Activation of a Diels-Alder reaction of a sterol 5,7-diene

Details Activation of a Diels-Alder reaction of a sterol 5,7-diene Activation of a Diels-Alder reaction of a sterol 5,7-diene

2001-03-15

2002-06-04

Badio, Barbara P. (Department: 1616)

Organic compounds -- part of the class 532-570 series

Organic compounds

Cyclopentanohydrophenanthrene ring system containing

C552S540000

Reexamination Certificate

active

06399796

ABSTRACT:

TECHNICAL FIELD
The invention relates to a method for the activation of a Diels-Alder reaction. More specifically, the invention relates to the activation of a Diels-Alder reaction in a sterol 5,7-diene.
BACKGROUND OF THE INVENTION
In certain processes for the commercial production of sterols, a Diels-Alder addition is useful as a protection step and also for purification. Diels-Alder addition has proven useful in the commercial production of 25-hydroxyvitamin D
3
. With the present state of the art, the economics of commercial 25-hydroxyvitamin D
3
production is strongly affected by the yield of the Diels-Alder reaction. It is therefore desirable to maximize the yield of the Diels-Alder reaction in commercial sterol production.
Diels-Alder reactions are the well-known chemical addition of a dienophile to a diene. Textbook methods to activate Diels-Alder reactions use electron-releasing groups to enhance the electron density of the pi-donor diene. Similarly, electron-withdrawing groups are used to decrease the electron density of the pi-acceptor, thus enhancing its electrophilicity. In some processes, such as commercial sterol production, it is not possible or practical to provide functional groups to activate the electron donor for a Diels-Alder reaction. Means to enhance a Diels-Alder reaction in commercial sterol production is sought.
In the art, a semisynthetic commercial production of 25-hydroxyvitamin D
3
involves the saponification of 5,7,24-cholestatrienyl esters from fermentation of a double mutant yeast to form the free 5,7,24-cholestatrienol (also known as 7-dehydrocholesterol or provitamin D
3
). The mutant yeast here contains the erg6 mutation in zymosterol-24-methyltransferase, and a mutation in the expression of ergosta-5,7,24(28)-trienol-22-dehydrogenase enzyme (erg5) as described in U.S. Pat. No. 5,460,949, whose disclosures are incorporated by reference.
Saponification procedures are common in the art, particularly where the hydrolysis of esters in organic matter is desired. Saponification reactions involve treatment with a strong base, typically with heating.
The saponification reaction product also includes squalene, fatty acids, including saturated and unsaturated fatty acids, and other sterols, which can include unconjugated dienols, and mono-unsaturated sterols. To separate the cholestatrienol and other sterols including lanosterol, 4,4,-dimethylzymosterol, 4-methylzymosterol, zymosterol, cholesta-7,24-diene-3&bgr;-ol from that mixture. The pH is typically adjusted to about 7-8 pH units, and the saponificate is extracted with heptane, and the heptane extract is washed with water. The washed heptane extract is concentrated, and mixed with ethyl acetate. This is called the “sterol extract”.
In a Diels-Alder protection and/or purification step of the art, the sterol extract is reacted with a dienophile to form a Diels-Alder adduct of the 5,7-diene of the cholestratrienol, while leaving the unconjugated dienols and other sterols unreacted. The dienophile is typically phthalhydrazine generated in situ from the reaction of aqueous bleach (sodium hypochlorite) on phthalhydrazide. The Diels-Alder adduct is then chromatographically separated from the unreacted yeast sterol mixture, the cholestatrienol is regenerated, further purified and used to make 25-hydroxyvitamin D
3
. This reaction sequence is illustrated in U.S. Pat. No. 5,391,777, whose disclosures are incorporated by reference.
U.S. Pat. No. 5,208,152 describes catalysts of Diels-Alder reactions where the substrates are cyclic conjugated dienes having a fugitive leaving group. Those dienes are unlike sterols.
U.S. Pat. No. 4,503,195 discloses the use of di- and tri-phenylated cation radical polymers as Diels-Alder catalysts, wherein the cation radical is a Group VA element (e.g. nitrogen, phosphorus or arsenic).
U.S. Pat. No. 4,413,154 and U.S. Pat. No. 4,384,153 disclose Diels-Alder reactions of 1,3-butadiene and 4-vinylcyclohexene over molecular sieves/zeolites.
BRIEF SUMMARY OF THE INVENTION
In accordance with the present invention, it has unexpectedly been found that the presence of at least a catalytic amount of an unsaturated C
12
-C
24
fatty carboxylic acid enhances the formation of Diels-Alder adduct between a steroid 5,7-diene and a dienophile (preferably generated in situ from an oxidizable dienophile precursor and an oxidant). According to the invention, at least a catalytic amount of an unsaturated C
12
-C
24
fatty carboxylic acid is exogenously provided to a Diels-Alder reaction.
The steroid 5,7-diene can be any such compound. Those that have been examined all undergo the Diels-Alder reaction with enhanced yield of adduct when at least a catalytic amount of a C
12
-C
24
unsaturated carboxylic acid is added. Ergosterol and 5,7,24-cholestatrienol are preferred steroid 5,7-dienes, with 5,7,24-cholestatrienol being particularly preferred.
The present invention also contemplates an improved method of forming a Diels-Alder adduct with a 5,7-diene sterol comprising the following steps. A 5,7-diene sterol is admixed with a dienophile to form a reaction mixture, and thereby converting the 5,7-diene sterol to a Diels-Alder adduct. The 5,7-diene sterol has the structural formula
wherein R
3
is selected from the group consisting of H and R
1
CO— wherein R
1
is monocyclic aryl of 5 to 7 carbon atoms or lower alkyl, and R
4
, R
5
and R
6
are independently selected from the group consisting of H, hydroxyl, and lower alkyl, and R
7
is a C
1
to C
10
hydrocarbyl group. In one preferred embodiment, the 5,7-diene steroid compound is cholesta-5,7,24-triene-3-ol. Further chemical conversion of the Diels-Alder adduct to provide a modified Diels-Alder adduct is optionally conducted. The Diels-Alder adduct or the modified Diels-Alder adduct is separated from the mixture.
The invention contemplates adding at least a catalytic amount of an ethylenically unsaturated C
12
to C
24
fatty acid to the Diels-Alder reaction mixture. Preferably, the ethylenically unsaturated C
12
to C
24
fatty acid is linolenic acid, linoleic acid, oleic acid or a mixture of two or all three acids.
In one embodiment, the dienophile has the structural formula X—R═R—Y wherein the R groups are both N or both C-Q where the Q groups are H or together form a third bond, and wherein X and Y are selected from electron-withdrawing groups themselves independently selected from the group consisting of —COOH, —CHO, —NO
2
, —CN, —COOR
8
and —COR
8
, where R
8
is lower alkyl, or wherein X and Y are linked together to form a —(CO)—Z—(CO)— bridge in which Z is lower alkylene, monocyclic arylene of 5 to 7 carbon atoms with up to 4 ring substituents, or —NR
2
wherein R
2
is lower alkyl, H or monocyclic aryl of 5 to 7 carbon atoms and up to 5 ring substituents, wherein the ring substituents are selected from the group consisting of —(CH
2
)
n
—NH
2
, —(CH
2
)
n
—COOH, —NO
2
, halogen and lower alkyl, where n is an integer that is zero to 6, inclusive.
In another embodiment, the dienophile is generated in situ from (i) an oxidizable dienophile precursor and (ii) an oxidizing agent effective to oxidize the precursor to form the dienophile, wherein the dienophile precursor has the structural formula X—NH—NH—Y wherein X and Y are selected from electron-withdrawing groups themselves independently selected from the group consisting of —COOH, —CHO, —NO
2
, —CN, —COOR
8
and —COR
8
where R
8
is lower alkyl, or wherein X and Y are linked together to form a —(CO)—Z—(CO)— bridge in which Z is lower alkylene, monocyclic arylene of 5 to 7 carbon atoms with up to 4 ring substituents, or —NR
2
wherein R
2
is lower alkyl, H or monocyclic aryl of 5 to 7 carbon atoms and up to 5 ring substituents, wherein the ring substituents are selected from the group consisting of lower alkyl, halogen, —NO
2
, —(CH
2
)
n
—NH
2
, and —(CH
2
)
n
—COOH, where n is an integer that is zero to 6, inclusive. Preferably, the oxidizable dienophile precursor is phthalhydrazine. Preferably, the oxidizing agent is hypochlorite (e.g. NaOCl or an aqueous solution of sodium hy

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