Synthesis and pharmacological evaluation of α4β2 nicotinic ligands with a 3-fluoropyrrolidine nucleus

Article abstract of DOI:10.1002/cmdc.201500073

Abstract Nicotinic acetylcholine receptors (nAChRs) play an important role in many central nervous system disorders such as Alzheimer's and Parkinson's diseases, schizophrenia, and mood disorders. The α₄β₂ subtype has emerged as an important target for the early diagnosis and amelioration of Alzheimer's disease symptoms. Herein we report a new class of α₄β₂ receptor ligands characterized by a basic pyrrolidine nucleus, the basicity of which was properly decreased through the insertion of a fluorine atom at the 3-position, and a pyridine ring carrying at the 3-position substituents known to positively affect affinity and selectivity toward the α₄β₂ subtype. Derivatives 3-(((2S,4R)-4-fluoropyrrolidin-2-yl)methoxy)-5-(phenylethynyl)pyridine (11) and 3-((4-fluorophenyl)ethynyl)-5-(((2S,4R)-4-fluoropyrrolidin-2-yl)methoxy)pyridine (12) were found to be the most promising ligands identified in this study, showing good affinity and selectivity for the α₄β₂ subtype and physicochemical properties predictive of a relevant central nervous system penetration. Basicity tuned by F: By combining structural features of niodene and compound B, we developed new α₄β₂ receptor ligands characterized by the 3-fluoropyrrolidine nucleus. Derivatives (2S,4R)-11 and (2S,4R)-12 were highlighted as the most promising ligands identified in this study, showing good affinity and selectivity for the α₄β₂ subtype and physicochemical properties predictive of good central nervous system penetration.

Full text of DOI:10.1002/cmdc.201500073

DOI: 10.1002/cmdc.201500073
Full Papers
Synthesis and Pharmacological Evaluation of a4b2 Nicotinic
Ligands with a 3-Fluoropyrrolidine Nucleus
Lucia Tamborini,[a] Andrea Pinto,*[a] Roberta Ettari,[b] Cecilia Gotti,[c] Francesca Fasoli,[c]
Paola Conti,[a] and Carlo De Micheli[a]
Nicotinic acetylcholine receptors (nAChRs) play an important
role in many central nervous system disorders such as Alzheim-
er’s and Parkinson’s diseases, schizophrenia, and mood disor-
ders. The a4b2 subtype has emerged as an important target for
the early diagnosis and amelioration of Alzheimer’s disease
symptoms. Herein we report a new class of a4b2 receptor li-
gands characterized by a basic pyrrolidine nucleus, the basicity
of which was properly decreased through the insertion of a flu-
orine atom at the 3-position, and a pyridine ring carrying at
the 3-position substituents known to positively affect affinity
and selectivity toward the a4b2 subtype. Derivatives 3-(((2S,4R)-
4-fluoropyrrolidin-2-yl)methoxy)-5-(phenylethynyl)pyridine (11)
and 3-((4-fluorophenyl)ethynyl)-5-(((2S,4R)-4-fluoropyrrolidin-2-
yl)methoxy)pyridine (12) were found to be the most promising
ligands identified in this study, showing good affinity and se-
lectivity for the a4b2 subtype and physicochemical properties
predictive of a relevant central nervous system penetration.
Introduction
Nicotinic acetylcholine receptors (nAChRs) play an important
role in many central nervous system (CNS) disorders such as
Alzheimer’s disease (AD), Parkinson’s disease (PD), schizophre-
nia, mood disorders, and nicotine dependence. The predomi-
nant nAChRs in the CNS are the heteromeric a4b2 subtype and
the homomeric a7 subtype. The a3b4 nAChR subtype is the
predominant subtype in restricted brain areas and in several
autonomic nervous system ganglia. The a4b2 subtype, which in
brain constitutes about 90% of the high-affinity heteromeric
receptors, has emerged as an important target for the early di-
agnosis and amelioration of AD symptoms.[1]
basicity of the secondary amine is an efficient strategy to
obtain ligands with faster brain imaging kinetics, as is the case
for [123I]niodene,[11] in which a five-membered 2,5-dihydro-1H-
pyrrole ring was introduced in place of the azetidine ring of
the model compound 5-[123I]A-85380 (Figure 1). Inspired by
3-Pyridyl ether compounds (e.g., A-84543 and A-85380) are
potent nAChR ligands that possess sub-nanomolar affinity for
brain nAChRs and differentially activate subtypes of neuronal
nAChRs.[2] Based on the structure of the azetidine derivative,
the radiolabeled high-affinity a4b2 agonist 5-[123I]A-85380 has
been developed for noninvasive imaging of nAChRs, using
single-photon emission computed tomography (SPECT).[3–9] Un-
fortunately, 5-[123I]A-85380 is characterized by slow imaging ki-
netics with scan times exceeding several hours, which causes
patient discomfort.[10] It has been reported that decreasing the
[a] Dr. L. Tamborini, Dr. A. Pinto, Prof. P. Conti, Prof. C. De Micheli
Dipartimento di Scienze Farmaceutiche
Università degli Studi di Milano
Via Mangiagalli 25, 20133 Milano (Italy)
E-mail: andrea.pinto@unimi.it
[b] Dr. R. Ettari
Figure 1. Structure of model compounds and target compounds 1–8.
Dipartimento di Scienze del Farmaco e Prodotti per la Salute
Università degli Studi di Messina
Viale Annunziata 98168, Messina (Italy)
this work, we designed new niodene analogues characterized
by a basic pyrrolidine nucleus. To attain optimal pKa, which
guarantees a correct balance between protonated and non-
protonated forms, respectively necessary for receptor interac-
[c] Dr. C. Gotti, Dr. F. Fasoli
Dipartimento di Biotecnologie Mediche e Medicina Traslazionale
CNR, Istituto di Neuroscienze, Università degli Studi di Milano
Via Vanvitelli 32, 20129 Milan (Italy)
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tion and blood–brain barrier permeability, we investigated the
possibility of tuning the basicity of the amine by the insertion
of electron-withdrawing groups at the 3-position of the pyrroli-
dine ring (Figure 1). As reported by Morgenthaler et al., the in-
troduction of a fluorine atom at the b-position to an amine
should produce a pKa shift of ~1.7 units for each fluorine
atom.[12] Therefore, we designed compounds 1–6, structurally
related to model compounds A-85380, A-84543, and niodene,
in which the basic nucleus is represented by a secondary or
tertiary amino group installed into a pyrrolidine nucleus, which
was differently functionalized in order to tune its basicity; in
particular, we inserted one or two fluorine atoms at the 3-posi-
tion of the pyrrolidine nucleus (Figure 1).
Notably, the presence of a single fluorine atom at the 3-posi-
tion generated a second stereogenic center; consequently,
both stereoisomers were prepared. On the basis of preliminary
binding assays, derivative 5 was selected as a model com-
pound for an in-depth structure–activity relationship (SAR) in-
vestigation. Modifications with groups known to influence af-
finity and selectivity at the a4b2 subtype were introduced at
the 3-position of the pyridine ring to generate derivatives 7–
13. As reported for model compound A-85380,[13] we replaced
the 3-bromo function with an iodo group (compounds 7–8,
Figure 1), as in niodene, or with a phenyl, phenylacetylene or
5-hexyn-1-ol groups, as in model compounds A, B, and saze-
tine-A (compounds 9–13, Figure 2).
Scheme 1. Reagents and conditions: a) 1m BH3/THF, D, 4 h.
sponding alcohols (2S)-16, (2S,4S)-18, and (2S,4R)-21, respec-
tively (Scheme 1).
Mitsunobu coupling of the properly functionalized alcohols
(2S)-16, (2S,4S)-18, and (2S,4R)-21 with 3-bromo-5-pyridinol
provided the desired ethers (2S)-22, (2S,4S)-23, and (2S,4R)-24
in very good yields (83–92%; Scheme 2). Compounds (2S)-22,
Figure 2. Structure of model compounds and target compounds 9–13.
Results and Discussion
Scheme 2. Reagents and conditions: a) DIAD, PPh3, THF, 24 h; b) TFA, CH2Cl2,
4 h; c) HCHO (37 wt.% in H2O), NaBH3CN, CH3CN, 2 h.
Chemistry
Synthesis of the target derivatives was performed starting
from enantiomerically pure acids (2S)-15, (2S,4S)-17, and
(2S,4R)-20, which were conveniently prepared by starting from
protected trans-4-hydroxy-l-proline (2S,4R)-14 or cis-4-hydroxy-
(2S,4S)-23, and (2S,4R)-24 were treated with a 30% solution of
trifluoroacetic acid in dichloromethane to afford (2S)-1, (2S,4S)-
3, and (2S,4R)-5, respectively. Finally, the N-methylpyrrolidine
derivatives were prepared by reductive amination, using form-
aldehyde and sodium cyanoborohydride, of secondary amines
(2S)-1, (2S,4S)-3, and (2S,4R)-5 to give (2S)-2, (2S,4S)-4, and
(2S,4R)-6 in 34–80% yields, respectively (Scheme 2). Using the
l-proline
(2S,4S)-19,
following
reported
procedures
(Scheme 1).[14] Reduction of the carboxylic acid function of (2S)-
15, (2S,4S)-17, and (2S,4R)-20 with 1n BH3/THF gave the corre-
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same synthetic strategy, alcohol (2S,4R)-21 was submitted to
Mitsunobu reaction with 3-iodo-5-pyridinol to afford intermedi-
ate (2S,4R)-25, which was deprotected at the nitrogen atom to
give compound (2S,4R)-7, which, in turn, was converted into
the corresponding tertiary amine (2S,4R)-8, through reductive
amination (Scheme 2).
Biology
The binding affinities of all new derivatives were determined in
binding competition studies using rat cortical membranes la-
beled with [3H]epibatidine for the a4b2 receptors, and rat hip-
pocampal membranes labeled with [125I]a-bungarotoxin for the
a7 subtype.[15] The compounds were also tested for their affini-
ty at human a3b4 subunits transfected in HEK 293 cells, using
[3H]epibatidine as radioligand.[15] All the results are reported in
Table 1. Interestingly, all the compounds show negligible affini-
ty for a7 nAChRs. Whereas the difluoro derivatives (2S)-1 and
(2S)-2 show only micromolar affinity for both the a4b2 and a3b4
receptors, the monofluoro derivatives (2S,4R)-3–6 display nano-
molar affinities for the a4b2 receptor subtype and notable se-
lectivity (one or two orders of magnitude) over the a3b4 sub-
type. The modest affinity displayed by (2S)-1 and (2S)-2 could
be explained by the presence of two fluorine atoms on the
pyrrolidine ring which may not be well tolerated in
the binding pocket; moreover, these may decrease
the basicity of the amine too much (i.e., compound
(2S)-2, pKa =5.15). Conversely, the insertion of only
one fluorine atom seems to be better tolerated and
confers a pKa value in the range 6.98–8.80, which
guarantees a correct balance between the protonat-
ed and non-protonated forms, necessary for both re-
ceptor interaction and membrane permeability. Con-
sidering the first set of derivatives 1–6, derivative
(2S,4R)-5 displayed the best affinity/selectivity profile,
and therefore, it was selected for further SAR studies
involving modifications at the pyridine nucleus, gen-
erating compounds 7–13. Within this second set of
derivatives, (2S,4R)-11 and (2S,4R)-12 showed higher
Compounds (2S,4R)-9 and (2S,4R)-10 were synthesized by
applying the Suzuki reaction to the key intermediate (2S,4R)-24
and phenylboronic acid or 4-fluorophenylboronic acid, respec-
tively, followed by Boc deprotection (Scheme 3). On the other
hand, phenylacetylene, 4-fluorophenylacetylene, or 5-hexyn-1-
ol were added to key intermediate (2S,4R)-24 under Sonoga-
shira reaction conditions to obtain derivatives (2S,4R)-28,
(2S,4R)-29, and (2S,4R)-30, respectively, which were finally treat-
ed with a 30% solution of trifluoroacetic acid in dichloro-
methane to afford the desired compounds (2S)-11, (2S,4R)-12,
and (2S,4R)-13 (Scheme 3).
Scheme 3. Reagents and conditions: a) Pd(PPh3)4, 2m Na2CO3, ArB(OH)2, toluene/EtOH
(3:1), 908C, 18 h; b) CuI, Pd(PPh3)2Cl2, Et3N, 908C, 18 h; c) TFA, CH2Cl2, 4 h.
Table 1. Binding affinities, ligand efficiencies, and predicted physicochemical properties of compounds 1–13.
[i]
Compd
Ki [nm][a]
a3b4
LE[e]
Mr [Da][f]
clogP[g]
PSA [Š2][h]
pKa1/pKa2
logBB[j]
[b]
[c]
[d]
a4b2
a7
234
[kcalmolÀ1
]
Nicotine[15b]
Niodene[11]
(2S)-1
10.0
256
ND
0.91
0.94
0.51
0.39
0.70
0.68
0.73
0.72
0.65
0.69
0.51
0.51
0.51
0.49
0.50
162.23
302.11
293.11
307.13
275.12
289.14
275.12
289.14
322.12
336.14
272.32
290.31
296.34
314.33
292.35
0.883
2.164
2.474
3.019
2.181
2.726
2.181
2.726
2.441
2.986
3.165
3.320
3.915
4.058
1.01
16.13
34.15
34.15
25.36
34.15
25.36
34.15
25.36
34.15
25.36
34.15
34.15
34.15
34.15
54.38
8.86/2.70
9.77/3.05
7.02/3.01
5.18/2.73
8.79/3.00
6.98/2.72
8.79/3.00
6.98/2.72
8.79/3.06
6.99/2.77
8.80/4.34
8.80/4.35
8.79/2.63
8.79/2.63
8.79/3.77
À0.234
À0.037
+0.010
+0.223
À0.035
+0.178
À0.035
+0.178
+0.005
+0.218
+0.115
+0.138
+0.229
+0.251
À0.112
0.27
1160Æ500
26500Æ14000
22.7Æ7.9
34.7Æ12.4
10.9Æ3.5
12.1Æ4.1
76.3Æ29.7
29.0Æ8.4
32.5Æ18.2
14.1Æ8
ND
34000Æ18700
11 2000Æ57120
383Æ137.8
1080Æ450
1430Æ800
143Æ61.4
126Æ49.1
53.3Æ18.1
497Æ114.3
95Æ38
>50”103
(2S)-2
>50”103
(2S,4S)-3
(2S,4S)-4
(2S,4R)-5
(2S,4R)-6
(2S,4R)-7
(2S,4R)-8
(2S,4R)-9
(2S,4R)-10
(2S,4R)-11
(2S,4R)-12
(2S,4R)-13
>50”103
>50”103
>50”103
>50”103
>50”103
>50”103
1800Æ684
3600Æ2600
16000Æ1400
36000Æ2880
34000Æ3000
7.4Æ2.8
306Æ125.5
475Æ156.7
430Æ141.9
7.5Æ3.6
24.1Æ13.5
[a] Values are the mean ÆSEM, and were calculated with the LIGAND program to fit the data obtained from three independent saturation and competition
binding experiments for each compound that, for each dilution, was tested in triplicate. All the compounds on each subtype have been tested in three
separate experiments. ND: not determined. [b] Determined in binding competition studies using rat cortical membranes labeled with [3H]epibatidine.
[c] Determined with human a3b4 subunits transfected in HEK 293 cells, using [3H]epibatidine as radioligand. [d] Determined with rat hippocampal mem-
branes labeled with [125I]a-bungarotoxin.[15] [e] Ligand binding efficiency was calculated according to the Hopkins equation: LE=1.372”[ÀlogKi (moles)]/N.
[f,g] Molecular weight and clogP were calculated from ChemBioDraw Ultra 11.0. [h,i] Polar surface area and pKa were calculated from www.chemicalize.org.
[j] logBB was calculated from Clark’s equation: logBB=À0.0148PSA+0.152clogP+0.139.
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spots were further evidenced by spraying with a dilute alkaline po-
tassium permanganate solution. Melting points were determined
on a model B540 Büchi apparatus and are uncorrected. MS analy-
ses were performed on a Varian 320-MS triple quadrupole mass
spectrometer with ESI source. Microanalyses (C, H, N) of new com-
pounds were within Æ0.4% of theoretical values.
General procedure for the synthesis of N-Boc-protected alco-
hols: To a stirred solution of the N-Boc-protected carboxylic acid
derivative (1.5 mmol) in THF (10 mL) was added 1m BH3 solution in
THF (3.2 mL). The resulting mixture was stirred at 708C for 4 h and
then AcOH (1 mL) was carefully added. EtOAc (50 mL) was added
and the organic layer was washed with brine (10 mL), dried over
anhydrous Na2SO4, filtered and evaporated to dryness. The residue
was purified by flash chromatography (cyclohexane/EtOAc).
affinity for the a4b2 receptor subtype (Ki =7.4 and 7.5 nm, re-
spectively) and good selectivity over a3b4 and a7 subtypes.
For all the tested derivatives, we calculated the ligand effi-
ciency (LE), which is an important metric in drug discovery and
has been used to measure the relationship between ligand af-
finity and molecular size.[16] LE is a useful optimization tool to
evaluate a ligand’s ability to bind effectively the target protein.
Considering the binding affinity (Ki) of the compounds, we cal-
culated the LE using the Hopkins equation.[17] LEꢀ0.3 is con-
sidered favorable and suggests that a compound is optimized
for receptor occupancy. All the new derivatives possess LE
values in the range of 0.4–0.7 kcalmolÀ1.
To identify the most promising derivatives in terms of good
CNS penetration, we evaluated a series of physicochemical
properties that influence blood–brain barrier (BBB) penetra-
tion,[18,19] such as molecular weight (Mr), polar surface area
(PSA), basicity (pKa), and lipophilicity (clogP). These parameters
were calculated for all new compounds and are listed in
Table 1. All the compounds in this report, with the exception
of (2S,4R)-13, have a Mr <350 Da, clogP>2, and PSA<40 Š2,
which taken together are predictive of good BBB penetration.
Using Clark’s equation, we also calculated the logBB, a model
for the prediction of blood–brain partitioning.[20–22] From this
analysis, we highlighted derivatives (2S,4R)-11 and (2S,4R)-12
as the most promising ligands identified in this study, since
beside their good affinity and selectivity profile for the a4b2
subtype, they showed improved physicochemical properties
over compound (2S,4R)-5. In particular, whereas compound
(S)-tert-Butyl
4,4-difluoro-2-(hydroxymethyl)pyrrolidine-1-car-
boxylate [(2S)-16]: Yield: 84% (colorless oil); Rf =0.70 (cyclohex-
1
ane/EtOAc 1:1); [a]2D0 =À34.0 (c=1.0 in CHCl3); H NMR (300 MHz,
CDCl3): d=4.26–4.02 (m, 1H), 3.90–3.52 (m, 4H), 2.55–2.38 (m, 1H),
2.30–2.04 (m, 1H), 1.48 ppm (s, 9H); 13C NMR (75 MHz, CDCl3): d=
156.0, 126.5 (t, J=249.1), 81.7, 65.6, 58.5, 54.2 (t, J=30.9), 36.7 (t,
J=23.8), 28.5 ppm; MS: 238.2 [M+H]+; Anal. calcd for
C10H17F2NO3: C 50.63, H 7.22, N 5.90, found: C 50.95, H 7.44, N 5.70.
(2S,4S)-tert-Butyl
4-fluoro-2-(hydroxymethyl)pyrrolidine-1-car-
boxylate [(2S,4S)-18]: Yield: 80% (colorless oil); Rf =0.40 (cyclohex-
1
ane/EtOAc 1:1); [a]2D0 =À22.6 (c=1.1 in CHCl3); H NMR (300 MHz,
CDCl3): d=5.30–5.00 (m, 1H), 4.20–4.00 (m, 1H), 3.85–4.75 (m, 1H),
3.70–3.40 (m, 3H), 2.40–1.95 (m, 2H), 1.40 ppm (s, 9H); 13C NMR
(75 MHz, CDCl3): d=156.8, 91.4 (d, J=176.3), 81.0, 67.6, 59.3, 54.2
(d, J=23.8), 35.2 (d, J=21.2), 28.5 ppm; MS: 220.1 [M+H]+; Anal.
calcd for C10H18FNO3: C 54.78, H 8.27, N 6.39, found: C 54.98, H
8.47, N 6.15.
(2S,4R)-5 was characterized by
a negative logBB value
(À0.035), the insertion of the additional phenyl acetylene
moiety conferred to derivatives (2S,4R)-11 and (2S,4R)-12 an in-
creased lipophilicity, resulting in positive logBB values (i.e.,
+0.229 and +0.225, respectively).
(2S,4R)-tert-Butyl
4-fluoro-2-(hydroxymethyl)pyrrolidine-1-car-
boxylate [(2S,4R)-21]: Yield: 80% (colorless oil); Rf =0.40 (cyclohex-
ane/EtOAc 1:1); [a]2D0 =À47.6 (c=1.4 in CHCl3); H NMR (300 MHz,
1
CDCl3): d=5.22–5.00 (m, 1H), 4.22–4.05 (m, 1H), 3.95–3.70 (m, 2H),
3.60–3.32 (m, 2H), 2.40–2.23 (m, 1H), 1.90–1.58 (m, 1H), 1.45 ppm
(s, 9H); 13C NMR (75 MHz, CDCl3): d=157.0, 91.4 (d, J=176.6), 81.2,
66.7, 59.1, 54.4 (d, J=23.2), 35.7 (d, J=22.0), 28.6 ppm; MS: 220.2
[M+H]+; Anal. calcd for C10H18FNO3: C 54.78, H 8.27, N 6.39, found:
C 54.70, H 8.42, N 6.20.
Conclusions
In summary, we have developed a novel series of a4b2 ligands
characterized by the 3-fluoropyrrolidine moiety, endowed with
nanomolar receptor affinity. Further decoration of the pyridine
nucleus has allowed significant improvements in physicochem-
ical properties important for BBB penetration, leading to deriv-
atives (2S,4R)-11 and (2S,4R)-12, which display single-digit
nanomolar affinity for the a4b2 receptor. Considering that com-
pound (2S,4R)-12 is characterized by the presence of a fluorine
atom at the para position of the aromatic ring, it is easy to
foresee the development of [18F](2S,4R)-12 as a radiolabeled
ligand for PET imaging applications.
General procedure for the Mitsunobu reaction: To a solution of
the N-Boc-protected alcohol (1.5 mmol), 3-bromo-5-hydroxypyri-
dine (1.7 mmol) and PPh3 (2.3 mmol) in dry THF (20 mL), stirred
under argon at 08C, diisopropyl azodicarboxylate (DIAD; 2.3 mmol)
was added. The reaction mixture was allowed to warm to room
temperature over a period of 2 h and was stirred for 24 h. The re-
action mixture was evaporated to dryness and the residue was pu-
rified by flash chromatography (cyclohexane/EtOAc).
(S)-tert-Butyl 2-((5-bromopyridin-3-yloxy)methyl)-4,4-difluoropyr-
rolidine-1-carboxylate [(2S)-22]: Yield: 83% (thick colorless oil);
Rf =0.60 (cyclohexane/EtOAc 7:3); [a]2D0 =À42.85 (c=1.0 in CHCl3);
1H NMR (300 MHz, CDCl3): d=8.26 (bs, 1H), 8.22 (d, J=2.7, 1H),
7.38 (bs, 1H), 4.46–4.26 (m, 1H), 4.26–3.55 (m, 4H), 2.66–2.42 (m,
2H), 1.44 ppm (s, 9H); 13C NMR (75 MHz, CDCl3): d=155.1, 154.1,
143.6, 136.7, 127.1 (t, J=250.8), 124.2, 120.6, 81.3, 68.2, 54.6, 54.1
(t, J=32.1), 36.5 (t, J=24.9), 28.5 ppm; MS: 393.1 [M+H]+; Anal.
calcd for C15H19BrF2N2O3: C 45.82, H 4.87, N 7.12, found: C 45.70, H
4.97, N 7.03.
Experimental Section
Materials and methods: All reagents were purchased from Sigma.
1H NMR and 13C NMR spectra were recorded with a Varian Mercu-
ry 300 (300 MHz) spectrometer. Chemical shifts (d) are expressed in
ppm, and coupling constants (J) are expressed in Hz. Optical rota-
tion determinations were carried out using a Jasco P-1010 spectro-
polarimeter, coupled with a Haake N3-B thermostat. TLC analyses
were performed on commercial silica gel 60 F254 aluminum sheets;
(2S,4S)-tert-Butyl
pyrrolidine-1-carboxylate [(2S,4S)-23]: Yield: 91%; (colorless
2-((5-bromopyridin-3-yloxy)methyl)-4-fluoro-
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solid); Rf =0.45 (cyclohexane/EtOAc 7:3); crystallized from n-
d=8.25 (d, J=1.7, 1H), 8.20 (d, J=2.7, 1H), 7.34 (dd, J=2.7, 1.7,
1H), 5.22 (ddd, J=54.5, 4.4, 4.4, 1H), 4.12–3.94 (m, 2H), 3.62–3.50
(m, 1H), 3.34 (dd, J=21.2, 13.0, 1H), 2.95 (ddd, J=35.5, 13.0, 4.4,
1H), 2.34–2.10 (m, 1H), 2.22 (bs, 1H), 1.93 ppm (ddd, J=28.3, 14.6,
4.4, 1H); 13C NMR (75 MHz, CDCl3): d=155.4, 143.4, 136.7, 124.2,
120.6, 94.7 (d, J=175.2), 71.5, 56.9, 54.1 (d, J=22.9), 36.1 ppm (d,
J=21.2); MS: 275.0 [M+H]+; Anal. calcd for C10H12BrFN2O: C 43.66,
H 4.40, N 10.18, found: C 43.52, H 4.58, N 9.95.
hexane/EtOAc as colorless prisms; mp: >118 8C (dec.); [a]D20
=
1
À53.60 (c=1.00 in CHCl3); H NMR (300 MHz, CDCl3): d=8.35–8.25
(m, 2H), 7.50–7.40 (m, 1H), 5.25 (bd, J=52.3, 1H), 4.44–4.22 (m,
2H), 4.00–3.85 (m, 1H), 3.80–3.50 (m, 2H), 2.56–2.38 (m, 1H), 2.34–
2.04 (m, 1H), 1.50 ppm (s, 9H); 13C NMR (75 MHz, CDCl3): d=155.4,
154.4, 143.3, 136.9, 124.1, 120.6, 93.1 (d, J=174.9), 80.8, 68.8, 55.3,
53.8 (d, J=23.8), 34.8 (d, J=19.5), 28.7 ppm; MS: 375.1 [M+H]+;
Anal. calcd for C15H20BrFN2O3: C 48.01, H 5.37, N 7.47, found: C
47.84, H 5.49, N 7.34.
3-(((2S,4R)-4-Fluoropyrrolidin-2-yl)methoxy)-5-bromopyridine
[(2S,4R)-5]: Yield: 85% (thick colorless oil); Rf =0.40 (CH2Cl2/MeOH
9:1); [a]2D0 = +21.60 (c=1.00 in CHCl3); 1H NMR (300 MHz, CDCl3):
d=8.24 (d, J=1.7, 1H), 8.20 (d, J=2.7, 1H), 7.34 (dd, J=2.7, 1.7,
1H), 5.22 (ddd, J=53.5, 3.9, 3.9, 1H), 3.96–3.85 (m, 2H), 3.85–3.75
(m, 1H), 3.30–3.15 (m, 1H), 3.05 (ddd, J=36.0, 12.6, 3.9, 1H), 2.35–
2.16 (m, 1H), 2.34 (bs, 1H), 1.73 ppm (dddd, J=39.3, 14.6, 8.5, 3.9,
1H); 13C NMR (75 MHz, CDCl3): d=155.5, 143.3, 136.6, 124.2, 120.5,
95.2 (d, J=174.6), 71.9, 55.9, 53.6 (d, J=22.6), 36.5 ppm (d, J=
21.1); MS: 275.0 [M+H]+; Anal. calcd for C10H12BrFN2O: C 43.66, H
4.40, N 10.18, found: C 43.62, H 4.48, N 10.02.
(2S,4R)-tert-Butyl
2-((5-bromopyridin-3-yloxy)methyl)-4-fluoro-
pyrrolidine-1-carboxylate [(2S,4R)-24]: Yield: 92%; (colorless
solid); Rf =0.45 (cyclohexane/EtOAc 7:3); crystallized from n-
hexane/EtOAc as colorless prisms; mp: 62–648C; [a]2D0 =À39.00
1
(c=1.00 in CHCl3); H NMR (300 MHz, CDCl3): d=8.25 (bs, 1H), 8.20
(d, J=2.7, 1H), 7.36 (bs, 1H), 5.20 (bd, J=53.0, 1H), 4.44–4.22 (m,
2H), 4.18–4.10 (m, 1H), 4.08–3.76 (m, 1H), 3.43 (ddd, J=35.7, 13.2,
3.6, 1H), 2.56–2.35 (m, 1H), 2.23 (dddd, J=37.7, 14.6, 7.7, 4.7, 1H),
1.45 ppm (s, 9H); 13C NMR (75 MHz, CDCl3): d=155.5, 154.8, 143.4,
137.0, 124.3, 120.6, 91.9 (d, J=172.3), 80.6, 69.8, 55.2, 54.1 (d, J=
22.9), 35.3 (d, J=20.6), 28.7 ppm; MS: 375.1 [M+H]+; Anal. calcd
for C15H20BrFN2O3: C 48.01, H 5.37, N 7.47, found: C 47.94, H 5.36, N
7.44.
3-(((2S,4R)-4-Fluoropyrrolidin-2-yl)methoxy)-5-iodopyridine
[(2S,4R)-7]: Yield: 85% (thick colorless oil); Rf: 0.30 (CH2Cl2/MeOH
9:1); [a]2D0 = +20.80 (c=1.00 in CHCl3); 1H NMR (300 MHz, CDCl3):
d=8.38 (d, J=1.6, 1H), 8.22 (d, J=2.5, 1H), 7.52 (dd, J=2.5, 1.6,
1H), 5.22 (ddd, J=53.9, 3.9, 3.9, 1H), 3.96–3.85 (m, 2H), 3.85–3.75
(m, 1H), 3.32–3.16 (m, 1H), 3.05 (ddd, J=36.0, 13.6, 3.9, 1H), 2.34–
2.18 (m, 1H), 2.28 (bs, 1H), 1.73 ppm (dddd, J=39.3, 14.3, 8.5, 3.9,
1H); 13C NMR (75 MHz, CDCl3): d=155.5, 148.3, 137.0, 129.7, 95.3
(d, J=174.1), 93.0, 71.9, 55.9, 53.6 (d, J=22.9), 36.5 ppm (d, J=
21.3); MS: 323.0 [M+H]+; Anal. calcd for C10H12FIN2O: C 37.29, H
3.75, N 8.70, found: C 37.39, H 3.97, N 8.49.
(2S,4R)-tert-Butyl 2-((5-iodopyridin-3-yloxy)methyl)-4-fluoropyr-
rolidine-1-carboxylate [(2S,4R)-25]: Yield: 86% (colorless solid);
Rf =0.40 (cyclohexane/EtOAc 75:25); crystallized from n-hexane/
EtOAc as colorless prisms; mp: 41–438C; [a]2D0 =À40.15 (c=1.00 in
1
CHCl3); H NMR (300 MHz, CDCl3): d=8.40 (bs, 1H), 8.21 (d, J=2.5,
1H), 7.36 (bs, 1H), 5.20 (bd, J=52.5, 1H), 4.42–4.22 (m, 2H), 4.18–
4.08 (m, 1H), 4.08–3.76 (m, 1H), 3.43 (ddd, J=35.7, 13.2, 3.3, 1H),
2.56–2.35 (m, 1H), 2.21 (dddd, J=37.7, 14.3, 7.7, 4.1, 1H), 1.44 ppm
(s, 9H); 13C NMR (75 MHz, CDCl3): d=155.5, 154.8, 148.4, 137.0,
129.8, 93.1, 91.0 (d, J=174.1), 80.5, 69.1, 55.2, 54.1 (d, J=22.9),
36.0 (d, J=21.3), 28.7 ppm; MS: 423.1 [M+H]+; Anal. calcd for
C15H20BrFN2O3: C 42.67, H 4.77, N 6.63, found: C 42.57, H 4.92, N
6.51.
3-(((2S,4R)-4-Fluoropyrrolidin-2-yl)methoxy)-5-phenylpyridine
[(2S,4R)-9]: Yield: 82%; (colorless oil); Rf =0.40 (CH2Cl2/MeOH 9:1);
[a]2D0 = +19.10 (c=1.05 in CHCl3); 1H NMR (300 MHz, CDCl3): d=
8.45 (d, J=1.9, 1H), 8.28 (d, J=2.7, 1H), 7.56–7.50 (m, 2H), 7.48–
7.32 (m, 4H), 5.25 (ddd, J=54.2, 4.0, 4.0, 1H), 4.03 (dd, J=9.1, 5.5,
1H), 3.98 (dd, J=9.1, 5.5, 1H), 3.90–3.80 (m, 1H), 3.28 (dd, J=24.5,
13.2, 1H), 3.12 (ddd, J=35.7, 13.2, 4.0, 1H), 2.64 (bs, 1H), 2.29
(ddd, J=23.1, 14.6, 7.1, 1H), 1.81 ppm (dddd, J=39.0, 14.6, 8.8, 4.0,
1H); 13C NMR (75 MHz, CDCl3): d=155.3, 141.0, 137.7, 137.6, 136.5,
129.3, 128.5, 127.4, 120.1, 95.2 (d, J=174.3), 71.5, 56.1, 53.6 (d, J=
22.6), 36.6 ppm (d, J=21.2); MS: 273.1 [M+H]+; Anal. calcd for
C16H17FN2O: C 70.57, H 6.29, N 10.29, found: C 70.35, H 6.57, N
9.90.
General procedure for N-Boc deprotection: Mitsunobu adduct
(1.0 mmol) was treated with a 30% CH2Cl2 solution of trifluoroace-
tic acid (TFA; 10.0 mmol) at 08C, and the solution was stirred at
room temperature for 4 h. The volatiles were removed under
vacuum, aqueous 1n NaOH (5 mL) was added, and the aqueous
layer was extracted with CH2Cl2 (3”20 mL). The organic layer was
dried over anhydrous Na2SO4, filtered, evaporated to dryness and
the residue was purified by flash chromatography (EtOAc or
CH2Cl2/MeOH).
3-(4-Fluorophenyl)-5-(((2S,4R)-4-fluoropyrrolidin-2-yl)methoxy)-
pyridine [(2S,4R)-10]: Yield: 84%; (colorless oil); Rf =0.40 (CH2Cl2/
MeOH 9:1); [a]2D0 = +17.30 (c=1.10 in CHCl3); 1H NMR (300 MHz,
CDCl3): d=8.39 (d, J=1.9, 1H), 8.26 (d, J=2.7, 1H), 7.52–7.45 (m,
2H), 7.31 (dd, J=2.7, 1.9, 1H), 7.16–7.08 (m, 2H), 5.25 (ddd, J=
54.2, 4.1, 4.1, 1H), 4.03 (dd, J=9.1, 5.2, 1H), 3.98 (dd, J=9.1, 5.8,
1H), 3.92–3.80 (m, 1H), 3.28 (dd, J=23.9, 13.2, 1H), 3.13 (ddd, J=
35.2, 13.2, 4.1, 1H), 2.77 (bs, 1H), 2.29 (dddd, J=22.8, 14.6, 6.9, 1.1,
1H), 1.81 ppm (dddd, J=39.0, 14.6, 8.8, 4.1, 1H); 13C NMR (75 MHz,
CDCl3): d=163.2 (d, J=248.2), 155.3, 140.8, 136.7, 136.5, 133.8 (d,
J=3.2), 129.1 (d, J=8.3), 120.0, 116.3 (d, J=21.7), 95.1 (d, J=
175.2), 71.4, 56.1, 53.6 (d, J=23.2), 36.5 ppm (d, J=21.5); MS: 291.1
[M+H]+; Anal. calcd for C16H16F2N2O: C 66.20, H 5.56, N 9.65,
found: C 66.15, H 5.80, N 9.35.
3-(((S)-4,4-Difluoropyrrolidin-2-yl)methoxy)-5-bromopyridine
[(2S)-1]: Yield: 75% (white solid); Rf =0.40 (EtOAc); crystallized
from n-hexane/EtOAc as colorless prisms; mp: 52–548C; [a]2D0 = +
1
8.83 (c=1.00 in CHCl3); H NMR (300 MHz, CDCl3): d=8.30 (d, J=
1.9, 1H), 8.23 (d, J=2.5, 1H), 7.37 (dd, J=1.9, 2.5, 1H), 4.03 (dd, J=
5.0, 9.4, 1H), 3.98 (dd, J=6.1, 9.4, 1H), 3.82–3.72 (m, 1H), 3.34
(ddd, J=12.4, 12.4, 12.4, 1H), 3.23 (ddd, J=12.4, 12.4, 12.4, 1H),
2.52–2.34 (m, 1H), 2.24 (bs, 1H), 2.22–2.00 ppm (m, 1H); 13C NMR
(75 MHz, CDCl3): d=155.2, 143.7, 136.5, 131.08 (t, J=250.8), 124.3,
120.6, 70.8, 56.1 (t, J=4.5), 54.5 (t, J=29.2), 38.3 ppm (t, J=25.0);
MS: 293.0 [M+H]+; Anal. calcd for C10H11BrF2N2O: C 40.98, H 3.78,
N 9.56, found: C 40.90, H 3.97, N 9.35.
3-(((2S,4S)-4-Fluoropyrrolidin-2-yl)methoxy)-5-bromopyridine
[(2S,4S)-3]: Yield: 82% (white solid); Rf =0.30 (CH2Cl2/MeOH 9:1);
crystallized from n-hexane/EtOAc as colorless prisms; mp: 69–
718C; [a]2D0 = +6.70 (c=0.7 in CHCl3); 1H NMR (300 MHz, CDCl3):
3-(((2S,4R)-4-Fluoropyrrolidin-2-yl)methoxy)-5-(phenylethynyl)-
pyridine [(2S,4R)-11]: Yield: 87%; (colorless solid); Rf =0.50 (CH2Cl2/
MeOH 9:1); crystallized from n-hexane/EtOAc as colorless prisms;
1
mp: 85–888C; [a]2D0 = +23.30 (c=1.10 in CHCl3); H NMR (300 MHz,
ChemMedChem 2015, 10, 1071 – 1078
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Full Papers
CDCl3): d=8.37 (d, J=1.1, 1H), 8.22 (d, J=2.7, 1H), 7.58–7.50 (m,
2H), 7.40–7.34 (m, 3H), 7.34–7.30 (m, 1H), 5.22 (ddd, J=54.2, 4.0,
4.0, 1H), 4.02–3.94 (m, 2H), 3.90–3.80 (m, 1H), 3.28 (dd, J=24.2,
13.2, 1H), 3.12 (ddd, J=35.5, 13.2, 4.0, 1H), 2.38–2.22 (m, 1H), 2.30
(bs, 1H), 1.79 ppm (dddd, J=39.0, 14.3, 8.5, 4.0, 1H); 13C NMR
(75 MHz, CDCl3): d=154.6, 145.0, 137.9, 131.9, 129.1, 128.7, 123.2,
122.7, 120.8, 95.2 (d, J=174.7), 92.7, 86.0, 71.6, 56.0, 53.6 (d, J=
22.9), 36.5 ppm (d, J=21.5); MS: 297.1 [M+H]+; Anal. calcd for
C18H17FN2O: C 72.95, H 5.78, N 9.45, found: C 72.63, H 6.10, N 9.07.
3-(((2S,4S)-4-Fluoro-1-methylpyrrolidin-2-yl)methoxy)-5-bromo-
pyridine [(2S,4S)-4]: Yield: 79% (colorless oil); Rf =0.40 (EtOAc);
1
[a]2D0 =À36.75 (c=0.8 in CHCl3); H NMR (300 MHz, CDCl3): d=8.26
(d, J=2.0, 1H), 8.23 (d, J=2.5, 1H), 7.36 (dd, J=2.5, 2.0, 1H), 5.11
(ddd, J=53.9, 4.4, 4.1 1H), 4.06 (dd, J=9.3, 5.2, 1H), 3.97 (dd, J=
9.3, 5.8, 1H), 3.35 (ddd, J=17.9, 11.8, 2.2, 1H), 2.80–2.68 (m, 1H),
2.56–2.32 (m, 2H), 2.47 (s, 3H), 2.10–1.90 ppm (m, 1H); 13C NMR
(75 MHz, CDCl3): d=155.5, 143.3, 136.6, 124.2, 120.6, 92.6 (d, J=
176.6), 71.5, 63.7 (d, J=21.2), 63.4, 41.6, 36.8 ppm (d, J=22.3); MS:
289.0 [M+H]+; Anal. calcd for C11H14BrFN2O: C 45.69, H 4.88, N
9.69, found: C 45.54, H 5.02, N 9.51.
3-((4-Fluorophenyl)ethynyl)-5-(((2S,4R)-4-fluoropyrrolidin-2-yl)-
methoxy)pyridine [(2S,4R)-12]: Yield: 88%; (colorless solid); Rf =
0.60 (CH2Cl2/MeOH 9:1); crystallized from n-hexane/EtOAc as color-
less prisms; mp: 102–1058C; [a]2D0 = +20.60 (c=1.10 in CHCl3);
1H NMR (300 MHz, CDCl3): d=8.36 (d, J=1.4, 1H), 8.26 (d, J=2.7,
1H), 7.55–7.48 (m, 2H), 7.30 (dd, J=2.7, 1.4, 1H), 7.10–7.03 (m,
2H), 5.28 (ddd, J=53.9, 4.0, 4.0, 1H), 4.05–3.95 (m, 2H), 3.92–3.82
(m, 1H), 3.30 (dd, J=25.0, 13.7, 1H), 3.14 (ddd, J=35.5, 13.7, 4.0,
1H), 2.30 (ddd, J=22.9, 14.3, 7.7, 1H), 2.08 (bs, 1H), 1.80 ppm
(dddd, J=39.0, 14.3, 8.8, 4.0, 1H); 13C NMR (75 MHz, CDCl3): d=
163.1 (d, J=250.5), 154.5, 145.0, 137.9, 133.9 (d, J=8.3), 123.2,
120.7, 118.8 (d, J=3.5), 116.3 (d, J=22.3), 95.0 (d, J=178.4), 91.7,
85.7, 71.3, 56.1, 53.5 (d, J=22.9), 36.5 ppm (d, J=21.2); MS: 315.1
[M+H]+; Anal. calcd for C18H16F2N2O: C 68.78, H 5.13, N 8.91,
found: C 68.63, H 5.45, N 8.58.
3-(((2S,4R)-4-Fluoro-1-methylpyrrolidin-2-yl)methoxy)-5-bromo-
pyridine [(2S,4R)-6]: Yield: 80% (colorless oil); Rf =0.40 (EtOAc);
1
[a]2D0 =À36.55 (c=0.8 in CHCl3); H NMR (300 MHz, CDCl3): d=8.27
(d, J=1.7, 1H), 8.23 (d, J=2.5, 1H), 7.36 (dd, J=2.5, 1.7, 1H), 5.30–
5.04 (m, 1H), 4.01 (dd, J=9.6, 5.0, 1H), 3.97 (dd, J=9.6, 5.0, 1H),
3.54 (ddd, J=26.1, 11.8, 5.8, 1H), 3.10–3.00 (m, 1H), 2.64 (dddd, J=
31.9, 11.8, 3.0, 1.1, 1H), 2.49 (s, 3H), 2.35–2.17 (m, 1H), 1.95 ppm
(dddd, J=33.6, 16.0, 9.9, 5.8, 1H); 13C NMR (75 MHz, CDCl3): d=
155.5, 143.4, 136.6, 124.3, 120.6, 92.4 (d, J=174.6), 70.4, 63.3 (d, J=
22.9), 62.9, 41.8, 37.3 ppm (d, J=21.5); MS: 289.0 [M+H]+; Anal.
calcd for C11H14BrFN2O: C 45.69, H 4.88, N 9.69, found: C 45.58, H
4.95, N 9.61.
3-(((2S,4R)-4-Fluoro-1-methylpyrrolidin-2-yl)methoxy)-5-iodopyri-
dine [(2S,4R)-8]: Yield: 51% (colorless oil); Rf =0.20 (EtOAc); [a]D20
=
6-(5-(((2S,4R)-4-Fluoropyrrolidin-2-yl)methoxy)pyridin-3-yl)hex-5-
yn-1-ol [(2S,4R)-13]: Yield: 74%; (colorless oil); Rf =0.20 (CH2Cl2/
MeOH 9:1); [a]2D0 = +6.70 (c=1.20 in CHCl3); 1H NMR (300 MHz,
CDCl3): d=8.20 (d, J=1.4, 1H), 8.16 (d, J=2.7, 1H), 7.17 (dd, J=
2.7, 1.4, 1H), 5.25 (ddd, J=53.9, 4.0, 4.0, 1H), 3.98 (dd, J=9.0, 5.2,
1H), 3.94 (dd, J=9.0, 5.8, 1H), 3.90–3.80 (m, 1H), 3.74–3.66 (m,
2H), 3.28 (ddd, J=25.0, 13.7, 3.6, 1H), 3.15 (ddd, J=25.5, 13.7, 4.0,
1H), 2.78 (bs, 2H), 2.50–2.42 (m, 2H), 2.30 (ddd, J=22.6, 14.6, 6.9,
1H), 1.81 (dddd, J=39.0, 14.6, 8.8, 4.0, 1H), 1.74–1.62 ppm (m, 4H);
13C NMR (75 MHz, CDCl3): d=154.4, 145.1, 137.1, 123.5, 121.5, 94.9
(d, J=174.6), 93.9, 77.4, 71.0, 62.4, 56.2, 53.4 (d, J=22.9), 36.3 (d,
J=20.6), 32.1, 25.1, 19.4 ppm; MS: 293.1 [M+H]+; Anal. calcd for
C16H21FN2O2: C 65.73, H 7.24, N 9.58, found: C 65.70, H 7.50, N 9.19.
À35.78 (c=1.10 in CHCl3); 1H NMR (300 MHz, CDCl3): d=8.27 (d,
J=1.7, 1H), 8.23 (d, J=2.5, 1H), 7.36 (dd, J=2.2, 1.7, 1H), 5.30–
5.04 (m, 1H), 4.04–3.92 (m, 2H), 3.54 (ddd, J=26.1, 11.8, 5.8, 1H),
3.10–2.98 (m, 1H), 2.74–2.56 (m, 1H), 2.49 (s, 3H), 2.34–2.18 (m,
1H), 1.95 ppm (dddd, J=33.6, 15.6, 10.1, 5.8, 1H); 13C NMR
(75 MHz, CDCl3): d=155.5, 148.4, 137.0, 129.8, 93.0, 92.4 (d, J=
175.5), 70.3, 63.3 (d, J=23.2), 62.9, 41.8, 37.4 ppm (d, J=21.5); MS:
337.0 [M+H]+; Anal. calcd for C11H14FIN2O: C 39.30, H 4.20, N 8.33,
found: C 39.26, H 4.39, N 8.14.
General procedure for Suzuki coupling reaction: To a solution of
(2S,4R)-24 (0.40 mmol) in toluene (6 mL ) and EtOH (2 mL) was
added phenylboronic acid (0.47 mmol), followed by 1.4 mL of 2m
Na2CO3 and tetrakis(triphenylphosphine)palladium (0.02 mmol).
The reaction was stirred for 18 h at 908C under nitrogen. Then, the
reaction was cooled to room temperature, diluted with water
(10 mL), and extracted with EtOAc (3”5 mL). The combined organ-
ic layers were dried over anhydrous Na2SO4, filtered and evaporat-
ed to dryness. The residue was purified by flash chromatography
(cyclohexane/EtOAc).
General procedure for reductive amination: To a solution of the
secondary amine (0.5 mmol) in CH3CN (3 mL) was added 37%
formaldehyde solution (2.5 mmol), and the mixture was stirred for
20 min at room temperature, then NaBH3CN (0.8 mmol) was added
in small portions. After 2 h, the solvent was evaporated at reduced
pressure, and 1n NaOH (5 mL) was added to the residue. The re-
sulting mixture was extracted with CH2Cl2 (3”20 mL). The com-
bined organic extracts were washed with water (10 mL), brine
(10 mL), dried with anhydrous Na2SO4 and evaporated to dryness.
The residue was purified by flash chromatography (cyclohexane/
EtOAc).
(2S,4R)-tert-Butyl 4-fluoro-2-((5-phenylpyridin-3-yloxy)methyl)-
pyrrolidine-1-carboxylate [(2S,4R)-26]: Yield: 98%; (colorless oil);
Rf =0.15 (cyclohexane/EtOAc 8:2); [a]2D0 =À32.20 (c=1.00 in CHCl3);
1H NMR (300 MHz, CDCl3): d=8.47 (bs, 1H), 8.28 (d, J=2.5, 1H),
7.58–7.52 (m, 2H), 7.50–7.34 (m, 4H), 5.22 (bd, J=53.1, 1H), 4.50–
4.26 (m, 2H), 4.24–4.16 (m, 1H), 4.08–3.76 (m, 1H), 3.46 (ddd, J=
36.2, 12.9, 3.6, 1H), 2.58–2.14 (m, 2H), 1.44 ppm (s, 9H); 13C NMR
(75 MHz, CDCl3): d=155.5, 154.8, 140.6, 137.8, 137.6, 136.4, 129.3,
128.6, 127.6, 120.2, 92.0 (d, J=177.8), 80.4, 69.2, 55.3, 53.9 (d, J=
23.2), 35.9 (d, J=20.9), 28.6 ppm; MS: 373.2 [M+H]+; Anal. calcd
for C21H25FN2O3: C 67.72, H 6.77, N 7.52, found: C 67.61, H 7.03, N
7.23.
3-(((S)-4,4-Difluoro-1-methylpyrrolidin-2-yl)methoxy)-5-bromo-
pyridine [(2S)-2]: Yield: 34% (white solid); Rf =0.50 (cyclohexane/
EtOAc 7:3); crystallized form n-hexane/EtOAc as colorless prisms;
mp: 41–438C; [a]2D0 =À36.01 (c=0.7 in CHCl3); 1H NMR (300 MHz,
CDCl3): d=8.31 (d, J=1.6, 1H), 8.25 (d, J=2.5, 1H), 7.38 (dd, J=
2.5, 1.6, 1H), 4.09 (dd, J=9.6, 5.0, 1H), 4.00 (dd, J=9.6, 5.2, 1H),
3.50–3.40 (m, 1H), 3.08–2.96 (m, 1H), 2.75 (ddd, J=16.0, 16.0, 11.0,
1H), 2.60–2.42 (m, 1H), 2.47 (s, 3H), 2.35–2.16 ppm (m, 1H);
13C NMR (75 MHz, CDCl3): d=155.3, 143.5, 136.3, 127.8 (dd, J=
246.0, 249.4), 124.6, 120.7, 69.8, 64.2 (t, J=29.0), 63.0, 41.0,
39.4 ppm (t, J=24.9); MS: 307.0 [M+H]+; Anal. calcd for
C11H13BrF2N2O: C 43.02, H 4.27, N 9.12, found: C 42.90, H 4.44, N
8.93.
(2S,4R)-tert-Butyl 4-fluoro-2-((5-(4-fluorophenyl)pyridin-3-yloxy)-
methyl)pyrrolidine-1-carboxylate [(2S,4R)-27]: Yield: 98%; (color-
less oil); Rf =0.25 (cyclohexane/EtOAc 7:3); [a]2D0 =À32.00 (c=1.00
1
in CHCl3); H NMR (300 MHz, CDCl3): d=8.42 (bs, 1H), 8.28 (d, J=
2.5, 1H), 7.56–7.48 (m, 2H), 7.44–7.28 (m, 1H), 7.20–7.10 (m, 2H),
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5.24 (bd, J=52.8, 1H), 4.50–4.28 (m, 2H), 4.28–4.16 (m, 1H), 4.14–
3.78 (m, 1H), 3.46 (ddd, J=35.5, 13.2, 3.6, 1H), 2.58–2.38 (m, 1H),
2.28 (ddd, J=37.4, 14.6, 4.4, 1H), 1.44 ppm (s, 9H); 13C NMR
(75 MHz, CDCl3): d=163.2 (d, J=248.2), 155.5, 154.8, 140.6, 136.8,
136.7, 133.6 (d, J=3.2), 129.2 (d, J=8.3), 120.2, 116.3 (d, J=21.8),
91.9 (d, J=175.7), 80.5, 69.2, 55.2, 54.0 (d, J=23.2), 36.0 (d, J=
21.5), 28.6 ppm; MS: 391.2 [M+H]+; Anal. calcd for C21H24F2N2O3: C
64.60, H 6.20, N 7.18, found: C 64.61, H 6.47, N 6.91.
Nicotinic receptor subtype binding: Frozen cortex and hippocam-
pus specimens were taken from adult male Sprague–Dawley rats
(Charles River, Calco, Italy). Tissue preparations as well as
[3H]epibatidine and [125I]a-bungarotoxin binding to a4b2, a3b4, and
a7 membrane subtypes were performed as previously described.[15]
For each compound, the experimental data obtained from the
three saturation and three competition binding experiments were
analyzed by a nonlinear least-squares procedure using the LIGAND
program as described by Tasso et al.[15a] When final compound con-
centrations up to 200 mm did not inhibit [125I]a-bungarotoxin bind-
ing, the Ki value was defined as being >50 mm based on the
Cheng–Prusoff equation.
General procedure for Sonogashira coupling reaction: The Mitsu-
nobu adduct (2S,4R)-24 (0.27 mmol), Pd(PPh3)2Cl2 (0.014 mmol),
and CuI (0.27 mmol) were placed in an oven-dried round-bottom
flask with nitrogen. After addition of Et3N (4 mL), the mixture was
stirred at room temperature for 5 min, the desired alkyne
(0.74 mmol) was added, and the resulting mixture was stirred at
room temperature for 10 min. Then, the mixture was heated at
908C for 18 h. After cooling to room temperature, the solution was
diluted with water (10 mL), and extracted with EtOAc (3”5 mL).
The combined organic layers were washed with brine (10 mL),
dried over anhydrous Na2SO4, filtered and evaporated to dryness.
The residue was purified by flash chromatography (cyclohexane/
EtOAc).
Acknowledgements
Financial support of this research by the Italian Ministry of Edu-
cation and Research (MIUR—Rome) and by the CNR Research
Project on Aging, Regione Lombardia Projects NUTEC ID
30263049 and MbMM-convenzione no. 18099/RCC (CG) is ac-
knowledged.
(2S,4R)-tert-Butyl 4-fluoro-2-((5-(phenylethynyl)pyridin-3-yloxy)-
methyl)pyrrolidine-1-carboxylate [(2S,4R)-28]: Yield: 98%; (color-
less solid); Rf =0.33 (cyclohexane/EtOAc 8:2); crystallized from n-
hexane/EtOAc as colorless prisms; mp: 92–948C; [a]2D0 =À16.53
Keywords: 3-fluoropyrrolidines
structure–activity relationships · a4b2 ligands · selectivity
·
nicotinic receptors
·
1
(c=1.00 in CHCl3); H NMR (300 MHz, CDCl3): d=8.38 (bs, 1H), 8.22
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Received: February 17, 2015
Revised: March 10, 2015
Published online on April 16, 2015
ChemMedChem 2015, 10, 1071 – 1078
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