Jan. 17, 1967 3,299,046 H. E. ALBURN ET AL METHOD OF PREPARING ANHYDROUS AMPICILLIN Filed June 23, 1964 3 Sheets—Sheet 1 mp 00m 3 .2 com 00a 2 N m o 2 02 0 0m o _0o0mv 0.0 0 INVENTORS: .-1 HARVEY E. ALBURN NORMAN H. GRANT ATTORNEY Jan. 17, 1967 3,299,046 HE. ALBURN ET AL METHOD OF PREPARING ANHYDROUS AMPICILLIN Filed June 23, 1964 3 Sheets-Sheet 2 3 2 00m 08 000 .NQE 0* m a n $E2ozm0>6<3 02 0 0m 0 0w o..o 2 8 INVENTORS: HARVEY E. ALBURN NORMAN H. GRANT MW K6690’ ATTORNEY ‘ Jan. 17, 1967 3,299,046 H. E. ALBURN ET Al METHOD OF PREPARING vANHYDROUS AMPICILLIN 3 Sheets-Sheet 3 Filed June 23, 1964 0.0 no: 880E0com9" cm8. 3 ow3.9. 8.00. 33 2 S Q : 2 o RR m R 0 m w m‘ 8.3% 8A0 W on.on J$5w20 MB;5 op0o0mw 0.0 NYW.H MmexumRN W E 12 W o H N TA. .H N T N We“, AT TORN EY United States Patent C) i lC€ 1 3,299,046 Patented Jan. 17, 1967 2. now U.S. Patent No. 3,144,445, we made the surprising discovery that ampicillin, i.e., D-6-(2-amino-2-phenyl 3,299,046 METHOD OF PREPARING ANHYDROUS AMPICHLLIN Harvey E. Album, West Chester, and Norman H. Grant, Wynnewood, ll’a., assignors to American Home Prod ucts Corporation, New York, N.Y., a corporation of Delaware acetamido) penicillanic acid, may be prepared in a pre viously unknown anhydrous form which unexpectedly shows highly advantageous storage stability charac teristics. The new anhydrous form of ampicillin is characterized by the fact that it has very little, if any, water. It is much less soluble in either water or dimethyl Filed June 23, 1964, Ser. No. 377,345 3 Claims. (Cl. 260-2391) This invention relates generally to the production of sulfoxide than is ampicillin monohydrate. The differing 10 structure of the anhydrous ampicillin form is demon strated by its infrared spectrograph, an example of which is shown in FIG. 1 of the drawings, when compared with derivatives of penicillanic acids and more particularly to a novel hydrated form of D-6-(2-amino-2-phenyl acetamido) penicillanic acid and to a novel method for the spectrograph of ampicillin monohydrate, an example of which is shown in FIG. 2. Moreover, X-ray diffrac converting said hydrated compound to the anhydrous 15 tion analyses of single crystals of anhydrous ampicillin compound. have indicated a molecular weight of 347.9 (the theoret The compound D - 6 - (Z-amino-2-phenyl-acetamido) ical molecular weight of the monomeric anhydrous com penicillanic acid per se, also now known by the generic pound being 349), as compared with the molecular weight term “ampicillin,” is of proven value in its broad spec of samples of one form of hydrated ampicillin of 36.7 trum antibacterial activity and is useful as a therapeutical 20 (precisely the theoretical weight of monohydrate) de agent in poultry and in mammals, and particularly in man, termined by osmometrical analyses, or 367i7, de in the treatment of infectious diseases caused by Gram termined by X-ray diffraction, the range being due to diffi culty in growing high quality monohydrate crystals. positive and Gram-negative bacteria, upon parenteral or The anhydrous ampicillin compound is further dis 25 tinguished in that it is of greater stability on storage than plement in animal feed. One hydrated form of D-6-r(2-amino-2—phenyl-acet— is hydrated ampicillin. Because of this stability, coupled amido) penicillanic acid, i.e., the monohydrate, is already with its denseness, the efficiency of production of the oral administration. It also has use as a nutritional sup known to the art from the USP 2,985,648, in which a com anhydrous compound in capsule dosage form is increased. paratively complex method for the preparation thereof is As a further advantage, linked to its lesser solubility in 30 water, anhydrous ampicillin exhibits slower absorption in disclosed. As disclosed in said USP 2,985,648, ampicillin mono the gut and hence provides prolonged blood levels and hydrate may be prepared by a method generally compris more effective action against intestinal pathogens. The ing the reaction of 6-amino penicillanic acid with the foregoing differences and advantages of anhydrous a-aminobenzyl acid chloride or anhydride in which the ampicillin over hydrate ampicilin are of enhanced sig amino group has previously been provided with a protect 35 ni?cance, since in utilizing them, therapeutic effectiveness ing acyl group, such as PhCH2OCO—, or some other is not at all sacri?ced. functionally equivalent protecting group. To recover the anhydrous ampicillin is substantially equal to that of On the contrary, effectiveness of desired ampicillin monohydrate, it is then necessary to re hydrated ampicillin on a weight-for-weight basis. move the protecting group by catalytic hydrogenation example, when mice were challenged intraperitoneally the penicillin nucleus. strain of S. typhosa and both types of ampicillin were ad ministered by the oral route to the separately infected For under sufficiently mild conditions to avoid destruction of 40 with a penicillin sensitive strain of S. aureus or a virulent Thereafter it is also necessary to separate the product from the catalyst by ?ltration and washing. Finally, the aqueous phase is concentrated at mice, equal protection was afforded by each drug. low temperatures and pressures. Further characterization of anhydrous ampicillin, in With respect to the mild conditions stated to be neces 45 cluding its storage stability under varying conditions of temperature, humidity and time; its antibiotic utility in human therapy; and its lack of affinity for water, and other properties; is set forth in the aforesaid copending sary for the method, the patent Warns that since some of the antibiotic substances obtained by the process disclosed therein are relatively unstable compounds which readily undergo chemical changes resulting in the loss of anti application U.S. Serial No. 247,394 now U.S. Patent No. biotic activity, it is desirable to choose the reaction con 50 3,144,445. ditions which are su?iciently moderate to avoid their As disclosed in said application, the method for pre paring the novel compound anhydrous ampicillin when the precursor is ampicillin monohydrate, comprises heat decomposition. In this connection, it is further stated in the patent that the temperature chosen for the process of preparation of the derivatives of penicillanic acid ing the hydrate in the presence of free water at a tem perature of from 40° to about 100° C., until the anhydrous able temperature is ambient temperature. Further in ampicillin crystals are formed. The heating may best this connection, each of the examples disclosed in the be carried out with the charge of hydrated ampicillin patent states the conditions under which recovery of the crystals plus water at a pH of from about 3.0 to 7.0. desired product is obtained is by evaporation in vacuum 60 Preferably the free water is present in amount that is at a temperature below 20° C. with the exception that at least 50% by weight of the charge, and the heating D - -6 - (2~amino-2-phenyl-acetamido) penicillanic acid is applied to the charge under vacuum until the dry an monohydrate may be obtained by evaporation in vacuum hydrous arnpicillin product is obtained. In an alterna at a temperature of 32° C. tive procedure, the required heat and water may be sup should not exceed 30° C., and that in many cases a suit 55 Hydrated ampicillin prepared by the foregoing pro 65 plied by directly steaming crystals of ampicillin monohy drate. It has been found most advantageous from the by the Karl Fischer method. This further indicates that standpoint of feasibility and of economic processing to cedures contains from 2.5% to 10% water as determined hydrated ampicillin as prepared heretofore has been in carry on the drying operation at a pH of from 5.0 to 5.5 and at a temperature within the range of 50°—55° C. the form of the hemihydrate or the monohydrate as re ferred to in USP 2,995,648, and possibly also as dihydrate. As disclosed and claimed in our copending U.S. patent The foregoing method has been found to be admirably suitable for preparing the highly desirable anhydrous am~ application Serial No. 247,394 ?led December 26, 1962, picillin product when ampicillin monohydrate is the pre 3,299,046 3 cursor. 4 Good results are also obtainable when the pre When an organic solvent of suitable water miscibility cursor comprises the hemihydrate and/or the dihydrate is selected, and sufficient water is totally available in the bound and free state, as referred to above; it has been found that the conversion of the trihydrate to the anhy drous form of ampicillin may be carried out within the broad pH range of from about 2.0 to about 8.0 (provided there is sufficient water present to render a pH determina tion possible). Optimum conversion yields appear to occur of ampicillin. We have since discovered that ampicillin may also be produced in crystalline form as the trihydrate. This form of ampicillin is characterized by containing about 13.4% bound water although ‘in some instances this form of the trihydrate has been found to have a vbound water content as low as about 12% and as high as about 15%. Be when the pH is maintained within the higher portion of cause it is a different entity, the trihydrate has been found 10 the range, although control of the pH is not absolutely to have a distinctive infrared spectrograph, which, as necessary to operability of the method, other than, at a shown in FIG. 3 of the drawings, is different from the higher pH than 8.0, destruction of the penicillin nucleus infrared spectrographs of anhydrous ampicillin and am tends to occur. Conversion itself is caused to occur by picillin monohydrate which are respectively disclosed in maintaining the organic solvent, water and crystalline ampicillin trihydrate systems described, within the tem FIGS. 1 and 2 of the drawings. The trihydrate, more over, has a molecular weight of about 403, as determined perature range of from about 50° C. to about 100° C. by X-ray diffraction analysis, in comparison to the molecu Generally, the new precursor (i.e., the trihydrate of lar weights of about 349 and about 367 for the anhydrous ampicillin) of the desired anhydrous form of ampicillin, may be prepared by admixing 6-amino penicillanic acid and D-phenylglycine N-carboxy anhydride in a weight and monohydrate forms of ampicillin, respectively. An other distinctive characteristic of this new form of am picillin is that it has been found to be less soluble in water than is the monohydrate. Accordingly, there is an inherent practical advantage in its production from a re action mixture in that its isolation from an aqueous medi 20 ratio of about 10 to 4.5 with about 100 to 150 parts by weight of water, adjusting the‘ pH of the aqueous system to within a preferred pH range of from about 4.8 to about 6.0 pH by addition of an alkaline material, e.g., um is facilitated. NaOH, and thereafter concentrating the reaction mixture However, it has been found, unexpectedly, that the method referred to previously for converting ampicillin monohydrate to anhydrous ampicillin is not effective, un der comparative conditions, for converting the newly dis covered ampicillin trihydrate to anhydrous ampicillin. For example, when a 20% suspension of ampicillin tri under vacuum to remove water. hydrate in water is heated for 30 minutes at 55° C., and the product is dried, it is still the trihydrate. Even The resulting precipi tate may then be removed and the ?ltrate passed through an anion exchange resin to separate excess 6-amino peni cillanic acid. Finally, the ampicillin-containing effluent 30 may be concentrated under vacuum until crystals of the trihydrate form. The trihydrate crystals may then be ?ltered off, and in accordance with well known recovery procedures, the concentration-?ltration cycle may be con tinued until ampicillin no longer precipitates from the ?ltrate. at 90° C., the trihydrate, when suspended in water at pH’s of from 5.5 to 7.0, is not converted to the anhydrous form. In 20 and 30% suspensions of the trihydrate, dried Speci?c modes for preparing ampicillin trihydrate and at 100° for 1 hour, the new compound has surprisingly transforming this compound to the anhydrate are given been converted to atypical forms of the monohydrate in Examples I and II below: with 4-5% water, and not to the anhydrous form. Example I In consideration of the foregoing, we have discovered 40 that the aqueous environment essential to the method of A mixture of 800 grams of é-aminopenicillanic acid obtaining anhydrous ampicillin from the previously known (6-APA) and 100 liters of water is adjusted to pH 5.0 hydrated forms of ampicillin, should, when the trihydrate with 10 N NaOH. During vigorous stirring, 325 grams is utilized as precursor, contain an organic solvent, which of D-phenylglycine-N-carboxyanhydride is added. After is preferably entirely miscible with water, or partially mis 4.5 1 hour the reaction mixture is concentrated under vacuum cible with water at least to the extent that the organic to 20 liters. The resulting precipitate is removed, and the solvent will retain 5% by volume of water in solution. ?ltrate is passed through an anion exchange resin to sep For example, acetone, ethanol, isopropanol, n-propanol, nabutanol, ethylene glycol, ethylene glycol monomethyl arate excess 6-APA. The ampicillin-containing effluent is concentrated under vacuum until a heavy crop of crys ether, and dioxane, which meet the aforesaid requirements 50 tals forms. This is ?ltered off, and the concentration with respect ‘to miscibility with water, have been found to ?ltration cycle continued until ampicillin no longer pre be eminently suitable for use as the requisite organic cipitates. The ?lter cakes are washed with 3 volumes of solvent, and, in certain instances, even when included in 85% isopropanol and, ?nally, slurried with one volume of proportions up to about 95% of the aqueous environment. 85% isopropanol without draining, and dried at 80°. The However, with respect to the last, it has been found product is ampicillin anhydrate, weighing 305 grams. that the total water present in the processing mixture con Example II taining the trihydrate crystals and furnished as both free A mixture of 1600 grams of 6-aminopenicillanic acid and chemically bound water, must constitute at least (6-APA) and 200 liters of water is adjusted to pH 5.0 about 10% by weight based on solids and total water present to obtain the desired conversion to the anhydrate. 60 with 10 N NaOH. During vigorous stirring, 650 grams of D-phenylglycine-N-carboxyanhydride is added. After 1 On the other hand, when the organic solvent is present hour the reaction mixture is concentrated under vacuum in the environment in concentrations of less than 20% to 40 liters. The precipitate is removed, and the ?ltrate by volume of said environment, the salutary effect of is passed through an anion exchange resin to separate the solvent for permitting transformation of the trihydrate to the desired anhydrous form is not in evidence. The surprising nature of the novel method of the in excess 6-APA. The e?luent is concentrated under vac uum until a heavy crop of ampicillin trihydrate crystals vention for converting ampicillin trihydrates to anhydrous forms. This is ?ltered off, and the process is repeated until ampicillin no longer precipitates. The ?lter cakes ampicillin is demonstrated by the fact that such other common organic solvents as n-amyl alcohol, n-hexyl alco are Washed with half volumes of ice-cold distilled water, hol, methyl isobutyl ketone, methyl amyl alcohol, and 70 and the product is dried at 20—25° under vacuum. The butyl acetate which do not meet the stated criteria with yield is 640 grams of 93% pure ampicillin, calculated as respect to miscibility with water, have not been found the anhydrate. The trihydrate is converted to the anhy suitable for the same purpose, although the ultimate reason drate by re?uxing a 15% suspension in 85 % isopropanol for such selectivity in the matter of operability is ob for 20 minutes, ?ltering, and drying at 50° in a Stokes scure. 75 oven. 3,299,046 6 5 The following are examples of conversions of ampicillin trihydrate to the anhydrous ‘form of ampicillin utilizing Hydroxymate assay (percent) ________ __ Moisture (K—F, percent) ____________ __ 1.5 various solvents, concentartions, and temperatures in ac cordance with the invention. Optical rotation ((1)1325 (degrees) _____ __ Form indicated by IR spectrum _______ __ +269 Anhydrate Yield of activity (percent) ___________ __ 87 Example III Suspend separate batches of 10 grams of ampicillin Appearance perature also designated in said table, to obtain anhydrous ampicillin in each case. TABLE A Organic Solvent Do Ethylene glycol monomethyl Temperature, Solvent, Percent degrees 75 50 50 85 85 50 20 75 50 85 50 85 69 71 60 71 85 25 ether ____________________ __ 75 85 Isopropanol _______________ __ 75 70 75 85 90 94 60 75 75 85 80 85 80 71 85 85 D _ Odor _____________________________ __ Off-white None We claim: 1. The method of preparing the substantially anhydrous crystalline form of D-6-(2-amino~2-phenyl-acetamido) penicillanic acid, which method comprises: (A) preparing a mixture comprising (1) the crystalline trihydrated form of D-6-(2-a-mino-2-phenyl-acet amido) penicillanic acid and (2) a reaction medium comprising, (a) a water-miscible organic solvent ca Concentration 01 75 60 60 75 75 75 _. ______________________ __ Fine powder Color _____________________________ __ trihydrate in 100 ml. of various mixtures of water and organic solvent, as set forth in Table A below, and, under atmospheric conditions, heat each mixture at the tem 10 93.2 pable of dissolving at least 5% by volume thereof of water, and present in amount that is at least 20% by volume of said medium, and (b) su?icient free water to bring the total amount of bound and free water in the mixture to :at least 10% by weight based on solids and total water present; (B) heating said mixture to a temperature of from about 50° C. to about 100° C.; and (C) drying the mixture to obtain crystals of the sub stantially anhydrous form of D-6-(2-amino-2-phenyl acetamido) penicillanic acid. 2. The method of preparing the substantially anhydrous 30 crystalline form of D-6-(2-amino-Z-phenyl-acetamido) penicillanic acid as de?ned in claim 1 wherein said water miscible organic solvent comprises from about 50% to about 95% by volume of said medium and said heating occurs in the temperature range of from about 60° C. to about 85° C. Example IV 3. The method of preparing the substantially anhydrous crystalline form of D-6-(Z-amino-Z-phenyl-acetarnido) Pump 85 liters of 99% isopropanol into a 50 gallon penicillanic acid as de?ned in claim 1 wherein said water stainless steel kettle. Add 15 litters of tap water. Start miscible solvent is isopropanol which comprises about the stirrer of the kettle, open the manhole, and warm the contents of the kettle to 80°. At 80°, with the 40 85% by volume of said medium and said heating occurs in the temperature range of from about 80° C. to about stirrer in operation, add 14.58 kg. of ampicillin trihydrate 85 ° C. at a rate of about 1.5 kg. per minute. Close the manhole References Cited by the Examiner and raise the temperature until re?uxing is observed (at a temperature reading of about 80° to 85° C.). Re?ux the system for 20 minutes at which time stop the heating, 45 continue the stirring and open the bottom valve of the kettle to release the contents to a 20‘ inch stainless steel table top Buchner with a canvas pad. Filter the suspen UNITED STATES PATENTS 2,985,648 5/1961 Doyle et al _______ __ 260—239.1 3,144,445 3,157,640 3,180,862 8/1964 11/1964 4/1965 Grant et al. _____ __ 260—239.1 Johnson et al _____ __ 260-2391 Siluestri et a1 _____ __ 260—239.1 SlOIl. Wash the ?lter cake with 20 liters of 85% isoproppanol, dry at 48° in a Stokes oven for 2 days, and then mill. The product, weighing 10.7 kg, has the following char acteristics: ALEX MAZEL, Primary Examiner. HENRY R. JILES, Examiner. J. W. ADAMS, Assistant Examiner.
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