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United States Patent 0 1 3,036,129 ICC Patented May 22, 1962 1 TABLE 3,036,129 PREPARATION OF 7-HALO-6-DEOXY TETRACYCLINES 7-Bromo-6- 7-Bromo-6- Organism Joseph John I-llavka, Valley Cottage, N.Y., and James Howard Boothe, Montvale, N.J., assignors to American 7-I0d0 Tetra- Deoxytetra- dernethyl-6~ 6~dcoxy~ cycline cycline deoxytctra- tetra cycline cycline Cyanamid Company, New York, N.Y., a corporation of Maine 10 This invention relates to the preparation of 7-halo-6 deoxytetracyclines which may be represented by the fol lowing general formula’ N(CHs)2 —OH ~CONH2 (at E ii 011 1 ATOC 607 _____________ __ 1 0. 5 l 0.25 0. 25 4 8 Staphylococcus aureus 209P. 2 1 1 2 Sarciua luteo 1001 _________ __ 2 1 0.5 2 Bacillus subtilis A’I‘CO 6633Streptococcus pyogeues C203. Streptococcus 'y N o. 11..-__ Staphylococcus albus N o. 69. 0. 5 0. 5 250 >250 250 0. 25 0. 25 4 8 0. 25 0. 5 4 8 0. 5 2 4 15 4 2 4 Streptococcus B N o. 80- ___- 15 112.1 H R2 R3 Mycobacterium rauae _____ __ lllycobacteriurn smeumatis No Drawing. Filed Oct. 9, 1959, Ser. No. 845,304 11 Claims. (Cl. 260-559) Staphylococcus aureus NY 104 2 1 1 _ __ 0. 25 0.25 0. 5 2 Pseudomouas acruginosa____ 15 250 31 >250 Proteus vulaaris 8427______ -_ 15 8 2 31 Escherichia coli ATCC 9637- 15 250 31 >250 Salmonella gallinarum .... __ Escherichia coli No. 22 ____ __ 8 4 250 15 31 8 >250 62 Bacillus cereus N o. 2 The 7-iododeoxytetracycline and 7-bromodeoxytetra cycline are particularly useful as diagnostic agents in the detection of cancer. The 7-iododeoxytetracycline and 7 bromodeoxytetracycline appear to concentrate in rapidly 25 proliferating tissue such as found in tumor growths. These compounds may be rendered radioactive by incorporation wherein R1 is Br, C1 or I and R2 is H or CH3 when R, is - of iodine 131 or bromine 82 in the 7-position of the ring H, and R2 is CH3 when R3 is OH. nucleus. These radioelabeled compounds by virtue of their gamma rays and beta particle emissions allow the The halodeoxytetracyclines of this invention may be prepared by dissolving asuitable 6~deoxytetracycline, i.e. 6-deoxytetracycline itself, 6-demethyl-?-deoxytetracycline 30 or 5-hydroxy~o-deoxytetracycline, in a suitable solvent,v detection, localization and diagnosis ‘of neoplastic tissue. ‘ The chemical methods for preparing these radio-labeled compounds are similar to those preparative methods al ready described for the preparation of non-radioactive compounds with the obvious exception that the radioiso as an N-haloamide, i.e., N-bromosuccinimide, N-bromo 35 tope of the appropriate halogens are used. such as concentrated mineral acid, i.e., hydrochloric acid, sulfuric acid, etc. and adding a‘halogenating agent such The invention will be described in greater detail in acetamide, N-bromophthalimide, N-chlorosuccinimide, N conjunction with the following speci?c examples. iodosuccinimide, etc. The reaction with the haloamide is preferably carried out at temperatures of from about 0° C. Example 1 to about 20° C. until the reaction is complete. Alterna tively, the bromination may be carried out with bromine in 40 PREPARATION OF 7-BROMO-G-DEOXYTETRACYCLINE a strong mineral acid, i.e., HBr and in a suitable organic To ‘a solution of 0.2 gram of 6-deoxytetracycline solvent such as a lower alkanoic acid, i.e. acetic acid, pro [J.A.C.S. 80, 5324 (1958)] in 10 milliliters of concen pionic acid, etc, at temperatures of from about 10° C. to trated sulfuric acid at 0° C. is added 76 milligrams of about 50° C. The halodeoxytetracycline so-formed is N-bromosuccinimide. The reaction mixture is kept at 0° isolated from the reaction mixture by any convenient 45 C. for 30 minutes, and then added slowly drop-wise to method as, for example, by precipitation with ethyl ether 200 milliliters of cold ether. A solid precipitates which or the like, and the product may be puri?ed by recrystal~ lization from an alcohol-acetone-ether solution in a stand ard manner. is ?ltered and dried. The product. weighs 160 milligrams. This product is crystallized from ethanol-acetone-ether solution to give 120 milligrams of puri?ed product. This The halodeoxytetracyclines are biologically active and 50 product is 1.2—1.5 times as active biologically as tetra~ cycline. have the broad-spectrum antibacterial activity of the Example 2 previously known tetracyclines. The antibacterial spec trum of certain of these compounds, representing the PREPARATION OF 7-BROMO-‘6~DEMETHYL-6-DEOXY amount required to inhibit the growth of various typical TETRACYCLINE bacteria, was determined in a standard manner by the 55 A solution of 200 milligrams (0.045 m mole) of 6 agar dilution streak technique which is commonly used in testing new antibiotics. The minimal inhibitory con centrations, expressed in gamrnas per milliliter, of 7 demethyl-6-deoxytetracycline hydrochloride [J.A.C.S. 80, For comparison purposes the antibacterial activity of tetracycline against the same organisms is also included. A portion (25 milligrams) of this material is recrystal lized from ethanol/ether yielding 19 milligrams of pure 51324 (1958)] and 80 milligrams (0.45 m mole) of N bromosuccinimide in 5.0 milliliters of concentrated sul furic acid is stored at ice-bath temperature for 30 minutes. tetracycline and 7-iodo - 6 - deoxy-tetracycline against 60 The reaction mixture is slowly added to 250 milliliters of various test organisms are reported in the table below. cold ether and the solid that separates weighs 0.2 gram. bromo-o-demethyl-6-deoxytetracycline, 7dbromo~6-deoxy 3,036,129 4 3 into 250 milliliters of cold ether. The solid that sepa rates weighs 0.157 gram. A portion (50 milligrams) of this material is crystallized from methyl cellosolve/ 7-brorno~6-demethyl-6-deoxytetracycline which is 2.2 to 2.5 times as active as tetracycline. Example 3 PREPARATION OF 7-CHLORO~6~DEOXYTETRACYCLINE chloroform; yield, 20 milligrams. 5 The procedure of Example 1 is followed except that N-chlorosuccinirnide is used as the halogenating agent. This application is a continuation-in-part of our co pending application Serial No. 795,609, ?led February 26, 1959, now abandoned. We claim: 1. The method of preparing 7-halo-6-deoxytetra The product is isolated as in Example 1 and 7-chloro-6 deoxyltetracycline is obtained. cyclines of the formula: Example 4 PREPARATION OF 7-CHLORO-6-DEMETHYL-6-DEOXY TETRACYCLINE I'M H Ra Ra N(CHs)2 The procedure of Example 2 is followed except that N-chlorosuccinimide is used as the halogenating agent. 15 The product is isolated as in Example 2 and 7-chloro-6 OH demethyl-6-deoxytetracycline is obtained. Example 5 PREPARATION OF 7-BROM0-6~DEOXYTETRACYCLINE —CONH2 l OH O 20 ‘ OH H O OH 85.6 milligrams of 6-deoxytetracycline is dissolved in 3.9 ‘milliliters of acetic acid and 1 milliliter of 30% HBr acetic acid is added. With stirring, 0.22 milliliter of 1 wherein R1 is a member of the group consisting of molar bromine in acetic acid is added (10% excess of 1 bromine, chlorine and iodine and R2 is a member of the equivalent). After standing about 65 hours at room 25 group consisting of hydrogen and methyl when R3 is hy temperature, 10 milliliters of ether are added and a yel drogen and R2 is methyl when R3 is hydroxy which com low crystalline solid slowly deposits. After 6 hours the prises treating a compound of the group consisting of solid is ?ltered off and dried; weight, 68.4 milligrams. 6-deoxytetracycline, 6~demethyl-6-deoxytetracycline and Example 6 30 5-hydroxy-6-deoxytetracycline with an N-haloamide of the group consisting of N-bromosuccinimide, N-bromo PREPARATION OF 7-BROMO-5~HYDROXY-6-DEOXY‘ TETRACYCLINE A solution of 200 milligrams (0.42 m mole) of S-hy droxy-6-deoxytetracycline hydrochloride [J.A.C.S. 80, 5324 (1958)] and 75 milligrams (0.42 m mole) of N bromosuccinimide in 5.0 milliliters of concentrated sul~ acetamide, N-bromophthalirnide, N-chlorosuccinimide, and N-iodosuccinimide in the presence of a concentrated mineral acid at a temperature of from about 0° C. to 35 about 20° C. 2. The method of preparing 7-bromo-6-deoxytetra— cycline which comprises treating 6-deoxytetracyc1ine with furic acid is stirred at ice-bath temperature for 10 min~ N-bromosuccinimide in the presence of a concentrated utes. The reaction solution is slowly poured into 250 mineral acid at a temperature of from about 0° C. to milliliters of cold ether. The solid is ?ltered and dried; 40 about 20° C. yield 1.8 grams. This material is converted to the free 3. The method of preparing 7-bromo-6-demethyl-6-de base by dissolving in water, adjusting the pH of the solu oxytetracycline which comprises treating 6-demethyl-6 tion to 6.5 with 1 N sodium carbonate and extraction deoxytetracycline with N-bromosuccinimide in the pres with n-butanol; yield 75 milligrams. Microbiological activity equals 45% of tetracycline (activity of starting S-hydroxy-6-deoxytetracycline equals 30% of tetracycline). Example 7 PREPARATION OF 7-IODO-6-DEOXYTETRACYCLINE SULFATE A solution of 200 milligrams (0.38 m mole) of 6-de oxytetracycline sulfate and 85.5 milligrams (0.35 m mole) ence of a concentrated mineral acid at a temperature of 45 from about 0° C. to ‘about 20° C. 4, The method of preparing 7-chloro-6-deoxytetra cycline which comprises treating 6-deoxytetracycline with N-chlorosuccinimide in the presence of a concentrated mineral acid at a temperature of from about 0° C. to 50 about 20° C. 5. The method of preparing 7-chloro-6-demethyl-6-de oxytetracycline which comprises treating 6-demethyl-6 deoxytetracycline with N-chlorosuccinimide in the pres of N-iodosuccinimide in 5.0 milliliters of concentrated ence of a concentrated mineral acid at a temperature of sulfuric acid is stirred ‘at 0° C. for forty minutes. The 55 from about 0° C. to about 20° C. mixture is added dropwise to 250 milliliters of cold ether. ‘6. The method of preparing 7-bromo-5-hydr0xy-6-de The sOlid that separates Weighs 0.16 grams. A portion oxytetracycline which comprises treating 5-hydroxy-6-de (50 milligrams) of this material is recrystallized from methyl cellosolve/chloroform; yield 22 milligrams. Example 8 PREPARATION OF 7-IODO-5-HYDROXY-6-DEOXYTETRA CYCLINE SULFATE The procedure of the preceding example is following except that 5-hydroxy-6-deoxytetracycline is used. 7 iodo-5-hydroxy-6-deoxytetracycline is obtained. Example 9 oxytetracycline with N-bromosuccinirnide in the presence of a concentrated mineral acid at a temperature of from 60 about 0° C. to about 20° C. 7. The method of preparing 7-iodo-6-deoxytetra cycline which comprises treating 6-ideoxytetracycline with N-iodosuccinimide in the presence of a concentrated mineral acid at a temperature of from about 0° C. to 65 about 20° C. 8. The method of preparing 7-iodo-6-demethyl-6-de oxytetracycline which comprises treating 6-demethy1-6 deoxytetracycline with N-iodosuccinimide in the presence of a concentrated mineral acid at a temperature of from 7-10DO-6-DEMETHYL~6~DEOXY'I‘ETRACYCLINE SULFATE 70 about 0° C. to about 20° C. 9. The method of preparing 7-iodo-5-hydroxy-6-de To a solution of 0.2 gram (0.39 m mole) of 6-de methyl-6-deoxytetracycline sulfate in 5.0 milliliters of cold (0° C.) concentrated sulfuric acid is added 88 milli oxytetracycline which comprises treating 5-hydroxy-6-de oxytetracycline with N-iodosuccinimide in the presence of a concentrated mineral acid at a temperature of from grams (0.39 m mole) of N-iodosuccinimide. The mix ture is stored at 0° C. for forty minutes and slowly poured 75 about 0° C. to about 20° C. 5 ,r 3,036,129 10. The method of preparing 7-bromo-6-deoxytetra cyclines of the formula: 6 an organic solvent at a temperature of from about 10° C. to about 50° C. References Cited in the ?le of this patent UNITED STATES PATENTS 2,736,725 Ritter _______________ .... Feb. 28, 1956 OTHER REFERENCES Geschickter: J. Am. Med. Assoc., Feb. '1, 1930, pages wherein R2 is a member of the group consisting of hy ‘ drogen and methyl when R3 is hydrogen and R2 is methyl when R3 is hydroxy which comprises reacting a com pound of the group consisting of 6-deoxytetracyc1ine, 6 demethyl-6-deoxytetracycline and S-hydroxy - 6 - deoxy tetracycline with bromine in a strong mineral acid, and 326—328. Fieser et 211.: Natural Products Related to Phenan threne, pages 388-389, 458; 533-536. Reinhold Pub. Co., New York (1949). Abbott Laboratories, “Diagnostic Procedures With Radioisotopes,” pages 23, 28 (January 1955). Migrdichian: Organic Synthesis, vol. II, page 924, in the presence of an organic solvent at a temperature of from about 10° C. to about 50° C. Reinhold Pub. Co., New York (1957). Rall et al.: “Journ. US. National Institute of Cancer,” 11. The method of preparing 7~bromo-6-déoxytetra 20 pages 79-83 (July 1957). cycline which comprises reacting 6-deoxytetracycline with Stephens et al.: I. Am. Chem. Soc., vol. 80, pages bromine in a strong mineral acid and in the presence of 5324-5 (October 1958).