U.S. patent application number 11/242615 was filed with the patent office on 2006-07-13 for methods for the synthesis of lutein.
Invention is credited to David C. Burdick, Yishu Du, Zhiqiang Fang, William B. Geiss, Henry L. Jackson, Samuel F. Lockwood, Geoff T. Nadolski, Peng Cho Tang, Richard Williams, Min Yang.
Application Number | 20060155150 11/242615 |
Document ID | / |
Family ID | 36042942 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060155150 |
Kind Code |
A1 |
Lockwood; Samuel F. ; et
al. |
July 13, 2006 |
Methods for the synthesis of lutein
Abstract
A method used for synthesizing intermediates for use in the
synthesis of carotenoids and carotenoid analogs, and/or carotenoid
derivatives. In some embodiments, the invention includes methods
for synthesizing optically active intermediates useful for the
synthesis of optically active carotenoids. Synthesis of optically
active carotenoids, in one embodiment, may be accomplished by
forming an optically active dihydroxy intermediate from
ketoisopherone. The optically active dihydroxy intermediate may be
converted into optically active lutein.
Inventors: |
Lockwood; Samuel F.; (Lago
Vista, TX) ; Tang; Peng Cho; (Moraga, CA) ;
Nadolski; Geoff T.; (Kaneohe, HI) ; Jackson; Henry
L.; (Honolulu, HI) ; Fang; Zhiqiang; (Hawthorn
Wds, IL) ; Du; Yishu; (Shanghai, CN) ; Yang;
Min; (Vernon Hills, IL) ; Geiss; William B.;
(Athens, NY) ; Williams; Richard; (Nashville,
TN) ; Burdick; David C.; (Guilderland, NY) |
Correspondence
Address: |
MEYERTONS, HOOD, KIVLIN, KOWERT & GOETZEL, P.C.
700 LAVACA, SUITE 800
AUSTIN
TX
78701
US
|
Family ID: |
36042942 |
Appl. No.: |
11/242615 |
Filed: |
October 3, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60615032 |
Oct 1, 2004 |
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60675957 |
Apr 29, 2005 |
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60691518 |
Jun 17, 2005 |
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60692682 |
Jun 21, 2005 |
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60699653 |
Jul 15, 2005 |
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60702380 |
Jul 26, 2005 |
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60712350 |
Aug 30, 2005 |
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Current U.S.
Class: |
568/346 |
Current CPC
Class: |
C07C 45/298 20130101;
C07F 9/113 20130101; C07C 49/713 20130101; C07C 45/511 20130101;
C07C 45/64 20130101; C07C 45/71 20130101; C07C 29/143 20130101;
C07C 45/71 20130101; C07D 319/06 20130101; C07C 29/143 20130101;
C07C 45/673 20130101; C07C 45/61 20130101; C07C 2601/16 20170501;
C07C 49/753 20130101; C07C 49/753 20130101; C07C 47/21 20130101;
C07C 49/603 20130101; C07C 49/713 20130101; C07C 35/08 20130101;
C07C 33/02 20130101; C07C 35/21 20130101; C07C 49/713 20130101;
C07C 49/713 20130101; C07C 49/753 20130101; C07C 49/713 20130101;
C07C 49/753 20130101; C07C 45/64 20130101; C07C 45/70 20130101;
C07C 49/753 20130101; C07C 403/08 20130101; C07C 45/64 20130101;
C07F 7/1892 20130101; C07C 29/143 20130101; C07C 29/143 20130101;
C07C 45/61 20130101; C07C 45/298 20130101; C07B 2200/07 20130101;
C12P 23/00 20130101; C07C 45/71 20130101; C07B 2200/09 20130101;
C07C 45/511 20130101; C07C 45/673 20130101; C07C 45/70 20130101;
C07C 47/21 20130101; C07F 9/5442 20130101; C07C 45/64 20130101;
C07F 7/188 20130101; C07C 403/24 20130101 |
Class at
Publication: |
568/346 |
International
Class: |
C07C 45/45 20060101
C07C045/45 |
Claims
1-32. (canceled)
33. A method of producing lutein comprising: contacting a diketone
compound having the structure: ##STR151## with a chiral catalyst,
to stereoselectively reduce the diketone to give a hydroxy product
having the structure: ##STR152## wherein the hydroxy product is
optically active; contacting the hydroxy product with a reducing
agent to form a dihydroxy compound having the structure: ##STR153##
wherein the dihydroxy compound is optically active; and performing
additional chemical processes to form lutein from the dihydroxy
compound.
34. The method of claim 33, wherein the chiral catalyst comprises
metal and an optically active chiral ligand.
35. The method of claim 33, wherein the chiral catalyst comprises
ruthenium and an optically active chiral ligand.
36. The method of claim 33, wherein the chiral catalyst comprises
ruthenium and an optically active amine.
37. The method of claim 33, wherein the chiral catalyst comprises
ruthenium and an optically active amino acid.
38. The method of claim 33, wherein the chiral catalyst comprises
ruthenium and an optically active amine having the structure
H.sub.2N--CHPh-CHPh-OH.
39. The method of claim 33, wherein the chiral catalyst comprises
ruthenium and an optically active amine having the structure
H.sub.2N--CHMe-CHPh-OH.
40. The method of claim 33, wherein the chiral catalyst comprises
ruthenium and an optically active amine having the structure
MeHN--CHMe-CHPh-OH.
41. The method of claim 33, wherein the reducing agent comprises a
borohydride reducing agent.
42. The method of claim 33, wherein the reducing agent comprises a
lithium trialkyl borohydride reducing agent.
43. The method of claim 33, wherein the reducing agent comprises an
aluminum hydride reducing agent.
44. The method of claim 33, wherein performing additional chemical
processes to form lutein comprises: converting the dihydroxy
compound into a protected ketone having the structure: ##STR154##
where R.sup.1 is alkyl, phenyl, aryl or silyl.
45. The method of claim 44, wherein performing additional chemical
processes to form lutein comprises: converting the protected ketone
into an unsaturated alkyne having the structure: ##STR155## where
R.sup.1 is alkyl, phenyl, aryl or silyl,
46. The method of claim 45, wherein converting the protected ketone
into the unsaturated alkyne comprises: reacting the protected
ketone with an acetylinic compound having the structure:
M.sup.+-C.ident.C--R.sup.2, where M is a metal and R.sup.2 is
##STR156## to give an addition product having the structure:
##STR157##
47. The method of claim 45, wherein performing additional chemical
processes to form lutein comprises: converting the unsaturated
alkyne into a compound having the structure: ##STR158## where
R.sup.1 is alkyl, phenyl, aryl or silyl; and R.sup.3 is
PR.sup.4.sub.3, P(O)R.sup.4.sub.3, SO.sub.2R.sup.4, or M.sup.+
where R.sup.4 is alkyl, phenyl, or aryl and M is Li, Na, or
MgBr.
48. The method of claim 47, wherein performing additional chemical
processes to form lutein comprises: reacting the compound having
the structure: ##STR159## where R.sup.1 is alkyl, phenyl, aryl or
silyl; and R.sup.3 is PR.sup.4.sub.3, P(O)R.sup.4.sub.3,
SO.sub.2R.sup.4, or M.sup.+ where R.sup.4 is alkyl, phenyl, or aryl
and M is Li, Na, or MgBr with an aldehyde having the structure:
##STR160## to form a lutein intermediate.
49. The method of claim 33, wherein lutein is formed as the 3R,
3'R,6R stereoisomer.
50-123. (canceled)
124. A method of producing lutein comprising: contacting a diketone
compound having the structure: ##STR161## with a chiral catalyst,
to stereoselectively reduce the diketone to give a hydroxy product
having the structure: ##STR162## wherein the hydroxy product is
optically active; contacting the hydroxy product with a reducing
agent to form a dihydroxy compound: ##STR163## wherein the
dihydroxy compound is optically active; and performing additional
chemical processes to form lutein from the dihydroxy compound.
125. The method of claim 124, wherein the chiral catalyst comprises
ruthenium and an optically active amine.
126. The method of claim 124, wherein lutein is formed as the
3S,3'S,6'S stereoisomer.
Description
PRIORTY CLAIM
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/615,032 entitiled "Methods for Synthesis of
Carotenoids, Including Analogs, Derivatives, and Synthetic and
Biological Intermediates" filed on Oct. 1, 2004; U.S. Provisional
Patent Application No. 60/675,957 entitiled "Methods for Synthesis
of Carotenoids, Including Analogs, Derivatives, and Synthetic and
Biological Intermediates" filed on Apr. 29, 2005; U.S. Provisional
Patent Application No. 60/691,518 entitiled "Methods for Synthesis
of Carotenoids, Including Analogs, Derivatives, and Synthetic and
Biological Intermediates" filed on Jun. 17, 2005; U.S. Provisional
Patent Application No. 60/692,682 entitiled "Methods for Synthesis
of Carotenoids, Including Analogs, Derivatives, and Synthetic and
Biological Intermediates" filed on Jun. 21, 2005; U.S. Provisional
Patent Application No. 60/699,653 entitiled "Methods for Synthesis
of Carotenoids, Including Analogs, Derivatives, and Synthetic and
Biological Intermediates" filed on Jul. 15, 2005; U.S. Provisional
Patent Application No. 60/702,380 entitiled "Methods for Synthesis
of Carotenoids, Including Analogs, Derivatives, and Synthetic and
Biological Intermediates" filed on Jul. 26, 2005; and U.S.
Provisional Patent Application No. 60/712,350 entitiled "Methods
for Synthesis of Carotenoids, Including Analogs, Derivatives, and
Synthetic and Biological Intermediates" filed on Aug. 30, 2005.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention generally relates to the fields of medicinal
and synthetic chemistry. More specifically, the invention relates
to the synthesis and use of carotenoids, including analogs,
derivatives, and intermediates.
[0004] 2. Description of the Relevant Art
[0005] Carotenoids are a group of natural pigments produced
principally by plants, yeast, and microalgae. The family of related
compounds now numbers greater than 700 described members, exclusive
of Z and E isomers. At least fifty (50) carotenoids have been found
in human sera or tissues. Humans and other animals cannot
synthesize carotenoids de novo and must obtain them from their
diet. All carotenoids share common chemical features, such as a
polyisoprenoid structure, a long polyene chain forming the
chromophore, and near symmetry around the central double bond.
Tail-to-tail linkage of two C.sub.20 geranyl diphosphate molecules
produces the parent C.sub.40 carbon skeleton. Carotenoids without
oxygenated functional groups are called "carotenes", reflecting
their hydrocarbon nature; oxygenated carotenes are known as
"xanthophylls." Cyclization at one or both ends of the molecule
yields 7 identified end groups (illustrative structures shown in
FIG. 1).
[0006] Documented carotenoid functions in nature include
light-harvesting, photoprotection, and protective and sex-related
coloration in microscopic organisms, mammals, and birds,
respectively. A relatively recent observation has been the
protective role of carotenoids against age-related diseases in
humans as part of a complex antioxidant network within cells. This
role is dictated by the close relationship between the
physicochemical properties of individual carotenoids and their in
vivo functions in organisms. The long system of alternating double
and single bonds in the central part of the molecule (delocalizing
the .pi.-orbital electrons over the entire length of the polyene
chain) confers the distinctive molecular shape, chemical
reactivity, and light-absorbing properties of carotenoids.
Additionally, isomerism around C.dbd.C double bonds yields
distinctly different molecular structures that may be isolated as
separate compounds (known as Z ("cis") and E ("trans"), or
geometric, isomers). Of the more than 700 described carotenoids, an
even greater number of the theoretically possible mono-Z and poly-Z
isomers are sometimes encountered in nature. The presence of a Z
double bond creates greater steric hindrance between nearby
hydrogen atoms and/or methyl groups, so that Z isomers are
generally less stable thermodynamically, and more chemically
reactive, than the corresponding all-E form. The all-E
configuration is an extended, linear, and rigid molecule. Z-isomers
are, by contrast, not simple, linear molecules (the so-called
"bent-chain" isomers). The presence of any Z in the polyene chain
creates a bent-chain molecule. The tendency of Z-isomers to
crystallize or aggregate is much less than all-E, and Z isomers may
sometimes be more readily solubilized, absorbed, and transported in
vivo than their all-E counterparts. This has important implications
for enteral (e.g., oral) and parenteral (e.g., intravenous,
intra-arterial, intramuscular, intraperitoneal, intracoronary, and
subcutaneous) dosing in mammals.
[0007] Carotenoids with chiral centers may exist either as the R
(rectus) or S (sinister) configurations. As an example, astaxanthin
(with 2 chiral centers at the 3 and 3' carbons) may exist as 3
possible stereoisomers: 3S,3'S; 3R,3'S and 3S,3'R (identical meso
forms); or 3R,3'R. The relative proportions of each of the stereois
vary by natural source. For example, Haematococcus pluvialis
microalgal meal is 99% 3S,3'S astaxanthin, and is likely the
predominant human evolutionary source of astaxanthin. Krill
(3R,3'R) and yeast sources yield different stereoisomer
compositions than the microalgal source. Synthetic astaxanthin,
produced by large manufacturers such as Hoffmann-LaRoche AG,
Buckton Scott (USA), or BASF AG, are provided as defined geometric
isomer mixtures of a 1:2:1 stereoisomer mixture (3S,3'S; 3R,3'S,
(meso); 3R,3'R) of non-esterified, free astaxanthin. Natural source
astaxanthin from salmonid fish is predominantly a single
stereoisomer (3S,3'S), but does contain a mixture of geometric
isomers. Astaxanthin from the natural source Haematococcus
pluvialis may contain nearly 50% Z isomers. As stated above, the Z
conformational change may lead to a higher steric interference
between the two parts of the carotenoid molecule, rendering it less
stable, more reactive, and more susceptible to reactivity at low
oxygen tensions. In such a situation, in relation to the all-E
form, the Z forms: (1) may be degraded first; (2) may better
suppress the attack of cells by reactive oxygen species such as
superoxide anion; and (3) may preferentially slow the formation of
radicals. Overall, the Z forms may initially be thermodynamically
favored to protect the lipophilic portions of the cell and the cell
membrane from destruction. It is important to note, however, that
the all-E form of astaxanthin, unlike .beta.-carotene, retains
significant oral bioavailability as well as antioxidant capacity in
the form of its dihydroxy- and diketo-substitutions on the
.beta.-ionone rings, and has been demonstrated to have increased
efficacy over .beta.-carotene in most studies. The all-E form of
astaxanthin has also been postulated to have the most
membrane-stabilizing effect on cells in vivo. Therefore, it is
likely that the all-E form of astaxanthin in natural and synthetic
mixtures of stereoisomers is also extremely important in
antioxidant mechanisms, and may be the form most suitable for
particular pharmaceutical preparations.
[0008] The antioxidant mechanism(s) of carotenoids, (e.g.,
astaxanthin), includes singlet oxygen quenching, direct radical
scavenging, and lipid peroxidation chain-breaking. The polyene
chain of the carotenoid absorbs the excited energy of singlet
oxygen, effectively stabilizing the energy transfer by
delocalization along the chain, and dissipates the energy to the
local environment as heat. Transfer of energy from triplet-state
chlorophyll (in plants) or other porphyrins and proto-porphyrins
(in mammals) to carotenoids occurs much more readily than the
alternative energy transfer to oxygen to form the highly reactive
and destructive singlet oxygen (.sup.1O.sub.2). Carotenoids may
also accept the excitation energy from singlet oxygen if any should
be formed in situ, and again dissipate the energy as heat to the
local environment. This singlet oxygen quenching ability has
significant implications in cardiac ischemia, macular degeneration,
porphyria, and other disease states in which production of singlet
oxygen has damaging effects. In the physical quenching mechanism,
the carotenoid molecule may be regenerated (most frequently), or be
lost. Carotenoids are also excellent chain-breaking antioxidants, a
mechanism important in inhibiting the peroxidation of lipids.
Astaxanthin can donate a hydrogen (H) to the unstable
polyunsaturated fatty acid (PUFA) radical, stopping the chain
reaction. Peroxyl radicals may also, by addition to the polyene
chain of carotenoids, be the proximate cause for lipid peroxide
chain termination. The appropriate dose of astaxanthin has been
shown to completely suppress the peroxyl radical chain reaction in
liposome systems. Astaxanthin shares with vitamin E this dual
antioxidant defense system of singlet oxygen quenching and direct
radical scavenging, and in most instances (and particularly at low
oxygen tension in vivo) is superior to vitamin E as a radical
scavenger and physical quencher of singlet oxygen.
[0009] Carotenoids, (e.g., astaxanthin), are potent direct radical
scavengers and singlet oxygen quenchers and possess all the
desirable qualities of such therapeutic agents for inhibition or
amelioration of ischemia-reperfusion injury. Synthesis of novel
carotenoid derivatives with "soft-drug" properties (i.e. active as
antioxidants in the derivatized form), with physiologically
relevant, cleavable linkages to pro-moieties, can generate
significant levels of free carotenoids in both plasma and solid
organs. In the case of non-esterified, free astaxanthin, this is a
particularly useful embodiment (characteristics specific to
non-esterified, free astaxanthin below):
[0010] Lipid soluble in natural form; may be modified to become
more water soluble;
[0011] Molecular weight of 597 Daltons (size <600 daltons (Da)
readily crosses the blood brain barrier, or BBB);
[0012] Long polyene chain characteristic of carotenoids effective
in singlet oxygen quenching and lipid peroxidation chain breaking;
and
[0013] No pro-vitamin A activity in mammals (eliminating concerns
of hypervitaminosis A and retinoid toxicity in humans).
[0014] The administration of antioxidants which are potent singlet
oxygen quenchers and direct radical scavengers, particularly of
superoxide anion, should limit hepatic fibrosis and the progression
to cirrhosis by affecting the activation of hepatic stellate cells
early in the fibrogenetic pathway. Reduction in the level of
"Reactive Oxygen Species" (ROS) by the administration of a potent
antioxidant can therefore be crucial in the prevention of the
activation of both "hepatic stellate cells" (HSC) and Kupffer
cells. This protective antioxidant effect appears to be spread
across the range of potential therapeutic antioxidants, including
water-soluble (e.g., vitamin C, glutathione, resveratrol) and
lipophilic (e.g., vitamin E, .beta.-carotene, astaxanthin) agents.
Therefore, a co-antioxidant derivative strategy in which
water-soluble and lipophilic agents are combined synthetically is a
particularly useful embodiment. Examples of uses of carotenoid
derivatives and analogs are illustrated in U.S. patent application
Ser. No. 10/793,671 filed on Mar. 4, 2004, entitled "CAROTENOID
ETHER ANALOGS OR DERIVATIVES FOR THE INHIBITION AND AMELIORATION OF
DISEASE" to Lockwood et al. published on Jan. 13, 2005, as
Publication No. US-2005-0009758 and PCT International Application
Number PCT/US2003/023706 filed on Jul. 29,2003, entitled
"STRUCTURAL CAROTENOID ANALOGS FOR THE INHIBITION AND AMELIORATION
OF DISEASE" to Lockwood et al. (International Publication Number WO
2004/011423 A2, published on Feb. 5, 2004) both of which are
incorporated by reference as if fully set forth herein.
[0015] Vitamin E is generally considered the reference antioxidant.
When compared with vitamin E, carotenoids are more efficient in
quenching singlet oxygen in homogeneous organic solvents and in
liposome systems. They are better chain-breaking antioxidants as
well in liposomal systems. They have demonstrated increased
efficacy and potency in vivo. They are particularly effective at
low oxygen tension, and in low concentration, making them extremely
effective agents in disease conditions in which ischemia is an
important part of the tissue injury and pathology. These
carotenoids also have a natural tropism for the heart and liver
after oral administration. Therefore, therapeutic administration of
carotenoids should provide a greater benefit in limiting fibrosis
than vitamin E.
[0016] Problems related to the use of some carotenoids and
structural carotenoid analogs or derivatives include: (1) the
complex isomeric mixtures, including non-carotenoid contaminants,
provided in natural and synthetic sources leading to costly
increases in safety and efficacy tests required by such agencies as
the FDA; (2) limited bioavailability upon administration to a
subject; and (3) the differential induction of cytochrome P450
enzymes (this family of enzymes exhibits species-specific
differences which must be taken into account when extrapolating
animal work to human studies). Selection of the appropriate analog
or derivative and isomer composition for a particular application
increases the utility of carotenoid analogs or derivatives for the
uses defined herein.
[0017] Synthesis of an appropriate analog or derivative and isomer
composition requires a supply of starting materials (e.g.,
carotenoids, carotenoid synthetic intermediates). Any new synthetic
route which is more efficient to a carotenoid analog or derivative
and/or synthetic intermediate would be beneficial. More efficient
synthetic routes would provide a more stable source of starting
materials (e.g., carotenoids) which may be difficult or expensive
to extract from natural sources. Synthetic routes to natural
products may facilitate the synthesis of analogs and derivatives of
the natural products.
SUMMARY
[0018] A synthetic route to a carotenoid, carotenoid analog or
derivative and/or synthetic intermediate is presented. In some
embodiments, methods and reactions described herein may be used to
synthesize naturally-occurring carotenoids. Naturally-occurring
carotenoids may include astaxanthin as well as other carotenoids
including, but not limited to, zeaxanthin, carotenediol,
nostoxanthin, crustaxanthin, canthaxanthin, isozeaxanthin,
hydroxycanthaxanthin, tetrahydroxy-carotene-dione, lutein,
lycophyll, and lycopene.
[0019] In one embodiment, a method of making lutein includes:
contacting a diketone compound having the structure ##STR1## where
each R is independently alkyl, phenyl, or aryl, with a chiral
catalyst, to stereoselectively reduce the ketone to give a hydroxy
product having the general structure: ##STR2## wherein R is alkyl,
phenyl, or aryl and wherein the "*" represents a chiral carbon atom
that exists, predominantly, as a single stereoisomer; and
contacting the hydroxy product with a reducing agent to form the
dihydroxy compound ##STR3## wherein the "*" represents chiral
carbon atoms that exist, predominantly, as a single
stereoisomer.
[0020] In some embodiments, each R is methyl. The chiral catalyst,
in some embodiments, includes a metal and an optically active
chiral ligand. The metal may be any transition metal. In some
embodiments, the metal is ruthenium. A chiral catalyst may include
ruthenium and an optically active chiral ligand. In some
embodiments, an optically active chiral ligand is an optically
active amine. Examples of optically active amines include: amino
acids, H.sub.2N--CHPh-CHPh-OH, H.sub.2N--CHMe-CHPh-OH,
MeHN--CHMe-CHPh-OH, H.sub.2N--CHPh-CHPh-OH, H.sub.2N--CHMe-CHPh-OH,
MeHN--CHMe-CHPh-OH.
[0021] The reducing agent may include any reducing agent capable of
reducing a ketone to a hydroxyl functional group. In some
embodiments the reducing agent is borohydride reducing agent. The
borohydride reducing agent may be a lithium trialkyl borohydride
reducing agent. In alternate embodiments, the reducing agent may be
an aluminum hydride reducing agent.
[0022] Use of a chiral catalyst to reduce the diketone starting
material may lead to optically active stereoisomers that include
the hydroxy ketone compound ##STR4## which may be further
transformed into the optically active dihydroxy compound
##STR5##
[0023] In an embodiment,the dihydroxy compound may be converted
into protected ketone having the structure ##STR6## where R.sup.1
is alkyl, phenyl, aryl or silyl.
[0024] The protected ketone may be converted into the unsaturated
ketone having the structure: ##STR7## where R.sup.1 is alkyl,
phenyl, aryl or silyl, and R.sup.2 is ##STR8## Formtaion of the
unsaturated ketone may be accomplished by reacting the protected
ketone with an acetylinic compound having the structure:
[0025] M.sup.+--C.ident.C--R.sup.2, where M is a metal and R.sup.2
is ##STR9## to give an addition product having the structure:
##STR10## and reacting the addition product with an oxidant to give
the unsaturated ketone. The unsaturated ketone may then be
converted into an aldehyde reactive compound having the structure:
##STR11## where R.sup.1 is is alkyl, phenyl, aryl or silyl; and
R.sup.3 is PR.sup.4.sub.3, P(O)R.sup.4.sub.3, SO.sub.2R.sup.4, or
M.sup.+where R.sup.4 is alkyl, phenyl, or aryl and M is Li, Na, or
MgBr. The aldehyde reactive compound may then be reacted with the
dialdehyde having the structure ##STR12## to form lutein. All
stereoisomers of lutein may be selectively formed using this method
including the 3R,3'R,6'R stereoisomer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The above brief description as well as further objects,
features and advantages of the methods and apparatus of the present
invention will be more fully appreciated by reference to the
following detailed description of presently preferred but
nonetheless illustrative embodiments in accordance with the present
invention when taken in conjunction with the accompanying
drawings.
[0027] FIG. 1 depicts a graphic representation of several examples
of "parent" carotenoid structures as found in nature.
[0028] While the invention is susceptible to various modifications
and alternative forms, specific embodiments thereof are shown by
way of example in the drawings and may herein be described in
detail. The drawings may not be to scale. It should be understood,
however, that the drawings and detailed description thereto are not
intended to limit the invention to the particular form disclosed,
but on the contrary, the intention is to cover all modifications,
equivalents and alternatives falling within the spirit and scope of
the present invention as defined by the appended claims.
DETAILED DESCRIPTION
[0029] Compounds described herein embrace both racemic and
optically active compounds. Chemical structures depicted herein
which do not designate specific stereochemistry are intended to
embrace all possible stereochemistries.
[0030] It will be appreciated by those skilled in the art that
compounds having one or more chiral center(s) may exist in and be
isolated in optically active and racemic forms. Some compounds may
exhibit polymorphism. It is to be understood that the present
invention encompasses any racemic, optically-active, polymorphic,
or stereoisomeric form, or mixtures thereof, of a compound. As used
herein, the term "single stereoisomer" refers to a compound having
one or more chiral center that, while it can exist as two or more
stereoisomers, is isolated in greater than about 95% excess of one
of the possible stereoisomers. As used herein a compound that has
one or more chiral centers is considered to be "optically active"
when isolated or used as a single stereoisomer.
[0031] The following definitions are used, unless otherwise
described. Halo, as used herein refers to fluoro, chloro, bromo, or
iodo. "Alkyl," "alkoxy," etc. denote both straight and branched
groups; but reference to an individual radical such as "propyl"
embraces only the straight chain radical, a branched chain isomer
such as "isopropyl" being specifically referred to.
[0032] Specific and preferred values listed below for radicals,
substituents, and ranges, are for illustration only; they do not
exclude other defined values or other values within defined ranges
for the radicals and substituents. Specifically, "alkyl" includes,
but is not limited to: methyl, ethyl, propyl, isopropyl, butyl,
iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl, octyl,
nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl or pentadecyl;
"alkenyl" includes but is not limited to vinyl, 1-propenyl,
2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl
3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,
5-hexenyl, 1-heptenyl, 2-hepteny, 3-heptenyl 4-heptenyl,
5-heptenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl,
6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl,
4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl;
1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl,
6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl,
1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl,
6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl,
11-dodecenyl, 1-tridecenyl, 2-tridecenyl, 3-tridecenyl,
4-tridecenyl, 5-tridecenyl, 6-tridecenyl, 7-tridecenyl,
8-tridecenyl, 9-tridecenyl, 10-tridecenyl, 11-tridecenyl,
12-tredecenyl, 1-tetradecenyl, 2-tetradecenyl, 3-tetradecenyl,
4-tetradecenyl, 5-tetradecenyl, 6-tetradecenyl, 7-tetradeceny,
8-tetradecenyl, 9-tetradecenyl, 10-tetradecenyl, 11-tetradecenyl,
12-tetradecenyl, 13-tetradeceny, 1-pentadecenyl, 2-pentadecenyl,
3-pentadecenyl, 4-pentadecenyl, 5-pentadecenyl, 6-pentadecenyl,
7-pentadecenyl, 8-pentadeceny, 9-pentadecenyl, 10-pentadecenyl,
11-pentadecenyl, 12-pentadecenyl, 13-pentadecenyl, 14-pentadecenyl;
"alkoxy" includes but is not limited to methoxy, ethoxy, propoxy,
isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy,
hexoxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy,
dodecyloxy, tridecyloxy, tetradecyloxy, or pentadecyloxy;
"cycloalkyl" includes, but is not limited to cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl.
"Aryl" includes but is not limited to phenyl, substituted benzene,
naphthyl, substituted naphthyl, anthracene, or substituted
anthracene.
[0033] The synthesis of certain naturally-occurring carotenoids is
presented herein. In some embodiments, methods and reactions
described herein may be used to synthesize naturally-occurring
carotenoids. Naturally-occurring carotenoids may include
astaxanthin as well as other carotenoids. Some of the other
carotenoids may include carotenoids such as, for example,
zeaxanthin, carotenediol, nostoxanthin, crustaxanthin,
canthaxanthin, isozeaxanthin, hydroxycanthaxanthin,
tetrahydroxy-carotene-dione, lutein, and lycopene. Carotenoids
having the general formula (I) below may be synthesized using the
methods described herein. ##STR13## Where X, Y, and Z are
independently --OH or .dbd.O.
[0034] The compound of formula I embraces "racemic" (e.g.
statistical mixture of stereoisomers), optically inactive (e.g.
meso forms) and optically active (e.g. enantiomeric) compounds. In
some embodiments, carotenoids may be isolated using methods
described herein with an enantiomeric excess of greater than 99%.
In some embodiments, carotenoids may be isolated using methods
described herein with an enantiomeric excess of greater than 95%.
In some embodiments, carotenoids may be isolated using methods
described herein with an enantiomeric excess of greater than
90%.
[0035] In some embodiments, Z is H, Y is --OH, and X is .dbd.O such
that the carotenoid has the general structure depicted below. The
carotenoid below is commonly referred to as astaxanthin.
##STR14##
[0036] In some embodiments, Z is H, Y is OH, and X is O such that
the carotenoid has the general structure depicted below. The
carotenoid below is commonly referred to as crustaxanthin.
##STR15##
[0037] In some embodiments, Z is H, Y is H, and X is .dbd.O such
that the carotenoid has the general structure depicted below. The
carotenoid below is commonly referred to as canthaxanthin.
##STR16##
[0038] In some embodiments, Z is H, Y is H, and X is --OH such that
the carotenoid has the general structure depicted below. The
carotenoid below is commonly referred to as isozeaxanthin.
##STR17##
[0039] In some embodiments, Z is OH, Y is H, and X is .dbd.O such
that the carotenoid has the general structure depicted below. The
carotenoid below is commonly referred to as hydroxycanthaxanthin.
##STR18##
[0040] In some embodiments, Z and Y are --OH and X is .dbd.O such
that the carotenoid has the general structure depicted below. The
carotenoid below is commonly referred to as
tetrahydroxy-carotene-dione ##STR19##
[0041] In an embodiment, carotenoids may be synthesized using the
general process shown in Scheme I below. ##STR20##
[0042] Where X, Y, and Z are independently --OH or .dbd.O; where
R.sup.3 is PR.sup.4.sub.3, SO.sub.2R.sup.4, or M.sup.+. R.sup.4 is
alkyl, phenyl, or aryl. M is Li, Na, or MgBr. Coupling of two "head
units" with the C.sub.10-aldehyde yields carotenoid. Coupling may
be accomplished using a Wittig coupling (R.sup.3 is
PR.sup.4.sub.3), sulphone coupling (R.sup.3 is SO.sub.2R.sup.4), or
condensation reaction (R.sup.3 is M.sup.+). The C.sub.10 aldehyde
is commercially available. Described herein are various methods of
synthesizing the appropriate headpiece. The following U.S. Patents,
all of which are incorporated herein by reference, describe the
synthesis of various carotene and carotenoid synthesis
intermediates: U.S. Pat. No. 4,245,109 to Mayer et al., U.S. Pat.
No. 4,283,559 to Broger et al, U.S. Pat. No. 4,585,885 to Bernhard
et al., U.S. Pat. No. 4,952,716 to Lukac et al., and U.S. Pat. No.
6,747,177 to Ernst et al.
[0043] In one embodiment, a headpiece useful for the synthesis of
astaxanthin may be formed using the process depicted in Scheme II.
##STR21##
[0044] While the compounds shown in Scheme II are generally
depicted as single stereoisomers, it should be understood that
Scheme II may be used to synthesize the racemic headpiece. An
intermediate in the synthesis of astaxanthin is shown below as
compound 108A. ##STR22## R.sup.1 may include hydrogen, alkyl, or
aryl. R.sup.3 may also include any alcohol protecting groups known
to one skilled in the art. Protecting groups may include, but are
not limited to, silyl protecting groups such as
tert-butyldimethylsilane (i.e., TBDMS). In some embodiments,
compound 108a may be synthesized from commercially available
keto-.alpha.-isopherone 109 having a general formula of
##STR23##
[0045] Keto-.alpha.-isopherone may be selectively reduced. The more
sterically hindered ketone may be reduced to an alcohol. The more
sterically hindered ketone A may be stereoselectively reduced to an
alcohol. In some embodiments, a complexing reagent may be used to
react with the less sterically hindered ketone. In so doing this,
the complexing agent may protect the less sterically hindered
ketone B from reacting with a reagent (e.g., a reducing agent),
thereby directing the reagent to react with the more sterically
hindered ketone A.
[0046] In some embodiments, a complexing agent may also be
optically pure or form an optically pure complex with an activating
metal, either of which may react with the less sterically hindered
ketone B, such that the reduction of more sterically hindered
ketone A results in an optically pure product. It should be noted
that within the description herein absolute terms or phrases used
(e.g., optically pure) are understood to include at least a range
typically acceptable to one skilled in the art. In one example, the
optically pure product referred to regarding the reduced ketone may
be >90% pure. In an example, the optically pure product referred
to regarding the reduced ketone may be >95% pure. In an example,
the optically pure product referred to regarding the reduced ketone
may be >99% pure. In an example, the optically pure product
referred to regarding the reduced ketone may be >99.9% pure.
[0047] In some embodiments a reduction catalyst may be a chiral
catalyst. A "chiral catalyst" a defined herein is a catalyst that
includes a single stereoisomer of a chiral molecule. In one
embodiment, a chiral catalyst includes a transition metal and an
optically active chiral ligand. Transition metals that may be used
to form a chiral catalyst for reduction of ketones include Ti, Zr,
Hf, V, Nb, Ta, Cr, Mo, W, Mn, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd,
Pt, Cu, Ag, and Au. In some embodiments, a ruthenium chiral
catalyst may be used to effect a stereoselective reduction of
keto-.alpha.-isopherone. The ruthenium chiral catalyst may be
formed from a mixture of [RuX.sub.2(.eta..sup.6-Ar)].sub.2 with an
optically active amine, where X represents a halogen (e.g., F, Cl,
Br, I) and Ar represents benzene or a substituted benzene (e.g.,
alkyl substituted benzene). In some embodiments, the optically
active amine includes both (S)- and (R)-amino acids, and other
optically active amines such as as H.sub.2N--CHPh-CHPh-OH,
H.sub.2N--CHMe-CHPh-OH, MeHN--CHMe-CHPh-OH. Reduction of
keto-.alpha.-isopherone with a chiral catalyst may yield the
optically active hydroxy ketone 116. While hydroxy ketone 116 is
depicted in the (R)-form, it should be understood that the (S)-form
may be formed by using the opposite optically active compound to
form a chiral catalyst. For example, forming a ruthenium catalyst
using (1R,2S)-(-)-norephedrine leads to the (R)-form of the hydroxy
ketone depicted below, while forming a ruthenium catalyst using
(1S,2R)-(+)-norephedrine leads to the (S)-form of the hydroxy
ketone below. Further details regarding the use of ruthenium
catalyst for the reduction of keto-.alpha.-isopherone may be found
in the paper "Synthesis of (R)- and (S)-hydroxyisophorone by
ruthenium-catalyzed asymmetric transfer hydrogenation of
ketoisopherone" by Henning et al., Tetrahedron:Asymmetry, 11 (2000)
1849-1858, which is incorporated herein by reference. ##STR24##
[0048] Compound 116 may be further reduced. The remaining ketone of
compound 116 may be reduced to an alcohol. The resulting alcohol to
which the remaining ketone of compound 116 has been reduced may be
optically pure. Any type of reducing agent suitable for reducing a
ketone to a hydroxy group may be used. The reducing agent may be a
chiral reducing agent or an a chiral reducing agent. The
stereoselectivity of the reduction at hydroxyl (D) is controlled,
at least in part, by the stereochemistry of the hydroxy group (C)
as depicted in 118.
[0049] In some embodiments, a borohydride reducing agent may be
used to reduce the ketone group of compound 116. In an embodiment,
a hindered borohydride reducing agent may be used to assist in
achieving an enantiomerically pure reduction of the remaining
ketone of compound 116. In an embodiment, the hindered borohydride
reducing agent is a lithium trialkyl borohydride. Examples of
lithium trialkyl borohydrides include, but are not limited to,
lithium tri-sec-butylborohydride and lithium trisiamylborohydride.
Reduction of the remaining ketone of 116 results in compound 118
having a general formula of ##STR25## Other types of hindered
reducing agents may be used such as hindered aluminum hydride
reducing agents may also be used to reduce ketone 116.
[0050] Alcohol D of compound 118 may be selectively protected using
any number of alcohol protecting groups known to one skilled in the
art to produce compound 120 having the general structure of
##STR26## where R.sup.1 is alkyl, phenyl, aryl or silyl. In some
embodiments, protecting groups may include sterically hindered
protecting groups. Examples of sterically hindered protecting
groups include hindered silyl protecting groups. Silyl protecting
groups may include, but are not limited to, trimethylsilane,
triethylsilane, triisopropylsilane, tert-butyl dimethyl silane
(i.e., TBDMS), and diphenyl-t-butylsilane. If R.sup.1 is a TBDMS
group, the resulting protected compound has the structure of 120a
##STR27##
[0051] Upon protecting less sterically hindered alcohol D, more
sterically hindered alcohol C may be oxidized to a ketone.
Oxidation of hydroxyl C may be accomplished using a variety of
oxidizing reagents such as chromium oxidants, manganese oxidants,
and selenium oxidants. In one embodiment, the oxidizing agent may
include, for example, pyridinium dichromate (PDC). Oxidation of
hydroxyl group C leads to optically active ketone 108a, where
R.sup.1 is alkyl, phenyl, aryl or silyl. ##STR28## In some
embodiments, R.sup.1 may include a protecting group (e.g., TBDMS)
such that 108a has a general structure of 108b ##STR29##
[0052] In some embodiments, an enantiomeric excess of compound 108a
may be determined. Enantiomeric excess may be determined by first
removing any protecting groups, then measuring the optical purity
using circular dichroism (CD) spectroscopy.
[0053] As depicted in Scheme II protected hydroxy ketone 108 may be
used to synthesize astaxanthin, as well as other carotenoid
derivatives, as described herein. In an embodiment, ketone 108 is
reacted with a nucleophilic acetylenic derivative to form an
addition product 112 depicted below ##STR30## where R.sup.1 is
alkyl, phenyl, aryl or silyl. Compound 112 may be formed by
reacting ketone 108 with a nucleophile. The nucleophile may
selectively react with the carbonyl group of compound 108,
transforming the carbonyl to an alcohol, as well as forming a new
substituent at the 2 position of the carbonyl. In a specific
embodiment compound 108 may be alkynylated. An alkyne may be
reacted with compound 108 in an inert solvent (e.g.,
tetrahydrofuran ("THF")). The reaction is preferably carried out at
low temperatures. Alkynes may include compounds having the general
formula H--C.ident.C--R.sup.2 where R.sup.2 includes: ##STR31## and
where R.sup.1 is alkyl, phenyl, aryl or silyl. In some embodiments,
R.sup.2 may include other substituents known to one skilled in the
art (e.g., H, silane substituents, alkynes, alkenes, alkyls, aryl
substituents, heteroaryl substituents). Addition of alkyne
H--C.ident.C--R.sup.2 to ketone may be accomplished by forming a
metal anion of the acetylene, to form the reactive nucleophilic
acetylenic compound M.sup.+-C.ident.C--R.sup.2, where M.sup.+ may
be, but is not limited to, Li, Na, MgBr, Cd, or Zn. A lithium salt
of alkyne H--C.ident.C--R.sup.2 may be formed by reacting the
alkyne with, for example, BuLi. Other metal salts of alkynes may be
made using methods known to one or ordinary skill in the art. The
nucleophilic acetylenic compound M.sup.+-C.ident.C--R.sup.2 may be
reacted with ketone 108 to form a coupling product 112 as depicted
below: ##STR32## where R.sup.2 includes: ##STR33## and where
R.sup.1 s alkyl, phenyl, aryl or silyl.
[0054] Compound 112 may be subjected to rearrangement conditions
and oxidized to be converted into unsaturated ketone 114, as
depicted below. ##STR34## where R.sup.2 includes: ##STR35## and
where R.sup.1 is alkyl, phenyl, aryl or silyl. Unsaturated ketone
114 may be formed by a two step process or in a novel one step
rearrangement oxidation. In one embodiment, compound 112 is
subjected to rearrangement conditions (e.g., treatment with aqueous
acid) to effect rearrangement of the alcohol to an allylic alcohol
(not shown). Subsequent oxidation of the allylic alcohol leads to
the unsaturated ketone 114. This two step procedure reduces the
efficiency of the process.
[0055] In an alternate embodiment, treatment of compound 112 with
an oxidant affords the unsaturated ketone 114. This ketone is
formed by simultaneous rearrangement and oxidation of the alcohol.
The oxidizing agent used in a one-step process may include, for
example, chromium oxidant (e.g., pyridinium dichlorochromate
(PDC)), selenium oxidant, or manganese oxidant.
[0056] Unsaturated ketone 114 may be reduced to olefin 104 as
depicted below. ##STR36## Compound 114 may be used to synthesize
compound 104. Treatment of compound 114 with an appropriate
reducing agent may reduce the alkyne substituent to give an
E-olefin as depicted above. Reducing metal reductions are
particularly suited for forming E-olefins from alkynes. Reducing
metal reductions may be accomplished using reagents such as
Li/NH.sub.3, Na/NH.sub.3 and Zn/acid. In some embodiments, zinc and
an acid may be used to reduce the alkyne to an alkene. The acid may
include, for example, glacial acetic acid, ammonium acetate and/or
ammonium chloride. The reduction yields the E-isomer predominantly.
In some embodiments, one or more protecting groups (e.g., alcohol
protecting groups (R.sup.1)) may be removed before partially
reducing the alkyne to an alkene.
[0057] Upon formation of conjugated alkene 104, the intermediate
may be converted into compound 102 having a functional group
capable of reacting with an aldehyde to form a double bond.
##STR37##
[0058] Examples of functionalities that may be reacted with an
aldehyde include PR.sup.4.sub.3, SO.sub.2R.sup.4, or M.sup.+ where
R.sup.4 is alkyl, phenyl, or aryl and M is Li, Na, or MgBr.
Coupling of two "head units" with a C.sub.10-aldehyde yields a
carotenoid. Coupling may be accomplished using a Wittig coupling
(R.sup.3is PR.sup.4.sub.3), sulphone coupling (R.sup.3 is
SO.sub.2R.sup.4), or condensation reaction (R.sup.3 is M.sup.+). A
phosphonium salt may be synthesized from compound 104. Phosphines
and acid may be used to synthesize the phosphonium salt. Phosphines
may have the general structure --PR.sup.5.sub.3 or
--CH.sub.2--P(.dbd.O)(OR.sup.5).sub.2 where R.sup.5 is alkyl,
phenyl, or aryl. Acids may include any of a number of acids known
to one skilled in the art. One example of an acid which may be used
is hydrogen bromide ("HBr").
[0059] Compound 102 may be reacted with a molecule containing an
aldehyde functionality. The functional group (e.g., the phosphonium
salt) may react with an aldehyde functionality under appropriate
conditions to couple compound 102 to the dialdehyde. Compound 102
may be reacted with a dialdehyde in order to perform a double
coupling as depicted below. ##STR38##
[0060] As shown above, the above-described sequence for the
formation of astaxanthin may be accomplished in a stereoselective
manner to give a single desired stereochemistry. While depicted as
a stereoselective synthesis, it should be understood that the above
described synthesize of astaxanthin may also be accomplished
without control of the stereochemistry to give a statistical
distribution of stereoisomers. In some embodiments, a method may
include analyzing the distribution of stereoisomers of a carotenoid
(e.g., astaxanthin). A method allowing analysis of the distribution
of possible stereoisomers of a carotenoid may be used to determine
the outcome of a synthetic method for preparing a carotenoid. The
method may also be useful for checking the purity of carotenoid
materials provided by chemical manufacturers. In one embodiment, a
chiral HPLC column may be used to determine the stereoisomeric
distribution of a carotenoid.
[0061] In an alternate embodiment, coupling of the headpiece unit
with a coupling agent may be accomplished by forming pendant
aldehyde groups on the headpiece and reacting them with a coupling
agent as depicted below. In some embodiments, a carotenoid, may be
synthesized by condensing a compound of the general formula
##STR39## with a compound of the general formula ##STR40##
[0062] Condensation reactions using compounds such as those
pictured above may, in some embodiments, be coupled under what are
commonly known as Wittig condensation conditions. For example, the
condensation may be carried out in the presence of an alkali metal
alcoholate (e.g., sodium methylate, lithium carbonate, or sodium
carbonate). The condensation may be carried out in the presence of
an alkyl substituted alkylene oxide (e.g., ethylene oxide,
1,2-butylene oxide). Appropriate solvents may be used, such as
alkanols (e.g., methanol, ethanol, isopropanol). The condensation
may be carried out over a range of temperatures. In some
embodiments, the condensation may be carried out below room
temperature (e.g., 0.degree. C.).
[0063] In one embodiment, intermediates used to synthesize
astaxanthin may also be used to synthesize other carotenoids such
as lutein and zeaxanthin. For example, lutein may be synthesized
using the scheme depicted below: ##STR41##
[0064] The synthesis of the intermediate 102 has been described
before with respect to the synthesis of astaxanthin. Synthesis of
the protected dialdehyde compound 130 and the unconjugated
headpiece unit 140, have been described in literature procedures.
The use of the synthetic methodologies described herein to obtain
headpiece 102 may increase the efficiency and/or yield of the
above-described synthesis of lutein.
[0065] In another embodiment, intermediates used to synthesize
astaxanthin may also be used to synthesize other carotenoids such
as zeaxanthin. For example, zeaxanthin may be synthesized using the
scheme depicted below: ##STR42##
[0066] The synthesis of the intermediate 150 is based on a modified
synthesis of the intermediate 102 used to make astaxanthin. As
shown above, the final coupling of intermediate 150 with a
dialdehyde yields zeaxanthin in an analogous manner to astaxanthin.
Synthesis of intermediate 150 may be accomplished using the scheme
depicted below. ##STR43##
[0067] In some embodiments, synthesis of the intermediate 150 may
be accomplished using the same synthetic techniques as have been
described above for astaxanthin to obtain intermediate 120.
Intermediate 120 may be converted into saturated ketone 160 using a
procedure that is modified from the process used in the synthesis
of astaxanthin. In an embodiment, a saturated ketone 160 may be
formed by a two step procedure by oxidizing the hydroxyl group and
reducing the double bond. Alternatively, the reduction of the
double bond may be performed prior to oxidation of the hydroxyl
group. The scheme for converting compound 120 to 150 is shown
below. ##STR44##
[0068] Upon protecting the less sterically hindered alcohol, the
more sterically hindered alcohol of compound 120 may be oxidized to
a ketone. Oxidation of the hydroxyl group may be accomplished using
a variety of oxidizing reagents such as chromium oxidants,
manganese oxidants, and selenium oxidants. In one embodiment, the
oxidizing agent may include, for example, pyridinium dichromate
(PDC). Oxidation of the hydroxyl group leads to an optically active
ketone 108a, where R.sup.1 is alkyl, phenyl, aryl or silyl.
Hydrogenation of the double bond using catalytic hydrogenation
(e.g., Raney Ni, Pd/H.sub.2, etc.) gives the intermediate 150. In
other embodiments, hydrogenation may be performed to reduce the
double bond followed by oxidation of the hydroxyl group to the
ketone to form intermediate 150.
[0069] Synthesis of zeaxanthin, as shown in the above-described
scheme, then proceeds in an analogous manner to the synthesis of
astaxanthin.
[0070] In an alternate method, intermediate 102 used to make
astaxanthin, may be formed using an alternate method. An alternate
method for making intermediate 120 is depicted below: ##STR45##
[0071] The method includes an initial step of oxidizing
ketoisopherone to hydroxylated ketoisopherone as depicted below:
##STR46## Suitable oxidants include chromium oxidants, manganese
oxidants and peroxide oxidants. For example, in some embodiments, a
cyclohexene derivative may be hydroxylated using hydrogen peroxide.
After the compound has been oxidized, the hydroxylated product is
reduced to form a dihydroxylated compound having the general
structure ##STR47## The method may also include protecting the
dihydroxylated compound. In some embodiments, a dihydroxylate may
be protected by reacting the dihydroxylated compound with a ketone
(e.g., acetone). A ketone may be reacted with the dihydroxylated
compound to form a protected dihydroxylated compound having the
general structure ##STR48## In some embodiments, R.sup.1 may be
alkyl (e.g., methyl), aryl or each R.sup.1 together forms a cyclic
ring. The method may include coupling an alkyne to the protected
dihydroxylate to form an intermediate coupled product. In some
embodiments, the intermediate coupled product may not be isolated.
Instead the intermediate product may be directly subjected to the
next reduction process to give a product having the structure:
##STR49## The intermediate coupled product may be transformed into
a phosphonium salt product. In some embodiments, R.sup.5 may be
alkyl or aryl.
[0072] In some embodiments, a method may include transforming a
hydroxylated product into a phosphonium salt product. Transforming
the hydroxylated product into a phosphonium salt product may
include reducing the hydroxylated product to form a dihydroxylated
compound having the general structure ##STR50## In some
embodiments, the hydroxylated compound may be reduced
stereoselectively.
[0073] The term "stereoselective reduction" may be generally
defined as stereochemical reduction by which one of a pair of
enantiomers, each having at least one asymmetric carbon atom, is
produced selectively, i.e., in an amount larger than that of the
other enantiomer. The stereo-differentiating reduction is
classified into enantioface- and diastereo-differentiating
reductions, by which optical isomers having one asymmetric carbon
atom and those having two asymmetric carbon atoms are produced,
respectively. The present reduction may be said to pertain to
stereo-differentiating hydrogenation of carbonyl compounds.
[0074] In some embodiments, a carbonyl may be stereoselectively
reduced such that the resulting chiral center comprises a
stereochemistry of R or S comprising a stereoselectivity of greater
than 50%. A stereoselectivity of a reduction may be greater than
75%. A stereoselectivity of a reduction may be greater than 90%. A
stereoselectivity of a reduction may be greater than 95%. A
stereoselectivity of a reduction may be greater than 99%.
[0075] A stereoselective reduction of a first carbonyl of
ketoisophorone (KIP) may proceed as depicted: ##STR51## In some
embodiments, compound 108c (S-phorenol) may be a useful
intermediate for the synthesis of certain carotenoids (e.g.,
zeaxanthin or astaxanthin).
[0076] Direct asymmetric reduction of KIP to 108c may save several
steps relative to syntheses previously reported. Use of catalytic
reagents for stereoselective reduction avoids expensive reagents
used in stoichiometric amounts for reduction. Compound 108c may be
useful for synthesis of carotenoids such as astaxanthin via
derivative 108b.
[0077] KIP is known and commercially available and therefore a
prime candidate for beginning a synthesis of some carotenoids
with.
[0078] Reduction of ketoisophorone can occur at C-1 and/or C-4
and/or at the double bond, thus problems of regioselectivity and
stereoselectivity must be solved.
[0079] 1,2-reduction at C-4 has been achieved with a stoichiometric
amount of the reagents sodium borohydride/cerium chloride (JOC,
1986, 491, incorporated herein by reference) to give racemic
product. 1,2-reduction at C-4 has been achieved with 2-propanol in
the presence of zirconium oxide catalyst to give racemic product
(Bull Chem Soc Jap, 1988, 3283, incorporated herein by
reference).
[0080] Compound 108c has been obtained by bioprocesses. Typical are
product mixtures from non-selective reduction and over-reduction.
See for example Agr Biol Chem, 1988, 2929 with Aspergillus niger,
incorporated herein by reference, the product was the undesired 4R
enantiomer. 108c has been obtained in up to 99% enantiomeric excess
by esterase hydrolysis of the racemic chloroacetate ester. The
maximum yield reported was 30%. The maximum theoretical yield is
50% (Tetr Assy. 1999, 3811, incorporated herein by reference). 108c
has been obtained in homochiral form by asymmetric catalytic
reduction of the enol acetate of KIP (U.S. Pat. No. 5,543,559 to
Broger et al., incorporated herein by reference). This requires
preparation of the enol acetate and hydrolysis of the product
acetate to obtain 108c.
[0081] Direct asymmetric catalytic reduction of KIP has been
reported using chirally modified ruthenium catalysts to obtain 108c
in low selectivity and maximum 76% ee (Tetr Assy, 2000, 1849,
incorporated herein by reference).
[0082] There are many methods known to one skilled in the art for
stereoselectively reducing a carbonyl group. Stereoselective
reductions may be carried out using catalytic reagents (e.g.,
chemical, biological). Biological catalysts may include for example
living organisms (e.g., yeast) capable of facilitating a reduction
of a carbonyl. Catalytic reagents may be used due to their
efficiency. Efficiency may be related to more than just a yield of
a reaction or turnover, but also may include cost of the reagent as
well as total cost of running the reaction (e.g., cost of catalyst,
mole percentage of catalyst required, ease of reclaiming catalyst).
Catalysts may be more attractive as possible reducing agents on an
industrial scale due to a reduction in related expenses.
[0083] In some embodiments, direct stereoselective reduction of KIP
(including derivatives and analogs of KIP) to the alcohol product
(including protected alcohols, such as ethers) may include the use
of reagents such as boranes. Boranes may include at least one B--H
bond (e.g., diborane, borane-THF complex, borane-methyl sulfide
complex, phenoxyboranes (such as catechol borane), amine-borane
complexes, or alkoxyboranes).
[0084] In some embodiments, borane reagents may include chiral
substituents. Chiral catalysts may include chiral derivatives which
form weak complexes with the borane reductant. Chiral catalysts
which form weak complexes with borane reductants may include amine
derivatives.
[0085] In some embodiments, chiral oxazaborolidine catalysts with
borane-THF may be used to stereoselectively reduce KIP and its
analogs and derivatives (as described by Prof E J Corey in U.S.
Pat. No. 4,943,635 and reviewed in Angew Chem Intl Engl, 1998, 37,
1986, both of which are incorporated herein by reference). In some
embodiments, oxazaborolidine catalysts may include a compound
having a general structure ##STR52## Using oxazaborolidine catalyst
202 with borane-THF as reductant, complete conversion may be
achieved with 100% regioselectivity of reduction of the carbonyl at
C-4 and a minimum of 25% enantiomeric excess.
[0086] Enantiomeric excesses of over 55% may be achieved using
compound 202. In some embodiments, regioselectivity and enatiomeric
excess may vary with temperature, the B--H source, and/or the
structure of the catalyst.
[0087] Enantiomeric excesses may be improved with purification
techniques known to one skilled in the art. In some embodiments, a
chiral product may be purified via crystallization. Compound 108c
is a crystalline solid whereas the racemate is typically obtained
as non-crystalline. Therefore crystallization of product to chiral
purity may be a useful means of achieving this end.
[0088] A stereoselective reduction of a first carbonyl of
substituted ketoisophorone (KIP) may proceed as depicted: ##STR53##
R.sup.3 may be SiR.sup.5.sub.3, H, alkyl, or aryl. Compound 1
(R.sup.3.dbd.H) is a known substance, found naturally and prepared
synthetically. The only other known example of structure 204 is the
methyl ether (R.sup.3.dbd.CH.sub.3) which was prepared as an
analytical derivative for characterization of natural product 204
(R.dbd.H).
[0089] 206 (R.sup.3.dbd.H) and 208 are known substances (racemic
and enantiomers) and demonstrated useful intermediates for the
synthesis of racemic or homochiral astaxanthins (Helv Chim Acta,
1981, 240, 2447, 2463, incorporated by reference herein).
Derivatives and analogs of 206 (R.sup.3.dbd.H) and 208 provide
useful intermediates for the synthesis of racemic or homochiral
carotenoids, as well as, other natural products and their
derivatives and analogs.
[0090] Preparation of 1 (R.sup.3.dbd.H) from ketoisophorone was
described in Helv Chim Acta, 1981, 2436, which is incorporated
herein by reference. Reduction of 204 to racemic 206
(R.sup.3.dbd.H) using zinc in acid or hydrogen and Raney nickel and
subsequent conversion to racemic 208 are also described therein.
Preparation of the pure enantiomers of 206 (R.sup.3.dbd.H) by
resolution of racemic 206 (R.sup.3.dbd.H) via diastereomeric
alpha-phenylamine salts are described therein.
[0091] Desirable is direct asymmetric reduction of 204 to 206 and
conversion to homochiral 208 for use in the synthesis of homochiral
carotenoids (e.g., astaxanthin). This sequence avoids the need for
problematic oxidation steps which are required when the 3-hydroxy
or 3-alkoxy substituents are absent. The presence of a C-3
substituent may facilitate the asymmetric reduction of the carbonyl
at C-4. ##STR54## The only prior asymmetric reduction of 204
reported is a bioreduction of 204 (R.sup.3.dbd.H) reported to give
the 4S isomer of 206a (R.sup.3.dbd.H) in 65% enantiomeric excess
(Helv Chim Acta, 1981, 240, 2447, incorporated by reference
herein).
[0092] In some embodiments, a method may include preparation of
epoxyketoisophorone from ketoisophorone. ##STR55## An epoxide of
ketoisophorone may be prepared using reagents including, but not
limited to, peroxides (e.g., hydrogen peroxide). There are many
epoxidation reactions known to one skilled in the art, many of
which include peroxides (e.g., m-ClC.sub.6H.sub.4CO.sub.3H). There
are other epoxidation reagents described in references such as
"Comprehensive Organic Transformations: A Guide to Functional Group
Preparations" Larock, R. C. VCH Publishers, Inc. pages 456-461,
which is incorporated herein by reference.
[0093] In some embodiments, a method may include preparation of
3-hydroxyketoisophorone from epoxyketoisophorone. ##STR56## A
hydroxide anion (e.g., sodium hydroxide), followed by acidification
of the solution may be employed to convert the epoxide to the
hydroxide.
[0094] In some embodiments, a method may include preparation of
3-methoxyketoisophorone from 3-hydroxyketoisophorone. ##STR57## A
base (e.g., sodium hydroxide, sodium carbonate) may be used to
deprotonate the hydroxide in a solvent (e.g., dimethyl formamide,
methanol). A methylating reagent (e.g., dimethylsulfate) may then
be added to the oxide anion in order to prepare the methoxy
substituent. The methyl group may act as a protecting group masking
the hydroxy group from reagents used in later transformations.
There are many protecting groups for hydroxy groups known to one
skilled in the art (e.g., silyl protecting groups).
[0095] In other embodiments, the hydroxy substituent of
3-hydroxyketoisophorone may be methylated using diazomethane. Other
alkylation methods may include going through an intermediate (e.g.,
a mesylate) which is subsequently subtituted with a methoxy
substitutent. An alkylation (e.g., methylation) may also be
accomplished by using a methylating agent such as trimethyl
orthoformate and an acid (e.g., trifluoroacetic acid) in a solvent
(e.g., methanol).
[0096] In some embodiments, the alkylation step may be circumvented
by opening the epoxy group of, for example, epoxyketoisophorone
with a methoxide salt (e.g., sodium methoxide) along with
simultaneous dehydration. ##STR58##
[0097] In some embodiments, a method may include preparation of
4-(S)-hydroxy-ketoisophorone from 3-methoxyketoisophorone.
##STR59## A hydrogen source (e.g., H.sub.2) may be used to reduce a
carbonyl to a hydroxide group. A catalyst may be used to catalyze
the reduction. In some embodiments, an enantiomeric excess of a
particular enantiomer may be achieved without the use of
stereoselective reagents. In some embodiments, stereoselective
reagents (e.g., chiral catalysts) may be used to produce a specific
enantiomer. In some embodiments, reagents which are not typically
stereoselective reagents may be used to reduce a carbonyl to a
hydroxy group. The reaction may not be stereoselective. The
reaction may be stereoselective, but may be stereoselective due to
the inherent nature of the molecule. For example sodium borohydride
may be used to reduce the carbonyl to the hydroxy compound.
[0098] In some embodiments, a method may include preparation of
4-hydroyxketoisophorone acetone ketal from
4-(S)-hydroxy-ketoisophorone. ##STR60##
[0099] A diol may be converted to an acetal using a ketone (e.g.,
acetone) and an acid (e.g., p-toluenesulfonic acid hydrate). The
acetal group may act as a protecting group masking the diol from
reagents used in later transformations. There are other protecting
groups for diols known to one skilled in the art. In some
embodiments, one or more of the synthetic steps of a method for
preparing 4-hydroyxketoisophorone acetone ketal may be combined
into a "one-pot reaction" and/or an intermediate may not be
isolated and/or purified before exposing it to another set of
reagents.
[0100] In some embodiments, a method may include stereoselectively
reducing a carybonyl 1 of a compound 210 having the general
structure ##STR61## to form a chiral center 2 of a compound 212
having the general structure ##STR62## R.sup.1 may be H or
OR.sup.3. R.sup.3 may be SiR.sup.5.sub.3, H, alkyl, or aryl.
R.sup.5 may be H, alkyl, or aryl.
[0101] In some embodiments, R.sup.3 and/or R.sup.5 of compound 1
may include alkyl, substituted alkyl, aryl. Alkyl may include alkyl
substituents, where alkyl comprises two or more carbons. Compound
1, where R.sup.3.dbd.H (or salts thereof) and R.sup.3=alkyl,
heteroalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, are
useful substrates for asymmetric reduction for preparation of
isomers of compound 2. R.sup.3 may include other substituents not
listed known to one skilled in the art, even substituents typically
unstable during reductive conditions may be used if protected
properly using known functional protection methodology.
[0102] In some embodiments, salts formed from compound 1
(R.sup.3.dbd.H) may include metals of period I or II or transition
metals compatible with the reductants, ammonia, or amines (e.g.,
alkyl, substituted alkyl, aryl, heteroaryl, primary, secondary, or
tertiary), or phosphines (e.g., alkyl, substituted alkyl, aryl,
heteroaryl, primary, secondary, or tertiary). Salts formed from
compound 1 (R.sup.3.dbd.H) may contain chirality in their
structures or as associated ligands.
[0103] In some embodiments of ethers of compound 1, R.sup.3 may be
any alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group
compatible with the reduction conditions. Any of the R groups may
contain chiral centers or associated chiral ligands. In certain
embodiments, R.sup.3 may be an alkyl group comprising from one to
eight carbons.
[0104] A reductant may be selected from among the classes of:
hydrogen, a non-gaseous hydrogen source (e.g., reduction with an
alcohol, formic acid, etc.), a nucleophilic metal hydride (e.g.,
NaBH.sub.4 etc.), a covalent metal hydride (e.g., Dibal), a
non-metal hydride (e.g., boranes or silanes) or metal catalyzed
transfer of hydride from alcohols (e.g., Meerwein-Pondorf-Verley
reduction). The reductant may be chiral. In certain embodiments,
reductants, may include hydrogen, formic acid, isopropanol, or
sec-butanol.
[0105] Catalysts for hydrogenation or transfer hydrogenation may be
chosen from among transition metals or metal ions (e.g., such as
nickel, cobalt, platinum, palladium, iridium, rhodium, and
ruthenium, modified with chiral ligands or surface modifiers)
capable of facilitating reduction of ketones selectively over
reduction of other moieties (e.g., esters). In certain embodiments,
catalysts for hydrogenation or transfer hydrogenation may be
complexes of rhodium (I) or Ruthenium (II) with C.sub.2-symmetric
ligands or platinum metal modified with chiral cinchona alkaloids.
Examples of ligands are known to one skilled in the art.
[0106] At least some of the intermediates 2 (R.sup.3=alkyl, etc.)
are found to be crystalline. It is a desirable feature that the
chiral purity of compound 2 may be upgraded by
recrystallization.
[0107] It has been previously reported that compound 2
(R.sup.3.dbd.H) may be converted to 3 by treatment with acetone or
acetone ketals in the presence of acid catalysts in either the
racemic or chiral series. It is reported here that compound 2
(R.sup.3=alkyl, etc.) may be converted to compound 2
(R.sup.3.dbd.H) under acidic hydrolytic conditions. It was
surprisingly found that compound 2 (R.sup.3=alkyl, etc.) may be
converted to compound 3 when treated with acetone in the presence
of acidic catalyst.
[0108] Compound 1 may be prepared from commercially available
ketoisophorone by several means: [0109] 1. alkylation of 1
(R.sup.3.dbd.H) with alkyl halides or sulfates in the presence of
base and solvent as appropriate. Preferred is dimethylsulfate with
sodium hydroxide in water, in the optional presence of methanol;
[0110] 2. treatment of 1 (R.sup.3.dbd.H) with alcohols or phenols
in the presence of an acid catalyst under conditions for physical
removal of water (e.g., distillation or azeotropic distillation) or
chemical removal of water (e.g., presence of a ketal or
orthoester); and [0111] 3. epoxidation of ketoisophorone followed
by treatment with alkoxide or phenoxide.
[0112] In some embodiments, a carbonyl may be stereoselectively
reduced, as for example: ##STR63## In some embodiments, R.sup.1 may
be R.sup.5, OSiR.sup.5.sub.3, or OR.sup.5. R.sup.3 may be
SiR.sup.5.sub.3, aryl, or alkyl. Alkyl may comprise two or more
carbons. R.sup.5 may be H, alkyl, or aryl. In some embodiments,
R.sup.5 may be methyl. R.sup.3 may be methyl or hydrogen. A
carbonyl may be stereoselectively reduced as below ##STR64##
[0113] In some embodiments, a method for reducing a carbonyl may
include selectively reducing a first carbonyl in the presence of a
second carbonyl. The second carbonyl may be chemically
distinguishable from the first carbonyl. For example the first
carbonyl may be electronically distinguishable from the second
carbonyl. The second carbonyl may not be reduced using the
described method for reducing the first carbonyl. For example the
second carbonyl may be described as a vinylic ester and/or and
ester. The second carbonyl may be sterically hindered. For reasons
such as these, a first carbonyl may be regioselectively
reduced.
[0114] In some embodiments a reduction catalyst may be a chiral
catalyst. In one embodiment, a chiral catalyst includes a
transition metal and an optically active chiral ligand. Transition
metals that may be used to form a chiral catalyst for reduction of
ketones include Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Re, Fe, Ru,
Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, and Au. In some embodiments, a
ruthenium chiral catalyst may be used to effect a stereoselective
reduction of keto-.alpha.-isopherone. The ruthenium chiral catalyst
may be formed from a mixture of [RuX.sub.2(.eta..sup.6-Ar)].sub.2
with an optically active amine, where X represents a halogen (e.g.,
F, Cl, Br, I) and Ar represents benzene or a substituted benzene
(e.g., alkyl substituted benzene). In some embodiments, the
optically active amine includes both (S)- and (R)-amino acids, and
other optically active amines such as as H.sub.2N--CHPh-CHPh-OH,
H.sub.2N--CHMe-CHPh-OH, MeHN--CHMe-CHPh-OH, and
TsNH--CHPh-CHPh-NH.sub.2.
[0115] In some embodiments, a chiral catalyst may include a
catalyst having the structure ##STR65##
[0116] In some embodiments, a method may include a stereoselective
reduction such as ##STR66##
[0117] In some embodiments, a solution of
(1S,2S)-N-p-toluenesulfonyl-1,2-diphenylethylenediamine may be
added to dichlorotp-cymene)ruthenium(II)dimer. The suspension may
be heated as necessary during which time the solids may go into
solution. The reaction may be cooled to room temperature, a
solution of 204b may be added followed by KOH.
[0118] The method may include protecting the dihydroxylated
compound. In some embodiments, a dihydroxylate may be protected by
reacting the dihydroxylate with a ketone (e.g., acetone). A ketone
may be reacted with the dihydroxylate compound to form a protected
dihydroxylate compound having the general structure ##STR67## In
some embodiments, R.sup.5 may be alkyl (e.g., methyl) or aryl. The
method may include coupling the protected diol to form an
intermediate coupled product. In some embodiments, the intermediate
coupled product may not be isolated. The intermediate coupled
product may include a compound having the general structure
##STR68## The intermediate coupled product may be transformed into
a phosphonium salt product having the general structure
##STR69##
[0119] In some embodiments, a synthetic sequence may include:
##STR70##
[0120] In some embodiments, an alkyne may be formed via the
following synthetic sequence ##STR71## In some embodiments, R.sup.3
may be SiR.sup.5.sub.3, H, alkyl, or aryl. R.sup.5 may be alkyl
(e.g., methyl) or aryl. R.sup.3 may include a protecting group,
such as the described silyl protecting group. There are many
protecting groups known to one skilled in the art for masking or
protecting hydroxy functionalities. Different protecting groups may
be used depending upon what conditions one wants to protect the
hydroxy group under and/or what conditions one desires to deprotect
and "unmask" the hydroxy group. The above synthetic sequence may
embody other types of optically active and/or non optically active
endproducts. In some embodiments, at least some of the synthetic
steps may be carried out in a similar manner to similar chemical
reactions as described in other synthetic schemes as described
herein above and/or in the Examples section.
[0121] In some embodiments, an isomer of the alkyne coupled to the
protected diol as described above may be employed to couple to the
protected diol. The isomer of the alkyne may include a compound
having the general structure ##STR72##
[0122] In some embodiments, the isomer of the alkyne may be
synthesized by coupling acetylene and methyl vinyl ketone. In some
embodiments, the acetylene may be added to the methyl vinyl ketone
via 1,2 addition. ##STR73## Due to the instability of methyl vinyl
ketone other synthetic routes my be employed to provide the desired
product. In some embodiments, stable chemical equivalents of methyl
vinyl ketone may be used. Stable equivalents may include
2-(beta-bromoethyl)-2-methyl-1,3-dioxolane.
[0123] In certain embodiments, carotenoids which may be synthesized
using methods described herein may include carotenoids based on a
chemical intermediate having the general structure ##STR74## The
compound depicted above embraces racemic, optically active
stereoisomers and optically inactive stereoisomers. In some
embodiments, R.sup.3 may be OR.sup.5, OSiR.sup.5.sub.3, H, alkyl,
or aryl. R.sup.5 may be H, alkyl, or aryl. In some embodiments,
R.sup.7 may include C--R.sup.3 or C.dbd.O. A method of synthesizing
such a compound may include transforming a halogenated derivative
having the general structure ##STR75## into a phosphorous compound
having the general structure ##STR76## In some embodiments, R.sup.5
may be alkyl or aryl. X may be a halogen (e.g., Br, Cl). The method
may include reacting the phosphorous compound with an aldehyde or
an aldehyde equivalent having a general structure ##STR77## to form
a alcohol coupling product having the general structure ##STR78##
The method may include transforming the alcohol coupling product
into a halogenated coupling product having the general structure
##STR79## In some embodiments, R.sup.5 may be alkyl or aryl. X may
be a halogen (e.g., Br, Cl).
[0124] In some embodiments, a method may include transforming the
halogenated coupling product into a phosphonium salt product having
the general structure ##STR80## R.sup.5 may be alkyl or aryl. X may
be a halogen (e.g., Br, Cl).
[0125] In some embodiments, a method may include reacting the
phosphonium salt product with a dialdehyde having the general
structure ##STR81## to form a carotenoid chemical intermediate
having the general structure ##STR82## In some embodiments, R.sup.3
may be OR.sup.5, OSiR.sup.5.sub.3, H, alkyl, or aryl. R.sup.5 may
be H, alkyl, or aryl. In some embodiments R.sup.7 may include
C--R.sup.3 or C.dbd.O.
[0126] In some embodiments, a carotenoid chemical intermediate may
include a compound having the general structure ##STR83##
[0127] In some embodiments, a synthetic sequence may include:
##STR84##
[0128] In some embodiments, carotenoid chemical intermediates may
be used to synthesize naturally occurring carotenoids as well as
carotenoid analogs and carotenoid derivatives. Carotenoid chemical
intermediates may be used to synthesize naturally occurring
carotenoids such as lycopene and lycophyll, and lycopene/lycophyll
analogs and lycopene/lycophyll derivatives.
[0129] In some embodiments of a method to synthesize lycopene and
lycophyll, and its derivatives and/or analogs, the chemical
intermediate pictured above having the general structure ##STR85##
may be coupled with a phosphonium salt product having the general
structure ##STR86## to form lycopene having the general structure
##STR87## In some embodiments, Y may include
--CH.sub.2--PR.sup.5.sub.3 or
--CH.sub.2--P(.dbd.O)(OR.sup.5).sub.2. R.sup.5 may be alkyl or
aryl.
[0130] In some embodiments, methodologies as described herein
(e.g., methods for synthesizing lycopene) may be used to prepare
acyclic carotenoids, as well as, derivatives and/or analogs of
acyclic carotenoids. Of course it is understood that at least some
of the intermediates used to synthesize acyclic carotenoids are
also useful in the preparation of carotenoids containing cyclic
rings (referred to herein sometimes as cyclic carotenoids, e.g.,
astaxanthin).
[0131] In some embodiments, a compound prepared by the method
described herein may include an enantiomeric excess of at least one
of the possible stereoisomers of the compound.
[0132] In some embodiments, a compound prepared by the method
described herein may include an excess of a stereoisomer relative
to the stereoisomer's statistical abundance.
[0133] In certain embodiments, carotenoids, carotenoid derivatives,
or carotenoid analogs which may be synthesized using methods
described herein may include carotenoids based on a chemical
intermediate having the general structure ##STR88## Compound 214
may be coupled to a phosphonium salt product 216 having the general
structure ##STR89## to form protected carotenoid 218 having the
general structure ##STR90## In some embodiments, Y may be
PR.sup.5.sub.3 or P(.dbd.O)(OR.sup.5).sub.2. R.sup.3 may be
SiR.sup.5.sub.3, H, alkyl, or aryl. R.sup.5 may be alkyl or aryl.
In some embodiments, a solution of LiOMe (e.g., in methanol) may be
used to couple the two compounds to prepare the protected
carotenoid.
[0134] In some of the phosphonium salt product 216 embodiments, Y
may be PR.sup.5.sub.3, R.sup.5 may be phenyl, and such that
phosphonium salt product 216 has the general structure ##STR91## In
some embodiments, X may be F, Cl, Br, or I. In some embodiments,
R.sup.3 may be methyl and X may be Br.
[0135] In some embodiments, a method may include reducing protected
carotenoid 218 to form carotenoid 220 having the structure
##STR92## In some embodiments, R.sup.3 may be SiR.sup.5.sub.3, H,
alkyl, or aryl. R.sup.5 may be alkyl or aryl. In some embodiments,
R.sup.3 may be H when protected carotenoid 218 is reduced to an
alcohol forming carotenoid 2H. Reducing agents (e.g., DIBAL or
Diisobutylaluminium hydride) known to one skilled in the art may be
used to reduce the protected carotenoid 218 to form the carotenoid
220. Other reducing agents known to one skilled in the art may be
used.
[0136] In some embodiments, carotenoid derivatives and analogs may
be synthesized from naturally occurring carotenoids. These
carotenoids may be synthetically produced and/or isolated from
natural sources.
[0137] In some embodiments, a method may include condensing
carotenoid 220 with succinic anhydride to prepare compound 222
having the general structure ##STR93## 222. In some embodiments,
R.sup.3 may be SiR.sup.5.sub.3, H, alkyl, or aryl. R.sup.5 may be
alkyl or aryl. In some embodiments, R3 may include a co-antioxidant
(e.g., Vitamin C, Vitamin C analogs and derivatives) and/or other
substituents described herein. A base (e.g.,
N,N-diisopropylethylamine in a solvent such as CH.sub.2Cl.sub.2)
may be used to facilitate condensation of carotenoid 220 to
succinic anhydride. A non-nucleophilic base may be used. The method
may include forming a salt 224 of compound 222 having a general
structure ##STR94## 224. wherein X is a counterion. In some
embodiments, X may be a counterion. X may include inorganic salts
and/or organic salts. X may include, but is not limited to, Li, Na,
or K. NaOMe may be used to convert the acid to the salt. Other
reagents such as LiOMe, NaOEt, as well as other based may be used
to prepare the salt.
[0138] In some embodiments, a method may include phosphorylating
carotenoid 220 to form compound 226 having the general structure
##STR95## 221. In some embodiments, Y may be PR.sup.5.sub.3 or
P(.dbd.O)(OR.sup.5).sub.2. R.sup.5 may be SiR.sup.5.sub.3, H,
alkyl, or aryl. R.sup.5 may be H, alkyl, benzyl, or aryl. The
method may include forming a salt 223 of compound 226 having a
general structure ##STR96## 223. In some embodiments, X may be a
counterion. X may include inorganic salts and/or organic salts. X
may include, but is not limited to, Li, Na, or K. NaOMe may be used
to convert the acid to the salt. Other reagents such as LiOMe,
NaOEt, as well as other bases may be used to prepare the salt.
[0139] In some embodiment, a method may include preparing
phosphonium salt product 216 by oxidizing ester 228 having the
general structure ##STR97## to form aldehyde 230 having the general
structure ##STR98## Selective oxidizing agents (e.g., SeO.sub.2 in
a solution of for example 95% ethanol) may be used to oxidize up to
the aldehyde. The method may include oxidizing aldehyde 230 to form
oxidized product 232 having the general structure ##STR99##
Selective oxidizing agents (e.g., NaClO.sub.2, Na.sub.2HPO.sub.4,
Me.sub.2C.dbd.CHMe, t-BuOH/H.sub.2O) may be used to oxidize up to
the acid and/or ester. Oxidized product 232 may be selectively
deprotected to form product 234 having the general structure
##STR100## Selective bases (e.g., K.sub.2CO.sub.3, MeOH/H.sub.2O)
may be used to convert oxidized product 232 (e.g., to the alcohol
and/or ether). Conversion of product 232 to product 234 may be
viewed as more of a deprotection of an alcohol. The method may
include halogenating product 234 to form halogenated product 236
having the general structure ##STR101## In an embodiment where
product 234 includes an alcohol, halogenation of alcohols may be
accomplished by a variety of methods (e.g., CBr.sub.4/Ph.sub.3P in
a polar solvent such as THF). Halogenated product 236 may be
converted to the phosphonium salt product 216. Conversion of the
halogen to the phosphonium salt may include using Ph.sub.3P in a
solvent such as EtOAc. In some embodiments, X may be a counterion.
X may include inorganic salts and/or organic salts. X may include
F, Cl, Br, or I. R.sup.3 may be SiR.sup.5.sub.3, H, alkyl, or aryl.
R.sup.5 may be alkyl, benzyl, or ary
[0140] In some embodiments, a multi-gram scale total synthesis of
lycophyll (16,16'-dihydroxy-lycopene;
.psi.,.psi.-carotene-16,16'-diol) may be based on a 2 (C10)+C20
synthetic methodology using the commercially available materials
geraniol (C10) and crocetindialdehyde (C20). A late-stage double
Wittig olefination of crocetindialdehyde may be used to form the
lycophyll scaffold. The double Wittig may generate a mixture of
polyenic geometric isomers that may be separated (e.g., using
HPLC). The all-trans lycophyll may be achieved in >95% purity
using about 8 linear synthetic steps. The disuccinate and
diphosphate sodium salts of the rare carotenoid may then be
prepared. Carotenoid derivatives and analogs (e.g., disuccinate and
diphosphate sodium salts) may be readily dispersible in water
without need for heat, detergents, co-solvents, or other additives.
Retrometabolic in design, these novel derivatives could find
utility in those applications where parenteral delivery of
therapeutically relevant forms of lycophyll are desired.
[0141] Studies in cultured human cells have shown that lycopene 2F,
the primary carotenoid in tomatoes, can be growth inhibitory
against transformed cells as well as normal prostatic epithelium,
alone and/or in combination with other antioxidants (e.g. vitamin
E). In animal studies, the results regarding protection against
proliferation of transformed cells induced with various
carcinogenic agents have been positive. For example, in the ferret,
the most representative model in terms of
absorption-distribution-metabolism-excretion (ADME) for humans,
lycopene was in fact protective against cigarette-smoke induced
lung pathology. Epidemiological studies in humans clearly support
an association between dietary consumption of lycopene-containing
food products and a lower risk of prostate cancer. These
lycopene-containing food products also contain lycophyll, albeit in
lower relative amounts. In some cases, the natural dietary mixture
of carotenoid compounds has efficacy in these settings, and
synthetically-prepared or naturally-isolated lycopene does not. In
some embodiments, a method of treating disease in a human subject
may include administering to the human subject a pharmaceutical or
nutraceutical composition including a predetermined ratio of two or
more geometric and/or stereoisomers of a structural analog or
derivative or synthetic intermediate of a carotenoid. In some
embodiments, a method of treating disease in a human subject may
include administering to the human subject a pharmaceutical or
nutraceutical composition including a predetermined ratio of two or
more structural analogs or derivatives or synthetic intermediates
of a carotenoid. Prospective, randomized clinical trials in humans
also demonstrate improved indices of proliferation and oxidative
stress across a range of oral doses in cancer patients. Delivery of
a highly potent radical scavenger to prostatic tissue may restore
or augment endogenous antioxidant levels.
[0142] Lycoxanthin 2G and lycophyll 2H, which can be isolated from
the red, ripe berries of Solanum dulcamara, as well as tomatoes and
watermelon, are C40 lycopene-like xanthophylls functionalized with
primary hydroxyl groups. The originally proposed chemical
structures of the xanthophylls however lacked complete assignment
and required further studies that were realized in the early
1970's. Utilizing high-resolution mass spectroscopy and NMR, the
regiochemistry of the hydroxyl groups was characterized.
Unambiguous confirmation of both structures were obtained
approximately one year later, facilitated by the total syntheses of
lycoxanthin and lycophyll reported by Kjosen and Liaaen-Jensen in
1972. The original total synthesis was based on a C10+C20+C10
synthetic paradigm, in part due to the commercial availability of
C20 dialdehyde (crocetindialdehyde). Up to the present, little
additional chemical or biological information has accumulated in
the primary literature for either compound.
[0143] Lycophyll was prepared by total synthesis at multiple gram
scale for the current testing and derivatization to novel
water-soluble, water-dispersible compounds. Isolation from natural
sources demonstrates high cost, significant manpower, and generally
low yields. Retrosynthetic analysis of the target xanthophyll
revealed an efficient methodology utilizing at least some
commercially available materials. In cases where commercial
material was not available, these intermediates were synthesized in
appropriate amounts. In some embodiments, commercially available
materials may include geranyl acetate, a protected form of geraniol
(C10), and/or crocetindialdehyde (C20). A method may include a
total synthesis of acyclic carotenoids (e.g., lycophyll). In some
embodiments, a synthesis of, for example, lycophyll may be realized
in about 8 synthetic steps (Schemes 1 and 2). Synthetic steps may
include an "endgame" double-Wittig olefination that successfully
forms the target C40 scaffold while generating a mixture of
geometric isomers (Scheme 2). The isomeric mixture may be
deconvoluted to yield the target all-trans lycophyll. Deconvolution
may include, but is not limited to, thermal or liquid
chromatographic methods. The methodology shown in Schemes 1 and 2
for synthesizing lycophyll may be used to synthesize other acyclic
carotenoids, carotenoid derivatives, and carotenoid analogs.
##STR102## Scheme 1. a. SeO.sub.2, 95% EtOH; b. NaClO.sub.2,
Na.sub.2HPO.sub.4, Me.sub.2C.dbd.CHMe, t-BuOH/H.sub.2O; c.
K.sub.2CO.sub.3, MeOH/H.sub.2O; d. CH.sub.3I, K.sub.2CO.sub.3,
DMF/H.sub.2O; e. CBr.sub.4/Ph.sub.3P, THF; f. Ph.sub.3P, EtOAc.
##STR103## Scheme 2. a. LiOMe in MeOH, toluene; b. DIBAL, THF.
[0144] Research has shown that targeted derivatization of
carotenoids can successfully increase the aqueous solubility and/or
dispersibility of the highly lipophilic natural scaffolds. These
compounds have demonstrated beneficial effects as direct aqueous
radical scavengers, as myocardial salvage agents in experimental
infarction models, as agents ameliorating and/or preventing chronic
liver injury, and/or as cancer chemopreventive agents. The
derivatives have shown increased utility as parenteral agents in
these settings, as well as improved oral bioavailability in model
animal studies. Currently, our efforts have extended along these
lines to include the derivatization of the rare xanthophyll
lycophyll, specifically directed by principles of retrometabolic
drug design. Acquisition of lycophyll through total synthesis
(Scheme 1 and 2) facilitated the generation of water-dispersible
lycophyll succinic and phosphoric diester salts (Scheme 3). These
novel compounds are readily dispersible in water without need of
heat, detergents, co-solvents, or other additives. Such derivatives
will likely find application in those indications in which
parenteral delivery of highly-potent radical scavengers possessing
the lycopene scaffold are necessary to achieve their intended
purpose. Specifically, these compounds will be evaluated for
efficacy in contemporary in vitro and in vivo cancer
chemoprevention models, utilizing the natural tissue tropism of
these compounds in mammals. ##STR104## Scheme 3. a. succinic
anhydride, N,N-diisopropylethylamine, CH.sub.2Cl.sub.2; b. NaOMe,
CH.sub.2Cl.sub.2/MeOH (4/1); c. benzyl alcohol, triethylamine,
Et.sub.2O; d. I.sub.2, CH.sub.2Cl.sub.2; e. pyridine,
CH.sub.2Cl.sub.2, then 14; f. bromotrimethylsilane,
N,O-bis(trimethylsilyl)acetamide, CH.sub.2Cl.sub.2 (254/222e
(1/4)), then reverse-phase HPLC; g. NaOMe, MeOH.
[0145] In some embodiments of a method to synthesize lycopene and
its derivatives and/or analogs, a phosphonium salt product having
the general structure ##STR105## may be coupled with an aldehyde
product having the general structure ##STR106## to form lycopene
having the general structure ##STR107## In some embodiments, Y may
include --CH.sub.2--PR.sup.5.sub.3 or
--CH.sub.2--P(.dbd.O)(OR.sup.5).sub.2. R.sup.5 may be alkyl or
aryl.
[0146] In some embodiments, a lycopene analog or a lycopene
derivative may include one or more substituents. At least one of
the substituents may include hydrophilic substituents. In some
embodiments, substituents may include chemically reactive
substituents which serve as chemical intermediates.
[0147] In some embodiments, carotenoid chemical intermediates may
be used to synthesize naturally occurring carotenoids such as
xanthophylls. A method may include coupling a phosphonium salt
product having the general structure ##STR108## with a dialdehyde
having the general structure ##STR109## to form a carotenoid having
the general structure ##STR110## In some embodiments, R.sup.1 and
R.sup.2 may be H or OR.sup.3. R.sup.3 may be SiR.sup.5.sub.3, H,
alkyl, or aryl. R.sup.5 may be alkyl or aryl. Y may include
--CH.sub.2--PR.sup.5.sub.3 or
--CH.sub.2--P(.dbd.O)(OR.sup.5).sub.2. R.sup.7 may include
C--OR.sup.3 or C.dbd.O. Examples of xanthophyll carotenoids than
may be synthesized using this methodology include, but are not
limited to, astaxanthin, lutein, zeaxanthin, and canthaxanthin.
[0148] In some embodiments, one or more of the conversions and/or
reactions discussed herein may be carried out within one reaction
vessel increasing the overall efficiency of the synthesis of the
final product. In some embodiments, a product of one reaction
during a total synthesis may not be isolated and/or purified before
continuing on with the following reaction. A reaction may instead
only partially be worked up. For example, solid impurities which
fall out of solution during the course of a reaction may be
filtered off and the filtrate washed with solvent to ensure all of
the resulting product is washed through and collected. In such a
case the resulting collected product still in solution may not be
isolated, but may then be combined with another reagent and further
transformed. In some cases multiple transformations may be carried
out in a single reaction flask simply by adding reagents one at a
time without working up intermediate products. These types of
"shortcuts" will improve the overall efficiency of a synthesis,
especially when dealing with large scale reactions (e.g., along the
lines of pilot plant scale and/or plant scale).
[0149] An example of increasing the overall efficiency of a
synthesis may include reducing the alkyne of compound 114 to an
alkene forming compound 104. In some embodiments, zinc and an acid
may be used to reduce the alkyne to an alkene. The acid may
include, for example, glacial acetic acid. The resulting zinc
acetate may then be filtered off, and the filter cake washed with
an organic solvent (e.g., methylene chloride) to ensure collection
of as much of the resulting product compound 104 as possible. The
resulting product compound 104, still in solution, may then be
added dropwise over a period of time (e.g., 30 minutes) to an
aqueous solution of acid (e.g., HBr) and the resulting mixture
stirred (e.g., for 10 minutes). The organic phase may be separated
from the aqueous phase and triphenylphosphine added to the organic
phase without isolating the previous product from solution. The
addition of triphenylphosphine may result in compound 102.
Dialdehyde compound 112 may be added to the resulting solution of
compound 102 and cooled down (e.g., to about 0.degree. C.). A base
in solution may be added to the solution (e.g., sodium methoxide in
methanol) dropwise. After stirring (e.g., about 5 hours), the
solution may be finally fully worked up to acquire the purified
isolated compound 104.
[0150] It has been stated that the compound of formula 100 embraces
racemic and optically active and optically inactive stereoisomers.
In some embodiments, a specific example of may include the
synthesis of astaxanthin having a general formula of ##STR111##
[0151] In an embodiment, carotenoid derivatives may be synthesized
from naturally-occurring carotenoids. The carotenoids may include
structures 2A-2F depicted in FIG. 1. In some embodiments, the
carotenoid derivatives may be synthesized from a
naturally-occurring carotenoid including one or more alcohol
substituents. In other embodiments, the carotenoid derivatives may
be synthesized from a derivative of a naturally-occurring
carotenoid including one or more alcohol substituents. The
synthesis may result in a single stereoisomer. The synthesis may
result in a single geometric isomer of the carotenoid derivative.
The synthesis/synthetic sequence may include any prior purification
or isolation steps carried out on the parent carotenoid. Synthesis
of carotenoid derivatives can be found in U.S. Published Patent
Application Nos. 2004-0162329 and 2005-0113372, both of which are
incorporated herein by reference.
EXAMPLES
[0152] Having now described the invention, the same will be more
readily understood through reference to the following example(s),
which are provided by way of illustration, and are not intended to
be limiting of the present invention.
Example 1
Preparation of (R)4-hydroxyisophorone(R)-116
[0153] ##STR112##
[0154] All solvents were free of O.sub.2. And the reactions were
done under N.sub.2. Benzeneruthenium (II) dimer (19.72 g, 39.42
mmol, 0.4 mol %) and (1R,2S)-(-)-norephedrine (99%) (24.14 g,
159.67 mmol, 1.62 mol %) were dissolved in a 12 L three-necked
flask containing 2-propanol (7.5 L). After stirring the red
solution for 45 min at 80.degree. C., the heat was removed. It was
transferred to a 50 L three-necked flask containing 2-propanol (28
L). 109 (1500 g, 9.86 mol) and 0.1 M potassium hydroxide in
2-propanol (3945 ml, 0.0.395 mol, 4 mol %) were added. After 3 h
(TLC showed the reaction was done), the red solution was filtered
through a short silica gel pad and the filtrate was evaporated to
dryness to obtain solids (about 1600 g). After five times
recrystallization from .sup.iPr.sub.2O (500 ml.times.5), 912 g of
(R)-116 was obtained. The yield: 60%. .sup.1H NMR (CDCl.sub.3, 300
MHz): .delta. 1.02 (s, 3H), 1.07 (s, 3H), 1.97 (s, 1H), 2.04 (t,
J=1.2 Hz, 3H), 2.21 (d, J=16.3 Hz, 1H), 2.39 (d, J=16.4 Hz, 1H),
4.03 (d, J=6.6 Hz, 1H), 5.86 (br s, 1H). .sup.13C NMR (CDCl.sub.3,
75 MHz): .delta. 21.22, 21.46, 26.89, 38.48, 48.97, 76.89, 126.29,
160.81, 198.79. [.alpha.].sub.D.sup.23+105.34 (c=1.006, MeOH),
literature [.alpha.].sub.D.sup.22+105.9 (c=1.00, MeOH).
Example 2
Preparation of (1R,4S)-2,6,6-trimethyl-2-cyclohexen-1,4-diol(1R,
4S)-118
[0155] ##STR113##
[0156] To a solution of L-Selectride (5674 mL, 1 M in THF, 1.25
equiv), a solution of compound (R)-116 (700 g, 4.54 mol, 1 equiv)
in THF (3000 mL) was added dropwise at -78.degree. C. After
stirring for 1.5 h, the mixture was sequentially treated with
H.sub.2O (600 mL), 4N NaOH (1450 mL). After extractions with AcOEt
(500 ml.times.5) an combined organic phase was dried and
concentrated. To the residue was charged 3000 mL of hexanes, then
the mixture was filtered. The solid was washed with hexanes (200
mL.times.3). The solid crude product was purified by flash
chromatography using Hexanes/AcOEt (3/1) as an eluent. 645 g of
compound (1R,4S)-118 was obtained (yield: 91%). Recrystallized from
1000 ml of EtOAc to obtain 504 g (70%) of (1R,4S)-118. .sup.1H NMR
(CDCl.sub.3, 500 MHz): .delta. 0.86 (s, 3H), 1.02 (s, 3H), 1.45
(dd, J=12.8, 9.5 Hz, 1H), 1.67 (ddt, J=12.8, 6.3, 1.1Hz, 1H), 1.84
(t, J=1.7 Hz, 3H), 3.34 (s, 1H), 4.18 (m, 1H), 5.54 (br s, 1H).
[.alpha.].sub.D.sup.23+68.63 (c=1.6000, CHCl.sub.3), literature
[.alpha.].sub.D.sup.24+67.4 (c=0.27, CHCl.sub.3).
Example 3
Preparation of
(1R,4S)-4-tert-Butyidimethylsilyloxy-2,6,6-trimethyl-2-cyclohexen-1-ol
(1R,4S)-120a
[0157] ##STR114##
[0158] A mixture of enantiomerically pure (1R,4S)-118 (1000 g, 6.40
mol), TBDMSCl (1194 g, 7.68 mol, 1.2 eq) and imidazole (566.37 g,
8.32 mol, 1.3 eq) in DMF (9 L) was stirred at room temperature for
1 hr and 20 min. Wate (2 L) was added, aqueous phase was extracted
with diethyl ether (2000 ml.times.3). The combined organic layer
was dried over Na.sub.2SO.sub.4. After concentration, the crude
product (1R,4S)-120a was subjects to next step without further
purification.
Example 4
Preparation of
(S)-4-tert-Butyidimethylsilyloxy-2-cyclohexenone(S)-108b
[0159] ##STR115##
[0160] (1R,4S)-120a (.about.6.40 mol) was added to a mixture of PDC
(3613 g, 1.5 eq) and DMF (8000 ml), which was cooled by ice-water.
And then, the mixture was stirred for 1 h and 10 min at rt. Ether
(8 L) was added. The mixture was passed through a pad of celite.
Then solution was washed with water (3 L.times.2). The organic
phase was dried over Na.sub.2SO.sub.4. 1718.2 g of (S)-108b (Yield:
100%, two steps from 118 to 108b) was obtained after column
chromatography (hexanes/ethyl acetate, 50/1.about.30/1). .sup.1H
NMR (CDCl.sub.3, 500 MHz): .delta. 0.12 (s, 3H), 0.13(s, 3H), 0.92
(s, 9H), 1.11 (s, 3H), 1.14 (s, 3H), 1.78 (br s, 3H), 1.87 (dd,
J=12.9, 9.8 Hz, 1H), 1.99 (ddd, J=12.9, 5.4, 1.8 Hz, 1H), 4.55 (m,
1H), 6.50 (br s, 1H).
Example 5
Determine the ee Value of Compound (S)-108b
[0161] ##STR116##
[0162] To a solution of compound 108b (40 g, 112 mmol) in THF (450
ml), .sup.nBU.sub.4NF (29.22 g, 112 mmol) in 150 ml of THF was
added. After 30 min, 200 ml of water and 500 ml of EtOAc was added.
The organic phase was then washed with a half-saturated brine (400
ml.times.2) and brine (400 mL). It was dried and concentrated and
subjected to column chromatography (hexane/ethyl acetate, 5/1) to
give 20.25 g of (S)-108c (92%). [.alpha.].sub.D.sup.23-48.0
(c=1.98, EtOH), literature [.alpha.].sub.D.sup.20-46.7 (c=1.0,
EtOH). The racemic 108c was separated using a chiral HPLC column;
baseline separation was not achieved. Reverse phase HPLC column,
Pirkle covalent, (S,S) Whelk-O 1, spherical silica; Eluent, 3:97
2-propanol:hexane, 1 ml/min. For racemic 108c, t.sub.1=14.07 min,
t.sub.2=14.67 min. Only one peak was detected for (S)-108c using
the same HPLC condition T.sub.s(108c)=14.74 min.
Example 6
Preparation of Compounds 112a
[0163] ##STR117##
[0164] A mixture of alkyne 110a (689.3 g, 4.10 mol, 1.1 eq) and THF
(13 L) was cooled to -78.degree. C., BuLi (1640 mL, 2.5 M, 4.10 mL,
1.1 eq) was added dropwise. After 2 h, compound (S)-108b (1000 g,
3.725 mol) in 2 L of THF was added dropwise. In the 4 hrs, the
temperature was allowed to raise from -78.degree. C. to -25.degree.
C. NH.sub.4Cl saturated solution (500 mL) and brine (500 mL) was
added and extracted with EtOAc (3000 mL.times.1). Dried. After
concentration, the crude product 112a (.about.1770 g) was subjected
to next step without further purification.
Example 7
Preparation of Compounds 114a
[0165] ##STR118##
[0166] A solution of compounds 112a (.about.3.75 mol) in DCM (2 L)
was added dropwise to a mixture of PDC (2101.86 g, 5.58 mol, 1.5
eq), NaOAc (458.16 g, 5.58 mol, 1.5 eq), 4 .ANG. MS (1000 g) and
DCM (10 L). After 24 h, ethyl acetate (2000 ml) was added and it
was subjected to a short silica gel pad and washed with ethyl
acetate. After concentration, the crude product 114a (1710 g) was
subjects to next step without further purification. .sup.1H NMR
(CDCl.sub.3, 300 MHz): .delta. 0.44 (s, 3H), 0.13 (s, 3H), 0.87 (s,
9H), 1.12 (td, J=6.9, 2.7 Hz, .about.3H), 1.21-1.32 (m,
.about.10H), 1.59 (s, .about.1.5H), 1.62 (s, .about.1.5H), 1.83-2.2
(m, .about.4H), 3.32-3.70 (m, .about.2H), 4.29 (dd, J=11.1, 6.9
Hz), 4.87 (q, J=5.4 Hz, .about.0.5H), 4.95 (q, J=5.4 Hz,
.about.0.5H), 5.18 (d, J=9.9 Hz, 1H), 5.49 (dd, J=17.4, 9.9 Hz,
1H), 5.83 (dd, J=17.1, 10.2 Hz, .about.0.5H), 5.95 (dd, J=17.1,
10.2 Hz, .about.0.5H). [.alpha.].sub.D.sup.26-106.13 (c=1.446,
1,4-dioxane)
Example 8
Preparation of Compounds 114b
[0167] ##STR119##
[0168] To a solution of compound 114a (1000 g, 2.303 mol) in 6000
mL of THF, was added 1450 ml of aq HCl (450 mL of con. HCl diluted
with 1000 mL of water). The mixture was stirred for 4 hrs, sodium
chloride (300 g) and 3 L of ethyl acetate were added. Organic phase
was separated and washed with water (2000 mL.times.1), the mix
solution of saturated NaHCO.sub.3 solution (2000 mL) and brine
(2000 mL). Combined aqueous phase was extracted with ethyl ether
(20000 mL.times.1). Washed with water (500 ml), brine (500 ml).
Dried. 980 g of compound 114b was obtained after column
chromatography (hexanes/ethyl acetate, 100/0 to 1/1). The yield was
57% (three steps, from 108b to 114b). .sup.1H NMR (CDCl.sub.3, 300
MHz): .delta. 1.27 (s, 3H), 1.32 (s, 3H), 1.64 (s, 3H), 1.77 (t,
J=13.8 Hz, 1H), 1.97 (s, 3H), 2.19 (dd, J=12.9, 6.0 Hz, 1H), 2.51
(br s, 3H), 3.62 (br s, 1H), 4.31 (dd, J=13.5, 5.4 Hz, 1H), 5.18
(d, J=10.2 Hz, 1H), 5.51(br d, J=17.1 Hz, 1H), 6.02(br dd, J=17.1,
10.2 Hz, 1H).
Example 9
Preparation of Phosphonium Salt (S)-102a from Alkynediol 114b
[0169] ##STR120##
[0170] 650 g of alkynediol 114b (2.62 mol) was added to a mixture
of 4300 mL of methylene chloride and 4300 mL of H.sub.2O. After
cooling to 0.degree. C., NH.sub.4Cl (280.07 g, 5.24 mol, 2 eq) and
Zn (256.67 g, 3.93 mol, 1.5 eq) was added.
[0171] Then the reaction mixture was stirred for 3.5 h at
0.about.5.degree. C. and the reaction was checked with HPLC. The
mixture was filtered through a pad-of celite, washed with DCM (500
ml.times.5). The organic phase was dried. To this solution, 387.6
mL of 48% aqueous HBr (3.4 mol, 1.3 eq) was added in two portions
at -8.degree. C. After 30 min, the reaction temperature was raised
to -2.degree. C. Water (1000 ml) was run in, and the organic phase
was separated off. The organic phase was washed with water (1000
ml.times.3). To the organic solution was added 23 mL of
1,2-epoxybutane. While cooling to .about.10.degree. C., 755.3 g
(2.88 mol, 1.1 eq) of triphenylphosphine was added. After PPh.sub.3
was dissolved, another 23 mL of 1,2-epoxybutane was added and the
mixture was stirred at rt for 3.5 h. Concentrated and .sup.tBuOMe
(1500 mL) was added to precipitate the phosphonium salt. The solids
were filtered and washed with .sup.tBuOMe (100 ml.times.2). 1000 g
of 102a (66%, three steps from 114b to 102a) was obtained.
Example 10
Preparation of (3S,3'S)-all-E-astaxanthin
[0172] ##STR121##
[0173] To a mixture of phosphonium salt (S)-102a (463 g, 0.804 mol,
2.2 eq), 4 .ANG. MS (100 g) and C.sub.10-dialdehyde
(2,7-dimethyl-2,4,6-octatrienedial 122)(60 g, 0.365 mol, 1 eq) in
DCM (7 L) at 0.degree. C., MeONa in 151 mL, 0.804 mol, 2.2 eq) was
added dropwise. After 4 h, the additional 42 g of phosphonium salt
(S)-102a (42 g, 0.2 mol) and 14 mL of MeONa in MeOH (30 wt %, 0.2
mol) was added. After 21 h, the mixture was filtered through a
silica gel pad (eluents: DCM/EtOAc.about.DCM/MeOH). Concentrated
and filtered to obtain 110 g of crude product. The crude product
(297 g) was mixed with 1000 ml of ethyl alcohol refluxed for 3 h.
After cooling, filtered and washed with ethyl alcohol (50
ml.times.2) to obtain 221 g of (3S,3'S)-all-E-astaxanthin. .sup.1H
NMR (CDCl.sub.3, 300 MHz): .delta. 1.21 (s, 6H), 1.32 (s, 6H),
1.81(t, J=13.2 Hz, 2H), 1.94 (s, 6H), 1.99 (s, 6H) and 2.00 (s,
6H), 2.15 (dd, J=12.6, 5.7 Hz, 2H), 4.32 (dd, J=13.8, 5.7 Hz, 2H),
6.18-6.72 (m, 14H).
Example 11
Preparation of Compounds 110a
[0174] ##STR122##
[0175] Ethyl vinyl ether (788 mL) was cooled to 5.degree. C. and
treated with PTSA (450 mg) followed by the slow addition of
compound 110b (freshly distilled, 450 g, 4.68 mol). After the
addition was done, the reaction mixture was kept at rt for 3 h,
quenched with triethylamine (3 mL), and then distilled to yield
acetal 110a (770 g, 97.8%). Bp: .about.80.degree. C./20 mmHg.
Example 12
Preparation of 2-(Triphenyl-phosphanylidene)-propionic Acid Ethyl
Ester
[0176] TABLE-US-00001 ##STR123## ##STR124## Quantity Raw Materials
FW Used Moles Ethyl 2-bromopropionate 181.03 1.0 Kg 5.52 mol
Triphenyl Phosphine 262.29 1.6 Kg 6.10 mol Potassium Carbonate
138.21 800 g 5.79 mol EtOAc 10 L MeOH 10 L
[0177] 1.6 Kg (6.10 mol) triphenyl phosphine was dissolved in 10 L
ethyl acetate and 1.0 Kg of ethyl 2-bromopropionate was added into
the above solution. The reaction mixture was stirred at room
temperature for 2 days. White solid was filtered off and the
precipitate was washed with ethyl acetate. The resulting compound
was dissolved in methanol and treated with saturated aqueous
potassium carbonate. After stirring for 2 h, the yellow solid was
filtered off and washed with water to give 1.5 Kg (75%) of desired
product.
Example 13
Preparation of 4Hydroxy-2-methyl-but-2-enoic Acid Ethyl Ester
[0178] TABLE-US-00002 ##STR125## ##STR126## ##STR127## Quantity Raw
Materials FW Used Moles 2-(Triphenyl-phosphanylidene)- 362.40 886 g
2.44 mol propionic acid ethyl ester Glycoaldehyde dimer 120.10 140
g 1.17 mol DCM 10 L
[0179] 886 g (2.44 mol) of 2-(triphenyl-phosphanylidene)-propionic
acid ethyl ester in methylene chloride (4 L) was added dropwise
into a refluxing solution of glycoaldehyde dimer (140 g, 1.17 mol)
in methylene chloride (6 L). After refluxing for 4 h, the solvent
was evaporated. Resulting crude product was fractionated (bp
108-114.degree. C. at 2 mmHg) to give 304 g (90%) pure product as
an oil. .sup.1H-NMR (300 Hz CDCl.sub.3) .delta. 6.88 (t, 1H, CH),
4.35 (d, 2H, CH.sub.2OH), 4.20 (q, 2H, OCH.sub.2), 1.85 (s, 3H,
CH.sub.3), 1.30 (t, 3H, CH.sub.3).
[0180] Note: This process was repeated and 660 g title compound was
collected.
Example 14
Preparation of 4-Bromo-2-methyl-but-2-enoic Acid Ethyl Ester
[0181] TABLE-US-00003 ##STR128## ##STR129## Quantity Raw Materials
FW Used Moles 4-Hydroxy-2-methyl-but-2-enoic acid 144.17 567 g 3.93
mol ethyl ester Carbon tetrabromide 331.63 1.44 kg 4.34 mol
Triphenyl phosphine 262.29 1.13 kg 4.30 mol THF 8 L
[0182] To a cooled solution (0.degree. C) of
4-hydroxy-2-methyl-but-2-enoic acid ethyl ester (567 g, 3.93 mol)
in THF(8 L) was added carbon tetrabromide followed by triphenyl
phosphine. The reaction mixture was slowly warmed to room
temperature and stirred overnight. White solid (identified as
compound 6) was isolated by filtering. The filtration was condensed
and added ether, the resulting white precipitated (identified as
triphenyl phosphate and triphenyl phosphine) was filtered off and
discarded. Ether was evaporated and the resulting crude product was
used without further purification in the next step.
[0183] Note: This process was repeated until 660 g of
4-hydroxy-2-methyl-but-2-enoic acid ethyl ester was consumed.
Example 15
Preparation of 2-Methyl-4-(triphenyl-phosphanyl)-but-2-enoic Acid
Ethyl Ester Bromide Salt
[0184] TABLE-US-00004 ##STR130## ##STR131## Quantity Raw Materials
FW Used Moles 4-Hydroxy-2-methyl-but-2-enoic acid 207.07 940 g 4.54
mol ethyl ester Triphenyl phosphine 262.29 1.34 Kg 5.11 mol EtOAc
10 L
[0185] 940 g (4.54 mol) of 4-hydroxy-2-methyl-but-2-enoic acid
ethyl ester was added into the solution of triphenyl phosphine
(1.34 Kg, 5.11 mol) in 10 L ethyl acetate. The reaction mixture was
stirred at room temperature for 2 days. The resulting white
precipitate was filtered and washed with ethyl acetate to give 2.11
kg (99%) of the title compound. .sup.1H-NMR (300 Hz DMSO-d.sub.6)
.delta. 7.78-7.95 (m, 15H, ArH), 6.40 (q, 1H, CH), 4.76 (q, 2H,
CH.sub.2P), 4.10 (q, 2H, CH.sub.2), 1.60 (d, 3H, CH.sub.3), 1.15
(t, 3H, CH.sub.3).
[0186] Note: This process was repeated and 4.2 Kg title compound
was collected
Example 16
Preparation of
2,6,11,15-Tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaenedioic Acid
Diethyl Ester
[0187] TABLE-US-00005 ##STR132## ##STR133## Quantity Raw Materials
FW Used Moles 2-Methyl-4-(triphenyl-phosphanyl)-but-2-enoic acid
469.35 2006.6 g 4.28 mol ethyl ester bromide salt
2,7-Dimethyl-octa-2,4,6-trienedial 164.20 234 g 1.43 mol NaOMe/MeOH
(30%) 54.02 749 mL 4.00 mol Methylene chloride 5 L
[0188] To a refluxing solution of
2-Methyl-4-(triphenyl-phosphanyl)-but-2-enoic acid ethyl ester
bromide salt (2006.6 g, 4.28 mol) and
2,7-Dimethyl-octa-2,4,6-trienedial (234 g, 1.43 mol) in DCM (5 L)
was added dropwise a solution of 30% by wt. NaOMe (749 mL, 4.00
mol) in methanol. The reaction mixture was refluxed for 3 hrs. The
mixture was pushed through a short column of silica and the solvent
was reduced in vacuo. The residue was redissolved in EtOH (3 L) and
heated to reflux for 3 hrs. Cooled and filtered. The solid was
washed with MeOH (100 mL.times.3) then diethyl ether (100 mL) and
dried to give 250 g of orange powder( 45%) .sup.1H NMR (300 Hz,
CDCl.sub.3) .delta. 7.28 (s, 1H, CH), 7.26 (s, 1H, CH), 6.60 (m,
8H, CH), 4.23 (q, 4H, CH.sub.2), 1.98 (s, 6H, CH.sub.3) 1.53 (s,
6H, CH.sub.3), 1.25 (t, 6H, CH.sub.3).
Example 17
Preparation of
2,6,11,1-Tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaene-1,16-diol
[0189] TABLE-US-00006 ##STR134## ##STR135## Quantity Raw Materials
FW Used Moles
2,6,11,15-Tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaenedioic acid
diethyl ester 384.51 200 g 0.52 mol Methylene chloride 3000 mL
Diisobutylaluminum hydride-(1.5 M, Toluene) 142.22 1533 mL 2.30
mol
[0190] To a solution of
2,6,11,15-tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaenedioic acid
diethyl ester (200 g, 0.52 mol) in 3000 mL of DCM was added
dropwise a solution of diisobutylaluminum hydride in toluene (1.5
M, 1533 mL, 2.30 mol) at -78.degree. C. The mixture was stirring at
0.degree. C. for 2 h. 100 mL of water was added NaOH was added to
quench the reaction. The suspension was filtered off and solid was
washed by a large amount of THF. The combined organic layer were
dried over Na.sub.2SO.sub.4 and evaporated to give 133.16 g (85%)
brown solid. .sup.1H NMR (300 Hz, CDCl.sub.3) .delta. 6.41 (m, 10H,
CH), 4.16 (s, 4H, CH.sub.2), 1.95 (m, 12H, CH.sub.3)
Example 18
Preparation of
2,6,11,15-Tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaenedial
[0191] TABLE-US-00007 ##STR136## ##STR137## Quantity Raw Materials
FW Used Moles
2,6,11,15-Tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaene-1,16-diol
300.44 50 g 0.166 mol Manganese dioxide 86.94 500 g 5.75 mol
Methylene chloride 3000 L
[0192] To a suspension of
2,6,11,15-tetramethyl-hexadeca-2,4,6,8,10,12,14-heptaene-1,16-diol
(50 g, 0.166 mol) in 3000 mL of DCM was added portionwise manganese
dioxide (500 g, 5.75 mol) at room temperature. The mixture was
After heated to reflux for 2 h, the solid was filtered via celite
and washed with CH.sub.2Cl.sub.2. The solvent was removed under
reduced pressure to give 36 g of pure product (73%). .sup.1H-NMR
(300 Hz DMSO-d.sub.6) .delta. 9.42 (s, 2H, CHO), 7.18 (s, 1H, CH),
7.16 (s, 1H, CH), 6.93 (m, 6H, CH), 2.01 (s, 6H, CH.sub.3), 1.82
(s, 6H, CH.sub.3)
Example 19
Preparation of
(S)-(-)-4-Hydroxy-3-methoxy-2,6,6-trimethyl-cyclohex-2-enone
[0193] ##STR138##
[0194] To a solution of
(1S,2S)-N-p-toluenesulfonyl-1,2-diphenylethylenediamine (26.8 mg,
0.073 mmol) in Argon sparged 2-propanol (10 mL) was added
dichloro(p-cymene)ruthenium(II)dimer (11.2 mg, 0.018 mmol). The
suspension was heated to 80.degree. C. for 30 min during which time
the solids went into solution. The reaction was cooled to room
temperature, a solution of 1 (670 mg, 3.67 mmol) in degassed
2-propanol (15 mL) was added followed by 0.1 M KOH in 2-propanol
(1.8 mL) and then stirred overnight. TLC analysis (1:1 ethyl
acetate:hexanes) showed the reaction was complete so the reaction
was neutralized with aq. citric acid, filtered through a small pad
of silica gel and then concentrated under vacuum. Purification by
column chromatography (silica gel, 20:80 ethyl acetate:hexanes to
40:60 ethyl acetate:hexanes over 30 min) provided compound 2 (589
mg, 87%) as a waxy solid
Example 20
Preparation of
(S)-2,2,4,6,6-Pentamethyl-7,7a-dihydro-6H-benzo[1,3]dioxol-5-one
[0195] ##STR139##
[0196] Compound 2 (587 mg, 3.18 mmol) was dissolved in acetone (5
mL), 2,2-dimethoxypropane (10 mL) and water (0.15 mL).
p-Toluenesulfonic acid monohydrate (30 mg, 0.157 mmol) was added
and the reaction was heated to reflux. After one hour the reaction
had only gone 10% so more water (0.15 mL) was added and the reflux
was continued. The reaction was monitored every hour and more water
(0.15 mL) was added until a total of 0.75 mL had been added. At
this point the reaction was cooled and allowed to stir over night.
The next morning all the enol ether had been hydrolyzed so the
reaction was heated to reflux for 1 hour to form the acetonide then
cooled and quenched with saturated aq. sodium bicarbonate (0.2 mL).
The volatile solvents were removed under reduced pressure then the
reaction was partitioned between ethyl acetate and water then
extracted with ethyl acetate, dried over sodium sulfate, filtered
and concentrated under vacuum. Purification by column
chromatography (silica gel, 15:85 ethyl acetate:hexanes to 25:75
ethyl acetate:hexanes over 20 column volumes) provided compound 3
(537 mg, 80%) as an oil. .sup.1H NMR (CDCl.sub.3) .delta. 4.88 (m,
1H), 2.20 (dd, J=11.5 Hz, J=5.5 Hz, 1H), 1.85 (dd, J=11.5 Hz,
J=11.5 Hz, 1H), 1.69 (s, 3H), 1.63 (s, 3H), 1.56 (s, 3H), 1.19 (s,
3H), 1.16 (s, 3H); ESI, m/z 211 [M+H].sup.+, 98.2% ee by HPLC.
HPLC Conditions:
[0197] Chiral method [0198] Mobile Phase 95:5 heptane:2-propanol
[0199] Column: CHIRALCEL OD column, 4.6 mm.times.250 mm, 10 um.
Part #14625 [0200] Flow: 1 mL/min [0201] Detection: UV@254 nm
Example 21
Preparation of Epoxyketoisophorone
[0202] ##STR140##
[0203] A buffer may be substituted for the controlled base feed and
the pH controller as described here. To a 500 ml 3-neck Morton
flask fitted with a bottom outlet, an addition funnel, a magnetic
stirrer and a thermometer were charged 30 g 5 wt % aqueous sodium
bicarbonate, 30 g 5 wt. % aqueous sodium carbonate, and 20 g
keto-isophorone (0.132 mole). To the stirred mixture was added
dropwise over one hour while maintaining the temperature between 20
and 25.degree. C. with water-ice bath cooling 15 g 35% hydrogen
peroxide (0.165 mole) and the mixture stirred an additional three
hours. TLC (e.g., ethyl acetate; heptane 30:70 v/v, silica, iodine
visualization, ketoisophorone Rf 0.70, epoxyketoisophorone Rf 0.77)
showed complete conversion. The mixture was allowed to separate,
the organic phase retained and the aqueous extracted three times,
each time with 100 ml dichloromethane. The combined organic and
dichloromethane phases were then washed with 50 ml 5 wt % sodium
bisulfite solution then with 50 ml 20 wt % sodium chloride solution
and the solution dried over anhydrous sodium sulfate. The filtered
solution was then concentrated in vacuo on a rotovac to furnish
19.7 g epoxyketoisophorone. Yield is estimated at 89%. NMR of this
product showed it to be >95% pure. 1H NMR: 1.08 (s, 3H), 1.3 (s,
3H), 1.55 (s, 3H), 2.26 (d, j=17, 1H), 3.05 (d, j=17, 1H), 3.52 (s,
1H).
Example 22
Preparation of 3-Hydroxyketoisophorone
[0204] ##STR141##
[0205] The epoxyketoisophorone product was converted to
3-hydroxyketoisophrone. To a 500 ml round bottom flask equipped
with an addition funnel, a thermometer, and a magnetic stirrer were
charged 30 ml water and 19.6 g epoxyketoisophorone and the mixture
stirred while adding dropwise over one hour 18 ml 28 wt % sodium
hydroxide solution while keeping the temperature between 30 and
35.degree. C. with a water ice cooling bath. The yellow mixture was
stirred another two hours, cooled to room temperature then
acidified by dropwise addition to pH 1 with 37% hydrochloric acid
during which a solid precipitated. The slurry was stirred for one
hour, then filtered over paper, washed to neutrality with water,
then dried at 50.degree. C. and 26 inches vacuum with a nitrogen
purge to furnish 17.6 g 3-hydroxyketoisophorone as a yellowish
solid. The yield is estimated at 90%. mp 137-139 (lit.
141-143).
Example 23
Preparation of 3-Methoxyketoisophorone
[0206] ##STR142##
[0207] 0.17 g epoxyketoisophorone (1.01 mmole) was dissolved in 2
mL dry methanol under an argon atmosphere. Sodium methoxide was
added to the reaction causing the reaction to darken, after an hour
at room temperature the reaction was heated to 50.degree. C. The
solvents were removed under reduced pressure, the reaction was
worked up with water and methylene chloride. The methylene chloride
phase was extracted with two portions of a sodium chloride solution
and dried over sodium sulphate. The product resulted as a yellow
oil (128 mg) with which the NMR spectra was consistent with the
desired product.
Example 24
Preparation of 3-Methoxyketoisophorone
[0208] ##STR143##
[0209] To a 3-neck round bottom flask fitted with a heating mantle,
an addition funnel, a magnetic stirrer, and a reflux condenser were
charged 1.68 g 3-hydroxyketoisophorone (10 mmole) and 10 mL
methanol and 11 mL 1 N sodium hydroxide and the mixture stirred to
furnish a yellow solution. To the solution was added dropwise 1.50
g dimethylsulfate which caused clouding. The resulting mixture was
stirred vigorously for 2 hours at 20.degree. C. then warmed to
reflux. The homogeneous solution was held at reflux for 4 hours.
TLC showed the reaction to be incomplete with no change after
another 2 hours. On cooling the reaction mixture was combined with
25 ml water then extracted three times, each time with 50 ml
dichloromethane. The combined dichloromethane phases were extracted
with 25 ml 5 wt % sodium carbonate and 25 ml 20 wt % sodium
chloride then dried over anhydrous sodium sulfate. The filtered
solution was stripped of solvent in a rotovac to 50.degree. C. and
26 inches vacuum to furnish a straw colored oil of 1.2 g
3-methoxyketoisophorone. Yield is estimated at 70%. NMR of the
product showed it to be >90% pure. 1H NMR: 1.25 (s, 6H), 1.90
(s, 3H), 2.70 (s, 2H), 4.00 (s, 3H).
Example 25
Preparation of 3-Methoxyketoisophorone
[0210] ##STR144##
[0211] 0.17 g 3-hydroxyketoisophorone (1.01 mmole) was dissolved in
3 mL methanol at 0.degree. C. Diazomethane in ether was added
dropwise to the solution to control foaming and spattering.
Addition was continued until a yellow color persisted (.about.8
mL). Reaction was allowed to continue for one hour, and solvents
removed. A yellow oil (186 mg) resulted with an NMR consistent with
the desired product.
Example 26
Preparation of 3-Methoxyketoisophorone
[0212] ##STR145##
[0213] 0.2 g 3-hydroxyketoisophorone (1.19 mmole) was dissolved in
methanol. 0.8 g trimethyl orthoformate and 0.04 g trifluoroacetic
acid were added to the solution. The reaction was heated to
50.degree. C.
Example 27
Preparation of 3-Methoxyketoisophorone
[0214] ##STR146##
[0215] 0.107 g epoxyketoisophorone (0.64 mmole) was dissolved in
1.0 mL methanol under an argon atmosphere. Trimethyl orthoformate
and trifluoroacetic acid were added to the solution. The reaction
was heated to 50.degree. C. and allowed to stir overnight.
Example 28
Preparation of 3-Methoxyketoisophorone
[0216] ##STR147##
[0217] 0.5 g 3-hydroxyketoisophorone (2.98 mmole) was dissolved in
2 mL dry pyridine and 9 mL methylene chloride under an argon
atmosphere. MesCl was added in one portion and the reaction was
stirred overnight. The reaction was stripped of solvents, and
methanol under argon was added (the precipitates were not
completely soluble). Sodium methoxide was added resulting in a dark
red color and the reaction was stirred at room temperature for six
hours. The reaction was checked by TLC, the product was present in
apparently low yields.
Example 29
Preparation of 4(S)-Hydroxy-ketoisophorone
[0218] ##STR148##
[0219] To a 50 ml round bottom flask fitted magnetic with a stirrer
and a septum with Argon purge was charged 10 ml isopropanol and
11.2 mg dichloro (p-cymene ) ruthenium dimer and 26.8 mg 1S,2S
(+)-N-p-luenesulfonyl,1,2-diphenylethylenediamine and the mixture
heated to 80.degree. C. for thirty minutes then cooled to
20.degree. C. To the mixture was charged 670 mg
3-methoxyketoisophorone in degassed 15 ml isopropanol and 1.8 ml
0.1 M potassium hydroxide in isopropanol and the reaction mixture
stirred overnight. The reaction mixture was neutralized by adding a
solution of 35 mg citric acid in 1 ml water, the mixture filtered
through a small pad of silica gel, them stripped to dryness on a
rotovac. The residue was chromatographed over 40 g silica gel using
a gradient of ethyl acetate-hexanes 20:80 to 40:60 v/v. On
concentration of fractions and stripping in vacuo was obtained 589
mg 4-hydroxyketoisophorone as a white crystalline solid. Yield was
estimated at 87%. 1H NMR: 1.10 (s, 3H), 1.22 (s, 3H), 1.75 (s, 3H)
2.2 (d,d, 1H) 4.0 (s, 3H), 4.7 (m, 1H).
Example 30
Preparation of 4Hydroxy-ketoisophorone
[0220] ##STR149##
[0221] 70 mg 3-methoxyketoisophorone (0.38 mmole) was dissolved in
methanol (2 mL) under an argon atmosphere. Sodium borohydride (50
mg) was added to the reaction in one portion and the reaction was
stirred at room temperature for 0.5 hours. Solvents were removed
under reduced pressure and worked up with water (0.5 mL) and
methylene chloride (2.0 mL). Methylene chloride fraction was dried
over sodium sulfate and the solvent removed under reduced pressure.
A colorless oil resulted (39 mg).
Example 31
Preparation of 4Hydroyxketoisophorone Acetone Ketal
[0222] ##STR150##
[0223] To a 50 ml round bottom flask fitted with a reflux condenser
and a magnetic stirrer were charged 587 mg 4-hydroxyketoisophorone
and 5 ml acetone and 10 ml 2,2-dimethoxypropane and 30 mg
p-toluenesulfonic acid hydrate and 150 mg water and the mixture
heated to reflux. At intervals of two hours an additional 150 mg
water were added each time and after the fourth addition reflux
continued for an additional two hours. The cooled reaction mixture
was neutralized by addition of 0.2 ml saturated sodium bicarbonate
solution then stripped in vacuo to near dryness. The residue was
extracted with ethyl acetate and water, the organic phase dried
with sodium sulfate then concentrated in vacuo. The residue was
chromatographed on 40 g silica gel eluting with ethyl
acetate-hexanes 15:85 to 25:75. The combined and stripped product
fractions furnished 537 mg ketal as an oil which later
crystallized. The yield is estimated at 80%. Chiral HPLC showed an
enantiomeric excess of 98%. 1H NMR: 1.18 (s, 3H), 1.20 (s, 3 H),
1.55 (s, 3H), 1.63 (s, 3H), 1.70 (s, 3H), 1.85 (d,d, 1H), 2.20 (d,
1H), 4.90 (m, 1H).
Example 32
Bulk Chromatographic Separation of the Diastereomeric Dicamphanic
Acid Ester(s) of Astaxanthin
[0224] Bulk chromatographic separation of the diastereomeric
dicamphanic acid ester(s) of synthetic astaxanthin at preparative
chromatography scale was performed to subsequently make gram-scale
quantities of each stereoisomer of disodium disuccinate ester
astaxanthin. A total of 135 g of astaxanthin dicamphanate esters
(ASTA-DCE) prepared by derivatization of racemic astaxanthin with
(-)-camphanic acid chloride were fractionated by preparative HPLC
(using a 77 mm i.d. 25 cm column formed by packing 550 g of 10
.mu.m Kromasil 60 .ANG. silica; Eka Chemicals, Marietta, Ga.) into
a Varian RamPak column packing station. After the dry column
packing material was mixed with 1200 mL of toluene/2-propanol
(50/50) and the resulting slurry was transferred to the 77 mm i.d.
column packing chamber, the column bed was formed using the dynamic
axial compression of the RamPak unit. The packing solvent was
flushed from the column bed for 50 min at a flow rate of 150 mL/min
using the preparative HPLC mobile phase consisting of 95% toluene
and 5% methyl ethyl ketone (MEK). The preparative HPLC system
consisted of a Waters Prep 4000 solvent delivery system and a
Waters model 486 variable UV detector fitted with a prep cell (3 mm
path length).
[0225] Sample solution was injected directly through the pump,
detection was at 580 nm, and the chromatogram was recorded on a
strip chart recorder. At the preparative flow rate of 280 mL/min,
the system backpressure was 840 psi. The laboratory was equipped
with yellow lights, and the windows were covered to avoid any
effects of light on the sample. A sample solution for preparative
HPLC was prepared by dissolving 30 g of ASTA-DCE in 90 mL of
methylene chloride and diluting the solution with 210 mL of
toluene. A portion of the resulting solution (272 mL) was further
diluted with 688 mL of preparative HPLC mobile phase to generate
the sample solution that was subsequently injected onto the
preparative HPLC system. The preparative HPLC injection consisted
of pumping 120 mL of this ASTA-DCE sample solution (3.4 g of
ASTA-DCE) through the pump and onto the preparative column. The
preparative loading was selected to optimize sample throughput, and
the resulting chromatogram consisted of three slightly overlapping
peaks with the 3R,3'R ester eluting at 14 min, the meso-(3R,3'S)
ester at 16.5 min, and the 3S,3'S ester at 21.5 min. To take
advantage of the blank section of the chromatogram for the first 10
min, subsequent injections were made 20 min into the previous run
at the valley between the meso and 3S,3'S peaks. Heart cuts of each
of the three peaks were collected in addition to the mixed
fractions at the overlap of the 3R,3'R/meso and the meso/3S,3'S
peaks.
[0226] A total of 40 preparative injections were processed using 84
L of mobile phase. Thirty-six (36) L of effluent were collected
among the five fractions. The preparative system was flushed with
100 mL of methylene chloride approximately every 6-8 injections or
whenever the chromatographic separation deteriorated due to effects
from mixing with mobile phase in the pump heads during the
injection process. Purified materials were recovered by removing
the solvents in a rotary evaporator protected from light to afford
25.4 g of 3R,3'R ester, 47.8 g of meso-(3R,3'S) ester, and 24.9 g
of 3S,3'S ester. The purified astaxanthin dicamphanate esters were
saponified to afford 8.5 g (79.8% purity by HPLC) of
3R,3'R-astaxanthin, 18.2 g (90.1% purity by HPLC) of
meso-astaxanthin, and 9.4 g (82.0% purity by HPLC) of
3S,3'S-astaxanthin. The major impurities of the saponification
reaction were the 13- and 9-cis isomers of astaxanthin, identified
by HPLC. The cis-isomers were thermally isomerized to all-trans by
refluxing in heptane to afford 8.5 g (87.3% purity by HPLC) of
3R,3'R-astaxanthin, 18.2 g (92.5% purity by HPLC) of
meso-astaxanthin, and 9.4 g (86.8% purity by HPLC) of
3S,3'S-astaxanthin.
Example 34
General Preparation of Lycophyll 2H
[0227] Crocetindialdehyde (238) was obtained from SynChem, Inc.
(Des Plaines, Ill.) as a brick-red solid and was used without
further purification. Lycopene was obtained from ChromaDex (Santa
Ana, Calif.) as a red solid and was used without further
purification. Acetic acid 3,7-dimethyl-8-oxo-octa-2,6-dienyl ester
(230a) (Liu and Prestwich 2002) was synthesized by literature
procedures from commercially available geranyl acetate (228a). All
other reagents and solvents used were purchased from Acros Organics
(Morris Plains, NJ) and Sigma-Aldrich (St. Louis, Mo.) and were
used without further purification. All reactions were performed
under a nitrogen atmosphere. All flash chromatographic
purifications were performed on Natland International Corporation
230-400 mesh silica gel using indicated solvents. LC/MS (APCI and
ESI+modes) were recorded on an Agilent 1100 LC/MSD VL system;
column: Zorbax Eclipse XDB-C18 Rapid Resolution (4.6.times.75 mm,
3.5 .mu.m); temperature: 25.degree. C.; flow rate: 1.0 mL/min.;
mobile phase (A=0.025% TFA in H.sub.2O, B=0.025% TFA in
acetonitrile). Gradient program (for intermediates 230a-236a and
216a): 70% A/30% B (start), step gradient to 50% B over 5 minutes,
step gradient to 100% B over 1.3 minutes, hold at 100% B over 4.9
minutes. Gradient program (for intermediates 218a, 2H): 70% A/30% B
(start), step gradient to 50% B over 5 minutes, step gradient to
98% B over 3.3 minutes, hold at 98% B over 16.9 minutes. All-trans
lycophyll was obtained from crude material using a Waters 996 Photo
Diode Array detector, Millipore 600E System Controller and Waters
717 Autosampler; column: YMC C30 Carotenoid S-5, (10.times.250 mm,
5 .mu.m column); temperature: 25.degree. C.; flow rate: 4.7 mL/min;
mobile phase (A=methanol (MeOH), B=methyl-t-butyl ether (MTBE))
Gradient program: 60% A/40% B (start), step gradient to 80% A over
1 minute, hold at 80% A over 119 minutes. Fractions were collected
from 55-66 minutes. Fraction analysis was performed on a YMC C30
Carotenoid S-5, (4.6.times.250 mm, 5 .mu.m column). Proton nuclear
magnetic resonance (NMR) spectra were obtained on a Varian Unity
INOVA 500 spectrometer operating at 500.111 MHz (megahertz).
Electronic absorption spectra were recorded on a Cary 50 Bio
UV-Visible spectrophotometer.
Example 35
Preparation of 8-Acetoxy-2,6-dimethyl-octa-2,6-dienoic Acid
(232a)
[0228] To a solution of aldehyde 230a (19.5 g, 92.7 mmol) in 300 mL
of tert-butyl alcohol was added 2-methyl-2-butene (98.0 mL, 925
mmol). To this was added a solution of sodium dihydrogen phosphate
(44.5 g, 371 mmol) in 300 mL of water. Sodium chlorite (33.6 g, 371
mmol) was added in several portions. The resulting mixture was
rapidly stirred overnight at room temperature. Ethyl acetate was
added and the aqueous layer was acidified to pH 3 by addition of 1
M HCl. The organic layer was separated, and the aqueous layer was
extracted with ethyl acetate (3.times.200 mL). The combined organic
extracts were washed with brine, dried over MgSO.sub.4, and reduced
to dryness in vacuo. The crude product (27.4 g, 121 mmol, >100%
yield) was used in the next step without further purification:
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 6.84(t of q, J=7.25 Hz,
J=1.50 Hz, 1H, .dbd.CH), 5.34 (t of q, J=7.00 Hz, J=1.50 Hz, 1H,
.dbd.CH), 4.56 (d, J=7.00 Hz, 2H, --CH.sub.2O--), 2.31 (q, J=7.50
Hz, 2H,--CH.sub.2--), 2.15 (t, J=7.50 Hz, 2H, --CH.sub.2--), 2.03
(s, 3H, --CH.sub.3), 1.81 (s, 3H, --CH.sub.3), 1.70 (s, 3H,
--CH.sub.3). LC/MS (ESI): m/z 249 [M+Na].sup.+.
Example 36
Preparation of 8-Hydroxy-2,6-dimethyl-octa-2,6-dienoic Acid
(234a)
[0229] To a solution of acid 232a (20.0 g, 88.4 mmol) in 400 mL of
methanol was added a solution of potassium carbonate (24.4 g, 177
mmol) in 100 mL of water. The resulting mixture was vigorously
stirred overnight at room temperature. The reaction was cooled to
0.degree. C., methylene chloride (200 mL) was added, and the
aqueous layer was acidified to pH 3 with 1 M HCl. The organic layer
was separated, and the aqueous layer was extracted with methylene
chloride (2.times.200 mL). The combined organic extracts were
washed with brine, dried over MgSO.sub.4, and reduced to dryness in
vacuo. The crude product (9.65 g, 52.4 mmol, 59% yield) was used in
the next step without further purification: 1H NMR (500 MHz,
CDCl.sub.3) .delta.: 6.86 (t of q, J=7.25 Hz, J=1.50 Hz, 1H,
.dbd.CH), 5.43 (t of q, J=7.00 Hz, J=1.50 Hz, 1H, .dbd.CH), 4.16
(d, J=7.00 Hz, 2H, --CH.sub.2O--), 2.33 (q, J=7.50 Hz, 2H,
--CH.sub.2--), 2.16 (t, J=7.50 Hz, 2H, --CH.sub.2--), 1.83 (s, 3H,
--CH.sub.3), 1.68 (s, 3H, --CH.sub.3). LC/MS (ESI): m/z 207
[M+Na].sup.+.
Example 37
Preparation of 8-Hydroxy-2,6-dimethyl-octa-2,6-dienoic Acid Methyl
Ester (234b)
[0230] To a solution of acid 234a (20.1 g, 109 mmol) in 400 mL of
DMF was added a solution of potassium carbonate (16.6 g, 120 mmol)
in 80 mL of water. The resulting mixture was vigorously stirred for
several minutes. To the mixture was added iodomethane (7.50 mL, 120
mmol) via syringe. The resulting mixture was vigorously stirred
overnight at room temperature. Ethyl acetate (400 mL) and water
(400 mL) were added and the aqueous layer was acidified to pH 3 by
addition of 1 M HCl. The organic layer was separated and the
aqueous layer was extracted with ethyl acetate (3.times.200 mL).
The combined organic extracts were washed with water (3.times.500
mL), saturated aqueous sodium carbonate, brine, and dried over
MgSO.sub.4. The solvent was removed under reduced pressure and the
crude product purified by flash chromatography
(MeOH/CH.sub.2Cl.sub.2, 1:49) to afford methyl ester 5 as a clear
oil (19.4 g, 90% yield): 1H NMR (500 MHz, CDCl.sub.3) .delta.: 6.72
(t of q, J=7.50 Hz, J=1.50 Hz, 1H, .dbd.CH), 5.43 (t of q, J=6.75
Hz, J=1.50 Hz, 1H, .dbd.CH), 4.16 (d, J=7.00 Hz, 2H,
--CH.sub.2O--), 3.73 (s, 3H, --CH.sub.3), 2.31 (q, J=7.50 Hz, 2H,
--CH.sub.2--), 2.15 (t, J=7.50 Hz, 2H, --CH.sub.2--), 1.83 (s, 3H,
--CH.sub.3), 1.69(s, 3H, --CH.sub.3). LC/MS (ESI): m/z 221
[M+Na].sup.+.
Example 38
Preparation of 8-Bromo-2,6-dimethyl-octa-2,6-dienoic Acid Methyl
Ester (236a)
[0231] To a 0.degree. C. solution of alcohol 234b (12.9, 64.9 mmol)
in 250 mL of anhydrous tetrahydofuran was added carbon tetrabromide
(23.8 g, 71.4 mmol) in several portions. The mixture was stirred
for a few minutes and then triphenylphosphine (18.7 g, 71.4 mmol)
was added and the mixture allowed to warm to room temperature and
stirred overnight. The solvent was removed under reduced pressure
and the resulting residue was suspended in diethyl ether. The
suspension was filtered through a pad of Celite. After solvent
removal under reduced pressure the resulting crude product
(contaminated with triphenylphosphine oxide) was used directly in
the next step: .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 6.61 (t
of q, J=7.50 Hz, J=1.50 Hz, 1H, .dbd.CH), 5 47 (t of q, J=8.00 Hz,
J=1.50 Hz, 1H, .dbd.CH), 3.92 (d, J=8.50 Hz, 2H, --CH.sub.2Br),
3.63 (s, 3H, --CH.sub.3), 2.22 (q, J=8.00 Hz, 2H, --CH.sub.2--),
2.10 (t, J=8.00 Hz, 2H, --CH.sub.2--), 1.75 (d, J=1.00 Hz, 3H,
--CH.sub.3), 1.66 (d, J=1.00 Hz, 3H, --CH.sub.3).
Example 39
Preparation of (2,6-Dimethyl-8-octa-2,6-dienoic Acid Methyl
Ester)Triphenylphosphonium Bromide (216a)
[0232] To a solution of bromide 236a (9.20 g, 35.2 mmol) in ethyl
acetate (200 mL) was added triphenylphosphine (10.2 g, 38.8 mmol).
The resulting mixture was vigorously stirred for a few minutes, at
which time an insoluble material began to oil out from the
solution, adhering to the sides of the flask. The reaction solution
was then decanted into a clean reaction vessel. This procedure was
repeated every 5 to 10 minutes until no more oily insoluble residue
was noted, at which time a white solid started to precipitate from
the solution. The cloudy mixture was then stirred overnight at room
temperature. The mixture was filtered and the filter cake was
rinsed with ethyl acetate and dried in vacuo to afford phosphonium
salt 7 as a white solid (9.60 g, 52% yield). .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta.: 7.88-7.84 (m, 6 arom. H), 7.79-7.75 (m, 3
arom. H), 7.68-7.64 (m, 6 arom. H), 6.51 (t of q, J=5.00 Hz, J=1.00
Hz, 1H, .dbd.CH), 5.10 (q, J=7.00 Hz, 1H, .dbd.CH), 4.70 (d of d,
J=15.0, J=8.00 Hz, 2H, --CH.sub.2PPh.sub.3Br), 3.67 (s, 3H,
--CH.sub.3), 2.16 (q, J=7.00 Hz, 2H, --CH.sub.2--), 2.08 (t, J=6.00
Hz, 2H, --CH.sub.2--), 1.70 (s, 3H, --CH.sub.3), 1.35 (d, J=4.00
Hz, 3H, --CH.sub.3). LC/MS (ESI): m/z 443 [M].sup.+.
Example 40
Preparation of Dimethyl .psi.,.psi.-Carotene-16,16'-dioate
(218a)
[0233] To a solution of crocetindialdehyde (238) (0.810 g, 2.74
mmol) and 216a (4.30 g, 8.21 mmol) in toluene (100 mL) was added 1
M LiOMe in MeOH (7.67 mL, 7.67 mmol) via syringe. The resulting
mixture was refluxed for 24 hours, cooled to room temperature, and
then water (100 mL) was added. The organic phase was collected,
extracted with water twice, and then dried over anhydrous sodium
sulfate. After filtration and removal of the solvent in vacuo, the
resulting residue was purified by flash chromatography (ethyl
acetate:toluene, 1:99) to afford dimethyl ester 240 as a red solid
(1.15 g, 67% yield). LC/MS (APCI): m/z 625 [M+H].sup.+.
Example 41
Preparation of .psi.,.psi.-Carotene-16,16'-diol (10)
[0234] To a solution of dimethyl ester 218a (1.14 g, 1.83 mmol) in
anhydrous tetrahydrofuran (100 mL) at 0.degree. C. was added DIBAL
(20% by wt. in toluene) (9.13 mL, 11.0 mmol) via syringe. The
mixture was warmed to room temperature and stirred for one hour.
The reaction was quenched by the sequential addition of H.sub.2O
(440 .mu.L), 15% aqueous NaOH (440 .mu.L), and H.sub.2O (1.10 mL).
The resulting mixture was stirred for 30 minutes and then dried
over anhydrous MgSO.sub.4. After filtration and removal of solvent
in vacuo, the resulting crude diol 2H (0.39 g, 38%) was used in the
next step without further purification. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta.: 6.63 (d of d, J=15.0 Hz, J=11.5 Hz, 2H, H11,
H11'), 6.63 (d, J=11.0 Hz, 2H, H15, H15'), 6.48 (d of d, J=15.0 Hz,
J=11.0 Hz, 2H, H7, H7'), 6.36 (d, J=15.0 Hz, 2H, H12, H12'), 6.25
(d, J=15.0 Hz, 2H, H8, H8'), 6.19 (d, J=11.5 Hz, 2H, H10, H10 '),
5.95 (d, J=11.0 Hz, 2H, H6, H6'), 5.40 (t of q, J=6.50 Hz, J=1.50
Hz, 2H, H2, H2'), 4.00 (s, 4H, --CH.sub.2O--), 2.19 (t, J=Hz, 4H,
--CH.sub.2--), 2.16 (t, J=Hz, 4H, --CH.sub.2--), LC/MS (APCI): m/z
569 [M+H].sup.+.
Example 42
General Preparation of Lycophyll Derivatives
[0235] LC/MS (APCI) and LC/MS (ESI) were recorded on an Agilent
1100 LC/MSD VL, PDA detector system; column: Zorbax Eclipse XDB-C18
Rapid Resolution (4.6.times.75 mm, 3.5 .mu.m); temperature:
25.degree. C.; flow rate: 1.0 mL/min; mobile phase (% A=0.025%
trifluoroacetic acid in H.sub.2O, % B=0.025% trifluoroacetic acid
in acetonitrile) Gradient program: 70% A/30% B (start), step
gradient to 50% B over 5 min, step gradient to 98% B over 8.30 hold
at 98% B over 25.20 min, step gradient to 30% B over 25.40 min. A
catalytic amount of trifluoroacetic acid is used in the eluents to
improve chromatographic resolution. The presence of trifluoroacetic
acid facilitates the protonation of synthesized lycophyll
dissucinate and diphosphate salts to give the free diacid forms (as
represented by the theoretical molecular ions M.sup.+=768 for
lycophyll disuccinate salt and M.sup.+=728 for lycophyll
disphosphate salt). LRMS: +mode; ESI: electrospray chemical
ionization, ion collection using quadrapole; APCI: atmospheric
pressure chemical ionization, ion collection using quadrapole.
Reverse-phase HPLC was performed on a Waters 996 HPLC with PDA
detector, Millipore 600E System Controller system; column: Zorbax
Eclipse XDB-C18 (9.4.times.250 mm, 5 .mu.m); temperature:
25.degree. C.; flow rate: 2.1 mL/min; mobile phase (% A=0.025%
trifluoroacetic acid in H.sub.2O, % B=0.025% trifluoroacetic acid
in MeOH) Isocratic program: 15% A/ 85% B. .sup.1H NMR analyses were
performed on a Varian spectrometer (300 MHz).
Example 43
Preparation of .psi.,.psi.-carotenyl 16,16'-disuccinate (222a)
[0236] To a solution of lycophyll (2H) (0.10 g, 0.176 mmol) in
CH.sub.2Cl.sub.2 (2 mL) was added N,N-diisopropylethylamine (0.613
mL, 3.52 mmol) and succinic anhydride (0.1761 g, 1.76 mmol). The
solution was stirred at room temperature overnight and then diluted
with CH.sub.2Cl.sub.2 and quenched with cold water/1 M HCl (9/1).
The aqueous layer was extracted two times with CH.sub.2Cl.sub.2 and
the combined organic layer was washed three times with cold water/1
M HCl (9/1), dried over Na.sub.2SO.sub.4, and concentrated to yield
disuccinate 222a (0.124 g, 92red hygroscopic solid; LC/MS (APCI):
11.59 min (65.17%), .lamda..sub.max 295 nm (28%), 362 nm (8%), 447
nm (72%), 472 nm (100%), 503 nm (93%), m/z 769 [M+H].sup.+ (100%),
668 [M-C.sub.4O.sub.3H.sub.4].sup.+ (9%), 651 (89%), 533 (30%);
12.13 min (33.69%), .lamda..sub.max 295 nm (26%), 362 nm (10%), 447
nm (77%), 472 nm (100%), 503 nm (91%), m/z 769 [M+H].sup.+ (28%),
651 (24%), 531 (8%), 261 (100%).
Example 44
Preparation of .psi.,.psi.-carotenyl 16,16'-disuccinate Sodium Salt
(224a)
[0237] To a solution of disuccinate 222a (0.124 g, 0.161 mmol) in
methanol (3 mL ) at 0.degree. C. was added dropwise sodium
methoxide (25% wt in methanol; 0.074 mL, 0.322 mmol). The solution
was stirred at room temperature overnight, then cooled to 0.degree.
C., and water was added. The red mixture was stirred for 5 min at
0.degree. C., and then methanol was removed in vacuo. The red,
aqueous solution was lyophilized to afford disuccinate salt 224a
(0.103 g, 88%) as a red hygroscopic solid; LC/MS (APCI): 11.58 min
(71.72%), .lamda..sub.max 295 nm (13%), 362 nm (9%), 44 nm (68%),
472 nm (100%), 503 nm (90%), m/z 769 [M+H].sup.+ (100%), 651 (42%),
533 (15%); 12.09 min (27.74%), .lamda..sub.max 295 nm (31%), 362 nm
(19%), 447 nm (80%), 472 nm (100%), 503 nm (88%), m/z 769
[M+H].sup.+ (100%), 669 [M-C.sub.4O.sub.3H.sub.4+H].sup.+ (12%),
651 (54%), 551 (8%), 533 (11%).
Example 45
Preparation of Tribenzyl Phosphite (13)
[0238] To a well-stirred solution of phosphorus trichloride (1.7
mL, 19.4 mmol) in Et.sub.2O (430 mL) at 0.degree. C. was added
dropwise a solution of triethylamine (8.4 mL, 60.3 mmol) in
Et.sub.2O (20 mL), followed by a solution of benzyl alcohol (8.1
mL, 77.8 mmol) in Et.sub.2O (20 mL). The mixture was stirred at
0.degree. C. for 30 min and then at room temperature overnight. The
mixture was filtered and the filtrate concentrated to give a
colorless oil. Silica chromatography (hexanes/Et.sub.2O/
triethylamine, 5.5/1/1%) of the crude product gave 13 (5.68 g, 83%)
as a clear, colorless oil that was stored under N.sub.2 at
-20.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.: 7.38
(15H, m), 4.90 (6H, d).
Example 46
Preparation of Dibenzyl Phosphoroiodidate (14)
[0239] To a solution of tribenzyl phosphite (0.708 g, 2.01 mmol) in
CH.sub.2Cl.sub.2 (5 mL) at 0.degree. C. was added I.sub.2 (0.49 g,
1.93 mmol). The mixture was stirred at 0.degree. C. for 10 min or
until the solution became clear and colorless. The solution was
then stirred at room temperature for 10 min and used directly in
the next step.
Example 47
Preparation of Mixture of 16,16'-Benzyl
Phosphoryloxy-.psi.,.psi.-carotenes (221a,221b,221c,221d)
[0240] To a solution of lycophyll (2H) (0.11 g, 0.193 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was added pyridine (0.624 mL 7.72 mmol).
The solution was stirred at 0.degree. C. for 5 min and then freshly
prepared 14 (1.93 mmol) in CH.sub.2Cl.sub.2 (5 mL) was added
dropwise to the mixture at 0.degree. C. The solution was stirred at
0.degree. C. for 1 h and then diluted with CH.sub.2Cl.sub.2 and
quenched with brine. The aqueous layer was extracted twice with
CH.sub.2Cl.sub.2 and the combined organic layer was washed once
with NaSSO.sub.4, once with brine, then dried over Na.sub.2SO.sub.4
and concentrated. Pyridine was removed from the crude red oil by
azeotropic distillation using toluene to yield a mixture of
benzyl-protected diphosphoric acid lycophyll derivatives
221a,221b,221c,221d used in the next step without further
purification; LC/MS (ESI) for 221a: 10.15 min (7.73%),
.lamda..sub.max 295 nm (21%), 362 nm (16%), 447 nm (72%), 472 nm
(100%), 503 nm (87%), m/z 819 [M+H].sup.+ (18%), 800
[M-H.sub.2O].sup.+ (11%), 672 (24%), 531 (10%); LC for 221b: 18.00
min (17.46%), .lamda..sub.max 295 nm (18%), 362 nm (13%), 447 nm
(74%), 472 nm (100%), 503 nm (85%); LC for 221c: 20.08 min
(20.00%), .lamda..sub.max 295 nm (18%), 362 nm (16%), 447 nm (74%),
472 nm (100%), 503 nm (86%); LC for 221d: 22.52 min (54.81%),
.lamda..sub.max 295 nm (19%), 362 rn (18%), 447 nm (73%), 472 nm
(100%), 503 nm (87%).
Example 48
Preparation of 16,16'-Diphosphoryloxy-.psi.,.psi.-carotene
(221e)
[0241] To a solution of a mixture of benzyl-protected diphosphoric
acid lycophyll derivatives 221a,221b,221c,221d (0.193 mmol) in
CH.sub.2Cl.sub.2 (15 mL) at 0.degree. C. was added dropwise
N,O-bis(trimethylsilyl)acetamide (0.479 mL, 1.93 mmol) and then
bromotrimethylsilane (0.203 mL, 1.54 mmol). The solution was
stirred at 0.degree. C. for 15 min, quenched with triethylamine,
and stirred at 0.degree. C. for 5 min. The red solution was then
diluted with CH.sub.2Cl.sub.2, Et.sub.2O, and MeOH (2/1/1), and
then concentrated. The resulting red oil was resuspended in a
minimum amount of MeOH and the cloudy solution was centrifuged to
remove insoluble reaction byproducts. The red supernatant was
concentrated to afford a mixture of monophosphate and diphosphate
lycophyll derivatives (254/221e) (1/4) contaminated with excess
reagents, and reaction and decomposition byproducts; LC/MS (ESI)
for 221e: 9.10 min (39.24%), .lamda..sub.max 295 nm (31%), 362 nm
(18%), 447 nm (74%), 472 nm (100%), 503 nm (88%), m/z 849 (25%),
827 (5%), 368(100%), 357 (11%), 317 (52%); 9.25 min (37.83%),
.lamda..sub.max 295 nm (31%), 362 nm (18%), 447 nm (75%), 472 nm
(100%), 503 nm (89%), m/z 849 (10%), 625 (8%), 581 (6%), 385 (20%),
368 (100%), 357 (28%); LC/MS (ESI) for 254: 10.21 min (18.50%),
.lamda..sub.max 295 nm (32%), 362 nm (24%), 447 (78%), 472 nm
(100%), 503 nm (89%), m/z 648 M.sup.+ (9%), 630 [M-H.sub.2O].sup.+
(5%), 568 (10%), 317 (100%); the crude mixture was subjected to
reverse-phase HPLC purification to give diphosphate 221e
(approximately 70% pure; 0.063 g, 45%) as a red oil, contaminated
with excess reagents, and reaction and decomposition byproducts;
LC/MS (ESI): 9.36 min (4.43%), .lamda..sub.max 295 nm (30%), 362 nm
(25%), 447 nm (79%), 472 nm (100%), 503 nm (82%), m/z 849 (16%),
619 (7%), 399 (23%), 368 (100%), 357 (10%), 317 (8%); 9.58 min
(46.42%), .lamda..sub.max 295 nm (30%), 362 nm (15%), 447 nm (80%),
472 nm (100%), 503 nm (92%), m/z 849 (19%), 619 (5%), 399 (21%),
368 (100%), 357 (10%), 317 (9%); 9.67 min (49.15%), .lamda..sub.max
295 nm (28%), 362 nm (12%), 447 nm (77%), 472 nm (100%), 503 nm
(95%), m/z 849 (15%), 619 (5%), 399 (20%), 368 (100%), 357 (8%),
317 (6%).
Example 49
Preparation of 16,16'-Diphosphoryloxy-.psi.,.psi.-carotene Sodium
Salt (223a)
[0242] To a solution of lycophyll diphosphate (221e) (approximately
70% pure; 0.04 g, 0.055 mmol) in methanol (2 mL) at 0.degree. C.
was added dropwise sodium methoxide (25% wt in methanol; 0.05 mL,
0.22 mmol). The solution was stirred at room temperature overnight,
then cooled to 0.degree. C., and water was added. The red mixture
was stirred for 5 min at 0.degree. C., and then methanol was
removed in vacuo. The red, aqueous solution was lyophilized to
yield diphosphate salt 223a (approximately 50% pure; 0.018 g, 43%)
as a red hygroscopic solid; LC/MS (ESI): 9.26 min (9.34%),
.lamda..sub.max 295 nm (28%), 362 nm (18%), 447 nm (81%), 472 nm
(100%), 503 nm (87%), m/z 897 (8%), 392 (100%), 381 (10%); 9.48 min
(46.98%), .lamda..sub.max 295 nm (29%), 362 nm (15%), 447 nm (80%),
472 nm (100%), 503 nm (91%), m/z 911 (10%), 849 (15%), 399 (87%),
368 (100%); 9.56 min (43.68%), .lamda..sub.max 295 (28%), 362
(12%), 447 nm (77%), 472 nm (100%), 503 nm (90%), m/z 849 (19%),
827 (5%), 368 (100%), 357 (8%).
[0243] In this patent, certain U.S. patents, U.S. patent
applications, and other materials (e.g., articles) have been
incorporated by reference. The text of such U.S. patents, U.S.
patent applications, and other materials is, however, only
incorporated by reference to the extent that no conflict exists
between such text and the other statements and drawings set forth
herein. In the event of such conflict, then any such conflicting
text in such incorporated by reference U.S. patents, U.S. patent
applications, and other materials is specifically not incorporated
by reference in this patent.
[0244] Further modifications and alternative embodiments of various
aspects of the invention will be apparent to those skilled in the
art in view of this description. Accordingly, this description is
to be construed as illustrative only and is for the purpose of
teaching those skilled in the art the general manner of carrying
out the invention. It is to be understood that the forms of the
invention shown and described herein are to be taken as the
presently preferred embodiments. Elements and materials may be
substituted for those illustrated and described herein, parts and
processes may be reversed, and certain features of the invention
may be utilized independently, all as would be apparent to one
skilled in the art after having the benefit of this description of
the invention. Changes may be made in the elements described herein
without departing from the spirit and scope of the invention as
described in the following claims.
Example 50
Separation of (3S,3'S)-all-E-astaxanthin
[0245] Analysis of the stereoisomeric distribution of astaxanthin
was accomplished using a chiral HPLC column. A Regis Pirkle
Covalent D-phenylglycine, 5 .ANG., 4.6.times.250 nm chiral HPLC
column was used. The detector was set at 474 nm. A 10 .mu.L sample
was injected into the column. The sample was passed through the
column using a mobile phase of 75% Heptane, 24% dichloromethane,
and 1% ethanol at a flow rate of 1.5 mL/min. Racemic astaxanthin
(e.g., 3S,3'S, meso (3R,3'S), and 3R,3'R in a 1:2:1 ratio) was run
through the chiral HPLC column and the retention time for 3S,3'S
("S,S")-astaxanthin was 32.763 min, meso-astaxanthin was 31.165,
and 3R, 3'R ("R,R")-astaxanthin was 29.937. The total run time was
60 minutes.
[0246] In this patent, certain U.S. patents, U.S. patent
applications, and other materials (e.g., articles) have been
incorporated by reference. The text of such U.S. patents, U.S.
patent applications, and other materials is, however, only
incorporated by reference to the extent that no conflict exists
between such text and the other statements and drawings set forth
herein. In the event of such conflict, then any such conflicting
text in such incorporated by reference U.S. patents, U.S. patent
applications, and other materials is specifically not incorporated
by reference in this patent.
[0247] Further modifications and alternative embodiments of various
aspects of the invention may be apparent to those skilled in the
art in view of this description. Accordingly, this description is
to be construed as illustrative only and is for the purpose of
teaching those skilled in the art the general manner of carrying
out the invention. It is to be understood that the forms of the
invention shown and described herein are to be taken as the
presently preferred embodiments. Elements and materials may be
substituted for those illustrated and described herein, parts and
processes may be reversed, and certain features of the invention
may be utilized independently, all as would be apparent to one
skilled in the art after having the benefit of this description to
the invention. Changes may be made in the elements described herein
without departing from the spirit and scope of the invention as
described in the following claims. In addition, it is to be
understood that features described herein independently may, in
certain embodiments, be combined.
* * * * *