Note: Descriptions are shown in the official language in which they were submitted.
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NON-PEPTIDE GnRH AGENTS, PHARMACEUTICAL COMPOSITIONS
AND METHODS FOR THEIR USE, AND
PROCESSES FOR PREPARING THEM AND THEIR INTERMEDIATES
TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY
OF THE INVENTION
This invention relates generally to compounds that affect the action of
human gonadotropin-releasing hormone (GnRH). More particularly, it relates to
non-
lo peptide GnRH antagonists or agonists and to their preparation. These non-
peptide
GnRH agents have advantageous physical, chemical, and biological properties,
and
are useful medicaments for diseases or conditions mediated by modulation of
the
pituitary-gonadal axis. The invention also relates to methods for treating
individuals
needing therapeutic regulation of GnRH--i.e., methods for treating diseases
and
15 conditions mediated by GnRH regulation. The invention further relates to
processes
for synthesizing intermediate compounds useful for making GnRH agents.
BACKGROUND OF THE INVENTION
Gonadotropin-Releasing Hormone (GnRII), also known as luteinizing
hormone-releasing hormone (LH-RH), plays a central role in the biology of
2o reproduction. A large variety of analogs have been used for an increasing
number of
clinical indications. The GnR_H_ decapeptide (pyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-
Arg-Pro-Gly-NH2 or p-EHWSYGLRPG-NH2) is produced in neurons of the medial
basal hypothalamus from a larger precursor by enzymatic processing. The
decapeptide is released in a pulsatile manner into the pituitary portal
circulation
2s system where GnR_H_ interacts with high-affinity receptors (7-Transmembrane
G-
Protein Coupled Receptors) in the anterior pituitary gland located at the base
of the
brain. In the pituitary, GnR_H_ triggers the release of two gonadotropic
hormones
(gonadotropins): luteinizing hormone (LH) and follicle-stimulating hormone
(FSH).
In testes and ovaries, LH stimulates the production of testosterone and
estradiol,
3o respectively. FSH stimulates follicle growth in women and sperm formation
in men.
When correctly functioning, the pulse-timed release and concentration levels
of
GnRH are critical for the maintenance of gonadal steroidogenesis and for
normal
functions of reproduction related to growth and sexual development.
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The pituitary response to GnRH varies greatly throughout life. GnRH and the
gonadotropins first appear in the fetus at about ten weeks of gestation. The
sensitivity
to GnR_H_ declines, after a brief rise during the first three months after
birth, until the
onset of puberty. Before puberty, the FSH response to GnRH is greater than
that of
LH. Once puberty begins, sensitivity to GnR_H_ increases, and pulsatile LH
secretion
ensues. Later in puberty and throughout the reproductive years, pulsatile
release of
GnRH occurs throughout the day, with LH responsiveness being greater than that
of
FSH. Pulsatile GnR_H_ release results in pulsatile LH and FSH release from the
pituitary, and hence, estosterone and estradiol release from the gonads. After
menopause, FSH and LH concentrations rise, and post-menopausal FSH levels are
higher than those of LH.
Chronic administration of GnRH agonists and antagonists to animals or to
man results in decreased circulating levels of both LH and FSH. GnRH agonists
are
compounds that mimic endogenous GnRH to stimulate receptors on the pituitary
15 gland, resulting in release of LH and FSH. After a transient rise in
gonadal hormone
production or "flare" response, chronic administration of GnRH agonists
results in a
down-regulation of GnRH receptors. GnRH receptor down-regulation and
a
desensitization of the pituitary results in a decrease of circulating levels
of LH and
FSH. In spite of the symptom-exacerbating hormonal flare experienced, GnRH
2o agonists have been the treatment of choice for sex-steroid-dependent
pathophysiologies. For example, GnRH agonists have been used to reduce
testosterone production, thereby reducing prostate volume in benign prostatic
hyperplasia (BPH) and slowing tumor growth in prostate cancer. These compounds
have also been used to treat breast and ovarian cancers.
2s Recently, GnitH antagonists have become available for clinical evaluation.
GnRH antagonists have an immediate effect on the pituitary without the
observed
flare associated with agonists. Use of GnRHantagonists (usually decapeptides)
has
been reported in the literature for treatment of breast, ovarian, and
prostatic cancers.
Other uses of antagonists, like agonists, include endometriosis (including
3o endometriosis with pain), uterine myoma, ovarian and mammary cystic
diseases
(including polycystic ovarian disease), prostatic hypertrophy, amenorrhea
(e.g.,
secondary amenorrhea), and precocious puberty. These compounds may also be
useful in the symptomatic relief of premenstrual syndrome (PMS). Furthermore,
antagonists may be useful to regulate the secretion of gonadotropins in male
mammals
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to arrest spermatogenesis (e.g., as male contraceptives), and for treatment of
male sex
offenders: Importantly, GnR_H_ antagonists (and agonists) have found utility
in
treatments where a reversible suppression of the pituitary-gonadal axis is
desired.
For over fifty years, androgen deprivation has been the most effective
systematic therapy for the treatment of metastatic carcinoma of the prostate.
The
rationale is simple the prostate gland requires androgens for proper growth,
maintenance, and function. Yet, prostate cancer and benign prostate
hyperplasia are
common in men and develop in an environment of continuous androgen exposure.
Thus, utilizing a GnR_H_ antagonist to interrupt the pituitary-gonadal axis
reduces
1o androgen production and results in tumor growth modulation. Furthermore,
GnRH
antagonists may have a direct effect on tumor growth by blocking receptors on
the
tumor cells. For those cancer types that respond both to sex hormones and to
GnRH
directly, antagonists should be effective in slowing tumor growth by these two
mechanisms. Since GnR_H_ receptors are present on many prostate and breast
cancer
1s cells, it has recently been speculated that GnR_H_ antagonists may also be
effective in
treating non-hormone-dependent tumors. Recent literature examples indicate
that
GnR_H_ receptors are present on a number of cancer cell lines, including:
~ prostate cancer: GnRH agonists exert both in vitro, and i~r vivo, a direct
inhibitory action on the growth of both androgen-dependent (LNCaP) and
2o androgen-independent (DU 145) human prostatic cancer cell lines
[Montagnani et al., Arch. Ital..: Urol. Androl., 69(4), 257-263 (1997);
"GnR_H_
Antagonist Inhibit the Growth of Androgen-Independent PC-3 Prostate Cancer
in Nude Mice," Jungwirth et al., Prostate, 32(3), 164-172 (1997)];
ovarian cancer: The demonstration of GnR_H_ receptors in human ovarian
2s cancers provides a rationale for the use of therapeutic approaches based on
GnR_H_ analogues in this malignancy [Srkalovic et al., hrt. J. ~hcol., 12(3),
489-498 (1998)].
~ breast cancer: Breast cancer is the most common type of cancer in women
over the age of forty and is the leading cause of cancer-related death in
3o women. Systematic endocrine intervention represents a major treatment
option for the management of advanced breast cancer, especially with
estrogen-dependent cancers. The genes for gonadotropin-releasing hormone
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and its receptor are expressed in human breast with fibrocystic disease and
cancer [Kottler et al., Iht. J. Cahce~, 71(4), 595-599 (1997)].
GnRIi agents may also be useful in treating cancer through generation of
thymus re-growth and therefore induction of the development of new T-cells.
See
s Norwood Abbey press release dated March 5, 2001. These white blood cells,
which
develop in the thymus gland, are a fundamental component of the immune
system's
involvement in a range of diseases, including viral infections, transplant
organ
rejection, cancer, and autoimmune diseases. Thus, for example, since the human
immunodeficiency virus (HIV) preferentially infects and destroys T-cells,
GnR_H_
to agents may be useful for treating HIV infection or acquired immune
deficiency
syndrome (AIDS). Additionally, GnRH agents may be useful in combating
infection
in tissue-transplant patients where immunosuppressive drugs, which remove T-
cells,
are being administered to counteract rejection of the transplanted tissue.
Similarly,
since adequate and effective T-cells help defend against cancer, and
chemotherapy
is and radiation regimens detrimentally impact T-cells, GnRH agents may be
useful in
treating cancer. Furthermore, GnRH agents may be useful for treating
autoimmune
diseases such as multiple sclerosis (MS), where T-cells are produced that
react against
a molecule surrounding nerve cells.
Heretofore, available GnR_H_ antagonists have primarily been peptide analogs
20 of GnRH. See, e.g., International Publication Nos. WO 93/03058, WO
99/50276,
WO 00/12521, and WO 00/12522; Koppan et al., Prostate, 38(2),151-8 (1999); and
Nagy et al., Proc Natl Acad Sci USA, 97(2),829-34 (2000). Though peptide
antagonists of peptide hormones are often quite potent, the use of peptide
antagonists
is typically associated with problems because peptides are degraded by
physiological
2s enzymes and often poorly distributed within the organism being treated.
The first non-peptide antagonist of the human leuteinizing hormone-releasing
hormone (LHRH) receptor was reported by Cho et al. (JMed Chem, 41(22), 4190
(1998). Since then, other non-peptide GnRH antagonists have been reported in
the
literature. For example, quinolone-6-carboxamides were reported by Walsh et
al. in
3o Bioorg & Med Chem Lt~s., 10, 443-447 (2000). Tricyclic diazepines and
cyclic
pentapeptides were reported in International Publication Nos. WO 96/38438 and
WO
96/34012, respectively. Tetrahydroisoquinoline derivatives were reported in
U.S.
Patent No. 5,981,521. For additional examples of non-peptide GnRH antagonists,
see
International Publication Nos. WO 97/21435, WO 97/21703, WO 97/21704, WO
4
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WO 02/098363 PCT/US02/17846
97/21707, WO 99/44987, WO 00/04013, WO 00/12522, WO 00/12521, WO
00/04013, and WO 00/20358.
Despite recent advances, there continues to be a need for non-peptide
antagonists of the peptide hormone GnR_H_ with desirable properties. For
example,
there is a need for non-peptide GnRH agents having advantageous physical,
chemical
and biological properties compared to peptides, which are useful medicaments
for
treating diseases mediated via the pituitary-gonadal axis and by directly
targeting the
receptor on tumor cells. There is also a need for GnR_H_ agents that act upon
these
receptors to treat both hormone-dependent and hormone-independent cancers.
1o SUM1VIARY OF THE INVENTION
In one general aspect, the invention is directed to compounds represented by
the following Formula I:
x
0
Ar~Z ~ ~ V~Y~Ri
wherein the variables are as defined in the claims.
is q In another general aspect, the invention is directed to compounds
represented
by Formula (II):
x
0
R ~Z ~ ~ V~YiArz
z
wherein the variables are as defined in the claims.
In a further general aspect, the invention is directed to compounds
20 represented by Formula (III):
wherein the variables are as defined in the claims.
In addition to compounds of Formulas I, II, and III, the invention is also
directed to pharmaceutically acceptable salts, pharmaceutically acceptable
prodrugs,
25 and pharmaceutically active metabolites of such compounds, and
pharmaceutically
5
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WO 02/098363 PCT/US02/17846
acceptable salts of such metabolites. Such compounds, salts, prodrugs and
metabolites are at times collectively referred to herein as "GnRH agents."
The invention also relates to pharmaceutical compositions each comprising a
therapeutically effective amount of a GnRT-T agent of the invention in
combination
with a pharmaceutically acceptable carrier or diluent. Moreover, the invention
relates
to methods for regulating the secretion of gonadotropins in mammals,
comprising
administering therapeutically effective amounts of GnR_H_ agents of the
invention.
Other aspects, features, and advantages of the invention will become apparent
from the detailed description of the invention and its preferred embodiments.
1o DETAILED DESCRIPTION OF INVENTION AND PREFERRED
EMBODIMENTS
As used herein, the terms "comprising" and "including" are used herein in
their open, non-limiting sense.
The term "alkyl" refers to a straight- or branched-chain alkyl group having
1s from 1 to 12 carbon atoms in the chain. Exemplary alkyl groups include
methyl (Me,
which also may be structurally depicted by ~, ethyl (Et), n-propyl, isopropyl,
butyl,
isobutyl, sec-butyl, tert-butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl,
isohexyl, and
the like.
The term "heteroalkyl" refers to a straight- or branched-chain alkyl group
2o having from 2 to 12 atoms in the chain, one or more of which is a
heteroatom selected
from S, O, and N. Exemplary heteroalkyls include alkyl ethers, secondary and
tertiary alkyl amines, alkyl sulfides, and the like.
The term "alkenyl" refers to a straight- or branched-chain alkenyl group
having from 2 to 12 carbon atoms in the chain. Illustrative alkenyl groups
include
25 prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, and
the like.
- The term "alkynyl" refers to a straight- or branched-chain alkynyl group
having from 2 to 12 carbon atoms in the chain. Illustrative alkynyl groups
include
prop-2-ynyl, but-2-ynyl, but-3-ynyl, 2-methylbut-2-ynyl, hex-2-ynyl, and the
like.
The term "aryl" (Ar) refers to a monocyclic, or fused or spiro polycyclic,
3o aromatic carbocycle (ring structure having ring atoms that are all carbon)
having from
3 to 12 ring atoms per ring. Illustrative examples of aryl groups include the
following
moieties:
6
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
° ~\
\ ~ \ \ ~ \ \ \
~ ~ , ~ ~ ~ , ~ ~ ,
\ \
~ , and the like.
The term "heteroaryl" (heteroAr) refers to a monocyclic, or fused or spiro
polycyclic, aromatic heterocycle (ring structure having ring atoms selected
from
carbon atoms as well as nitrogen, oxygen, and sulfur heteroatoms) having from
3 to
12 ring atoms per ring. Illustrative examples of aryl groups include the
following
moieties:
NON I \ N \ S \ N
~N ~ NON , / ~ , I ,i ~ , I ~ ~~ ,
N
~N~ ~S~ ~O N\O~ ~N ~S N\S~
U,U~~N ,U,UN ~UN ,U~
N~N O Nw \ N~ N~ N
\ ~ ~ ~ ~ / NI ~ C ~ I / N ~N
> > > 'N , N > >
io
S
N ~ ~ ~N
i / , and the like.
S N
The term "cycloalkyl" refers to a saturated or partially saturated, monocyclic
or fused or spiro polycyclic, carbocycle having from 3 to 12 ring atoms per
ring.
Illustrative examples of cycloalkyl groups include the following moieties:
7
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
> > > '
D~ 0 ' > >
U ~.
> > >
s
\ , and the like.
A "heterocycloalkyl" refers to a monocyclic, or fused or spiro polycyclic,
ring
structure that is saturated or partially saturated and has from~3 to 12 ring
atoms per
1o ring selected from C atoms and N, O, and S heteroatoms. Illustrative
examples of
heterocycloalkyl groups include:
O O 0 O O O
~Si N
S N~N N O O O
~U, ~ > > >~U~~S~
N N // O O // CO N
U ' ~N ~ U , ~N > > U ~ N-N
O
O S
N N~O 0
U c~ c~
, ~ ~~C>> >UU
N N N N N
~O
N~S;O N N ~ O
N , I / ~ , and the like.
J 0
IS
The term "halogen" represents chlorine, fluorine, bromine or iodine. The term
"halo" represents chloro, fluoro, bromo or iodo.
8
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WO 02/098363 PCT/US02/17846
The term "substituted" means that the specified group or moiety bears one or
more substituents. The term "unsubstituted" means that the specified group
bears no
substituents. The term "optionally substituted" means that the specified group
is
unsubstituted or substituted by one or more substituents.
Preferred GnRH agents of the invention include those having a I~1 value of
about 10 wM or less. Especially preferred GnRH agents are those having a I~;
value in
the nanomolar range.
It is understood that while a compound may exhibit the phenomenon of
tautomerism, the formula drawings within this specification expressly depict
only one
of the possible tautomeric forms. It is therefore to be understood that a
formula is
intended to represent any tautomeric form of the depicted compound and is not
to be
limited merely to a specific compound form depicted by the structural formula.
It is also understood that a compound of Formula I, II or III may exist as an
"E" or "Z" configurational isomer, or a mixture of E and Z isomers. It is
therefore to
1s be understood that a formula is intended to represent any configurational
form of the
depicted compound and is not to be limited merely to a specific compound form
depicted by the formula drawings.
Some of the inventive compounds may exist as single stereoisomers (i.e.,
essentially free of other stereoisomers), racemates, and/or mixtures of
enantiomers
2o and/or diastereomers. All such single stereoisomers, racemates and mixtures
thereof
are intended to be within the scope of the present invention. In one preferred
embodiment, the inventive compounds that are optically active are used in
optically
pure form.
As generally understood by those skilled in the art, an optically pure
2s compound having one chiral center (i.e., one asymmetric carbon atom) is one
that
-- consists essentially of one of the two possible enantiomers (i.e., is
enantiomerically
pure), and an optically pure compound having more than one chiral center is
one that
is both diastereomerically pure and enantiomerically pure. Preferably, the
compounds
of the present invention are used in a form that is at least 90% optically
pure, that is, a
3o form that contains at least 90% of a single isomer (80% enantiomeric excess
("e.e.")
or diastereomeric excess ("d.e.")), more preferably at least 95% (90% e.e. or
d.e.),
even more preferably at least 97.5% (95% e.e. or d.e.), and most preferably at
least
99% (98% e.e. or d.e.).
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As indicated above, GnRH agents in accordance with the invention also
include active tautomeric and stereoisomeric forms of the compounds of Formula
I, II
or III, which may be readily obtained using techniques known in the art. For
example, optically active (R) and (S) isomers may be prepared via a
stereospecific
s synthesis, e.g., using chiral synthons and chiral reagents, or racemic
mixtures may be
resolved using conventional techniques.
Additionally, Formulas I, II, and III are intended to cover, where applicable,
solvated as well as unsolvated forms of the compounds. Thus, each formula
includes
compounds having the indicated structure, including the hydrated as well as
the non-
to hydrated forms.
In addition to compounds of the Formulas I, II, and III, the GnR_H_ agents of
the invention include pharmaceutically acceptable salts, prodrugs, and active
metabolites of such compounds, and pharmaceutically acceptable salts of such
metabolites. Such non-peptide agents are pharmaceutically advantageous over
1s peptide agents since they provide better biodistribution and tolerance to
degradation
. by physiological enzymes.
A "prodrug" is a compound that may be converted under physiological
conditions or by solvolysis to the specified compound or to a pharmaceutically
acceptable salt of such compound. An "active metabolite" is a
pharmacologically
2o active product produced through metabolism in the body of a specified
compound or
salt thereof. Prodrugs and active metabolites of a compound may be identified
using
routine techniques known in the art. See, e.g., Bertolini et al., J. Med.
Chem., 40,
2011-2016 (1997); Shan et al., J. Pharm. Sci., 86 (7), 765-767; Bagshawe, Drug
Dev.
Res., 34, 220-230 (1995); Bodor, Advances i~ Drug Res., 13, 224-331 (1984);
25 Bundgaard, Design of Prodrugs (Elsevier Press 1985); and Larsen, Design and
Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al.
eds., Harwood Academic Publishers, 1991); Dear et al., J. Chromatogr. B, 748,
281-
293 (2000); Sprain et al., J. Pharmaceutical & Biomedical Av~alysis, Vol. 3,
No. 2,
103-112 (1992).
3o The term "pharmaceutically acceptable salts" refers to salt forms that are
pharmacologically acceptable and substantially non-toxic to the subject being
administered the GnRH agent. Pharmaceutically acceptable salts include
conventional acid-addition salts or base-addition salts formed from suitable
non-toxic
organic or inorganic acids or inorganic bases. Exemplary acid-addition salts
include
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WO 02/098363 PCT/US02/17846
those derived from inorganic acids such as hydrochloric acid, hydrobromic
acid,
hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric
acid, and
those derived from organic acids such as p-toluenesulfonic acid,
methanesulfonic
acid, ethane-disulfonic acid, isethionic acid, oxalic acid, p-
bromophenylsulfonic acid,
carbonic acid, succinic acid, citric acid, benzoic acid, 2-acetoxybenzoic
acid, acetic
acid, phenylacetic acid, propionic acid, glycolic acid, stearic acid, lactic
acid, malic
acid, tartaric acid, ascorbic acid, malefic acid, hydroxymaleic acid, glutamic
acid,
salicylic acid, sulfanilic acid, and fumaric acid. Exemplary base-addition
salts include
those derived from ammonium hydroxides (e.g., a quaternary ammonium hydroxide
1o such as tetramethylammonium hydroxide), those derived from inorganic bases
such as
alkali or alkaline earth-metal (e.g., sodium, potassium, lithium, calcium, or
magnesium) hydroxides, and those derived from organic bases such as amines,
benzylamines, piperidines, and pyrrolidines.
If the inventive compound is a base, the desired pharmaceutically acceptable
salt may be prepared by any suitable method available yin the art, for
example,
treatment of the free base with an inorganic acid, such as hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or
with an
organic acid, such as acetic acid, malefic acid, succinic acid, mandelic acid,
fumaric
acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid,
a
2o pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-
hydroxy acid,
such as citric acid or tartaric acid, an amino acid, such as aspartic acid or
glutamic
acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic
acid, such as
p-toluenesulfonic acid or ethanesulfonic acid, or the like.
If the inventive compound is an acid, the desired pharmaceutically acceptable
2s salt may be prepared by any suitable method, for example, treatment of the
free acid
with an inorganic or organic base, such as an amine (primary, secondary or
tertiary),
an alkali metal hydroxide or alkaline earth metal hydroxide, or the like.
Illustrative
examples of suitable salts include organic salts derived from amino acids,
such as
glycine and arginine, ammonia, primary, secondary, and tertiary amines, and
cyclic
3o amines, such as piperidine, morpholine and piperazine, and inorganic salts
derived
from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,
aluminum and lithium.
In the case of agents that are solids, it is understood by those skilled in
the art
that the inventive compounds, agents and salts may exist in different crystal
or
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WO 02/098363 PCT/US02/17846
polymorphic forms, all of which are intended to be within the scope of the
present
invention and specified formulas.
A variety of known assays and techniques may be employed to determine the
level of activity of various forms of the compounds in the GnRH system. Ligand
s binding assays are used to determine interaction with the receptor of
interest. Where
binding is of interest, a labeled receptor may be used, where the label is a
fluorescer,
enzyme, radioisotope, or tfe like, which registers a quantifiable change upon
the
binding of the receptor. Alternatively, the artisan may provide for an
antibody to the
receptor, where the antibody is labeled, which may allow for amplification of
the
l0 signal. Binding may also be determined by competitive displacement of a
ligand
bound to the receptor, where the ligand is labeled with a detectable label.
Where
agonist and/or antagonist activity is of interest, an intact organism or cell
may be
studied, and the change in an organismic or cellular function in response to
the
binding of the compound of interest may be measured. Various devices are
available
1s for detecting cellular response, such as a microphysiometer available from
Molecular-
Devices, Redwood City, California. In vitNO and in vivo assays useful in
measuring
GnR_H_ antagonist activity are known in the art. See, e.g., Bowers et al., "LH
suppression in cultured rat pituitary cells treated with 1 ng of LHRH,"
Endocrinology,
1980, 106:675-683 (in vitro,) and Corbin et al., "Antiovulatory activity (AOA)
in
20 rats," Endoc~~. Res. Commun., 2:1-23 1975.. Particular test protocols that
may be used
are described below.
For example, GnRH-receptor 7antagonists may be functionally assessed by
measurement of change in extracellular acidification rates as follows. The
ability of
compounds to block the extracellular rate of acidification mediated by GnRH in
HEK
2s 293 cells expressing human GnRH receptors is determined as a measure of the
-- compound's antagonist activity i~ vit~~o. Approximately 100,000
cellslchamber are
immobilized in agarose suspension medium (Molecular Devices) and perfused with
unbuffered MEM media utilizing the Cytosensor~ Microphysiometer (Molecular
Devices). Cells are allowed to equilibrate until the basal acidification rate
remains
30 stable (approximately one hour). Control dose-response curves are performed
to
GnR_H_ (10-11 M to 10-' M). Compounds are allowed to incubate 15 minutes prior
to
stimulation with GnRH, and are assessed for antagonist activity. After
incubation
with test compounds, repeat dose-response curves to GnRH in the presence or
absence of various concentrations of the test compounds are obtained. Schild
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regression analysis is performed on compounds to determine whether compounds
antagonize GnRH-mediated increases in extracellular acidification rates
through a
competitive interaction with the GnR_H_ receptor.
In another test, accumulation of total inositol phosphates may be measured by
formic acid extraction from cells, followed by separation of the phosphates on
Dowex
columns. Cells are split using trypsin into two 12-well plates and pre-labeled
with
3H-myoinositol (0.5 Ci to 2 mCi per mL) for 16-18 hours in inositol-free
medium.
The medium is then aspirated and the cells rinsed with either 1X HBSS, 20 mM
HEPES (pH 7.5), or serum-free DMEM, 1X HBSS, 20mM HEPES (pH 7.5)
to containing test compound, and 20 mM LiCI is then added and the cells are
incubated
for the desired time. The medium is aspirated and the reaction stopped by
addition of
ice-cold 10 mM formic acid, which also serves to extract cellular lipids.
lnositol
phosphates are separated by ion-exchange chromatography on Dowex columns,
which
are then washed with 5 mL of 10 mM myoinositol and 10 mM formic acid. The
1s columns are then washed with 10 mL of 60 mM sodium formate and 5 mM borax,
and total inositol phosphates are eluted with 4.5 mL 1M ammonium formate, O.1M
formic acid.
It will be appreciated that the actual dosages of the agents of this invention
will vary according to the particular agent being used, the particular
composition
2o formulated, the mode of administration, and the particular site, host, and
disease being
treated. Optimal dosages for a given set of conditions may be ascertained by
those
skilled in the art using conventional dosage-determination tests in view of
the
experimental data for a given compound. For oral administration, an exemplary
daily
dose generally employed will be from about 0.001 to about 1000 mg/kg of body
25 weight, with courses of treatment repeated at appropriate intervals.
Administration of
prodrugs may be dosed at weight levels that are chemically equivalent to the
weight
levels of the fully active compounds.
To treat diseases or conditions mediated by GnR_H_ agonism or antagonism, a
pharmaceutical composition of the invention is administered in a suitable
formulation
so prepared by combining a therapeutically effective amount (i.e., a GnR_H_
modulating,
regulating, or inhibiting amount effective to achieve therapeutic efficacy) of
at least
one GnR_H_ agent of the invention (as an active ingredient) with one or more
pharmaceutically suitable carriers, which may be selected from diluents,
excipients
and auxiliaries that facilitate processing of the active compounds into the
final
13
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
pharmaceutical preparations. Optionally, one or more additional active
ingredients,
such as a second GnRH agent, may be employed in a pharmaceutical composition
according to the invention.
The pharmaceutical carriers employed may be either solid or liquid.
Exemplary solid carriers are lactose, sucrose, talc, gelatin, agar, pectin,
acacia,
magnesium stearate, stearic acid and the like. Exemplary liquid carriers are
syrup,
peanut oil, olive oil, water and the like. Similarly, the inventive
compositions may
include time-delay or time-release material known in the art, such as glyceryl
monostearate or glyceryl distearate alone or with a wax, ethylcellulose,
1o hydroxypropylmethylcellulose, methylmethacrylate or the like. Further
additives or
excipients may be added to achieve the desired formulation properties. For
example,
a bioavaliability enhancer, such as Labrasol, Gelucire or the like, or
formulator, such
as CMC (carboxymethylcellulose), PG (propyleneglycol), or PEG
(polyethyleneglycol), may be added. Gelucire~, a semi-solid vehicle that
protects
1s active ingredients from light, moisture and oxidation, may be added, e.g.,
when
preparing a capsule formulation.
If a solid carrier is used, the preparation can be tableted, placed in a hard
gelatin capsule in powder or pellet form or in the form of a troche or
lozenge. The
amount of solid carrier may vary, but generally will be from about 25 mg to
about 1 g.
2o If a liquid carrier is used, the preparation may be in the form of syrup,
emulsion, soft
gelatin capsule, sterile injectable solution or suspension in an ampoule or
vial or non-
aqueous liquid suspension. If a semi-solid carrier is used, the preparation
may be in
the form of hard and soft gelatin capsule formulations. The inventive
compositions
are prepared in unit-dosage form appropriate for the mode of administration,
e.g.,
25 parenteral or oral administration.
To obtain a stable water-soluble dose form, a pharmaceutically acceptable salt
of an inventive agent may be dissolved in an aqueous solution of an organic or
inorganic acid, such as 0.3 M solution of succinic acid or citric acid. If a
soluble salt
form is not available, the agent may be dissolved in a suitable cosolvent or
3o combinations of cosolvents. Examples of suitable cosolvents include
alcohol,
propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the
like in
concentrations ranging from 0-60% of the total volume. In an exemplary
embodiment, a compound of Formula I, II, or III is dissolved in DMSO and
diluted
with water. The composition may also be in the form of a solution of a salt
form of
14
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
the active ingredient in an appropriate aqueous vehicle such as water or
isotonic saline
or dextrose solution.
Proper formulation is dependent upon the route of administration chosen. For
injection, the agents of the invention may be formulated into aqueous
solutions,
s preferably in physiologically compatible buffers such as Hanks solution,
Ringer's
solution, or physiological saline buffer. For transmucosal administration,
penetrants
appropriate to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by
1o combining the active compounds with pharmaceutically acceptable carriers
known in
the art. Such carriers enable the compounds of the invention to be formulated
as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like,
for oral ingestion by a patient to be treated. Pharmaceutical preparations for
oral use
can be obtained using a solid excipient in admixture with the active
ingredient (agent),
1s optionally grinding the resulting mixture, and processing the mixture of
granules after
adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable
excipients include: fillers such as sugars, including lactose, sucrose,
mannitol, or
sorbitol; and cellulose preparations, for example, maize starch, wheat starch,
rice
starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-
cellulose,
20 sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone,
agar,
or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally contain gum
arabic,
25 polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium
dioxide,
-- lacquer solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or
pigments may be added to the tablets or dragee coatings for identification or
to
characterize different combinations of active agents.
Pharmaceutical preparations which can be used orally include push-fit
so capsules made of gelatin, as well as soft, sealed capsules made of gelatin
and a
plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain
the active
ingredients in admixture with fillers such as lactose, binders such as
starches, and/or
lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
In soft
capsules, the active agents may be dissolved or suspended in suitable liquids,
such as
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
fariy oils, liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may
be added. All formulations for oral administration should be in dosages
suitable for
such administration. For buccal administration, the compositions may take the
form
of tablets or lozenges formulated in conventional manner.
s For administration intranasally or by inhalation, the compounds for use
according to the present invention may be conveniently delivered in the form
of an
aerosol spray presentation from pressurized packs or a nebuliser, with the use
of a
suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a
'pressurized aerosol the dosage unit may be determined by providing a valve to
deliver
a~ metered amount. Capsules and cartridges of gelatin for use in an inhaler or
insufflator and the like may be formulated containing a powder mix of the
compound
and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection,
e.g., by bolus injection or continuous infusion. Formulations fox injection
may be
presented in unit-dosage form, e.g., in ampoules or in mufti-dose containers,
with an
added preservative. The compositions may take such forms as suspensions,
solutions
or emulsions in oily or aqueous vehicles, and may contain formulatory agents
such as
suspending, stabilizing andlor dispersing agents.
2o Pharmaceutical formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form. Additionally,
suspensions
of the active agents may be prepared as appropriate oily injection
suspensions.
Suitable lipophilic solvents or vehicles include fatty oils such as sesame
oil, or
synthetic fatty acid esters, such as ethyl oleate or triglycerides, or
liposomes.
2s Aqueous injection suspensions may contain substances which increase the
viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or agents
which
increase the solubility of the compounds to allow for the preparation of
highly
concentrated solutions.
3o For administration to the eye, a GnR_u_ agent may be delivered in a
pharmaceutically acceptable ophthalmic vehicle such that the compound is
maintained in contact with the ocular surface for a sufficient time period to
allow the
compound to penetrate the corneal and internal regions of the eye, including,
for
example, the anterior chamber, posterior chamber, vitreous body, aqueous
humor,
16
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
vitreous humor, cornea, irislcilary, lens, choroid/retina and selera. The
pharmaceutically acceptable ophthalmic vehicle may be an ointment, vegetable
oil, or
an encapsulating material. A compound of the invention may also be injected
directly
into the vitreous and aqueous humor.
s Alternatively, the active ingredient may be in powder form for constitution
with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The
compounds
may also be formulated in rectal compositions such as suppositories or
retention
enemas, e.g, containing conventional suppository bases such as cocoa butter or
other
glycerides.
1o In addition to the formulations described above, the compounds may also be
formulated as a depot preparation. Such long-acting formulations may be
administered by implantation (for example, subcutaneously or intramuscularly)
or by
intramuscular injection. Thus, for example, the compounds may be formulated
with
suitable polymeric or hydrophobic materials (for example, as an emulsion in an
15 acceptable oil) or ion-exchange resins, or as sparingly ~ soluble
derivatives, for
example, as a sparingly soluble salt.
A pharmaceutical carrier for hydrophobic compounds is a cosolvent system
comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic
polymer,
and an aqueous phase. The cosolvent system may be a VPD co-solvent system. VPD
2o is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant
polysorbate
80, and 65% w/v polyethylene glycol 300, made up to volume in absolute
ethanol.
The VPD co-solvent system (VPD: 5W) contains VPD diluted 1:1 with a 5%
dextrose
in water solution. This co-solvent system dissolves, hydrophobic compounds
well,
and itself produces low toxicity upon systemic administration. The proportions
of a
2s co-solvent system may be suitably varied without destroying its solubility
and toxicity
characteristics. Furthermore, the identity of the co-solvent components may be
varied: for example, other low-toxicity nonpolar surfactants may be used
instead of
polysorbate 80; the fraction size of polyethylene glycol may be varied; other
biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl
pyrrolidone;
3o and other sugars or polysaccharides may be substituted for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical
compounds may be employed. Liposomes and emulsions are known examples of
delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents
such as
dimethylsulfoxide also may be employed, although usually at the cost of
greater
17
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
toxicity. Additionally, the compounds may be delivered using a sustained-
release
system, such as semipermeable matrices of solid hydrophobic polymers
containing
the therapeutic agent. Various sustained-release materials have been
established and
are known by those skilled in the art. Sustained-release capsules may,
depending on
s their chemical nature, release the compounds for a few weeks up to over 100
days.
Depending on the chemical nature and the biological stability of the
therapeutic
reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid- or gel-
phase carriers or excipients. Examples of such carriers or excipients include
calcium
1o carbonate, calcium phosphate, sugars, starches, cellulose derivatives,
gelatin, and
polymers such as polyethylene glycols.
Some of the compounds of the invention may be provided as salts with
pharmaceutically compatible counter ions. Pharmaceutically compatible salts
may be
formed with many acids, including hydrochloric, sulfuric, acetic, lactic,
tartaric,
15 malic, succinic, etc. Salts tend to be more soluble in aqueous or other
protonic
solvents than are the corresponding free-base forms.
The compounds of Formulas I, II and III and their intermediates may be
prepared by advantageous processes described below. Preferred intermediates
useful
for synthesizing the inventive compounds include 5-(2-methylphenoxy)-2-furoic
acid,
20 5-[2-bromo-5-(tert-butyl)phenoxy]-2-furoic acid, 5-[(3,3,6-trimethyl-2,3-
dihydro-1H-
inden-5-yl)oxy]-2-furoic acid, 5-(4-chloro-5-isopropyl)-2-methylphenoxy)-2-
furoic
acid, 5-[(4-bromo-3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furoic
acid, and
6-methyl-2-[(3, 5, 5, 8, 8-pentamethyl-5, 6, 7, 8-tetrahydro-2-
naphthalenyl)oxy]-4-
pyrimidinecarboxylic acid. Additional preferred nitro and amine intermediates
useful
25 for synthesizing of GnRH agents of the present invention are:
__ ~p ~O ~O
H N N ~O H2N~ 'N HZN 'N
I N~N J w0~~0~0~ ~ ~'~ OH
O ~ , O N 0~
~O ~O
O~O I N~ HaN I 'N
CI and
CI
Methods for electrophilic aromatic nitration are described in the art. See,
for
example, Coon et al., J. O~g. Chem., 38: 4243 (1973); Yarbro et al., J.
Fluo~ihe
18
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
Chem. 6:187 (1975); Hakimelahi et al., Hel. Chim. Acta 61: 906 (1984); Suri et
al.,
Synthesis 743(1988); Umenoto et al, Tetrahed~o~c Lett. 31:3579 (1990);
Shackelford
et al., Abstracts of the 11 th Rocky Mou~taih Regional America~r. Chemical
Society
Meeting, Albuquerque, NM, July 10-12, 1992; and Adams et al., Tetf ahedroh
Lett.
s 34: 6669 (1993). These methods have drawbacks, however. For example, the
Yarbro
et al. and Hakimelahi et al. methods generate anhydrous nitronium triflate
using toxic,
gaseous nitryl chloride, NOZCI, and corrosive triflic chloride (acid
chloride).
Various drawbacks of prior methods have been overcome by the nitration
process of the present invention, which comprises forming a nitrating reagent
by
to adding trifluoromethanesulfonic anhydride to 2-tetramethylammonium nitrate
in a
polar solvent and reacting the nitrating reagent with an aryl or heteroaryl
compound.
Advantages of the present method are, e.g.: a) simplified and more rapid
aqueous work-up, thereby eliminating chromatographic column separation and/or
plug filtration; b) improved reactant solubility and reduced byproduct
formation as a
1s result of organic solvents rather than aqueous or easily hydrolyzed,
corrosive
anhydride solvents; c) facilitated synthesis of regioisomeric nitrated
products
sometimes not available when .using other nitration procedures; d) enables the
preparation of novel nitroaromatic and nitroheterocyclic compounds; e)
achieves
selective and exclusive mono-nitration from mild reaction conditions; ~
provides
2o higher product yields than many conventional nitration procedures; g)
higher crude
nitrated product purity; and h) scaleable over a wide range to provide small
or large
product quantities.
This nitration method described herein may be used to provide nitrated
benzene derivatives (e.g., Compound II) and aromatic heterocyclic
intermediates.
2s Such nitrated intermediates are reduced (e.g., Compound II to Compound III)
to
provide intermediates useful for preparing final GnR_H_ agents of the
invention (e.g.,
Compound V--the compound of Example B52).
19
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WO 02/098363 PCT/US02/17846
N02 NH2
O O O\ O O~O
NITRATION / I~~ \ REDUCTION /
NYN '~ NYN ~ NYN
CI ICI CI
NH
.O ~ ~ ?O.
NYN l
O CI O O O
O III ~ N CI
OH HATU,TEA I ~ I / HN /
IV V O\
General Procedure for the Tetramethylammonium Nitrate-Generated Nitronium
Triflate Nitration of Aromatic Compounds (General Procedurel:
R
R
00 0o OOo Oo
a a cNZc~z a a
(CH3)QNN03 + CF3SOSCF3 RT,--t~ NOZOSCF3 -1- N(CH3)qOSCF3 a,~B~CtoRT ~ + HOSCF3
O O ~ ~ cHZci2 N02 O
[HBO soluble] [H20 soluble]
Under nitrogen at room temperature, 2.26-2.39 g trifluoromethanesulfonic
anhydride was added dropwise from a pressure-equalized addition funnel to a
stirred
suspension of 1.12 g tetramethylammonium nitrate (96%) in 24 mL commercial
anhydrous (or low water) dichloromethane (DCM) with a slight temperature rise
1o resulting. The addition funnel was rinsed with 8 mL anhyd. DCM, and this
rinse was
added to the reaction suspension. After stirring for at least 1.5 hours, the
stirred
suspension was cooled in a dry ice/acetone bath to at least -69°C. The
aromatic
substrate, 7.50 mmol of in 10 mL DCM, was added dropwise to the stirred
nitronium
triflate suspension keeping the reaction temperature at -65.0°C or
less. The addition
15 funnel was then rinsed with 2 mL anhyd. DCM; this rinse also was added to
the
stirred reaction suspension. The dry ice/acetone bath could then be removed;
however, many reactions proceeded more cleanly if the bath was kept in place
30 to
60 minutes and was then allowed to warm unattended to room temperature over
the
next 15 to 48 hours. In several reactions, the acetone bath was kept in place
one hour
2o and then was replaced with a dry ice/acetonitrile cold bath. The reaction
was
quenched by adding 15 mL of 5% sodium bicarbonate solution and stirring until
bubbling ceased with an aqueous layer pH = 8 (With acidic compounds containing
phenolic and carboxylic acid groups, only water was added to maintain an
acidic pH).
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
The reaction contents were transferred with 25 mL DCM and 50 mL H20. The lower
DCM layer was separated and washed with 5 x 25 mL H20. The combined H20
washes were back-extracted with 25 ml DCM, and the combined DCM portions dried
over anhyd. MgS04. DCM removal by rotary evaporation gave crude product. This
s procedure proportionally can be scaled to larger quantities by directly
increasing
reactant and solvent amounts while keeping reaction time periods the same.
Compound II, shown below, was obtained in 98% crude yield with an isolated
99% crude product purity from reactant I. Originally synthesized on a 7.5
mmole
scale, its synthesis has been scaled up to 2864 mmoles, again, with a
resultant 98%
1o crude product yield. One preliminary attempt to synthesize Compound II by
the
traditional HN03/acetic anhydride solvent procedure was not successful.
N02 NH2
H3C0 I ~ OCH3 H3CO~OCH3 H3CO~OCH3
NYN INY'IN NYN
CI CI CI
2-chloro-4,6-dimethogy-5-nitropyrimidine (Compound I1J: Reacted 1.31g
(7.50mmo1) I with 1.50 equivalents of nitronium triflate for 48 hours at room
15 temperature to obtain 1.61g II, (98%). FW = 219.6; 1H NMR (DMSO-d6) 8 4.06
(s,
6H); GCIMS (CI, m/z) 220 (Mk and base peak); FTIR (KBr) 3126, 3073, (aromatic
CH); 3000, 2958, 2924, 2852 (aliphatic CH); 1524, 1354, (NOa), crri 1; mp
uncorrected = 131.8 to 132.0 °C; Elem. Anal. for C6H6C1N3O4 (crude
product)
calculated C 32.82%, H 2.75%, N 19.14%, Cl 16.15, found C 33.12 %, H 2.81%, N
20 18.95%, Cl 16.43.
2-chloro-4,6-trimethogypyrimidin-5-amine (Compound III): A 500 mL three-
__ necked, round bottom flask containing a Teflon-coated magnetic stirring bar
and a
thermometer was charged with 10.00 g (45.54 mmol) 2-chloro-4,6-dimethoxy-5-
nitropyrimidine, 100 mL 190 proof ethanol, and 50 mL saturated, aqueous
ammonium
25 chloride solution. The resultant suspension was stirred at room temperature
for
several minutes while iron powder (-325 mesh) was added in several portions
over a
22 minute period such that the reaction temperature from a slow rising
exotherm did
not exceed 56 °C. After addition of all the iron powder, the reaction
was stirred for
two and one-half hours in a room temperature environment. The reaction
suspension
3o was suction filtered, and the isolated iron powder was washed with 2 x 25
mL of
21
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
ethanol, followed by 3 x 25 mL of ethyl acetate. Too the organic filtrate was
added
200 mL water and 50 mL more ethyl acetate to effect separation. The upper
organic
layer was separated, and the lower aqueous layer was extracted with 3 x 50 mL
of
ethyl acetate. All ethyl acetate portions were combined and dried over
anhydrous
magnesium sulfate. Suction filtration, washing the spent magnesium sulfate
with 15
mL ethyl acetate, and rotary evaporation left a pinkish, wet solid. The wet
solid was
dissolved in 75 mL dichloromethane, and the dichloromethane was washed with 3
x
25 mL of water. The combined water washings were back-extracted with 25 mL
dichloromethane. The combined dichloromethane portions were dried over
anhydrous magnesium sulfate. Suction filtration, washing the spent magnesium
sulfate with 25 mL dichloromethane, and rotary evaporation afforded 8.21 g
(95%) of
a cream-colored solid shown to be 98% pure by HPLC analysis. The NMR and mass
spectrometry data consistent with the desired product is as follows: FW =
189.6; 1H
NMR (DMSO-d6) 8 4.59 (s, 2H), 3.89 (s, 6H); FILMS (APCI, nZ/z) 192.0, 190.0
is (M++1); FTIR (ATR film) 3420, 3342, 3290 (NH) 3180, 3010, (aromatic CH);
2960,
2871 (aliphatic CH); 1587, 1488, 1458, 1398, 1375, 1314, 1197, 1067, 942
(fingerprint) cm 1 ; Elem. Anal. for C6H8C1N302 calculated: C 38.01%, H 4.25%,
Cl
18.70, N 22.16%, found C 38.24 %, H 4.32%, Cl 18.70, N 22.09%.
NOp
H3CO~OCH3 H3CO~OCH3
N~.N INI~.NI
OCH3 OCH3
VI VII
2o The synthesis of VII was also achieved by direct nitronium triflate
nitration of VI
in a 73% crude yield with 85% crude yield purity. This represents the first
synthesis of VII by direct nitration. Use of the acetic anhydride/nitric acid
nitration procedure gave several byproducts plus VII in only 16% purity. The
only other literature preparation appearing in the Beilstein electronic data
base for
2s VII produced it from VI and gave a 66% yield [Cherkasov, V. M.; Remennikov,
G. Ya.; Kisilenleo, A. A. Chena. Heterocycl. Compd. (Engl. Transl.); EN; 526
(1982)].
2,4,6-trimethogy-5-nitropyrimidine (Compound VIIJ: Into a 5-L three
necked, round bottom flask fitted with an overhead mechanical stirrer, was
placed 920
3o g (0.675 mole) of tetramethylammonium nitrate and 1 L DCM. The reaction
flask
was fitted with a thermometer and addition funnel. The suspension was stirred
under
22
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
nitrogen gas for 15 minutes at room temperature. Into the addition funnel was
placed
190 g (0.675 mole, 118mL) of trifluoromethanesulfonic anhydride that was added
dropwise to the stirred suspension over a 35 minute duration so the
temperature did
not rise more than 5 °C. The addition funnel was rinsed with a small
amount of DCM,
s and this rinse was added to the stirred reaction suspension. The resultant
suspension
was stirred at room temperature for 1.5 hours. The addition funnel was charged
with
76.9 g (0.452 mole) dissolved in a minimum amount of DCM and was added
dropwise at room temperature over 85 minutes such that the reaction
temperature did
not rise more than 5 °C, and a bright crimson red suspension resulted.
The reaction
1o was stirred overnight at room temperature. Work up entailed adding 5 kg of
ice to the
stirred reaction suspension followed by 10% NaHC03 solution until a pH 8 was
reached and the reaction turned from a burgundy to purple to blue to green to
yellow
color change. The lower DCM layer was separated and washed with 3 x 1.5 L of
water. The DCM portion was then dried sodium sulfate, filtered, and the DCM
15 solvent was removed by rotary evaporation giving ca. 70 g of green solid.
Recrystallization from methanol/water in two crops afforded 50.5 g of purified
product for an overall 52% yield. The NMR and mass spectrometry data
consistent
with the desired product is as follows: FW = 215.2; 1H NMR (CDC13) 8 4.06 (s,
6H),
4.02 (s, 3H); FI/MS (APCI, m/z) 216 (M++1 and base peak); FTIR (ATR film)
3047,
20 3009, 2999 (aromatic CH); 2957, 2877, 2847 (aliphatic CH); 1578, 1338
(N02), cm 1;
Elem. Anal. for C~H9N305 calculated: C 39.07%, H 4.22%, N 19.53%, found C
39.14
%, H 4.19%, N 19.61%.
O
02N O
OCH3
VIII
Compound VIII represents another class of aromatic heterocycle product
2s that was obtained by the above mentioned nitronium triflate method and
demonstrates that a pendant ester group also is not attacked nor modified by
these
nitration conditions.
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WO 02/098363 PCT/US02/17846
Anhydrous Electrophilic Aromatic Nitration with Tetramethvlammonium Nitrate:
Nitration of non-heterocyclic aromatic compounds are also achieved with this
anhydrous, one-pot, two-step method in which the nitronium triflate XVI
nitrating
reagent is generated ih-situ under a static nitrogen gas blanket from
s tetramethylammonium nitrate XIV and trifluoromethanesulfonic anhydride
(triflic
anhydride) XV. The aromatic compound to be nitrated is then introduced XVII.
Nitration occurs to produce the desired product XVIII plus the
tetramethylammonium tiflate salt byproduct XIX.
Step 1 R Step 2
+ ~ ~ CH2CI2 + - ~ ~ CHZCI2
(CH3)4NN03 + CF3SOSCF3 ~ N020SCF3 + ~ , N as
O O N2 gas 2 9
RT New ReaCgent -7$ °C to Reflex
Generation Method
XIV XV XVI XVII
R O
+- ii
\ + (CH3)4NOSCF3
' \NO2 O
XVIII XIX
1o When using the tetramethylammonium nitrate reagent XIV, the resulting salt
byproduct XIX is water soluble and is removed during an aqueous work-up
leaving
only the desired nitrated product XVIII after drying. Under similar reaction
conditions, higher homologues of XIV (e.g. tetra-rc-butylammonium nitrate and
tetraethylammonium nitrate) require additional, time intensive purification by
column
is chromatography or short column Silica Gel filtration to remove the
analogous XIX
byproduct. The one lower homolog, ammonium nitrate, gives irreproducible
results
under analogous nitration conditions.
Most reactions have been conducted in methylene chloride solvent at room
temperature. However, chloroform, dichloroethane, and nitromethane also would
be
2o suitable solvents and would permit higher temperatures to be achieved by
refluxing
the reaction in step 2 with electron-deficient aromatic ring systems or when
attempting to effect aromatic dinitration.
From 1.05 to 1.50 equivalents of nitronium triflate have been used for
mononitration of the aromatic or heterocyclic reactant with excess reagent
having no
2s deleterious effect. The less reactive the reactant, the larger the excess
of nitronium
24
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
triflate is needed for complete conversion. More than 1.5 equivalents of
nitronium
triflate could be used; no upper limit has been determined.
This reaction method (General Procedure) has been scaled linearly from the
7.5 mmole size (aromatic compound to be nitrated) to 92.9 mmoles. To date, no
s upper limit has been established. It appears this method is limited only by
the size of
equipment available.
Additional Nitration Examples:
The following mono-substituted benzene compounds were evaluated for
directing effects and substituent stability during the reaction conditions of
this
nitration method (ND = not detected). Table 1 illustrates the wide scope of
mono-
substituted benzenes nitrated with this method and reveals the directional
susceptibility of nitronium triflate nitration to aromatic pendant group
effects.
The nitrations were conducted with 1.05 equivalents of nitronium triflate as
described by the General Procedure. Reaction conditions were not optimized for
1s these exploratory reactions, and isomeric percentages were determined by
proton
NMR analysis of the isolated crude products.
Table 1. Tetramethylammonium Nitrate Nitration of Mono-Substituted
Benzenes (7.5 mmol scale).
Compound No. Conversion Isolated o/m/t~-IsomersTime fhrs.l
(%1 Yield (%1 (%1
R = OCH3 1 100 97 63/5/32 24
OH 2 100 74 9010/10 23
CH3 3 100 99 62/0/38 24
Br 4 95 90 33/0/67 26
CHO 5 ND 70 31/63/6 97
CF3 6 ND 68 8/88/4 24
____ 6 ___ 68 7/89/4 48
COOH 7 71 52 13/7816 26
S02CH3 8 57 89" 13/84/3 102
ND = not determined because of reactant volatility and its loss during solvent
removal.
A= Actual yield in a pure isolated mixture contained only reactant (43%) and
product
isomers (57%).
OMe OH CH3 Br HO CF3 C02H S02CH3
\ \ \ \ \ \ \ \
1 2 3 4 5 6 7
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This one-pot tetramethylammonium nitrate-based nitration also was
applied to multiple-substituted aromatic compounds under the same reaction
conditions. Table 2 displays the results obtained.
Table 2. Tetramethylammonium Nitrate Nitration of Multiple-Substituted
Benzenes
(7.5 mmol scale).
Com op and % Converted Product s Reaction Time Isolated Yield (%1 Isolated
Puritv (961
OCH3 OCH3
100 % I ~ 17 hrs. 71 % 92~
~NOz
OCH3 OCH3
8 9
OCH3 OCI-~ OCH3
100 % ~ I ~ NOz _____
hrs. 82%
H3C0 ~ H3C0 ~ ~ OCH3
10 No2
95% 5%
11a 11b
OH OH
NOZ
100 °~ I ~ 27 hrs. 94% 91%
0 CHZBr O CHZBr
12 13 7
H3co cN ocH3 58 °~A H3co ~N ocH3 15 hrs. 43% -'-"
I i 96 °~A I ~ NOz 46 hrs. 80 % 81
OCH3 100 °!°B oCH3 53 hrs. 82% 95%
14 15
A. NOzOTf equivalents = 1.05 relative to the reactant 14.
B. NOZOTf equivalents = 1.50 relative to the reactant 14.
Compound purity was determined by HPLC analysis. Crude product 9
was purified further by preparative HPLC to greater than 99%. Compound 13 was
purified by recrystallization from hexane. Both 9 and 13 gave acceptable
elemental analyses. The only previous synthesis of 13 by direct nitration used
fuming nitric acid at 5 °C for one hour, and gave a 70% crude product
yield with
an apparent purification obtained from an ethanol recrystallizati0n as
described by
Garg et al., J. Chem. Soc., 607, (1969). Compound 10 and its products lla and
llb reveal that the sterically crowded position between 1,3-disubstituted
methoxy
groups is not attacked when less sterically hindered positions are available.
When
2o no alternative is available, nitration readily occurs between two 1,3-di-
substituted
methoxy groups as shown by 2,4,6-trimethoxybenzonitrile 14 where product 15 is
obtained in good yield and purity. Other than the equivalents of NOZOTf, used,
the main difference in reaction conditions between the latter two runs of
reactant
14 was reactant 14 had a slower warming profile that gave the highest crude
yield
2s and purity for product 15. In the first two reactions with 14, the dry
ice/acetone
26
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WO 02/098363 PCT/US02/17846
cooling bath was removed immediately after adding reactant 14 like that
described
in the General Procedure. The better result in the third reaction of 14 was
obtained by replacing the dry ice/acetone cooling bath with a dry
ice/acetonitrile
cooling bath for 2.5 hours (-50°C to -35°C), allowing the bath
to warm to 9°C over
S the next 2.5 hours, then removing the cooling bath completely and stirring
the
reaction for 48 hours. Product IS has not previously been reported in the
chemical literature and represents a new compound.
This General Procedure scales directly from 7.5 to 100 millimoles by
proportionally increasing the amounts of reagent and solvent while keeping
to reaction times the same. Results of large-scale reactions appear in Table
3.
Unless stated otherwise, product particles were determined by HPLC analysis.
Table 3. Tetramethylammonium
Nitrate Nitration
at Large-Scale
(86 to 100
mmol
scale).
Reactant Scale Eauiv. of 3 Product IsolatedIsolated
s Reaction Time Yeld Puritv
OH H
~
NO2 91 % 88/ (nmr)
93 mmoles 1.1 I i 23 hrs.
CH2Br CHzBr
O O
12 13
CH3 CH3 CH3
24 hrs. 94% 93% (nmr)
05 I ~ I ~
86
l
1
~N02
NO
es
.
mmo
z
H3C-C-CH3 H3C-C-CH3 H3C-C-CH3
CH3 CH3 CH3
23 100% 0%
24a 24b
CN CN
H3CO~OCH3 H3CO~OCH3
90-100 1.5-1.7 ~ 48-52 hrs.2-84 0-95 %
%
mmoles
H Ox
OCH OC
3 3
14 - 15
CN CN CN
H3C0 ~ H3C0 ~ H3C0
100 mmoles 1.5 I ~ N02 OZN 91-96/ 78-82 %
I ~ 52 hrs.
OCH3 OCH3 O
CH3
25 O
100
26a 26b
- CN CN
~ OCH3 H3C0
H3C0 ~ OCH3
I I 100 97 %
100 mmoles 1.5 ~ NOz 48-52 %
hrs.
LS 27 28
As shown in Tables 2 and 3, the brominated acetyl group of reactant I2
was stable to the mild nitronium triflate electrophilic nitration conditions.
The
carbonyl group in aldehyde, and carboxylic actid groups are compatible with
this
nitration procedure (Table 1), as is an ester group, and the unsaturated cyano
2o group is compatable with this reaction.
27
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In the smaller 7.5 milligram scale nitration of reactant 10, nine percent of
product isomer llb was obtained and was removed by recrystallization. No
formation of product 11b was obtained in the larger scale reaction, see Table
3.
Compound 23 selectively produced the one isomeric product 24a in an excellent
yield. In contrast, a literature nitration of 23 using the nitronium
tetrafluoroborate
reagent (N02+ BF4 ) produced 5% 24b by product as an impurity as described in
Olah et al, J. Am. Chem. Soc., 86, 1067, (1964).
Compound 29 nitrated by this method gave different results when compared
with the standard acetic anhydride/nitric acid nitration system. Each method
gave the
different xegioisomer in exclusive or a nearly exclusive yield. The nitronium
triflate
method heavily favored o>"tho-nitration with respect to the methylsulfamide
group
30a, while the standard acetic anhydridelnitric acid method provided exclusive
papa-
nitration 30b.
H3C0 N02
NOZ* OS02CF3
1.05 eq., CHZCIz, Nz (g) ~ ~ N-S02CH3
H3C0 -72°C toRT HCO
3
H 30a + pace of 30b
N-SO2CH3 -
0 O
H3C0 CH3COCC1-lo / HN03 HaCO
H
29 RT 02N ~ ~ N-S02CH3
H3C0 30b
In the oxidative environment of this in-situ nitration method, axomatic
alkylsulfides 31 are cleanly oxidized to their analogous sulfoxide 32, and the
sulfoxide 32 is then oxidized to a sulfone 33 in an apparent stepwise fashion.
Once the fully oxidized sulfone 33 is obtained, nitration can then occur to
provide
the mono-nitrated sulfone 34. This is illustrated by the three following non-
optimized reactions.
O~ ~CHg
CH3
O
+ - II CH2CIz, N2 (g) ~ 100 % CONVERSION
I / ~.. N02 O ii CF3 _7a °C~ I ~ g7 % CRUDE YIELD
31 ~ 32
1.05 equivalents
CH3
__ _ ~ CH
O CH3 + - S CH2C12, Nz (g) ' O S O O~' 3
+ N02 OSOCF
3 -75 °C to RT
I / ~ I / "f I / 64 % CONVERSION
NOT OPTIMIZED
32 1.05 equivalents 64 % 36
33 32
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CH3 CH3 CH3
O=s=OO O=S=O O=s=O
\ -I- - CHzCIz, Nz (g) \
\
+ ~ ~ / 56 % CONVERSION
/ N02 OSOCF3 _73 G~ ~ / NOT OPTIMIZED
II NO2
O
33 1.05 equivalents56 % 44
50LE ISOMER
33
34
In each reaction step, only the product and unconverted reactant were detected
with no byproducts present. With the correct number of nitronium triflate
s equivalents, unique aromatic sulfones could be made during a clean, one-pot
reaction.
SR
O O=S=O
\ -F - I I CH2CIz, Nz
(g) \
NOp O ~~ CF3 -74Ct~
O /
multiule equivalents NOZ
d
HO~OH + _ ~ GH2ch, Nz (g) \ OH
Fi0
I YN ..f. NOp O ii CF3 I
a4C to RT NYN
SCH3 O O=S=O
mul- tiule equivalentsCH3
The nitrated products obtained by this nitration method shown in Tables 2
and 3 were characterized as follows:
l0 1,4-Dimethogy-2-nitrobenzene (9): Reacted 1.04 g (7.50 mmol) 8 with 1.05
equivalents of nitronium triflate to obtain an isolated yield of 0.97 g (71%).
FW =
183.1; 1H NMR (DMSO-d6) 8 7.46 (s, 1H), 7.28 (m, 2H), 3.93 (s, 3H), 3.86 (s,
3H).
GC/MS (CI, m/z) 183 (M+ and base peak); FTIR (KBr,) 3071, 3024 (aromatic CH),
2982, 2946, 2844 (aliphatic CH), 1528, 1355 (N02) cni l; mp uncorrected,
(crude =
15 68.6 to 70.0°C), (hplc purified = 70.8 to 71.2 0 °C), lit. mp
= 71-73°C, 68-70 °C, 71
°C; Elem Anal for C$H9N04 calcd C 52.46%, H 4.95%, N 7.65%, found C
52.55%,
H 4.94%, N 7.63.
1,3-Dimethogy-4-nitrobenzene (11a): Reacted 1.04 g (7.50 mmol) 10 with 1.05
equivalents of nitronium triflate to obtain an isolated yield of 1.13 g (82%
with 5%
20 llb isomer). FW = 183.1; 1H NMR (DMSO-d6) 8 7.97 (d, J = 9Hz, 1H), 6.80 (d,
J =
2Hz, 1H), 6.67 (dd, Jl = 9Hz, J2 = 3Hz, 2H), 3.86 (s, 3H), 3.78 (s, 3H); GC/MS
(CI,
n~/z) 183 (MF and base peak).
1,3-Dimethogy-2-nitrobenzene (11b): FW = 183.1; 1H NMR (DMSO-d6) 8
7.48 (t, 1H), 6.89 (d, 2H), 3.86 (s, 6H); GC/MS, (CI, m/z) 183 (M'~ and base
peak).
2s 2-Bromo-1-(4-hydroxy-3-nitrophenyl)ethanone (13): Small Scale: Reacted
1.70 g (97.91 mmol) 12 with 1.15 equivalents of nitronium triflate to obtain
an
29
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WO 02/098363 PCT/US02/17846
isolated yield of 1.93 g (93%). Lame Scale: Reacted 19.98 g 12 with 1.10
equivalents of nitronium triflate to obtain an isolated yield of 21.90 g
(91%).
Recrystallized from hot hexane. FW = 260.0; 1H NMR (DMSO-d6) 8 12.18 (brd. s,
1H), 8.49 (s, 1H), 8.13 (d, 1H), 7.24 (d, 1H), 4.89 (s, 2H); ES/MS (CI, m/z)
260
s (M'), 258 (base peak); FTIR (I~Br) 3279 (OH), 3086, (aromatic CH); 2997,
2940
(aliphatic CH), 1695 (C=O), 1568, 1329 (NOa) crri 1; mp uncorrected = 87.8 to
89.4
°C, (lit. mp = 91.5 to 92.0°C), Elem Anal for C$H6BrN04 calcd C
36.95%, H 2.33%,
N 5.39%, Br 30.73%, found, C 37.28 %, H 2.34%, N 5.44%, Br 30.90%.
2,4,6-Trimethogy-3-nitrobenzonitrile (15): Small Scale: Reacted 1.48 g (7.51
1o mmol) 14 (98%) with 1.50 equivalents of nitronium triflate to obtain an
isolated yield
of 1.54 g (86%). Large Scale: Reacted 17.80 g (90.29 mmol) 14 (98%) with 1.66
equivalents of nitronium triflate to obtain an isolated yield of 17.59 g
(82%).
Recrystallized from methanol. FW = 238.2; 1H NMR (DMSO-d6) 8 6.79 (s, 1H),
4.02 (s, 6H), 4.01 (s, 1H); FI/MS (APCI, mlz) 209 (Mh-30 and base peak); FTIR
is (ATR film) 3114, (aromatic CH); 2994, 2957, 2924, 2984 (aliphatic CH), 2228
(CN),
1529, 1349 (N02) cm 1; mp uncorrected = 195.2 to 195.6 °C; Elem Anal
for
CloHION2O5 calcd C 50.42%, H 4.23%, N 11.76%, found C 50.60 %, H 4.18%, N
11.80%.
4-tart-Butyl-2-nitrophenol (24a): FW = 193.3; Reacted 12.2 g (82.3 mmol) 23
20 with 1.04 equivalents of nitronium triflate to obtain an isolated yield of
15.0 g (94%).
1H NMR (CDC13) 8 7.84 (s, 1H), 7.40 (d, 1H), 7.12 (d, 1H), 2.42 (s 3H), 1.21
(s, 9H);
GC/MS (CI, m/z) 194 (M++1), 178 (base peak).
2,4-Dimethoay-5-nitrobenzonitrile (26a): Small Scale: Reacted 1.23 g (7.54
mmol) 25 (98%) with 1.25 equivalents of nitronium triflate to obtain an
isolated yield
2s of 1.40 g (89% with 9% 26b isomer)._Lar eg Scale: Reacted 16.65 g (100.0
mmol) 25
(98%) with 1.50 equivalents of nitronium triflate to obtain an isolated yield
of 18.99 g
(91%). Recrystallized from methanol. FW = 208.2; 1H NMR (DMSO-d6) 8 8.46 (d, J
= 3Hz, 1H), 6.99 (s, J = 3Hz, 1H), 4.06 (s, 6H); GC/MS (CI, mlz) 208 (M~, 161
(base peak); FTIR (ATR film): 3126, 3073 (aromatic CH) 3000, 2958, 2924, 2852,
30 (aliphatic CH), 2232 (CN), 1524, 1354 (N02) cm 1; mp uncorrected = 195.8 to
196.4
°C; Elem Anal for C9H8N204 calcd C 51.93%, H 3.87%, N 13.46%, found, C
51.93
%, H 3.85%, N 11.45%.
2,6-Dimethogy-3-nitrobenzonitrile (28): Reacted 16.82 g (100.0 mmol) 27
(97%) with 1.50 equivalents of nitronium triflate to obtain an isolated yield
of 20.85 g
3s (100%). Recrystallized from methanol. FW = 208.2; 1H NMR (DMSO-d6) b 8.34
(d,
1H), 7.18 (d, 1H), 4.03 (s, 6H); FI/MS (APCI, m/z) 209 (M'-+1), 208 (M'~, 179
(base
peak); GC/MS (CI, m/z) 208 (M'~), 178 (base peak); FTIR (ATR film) 3114
(aromatic
CH), 2994, 2957, 2924, 2854 (aliphatic CH), 2228 (CN), 1529, 1349 (NOa) cm 1;
mp
uncorrected, crude = 115.4 to 115.6 °C); Elem Anal for C9H$N204 calcd C
50.42%, H
40 4.23%, N 11.76%, found C 50.60 %, H 4.18%, N 11.80%.
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
N-(3,5-dimethoxy-2-nitrophenyl)methanesulfonamide (30a): Reacted 1.75 g
(7.51 mmol) 29 with 1.05 equivalents of nitronium triflate to obtain an HPLC
purified
yield of 0.25 g (11%). FW = 276.3; 1H NMR (CDC13) 8 8.31 (s, 1H), 6.88 (d, J =
3Hz, 1H), 6.32 (d, J = 3Hz, 1H), 3.90 (s, 3H), 3.87 (s, 3H), 3.03 (s 3H); 13C
NMR
(CDC13, 300 MHz) 164.01 (1C), 156.35 (1C), 134.75 (1C), 97.65 (1C), 96.45
(1C),
57.18 (1C), 56.47 (1C), 40.57 (1C); FI/MS (ANCI, m/z) 275 (M'-1).
N-(3,5-dimethoxy-4-nitrophenyl)methanesulfonamide (30b): Reacted 0.544 g
(2.35 mmol) 29 with fuming nitric acid in acetic anhydride in an ice bath for
30
minutes, removed ice bath for 4 hrs, and worked up reaction to obtain an HPLC
1o purified yield of 0.070 g (10%). FW = 276.3; 1H NMR (CDC13) 8 6.48m (s,
2H),
6.87 (s, 6H), 3.07 (s, 3H); 13C NMR (CDCl3, 300 MHz) 153.49 (2C), 140.26 (1C),
126.28 (1C), 95.85 (2C), 57.07 (2C), 40.12 (1C); FI/MS (APCI, m/z) 277 (MF+1).
Procedural Notes:
Reactant 14 (Table 2): the first two runs were conducted by removing the dry
ice/acetone bath as described in the General Procedure. The best small-scale
result
with 14 used a slower warming profile by replacing the dry ice/acetone cooling
bath
with a dry ice/acetonitrile cold bath fro 2.5 hours (-49.6°C to -
35.0°C), allowing the
bath to warm to 9.0°C over the next 2.5 hours (-35.0°C-
9.0°C), then removing the bath
2o completely and stirring for 48 hours. This led to the following temperature
warming
modification for the large-scale nitrations with reactants 14, 25, and 27.
After
addition of aromatic reactant, the reaction was kept one hour in the dry
ice/acetone
bath; then, it was then replaced with a dry ice/acetonitrile bath (ca. --
45°C) for three
hours, after which, the bath was permitted gradually to warm unattended to
room
2s temperature (RT) over the nexr 48 hours. For compound 23, the dry
ice/acetone bath
was removed as soon as addition was complete arid was stirred in a RT
environment
for 24 hours. For compound 12 (large scale), the dry ice/acetone bath was not
removed following its addition, but was left in place to warm gradually,
unattended to
RT over the next 23 hours.
Synthesis Of GnR_H_ Reagents And Compounds:
The inventive agents may be prepared using the reaction routes and synthesis
schemes as described below, employing the techniques available in the art
using
starting materials that are readily available. The preparation of preferred
compounds
3s of the present invention is described in detail in the following examples,
but the
artisan will recognize that the chemical reactions described may be readily
adapted to
prepare a number of other protein kinase inhibitors of the invention. For
example, the
synthesis of non-exemplified compounds according to the invention may be
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WO 02/098363 PCT/US02/17846
successfully performed by modifications apparent to those skilled in the art,
e.g., by
appropriately protecting interfering groups, by changing to other suitable
reagents
known in the art, or by making routine modifications of reaction conditions.
Alternatively, other reactions disclosed herein or known in the art will be
recognized
as having applicability for preparing other compounds of the invention.
Reagents useful for synthesizing compounds may be obtained or prepared
according to techniques known in the art. For example, the preparation of free
amines
from common salt forms and stock reagent solutions can be useful for small-
scale
reactions. See also Abdel-Magid et al., "Reductive Amination of Aldehydes and
1o Ketones with Sodium Triacetoxyborohydride," J. O~g. Chem. 61: 3849 (1996).
Methanolic solutions of the free bases can be prepared from hydrochloride,
dihydrochloride, hydrobromide, or other salts when the free base is soluble in
methanol. In this procedure, once the sodium methoxide is added, care should
be
taken to prevent exposure to air, since amine free bases, particularly primary
amines,
absorb carbon dioxide from the air to form salts. A 10~-mL quantity of a O.1M
solution of a free base in methanol may be prepared as follows. Weigh 1.0 mmol
of a
monohydrochloride salt into a tared Erlenmeyer flask containing a stirring
bar, and
ad~i 7 mL of methanol. To the stirred slurry, add 229 mL (1.0 mmol, 1 equiv.)
of
sodium methoxide in methanol (25 wt %, 4.37 M), stopper the flask, and stir
the
2o mixture vigorously for 2 hours. The slurry will sometimes change in
appearance as a
finer, milky precipitate of sodium chloride is formed. Filter the slurry
through a 15-
mL medium fritted glass funnel, wash the filter case with 1-2 mL methanol,
transfer
the filtrate to a 20-mL vial, and dilute to 10 mL with methanol. The
theoretical yield
of sodium chloride is nearly 59 mg, but the recovery is usually not
quantitative, owing
2s to a slight solubility in methanol. For a dihydrochloride salt, a second
equivalent of
sodium methoxide is required (458 mL).
A 0.5 M solution of sodium borohydride in ethanol may be prepared as
follows. Sodium borohydride (520 mg, 13.8 mmol) is stirred in pure (non-
denatured)
anhydrous ethanol (25 mL) for ~2-3 minutes. The suspension is filtered through
a
3o medium fritted glass funnel to remove a small amount of undissolved solid
(typically
about 5% of the total mass of borohydride, or 25 mg). The filtrate should
appear as a
colorless solution that evolves only a little hydrogen. This solution should
be used
immediately, as it decomposes significantly over a period of a few hours,
resulting in
the formation of a gelatinous precipitate. Sodium borohydride is hygroscopic,
so
32
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WO 02/098363 PCT/US02/17846
avoid exposure to air by making the solution at once after weighing the solid.
Sodium
borohydride has a solubility of about 4% in ethanol at room temperature. This
corresponds to a little over 0.8 M. However, sometimes a small percentage of
the
solid remains undissolved regardless of the concentration being prepared, even
after
stirring for > 5 minutes.
Material and Methods:
In the examples described below, unless otherwise indicated, all temperatures
in the following description are in degrees Celsius and all parts and
percentages are by
weight, unless indicated otherwise.
1o Various starting materials and other reagents were purchased from
commercial
suppliers, such as Aldrich Chemical Company or Lancaster Synthesis Ltd., and
used
without further purification, unless otherwise indicated. Tetrahydrofuran
(THF) and
N,N-dimethylformamide (DMF) were purchased from Aldrich in SureSeal~ bottles
and used as received. All solvents were purified by using standard methods in
the art,
1s unless otherwise indicated.
The reactions set forth below were performed under a positive pressure
of nitrogen or with a drying tube, at ambient temperature (unless otherwise
stated), in
anhydrous solvents, and the reaction flasks are fitted with rubber septa for
the
introduction of substrates and reagents via syringe. Glassware was oven-dried
and/or
Exemplary GnR_H_ Agents:
The following compounds were prepared according to Scheme A set forth
below:
Scheme A
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11
\ H KOH ~ \ OK
R i / R i / . R ~ / ~O-'
DMSO 12
-
\ ~z
RI
R
NaOH, CH3OH \ O _ R i
H EDCI or II?.TU /
13
SOCIz, benzene
Catalytic DMF
~~2
R [~'I
i /
\ O
R
C1
TEA, DCM
14
Potassium phenogide 10: A mixture of potassium hydroxide (2.55g, 44.8
mmol) and the appropriate phenol 9_ (52.9 mmol) was heated in an oil bath at
150-155
°C for 1-2 hours. The dark colored liquid was then evacuated at 130-140
°C to
s remove water. The residue (potassium phenoxide ~ was dried i~c vacuo
overnight.
Alternatively, the phenoxide 10 may be prepared by reaction with potassium t
butoxide in tetrahydrofuran.
Condensation 12: A mixture of potassium phenoxide 10 (7 mmol), prepared
as described above, and methyl 5-bromo-2-furoate 11 (5.8 mmol) in DMSO (10 mL)
to was heated at 85 °C under nitrogen atmosphere. The reaction mixture
was then
diluted with water, and the aqueous mixture was acidified with concentrated
HCI, and
then extracted with diethyl ether. The combined ether extracts were
concentrated and
the product 12 was purified by silica gel chromatography, eluting with a
mixture of
ethyl acetate and hexanes (1:5 to 1:1 v/v). Yield was in the range of 50-80%.
Saponification 13: The methyl ester 12 obtained from above was dissolved in
methanol (4 mmol in 15 mL of solvent). An aqueous solution of sodium hydroxide
(0.7g in 5 mL water) was added. The mixture was monitored by TLC for
completion
of reaction. It was concentrated, diluted with water, and extracted with
diethyl ether.
The aqueous layer was then acidified with concentrated HCI, and extracted with
ethyl
2o acetate. The ethyl acetate extracts were washed with brine, dried over
magnesium
sulfate and concentrated to give a solid residue. The product 5-substituted-2-
furoic
acid 13 may be purified, if necessary, by silica gel chromatography. Yield was
greater
than 90%.
Amide Formation: Procedure l: The furoic acid 13 from above (1 mmol) was
dissolved in dimethylformamide (5 mL). To this solution was added 1-ethyl-3-(3-
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WO 02/098363 PCT/US02/17846
dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 1 mmol), followed by
the
addition ofthe appropriate aniline or aniline hydrochloride (1 mmol), and
triethylamine (1.1 mmol). The reaction mixture was stirred'at room temperature
overnight. DIVIF' was removed on a rotovap. The residue was suspended in ethyl
acetate, and washed with 10% HCl (aqueous), aqueous sodium bicarbonate, brine,
and
dried over magnesium sulfate. The solvent was removed on a rotovap. The
product
15 was purified by silica gel chromatography using a mixture of ethyl
acetate/hexanes
(2/1 v/v) as the eluting solvent.
Procedure 2: The furoic acid 13 from above (1 mmol) was dissolved in
to dimethylformamide (5 mL). To this solution was added HATU (1 mmol),
followed
by the addition of the appropriate aniline or aniline hydrochloride ( 1 mmol),
and
triethylamine (2-3 mmol). The reaction mixture was stirred at room temperature
overnight. D1VIF was removed on a rotovap. The residue was suspended in ethyl
acetate, and washed with 10% HCl (aqueous), aqueous sodium bicarbonate, brine,
and
1s dried over magnesium sulfate. The solvent was removed on~a rotovap. The
product
15 was purified by silica gel chromatography using a mixture of ethyl
acetate/hexanes
(2/1 v/v) as the eluting solvent.
This procedure may be applied to the synthesis of anilides of the present
invention. Alternatively, the appropriate amide 15 may be prepared via
carboxylic
20 acid chloride 14. A suspension of 13 (300mmol) in anhydrous benzene (100m1)
containing a ew drops of anhydrous DMF was heated to relax under nitrogen as
thionyl chloride(l.l eq.) in benzene (35m1) was added dropwise. The solution
was
refluxed for 10 hours and then cooled to room temperature and concentrated
under
vacuum to give 14. A mixture of the appropriate aniline or aniline
hydrochloride salt
25 (l.2eq.) and triethylamine (2.Se~ in dichloromethane was stirred at
0°C under
nitrogen as a solution of 14 in dichloromethane was added dropwise. The
solution
was allowed to warm to room temperatureand further stirred for 12 hours. The
resulting suspension was washed several times with 2N hydrochloric acid,
saturated
soduim bicarbonate, brine and water successively. The organic layer was dried
over
3o anhydrous sodium sulfate, concentrated under vacuum to give 15 which was
then
purified by column chromatography.
Example Al: 5-(3,5-dichlorophenogy)-N-(2,4,6-trimethogyphenyl)-2-furamide
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
I
00 ,
ci I ~ o 0
\/
A1 c~
Compound A1 was made according to Scheme A where:
\o
c1 ~ off
H2N
\o ~ i o~
and
CI
were used as the starting materials Yield of the purified product was 70-85%.
NMR
and mass spectrometry data consistent with the desired title product were as
follows:
1H NMR (300 MHz, CDC13): 8 7.39 (s, 9H), 5.78 (d, 1H), 6.15 (s, 2H), 7.00 (s,
2H),
7.16 (d, 2I~, 7.24 (s, 1H), APCI-MS m/z 438 (M+H)+.
Example A2: 5-(2,6-dimethylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
o\
0
\ o o \
I H
/O
Compound A2 was synthesized in a manner analogous to that of A1,
according Scheme A. Yield of the purified product was 17%. NMR and mass
spectrometry data consistent with the desired title product were as follows:
1H NMR
(300 MHz, CDCl3): 8 2.24 (s, 6H), 3.83 (s, 3H), 3.85 (s, 6H), 4.96 (d, 1H),
6.20 (s,
2H), 7.05 (d, 1H), 7.10 (s, 3H), 7.20 (lir s, 1H), APCI-MS m/z 398.1 (M+H)+.
15 Example A3: 5-(5-isopropyl-2-methylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
i
0
H \
/o
Compound A3 was synthesized in a manner analogous that of Al, according
to Scheme A. The yield of the purified product was 48%. NMR and mass
2o spectrometry data consistent with the desired title product were as
follows: 1H NMR
(300 MHz, CDC13): ~ 1.25 (d, 6H), 2.26 (s, 3H), 2.88 (m, 1H), 3.81 (s, 9H),
5.38 (d,
1H), 6.18 (s, 2H), 6.95 (br s, 1H), 7.0 - 7.21 (m, 4H), APCI-MS m/z 426.1
(M+H)+.
36
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Example A4: 5-(2-methyl-6-propylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
I
00
0 0
\/
A4 -o
Compound A4 was synthesized in a manner analogous to that of Al,
according to Scheme A. NMR data consistent with the desired title product were
as
follows: 1H NMR (300 MHz, CDC13): ~ 0.95 (t, 3H), 1.58 (sextet, 2H), 2.26 (s,
3H),
2.56 (t, 2H), 3.81 (s, 3H), 3.83 (s, 6H), 4.96 (d, 1H), 6.18 (s, 2H), 7.10 (d,
1H), 7.13
(s, 3H), 7.22 (br s, 1H), APCI-MS m/z 426.1 (M+H)+.
Example A5: 5-[(1-bromo-3,8,8-trimethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)oxy]-N-(2,4,6-trimethoxypyrimidin-5-yl)-2-furamide
I I
e~ o ° %~ °
O O N
I ~ \ ~ " O
A
Compound AS was synthesized in a manner analogous to that of Al, using
similar starting compounds and where the pyrimidine is made from nitro
compound
VII which was further reduced to the amine in a manner analogous to that of
compound III. NMR and mass spectrometry data consistent with the desired title
product were as follows: 1H NMR (CH30D): S 1.18 (s, 6H), 1.57 (d, 2H, J = 6.04
Hz), 1.77 (d, 2H, J = 6.42, 3.40 Hz), 2.27 (s, 3H), 2.68 (m, 2H, J = 6.80,
6.42 Hz),
3.86 (s, 6H), 3.91 (s, 3H), 5.32 (d, 1H, J = 3.40 Hz), 7.09 (d, 1H, J = 4.91
Hz), APCI-
MS m/z 546 (M+H)+.
2o Example A6: 5-(4-bromo-2,6-dimethylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
0 0
I ~ ~ I~I ~ \ / ~\
0
A6 \
Compound A6 was synthesized in a manner analogous to that of Al,
according to Scheme A, via the acid chloride as set forth above. The yield of
the
2s purified product was 35%. NMR and mass spectrometry data consistent with
the
desired title product were as follows: 1H NMR (300 MHz, CDC13): ~ 2.15 (s,
6H),
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WO 02/098363 PCT/US02/17846
3.76 (s, 3H), 3.77 (s, 6H), 4.92 (d, 1H), 6.12 (s, 2H), 6.98 (d, 1H), 7.11 (br
s, 1H),
7.19 (d, 2H), APCI-MS nalz 475.9 (M+H)+.
Example A7: 5-[(1-bromo-3,8,8-trimethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)oxy]-N-(2,6-dimethoxyphenyl)-2-furamide
I
°
°
°
A7 ~
Compound A7 was synthesized in a manner analogous to that of Al, using
similar starting compounds and reaction conditions. NMR and mass spectrometry
data consistent with the desired title product were as follows: 1H NMR
(CH30D):
8 1.18 (s, 6H), 1.56 (m, 2H), 1.78 (m, 2H), 2.28 (s, 3H), 2.68 (d, 2H, J =
6.42 Hz),
3.75 (s, 6H), 5.31 (d, 1H, J= 3.78 Hz), 6.61 (d, 2H, J = 8.69 Hz), 7.06 (d,
1H, J =
3.40 Hz), 7.10 (s, 1H), 7.1? (t, 1H, J = 8.69, 8.31 Hz), APCI-MS m/z 515
(M+H)~".
Example A8: 5-[(3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamic~e
I
o I
w ~b
A8 ~
1s Compound A8 was synthesized according to Scheme A. The yield of the
purified product was 25%. NMR and mass spectrometry data consistent with the
desired title product were as follows: 1H NMR (300 MHz, CDCl3): 81.22 (s, 6H),
1.28
(s, 6H), 1.66 (s, 4H), 3.80 (s, 9H), 5.41 (d, 1H), 6.15 (s, 2H), 7.06 (s, 1H),
7.18 (d,
1H), 7.51 (s, 1H), APCI-MS m/z 558.3 (M+H)+.
2o The requisite phenol, 3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-
naphthalenol, was prepared as follows:
/ C CI~~~\~I //~~// 7.5 mol% AICI° / C BBr3, CHZCI~ / OH
I Br + ~CI CH ~ I gr -78 °~ ~ I Br
101
6-bromo-7-methoxy-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene
(~: A solution of 2-bromoanisole, 2,5-dichloro-2,5-dimethylhexane (1.l equiv.)
in
25 dichloromethane (2 mL/mmol) was stirred at 0 °C under nitrogen as
anhydrous A1C13
(7.5 mol%) was added portionwise while keeping the temperature below 5
°C. The
suspension was allowed to warm to room temperature and further stirred for
about 15
38
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WO 02/098363 PCT/US02/17846
hours. The resulting white suspension was poured into ice water (50 mL) and
the
aqueous l~.yer was extracted with ethyl acetate (2 x 50 mL). The combined
organic
extracts were washed with water and brine, dried over anhydrous Na2SO4 and
concentrated. The white solid thus obtained was recrystallized from toluene to
give 6-
s bromo-7-methoxy-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene in 78%
isolated
yield. NMR data consistent with the desired title product were as follows: 1H
NMR
(300 MHz, CDCl3): b 1.21 (s, 6H), 1.27 (s, 6H), 1.61 (s, 4H), 3.80 (s, 3H),
6.80 (s,
1H), 7.40 (s, 1H).
3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenol ii : 6-
1o Bromo-7-methoxy-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene in CH2C12
(3
mL/mmol) was demethylated by adding borontribromide (1.2 equiv.) in CHZCl2 at-
78 °C. The reaction mixture was allowed to warm to room temperature and
further
stirred for 15 hours. The solution was diluted with CH2Clz, washed with
saturated
NaHCO3. The organic layer was dried (Na2S04) and concentrated to give 3-bromo-
1s 5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenol in 90% yield. NMR
data
consistent with the desired title product was as follows: 1H NMR (CDC13): 8
1.25 (s,
12H), 1.68 (s, 4H), 5.28 (s, 1H), 6.94 (s, 1H), 7.32 (s, 1H).
Example A9: N-(2,6-dimethoxyphenyl)-5-(3,5,5,8,8-pentamethyl-5,6,7,8-
tetrahydro-2-naphthalenyl)oxy)-2-furamide
o
_0 0
I ~ I I " \
2o A9 \
Compound A9 was synthesized according to Scheme A via HATU. The yield
for purified product was 27%. NMR and mass spectrometry data consistent with
the
desired title product were as follows: 1H NMR (CDC13): & 1.23 (s, 6H), 1.28
(s, 6H),
1.67 (s, 4H), 3.85 (s, 3H), 5.34 (d, 1H), 6.62 (d, 2H), 7.01 (s, 1H), 7.13 (d,
1H), 7.15
25 (s, 1H), 7.23 (s, 1H), 7.26 (s, 1H), 7.34 (br s, 1H), APCI-MS m/z 464.1
(M+H)+.
OH CI~ ~ / 0 °C to rt, 2 h ~ OH
+ %~CI + AICI3 CH2CI2
v
1.1 equiv 10 mol%
102
Example A10: 6-methogy-3,3-dimethyl-1-ogo-N-(2,4,6-trimethoxypehnyl)-5-
indanecarboxamide
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WO 02/098363 PCT/US02/17846
A10
Compound A10 was synthesized in a manner analogous to that of Al,
according to Scheme A, using similar starting compounds and reaction
conditions.
The overall yield is 5%. NMR and mass spectrometry data consistent with the
desired
s title product were as follows:1H NMR (300 MHz, CDC13): ~ 1.48 (s, 6H), 2.62
(s,
2H), 3.81 (s, 9H), 3.92 (s, 3H), 5.69 (d, 1H), 6.24 (s, 2H), 7.20 (s, 1H),
7.32 (s, 1H),
APCI-MS m/z 482.1 (M+H)+.
OH / OH
I OH ~O PPA O
O O
116
Example All: N-(benzyloxy)-5- f (3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
1o naphthalenyl)oxy}-2-furamide
0 0 0
HN-~O
All
Compound Al l was synthesized in a manner analogous to that of Al,
according to Scheme A, using similar starting compounds and reaction
conditions.
The overall yield was 33%. NMR and mass spectrometry data consistent with the
15 desired title product were as follows: 1HNMR(300 MHz, CDC13): S 1.20 (s,
6H), 1.26
(s, 6H), 1.78 (s, 4H), 2.21 (s, 3H), 5.00 (s, 2H), 5.30 (d, 2H), 6.90 (s, 1H),
7.10 (s,
1H), 7.14 (d, 1H), 7.40 (m, SH), 8.56 (br s, 1H), APCI-MS m/z 434.1 (M+H)+.
Example A12: 5-[(7-chloro-1,4,4-trimethyl-2-oxo-1,2,3,4-tetrahydro-6-
quinolinyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
i
0
~ ° \ / H \ / °v
O N ~ CI
2o A12
Compound A12 was synthesized in a manner analogous to that of A1,
according to the following scheme:
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
OMe OH
I OMe + O / E>3N~ THF ~ ~ ~ CI AICo3 _ I
CI HN 130 C CI
H2N~C1 ~ O H
3A 3B 3C 3D (49% yield
from 3A)
O HO ~ O O
general scheme 1A I ~ O \O/ H ~ / O\ Mel~NaH I ~ O \O/ H O / O\
O 0 DMF
H OI \ O N CI
A12
To a solution of Compound 3A (10g) and 3B (7.5g) in THF (200 ml) was added
EtN3
(6.5g). The solution was stirred at room temperature overnight. The reaction
mixture
s was extracted with EtOAc, dried and concentrated to give 16 g of 3C as brown
oil...The residue was dissolved in 100 ml of CHZC12. To this solution was
added
A1C13 (33g). The solution was concentrated. The mixture was heated to
130°C in an
oil bath under NZ overnight. The mixture was cooled to room temperature and
extracted with EtOAC. Compound 3D was precipitated in ~H3CN (7.3g). To a
to solution of Compound 3A (10g) and 3B (7.5g) in THF (200 ml) was added EtN3
(6.5g). The solution was stirred at room temperature overnight. The reaction
mixture
was extracted with EtOAc, dried and concentrated to give 16 g of 3C as brown
4
oil...The residue was dissolved in 100 ml of CH2Clz. To this solution was
added
A1C13 (33g). The solution was concentrated. The mixture was heated to
130°C in an
~s oil bath under N2 overnight. The mixture was cooled to room temperature and
extracted with EtOAC. Compound 3D was precipitated in CH3CN (7.3g). NMR and
mass spectrometry data consistent with the desired title product were as
follows: 1H
NMR (MeOD): 8 1.29 (s, 6H), 2.54 (s, 2H), 3.41 (s, 3H), 3.88 (s, 9H), 5.45 (d,
1H),
6.21 (s, 2H), 7.10 (s, 1H), 7.14 (d, 1H), 7.19 (s, 1H), APCI-MS rrmlz 515.2
(M+H)+.
2o Example A13: 5-[(7-chloro-1,4,4-trimethyl-1,2,3,4-tetrahydro-6-
quinolinyl)oxy]-
N-(2,4,6-trimethoxyphenyl)-2-furamide
/
0o i I o\
I w o \o/ H
~cl
A13
Compound A13 was synthesized in a manner analogous to that of Al,
according to the following scheme:
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WO 02/098363 PCT/US02/17846
~ ~(7~ ~0
LAFi ~ OH ~~~OMe
~~ general scheme 1A ~~~ \ /
n ni ci mo .oa,rv ~ N CI
_. , . , ..,.,.
1o3 104
O O i
l.Mel, ICzC03, DMF
\O/ OHgeneralschemelA I ~ O \O/
2. NaOH N ~ CI N ~ CI
I
A13
NMR and mass spectrometry data consistent with the desired title product were
as
follows: lHhIMR (DMSO-d6): 8 1.20 (s, 6H), 1.69 (t, 2H), 3.22 (t, 2H), 3.71
(s, 6H),
3.78 (s, 3H), 5.29 (d, 1H), 6.26 (s, 2H), 6.63 (s, 1H), 7.21 (d, 1H), 8.9 (s,
1H), APCI-
MS m/z 501.1 (M+I~~.
Example A14: 5-[(1-acetyl-7-chloro-4,4-dimethyl-1,2,3,4-tetrahydro-6-
quinolinyl)oxy]-N-(2,6-dimethoxyphenyl)-2-furamide
a
" .0 0
N I ~ CI I~/ HN
A14 ~o -o
Compound A14 was synthesized in a manner analogous to that of Al,
1o according to Scheme A, using similar starting compounds and reaction
conditions:
NMR and mass spectrometry data consistent with the desired title product were
as
follows:1HNMR (DMSO-d6): 8 1.17 (s, 6H), 1.68 (t, 2H, J = 6.04 Hz), 2.15 (s,
3H),
3.64 (s, 2H), 3.65 (m, 6H), 5.54 (d, 1H, J = 3.40 Hz), 6.63 (d, 2H, J = 8.50
Hz), 7.17
(d, 2H, J = 16.81 Hz), 7.35 (s, 1H), 7.83 (s, 1H), 9.04 (s, 1H), APCI-MS m/z
499
is (M+H)+.
OMe II CI O \ I OMe ~Br ~ \ I OMe
~ N CI
H2N- v 'CI E N, CH2C12 ~HN~CI LDA, THF
rt, 5h
2A 2B
2C (66°~ yield
AICI3 I ~ OH from 2A)
130°C ' ~ CI
O
2D(87%)
To a solution of 2A (11 g) and triethylamine (8.5 g) in CH2C12 was added
acetylchlorode (6.6 g) slowly at room temperature. The solution was stirred
for I hour,
2o extracted with CH2Cla and concentrated to give compound 2B. Without
purification
the crude product was dissolved in THF (100 mL). To this solution was added
LDA
(1.3 eq.) followed by addition of allylbromide (11.3 g) at rt. The solution
was stirred
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WO 02/098363 PCT/US02/17846
overnight. Compound 2C (12.4g) was isolated by column chromatography
(hexaneBtoAC 2/1) Compound 2C (9g, 33.7 mmol) and A1C13 (9.1g, 67.4 mmol) was
dissolved in 100 ml of CH3N02. The solvent was evaporated and the dried
mixture
was heated to 135°C for 1.5 hours. The mixture was cooled to room
temperature,
dissolved in CH3NO2, poured into ice slow water and extracted with EtOAc.
Column
chromatography (hexane: EtOAc: 2:1) gave 2D (7.8g) in 87% yield.
Example A15: 5-[(1-methoxy-3,8,8-trimethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
0
o~ o
0 0
\ / H \ /
0
A15
to Compound A15 was synthesized in a manner analogous to that of Al,
according to Scheme A and the scheme set forth below:
Br Br O
OH BrZ AcOH I W OH general scheme 1A I w O \O/ OH
>90°/
30 105
O
Br O OMe O OMe 0 ~
I ~ O \O/ H NaOMe_ I ~ O ~O/ OH general scheme 1A I ~ O \O/
CuBr
A15
NMR and mass spectrometry data consistent with the desired title product
1s were as follows: 1H NMR (CH30D): 8 1.37 (s, 6H), 1.64 (d, 2H, J = 10.20
Hz), 1.77
(d, 2H, J = 9.07 Hz), 2.17 (s, 3H), 2.71 (dd, 2H, J = 6.04, 5.67 Hz), 3.81 (s,
6H), 3.85
(s, 3H), 3.90 (s, 3H), 5.07 (d, 1H, J = 3.78 Hz), 6.26 (s, 2H), 6.75 (s, 1H),
7.11 (s,
1H), APCI-MS fnlz 496 (M+H)+.
Example A16: N-(2,6-dimethoxyphenyl)-5-[(1-methoxy-3,8,8-trimethyl-5,6,7,8-
2o tetrahydronaphthalen-2-yl)oxy]-2-furamide
I
of ° ° i
° N
I / \ I H O
A16
Compound A16 was synthesized in a manner analogous to that of A15, using
similar starting compounds and reaction conditions. NMR and mass spectrometry
data consistent with the desired title product were as follows: 1H NMR
(CH30D): 8
25 1.12 (s, 6H), 1.55 (d, 2H, J = 5.67 Hz), 1.72 (d, 2H, J = 4.15 Hz), 2.10
(s, 3H), 2.65
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(dd, 2H, J = 6.42, 6.04 Hz), 3.63 (s, 3H), 3.73 (s, 6H), 5.32 (d, 1H, J = 3.78
Hz), 6.60
(m, 2H), 6.84 (s, 1H), 7.02 (s, 1H), 7.17 (t, 1H, J = 8.69, 8.31 Hz), APCI-MS
m/z 466
(M+~+.
Example A17: 5-[(1-bromo-3,8,8-trimethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
I I
o ~ o
sr o
° ° \
\ / H
A17 °w
Compound A17 was synthesized in a manner analogous to that of A15, using
similar starting compounds and reaction conditions. NMR and mass spectrometry
data consistent with the desired title product were as follows: 1H NMR
(CH30D): 8
1.18 (s, 6H), 1.56 (m, 2H), 1.77 (m, 2H), 2.29 (s, 3H), 2.69 (t, 2H, J = 6.42
Hz), 3.70
(s, 6H), 3.75 (s, 3H), 5.32 (d, 1H, J = 3.78 Hz), 6.22 (s, 2H), 7.04 (d, 1H, J
= 3.02 Hz),
7.10 (s, 1H), APCI-MS m/z 553 (M+H)+.
k
The following compounds were prepared according to Scheme B set forth
below:
Scheme B
Br 11
I \ H / O i \ NaOH, CH30H
R-
R I / CszC03. DMF / O
90-120°C 12
\ ~2
I / I \
J'
R I / ~ / OH EDCI or HATU /
SOCIy benzene
Catalytic DMF
~~z
R I~'~I
O
RI
I / ~ / C1
14 DCM or DMF
Scheme B is a modification of Scheme A. Numbered compounds and
identified reagents of Scheme B are analogous to those similarly identified
compounds and reagents of Scheme A.
2o Example Bl: N-(2,4,6-trimethoxyphenyl)-5-[(3,8,8-trimethyl-5,6,7,8-
tetrahydro-
2-naphthalenyl)oxy]-2-furamide
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WO 02/098363 PCT/US02/17846
I
~I ~
C><~ H
I I N o
B1
Compound B1 was made according to Scheme B wherein:
~o
OH H2N
and ~O ~ ~ O/
were used as the starting materials and the synthesis of the phenol is shown
below.
5 NMR and mass spectrometry data consistent with the desired title product
were as
follows: 1HNMR (300 MHz, CD30D): 8 7.38 (1H, d, J = 3.6 Hz), 7.32 (1H, s),
7.21
(1H, s), 6.52 (2H, s), 5.54 (1H, d, J = 3.6 Hz), 4.07 (3H, s), 4.05 (6H, s),
2.97 (2H, t, J
= 6.23 Hz), 2.45 (3H, s), 2.10-1.89 (4H, m), 1.49 (6H, s), APCI-MS m/z: 466.2
(M+H)+.
10 The requisite phenol was synthesized according to the procedure shown and
described below:
~ b. ~ Br c.
/ ~/
27
d.
a. 1y, heat, 110°C, 30mtn., neat '
b. Brz, AICI3, CH3NCz
c. NaOMe, Cu(I)Br,EtOAc, reflux,12h H
d. BBr3, -78°C, ovar night, quench with MeOH
v- 30
7-bromo-1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalene 29: 1,1,6-
trimethyl-1,2,3,4-tetrahydronaphthalene was synthesized from beta-ionone as
1s discussed in Parlow, TETRAB; Tetrahedron, EN, 49; 13; 2577-2588 (1993). To
a
solution containing 1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalene 28 (1.0 eq,
7.5 g,
43.1 mmol, 0.2 M) in nitromethane, bromine (1.0 eq, 2.21 mL, 43.1 mmol) was
added
dropwise over 2 minutes. The solution was then stirred vigorously and aluminum
trichloride (0.07 eq, 375 mgs, 2.81 mmol) was added solid. The mixture was
stirred
20 overnight and quenched with sodium thiosulfate and extracted with methylene
chloride. The organic solvent was evaporated in vacuo and the crude mixture
was
dissolved in minimal amount of hexanes. The resulting liquid was loaded onto a
silicagel (700 mL) plug column and eluted with hexanes to yield 50% 7-bromo-
1,1,6-
trimethyl-1,2,3,4-tetrahydronaphthalene 29 (5.50 g).
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7-methoxy-1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalene 76: To a flask
containing 7-bromo-1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalene (1.0 eq, 60
g, 228
mmol), sodium methoxide (521 mL) was added along with ethyl acetate (80 mL)
and
Cu(I)Br (0.03 eq, 1g, 7.0 mmol). The solution was refluxed 24 hours and
quenched
with concentrated HCI. The solution was diluted with water and extracted with
ethyl
acetate. The crude was purified by silica gel using ethyl acetate hexane
elution to
yield 7-methoxy-1,1,6-trimethyl-1,2,3,4-tetrahydronaphthalene 76 (19.88 g, 97
mmol,
43% yield).
3,8,8-trimethyl-5,6,7,8-tetrahydro-2-naphthalenol 30: 7-methoxy-1,1,6-
~ trimethyl-1,2,3,4-tetrahydronaphthalene (1.0 eq, 5.67 g, 27.8 mmol) was
dissolved in
methylene chloride (0.2 M) and cooled to -78°C. To this solution, BBr3
(1.0 eq, 1 M,
27 mL) was added at once and stirred overnight slowly bringing the solution to
room
temperature. The solution was then quenched with methanol and passed through a
silicagel plug to yield 3,8,8-trimethyl-5,6,7,8 tetrahydro-2-naphthalenol 30
(4.68 g,
88%).
Example B2: 5-(3-Benzenesulfonylamino-phenoxy)-furan-2-carboxylic acid
(2,4,6-trimethoxy-phenyl)-amide
B2
2o Compound B2 was synthesized in a manner analogous to that of B 1, according
to Scheme B, using similar starting compounds and reaction conditions.
Example B3: 5- f [(6s)-3,5,5,6,88-hexamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl] oxy}-N-(2,4,6-trimethoxyphenyl)-2-furamide
0
\ / °\
B3
Compound B3 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
Yield of purified product was 89%. NMR data consistent with the desired title
product were as follows: 1H (300 MHz, CDCl3): 8 0.94 (d, 3H, J = 6.42 Hz),
1.08,
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1.22, 1.25, 1.30 (4s, 3H each), 1.30 - 1.37 (m, 1H), 1.63 (t, J =12.8 Hz),
1.78-1.93
(m, 1H), 2.23 (s, 3H), 4.6 (s, 1H, OH), 6.75 and 7.15 (2s, 1H each).
The requisite phenol was prepared by the following scheme:
O mCPBA ~ OH
Ii Ii
106
3,5,5,6,8,8-hexamethyl-5,6,7,8-tetrahydro-2-naphthalenol: To the solution
of fixolide (25.84 g, 100 mmol) in dichloromethane (500 mL) was added m-
chloroperbenzoic acid (57-86 % taken mean value of 71%, 12.08 g, 100 mmol).
The
resulting mixture was stirred for 16 hours at room temperature. The solvent
was
evaporated, and the residue was dissolved in methanol (200 mL). Sodium
methoxide
(457 mL of 25%, 200 mmol) was added. The mixture was stirred for 2 hours, and
methanol evaporated. The residue was diluted with water and neutralized with
dilute
hydrochloric acid, and extracted with ethyl acetate. The ethyl acetate layer
was
filtered through a plug of silica gel. The solvent was evaporated and the
residue on
crystallization with ethyl acetate-hexane mixture gave white powder, 20.9 g.
Example B4: N-(2,6-dimethoxyphenyl)-5-[(5,5,8,8-tetramethyl-5,6,7,8-
s
tetrahydro-2-naphthalenyl)oxy]-2-furamide
a o
i
B4
Compound B4 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the desired title product were as follows:
1H NMR
(300 MHz, CDC13): 8 1.26 (s, 12H), 1.68 (s, 4H), 3.83 (s, 6H), 5.53 (d, 1H),
6.60 (d,
2H), 6.90 (d, 1H), 7.15 (s, 1H), 7.19 (d, 1H), 7.22 (t, 1H), 7.28 (d, 1H),
APCI-MS m/z
450.3 (M+I~+.
Example B5: ethyl 4-[(5-~[5-(4-chloro-3-isopropyl-2-methoxy-6-methylphenoxy)-
2-furoyl]amino)-4,6-dimethoxy-2-pyrimidinyl)amino]butanoate
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o~ o
-N
~O~ H ~ /~NH O
N
CI ~ O
0
B5
Compound BS was synthesized by coupling of the pyrimidine derivative, the
preparation of which is described below, to a substituted furoic acid
according to
s Scheme B. NMR and mass spectrometry data consistent with the desired title
product
were as follows: 1HNMR (CDC13): 8 1.26 (3H, t), 1.40 (6H, d), 1.98 (2H,
sextet),
2.19 (3H, s), 2.39 (2H, t), 3.48 (2H, c~, 3.58 (1H, heptet), 3.83 (3H, s),
3.89 (6H, s),
4.12 (2H, c~, 5.08 (1H, br t); 5.11(1H, d), 6.91 (1H, s), 7.03 (1H, s), 7.08
(1H, d), FI-
PCI m/z 592.2 & 593.2 (M+H)+.
o ~ o
i0 I N~C~ ,O N~N~LO~ i0 NYN O
N ~ SIN I iN
OaN OaN H2N
i0 i0 i0
~~ 117 118
Displacement of 4-chloride: To a solution of 4-chloro-2,4-dimethoxy
nitropyrimidine (3.28 g, 15 mmol) in DMF (30 mL) was added triethylamine (2.09
mL, 15 mol) and ethyl 4-aminobutyrate hydrochloride (2.51 g, 15 mmol). The
reaction mixture was stirred at room temperature overnight. Most of DMF was
removed on a rotovap. The concentrate was redissolved in ethyl acetate, washed
with
water, brine, dried over magnesium sulfate. The solvent was removed on a
rotovap.
The product was purified by flash chromatography (solvent: 1 ethyl acetate: 3
hexanes
to 1 ethyl acetate: 2 hexanes): 2.38 g (50.5%). NMR data consistent with the
desired
title product were as follows:1H NMR (CDCl3): 8 1.27 (3H, t), 1.98 (2H,
sextet), 2.41
(2H, t), 3.49 (2H, c~, 3.94 (3H, s), 4.01 (3H, s), 4.15 (2H, ~, 5.50 (1H, br
t).
Hydrogenation: The nitro pyrimidine compound obtained from above
(2.38 g, 7.58 mmol) was dissolved in methanol (25 mL). 10% palladium on
carbon catalyst (.4 g) was carefully added to the methanolic solution and the
mixture was hydrogenated at 45-50 psi overnight. The mixture was then filtered
through Celite and then washed with methanol. The combined filtrate was
concentrated to a dark oil, 1.76 g (82%).
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8r O
I ~ OH NBS, (iPrhNH OH NaOMe, CuBr OH
CH CI I ~ CH OH
2 2 3
SOZCIZ
--~ ~OH
CHC13
CI
NMR and Mass spectrometry data consistent with the title product were as
follows: 1H NMR (CDCl3): ~ 1.24 (3H, t), 1.98 (2H, sextet), 2.39 (2H, t), 2.49
(2H, br s), 3.48 (2H, q), 3.90 (6H, s), 4.12 (2H, q), 4.60 (1H, br t), FI-PCI
m/z
285.2 (M+H)+.
Example B6: 4-bromo-5-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
o i
0
0
B6 Br
Compound B6 was synthesized in a manner analogous to that of B 1, according
1o to Scheme B. NMR and mass spectrometry data consistent with the desired
title
product were as follows: IH NMR (300 MHz, CD30D): 8 1.52 (s, 12H), 1,96 (s,
4H),
4.06 (s, 6H), 6.94 (d, 2H), 7.09 (d, 1H), 7.28 (s, 1H), 7.54 (t, 2H), 7.62 (d,
1H), APCI-
MS m/z 528.3 (M+H)+.
Example B7: 4-bromo-5-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-
15 naphthalenyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
o ,
O
Fi
O\
B7 Br
Compound B7 was synthesized in a manner analogous to that of B l, according
to Scheme B, using similar starting compounds and reaction conditions. NMR and
mass spectrometry data consistent with the desired title product were as
follows: IH
2o NMR (300 MHz, CD30D): 8 1.26 (s, 12H), 1,70 (s, 4H), 3.77 (s, 3H), 3.80 (s,
6H),
6.25 (s, 2H), 6.81 (d, 1H), 7.01 (s, 1H), 7.27 (s, 1H), 7.32 (d, 1H), APCI-MS
mlz
558.4 (M+H)+.
Example B8: 5-[(3,5,5,8',8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-
N-(2H-tetrazol-5-yl)-2-furamide
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0
a o
NH
B8
Compound B8 was synthesized in a manner analogous to that of B1, according
to Scheme B, using similar starting compounds and reaction conditions. NMR
data
consistent with the desired title product were as follows: 1H NMR (CD30D-d4):
8
1.25 (s, 6H), 1.30 (s, 6H), 1.72 (s, 4H), 2.23 (s, 3H), 5.41(d, 1H), 7.08 (s,
1H), 7.28 (s,
1H), 7.45 (d, 1H), LC-MS, APCI, (M+H)+: 396.
Example B9: 5-[(2,2,5,7,8-pentamethyl-3,4-dihydro-2h-chromen-6-yl)oxy]-n-
(2,4,6-trimethoxyphenyl)-2-furamide
I
° ° \ °\
° ° ~ i
~I H
O~ O\
B9
1o Compound B9 was synthesized in a manner analogous to that of B 1, according
to Scheme B, using similar starting compounds and reaction conditions. NMR and
mass spectrometry data consistent with the desired title product were as
follows: 1H
NIvIR (300 MHz, CDC13): ~ 1.32 (s, 6H), 1.81 (t, 2H), 2.05 (s, 3H), 2.11 (s,
6H), 2.62
(t, 2H), 3.81 (s, 9H), 4.91 (d, 1H), 6.19 (s, 2H), 7.08 (d, 1H), 7.22 (s, 1H),
APCI-MS
m/z 496.1 (M+H)+.
Example 810: N-(2,4,6-trimethoxyphenyl)-5-[(4,4,7-trimethyl-3,4-dihydro-2h-
chromen-6-yl)oxy]-2-furamide
0
p ~b
B10 ° ~ -°
Compound B 10 was synthesized in a manner analogous to that of B 1,
2o according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 26%. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H NMR (300 MHz,
CDCl3):
~ 1.32 (s, 6H), 1.84 (dd, 2H, J = 4.14, 6.23 Hz), 2.21 (s, 3H), 3.81 (br s,
H20), 3.81 (s,
9H), 4.2 (dd, 2H, J = 4.23, 6.23 Hz), 5.22 (d, 1H, J = 3.4 Hz), 6.19 (s, 2H),
6.69 (s,
1H), 7.03 (s, 1H), 7.17 (d, 1H, J = 3.02 Hz), 7.31 (br s, 1H), APCI-MS m/z
468.2
(M+H)+.
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Example B11: N-(2,4,6-trimethoxyphenyl)-5-[(4,4,8-trimethyl-3,4-dihydro-2h-
chromen-6-yl)oxy]-2-furamide
d
H~ ,
Bll
Compound B 11 was synthesized in a manner analogous to that of B 1,
s according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 26%.
The requisite chromanophenol was synthesized according to the following
method:
CHCI3-MeOH(9:1) TBDMS-CI,
OH / reilux, 16 h Imidazale, DMF
HO I ~ + ' 16% of inseparable mixture
0
~ O~ CH3MgI, 81%
/~ + s
~O SI~O i Non-separable mixture
O
~ f~H
O~SI~ \ O~ AICI9,CH3N02 ,90%
~p I ~ / ~ + ~gp0 ~ / Non-separable mixture
-i-OH
\ OH ~~OH
> ~~i + o l i
0
107 108
1o The mixture of these chromanophenols was treated with methyl bromofuroate.
The final products were separated using HPLC. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H NMR (300 MHz,
CDCl3): 8 1.24 (s, 6H), 1.75 (t, 2H, J = 5.29 Hz), 2.10 (s, 3H), 3.73 (s, 9H),
4.13 (t,
2H, J = 5.29 Hz), 4.25 (br s, HZO), 5.32 (d, 1H, J = 3.59 Hz), 6.08 (s, 2H),
6.69 (d,
15 1H, J = 3.03 Hz), 6.84 (d, 1H, J = 3.03 Hz), 7.10 (d, 1H, J = 3.59 Hz),
7.31 (br s, 1H),
APCI-MS m/z 468.2 (M+H)+.
Example 812: N-[(5-methyl-2-pyrazinyl)methyl]-5-[(4,4,7-trimethyl-3,4-dihydro-
2H-chromen-6-yl)oxy]-2-furamide
~ '~ O O O H
B12 a' v \
2o Compound B 12 was synthesized in a manner analogous to that of B 11,
according to Scheme B, using similar starting compounds and reaction
conditions.
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The yield of the purified product was 12%. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H NMR (300 MHz,
CDCl3):
~ 1.30 (s, 6H), 1.83 (t, 2H, J = 5.48 Hz), 2.18, 2.62 (2s, 3H each), 4.19 (t,
2H, J = 5.48
Hz), 5.06 (br s, H20), 5.19 (d, 1H, J = 3.58 Hz), 6.68 (s, 1H), 6.99 (s, 1H),
7.11 (d,
1H, J = 3.58 Hz), 7.13 (br s, 1H), 8.48, 8.60 (2s, 1H each), APCI-MS m/z 408.1
(M+H)+.
Example B13: N-[(5-methyl-2-pyrazinyl)methyl]-5-[(4,4,8-trimethyl-3,4-dihydro-
2H-chromen-6-yl)oxy]-2-furamide
H
O'
B1 I3
1o Compound B 13 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 11%. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H NMR (300 MHz,
CDC13):
8 1.32 (s, 6H), 1.85 (dd, 2H, J = 5.29, 5.47 Hz), 2.18, 2.63 (2s, 3H each),
4.23 (dd,
1s 2H, J = 5.29, 5.48 Hz), 4.78 (d, 2H, J = 5.47 Hz), 5.39 (d, 1H, J = 3.59
Hz), 6.14 (br s,
H20), 6.66 (d, 1H, J = 2.83 Hz), 6.92 (d, 1H, J = 3.02 Hz), 7.13 (d, 1H, J =
3.58 Hz),
7.17 (br s, 1H), 8.50, 8.62 (2s, 1H each), APCI-MS m/z 408.1 (M+H)+.
Example B14: N-(2,6-dimethoxyphenyl)-5-[(4,4,7-trimethyl-3,4-dihydro-2H-
chromen-6-yl)oxy]-2-furamide
2o B14
Compound B 14 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 66%. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H NMR (CDC13): S
1.29 (s,
2s 6H), 1.81 (t, 2H, J = 5.48 Hz), 2.19 (s, 3H), 3.84 (s, 6H),'4.17 (t, 2H, J
= 5.29 Hz),
5.21 (d, 1H, J = 3.59 Hz), 6.62 (m, 2H), 7.01 (s, 1H), 7.15 (d, 1H, J = 3.4
Hz), 7.21 (t,
1H, J = 8.5 Hz), 7.40 (br s, 1H), MS m/z 438.2 (M+H)+.
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Example B15: 5-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-
N-(3-{ [(2-{ [(2R)tetrahydro-2-furanylmethyl] amino}-4-
pyrimidinyl)amino]methyl}benzyl)-2-furamide
B15
Compound B 15 was synthesized according to Scheme B.
~N
THF Op N"', O
CI~N~CI H2N U r.t
O O
O I,
HZN I W NI12 + ~ ~ ~ FsC~H I ~ NHZ CI~'O~
g
p ~N
O O ~ N~O~ CI~:N~H ~V
II II [(ZG~g
F30~H I \ H~O~ I H ~CI
/ I,
NHZ
~N
~I I /~ /'~..
~Hi°,.~ ICOH H2N I / H I N H
HO'~'OH,fl 109
Preparation of 2-chloro-N-[(2R)-tetrahydro-2-furanmethyl]-4-pyrimidinamine & 4-
chloro-N-[(2R)-tetrahydro-2-furanmethyl]-2-pyrimidinamine: To a 250 mL round
bottom
flask was placed 2,4-dichloropyrimidine (5.0g, 33.56 mmol) and 200 mL THF. To
this
solution was added triethylamine (14.0 mL, 100.68 mmol) and [R]-
tetrahydrofurfiuylamine.
The solution was stirred overnight. The reaction mixture was poured into water
and extracted
with methylene chloride. The separated organic layer was washed with brine,
dried over
magnesium sulfate, and concentrated on a rotary evaporator. The crude compound
was
purified by silica gel chromatography with hexane/ ethyl acetate (4:1 v/v to
1:1 v/v) to yield
chloro-N-[(2R)-tetrahydro-2-furanmethyl]-2-pyrimidinamine (1.3 g) and 2-chloro-
N-[(2R)-
tetrahydro-2-furanmethyl]-4-pyrimidinamine (3.98 g). Preparation of N-[3-
(aminomethyl)benzyl]-2,2,2-trifluoroacetamide: To a solution of m-xylene
diamine (28.76g,
211.15 mmol) in THF (300 mL, .7IV1) was added dropwise a solution of ethyl
trifluoroacetate
(10g, 70.38 mmol) in THF (50 mL, 1.41V1]. The solution was stirred at room
temperature
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overnight. The reaction was monitored by TLC. The solvent was concentrated and
residue
was acidified to pH 2 with 4N HCl and dissolved in water and washed with ethyl
acetate. The
separated aqueous layer was basified to pH 11 using NH40H and compound was
extracted
with dichloromethane. The separated organic layer was wash with water/brine,
dried over
magnesium sulfate and concentrated to yield N-[3-(aminomethyl)benzyl]-2,2,2-
trifluoroacetamide ( 8.718, 53% yield). Preparation of ethyl 3-
(aminomethyl)benzylcarbamate: To a solution of N-[3-(aminomethyl)benzyl]-2,2,2-
trifluoroacetamide (10.6g, 43.1 mmol) was added ethyl chloroformate (leq.)
followed by
triethylamine.. Reaction was stirred at room temperature for 30 min. Crude
product was
extracted with methylene chloride and concentrated to give ethyl 3-
{[(trifluoroacetyl)amino]methyl} benzylcarbamate 4. This crude product was
dissolved in
methanol (100 mL) and 2N I~zC03 ( 100 mL) and stirred overnight. Reaction
mixture was
basified to pH 14 with 20% NaOH, extracted with methylene chloride, wash with
brine and
dried over magnesium sulfate to yield ethyl 3-(aminomethyl)benzylcarbamate
(5.2g).Prepaxation of ethyl 3-~[(2-f [(2R)-tetrahydro-2furanylmethyl]amino}-4-
pyrimidinyl)amino]methyl}benzylcarbamate: To a solution of ethyl 3-
(aminomethyl)benzylcarbamate and 4-chloro-N-[(2R)-tetrahydro-2-furarnnethyl]-2-
pyrimidinamine in chlorobenzene was added triethylamine. Reaction mixture was
reflux
overnight. The solution was cooled to room temperature and loaded on a silica
gel column
and eluted with hexane/ethyl acetate (1:l v/v) to yield ethyl 3-([(2-{[(2R)-
tetrahydro-2-
furanylmethyl]amino}-4-pyrimidinayl)amino]methyl}benzylcarbamate (73% yield).
Ethyl 3-
f [(2-{[(2R)-tetrahydro-2-furanylinethyl]amino}-4-pyrimidinayl)
amino]methyl}benzylcarbamate was dissolved in ethylene glycol and potassium
hydroxide
(1:1 v/v). The solution washeated to 100°C overnight. The mixture was
cooled to room
temperature and extracted with chloroform, washed with brine, and dried over
magnesium
sulfate to yield N4-[3-(aminomethylObenzyl]-NZ-[(2R)-tetrahydro-2-
furanylmethyl]-2,4-
pyrimidinediamine (82 % yield). NMR and mass spectrometry data consistent with
the
desired title product were as follows: 1HNMR (CDC13): 8 1.20 (s, 6H), 1.25 (s,
6H),
1.66 (s, 4H), 1.87-1.99 (m, 4H), 2.21 (s, 3H), 3.74-3.89 (m, 2H), 3.75 (t,
1H), 3.87 (t,
1H), 3.88 (m, 1H), 4.5 (d, 2H), 4.58 (d, 2H), 5.30 (d, 1H), 5.73 (d, 1H), 6.57
(t, 1H),
6.94 (s, 1H), 7.08 (d, 1H), 7.14 (s, 1H), ?.23-7.34 (m, SH) (s, 1H), 7.76 (d,
1H),
APCI-MS ynlz 624.4 (M+H)+.
Example B16: 5-[(3-isopropyl-1,1,2,6-tetramethyl-2,3-dihydro-1H-inden-5-
yl)oxy]-N-(2,4,6-trimethoxy-5-pyrimidinyl)-2-furamide
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0 0
o~
B16
Compound B 16 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds where the nitro-
pyrimidine
is compound VII which was reduced to the amine in a manner analogous to that
of
s compund III and the synthesis of the phenol is shown below. The yield of the
purified
product was 61%.
The requisite phenol, 3-isopropyl-1,1,2,6-tetramethyl-5-indanol, was
synthesized according to the following method:
0
1. mCPBA I ~ OH
i 2. NaOMe
110
1o To the solution of transeolide (5.16 g, 20 mmol) in dichloromethane (100
mL)
was added m-chloroperbenzoic acid (57-86% taken mean value of 71%, 5.83 g, 24
mmol) and sodium bicarbonate (1.7 g, 20 mmol). The reaction mixture was
stirred
fox 16 hours at room temperature. The solvent evaporated, the residue was
dissolved
in methanol (100 mL). Sodium methaoxide (13.72 mL of 25% methanolic soution,
60
is mmol) was added and the mixture was stirred at room temperature for 2
hours, and
solvent evaporated. The residue was diluted with water (150 mL), neutralized
with
diluted hydrochloric acid, and extracted with ethyl acetate (500 mL). The
organic
layer was dried and filtered through a small plug of silica gel to give
desired phenol as
white solid (4.29 g, 92%). NMR and mass spectrometry data consistent with the
2o desired title product were as follows: 1H (300 MHz, CDC13): 8 0.96 (d, 6H,
J = 9.8
Hz), 1.01 (s, 3H), 1.09 (d, 3H, J = 9.0 Hz), 1.27 (s, 3H), 1.8-1.95 and 2.1-
2.25 (2m,
1H each), 2.25 (s, 3H), 2.73 (d, 1H, J = 9.82 Hz), 3.95 (s, 6H), 3.98 (s, 3H),
5.32 (d,
1H, J = 3.58 Hz), 6.91 (s, 1H), 7.05 (s, 1H), 7.15 (d, 1H, J = 3.59 Hz), APCI-
MS m/z
510.3 (M+I~+.
25 Example B17:4-[(5-~[5-(4-chloro-3-isopropyl-2-metho~ry-6-methylphenoxy)-2-
furoyl]amino}-4,6-dimethogy-2-pyrimidinyl)amino]butanoic acid
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
o~ o /
\ / " ~ ~'~~ ~a~
a ~,//\
OH
B17
Compound B17 was synthesized from Compound B5. Compound BS (53 mg,
0.089 mmol) was dissolved in ethanol (1 mL) and an aqueous solution of sodium
hydroxide (2 equivalents) was added. The reaction was monitored by TLC
(developing solvent: 5% methanol in methylene chloride) for completion of
reaction.
After saponification was complete, the mixture was concentrated. The residue
was
diluted with water and washed with ether. The aqueous layer was then carefully
acidified to pH 2 with 10% HCl and the product was extracted with methylene
1o chloride. The title product was purified by silica gel chromatography
(solvent: 10%
methanol in methylene chloride). NMR and Mass spectrometry data consistent
with
the desired title product were as follows: 1H NMR (CDCl3):a8 1.35 (6H, d),
1.95 (2H,
sextet), 2.19 (3H, s), 2.43 (2H, t), 3.47 (2H, q), 3.58 (1H, heptet), 3.83
(3H, s), 3.89
(6H, s), 5.11(1H, d), 5.30 (1H, br t); 6.93 (1H, s), 7.01 (1H, s), 7.08 (1H,
d); FI-PCI
15 rn/z 564.2 & 565.2 (M+I~+.
s
Example B18: 5-[(3-isopropyl-1,1,2,6-tetramethyl-2,3-dihydro-1H-inden-5-
yl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
0
0 0
~ /
O\
B18
Compound B 18 was synthesized in a manner analogous to that of B 1,
20 according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 55%. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H (300 MHz, CH30H-
d4): 8
0.96 (d, 6H, ~J = 8.87 Hz), 0.99 (s, 3H), 1.01 (d, 3H, J = 6.98 Hz), 1.28 (s,
3H), 1.70-
1.95 and 2.1-2.25 (2m, 1H each), 2.26 (s, 3H), 2.71 (d, 1H, J = 9.25 Hz), 3.80
(2s, 3H
25 each), 3.82 (s, 3H), 5.31 (d, 1H, J = 3.58 Hz), 6.27 (2s, 1H each), 6.92
and 7.06 (2s,
1H each), 7.15 (br s, 1H), APCI-MS rnlz 508.2 (M+H)+.
Example B19: N-(2,6-dimethoxy-3-pyridinyl)-5-((3-isopropyl-1,1,2,6-tetramethyl-
2,3-dihydro-1H-inden-5-yl)oxy]-2-furamide
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0 0 -_ /
0
B19 \ _ - -o
Compound B 19 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 28 %. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H (300 MHz, CH30H-
d4): 8
0.95 (d, 6H, J = 7.18 Hz), 1.00 (s, 3H), 1.09 (d, 3H, J = 6.98 Hz), 1.27 (s,
3H), 1.80-
1.95 and 2.1 - 2.25 (2m, 1H each), 2.25 (s, 3H), 2.71 (d, 1H, J = 8.31 Hz),
3.90 and
4.0 (2s, 3H each), 5.33 (d, 1H, J = 3.58 Hz), 6.33 (d, 1H, J = 8.31 Hz), 6.92
and 7.06
(2s, 1H each), 7.16 (d, 1H, J = 3.78 Hz), 8.05 (d, 1H, J = 8.5 Hz), APCI-MS
m/z 479.2
io (M+H)+.
Example B20: N-[2-(2,4-difluorophenoxy)-3-pyridinyl]-5-[(3-isopropyl-1,1,2,6-
tetramethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furamide
B20
Compound B20 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was~2 %. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H (300 MHz, CH30H-
d4): S 0.91 (d, 6H, J = 6.99 Hz), 1.00 (s, 3H), 1.04 (d, 3H, J = 6.80 Hz),
1.24 (s,
3H), 1.80-1.95 and 2.1-2.25 (2m, 1H each), 2.22 (s, 3H), 2.65 (d, 1H, J = 9.07
2o Hz), 5.36 (d, 1H, J = 3.78 Hz), 6.91 (s, 1H), 6.95-7.15 (m, 4H), 7.26 (d,
1H, J =
3.78 Hz), 7.26-7.37 (m, 1H), 7.80 (dd, 1H, J = 6.6, 1.7 Hz), 8.47 (dd, 1H, J =
9.44
and 1.70 Hz), APCI-MS m/z 547.2 (M+H)+.
Example B21: 4-bromo-5-[(3,5,5,6,8,8-hexamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
\ l °\
2s B21
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Compound B21 was synthesized in a manner analogous to that of B1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 16 %. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H (300 MHz,
CDCl3): b
0.97 (d, 3H, J = 6.61 Hz), 1.05, 1.20, 1.22, 1.31 (4s, 3H each), 1.30-1.45 (m,
1H),
1.50-1.70 (m, 1H), 1.80-2.0 (m, 1H), 2.33 (s, 3H), 3.79 (2s, 6H), 3.81 (s,
3H), 5.34 (d,
1H, J = 3.4 Hz), 6.15 (s, 2H), 6.79 (s, 1H), 7.08 (br s, 1H), 7.18 and 7.19
(2s, 1H
each), APCI-MS nalz 586.2 and 588.2 (M+H)+.
Example B22: 4-bromo-N-(2,4,6-trimethoxyphenyl)-5-[(3,8,8-trimethyl-5,6,7,8-
l0 ' tetrahydro-2-naphthalenyl)oxy]-2-furamide
0
0 0
\ / ~ \ / °\
i
B22 Br o\
Compound B22 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 18%. NMR and mass spectrometry data
1s consistent with the desired title product were as follows: 1H (300 MHz,
CDC13): 8
1.21 (s, 6H), 1.55 -1.66 (m, 2H), 1.73 - 1.83 (m, 2H), 2.29 (s, 3H), 2.70 (t,
1H, J =
6.24 Hz), 3.78 (s, 6H), 3.81 (s, 3H), 6.16 (s, 2H), 6.85,6.90, 7.07 (br) and
7.19 (4s, 1H
each), APCI-MS mlz 544.2 and 545.2 (M+H)+.
Example B23: 4-bromo-5-[(3,5,5,6,8,8-hexamethyl-5,6,7,5-tetrahydro-2-
20 naphthalenyl)oxy]-N-(2,4,6-trimethoxy-5-pyrimidinyl)-2-furamide
0
0 0
\ ~\
°\
B23
Compound B23 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 8%. NMR and mass spectrometry data
25 consistent with the desired title product were as follows: 1H (300 MHz,
CDCl3): S
0.98 (d, 3H, J = 6.61 Hz), 1.05, 1.20, 1.21, 1.32 (4s, 3H each), 1.30-1.45 (m,
1H), 1.61
(dd, 1H, J =12.8, 13.2 Hz), 1.77 - 1.92 (m, 1H), 2.32 (s, 3H), 3.94 (s, 6H),
3.94 (s,
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3H), 6.77 (s, 1H), 6.93 (br s, 1H), 7.19 and 7.22 (2s, 1H each), APCI-MS m/z
588.2
and 590.2 (M+H)+.
Example B24: 4-bromo-N-(2,4,6-trimethoxyphenyl)-5-[(3,3,6-trimethyl-1,3-
dihydro-2-benzofuran-5-yl)oxy]2-furamide
0 0
o I ~ ~ / b \ /
B24 g' °\
Compound B24 was synthesized in a manner analogous to that of B l,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 21 %.
.M~ ASH
HO I ~ ~ O I ~ \ I ~ \ O I ~ H
HCI THF NaH
refluxovernight overnight DMF reflux
OC to reflux
NMR and mass spectrometry data consistent with the desired title product were
as
follows: 1H (300 M>=Iz, CDC13): 8 1.46 and 2.37 (2s, 6H and 3H respectively),
3.81 (s,
6H), 3.99 (s, 3H), 5.01, 6.16 (2s, 2H each), 6.62 and 7.05 (2s, 1H each), 7.09
(brs,
1H), 7.20 (s, 1H), APCI-MS m/z 533 and 534 (M+H)+.
Example B25: 4-bromo-N-methyl-N-(2,4,6-trimethoxyphenyl)-5-[(3,3,6-
trimethyl-1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furamide
0
0 o I
o I ~ ~ / N \ / °
far o\
B25
Compound B25 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
2o The yield of the purified product was 20%. NMR and mass spectrometry data
consistent with the desired title product were as follows: 1H (300 MHz,
CDC13): 8
1.41 (s, 6H), 2.24 and 3.17 (2s, 3H each), 3.70 (s, 6H), 3.82 (s, 3H), 4.98,
6.04 (2s, 2H
each), 6.08 (s, 2H), 6.50 and 6.97 (2s, 1H each), APCI-MS m/z 533 and 546 and
548
(M+H)+.
Example B26: 5-(3,3,6-Trimethyl-indan-5-yloxy)-furan-2-carboxylic acid (2-
chloro-4,6-dimethoxy-pyrimidin-5-yl)-amide
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o N\Yci
p O N ~ /N
/
/O
B26
Compound B26 was synthesized in a manner analogousto that of B 1,
according to scheme B.
Example B27: 5-(4-chloro-3-isopropyl-2-methoxy-6-methylphenoxy)-N-(2- f [3-
(dimethylamino)propyl]amino}-4,6-dimethoxy-5-pyrimidinyl)-2-furamide
O~ '~NYN~N~
O O N~N
/ ~/ TO
CI v~ ~
B27
Compound B27 was synthesized in a manner analogousto that of B 1,
1o according to scheme B.
Example B28: N-{4,6-dimethoxy-2-[(pyridin-2-ylmethyl)amino]pyrimidin-5-yl}-
5-[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furamide
0 00
HN ~ ~>--NH N-
N
O
B28
Compound B28 was synthesized in manner analogous to that of B52. NMR
~s and mass spectrometry data consistent with the title product were as
follows: 1H NMR
(300 MHz, CDC13): 8 1.21 (s, 6H), 1.93(dd, 2H, J =14.35, 7.18, Hz), 2.24 (s,
3H),
2.84 (t, 2H, J = 14.35 Hz), 3.86 (s, 6H), 4.72 (d, 2H, J = 6.04 Hz), 5.28 (s,
1H), 5.84
(dd, 1H, J = 11.33, 5.67 Hz), 6.82 (s, 1H), 6.94 (s, 1H), 7.05 (s, 1H), 7.09
(d, 1H, J =
3.78 Hz), 7.18 (d, 1H, J =11.71 Hz), 7.35 (d, 1H, J = 7.93 Hz), 7.66 (dd, 1H,
J'=
20 15.49, 7.93 Hz), 8.56 (d, 1H, J = 4.91 Hz), APCI-MS m/z 530.6 (M+H)+.
Example 829: N- f 4,6-dimethoxy-2-[(2-morpholin-4-ylethyl)amino]pyrimidin-5-
yl~-5-[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furamide
0 0 0 0
_N
I / I ~ HN ~ /~NH
~N ~N
B29
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Compound B29 was synthesized in manner analogous to that of B52. NMR
and mass spectrometry data consistent with the title product were as follows:
1H NMR
(300 MHz, CDC13): 8 1.21 (s, 6H), 1.93 (dd, 2H, J =1 4.35, 7.18 Hz), 2.24 (s,
3H),
2.50 (s, 4H), 2.59 (t, 2H, J = 12.09 Hz), 2.84 (dd, 2H, J =14.35, 7.18 Hz),
3.49 (dd,
1H, J =11.71, 5.67 Hz), 3.75 (tt, 4H, J = 9.07, 4.53 Hz), 3.88 (s, 6H),
5.27(d, 1H, J =
3.78 Hz), 5.36 (d, 1H, J = 10.20 Hz), 6.82 (s, 1H), 6.95 (s, 1H), 7.05 (s,
1H), 7.10 (d,
1H, J=3.40 Hz), APCI-MS m/z 552.6 (M+H)+.
Example B30: N (2,4,6-trimethoxyphenyl)-5-[(1,1,5-trimethyl-1H inden-6-
yl)oxy]-2-furamide
CH3
O
HaC Hs -
NH \ / oCH3
CH3 O
to B30 ~"3
Compound B30 was synthesized in a manner analogous to that of B l,
according to Scheme B, using similar starting compounds and reaction
conditions.
CsZCOs, DMF, 120 °C ~ O O O -E- ~ O O-
w OH \ 1i I/ O_ I~ I/ O
1i O O ,
Br ~ / O'
Note: the reaction content were not degassed
before heating
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDC13): b 8.89 (bs, 1H), 7.24 (s, 2H), 7.18 (bd, 1H, J = 3 Hz), 6.63 (d,
1H, J
= 6 IIz), 6.45 (d, 1H, J = 6 Hz), 6.26 (s, 2H), 5.47 (d, 1H, J = 3 Hz), 3.79
(s, 3H),
3.71 (s, 6H), 2.24 (s, 3H), 1.23 (s, 6H).
Example B31: N (2,4,6-trimethoxyphenyl)-5-[(3,3,6-trimethyl-2,3-dihydro-1H-
inden-5-yl)oxy]-2-furamide
CH3
O 0
H3/~ ~ O ~0~ NH \ ~ oCH3
CH3 0
2o B31 cH3
Compound B31 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The requisite phenol was synthesized according to the following method:
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O O OH
OH ~OH ~ OH I ~ Hz, Pd/C ~ OH
cat. H,SO,, _ I
polyphosphoric acid '\~ O
60 110 °C major
61 6z 63
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm,
CDC13): 8 7.17 (bs, 1H), 7.10 (d, 1H, J = 3 Hz), 7.05 (s, 1H), 6.84 (s, 1H),
6.17 (s,
2H), 5.29 (d, 1H, J = 3.6 Hz), 3.81 (s, 9H), 2.84 (t, 2H, J = 7.2, 14.4 Hz),
2.25 (s, 3H),
s 1.93 (t, 2H, J = 7.2, 14.4 Hz), 1.22 (s, 6H).
Example B32: N (2,6-dimethoxyphenyl)-5-[(1,1,3,3,6-pentamethyl-2,3-dihydro-
1H-inden-5-yl)oxy]-2-furamide
H3
O O /
H3C CHa
/ O O ~ NH \
O
CHa ~CHg
B32 Hay cHa
Compound B32 was synthesized in a manner analogous to that of B 1,
1o according to Scheme B, using similar starting compounds arid reaction
conditions.
OH HCI (conc) OI ~ ~ OH AICI3 ~ ~ OH
OH 30 min OI ~ CH3N02 '
min
119
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDC13): 8 7.33 (bs, 1H), 7.19 (t, 1H, J = 8.31, 8.35 Hz), 7.13 (d, 1H, J
= 3.6
1s Hz), 6.96 (s, 1H), 6.81 (s, 1H), 6.62 (s, 1H), 6.59 (s, 1H), 5.34 (d, 1H, J
= 3.4 Hz),
3.84 (s, 6H), 2.28 (s, 3H), 1.92 (s, 2H), 1.30 (s, 6H), 1.26 (s, 6H).
Example B33: N (2,6-dimethoxyphenyl)-5-[(1,3,5,5,8,8-hexamethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)oxy]-2-furamide
~ Ha
O
H3C CH3 CH3 O
O ~O~ NH
~CH3 O
B33 H'C CH3 CH
3
2o Compound B33 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The requisite phenol was prepared according to the following method:
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~OH A1CI3, CH3NOZ ~'~OH
[~ i~' ~ fl 'T~/
CI CSI
114
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm,
CDC13): ~ 7.35 (s, 1H), 7.20 (t, 1H, J = 9 Hz), 7.06 (d, 2H, J = 3 Hz), 6.63
(d, 2H, J =
9 Hz), 4.98 (d, 1H, J = 3 Hz), 3.86 (s, 6H), 2.36 (s, 3H), 2.17 (s, 3H), 1.66
(m, 4H),
1.40 (s, 6H), 1.28 (s, 6H).
Example B34: N-(4,6-dimethoxy-2-{[3-(4-methylpiperazin-1-
yl)propyl]amino~pyrimidin-5-yl)-5-[(3,8,8-trimethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)oxy]-2-furamide
I~ /~ N
O O ~N
N N~
N~,
1o B34
Compound B34 was synthesized in a manner analogous to that of compound
B 1, according to scheme B, using similar starting compounds and reaction
conditions.
Example 835: 5-(2-bromo-5-tert-butylphenoxy)-N-(2,4,6-trimethoxy-5-
pyrimidinyl)-2-furamide
CH3
O
H3C CH3 O N CH3
Ii ~ ~ O ~ O ~ NH ~ ~>-O
-N
Br O
1s B35 .~_ cH3
Compound B35 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
OH Br2/CCI4 / I OH
0 °C to rt ~ Br
120
NMR data consistent with the desired title product were as follows: 1H NMR
20 (ppm, CDCl3): 8 7.54 (d, 1H, J = 6 Hz), 7.19-7.15 (m, 3H), 7.02 (bs, 1H),
5.46 (d,
1H, J = 3.6 Hz), 3.97 (s, 9H), 1.29 (s, 9H).
Example 836: N-(2,4,6-trimethoxy-5-pyrimidinyl)-5-[(3,3,6-trimethyl-2,3-
dihydro-1H-inden-5-yl)oxy]-2-furamide
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CH3
0 0
CHg
H3C ~ \ 0 ~ O 1 NH ~ ~~--O
/ -N CH3
CH3 O
B36 ~H3
Compound B36 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDC13): ~ 7.13 (d, 1H, J = 3.6 Hz), 7.06 (s, 1H), 7.0 (bs, 1H), 6.83 (s,
1H),
5.30 (d, 1H, J = 3.6 Hz), 3.97 (s, 9H), 2.85 (t, 2H, J =14.54 Hz), 2.24 (s,
3H), 1.93
(t, 2H, J = 7.18 Hz), 1.22 (s, 6H).
Example B37: 5-[(1,3,5,5,8,8-hexamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)oxy]-
N (2,4,6-trimethoxyphenyl)-2-furamide
H3
H3C CH3 H3 0 ~ I O\CH3
\ O 0 NH~
~CH3 CH3
B37 H3C CH3
Compound B37 was synthesized in a manner analogous to that of Bl,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDCl3): ~ 7.19 (s, 1H), 7.05 (s, 1H), 7.04 (d, 1H, J = 3.6 Hz), 6.19 (s,
2H),
is 4.96 (d, 1H, J = 3.6 Hz), 3.83 (s, 6H), 3.82 (s, 3H), 2.35 (s, 3H), 2.16
(s, 3H), 1.40
(s, 6H), 1.28 (s, 6H).
Example B38: 5-(2,4-dibromo-5-tent butylphenoxy)-N (2,4,6-
trimethoxypyrimidin-5-yl)-2-furamide
CH3
H C CH3 O O N
HsC ~ \ O ~ O ~ NH ~ ~ O
N~ CH3
Br ~ Br O
B38 ~"3
2o Compound B38 was synthesized in a manner analogous to that of Bl,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDC13): ~ 7.85 (s, 1H), 7.23 (s, 1H), 7.16 (d, 1H, J = 3.6 Hz), 7.01
(bs, 1H),
5.51 (d, 1H, J = 3.6 Hz), 3.97 (s, 9H), 1.46 (s, 9H).
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Example B39: 5-[(1,3,5,5,8,8-hexamethyl-5,6,7,8-tetrahydronaphthalen-1-yl)oxy]-
N (2,4,6-trimethoxypyrimidin-5-yl)-2-furamide
CH3
O
H3C CH3CH3 O N CH
\ O I O I NH ~ ~~-p s
-N
CH3 O
B39 "30 CH3 CH3
Compound B39 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDC13): 8 7.07 (d, 1H, J = 3.8 Hz), 7.03 (s, 1H), 4.98 (d, 1H, J = 3.6
Hz),
3.99 (s, 6H), 3.98 (s, 3H), 2.35 (s, 3H), 2.15 (s, 3H), 1.66 (m, 4H, J =1.32
Hz),
1.40 (s, 6H), 1.28 (s, 6H).
1o Example B40: N-(2-{[3-(dimethylamino)propyl]amino}-4,6-
dimethoxypyrimidin-5-yl)-5-[(3,8,8-trimethyl-5,6,7,8-tetrahydronaphthalen-
2-yl)oxy]-2-furamide
Ij /~ N .
0 0
o ~J~.
N NON
B40
is Compound B40 was synthesized in a manner analogous to that of
compound B 1, according to scheme B, using similar starting compounds and
reaction conditions.
Example 841: 5-(2-bromo-5-tent-butylphenoxy)-N (2,4,6-trimethoxyphenyl)-2-
furamide
OH3
H C ~H3 O
HsC ~ \ O ~ O ~ NH ~ ~ O
CH3
Br O
2o B41 ~"3
Compound B41 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDC13): 8 7.54 (d, 1H, J = 8.5 Hz), 7.18 (d, 1H, J = 3.6 Hz), 7.14-7.12
(m,
25 2H, J = 2.27, 4.72 Hz), 6.17 (s, 2H), 5.47 (d, 1H, J = 3.6 Hz), 3.81 (s,
9H), 1:28 (s,
9H).
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Example 842: 5-[2-bromo-5-tent-butyl-4-(2,4-dibromo-5-tert-
butylphenoxy)phenoxy]-N (2,4,6-trimethoxypydrimidin-5-yl)-2-furamide
B42
Compound B42 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
I \ OH CsZC03, DMF, 120 °C 0 O 0 ~ 0 p O
_ -I- ~ ~ / _
I ~ ~ / O Br ~ Br O
Br Br 0 O Br
Br ~ O, Br \ I
Br
NMR data consistent with the desired title product were as follows:1H NMR
(ppm, DMSO - d6): 8 9.24 (bs, 1H), 8.00 (s, 1H), 7.37 (s, 1H), 7.24 (d, 1H, J
= 3.6
Hz), 7.06 (s, 2H), 7.00 (s, 1H), 5.64 (d, 1H, J = 3.6 Hz), 3.91 (s, 3H), 3.87
(s, 6H),
1.38 (s, 9H), 1.37 (s, 9H).
Example B43: 5-(2-bromo-5-tent-butylphenoxy)-N (4-ethoxypyridin-3-yl)-2-
furamide
/N
H3C CH9 r
O O /
H3C ~ ~ ~ NH~
/ O
/%~B~
B43 --,_ 1 H
Compound B43 was synthesized in a manner analogous to that of B 1,
1s according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDC13): ~ 9.57 (bs, 1H), 8.30 (s, 1H), 8.27 (d, 1H, J = 5.48 Hz), 7.55
(d,
1H, J = 8.31 Hz), 7.21 (m, 2H), 7.15 (t, 1H, J = 2.17 Hz), 6.80 (d, 1H, J =
5:67
Hz), 5.46 (m, 1H), 4.20 (d, 2H, J =13.98 Hz), 1.48 (dd, 3H, J =13.98, 6.99
Hz),
1.28 (s, 9H).
Example B44: 5-(2-bromo-5-tert-butylphenoxy)-N quinolin-3-yl-2-furamide
I
CH3 O
H3C
H I ~ O \ O' NH /N
B44
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Compound B44 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the desired title product were as follows:1H NMR
(ppm, CDC13): 8 8.86 (t, 1H, J = 7.18 Hz), 806 (d, 1H, J = 8.31 Hz), 7.83 (d,
1H, J
= 8.12 Hz), 7.65 (d, 1H, J = 8.31 Hz), 7.55 (ddd, 2H, J =12.84, 8.31, 7.18
Hz),
7.27 (d, 1H, J = 3.59 Hz), 7.22 (m, 1H), 7.18 (d, 1H, J = 10.58 Hz), 5.47 (d,
1H, J
= 3.6 Hz), 1.30 (s, 9H).
Example B45: N (2-chloro-4,6-dimethoxypyrimidin-5-yl)-5-[(3,3,6-trimethyl-2,3-
dihydro-IH inden-5-yl)oxy]-2-furamide
CH3
O O N"CI
H C CH3
O O NH~N
/ ,o
HC
CHI
1o B45
Compound B45 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the desired title product were as follows: 1H NMR
(ppm, CDC13): 8 7.15 (d, 1H, J = 3.59 Hz), 7.07 (s, H), 6.83 (s, 1H), 5.29 (d,
1H, J
is 3.59 Hz), 4.01 (s, 6H), 2.85 (d, 2H, J =10.01 Hz), 2.24 (s, 3H), 1.94 (t,
2H, J =
7.27 Hz), 1.22 (s, 6H).
Example 847: 5-(2-bromo-5-tert-bntylphenoxy)-N-(2,6-dimethoxypyridin-3-
yl)-2-furamide
CH3 O / O~CH3
H3C O~~ \ IN
H3C ~ '(~~/ NH
O
B47 / Br H3c'
2o Compound B47 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR data consistent with the title product were as follows: 1H NMR (ppm,
DMSO-d6): 8 9.26 (s, 1H) 7.81 (d, 1H, J = 8.31 Hz), 7.67 (d, 1H, J = 8.69 Hz),
7.34 (d, 1H, J = 1.89 Hz), 7.27 (d, 2H, J = 8.69 Hz), 6.38 (d, 1H, J = 8.31
Hz),
2s 5.64 (m, 1H), 3.90 (s, 3H), 3.85 (s, 3H), 1.25 (s, 9H).
Example 848: 5-(5-Chloro-1,1,7-trimethyl-indan-4-yloxy)-furan-2-carboxylic
acid [2-(3-dimethylamino-propylamino)-4,6-dimethoxy-pyrimidin-5-yl]-amide
acetic acid salt
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0
\ O 0 O ~N~N~N
N \ N \
ci
0
B48
Compound B48 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
Example 849: 5-[(3-isopropyl-1,1,4,6-tetramethyl-2,3-dihydro-1H-inden-5-
yl)oxy] N (2,4,6-trimethoxyphenyl)-2-furamide
B49
Compound B49 was synthesized in a manner analogous to that of B l,
according to Scheme B, using similar starting compounds and reaction
conditions.
~ OH Cs2C03, DMF, 100 °C ~ O O O O + w O O O O
~i ~ ~~ ~~ HN ~ ~ ~ ~~ ~~ HN \ / O
O O O ~ ~ O O'
114 Br ~ ~ H
- O'
NMR data consistent with the title product were as follows: 1H NMR (ppm,
CDC13): ~ 7.19 (s, 1H), 7.04 (d, 1H, J = 3.59 Hz), 6.81 (s, 1H), 6.19 (s, ZH),
4.92
(d, 1H, J = 3.59 Hz), 3.83 (s, 6H), 3.82 (s, 3H), 2.20 (s, 3H), 2.17 (s, 3H),
1.33(s,
3H), 1.15 (s, 3H), 0.96 (d, 3H, J = 6.80 Hz), 0.60 (d, 3H, J = 6.80 Hz).
Example 850: 5-[(3-isopropyl-1,1,4,6-tetramethyl-2,3-dihydro-1H-inden-5-
yl)oxy] N (2,4,6-trimethoxypyrimidin-5-yl)-2-furamide
B50
Compound B50 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
2o NMR data consistent with the title product were as follows: 1H NMR (ppm,
68
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CDCl3): 8 7.06 (d, 1H, J= 3.59 Hz), 7.03 (s, 1H), 6.80 (s, 1H), 3.98 (s, 6H),
3.97
(s, 3H), 4 ;92 (m, 1H), 2.90 (m, 2H, J =19.83, 11.90, 11.52, 4.72 Hz), 2.18
(s, 3H),
2.16 (s, 3H), 1.84 (dt, 1H, J =13.22, 8.88 Hz), 1.32 (s, 3H), 1.14 (s, 3H),
0.95 (m,
3H), 0.60 (d, 3H, J = 6.80 Hz).
Example 851: 5-(4-chloro-5-isopropyl-2-methylphenoxy)-N (2,4,6-
trimethoxypyrimidin-5-yl)-2-furamide
CH3
H3 O
-N
H3C ~ ~ \O/ NH
/ ~N CH3
CI CH3 0
B51 ~"3
Compound B51 was synthesized in a manner analogous to that of Bl,
according to Scheme B, using similar starting compounds and reaction
conditions.
1o NMR data consistent with the title product were as follows: 1H NMR (ppm,
CDC13): 8 7.24 (s, 1H), 7.14 (d, 1H, J = 3.40 Hz), 6.98 (s, 2H), 5.35 (d, 1H,
J =
3.40 Hz), 3.97 (s, 9H), 3.33 (d, 1H, J = 6.80 Hz), 2.24 (s, 3H), 1.20 (s, 3H),
1.18
(s, 3H).
Example B52: N {4,6-dimethoxy-2-[(3-morpholin-4-ylpropyl)amino]pyrimidin-5-
~s yl}-5-[3,3,6-trimthyl-2,3-dihydro-IH inden-5-yl)oxy]-2-furamide
CH3
CH3
HaC I w C ~~ NH~N~--NH'W/~ ~O
CH3 p
B52 ~H3
Compound B52 was synthesized according to a method analogous to Scheme
B, using similar starting compounds and reaction conditions as shown and
described
below. NMR data consistent with the title product were as follows: 1H NMR
(ppm,
2o CDC13): ~ 7.09 (d, 1H, J = 3.40 Hz), 7.05 (s, 1H), 6.95 (s, 1H), 6.82 (s,
1H), 5.72 (bs,
1H), 5.29 (d, 1H, J = 3.40 Hz), 3.87 (s, 6H), 3.75 (dd, 4H, J = 9.07, 4.53
Hz), 3.47 (m,
2H), 2.84 (d, 2H, J = 7.18 Hz), 2.51 (d, 6H, J = 6.42 Hz), 2.23 (s, 3H), 1.93
(d, 2H, J =
7.18 Hz), 1.78 (t, 2H, J = 6.42 Hz), 1.21 (s, 6H).
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0 0
OH I \ Og I
\ i ~
off OH
i i
~--~
polyphosphori c acid
los 61 O
C 62
major
60
IHZ,
Pd/C
~'I~cst.
H2SOq
O CsiC03, ~
p DMF OH
O Br~ p p ~
i i
~
~
O- ~ ~ 63
65 O .
- _64
NaOH,
MeOH,
tt.
O DMF, HATU.
O r.t ~
O ~ O O
OH O O ~O
I ~ I /
HN \ N~NMN
~
~O N
H
66 -
HZN 68
N (CompoundB52)
O N
wp
~
~
67
SOC12,DCM,
refluxed 120 C, 20
min. SmithSynthesize5
CsF, CH3CN,
' [N~NHz
O
_7z
~O /
O _ ~
O HZN J~.~ w
O ~ O
~~ O
O
O
~
~
~
N
,,, HN
CI \
O ~-CI
~
CI N
EtOAc,
69 DIEA,
r.2. -O
or 120
~C, 10
min. SmithSynthesizer
5-hydrogy-3,3,6-trimethyl-1-indanone 62: To a three-necked round bottom
flask assembled with a condenser, thermometer and mechanic stirrer under
nitrogen,
s o-cresol (1197 mmol, 124 ml) and 3,3-dimethylacrylic acid (1520 mmol, 154 g)
were
added. The mixture was gently stirred and heated at 40 °C while adding
polyphosphoric acid (3.9 L). After the addition of polyphosphoric acid was
completed, the contents were rapidly heated to 105 °C, and the heating
mantel was
removed. The reaction mixture was monitored by TLC (1:3 ethyl acetate: hexane)
1o showing no starting materials. The reaction was quenched by pouring the hot
mixture
into a large bucket of ice water with constant stirring. The aqueous layer was
extracted with ethyl acetate. The organic phase was concentrated, and the
crude
product was crystallized with ethyl acetate to obtain pure Compound 62 (21 g,
9.5 %).
NMR data consistent with the title product were as follows: II~TMR (300 MHz,
15 CDC13): 8 7.50 (s, 1H), 7.83 (s, 1H), 6.06 (bs, 1H), 2.55 (s, 2H), 2.27 (s,
3H), 1.36 (s,
6H).
3,3,6-trimethyl-5-indanol 63:A solution of 5-hydroxy-3,3,6-trimethyl-1-
indanone (21.5 mmol, 4.1 g) and sulfuric acid (290 ~.~.1) in methanol (150 ml)
were
7o
CA 02449843 2003-12-05
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degassed with nitrogen for at least twenty minutes following by the addition
of
catalyst palladium on carbon (4.3 mmol, 0.63 g). The ketone was reduced under
40
psi of H2 overnight. The contents were filtered over celite. Methanol was
removed in
vacuum to give brown oil residue that was redissolved in ethyl acetate and
washed
with water until neutral and brine. The organic layer was dried over sodium
sulfate
and brought to dryness given light yellow oil. The crude product was purified
by plug
column chromatography (1:3 ethyl acetate: hexane) to give light yellow solid
Compound 63 (3.6 g, 93%). NMR data consistent with the title product were as
follows: iHNNIR (300 MHz, CDCl3): 8 6.94 (s, 1H), 6.57 (s, 1H), 4.58 (s, 1H),
2.78
(t, 2H), 2.21 (s, 3H), 1.89 (t, 2H), 1.21 (s, 6H).
Methyl 5-[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furoate 65:
In a one-necked round-bottom flask assembled with a condenser and gas outlet,
a
solution of 63 (22.73 mmol, 4.0 g), 64 (17.6 mmol, 3.6 g) and cesium carbonate
( 22.8
mmol, 7.4 g) in DMF ( 45 mL) was degassed with nitrogen gas for 20 minutes
then
1s heated to 100 °C for 7 hours under N2. The mixture was cooled down
to room
temperature and quenched with 1M HCI. The content was extracted with ethyl
acetate, water and brine. The organic phase was dried over sodium sulfate. The
crude
prbduct was purified by plug column chromatography (1:5 ethyl acetate: hexane)
to
obtain yellow oil Compound 65 (5.8 g, 84%). Note: The material from the
baseline of
2o the column was 66. NMR and mass spectrometry data consistent with the title
product were as follows: 1HNMR (300 MHz, CDCl3): S 1.21 (s, 6H), 1.89 (t, 2H),
2.20 (s, 3IT), 2.77 (t, 2H), 3.86 (s, 3H), 5.22 (d, 2H), 6.56 (s, 1H), 6.93
(s, 1H), 7.12
(d, 1H), APCI-MS mlz 302.2 (M+H)+.
5[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furoic acid 66: To a
25 solution of 65 (19.3 mmol, 5.8 g) in methanol (10 ml) in a one-necked round
bottom
flask assembled with a stir bar, 4M NaOH aqueous solution (20 ml) was added.
The
contents were stirred at room temperature overnight. The clear brown solution
was
acidified with 2M HCl and stirred for 3 hours. The aqueous layer was extracted
with
ethyl acetate and brine then dried over sodium sulfate. The crude product was
3o crystallized with CH3CN to give pure light yellow solid Compound 66 (3.18
g, 57 %).
NMR data consistent with the title product were as follows: 1HNMR (CDCl3,
ppm): 8
7.26 (d, 1H), 7.05 (s, 1H), 6.85 (s, 1H), 2.84 (t, 2H), 2.20 (s, 3H), 1.93 (t,
2H), 1.21 (s,
6H).
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N-~4,6-dimethogy-2-[(3-morpholin-4-ylpropyl)amino] pyrimidin-5-yl}-5-
[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)ogy]-2-furamide 68 (Compound
B52): Procedure 1: To a solution of 66 (9.74 mmol, 2.8 g), 67 (8.0 mmol, 2.4g)
and
HATU (10.2 mmol, 3.4 g) in DMF (18 ml) prepared in a one-necked flask
assembled
with a gas outlet and a stir bar, diisopropylethylamine (33.5 mmol, 6 ml) was
added
slowly via a syringe under N2. The reaction mixture was stirred under N2
overnight.
The solvent was removed under vacuum pressure then purified by HPLC (32-95 70
min. - CH3CN: O.1M NH4oAC) without further work-up to give white solid product
68 (2.84 g, 52%). Compound 6~ was dissolved in methylene chloride and then
1o dried in vacuum, yielding an amorphous solid. NMR data consistent with the
title
product were as follows: 1HNMR (300 MHz, CDC13): 8 17.10 (d, 1H), 7.05 (s,
1H),
6.95 (s, 1H), 6.82 (s, 1H), 5.72 (bs, 1H), 5.29 (d, 1H), 3.87 (s, 6H), 3.75
(t, 4H), 3.48
(bt, 2H), 2.84 (t, 2H), 2.49 (m, 6H), 2.23 (s, 3H), 1.93 (t, 2H), 1.81 (t,
4H), 1.21 (s,
6H).
Procedure 2: Compound 68 was also be synthesized from 71. In a 5 ml
microwave tube assembled with a stir bar, N (2-chloro-4,6-dimethoxypyrimidin-5-
yl)-
5-[(3,3,6-trimethyl-2,3-dihydro-1H inden-5-yl)oxy]-2-furamide 71( 0.35 g, 0.76
mmol), cesium fluoride (0.29 g, 1.9 mmol), acetonitrile (3.8 ml) and amine
(0.14 g,
0.91 mmol) were added. The mixture was heated to 120 °C for 20 minutes
by Smith
2o Synthesizer. Without work-up the crude product was purified by HPLC.
5-[(3,3,6-trimethyl-2,3-dihydro-1H inden-5-yl)oxy]-2-furoyl chloride 69:
In a one-necked round bottom flask assembled with a stir bar, condenser and
gas inlet was added 5[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furoic
acid
66 ( 18.6 mmol), thionyl chloride (6 ml), and methylene chloride (100 ml). The
2s mixture was refluxed overnight. After cooling down to room temperature, the
contents were washed with water and brine to quench the excess thionyl
chloride.
Organic solution was dried over Na2S04. Reddish-brown oil product was passed
over a short silica gel plug with 1:1 (ethyl acetate: hexane). NMR data
consistent
with the title product were as follows: 1HNMR (300 MHz, CDC13): b 1.22 (s,
6H),
30 1.91-197 (m, 2H), 2.19 (s, 3H), 2.85 (t, 2H), 5.31 (d, 1H), 6.86 (s, 1H),
7.07 (s, 1H),
7.45 (d, 1H).
N (2-chloro-4,6-dimethogypyrimidin-5-yl)-5-[(3,3,6-trimethyl-2,3-
dihydro-1H inden-5-yl)oxy]-2-furamide 71: 5-[(3,3,6-trimethyl-2,3-dihydro-
lII inden-5-yl)oxy]-2-furoyl chloride 69 (2.66 g, 8.71 mmol) and 2-chloro-4,6-
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WO 02/098363 PCT/US02/17846
dimethoxypyrimidin-5-amine (1.65 g, 8.71 mmol) were added into a 25-ml round-
bottam flask containing a stir bar and ethyl acetate (17.5 ml) following by
slow
addition of diisopropylethyl amine (3.2 ml). The mixture was stirred at room
temperature overnight. An alternate route was to heat the mixture to 120
degree
Celsius for 10 minutes by Smith Synthesizer microwave. Classical work up was
carried out. Crude product was purified by flask chromatography (silica gel
1:5 ethyl
acetate: hexane). Light brown product 71 was obtained (0.5 g, 13%). NMR data
consistent with the title product were as follows: 1HNMR (300 MHz, CDC13): 8
1.22
(s, 6H), 1.94 (t, 2H), 2.24 (s, 3H), 2.85 (t, 2H), 4.01 (s, 6H), 5.30 (d, 1H),
6.83 (s, 1H),
7.08 (d, 2H), 7.15 (d, 1H).
Compound 67 was synthesized according to the nitration scheme below:
w
W N CF3S020501,CFa _ OpN ~ DMP, r.t 02N W N
/~ (CH3)4~°a,DCM N
~O N"OI ~ N_"CI OJN~NHZ \O N HEN
~O
73 74 75
HZ, Pd/C. MeOH H2N
~O N H~N
67
2-chloro-4,6-dimethogy-5-nitropyrimidine 74: In a 12-L flask assembled
with an overhead stirrer, thermometer, N2 inlet and addition funnel,
tetramethylammonium nitrate (587 g, 4.31 mol) and dichloromethane (4 L) were
added. The contents were stirred under N2 for 1 hour at room temperature
(20°C).
Triflic anhydride (1.216 g, 4.31 mol, 725 ml) was added dropwise over a period
of 45
minutes so that the temperature remained below 25°C. The addition
funnel was rinsed
with 100 ml of dichloromethane, and the dichloromethane was added to the
reaction.
2o The contents were stirred at room temperature under N2 for 2 hours. The
reaction
mixture was then cooled to -78°C in a dry ice/acetone bath. The 2-
chloro-4,6-
dimethoxypyrimidine 73 (500 g, 2.87 mol) was dissolved in minimal amount of
dichloromethane (3L). The solution of 73 was added dropwise aver a period of
1.5
hours. The addition rate was important to ensure the temperature did not rise
above
5°C. After the addition of solution 73, the addition funnel was rinsed
with 100 ml of
dichloromethane and the rinse was added to the reaction. The acetone/dry bath
was
removed and the reaction was stirred for 38 hours under N2 as it warmed to
room
temperature. The reaction was monitored by TLC (3:1 CHCL3: Hexanes) and
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WO 02/098363 PCT/US02/17846
quenched by pouring reaction mixture into N2 kg of ice. The contents were
neutralized with NaHC03 aqueous solution (pH = 8) and the dichloromethane
layer
was separated. The aqueous layer was extracted with 3x100 ml of
dichloromethane.
The dichloromethane portions were combined and washed with 2xlL of H20. The
combined dichloromethane portion was dried over MgS04, and brought to dryness.
Compound 74 was a white solid (615 g, 98 %).
4,6-dimethoxy-N-(3-morpholin-4-ylpropyl)-5-nitropyrimidine-2-amine 75:
A mixture of 2-chloro-4,6-dimethoxy-5-nitropyrimidine 74 (92.32 mmol, 20.2 g)
in
DMF -(23 ml) was cooled down to 0°C. 4-(3-Aminopropyl) morpholine
72 (92.4
to mmol, 13.5 ml) was added drop by drop via a syringe into the mixture 74.
The
contents were warmed up to room temperature and stirred overnight under N2.
The
organic solvents were removed by high vacuum, and the crude product was
purified
by flash column chromatography (2:5 methanol: ethyl acetate) without aqueous
work
up. Compound 75 had bright yellow solid (18 g, 60 %).
1s N-(5-amino-4,6-dimethoxy-2-pyrimidinyl)-N-(3-(~-
morpholinyl)propyl]amine 67: A solution of 4,6-dimethoxy-N-(3-morpholin-4-
ylpropyl)-5-nitropyrimidine-2-amine 75 (55 mmol, 18 g) in methanol (500 ml)
was
ddgassed for 15 minutes followed by the addition of catalyst Pd/C (5.5 mmol,
0.8 g).
The nitro-group was reduced under 30 psi of H2 overnight. The contents were
filtered
20 over Celite, and the organic solvent was removed in vacuum. The product was
dried
under high vacuum to give brown solid Compound 67 (15 g, 90 %).
Exapample B53: 5-[(5-chloro-1,1,7-trimethyl-2,3-dihydro-1H-inden-4-yl)oxy]-N-
(4,6-dimethoxy-2{[3-(4-methyl-1-piperazinyl)propyl]amino}-5-pyrimidinyl)-2-
furamide Acetic acid Salt
i
0 0
0 0 ~
I N \ ~N~
CI
B53
Compound B53 was synthesized in a manner analogous to that of B1,
according to Scheme B, using similar starting compounds and reaction
conditions.
Example B54: 5-[(4-bromo-3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-N
(2,4,6-trimethoxyphenyl)-2-furamide
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WO 02/098363 PCT/US02/17846
CH3
CH3 Br O O
HaC O O CHs
I I NH ~ ~ O
~CH
BS4 3 H3C-O
Compound B54 was synthesized in a mamier analogous to that of B1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The requisite phenol was prepared according to the following method:
I \ OH Br2, CCI4, 0 °C ~OH
,~ \
I
63 150
NMR data consistent with the title product were as follows: 1H NMR (ppm,
CDC13): 8
7.20 (bs, 1H), 7.05 (d, 1H, J = 3.40 Hz), 7.00 (s, 1H), 6.18 (s, 2H), 5.04 (d,
1H, J =
3.78 Hz), 3.83 (s, 6H), 3.82 (s, 3H), 2.85 (t, w2H, J = 7.55 Hz), 2.23 (s,
3H), 1.98 (dd,
2H, J = 14.73, 7.55 Hz), 1.42 (s, 6H).
1o Example B55: 5-[(4-bromo-3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy] N
(4,6-dimethoxy-2- f [3-(4-methylpiperazin-1-yl)propyl]amino~pyrimidin-5-yl)-2-
furamide
CH3
CH3 Br O O _N ~N-CH3
HsC I i O 101 NH I-'N NH,/~N
CH3 O
B55 CH3
Compound B55 was synthesized in a manner analogous to that of B1,
1s according to Scheme B, using similar~starting compounds and reaction
conditions.
NMR data consistent with the title product were as follows: 1H NMR (ppm,
CDC13): 8 7.04 (d, 1H, J = 3.78 Hz), 7.00 (s, 1H), 6.97 (s, 1H), 5.70 (bs, 1H)
5.03
(d, 1H, J = 3.78 Hz), 3.89 (s, 6H), 3.46 (d, 2H, J = 5.29 Hz), 2.85 (dd, 2H, J
=
14.73, 7.18 Hz), 2.56 (dd, 8H, J = 7.55, 6.80 Hz), 2.35 (s, 3H), 2.22 (s, 3H),
1.98
20 (t, 2H, J = 7.55 Hz), 1.79 (d, 2H, J = 6.42 Hz), 1.42 (s, 6H).
Example B56: 5-[(4-bromo-3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-N
{4,6-dimethoxy-2-[(3-morpholin-4-ylpropyl)amino]pyrimidin-5-yl}-2-furamide
OH3 er ; H3
Hs0 O O O O
NH ~ ~NH
CH3 N/ ~ O
N'
B56 H3~- V
Compound B56 was synthesized in a manner analogous to that of B 1,
2s according to Scheme B, using similar starting compounds and reaction
conditions.
CA 02449843 2003-12-05
WO 02/098363 PCT/US02/17846
NMR data consistent with the title product were as follows: 1H NMR (ppm,
CDC13): 8 7.13 (s, 1H), 7.08 (d, 1H, J = 3.40 Hz), 7.01 (s, 1H), 5.78 (bs,
1H), 5.05
(d, 1H, J = 3.78 Hz), 3.90 (s, 10H), 3.53 (s, 2H), 3.06 (s, 6H) 2.84 (t, 2H, J
= 7.18
Hz), 2.22 (s, 3H), 1.95 (d, 4H, J = 7.55 Hz), 1.41 (s, 6IT).
Example B57: 5-(2-bromo-5-tent-butylphenoxy)-N (4,6-dimethoxy-2-{[3-(4-
methylpiperazin-1-yl)propyl]amino}pyrimidin-5-yl)-2-furamide
CH3
CH3
HsC C p 0
_N
H3C I / I ~NH
N ~~H ~~--------~~
Br ~ N ~N-CH3
'~~JJ/N
B57 H3c--o
Compound B57 was synthesized in a manner analogous to that of B1,
according to Scheme B, using similar starting compounds and reaction
conditions.
to The requisite phenol was synthesized according to the following method:
Br o'
I ~ OH Br _ I ~ OH NaOCHz/MeOH I ~ OH
z
/ Acetic Acid / CuBr, EtOAc, D /
102 92 k Yleld 89.57 h Yleld
105 121
1-Bromo-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenol: In a
500 mL round bottom flask, 3,5,5,8,8-pentamethyl-5,6;7,8-tetrahydro-2-
naphthalenol
(10.2 g, 46.72 mmol) was dissolved in 100mL acetic acid. To this solution
Bromine
15 (8.2 g, 51.39 mmol) was added. The reaction was stirred at room temperature
for 20
minutes. The reaction mixture was poured into water and extracted with ethyl
acetate.
The separated organic layer was washed with brine, dried over magnesium
sulfate and
concentrated. The crude product was purified by silica gel chromatography
eluted
with hexane to yield 1-Bromo-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
2o naphthalenol (12.8 g, 92% yield). NMR data consistent with the title
product were as
follows: 1H NMR (CDC13): 8 1.25 (s, 6H), 1.52 (s, 6H), 1.58-1.63 (m, 2I~, 1.68-
1.71
(m, 2H), 2.25 (s, 3H), 5.99 (s, 1H), 7.05 (s, 1H).
1-Methoxy-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenol: In
1000 mL round-bottom flask, 1-bromo-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
2s naphthalenol (12.8 g, 43.06 mmol) and Sodium methoxide in methanol (5.0
IVIJ were
combined. To this solution CuBr (1.24 g, 8.61mmo1) was added followed by ethyl
acetate (2.5 mL). The reaction was stirred and heated to reflux for 16 hours.
The
reaction mixture was cooled to room temperature then poured into water and
extracted
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with ethyl acetate. The separated organic layer was washed with brine, dried
over
magnesium sulfate and concentrated to yield 1-Methoxy-3,5,5,8,8-pentamethyl-
5,6,7,8-tetrahydro-2-naphthalenol (9.58 g, 90% yield). NMR data consistent
with the
title product were as follows: IIiNMR (CDC13): 8 1.25 (s, 6H), 1.39 (s, 6H),
1.52-1.61
(m, 4H), 2.21 (s, 3H), 3.81 (s, 3H), 5.08 (s, 1H), 7.01 (s, 1H).
5-[(1-Methoxy-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2
naphthalenyl)oxy]-2-furoic acid: 1-Methoxy-3,5,5,8,8-pentamethyl-5,6,7,8
tetrahydro-2-naphthalenol (2.0 g, 8.05 mmol), methyl 5-bromo-2-furoate (1.65
g, 8.05
mmol), and Cs2C03 were dissolved in DMF (20 mL). The solution was placed under
1o nitrogen, stirred and heated to 70°C overnight. The cooled reaction
mixture was
poured into water, acidify with 6N HCl (100 mL) and extracted with ethyl
acetate.
The separated organic layer was washed with brine, dried over magnesium
sulfate and
concentrated. The crude product was purified by silica gel chromatography
eluted
with hexane/ethyl acetate (2:1 v/v) to yield 5-[(1-methoxy-3,5,5,8,8-
pentamethyl-
1s 5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-2-furoic acid (1.1 g, 37 % yield).
NMR and
mass spectrometry data consistent with the title product were as follows: 1H
NMR
(CDC13): 8 1.26 (s, 6H), 1.35 (s, 6H), 1.60 (m, 4H), 2.17 (s, 3H), 3.84 (s,
3H), 5.09 (d,
ll~, 6.92 (s, 1H), 7.24 (d, 1H), APCI-MS m/z 373.1 (M+H)+. NMR data consistent
with the title product were as follows: 1(ppm, CDCl3): S 7.53 (d, 1H, J = 8.31
2o Hz), 7.17 (d, 1H, J =1.89 Hz), 7.13 (d, 2H, J = 5.29 Hz), 6.97 (s, 1H),
5.71 (bs, 1H),
5.44 (d, 1H, J = 3.40 Hz), 3.87 (s, 6H), 2.57 (dd, 8H, J = 13.60, 6.80 Hz),
2.36 (s, 3H),
2.10 (d, 4H, J = 18.89 Hz), 1.78 (d, 2H, J =13.22 Hz), 1.28 (s, 9H).
Example B58: 5-[(1-Methoxy-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
I
o' o o , o~
I
I w o \o/ H
i ~ o~
25 B58
Compound B58 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H NMR (DMSO-d6): 8 1.29 (s, 6H), 1.39 (s, 6H), 1.68 (m, 4H), 2.22
(s,
30 3H), 3.82 (s, 6H), 3.84 (s, 3H), 3.88 (s, 3H), 5.10 (d, 1H), 6.29 (s, 2H),
7.05 (s,
1H), 7.11 (d, 1H), APCI-MS m/z 524 (M+H)+
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1e 859: N-(2,6-dimethoxyphenyl)-5-[(1-methoxy-3,5,5,8,8-pentamethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)oxy]-2-furamide
o~ o o _
\/
.. _ o
859 '
Compound B59 was synthesized in a manner analogous to that of B1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (acetonitrile-d3): 8 1.29 (s, 6H), 1.38 (s, 6H), 1.67 (s. 4H), 2.25 (s,
3H), 3.82 (s,
6H), 3.87 (s, 3H), 5.15 (d, 1H), 6.71 (s, 1H), 6.73 (s, 1H), 7.03 (d, 1H),
7.09 (s, 1H),
7.29 (t, 1H) 7.62 (s, 1H), APCI-MS m/z 494 (M+H)+.
to Example B60: 5-[(1-methoxy-3,5,5,8,8-pentamethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)oxy]-N-(2,4,6-trimethoxypyrimidin-5-yl)-2-furamide
I I
o~ o o~Yo
O O N-~wYN
B60
Compound B60 was synthesized in a manner analogous to that of B l,
according to Scheme B, using similar starting compounds and reaction
conditions.
~s NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (MeOD): 8 1.20 (s, 6H), 1.29 (s, 6H), 1.58 (d, 4H, J = 2.27 Hz), 2.11 (s,
3H),
3.78 (s, 3H), 3.89 (s, 6H), 3.91 (s, 3I~; 5.01 (d, 1H, J = 3.78 Hz), 6.95 (s,
1H), 7.03
(d, 1H, J = 3.40 Hz), API-MS m/z 526 (M+H)+.
Example B61: 5-[5-(tert-butyl)-2-methylphenoxy]-N-(4,6-diemthoxy-2- f [3-(4-
20 methyl-1-piperazinyl)propyl]amino]-5-pyrimidinyl)-2-furamide
N~
O O N~N~
O O N ~ N
B61
Compound B61 was synthesized in a manner analogous to that of B 1,
according to Scheme B (HBTIJ coupling), using similar starting compounds and
reaction conditions. NMR and mass spectrometry data consistent with the title
2s product were as follows: 1H NMR (CDC13): 8 1.30 (s, 9H), 1.80 (m, 6H), 2.26
(s, 3H),
2.50 (s, 3H), 2.60 (br m, 6H), 3.64 (t, 2H), 3.88 (s, 61H), 5.35 (d, 1H), 5.82
(t, 1H),
6.98 (s, 1H), 7.10 (s and d, 2H), 7.17 (m, 2H), APCI-MS nZ/z 567.2 (M+I~+'
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Example B62: 5-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-
N-(2,4,6-trimethoxy-5-pyrimidinyl)-2-furamide
I
O O NYO\
O O ~N
H
w \
B62 ~
Compound B62 was synthesized in a manner analogous to that of B 1,
according to Scheme B (HBTIJ coupling), using similar starting compounds and
reaction conditions. NMR and mass spectrometry data consistent with the title
product were as follows:1H NMR (d4-CH30H): 81.23 (s, 6H), 1.27 (s, 6H), 1.69
(s,
4H), 2.22 (s, 3H), 3.95 (s, 6H), 3.98 (s, 3H), 5.34 (d, 1H), 7.02 (s, 1H),
7.14 (d, 1H),
7.23 (s, 1H), APCI-MS m/z 496.5 (M+H)+.
1o Example B63: 5-(5-tert-butyl-2-methylphenoxy)-N-(2,4,6-trimethoxy-5-
pyrimidinyl)-2-furamide
0
0
O \O/ H \ N
B63
Compound B63 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H NMR
(CDCl3):
1s 8 1.29 (s, 9H), 2.24 (s, 3H), 3.95 (s, 6H), 3.98 (s, 3H), 5.36 (d, 1H),
7.12 (s, 1H), 7.15
(d, 1H), 7.22 (m, 2H), APCI-MS m/z 442.1 (M+H)+.
Example B64: N (2-chloro-4,6-dimethoxypyrimidin-5-yl)-5-[(3,3,6-trimethyl-1,3-
dihydro-2-benzofuran-5-yl)oxy]-2-furamide
0 j YG
0 0 N
0 ~ / ~ ~ 0
B64
20 Compound B64 was synthesized according to Scheme B. NMR data
consistent with the title product were as follows: 1H NMR (CDC13): 8 1.46 (s,
6H),
2.29 (s, 3H), 4.01 (s, 6H), 5.02 (s, 2H), 5.37 (d, 1H, J = 3.40 Hz), 6.81 (s,
1H), 7.08 (s,
1H), 7.16 (d, 1H, J = 3.59 Hz).
Example B65: N-(2,4,6-trimethoxy-5-pyrimidinyl)-5-[(3,8,8-trimethyl-5,6,7,8-
25 tetrahydro-2-naphthalenyl)oxy]-2-furamide
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I
0 0~~~0
I ~ \ / JT~Io
i
B65
Compound B65 was synthesized in a manner analogous to that of B 1,
according to Scheme B (HBTU coupling), using similar starting compounds and
reaction conditions. Yield of purified product was 33%. NMR and mass
s spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): 8 1.24 (s, 6H), 1.68 (m, 2H), 1.78 (m, 2H), 2.19 (s, 3H), 2.72 (t,
2H, J =
6.32 Hz), 3.95 (s, 6H), 3.98 (s, 3H), 5.29 (d, 1H, J = 3.59 Hz), 6.96,(d,
1H,), 7.06
(s, 1H), 7.14 (d, 1H, J = 3.59 Hz), Mass APCI 468.5.
Example 866: 5-[(3,6,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-N-
(2,4,6-trimethoxyphenyl)-2-furamide
0
\/
i
0
B66
Compound B66 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The requisite phenol was synthesized according to the following method:
0
a.
I~ I~
b
d. I ~ OS~ c. I ~ off
i
O O
O
e.
r
\/ f. H
O.
SIB
122
a. AIC13, AcylChlodde, CH3N02,0°C, 7 hrs.
b. McPBA, NaHC09(aq), CHzCIZ, air 423-120
o. TBDMS, DMF, imldazole
d. 1. LDA, -78°C, THF, 30 min
2. Mel, 12h, RT
e, hydrogentation
1$ f. acid
NMR and mass spectrometry data consistent with the title product were as
follows:
1HNMR (dmso-d6): b 1.00 (d, 3H, J = 6.42 Hz), 1.14 (s, 3H), 1.21 (s, 3H), 1.29
(d,
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1H, J =12.65 Hz), 1.57 (d, 1H, J = 13.03 Hz), 1.87 (s, 1H), 2.14 (s, 3H), 2.25
(dd, 1H,
J = 12.09,, 8.50 Hz), 2.72 (d, 1H, J = 16.43 Hz), 3.72 (s, 6H), 3.79 (s, 3H),
5.40 (d, 1H,
J = 3.40 Hz), 6.26 (s, 2H), 6.97 (s, 1H), 7.11(s, 1H), 7.17 (d, 1H, J = 3.21
Hz), 8.90 (s,
1H), APCI mass 480.2.
Example B67: N-(2-methoxy-3-pyridinyl)-5-[(3,8,8-trimethyl-5,6,7,8-tetrahydro-
2-naphthalenyl)oxy]-2-furamide
0 0 I /N
~I I I b 0
B67
Compound B67 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
1o NMR and mass spectrometry data consistent with the title product were as
follows:
1I3N1VIR (CD30D): 8 1.23 (s, 6H), 1.66 (m, 2H), 1.81 (m, 2H), 2.19 (s, 3H),
2.72 (t,
2H, J = 6.23 Hz), 4.03(s, 3H), 5.34 (d, 1H, J = 3.59 Hz), 6.96 (m, 2H, J =
5.10, 4.91,
3.02, 1.89 Hz), 7.09 (s, 1H), 7.22 (d, 1H, J = 3.59 Hz), 7.89 (td, 1H, J =
5.10, 1.70
Hz), 8.42 (dd, 1H, J = 7.74, 1.70 Hz), APCI mass 407.2.
15 Example B68: N-(2,4-dimethoxy-3-pyridinyl)-5-[(3,8,8-trimethyl-5,6,7,8-
tetrahydro-2-naphthalenyl)oxy]-2-furamide
I
0
0 0 ~ \ I
I~ I I o
\
B68
Compound B68 was synthesized in a manner analogous to that of B1,
according to Scheme B, using similar starting compounds and reaction
conditions.
2o NMR and mass spectrometry data consistent with the title product were as
follows:
1HNMR (CDC13): 81.44 (s, 6H), 1.85 (m, 2H), 2.00 (m, 2H), 2.43 (s, 3H), 2.92
(t,
2H, J = 6.33 Hz), 4.10 (s, 3H), 4.18 (s, 3H), 5.50 (d, 1H, J = 3.59 Hz), 6.81
(d, 1H, J =
.85 Hz), 7.14 (s, 1H), 7.35 (d, 1H, J = 3.59 Hz), 7.47 (s, 1H), 8.22 (d, 1H, J
= 5.85
Hz), APCI mass 437.2.
25 Example B69: 5-(4-chloro-5-isopropyl-2-methylphenoxy)-N-[2-(2-
hydroxyethoxy)-4,6-dimethoxy-5-pyrimidinyl]-2-furamide
O N O
O ~ ~ ~OH
O 0 ~ N
I "
o~
ci
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B69
Compound B69 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
I I
O N\ CI RAH O NYO.R O I NYO.R
O N I N ~ 02N I i NI ~ H2N~N
2 O
O~
NMR and mass spectrometry data consistent with the title product were as
follows:
1HNMR (CDC13): 8 1.19 (d, 6H, J = 7.18 Hz), 2.24 (s, 3H), 3.33 (dq, 1H, J =
7.18,
6.80 Hz), 3.97 (m, 8H), 4.48 (dd, 2H, J = 4.91, 4.53 Hz), 5.35 (d, 1H, J =
3.40 Hz),
6.97 (s, 1H), 7.02 (s, 1H), 7.14 (d, 1H, J = 3.78 Hz), 7.25 (m, 1H), APCI Mass
492.1.
Example B70: 5-(4-chloro-5-isopropyl-2-methylphenoxy)-N-[4,6-dimethoxy-2-(2-
l0 methoxyethoxy)pyrimidin-5-yl]-2-furamide
,o
(,0
0 0 ~y
~0
N
CI
B70
Compound B70 was synthesized in a manner analogous to that of B 1,
1s according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows:
IIiNMR (CDC13): 8 1.16 (d, 6H, J = 6.80 Hz), 2.23 (s, 3H), 3.33 (m, 1H, J =
6.80,
6.80, 6.80, 6.80 Hz), 3.42 (s, 3H), 3.76 (t, 2H, J = 4.91 Hz), 3.96 (s, 6H),
4.50 (dd,
2H, J = 5.29, 4.91 Hz), 5.34 (d, 1H, J = 3.40 Hz), 6.99 (s, 2H), 7.14 (d, 1H,
J = 3.78
2o Hz), 7.23 (s, 1H), APCI Mass 506.1.
Example B71: 5-(4-chloro-5-isopropyl-2-methylphenoxy)-N-(4,6-dimethoxy-2-
phenoxypyrimidin-5-yl)-2-furamide
0 0 / \
N
/ N
CI I \ O \O/ N~ ~ O
O\
B71
2s Compound B71 was synthesized in a manner analogous to that of B l,
according to Scheme B, using similar starting compounds and reaction
conditions.
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NMR and mass spectrometry data consistent with the title product were as
follows:
1HNMR (CDC13): S 1.19 (d, 6H, J = 7.18 Hz), 2.24 (s, 3H), 3.34 (m, 1H, J =
7.18,
6.80, 6.80, 6.42 Hz), 3.85 (s, 6H), 5.35 (d, 1H, J = 3.40 Hz), 6.98 (s, 1H),
6.99 (s, 1H),
7.02 (s, 1I~, 7.14 (d, 1H, J = 3.40 Hz), 7.21 (d, 2H, J = 8.31 Hz), 7.37 (d,
1H, J = 8.31
Hz), 7.39 (dd, 1H, J = 8.31, 7.18 Hz), APCI Mass 522.2.
Example B72: 5-(5-isopropyl-2-methylphenoxy)-N-(2,4,6-trimethoxy-5-
pyrimidinyl)-2-furamide
I
00
\ N
B72
Compound B72 was synthesized in a manner analogous to that of B 1,
1o according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H NMR (CDCl3): 8 1.22 (6H, d), 2.27 (3H, s), 2.87 (1H, hep), 3.94
(9H,
s), 5.35 (1H, d), 6.93 (1H, s), 7.02 (2H, br s), 7.15 (2H, m), FI-PCI m/z
428.2
(M+H)+.
15 Example B73: 5-(4-chloro-3-isopropyl-2-methoxy-6-methylphenoxy)-N-(4,6-
dimethoxy-2-{ [3-(4-methyl-1-piperazinyl)propyl] amino}-5-pyrimidinyl)-2-
furamide
I H~/\
o. o~ ~YN~
CI I \ \ / N \ \ N
H O
N
B73
Compound B73 was synthesized in a manner analogous to that of B 1,
2o according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (CDC13): 8 1.34 (6H, d), 1.78 (2H, m), 2.19 (3H, s), 2.31 (3H, s), 2.51
(10H, br
m), 3.38 (2H, m), 3.55 (1H, hep), 3.83 (3H, s), 3.89 (6H, s), 5.10 (1H, d, J =
4.8 Hz),
5.82 (1H, br t), 6.88 (1H, s, amide NH], 7.01 (1H, s), 7.06 (1H, d, J = 4.8
Hz), FI-PCI
2s m/z 618.4, 619.4 (M+H)+.
Example B74: 5-[(2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)oxy]-N-(2,4,6-
trimethoxyphenyl)-2-furamide
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0
B74
Compound B74 was synthesized in a manner analogous to that of B l,
according to Scheme B, using similar starting compounds and reaction
conditions.
The yield of the purified product was 41%. NMR and mass spectrometry data
s consistent with the title product were as follows: 1H NMR (300 MHz, CDC13):
8 1.51
(s, 6H), 3.12 (s, 2H), 3.80 (s, 9H), 5.48 (d, 1H), 6.20 (s, 2H), 6.81 (t, 1H),
6.98 (dd,
2H), 7.15 (d, 1H), 7.20 (br s, 1H), APCI-MS m/z 440.1 (M+H)+.
Example B75: 5-(3-chloro-2-isopropyl-5-methylphenoxy)-N-(2,6-
dimethoxyphenyl)-2-furamide
to B75
Compound B75 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (300 MHz, CDC13): ~ 1.25 (s, 3H), 1.27 (s, 3H), 2.34 (s, 3H), 3.28
(septet, 1H, J
15 = 9Hz), 3.87 (s, 6H), 5.44 (d, 1H, J =~3Hz), 6.62 (s, 1H), 6.65 (s, 1H),
6.95 (s, 1H),
7.17 (d, 1H), 7.23 (t, 1H), 7.31 (s, 1H), 7.37 (s, 1H), APCI-MS mlz 430.1
(M+H)+.
Example B76: 5-(3-chloro-2-isopropyl-5-methylphenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
B76
2o Compound B76 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows:
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1HNMR (300 MHz, CDC13): 8 1.24 (s, 3H), 1.26 (s, 3H), 2.34 (s, 3H), 3.27
(septet,
1H, J = 9 Hz, J = 6 Hz), 5.44 (d, 1H, J = 3 Hz), 6.20 (s, 2H), 6.94 (s, 1H),
7.16 (d, 1H,
J = 6 Hz), 7.23 (s, 1H), 7.30 (s, 1H), APCI-MS m/z 460.2 (M+H)+.
Example B77: 4-bromo-N-(2,6-dimethoxyphenyl)-5-[(3,5,5,8,8-pentamethyl-
5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-2-furamide
B77
Compound B77 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
1o NMR (300 MHz, DMSO-d6): 8 1.16 (s, 6H), 1.24 (s, 6H), 1.61 (s, 4H), 2.26
(s, 3H),
3.72 (s, 6H), 6.70 (d, 2H), 6.81 (s, 1H), 7.21 (t, 1H), 7.26 (s, 1H), 7.46 (s,
1H), 9.25
(s, 1H), APCI-MS m/z 498.3 (M+H)+.
Example B78: N-(4,6-dimethoxy-2- f [3-(4-methyl-1-piperazinyl)propyl]amino)-5-
pyrimidinyl)-5-[(2,2,4,6-tetramethyl-2,3-dihydro-1-benzofuran-7-yl)oxy]-2-
furamide
B78
Compound B78 was synthesized in a manner analogous to that of compound
B 1, according to scheme B, using similar starting materials and reaction
conditions.
2o Example B79: 4-bromo-5-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-n-(2,4,6-trimethylphenyl)-2-furamide
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o-
/ \
i
H O-
O
8r
O
(/
B79
Compound B79 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows:
1HIVMR (300 MHz, DMSO-d6): 8 1.15 (s, 6H), 1.22 (s, 6H), 1.60 (s, 4H), 2.25
(s,
3H), 3.69 (s, 6H), 3.78 (s, 3H), 6.28 (s, 2H), 6.82 (s, 1H), 7.24 (s, 1H),
7.43 (s, 1H),
9.08 (s, 1H), APCI-MS m/z 572.4 (M+H)+.
Example B80: N-{4,6-dimethoxy-2-[(3-morpholin-4-ylpropyl)amino]pyrimidin-5-
yl~-5-[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furamide acetate
O N~/ N~
\ O O N
/
~ /° ~O
B80
Compound B78 was synthesized in a manner analogous to that of compound
B 1, according to scheme B, using similar starting materials and reaction
conditions.
Example B81: 5-[(3-chloro-8,8-dimethyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-
N-(2,4,6-trimethoxyphenyl)-2-furamide
0
0 0 -
I~ \/ H ~ ~ °v
ci o
B81
Compound B81 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
The requisite phenol was synthesized according to the following method:
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0 0
O CI / OH MeOH CI OMe
CI~ _ ~ ~ I O
, HO ~ HO ~ I O TsOH HO
AICI3, nitrobenzene
Et3SiH TFA
OMe
OH 01 / OH MeMgCI (3.5 eq.) 01
AICI3,~ CH3N02 I ~ I O
HO
I HO THF
123 >95% 88%
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (DMSO-d6): b 1.21 (s, 6H), 1.61 (m, 2H), 1.73 (m, 2H), 2.71 (t, 2H), 3.71
(s,
6H), 3.78 (s, 3H), 5.51 (d, 1H), 6.26 (s, 2H), 7.18 (s, 1H), 7.29 (s, 1H),
7.37 (s, 1H),
5 8.92 (s, 1H), APCI-MS m/z 486.3 (M+H)+.
Example B82: 5-[(3-methoxy-1,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
I
0 0 ~ o~
I
I ~ o \ / H
q ,0
B82
Compound B82 was synthesized in a manner analogous to that of B1,
Io according to Scheme B, using similar starting compounds and reaction
conditions.
The requisite phenol was synthesized according to the following method:
c1
OH OMe O
I ~ OH Mel (1 eq.) ~:z_ I w OH 01 I
_~zC M / AICI3 OMe
48% 26
121
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (DMSO-d6): ~ 1.24 (s, 6H), 1.39 (s, 6H), 1.66 (s, 3H), 3.76 (s, 9H), 3.82
(s,
15 3H), 5.10 (d, 1H), 6.37 (s, 2H), 7.14 (s, 1H), 7.48 (d, 1H), 8.94 (s, 1H),
APCI-MS m/z
524.3 (M+H)+.
Example B83: 5-[(3-methoxy-1,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-N-(2,4,6-trimethoxy-5-pyrimidinyl)-2-furamide
I
0 0 ~~o~
O \O/ H~N
i _ ~ ,O
B83 v q
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Compound B83 was synthesized in a manner analogous to that of Bl,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H NMR (DMSO-d6): ~ 1.27 (s, 6H), 1.34 (s, 6H), 1.61 (s, 4H), 2.30
(s,
3H), 3.71 (s, 3H), 3.86 (s, 6H), 3.90 (s, 3H), 5.08 (d, 1H), 6.94 (s, 1H),
7.09 (d,
1H), 9.14 (s, 1H), APCI-MS m/z 526.3 (M+H)+.
Example B84: 5-[(7-chloro-4,4-dimethyl-1,2,3,4-tetrahydro-6-quinolinyl)oxy]-N-
(2,4,6-trimethoxyphenyl)-2-furamide
0 0
0 0
\ / H \ /
,c1 o
B84
1o Compound B84 was synthesized in a manner analogous to that of B 1,
according to Scheme B, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H NMR (CDCl3): 8 1.25 (s, 6H), 1.71 (t, 2H), 3.31 (t, 2H), 3.81 (s,
9H),
5.22 (d, 1H), 6.18 (s, 2H), 6.50 (s, 1H), 7.06 (s, 1H), 7.08 (d, 1H), 7.19 (s,
1H),
15 APCI-MS m/z 487.2 (M+H)+.
Example B85: 5-[(1-acetyl-7-chloro-4,4-dimethyl-1,2,3,4-tetrahydro-6-
quinolinyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
0
o /o\ r"v \ /
NCI C
B85 ~O
Compound B85 was synthesized in a manner analogous to that of B1,
2o according to Scheme B, using similar starting compounds and reaction
conditions.
The requisite phenol was synthesized as follows:
OMe II CI / I CMe ~B , I OMe
C
HZN ~ I CI ESN, CHZCIz ~HN~CI LDA, THF N CI
100 rt, Sh
92~
AIC13 I ~ Dli NaOH I ~ OH
130°C ~ ~ CI H ~ CI
D 104
100
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NMR and mass spectrometry data consistent with the title product were as
follows:1H
NMR (CDC13): 8 1.26 (s, 6H), 1.78 (t, 2H, J = 6.318 Hz), 2.28 (s, 3H), 3.76(t,
2H),
3.78 (s, 9H), 5.48 (d, 1H, J = 3.546 Hz), 6.17 (s, 2H), 7.13 (s, 2H), 7.15 (s,
1H),
APCI-MS rnlz 529 (M+H)+.
Example B86: 5-[(1-acetyl-7-chloro-4,4-dimethyl-1,2,3,4-tetrahydro-6-
quinolinyl)oxy]-N-(2,4,6-trimethoxy-5-pyrimidinyl)-2-furamide
\ O O O O N~
/ HN ~ // O
N CI N
B86 ~o -O
Compound B 86 was synthesized in a manner analogous to that of B l,
according to Scheme B, using similar starting compounds and reaction
conditions.
1o NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (DMSO-d6): ~ 1.15 (s, 6H), 1.67 (t, 2H, J = 6.04 Hz), 2.17 (s, 3H), 3.66
(t, 2H,
J = 6.14 Hz), 3.81 (s, 6H), 3.85 (s, 3H), 5.55 (d, 1H, J = 3.59 Hz), 7.15 (d,
1H, J =
3.40 Hz), 7.36 (s, 1H), 7.81(s, 1H), 9.16 (s, 1H), APCI-MS m/z 532 (M+H)+.
Example B87: 5-[(4,6-dibromo-2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl)oxy]-
15 N-(4,6-dimethoxy-2-~[3-(4-methylpiperazin-1-yl)propyl]amino]pyrimidin-5-yl)-
2-
2
furamide
j ~NH~\~N~
O ~ ~IO
N~
\ O N
0
er er
B87
2o Compound B87 was synthesized according to Scheme B.
0 0
I \ OH grz I ~ OH
Br ~ Br
CC14
r.t. 126
To a solution of phenol (3.28 g, 20 mmol) in carbon tetrachloride (50 mL) was
added
bromine (2.06 mL, 40 mmol) dropwise. The reaction mixture was stirred at r.t.
overnight. It was washed with water, saturated sodium bicarbonate, brine,
dried
2s (sodium sulfate) and evaporated to a dark oil. The dibrominated product was
purified
by flash chromatography (eluting solvent: lethyl acetate/10 hexanes to lethyl
acetate/Shexanes): 4.5 g (70%). NMR and mass spectrometry data consistent with
the
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title product were as follows: 1H NMR (CDCl3): 8 1.55 (6H, d), 1.90 (2H,
sextet),
2.28 (3H,,s), 2.46 - 2.51 (lOH,m), 3.10 (2H, s), 3.44 (2H, m), 3.87 (6H, s),
5.52(1H, d,
J = 3.78 Hz), 5.82 (1H, t), 6.93 (1H, s), 7.14 (1H, d, J = 3.78 Hz), 7.26 (1H,
s), FI-PCI
mlz 725.1 and 727.1 (M+I~+.
Example B88: N-(2-anilino-4,6-dimethoxypyrimidin-5-yl)-5-(4-chloro-5-
isopropyl-2-methylphenoxy)-2-furamide
00
O r~ N~N
I / H~N
CI
B88
Compound B88 was synthesized according to Scheme B. NMR and mass
1o spectrometry data consistent with the title product were as follows: IIiNMR
(CDC13):
8 1.19 (d, 6H, J = 6.80 Hz), 2.24 (s, 3H), 3.33 (dd, 1H, J = 7.18, 6.42 Hz),
3.98 (s,
6H), 5.35 (d, 1H, J = 3.78 Hz), 6.98 (s, 2H,) 7.04 (t, 1H, J = 7.55 Hz), 7.14
(d, 1H, J =
3.40 Hz), 7.32 (dd, 2H, J = 8.31, 7.55 Hz), 7.62 (d, 2H, J = 8.31 Hz), Mass (M-
H)' _
521.3.
is Example B89: N-(4,6-dimethoxy-2- f [3-(4-methylpiperazin-1-
yl)propyl] amino~pyrimidin-5-yl)-5-[(3.3.6-trimethyl-2,3-dihydro-1H-inden-5-
yl)oxy]-2-furamide
~ O ~ -N N -
HN~ ~~--NH
~~-N
O
B89 - \
Compound B89 was synthesized in manner analogous to that of B52. NMR
2o and mass spectrometry data consistent with the title product were as
follows: 1H NMR
(300 MHz, CDCl3): 1H NMR (300 MHz, CDC13): 8 1.21 (s, 6H), 1.76 (dd, 2H, J =
12.84, 6.42 Hz), 1.92 (t, 2H, J = 7.18 Hz), 2.24 (s, 3H), 2.34 (s, 3H), 2.54
(dd, l OH, J
= 7.18, 6.42 Hz), 2.84 (d, 2H, J = 7.18 Hz), 3.45 (dd, 2H, J = 12.09, 6.04
Hz), 3.87 (s,
6H), 5.29 (d, 1H, J = 6.80 Hz), 5.78 (bs, 1H), 6.82 (s, 1H), 6:94 (s, 1H),
7.05 (s, 1H),
25 7.09 (m, 1H), APCI-MS m/z 579.7 (M+H)+.
Example B90: N-(2-{[2-(dimethylamino)ethyl]amino-4,6-dimethoxypyrimidin-5-
yl)-5-[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furamide
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0 00
HN \ N>--NH
N
O N-
B9
Compound B90 was synthesized in manner analogous to that of B52. NMR
and mass spectrometry data consistent with the title product were as follows:
1H NMR
(300 MHz, CDC13): 8 1.21 (s, 6H), 1.92 (dd, 2H, J = 14.73, 7.18 Hz), 2.23 (s,
3H),
s 2.28 (s, 6H), 2.51 (t, 2H, J = 6.04 Hz), 2.84 (dd, 2H, J =14.35, 7.18 Hz),
3.48 (ddd,
2H, J = 11.71, 11.33, 5.67 Hz), 3.87 (s, 6H), 5.28 (m, 1H), 5.42 (t, 1H, J =
10.20 Hz),
6.82 (s, 1H), 6.97 (s, 1H), 7.05 (s, 1H), 7.10 (d, 1H, J = 3.40 Hz), APCI-MS
rn/z
579.7 (M+H)+.
Example B91: 5-[(3,5,5,7,8,8-hexamethyl-5,6,7,8- tetrahydro-2-
1o naphthalenyl]oxy~-N-(2,4,6-triethyl-5-pyrimidinyl)-2-furamide
~ / ~ N
~N
'-
N
B91
Compound B91 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
1s MeOH-d4): 8 0.90 (d, 3H, J = 6.6 Hz), 0.96, 1.12, 1.14, 1.23 (4s, 3H each),
1.29 (dd,
1H, J = 2.4, 13.5 Hz), 1.55 (dd, 1H, J =12.8, 13.4 Hz), 1.72-1.84 (m, 1H),
2.13 (s,
3H), 3.85 (s, 6H), 3.88 (s, 3H), 5.25 (d, 1H, J = 3.59 Hz), 6.91 (s, 1H), 7.05
(d, 1H, J
= 3.59 Hz), 7.18 (s, 1H), 7.20-7.31 (m, 2H), APCI-MS nalz 510.4 (M+H)+.
Example B92: N-(2,6-dimethoxyphenyl)-5-[(3,5,5,6,8,8-hexamethyl-5,6,7,8-
2o tetrahydro-2-naphthalenyl]oxy}-2-furamide
I w o /o \ r"~ w
i o I i
B92 \ I
Compound B92 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
MeOH-d4): 8 0.99 (d, 3H, J = 6.8 Hz), 1.03, 1.22, 1.23, 1.32 (4s, 3H each),
1.38 (dd,
2s 1H, J = 2.4,13.4 Hz), 1.64 (dd, 1H, J = 13.0, 13.2 Hz), 1.80-1.95 (m, 1H),
2.23 (s,
3H), 3.80 (s, 6H), 5.34 (d, 1H, J = 3.59 Hz), 6.68 (d, 1H, J = 8.3 Hz), 7.01
(s, 1H),
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7.14 (d, 1H, J = 3.59 Hz), 7.22 (d, 1H, J = 8.5 Hz), 7.27 (s, 1H), APCI-MS m/z
478.2
(~'I+~+.
Example B93: 5-[(3,5,5,6,8,8-hexamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl] oxy~-N-(1H-indol-5-yl)-2-furamide
I ~ o / \
0
i o I i
i NH
s B93
Compound B93 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
MeOH-d4): S 0.90 (d, 3H, J = 6.8 Hz), 0.96, 1.11, 1.13, 1.23 (4s, 3H each),
1.28 (dd,
1H, J = 2.6, 13.6 Hz), 1.54 (dd, 1H, J = 13.0, 13.2 Hz), 1.70-1.85 (m, 1H),
2.13 (s,
3H), 5.26 (d, 1H, J = 3.39 Hz), 6.32 (d, 1H, J = 3.59 Hz), 6.92 (s, 1H), 7.08
(s, 1H, J =
3.59 Hz), 7.12 (d, 1H, J = 3.0 Hz), 7.15-7.25 (m, 2H), 7.25-7.30 (m, 1H) 7.70-
7.75
(m, 1H), APCI-MS m/z 457.4 (M+H)+.
a
Example B94: 5-[(3,5,5,6,8,8-hexamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl] oxy}-N-(6-quinolinyl)-2-furamide
i
O / N
O O
I / \ /
1s B94 ~'
Compound B94 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
DMSO-d6): 8 0.93 (d, 3H, J = 6.61 Hz), 1.00, 1.15, 1.18, 1.30 (4s, 3H each),
1.32 (dd,
1H, J = 2.2, 13.6 Hz), 1.54 (dd, 1H, J =13.0, 13.2 Hz), 1.72-1.85 (m, 1H),
2.19 (s,
3H), 5.53 (d, 1H, J = 3.59 Hz), 7.05 (s, 1H), 7.32 (s, 1H), 7.38 (d, 1H, J =
3.58 Hz),
7.42-7.52 (m, 2H), 7.92-8.06 (m, 2H), 8.27 (d, 1H, J = 7.55 Hz), 8.43 (d, 1H,
J = 1.8
Hz), 8.77 (dd, 1H, J = 1.7, 4.3 Hz), 10.32 (br s, 1H, NI-~, APCI-MS m/z 469.4
(M+H)+.
Example B95: N-(1H-benzimidazol-2-ylmethyl)-5-[(4,4,8-trimethyl-3,4-dihydro-
2H-chromen-6-yl)oxy]-2-furamide
HI
N L N
B95 l OO
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Compound B95 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
MeOH-d4): S 1.32 (s, 6H), 1.85 (dd, 2H, J = 5.29, 5.48 Hz), 2.15 (s, 3H), 4.22
(dd,
2H, J = 4.34, 5.28 Hz), 4.98 (s, 2H), 5.52 (d, 1H, J = 3.78 Hz), 6.79 (d, 1H,
J = 2.2
s Hz), 6.99 (d, 1H, J = 2.8 Hz), 7.21 (d, 1H, J = 3.59 Hz), 7.6-7.69 (m, 2H),
7.78 -
7.89(m, 2H), APCI-MS m/z 432.4 (M+H)+.
Example B96: N-(1H-benzimidazol-2-ylmethyl)-5-[(4,4,7-trimethyl-3,4-dihydro-
2H-chromen-6-yl)oxy]-2-furamide
H
B96
1o Compound B96 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
MeOH-d4): 8 1.31 (s, 6H), 1.84 (dd, 2H, J = 5.29, 5.48 Hz), 2.16 (s, 3H), 4.18
(dd,
2H, J = 4.15, 5.29 Hz), 5.0 (s, 2H), 5.28 (d, 1H, J = 3.59 Hz), 6.69 (s, 1H),
7.08 (s,
1H), 7.19 (d, 1H, J = 3.59 Hz), 7.55 - 7.65 (m, 2H), 7.70-7.85(m, 2H), APCI-MS
m/z
1s 432.3 (M+H)+.
Example 97: 5-[(3-methyl-8-phenyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-N-
(2,4,6-trimethoxyphenyl)-2-furamide
I
0 0
w o 0 0~ v
I.% I/ N wI
H
B97 Ow
Compound B97 was synthesized according to Scheme B.
o I~ I
i i
OH O AICI3, CH3CN / OH (CH3CH~3SiH / OH
rt, overnight W I TFA, rt, 8h w I
4A O
4B ° 4D 98%
4C 21
20 127 128
A solution of 4A (3g), 4B (4.5g) and A1C13 (5.6g) was stirred at room
temperature overnight. The solution was extracted with EtOAc. Compound 4C
(1.5g) was purified by column (hexane: EtOAC 2:1). To a solution of compound
4C
(1.3g) in TFA (5 ml) was added (CH3CH2)3SiH at 0°C. The solution was
stirred for 2
2s hours. The solution was warmed up to room temperature and stirred
overnight. The
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solution was extracted with EtOAC, concentrated to give compound 4D (1.2g).
NMR
and mass spectrometry data consistent with the title product were as follows:
1H NMR
(DMSO-d6): 8 1.55 - 1.75 (m, 3H), 1.92-2.05 (m, 1H), 2.03(s, 3H), 2.7-2.85 (m,
2H),
3.65 (s, 6H), 3.72 (s, 3H), 4.02 (t, 1H), 5.6 (d, 1H), 6.20 (s, 2H), 6.6 (s,
1H), 6.8 (s,
1H), 7.02 (d, 2H), 7.1 - 7.2 (m, 2H), 7.25-7.3 (t, 2H), 8.85 (s, 1H), APCI-MS
m/z
514.3 (M+H)+.
Example 898: 5-[(7-chloro-4,4-trimethyl-2-oxo-1,2,3,4-tetrahydro-6-
quinolinyl)ogy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
0
j \ / H \ / °\
0 H CI
1o B98
Compound B98 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as fellows: 1H NMR
(DMSO): 8 1.21 (s, 6H), 2.38 (s, 2H), 3.71 (s, 6H), 3.78 (s, 3H), 5.52 (d,
1H), 6.26 (s,
2H), 7.05 (s, 1H), 7.18 (d, 1H), 7.35 (s, 1H), 8.92 (s, 1H), 10.31 (s, 1H),
APCI-MS
1s tnlz 501.2 (M+I~~.
Example B99: 5-[2,4-dimethyl-5-(1,I,3,3-tetramethylbutyl)phenoxy]-N (2,4,6-
trimethoxylphenyl)-2-furamide
/ NN \ /
0
\
B99
2o Compound B99 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H NMR
(MeOD):
8 0.97 (s, 9H), 1.64 (s, 6H), 2.15 (s, 2H), 2.35 (s, 3H), 2.56 (s, 3H), 4.05
(s, 6H), 4.06
(s. 3H), 5.32 (d, J = 6 Hz, 1H), 6.51 (s, 2H), 7.22 (s, 1H), 7.36 - 7 .40 (br,
2H), LC/
MS (M+H)+: 510.
25 Example 8100: N-(1H-benzimidazol-2-ylmethyl)-5-[(3,5,5,6,8,8-hexamethyl-
5,6,7,8-tetrahydro-2-naphthalenyl]oxy~-2-furamide
0
H
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B100
Compound B 100 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
CDCl3): 8 0.99 (d, 3H, J = 6.8 Hz), 1.06, 1.18, 1.20, 1.33 (4s, 3H each), 1.30
- 1.45
s (m, 1H), 1.62 (dd, 1H, J = 12.8, 13.4 Hz), 1,.75 - 1.95 (m, 1H), 2.20 (s,
3H), 5.25-5.32
(m, 1H, NH), 5.32 (d, 1H, J = 3.58 Hz), 6.97 (s, 1H), 7.20 (s, 1H), 7.41 - 7.5
(m, 3H),
7.80 - 7.90 (m, 2H), 10.05 (br s, 1H, NH), APCI-MS m/z 472.3 (M+H)+.
Example B101: N-(2,4,6-trimethoxy-5-pyrimidinyl)-5-[(4,4,7-trimethyl-3,4-
dihydro-2H-chromen-6-yl)oxy]-2-furamide
0
Fi
to
B101
Compound B 101 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
MeOH-d4): 8 1.29 (s, 6H), 1.80 (dd, 2H, J = 5.2, 5.4 Hz), 2.15 (s, 3H), 3.95
(s, 6H),
15 3.9.7 (s, 3H), 4.15 (dd, 2H, J = 5.2, 5.4 Hz), 5.22 (d, 1H, J = 3.59 Hz),
6.65 (s, 1H),
7.07 (s, 1H), 7.12 (d, 1H, J = 3.59 Hz), APCI-MS m/z 470.1 (M+H)+.
Example B102: N-(2,4,6-trimethoxy-5-pyrimidinyl)-5-[(4,4,8-trimethyl-3,4-
dihydro-2H-chromen-6-yl)oxy]-2-furamide
/
0
o I ~ -
p Fi
O' Y
O\
2o B102
Compound B 102 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
CDC13): 8 1.31 (s, 6H), 1.82 (dd, 2H, J = 5.2, 5.4 Hz), 2.16 (s, 3H), 3.97 (s,
9H), 4.20
(d, 2H, J = 5.0, 5.4 Hz), 5.39 (d, 1H, J = 3.59 Hz), 6.74 (d, 1H, J = 3.0 Hz),
6.90 (d,
25 1H, J = 3.0 Hz), 6.99 (s, 1H), 7.12 (d, 1H, J = 3.59 Hz), APCI-MS m/z 470.2
(M+H)+.
Example B103: N-~4,6-dimethoxy-2-[methyl(pyridin-2-
ylmethyl)amino]pyrimidin-5-yl)-5-[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-
yl)oxy]-2-furamide
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N'
N ~I
O O N~N
\ / O~
B103
Compound B 103 was synthesized in a manner analogous to that of compound
B 1, according to scheme B, using similar starting materials and reaction
conditions.
Example B104: 5-[(6-methoxy-3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-N-
(2,4,6-trimethoxy-5-pyrimidinyl)-2-furamide
I
o ~~'°'
N
H
O
B104 I
Compound B 104 was synthesized according to Scheme B.
OH O PPA, 100-110 °C ~ I OH pd~C / I OH
O cat HzS04 ~ O
I OH O I CH30H I
129 130
1o NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (d4-CH30H): ~ 1.15 (s, 6H), 1.87 (t, 2H), 2.81 (t, 2H), 3.71 (s, 3H), 3.87
(s,
6H), 3.90 (s, 3H), 5.14 (d, 1H), 6.89 (s, 2H), 7.02 (d, 1H), APCI-MS m/z 470.2
(M+H)+.
Example B105: 5-[(2,5-dimethyl-1,3-benzothiazol-6-yl)oxy]-N-(2,6-
15 dimethoxyphenyl)-2-furamide
° O O
s O
HN
N
O
B105
Compound B105 was synthesized according to Scheme B. NMRand mass
spectrometry data consistent with the title product were as follows: 1H NMR
(DMSO-
d6): ~ 2.38 (s, 3H), 2.76 (s, 3H), 3.72 (s, 6H), 5.71 (d, J = 6 Hz, 1H),
6.69(d, J = 9 Hz,
20 2H), 7.21 - 7.26 (m, 2H), 7.86 (s, 1H), 8.87 (s, 1H), 9.10 (s, 1H), LC/ MS
(M+H)+:
425.
Example B106: 5-[(4,4,7,8-tetra-mehtyl-1,2,3,4-tetrahydroquinolin-6y1)oxy]-N-
(2,4,6-trimethoxyphenyl)-2-furamide
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0
\ I r, \ /
-0
B106
Compound B106 was synthesized according to Scheme B.
OMe
OMe / AICI3 ~ OH
Et3N, THF ~HN \ I 130°C O N I /
H2N~ J~ /~/~ H
49%
131
LAH I ~ OH
THF
132
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (DMSO-d6): 8 1.20 (s, 6H), 1.61 (t, J = 6 Hz, 2H), 1.97 (s, 3H), 2.06 (s,
3H),
3.25 (br. 2H), 3.72 (s, 6H), 3.79 (s, 3H), 5.14 (d, J = 3 Hz, 1H), 6.27 (s,
2H), 6.89 (s,
1H), 7.10 (d, J = 3 Hz, 1H), 8.81 (s, 1H), LC/ MS (M+H)+: 4.81.
Example 8107: 5-[5-(1-cyano-1-methylethyl)-2-methylphenoxy]-N-(2,4,6-
l0 trimethoxyphenyl)-2-furamide
0 0 0
NC ~ ~ _
/ HN ~ / o
O
B107
Compound B 107 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H NMR
(DMSO-
d6): 8 1.58 (s, 6H), 2.18 (s, 3H), 3.63 (s, 6H), 3.70 (s, 3H), 5.53 (d, J = 3
Hz, 1H),
6.18 (s, 2H), 7.11 (br. 1H), 7.20 (d, J = 1.8 Hz, 1H), 7.26 (dd, J = 1.8 Hz, J
= 9 Hz,
1H), 7.34 (d, J = 9 Hz, 1H), 8.84 (s, 1H), LC/ MS (M+H)+: 451.
Example B108: 4-~5-[acetyl(methyl)amino]2-methylphenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
0
0
/ HN
O,
B108
Compound B 108 was synthesized according to Scheme B.
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H
O2N ~ O\ H2, Pd/C HzN ~ \ O\ MeCOCI O~N ~ ~ O\
NEt3
1. MeI, NaH O ~ OH
2. BBr3
133
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (MeOD): S 1.87 (s, 3H), 2.36 (s, 3H), 3.22 (s, 3H), 3.81 (s, 6H), 5.62 (d,
J = 3.3
Hz, IH), 6.69 (d, J = 8.4 Hz, 2H), 7.08-7.13 (br. d, 2H), 7.18(d, J = 3.3 Hz,
1H), 7.26
(t, J = 8.4 Hz, 1H), 7.41 (d, J = 7.6 Hz, IH), LC/MS (M+H)+: 425.
Example B109: 5-[(2,5-dimethyl-1,3-benzothiazol-6-yl)oxy]-N-(2,4,6-
trimethoxyphenyl)-2-furamide
I ~ I / "~ ~-
\ /
8109
1o Compound B 109 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H NMR
(DMSO-
d6): 8 2.38 (s, 3H), 2.76 (s, 3H), 3.71 (s, 6H), 3.78 (s, 3H), 5.70 (d, J =
3.3 Hz, 1H),
6.26 (s, 2H), 7.22 (d, J = 3.3 Hz, 1H), 7.85 (s, 1H), 7.87 (s, 1H), 8.96 (s,
1H), LC/ MS
(M+I~~: 455. .~._
Example B110: 4-[(7-clrloro-4,4-dimethyl-1,2,3,4-tetrahydroquinolin-6-yl)oxy]-
N-
(2,4,6-trimethoxypyrimidin-5-yl)-2-furamide
\ O O 0 O N
N ~ / CI ~ ~ HN ~ ~O
H
-~O
B110
Compound B 110 was synthesized according to Scheme B. NMR and mass
2o spectrometry data consistent with the title product were as follows: 1H NMR
(CD3CI~: 8 1.24 (s, 6H), 1.67 (t, 2H,), 3.2 (t, 2H), 3.92 (s, 6H), 3.96 (s,
3H), 4.81 (s,
1H), 5.28 (d, 1H), 6.59 (s, 1H), 7.03 (d, 1H), 7.186 (s, 1H), 7.57 (s, 1H),
APCI-MS
m/z 489.2 (M+H)+.
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Example 8111: 5-{5-[isopropyl(methyl)amino]-2-methylphenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
0
'N
HN
O
8111
Compound B 111 was synthesized according to Scheme B.
OzN ~ ~ O~ ~ PAC H2N ~ ~ O~ Aceton _ \ 'N ~ \ O~
BH IY3
1. MeI, KZC03 ~ \ OH
2. HBr
134
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (MeOD): 8 1.28 (s, 3H), 1.30 (s, 3H), 2.38 (s, 3H), 3.17 (s, 3H), 3.81 (s,
6H),
3.84 (s, 3H), 3.9-4.0 (m, 1H), 5.75 (d, J = 3.4 Hz, 1H), 6.29 (s, 2H), 7.11-
7.30 (m,
3H), 7.49 (d, J = 9 Hz, 1H), LC/ MS (M+H)+: 455.
to Example B112: 5-~5-(diethylamino)-2-methylphenoxy]-N-(4,6-dimethoxy-2-{[3-
(4-methylpiperazin-1-yl)-propyl]amino) pyrimidin-5-yl)-2-furamide
1
HN
B112
Compound B 112 was synthesized according to Scheme B.
H2N ~ O~ 1. EtI, KZC03 ~ OH
-~ ~ \
z. HBr
135
is NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (MeOD): 8 1.10 (dd, J = 7.18, 6.80 Hz, 6H), 1.86 (m, 2H), 2.13 (s, 3H),
2.53 (s,
3H), 2.58 - 2.95 (m, l OH), 3.45 (dd, J = 6.80, 6.42 Hz, 2H), 3.88 (s, 6H),
5.37 (d, J =
3.78 Hz, 1H), 6.40 (d, J = 2.27 Hz, 1H), 6.53 (dd, J = 8.31, 2.27 Hz, 1H),
7.06 (d, J =
8.31 Hz, 1H), 7.13 (d, J = 3.40 Hz, 1H), LC/ MS (M+H)+: 582.
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Example B113: 5-[5-(isopropylamino)-2-methylphenoxy]-N-(2,4,6-
trimethoxyphenyl)-2-furamide
0 0 0 /
~ l HN ~ /
B113
Compound B 113 was synthesized according to Scheme B.
02N \ O\ g2, Pd/C HEN ~ \ O\ Aceton ' \ 'N ~ \ O\
BH ~I'3
H$r N \ OH
136
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (MeOD): ~ 1.49 (d, J = 9 Hz, 6H), 2.55 (s, 3H), 3.90 (m, 1H), 4.01 (s,
6H), 4.04
(s, 3H), 5.94 (d, J = 3 Hz, 1H), 6.49 (s, 2H), 7.14 (s, 1H), 7.22 (d, J = 6
Hz, 1H), 7.42
(s, 1H), 7.62 (d, J = 6 Hz, 1H), LC/ MS (M+H)+: 441.
to Example B114: 5-(2-methyl-5-tert-pentylphenoxy)-N-(2,4,6-trimethoxylphenyl)-
2-furamide
0
/ HN
8114
Compound B 114 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H NMR
(MeOD):
1s ~ 0.67 (t, J = 9 Hz, 3H), 1.25 (s, 6H), 1.63 (q, J = 9 Hz, 2H), 2.25 (s,
3H), 3.79 (3,
6H), 3.81 (s, 3H), 5.32 (d, J = 3 Hz, 1H), 6.26 (s, 6H), 7.06 (s, 1H), 7.12-
7.17 (br.d,
2H), 7.23 (d, J = 9 Hz, 1H), LC/ MS (M+H)+: 454.
Example B115: N-(4,6-dimethoxy-2-}[3-(4-methylpiperazin-1-
yl)propyl] amino } pyrimidin-5-yl)-5-[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-
20 yl)oxy]-2-furamide acetic acid salt
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B115
Compound B 115 was synthesized in a manner analogous to that of compound
B l, according to scheme B, using similar starting materials and reaction
conditions.
Example B116: N-(4,6-dimethoxy-2- f [3-(4-moprpholinyl)propyl]amino}-5-
pyrimidinyl)-5-[(3,5,5,6,8,8-hexamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-
2-furamide
o l Vo
-N
I H ~ /~NH
N
B116
1o Compound B I 16 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
CDC13): ~ 0.99 (d, 3H, J = 6.79 Hz), 1.05, 1.20, 1.24, 1.31 (4s, 3H each),
1.36 (dd,
1H, J = 2.2, 13.4 Hz), 1.62 (dd, 1H, J =13.0, 13.2 Hz), 1.75-1.95 (m, 3H),
2.24 (s,
3H), 2.40-2.60 (m, 6H), 3.48 (dd, 2H, J = 6.0, 6.2 Hz), 3.70-3.81 (m, 4H),
3.87 (s,
1s 6H), 5.32 (d, 1H, J = 3.59 Hz), 5.73 ~t, 1H, J = 5.4, NH), 6.95 (s, 2H),
7.10 (d, 1H, J =
3.4 Hz), 7.19 (s, 1H), APCI-MS m/z 622.3 (M+H)+.
Example B117: N-(4,6-dimethoxy-2-}[3-(4-morpholinyl)propyl]amino}-5-
pyrimidinyl)-5-[(3,3,6-trimethyl-1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furamide
/ n
~N~O
.N
I H ~ N~NH
2o B117
Compound B 117 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the desired product were as follows: 1H (300
MHz,
CDC13): 8 1.45 (s, 6H), 1.72-1.82 (m, 2H), 2.23 (s, 3H), 2.45-2.55 (m, 6H),
3.47 (dd,
2H, J = 6.0, 6.23 Hz), 3.74 (t, 4H, J = 4.54 Hz), 3.86 (s, 6H), 5.01 (s, 2H),
5.36 (d, 1H,
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J= 3.59 Hz), 5.77(t, 1H, J= 5.48 Hz, NH), 6.79 (s, 1H), 6.90(d, 1H), 7.06 (s,
1H),
7.10 (d, 1H, J = 3.59 Hz), APCI-MS m/z 568.3 (M+H)+.
Example B118: N-(4,6-dimethoxy-2-{[3-(4-morpholinyl)propyl]amino}-5-
pyrimidinyl)-S-[(1-methoxy-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-2-furamide
O O /-N
0
H ~ N~NH
..
B118
Compound B 118 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CDC13):
l0 8 1.26 (s, 6H), 1.35 (s, 6H), 1.60-1.67 (m, 4H), 2.12-2.16 (m, 2H), 2.19
(s, 3H), 2.85 -
2.94 (m, 2H), 3.16 - 3.21 (m, 2H), 3.49-3.57 (m, 4H), 3.86 (s, 3H), 3.94 (s,
6H), 3.98 -
4.01 (m, 4H), 5.10 (d, 1H, J = 3.78 Hz), 6.93 (s, 1H), 6.98 (s, 1H), 7.10 (d,
1H, J =
3.40 Hz), MS (APCI): 638.4 (M+H)+.
Example B119: 4-[(5-{[5-(5-tert-butyl-2-methylphenoxy)-2-furoyl]amino}-4,6-
1s dimethoxypyrimidin-2-yl)amino]piperidine-1-carboxylate
B119
Compound B 119 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the desired product were as follows: IH NMR
(d4-
2o CH30H): 8 1.29 (s, 9H), 1.45 (s, 9H), 1.95 (dd, 2H), 2.24 (s, 3H), 2.97 (t,
4H), 3.30 (s,
3H), 3.93 - 4.03 (m, SH), 5.35 (d, 2H), 7.11 (s, 1H), 7.12 (d, 1H), 7.21 (dd,
2H),
APCI-MS f~z/z 610.1 (M+H)+.
Example B120: 5-[(7-chloro-4,4-dimethyl-1,2,3,4-tetrahydro-6-quinolinyI)oxy]-N-
{2,6-dimethoxy-4-[3-(4-morpholinyl)propoxy] pyrimidinyl}-2-furamide
i <-o
O O N H
~NwN~
~ N
O
25 H ~ ci
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B120
Compound B 120 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H NMR
(CDC13):
8 1.24 (s, 6H), 1.61-1.9 (m, 4H), 2.45-2.49 (m, 6H), 3.30 (t, 2H), 3.47 (m,
2H), 3.74
(t, 4H), 3.87 (s, 6H), 5.21 (d, 1H), 5.72 (t, 1H), 6.49 (s, 1H), 6.96 (s, 1H),
7.03 (d,
1H), APCI-MS m/z 601.1 (M+H)+.
Example B121: 5-(2-bromo-5-tert-butylphenoxy)-N-{4,6-dimethoxy-2-[(3-
morpholin-4-ylpropyl)amino]pyrimidin-5-yl}-2-furamide
0
N
H
/0
1o B121
Compound B 121 was synthesized according to Scheme B. NMR data
consistent with the title product were as follows: 1HNMR (~00 MHz,.CD30D): S
1.30
(s, 9H), 2.10 (m, 2H), 3.18 (m, 26H), 3.24 (m, 2H), 3.48 (m, 4H), 3.72 (m,
2H), 4.03
(s, 6H), 4.07 (m, 2H), 5.48 (d, 1H, J = 3 Hz), 7.15 (d, 3H, J = 3 Hz), 7.27
(m, 2H),
1s 7:59 (d, 1H, J = 6 Hz).
Example B122: N- f 4,6-dimethoxy-2-[(3-morpholin-4-ylpropyl)amino] pyrimidin-
5-yl)-5-[(4,4,7-trimethyl-3,4-dihydro-2H-chromen-6-yl)oxy]-2-furamide
o ~ N~o
-N
I H ~ N~NH
B122
2o Compound B 122 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1H (300
MHz,
DMSO-d6): ~ 1.22 (s, 6H), 1.73 (dd, 2H, J = 4.91, 5.28 Hz), 2.10 (s, 3H), 2.25
- 2.45
(m, 4H), 3.20 - 3.35 (m, 4H), 4.10 (t, 2H, J = 4.91 Hz), 5.30 (d, 1H, J = 3.4
Hz), 6.66
(s, 1H), 7.11 (d, 1H, J = 3.4 Hz), 7.13 (s, 1H), 8.82 (s, 1H, NH), APCI-MS m/z
582.3
2s (M+H)+.
Example B123: ethyl 4-[(5-{[5-(5-tert-butyl-2-methoxyphenoxy)-2-furoyl]amino)-
4,6-dimethoxy-2-pyrimidinyl)amino]butanoate
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0 0
H \ N NH O
B123
Compound B 123 was synthesized according to Scheme B, using HBTU as the
coupling agent.
/ OH
OFi Br2/CC14 / I OFi CuBr, NaOMe
0 °C to rt ' ~ gr cat. EtOAc, MeOH ~ O
reflux
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (CDC13): b 1.26 (t, 3H), 1.28 (s, 9H), 1.90 (quint, 2H), 2.45 (t, 2H),
3.40 (q,
3H), 3.85 (s, 3H), 3.87 (s, 6H), 5.00 (t, 1H), 5.32 (d, 1H), 6.92 (d, 1H),
6.94 (d, 1H),
7.08 (d, 1H), 7.19(s, 1H), 7.21 (s, 1H), APCI-MS m/z 557.3 (M+H)+.
1o Example B124: 5-(5-tert-butyl-2-methoxyphenoxy)-N-(2-~[3-
(dimethylamino)propyl]amino)-4,6-dimethoxy-5-pyrimidinyl)-2-furamide
/
0
-N
I H \ N~NH
O
/ ~ \ \
B124
Compound B 124 _was synthesized according to Scheme B, using HBTU as the
~s coupling agent. NMR and mass spectrometry data consistent with the title
product
were as follows: IH NMR (d4_CH30H): 8 1.20 (s, 9H), 1.70 (quint, 2H), 2.17 (s,
6H),
2.30 (t, 2H), 3.24 (t, 2H), 3.71 (s, 3H), 3.77 (s, 6H), 5.10 (d, 1H), 6.94 (d,
1H), 6.98
(d, 1H), 7.12 (s, 1H), 7.14 (d, 1H), APCI-MS m/z 528.3 (M+H)+.
Example B125: [5-(tert-butyl)-2-methoxyphenoxy]-N-(4,6-dimethoxy]-2-f [3-(4-
20 morpholinyl)propyl]amino)-5-pyrimidinyl)-2-furamide
0 0
-N
H ~ /~NH
O N ~ /-\
O\ N~
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B125
Compound B 125 was synthesized according to Scheme B, using HBTU as the
coupling agent. NMR and mass spectrometry data consistent with the title
product
were as follows: 1H NMR (d4_CH30H): S 1.48 (s, 9H), 2.02 (t, 2H), 2.67 (m,
6H),
3.49 (t, 2H), 3.89 (t, 4H), 4.00 (s, 3H), 4.06 (s, 6H), 5.45 (d, 1H), 7.20 (d,
1H), 7.24
(d, 1H), 7.12 (s, 1H), 7.46 (s, 1H), 7.48 (s, 1H), APCI-MS m/z 570.3 (M+H)~.
Example B126: N-(4,6-dimethoxy-2-~[3-(4-methyl-1-piperazinyl)propyl]amino}-
5-pyrimidinyl)-5-[(6-methoxy-3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)oxy-2-
furamide
to
B126
Compound B126 was synthesized according to Scheme B, using HBTU as the
coupling agent. NMR and mass spectrometry data consistent with the title
product
were as follows: 1H NMR (d4_CH30H): 8 1.21 (s, 6H), 1.76 (quintet, 2H), 1.94
(t,
~s 2H), 2.30 (s, 3H), 2.47 - 2.54 (m, 10H), 2.87 (t, 2H), 3.43 (m, 2H), 3.81
(s, 3H), 3.86
(s, 6H), 5.26 (d, 1H), 5.72 (br s, 1H), 6.84 (s, 1H), 6.90 (s, 1H), 6.96 (s,
1H), 7.08 (d,
1H), APCI-MS m/z 595.3 (M+H)+.
Example B127: N-(4,6-dimethoxy-2-~[3-(4-methyl-1-piperazinyl)propyl]amino}-
5-pyrimidinyl-5-[(5-methoxy-1,1-dimethyl-1H-inden-6-yl)oxy]-2-furamide
N N~
O
H O
~~O
20 8127
Compound B 127 was synthesized according to Scheme B, using HBTU as the
coupling agent. NMR and mass spectrometry data consistent with the title
product
were as follows: 1H NMR (CDC13): b 1.29 (s, 6H), 1.92 (quintet, 2H), 2.34 (s,
3H),
2.52 - 2.59 (m, 6H), 2.87 (t, 2H), 3.87 (s, 3H), 3.88 (s, 6H), 5.30 (d, 1H),
5.70 (br s,
25 1H), 6.43 (s, 1H), 6.57 (d, 1H), 6.97 (s, 1H), 7.11 (d, 1H), 7.11 (s, 1H),
APCI-MS m/z
593.3 (M+H)+.
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Example B128: N-(4,6-dimethoxy-2-{[3-(4-morpholinyl)propyl]amino)-5-
pyrimidinyl)-5-[(6-methoxy-3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-
furamide
0
N
0
8128
Compound B128 was synthesized according to Scheme B, using HBTU as the
coupling agent. NMR and mass spectrometry data consistent with the title
product
were as follows: 1H NMR (CDC13): 8 1.23 (s, 6H), 1.95 (quintet, 2H), 1.98 (t,
2H),
2.51 (m, 6H), 2.88 (t, 2H), 3.40 (t, 2H), 3.74 (t, 4H), 3.83 (s, 3H), 3.88 (s,
6H), 5.28
to (d, 1H), 5.72 (br s, 1H), 6.86 (s, 1H), 6.91 (s, 1H), 6.98 (s, 1H), 7.10
(d, 1H), A.PCI-
MS m/z 582.3 (M+H)+.
Example B129: N-(4,6-dimethoxy-2-{[3-(4-morpholinyl)propyl]amino)-5-
pyrimidinyl)-5-[(5-methoxy-1,1-dimethyl-1H-inden-6-yl)oxy]-2-furamide
N~
O ~~
0
8129
15 Compound B129 was synthesized according to Scheme B, using HBTU as the
coupling agent. NMR and mass spectrometry data consistent with the title
product
were as follows: 1H NMR (CDC13): 8 1.29 (s, 6H), 1.86 (quintet, 2H), 2.53 (m,
6H),
2.88 (t, 2H), 3.40 (t, 2H), 3.78 (t, 4H), 3.87 (s, 3H), 3.88 (s, 6H), 5.30 (d,
1H), 5.74
(br s, 1H), 6.43 (d, 1H), 6.57 (d, 1H), 6.97 (s, 1H), 6.97 (s, 1H) 7.11 (d,
1H)~ 7.11 (d,
20 1H), APCI-MS m/z 580.3 (M+H)+.
Example B130: N-(2,6-dimethoxy-4-{[3-(4-methyl-1-
piperazinyl)propyl] amino) phenyl)-5-[(6-methoxy-3,3-dimethyl-2,3-dihydro-1H-
inden-5-yl)oxy]-2-furamide
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B130
Compound B 130 was synthesized according to Scheme B, using HBTU as the
coupling agent. NMR and mass spectrometry data consistent with the title
product
were as follows: 1H NMR (CDCl3): 8 1.24 (s, 6H), 1.81 (quintet, 2H), 1.96 (t,
2H),
2.52 (m, 10H), 2.88 (t, 2H), 3.19 (t, 2H), 3.78 (s, 6H), 3.78 (s, 3H), 3.83
(s, 6H), 5.29
(d, 1H), 5.84 (s, 2H), 6.85 (s, 1H), 6.92 (s, 1H), 6.92 (s, 1H), 7.04 (d, 1H),
7.12 (d,
1H), APCI-MS m/z 593.3 (M+H)+.
Example 8131: N-(2-{[3-dimethylamino)propyl]amino}-4,6-dimethoxy-5-
to pyrimidinyl)-5-[(5-methoxy-1,1-dimethyl-1H-inden-6-yl)oxy]-2-furamide
y
\/ o
I
B131
Compound B 131 was synthesized according to Scheme B, using HBTU as the
coupling agent. NMR and mass spectrometry data consistent with the title
product
1s were as follows: 1H NMR (CDCl3): 8 1.29 (s, 6H), 1.96 (quintet, 2H), 2.51
(s, 6H)~
2.88 (t, 2H), 3.40 (t, 2H), 3.87 (s, 3H), 3.88 (s, 6H), 5.30 (d, 1H), 5.40 (br
s, 1H),
6.43 (d, 1H), 6.57 (d, 1H), 6.97 (s, 1H), 6.99 (s, 1H), 7.11 (d, 1H), 7.11 (s,
1H), APCI-
MS m/z 538.2 (M+H)+.
Example B132: N-(2-~[3-(dimethylamino)propyl]amino,-4,6-dimethoxy-5-
2o pyrimidinyl)-5-[(6-methoxy-3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-
furamide
o ° ,~W
I
\/
o~
I
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B132
Compound B 132 was synthesized according to Scheme B, using HBTU as the
coupling agent. NMR and mass spectrometry data consistent with the title
product
were as follows: 1H NMR (CDC13): S 1.23 (s, 6H), 1.82 (quintet, 2H), 1.95 (t,
2H),
s 2.36 (s, 6H), 2.48 (t, 2H), 2.88 (t, 2H), 3.44 (t, 2H), 3.83 (s, 3H), 3.88
(s, 6H), 5.27
(d, 1H), 5.31 (br s, 1H), 6.85 (s, 1H), 6.91 (s, 1H), 6.98 (s, 1H), 7.11 (d,
1H), APCI-
MS tnlz 540.3 (M+H)+.
Example B133: 5-(2-bromo-5-tert-butylphenoxy)-N-(2- f [3-(1H-imidazol-1-
yl)propyl]amino}-4.6-dimethoxypyrimidin-5-yl)-2-furamide
o ~~
\ 0 0 ~~N
0
l0 Br
B133
Compound B133 was synthesized according to Scheme B. NMRand mass
spectrometry data consistent with the title product were as follows: 1HNMR
(300
MHz, CDC13): 8 1.28 (s, 9H), 2.12 (m, 2H), 3.43 (m, 2H), 3:86 (s, 6H), 4.10
(m, 2H),
15 4.98 (m, 1H), 5.45 (d, 1H, J = 3 Hz), 6.97 (s, 1H), 7.15 (m, 3H), 7.55(d,
2H, J = 9
Hz), 7.71 (s, 1H), APCI (M+H)~: 599.
Example B134: 5-(2-bromo-5-tertbutylphenoxy)-N-X4,6-dimethoxy-2-[(2-
pyrrolidin-1-ylethyl)amino]pyrimidin-5-yl}-2-furamide
I H
\ \ IN
H
20 8134
Compound B 134 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: ~HNMR
(300
MHz, CDC13): 8 1.28 (s, 9H), 1.90 (m, 4H), 2.85 (m, 4H), 3.64 (m, 2H), 3.87
(s, 6H),
5.45 (d, 1H, J = 3 Hz), 5.63 (m, 1H), 6.97 (s, 1H), 7,15 (m, 3H), 7.53(d, 1H,
J = 9
25 Hz), APCI (M+H)+: 588.
Example B135: S-(2-bromo-5-tert-butylphenoxy)-N-(2-~[3-
(dimethylamino)propyl]amino}-4,6-dimethoxypyrimidin-5-yl)-2-furamide
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O j ~b~N\
\ 0 0 ~~~N
/ Br O\
B135
Compound B135 was synthesized according to Scheme B. NMRand mass
spectrometry data consistent with the title product were as follows: IIINMR
(300
s MHz, CDCl3): 8 1.16(s, 3H), 1.26 (s, 3H), 1.31 (s, 3H), 2.12 (dd, 2H, J
=18.51, 7.18
Hz), 2.79 (s, 6H), 3.09 (dd, 2H, J = 7.18, 6.80 Hz), 3.56 (dd, 2H, J = 16.24,
6.04 Hz),
5.39 (s, 1H), 5.47 (d, 1H, J = 3.40 Hz), 6.97 (s, 1H), 7.11 (m, 1H), 7.13 (m,
1H), 7.18
(m, 1H), 7.55 (d, 1H, J = 8.31 Hz), APCI (M+H)+: 577.
Example B136: 5-(2-bromo-5-tert-butylphenoxy)-N-(4,6-dimethoxy-2- f [2-
l0 (propylamino)ethyl]amino~pyrimidin-5-yl)-2-furamide
o ~~b~p~
H IN V
/ ~\
9r
B136
1s Compound B136 was synthesized according to Scheme B. NMRand mass
spectrometry data consistent with the title product were as follows: 1HNMR
(300
MHz, CDCl3): ~ 0.96 (dt, 3H, J = 7.66, 7.18 Hz), 1.29 (s, 9H), 1.66 (dd, 2H, J
= 7.55,
7.18 Hz), 2.75 (d, 2H, J = 7.93 Hz), 3.02 (dd, 2H, J = 6.57, 5.29 Hz), 3.65
{m, 2H),
3.87 (s, 6H), 5.43 (d, 1H, J = 3.78 Hz), 5.75 (s, 1H), 6.98 (s, 1H), 7,12 (tt,
2H, J =
20 3.78, 3.40 Hz), 7.17(m, 1H), 7.53 (d, 1H, J = 8.31 Hz), APCI (M+H)+: 577.
Example B137: 5-[(6-chloro-3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-N-(4,6-
dimethoxy-2-{[3-(4-methyl-1-piperazinyl)propyl]amino~-5-pyrimidinyl)-2-
furamide
o ~ ~p~N~
Y N\
\ O O ~~N
H. YO
~CI \
2s B137
Compound B 137 was synthesized according to Scheme B.
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I OH + ~ H3 I / I OH + HO / I OH
\ \
CI HO rt, overnight CI CI
5A 5B 5C 5D
AIC13/CHpCl2 ~'~OH
5E (from 5A 48%)
138
Compound 5A (30g) was dissolved in H2P03 (100 ml). To the solution was
added compound 5B (26g). The mixture was stirred at room temperature
overnight.
The solution was extracted with EtOAC, washed with water, dried (Mg2S04) and
s concentrated to give a crude mixture of 5C and SD (50g). The mixture was
dissolved
in 300 ml of CH2C12 and AlCl3 (31g) was added into. After being stirred at
room
temperature fox 1.5 hour the solution was poured into ice water and extracted
with
EtOAC. Column chromatography (hexane:EtOAC 18:1) gave compound 5E (22g).
NMR and mass spectrometry data consistent with the title product were as
follows:
IIiNMR (CDC13): 8 1.19 (s, 6H), 1.89 (d, 2H, J = 7.18 Hz), 1.94 (d, 2H, J
=14.73
Hz), 2.82 (s, 3H), 2.86 -2.88 (m, 2H), 3.08-3.18 (m, 4H), 3.3-3.37 (m, 4H),
3.5 - 3.65
(m, 3H), 3.79 (s, 6H), 5.50 (d, 1H, J = 3.78 Hz), 7.18 (d, 1H, J = 3.40 Hz),
7.21 (s,
1H), 7.26-7.32 (m, 1H), 7.44 (s, 1H), 8.98 (s, 1H), MS (APCI): 599.3 (M+IT)+.
Example B138: N-f2-[(2-aminoethyl)(propyl)amino]-4,6-dimethoxypyrimidin-5-
yl}-5-(2-bromo-5-tert-butylphenoxy)-2-furamide
~d N
0 0 ~~N Hz
N
Br
B138
Compound B138 was synthesized according to Scheme B. NMRand mass
spectrometry data consistent with the title product were as follows: IIiNMR
(300
2o MHz, CDC13): 8 0.88 (dt, 3H, J = 7.66, 7.18 Hz), 1.28 (s, 9H), 1.66 (dd,
2H, J = 7.55,
7.18 Hz), 3.23 (m, 2H), 3.48 (m, 2H), 3.86 (m, 8H), 5.43 (d, 1H, J = 3 Hz),
7.15(m,
4H), 7.53 (d, 1H, J = 8.31 Hz), APCI (M+H)+: 577.
Example B139: 5-(2-bromo-5-tert-butylphenoxy)-N-(2-chloro-4,6-
dimethoxypyrimidin-5-yl)-2-furamide
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O ,N' /CI
~'O
O O ~ N
0
Br
B139
Compound B I39 was synthesized according to Scheme B. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(300
s MHz, CDC13): 8 1.29 (s, 9H), 3.99 (s, 6H), 4.01 (s, 6H), 5.43 (d, 1H, J =
3.40 Hz),
7.14 (dd, 3H, J = 3.78, 2.27 Hz), 7.15 (s, 3H), 7.53 (d, 1H, J = 8.31 Hz).
APCI
(M+I~+: 510.
Example B140: N (2-chloro-4,6-dimethoxypyrimidin-5-yl)-5-[(3,3,6-trimethyl-
1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furamide
OH,OO
~~OCHy
~O
N ~ N
HacO
B140
Compound B140 was synthesized according to Scheme B. The overall yield
was 21%.
1. MCPBA ,NaHC03
H20:DCM reflux \ off
\ w
/ ~/
2. NaOCH3, MeOH
139
15 NMR data consistent with the title product were as follows: 1H NMR, (DMSO):
b
0.94 (d, 3H, J = 7.37 Hz), 0.99 (s, 3H), 1.01(s, 3H), 1.19 (s, 3H), 1.23 (s,
3H), 1.79 (q,
1H, J = 7.37 Hz), 2.22 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 5.51 (d, 1H, J =
3.59 Hz),
6.97 (s, 1H), 7.14 (s, 1H), 7.20 (d, 1H, J = 3.59 Hz).
Example B141: N-~4,6-dimethoxy-2-[(3-piperidin-1-ylpropyl)amino]pyrimdine-
20 5-yl~-5-[(3,3,6-trimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-2-furamide
0 0 0 0
N ~
B141
Compound B 141 was synthesized according to scheme B.
Example B142: N [4,6-dimethoxy-2-(2-methoxyethoxy)pyrimidin-5-yl]-S-[(3,3,6-
25 trimethyI-1,3-dihydro-Z-benzofuran-5-yl)oxy]-2-furamide
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CHa
CHa
HaC O O 0
O N
'\~NH //
O ~ ~ CHa ~ ~ N O
-CHa
HaC-O
B142
Compound B 142 was synthesized according to Scheme B. The requisite
phenol was prepared according to the following method:
ASH
HO I \ O ~ ~ O ~ ~ ~ ~ ~ OH
s i i i
Ha THF NaH
refwxovemi9ht overnight DMF reflux
OC to reflux 111
NMR data consistent with the title product were as follows: 1H NMR (MeOD): 8
1.40
(s, 6H), 2.24 (s, 3H), 3.36 (s, 3H), 3.70 (t, 2H, J = 4.53 Hz), 3.90 (s, 6H),
4.45 (dd,
2H, J = 4.91, 4.53 Hz), 4.95 (s, 2H), 5.38 (d, 1H, J = 3.40 Hz), 6.92 (s, 1H),
7.12 (m,
2~.
1o Example B143: N (4,6-dimethoxy-Z-phenoxypyrimidin-5-yl)-5-[(3,3,6-trimethyl-
1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furamide
HaC Ha OHaCO
o \ N ~
H
CHa
H9C0
B143
Compound B 143 was synthesized according to Scheme B. NMR data
consistent with the title product were as follows: 1H NMR, (MeOD): 8 I .40 (s,
6H),
2.24 (s, 3H), 3.78 (s, 6H), 4.95 (s, 2H), 5.38 (d, 1H, J = 3.40 Hz), 6.92 (s,
1H), 7.16
(m, 5H), 7.37 (dd, 2H, J = 7.93, 7.55 Hz).
Example B144: 4-(Methoxymethyl)-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl
5-[(3,3,6-trimethyl-1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furoate
B144
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Compound B 144 was synthesized according to Scheme B. NMR data
consistent with the title product were as follows:1H NMR, (DMSO): S 1.40 (s,
6H),
2.23 (s, 3H), 2.56 (s, 3H), 3.41 (s, 3H), 3.89 (s, 3H), 4.89 (s, 2H), 4.93 (s,
2H), 5.68
(d, 1H, J = 3.78 Hz), 6.99 (s, 1H), 7.21 (s, 1H), 7.24 (s, 1H), 7.65 (d, 1H, J
= 3.78 Hz).
Example B145: 1-tent-butyl-3,4-dimethyl-1H pyrazolo[3,4-b]pyridin-6-yl 5-
[(3,3,6-trimethyl-1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furoate
N-
0
B145
Compound B 145 was synthesized according to Scheme B. NMR data
1o consistent with the title product were as follows: 1H NMR, (DMSO): S 1.39
(s, 6H),
1.67 (s, 9H), 2.23 (s, 3H), 2.59 (s, 3H), 2.66 (s, 3H), 4.93 (s, 2H), 5.67 (d,
1H, J =
3.78 Hz), 6.82 (s, 1H), 7.21 (s, 1H), 7.24 (s, 1H), 7.65 (d, 1H, J = 3.40 Hz).
Example B146: N-[2-(2-hydroxyethoxy)-4,6-dimethoxypyrimidin-5-yl]-5-[(3,3,6-
trimethyl-1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furamide
H~CO
H3C Hs 0 \ ~
O ~ ~ O ~ \ ~O~OH
H3C0
15 v 'CH3
B146
Compound B 146 was synthesized according to Scheme B. NMR data
consistent with the title product were as follows: 1H NMR, (MeOD): 8 1.40 (s,
6H),
2.24 (s, 3H), 3.82 (dd, 2H, J = 5.29, 4.53 Hz), 4.39 (t, 2H, J = 4.91 Hz),
4.95 (s, 2H),
20 5.38 (d, 1H, J = 3.40 Hz), 6.92 (s, 1H), 7.11(m, 2H).
Example B147: N (2-anilino-4,6-dimethoxypyrimidin-5-yl)-5-[(3,3,6-trimethyl-
1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furamide
0 0
o W ~ ~ b y--b
I
B147
z5 Compound B 147 was synthesized according to Scheme B. NMR data
consistent with the title product were as follows: 1H NMR, (DMSO): 8 1.37 (s,
6I-~,
2.23 (s, 3H), 3.87 (s, 6H), 4.90 (s, 2H), 5.54 (d, 1H, J = 3.40 Hz), 6.94 (dd,
1H, J =
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7.55, 7.18 Hz), 7.10 (s, 1H), 7.19 (d, 2H, J = 4.15 Hz), 7.27 (dd, 2H, J =
7.93, 7.55
Hz), 7.76 (d, 2H, J = 7.93 Hz).
Example B148: N-(4,6-dimethoxy-2-}[2-(methyl}5-[(3,3,6-trimethyl-1,3-dihydro-
2-benzofuran-S-yl)oxy]-2-furoyl}amino)ethyl]amino}pyrimidin-5-yl)-5-[(3,3,6-
trimethyl-1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furamide
B148
Compound B 148 was synthesized according to acyl chloride coupling method
of Scheme B where the amine was:
~O N~ N~Ni
H2N I i N
and reacted at both the primary and alkyl-secondary amines to give the dimer B
148.
NMR data consistent with the title product were as follows: 1H NMR, (MeOD): 8
1.35 (s, 6H), 1.39 (s, 6H), 2.19 (s, 3H), 2.23 (s, 3H), 3.14 (s, 3H), 3.55 (t,
2H, J =
6.04, 5.67 Hz), 3.78 (t, 2H, J = 6.80, 6.04 Hz), 3.83 (s, 6H), 4.91 (s, 2H),
4.94 (s, 2H),
5.33 (d, 1H, J = 3.40 Hz), 5.37 (d, 1H, J = 3.40 Hz), 6.82 (s, 1H), 6.90 (s,
1H), 6.96
(d, 1H, J = 3.40 Hz), 7.07 (d, 2H, J = 4.15 Hz), 7.11 (s, 1H).
Example B149: 1,3,4-trimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl 5-[(3,3,6-
trimethyl-1,3-dihydro-2-benzofuran-5-yl)oxy]-2-furoate
N-' \
i
0 ~ ~ O N~
O ~ ~ 0
2o B149
Compound B 149 was synthesized according to Scheme B. NMR data
consistent with the title product were as follows: 1HNM>ZR, (MeOD): 8 1.40 (s,
6H),
2.22 (s, 3H), 2.59 (s, 3H), 2.66 (s, 3H), 3.87 (s, 3H), 4.95 (s, 2H), 5.44 (d,
1H, J =
3.78 Hz), 6.72 (s, 1H), 6.98 (s, 1H), 7.13 (s, 1H), 7.44 (d, 1H, J = 3.40 Hz).
Example B150: N (2,4,6-trimethoxypyrimidin-5-yl)-5-[(3,3,6-trimethyl-1,3-
dihydro-Z-benzofuran-5-yl)oxy]-2-furamide
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o f ~~o\
O O N
O I \ ~ ~ O
B150
Compound B 150 was synthesized according to Scheme B. Yield of purified
product was 36%. NMR data consistent with the title product were as follows:
1H
NMR (CDC13): 8 1.45 (s, 6H), 2.29 (s, 3H), 3.96 (s, 6H), 5.01 (s, 2H), 5.37
(d, 1H, J =
3.59 Hz), 6.80 (s, 1H), 7.07 (s, 1H), 7.14 (d, 1H, J = 3.59 Hz).
Example B151: 5-(2-bromo-5-tert-butyIphenoxy)-N-(4,6-dimethoxy-2-~[3-(4-
methylpiperazin-1-yl)propyl]amino)pyrimidin-5-yl)-2-furamide acetate salt
\ 0 0 00 0
~ n
Br N~~--N~N~1-
-N
-O
1o B151
a
Compound B 151 was synthesized according to scheme B.
Example B152: 5-[(5-chloro-1,1,7-trimethyl-2,3-dihydro-1H-inden-4-yl)oxy]-N-
(2,6-dimethoxyphenyl)-2-furamide
I
0
° o
\ / N
ci °\
1s B152
Compound B152 was synthesized according to scheme B wherein the phenol
was synthesized according to the following scheme.
O KzC03
I ~ CI CI v \ DMF
r.t., 30 min.
O
A1C13 NaCI (Et)3SiH
\ OH \ OH
210°C 15 min. ' I , TFA I ,
' ~ ~CI r.t., overnight CI
Example B153: 5-[(5-chloro-1,1,7-trimethyl-2,3-dihydro-1H-inden-4-y1)oxy]-N-
20 f 4,6-dimethoxy-2-[(3-morpholin-4-ylpropyl)amino]pyrimidin-S-yl)-2-furamide
acetate
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O O NY N~
W O O N
/ 0 0
ci
0
8153
Compound B 153 was synthesized in a manner analogous to that of compound
B 152, according to scheme B.
Example B154: 5-(2-bromo-5-tert-butylphenoxy)-N-(4,6-dimethoxy-2- f [2-
(propylamino)ethyl]amino]pyrimidin-5-yl)-2-furamide acetate salt
O IO OO--N
Br N~N N
O~-
O N~.
B154
Compound B 154 was synthesized in a manner analogous to that of compound
to B1, according to scheme B, using similar starting materials and reaction
conditions.
Example B155: N-(2-chloro-4,6-dimethoxypyrimidin-5-yl)-5-[(3,5,5,6,8,8-
hexamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)oxy]-2-furamide
~ O O O ~ -N
t , t / N,..r N CI
.«- OO
B155
is Compound B 155 was synthesized in a manner analogous to that of compound
B l, according to scheme B, using similar starting materials and reaction
conditions:
The following compounds were prepared according to Scheme C set forth
below:
Scheme C
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0
O ~ ~ NH2 (HCI)
33
B ' ~ H SOCIZ, cat. DMF B , ~ I
a
benzene, refiux (Et)3N, CHZCI2, rt
31 98%
OH 32
OO p~ ~~ O~ / O
R
Br O N ~ 35 ' ~ O O N
H O- Cs2C03 (1.3 equiv.) R'~ ~ / H
34 600 DMF, 145 °C 60-65%
15-24 h 36
5-bromo-2-furoyl chloride 32: A suspension of 5-bromo-2-furoic acid (31,
57.3 g, 300 mmol) in anhydrous benzene (100 mL) containing few drops of
anhydrous DMF' was heated to reflux under nitrogen as thionyl chloride (1.1
equiv.,
s 24.6 mL) in benzene (35 mL) was added dropwise. The resulting pale brown
solution
was further reflux for an additional 10 hours. The solution was cooled to room
temperature and concentrated under vacuum to give 61.5 g (98%) of crude acid
chloride. The 1HNMR of the crude showed >95% purity. The crude acid chloride
32
thus obtained was used in the next step without further purification. NMR data
1o consistent with the title product were as follows: 1HNMR (CDCl3): 8 6.74
(d, 1H),
7.38 (d, 1H).
5-bromo-N-(2,4,6-trimethoxyphenyl)-2-furamide 34: A mixture of
trimethoxyaniline hydrochloride (33 1.2 equiv., 21.1 g, 96 mmol) and triethyl
amine
(2.5 equiv., 27.9 mL, 200 mmol) in dichloromethane (200 mL) was stirred at
0°C
15 under nitrogen as a solution of 5-bromo-2-furoyl chloride (I6.8 g, 80 mmol)
in
dichloromethane (120 mL) was added dropwise. The solution was allowed to warm
to room temperature and further stirred for 12 hours. The resulting suspension
was
washed several times with 2N hydrochloric acid, saturated sodium bicarbonate,
brine
and water successively. The organic layer was dried over anhydrous sodium
sulfate,
2o concentrated under vacuum to give about 25 g of brown colored crude
material. The
crude material (in two equal batches) was subjected to silica gel
chromatography
using ethyl acetate/hexane 3:2 as the eluant to give 17.1 g (60%) of the
desired
bromofuranamide 34. NMR data consistent with the title product were as
follows: 1H
NMR (CDC13): 8 3.82 (s, 9H), 6.18 (s, 2H), 6.47 (d, 1H), 7.19 (d, 1H), 7.29
(br s, 1H).
25 5-aryloxy-2-furamide 36: A suspension of a phenol (35, 2.85 g, 13 mmol)
and cesium carbonate (1.3 equiv., 4.24 g, 13 mmol) in anhydrous DMF (20 mL)
was
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stirred at room temperature under nitrogen for 15 minutes before adding 5-
bromo-N-
(2,4,6-trimethoxyphenyl)-2-furamide (34, 3.57 g, 10 mmol) at once. The
resulting
suspension was stirred at 145 °C (bath temperature) for 18 hours. The
progress of the
reaction was monitored by TLC and FIMS. The suspension was quenched with cold
water and extracted several times with chloroform. The organic layer was
washed
with brine and water, dried (anhydrous sodium sulfate) and concentrated. The
crude
brown oil was further dried under high vacuum to remove last traces of
solvents. The
residue thus obtained was either recrystallized from ether or subjected to
chromatography and dried overnight at 45 °C under vacuum to give title
compound
(60-65%) as a white solid which showed >95% purity by I~PLC analysis.
Example Cl: 5-[(6-methoxy-3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)oxy]-N-
(2,4,6-trimethoxyphenyl)-2-furamide
o i
°
o °
d
o o~
C1
Compound Cl was synthesized according to Scheme C, using similar starting
ci~mpounds and reaction conditions, with the exception that the residue
obtained from
the reaction between 5-bromo-N-(2,4,6-trimethoxyphenyl)-2-furamide and
3,5,5,8,8-
pentamethyl-5,6,7,8-tetrahydro-2-naphthalenol was subjected to chromatography
to
give Compound C 1 and dried overnight at 45 °C under vacuum to give
title
compound (60-65%) as a white solid, which showed >95% purity by HPLC analysis.
2o The requisite phenol, 6-methoxy-3,3-dimethyl-5-indanol, was prepared
starting from 2-methoxyphenol via Fries rearrangement as described below:
OH + ~O PPA _ / I OH Pd/C _ / I OH
0 0H 45 to 110 °C ~ O conc. H S04 ~ O
I 2 h O I CH301-~ I
iii iv
5-hydroxy-6-methoxy-3,3-dimethyl-1-indanone iii : A mixture of 3,3-
dimethylacrylic acid, 2-methoxyphenol (1.25 equiv.), and polyphosphoric acid
(3
g/mmol) was heated to 45 °C for 0.5 hours. The reaction temperature was
gradually
increased to 110 °C and kept at that temperature for 2 hours. The
viscous oil was
cooled to 55-60 °C and extracted extensively with ethyl acetate. The
organic layer
was washed with saturated NaHC~3, dried over Na2S04 and concentrated. The
crude
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residue was recrystallized from ether to afford 5-hydroxy-6-methoxy-3,3-
dimethyl-1-
indanonesin 30-35% isolated yield. NMR data consistent with the title product
were
as follows: 1H NMR (CDC13): S 1.48 (s, 6H), 2.51 (s, 2H), 3.91 (s, 3H), 6.35
(s, 1H),
6.98 (s, 1H), 7.16 (s, 1H).
s 6-methoxy-3,3-dimethyl-5-indanol iv : 5-hydroxy-6-methoxy-3,3-dimethyl-
1-indanone was hydrogenated with 10% Pd on carbon (25 mg/mmol), in the
presence
of catalytic amount of concentrated H2SO4 ( 25 mg/mmol) in methanol (2
mL/mmol)
for 15 hours. The resulting suspension was filtered through Celite and washed
the
precipitate with ethyl acetate. The filtrate was dried over Na2S04 and
concentrated to
1o give 6-methoxy-3,3-dimethyl-5-indanol in 90% yield. NMR data consistent
with the
title product were as follows: ~H NMR (CDC13): ~ 1.10 (s, 6H), 1.78 (t, 2H),
2.70 (t,
2H), 3.74 (s, 3H), 5.41 (br s, 1H), 6.58 (s, 2H). NMR and mass spectrometry
data
consistent with the title product were as follows: 1H NMR (CDC13): 8 1.25 (d,
6H),
2.36 (s, 3H), 3.85 (s, 9H), 4.25 (m, 1H), 5.44 (d, IH), 5.9I (br d, 1H), 6.17
(s, 2H),
~s 7.11 (d, 1H), 7.I8 (s, 1H), 7.32 (d, 1H), 7.48 (d, 1H), 7.51 (s, 1H), MS
(APCI+):
469.1 (M+ H)+.
Example C2: 5-(5-tert-butyl-2-methylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
I
° °
° °
I ~ I I " ~
C2 °
2o Compound C2 was synthesized in a manner analogous to that of C1,
according to Scheme C.
The requisite phenol for the synthesis of Compound C2, 5-tert-butyl-2-methyl-
phenol, was prepared as follows:
(OHa)aNN03_ / I Pd/C i I - NaN02_
N02 NH~NH2 ~ NH2 H2S04 ~ OH
warm
143
25 5-tert-butyl-2-vitro-toluene was synthesized from 4-tert-butyltoluene in
90%
yield using tetramethylammonium nitrate according to the nitration procedure
discussed in detail above. The regiochemistry of the nitration product was
confirmed
by NOE studies. NMR data consistent with the title product were as follows: 1H
NMR (CDC13): 8 1.21 (s, 9H), 2.42 (s, 3H), 7.12 (d, 1H), 7.40 (d, 1H), 7.84
(s, 1H).
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The reduction of nitro compound to corresponding aniline was
accomplished by hydrogenation with excess hydrazine (N50 equiv.) in the
presence of Pd/C (10 mol %) in ethanol (2.5 mL/mmol) at room temperature for
15 hours. The aniline derivative was isolated as its HCl salt in 85-90% yield.
NMR data consistent with the title product were as follows: 1H NMR (CDC13): 8
1.15 (s, 9H), 2.00 (s, 3H), 6.50 (s, 1H), 6.56 (d, 1H), 6.82 (d, 1H).
5-tert-butyl-2-methylaniline was diazotized under standard conditions
(NaN02, l .I equiv.. concentrated HZS04 0.25 mL/mmol; H2O, 1.7 mL/mmol, 0
°C)
which upon heating at 50 °C for 2 hours afforded the requisite phenol
in 30-35%
yield. NMR data consistent with the title product were as follows: 1H NMR
(CDC13):
~ 1.38 (s, 9H), 2.43 (s, 3H), 4.82 (br s, 1H), 6.54 (d, 1H), 6.83 (s, 1H),
7.00 (d, 1H).
NMR and mass spectrometry data consistent with the title product were as
follows:1H
NMR (d6-DMSO): ~ 1.23 (s, 9H), 2.20 (s, 3H), 3.70 (s, 3H), 3.77 (s, 6H), 5.50
(d,
1H), 6.25 (s, 2H), 7.I I (s, 1H), 7.32-7.50 (m, 3H), 8.88 (s, 1H). The overall
yield was
is 15%, APCI-MS m/z 440.1 (M+H)+.
Example C3: 5-(5-amino-2-methylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
1
0
I
HaN
I / ~ ~ H
C3
Compound C3 was synthesized in a manner analogous to that of C1, according
2o to Scheme C. The overall yield was 60%. NMR and mass spectrometry data
consistent with the title product were as follows: 1H NMR (d4-CH30H): 8 2.16
(s,
3H), 3.78 (s, 3H), 3.82 (s, 6H), 5.46 (d, 1H), 6.28 (s, 2H), 6.50 (d, 1H),
6.52-6.56 (dd,
1H), 7.01 (d, 1H), 7.15 (d, 1H), APCI-MS m/z 399.2 (M+H)+.
Example C4: 5-[2,4-dibromo-5(tert-butyl)phenoxy]-N-(2,6-dimethoxyphenyl)-2-
25 furamide
0
\ /
B .. _Br O
C4
Compound C4 was synthesized in a manner analogous to that of C1, according
to Scheme C. Compound C4 was obtained as white solid (23 mg, 6.5 %). The
requisite phenol was prepared as follows:
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OH Br~/FIOAC \ OH
\ _
Br ~ ~ Br
144
To a solution of 3-tert-butylphenol (1.5 g, 10 mmol) in HOAc (4 mL) was
added Br2 (2 mL, 15 mmol). The reaction mixture was stirred at room
temperature ..
over night. It was quenched with ascorbic acid. The crude product was
extracted with
EtOAc, washed with brine, dried over Na2S0ø and taken to dryness. Column
chromatography with EtOAc and hexane (1:10) offered white solid product (0.42
g,
14%). NMR and mass spectrometry data consistent with the title product were as
follows: 1H NMR (300 MHz, CDC13): 8 1.46 (9H, s), 5.34 (1H, s), 7.11 (1H, s),
7.65
(1H, s), APCI-MS m/z 305 (M-H)-.
5-bromo-N-(2,6-dimthyloxyphenyl)-2-furamide (0.2 g, 0.49 mmol) and
cesium carbonate (0.21 g, 0.63 mmol) were added in the flask containing DMF (2
mL). The reaction mixture was gently heated at 130°C overnight. It was
quenched
with water, extracted with EtOAc and washed with water and brine. Organic
layer
was dried over Na2S04 and taken to dryness. Crude product was purified by
HPLC.
is NMR and mass spectrometry data consistent with the title product were as
follows:
1HNMR (300 MHz, DMSO): ~ 1.48 (9H, s), 3.78 (6H, s), 5.86 (1H, d, J = 3 Hz),
6.75
(2H, d, J = 9 Hz), 7.26 - 7.28 (2H, m), 7.39 (1H, s), 8.07 (1H, s), 9.20 (1H,
s), APCI-
MS m/z 552 (M+H)+.
Example C5: 5-[2,4-dibromo-5(tert-butyl)phenoxy]-N-(2,4,6-trimethoxyphenyl)-
2-furamide
i
I ~ ~ \ / ~
~r ~ Br
CS
Compound CS was synthesized in a manner analogous to that of C1, according
to Scheme C. NMR and mass spectrometry data consistent with the title product
were
as follows: 1H NMR (300 MHz, DMSO): ~ 1.48 (s, 9H), 3.76-3.84 (m, 9H), 5.84
(d,
2s 1H, J = 3 Hz), 6.32 (s, 2H), 7.27 (brs, 1H), 7.38 (s, 1H), 8.07 (s, 1H),
9.03 (s, 1H),
APCI-MS m/z 584 (M+H)+.
Example C7: N-(2,6-dimethoxyphenyl)-5-[2-methyl-4-(1,1,3,3-
tetramethylbutyl)phenoxy]-2-furamide
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I ~ \ / \ /
- - o
C7
Compound C7 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H NMR (CDC13): b 0.73 (9H, s), 1.36 (6H, s), 1.72 (2H, s), 2.30 (3H,
s),
3.85 (6H, s), 5.27-5.28 (1H, d, J = 3.59 Hz), 6.59-6.62 (2H, d, J = 8.31 Hz),
6.97-
7.00 (1H, d, J = 8.50 Hz), 7.14-7.15 (1H, d, J = 3.59 Hz), 7.18-7.21 (1H, d, J
=
8.31 Hz), 7.19-7.21 (1H, d, J = 8.50 Hz), 7.24 (1H, s); m/z 466.
Example C8: 5-[2-Methyl-4-(1,1,3,3-tetramethylbutyl)phenoxy]-N-(2,4,6-
1o trimethoxyphenyl)-2-furamide
0 0
o \o/ ~ \ /
- o
C8
Compound C8 was synthesized in a manner analogous to that of C1, according
to Scheme C, using similar starting compounds and reaction conditions. NMR and
mass spectrometry data consistent with the title product were as follows: 1H
NMR
~s (CDCl3): 8 0.72 (9H,s), 1.35 (6H, s)~1.71(2H, s), 2.28 (3H, s), 3.81(9H,
s), 5.25-5.26
(1H, d, J = 3.59 Hz), 6.16 (2H, s), 6.96-6.99 (1H, d, J = 8.49 Hz)~ 7.13-7.14
(1H, d, J
= 3.02 Hz), 7.18-7.21 (1H, d, J = 8.31 Hz), 7.23 (1H, s); m/z 496.
Example C9: 5-(3-hydroxy-2-mehtylphenoxy)-N-(2,4,6-trimethyoxyphenyl)-2-
furamide
/
0 0
I ~ \o/ \ / °
0
2o Cg o" \
Compound C9 was synthesized in a manner analogous to that of C 1,
according to Scheme C, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows: 1H NMR, (MeOD): 8 2.14 (3H, s), 3.81 (6H, s), 3.82 (3H, s), 5.41-5.42
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(IH, d, J = 3.59 Hz), 6.27 (2H, s), 6.58-6.61 (1H, d, J = 4.91 Hz), 6.67-6.69
(1H,
d, J = 8.31 Hz), 7.01-7.06 (1H, t, J = 5.85 Hz), 7.13 (1H, d, J = 3.02 Hz);
m/z 400.
Example C10: 5-[(1,1,3,3,6-pentamethyl-2,3-dihydro-1H-inden-5-yl)oxy]-N-
(2,4,6-trimethoxyphenyl)-2-furamide
0
\ /
i
v _ y
C10
Compound C10 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
Yield of purified product was 4%. NMR data consistent with the title product
were as follows: 1H NMR, (CDCl3): 8 1.27 (6H,s), 1.30 (6H, s), 1.92 (2H, s),
2.27
(3H, s), 3.81 (9H, s), 5.33-5.35 (1H, d, J= 3.40 Hz), 6.17 (2H, s), 6.18 (1H,
s),
6.69 (1H, s), 7.15-7.16 (1H, d, J = 3.40 Hz); m/z 480.
Example Cll: 5-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-N-
(2,4,6-trimethoxyphenyl)-2-furamide
/
o ° i o.
~ o o N ~ I
~ \/ H
C11
~s Compound C11 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
Yield of purified product was 4%. NMR and mass spectrometry data consistent
with
the title product were as follows: 1H NMR (d6-DMSO): ~ 1.30 (s, 12H), 1.70 (s,
4H),
3.78 (s, 6H), 3.85 (s, 3H), 5.85 (d, 1H), 6.33 (s, 2H), 6.95-6.99 (dd, 1H),
7.19 (d, 1H),
7.43 (d, 1H), 8.98 (s, 1H), APCI-MS m/z 480.1 (M+H)+.
Example C12: 5-[(5-methoxy-1,1-dimethyl-1H-inden-6-yl)oxy]-N-(2,4,6-
trimethoxyphenyl)-2-furamide
I ~ o \ / ~ of
C12 °
Compound C12 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
follows:1H
NMR (d4-CH30H): 8 I.28 (s, 6H), 3.80 (s, 3H), 3.8I (s, 3H), 3.84 (s, 6H), 5.24
(d,
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1H), 6.26 (s, 2H), 6.44 (d, 1H), 6.62 (d, 1H), 7.10 (d, 1H), 7.10 (s, 1H),
7.20 (s, 1H),
APCI-MS m/z 466.2 (M+H)+.
Example CI3: 5-[(3-formyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
I
o°
I ~ o o ~ I
\ / H
C13 0
Compound C13 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
00 ~ o. oo ~ o~
\O/ N ~ I tBuLi, DMF I ~ O ~ / N
O~ i O~
II
O
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
1o NMR (CDC13): 8 1.18 (s, 6H), 1.23 (s, 6H), 1.62 (s, 4H), 3.73 (s, 9H), 5.55
(d, 1H),
6.08 (s, 2H), 6.89 (d, 1H), 7.08 (d, 1H), 7.09 (s, 1H), 7.82 (s, 1H), 10.32
(s, 1H),
APCI-MS m/z 508.2 (M+H)+.
)Jxample C14: 5-{4-[2-(dimethylamino)ethoxy]-5-isopropyl-2-methylphenoxy}-N-
(2,4,6-trimethoxyphenyl)-2-furamide
I
_ o o ~ o~
I
I ~ ~ ° \o/
1s C14'N'~o
Compound C14 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
NaOH \ \ I OH
/N ~°
145
NMR and mass spectrometry data consistent with the title product were as
2o follows: 1H NMR (CDC13): 8 1.16 (s, 3H), 1.18 (s, 3H), 2.24 (s, 3H), 2.44
(s, 6H),
2.90 (t, 2H), 3.29-3.35 (s, m), 3.79 (s, 6H), 3.81 (s, 3H), 4.14 (t, 2H), 5.21
(d, 1H),
6.26 (s, 2H), 6.87 (s, 1H), 6.99 (s, 1H), 7.11 (d, 1H), APCI-MS rnlz 513.4
(M+H)+.
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Example C15: 5-(3-tert-butyl-5-methoxyphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
I
o°
I~ o o ~I
\ / H o
0
C15
Compound C15 was synthesized in a manner analogous to that of Cl,
according to Scheme C, using similar starting compounds and reaction
conditions.
/ OH
/ I OH Br2/CC14 ~ I OH CuBr, NaOMe I
0 0~ ~ Br cat. EtOAc, MeOH
reflux
120 137
NMR and mass spectrometry data consistent with the title product were as
follows: 1H NMR (d6-DMSO): ~ 1.26 (s, 9H), 3.72 (s, 6H), 3.79 (s, 6H), 5.35
(d,
1H), 6.27 (s, 2H), 7.14 (d, 1H), 7.14 (d, 1H), 7.2 (d, 1H), 7.25 (s, 1H), 8.86
(s,
1H), APCI-MS m/z 456.1 (M+H)+.
Example C16: 5-[5-(dimethylamino)-2-methylphenoxy]-N-(2,4,6-
trimethoxyphenoxy)-2-furamide
I
o°, o
iN ~ O O N
\ / H
C16
Compound C16 was synthesized in a manner analogous to that of Cl,
according to Scheme C, using similar starting compounds and reaction
conditions.
I
H2N~OH aq, HCHO iN / I OH
Pd/C (10 mold)
151
NMR and mass spectrometry data consistent with the title product were as
follows: IH NMR (d4-CH30H): 8 2.26 (s, 3H), 3.05 (s, 6H), 3.79 (s, 6H), 3.82
(s,
3H), 5.50 (d, 1H), 6.27 (s, 2H), 6.80 (d, 1H), 6.90 (d, 1H), 7.25 (s, 1H),
7.35 (d,
1H), APCI-MS m/z 427.2 (M+H)+.
Example C17: 5-(5-tert-butyl-2,4-dimethylphenoxy)-N-(2,4,6-trimethoxyphenyl)-
2-furamide
I
I
I w o \o/ H
o~
C17
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Compound C17 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
I
I OH Br2, CCI4 / I OH K2C03, CH31 ~ I O tBuLi, CH31
heat gr ~ Br acetone Br ~ Br
144
BBr3 , I OH
148
NMR and mass spectrometry data consistent with the title product were as
s ~ follows: 1H NMR (CDCl3): 8 1.36 (s, 9H), 2.21 (s, 3H), 2.49 (s, 3H), 3.81
(s, 9H),
5.25 (d, 1H), 6.18 (s, 2H), 6.99 (s, 1H), 7.09 (d, 1H), 7.09 (s, 1H), 7.17 (s,
1H),
APCI-MS r~alz 454.3 (M+H)+.
Example C18: 5-[(3-Chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-furamide
0
0 0
\/ H
~cl o
1o C18
Compound C18 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
The requisite phenol was synthesized according to the following scheme:
OFi CI CI AICI3 I ~ OH
I ~ CI ~ ~~~ GH3N02 ~ CI
rt
38% yield
15 NMR and mass spectrometry data consistent with the title product were as
follows:1H NMR (CDC13): S 1.22 (s, 6H), 1.27 (s, 6H), 1.67 (s, 4H), 3.81 (s,
9H),
5.43 (d, 1H), 6.17 (s, 2H), 7.11 (d, 1H), 7.1~ (s, 1H), 7.34 (s, 1H), APCI-MS
m/z
514.4 (M+H)+.
Example C19: 5-[(6-chloro-3,3-dimethyl-2,3-dihydro-1H-indol-5-yl)oxy]-N-
20 (2,4,6-trimethoxyphenyl)-2-furamide(8783)
i
0 0
0 0
\/ H
C~ 0
C19
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Compound C19 was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
The requisite phenol was synthesized according to the following scheme:
0
OMe ~ D ~ I OMe
i I Me0 CI
H2N ~ CI Et3N, CHZCIz Me0 HN CI LDA, THF
45~ rt, 5h
OMe
' 'N ~ I CI AIC13 I ~ OH
130°C H ~ CI
96~
149
NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (DMSO-d6): S 1.23 (s, 6H), 2.60 (s, 1H) 3.33 (s, 2H), 3.39 (s, 2H), 3.75
(s, 9H),
5.27 (d, 1H, J = 3.59 Hz), 6.10 (s, 2H), 6.70 (s, 1H), 6.84(s, 1H), 7.06 (d,
1H, J = 3.40
Hz), 7.19 (s, 1H), 7.22 (s, 1H), APCI-MS m/z 473 (M+H)+.
Example C20: 5-[(7-chloro-4,4-dimethyl-1,2,3,4-tetrahydroquinolin-6-yl)oxy]-N-
1o (2,6-dimethoxyphenyl)-2-furamide
0
0
w o o N w I
I~ \/
C20 H c1
Compound C20 was synthesized in a manner analogous to that of Cl,
according to Scheme C, using similar starting compounds and reaction
conditions.
NMR and mass spectrometry data consistent with the title product were as
~s follows: 1H NMR (DMSO-d6): 8 1.11 (s, 6H), 1.53 (t, 2H, J = 5.67 Hz), 3.10
(t,
2H, J = 5.67 Hz), 3.68 (s, 6H), 5.23 (d, 1H, J = 3.59 Hz), 6.52 (s, 1H), 6.63
(m,
2H), 7.07 (d, 1H, J = 3.40 Hz) 7.12 (s, 1H), 7.19 (d, 1H, J = 4.15 Hz), 8.96
(s,
1H), Al'CI-MS m/z 456 (M+H)+.
Example C21: N-(2,4,6-trimethoxyphenyl)-5-[(4,4,7-trimethyl-1,2,3,4-tetrahydro-
20 6-quinolinyl)oxy]-2-furamide
I
D °
0 0
\ / H
~o
C21
Compound C2I was synthesized in a manner analogous to that of C1,
according to Scheme C, using similar starting compounds and reaction
conditions.
The particular procedure utilized is shown below:
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OII Br Br
\ I Br ~CI ~HN / I \ Br ~N
HzN Et3N, CHzCl2 LDA, THF
45% rt, overnight
83%~
O
AICI3 ~ Br NaOMe ~~~OMe ~CI ~ OMe
- o ' N ~ / ~ < ~~/ ~ N I
130 C CuBr, DMF N Et3N, CH2CI2
~0 H ~O
7go~ 55%
O O / O\
BBrs/CH2CIz I ~ OH general scheme 2A ~ O ~O~ H \ I
/
O O
70% 71
00 / ~ O\
NaOH/EtOH
reflux I / O~
N
H
C21
NMR and mass spectrometry data consistent with the title product were as
follows:
1HNMR (DMSO-d6): 8 1.24 (s, 6H), 1.70 (t, 2H, J = 5.76 Hz), 2.15 (s, 3H), 3.29
(t,
2H, J = 5.76 Hz), 3.83 (s, 9H), 5.15 (d, 1H, J = 3.59 Hz), 6.20 (s, 2H), 6.32
(s, 1H),
6.95 (s, 1H), 7.07 (d, 1H, J = 3.59 Hz), 7.18 (s, 1H), APCI-MS m/z 467 (M+I~+.
Example C22: [3,S-dimethoxy-2-(]S-[(3,S,S,8,8-pentamethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)oxy]-2-furoyl}amino)phenoxy]acetic acid
z.._ p
HO~
OO
p O
/ H O
C22
Compound C22 was synthesized in a manner analogous to that of C1,
to according to Scheme C, using similar starting compounds and reaction
conditions.
The particular procedure utilized is shown below:
OH
O
O / O\ ~ / O\
O O I 1. BBr3 O O
~ I N~ 2. ~ ~ I H
~H O O ~ / p
/ w gr~
OMe
3, NaOH
C22
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NMR and mass spectrometry data consistent with the title product were as
follows: 1H
NMR (CH30D): 8 1.13(s, 6H), 1.19 (s, 6H), 1.59 (s, 4H), 2.13 (s, 3H), 3.68 (s,
3H),
3.72 (s, 3H), 4.57 (s, 2H), 5.21 (d, 1H, J = 3.78 Hz), 6.01 (d, 1H, J = 2.64
Hz), 6.12
(d, 1H, J = 2.64 Hz), 6.94 (s, 1H), 7.09 (d, 1H, J = 3.40 Hz), 7.15 (s, 1H),
APCI-MS
m/~ 538 (M+H)+.
Example C23: [(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)oxy]-
N(2,4,6-trimethoxyphenyl)-2-furamide
°
I pil
C23
Compound C23 was synthesized according to Scheme C. The requisite phenol
1o was synthesized according the following method:
OH 1 DCIv~I' ~ OH
Cl /
34 35
3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenol 36: A solution of
o-cresol (17.3 g, 160 mmol), 2,5-dichloro-2,5-dimethylhexane (32.1 g, 175
mmol) in
dichloromethane (80 mL) was stirred at 0°C under nitrogen as anhydrous
AlCl3 (2.34
1s g, 17.5 mmol) was added portionwise while keeping the temperature below
5°C. The
suspension was allowed to warm to room temperature and further stirred for
about 15
hours. The resulting white suspension was poured into ice water (50 mL) and
the
aqueous layer was extracted with ethyl acetate (2 x 50 mL). The combined
organic
extracts were washed with water and brine, dried over anhydrous Na2S04 and
2o concentrated. The white solid thus obtained was recrystallized from toluene
to give
28 g (80%) of the desired phenol. NMR data consistent with the title product
were as
follows: 1HNMR (CDCl3): 8 1.25 (s, 12H), 1.60 (s, 4H), 2.25 (s, 3H), 4.73 (s,
1H),
6.69 (s, 1H), 7.03 (s, 1H).
Compound C23: A suspension of phenol 3f (2.85 g, 13 mmol) and cesium
2s carbonate (1.3 equiv., 4.24 g, 13 mmol) in anhydrous DMF (20 mL) was
stirred at
room temperature under nitrogen for 15 minutes before adding 5-Bromo-N-(2,4,6-
trimethoxyphenyl)-2-furamide (3.57 g, 10 mmol) at once. The resulting
suspension
was stirred at 145 °C (bath temperature) for 18 hours. The progress of
the reaction
was monitored by TLC and FIMS. The suspension was quenched with cold water and
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extracted several times with chloroform. The organic layer was washed with
brine
and water, dried (anhydrous sodium sulfate) and concentrated. The crude brown
oil
was further dried under high vacuum to remove last traces of solvents. The
residue
thus obtained was either recrystallized (Compound C23) from ether and dried
s overnight at 45 °C under vacuum to give title compound (60-65%) as a
white solid
which showed >95% purity by HPLC analysis. NMR and mass spectrometry data
consistent with the title product were as follows: IIINMR (300 MHz, CDC13): 8
1.16
(s, 6H), 1.20 (s, 6H), 1.60 (s, 4H), 2.18 (s, 3H), 3.75 (s, 9H), 5.25 (d, 1H),
6.11 (s,
2H), 6.92 (s, 1H), 7.04 (d, 1H), 7.08 (s, 1H), 7.13 (br s, 1H), APCI-MS rralz
494.2
io (M+H)+.
The following compounds were prepared according to Scheme D set forth
below:
Scheme D
/ /
H ~ - CszC03 DMF
+B ~ \ / ~(OT~2> Cat ~ I / ~ \
16
NaOH _ H I ~ I / - R~1F-I2(excess)
MeOH ~ ~ \ / HATU,DMF
19
RH I j I ~ /
20 O~
is
Condensation 18: A DMF solution (10 mL) containing 16 (0.365 g, 2.2
mmol), 17 (0.712 g, 2.0 mmol), cesium carbonate (0.716 g, 2.2 mmol), and
copper
triflate (0.036 g, 5 mol%), was heated at 100°C for 16 hours. DMF was
then removed
under reduced pressure. The crude was redissolved in ethylacetate and washed
with
20 10%HCI, brine, dried (magnesium sulfate) and evaporated. The product 18 was
purified by flash chromatography (1:1 EtOAc/Hexanes to EtOAc): 0.13 g.
Saponification 19: Compound 18 (0.126 g, .28 mmol) was saponified in
methanolic NaOH. The reaction was monitored by TLC (1:1 EtOAc/Hexanes) and
NaOH was added as needed. The reaction mixture was then concentrated. The
crude
2s was redissolved in water and extracted with diethyl ether. The aqueous
layer was
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acidified to pH 2-3 with concentrated HCl and extracted with methylene
chloride.
The methylene chloride extract was dried (magnesium sulfate) and evaporated to
give
the desired acid 19: 0.10 g.
HATU coupling 20: To a DMF solution (1 mL) containing 19 (0.056 g, .12
mmol) was added HATU (0.046 g, 0.121 mmol), and isopropylamine (9 molar
excess). The reaction mixture was stirred at room temperature overnight. It
was then
diluted with ethyl acetate, washed with 10%HCl, saturated sodium bicarbonate,
brine,
dried (magnesium sulfate), and concentrated. The product 20 (Compound Dl) was
purified by prep TLC (5% MeOH m CH2C12): 0.019 g.
to Example D1: 5-~5-[(isopropylamino)carbonyl]-2-methylphenoxy}-N-(2,4,6-
trimethoxyphenyI)-2-furamide
I
0.
~H ' i ~ ~ H
D1 °'
Compound D1 was synthesized according to Scheme D above. NMR and
mass spectrometry data consistent with the title product were as follows: 1H
NMR
1s (CDCl3): 8 1.25 (d, 6H), 2.36 (s, 3H), 3.85 (s, 9H), 4.25 (m, 1H), 5.44 (d,
1H), 5.91
(br d, 1H), 6.17 (s, 2H), 7.11 (d, 1H), 7.18 (s, 1H), 7.32 (d, 1H), 7.48 (d,
1H), 7.51 (s,
1H), MS (APCI ~: 469.1 (M+IT)+.
Example D2: 5-carboxy-(2-methylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
FI° / ~ ° I
2o D2
Compound D2 was synthesized in a manner analogous to that of D1,
according to Scheme D. The yield of the purified product was 11 %. NMR and
mass
spectrometry data consistent with the title product were as follows: 1H NMR
(CDCl3): 8 2.39 (3H, s), 3.82 (9H, s), 5.48 (1H, d), 6.16 (2H, s), 7.14 (1H,
d), 7.20
25 (1H, s), 7.35 (1H, d), 7.76 (1H, s), 7.85 (1H, d), FI-NCI m/z 426.1 (M-H).
Example D3: 5-~5-[(diethylamino)carbonyl]-2-methylphenoxy]-N-(2,4,6-
trimethoxyphenyl)-2-furamide
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0 0 ° ~
0
H
/ ,o
J
D3
Compound D3 was synthesized in a manner analogous to that of D1,
according to Scheme D, with the exception that compound 18 was coupled with
diethylamine to give Compound D3, 5-~5-[(diethylamino)carbonyl]-2-
s methylphenoxy}-N-(2,4,6-trimethoxyphenyl)-2-furamide. NMR and mass
spectrometry data consistent with the title product were as follows: 1H NMR
(300
MHz, CDC13): 8 1.15 (br, 6H), 2.38 (s, 3H), 3.28 (br, 2H), 3.50 (br, 2H), 3.82
(s, 9H),
5.51 (d, 1H), 6.18 (s, 2H), 7.05 (s, 1H), 7.15 (m, 3H), 7.28 (s, 1H), APCI-MS
m/z
483.1 (M+H)+.
to
Diarylthio ether analogs of the present invention, such as Compound F1, can
be synthesized according to Scheme F shown and described below:
Scheme F
'/\~ 0 0
R-SH + B~~O~ NaH S O ~ NaOH S O
~J ---RB \I o ~R' \/ OH
40 11 41 42
O
_HATU S O
R- H R~ \ / NHR
44
OR
° SOC12 ° R-NHZ O
S ° ~ S O ~ S O
R \ I OH R~ \ I C~ ~ R' \ I NHR
42 43
1s Methyl 5-thioaryl-2-furoate 41: A suspension of thiol 40 (1 mmol), NaH
(60% dispersion in mineral oil, 1.1 mmol) in anhydrous N,N-dimethylacetamide
(DMA, 3 mL/mmol) was heated under nitrogen at 60-70 °C with stirring
for 30
minutes. A solution of methyl 5-bromo-2-furoate 11 (1 mmol) in anhydrous DMA
(1
mL/mmol) was added dropwise to the hot solution. The temperature of the
reaction
20 mixture was gradually increased to 145°C. The reaction mixture was
stirred at this
temperature for an additional I S hours. The solution was cooled and
concentrated to
about one quarter of its original volume in vacuo before adding to cold water
(10 mL).
The solution was extracted with ether (3 X 10 mL). The combined ether layer
was
washed with saturated NaHCO3 (2 X 5 mL) and brine (2 X 5 mL), dried (Na2S04)
and
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concentrated in vacuo to give pure methyl 5-thioaryl-2-furoate 41.
Acidification of
the aqueous layer resulted a mixture consisted of small amounts of 5-thioaryl-
2-furoic
acid and 5-bromo-2-furoic acid. Yield of 41: 40-75%.
Saponification 42: Compound 41 was saponified to the acid in ethanolic
s NaOH solution under similar reaction conditions as described above.
HATU coupling 45: The acid 42 was dissolved with DMF and coupled with
the amine 44 under similar reaction conditions as described above to produce
the
Compound 45.
Acylhalide route 45: The acylhalide route via 43 can also be used to produce
1o Compound 45.
Example Fl: 5-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-
naphthalenyl)sulfanyl]-N-(2,4,6-trimethoxyphenyl)-2-furamide
o
\ / ~ \ /
- - o
F1 \
Compound Fl was synthesized according to Scheme F. The overall yield was
1s 2,0%. NMR and mass spectrometry data consistent with the title product were
as
follows: 1H NMR (300 MHz, CDCl3): 8 1.18 (s, 6H), 1.25 (s, 6H), 1.63 (s, 4H),
2.93
(s, 3H), 3.78 (s, 6H), 3.80 (s, 3H), 5.17 (br s, 2H, hydrated water), 6.14 (s,
2H), 6.58
(d, 1H), 7.12-7.13 (d, 2H), 7.24 (d, 1H), 7.46 (s, 1H), MS (APCI) m/z 510.1
(M+H)+.
Example F2: 5-[(2,5-dimethoxyphenyl)sulfanyl]-N-(2,4,6-trimethoxyphenyl)-2-
2o furamide
F2
Compound F2 was synthesized in a manner analogous to that of F 1, according
to Scheme F. NMR and mass spectrometry data consistent with the title product
were
as follows: 1F~MR (300 MHz, CDCl3): 8 3.72 (s, 3H), 3.82 (s, 6H), 3.83 (s,
3H),
2s 3.89 (s, 3H), 6.18 (s, 2H), 6.54 (d, 1H), 6.75 (dd, 1H), 6.84 (d, 1H), 6.85
(d.lH), 7.28
(s, 1H), 7.49 (s, 1H), APCI-MS mlz 446.0 (M+IT)+. The overall yield was 45%.
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Example F3: 5- f [5-(tert-butyl)-2-methylphenyl]sulfanyl,~-N-(2,4,6-
trimethoxyphenyl)-2-furamide
F3
Compound F3 was synthesized in a manner analogous to that of F1, according
s to Scheme F. The overall yield was 45%. NMR and mass spectrometry data
consistent with the title product were as follows: 1H NMR (300 MHz, CDC13): 8
1.26
(s, 9H), 2.35 (s, 3H), 1.70 (s, 6H), 1.72 (s, 3H), 6.10 (s, 2H), 6.60 (d, 1H),
7.04 - 7.10
(m, 3H), 7.13 (d, 1H), 7.21 (s, 1H), APCI-MS m/z 456.1(M+H)+.
1o The following compounds were prepared according to Scheme H set forth
below:
Scheme H
\ off /
s
+
H \ ~ ° / \ /
53 54
1-methyl-2-pyn'olidinone / /, /
Microwave
- HO
~ ~ / \
P
Synthesis of Compound 55: Phenol, bromide, and Cs2C03 were semi-
15 dissolved in minimum I-methyl-2-pyrrolidinone. Mixture was placed in
microwave
for 2 minutes at 1100 W then allowed to cool for 1 minute before placing the
mixture
in microwave for an additional 2 minutes. Reaction was quenched with water/HCl
and extracted with ethyl acetate.
Example Hl: N-(2,6-dimethoxyphenyl)-5-[(6'-hydroxy-4,4,4',4',7,7'-hexamethyl-
20 2,2'-spirochromen-6-yl)oxy]-2-furamide
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0 0
O I / O ~O/
'~I \y/~O~/
Hl ° /
Compound H1 was synthesized according to Scheme H. Purification was
done by HPLC to give a 26% overall yield. NMR and mass spectrometry data
consistent with the title product were as follows: IHhIM>ZR, (MEOD): 8 1.30
(6H, s),
1.31 (6H, s), 1.56 (6H, s), 1.58 (6H, s), 1.92 - 1.98 (2H, dd, J = 4.35 Hz,
14.14 Hz),
2.03 (3H, s), 2.04 - 2.11 (2H, dd, J = 6.04 Hz, 14.14 Hz), 2.13 (3H, s), 3.82
(6H, s),
5.29 (1H, d, J = 3.77 Hz), 6.29 (1H, s), 6.52 (1H, s), 6.68 - 6.71 (2H, d, J =
8.69 Hz),
6.72(lH,s),7.13(lH,s),7.13-7.14(lH,d,J=2.61Hz),7.22-7.28(lH,t,J=8.50
Hz); m/z 614.
to Example H2: 5-~[7-( f 5-[(2,6-dimethoxy anilino)carbonyl]-2-furyl~oxy)-
4,4,4',4',7,7'-hexamethylbis-2,2'-spirochromen] oxy}-N-(2,6-dimethoxyphenyl)-2-
furamide
p ° 00
O I I I HN
~~ /O\ O ~ I O~ ~'0
O
0
H2
Compound H2 was synthesized in a manner analogous to that of Hl,
1s according to Scheme H, using similar starting compounds and reaction
conditions.
The yield of the title product was 31 %. NMR and mass spectrometry data
consistent
with the title product were as follows: 1H NMR, (MEOD): 8 1.34 (6H, s), 1.61
(6H,
s), 1.99 - 2.04 (2H, d, J = 14.16 Hz), 2.14 (6H, s), 2.15 - 2.17 (2H, d, J =
14.16 Hz),
3.82 (12H, s), 5.31 (2H, d, J = 3.58 Hz), 6.55 (2H, s), 6.67-6.72 (4H, d, J =
8.49 Hz),
20 7.12-7.15 (2H, d, J = 3.58 Hz), 7.16 (2H, s), 7.22-7.28 (2H, t, J = 8.40
Hz); m/z 859.
The following compounds were synthesized via the phenol coupling procedure
set forth below in Scheme I:
Scheme I
00 /
R X Br ' ' a. R~ \ O O O O -
I / + ~ O/ H ~ / O\ ~ I / ~ / ~ ~ / O\
O
\ O\
X is SH or OH
25 a. CsC03, DMF, microwave 220°C, iminute; 180°C, l0minutes
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To an empty glass tube (specifically manufactured for the Smith synthesizer),
phenol (77.6 mgs, 0.421 mmol, 1.5 ec~, 5-bromo-N (2,4,6-trimethoxyphenyl)-2-
furamide (100 mgs, 0.28 mmol, l.Oec~, and Cesium carbonate (183 mgs, 0.56
mmol,
2.0 e~ were suspended in 2.25 mL of anhydrous DMF. A Teflon coated stir bar
was
added. The tube was crimp-sealed and placed in the Smith Synthesizer microwave
then heated for 1 minute at 220°C, and 10 minutes further at
180°C.
Example Tl: 5-(1,3-benzodioxol-5-yloxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
!~
0
o i I o\
\/
I1
Compound I1 was synthesized by Scheme I. Product was purified by HPLC
to to yield compound in 10-50% yield. NMR and mass spectrometry data
consistent
with the title product were as follows: 1HNMR (CD30D): 8 3.79 (s, 9H), 3.81
(s, 3H),
5.57 (d, 1H, J = 3.59 Hz), 5.99 (s, 2H), 6.26 (s, 2H), 6.67 (dd, 1H, J = 8.50,
2.27 Hz),
6.76 (dd, 1H, J = 2.27 Hz), 6.82 (d, 1H, J = 8.50 Hz), 7.13 (d, 1H, J = 2.64
Hz), Mass
APCI 414.1.
15 Example I2: 5-(3-morpholin-4-ylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
I w r~ /o\
\ I N 1
~IO
Compound I2 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
2o spectrometry data consistent with the title product were as follows: 1HNMR
(CD3OD): 8 3.17 (t, 4H, J = 4.82 Hz), 3.79 (s, 9H), 3.81 (t, 4H), 5.66 (d, 1H,
J = 3.59
Hz), 6.26 (s, 2H), 6.63 (dd, 1H, J = 7.93, 1.89 Hz), 6.78 (t, 1H, J = 2.17
Hz), 6.84 (d,
1H, J = 7.93 Hz), 7.15 (d, 1H, J = 2.64 Hz), 7.27 (t, 1H, J = 8.22 Hz), Mass
APCI
455.2.
25 Example I3: 5-(4-isopropyl-3-methylphenogy)-N-(2,4,6-trimethylphenyl)-2-
furamide
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I
o ~ o~
0 0
\ / b o
I3
Compound I3 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
s (CD30D): 8 1.18 (m, 3H), 1.23 (s, 3H), 2.32 (s, 3H), 3.16 (t, 1H, J = 6.80
Hz), 3.79 (s,
6H), 3.81 (s, 3H), 5.59 (d, 1H, J = 3.40 Hz), 6.24 (s, 2H), 6.96 (m, 2H), 7.14
(s, 1H),
7.28 (d, 1H, J = 9.44 Hz), APCI 426.2.
Example I4: 5-(4-chloro-5-isopropyl-2-methylphenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
I
0 0
p \/ o
a w
l0 I4
Compound I4 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1
(CD30D): b 1.20 (d, 6H, J = 6.99 Hz), 2.25 (s, 3H), 3.37 (m, 1H, J = 6.89 Hz),
3.78
15 (s, 6H), 3.83 (s, 3H), 5.46 (d, 1H, J = 3.59 Hz), 6.26 (s, 2H), 7.06 (s,
1H), 7.15 (d, 1H,
J = 2.83 Hz), 7.31 (s, 1H), Mass APCI 460.1.
Example I5: 5-(3-isopropylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
b / \ ~ I
0
IS I
Compound IS was made in a manner analogous to Il, according to Scheme I,
2o using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: lI3NMR
(CD30D): 8 1.23 (d, 6H, J = 6.99 Hz), 2.91 (q, 1H, J = 6.92 Hz), 3.77 (s, 6H),
3.80 (s,
3H), 5.64 (d, 1H, J = 3.59 Hz), 6.25 (s, 2H), 6.95 (d, 1H, J = 8.12 Hz), 7.04
(s, 1H),
7.10 (d, 1H, J = 7.74 Hz), 7.16 (s, 1H), 7.31 (t, 1H, J = 7.93 Hz), APCI mass
412.1.
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Example I6: 5-[4-(cyanomethyl)phenoxy]-N-(2,4,6-trimethoxyphenyl)-2-
furamide
b w
o I~
~I
N
I6 ''
Compound I6 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): 8 3.78 (s, 6H), 3.81 (s, 3H), 3.90 (s, 2H), 5.74 (d, IH, J = 3.59
Hz), 6.26 (s,
2H), 7.19 (t, 3H, J = 8.50 Hz), 7.42 (d, 2H, J = 8.69 Hz), APCI 409.1.
Example I7: 5-(4-benzylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
to I7
Compound I7 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: IIINMR
(CD3OD): b 3.76 (s, 6H), 3.79 (s, 3H), 3.94 (s, 2H), 5.58 (d, 1H, J = 3.40
Hz), 6.24 (s,
1s 2H), 7.14 (m, IOH), Mass APCI 460.1.
Example I8: 5-(1,1'-biphenyl-4-yloxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
I8
Compound I8 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
2o spectrometry data consistent with the title product were as follows: 1HNMR
(DMSO-
d6): 8 3.71 (s, 6H), 3.78 (s, 3H), 5.94 (d, 1H, J = 3.59 Hz), 6.27 (s, 2H),
7.26 (d, 3H, J
= 8.69 Hz), 7.36 (t, 1H, J = 7.27 Hz), 7.46 (dd, 2H, J = 7.74, 7.18 Hz), 7.65
(d, 2H, J =
7.37 Hz), 7.73 (d, 2H, J = 8.88 Hz), 8.97 (s, 1H), APCI mass 446.4.
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Example I9: 5-(3,4-dimethoxyphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
/\
0
of ,
0
I9 ~
Compound I9 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): b 3.79 (s, 6H), 3.81 (s, 3H), 3.83 (s, 6H), 5.57 (d, 1H, J = 3.59
Hz), 6.26 (s,
2H), 6.74 (dd, 1H, J = 8.69, 2.64 Hz), 6.87 (d, 1H, J = 2.64 Hz), 6.96 (d, 1H,
J = 8.88
Hz), 7.14 (d, 1H, J = 2.64 Hz), APCI mass 430.2.
Example I10: S-~3-[(1S)-1-hydroxy-2-(methylamino)ethyl]phenoxy}-N-(2,4,6-
1o trimethoxyphenyl)-2-furamide
Chirel
/
0
o I i
b
I10 0"
Compound I10 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
15 (CD30D): 8 2.73 (s, 3H), 3.11 (dt, 1H, J =12.46, 9.82 Hz), 3.23 (m, 1H, J
=12.65,
9.25, 3.40, 3.40 Hz), 3.78 (s, 6H), 3.82 (s, 3H), 4.97 (dt, 1H, J = 9.63, 3.21
Hz), 5.76
(d, 1H, J = 3.59 Hz), 6.26 (s, 2H), 7.17 (s, 2H), 7.29 (s, 2H), 7.45 (s, 1H),
APCI mass
443.3.
Example Ill: S-(dibenzo[b,d]furan-2-yIoxy)-N-(2,4,6-trimethoxyphenyl)-2-
2o furamide
o ,
/ \ ~ I o /o\ b ~
I
I11
Compound I11 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
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(CD30D): 8 3.76 (s, 6H), 3.78 (s,, 3H), 5.62 (d, 1H, J = 3.40 Hz), 6.23 (s,
2H), 7.16 (d,
1H, J = 2e 83 Hz), 7.30 (d, 1H, J = 5.29), 7.34 (d, 1H, J = 7.74 Hz), 7.48
(dd, 1H, J =
8.12, 7.18 Hz), 7.57 (dd, 2H, J = 8.50, 6.61 Hz), 7.83 (s, 1H), 7.97 (d, 1H, J
= 7.74
Hz), Mass APCI 460.4.
Example I12: 5-(4-amino-3-chlorophenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
/\ p
0
I
~I o
I12 N"
Compound I12 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
1o spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): 8 3.79 (s, 6H), 3.81 (s, 3H), 5.52 (d, 1H, J = 3.59 Hz), 6.26 (s,
2H), 6.89 (d,
1H, J = 8.88 Hz), 6.98 (d, 1H, J = 8.88 Hz), 7.13 (s, 1H), 7.15 (d, 1H, J =
2.64 Hz),
Mass APCI 419.4.
Example I13: 5-(quinolin-6-yloxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
/\ p
0
i o I i
~I
,J
I13 ~~
Compound I13 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD3OD): 8 3.78 (s, 6H), 3.79 (s, 3H), 6.04 (d, 1H, J = 3.59 Hz), 6.24 (s,
2H), 7.28 (d,
1H, J = 3.02 Hz), 7.86 (m, 3H), 8.22 (d, 1H, J = 9.06 Hz), 8.81 (d, 1H, J =
8.31 Hz),
9.02 (d, 1H, J = 6.42 Hz), Mass APCI 422.1.
Example I14: Ethyl 7-[(5-~[(2,4,6-trimethoxyphenyl)amino]carbonyl-2-
furyl)oxy]-1H-indole-2-carboxylate
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I14
Compound I14 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNNIR
(CD30D): ~ 1.40 (t, 3H, J = 7 Hz), 3.79 (s, 3H), 3.81 (s, 6H), 4.37 (q, 2H, J
= 6.99
Hz), 5.49 (d, 1H, J = 3.4 Hz), 6.26 (s, 2H), 7.17 (m.3H), 7.48 (m, 2H), APCI
mass
481.6.
Example I15: 5-(1-Naphthyloxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
0
o \o/ ~ / \ o
0
I15 ~
Compound I15 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): 8 3.80 (s, 6H), 3.81 (s, 3H), 5.66 (d, 1H, J = 3.59 Hz), 7.17 (d, 1H,
J = 3.59
Hz), 7.23 (d, 1H, J = 7.74 Hz), 7.47 (t, 1H, J = 8.12 Hz), 7.57 (m, 2H), 7.75
(d, 1H, J
1s = 8.12 Hz), 7.93 (m, 1H), 8.18 (m, 1H), APCI mass 421Ø
Example I16: 5-(4-phenoxyphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
0
i I I ~ o \o/ (~ / \ o
0
I16 \
Compound I16 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
2o spectrometry data consistent with the title product were as follows: iHNMR
(CD30D): 8 3.78 (s, 6H), 3.81 (s, 3H), 5.65 (d, 1H, J = 3.59 Hz), 6.26 (s,
2H), 7.00
(m, 4), 7.10 (t, 1H, J = 7.37 Hz), 7.19 (m, 2H), 7.34 (dd, 2H, J = 8.50, 7.37
Hz), APCI
mass 463.1.
Example I17: 5-(quinolin-8-yloxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
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° ~ / \ o
I
I wN
I17
Compound I17 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): 6 3.79 (s, 6H), 3.81 (s, 3H), 5.91 (d, 1H, J = 3.59 Hz), 6.25 (s,
2H), 7.23 (d,
1H, J = 3.02 Hz), 7.69 (d, 1H, J = 7.74 Hz), 7.78 (t, 1H, J = 8.03 Hz), 7.88
(t, 1H, J =
4.82 Hz), 7.99 (d, 1H, J = 8.31 Hz), 8.82 (d, 1H, J = 8.50 Hz), 9.04 (d, 1H, J
= 4.72
Hz), APCI mass 421.8.
Example I18: 5-(3-{[(4-chlorophenyl)sulfonyl]amino~phenoxy)-N-(2,4,6-
to trimethoxyphenyl)-2-furamide
I18
Compound I18 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HIVMRR
1s (CD30D): ~ 3.79 (s, 6H), 3.81 (s, 3H), 5.68 (d, 1H, J = 3.21 Hz), 6.26 (d,
1H, J = 3.21
Hz), 6.91 (m, 3H), 7.18 (d, 1H, J = 1.89 Hz), 7.26 (t, 1H, J = 8.40 Hz), 7.51
(d, 2H, J
= 8.50 Hz), 7.74 (d, 2H, J = 8.31 Hz), APCI mass 559.5.
Example I19: 5-(2-naphthyloxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
o °
0 0
\ / (~ \ / °\
0
I19 \
2o Compound I19 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD3OD): 8 3.78 (s, 3H), 3.83 (s, 6H), 5.77 (d, 1H, J = 3.59 Hz), 6.25 (s,
2H), 7.21 (s,
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1H), 7.36 (dd, 1H, J = 9.06, 2.27 Hz), 7.48 (m, 2H), 7.58 (s, 1H), 7.83 (d,
1H, J = 7.74
Hz), 7.88 (d, 1H, J = 7.74 Hz), 7.95 (d, 1H, J = 8.88 Hz), APCI mass 420.9.
Example I20: 5-{4-[(2-chlorobenzoyl)amino]phenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
0
G
0
s I20 °\
Compound I20 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: IHNMR
(DMSO-
d6): 8 3.71 (s, 6H), 3.78 (s, 3H), 5.79 (d, 1H, J = 3.40 Hz), 6.27 (s, 2H),
7.23 (m, 3H),
7.52 (m, 3H), 7.78 (m, 2H), 8.93 (s, 1H), 10.59 (s, 1H), APCI mass 523.6.
Example T21: 5- f (6-amino-1-naphthyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-
furamide
/
0 0
\ / b \ / °\
I~
NHs
Compound I21 was made in a manner analogous to I1, according to Scheme I,
1s using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with therytitle product were as follows: 1HNMR
(CD30D): 8 4.52 (s, 6H), 4.59 (s, 3H), 6.61 (d, 1H, J = 3.40 Hz), 7.08 (s,
2H), 7.67 (d,
1H, J = 7.55 Hz), 7.85 (s, 1H), 7.91 (dd, 1H, J = 9.07, 2.27 Hz), 8.04 (s,
1H), 8.12 (t,
1H, J = 7.93 Hz), 8.27 (d, 1H, J = 8.31 Hz), 8.72 (d, 1H, J = 9.07 Hz), 9.77
(s, 1H),
2o APCI mass 435.1.
Example I22: 5-][3-(2-hydroxyethyl)-1H-indol-7-yl]oxy]-N-(2,4,6-
trimethoxyphenyl)-2-furamide
° \°/ <i \ / °~
H
~2 off
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Compound I22 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: IIfNMR
(DMSO-
d6): 8 2.80 (d, 2H, J = 6.99 Hz), 3.61 (t, 2H, J = 7.18 Hz), 3.72 (s, 6H),
3.79 (s, 3H),
5.53 (d, 1H, J = 3.59 Hz), 6.27 (s, 2H), 6.96 (d, 1H, J = 8.69 Hz), 7.16 (s,
1H), 7.23 (s,
1H), 7.35 (s, 1H), 7.37 (d, 1H, J = 9.06 Hz), 8.87 (s, 1H), 10.94 (s, 1H),
APCI mass
453.1.
Example I23: 5-[(6-benzoyl-1-naphthyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-
furamide
1o I23
Compound I23 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNM1ZR
(dmso-
d6): 8 3.71 (s, 6H), 3.79 (s, 3H), 6.07 (d, 1H, J = 3.40 Hz), 6.27 (s, 1H),
7.52 (d, 1H, J
1s = 9.07 Hz), 7.59 (dd, 2H, J = 7.55, 7.18 Hz), 7.70 (dd, 1H, J = 7.55, 7.18
Hz), 7.76
(m, IH), 7.81 (d, 2H, J = 7.18 Hz), 7.89 (d, 1H, J = 8.31 Hz), 8.10 (d, 1H, J
= 8.69
Hz), 8.24 (d, 1H, J = 9.07 Hz), 8.36 (s, 1H), 9.01 (s, 1H), APCI mass 524.2.
Example I24: 5-[3-(diethylamino)phenoxy]-N-(2,4,6-trimethoxyphenyl)-2-
furamide
I
~N ~ 0 O I
O
2o I24
Compound I24 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): S 1.15 (t, 6H, J = 7.18 Hz), 3.50 (t, 4H, J = 7.18 Hz), 3.78 (s, 6H),
3.82 (s,
2s 3H), 5.77 (d, 1H, J = 3.02 Hz), 6.26 (s, 2H), 6.84 (s, 3H), 7.19 (s, 1H),
7.38(t, 1H, J =
8.31 Hz), APCI mass 441.1.
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Example I25: 5-(2-iodophenoxy)-N-(2,4,6-trimethoxyphenyl)-2-furamide
I
~ o
\/
,o
IZS ~'
Compound I25 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): ~ 3.79 (s, 6H), 3.82 (s, 3H), 5.57 (d, 1H, J = 3.40 Hz), 6.26 (s,
2H), 7.02 (t,
1H, J = 7.55 Hz), 7.16 (s, 1H), 7.20 (d, 1H, J = 8.69 Hz), 7.43 (dd, 1H, J =
6.80, 1.51
Hz), 7.91 (dd, 1H, J = 7.93, 1.51 Hz), APCI mass 496.1.
Example I26: 5-[(2-methyl-1-naphthyl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-
to furamide
I
w o o b w I
I \/
,o
I26
Compound I26 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNIVIR
(dmso-
1s d6): 8 2.38 (s, 3H), 3.70 (s, 6H), 3.81 (s, 3H), 5.06 (d, 1H, J = 3.40 Hz),
6.28 (s, 2H),
7.10 (s, 1H), 7.54 (m, 3H), 7.87 (d, 2H, J = 8.31 Hz), 7.99 (d, 1H, J = 8.31
Hz), 8.91
(s, 1H), APCI mass 434.1.
Example I28: 5-(2,3-dihydro-1H-inden-5-yloxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
O O H3C0
OCH3
HN
20 I28 H3co
Compound I28 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CD30D): 8 2.10 (m, 2H, J = 7.37, 7.55, 7.37, 7.37 Hz), 2.89 (q, 4H, J = 7.49
Hz),
25 3.78 (s, 6H), 3.82 (s, 3H), 5.56 (d, 1H, J = 3.40 Hz), 6.26 (s, 2H), 6.92
(d, 1H, J = 8.12
Hz), 7.02 (s, 1H), 7.13 (s, 1H), 7.22 (d, 1H, J = 8.12 Hz), APCI mass 410.1.
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Example I29: 5-[3-(dimethylamino)phenoxy]-N-(2,4,6-trimethoxyphenyl)-2-
furamide
C H3C0
N
/ / OCH3
HN
I29 H,oo
Compound I29 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: IIiNMR
(CD30D): 8 2.99 (s, 6H), 3.77 (s, 6H), 3.82 (s, 3H), 5.69 (d, 1H, J = 3.59
Hz), 6.26 (s,
1H), 6.60 (d, 1H, J = 8.12 Hz), 6.69 (s, 1H), 6.76 (d, 1H, J = 8.31 Hz), 7.16
(d, 1H, J =
2.83 Hz), 7.28 (t, 1H, J = 8.22 Hz), APCI mass 413.1.
to Example I30: 5-(3-piperidin-4-ylphenoxy)-N-(2,4,6-trimethoxyphenyl)-2-
furamide
HN
H~CO
/ / ~ ~ OCH3
HN
I30 H3~o
Compound I30 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
1s spectrometry data consistent with the title product were as follows: IIiNMR
(CD30D): 8 1.87 (t, 2H, J = 12.84 Hz), 2.09 (d, 2H, J =13.98 Hz), 2.95 (m, 1H,
J =
12.09, 11.33, 3.78, 3.40 Hz), 3.12 (dd; 2H, J = 12.84, 12.09 Hz), 3.49 (d, 2H,
J =
10.95 Hz), 3.77 (s, 6H) 3.82 (s, 3H), 5.70 (d, 1H, J = 3.02 Hz), 6.25 (s, 2H),
7.12 (M,
3H), 7.39 (M, 1H), APCI mass 453.2.
2o Example I31: 5- f 4-[(lE)-3-oxobut-1-enyl]phenoxy]-N-(2,4,6-
trimethoxyphenyl)-
2-furamide
0
O H3C0
/ ~ / ~ ~ HN ~ ~ CHa
I31 H3oo
Compound I31 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
25 spectrometry data consistent with the title product were as follows: 1HNMR
(CD3OD): ~ 2.37 (s, 3H), 3.77 (s, 6H), 3.83 (s, 3H), 5.85 (d, 1H, J = 3.40
Hz), 6.27 (s,
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2H), 6.76 (d, 1H, J = 16.24 Hz), 7.13 (bs, 1H), 7.21 (d, 2H, J = 6.23 Hz),
7.62 (d, 1H,
J = 16.24 Hz), 7.74 (s, 2H), APCI mass 438.1.
Exmple I32: 5-(4-Ethyl-phenylsulfanyl)-furan-2-carboxylic acid (2,4,6-
trimethoxy-phenyl)-amide
I
o , o~
\I
\/ H
~ io
I32
Example I33: 5-[(4-methylpyridin-2-yl)oxy]-N-(2,4,6-trimethoxyphenyl)-2-
furamide
0 HsC
SN ~ ~ ~ HN ~ ~ OCHy
I33 H3~o
1o Compound I33 was made in a manner analogous to I1, according to.Scheme h
using similar starting compounds and reaction conditions. NMR and mass
spectrometry data consistent with the title product were as follows: 1HNMR
(CDC13):
8 2.27 (s, 3H), 3.77 (s, 6H), 3.84 (s, 3H), 6.27 (s, 2H), 6.38 (d, 1H, J =
7.18 Hz), 6.46
(s, 1H), 6.92 (s, 1H), 7.29 (s, 1H), 8.02 (d, 1H, J = 6.80 Hz), APCI mass
385.1.
Example I34: 5-(4-amino-5-isopropyl-2-methylphenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
I
O O O \.I Ow
O
H
I34 H2N
Compound I34 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. NMR and mass
2o spectrometry data consistent with the title product were as follows: 1H NMR
(CDCl3):
8 1.22 (6H, d), 2.17 (3H, s), 2.86 (1H, hep), 3.60 (2H, br s), 3.82 (9H, s),
5.15 (1H, d),
6.18 (2H, s), 6.54 (1H, s), 6.90 (1H, s), 7.07 (1H, d), 7.18 (1H, s), FI-PCI
m/z 441.2
(M+H)+.
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Br H pd/C
Br OH N02+'OTf ~ OH z~
OH NBS/iPr2NH ~
-" ~x
CH C1 I ~ CHZC12 02N' v ' 9515:EtOH/
2 2
aq.l0%HC1
OH
.HC1
HzN
155
Example I35: 5-(5-isopropyl-4-methoxy-2-methylphenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
I
0
0 0
I \/ o
i
I35
Compound I35 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions. i
NaOCH3
OH Brz I ~ OH CuBr I ~ OH
CC14 Br ~ CH30H O
0°C - r.t. reflex 156
OH
I35
O
I
NaOCH3
I ~ OH Br2 I ~ OH CuBr f ~ OH
CC14 Br ~ CH30H O
0C reflex
-
r.t.
to To a carbon tetrachloride solution (25 mL) containing 5-isopropyl-2-
methylphenol
(1.5 g, 10 mmol) was added bromine (.550 mL, 10 mmol) dropwise at 0°C.
The
mixture was allowed to warm to r.t. and was stirred overnight. It was diluted
with
dichloromethane, washed with aq. Sodium bicarbonate, brine, dried (magnesium
sulfate) and evaporated to a liquid: 2.41 g (94%). 1H NMR (CDCl3) 8 1.21 (6H,
d),
2.18 (3H, s), 3.22 (1H, hep), 4.66 (1H, s), 6.69 (1H, s), 7.25 (1H, s). To a
RB flask
was charged with 4-bromo-5-isopropyl-2-methylphenol (1.82 g, 8 mmol),
ethylacetate
(.7 mL), cuprous bromide (.23 g, 1.6 mmol), and 25 wt% (~SlVn sodium methoxide
in
methanol (16 mL). The mixture was heated gradually in an oil bath to reflex
(oil bath
temperature 90-95oC) under argon atmosphere for 16 h. After cooling to r.t.,
the
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reaction mixture was acidified with conc. HCl to pH 2. The acidified mixture
was
concentrated on a rotovap. The aqueous solution was extracted with diethyl
ether 3
times. The combined ether extracts were washed with brine, dried over
magnesium
sulfate, and evaporated to an orange-colored liquid. The desired product was
purified
by flash chromatography (eluting solvents: hexanes to 10% ethylacetate in
hexanes):
1.05 g (73%). 1H NMR (CDC13) 8 1.35 (6H, d), 2.42 (3H, s), 3.42 (1H, hep),
3.95
(3H, s), 4.80 (1H, s), 6.83 (1H, s), 6.84 (1H, s). NMR and mass spectrometry
data
consistent with the title product were as follows: 1H NMR (CDC13): S 1.16 (6H,
d),
2.25 (3H, s), 3.52 (1H, hep), 3.82 (12H, s), 5.21 (1H, d), 6.18 (2H, s), 6.71
(1H, s), 7.0
(1H, s), 7.12 (1H, d), 7.19 (1H, s), FI-PCI mlz 456.2 (M+H)+.
Example I36: 5-[4-(dimethylamino)-5-isopropyl-2-methylphenoxy]-N-(2,4,6-
trimethoxyphenyl)-2-furamide
I
0 0 00 ~ I o~
N ~
N
I36 I
Compound I36 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. The specific method
utilized for the synthesis of I36 is depicted as follows:
Br
Br OH NOz+-OTf ~OH HzCO
OH NBSIiPrzNH ~ ~ I
O N ~ Ha Pd/C
CHZCIz a
MeOH
I
8r O O N W I O o , Ow
~ a H ,0 0 0
~~ I~ W
N i0
N
CszC03, DMF I
microwave, 200°C, 2x10 min. >36
(Scheme 7)
Bromination: A solution of 5-isopropyl-2-methylphenol (3.0 g, 20 mmol) in
methylene chloride (5 mL) was mixed with diisopropylamine (.280 mL, 2 rnmol)
in a
500 mL 1tB flask. To this mixture was added dropwise a solution of N-
bromosuccinimide (3.56 g, 20 mmol) in methylene chloride (120 mL). The
reaction
mixture was stirred at room temperature overnight. It was washed with
saturated
sodium bicarbonate, brine, dried over magnesium sulfate and the solvent was
then
evaporated to give the product 6-bromo-5-isopropyl-2-methylphenol: liquid,
4.22 g
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(92.6%). NMR data consistent with the title product were as follows: 1H NMR
(CDC13): eS 1.22 (6H, d), 2.26 (3H, s), 3.27 (1H, hep), 5.73 (1H, s), 6.75
(1H, d), 7.02
(1H, d).
Nitration: Into a RB flask containing a stirred suspension of
tetramethylammonium nitrate (96%, Aldrich) (1.288 g, 9.07 mmol) in anhydrous
methylene chloride (25 mL) was added trifluoromethane sulfonic anhydride (1.58
mL/2.65 g, 9.31 mmol) dropwise at room temperature, under argon. The
suspension
was stirred at room temperature for 1.5 hours. The flask was then placed in a
dry
ice/acetone cold bath. A methylene chloride solution (10 mL) of 6-bromo-5-
isopropyl-2-methylphenol (1.96 g, 8.63 mmol) was added via a syringe. The
reaction
mixture was allowed to warm to room temperature and stirred at room
temperature
overnight. It was diluted with methylene chloride, washed with water until
neutral pH
was reached, followed by brine, dried (magnesium sulfate) and evaporated. The
product 6-bromo-4-vitro-5-isopropyl-2-methylphenol was purified by flash.
chromatography on silica gel (eluting solvent: 1 ethyl acetate/5 hexanes):
0.60 g,
(25.4%). NMR data consistent with the title product were as follows: 1H NMR
(CDCl3): 8 1.40 (6H, d), 2.30 (3H, s), 3.50 (1H, hep), 6.21 (1H, s), 7.36 (1H,
s).
Hydrogenation: A mixture of 6-bromo-4-vitro-5-isopropyl-2-methylphenol
(0.60 g, 2.19 mmol), formaldeyde solution (40% aqueous solution, 700 mL, 8.80
2o mmol), 10% palladium on carbon (0.2 g), in methanol (20 mL) was
hydrogenated in a
Parr apparatus at 45 psi for 16 hours. The mixture was then filtered through a
pad of
Celite. The filtrate was concentrated on a rotovap to give the product 4-N,N-
dimethylamino-5-isopropyl-2methylphenol. HBr as a dark solid residue: 0.522 g
(87%). NMR and mass spectrometry data consistent with the desired title
product is
2s as follows: 1H NMR (CDCl3): 8 1.28 (6H, d), 2.25 (3H, s), 3.22 (6H, s),
3.80 (1H,
hep), 5.85 (1H, br s), 6.92 (1H, s), 7.18 (1H, s).
Phenol coupling: The microwave protocol of Scheme I was followed.
Condition: DMF, 200°C, 2x10 minutes. NMR and mass spectrometry data
consistent
with the title product were as follows: 1H NMR (CDCl3): 8 1.16 (6H, d), 2.25
(3H, s),
30 2.66 (6H, s), 3.52 (1H, hep), 3.82 (9H, s), 5.30 (1H, d), 6.18 (2H, s),
6.96 (2H, s), 7.12
(1H, d), 7.21 (1H, s), FI-PCI m/z 469.2 (M+H)+.
Example I37: 5-(4-bromo-5-isopropyl-2-methylphenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
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I
00 ,
B o lo/ H ~I
0
I37 Br w
Compound I37 was made in a manner analogous to Il, according to Scheme I,
using similar starting compounds and reaction conditions.
OH gr2 ~ OH
CC14 gr
0°C - r.t. 157
s To a carbon tetrachloride solution (25 mL) containing 5-isopropyl-2-
methylphenol
(1.5 g, 10 mmol) was added bromine (.550 mL, 10 mmol) dropwise at 0°C.
The
mixture was allowed to warm to r.t. and was stirred overnight. It was diluted
with
dichloromethane, washed with aq. Sodium bicarbonate, brine, dried (magnesium
sulfate) and evaporated to a liquid: 2.41 g (94%). 1H NMR (CDC13) b 1.21 (6H,
d),
2.18 (3H, s), 3.22 (1H, hep), 4.66 (1H, s), 6.69 (1H, s), 7.25 (1H, s). NMR
and mass
spectrometry data consistent with the title product were as follows: 1H NMR
(DMSO-
d6): 8 1.09 (6H, d), 2.15 (3H, s), 3.13 (1H, hep), 3.65 (6H, s), 3.7 (3H, s),
5.54 (1H, d),
6.20 (2H, s), 7.07 (1H, s), 7.12 (1H, br d), 7.53 (1H, s), 8.85 (1H, br s). FI-
PCI m/z
504.1, 506.0 (M+H)+.
i5 Example I38: 5-(4-chloro-3-isopropyl-2-methoxy-6-methylphenoxy)-N-(2,4,6-
trimethoxyphenyl)-2-furamide
I
0 0 , o~
\ / H w
I38 c~
Compound I38 was made in a manner analogous to I1, according to Scheme I,
using similar starting compounds and reaction conditions. The specific method
2o utilized for the synthesis of I38 is depicted as follows:
gr o. o
OH NaOMe ~ OH SOZC12 ~ OH
I,
CuBr
MeOH
152
Br 'O/ O N o I O\ of O O ~ I O
O
I~
CI
CszCOs, DMF
microwave, 200°C, 2x10 min. 13g
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Displacement of bromine by methogide: A mixture consisting of 6-bromo-
5-isopro~yl-2-methylphenol (2.28 g, 10 mmol), ethyl acetate (0.8 mL), cuprous
bromide (0.286 g, 2 mmol), and a 25wt% solution of sodium methoxide (20 mL)
was
heated at reflux (oil bath 90 - 95°C) under argon for 16 hours. The
reaction mixture
was cooled to room temperature, and was acidified with concentrated HCl to pH
2-3.
The mixture was then diluted with methanol and filtered through Celite. The
filtrate
was concentrated. The concentrate was redissolved in diethyl ether, washed
with
aqueous 10% HCI, brine, dried (magnesium sulfate), and evaporated to a solid
residue
6-methoxy-5-isopropyl-2-methylphenol: 1.5 g (83%). NMR data consistent with
the
to desired title product is as follows:1H NMR (CDCl3): 8 1.22 (6H, d), 2.22
(3H, s), 3.23
(1H, hep), 3.78 (3H, s), 5.64 (1H, s), 6.68 (1H, d), 6.85 (1H, d).
Chlorination: Sulfuryl chloride (.442 mL, 5.5 mmol) was added to a
chloroforni solution (25 mL) of 6-methoxy-5-isopropyl-2-methylphenol (1.05 g,
5
mmol). The reaction mixture was heated at 60°C for 2 hours, then was
stirred at room
1s temperature for 16 hours. The reaction mixture was washed with saturated
sodium
bicarbonate, brine, dried over magnesium sulfate and evaporated to an oil
(1.33 g).
The product 6-methoxy-4-chloro-5-isopropyl-2-methylphenol was purified by
flash
chromatography on silica gel: 1.03 g (96%). NMR data consistent with the title
product were as follows: 1H NMR (CDC13) 0 1.41 (6H, d), 2.19 (3H, s), 3.51
(1H,
2o hep), 3.76 (3H, s), 5.53 (1H, s), 6.88 (1H, s).
Phenol coupling: The microwave protocol of Scheme I was followed.
Condition: DMF, 200°C, 2x10 minutes. NMR and mass spectrometry data
consistent
with the title product were as follows: 1H NMR (MeOD-d4): 8 1.35 (6H, d), 2.21
(3H,
s), 3.61 (1H, hep), 3.80 (6H, s), 3.81 (3H, s), 3.85 (3H, s), 5.16 (1H, d),
6.26 (2H, s),
2s 7.10 (2H, s), FI-PCI mlz 490.1, 492.1 (M+H)+.
Example I39: 5-o-Tolylogy-furan-2-carboxylic acid (2,4,6-trimethogy-phenyl)-
amide
0
0 0
\ / ~ \ / o\
o\
I39
so Compound I39 was synthesized in a manner analogous to compound Il,
according to scheme I, using similar starting materials and reaction
conditions.
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Example I40: 5-(1-Methyl-1H-indazol-6-yloxy)-furan-2-carboxylic acid (2,4,6-
trimethoxy-phenyl)-amide
0
0 0 -
H
-0
I40
Compound I40 was synthesized in a manner analogous to compound I1,
according to scheme I, using similar starting materials and reaction
conditions.
Example I41: 5-(3-text-Butyl-phenoxy)-furan-2-carboxylic acid (2,4,6-
trimethoxy-phenyl)-amide
I41
to Compound I41 was synthesized in a manner analogous to compound I1,
according to scheme I, using similar starting materials and reaction
conditions.
Example I42: 5-(3-Trifluoromethyl-phenylsulfanyl)-furan-2-carboxylic acid
(2,4,6 trimethoxy-phenyl) -amide
F
F /
O
S 0 -
H
1s I42
Compound I42 was synthesized in a manner analogous to compound I1,
according to scheme I, using similar starting materials and reaction
conditions.
Example I43: 5-(4-Isopropyl-phenylsulfanyl)-furan-2-carboxylic acid (2,4,6-
trimethoxy-phenyl)-amide
I
o / o
S O
I
/O
I43
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Compound I43 was synthesized in a manner analogous to compound I1,
according to scheme I, using similar starting materials and reaction
conditions.
Example I44: 5-(3,4-Dimethoxy-phenylsulfanyl)-furan-2-carboxylic acid (2,4,6-
trimethoxy-phenyl)-amide
I
0
\ S O N \ I
l H
O /O
~o
I44
Compound I44 was synthesized in a manner analogous to compound Il,
according to.scheme I, using similar starting materials and reaction
conditions.
to Scheme J
CI N N~ NaOH
CI N\ CI O O CsZCO3 ~ i _
1// o
N~ + HZN ~ / o DMF N / ~ CH30H
110°C, 20 h.
NH2
rO~Ow
H O INI~ IN H 00Y N~NHR
CIYN\ N \O/ OH NHR OI\'N\ N 'O/ H~N
- N~ TNI O~w
HA'I'IT
Example Jl: 5-[(2-chloro-5-methylpyrimidin-4-yl)amino]-N-(4,6-dimethoxy-2-
~[3-(4-methylpiperazin-1-yl)propyl]amino~pyrimidin-5-yl)-2-furamide acetate
N
O N~
CI !~ N O N
1i~ v / o
0
1s Jl
Compound JI was synthesized according to scheme J, using similar starting
materials and reaction conditions.
Exmaple J2: 5-[(2-chloro-5-methylpyrimidin-4-yl)amino]-N-(2-([3-
(dimethylamino)propyl]amino-4,6-dimethogypyrimidin-5-yl)-2-furamide
20 acetate
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p ~1~N~N~
O ~
CI~N~ N O N~N
O
N / \ / O~
O
J2
Compound J2 was synthesized according to scheme J, using similar starting
materials and reaction conditions.
s Example J3: 5-((2-chloro-5-methylpyrimidin-4-yl)amino]-N-{4,6-dimethoxy-2-
[(3-morpholin-4-ylpropyl)amino]pyrimidin-5-yl)-2-furamide acetate
o ,J'1 N~
CI N N O N
\/
N~ O~
O
J3
Compound J3was synthesized according to scheme J, using similar starting
1o materials and reaction conditions.
Exmaple J4: 5-[(2-chloro-5-methylpyrimidin-4-yl)amino]-N-(2,6-
dimethoxyphenyl)-2-furamide
I
o /
CI N~ N 0 N
N~ O~
"J4
1s Compound J4 was synthesized according to scheme J, using similar starting
materials and reaction conditions.
Scheme I~
O ~OH SOCl2 0
Br O CI N~ -~ Br \O/ N
CHZC12 0°C -~ r.t.
2 h. 62% (2 steps)
OH
O I
O
W O~ ~N
CszC03, DMF O I i
120°C, 16 h.
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Example Kl: 4,4-Dimethyl-2-[5-(3,3,6-trimethyl-1,3-dihydro-isobenzofuran-5-
yloxy)-faran-2-yl]-4,5-dihydro-oxazole
0 0
o I i \ / N
K1
Compound I~1 was synthesized according to scheme K.
Biological Testing And Enzvme Assays
Ih Vitro Assays:
Assessment of GnRU Receptor Activation Using Microphysiometrv
1o By performing exemplary assays described below, the functionality of the
compounds of the invention as GnRH antagonists may be confirmed.
Materials and Methods.
GnRH, Ac-D-2-Nal-p-chloro-D-Phe-(3-(3-pyridyl)-D-Ala-Ser-Lys(nicotinoyl)-
D-Lys(nicotinoyl)-Leu-Lys(isopropyl)-Pro-D-Ala-NH2 (amide), the superagonist
peptide [D-Ala6, des-Gly1°]proethylamide9-LHRH (GnRH-A), and TRH may be
purchased from Bachem (Torrance, CA). Cell Culture media and forskolin may be
purchased from Sigma (St. Louis, MO). Fetal bovine serum (FBS) and
penicillin/streptomycin are available from Omega Scientific, Inc. (Tarzana,
CA).
6418 may be obtained from Gemini (Calabasas, CA). Staurospoxine, Rp-adenosine
3',5'-cyclic monophosphothioate triethylamine (Rp-CAMPS), PMA, and 5-(N-
methyl-N-isobutyl)-amiloride (MIA) are available from RBI (Natick, MA). 2-[1-
(3-
Dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF 109203 may be
purchased from Tocris (Ballwin, MO).
Cell Culture. GGH3 cells (Dr. William Chin, Harvard Medical School,
Boston, MA) are grown in low glucose Dulbecco's modified Eagle's medium
(DMEM) containing 100U/mL penicillin/streptomycin, 0.6 g/L G418 and 10% heat-
inactivated FBS.
Total Inositol Phosphates Measurement.
The activity of various GnRH peptide agonists is initially assessed utilizing
an
3o assay that measures accumulation of total inositol phosphates.
Approximately
200,000 GGH3 cells/well are plated onto 24-well tissue culture plates using
DMEM
media. The following day, cells are loaded with [3H]myoinositol (0.5 Ci/ml)
for 16-18
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hours in inositol-free medium. The medium is aspirated and the cells rinsed
with
serum-free DMEM. Cells are stimulated with GnR_H_ (0.1 nM-1 p,M) or the
superagonist, GnRH-A (0.01 nM-100 nNl) dissolved in DMEM media in a total
volume of 1 mL containing 10 mM LiCI at 37°C for 45 minutes. The media
is
replaced with 1 mL ice-cold 10 mM formic acid, which stops the reaction and
also
serves to extract cellular lipids. Inositol phosphates are separated by ion-
exchange
chromatography on Dowex columns, which are washed with 2.5 mL of 10 mM
myoinositol and 10 mM formic acid. The columns are then washed with 5 mL of 60
mM sodium formate and 5 mM borax, and total inositol phosphates are eluted
with 5
1o mL 1M ammonium formate, 0.1 M formic acid. The column eluates are added to
liquid scintillation vials containing 15 ml of scintillation cocktail and are
counted by
liquid scintillation counting.
Preparation of l2sl-GnRH-A radioligand.
The radioiodinated agonist analog of GnRH, lzsl-GnRH-A, is used as the
1s radioligand. One ~g of GnRH-A diluted in O.1M acetic acid is added to an
Iodogen~-
coated borosilicate glass tube (Pierce) containing 35 p1 of 0.05 M phospate
buffer (pH
7.4-7.6) and I rnCi of Na[lzsl]. The reaction mixture is vortexed and
incubated for I
min at room temperature. 2 ml of 0.5 M acetic acid is added to the reaction
tube and
the mixture is added to a C18 Sep-Pak cartridge. The cartridge is washed with
2o subsequent washes of 5 ml H20 and 5 ml O.SM acetic acid and then eluted
with 5 x 1.
ml of 60% CH3CN/40% O.SM acetic acid. The eluate is diluted with 3x volume of
HPLC buffer A (0.1 % TFA in H20)° and loaded onto a C 18 column. The
iodinated
product is eluted over 20-25 min with a gradient of 25-100% CH3CN containing
0.1%TFA. The radioactive fractions (750 p,l/fraction) are collected into clean
25 polypropylene tubes containing 100 ~1 of 10% BSA. Fractions are assessed
for
biological activity by radioligand binding.
Competition Radioligand Binding.
Approximately two million GGH3 cells/tube are utilized for radioligand
binding. Izsl-GnRH-A (approximately 0.1-0.3 nM) is incubated with cells in the
3o presence or absence of competing agents in a final volume of 300 p,1
binding assay
buffer [50 mM HEPES (pH 7.4), 1 mM EDTA, 2.5 mM MgClz, and 0.1% BSA~ to
test the ability of compounds to displace agonist binding. Reactions are
performed on
ice for 2 hr and stopped by the addition of 2 mI of ice-cold PBS wash buffer
(SO mM
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NaP04, 0.9% NaCI, 2 mM MgCl2, and 0.02% NaN3, pH 7.4) and rapid filtration
onto
GF/C filters presoaked with 0.05% polyethylenimine utilizing a Brandcl cell
harvester. Filters are counted on a gamma counter.
Microphysiometry.
s 'The Cytosensor~ Microphysiometer (Molecular Devices, Sunnyvale, CA) is a
real-time, noninvasive, nonradioactive semiconductor-based system for
monitoring
the cellular responses to various stimuli. It is based on a pH-sensitive
silicon sensor,
the light-addressable potentiometric sensor which forms part of a microvolume
flow
chamber in which cultured cells are immobilized (14, 15, I7). GGH3 cells are
seeded
1o in low-buffered minimal essential media (MEM, Sigma) containing 25 mM NaC1
and
0.1% BSA at a density of 500,000 cells/capsule onto the polycarbonate membrane
(3
pm porosity) of cell capsule cups (Molecular Devices, Sunnyvale, CA). Capsule
cups
are transferred to sensor chambers where cells are held in close apposition to
a silicon
sensor within a sensor chamber, which measures small changes in pH in the
15 microvolume of the sensor chamber. Low-buffered medium is pumped
continuously
across the cells at a rate of approximately 100 ~,1/min from one of two fluid
reservoirs.
A selection valve determines which reservoir from which fluid is perfused onto
the
cells.
The Cytosensor~Microphysiometer generates a voltage signal, which is a
20 linear function of pH, every second. In order to measure acidification
rates, flow to
the sensor chamber containing the cells is periodically interrupted, allowing
excreted
acidic metabolites to build up in the extracellular fluid of the cells. Cells
are
maintained at 37 °C on a two-minute flow cycle with cells being
perfused with media
for ~0 seconds followed by 40 seconds in which the flow of media is stopped.
During
25 this 40-second interval, acidification rates are measured for a 30 sec
interval. In this
fashion, a single acidification rate is calculated every two min. The
Cytosensor
Microphysiometer device contains eight such sensor units, allowing for eight
simultaneous experiments to be performed. Each unit is individually programmed
utilizing a computer linked to the system.
3o GGH3 cells are initially equilibrated in the low-buffered MEM media for a
period of 30-60 min in which basal acidification rates (measured as ~,V/sec),
in the
absence of any stimuli, are monitored. When the basal rate of acidification
changes
by less than ten percent over a period of twenty minutes, experiments are
initiated.
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Time course experiments are performed to determine the optimal time for
agonist
exposure prior'to acidification rate measurement and the duration of exposure
needed
to obtain peak acidification responses to various agonists. From these time
course
experiments, it has been determined that cells should be exposed to GnRH
peptide
agonists at least one minute prior to collection of acidification rate data.
Peak
acidification rates usually occur in the first two-rnin exposure cycle. When
the
effects of various inhibitors are measured, cells are pretreated for 20 min
with test
compound diluted in low-buffered MEM containing 1% DMSO final concentration
prior to exposure of the cells for 4 min to a solution containing GnR_H_ or
PMA at
appropriate concentration in the presence of inhibitor.
Cyclic AMP Measurement.
The ability of various compounds to increase basal CAMP formation in GGH3
cells is assessed utilizing 96-well adenylyl cyclase flashplates purchased
from New
England Nuclear (NEN, Boston, MA). Cells (approximately 50,000 cells/well) are
incubated with either forskolin (10 nM-10 ~, GnIZ_H_ (1 nM-1 pM) or GnRH-A
(0.1 nM-100 nM) in a total volume of 100 p1 on flashplates for 20 minutes at
room
temperature to assess for agonist activity. 100 p,1 of detection mix
containing 125I-
cAMP is added to quench reactions according to the manufacturer's
instructions.
Plates are counted on a Packard TopCount after approximately two hours. Cyclic
2o AMP levels are determined from standard curves generated to non-radioactive
cAMP
standards (10 nM-1 ~.
Data Analysis.
Cytosensor ° Microphysiometer data are normalized utilizing
Cytosoft~
software (Molecular Devices, Sunnyvale, CA). ECSO values for agonists and TCSo
values for inhibitors are generated utilizing PrismTM (version 2.01, GraphPad
Software, San Diego, CA), a computer graphics and statistics program. Values
for
multiple experiments are presented as means ~ SE of at least three replicate
experiments.
Effect of Compounds on lzSI-GnRH-A Binding to GGH~ Cells.
3o In order to assess the specific functionality, compounds were assessed for
their
ability to inhibit lzSZ-GnRH-A binding to GGH3 cells. The peptide ligands
GnRH,
GnRH-A, and Antide, but none of the tested compounds of the invention, blocked
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iasl-GnRH-A binding to these cells. Thus, the compounds of the invention are
GnRH
antagonists.
Determination of Binding Inhibition Constants
Using the assay described below, Ki values for compounds of the invention
were determined.
Chemicals and Reagents.
GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), was purchased
from Sachem (Torrance, CA). Cell Culture media was purchased from Sigma (St.
Louis, MO). Fetal bovine serum (FBS) was from Omega Scientific, Inc. (Tarzana,
1o CA). 6418 and penicillin/streptomycin were from Gemini (Calabasas, CA).
Newborn calf serum was from Summit Biotech (Fort Collins, CO). All other
reagents
were of the highest quality from standard sources.
Cell Culture.
HEK 293 cells stably transfected with mouse or human GnRH receptors as
1s described above were grown in Dulbecco's high-glucose, modified Eagle's
medium
(DMEM) supplemented with 0.2% 6418, 10% fetal bovine serum (FBS) and
100U1mL penicillin/streptomycin. GH3 cells stably transfected with the rat
GnR_H_
receptor (GGH3) were provided by Dr. William Chin (Harvard Medical School,
Boston, MA). These cells have been extensively characterized previously
(Kaiser et
2o al., 1997). The cells were grown in low glucose DMEM containing: 100U/mL
penicillin/streptomycin, 0.6% 6418 and 10% heat-inactivated FBS.
Cell Membrane Preparation.
HEK 293 cells containing mouse or human receptors, or rat pituitaries (Pel
Freez Biologicals, Rogers, AR) were homogenized in buffer A containing: 50 mM
2s Tris (pH 7.4), 0.32 M sucrose, 2 mM EGTA, 1 mM PMSF, 5 wg/ml aprotinen, 5
p,g/ml Pepstatin A, and 1 pg/ml leupeptin. Homogenized cells were centrifuged
at
4°C at 20,000 x g for 25 minutes, re-suspended in buffer A and re-
centrifuged at 4°C
at 20,000 x g for an additional 25 minutes. Total membrane protein was
determined
with a BCA kit (Pierce, Rockford, IL). Membranes were stored at -70°C
at a final
3o membrane protein concentration of approximately 5 mg/ml.
Radioli and d Preparation.
The radioiodinated agonist analog of GnRH, [des-Glyl°,D-Ala6)GnRH
ethylamide (l2sI-GnRH-A), was used as the radioligand. One ~g of GnRH-A
diluted
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CA 02449843 2003-12-05
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in 0.5 M phosphate buffer (pH 7.4) was added to an Iodogen~-coated
borosilicate
glass tube (Pierce, Rockford, IL) containing 35 p1 of 0.05 M phosphate buffer
(pH
7.4-7.6) and 1 mCi of Na[lasl]. The reaction mixture was vortexed and
incubated for
1 minute at room temperature. After one minute, the mixture was vortexed and
allowed to incubate for an additional minute. 2 ml of 0.5 M acetic acid/1% BSA
was
added to the reaction tube and the mixture was added to a C18 Sep-Pak
cartridge.
The cartridge was washed with subsequent washes of 5 ml H20 and 5 ml 0.5 M
acetic
acid and then eluted with 5 x 1 ml of 60%CH3CN/40% 0.5 M acetic acid. The
eluate
was diluted with 3x volume of HPLC buffer A (0.1% TFA in H20) and loaded onto
a
1o C18 column. The iodinated product was eluted over 20-25 min with a gradient
of 25-
100% CH3CN containing 0.1% TFA. The radioactive fractions (750 p,l/fraction)
were
collected into clean polypropylene tubes containing 100 ~,1 of 10% BSA.
Fractions
were assessed for biological activity by radiolig and binding. Specific
activity of the
radioligand was approximately 2200 Ci/mmol.
Radioli~and Binding_Assays.
Membranes were diluted to 0.01-0.5 mg/ml (depending upon the species of
receptor) with assay buffer containing 50 mM HEPES (pH 7.4), 1 mM EDTA, 2.5
mM MgCl2, and 0.1% BSA. Membranes (diluted to utilize similar receptor numbers
between assays) were incubated with approximately 0.04-0.06 nM lasl-GnRH-A in
2o the presence or absence of competing agents (0.1- 10,000 nM) in a total
volume of
200 ~,1 in 96-well polypropylene.plales for 1 hour at room temperature. Assays
were
stopped by rapid filtration onto 96-well GF/C filters soaked in 0.1 %
polyethylenimine
(PEI) utilizing a Packard 96-well cell harvester. Filters were washed three
times with
ice-cold PBS (50 mM NaP04, 0.9% NaCI, 2 mM MgCl2, and 0.02% NaN3, pH 7.4).
2s 35 ~,1 of scintillation cocktail was added to each filter well and filters
were counted on
a Packard Topcount. Control dose-response curves were generated to GnRH (0.1
nM-
100 nM] in each competition binding experiment. Binding inhibition constants
(K;)
for the GnRH agents were calculated and are provided in Table 2 below. Ki
values
were calculated from IC50 values according to Cheng et al., Biochemical
Pharmacol.
so 22: 3099-3108, 1973.
IC~n
K; = ~1 + [ligand]
Kd of ligand
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Table 2
K; for GnRH Agents:
Tnhibition Binding of lzsI-GnRH-A to GnRH Receptors of Various Species
Example GnRH ReceptorK; (nNn
No.
A1 Human >10000
Mouse ND
Rat ND
A2 Human 180
Mouse 87
Rat 69
A3 Human 42
Mouse 16
Rat 14
A4 Human 189
Mouse 101
Rat 84
AS ~ Human 42
Mouse 134
Rat 46
A6 Human 455
Mouse ND
Rat ND
A7 Human 244
Mouse 435
Rat 191
A8 Human 3
Mouse 2
Rat 4
A9 Human 2
Mouse 3
Rat 11
A10 Human 649
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Example GnRH ReceptorK; (n1V>)
No.
Mouse ND
Rat ND
All Human 460
Mouse ND
Rat ND
A12 Human 2716
Mouse ND
Rat ND
A13 Human 78
Mouse 2S
Rat 80
A14 Hurnan 844
Mouse ND ,
Rat ND
A15 Human 2
Mouse 2
Rat 2
A16 Human 47S
Mouse ND
Rat ND
A17 Human 1S3
Mouse 74
Rat 126
Bl Human 0.42
Mouse 1
Rat 4
B2 Human >1000
Mouse ND
Rat ND
B3 Human 3
Mouse 2
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Example GnRH ReceptorK; (nlVn
No.
Rat 7
B4 Human 103
Mouse 157
Rat 563
BS Human 14
Mouse ND
Rat 14
B6 Human 999
Mouse ND
Rat ND
B7 Human 561
Mouse ND
Rat ND
B8 Human >10000
Mouse ND
Rat ND
B9 Human ~0
Mouse 69
Rat 203
B10 I4uman 4
Mouse 6
Rat 5
Bll Human 403
Mouse ND
Rat ND
B12 Human 520
Mouse ND
Rat ND
B13 Human >10000
Mouse ND
Rat ND
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Example GnRH ReceptorK; (nlV~
No.
B14 Human 5
Mouse 29
Rat 32
B15 Human 11
Mouse 7
Rat 10
B16 Human 2
Mouse 2
Rat 4
B17 Human 251
Mouse ND
Rat ND
B18 Human 6
Mouse 3
Rat 4
B19 Human 138
Mouse 84
Rat 72
B20 Human 4906
Mouse ND
Rat ND
B21 Human 14
Mouse 11
Rat 24
B22 Human 16
Mouse 20
Rat 42
B23 Human 5
Mouse 6
Rat 13
B24 Human 457
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Example GnRH ReceptorK; (nlVn
No.
Mouse ND
Rat ND
B25 Human 1180
Mouse ND
Rat ND
B26 Human 1.4
Mouse 1.1
Rat 1.7
B27 Human 0.9
Mouse ND
Rat 0.7
B28 Human 4
Mouse ND ~
Rat 1
B29 Human 1
Mouse ND
Rat 0.69
B30 Human 2
Mouse 1.2
Rat 1
B31 Human 2
Mouse 0.74
Rat 1
B32 Human 2
Mouse 2
Rat 5
B33 Human 6
Mouse 7
Rat 9
B34 Human 0.2
Mouse ND
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Example GnRH ReceptorK; (nlV~
No.
Rat 0.3
B35 Human 3
Mouse 6
Rat 3
B36 Human 0.18
Mouse 0.26
Rat 0.43
B37 Human 5
Mouse 4
Rat 4
B38 Human 7
Mouse 6
Rat 9
B39 Human 2
Mouse 2
Rat 3
B40 Human 0.2
Mouse ND
Rat 0.4
B41 I~uman ND
Mouse ND
Rat ND
B42 Human 31
Mouse 21
Rat 96
B43 Human 63
Mouse 411
Rat 711
B44 Human 1362
Mouse ND
Rat ND
167
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Example GnRH ReceptorK; (nlVn
No.
B45 Human 1
Mouse 0.96
Rat Z
B46 Human 0.5
Mouse ND
Rat 0.6
B47 Human 1402
Mouse ND
Rat ND
B48 Human 11
Mouse ND
Rat 15
B49 Human 4
Mouse 4
Rat 16
B50 Human I
Mouse 3
Rat 2
B51 Human 3
Mouse 0.13
Rat 6
B52 Human 0.1
Mouse 0.18
Rat 0.13
B53 Human 10
Mouse ND
Rat 8
B54 Human 13
Mouse ND
Rat 7
B55 Human 2
168
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Example GnRH ReceptorK; (nM~
No.
Mouse ND
Rat 3
B56 Human 2
Mouse ND
Rat 3
B57 Human 0.96
Mouse ND
Rat 0.24
BS8 Human 2
Mouse 0.93
Rat 2
B59 Human 3
Mouse . 6.4 $
. Rat 2
B60 Human 0.67
Mouse 2
Rat 0.8
B61 Human 3
Mouse ND
Rat 0.34
B62 Human 0.58
Mouse 0.8
Rat 0.7
B63 Human 3
Mouse 2
Rat 3
B64 Human 25
Mouse 21
Rat 29
B65 Human 0.41
Mouse 0.4
169
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Example GnRH ReceptorK; (nN~
No.
Rat 0.33
B66 Human 3
Mouse 2
Rat 5
B67 Human 350
Mouse ND
Rat ND
B68 Human 0.86
Mouse 1
Rat 1
B69 Human 5
Mouse 3
Rat 7
B70 Human 1I
Mouse ND
Rat 13
B71 Human 11
Mouse ND
Rat 38
B72 vIuman 5
Mouse 3
Rat 5
B73 Human 2
Mouse ND
Rat 1
B74 Human 2117
Mouse ND
Rat ND
B75 Human 3096
Mouse ND
Rat ND
170
CA 02449843 2003-12-05
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Example GnRH ReceptorK; (nlVn
No.
B76 Human 1909
Mouse ND
Rat ND
B77 Human 16
Mouse 30
Rat 34
B78 Human 1010
Mouse ND
Rat ND
B79 Human 29
Mouse 7
Rat 33
B80 Human ND
Mouse ND
Rat ND
B81 Human 9
Mouse 2
Rat 4
B82 Human 29
Mouse 25
Rat 34
B83 Human 5
Mouse 10
Rat 13
B84 Human 10
Mouse 6
Rat 5
B85 Human 1401
Mouse ND
Rat ND
B86 Human 196
171
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E$ample GnRH ReceptorK; (nlV~
No.
Mouse ND
Rat ND
B87 Human 580
Mouse ND
Rat ND
B88 Human 4
Mouse ND
Rat 6
B89 Human 0.24
Mouse 0.22
Rat 0.14
B90 Human 0.42
Mouse ND d
Rat 0.52
B91 Human 0.42
Mouse 0.97
Rat 0.71
B92 Human 0.96
Mouse 2
Rat 3
B93 Human 24
Mouse 51
Rat 39
B94 Human 102
Mouse 75
Rat 154
B95 Human >10000
Mouse ND
Rat ND
B96 Human 987
Mouse ND
172
CA 02449843 2003-12-05
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Example GnRH ReceptorK; (nlVn
No.
Rat ND
B97 Human 53
Mouse 18
Rat 65
B98 Human 2751
Mouse ND
Rat ND
B99 Human 1268
Mouse ND
Rat ND
B100 Human 261
Mouse 22
Rat 26
B101 Human 11
Mouse 4
Rat 3
8102 Human 372
Mouse ND
Rat ND
B103 I4uman ND
Mouse ND
Rat ND
B104 Human 2
Mouse 3
Rat 4
B10S Human 1533
Mouse ND
Rat ND
B106 Human 478
Mouse ND
Rat ND
173
CA 02449843 2003-12-05
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Example GnRli ReceptorK; (nlVn
No.
B107 Human 94
Mouse 43
Rat 41
B108 Human >10000
Mouse ND
Rat ND
B109 Human 2804
Mouse ND
Rat ND
B110 Human 1
Mouse 0.8
Rat 1
Blll Human 55
Mouse 13
Rat 16
B112 Human 5
Mouse ND
Rat 3
B113 Human 703
Mouse ND
Rat ND
B114 Human 7
Mouse ND
Rat 5
B115 ~ Human ND
Mouse ND
Rat ND
B116 Human 0.84
Mouse ND
Rat 0.67
B117 Human 6
174
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Example GnRH ReceptorK; (nlV~
No.
Mouse ND
Rat 2
B118 Human 0.57
Mouse ND
Rat 1.1
8119 Human 3
Mouse ND
Rat 3
B120 Human 0.5
Mouse ND
Rat 1
B121 Human 2
Mouse ND ~
Rat 0.73
B122 Human 1
Mouse ND
Rat 0.57
B123 Human 6.6
Mouse ND
Rat 4
B124 Human 1
Mouse ND
Rat 0.57
B125 Human 1
Mouse ND
Rat 0.49
8126 Human 0.72
Mouse ND
Rat 0.53
B127 Human 0.92
Mouse ND
175
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Example GnRH ReceptorK; (nlVn
No.
Rat 0.47
8128 Human 0.56
Mouse ND
Rat 0.23
B129 Human 0.70
Mouse ND
Rat 0.62
B130 Human 5
Mouse ND
Rat 4
8131 Human 1
Mouse ND
Rat 2
8132 Human 0.3 7
Mouse ND
Rat 0.47
8133 Human 4
Mouse ND
Rat 4
B134 ~I4uman 0.79
Mouse ND
Rat 0.5
B135 Human 1
Mouse ND
Rat 1
B136 Human 0.21
Mouse 0.35
Rat 0.6
B137 Human 0.7
Mouse ND
Rat 0.22
176
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Example GnRH ReceptorK; (nNn
No.
B138 Human 4.7
Mouse ND
Rat 3.7
B139 Human 9.2
Mouse ND
Rat ND
B140 Human 0.66
Mouse 0.35
Rat 0.25
B141 Human >1000
Mouse ND
Rat ND
B142 Human 73
Mouse ND
Rat 32
B143 Human 10
Mouse ND
Rat 21
8144 Human 3093
Mouse ND
Rat ND
B145 Human 650
Mouse ND
Rat ND
B146 Human 28
Mouse ND
Rat 18
B147 Human 3
Mouse ND
Rat 3
B148 Human 27
177
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EaampIe GnRH ReceptorK; (nll~
No.
Mouse ND
Rat ND~
B149 Human 198
Mouse ND
Rat ND
B150 Human 13
Mouse 14
Rat 11
B151 Human ND
Mouse ND
Rat ND
B152 Human 21
Mouse ND ,
Rat 170
B153 Human 4.9
Mouse ND
Rat 2
B154 Human ND
Mouse ND
Rat ND
B155 Human ND
Mouse ND
Rat ND
C1 Human 2
Mouse 1
Rat 4
C2 Human 7
Mouse 4
Rat 4
C3 Human 3669
Mouse ND
178
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Example GnRH ReceptorK; (nlV~
No.
Rat ND
C4 Human 25
Mouse 51
Rat 76
CS Human 17
Mouse 11
Rat 27
C6 Human >10000
Mouse ND
Rat ND
C7 Human 12
Mouse 26
Rat 51
C8 Human 51
Mouse 31
Rat 3 8
C9 Human > 10000
Mouse ND
Rat ND
C10 -Human 1
Mouse 1
Rat 2
C1I Human 220
Mouse 154
Rat 198
C12 Human 12
Mouse 3
Rat 11
C13 Human 15
Mouse 5
Rat 17
179
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Example GnRH ReceptorK; (nlVn
No.
C14 Human 693
Mouse ND
Rat ND
C15 Human 11
Mouse 5
Rat 15
CI6 Human 450
Mouse ND
Rat ND
C17 Human 14
Mouse 75
Rat 154
C18 Human 5
Mouse S
Rat 7
C19 Human 59
Mouse 36
Rat 16
C20 Human 10
Mouse 29
Rat 15
C21 Human 29
Mouse 13
Rat 11
C22 Human 74
Mouse 97
Rat 142
C23 Human 4
Mouse 3
Rat 0.34
Dl Human >10000
180
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Example GnRH ReceptorK; (nlV1)
No.
Mouse ND
Rat ND
D2 Human 841
Mouse ND
Rat ND
D3 Human 3747
Mouse ND
Rat ND
Fl Human 31
Mouse 36
Rat 65
F2 Human 6430
Mouse ND ,
9
Rat ND
F3 Human 380
Mouse ND
Rat ND
Hl Human 27
Mouse 14
Rat 115
H2 Human 7
Mouse 20
Rat 207
Il Human >10000
Mouse ND
Rat ND
I2 Human 6540
Mouse ND
Rat ND
I3 Human 4910
Mouse ND
181
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E$ample GnRH ReceptorI~; (nlV1)
No.
Rat ND
I4 Human 8
Mouse 8
Rat 6
IS Human 455
Mouse ND
Rat ND
I6 Human >10000
Mouse ND
Rat ND
I7 Human 3654
Mouse ND
Rat ND
I8 Human 7379
Mouse ND
Rat ND
I9 Human >10000
Mouse ND
Rat ND
I10 ~~uman >10000
Mouse ND
Rat ND
Ill Human 3226
Mouse ND
Rat ND
I12 Human >10000
Mouse ND
Rat ND
I13 Human >10000
Mouse ND
Rat ND
182
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Ezample GnRH ReceptorK; (nlV1)
No.
I14 Human 2474
Mouse ND
Rat ND
I15 Human 2740
Mouse ND
Rat ND
I16 Human 2204
Mouse ND
Rat ND
I17 Human >10000
Mouse ND
Rat ND
I18 Human >10000
Mouse ND
Rat ND
I19 ~ Human 8006
Mouse ND
Rat ND
IZO Human >10000
Mouse ND
Rat ND
I21 Human >10000
Mouse ND
Rat ND
I22 Human >10000
Mouse ND
Rat ND
I23 Human 1313
Mouse ND
Rat ND
I24 Human 88
183
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Example GnRH ReceptorK; (nlV1)
No.
Mouse 64
Rat 76
I25 Human 992
Mouse ND
Rat ND
I26 Human 113
Mouse 67
Rat 93
I28 Human 12361
Mouse ND
Rat ND
I29 Human 3201
Mouse ND ~
Rat ND
I30 Human >10000
Mouse ND
Rat ND
I31 Human >10000
Mouse ND
Rat ND
I32 Human 3700
Mouse ND
Rat ND
I33 Human 8400
Mouse ND
Rat ND
I34 Human 99
Mouse 24
Rat 61
I35 Human 44
Mouse 16
184
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Example GnRH ReceptorK; (nlV1)
No.
Rat 29
I36 Human 6
Mouse 9
Rat 9
I37 Human 13
Mouse 6
Rat 10
I38 Human 6
Mouse 4
Rat 9
I39 Human ND
Mouse ND
Rat ND
I40 Human ND
Mouse ND
Rat ND
I41 Human ND
Mouse ND
Rat ND
I42 Human 6870
Mouse ND
Rat ND
I43 Human >1000
Mouse ND
Rat ND
I44 Human >1000
Mouse ND
Rat ND
Jl Human > 1000
Mouse ND
Rat ND
185
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Example GnRH ReceptorK; (nIV~
No.
J2 Human >1000
Mouse ND
Rat ND
J3 Human >1000
Mouse ND
Rat ND
J4 Human > 1000
Mouse ND
Rat ND
K1 Human 1620
Mouse ND
Rat ND
ND = not determined
Micr~hysiometry.
GGH3 cells were seeded~in low-buffered minimal essential media (MEM,
Sigma) containing 25 mM NaCI and 0.1°1o BSA at a density of 500,000
cellslcapsule
onto the polycarbonate membrane (3 pxn porosity) of cell capsule cups
(Molecular
Devices, Sunnyvale, CA). Capsule cups were transferred to sensor chambers
where
cells were held in close apposition to a silicon sensor within a sensor
chamber, which
measures small changes in pH in the microvolume of the sensor chamber. Low-
no buffered medium was pumped continuously across the cells at a rate of
approximately
100 ul/min from one of two fluid reservoirs. A selection valve determined
which
reservoir from which fluid was perifused onto the cells.
The Cytosensor~Microphysiometer generates a voltage signal, which is a
linear function of pH, every second. In order to measure acidification rates,
flow to
1s the sensor chamber containing the cells was periodically interrupted,
allowing for
excreted acidic metabolites to build up in the extracellular fluid of the
cells. In these
experiments, cells were maintained at 37 °C on a two-minute flow cycle
with cells
being perfused with media for 80 seconds followed by 40 seconds in which the
flow
of media was stopped. During this 40-second interval, acidification rates were
2o measured for a 30-second interval. In this fashion, a single acidification
rate was
186
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calculated every two minutes. The Cytosensor~ Microphysiometer unit contains
eight
such sensor units, allowing for eight simultaneous experiments to be
performed. Each
unit was individually programmed utilizing a computer linked to the system.
GGH3 cells were initially equilibrated in the low-buffered MEM media for a
period of 30-60 minutes in which basal acidification rates (measured as
uV/sec), in
the absence of any stimuli, were monitored. When the basal rate of
acidification
changed by less than ten percent over a period of twenty minutes, experiments
were
initiated. Cells were pretreated for 20 minutes with cell media containing
vehicle (1%
DMSO) or test compounds at various concentrations (in 1% DMSO final
1o concentration) prior to stimulation with CmRH at various concentrations.
Total Inositol Phosphates Measurement.
Approximately 200,000 GGH3 cells or 293 cells containing human GnRH
receptors were plated onto 24-well tissue culture plates using DMEM media. The
following day, cells were loaded with [3H] myoinositol (0.5 Ci/ml) for 16-1 ~
hours in
15 inositol-free medium. The medium was aspirated and the cells rinsed with
serum-free
DMEM. Cells were pretreated with various compounds (dissolved in I% DMSO
final concentration) for 30 minutes and were then stimulated for 45 minutes
with
(inRH (0.1 nM-1 ~M) dissolved in DMEM media in a total volume of 1 mL
containing IO mM LiCI at 37°C. The media was replaced with 1 mL ice-
cold 10 mM
2o formic acid, which stopped the reaction and also served to extract cellular
lipids.
Inositol phosphates were separated by ion-exchange chromatography on Dowex
columns, which were washed with 2.5 mL of 10 mM myoinositol and 10 mM formic
acid. The columns were then washed with 5 mL of 60 mM sodium formate and 5 mM
borax, and total inositol phosphates were eluted with 5 mL of 1 M ammonium
2s formate, 0.1 M formic acid. The column eluates were added to liquid
scintillation
vials containing 15 ml of scintillation cocktail and were counted by liquid
scintillation
counting.
Pharmacokinetics and Metabolism:
Pharmacokinetics.
3o Rats (male or female, 200-225 g) were prepared with indwelling jugular vein
cannula as described by Harms et al., Applied Playsiol. 36:391-398 (1974), and
allowed to recover overnight with free access to the standard vivarium chow
and
water. The compounds were administered to female rats at 5 mg/lcg i.v. and 10
mg/kg
187
CA 02449843 2003-12-05
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p.o. as solutions in 10% DMSO+10% cremophor+80% saline or 10%
cremophor+90% saline. The male rats were dosed orally at 50 mg/kg in the
vehicles
specified in Table 3. The blood samples were withdrawn at specific times,
plasma
was immediately separated and compound extracted with ethyl acetate. The
samples
were analyzed by LC-MS using 30-90% gradient of ACN in 50 mM ammonium
acetate.
The pharmacokinetic parameters were calculated using WinNonlin software
(Scientific Consulting Inc.). The bioavailability was calculated as
AUCp.o./AUCi.v.,
where AUCp.o. and AUC i.v. are areas under the plasma concentration-time curve
1o after oral and i.v. administration, respectively.
In vitro metabolism.
Human, rat, dog, and monkey liver microsomes were isolated by differential
centrifugation. Specimens of human liver were obtained from the International
Institute for the Advancement of Medicine (Scranton, PA). The disappearance of
the
1s parent compound was studied in a mixture containing 5 uM compound, 0.5
mg/ml
microsomal protein, and 2 mM NADPH in 50 mM I~ Phosphate buffer, pH 7.4.
Samples were incubated for 30 minutes at 37°C. The reaction was
terminated by the
addition of acetonitrile and compounds analyzed by LC-MS as described above.
188
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Table 3
Compound
No. Human M F Rat Dog MonkeyH Solubility
a a S
I m A
a a
R 1
a a
t R
a
t
rem. % TvzCm~T~ Faa% TvzCm~ Tm~FØplasma% % columnug/ml
5' rem. hrpM hr rem. hrwM hr % rem.rem. a pH2
30' ' 5' remain30' 30' pH6.5
30' 30'
A10 ND 45 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
A2 ND 40 NDNDND ND ND ND 280.63.340.523%'ND ND ND ND NDND
A3 ND 51 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
A4 ND 55 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
A6 ND 35 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
A8 ND 30 NDNDND ND ND ND 682.11 1 7/z 76 0 ND >30 ND0.3
,
A9 ND 36 NDNDND ND ND ND 862 0.2 0.54%' 9 ND ND 18 ND0.7
Bl ND 5070 16ND0.31 ND ND 893.21.7 1 41%'79 8 1 12 NDQ.9
B10 ND 23 NDNDND ND ND ND 78NDND NDND 100 0 ND 3 ND1.9
Bll ND 24 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
B12 ND 35 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
B13 ND 42 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
B14 ND 30 NDNDND ND ND ND 574 ND ND** 1 1 ND 2 ND3.5
B15 ND 58 NDNDND ND ND ND 45NDND NDND ND ND ND ND NDND
B3 ND 24 1 2.81 1 15%'ND 382.94 3 74%'100 1 0 11 ND0.8
B4 ND 50 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
B7 ND 60 NDNDND ND ND ND NDNDND NDND ND ND Nb ND NDND
B74 ND ND NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
B75 ND 80 NDNDND ND ND ND NDNbND NDND ND ND ND ND NDND
B76 ND 76 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
B77 ND 46 NDNDND ND ND ND 43NDND NDND ND ND ND ND NDND
B79 ND 57 NDNDND ND ND ND 742 1 1 10%zND ND ND ND NDND
B9 ND 78 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
C10 ND 32 NDNDND ND ND ND 74NDND NDND 92 ND ND 15 ND0.7
C18 ND 5763 7 ND1.51 ND ND 881.71 1 21%z82 4 ND >30 ND0.5
C23 88 4255 152.50.41 17%z95 871.61.5 1 33%z76 0 ND 26 ND5.9
C4 ND 85 NDNDND ND ND ND NDNDND NDND 0 ND ND ND NDND
C7 ND 16 NDNDND ND ND ND 34NDND NDND 100 5 ND ND ND0.4
CS ND 25 NDNDND ND ND ND ND~NDND NDND ND ND ND ND NDND
C9 ND 43 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
D3 ND 49 NDNDND ND ND Nb NDNDND NDND ND ND ND ND NDND
H1 ND 86 NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
H2 ND 100 NDNDND ND ND ND ND1.50.1 1 0.1%'100 ND ND ND ND0.4
Compound
No. HumanM F Rat Dog MonkeyH Solubility
a a S
I m A
a a
R 1
a a
t R
a
t
rem. % Tz,z FRa% Tl,zCm~ Tm~ plasma% % columnlZg/ml
rem. Cm~ rem. FP,, rem.rem.
Tm~
189
CA 02449843 2003-12-05
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5' 30'' 30'hrIcMhr 5' 30'hr~M hr % 30' 30' a pH2pH6.5
remain
B101 ND 39ND 3 NDND ND ND ND 601.81.2 0.529%'100 1 1 2 NDND
B104 ND 37ND 11NDND ND ND ND 69NDND NDND ND 45 1 3 5.84.4
B140 ND 49ND 1 NDND ND ND ND 871.10.4 1 7%'zND 2 1 22 0.70.7
B16 ND 6080 7 NDND ND ND ND 862.52.3 2 37%'z84 10 3 >30 1.20.4
B18 ND 7272 13NDND ND ND ND 862.62 2 17%'388 11 1 >30 ND0.8
B21 ND 93ND 31NDND ND ND ND 842.52.4 I 8/' 100 25 7 >30 NDND
B22 ND 58ND 11NDND ND ND ND 941.7L8 1 19%'15 32 1 >30 NDND
B30 ND 75ND NDNDND ND ND ND 953.20.5 0.535/'100 45 0 6 ND1.4
B31 ND 8573 18ND0.51 ND ND 923.40.9 1 34%'100 36 2 5 ND0.7
B32 ND 31ND 0 NDND ND ND ND 741.60.3 0.258/' 7 1 1 15 0.91.4
B33 ND 73ND 1 NDND ND ND ND 82NDND NDND 42 3 1 23 ND0.8
B35 ND 6764 32ND1.12 ND ND 875.30.9 3 31 100 63 2 7 ND1
%"
B36 ND 6766 191.10.2I 8%$ND 762.21.4 0.527%'100 43 1 6 ND1
B37 ND 6560 12ND1.22 ND ND 872.51.8 2 52%'3100 17 0 37 ND0.5
B38 ND 51ND 31ND4.52 ND ND 743.83.7 2 46/'I00 45 3 26 ND0.9
B39 ND 60ND 1 ND1.33 ND ND 1002.50.8 2 17%'z100 14 1 >30 ND0.7
B62 ND 28ND 8 1.3<0.03ND <I%'ND 831.71.4 1 24%'100 2 2 14 ND0.4
B63 ND 7661 13NDND ND ND ND 852.80.7 1 32%';100 74 11 4 ND2.8
B65 ND 62 NDNDND ND ND ND 892.40.351 50%'100 6 1 10 ND0.9
B66 ND 7080 21NDND ND ND ND 802.80.8 2 35%'"100 18 3 25 ND0.8
B68 ND 41ND 4 NDND ND ND ND 702.30.6 2 15%z'1 10 0 6 7.22.2
B72 "ND83ND 0 NDND ND ND ND 493.4LI 2 65%z'100 43 0 3 ND1.8
B81 ND 90ND 12NDND ND ND ND 812.72.7 1 57/'100 20 1 14 ND0.8
B83 ND 37ND 1 NDND ND ND ND 823.60.3 1 5%' 100 4 I 13 ND0.8
B84 ND 60ND 13ND0.11 ND ND 861.70.6 1 47%'''100 18 2 5 ND2.1
B91 ND 62ND 11NDND ND ND ND 791.90.9 1 14%'100 2 5 8 0.30.4
B92 ND 58ND 3 NDND ND ND ND 681.10.7 1 10%z21 18 1 >30 NDND
C12 ND 55ND 13NDND ND ND ND 593.54.6 0.534%'ND 20 0 3 7 ND
C13 ND 63ND 3 NDND ND ND ND 50NDND NDND 46 ND ND 8 0.3ND
C14 ND 82ND NDNDND ND ND ND NDNDND NDND ND ND ND ND NDND
C17 ND 6479 214.10.41 15%'ND 772.94.4 1 75%'80 47 1 2 0.9LS
C2 ND 8077 30ND3.22 ND ND 852.91.7 1 57%'24 65 2 5 ND0.7
C5 ND 86 NDNDND ND ND ND NDNDND NDND ND ND 25 ~ ND
ND
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Table 3 (cont'd.)
Compound
No. Human M F Rat Dog MonkeyH Solubility
. a a S
I m A
a a
R 1
a a
t R
a
t
rem. % TvzCm~T~ Fa0.% TvzCm~Tm~Fp,0.plasma% % columnpglml
rem. rem. rem.rem. '
5' 30'' 30'hr1tMhr S' 30'hr plvlhr % 30' 30' a pH2pH6.5
remain
B34 ND 83 10NDND ND ND ND 30ND ND ND ND ND 8 18 ND NDND
B40 ND 85 72NDND ND ND ND 83ND ND ND ND ND 68 47 ND NDND
B46 ND 85 62NDND ND ND ND 75ND ND ND ND ND 76 57 ND NDND
B82 ND 82 71NDND ND ND ND 76ND ND ND ND ND 80 56 ND NDND
B53 ND 81 13NDND ND ND ND 50ND ND ND ND ND 8 26 Nb NDND
A15 ND 52 3 NDND ND ND ND 82ND ND ND ND ND 1 0 12 ND0.6
B114 ND 51 25NDND ND ND ND 82ND ND ND ND ND 22 0 ND ND1.5
B143 ND 69 17NDND ND ND ND 70ND ND ND ND ND 11 4 4 ND0.3
B147 ND 57 19NDND ND ND ND 89ND ND ND ND ND 35 11 ND ND0.4
B41 ND 57 29ND3.61 ND ND 793.64 2 64%'ND 58 1 8 NDI
B45 ND ND NDNDND ND ND ND NDND ND ND ND ND ND ND ND ND1.5
B49 ND 59 61NDND ND ND ND 952.62.31 17%'83 63 0 >30 ND0.5
BSO ND 50 70NDND ND ND ND 822.25 2 15%'100 77 0 >30 ND0.5
B51 ND 86 2 NDND ND ND ND SO2.30.21 4%'94 27 1 7 ND0.6
BS2 ND 57 43ND0.93 ND ND 906.60.42 27%'100 33 0 4 ND8
B54 ND 38 1 NDND ND ND ND 77ND ND ND ND ND 25 2 ND ND0.5
B55 ND 75 20NDND ND ND ND 60ND ND ND ND ND 12 30 >10 ND4.2
B56 ND 57 54NDND ND ND ND 88ND ND ND ND ND 31 0 >20 ND2.8
B57 ND 76 24ND4.87 ND ND 61>101.25 46%'100 33 48 >20 ND>I5
BS8 ND 63 40NDND ND ND ND 91ND ND ND ND ND 18 0 21 ND1
B59 ND 38 1 NDND ND ND ND 73ND ND ND ND ND 2 0 18 NDND
B60 ND 55 4 NDND ND ND ND 80ND ND ND ND ND 3 0 16 NDND
B61 ND 86 15ND2.37 ND ND 436.20.75 34/100 13 54 5 ND12
1
B64 ND 69 0 NDND ND ND ND 71ND ND ND ND ND 0 0 0.9 ND7
B69 ND 80 6 NDND ND ND ND 1002.45.21 53%'ND 75 1 4 ND2.5
B70 ND 61 8 NDND ND ND ND 82ND ND ND ND ND 31 0 5 ND1
B71 ND 72 23NDND ND ND ND 76ND ND ND ND ND 25 7 >10 ND0.4
B73 ND 82 4 NDND ND ND ND 20ND ND ND ND ND 26 14 28 ND14
B88 ND 74 46NDND ND ND ND 8IND ND ND ND ND 54 20 14 ND0.4
C15 ND 59 16ND3.23 ND ND 892 1.70.531/'ND 52 0 2 NDND
C20 ND 28 0 NDND ND ND ND 171 <0.1ND <1/'0 1 1 5 ND4,5
C22 ND 5 NDNDND ND ND ND NDND ND ND ND ND ND ND ND NDND
I26 ND 44 NDNDND ND ND ND NDND ND ND ND ND ND ND ND NDND
136 ND 26 0 NDND ND ND ND 823.20.40.520/'100 0 0 3 ND4
I37 ND 66 NDNDND ND ND ND 893.40.71 25/'ND ND ND 21 ND0.8
I38 ND 82 17NDND ND ND ND 822.10.92 20%'24 7 2 8 ND1
T4 ND 68 31NDND ND ND ND 893.10.72 21%'81 30 1 10 ND0.2
Table 3 (cont'd.)
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Compound
No. Human M F Rat Dog MonkeyH Solubility
a a S
1 m A
a a
R 1
a a
t R
a
t
rem. % T1,2Cm~Tm~FP.,% TvaCm~ Tm~Fp,,plasma% % columnpg/ml
rem. rem. rem.rem.
5' 30'' 30'hrpM hr 5' 30'hr~M hr % 30' 30' a pH2pH6.5
remain
Bil2 ND 73 11 NDND ND ND ND 51NDND ND ND ND 15 18 ND NDND
B116 ND 78 63 NDND ND ND ND 100NDND ND ND 100 33 0 >30 ND1.6
BII7 ND 56 9 NDND ND ND ND 59NDND ND ND ND 5 0 2 ND>15
B118 ND 60 31 NDND ND ND ND 845.90.3 3 8/'100 15 1 >4 ND3.8
BI19 ND 72 80 NDND ND ND ND 962.91.2 1 11/'95 82 ND >30 ND0.5
B120 ND 51 13 NDND ND ND ND 571.81 1 23%'100 12' 1 >4 ND10
BI21 ND 71 29 NDND ND ND ND 645.20.9 7 45%'86 36 1 >30 ND9
B122 ND 45 50 NDND ND ND ND 84NDND ND ND ND 32 0 ND NDND
B123' ND 12 0 NDND ND ND ND 0 NDND ND ND ND 4 0 ND NDND
B124 ND 95 76 NDND ND ND ND 774.60.9 3 35%1ND 65 59 5 NDND
BI25 ND 68 33 NDND ND ND ND 77NDND ND ND ND 23 1 ND NDND
B126 ND 92 15 NDND ND ND ND 53NDND ND ND ND 12 30 ND NDND
B127 ND 86 15 NDND ND ND ND 50NDND ND ND ND 12 25 ND NDND
B128 ND 43 21 NDND ND ND ND 78NDND ND ND ND 1 0 ND NDND
B129 ND 47 12 NDND ND ND ND 84NDND ND ND ND 2 0 ND NDND
BI30 ND 97 48 NDND ND ND ND 70NDND ND ND ND 45 78 ND NDND
B131 ND 95 87 NDND ND ND ND 91NDND ND ND ND 51 53 ND NDND
BI32 ND 86 83 NDND ND ND ND 867.90.6 3 31%'ND 58 51 7 NDND
B133 ND 90 35 NDND ND ND ND 91NDND ND ND ND 72 73 ND NDND
B136 ND 82 48 NDND ND ND ND 55NDND ND ND ND 51 54 ND NDND
B28 ND 53 24 NDND ND ND ND 85NDND ND ND ND 25 1 ND NDND
B29 ND 27 49 NDND ND ND ND 85NDND ND ND ND 35 0 ND NDND
B89 ND 99 40 ND2.65 ND ND 788.90.7 7 31%'100 44 55 >10 ND50
B90 ND 91 65 NDND ND ND ND 80NDI ND ND ~ I ~ I ~ ~
ND ND 70 44 ND NDND
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Table 3 Notes:
' 10 mg/kg as 5 mg/ml solution in 10% DMSO 10% cremophor 80% saline
2 IO mg/kg as 5 mg/ml solution in 10% cremophor 90% saline
' SOmg/kg as 25mg/ml solution in 50%Labraso150%H20(Cmax-l.4uM,Tmax-
lhras100mg/ml
inLabrasol;Cmax<O.OSuMas25mg/ml in0.5%CMC)
4 50 mg/kg as 25 mg/ml solution in 50%Labrasol 50%H20 (Cmax - 0.04uM, Tmax -
2hr as 25 mg/ml in 0.5%CMC)
s 50 mg/kg as 50 mg~ml solution in 50%Labrasol 50%H20 (Cmax - 0.6uM, Tmax -
lbr as 100 mg/ml in PEG400;
Cmax - 0.6uM, Tmax - Shr as 100mg/ml in PG; Cmax - 0.3uM Tmax - 3hr as 25
mg/ml in 0.5%CMC)
6 50 mg/kg as 25 mgJml solution in 50%Labrasol 50%H20 (Cmax - O.IuM, Tmax -
lhr as 100mg/ml in Labrasol;
Cmax<O,luM as 100mg/ml in PEG400 or 25 mg/ml in 0.5%CMC)
' S0 mg/kg as 25 mg/ml solution in 50%Labrasol 50%H20 (Cmax - 0.2uM, Tmax -
Shr as 100 mg/ml in PEG400;
Cmax - 0.4uM, Tmax - Shr as 25 mg/ml solution in 0.5%CMC)
$ 20 mg/kg as 10 mg/ml solution in 50% Labrasol 50% H20 (at SOmg/kg dose: Cmax
-O.luM Tmax - 2hr as 25 mg/ml in
50%Labrasol 50%H20; Cmax<O.OSuM as 100mgJm1 in PEG400 or 25 mg/ml in 0.5%CMC)
0 20 mg/kg as 10 mg/ml solution in 10% DMSO 10% cremophor 80% saline (when
given as 40 mg/ml in PEG400 or PG, FP,°_ was 0%)
'° 10 mg/lcg as 5 mg/ml solution in 10% DMSO 20% cremophor 70% saline
" 6 mg/kg as 3 mg/ml solution in 10% DMSO 10% cremophor 80% saline
'z 10 mg/kg as 5 mg/ml solution in 30% Labrasol 70% saline
"10 mg/kg as 5 mg/ml solution in 50% Labrasol 50% H20
'45 mg/kg as 2.5 mg/ml solution in 10% DMSO 10% cremophor 80% saline
's 50 mg/kg as 50 mg/ml solution in 50%Labrasol 50%H20 (Cmax - 3.4uM, Tmax -
lhr as 25 mg/ml in 0.5% CMC;
Cmax - 0.4uM, Tmax - 2hr as 100 mgJml in PEG400)
'620 mg/kg as 10 mg/ml solution in 10% DMSO 10% cremophor 80% saline (could
not dissolve it in PEG, Labrasol or PG at 50 mg/ml)
9
"50 mg/kg as 25 mg/ml solution in 50%Labrasol 50%H20 (Cmax - 0.07uM, Tmax -
3hr as 25 mg/ml in 0.5% CMC)
'B 50 mg/lcg as 100 mg/ml in PEG400 or as 25 mg/ml in 0.5% CMC (Cmax - 0.2uM,
Tmax - 0.25 hr as
50 mg/ml solution in 50%Labrasol 50%H20)
'9 50 mg/kg as 50 mg/ml solution in 50%habrasol 50%H20 (Cmax - 0.9uM Tmax -
lhr as 100 mg/ml in PEG400;
Cmax - 0.9uM Tmax - O.Shr as 100 mg/ml in PG; Cmax - 0.4uM Tmax- lhr as 25
mg/ml in 0.5%CMC)
zo 50 mg/Icg as 50 mg/ml solution in 50%Labrasol 50%H20 (Cmax - O.SuM; Tmax -
3hr as 25 mg/ml in 0.5%CMC)
z' S0 mg/kg as 50 mg/ml solution in Labrasol/H201/1 (Cmax - I .2uM, Tmax - 7hr
as 100 mg/ml in PEG400;
Cmax - 0.7uM, Tmax - 3hr as 25 mg/ml in 0.5% CMC; Cmax -1 uM, Tmax - 7hr as I
OOmg/ml in PG)
zz 50 mg/kg as 25 mg/mI suspention in 0.5%CMC (Cmax - 3.3uM, Tmax - Shr as 50
mg/ml in Labrasol/H201/1)
z' S0 mg/kg as 25mg/ml solution in Labrasol/H201/1 (Cmax-2.SuM, Tmax-7hr as
100 mg/ml in PG;
Cmax-3.6uM, Tmax-7hr as 25 mg/ml in 0.5%CMC)
z° 50 mp~kg as 50 mg/ml in 50%Labrasol 50%H20 (Cmax -l.9uM, Tmax - Shr
as 25 mg/ml in 1%CMC)
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In Tlivo Tests
Materials and Methods:
Adult male Sprague-Dawley rats were purchased from Harlan Sprague
Dawley (San Diego). Animals were housed two per cage and maintained in a
temperature- controlled room (22 ~ 2°C) with a photoperiod of 12 hours
light/12
hours dark (lights on at 0600 hours). Rat chow (Teklad LM-485 rat diet,
Madison,
WI) and tap water were provided ad libitum.
Animal models to assess activity of GnR_u_ anta onists:
Model # 1: Castrated Male Rat Model
1o Surgical removal of the gonads removes circulating testosterone and
eliminates the negative feedback of testosterone on the hypothalamus. As a
result,
GnRH is elevated and consequently elevates LH. A GnRH antagonist would be
expected to reduce GnR_H_ mediated elevations of LH levels. Amide, a GnR_H_
peptide
antagonist, reduces LH levels in castrated rats. The model seems suitable for
is evaluating small molecule GnRH antagonists.
Male Sprague-Dawley (200-225 g) rats were castrated via the scrotal approach
under halothane anesthesia. Animals were allowed 14 days post operative
recovery
prior to study. Thirteen days following castration, animals were anesthetized
with
halothane and instrumented with indwelling jugular vein cannula. Details ofthe
2o cannulation procedure have been described previously (Harms et al., Applied
Physiol.
36:391-398 (1974)).
On study day, animals were .allowed to acclimate to the procedure room while
residing in their home cage. Basal blood samples were drawn from all animals.
Immediately following basal sampling, vehicle or test compounds were
administered
25 by intravenous (iv), intraperitoneal (ip), intramuscular (im) or oral (po)
routes. Test
compounds were formulated in vehicles specified in Table 3. Blood samples were
drawn into heparin containing tubes at multiple time points post treatment.
Blood was
centrifuged immediately, plasma collected and stored in -20° freezer
until assayed.
Plasma samples were analyzed using DSL-4600 ACTIVE LH coated-tube
3o immunoradiometric assay kit from Diagnostic Systems Laboratories, Inc.
Webster,
Texas. Cremophor EL obtained from Sigma, St. Louis, MO.
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The results of the GnRH antagonist experiments with various concentrations
(1.0-100 mg/kg) in different administration routes (iv, im, and po) of GnRH
agents,
Compounds C23, and B52 in the above castrated rat model are shown in Figures 1-
4.
Model #2: Intact Male Rat
Testosterone is a hormone regulated by the hypothalamic-pituitary-gonadal
axis. GnRH is secreted in pulses from the hypothalamus and stimulates the
anterior
pituitary gland to release gonadotropic hormones LH and FSH. Testosterone is
produced when the testes are stimulated by LH. A GnRH antagonist is expected
to
reduce testosterone levels by inhibiting GnRH stimulation of LH release.
' Male Sprague-Dawley (250-275 g) rats were single-housed and allowed to
acclimate for 1 week prior to study. On study day animals were treated with
vehicles
specified in Table 3) or test compound. Blood samples were obtained via
indwelling
jugular vein cannulae implanted 5 days prior to study (Harms et al., Applied
Physiol.
36:391-398 (1974)). Blood samples were drawn into heparin containing tubes at
multiple time points post treatment. Blood was centrifuged immediately, plasma
collected and stored in -20° freezer until assayed. Plasma samples were
analyzed
using DSL-4000 ACTIVE Testosterone coated-tube radioimmunoassay kit from
Diagnostic Systems Laboratories, Inc. Webster, TX.
The results of the GnRH antagonist experiments with various concentrations
(1.0 -100 mg/lcg) in administration routes (iv, im, and po) of GnR_H_ agents,
Compounds C23, B3, C2, B52, and B89 in the above intact rat model are shown in
Figures 5-10.
Pharmaceutical Compositions
The exemplary compounds described above may be formulated into
2s pharmaceutical compositions according to the following general examples.
Parenteral Composition
To prepare a parenteral pharmaceutical composition suitable for
administration by injection, 100 mg of a water-soluble salt of a compound of
the
Formula I, II, or III is dissolved in DMSO and then mixed with 10 mL of 0.9%
sterile
3o saline. The mixture is incorporated into a dosage unit form suitable for
administration
by injection.
Oral Composition
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To prepare a pharmaceutical composition for oral delivery, 100 mg of a
compound of Formula I, II or III is mixed with 750 mg of lactose. The mixture
is
incorporated into an oral dosage unit for, such as a hard gelatin capsule,
which is
suitable for oral administration.
Intxaocular Composition
To prepare a sustained release pharmaceutical composition for intraocular
delivery, a compound of Formula I, II or III is suspended in a neutral,
isotonic
solution of hyaluronic acid (1.5% cone) in a phosphate buffer (pH 7.4) to form
a 1%
suspension, which is suitable for intraocular administration.
It is to be understood that the foregoing description is exemplary and
explanatory in nature, and is intended to illustrate the invention and its
preferred
embodiments. Thus, scope of the invention should be understood to be defined
not by
the foregoing description, but by the following claims and their equivalents.
1s
h
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