
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gave the intermediate 2ꢀalkylaminoꢀ3ꢀbenzoylpyridines 12. Bromination with NBS followed by
Suzuki coupling then gave the desired compounds 7pꢀz and 7aa. This method generally worked
well except in the case of 3ꢀ(3,4ꢀdifluorobenzoyl)ꢀ2ꢀfluoropyridine (14, Scheme 4). Addition of
amines to 14 resulted in the formation of the desired compound 15 along with significant
displacement of the 4ꢀfluorine on the benzoyl group to give 16 (identified by mass
spectroscopy). The two products were separated by using flash chromatography. This sideꢀ
reaction was not observed with 4ꢀchloroꢀ or 4ꢀfluorobenzoyl groups.
Scheme 4. Reaction of 3ꢀ(3,4ꢀdifluorobenzoyl)ꢀ2ꢀfluoropyridine (14) with amines.
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In vitro Pharmacology. Compounds were tested for in vitro activity at human α7 nAChRs
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expressed in frog oocytes at rt using previously published protocols. Compounds were inactive
in the absence of direct agonists, generally nicotine at an EC concentration (concentration that
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evokes 5% of the maximum nicotine response (Imax)), confirming that the compounds are
allosteric modulators. Acetylcholine (ACh) and choline responses were also enhanced in the
presence of the modulators. Concentrationꢀresponse curves were generated for the compounds in
Tables 1, 2, 3 and 4 and the maximum modulation (generally at 10 ꢀM or the limit of solubility)
and EC (concentration at which half the maximum modulation is observed) values determined.
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arylpryidinꢀ3ꢀylmethanones. The unsubstituted 2ꢀamino compound 7p was active as were
compounds incorporating methyl, ethyl or propyl groups as in 7b, q, r and 7a. Increasing the size
of the 2ꢀalkylamino group generally led to increases in potency with the ethyl and propylamines
showing 15ꢀ and 18ꢀfold improvements over the unsubstituted amino group (7p) in EC ,
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respectively. Attempted substitution with larger 2ꢀalkylamines, including 3ꢀpentyl (7c), 2,5ꢀ
difluorobenzylꢀ (7d) and phenethylamine (7e) gave inactive compounds. The tertiary amine 7i
was also found to be inactive.
Table 1. In vitro activity of [2ꢀalkylaminoꢀ5ꢀ(4ꢀethoxyphenyl)pyridinꢀ3ꢀyl]arylmethanones in
a
oocytes expressing human α7 nAChRs.
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Compound
R1
R2
EC (ꢀM) Max. mod. (% at 10 ꢀM)
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7
7
p
b
H
Cl
F
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Me
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r
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phenethyl
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i
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Data are from 2ꢀ5 individual oocytes treated with a nicotine EC (concentration that evokes 5% of the maximum
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nicotine response). The EC50 is the drug concentration that gives half of the maximum modulation (max mod). 95%
Confidence intervals are given in supplementary information, ND = Not determined, IA = inactive
Modifications to the substitution on the benzoyl group were made within the 5ꢀ(4ꢀethoxyphenyl)ꢀ
1
7
2ꢀpropylaminopyridine series using a Topliss tree approach . In vitro data are given in Table 2.
Moving the 4ꢀchlorine in 7a to the 2ꢀposition gave compound 7s which was poorly active as a
modulator of α7 nAChRs with maximum modulation at 10 ꢀM of only 35%. The 2ꢀmethyl
compound (7f) was found to be inactive. Similarly, 2,4ꢀdichloro substitution as in 7t gave poor
maximum modulation of 40%. The isomeric 2ꢀ, 3ꢀ and 4ꢀfluorobenzoyl compounds were
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synthesized and tested for activity. While the 3ꢀ and 4ꢀfluoro groups (7h and 7g) were active and
exhibited similar potencies and maximum modulations, moving the fluorine atom to the 2ꢀ
position dramatically reduced activity (7u). Adding a 3ꢀfluorine to 7g gave the 3,4ꢀdifluoride 7v
which exhibited excellent activity with maximum modulation of 1200% and EC50 = 0.18 ꢀM.
The isomeric 2,5ꢀdifluoride, 7w, was poorly active with maximum modulation of only 200% and
an EC = 3 ꢀM. Substitutions with fluorine at the 3ꢀ and 5ꢀpositions gave compound 7x with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
poor maximum modulation of 130%. With no substitution on the benzoyl group as in 7y, potent
modulation was observed (EC = 0.21 ꢀM) with modest maximum modulation (300%).
5
0
Table 2. In vitro activity of aryl[5ꢀ(4ꢀethoxyphenyl)ꢀ2ꢀ(propylamino)pyridinꢀ3ꢀyl]methanones 7
a
in oocytes expressing human α7 nAChR.
Compound
R2
EC (ꢀM)
Max. mod. (%, 10 ꢀM)
50
7
a
4ꢀCl
2ꢀCl
0.14
ND
600
35
7s
7
f
2ꢀMe
2,4ꢀdiCl
4ꢀF
ND
IA
7t
ND
40
7
g
0.13
0.45
600
700
7
h
3ꢀF
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7
u
2ꢀF
3,4ꢀdiF
2,5ꢀdiF
3,5ꢀdiF
H
ND
0.18
3.0
75
1200
200
130
300
7v
7
w
10
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54
55
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57
58
59
60
7
7
x
y
ND
0.21
a. See footnote to Table 1. ND = Not determined, IA = inactive
Next, modifications to the 4ꢀethoxy group were made in order to explore the role of this group in
the activity of the PAMs (Table 3). Replacement of the 4ꢀethoxy in 7a with a 4ꢀchloride (7j)
resulted in essentially complete loss of activity. Within the 4ꢀfluorobenzoyl series, activity was
the best for 4ꢀethoxy group as in 7g while replacement of the 4ꢀethoxy group with
trifluoromethoxy, propyl, phenoxy or ethylthio groups abolished activity (7l, m, n and o,
respectively). It was found, however, that the ethoxy group could be replaced with a 4ꢀfluorine as
in 7k, resulting in good potency (EC = 0.22 ꢀM) but with a 2ꢀfold loss in maximum efficacy
5
0
compared to 7a.
Table 3. In vitro activity of aryl[5ꢀarylꢀ2ꢀ(propylamino)pyridinꢀ3ꢀyl]methanones 7 in oocytes
a
expressing human α7 nAChRs.
O
NHPr
N
R2
R3
7
Compound
R2
R3
EC (ꢀM)
Max. mod. (%, 10 ꢀM)
50
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5
5
5
5
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7
a
Cl
Cl
F
OEt
Cl
0.14
ND
600
40
7j
7g
OEt
OCF
Pr
0.13
ND
600
55
0
1
2
3
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7
8
9
0
1
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7
8
9
0
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2
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9
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9
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7
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9
0
7
l
F
3
7m
F
ND
30
7n
F
OPh
SEt
F
0.56
ND
30
7o
F
40
7k
F
0.22
300
a. See footnote to Table 1, ND = not determined.
Based on the compounds presented in Tables 1, 2 and 3, 7v emerged as a compound with both
potent activity as a modulator of α7 nAChRs (EC50 = 0.18 ꢀM) and excellent maximum
modulation (1200%). Close analogs were prepared to examine the effect of changes in the 2ꢀ
alkylamino group on the activity of this compound. In vitro data are given in Table 4. The
methyl and ethyl analogs, 7z and 7aa, showed large losses in potency compared to 7v and were
less efficacious with maximum modulations of 770 and 470%, respectively.
Table 4. In vitro activity of [2ꢀalkylaminoꢀ5ꢀ(4ꢀethoxyphenyl)pyridinꢀ3ꢀyl](3,4ꢀdifluorophenyl)ꢀ
methanones (7v, z and aa) in
Xenopus oocytes expressing human
a
α7 nAChRs.
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Compound
R
1
EC (ꢀM)
Max. mod. (%, 10 ꢀM)
50
7z
Me
Et
1.2
4.9
770
470
7aa
10
11
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57
58
59
60
7v
Pr
0.18
1200
a. See footnote to Table 1.
Unfortunately, initial testing in oocytes indicated that 7z was more potent as a modulator of α7
nAChRs than 7v leading to the fuller characterization of 7z in vitro before additional oocyte data
gave the relative EC ’s reported in Table 4. Compound 7z at its EC concentration of 1 ꢀM
5
0
50
induced a leftward shift in the concentrationꢀresponse curve for ACh as shown in Figure 4. The
EC value for ACh was reduced from 206 ꢀM in the absence of 7z to 123 ꢀM in the presence of
5
0
the modulator, although the change was not significant. At all concentrations of ACh tested, 7z
significantly increased modulation of control currents. At 10 ꢀM, 7z did not significantly inhibit
the binding of the α7 nAChR antagonist αꢀbungarotoxin (percent inhibition 5 ± 5, n = 2),
consistent with its action as a PAM of α7 nAChRs. Compound 7z was tested for selectivity
among nAChRs and for activity at α β γ GABA Rs (Table 5). The compound had little
1
2 2L
A
activity at α β and α β nAChRs and unlike the enaminone amides and esters from which it was
4
2
3 4
derived, poor activity was observed with α β γ GABA Rs.
1
2 2L
A
Figure 4. Acetylcholine (ACh) concentrationꢀresponse curves in the presence and absence of an
EC concentration of compound 7z.
50
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5
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5
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9
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5
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7
8
9
0
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2
3
4
5
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7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. Activity of 7z at human α β nAChRs, rat α β nAChRs and human α β γ GABA Rs
4
2
3
4
1
2 2L
A
expressed in Xenopus oocyctes.
Receptor
α β nACh
Max. mod. (% at 10 ꢀM)
ꢀ2 ± 5
ꢀ11 ± 3
71 ± 12
4
2
α β nACh
3
4
α β γ GABA
1
2 2L
A
The pyridines were found to be Type I modulators that do not alter the rapid desensitization
kinetics of the native channel. (see Figure 5 for a representative current trace). Compound 7z did
not block the desensitization of the channel initiated by 100 ꢀM nicotine (see Figure 6). This
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behavior is in stark contrast to Type II modulators such as PNUꢀ120596 (Nꢀ(5ꢀchloroꢀ2,4ꢀ
1
8
9
dimethoxyphenyl)ꢀN ꢀ(5ꢀmethylꢀ3ꢀisoxazolyl)urea) that can reopen the desensitized channel.
Figure 5. Modulation of nicotine EC evoked currents in Xenopus oocytes expressing human α7
5
10
11
12
13
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22
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49
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59
60
nAChRs with increasing concentrations of 7z.
Figure 6. Current trace of 1ꢁM 7z in the presence of 100 ꢀM nicotine showing no reversal of
desensitization
In vivo Pharmacology
As mentioned previously, initial in vitro studies had overꢀestimated the potency of 7z leading to
its testing in vitro and in vivo. A pharmacokinetic (PK) study with compound 7z was undertaken
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5
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to establish that the compound was able to cross the bloodꢀbrain barrier (BBB) after
intraperitoneal (i.p.) administration. When given to CD1 mice, a 3 mg/kg (8.1 ꢀmol/kg) i.p. dose
of 7z in 80% PEG 400/20% saline gave brain levels at 30, 60 and 120 min of 0.64 (± 0.03) ꢂM,
0.26 (± 0.04) ꢂM and 0.012 (± 0.007) ꢂM, respectively. The corresponding plasma levels were
found to be 1.75 (± 0.14) ꢂM, 0.34 (± 0.07) ꢂM and 0.06 (± 0.02) ꢂM. With confirmation of
exposure to the CNS, compound 7z was tested for activity in the novel object recognition (NOR)
0
1
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0
19
paradigm. CD1 mice were dosed with 0.7 mg/kg (2.1 ꢀmol/kg) subcutaneous (s.c.)
scopolamine hydrochloride to disrupt their ability to distinguish between a novel and a familiar
object. A dose response of 7z (formulated in 80% PEG 400/20% saline) is shown in Figure 7
where doses greater than 0.3 mg/kg (0.81 ꢀmol/kg) reversed the effect of scopolamine. The lack
of a change in activity for the active doses can be explained by the nature of the NOR paradigm
where activity shows a threshold phenomenon. The effect of 3 mg/kg (8.1 ꢀmol/kg) 7z was
reversed by 3 mg/kg (3.4 ꢀmol/kg) MLA.
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***
8
0
0
*
*
**
**
6
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54
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56
57
58
59
60
40
20
0
N = 40
N = 8
N = 8
N = 16
N = 8
N = 8
Figure 7. Effect of 7z (0.3 to 10 mg/kg, 0.81 to 27 ꢀmol/kg) i.p. in 80% PEG 400/20% saline on
the % time spent with a novel object (% novel time) in the mouse (CDꢀ1) NOR paradigm in the
presence of 0.7 mg/kg (2.1 ꢀmol/kg) s.c. scopolamine hydrochloride (Scop) versus those treated
with drug, vehicle and Scop alone. All animal groups received Scop. Significant Oneꢀway ANOVA
P<0.0001. Post Hoc Dunnet’s Multiple Comparison Test **P<0.01. The effect of 3 mg/kg (8.1
ꢀ
mol/kg) 7z is reversed by 3 mg/kg (3.4 ꢀmol/kg) MLA (far right bar). Post Hoc Bonferroni’s
Multiple Comparison Test ***P<0.001.
Discussion
Conformational restriction of the presumed active isomer of the enaminone amide 1c resulted in
a series of arylpyridꢀ3ꢀylmethanones that maintained activity as Type I PAMs of human α7
nAChRs. Unlike 1c, however, the methanones were poorly active at α β γ GABA Rs. In
1
2 2L
A
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2
2
2
2
2
2
2
2
3
3
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3
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3
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
addition, they were inactive at α β and α β nAChRs at 10 ꢀM. Three areas of the pyridine
4
2
3 4
structure were varied as part of SAR studies: (1) changes to the 2ꢀamino group (2) changes in
substitutions to the 3ꢀbenzoyl group and (3) changes in substitutions to the 5ꢀaryl group. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
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3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
potency of the pyridines as PAMs generally increased as the 2ꢀamino substitutient (R ) increased
1
in size from H (7p, EC = 2.5 ꢀM) to Me (7q, EC = 0.38 ꢀM), Et (7r, EC = 0.16 ꢀM) and Pr
5
0
50
50
(
7a, EC = 0.14 ꢀM). This trend was also observed within the series with R = 4ꢀF and 3,4ꢀdiF
5
0
2
where activity improved from R = Me (7b and 7z, EC = 1.9 ꢀM and 1.2 ꢀM, respectively) to
1
50
R = Pr (7g and 7v, EC = 0.13 ꢀM and 0.18 ꢀM, respectively). With Et substitution as in 7aa, a
1
50
4ꢀfold loss in potency was observed compared to Me substitution (7z). Improvements in the
maximum modulation measured were striking for 7q (1200%) versus compounds with small
alkyl group substitution that generally showed maximum modulations of 500ꢀ700% (Table 1). A
limit on the size of R was noted in the loss of activity for R groups larger than propyl, including
1
1
3ꢀpentyl, 2,5ꢀdifluorobenzyl and phenethyl (7c, 7d and 7e). The tertiary amine 7i was also
devoid of activity, indicating either that 7i is unable to act as a Hꢀbond donor that is required for
activity or the Nꢀmethylpropylamino group forces the benzoyl group further out of the plane of
the pyridine ring resulting in poor activity.
Changes to the 3ꢀbenzoyl group indicated that ortho substitution (R = 2ꢀF, 2ꢀMe, 2ꢀCl and 2,4ꢀ
2
diCl as in 7u, 7f, 7s and 7t) is not accommodated by the α7 nAChR. In the case where the ortho
substituent is Me or Cl, this is likely a steric effect that twists the phenyl ring of the benzoyl
group further out of the plane of the pyridine ring. With 2ꢀfluorine substitution, the strong CꢀF
dipole can orient to reduce the overall dipole of the molecule by at least partially opposing the
dipole of the carbonyl group. This change in conformation would be less important in the 3ꢀ and
4ꢀfluorobenzoyl groups (7h and 7g). This effect of fluorine position on conformational
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preference has been noted in the isomeric fluoroacetophenones where the 2’ꢀisomer has a large
barrier for rotation about the phenylꢀcarbonyl bond and the molecule exists almost completely as
the sꢀtrans conformer (dipoles of the CꢀF and carbonyl bond opposed to each other). With the 3’ꢀ
and 4’ꢀfluoroacetophenones free rotation about the arylꢀcarbonyl bond is observed and the
10
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2
0
molecules exist in both the sꢀcis and sꢀtrans conformers . In order to better understand the effect
of the 2ꢀfluorobenzoyl group on the α7 nAChR activity of 7u, a comparison with the
unsubstituted benzoyl compound 7y and the 3,4ꢀdifluoro analog 7z was made using
conformational analysis. Rotation about the bond between the phenyl group and carbonyl carbon
of the benzoyl group (ψ in Figure 8) confirmed that the barrier to rotation is much larger for 7u
than for 7y or 7z (see supporting material). The preferred conformation for activity at the α7
nAChR is likely one that is somewhat removed from the minima found in the conformational
analysis. 7y and 7z can more easily access this active conformation because its energy is closer
to the energy of the lowest energy conformer, while 7u cannot.
Figure 8. Dihedral angle (ψ) varied in conformational analysis
In the isomeric difluoro compounds, 3,4ꢀsubstitution as in 7v resulted in the most potent and
efficacious compound tested (EC = 0.18 ꢀM, maximum modulation = 1200%) while the 2,5ꢀ
5
0
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2
2
2
2
2
2
2
2
2
3
3
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3
3
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3
3
3
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and 3,5ꢀisomers were less active (7w and 7x). The improvement in activity in 7v compared to the
2
1
3
ꢀ and 4ꢀmonofluoro analogs may involve a specific Hꢀbonding interaction . The poor activity
for the 2,5ꢀisomer may involve similar dipole interactions as noted with mono fluorination at the
ꢀposition.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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0
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0
1
2
3
4
5
6
7
8
9
0
2
Modifications to the 5ꢀaryl group showed that 4ꢀethoxy substitution gave the most efficacious
compounds. Replacing the ethoxy group with other groups including trifluoromethoxy, propyl,
phenoxy and ethylthio abolished activity. The only other compound that showed similar potency
to the 4ꢀethoxy group (7g) was the 4ꢀfluoro (7k) but exhibited a 2ꢀfold loss in efficacy. It is clear
from these data that the size of the ethoxy group is not the deciding factor in its activity because
the propyl analog (7m) is inactive. It is likely that the ether oxygen is acting as a Hꢀbond
acceptor. This may also explain the activity of the 4ꢀfluoro substituted analog (7k) that may also
2
1
benefit from a Hꢀbonding interaction with the receptor .
Based on these SAR studies and PK studies that showed that compound 7z crossed the BBB, 7z
was tested for activity in the NOR paradigm after i.p. dosing. Enhancement of NOR performance
was observed with a minimum effective dose i.p. of 1.0 mg/kg (2.7 ꢀmol/kg). This effect was
reversed by the selective α7 nAChR antagonist MLA, which is consistent with activity mediated
by α7 nAChRs.
Conclusions
A series of substituted arylpyridꢀ3ꢀylmethanones (7) were designed from enaminone esters and
9
amides (1aꢀc) that had been described as allosteric modulators of both GABA and α7 nAChRs.
A
The methanones were found to be Type I PAMs at α7 nAChRs that maintain the native kinetics
of the receptor and unlike Type II PAMs do not reverse receptor desensitization. The compounds
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were poorly active at α β γ GABA Rs and inactive at α β and α β nAChRs. SAR studies
1
2 2L
A
4
2
3 4
resulted in the synthesis of compound 7v that exhibited potent and efficacious modulation of
nicotine EC responses (EC50 = 0.18 ꢀM, max. mod. = 1200%). Compound 7z was found to
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penetrate the CNS after i.p. dosing in mice and was active in the NOR paradigm with a minimum
effective dose i.p. of 1.0 mg/kg (2.7 ꢀmol/kg). Additional studies are underway to more fully
characterize the activity of these compounds in other animal models relevant to the treatment of
neuropsychiatric disorders including schizophrenia, ADHD and Alzheimer’s disease.
Experimental Section
1
13
Chemistry. H and C NMR spectra were recorded on a Varian Unity Inova spectrometer at 400
and 100 MHz, respectively, or with a Bruker 400 or 500 MHz NMR in CDCl referenced to
3
CHCl (7.26 ppm) or to tetramethylsilane (0.00 ppm) and to CDCl at 77.00 ppm or DMSOꢀd
6
3
3
referenced to DMSOꢀd (2.50 ppm). Melting points were determined on an Electrothermal MELꢀ
5
TEMP 3.0 apparatus (Barnstead International, Dubuque, IA) and are not corrected. Compounds
were determined to have purity >95% using elemental analysis performed by Robertson Microlit
Laboratories, Madison, NJ and are within 0.4% of theoretical values unless otherwise noted (see
22
supporting information). Flash chromatography was carried out by using the method of Still et
al., on 230ꢀ400 mesh silica gel (silica gel 60, Geduran) obtained from EMD. Solvents were
HPLC grade and were obtained from EMD. DMSO, nicotine, GABA, scopolamine
hydrochloride (Scop) and anhydrous solvents were obtained from Aldrich Chemical Co.
(Milwaukee, WI). Methyllycaconitine citrate (MLA) was obtained from Tocris Biosciences
(Bristol, UK). Reactions were carried out under an atmosphere of dry nitrogen gas. Inhibition of
αꢀbungarotoxin binding by 7z was carried out by CEREP (Redmond, WA).
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Method
A:
Synthesis
of
(4ꢀchlorophenyl)[5ꢀ(4ꢀethoxyphenyl)ꢀ2ꢀpropylaminoꢀ3ꢀ
pyridinyl]methanone (7a) starting from 4ꢀethoxyphenylacetic acid.
αꢀ[(Dimethylamino)methylene]ꢀ4ꢀethoxybenzeneacetaldehyde (3a). Phosphorus oxychloride
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o
(6.4 mL) was slowly added to DMF (8 mL) at 0 C. To the mixture was added 4ꢀ
o
ethoxyphenylacetic acid (Acros; 4.53 g; 25.1 mmol) and the reaction was heated at 75 C for 6 h.
After cooling to rt the reaction was added to iceꢀwater and brought to pH 11 with 13.0 g of solid
o
NaOH. After heating at 100 C for 1h, the mixture was allowed to cool to rt. The resulting solid
was collected by filtration, washed with cold water and air dried. The crude product was treated
with 100 mL of CH Cl , filtered to remove insoluble material, dried (MgSO ), filtered and conc.
2
2
4
to dryness. The residue was dissolved with heating in 100% EtOAc and subjected to flash
chromatography (elution with 100% EtOAc) to give 3.57 g (65% yield) of the title compound as
o
+
+
1
a light yellow solid, mp 101ꢀ102 C. TOF MS ES m/z 220 (M + H ). H NMR (500 MHz,
CDCl ) δ 9.08 (s, 1H), 7.08 (d, 2H, J = 8.5 Hz), 6.87 (d, 2H, J = 8.5 Hz), 6.76 (br s, 1H), 4.03 (q,
3
2H, J = 6.8 Hz), 2.83 (br s, 6H), 1.41 (t, 3H, J = 7.0 Hz).
1,2ꢀDihydroꢀ5ꢀ(4ꢀethoxyphenyl)ꢀ2ꢀoxoꢀ3ꢀpyridinecarbonitrile (4a). Sodium metal (0.70 g, 30
mmol) was added to 50 mL of MeOH and the resulting solution was treated with
cyanoacetamide (1.26 g, 15 mmol). The mixture was stirred at rt for 1 h, and solid αꢀ
[
(dimethylamino)methylene]ꢀ4ꢀethoxybenzeneacetaldehyde (2.50 g, 11.0 mmol) was added. The
mixture was heated for 4 h at reflux and allowed to cool to rt. The resulting yellow precipitate
was collected and washed with cold MeOH. The solid was then dissolved in hot water (150
mL), filtered, and the filtrate was acidified with a 1N aqueous HCl solution. The resulting white
solid was collected, washed with cold water, and dried, affording 1.90 g (70% yield) of 4a. TOF
+
1
MS ES m/z 241, 263. H NMR (400 MHz, DMSOꢀd ) δ 12.40 (s, 1H), 8.47 (s, 1H), 7.99 (s,
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H), 7.49 (d, 2H, J = 8.5 Hz), 6.91 (d, 2H, J = 8.5 Hz), 4.00 (q, 2H, J = 7.0 Hz), 1.28 (t, 3H, J =
1
23
7
.0 Hz). H NMR matches lit data .
2ꢀChloroꢀ5ꢀ(4ꢀethoxyphenyl)ꢀ3ꢀpyridinecarbonitrile (5a). 4a (515 mg, 2.14 mmol) was
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o
heated in phosphorus oxychloride (3 mL) at 120ꢀ130 C overnight. The resulting solution was
evaporated to dryness and the residue was treated with toluene and conc. in vacuo. This material
was partitioned between a sat. aq. NaHCO solution and CH Cl . The aqueous layer was washed
3
2
2
twice with CH Cl and the pooled organic layers were extracted with a sat. aq. NaHCO solution
2
2
3
and brine. The emulsion that formed was filtered through Celite. The CH Cl layer was then
2
2
dried (MgSO ), filtered and conc. to dryness. The solid that formed was dissolved in CH Cl and
4
2
2
subjected to flash chromatography. Elution with CH Cl gave 340 mg (61% yield) of 5a as a
2
2
o
+
1
white solid, mp 162ꢀ162.5 C. TOF MS ES m/z 259, 261. H NMR (500 MHz, CDCl ) δ 8.75 (d,
3
1
H, J = 2.5 Hz), 8.11 (d, 1H, J = 2.5 Hz), 7.47 (d, 2H, J = 9.0 Hz), 7.02 (d, 2H, J = 8.5 Hz), 4.10
(q, 2H, J = 7.0 Hz), 1.46 (t, 3H, J = 7.0 Hz).
5ꢀ(4ꢀEthoxyphenyl)ꢀ2ꢀpropylaminoꢀ3ꢀpyridinecarbonitrile (6a). A solution of 2ꢀchloroꢀ5ꢀ(4ꢀ
ethoxyphenyl)ꢀ3ꢀpyridinecarbonitrile (0.30 g 1.16 mmol) in 3 mL of DMSO was treated with
o
neat propylamine (0.47 mL, 5.8 mmol). The mixture was heated at 60ꢀ70 C overnight and then
poured into iceꢀwater. The resulting precipitate was collected, washed with cold water, and dried
o
+
to give 0.28 g (90% yield) of 6a as a light yellow solid with mp 103.6ꢀ104.6 C. TOF MS ES
1
m/z 282. H NMR (500 MHz, CDCl ) δ 8.48 (d, 1H, J = 2.5 Hz), 7.80 (d, 1H, J = 2.5 Hz), 7.34
3
(
d, 2H, J = 8.5 Hz), 6.96 (d, 2H, J = 8.5 Hz), 5.18 (br s, 1H), 4.07 (q, 2H, J = 7.0 Hz), 3.50 (q,
2
H, J = 6.7 Hz), 1.44 (t, 3H, J = 7.0 Hz), 1.02 (t, 3H, J = 7.2 Hz).
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(
4ꢀChlorophenyl)[5ꢀ(4ꢀethoxyphenyl)ꢀ2ꢀpropylaminoꢀ3ꢀpyridinyl]methanone
(7a).
A
solution of 6a (56 mg, 0.20 mmol) in THF was treated with a solution of 4ꢀ
chlorophenylmagnesium bromide (1 mL of 1M solution in ether; 1.0 mmol) giving a yellow
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precipitate. After heating for 20 h at 70 C, the resulting solid was allowed to cool and then
treated with 3 mL of an aqueous 6N HCl solution. After heating at reflux for 30 min, the reaction
was diluted with water, neutralized with solid K CO (0.5 g) and extracted with EtOAc (3 x 5
2
3
mL). The organic layers were pooled, dried (Na SO ), and concentrated. Purification by flash
2
4
chromatography eluting with CH Cl , followed by crystallization from MeOH gave 7a (10 mg,
2
2
+
1
1
2% yield) as a yellow solid. TOF MS ES m/z 395, 397. H NMR (CDCl , 500 MHz) δ 8.70 (t,
3
J = 5.1 Hz, 1H) 8.50 (s, 1H), 7.77 (s, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H), 7.24
(d, J = 8.9 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 3.98 (q, J = 7.0 Hz 2H), 3.52 (q, J = 7.0 Hz, 2H),
13
1
1
1
1
.68 (sxt, J = 7.2 Hz, 2H), 1.35 (t, J = 7.0 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (CDCl3,
25 MHz) 196.82 (1C), 158.32 (1C), 157.98 (1C), 152.71 (1C), 140.68 (1C), 137.49 (1C),
37.12 (1C), 130.49 (2C), 129.87 (1C), 128.11 (2C), 127.11 (2C), 123.40 (1C), 115.09 (2C),
11.58 (1C), 63.61 (1C), 42.99 (1C), 22.80 (1C), 14.89 (1C), 11.76 (1C) ppm. Anal.
(C H ClN O ) C, H, N.
2
3
23
2
2
Compounds 7bꢀk were prepared by using Method A and the appropriate starting
arylacetic acids, alkylamines and arylmagnesium halides:
[5ꢀ(4ꢀEthoxyphenyl)ꢀ2ꢀmethylaminoꢀ3ꢀpyridinyl](4ꢀfluorophenyl)methanone (7b). The title
compound was prepared by using the procedure described for the synthesis of 7a except that 4ꢀ
chlorophenylmagnesium bromide was replaced with 4ꢀfluorophenylmagnesium bromide and
+
1
propylamine was replaced with methylamine. TOF MS ES m/z 351, 352. H NMR (CDCl , 500
3
MHz) δ 8.60 (m, 2H), 7.86 (s, 1H), 7.66 (m, 2H), 7.32 (d, J = 8.8 Hz 2H), 7.16 (t, J = 8.6 Hz,
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H), 6.92 (d, J = 8.7 Hz, 2H), 4.03 (q, J = 7.0 Hz, 2H), 3.16 (d, J = 4.9 Hz 3H), 1.42 (t, J = 7.0
13
Hz, 3H). C NMR (CDCl , 125 MHz) 196.68 (1C), 165.70 (1C), 163.69 (1C), 158.36 (1C),
3
1
52.37 (1C), 140.63 (1C), 135.61 (1C), 131.60 (1C), 131.53 (1C), 129.96 (1C), 127.15 (2C),
23.47 (1C), 115.69 (1C), 115.52 (1C), 115.09 (2C), 112.19 (1C), 63.61 (1C), 28.09 (1C), 14.89
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1
(1C) ppm. Anal. (C H FN O ) C, H, N.
2
1
19
2
2
(4ꢀChlorophenyl)[5ꢀ(4ꢀethoxyphenyl)ꢀ2ꢀ[(1ꢀethylpropyl)amino]ꢀ3ꢀpyridinyl]methanone (7c).
7c was prepared as described above for the synthesis of 7a except that propylamine was replaced
1
with 1ꢀethylpropylamine. H NMR (CDCl , 500 MHz) δ 8.64 (t, J = 8.2 Hz, 1H) 8.47 (s, 1H),
3
7.76 (s, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 6.84 (d,
J = 8.7 Hz, 2H), 4.23 (sxt, J = 6.1 Hz 1H), 3.97 (q, J = 7.1 Hz, 2H), 1.64 (p, J = 7.1 Hz, 2H),
1
3
1.55 (p, J = 7.1 Hz, 2H), 1.35 (t, J = 7.0 Hz, 3H), 0.91 (t, J = 7.4 Hz, 6H). C NMR (CDCl , 125
3
MHz) 197.40 (1C), 158.20 (1C), 152.91 (1C), 140.79 (1C), 137.97 (1C), 137.63 (1C), 130.51
(2C), 130.44 (1C), 130.25 (1C), 130.05 (1C), 128.72 (2C), 127.06 (2C), 124.26 (1C), 123.09
(1C), 115.07 (2C), 63.60 (1C), 52.82 (1C), 27.05 (1C), 17.68 (1C), 14.89 (1C), 10.25 (1C) ppm.
Anal. (C H ClN O ) C, H, N.
2
5
27
2
2
(4ꢀChlorophenyl)[2ꢀ[(2,5ꢀdifluorobenzyl)amino]ꢀ5ꢀ(4ꢀethoxyphenyl)ꢀ3ꢀpyridinyl]methanone
(7d). 7d was prepared as described above for the synthesis of 7a except that propylamine was
replaced with 2,5ꢀdifluorobenzylamine. The compound was isolated as a yellow solid with mp
o
1
1
40.5ꢀ143 C. H NMR (CDCl , 500 MHz) δ 9.08 (t, J = 5.9 Hz, 1H), 8.63 (s, 1H), 8.41 (s, 1H),
3
7.67 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.20 (ddd, J = 3.1,
5
6
1
.3, 8.8 Hz, 1H), 7.08 (sxt, J = 4.4 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 6.97 (m, 1H), 4.96 (d, J =
1
3
.0 Hz, 2H), 4.11 (q, J = 7.0 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H).
C NMR (CDCl , 125 MHz)
3
96.88 (1C), 159.87 (1C), 158.48 (1C), 157.52 (1C), 156.33 (1C), 155.21 (1C), 152.43 (1C),
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40.61 (1C), 137.99 (1C), 137.54 (1C), 130.59 (2C), 129.67 (1C), 128.81 (2C), 127.71 (1C),
27.24 (2C), 124.59 (1C), 116.42 (1C), 115.12 (2C), 114.75 (1C), 112.16 (1C), 63.61 (1C),
8.51 (1C), 14.89 (1C) ppm. Anal. (C H F N O ) C, H, N.
2
7
21
2
2
2
0
1
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(4ꢀChlorophenyl)[5ꢀ(4ꢀethoxyphenyl)ꢀ2ꢀ(phenethylamino)ꢀ3ꢀpyridinyl]methanone (7e). 7e
was prepared as described above for the synthesis of 7a except that propylamine was replaced
1
with phenethylamine. H NMR (CDCl , 500 MHz) δ 8.70 (t, J = 5.0 Hz, 1H) 8.57 (s, 1H), 7.85
3
(s, 1H), 7.65 (m, 2H), 7.32 (m, 5H), 7.22 (m, 2H), 7.16 (t, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz,
2H), 4.05 (q, J = 7.0 Hz 2H), 3.88 (q, J = 7.0 Hz, 2H), 3.02 (t, J = 7.2 Hz, 2H), 1.42 (t, J = 7.0
13
Hz, 3H). C NMR (CDCl , 125 MHz) 196.55 (1C), 165.62 (1C), 163.61 (1C), 158.29 (1C),
3
1
57.71 (1C), 152.31 (1C), 140.54 (1C), 139.46 (1C), 135.55 (1C), 131.52 (1C), 129.91 (1C),
128.84 (2C), 128.52 (2C), 127.09 (2C), 126.34 (1C), 123.59 (1C), 115.61 (1C), 115.44 (1C),
15.04 (2C), 111.96 (1C), 63.56 (1C), 42.69 (1C), 35.87 (1C), 14.83 (1C) ppm. Anal.
C H FN O ) C, H, N.
1
(
2
8
25
2
2
[5ꢀ(4ꢀEthoxyphenyl)ꢀ2ꢀpropylaminoꢀ3ꢀpyridinyl](2ꢀmethylphenyl)methanone (7f). 7f was
prepared as described above for the synthesis of 7a except that 2ꢀchlorophenylmagnesium
1
chloride was replaced with 2ꢀtolylmagnesium chloride. H NMR (CDCl , 500 MHz) δ 9.14 (t, J
3
= 4.9 Hz, 1H), 8.54 (s, 1H), 7.63 (s, 1H), 7.36 (t, J = 7.0 Hz, 1H), 7.28 (d, J = 7.6 Hz 2H), 7.25
(m, 3H), 6.88 (d, J = 8.8 Hz, 2H), 4.02 (q, J = 7.0 Hz, 2H), 3.62 (q, J = 7.0 Hz 2H), 2.29 (s, 3H),
1
3
1.77 (sxt, J = 7.3 Hz, 2H), 1.40 (t, J = 7.0 Hz, 3H), 1.07 (t, J = 7.4 Hz, 3H). C NMR (CDCl ,
3
125 MHz) 200.36 (1C), 158.20 (1C), 157.84 (1C), 152.94 (1C), 141.24 (1C), 139.51 (1C),
1
35.30 (1C), 130.82 (1C), 129.92 (1C), 129.59 (1C), 127.29 (1C), 127.10 (2C), 125.42 (1C),
23.58 (1C), 114.96 (2C), 112.49 (1C), 63.53 (1C), 42.91 (1C), 22.77 (1C), 19.59 (1C), 14.82
1
(1C), 11.73 (1C) ppm. Anal. (C H N O ) C, H, N.
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[
5ꢀ(4ꢀEthoxyphenyl)ꢀ2ꢀpropylaminoꢀ3ꢀpyridinyl](4ꢀfluorophenyl)methanone (7g). 7g was
prepared by using the procedure described for the synthesis of 7a except that 4ꢀ
chlorophenylmagnesium bromide was replaced with 4ꢀfluorophenylmagnesium bromide. The
10
11
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o
+
1
compound was isolated as a yellow solid with mp 104.2ꢀ104.7 C. TOF MS ES 379. H NMR
(CDCl , 500 MHz) δ 8.72 (t, J = 5.0 Hz, 1H) 8.56 (s, 1H), 7.86 (s, 1H), 7.66 (m, 2H), 7.33 (d, J
3
=
8.8 Hz, 2H), 7.17 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 4.04 (q, J = 7.0 Hz 2H), 3.59
1
3
(
q, J = 7.1 Hz, 2H), 1.74 (t, J = 7.3 Hz, 2H), 1.42 (t, J = 7.0 Hz, 3H), 1.06 (t, J = 7.4 Hz, 3H). C
NMR (CDCl , 125 MHz) 196.64 (1C), 165.62 (1C), 163.61 (1C), 158.25 (1C), 157.43 (1C),
3
1
52.43 (1C), 140.63 (1C), 135.63 (1C), 131.50 (1C), 129.96 (1C), 127.05 (2C), 123.29 (1C),
15.62 (1C), 115.45 (1C), 115.03 (2C), 111.71 (1C), 63.55 (1C), 42.94 (1C), 22.75 (1C), 14.83
1
(1C), 11.71 (1C) ppm. Anal. (C H FN O ) C, H, N.
2
3
23
2
2
[
5ꢀ(4ꢀEthoxyphenyl)ꢀ2ꢀpropylaminoꢀ3ꢀpyridinyl](3ꢀfluorophenyl)methanone (7h). 7h was
prepared by using the procedure described for the synthesis of 7a except that 4ꢀ
chlorophenylmagnesium bromide was replaced with 3ꢀfluorophenylmagnesium bromide. TOF
+
1
MS ES 379. H NMR (CDCl , 500 MHz) δ 8.72 (t, J = 5.0 Hz, 1H) 8.56 (s, 1H), 7.86 (s, 1H),
3
7
.65 (dd, J = 8.7, 5.5 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 7.17 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7
Hz, 2H), 4.05 (q, J = 7.0 Hz 2H), 3.59 (q, J = 7.1 Hz, 2H), 1.75 (t, J = 7.3 Hz, 2H), 1.42 (t, J =
13
7
.0 Hz, 3H), 1.06 (t, J = 7.4 Hz, 3H). C NMR (CDCl , 125 MHz) 196.73 (1C), 165.69 (1C),
3
163.68 (1C), 158.32 (1C), 157.99 (1C), 152.50 (1C), 140.71 (1C), 135.68 (1C), 131.57 (1C),
1
30.03 (1C), 127.12 (2C), 123.36 (1C), 115.69 (2C), 115.10 (2C), 111.78 (1C), 63.61 (1C),
3.00 (1C), 22.80 (1C), 14.89 (1C), 11.77 (1C) ppm. Anal. (C H FN O ꢀ½MeOH) C, H, N.
4
2
3
23
2
2
[
5ꢀ(4ꢀEthoxyphenyl)ꢀ2ꢀ(Nꢀmethylpropylamino)ꢀ3ꢀpyridinyl](4ꢀfluorophenyl)methanone
(
7i). 7i was prepared as described above for the synthesis of 7a except that 4ꢀ
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