IL38383A - Manufacture of 6-methylenetetracycline - Google Patents

Description

Manufacture of 6-meth lenetetracycline Rachelle Laboratories Italia, S.p.A Blackvood et al, in U.S.Patent No. 2,984,686, assigned to Chas.Pfizer & Co., Inc. and issued May 16, 1961, disclose the production of 6-tnethylene-5-oxytetracycline and its 7-halo derivatives; the same procedure is shown for other tetracyclines by Blackvood et al, vith one further collaborator, in the Journal of the American Chemical Society, Vol. 85(1965) at pages 3943 to 3953. All of the products have antibiotic properties of varying degree, generally being at least as strong as the starting product; 6-methylene-5-oxytetracycline is particularly useful, possessing better properties than the Initial reactant, oxytetracycline.

Blackvood et al's basic process involves (1) lla-halogenation of a tetracycline, eithe in base or salt form, to produce an lla-halo-6,12-hetaiketal; (2Rehydration of. y the hemiketal to the lla-halo-6-methylcne derivative; and ^ (3) reduction of this halo-methylene derivative, in the salt form, to the end product. The process as described is characterized by low over-all yields, resulting in high costs.

OBJECT OF THE INVENTION The principal object of this invention is the production of 6-methylene-5-oxytetracycline and other 6-methylenetetracyclines, of high purity and biological potency, in substantially higher yields and at lower costs than with prior art methods.

SUMMARY OF THE INVENTION It has now been found that 6-methylen.e-5-oxytetracy-cline and other 6-methylenetetracyclines can be prepared in substantially higher yields and purities than heretofore, by halogenating the 5-oxytetracycline or other tetracycline re-actant under conditions sufficient to spontaneously precipitate the lla-halo-6 , 12-hemiketal base in the enolic form and converting the hemiketal base to the hemiketal acid salt, prior to dehydrating the latter material to the corresponding 6-methylene compound. This is accomplished by conducting the halogenation of a solution of the tetracycline base or salt at a sub-ambient temperature, while maintaining the pH between about 3.0 and 5, to spontaneously initiate precipitation of the enolic form of the hemiketal, then reacting the hemiketal to produce the hemiketal acid salt at temperatures from -10° to 20° C, crystallizing the hemiketal acid salt in the enolic form, and dehydrating the salt to form lla-halo- 6-methylene - 5- oxytetracycline or the corresponding derivatives of other 6-methylenetetracyclines. The lla-halo intermediate may then be dehalogenated in known fashion to the deshalogenated 6- - - - In accordance with a further feature of this invention, the free base form of the final 6-methylenetetracycline product may be prepared in substantially higher yields and in greater purity than by hitherto known methods, by neutralizing an aqueous solution of the salt at about 50° to 90° C. to the neutral point (pH about 6.5 to 7.5 at ambient temperatures about 20° C.) with suitable alkali. The elevated temperature, combined with the control of pH, insures recovery of the free base in superior yield and purity.

While the present invention is principally described herein in terms of the conversion of oxytetracycline to metha-cycline it will be understood that it is also applicable to the production of other 6-methylenetetracyclines from the corresponding tetracyclines. Accordingly, the following description of the preferred parameters utilized in the syntheses of methacycline should be regarded as illustrative rather than limiting. Further, all parts and percentages referred to in the following description are given by weight unless otherwise indicated.

As indicated hereinabove, the principal elements of the present invention comprise the spontaneous precipitation of the lla-halo hemiketals, and the subsequent conversion of such materials to the corresponding hemiketal acid salts prior to dehydration to form the lla-halo-6-methylenetetra-cyclines therefrom. It is most desirable to produce the lla-halo-6 , 12-hemiketals in the enolic rather than in the ketonic form (viz., that incorporating a free ketone group at C-12). Such is the case since the latter form does not crystallize, is therefore difficult to recover and moreover, is subject to appreciable degradation. technique contain substantial impurities in the undesirable ketonic form, as evidenced by the determination of infrared absorption bands below 6 microns in materials so produced. The spontaneous precipitation of the hemiketals in the enolic form without co-production of substantial amounts of the soluble ketonic form, and the stabilization of the enol during the subsequent dehydration reaction are, therefore, important elements of the present invention.

In particular, it has been found that the lla-chloro-5 -oxytetracycline-6 , 12 -hemiketal base, prepared as described in Example XV of the aforesaid Blackwood et al patent by halogenation in 1 , 2-dimethoxyethane and precipitation in water, exhibits an infrared absorption band below 6 microns thus indicating the presence of the ketonic form of the desired hemiketal. When, on the other hand, the lla-halogena-tion is conducted at the particular temperature and acidity conditions specified hereinabove, the enolic form of the hemiketal spontaneously crystallizes, even in the presence of an aqueous solvent medium. Infrared analysis of the product thus produced shows no carbonyl absorption and thus confirms formation of the enol to the substantial exclusion of the undesirable ketonic material.

It has been further found that conversion of the enolic form of the hemiketal base thus produced to the corresponding hemiketal acid salt, e.g., the hydrochloride, prior to dehydration to the lla-halo-6-methylenetetracycline , increases both the yield and purity of the product subsequently recovered. Thus, the enolic form of the hemiketal salt possesses markedly higher stability than the corresponding carbonyl compound to attack by the hydrogen fluoride or in higher purities than the hemiketal base as a result of the differing solubility characteristics thereof. The formation of the hemiketal salt in the synthesis reaction is, therefore, a further important aspect of the present invention.

As indicated hereinafter, methacycline hydrochloride has been prepared by the procedures described in Examples I, II and XV of the aforesaid Blackwood et al patent, obtaining such product in yields (based upon the weights of oxytetracy-cline base reacted and methacycline hydrochloride recovered) of only from 25-291. On the other hand, when methacycline hydrochloride was prepared by the spontaneous -precipitation of the hemiketal base in the enolic form and the subsequent conversion of such base to the hemiketal hydrochloride prior to dehydration and lla-dehalogenation , yields of from about 45-49% were achieved. Such difference in yields is particularly important in those instances in which it may be desired to further convert the methacycline product to doxycycline ( (X-6-deoxy-5-oxytetracycline) .

PREFERRED EMBODIMENTS OF THE INVENTION The preferred mode of carrying out the method of the invention is described below; such description may be considered in the light of the attached drawing, incorporating a flow sheet of the synthesis (illustrated in general terms on the left-hand side of the drawing in connection with one preferred embodiment of the invention on the right-hand side thereof).

As indicated hereinabove, the raw material utilized in the present synthesis is a tetracycline, e.g., oxytetra-cycline. Such material may be used as the free base, or in r' should first be solubilized by refluxing in an apolar solvent ^ such as tetrahydrofuran , acetone or dioxane; the small amount of insolubles are filtered off, and the base recovered from solution. The tetracycline base may then be dissolved in an organic solvent, e.g., a saturated monohydric alcohol having from about 1 to 5 carbon atoms, suitably isopropanol; a ketone having from 3 to 10 carbon atoms, suitably acetone; dioxane; tetrahydrofuran; or a lower alkyl ether of ethylene or di-ethylene glycol, suitably isopropyl glycol.

When the tetracycline reactant is employed in the salt form, e.g., as oxytetracycline hydrochloride in the case of the synthesis of methacycline , such material may suitably be dissolved in an aqueous solvent system. Such systems may include mixtures of any of the above organic solvents with water, particularly good results having been obtained with mixtures of varying proportions of isopropyl glycol and water. In the case of the production of methacycline it has been found that higher yields of the hemiketal may be obtained by the halogenation of oxytetracycline hydrochloride rather than oxytetracycline base; accordingly, it is particularly preferred to utilize the acid salt reactant in such synthesis.

The tetracycline reactant, whether in the base or salt form and whether in an anhydrous or aqueous reaction medium, is halogenated in accordance with the present invention under conditions designed to produce the hemiketal base in the enolic form to the exclusion of the ketonic form thereof. For this purpose the reaction mixture is maintained at sub-ambient temperatures, preferably below about 0° C, and desirably from about -15° to -4° C, while the acidity of the reaction mixture is regulated to between about pll 3.0 and critical to produce the enolic form of the hemiketal base.

Since the enol possesses a higher free energy than the corresponding ketonic form and is, therefore, inherently less stable, the low reaction temperature must be maintained.

Similarly, control of the acidity of the reaction mixture is required to prevent formation of the carbonyl moiety.

The lla-halogenation of the tetracycline base or acid salt may be effected by any halogenating agents conventionally employed in the art including, for example, those chlorinating agents described in the aforesaid Blackwood et al patent. Halogenating agents which may be so utilized include chlorine; N-chloro lower alkanoic acid amides, .e.g., N-chloroacetamide ; hydrocarbon dicarboxylic acid imides, e.g., N-chlorosuccinimide , phthalimide and the like; N-lower-alkanoylanilides , e.g., N-chloroacetanilide , propionanalide 1,3 and the like; 3-chloro and -3-j-S -dichloro , 5 , 5 -dimethylhydantoin and the corresponding bromo-substituted hydantoins; pyridinium perchloride hydrohalides , e.g., pyridinium perchloride hydrochloride and lower alkyl hypochlorites, e.g., t-butylhypo-chlorite. Particularly satisfactory results have been obtained by the use of the hydrocarbon dicarboxylic acid imides, such as N-chlorosuccinimide, as the halogenating agent.

The reaction is suitably conducted with a slight excess of the halogenating agent, generally from about 10% to 80% over theoretical. In the case of the halogenation of a tetracycline base with N-chlorosuccinimide in an anhydrous reaction medium from about 1.1 to about 1.4 moles, preferably about 1.25 moles, of the halogenating agent are reacted per mole of base. On the other hand, in the case of the halogenation of tetracycline acid salts in aqueous media it is neces V' of N-chlorosuccinimide with oxytetracycl ne hydrochloride from about 1.5 to 1.8 moles, preferably about 1.7 moles, of the N-chlorosuccinimide are employed per mole of oxytetracycline hydrochloride, and from about 2.4 to about 2.6 moles, preferably about 2.5 moles, of an organic base, e.g., triethylamine or N , 1 -dimethylformamide , are utilized to correct the acidity of the solution to about pH 4-4.5.

For optimum results it is also desirable, when halogenating a tetracycline base, to utilize reaction mixtures incorporating at least 2.9 moles of the base per liter of solution reacted. It has been found that higher product yields are obtained when such reactants are emp.loyed as compared, for example, with the relatively lower concentration reaction mixtures referred to in the aforesaid Blackwood et al patent.

After spontaneous crystallization of the enolic form of the hemiketal base is initiated, the reaction mixture is filtered, water or ethyl ether, for. example, being added to assist with the filtration if desired, and the product recovered for further reaction.

As previously indicated, the hemiketal base is there after converted to the corresponding hemiketal acid salt, e.g. the hydrochloride, the sulfosalicylate , or the p-toluene^sul -fonate, to stabilize the enol and to facilitate recovery of the product in high purities. The hemiketal salt is crystallized from a solution of the base in a solvent, e.g., a low boiling alcohol such as methanol, ethanol or isopropanol, by reaction with the appropriate acid, e.g., hydrochloric acid. The use of gaseous hydrogen chloride in 5% to 15% concentrations in methanol has been found suitable for this purpose. - relatively short period of time, e.g., within from about 2-3 hours. The use of higher temperatures or substantially higher acid concentrations provokes the opening of the oxygen bridge and the formation of the undesired ketonic material.

By converting the hemiketal base to the hemiketal hyddrochloride or other acid salt prior to dehydrating the same to the lla-halo- 6-methylenetetracycline intermediate, the final product may be recovered in substantially higher yields and purities than heretofore possible. The improved results thus obtained are demonstrated in the experiments described below. It is believed that improved product yields and purities are achieved by formation and reaction of the hemiketal acid salt since the ketal. bridge of such material is more stable to thermal attack in the subsequent dehydration reaction than is the corresponding bridge of the hemiketal base. Moreover, dehydration of the hemiketal acid salt is less exothermic than dehydration of the hemiketal base. It will, however, be appreciated that the reaction mechanism postulated should not be construed as limiting, but only as a possible explanation, in part, of the improved results obtained in accordance herewith.

After recovering the hemiketal hydrochloride, as by filtration, it is dehydrated to form the lla-halo- 6-methylenetetracycline , e.g., lla-chloro-6-methylene-5-oxytetracycline . The hemiketal hydrochloride may thus be treated with a strong, dehydrating acid such as trifluoroacetic or hydrofluoric acid, the latter being preferred. Employing hydrofluoric acid, the dehydration is carried out at temperatures of from about -10° to +19° C, preferably from about -5° to- +5° C, for periods of from about 3-4 hours. The dehydrating acid is reacted in The lla-chloro- 6 -methylene product may be recovered directly as the hydrofluor ide salt or alternatively as, for example, a perchlorate, hydrochloride, p-toluene sulfonate, or sulfosalicylate salt. Preferably, the dehydrated material is recovered as the perchlorate, e.g., by reacting perchloric acid with the dehydration reaction mixture and crystallizing the perchlorate salt therefrom. The perchloric acid is reacted in excess of that stoichiometrically required for salt formation, desirably in about twice such amount. Suitably the resulting aqueous solution of perchloric acid is cooled to about 5° C. prior to combining with the dehydration reaction mixture. Upon combining such materials and agitating the resulting mixture the perchlorate salt precipitates and may be separated and recovered by filtration or otherwise.

It is also feasible, as noted above, to recover the hydrofluoride salt formed in the dehydration reaction as such, if so desired. In this instance the lla-chloro-6-methylene material may be precipitated from a suitable cold non-solvent, e.g., ethyl or isopropyl ether, maintained at temperatures below 0° C. The product may thereafter be separated from the solvent by filtration or the like and washed with ether or other non-solvent to remove any remaining acid.

As previously indicated, it is particularly preferred to recover the lla-chloro-6-methylene product as the perchlorate, since such material readily precipitates in pure crystalline form as compared, for example, with the fluoride salt recovery by addition of an ether or other non-solvent. Moreover, formation of the perchlorate obviates the necessity for the addition of an ether, thus decreasing the volumes of volatile liquids to be handled and, consequently, Whichever lla-chloro-6-methylenetetracycline salt is, however, recovered, such material may subsequently be reduced to remove the lla-halogen and produce methacycline in the manner known in the art. The lla-dehalogenat ion may be effected with any standard reducing agent including an alkali metal hydrosulfite , e.g., sodium hydrosulfite in aqueous media; an active metal, e.g., zinc or iron in a mineral acid such as dilute hydrochloric acid; or sodium iodide in a halogen-acceptor solvent, e.g., acetone or methanol, and preferably in the presence of zinc metal. The reduction is suit ably carried out in solution in an aqueous medium incorporating a water-miscible solvent, e.g., the isopropyl ether of ethylene glycol, with sufficient acid present in the medium to maintain the same at pH values within the range of from about 1.5-6.5, preferably from about 4.5-5. The reducing agent, desirably sodium hydrosulfite, is reacted with the lla-halo-6-methylenetetracycline in amounts of from about 1.1 to 1.5 moles, desirably about 1.2 moles, per mole of the latter.

The reaction mixture containing the dehalogenated product, e.g., methacycline, is, desirably, then brought to a pH of from about 7.5 to 7.7 to completely solubilize the 6-methylenetetracycline and facilitate separation of sulfur or other impurities formed therein. The bioactive material may thereafter be precipitated from the reaction mixture as the acid salt by the addition of excess acid, e.g., hydrochloric acid, and the product recovered, e.g., by filtration or the like.

In accordance with a further feature of the invention the methacycline hydrochloride or other 6-methylenetetra c cline acid salt ma be converted to the corres ondin free heretofore possible by neutralizing an aqueous solution of the salt at temperatures within the previously indicated range of from about 50°-90° C, preferably at temperatures of from about 60°-70° C. , and at pH values of from about 6.5-7.5, desirably from about 6.7-7.2. The salts may be neutralized with alkali metal hydroxides, carbonates or bicarbonates or with suitable organic bases, the use of any desired alkaline material being permissible so long as the neutralization is carried out in aqueous medium at the temperature and acidity conditions specified hereinabove.

As noted in Examples V and VI below, it has been found that tetracycline bases produced in this .manner may be. recovered in yields of from about 84-87% and conversions of from about 91-941, and with chemical assays of from about 96-97 , whereas bases recovered by the neutralization technique described, for example, in the aforesaid Blackwood et al J.A.C.S. article may only be obtained in yields of from about 40-45% and conversions of from about 43.4-48.6%, and with chemical assays of about 89%. The present technique thus additionally provides a markedly improved procedure for converting the 6-methylenetetracycline acid salts to the corresponding free bases, whether such salts have been prepared from the corresponding tetracyclines by the synthesis described hereinabove or by a previously known technique.

The following examples illustrate preferred forms of the present method for the synthesis of methacycline from oxytetracycline , and evidence the marked differences between the process of this invention and previously described syntheses of 6-methylenetetracyclines . It should be understood that the preferred embodiments of the invention described As used in the following Examples or otherwise specified herein, the percentage yield refers to the weight of product formed as a percentage of the weight of the initial reactant, whereas the percentage conversion refers to the amount of product formed as a percentage of the amount of product theoretically producible by stoichiometric reaction. As further specified, the percentage purity refers to the "titer", viz., the weight percent of the particular tetracycline in the material analyzed, calculated as the base (U.S. Code of Federal Regulations, Title 21, Part 148y) .

' EXAMPLE I METHACYCLINE HYDROCHLORIDE FROM OXYTETRACYCLINE HYDROCHLORIDE (a) Preparation of Hemiketal Base 250 grams of oxytetracycline hydrochloride were dis solved in a mixture of 2500 ml of water and 2500 ml of the isopropyl ether of ethylene glycol at a temperature of -8° C. After dissolution, 90 ml of triethylamine were added, and immediately after 110 grams of N-chlorosuccinimide were added to adjust the pH to 4-4.5. Strong agitation was maintained for 10 minutes; spontaneous precipitation of product started in two minutes. After ten minutes, when precipitation seemed complete, 12.5 liters of water were added to facilitate filtration, and the product was recovered. Yield 198 g. (79.3%) There were no infrared absorption bands below 6 microns; U.V. in methanol, 0.01 N HC1 = 266 and 345, identifying the product as the enolic form of lla-chloro- 5-oxytetracycl ine- 6 , 12 -hemiketal . (b) Preparation of Hemiketal Hydrochloride 60 grams of the hemiketal base thus prepared were (c) Preparation of Methacycline Hydrochloride The hemiketal hydrochloride thus formed was added to 130 ml of anhydrous HF at -5° C, and stirred for 4 hours. The reaction mixture was then treated with 1300 ml of isopropyl ether, agitated, and filtered, 43 grams of lla-chloro-6- ethylene- 5-oxytetracycline hydrofluoride being recovered. 42 grams of the dehydrated product thus formed was dissolved in a mixture of 168 ml of the isopropyl ether of ethylene glycol, 84 ml of water and 4.2 ml of 37 % hydrochloric acid. To this was added a solution of 21 grams of sodium hydrosulfite in 84 ml of water. After 3 hours of agitation, triethylamine was added in an amount sufficient to adjust the pH of the mixture to 7.5 and the solution was filtered. The filter cake was then washed with 42 ml of a mixture of equal proportions of water and the isopropyl ether of ethylene glycol. To the filtrate, 176 ml of concentrated hydrochloric acid were added, and the mixture stirred for 3 hours while maintaining it at a temperature of 5° C. The methacycline salt crystallized, was filtered, and washed with isopropanol and acetone. Yield 29 grams (48.5% based on the hemiketal). Biological assay 865 mcg/mg; chemical assay 91.3% (expressed as methacycline base). Infrared analysis confirmed a structure consistent with that of methacycline hydrochloride.

EXAMPLE II METHACYCLINE HYDROCHLORIDE FROM OXYTETRACYCLINE HYDROCHLORIDE (a) Preparation of Hemiketal Hydrochloride 198 grams of the hemiketal base prepared as described in Example 1 were dissolved in 590 ml of anhydrous methanol containing 10-121 HC1. The mixture was stirred and ° The latter was recovered by filtration in an amount of 177.3 grams (71% yield based upon the initial oxytetracycline hydrochloride reacted) . (b) Preparation of Methacycline Hydrochloride 177 grams of the hemiketal hydrochloride thus produced was added to 500 ml of anhydrous hydrogen fluoride at -5° C. and stirred for 4 hours. The reaction mixture was then treated with 5000 ml of isopropyl ether, agitated and filtered, 174.9 grams of lla-chloro-6-methylene-5-oxytetracycline hydro-fluoride being recovered.

The dehydrated product thus formed was dissolved in a mixture of 700 ml of the isopropyl ether of ethylene glycol, 350 ml of water and 17.5 ml of 371 hydrochloric acid. To this was added a solution of 87.5 grams of sodium hydrosulfite in 350 ml of water. After three hours of agitation, triethyl-amine was added in an amount sufficient to adjust the pH of the mixture to 7.5 and the solution was filtered. The filter cake was then washed with 175 ml of a mixture of equal proportions of water and isopropyl ether of ethylene glycol. To the filtrate, 745 ml of concentrated hydrochloric acid were added, and the mixture stirred for three hours while maintaining it at a temperature of 5° C. The methacycline salt crystallized, was filtered, and washed with isopropanol and acetone. Yield 121 grams (48.51 based on the oxytetracycline hydrochloride).

Biological assay 865 mcg/mg; chemical analysis 91.5% (expressed moisture content as methacycline base); humidify (K.F.) of 0.5%.

EXAMPLE III PREPARATION OF THE HEMIKETAL BASE FROM OXYTETRACYCLINE BASE The hemiketal base may be prepared from oxytetracycline base rather than an oxytetracycline salt, as follows: 46 grams of oxytetracycline base, pretreated by reflux in dioxahe, were dissolved in 350 ml of 1,2-dimethoxy-ethane at -5°C. t and 16 grams of N-chlorosuccinimide were admixed w&ile maintaining the temperature at -5°C.Acidity was ■ equal to pH 3-^4 (apparent value In anhydrous solvent). After 2-1/2 minutes, the product started to crystallize* Stirring was maintained for several minutes; the product was filtered, washed with more 1,2-dimethoxyethane, and dried. Yield 21 grams (45.6$)· Upon infrared analysis (KBr at 1 concen ration) the product did not exhibit any absorption bands below 6 microns; _ 266 λ 344 U.V.in methanol, 0.01 N HC1 = A 216, > 35 /. The product was thus identified as the enolic form of lla-chloro-5-oxytetracycline-^,12,-hemiketai.

EXAMPLE IV RECOVERY OP THE lla-HALO-6-I^THYLElJETETRACYCLIKE SALT AS THE PERCHLORATE A further quantity of methacycline hydrochloride was formed from the hemiketai hydrochloride made in accordance with Example II above, by the following operations in which the lla-chloro-6-methylenetetracycline salt formed by dehydration of the hemiketai hydrochloride was recovered as the perchlorate.

Initially t 100 grams of the hemiketai hydrochloride made as described above was added to 300 ml of anhydrous hydrogen fluoride at ^5°C and stirred for 4 hours. Concurrently, 60 ml of 70$ perchloric acid was mixed in 900 ml of water. The two solutions were combined: the temperature of the combined perchloric ° ° by filtration. The crystals were washed and dried, 78.5 grams of lla-chloro-6-methylene-5-oxytetracycline perchlorate resulting .

The dehydrated perchlorate product thus formed was further reduced to methacycline hydrochloride in the manner described in Example II.

EXAMPLE V METHACYCLINE BASE FROM METHACYCLINE HYDROCHLORIDE grams of methacycline hydrochloride were mixed with 0.54 grams of anhydrous sodium carbonate. The mixture was slowly added, with stirring, to 25 ml of water maintained at a temperature of 60-70° C. The mixture was maintained at such temperature, with agitation, for from 10 to 15 minutes after completion of the addition of the methacycline hydrochloride- sodium carbonate mixture thereto. The mixture was then cooled to 5° C. and held at such temperature, with agitation, for a further hour. The resulting suspension had a pH of 6.7-7.2. It was filtered and the product washed with water .

The dry methacycline base thus produced weighed 4.25 grams, had a titer (based on methacycline base) of 97.8% and moisture content a ¼«»i.di.fcy (K.F.) of 3.89%. The product was thus produced in a yield of 851, equivalent to a conversion of 90.5%.

EXAMPLE VI METHACYCLINE BASE FROM METHACYCLINE HYDROCHLORIDE grams of methacycline hydrochloride (98%) were added to 25 ml of water, and the mixture heated to a temperature of 60-70° C. The mixture was agitated and triethylamine added thereto to a final H of 6.7-7.2. A itation was con to stand for an hour, after which it was filtered, the being washed with cold water.

The dry methacycline base thus produced weighed 4.38 grams, had a titer (based on methacycline base) of 96.6% moisture content and a -mamird-Tty (K.F.) of 4.33%. The product was thus produced in a yield of 87.5%, equivalent to a conversion of 92%.

For purposes of comparison, and employing the same methacycline hydrochloride utilized in Examples V and VI, methacycline base was prepared in the manner described at 8 f> in 85, J.A.C.S. ,/ 3950 , column 2, lines 15-20.

Specifically, 5 grams of the methacycline hydrochloride (98% titer) were dissolved in 150 ml of methanol and 4 ml of water containing 1.42 ml of triethylamine . After stirring for a brief period the solution was immediately filtered. The product crystallized over night at 5° C. and was thereafter recovered by filtration and washed with methanol and cold water .

The dry methacycline base thus produced weighed 2.36 grams, had a titer (based on methacycline base) of 88.6% moisture content and a-h-tmidity- (K . F . ) of 5.02%. The product was thus produced in a yield of 47%, equivalent to a conversion of 45.5%.

METHACYCLINE HYDROCHLORIDE BY SPONTANEOUS CRYSTALLIZATION OF HEMIKETAL BASE, WITHOUT FORMATION OF HEMIKETAL HYDROCHLORIDE Example I was repeated, using an identical 60 grams of hemiketal base as the initial reactant, and omitting the intermediate step involving preparation of the hemiketal salt.

The subsequent dehydration and reduction steps were carried out on the 60-gram sample of hemiketal base employing, however, approximately 1.5-fold the amount of each reagent specified therein. There were thus obtained 22 grams of methacycline ETHACYCLINE HYDROCHLORIDE BY PARTIAL CRYSTALLIZATION OF HEMIKETAL BASE, FOLLOWED BY FORMATION OF HEMIKETAL HYDROCHLORIDE (a) Preparation of Hemiketal Base For purposes of comparison the hemiketal base, pre-treated by refluxing as indicated in Example IV, was prepared in the manner described in part in Example XV of Blackwood et al Patent No. 2,984,686 by dissolving 9.2 grams of anhydrous oxytetracycline base in 100 ml of 1 , 2 -dimethoxyethane , followed by the addition of 3.2 grams of N-chlorosuccinimide.

The mixture was stirred for three minutes, and the crystallized portion recovered by filtration. 2.1 grams of product (m.p. 180° C, yield 22.8%) were thus recovered which did not show any I.R. absorption bands prior to 6 microns. The product exhibited ultraviolet peaks (in 0.01 N HCl in methanol) at ^ 264 and ^344.

The mother liquor obtained by filtration was poured into 0.400 liters of water, 2 grams of further product (m.p. 180° C.) being separated and recovered by filtration therefrom. The additional product (m.p. 180° C.) showed carbonyl I.R. absorption at 5.65 microns. U.V. peaks were exhibited at /S266 and ^ 345. The total yield of hemiketal base, in both enol and ketone forms was 4.1 grams (44.5%). (b) Preparation of Hemiketal Hydrochloride grams of hemiketal base prepared in the preceding manner were dissolved in 60. ml of anhydrous methanol containing 10% HCl. The mixture was maintained at 20-22° C. and at a pH of about 1 to permit crystallization of the hemiketal acid salt. The hydrochloride was recovered by filtration, 18 grams being re covered after washing and drying the filter cake. agitated for 3.5 hours. The reaction mixture was then treated with 540 ml of isopropyl ether at 0° C. to precipitate the lla-chloro-6-methylenetetracycline intermediate. The product was separated by filtration, and washed with further ether to remove the remaining acid. 18 grams of the dehydrated product, as the hydrofluor ide , were thus obtained. 18 grams of the intermediate were thereafter reduced in a mixture containing 72 ml isopropyl ether of ethylene glycol, 36 ml water and 1.8 ml of HCl (37%), to which 9 grams of sodium hydrosulfite in 36 ml of water were first added. The reaction mixture was maintained at a temperature of 20° C. for a period of 3 hours. Triethylamine was added to the stirred mixture during such period to adjust the pH to 7.5.

The reaction mixture was then filtered and 75 ml of concentrated HCl were added to the filtrate to precipitate the desired dehalogenated 6-methylenetetracycline product as the hydrochloride. After 3 hours, the product was recovered by filtration, and washed with isopropanol and acetone. 14.4 grams of product (melting point 203° C, chemical assay 91.3%, and biological assay 865 mcg/mg were thus obtained; 32 % total yield (w/w) .

BLACKWOOD ET AL EXAMPLE XV The hemiketal base prepared in the manner described in Example XV of Blackwood et al U.S. Patent No. 2,984,686 (as indicated in the above experiment) was further converted to methacycline hydrochloride by the procedures described in Examples I and II of the said Blackwood et al patent (without intermediate formation of the hemiketal acid salt). For such purposes 10 grams of the hemiketal were added to 30 ml of dry, obtain the dehydrated product in the form of the hydrofluoride salt .

The crude hydrofluoride product was purified by dissolving the same in water, and adding 70% perchloric acid ate dropwise to precipitate perchlor44« salt therefrom. Alternatively, the crude hydrofluoride was purified by dissolving the same in acetone, and precipitating the product as the hydrio-dide salt by the addition of 471 hydriodic acid.

The lla-chloro-6-methylene-5-oxytetracycline was thereafter lla-dehalogenated in aqueous solution in the presence of hydrochloric acid and zinc dust. 12 grams of the dehydrated product (in the form of the hydriodide.) were thus dissolved in 300 ml of 0.7% HC1 and 4 grams of zinc dust were added thereto at room temperature. After stirring for 10-15 minutes the zinc was removed by filtration, the filtrate adjusted to a pH of 0.6-0.8 and extracted with butanol. The butanol extract was concentrated under reduced pressure and the residue triturated with ether and recrystallized from methanol -acetone-concentrated HCl-ether to obtain the product as the partial ester. 5;8 grams of the product were thus obtained, corresponding to a yield of 261 as the methacycline hydrochloride .

For purposes of further comparison an additional sample of hemiketal base was prepared as described in Example XV of the above Blackwood et al patent, reacting the proportions of anhydrous oxytetracycline base and N-chlorosuccinimide in 1 , 2 -dimethoxyethane as specified in the last experiment. After the initial precipitation, the remaining filtrate was poured into 1500 ml of ethyl ether, rather than water, in an effort to increase product yield. 5.6 grams of product (60% ield were thus obtained the roduct dis la in an IR band maxima at \ 266 and A 345. The product thus contained a substantial proportion of the ketonic form of the hemiketal base in addition to the desired enolic form thereof.

By way of summary, the results obtained in Examples I and II above are compared below with those obtained in the preceding experiments involving either the partial crystallization of the hemiketal base, the spontaneous crystallization and subsequent dehydration of such base without formation of the hemiketal hydrochloride, or the use of both suc procedures as disclosed in the aforesaid Blackwood et al U.S. Patent No. 2,984,686. The respective experiments have been compared on the basis of the conversions of the initial oxytetracycline reactant to final methacycline hydrochloride product.

It will be seen from the following tabulation that use of the method of the present invention, involving spontaneous crystallization of the hemiketal base followed by formation of the hemiketal acid salt and subsequent dehydration of the latter material, provides markedly superior product yields and purities as compared with syntheses which do not involve such steps.

Equivalent Procedure Convcrs ion Pu ity Method of the Invention Example I II Spontaneous Crystallization of Hemiketal Base, Without Formation of Hemiketal Hydrochloride Partial Crystallization of Hemiketal Base, Followed By Formation of Hemiketal Hydrochloride (on total hemiketal base) 32. % 865 mcg/mg (on hemiketal base in enol form) 16.4% · 91.3% Blackwood et al , Example Impure It is apparent that various changes may be made in the preferred embodiments described hereinabove without departing from the scope of the present invention. The scope our of-»)t improved method for manufacture of 6-methylenetetracy-cline will rather be apparent from consideration of the following claims.

Claims (6)

I CLAIM:

1. A method for the preparation of a 6-meth lcne-tetracyclinc which comprises halogcnating a tetracycline base or an acid salt thereof to form the corresponding lla-halo- 6 , 12-hemiketal , dehydrating the hemiketal to form the corresponding lla-halo-6-methylenetetracycline, and dehalogenating the lla-halo- 6-methylenetetracycline to provide the desired 6-methylenetetracycline , characterized in halogenating the tetracycline base or acid salt thereof in solution at sub-ambient temperatures while maintaining the acidity of the reaction mixture between pH 3.0 and 5 to spontaneously crystallize the lla-halo-6,12-hemiketal substantially entirely in the enolic form, converting the lla-halo- 6 , 12 -hemiketal base to the corresponding hemiketal acid salt, and dehydrating said salt to the corresponding lla-halo-6-methylenetetra-cycline .

2. The method of claim 1 characterized in that the conversion to the salt form is carried out by treatment with acid at temperatures from -10° to 20° C. , and the hemiketal acid salt is crystallized substantially entirely in the enolic form.

3. The method of claim 1 characterized in that the hemiketal acid salt is dehydrated and converted to lla-halo-6-methylenetetracycline perchlorate , and such salt is thereafter deJialogenated to the desired 6-methylenetetracycline.

4. The method of claim 1 characterized in that the 6-methylcnctctracyclinc is 6-methylcne-5-oxytetracycline , and in which the initial tetracycline base or acid salt reactant is 5-oxy tetracycline.

5. The method of claim 1 characterized in that the lla-lialo-6 , 12-hemikcta.l base is formed by halogenating the tetracycline base or acid salt thereof in solution at temperatures below about 0° C. while maintaining the acidity of the reaction mixture between pH 3.0 and 5 to spontaneously crystallize the enolic lla-halo-6 , 12-hemiketal base.

6. The method of claim 1 characterized in that an acid salt of the 6-methylenetetracycline is produced by de-halogenating said lla-halo- ό-methylenetetracycline , and in which said acid salt is converted to the free base of the desired 6-methylenetetracycline by neutralizing the same at temperatures of from 50° to 90° C. in an aqueous solution maintained at from pH 6.5 to 7.5. For the Applicants Dr.Yitzh Hess