US3778469A - Isolation of prostaglandins from plexaura homomalla - Google Patents

Isolation of prostaglandins from plexaura homomalla Download PDF

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US3778469A
US3778469A US00307721A US3778469DA US3778469A US 3778469 A US3778469 A US 3778469A US 00307721 A US00307721 A US 00307721A US 3778469D A US3778469D A US 3778469DA US 3778469 A US3778469 A US 3778469A
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prostaglandin
nitroalkane
cortex
prostaglandins
plexaura homomalla
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J Baker
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Wyeth Holdings LLC
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American Cyanamid Co
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C405/00—Compounds containing a five-membered ring having two side-chains in ortho position to each other, and having oxygen atoms directly attached to the ring in ortho position to one of the side-chains, one side-chain containing, not directly attached to the ring, a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, and the other side-chain having oxygen atoms attached in gamma-position to the ring, e.g. prostaglandins ; Analogues or derivatives thereof

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  • This invention relates to a method of recovering a prostaglandin enriched isolate consisting essentially of prostaglandin A 15-'O-acetyl methyl ester, 15-epiprostaglandin A IS-O-acetyl methyl ester, prostaglandin A methyl ester and l5-epi-prostaglandin A methyl ester from the cortex of the gorgonid Plexaura homomalla. More particularly, this invention involves the partition of the material obtained by hexane extraction of dried, comminuted cortex of Plexaura homomalla, after removal of solvent, between two immiscible solvent systems.
  • One phase consists of a nitroalkane solvent system selected from the group consisting of nitroalkanes having one or two carbon atoms, and mixtures thereof.
  • the other phase consists of a nitroalkane-immiscible solvent system selected from the group consisting of alkanes having from six to eight carbon atoms, cycloalkanes having from five to seven carbon atoms, and mixtures thereof.
  • Undesired impurities are concentrated in the nitroalkane-immiscible solvent system whereas the desired prostaglandin A derivatives are found in the nitroalkane solvent system and may be recovered therefrom by removal of solvent.
  • prostaglandins are an important group of substances playing a significant, although as yet not fully defined role in many areas of human biochemistry and physiology.
  • the value of certain prostaglandins, in particular prostaglandin E and prostaglandin F for the induction of labor and as abortifacients has been demonstrated in humans and is under active clinical study.
  • prostaglandin E has been shown to be a potent bronchodilator.
  • the cortex of the sea coral Plexaura homomalla contains relatively large amounts of either prostaglandin A 15(S)-O-acetyl methyl ester or the corresponding compound in the 15(R) configuration, or both.
  • prostaglandin A methyl ester and its IS-epi isomer as well as the prostaglandin A carboxylic acids.
  • the gorgonid Plexaura homomalla is by far the richest source of prostaglandins yet reported.
  • the transformation of the prostaglandin A derivatives of either the 15(8) or 15(R) configuration to 15(S)-prostaglandins E and F has been reported as has the transformation of prostaglandin E to prostaglandin E and thence to prostaglandin P
  • the cortex of Plexaura homomalla is relatively rich in prostaglandins
  • the prostaglandin content is nevertheless only about 12% of the total.
  • the chemical literature describes column chromatography procedures for the final purification of the prostaglandins following ice an initial extraction of the cortex with hexane.
  • the starting material for this initial extraction is the outer sheath or cortex of the animal which is peeled or stripped off.
  • the cortex is then comminuted by grinding or by mincing in the usual manner.
  • the fresh cortex may be frozen and then ground to prevent any possibility of loss of prostaglandin content.
  • the comminuted cortex is then dried by any convenient means such as by air-drying on trays in sunshine or by drying in low temperature ovens. 'If the dried comminuted cortex must be stored for any considerable period of time, it is preferable to store it under nitrogen in order to avoid oxidation of the prostaglandin content thereof.
  • the initial hexane extraction of the dried comminuted cortex may be carried out with any hexane such as nhexane, isohexane, 3-methylpentane, 2-ethylbutane, 2,3- dimethylbutane, etc., or with various mixtures of these isomeric hexanes. Best results are obtained when 3-5 liters of isomeric hexanes are employed per kilogram of cortex.
  • the extraction may be carried out at from about 0 C. to about 60 C., preferably at ambient temperature and with agitation, over a period of time of 2-24 hours.
  • the hexane extract is then separated from the insoluble solid residue by any convenient means such as filtration or centrifugation.
  • the solvent is then removed from the hexane extract by evaporation, distillation under vacuum, or freeze drying to provide an initial prostaglandin-containing material.
  • This initial hexane extraction enriches the prostaglandin content of initial prostaglandin-containing material to about 10% from the original 12% of the cortex.
  • the present invention comprises a novel useful, and convenient process for the further enrichment of the prostaglandin content of the initial hexane extract to about 40-60%, thus substantially reducing the complexity, amount of adsorbent, solvent volume, and time necessary for the final chromatographic purification.
  • these improvements substantially reduce the economic cost.
  • the present invention involves the partition of the material obtained by hexane extraction between two immiscible solvent systems.
  • the first phase consists of a nitroalkane solvent system selected from the group consisting of nitroalkane having one or two carbon atoms, and mixtures thereof.
  • the second phase consists of a nitroalkaneimmiscible solvent system selected from the group consisting of alkanes having from six to eight carbon atoms, cycloalkanes having from five to seven carbon atoms, and mixtures thereof.
  • Suitable second phase solvent systems are, for example, a mixture of isomeric hexanes, a mixture of isomeric heptanes, a mixture of isomeric octanes, cyclopentane, cyclohexane, cycloheptane, a 1:1 by volume mixture of isomeric hexanes and isomeric octanes, a 1:1 by volume mixture of cyclopentane and isomeric hexanes, and the like.
  • the partition of the ca. 10% prostaglandin-containing material is best carried out with about equal volumes of first and second phase solvent systems, although considerable variation is permissible with this parameter. Preferably, about 3-5 liters sum total of the solvent phases is employed per kilogram of the hexane extract material. Partition is carried out, with agitation, at a temperature of from about 0 C. to about 60 C., preferably at ambient temperature, over a period of time of 2 minutes to one hour. Thereupon, the nitroalkane-immiscible second phase solvent system is separated and discarded.
  • Solvent is then removed from the nitroalkane first phase solvent system by evaporation, vacuum distillation, or freeze drying to provide a prostaglandin enriched material containing about 40-60% of prostaglandins.
  • the 15(S) and/or 15(R) prostaglandin A IS-O-acetyl methyl esters, as well as the corresponding prostaglandin A methyl esters, and sometimes the corresponding prostaglandin A carboxylic acids, are found in this final concentrate and may be subsequently isolated and purified by column chromatography.
  • This process in addition to the removal of unwanted materials, largely fatty esters, also has the benefit of selectively removing the sterols that have nearly the same Rf. index on silica gel as that of the methyl ester of li-O-acetylprostaglandin A
  • Purification of the residue via dry column chromatography using acid washed silica gel and ethyl acetate as elutant yields the 1S-O-acetyl-prostaglandiu A methyl ester and prostaglandin A methyl ester.
  • the combined yield of the two prostaglandins is 12%.
  • the configuration of the C-15 carbon is estabilshed by degradation with ozone followed by chromic acid oxidation to yield u-acetoxyheptanoic acid.
  • the configuration of the acetoxy grouping is established by comparison of the circular dichromism curves of u-acetoxyheptanoic acid and D-a-acetoxypropanoic acid prepared from the calcium salt of D-lactic acid (R-configuration). Compounds of like configuration will display curves of the same sign.
  • D-a-acetoxypropanoic acid displays a negative circular dichromism curve whereas the degradation product, a-acetoxyheptanoic acid gives a positive circular dichromism curve.
  • a prostaglandin enriched material consisting essentially of prostaglandin A 15-O- acetyl methyl ester, IS-epi-prostaglandin A IS-O-acetyl methyl ester, prostaglandin A -methyl ester and l5-epiprostaglandin A methyl ester, from cortex of the gorgonid Plexaura homomalla (esper) containing them which comprises the steps of:
  • nitroalkane-immiscible solvent selected from the group consisting of alkanese having from six to eight carbon atoms, cycloalkanes having from five to seven carbon atoms, and mixtures thereof at a temperature of from about 0 C. to about 60 C. for a period of time sufiicient for a substantial degree of extraction of non-prostaglandin material into the nitroalkaneimmiscible solvent phase to occur;
  • nitroalkane is nitromethane and the nitroalkane-immiscible solvent is cyclohexane.
  • nitroalkane is nitromethane and the nitroalkane-immiscible solvent is a mixture of isomeric hexanes.
  • nitroalkane is nitromethane and the nitroalkane-immiscible solvent is a mixture of isomeric hepanes.
  • nitroalkane is nitromethane and the nitroalkane-immiscible solvent is a mixture of isomeric octanes.
  • nitroalkane is nitroethane and the nitroalkane-immiscible solvent is a mixture of isomeric heptanes.

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  • Organic Chemistry (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

THIS DISCLOSURE DESCRIBES A MULTISTEP PROCESS FOR RECOVERING A PROSTAGLANDIN ENRICHED ISOLATE FROM THE CORTEX OF THE SEA CORAL PLEXAURA HOMOMALLA.

Description

United States Patent 3,778,469 ISOLATION OF PROSTAGLANDINS FROM PLEXAURA HOMOMALLA Jesse Lee Baker, Emerson, N.J., assignor to American Cyanamid Company, Stamford, Conn. No Drawing. Filed Nov. 17, 1972, Ser. No. 307,721 Int. Cl. C07c 67/06 US. Cl. 260-499 6 Claims ABSTRACT OF THE DISCLOSURE This disclosure describes a multistep process for recovering a prostaglandin enriched isolate from the cortex of the sea coral Plexaura homomalla.
BRIEF SUMMARY OF THE INVENTION This invention relates to a method of recovering a prostaglandin enriched isolate consisting essentially of prostaglandin A 15-'O-acetyl methyl ester, 15-epiprostaglandin A IS-O-acetyl methyl ester, prostaglandin A methyl ester and l5-epi-prostaglandin A methyl ester from the cortex of the gorgonid Plexaura homomalla. More particularly, this invention involves the partition of the material obtained by hexane extraction of dried, comminuted cortex of Plexaura homomalla, after removal of solvent, between two immiscible solvent systems. One phase consists of a nitroalkane solvent system selected from the group consisting of nitroalkanes having one or two carbon atoms, and mixtures thereof. The other phase consists of a nitroalkane-immiscible solvent system selected from the group consisting of alkanes having from six to eight carbon atoms, cycloalkanes having from five to seven carbon atoms, and mixtures thereof. Undesired impurities are concentrated in the nitroalkane-immiscible solvent system whereas the desired prostaglandin A derivatives are found in the nitroalkane solvent system and may be recovered therefrom by removal of solvent.
DETAILED DESCRIPTION OF THE INVENTION The prostaglandins are an important group of substances playing a significant, although as yet not fully defined role in many areas of human biochemistry and physiology. The value of certain prostaglandins, in particular prostaglandin E and prostaglandin F for the induction of labor and as abortifacients has been demonstrated in humans and is under active clinical study. In limited experiments with humans, with and without a history of bronchial asthma, prostaglandin E has been shown to be a potent bronchodilator. I
Thus, an inexpensive and convenient source of supply for relatively large amounts of the various prostaglandins is of great importance. The cortex of the sea coral Plexaura homomalla contains relatively large amounts of either prostaglandin A 15(S)-O-acetyl methyl ester or the corresponding compound in the 15(R) configuration, or both. Depending upon how the collected samples are handled, there may also be present prostaglandin A methyl ester and its IS-epi isomer as well as the prostaglandin A carboxylic acids. The gorgonid Plexaura homomalla is by far the richest source of prostaglandins yet reported. Furthermore, the transformation of the prostaglandin A derivatives of either the 15(8) or 15(R) configuration to 15(S)-prostaglandins E and F has been reported as has the transformation of prostaglandin E to prostaglandin E and thence to prostaglandin P Although the cortex of Plexaura homomalla is relatively rich in prostaglandins, the prostaglandin content is nevertheless only about 12% of the total. The chemical literature describes column chromatography procedures for the final purification of the prostaglandins following ice an initial extraction of the cortex with hexane. The starting material for this initial extraction is the outer sheath or cortex of the animal which is peeled or stripped off. The cortex is then comminuted by grinding or by mincing in the usual manner. Also, the fresh cortex may be frozen and then ground to prevent any possibility of loss of prostaglandin content. The comminuted cortex is then dried by any convenient means such as by air-drying on trays in sunshine or by drying in low temperature ovens. 'If the dried comminuted cortex must be stored for any considerable period of time, it is preferable to store it under nitrogen in order to avoid oxidation of the prostaglandin content thereof.
The initial hexane extraction of the dried comminuted cortex may be carried out with any hexane such as nhexane, isohexane, 3-methylpentane, 2-ethylbutane, 2,3- dimethylbutane, etc., or with various mixtures of these isomeric hexanes. Best results are obtained when 3-5 liters of isomeric hexanes are employed per kilogram of cortex. The extraction may be carried out at from about 0 C. to about 60 C., preferably at ambient temperature and with agitation, over a period of time of 2-24 hours. The hexane extract is then separated from the insoluble solid residue by any convenient means such as filtration or centrifugation. The solvent is then removed from the hexane extract by evaporation, distillation under vacuum, or freeze drying to provide an initial prostaglandin-containing material. This initial hexane extraction enriches the prostaglandin content of initial prostaglandin-containing material to about 10% from the original 12% of the cortex.
The present invention comprises a novel useful, and convenient process for the further enrichment of the prostaglandin content of the initial hexane extract to about 40-60%, thus substantially reducing the complexity, amount of adsorbent, solvent volume, and time necessary for the final chromatographic purification. In the preparation of relatively large quantities of prostaglandins from the cortex of Plexaura homomalla, these improvements substantially reduce the economic cost. More particularly, the present invention involves the partition of the material obtained by hexane extraction between two immiscible solvent systems. The first phase consists of a nitroalkane solvent system selected from the group consisting of nitroalkane having one or two carbon atoms, and mixtures thereof. The second phase consists of a nitroalkaneimmiscible solvent system selected from the group consisting of alkanes having from six to eight carbon atoms, cycloalkanes having from five to seven carbon atoms, and mixtures thereof. Suitable second phase solvent systems are, for example, a mixture of isomeric hexanes, a mixture of isomeric heptanes, a mixture of isomeric octanes, cyclopentane, cyclohexane, cycloheptane, a 1:1 by volume mixture of isomeric hexanes and isomeric octanes, a 1:1 by volume mixture of cyclopentane and isomeric hexanes, and the like.
. The partition of the ca. 10% prostaglandin-containing material is best carried out with about equal volumes of first and second phase solvent systems, although considerable variation is permissible with this parameter. Preferably, about 3-5 liters sum total of the solvent phases is employed per kilogram of the hexane extract material. Partition is carried out, with agitation, at a temperature of from about 0 C. to about 60 C., preferably at ambient temperature, over a period of time of 2 minutes to one hour. Thereupon, the nitroalkane-immiscible second phase solvent system is separated and discarded. Solvent is then removed from the nitroalkane first phase solvent system by evaporation, vacuum distillation, or freeze drying to provide a prostaglandin enriched material containing about 40-60% of prostaglandins. The 15(S) and/or 15(R) prostaglandin A IS-O-acetyl methyl esters, as well as the corresponding prostaglandin A methyl esters, and sometimes the corresponding prostaglandin A carboxylic acids, are found in this final concentrate and may be subsequently isolated and purified by column chromatography.
The invention will be described in greater detail in conjunction with the following specific example.
EXAMPLE 1 Isolation of the methyl ester of IS-O-acetylprostaglandin A from Plexaura homomalla (esper) Specimens of Plexaura homomalla (esper) were collected in Puerto Rican waters, air dried and stored under a nitrogen atmosphere at C. The cortex was removed, ground and extracted with isomeric hexanes. The organic solvent was evaporated in vacuo to alford a prostaglandin containing residue. The residue was dissolved in nitromethane and extracted with isomeric hexanes. The nitromethane solution was evaporated to give a residue that was approximately 50% by weight of the residue prior to the nitromethane treatment. This process, in addition to the removal of unwanted materials, largely fatty esters, also has the benefit of selectively removing the sterols that have nearly the same Rf. index on silica gel as that of the methyl ester of li-O-acetylprostaglandin A Purification of the residue via dry column chromatography using acid washed silica gel and ethyl acetate as elutant yields the 1S-O-acetyl-prostaglandiu A methyl ester and prostaglandin A methyl ester. The combined yield of the two prostaglandins is 12%.
The configuration of the C-15 carbon is estabilshed by degradation with ozone followed by chromic acid oxidation to yield u-acetoxyheptanoic acid. The configuration of the acetoxy grouping is established by comparison of the circular dichromism curves of u-acetoxyheptanoic acid and D-a-acetoxypropanoic acid prepared from the calcium salt of D-lactic acid (R-configuration). Compounds of like configuration will display curves of the same sign. D-a-acetoxypropanoic acid displays a negative circular dichromism curve whereas the degradation product, a-acetoxyheptanoic acid gives a positive circular dichromism curve.
What is claimed is:
1. The process of recovering a prostaglandin enriched material, consisting essentially of prostaglandin A 15-O- acetyl methyl ester, IS-epi-prostaglandin A IS-O-acetyl methyl ester, prostaglandin A -methyl ester and l5-epiprostaglandin A methyl ester, from cortex of the gorgonid Plexaura homomalla (esper) containing them which comprises the steps of:
(a) digesting dried, comminuted cortex of Plexaura homomalla with n-hexane or isomeric hexanes at a temperature of from about 0 C. to about 60 C. for a period of time sufiicient for a substantial degree of extraction of prostaglandin material into hexane to occur;
(b) removing insoluble solid material from the digested mixture to provide a hexane solution containing prostaglandin material;
(c) removing hexane from the hexane solution to provide prostaglandin containing material;
(d) dissolving the prostaglandin containing material in a nitroalkane having one or two carbon atoms or mixtures thereof;
(e) extracting the nitroalkane solution with a nitroalkane-immiscible solvent selected from the group consisting of alkanese having from six to eight carbon atoms, cycloalkanes having from five to seven carbon atoms, and mixtures thereof at a temperature of from about 0 C. to about 60 C. for a period of time sufiicient for a substantial degree of extraction of non-prostaglandin material into the nitroalkaneimmiscible solvent phase to occur;
(f) separating the nitroalkane-immiscible solvent phase from the nitroalkane phase; and
(g) removing nitroalkane from the nitroalkane phase to provide prostaglandin enriched material.
2. A process in accordance with claim 1 wherein the nitroalkane is nitromethane and the nitroalkane-immiscible solvent is cyclohexane.
3. A process in accordance with claim 1 wherein the nitroalkane is nitromethane and the nitroalkane-immiscible solvent is a mixture of isomeric hexanes.
4. A process in accordance with claim 1 wherein the nitroalkane is nitromethane and the nitroalkane-immiscible solvent is a mixture of isomeric hepanes.
5. A process in accordance with claim 1 wherein the nitroalkane is nitromethane and the nitroalkane-immiscible solvent is a mixture of isomeric octanes.
6. A process in accordance with claim 1 wherein the nitroalkane is nitroethane and the nitroalkane-immiscible solvent is a mixture of isomeric heptanes.
References Cited UNITED STATES PATENTS 3,504,019 3/1970 Lapidus et al 260-514 D 3,598,858 8/1971 Bergstrom et a1. 260-514 D 3,706,789 12/ 1972 Bergstrom et al. 260-514 D VIVIAN GARNER, Primary Examiner US. Cl. X.R.
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