Note: Descriptions are shown in the official language in which they were submitted.
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HETEROCYCLIC COMPOUNDS WITH AFFINITY TO MUSCARINIC RECEPTORS
Field of the invention
This invention relates to new heterocyclic compounds having affinity to
muscarinic
receptors, a pharmaceutical composition containing said compounds, as well as
the
use of said compounds for the preparation of a medicament for treating,
alleviating or
preventing muscarinic receptor mediated diseases and conditions.
Background of the invention
Muscarinic cholinergic receptors mediate the actions of the neurotransmitter
acetylcholine in the central and peripheral nervous systems. Muscarinic
receptors
comprise five distinct subtypes, denoted as muscarinic M1, M2, M3, M4 and M5
receptors. Each subtype has a unique distribution in the central and
peripheral
nervous systems. The M1 receptor is predominantly expressed in the cerebral
cortex
and is believed to be involved in the control of higher cognitive functions;
the M2
receptor is the predominant subtype found in heart and is involved in the
control of
heart rate; the M3 receptor is widely expressed in many peripheral tissues and
is
believed to be involved in gastrointestinal and urinary tract stimulation as
well as
sweating and salivation; the M4 receptor is present in the brain and may be
involved
in locomotion and antipsychotic effects; the M5 receptor is located in the
brain and
may be involved in compound addition and in psychotic conditions such as
schizophrenia. In view of the key physiological roles attributed to each of
the
muscarinic receptor subtypes, extensive efforts have been made to generate new
compounds showing selective agonistic or antagonistic properties (see for
example
EP 0296721; EP 0316718; Sauerberg, P. et al., J. Med. Chem., 1992, Vol. 35,
No.
22, 2274-2283; Ward, J.S. et al., 1992, J. Med. Chem., Vol. 35, No. 22, 4011-
4019;
US 5,527,813; Zlotos, D.P. et al., Exp. Opin. Ther. Patents, Vol. 9, No. 8,
1999,
1029-1053; Plate, R., et al., Bioorg. Med. Chem. 4 (1996), 227-237; Plate, R.
et al.,
Bioorg. Med. Chem. 8 (2000), 449-454; Del Guidice, M.R. et al., Arch. Pharm.
Med.
Chem. 2003, 336, 143-154).
A well known example of a M1/M4 preferring muscarinic receptor agonist is the
thiadiazole compound xanomeline which in preclinical studies has a desirable
profile,
however, in clinical studies displays a unfavourable side effects (see for
example the
review by Eglen, R.M., Progress in Medicinal Chemistry, 2005, 43, p.105-136
and US
5,376,668), which seem to be related to M2 receptor mediated activity (e.g.
heart rate
effects). In addition, xanomeline has a relatively low (in vitro) metabolic
stability.
Xanomeline related compounds are further disclosed in US 5,527,813. However,
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2
representative compounds display unfavourable side effects which seem to be
related to M2 and M3 receptor mediated activity (e.g. heart rate effects and
salivation, respectively).
Although further research is ongoing to develop therapeutics that have the
selective
M1/M4 profile, this has not yet resulted in succesfull candidates. Therefore,
there is a
need for new selective compounds with the desired properties.
Description of the invention
It has now been found that heterocyclic compounds of the formula (I)
Z, Y
~~ \X~
~ ~ (I)
5c2
R1
wherein
- the heterocycle comprises two double bonds which may be present at several
positions, represented by the dashed lines (---);
- the heterocycle contains two heteroatoms,
- W being N or NH;
- Y being CH, 0 or NH, wherein
if Y is 0, X, is CH and X2 is the residue C-Z-R2 or C-R3, wherein Z is NH, 0,
or S; and
if Y is CH or NH, one of X, and X2 is CH or N, the other being the residue
C-Z-R2 or C-R3, wherein Z is NH or S;
- R1 is selected from the structures (a), (b) and (c):
(a), IJ(b)and IIII5(C)
r
N N N
CH3
- R2 is selected from (C,-C,o)alkyl, (C2-C,o)alkenyl and (C2-C,o)alkynyl,
optionally
independently substituted with one or more substituents selected from halogen,
hydroxy, cyano, oxo, (C,-C6)alkoxy, (C,-C6)alkylthio, (Cl-Cs)alkenyloxy,
(C,-C6)alkenylthio, (C,-C4)alkoxy(C,-C4)alkoxy, (C5-C,)cycloalkyl, a 5-
membered
unsaturated heterocycle (optionally substituted with halogen), phenyl,
phenyloxy
and phenylthio, wherein the phenyl group is optionally substituted with
halogen;
or
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- R2 is an unbranched (C2-C8)alkyl substituted at the Za-symbol of a group
with
the formula (Ia)
Ya
Wa/ \Xia
-/ (la)
X2a
Ria
wherein if X, is CH or N, X,a is CH or N and X2a is C-Za-, or
if X, is C-Z-R2, X,a is C-Za- and X2a is CH or N; and
the symbols Wa, Ya and Za and the substituent R1a have the same meanings as
defined previously for the symbols W, Y and Z and the substituent R1 and are
not
independently selected, each of the symbols Wa, Ya and Za and the substituent
R1 a representing identical symbols and substituents, respectively, as the
symbols
W, Y and Z and the substituent R1 in the other part of the structure of
formula (I);
- R3 is selected from (C4-Clp)alkyl, (C2-Clo)alkenyl and (C2-Clo)alkynyl,
optionally
independently substituted with one or more substituents selected from halogen,
hydroxy, cyano, (Cl-Cs)alkoxy, (Cl-Cs)alkylthio, (Cl-Cs)alkenyloxy,
(C,-C6)alkenylthio, (C,-C4)alkoxy(Cj-C4)alkoxy, (C5-C,)cycloalkyl, a 5-
membered
unsaturated heterocycle optionally substituted with halogen, phenyl, phenyloxy
and phenylthio, wherein the phenyl group is optionally substituted with
halogen;
or pharmaceutically acceptable salts, solvates or hydrates thereof
display affinity to muscarinic receptors, in particular to Ml and/or M4
receptors,
having muscarinic receptor modulating, in particular (partially) agonistic,
properties.
In addition, compounds of this invention display a higher (in vitro) metabolic
stability
than the prior art compound xanomeline.
The compounds of the invention are useful for treating, alleviating and
preventing
muscarinic receptor mediated diseases and conditions. Preferred compounds are
Ml
and M4 receptor agonists and may be used in the treatment of muscarinic M1/M4
mediated diseases and conditions, e.g. -but not limited to- Alzheimer's
disease,
cognitive impairment, Sjogren's disease, Schizophrenia and antinociception. In
particular, the compounds of the present invention may be used to treat,
alleviate or
prevent cognitive impairment and psychotic disorders.
In an embodiment of the invention, the compounds have formula (I) wherein
wherein
R2 is selected from (Cl-C,o)alkyl, (C2-C,o)alkenyl and (C2-C,o)alkynyl,
optionally
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independently substituted with one or more substituents selected from halogen,
hydroxy, cyano, oxo, (C,-C6)alkoxy, (Cl-Cs)alkylthio, (Cl-Cs)alkenyloxy,
(C,-C6)alkenylthio, (C1-C4)alkoxy(C,-C4)alkoxy, (C5-C,)cycloalkyl, a 5-
membered
unsaturated heterocycle (optionally substituted with halogen), phenyl,
phenyloxy and
phenylthio, wherein the phenyl group is optionally substituted with halogen.
Preferably, R2 is selected from (C,-Cs)alkyl, (C2-C8)alkenyl and (C2-
Cs)alkynyl,
optionally independently substituted with one or more substituents selected
from
halogen, hydroxy, cyano, oxo, (C,-C6)alkoxy, (C1-C4)alkoxy(C,-C4)alkoxy,
(C5-C7)cycloalkyl, tetrahydrofuranyl and phenyl, wherein the phenyl group is
optionally substituted with halogen. In particular preferred are compounds of
formula
(I) wherein R2 is selected from (C,-Cs)alkyl, (CrCs)alkenyl, optionally
substituted
with one or more halogen or (C,-C6)alkoxy.
Further, in an embodiment of the invention, R3 is selected from (C4-C,o)alkyl,
(CZ-C,o)alkenyl and (C2-C,o)alkynyl, optionally substituted with a substituent
selected
from (C5-C,)cycloalkyl or phenyl, wherein the phenyl group is optionally
substituted
with halogen.
In a further embodiment of the invention, R1 has the structure (a) or (b), in
particular
(a).
In another embodiment, the compounds have formula (I) wherein W is N and Y is
NH, in particular when X, is CH and X2 is the residue C-Z-R2 or C-R3, and Z is
0 or
S and preferably X2 is the residue C-Z-R2. Z preferably is S.
In a further embodiment Y is 0 and Z is 0 or S, and preferably Z is S.
The term halogen refers to fluoro, chloro, bromo, or iodo. Preferred is
fluoro.
The term (Cl-Clo)alkyl means a branched or unbranched alkyl group having 1-10
carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, n-pentyl,
sec-pentyl,
hexyl, octyl. In particular in the residue C-Z-R2 when Z is 0 or S,
unsubstituted n-
pentyl is a preferred alkyl group. Preferred substituted R2 alkyl groups are
ethoxyethyl, when Z is 0 or S, and -(CH2)3CF3 when Z is S.
The term (C,-C6)alkoxy means an alkoxy group having 1-6 carbon atoms, wherein
the alkyl moiety is as defined above. The term (Cl-C6)alkylthio has a similar
meaning.
The term (C1-C4)alkoxy(Cj-C4)alkoxy means a(Cl-C4)alkoxy group, the alkyl
moiety
of which is in turn substituted with (Cl-C4)alkoxy.
The term (C2-Cs)alkenyl means a branched or unbranched alkenyl group having 2-
8
carbon atoms wherein the double bond(s) may be present at different parts of
the
group, for example vinyl, allyl, butenyl, n-pentenyl, sec-pentenyl, hexenyl,
octenyl,
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etc. In the residue C-Z-R2, when Z is 0 or S, a preferred alkenyl group is 4-
pentenyl
and a preferred substituted alkenyl group is 4,4-difluoro-but-3-enyl.
The term (Cl-C6)alkenyloxy means an alkenyloxy group having 1-6 carbon atoms,
wherein the alkenyl moiety is as defined above. The term (C,-C6)alkenylthio
has a
5 similar meaning.
The term (C2-Cs)alkynyl means a branched or unbranched alkynyl group having 2-
8
carbon atoms wherein the triple bond(s) may be present at different parts of
the
group, for example ethynyl, propargyl, 1-butynyl, 2-butynyl, etc.
The term (C5-C7)cycloalkyl means a cyclic alkyl group having 5-7 carbon atoms,
thus
cyclopentyl, cyclohexyl, cyclopheptyl or cyclooctyl.
The term 5-membered unsaturated heterocycle in the definition of R2 means a
heterocycle containing 5 atoms, wherein at least one atom is a heteroatom
selected
from 0, N and S, the other atoms being carbon atoms, wherein the heterocycle
further at least contains one double bond. Examples are furanyl and pyrrollyl
groups.
With reference to substituents, the term "independently" means that the
substituents
may be the same or different from each other.
The compounds of the invention may suitably be prepared by methods available
in
the art, and as illustrated in the experimental section of this description.
Some novel
and useful intermediates have been found for the preparation of the compounds
of
this invention.
Thus, another embodiment of the invention is a heterocyclic compound of the
formula
(II)
Y*
W*/ X
-5<2*
N
wherein
- the heterocycle comprises two double bonds which may be present at several
positions, represented by the dashed lines (---);
- the heterocycle comprises two heteroatoms,
- W* being N, NH or N-2-(trimethylsilyl)ethoxymethyl;
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- Y* being CH, 0, N or NR4, wherein R4 is selected from H, 2-(trimethylsilyl)-
ethoxymethyl, -SOZN(CH3)Z and -SOZphenyl; wherein
if Y* is 0, X,* is CH and X2* is the residue C-Z*-R2* or C-R3*, wherein Z* is
NH, 0, or S; and
if Y* is CH or NH, one of X,* and X2* is CH or N, the other being the residue
C-Z*-R2* or C-R3*, wherein Z* is NH or S;
- R2* is selected from (C,-C$)alkyl, (Cz-C$)alkenyl and (Cz-C$)alkynyl,
optionally
independently substituted with one or more: halogen, hydroxy, cyano, oxo,
P-Cs)alkoxy, P-Cs)alkylthio, P-Cs)alkenyloxy, P-Cs)alkenylthio,
(C,-C4)alkoxy(C,-C4)alkoxy, (C5-C,)cycloalkyl, a 5-membered unsaturated
heterocycle (optionally substituted with halogen), phenyl, phenyloxy or
phenylthio,
wherein the phenyl group is optionally substituted with halogen;
or
- R2* is an unbranched (C2-C$)alkyl substituted at the Z*a-symbol of a group
with the
formula (Ila)
W*a/X,a
Y *
(Ila)
- - - X2*a
~ ~
-N
wherein if X,* is CH or N, X1 *a is CH or N and X2*a is C-Z*a-, or
if X,* is C-Z*-R2*, Xi*a is C-Z*a- and X2*a is CH or N; and
the symbols W*a, Y*a and Z*a have the same meanings as defined previously for
the
symbols W*, Y* and Z* and are not independently selected, each of the symbols
W*a, Y*a and Z*a representing identical symbols, respectively, as the symbols
W*,
Y* and Z* in the other part of the structure of formula (II); and
- R3* is selected from (C4-C,o)alkyl, (CrC,o)alkenyl and (C2-C,o)alkynyl,
optionally
independently substituted with one or more substituents selected from halogen,
hydroxy, cyano, (C,-C6)alkoxy, (C,-C6)alkylthio, (C,-C6)alkenyloxy, (C,-
C6)alkenylthio,
(C,-C4)alkoxy(C,-C4)alkoxy, (C5-C,)cycloalkyl, a 5-membered unsaturated
heterocycle optionally substituted with halogen, phenyl, phenyloxy and
phenylthio,
wherein the phenyl group is optionally substituted with halogen,
which compound is useful in the preparation of compounds of formula(I) wherein
R1
has the structure (a). The preferred substitution pattern in the compound of
formula
(II) corresponds to the preferred substitution pattern of compounds of formula
(I).
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Also an embodiment of this invention is a heterocyclic compound of the formula
(III)
N-O
R5
R6 (III)
N
wherein R5 is H and R6 is Br,
or R5 is -Si(CH3)3 and R6 is Br or -Si(CH3)3,
which compound is useful in the preparation of compounds of formula (I)
wherein R1
has the structure (a).
The compounds of the present invention may contain one or more asymmetric
centers and can thus occur as racemates and racemic mixtures, single
enantiomers,
diastereomeric mixtures and individual diastereomers. Additional asymmetric
centers
may be present depending upon the nature of the various substituents on the
molecule. Each such asymmetric center will independently produce two optical
isomers and it is intended that all of the possible optical isomers and
diastereomers
in mixtures and as pure or partially purified compounds are included within
the ambit
of this invention. The present invention is meant to comprehend all such
isomeric
forms of these compounds. The independent syntheses of these diastereomers or
their chromatographic separations may be achieved as known in the art by
appropriate modification of the methodology disclosed herein. Their absolute
stereochemistry may be determined by the x-ray crystallography of crystalline
products or crystalline intermediates which are derivatized, if necessary,
with a
reagent containing an asymmetric center of known absolute configuration. If
desired,
racemic mixtures of the compounds may be separated so that the individual
enantiomers are isolated. The separation can be carried out by methods well
known
in the art, such as the coupling of a racemic mixture of compounds to an
enantiomerically pure compound to form a diastereomeric mixture, followed by
separation of the individual diastereomers by standard methods, such as
fractional
crystallization or chromatography.
Compounds may exist as polymorphs and as such are intended to be included in
the
present invention. In addition, compounds may form solvates with water (i.e.,
hydrates) or common organic solvents, and such solvates are also intended to
be
encompassed within the scope of this invention.
Isotopically-labeled compound of formula (I) or pharmaceutically acceptable
salts
thereof, including compounds of formula (I) isotopically-labeled to be
detectable by
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PET or SPECT, also fall within the scope of the invention. The same applies to
compounds of formula (I) labeled with [13C]_, [14C]_ [3H]_ [18F]_ [1251]- or
other
isotopically enriched atoms, suitable for receptor binding or metabolism
studies.
The term "pharmaceutically acceptable salt" refers to those salts that are,
within the
scope of sound medical judgment, suitable for use in contact with the tissues
of
humans and lower animals without undue toxicity, irritation, allergic
response, and
the like, and are commensurate with a reasonable benefit/risk ratio.
Pharmaceutically
acceptable salts are well-known in the art. They can be prepared in situ when
finally
isolating and purifying the compounds of the invention, or separately by
reacting
them with pharmaceutically acceptable non-toxic bases or acids, including
inorganic
or organic bases and inorganic or organic acids.
The compounds of the invention may be administered enterally or parenterally.
The
exact dose and regimen of these compounds and compositions thereof will be
dependent on the biological activity of the compound per se, the age, weight
and sex
of the patient, the needs of the individual subject to whom the medicament is
administered, the degree of affliction or need and the judgment of the medical
practitioner. In general, parenteral administration requires lower dosages
than other
methods of administration which are more dependent upon adsorption. However,
the
dosages for humans are preferably 0.001 - 10 mg per kg body weight, more
preferably 0.01 - 1 mg per kg body weight. In general, enteral and parenteral
dosages will be in the range of 0.1 to 1,000 mg per day of total active
ingredients.
The medicament manufactured with the compounds of this invention may also be
used as adjuvant in therapy. In such a case, the medicament or is administered
in a
combination treatment with other compounds useful in treating such disease
states.
Also pharmaceutical combination preparations comprising at least one compound
of
the present invention and at least one other pharmacologically active
substance are
considered in this respect.
Mixed with pharmaceutically suitable auxiliaries, e.g. as described in the
standard
reference "Remington, The Science and Practice of Pharmacy" (21S` edition,
Lippincott Williams & Wilkins, 2005, see especially Part 5: Pharmaceutical
Manufacturing) the compounds may be compressed into solid dosage units, such
as
pills or tablets, or be processed into capsules or suppositories. By means of
pharmaceutically suitable liquids the compounds can also be applied in the
form of a
solution, suspension or emulsion.
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For making dosage units, e.g. tablets, the use of conventional additives such
as
fillers, colorants, polymeric binders and the like, is contemplated. In
general, any
pharmaceutically suitable additive which does not interfere with the function
of the
active compounds can be used.
Suitable carriers with which the compounds of the invention can be
administered
include for instance lactose, starch, cellulose derivatives and the like, or
mixtures
thereof, used in suitable amounts. Compositions for intravenous administration
may
for example be solutions of the compounds of the invention in sterile isotonic
aqueous buffer. Where necessary, the intravenous compositions may include for
instance solubilizing agents, stabilizing agents and/or a local anesthetic to
ease the
pain at the site of the injection.
Pharmaceutical compositions of the invention may be formulated for any route
of
administration and comprise at least one compound of the present invention and
pharmaceutically acceptable salts thereof, with any pharmaceutically suitable
ingredient, excipient, carrier, adjuvant or vehicle.
By "pharmaceutically suitable" it is meant that the carrier, diluent or
excipient must be
compatible with the other ingredients of the formulation and not deleterious
to the
recipient thereof.
In an embodiment of the invention, a pharmaceutical pack or kit is provided
comprising one or more containers filled with one or more pharmaceutical
compositions of the invention. Associated with such container(s) can be
various
written materials such as instructions for use, or a notice in the form
prescribed by a
governmental agency regulating the manufacture, use or sale of pharmaceuticals
products, which notice reflects approval by the agency of manufacture, use, or
sale
for human or veterinary administration.
Unless otherwise defined, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which
this invention pertains. Although methods and materials similar or equivalent
to those
described herein can be used in the practice or testing of the present
invention,
suitable methods and materials are described in this document. All
publications,
patent applications, patents, and other references mentioned herein are
incorporated
by reference in their entirety. In case of conflict, the present
specification, including
definitions, will control.
The following examples are only intended to further illustrate the invention
in more
detail, and therefore these examples are not deemed to restrict or limit the
scope of
the invention in any way.
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EXAMPLES
1. MATERIALS AND METHODS
5
Nuclear magnetic resonance spectra ('H NMR and 13C NMR, APT) were
determined in the indicated solvent using a Bruker ARX 400 (1H: 400 MHz, 13C:
100
MHz) at 300 K, unless indicated otherwise. 19F NMR and13C NMR experiments were
carried out on a Varian Inova 500 spectrometer operating at 11.74 T (499.9 MHz
for
10 'H; 125.7 MHz for13C; 50.7 Mhz, 470.4 MHz for 19F) using a 5 mm SW probe.
The
spectra were determined in deuterated chloroform or dichloromethane obtained
from
Cambridge Isotope Laboratories Ltd. Chemical shifts (8) are given in ppm
downfield
from tetramethylsilane (1 H, 13C) or CCI3F (19F). Coupling constants J are
given in
Hz. Peakshapes in the NMR spectra are indicated with the symbols `q'
(quartet), `dq'
(double quartet), 't' (triplet), 'dt' (double triplet), 'd' (doublet), 'dd'
(double doublet), `s'
(singlet), `bs' (broad singlet) and `m' (multiplet). NH and OH signals were
identified
after mixing the sample with a drop of D20.
Flash chromatography refers to purification using the indicated eluent and
silica gel
(either Acros: 0.030-0.075 mm or Merck silica gel 60: 0.040-0.063 mm).
Column chromatography was perfiormed using silica gel 60 (0.063-0.200 mm,
Merck).
Melting points were recorded on a Buchi B-545 melting point apparatus.
Mass spectra (MS) were recorded on a Micromass QTOF-2 instrument with
MassLynx application software for acquisition and reconstruction of the data.
Exact
mass measurement was done of the quasimolecular ion [M+H]+. Accurate mass
measurements were performed using a JEOL JMS-SX/SX 102 A Tandem Mass
Spectrometer using Fast Atom Bombardement (FAB). A resolving power of 10,000
(10% valley definition) for high resolution FAB mass spectrometry was used.
All reactions involving moisture sensitive compounds or conditions were
carried out
under an anhydrous nitrogen atmosphere.
Reactions were monitored by using thin-layer chromatography (TLC) on silica
coated plastic sheets (Merck precoated silica gel 60 F254) with the indicated
eluent.
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Spots were visualised by UV light (254 nm) or 12.
Extinction coefficients were determined with a HP 8453 UV-Vis
spectrophotometer.
Analytical high performance liquid chromatography (HPLC) was performed on a
C18 column (Inertsil ODS-3, particle size 3 mm; 4.6mm 50mm) using the
following
elution gradient: linear gradient of 5 % to 95 % aqueous CH3CN containing 0.04
%
HCO2H over 5 min, then 95 % aqueous CH3CN containing 0.04 % HCO2H for 2 min
at 2.0 ml min-'. Products were detected at k = 254 nm.
Liquid Chromatography- Mass Spectrometrry (LC-MS), method A
The LC-MS system consists of 2 Perkin elmer series 200 micro pumps. The pumps
are connected to each other by a 50 pl tee mixer, connected to a Gilson 215
auto
sampler. The method is as follows:
step total time flow (ul/min) A(%) B(%)
0 0 2000 95 5
1 1.8 2000 0 100
2 2.5 2000 0 100
3 2.7 2000 95 5
4 3.0 2000 95 5
A= 100% Water with 0.025% HCOOH and 10mmol NH4HCO0 pH= +/- 3
B= 100% ACN with 0.025% HCOOH
The auto sampler has a 2 pl injection loop. The auto sampler is connected to a
Waters Atlantis C18 30*4.6 mm column with 3 pm particles. The column is thermo
stated in a Perkin Elmer series 200 column oven at 40 C. The column is
connected
to a Perkin Elmer series 200 UV meter with a 2.7 ial flowcel. The wavelength
is set to
254 nm. The UV meter is connected to a Sciex API 150EX mass spectrometer. The
mass spectrometer has the following parameters:
Scanrange:150-900 a.m.u.; polarity: positive; scan mode: profile ; resolution
Q1:
UNIT ; step size: 0.10 a.m.u.; time per scan: 0.500 sec; NEB: 10; CUR: 10
IS: 5200; TEM: 325; DF: 30; FP: 225 and EP: 10.
The light scattering detector is connected to the Sciex API 150. The light
scattering
detector is a Sedere Sedex 55 operating at 50 C and 3 bar N2.
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The complete system is controlled by a G3 powermac.
Liquid Chromatography- Mass Spectrometry (LC-MS), method B
The LC-MS system consists of an Agilent series 1100 system consisting of the
following components:
- G1379A Degasser
- G1312A Binary Pump
The pumps are connected to a G1313AALS auto sampler.
The method is as follows:
Step total flow A (%) B(%)
Time (min) (ml/min)
0 0 1.0 2 98
1 10.5 1.0 98 2
2 18.0 1.0 98 2
3 18.1 1.0 2 98
4 24.0 1.0 2 98
A: Acetonitrile with 0.1% HCOOH or Acetonitrile with 10 mM NH3
B: Water with 0.1% HCOOH or Water with 10 mM NH3
The auto sampler is connected to a Zorbax Extend C18 column 150x4.6 mm with
3.5
um particles.
The column is thermo stated in a G1316A Colcomm column oven at 35 C.
The column is connected to a G1315B DAD diode array detector. The wavelength
range is set from 220 to 320 nm. The UV meter is connected to G1946D MSD mass
spectrometer, operating in electron spray mode.
The mass spectrometer has the following parameters:
Scan range: 100 - 800 amu
Polarity: Positive & Negative
Mode: Scan
Step size: 0.20
Cycle time: 1.04 sec
%Cycle time: 50%
Drying gas: Nitrogen
Gas flow: 10 I/min
Gas temp.: 300 C
Neb. Press.: 30 psi
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13
Capillary Volt.: 3000 V
An Alltech ELSD 2000 detector is connected parallel with the MSD. The flow is
split
after the DAD.
The ELSD has the following parameters:
Drying gas: Nitrogen
Gas flow: 1.5 I/min
Drift tube temp.: 39 C
Impactor: On
2. ABBREVIATIONS
n-BuLi n-butyl lithium
t-BuOH t-butanol
dba dibenzylideneacetone
DCM dichloromethane
DMF N,N'-dimethylformamide
DMF-DMA N,N'-dimethylformamide dimethyl acetal
DMSO dimethylsulfoxide
EtOH ethanol
Et20 diethyl ether
g gram(s)
h hour(s)
Me methyl
Mel methyl iodide
MeOH methanol
mg milligram(s)
min minute(s)
ml milliliter(s)
m.p. melting point c.q. melting range
NBS N-bromosuccinimide
NIS N-iodosuccinimide
PE petroleum ether (40-65 oC)
Rt retention time (LC/MS)
SEM-CI (2-Chloromethoxy-ethyl)-trimethylsilane
TBAF tetrabutylammonium fluoride
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THF tetrahydrofuran
3. GENERAL ASPECTS OF SYNTHESES
Different synthetic routes for the preparations of compounds of the present
invention
illustrated in formula I are described and easily prepared from readily
available
starting materials. More general information on pyrazole, imidazole and
isoxazole
chemistry, see for example: J.A. Joule, K. Mills and G.F. Smith, "Heterocyclic
Chemistry", third edition, Stanley Thornes (Publishers) Ltd., Cheltenham,
1998. More
information on addition and subsequent removal of protective groups in organic
synthesis can be found in : T.W. Greene and P.G.M. Wuts, "Protective Groups in
Organic Synthesis", third edition, John Wiley & Sons, Inc., New York, 1999.
The selection of the particular method depends on factors such as the
compatibility
of functional groups with the reagent used, the possibility to use protecting
groups,
catalysts, activating and coupling reagents and the ultimate structural
features
present in the final compound being prepared.
In an example of the general procedure (scheme 1), nicotinoyl chloride
hydrochloride
(1) is converted to the N-methyl-N-methoxyamide (2) in the presence of a base
and
reacted with hexyl-lithium (J. Med. Chem., 35, 1992, 2392-2406) to produce 1-
pyridin-3-yl-heptan-l-one (3).
Mild a-methylenation of compound 3 (J. Org. Chem., 71, 2006, 2538-2541)
afforded
2-methylene-1 -pyridin-3-yl-heptan-1 -one (4), which was reacted with
hydrazine
(Synthesis, 1989, 320-321) to produce 1-(4-pentyl-3-pyridin-3-yl-4,5-
dihydropyrazol-
1-yl)-ethanone (5). Oxidation of a 2-pyrazoline to a pyrazol can be
accomplished
using methods well known to those skilled in the art. Specific conditions are:
activated Mn02 in dichloroethane (EP0094555) to produce compound 6, which was
deprotected under basic conditions to afford compound 7. The 3-(4-pentyl-1 H-
pyrazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine derivate (9) was obtained
from
compound 8 by quarternizing the pyridine moiety with CH31 and reducing the
corresponding pyridinium salt with NaBH4 (Arch. Pharm. Pharm. Med. Chem., 336,
2003, 143-154).
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Scheme 1
0 0 0
(N) CI NHCH3(OCH3).HCI I\ i 'O\ .HCI Pyridine N THF
N
1 2 \ 3 O
C i-N i-N
HCHO I NH2NH2 \ MnOz \
HOAc
Piperidine
MeOH N CH3COOH N Dichloroethane N
4 5 6
H
N-N N-N N-N
~
2M NaOH/ EtOH 1:1 \ Mel ~/ NaBH4 rN
~ Acetone I MeOH
7 1 I+ 8 I 9
In another example of the general procedure (scheme 2), readily available
pyridin-3-
yl-acetic acid (10) is converted to the N-methyl-N-methoxyamide derivate (12),
which
5 is reacted with BuLi to produce 1-pyridin-3-yl-hexan-2-one (13). N,N-
dimethyl-
formamide dimethylacetal treatment of 13 furnished the enamine (14) which is
converted to the pyrazol (15). The synthesis of 3-(4-Butyl-1 H-pyrazol-3-yl)-
1,2,5,6-
tetrahydro-l-methylpyridine (16) is illustratated in scheme 2 according to the
two step
sequence shown in scheme 1.
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16
Scheme 2
(COCI)2 ~
0 DMF _ \ O NHCH3(OCH3).HCI N,
O I
I i OH DCM I i CI pyridine
N N
.HCI
11 12
NI-I
DMFDMA O
\ t-BuOH IN
THF N ~ O -
13 14
H H
N-N N-N
NH2NH2.H20 Mel NaBH4 \
EtOH ~ C"Y
I ~ Acetone MeOH
N i
16
In yet another example of the general procedure (scheme 3), 3-(4-bromo-1 H-
pyrazol-
3-yl)-pyridine (20A) or its iodo analog (20B) (Bioorganic & Medicinal
Chemistry, 4,
1996, 227-237) is used as precursor for the synthesis of compounds of the
general
5 formula I. The di-lithio derivate (Bioorganic & Medicinal Chemistry, 8,
2000, 2317-
2335) of 20A, prepared on multigram scale by pyrazol-NH deprotonation and
bromine-lithium exchange (2,1 equiv. n-Buli, THF, -78 C, 2 hr), was trapped
with a
disulfide (for example methyldisulfanylmethane) affording 3-(4-methylsulfanyl-
1 H-
pyrazol-3-yl)-pyridine 21A, which was converted to the 1,2,5,6-tetrahydro-l-
10 methylpyridine derivate 22A according to the two step sequence shown in
scheme 1.
The generation of anions at the ortho position of the aromatic systems
employed in
the synthetic procedures described in this application is performed according
to a
general synthetic strategy known as Directed Ortho Metalation (DOM). Within
this
15 area, a number of functional groups known as Directed Metalation Groups
(DMG's)
have been studied for this purpose.
The dimethylsulfonamide group as Directing Metalation Group (DMG) in the Ni-
position of 3-pyridin-3-yl-pyrazole-l-sulfonic acid dimethylamide (23) enables
the
lithiation of the 5-position and thereby its functionalisation (Chem. Ber.,
124, 1991,
1639-1650). The 5-lithioderivate of 23, prepared on multigram scale by a-
metallation
(1,0 equiv, t-Buli, THF, -78 C, 1 hr), was trapped (J. Org. Chem., 64, 1999,
5366-
5370) with a disulfide (for example 1-butyldisulfanylbutane) to afford 5-
butylsulfanyl-
3-pyridin-3-yl-pyrazol-l-sulfonic acid dimethylamide 24, which was deprotected
(25)
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and converted to the 1,2,5,6-tetrahydro-l-methylpyridine derivate 26 according
to the
two step sequence shown in scheme 1.
Scheme 3
H
O O N-N
DMFDMA HZNNHZ.HZO
(N) EtOH I\ EtOH (N)
N 17 18 19
NBS N-N i-N N-N
DMF 2.1 eq BuLi
-400C_ CH3SSCH3 cr /
N -~
Br THF N S N /S
20A 21A 22A
H
NIS N-N
DMF
0 C \
19 _ I ~ I
N
20B
N-N SO2N(CH3)2 SO2N(CH3)Z
N-N t-BuLi N-
N
\ I~ CISOzN(CH3)2 BuSSBu I/
S
I \ THF
Pyridine
i -~
N N N
19 23 24
N_N _N S
\ I ~ S
2 M KOH
26
CN 25 N
Another synthetic route for the preparations of compounds of the present
invention
illustrated in formula I is described in scheme 4. The introduction of the 2-
(trimethylsilyl)-ethoxymethyl group (SEM) as a protective group (Tetrahedron
Letters,
39, 1998, 5171-5174) afforded a mixture of compounds 27A and 27B. Subsequent
bromine-lithium exchange (1,1 equiv. n-Buli, THF, -78 C, 1 hr) and reacting
this 4-
lithioderivate of 27A/B with S8 generates the intermediate lithium aryl
thiolate (J. Org.
Chem., 69, 2004, 3236-3239) of 3-pyridin-3-yl-1-(2-trimethylsilanyl-
ethoxymethyl)-1 H-
pyrazol-4-thiol. This intermediate was trapped with 4-bromo-1,1,1-trifluoro-
butane to
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18
afford a mixture of 28A and 28B. Subsequent removal of the SEM protecting
group
resulted in the desired 3-[4-(4,4,4-trifluoro-butylsulfanyl)-1 H-pyrazol-3-yl]-
pyridine
21B, which was converted to the 1,2,5,6-tetrahydro-l-methylpyridine derivate
22B
according to the two step sequence shown in scheme 1.
Scheme 4
H SEM
N'N NaH NN SEMN_N
SEM-CI
THF
iXr N Br N + Br
N
20A 27A 27B
SEM
~ SEM
N-N ~N
1.BuLi THF
2.S8 \
N S + I N S
4-Bromo-1,1,1-trifluoro-butane
28A 28B
F F
F F
H
H F N-N F
N-N
TBAF \ I ~ \
THF S
S N
N
21B 22B
F F
F F
F
F
In another aspect, 3-(4-iodo-1 H-pyrazol-3-yl)-pyridine (20B, scheme 5) is
employed
as starting material for compounds of the present invention illustrated in
formula I.
The 4-lithioderivate of the SEM protected derivative 29A/B, prepared according
to the
corresponding compounds 27A/B (scheme 4), was reacted with trimethyl borate
followed by in situ hydrogen peroxide oxidation (J. Heterocyclic Chem., 31,
1994,
1377-1380) to afford the corresponding 3-pyridin-3-yl-1-(2-trimethylsilanyl-1-
ethoxymethyl)-1 H-pyrazol-4-ol (30, one isomer given). Alkylation of the 4-
hydroxy
derivate 30 can be accomplished using methods well known in the art, for
example,
by reacting compound 30 with K2CO3 in DMF in the presence of a variety of
(aryl)alkyl halides, for example (3-bromo-propyl)-benzene, to generate
compound
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31A (one isomer given). Subsequent removal of the SEM group resulted in 3-[4-
(3-
phenyl-propoxy)-l H-pyrazol-3-yl]-pyridine (32A), which was converted to the
1,2,5,6-
tetrahydro-l-methylpyridine derivate 33A according to the two step sequence
shown
in scheme 1.
Scheme 5
H SEM
SEM
_N NaH _N N
SEM-CI N
THF \ \ ~
I ~ I ~ I i I + I ~ I
N N N
20B 29A 29B
SEM SEM
N-N N-N
BuLi ~~ B \ I ~~
THF \
B(OMe)3 ()H K2C03 0
H202 N DMF N
30 31A
H H
i i
N-N N-N ~
TBAF
THF
N N
32A I 33A
~
Scheme 6 illustrates two alternative methods of preparing 3-(4-alkoxy-1 H-
pyrazol-3-
yl)-1,2,5,6-tetrahydro-l-methylpyridine compounds.
The mixture of readily available SEM protected pyrazoles 29A/B was converted
to a
mixture of 34A/B by the efficient two step sequence described in scheme 1
(quarternizing the pyridine moiety with CH31 and reducing the corresponding
pyridinium salt with NaBH4).
Techniques for the formation of C-0 bonds have been reported (e.g. J. Am.
Chem.
Soc., 123, 2001, 10770). More precisely, an efficient transformation of
primary
alcohols with the 4-iodo-pyrazole analog (34A/B) could be achieved using the
Cul/1,10-phenanthroline catalyzed cross-coupling methodology (Organic Letters,
4,
2002, 973-976). Subsequent deprotection of compound 35A (one isomer given)
yielded 3-(4-hexyloxy-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine
(33B).
In an alternative synthetic sequence (scheme 6), C-O bond formation can be
accomplished using the aforementioned Cul/1,10-phenanthroline catalyzed cross-
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coupling methodology to generate compound 31G. The synthesis of compound 33G
has been illustrated in scheme 6, according to the procedures illustrated in
scheme
5.
Scheme 6
SEM MES
~'N \N
29A + 29B
+ I
34A ~ 34B
SEM H
N-N N-N
I TBAF
Cs ~
2CO3 THF
0 O
1,10-Phenanthroline
C6H130H I I
35A L 33B
SEM H
29A + 29B N-N N-N N-H
N
Cul
Cs2CO3 ~ I/ TBAF
c THF_
C5H110H / O / O - O
1,10-Phenanthroline N
I
IL IL N
31 G 32G 33G
One aspect of the invention relates to bis 3-(4-alkylsulfanyl-1 H-pyrazol-3-
yl)-1,2,5,6-
tetrahydro-l-methylpyridine derivatives (for example compound 22C, scheme 7)
that
bind to and can activate muscarinic receptors (J. Med. Chem., 44, 2001, 4563-
4576).
10 Introdution of the phenylsulfonyl as protective group (starting from 20A)
was
accomplished regio-selectively to generate 3-(1 -phenylsulfonyl-4-bromo-1 H-
pyrazol-
3-yl)-pyridine (36A from 20A).
The cross-coupling of aliphatic and aromatic thiols and aryl bromides can be
mediated by a Pd2(dba)3/Xantphos catalytic system in refluxing xylene to
afford the
15 corresponding aryl thioethers (Organic Lettters, 6, 2004, 4587-4590,
Tetrahedron,
61, 2005, 5253-5259).
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Using this methodology, 36A was converted to the protected bis-alkylsulfanyl-
pyrazol
derivative 37. Removal of the N,-phenylsulfonyl group can be accomplished
using
methods well known to those skilled in the art, for example, optionally
reacting
compound 37 with potassium hydroxide in diethylene glycol in the presence of
hydrazine. Quarternizing the (bis)-pyridine moiety with CH31 and reducing the
corresponding (bis)-pyridinium salt with NaBH4 afforded pyrazol derivative
22C.
Scheme 7 illustrates an alternative method for preparing 3-(4-alkylsulfanyl-1
H-
pyrazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine derivatives, so can be used
to
synthezise compounds presented in scheme 3 & 4 (22A and 22B respectively), the
main feature being the availability of the parent thiol.
Scheme 7
H SO2Ph
N-N N-N
NaHITHF
I i Br CISOZPh I ~ Br
N N
20A SO2Ph 36A H
N-N N-N
Y deprotection Y
K2CO3 I i S N S
Pd2(dba)3 N scheme 1 I
Xantphos 37 22C
Xylene
5 S
N-SO2Ph NH
N N
N `N
Another illustration of the preparation of compounds of the present invention
of
formula I is shown in scheme 8.
Straightforward nitration (Chem. Ber., 88, 1955, 1577) of pyrazole derivative
19,
afforded 3-(4-nitro-1 H-pyrazol-3-yl)-pyridine (38), which was reduced to the
corresponding 3-pyridin-3-yl-1 H-pyrazol-4-ylamine (39) and reacted with an
acid
chloride, for example butyryl chloride, to generate the amide (40). Subsequent
conversion to the 1,2,5,6-tetrahydro-l-methylpyridine derivate 41 was done
accordingly to the two step sequence shown in scheme 1. Subsequent LiAIH4
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reduction of the amide generates butyl-[3-(1-methyl-1,2,5,6-tetrahydro-pyridin-
3-yl)-
1 H-pyrazol-4-yl]-amine (42).
Scheme 8
H H H
N-N H SO N-N N-N
I\ I~ HNO34 I Pd/ C~ \ ~~
N02 MeOH ~ NH2
N N N
19 38 39
H
Et3N i-N N-N N
N-
~~~ \ / \ I / LiAIH4 \ I ~
CH3CN N HN N HN THF HN
N
40 I 41 ~ 42
Scheme 9 illustrates the preparation of 3-(4-alkynyl (and alkenyl)-1 H-pyrazol-
3-yl)-
1,2,5,6-tetrahydro-1-methylpyridine derivatives as compounds of the formula I.
3-(1-Phenylsulfonyl-4-bromo-1 H-pyrazol-3-yl)-pyridine (36A, scheme 7) or its
iodo
analog 36B (scheme 9) are excellent substrates for Sonogashira couplings with
terminal acetylenes (Tetrahedron Letters, 38, 1997, 7835-7838., Eur J. Org.
Chem.,
2006, 3283-3307). Catalysis with PdCl2(PPH3)2 in the presence of Cul (excess
Et3N,
DMF, 80 C, 2 hr) and (for example) hex-1-yne generates compound 43A.
Subsequent deprotection (accordingly to scheme 7), followed by the two step
sequence shown in scheme 1, afforded 3-(4-hex-1-ynyl-1H-pyrazol-3-yl)-1,2,5,6-
tetrahydro-1-methylpyridine (45A).
The scope and reactivity of compound 36A (or 36B) is further illustrated by
the
Suzuki-Miyaura coupling reactions with (for example) alkenylboronic acids.
Catalysis
with Pd(OAc)2 and the effective S-Phos in the presence of K3PO4 and (for
example)
(E)-hexen-1-ylboronic acid (J. Am. Chem. Soc., 127, 2005, 4685-4696) afforded
the
alkenyl 46A. Subsequent deprotection, followed by the two step sequence shown
in
scheme 1 afforded 3-(4-hex-1-enyl-1H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methyl-
pyridine (47A).
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Scheme 9
H SO2Ph SO2Ph
N-N N-N N-N
PhsoZCi i I NaH/THF I ~ I PdCl2(PPh3)2 I i
N N PPh3 N
20B 36B Et3IN 43A
DMF
H H
N-N N-N
2-(2-Hydroxy-ethoxy)-ethanol ~ I KoH N \\ N
HZNNHZ.HZO I/
44A 45A
SO2Ph H
N
B(OH)2 I-N i-
36A - ~ - ~
K3P04
Pd(OAc)2
S-Phos N
Toluene 46A 47A
In another aspect, 3-(4-iodo-1 H-pyrazol-3-yl)-1-azabicyclo[2.2.2]octane (48,
Bioorganic & Medicinal Chemistry 8, 2000, 449-454) is employed as starting
material
for compounds of the present invention of formula I (scheme 10).
Referring to scheme 3, the di-lithio derivative (Bioorganic & Medicinal
Chemistry, 8,
2000, 2317-2335) of 48, (2,1 equiv. n-Buli, THF, -78 C, 2 hr), was trapped
with a
disulfide (for example 1-butyldisulfanyl-butane) to afford the corresponding 3-
(4-
butylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclo[2.2.2]octane 49A.
Scheme 10 illustrates an alternative - but also general - method of preparing
3-(4-
alkylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclo[2.2.2]octane derivatives. So,
compound
48 could be converted by the Pd2(dba)3/Xantphos catalytic system (analogous to
scheme 7, but in DMF at 120 C) yielding the corresponding aryl thioether 49B,
in a
single step without protection.
The mixture of readily available SEM protected pyrazoles 50 (one isomer given)
was
converted to a mixture of 51A by the Cul/1,10-phenanthroline catalyzed cross-
coupling methodology as described in scheme 6, however using different
conditions.
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Subsequent deprotection of 51A yields the corresponding 3-(4-butoxy-1 H-
pyrazol-3-
yl)-1-azabicyclo[2.2.2]-octane 52A.
Scheme 10
H
H 2.1 eq BuLi N-N
N-N C4H9SSC4H9 n--y,
N THF
N S
648 49A
Hs H
N-N
NaH K2CO3 ~
THF Pd2(dba)3 /
SEM-CI
Xantphos S
DMF N ~
49B
SEM Cul ,SEM H
N-N CsZCO3 NI-N TBAF NI N
C4H9OH ~ THF
1,10-Phenanthroline
p & -
N 150 C N O N O
Microwave
50 51A - 52A
A further illustration of the preparation of compounds of the present
invention of
formula I is shown in scheme 11.
The readily available 1-aza-bicyclo[3.2.1]octan-6-one (53), (J. Med. Chem.,
36, 1993,
683-689) was converted to 6-(1 H-pyrazol-3-yl)-1-azabicyclo[3.2.1]-6-o1 (55),
analogous to an efficient two step sequence (Bioorganic & Medicinal Chemistry
8,
2000, 449-454). Attempts to improve the yield of dehydratation of the alcohol
(55)
was accomplished by acylation (56) (Heterocycles, 24, 1986, 971-977) and
subsequent elimination in the heat (185 C). Reduction of the enamine (57) gave
the
anticipated endo 1-azabicyclo[3.2.1] derivative (58). lodination (Bioorganic &
Medicinal Chemistry, 4, 1996, 227-237) and introduction of pentane-l-thiol by
the
Pd2(dba)3/Xantphos catalytic system (accordingly to scheme 7) afforded endo-6-
(4-
pentylsulfanyl-l-H-pyrazol-3-yl)-1-azabicyclo[3.2.1 ]octane (60A).
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Scheme 11
C
H
O O----, / o i-N
H2NNHZ.HCI ~
CH
-~ ~ -~ H
t-BuOK EtOH
THF CN)<
CN?
53 5
4 55
H
~ N-N N-N
(CH3C0)ZO 185 C Pd/ C ON CN~
Pyridine N Benzene 0 MeOH
56 57 58
H
N-N Pd2(dba)3 N-N
Xantphos
NIS SH
DMF 11 ~ S
N
C N Xylene/DMF
59 KzC03 60A
In scheme 12, yet another illustration of the preparation of compounds of the
present
invention of formula I is shown.
Commercially available 3-pyridinealdoxime (61) is converted to its
5 chlorohydroxyimino derivative (US 2004/0157900) which is converted to
nicotinonitrile oxide (Tetrahedron, 61, 2005, 4363-4371) "in situ" and reacted
with
1,2-bis-trimethylsilanyl-ethyne (Chem. Ber., 107, 1974, 3717-3722) to afford
the 1,3-
dipolar cycloaddition product 3-(4,5-bis-trimethylsilanyl-isoxazol-3-yl)-
pyridine (62).
Halogen-induced ipso desilylation resulted in the 4-bromo-5-trimethylsilanyl
10 derivative (63). Subsequent desilylation with NH4OH (Chem. Ber., 112, 1979,
2829-
2836) generates 3-(4-bromo-isoxazol-3-yl)-pyridine (64).
The isoxazole-pyridine derivatives 62, 63 and 64 are new compounds and, as
such,
embodiments of the present invention.
Introduction of (for example) butane-1-thiol to compound 64 by the
15 Pd2(dba)3/Xantphos catalytic system (accordingly to scheme 7, 10 and 11)
afforded
3-(4-butylsulfanyl-isoxazol-3-yl)-pyridine (65A). Quarternizing the pyridine
moiety,
preferentially with sulfuric acid dimethyl ester and reducing the
corresponding
pyridinium salt with NaBH4, generates the corresponding 3-(4-butylsulfanyl-
isoxazol-
3-yl)-1,2,5,6-tetrahydro-l-methylpyridine (66A).
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Scheme 12
~OH
N N-O N-O
NCS SiMe3 Br2 SiMe3
Me3Si - SiMe3 CCI4
Et N I i SiMe3 I ~ Br
THF N N
61 62 63
O Pd(OAc)2 O
N -Xantphos i
NH OH butane-l-thiol
4
EtOH Dioxane
~ I i Br - I i S
N N
64 65A
N-O
NaBH4
CH3OSO2OCH3 MeOH
Acetone
N S
I
66A
Scheme 13 illustrates the preparation of 3-(4-alkynyl (and alkenyl)-isoxazol-3-
yl)-
1,2,5,6-tetrahydro-l-methylpyridine derivatives as compounds of the formula I.
3-(4-Bromo-isoxazol-3-yl)-pyridine (64) is an excellent substrate for
Sonogashira
couplings with (terminal) acetylenes using an analog of the methodology
described in
scheme 9.
Subsequent conversion of these alkynyl derivatives (scheme 13, for example
69A)
using the quarternizing and reduction conditions described in scheme 12,
afforded
the corresponding 3-(4-alkynyl-isoxazol-3-yl)-1,2,5,6-tetrahydro-1-
methylpyridine
derivatives (for example 70A).
The scope and reactivity of compound 64 is further illustrated by the Suzuki-
Miyaura
coupling reactions with (for example) alkenylboronic acids using the
methodology
described in scheme 9. Subsequent conversion of these alkenyl derivatives
(scheme
13, for example 67A) using the quarternizing and reduction conditions
described in
scheme 12, afforded the corresponding 3-(4-alkenyl-isoxazol-3-yl)-1,2,5,6-
tetrahydro-
1-methylpyridine derivatives (for example 68A).
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Scheme 13
Pd(OAc)2
N_O S-phos N-O N-O
K3PO4
\ I / THF I N Br B(OH)zN
64 67A 68A
Pd(OAc)2
PPh3 O
N-O KOAc N'O N'
TBAF
DMF
Br
N
N
64 ~'Si 69A 70A
I
The preparation of compounds of the present invention of formula I is further
shown
in scheme 14.
3-Trimethylstannanyl-pyridine (71), (Eur. J. Org. Chem., 2002, 2126), obtained
from
3-bromo-pyridine using Knochel methodology (Angew. Chem., Int. Ed., 39, 2000,
4414-4435) is coupled under Stille conditions (Toluene, 120 C, PdCl2(PPH3)2
with
4,5-dibromo-1-(2-trimethylsilanyl-ethoxymethyl)-1 H-imidazole (72)
(Tetrahedron
Letters, 39, 1998, 5171-5174) to afford the 3-[5-bromo-3-(2-trimethylsilanyl-
ethoxymethyl)-3H-imidazol-4-yl]-pyridine (73). Introduction of (for example)
pentane-
1-thiol by the Pd2(dba)3/Xantphos catalytic system (accordingly to scheme 12)
afforded the corresponding 5-pentylsulfanyl-isoxazole derivative (74A).
Quarternizing the pyridine moiety (CH31) and reducing the corresponding
pyridinium
salt with NaBH4 (75A) followed by subsequent removal of the SEM group,
generates
3-[5-pentylsulfanyl-3H-imidazol-4-yl]-1,2,5,6-tetrahydro-l-methylpyridine
(76A).
Alternative to the conversion of 74A to 76A, deprotection of 74B (scheme 14,
compound 77) followed by quarternization and reduction generates the desired 3-
[5-
hexylsulfanyl-3H-imidazol-4-yl]-1,2,5,6-tetrahydro-l-methylpyridine (76B).
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Scheme 14
SEM
MgCI ~ PdC12(PPh3)2 NCIBr \ SnCI(Me)3- I\ S\\ SEM,N^N Toluene
\
N THF N 71 ~ i Br
Br Br N
71 72 73
Pd2(dba)3 SEM
K Xantphos N~ SEM N~ HN
zCOs N N TBAF N
Xylene ac~Mel etone THF
S S S
HS N NaBH4 N
MeOH
74A 75A 76A
Pdz(dba)3
Xantphos SEM
K2C03 N HN HN
Xylene N TBAF \ N C_JJN
Mel
73 S THF I acetone
N N S B 4 CN S
MeOH
74B 77 76B
4. SYNTHESES OF SPECIFIC COMPOUNDS
N-Methoxy-N-methyl-nicotinamide.
(compound 2, Scheme 1)
Nicotinoyl chloride hydrochloride (compound 1) (10 g, 56 mmol) and 6.28 g of
N,O-
dimethyl-hydroxylamine.HCI (72.8 mmol) were combined in 200 ml
dichloromethane.
To this mixture was added 18.14 ml of pyridine (in 15 minutes at 0 C). The
reaction
mixture was subsequently stirred for 4 hours at room temperature. The reaction
was
concentrated in vacuo. The resulting residue was taken up in dichloromethane
and
H20 (0 C), washed with a 2N NaOH solution followed by brine, dried (Na2SO4),
filtered and concentrated in vacuo. Purification by flash chromatography
(MeOH/triethylamine 97/3) afforded compound 2 as an oil (6.92 g, 74%). 'H- NMR
(200 MHz, CDCI3) 5 8.96 (d, J = 2 Hz, 1 H), 8.69 (d, J = 5 Hz, 2 Hz, 1 H),
8.04 (dt, J =
8 Hz, 2 Hz, 1H), 7.41-7.32 (m, 1H), 3.56 (s, 3H), 3.40 (s, 3H). (TLC
MeOH/triethylamine Rf 0.19).
1-Pyridin-3-yl-heptan-1-one.
(compound 3, Scheme 1)
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To a solution of anhydrous THF (15 ml) containing compound 2 (1.0 g, 6.02
mmol)
was added 3.08 ml (7.66 mmol) of hexyl-lithium (2.5 M in hexane) dropwise at -
78 C
under N2. After the addition, the resulting solution was stirred for 30
minutes at -78 C.
The mixture was allowed to warm to ambient temperature and poured into a NH4CI
solution (10 g/50 ml H20, 0 C). Ethyl acetate was added and the organic layer
was
washed with a 5% NaHCOs solution, dried (Na2SO4), filtered and concentrated in
vacuo. The resulting residue was purified by flash chromatography (diethyl
ether/PE
1:1) to give compound 3 as an oil (0.91 g, 78%).1H-NMR (200 MHz, CDC13) : 5
9.18
(d, J = 2 Hz, 1 H), 8.78 (d, J = 5 Hz, 2 Hz, 1 H), 8.24 (dt, J = 8 Hz, 2 Hz, 1
H), 7.47-
7.37 (m, 1 H), 2.99 (t, J = 7 Hz, 2H), 1.84-1.65 (m, 2H), 1.47-1.25 (m, 6H),
0.90 (bt, J
= 7 Hz, 3H).
2-Methylene-1-pyridin-3-yl-heptan-1-one.
(compound 4, Scheme 1)
To 1 g (5.2 mmol) of compound 3, dissolved in 10 ml of MeOH, was added 0.1 ml
of
piperidine, 0.1 ml of acetic acid and 3 ml of an aqueous formaldehyde solution
(37%
formaldehyde in water). The mixture was heated to reflux for 48 hour.
The mixture was cooled and concentrated in vacuo. Ethyl acetate was added and
the
organic layer was washed with a 5% NaHCOs solution, dried (Na2SO4), filtered
and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(diethyl ether/PE 1/1) to give compound 4 as an oil (1.05 g, 95%). 1H-NMR (200
MHz,CDCl3):b8.94(d,J=2Hz, 1 H), 8.76 (d, J = 5 Hz, 2 Hz, 1 H), 8.05 (dt, J = 8
Hz, 2 Hz, 1 H), 7.46-7.35 (m, 1 H), 5.94 (s, 1 H), 5.64 (s, 1 H), 2.48 (bt, J
= 7 Hz, 2H),
1.60-1.25 (m, 6H), 0.99-0.82 (m, 3H).
1-(4-Pentyl-3-pyridin-3-y1-4,5-dihydropyrazol-1-yl)-ethanone.
(compound 5, Scheme 1)
Compound 4 (3.37 g, 16.6 mmol) and 5.89 ml of hydrazine hydrate were dissolved
in
50 ml of acetic acid and heated to reflux for 1.5 hour. The mixture was cooled
and
concentrated in vacuo. Ethyl acetate was added and the organic layer was
washed
with a 5% NaHCOs solution, dried (Na2SO4), filtered and concentrated in vacuo.
The
resulting residue was purified by flash chromatography (ether/ethyl acetate
1/1) to
give compound 4. (amorphous, 2.92 g, 68%). 1H-NMR (600 MHz, D6DMSO) : 5 8.92
(d, J 2 Hz, 1 H), 8.67-8.64 (m, 1 H), 8.07 (bd, J = 8 Hz, 1 H), 7.39-7.36 (m,
1 H), 4.04
(t, J 10 Hz, 1 H), 3.96 (dd, J = 10 Hz, J = 5 Hz, 1 H), 3,67-3.61 (m, 1 H),
2.40 (s,
3H), 1.76-1.69 (m, 1 H), 1.50-1.42 (m, 1 H), 1.39-1.22 (m, 6H), 0.87 (bt, J =
7 Hz, 3H).
3-(4-Pentyl-1 H-pyrazol-3-yl)-pyridine.
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(compound 7, Scheme 1)
Compound 5 (0.9 g, 3.47 mmol) and 3.01 g of Mn02 (10 eq.) were combined in
dichloroethane (100 ml) and warmed to reflux for 2 hours (Dean Stark
conditions).
Additional Mn02 (6.02 g) was added and the mixture was refluxed for another 12
5 hours. The mixture was cooled, filtered and the filtrate was washed
thoroughly with
dichloroethane/isopropyl alcohol (1/1). This mixture was concentrated in vacuo
to
afford the oxidation product (6) (TLC ethyl acetate Rf 0.20), contaminated
with some
starting material (5) (TLC ethyl acetate Rf 0.27) and already deacylated
product (7)
(TLC ethyl acetate Rf 0.12). This mixture (0.64 g) was used as in the next
step
10 without further purification.
The aforementioned material was dissolved in 5 ml of EtOH and 5 ml of 2 N NaOH
and the reaction mixture was refluxed for 4 hours. The mixture was cooled and
concentrated in vacuo. Ethyl acetate was added and the organic layer was
washed
with a 5% NaHCOs solution, dried (Na2SO4), filtered and concentrated in vacuo.
The
15 resulting residue was purified by flash chromatography (ethyl acetate) to
afford the
title compound (7) as an oil (344 mg, 1.6 mmol, 46% (overall)).'H- NMR (200
MHz,
CDC13) : 5 8.88 (d, J = 2 Hz, 1 H), 8.60 (dd, J = 5 Hz, 2 Hz, 1 H), 7.92 (dt,
J = 8 Hz, 2
Hz, 1 H), 7.47 (bs, 1 H), 7.38-7.33 (m, 1 H), 2.62 (t, J = 7 Hz, 2H), 1.64-
1.55 (m, 2H),
1.36-1.28 (m, 6H), 0.85 (bt, J = 7 Hz, 3H).
3-(4-Pentyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 9, Scheme 1)
lodomethane (0.08 ml, 1.28 mmol) was added to a solution of 7 (130 mg, 0.6
mmol)
in acetone (10 ml). After heating for 12 hours, the reaction mixture was
cooled and
the precipitated crystals were filtered, washed with diethyl ether and dried
to afford
compound 8. To a cooled (-30 C) suspension of this pyridinium iodide
derivative (8)
in MeOH (15 ml), sodium borohydride (90 mg, 2.4 mmol) was added in small
portions. The mixture was allowed to warm to ambient temperature and poured
into a
saturated NH4CI solution (0 C). The solvent was (partly) removed under reduced
pressure. Ethyl acetate was added and the organic layer was washed with a
concentrated NaHCOs solution, dried (Na2SO4), filtered and concentrated in
vacuo.
The resulting residue was purified by flash chromatography (MeOH/triethylamine
97/3) to afford the title compound 9 (amorphous, 63 mg, 45% (overall)). 1H-NMR
(200 MHz, CDC13) : 5 7.35 (bs, 1 H), 6.07-6.00 (bs, 1 H), 3.33-3.25 (m, 2H),
2.64-2.35
(m, 6H), 2.45 (s, 3H), 1.68-1.50 (m, 2H), 1.41-1.28 (m, 4H), 0.90 (bt, J = 7
Hz, 3H).
N-Methoxy-N-methyl-2-pyridine-3-yl-acetamide.
(compound 12, Scheme 2)
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To a solution of anhydrous dichloromethane (200 ml) containing compound 10
(15.35
g, 88.4 mmol) was added 14.93 ml (163.3 mmol) of oxalyl chloride and a few
drops
of DMF. The mixture was gently refluxed for 8 hours under N2. The mixture was
cooled and concentrated, re-dissolved in dichloromethane and concentrated. The
residue was dissolved in 200 ml of anhydrous dichloromethane and 11.09 g
(113.7
mmol) of N,O-Dimethyl-hydroxylamine.HCI was added. To this mixture (0 C) was
added 2.73 ml of pyridine (in 15 minutes). The reaction mixture was
subsequently
stirred for 4 hours at room temperature. The reaction was concentrated in
vacuo. The
resulting residue was taken up in dichloromethane and H20 (0 C), washed with a
2N
NaOH solution followed by brine, dried (Na2SO4), filtered and concentrated in
vacuo.
Purification by flash chromatography (ethyl acetate) afforded compound 12 as
an oil
(5.2 g, 33%). 'H- NMR (400 MHz, CDC13) :6 8.53-8.49 (m, 2H), 7.66 (bd, J = 8
Hz,
1 H), 7.29-7.24 (m, 1 H), 3.78 (s, 2H), 3.68 (s, 3H), 3.20 (s, 3H).
1-Pyridin-3-yl-hexan-2-one.
(compound 13, Scheme 2)
To a solution of anhydrous THF (15 ml) containing compound 12 (1.0 g, 5.5
mmol)
was added 2.6 ml (6.5 mmol) of n-Buli (2.5 M in hexanes) dropwise at -50 C
under
N2. After the addition, the resulting solution was stirred for 30 minutes at -
50 C. The
mixture was allowed to warm to ambient temperature and poured into a NH4CI
solution (10 g/50 ml H20, 0 C). Ethyl acetate was added and the organic layer
was
washed with a 5% NaHCO3 solution, dried (Na2SO4), filtered and concentrated in
vacuo. The resulting residue was purified by flash chromatography (ethyl
acetate) to
give compound 13 as an oil (0.21 g, 25%).1H-NMR (400 MHz, CDC13) : 6 8.50 (dd,
J
= 5 Hz, 2 Hz, 1 H), 8.45 (d, J = 2 Hz, 1 H), 7.54 (dt, J = 8 Hz, J = 2Hz, 1
H), 7.29-7.24
(m, 1 H), 3.70 (s, 2H), 2.50 (t, J = 7 Hz, 2H), 1.62-1.53 (m, 2H), 1.34-1.24
(m, 2H), 0.9
(bt, J = 7 Hz, 3H).
1-Dimethylamino-2-pyrid in-3-yl-hept-l-en-3-one.
(compound 14, Scheme 2)
A solution of 13 (2.0 g, 11 mmol) and DMFDMA (2.5 ml, 14.6 mmol) in dry t-BuOH
was refluxed for 18 hours under N2. The solution was allowed to attain room
temperature and subsequently concentrated in vacuo. The resulting residue was
purified by flash chromatography (ethyl acetate) to give compound 14 as an oil
(1.85
g, 62%). 1H-NMR (400 MHz, CDC13) : b 8.51 (dd, J = 5 Hz, 2 Hz, 1 H), 8.45 (d,
J = 2
Hz, 1 H), 7.66 (s, 1 H), 7.53 (dt, J = 8 Hz, J = 2Hz, 1 H), 7.28-7.24 (m, 1
H), 2.72 (bs,
6H), 2.20 (t, J = 7 Hz, 2H), 1.54-1.46 (m, 2H), 1.26-1.16 (m, 2H), 0.9 (bt, J
= 7 Hz,
3H).
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3-(4-Butyl-1 H-pyrazol-3-yl)-pyridine.
(compound 15, Scheme 2)
Compound 14 (0.95 g, 4 mmol) and 0.46 ml hydrazine hydrate (9.4 mmol) were
dissolved in anhydrous ethanol (25 ml) and heated to reflux for 2 hours. The
mixture
was cooled and concentrated in vacuo. The resulting residue was purified by
flash
chromatography (ethyl acetate) to give compound 15. (amorphous, 0.7 g, 85%).
'H-
NMR (200 MHz, CDC13) :6 8.65 (d, J = 2 Hz, 1H), 8.52 (dd, J = 5 Hz, 2 Hz, 1H),
7.72-7.67 (m, 2H), 7.35-7.31 (m, 1 H), 2.82 (t, J = 7 Hz, 2H), 1.70-1.62 (m,
2H), 1.42-
1.33 (m, 2H), 0.9 (bt, J = 7 Hz, 3H).
3-(4-Butyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 16, Scheme 2)
lodomethane (0.9 ml, 14 mmol) was added to a solution of 15 (600 mg, 3 mmol)
in
acetone (50 ml). After heating for 12 hours, the reaction mixture was cooled
and the
precipitated crystals were filtered, washed with diethyl ether and dried to
afford the
corresponding pyridinium iodide derivative. To a cooled (-30 C) suspension of
this
pyridinium iodide derivative in MeOH (100 ml), sodium borohydride (0.5 g, 18.9
mmol) was added in small portions. The mixture was allowed to warm to ambient
temperature and poured into a saturated NH4CI solution (0 C). The solvent was
(partly) removed under reduced pressure. Ethyl acetate was added and the
organic
layer was washed with a concentrated NaHCO3 solution, dried (Na2SO4), filtered
and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(MeOH/triethylamine 97/3) to afford the title compound 16 (amorphous, 500 mg,
70%
(overall)).'H-NMR (400 MHz, CDC13) : b 7.40 (bs, 1H), 5.78-5.74 (bs, 1H), 3.15-
3.05
(m, 2H), 2.71 (bt, J = 7 Hz, 2H), 2.56 (t, J = 6 Hz, 2H), 2.43 (s, 3H), 2.38-
2.32 (m,
2H), 1.68-1.60 (m, 2H), 1.43-1.36 (m, 2H), 0.96 (t, J = 7 Hz, 3H).
3-(4-Methylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 21A, Scheme 3)
To a solution of anhydrous THF (150 ml) containing compound 20A (3.0 g, 13.4
mmol, prepared accordingly to Bioorganic & Medicinal Chemistry, 4, 1996, 227-
237)
was added 2.1 eq n-BuLi (11.2 ml, 2.5 M in hexane) dropwise at -78 C under N2.
After the addition, the resulting solution was stirred for 2 hours at -78 C.
At this
temperature 1.1 eq methyldisulfanyl methane (1.33 ml) was added and the
resulting
solution was stirred for 1 hour at -78 C and subsequently allowed to warm to
ambient
temperature overnight. Then the mixture was quenched with a saturated NH4CI
solution at 0 C and concentrated in vacuo. Ethyl acetate was added and the
organic
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layer was washed with 5% NaHCOs, dried (Na2SO4), filtered and concentrated in
vacuo. Purification by flash chromatography (ethyl acetate) afforded compound
21A
(oil, 1.71 g, 67%).'H-NMR (200 MHz, CDC13) : b 9.18 (d, J = 2Hz, 1 H), 8.60
(dd, J= 5
Hz, 2Hz, 1 H), 8.28 (dt, J = 8 Hz, 2 Hz, 1 H), 7.70 (s, 1 H), 7.42-7.33 (m, 1
H), 2.38 (s,
3H)
3-(4-Methylsu Ifanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 22A, Scheme 3)
2.5 eq iodomethane (1.39 ml, 22.37 mmol) were added to a solution of 21A (1.71
g,
8.95 mmol) in acetone (100 ml) and the mixture was stirred for 18 hours. The
precipitated crystals were filtered, washed with diethyl ether and dried to
afford the
corresponding pyridinium iodide derivative. To a cooled (-30 C) suspension of
this
pyridinium iodide derivative in MeOH (100 ml), sodium borohydride (1.35 g,
35.5
mmol) was added in small portions. The mixture was allowed to warm to ambient
temperature and poured into a saturated NH4CI solution (0 C). The solvent was
(partly) removed under reduced pressure. Ethyl acetate was added and the
organic
layer was washed with a concentrated NaHCO3 solution, dried (Na2SO4), filtered
and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(MeOH) to afford the title compound 22A. (solid, 1.39 g, 74% (overall)). mp
131.5 C.
LCMS (method A) ; Rt: 0.96 min, ([M+H]+ = 210).'H-NMR (400 MHz, CDCI3) : 5
7.52
(s, 1 H), 6.56-6.46 (bs, 1 H), 3.40-3.36 (m, 2H), 2.62 (bt, J = 6 Hz, 2H),
2.46 (s, 3H),
2.45-2.38 (m, 2H), 2.30 (s, 3H).
3-[4-(4,4,4-trifluoro-butylsulfanyl)-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-1-
methylpyridine.
(compound 22B, Scheme 4)
A 60% dispersion of NaH in mineral oil (0.54 g, 13.64 mmol) was added to a
solution
of anhydrous THF (100 ml) containing compound 20A (2.79, 12.4 mmol) under N2.
The resulting mixture was stirred for 2 hours at room temperature and
subsequently
treated with 13.64 mmol (2.41 ml) of (2-Chloromethoxy-ethyl)-trimethylsilane
(SEM-
CI). The resulting mixture was stirred for 18 hours at room temperature. Ethyl
acetate
was added to the mixture and the organic layer was washed three times with a
saturated NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The
resulting
residue was purified by flash chromatography (diethyl ether/PE 1/1) to give a
mixture
of 27A (major component) and 27B as an oil (3.19 g, 73%). Carefully controlled
purification by flash chromatography (diethyl ether/PE 1/1) gave 27B and
subsequently 27A. Compund 27B (oil).'H-NMR (400 MHz, CDCI3) : b 9.15 (d, J = 2
Hz, 1 H), 8.60 (dd, J = 5 Hz, 2 Hz, 1 H), 8.20 (dt, J = 8 Hz, 2 Hz, 1 H), 7.71
(s, 1 H),
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7.38-7.34 (m, 1 H), 5.44 (s, 2H), 3.64 (t, J = 8 Hz, 2H), 0.94 (bt, J = 8 Hz,
2H), 0.02 (s,
9H). Compound 27A (oil).'H-NMR (400 MHz, CDCI3) : b 8.90 (d, J = 2 Hz, 1H),
8.75
(dd, J = 5 Hz, 2 Hz, 1 H), 7.96 (dt, J = 8 Hz, 2 Hz, 1 H), 7.63 (s, 1 H), 7.47-
7.42 (m,
1H), 5.34 (s, 2H), 3.70 (t, J = 8 Hz, 2H), 0.92 (bt, J = 8 Hz, 2H), 0.02 (s,
9H).
NOESYPHSW and HMBCGP analyses were used to confirm both compounds.
To a solution of anhydrous THF (50 ml) containing a mixture of 27A/B (0.92 g,
2.6
mmol) was added 1.14 ml (1.1 eq) of n-BuLi (2.5 M in hexane) dropwise (-78 C
under N2). After the addition, the resulting solution was stirred for 60
minutes at
-78 C. At this temperature, sulfur powder (2.6 mmol, 0.083 g) was added and
the
reaction mixture was stirred for another 2 hours (-78 C). The reaction was
monitored
by thin-layer chromatography. After the addition of 4-bromo-1,1,1-trifluoro-
butane
(1.1 eq, 0.54 ml), the mixture was allowed to warm to ambient temperature
(overnight) and poured into a saturated NH4CI solution (0 C). The solvent was
(partly) removed under reduced pressure. Ethyl acetate was added and the
organic
layer was washed with a concentrated NaHCO3 solution, dried (Na2SO4), filtered
and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(diethyl ether/PE 1/1)) to afford a mixture of (predominantly) 28A and 28B.
(oil, 0.49
g, 45%).1H-NMR (data of 28A are described, 400 MHz, CDC13) : 6 8.80 (d, J = 2
Hz,
1 H), 8.70 (dd, J = 5 Hz, 2 Hz, 1 H), 7.96 (dt, J = 8 Hz, 2 Hz, 1 H), 7.68 (s,
1 H), 7.48-
7.43 (m, 1 H), 5.35 (s, 2H), 3.72 (bt, J 8 Hz, 2H), 2.58 (t, J = 7 Hz, 2H),
2.10-1.97
(m, 2H), 1.71-1.59 (m, 2H), 0.93 (bt, J 8 Hz, 2H), 0.00 (s, 9H).
To a solution of anhydrous THF (20 ml) containing a mixture of 28A/B (0.49 g,
1.18
mmol) was added 3.54 ml (3.0 eq) of TBAF (1.0 M in THF) under N2. After the
addition, the resulting solution was refluxed for 18 hours and subsequently
concentrated in vacuo. Ethyl acetate was added and the organic layer was
washed
with a concentrated NaHCO3 solution, dried (Na2SO4), filtered and concentrated
in
vacuo. The resulting residue was purified by flash chromatography (diethyl
ether) to
afford 3-[4-(4,4,4-Trifluoro-butylsulfanyl)-1 H-pyrazol-3-yl]-pyridine (21 B).
(oil, 0.32 g,
95%).'H-NMR (400 MHz, CDC13) : 5 9.20 (d, J = 2 Hz, 1H), 8.65 (dd, J = 5 Hz, 2
Hz,
1 H), 8.28 (dt, J = 8 Hz, 2 Hz, 1 H), 7.73 (s, 1 H), 7.42-7.37 (m, 1 H), 2.61
(t, J = 7 Hz,
2H), 2.19-2.08 (m, 2H), 1.75-1.65 (m, 2H).
Compound 21 B (0.3 g, 1.49 mmol) was converted to compound 22B, using the
methodology described for the conversion of 21 A to 22A. Yield 0.131 g
(amorphous,
72% overall). LCMS (method A) ; R, : 1.64 min, ([M+H]+ = 306). 1H-NMR (400
MHz,
CDC13) : 6 7.57 (s, 1 H), 6.67-6.60 (bs, 1 H), 3.43-3.39 (m, 2H), 2.71-2.62
(m, 4H),
2.49 (s, 3H), 2.48-2.43 (m, 2H), 2.28-2.19 (m,2H), 1.81-1.75 (m, 2H).
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bis-[3-(1-Methyl-1,2,5,6-tetrahydro-pyridin-3-yl)-1 H-pyrazol-4-yl)-2-
sulfanylethyll-
methane.
(compound 22C, Scheme 7)
A 60% dispersion of NaH in mineral oil (0.73 g, 18.3 mmol) was added to a
solution
5 of anhydrous THF (100 ml) containing compound 20A (3.71, 16.6 mmol) under
N2.
The resulting mixture was stirred for 2 hours at room temperature and
subsequently
treated with 18.3 mmol (2.33 ml) Phenylsulfonyl chloride. The resulting
mixture was
stirred for 18 hours at room temperature. Ethyl acetate was added to the
mixture and
the organic layer was washed three times with a saturated NaHCO3 solution,
dried
10 (Na2SO4), filtered and concentrated. The resulting residue was purified by
flash
chromatography (diethyl ether to afford 3-(1-phenylsulfonyl-4-bromo-1 H-
pyrazol-3-
yl)-pyridine (36A). (TLC ethyl acetate Rf 0.7) (amorphous, 5.34 g, 89%). 'H-
NMR
(400 MHz, CDC13) : b 9.1 (d, J = 2 Hz, 1 H), 8.65 (dd, J = 5 Hz, 2 Hz, 1 H),
8.24 (s,
1 H), 8.16 (dt, J = 8 Hz, 2 Hz, 1 H), 8.10-8.05 (m, 2H), 7.70 (bt, J = 7 Hz,
2H), 7.62-
15 7.56 (m, 2H), 7.39-7.34 (m, 1 H).
To a degassed solution of xylene (20 ml) containing 36 (0.7 g, 1.92 mmol),
were
added 0.45 eq (0.12 ml, 0.86 mmol) of pentane-1,5-di-thiol and 0.5 eq of K2CO3
(0.137 g, 0.96 mmol). The resulting mixture was stirred for another 2 hours
under N2.
Successively were added 0.192 mmol of Pd2(dba)3 (176 mg) and 0.384 mmol of
20 Xantphos (222 mg). After the addition, the resulting solution was refluxed
for 18
hours under N2. After cooling to room temperature, the mixture was diluted
with ethyl
acetate, washed three times with a saturated NaHCO3 solution, dried (Na2SO4),
filtered and concentrated. The resulting residue was purified by flash
chromatography
(ethyl acetate) to afford compound 37 as an oil (0.46 g, 68%). (TLC ethyl
acetate Rf
25 0.29).
Compound 37 (0.46 g, 0.65 mmol), 0.7 g KOH and 1 ml NH2NH2.H2O were combined
in diethylene glycol (10 ml) and warmed to reflux for 1 hour under N2. The
mixture
was cooled, concentrated and re-dissolved in MeOH. Filtration over 5 g of SCX-
2
(MeOH followed by 1 N NH3/MeOH) and subsequent purification by flash
30 chromatography (ethyl acetate) afforded bis-[3-pyridin-3-yl-1 H-pyrazol-4-
yl)-2-
sulfanylethyl]-methane as the deprotected analog of 37 (amorphous, 0.23 g,
83%).
'H-NMR (400 MHz, CDC13) : 6 9.15 (d, J = 2 Hz, 2H), 8.57 (dd, J = 5 Hz, 2 Hz,
2H),
8.31 (dt, J = 8 Hz, 2 Hz, 2H), 7.66 (s, 2H), 7.36-7.32 (m, 2H), 2.50-2.42 (m,
4H),
1.34-1.26 (m, 6H).
35 The deprotected analog of compound 37 (0.23 g, 0.54 mmol) was converted to
compound 22C, using the methodology described for the conversion of 21 A to
22A.
Yield 0.2 g (oil, 80% overall). LCMS (method A) ; Rt : 1.66 min, ([M+H]+ =
459). 'H-
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NMR (400 MHz, CDC13) : 6 7.50 (s, 2H), 6.92-6.85 (bs, 2H), 3.90-3.80 (m, 4H),
3.07
(bt, J = 7 Hz, 4H), 2.77 (s, 6H), 2.66-2.54 (m, 8H), 1.54-1.43 (m, 6H).
3-(4-Ethylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 21 D)
Compound 21 D was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using ethyldisulfanyl-ethane as the disulfide
and
3-(4-bromo-1 H-pyrazol-3-yl)-pyridine (compound 20A).
Yield : 76%. (oil). LCMS (Method A) ; R, : 1.56 min, ([M+H]+ = 206). 'H-NMR
(200
MHz, CDCI3) : b 9.11 (d, J = 2Hz, 1 H), 8.64 (dd, J= 5 Hz, 2Hz, 1 H), 8.18
(dt, J = 8
Hz, 2 Hz, 1 H), 7.71 (s, 1 H), 7.39-7.35 (m, 1 H), 2.64 (q, J = 7 Hz, 2H),
1.26 (t, J = 7
Hz, 3H).
3-(4-Ethylsulfanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 22D)
Compound 22D was prepared from compound 21D following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 73% (solid). mp 95-97 C. LCMS (method A) ; Rt : 1.15 min, ([M+H]+ =
224).
'H-NMR (mixture of rotational isomers (3 /1), major one described, 400 MHz,
CDC13):
5 7.53 (s, 1 H), 6.55-6.46 (bs, 1 H), 3.40-3.37 (m, 2H), 2.67-2.60 (m, 4H),
2.46 (s, 3H),
2.43-2.36 (m, 2H), 1.19 (3H).
3-(4-Propylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 21 E)
Compound 21 E was prepared following the procedure as described for the
synthesis
of compound 21A using (see scheme 3) 1-propyldisulfanyl-propane as the
disulfide
and 3-(4-bromo-1 H-pyrazol-3-yl)-pyridine (compound 20A). (flash
chromatography
conditions ethyl acetate/diethyl ether 5/1).
Yield : 76%. (oil).'H-NMR (200 MHz, CDC13) : b 9.20 (d, J = 2Hz, 1 H), 8.60
(dd, J= 5
Hz, 2Hz, 1 H), 8.31 (dt, J = 8 Hz, 2 Hz, 1 H), 7.68 (s, 1 H), 7.39-7.35 (m, 1
H), 2.56 (t, J
= 7 Hz, 2H), 1.54-1.43 (m, 2H), 0.90 (t, J = 7 Hz, 3H).
3-(4-Propylsulfanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 22E)
Compound 22E was prepared compound from 21E following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 77% (solid). mp 111 C. LCMS (method A) ; Rt: 1.39 min, ([M+H]+ =
238).'H-
NMR (mixture of rotational isomers 8/1), major one described, 400 MHz, CDC13)
: 5
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7.55 (s, 1 H), 6.61-6.55 (bs, 1 H), 3.43-3.39 (m, 2H), 2.68-2.60 (m, 4H), 2.48
(s, 3H),
2.46-2.39 (m, 2H), 1.58 (dq, J = 7 Hz, 7 Hz, 2H), 0.98 (t, J = 7 Hz, 3H).
3-(4-Butylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 21 F)
Compound 21 F was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 1-butyldisulfanyl-butane as the disulfide
and
3-(4-iodo-1 H-pyrazol-3-yl)-pyridine (compound 20B). (flash chromatography
conditions ethyl acetate/diethyl ether 3/1).
Yield : 18.4%. (oil).'H-NMR (200 MHz, CDC13) : b 9.20 (d, J = 2Hz, 1H), 8.60
(dd, J=
5 Hz, 2Hz, 1 H), 8.31 (dt, J = 8 Hz, 2 Hz, 1 H), 7.68 (s, 1 H), 7.39-7.34 (m,
1 H), 2.58 (t,
J = 7 Hz, 2H), 1.48-1.40 (m, 2H), 1.36-1.25 (m, 2H), 0.90 (t, J = 7 Hz, 3H).
3-(4-Butylsulfanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 22F)
Compound 22F was prepared from compound 21 F following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 73% (amorphous). Compound 22A was reacted with 1 equivalent of fumaric
acid in EtOH and concentrated. Recrystallization from EtOH/ethyl acetate
afforded a
solid (free base/fumaric acid 1/1), mp 120-121 C. LCMS (method A) ; Rt : 1.16
min,
([M+H]` = 252).
3-(4-Pentylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 21 G)
Compound 21 G was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 1-pentyldisulfanyl-pentane as the
disulfide
and 3-(4-bromo-1 H-pyrazol-3-yl)-pyridine (compound 20A).
Yield : 71%. (oil).'H-NMR (400 MHz, CDC13) : b 9.18 (d, J = 2Hz, 1H), 8.60
(dd, J= 5
Hz, 2Hz, 1 H), 8.32 (dt, J = 8 Hz, 2 Hz, 1 H), 7.66 (s, 1 H), 7.39-7.34 (m, 1
H), 2.57 (t, J
= 7 Hz, 2H), 1.48-1.41 (m, 2H), 1.30-1.14 (m, 4H), 0.81 (t, J = 7 Hz, 3H).
3-(4-Pentylsulfanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 22G)
Compound 22G was prepared from compound 21G following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 77% (solid). mp 100-101 C. LCMS (method A) ; Rt : 1.67 min, ([M+H]+ =
266).
'H-NMR (400 MHz, CDC13) : 6 7.51 (s, 1H), 6.56-6.47 (bs, 1H), 3.40-3.36 (m,
2H),
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2.65-2.59 (m, 4H), 2.45 (s, 3H), 2.44-2.38 (m, 2H), 1.57-1.47 (m, 4H), 1.39-
1.24 (m,
4H) 0.88 (t, J = 7 Hz, 3H).
3-(4-Hexylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 21 H)
Compound 21 H was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 1-hexyldisulfanyl-hexane as the disulfide
and 3-(4-iodo-1 H-pyrazol-3-yl)-pyridine (compound 20B).
Yield : 37%. (oil).'H-NMR (400 MHz, CDC13) : 5 9.18 (d, J = 2Hz, 1 H), 8.62
(dd, J= 5
Hz, 2Hz, 1 H), 8.31 (dt, J = 8 Hz, 2 Hz, 1 H), 7.70 (s, 1 H), 7.40-7.35 (m, 1
H), 2.59 (t, J
= 7 Hz, 2H), 1.50-1.42 (m, 2H), 1.34-1.12 (m, 6H), 0.81 (t, J = 7 Hz, 3H).
3-(4-Hexylsulfanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine.
(compound 22H)
Compound 22H was prepared from compound 21H following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 49% (amorphous). LCMS (method A) ; Rt : 1.81 min, ([M+H]+ = 280).1H-
NMR
(400 MHz, CDC13) : b 7.51 (s, 1 H), 6.60-6.50 (bs, 1 H), 3.40-3.36 (m, 2H),
2.65-2.59
(m, 4H), 2.45 (s, 3H), 2.44-2.38 (m, 2H), 1.56-1.48 (m, 2H), 1.39-1.20 (m, 6H)
0.88 (t,
J= 7 Hz, 3H).
3-[4-(3-Phenyl-propylsulfanyl)-1 H-pyrazol-3-yll-pyridine. (compound 211)
Compound 211 was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 3-phenyl-propyldisulfanyl-3-propylbenzene
as the disulfide (prepared according to the methodology described in
Tetrahedron
Letters, 42, 2001, 6741-6743) and 3-(4-bromo-1 H-pyrazol-3-yl)-pyridine
(compound
20A). (conditions flash chromatography (ethyl acetate)) Yield : 22% (oil). 1H-
NMR
(400 MHz, CDCI3) : b 9.18 (d, J = 2Hz, 1 H), 8.62 (dd, J= 5 Hz, 2Hz, 1 H),
8.31 (dt, J =
8 Hz, 2 Hz, 1 H), 7.69 (s, 1 H), 7.40-7.36 (m, 1 H), 7.24 (bt, J = 7 Hz, 2H),
7.17 (bt, J =
7 Hz, 1 H), 7.04 (bd, J = 7 Hz, 2H), 2.65-2.57 (m, 4H), 1.84-1.75 (m, 2H).
3-[4-(3-Phenyl-propylsulfanyl)-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-1-
methylpyridine.
(compound 221)
Compound 221 was prepared from compound 211 following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 59% (amorphous). 1H-NMR (400 MHz, CDC13) : 5 7.51 (s, 1 H), 7.30-7.22
(m,
2H), 7.21-7.12 (m, 3H), 6.66-6.46 (bs, 1H), 3.39-3.34 (m, 2H), 2.70-2.55 (m,
6H),
2.44 (s, 3H), 2.43-2.36 (m, 2H), 1.89-1.80 (m, 2H).
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3-f4-(4,4-Difluoro-but-3enylsulfanyl)-1 H-pyrazol-3-yll-pyridine.
(compound 21J)
Compound 21J was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 4-(4,4-difluoro-but-3-enyldisulfanyl)-1,1-
difluoro-but-l-ene as the disulfide (Tetrahedron Letters, 42, 2001, 6741-6743)
and 3-
(4-bromo-1 H-pyrazol-3-yl)-pyridine (compound 20A). (conditions flash
chromatography (ethyl acetate)) Yield : 58% (oil).'H-NMR (400 MHz, CDC13) : 5
9.18
(d, J = 2Hz, 1H), 8.62 (dd, J= 5 Hz, 2Hz, 1H), 8.31 (dt, J = 8 Hz, 2 Hz, 1H),
7.72 (s,
1 H), 7.42-7.36 (m, 1 H), 4.19-4.06 (m, 1 H), 2.59 (t, J = 7 Hz, 2H), 2.18-
2.09 (m, 2H).
3-f4-(4,4-Difluoro-but-3enylsulfanyl)-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-1-
methyl-
rpy idine.
(compound 22J)
Compound 22J was prepared from compound 21H following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 90% (amorphous). 'H-NMR (400 MHz, CDC13) : b 7.54 (s, 1H), 6.68-6.44
(bs,
1 H), 4.27-4.15 (m, 1 H), 3.40-3.36 (m, 2H), 2.66-2.60 (m, 4H), 2.47 (s, 3H),
2.45-2.38
(m, 2H), 2.22-2.14 (m, 2H).
3-f4-(3-Phenyl-allylsulfanyl)-1 H-pyrazol-3-yll-pyridine.
(compound 21 K)
Compound 21 K was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 3-phenyl-allyldisulfanyl-3-allylbenzene
as
the disulfide (Tetrahedron Letters, 42, 2001, 6741-6743) and 3-(4-bromo-1H-
pyrazol-
3-yl)-pyridine (compound20A). (conditions flash chromatography (ethyl
acetate)).
Yield : 22%. (oil), (TLC ethyl acetate Rf 0.45).
3-f4-(3-Phenyl-allylsulfanyl)-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-1-
methylpyridine.
(compound 22K)
Compound 22K was prepared from compound 21K following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 72% (amorphous). 1H-NMR (400 MHz, CDC13) : 5 7.52 (s, 1H), 7.30-7.18
(m,
5H), 6.70-6.20 (bs, 1H), 6.20-6.07 (m, 2H), 3.37 (d, J = 7 Hz, 2H), 3.33-3.28
(m, 2H),
2.54 (bt, J = 7 Hz, 2H), 2.40 (s, 3H), 2.39-2.30 (m, 2H).
3-f4-Pent-4-enylsulfanyl)-1 H-pyrazol-3-yll-pyridine.
(compound 21 L)
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Compound 21 L was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 5-pent-4-enyldisulfanyl-pent-l-ene as the
disulfide (Tetrahedron Letters, 42, 2001, 6741-6743) and 3-(4-iodo-1 H-pyrazol-
3-yl)-
pyridine (compound20B). (conditions flash chromatography (ethyl acetate))
Yield :
5 28%. (oil). LCMS (method A) ; Rt : 2.21 min, ([M+H]+ = 246). 1H-NMR (400
MHz,
CDC13) : 6 9.20 (d, J = 2Hz, 1 H), 8.62 (dd, J= 5 Hz, 2Hz, 1 H), 8.31 (dt, J =
8 Hz, 2
Hz, 1 H), 7.71 (s, 1 H), 7.41-7.35 (m, 1 H), 5.73-5.59 (m, 2H), 4.97-4.89 (m,
2H), 2.59
(t, J = 7 Hz, 2H), 2.10-2.03 (m, 2H), 1.61-1.54 (m, 2H).
10 3-[4-Pent-4-enylsulfanyl)-1 H-pyrazol-3-yll-1,2,5,6-tetrahydro-1-
methylpyridine.
(compound 22L)
Compound 22L was prepared from compound 21 L following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 61% (amorphous). LCMS (method A) ; Rt : 1.84 min, ([M+H]+ = 264).1H-
NMR
15 (400 MHz, CDC13) : b 7.52 (s, 1 H), 6.70-6.40 (bs, 1 H), 5.81-5.68 (m, 1
H), 5.04-4.94
(m, 2H), 3.40-3.36 (m, 2H), 2.66-2.57 (m, 4H), '2.45 (s, 3H), 2.45-2.37 (m,
2H), 2.17-
2.09 (m, 2H), 1.66-1.58 (m, 2H).
3-[4-(Furan-2-ylmethylsulfanyl)-1 H-pyrazol-3-yll-pyridine.
20 (compound 21 M)
Compound 21 M was prepared following the procedure as described for the
synthesis
of compound 21A (see scheme 3) using difurfuryl-disulfide and 3-(4-iodo-1 H-
pyrazol-
3-yl)-pyridine (compound 20B). (conditions flash chromatography (ethyl
acetate))
Yield : 54%. (oil).'H-NMR (200 MHz, CDC13) : 6 9.20 (d, J = 2Hz, 1 H), 8.59
(dd, J= 5
25 Hz, 2Hz, 1 H), 8.20 (dt, J = 8 Hz, 2 Hz, 1 H), 7.54 (s, 1 H), 7.38-7.32 (m,
1 H), 7.21-
7.19 (m, 1 H), 6.16-6.12 (m, 1 H), 5.88-5.84 (m, 1 H), 3.75 (s, 2H).
3-[4-(Furan-2-yl-methylsulfanyl)-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-1-
methylpyridine.
(compound 22M)
30 Compound 22M was prepared from compound 21M following the procedure as
described for the synthesis of compound 22A (see Scheme 3).
Yield : 65% (amorphous). LCMS (method A) ; Rt : 1.04 min, ([M+H]+ = 276).1H-
NMR
(200 MHz, CDC13) : b 7.36 (s, 1 H), 7.33-7.31 (m, 1 H), 6.38-6.26 (bs, 1 H),
6.24-6.20
(m, 1H), 5.93-5.90 (m, 1H), 3.78 (s, 2H), 3.30-3.22 (m, 2H), 2.60 (bt, J = 7
Hz, 2H),
35 2.44 (s, 3H), 2.41-2.33 (m, 2H).
3-(4-Benzylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 21 N)
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Compound 21 N was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using dibenzyl-disulfide and 3-(4-iodo-1 H-
pyrazol-
3-yl)-pyridine (compound 20B). (conditions flash chromatography (ethyl
acetate))
Yield : 33%. (oil).'H-NMR (400 MHz, CDC13) : 5 9.0 (d, J = 2Hz, 1H), 8.58 (dd,
J= 5
Hz, 2Hz, 1 H), 8.10 (dt, J = 8 Hz, 2 Hz, 1 H), 7.43 (s, 1 H), 7.33-7.28 (m, 1
H), 7.17-
7.11 (m, 3H), 7.02-6.96 (m, 2H), 3.72 (s, 2H).
3-(4-Benzylsulfanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 22N)
Compound 22N was prepared from compound 21N following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 48% (amorphous). LCMS (method A) ; Rt : 1.55 min, ([M+H]+ = 286).
Compound 22N was reacted with 1 equivalent of fumaric acid in EtOH and
concentrated. 'H-NMR (600 MHz, D6DMS0) : 6 7.63 (s, 1 H), 7.28 (t, J = 7 Hz,
2H),
7.28 (t, J = 7 Hz, 2H), 7.24 (bt, J = 7 Hz, 1 H), 7.17 (bd, J = 7 Hz, 2H),
6.82-6.79 (bs,
1 H), 6.66 (s, 2H), 4.28 (bd, J = 15 Hz, 1 H), 4.05 (dd, J = 16 Hz, 6 Hz, 2H),
3.86-3.80
(m, 1H), 3.55-3.50 (m, 1H), 3.18-3.10 (m, 1H), 2.94 and 2.93 (2 x s, 3H), 2.68-
2.46
(m, 2H).
3-[4-(2-Ethoxy-ethylsulfanyl)-1 H-pyrazol-3-yll-pyridine.
(compound 210)
Compound 210 was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 1-ethoxy-2-(2-ethoxy-ethyldisulfanyl)-
ethane
as the disulfide (Tetrahedron Letters, 42, 2001, 6741-6743) and 3-(4-iodo-lH-
pyrazol-3-yl)-pyridine (compound 20B). (conditions flash chromatography (ethyl
acetate)) Yield : 28%. (oil). LCMS (method A) ; Rt : 2.21 min, ([M+H]+ = 246).
'H-
NMR (400 MHz, CDC13) : b 9.20 (d, J = 2Hz, 1H), 8.62 (dd, J= 5 Hz, 2Hz, 1H),
8.33
(dt, J 8 Hz, 2 Hz, 1H), 7.75 (s, 1H), 7.41-7.36 (m, 1H), 3.46 (t, J = 7 Hz,
2H), 3.38
(q,J=7Hz,2H),2.78(t,J=7Hz,2H),1.14(t,J=7Hz,3H),
3-[4-(2-Ethoxy-ethylsulfanyl)-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-l-
methylpyridine.
(compound 220)
Compound 220 was prepared from compound 210 following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 63% (amorphous). 'H-NMR (400 MHz, CDC13) : 6 7.56 (s, 1 H), 6.70-6.50
(bs,
1 H), 3.55-3.43 (m, 4H), 3.40-3.36 (m, 2H), 2.81 (t, J = 7 Hz, 2H), 2.61 (bt,
J = 7 Hz,
2H), 2.46 (s, 3H), 2.44-2.38 (m, 2H), 1.18 (t, J = 7 Hz, 3H).
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3-{4-[2-(2-Methoxy-ethoxy)-ethylsulfanyll-1 H-pyrazol-3-yl}-pyridine.
(compound 21 P)
Compound 21 P was prepared following the procedure as described for the
synthesis
of compound 211 (see Scheme 3) using 1-methoxy-2-{2-[2-((2-methoxy-ethoxy)-
ethyldisulfanyl]-ethoxy}-ethane as the disulfide (Tetrahedron Letters, 42,
2001, 6741-
6743) and 3-(4-iodo-1 H-pyrazol-3-yl)-pyridine (compound 20B). (conditions
flash
chromatography (ethyl acetate)) Yield : 36%. (oil). 'H-NMR (400 MHz, CDC13) :
b
9.18 (d, J = 2Hz, 1 H), 8.58 (dd, J= 5 Hz, 2Hz, 1 H), 8.35 (dt, J = 8 Hz, 2
Hz, 1 H), 7.78
(s, 1 H), 7.39-7.32 (m, 1 H), 3.51-3.44 (m, 6 H), 3.34 (s, 3H), 2.78 (t, J = 7
Hz, 2H).
3-{4-[2-(2-Methoxy-ethoxy)-ethylsulfanyll-1 H-pyrazol-3-yl}-1,2,5,6-tetrahydro-
1-
methylpyridine.
(compound 22P)
Compound 22P was prepared from compound 21P following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 33% (amorphous). 'H-NMR (400 MHz, CDC13) : 6 7.57 (s, 1H), 6.74-6.69
(bs,
1H), 3.59-3.51 (m, 8H), 3.38 (s, 3H), 2.83 (t, J = 7 Hz, 2H), 2.77 (bt, J = 7
Hz, 2H),
2.55 (s, 3H), 2.52-2.45 (m, 2H).
3-(4-Allylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 21 Q)
Compound 21Q was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 3-allyldisulfanyl-propene as the
disulfide
and 3-(4-iodo-1 H-pyrazol-3-yl)-pyridine (compound 20B). (conditions flash
chromatography (ethyl acetate)) Yield : 27%. (oil). 'H-NMR (200 MHz, CDC13) :
6
9.20 (d, J = 2Hz, 1 H), 8.60 (dd, J= 5 Hz, 2Hz, 1 H), 8.29 (dt, J = 8 Hz, 2
Hz, 1 H), 7.70
(s, 1 H), 7.40-7.36 (m, 1 H), 5.77-5.66 (m, 1 H), 4.94 (bdd, J = 11 Hz, 1 Hz,
1 H), 4.83
(bdd, J = 17 Hz, 1 Hz, 1 H), 3.19 (bd, J = 8 Hz, 2 H).
3-(4-Allylsulfanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 22Q)
Compound 22Q was prepared from compound 21Q following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 17% (amorphous). 'H-NMR (200 MHz, CDC13) : b 7.52 (s, 1H), 6.62-6.48
(bs,
1 H), 5.84-5.74 (m, 1 H), 4.98 (bdd, J = 11 Hz, 1 Hz, 1 H), 4.90 (bdd, J = 17
Hz, 1 Hz,
1 H), 4.00-3.95 (m, 2H), 3.23 (bd, J = 8 Hz, 2 H), 2.61 (bt, J = 7 Hz, 2H),
2.46 (s, sH),
2.44-2.39 (m, 2H).
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3-(3-Pyridin-3-yl-1 H-pyrazol-4-ylsulfanyl)-propionitrile.
(compound 21 R)
Compound 21 R was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 3-(2-cyano-ethyldisulfanyl)-propionitrile
as
the disulfide and 3-(4-iodo-1 H-pyrazol-3-yl)-pyridine (compound 20B).
(conditions
flash chromatography (ethyl acetate)) Yield : 62%. (oil).'H-NMR (200 MHz,
CDC13) :
b 9.20 (d, J = 2Hz, 1H), 8.60 (dd, J= 5 Hz, 2Hz, 1H), 8.33 (dt, J = 8 Hz, 2
Hz, 1H),
7.82 (s, 1 H), 7.43-7.37 (m, 1 H), 2.76 (t, J = 7 Hz, 2H), 2.45 (t, J = 7 Hz,
2H).
343-(1-Methyl-1,2,5,6-tetrahydro-pyridin-3-yl)-1 H-pyrazol-4-ylsulfanyll-
propionitrile.
(compound 22R)
Compound 22R was prepared from compound 21R following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 8% (amorphous). 'H-NMR (200 MHz, CDC13) : 6 7.58 (s, 1 H), 6.60-6.50
(bs,
1H), 3.40-3.36 (m, 2H), 2.79 (t, J = 7 Hz, 2H), 2.66 (bt, J = 7 Hz, 2H), 2.50
(t, J = 7
Hz, 2H), 2.47 (s, 3H), 2.45-2.38 (m, 2H).
344-(3-methyl-butylsulfanyl)-1 H-pyrazol-3-yll-pyridine.
(compound 21 S)
Compound 21S was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 3-Methyl-l-(3-methyl-butyldisulfanyl)-
butane
as the disulfide and 3-(4-iodo-1 H-pyrazol-3-yl)-pyridine (compound 20B).
(conditions
flash chromatography (ethyl acetate)) Yield : 39%. (oil).'H-NMR (200 MHz,
CDC13) :
b 9.18 (d, J = 2Hz, 1H), 8.65 (dd, J= 5 Hz, 2Hz, 1H), 8.35 (dt, J = 8 Hz, 2
Hz, 1H),
7.78 (s, 1 H), 7.39-7.35 (m, 1 H), 2.60 (t, J = 7 Hz, 2H), 1.65-1.57 (m, 1 H),
1.42-1.33
(m, 2H), 0.81 (d, J = 7 Hz, 6H).
3-f4-(3-methyl-butylsulfanyl)-1 H-pyrazol-3-yll-1,2,5,6-tetrahydro-1 -
methylpyridine.
(compound 22S)
Compound 22S was prepared from compound 21S following the procedure as
described for the synthesis of compound 22A (from 21A) (see Scheme 3).
Yield : 73% (amorphous). 1H-NMR (200 MHz, CDCI3) : 6 7.5 (s, 1 H), 6.60-6.48
(bs,
1 H), 3.40-3.36 (m, 2H), 2.66-2.59 (m, 4H), 2.45 (s, 3H), 2.44-2.39 (m, 2H),
1.72-1.62
(m, 1 H), 1.45-1.39 (m, 2H), 0.86 (d, J = 7 Hz, 6H).
3-Pyridin-3-yl-pyrazole-l-sulfonic acid dimethylamide.
(compound 23, Scheme 3)
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Compound 19 (3.0 g, 20.7 mmol) and 2.22 ml of phenylsulfonyl chloride (20.7
mmol)
were combined in pyridine (100 ml) and stirred for 18 hours at reflux. The
reaction
was concentrated in vacuo. The residue was taken up in ethyl acetate, washed
three
times with a saturated NaHCO3 solution, dried (Na2SO4), filtered and
concentrated in
vacuo. The resulting residue was purified by flash chromatography (diethyl
ether/PE
2/1) to afford the afford compound 23 (amorphous, 2.86 g, 55%).1H-NMR (200
MHz,
CDC13) : b 9.1 (d, J = 2Hz, 1H), 8.6 (dd, J= 5 Hz, 2Hz, 1H), 8.16 (dt, J = 8
Hz, 2 Hz,
1 H), 8.05 (d, J = 3 Hz, 1 H), 7.39-7.34 (m, 1 H), 6.75 (d, J = 3 Hz, 1 H),
3.02 (s, 6H).
5-Butylsulfanyl-3-pyridin-3-yl-pyrazol-l-sulfonic acid dimethylamide.
(compound 24, Scheme 3)
To a solution of anhydrous THF (50 ml) containing compound 24 (1.0 g, 4 mmol)
was
added 1 eq of n-BuLi (2.35 ml, 1.7 M in pentane) dropwise at -78 C under N2.
After
the addition, the resulting solution was stirred for 1 hour at -78 C. At this
temperature
1.1 eq 1-butyldisulfanyl-butane (0.79 ml) was added and the resulting solution
was
stirred for 1 hour at -78 C and subsequently allowed to warm to ambient
temperature
overnight. Then the mixture was quenched with a saturated NH4CI solution at 0
C
and concentrated in vacuo. Ethyl acetate was added and the organic layer was
washed with a 5% NaHCOs solution, dried (Na2SO4), filtered and concentrated in
vacuo. Purification by flash chromatography (diethyl ether/PE 5/1) to ethyl
acetate/diethyl ether 1/1) afforded compound 24 (oil, 0.93 g, 70%).1H-NMR (200
MHz, CDC13) : b 9.1 (d, J = 2Hz, 1 H), 8.6 (dd, J= 5 Hz, 2Hz, 1 H), 8.15 (dt,
J = 8 Hz, 2
Hz, 1 H), 7.39-7.34 (m, 1 H), 6.50 (s, 1 H), 3.02 (s, 6H), 3.02 (t, J = 7 Hz,
2H), 1.82-
1.67 (m, 2H), 1.60-1.42 (m, 2H), 0.97 (t, J = 7 Hz, 3H).
3-(5-Butylsulfanyl-1 H-pyrazol-3-yl)-pyridine.
(compound 25, Scheme 3)
Compound 24 (0.92 g, 2.71 mmol) was dissolved in 50 ml of n-BuOH. To this
solution was added 2 g of KOH dissolved in 50 ml of H20 and the reaction
mixture
was stirred for 18 hours at room temperature under N2. The reaction mixture
was
concentrated in vacuo. The resulting residue was taken up in ethyl acetate,
washed
with a 5% NaHCO3 solution, dried (Na2SO4), filtered and concentrated in vacuo.
Purification by flash chromatography (diethyl ether/PE 4/1) afforded compound
25
(amorphous, 0.44 g, 70%). (TLC ethyl acetate Rf 0.34).
3-(5-Butylsulfanyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 26, Scheme 3)
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Compound 26 was prepared following the procedure as described for the
synthesis
of compound 22A (from 21A).
Yield : 70% (amorphous). 'H-NMR (200 MHz, CDC13) : 6 6.27 (s, 1H), 6.22-6.18
(bs,
1H), 3.31-3.27 (m, 2H), 2.82 (t, J = 7 Hz, 2H), 2.58 (bt, J = 7 Hz, 2H), 2.44
(s, 3H),
5 2.39-2.32 (m, 2H), 1.62-1.54 (m, 2H), 1.44-1.37 (m, 2H), 0.86 (t, J = 7 Hz,
3H).
3-f4-(3-Phenyl-propoxy)-1 H-pyrazol-3-y11-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 33A, Scheme 5)
A 60% dispersion of NaH in mineral oil (3 g, 1.1 eq) was added to a solution
of
10 anhydrous THF (100 ml) containing compound 20B (18.36 g, 68.74 mmol) under
N2.
The resulting mixture was stirred for 2 hours at room temperature and
subsequently
treated with 13.37 ml (1.1 eq) of (2-chloromethoxy-ethyl)-trimethylsilane (SEM-
CI).
The resulting mixture was stirred for 18 hours at room temperature. Ethyl
acetate
was added to the mixture and the organic layer was washed three times with a
15 saturated NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The
resulting
residue was purified by flash chromatography (diethyl ether/PE 1/1) to give a
(variable) mixture of 29A and 29B as an oil (23.09 g, 83%). 'H-NMR (400 MHz,
CDC13) : The NMR shows great resemblance with the NMR of the mixture of 27A/B
(see Scheme 4), major isomer (presumably 29A) is given : 6 9.15 (d, J = 2 Hz,
1 H),
20 8.65 (dd, J = 5 Hz, 2 Hz, 1 H), 8.17 (dt, J = 8 Hz, 2 Hz, 1 H), 7.74 (s, 1
H), 7.39-7.34
(m, 1H), 5.46 (s, 2H), 3.71-3.62 (m, 4H, both isomers), 0.98-0.84 (m, 4H, both
isomers), 0.02 (both isomers, Si(CH3)3).
To a solution of anhydrous THF (250 ml) containing a mixture of 29A/B (10 g,
25
mmol) was added 10.5 ml (1.1 eq) of n-BuLi (2.5 M in hexane) dropwise (-78 C
25 under N2). After the addition, the resulting solution was stirred for 60
minutes at
-78 C. At this temperature, trimethyl borate (3 eq, 8.50 ml) was added
(dropwise in
15 minutes) and the reaction mixture was stirred for another 2 hours at -78 C.
The
reaction mixture was then allowed to warm to ambient temperature (overnight).
The temperature of the reaction mixture was lowered to -10 C and 2.2 ml (1.5
eq) of
30 acetic acid was added. Subsequently, 1.1 eq of a 30% H202 solution (2.93
ml) was
added dropwise, while keeping the temperature <-5 C. The mixture was allowed
to
warm to ambient temperature and stirred for another 4 hours. To the reaction
mixture
was added 10 ml of H20 and subsequently ethyl acetate (500 ml). The organic
layer
was washed with a 5% NaHCO3 solution, dried (Na2SO4), filtered and
concentrated in
35 vacuo. The resulting residue was purified by flash chromatography (diethyl
ether
followed by ethyl acetate) to afford 3-pyridin-3-yl-1-(2-trimethylsilanyl-1-
ethoxymethyl)-1 H-pyrazol-4-ol (30), as a (variable) mixture of the SEM
protected
product isomers. (amorphous, 2.9 g, 40%). 'H-NMR ( 400 MHz, CDCI3, mixture of
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isomers -1/1) : b 9.22 and 8.90 (2 x bs, 1 H), 8.47-8.18 (m, 2H), 7.45-7.34
(m, 1 H),
7.36 and 7.31 (2 x s, 1H), 5.36 and 5.35 (2 x s, 2H), 3.74-3.69 and 3.64-3.59
(2 x m,
2H), 0.99-0.90 (m, 2H), 0.02 and 0.01 (2 x s, 9H)
To a solution of anhydrous DMF (50 ml) containing compound 30 (2.03 g, 6.98
mmol)
were added 1.5 eq of K2CO3 (1.45 g) and the mixture was stirred for 1 hour
under N2.
After the addition of 3-bromo-propylbenzene (1,1 eq, 1.17 ml), the resulting
solution
was stirred for 18 hours at 45 C and allowed to reach ambient temperature.
Ethyl
acetate was added and the organic layer was washed with a concentrated NaHCO3
solution, dried (Na2SO4), filtered and concentrated in vacuo. The resulting
residue
was purified by flash chromatography (diethyl ether) to afford 31A as mixture
of SEM
isomers. (oil, 1.92 g, 67%).'H-NMR (400 MHz, CDCI3, major isomer is given) : 6
8.96
(d, J = 2Hz, 1 H), 8.61 (dd, J= 5 Hz, 2Hz, 1 H), 8.05 (dt, J = 8 Hz, 2 Hz, 1
H), 7.42-7.39
(m, 1H), 7.39 (s, 1H), 7.30-7.14 (m, 5H), 5.37 (s, 2H), 3.98 (t, J = 7 Hz,
2H), 3.74-
3.69 (m, 2H), 2.74 (t, J = 7 Hz, 2H), 2.10-2.02 (m, 2H), 0.98-0.92 (m, 2H),
0.01 (s,
9H).
To a solution of anhydrous THF (50 ml) containing 31A (1.93 g, 4.71 mmol) was
added 14.16 ml (3.0 eq) of TBAF (1.0 M in THF) under N2. After the addition,
the
resulting solution was refluxed for 18 hours and subsequently concentrated in
vacuo.
Ethyl acetate was added and the organic layer was washed with a concentrated
NaHCO3 solution, dried (Na2SO4), filtered and concentrated in vacuo. The
resulting
residue was purified by flash chromatography (ethyl acetate) to afford the
desired 3-
[4-(3-phenyl-propoxy)-1 H-pyrazol-3-yl]-pyridine (32A). (oil, 1.32 g, 73%). 1H-
NMR
(400 MHz, CDCI3) : b 9.15 (d, J = 2Hz, 1 H), 8.54 (dd, J= 5 Hz, 2Hz, 1 H),
8.24 (dt, J =
8 Hz, 2 Hz, 1 H), 7.35-7.31 (m, 1 H), 7.30-7.25 (m, 2H), 7.22-7.17 (m, 3H),
3.96 (t, J =
7 Hz, 2H), 2.83 (t, J = 7 Hz, 2H), 2.19-2.11 (m, 2H)
Compound 32A (0.3 g, 1.49 mmol) was converted to compound the title compound 3-
[4-(3-phenyl-propoxy)-1 H-pyrazol-3-yl]-1,2,5,6-tetrahydro-1-methylpyridine
(33A),
using the methodology described for the conversion of 21A to 22A (see Scheme
3).
Yield : 70% (amorphous, 72%). LCMS (method A) ; Rt : 1.52 min, ([M+H]` =
298).'H-
NMR (400 MHz, CDCI3) : b 7.31-7.25 (m, 2H), 7.22-7.17 (m, 3H), 7.16-7.11 (bs,
1H),
6.62-6.44 (bs, 1 H), 3.87 (t, J = 7 Hz, 2H), 3.41-3.36 (m, 2H), 2.79 (t, J = 7
Hz, 2H),
2.58 (t, J = 7 Hz, 2H), 2.44 (s, 3H), 2.42-2.35 (m, 2H), 2.11-2.04 (m, 2H).
3-(4-Hexyloxy-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 33B, Scheme 6)
Compound 29A/B was converted to 3-(4-iodo-1-(2-trimethylsilanyl-ethoxymethyl)-
1 H-
pyrazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine (compound 34NB), using the
methodology described for the conversion of 21A to 22A (see Scheme 3).
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Yield : 47%.(amorphous). LCMS (method A) ; Rt : 2.66 min, ([M+H]+ = 420) and
Rt
2.74 min, ([M+H]+ = 420).
A mixture of compound 34A/B (0.75 g, 1.79 mmol), Cul (34 mg, 0.179 mmol),
Cs2CO3 (1.18 g, 3.58 mmol), 1,10-phenanthroline (0.07 g, 0.358 mmol) and 1-
hexanol (5 ml, 40 mmol) was heated at 140 C for 18 hours (under air).
The mixture was cooled to room temperature. Ethyl acetate was added and the
organic layer was washed met a 5% NaHCO3 solution, dried (Na2SO4), filtered
and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(ethyl acetate/PE 1:1) to afford compound 35A as an oil (0.24 g, 34%). LCMS
(Method A) ; R, : 2.50 min, ([M+H]+ = 394).(TLC ethyl acetate/PE 1/1, Rf
0.07).
To a solution of anhydrous THF (20 ml) containing a mixture of 35A (0.24 g,
0.6
mmol) was added 1.52 ml (2.5 eq) TBAF (1.0 M in THF) under N2. After the
addition,
the resulting solution was refluxed for 18 hours and subsequently concentrated
in
vacuo. Ethyl acetate was added and the organic layer was washed with a
concentrated NaHCO3 solution, dried (Na2SO4), filtered and concentrated in
vacuo.
The resulting residue was purified by flash chromatography (ethyl acetate/MeOH
(1/1)) to afford the title compound 33B. (oil, 0.12 g, 75%). LCMS (Method A) ;
Rt
1.99 min, ([M+H]+ = 264).
Compound 33B was reacted with 1 equivalent of fumaric acid in EtOH and
concentrated (amorphous). 1H-NMR (600 MHz, D6DMSO) : 6 7.50 (bs, 1 H), 6.58
(s,
2H), 6.50 (bs, 1 H), 3.88 (t, J = 7 Hz, 2H), 3.75 (bs, 2H), 3.02 (bt, J = 7
Hz, 2H), 2.69
(s, 3H), 2.48-2.42 (m, 2H), 1.75-1.67 (m, 2H), 1.45-1.38 (m, 2H), 1.36-1.28
(m, 4H),
0.89 (t, J = 7 Hz, 3H).
3-(4-Butyloxyy-1 H-pyrazol-3-yl)-pyridine.
(compound 32C)
Compound 32C was prepared following the procedure as described for the
synthesis
of compound 32A (see Scheme 5) using 1-bromo-butane as the alkyl halogenide
and
3-pyridin-3-yl-1-(2-trimethylsilanyl-1-ethoxymethyl)-1 H-pyrazol-4-ol (30).
Work-up and flash chromatography (ethyl acetate/diethyl ether 1/1) afforded
compound 31 C. Yield 30% (oil). 1H-NMR (major isomer is given , 400 MHz,
CDCI3)
6 8.9(d,J=2Hz,1H),8.6(dd,J=5Hz,2Hz,1H),8.05(dt,J=8Hz,2Hz,1H),7.42
(s, 1 H), 7.41-7.37 (m, 1 H), 5.37 (s, 2H), 3.98 (t, J = 7 Hz, 2H), 3.74-3.68
(m, 2H),
1.74-1.66 (m, 2H), 1.49-1.39 (m, 2H), 0.92 (t, J = 7 Hz, 3H), 0.02 (s, 9H).
The mixture of SEM-isomers was deprotected (TBAF/THF) to afford 3-(4-butyloxyy-
1 H-pyrazol-3-yl)-pyridine (32C).
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Yield : 70% (amorphous).'H-NMR (400 MHz, CDC13) : b 9.25 (d, J = 2Hz, 1H),
8.53
(dd, J= 5 Hz, 2Hz, 1 H), 8.23 (dt, J = 8 Hz, 2 Hz, 1 H), 7.35-7.31 (m, 1 H),
7.30 (s, 1 H),
3.77 (t, J = 7 Hz, 2H), 1.84-1.77 (m, 2H), 1.57-1.47 (m, 2H), 0.98 (t, J = 7
Hz, 3H).
3-(4-Butyloxyy-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine.
(compound 33C)
Compound 32C was converted to the title compound (33C), using the methodology
described for the conversion of 21A to 22A (see Scheme 3).
Yield : 77% (amorphous). LCMS (method A); Rt : 1.48 min, ([M+H]+ = 236).1H-NMR
(400 MHz, CDC13) : b 7.19 (bs, 1 H), 6.52-6.42 (bs, 1 H), 3.88 (t, J = 7 Hz,
2H), 3.38-
3.34 (m, 2H), 2.57 (t, J = 7 Hz, 2H), 2.44 (s, 3H), 2.41-2.36 (m, 2H), 1.79-
1.71 (m,
2H), 1.53-1.43 (m, 2H), 0.97 (t, J = 7 Hz, 3H).
3-(4-But-3-enyloxy-1 H-pyrazol-3-yl)-pyridine.
(compound 32D)
Compound 32D was prepared following the procedure as described for the
synthesis
of compound 32A (see Scheme 5) using 4-bromo-but-l-ene as the alkyl halogenide
and 3-pyridin-3-yl-1-(2-trimethylsilanyl-1-ethoxymethyl)-1 H-pyrazol-4-ol
(30).
Work-up and flash chromatography (diethyl ether) afforded compound 31D. Yield
43% (oil, TLC diethyl ether Rf 0.37), which was subsequently deprotected
(TBAF/THF) to afford 3-(4-but-3-enyloxy-1 H-pyrazol-3-yl)-pyridine (32D).
Yield : 75% (amorphous).'H-NMR (400 MHz, CDC13) : b 9.15 (d, J = 2Hz, 1H),
8.58
(dd, J= 5 Hz, 2Hz, 1 H), 8.24 (dt, J = 8 Hz, 2 Hz, 1 H), 7.35-7.30 (m, 1 H),
7.32 (s, 1 H),
5.96-5.85 (m, 1H), 5.22-5.11 (m, 2H), 4.02 (t, J = 7 Hz, 2H), 2.62-2.55 (m,
2H).
3-(4-But-3-enyloxyy-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 33D)
Compound 32D was converted to the title compound (33D), using the methodology
described for the conversion of 21A to 22A (see Scheme 3).
Yield : 94%.(amorphous). LCMS (method A); Rt : 1.33 min, ([M+H]+ = 234).1H-NMR
(400 MHz, CDC13) : b 7.20 (bs, 1 H), 6.60-6.42 (bs, 1 H), 5.94-5.83 (m, 1 H),
5.19-5.07
(m, 2H), 3.94 (t, J = 7 Hz, 2H), 3.38-3.33 (m, 2H), 2.59-2.50 (m, 4H), 2.44
(s, 3H),
2.41-2.36 (m, 2H).
3-(4-Heptyloxy-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 33E)
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A mixture of compound 34A/B (0.75 g, 1.79 mmol) (see Scheme 6), Cul (34 mg,
0.179 mmol), CszCO3 (1.18 g, 3.58 mmol), 1,10-phenanthroline (0.07 g, 0.358
mmol)
and 1-heptanol (5 ml) was heated at 140 C for 18 hours (under air).
The mixture was cooled to room temperature. Ethyl acetate was added and the
organic layer was washed with a 5% NaHCO3 solution, dried (Na2SO4), filtered
and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(ethyl acetate/PE 1/1) to afford compound 35E (heptyl analogue of 35A, Scheme
6)
as an oil (0.22 g, 30%). LCMS (method A); Rt : 2.60 min, ([M+H]+ = 408).
To a solution of anhydrous THF (20 ml) containing a mixture of 35E (0.22 g,
0.54
mmol) was added 1.35 ml (2.5 eq) of TBAF (1.0 M in THF) under N2. After the
addition, the resulting solution was refluxed for 18 hours and subsequently
concentrated in vacuo. Ethyl acetate was added and the organic layer was
washed
with a concentrated NaHCO3 solution, dried (Na2SO4), filtered and concentrated
in
vacuo. The resulting residue was purified by flash chromatography (ethyl
acetate/MeOH 1/1) to afford the title compound 33E. (oil, 0.08 g, 53%). LCMS
(method A) ; R, : 2.23 min, ([M+H]+ = 278).
Compound 33E was reacted with 1 equivalent of fumaric acid in EtOH and
concentrated (amorphous). 1H-NMR (600 MHz, D6DMSO) : b 7.37 (bs, 1 H), 6.58
(s,
2H), 6.43 (bs, 1 H), 3.84 (t, J = 7 Hz, 2H), 3.51 (bs, 2H), 2.77 (bt, J = 7
Hz, 2H), 2.52
(s, 3H), 2.39-2.33 (m, 2H), 1.72-1.66 (m, 2H), 1.45-1.22 (m, 8H), 0.87 (t, J =
7 Hz,
3H).
3-(4-Pent-4-enyloxy-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 33F)
A mixture of compound 34A/B (0.75 g, 1.79 mmol) (see Scheme 6), Cul (34 mg,
0.179 mmol), Cs2CO3 (1.18 g, 3.58 mmol), 1,10-phenanthroline (0.07 g, 0.358
mmol)
and pent-4-en-l-ol (5 ml) was heated at 140 C for 18 hours (under air).
The mixture was cooled to room temperature. Ethyl acetate was added and the
organic layer was washed met a 5% NaHCO3 solution, dried (Na2SO4), filtered
and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(ethyl acetate/PE 1:1) to afford compound 35F (pent-4-enyl analogue of 35A,
Scheme 6) as an oil (0.41 g, 60%). LCMS (Method A); Rt : 2.33 min, ([M+H]+ =
378).
To a solution of anhydrous THF (20 ml) containing a mixture of 35F (0.22 g,
0.54
mmol) was added 1.35 ml (2.5 eq) of TBAF (1.0 M in THF) under N2. After the
addition, the resulting solution was refluxed for 18 hours, cooled and
subsequently
concentrated in vacuo. Ethyl acetate was added and the organic layer was
washed
with a concentrated NaHCO3 solution, dried (Na2SO4), filtered and concentrated
in
vacuo. The resulting residue was purified by flash chromatography (ethyl
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acetate/MeOH 1/1) to afford the title compound 33F. (oil, 0.08 g, 53%). LCMS
(Method A); Rt : 1.83 min, ([M+H]+ = 248).
Compound 33F was reacted with 1 equivalent of fumaric acid in EtOH and
concentrated (amorphous). 1H-NMR (600 MHz, D6DMSO) : 5 7.38 (bs, 1 H), 6.56
(s,
5 2H), 6.48-6.44 (m, 1 H), 5.88-5.80 (m, 1 H), 5.06-4.95 (m, 2H), 3.86 (t, J =
7 Hz, 2H),
3.60 (bs, 2H), 2.86 (bt, J = 7 Hz, 2H), 2.58 (s, 3H), 2.42-2.37 (m, 2H), 2.20-
2.15 (m,
2H), 1.83-1.77 (m, 2H).
3-(4-Pentyloxy-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
10 (compound 33G, Scheme 6)
A mixture of compound 29A/B (1.0 g, 2.49 mmol) (see Scheme 5), Cul (0.05 g,
0.249
mmol), Cs2CO3 (1.62 g, 4.98 mmol), 1,10-phenanthroline (0.09 g, 0.498 mmol)
and
pentanol (7 ml) was heated at 140 C for 18 hours (under air).
The mixture was cooled to room temperature. Ethyl acetate was added and the
15 organic layer was washed met a 5% NaHCO3 solution, dried (Na2SO4), filtered
and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(ethyl acetate/PE 1:2) to afford compound 31G as an oil (0.24 g, 27%). LCMS
(method A) ; Rt : 2.76 min, ([M+H]+ = 362). 'H-NMR (400 MHz, mixture of
isomers,
CDC13) : b 9.25 and 8.90 (d, J = 2Hz, 1 H), 8.61 and 8.52 (dd, J= 5 Hz, 2Hz, 1
H), 8.28
20 and 8.06 (dt, J = 8 Hz, 2 Hz, 1 H), 7.43 and 7.29 (s, 1 H), 7.41-7.37 and
7.35-7.31 (m,
1 H), 5.39 and 5.37 (s, 2H), 4.0-3.92 (m, 2H), , 3.71 and 3.60 (bt, J = 7 Hz,
2H), 1.88-
1.80 and 1.76-1.69 (m, 2H), 1.52-1.25 (m, 6H), 0.97-0.90 (m, 3H).
To a solution of anhydrous THF (25 ml) containing 31G (0.23 g, 0.63 mmol) were
added 1.59 ml (2.5 eq) of TBAF (1.0 M in THF) under N2. After the addition,
the
25 resulting solution was refluxed for 18 hours and subsequently concentrated
in vacuo.
Ethyl acetate was added and the organic layer was washed with a concentrated
NaHCOs solution, dried (Na2SO4), filtered and concentrated in vacuo. The
resulting
residue was purified by flash chromatography (ethyl acetate) to afford 3-(4-
pentyloxy-
1H-pyrazol-3-yl)-pyridine 32G. (oil, 0.14 g, 95%). LCMS (method A) ; Rt : 2.27
min,
30 ([M+H]+ = 232). 'H-NMR (400 MHz, CDC13) : b 9.25 (d, J = 2Hz, 1H), 8.53
(dd, J= 5
Hz, 2Hz, 1 H), 8.23 (dt, J = 8 Hz, 2 Hz, 1 H), 7.36-7.29 (m, 2H), 3.96 (bt, J
= 7 Hz, 2H),
1.87-1.78 (m, 2H), 1.51-1.34 (m, 6H), 0.94 (t, J = 7 Hz, 3H).
Compound 32G was converted to the title compound (33G), using the methodology
described for the conversion of 21A to 22A (see Scheme 3).
35 Yield : 85% (amorphous). LCMS (Method A); Rt : 1.83 min, ([M+H]+ = 250).1H-
NMR
(400 MHz, CDC13): 5 7.19 (bs, 1 H), 6.54-6.50 (bs, 1 H), 3.88 (t, J = 7 Hz,
2H), 3.40-
3.36 (m, 2H), 2.59 (t, J = 7 Hz, 2H), 2.45 (s, 3H), 2.43-2.37 (m, 2H), 1.81-
1.75 (m,
2H), 1.46-1.34 (m, 4H), 0.93 (t, J = 7 Hz, 3H).
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Butyl-[3-(1-Methyl-1,2,5,6-tetrahydro-pyridin-3-yl)-1 H-pyrazol-4-yll-amine.
(compound 42, Scheme 8)
To a solution of concentrated (10 ml) H2SO4 containing compound 19 (0.725 g, 5
mmol) was added a mixture of 5 ml H2SO4 and 5 ml HNO3 dropwise at -10 C under
N2. After the addition, the resulting solution was stirred for 3 hours at room
temperature. The mixture was poured into ice followed by subsequent addition
of 2 N
NaOH. Ethyl acetate was added and the organic layer was washed with brine,
dried
(Na2SO4), filtered and concentrated in vacuo. The resulting residue was
purified by
flash chromatography (ethyl acetate) to give compound 38 as an oil (0.57 g,
60%).
'H-NMR (200 MHz, CDC13 and D6DMSO 1/1) : b 8.9 (d, J = 2 Hz, 1H), 8.7 (d, J =
5
Hz, 2 Hz, 1 H), 8.05 (dt, J = 8 Hz, 2 Hz, 1 H), 7.60 (s, 1 H), 7.48-7.40 (m, 1
H).
Compound 38 (0.37 g, 1.9 mmol) was dissolved in MeOH (containing Pd(OH)2/C
(0.03 g)). Hydrogenation was accomplished within 3 hours (1 atm) at room
temterature. The reaction mixture was filtered, washed with ethyl acetate/MeOH
(1/1)
and concentrated in vacuo. The resulting residue was purified by flash
chromatography (ethyl acetate followed by ethyl acetate/MeOH 1/1) to afford
compound 39 as an oil (0.29 g, 95%).1H-NMR (200 MHz, CDC13 and D6DMSO 1/1) :
6 9.05(d,J=2Hz, 1H),8.51 (d, J = 5 Hz, 2 Hz, 1 H), 8.09 (dt, J = 8 Hz, 2 Hz,
1H),
7.43 (s, 1 H), 7.37-7.32 (m, 1 H).
To a solution of anhydrous CH3CN (15 ml) containing a mixture of compound 39
(0.21 g, 1.3 mmol) and 0.27 ml (1.5 eq) of triethyl amine was added 0.14 ml of
butyryl
chloride under N2. After the addition, the resulting solution was stirred for
3 hours at
room temperature and subsequently concentrated in vacuo. Ethyl acetate was
added
and the organic layer was washed with a concentrated NaHCO3 solution, dried
(Na2SO4), filtered and concentrated in vacuo. The resulting residue was
purified by
flash chromatography (ethyl acetate) to afford N-(3-pyridin-3-yl-1 H-pyrazol)-
butyramide 40 (oil, 0.19 g, 77%).(TLC ethyl acetate Rf 0.16).
Compound 40 was converted to compound 41, using the methodology described for
the conversion of 21A to 22A (see Scheme 3).
Yield : 65% (amorphous). LCMS (Method A); Rt : 0.73 min, ([M+H]+ = 249).1H-NMR
(600 MHz, CDC13): b 8.4 (bs, 1 H), 7.5 (bs, 1 H), 6.0-5.96 (m, 1 H), 3.27 (bs,
2H), 2.63
(t, J = 7 Hz, 2H), 2.43 (s, 3H), 2.43-2.37 (m, 2H), 2.32 (t, J = 7 Hz, 2H),
1.76-1.70
(m, 2H), 0.98 (t, J = 7 Hz, 3H).
To a solution of anhydrous THF (25 ml) containing compound 41 (0.27 g, 1.09
mmol)
was added 0.04 g (1.0 eq) LiAIH4 under N2. After the addition, the resulting
solution
was refluxed for 18 hours and allowed to warm to ambient temperature. To the
reaction mixture was added 0.04 ml of H20, followed by 0.08 ml of 2N NaOH and
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0.04 ml of H20. The resulting mixture was warmed for 10 minutes (60 C), cooled
to
room temperature and filtrated. Ethyl acetate was added and the organic layer
was
washed with a concentrated NaHCO3 solution, dried (Na2SO4), filtered and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(ethyl acetate/MeOH 1/1) to afford the title compound 42. (oil, 0.21 g, 95%).
LCMS
(method A); Rt : 1.05 min, ([M+H]+ = 235).1 H-NMR (600 MHz, CDC13): b 7.1 (bs,
1H),
6.17-6.14 (m, 1 H), 3.4-3.37 (m, 2H), 2.99 (t, J = 7 Hz, 2H), 2.64 (t, J = 7
Hz, 2H),
2.48 (s, 3H), 2.47-2.42 (m, 2H), 1.66-1.60 (m, 2H), 1.47-1.40 (m, 2H), 0.97
(t, J = 7
Hz, 3H).
3-(4-Hex-1-ynyl-1 H-pyrazol-3-yl)-pyridine.
(compound 44A, Scheme 9).
Compound 20B (10.49 g, 38.6 mmol) was converted to 3-(1-phenylsulfonyl-4-iodo-
1 H-pyrazol-3-yl)-pyridine (36B), using the methodology described for the
conversion
of 20 A to 36A (see Scheme 7). Yield 11.65 g (amorphous, 74%). (TLC diethyl
ether
Rf 0.4) 'H-NMR (400 MHz, CDC13) : b 9.05 (d, J = 2 Hz, 1H), 8.65 (dd, J = 5
Hz, 2
Hz, 1 H), 8.27 (s, 1 H), 8.12 (dt, J = 8 Hz, 2 Hz, 1 H), 8.10-8.06 (m, 2H),
7.73-7.67 (m,
1 H), 7.58 (bt, J = 7 Hz, 2H), 7.39-7.34 (m, 1 H).
To triethyl amine (10 ml) and DMF (4 ml) containing 36B (0.97 g, 2.36 mmol),
was
added 3 eq of hex-1-yne (0.81 ml). The resulting mixture was stirred for
another 2
hours under N2. Successively were added 10 mol % of Cul (45 mg), 5 mol % of
PdCl2(PPH3)2 (83 mg) and 18 mol% of PPH3 (111 mg). After the addition, the
resulting solution was warmed at 70 C for 18 hours under N2. After cooling to
room
temperature, the mixture was diluted with ethyl acetate, washed three times
with a
saturated NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The
resulting
residue was purified by flash chromatography (diethyl ether/PE (1/1)) to
afford 3-(1-
phenylsulfonyl-4-hex-1-ynyl-1 H-pyrazol-3-yl)-pyridine (43A) as an oil (0.66
g, 77%).
'H-NMR (400 MHz, CDC13) : b 9.5-9.2 (bs, 1H), 8.8-8.4 (bs, 1H), 8.34 (bd, J =
8 Hz,
1 H), 8.21 (s, 1 H), 8.08-8.04 (m, 2H), 7.67 (bt, J = 7 Hz, 1 H), 7.57 (bt, J
= 7 Hz, 2H),
7.41-7.32 (bs, 1H), 2.42 (t, J = 7 Hz, 2H), 1.62-1.53 (m, 2H), 1.49-1.39 (m,
2H), 0.93
(t, J = 7 Hz, 3H).
Compound 43A (0.66 g, 1.81 mmol), 1.3 g of KOH and 2 ml of NH2NH2.H20 were
combined in diethylene glycol (20 ml) and warmed to reflux for 1 hour under
N2. The
mixture was cooled, concentrated and redissolved in MeOH. Filtration over 25 g
of
SCX-2 (MeOH followed by 1 N NH3/MeOH) and subsequent purification by flash
chromatography (ethyl acetate) afforded the title compound 44A. Yield 0.37 g
(90%).
1 H-NMR (400 MHz, CDC13) : 6 9.25 (d, J = 2 Hz, 1H), 8.60 (dd, J = 5 Hz, 2 Hz,
1H),
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8.34 (dt, J = 8 Hz, 2 Hz, 1 H), 7.73 (s, 1 H), 7.39-7.34 (m, 1 H), 2.43 ((t, J
= 7 Hz, 2H),
1.64-1.56 (m, 2H), 1.52-1.42 (m, 2H), 0.94 (t, J = 7 Hz, 3H).
3-(4-Hex-1-ynyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 45A, Scheme 9).
Compound 44A was converted to compound 45A, using the methodology described
for the conversion of 21 A to 22A (see Scheme 3). Yield : 90% (amorphous).
LCMS
(method A); Rt : 1.79 min, ([M+H]' = 244).1 H-NMR (400 MHz, CDC13): b 7.57 (s,
1H),
6.78-6.66 (bs, 1 H), 3.42-3.38 (m, 2H), 2.59 (bt, J = 7 Hz, 2H), 2.45 (s, 3H),
2.44-2.37
(m, 2H), 1.62-1.43 (m, 4H), 0.94 (t, J = 7 Hz, 3H).
3-(4-Hept-1-ynyl-1 H-pyrazol-3-yl)-pyridine.
(compound 44B).
Compound 44B was prepared following the procedure as described for the
synthesis
of compound 44A (see Scheme 9) using hept-1-yne and 3-(1-phenylsulfonyl-4-
bromo-1 H-pyrazol-3-yl)-pyridine (36A). (flash chromatography with diethyl
ether) to
afford 3-(1-phenylsulfonyl-4-hept-1-ynyl-1 H-pyrazol-3-yl)-pyridine (43B) as
an oil
(90%). 1H-NMR (400 MHz, CDC13) : 5 9.25 (bs, 1 H), 8.6 (bs, 1 H), 8.34 (bd, J
= 8 Hz,
1 H), 8.21 (s, 1 H), 8.06 (bd, J = 8 Hz, 2H), 7.67 (bt, J = 7 Hz, 1 H), 7.57
(bt, J = 7 Hz,
2H), 7.36-7.30 (m, 1 H), 2.42 (t, J = 7 Hz, 2H), 1.63-1.55 (m, 2H), 1.44-1.28
(m, 4H),
0.89 (t, J = 7 Hz, 3H). Compound 43B was converted to the title compound 44B,
using the methodology described for the conversion of 43A to 44A (see Scheme
9).
Yield :98% (oil).'H-NMR (400 MHz, CDC13) : b 9.25 (d, J = 2 Hz, 1 H), 8.59
(dd, J = 5
Hz, 2 Hz, 1 H), 8.37 (dt, J = 8 Hz, 2 Hz, 1 H), 7.72 (s, 1 H), 7.38-7.33 (m, 1
H), 2.42 ((t,
J = 7 Hz, 2H), 1.65-1.57 (m, 2H), 1.46-1.30 (m, 4H), 0.90 (t, J = 7 Hz, 3H).
3-(4-Hept-l-ynyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine.
(compound 45B).
Compound 44B was converted to compound 45B, using the methodology described
for the conversion of 21 A to 22A (see Scheme 3). Yield : 44% (amorphous).
LCMS
(method A) ; Rt : 1.84 min, ([M+H]+ = 258). 'H-NMR (400 MHz, CDC13) : b 7.57
(s,
1 H), 6.78-6.66 (bs, 1 H), 3.42-3.38 (m, 2H), 2.59 (bt, J = 7 Hz, 2H), 2.45
(s, 3H), 2.44-
2.37 (m, 2H), 1.62-1.43 (m, 4H), 0.94 (t, J = 7 Hz, 3H).
3-(4-Non-1-ynyl-1 H-pyrazol-3-yl)-pyridine.
(compound 44C).
Compound 44C was prepared following the procedure as described for the
synthesis
of compound 44A (see Scheme 9) using non-1-yne and 3-(1-phenylsulfonyl-4-iodo-
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1 H-pyrazol-3-yl)-pyridine (36B). (flash chromatography with diethyl ether/PE
1/1) to
afford 3-(1-phenylsulfonyl-4-non-1-ynyl-lH-pyrazol-3-yl)-pyridine (43C) as an
oil
(80%). (TLC diethyl ether Rf 0.44).
Compound 43C was converted to the title compound 44C, using the methodology
described for the conversion of 43A to 44A (see Scheme 9). Yield : 78% (oil).
'H-
NMR (400 MHz, CDCI3): b 9.25 (d, J = 2 Hz, 1 H), 8.61 (dd, J = 5 Hz, 2 Hz, 1
H), 8.35
(dt, J = 8 Hz, 2 Hz, 1 H), 7.73 (s, 1 H), 7.39-7.34 (m, 1 H), 2.42 ((t, J = 7
Hz, 2H), 1.65-
1.56 (m, 2H), 1.47-1.38 (m, 2H), 1.37-1.22 (m, 6H), 0.90 (t, J = 7 Hz, 3H).
3-(4-Non-1-ynyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 45C).
Compound 44C was converted to compound 45C, using the methodology described
for the conversion of 21 A to 22A (see Scheme 3). Yield : 79% (amorphous).
LCMS
(method A) ; Rt : 2.17 min, ([M+H]+ = 286). 'H-NMR (400 MHz, CDC13) : 6 7.57
(s,
1 H), 6.74-6.69 (bs, 1 H), 3.40-3.36 (m, 2H), 2.58 (bt, J = 7 Hz, 2H), 2.44
(s, 3H), 2.43-
2.36 (m, 2H), 1.63-1.55 (m, 2H), 1.48-1.39 (m, 2H), 1.36-1.23 (m, 6H), 0.89
(t, J = 7
Hz, 3H).
344-(5-Phenyl-pent-l-ynyl)-1 H-pyrazol-3-yll-pyridine.
(compound 44D).
Compound 44C was prepared following the procedure as described for the
synthesis
of compound 44A (see Scheme 9) using pent-4-ynyl-benzene and 3-(1-
phenylsulfonyl-4-iodo-1 H-pyrazol-3-yl)-pyridine (36B). (flash chromatography
with
diethyl ether/PE (1/1)) to afford 3-[1-phenylsulfonyl-4-(5-phenyl-pent-1-ynyl)-
1H-
pyrazol-3-yl]-pyridine (43D) as an oil (80%).1H-NMR (400 MHz, CDCI3) : 6 9.3
(d, J =
2 Hz, 1 H), 8.6 (dd, J = 5 Hz, 2 Hz, 1 H), 8.33 (dt, J = 8 Hz, 2 Hz, 1 H),
8.22 (s, 1 H),
8.09-8.05 (m, 2H), 7.68 (bt, J = 8 Hz, 1 H), 7.57 (bt, J 8 Hz, 2H), 7.35-7.25
(m, 3H),
7.21-7.15 (m, 3H), 2.74 ((t, J = 7 Hz, 2H), 2.42 ((t, J 7 Hz, 2H), 1.96-1.87
(m, 2H).
(TLC diethyl ether Rf 0.56).
Compound 43D was converted to the title compound 44D, using the methodology
described for the conversion of 43A to 44A (see Scheme 9). Yield : 86%
(oil).'H-
NMR (400 MHz, CDCI3) : b 9.25 (d, J = 2 Hz, 1 H), 8.6 (dd, J = 5 Hz, 2 Hz, 1
H), 8.35
(dt, J 8 Hz, 2 Hz, 1 H), 7.74 (s, 1 H), 7.40-7.17 (m, 6H), 2.77 ((t, J = 7 Hz,
2H), 2.44
((t, J 7 Hz, 2H), 1.99-1.89 (m, 2H).
344-(5-Phenyl-pent-l-ynyl)-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-1-
methylpyridine.
(compound 45D).
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Compound 44D was converted to compound 45D, using the methodology described
for the conversion of 21 A to 22A (see Scheme 3). Yield : 95% (amorphous).
LCMS
(method A); R, : 1.99 min, ([M+H]+ = 306). 1H-NMR (400 MHz, CDC13) : 5 7.58
(s,
1H), 7.31-7.18 (m, 5H), 6.79-6.73 (bs, 1H), 3.43-3.40 (m, 2H), 2.78 (t, J 7
Hz, 2H),
5 2.60 (bt, J = 7 Hz, 2H), 2.45-2.37 (m, 7H), 1.95-1.87 (m, 2H).
3-[4-(5-Cyclohexyl-pent-1-ynyl)-1 H-pyrazol-3-yll-pyridine.
(compound 44E).
Compound 44E was prepared following the procedure as described for the
synthesis
10 of compound 44A (see Scheme 9) using pent-4-ynyl-cyclohexane and 3-(1-
phenylsulfonyl-4-iodo-1 H-pyrazol-3-yl)-pyridine (36B). (flash chromatography
with
diethyl ether/PE 1/1) to afford 3-[1-phenylsulfonyl-4-(5-cyclohexyl-pent-1-
ynyl)-1H-
pyrazol-3-yl]-pyridine (43E) as an oil (90%).1H-NMR (400 MHz, CDC13) : b 9.25
(d, J
=2Hz, 1 H), 8.6 (dd, J = 5 Hz, 2 Hz, 1H),8.33(dt,J=8Hz,2Hz, 1H),8.21 (s, 1H),
15 8.08-8.04 (m, 2H), 7.68 (bt, J = 8 Hz, 1 H), 7.57 (bt, J 8 Hz, 2H), 7.35-
7.31 (m, 1 H),
2.38 (t, J = 7 Hz, 2H), 1.72-1.55 (m, 6H), 1.31-1.10 (m, 7H), 0.95-0.8 (m,
2H).
Compound 43E was converted to the title compound 44E, using the methodology
described for the conversion of 43A to 44A (see Scheme 9). Yield : 94%
(oil).'H-
NMR (400 MHz, CDCI3) : b 9.25 (d, J = 2 Hz, 1 H), 8.6 (dd, J = 5 Hz, 2 Hz, 1
H), 8.35
20 (dt, J = 8 Hz, 2 Hz, 1 H), 7.72 (s, 1 H), 7.39-7.34 (m, 1 H), 2.40 (t, J =
7 Hz, 2H), 1.73-
1.57 (m, 6H), 1.35-1.10 (m, 7H), 0.93-0.75 (m, 2H).
3-[4-(5-Cyclohexyl-pent-1-ynyl)-1 H-pyrazol-3-yl1-1,2,5,6-tetrahydro-1-
methylpyridine.
(compound 45E).
25 Compound 44E was converted to compound 45E, using the methodology described
for the conversion of 21 A to 22A (see Scheme 3). Yield : 90% (amorphous).
LCMS
(method A); R, : 2.28 min, ([M+H]+ = 312). 1H-NMR (400 MHz, CDC13) : b 7.57
(s,
1 H), 6.76-6.71 (bs, 1 H), 3.41-3.37 (m, 2H), 2.59 (bt, J = 7 Hz, 2H), 2.45-
2.35 (m, 7H),
1.75-1.55 (m, 6H), 1.35-1.10 (m, 7H), 0.94-0.8 (m, 2H).
3-(4-Hex-1 -enyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 47A, Scheme 9).
To toluene (20 ml) containing 36A (0.67 g, 1.84 mmol) (see Scheme 7), was
added
1.5 eq of (E)-hexene-1-ylboronic acid (0.35 g). The resulting mixture was
stirred for 2
hours under N2. Successively were added 2 eq of K3PO4 (0.78 g), 4 mol % of
Pd(OAc)2 (16.5 mg) and 8 mol% of S-Phos (60.4 mg). After the addition, the
resulting
solution was warmed at 90 C for 18 hours under N2. After cooling to room
temperature, the mixture was diluted with ethyl acetate, washed three times
with a
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saturated NaHCOs solution, dried (Na2SO4), filtered and concentrated. The
resulting
residue was purified by flash chromatography (diethyl ether/PE 1/1) to afford
3-(1-
phenylsulfonyl-4-hex-l-enyl-lH-pyrazol-3-yl)-pyridine (46A) as an oil (0.38 g,
61%).
'H-NMR (400 MHz, CDC13) : 5 8.8 (d, J = 2 Hz, 1H), 8.6 (dd, J = 5 Hz, 2 Hz,
1H),
8.15 (s, 1 H), 8.10-8.05 (m, 2H), 7.94 (dt, J = 8 Hz, 2 Hz, 1 H), 7.70-7.64
(m, 1 H), 7.56
(bt, J = 8 Hz, 2H), 7.37-7.33 (m, 1 H), 6.19-6.02 (m, 1 H), 2.2-2.13 (m, 2H),
1.45-1.29
(m, 4H), 0.90 (t, J = 7 Hz, 3H).
Compound 46A (0.34 g, 0.97 mmol), 0.7 g of KOH and 1 ml of NH2NH2.H2O were
combined in diethylene glycol (10 ml) and warmed to reflux for 1 hour under
N2. The
mixture was cooled, concentrated and redissolved in MeOH. Filtration over 25 g
of
SCX-2 (MeOH followed by 1 N NH3/MeOH) and subsequent purification by flash
chromatography (ethyl acetate) afforded 3-(4-hex-l-enyl-1 H-pyrazol-3-yl)-
pyridine
(the deprotected analog of 46A). Yield 0.18 g (86%). (TLC diethyl ether Rf
0.18).
3-(4-Hex-1-enyl-1 H-pyrazol-3-yl)-pyridine was converted to the title compound
47A,
using the methodology described for the conversion of 21 A to 22A (see Scheme
3).
Yield : 50% (amorphous). LCMS (method A); Rt: 2.30 min, ([M+H]+ = 246).1H-NMR
(400 MHz, CDCI3) : b 7.59 (s, 1 H), 6.22 (bd, J = 16 Hz, 1 H), 6.03-5.98 (bs,
1 H), 5.98-
5.88 (m, 1 H), 3.26-3.21 (m, 2H), 2.63 (bt, J = 7 Hz, 2H), 2.44 (s, 3H), 2.43-
2.36 (m,
2H), 2.19-2.11 (m, 2H), 1.46-1.29 (m, 4H), 0.91 (t, J = 7 Hz, 3H).
3-{4-2[-(3-Fluor-phenyl)-vinyll-1 H-pyrazol-3-yl}-1,2,5,6-tetrahydro-1-
methylpyridine.
(compound 47B).
To toluene (20 ml) containing 36A (0.48 g, 1.32 mmol) (see Scheme 7), was
added
1.5 eq of (E)-2-(3-fluorphenyl)-vinyl boronic acid (0.33 g). The resulting
mixture was
stirred for 2 hours under N2. Successively were added 2 eq of K3PO4 (0.56 g),
4 mol
% of Pd(OAc)2 (8.8 mg) and 8 mol % of S-Phos (32 mg). After the addition, the
resulting solution was warmed at 90 C for 18 hours under N2. After cooling to
room
temperature, the mixture was diluted with ethyl acetate, washed three times
with a
saturated NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The
resulting
residue was purified by flash chromatography (diethyl ether/PE 1/1) to afford
3-(1-
phenylsulfonyl-4-[2-(3-fluoro-phenyl)-vinyl]-1 H-pyrazol-3-yl)-pyridine (46B)
as an oil
(0.39 g, 73%). 1H-NMR (400 MHz, CDC13) : b 8.85 (d, J = 2 Hz, 1 H), 8.65 (dd,
J = 5
Hz, 2 Hz, 1 H), 8.36 (s, 1 H), 8.13-8.08 (m, 2H), 7.95 (dt, J = 8 Hz, 2 Hz, 1
H), 7.72-
7.67 (m, 1 H), 7.58 (bt, J = 8 Hz, 2H), 7.41-7.37 (m, 1 H), 7.33-7.27 (m, 1
H), 7.16 (bd,
J = 7 Hz, 1 H), 7.12-7.07 (m, 1 H), 7.0-6.94 (m, 1 H), 6.92 (d, J = 16 Hz, 1
H), 6.82 (d, J
=16Hz,1H).
Compound 46B (1.06 g, 2.62 mmol), 1.3 g of KOH and 2 ml NH2NH2.H2O were
combined in diethylene glycol (25 ml) and warmed to reflux for 1 hour under
N2. The
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mixture was cooled, concentrated and redissolved in MeOH. Filtration over 25 g
of
SCX-2 (MeOH followed by 1 N NH3/MeOH) and subsequent purification by flash
chromatography (ethyl acetate) afforded 3-{4-[2-(3-fluor-phenyl)-vinyl]-1 H-
pyrazol-3-
yl}-pyridine (the deprotected analog of 46B). Yield 0.42 g (60.4%). 1H-NMR
(400
MHz, CDC13) : b 8.85 (d, J = 2 Hz, 1 H), 8.65 (dd, J = 5 Hz, 2 Hz, 1 H), 7.95-
7.90 (m,
2H), 7.45-7.41 (m, 1 H), 7.32-7.27 (m, 1 H), 7.20-7.16 (m, 1 H), 7.14-7.09 (m,
1 H),
6.99 (d, J = 16 Hz, 1 H), 6.96-6.86 (m, 2H).
3-{4-[2-(3-Fluor-phenyl)-vinyl]-1 H-pyrazol-3-yl}-pyridine was converted to
the title
compound 47B, using the methodology described for the conversion of 21A to 22A
(see Scheme 3). Yield : 67% (amorphous). LCMS (method A); Rt : 1.79 min,
([M+H]+
= 284).1H-NMR (400 MHz, CDC13) : b 7.78 (s, 1H), 7.31-7.25 (m, 1H), 7.20-7.10
(m,
2H), 7.0-6.88 (m, 2H), 6.81 (d, J = 16 Hz, 1 H), 6.07-6.01 (m, 1 H), 3.33-3.25
(m, 2H),
2.69 (bt, J = 7 Hz, 2H), 2.50-2.42 (m, 5H).
3-(4-Oct-l-enyl-1 H-pyrazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine.
(compound 47C).
To toluene (20 ml) containing 36A (0.91 g, 2.5 mmol) (see Scheme 7), was added
1.5 eq (E)-octene-1-ylboronic acid (0.59 g). The resulting mixture was stirred
for 2
hours under N2. Successively were added 2 eq of K3PO4 (1.06 g), 4 mol % of
Pd(OAc)2 (22.4 mg) and 8 mol % of S-Phos (82 mg). After the addition, the
resulting
solution was warmed at 90 C for 18 hours under N2. After cooling to room
temperature, the mixture was diluted with ethyl acetate, washed three times
with a
saturated NaHCOs solution, dried (Na2SO4), filtered and concentrated. The
resulting
residue was purified by flash chromatography (diethyl ether/PE 1/1) to afford
3-(1-
phenylsulfonyl-4-oct-l-enyl-1 H-pyrazol-3-yl)-pyridine (46C) as an oil (0.31
g, 32%).
'H-NMR (400 MHz, CDC13) : 5 8.85 (d, J = 2 Hz, 1H), 8.65 (dd, J = 5 Hz, 2 Hz,
1H),
8.15 (s, 1 H), 8.08-8.05 (m, 2H), 7.93 (dt, J = 8 Hz, 2 Hz, 1 H), 7.69-7.64
(m, 1 H),
7.59-7.52 (m, 2H), 7.37-7.33 (m, 1 H), 6.19 (d, J = 16 Hz, 1 H), 6.10-6.03 (m,
1 H),
2.19-2.12 (m, 2H), 1.45-1.37 (m, 2H), 1.36-1.23 (m, 6H), 0.90 (t, J = 7 Hz,
3H).
Compound 46C (0.35 g, 0.89 mmol), 0.7 g of KOH and 1 ml of NH2NH2.H2O were
combined in diethylene glycol (10 ml) and warmed to reflux for 1 hour under
N2. The
mixture was cooled, concentrated and re-dissolved in MeOH. Filtration over 25
g of
SCX-2 (MeOH followed by 1 N NH3/MeOH) and subsequent purification by flash
chromatography (ethyl acetate) afforded 3-(4-oct-l-enyl-1 H-pyrazol-3-yl)-
pyridine
(the deprotected analog of 46C). Yield 0.19 g(95%).'H-NMR (400 MHz, CDC13) : b
8.85 (d, J = 2 Hz, 1 H), 8.65 (dd, J = 5 Hz, 2 Hz, 1 H), 7.91 (dt, J = 8 Hz, 2
Hz, 1 H),
7.71 (s, 1 H), 7.41-7.36 (m, 1 H), 6.27 (d, J = 16 Hz, 1 H), 6.07-5.98 (m, 1
H), 2.19-2.12
(m, 2H), 1.46-1.38 (m, 2H), 1.37-1.23 (m, 6H), 0.89 (t, J = 7 Hz, 3H).
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3-(4-Oct-l-enyl-1 H-pyrazol-3-yl)-pyridine was converted to the title compound
47C,
using the methodology described for the conversion of 21 A to 22A (see Scheme
3).
Yield : 95% (amorphous). 1H-NMR (400 MHz, CDC13) :6 7.58 (s, 1H), 6.24 (bd, J
=
16 Hz, 1 H), 6.07-6.02 (m, 1 H), 5.98-5.89 (m, 1 H), 3.29-3.25 (m, 2H), 2.63
(bt, J = 7
Hz, 2H), 2.46 (s, 3H), 2.44-2.38 (m, 2H), 2.18-2.12 (m, 2H), 1.46-1.38 (m,
2H), 1.38-
1.24 (m, 6H), 0.91 (t, J = 7 Hz, 3H).
3-(4-Butylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclo[2.2.2loctane.
(compound 49A, Scheme 10)
To a solution of anhydrous THF (150 ml) containing compound 48 (0.5 g, 1.65
mmol), prepared according to Biorganic & Medicinal Chemistry, 8, 2000, 449-
454,
were added 2.1 eq of n-BuLi (1.39 ml, 2.5 M in hexane) dropwise at -78 C under
N2.
After the addition, the resulting solution was stirred for 1.5 hours at -78 C.
At this
temperature 1.1 eq of ethyldisulfanylethane (0.35 ml) were added and the
resulting
solution was stirred for 1 hour at -78 C and subsequently allowed to warm to
ambient
temperature overnight. Then the mixture was quenched with a saturated NH4CI
solution at 0 C and concentrated in vacuo. Ethyl acetate was added and the
organic
layer was washed with 2 N NaOH, dried (Na2SO4), filtered and concentrated in
vacuo. Purification by flash chromatography (MeOH) afforded the title compound
49A
(amorphous, 0.15 g, 35%). Compound 49A was reacted with 1 equivalent of
fumaric
acid in EtOH and concentrated. (solid). mp 162-164 C. LCMS (method A) ; Rt :
1.62
min, ([M+H]+ = 266). 'H-NMR (400 MHz, D6DMSO) : 6 7.83 (s, 1H), 6.43 (s, 2H),
3.60-3.39 (m, 3H), 3.25-3.00 (m, 4H), 2.58 (bt, J = 7 Hz, 2H), 2.10-2.05 (m, 1
H),
1.98-1.83 (m, 2H), 1.78-1.68 (m, 1 H), 1.48-1.30 (m, 4H), 0.84 (t, J = 7 Hz,
3H).
3-(4-Pentylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclo[2.2.2]octane.
(compound 49B, Scheme 10)
Compound 48 (2.50 g; 8.25 mmol), K2CO3 (1.82 g; 13.2 mmol) and pentane-l-thiol
(1.53 ml, 12.4 mmol) were dissolved in 30 ml of DMF and the solution was
degassed
for 45 minutes with argon. To this solution were added Pd2(dba)3 (755 mg; 0.83
mmol) and Xantphos (953 mg; 1.65 mmol). After the addition, the reaction
mixture
was heated up to 120 C and stirred for 20 hours under N2. The mixture was
cooled,
concentrated and re-dissolved in MeOH. Filtration over 25 g of SCX-2 (MeOH
followed by 1 N NH3/MeOH) and subsequent purification by flash chromatography
(EtOH) afforded the title compound 49B as an oil. Yield 395 mg (17%). Compound
49B was reacted with 1 equivalent of fumaric acid in EtOH and concentrated
(solid).
mp 142-145 C.'H-NMR (600 MHz, D6DMSO) : 6 7.83 (s, 1 H), 6.48 (s, 2H), 3.58-
3.51
(m, 1 H), 3.48-3.41 (m, 2H), 3.25-3.15 (m, 3H), 3.11-3.03 (m, 1 H), 2.61-2.53
(m, 2H),
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59
2.11-2.06 (m, 1 H), 1.97-1.85 (m, 2H), 1.77-1.69 (m, 1 H), 1.59-1.52 (m, 1 H),
1.48-
1.41 (m, 2H), 1.34-1.22 (m, 4H), 0.83 (t, J = 7 Hz, 3H).
3-(4-Methylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclof2.2.2loctane.
(compound 49C)
Compound 48 (2.50 g; 8.25 mmol)(see Scheme 10) was dissolved in 30 ml of DMF
and the solution was degassed for 45 minutes with argon. To this solution were
added NaSMe (867 mg, 12.4 mmol), Pd2(dba)3 (755 mg; 0.83 mmol) and Xantphos
(953 mg; 1.65 mmol). After the addition, the reaction mixture was heated to
120 C
and stirred for 20 hours under N2. The mixture was cooled, concentrated and re-
dissolved in MeOH. Filtration over 25 g of SCX-2 (MeOH followed by 1 N
NH3/MeOH)
and subsequent purification by flash chromatography (EtOH) afforded compound
49C (oil). Crystallization from diethyl ether gave the title compound
(solid).mp 145-
147 C Yield : 290 mg (16%). 1H-NMR (600 MHz, D6DMSO) : b 7.70 (s, 1 H), 3.21-
3.12 (m, 1 H), 3.10-3.01 (m, 2H), 2.92-2.85 (m, 1 H), 2.81-2.73 (m, 2H), 2.68-
2.61 (m,
1 H), 2.21 (s, 3H), 1.81-1.78 (m, 1 H), 1.68-1.55 (m, 3H), 1.25-1.18 (m, 1 H).
3-(4-Ethylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclof2.2.2loctane.
(compound 49D)
Compound 49D was prepared following the procedure as described for the
synthesis
of compound 49B (see Scheme 10) using ethanethiol as reagent. Subsequent
purification by flash chromatography (EtOH to EtOH/triethylamine 99/1)
afforded
compound 49D.
Yield : 30% (oil). 'H-NMR (600 MHz, D6DMSO) : 6 7.69 (s, 1H), 3.16-2.99 (m,
3H),
2.92-2.84 (m, 1H), 2.80-2.71 (m, 2H), 2.67-2.60 (m, 1H), 2.56-2.51 (m, 2H),
1.78-
1.75 (m, 1 H), 1.66-1.54 (m, 3H), 1.25-1.18 (m, 1 H), 1.08 (t, J = 7 Hz, 3H).
3-(4-Propylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclof2.2.2loctane.
(compound 49E)
Compound 49E was prepared following the procedure as described for the
synthesis
of compound 49B (see Scheme 10) using propane-l-thiol as reagent. Subsequent
purification by flash chromatography (EtOH to EtOH/triethylamine 99/1)
afforded
compound 49E.
Yield : 25% (amorphous).'H-NMR (600 MHz, D6DMSO) : 6 7.72-7.64 (bs, 1H), 3.16-
2.99 (m, 3H), 2.93-2.84 (m, 1 H), 2.80-2.71 (m, 2H), 2.67-2.60 (m, 1 H), 2.54-
2.48 (m,
2H + DMSO), 1.78-1.75 (m, 1 H), 1.66-1.54 (m, 3H), 1.47-1.40 (m, 2H), 1.25-
1.18 (m,
1 H), 0.90 (t, J = 7 Hz, 3H).
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3-(4-Hexylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclof2.2.2loctane.
(compound 49F)
Compound 49F was prepared following the procedure as described for the
synthesis
of compound 49B (see Scheme 10) using hexane-l-thiol as reagent. Subsequent
5 purification by flash chromatography (EtOH) afforded compound 49F.
Yield : 25% (oil). Compound 49F was reacted with 1 equivalent of fumaric acid
in
EtOH and concentrated (solid). mp 130 C.'H-NMR (600 MHz, D6DMSO) : b 7.81 (s,
1 H), 6.46 (s, 2H), 3.55-3.49 (m, 1 H), 3.45-3.39 (m, 2H), 3.22-3.13 (m, 3H),
3.08-3.02
(m, 1 H), 2.60-2.52 (m, 2H), 2.09-2.05 (m, 1 H), 1.95-1.83 (m, 2H), 1.75-1.68
(m, 1 H),
10 1.57-1.51 (m, 1 H), 1.46-1.40 (m, 2H), 1.35-1.29 (m, 2H), 1.27-1.17 (m,
4H), 0.83 (t, J
= 7 Hz, 3H).
3-(4-Phenethylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclof2.2.2loctane.
(compound 49G)
15 Compound 49G was prepared following the procedure as described for the
synthesis
of compound 49B (see Scheme 10) using 3-phenyl-l-propanethiol as reagent.
Subsequent purification by flash chromatography (EtOH) afforded compound 49G.
Yield : 11% (oil). Compound 49G was reacted with 1 equivalent of fumaric acid
in
EtOH and concentrated (amorphous).'H-NMR (600 MHz, D6DMSO) : b 7.86 (s, 1H),
20 7.26 (t, J = 8 Hz, 2H), 7.19-7.14 (m, 3H), 6.49 (s, 2H), 3.56-3.50 (m, 1
H), 3.46-3.39
(m, 2H), 3.24-3.14 (m, 3H), 3.10-3.03 (m, 1H), 2.66 (t, J = 7 Hz, 2H), 2.60-
2.53 (m,
2H), 2.03-2.00 (bs, 1 H), 1.94-1.82 (m, 2H), 1.77-1.68 (m, 3H), 1.56-1.50 (m,
1 H).
3-f4-(3-Methyl-butylsulfanyl)-1 H-pyrazol-3-yll-1-azabicyclof2.2.2loctane.
25 (compound 49H)
Compound 49H was prepared following the procedure as described for the
synthesis
of compound 49B (see Scheme 10) using 3-methyl-l-butanethiol as reagent.
Subsequent purification by flash chromatography (EtOH) afforded compound 49G.
Yield : 20% (oil). Compound 49G was reacted with 1 equivalent of fumaric acid
in
30 EtOH and concentrated (solid). mp 157-159 C.'H-NMR (600 MHz, D6DMSO) : b
7.83
(s, 1H), 6.47 (s, 2H), 3.56-3.50 (m, 1H), 3.46-3.40 (m, 2H), 3.24-3.15 (m,
3H), 3.09-
3.03 (m, 1 H), 2.58 (t, J = 7 Hz, 2H), 2.09-2.06 (bs, 1 H), 1.96-1.84 (m, 2H),
1.76-1.68
(m, 1 H), 1.67-1.59 (m, 1 H), 1.58-1.52 (m, 1 H), 1.38-1.30 (m, 2H), 0.83 (d,
J = 7 Hz,
6H).
3-[4-(4,4-Difluoro-but-3-enylsulfanyl)-1 H-pyrazol-3-yll-l-
azabicyclof2.2.2loctane.
(compound 491)
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Compound 491 was prepared following the procedure as described for the
synthesis
of compound 21A (see Scheme 3) using 3-phenyl-propyldisulfanyl-3-propylbenzene
as the disulfide (prepared according to the methodology described in
Tetrahedron
Letters, 42, 2001, 6741-6743) and 3-(4-iodo-1 H-pyrazol-3-yl]-1-
azabicyclo[2.2.2]octane (compound 48, see Scheme 10). Purification conditions
were: PrepHPLC (system CHSLCP02), Column : Inertsil ODS-3, 8 um. Eluens
10%/90% CH3CN/H20 + HCOOH, 50 ml/min. LCMS (method A) : Rt : 1.16 min,
([M+H]` = 300). Yield : 4.5%. (oil).'H-NMR (600 MHz, CDC13) : 5 7.63 (s, 1H),
4.24
and 4.17 (ddt, J = 7 Hz, 26 Hz, 3Hz, 1 H), 3.91-3.84 (m, 1 H), 3.51-3.37 (m,
3H), 3.32-
3.19 (m, 2H), 3.12-3.03 (m, 1H), 2.57 (t, J = 7 Hz, 2H), 2.23-2.15 (m, 3H),
2.12-2.01
(m, 1 H), 2.01-1.89 (m, 2H), 1.61-1.51 (m, 1 H).
3-(4-Butoxy-1 H-pyrazol-3-yl)-1-azabicyclo[2.2.2loctane.
(compound 52A, Scheme 10)
A 60% dispersion of NaH in mineral oil (1.5 g, 38 mmol) was added to a
solution of
anhydrous THF (300 ml) containing 3-(4-iodo-1 H-pyrazol-3-yl]-1-
azabicyclo[2.2.2]octane (compound 48, 9.6 g, 31.7 mmol) under N2. The
resulting
mixture was stirred for 2 hours at room temperature until all solids had
dissolved. The
reaction mixture was subsequently treated with 38.4 mmol (6.74 ml) of 2-
ch loromethoxy-ethyl-trimethylsi lane (SEM-CI). The resulting mixture was
stirred for
20 hours at room temperature. Because of partial quarternization of the
desired
product (50), TBAF (1 M solution in THF, 45 ml, 45 mmol) was added and the
mixture was stirred for 20 hours at room temperature. Ethyl acetate was added
to the
mixture and the organic layer was washed with a 2 N NaOH solution followed by
brine, dried (Na2SO4), filtered and concentrated to afford 3-[4-iodo-l-(2-
trimethylsilanyl-ethoxymethyl)-1H-pyrazol-3-yl]-1-azabicyclo[2.2.2]octane
(50). Yield
11.6 g, 26.7 mmol, 84%. LCMS (method B) ; Rt : 3.11 min, ([M+H]+ = 434).
A mixture of compound 50 (2.5 g, 5.77 mmol), Cul (1.37 g, 7.19 mmol), Cs2CO3
(3.90
g, 12 mmol), 1,10-phenanthroline (2.60 g, 14.4 mmol) and butanol (25 ml) was
heated at 150 C for 4 hours in the microwave.
The mixture was cooled to room temperature. Ethyl acetate was added and the
organic layer was washed met a 2 N NaOH solution, dried (NazSO4), filtered and
concentrated in vacuo. The resulting residue was purified by flash
chromatography
(gradient EtOH to EtOH/triethylamine 300/1) followed by a second flash
chromatography (EtOH/ethyl acetate/triethylamine 25/75/1) to afford compound
51A
as an oil (0.33 g, 15%). LCMS (method B) ; Rt : 3.27 min, ([M+H]+ = 380).
To a solution of anhydrous THF (5 ml) containing compound 51A (0.33 g, 0.86
mmol)
were added 3.5 ml of TBAF (1.0 M in THF) under N2. After the addition, the
resulting
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solution was stirred for 20 hours at room temperature and subsequently
concentrated
in vacuo. Ethyl acetate was added and the organic layer was washed with a
concentrated NaHCO3 solution, dried (Na2SO4), filtered and concentrated in
vacuo.
The resulting residue was purified by flash chromatography (gradient EtOH to
EtOH/triethylamine 97/3), followed by a preparative HPLC purification :
PrepHPLC
(system CHSLCP02), Column : Inertsil ODS-3, 8 um. Eluens 10%/90% CH3CN/H20
+ HCOOH, 50 ml/min, to afford the title compound 52A. (oil, 0.1 g, 45%). LCMS
(method A) : Rt : 1.0 min, ([M+H]+ = 250). 'H-NMR (600 MHz, D6DMSO + a few
drops of HCOOH) : 5 8.63 (s, -1H), 7.2 (s, 1H), 3.99-3.93 (m, 1H), 3.85 (t, J
= 7 Hz,
2H), 3.54-3.26 (m, 5H), 3.19-3.12 (m, 1 H), 2.36-2.32 (m, 1 H), 2.12-1.95 (m,
3H),
1.76-1.70 (m, 2H), 1.67-1.60 (m, 1 H), 1.50-1.42 (m, 2H), 0.98 (t, J = 7 Hz,
3H).
3-(4-Propoxy-1 H-pyrazol-3-yl)-1-azabicyclo[2.2.2]octane.
(compound 52B)
Compound 52B was prepared following the procedure as described for the
synthesis
of compound 52A (see Scheme 10) using propanol and 3-[4-iodo-l-(2-
trimethylsilanyl-ethoxymethyl)-1 H-pyrazol-3-yl]-1-azabicyclo[2.2.2]octane
(50). Work-
up and flash chromatography (gradient EtOH to EtOH/triethylamine 300/1)
followed
by a second flash chromatography (EtOH/ethyl acetate/triethylamine 25/75/1)
afforded compound 51 B as an oil (14%). LCMS (method B) : Rt: 7.15 min,
([M+H]' =
366). Compound 51A was subsequently deprotected (TBAFITHF) and purified by
flash chromatography (gradient EtOH to EtOH/triethylamine 97/3), followed by a
preperative TLC purification (CH3CN/H20) to afford the title compound 52B as
an oil.
(51%). LCMS (method A) ; Rt : 0.91min, ([M+H]+ = 236). 'H-NMR (600 MHz,
D6DMSO) : 5 7.34 (s, 1 H), 3.74 (t, J = 7 Hz, 2H), 3.24-3.19 (m, 1 H), 3.03-
2.96 (m,
1 H), 2.92-2.87 (m, 1 H), 2.86-2.74 (m, 3H), 2.69-2.62 (m, 1 H), 1.88-1.84 (m,
1 H),
1.67-1.55 (m, 5H), 1.26-1.20 (m, 1 H), 0.92 (t, J = 7 Hz, 3H).
3-(4-Pentyloxy-1 H-pyrazol-3-yl)-1-azabicyclo[2.2.2loctane.
(compound 52C)
Compound 52C was prepared following the procedure as described for the
synthesis
of compound 52A (see Scheme 10) using pentanol (25 ml) and 3-[4-iodo-1-(2-
trimethylsilanyl-ethoxymethyl)-1H-pyrazol-3-yl]-1-azabicyclo[2.2.2]octane
(50), (2 g,
4.61 mmol). Work-up and flash chromatography (gradient EtOH to
EtOH/triethylamine 300/1) followed by a second flash chromatography
(EtOH/ethyl
acetate/triethylamine 25/75/1) afforded compound 51C as an oil (0.46 g, 15%).
LCMS (method B) ; R, : 3.30 min, ([M+H]+ = 394).
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To a solution of SEM-protected pyrazole 51 C(0.46 g, 1.2 mmol) in EtOH (5 ml)
was
added HCI (4 M in dioxane, 1 ml, 4 mmol) and the reaction mixture was stirred
at
75 C for 20 hours. Subsequent purification by flash chromatography (gradient
EtOH
to EtOH/triethylamine 97/3), followed by a preparative HPLC purification :
PrepHPLC
(system CHSLCPO2), Column : Inertsil ODS-3, 8 um. Eluens 10%/90% CH3CN/H2O
+ HCOOH, 50 ml/min, afforded the title compound 52C. (oil, 60 mg, 19%).'H-NMR
(600 MHz, D6DMSO + a few drops of HCOOH) : 6 8.65 (s, -1 H), 7.2 (s, 1 H),
3.96-
3.90 (m, 1H), 3.84 (t, J = 7 Hz, 2H), 3.54-3.46 (m, 1H), 3.45-3.26 (m, 4H),
3.19-3.12
(m, 1 H), 2.36-2.32 (m, 1 H), 2.12-1.95 (m, 3H), 1.79-1.74 (m, 2H), 1.67-1.60
(m, 1 H),
1.45-1.35 (m, 4H), 0.95 (t, J = 7 Hz, 3H).
Exo-6-(1 H-pyrazol-3-yl)-1-azabicyclo[3.2.1 loctan-6-ol.
(compound 55, Scheme 11)
A suspension of propargylaldehyde diethyl acetale (14.62 g, 114 mmol) and t-
BuOK
(14.92 g, 133 mmol) in 350 ml of anhydrous THF was stirred for 1 hour at -10 C
under N2. Then a suspension of 1-aza-bicyclo[3.2.1]octan-6-one (53, J. Med.
Chem.,
36, 1993, 683-689) (11.92 g, 95 mmol) in 100 ml of THF was added and the
resulting homogeneous reaction mixture was stirred for another 2 hours at O C.
Then
the mixture was quenched with an aqueous acetic acid solution at 0 C and
concentrated in vacuo. Ethyl acetate was added and the organic layer was
washed
with 2 N NaOH, dried (Na2SO4), filtered and concentrated in vacuo.
Purification by
flash chromatography (dichloromethane/MeOH/NH4OH 93/7/0.5) afforded 6-(3,3-
diethoxy-prop-1-ynyl)-1-azabicyclo[3.2.1]octan-6-ol (54) as an oil (20.87 g,
86%).
LCMS (Method A) : Rt: 1.18 min, ([M+H]+ = 254).'H-NMR (400 MHz, CDCI3) : b
5.28
(s, 1 H), 3.74-3.65 (m, 2H), 3.61-3.52 (m, 2H), 3.45 (d, J = 13 Hz, 1 H), 3.12-
3.04 (m,
1 H), 3.0-2.82 (m, 4H), 2.22-2.18 (m, 1 H), 2.17-2.02 (m, 1 H), 1.98-1.89 (m,
1 H), 1.71-
1.61 (m, 1 H), 1.39-1.30 (m, 1 H), 1.22 (t, J = 7 Hz, 6H).
Compound 54 (15.39 g, 60.7 mmol) and 7.02 g of hydrazine dihydrochloride were
combined in EtOH/H20 (3/2, 250 ml) and warmed to reflux for 18 hour under N2.
The
mixture was cooled, concentrated and re-dissolved in MeOH. To the reaction
mixture
was added Amberlyte IRA-95 (basic) and subsequently stirred for 18 hours at
room
temperature. The mixture was filtrated, concentrated and re-dissolved in MeOH.
Filtration over 100 g of SCX-2 (MeOH followed by 1 N NH3/MeOH) afforded the
title
compound 55 (amorphous). Yield 8.45 g (72%). 1H-NMR (600 MHz, D6DMSO + a
few drops of CF3COOH) : b 7.70 (d, J = 2 Hz, 1 H), 6.42 (d, J = 2 Hz, 1 H),
4.24-4.20
(m, 1 H), 3.41-3.31 (m, 4H), 3.25-3.21 (m, 1 H), 2.45-2.42 (m, 1 H), 2.40-2.30
(m, 1 H),
2.10-2.04 (m, 1H), 1.74-1.64 (m, 2H), used techniques are DEPT, HSQC, COSY,
HMBC, ROESY and NOESY
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64
Endo-6-(1 H-pyrazol-3-yl)-1-azabicyclo[3.2.1]octane.
(compound 58, Scheme 11)
Compound 55 (7.83 g, 41 mmol) and 9.81 ml (120 mmol) of pyridine were combined
in benzene (150 ml). Acetic anhydride was added (11.48 ml, 120 mmol) and the
reaction mixture was warmed to 40 C for 72 hour under N2. The mixture was
cooled,
concentrated and re-dissolved in MeOH. Filtration over 100 g of SCX-2 (MeOH
followed by 1 N NH3/MeOH) afforded compound 56 (amorphous, 9.6 g, - 100%),
LCMS (method A) : Rt : 0.98 min, ([M+H]+ = 236), which was used without
further
purification.
Compound 56 (1.2 g, 5.1 mmol) was heated (in the neat) at 200 C for 5 min
under
reduced pressure (23 mbar) and subsequently cooled to room temperature.
Purification by flash chromatography (dichloromethane/MeOH/NH4OH 85/15/1)
afforded 6-(1H-pyrazol-3-yl)-1-azabicyclo[3.2.1]oct-6-ene (57) as an oil (0.49
g,
49%). LCMS (Method A) ; Rt : 1.27 min, ([M+H]+ = 176).1H-NMR (400 MHz, CDCI3)
:
5 7.56 (d, J = 2 Hz, 1 H), 6.43 (s, 1 H), 6.36 (d, J = 2 Hz, 1 H), 3.50-3.45
(m, 1 H), 3.06-
2.97 (m, 1 H), 2.93 (d, J = 10 Hz, 1 H), 2.86-2.79 (m, 2H), 2.06-1.93 (m, 1
H), 1.78-
1.72 (m, 1 H), 1.49-1.41 (m, 1 H).
Compound 57 (3.84 g, 21.9 mmol), 6.9 g of ammonium formate (110 mmol) and 20%
Pd(OH)2/C (340 mg) were combined in MeOH (150 ml) and warmed to reflux for 1
hour. The mixture was cooled, filtered, concentrated and re-dissolved in MeOH.
Filtration over 40 g of SCX-2 (MeOH followed by 1 N NH3/MeOH) and subsequent
purification by flash chromatography (MeOH/triethylamine 90/3) afforded endo-6-
(1 H-
pyrazol-3-yl)-1-azabicyclo[3.2.1]octane (58), (amorphous, 2.8 g, 73%). LCMS
(method A) ; Rt : 0.80 min, ([M+H]+ = 178). 1H-NMR (600 MHz, D6DMSO/ CDCI3
(1 /1)) : 5 7.54 (d, J = 2 Hz, 1 H), 6.16 (d, J = 2 Hz, 1 H), 3.98-3.92 (m, 1
H), 3.90-3.84
(m, 1 H), 3.82-3.77 (m, 1 H), 3.53-3.48 (m, 1 H), 3.32-3.24 (m, 3H), 2.82-2.77
(m, 1 H),
2.03-1.94 (m, 1 H), 1.68-1.60 (m, 1 H), 1.58-1.52 (m, 1 H), 1.51-1.47 (m, 1
H), used
techniques are DEPT, HSQC, COSY, HMBC, ROESY and NOESY.
Endo-6-(4-pentylsulfanyl-1 H-Pyrazol-3-yl)-1-azabicyclo[3.2.1]octane.
(compound 60A, Scheme 11)
To a solution of 58 (1.49 g, 8.41 mmol) in anhydrous DMF (60 ml) at -10 C were
added 2.68 g (10.93 mmol) of N-iodosuccinimide. The reaction mixture was
stirred
for 2 hours at -10 C and 1 hour at room temperature. The solvent was (partly)
removed under reduced pressure. MeOH was added and the resulting reaction
mixture was filtrated over 120 g of SCX-2 (MeOH followed by 1 N NH3/MeOH) to
afford endo-6-(4-iodo-1 H-pyrazol-3-yl)-1-azabicyclo[3.2.1 ]octane (59),
(amorphous,
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1.73 g, 67%). LCMS (Method A) ; Rt : 1.25 min, ([M+H]+ = 304).'H-NMR (600 MHz,
D6DMSO + CF3COOH) : b 7.72 (s, 1 H), 4.08-4.03 (m, 1 H), 3.80-3.75 (m, 1 H),
3.73-
3.68 (m, 1 H), 3.50-3.45 (m, 1 H), 3.34-3.25 (m, 3H), 3.04-3.0 (m, 1 H), 2.24-
2.14 (m,
1H), 1.64-1.57 (m, 1H), 1.50-1.42 (m, 1H), 1.22-1.17 (m, 1H), used technics
are
5 DEPT, HSQC, COSY, HMBC, ROESY and NOESY.
Compound 59 (0.50 g; 1.65 mmol), K2COs (0.3 g; 2.14 mmol) and pentane-l-thiol
(0.26 ml, 2.06 mmol) were dissolved in 20 ml of xylene/DMF (9/1) and the
solution
was degassed for 45 minutes with argon. To this solution were added Pd2(dba)3
(150
mg; 0.165 mmol) and Xantphos (190 mg; 0.33 mmol). After the addition, the
reaction
10 mixture was heated up to 130 C and stirred for 20 hours under N2. The
mixture was
cooled, concentrated and re-dissolved in MeOH. Filtration over 75 g of SCX-2
(MeOH followed by 1 N NH3/MeOH) and subsequent purification by flash
chromatography (MeOH/triethylamine (97/3)) afforded the title compound 60A as
an
oil. Yield 150 mg (32%). Compound 60A was reacted with 1 equivalent of fumaric
15 acid in EtOH and concentrated (amorphous).
LCMS (method A) ; Rt: 1.39 min, ([M+H]' = 280).1H-NMR (600 MHz, D6DMSO) : 5
7.85 (s, 1H), 6.43 (s, 2H), 3.80-3.70 (m, 2H), 3.63-3.58 (m, 1H), 3.28-3.22
(m, 1H),
3.20-3.19 (m, 3H), 2.82-2.78 (m, 1 H), 2.63-2.55 (m, 2H), 2.12-2.01 (m, 1 H),
1.65-
1.56 (m, 1 H), 1.50-1.44 (m, 2H), 1.37-1.15 (m, 6H), 0.85 (t, J = 7 Hz, 3H).
Endo-6-(4-butylsulfanyl-1 H-pyrazol-3-yl)-1-azabicyclo[3.2.1 loctane.
(compound 60B)
Compound 60B was prepared following the procedure as described for the
synthesis
of compound 60A (see Scheme 11) using butane-l-thiol and endo-6-(4-iodo-1 H-
pyrazol-3-yl)-1-azabicyclo[3.2.1]-octane (50). Compound 60B was reacted with 1
equivalent of fumaric acid in EtOH and concentrated. Yield : 49% (amorphous).
LCMS (method A) ; Rt : 1.71 min, ([M+H]+ = 266).1H-NMR (600 MHz, D6DMSO) : b
7.80 (s, 1 H), 6.46 (s, 2H), 3.94-3.89 (m, 1 H), 3.86-3.81 (m, 1 H), 3.79-3.74
(m, 1 H),
3.45-3.41 (m, 1H), 3.31-3.22 (m, 3H), 2.93-2.89 (m, 1H), 2.62-2.55 (m, 2H),
2.21-
2.11 (m, 1 H), 1.66-1.59 (m, 1 H), 1.49-1.42 (m, 2H), 1.40-1.33 (m, 2H), 1.26-
1.20 (m,
2H), 0.86 (t, J = 7 Hz, 3H).
3-(4-B romo-isoxazol-3-yl)-pyrid ine.
(compound 64, Scheme 12).
To a solution of anhydrous THF (250 ml) containing 3-pyridinealdoxim (61)
(18.87 g,
154.7 mmol) were added 20.57 g (1.0 eq) of N-chlorosuccinimide. After the
addition,
the resulting solution was stirred for 18 hours at 65 C. Then the temperature
of the
reaction mixture was lowered to -30 C and 1,2-bis-trimethylsilanyl-ethyne was
added
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66
(26.4 g, 170.4 mmol), subsequently followed by triethylamine (2 eq, 42 ml,
dropwise),
while keeping the temperature <-25 C. After stirring for another 30 minutes,
the
mixture was allowed to warm to ambient temperature, diluted with ethyl
acetate,
washed three times with a saturated NaHCO3 solution, dried (Na2SO4), filtered
and
concentrated. The resulting residue was purified by flash chromatography
(diethyl
ether/PE 111) to afford 3-(4,5-bis-trimethylsilanyl-isoxazol-3-yl)-pyridine
(62) as an oil
(4.45 g, 17%).1H-NMR (400 MHz, CDC13) : b 8.70 (dd, J =5 Hz, 2 Hz, 1H), 8.68
(d, J
= 2 Hz, 1 H), 7.76 (dt, J = 8 Hz, 2 Hz, 1 H), 7.41-7.37 (m, 1 H), 0.46 (s,
9H), .011 (s,
9H).
To a solution of anhydrous CCI4 (80 ml) containing compound 62 (4.45 g, 15.4
mmol)
was added (1.1 eq, 1.04 ml) Br2. After the addition, the resulting solution
was stirred
for 18 hours at 40 C. The reaction mixture was cooled and concentrated in
vacuo.
Ethyl acetate was added and the organic layer was washed with with a saturated
NaHCOs solution, dried (Na2SO4), filtered and concentrated in vauo.
Purification by
flash chromatography (diethyl ether) afforded 3-(4-bromo-5-trimethylsilanyl-
isoxazol-
3-yl)-pyridine (63) as an oil (4.5 g, -100%).1H-NMR (400 MHz, CDC13) : 6 9.08
(d, J
= 2 Hz, 1 H), 8.73 (dd, J =5 Hz, 2 Hz, 1 H), 8.14 (dt, J = 8 Hz, 2 Hz, 1 H),
7.46-7.41 (m,
1 H), 0.40 (s, 9H).
To a solution of EtOH (20 ml) containing compound 63 (4.5 g, 15.4 mmol) were
added 5 ml of 25% NH4OH. After the addition, the resulting solution was
stirred for 10
minutes at room temperature. The reaction mixture was concentrated in vacuo.
Ethyl
acetate was added and the organic layer was washed with a saturated NaHCO3
solution, dried (Na2SO4), filtered and concentrated in vauo. Purification by
flash
chromatography (diethyl ether) afforded the title compound (64) as an oil
(2.76 g,
82%). (TLC diethyl ether Rf 0.4). 'H-NMR (400 MHz, CDC13) : 6 9.14 (d, J = 2
Hz,
1 H), 8.76 (dd, J = 5 Hz, 2 Hz, 1 H), 8.58 (s, 1 H), 8.19 (dt, J = 8 Hz, 2 Hz,
1 H), 7.48-
7.43 (m, 1 H).
3-(4-Butylsulfanyl-isoxazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 66A, Scheme 12).
To a degassed solution of dioxane (40 ml) containing 64 (1.0 g, 4.44 mmol),
were
added 1.2 eq (0.62 ml) of butane-l-thiol and 2 eq of triethylamine (1.52 ml).
The
resulting mixture was stirred for another 2 hours under N2. Successively were
added
2.5 mol % of Pd2(dba)3 (100 mg) and 5 mol % of Xantphos (128 mg). After the
addition, the resulting solution was stirred for 18 hours at 95 C under N2.
After
cooling to room temperature, the mixture was diluted with ethyl acetate,
washed with
a saturated NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The
resulting residue was purified by flash chromatography (diethyl ether) to
afford 3-(4-
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67
butylsulfanyl-isoxazol-3-yl)-pyridine (65A) as an oil (0.26 g, 25%). (TLC
diethyl ether
Rf 0.54). 'H-NMR (400 MHz, CDC13) : b 9.24 (d, J = 2 Hz, 1 H), 8.72 (dd, J = 5
Hz, 2
Hz, 1 H), 8.49 (s, 1 H), 8.31 (dt, J = 8 Hz, 2 Hz, 1 H), 7.45-7.41 (m, 1 H),
2.61 (t, J = 7
Hz, 2H), 1.52-1.43 (m, 2H), 1.39-1.29 (m, 2H), 0.83 (t, J = 7 Hz, 3H).
1 Eq of sulfuric acid dimethyl ester (0.14 ml, 1.5 mmol) was added to a
solution of
65A (0.35 g, 1.5 mmol) in acetone (20 ml) and the mixture was stirred for 18
hours at
room temperature. The precipitated crystals were filtered, washed extensively
with
diethyl ether and dried to afford the corresponding pyridinium sulfuric acid
mono
methyl ester derivative. To a cooled (-30 C) suspension of this compound in
MeOH
(25 ml), sodium borohydride (0.17 g, 4.5 mmol) was added in small portions.
The
mixture was allowed to warm to ambient temperature and poured into a saturated
NH4CI solution (0 C). The solvent was (partly) removed under reduced pressure.
Ethyl acetate was added and the organic layer was washed with a saturated
NaHCOs solution, dried (Na2SO4), filtered and concentrated in vacuo. The
resulting
residue was purified by flash chromatography (MeOH) to afford the title
compound
66A. (amorphous, 1.09 g, 73% (overall)). LCMS (method A) : Rt: 1.58 min,
([M+H]' =
253). 1H-NMR (600 MHz, CDCI3) : 6 8.23 (s, 1H), 7.06-7.04 (m, 1H), 3.40-3.38
(m,
2H), 2.68 (t, J = 7 Hz, 2H), 2.59 (t, J = 7 Hz, 2H), 2.48-2.42 (m, 5H), 1.57-
1.51 (m,
2H), 1.44-1.37 (m, 2H), 0.90 (t, J = 7 Hz, 3H).
3-(4-Hexylsulfanyl-isoxazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine.
(compound 66B).
To a degassed solution of dioxane (30 ml) containing 64 (0.6 g, 2.66 mmol)
(see
Scheme 12), were added 1.2 eq (0.62 ml) of hexane-l-thiol and 2 eq of
triethylamine
(0.92 ml). The resulting mixture was stirred for another 2 hours under N2.
Successively were added 2.5 mol % of Pd2(dba)3 (61 mg) and 5 mol % Xantphos
(77
mg). After the addition, the resulting solution was stirred for 18 hours at 95
C under
N2. After cooling to room temperature, the mixture was diluted with ethyl
acetate,
washed with a saturated NaHCO3 solution, dried (Na2SO4), filtered and
concentrated.
The resulting residue was purified by flash chromatography (diethyl ether) to
afford 3-
(4-Hexylsulfanyl-isoxazol-3-yl)-pyridine (65B) as an oil (0.25 g, 37%). 'H-NMR
(400
MHz, CDC13) : b 9.24 (d, J = 2 Hz, 1 H), 8.73 (dd, J = 5 Hz, 2 Hz, 1 H), 8.49
(s, 1 H),
8.31 (dt, J = 8 Hz, 2 Hz, 1 H), 7.45-7.41 (m, 1 H), 2.61 (t, J = 7 Hz, 2H),
1.52-1.43 (m,
2H), 1.35-1.13 (m, 6H), 0.85 (t, J = 7 Hz, 3H).
1 Eq of sulfuric acid dimethyl ester (0.09 ml, 0.95 mmol) was added to a
solution of
65A (0.25 g, 0.95 mmol) in acetone (20 ml) and the mixture was stirred for 18
hours
at room temperature. The precipitated crystals were filtered, washed
extensively with
diethyl ether and dried to afford the corresponding pyridinium sulfuric acid
mono
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68
methyl ester derivative. To a cooled (-30 C) suspension of this compound in
MeOH
(25 ml), sodium borohydride (0.144 g, 4 eq.) was added in small portions. The
mixture was allowed to warm to ambient temperature and poured into a saturated
NH4CI solution (0 C). The solvent was (partly) removed under reduced pressure.
Ethyl acetate was added and the organic layer was washed with a saturated
NaHCOs solution, dried (Na2SO4), filtered and concentrated in vacuo. The
resulting
residue was purified by flash chromatography (MeOH) to afford the title
compound
66B. (amorphous, 0.72 g, 65% (overall)). LCMS (method A) ; Rt : 1.92 min,
([M+H]' =
281). 1H-NMR (400 MHz, CDCI3) : 5 8.23 (s, 1H), 7.06-7.04 (m, 1H), 3.40-3.38
(m,
2H), 2.67 (t, J = 7 Hz, 2H), 2.59 (t, J = 7 Hz, 2H), 2.48-2.42 (m, 5H), 1.59-
1.51 (m,
2H), 1.41-1.21 (m, 6H), 0.85 (t, J = 7 Hz, 3H).
3-(4-Hex-1-enyl-isoxazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 68A, Scheme 13).
To anhydrous THF (30 ml) containing compound 64 (0.64 g, 2.84 mmol), were
added
1.5 eq of (E)-hexene-1-ylboronic acid (0.55 g). The resulting mixture was
stirred for 2
hours under N2. Successively were added 2 eq of K3PO4 (1.2 g), 2 mol % of
Pd(OAc)2 (13 mg) and 4 mol % of S-Phos (47 mg). After the addition, the
resulting
solution was warmed at 65 C for 18 hours under N2. After cooling to room
temperature, the mixture was diluted with ethyl acetate, washed with a
saturated
NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The resulting
residue
was purified by flash chromatography (diethyl ether/PE 111) to afford 3-(4-hex-
l-enyl-
isoxazol-3-yl)-pyridine (compound 67A) as an oil (0.47 g, 69%). 1H-NMR (400
MHz,
CDC13):6 8.9(d,J=2Hz, 1 H), 8.72 (dd, J = 5 Hz, 2 Hz, 1H),8.49(s, 1H),8.0(dt,J
= 8 Hz, 2 Hz, 1 H), 7.45-7.41 (m, 1 H), 6.11-5.99 (m, 2H), 2.22-2.14 (m, 2H),
1.46-1.29
(m, 4H), 0.91 (t, J = 7 Hz, 3H).
3-(4-Hex-1-enyl-1 H-pyrazol-3-yl)-pyridine (67A) was converted to the title
compound
68A, using the methodology described for the conversion of 65A to 66A (see
Scheme
12). Yield : 95% (amorphous). LCMS (method A) : R, : 1.68 min, ([M+H]' =
247).'H-
NMR (400 MHz, CDC13) : b 8.28 (s, 1H), 6.36-6.30 (m, 1H), 6.09-5.93 (m, 2H),
3.46-
3.41 (m, 2H), 2.70 (t, J = 7 Hz, 2H), 2.52 (s, 3H), 2.51-2.45 (m, 2H), 2.21-
2.14 (m,
2H), 1.47-1.31 (m, 4H), 0.92 (t, J = 7 Hz, 3H).
3-(4-Oct-l-enyl-isoxazol-3-yl)-1,2,5,6-tetrahydro-l-methylpyridine.
(compound 68B).
To anhydrous THF (20 ml) containing 64 (0.49 g, 2.17 mmol) (see Scheme 13),
were
added 1.5 eq of (E)-octene-1-ylboronic acid (0.51 g). The resulting mixture
was
stirred for 2 hours under N2. Successively were added 2 eq of K3PO4 (0.92 g),
2 mol
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69
% of Pd(OAc)2 (10 mg) and 4 mol % of S-Phos (36 mg). After the addition, the
resulting solution was warmed at 65 C for 18 hours under N2. After cooling to
room
temperature, the mixture was diluted with ethyl acetate, washed with a
saturated
NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The resulting
residue
was purified by flash chromatography (diethyl ether/PE 1/1) to afford 3-(4-Oct-
l-enyl-
isoxazol-3-yl)-pyridine (compound 67B) as an oil (0.19 g, 34%). 1H-NMR (400
MHz,
CDC13):6 8.9(d,J=2Hz, 1 H), 8.72 (dd, J = 5 Hz, 2 Hz, 1H),8.49(s, 1H),7.9(dt,J
= 8 Hz, 2 Hz, 1H), 7.45-7.40 (m, 1H), 6.11-5.99 (m, 2H), 2.21-2.13 (m, 2H),
1.46-1.38
(m, 2H), 1.37-1.21 (m, 6H), 0.91 (t, J = 7 Hz, 3H).
3-(4-Oct-1-enyl-1 H-pyrazol-3-yl)-pyridine (67B) was converted to the title
compound
68B, using the methodology described for the conversion of 65A to 66A (see
Scheme
12). Yield : 54% (amorphous). LCMS (method A) ; Rt : 2.0 min, ([M+H]+ = 275).
'H-
NMR (400 MHz, CDC13) : b 8.28 (s, 1H), 6.32-6.27 (m, 1H), 6.09-5.93 (m, 2H),
3.37-
3.33 (m, 2H), 2.61 (t, J = 7 Hz, 2H), 2.45 (s, 3H), 2.44-2.39 (m, 2H), 2.20-
2.12 (m,
2H), 1.48-1.38 (m, 2H), 1.37-1.24 (m, 6H), 0.85 (t, J = 7 Hz, 3H).
3-(4-Hept-1-ynyl-isoxazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
(compound 70A, Scheme 13).
To anhydrous DMF (20 ml) containing compound 64 (1.08 g, 4.8 mmol), were added
1.5 eq of hept-1-ynyl-trimethyl-silane (1.21 g), 3 eq of KOAc (1.41 g) and 1
eq of
TBAF (1.0 M in THF). The resulting mixture was stirred for another 2 hours
under N2.
Successively were added 10 mol % of Pd(OAc)2 (108 mg) and 20 mol % of PPH3
(251 mg). After the addition, the resulting solution was warmed at 90 C for 18
hours
under N2. After cooling to room temperature, the mixture was diluted with
ethyl
acetate, washed three times with a saturated NaHCO3 solution, dried (Na2SO4),
filtered and concentrated. The resulting residue was purified by flash
chromatography
(diethyl ether/PE 1/1) to afford 3-(4-Hept-1-ynyl-isoxazol-3-yl)-pyridine
(69A) as an oil
(0.34 g, 30%).1H-NMR (400 MHz, CDC13) : b 9.3 (d, J = 2 Hz, 1H), 8.7 (dd, J =
5 Hz,
2 Hz, 1 H), 8.58 (s, 1 H), 8.34 (dt, J = 8 Hz, 2 Hz, 1 H), 7.44-7.39 (m, 1 H),
2.42 (t, J = 7
Hz, 2H), 1.65-1.57 (m, 2H), 1.45-1.29 (m, 4H), 0.91 (t, J = 7 Hz, 3H).
3-(4-Hept-1 -ynyl-1 H-pyrazol-3-yl)-pyridine (69A) was converted to the title
compound
70A, using the methodology described for the conversion of 65A to 66A (see
Scheme
12). Yield : 50% (amorphous). LCMS (method A) : R, : 1.78 min, ([M+H]' =
259).'H-
NMR (400 MHz, CDC13) : b 8.40 (s, 1 H), 7.18-7.13 (m, 1 H), 3.41-3.36 (m, 2H),
2.59
(t, J = 7 Hz, 2H), 2.45 (s, 3H), 2.45-2.38 (m, 2H), 1.64-1.55-2.12 (m, 2H),
1.46-1.30
(m, 4H), 0.91 (t, J = 7 Hz, 3H).
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345-Bromo-3-(2-trimethylsilanyl-ethoxymethyl)-3H-im idazol-4-yll-pyridine.
(compound 73, Scheme 14)
To anhydrous THF (100 ml) containing 3-bromo-pyridine (3.29 g, 20.82 mmol),
were
added 1.2 eq of iPrMgCl (12.49 ml, 2M in THF) and the resulting mixture was
stirred
5 for 2 hours under N2 (10 C). Subsequently 1.2 eq of (CH3)3SnCl were added
and the
reaction mixture was stirred for 18 hours at room temperature (under N2). The
reaction mixture was quenched with a saturated NH4CI solution, diluted with
ethyl
acetate, washed three times with a saturated NaHCO3 solution, dried (Na2SO4),
filtered and concentrated. The resulting residue was purified by flash
chromatography
10 (diethyl ether/PE 1/1) to afford 3-trimethylstannanyl-pyridine (71) (3.32
g, 66%).
LCMS (method A) : Rt : 2.09 min, ([M+H]+ = 242). 'H-NMR (400 MHz, CDC13) : b
8.62 (bs, 1 H), 8.52 (dd, J = 5 Hz, 2 Hz, 1 H), 7.76 (dt, J = 8 Hz, 2 Hz, 1
H), 7.25-7.21
(m, 1 H), 0.33 (s, 9H), with Sn satellites at 0.41 and 0.27 .
To a solution of 71 (0.82 g, 3.4 mmol) in anhydrous toluene (50 ml) were added
0.9
15 eq (1.10 g) of 4,5-dibromo-1 -(2-trimethylsilanyl-ethoxymethyl)-1 H-
imidazole
(compound 72, LCMS (method A) : Rt : 3.60 min, ([M+H]+ = 357)) and the
resulting
mixture was stirred for 2 hours under N2 (room temperature). To this reaction
mixture
were added 10 mol % of PdCl2(PPH3)2 (240 mg). After the addition, the
temperature
was raised to 100 C and the resulting solution was stirred for 18 hours under
N2. The
20 reaction mixture was cooled, diluted with ethyl acetate, washed three times
with a
saturated NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The
resulting
residue was purified by flash chromatography (diethyl ether followed by ethyl
acetate)
to afford 3-[5-bromo-3-(2-trimethylsilanyl-ethoxymethyl)-3H-imidazol-4-yl]-
pyridine
(73) as an oil (385 mg, 36%). LCMS (method A) : Rt : 3.40 min, ([M+H]+ = 355).
'H-
25 NMR (400 MHz, CDC13 and HMBC) : b 8.79 (d, J = 2 Hz, 1 H), 8.66 (dd, J = 5
Hz, 2
Hz, 1 H), 7.91 (dt, J = 8 Hz, 2 Hz, 1 H), 7.66 (s, 1 H), 7.44-7.40 (m, 1 H),
5.17 (s, 2H),
3.56-3.50 (m, 2H), 0.94-0.87 (m, 2H), 0.0 (s, 9H).
3-(5-Pentylsulfanyl-3H-imidazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine.
30 (compound 76A, Scheme 14).
To a degassed solution of xylene (100 ml) containing 73 (1.82 g, 5.16 mmol),
were
added 1.25 eq (0.80 ml) of pentane-l-thiol and 1.25 eq of KZC03 (0.8 g). The
resulting mixture was stirred for another 2 hours under N2. Successively were
added
10 mol % of Pd2(dba)3 (470 mg) and 20 mol % mmol Xantphos (600 mg). After the
35 addition, the resulting solution was stirred for 18 hours at 130 C under
N2. After
cooling to room temperature, the mixture was diluted with ethyl acetate,
washed with
a saturated NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The
resulting residue was purified by flash chromatography (diethyl ether followed
by
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71
ethyl acetate) to afford 3-[5-pentylsulfanyl-3-(2-trimethylsilanyl-
ethoxymethyl)-3H-
imidazol-4-yl]-pyridine (74A) as an oil (530 mg, 30%) and starting material
(73, 380
mg, 21%),1 H-NMR (400 MHz, CDC13) : 5 9.38 (d, J = 2 Hz, 1 H), 8.53 (dd, J = 5
Hz, 2
Hz, 1 H), 8.42 (dt, J = 8 Hz, 2 Hz, 1 H), 7.82 (s, 1 H), 7.35-7.30 (m, 1 H),
5.43 (s, 2H),
3.59-3.54 (m, 2H), 2.66 (t, J = 7 Hz, 2H), 1.47-1.38 (m, 2H), 1.28-1.10 (m,
4H), 0.96-
0.89 (m, 2H), 0.80 (t, J = 7 Hz, 3H), 0.0 (s, 9H).
3-[5-Pentylsulfanyl-3-(2-trimethylsilanyl-ethoxymethyl)-3 H-im idazol-4-yl]-
pyridine
(74A) was converted to 3-[5-pentylsulfanyl-3-(2-trimethylsilanyl-ethoxymethyl)-
3H-
imidazol-4-yl]-1,2,5,6-tetrahydro-l-methylpyridine (75A) using the methodology
described for the conversion of 21A to 22A (see scheme 3). Yield : 75%
(amorphous).'H-NMR (400 MHz, CDC13) : b 7.68 (s, 1 H), 6.75-6.71 (m, 1 H),
5.37 (s,
2H), 3.54-3.46 (m, 4H), 2.67 (t, J = 7 Hz, 2H), 2.57 (t, J = 6 Hz, 2H), 2.46
(s, 3H),
2.44-2.38 (m, 2H), 1.55-1.46 (m, 2H), 1.37-1.24 (m, 4H), 0.94-0.85 (m, 5H),
0.0 (s,
9H).
To a solution of anhydrous THF (20 ml) containing 75A (0.42 g, 1.06 mmol) were
added 3.18 ml (3 eq) of TBAF (1.0 M in THF) under N2. After the addition, the
resulting solution was refluxed for 18 hours and subsequently concentrated in
vacuo.
Ethyl acetate was added and the organic layer was washed with a concentrated
NaHCOs solution, dried (Na2SO4), filtered and concentrated in vacuo. The
resulting
residue was purified by flash chromatography (MeOH), followed by a further
purification on (25 g) SCX-2 (MeOH followed by 1 N NH3/MeOH) to afford the
title
compound 76A. (oil, 0.2 g, 73%). LCMS (Method A) : Rt : 1.0 min, ([M+H]+ =
266).'H-
NMR (400 MHz, CDC13) : b 7.54 (s, 1 H), 6.36-6.31 (m, 1 H), 3.49-3.46 (m, 2H),
2.75
(t, J = 7 Hz, 2H), 2.64 (t, J = 6 Hz, 2H), 2.47 (s, 3H), 2.43-2.38 (m, 2H),
1.55-1.49 (m,
2H), 1.35-1.23 (m, 4H), 0.85 (t, J = 7 Hz, 3H).
3-(5-Hexylsulfanyl-3H-imidazol-4-yl)-1,2,5,6-tetrahydro-l-methylpyridine.
(compound 76B, Scheme 14).
To a degassed solution of xylene (30 ml) containing compound 73 (0.6 g, 1.70
mmol), were added 1.25 eq (0.30 ml) of hexane-l-thiol and 1.25 eq of K2CO3
(0.29
g). The resulting mixture was stirred for another 2 hours under N2.
Successively were
added 10 mol % of Pd2(dba)3 (160 mg) and 20 mol % of Xantphos (200 mg). After
the addition, the resulting solution was stirred for 18 hours at 130 C under
N2. After
cooling to room temperature, the mixture was diluted with ethyl acetate,
washed with
a saturated NaHCO3 solution, dried (Na2SO4), filtered and concentrated. The
resulting residue was purified by flash chromatography (diethyl ether) to
afford 3-[5-
hexylsulfanyl-3-(2-trimethylsilanyl-ethoxymethyl)-3H-imidazol-4-yl]-pyridine
(74B) as
an oil (120 mg, 18%) and starting material (73, 380 mg),'H-NMR (400 MHz,
CDCI3) :
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72
6 9.38(d,J=2Hz, 1 H), 8.53 (dd, J = 5 Hz, 2 Hz, 1 H), 8.42 (dt, J = 8 Hz, 2
Hz, 1H),
7.82 (s, 1H), 7.35-7.30 (m, 1H), 5.43 (s, 2H), 3.59-3.54 (m, 2H), 2.66 (t, J =
7 Hz,
2H), 1.46-1.37 (m, 2H), 1.28-1.06 (m, 8H), 0.96-0.89 (m, 2H), 0.80 (t, J = 7
Hz, 3H),
0.0 (s, 9H).
To a solution of anhydrous THF (30 ml) containing 74B (0.29 g, 0.74 mmol) were
added 2.2 ml (3 eq) of TBAF (1.0 M in THF) under N2. After the addition, the
resulting
solution was refluxed for 18 hours and subsequently concentrated in vacuo.
Ethyl
acetate was added and the organic layer was washed with a concentrated NaHCO3
solution, dried (Na2SO4), filtered and concentrated in vacuo. The resulting
residue
was purified by flash chromatography (ethyl acetate) to afford 3-(5-
hexylsulfanyl-3H-
imidazol-4-yl)-pyridine. (77) as an oil (0.14 g, 71%). 'H-NMR (400 MHz, CDC13)
: b
9.25 (bs, 1 H), 8.53 (dd, J = 5 Hz, 2 Hz, 1 H), 8.42-8.35 (m, 1 H), 7.78 (s, 1
H), 7.38-
7.34 (m, 1 H), 2.79-2.69 (m, 2H), 1.53-1.44 (m, 2H), 1.33-1.09 (m, 6H), 0.80
(t, J = 7
Hz, 3H).
3-(5-Hexylsulfanyl-3H-imidazol-4-yl]-pyridine (77) was converted to the title
compound (76B) using the methodology described for the conversion of 21A to
22A
(see Scheme 3) (the quarternization however was done at room temperature with
a
slight excess of CH31). Yield : 70% (oil). LCMS (method A) ; Rt : 1.21 min,
([M+H]+ =
280). 'H-NMR (400 MHz, CDC13) : b 7.53 (s, 1H), 6.40-6.29 (m, 1H), 3.45-3.41
(m,
2H), 2.79-2.72 (m, 2H), 2.59 (t, J 6 Hz, 2H), 2.45 (s, 3H), 2.41-2.37 (m, 2H),
1.55-
1.49 (m, 2H), 1.38-1.31 (m, 2H), 1.30-1.19 (m, 4H), 0.85 (t, J = 7 Hz, 3H).
5. PHARMACOLOGICAL TESTS
(I) ASSAY METHOD FOR MUSCARINIC RECEPTOR LIGAND SCREENING
(In vitro; functional assay)
Test Substance
Compounds were dissolved in DMSO (10 mM) and diluted in assay buffer to test
concentration. Prime testing was at 1 pM; for actives in antagonist mode (PI,
percent
inhibition with respect to reference agonist and blank > 30%) as well as
actives in
agonist mode (PS, percent stimulation with respect to reference agonist and
blank >
30%) testing was continued at lower concentrations in 10-fold dilutions: 0.1
pM, 0.01
pM, etc.
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73
Assay Characteristics: Target, Species, Tissue
Assay Target Species Tissue
Muscarine M1 GPCR-A-MA-ACH M1 Human CHO cells
Muscarine Ml GPCR-A-MA-ACH Ml Rabbit vas deferens
(field-stimulated)
Muscarine M2 GPCR-A-MA-ACH M2 Guinea Pig left atrium (electrically paced)
Muscarine M3 GPCR-A-MA-ACH M3 Guinea Pig ileum
Muscarine M4 GPCR-A-MA-ACH M4 Human CHO cells
Assay Characteristics: Ligand (Kd, Concentration), Non Specific Binding
(Compound, Concentration)
Ligand Ligand
Reference Reference
Assay gonist EC50 Concentration L ntagonist Response
(nM) (nM)
Muscarine M1 acetylcholine 0.8 100 (ago mode) pirenzepine Ca2+ - FLIPR
3 (anta mode) Fluorimetry
inhibition of
Muscarine M1 McN-A-343 250 3000(ago mode) pirenzepine twitch
1000 (anta mode)
contraction
Muscarine M2 carbachol 150 3000 (ago mode) methoctramine negative
1000 (anta mode) inotropic effect
Muscarine M3 carbachol 125 3000 (ago mode) 4-DAMP contraction
1000 (anta mode)
10000 (ago mode) pirenzepine Ca2+ - Aequorin
Muscarine M4 oxotremorine 40
10000 (anta mode) luminescence
Assay Characteristics: Method, Bibliography
Assay Method (see below) Bibliography (see below)
Muscarine M1 cell-based assay Sur et al. (2003)
Muscarine M1 isolated organ Eltze (1988)
Muscarine M2 isolated organ Eglen at al. (1988)
Muscarine M3 isolated organ Clague et al. (1985)
Muscarine M4 cell-based assay Stables et al. (1997)
Assay Procedures & Calculations
Assay arocedures
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74
Isolated Organ Assays, Agonist Mode:
The tissues were exposed to a maximal concentration of the respective
reference
agonist to verify responsiveness and to obtain a control response. Following
extensive
washings and recovery of the initial state, the tissues were exposed to the
test
compounds or the same agonist. In the Ml and M2 receptor assays, the compounds
were left in contact with the tissues until a stable response was obtained or
for a
maximum of 15 min. When several concentrations were tested, they were added
cumulatively. In the M3 receptor assay, the compounds were left in contact
with the
tissues for a time sufficient to obtain a peak response or for a maximum of 10
min, then
washed out. When several concentrations were tested, they were added
consecutively
at 40-min intervals. Where an agonist-like response was obtained, the
respective
reference antagonist was tested against the highest concentration of the
compounds to
confirm the involvement of the receptor studied in this response.
Isolated Organ Assays, Antagonist Mode:
The tissues were exposed to a submaximal concentration of the respective
reference
agonist to obtain a control response. In the Ml and M2 receptor assays, the
test
compounds or the reference antagonists were added after stabilization of the
agonist-
induced response then left in contact with the tissues until a stable effect
was obtained
or for a maximum of 15 min. When several concentrations were tested, they were
added cumulatively. In the M3 receptor assay, the test compounds or the
reference
antagonist were added 30 min before re-exposure to the agonist which was added
at
40-min intervals. Where it occurred, an inhibition of the agonist-induced
response
produced by the compounds indicated an antagonist activity at the receptor
studied.
Each compound was investigated in the three assays for agonist and antagonist
activities at one or several concentrations in three separate tissues. In each
assay, the
reference agonist and antagonist were tested at several concentrations in
three
separate tissues to obtain concentration-response curves.
Cell-based Assays:
Cells were incubated with compound and the response indicated was measured.
Response and Calculation of Results
Isolated Organ Assays:
The parameters measured were the maximum change in the amplitude of the
electrically-evoked contractions (Ml and M2 receptor assays) or in tension (M3
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receptor assay) induced by each compound concentration. The results are
expressed
as a percent of the control agonist-induced response. The EC50 values of the
reference agonists (concentration producing a half-maximum response) and IC50
values of the reference antagonists (concentration producing a half-maximum
inhibition
5 of the agonist-induced response) were calculated by linear regression
analysis of their
concentration-response curves.
Cell-based Assays:
The results are expressed as a percent of reference agonist values and blanks
in the
10 presence of the test compound, percent stimulation for agonist mode; for
the
antagonist mode (test compound in the presence of reference agonist) as
percent
inhibition. The EC50 values (concentration causing a half-maximal stimulation
of
control values), IC50 values (concentration causing a half-maximal inhibition
of control
values) were determined by non-linear regression analysis of the concentration-
15 response curves using Hill equation curve fitting.
Bibliography
CLAGUE, R.U., EGLEN, R.M., STRACHAN, A.C. and WHITING, R.L. (1985) Action
of agonists and antagonists at muscarinic receptors present on ileum and atria
in
20 vitro. Brit. J. Pharmacol., 86: 163-170.
EGLEN, R.M., MONTGOMERY, W.W., DAINTY, I.A., DUBUQUE, L.K. and
WHITING, R.L. (1988) The interaction of methoctramine and himbacine at atrial,
smooth muscle and endothelial muscarinic receptors in vitro. Brit. J.
Pharmacol., 95 :
1031-1038.
25 ELTZE, M. (1988) Muscarinic Ml- and M2-receptors mediating opposite effects
on
neuro-muscular transmission in rabbit vas deferens. Eur. J. Pharmacol., 151 :
205-
221.
STABLES, J., GREEN, A., MARSHALL, F., FRASER, N., KNIGHT, E., SAUTEL, M.,
MILLIGAN, G., LEE, M., and REES, S. (1997) A bioluminescent assay for agonist
30 activity at potentially any G-Protein-Coupled Receptor. Anal. Biochem. 252:
115-126.
SUR, C., MALLORGA, P.J., WITTMANN, M., JACOBSON, M.A., PASCARELLA, D.,
WILLIAMS, J.B., BRANDISH, P.E., PETTIBONE, D.J., SCOLNICK, E.M. and CONN,
P.J. (2003) N-desmethylclozapine, an allosteric agonist at muscarinic 1
receptor,
potentiates N-methyl-D-aspartate receptor activity. PNAS, 100: 13674-13679.
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76
(II) ASSAY METHOD FOR MUSCARINIC RECEPTOR LIGAND SCREENING
(In vitro; receptor binding assay)
Test Substance
Compounds were dissolved in DMSO (10 mM) and diluted in assay buffer to test
concentration. Prime testing was at 10 pM; for actives (PI, percent inhibition
with
respect to total and non-specific binding > 40%) testing was continued at
lower
concentrations in 10-fold dilutions: 1 pM, 0.1 pM, etc.
Assay Characteristics: Target, Species, Tissue
Assay Target Species Tissue
Muscarine M non-
GPCR-A-MA-ACH M Rat cerebral cortex
selective
Muscarine Ml GPCR-A-MA-ACH Ml Human CHO cells
Muscarine M2 GPCR-A-MA-ACH M2 Human CHO cells
Muscarine M3 GPCR-A-MA-ACH M3 Human CHO cells
Muscarine M4 GPCR-A-MA-ACH M4 Human CHO cells
Assay Characteristics: Ligand (Kd, Concentration), Non Specific Binding
(Compound, Concentration)
Ligand Non Specific Non Specific
Assay Ligand Ligand Concentrati Binding Binding
Kd (nM) Concentration
on L (nM) Compound
(pM)
Muscarine M non-
3H-QNB 0.01 0.05 Atropine 1 pM
selective
Muscarine Ml 3H-Pirenzepine 13 2 Atropine 1 pM
Muscarine M2 3H-AFDX384 4.3 2 Atropine 1 pM
Muscarine M3 3H-4DAMP 0.5 0.2 Atropine 1 pM
Muscarine M4 3H-Oxotremorine 4.5 6 Atropine 1 pM
Muscarine M4 3H-4DAMP 0.332 0.2 Atropine 1 pM
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Assay Characteristics: Incubation Conditions (period / temperature),
Bibliography
Incubation Conditions
Assay Bibliography (see below)
period / temperature
Muscarine M non-
selective 120 min/22 C Richards (1990)
Muscarine Ml 60 min/22 C Dorje et al. (1991)
Muscarine M2 60 min/22 C Dorje et al. (1991)
Muscarine M3 60 min/22 C Peralta et al. (1987)
Muscarine M4 30 min/25 C Dorje et al. (1991)
Muscarine M4 60 min/22 C Dorje et al. (1991)
Assay Procedures & Calculations
Assay Procedure
Following incubation of compound with the receptor preparation (from `Tissue')
and the
Ligand at the time and temperature indicated, the receptor preparations were
rapidly
filtered under vacuum through glass fibre filters; the filters were washed
extensively
with an ice-cold buffer using a harvester. Bound radioactivity was measured by
scintillation counting using a liquid scintillation cocktail.
Response and Calculation of Results
Results were expressed as percentage of total Ligand binding and that of Non
Specific
Binding, per concentration of Compound tested (duplicates); from the
concentration -
displacement curves IC50 values were determined by non-linear regression
analysis
using Hill equation curve fitting. The inhibition constants (Ki) were
calculated from the
Cheng-Prushoff equation Ki=1C50/(1+L/Kd), where L is the concentration of
radioligand
in the assay, and Kd the affinity of the radioligand for the receptor. Results
were
expressed as pKi's, means SD of at least 2 separate experiments; i.e.
outliers
(outside +/- 1 std of mean) and discrepancies were excluded. Compounds with no
significant affinity at concentrations of 10 pM and higher were concluded to
be
"inactive" denoted by pKi of "<5.0".
Bibliography
DORJE, F., WESS, J., LAMBRECHT, G., TACKE, R., MUTSCHLER, E. and BRANN,
M.R. (1991) Antagonist binding profiles of five cloned human muscarinic
receptor
subtypes. J. Pharmacol. Exp. Ther., 256: 727-733.
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78
RICHARDS, M.H. (1990) Rat hippocampal muscarinic autoreceptors are similar to
the M2 (cardiac) subtype : comparison with hippocampal M1, atrial M2 and ileal
M3
receptors. Brit. J. Pharmacol., 99 : 753-761.
PERALTA, E. G., ASHKENAZI, A., WINSLOW, J. W., SMITH, D. H.,
RAMACHANDRAN, J. and CAPON, D. J. (1987) Distinct primary structures, ligand-
binding properties and tissue-specific expression of four human muscarinic
acetylcholine receptors. EMBO. J., 6: 3923-3929.
DETERMINATION OF METABOLIC STABILITY (in vitro)
Method used according to procedures described by DI, L. et al., Journal of
Biomolecular Screening, Vol. 8, No. 4, 453-462 (2003).
6. PHARMACOLOGICAL DATA
TABLE 1. Affinity to Ml and M4 receptors and efficacy.
(RB = receptor binding; n. d. = not done)
Compound RB RB Cell funct. RB Cell funct.
No Musc. M4 Musc. M4 Musc. M4 Musc. Ml Musc. M1
3H-DAMP 3H- Agonism 3H- Agonism
Oxotremorine Pirenzepine
pKi pKi pEC50 pKi pEC50
9 6.5 6.4 <5.0 5.9 6.9
16 n.d. n.d. <5.0 n.d. n.d.
22F 7.4 7.2 6.9 6.5 6.7
22M 6.4 6.6 <5.0 6.3 n.d.
26 6.7 6.6 <5.0 6.8 n.d.
49A 7.8 7.6 7.4 6.9 8.0
22N 6.6 6.6 <5.0 6.4 n.d.
22B 6.9 6.4 6.3 6.3 7.1
41 <5 <5 <5.0 <5 n.d.
22A 6.4 6.6 <5.0 5.8 n.d.
22E 6.5 7 <5.0 6.4 n.d.
42 5.3 5.4 <5.0 5.1 n.d.
22G 7.3 6.7 6.5 6.7 7.4
22D 6.4 6.5 <5.0 5.8 n.d.
33C 6.5 5.8 5.9 5.9 6.9
22Q 6.3 6.9 <5.0 6.1 n.d.
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22R 5.4 5.7 <5.0 4.9 n.d.
22S 7.3 6.8 <5.0 6.9 6.4
33A 6.6 6.3 6.1 5.9 6.9
22H 7.2 6.9 8.0 6.7 6.9
33D 6.0 5.8 5.9 5.6 7.0
66B 7.0 6.8 6.3 6.2 n.d.
66A 7.4 7.1 <5.0 6.6 n.d.
68B 6.0 6.1 6.1 5.3 6.1
70A 6.7 6.6 6.2 6.0 6.6
47A 6.1 6.6 6.3 5.6 6.8
68A 6.6 6.2 6.1 5.8 6.7
45B 5.9 6.3 6.5 5.7 6.9
49D 7.0 6.9 6.8 6.1 7.4
60B 6.8 6.6 <5.0 6.4 <6.0
49E 7.4 7.1 7.1 6.5 8.0
45C 6.1 6.3 5.7 6.1 <6.0
45A 6.2 6.4 6.1 5.8 6.9
47B 6.5 6.8 <5.0 6.1 n.d.
45E 5.8 5.8 <5.0 5.8 <6.0
22C 7.2 6.8 <5.0 6.6 <6.0
45D 6.2 6.3 5.9 5.7 6.1
47C 6.1 6.6 6.2 6.1 6.1
221 6.6 7.0 6.6 6.1 7.0
22J 6.6 7.2 6.6 6.2 7.4
22K 6.2 6.4 5.9 5.9 7.0
49F 7.3 7.9 7.9 6.6 6.1
49B 7.6 8.1 8.2 6.8 8.0
49H 7.4 7.5 <5.0 6.8 7.3
33B 6.5 6.7 6.3 6.0 7.0
33G 6.7 6.9 6.2 5.8 7.6
49C 6.9 6.9 6.3 6.0 7.1
22L 7.0 7.2 7.2 6.1 8.0
220 6.1 6.4 6.2 5.1 7.5
33E 6.5 6.8 6.2 6.0 6.7
33F 6.3 6.5 6.1 5.4 7.4
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59 7.0 6.7 <5.0 6.0 7.1
49G 7.0 7.6 8.0 6.2 7.3
52C 6.6 7.2 n.d. 6.0 7.9
52A 6.6 6.9 n.d. 6.1 7.7
52B 6.9 7.2 7.0 6.2 8.1
491 7.2 7.9 7.7 6.5 8.5
22P 5.2 5.2 5.9 <5.0 6.4
76B 6.3 6.0 6.0 5.4 7.1
76A 6.5 6.2 5.1 5.6 6.9
60A 6.6 7.2 <5.0 6.4 n.d.
TABLE 2. Affinity to M2 and M3 receptors and efficacy.
(RB = receptor binding)
Compound RB Musc Cell funct. Cell RB Musc Cell funct. Cell
No M2 Musc. M2 funct. M3 Musc. M3 funct.
methoctra Agonism Musc. M2 4-DAMP Agonism Musc. M3
mine pEC50 Antagonism pEC50 Antagonism
pA2 pA2
22J 5.5 <6 <6 6.5 <6 <6
33G 5.4 <6 <6 6.1 <6 <6
33B 5.9 <6 <6 6.4 <6 <6
22L 5.7 <6 <6 6.5 <6 <6
33F 5.3 <6 <6 6.0 <6 <6
220 5.0 <6 7.7 5.6 <6 <6
49G 6.1 <6 <6 6.5 <6 <6
52A 5.4 <6 <6 6.1 <6 <6
52C 5.6 <6 <6 6.2 <6 <6
52B 5.9 <6 7.6 6.5 <6 <6
76B 5.2 <6 <6 5.6 <6 <6
76A 5.3 <6 <6 6.1 <6 <6
49E 6.3 6.1 <6 6.9 <6 <6
22S 5.7 <6 <6 7.3 <6 7.0
22C 6.4 <6 <6 7.5 <6 <6
49F 6.4 <6 <6 7.0 <6 <6
49B 6.2 <6 <6 7.2 <6 <6
Methoctramine 7.5 7.9
4-DAMP 9.4
Carbachol 6.8
McN-A-343 6.9
Pirenzepine 8.9
CA 02682994 2009-10-05
WO 2008/129054 PCT/EP2008/054897
81
TABLE 3. Stability in human liver homogenate / selected compounds
Compound t'/2 (minutes)
22B 52
22G 50
22J 129
22L 56
220 245
33F 230
33G 56
Xanomeline (reference) 17
