Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL BIOACTIVE DIPHENYL ETHENE COMPOUNDS AND THEIR THERAPEUTIC APPLICATIONS
Background of the Invention
Stilbene derivatives are well-known in the art to have a wide range of
activities and are widely
distributed in nature. There is a growing interest in stilbene derivatives
because of a range of activities
that have been observed in some of the naturally occurring as well as some of
the synthetic stilbenes. A
stilbene derivative, 3,5,4'-trihydroxystilbene, commonly known as resveratrol
in which both isomers (cis
or trans) has been reported to have a range of biological functions, such as
mediating inflammation and
cancer chemoprevention (Jang, et al. 1997, Science, 275, 218, US6,008,260).
It is known in the art that substitution on the various position on the two
phenyl rings of the basic
stilbene structure results in a great diversity of compounds, including those
with one or two substituents
on one or both of the phenyl rings of the stilbene structure(Shudo K., 1988,
US4723028; Hensley, K.L.,
et al., WO99/59561, Kunihiro N., 1983, JP58159410; Genji L, 1995, JP07053359
and GB1465661) and
those with three or more substituents on the phenyl rings (Koichi, S. et al.,
1986, EP0170105; Shozo Y.,
et al., 1986, JP08337523; and Charpentier B. et al., 1992, W092/19583).
Compounds with other
substitution on the phenyl ring, such as derivatives of vitamine A (Ney, U.M.,
et al. 1987, Dermatologica,
175:93-99) and vitamine D (WO 00/26167) are well-known in the art. Several
publications
(W092/16486, W099/40056, WO01/95859 and Cuslunan M. et. al. (1992, J. Med.
Chem., 35:2293-
2306) disclosed compounds that are derived from 3, 4, 5-trimethoxyl stilbene.
These compounds showed
anti-neoplastic activity and modest activity of modulating cytokines
(WO01/95859).
Recently, a group of stilbenes with a unique substitute pattern of two
hydroxyl groups, or their
derivatives, in position 3 and 5 and a substituent in between have been
disclosed. Pending applications of
the inventors are directed to compounds having inhibitory activity against
kinases, anti-inflammatory
activity (WO 01/42231), having effect on T lymphocytes, macrophages,
neutrophils and mast cells and
modulating a variety of immune and inflammatory activities (WO 02/057219).
However, it is not
discovered until now that with the unique substitution pattern on one phenyl
group the substitution on the
other phenyl group with a range of specific substituents, in particular fluoro
atoms, resulted in
compounds that have surprising immune-modulating activity. The present
invention is related to these
novel stilbene compounds, their synthesis, their unexpected activity,
pharmaceutical composition and
their use for treatment of disorders associated with these activities.
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Summary of the Invention
The invention disclosed herein relates to compounds of Formula I,
pharmaceutically acceptable salts
thereof, pharmaceutical composition of these compounds that have been found
useful as immune-
modulating agents.
R3~
R4 Rs
R1 ~ ~ \ _
R2O ~ ~ R6 I
R8 R'
Detailed Description of the Invention
The invention covers new compounds of general formula I,
R30
R4 Rs
R1
R2O - ~ > ~ R6 1
R$ R'
wherein R' is selected from the group consisting of unsubstituted or
substituted alkyl, cycloalkyl, alkenyl,
alkynyl, aryl or aralkyl group, halo, or COR9;
RZ and R3 are independently selected from the group consisting of H,
unsubstituted or substituted alkyl,
cycloalkyl, aryl, aralkyl or acyl;
R4, R5, R6, R' and R$ are not H simultaneously and are independently selected
from the group
consisting of H, unsubstituted or substituted alkyl, alkenyl, alkynyl, aryl or
aralkyl group, halo, nitro, CN,
CORD, NRl°R", S(O)ZN R'°Rll, S(O)"Rl°, n = 0-2, OR12, a
cyclic, or a heterocyclic group; with the
proviso that R~ is not hydroxy or alkyoxy group when R' is an unsaturated
group comprising of 1-3
isoprene unit(s);
R9 is selected from H, unsubstituted or substituted alkyl, cycloalkyl, aryl,
or aralkyl, or NR'°R11, or
ORIO;
Rl° and Rll are selected from H, unsubstituted or substituted alkyl,
cycloalkyl, aryl or aralkyl;
R'z is selected from H, unsubstituted or substituted alkyl, cycloalkyl, aryl,
aralkyl or acyl.
In particular, new compounds of general formula I, wherein
R4, R5, R6, R' and R8 are independently selected from the group consisting of
H, unsubstituted or
substituted alkyl, alkenyl, alkynyl, aryl or aralkyl group, halo, nitro, CN,
COR9, NRl°Rll, S(O)ZN
R'°RI1, S(O)"R'°, n = 0-2, OR12, a cyclic, or a heterocyclic
group and one or more than one of R4, R5,
R6, R' and R8 is F. The configuration of the double bond of the compound of
formula I is E or ~.
Highly preferred compounds include the following:
4-[2-(3.5-Dihydroxy-4-i-propylphenyl)ethenyl]benzoic acid (6).
3-[2-(3.5-Dihydroxy-4-i-propylphenyl)ethenyl]benzoic acid (7).
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5-[2-(4-Hydroxyphenyl)ethenyl]-2-i-propyl-1,3-benzenediol (13).
5-[2-(3,5-Dihydroxyphenyl)ethenyl]-2-i-propyl-1,3-benzenediol (15).
5-[2-(2-Fluorophenyl)ethenyl]-2-i-propyl-1,3-benzenediol (37).
5-[2-(3-Fluorophenyl)ethenyl]-2- i-propylphenyl-1,3-diol (38).
5-[2-(4-Fluorophenyl)ethenyl]-2- i-propylphenyl-1,3-diol (39).
5-[2-(3,5-Difluorophenyl)ethenyl]-2- i-propylphenyl-1,3-diol (40).
5-[2-(2,4-Difluorophenyl)ethenyl]-2-i-propyl-1,3-benzenediol (41).
S-[2-(2,6-Difluorophenyl)ethenyl]-2-i-propyl-1,3-benzenediol (42).
2-i-Propyl-5-[2-(2,4,6-trifluorophenyl)ethenyl]-1,3-benzenediol (43).
5-[2-(2,3,4,5,6-Pentafluorophenyl)ethenyl]-2-i-propyl-1,3-benzenediol (44).
The invention also covers use of the compounds of general formula I as immune-
modulating agents.
The compounds of this invention may be synthesized using general procedures
disclosed in patent
publication W002/057219 with specific modifications. Examples given herein are
illustrative only, and
are not considered as limitations of this invention. In general, the stilbene
structures of the compounds of
the invention are constructed via Wittig olefination (Scheme 1) and Heck
reaction (Scheme 2). The
corresponding 1,3-benezendiol can be obtained by a deprotection reaction.
Scheme 1. Wittig olefination:
H O
-r- ~P(O)(OEt)2
R20 ~ 3 Ar
OR
RI RIO
O Rl
P(OEt)2 \ Ar
O R30
II
'Ar
R20 \ OR3
R1
Scheme 2. Heck reaction:
i Pd-Cat R3O
R O ~ I 2 + ArBr RI
\OR 2 \ Ar
RO
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Scheme 3. Modification:
R3
BuLi HO / \
R'CHO R' Rzo
R30
R3 Ar'B(OH~ ~,, / \
\ Ar
~r / \ ~ R2O
'-Ar
RzO Rs0
BuLi OHC / \
DMF Rz0
Etc.
One modification of Rl is to start with a bromostilbene (Scheme 3). The
bromide can be converted
to other functional groups by Suzuki coupling or a bromo-lithium exchange
followed by reacting with an
electrophile.
The compounds utilized in accordance with the present invention have Z or E
configuration of the
double bonds resulting in traps and cis isomers. The scope of the present
invention is intended to cover
all such isomers as well as mixtures of cis and traps isomers.
A pharmaceutically acceptable salt may be prepared for any compounds in this
invention having a
functional capability of forming such salt. Pharmaceutically acceptable salts
may be formed with
inorganic and/or organic acids and bases. Suitable acids include, for example,
hydrochloric, sulfuric,
nitric, benzenesulfonic, acetic, malefic, tartaric and the like, which are
pharmaceutically acceptable.
While pharmaceutically acceptable salts are preferred, particularly when
employing the compounds of
the invention as medicaments, other salts find utility, for example, in the
production of these compounds,
or where non-medicament-type uses are contemplated.
Compounds of the present invention have shown a range of immune-modulating
activities that are
demonstrated and confirmed in the forthcoming examples. Compounds which have
immune-modulating
activity are well-known in the art, and are described in numerous patent and
scientific publications. It is
generally known and accepted in the art that immune-modulating activity is
useful for treating numerous
diseases and conditions of animals, including humans. It is generally known in
the art that
pharmaceuticals having a compound or compounds with immune-modulating
activity, such as those
disclosed herein, as the active ingredient are useful agents for the treatment
of disorders such as: clinical
transplants (such as organ transplant, acute transplant or heterograft or
homograft (such as is employed in
burn treatment)) rejection; protection from ischemic or reperfusion injury
such as ischemic or reperfusion
injury incurred during organ transplantation, myocardial infarction, stroke or
other causes;
transplantation tolerance induction; arthritis (such as rheumatoid arthritis,
psoriatic arthritis or
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osteoarthritis); multiple sclerosis; inflammatory bowel disease, including
ulcerative colitis and Crohn's
disease; lupus (systemic lupus erythematosis); graft vs. host disease; T-cell
mediated hypersensitivity
diseases, including contact hypersensitivity, delayed-type hypersensitivity,
and gluten-sensitive
enteropathy (Celiac disease); psoriasis; contact dermatitis (including that
due to poison ivy); Hashimoto's
thyroiditis; Sjogren's syndrome; Autoimmune Hyperthyroidism, such as Graves'
Disease; Addison's
disease (autoimmune disease of the adrenalglands); Autoimmune polyglandular
disease (also known as
autoimmune polyglandular syndrome); autoimmune alopecia; pernicious anemia;
vitiligo; autoimmune
hypopituatarism; Guillain-Barre syndrome; other autoimmune diseases;
glomerulonephritis, serum
sickness; uticaria; allergic diseases such as respiratory allergies (asthma,
hay fever, allergic rhinitis) or
skin allergies; scleracierma; mycosis fungoides; acute inflammatory responses
(such as acute respiratory
distress syndrome and ishchemia/reperfusion injury); dennatomyositis; alopecia
areata; chronic actinic
dermatitis; eczema; Behcet's disease; Pustulosis palmoplanteris; Pyoderma
gangrenum; Sezary's
syndrome; atopic dermatitis; systemic schlerosis; and morphea. In particular,
the activity against VEGF
expression finds utility in treating cancers and VEGF associated disorders.
The inhibition of LTBd
induced cell migration is useful as anti-inflammatory agents.
The present invention thus provides methods for the treatment of disorders
associated with the
abovementioned activities, comprising the step of administering to a subject
in need thereof at least one
compound of the formula I in an amount effective therefore. Other therapeutic
agents such as those
known to the skilled in the art may be employed with the inventive compounds
in the present methods. W
the methods of the present invention, such other therapeutic agents) may be
administered prior to,
simultaneously with or following the administration of the compounds) of the
present invention.
Examples of pharmaceutical compositions include any solid (tablets, pills,
capsules, granules,
powder, suppositories etc.) or liquid (solutions, suspensions or emulsions) in
a suitable composition for
oral, topical, parenteral or rectal administration. These formulations may
contain the pure compound or
be in combination with a carrier or some other pharmaceutically active
compound. These compositions
may need to be sterile when administered parenterally.
For topical use, it will be preferred to use in the form of creams, ointments,
jellies, solutions or
suspensions, etc., containing the compound of Formula I (For purposes of this
application, topical
application shall include mouth washes and gargles.)
Dosage levels of the order of from about 0.01 mg to about 140 mg/kg of body
weight per day are
useful in the treatment of the above-indicated conditions, or alternatively
about 0.5 mg to about 7 g per
patient per day. For example, inflammation may be effectively treated by the
administration of from
about 0.01 to about 50 mg of the compound per kilogram of body weight per day,
or alternatively about
0.5 mg to about 3.5 g per patient per day, preferably 2.5 mg to 1 g per
patient per day.
The amount of active ingredient that may be combined with the carrier
materials to produce a single
dosage form will vary depending upon the host treated and the particular mode
of administration. For
example, a formulation intended for the oral administration of humans may
contain from 0.5 mg to 5 g of
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active agent compounded with an appropriate and convenient amount of carrier
material that may vary
from about 5 to about 95 percent of the total composition. Dosage unit forms
will generally contain
between from about 1 mg to about 500 mg of an active ingredient, typically 25
mg, 50 mg, 100 mg, 200
mg, 300 mg, 400 mg, 50,0 mg, 600 mg, 800 mg, or 1000 mg.
It will be understood, however, that the specific dose level for any
particular patient will depend
upon a variety of factors including the age, body weight, general health, sex,
diet, time of administration,
route of administration, rate of excretion, drug combination and the severity
of the particular disease
undergoing therapy.
The invention is now described in greater detail by reference to the following
non-limiting examples.
Synthesis of compounds
Example 1. 4-[2-(3.5-Dimethoxy-4-i-propylphenyl)ethenyl]benzoic acid (1).
a). Methyl 3,5-dimethoxy-4-i-propylbenzoate.
This compound was obtained using method described in WO 01/42231. 1HNMR
(CDC13, ppm): b
1.32 (d, J = 7.2 Hz, 6H), 3.66 (hept, J = 7.2 Hz, 1H), 3.82 (s, 6H), 3.95 (s,
3H), 7.25 (s, 2H).
b). 3,5-Dimethoxy-4-i-propylbenzyl alcohol.
To a suspension of LiAlH4 (95%) (S.OOg, 125mmol) in dry ether (100mL) at
0°C was added a
solution of methyl 3,5-dimethoxy-4-i-propylbenzoate (15.7g, 90.1mmo1), in
ether (300mL) under Nz.
The suspension was stirred at 0°C for one hour then for an additional
hour at room temperature. The
reaction was quenched by slow addition of a saturated Na2S04 aqueous solution
(1 OmL) at 0°C. The
mixture was stirred overnight. The solid was filtered off and the filtrate was
evaporated to dryness to
give the desired alcohol (13.8g, 88% yield) as white crystals. IHNMR (CDCl3.
ppm): 8 1.34 (d, J =
7.2Hz, 6H), 3.65 (kept., J = 7.2Hz, 1H), 3.88 (s, 6H), 4.70 (s, 2H), 6.62 (s,
2H).
c). 3,5-Dimethoxy-4-i-propylbenzyl aldehyde.
A mixture of 3,5-dimethoxy-4-i-propylbenzyl alcohol (13.OSg, 62.lmmol) and
pyridinium
chlorochromate (33.92g, 157mmo1) was stirred in CHzCl2 (100mL) in the presence
of KzCO3 (4.18g,
30mmo1) for 30 min. Ether (300mL) was added to quench the reaction. The
mixture was passed
through a short pad of Florisil and the pad was washed thoroughly with ether.
Evaporation of the
solvent gave 3,5-dimethoxy-4-i-propylbenzyl aldehyde (11.89g. 92% yield) as a
yellowish crystal.
1HNMR (CDCl3, ppm): ~ 1.32 (d, J = 7.2Hz, 6H), 3.68 (hept., J = 7.2Hz, 1H),
3.92 (s, 6H), 7.12 (s,
2H), 9.96 (s, 1H).
d). (3,5-Dimethoxy-4-i-propylphenyl)ethene.
To a suspension of methyltriphenylphosphonium bromide (6.89g, 19.3mmo1) in THF
(100mL) under
argon was added BuLi (7.7m1, 2.5M in hexane, 19.3mmol) at room temperature.
The resultant red
solution was stirred for 10 min. and then 3,5-dimethoxy-4-i-propylbenzyl
aldehyde (4.02g, 19.3mmo1)
in THF (20mL) was added. After 2 hours, the reaction was quenched with water
(20mL). The
mixture was extracted with ether (3 x 100mL). The extract was washed with
saturated saline solution
(3 x 30mL) and dried over sodium sulphate. Evaporation of ether followed by
flash chromatography
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using 3% ethyl acetate in hexane afforded pure (3,5-dimethoxy-4-i-
propylphenyl)ethene (2.64g, 66%
yield) as a colorless solid. II~IMR (CDCl3, ppm): 8 1.31 (d, J = 7.lHz, 6H),
3.61 (qint, J = 7.lHz,
1H), 3.86 (s, 6H), 5.25 (d, J = llHz, 1H), 5.73 (d, J = l7Hz, 1H), 6.64 (s,
2H), 6.70 (dd, J = 11, l7Hz,
1H).
e). 4-[2-(3.5-Dimethoxy-4-i-propylphenyl)ethenyl]benzoic acid (1).
A mixture of (3,5-dimethoxy-4-i-propylphenyl)ethene (0.303g, l.SOmmol), 4-
bromobenzoic acid
(0.269g, 1.30mmol), dihydrogen di-p.-chlorotetrkis(di-test-butylphosphinito-
xP)dipalladate (0.0625g,
0.067mmol), Bu4NI (0.245g, 0.67mmo1) and KzC03 (0.614g, 4.40mmol) in DMF (7mL)
was heated
at 140°C under argon. After the reaction was complete (Sh), the
reaction mixture was poured into
water (100m1). This was washed with ether. The aqueous phase was acidified
with 6NHCl and
extracted with ether (2 X100mL). The extract was washed with saturated sodium
chloride and then
dried over anhydrous Na2SO4. Evaporation of ether gave the pure acid 1
(0.345g, 71% yield). 1~INMR
(CDCl3, ppm): S 1.32 (d, J = 7.lHz, 6H), 3.63 (qint, J = 7.lHz, 1H), 3.90 (s,
6H),6.76 (s, 2H), 7.08 (d,
J = l7Hz, 1H), 7.27 (d, J = l7Hz, 1H), 7.63 (d, J = 8Hz, 2H), 8.13 (d, J =
8Hz, 2H).
Example 2. 3-[2-(3.5-Dimethoxy-4-i-propylphenyl)ethenyl]benzoic acid (2).
This compound was synthesized from (3,5-dimethoxy-4-i-propylphenyl)ethene and
3-bromobenzoic
acid in 77% yield in the same way as described in preparation of 1. 1HNMR
(CDCl3, ppm): 8 1.32 (d,
J = 7.lHz, 6H), 3.63 (qint, J = 7.lHz, 1H), 3.90 (s, 6H), 6.76 (s, 6H), 7.08
(d, J = l7Hz, 1H), 7.25 (d,
J = l7Hz, 1H), 7.50 (t, J = 7.7Hz, 1H), 7.79 (d, J = 7.7Hz, 1H), 8.04 (d, J =
7.7Hz, 1H), 8.31 (s, 1H).
Example 3. 4-[2-(3.5-Dihydroxy-4-i-propylphenyl)ethenyl]benzoic acid (6).
A mixture of 4-[2-(3.5-dimethoxy-4-i-propylphenyl)ethenyl]benzoic acid
(0.289g, 0.886mmol) and
pyridine hydrochloride (0.678, 5.9 mmol) was heated at 200°C for 2 h
under a stream of argon. The
reaction mixture was cooled to room temperature. 2NHC1 (IOmL) and ether (SOmL)
was added. The
organic layer was separated and the aqueous mixture was extracted with ether
(2 x SOmL). The
extract was washed with saturated brine and dried over anhydrous NaaS04.
Evaporation of ether
followed by flash chromatography using ethyl acetate/hexane/acetic acid
(40/60/1) afforded the pure
acid 6 (0.03g, 11% yield).'HNMR (DMSO-d6, ppm): 8 1.22 (d, J = 7.OHz), 6.49
(s, 2H), 6.90 (d, J =
l8Hz, 1H), 7.19 (d, J = l8Hz, 1H), 7.67 (d, J = 8Hz, 2H), 7.90 (d, J = 8Hz,
2H), 9.14 (s, 2H).
Example 4. 3-[2-(3.5-Dihydroxy-4-i-propylphenyl)ethenyl]benzoic acid (7).
This material was prepared from 3-[2-(3.5-dimethoxy-4-i-
propylphenyl)ethenyl]benzoic acid 2 and
pyridine hydrochloride in 86% yield in the same way as described in example 3.
'I~~1MR (DMSO-d6,
ppm): 8 1.22 (d, J = 7.OHz, 6H),6.48 (s, 2H), 7.03 (d, J = l7Hz, 1H), 7.12 (d,
J = l7Hz, 1H), 7.46 (t, J
= 7.SHz, 1H), 7.7-7.9 (m, 2H), 8.06 (s, 1H), 9.12 (s, 2H).
Example 5. 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-phenylethene (19).
a). Diethyl benzylphosphonate.
The mixture of benzyl bromide (l2mL, lOlmmol) and triethyl phosphite (25mL,
146mmol) was
heated at 110-130°C in the presence of Bu4NI (O.OSg) overnight. The
excess triethyl phosphite was
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removed under reduced pressure at 110°C. The phosphonate (23g) was
obtained quantitatively as a
colorless liquid. 1HNMR (CDC13, ppm): 8 1.28 (t, J = 7.2Hz, 6H), 3.20 (d, J =
21.9Hz, 2H), 4.10 (dt.,
J = 7.2Hz, 7.2Hz, 4H), 7.30 (s, SH).
b). 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-phenylethene (19).
To a solution of diethyl benzylphosphonate obtained above (11.39g, 54.7mmol)
in THF (100mL) at
0°C was added NaH (60% in mineral oil) (4.68g, 115mmol) under N2. After
the addition was
completed, the suspension was stirred at 0°C for 1 h and 3,5-dimethoxy-
4-i-propylbenzyl aldehyde
obtained in example 1(c) (11.39g, 54.7 mmol) in THF (100mL) was added. The
reaction was kept at
0°C for 1 h and then at ,45-50°C for 5 h. The reaction was
cooled to 0°C. Water was added slowly to
quench the reaction followed by addition of 2N HCl (75mL). The mixture was
extracted with ether (3
ae 200mL). The extract was dried over anhydrous NazSOd. Evaporation of ether
gave crude 5-(2-
phenylethenyl)-2-i-propyl-1,3-dimethoxy benzene (18.07g). This was used for
the next reaction
without further purification. A small amount of the crude product was purified
by flash
chromatography using 10% ethyl acetate in hexane to afford pure product.'HNMR
(CDC13, ppm): 8
1.28 (d, J= 7.0 Hz, 6H), 3.58 (hept, J= 7.0 Hz, 1 H), 3.85 (s, 6 H), 6.69 (s,
2 H), 7.05 (s, 2 H), 7.25
(m, 1 H), 7.3 5 (m, 2 H), 7.25 (m, H).
Example 6. 5-(2-Phenylethenyl)-2-i-propyl-1,3-benzenediol (20).
To the crude 1-(3,5-dimethoxy-4-i-propylphenyl)-2-phenylethene (18.07g) in dry
CHZC12 (100mL) at
-78°C under NZ was added BBr3 (5.2mL, SSmmol) dropwise. After the
reaction was stirred at -78°C
for 1 h, the temperature was allowed to rise to room temperature and the
reaction mixture was stirred
at room temperature for 2 days. Water was added to quench the reaction,
followed by 20% NaOH to
adjust pH > 12. The organic layer was removed and the aqueous layer was washed
with hexane (2 x
100mL). The aqueous layer was acidified with 6N HCl to pH 1 and extracted with
ether (3 x 200mL).
The organic layer was separated and washed with water (SOmL) and brine (SOmL)
and dried over
anhydrous Na2SO4. Evaporation of ether gave a red syrup. Recrystallization
with chloroform yielded
pure stilbene product 20 (6.92g) as a white' crystal. The mother liquid was
concentrated and the
residue was recrystallized once more to afford an additional 2.Sg of 20 (total
9.42g, 67.7% over two
steps).'HNMR (CDCl3, ppm): 8 1.38 (d, J = 7.3Hz, 6H), 3.46 (kept., J = 7.3Hz,
1H), 4.80 (s, 2H),
6.50 (s, 2H), 6.92 (d, J = 17.2Hz, 1H), 6.97 (d, J = 17.2Hz, 1H), 7.25 (m,
1H), 7.34 (m, 2H), 7.52 (m,
2H).
Example 7. 3-Acetoxy-5-(2-phenylethenyl)-2-i-propylphenyl acetate (10).
To 5-(2-Phenylethenyl)-2-i-propyl-1,3-benzenediol obtained in example 11
(l.OOg, 3.93mmol) and
triethylamine (l.SmL, 10.8mmol) in dichloromethane (100mL) at 0°C was
added acetyl chloride
dropwise. The reaction was monitored by TLC. Water (SOmL) was added after the
reaction was
complete (~30 min.). The organic layer was separated and washed with 2NHCl
(30mL), H20 (SOmL),
saturated NaHC03 (SOmL), H20 (SOmL) and brine (SOmL), and dried over anhydrous
sodium sulfate.
Evaporation of the solution followed by flash chromatography using 5% ethyl
acetate in hexane
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yielded 3-acetoxy-5-(2-phenylethenyl)-2-i-propylphenyl acetate. (1.32g, 92%)
as a white solid.
'HNMR (CDC13, ppm): 8 1.26 (d, J = 7.OHz, 6H), 2.35 (s, 6H), 3.08 (hept., J =
7.OHz, 1H), 6.98 (d, J
=17.4Hz, 1H), 7.04 (d, J = 17.4Hz, 1H), 7.07 (s, 2H), 7.24-7.29 (m, 1H), 7.34-
7.38 (m, 2H), 7.45-7.49
(m, 2H).
Example 8. 3-Chloroacetoxy-5-(2-phenylethenyl)-2-i-propylphenyl
chloroacetate(11).
This material was synthesized from anhydrous chloroacetic and 5-(2-
Phenylethenyl)-2-i-propyl-1,3-
benzenediol obtained in example 11 in 72%yield by the same procedure as
described in example 12.
'HNMR (CDC13, ppm): b 1.30 (d, J = 7.OHz, 6H), 3.08 (hept, J=7.OHz, 1H), 4.39
(s, 4H), 6.96 (d, J =
l7Hz, 1H), 7.14 (d, J = l7Hz, 1H) 7.17 (s, 2 H), 7.2-7.5 (m, 5 H).
Example 9. 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-(4-methoxyphenyl)ethene (12).
a). 3,5-Dimethoxy-4-isopropyl benzyl bromide
To 3,5-Dimethoxy-4-i-propylbenzyl alcohol (12.57g, 59.8mmo1) in dry ether
(100mL) at 0°C was
added PBr3 (3.OmL, 31.2mmo1) dropwise under nitrogen. The reaction was
monitored by TLC. After
the reaction was completed (~4h), water (180mL) was added. The organic layer
was separated and
the aqueous layer was extracted with ether (3 ae SOmL). The extract was washed
with water (20mL),
sat. NazC03 (20mL), water (20mL) and brine (20mL), and dried over anhydrous
sodium sulfate.
Evaporation of the solution yielded pure bromide (14.93g, 91.4%) as a white
solid.'HNMR (CDC13,
ppm): 8 1.29 (d, J = 7.lHz, 6H), 3.64 (hept, J = 7.lHz, 1H), 3.84 (s, 6H),
4.50 (s, 2H), 6.60 (s, 2H).
b). Diethyl (3,5-dimethoxy-4-i-propylbenzyl)phosphonate.
The mixture of 3,5-dimethoxy-4-i-propylbenzyl bromide (S.Olg, 18.3mmol) and
triethyl phosphite
(4.7mL, 27.4mmo1) was heated at 110-130°C in the presence of BudNI
(O.OSg) overnight. The excess
triethyl phosphite was removed under reduced pressure at 110°C to give
the phosphonate (5.58g,
92%). 1HNMR (CDC13, ppm): h 1.27 (d, J = 7. lHz, 6H), 1.29 (t, J = 7.OHz, 6H),
3.12 (d, J =2l.SHz,
2H), 3.4-3.7 (m, 1H), 3.80 (s, 6H), 4.06 (dt, J =7.1, 7.lHz, 4H), 6.50 (d, J =
2.6Hz, 2H).
c). 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-(4-methoxyphenyl)ethene (12).
This material was prepared from diethyl (3,5-dimethoxy-4-i-
propylbenzyl)phosphonate and 4-
anisaldehyde in 63% yield as the same procedure as described in example
5(b).'HNMR (CDC13,
ppm): 8 1.31 (d, J=7.lHz, 6H), 3.51-3.74 (m, 1H), 3.86 (s, 3H), 3.91 (s, 6H),
6.71 (s, 2H), 6.84-7.09
(m, 4H), 7.39-7.60 (m, 2H).
Example 10. 5-[2-(4-Hydroxyphenyl)ethenyl]-2-i-propyl-1,3-benzenediol (13).
This material was prepared from 1-(3,5-dimethoxy-4-i-propylphenyl)-2-(4-
methoxyphenyl)ethene and
pyridine hydrochloride in 30% yield in the same way as described in example 3.
'H NMR (DMSO-d6,
ppm): 8 1.22 (d, J=7.OHz, 6H), 3.41 (m, 1H), 6.40 (s, 2H), 6.73 (d, J = 6.3Hz,
4H), 7.33 (s, 1H), 7.41
(s, 1H), 8.98 (s, 2H), 9.51 (s, 1H).
Example 11. 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-(3,5-dimethoxyphenyl)ethene
(14).
This material was prepared from diethyl (3,5-dimethoxy-4-i-
propylbenzyl)phosphonate and 3,5-
dimethoxybenzaldehyde in 25%yield as the same procedure as described in
example 5(b)
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Example 12. 5-[2-(3,5-Dihydroxyphenyl)ethenyl]-2-i-propyl-1,3-benzenediol
(15).
This material was prepared from 1-(3,5-dimethoxy-4-i-propylphenyl)-2-(3,5-
dimethoxyphenyl)ethene
and BBr3 by the same procedure as described in example 11.
Example 13. 1-(4-Bromo-3,5-dimethoxyphenyl)-2-phenylethene (21).
a). Methyl 4-bromo-3,5-dimethoxybenzoate.
This material was synthesized from 4-bromo-3,5-dihydroxybenzoic acid and
MeaS04 in 95% yield by
the same method as described in example 1 (a). 1HNMR (CDCl3, ppm): 8 3.96 (s,
3 H), 3.99 (s, 6 H),
7.28 (s, 2 H).
b). 4-Bromo-3,5-dimethoxybenzyl alcohol.
This material was synthesized from methyl 4-bromo-3,5-dimethoxybenzoate
obtained above in 85%
yield by the same method as described in example 1(b). IHNMR (CDCl3, ppm):
81.95 (s, 1H), 3.93 (s,
6H), 4.69 (s, 2H), 6.61 (s, 2H).
c). 4-Bromo-3,5-dimethoxybenzaldehyde.
This material was synthesized from 4-bromo-3,5-dimethoxybenzyl alcohol in 75%
yield by the same
method as described in example 1(c). ~HNMR (CDCl3, ppm): 8 4.02 (s, 6 H). 7.11
(s, 2H), 9.97 (s, 1
H).
d). 1-(4-Bromo-3,5-dimethoxyphenyl)-2-phenylethene (21).
This material was synthesized from 4-bromo-3,5-dimethoxybenzyl aldehyde and
diethyl
benzylphosphonate in 70% yield by the same method as described in example
5(b).'HNMR (CDCl3,
ppm): S 3.96 (s, 6 H), 6.72 (s, 2 H), 7.06 (d, J = l7Hz, 1H), 7.11 (d, J =
l7Hz, 1H), 7.28 (m, 1 H),
7.3 7 (m, 2 H), 7.5 5 (m, 2 H).
Example 14. 2-Bromo-5-(2-phenylethenyl)-1,3-benzenediol (22).
This material was synthesized from 1-(4-bromo-3,5-dimethoxyphenyl)-2-
phenylethene (21) and BBr3
in 90% yield by the same method as described iii example 6. 1HNMR (CDCl3,
ppm): ~ 5.39 (s, 2H),
6.81 (s, 2H), 7.06 (d, J = l7Hz, 1H), 7.11 (d, J = l7Hz, 1H), 7.28 (m, 1H),
7.37 (m, 2H), 7.55 (m, 2H).
Example 15. 1-[2,5-Dimethoxy-4-(2-phenylethenyl)]phenyl-1-phenylmethanol (16).
To a solution of 1-(4-bromo-3,5-dimethoxyphenyl)-2-phenylethene (0.2185g.
0.6845mmol) in dry
THF (lOmL) at-78°C was added BuLi (0.3mL, 2.SM in hexane, 0.7530mmol).
One hour after the
addition, benzaldehyde (0.07mL, 0.69mmo1) was added. The reaction mixture was
stirred at -78°C
for another 4 hours and then water (l2mL) was added to quench the reaction.
This was extracted with
ether (3 x 20mL). The extract were combined and dried over anhydrous NaaS04.
Evaporation of
solvent followed by flash chromatography using 5% ethyl acetate in hexane
afforded pure 16 (0.203,
86% yield) as a yellow solid. The 1HNMR (CDCl3, ppm): S 3.88 (s, 6H), 4.26 (d,
J = 5.6Hz, 1H),6.40
(br, 1H), 6.79 (s, 2H), 7.12 (s, 2H), 7.2-7.6 (m, lOH).
Example 16. 2,5-Dimethoxy-4-(2-phenylethenyl) benzaldehyde (17).
This compound was synthesized from 1-(4-bromo-3,5-dimethoxyphenyl)-2-
phenylethene, BuLi and
N,N-dimethylformamide in 38% yield by the same method as described in example
15. I~
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(CDC13, ppm): S 3.9'4 (s, 3H), 4.00 (s, 3H), 6.75 (s, 2H), 7.14 (s, 2H), 7.3-
7.5 (m, SH), 10.52 (s, 1H).
Example 17. 1-(3,5-Dimethoxy-4-ethylphenyl)-2-phenylethene (23).
To a solution of 1-(4-bromo-3,5-dimethoxyphenyl)-2-phenylethene (0.53g,
l.7mmo1) in THF (lOmL)
was added t-Butyl Li (1.1 mL, 1M in THF) at - 78 °C. After the addition
complete, the solution was
slowly heated to reflux for 30 min and then cooled down to - 78 °C.
Ethyl iodide (1.2 eq, 0.27 mL)
was added to the solution. Water (1 OmL) was added after the completion of the
reaction. THF was
evaporated and the mixture was extracted with CHZC12 (3 x 5 mL). The extract
was combined and
dried over anhydrous magnesium sulfate. Evaporation of the solution followed
by flash
chromatography using 20% ether in hexane gave 1,3-dimethoxy-2-ethyl-5-(2-
phenylethenyl)benzene
in 70% yield. IHNMR (CDC13, ppm): 8 1.12 (t, J= 7.2 Hz, 6H), 2.70 (q, J= 7.2
Hz, 2H), 3.91 (s, 6H),
6.74 (s, 2H), 7.07 (s, 2 H), 7.26 (m, 1H), 7.36 (m, 2H'), 7.52 (m, 2 H).
Example 18. 2-Ethyl-5-(2-phenylethenyl)-1,3-benzenediol (24).
This material was synthesized from 1-(3,5-dimethoxy-4-ethylphenyl)-2-
phenylethene and BBr3 in
91% yield by the same method as described in example 6. 1HNMR (CDC13, ppm): S
1.22 (t, J= 7.SHz,
6H), 2.70 (q, J= 7.SHz, 2H), 4.81 (s, 2H), 6.60 (s, 2H), 7.00 (s, 2H), 7.26
(m, 1H), 7.36 (m, 2H), 7.52
(m, 2H).
Example 19. 1-(3,5-Dimethoxy-4-n-tetradecanylphenyl)-2-phenylethene (25).
This material was prepared from 2-bromo-1,3-dimethoxy-5-(2-
phenylethenyl)benzene and 1-bromo-
n-tetradecane by the same procedure as described in example 15. 1HNMR (CDCl3,
ppm): 8 0.91 (m,
6H), 1.29 (m, 22 H), 2.65 (m, 2H), 3.90 (s, 6H), 6.73 (s, 2H), 7.10 (s, 2H),
7.26 (m, 1H), 7.36 (m, 2H),
7.52 (m, 2H).
Example 20. 5-(2-Phenylethenyl)-2-n-tetradecanyl-1,3-benzenediol (26).
This material was synthesized from 1-(3,5-dimethoxy-4-n-tetradecanylphenyl)-2-
phenylethene and
BBr3 by the same method as described in example 6.'HNMR (CDC13, ppm): b 0.95
(m, 6H), 1.30 (m,
22H), 2.65 (m, 2H), 4.80 (s, 2H), 6.60 (s, 2H), 7.00 (s, 2H), 7.26 (m, 1H),
7.36 (m, 2H), 7.52 (m, 2H).
Example 21. 2-(3,5-Dimethoxy-4-i-propylphenyl)-1-(2-fluorophenyl)ethene (27).
To a solution of diethyl (3,5-dimethoxy-4-i-propylbenzyl)phosphonate (O.SOg,
l.Smmol) in THF
(IOmL) at 0°C was added NaH (60% in mineral oil) (0.14g, 3.Smmo1) under
N2. After the addition
was completed, the suspension was stirred at 0°C for 1 h and then 2-
fluorobenzaldehyde (0.2mL, 1.9
mmol) in THF (lOmL) was added. The reaction was kept at 0°C for 1 h and
then at 50°C for Sh. The
reaction was cooled to 0°C. Water (SmL) was added slowly to quench the
reaction followed by
addition of 2N HCl (8mL). The mixture was extracted with ether (3 x 20mL). The
extract was dried
over anhydrous NazS04. Evaporation of ether followed by flash chromatography
using 5% ethyl
acetate in hexane as eluent afforded 2-(3,5-dimethoxy-4-i-propylphenyl)-1-(2-
fluorophenyl)ethene (1).
(0.31 g, 68%) as a yellow crystal. 1HNMR (CDC13, ppm): 8 1.34 (d, J = 7.lHz,
6H), 3.60 (qint. J=
7.lHz, 1H), 3.89 (s, 6H), 6.74 (s, 2H), 7.0-7.2 (m, SH), 7.4-7.6 (m, 1H).
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Example 22. 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-(3-fluorophenyl)ethene (28).
This material was prepared from diethyl (3,5-dimethoxy-4-i-
propylbenzyl)phosphonate and 3-
fluorobenzaldehyde in the same way as described in example 21.
Example 23. 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-(4-fluorophenyl)ethene (29).
This material was prepared from diethyl (3,5-dimethoxy-4-i-
propylbenzyl)phosphonate and 4-
fluorobenzaldehyde in the same procedure as described in example 21.
Example 24. 2-(3,5-Difluorophenyl)-1-(3,5-dimethoxy-4-i-propylphenyl)ethene
(30).
This material was prepared from (3,5-dimethoxy-4-i-propylbenzyl)phosphonate
and 3,5-
difluorobenzaldehyde in 27% yield in the same way as described in example 21.
1HNMIZR (CDC13,
ppm): 8 1.32 (d, J = 7.OHz, 6H), 3.66 (qint., J = 7.OHz, 1H), 3.90 (s, 6H),
6.72 (s, 2H), 6.8-7.2 (m,
SH).
Example 25. 1-(2,4-Difluorophenyl)-2-(3,5-dimethoxy-4-i-propylphenyl)ethene
(31)
(3,5-Dimethoxy-4-i-propylphenyl)ethane.
To a suspension of methyltriphenylphosphonium bromide (6.89g, 19.3mmol) in THF
(100mL) under
argon was added BuLi (7.7m1, 2.SM in hexane, 19.3mmol) at room temperature.
The resultant red
solution was stirred for 10 min. and then 3,S-dimethoxy-4-i-
propylbenzylaldehyde (4.02g, 19.3mmol)
obtained above in THF (20mL) was added. After 2 hours, the reaction was
quenched with water
(20mL). The mixture was extracted with ether (3 x 100mL). The extract was
washed with saturated
saline solution (3 x 30mL) and dried over sodium sulphate. Evaporation of
ether followed by flash
chromatography using 3% ethyl acetate in hexane afforded pure (3,5-dimethoxy-4-
i-
propylphenyl)ethene (2.64g, 66% yield) as a colorless solid.'HNMR (CDCl3,
ppm): S 1.31 (d, J =
7.lHz, 6H), 3.61 (qint, J = 7.lHz, 1H), 3.86 (s, 6H), 5.25 (d, J = llHz, 1H),
5.73 (d, J = l7Hz, 1H),
6.64 (s, 2H), 6.70 (dd, J = 1 l, l7Hz, 1H).
A mixture of (3,5-dimethoxy-4-i-propylphenyl)ethene (0.649g, 3.15mmol), 1-
bromo-2,4-
diflurobenzene (1.23g, 6.37mmol), dihydrogen di-p,-chlorotetrkis(di-tart-
butylphosphinito-
~cP)dipalladate (0.1409g, 0.151mmol), Bu4NI (0.582g, 1.58mmol) and I~ZC03
(1.45g, 10.5mmo1) in
DMF (lOmL) was heated at 140°C under argon. After the reaction was
complete (6h), the reaction
mixture was poured into water (lOml).. The aqueous was acidified with 2NHCl
and extracted with
ether (2 X SOmL). The extract was washed with saturated sodium chloride and
then dried over
anhydrous NazS04. Evaporation of ether followed by flash chromatography using
2% ethyl acetate in
hexane afforded 1-(2;4-difluorophenyl)-2-(3,5-dimethoxy-4-i-
propylphenyl)ethene (31)
quantitatively as a yellowish crystal. II~IMR (CDC13, ppm): 8 1.32 (d, J =
7.lHz, 6H), 3.63 (qint, J =
7.lHz, 1H), 3.90 (s, 6H), 6.76 (s, 2H), 7.08 (d, J = l7Hz, 1H), 7.27 (d, J =
l7Hz, 1H), 7.63 (d, J =
BHz, 2H), 8.13 (d, J = 8Hz, 2H).
Example 26. 1-(2,6-Difluorophenyl)-2-(3,5-dimethoxy-4-i-propylphenyl)ethene
(32).
This compound was synthesized from (3,5-dimethoxy-4-i-propylphenyl)ethene and
1-bromo-2,6-
diflurobenzene quantitatively in the same procedure as described in
preparation of 31. II~IMR
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(CDC13, ppm): 8 1.32 (d, J = 7.lHz, 6H), 3.62 (qint, J = 7.lHz, 1H), 3.90 (s,
6H), 6.73 (s, 2H), 6.8-7.2
(m, 4H), 7.41 (d, J = 16.6Hz, 1H).
Example 27. 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-(2,4,6-trifluorophenyl)ethene
(33).
This compound was synthesized from (3,5-dimethoxy-4-i-propylphenyl)ethene and
1-bromo-2,4,6-
triflurobenzene in 58% yield in the same procedure as described in preparation
of 31. 1HNMR (CDC13,
ppm): 8 1.32 (d, J = 7.OHz, 6H), 3.62 (qint, J = 7.lHz, 1H), 3.89 (s, 6H),
6.73 (s, 2H), 6.79-7.55 (m,
4H).
Example 28. 1-(3,5-Dimethoxy-4-i-propylphenyl)-2-(2,3,4,5,6-
pentafluorophenyl)ethene (34).
This compound was synthesized from (3~5-dimethoxy-4-i-propylphenyl)ethene and
1-bromo-
2,3,4,5,6-triflurobenzene in the same procedure as described in preparation of
31.
Example 29. 5-[2-(2-Fluorophenyl)ethenyl]-2-i-propyl-1,3-benzenediol (37).
A mixture of 2-(3,5-dimethoxy-4-i-propylphenyl)-1-(2-fluorophenyl)ethene (27)
(0.308g, 1.03mmol)
and pyridine hydrochloride (0.72g, 6.2 mmol) was heated at 200°C for 4
h under a stream of argon.
The reaction mixture was cooled to room temperature. 2NHC1 (lOmL) and ether
(lSmL) was added.
The organic layer was separated and the aqueous layer was extracted with ether
(3 ae 1 OmL). The
extract was dried over anhydrous NazS04. Evaporation of ether followed by
flash chromatography
using 15% ethyl acetate in hexane afforded pure 5-[2-(2-fluorophenyl)ethenyl]-
2-i-propyl-1,3-
benzenediol (37) (0.269g, 95% yield) as an off white solid. 1HNMR (CDCl3,
ppm): 8 1.41 (d, J =
7.2Hz, 6H), 3.51 (qint., J = 7.2Hz, 1H), 5.01 (b, 2H), 6.56 (s, 2H), 6.98 (d,
J = 17.6Hz, 1H), 7.0-7.3
(m, 4H), 7.60 (ddd, J = 7.5, 7. S, 2.2Hz, 1 H).
Example 30. 5-[2-(3-Fluorophenyl)ethenyl]-2- i-propylphenyl-1,3-diol (38).
This material was prepared from 1-(3,5-dimethoxy-4-i-propylphenyl)-2-(3-
fluorophenyl)ethene (28)
and pyridine hydrochloride in the same procedure as described in example 34.
1HNMR (CDC13, ppm):
8 1.41 (d, 7.2Hz, 6H), 3.49 (qint., J = 7.2Hz, 1H), 6.53 (s, 2H), 6.9-7.5 (m,
6H).
Example 31. 5-[2-(4-Fluorophenyl)ethenyl]-2- i-propylphenyl-1,3-diol (39).
This material was prepared from 1-(3,5-dimethoxy-4-i-propylphenyl)-2-(4-
fluorophenyl)ethene 29
and pyridine hydrochloride (38% yield over 2 steps) in the same procedure as
described in example
34. IHNMR (CDCl3, ppm): 8 1.41 (d, 7.2Hz, 6H), 3.48 (qint., J = 7.2Hz, 1H),
6.52 (s, 2H), 6.81 (d, J
= l7Hz, 1H), 7.00 (d, J = l7Hz, 1H), 7.0-7.2 (m, 2H), 7.4-7.6 (m, 2H); 1HNMR
(DMSO-d6, ppm): b
1.22 (d, J = 7.lHz, 6H), 3.35 (qint., J = 7.lHz, 1H), 6.45 (s, 2H), 6.81 (d, J
= 16.7Hz, 1H), 6.99 (d, J =
16.7Hz, 1 H), 7.17 (dd, J = 8.8, 8.8Hz, 2H), 7.61 (dd, J = 8.8Hz, 5.6Hz, 2H),
9.05 (s, 2H).
Example 32. 5-[2-(3,5-Difluorophenyl)ethenyl]-2- i-propylphenyl-1,3-diol (40).
This material was prepared from 1-(3,5-dimethoxy-4-i-propylphenyl)-2-(3,5-
difluorophenyl)ethene
and pyridine hydrochloride in 70% yield in the same procedure as described in
example 34. 1HNMR
(CDC13, ppm): 8 1.40 (d, J = 7.lHz, 6H), 3.56 (qint., J = 7.2Hz, 1H), 4.90 (s,
2H), 6.52 (s, 2H), 6.2-
7.1 (m, SH).
Example 33. 5-[2-(2,4-Difluorophenyl)ethenyl]-2-i-propyl-1,3-benzenediol (41).
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This material was prepared from 1-(2,4-difluorophenyl)-2-(3,5-dimethoxy-4-i-
propylphenyl)ethene
and pyridine hydrochloride in 44% yield in the same way as described in
example 34, II~lMR
(CDC13, ppm): ~ 1.41 (d, J=7.lHz, 6H), 3.49 (qint, J = 7.lHz, 1H), 4.78 (br,
2H), 6.54 (s, 2H), 6.69-
7.02 (m, 3H), 7.13 (d, J=l6Hz, 1H), 7.41-7.75 (m, 1H).
Example 34. 5-[2-(2,6-Difluorophenyl)ethenyl]-2-i-propyl-1,3-benzenediol (42).
This material was prepared from 1-(2,6-difluorophenyl)-2-(3,5-dimethoxy-4-i-
propylphenyl)ethene
and pyridine hydrochloride in 29% yield in the same way as described in
example 34. IHI~tMR
(CDCl3, ppm): S 1.42 (d, J = 7.lHz, 6H), 3.50 (qint, J= 7.lHz, 1H), 4.77 (br,
2H), 6.57 ( s, 2H), 6.8-
7.4 (m, SH).
Example 35. 2-i-Propyl-5-[2-(2,4,6-trifluorophenyl)ethenyl]-1,3-benzenediol
(43).
This material was prepared from 1-(3,5-dimethoxy-4-i-propylphenyl)-2-(2,4,6-
trifluorophenyl)ethene
and pyridine hydrochloride in 14% yield in the same way as described in
example 29. 1FINMR
(CDC13, ppm): b 1.42 (d, J=7.lHz, 6H), 3.50 (qint, J = 7.lHz, 1H), 4.77 (br,
2H), 6.55 (s, 2H), 6.59-
7.24 (m, 4H).
Example 36. 5-[2-(2,3,4,5,6-Pentafluorophenyl)ethenyl]-2-i-propyl-1,3-
benzenediol (44).
This material was prepared from 1-(2,3,4,5,6-pentafluorophenyl)-2-(3,5-
dimethoxy-4-i-
propylphenyl)ethene and pyridine hydrochloride in 21% yield in the same way as
described in
example 34. 1HNMR (CDC13, ppm): b 1.40 (d, J=7.2Hz, 6H), 3.53 (d, J=7.2Hz,
6H), 4.91 (s, 2H),
6.55 (s, 2H), 6.86 (d, J = l7Hz, 1H), 7.28 (d, J = l7Hz, 1H).
The standard pharmacological procedures, described fully in the examples
hereafter, show the
compounds of the invention to inhibit T-cell, keratinocyte proliferation, cell
migration induced by
leukotriene B4 and to inhibit IFN-y secretion and VEGF expression ih vitr~o as
well as to inhibit TNF-oc
and edema ih vivo.
Example 37. Biological activity of novel compounds.
These assays for the following biological activities are well-established and
known in the art, brief
descriptions are provided herein for clarity.
(a). Effect on proliferation and IFN-y production of human peripheral blood
mononuclear cells
(PBMC) stimulated by phytochemagglutinin (PHA).
Experiment:PBMC were cultured with PHA and cultured with titrated
concentrations of compounds
or solvent, or media alone using standard cell culture techniques. The MTT
assay was performed after
48 hours of culture. Supernatants were collected after 48 hours of culture and
levels of IFN-y were
assayed by ELISA.
Results: 5-[2-(4-Hydroxyphenyl)ethenyl]-2-i-propyl-1,3-benzenediol (13) of the
present invention
had an ICSO of 2.97 against human PBMC proliferation while resveratrol had an
ICSO of > 50.
Compound 13 is 20 times more potent in inhibiting PBMC proliferation (Table
1). Similarly,
compound 13 is more than 15 times more potent than is resveratrol in
inhibition IFN-y production
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(Table 2). Similarly, the three fluorinated compounds, 37, 38 and 39 had ICso
< 10 ~M whereas that
of resveratrol was > 50 ~M the highest concentration tested. The fluorinated
compounds had superior
activity in inhibiting PBMC proliferation to that of resveratrol with >5 times
more potency (Table 1).
Similarly, the ICso value of resveratrol is more than 9 times higher than that
of the three fluorinated
compounds, indicating that the fluorinated compounds are over 9 times more
potent than resveratrol
in inhibiting IFN-y production by human PBMC (Table 2).
Table 1. Effect of the novel compounds and resveratrol against human PBMC
proliferation.
Compound ICSO (~,M)
13 2.97
37 5.62
38 9.91
39 7.36 _
Resveratrol > 50
Table 2. Effect of the novel compounds and resveratrol on IFN-y production by
human PBMC
Compound IFNy ICso (~M)
13 2.55
37 3.80
38 4.29
39 4.16
Resveratrol ~ 39.2
(b).
Effect
on human
keratinocyte
proliferation
Human keratinocytes were cultured in the presence of IFN-y and titrated
concentrations of drug or the
vehicle. The MTT assay was performed after 48 hours of culture.
Results: Compound 13 had an ICSO of 4.3 ~M compared to that of resveratrol of
>50, indicating
compound 13 is more than 10 times potent than is resveratrol (Table 3).
Table 3. Effect of the novel compound 13 and resveratrol on human keratinocyte
proliferation.
Compound ICso (N.M)
13 4.3
resveratrol > 50
(c).
Effect
on
migration
of
human
white
blood
cells
(WBC)
induced
by
leukotriene
B4
Experiment:WBC collected from donors was mixed with equal volume of 3% dextran
(in O.15M
NaCI). The red blood cells were sedimented (45 minutes, room temperature) and
removed. Any
remaining red blood cells in the plasma were removed by adding 150mM of Tris-
NH4Cl. The
leukocyte-rich plasma was washed twice in Hanks balanced salts solution
containing 20 mM HEPES.
The WBC was then transferred to RPMI-1640 medium and adjusted to a density of
5x10' cellslml.
An agarose plate assay system (Nelson et al. 1978)was used to measure the WBC
migration. Briefly,
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WO 2004/031117 PCT/CA2003/001497
a 0.8% agarose solution was prepared with complete RPMI-1640 cell culture
medium. About 3.5 ml
of this agarose solution was transferred to a glass slide before it
solidified. Wells were made on the
slide in a 3~e6 array fashion (~2 mm, inter-well distance 3mm) once the
agarose had solidified. LTB4
was dissolved in anhydrous ethanol to 104 ng/ml and further diluted with the
RPMI-1640 medium
to 10 ng/ml for the test. Compound 39 was dissolved in DMSO, diluted with RPMI-
1640 to 103 p,g/ml
and tested at the following concentrations:100, 10, 1, 0.1 and 0.01 p,g/ml.
Ten microlitres of cell
suspension with different concentrations of compound 39 was added to each well
of the center row of
the three rows of wells. The same volume of LTBQ in RPMI-1640 medium or the
medium alone was
added to wells in the other rows and served as controls. After 5 h incubation
(5% COa, 37°C) the test
slides were fixed with 100% methanol (30 min) and dried at 4°C
(overnight). The slides were then
examined microscopically. The migration index was defined as the average
distances that cells
migrated towards the positive LTBd well divided by that of spontaneous
migration. The percentage
migration was compared between treatment and the non-drug control. The dose-
effect relationship
was determined by plotting the percentage chemotaxis vs concentration for ICso
values.
Results: Compound 39 inhibited the migration of WBC towards LTB4 in a dose-
dependent manner
(Table 4).
Table 4. Effect of Compound 39 on human white blood cell migration towards
LTB4.
Concentration (pM) % Migration
40 13.075.8
8 58.464.3
1.6 83.8515.9 -
0.32 88.4618.6
0 100
Conclusion:
Compound
39
showed
potent
inhibitory
activity
against
WBC
migration
induced
by
leukotriene B4, a mediator that plays important role in inflammation,
including the auto-immune
response.
(d). The effect on vascular endothelial growth factor (VEGF) protein
expression
Experiment: Compound 39 was dissolved in DMSO, diluted with keratinocyte-serum-
free medium
(KC-SFM) to 103 p,g/ml, further diluted with the culture medium and tested at
the following
concentrations: 10, 1, 0.1 and 0.01 p,g/ml. Prime cultures of keratinocytes
were obtained from a
Z5 commercial source and maintained with KC-SFM at a cell density of 106/ml.
In the test, cells were
cultured in 24-well plates and incubated at 37°C in 5% COZ first for 4
h, and then treated with rhTGF-
a (final concentration 100 ng/ml) and the test compound at different
concentrations (0.01-10 p,g/ml).
Medium without test compound was the negative control. The culture supernatant
from each well was
separately collected after an additional 24 h incubation and centrifuged at
2000rpm for 5 minutes
before measuring the VEGF concentration. VEGF concentration in the supernatant
in each well was
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WO 2004/031117 PCT/CA2003/001497
calculated based on measurements taken using an ELISA kit, according to the
manufacturer's
instructions.
Results: Compound 39 showed a dose-dependent effect on the VEGF concentration
in the cell
supernatant of keratinocytes induced by rhTGF-a after 24-h treatment. This
effect increased
substantially and the protein concentration decreased 100% when compound 39
concentration
increased to 40 M (Table 5).
Table 5. Effect of Compound 39 on VEGF expression of human keratinocytes
induced by rhTGF-cc.
Concentration (pM)VEGF(pg/ml)
40
00
8 33.61.8
1.6 34.42.0
0 3 8.92.8
Conclusion:
Compound
39
had
a
significant
inhibitory
effect
on
VEGF
expression
in
human
keratinocytes.
(e) In vivo efficacy in endotoxemia mouse model.
Experiment: Test compounds were dissolved and formulated in 50% PEG-400 in
water. Female
Balb/c mice (~20g) were first injected separately intraperitoneally (IP) with
25 mg/kg of each test
compound, then challenged by injection with 40 mglkg lipopolysaccharide (LPS)
(IP) 30 minutes
later. One drug injection with 12.5 mg/kg of test compound was done at the
same time as (LPS
challenge and two subsequent sequential injections at 30 minutes intervals.
Positive control of
dexamethasone was administered in a similar manner starting at 0.4 mg/kg and
subsequently 0.2
mg/kg for three additional injections. Mice were sacrificed and blood
collected by cardiac puncture
150 minutes after LPS challenge. The serum TNF-a levels were determined by
ELISA. Each test
group was comprised of six mice. Group of mice injected with the vehicle alone
was used as negative
control.
Results: Compound 37 and 39 decreased significantly (P<0.05) TNF-a levels in
mice blood induced
by LPS (Table 6).
Table 6. Effect of the novel compounds, 37 and 39 on TNF-a levels induced by
LPS in a mouse
model.
Compound TNF-a (pg/mL)P-value
37 638.9273.0 0.03
39 601.6211.9 0.01
Carrier 1126.63 96.4
Dexamethasone 281.367.2 0.0004
ZS P-values calculated with Student's t-test (unpaired, two-tailed)
Conclusions:The fluorinated compounds, compound 37 and 39 significantly
decreased levels of TNF-
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a that modulate a broad range of activities in mice, resulting in reduced
inflammatory reactions in
animals.,
(f). Efficacy on TPA induced edema.
Experiment: Three representative compounds, 5-(2-phenylethenyl)-2-i-propyl-1,3-
benzenediol, a s
previously reported, a closely related stilbene derivative (WO 0142231) and
compound 39, a novel
compound of the current invention, were assayed against the edema on female
mice (Balblc) aged 10-
12 weeks, using 0.01% Calcitriol (a commercial standard) as a positive
control. Phorbol-12-myristate-
13-acetate (TPA) was used as the edema inducer. TPA and the test compounds
were all dissolved in
100% ethanol and 20 p,l applied on the right ear of the mouse with six mice
per group. The TPA
concentration used was 0.01 % (w/v). Ear thickness was measured 6 hours after
TPA treatment to
determine if edema was decreased. In each experiment replicated groups of TPA
treated mice were
treated with either 5-(2-phenylethenyl)-2-i-propyl-1,3-benzenediol,
Calcitriol, compound 39 or only
ethanol, and the levels of inhibition was obtained by measuring the thickness
of the ear and
expressing the difference in thiclmess of the treated ear from that of the
ethanol treated ear, as a
percentage.
Results: The fluorinated compound reduces the edema significantly. With one of
H atoms of the
previously reported stilbene, 5-(2-phenylethenyl)-2-i-propyl-1,3-benzenediol,
replaced by a F to the
novel compound 39 of the current invention, the inhibition of edema is
increased from 8% to 85%
while inhibition of Calcitriol was 31%, demonstrating the surprisingly high
levels of activity of the
novel compound 39 of the current invention.
Table 6. Anti-inflammatory activity of stilbene compound after a single
topical administration in the
TPA-induced ear edema model
Treatment % edema inhibition
TPA(0.01%) +
5-(2-phenylethenyl)-2-i-propyl-1, 8.0
3-benzenediol
(0.3%)
TPA(0.01%) +
Compound 39 (0.3%) 85.2
TPA(0.01%) +
Calcitriol (0.01%) 31.2
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