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1.ApplicationNumber: US-73245358-A
1.PublishNumber: US-2904560-A
2.Date Publish: 19590915
3.Inventor: DODSON RAYMOND M.
TWEIT ROBERT C.
4.Inventor Harmonized: DODSON RAYMOND M()
TWEIT ROBERT C()
5.Country: US
6.Claims:
7.Description:
(en)7 ACYLTHIO- l-PREGN ENE-3,20-DIONES Raymond M; Dodson, Park Ridge, and Robert. C. Tweit, Wilmette,..lll., assignors to G.D. Searle & Co., Chicago, 111., a; corporation of Delaware No Drawing. Application May 2, 1958' Serial No. 732,453
11 Claims. (Cl. 2 60-'-397.3)
Thepresent invention is concerned with 7ot-acylthio substitution products of 4-pregnene-3,20-diones which can be represented by the general structural formula CHzR I XI
in which R" represents hydrogen or a lower alkyl radical, to a 4,6-pregnadiene-3,ZO-dione which can be represented by the structural formula in which R, R, X, X, and Y have the meanings as aforesaid. The addition reaction proceeds fairly rapidly at elevated temperatures, such as those in excess of 50 and can be carried out optionally under the influence of ultraviolet light. H In carrying out the addition reaction with lower boiling thioalkanoic acids, such asthioacetic acid, it is convenient to conduct the reaction at the reflux temperature of the reaction mixture; whereas with higher boiling, thioalkanoic acids it is convenient'to conduct the. reaction ata temperature of about 90 -100. Heating periods of up to afew hours are suflicientjto achieve-substantial conversion to the desired. reaction productsiw Typically, the desired co'mpound is obtained 2,904,560 Patented Sept, 15 1959 Since thioalkanoic acids are effective acylating agent s,
' employmentas a starting material of a 4,6-Pregnadiene- 3,20-dione having a-free hydroxyl group at position 21 leads to the formation of a substantial amount of a 21 acyloxy derivative after a prolonged reaction time. In.
1 general, however, reaction products containing esterified hydroxyl groups are best prepared by using a starting material containing the desired esterified hydroxyl groups. in position and then conducting the addition reaction of the thioalkanoic acid as set out hereinbelow. Alternatively, however, starting materials containing free hydroxyl groups can be subjected to the addition reaction of the, thioalkanoic acid followed by esterification. procedures known in the art such as by the use of the corresponding (lower) alkanoic acid anhydrides and pyridine.
The addition of thioalkanoic acids to the 6,7-double bond of the starting materials employed in the practice of this invention proceeds under *steric influences such that of the stereoisomersformed, one is obtained in predominant amount, which, in each case, has been characterized herein as possessing the tat-configuration of the.7- acylthio group. This configuration has been. designated in order to provide a more complete exposition of the present invention, and in order that the specification shall constitute a more useful contribution to the art. However, the designated configuration of the 7-acylthio group is based upon an analysis of molecular rotation data presently appearing in the chemical literature, and'is therefore not to be interpreted except in relation to the state of theart presently known to organic chemists. It will be apparent that no part of the specification will be materially ,defective'if it should later be established that the configuration is the opposite of that deducible from data presently available to workers in the field. I
.The compounds of the present invention have useful pharmacological properties in con-sequence of their hormonal and anti-hormonal activities. They are antiinflammatory agents of the cortisone type and'progestational agents. They also inhibit the sodium retention characteristic of desoxycorticosterone. This application is a continuation-in-part of our application Serial No; 706,260, filed December 3-1, 1957, and now abandoned.
This invention will appear more fully from the examples which follow. These examples are set forth by way of illustration only and it will be understood that the invention is not to be construed as limited in spirit or in scope by the details contained therein, as many modifications in materi-als and in methods will be apparent from this disclosure to those skilled in the art. In these examples, temperatures are given in degrees centigrade C.) and quantities of materials are expressed in parts by weight and-parts by volume which b'ear the same relation one to the other as kilograms to liters.
, Example 1" .f'Aj stainless steel fermentation tank: having a capacity of about 510,000 volumes is charged with a solution of 1000 parts by weight of dextrose, 200 part's by weight of a commercial cotton seed meal flour, 9.0. partsby volume of corn steep liquor and 12 parts by volume of concentrated hydrochloric acidin 25,000 parts by Volume of in a satisfactory state of purity by direct filtration of the cooled reaction mixture, preceded, where necessary by removalof excessthioalkanoie acid. If. desired, however, the-reaction product can bepurified by chromatographic techniques; using adsorbents such as. silica gel followed by elution: with mixtures of ethyl acetate in benzene.
" stream of air which has-been sterilized by p'assage through a glass wool filter is introduced through an inlet tube into the contents of the fermentor at a rate of from 20,000 to 35,000 volumes of air per minute. This rate of aeration is measured by means of a rotameter placed in the sterile portion of the air line. Growth of the microorganism is allowed to continue for 46 hours at a temperature of about 25. During this period of incubation, additional small portions of anti-foam agent are added as necessary. A solution of 10 parts by weight of progesterone in 750 parts by volume of ethanol is added and fermentation 1n the presence of the steroid substrate is continued for an additional 20 hours at 25 with the same rates of stirring and aeration. The contents of the fermentor are st1rred with 18,000 parts by volume of methylene chloride for one hour. The mycelium is then separated by filtration and washed with about twice its volume of methylene chloride. The methylene chloride extracts are combined and concentrated under reduced pressure to about 600 parts by volume, filtered and further concentrated to dryness. The residue is triturated with ether, followed by warm acetone-ether to remove the 6,14-isomer and the residue crystallized from methanol-acetone to yield 7a,14u-dihydroxyprogesterone, M.P. 234238.
A solution of 2 parts by weight of 7a,l4a-dihydroxyprogesterone, 3 parts by weight of potassium hydroxide and 500 parts by volume of methanol is allowed to stand overnight. The reaction mixture is made neutral with acetic acid, concentrated in vacuo to 25 parts by volume, diluted with 100 parts by volume of water and the precipitate of l4oc-hydroxy4,6-pregnadiene-3,20-dione WhlCh forms recovered; melting point 177-17 8.
A solution of 0.9 part by weight of 14a-hydroxy- 4,6-pregnadiene-3,20-dione in 2 parts by volume of theacetic acid is heated for 6 hours while irradiating with an ultraviolet light. The solution is allowed to stand overnight, diluted with 10 parts by volume of ether and 40 parts by volume of methylene chloride, washed with water followed by dilute sodium hydroxide solution and concentrated to dryness in vacuo. The residue is dissolved in 50 parts by volume of benzene and chromatographed on silica gel. The chromatographic column is eluted with 6000 parts by volume of 20% ethyl acetate in benzene, the solvent removed from the eluate, the residue collected and crystallized from acetone-ether to yield 7a-acetylthio-l4a-hydroxy-4-pregnene 3,20 dione; melting point l88-l91 (dec.); ultraviolet extinction coefiicient 18,300 at 238.5 millimicrons.
Example 2 A solution of 2 parts of 17u-l1ydroxy21-acetoxy-4,6- pregnadiene-3,l1,20-trione in 2 parts of thioacetic acid is heated under reflux and irradiated with ultraviolet light for 2 hours. The solid product which results is collected on a filter and washed with ether. This product is dissolved in 'a minimum quantity of methylene chloride, and recrystallized by adding 20 parts of methanol and concentrating the resulting solution. The compound thus obtained is 7a-acety1thio-17u-hydroxy-2l-acetoxy-4-pregnene-3,11,20-trione which melts at about 234-235 has an ultraviolet absorption maximum at 235.5 millimicrons with a molecular extinction coefiicient of about 16,850, and has a specific rotation of about +1025" in chloroform solution. The structural formula is 4 Example 3 A stainless steel fermentation tank having a capacity of about 400,000 volumes is charged with a solution prepared by boiling 1200 parts by Weight of a commercial cotton seed meal flour, 500 parts by volume of corn steep liquor and 50 parts by volume of concentrated hydrochloric acid in about 20,000 parts by volume of tap Water. The resulting solution is cooled and filtered. Five thousand parts by weight of dextrose and 10 parts by weight of an anti-foam agent, suitably of a silicone type, are added and the volume is brought up to 180,000 Volumes in the fermentor. The contents of the vessel are sterilized by the addition of live steam under pressure to a temperature of about to The contents of the fermentor are cooled and inoculated with a spore suspension of Penicillium sp., ATCC 12,556. The contents of the fermentor are kept agitated by a stirrer operating at about revolutions per minute and a stream of air sterilized by passage through a glass wool filter is introduced into the medium through an inlet tube at the rate of from 40,000 to 50,000 volumes of air per minute. This rate of aeration is measured by means of a rotameter placed in the sterile portion of the air line. Growth of the microorganism is allowed to continue for 52.5 hours at a temperature of about 25 During this period, additional amounts of anti-foam agent are added as required. A solution of 50 parts by weight of progesterone in 1500 parts by volume of ethanol is added and fermentation in the presence of the steroid substrate is continued for an additional 13 hours. The mycelium is separated by filtration in a basket type centrifuge and washed with about twice its volume of methylene chloride. The aqueous filtrate is extracted twice by stirring with approximately 100,000 parts by volume portions of methylene chloride. The methylene chloride extracts are combined and reduced to about 8000 volumes by distillation of the solvent at atmospheric pressure. The concentrate is filtered and reduced to about 670 volumes by distillation at atmospheric pressure and then in vacuo to dryness. The residue is triturated with petroleum ether, ether and acetone and the residue is crystallized from acetone.
A solution of 2 parts by weight of this residue and 2 parts by weight of sodium hydroxide in 500 parts by volume of methanol is allowed to stand for 60 hours. The reaction mixture is made neutral with 3 parts by volume of acetic acid and concentrated in vacuo. The residue is diluted with water and the solid collected and crystallized from acetone to yield l5 8-hydroxy-4,6-pregnadiene-3,20-dione; melting point 214-216"; ultraviolet extinction coefficient 26,300 at 284 millimicrons.
A mixture of 0.5 part by weight of 15 8-hydroxy-4,6- pregnadiene-3,20-dione and 0.5 part by volume of thioacetic acid is heated for one-half hour while being irradiated with an ultraviolet light. Another 0.5 part by volume of thioacetic acid is added and heating continued for a total of 1.5 hours. After standing overnight the solution is poured into water, neutralized with sodium bicarbonate and extracted several times with 25 parts by volume portions of methylene chloride. The extracts are combined, dried over magnesium sulfate, decanted from the drying agent and concentrated to dryness. The residue is collected, dissolved in 50 parts by volume of ben- Zene and the solution chromatographed on 20 parts by weight of silica gel. The column is eluted with 250 parts by volume of 30% ethyl acetate in benzene, the eluate evaporated to dryness and the residue crystallized from acetone-ether to yield 7u-acetylthio-15/9-hydroxy-4-pregnene-3,20-dione; melting point 218-221 (dec.); ultraviolet extinction coefiicient 21,000 at 238 millimicrons.
Example 4 A mixture of 1.5 parts by weight of 4,6-pregnadiene- 3,20-dione and 1.5 parts by volume of thiopropionic acid is heated on the steam bath for 3 hours. After standing overnight, the reaction mixture is diluted with 25 parts by volume'of'ether and the crystals which form om emu ing are separated and recrystallized from acetone ether to yield 7a-propionylthioprogesterone; melting point 134 136; [a] +7.6';' ultraviolet extinctiort coefficient 17,700 at 238 millimicrons. i
Exa p 5. Y 7 Y A- mixture of 1.5 parts by Weight of '2l-ace't'oxy-1745chydroxy-4,6-pregnadiene-3,20-dione and 1.5 parts by volume of thioacetic acid is heated for 2 hours. The reaction mixture is concentratedunder a nitrogen atmosphere to 2 parts by volume. The crystals which form onstanding are collected, washed with ether and recrystallized from methylene chloride-acetone to'yield 21"-acetoxy'-1-7uhydroxy 70c acetylthio- 4preg'nene'-3,20 dione; melting point 211-213 (dec.); [a] =+3'; ultraviolet extinction coefiicient 20,300 at 238millimicrons.
Example 6 A mixture of 1.1 parts by weight of- 2'1-acetoxy- 11e,17a-dihydroxy-4,6-pregnadiene-3,ZO-dione and 1;.1- parts by volume of thioacetic acid is heated for one-halfhour on the steam bath. After standing at room temperature overnight, the reaction mixture is; concentrated in vacuo to 1.5 parts by volume and then diluted" with ether. The solid which forms is collected and recrystallized from acetone to yield 21-acetoxy-7a-acetylthiol118,17a-dihydroxy-4-pregnene-3,20-dione; melting point 181-182 (dec.); [a] =+5O.5; ultraviolet extinction coeflicient 17,900 at 238.5 millimicrons.
Example 7 A mixture of 1.1 parts by weight of 21-acetoxy-4,6- pregnadiene-3,20-dione and 1 partby volume of thiopropionic acid is heated on the steam bath for 3 hours. The reaction mixture is concentrated in vacuo to 1.5 parts by volume, cooled and diluted with ether. The crystalline residue which forms is collected and recrystallized from acetone-ether to yield 21-acetoxy-7'apropionylthio 4 preg n'en'e-3,20-dion'e;v melting point 92-96; [111 +276; ultraviolet extinction coefiicient 17,800 at 23.8 millimicrons.
Example 8 A mixture of 2 parts by weight of 17a-hydroxy4,6- pregnadiene-3,20-dione and 2 parts by, volume of thioacetic acid is heated o'n-the steam bath for 3-hours. The reaction mixture is cooled, stirred and diluted with 20 parts by volume of ether. The solid residue which separates is collected and washed with ether to yield 7-a-a'cetylthio-17a-hydroxy-4-pregnene-3,20-dione; melting point 227'-229 (dec.); [a]j' =38; ultravioletextinction 'coefficient 19,200 at 238.5 millimicron's.
Example 9 A solution of 10 parts by weight of 4-preg'nene-3',11, 20-trione and .10 parts by weight ofv chloranil in 1000 parts by volume of xylene is refiuxedfor- 2 hours. The reaction mixture is cooled and chromatographed'over 600 parts by weight of silica gel. The chromatographic column is washe'dsuc'cessively with 6000 parts by volume portions of benzene, 10%. ethylacetate in benzene and 20% ethyl acetate in benzene and'eluted with 10,000 parts by volume of 30% ethyl acetate in benzene. The solvent is removed from the eluate in vacuo and the residue which remains is crystallized from acetonepetroleum ether to yield 4,6-pregnadiene-3,l1,20-trione; melting point 152454"; [a] =|-269.5; ultraviolet extinction coefficient 24,300 at 281 millimicrons.
A mixture of 0.8 part by weight of 4,6-pregnadiene- 3,11,20-trione and 1 part by volume of thiopropionic acid is heated for 3 hours on the steam bath. The reaction mixture is concentrated in vacuo and the crystalline residue thus obtained is recrystallized from acetone to yield 7a-propionylthio-4-pregnene-3,11,20-trione; melting point 153-1552 [ab-- +.4 ultraviolet extinction coefii cient 18,000 at 236 millimicrons.
' Example 10 A mixture of 1 .13 parts by weight oflh-hydroxy- 4,6 -pregnadiene-.3,20 dione, 1.5 parts byvolume ofv propionic anhydride, 0.2 part by weight of p-toluenesulfonic acid monohydrate and 25 parts by volume of benzene is heated on the steam bath and allowed to st-and for 4. daysat room temperature. The reaction mixture is poured into-water, the benzene layer is separated, washed with sodium bicarbonate solution and concentrated in vacuo. The oily residue is triturated with 10 parts by volume of ether and the solid which forms is collected to yield 170: propionoxy-4,6-pregnadiene-3,20-dione; melting point 170-172.
The 17u-propionoxy-4,6-pregnadiene-3,20-dione obtained above is mixed with 0.7 part by volume of thioacetic acid, warmed on the steam bath for 2 hours, cooled, diluted with 20 parts by volume of ether and the crystalline residue thus formed is collected and washed: with ether to yield 7 a-acetylthio-17a-propionoxy- 4-pregnene-3,20.-dione; melting point 20921'1 (dec.); [a]j =46.l; ultraviolet extinction coetficient 19,500 at 238- millimicrons.
Example 11 A solution of 1 part by weight of 11B',17a-dihydroxyprogesterone, 1 part by Weight of chloranil and a trace of p' -tbluene'sulfonic acid monohydrate in 125 parts by volume of xylene isrefl'uxed' for 48 hours, cooled and chromatographed over silica gel; The chromatographic column iswashed successively with 2000 parts by volume portionsfof 5% ethyl acetate in benzene, 10% ethyl acetate in benzene, and 15 ethyl acetate in benzene and then eluted with] 10,000. parts by volume of 20% ethyl acetate in benzene. The solvent is removed, from the eluate and theresidue collected" to yield 11,8,17m dihyd roxy-4,6-p'regnadiene=3,20-dione; ultraviolet maximum at 283 millimicrons.
A mixture of 0.7 partbyweight of 11,8,17u-dihydroxy- 4,6-pregnadiene-3,20-dione and 1 part by volume of thioacetic acid is heated for 1 hour on the steam bath. The reactionmixture is evaporated in vacuo, the residue dissolved in 50 parts by 'volume of benzene *and chromatographed on silica gel. The column is washed successively with. 2000 parts by volume portions of benzene, 5%, 10% and 15% ethylacetate in benzene, and eluted with, 10,000 parts by volume of 20% ethyl acetate.v in benzene. vThe eluate is evaporated to dryness in vacuo. The. residue is crystallized from acetone-ether to yield 7a-acetylthio- 1113,1700 dihydroxy 4 pregnene- 3,20- dione; M.P. 233-4236 (dec.); ultraviolet extinction coefli'eient 19,600 at 238 millimicrons.
Example 12 A mixture of 1 part by weight of 17a,21-dihydroxy 4,6-pregnadiene-3,l1,20-trione and 1 part by 'volume of 3 -methylbutanethiolic acid-.is heated for20 minutes on the steam bath. Then-the excess thiolic acid is removed invacuo, the residue dissolved in parts by volume of benzeneand, chromatographedonsilica gel. The column is washed successively with 6000. parts. by volume portions of benzene, 5% ethyl acetate in benzene, 10% ethyl acetate in benzene, and 25% ethyl acetate in benzene and the product is eluted with 8000 parts by volume of 40% ethyl acetate in benzene. The solvents are removed from the eluate to yield 17a,21-dihydroxy-7a- (3-methylbutyrylthio) 4-pregnene 3,11,20-trione as a glass. The compound has a maximum in the ultraviolet absorption spectrum at 235 millimicrons and infrared absorption maxima at 2.9, 5.8, 5.9 and 6.2 microns.
Example 13 A mixture of 1 part by weight of 17a,21-dihydroxy- 7 0c-( 3-methylbutyrylthio) 4 pregnene 3,11,20 trione,
7 4 parts by volume of isobutyric anhydride and 8 parts by volume of pyridine is warmed on the steam bath until homogeneous. The solution is allowed to stand overnight and then is diluted with water and extracted with methylene chloride. The methylene chloride solution is washed with sodium carbonate solution and then concentrated in vacuo. The residue is dissolved in 50 parts by volume of benzene and chromatographed on silica gel. The chromatographic column is Washed successively with 1000 parts by volume portions of benzene, ethyl acetate in benzene and 10% ethyl acetate in benzene. The product is eluted with 2000 parts by volume of 20% ethyl acetate in benzene. The solvents are removed from the eluate, leaving l7u-hydroxy-2l-isobutyroxy 7a (3-methylbutyrylthio)-4-pregnene-3,ll,20- trione. The compound has a maximum in the ultraviolet absorption spectrum at 235 millimicrons and infrared absorption maxima at 2.9, 5.8, 5.9, 6.0 and 6.2 microns.
Example 14 A solution of 3 parts by weight of 15fi-hydroxy-4,6- pregnadiene-3,20-dione and 40 parts by volume of pyridine is added to a mixture of 3 parts by weight of chromium trioxide in 30 parts by volume of pyridine. The resulting dark mixture is allowed to stand 24 hours, diluted with 400 parts by volume of water, and filtered through diatomaceous earth. The residue and filtrate are both extracted three times with a total of 500 parts by volume of 1:1 ether:benzene. The combined extracts are washed with water and concentrated to dryness in vacuo to yield 4,6-pregnadiene-3,15,20-trione which melts at 197199 after crystallization from acetone.
A mixture of one part by weight of 4,6-pregnadiene-3, 15,20-trione and one part by volume of thioacetic acid is heated on the steam bath for one and one-half hours. The excess thioacetic acid is removed in vacuo, ether is added and on scratching a solid forms which is recrystallized from acetone-ether to yield 7a-acetylthio-4-pregnene-3,15,20-trione melting at 187-189".
Example 15 A mixture of one part by weight of l1a-hydroxy-4,6- pregnadiene-3,20-dione and one part by volume of thioacetic acid is heated on the steam bath for minutes. The resulting solution is diluted with 50 parts by volume of benzene and chromatographed on 50 parts by weight of silica gel. The chromatographic column is washed with 1000 parts by volume portions of benzene, 10% acetate in benzene, and 20% ethyl acetate in benzene. Then the column is eluted with 200 parts by volume of 50% ethyl acetate in benzene. The solvent is removed from the eluate in vacuo to yield 7e-acetylthio-lla-hydroxy-4-pregnene,-3,20-dione which exhibits absorption maxima in the ultraviolet absorption spectrum at 238 millimicrons and in the infrared absorption spectrum at 2.9, 5.9, 6.0, 6.2, 8.8 and 9.1 microns.
Example 16 A solution of 5 parts by weight of 15a-hydroxy-progesterone, 5 parts by Weight of chhoranil and 0.1 part by weight of p-toluenesulfonic acid monohydrate in 500 parts by volume of xylene is refluxed for 3 hours. The reaction mixture is chromatographed on 250 parts by weight of silica gel and the chromatographic column washed successively with 3000 parts by volume portions of benzene, 10% ethyl acetate in benzene and 25% ethyl acetate in benzene, Then the column is eluted with 6000 parts by volume of 40% ethyl acetate in benzene and the solvent removed from the eluate in vacuo to yield 15a-hydroxy-4,6-pregnadiene-3,20-dione which exhibits absorption maxima in the ultraviolet absorption spec trum at 2.9, 5.85, 6.0, 6.2 and 6.3 microns.
A mixture of 2 parts by weight of 15u-hydroxy-4,6- pregnadiene-3,20-dione and 2 parts by volume of thiopropionic acid is heated on the steam bath for 15 minutes. The resulting solution is diluted with 50 parts by volume of benzene and chromatographed on parts by weight of silica gel. The chromatographic column is washed successively with 2500 parts by volume portions of benzene, 10% ethyl acetate in benzene and 20% ethyl acetate in benzene. The column is eluted with 5000 parts by volume of 40% ethyl acetate in benzene and the solvent removed from the eluate in vacuo to yield 7a-propionylthio-15a-hydroxy-4-pregnene-3,20-dione which exhibits absorption maxima in the ultraviolet absorption spectrum at 238 millimicrons and in the infrared absorption spectrum at 2.9, 5.9, 6.0, 6.2 and 10.7 microns.
What is claimed is:
1. A compound of the formula in which R and R are selected from the group consisting of hydrogen, hydroxyl and (lower)alkanoyloxy, X and X are selected from the group consisting of methylene, hydroxymethylene and carbonyl, and Y is hydrogen when X is oxygenated and selected from the group consisting of hydrogen and hydroxyl when X is methylene.
2. 21 acetoxy 7 acetylthio 17cc hydroxy 4- pregnene-3 ,1 1,20-trione.
3. 7 acetylthio 15 hydroxy 4 pregnene 3,20- dione.
4. 7-propionylthioprogesterone.
5. 21 acetoxy 7 acetylthio 17cc hydroxy 4- pregnene-3,20-dione.
6. 21 acetoxy 7 acetylthio 11,17 dihydroxy- 4-pregnene-3,20-dione.
7. 21 acetoxy 7 propionylthio 4 pregnene- 3,20-dione.
8. 7 acetylthio 17a hydroxy 4 pregnene 3,20- dione.
9. 7 -propionylthio-4-pregnene-3,1l-20-trione.
10. 7 acetylthio 17a propionoxy 4 pregnene- 3,20-dione.
11. 7oc-acety1thio-4-pregnene6,15,20-trione.
No references cited.
UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No 2,904,560 September 15, 1959 Raymond M, Dodson et a1,
It is hereby certified that error appears in the printed specification of the above numbered patent requiring correction and that the said Letters Patent should readas corrected below.
Column '7, lines 48 and 49, for "10% acetate" reed 10% ethyl acetate line 50, for "200 parts" read 2000 parts column 8, line 9, after "at" insert 283 millimierons and in the infrared absorption spectrum at Attest:
KARL Ho .AXLINE Attesting Oflicer ROBERT C. WATSON Commissioner of Patents
1.PublishNumber: US-2904560-A
2.Date Publish: 19590915
3.Inventor: DODSON RAYMOND M.
TWEIT ROBERT C.
4.Inventor Harmonized: DODSON RAYMOND M()
TWEIT ROBERT C()
5.Country: US
6.Claims:
7.Description:
(en)7 ACYLTHIO- l-PREGN ENE-3,20-DIONES Raymond M; Dodson, Park Ridge, and Robert. C. Tweit, Wilmette,..lll., assignors to G.D. Searle & Co., Chicago, 111., a; corporation of Delaware No Drawing. Application May 2, 1958' Serial No. 732,453
11 Claims. (Cl. 2 60-'-397.3)
Thepresent invention is concerned with 7ot-acylthio substitution products of 4-pregnene-3,20-diones which can be represented by the general structural formula CHzR I XI
in which R" represents hydrogen or a lower alkyl radical, to a 4,6-pregnadiene-3,ZO-dione which can be represented by the structural formula in which R, R, X, X, and Y have the meanings as aforesaid. The addition reaction proceeds fairly rapidly at elevated temperatures, such as those in excess of 50 and can be carried out optionally under the influence of ultraviolet light. H In carrying out the addition reaction with lower boiling thioalkanoic acids, such asthioacetic acid, it is convenient to conduct the reaction at the reflux temperature of the reaction mixture; whereas with higher boiling, thioalkanoic acids it is convenient'to conduct the. reaction ata temperature of about 90 -100. Heating periods of up to afew hours are suflicientjto achieve-substantial conversion to the desired. reaction productsiw Typically, the desired co'mpound is obtained 2,904,560 Patented Sept, 15 1959 Since thioalkanoic acids are effective acylating agent s,
' employmentas a starting material of a 4,6-Pregnadiene- 3,20-dione having a-free hydroxyl group at position 21 leads to the formation of a substantial amount of a 21 acyloxy derivative after a prolonged reaction time. In.
1 general, however, reaction products containing esterified hydroxyl groups are best prepared by using a starting material containing the desired esterified hydroxyl groups. in position and then conducting the addition reaction of the thioalkanoic acid as set out hereinbelow. Alternatively, however, starting materials containing free hydroxyl groups can be subjected to the addition reaction of the, thioalkanoic acid followed by esterification. procedures known in the art such as by the use of the corresponding (lower) alkanoic acid anhydrides and pyridine.
The addition of thioalkanoic acids to the 6,7-double bond of the starting materials employed in the practice of this invention proceeds under *steric influences such that of the stereoisomersformed, one is obtained in predominant amount, which, in each case, has been characterized herein as possessing the tat-configuration of the.7- acylthio group. This configuration has been. designated in order to provide a more complete exposition of the present invention, and in order that the specification shall constitute a more useful contribution to the art. However, the designated configuration of the 7-acylthio group is based upon an analysis of molecular rotation data presently appearing in the chemical literature, and'is therefore not to be interpreted except in relation to the state of theart presently known to organic chemists. It will be apparent that no part of the specification will be materially ,defective'if it should later be established that the configuration is the opposite of that deducible from data presently available to workers in the field. I
.The compounds of the present invention have useful pharmacological properties in con-sequence of their hormonal and anti-hormonal activities. They are antiinflammatory agents of the cortisone type and'progestational agents. They also inhibit the sodium retention characteristic of desoxycorticosterone. This application is a continuation-in-part of our application Serial No; 706,260, filed December 3-1, 1957, and now abandoned.
This invention will appear more fully from the examples which follow. These examples are set forth by way of illustration only and it will be understood that the invention is not to be construed as limited in spirit or in scope by the details contained therein, as many modifications in materi-als and in methods will be apparent from this disclosure to those skilled in the art. In these examples, temperatures are given in degrees centigrade C.) and quantities of materials are expressed in parts by weight and-parts by volume which b'ear the same relation one to the other as kilograms to liters.
, Example 1" .f'Aj stainless steel fermentation tank: having a capacity of about 510,000 volumes is charged with a solution of 1000 parts by weight of dextrose, 200 part's by weight of a commercial cotton seed meal flour, 9.0. partsby volume of corn steep liquor and 12 parts by volume of concentrated hydrochloric acidin 25,000 parts by Volume of in a satisfactory state of purity by direct filtration of the cooled reaction mixture, preceded, where necessary by removalof excessthioalkanoie acid. If. desired, however, the-reaction product can bepurified by chromatographic techniques; using adsorbents such as. silica gel followed by elution: with mixtures of ethyl acetate in benzene.
" stream of air which has-been sterilized by p'assage through a glass wool filter is introduced through an inlet tube into the contents of the fermentor at a rate of from 20,000 to 35,000 volumes of air per minute. This rate of aeration is measured by means of a rotameter placed in the sterile portion of the air line. Growth of the microorganism is allowed to continue for 46 hours at a temperature of about 25. During this period of incubation, additional small portions of anti-foam agent are added as necessary. A solution of 10 parts by weight of progesterone in 750 parts by volume of ethanol is added and fermentation 1n the presence of the steroid substrate is continued for an additional 20 hours at 25 with the same rates of stirring and aeration. The contents of the fermentor are st1rred with 18,000 parts by volume of methylene chloride for one hour. The mycelium is then separated by filtration and washed with about twice its volume of methylene chloride. The methylene chloride extracts are combined and concentrated under reduced pressure to about 600 parts by volume, filtered and further concentrated to dryness. The residue is triturated with ether, followed by warm acetone-ether to remove the 6,14-isomer and the residue crystallized from methanol-acetone to yield 7a,14u-dihydroxyprogesterone, M.P. 234238.
A solution of 2 parts by weight of 7a,l4a-dihydroxyprogesterone, 3 parts by weight of potassium hydroxide and 500 parts by volume of methanol is allowed to stand overnight. The reaction mixture is made neutral with acetic acid, concentrated in vacuo to 25 parts by volume, diluted with 100 parts by volume of water and the precipitate of l4oc-hydroxy4,6-pregnadiene-3,20-dione WhlCh forms recovered; melting point 177-17 8.
A solution of 0.9 part by weight of 14a-hydroxy- 4,6-pregnadiene-3,20-dione in 2 parts by volume of theacetic acid is heated for 6 hours while irradiating with an ultraviolet light. The solution is allowed to stand overnight, diluted with 10 parts by volume of ether and 40 parts by volume of methylene chloride, washed with water followed by dilute sodium hydroxide solution and concentrated to dryness in vacuo. The residue is dissolved in 50 parts by volume of benzene and chromatographed on silica gel. The chromatographic column is eluted with 6000 parts by volume of 20% ethyl acetate in benzene, the solvent removed from the eluate, the residue collected and crystallized from acetone-ether to yield 7a-acetylthio-l4a-hydroxy-4-pregnene 3,20 dione; melting point l88-l91 (dec.); ultraviolet extinction coefiicient 18,300 at 238.5 millimicrons.
Example 2 A solution of 2 parts of 17u-l1ydroxy21-acetoxy-4,6- pregnadiene-3,l1,20-trione in 2 parts of thioacetic acid is heated under reflux and irradiated with ultraviolet light for 2 hours. The solid product which results is collected on a filter and washed with ether. This product is dissolved in 'a minimum quantity of methylene chloride, and recrystallized by adding 20 parts of methanol and concentrating the resulting solution. The compound thus obtained is 7a-acety1thio-17u-hydroxy-2l-acetoxy-4-pregnene-3,11,20-trione which melts at about 234-235 has an ultraviolet absorption maximum at 235.5 millimicrons with a molecular extinction coefiicient of about 16,850, and has a specific rotation of about +1025" in chloroform solution. The structural formula is 4 Example 3 A stainless steel fermentation tank having a capacity of about 400,000 volumes is charged with a solution prepared by boiling 1200 parts by Weight of a commercial cotton seed meal flour, 500 parts by volume of corn steep liquor and 50 parts by volume of concentrated hydrochloric acid in about 20,000 parts by volume of tap Water. The resulting solution is cooled and filtered. Five thousand parts by weight of dextrose and 10 parts by weight of an anti-foam agent, suitably of a silicone type, are added and the volume is brought up to 180,000 Volumes in the fermentor. The contents of the vessel are sterilized by the addition of live steam under pressure to a temperature of about to The contents of the fermentor are cooled and inoculated with a spore suspension of Penicillium sp., ATCC 12,556. The contents of the fermentor are kept agitated by a stirrer operating at about revolutions per minute and a stream of air sterilized by passage through a glass wool filter is introduced into the medium through an inlet tube at the rate of from 40,000 to 50,000 volumes of air per minute. This rate of aeration is measured by means of a rotameter placed in the sterile portion of the air line. Growth of the microorganism is allowed to continue for 52.5 hours at a temperature of about 25 During this period, additional amounts of anti-foam agent are added as required. A solution of 50 parts by weight of progesterone in 1500 parts by volume of ethanol is added and fermentation in the presence of the steroid substrate is continued for an additional 13 hours. The mycelium is separated by filtration in a basket type centrifuge and washed with about twice its volume of methylene chloride. The aqueous filtrate is extracted twice by stirring with approximately 100,000 parts by volume portions of methylene chloride. The methylene chloride extracts are combined and reduced to about 8000 volumes by distillation of the solvent at atmospheric pressure. The concentrate is filtered and reduced to about 670 volumes by distillation at atmospheric pressure and then in vacuo to dryness. The residue is triturated with petroleum ether, ether and acetone and the residue is crystallized from acetone.
A solution of 2 parts by weight of this residue and 2 parts by weight of sodium hydroxide in 500 parts by volume of methanol is allowed to stand for 60 hours. The reaction mixture is made neutral with 3 parts by volume of acetic acid and concentrated in vacuo. The residue is diluted with water and the solid collected and crystallized from acetone to yield l5 8-hydroxy-4,6-pregnadiene-3,20-dione; melting point 214-216"; ultraviolet extinction coefficient 26,300 at 284 millimicrons.
A mixture of 0.5 part by weight of 15 8-hydroxy-4,6- pregnadiene-3,20-dione and 0.5 part by volume of thioacetic acid is heated for one-half hour while being irradiated with an ultraviolet light. Another 0.5 part by volume of thioacetic acid is added and heating continued for a total of 1.5 hours. After standing overnight the solution is poured into water, neutralized with sodium bicarbonate and extracted several times with 25 parts by volume portions of methylene chloride. The extracts are combined, dried over magnesium sulfate, decanted from the drying agent and concentrated to dryness. The residue is collected, dissolved in 50 parts by volume of ben- Zene and the solution chromatographed on 20 parts by weight of silica gel. The column is eluted with 250 parts by volume of 30% ethyl acetate in benzene, the eluate evaporated to dryness and the residue crystallized from acetone-ether to yield 7u-acetylthio-15/9-hydroxy-4-pregnene-3,20-dione; melting point 218-221 (dec.); ultraviolet extinction coefiicient 21,000 at 238 millimicrons.
Example 4 A mixture of 1.5 parts by weight of 4,6-pregnadiene- 3,20-dione and 1.5 parts by volume of thiopropionic acid is heated on the steam bath for 3 hours. After standing overnight, the reaction mixture is diluted with 25 parts by volume'of'ether and the crystals which form om emu ing are separated and recrystallized from acetone ether to yield 7a-propionylthioprogesterone; melting point 134 136; [a] +7.6';' ultraviolet extinctiort coefficient 17,700 at 238 millimicrons. i
Exa p 5. Y 7 Y A- mixture of 1.5 parts by Weight of '2l-ace't'oxy-1745chydroxy-4,6-pregnadiene-3,20-dione and 1.5 parts by volume of thioacetic acid is heated for 2 hours. The reaction mixture is concentratedunder a nitrogen atmosphere to 2 parts by volume. The crystals which form onstanding are collected, washed with ether and recrystallized from methylene chloride-acetone to'yield 21"-acetoxy'-1-7uhydroxy 70c acetylthio- 4preg'nene'-3,20 dione; melting point 211-213 (dec.); [a] =+3'; ultraviolet extinction coefiicient 20,300 at 238millimicrons.
Example 6 A mixture of 1.1 parts by weight of- 2'1-acetoxy- 11e,17a-dihydroxy-4,6-pregnadiene-3,ZO-dione and 1;.1- parts by volume of thioacetic acid is heated for one-halfhour on the steam bath. After standing at room temperature overnight, the reaction mixture is; concentrated in vacuo to 1.5 parts by volume and then diluted" with ether. The solid which forms is collected and recrystallized from acetone to yield 21-acetoxy-7a-acetylthiol118,17a-dihydroxy-4-pregnene-3,20-dione; melting point 181-182 (dec.); [a] =+5O.5; ultraviolet extinction coeflicient 17,900 at 238.5 millimicrons.
Example 7 A mixture of 1.1 parts by weight of 21-acetoxy-4,6- pregnadiene-3,20-dione and 1 partby volume of thiopropionic acid is heated on the steam bath for 3 hours. The reaction mixture is concentrated in vacuo to 1.5 parts by volume, cooled and diluted with ether. The crystalline residue which forms is collected and recrystallized from acetone-ether to yield 21-acetoxy-7'apropionylthio 4 preg n'en'e-3,20-dion'e;v melting point 92-96; [111 +276; ultraviolet extinction coefiicient 17,800 at 23.8 millimicrons.
Example 8 A mixture of 2 parts by weight of 17a-hydroxy4,6- pregnadiene-3,20-dione and 2 parts by, volume of thioacetic acid is heated o'n-the steam bath for 3-hours. The reaction mixture is cooled, stirred and diluted with 20 parts by volume of ether. The solid residue which separates is collected and washed with ether to yield 7-a-a'cetylthio-17a-hydroxy-4-pregnene-3,20-dione; melting point 227'-229 (dec.); [a]j' =38; ultravioletextinction 'coefficient 19,200 at 238.5 millimicron's.
Example 9 A solution of 10 parts by weight of 4-preg'nene-3',11, 20-trione and .10 parts by weight ofv chloranil in 1000 parts by volume of xylene is refiuxedfor- 2 hours. The reaction mixture is cooled and chromatographed'over 600 parts by weight of silica gel. The chromatographic column is washe'dsuc'cessively with 6000 parts by volume portions of benzene, 10%. ethylacetate in benzene and 20% ethyl acetate in benzene and'eluted with 10,000 parts by volume of 30% ethyl acetate in benzene. The solvent is removed from the eluate in vacuo and the residue which remains is crystallized from acetonepetroleum ether to yield 4,6-pregnadiene-3,l1,20-trione; melting point 152454"; [a] =|-269.5; ultraviolet extinction coefficient 24,300 at 281 millimicrons.
A mixture of 0.8 part by weight of 4,6-pregnadiene- 3,11,20-trione and 1 part by volume of thiopropionic acid is heated for 3 hours on the steam bath. The reaction mixture is concentrated in vacuo and the crystalline residue thus obtained is recrystallized from acetone to yield 7a-propionylthio-4-pregnene-3,11,20-trione; melting point 153-1552 [ab-- +.4 ultraviolet extinction coefii cient 18,000 at 236 millimicrons.
' Example 10 A mixture of 1 .13 parts by weight oflh-hydroxy- 4,6 -pregnadiene-.3,20 dione, 1.5 parts byvolume ofv propionic anhydride, 0.2 part by weight of p-toluenesulfonic acid monohydrate and 25 parts by volume of benzene is heated on the steam bath and allowed to st-and for 4. daysat room temperature. The reaction mixture is poured into-water, the benzene layer is separated, washed with sodium bicarbonate solution and concentrated in vacuo. The oily residue is triturated with 10 parts by volume of ether and the solid which forms is collected to yield 170: propionoxy-4,6-pregnadiene-3,20-dione; melting point 170-172.
The 17u-propionoxy-4,6-pregnadiene-3,20-dione obtained above is mixed with 0.7 part by volume of thioacetic acid, warmed on the steam bath for 2 hours, cooled, diluted with 20 parts by volume of ether and the crystalline residue thus formed is collected and washed: with ether to yield 7 a-acetylthio-17a-propionoxy- 4-pregnene-3,20.-dione; melting point 20921'1 (dec.); [a]j =46.l; ultraviolet extinction coetficient 19,500 at 238- millimicrons.
Example 11 A solution of 1 part by weight of 11B',17a-dihydroxyprogesterone, 1 part by Weight of chloranil and a trace of p' -tbluene'sulfonic acid monohydrate in 125 parts by volume of xylene isrefl'uxed' for 48 hours, cooled and chromatographed over silica gel; The chromatographic column iswashed successively with 2000 parts by volume portionsfof 5% ethyl acetate in benzene, 10% ethyl acetate in benzene, and 15 ethyl acetate in benzene and then eluted with] 10,000. parts by volume of 20% ethyl acetate in benzene. The solvent is removed, from the eluate and theresidue collected" to yield 11,8,17m dihyd roxy-4,6-p'regnadiene=3,20-dione; ultraviolet maximum at 283 millimicrons.
A mixture of 0.7 partbyweight of 11,8,17u-dihydroxy- 4,6-pregnadiene-3,20-dione and 1 part by volume of thioacetic acid is heated for 1 hour on the steam bath. The reactionmixture is evaporated in vacuo, the residue dissolved in 50 parts by 'volume of benzene *and chromatographed on silica gel. The column is washed successively with. 2000 parts by volume portions of benzene, 5%, 10% and 15% ethylacetate in benzene, and eluted with, 10,000 parts by volume of 20% ethyl acetate.v in benzene. vThe eluate is evaporated to dryness in vacuo. The. residue is crystallized from acetone-ether to yield 7a-acetylthio- 1113,1700 dihydroxy 4 pregnene- 3,20- dione; M.P. 233-4236 (dec.); ultraviolet extinction coefli'eient 19,600 at 238 millimicrons.
Example 12 A mixture of 1 part by weight of 17a,21-dihydroxy 4,6-pregnadiene-3,l1,20-trione and 1 part by 'volume of 3 -methylbutanethiolic acid-.is heated for20 minutes on the steam bath. Then-the excess thiolic acid is removed invacuo, the residue dissolved in parts by volume of benzeneand, chromatographedonsilica gel. The column is washed successively with 6000. parts. by volume portions of benzene, 5% ethyl acetate in benzene, 10% ethyl acetate in benzene, and 25% ethyl acetate in benzene and the product is eluted with 8000 parts by volume of 40% ethyl acetate in benzene. The solvents are removed from the eluate to yield 17a,21-dihydroxy-7a- (3-methylbutyrylthio) 4-pregnene 3,11,20-trione as a glass. The compound has a maximum in the ultraviolet absorption spectrum at 235 millimicrons and infrared absorption maxima at 2.9, 5.8, 5.9 and 6.2 microns.
Example 13 A mixture of 1 part by weight of 17a,21-dihydroxy- 7 0c-( 3-methylbutyrylthio) 4 pregnene 3,11,20 trione,
7 4 parts by volume of isobutyric anhydride and 8 parts by volume of pyridine is warmed on the steam bath until homogeneous. The solution is allowed to stand overnight and then is diluted with water and extracted with methylene chloride. The methylene chloride solution is washed with sodium carbonate solution and then concentrated in vacuo. The residue is dissolved in 50 parts by volume of benzene and chromatographed on silica gel. The chromatographic column is Washed successively with 1000 parts by volume portions of benzene, ethyl acetate in benzene and 10% ethyl acetate in benzene. The product is eluted with 2000 parts by volume of 20% ethyl acetate in benzene. The solvents are removed from the eluate, leaving l7u-hydroxy-2l-isobutyroxy 7a (3-methylbutyrylthio)-4-pregnene-3,ll,20- trione. The compound has a maximum in the ultraviolet absorption spectrum at 235 millimicrons and infrared absorption maxima at 2.9, 5.8, 5.9, 6.0 and 6.2 microns.
Example 14 A solution of 3 parts by weight of 15fi-hydroxy-4,6- pregnadiene-3,20-dione and 40 parts by volume of pyridine is added to a mixture of 3 parts by weight of chromium trioxide in 30 parts by volume of pyridine. The resulting dark mixture is allowed to stand 24 hours, diluted with 400 parts by volume of water, and filtered through diatomaceous earth. The residue and filtrate are both extracted three times with a total of 500 parts by volume of 1:1 ether:benzene. The combined extracts are washed with water and concentrated to dryness in vacuo to yield 4,6-pregnadiene-3,15,20-trione which melts at 197199 after crystallization from acetone.
A mixture of one part by weight of 4,6-pregnadiene-3, 15,20-trione and one part by volume of thioacetic acid is heated on the steam bath for one and one-half hours. The excess thioacetic acid is removed in vacuo, ether is added and on scratching a solid forms which is recrystallized from acetone-ether to yield 7a-acetylthio-4-pregnene-3,15,20-trione melting at 187-189".
Example 15 A mixture of one part by weight of l1a-hydroxy-4,6- pregnadiene-3,20-dione and one part by volume of thioacetic acid is heated on the steam bath for minutes. The resulting solution is diluted with 50 parts by volume of benzene and chromatographed on 50 parts by weight of silica gel. The chromatographic column is washed with 1000 parts by volume portions of benzene, 10% acetate in benzene, and 20% ethyl acetate in benzene. Then the column is eluted with 200 parts by volume of 50% ethyl acetate in benzene. The solvent is removed from the eluate in vacuo to yield 7e-acetylthio-lla-hydroxy-4-pregnene,-3,20-dione which exhibits absorption maxima in the ultraviolet absorption spectrum at 238 millimicrons and in the infrared absorption spectrum at 2.9, 5.9, 6.0, 6.2, 8.8 and 9.1 microns.
Example 16 A solution of 5 parts by weight of 15a-hydroxy-progesterone, 5 parts by Weight of chhoranil and 0.1 part by weight of p-toluenesulfonic acid monohydrate in 500 parts by volume of xylene is refluxed for 3 hours. The reaction mixture is chromatographed on 250 parts by weight of silica gel and the chromatographic column washed successively with 3000 parts by volume portions of benzene, 10% ethyl acetate in benzene and 25% ethyl acetate in benzene, Then the column is eluted with 6000 parts by volume of 40% ethyl acetate in benzene and the solvent removed from the eluate in vacuo to yield 15a-hydroxy-4,6-pregnadiene-3,20-dione which exhibits absorption maxima in the ultraviolet absorption spec trum at 2.9, 5.85, 6.0, 6.2 and 6.3 microns.
A mixture of 2 parts by weight of 15u-hydroxy-4,6- pregnadiene-3,20-dione and 2 parts by volume of thiopropionic acid is heated on the steam bath for 15 minutes. The resulting solution is diluted with 50 parts by volume of benzene and chromatographed on parts by weight of silica gel. The chromatographic column is washed successively with 2500 parts by volume portions of benzene, 10% ethyl acetate in benzene and 20% ethyl acetate in benzene. The column is eluted with 5000 parts by volume of 40% ethyl acetate in benzene and the solvent removed from the eluate in vacuo to yield 7a-propionylthio-15a-hydroxy-4-pregnene-3,20-dione which exhibits absorption maxima in the ultraviolet absorption spectrum at 238 millimicrons and in the infrared absorption spectrum at 2.9, 5.9, 6.0, 6.2 and 10.7 microns.
What is claimed is:
1. A compound of the formula in which R and R are selected from the group consisting of hydrogen, hydroxyl and (lower)alkanoyloxy, X and X are selected from the group consisting of methylene, hydroxymethylene and carbonyl, and Y is hydrogen when X is oxygenated and selected from the group consisting of hydrogen and hydroxyl when X is methylene.
2. 21 acetoxy 7 acetylthio 17cc hydroxy 4- pregnene-3 ,1 1,20-trione.
3. 7 acetylthio 15 hydroxy 4 pregnene 3,20- dione.
4. 7-propionylthioprogesterone.
5. 21 acetoxy 7 acetylthio 17cc hydroxy 4- pregnene-3,20-dione.
6. 21 acetoxy 7 acetylthio 11,17 dihydroxy- 4-pregnene-3,20-dione.
7. 21 acetoxy 7 propionylthio 4 pregnene- 3,20-dione.
8. 7 acetylthio 17a hydroxy 4 pregnene 3,20- dione.
9. 7 -propionylthio-4-pregnene-3,1l-20-trione.
10. 7 acetylthio 17a propionoxy 4 pregnene- 3,20-dione.
11. 7oc-acety1thio-4-pregnene6,15,20-trione.
No references cited.
UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No 2,904,560 September 15, 1959 Raymond M, Dodson et a1,
It is hereby certified that error appears in the printed specification of the above numbered patent requiring correction and that the said Letters Patent should readas corrected below.
Column '7, lines 48 and 49, for "10% acetate" reed 10% ethyl acetate line 50, for "200 parts" read 2000 parts column 8, line 9, after "at" insert 283 millimierons and in the infrared absorption spectrum at Attest:
KARL Ho .AXLINE Attesting Oflicer ROBERT C. WATSON Commissioner of Patents
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