BE542223A - - Google Patents

Description

       

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   The present invention relates to a process for the preparation and refining of quatrimycins, which constitute a new group of antibiotics exhibiting useful antibacterial characteristics.



   Quatrimycins are believed to be des-isomers of tetracycline antibiotics, in that they have the same chemical composition, molecular weight, and
 EMI1.1
 that they can derive from it by a process of discord. Each of the tetracyclines - including tetracycline, chlorotetracycline, bromotetracycline, and oxytetracycline - has a corresponding quatrimycin isomer, which is called quatrimycin, chloroquatrimycin, bromoquatrimycin, and (oxyquatrimycin, respectively.



   Tetracycline is chemically referred to as
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 the name of l-dimethylâb'lino-4,6-dioxo., - methyl-2, .a5,7, i1.- pe iit aliydroxy-l,., la, 6, la, 12, 7.2ao ct ahydronapht a ene-3 - car-. boxamide. It is not currently possible to refer to quatrimycins by a similar chemical name, because the exact structure of these bodies is not known. It appears from the ultraviolet absorption spectrum of the fourth mycins that they differ from the corresponding tetracyclines by a rearrangement of atoms relative to positions 1, 2 and 3 or 4 of the tetracycline ring. . Other explanations as to the structure of the isomers can be made, and the invention will not be bound by any hypothetical configuration of the products in question.



   Quatrimycin analogs differ markedly from their respective tetracycline isomers in their properties.
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 tees) llysinxles, chemical and biological. The products are

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 stable and generally more soluble in water than their tetracycline isomers. They have markedly different ultraviolet and infra-red absorption spectra, as can be seen from the accompanying drawings which will be explained later.

   Quatrimycins have significantly lower antibacterial activity in vitro than their tetracycline isomers against the microorganisms that have been tested, but they have comparable activity in vivo, and can be used. in the treatment of the disease in a manner very similar to that adopted for the use of tetracyclines. Dosage forms and routes of administration will best be determined by the attending physician. The different solubilities and the different physiological characteristics of the antibiotics considered make them more interesting than the tetracyclines in various special cases.



   It is possible that quatrimycins are formed directly by fermentation with Streptomyces species because they have been isolated from these fermentation liquors when processed under special conditions. But it is best to prepare them directly from the corresponding tetracycline, by an isomerization process comprising certain controlled conditions which will be described below. In other words, one can obtain = quatrimycin from tetracycline, one can obtain chloroquatri-mycin from chlorotetracycline, etc.



   The process of isomerization of tetracyclines to quatrimycins occurs when a tetracycline is dissolved in a suitable solvent, at certain pH, and when it is allowed to stand. At rest, isomerization takes place until an equilibrium is reached for which, apparently, one ob-
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 t? 8nt den equal amounts aDT) roxirntivel.1ent of the tetra.-.

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 cyclin and quatrimycin. The quatrimycin can then be recovered from the solution and separated from the initial tetracyclin.



   The pH of the solution appears to be the most critical factor in isomerization. The highest quatrimycin yields are obtained in the shortest time when the pH is between 3.5 and 4.5, although pHs in a wider range also lead to appreciable conversion of the tetracyclines. in corresponding quatrimycins, in longer times. The isomerization can take place in distilled water adjusted to a pH within the preferential range, but it is very slow. One of the factors which explains this low conversion rate is the low concentration of tetracycline in the solution. Low tetracycline concentrations lead to low conversion rates.

   Therefore, it is derisory that the concentration of the solution is as high as possible with respect to the tetracycline raw material.



  Solvent systems in which the tetracyclines are most soluble are therefore preferable.



   Isomerization most conveniently takes place at room temperature, although a higher conversion rate is obtained at higher temperatures.



  It also appears that some salts and acids catalyze or increase the rate of conversion. The role these bodies play in catalyzing isomerization is not known, but may arise from the action of anions or cations in the isomerization process. Buffers are preferable because they play the additional role of maintaining the pH of the solution near the optimum value.

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  It has also been noted that by increasing the concentration of the buffer, the rate of conversion of tetracyclines to quatrimycins is also increased.
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  We. examined a number of solvent and buffer systems for the conversion of. fourth-mycin tetracycline. A typical group is shown in the following table. To obtain these results, a 20% solution of tetracycline hydrochloride in the solvent system is prepared, and the solution is allowed to equilibrate at room temperature for the period of time indicated in the table.



  -In a few cases the solubility limit was lower
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 to 200, and the results were determined.: for, a .: saturated soutiol1. In Tables I and II, Me denotes CH3 and Bu denotes C4H9.



   TABLE 1
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<tb> Isomerization <SEP>% <SEP> of <SEP> quatrimycin
<tb>
<tb> System <SEP> pH <SEP> time <SEP> (h)
<tb>
 
 EMI4.4
 PO49 2Na 1M: 6sl..pe 4.2 14e0H (1; 1: l) 3.5 72 51.0 P04H2Na 4M: Mea (112) 3.5 24 35.5 PO e211 a U4: Acetone (1: 2 ) 3.5 24 '3810 POe2Na 4M: etos 1:12) 3.5 24 23.0 P04H2Na]} I: MeOH (H2) 3.5 24' 50.5
 EMI4.5
 
<tb> P04H2Na <SEP> 1M <SEP>: BuOH <SEP>: MeOH
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> (45: 1) <SEP> 3.5 <SEP> 24 <SEP> 50.5
<tb>
 
 EMI4.6
 P04H2Na 1M: MeOH (1: 2) 3.5 24 55.5 Hz0 (206 -JI cm3) 3.5 120 12 Xie0H (neutral tetracycline 169 f crrt) - 120 17 From a monk, the conversion of chloro-
 EMI4.7
 tf - 'tracycline to chloroquatrimycin in various solvents, and the results are shown in the following table:

   

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 'TOBLIAU 11. -
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<tb> System <SEP> Time <SEP> of iso- <SEP> Chloroqua-
<tb>
<tb>
<tb>
<tb> merization <SEP> trimycin
<tb>
<tb>
<tb> bear) ¯¯ <SEP> @
<tb>
 
 EMI5.3
 2-ethoxyethanol: P04H2Na 1M (2: 1) 7 43.0 2-rnethoxypropano1: P04H2Na 1M (2: 1) 1 31.1 2methoxypropanol: P04H2Na lM (2: 1) pH 2.5 1 36.4 tetralihydrofuran PO2Na 1M (2: 1) 1 40.7 thanol P04H2Na IN (2: 1) pH 2.5 2 51.0 MEPH, (MgCl2):

   P002 1M Na (2: 1). 1 19.3
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<tb> Boric <SEP> <SEP> to <SEP> 5% <SEP> 3 <SEP> 4.5
<tb>
<tb>
<tb> cide <SEP> citric <SEP> to <SEP> 5% <SEP> 3 <SEP> 36.2
<tb>
<tb>
<tb>
<tb> Ammonium oxalate <SEP> <SEP> to <SEP> 5% <SEP> 3 <SEP> 54.0
<tb>
<tb>
<tb>
<tb> Acetic acid <SEP> <SEP>: <SEP> MeOH <SEP> (1: <SEP> 1) <SEP> 3 <SEP> 31.0
<tb>
<tb>
<tb>
<tb> PO <SEP> H @ Na <SEP> 1M: <SEP> dimethylformamide <SEP>: <SEP>
<tb>
<tb>
<tb> 42 <SEP> tetrahydrofuran <SEP> (1: 1: 1) <SEP> 3 <SEP> 46.5
<tb>
<tb>
<tb> PO4H2Na <SEP> 1M <SEP>: <SEP> tetrahydrofuran <SEP> (1: 2) <SEP> 3 <SEP> 54.0
<tb>
 
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 P l.H2Na lu: dimethylformamide (1: 2) 1. $, 0
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<tb> PO4H2Na <SEP> 1M <SEP>:

   <SEP> MeOH <SEP> (1: 2) <SEP> 3 <SEP> 50
<tb>
 We observe similar results when we
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 converts bromotetracycline to bromoquatrimycin and Lloyy- .. tetracycline to oxyquatrimycin.
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 Another Berry of experiences, indicated in t.b'Ia.



    III, shows the effect of buffer concentration on the conversion of tetracycline to quatrimycin. In these experiments, the concentration of tetracycline in the solu-
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 primitive ration was between 186 and 206 microgammas per cm3.

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    TABLE III.-
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 Time (days)) 1 quatrimycin PO H Na P0¯ H2Na amformate of a- 3 031, fi monium 0.1M monimn 0.01 .: 0.31 pH 3 'Hlr3J pH 3y8 piu 3.5
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<tb> 28 <SEP> 13 <SEP> 27 <SEP> 17
<tb>
<tb> 2 <SEP> 37 <SEP> 15 <SEP> 31 <SEP> 22
<tb>
<tb> 5- <SEP> 37 <SEP> 34 <SEP> 33
<tb>
 
Various buffers which maintain the pH of the solution in the preferred range can be used in addition to those indicated in the tables above for the conversion of the tetracyclines to the corresponding quatrimycins.
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  The quatrimycins can be recovered at the expense of solutions in which they exist, by a variety of techniques. Generally, they can be recovered at the same time as the corresponding tetracyclines with which they are associated in the same way as the tetracyclines can be recovered in similar solutions. We can
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 separating quatrimycin from tgtracycline by several methods based on differences in physical properties. For example, quatrimycins are considerably more soluble in various solvents than the corresponding tetracyclines and can be recovered by fractional crystallization.

   They can also be separated from the tetracyclines by countercurrent extraction processes such as Craig's, or by chromatographic adsorption or separation chromatography.



   A preferred method of recovering the fourth mycins and separating them from the associated tetracyclines consists of

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 treating a solution of the material to remove insoluble material and inorganic salts, preparing a concentrated aqueous solution of the material by evaporation of organic solvents, if necessary, and part of the water ; and precipitating the aqueous solution with an alkali such as ammonia, triethylamine, potassium carbonate, sodium hydroxide, etc. At a pH of about 8, the product contains a high proportion of quatrimycin and a low proportion of tetracyclin, but the yield is relatively low. At higher pHs, more complete recovery of quatrimycin is obtained, but the product also contains more tetracycline.

   Repeated precipitation and recrystallization produces pure quatrimycin. These methods will be illustrated in the examples which follow.



   In general, quatrimycins in their neutral form, also called the free base, are soluble in water, lower alcohols, wet polar solvents, acids and bases. They are insoluble in hydrocarbons, esters, ethers and chlorocarbons.



   The hydrochlorides are soluble in water, lower alcohols, 2-ethoxyethanol, wet polar solvents and pyridine bases. They are insoluble in dry butanol, acetone, esters, ethers and nonpolar solvents.



   Drammonium salts are soluble in water, wet acetone, methanol, wet ethanol, propanol and butanol, wet 2-ethoxyethanol, pyridine bases, dimethylformamide and acetic acid. . They are insoluble in dry ketones, aqueous ammonia, dry higher alcohols, hydrocarbons, chlorinated hydrocarbons, esters and ethers.



   Quatrimycins form salts and complexes

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 EMI8.1
 similar to those formed by tetracycline, varotevra-cycline, bromotetracycline and oxytetracycline. These salts include acid addition salts, such as hydrochloride, sulfate, phosphate, ascorbate, acetate, citrate, etc.
 EMI8.2
 as well as cation salts such as sodium, calcium, magnesium, ammonium, and alkylamide salts, and those of other alkali metals, alkaline earth metals and amines.



   As noted above, quatrimycins have. the same chemical composition as their corresponding tetracycline isomers. But we can distinguish them by other ca-
 EMI8.3
 characteristic, partly by their infra-red and ultra-violet spectra.



   . In Figure 1- attached, we see the spectrum in-
 EMI8.4
 fra-rouge de-la quatr; mycin obtained with a Perkin-Elmer model 21 double beam infrared recorded spectro-photometer - including a sample of pure fourth mycin hydrochloride. For comparison, and. To show its pa-
 EMI8.5
 This figure also shows a recording made on the same instrument for a sample of tetracycline hydrochloride prepared under the same conditions. The drawing is a plot of the actual data from the Ferkin-Elmer Model 21 spectrophotometer.



  In FIG. 1, as also in FIG. 2, the wavelength in microns and the frequency in cm −1 are indicated on the abscissa below and above respectively. diagram, while the transmission in% is given on the ordinate. the solid line curve relates to tetra cyclin hydrochloride, while the dotted line relates to quatrimycin hydrochloride.



   Figure 2 shows an absorption spectrum of in-
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 fra-rouge for the al! lInonium salt of quatrymicin. This conbe is obtained for a crystalline solid phase of salt

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 of ammonium in a plate of potassium bromide um. The characteristic absorption lines, expressed in cm-1, are found in the vicinity of:

   
3623 1117
3412 1091
3278 1071
2941 1054
2881 1049
2105 1022
1607 995
1503 952
1459 938 1388 921
1353 884
1333 852
1290 822
1265 812
1237 790 1206 '787
1190 773
1173 756 '1161 722 1149 701
1136 685
1126
In figures 3 to 5 attached, we see the ultraviolet spectrum of quatrimycin (solid lines) compared to the corresponding spectra of clilorotetracyclint (figure 3) of oxytetracycline (figure 4) and of tetracyclin (figure 5) (In these figures, the wavelength in millimicrons is indicated on the abscissa and the absorbance on the ordinate).

   Each of these spectra was taken from the acid

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 0.1N sulfuric acid in water, at an antibiotic concentration of about 50 mg / liter as the hydrochloride. It will be noted that the four curves are similar in general configuration, each having three points in the band of 200 to 400 millimicrons. The absolute absorption for an arbitrary wavelength can be used as a concentration index of one of these antibiotics.



   Optical densities are measured by dissolving a sample in 0.1N sulfuric acid at a dilution of about 50 miurograms per cm 3. The absorbance or optical density is measured at room temperature. 'The curves are measured on a Cary recording spectrophotometer, and they give the graph of the optical density or absorbance
1. 'which is the decimal logarithm of 1. if we call 1. the intensity of the incident radiation and 1 the intensity of the radiation transmitted for a given wavelength. One centimeter quartz cell is used for the measurement. In the figures the graph of absorbance versus wavelength is shown for quatrimycin recrystallized twice as the ammonium salt.
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  The curves for chlorotetracyc.zne, oxytetracycline and tetracycline relate to hydrochlorides at a concentration of 50 micrograms per cm3.



   Figure 6 shows the ultraviolet absorption spectra of chloroquatrimycin (solid line curve and chlorotetracycline (dashed line) for the wavelength shown. In this figure 6, as well as in figure 7, the wavelength in millimicrons is plotted on the abscissa and the absorbance on the ordinate.



   Figure 7 represents the absorption of ultraviolet
 EMI10.2
 oxyquatrimycin (solid line) and oxytetracycline (dotted line.

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 6 and 7, the antibiotics were dissolved in the sulfuric acid
 EMI11.1
 rique 0.1N at a concentration of 30 microgranune s per cm3 of solution.



   Ultraviolet absorption spectra and these antibiotics as shown in Figures 3-7 are very useful analytical instruments in determining the amount of a particular quatrinycin in an aqueous solution. The small scale of the curves reproduced makes it difficult to determine the ratios of the opic densities of the various antibiotics, and consequently, the following table is given, with the optical ratios of the most important quatrimycines and tetracyclines at different lengths of. waves.



   TABLE IV.-
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 255m z55m, 255m, 4 357m (Ú 357mgr 268 m, 4.i, 357m 300m, .t4 300m 217mk QuatrîmyPi; ie 1.07 1.10 2.34 2.12 ', 1.07 Chloroquatrimy- five 1, 12 2.03 3.24 a., 5 0.57 0xyquat;] inycin 1.14 1.16, 2.07 1.80 r, Ol Tetracycline 0, $$ 1.09 l, $ 6 1.69 1. 05 Ch10rQtétr4 \ cy- cjjqo o, 9 - ¯ 1.76 2.52 lt43 0.72 Terrartcre 0.90 1.24 1.92 1.55 0.96
The infrared absorption spectra of chloroquatrimycin hydrochloride (solid line) and of chlorotetracycline hydrochloride (broken line) are shown comparatively in Figure 8 (abs-
 EMI11.3
 c.sses: turkey length in microns and frequency in Z box; ordon- TH1es: transmission in 50).

   Likewise, the ultra-red absorption spectra of oxyquatrimycin hydrochloride (solid curve) and o jt4tracficline hydrochloride (dashed line) are shown on 1u fl! Llr0 9 (1I1tllleS abscissa and ordinate than in Figure 8).

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   Colorimetric tests with lucid sulfuric acid on quatrimycin resemble those for tetracycline and give a purplish color for both quatrimycin and quatrimycin borate.



   Additional typical properties of the fourth mycins and tetracyclines are shown in Table v, in which the specific rotation is shown.



     TABLE V.-
 EMI12.1
 
<tb> Compound <SEP> Solvent <SEP> Rotation <SEP> spec-
<tb>
<tb> ¯¯¯¯¯ <SEP> cific
<tb>
<tb>
<tb>
<tb> Ammonium <SEP> salt <SEP> of <SEP> quatrimycin <SEP> HCl <SEP> 0.2N <SEP> - <SEP> 319 <SEP> 4
<tb>
<tb>
<tb>
<tb>
<tb> Quatrimycin <SEP> neutral <SEP> HCl <SEP> 0.2N <SEP> - <SEP> 333.0
<tb>
<tb>
<tb>
<tb> <SEP> ammonium salt <SEP> of <SEP> chloroqua-
<tb>
<tb>
<tb> trimycin <SEP> SO4H2 <SEP> 0.1N <SEP> .- <SEP> 353.1
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Chloroquatrimycin <SEP> neutral <SEP> SO4H2 <SEP> 0.1N <SEP> - <SEP> 275.9 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> Chloroquatri- <SEP> hydrochloride <SEP>
<tb>
<tb>
<tb> mycine <SEP> H20 <SEP> -223,

  6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> Tetracycline Hydrochloride <SEP> H2O <SEP> -227
<tb>
<tb>
<tb>
<tb>
<tb> Chlorotetra <SEP> hydrochloride <SEP>
<tb>
<tb>
<tb> cyclin <SEP> H20 <SEP> -241
<tb>
 
The melting points of quatrimycins and tetracyclines are not sharp. However, the following melting and / or decomposition temperatures are shown in Table VI.

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  TABLE VI.-
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<tb> composed <SEP> Point <SEP> of <SEP> fusion, <SEP> C-,
<tb>
<tb>
<tb> Ammonium <SEP> salt <SEP> of <SEP> quatrimycin <SEP> background <SEP> to <SEP> 1700
<tb>
<tb>
<tb> Quatrimycin <SEP> neutral <SEP> background <SEP> to <SEP> 178.5 C
<tb>
 
 EMI13.2
 Atlawniuni salt of chloroquatrimycin turns brown to 156 (> background to J-lÓ4 'Neutral chloroquatrimycin turns brown to 19,' background to U, '- 1.3'
 EMI13.3
 
<tb> <SEP> chloroquatrimycin hydrochloride <SEP> <SEP> turns brown <SEP> to <SEP> 188,

   <SEP> background <SEP> to <SEP> 211-214
<tb>
<tb> Tetracycline <SEP> neutral <SEP> background <SEP> to <SEP> 170-173
<tb>
<tb> Chlorotetracycline <SEP> neutral <SEP> background <SEP> to <SEP> 168-169
<tb>
<tb> <SEP> Tetracycline Hydrochloride <SEP> <SEP> decomposes <SEP> to <SEP> 214
<tb>
<tb> Chlorotetracycline <SEP> hydrochloride <SEP> <SEP> breaks down <SEP> to <SEP> 210
<tb>
 
Quatrimycin is more soluble in water than tetracycline. For example, under similar conditions, when a solution of tetracycline hydrochloride is adjusted to pH 5.5 with sodium hydroxide, it is formed in a solution.
 EMI13.4
 tion originally containing 11-100 miur, branched by wax3, crystals of tetracycline free base, and there remains a solu-
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 exhaustion with a concentration of 390 micrograms per cm3.

   A similar solution of quatrimycin, at the same pH and under the same conditions, remains as a clear solution at pH 5.5, which shows that at pH 5.5 the base li-
 EMI13.6
 bre tetracycline is soluble in an amount of 0.039 '- "&, while quatramycin and many of its salts, in particular the hydrochloride, the bronihydrate, the sulfate, the phosphate, the potassium salt and the ethylene salt. are extremely soluble in water and form syrups and viscous masses
 EMI13.7
 treuses when we increase the. concentration.

   The close chemical resemblance between tetracycline and quatrimycin is
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 confirmed by a tendency of quatrimycin to crystallize together with tetracycline from <1.1 aqueous outflows, even if they are at lJllf 'conc (nt1''t.Loll

 <Desc / Clms Page number 14>

 such that quatrimycin alone would remain in solution in the aqueous phase.



   Potency in vivo is shown by the injection of quatrimycin hydrochloride into mice which have come in contact with Streptococcus hemolyticus C-203. We
18 inoculated mice weighing / + 2 grams with one thousand lethal doses of S. hemopyticus C-203, and were simultaneously treated intraperitoneally with quatrimycine hydrochloride. At a dose of 10 mg / kg, 90% survive; at 5 mg / kg, 40%, survive.



   Likewise, mice brought into contact with Klebsiella pneumoniae K-AD, and protected by quatrimycin hydrochloride, exhibit the following survivals:
 EMI14.1
 
<tb> Dose, <SEP> in <SEP> mg / kg <SEP>% <SEP> of <SEP> survivors <SEP>
<tb> 80 <SEP> 90 <SEP> '<SEP>
<tb>
<tb> 40 <SEP> 70
<tb>
<tb>
<tb> 10 <SEP> 10
<tb> 5 <SEP> 0
<tb>
 The seven-day-old hen's eggs are contacted with Meningopneumonitis Cal.10. Unprotected embryos die Ten out of ten embryos protected by 0.15 mg of quatrimycin hydrochloride per embryo survive.



   EXAMPLE I. - Quatrimycin obtained from the fermentation liquor.
A liquor resulting from the fermentation of an aqueous nutrient medium with a strain of S. aureofaciens and containing tetracycline and quatrimycin is taken, it is adjusted to pH 7.1 with 50% sodium hydroxide. 1 1/4 kg of synthetic magnesium silicate (Magnesol) are added, the mixture is stirred for 30 minutes and then filtered. The cake weighs 33.4 kg and contains 95.4% of the activity of the fermentation mass, titrated spectrophotometrically. An extract on

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 cake three times, each time with 70 liters of butanol at pH 2.0, acidifying the fermentation mass with hydrochloric acid.

   7 liters of water are added during the second and third extraction to keep the butanol phase saturated with water. The butanol extracts are combined and 0.2% decolorizing carbon (Darco) and 1% sodium chloride, calculated by weight per volume, are added thereto, and the extract is then filtered. The filtrate is allowed to settle for 10 h and the aqueous layer is removed.



   The clarified extract is concentrated in vacuo at 23-
29 C, up to 16.8 liters grading 2,600 micrograms per cm3. This butanol concentrate is extracted three times with its own volume of water at a pH of 2.6. The aqueous extracts, measuring 52.0 liters, are combined and adjusted to pH 5.5 with 50% sodium hydroxide. , it is left to age 10 1/2 hours and filtered. The crude crystals contain tetracycline and quatrimycin, washed with water saturated with butanol and dried in vacuo for 16 h at 45 ° C to obtain 1068 g of product assaying 632 micrograms per milligram. . The yield from the fermentation mass is 64% of the activity.



   A mixture of 80 liters of butanol, 20 liters of chloroform, 20 liters of distilled water and 20 cm3 of concentrated hydrochloric acid is prepared. After stirring and separation, the two layers formed are separated. The pH of the aqueous layer is 2.0. 2.35 liters of the aqueous phase are mixed with 4.7 kg of acid washed diatomaceous earth (Celite 545), and this mixture is strongly packed in a glass column 15 cm in diameter, to form a column of envi roll 1.10 m high. a 40g dilute of the mixed crystals of set 1 with 250 cm3 of the aqueous phase. One adjusts the pH to 2.0 with hydrochloric acid.

   Onfilitre la @@@ il @e

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 on diatomaceous earth as a filter aid, and 240 cm3 of the saturated solution containing a total of 12.75 g of the antibiotic are obtained. This solution is mixed with 480 g of Celite 545 and the Celite which is already in the column is packed over it. The column is developed with 32.2 liters of the solvent phase of the mixture. Portions of 730 cc each are taken and titrated spectrophotometrically to 365 millimicrons. Portions 28 to 35 which contain 2.68 g of activity, mainly quatrimycin hydrochloride, are combined and concentrated in vacuo in the presence of eua to give a residue of 100 cm 3 of aqueous solution.

   This solution is clarified by filtration with diatomaceous earth, frozen, and dried.



  A yield of 2.47 g was obtained which titrated about 80% of quatrimycin hydrochloride.



   EXAMPLE II. -
Ammonium salt as quatrimycin.-
The above frozen and dried amorphous material was crystallized by diluting it with 12.4 cc of concentrated ammonia. The thick crystalline paste obtained was centrifuged and washed with another 12.4 cc portion of concentrated ammonia. The crystals, moist with ammonia, are dissolved in 20 cm3 of water and 100 cm3 of acetone are added. Crystallization begins quickly. After aging for 2 h at -10 C, the slurry is filtered. The crystals were washed with a 5: 1 mixture of acetone and water, followed by acetone, then dried in vacuo at room temperature. The quatrimycin ammonium gel crystals thus formed weigh 1.87 g.



   A portion of the ammonium salt of quatrimycin obtained above, and recrystallized twice with the water-acetone-ammonia mixture, has a water content, by

 <Desc / Clms Page number 17>

 the Karl Fischer method, 3.98 and 3.78%. The ultra- spectrum
 EMI17.1
 violet of this product in 0.1N sulfuric aid is represented by figures 3, 4 and 5.



     EXAMPLE III. -
A slurry of 56.5 g of tetracycline hydrochloride in 80 cm3 of PO 4 H 2 Na 1M and 160 cm3 of methanol is left to stand for one hour, and it is filtered to remove the insoluble bodies. The clear filtrate is left to stand at room temperature under a nitrogen atmosphere for 24 hours.
 EMI17.2
 res; at this time, the solution titrated 5,000 microgranunes per cm3 of quatrimycin, by the optical density ratio (ODR) method.



   A 10 cc portion of the solution is mixed with 20 cc of water and concentrated in vacuo and nitrogen atmosphere until the methanol is removed. One brings the concentrate to 30 cm3 with water, and the pH 2.7 is adjusted to pH 5.3 with sodium hydroxide. The mixture is seeded with tetracycline, allowed to age for 15 minutes and centrifuged to remove solids, which contain 36% quatrimycin. Drying the mother liquor by freezing
 EMI17.3
 gives 1.25 g of a solid assaying 428 micrograms per mg of quatrimycin.



   A 39.4 mg portion of the freeze-dried material is mixed with 0.25 cc of concentrated ammonia, and the crystals which immediately form are centrifuged, washed with concentrated ammonia and acetone, and they are dried under vacuum. A yield of 26.5 mg of the ammonium salt of quatrimycin is obtained.



    EXAMPLE IV. - leave to stand at room temperature for
 EMI17.4
 before. 1 ± hours a solution of 200 r; "of ch] ', ll'hycJl' 11,1 'ne tetracycline (976 1 ;; icr; .jranuT e # par ri;) <1 ;; ii :;,>> <;, i 1 <,

 <Desc / Clms Page number 18>

 
 EMI18.1
 P 4-H2. \ La. 'h) and 670 cm3 of methanol. The equilibrated solution is treated with 1500 cm3 of acetone and left to stand at 4 ° C. for 2 h; at the end of this time, the precipitated salts are removed by filtration.



   The solution is then concentrated under vacuum up to 400 cm3
 EMI18.2
 We splash c3. 1% ammonia gas in a 100 cc portion of the concentrate. The precipitate which forms is filtered off, washed with concentrated ammonia, then with acetone and dried under vacuum. A yield of 41.47 g of the ammonium salt of quatrimycin is obtained.



   EXAMPLE V.-
 EMI18.3
 separation of the ammonium salt of quatrimycin and conversion to hydrochloride.- -Or purify 40 g of crude neutral quatrimycin (632 micrograms per mg) by the method of chromatography.
 EMI18.4
 that the following to eliminate the head! 1. "afQy'Cli # and the others impregnated" is: s A mixture of 80 liters of bentanol is prepared; 20 liters of chloroform; 20 iters iëdti ë 20 em3 of concentrated hydrochloric acid. After stirring and decaiitatiofii the two layers are separated. The pH of the aqueous phase is 2.0. 2.35 liters of this aqueous phase and 4.7 kg of acid washed diatomaceous earth (Celite 545) are made a mixture, and packed tightly into a 15 cm glass column. in diameter to form a column about 1.10 m high.



   40 g of the crude quatrimycin are mixed with 250 cm3 of the aqueous phase prepared above and the pH is adjusted.
 EMI18.5
 at 2, L1 with L5 hydrochloric acid. The slurry is filtered and the filtrate (natural solution containing a total of 2r q5 g ri 'arxt zbiotia ue) is mixed with / d80 Celite 54, and the mixture is mixed.

 <Desc / Clms Page number 19>

 stand on top of the column content.



   The column is developed with 32.2 liters of the solvent phase. Portions of 730 cm 3 are taken and titrated spectrophotometrically. We combine portions 28 to
35, which contained 2.68 g of activity, mainly fourth-mycin, and were concentrated in vacuo in the presence of water to give a residue of 100 cm 3 of aqueous solution. This solution is filtered, frozen and dried to give 2.47 g of an amorphous product containing about 80% quatrimycin. The crystallization is carried out by diluting with 12.4 cm3 of concentrated ammonia.

   The crystals are centrifuged, washed with ammonia, then recrystallized by dissolving in 20 cm3 of water and adding 100 cm3 of acetone,
After aging for 2 hours at -10 ° C., the crystalline ammonium salt of quatrimycin is filtered off, washed with 5: 1 acetone-water mixture, then with acetone, and dried in vacuo.



  A yield of 1.87 g of a product assaying 935 micrograms per mg is obtained.



   A portion of this material is re-chromatographed by the method described above. Appropriate portions of the column flow liquid are concentrated in vacuo, in the presence of water, to give an aqueous concentrate which is freeze dried. The product is converted to the crystalline ammonium salt in ammonia, and the resulting product is re-chromatographed, as before, by passing an acidic solution (HCl), pH 2.0, through a column of. Celite.

   Concentrate the appropriate portions in the presence of water to obtain an aqueous solution which is freeze dried to give amorphous quatrimycin hydrochloride.

 <Desc / Clms Page number 20>

 
EXAMPLE VI.- Neutral quatrimycin.-
 EMI20.1
 A mixture of the crude ammonium salt of quatrimycin was prepared, totaling 1,171 g.



  The crude products are obtained by -a -méthod similar to that described in Example III. The mixture is dissolved in 4 liters of water by adding 150 cm3 of concentrated hydrochloric acid to pH 6.5. After having filtered out the insoluble impurities, 160 cm3 of concentrated ammonia is added to raise the pH to 8.0 and crystallization occurs. The mixture is stirred and the product is filtered, washed with acetone. -water 4: 1, water, acetone-water 2: 1, and vacuum dried.



   A solution of 300 g of this ammonium salt in two liters of water is taken, adjusted to pH 5.75 with 48 cm3 of concentrated hydrochloric acid. The crystalline product is filtered off and washed with water.



   Take a slurry of the wet product in 300 cc of water, treat with hydrochloric acid to dissolve to pH 4.0, then adjust to pH 7.0 with concentrated ammonia. The resulting crystal boil is allowed to age and is filtered, the product washed and dried in vacuo. A yield of 62.8 g is obtained.



   2 g of neutral quatrimycin are stirred in 25 cm3 of water, and the pH is adjusted to 1.20 with concentrated hydrochloric acid. The resulting solution is filtered and adjusted to pH 5.75 with concentrated ammonia. The crystalline product is filtered, washed with water and acetone, and dried in vacuo. Analysis: calculated for quatrimycin neu-
 EMI20.2
 be C2zH = 120): C: 5g, &; H: 5.44; It 6.32; 0: $ 2, $. Effective: C T: 57988; ; H: 5th58; i1 6.02; .0: 30.52 (diff);

 <Desc / Clms Page number 21>

 loss on desiccation: 2.78; ash: 0.



   EXAMPLE VII. - Preparation of the ammonium salt of chloroquatrimycin.
A solution of 100 g of chlorotetracycline hydrochloride in 500 cm3 of a 1: 2 mixture of
 EMI21.1
 PO4H2N'a 114 and dimethylformalide, place on a rotary stirrer at room temperature for about 16 hours and filter to remove the insoluble portion. The inorganic salts are precipitated by the addition of 750 cm3 of acetone and they are removed by filtration. Concentrated ammonia is added to adjust the pH to 8.0 and the mixture is stirred for one hour to allow complete crystallization. The product is filtered, washed with a mixture of solvents comprising 2 parts by volume of acetone and 1 part by volume of dimethylformamide; then with acetone, and dried.

   The product is the ammonium salt of chloroquatrimycin ...



   A second crop is obtained by adding a further 250 cm3 of acetone. After filtration, washing and drying, the product weighs 7.89 g.



   EXAMPLE VIII. -
 EMI21.2
 Preparation of the ammonium salt of cliloroquatrimycin.
A solution is prepared by dissolving 20 g of chlorotetracyline hydrochloride in 400 cm3 of 1M PO4H2Na and 800 cm3 of methanol. Some crystals form at the expense of the solution, and they are removed by filtration. The clear solution is left to stand at room temperature under a nitrogen atmosphere for 3 days.



   To remove methanol, the equilibrated solution is concentrated in vacuo, with nitrogen, to a volume of 194 cm3. The concentrate is then extracted five times with 50 cm3

 <Desc / Clms Page number 22>

 benzyl alcohol, and the extract was dried over anhydrous sodium sulfate.



   The activity is precipitated by gradual addition of the extract to 6 liters of ether. The precipitate is filtered, washed with ether, then with carbon tetrachloride, and dried in vacuo to obtain 10.47 g of product.



   The amorphous precipitate is stirred with 52.5 cc of concentrated ammonia to produce immediate crystallization.



  The product is centrifuged. It is washed with concentrated ammonia, then with acetone, and dried in vacuo. A yield of 5.2 g is obtained.



   EXAMPLE IX Preparation of chloroquatrimycin hydrochloride
A sample of 209 mg of the se) 1, of ammonium prepared in Example VIII is taken, it is stirred in 2 cm3 of a mixture of 98% cellosolve and 2% water, and it is treated with concentrated hydrochloric acid. until the pH is less than 2. The amorphous insolubles are removed by centrifugation, and the supernatant liquor is allowed to crystallize for 4 1/2 hours at room temperature. The product is filtered, washed with water, then with anhydrous ethanol, and dried in vacuo. Yield 65.6 mg.



     EXAMPLE X Preparation of neutral chloroquatrimycin.



   A 50 mg sample of the ammonium salt prepared in Example VIII is taken, diluted with 0.3 cm3 of 0.1N HCl and treated with a 'drop of concentrated hydrochloric acid'. produce the solution at a pH of about 5 '.



    A crystalline precipitate readily forms, it is centrifuged, washed with water and dried by freezing.

 <Desc / Clms Page number 23>

 



   The optical properties of quatrimycins are not easy to determine because the crystals are very small and are not well defined. However, chloroquatrimycin spus' free base form has an alpha refractive index of 1.607 0.003 and a beta refractive index of 1.688 ¯ 0.003. The corresponding refractive indices for chlo tetracycline free base are alpha 1.650 + 0.005 and beta 1.672 + 0.005. Chloroquatrimycin hydrochloride has a beta refractive index of 1.694¯ 0.003 with the optical sign being positive. Chlorotetracycline hydrochloride has a beta index of 1.705¯ 0.003 and a negative optical sign.



    EXAMPLE XI. -
Crude ammonium salt of chloroquatrimycin was prepared by a method similar to Example III. A 1.43 kg portion of the material was dissolved in 2.5 liters of water to form a solution at pH 7.6. The addition of 250 cm3 of concentrated ammonia brings the pH to 9.3 and crystallization begins. After stirring for 1 hour, the product is filtered, washed with a 4: 1 acetone-water mixture, then with acetone, and dried in vacuo.

   A yield of 444 g of ammonium salt of chloroquatrimycin is obtained. f
A solution of 49.7 gr of the above product in 400 cm3 of water is prepared, and the mixture is crystallized.
 EMI23.1
 neutral chloroquatrimycin by addition of 14.2 cm3 of concentrated hydrochloric acid up to pH 5.5. After aging for two hours, the product is filtered, washed with acetone and dried in vacuo.



   1 g of this product is dissolved in 30 cm3 of methanol, quickly filtered and crystallization is initiated by scraping the wall of the vessel. The crystalline neutral chloroquatamycin is filtered off, washed with water and methanol, and dried in vacuo.

 <Desc / Clms Page number 24>

 



  Analysis: Calculated for C22H23N2C108:
 EMI24.1
 : 55,; H: t., 0 Pd: 5, $ c-; Cl 7.41; 0: 26.7. Effective: C: 55.03; H: 4ego; TvT: 5.3; C1 7.36; 0.26, Se (diff), ash 0.1%; loss on drying 0.37%.
 EMI24.2
 EXIàIPIE XII. - Preparation of oxyquatrimycin.Beutre.-, .'l] A solution of 20.2 g of neutral oxytetracycline in 100 cm3 of glacial acetic acid is taken, it is stirred
 EMI24.3
 under nitrogen atmas-ohexe for about 24 hours, at this time it titrates 60..00 mfcrograms of oxyquatrimycin-per cm3.

   The solution is mixed with 500 cm3 of water and concentrated in vacuo under a nitrogen atmosphere to a volume of 100 cm3.
 EMI24.4
 Add a second, 500 cc portion of water, and repeat the concentration. Recrystallization, which begins shortly
 EMI24.5
 te! np & apréw, 3a second, addition of water, continues throughout the remainder of the concentration. The crystals (mainly oxvjSetracycline) are filtered off and the filtrate is dried with c; é.t pr, h.¯'en3r 6.15 g of product containing 660 microgranunew with rozyquatrimycin per mg.



   The addition of 30 cm3 of water to the amorphous material causes the immediate formation of crystals, which are dissolved by adding 3.1 cm3 of concentrated ammonia to pH 9.2.
 EMI24.6
 



  When o t-ra: ite; with 2.3 cm3 of 6N hydrochloric acid, up to the pX 'to # ..,.: Slow crystallization appears. After 30 minutes, the product is filtered off and washed with water. and dried in vacuo Yield: 2.35 g, 920 micrograms of oxy-quatrimycin per mg.



   A second crop of crystals is isolated from the mother liquor by further adjusting the pH to 5.6 with 1.8 cm3 of 6N hydrochloric acid. After washing and drying, the
 EMI24.7
 product weighs 2.79 as 5 ± 0 micrograms of oxyqu,). trL, -, y- eine per mg.

 <Desc / Clms Page number 25>

 



  EXAMPLE XIII. -
 EMI25.1
 Renewal of the sodium salt of oxygenatrimycin. A suspension of 202 mg of neutral oxyquatrimycin in 7.5 cm3 of methanol is prepared. Partial dissolution occurs, but this is followed by reprecipitation.



  When 50 mg of sodium hydroxide dissolved in 2.5 cm3 of methanol are added, the solids have completely dissolved, but after standing for 2 1/2 hours at 4 ° C., the sodium salt no longer crystallizes. Another 20 mg of sodium hydroxide, dissolved in 1 cm3 of methanol is added. After 18 haures at -11 C, there is some-
 EMI25.2
 quen 9 = 5 jamac! of crystals, when divided, abundant crystallization follows. The slurry is allowed to age for an additional 4 hours at -11 C, and filtered. The product is washed with methanol and dried in vacuo. Yield: 186 mg.



   EXAMPLE XIV.-
 EMI25.3
 '. at.,. ^ ss:.:, d, u oxyquatrimycin hydrochloride .... take a solution of 98.5 mg of neutral oxyquatrimycin in 0.5 cm3 of 3N hydrochloric acid, and it is ened-
 EMI25.4
 this with xyq {uatrimycin hydrochloride, and rapid crystallization occurs. A further 0.3 cc of 3N hydrochloric acid is added to dilute the slurry and facilitate filtration. The filtered product was washed with acetone and dried in vacuo to obtain 47.3 mg; assaying 925 micrograms of oxy. quatrimycin per mg.



    EXAMPLE XV.-
 EMI25.5
 Preparation of neutral uquatrinyein. -
A slurry of 10.06 g of oxytetracycline hydrochloride and 2.06 g of potae sium acetate in 50 cm3 of glacial acetic acid is stirred for 4 hours until dissolution occurs, and the mixture is filtered. slight insoluble residue. The solution is left to stand overnight under

 <Desc / Clms Page number 26>

   of nitrogen, mixed with 250 cm3 of water, and concentrated in vacuo to 50 cm3. Another 250 cm3 portion of water is added, and the concentration step is repeated.



   The crystalline precipitate, which is mainly oxytetracycline, is filtered off and washed with water. The filtrate and wash liquor are combined, freeze-dried to give 3.33 g of crude oxyquatrimycin.



   The amorphous product is dissolved in 16.7 cm3 of water and the insoluble matters are removed by centrifugation.



  The supernatant liquor is adjusted to pH 6 by addition of concentrated ammonia, and stirred for 20 minutes. The crystalline product is filtered off, washed with water and dried under vacuum to give 1.7 g.



   The product is recrystallized four times with water, dissolving it at pH 9.2 and using ammonia for pH adjustment, and reprecipitating by adding hydrochloric acid to pH. 8. C:
Analysis: calculated for C22H24N2O94H2O: / 49.6; H, 6.06; N: 5.26; 0: 3.91; H2O; 13.5. Effective: C: 48.51; 48.50; H: 6.16; 6.05; N: 4,. $ 2; 5.04; 0: 40.51; 40.41; (by difference); H2O; 12.40; .12.42.



   EXAMPLE XVI. - Reduction of chloroquatrimycin.
5 g of chloroquatri-mycin ammonium salt are dissolved in 45.0 cm3 of water, and hydrogenated on a Barr stirrer in the presence of 1.4 g of 5% Pd on C. In the space of 20 minutes. minutes, the reduction stops, one equivalent of hydrogen having been absorbed. The mixture is filtered and added to the clarified solution of concentrated ammonia, raising the pH to 9.0. Add an equal volume of acetone, and more ammonia to readjust the pH to 8.5-9.0. We filter the preci-

 <Desc / Clms Page number 27>

 pit6 obtained after 25 minutes, washed with acetone and dried in vacuo at room temperature for 1 h.



   A yield of 1.0 g of quatrimycin ammonium salt is obtained.



   EXAMPLE XVII. - Conversion of quatrimycin to tetracycline.
50 mg of the ammonium salt of quatrimycin is dissolved in 1.0 cc of water and the pH is adjusted with formic acid to 4.25. After standing overnight at room temperature, the solution precipitates a pale yellow crystalline substance.



   About 20 mg of the crystalline product is collected by centrifugation, washed with water and dried in vacuo. The ultraviolet spectrum of the product indicates that it is a mixture of tetracycline and quatrimycin containing about 70% tetracyclin. The product is dissolved in a drop of 2-ethoxyethanol, acidified with hydrochloric acid and allowed to evaporate to dryness. When the dried glassy hydrochloride is rewetted with 2-ethoxyethanol, and when seeded with a minimal amount of tetracycline hydrochloride, the glassy mass crystallizes giving a good yield of crystalline tetracycline hydrochloride. . The product is identified by the ultraviolet spectrum and the infrared spectrum, both of which are identical to the spectra of an authentic specimen of tetracycline hydrochloride.



   The composition of the fenmentatino medium, the fermentation conditions, the strains cultivated, and the recovery methods, can vary widely while remaining within the scope of the present invention.

** ATTENTION ** end of DESC field can contain start of CLMS **.


    

Claims (1)

CLAIMS.- 1.- Process for prenaration of quatrimycins by isomerization of tetracyclines, in particular tetracycline, ohlo- <Desc / Clms Page number 28> rotetracycline, bromotetracycline and oxytetracycline and their acid and base salts, in which process a concentrated solution of a tetracycline is prepared, the pH is adjusted to about 3.0 and 5.0 and allowed to stand until that an appreciable proportion of the tetracycline is isomerized to a quatrimycin, and if desired, this is reacted with an acid or a base to obtain salts. 2. A method according to claim 1, wherein the solution is allowed to stand for at least one day. 3. A process according to either of claims 1 and 2, wherein the solvent system contains an oxygenated polar organic solvent in which the tetracyclines are more soluble than in water. 4. A process according to either of claims 1, 2 and 3, in which the solution is allowed to stand at a temperature above room temperature. 5. A method according to any of claims 1 to 4, in which buffers such as NaH2PO4 are added to the solution. 6. A process according to any of claims 1 to 5, wherein, after standing, insoluble matter is removed from the solution, concentrated if desired, the pH is adjusted above about 8, and the quatrimycin product which precipitates is recovered. 7. A process according to claim 6, in which the quatrimycin is precipitated, above approximately pH 8, by addition of ammonia. 8. A process according to either of claims (, and 7, wherein the quatrimycin product is redissolved in an acidified solvent, and reprecipitated by adjusting the pH to above about 8. <Desc / Clms Page number 29> 9.- Process for the preparation of quatrimycin by isomerization of tetracyclines, in particular tetracycline, chlo-rotetracycline, breotetracycline, and oxytetracycline and their. salts of acids and bases, in substance, as described above. 10. - Quatrimycins by isomerization of tetracyclines, in particular tetracycline, chlorotetrapycline, bromotetracycline and oxytetracycline and their salts of acids and bases, when they are obtained by the process according to one or other of the preceding claims . He. - Process for converting quatrimycins to isomeric tetracyclines, comprising preparing a concentrated solution of a quatrimycin, adjusting the pH to between about 3.0 and 5.0, and allowing the solution to stand until a sufficient proportion appreciable of quatrimycin is isomerized to tetracycline. 12.- As new products, quatrimycin <in particular quatrimycin, chloroquatrimycin, bromoquatrimycin and oxyquatrimycin, as well as their salts of acids and bases.