U.S. patent application number 17/111146 was filed with the patent office on 2021-03-25 for substituted aromatic compounds and pharmaceutical compositions for tissue self-repair and regeneration.
The applicant listed for this patent is LIMINAL BIOSCIENCES LIMITED. Invention is credited to Lyne GAGNON, Pierre LAURIN.
Application Number | 20210085626 17/111146 |
Document ID | / |
Family ID | 1000005253450 |
Filed Date | 2021-03-25 |
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United States Patent
Application |
20210085626 |
Kind Code |
A1 |
GAGNON; Lyne ; et
al. |
March 25, 2021 |
SUBSTITUTED AROMATIC COMPOUNDS AND PHARMACEUTICAL COMPOSITIONS FOR
TISSUE SELF-REPAIR AND REGENERATION
Abstract
Described herein are compounds of Formula I, or pharmaceutically
acceptable salts thereof, or combinations thereof, as well as uses
thereof. Such uses include promoting tissue self-repair or tissue
regeneration of an organ, stimulating the generation of tissue
growth, modulating (e.g., increasing) the level of a tissue-repair
marker, treating physical injury in an organ, tissue or cell,
promoting wound healing, as well as anti-aging applications.
Corresponding compositions, methods, kits, and uses are also
described. Formula I wherein A is C.sub.5 alkyl, C.sub.6 alkyl,
C.sub.5 alkenyl, C.sub.6 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3
or CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3 or 4; R.sub.1
is H, F of OH; R.sub.2 is H, F, OH, C.sub.5 alkyl, C.sub.6 alkyl,
C.sub.5 alkenyl, C.sub.6 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3
or CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3 or 4; R.sub.3
is H, F, OH, or CH.sub.2Ph; R.sub.4 is H, F or OH; Q is 1)
(CH.sub.2), C(O)OH wherein m is 1 or 2 2) CH(CH.sub.3)C(O)OH, 3)
C(CH.sub.3).sub.2C(O)OH, 4) CH(F)--C(O)OH, 5) CF.sub.2--C(O)OH or
6) C(O)--C(O)OH.
Inventors: |
GAGNON; Lyne; (Laval,
CA) ; LAURIN; Pierre; (Ville Mont-Royal, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIMINAL BIOSCIENCES LIMITED |
Cambridge |
|
GB |
|
|
Family ID: |
1000005253450 |
Appl. No.: |
17/111146 |
Filed: |
December 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
16510581 |
Jul 12, 2019 |
10869849 |
|
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17111146 |
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15526405 |
May 12, 2017 |
10391073 |
|
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PCT/CA2015/000572 |
Nov 12, 2015 |
|
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|
16510581 |
|
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62078704 |
Nov 12, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 59/52 20130101;
A61P 17/02 20180101; A61K 8/36 20130101; A61K 8/365 20130101; A61K
31/192 20130101; A61Q 19/08 20130101; A61P 19/08 20180101; C07C
57/58 20130101; C07C 57/30 20130101; C07C 57/32 20130101; C07C
59/84 20130101 |
International
Class: |
A61K 31/192 20060101
A61K031/192; A61P 17/02 20060101 A61P017/02; A61P 19/08 20060101
A61P019/08; A61K 8/36 20060101 A61K008/36; A61K 8/365 20060101
A61K008/365; A61Q 19/08 20060101 A61Q019/08; C07C 57/30 20060101
C07C057/30; C07C 57/32 20060101 C07C057/32; C07C 57/58 20060101
C07C057/58; C07C 59/52 20060101 C07C059/52; C07C 59/84 20060101
C07C059/84 |
Claims
1. (canceled)
2. A method for stimulating tissue growth or regeneration in an
organ or tissue of a subject in need thereof, wherein the organ or
tissue is bladder, ovary, prostate, spleen, breast, blood vessel or
a neural tissue, the method comprising administering to the subject
an effective amount of a compound represented by Formula I or a
pharmaceutically acceptable salt thereof: ##STR00081## wherein A is
C.sub.5 alkyl, C.sub.6 alkyl, C.sub.5 alkenyl, C.sub.6 alkenyl,
C(O)--(CH.sub.2).sub.n--CH.sub.3 or
CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3 or 4; R.sub.1 is
H, F or OH; R.sub.2 is H, F, OH, C.sub.5 alkyl, C.sub.6 alkyl,
C.sub.5 alkenyl, C.sub.6 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3
or CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3 or 4; R.sub.3
is H, F, OH or CH.sub.2Ph; R.sub.4 is H, F or OH; Q is 1)
(CH.sub.2).sub.mC(O)OH wherein m is 1 or 2, 2) CH(CH.sub.3)C(O)OH,
3) C(CH.sub.3).sub.2C(O)OH, 4) CH(F)--C(O)OH, 5) CF.sub.2--C(O)OH,
or 6) C(O)--C(O)OH.
3. The method according to claim 2, wherein A is C.sub.5 alkyl or
C.sub.6 alkyl.
4. The method according to claim 2, wherein R.sub.2 is H, F, OH,
C.sub.5 alkyl or C.sub.6 alkyl.
5. The method according to claim 2, wherein R.sub.3 is H, OH or
CH.sub.2Ph.
6. The method according to claim 2, wherein Q is
(CH.sub.2).sub.mC(O)OH where m is 1 or 2.
7. The method according to claim 2, wherein A is C.sub.5 alkyl or
C.sub.6 alkyl; R.sub.1 is H, F or OH; R.sub.2 is H, F, OH, C.sub.5
alkyl or C.sub.6 alkyl; R.sub.3 is H, OH or CH.sub.2Ph; R.sub.4 is
H, F or OH; and Q is (CH.sub.2).sub.mC(O)OH where m is 1 or 2.
8. The method according to claim 2, wherein A is C.sub.5 alkyl;
R.sub.1 is H; R.sub.2 is H or C.sub.5 alkyl; R.sub.3 is H; R.sub.4
is H; and Q is (CH.sub.2).sub.mC(O)OH where m is 1.
9. The method according to claim 2, wherein said compound or
pharmaceutically acceptable salt thereof is: ##STR00082##
##STR00083## ##STR00084## ##STR00085## or a pharmaceutically
acceptable salt thereof.
10. The method according to claim 9, wherein said compound or
pharmaceutically acceptable salt thereof is: ##STR00086## or
pharmaceutically acceptable salt thereof.
11. The method according to claim 9, wherein said compound or
pharmaceutically acceptable salt thereof is: ##STR00087## or
pharmaceutically acceptable salt thereof.
12. The method according to claim 2, wherein the pharmaceutically
acceptable salt is a base addition salt comprising a metal
counterion selected from the group consisting of sodium, potassium,
calcium, magnesium, lithium, ammonium, manganese, zinc, iron, or
copper.
13. The method according to claim 12, wherein the pharmaceutically
acceptable salt is sodium.
14. The method according to claim 2, wherein the organ or tissue is
an injured organ or tissue.
15. A method for increasing the expression of a tissue self-repair
marker or a tissue regeneration marker in an organ or tissue of a
subject, wherein the organ or tissue is bladder, ovary, prostate,
spleen, breast, blood vessel or a neural tissue, the method
comprising the step of administering to a subject in need of tissue
regeneration a compound represented by Formula I or a
pharmaceutically acceptable salt thereof: ##STR00088## wherein A is
C.sub.5 alkyl, C.sub.6 alkyl, C.sub.5 alkenyl, C.sub.6 alkenyl,
C(O)--(CH.sub.2).sub.n--CH.sub.3 or
CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3 or 4; R.sub.1 is
H, F or OH; R.sub.2 is H, F, OH, C.sub.5 alkyl, C.sub.6 alkyl,
C.sub.5 alkenyl, C.sub.6 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3
or CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3 or 4; R.sub.3
is H, F, OH or CH.sub.2Ph; R.sub.4 is H, F or OH; Q is 1)
(CH.sub.2).sub.mC(O)OH wherein m is 1 or 2, 2) CH(CH.sub.3)C(O)OH,
3) C(CH.sub.3).sub.2C(O)OH, 4) CH(F)--C(O)OH, 5) CF.sub.2--C(O)OH,
or 6) C(O)--C(O)OH.
16. The method according to claim 15, wherein said compound or a
pharmaceutically acceptable salt thereof is: ##STR00089##
##STR00090## ##STR00091## ##STR00092## and pharmaceutically
acceptable salts thereof.
17. The method according to claim 16, wherein said compound or
pharmaceutically acceptable salt thereof is: ##STR00093## or
pharmaceutically acceptable salt thereof.
18. The method according to claim 16, wherein said compound or
pharmaceutically acceptable salt thereof: ##STR00094## or
pharmaceutically acceptable salt thereof.
19. The method according to claim 15, wherein the marker is a
metalloproteinase or a growth factor.
20. The method according to claim 15, wherein the marker is
hepatocyte growth factor (HGF) or Serpin A1 (AAT).
21. The method according to claim 15, wherein the marker is Serpin
A1 (AAT) and wherein the tissue or a neural tissue.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/510,581, filed Jul. 12, 2019, which is a
continuation of U.S. patent application Ser. No. 15/526,405, filed
May 12, 2017, now U.S. Pat. No. 10,391,073, which is a national
stage application under 35 U.S.C. .sctn. 371 of PCT Application No.
PCT/CA2015/000572, filed Nov. 12, 2015, which claims the priority
benefit of U.S. Provisional Patent Application Ser. No. 62/078,704,
filed Nov. 12, 2014.
FIELD OF INVENTION
[0002] The present invention relates to the field of medicine.
Particular aspects of the invention relates to compounds,
pharmaceutical compositions and uses thereof for the tissue
self-repair and/or the tissue regeneration of an injured organ, for
stimulating the generation of tissue growth, and/or for modulating
the expression of tissue self-repair markers and/or tissue
regeneration markers such as metalloproteinases and growth
factors.
BACKGROUND OF INVENTION
[0003] Tissue regeneration involves known markers such as
metalloproteinases and growth factors, including without limitation
HGF (hepatocyte growth factor), LOX (lysyl oxidase), MMP1, MMP2,
MMP9, MMP13, PLAT (tPA), PLAU (uPA), Serpin A1 (AAT), Serpin E1
(PAI-1), TIMP3, ILK (integrin-linked kinase).
[0004] The impact of HGF in tissue repair and regeneration is well
described in the scientific review: The discovery of Hepatocyte
Growth factor (HGF) and its significance for cell biology, life
sciences and clinical medicine from Nakamura and Mizuno, Proc. Jpn.
Acad. Ser B86 (2010). This review article describes the role of HGF
in tissue regeneration in liver, kidney, heart, and lung. Also, HGF
is required for self-repair after injuries of skin, stomach,
intestine, muscle and cartilage and is also involved in organ
development (organogenesis including mitogenesis, motogenesis and
morphogenesis). HGF is also implicated in the regeneration of
injured tissue by its modulation of regeneration enzyme
(metalloproteinases) and also by inhibiting apoptosis. Furthermore,
recent reports suggest that HGF has an anti-inflammatory action and
attenuated cellular senescence. Thus, HGF gene therapy or compound
increasing HGF expression and secretion might be an anti-aging
therapy in cardiovascular diseases (Nakagami, Morishita, 2009). HGF
is also known to accelerate would healing (Li et al., BioMed
Research International, Volume 2013 (2013), Article ID 470418.
[0005] Regeneration enzymes (including metalloproteinases) are also
very important in repair and regeneration of injured organs.
[0006] A recent publication (abstract presented at Plastic surgery
meeting 2014 by Radtke et al. entitled Single treatment With
Alpha-1 antitrypsin Enhances Nerve Regeneration After Peripheral
Nerve Injury) has demonstrated that AAT improves peripheral nerve
regeneration. The application of AAT into an acute axotomy model
led to the significantly improved axonal regeneration and
re-myelination than compared control animals. Moreover, not only
histological, but also functional improvement was observed
following direct injection of AAT after acute peripheral nerve
lesion. Their results indicate that AAT delivered into injured
peripheral nerve participate in neural repair.
[0007] Cutaneous aging is a complex phenomenon responsible for
progressive changes of the skin. Aging of the skin results from two
processes: (1) an intrinsic process, corresponding to chronological
aging, and (2) an extrinsic process resulting mainly from the
deleterious effect of exposure environmental stresses. Genetic, UV
exposure, climatic factors (harshness/wind/cold/warm), pollution
(chemical, free radicals, contaminant, nitrogen oxide, metals),
alcohol consumption or smoking are factors involved in cutaneous
aging.
[0008] Exposure to irritants compromises the barrier function of
the stratum corneum and decreases its ability to protect the skin
against environmental stresses (e.g., ultraviolet irradiation,
infections agents, etc.). Repeated and prolonged exposition to
environmental irritants results in denatured skin proteins,
disorganization of the lipid lamellae layers, removal of the
protective intercellular lipids, loss of natural moisturizing
factors and decreased cohesion between cells. These damages are
also responsible for the loss of function of the enzymes
responsible for desquamation of corneocytes. There is accentuation
of these problems with exposure to pollution, cold, sun, wind, low
humidity or chemical agents. An irritant is any agent that is
capable of producing cell damage if there exposure for sufficient
time and in sufficient concentrations. The severity of the damage
is dependent of the type and intensity of exposure to these
irritating factors. There are also endogenous factors that make one
susceptible to damaged skin by external factors. These factors
include having active skin disease such as eczema, inherited dry
skin conditions, a previous history of skin disease, sensitive skin
and/or older age.
[0009] Novel compounds and medicaments are needed to stimulate the
tissue self-repair and the tissue regeneration in injured
organ.
BRIEF SUMMARY OF THE INVENTION
[0010] General aspects of the invention relate to the
pharmaceutical use of compounds according to Formula I as defined
herein and pharmaceutically acceptable salts thereof.
[0011] Particular aspects of the invention relates to the use of
compounds and compositions for the tissue self-repair and/or the
tissue regeneration of an injured organ, and/or for modulating the
expression of tissue self-repair markers and/or tissue regeneration
markers such as metalloproteinases and growth factors, including
without limitation HGF, LOX (Lysyl oxidase), MMP1, MMP2, MMP9,
MMP13, PLAT (tPA), PLAU (uPA), Serpin A1 (AAT), Serpin E1 (PAI-1),
TIMP3, and ILK (integrin-linked kinase).
[0012] A method for tissue self-repair or tissue regeneration of an
organ in a subject in need thereof, comprising the step of
administering to a subject in need thereof a compound represented
by Formula I or a pharmaceutically acceptable salt thereof.
[0013] According to another aspect, the invention relates to a
method for tissue self-repair or tissue regeneration of an organ in
a subject in need thereof, comprising administering a compound
represented by Formula I or a pharmaceutically acceptable salt
thereof as defined herein to said subject. In an embodiment, the
invention relates to a method for tissue self-repair of an organ in
a subject in need thereof, comprising administering a compound
represented by Formula I or a pharmaceutically acceptable salt
thereof as defined herein to said subject. In an embodiment, the
invention relates to a method for tissue remodelling of an organ in
a subject in need thereof, comprising administering a compound
represented by Formula I or a pharmaceutically acceptable salt
thereof as defined herein to said subject. In an embodiment, the
invention relates to a method for tissue regeneration of an organ
in a subject in need thereof, comprising administering a compound
represented by Formula I or a pharmaceutically acceptable salt
thereof as defined herein to said subject.
[0014] According to another aspect, the invention relates to a
method for stimulating the generation of tissue growth, with a
compound represented by Formula I or a pharmaceutically acceptable
salt thereof as defined herein.
[0015] According to another aspect, the invention relates to a
method for stimulating the expression of tissue self-repair markers
and/or tissue regeneration markers, with a compound represented by
Formula I or a pharmaceutically acceptable salt thereof as defined
herein. More particularly, said markers includes without limitation
metalloproteinases, growth factors, hepatocyte growth factor (HGF),
LOX (Lysyl oxidase), MMP1, MMP2, MMP9, MMP13, PLAT (tPA), PLAU
(uPA), Serpin A1 (AAT), Serpin E1 (PAI-1), TIMP3, and ILK
(integrin-linked kinase).
[0016] According to another aspect, the invention relates to a
method for increasing HGF level in an organ, comprising the step of
administering to said organ, a compound represented by Formula I or
a pharmaceutically acceptable salt thereof as defined herein. The
organ includes without limitation kidney, heart, liver, lung, skin,
stomach, intestine, muscle and cartilage.
[0017] According to another aspect, the invention relates to a
method for increasing AAT level in an organ, comprising the step of
administering to said organ, a compound represented by Formula I or
a pharmaceutically acceptable salt thereof as defined herein.
[0018] Further aspects of the invention will be apparent to a
person skilled in the art from the following description, claims,
and generalizations herein.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 is an illustration of the effect of Compound I on the
increase of mRNA expression of Hepatocyte Growth Factor (HGF), a
growth factor involved in tissue self-repair and regeneration.
[0020] FIG. 2 is an illustration of the effect of Compound I on the
modulation of regeneration markers expressed in injured fibroblast
(NHDF) involved in self-repair and regeneration of tissue.
[0021] FIG. 3 is an illustration of the effect of Compound I on the
modulation of regeneration markers expressed in injured epithelial
cells (HK-2) involved in self-repair and regeneration of
tissue.
[0022] FIG. 4 demonstrates that Compound I can increase mRNA
expression of Serpin A1 (AAT) involved in nerve generation.
[0023] FIG. 5 is a representation of the increase in organ function
(GFR) observed with Compound I and indicating tissue regeneration
of an injured kidney.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present discloses compounds of Formula I,
pharmaceutically acceptable salts thereof, compositions comprising
same and uses thereof. Various embodiments of the present invention
include:
Compounds of the Invention
[0025] According to one aspect, the invention concerns the
pharmaceutical uses of compounds represented by Formula I, or
pharmaceutically acceptable salts thereof:
##STR00001##
wherein A is C.sub.5 alkyl, C.sub.6 alkyl, C.sub.5 alkenyl, C.sub.6
alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3 or
CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3 or 4; or is
C.sub.5 alkyl, C.sub.5 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3 or
CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3; or is C.sub.6
alkyl, C.sub.6 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3 or
CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 4;
R.sub.1 is H, F or OH; or is H or OH;
[0026] R.sub.2 is H, F, OH, C.sub.5 alkyl, C.sub.6 alkyl, C.sub.5
alkenyl, C.sub.6 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3 or
CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3 or 4; or is
C.sub.5 alkyl, C.sub.5 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3 or
CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 3; or is C.sub.6
alkyl, C.sub.6 alkenyl, C(O)--(CH.sub.2).sub.n--CH.sub.3 or
CH(OH)--(CH.sub.2).sub.n--CH.sub.3 wherein n is 4
[0027] R.sub.3 is H, F, OH or CH.sub.2Ph; or is H, F or OH; or is H
or OH;
[0028] R.sub.4 is H, F or OH; or is H or OH;
[0029] Q is
1) (CH.sub.2).sub.mC(O)OH wherein m is 1 or 2,
2) CH(CH.sub.3)C(O)OH,
[0030] 3) C(CH.sub.3).sub.2C(O)OH,
4) CH(F)--C(O)OH,
5) CF.sub.2--C(O)OH, or
6) C(O)--C(O)OH.
[0031] According to a particular embodiment, A is C.sub.5 alkyl or
C.sub.6 alkyl. Preferably, C.sub.5 alkyl is a straight chain
C.sub.5 alkyl.
[0032] According to a particular embodiment, R.sub.1 is H or
OH.
[0033] According to a particular embodiment, R.sub.2 is H, F, OH,
C.sub.5 alkyl or C.sub.6 alkyl.
[0034] According to a particular embodiment, R.sub.3 is H or
OH.
[0035] According to a particular embodiment, R.sub.4 is H or
OH.
[0036] According to a particular embodiment, Q is:
1) (CH.sub.2).sub.mC(O)OH wherein m is 1 or 2,
2) CH(F)--C(O)OH,
3) CF.sub.2--C(O)OH, or
4) C(O)--C(O)OH.
[0037] According to a particular embodiment, Q is
(CH.sub.2).sub.mC(O)OH where m is 1 or 2.
[0038] According to another embodiment, the compound is of Formula
I, wherein A is C.sub.5 alkyl or C.sub.6 alkyl; R.sub.1 is H, F or
OH; R.sub.2 is H, F, OH, C.sub.5 alkyl or C.sub.6 alkyl; R.sub.3 is
H, OH or CH.sub.2Ph; R.sub.4 is H, F or OH; and Q is
(CH.sub.2).sub.mC(O)OH where m is 1 or 2.
[0039] According to another embodiment, the compound is of Formula
I; wherein A is C.sub.5 alkyl; R.sub.1 is H; R.sub.2 is H or
C.sub.5 alkyl; R.sub.3 is H; R.sub.4 is H; and Q is
(CH.sub.2).sub.mC(O)OH where m is 1.
[0040] As used herein, the term "alkyl" is intended to include a
straight chain saturated aliphatic hydrocarbon group having the
specified number of carbon atoms in a linear arrangement, and a
branched chain saturated aliphatic hydrocarbon group having the
specified number of carbon atoms in a non-linear arrangement, or a
cyclic chain saturated aliphatic hydrocarbon group having the
specified number of carbon atoms in a cyclic arrangement.
[0041] As used herein, the term, "alkenyl" is intended to mean
unsaturated straight chain hydrocarbon groups having the specified
number of carbon atoms therein, and in which at least two of the
carbon atoms are bonded to each other by a double bond, and having
either E or Z regiochemistry and combinations thereof.
[0042] Examples of compounds of Formula I include, but are not
limited to, Compounds I to XXXIII and acid form thereof listed in
Table 1 hereinbelow.
TABLE-US-00001 TABLE 1 Representative compounds of Formula I and
acid form thereof Compound Sodium Salt Acid Form I ##STR00002##
##STR00003## II ##STR00004## ##STR00005## III ##STR00006##
##STR00007## IV ##STR00008## ##STR00009## V ##STR00010##
##STR00011## VI ##STR00012## ##STR00013## VII ##STR00014##
##STR00015## VIII ##STR00016## ##STR00017## IX ##STR00018##
##STR00019## X ##STR00020## ##STR00021## XI ##STR00022##
##STR00023## XII ##STR00024## ##STR00025## XIII ##STR00026##
##STR00027## XIV ##STR00028## ##STR00029## XV ##STR00030##
##STR00031## XVI ##STR00032## ##STR00033## XVII ##STR00034##
##STR00035## XVIII ##STR00036## ##STR00037## XIX ##STR00038##
##STR00039## XX ##STR00040## ##STR00041## XXI ##STR00042##
##STR00043## XXII ##STR00044## ##STR00045## XXIII ##STR00046##
##STR00047## XXIV ##STR00048## ##STR00049## XXV ##STR00050##
##STR00051## XXVI ##STR00052## ##STR00053## XXVII ##STR00054##
##STR00055## XXVIII ##STR00056## ##STR00057## XXIX ##STR00058##
##STR00059## XXX ##STR00060## ##STR00061## XXXI ##STR00062##
##STR00063## XXXII ##STR00064## ##STR00065## XXXIII ##STR00066##
##STR00067##
Salts
[0043] As used herein, the term "pharmaceutically acceptable salt"
is intended to mean base addition salts. Example of
pharmaceutically acceptable salts are also described, for example,
in Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 66, 1-19
(1977). Pharmaceutically acceptable salts may be synthesized from
the parent agent that contains an acidic moiety, by conventional
chemical methods. Generally, such salts are prepared by reacting
the free acid forms of these agents with a stoichiometric amount of
the appropriate base in water or in an organic solvent, or in a
mixture of the two. Salts may be prepared in situ, during the final
isolation or purification of the agent or by separately reacting a
purified compound of the invention in its free acid form with the
desired corresponding base, and isolating the salt thus formed.
[0044] The pharmaceutically acceptable salt of the compounds of
Formula I may be selected from the group consisting of base
addition salts of sodium, potassium, calcium, magnesium, lithium,
ammonium, manganese, zinc, iron, or copper. In preferred
embodiments, the pharmaceutically acceptable salt of the compounds
according to the invention may be the sodium, potassium, calcium,
magnesium or lithium salt. More preferably the pharmaceutically
acceptable salt is sodium.
[0045] The compounds of Formula I disclosed herein may be in any
form, including any acid, salt or other ionic and non-ionic forms.
For example, if a compound is shown as an acid herein, the salt
forms of the compound are also included. Likewise, if a compound is
shown as a salt and the acid forms are also included.
Prodrugs
[0046] In certain embodiments, the compounds of Formula I disclosed
herein, wherein said compounds are present in the free carboxylic
acid form, may also include all pharmaceutically acceptable salts,
isosteric equivalents such as tetrazole and prodrug forms thereof.
Examples of the latter include the pharmaceutically acceptable
esters or amides obtained upon reaction of alcohols or amines,
including amino acids, with the free acids defined by Formula
I.
Chirality
[0047] The compounds of Formula I disclosed herein, their
pharmaceutically acceptable salts, or prodrugs thereof, may contain
one or more asymmetric centers, chiral axes and chiral planes and
may thus give rise to enantiomers, diastereomers, and other
stereoisomeric forms and may be defined in terms of absolute
stereochemistry, such as (R)- or (S)-. The present invention is
intended to include all such possible isomers, as well as, their
racemic and optically pure forms. Optically active (+) and (-),
(R)- and (S)-, isomers may be prepared using chiral synthons or
chiral reagents, or resolved using conventional techniques, such as
reverse phase HPLC. The racemic mixtures may be prepared and
thereafter separated into individual optical isomers or these
optical isomers may be prepared by chiral synthesis. The
enantiomers may be resolved by methods known to those skilled in
the art, for example by formation of diastereoisomeric salts which
may then be separated by crystallization, gas-liquid or liquid
chromatography, selective reaction of one enantiomer with an
enantiomer specific reagent. It will also be appreciated by those
skilled in the art that where the desired enantiomer is converted
into another chemical entity by a separation technique, an
additional step is then required to form the desired enantiomeric
form. Alternatively specific enantiomers may be synthesized by
asymmetric synthesis using optically active reagents, substrates,
catalysts, or solvents or by converting one enantiomer to another
by asymmetric transformation.
[0048] Certain compounds of Formula I or pharmaceutically
acceptable salts thereof disclosed herein may exist in Zwitterionic
form and the present invention includes the use of Zwitterionic
forms of these compounds and mixtures thereof.
Hydrates
[0049] In addition, the compounds of Formula I or pharmaceutically
acceptable salts thereof disclosed herein may also exist in
hydrated and anhydrous forms. The present invention includes the
use of hydrates of any of the compounds of Formula I or
pharmaceutically acceptable salts thereof described herein, which
may exist as a monohydrate or in the form of a polyhydrate.
Methods of Preparation
[0050] In general, all compounds of Formula I or pharmaceutically
acceptable salts thereof disclosed herein may be prepared by any
conventional methods, using readily available and/or conventionally
preparable starting materials, reagents and conventional synthesis
procedures. Of particular interest is the work of Hundertmark, T.;
Littke, A. F.; Buchwald, S. L.; Fu, G. C. Org. Lett. 12, 1729-1731
(2000).
[0051] The exemplification section hereinafter provides general
schemes and specific, but non limitative, examples for the
synthesis of Compounds I-XXXIII
Pharmaceutical Uses
[0052] The Compounds of Formula I or pharmaceutically acceptable
salts thereof (or a composition comprising same) disclosed herein
are useful: in the tissue self-repair and/or the tissue
regeneration of an injured organ, tissue or cell, in stimulating
the generation of new cells in an in vitro cell culture, and/or in
modulating the expression of tissue self-repair markers and/or
tissue regeneration markers such as metalloproteinases and growth
factors. According to an embodiment, the Compounds of Formula I or
pharmaceutically acceptable salts thereof disclosed herein are
useful for an anti-aging treatment. In an embodiment, the treatment
preferably comprises the administration of a Compound of Formula I
or pharmaceutically acceptable salts thereof disclosed herein or a
combination thereof, or a pharmaceutical composition comprising a
therapeutically effective amount one or more of the compounds of
Formula I or pharmaceutically acceptable salts thereof disclosed
herein. The expressions "tissue self-repair" and "tissue
regeneration" used herein may also refer to processes involved in
an anti-aging treatment. Representative Compounds according to
Formula I disclosed herein have been found to stimulate the
expression of known markers associated with anti-aging, tissue
regeneration and tissue self-repair, and to stimulate the
generation of new cells.
[0053] In an embodiment, the injured organ, tissue or cell is not
an organ, tissue or cell injured by an inflammatory-related
disease. In an embodiment, the injured organ, tissue or cell is not
an organ, tissue or cell injured by a cancer.
[0054] In an embodiment, the organ, tissue or cell injury results
from a physical injury (i.e. following an acute exposure to an
external agent or stress that results in some form of damage/injury
to the organ, tissue or cell), for example an organ, tissue or cell
injured by a physical trauma/insult (e.g., cut, bite, shock, tear,
puncture, perforation, burn (heat or chemical), freezing,
radiations, electrocution, physical overexertion), or a surgery.
Physical injury as used herein excludes organ, tissue or cell
damages resulting from (i.e. in which the primary cause of the
organ, tissue or cell damages is) an underlying disease, for
example inflammatory or autoimmune diseases such as inflammatory
bowel diseases, glomerulonephritis, vasculitis, psoriatic
arthritis, systemic lupus erythematoses (SLE), idiopathic
thrombocytopenic purpura (ITP), psoriasis, Crohn's disease,
inflammatory bowel disease, ankylosing spondylitis, Sjogren's
syndrome, Still's disease (macrophage activation syndrome),
uveitis, scleroderma, myositis, Reiter's syndrome, and Wegener's
syndrome. However, the Compounds of Formula I or pharmaceutically
acceptable salts thereof (or composition comprising same) disclosed
herein may be used to promote tissue self-repair and/or the tissue
regeneration to treat secondary tissue damages/injuries that result
from the initial physical injury, for example secondary tissue
damages/injuries caused by inflammation that may occur following
the initial physical injury.
[0055] Thus, in another aspect, the present invention provides a
method for treating a physical injury in an organ, tissue or cell
(e.g., for promoting self-repair and/or tissue regeneration of the
injured organ, tissue or cell), the method comprising contacting
the organ, tissue or cell with an effective amount of the compound
of Formula I or pharmaceutically acceptable salt thereof (or a
composition comprising same) disclosed herein.
[0056] In another aspect, the present invention provides the use of
the compound of Formula I or pharmaceutically acceptable salt
thereof (or a composition comprising same) disclosed herein for
treating a physical injury in an organ, tissue or cell (e.g., for
promoting self-repair and/or tissue regeneration of the injured
organ, tissue or cell). In another aspect, the present invention
provides the compound of Formula I or pharmaceutically acceptable
salt thereof (or a composition comprising same) disclosed herein
for use in treating a physical injury in an organ, tissue or cell
(e.g., for promoting self-repair and/or tissue regeneration of the
injured organ, tissue or cell).
[0057] In an embodiment, the (physically) injured organ, tissue or
cell is not a kidney or kidney tissue. In another embodiment, the
(physically) injured organ, tissue or cell is not a bone or bone
tissue. In an embodiment, the (physically) injured organ, tissue or
cell is skin, muscle, tendon, ligament, liver, heart, pancreas, an
organ/tissue of the digestive/gastrointestinal tract (e.g., mouth,
esophagus, stomach, intestines), gallbladder, liver, an organ of
the respiratory tract (e.g., lung), spinal cord, spleen, breast,
ocular tissue, a blood vessel, a periodontal tissue, mucosa (e.g.,
oral mucosa, nasal mucosa) and/or cartilage.
[0058] In an embodiment, the compounds of Formula I or
pharmaceutically acceptable salts thereof (or composition
comprising same) disclosed herein are used/administered acutely,
i.e. shortly after the injury. In an embodiment, the compounds of
Formula I or pharmaceutically acceptable salts thereof (or
composition comprising same) disclosed herein are used/administered
to promote tissue self-repair and/or the tissue regeneration prior
to the development of fibrosis in the injured organ, tissue or
cell, e.g. prior to the development of a fibrotic disease.
[0059] In an embodiment, the compounds of Formula I or
pharmaceutically acceptable salts thereof (or composition
comprising same) disclosed herein are useful for promoting wound
healing.
[0060] In another embodiment, the injured organ, tissue or cell is
an organ, tissue or cell of the nervous system (e.g., a neural
tissue), for example an organ, tissue or cell of the central
nervous system or peripheral nervous system. In an embodiment, the
compounds of Formula I or pharmaceutically acceptable salts thereof
(or composition comprising same) disclosed herein are useful for
tissue self-repair and/or tissue regeneration following neural
injury, for example spinal cord injury, peripheral nerve injury, or
neural injury associated with multiple sclerosis.
[0061] In an embodiment, the compounds of Formula I or
pharmaceutically acceptable salts thereof (or composition
comprising same) disclosed herein are useful for tissue self-repair
and/or tissue regeneration in the skin, for example following a
skin cut, puncture, bruise or burn.
[0062] In an embodiment, the injured organ, tissue or cell is an
organ, tissue or cell of the respiratory system, for example
lungs.
[0063] In an embodiment, the injured organ, tissue or cell is liver
or a liver tissue.
[0064] In an embodiment, the injured organ, tissue or cell is
bladder or a bladder tissue.
[0065] In an embodiment, the injured organ, tissue or cell is an
ovary or an ovarian tissue.
[0066] In an embodiment, the injured organ, tissue or cell is
prostate or a prostate tissue.
[0067] In an embodiment, the injured organ, tissue or cell is
spleen or a spleen tissue.
[0068] In an embodiment, the injured organ, tissue or cell is
breast or a breast tissue.
[0069] In an embodiment, the injured organ, tissue or cell is a
muscle, for example a muscle injured by muscle strain, muscle tear
and/or any other type of physical muscle injury.
[0070] In an embodiment, the injured organ, tissue or cell is a
blood vessel (e.g., an artery).
[0071] In an embodiment, the injured organ, tissue or cell is an
organ/tissue of the digestive/gastrointestinal tract (e.g., mouth,
esophagus, stomach, intestines).
[0072] In particular embodiments, the methods and used described
herein are not for bone remodelling and/or regeneration of Islets
of Langerhans. In a particular embodiment, the tissue is not a
bone. In an embodiment, the tissue is not a pancreatic tissue.
[0073] The present inventors have shown that representative
compounds of formula I or pharmaceutically acceptable salts thereof
(or composition comprising same) disclosed herein increase markers
that stimulate tissue self-repair and tissue regeneration of an
injured organ in a subject. In an embodiment, the compounds of
formula I described herein exert a tissue regenerative
activity.
[0074] In another aspect, the present invention relates to a
cosmetic composition comprising a compound of formula I or
pharmaceutically acceptable salts thereof (or composition
comprising same) disclosed herein. In another aspect, the present
invention relates to a skin care composition comprising a compound
of formula I or pharmaceutically acceptable salts thereof (or
composition comprising same) disclosed herein. In another aspect,
the present invention relates to an anti-aging skin care
composition comprising a compound of formula I or pharmaceutically
acceptable salts thereof (or composition comprising same) disclosed
herein.
[0075] In another aspect, the present invention relates to the
above-mentioned compound of formula I or pharmaceutically
acceptable salts thereof (or composition comprising same) for use
in anti-aging skin care. In another embodiment, the above-mentioned
compound of formula I or composition comprising same is for use in
stimulating skin repair and/or regeneration following skin damage
associated with aging. In another embodiment, the above-mentioned
compound or composition is for use in stimulating skin repair
and/or regeneration following skin damage or injury. In an
embodiment, the skin damage or injury results from exposure to UV
irradiation, e.g. exposure to sun (e.g., sunburns).
[0076] In an embodiment, the methods and uses disclosed herein
further comprise identifying a subject having an injured organ,
tissue or cell and who is in need of a treatment with the
above-mentioned compound of formula I or pharmaceutically
acceptable salts thereof (or composition comprising same) for
promoting tissue self-repair and/or tissue regeneration in the
injured organ, tissue or cell. The method may comprise identifying
in a sample from a subject, such as an organ, tissue or cell
sample, a decreased level of one or more tissue self-repair and/or
tissue regeneration markers, such as metalloproteinases and growth
factors, including without limitation HGF, LOX (Lysyl oxidase),
MMP1, MMP2, MMP9, MMP13, PLAT (tPA), PLAU (uPA), Serpin A1 (AAT),
Serpin E1 (PAI-1), TIMP3, and ILK (integrin-linked kinase), and
contacting the organ, tissue or cell with an effective amount of
the compound of formula I or pharmaceutically acceptable salts
thereof (or composition comprising same) disclosed herein.
[0077] The term "subject" includes living organisms in need of a
treatment as disclosed herein, for example in which an organ is
injured. The term "subject" includes animals such as mammals or
birds. Preferably, the subject is a mammal, including but not
limited to human, horse, dog and cat. In some embodiments, the
mammal is not a mouse. More preferably, the subject is a human.
Pharmaceutical Compositions and Formulations
[0078] In an embodiment, the compounds of Formula I or
pharmaceutically acceptable salts thereof described herein are
comprised in pharmaceutical compositions comprising a
therapeutically effective amount of the compounds or
pharmaceutically acceptable salts thereof. As indicated
hereinbefore, the pharmaceutical compositions may be useful: in the
tissue self-repair and/or the tissue regeneration of an injured
organ, in stimulating the generation of new cells in an in vitro
cell culture, and/or in modulating the expression of tissue
self-repair markers and/or tissue regeneration markers such as
metalloproteinases and growth factors.
[0079] As used herein, the term "therapeutically effective amount"
means the amount of compound that, when administered to a subject
for treating or preventing a particular disorder, disease or
condition, or for exerting a biological effect (e.g., to stimulate
tissue self-repair and/or the tissue regeneration of an injured
organ, to stimulate the generation of new cells in an in vitro cell
culture, and/or to modulate (increase) the expression of tissue
self-repair markers and/or tissue regeneration markers), is
sufficient to effect such treatment or prevention of that disorder,
disease or condition, or to exert the biological effect. Dosages
and therapeutically effective amounts may vary for example,
depending upon a variety of factors including the activity of the
specific agent employed, the age, body weight, general health,
gender, and diet of the subject, the time of administration, the
route of administration, the rate of excretion, and any drug
combination, if applicable, the effect which the practitioner
desires the compound to have upon the subject, the properties of
the compounds (e.g., bioavailability, stability, potency, toxicity,
etc.), and the particular disorder(s) the subject is suffering
from. In addition, the therapeutically effective amount may depend
on the subject's blood parameters (e.g., calcium levels, lipid
profile, insulin levels, glycaemia), the severity of the disease
state, organ function, or underlying disease or complications. Such
appropriate doses may be determined using any available assays
including the assays described herein. When one or more of the
compounds of Formula I or pharmaceutically acceptable salts thereof
disclosed herein is to be administered to humans, a physician may
for example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. The
dose to be administered will ultimately be at the discretion of the
health care professional. In general, however, it is envisioned
that the dose for the compounds of Formula I or pharmaceutically
acceptable salts thereof disclosed herein may be in the range of
about 1 to about 50 mg/kg per day in human. In selected
embodiments, the range may be between 1 to 30 mg/kg per day in
human. In selected embodiments, the range may be between 1 to 20
mg/kg per day in human. In selected embodiments, the range may be
between 5 to 18 mg/kg per day in human. In selected embodiments,
the range may be between 1 to 18 mg/kg per day in human.
[0080] As used herein, the term "pharmaceutical composition" refers
to the presence of at least one compound according to Formula I or
pharmaceutically acceptable salts thereof as defined herein and at
least one pharmaceutically acceptable carrier, diluent, vehicle or
excipient. As used herein, the term "pharmaceutically acceptable
carrier", "pharmaceutically acceptable diluent" or
"pharmaceutically acceptable excipient" is intended to mean,
without limitation, any adjuvant, carrier, excipient, glidant,
sweetening agent, diluent, preservative, dye/colorant, flavor
enhancer, surfactant, wetting agent, dispersing agent, suspending
agent, stabilizer, isotonic agent, solvent, emulsifier, or
encapsulating agent, such as a liposome, cyclodextrins,
encapsulating polymeric delivery systems or polyethyleneglycol
matrix, which is acceptable for use in subjects, preferably humans.
It preferably refers to a compound or composition that is approved
or approvable by a regulatory agency of the Federal or State
government or listed in the U.S. Pharmacopoeia or other generally
recognized pharmacopoeia for use in animals and more particularly
in humans. The pharmaceutically acceptable vehicle can be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol), suitable mixtures thereof, and vegetable
oils. Additional examples of pharmaceutically acceptable vehicles
include, but are not limited to: Water for Injection USP; aqueous
vehicles such as, but not limited to, Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium
Chloride Injection, and Lactated Ringer's Injection; water-miscible
vehicles such as, but not limited to, ethyl alcohol, polyethylene
glycol, and polypropylene glycol; and non-aqueous vehicles such as,
but not limited to, corn oil, cottonseed oil, peanut oil, sesame
oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
Prevention of the action of microorganisms can be achieved by
addition of antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like. In many cases, isotonic agents are included, for example,
sugars, sodium chloride, or polyalcohols such as mannitol and
sorbitol, in the composition. Prolonged absorption of injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0081] The composition of the present invention may include one or
more compounds of Formula I as defined herein or pharmaceutically
acceptable derivatives, salts, prodrugs, analogues, isomers or
enantiomers thereof. Formulations of the active compound may be
prepared so as to provide a pharmaceutical composition in a form
suitable for enteral, mucosal (including oral, sublingual,
ophthalmic, nasal, pulmonary and rectal), parenteral (including
intramuscular, intradermal, subcutaneous and intravenous) or
topical (including ointments, creams, lotions or drops)
administration. The formulation may, where appropriate, be
conveniently presented in discrete dosage units and may be prepared
by any of the methods well-known in the art of pharmaceutical
formulation. All methods include the step of bringing together the
active pharmaceutical ingredient with liquid carriers or finely
divided solid carriers or both as the need dictates. When
appropriate, the above-described formulations may be adapted so as
to provide sustained release of the active pharmaceutical
ingredient. Sustained release formulations well-known to the art
include the use of a bolus injection, continuous infusion,
biocompatible polymers or liposomes.
[0082] The above-mentioned compound or composition may be
formulated in a topically applicable cosmetic composition (e.g., a
topical formulation). Non-limitative examples of such topically
applicable compositions include skin care cream, cleansing cream,
ointment, skin care lotion, skin care gel, skin care foam, sun care
composition, sunscreen skin care, make-up removal cream, make-up
removal lotion, foundation cream, liquid foundation, bath and
shower preparation, deodorant composition, antiperspirant
composition, shaving products composition, after-shave gel or
lotion, beauty aids composition, depilatory cream, soap
composition, hand cleaner composition, cleansing bar, baby care,
hair care, shampoo, setting lotion, treatment lotion, hair cream,
hair gel, colouring composition, restructuring composition,
permanent composition, or any other composition which is adapted
for the use in a topical cosmetic regimen. Such compositions may
further comprise one or more cosmeceutically acceptable
vehicles.
[0083] Creams, as is well known in the arts of pharmaceutical and
cosmeceutical formulation, are viscous liquids or semisolid
emulsions, either oil-in-water or water-in-oil. Cream bases are
water-washable, and contain an oil phase, an emulsifier, and an
aqueous phase. The oil phase, also called the "internal" phase, is
generally comprised of petrolatum and a fatty alcohol such as cetyl
or stearyl alcohol. The aqueous phase usually, although not
necessarily, exceeds the oil phase in volume, and generally
contains a humectant. The emulsifier in a cream formulation is
generally a non-ionic, anionic, cationic or amphoteric
surfactant.
[0084] Lotions are preparations to be applied to the skin surface
without friction, and are typically liquid or semi liquid
preparations in which solid particles, including the active agent,
are present in a water or alcohol base. Lotions are usually
suspensions of solids, and preferably, for the present purpose,
comprise a liquid oily emulsion of the oil-in-water type. Lotions
are preferred formulations for treating large body areas, because
of the ease of applying a more fluid composition. It is generally
necessary that the insoluble matter in a lotion be finely divided.
Lotions will typically contain suspending agents to produce better
dispersions as well as compounds useful for localizing and holding
the active agent in contact with the skin, e.g., methylcellulose,
sodium carboxymethyl-cellulose, or the like.
[0085] Solutions are homogeneous mixtures prepared by dissolving
one or more chemical substances (solutes) in a liquid such that the
molecules of the dissolved substance are dispersed among those of
the solvent. The solution may contain other cosmeceutically
acceptable chemicals to buffer, stabilize or preserve the solute.
Common examples of solvents used in preparing solutions are
ethanol, water, propylene glycol or any other cosmeceutically
acceptable vehicles.
[0086] Gels are semisolid, suspension-type systems. Single-phase
gels contain organic macromolecules distributed substantially
uniformly throughout the carrier liquid, which is typically
aqueous, but also, preferably contain an alcohol, and, optionally,
oil. "Organic macromolecules," i.e., gelling agents, are
crosslinked acrylic acid polymers such as the "carbomer" family of
polymers, e.g., carboxypolyalkylenes that may be obtained
commercially under Carbopol.TM.. Other examples are hydrophilic
polymers such as polyethylene oxides,
polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol;
cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl
cellulose, hydroxypropyl methylcellulose, hydroxypropyl
methylcellulose phthalate, and methyl cellulose; gums such as
tragacanth and xanthan gum; sodium alginate; and gelatin. In order
to prepare a uniform gel, dispersing agents such as alcohol or
glycerin can be added, or the gelling agent can be dispersed by
trituration, mechanical mixing or stirring, or combinations
thereof.
[0087] Ointments are semisolid preparations that are typically
based on petrolatum or other petroleum derivatives. The specific
ointment base to be used, as will be appreciated by those skilled
in the art, is one that will provide for a number of desirable
characteristics, e.g., emolliency or the like. As with other
carriers or vehicles, an ointment base should be inert, stable, no
irritating, and no sensitizing. As explained in Remington: The
Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack
Publishing Co., 1995), at pages 1399-1404, and ointment bases may
be grouped in four classes: oleaginous bases; emulsifiable bases;
emulsion bases; and water-soluble bases. Oleaginous ointment bases
include, for example, vegetable oils, fats obtained from animals,
and semisolid hydrocarbons obtained from petroleum. Emulsifiable
ointment bases, also known as absorbent ointment bases, contain
little or no water and include, for example, hydroxystearin
sulfate, anhydrous lanolin, and hydrophilic petrolatum. Emulsion
ointment bases are either water-in-oil (W/O) emulsions or
oil-in-water (O/W) emulsions, and include, for example, cetyl
alcohol, glyceryl monostearate, lanolin, and stearic acid.
Preferred water-soluble ointment bases are prepared from
polyethylene glycols of varying molecular weight; again, see
Remington: The Science and Practice of Pharmacy for further
information.
[0088] Pastes are semisolid dosage forms in which the active agent
is suspended in a suitable base. Depending on the nature of the
base, pastes are divided between fatty pastes or those made from
single-phase aqueous gels. The base in a fatty paste is generally
petrolatum or hydrophilic petrolatum or the like. The pastes made
from single-phase aqueous gels generally incorporate
carboxymethylcellulose or the like as a base.
[0089] Formulations may also be prepared with liposomes, micelles,
and microspheres. Liposomes are microscopic vesicles having a lipid
wall comprising a lipid bilayer, and, in the present context,
encapsulate one or more components of the anti-aging formulations.
Liposomal preparations herein include cationic (positively
charged), anionic (negatively charged), and neutral preparations.
Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are available under the tradename Lipofectin.TM. (GIBCO BRL, Grand
Island, N.Y.). Similarly, anionic and neutral liposomes are readily
available as well, e.g., from Avanti Polar Lipids (Birmingham,
Ala.), or can be easily prepared using readily available materials.
Such materials include phosphatidyl choline, cholesterol,
phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with DOTMA in appropriate ratios. Methods for making
liposomes using these materials are well known in the art.
[0090] Micelles are known in the art as comprised of surfactant
molecules arranged so that their polar head groups form an outer
spherical shell, while the hydrophobic, hydrocarbon chains are
oriented towards the centre of the sphere, forming a core. Micelles
form in an aqueous solution containing surfactant at a high enough
concentration so that micelles naturally result. Surfactants useful
for forming micelles include, but are not limited to, potassium
laurate, sodium octane sulfonate, sodium decane sulfonate, sodium
dodecane sulfonate, sodium lauryl sulfate, docusate sodium,
decyltrimethylammonium bromide, dodecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium
chloride, dodecylammonium chloride, polyoxyl-8 dodecyl ether,
polyoxyl-12 dodecyl ether, nonoxynol 10, and nonoxynol 30.
[0091] Microspheres, similarly, may be incorporated into the
present formulations. Like liposomes and micelles, microspheres
essentially encapsulate one or more components of the present
formulations. They are generally although not necessarily formed
from lipids, preferably charged lipids such as phospholipids.
Preparation of lipidic microspheres is well known in the art and
described in the pertinent texts and literature.
Kits
[0092] The compound(s) of Formula I or pharmaceutically acceptable
salts thereof disclosed herein may be packaged as part of a kit,
optionally including a container (e.g., packaging, a box, a vial,
etc.). The kit may be commercially used according to the methods
described herein and may include instructions for use in a method
disclosed herein. Additional kit components may include acids,
bases, buffering agents, inorganic salts, solvents, antioxidants,
preservatives, or metal chelators. The additional kit components
are present as pure compositions, or as aqueous or organic
solutions that incorporate one or more additional kit components.
Any or all of the kit components optionally further comprise
buffers.
[0093] The compound(s) of Formula I or pharmaceutically acceptable
salts thereof disclosed herein may or may not be administered to a
patient at the same time or by the same route of administration.
Therefore, the methods of the invention encompass kits which, when
used by the medical practitioner, can simplify the administration
of appropriate amounts of two or more active ingredients to a
patient.
[0094] A typical kit of the invention comprises a unit dosage form
of at least one compound of Formula I as defined herein, or a
pharmaceutically acceptable salt thereof, and a unit dosage form of
at least one additional active ingredient. Examples of additional
active ingredients that may be used in conjunction with the
compounds of the invention include, but are not limited to, any of
the drugs indicated hereinbefore that could be used in combination
with the compound(s) Formula I or pharmaceutically acceptable salts
thereof as defined herein.
[0095] Kits of the invention can further comprise pharmaceutically
acceptable vehicles that can be used to administer one or more
active ingredients. For example, if an active ingredient is
provided in a solid form that must be reconstituted for parenteral
administration, the kit can comprise a sealed container or a
suitable vehicle in which the active ingredient can be dissolved to
form a particulate-free sterile solution that is suitable for
parenteral administration. Examples of pharmaceutically acceptable
vehicles are provided hereinbefore.
EXAMPLES
[0096] The following examples further illustrate the practice of
this invention but are not intended to be limiting thereof.
Example 1: Experimental Procedures for the Preparation Certain
Representative Compounds
[0097] All HPLC chromatograms and mass spectra were recorded on an
HP 1100 LC-MS Agilent.TM. instrument using an analytical C18 column
(250.times.4.6 mm, 5 microns) with a gradient over 5 min of 15-99%
CH.sub.3CN--H.sub.2O with 0.01% TFA as the eluent and a flow of 2
mL/min.
Compound I: Synthesis of Sodium Salt of (3-pentylphenyl)acetic acid
Using a Modified Sonogashira Procedure
##STR00068##
[0098] Step 1
[0099] To a solution/suspension of 3-bromophenylacetic acid (5.02
g, 23.33 mmol) in ethanol (100 mL) at room temperature was added
concentrated sulfuric acid (1 mL). The colorless solid was then
stirred overnight at 80.degree. C. The solution was concentrated
under reduced pressure. The residue was diluted with ethyl acetate
(25 mL), water (25 mL) and the two layers were separated. The
aqueous layer was extracted with ethyl acetate (2.times.25 mL) and
brine (20 mL). The combinated organic layers were washed with
saturated solution of NaHCO.sub.3 (2.times.25 mL), brine (25 mL)
and dried over sodium sulfate. After filtration the solution it was
evaporated to dryness. This gave a light yellow oil (5.4 g, 95%).
.sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.26 (t, J=4.7 Hz, 3H),
3.57 (s, 2H), 4.15 (Q, J=7.0 and 14.3 Hz, 2H), 7.17-7.26 (m, 2H),
7.38-7.44 (m, 1H), 7.44 (d, J=1.56 Hz, 1H).
Step 2
[0100] A mixture of ethyl (3-bromophenyl)acetate (0.3 g, 1.24 mmol)
and tetrabutylammonium fluoride hydrate (0.97 g, 3.72 mmol), was
treated with PdCl.sub.2(PPh.sub.3).sub.2 (26 mg, 0.037 mmol; 3 mole
%) and 1-pentyne (367 .mu.L, 3.72 mmol) in a sealed tube. The tube
was heated at 80.degree. C. for 2 h. The mixture was treated with
water, and was extracted with diethyl ether. The organic extract
was dried over sodium sulfate, filtered and evaporated in vacuo to
give the crude product. Purification on a Biotage.TM. 25 M column
(silica), eluting with ethyl acetate/hexane 0:1 to 2:98, gave ethyl
(3-(pentyne-1-yl)phenyl)acetate as a pale yellow oil (0.23 g,
79%).
Step 3
[0101] To ethyl[3-[pentyne-1-yl]phenyl]-acetate (0.23 g, 0.98 mmol)
in ethanol (5 mL) under nitrogen atmosphere was added Pd on carbon
(10%, 25 mg, 10% w/w). The mixture was vigorously stirred under
hydrogen atmosphere at room temperature overnight. The solution was
filtered and the palladium/carbon was washed with ethanol (20 mL).
The filtrate was concentrated with silica gel. The crude product
was purified by flash chromatography using a mixture of 10%
hexanes/ethyl acetate. A clear oil was obtained (0.21 g, 90%).
Step 4
[0102] To a solution of the ester (0.2 g, 0.9 mmol) in
tetrahydrofuran (5 mL), methanol (1.5 mL) and water (1.5 mL) was
added lithium hydroxide (0.09 g, 3.6 mmol) at 0.degree. C. The
reaction mixture was stirred overnight at room temperature.
Insolubles were filtered and the filtrate was concentrated under
reduced pressure. The residue was then treated with 2 M HCl and
extracted with ethyl acetate. The organic phase was dried over
sodium sulfate and evaporated under reduced pressure. The crude
material was purified on a 40 L Biotage column (silica) using ethyl
acetate/hexanes (0:10 to 4:6) as eluant. This gave pure
(3-pentylphenyl)acetic acid (0.19 g, 99%) as a white gummy solid.
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 0.90 (t, J=7.0 Hz, 3H),
1.28-1.38 (m, 4H), 1.61 (qt, J=7.6 Hz, 15.0 Hz, 2H), 2.58 (t, J=7.6
Hz, 2H), 3.56 (s, 2H), 7.07 (m, 3H), 7.20 (m, 1H); LRMS (ESI): m/z
207 (MH.sup.+); HPLC: 4 min.
Step 5
[0103] To a stirred solution of the acid (0.19 g, 0.82 mmol) in
ethanol (4 mL) and water (1 mL) was added sodium bicarbonate (0.07
g, 0.82 mmol). The reaction mixture was stirred at room temperature
overnight. The solvent was evaporated and the white gummy solid was
dissolved in water and the solution was lyophilized. This gave pure
sodium salt of (3-pentylphenyl)acetic acid (0.17 g, 92%) as a white
solid. mp 110-112.degree. C.; .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 0.89 (t, J=6.8 Hz, 3H), 1.28-1.37 (m, 4H), 1.60 (qt, J=7.4
Hz, 15.0 Hz, 2H), 2.56 (t, J=7.6 Hz, 2H), 3.43 (s, 2H), 6.96 (m,
1H), 7.12 (m, 3H); LRMS (ESI): m/z 207 ((MH.sup.+); HPLC: 4
min.
Compound II: Sodium Salt of 3-(3-pentylphenyl)propionic Acid
[0104] The above compound was prepared as for Compound I starting
with 3-Oxo-3-bromophenylpropionic acid ethyl ester. The ketone
group and the double bond were simultaneously reduced using
palladium/carbon in ethanol under hydrogen pressure. White solid;
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.14-7.10 (m, 1H),
7.04-7.00 (m, 2H), 6.95-6.93 (m, 1H), 2.88-2.84 (m, 2H), 2.55 (t,
J=7.4 Hz, 2H), 2.44-2.40 (m, 2H), 1.63-1.55 (m, 2H), 1.35-1.28 (m,
4H), 0.90 (m, 3H); .sup.13C NMR (101 MHz, CD.sub.3OD): .delta.
179.3, 141.2, 140.8, 126.7, 126.4, 124.0, 123.8, 38.6, 34.2, 31.2,
29.9, 29.8, 20.9, 11.7; LRMS (ESI): m/z 203 (MH.sup.+-CO-NaOH);
HPLC: 4.5 min.
Compound III: Sodium Salt of 3-(3-butylphenyl)propionic Acid
##STR00069##
[0105] Step 1
[0106] In a round bottom flask (250 mL) was weight
isophthalaldehyde (1.0 g, 7.5 mmol), followed by dichloromethane
(100 mL). Via a separatory funnel with pressure equilibrium was
added the Methyl (triphenyl-phosphoranylidene) acetate (2.7 g, 8.2
mmol) in dichloromethane (25 mL) at room temperature. The reaction
was stirred at room temperature overnight. The mixture was filtered
over a small pad of silica gel, and washed with dichloromethane
(150 mL). The solvent was then evaporated under reduced pressure
and the crude product was used in the next step without further
purification.
Step 2
[0107] The Propyl triphenylphosphonium Bromide (3.2 g, 8.2 mmol)
was placed in a round bottom flask, under nitrogen, and dry THF (5
mL) was added. The flask is cooled in an ice/acetone (-10.degree.
C.) bath, and nButyllithium (2.5 M in Hexanes, 3.28 mL, 8.2 mmol)
was added slowly. The mixture turn dark colored with stirring for
30 minutes. In an ice/acetone (-10.degree. C.) bath was placed the
crude reaction mixture from the previous step in dry THF (5 mL)
under nitrogen. The phosphonium solution was added slowly to the
aldehyde solution at -10.degree. C., and the reaction mixture was
warmed slowly to room temperature and stirred for 4 h. Saturated
ammonium chloride solution (10 mL) was added and the organic layer
was extracted with ethyl acetate (3.times.). The organic layer was
dried over anhydrous sodium sulfate, filtered and silica gel is
added to obtain a drypack. Compound was purified with the SP1
(ethyl acetate/hexanes). This gave the expected product (8.8 g,
54%). .sup.1H NMR (400 MHz, CDCl.sub.3): .quadrature. 7.70-7.65 (m,
1H), 7.45-7.24 (m, 4.5H), 6.45-6.28 (m, 2.5H), 5.70-5.67 (m, 0.5H),
3.78 (m, 3H), 2.34-2.20 (m, 2H), 1.10-1.03 (m, 3H).
Step 3
[0108] In a round bottom flask (25 mL) is placed the unsaturated
ester (140 mg, 0.65 mmol), dissolved in ethyl acetate (10 mL). To
this solution was added 10% palladium on activated charcoal Pd/C
(10 mg). The flask was capped with a septa, and a hydrogen balloon
was placed on top. The flask was purged three times with hydrogen,
and the reaction was stirred at room temperature overnight. The
solid was then filtered over Celite.TM.. Silica gel was added and a
drypack is prepared. Purification by flash chromatography using
0-20% ethyl acetate/hexanes gave the desired product (124 mg, 87%).
LRMS (ESI): m/z 221 (MH.sup.+); HPLC: 5.0 min.
Step 4
[0109] In a round bottle flask was placed the ester (124 mg, 0.56
mmol) followed by methanol (4 mL) and lithium hydroxide (118 mg,
2.8 mmol). Water (1 mL) was added and the reaction was heated at
50.degree. C. with agitation for 17 h. The reaction is transferred
into a separatory funnel, acidified to pH lower than 4 with HCl
(1M), and extracted with ethyl acetate (3.times.). The organic
layer was dried over anhydrous sodium sulfate, filtered and
evaporated. The crude material was purified by HPLC/Waters. This
gave a white solid (80 mg, 70%). .sup.1H NMR (400 MHz, CD.sub.3OD):
.quadrature. 7.16-7.12 (m, 1H), 7.01-6.96 (m, 3H), 2.88-2.84 (m,
2H), 2.57-2.53 (m, 4H), 1.60-1.52 (m, 2H), 1.37-1.28 (m, 2H), 0.91
(t, 3H, J=7.3 Hz); LRMS (ESI): m/z 205 (M-H); HPLC: 4.2 min.
Step 5
[0110] In a flask (20 mL) was placed the acid (80 mg, 0.39 mmol)
followed by NaHCO.sub.3 (33 mg, 0.39 mmol) and water (8 mL). To the
mixtures was added acetonitrile (3 mL) and the reaction was
sonicated, heated and agitated until almost all the solids were in
solution. The solution was filtered over a nylon filter. The water
is solidified by plunging the vial in a dry ice/acetone bath, and
lyophilized overnight. This gave the desired product as a white
solid. .sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 7.14-7.10 (m,
1H), 7.04-6.93 (m, 3H), 2.88-2.84 (m, 2H), 2.57-2.54 (m, 2H),
2.44-2.40 (m, 4H), 1.61-1.53 (m, 2H), 1.39-1.30 (m, 2H), 0.93 (t,
3H, J=7.3 Hz); .sup.13C NMR (101 MHZ, CD.sub.3OD): .delta. 142.7,
142.4, 128.2, 128.0, 125.6, 125.4, 125.3, 40.1, 35.5, 33.9, 32.7,
22.2, 13.1; LRMS (ESI): m/z 251.0 (m, MNa.sup.+), 229.0 (w,
MH.sup.+), 189.2 (100%, acylium ion [M-Na.sup.+2H.sup.+-H2O]);
HPLC: 4.1 min.
Compound IV: Sodium Salt of E-(3-pent-1-enyl-phenyl)acetic Acid
[0111] The above compound was prepared as for Compound I starting
with E-(3-pent-1-enyl-phenyl)acetic acid methyl ester. The latter
was prepared by reacting 3-bromophenyl acetic acid methyl ester
with trans-1-pentenylboronic acid pinacol ester under Suzuki
conditions. White solid; .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta.=7.32 (s, 1H), 7.11-7.18 (m, 3H), 6.35 (d, J=15.7 Hz, 1H),
6.20-6.27 (m, 1H), 3.44 (s, 2H), 2.19 (m, 2H), 1.45-1.54 (m, 2H),
0.96 (t, J=7.4, 3H); .sup.13C NMR (101 MHz, CD.sub.3OD):
.delta.=179.26, 138.25, 137.92, 130.32, 130.04, 128.06, 127.59,
126.60, 123.52, 45.21, 35.06, 22.52, 12.89; LRMS (ESI): m/z 205
(MH.sup.+); HPLC: 4.1 min.
Compound V: Sodium Salt of 2-(3-(Hex-1-enyl]phenyl)acetic Acid
[0112] The above compound was prepared by Suzuki coupling of methyl
2-(3-bromophenyl)acetate and (E)-hex-1-enylboronic acid pinacol
ester as for Compound VII; followed by ester hydrolysis and sodium
salt formation as for Compound I. White solid: .sup.1H NMR (400
MHz, CD.sub.3OD): .delta. 7.33 (s, 1H), 7.12-7.19 (m, 3H), 6.35 (d,
J=15.8 Hz, 1H), 6.20 (dt, J=15.8, 6.8 Hz, 1H), 3.46 (s, 2H),
2.17-2.22 (m, 2H), 1.33-1.49 (m, 4H), 0.93 (t, J=7.2 Hz, 3H);
.sup.13C NMR (101 MHz, CD.sub.3OD): .delta. 179.35, 138.27, 137.95,
130.27, 130.16, 128.10, 127.61, 126.64, 123.56, 45.24, 32.66,
31.67, 22.16, 13.22; LRMS (ESI): m/z 263.1 (100%, M+Na.sup.+);
HPLC: 4.4 min.
Compound VI: Sodium Salt of 2-(3-hexylphenyl)acetic Acid
[0113] The above compound was prepared by Suzuki coupling of methyl
2-(3-bromophenyl)acetate and (E)-hex-1-enylboronic acid pinacol
ester as for Compound VII; followed by hydrogenation, ester
hydrolysis and sodium salt formation as for Compound I. White
solid; .sup.1H NMR (400 MHz, D.sub.2O): .delta. 7.14 (dd, J=7.8,
7.6 Hz, 1H), 7.01 (s, 1H), 7.00 (d, J=7.8 Hz, 1H), 6.96 (d, J=7.6
Hz, 1H), 3.34 (s, 2H), 2.46 (d, J=7.5 Hz, 2H), 1.41-1.48 (m, 2H),
1.10-1.18 (m, 6H), 0.70 (t, J=6.8 Hz, 3H); .sup.13C NMR (101 MHz,
D.sub.2O): .delta. 181.23, 143.98, 137.46, 129.47, 128.73, 126.63,
126.48, 44.58, 35.14, 31.12, 30.94, 28.23, 22.13, 13.53; LRMS
(ESI): m/z 265 (100%, M+Na.sup.+); HPLC: 4.6 min.
Compound VII: Sodium Salt of 3-hydroxy-5-pentylphenylacetic
Acid
##STR00070##
[0114] Step 1
[0115] A solution of methyl [3,5-dihydroxyphenyl]acetate (2.1 g,
11.5 mmol) in acetone (100 mL) was treated with potassium carbonate
(2.4 g, 17.4 mmol), potassium iodide (383 mg, 2.31 mmol) and benzyl
bromide (1.5 mL, 12.7 mmol), and the mixture was stirred at room
temperature overnight. The reaction was diluted with water and
extracted with dichloromethane (.times.3). Combined organic
extracts were dried over sodium sulfate and evaporated in vacuo.
The crude material was purified on a Biotage.TM. 40M column
(silica), eluting with 40% ethyl acetate/hexane, to give methyl
[3-benzyloxy-5-hydroxyphenyl]acetate (1.0 g, 33%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 7.32-7.42 (m, 5H), 6.48 (d, J=1.4 Hz,
1H), 6.38-6.39 (m, 2H), 4.99 (s, 2H), 3.69 (s, 3H), 3.53 (s,
2H).
Step 2
[0116] A solution of the benzyl ether (1.04 g, 3.8 mmol) in
dichloromethane (15 mL) at 0.degree. C., was treated with
N-phenyl-bis(trifluorosulfonyl)imide (1.40 g, 3.9 mmol), and then
triethylamine (0.6 mL, 4.1 mmol) was added slowly. The reaction was
stirred at 0.degree. C. for 1 h, and then at room temperature for 1
h. The reaction mixture was diluted with water, and then extracted
with diethylether (.times.2). Combined organic extracts were washed
with 1M aqueous sodium hydroxide, water (.times.2) and saturated
aqueous sodium chloride, then dried over sodium sulfate, filtered
and evaporated in vacuo, to give the crude product. Purification on
a Biotage.TM. 40M column (silica), eluting with 25% ethyl
acetate/hexane, gave methyl
[3-benzyloxy-5-trifluoromethanesulfonyloxyphenyl]acetate (1.2 g,
79%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.36-7.46 (m, 5H),
6.98 (s, 1H), 6.97 (s, 1H), 6.84 (s, 1H), 5.06 (s, 2H), 3.72 (s,
3H), 3.63 (s, 2H).
Step 3
[0117] A solution of E-1-penten-1-ylboronic acid pinacol ester (0.8
g, 3.9 mmol) in dimethoxyethane (5 mL) was treated with a solution
of the triflate (1.2 g, 3.0 mmol) in dimethoxyethane (5 mL). The
solution was treated with palladium zero (0.7 g, 0.6 mmol) and 2M
aqueous sodium carbonate (1.3 mL, 2.6 mmol). The mixture was then
heated at 90.degree. C. for 3 days. The reaction was cooled to room
temperature and filtered through Celite.TM.. The filtrate was
evaporated in vacuo, and the crude material was purified on a
Biotage.TM. 25M column (silica), eluting with 5% ethyl
acetate/hexane, to give methyl
[3-benzyloxy-5-[pent-1-enyl]phenyl]acetate (0.4 g, 40%). .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 7.36-7.47 (m, 5H), 6.90-6.92 (m,
2H), 6.79 (dd, J=2.0, 2.0 Hz, 1H), 6.35 (d, J=15.9 Hz, 1H), 6.24
(dt, J=15.9, 6.8 Hz, 1H), 5.07 (s, 2H), 3.70 (s, 3H), 3.59 (s, 2H),
2.20 (td, J=7.4, 6.8 Hz, 2H), 1.51 (dt, J=7.4 Hz, 2H), 0.98 (t,
J=7.4 Hz, 3H).
Step 4
[0118] A solution of the alkene (0.4 g, 1.2 mmol) in ethanol (13
mL) was treated with 1% palladium on carbon (40 mg). The mixture
was stirred under 1 atm. of hydrogen at room temperature overnight.
The reaction was filtered, evaporated in vacuo, and purified on a
Biotage.TM. 25S column (silica), eluting with 15% ethyl
acetate/hexane, to give methyl [3-hydroxy-5-pentylphenyl]acetate
(0.3 g, 93%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 6.64 (s,
1H), 6.58-6.60 (m, 2H), 3.70 (s, 3H), 3.55 (s, 2H), 2.51 (t, J=7.7
Hz, 2H), 1.55-1.59 (m, 2H), 1.28-1.34 (m, 4H), 0.88 (t, J=7.0 Hz,
3H).
Step 5
[0119] A solution of the ester (0.3 g, 1.3 mmol) in ethanol (12 mL)
was treated with water (3 mL) and lithium hydroxide (155 mg, 6.4
mmol), and the mixture was stirred vigorously at room temperature
overnight. The reaction mixture was diluted with water (100 mL);
washed with dichloromethane; then acidified to pH 1 with 1M aqueous
hydrochloric acid and extracted with dichloromethane (.times.3).
Combined organic extracts were dried over sodium sulfate (0.3 g,
95%). This material was used without further purification. .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 6.66 (s, 1H), 6.58-6.59 (m, 2H),
3.55 (s, 2H), 2.52 (t, J=7.7 Hz, 2H), 1.55-1.59 (m, 2H).
Step 6
[0120] A solution of the acid (0.27 g, 1.23 mmol) in ethanol (6 mL)
and water (6 mL) was treated with a sodium bicarbonate (0.1 g, 1.2
mmol), and the reaction was stirred at room temperature for a few
hours. Solvent was concentrated in vacuo, and the solution was
diluted with water, filtered (0.2 .mu.m), and lyophilized to give
sodium [3-hydroxy-5-pentylphenyl]acetate as a white solid (0.3 g,
95%). mp 63-66.degree. C.; .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 6.63 (s, 1H), 6.58 (s, 1H), 6.42 (s, 1H), 3.36 (s, 2H),
2.48 (t, J=7.6 Hz, 2H), 1.55-1.62 (m, 2H), 1.26-1.38 (m, 4H), 0.89
(t, J=6.8 Hz, 3H); .sup.13C NMR (101 MHz, CD.sub.3OD): .delta.
177.79, 155.31, 142.36, 137.62, 119.08, 111.66, 111.18, 43.70,
34.17, 29.95, 29.56, 20.87, 11.64; LRMS (ESI): m/z 445.2
(2M-2Na.sup.++3H.sup.+), m/z 223 (M-Na.sup.+2H.sup.+); HPLC: 3.5
min.
Compound VIII: Sodium Salt of 2-(4-Hydroxy-3-pentylphenyl)acetic
Acid
[0121] The above compound was prepared by Suzuki coupling of benzyl
2-(4-(benzyloxy)-3-bromophenyl)acetate and (E)-pent-1-enylboronic
acid pinacol ester as for example VII; followed by hydrogenation.
White solid; melting point 192-195.degree. C.; .sup.1H NMR (400
MHz, CD.sub.3OD): .delta. 7.01 (d, J=2.3 Hz, 1H), 6.93 (dd, J=8.2,
2.3 Hz, 1H), 6.64 (d, J=8.2 Hz, 1H), 3.35 (s, 2H), 2.53 (t, J=7.7
Hz, 2H), 1.54-1.61 (m, 2H), 1.30-1.37 (m, 4H), 0.90 (t, J=7.2 Hz,
3H); .sup.13C NMR (101 MHz, CD.sub.3OD): .delta. 180.25, 153.20,
130.54, 128.80, 128.76, 127.10, 114.49, 44.45, 31.84, 30.10, 29.73,
22.52, 13.31; LRMS (ESI): m/z 245.2 (55%, MH.sup.+), 177.4 (100%,
M-CO.sub.2Na); HPLC: 1.9 min.
Compound IX: Sodium Salt of 2-(2-Hydroxy-3-pentylphenyl)acetic
Acid
##STR00071##
[0122] Step 1
[0123] A solution of 2-(2-hydroxyphenyl)acetic acid (3.00 g, 19.7
mmol) in methanol (40 mL) was treated with sulfuric acid (0.95 mL,
17.8 mmol) and the reaction was stirred at room temperature for 18
hours. The reaction mixture was diluted with ethyl acetate (250
mL), and the solution was washed with water (2.times.150 mL) and
with saturated aqueous sodium chloride (150 mL); dried over sodium
sulfate; filtered and evaporated in vacuo to give the crude
product. Recrystallization from hot hexanes gave methyl
2-(2-hydroxyphenyl)acetate (2.83 g, 87%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 7.20 (ddd, J=7.7, 7.4, 1.8 Hz, 1H), 7.09-7.11
(m, 1H), 6.94 (dd, J=8.0, 1.2 Hz, 1H), 6.88 (ddd, J=7.4, 7.4, 1.2
Hz, 1H), 3.75 (s, 3H), 3.69 (s, 2H).
Step 2
[0124] A solution of methyl 2-(2-hydroxyphenyl)acetate (1.00 g, 6.0
mmol), triphenylphosphine (2.37 g, 9.0 mmol) and pent-1-en-3-ol
(0.78 g, 9.0 mmol) in tetrahydrofuran (30 mL) was cooled to
0.degree. C. under nitrogen, and diisopropyl azodicarboxylate (1.86
mL; 9.0 mL) was added dropwise over 10 minutes. The reaction was
then heated to 60.degree. C. for 21.5 hours. Solvent was evaporated
in vacuo and the residue was extracted with 5% ethyl acetate in
hexanes. The extract was filtered and evaporated in vacuo to give
the crude product. Purification on a Biotage.TM. SP1 system (120 g
silica cartridge), eluting with 0-3% ethyl acetate in hexanes, gave
methyl 2-(2-(pent-1-en-3-yloxy)phenyl)acetate (0.39 g, 28%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.21-7.26 (m, 1H), 7.20
(d, J=7.6 Hz, 1H), 6.91 (ddd, J=7.4, 7.4, 1.0 Hz, 1H), 6.87 (d,
J=8.0 Hz, 1H), 5.84 (ddd, J=17.4, 10.7, 6.0 Hz, 1H), 5.26 (d,
J=17.4 Hz, 1H), 5.22 (d, J=10.7 Hz, 1H), 4.63 (dt, J=6.0, 6.0 Hz,
2H), 3.70 (s, 3H), 3.68 (s, 2H), 1.71-1.87 (m, 2H), 1.02 (t, J=7.5
Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3): .delta. 172.58,
156.28, 137.75, 131.19, 128.50, 123.87, 120.52, 116.66, 113.18,
79.76, 52.00, 36.61, 28.71, 9.62.
Step 3
[0125] A solution of methyl 2-(2-(pent-1-en-3-yloxy)phenyl)acetate
(0.24 g, 1.0 mmol) in N-methyl-2-pyrrolidone (1.0 mL) was
irradiated with microwave radiation in a Biotage Initiator at
180.degree. C. for 30 minutes, then for 15 minutes. The solution
was diluted with ethyl acetate (25 mL), then washed with water
(4.times.25 mL) and with saturated aqueous sodium chloride (25 mL);
dried over sodium sulfate; filtered and evaporated in vacuo to give
the crude product. Purification on a Biotage.TM. SP1 system (40 g
silica cartridge), eluting with 0-7% ethyl acetate in hexanes, gave
methyl (E)-2-(2-hydroxy-3-(pent-2-enyl)phenyl)acetate (0.89 g,
37%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.09 (s, 1H), 7.08
(dd, J=7.4, 1.6 Hz, 1H), 7.01 (dd, J=7.6, 1.6 Hz, 1H), 6.85 (dd,
J=7.6, 7.4 Hz, 1H), 5.59-5.70 (m, 2H), 3.75 (s, 3H), 3.69 (s, 2H),
3.41 (d, J=4.7 Hz, 2H), 2.04-2.11 (m, 2H), 1.01 (t, J=7.4 Hz, 3H);
.sup.13C NMR (101 MHz, CDCl.sub.3): .delta. 174.31, 153.53, 134.44,
129.86, 129.32, 128.62, 127.13, 121.08, 120.82, 52.79, 37.59,
34.17, 25.77, 13.97.
Step 4
[0126] Methyl (E)-2-(2-hydroxy-3-(pent-2-enyl)phenyl)acetate (0.14
g, 0.6 mmol) was hydrogenated as for Compound I, step 3, but using
methanol as solvent, to give methyl
2-(2-hydroxy-3-pentylphenyl)acetate (0.11 g, 76%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 7.57 (s, 1H), 7.11 (dd, J=7.4, 1.6 Hz,
1H), 6.96 (dd, J=7.4, 1.6 Hz, 1H), 6.84 (dd, J=7.4, 7.4 Hz, 1H),
3.76 (s, 3H), 3.70 (s, 2H), 2.68 (t, J=7.8 Hz, 2H), 1.61-1.67 (m,
2H), 1.36-1.43 (m, 4H), 0.93 (t, J=7.0 Hz, 3H); .sup.13C NMR (101
MHz, CDCl.sub.3): .delta. 175.01, 153.48, 131.75, 129.98, 128.75,
120.74, 120.60, 53.01, 38.30, 32.10, 30.50, 29.91, 22.87,
14.34.
Step 5
[0127] Methyl 2-(2-hydroxy-3-pentylphenyl)acetate (0.11 g, 0.5
mmol) was hydrolysed as for Compound I, step 4, using
acetonitrile/water (4:1) as solvents, to give
2-(2-hydroxy-3-pentylphenyl)acetic acid (0.57 g, 57%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 8.70 (br s, 1H), 7.09 (dd, J=7.6,
1.6 Hz, 1H), 6.98 (dd, J=7.4, 1.6 Hz, 1H), 6.84 (dd, J=7.6, 7.4 Hz,
1H), 3.68 (s, 2H), 2.62 (t, J=7.8 Hz, 2H), 1.57-1.65 (m, 2H),
1.31-1.40 (m, 4H), 0.91 (t, J=7.0 Hz, 3H); .sup.13C NMR (101 MHz,
CDCl.sub.3): .delta. 179.89, 152.79, 130.92, 130.04, 128.98,
121.08, 120.24, 37.74, 32.02, 30.34, 29.78, 22.80, 14.30.
Step 6
[0128] 2-(2-Hydroxy-3-pentylphenyl)acetic acid (22 mg, 0.098 mmol)
was converted to the sodium salt as for Compound I, step 5 to give
sodium 2-(2-hydroxy-3-pentylphenyl)acetate (24 mg, 98%). .sup.1H
NMR (400 MHz, CD.sub.3OD): .delta. 6.91 (dd, J=7.5, 1.6 Hz, 1H),
6.87 (dd, J=7.5, 1.6 Hz, 1H), 6.66 (dd, J=7.5, 7.5 Hz, 1H), 3.49
(s, 2H), 2.59 (t, J=7.7 Hz, 2H), 1.55-1.62 (m, 2H), 1.28-1.38 (m,
4H), 0.90 (t, J=7.0 Hz, 3H); .sup.13C NMR (101 MHz, CD.sub.3OD):
.delta. 180.26, 154.27, 130.75, 128.21, 127.90, 124.24, 119.23,
42.91, 31.83, 30.21, 29.82, 22.51, 13.29; LRMS (ESI negative): m/z
220.8 (100%, M-Na.sup.+); UPLC (System A): 5.0 min. UPLC System A:
Mobile phase A=10 mM aqueous ammonium formate; mobile phase
B=acetonitrile; solid phase=HSS T3 column; gradient=5-100% B in A
over 10 minutes.
Compound X: Sodium Salt of 2-(3-fluoro-5-pentylphenyl)acetic
Acid
##STR00072##
[0129] Step 1
[0130] A solution of 3-bromo-5-fluorobenzoic acid (2.74 g, 12.5
mmol) in tetrahydrofuran (6 mL), at 0.degree. C. under nitrogen,
was treated with borane-tetrahydrofuran complex (1M, 15 mL, 15
mmol) in small portions over 12 min, and the reaction was then
stirred at 0.degree. C. for 70 minutes, and at room temperature for
22 h. The reaction was quenched by addition of methanol (10 mL),
and the methanolic mixture was stirred at room temperature for 3 h,
and then evaporated in vacuo, with co-evaporation from methanol,
then from ethyl acetate, to give the crude product. The material
was dissolved in ethyl acetate (200 mL), and the solution was
washed with 0.5M aqueous sodium hydroxide (200 mL), and with
saturated aqueous sodium chloride (100 mL); then dried over sodium
sulfate; filtered and evaporated in vacuo to give
3-bromo-5-fluorobenzyl alcohol (1.79 g, 67%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 7.29 (s, 1H), 7.15 (ddd, J.sub.HF=8.2 Hz,
J.sub.HH=2.2, 1.8 Hz, 1H), 7.00-7.02 and 7.02-7.04 (dm,
J.sub.HF=9.2 Hz, J.sub.HH=unresolved, 1H), 4.66 (s, 2H), 2.04 (br
s, 1H); .sup.19F NMR (377 MHz, CDCl.sub.3): .delta. -111.05 (dd,
J.sub.HF=9.3, 8.0 Hz, 1F); .sup.13C NMR (101 MHz, CDCl.sub.3):
.delta. 162.87 (d, J.sub.CF=250.6 Hz), 145.42 (d, J.sub.CF=6.9 Hz),
125.45 (d, J.sub.CF=3.1 Hz), 122.69 (d, J.sub.CF=9.2 Hz), 118.01
(d, J.sub.CF=24.6 Hz), 112.51 (d, J.sub.CF=21.5 Hz), 63.60 (d,
J.sub.CF=2.3 Hz).
Step 2
[0131] A solution of 3-bromo-5-fluorobenzyl alcohol (1.79 g, 8.39
mmol) and triphenylphosphine (3.65 g, 10.10 mmol) in
dichloromethane (45 mL), was treated with carbon tetrabromide (3.34
g, 10.10 mmol) in small portions over 10 min, and the reaction was
then stirred at room temperature overnight. Solvent was evaporated
in vacuo, and the residue was treated with diethyleher (50 mL). The
resultant white slurry was stirred at room temperature, and then
filtered through Celite.TM.. The residue was washed with
diethylether (2.times.50 mL), and the combined filtrate and
washings were evaporated in vacuo to give the crude product.
Purification on a silica pad, eluting with 2% ethyl acetate/hexane,
gave 3-bromo-5-fluorobenzyl bromide (2.21 g, 98%). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 7.33 (s, 1H), 7.18 (ddd, J.sub.HF=8.2 Hz,
J.sub.HH=2.0, 2.0 Hz, 1H), 7.05 (ddd, J.sub.HF=9.0 Hz,
J.sub.HH=1.8, 1.6 Hz, 1H), 4.38 (s, 2H); .sup.19F NMR (377 MHz,
CDCl.sub.3): .delta. -110.19 to -110.14 (m, 1F); .sup.13C NMR (101
MHz, CDCl.sub.3): .delta. 162.67 (d, J.sub.CF=252.1 Hz), 141.61 (d,
J.sub.CF=8.5 Hz), 128.17 (d, J.sub.CF=3.1 Hz), 122.94 (d,
J.sub.CF=10.0 Hz), 119.39 (d, J.sub.CF=24.6 Hz), 115.34 (d,
J.sub.CF=22.3 Hz), 31.31 (d, J.sub.CF=2.3 Hz).
Step 3
[0132] A suspension of sodium cyanide (0.38 g, 7.73 mmol) in water
(0.35 mL) was treated with a solution of 3-bromo-5-fluorobenzyl
bromide (1.38 g, 5.15 mmol) in dimethylformamide (2.6 mL), and the
reaction was heated at 75.degree. C. in a sealed tube for 3 h. The
reaction was cooled to room temperature and was partitioned between
ethyl acetate (50 mL) and 2.5% w/v aqueous sodium bicarbonate (100
mL). The aqueous phase was extracted with a further portion of
ethyl acetate (50 mL); and the combined extracts were washed with
water (2.times.50 mL) and with saturated aqueous sodium chloride
(50 mL); dried over sodium sulfate; filtered, and evaporated in
vacuo to give the crude product. Purification on a Biotage.TM. 40
iM column (silica), eluting with 10% ethyl acetate/hexane, gave
2-[3-bromo-5-fluorophenyl]acetonitrile (0.64 g, 58%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 7.26-7.28 (m, 1H), 7.17-7.19 &
7.19-7.21 (dm, J.sub.HF=8.0 Hz, J.sub.HH=unresolved, 1H), 6.98-7.00
& 7.00-7.02 (dm, J.sub.HF=8.8 Hz, J.sub.HH=unresolved, 1H),
3.73 (s, 2H); .sup.19F NMR (377 MHz, CDCl.sub.3): .delta. -109.46
(dd, J.sub.HF=8.0, 8.0 Hz, 1F); .sup.13C NMR (101 MHz, CDCl.sub.3):
.delta. 162.90 (d, J.sub.CF=252.1 Hz), 133.95 (d, J.sub.CF=8.5 Hz),
127.24 (d, J.sub.CF=3.8 Hz), 123.53 (d, J.sub.CF=10.0 Hz), 119.22
(d, J.sub.CF=23.8 Hz), 117.00, 114.50 (d, J.sub.CF=23.1 Hz), 23.30
(d, J.sub.CF=1.5 Hz).
Step 4
[0133] A solution of the aryl bromide (0.55 g, 2.58 mmol) and
(E)-1-penten-1-ylboronic acid pinacol ester (0.61 g, 3.13 mmol) in
dimethoxyethane (13 mL) was treated with a solution of sodium
carbonate (0.55 g, 5.17 mmol) in water (3 mL). The solution was
deoxygenated with nitrogen, and was treated with
tetrakis(triphenylphosphine)palladium (0.15 g, 0.13 mmol; 5 mole
%). The mixture was then heated at 90.degree. C., in a sealed tube
for 17 h. The reaction was cooled to room temperature and was
partitioned between ethyl acetate (50 mL) and 1M aqueous
hydrochloric acid (50 mL). The organic phase was washed with
saturated aqueous sodium chloride (30 mL); dried over sodium
sulfate; filtered, and evaporated in vacuo to give the crude
product. Purification on a Biotage.TM. 40 iM column (silica),
eluting with (3%) ethyl acetate/hexane, gave
(E)-2-[3-fluoro-5-[pent-1-enyl]phenyl]acetonitrile (0.43 g, 82%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.04 (s, 1H), 6.97 (ddd,
J.sub.HF=9.8 Hz, J.sub.HH=2.0, 1.5 Hz, 1H), 6.82-6.85 (m, 1H), 6.31
(d, J=15.8 Hz, 1H), 6.25 (ddd, J=15.8, 5.9, 0 Hz, 1H), 3.68 (s,
2H), 2.18 (td, J=7.2, 5.4 Hz, 2H), 1.49 (qt, J=7.4, 7.4 Hz, 2H),
0.95 (t, J=7.4 Hz, 3H); .sup.19F NMR (377 MHz, CDCl.sub.3): .delta.
-112.93 (dd, J.sub.HF=10.6, 9.3 Hz, 1F); .sup.13C NMR (101 MHz,
CDCl.sub.3): .delta. 163.43 (d, J.sub.CF=246.0 Hz), 141.44 (d,
J.sub.CF=8.5 Hz), 133.99, 132.37 (d, J.sub.CF=8.5 Hz), 128.42 (d,
J.sub.CF=2.3 Hz), 121.60 (d, J.sub.CF=3.1 Hz), 117.66, 113.40 (d,
J.sub.CF=23.1 Hz), 112.21 (d, J.sub.CF=22.3 Hz), 35.22, 23.49 (d,
J.sub.CF=2.3 Hz), 22.51, 13.94.
Step 5
[0134] A solution of the phenylacetonitrile derivative (0.43 g,
2.10 mmol) in methanol (42 mL) was treated with aqueous sodium
hydroxide (5M; 21 mL, 105 mmol), and the mixture was heated at
75.degree. C. in a sealed tube for 4.5 h. The reaction mixture was
cooled to room temperature, and was quenched with 6M aqueous
hydrochloric acid (21 mL); stirred at room temperature for 10 min;
then extracted with ethyl acetate (2.times.75 mL). The organic
extract was washed with saturated aqueous sodium chloride (75 mL);
dried over sodium sulfate; filtered, and evaporated in vacuo to
give the crude product. Purification on a Biotage.TM. 40 iM column
(silica), eluting with 70% ethyl acetate/hexane, gave the methyl
ester of the desired product (0.09 g, 18%), and .about.95% pure
(E)-2-[3-fluoro-5-[pent-1-enyl]phenyl]acetic acid (0.22 g, 48%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 11.17 (br s, 1H), 7.02
(s, 1H), 6.98 (ddd, J.sub.HF=9.8 Hz, J.sub.HH=2.0, 1.8 Hz, 1H),
6.85 (ddd, J.sub.HF=9.0 Hz, J.sub.HH=1.8, 1.6 Hz, 1H), 6.33 (d,
J=15.8 Hz, 1H), 6.25 (dt, J=15.8, 6.4 Hz, 1H), 3.62 (s, 2H),
2.17-2.22 (m, 2H), 1.51 (qt, J=7.4, 7.4 Hz, 2H), 0.96 (t, J=7.4 Hz,
3H); .sup.19F NMR (377 MHz, CDCl.sub.3): .delta. -114.10 (dd,
J.sub.HF=9.3, 9.3 Hz, 1F).
Step 6
[0135] A solution of the partially-purified acid (0.28 g, 1.26
mmol) in acetone (5 mL) was treated with potassium carbonate (0.26
g, 1.90 mmol), potassium iodide (0.04 g, 0.25 mmol) and benzyl
bromide (0.18 mL, 1.5 mmol), and the reaction was stirred at room
temperature for 18 h. The reaction mixture was partitioned between
ethyl acetate (25 mL) and 1M aqueous hydrochloric acid (25 mL). The
organic phase was then washed with saturated aqueous sodium
chloride (25 mL); dried over sodium sulfate; filtered, and
evaporated in vacuo to give the crude product. Purification on a
Biotage.TM. 40 iM column (silica), eluting with 5% ethyl
acetate/hexane gave benzyl
(E)-2-[3-fluoro-5-[pent-1-enyl]phenyl]acetate (0.3 g, 75%). .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 7.32-7.40 (m, 5H), 7.03 (s, 1H),
6.97 (ddd, J.sub.HF=10.0 Hz, J.sub.HH=2.3, 1.5 Hz, 1H), 6.86 (ddd,
J.sub.HF=9.0 Hz, J.sub.HH=2.0, 1.7 Hz, 1H), 6.33 (d, J=15.8 Hz,
1H), 6.23 (dt, J=15.8, 6.5 Hz, 1H), 5.16 (s, 2H), 3.64 (s, 2H),
2.17-2.23 (m, 2H), 1.52 (qt, J=7.4, 7.4 Hz, 2H), 0.97 (t, J=7.4 Hz,
3H); .sup.19F NMR (377 MHz, CDCl.sub.3): .delta. -114.34 (dd,
J.sub.HF=9.3, 9.3 Hz, 1F); .sup.13C NMR (101 MHz, CDCl.sub.3):
.delta. 171.08, 163.32 (d, J.sub.CF=244.4 Hz), 140.65 (d,
J.sub.CF=7.7 Hz), 136.17 (d, J.sub.CF=8.5 Hz), 135.93, 133.05,
128.95 (d, J.sub.CF=3.1 Hz), 128.84, 128.52 (d, J.sub.CF=9.2 Hz),
128.48, 123.09 (d, J.sub.CF=2.3 Hz), 114.78 (d, J.sub.CF=22.3 Hz),
111.46 (d, J.sub.CF=22.3 Hz), 67.04, 41.26 (d, J.sub.CF=1.5 Hz),
35.27, 22.63, 14.00.
Step 7
[0136] A solution of the benzyl ester (0.16 g, 0.50 mmol) in ethyl
acetate (2 mL) was treated with palladium on carbon (1% w/w Pd; 15
mg). The mixture was degassed with hydrogen, and was stirred under
1 atmosphere of hydrogen at room temperature overnight. The
reaction was filtered, and evaporated in vacuo to give
2-[3-fluoro-5-pentylphenyl]-acetic acid (0.11 g, 97%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 11.47 (br s, 1H), 6.89 (s, 1H),
6.81-6.86 (m, 2H), 3.62 (s, 2H), 2.60 (t, J=7.8 Hz, 2H), 1.58-1.66
(m, 2H), 1.28-1.41 (m, 4H), 0.92 (t, J=6.8 Hz, 3H); .sup.19F NMR
(377 MHz, CDCl.sub.3): .delta. -114.34 (dd, J.sub.HF=9.3, 9.3 Hz,
1F); .sup.13C NMR (101 MHz, CDCl.sub.3): .delta. 178.15, 163.08 (d,
J.sub.CF=246.0 Hz), 145.02 (d, J.sub.CF=7.7 Hz), 135.04 (d,
J.sub.CF=8.5 Hz), 125.49 (d, J.sub.CF=2.3 Hz), 114.49 (d,
J.sub.CF=20.8 Hz), 113.83 (d, J.sub.CF=22.3 Hz), 41.01 (d,
J.sub.CF=1.5 Hz), 35.87 (d, J.sub.CF=1.5 Hz), 31.67, 31.03, 22.74,
14.24.
Step 8
[0137] A solution of the acid (0.11 g, 0.49 mmol) in ethanol (3 mL)
was treated with a solution of sodium bicarbonate (0.041 g, 0.49
mmol) in water (0.75 mL), and the reaction was stirred at room
temperature for 17 h. Ethanol was evaporated in vacuo, and the
residual aqueous syrup was diluted with water (10 mL), filtered
(0.2 .mu.m), and lyophilised to give sodium
2-[3-fluoro-5-pentylphenyl]acetate as a white solid (0.12 g, 99%).
mp 120-123.degree. C.; .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
6.94 (s, 1H), 6.87 (ddd, J.sub.HF=9.8 Hz, J.sub.HH=2.0, 2.0 Hz,
1H), 6.70 (ddd, J.sub.HF=10.0 Hz, J.sub.HH=2.0, 2.0 Hz, 1H), 3.45
(s, 2H), 2.56 (t, J=7.7 Hz, 2H), 1.58-1.63 (m, 2H), 1.26-1.39 (m,
4H), 0.90 (t, J=7.0 Hz, 3H); .sup.19F NMR (377 MHz, CD.sub.3OD):
.delta. -117.54 (dd, J.sub.HF=10.0, 10.0 Hz, 1F); .sup.13C NMR (101
MHz, CD.sub.3OD): .delta. 178.66, 163.04 (d, J.sub.CF=242.9 Hz),
145.07 (d, J.sub.CF=7.7 Hz), 140.42 (d, J.sub.CF=8.5 Hz), 125.03
(d, J.sub.CF=2.3 Hz), 112.99 (d, J.sub.CF=22.3 Hz), 112.30 (d,
J.sub.CF=20.8 Hz), 44.96, 35.53 (d, J.sub.CF=1.5 Hz), 31.46, 31.00,
22.45, 13.30; HPLC: 1.2 min.
Compound XI: Sodium Salt of 2-(2-Fluoro-3-pentylphenyl)acetic
Acid
[0138] The above compound was prepared as for Compound X, starting
with 3-bromo-2-fluorobenzoic acid. White solid; .sup.1H NMR (400
MHz, CD.sub.3OD): .delta. 7.13 (ddd, J.sub.HF=7.0 Hz, J.sub.HH=7.4,
1.9 Hz, 2H), 7.03 (ddd, J.sub.HF=7.0 Hz, J.sub.HH=7.4, 1.9 Hz, 1H),
6.97 (dd, J.sub.HH=7.4, 7.4 Hz, 1H), 3.51 (d, J.sub.HF=1.4 Hz, 2H),
2.61 (t, J=7.6 Hz, 2H), 1.56-1.63 (m, 2H), 1.28-1.40 (m, 4H), 0.90
(t, J=6.9 Hz, 3H); .sup.13C NMR (101 MHz, CD.sub.3OD): .delta.
178.21, 159.70 (d, J.sub.CF=242.9 Hz), 129.07 (d, J.sub.CF=4.6 Hz),
128.88, 128.43 (d, J.sub.CF=5.4 Hz), 125.02 (d, J.sub.CF=17.7 Hz),
123.31 (d, J.sub.CF=4.6 Hz), 37.89 (d, J.sub.CF=3.8 Hz), 31.55,
29.98, 28.91 (d, J.sub.CF=3.1 Hz), 22.41, 13.26; .sup.19F NMR (377
MHz, CD.sub.3OD): .delta. -126.09 to -126.05 (m, 1F); LRMS (ESI):
m/z 220.0 (M-CO.sub.2Na+acetonitrile), 179.4 (M-CO.sub.2Na); HPLC:
1.2 min.
Compound XII: Sodium Salt of 2-(4-Fluoro-3-pentylphenyl)acetic
Acid
[0139] The above compound was prepared from methyl
2-(3-bromo-4-fluorophenyl)acetate by Suzuki coupling as for
Compound VII; followed by hydrogenation, ester hydrolysis and salt
formation as for Compound I. The starting ester was prepared by
reaction of 2-(3-bromo-4-fluorophenyl)acetic acid with methanol in
the presence of sulfuric acid. White solid; .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 7.16 (dd, J.sub.HF=7.4 Hz, J.sub.HH=2.3 Hz,
2H), 7.08 (ddd, J.sub.HF=5.0 Hz, J.sub.HH=8.3, 2.3 Hz, 1H), 6.88
(dd, J.sub.HF=10.1 Hz, J.sub.HH=8.3 Hz, 1H), 3.40 (s, 2H), 2.59 (t,
J=7.7 Hz, 2H), 1.55-1.63 (m, 2H), 1.28-1.40 (m, 4H), 0.90 (t, J=7.0
Hz, 3H); .sup.13C NMR (101 MHz, CD.sub.3OD): .delta. 179.12, 159.88
(d, J.sub.CF=240.6 Hz), 133.88 (d, J.sub.CF=3.8 Hz), 131.26 (d,
J.sub.CF=4.6 Hz), 128.78 (d, J.sub.CF=16.1 Hz), 127.96 (d,
J.sub.CF=8.5 Hz), 114.26 (d, J.sub.CF=23.1 Hz), 44.38, 31.51,
30.00, 28.76 (d, J.sub.CF=1.5 Hz), 22.36, 13.18; .sup.19F NMR (377
MHz, CD.sub.3OD): .delta. -126.45 to -126.40 (m, 1F); LRMS (ESI):
m/z 225.2 (M-Na.sup.+2W); HPLC: 1.9 min.
Compound XIII: Sodium Salt of
(RS)-2-Fluoro-2-(3-pentylphenyl)acetic Acid
[0140] The above compound was prepared from ethyl
2-fluoro-2-(3-pentylphenyl)acetate as for Compound I. The ester was
prepared by reaction of ethyl 2-(3-pentylphenyl)acetate with
lithium diisopropylamide and N-fluorobenzenesulfonimide at
-78.degree. C. in Tetrahydrofuran. White solid; .sup.1H NMR (400
MHz, CD.sub.3OD): .delta. 7.34 (s, 1H), 7.30 (dd, J=7.6, 1.4 Hz,
1H), 7.24 (dd, J=7.6, 7.6 Hz, 1H), 7.13 (dd, J=7.4, 1.0 Hz, 1H),
5.53 (d, J.sub.HF=51.3 Hz, 1H), 2.60 (t, J=7.7 Hz, 2H), 1.59-1.65
(m, 2H), 1.27-1.39 (m, 4H), 0.76 (t, J=6.9 Hz, 3H); .sup.13C NMR
(101 MHz, CD.sub.3OD): .delta. 173.73 (d, J.sub.CF=23.9 Hz),
141.34, 136.37 (d, J.sub.CF=20.0 Hz), 126.79 (d, J.sub.CF=2.3 Hz),
126.40, 125.41 (d, J.sub.CF=5.4 Hz), 122.84 (d, J.sub.CF=5.4 Hz),
90.34 (d, J.sub.CF=183.4 Hz), 34.13, 29.91, 29.65, 20.85, 11.64;
.sup.19F NMR (377 MHz, CD.sub.3OD): .delta. -168.83 (d,
J.sub.HF=51.7 Hz, 1F); LRMS (ESI negative): m/z 223.0 (100%,
M-Na.sup.+); HPLC: 4.1 min.
Compound XIV: Sodium 2-[3,5-Dipentylphenyl] acetate
##STR00073##
[0141] Step 1
[0142] A suspension of methyl 2-[3,5-dihydroxyphenyl]acetate (1.00
g, 5.49 mmol) and N-phenyl-bis(trifluoromethylsulfonyl)imide (4.31
g, 12.1 mmol) in dichloromethane (20 mL), at 0.degree. C. under
nitrogen, was treated with triethylamine (1.68 mL, 12.1 mmol). A
clear solution formed. The reaction was then stirred under nitrogen
at 0.degree. C. for 2 h, and at room temperature for 21 h. The
reaction was diluted with ethyl acetate (100 mL), and the solution
was washed with 0.5M aqueous sodium hydroxide (2.times.100 mL), and
with saturated aqueous sodium chloride (75 mL); then dried over
sodium sulphate; filtered and evaporated in vacuo to give the crude
product. Purification on a Biotage.TM. 40 iM column (silica),
eluting with ethyl acetate/hexane 0:1 to 1:9, gave methyl
2-[3,5-bis(trifluoromethylsulfonyloxy)phenyl]acetate (2.23 g, 91%)
as pale oil. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.32 (d,
J=2.2 Hz, 2H), 7.18 (dd, J=2.2, 2.2 Hz, 1H), 3.72 (s, 5H); 19F NMR
(377 MHz, CDCl.sub.3): .delta. -73.20 (s, 3F); .sup.13C NMR (101
MHz, CDCl.sub.3): .delta. 170.05, 149.48, 139.01, 122.95, 118.87
(q, J.sub.CF=320.5 Hz), 114.42, 52.62, 40.29.
Step 2
[0143] A solution of the aryl bis(triflate) (2.23 g, 4.99 mmol) and
(E)-1-penten-1-ylboronic acid pinacol ester (2.45 g, 12.5 mmol) in
1,2-dimethoxyethane (25 mL) was treated with a solution of sodium
carbonate (1.59 g, 15.0 mmol) in water (8 mL). The solution was
deoxygenated with nitrogen, and was then treated with
Tetrakis(triphenylphosphine) palladium (0.58 g, 0.50 mmol). The
mixture was heated at 90.degree. C., in a sealed tube for 17 h. The
reaction was cooled to room temperature and was partitioned between
ethyl acetate (200 mL) and 1M aqueous hydrochloric acid (150 mL).
The organic phase was washed with 5% aqueous sodium bicarbonate
(150 mL), and with saturated aqueous sodium chloride (150 mL); then
dried over sodium sulphate; filtered, and evaporated in vacuo to
give the crude product. Purification on a Biotage.TM. 40 iL column
(silica), eluting with ethyl acetate/hexane 0:1 to 3:97, gave
methyl 2-[3,5-di[(E)-1-pent-1-enyl]phenyl] acetate as an
inseparable 10:4 mixture with excess (E)-1-penten-1-ylboronic acid
pinacol ester (1.12 g, 61%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 7.21 (s, 1H), 7.10 (d, J=1.3 Hz, 2H), 6.34 (d, J=15.8 Hz,
1H), 6.22 (dd, J=15.8, 6.7 Hz, 1H), 3.65 (s, 3H), 3.55 (s, 2H),
2.18 (tdd, J=6.8, 6.8, 1.0 Hz, 2H), 1.49 (qt, J=7.4, 7.2 Hz, 2H),
0.96 (t, J=7.4 Hz, 3H); .sup.13C NMR (101 MHz, CDCl.sub.3): .delta.
172.04, 138.59, 134.47, 131.34, 129.97, 125.57, 122.75, 52.07,
41.32, 35.39, 22.77, 13.97.
Step 3
[0144] A solution of the unsaturated compound (1.12 g, 78.5% w/w,
3.07 mmol) in ethyl acetate (1 mL) and methanol (1 mL) was treated
with palladium on carbon (10% w/w Pd; 0.12 g). The mixture was
degassed with hydrogen, and was stirred under 1 atm. of hydrogen at
room temperature for 22 h. The reaction was filtered, and
evaporated in vacuo to give methyl 2-[3,5-dipentylphenyl] acetate
as an inseparable 10:4 mixture with pentylboronic acid pinacol
ester (0.86 g, 76%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
6.93 (s, 3H), 3.70 (s, 3H), 3.59 (s, 2H), 2.58 (t, J=7.9 Hz, 2H),
1.58-1.66 (m, 2H), 1.32-1.38 (m, 4H), 0.91 (t, J=6.8 Hz, 3H).
Step 4
[0145] A solution of the methyl ester (0.86 g, 79% w/w, 2.34 mmol)
in acetonitrile (24 mL) was treated with a solution of lithium
hydroxide (0.28 g, 11.7 mmol) in water (6 mL), and the reaction was
stirred at room temperature for 22 h. The reaction was quenched
with 1M aqueous hydrochloric acid (55 mL), and then extracted with
ethyl acetate (100 mL). The organic extract was washed with
saturated aqueous sodium chloride (50 mL); then dried over sodium
sulphate; filtered, and evaporated in vacuo to give the crude
product. Purification on a SiliaSep silicon oxide column, eluting
with ethyl acetate/hexane 0:1 to 1:4, gave 2-[3,5-dipentyl]phenyl]
acetic acid as a colorless oil (0.55 g, 84%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 6.99 (s, 3H), 3.65 (s, 2H), 2.63 (t, J=7.8 Hz,
2H), 1.64-71 (m, 2H), 1.36-1.44 (m, 4H), 0.97 (t, J=6.9 Hz, 3H);
.sup.13C NMR (101 MHz, CDCl.sub.3): .delta. 178.96, 143.55, 133.21,
127.93, 127.06, 41.47, 36.13, 31.94, 31.47, 22.86, 14.34.
Step 5
[0146] A solution of the acid (0.48 g, 1.75 mmol) in ethanol (12
mL) was treated with a solution of sodium bicarbonate (0.15 g, 1.75
mmol) in water (3 mL), and the reaction was stirred at room
temperature for 3 d. Ethanol was evaporated in vacuo, and the
residual aqueous syrup was diluted with water (50 mL), filtered
(PES, 0.2 .mu.m), and lyophilised to give sodium
2-[3,5-dipentylphenyl] acetate as a white solid (0.52 g,
quantitative). mp 225-230.degree. C.; .sup.1H NMR (400 MHz,
CD.sub.3OD+D.sub.2O): .delta. 6.92 (s, 2H), 6.76 (s, 1H), 3.41 (s,
2H), 2.50 (t, J=7.5 Hz, 2H), 1.52-1.59 (m, 2H), 1.23-1.33 (m, 4H),
0.85 (t, J=6.9 Hz, 3H); .sup.13C NMR (101 MHz,
CD.sub.3OD+D.sub.2O): .delta. 179.99, 142.66, 137.63, 126.66,
126.16, 45.11, 35.61, 31.36, 31.19, 22.41, 13.47; LRMS (ESI): m/z
277.5 (w, [M-Na++2H+]), 231.1 (100%, tropylium ion from loss of
carboxy group); HPLC: 3.0 min.
Compound XV: Sodium Salt of 2-(3,5-Dihexylphenyl)acetic Acid
[0147] The above compound was prepared from (E)-hex-1-enylboronic
acid pinacol ester as for Compound XIV. White solid; .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 6.96 (s, 2H), 6.79 (s, 1H), 3.43 (s,
2H), 2.54 (d, J=7.7 Hz, 4H), 1.55-1.63 (m, 4H), 1.28-1.36 (m, 12H),
0.89 (t, J=6.8 Hz, 6H); 13C NMR (101 MHz, CD.sub.3OD): .delta.
179.68, 142.38, 137.82, 126.55, 126.07, 45.30, 35.87, 31.83, 31.67,
29.02, 22.61, 13.42; LRMS (ESI): m/z 322.0 (100%,
M-Na++H++NH.sub.4+) and 259.0 (35%, M-CO.sub.2Na); UPLC (System A):
8.9 min. UPLC System A: Mobile phase A=10 mM aqueous ammonium
bicarbonate; mobile phase B=acetonitrile; solid phase=HSS T3
column; gradient=5-100% B in A over 10 minutes.
Compound XVI: Sodium Salt of 2-(2-Hydroxy-3,5-dipentylphenyl)acetic
Acid
##STR00074## ##STR00075##
[0148] Step 1
[0149] A solution of 2,4-dibromo-6-(bromomethyl)phenol (3.5 g, 10.0
mmol) in acetonitrile (17 mL) was treated with a solution of sodium
cyanide (2.5 g, 50.0 mmol) and the reaction was heated at
100.degree. C. under reflux for 1 h. The reaction mixture cooled to
room temperature and was poured into water (100 mL). The pH was
adjusted from 10 to 8 with 1M aqueous hydrochloric acid, and the
mixture was extracted with ethyl acetate (3.times.250 mL). Combined
extracts were washed with 1M aqueous hydrochloric acid (250 mL) and
with saturated aqueous sodium chloride (250 mL); dried over sodium
sulfate; filtered and evaporated in vacuo to give the crude
product. Extraction with acetone; filtration; and evaporation in
vacuo gave 2-(3,5-dibromo-2-hydroxyphenyl)acetonitrile (2.6 g,
90%). .sup.1H NMR (400 MHz, d6-acetone): .delta. 8.75 (br s, 1H),
7.69 (d, J=2.3 Hz, 1H), 7.54 (d, J=2.3 Hz, 1H), 3.92 (s, 2H);
.sup.13C NMR (101 MHz, d6-acetone): .delta. 151.31, 134.51, 131.92,
122.80, 117.43, 111.89, 111.53, 18.70.
Step 2
[0150] 2-(3,5-Dibromo-2-hydroxyphenyl)acetonitrile (2.6 g, 9.0
mmol) was treated with a mixture of sulfuric acid (2.5 mL), acetic
acid (2.5 mL) and water (2.5 mL), and the reaction was heated at
125.degree. C. under reflux for 2 h. The reaction mixture was
cooled to room temperature and was poured into a mixture of ice (50
mL) and water (50 mL), and was then stirred until the ice had
melted. The mixture was extracted with ethyl acetate (250 mL); and
the extract was then washed with water (100 mL) and with saturated
aqueous sodium chloride (100 mL); dried over sodium sulfate;
filtered and evaporated in vacuo to give the crude
2-(3,5-dibromo-2-hydroxyphenyl)acetic acid (3.1 g). This material
was used directly in the next step without further purification or
characterization.
Step 3
[0151] A solution of crude 2-(3,5-dibromo-2-hydroxyphenyl)acetic
acid (3.1 g, 9.0 mmol) in methanol (17 mL) was treated with
sulfuric acid (0.43 mL, 8.1 mmol) and the reaction was stirred at
ambient temperature for 16 h. Methanol was evaporated in vacuo, and
the residue was dissolved in ethyl acetate (270 mL). The solution
was washed with water (2.times.200 mL) and with saturated aqueous
sodium chloride (130 mL); dried over sodium sulfate; filtered and
evaporated in vacuo to give the crude product. Purification on a
Biotage.TM. SP1 system (120 g silica cartridge), eluting with 0-20%
ethyl acetate in hexanes, gave methyl
2-(3,5-dibromo-2-hydroxyphenyl)acetate (1.4 g, 49%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 7.52 (d, J=2.2 Hz, 1H), 7.23 (d,
J=2.2 Hz, 1H), 6.42 (br s, 1H), 3.72 (s, 3H), 3.65 (s, 2H); 13C NMR
(101 MHz, CDCl.sub.3): .delta. 172.06, 150.60, 133.74, 133.50,
123.94, 112.62, 111.77, 52.78, 36.61.
Step 4
[0152] A solution of methyl 2-(3,5-dibromo-2-hydroxyphenyl)acetate
(0.5 g, 1.54 mmol) in acetone (5 mL) was treated with potassium
carbonate (0.26 g, 1.86 mmol), potassium iodide (0.05 g, 0.32 mmol)
and benzyl bromide (0.20 mL, 1.7 mmol), and the reaction was
stirred at room temperature for 1 h. Acetone was evaporated in
vacuo, and the residue was partitioned between ethyl acetate (50
mL) and 1M aqueous hydrochloric acid (50 mL). The organic phase was
washed with saturated aqueous sodium chloride (50 mL); dried over
sodium sulfate; filtered and evaporated in vacuo to give the crude
product. Purification on a Biotage.TM. SP1 system (40 g silica
cartridge), eluting with 0-10% ethyl acetate in hexanes, gave
methyl 2-(2-(benzyloxy)-3,5-dibromophenyl)acetate (0.6 g, 95%).
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.67 (d, J=2.4 Hz, 1H),
7.48-7.51 (m, 2H), 7.37 (d, J=2.4 Hz, 1H), 7.34-7.43 (m, 3H), 4.99
(s, 2H), 3.66 (s, 3H), 3.60 (s, 2H); 13C NMR (101 MHz, CDCl.sub.3):
.delta. 171.26, 153.79, 136.56, 135.38, 133.57, 132.04, 128.82,
128.64, 128.52, 118.69, 117.56, 75.53, 52.50, 35.86.
Step 5
[0153] Methyl 2-(2-(benzyloxy)-3,5-dibromophenyl)acetate (0.3 g,
0.73 mmol) and (E)-pent-1-enylboronic acid pinacol ester (0.4 g,
1.79 mmol) were coupled as for Compound I, step 2, to give methyl
2-(2-(benzyloxy)-3,5-di((E)-pent-1-enyl)phenyl)acetate (0.21 mg,
72%). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.50 (d, J=7.2 Hz,
2H), 7.44 (dd, J=7.2, 7.2 Hz, 2H), 7.43 (d, J=2.1 Hz, 1H), 7.38
(dd, J=7.2, 7.2 Hz, 1H), 7.18 (d, J=2.1 Hz, 1H), 6.72 (d, J=15.8
Hz, 1H), 6.39 (d, J=15.8 Hz, 1H), 6.32 (dt, J=15.8, 7.0 Hz, 1H),
6.22 (dt, J=15.8, 6.8 Hz, 1H), 4.87 (s, 2H), 3.69 (s, 3H), 3.67 (s,
2H), 2.20-2.29 (m, 4H), 1.50-1.60 (m, 4H), 1.01 (t, J=7.3 Hz, 3H),
1.00 (t, J=7.4 Hz, 3H); 13C NMR (101 MHz, CDCl.sub.3): .delta.
172.49, 153.59, 137.58, 134.35, 132.91, 131.91, 130.84, 129.53,
128.78, 128.32, 128.30, 128.24, 127.26, 125.21, 123.89, 75.89,
52.21, 35.94, 35.74, 35.42, 22.87, 22.77, 14.07, 14.06.
Step 6
[0154] Methyl
2-(2-(benzyloxy)-3,5-di((E)-pent-1-enyl)phenyl)acetate (0.2 g, 0.53
mmol) was hydrogenated as for Compound I, step 3, to give methyl
2-(2-hydroxy-3,5-dipentylphenyl)acetate (0.12 g, 73%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 7.37 (s, 1H), 6.92 (d, J=2.1 Hz,
2H), 6.77 (d, J=2.1 Hz, 1H), 3.76 (s, 3H), 3.67 (s, 2H), 2.65 (t,
J=7.8 Hz, 2H), 2.51 (t, J=7.8 Hz, 2H), 1.58-1.66 (m, 4H), 1.31-1.41
(m, 8H), 0.93 (t, J=7.0 Hz, 3H), 0.92 (t, J=6.9 Hz, 3H); 13C NMR
(101 MHz, CDCl.sub.3): .delta. 175.01, 151.27, 135.14, 131.48,
129.92, 128.52, 120.30, 52.95, 38.35, 35.34, 32.15, 31.86, 31.74,
30.61, 30.03, 22.87, 22.83, 14.34, 14.31.
Step 7
[0155] Methyl 2-(2-hydroxy-3,5-dipentylphenyl)acetate (0.2 g, 0.53
mmol) was hydrolysed as for Compound I, step 4, to give the crude
product mixed with lactonised material. A small portion was
purified on a Biotage.TM. SP1 system (120 g silica cartridge),
eluting with 0-100% ethyl acetate in hexanes, to give
2-(2-hydroxy-3,5-dipentylphenyl)acetic acid (13.5 mg). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 10.5 (br s, 1H), 6.89 (d, J=2.2 Hz,
1H), 6.78 (d, J=2.2 Hz, 1H), 6.32 (br s, 1H), 3.66 (s, 2H), 2.58
(t, J=7.9 Hz, 2H), 2.48 (t, J=7.8 Hz, 2H), 1.52-1.63 (m, 4H),
1.26-1.37 (m, 8H), 0.90 (t, J=7.0 Hz, 3H), 0.88 (t, J=6.8 Hz,
3H).
Step 8
[0156] 2-(2-Hydroxy-3,5-dipentylphenyl)acetic acid (13.5 mg, 0.046
mmol) was converted to the sodium salt as for Compound I, step 5 to
give sodium 2-(2-hydroxy-3,5-dipentylphenyl)acetate (11 mg, 77%).
.sup.1H NMR (400 MHz, CD.sub.3OD): .delta. 6.72 (d, J=2.0 Hz, 1H),
6.69 (d, J=2.0 Hz, 1H), 3.46 (s, 2H), 2.56 (t, J=7.6 Hz, 2H), 2.44
(t, J=7.6 Hz, 2H), 1.50-1.61 (m, 4H), 1.25-1.37 (m, 8H), 0.90 (t,
J=6.8 Hz, 3H), 0.88 (t, J=7.0 Hz, 3H); 13C NMR (101 MHz,
CD.sub.3OD): .delta. 180.33, 151.94, 133.47, 130.37, 128.21,
127.81, 123.99, 42.90, 34.97, 31.81, 31.60, 31.40, 30.25, 29.88,
22.51, 22.45, 13.29, 13.24; LRMS (ESI negative): m/z 291.2 (100%,
M-Na+); UPLC (System B): 7.7 min. UPLC System B: Mobile phase
A=0.1% aqueous formic acid; mobile phase B=0.1% formic acid in
acetonitrile; solid phase=HSS T3 column; gradient=5-100% B in A
over 10 minutes.
Compound XVII: Sodium Salt of 2-(3,5-Dihexyl-2-hydroxyphenyl)acetic
Acid
[0157] The above compound was prepared as for Compound XVI, using
(E)-hex-1-enylboronic acid pinacol ester. .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 6.72 (d, J=2.0 Hz, 1H), 6.69 (d, J=2.0 Hz,
1H), 3.46 (s, 2H), 2.56 (t, J=7.6 Hz, 2H), 2.44 (t, J=7.5 Hz, 2H),
1.50-1.60 (m, 4H), 1.27-1.37 (m, 12H), 0.89 (t, J=6.6 Hz, 3H), 0.88
(t, J=6.80 Hz, 3H); LRMS (ESI negative): m/z 319 (100%, M-Na+);
UPLC (System B): 8.7 min. ULC System B: Mobile phase A=0.1% aqueous
formic acid; mobile phase B=0.1% formic acid in acetonitrile; solid
phase=HSS T3 column; gradient=5-100% B in A over 10 minutes.
Compound XVIII: Sodium Salt of
2-(4-Hydroxy-3,5-dipentylphenyl)acetic Acid
[0158] The above compound was prepared as for Compound XVI, from
2-(3,5-dibromo-4-hydroxyphenyl)acetic acid. .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 6.87 (s, 2H), 3.33 (s, 2H), 2.55 (t, J=7.7 Hz,
4H), 1.53-1.61 (m, 4H), 1.31-1.37 (m, 8H), 0.90 (t, J=7.0 Hz, 6H);
LRMS (ESI negative): m/z 291.1 (100%, M-Na+); UPLC (System B): 6.8
min. UPLC System B: Mobile phase A=0.1% aqueous formic acid; mobile
phase B=0.1% formic acid in acetonitrile; solid phase=HSS T3
column; gradient=5-100% B in A over 10 minutes.
Compound XIX: Sodium Salt of 2-(3,5-Dihexyl-4-hydroxyphenyl)acetic
Acid
[0159] The above compound was prepared as for Compound XVI, from
2-(3,5-dibromo-4-hydroxyphenyl)acetic acid, and
(E)-hex-1-enylboronic acid pinacol ester. .sup.1H NMR (400 MHz,
CD.sub.3OD): .delta. 6.72 (d, J=2.0 Hz, 1H), 6.69 (d, J=2.0 Hz,
1H), 3.46 (s, 2H), 2.56 (t, J=7.6 Hz, 2H), 2.44 (t, J=7.5 Hz, 2H),
1.50-1.60 (m, 4H), 1.27-1.37 (m, 12H), 0.89 (t, J=6.6 Hz, 3H), 0.88
(t, J=6.8 Hz, 3H); LRMS (ESI negative): m/z 319.1 (100%, M-Na+);
UPLC (System B): 7.6 min. UPLC System B: Mobile phase A=0.1%
aqueous formic acid; mobile phase B=0.1% formic acid in
acetonitrile; solid phase=HSS T3 column; gradient=5-100% B in A
over 10 minutes.
Compound XX: Sodium Salt of 2-(4-Fluoro-3,5-dihexylphenyl)acetic
Acid
[0160] The above compound was prepared as for Compound XVI,
starting from 3,5-dibromo-4-fluorobenzyl bromide and
(E)-hex-1-enylboronic acid pinacol ester.
3,5-Dibromo-4-fluorobenzyl bromide was prepared by bromination of
3,5-dibromo-4-fluorotoluene with N-bromosuccinimide and
azobisisobutyronitrile in acetonitrile at 80.degree. C. .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 6.98 (d, J.sub.HF=7.0 Hz, 2H), 3.38
(s, 2H), 2.57 (t, J=7.7 Hz, 4H), 1.54-1.61 (m, 4H), 1.28-1.37 (m,
12H), 0.89 (t, J=6.7 Hz, 6H); 19F NMR (377 MHz, CD.sub.3OD):
.delta. -132.17 (d, J.sub.HF=6.6 Hz, 1F); 13C NMR (101 MHz,
CD.sub.3OD): .delta. 179.44, 158.11 (d, J.sub.CF=239.8 Hz), 133.26
(d, J.sub.CF=3.8 Hz), 128.73 (d, J.sub.CF=5.4 Hz), 128.56 (d,
J.sub.CF=16.9 Hz), 44.52, 31.69, 30.35 (d, J.sub.CF=1.5 Hz), 28.98,
28.97 (d, J.sub.CF=3.1 Hz), 22.51, 13.29; LRMS (ESI negative): m/z
321.0 (100%, M-Na+); UPLC (System B): 9.2 min. UPLC System B:
Mobile phase A=0.1% aqueous formic acid; mobile phase B=0.1% formic
acid in acetonitrile; solid phase=HSS T3 column; gradient=5-100% B
in A over 10 minutes.
Compound XXI: Sodium Salt of 2-(4-Fluoro-3,5-dipentylphenyl)acetic
Acid
[0161] The above compound was prepared as for Compound XVI,
starting from 3,5-dibromo-4-fluorobenzyl bromide. .sup.1H NMR (400
MHz, CD.sub.3OD): .delta. 6.98 (d, J.sub.HF=6.8 Hz, 2H), 3.37 (s,
2H), 2.57 (t, J=7.6 Hz, 4H), 1.54-1.62 (m, 4H), 1.28-1.37 (m, 8H),
0.90 (t, J=7.0 Hz, 6H); 19F NMR (377 MHz, CD.sub.3OD): .delta.
-132.34 (d, J.sub.HF=6.6 Hz, 1F); 13C NMR (101 MHz, CD.sub.3OD):
.delta. 179.41, 158.10 (d, J.sub.CF=239.8 Hz), 133.26 (d,
J.sub.CF=3.8 Hz), 128.72 (d, J.sub.CF=4.6 Hz), 128.56 (d,
J.sub.CF=16.9 Hz), 44.51, 31.54, 30.07, 28.92 (d, J.sub.CF=3.1 Hz),
22.38, 13.22; LRMS (ESI negative): m/z 293.0 (100%, M-Na+); UPLC
(System B): 8.4 min. UPLC System B: Mobile phase A=0.1% aqueous
formic acid; mobile phase B=0.1% formic acid in acetonitrile; solid
phase=HSS T3 column; gradient=5-100% B in A over 10 minutes.
Compound XXII: Sodium Salt of 2-(2-Benzyl-3,5-dipentylphenyl)acetic
Acid
[0162] The title compound was prepared as for Compound XIV, from
methyl 2-(2-benzyl-3,5-di((E)-pent-1-enyl)phenyl)acetate. The
latter was isolated as a side product (1.1% yield) from the
scale-up of Compound XIV. .sup.1H NMR (400 MHz, CD.sub.3OD):
.delta. 7.17 (dd, J=7.3, 7.3 Hz, 2H), 7.09 (dd, J=7.3, 7.3 Hz, 1H),
6.97-6.99 (m, 3H), 6.86 (d, J=1.8 Hz, 1H), 4.13 (s, 2H), 3.40 (s,
2H), 2.55 (t, J=7.7 Hz, 2H), 2.49 (t, J=7.8 Hz, 2H), 1.59-1.67 (m,
2H), 1.31-1.45 (m, 6H), 1.21-1.26 (m, 4H), 0.91 (t, J=7.0 Hz, 3H),
0.82 (t, J=7.0 Hz, 3H); .sup.13C NMR (101 MHz, CD.sub.3OD): .delta.
179.48, 141.46, 141.24, 140.47, 137.46, 133.70, 128.36, 128.05,
127.86, 127.75, 125.42, 43.25, 35.54, 33.90, 33.61, 31.86, 31.65,
31.25, 30.96, 22.49, 22.40, 13.31, 13.23; LRMS (ESI negative): m/z
365.0 (20%, M-Na.sup.+), 321.1 (100%, M-CO.sub.2Na); UPLC (System
B): 9 min. (UPLC System B: Mobile phase A=0.1% aqueous formic;
mobile phase B=0.1% formic in acetonitrile; solid phase=HSS T3;
gradient=5-100% B in A over 10 min.)
Compound XXIII: Sodium 2-[3,5-Di[(E)-Pent-1-enyl]phenyl]acetate
[0163] The title compound was prepared using the same procedure as
for Compound XIV, but with the omission of the hydrogenation step.
mp 226-30.degree. C.; .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.
7.18 (d, J=1.2 Hz, 2H), 7.11 (d, J=1.2 Hz, 1H), 6.34 (d, J=15.9 Hz,
2H), 2.23 (dt, J=15.9, 6.7 Hz, 2H), 3.44 (s, 2H), 2.14-2.19 (m,
4H), 1.49 (tq, J=7.4, 7.4 Hz, 4H), 0.95 (t, J=7.3 Hz, 6H); .sup.13C
NMR (101 MHz, CD.sub.3OD): .delta. 179.41, 138.34, 138.06, 130.30,
130.16, 125.26, 121.60, 45.24, 35.10, 22.55 & 12.98; LRMS
(negative mode): m/z 271 (w, [M-Na.sup.+]), 227.2 (100%,
[M-Na.sup.+-CO.sub.2]); UPLC: 8 min. (UPLC; Conditions solvent
A=0.1% formic acid in water; Solvent B=0.1% formic acid in
acetonitrile; Gradient: 5-100% B in A over 10 m in at 0.7
mL/min.)
Compound XXIV: Sodium 3-[3,5-Dipentylphenyl]propanoate
[0164] The title compound was prepared using the same procedure as
for Compound XIV starting from 3-[3,5-dibromophenyl]propanoic acid.
mp 211-217.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
6.73 (s, 1H), 6.68 (s, 2H), 2.73-2.77 (m, 2H), 2.42-2.46 (m, 2H),
2.38 (t, J=7.8 Hz, 4H), 1.43-1.51 (m, 4H), 1.19-1.28 (m, 8H), 0.83
(t, J=6.9 Hz, 6H); .sup.13C NMR (101 MHz, CDCl.sub.3): .delta.
182.55, 142.93, 141.85, 125.96, 125.77, 39.80, 36.13, 32.77, 31.99,
31.47, 22.79 & 14.27; LRMS (negative mode): m/z 289.4 (100%,
[M-Na.sup.+]); UPLC: 9 min. (UPLC: Conditions solvent A=0.1% formic
acid in water, solvent B=0.1% formic acid in acetonitrile,
Gradient: 5-100% B in A over 10 min at 0.7 mL/min.
Compound XXV: Sodium Salt of 2-Methyl-2-(3-pentylphenyl)propanoic
Acid
[0165] The tittle compound was prepared from methyl
2-[3-bromophenyl]acetate as for compound XIV, with the additional
step of alkylation of the methyl 2-[3-pentylphenyl]acetate
intermediate with sodium hydride and methyl iodide; and with the
temperature of the ester hydrolysis step being raised to 50.degree.
C. Off-white solid: 1H NMR (400 MHz, D2O): .delta. 7.11 (dd, J=7.7,
7.7 Hz, 1H), 7.07 (s, 1H), 7.02 (d, J=7.6 Hz, 1H), 6.95 (d, J=7.4
Hz, 1H), 2.44 (t, J=7.7 Hz, 2H), 1.43 (tt, J=7.4, 7.4 Hz, 2H), 1.28
(s, 6H), 1.09-1.17 (m, 4H), 0.68 (t, J=7.0 Hz, 3H); 13C NMR (101
MHz, D2O): .delta. 186.51, 148.17, 143.67, 128.48, 126.27, 126.24,
123.26, 48.67, 35.33, 30.90, 30.77, 27.20, 22.01, 13.46; LRMS
(ESI+ve): m/z 189.1 (100%, MH+-CO2Na); HPLC: 5 min (15-99%
acetonitrile in water over 5 min (trifluoroacetic acid in both
solvents).
Compound XXVI: Sodium Salt of (RS)-2-(3-Pentylphenyl)propanoic
Acid
##STR00076##
[0166] Step 1
[0167] A mixture of copper(I) iodide (17 mg, 0.09 mmol),
2-picolinic acid (22 mg, 0.18 mmol) and cesium carbonate (1.7 g,
5.30 mmol), under argon, was treated with anhydrous 1,4-dioxane (3
ml), diethyl malonate (0.54 ml, 3.5 mmol) and 1-bromo-3-iodobenzene
(0.23 ml, 1.77 mmol). The reaction was then heated at 70.degree.
C., under argon, for 15 h. The crude reaction mixture was
evaporated onto silica gel and purified on a SiliaSep SiO2 column,
eluting with ethyl acetate in hexanes (0-12%) to give diethyl
2-[3-bromophenyl]malonate (0.34 g, 64%). 1H NMR (400 MHz, CDCl3):
.delta. 7.30-7.47 (m, 3H), 7.20-7.26 (m, 1H), 4.16-4.24 (m, 4H),
3.36 (s, 1H), 1.23-1.29 (m, 6H).
Step 2
[0168] A suspension of sodium hydride (60% w/w; 0.53 g, 13.3 mmol)
in anhydrous THF (16 ml) was cooled to 0.degree. C. under argon,
and was treated with a solution of diethyl
2-[3-bromophenyl]malonate (3.0 g, 9.52 mmol) in anhydrous THF (20
ml). The reaction mixture was stirred at 0.degree. C. for 30 min,
and was then treated dropwise with methyl iodide (0.8 ml, 13.3
mmol). The reaction mixture was then warmed to room temperature,
and was stirred at room temperature, under argon, overnight. The
reaction was quenched with saturated aqueous ammonium chloride
solution (100 ml), and the mixture was extracted with ethyl acetate
(3.times.100 ml). The combined organic extracts were dried
(magnesium sulfate), and evaporated in vacuo to give the crude
compound. Purification on a SiliaSep SiO2 column, eluting with
ethyl acetate in hexanes (0-5%) gave diethyl
2-[3-bromophenyl]-2-methylmalonate (2.6 g, 82%). 1H NMR (400 MHz,
CDCl3): .delta. 7.52 (ddd, J=1.9, 1.9, 0.4 Hz, 1H), 7.43 (ddd,
J=7.9, 1.9, 1.0 Hz, 1H), 7.31 (ddd, J=8.0, 1.9, 1.0 Hz, 1H), 7.20
(ddd, J=7.9, 7.9, 0.4 Hz, 1H), 4.21-4.26 (m, 4H), 1.84 (s, 3H),
1.26 (t, J=7.2 Hz, 6H).
Step 3
[0169] Diethyl 2-[3-bromophenyl]-2-methylmalonate (2.6 g, 7.8 mmol)
was coupled with (E)-1-penten-1-ylboronic acid pinacol ester (2.1
g, 10.9 mmol) using the method described for compound X, Step 4, to
give diethyl (E)-2-methyl-2-[3-[pent-1-enyl]phenyl]malonate (1.7 g,
68%). 1H NMR (400 MHz, CDCl3): .delta. 7.24-7.32 (m, 3H), 7.21
(ddd, J=7.1, 1.9, 1.9 Hz, 1H), 6.37 (d, J=15.9 Hz, 1H), 6.20 (dt,
J=15.9, 6.9 Hz, 1H), 4.21-4.26 (m, 4H), 2.15-2.21 (m, 2H), 1.87 (s,
3H), 1.49 (tt, J=7.3, 7.3 Hz, 2H), 1.26 (t, J=7.2 Hz, 6H), 0.95 (t,
J=7.4 Hz, 3H).
Step 4
[0170] Diethyl (E)-2-methyl-2-[3-[pent-1-enyl]phenyl]malonate (1.4
g, 4.27 mmol) was hydrogenated using the method described for
compound I, Step 3, to give diethyl
2-methyl-2-[3-pentylphenyl]malonate (1.2 g, 91%). 1H NMR (400 MHz,
CDCl3): .delta. 7.24 (dd, J=7.3, 7.3 Hz, 1H), 7.16 (d, J=7.3 Hz,
1H), 7.15 (s, 1H), 7.10 (d, J=7.6 Hz, 1H), 4.20-4.25 (m, 4H), 2.59
(t, J=7.9 Hz, 2H), 1.85 (s, 3H), 1.49 (tt, J=7.6, 7.6 Hz, 2H),
1.28-1.34 (m, 4H), 1.25 (t, J=7.0 Hz, 6H), 0.88 (t, J=7.0 Hz,
3H).
Step 5
[0171] A solution of diethyl 2-methyl-2-[3-pentylphenyl]malonate
(1.1 g, 3.5 mmol) in acetonitrile (9 ml), methanol (3 ml) and water
(3 ml), was treated with lithium hydroxide (1.3 g, 52.8 mmol), and
the mixture was heated at 50.degree. C. for 48 h. The reaction
mixture was concentrated in vacuo, diluted with water (10 ml), and
then washed with dichloromethane (15 ml). The pH of the aqueous
phase was then adjusted to pH 4 with 1M aqueous hydrochloric acid,
and the mixture was extracted with dichloromethane (3.times.25 ml).
The combined organic extracts were dried (magnesium sulphate) and
evaporated in vacuo to give the crude compound. Purification on a
SiliaSep SiO2 column, eluting with ethyl acetate in hexanes (0-20%)
gave (RS)-2-[3-pentylphenyl]propanoic acid (0.4 g, 52%). 1H NMR
(400 MHz, CD3OD): .delta. 7.20 (dd, J=7.6, 7.6 Hz, 1H), 7.03-7.12
(m, 3H), 3.66 (q, J=7.1 Hz, 1H), 2.58 (t, J=7.8 Hz, 2H), 1.60 (tt,
J=7.6, 7.6 Hz, 2H), 1.42 (d, J=7.1 Hz, 3H), 1.27-1.38 (m, 4H), 0.90
(t, J=7.1 Hz, 3H).
Step 6
[0172] (RS)-2-[3-Pentylphenyl]propanoic acid (0.4 g, 1.8 mmol) was
converted to the sodium salt using the method described for
compound I, Step 5, to give sodium
(RS)-2-[3-pentylphenyl]propanoate (0.44 g, quantitative). 1H NMR
(400 MHz, CD3OD): .delta. 7.19 (s, 1H), 7.14-7.17 (m, 1H), 7.13
(dd, J=7.5, 7.5 Hz, 1H), 6.95 (d, J=6.9 Hz, 1H), 3.54 (q, J=7.1 Hz,
1H), 2.56 (t, J=7.8 Hz, 2H), 1.60 (tt, J=7.5, 7.5 Hz, 2H), 1.39 (d,
J=7.2 Hz, 3H), 1.29-1.35 (m, 4H), 0.90 (t, J=7.0 Hz, 3H); 13C NMR
(101 MHz, CD3OD): .delta. 182.18, 144.23, 142.49, 127.76, 127.55,
125.82, 124.73, 49.17, 35.85, 31.54, 31.33, 22.43, 18.95, 13.22;
HPLC: 5 min (15-99% acetonitrile in water over 5 min
(trifluoroacetic acid in both solvents).
Compound XXVII: Sodium Salt of 2-(2-Hydroxy-5-pentylphenyl)acetic
Acid
[0173] The above compound was prepared in the same manner as
compound VII, Steps 3-6, using methyl
2-[2-(benzyloxy)-5-bromophenyl]acetate (prepared in 2 steps from
2-[5-bromo-2-hydroxyphenyl]acetic acid. White solid: 1H NMR (400
MHz, CD3OD): .delta. 6.82-6.88 (m, 2H), 6.69 (d, J=8.6 Hz, 1H),
3.47 (s, 2H), 2.47 (t, J=7.7 Hz, 2H), 1.51-1.59 (m, 2H), 1.24-1.36
(m, 4H), 0.89 (t, J=7.0 Hz, 3H); 13C NMR (101 MHz, CD3OD): .delta.
180.04, 154.04, 134.05, 130.25, 127.36, 124.15, 116.57, 42.50,
34.90, 31.59, 31.42, 22.44, 13.23; LRMS (ESI-ve): m/z 221.1 (100%,
M-Na+), 177.1 (m, M-Na+-CO2); HPLC: 2 min (Gradient uses 70-99%
acetonitrile in water over 5 min and trifluoroacetic acid in both
solvents).
Compound XXVIII: Sodium Salt of 2-Oxo-2-[3-pentylphenyl]acetic
Acid
##STR00077##
[0174] Step 1:
[0175] i) A solution of methyl 2-[3-pentylphenyl]acetate (0.5 g,
2.0 mmol) in acetonitrile (15 ml), under nitrogen, was treated with
1,8-diazabicyclo[5.4.0]undec-7-ene (0.22 ml, 1.5 mmol) and the
reaction was stirred at room temperature for 15 min. The reaction
was cooled to 0.degree. C., and 4-acetamidobenzenesulfonyl azide
(0.6 g, 2.4 mmol) was added slowly. The reaction was then warmed to
room temperature, and was stirred, under nitrogen, for 22.5 h.
[0176] ii) This solution of the methyl
2-diazo-2-[3-pentylphenyl]acetate intermediate was diluted with
toluene (15 ml), acetone (11 ml), and water (15 ml), and was then
treated with sodium bicarbonate (6.4 g, 75.7 mmol). Oxone (12.1 g,
19.7 mmol) was added slowly, and the reaction mixture was then
stirred vigorously at room temperature for 25 min. The reaction was
diluted with water (30 ml), and then extracted with ethyl acetate
(3.times.30 ml). The combined extracts were washed with saturated
aqueous sodium chloride (30 ml), dried over sodium sulphate, and
evaporated in vacuo to give the crude product. Extraction with
dichloromethane and purification on a SiliaSep SiO2 column, eluting
with ethyl acetate in hexanes (0-2%) gave methyl
2-oxo-2-[3-pentylphenyl]acetate (0.13 g, 30%). 1H NMR (400 MHz,
CDCl3): .delta. 7.79-7.82 (m, 2H), 7.47 (d, J=7.6 Hz, 1H), 7.66
(dd, J=7.6, 7.6 Hz, 1H), 3.97 (s, 3H), 2.66 (d, J=7.8 Hz, 2H),
1.58-1.64 (m, 2H), 1.27-1.35 (m, 4H), 0.88 (t, J=6.9 Hz, 3H); 13C
NMR (101 MHz, CDCl3): .delta. 186.61, 164.48, 144.17, 135.53,
132.61, 129.88, 129.01, 127.97, 52.96, 35.87, 31.58, 31.18, 22.70,
14.22.
Step 2
[0177] Methyl 2-oxo-2-[3-pentylphenyl]acetate (64 mg, 0.8 mmol) was
hydrolysed as described for Compound IX, Step 5, to give
2-oxo-2-[3-pentylphenyl]acetic acid (60 mg, quant.). 1H NMR (400
MHz, CDCl3): .delta. 10.32 (br s, 1H), 7.98 (d, J=7.4 Hz, 1H), 7.96
(s, 1H), 7.43 (d, J=7.5 Hz, 1H), 7.36 (dd, J=7.4, 7.4 Hz, 1H),),
2.60 (d, J=7.7 Hz, 2H), 1.52-1.59 (m, 2H), 1.20-1.29 (m, 4H), 0.81
(t, J=6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3): .delta. 185.51,
164.18, 144.28, 136.10, 132.04, 130.81, 129.12, 128.85, 35.90,
31.59, 31.19, 22.71, 14.23.
Step 3
[0178] 2-Oxo-2-[3-pentylphenyl]acetic acid (57 mg, 0.3 mmol) was
converted to the sodium salt using the method described for
compound I, Step 5, to give sodium 2-oxo-2-[3-pentylphenyl]acetate
(51 mg, 95%). 1H NMR (400M Hz, CD3OD): .delta. 7.79-7.81 (m, 2H),
7.45 (ddd, J=7.6, 1.5, 1.5 Hz, 1H), 7.41 (ddd, J=7.8, 7.8, 1.0 Hz,
1H), 2.67 (t, J=7.6 Hz, 2H), 1.64 (tt, J=7.5, 7.5 Hz, 2H),
1.28-1.39 (m, 4H), 0.90 (t, J=7.1 Hz, 3H); 13C NMR (101 MHz,
CD3OD): .delta. 196.19, 172.77, 143.54, 133.89, 133.76, 129.34,
128.47, 127.03, 35.45, 31.32, 31.06, 22.38, 13.20; LRMS (ESI-ve):
m/z 219.1 (100%, M-Na+); HPLC: 3.3 min (Gradient uses 15-99%
acetonitrile in water over 5 min and trifluoroacetic acid in both
solvents).
Compound XXIX: Sodium Salt of
(E)-2-[2-Fluoro-5-[pent-1-enyl]phenyl]acetic Acid
[0179] The above compound was prepared from methyl
2-[2-fluoro-5-bromophenyl]acetate as for compound XIV, with the
omission of the hydrogenation step. White solid; 1H NMR (400 MHz,
CD3OD): .delta. 7.32 (dd, JHF=7.4 Hz, JHH=2.1 Hz, 1H), 7.15-7.18
(m, 1H), 6.92 (dd, JHF=9.4 Hz, JHH=8.8 Hz, 1H), 6.33 (d, J=15.8 Hz,
1H), 6.16 (dd, J=15.8, 7.0 Hz, 1H), 2.16 (td, J=7.1, 7.1 Hz, 2H),
1.48 (tt, J=7.3, 7.3 Hz, 2H), 0.95 (t, J=7.3 Hz, 3H); 19F NMR (377
MHz, CD3OD): .delta. -122.74 to -122.26 (m, 1F), 13C NMR (101 MHz,
CD3OD): .delta. 177.91, 160.51 (d, JCF=243.6 Hz), 134.08 (d,
JCF=3.8 Hz), 129.87 (d, JCF=1.5 Hz), 129.23, 128.94 (d, JCF=4.6
Hz), 125.09-125.26 (m, 2C), 114.63 (d, JCF=22.3 Hz), 37.75 (d,
JCF=1.5 Hz), 35.00, 22.50, 12.87; LRMS (ESI-ve): m/z 176.9 (100%,
M-Na+-CO2); HPLC: 6 min (UPLC Gradient: Mobile phase A=0.1% formic
acid in water; mobile phase B=0.1% formic acid in acetonitrile;
solid phase=HSS T3; gradient=5-100% B in A over 10 min).
Compound XXX: Sodium Salt of 2-[2-Benzyl-5-pentylphenyl]acetic
Acid
##STR00078##
[0180] Step 1
[0181] Compound XXVII (2.4 g, 10.0 mmol) was esterified in the same
manner as compound IX, Step 1, to give methyl
2-[2-hydroxy-5-pentylphenyl]acetate (2.3 g, 96%). 1H NMR (400 MHz,
CDCl3): .delta. 7.24 (br s, 1H), 6.98 (dd, J=8.2, 2.3 Hz, 1H), 6.90
(d, J=2.3 Hz, 1H), 6.83 (d, J=8.2 Hz, 1H), 3.73 (s, 3H), 3.65 (s,
2H), 2.50 (t, J=7.9 Hz, 2H), 1.52-1.60 (m, 2H), 1.25-1.36 (m, 4H),
0.86-0.90 (m, 3H).
Step 2
[0182] Methyl 2-[2-hydroxy-5-pentylphenyl]acetate (2.3 g, 9.6 mmol)
was converted to the trifluoromethanesulfonate-derivative as
described for Compound VII, Step 2, to give methyl
2-[5-pentyl-2-(trifluoromethylsulfonyloxy)phenyl]acetate (3.4 g,
97%). 1H NMR (400 MHz, CDCl3): .delta. 7.20 (d, J=8.6 Hz, 1H), 7.18
(d, J=2.4 Hz, 1H), 7.16 (dd, J=8.6, 2.4 Hz, 1H), 3.72 (s, 3H), 3.71
(s, 2H), 2.60 (t, J=7.8 Hz, 2H), 1.56-1.64 (m, 2H), 1.27-1.37 (m,
4H), 0.89 (t, J=6.9 Hz, 3H); 19F NMR (377 MHz, CDCl3): .delta.
-73.92 (s, 3F); 13C NMR (101 MHz, CDCl3): .delta. 170.59, 146.25,
143.76, 132.42, 129.30, 126.95, 121.31, 118.76 (q, J.sub.CF=319.8
Hz), 52.38, 35.70, 35.40, 31.62, 31.08, 22.66, 14.10.
Step 3
[0183] A nitrogen-flushed pressure vessel was charged sequentially
with tribasic potassium phosphate (5.4 g, 25.3 mmol), palladium(II)
acetate (74 mg, 0.33 mmol),
2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl (0.14 g, 0.33
mmol), a solution of methyl
2-[5-pentyl-2-(trifluoromethylsulfonyloxy)phenyl]acetate (3.1 g,
8.3 mmol) in anhydrous tetrahydrofuran (20 ml) and a 0.5M solution
of 9-benzyl-9-borabicyclo[3.3.1]nonane in tetrahydrofuran (34 ml,
17 mmol). The vessel was then sealed, and the reaction was heated
at 60.degree. C. After 17 h, the reaction mixture was cooled to
room temperature and was partitioned between ethyl acetate (300 ml)
and 0.5M aqueous sodium hydroxide (250 ml). The organic phase was
washed with saturated aqueous sodium chloride (200 ml), dried over
sodium sulphate, filtered and evaporated in vacuo to give the crude
compound. Purification on a SiliaSep SiO2 column, eluting with
ethyl acetate in hexanes (0-2%) gave methyl
2-[2-benzyl-5-pentylphenyl]acetate (2.5 g, 96%). 1H NMR (400 MHz,
CDCl3): .delta. 7.29 (dd, J=7.4, 7.0 Hz, 2H), 7.21 (dd, J=7.4, 7.0
Hz, 1H), 7.13-7.15 (m, 2H), 7.08-7.09 (m, 3H), 4.04 (s, 2H), 3.63
(s, 3H), 3.60 (s, 2H), 2.61 (t, J=7.8 Hz, 2H), 1.61-1.68 (m, 2H),
1.34-1.39 (m, 4H), 0.93 (t, J=7.1 Hz, 3H); 13C NMR (101 MHz,
CDCl3): .delta. 172.29, 141.66, 140.65, 136.61, 132.84, 131.21,
130.87, 129.03, 128.67, 127.83, 126.28, 52.19, 39.01, 39.00, 35.73,
31.89, 31.40, 22.84, 14.35.
Step 4
[0184] Methyl 2-[2-benzyl-5-pentylphenyl]acetate (2.9 g, 9.3 mmol)
was hydrolysed as described for Compound IX, Step 5, to give
2-[2-benzyl-5-pentylphenyl]acetic acid (2.48 g, 90%). 1H NMR (400
MHz, CDCl3): .delta. 7.26 (dd, J=7.3, 7.3 Hz, 2H), 7.16 (dd, J=7.5,
7.5 Hz, 1H), 7.10-7.13 (m, 2H), 7.05-7.07 (m, 3H), 4.01 (s, 2H),
3.58 (s, 2H), 2.58 (t, J=7.8 Hz, 2H), 1.57-1.65 (m, 2H), 1.30-1.37
(m, 4H), 0.90 (t, J=7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3): .delta.
178.67, 141.80, 140.51, 136.84, 132.20, 131.37, 130.95, 129.08,
128.75, 128.13, 136.39, 39.07, 38.98, 35.74, 31.93, 31.41, 22.87,
14.38.
Step 5
[0185] 2-[2-Benzyl-5-pentylphenyl]acetic acid (2.5 g, 8.4 mmol) was
converted to the sodium salt using the method described for
compound I, Step 5, to give sodium
2-[2-benzyl-5-pentylphenyl]acetate (2.5 g, 93%). 1H NMR (400 MHz,
CD3OD): .delta. 7.22 (dd, J=8.4, 7.4 Hz, 2H), 7.09-7.15 (m, 3H),
6.92-6.93 (m, 3H), 4.03 (s, 2H), 3.47 (s, 2H), 2.55 (t, J=7.8 Hz,
2H), 1.57-1.65 (m, 2H), 1.28-1.38 (m, 4H), 0.90 (t, J=7.0 Hz, 3H);
13C NMR (101 MHz, CD3OD): .delta. 179.25, 141.25, 140.60, 136.90,
136.48, 130.45, 129.78, 128.83, 128.13, 126.13, 125.64, 42.70,
38.49, 35.49, 31.64, 31.32, 22.51, 13.35; LRMS (ESI-ve): m/z 295.2
(40%, M-Na+), 251.2 (100%, M-Na+-CO2); HPLC: 5.0 min (Gradient uses
70-99% MeCN in water over 5 min and trifluoroacetic acid in both
solvents).
Compound XXXI: Sodium Salt of
2-(3,5-Di((E)-hex-1-enyl)phenyl)acetic Acid
[0186] The title compound was prepared in the same manner as
compound II, but with the omission of the hydrogenation step.
Off-white solid: 1H NMR (400 MHz, CD3OD): .delta. 7.17 (d, J=1.1
Hz, 2H), 7.10 (s, 1H), 6.33 (d, J=15.8 Hz, 2H), 6.22 (dt, J=15.8,
6.7 Hz, 2H), 3.44 (s, 2H), 2.16-2.21 (m, 4H), 1.34-1.46 (m, 8H),
0.93 (t, J=7.3 Hz, 6H); 13C NMR (101 MHz, CD3OD): .delta. 179.44,
138.34, 138.07, 130.37, 130.13, 125.27, 121.60, 45.26, 32.70,
31.67, 22.19, 13.27; LRMS (ESI negative mode): m/z 299.2 (m, M-Na+)
and 255.2 (100%, M-Na+-CO2); UPLC: 8.7 min. (UPLC conditions
solvent A=0.1% formic acid in water; mobile phase B=0.1% formic
acid in acetonitrile; solid phase=HSS T3; gradient=5-100% B in A
over 10 min)
Compound XXXII: Sodium Salt of
2-(2-Fluoro-3,5-dipentylphenyl)acetic Acid
##STR00079## ##STR00080##
[0187] Step 1:
[0188] Methyl 2-amino-3,5-dibromobenzoate (10.0 g, 32.4 mmol) was
coupled with (E)-1-penten-1-ylboronic acid pinacol ester (15.2 g,
77.7) using the method described for compound I to give methyl
2-amino-3,5-di[(E)-pent-1-enyl]benzoate (6.00 g, 64%). 1H NMR (400
MHz, CDCl3): .delta. 7.76 (d, J=2.2 Hz, 1H), 7.37 (d, J=2.2 Hz,
1H), 6.35 (d, J=15.4 Hz, 1H), 6.26 (d, J=15.8 Hz, 1H), 6.08 (dt,
J=15.6, 7.0 Hz, 1H), 6.06 (dt, J=15.8, 7.0 Hz, 1H), 5.5-6.5 (br s,
2H), 3.87 (s. 3H), 2.19-2.25 (m, 2H), 2.13-2.18 (m, 2H), 1.43-1.56
(m, 8H), 0.97 (t, J=7.3 Hz, 3H), 0.94 (t, J=7.3 Hz, 3H).
Step 2:
[0189] Methyl 2-amino-3,5-di[(E)-pent-1-enyl]benzoate (5.7 g, 19.9
mmol) was hydrogenated as described for compound I to give methyl
2-amino-3,5-dipentylbenzoate (5.50 g, 95%). 1H NMR (400 MHz,
CDCl3): .delta. 7.50 (d, J=2.2 Hz, 1H), 6.95 (d, J=2.2 Hz, 1H),
5.5-6.1 (br s, 2H), 3.79 (s. 3H), 2.40 (t, J=7.2 Hz, 4H), 1.45-1.58
(m, 4H), 1.20-1.32 (m, 8H), 0.84 (t, J=7.2 Hz, 3H), 0.82 (t, J=7.1
Hz, 3H).
Step 3:
[0190] Methyl 2-amino-3,5-dipentylbenzoate (4.5 g, 15.4 mmol) was
treated with aqueous tetrafluoroboric acid (5.5M, 3.7 ml, 20 mmol)
and aqueous hydrochloric acid (8.5M, 3.3 ml, 28 mmol). The mixture
was cooled to 0.degree. C., and was then treated dropwise with an
aqueous solution of sodium nitrite (2.1M, 8.8 ml, 18.5 mmol) over 2
minutes. After 60 minutes at 0.degree. C., the reaction mixture was
extracted with xylenes (30 ml). The xylenes extract was dried over
sodium sulfate, and was then heated from 60.degree. C. to
120.degree. C. over 55 minutes. Filtration and evaporation of
xylenes in vacuo gave the crude compound, which was purified on a
SiliaSep SiO2 column, eluting with ethyl acetate in hexanes (0-5%)
to give methyl 2-fluoro-3,5-dipentylbenzoate (3.1 g, 69%). 1H NMR
(400 MHz, CDCl3): .delta. 7.50 (dd, JHF=6.5 Hz, JHH=2.4 Hz, 1H),
7.15 (dd, JHF=6.5 Hz, JHH=2.4 Hz, 1H), 3.91 (s. 3H), 2.62 (td,
JHH=7.7 Hz, JHF=1.2 Hz, 2H), 2.56 (t, J=7.7 Hz, 2H), 1.55-1.63 (m,
4H), 1.26-1.37 (m, 8H), 0.89 (t, J=7.0 Hz, 6H); 19F NMR (377 MHz,
CDCl3): .delta. -121.31 (dd, JHF=6.6, 6.6 Hz, 1F).
Step 4:
[0191] A solution of methyl 2-fluoro-3,5-dipentylbenzoate (3.1 g,
10.6 mmol) in anhydrous tetrahydrofuran (60 ml) was cooled to
-78.degree. C., and was treated slowly with lithium aluminium
hydride (0.5 g, 13.8 mmol). The reaction mixture was stirred at
-78.degree. C. for 25 minutes, then at 0.degree. C. for 30 minutes.
The reaction was quenched by addition of ethyl acetate. The mixture
was washed with aqueous potassium sodium tartrate (1M, 100 ml), and
with saturated aqueous sodium chloride (100 ml); and was then dried
over sodium sulfate, filtered and evaporated in vacuo to give the
crude compound. Purification on a SiliaSep SiO2 column, eluting
with ethyl acetate in hexanes (3-20%) gave
2-fluoro-3,5-dipentylbenzyl alcohol (1.8 g, 65%). 1H NMR (400 MHz,
CDCl3): .delta. 7.02 (dd, JHF=6.8 Hz, JHH=2.3 Hz, 1H), 6.92 (dd,
JHF=7.1 Hz, JHH=2.4 Hz, 1H), 4.71 (s. 2H), 2.59 (td, JHH=7.6 Hz,
JHF=1.2 Hz, 2H), 2.54 (t, J=7.8 Hz, 2H), 1.73 (s, 1H), 1.54-1.62
(m, 4H), 1.25-1.36 (m, 8H), 0.894 (t, J=7.0 Hz, 3H), 0.890 (t,
J=7.1 Hz, 3H); 19F NMR (377 MHz, CDCl3): .delta. -131.25 (dd,
JHF=6.7, 6.6 Hz, 1F); 13C NMR (101 MHz, CDCl3): .delta. 157.41 (d,
JCF=242.9 Hz), 138.48 (d, JCF=4.3 Hz), 130.07 (d, JCF=5.4 Hz),
129.33 (d, JCF=16.2 Hz), 127.33 (d, JCF=15.6 Hz), 126.67 (d,
JCF=4.6 Hz), 59.84 (d, JCF=5.4 Hz), 35.50, 31.86, 31.77, 31.62,
30.21, 29.21 (d, JCF=2.4 Hz), 22.80, 22.74, 14.28 (2C).
Step 5:
[0192] A solution of 2-fluoro-3,5-dipentylbenzyl alcohol (1.4 g,
5.3 mmol) in anhydrous dichloromethane (35 ml) was cooled to
0.degree. C., and was treated dropwise with methanesulfonyl
chloride (0.5 ml, 5.8 mmol) over 10 minutes. The reaction was
stirred at 0.degree. C. for 20 minutes, and was then quenched by
addition of ice-cold water (35 ml). The organic phase was washed
with aqueous hydrochloric acid (1M, 35 ml), saturated aqueous
sodium bicarbonate (35 ml) and with saturated aqueous sodium
chloride (35 ml); and was then dried over sodium sulfate, filtered
and evaporated in vacuo to give the crude
2-fluoro-3,5-dipentylbenzyl methanesulfonate (1.7 g, 93%). This
material was used in the next step without purification. 1H NMR
(400 MHz, CDCl3): .delta. 7.02-7.05 (m, 2H), 5.26 (d, JHF=1.0 Hz,
2H), 2.98 (s. 3H), 2.52-2.63 (m, 2H), 2.54 (t, J=7.8 Hz, 2H),
1.54-1.62 (m, 4H), 1.27-1.37 (m, 8H), 0.892 (t, J=7.0 Hz, 3H),
0.888 (t, J=7.0 Hz, 3H).
Step 6:
[0193] The pH of a solution of sodium cyanide (0.4 g, 7.4 mmol) in
water (5 ml) was adjusted to pH 10 with 6M aqueous hydrochloric
acid. A solution of 2-fluoro-3,5-dipentylbenzyl methanesulfonate
(1.7 g, 4.9 mmol) in acetonitrile (25 ml) was then added, and the
reaction was heated at 60.degree. C. for 2 h. The reaction mixture
was concentrated to 15 ml in vacuo, and was extracted with ethyl
acetate (100 ml). The organic extract was washed with water (100
ml), and with saturated aqueous sodium chloride (100 ml); and was
then dried over sodium sulfate, filtered and evaporated in vacuo to
give the crude compound. Purification on a SiliaSep SiO2 column,
eluting with ethyl acetate in hexanes (1-10%) gave
2-[2-fluoro-3,5-dipentylphenyl]acetonitrile (0.7 g, 55%). 1H NMR
(400 MHz, CDCl3): .delta. 7.04 (dd, JHF=6.9 Hz, JHH=2.2 Hz, 1H),
6.96 (dd, JHF=7.1 Hz, JHH=2.2 Hz, 1H), 3.72 (s. 2H), 2.59 (td,
JHH=7.7 Hz, JHF=0.9 Hz, 2H), 2.55 (t, J=7.8 Hz, 2H), 1.54-1.62 (m,
4H), 1.27-1.37 (m, 8H), 0.90 (t, J=7.0 Hz, 6H); 19F NMR (377 MHz,
CDCl3): .delta. -131.25 (ddd, JHF=7.0, 7.0, 0.8 Hz, 1F); 13C NMR
(101 MHz, CDCl3): .delta. 157.02 (d, JCF=244.5 Hz), 139.16 (d,
JCF=4.7 Hz), 130.84 (d, JCF=4.6 Hz), 129.93 (d, JCF=16.1 Hz),
126.97 (d, JCF=3.1 Hz), 117.52, 116.79 (d, JCF=16.2 Hz), 35.38,
31.74, 31.66, 31.54, 30.06, 29.16 (d, JCF=2.4 Hz), 22.74, 22.68,
17.90 (d, JCF=6.1 Hz), 14.26, 14.23.
Step 7:
[0194] A mixture of 2-[2-fluoro-3,5-dipentylphenyl]acetonitrile
(0.7 g, 2.7 mmol), acetic acid (4 ml) and water (4 ml) was treated
dropwise with concentrated sulfuric acid (4 ml); and the mixture
was then heated at 125.degree. C. for 3.5 h. The reaction was
cooled to room temperature and was then quenched by addition of ice
(40 ml). The mixture was extracted with ethyl acetate (40 ml), and
the organic extract was then washed with saturated aqueous sodium
chloride (40 ml); dried over sodium sulfate, filtered and
evaporated in vacuo to give 2-[2-fluoro-3,5-dipentylphenyl]acetic
acid (537 mg, 67%). 1H NMR (400 MHz, CDCl3): .delta. 6.84 (dd,
JHF=7.0 Hz, JHH=2.3 Hz, 1H), 6.80 (dd, JHF=6.8 Hz, JHH=2.2 Hz, 1H),
3.59 (d, JHF=1.2 Hz, 2H), 2.52 (t, J=7.5 Hz, 2H), 2.45 (t, J=7.8
Hz, 2H), 1.46-1.55 (m, 4H), 1.20-1.30 (m, 8H), 0.80-0.84 (m,
6H).
Step 8:
[0195] 2-[2-Fluoro-3,5-dipentylphenyl]acetic acid (537 mg, 1.8
mmol) was converted to the sodium salt as described for compound I
to give sodium 2-[2-fluoro-3,5-dipentylphenyl]acetate (465 mg, 81%)
as a pale brown, sticky solid: 1H NMR (400 MHz, CD3OD): .delta.
6.94 (dd, JHF=6.9 Hz, JHH=2.2 Hz, 1H), 6.83 (dd, JHF=7.0 Hz,
JHH=2.3 Hz, 1H), 3.48 (d, JHF=1.1 Hz, 2H), 2.58 (t, J=7.6 Hz, 2H),
2.51 (t, J=7.6 Hz, 2H), 1.54-1.62 (m, 4H), 1.28-1.38 (m, 8H), 0.90
(t, J=7.0 Hz, 3H), 0.89 (t, J=7.0 Hz, 3H); 19F NMR (377 MHz,
CD3OD): .delta. -130.71 (dd, JHF=6.6, 6.6 Hz, 1F); 13C NMR (101
MHz, CD3OD): .delta. 178.31, 157.95 (d, JCF=240.6 Hz), 137.64 (d,
JCF=3.8 Hz), 128.72 (d, JCF=4.6 Hz), 128.42 (d, JCF=17.7 Hz),
128.21 (d, JCF=5.4 Hz), 124.50 (d, JCF=17.7 Hz), 37.94 (d, JCF=3.1
Hz), 35.05, 31.52, 31.45, 31.37, 30.00, 28.96 (d, JCF=2.3 Hz),
22.43, 22.38, 13.23, 13.21; LRMS (ESI negative mode): m/z 293 (w,
M-Na+) and 249.1 (100%, M-Na+-CO2); UPLC: 8.4 min (UPLC conditions
Mobile phase A=0.1% formic acid in water; mobile phase B=0.1%
formic acid in acetonitrile; solid phase=HSS T3; gradient=5-100% B
in A over 10 min.
Compound XXXIII: Sodium Salt of
2-(3,5-Dipentylphenyl)-2-methylpropanoic Acid
[0196] The above compound was prepared in the same manner as
compound I, with the additional step of alkylation of the methyl
2-[3,5-dipentylphenyl]acetate intermediate with sodium hydride and
methyl iodide; and with the temperature of the ester hydrolysis
step being raised to 100.degree. C. Off-white solid: .sup.1H NMR
(400 MHz, CD.sub.3OD): .delta. 7.04 (d, J=1.3 Hz, 2H), 6.76 (s,
1H), 2.54 (t, J=7.7 Hz, 4H), 1.55-1.63 (m, 4H), 1.46 (s, 6H),
1.27-1.38 (m, 8H), 0.90 (t, J=7.0 Hz, 6H); .sup.13C NMR (101 MHz,
CD.sub.3OD): .delta. 184.58, 148.51, 141.98, 125.57, 123.46, 36.02,
48.26, 31.59, 31.42, 27.57, 22.47, 13.29; LRMS (ESI negative mode):
m/z 303.1 (100%, M-Na.sup.+); UPLC: 8.9 min (UPLC conditions mobile
phase A=0.1% formic acid in water; mobile phase B=0.1% formic acid
in acetonitrile; solid phase=HSS T3; gradient=5-100% B in A over 10
min).
Example 2: Effect of Representative Compounds of Formula I on
Expression of Hepatocyte Growth Factor (HGF), for Tissue
Self-Repair, Regeneration and Anti-Aging
[0197] Experiments were undertaken to determine the effect of
compounds on hepatocyte growth factor expression in vitro normal
human dermal fibroblasts (NHDF) from adult donor (Clonetics
#CC-2511). NHDF were starved overnight in DMEM/F12+0.5% FBS and
treated with or without rhTGF-.beta.1 (10 ng/ml) and compound I
(500 .mu.M) for 24 h. RNA was isolated with miRNeasy.RTM. kit
(QIAGEN.RTM.), including on-column DNase digestion step. cDNA
synthesis was done (0.5 .mu.g RNA/reaction) using the RT.sup.2
First Strand kit (QIAGEN.RTM. #330401). Real-Time PCR was performed
as described in the RT.sup.2 Profiler PCR Array handbook on a
AB-7900HT real-time cycler. Real-Time PCR data was analyzed using
the .DELTA..DELTA.Ct method on the RT.sup.2 Profiler PCR Array Data
Analysis Web Portal. All Ct values >35 or non amplified were
changed to the cut-off value of 35. The housekeeping genes used for
normalization are GAPDH and RPLP0. The control group is TGF-.beta.1
treated cells.
[0198] As illustrated in FIG. 1, Compound I increases the
expression of HGF, growth factor associated with tissue repair,
regeneration and anti-aging. The following Table 2 shows that HGF
expression in NHDF cells (Untreated) is reduced by TGF-.beta.1
which is corrected or increased with representative Compounds of
formula I disclosed herein (Compound #).
TABLE-US-00002 TABLE 2 Compound Concentration HGF Relative Cells
(.mu.M) Quantitation Untreated -- 7.23 TGF-.beta.1 -- 1.00
TGF-.beta.1 + Compound I 500 4.23 TGF-.beta.1 + Compound XVIII 25
1.40 TGF-.beta.1 + Compound XXXIII 6 1.54 TGF-.beta.1 + Compound
XXXII 10 1.73 TGF-.beta.1 + Compound IV 500 3.80 TGF-.beta.1 +
Compound III 500 2.41 TGF-.beta.1 + Compound II 250 1.37
TGF-.beta.1 + Compound XII 500 2.47 TGF-.beta.1 + Compound V 100
2.73 TGF-.beta.1 + Compound VI 100 2.77 TGF-.beta.1 + Compound XIII
500 1.71 TGF-.beta.1 + Compound VII 500 2.66 TGF-.beta.1 + Compound
VIII 500 1.44 TGF-.beta.1 + Compound XI 250 3.38 TGF-.beta.1 +
Compound X 250 3.06
[0199] An experiment was undertaken to determine the effect of
compounds on the expression of regeneration markers. This
experiment was performed with NHDF (Normal Human Dermal
Fibroblasts) and human epithelial cells (renal tubular epithelial
cells, HK-2) involved in tissue regeneration after single, multiple
or constant injury. Injury was simulated by incubation of the cells
with TGF-.beta.1. NHDF was used as previously described and HK-2
human epithelial proximal tubule cells (ATCC #CRL-2190) were
starved overnight in DMEM/F12+0.2% FBS and treated with or without
rhTGF-.beta.1 (10 ng/ml) and compound I (500 .mu.M) for 24 h.
Results indicated that compound I brings the expression level of
the regeneration markers at a normal control level indicating a
self-repair mechanism of the injured cells. In NHDF (FIG. 2), LOX,
MMP13, PLAU (uPA), serpin E1, TIMP3 and ILK are all expressed at a
normal level, additionally in HK-2 cells (FIG. 3), LOX, MMP1, MMP2,
MMP9, MMP13, TIMP3 and PLAT (tPA) are also all expressed at a level
close to the normal level observed in healthy cells.
Example 3: Effect of Compound I on Endogenous Production of AAT and
Regeneration of Nerve Tissue
[0200] As mentioned above, AAT can induce nerve regeneration.
Through a qPCR-panel on NHDF (method described in Example 2),
Compound I has demonstrated an ability to increase AAT mRNA
expression (FIG. 4) in injured cells, indicating that Compound I
can increase nerve regeneration or other injured tissues. Compound
I is representative of the compounds of formula I disclosed herein.
Therefore, the compounds of formula I disclosed herein may increase
regeneration of nerves via the production of endogenous AAT at the
site of injury.
[0201] Headings are included herein for reference and to aid in
locating certain sections These headings are not intended to limit
the scope of the concepts described therein, and these concepts may
have applicability in other sections throughout the entire
specification Thus, the present invention is not intended to be
limited to the embodiments shown herein but is to be accorded the
widest scope consistent with the principles and novel features
disclosed herein.
[0202] The singular forms "a", "an" and "the" include corresponding
plural references unless the context clearly dictates
otherwise.
[0203] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, concentrations,
properties, and so forth used in the specification and claims are
to be understood as being modified in all instances by the term
"about". At the very least, each numerical parameter should at
least be construed in light of the number of reported significant
digits and by applying ordinary rounding techniques. Accordingly,
unless indicated to the contrary, the numerical parameters set
forth in the present specification and attached claims are
approximations that may vary depending upon the properties sought
to be obtained. Notwithstanding that the numerical ranges and
parameters setting forth the broad scope of the embodiments are
approximations, the numerical values set forth in the specific
examples are reported as precisely as possible. Any numerical
value, however, inherently contain certain errors resulting from
variations in experiments, testing measurements, statistical
analyses and such.
[0204] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the
present invention and scope of the appended claims.
* * * * *