United States Patent Office
`3,703,544
`Patented Nov. 21, 1972
`
`1
`
`2
`
`3,703,544
`PROCESS FOR PREPARING THE TRIS(HYDROXY-
`METHYL)-AMINOMETHANE SALT OF PGE2
`Walter Moi-ozowich, Kalamazoo, Mich., assignor to The
`Upjohn Company, Kalamazoo, Mich.
`No Drawing. Continuation-in-part of application Ser. No.
`42,458, June 1, 1970. This application May,10, 1971,
`Ser. No. 142,019
`Int. Cl. C07c 9] /02
`U.S. Cl. 260—501.l7
`
`
`3 Claims
`
`ABSTRACT OF THE DISCLOSURE
`
`Free flowing crystals of the tris(hydroxymethy1)amino-
`methane salts of PGE2 and PGF2.., and processes for
`producing those, are disclosed. Those crystals are useful
`for the same pharmacological and medical purposes as
`PGE2 and PGF2,,, and are also useful as a means for
`purifying PGE2 and PGF_-4,.
`
`
`CROSS REFERENCE TO RELATED
`‘
`APPLICATION
`
`This application is a continuation-in-part of my co-
`pending application Ser. No. 42,548, filed June 1, 1970
`now Pat. No. 3,657,327.
`DESCRIPTION OF THE INVENTION
`
`This invention relates to novel manufactures and to
`novel methods for producing those. In particular,
`this
`invention relates to free flowing crystals of the tris(hy-
`droxymethy1)aminomethane salt of a compound of the
`formula:
`
`
`
`wherein X is =0 or
`
`/Hs‘
`‘ OH
`
`1
`
`_
`and to methods for making those crystals.
`The compound of Formula I wherein X is =O.lS an
`optically active compound known as prostaglandin E2
`(PGE2). The compound of Formula I wherein X is
`
`(H
`‘OH
`
`is an optically active compound known as prostaglandin
`F2, (PGF2,). These compounds are known to be useful
`for a variety of pharmacological and medical purposes,
`for example, labor induction and abortion in pregnant
`animals, including humans, and menstrual regulation in
`
`including hu-
`both pregnant and non-pregant animals,
`mans. For these purposes, the usual route of administra-
`tion is intravenous injection or infusion, although the
`oral route is also used in labor" induction and the vaginal
`and intrauterine routes are also used for abortion and
`
`_
`menstrual regulation.
`It is difficult to formulate these Formula I carboxylic
`acids into compositions suitable for pharmacological and
`medical uses. For example, they do not dissolve readily
`in water or in the isotonic solutions necessary for intra-
`
`venous injection or infusion. Preliminary treatment of
`the prostaglandin with a water-miscible organic solvent
`and/or an aqueous solution of a base, for example, so-
`dium hydroxide or sodium carbonate, is usually necessary
`before an isotonic aqueous solution of the proper con-
`centration can be formed. Moreover, PGF2,
`is a low-
`melting waxy solid which is difficult to solidify and purify,
`and which is slow in dissolving in aqueous base solutions.
`Although PGE2 is a crystalline solid, it also is waxy and
`slow to dissolve in aqueous base solutions.
`PGE2 and PGF2, are carboxylic acids, and salt forma-
`tion is involved when PGE2 or PGF2,
`is dissolved in
`aqueous base solutions as a preliminary step in the known
`formulation procedures. See. also British specification
`1,040,544 where pharmacological and medical use of
`PGE2 and PGF2,
`in pharmacologically acceptable salt
`form is suggested. Among the pharmacologically accept-
`able cations suggested there are those derived from the
`alkali and alkaline earth metals, ammonia, and various
`amines.
`
`There would be substantial advantage in having avail-
`able PGE2 and PGF-2,, each in the form of a stable, crys-
`talline, high-melting salt which is rapidly soluble in water
`or in the isotonic solutions necessary for intravenous ad-
`ministration. These salts would also be useful in formula-
`tions intended for other routes of administration, for
`example, oral, buccal,
`intravaginal, and intrauterine.
`There would also be substantial advantage in being able
`to recrystallize those same salts to produce preparations
`of the desired degree of purity. This would be especially
`desirable for PGF2, which, as mentioned above, is diffi-
`cult to purify as a free acid.
`I have now made the surprising and unexpected dis-
`covery that free-flowing crystals of the tris(hydroxymeth-
`yl)aminomethane salts of PGE2 and PGF2, are produced
`by mixing a dilute acetonitrile solution of the prostag-
`landin in the range 65° to 85° C. with a concentrated
`aqueous solution of an equivalent amount of tris(hydroxy-
`methyl)aminomethane, cooling the resulting mixture to
`the range 20° to 30° C., maintaining the mixture in the
`range 20° to 30° C. until crystals are formed, and collect-
`ing said crystals. These free flowing salt crystals are non-
`hygroscopic, easily dried, free of water and acetonitrile
`solvate molecules, and recrystallizable. They also dis-
`solve rapidly and completely in water and in the usual
`isotonic solutions used for intravenous injection or in-
`fusion, and are useful for the same pharmacological and
`medical purposes as PGE2 and PGF2,.
`In carrying out this novel process, it is desirable to use
`equivalent amounts of the prostaglandin and the tris(hy-
`droxymethyl)aminomethane. The latter is rather insoluble
`in acetonitrile and an excess will precipitate with and con-
`taminate the desired salt. Using less than an equivalent
`amount of the amine will result in part of the prostag-
`landin staying in the acetonitrile.
`As mentioned abve, the salt formation and crystalliza-
`tion occur in a mixture of acetonitrile and water. It is
`important that the proportions of acetonitrile and water
`be such that the maximum amount of salt crystallizes
`from the solvent mixture. Enough water must be used,
`of course, to form and transfer a homogeneous solution
`of tris(hydroxymethyl)aminomethane to the acetonitrile
`solution of the prostaglandin. But use of water substan-
`tially above that minimum amount will require use of
`excessive amounts of acetonitrile. For each volume of
`water about 100 to 200 volumes of acetonitrile should
`be used. Use of substantially less acetonitrile will result
`in a mixture of acetonitrile and water which will unnec-
`essarily retain the desired salt in solution. Use of larger
`amounts of acetonitrile would be unnecessary and waste-
`ful of the acetonitrile. A convenient and suitable amount
`
`10
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`20
`
`30
`
`35
`
`40
`
`50
`
`OX01
`
`60
`
`65
`
`70
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`SteadyMed - Exhibit 1015 - Page 1
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`SteadyMed - Exhibit 1015 - Page 1
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`3,703,544
`
`3
`of water is about 3 milliliters per gram of tris(hydroxy-
`methyl)aminomethane. Not much less can be used be-
`cause of the solubility of that amine in water (about 0.5
`gram per ml. at 25° C.). Use of more water will require
`excessive amounts of acetonitrile.
`The acetonitrile solution of the prostaglandin should
`be in the range 65° to 85° C. when it is mixed with the
`aqueous tris(hydroxymethyl)aminomethane solution. It
`it advantageous to heat the acetonitrile solution to about
`its boiling point, i.e., about 80° C. at atmospheric pres-
`sure. To minimize degradation of the prostaglandin, heat-
`ing should be rapid and mixing with the aqueous amine
`solution should be done promptly when the acetonitrile
`solution is at the desired temperature. It is advantageous
`though not necessary to warm the aqueous amine solu-
`tion to the range 50° to 80° C. before mixing.
`The aqueous amine solution is advantageously added
`slowly and with vigorous stirring to the hot acetonitrile
`solution. The resulting hot mixture is
`then cooled to
`room temperature (about 20° to 30° C.).
`When this process is first carried out, it is advantageous
`to do it in a glass vessel, the inner walls of the vessel
`being scratched vigorously with a glass rod when the mix-
`ture just starts to become cloudy. Doing that will hasten
`crystal formation. In subsequent operations, a few crys-
`tals from this first crystallization can be added as seeds
`at the same point of initial clouding to hasten crystal
`formation, and a glass vessel need not be used.
`After this initial cooling, the mixture «is maintained at
`room temperature (about 20° to 30° C.) until no further
`crystallization occurs. The crystals are then collected by
`conventional techniques, e.g., filtration or centrifugation,
`and washed with a small amount of acetonitrile. Most
`of the acetonitrile is removed either under reduced pres-
`sure or in a current of warm nitrogen or argon. The crys-
`tals are then ground to a finer particle size if desired,
`and drying is completed by heating in the range 50° to
`75° C.
`the tris(hydroxymethyl)
`When recrystallization of
`aminomethane salt of PGE2 or PGF2, is desired, that is
`advantageously done by dissolving the salt
`in a small
`amount of water (1. to 3 ml. of water per g. of salt),
`adding that solution with vigorous stirring to hot aceto-
`nitrile (65° to 85° C.; about 200 ml. per g. of salt),
`and cooling the resulting mixture to room temperature
`(20° to 30° C.), advantageously adding a few crystals
`at the cloud point to assure prompt crystallization. The
`crystals are collected, washed, and dried as above.
`I have also made the surprsing and unexpected dis-
`covery that free flowing crystals of the tris(hydroxymeth-
`yl/aminomethane salt of PGE2 are also produced by a
`process which comprises the steps, (1) mixing a concen-
`trated solution of equivalent amounts of PGE2 and tris-
`(hydroxymethyl)aminomethane
`in
`a water-miscible,
`polar, normally liquid organic compound below about 40°
`C. with sufiicient of a less polar, normally liquid organic
`compound miscible with said polar compound to cause
`at least part of said salt to precipitate in the range —20°
`to 40° C., (2) maintaining the resulting mixture in the
`range —20° to 40° C. until crystals have formed, and
`(3) collecting said crystals.
`This is a preferred process for producing this free flow-
`ing crystalline salt of PGE2.
`the term “normally liquid”
`In this preferred process,
`means a compound which is a liquid at 40° C. and under
`one atmosphere of pressure (760 mm. of mercury). Ex-
`amples of suitable water-miscible, polar, normally liquid
`organic compounds are dimethyl sulfoxide, dimethylform-
`amide,
`tetramethylurea, sulfolane (tetramethylene sul-
`fone or
`tetrahydrothiophene-1,1-dioxide),
`and meth-
`anol. Especially preferred is dimethyl sulfoxide.
`Examples of suitable less polar, normally liquid or-
`ganic compounds miscible with said polar compound are
`acetonitrile, propionitrile, butyronitrile, dichloromethane,
`methyl propyl ketone, diethyl ether, and chloroform.
`
`10
`
`20
`
`30
`
`35
`
`40
`
`U!U!
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`60
`
`,...
`H)
`
`4
`Nitriles are preferred less polar compounds in this proc-
`ess, especially acetonitrile.
`An especially preferred combination of polar and less
`polar compounds is dimethyl sulfoxide and acetonitrile,
`respectively.
`As for the previous novel process, in this second and
`preferred novel process also, it is desirable to use equiva-
`lent amounts of PGE2 and the tris(hydroxymethyl)
`aminoethane.
`In this second novel process, the salt formation occurs
`when the PGE2 and the tris(hydroxymethyl)amino-
`methane are mixed together to form a solution in the
`water-miscible, polar, normally liquid organic compound.
`Said salt is then caused to precipitate in the form of the
`desired free flowing crystals by addition of suflicient of
`the less polar compound to said solution and maintaining
`the resulting mixture in the above-mentioned tempera-
`ture range of ——20°
`to 40° C. It is desirable that the
`minimum amount of the polar compound be used to form
`and dissolve the PGE2 and tris(hydroxymethyl)amino-
`methane and the resulting salt. Since this amount of
`polar compound is quite small
`in some instances,
`it is
`advantageous in those instances to have also present a
`small amount of the intended less polar compound at the
`time the PGE2 and tris(hydroxymethyl)aminomethane
`are first mixed. Then, sufficient additional nonpolar com-
`pound is added to cause the desired precipitation.
`As for the first process, also in this second preferred
`process, it is ad-vantageous to carry out the process the
`first time in a glass vessel, the inner walls being scratched
`vigorously with a glass rod while step 2 is being carried
`out. Doing that
`tends to hasten crystal formation. In
`subsequent operations, a few crystals from the first
`crystallization can be added as seeds at the same point
`in the process to hasten crystal formation, and a glass
`vessel
`is not needed.
`The amount of less polar compound necessary to cause
`precipitation of the free flowing crystalline salt will vary
`somewhat according to the combination of the particular
`polar compound and less polar compound that is used.
`The appearance of turbidity during addition of the less
`polar compound is a useful indication that the minimum
`amount of the less polar compound has been added. Ad-
`ditional
`less polar compound beyond this amount
`is
`usually necessary,
`the exact
`total amount
`to be used
`being easily determined by one of ordinary skill in this
`art. Use of an excessive amount of less polar compound
`will result in an oily salt rather than a crystalline salt.
`Although satisfactory results in this second process
`are usually obtained by carrying out the entire process
`in the range of 20° to 30° C., there is frequently ad-
`vantage in cooling during the second step of the process
`below 20°, preferably down to 0° C. or even as low as
`——20‘’ C., and then allowing the cooled mixture to warm
`slowly to the range 20° to 30° C. Doing this at least
`once and in some cases two or more times tends to
`hasten the crystallization.
`is col-
`the salt
`When crystal formation is complete,
`lected, washed, and dried as described above for the first
`process.
`the tris(hydroxymethyl)-
`When recrystallization of
`aminomethane salt of PGE2 is desired, a preferred man-
`ner of doing that is by solution of said salt in a minimum
`of one of the polar liquids mentioned above, preferably
`dimethyl sulfoxide, and then addition of sufficient of
`one of the less polar liquids mentioned above, preferably
`acetonitrile,
`to cause precipitation of the free flowing
`crystalline salt in the range ---20'’ to 40° C. The crystals
`are collected, washed, and dried as described above.
`It is advantageous although not essential to carry out
`all of the above operations, both first and second proc-
`esses, with minimum exposure to oxygen by replacing
`air with an inert gas, for example, nitrogen or argon.
`When recovery of PGE2 or PGF2, from their respec-
`tive tris(hydroxymethyl)aminomethane salts is desired,
`
`SteadyMed - Exhibit 1015 - Page 2
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`SteadyMed - Exhibit 1015 - Page 2
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`

`
`3,703,544.
`
`5
`that is accomplished by disolving the salt in water (10
`ml. per g. of salt), adjusting the pH of that solution to
`the range 6 to 7, and extracting the solution repeatedly
`with ethyl acetate. The ethyl acetate extracts are com-
`bined, washed successively with water and saturated
`aqueous sodium chloride solution, dried, and evaporated
`to give POE; or PGF-2,,.
`The invention can be more fully understood by the
`following examples:
`
`EXAMPLE 1
`
`Free flowing crystals of the tris(hydrovcymethyl)-
`aminomethane salt of PGFza
`A solution
`of
`tris(hydroxymethyl)aminomethane
`(1.645 g.) in 3.0 ml. of water at 60° C. is added with
`vigorous stirring to a solution of PGFZ, (5.00 g.) in 700
`ml. of acetonitrile which has just been brought to its
`boiling point. The vessel which contained the aqueous
`amine solution is rinsed with three 0i.66—ml. portions of
`water, each rinsing being added with vigorous stirring to
`the acetonitrile solution. The mixture is then cooled to
`about 55° C. by immersion of the vessel in cool water,
`and then to 25° C. under ambient conditions. At
`the
`cloud point (about 50° C.), the vessel wall (glass) below
`the liquid surface is scratched vigorously with a glass rod.
`The mixture is then maintained at 25° C. for 12 hours.
`The resulting crystals are collected by filtration under
`nitrogen, washed on the filter with 50 ml. of acetonitrile,
`and then dried by passing nitrogen at 50° C.
`through
`the filter cake for one hour. Drying is completed in an
`oven at 70° C. for 2 hours to give 5.965 g. of the tris-
`(hydroxymethyl)aminomethane salt of PGF2,
`in free
`flowing crystalline form; M.P. 100-101° C.
`Following the procedure of Example 1 but using a
`few crystals of the product of Example 1 in place of
`scratching with a glass rod, the same salt in free flowing
`crystalline form and with the same melting point is ob-
`tained.
`
`‘EXAMPLE 2
`
`Free flowing crystals of the trz's(lzydroxymethyl)-
`aminomethane salt 0]‘ PGF2
`Following the procedure of Example 1 but using PGE2
`in place of PGF2,,, free flowing crystals of the tris(hy-
`droxymethyl)aminomethane salt of PGE2 are obtained.
`-EXAMPLE 3
`
`Free flowing crystals of the trz's(hydroxymethyl)-
`aminomethane salt of PGF2
`A solution
`of
`tris(hydroxymethyl)aminomethane
`(36.5 mg.) in 0.4 ml. of dimethyl sulfoxide is added to
`a solution of -PGEZ (116 mg.) in one ml. of acetonitrile
`at 25° C. Acetonitrile (3 ml.)
`is added gradually to
`this mixture with stirring. At this point, the mixture is
`slightly turbid. Then, additional acetonitrile (15 ml.)
`is added with stirring, and the mixture is cooled to 0°
`C. and then allowed to warm slowly to 25° C. The
`fiocculant, free flowing crystals which form are sepa-
`rated from the mixture by pressure filtration (nitrogen),
`washed on the filter with acetonitrile, and then dried by
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`50
`
`55
`
`6
`passing nitrogen through the filter cake at 25 ° C. for 15
`minutes. Drying is completed under reduced pressure
`at 30° C. for 24 hours to give 80 mg. of the tris(hydroxy-
`methyl)am.inomethane salt of PGE2 in free flowing
`crystalline form; R; 0.3——TLC—EtOAc+3% HOAC.
`Following the procedure of Example 3 but using
`separately, dimethylformamide, methanol,
`and tetra-
`methylurea in place of the dimethyl sulfoxide, the same
`free flowing crystalline salt is obtained.
`Also following the procedure of Example 3 but using
`separately, methyl pr-opyl ketone, diethyl ether, chloro-
`form, dichloromethane, and butyronitrile in place of the
`acetonitrile,
`the same free flowing crystalline salt
`is
`obtained.
`
`EXAMPLE 4
`
`Free flowing crystals of the tris(hydroxymethyl)
`aminomethane salt of PGE2
`is
`Tris(hydroxymethy1)aminomethane
`(48.4 mg.)
`added with stirring to a solution of PGE2 (140.8 mg.)
`in 0.8 ml. of dimethylformamide. Acetonitrile (150 ml.)
`is added gradually to the resulting solution with stirring
`at 25 ° C. The mixture is then maintained at 25° C. for
`24 hours under nitrogen. The crystals which formed are
`separated, washed, and dried as described in Example 3
`to give 50 mg. of the tris(hydroxymethyl)aminomethane
`salt of PGE2 in free flowing crystalline form; M.P. 94.2-
`95° C.
`Following the procedure of Example 4 but using sul-
`folane in place of the dimethylformamide, the same free
`flowing crystalline salt is obtained.
`I claim:
`1. A process for producing free flowing crystals of the
`tris (hydroxymethyl)aminomethane salt of PGE2 which
`comprises the steps, (1) mixing a concentrated solution
`of equivalent amounts of PGEZ and tris(hyd:roxymethyl)
`aminomethane in a first
`solvent
`selected from the
`group consisting of dimethyl sulfoxide, dimethylform-
`amide,
`tetramethylurea, sulfolane, and methanol below
`about 40° C. with sufficient second solvent selected from
`the group consisting of acetonitrile, propionitrile, butyro-
`nitrile, dichloromethane, methyl propyl ketone, diethyl
`ether and chloroform to cause at least part of said salt
`to precipitate in the range —20° to 40° C., (2) main-
`taining the resulting mixture in the range —20° to 40° C.
`until crystals have formed, and (3) collecting said crys-
`tals.
`2. A process according to claim 1 wherein said second
`solvent is acetonitrile.
`3. A process according to claim 2 wherein said first
`solvent is dimethyl sulfoxide.
`
`References Cited
`UNITED STATES PATENTS
`4/1972 Morozowich ______ 260—-50=1.17
`8/1971 Bergstrom et al. ____.. 260-468
`
`3,657,327
`3,598,858
`
`LEON ZITVER, Primary Examiner
`M. W. GLYNN, Assistant Examiner
`
`SteadyMed - Exhibit 1015 - Page 3
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`SteadyMed - Exhibit 1015 - Page 3