U.S. patent application number 11/913729 was filed with the patent office on 2009-03-19 for use of the irritating principal oleocanthal in olive oil, as well as structurally and functionally similar compounds.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to Gary K. Beauchamp, Paul A.S. Breslin, Qiang Han, Russell S.J. Keast, Jianming Lin, Amos B. Smith, III.
Application Number | 20090076142 11/913729 |
Document ID | / |
Family ID | 37397244 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090076142 |
Kind Code |
A1 |
Han; Qiang ; et al. |
March 19, 2009 |
USE OF THE IRRITATING PRINCIPAL OLEOCANTHAL IN OLIVE OIL, AS WELL
AS STRUCTURALLY AND FUNCTIONALLY SIMILAR COMPOUNDS
Abstract
The invention provides methods of synthesizing the purified
enantiomers of oleocanthal. The invention further provides methods
of using oleocanthals in various formulations including, food
additives; pharmaceuticals; cosmetics; animal repellants; and
discovery tools for mammalian irritation receptor genes, gene
products, alleles, splice variants, alternate transcripts and the
like.
Inventors: |
Han; Qiang; (Levittown,
PA) ; Smith, III; Amos B.; (Merion, PA) ;
Beauchamp; Gary K.; (Philadelphia, PA) ; Breslin;
Paul A.S.; (Highland Park, NJ) ; Keast; Russell
S.J.; (Victoria, AU) ; Lin; Jianming; (West
Windsor, NJ) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR, 2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
The Trustees of the University of
Pennsylvania
Philadelphia
PA
Monell Chemical Sense Center
Philadelphia
PA
|
Family ID: |
37397244 |
Appl. No.: |
11/913729 |
Filed: |
May 9, 2006 |
PCT Filed: |
May 9, 2006 |
PCT NO: |
PCT/US06/17937 |
371 Date: |
November 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60679136 |
May 9, 2005 |
|
|
|
60703565 |
Jul 29, 2005 |
|
|
|
Current U.S.
Class: |
514/532 ;
426/534; 426/654; 435/184; 435/455; 435/6.13; 506/9; 560/61 |
Current CPC
Class: |
C07C 67/56 20130101;
A61P 11/06 20180101; A61P 21/00 20180101; A61P 1/00 20180101; A61P
1/02 20180101; C07C 67/56 20130101; A61P 27/02 20180101; C07C
69/738 20130101; C07C 69/738 20130101; A61P 29/00 20180101; C07C
69/738 20130101; A61P 31/02 20180101; A61P 39/06 20180101; A61P
3/10 20180101; A61K 31/222 20130101; C07C 67/58 20130101; A61P
17/06 20180101; A61P 17/00 20180101; A61P 11/00 20180101; A61P
35/00 20180101; A61P 25/00 20180101; A61P 9/10 20180101; A61P 1/04
20180101; A61P 21/04 20180101; A61P 9/00 20180101; A61P 11/04
20180101; A61P 31/04 20180101; C07C 67/313 20130101; A61P 27/16
20180101; C07C 67/313 20130101; A61P 43/00 20180101; A61P 13/12
20180101; A61P 7/06 20180101; A61P 25/28 20180101; A61P 31/00
20180101; A61P 17/02 20180101; A61P 25/06 20180101; C07C 67/58
20130101 |
Class at
Publication: |
514/532 ; 560/61;
435/184; 426/534; 426/654; 506/9; 435/6 |
International
Class: |
A61K 31/216 20060101
A61K031/216; C07C 69/612 20060101 C07C069/612; A23L 1/226 20060101
A23L001/226; C40B 30/04 20060101 C40B030/04; A61P 25/28 20060101
A61P025/28; A01P 17/00 20060101 A01P017/00; A61P 31/04 20060101
A61P031/04; C12Q 1/68 20060101 C12Q001/68; A23L 1/48 20060101
A23L001/48; C12N 9/99 20060101 C12N009/99 |
Claims
1. A method of synthesizing a purified (-) enantiomer of a compound
having the formula: ##STR00017## comprising (a) converting D-ribose
into a compound of Formula I with a strong acid in a suitable
solvent; (b) contacting the compound of Formula I with a
halogenation reagent followed by metal-halogen exchange induced
ring opening to yield a compound of Formula IIa; (c) contacting the
compound of Formula IIa with CH.sub.2.dbd.CH--MgBr in a suitable
solvent to yield a compound of Formula IIIa; (d) contacting the
compound of Formula IIIa with Grubbs catalyst in a suitable solvent
followed by oxidation to yield a compound of Formula IVa; (e)
contacting the compound of Formula IVa with hydrogen, palladium
catalyst in a suitable solvent to yield (-)-cyclopentanone (Formula
Va); (f) contacting the (-)-cyclopentanone with lithium
hexamethyldisilazide in a suitable solvent followed by
hexamethylphosphoramide (HMPA) dimethyl zinc and alkyl bromoacetate
to yield a compound of Formula VIa; (g) subjecting the compound of
Formula VIa to a Wittig ethylnation using ethyltriphenylphosphine
bromide at reduced temperature; (h) hydrolyzing the ester to yield
a compound of Formula VIIIa; (i) contacting the compound of Formula
VIIIa with 4-hydroxyphenethyl alcohol under conditions to perform
an esterification to yield a compound of Formula IXa; (j)
liberating the vicinal diol moiety; and (k) performing an oxidative
cleavage to yield (-)-oleocanthal (Formula Xa).
2. A method of synthesizing a purified (+) enantiomer of a compound
having the formula: ##STR00018## comprising: (a) converting
D-ribose into a compound of Formula XI with a strong acid in
acetone; (b) contacting the compound of Formula XI with a
methyltriphenylphosphine bromide, followed by oxidative cleavage to
yield a compound of Formula IIb; (c) contacting the compound of
Formula IIb with CH.sub.2.dbd.CH--MgBr in a suitable solvent to
yield a compound of Formula IIIb; (d) contacting the compound of
Formula IIIb with Grubbs catalyst, in a suitable solvent, followed
by oxidation to yield a compound of Formula IVb; (e) contacting the
compound of Formula IVb with hydrogen, palladium catalyst in a
suitable solvent to yield (+)-cyclopentanone (Formula Vb); (f)
contacting the (+)-cyclopentanone with lithium hexamethyldisilazide
in a suitable solvent followed by hexamethylphosphoramide (HMPA),
dimethyl zinc and methyl bromoacetate to yield a compound of
Formula VIb; (g) subjecting the compound of Formula VIb to a Wittig
ethylnation using ethyltriphenylphosphine bromide at reduced
temperature; (h) hydrolyzing the ester to yield a compound of
Formula VIIIb; (i) contacting the compound of Formula VIIIb with
4-hydroxyphenethyl alcohol under conditions to perform an
esterification to yield a compound of Formula IXb; (j) liberating
the vicinal diol moiety; and (k) performing an oxidative cleavage
to yield (+)-oleocanthal (Formula Xb).
3. A compound comprising the formula: ##STR00019## wherein: R.sub.1
and R.sub.4 are independently H or OR.sub.5 R.sub.2 and R.sub.3 are
independently CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5
alkyl, or a glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3,
or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5; wherein said compound is other than
oleocanthal.
4. A method of inhibiting COX-1, COX-2, COX-3 or lipoxygenase
comprising administering an effective amount of a compound having
the formula: ##STR00020## wherein: R.sub.1 and R.sub.4 are
independently H or OR.sub.5 R.sub.2 and R.sub.3 are independently
CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5 alkyl, or a
glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3, or
CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5.
5. The method of claim 4 wherein said compound is oleocanthal.
6. The method of claim 5 wherein said oleocanthal is the
(-)-enantiomer.
7. The method of claim 5 wherein said oleocanthal is the
(+)-enantiomer.
8. An anti-inflammatory composition comprising a therapeutically
effective amount of a compound having the formula: ##STR00021##
wherein: R.sub.1 and R.sub.4 are independently H or OR.sub.5
R.sub.2 and R.sub.3 are independently CHO, or COOR.sub.5 R.sub.5 is
a H, C.sub.1-C.sub.5 alkyl, or a glycoside X is O, NH or CH.sub.2 Y
is C.dbd.CHCH.sub.3, or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5
A is CH.sub.2, or CH--COOR.sub.5; and a pharmaceutically acceptable
carrier.
9. The composition of claim 8 wherein said compound is
oleocanthal.
10. The composition of claim 9 wherein said oleocanthal is the
(-)-enantiomer.
11. The method of claim 9 wherein said oleocanthal is the
(+)-enantiomer.
12. An antioxidant composition comprising a therapeutically
effective amount of a compound having the formula: ##STR00022##
wherein: R.sub.1 and R.sub.4 are independently H or OR.sub.5
R.sub.2 and R.sub.3 are independently CHO, or COOR.sub.5 R.sub.5 is
a H, C.sub.1-C.sub.5 alkyl, or a glycoside X is O, NH or CH.sub.2 Y
is C.dbd.CHCH.sub.3, or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5
A is CH.sub.2, or CH--COOR.sub.5; and a pharmaceutically acceptable
carrier.
13. The composition of claim 12 wherein said compound is
oleocanthal.
14. The composition of claim 13 wherein said oleocanthal is the
(-)-enantiomer.
15. The method of claim 13 wherein said oleocanthal is the
(+)-enantiomer.
16. A method of enhancing the flavor of food comprising adding an
effective amount of a compound of the formula: ##STR00023##
wherein: R.sub.1 and R.sub.4 are independently H or OR.sub.5
R.sub.2 and R.sub.3 are independently CHO, or COOR.sub.5 R.sub.5 is
a H, C.sub.1-C.sub.5 alkyl, or a glycoside X is O, NH or CH.sub.2 Y
is C.dbd.CHCH.sub.3, or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5
A is CH.sub.2, or CH--COOR.sub.5.
17. The method of claim 16 wherein said compound is
oleocanthal.
18. The method of claim 17 wherein said oleocanthal is the
(-)-enantiomer.
19. The method of claim 17 wherein said oleocanthal is the
(+)-enantiomer.
20. An animal repellent comprising an effective amount of a
compound of the formula: ##STR00024## wherein: R.sub.1 and R.sub.4
are independently H or OR.sub.5 R.sub.2 and R.sub.3 are
independently CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5
alkyl, or a glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3,
or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5.
21. The animal repellent of claim 20 wherein said compound is
oleocanthal.
22. The animal repellent of claim 21 wherein said oleocanthal is
the (-)-enantiomer.
23. The animal repellent of claim 21 wherein said oleocanthal is
the (+)-enantiomer.
24. The animal repellent of claim 20 wherein said repellent is in
the form of a propellent spray.
25. A method for treating a sore throat comprising administering to
a patient with a sore throat an effective amount of a composition
comprising a compound of the formula: ##STR00025## wherein: R.sub.1
and R.sub.4 are independently H or OR.sub.5 R.sub.2 and R.sub.3 are
independently CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5
alkyl, or a glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3,
or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5; and a pharmaceutically acceptable carrier.
26. The method of claim 25 wherein said compound is
oleocanthal.
27. The method of claim 26 wherein said oleocanthal is the
(-)-enantiomer.
28. The method of claim 26 wherein said oleocanthal is the
(+)-enantiomer.
29. The method of claim 25 wherein said composition is in the form
of a lozenge.
30. The method of claim 25 wherein said composition is in the form
of a spray.
31. A method of preserving food comprising contacting a food with
an effective amount of a compound of the formula: ##STR00026##
wherein: R.sub.1 and R.sub.4 are independently H or OR.sub.5
R.sub.2 and R.sub.3 are independently CHO, or COOR.sub.5 R.sub.5 is
a H, C.sub.1-C.sub.5 alkyl, or a glycoside X is O, NH or CH.sub.2 Y
is C.dbd.CHCH.sub.3, or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5
A is CH.sub.2, or CH--COOR.sub.5.
32. The method of claim 31 wherein said compound is
oleocanthal.
33. The method of claim 32 wherein said oleocanthal is the
(-)-enantiomer.
34. The method of claim 32 wherein said oleocanthal is the
(+)-enantiomer.
35. The method of claim 31 wherein said compound is incorporated
into a film.
36. The method of claim 31 wherein said compound is coated onto a
packaging material which is in contact with said food.
37. The method of claim 31 wherein said compound is added directly
to said food.
38. A method of repelling animals from an edible source comprising
adding to an edible source an effective amount of a compound
comprising the formula: ##STR00027## wherein: R.sub.1 and R.sub.4
are independently H or OR.sub.5 R.sub.2 and R.sub.3 are
independently CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5
alkyl, or a glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3,
or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5.
39. The method of claim 38 wherein said compound is
oleocanthal.
40. The method of claim 39 wherein said oleocanthal is the
(-)-enantiomer.
41. The method of claim 39 wherein said oleocanthal is the
(+)-enantiomer.
42. The method of claim 38 wherein said compound is added to an
edible source otherwise susceptible to consumption by birds.
43. The method of claim 38 wherein said compound is added to an
edible source otherwise susceptible to consumption by non-human
mammals.
44. The method of claim 38 wherein said compound is added to an
edible source which is toxic to animals upon consumption.
45. The method of claim 38 wherein said edible source is
antifreeze.
46. A method of inhibiting sweetness perception in an edible source
comprising adding to an edible source a sweetness inhibiting amount
of a compound of the formula: ##STR00028## wherein: R.sub.1 and are
independently H or OR.sub.5 R.sub.2 and R.sub.3 are independently
CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5 alkyl, or a
glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3, or
CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5.
47. The method of claim 46 wherein said compound is
oleocanthal.
48. The method of claim 47 wherein said oleocanthal is the
(-)-enantiomer.
49. The method of claim 47 wherein said oleocanthal is the
(+)-enantiomer.
50. A method of treating a cold comprising administering to a
patient in need of treatment a composition comprising an effective
amount of a compound of the formula: ##STR00029## wherein: R.sub.1
and R.sub.4 are independently H or OR.sub.5 R.sub.2 and R.sub.3 are
independently CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5
alkyl, or a glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3,
or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5.
51. The method of claim 50 wherein said compound is
oleocanthal.
52. The method of claim 51 wherein said oleocanthal is the
(-)-enantiomer.
53. The method of claim 51 wherein said oleocanthal is the
(+)-enantiomer.
54. The method of claim 50 wherein said compound is administered as
a nasal lavage or mist.
55. A method of treating a patient with an inflammatory disorder
comprising administering to a patient with an inflammatory disorder
an effective amount of a composition comprising a compound of the
formula: ##STR00030## wherein: R.sub.1 and R.sub.4 are
independently H or OR.sub.5 R.sub.2 and R.sub.3 are independently
CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5 alkyl, or a
glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3, or
CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5; wherein said composition alleviates inflammation in
said patient.
56. The method of claim 55 wherein said compound is
oleocanthal.
57. The method of claim 56 wherein said oleocanthal is the
(-)-enantiomer.
58. The method of claim 56 wherein said oleocanthal is the
(+)-enantiomer.
59. (canceled)
60. The method of claim 55 wherein said inflammatory disorder is
selected from the group consisting of psoriasis, cancer, asthma,
allergic rhinitis, respiratory distress syndrome, inflammatory
bowel disease, Chron's disease, gastritis, irritable bowel
syndrome, ulcerative colitis, migraine, periarteritis nodosa,
thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, type I
diabetes, myasthenia gravis, multiple sclerosis, sorcoidosis,
ischemic kidney disease, nephrotic syndrome, Bechet's syndrome,
polymyositis, gingivitis, conjunctivitis, vascular disease
myocardial ischemia, heart disease, and stroke.
61. A method of inhibiting growth of microorganisms comprising
contacting microorganisms with an effective amount of a composition
comprising a compound of the formula: ##STR00031## wherein: R.sub.1
and R.sub.4 are independently H or OR.sub.5 R.sub.2 and R.sub.3 are
independently CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5
alkyl, or a glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3,
or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5; wherein said composition inhibits the growth of
microorganisms.
62. The method of claim 61 wherein said compound is
oleocanthal.
63. The method of claim 62 wherein said oleocanthal is the
(-)-enantiomer.
64. The method of claim 62 wherein said oleocanthal is the
(+)-enantiomer.
65. The method of claim 61 wherein said composition is incorporated
into sutures or bandages.
66. The method of claim 61 wherein said composition is applied to a
wound.
67. A method for screening for genes associated with oleocanthal
sensitivity comprising contacting an expression library formed from
an oleocanthal-responsive cell with oleocanthal conjugated to a
detectable label; identifying an expression clone that binds said
oleocanthal; and sequencing the polynucleotide that encodes the
expression clone, thereby identifying a gene associated with
oleocanthal sensitivity.
68. A method of screening for candidate genes associated with
oleocanthal sensitivity comprising identifying differentially
expressed genes in oleocanthal-responsive cells, obtaining
sequences of said differentially expressed genes, comparing
sequences of said differentially expressed genes and correlating
similarity of said sequences to known that of taste receptors,
wherein high homology to a taste receptor thereby identifies a
candidate gene associated with oleocanthal sensitivity.
69. A purified (-) isomer of the formula: ##STR00032##
70. A purified (+) isomer of the formula: ##STR00033##
71. A method of treating pain in a patient comprising administering
to a patient an effective amount of a composition comprising a
compound of the formula: ##STR00034## wherein: R.sub.1 and R.sub.4
are independently H or OR.sub.5 R.sub.2 and R.sub.3 are
independently CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5
alkyl, or a glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3,
or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5; wherein said composition alleviates inflammation in
said patient.
72. The method of claim 71 wherein said compound is
oleocanthal.
73. The method of claim 72 wherein said oleocanthal is the
(-)-enantiomer.
74. The method of claim 72 wherein said oleocanthal is the
(+)-enantiomer.
75. A method of purifying oleocanthal comprising extracting olive
oil with 80:20 volume/volume methanol:water to obtain a phenolic
extract; pre-fractionating said phenolic extract on a C18 solid
phase extraction cartridge; separating oleocanthal by
reversed-phase HPLC in a weak gradient and selecting a fraction
containing throat irritating activity, thereby purifying
oleocanthal.
76. A method of preventing a neurodegenerative disorder comprising
administering to a patient an effective amount of a composition
comprising a compound of the formula: ##STR00035## wherein: R.sub.1
and R.sub.4 are independently H or OR.sub.5 R.sub.2 and R.sub.3 are
independently CHO, or COOR.sub.5 R.sub.5 is a H, C.sub.1-C.sub.5
alkyl, or a glycoside X is O, NH or CH.sub.2 Y is C.dbd.CHCH.sub.3,
or CH--COOR.sub.5 Z is C.dbd.O or CH--OR.sub.5 A is CH.sub.2, or
CH--COOR.sub.5.
77. The method of claim 76 wherein said compound is
oleocanthal.
78. The method of claim 77 wherein said oleocanthal is the
(-)-enantiomer.
79. The method of claim 77 wherein said oleocanthal is the
(+)-enantiomer.
80. The method of claim 76 wherein said composition prevents
production of A.beta.42 in said patient.
81. The method of claim 76 wherein said neurodegenerative disorder
is selected from the group consisting of Alzheimer's disease and
cognitive impairment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 60/679,136, filed May 9, 2005 and U.S. Provisional
Application No. 60/703,565, filed Jul. 29, 2005, the disclosures of
which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the active principal in olive oil,
termed oleocanthal, and methods of using oleocanthals in various
formulations including, food additives, pharmaceuticals, cosmetics,
animal repellants, and discovery tools for mammalian irritation
receptor genes, gene products, alleles, splice variants, alternate
transcripts and the like.
BACKGROUND OF THE INVENTION
[0003] Over forty years ago, Fisher and Griffin suggested that the
human oral cavity could be regarded as a pharmacological
preparation in situ. They proposed that the perceived bitterness
intensity of a compound reflects the compound's pharmacological
activity and potency. As support for this idea, they pointed out
that for several drugs, the active isomer was more bitter than the
inactive one. There is also a rough correlation between the bitter
potency of selected toxins and their LD.sub.50 values.
[0004] In addition to the quality and intensity of a sensation, the
perceived location may have pharmacological implications. Many
compounds when put in the oral cavity elicit irritation (e.g.,
burning, stinging, cooling) and, just as for bitter taste, the
irritation may serve as a signal of potential danger.
[0005] Some compounds with site-specific irritation have a
beneficial effect. A desirable attribute of many premium olive oils
is the distinctive irritation or pungency that is unusual because
it is almost exclusively perceived on the pharynx and not in the
mouth.
[0006] In 1993, Montedoro and co-workers reported the isolation of
a new class of phenolic compounds (1-4), including the dialdehydic
and aldehydic forms of ligstroside (5) and oleuropeine (6) from
virgin olive oils (Montedoro, G. et al. (1993) J. Agric. Food Chem.
41:2228-2234) (See FIG. 1 for structures). These phenolic compounds
comprise important minor constituents of virgin olive oils that
have been implicated in the organoleptic characteristics including
bitterness, pungency, and astringency (Andrewes, P. et al. (2003)
J. Agric. Food Chem. 57:1415-1420). In addition, these agents have
been suggested to contribute to the oxidative stability of virgin
olive oil and as such are associated with health benefits of olive
oils, specifically their antioxidant/anticancer activities (Owen,
R. W. et al. (2000) Food Chem. Toxicology 38:647-659; Owen, R. W.
et al. (2000) Eur. J. Cancer 36 (10):1235-1247; Baldioli, M. et al.
(1996) J. Am. Oil Chem. Soc. 73 (11):1589-1593; Manna, C. et al
(2002) J. Agric. Food Chem. 50 (22):6521-6526). Similar structural
features have been reported in the constituents of the Jasminum
(Somanadhan, B. et al. (1998) Planta Medica 64:246-50; Takenaka, Y.
et al. (2002) Chem. & Pharm. Bull 50 (3):384-389) and related
plant species (Takenaka, Y. et al. (2002) Phytochemistry 59
(7):779-787). It has been shown that both ibuprofen and a
Mediterranean diet (i.e., high in olive oil) both decrease the
risk/incidence for breast and lung cancer.
[0007] In 2003, Busch and co-workers at Unilever Research and
Development Vlaardingen (The Netherlands) identified
deacetoxydialdehydic ligstroside aglycone as a principal
contributor to the potent pungent (burning) sensation at the back
of throat associated with high quality virgin olive oils (Andrewes,
P. et al. (2003) J. Agric. Food Chem. 57:1415-1420). Studies at
Firmenich, Inc., reached the same conclusion (Firmenich, Inc.
study). The structure of 1 was assigned,
##STR00001##
employing a series of 1 and 2D NMR experiments (Andrewes, P. et al.
(2003) J. Agric. Food Chem. 57:1415-1420), in conjunction with
comparison to literature data (Montedoro, G. et al. (1993) J.
Agric. Food Chem. 41:2228-2234). The absolute stereochemistry
remained undetermined. That 1 was responsible for the strong
pungent (burning) sensation at the back of the throat was based on
an extensive series of HPLC fraction analysis, omission analysis
and correlation, and hydrolysis studies, in conjunction with human
sensory studies. Andrewes et al., however, acknowledged that "a
coelution compound causing the burning sensation" could not be
eliminated without completing a synthesis of 1, which they stated
to be "extremely challenging."
SUMMARY OF THE INVENTION
[0008] The invention provides the enantioselective total syntheses
of both enantiomers of oleocanthal 1 (FIG. 1), which not only
confirms the structure, but also permits the assignment of absolute
stereochemistry of the olive oil irritant. The synthesis provides
an effective route to both enantiomers for further
biological/sensory evaluation. Studies demonstrate that the
levorotary (-)-enantiomer of 1 (FIG. 1) is responsible for the
organoleptic properties experienced with premium olive oils at back
of the throat.
[0009] The invention therefore provides isolated and purified
deacetoxydialdehydic ligstroside aglycone, which we term
oleocanthal. The invention also provides functional derivatives of
oleocanthal having the general formula:
##STR00002##
[0010] wherein:
[0011] R.sub.1 and R.sub.4 are independently H or OR.sub.5
[0012] R.sub.2 and R.sub.3 are independently CHO, or COOR.sub.5
[0013] R.sub.5 is a H, C.sub.1-C.sub.5 alkyl, or a glycoside
[0014] X is O, NH or CH.sub.2
[0015] Y is C.dbd.CHCH.sub.3, or CH--COOR.sub.5
[0016] Z is C.dbd.O or CH--OR.sub.5
[0017] A is CH.sub.2, or CH--COOR.sub.5
[0018] The compounds of Formula I, including oleocanthal, are
collectively referred to herein as "oleocanthals." The Term
"oleocanthal" specifically refers to deacetoxydialdehydic
ligstroside aglycone.
[0019] The invention provides methods of synthesizing the purified
enantiomers of oleocanthal.
[0020] The invention further provides methods of using oleocanthals
in various formulations including, food additives (e.g., flavor
enhancers, sweetness inhibitors, spices, flavorings, and
preservatives); pharmaceuticals (e.g., antioxidants, micro-G
protein and associated kinase inhibitors, A.beta.42 inhibitors,
presenilin modifiers, .gamma.-secretase inhibitors, non-steroidal
anti-inflammatories, anti-pyretics, cold and flu symptom relievers,
COX-1, Cox-2 inhibitors, Cox-3 inhibitors, lipoxygenase inhibitors,
and wound healers); cosmetics; animal repellants; and discovery
tools for mammalian irritation receptor genes, gene products,
alleles, splice variants, alternate transcripts and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows phenolic compounds (1-4), including the
dialdehydic and aldehydic forms of ligstroside (5) and oleuropeine
(6).
[0022] FIG. 2 shows a graph of the irritation intensity of various
olive oils plotted against their concentrations of oleocanthal.
[0023] FIG. 3 shows the synthetic scheme of (-)-oleocanthal.
[0024] FIG. 4 shows the synthetic scheme of (+)-oleocanthal.
[0025] FIG. 5 shows the scheme of a Structure Activity Relationship
(SAR) Study.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0026] The reference works, patents, patent applications, and
scientific literature that are referred to herein establish the
knowledge of those with skill in the art and are hereby
incorporated by reference in their entirety to the same extent as
if each was specifically and individually indicated to be
incorporated by reference. Any conflict between any reference cited
herein and the specific teachings of this specification shall be
resolved in favor of the latter.
[0027] Various definitions are made throughout this document. Most
words have the meaning that would be attributed to those words by
one skilled in the art. Words specifically defined either below or
elsewhere in this document have the meaning provided in the context
of the present invention as a whole and as are typically understood
by those skilled in the art. Any conflict between an art-understood
definition of a word or phrase and a definition of the word or
phrase as specifically taught in this specification shall be
resolved in favor of the latter. Headings used herein are for
convenience and are not to be construed as limiting.
[0028] Standard reference works setting forth the general
principles of chemical synthesis are well known to those of skill
in the art and include, for example, A. I. Vogel, VOGEL'S TEXTBOOK
OF PRACTICAL ORGANIC CHEMISTRY (5.sup.TH EDITION) WILEY, N.Y. 1989;
and ORGANIC SYNTHESES. 9 collective volumes; Index for vol. 1-8;
Wiley, N.Y.
[0029] Standard reference works setting forth the general
principles of recombinant DNA technology known to those of skill in
the art include Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley & Sons, New York, 1998; Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED., Cold Spring Harbor
Laboratory Press, Plainview, N.Y., 1989; Kaufman et al., Eds.,
HANDBOOK OF MOLECULAR AND CELLULAR METHODS IN BIOLOGY AND MEDICINE,
CRC Press, Boca Raton, 1995; McPherson, Ed., DIRECTED MUTAGENESIS:
A PRACTICAL APPROACH, IRL Press, Oxford, 1991.
[0030] As used herein, "taste perception" refers to a response
(e.g., biochemical, behavioral) or sensitivity to a taste stimulus.
"Taste stimulus" as used herein refers to any compound that
elicits, for example at the biochemical level (e.g., activation or
inhibition of a taste receptor) or behavioral level (e.g.,
preference, indifference, or distaste), a taste response which
would be perceived by a mammal as at least one of the five taste
elements, including sweet, salty, sour, bitter, and umami. "Taste
perception" or "taste stimulus," or variants thereof, does not
require, though it does include, transmission of a neural signal
resulting in in vivo sensation of taste by a mammal. Modification
of taste perception includes an alteration of (enhancement of,
reduction to, or change to) a biochemical response, an ingestive
response, a taste preference, or general behavior of a mammal in
response to a compound.
[0031] "Acyl" refers to a straight or branched alkyl-C.dbd.O group.
"Thioacyl" refers to a straight or branched alkyl-C.dbd.S group.
Preferred acyl and thioacyl groups are lower alkanoyl and lower
thioalkanoyl having from 1 to about 6 carbon atoms in the alkyl
group, and all combinations and subcombinations of ranges
therein.
[0032] "Alkyl" refers to a saturated aliphatic hydrocarbon group
which may be straight or branched and having from 1 to about 20
carbon atoms in the chain, and all combinations and subcombinations
of ranges therein. Preferred alkyl groups may be straight or
branched and have from 1 to about 10 carbon atoms in the chain.
Branched means that a lower alkyl group such as, for example,
methyl, ethyl or propyl, is attached to a linear alkyl chain.
[0033] "Lower alkyl" refers to an alkyl group having from 1 to
about 6 carbons, and all combinations and subcombinations of ranges
therein.
[0034] "Cycloalkyl" refers to an aliphatic ring having from about 3
to about 10 carbon atoms in the ring, and all combinations and
subcombinations of ranges therein. Preferred cycloalkyl groups have
from about 4 to about 7 carbon atoms in the ring.
[0035] "Carbamoyl" refers to an H.sub.2N--C.dbd.O group.
Alkylcarbamoyl and dialkylcarbamoyl means that the nitrogen of the
carbamoyl is substituted by one or two alkyl groups,
respectively.
[0036] "Carboxyl" refers to a COOH group.
[0037] "Alkoxy" refers to an alkyl-O group in which "alkyl" is as
previously described. Lower alkoxy groups are preferred. Exemplary
alkoxy groups include, for example, methoxy, ethoxy, n-propoxy,
i-propoxy and n-butoxy.
[0038] "Alkoxyalkyl" refers to an alkyl group, as previously
described, substituted by an alkoxy group, as previously
described.
[0039] "Alkoxycarbonyl" refers to an alkoxy-C.dbd.O group.
[0040] "Aryl" refers to an aromatic carbocyclic radical containing
from about 6 to about 10 carbons, and all combinations and
subcombinations of ranges therein. Exemplary aryl groups include
phenyl and naphthyl.
[0041] "Aralkyl" means an alkyl group substituted by an aryl
radical. "Optionally substituted aralkyl" and "optionally
substituted aryl" means that the aryl group, or the aryl group of
the aralkyl group, may be substituted with one or more substituents
which include, for example, alkyl, alkoxy, amino, nitro, carboxy,
carboalkoxy, cyano, alkyl amino, halo, hydroxy, hydroxyalkyl,
mercaptyl, alkylmercaptyl, trihaloalkyl, carboxyalkyl or
carbamoyl.
[0042] "Aralkoxycarbonyl" refers to an aralkyl-O--C.dbd.O
group.
[0043] "Aryloxycarbonyl" refers to an aryl-O--C.dbd.O group.
[0044] "Carbalkoxy" refers to a carboxyl substituent esterified
with an alcohol of the formula C.sub.nH.sub.2n+1OH, wherein n is
from 1 to about 6.
[0045] "Halogen" (or "halo") refers to chlorine (chloro), fluorine
(fluoro), bromine (bromo) or iodine (iodo). Preferred among the
halogens (or halos) is chlorine (or chloro).
[0046] "Heterocyclyl" refers to a ring structure containing from
about 4 to about 10 members in which one or more of the atoms in
the ring is an element other than carbon, e.g., N, O or S.
Heterocyclyl groups may be aromatic or non-aromatic, i.e., the
rings may be saturated, partially unsaturated, or fully
unsaturated. Preferred heterocyclyl groups include, for example,
pyridyl, pyridazinyl, pyrimidinyl, isoquinolinyl, quinolinyl,
quinazolinyl, imidazolyl, pyrrolyl, furanyl, thienyl, thiazolyl,
benzothiazolyl, piperidinyl, pyrrolidinyl, tetrahydrofuranyl,
tetrahydropyranyl, and morphonlinyl groups.
[0047] "Optionally substituted heterocyclyl" means that the
heterocyclyl group may be substituted by one or more substituents
wherein the substituents include, for example, alkoxy, alkylamino,
aryl, carbalkoxy, carbamoyl, cyano, halo, heterocyclyl,
trihalomethyl, hydroxy, mercaptyl, alkylmercaptyl and nitro.
[0048] "Hydroxyalkyl" refers to an alkyl group substituted by a
hydroxy group. Hydroxy lower alkyl groups are preferred. Exemplary
preferred groups include, for example, hydroxymethyl,
2-hydroxyethyl, 2-hydroxypropyl and 3-hydroxypropyl.
[0049] "Hydrogenation catalyst" refers to any compounds known in
the art of organic synthesis to facilitate the addition of
hydrogen. Hydrogenation catalysts include, but are not limited to
palladium on carbon, palladium hydroxide on carbon, palladium on
calcium carbonate poisoned with lead, and platinum on carbon.
[0050] "Sulfonating agent" refers to any reagents known in the art
of organic synthesis to react with an alcohol to provide a
sulfonate ester. Examples include, but are not limited to
methanesulfonyl chloride, methanesulfonic anhydride,
trifluoromethane sulfonyl chloride, trifluoromethane sulfonic
anhydride, benzene sulfonyl chloride, p-toluenesulfonyl chloride, a
p-toluenesulfonyl anhydride. "Sulfonate ester" includes groups
which result when a sulfonating agent is reacted with an alcohol in
the presence of an acid scavenger to give a compound of form --OA,
wherein A is SO.sub.2R', with R' deriving from the sulfonating
agent.
[0051] "Reducing agent" refers to any reagents known in the art of
organic synthesis to reduce the oxidation state of a carbon atom,
for example, by reducing a ketone to an alcohol. Reducing agents
include, but are not limited to hydride derivatives, such as
borohydrides, including lithium borohydride and sodium
borohydrides.
[0052] "Methylating agent" refers to any reagent known in the art
of organic synthesis to donate a methyl group to an alcohol to form
an ether. Methylating agents include, but are not limited to
methylhalides such as methyliodide, methylchloride, methylbromide,
and dimethylsulfate.
[0053] "Acid scavenger" refers to any species known in the art of
organic synthesis capable of accepting a proton without reacting
with the starting material or product.
[0054] "Concatenated" refers to multi-step processes (i.e.,
processes containing two or more steps) wherein the steps may be
performed in a substantially continuous or sequential manner,
preferably without the necessity for interim isolation and/or
purification of the intermediate compounds.
[0055] "Pharmaceutically acceptable" refers to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem complications
commensurate with a reasonable benefit/risk ratio.
[0056] "Pharmaceutically acceptable salts" refer to derivatives of
the disclosed compounds wherein the parent compound is modified by
making acid or base salts thereof. Examples of pharmaceutically
acceptable salts include, but are not limited to, mineral or
organic acid salts of basic residues such as amines; alkali or
organic salts of acidic residues such as carboxylic acids; and the
like. Thus, the term "acid addition salt" refers to the
corresponding salt derivative of a parent compound which has been
prepared by the addition of an acid. The pharmaceutically
acceptable salts include the conventional non-toxic salts or the
quaternary ammonium salts of the parent compound formed, for
example, from non-toxic inorganic or organic acids. For example,
such conventional non-toxic salts include those derived from
inorganic acids such as hydrochloric, hydrobromic, sulfuric,
sulfamic, phosphoric, nitric and the like; and the salts prepared
from organic acids such as acetic, propionic, succinic, glycolic,
stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,
hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,
sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic, and the
like. Certain acidic or basic compounds may exist as zwitterions.
All forms of the compounds, including free acid, free base and
zwitterions, are contemplated to be within the scope of the present
invention.
[0057] The reactions of the synthetic methods described and claimed
herein may be carried out in suitable solvents which may be readily
selected by one skilled in the art of organic synthesis. Generally,
suitable solvents are solvents which are substantially non-reactive
with the starting materials (reactants), the intermediates, or
products at the temperatures at which the reactions are carried
out, i.e., temperatures which may range from the solvent's freezing
temperature to the solvent's boiling temperature. A given reaction
may be carried out in one solvent or a mixture of more than one
solvent. Depending on the particular reaction, suitable solvents
for a particular work-up following the reaction may be selected.
Suitable solvents, as used herein may include, by way of example
and without limitation, chlorinated solvents, hydrocarbon solvents,
aromatic solvents, ether solvents, protic solvents, polar aprotic
solvents, and mixtures thereof.
[0058] Suitable halogenated solvents include, but are not limited
to carbon tetrachloride, bromodichloromethane,
dibromochloromethane, bromoform, chloroform, bromochloromethane,
dibromomethane, butyl chloride, dichloromethane,
tetrachloroethylene, trichloroethylene, 1,1,1-trichloroethane,
1,1,2-trichloroethane, 1,1-dichloroethane, 2-chloropropane,
hexafluorobenzene, 1,2,4-trichlorobenzene, o-dichlorobenzene,
chlorobenzene, fluorobenzene, fluorotrichloromethane,
chlorotrifluoromethane, bromotrifluoromethane, carbon
tetrafluoride, dichlorofluoromethane, chlorodifluoromethane,
trifluoromethane, 1,2-dichlorotetrafluorethane and
hexafluoroethane.
[0059] Suitable hydrocarbon solvents include, but are not limited
to alkane or aromatic solvents such as cyclohexane, pentane,
hexane, toluene, cycloheptane, methylcyclohexane, heptane,
ethylbenzene, m-, o-, or p-xylene, octane, indane, nonane, benzene,
ethylbenzene, and m-, o-, or p-xylene.
[0060] Suitable ether solvents include, but are not limited to
dimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan,
diethyl ether, ethylene glycol dimethyl ether, ethylene glycol
diethyl ether, diethylene glycol dimethyl ether, diethylene glycol
diethyl ether, triethylene glycol diisopropyl ether, anisole, or
t-butyl methyl ether.
[0061] Suitable protic solvents include, but are not limited to
water, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol,
2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol,
2-methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butyl
alcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2-, or 3-pentanol,
neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl
ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl
alcohol, phenol, and glycerol.
[0062] Suitable aprotic solvents include, but are not limited to
dimethylformamide (DMF), dimethylacetamide (DMAC),
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),
1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP),
formamide, N-methylacetamide, N-methylformamide, acetonitrile
(ACN), dimethylsulfoxide (DMSO), propionitrile, ethyl formate,
methyl acetate, hexachloroacetone, acetone, ethyl methyl ketone,
ethyl acetate, isopropyl acetate, t-butyl acetate, sulfolane,
N,N-dimethylpropionamide, nitromethane, nitrobenzene, and
hexamethylphosphoramide.
[0063] The term "substantially pure form," as used herein, means
that the compounds prepared using the present processes may
preferably be substantially devoid of organic impurities. The term
"organic impurities," as used herein, refers to organic materials,
compounds, etc., other than the desired product, that may be
typically associated with synthetic organic chemical
transformations including, for example, unreacted starting
reagents, unreacted intermediate compounds, and the like. In
preferred form, the present processes may provide compounds that
are at least about 75% pure, as measured by standard analytical
techniques such as, for example, HPLC. Preferably, the compounds
prepared using the present processes may be at least about 80%
pure, with a purity of at least about 85% being more preferred.
Even more preferably, the compounds prepared using the present
processes may be at least about 90% pure, with a purity of at least
about 95% being more preferred. In particularly preferred
embodiments, the compounds prepared using the present processes may
be more than about 95% pure, with a purity of about 100% being
especially preferred.
[0064] Typically, substituted chemical moieties include one or more
substituents that replace hydrogen. Exemplary substituents include,
for example, halo (e.g., F, Cl, Br, I), alkyl, alkenyl, alkynyl,
aralkyl, aryl, heteroaryl, heterocyclyl, hydroxyl (OH), nitro
(NO.sub.2), nitrosyl (NO), cyano (CN), cyanato (CNO), thiocyanato
(SCN), amino (e.g., NH.sub.2, NHR', NR'.sub.2), azido (N.sub.3),
carboxyl (COOH), C(O)R', OR', C(O)OR', NHC(O)R', aminocarbonyl,
thiol, thiolato (SR'), sulfonic acid (SO.sub.3H), phosphonic acid
(PO.sub.3H), SO.sub.2R', phosphino (PH.sub.2, PHR', PR'.sub.2),
silyl (SiR'.sub.3, SiHR'.sub.2, SiH.sub.2R', SiH.sub.3) and the
like. In relation to the aforementioned substituents, each moiety
R' can be, independently, any of H, alkyl, aryl, aralkyl,
heteroaryl, or heterocyclyl, for example.
[0065] Processes of the present invention may yield mixtures of
diastereomers. Thus, in some embodiments, processes may, if
desired, include a separation step to isolate diastereomers.
Methods for separation of diastereomers are well known in the art
and include, for example, chiral column chromatography, HPLC,
re-crystallization, or classical resolution methods involving
selective reactivity. In some embodiments, asymmetric synthesis may
be used to produce a specific diastereomer.
[0066] As used herein "polynucleotide" refers to a nucleic acid
molecule and includes genomic DNA, cDNA, RNA, mRNA, mixed polymers,
recombinant nucleic acids, fragments and variants thereof, and the
like. Polynucleotide fragments of the invention comprise at least
10, and preferably at least 12, 14, 16, 18, 20, 25, 30, 35, 40, 45,
50, 75, or 100 consecutive nucleotides of a reference
polynucleotide. The polynucleotides include sense and antisense
strands. The polynucleotides may be naturally occurring or
non-naturally occurring polynucleotides. A "synthesized
polynucleotide" as used herein refers to polynucleotides produced
by purely chemical, as opposed to enzymatic, methods. "Wholly"
synthesized DNA sequences are therefore produced entirely by
chemical means, and "partially" synthesized DNAs embrace those
wherein only portions of the resulting DNA were produced by
chemical means. The polynucleotides of the invention may be single-
or double-stranded. The polynucleotides of the invention may be
chemically modified and may contain non-natural or derivatized
nucleotide bases as will be readily appreciated by those skilled in
the art. Such modifications include, for example, labels,
methylation, substitution of one or more nucleotides with an
analog, internucleotide modifications such as uncharged linkages
(e.g., methyl phosphonates, phosphotriesters, phosphoramidates,
carbamates, etc.), charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), pendent moieties (e.g., polypeptides,
etc.), intercalators (e.g., acridine, psoralen, etc.), chelators,
alkylators, and modified linkages (e.g., alpha anomeric nucleic
acids, etc.). Also included are synthetic molecules that mimic
polynucleotides in their ability to bind to a designated sequence
via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in
which peptide linkages substitute for phosphate linkages in the
backbone of the molecule.
[0067] "Recombinant nucleic acid" is a nucleic acid generated by
combination of two segments of nucleotide sequence. The combination
may be, for example, by chemical means or by genetic
engineering.
[0068] As used herein, "polynucleotide amplification" refers to a
broad range of techniques for increasing the number of copies of
specific polynucleotide sequences. Typically, amplification of
either or both strand(s) of the target nucleic acid comprises the
use of one or more nucleic acid-modifying enzymes, such as a DNA
polymerase, ligase, RNA polymerase, or RNA-dependent reverse
transcriptase. Examples of polynucleotide amplification include,
but are not limited to, polymerase chain reaction (PCR), nucleic
acid sequence based amplification (NASB), self-sustained sequence
replication (3SR), strand displacement activation (SDA), ligase
chain reaction, Q.beta. replicase system, and the like. A wide
variety of alternative cloning and in vitro amplification
methodologies are well known to those skilled in the art. Examples
of these techniques are found in, for example, Berger et al., Guide
to Molecular Cloning Techniques, METHODS IN ENZYMOLOGY 152,
Academic Press, Inc., San Diego, Calif. (Berger), which is
incorporated herein by reference in its entirety.
[0069] As used herein, the term "oligonucleotide" or "primer"
refers to a series of linked nucleotide residues which has a
sufficient number of bases to be used in a polymerase chain
reaction (PCR). This short sequence is based on (or designed from)
a genomic or cDNA sequence and is used to amplify, confirm, or
reveal the presence of an identical, similar, or complementary DNA
or RNA in a particular cell or tissue. Oligonucleotides comprise
portions of a nucleic acid sequence having at least about 10
nucleotides and as many as about 50 nucleotides, often about 12 or
15 to about 30 nucleotides. They are chemically synthesized and may
be used as probes. "Primer pair" refers to a set of primers
including a 5' upstream primer that hybridizes with the 5' end of a
target sequence to be amplified and a 3' downstream primer that
hybridizes with the complement of the 3' end of the target sequence
to be amplified.
[0070] As used herein, the term "probe" refers to nucleic acid
sequences of variable length, for example between at least about 10
and as many as about 8,500 nucleotides, depending on use. Probes
are used in the detection of identical, similar, or complementary
target nucleic acid sequences, which target sequences may be
single- or double-stranded. Longer probes are usually obtained from
a natural or recombinant source, are highly specific, and are much
slower to hybridize than oligomers, or shorter probes. They may be
single- or double-stranded and are carefully designed to have
specificity in PCR, hybridization membrane-based, or ELISA-like
technologies.
[0071] As used herein, the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which a probe, primer, or oligonucleotide will hybridize to its
target sequence, but to a minimal number of or no other sequences.
Stringent conditions are sequence-dependent and will be different
in different circumstances. Longer sequences will hybridize with
specificity to their proper complements at higher temperatures.
Generally, stringent conditions are selected to be about 5.degree.
C. lower than the thermal melting point (T.sub.m) for the specific
sequence at a defined ionic strength and pH. The T.sub.m is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present in excess, at
T.sub.m, 50% of the probes are hybridized to their complements at
equilibrium. Stringent temperature conditions will generally
include temperatures in excess of 30.degree. C., typically in
excess of 37.degree. C., and may be in excess of 45.degree. C.
Stringent salt conditions will ordinarily be less than 1.0 M,
typically less than 0.5 M, and may be less than 0.2 M. Typically,
stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is
at least about 30.degree. C. for short probes, primers, or
oligonucleotides (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for longer probes, primers, or oligonucleotides.
Stringent conditions may also be achieved with the addition of
destabilizing agents, such as formamide.
[0072] As used herein "antisense oligonucleotide" refers to a
nucleic acid molecule that is complementary to at least a portion
of a target nucleotide sequence of interest and specifically
hybridizes to the target nucleotide sequence under physiological
conditions. The term "double stranded RNA" or "dsRNA" as used
herein refers to a double-stranded RNA molecule capable of RNA
interference, including small interfering RNA (siRNA) (see for
example, Bass (2001) Nature 411:428-429; Elbashir et al. (2001)
Nature, 411:494-498).
[0073] As used herein, the term "complementary" refers to
Watson-Crick base pairing between nucleotide units of a nucleic
acid molecule.
[0074] The term "marker gene" or "reporter gene" refers to a gene
encoding a product that, when expressed, confers a phenotype at the
physical, morphologic, or biochemical level on a transformed cell
that is easily identifiable, either directly or indirectly, by
standard techniques and includes, but is not limited to, genes
encoding proteins that confer resistance to toxins or antibiotics
such as ampicillin, neomycin, and methotrexate; genes encoding
proteins that complement auxotrophic deficiencies; and genes
encoding proteins that supply critical components not available
from complex media. Examples of marker genes include green
fluorescent protein (GFP), red fluorescent protein (DsRed),
alkaline phosphatase (AP), .beta.-lactamase, chloramphenicol
acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside
phosphotransferase (NEOr, G418r) dihydrofolate reductase (DHFR),
hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ
(encoding .beta.-galactosidase), .beta.-lactamase, luciferase
(luc), and xanthine guanine phosphoribosyltransferase (XGPRT). As
with many of the standard procedures associated with the practice
of the invention, skilled artisans will be aware of additional
sequences that can serve the function of a marker or reporter.
Thus, this list is merely meant to show examples of what can be
used and is not meant to limit the invention.
[0075] As used herein, the term "promoter" refers to a regulatory
element that regulates, controls, or drives expression of a nucleic
acid molecule of interest and can be derived from sources such as
from adenovirus, SV40, parvoviruses, vaccinia virus,
cytomegalovirus, or mammalian genomic DNA. Examples of suitable
promoters include, but are not limited to, CMV, MSH2, trp, lac,
phage, and TRNA promoters. Suitable promoters that can be used in
yeast include, but are not limited to, such constitutive promoters
as 3-phosphoglycerate kinase and various other glycolytic enzyme
gene promoters such as enolase or glyceraldehydes-3-phosphate
dehydrogenase, or such inducible promoters as the alcohol
dehydrogenase 2 promoter or metallothionine promoter. Again, as
with many of the standard procedures associated with the practice
of the invention, skilled artisans will be aware of additional
promoters that can serve the function of directing the expression
of a marker or reporter. Thus, the list is merely meant to show
examples of what can be used and is not meant to limit the
invention.
[0076] "Operably linked" refers to juxtaposition wherein the
components are in a functional relationship. For example, a
promoter is operably linked or connected to a coding sequence if it
controls the transcription or expression of the sequence.
[0077] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein. "Polypeptide" refers to a polymer of amino
acids without referring to a specific length. Polypeptides of the
invention include peptide fragments, derivatives, and fusion
proteins. Peptide fragments preferably have at least about 10, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids.
Some peptide fragments of the invention are biologically active.
Biological activities include immunogenicity, ligand binding, and
activity associated with the reference peptide. Immunogenic
peptides and fragments of the invention generate an
epitope-specific immune response, wherein "epitope" refers to an
immunogenic determinant of a peptide and preferably contains at
least three, five, eight, nine, ten, fifteen, twenty, thirty,
forty, forty-five, or fifty amino acids. Some immunogenic peptides
of the invention generate an immune response specific to that
peptide. Polypeptides of the invention include naturally occurring
and non-naturally occurring peptides. The term includes modified
polypeptides (wherein examples of such modifications include
glycosylation, acetylation, phosphorylation, carboxylation,
ubiquitination, labeling, etc.), analogs (such as non-naturally
occurring amino acids, substituted linkages, etc.), and functional
mimetics. A variety of methods for labeling polypeptides are well
known in the art and include radioactive isotopes such as .sup.32P
or .sup.35S, ligands that bind to labeled antiligands (e.g.,
antibodies), fluorophores, chemiluminescent agents, enzymes, and
antiligands.
[0078] As used herein, the term "amino acid" denotes a molecule
containing both an amino group and a carboxyl group. In some
embodiments, the amino acids are .alpha.-, .beta., .gamma.- or
.delta.-amino acids, including their stereoisomers and racemates.
As used herein the term "L-amino acid" denotes an .alpha.-amino
acid having the L configuration around the .alpha.-carbon, that is,
a carboxylic acid of general formula CH(COOH)(NH.sub.2)-(side
chain), having the L-configuration. The term "D-amino acid"
similarly denotes a carboxylic acid of general formula
CH(COOH)(NH.sub.2)-(side chain), having the D-configuration around
the .alpha.-carbon. Side chains of L-amino acids include naturally
occurring and non-naturally occurring moieties. Non-naturally
occurring (i.e., unnatural) amino acid side chains are moieties
that are used in place of naturally occurring amino acid side
chains in, for example, amino acid analogs. Amino acid substituents
may be attached, for example, through their carbonyl groups through
the oxygen or carbonyl carbon thereof, or through their amino
groups, or through functionalities residing on their side chain
portions.
[0079] The amino acid sequences are presented in the amino (N) to
carboxy (C) direction, from left to right. The N-terminal
.alpha.-amino group and the C-terminal .beta.-carboxy groups are
not depicted in the sequence. The nucleotide sequences are
presented by single strands only, in the 5' to 3' direction, from
left to right. Nucleotides and amino acids are represented in the
manner recommended by the IUPAC-IUB Biochemical Nomenclature
Commission, or amino acids are represented by their three letters
code designations.
[0080] As used herein, the term "binding" means the physical or
chemical interaction between two proteins or compounds or
associated proteins or compounds or combinations thereof. Binding
includes ionic, non-ionic, Hydrogen bonds, Van der Waals,
hydrophobic interactions, etc. The physical interaction, the
binding, can be either direct or indirect, indirect being through
or due to the effects of another protein or compound. Direct
binding refers to interactions that do not take place through or
due to the effect of another protein or compound but instead are
without other substantial chemical intermediates. Binding may be
detected in many different manners. As a non-limiting example, the
physical binding interaction between two molecules can be detected
using a labeled compound. Other methods of detecting binding are
well-known to those of skill in the art.
[0081] As used herein, the term "contacting" means bringing
together, either directly or indirectly, a compound into physical
proximity to a molecule of interest. Contacting may occur, for
example, in any number of buffers, salts, solutions, or in a cell
or cell extract.
[0082] As used herein, the terms "modulates" or "modifies" means an
increase or decrease in the amount, quality, or effect of a
particular activity or protein. "Modulators" refer to any
inhibitory or activating molecules identified using in vitro and in
vivo assays for, e.g., agonists, antagonists, and their homologues,
including fragments, variants, and mimetics, as defined herein,
that exert substantially the same biological activity as the
molecule. "Inhibitors" or "antagonists" are modulating compounds
that reduce, decrease, block, prevent, delay activation,
inactivate, desensitize, or downregulate the biological activity or
expression of a molecule or pathway of interest. "Inducers,"
"activators," or "agonists" are modulating compounds that increase,
induce, stimulate, open, activate, facilitate, enhance activation,
sensitize, or upregulate a molecule or pathway of interest. In some
preferred embodiments of the invention, the level of inhibition or
upregulation of the expression or biological activity of a molecule
or pathway of interest refers to a decrease (inhibition or
downregulation) or increase (upregulation) of greater than about
50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99%. The inhibition or upregulation may be direct,
i.e., operate on the molecule or pathway of interest itself, or
indirect, i.e., operate on a molecule or pathway that affects the
molecule or pathway of interest.
[0083] A "purified" or "substantially purified" polynucleotide or
polypeptide is substantially separated from other cellular
components that naturally accompany a native (or wild-type) nucleic
acid or polypeptide and/or from other impurities (e.g., agarose
gel). A purified polypeptide or protein will comprise about 60% to
more than 99% w/w of a sample, and may be about 90%, about 95%, or
about 98% pure. As used herein, the term "isolated" refers to a
molecule that has been removed from its native environment.
Examples of isolated nucleic acid molecules include, but are not
limited to, recombinant DNA molecules contained in a vector,
recombinant DNA molecules maintained in a heterologous host cell,
partially or substantially purified nucleic acid molecules, and
synthetic DNA or RNA molecules.
[0084] "About" as used herein refers to +/-10% of the reference
value.
[0085] As used herein, "variant" nucleotide or amino acid sequences
refer to homologues, including, for example, isoforms, species
variants, allelic variants, and fragments of the sequence of
interest. "Homologous nucleotide sequence" or "homologous amino
acid sequence," or variations thereof, refers to sequences
characterized by a relative identity, at the nucleotide level with
respect to a reference sequence, or homology at the amino acid
level, of at least about 60%, at least about 70%, at least about
75%, at least about 80%, at least about 81%, at least about 82%, at
least about 83%, at least about 84%, at least about 85%, preferably
at least about 90%, at least about 95%, at least about 98%, or at
least about 99%, and more preferably 100%, to a reference sequence,
or portion or fragment thereof encoding or having a functional
domain.
[0086] As is well known in the art, because of the degeneracy of
the genetic code, there are numerous DNA and RNA molecules that can
code for the same polypeptide as that encoded by a nucleotide
sequence of interest. The present invention, therefore,
contemplates those other DNA and RNA molecules which, on
expression, encode a polypeptide encoded by the nucleic acid
molecule of interest. DNA and RNA molecules other than those
specifically disclosed herein characterized simply by a change in a
codon for a particular amino acid, are within the scope of this
invention.
[0087] Amino acid "insertions," "substitutions" or "deletions" are
changes to or within an amino acid sequence. The variation allowed
in a particular amino acid sequence may be experimentally
determined by producing the peptide synthetically or by
systematically making insertions, deletions, or substitutions of
nucleotides in the nucleic acid sequence using recombinant DNA
techniques. Alterations of the naturally occurring amino acid
sequence can be accomplished by any of a number of known
techniques. For example, mutations can be introduced into the
polynucleotide encoding a polypeptide at particular locations by
procedures well known to the skilled artisan, such as
oligonucleotide-directed mutagenesis.
[0088] A chemical variant of the present invention may exhibit
substantially the biological activity of a naturally occurring
oleocanthal, or have improved activity. "Biological activity" as
used herein refers to the level of a particular function (for
example, antioxidant activity, anti-inflammatory activity, etc.) of
a molecule or pathway of interest in a biological system.
"Wild-type biological activity" refers to the normal level of
function of a molecule or pathway of interest. "Reduced biological
activity" refers to a decreased level of function of a molecule or
pathway of interest relative to a reference level of biological
activity of that molecule or pathway. "Increased biological
activity" refers to an increased level of function of a molecule or
pathway of interest relative to a reference level of biological
activity of that molecule or pathway. For example, increased
biological activity may refer to an increased level of biological
activity relative to the wild-type biological activity of a
molecule or pathway of interest. Reference to exhibiting
"substantially the biological activity of naturally-occurring
oleocanthal" indicates that variants within the scope of the
invention can comprise substitutions, meaning that one or more
chemical moieties of oleocanthal are replaced by different chemical
moieties and such compounds retain the biological activity of
oleocanthal, have substantially the same biological activities of
oleocanthal, or have improved biological activity as compared to
naturally-occurring oleocanthal.
[0089] A nucleotide and/or amino acid sequence of a nucleic acid
molecule or polypeptide identified by the screening method of the
invention may be used to search a nucleotide and amino acid
sequence databank for regions of similarity using Gapped BLAST
(Altschul, et al. (1997) Nucl. Acids Res. 25:3389). Briefly, the
BLAST algorithm, which stands for Basic Local Alignment Search Tool
is suitable for determining sequence similarity (Altschul, et al.
(1990) J. Mol. Biol. 215:403-410). Software or performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul, et al. (1990) J.
Mol. Biol. 215:403-410). These initial neighborhood word hits act
as seeds for initiating searches to find HSPs containing them. The
word hits are extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Extension for the word hits in each direction are halted when: (1)
the cumulative alignment score falls off by the quantity X from its
maximum achieved value; (2) the cumulative score goes to zero or
below, due to the accumulation of one or more negative-scoring
residue alignments; or (3) the end of either sequence is reached.
The BLAST algorithm parameters W, T, and X determine the
sensitivity and speed of the alignment. The BLAST program uses as
defaults a word length (W) of 11, the BLOSUM62 scoring matrix
(Henikoff, et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919)
alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a
comparison of both strands. The BLAST algorithm (Karlin, et al.
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877) and Gapped BLAST
perform a statistical analysis of the similarity between two
sequences. One measure of similarity provided by the BLAST
algorithm is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. For
example, a nucleic acid is considered similar to a gene or cDNA if
the smallest sum probability in comparison of the test nucleic acid
to the reference nucleic acid is less than about 1, preferably less
than about 0.1, more preferably less than about 0.01, and most
preferably less than about 0.001.
[0090] The term "mimetic" as used herein refers to a compound that
is sterically similar to a reference compound. Mimetics are
structural and functional equivalents to the reference
compounds.
[0091] The terms "patient" and "subject" are used interchangeably
herein and include, but are not limited to amphibians, birds, dogs,
cats, cattle, horses, buffalo, llama, sheep, goats, pigs, rodents,
monkeys, apes, and humans. "Host cell" includes, for example,
prokaryotic cells, such as bacterial cells; eukaryotic cells, such
as yeast cells and animal cells, including, but not limited to
invertebrate cells (e.g., insect cells and nematode cells),
amphibian cells (e.g., frog cells), particularly mammalian cells
(e.g., human, rodent, canine, feline, caprine, ovine, bovine,
equine, porcine, simian); or plant cells. "Rodents" include, for
example, rats and mice. Mammalian cell lines available as hosts for
expression are known in the art and include many immortalized cell
lines available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells
(COS), N1E-115 (Liles et al., (1986) J. Biol. Chem. 261:5307-5313),
PC 12 human hepatocellular carcinoma cells (e.g., Hep G2).
[0092] The term "treatment" as used herein refers to any indicia of
success of prevention, treatment, or amelioration of a disease or
condition. Treatment includes any objective or subjective
parameter, such as, but not limited to, abatement, remission,
normalization of receptor activity, reduction in the number or
severity of symptoms or side effects, or slowing of the rate of
degeneration or decline of the patient. Treatment also includes a
prevention of the onset of symptoms in a patient that may be at
increased risk for or is suspected of having a disease or condition
but does not yet experience or exhibit symptoms thereof.
[0093] As used herein, the term "compound" means any identifiable
chemical or molecule, including, but not limited to a small
molecule, peptide, protein, sugar, nucleotide, or nucleic acid.
Such compound can be natural or synthetic.
[0094] As used herein, "bitter" refers to a basic taste
characterized by solutions of such compounds as quinine, caffeine,
and certain other alkaloids, that are sensed in humans primarily by
taste buds at the back of the tongue, which are perceived as acrid,
sharp, pungent, or harsh.
[0095] As used herein, "sweet" refers to a basic taste
characterized by solutions of sugars (e.g., sucrose and glucose),
alcohols, glycols, some small molecules and some amino acids that
are sensed in humans primarily by taste buds on the tip of the
tongue, which are perceived as agreeable or pleasing.
[0096] As used herein, "sour" refers to a basic taste characterized
by solutions of vinegar and the juices of most unripe fruits and
having a acid or sharp, tart, or biting taste.
[0097] Oleocanthals have the general formula:
##STR00003##
[0098] wherein:
[0099] R.sub.1 and R.sub.4 are independently H or OR.sub.5
[0100] R.sub.2 and R.sub.3 are independently CHO, or COOR.sub.5
[0101] R.sub.5 is a H, C.sub.1-C.sub.5 alkyl, or a glycoside
[0102] X is O, NH or CH.sub.2
[0103] Y is C.dbd.CHCH.sub.3, or CH--COOR.sub.5
[0104] Z is C.dbd.O or CH--OR.sub.5
[0105] A is CH.sub.2, or CH--COOR.sub.5
[0106] "Oleocanthal" is specifically deacetoxydialdehydic
ligstroside aglycone, which exists as a single isomer (enantiomer).
The (-)-enantiomer is the natural product and has the following
chemical formula:
##STR00004##
[0107] The enantiomers of oleocanthal may be synthesized and
purified by the following methods:
##STR00005## ##STR00006##
D-ribose may be converted to Formula I with a strong acid (e.g.,
hydrochloric acid) in acetone and methanol to yield Formula Ia. The
compound of Formula Ia may be treated with a halogenation reagent
(e.g., iodine), phosphine (PPh.sub.3) imidazole followed by metal
halogen exchange (e.g., BuLi or Zn) induced ring opening to yield
an aldehyde of Formula IIa. Thereafter the compound of Formula IIa
may be contacted with a CH.sub.2.dbd.CH--MgBr in a suitable solvent
(e.g., tetrahydrofuran) to yield a compound of Formula IIIa which
is converted to a compound of Formula IVa by treatment with Grubbs
catalyst in a suitable solvent (e.g., dichloromethane (DCM))
followed by treatment with an oxidizing reagent (e.g., pyridinium
chlorochromate (PCC)). The compound of Formula IVa is contacted
with hydrogen, palladium in a suitable solvent (e.g., ethyl acetate
(EtOAc)) to yield (-)-cyclopentanone (Formula Va). The
(-)-cyclopentanone (Formula Va) is treated with lithium
hexamethyldisilazide (LHMDS) followed by hexamethylphosphoramide
(HMPA), dimethyl zinc and allyl bromoacetate (e.g., methyl, ethyl,
tert-butyl) to yield (-)-(3,4-dimethoxy-2-oxo-cyclopentyl)-acetic
acid ester (Formula VIa). The compound of Formula VIa is subjected
to a Wittig ethylnation using ethyltriphenylphosphine bromide (or
iodide) at reduced temperature, preferably -40.degree. C. or less.
The ester is hydrolyzed (Formula VIIIa) and the compound of formula
VIIIa is contacted with 4-hydroxyphenethyl alcohol in the presence
of phosphine, dialkyl azodicarboxylate (e.g., diethyl or
diisopropyl)(DEAD or DIAD) to give Formula IXa. The vicinal diol
moiety is liberated and oxidative cleavage yields the
(-)-oleocanthal (Formula Xa). See also FIG. 3.
##STR00007## ##STR00008##
D-ribose may be converted to Formula XI with a strong acid (e.g.,
hydrochloric acid) in acetone to yield Formula XI. The compound of
Formula XI may be treated with methyltriphenylphosphine bromide (or
iodide) followed by oxidative cleavage of the diol to yield a
compound of Formula IIb. Thereafter the compound of Formula IIb may
be contacted with a CH.sub.2.dbd.CH--MgBr in a suitable solvent
(e.g., tetrahydrofuran) to yield a compound of Formula IIIb which
is converted to a compound of Formula IVb by treatment with Grubbs
catalyst in a suitable solvent (e.g., dichloromethane (DCM))
followed by treatment with an oxidizing reagent (e.g., pyridinium
chlorochromate) (PCC) or MnO.sub.2). The compound of Formula IVb is
contacted with hydrogen, catalyst in a suitable solvent (e.g.,
ethyl acetate (EtOAc)) to yield (+)-cyclopentanone (Formula Vb).
The (+)-cyclopentanone (Formula Vb) is treated with lithium
hexamethyldisilazide (LHMDS) followed by hexamethyl phosphoramide
(HMPA), dimethyl zinc and alkyl bromoacetate (eg., methyl, ethyl,
tert-butyl) to yield (+)-(3,4-dimethoxy-2-oxo-cyclopentyl)-acetic
acid ester (Formula VIb). The compound of Formula VIb is subjected
to a Wittig ethylnation using ethyltriphenylphosphine bromide (or
iodide) at reduced temperature, preferably -40.degree. C. or less.
The ester is hydrolyzed (Formula VIIIb) and the compound of formula
VIIIb is contacted with 4-hydroxyphenethyl alcohol in the presence
of phosphine, dialkyl azodicarboxylate (e.g., diethyl or
diisopropyl) (DEAD or DIAD) to give the Formula IXb. The vicinal
diol moiety is liberated and oxidative cleavage yields the
(+)-oleocanthal (Formula Xb). See also FIG. 4.
[0108] The invention contemplates mimetics of oleocanthal that have
the general formula shown above. Mimetics or mimics of oleocanthal
(sterically similar compounds formulated to mimic the key portions
of the structure) may be designed for pharmaceutical use. Mimetics
may be used in the same manner as oleocanthal, and hence are
functional equivalents. The generation of a structural-functional
equivalent may be achieved by the techniques of modeling and
chemical design known to those of skill in the art. It will be
understood that all such sterically similar constructs fall within
the scope of the present invention.
[0109] The design of mimetics to a known pharmaceutically active
compound is a known approach to the development of pharmaceuticals
based on a "lead" compound. This is desirable where, for example,
the active compound is difficult or expensive to synthesize, or
where it is unsuitable for a particular method of administration,
e.g., some peptides may be unsuitable active agents for oral
compositions as they tend to be quickly degraded by proteases in
the alimentary canal.
[0110] There are several steps commonly taken in the design of a
mimetic. First, the particular parts of the compound that are
critical and/or important in determining its organoleptic
properties are determined. In the case of oleocanthal, this can be
done, for example, by systematically varying the R groups of the
general formula and testing for anti-inflammatory activity, such
as, for example, by the assays described in the Examples.
[0111] Once the active region of the compound has been identified,
its structure is modeled according to its physical properties,
e.g., stereochemistry, bonding, size, and/or charge, using data
from a range of sources, such as, but not limited to, spectroscopic
techniques, X-ray diffraction data, and NMR. Computational
analysis, similarity mapping (which models the charge and/or volume
of the active region, rather than the bonding between atoms), and
other techniques known to those of skill in the art can be used in
this modeling process. In a variant of this approach, the
three-dimensional structure of the compound is modeled.
[0112] A candidate general formula is selected onto which chemical
groups that mimic the oleocanthal can be grafted. The general
formula and the chemical groups grafted onto it can conveniently be
selected so that the mimetic is easy to synthesize, is
pharmacologically acceptable, and does not degrade in vivo, while
retaining the biological activity of oleocanthal. Further
optimization or modification can then be performed to arrive at one
or more final mimetics for in vivo or clinical testing.
Uses of Oleocanthals
[0113] A. As a Food Additive:
[0114] The oleocanthals of the invention provide the characteristic
irritation sensation found in premium olive oils. The oleocanthals
may be added to lower grade oils to provide for an oil that tastes
like premium extra virgin olive oil. As such, the oleocanthals act
as a flavorant or flavor enhancer. The oleocanthals and
formulations of the invention may also be added to other foods to
enhance the flavor or the food by providing a pleasing irritation
sensation of olive oil.
[0115] The oleocanthals of the invention may be added to foods and
oral pharmaceutical preparations and oral hygiene products such as
toothpaste, mouthwash, breath-fresheners, films, candies, lozenges
to provide an irritant for the oral product's sensory-irritation
experience.
[0116] Oleocanthals may also provide sweetness inhibition, or allow
the structural design of other sweetness inhibitors. Such sweetness
inhibitors are useful when carbohydrates are added for bulking and
altering food body and texture.
[0117] Finally, oleocanthals may be used to add an irritant to food
for enhancing the flavor and gastronomic experience in a similar
fashion to other spices such as chilis, mustards, onions, Szechwan
pepper, and ginger, for example.
[0118] B. Preservative:
[0119] The oleocanthal and formulations of the invention may be
added directly to food items to act as a preservative. The food
items may be for human consumption or animal consumption.
Especially preferred food items for the method of preservation are
items which are customarily stored in oil. In this method a
suitable and effective amount of oleocanthal or a formulation
thereof is added directly to the food item or oil in which the food
item is stored.
[0120] In another embodiment of the invention, the oleocanthal or
formulation thereof is used to coat the food item prior to
packaging. The formulation may be sprayed onto the food item or the
food item may be dipped in the formulation. In another embodiment,
the oleocanthal or formulation thereof is applied to the inside
surface of packaging material that is in contact with the food item
to prevent spoilage. The coating may be a thin film sprayed onto
the inner surface or laminated onto the inner surface, for example.
In another embodiment of the invention, the packaging material used
to store the food item is impregnated with oleocanthal or a
formulation thereof. All of the embodiments for incorporating a
preservative into packaging materials or for incorporating a
preservative in food are well-known in the art, and any suitable
means may be employed. Without wishing to be bound by any
particular theory of operation, the preservative formulations and
oleocanthals possess anti-bacterial and antifungal properties which
allow them to act as preservatives.
[0121] C. Pharmaceutical Formulations
[0122] When employed as pharmaceuticals, the oleocanthals of this
invention are usually administered in the form of pharmaceutical
compositions. These compounds can be administered by a variety of
routes including oral, rectal, transdermal, subcutaneous,
intravenous, intramuscular, and intranasal. These compounds are
effective as both injectable and oral compositions. Such
compositions are prepared in a manner well known in the
pharmaceutical art and comprise at least one active compound.
[0123] This invention also includes pharmaceutical compositions
which contain other active ingredients in addition to the
oleocanthal compound(s) with pharmaceutically acceptable carriers.
In making the compositions of this invention, the active ingredient
is usually mixed with an excipient, diluted by an excipient or
enclosed within such a carrier which can be in the form of a
capsule, sachet, paper or other container. When the excipient
serves as a diluent, it can be a soled, semi-solid, or liquid
material, which acts as a vehicle, carrier or medium for the active
ingredient. Thus, the compositions can be in the form of tablets,
pills, powders, lozenges, sachets, cachets, elixirs, suspensions,
emulsions, solutions, syrups, aerosols (as a solid or in a liquid
medium), ointments containing, for example, 1-10% by weight of the
active compound, soft and hard gelatin capsules, suppositories,
sterile injectable solutions, and sterile packaged powders.
[0124] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, sterile water, syrup, and methyl cellulose. The
formulations can additionally include: lubricating agents such as
talc, magnesium stearate, and mineral oil; wetting agents;
emulsifying and suspending agents; preserving agents such as
methyl- and propylhydroxy-benzoates; sweetening agents; and
flavoring agents. The compositions of the invention can be
formulated so as to provide quick, sustained or delayed release of
the active ingredient after administration to the patient by
employing procedures known in the art.
[0125] The compositions are preferably formulated in a unit dosage
form, each dosage containing from about 0.001 to about 1 g, more
usually about 1 to about 30 mg, of the active ingredient. The term
"unit dosage forms" refers to physically discrete units suitable as
unitary dosages for human subjects and other mammals, each unit
containing a predetermined quantity of active material calculated
to produce the desired therapeutic effect, in association with a
suitable pharmaceutical excipient. Preferably, the compound of
formula I above is employed at about 20 weight percent of the
pharmaceutical composition or less, more preferably about 15 weight
percent or less, with the balance being pharmaceutically inert
carrier(s).
[0126] The active compound is effective over a wide dosage range
and is generally administered in a pharmaceutically effective
amount. It, will be understood, however, that the amount of the
compound actually administered will be determined by a physician,
in the light of the relevant circumstances, including the condition
to be treated, the chosen route of administration, the actual
compound administered and its relative activity, the age, weight,
and response of the individual patient, the severity of the
patient's symptoms, and the like.
[0127] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical
excipient to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention. When
referring to these preformulation compositions as homogeneous, it
is meant that the active ingredient is dispersed evenly throughout
the composition so that the composition may be readily subdivided
into equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation is then subdivided into unit
dosage forms of the type described above containing from, for
example, 0.1 to about 500 mg of the active ingredient of the
present invention.
[0128] The tablets or pills of the present invention may be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0129] Of course, additionally, the compositions of the present
invention may be formulated in sustained release form to provide
the rate controlled release of any one or more of the components to
optimize the therapeutic effects while minimizing undesirable side
effects. Suitable dosage forms for sustained release include
layered tablets containing layers of varying disintegration rates
or controlled release polymeric matrices impregnated with the
active components and shaped in tablet form or capsules containing
such impregnated or encapsulated porous polymeric matrices.
[0130] The liquid forms in which the novel compositions of the
present invention may be incorporated for administration orally or
by injection include aqueous solutions, suitably flavored syrups,
aqueous or oil suspensions, and flavored emulsions with edible oils
such as corn oil, cottonseed oil, sesame oil, olive oil, coconut
oil, or peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0131] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable carrier materials. Preferably the compositions are
administered by the oral or nasal respiratory route for local or
systemic effect. Compositions in preferably pharmaceutically
acceptable solvents may be nebulized by use of inert gases.
Nebulized solutions may be inhaled directly from the nebulizing
device or the nebulizing device may be attached to a face mask
tent, or intermittent positive pressure breathing machine.
Solution, suspension, or powder compositions may be administered,
preferably orally or nasally, from devices which deliver the
formulation in an appropriate manner.
[0132] (1) Cold Symptom Relief:
[0133] The oleocanthals of the invention may be used in a method
for treat the symptoms of the cold or flu. Formulations may be
prepared containing oleocanthals as the active ingredient, or in
combination with other active ingredients to be taken orally,
rectally, intranasally or as an inhalant, for example.
[0134] When taken orally, the oleocanthal formulation may be in the
form of a lollipop, quick-dissolving film, tablet, syrup, liquid,
liqui-gel, capsule, or the like.
[0135] The amount of oleocanthals in the preparation may be
adjusted by a physician of skill in the art for suitable dosages
for adults or pediatric use, or by a veterinarian of skill in the
art for use in various animals. The dosage of drug may be
determined based on the weight of the subject or based on surface
area. Any method of determining proper dosages is acceptable.
[0136] The oleocanthals are preferably formulated with a
pharmaceutically acceptable diluent, excipient or carrier
(collectively referred to herein as "carrier" materials) as
described above.
[0137] (2) Counter-Irritant for Sore Throat:
[0138] The oleocanthals of the invention are useful as
counter-irritants for sore throat which may accompany a cold or
flu, for example. The oleocanthal may be applied in combination
with other ingredients for sore throat relief or may be provided as
the sole active ingredient. The oleocanthal-based sore throat
formulations may be in the form of a tablet, lozenge, lollipop,
chewing gum, or throat spray. The formulation may be prepared and
packaged by any means known in the art.
[0139] For example, solid dosage forms may contain other
ingredients known in such dosage forms such as acidity regulators,
opacifiers, stabilizing agents, buffering agents, flavorings,
sweeteners, coloring agents, and preservatives. For example, a
lozenge may be prepared as by heating the lozenge base (e.g., a
mixture of sugar and liquid glucose) under a vacuum to remove
excess water and the remaining components are then blended into the
mixture. The resulting mixture is then drawn into desired shape.
The lozenges are cooled, and packaged into suitable packaging.
Lozenges will normally be sucked by the patient to release the
oleocanthal. Chewable solid dose formulations may be made by the
methods used to prepare chewable candy products or chewing gums.
For example, a chewable solid dosage form may be prepared from an
extruded mixture of sugar and glucose syrup to which the
oleocanthal has been added with optional addition of whipping
agents, humectants, lubricants, flavors and colorings. (See
Pharmaceutical Dosage Forms: Tablets, Volume 1, Second Edition
edited by H A Lieberman, L Lachman and J B Schwartz published in
1989).
[0140] Spray formulations may be prepared by dissolving or
suspending the oleocanthal in a liquid medium which may also
contain other ingredients such as stabilizing agents, buffering
agents, flavorings, sweeteners, coloring agents, and preservatives.
For example, a spray may be prepared by dissolving water soluble
components in water and non-water soluble ingredients in a
co-solvent (e.g., alcohol). The two phases are then mixed and the
resulting mixture filtered and placed into dispensing containers.
The dispensing containers may be fitted with a metered,
manually-operated spray mechanism or the dispenser may contain a
pressurized propellant and be fitted with a suitable dispensing
valve.
[0141] (3) Nasal Decongestant:
[0142] The oleocanthals of the invention are useful as a nasal
decongestant. The oleocanthal may be applied in combination with
other nasal decongestants or may be provided as the sole active
ingredient. The oleocanthal-based nasal formulations may be in the
form of a lavage or nasal mist. The formulation may be prepared and
packaged by any means known in the art for nasal lavages and
mists.
[0143] (4) Antioxidant:
[0144] Oleocanthals are believed to have anti-oxidant activity and
as such may be used to treat or prevent various conditions
including cancer. The oleocanthals may also be used to promote
wound healing, either by application directly onto wounds, or as a
coating or impregnation of bandages, sutures and the like.
[0145] The antioxidant effects of oleocanthals may also be
exploited in the formulation of cosmetics. The compositions can
protective of skin or hair or as an anti-solar composition. In
accordance with the invention the compound of formula (I), and
preferably oleocanthal is generally present in an amount ranging
from 1 to 1,000 mg. In some embodiments, oleocanthal or an
oleocanthal derivative is present in an amount of about 5 to 800
mg. In other embodiments, oleocanthal or an oleocanthal derivative
is present in an amount of about 10 to 750 mg. In other
embodiments, oleocanthal or an oleocanthal derivative is present in
an amount of about 25 to 600 mg. In other embodiments, oleocanthal
or an oleocanthal derivative is present in an amount of about 50 to
500 mg. In certain embodiments, the oleocanthal or derivative
thereof is present in 1, 5, 10, 20, 25, 50, 75, 100, 125, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950 or 1,000 mg.
[0146] In the compositions according to the present invention, the
compound of formula (I) acts as an antioxidant agent. These
compositions can be capillary compositions such as hair lacquers,
hair setting lotions or hair treating or disentangling lotions,
shampoos, coloring shampoos, hair dye compositions, makeup products
such as nail enamels, skin treating creams and oils, foundations,
lipsticks, compositions for the care of the skin such as bath oils
or creams as well as any other cosmetic composition capable of
exhibiting, because of their components, oxidation stability
problems during storage.
[0147] (5) Pain Relief:
[0148] The oleocanthals of the invention may be used as to treat
and prevent pain. The compounds are useful for the relief of pain
associated with a variety of conditions including, but not limited
to influenza or other viral infections, common cold, low back and
neck pain, dysmenorrhea, headache, toothache, sprains and strains,
myositis, neuralgia, synovitis, arthritis, including rheumatoid
arthritis, degenerative joint diseases (osteoarthritis), gout and
ankylosing spondylitis, bursitis, burns, injuries, cancer and for
pain associated with surgical and dental procedures.
[0149] (6) Anti-Inflammatory:
[0150] The oleocanthals of the invention may be used as
anti-inflammatory agents. The oleocanthals may be used in a method
for treating or preventing diseases marked by inflammation,
including but not limited to psoriasis, cancer, asthma, allergic
rhinitis, respiratory distress syndrome, inflammatory bowel
disease, Crohn's disease, gastritis, irritable bowel syndrome,
ulcerative colitis, migraine, periarteritis nodosa, thyroiditis,
aplastic anemia, Hodgkin's disease, scleroderma, type I diabetes,
myasthenia gravis, multiple sclerosis, sorcoidosis, ischemic kidney
disease, nephrotic syndrome, Bechet's syndrome, polymyositis,
gingivitis, conjunctivitis, vascular disease myocardial ischemia,
heart disease, stroke, and hypertension.
[0151] (7) Micro-G Protein and Associated Kinase Inhibitor:
[0152] The oleocanthals of the invention may also be formulated for
treatment or prevention of the development of A.beta.42 associated
Alzheimer's plaques and tangles in a manner similar to that found
for non-steroidal anti-inflammatory drugs such as ibuprofen.
Without wishing to be bound by any particular theory of operation,
it is believed that ibuprofen, and oleocanthal inhibit micro-G
proteins and associated kinases, for example Ras and Rock, which
have been associated with the development of A.beta.42 associated
plaques and tangles in the brains of Alzheimer's patients.
Oleocanthals also acts to inhibit .gamma.-secretases and alter
presenilin conformations of which both activities are associated
with reducing A.beta.42 associated Alzheimer's plaques and
tangles.
[0153] It is believed that certain non-steroidal anti-inflammatory
drugs inhibit .gamma.-secretases without significantly altering
other activities in the A.beta. amyloid precursor protein (APP)
processing pathway. In patients with certain mutations in APP and
all mutations known for presenilin, APP is processed such that
there is a large increase in the amount of a proteolytic fragment
of 40-42 residues (A.beta.42) (Weggen et al. (2001) Nature 414
(8):212). Certain NSAIDs appear to have an effect of reducing the
production of A.beta.42 by a mechanism that is independent of the
cyclooxygenase activity associated with the anti-inflammatory
activity of the NSAIDs. It has been shown that for many NSAIDs,
which are administered as racemic mixtures of the active compounds,
that a specific enantiomer (the S-enantiomer) appears to be
responsible for the inhibition of cyclooxygenase activity, and
hence the anti-inflammatory effect (Weggen et al. (2001) Nature 414
(8):212). It has also been shown that the R-enantiomer of the
NSAIDs may mediate reduction of A.beta.42 production and may be
responsible for the decreased risk in Alzheimer's and cognitive
impairment seen with long term use of NSAIDs (Morihara et al.
(2002) J. Neurochem. 83:1009-1012).
[0154] Also correlating with lower risk of developing Alzheimer's
and cognitive impairment is the so-called Mediterranean diet, which
is typically high in consumption of, among other things, olive oil.
Thus, the observation made herein of the association with the
organoleptic properties of oleocanthal and the similarity to
ibuprofen and the observations associated with long term use of
NSAIDs and dietary intake of olive oil suggest that oleocanthal may
be used for the treatment and prevention of neurodegenerative
disorders (e.g., Alzheimer's and other cognitive impairment
associated with amyloid plaques and tangles). The treatment and
prevention of such neurodegenerative disorders may be performed
using a racemic mixture of oleocanthal, or may be using one of the
purified enantiomers of oleocanthal.
[0155] (8) For Oral Surgery and Oral Irradiation Treatment of
Cancer:
[0156] The oleocanthals of the present invention are also useful as
treatments for use in conjunction with oral surgery and oral
irradiation treatment of cancer. While not wishing to be bound by
any particular theory of operation, it is believed that the
oleocanthals, with their attendant anti-inflammatory activity, act
to inhibit the inflammation that occurs in the oral cavity as a
result of surgery or oral irradiation. The oleocanthals may be
formed as an oral rinse which can be administered before the
procedure, after the procedure, or during the procedure, or a
combination of these treatment regimens. The amount of oleocanthal
in the rinse is a therapeutically effective amount which is readily
determined by one of skill in the art.
[0157] D. Animal Repellant:
[0158] It is believed that oleocanthals, with their organoleptic
qualities, are useful as animal repellents. The compounds may be
used to repel carnivorous and omnivorous animals and birds,
including domestic cats, rodents, raccoons, dogs, other canids such
as coyotes.
[0159] The method of this invention comprises applying an
effective, repellent amount of the oleocanthals, either alone or in
combination with a suitable carrier, to the locus from which the
animals are to be repelled. Suitable carriers would include liquid
diluents such as water, hydrocarbons, alcohols, emulsifiers and
other liquids generally found in household spray formulations or
pharmaceutical preparations so as to be acceptable from a human
safety viewpoint. Inert solid carriers such as starches may also be
of use, and it might be desirable to incorporate the compounds into
a controlled-release formulation.
[0160] It may be desirable to apply the oleocanthals to containers
for discarded edible refuse, such as metal or plastic garbage cans,
plastic bags, paper and cardboard boxes and the like. Further, the
repellent compounds disclosed herein might be incorporated into
various potentially-edible compositions which, if consumed, could
injure or kill an animal. An example of such a composition would be
liquid antifreeze.
[0161] Another aspect of this invention provides methods for
repelling birds from consuming or utilizing a material otherwise
susceptible to consumption or utilization by birds, comprising
providing to the material an avian repellent amount of at least one
oleocanthal.
[0162] Liquid carriers may be employed and the repellent may be
sprayed on the material. See e.g., U.S. Pat. No. 2,967,128 which
patent is incorporated by reference as if fully set forth herein.
The compound may be dispersed in the liquid from which the birds
are to be repelled. The repellent may be at least partially trapped
in a solid vehicle to improve its persistency such as disclosed in
U.S. Pat. No. 4,790,990. The vehicle may be a modified starch, oil
or polymer which at least partially encapsulates, emulsifies or
substantially uniformly disperses the aversive agent. The repellent
compound and vehicle may be dispersed throughout solids consumed by
avian species to reduce the likelihood that they will eat the
treated edible.
[0163] Certain embodiments of the present invention are directed to
methods of repelling birds from consuming or utilizing non-potable
liquids such as industrial or agricultural waste water, mine
tailing ponds, and freestanding water on artificial surfaces like
airport runways and parking lots. "Non-potable" refers to liquids
or aquatic habitats wherein said liquid may be consumed or utilized
by birds to the detriment of man or the birds.
[0164] E. Discovery
[0165] Knowledge of the absolute structure of oleocanthal allows
the identification of the oleocanthal receptor and related genes.
Screening assays for receptors, well-known in the art, may be
employed to determine the oleocanthal receptor. Tissue from the
back of the throat, known to interact with oleocanthal may be
isolated and subjected to various assays to determine the binding
of oleocanthal to cells and the molecular signaling pathway of
oleocanthals.
[0166] Labeled oleocanthal may be used in tissue binding studies to
determine the cell types that contain a presumptive oleocanthal
receptor. Cells that have bound labeled oleocanthal may be
visualized by any method known in the art. For example, but not by
way of limitation, oleocanthal may be labeled with a radiolabel
(e.g., .sup.125I, .sup.35S, .sup.32P, .sup.33P, .sup.3H), a
fluorescence label, a chemiluminescent label, an enzymatic label,
or an immunogenic label. In other embodiments, luminescent or
fluorescent molecules may be conjugated to the oleocanthal
molecule. The labeled oleocanthal may be allowed to bind to cells
in situ and visualized under a microscope. Alternatively, cells in
suspension may be labeled with the labeled oleocanthal and labeled
cells may be separated from unlabeled cells by flow cytometry or
using a sorter, such as a fluorescence-activated cell sorter
(FACS). Labeled cells may be collected for subsequent genetic
analysis, for example.
[0167] In some embodiments, a molecule is conjugated to oleocanthal
that allows the conjugated oleocanthal to be cross-linked to its
receptor upon binding. This may be performed by any means known in
the art. Thereafter the cross-linked receptor may be isolated from
the cells, purified and subjected to N-terminal amino acid
sequencing. With the identity of the N-terminal amino acids,
degenerate oligonucleotides may be synthesized based on the
possible combinations of oligonucleotides encoding the amino acid
sequence and the oligonucleotides may be used in various ways to
identify the gene encoding the oleocanthal receptor. In some
embodiments, the degenerate oligonucleotides are used to probe gene
libraries. The gene library may a library formed from animal cells,
particularly human cells, or it may be a specific cell-type library
from animal cells known to be responsive to oleocanthal. In other
embodiments, the library may be a subtractive library formed by
removing commonly expressed genes from oleocanthal-responsive and
oleocanthal-unresponsive cells, such that the library consists of a
subset of genes reflecting unique sequences of the
oleocanthal-responsive cells. In another embodiment, the degenerate
oligonucleotides are paired with a second set of oligonucleotides
to allow rt-PCR amplification of polynucleotides containing the
sequences encoding the amino acid sequence of the oleocanthal
receptor. Such second set of oligonucleotides may include, for
example, oligo-dT which anneals to poly-adenosine tracts of mRNA.
The rt-PCT reaction may be performed on RNA extracted from
oleocanthal responsive cells. The methods and techniques for such
genetic analysis are well-known in the art and may be found in the
references and texts referred to herein.
[0168] Further aspects of the invention are exemplified below,
however, the examples are merely illustrative of the invention and
the scope of the invention is not to be limited thereto or
thereby.
EXAMPLES
Example 1
Isolation of Deacetoxydialdehydic Ligstroside Aglycone
"Oleocanthal"
[0169] A. Synthesis of Oleocanthal
[0170] Retrosynthetically, we envisioned both enantiomers of (1) to
derive from the enantiomeric forms of cyclopentanediols (7) via
oxidative cleavage of the diol moiety (Scheme 1). The requisite
cyclopentanediols (7) in turn would be prepared from
cyclopentanones (+)- and (-)-(10), via alkylation to introduce
stereoselectively the side chain from the convex face, followed by
stereoselective Wittig ethylnation and removal of the acetonide
moiety (Scheme 1).
##STR00009##
(5) Initially (+)- and (-)-cyclopentanones (10) were prepared via
the sulfoximine and/or enzymatic protocols introduced and developed
by Johnson (Johnson, C. R. and T. Penning (1988) J. Am. Chem. Soc.
110:4726-4735; Johnson, C. R. (1998) Acc. Chem. Res. 31:333-341).
Although effective on modest scale (10-100 mg), the requirement for
gram quantities of the oleocanthals demanded that we secure for
more scalable routes to (10). Towards this end, we optimized a
hybrid of synthetic approaches (Moon, H. et al. (2002) Tetrahedron:
Asym. 13 (11):1189-1193; Jin, Y. et al. (2003) J. Org. Chem. 68
(23):9012-9018; Yang, M. (2004) J. Org. Chem. 69 (11):3993-3996;
Palmer, A. et al. (2001) Eur. J. Org. Chem. 66 (7):1293-1308;
Paquette, L. and S. Bailey (1995) J. Org. Chem. 60:7849-7856) as
outlined in Scheme 2. Importantly, both enantiomers of (10) could
be prepared in multi-gram quantities in 7 steps, with an overall
efficiency of 40% from inexpensive D-(-)-ribose. Key elements of
both sequences entailed vinyl Grignard addition to the enantiomers
of aldehyde (12), followed in turn by ring closing metathesis
(RCM), PCC oxidation and hydrogenation (Scheme 2).
##STR00010## ##STR00011##
[0171] Alkylation of (+)- and (-)-cyclopentanone (10) with methyl
bromoacetate was then anticipated to proceed from the less hindered
convex face of the bicyclic skeleton to install the side chain in a
stereoselective fashion. Initial attempts however to alkylate
(-)-(8) with methyl bromoacetate employing LDA in the presence of
HMPA furnished only a complex mixture containing only trace amounts
of (-)-(16). Neither addition of Cu(I) (Johnson, C. R. and T.
Penning (1988) J. Am. Chem. Soc. 110:4726-4735) reportedly to
suppress side reactions, nor the use of the corresponding tin
enolate [generated by treatment of (-)-(10) in THF with LDA,
followed by HMPA and tributyltin chloride (Suzuki, M. et al. (1985)
J. Am. Chem. Soc. 107:3348; Nishiyama, H. et al. (1984) Tetrahedron
Lett. 25:223)] improved the situation. Alkylation of the zinc
enolate of (-)-(10) [generated by treatment of (-)-(10) in THF with
1.1 eq. LHMDS, followed in turn by HMPA (3.0 eq.) and dimethyl zinc
(Morita, Y. et al. (1989) J. Org. Chem. 54:1787-1788) (1.0 eq.)]
with methyl bromoacetate, however consistently furnished (-)-(16)
in 55-60% yield as a single diastereomer (this reaction was fairly
clean except some baseline materials. Using t-butyl bromoacetate
instead of methyl bromoacetate did not improve the yield) (Scheme
3).
##STR00012##
[0172] Wittig ethylnation of (-)-(16) was next achieved with
ethyltriphenylphosphine bromide. Best results were obtained
employing LDA as the base at -45.degree. C. Although excellent
stereoselectivity (ca., 10:1 E:Z) favoring the E-isomer (-)-(17)
was achieved, the yield was only modest (42%), presumably due to
the ease of enolization of (-)-(16) (Edmunds, M. "The Wittig
Reaction" In MODERN CARBONYL OLEFINATION, Takeda, Ed., John Wiley
& Sons, New Jersey, 2004). Interestingly, the stereoselectivity
varied dramatically with reaction temperature. At 0.degree. C., the
E:Z selectivity was 3.3:1, while at room temperature the
selectivity was 1.6:1. Assignment of the E geometry of the olefin
was based on NMR NOE analysis (Scheme 4).
##STR00013##
[0173] Hydrolysis of ester (-)-(17) (LiOH/THF/H.sub.2O) next
afforded acid (-)-(18), which was subjected to Mitsunobu
esterification (Mitsunobu, O. (1981) Synthesis 1-28) with
4-hydroxyphenethyl alcohol to furnish phenol (-)-(19) in 92% yield.
As expected, the Mitsunobu reaction proceeded with complete
chemoselectivety at the primary hydroxyl (Appendino, G. et al.
(2002) Org. Lett. 4:3839-3841). Completion of the synthesis of
(-)-oleocanthal (1) was then achieved via liberation of the vicinal
diol moiety (4N HCl/acetonitrile), followed by oxidative cleavage
(NaIO.sub.4); (-)-oleocanthal (1) was identical in all respects
(e.g., .sup.1H and .sup.13C NMR, IR and HRMS) with an authentic
sample isolated from virgin olive oil, the latter possessing
spectral data identical to that reported in the literature
(Montedoro, G. et al. (1993) J. Agric. Food Chem. 41:2228-2234).
The structural assignment of (1) was also confirmed by COSY NMR
analysis. Synthetic (-)-(1) displayed a small negative optical
rotation ([.alpha.].sup.25.sub.D -0.78, c=0.9, CHCl.sub.3)
identical to that obtained from a sample isolated from virgin olive
oil ([.alpha.].sup.25.sub.D -0.9, c=2.0, CHCl.sub.3). Thus the
stereochemistry of (-)-oleocanthal (1) is 3S, 4E. The enantiomer of
the natural product (+)-(1) was prepared via a similar reaction
sequence beginning with (+)-(10) to furnish (+)-1 ([a].sup.25.sub.D
+0.73, c=0.55, CHCl.sub.3) (Scheme 5).
##STR00014##
[0174] In summary, an effective, scalable synthesis of both
enantiomers of oleocanthal (1) has been achieved, each hi 13 steps
(7% overall yield) from inexpensive (D)-(-)-ribose, requiring only
6 chromatographic separations. The structural similarity of
oleocanthal to a number of related natural products (Somanadhan, B.
et al. (1998) Planta Medica 64:246-50; Takenaka, Y. et al. (2002)
Chem. & Pharm. Bull. 50 (3):384-389; Takenaka, Y. et al. (2002)
Phytochemistry 59 (7):779-787) suggests that the synthetic approach
presented here should also be applicable to their construction.
[0175] B. Functional Studies of Oleocanthal
[0176] This restricted throat irritation of oleocanthal is
remarkably similar to that elicited by ibuprofen. Due to the
observed organoleptic similarity, we isolated and then synthesized
oleocanthal from olive oil. Sensory and chemical evaluation of 10
commercially available olive oils revealed a strong positive
relationship between throat irritation intensity and oleocanthal
concentration. Cyclooxygenase and lipoxygenase assays with
synthesized oleocanthal demonstrated that it is a NSAID with an
anti-inflammatory profile strikingly similar to that of ibuprofen,
in accordance with its sensory properties. Oleocanthal may play a
significant role in the well-known health benefits associated with
a diet high in olive oil. Moreover, identification of other
pharmacologically important compounds is hereby facilitated by
attention to similarities of sensory properties.
[0177] Recent studies in our laboratories have demonstrated that
ibuprofen, as well as some other non-steroidal anti-inflammatory
drugs (NSAIDs), have the unusual sensory property of stinging
almost exclusively in the throat, unlike for example, capsaicin and
piperine that also burn the mouth and lips. While tasting
newly-pressed Sicilian olive oil, it was observed that the
throat-irritating sensation appeared identical to that of
ibuprofen. Indeed, high quality extra virgin olive oils are often
characterized by a stinging or burning sensation akin to that felt
when swallowing ibuprofen. With olive oil, this sensation often
elicits a small cough or throat-clearing when olive oil is
swallowed neat. Olive oil enthusiast categorize oils as 0, 1, 2 or
3 cough oils with the higher numbers being superior. The entity
responsible for this sensory property has recently been reported to
be deacetoxy-dialdehydic ligstroside aglycone, one of many
polyphenols found in olive oil.
[0178] Based on their similar oro-sensory properties, we reasoned
that oleocanthal might also share pharmacologic properties of
ibuprofen. To test this thesis, we first had to verify and
definitely prove the identity of oleocanthal. This required
development of an efficient analytical method for isolating and
quantifying it. Two approaches to verify the identity of the
compound and its properties were taken. First, we undertook
psychophysical experiments with oleocanthal, correlating the amount
of identified compound with the degree of burn in commercial olive
oils. Second, we synthesized oleocanthal and tested the
psychophysical properties of the synthesized material. Finally, to
examine oleocanthal for pharmacological activity that might mimic
ibuprofen, cyclooxygenase, lipoxygenase and lipid peroxidation
assays with synthetic material were conducted.
[0179] To isolate and purify oleocanthal, we employed a systematic
sensory-directed approach. That is, we used taste analysis as a
tool to monitor the presence of the throat-irritation compound in
each step of an isolation and purification procedure similar to
that used by Andrewes et al. Briefly, the irritant was first
extracted from olive oil with methanol/water (80/20, v/v). The
phenolic extract was separated into 15 fractions, only one of which
was irritating, using a reversed-phase HPLC method. To obtain pure
material, we pre-fractioned the olive oil phenolic extract on a C18
solid phase extraction cartridge. Retention information about the
throat-irritating principal from the HPLC method allowed us to
separate it from the majority of the other co-extracted phenolic
compounds using methanol and water solvent mixtures at three
different ratios of eluting solvents. HPLC analysis of the throat
irritating fraction revealed the presence of several unresolved
compounds. A new HPLC gradient was thus developed and only one
well-resolved peak was throat-irritating. A detailed NMR (1D and
2D) analysis was conducted with this material. Although .sup.1H-NMR
spectra indicated the presence of minor impurities, the structure
of the major compounds was readily identified to be
2-(4-hydroxyphenyl)ethyl, 4-formyl-3-(2-oxoethyl)-4-hexenoic acid
ester, the deacetoxy-dialdehydic ligstroside aglycone, as first
identified in olive oil by Montedoro and recently reported as the
throat irritant. Optical rotation measurements of oleocanthal
revealed the natural enantiomer to be levorotary.
[0180] Olive oils differ markedly in their ability to elicit throat
irritation. If oleocanthal is primarily responsible for this
sensory property there should be a positive relation between
compound concentration and degree of throat irritation. To test
this hypothesis, we purchased 10 different olive oils with widely
varying degrees of throat irritation based on informal evaluation.
The amount of oleocanthal in each was then quantified. The compound
was extracted from small amounts of each of the 10 oils (1 g) by
hexane-acetonitrile (liquid-liquid) extraction. The solvent extract
was analyzed by reverse-phase HPLC with UV detection at 278 nm.
Oleocanthal was chromatographically separated from the other
extracted compounds with an elution gradient of acetonitrile and
water. All analyses were done in duplicate using solutions of pure,
isolated oleocanthal as the external standard. When the compound
was later synthesized, this was also used as a standard to confirm
these methods. Overall, the reproducibility was high (RSD=4.7%),
recovery was good (95%), the calibration curve was linear
(r.sup.2=0.9999) and the limit of quantitation was <1 ppm.
[0181] The degree of throat irritation of these 10 oils was
quantified by 17 volunteers. Each subject was tested only 2 times
per day with two different olive oils samples with 1-2 hours
separating each test since the irritation may be sensitive to
shorter inter-trial intervals. Subjects wore nose clips to
eliminate olfactory cues. Tasting consisted of placing
approximately 3.5 ml of olive oil in the mouth, holding it there
for 3 seconds and then swallowing it in two aliquots so as to
insure the throat would be stimulated. After 45 seconds passed,
subjects were asked to rate the peak throat irritation sensitivity
using the general labeled magnitude scale, a sensory scale
developed to generate magnitude estimation-like quality data. Each
subject was tested twice with all ten oils.
[0182] The concentrations of oleocanthal in the 10 olive oils and
their degree of throat irritation proved statistically significant
(r=0.90; FIG. 2) providing additional evidence that oleocanthal is
responsible for the majority of the throat irritation in the olive
oils tested.
[0183] These studies strongly implicate oleocanthal as the major
throat irritating compound in olive oil. Nevertheless, as noted by
Andrewes et al., co-elution of a minor component or mixture of
components causing the burning sensation cannot be eliminated as a
possible source of irritation without completing a de novo total
synthesis followed by organoleptic analysis. Since the structure of
oleocanthal possesses a stereospecific center, we synthesized both
enantiomers from readily available D-ribose. The synthesis of both
(+) and (-)-oleocanthal required 13 steps as outlined for the
recovery of levorotary (-)-enantiomer in FIGS. 3 and 4. Both
syntheses proved scalable, proceeding in 7% overall yield and
thereby providing ample material for sensory and pharmacological
evaluation. The levoratory (-)-enantiomer of synthetic oleocanthal
displayed the same sign and magnitude of the optical rotation as
the natural material. Thus the absolute stereodirection of variant
(1) is as depicted in FIG. 2.
[0184] Three individuals experienced in tasting olive oils and
ibuprofen, using a standard 2 alternative forced-choice method,
evaluated the synthetic compound (the natural (-)-isomer only)
dispersed in non-irritating corn oil at approximately twice the
concentration found in Falconaro olive oil, the most potent olive
oil we have evaluated (FIG. 1).
[0185] Testing was double-blind and each was exposed to three sets
of two samples, one of which was added synthesized oleocanthal and
the other served as the blank control. The task was to indicated
which of the pair was more irritating. Each of the three evaluators
correctly identified the sample on each of the three presentations
(9 of 9 correct, p<0.01) and all three identified the distinct
back of the throat irritation with the cough-eliciting sensation
characteristic of both olive oil and ibuprofen. As predicted, the
throat irritation of synthetic (-)-oleocanthal was identical to
oleocanthal isolated from premium olive oil. Importantly, the
effect was dose dependent (FIG. 2 open triangles, dashed line). Ten
subjects were tested with non-irritating commercial corn oils
presented neat and mixed with either synthesized (-)-oleocanthal or
the bitter agent sucrose octaacetate (SOA) (Sigma-Aldrich). The
addition of SOA enabled forced-choice trials to be conducted
without revealing to subjects the identity of the irritating
samples due to bitterness or other non-irritating cues.
(-)-Oleocanthal was tested at the highest concentration identified
in the ten rated oils, 200 .mu.g/ml, and at one half and whole log
steps more dilute 63.25 and 20 .mu.g/ml. SOA was added to the corn
oil (4.times.10.sup.-4, 1.times.10.sup.-4, 5.times.10.sup.-5 M) to
intensity match the irritation of the three levels of
(-)-oleocanthal. Subjects participated in two-alternative
forced-choice (2AFC) trials (four trials at every concentration for
each subject) and in intensity ratings sessions (four ratings per
each oil). For the 2AFC trials subjects were presented with two 3.0
ml corn oil samples with matching intensities of SOA and
(-)-oleocanthal in ascending order, and were required to sample
oils as described above. While blind to stimulus position, subjects
were asked two questions on each trial, "Which of the two oils was
more irritating in the throat?" and "Which one was more bitter?" At
the 20 .mu.g/ml & 5.times.10.sup.-5 M level most subjects
reported on some trials that the same oil was both the more
irritating and the more bitter of the two. This demonstrates that
participants were willing to select the one oil as stronger on both
traits within a trial. Subjects performed at chance when selecting
among two unadulterated corn oils, when the correct choice was
randomly assigned prior to testing. At 20 .mu.g/ml subjects were
correct 24 out of 40 trials, indicating that this concentration is
near detection threshold levels in corn oil. The other two
concentrations were correct 39/40 and 40/40 trials. For the
intensity rating trials subjects were presented with all eight oils
in ascending order, counterbalanced for stimulus order and asked to
rate the throat irritation and bitterness of every oil on a general
labeled magnitude scale.
[0186] Assuming the quality and locus of irritation provides a
signal of pharmacological activity, then oleocanthal should mimic
at least some of the pharmacological properties of ibuprofen, a
potent modulator of inflammation. To test this we chose to evaluate
inhibition of cyclooxygenase (COX) and lipoxygenase (LO), two
enzymes central to the inflammatory process. Ibuprofen is a potent
COX-1 and COX-2 inhibitor but does not inhibit lipoxygenase. The
concentration dependence of oleocanthal for inhibition of ovine
COX-1, human recombinant COX-2 and soybean 15-lipoxygenase
activities was measured using commercially available kits (Cayman
Chemicals). Indomethacin was used as a positive (inhibitory)
control in the cyclooxygenase assays and nordihydroguaracetic acid
(NDGA) and caffeic acid were used as positive (inhibitory) controls
in the lipoxygenase assays. Both enantiomers of oleocanthal,
exhibited a dose-dependent inhibition of both COX-1 and COX-2
activities, with no effect on lipoxygenase activity, much as
observed with ibuprofen (Table 1). The calculated IC.sub.50 (least
squares regression analysis of inhibition vs. concentration) for
oleocanthal (-) was 21.4 .mu.m and 29.4 .mu.m for COX-1 and COX-2,
respectively. The IC.sub.50 for oleocanthal (+) was 27.9 .mu.m and
40.5 .mu.m for COX-1 and COX-2, respectively. In these experiments,
both enantiomers of oleocanthal were more potent at equimolar
concentrations than ibuprofen in inhibiting COX-1 and COX-2. Both
enantiomers of oleocanthal inhibited the peroxidation of serum
lipids induced by metal ions in vitro to a similar degree as
equimolar alpha-tocopherol (data not shown). Thus, oleocanthal
exhibits antioxidant activity comparable to alpha-tocopherol and
has an arachidonic acid inhibitory profile (cyclooxygenase
inhibition without lipoxygenase inhibition) indicating that both
enantiomers of oleocanthal are classic NSAIDs, with potency
superior to that of ibuprofen.
[0187] Taken together, these data are consistent with our
hypothesis that the throat irritating compound in olive oil is an
ibuprofen-like anti-inflammatory agent. Importantly, the
oleocanthal results provide an example of how sensations from the
mouth may serve as an in vivo pharmacological assay. These results
further suggest an additional basis for the health benefits of
olive oil consumption have been attributed to a combination of the
lipid profile, the antioxidant activity of many of the polyphenols
present and the anti-inflammatory agents that inhibit lipoxygenase.
We suggest here an additional benefit: long-term consumption of
oleocanthal, with anti-inflammatory ibuprofen-like activity may
enhance health and well-being. Assuming that an olive oil consumer
in the high normal range ingest about 50 g of olive oil per day and
that this oil contains up to 200 .mu.g/ml of oleocanthal, the
person would then consume approximately 10 mg/day. Although this
dose is relatively low (-10% of the dosage of ibuprofen recommended
for adult pain relief), chronic low doses of other COX inhibitors
(e.g., aspirin) are known to have important health benefits,
chiefly a reduction in heart attack risk and at slightly higher
doses a reduction in both heart attack and stroke risk.
[0188] In addition to anti-inflammatory activity, ibuprofen has
recently been shown to have a COX-independent ability to decrease
the highly amyloidogenic AB42 peptide, perhaps accounting for
epidemiologic evidence that Alzheimer's disease. Thus, it would be
important to determine whether oleocanthal has similar
activity.
[0189] The initial hypothesis that the throat irritating compound
in olive oil might have pharmacological activity was based on the
oro-sensory similarities of ibuprofen and olive oil. This implies a
similar sensory mechanism but exactly how ibuprofen (or
oleocanthal) elicits almost exclusive throat irritation remains
elusive. One possible explanation is that there is a currently
unknown receptor system that is responsible to both ibuprofen and
olive oil. Alternatively, or additionally, both compounds could
have particularly easy access to free nerve endings in the throat,
but why this would occur preferentially in the throat is unknown.
It is also unclear why other lipophilic irritants such as lactic
acid or capsaicin would not stimulate the throat exclusively as
well, if the mechanism were simply one of ready access to free
nerve endings. Elucidation of the sensory mechanism may assist in
determining the common pathway for the anti-inflammatory activities
of these molecules, or vice versa. The sensory properties of foods,
spices and flavors may provide clues to pharmacological activity
and thus serve not only to provide pleasure but also to enhance
health.
TABLE-US-00001 TABLE 1 Percent inhibition of COX-1, COX-2 and 15-LO
by Oleocanthal (-), (+) Concen- tration Agent (uM) COX-1 COX-2
15-LO Oleocanthal (-) 100 83.5 .+-. 3.5 70.9 .+-. 8.6 0.4 .+-. 0.8
25 56.1 .+-. 3.2 56.6 .+-. 9.5 0.0 .+-. 0.0 7 24.6 .+-. 7.3 14.5
.+-. 2.3 0.0 .+-. 0.0 Oleocanthal (+) 100 68.0 .+-. 15.2 69.6 .+-.
3.9 3.5 .+-. 5.5 25 54.5 .+-. 4.6 41.3 .+-. 15.9 0.7 .+-. 1.0 7
24.6 .+-. 7.5 6.1 .+-. 4.2 0.0 .+-. 0.0 Ibuprofen 25 17.8 .+-. 2.3
12.7 .+-. 3.6 0.2 .+-. 0.3 7 0.0 1.3 n.d. Indomethacin 25 45.8 .+-.
4.4 77.6 .+-. 10.2 0.1 .+-. 0.9 7 33.0 .+-. 6.1 71.6 .+-. 7.3 0.5
.+-. 0.1 NDGA 25 n.d. n.d. 63.1 .+-. 0.8 7 n.d. n.d. 52.5 .+-. 1.1
Caffeic Acid 25 n.d. n.d. 25.2 .+-. 2.2 7 n.d. n.d. 19.8 .+-. 1.3 *
Data are presented as mean % inhibition .+-. SEM for three
independent experiments. N.d. = not determined.
Example 2
Structure Activity Relationship (SAR) Study
[0190] A Structure Activity Relationship (SAR) Study may be
conducted to determine the functional relative activities of
oleocanthal derivatives. As shown in FIG. 5, a compound having the
structure:
##STR00015##
is reacted with a compound selected from the following:
##STR00016##
to produce oleocanthal derivatives. These compounds are then tested
for activity as described above. Relative efficacies and potencies
of the oleocanthal derivatives may be assigned to each compound and
structural-functional information may be derived for rational drug
design of oleocanthals.
* * * * *