BE556763A - - Google Patents

Description

       

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   The present invention relates to antibiotic substances and to methods for synthesizing them. It relates more particularly to the synthesis of novobiocin and related antibiotic substances.



   Novobiocin (generic name of an antibiotic, a trademark of which is referred to as "Cathomycine") is produced by the growth, under controlled conditions, of a previously unknown species of microorganism, which has been called Streptomyces spheroides. This microorganism, which was isolated from a soil sample from an old grassy pasture in Vermont, has been designated as Streptomyces spheroides MA 319 in the crop collection of the Merck Company. # CO., Inc., Rahway, Mew Jersey.

   A viable culture of this microorganism has been. deposited in the fermentation section of the Northern Utilization Research Bureau of the United States Department of Agriculture in Peoria, Illinois, and added to its permanent collection of cultures. under No. NRRL 2449. In aqueous media suitable for Aerobic cultivation of strains of Streptomyces Spheroides to produce novobiocin are generally suitable for the production of other antibiotics by culturing other organisms of the genus Streptomyce.

   These media contain sources of assimilable carbon, such as carbon hydrate, sources of assimilable nitrogen, such as corn maceration liquor, casein hydrolyzate, distiller solutions or the like, and salts. inorganic, as well as traces of metaxu reduced to ensure a meta-

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   suitable bolisBia of the microorganism. Preferably, the medium is maintained at a temperature of 24-20 C during the period, usually about 1 to 7 days,

   during which the microorganism is cultivated and aeration is provided to ensure optimum growth of the organism and the production of novobiocin. Fermented broths produced in this way have an activity of about 150-2000 units of novobiocin, as defined below, per cc and the solids of the fermentation broth have an activity of about 2.25 units. of novobiocin per mgr of solids. The antibioactive material can be purified and recovered in a purer form by several methods.



   Thus, any broth can be filtered at the hydrogen ion concentration of the crop, usually about pH 7.0-8.0; the filtrate can be extracted at a concentration of hydrogen ions in the acid region below about pH 7.0, using a substantially neutral, only slightly polar, liquid organic solvent immiscible with the acid. water, soluble in cold concentrated sulfuric acid and in cold syrupy orthophosplioric acid.

   The organic extract can be extracted with an aqueous alkaline buffer solution at a hydrogen ion concentration of at least pH 8.5, so as to obtain a solution containing a substantial content of novobiocin salt. The two extraction stages can be repeated in succession, so as to obtain a still concentrated glue solution, from which the active novobiokinic material can be recovered by acid precipitation. The product thus obtained can be purified by recrystallization from an aqueous acidic alcoholic solution.



   It should be noted that in addition to Streptomycdes spheroides

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 NRR 2449, other CI producer strains (: novobiocin de.



  Streptomyces spheroides can be used to obtain novobiocin; thus, mutants of the organism described, obtained by natural selection, or those produced by a mutating agent, for example x-ray irradiation, ultraviolet ray irradiation, nitrogen mustards and the like, can be used to produce novobiocin.



   The new antibiotic, novobiocin, comprises the following components: carbon, hydrogen, nitrogen and oxygen combined into a substance having approximately the formula C31H36N2O11 according to the data currently available. It reacts as an acidic organic compound with alkaline reagents, such as aqueous solutions of carbonate hydroxides and alkali metal bicarbonates, and is soluble in these reagents; it has two linking groups with bases and can be precipitated from its. solution in alkalis by acidification.

   It is soluble in lower alkanils, lower aliphatic ketones, acetic acid, ethyl acetate, dioxane; it is insoluble or only slightly soluble in ether, benzene, chloroform, carbon tetrachloride, ethylene dichloride, water and hydrochloric acid.



   Substantially pure novobiocin was obtained in two crystalline forms: one crystallizing in the form of rosettes and melting at about 152-154 C and another form having the appearance of flat needles melting at about 170-172 C. Each of these forms crystals of the antibiotic can be converted into a so-called standardized form, or can be an amorphous or sub-microcrystalline form by dissolving the crystals in acetone, adding ranide to this solution of a relatively large volume.

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 large petroleum ether, and by recovery of the precipitated standard material by filtration.



   Aqueous alkaline solutions of novobiocin and. suspensions in mineral oil of the standardized form of the antibiotic show characteristic absorption, the former in the ultraviolet functions and the latter in the infrared fractions of the spectrum. A solution of substantially pure novobiocin in 0.1JN aqueous sodium hydroxide exhibits a characteristic ultraviolet absorption peak at 3070 this absorption peak is the indication of a substantially pure material. having a specific absorbance of 60U, measured at this wavelength, using a solution containing one gram of pure novobiocin per 100 cc of the solution, contained in a cell having an absorption path of one centimeter.

   A solution of pure novobiocin in 0.1N aqueous methanolic hydrochloric acid exhibits a characteristic ultraviolet absorption peak at
3240 A with e1 = 390. A suspension in mineral oil of substantially pure standardized novobiocin pre-
 EMI4.1
 feels characteristic infrared absorption peaks at the following wavelengths, expressed in microns: 5.8-6.0 (broad), 6.10, or1, o30 $ u, 4Y, 6 63e 7, 4- 7, ô (wide shoulder), '/,' / +, '1,') 6, 8,7 (reliable), $, 6U (shoulder), 8,7 (Shoulder.emetlt), a, 13 , Y, 40.1U, 0-10.1.



  (broad), 10.2.1U, uO (1; irge), 12.0-12.30 (broad), 12.0-. 11, -1.75 (broad), 13.07 and 13.3 '.



   Novobiocin units indicate the microbiological activity of the substantially pure crystalline novobiocne;
 EMI4.2
 activity 1 <'microbiolo.xiqu ;. substantially pure novobiocin was arbitrarily set at 5000 units per mgr and determined by cuvette plate diffusion methods
 EMI4.3
 standard, PIl ubilisnnt du Hacillun nubti lis lrl ". The 1.-3: =

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   as a test organism.



   Briefly, in this method, B. subtilis ATTC 12.432 is grown on agar heart and brain infusion slant culture media (Difco Manual, 9th Edition, pages 90,91) for 24 hours at 37 c and the culture is then stored at 5 C for periods not exceeding one month. To prepare spores, an inoculum is prepared by adding 5 cc of sterile, distilled water to a strain freshly grown on a slant of B. subtilis.



   The cells are aseptically scraped from the slant culture medium; mixed well and transferred to 50 cc of sterile distilled water in an Erlenmeyer flask. 2 cc of the bacterial suspension are introduced, in the form of noculu, into a Roux flask containing a medium comprising 3% of flour of soybean, 0.1, 2% NaCl,, 4% distiller solubles, 0.8% NaCl and 2.0% agar.



   After incubation for 7 days at 37 ° C., the bacterial culture obtained is suspended in 50 cc of sterile distilled water and pasteurized at 65 ° C. for 30 minutes.



   4 cc of a 1:50 dilution of this spore suspension; used per liter of test medium containing 0.5% peptone, 0.3% bug extract, 0.3% yeast extract and 1.5% agar. a pH of 5.9-6.1. Fractions of
15 cc of inoculated medium are distributed in deep petri boots with a flat bottom. i
Six stainless steel cylinders are placed on the seeded agar.

   Three of these cylinders are filled with a standard solution of 4 mecrograms of novobiocin per cc (cc which corresponds to 20 de novo biocin units / cc) and the other three cylinders are filled with the unknown solution. diluted to approximately the same activity using a phosphate buffer / M / 20

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 EMI6.1
 at pIl die, 0 / Ume COurbE 'daily standard is plotted with pure bovobicoine diluted at various concentrations ranging from to 16ugr / cc.
 EMI6.2
 After 1.L hr1! D! 'ES incubation at 8 t the diameters of the zones of inhibition of the unknown and standard solutions on each plate are measured.

   The activity of the unknown product is determined from a Homograph based on the degree of response to various concentrations established from the daily standard curve.
 EMI6.3
 



  Novdbiocin is optically active, C41 21¯ -27 "(c = 1. In IN sodium hydroxide) and L i7 5 = -44 '(c = 1 in pyridine).



   Novobione is active in that it inhibits
 EMI6.4
 It is mainly the growth of gram-positive microorganisms, although it also shows some activity against gram-negative microorganisms. In particular, it inhibits the growth of the following organisms:
 EMI6.5
 D¯T ... yo, enes var. albus. pyogenes var. aureus iplococcus pneumoniae, ...



  Uoryllebacterium diphteriae type gravis uoryiiebacteriiti dinhteriae t pe intertitedius Coryne bacteri u) tt diphteriae type mitis C oryne b ac t e r i um xerose Coryilebacteriani renale Neisseria meningtidis Sarcina l.utea (VD0
 EMI6.6
 0 .. Eyo / 3enes var. aureus resisting to 1. ' aureomycin. pyosenes var. nurcus resistant to: Streptomycin-streptothricin
 EMI6.7
 . J1yoelles var. penicillin resistant aureus

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Novobiocin salts also have activity - antibiotic.

   Thus, it has been found that the sodium salt of novobicoin inhibits when tested by the dilute test. tion on agar, the growth of various strains of M.
 EMI7.1
 pyogeties var. aureus, ldeisser ia plenin '\ itid: ls (% 274) and Sarchina lutea (VD) at concentrations below 0.5 mcrgr per cc. Other microorganisms are also affected by novobiocin or its salts to varying degrees.



   It has now been found, in accordance with the present invention, that novobiocin and related substances can be prepared by reacting sodium chloride.
 EMI7.2
 2-acyloxy-3-carbainyl-4-methyInovobiosyl (XIIR) with a 3-acylamido-4) 7-dihydroxy-ë-methylcou! Rarine (VR) in the presence of silver oxide, then hydrolyzing the product resulting reaction. This reaction can be represented as follows:
 EMI7.3
 

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 R representing an acyl radical /
 EMI8.1
 Thus, when s- (3- / p Y -dimethyla11.y1? -4-acy7.o: çybonzarnido) 4â-dihydroxy - $ - methylcouMarine (IX R) is reacted with 2-acyloxy- chloride 3-carbamyl-4-methylnovobiosyl in the presence of silver oxide and when
 EMI8.2
 hydrolyzes the ndensation product, we boil novobiocin.

   Likewise, when 3- (3- 3-methyl -butyl-acyLoxybetxzamido) -, 7-dihydroxy - $ .. methylcoumamine (IXAR) is reacted with 2-aoyloxy-3-oarbamyl-4- chloride methylnovobiosyl and when the intermediate acyl derivative is hydrolyzed, dihydronovobiocin is obtained. In this way, other compounds related to novobiocin and dihydronovobiocin and having antibiotic activity can be produced.

   Thus, when we react to the 3- (2-
 EMI8.3
 2-dimethylchroman-6-carboxamido) -4 7-dihydroxy-8-methylcoumamine (IX B) and 3-acetarttido-7-dihydroxy - $ - methylcoumarin (VB) with 2-acyloxy-3-carbamyyl- chloride 4-methyltiovobiosyl and when the intermediate condensation products are hydrolyzed, cyclonovobiocin (XIII B) and 3-acetamido-7- (3-carbami- ... do-4-methylnovobiosylcxy) -li are respectively obtained .-ltydroxy - $ - triPthlcournariue (XIII C) The reactions can be schematized as follows:.

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 EMI9.1
 

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 EMI10.1
 



  R?: Both an acyl madical.



     For the execution of this method of preparation of
 EMI10.2
 novobiociac and related compounds according to the invention, 3-acjri.nmi <io-1F, 7-di> iydroxy-8-iuôtliylcouinarin is dissolved in a suitable solvent, such as dioxane, and

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 silver oxide is added to it. To the resulting suspension is then added 2-acyloxy-3-carbamyl- chloride.
4-methylnovobiosyl and the reaction mixture is stirred for a sufficient time to complete the formation of the condensation product. Usually, it is found that stirring at room temperature for 5-10 hours is sufficient to complete the reaction.



   In general, it is desirable to run the reaction in the absence of light and under substantially anhydrous conditions, in order to obtain optimum yields of the desired reaction product. It is also found that the reaction proceeds faster if a small amount of iodine is added to the reaction mixture, although the addition of iodine is not essential for the condensation reaction.



   When the reaction reaction is complete, the acyl substituent of the novobiosyl part is readily. removed by hydrolysis with alkali. Thus, after filtration of the reaction mixture to separate insoluble by-products and after reduction of the excess iodine with sodium bisulfite, the filtrate is concentrated to small volume at reduced pressure and treated with l. sodium hydroxide, to a pH of about 7, so as to separate the acyl substituent from the novobiosyl part by hydrolysis.



   The novobiocin or the compound analogous to novobiocin is advantageously recovered from the neutralized solution by lyophilization. The product thus obtained can be further purified by recrystallization from suitable solvents or mixtures of suitable solvents.



   According to another feature of the present invention, it has now been found that 2-acyl chloride
 EMI11.1
 Oxy-3-carbamyl-4-methylnovobiosyl (XII R) used in

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 condensation reactions described above can be
 EMI12.1
 prepared by acylating 3-carba) nyl-4-methylnovobiose (X) so as to form the diacyle derivative, 1,2-diacyloxy-3-ear-bamyl-4-methylhovobiose (XI R), and making react this diacylated compound with hydrogen chloride under anhydrous conditions, so as to produce
 EMI12.2
 2-acyloxy-.3-carballlyl-4-methylnovobiosyl (XII R). These reactions can be schematized as follows:
 EMI12.3
 
R representing an acyl radical.



    . When running the. first stage of the foregoing process '' it is preferred to use the acyl derivatives, in which the acyl groups are derived from lower aliphtaic acids having no more than eight carbon atoms, although other acyl groups can also be used as well. this end. This step is advantageously carried out, using a lower aliphatic acid anhydride as the acylating agent. Thus, the diacetyl derivative 'is easily prepared by
 EMI12.4
 reacting 3-carbarnyl-4-methyluovobiose with an excess of acetal anhydride, in the presence of sodium acetate at 100 ° C. for approximately 1 hour.

   When the reaction is complete, the resulting mixture is diluted with ice and water and neutralized with sodium bicarbonate.



  The acetylated derivative is recovered by extraction with an inappropriate solvent, such as chloroform, and evaporation of

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 EMI13.1
 chloromnic extract; , - According to another embodiment of this
 EMI13.2
 invention, it has been found that, 3-acylamido-4t7-dihydroxy-6-methylcoumarin (V R) used as a starting material in the processes described above can be. prepared in
 EMI13.3
 3-amino-4. acylant, t-d.Piydroxy - $ - methylcoumaritle. This reaction can be schematized as follows:
 EMI13.4
 R representing an acyl substituent.
 EMI13.5
 



  Thus, 3-amino-4,7-dihydroxy-8-niethylcownarin can be caylée by reaction with an acid anhydride in the presence of pyridine, for example, acetic anhydride = in the presence of pyridine, so to produce the derivative
 EMI13.6
 acylated corresponding.

   Or, 3-arnino -., 7-dihydroxy - $ - coumarin can be acylated by reaction with an acid chloride. For example, when 3-amino-
 EMI13.7
 4,7 ... dihydroxy - $ - methylcoumarin with 3- (', -dirnethyl, a.lyl) -.-' acyloxybenzoy.e (VIII R) chloride 3- (3-ethylbutyl) '- 4-acyloxybelizoyl (VIII AR) or 2,2-dinrethylchroman-6-carbonyl chloride (VIII B) in the presence of sodium acetate, we obtain respectively 3- (3- ± -y-, V- dimnthy1a7 .1y.7-4-acyloxybetzarrrido) -., 7dihydroxy-8-1I1ethylcoullwrine (IX R), 3- (3 - nualzylbuty -4-acy loxy-be uzanrido) -r, 7-dilhydroxy-8-mathylcoumarin (IX AR) and 3- (1,2-.dimethylcliromatl-6-caxboxar: rido) -., '7d: klycrroxy-8-m .; thyZ.coumarin (IX L3).

   These reactions can be schematized as follows:

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 EMI14.1
 
R representing an acyl group.



   These reactions are; preferably, carried out by suspending the coumarin compound in water, adding sodium acetate thereto, and then adding acyl chloride in small amounts, while stirring the reaction mixture. When the reaction is complete, the desired product is obtained by acidifying the aqueous solution to a pH of about 2. At this pH, the product is insoluble.

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 and can be recovered by filtration. If desired, the new product can be further purified by crystallization from suitable solvents.



   Another execution volume of the. present invention, it has now been discovered that the hydrochloride
 EMI15.1
 of 3-antitzo -., 7-dihydroxy - $ - rnëthylcounraritze (V) can be prepared in several stages, starting from 2-methylresorcinol.

   This process involves the air reacting of 2-methylresorcinol with an ester of cyanoacetic acid in the presence of zinc chloride and hydrogen chloride under anhydride conditions, thereby producing 7-hydroxy-
 EMI15.2
 4-imino-6-methyl-2-oxochroman (II) to be heated, this compound, imino with 50 µl sull'uric acid, so as to obtain 4,7-dihydroxy-ë-methylcou) ttarine ( III) j to treat this compound with nitrous acid so as to produce 2,4-dioxo-7-hydroxy-8-methyl-3-oximinochroman (IV), and to reduce '* this product with hydrogen in the presence of a catalyst consisting of a noble metal, such as dU pal.la- dium on charcoal,

   so as to produce the
 EMI15.3
 3-amitzo hydrochloride = G., 7dihydroxy-F3-netlzyl.couinarine (V). These reactions can be schematized as follows:
 EMI15.4
 

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 EMI16.1
 
R1 representing a hydrocarbon group.



   In order to carry out the first stage of the reactions described above, any cya, no-acetate ester can be used, although it is preferred to use a lower alkyl ester in this reaction, for example desesters. lower alkanols having 1 to 8 carbon atoms. Thus, this reaction is advantageously carried out using ethyl cyanoacetate;

     When this ester is used, the condensation is easily carried out by reacting the
2-methylresorcoinal with ethyl cyanoacetate in a suitable solvent, such as ether, cooling this solution, adding zinc chloride and finally passing dry hydrogen chloride through the cold mixture. When the reaction is. Completed, the temperature is allowed to rise to room temperature and the reaction mixture is left for about 1 day. to ensure the completion of the reaction.

   The reaction product, namely 7-hydroxy-4-imino- hydrochloride
8-methyl1-2-oxochroman, senates from the mixture and is easily recovered by filtration. The recovered product can be further purified, if desired, by crystallization in suitable solvents or mixtures of suitable solvents.



   The second stage of the process described above is easily carried out by heating a solution of the imino compound (11) in a large volume of 50% sulfuric acid at about 100 ° C. The product is recovered by cooling the product.

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 EMI17.1
 mixture reacts 1 and filtration of., 7-dihroxy - $ - methßlc oumarine (III).

   ,] The conversion stage of -, '7-dihydroxy - $ - lnethylcoumarin to 2,.-Dioxo - kiyd.axy t-rnethyl-3-owcimiuo-chromane (IV) is easily carried out by mst-ting the ma .tüiêrwa starting in suspension in water and adding a solu-
 EMI17.2
 To ensure the completion of the reaction, the mixture is stirred frequently for 3 hours, during which time the coumarin compound gradually dissolves. After filtration of this reaction mixture, it is cooled in an ice bath, so as to cause precipitation of the oximino compound in crystalline form, this compound being able to be easily recovered by filtration.



   The final stage of the process for preparing chlor-
 EMI17.3
 3-amino hydrate -., 7-dihydroxy - $ - methylcoturtaritie is advantageously carried out by dissolving 2,4-dioxo-7-hydroxy - $ - me '.: thyl-3-oxirnino, coumarin in a suitable solvent) such as that ethanol, by adding a catalyst consisting of a noble metal, and at least one dihydrochloric acid equivalent, and hydrogenating the resulting reaction mixture.



  The hydrogenation is continued until the required amount of hydrogen has been absorbed. For carrying out this reduction, it is preferred to use palladium on charcoal, although other forms of noble materials or noble metals coated on inert supports can be used for this purpose. When the reaction is carried out in this manner, the product is easily recovered by removing the catalyst by filtration and evaporating the resulting solution to dryness.



     According to a further embodiment of the present invention, it has now been found that

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 3-18. 8-Dimethylallyl) -4-acyloxybenzolyl (VIII R) used as a starting material in, the above-described process can be prepared by acylating 3 - (#, # -dimethylaollyl) -4-hydroxybeuzoic acid (VI), and by processing the sodium salt of the product acylated with oxalyl chloride. These reactions can be schematized as follows:
 EMI18.1
 
R representing an acyl radical.



  To accomplish the acylation of 3 - (#, # -dimethylallyl) -4-hydroxybenzoic acid, any of the various methods used to acylate phenolic compounds can be applied. It has been found that the + 6 'acylated derivatives of lower aliphatic carboxylic acids having 2 to 8 carbon atoms are particularly advantageous in the process described and constituting, therefore, preferred intermediates. These acylated derivatives are easily prepared by reaction with acid anhydride in the presence of pyridine.

   For example, the acetoxylated derivative is advantageously obtained by mixing the Ilydrgxybelizoic acid with acetic anhydride in the presence of dry pyridine and leaving the resulting reaction mixture overnight. The acetylated product can be recovered by conventional methods, in particular by treating the reaction mixture with water, acidifying the resulting solution.

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 aunt and. and) recovering the precipitated product by filtrat-ion *
The.

   second stage of the process for preparing chloru-
 EMI19.1
 3- (- Dimethylalyl) -4-acetoxybenzoyl re is easily accomplished by treating the sodium salt of the acylated product with oxalyl chloride at low temperatures in a suitable solvent such as benzene. When the reaction is complete, the precipitated sodium chloride is separated and the filtrate is. concentrated under reduced pressure, so that the acid chloride is obtained.



   Next, another, embodiment of the present
 EMI19.2
 Invention 3- (3- / 3-In6tliylbutyl7-4-acyloxyberizoylchloride (VIIIAR) is obtained in a manner similar to that described above for:
 EMI19.3
 .. 3- (', ".: -Dimethylally1") -4-acyloxybenzoyl, using 3- (3-methylbutyl) -4-hydroxybenzoic acid, (VI A) as the starting material. So, the starting material is first. acylated so as to form the corresponding acylated derivative and
 EMI19.4
 this acyl derivative is reacted with thionyl chloride, so as to produce 3- (3-m- ¯-thylbutyl) -4-a, cyloxy-belJ; zoyie (VIII AR) chloride.

   These reactions can be schematized as follows::
 EMI19.5
   . R representing an acyl group.
 EMI19.6
 



  The 2-dinth, ylcriromatl - carbotlβJ.e chloride (VIII B) used in the process described above can be prepared by reacting 2,2-dirtualiylchroman-6-carboxylate with ttÜol1yl chloride at room temperature. . After completion of the action, the mixture is concentrated under / 1.

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 pressure, reduced to obtain the desired product in solid form.
 EMI20.1
 The 3-acylatuitro-, 7-dihydroxy- -m> iNi filco alnarin used as the starting material in the reactions described above is easily prepared by reacting -la
 EMI20.2
 3-anrito, -dihydroy-mëthyleoutrtarïne with a tacylating agent, so as to produce the diacyl compound which is converted by selective hydrolysis to 3-acy: Latnino -. '7-dihydroxy-, 8-iné-tliylcownarin (BV ).

   These reactions can be schematized as follows:
 EMI20.3
 
 EMI20.4
 R representing an acyl group.



   When preparing compound V B thus, it is preferred to use esters of lower aliphatic acids for this purpose. Thus, the diacyl derivative is advantageously obtained by reacting the starting material in the presence of pyridine with an anhydride of a lower aliphatic acid having 2 to 8 carbon atoms. The diacylated derivative thus
 EMI20.5
 obtained is selected for hydrolysis by (; actl, ol1 with a solution of an alkali, such as sodium hydroxide.
 EMI20.6
 



  In the rf actioun described above, 3-ainino-4, -ditiydro: y-t.-lnethylcoarnaritle (V) and its derivatives i, -acyl (s,

 <Desc / Clms Page number 21>

 such. that the compounds V B, IX, IX. A and IX B, have been described and. shown as coumarin compounds. However, these compounds can exist and react as chromone compounds in which the positions of the 4-hydroxy group and the keto group are reversed. Thus, these compounds can be represented as follows;
 EMI21.1
 
R1 representing 'hydrogen or an acyl group.



   Novo, biocin and its salts are valuable antimicorbin agents. They can, for example, be used to remove susceptible microorganisms from pharmaceutical equipment and the like or to separate. certain microorganisms from solutions containing mixtures of various microorganisms. Novobiocin and its salts are also useful for the treatment of animals infected with microorganisms sensitive to the action of the novel antibiotic. Thus, novobiocin has been shown to be very useful in the treatment of mastitis in cows. Ointments containing 20 to 100 mg per 7.5 g of sodium novobiocin are suitable for this purpose.



   Novobiocin and its salts are active against penicillin resistant staphylococci, as well as against pneumococci and streptococci. 1 !: Since these microorganisms are the cause of most bacterial respiratory infections, novobiocin can be used for the treatment of these infections in humans. For this purpose, salt. Novobiocin sodium can be administered by mouth in capsule form containing, for example, about 100 to 500 mg of the antibiotic

 <Desc / Clms Page number 22>

 
 EMI22.1
 at a daily dose of 1 to 21 grlwiii; s.

   Ain '; i, a new aonropric dosage to be: 11 "(pure by cleaning 2i50: ,,: r of monosodirue gel in a capsule of. Latin ig. Mole 11 1 ... The llOV, 0biochie and its sets These bones can be prepared in the form of tablets. These bones can be prepared in! nel8il.; cnn.:b of In novobiocin sodium. powder;

   a small ('u [111t.Lt6 of a 5 / -de, htr: .at (cellulose acetate hydrogen d [! ns a 1: 1''lale (.iu: 50) solution of! 11 ( tI1Jt107. and acetone, etr, granulating the iiiatlùrc on a tc.au.ic 11 tI 0, drying the resulting ;; r: 'l1ules, in' %% - 11.1..SFlilt the dry granules
 EMI22.2
 
 EMI22.3
 on a n ° 1 coppice and in c01'lpr1 Dnt the resulting granules with the addition of a small C1LC ?lltlt 'of magnesium stearate, .. of' den fi. form tablets containing 50 mgr of sodium novobicoin. Novobiocin is highly effective in the treatment and control of plant diseases. Thus, it may be used to control bean niello caused by .la ', aiitlloi-toii <i ,, 4 l2! .Laseaoli.

   To this end, we spray
 EMI22.4
 se the plants using an aqueous solution containing en-
 EMI22.5
 About 11-u parts per 1n, Ll1.ioll of novobiocin sodium salt. Spray products may contain various agents
 EMI22.6
 wetting or spreading and / or other active agents
 EMI22.7
 and nr'uvent #ttrP prepared by methods well known in
 EMI22.8
 the technique.
 EMI22.9
 



  Dil1ydronovobiocinc and its salts 1 have been found to be less as active and, in most cases, more active than novobiocin towards cnsai microorganisms, particularly> lJLrr V 1.:-lV.L ; 1 d '.lI.lDrwt: llt.; 1 t) rf) (' 9 de Jlicroorr; [\ i1i: 3lneS t7a til0 ^, alt: S., Ilns.l, ln Cl.l.ilyC1rO110V?). LOC.lIIC ecib more than dt- <5t times ai> zr; 1 nctive V.;: -.-VL :. (the 'ii <.ur of these t'I: LCX OOr ;; =' 117.I11C ^ namely G. pocudotl1br'rcu ', 0: 113 5 / éj and G. psoudodiphthc1 "ificulIl 5 <J. Co) npar''ca In 110Vr) I) i.OC: .i.tlf dihydronovobiocin is very much pl, v>; active in vlvo vi.;> - 1-vi.s of infectLonn due tz;

 <Desc / Clms Page number 23>

 
 EMI23.1
 Proteus sn. 1-licrococcus nyogenes var. aureas i3mith, Streptococcus oyol; eues and Diplococcus pueuntotl: resistant r.ae ..léry; t: hramyciue.



  'Si1Uilaireme.lït cyclonovobiocin and 3-acetamido- 7- (3 = c arbawyfl - * - mnôtkyInovobio ssr loxsr) -4-hydroxy- 8-methyl.coumariiie are valuable allrtimicrobial age.nts and inhibit the growth of various mii.'croorganisms pathogens.



  The following examples illustrate embodiments of the invention.



  E1T'ïL; 1. Preparation of 3-amino-4,7-dihydroxy hydrochloride: 8-methylcoumarin (V) from 2-methylresoroinol (I) A. - Preparation: of 7-hydroxy-h-imino-8-methyl- 2-oxochroman ##. ## - # JEL ####### .- ## - # 37 gr of -; rnethylrésorc: io1 (I) (RC Shah and Ivi.C.



  Laiwalla, J. Chem. Joc. -1) 3c "d, 1828) and 34 g. Of ethyl cyaiioacetate are dissolved in 150 cc of ether. The solution cooled in an ice bath and 20 g of molten zinc chloride are added. In portions with stirring, then dry HCl gas is passed through the ice-cold mixture for 2 hours. The ice bath is removed and the mixture is allowed to come to room temperature and left to stand.
 EMI23.2
 for 2 days. The solid product 7-hydroxy-4.-arnitlo - trtéthy: L-2-oxochromane is separated by filtration, washed with a little cold water and dried in a vacuum desiccator over phosphorus pentoxide.
 EMI23.3
 



  B.- Preparation of i +, 7-dihydroxxy-h-métliy7.coumar ine (III) .a solution of 1'c p; of 7-hydroxy-4-intino-8-methyl-2-oxochroman (II) in 400 cc of 50% sulfuric acid is heated in a steam bath for 2U hours. The mixture
 EMI23.4
 is cooled. in an ice bath and the 4, '/ - d: The solid 8-hydroxymethylcoumarin is collected on a filter, washed

 <Desc / Clms Page number 24>

 with ice water and dried in a vacuum desiccator over phosphorus pentoxide.
 EMI24.1
 



  C.- Preparation of 2,4.-dioxo-7-lydro.> Y - ntthyl-3-oxitnitlochroiiiaiie 1- (IV-. ------------------- --- --------- To a suspension of 0.54 gr of 4, '? - diliydroxy-8-methylcoumarin (III) in 100 cc of water, a solution of 0.20 is added g of sodium nitrite in 5 cc of water The mixture is kept at room temperature and stirred frequently for 3 hours, during which time the cownarin dissolves slowly. The clear greenish-brown solution is filtered to remove a trace. of solid material and is then cooled in an ice bath and acidified with acetic acid.

   When crystallization is
 EMI24.2
 Completed, the 2,4L, dioko-7-hyàrôxy-8-métJthyl-3-oxiininochroinane is collected on a filter, washed with a little ice water and dried in a vacuum desiccator over phosphorus pentoxide.
 EMI24.3
 



  D.- Preparation of hydrochloride of 3-amino-4,7-dihydroxy-8methylcownaril (V) .. - A solution of 100 mg, of 2,1-d3.oxo-7.-hydroxy- $ methyl-3-oximinochroniane (1V} in 20 cc of absolute ethanol is added to a suspension of 0.5 g of prereduced palladium on Darco (10%) in 20 cc of absolute alcohol and 0.6 cc of 2.5 N HCl. The mixture is stirred with hydrogen at 25 ° C. until the calculated amount is absorbed. The catalyst is filtered off and the filtrate is evaporated to dryness under reduced pressure. - mine is recrystallized in alcohol.

 <Desc / Clms Page number 25>

 
 EMI25.1
 



  Xlu: .aPh; 2e- Preparation of 3- (Y, Y-dznuethyl.a11.y1) -r-hydroxybenL-zoic acid starting from p-hydro.ybet2ôate dtethyl.A.- Preparation of 3- {y, (-c1! IJ .n6hYlallYl} - / + - ethyl hYdrOXYbenzoa "te - ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯
A mixture of 7 g of metallic sodium cut into small pieces, 300 cc of toluene, dry and 50 g of p-hydroxy-. Ethyl benzoate is heated under reflux and stirred for a few hours. The mixture is cooled in an ice bath and stirred, while 50 g of (-dimé '.-; thylallyl bromide are added dropwise over 2 hours. The reaction mixture is stirred for 15 hours. at room temperature, then heated to 50 C for 1/2 hour.

   After filtration, to separate the sodium bromide, the toluene solution is concentrated under reduced pressure to 100 cc and extracted three times with 100 cc portions of 2.5N sodium hydroxide. The alkaline extracts are combined, cooled to 0 C and carefully acidified to pH 6 with 2N sulfuric acid. The heavy oil which separates is extracted with ether, dried over magnesium sulfate and concentrated in vacuo; weight:
42 gr. This oil is dissolved in cyclohexane and extracted with a saturated solution of sodium carbonate,
 EMI25.2
 to remove the small fraction of unreacted ethyl p-hydroxy-benzoate. The cyclohexane layer is dried over magnesium sulfate and concentrated in vacuo.

   The residue is dissolved in a mixture boiling in equal parts
 EMI25.3
 cyclohexane and petroleum ether (P.L. 3U-fi0). After cooling to room temperature for a few: hours and at 3 until the next day, the.! colorless prisms
 EMI25.4
 are received, l-1.llis on a filter; P.l ''. 62-6o '. By recrystallization in a methyl? Njge dp cyclohexane and <1 '<' petroleum tlier

 <Desc / Clms Page number 26>

 (P.E.: 30-60) 18.9 g of colorless prisms are obtained; P.F. 66-69 c.
 EMI26.1
 B.- Preparation of 3- (), 6-dimethylallyl) -4-hydroxy-benzoic acid.
A solution of 7 g of ethyl 3- (#, # -dimethylallyl) - 4-hydroxybenzoate in 30 cc of 4N sodium hydroxide is heated on a steam tank for 4 hours.



  After cooling in an ice bath, the solution
 EMI26.2
 The resulting sodium salt of 3- (f, (-dimethylallyl) - 4-hydroxybenzolque acid is acidified with dilute hydrochloric acid and extracted with ether. The combined ether extracts are washed with water, dried over magnesium sulfate, filtered and concentrated in vacuo. The residue is dissolved in hot benzene and crystallized slowly by adding cyclohexane; mp 80-84. Recrystallization from a mixture of benzene and cyclohexane gives -
 EMI26.3
 my colorless melting at '94-95 C.

   3- ((, "(-dimethyl-allyl) -4-hydroxybenzolque acid can be purified by dissolving it in a sodium carbonate solution and extracting with ether. After acidification of the ice-cold solution of carbonate. sodium bonate using hydrochloric acid, the product is collected on a filter and laced with cold water.

   By recrystallization from a mixture of methanol and water, colorless needles are obtained; M.P. 101-108 C
EXAMPLE 3. -
 EMI26.4
 Preparation of 3- (ie t -di rnethylallyl) -4-acetoxybenzoyl (VIII) chloride from 3- (, -d.methylal.lyl) 4-acetoxy benzoic acid (VII) .A.- Preparation d '3- (((, {-dimcthylallyD-4-acetoxy- ¯¯¯¯¯¯ benzoic acid (VII) .- ---------------------- ------------------------------ A solution of 2U, E gr (0,; 1. Mole) of acid 3- (ysydil "4thYlaîl-Yl) -4-ilY <IrO? ty'Jenzotque (Vil 'in 100 cc of pyri-

 <Desc / Clms Page number 27>

 
 EMI27.1
 The dry dine is treated with c 12 J S gr z 5 mol) of acetic anhydride and kept at room temperature overnight. The reaction mixture is diluted with 400 cc of ice and water and acidified with concentrated hydrochloric acid.

   The precipitated product is separated by filtration.



  The crude product is recrystallized from petro- ether.
 EMI27.2
 (P.D. 5-10U), whereby purified 3- (r, -ditnethylallyl) -4-acetoxybenzoic acid is obtained.



  B.- Preparation of chloride, 3- (, -dimethylallyD-4acetoxybenzoyie (VIII) .- ¯¯¯ ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ A suspension of 24 J g gr (U , 1 mole) of 3- (1 -diinetliylallyl) -4-acetoxybenzotque (VII) acid in 50 cc of water is treated with 40 cc of 2.5 N sodium hydroxide. The resulting solution is lyophilized. to produce a residue
 EMI27.3
 of sodium 3- (, ydintetriylallyl) -4-acetoxybetizoate. The sodium salt is added in portions, while stirring, at ice temperature, to a solution of 127 gr (1 mol])) of oxalyl chloride in 400 cc of dry benzene. After the final addition, stirring is continued for about 2 hours. The precipitated sodium chloride is separated and the filtrate is concentrated under reduced pressure.

   The residue is redissolved in benzene and reconcentrated 'to remove the last strands' of oxalyl chloride from 3 - (#,
 EMI27.4
 1 -dimethyla l lyl) -4- ao et oxybe nz oyl q, J.!; 1ilil'lPLJ 4 .....



  Preparation of 3-carbaittyl -.- nétliyhlovobiosa.-, a solution of 20 gr of novobiocin in 1 liter
 EMI27.5
 of methyl alcohol, 1U cc of hydrochloric acid (density 1.19) is added. The resulting solution is heated at reflux for about 1 1/4 hour, after which it is diluted to volume with 1 / equivalent of water and cooled. The resulting precipitate is collected and the filtrate is concentrated, to approx.

 <Desc / Clms Page number 28>

 ron 300 cc under reduced pressure. After filtration, the concentrated solution is made alkaline with sodium bicarbonate and freeze-dried.



   Residual solids are fractionated with about 300 cc of acetone and the combined acetone extracts are concentrated to about 25 cc. Methyl-
 EMI28.1
 3-carbamyl-4-methylnovobiosirl is installed in the resulting solution, and is collected and washed with acetone. After recrystallization from hot acetone, the product melts at 190-192 C.
 EMI28.2
 



  1.5 gr of methyl 3-carbamyl-4-mbthylnovobioside is heated on a steam bath in 150 cc of 0.1N hydrochloric acid for about 45 minutes, until the rota-? tion of the solution reaches a constant value. L burps (- tion of the solution observed at the end of this period is; '
 EMI28.3
 of + 0.501 in a 1 cm tube (D li; useful sodium tie} éà.1 The resulting solution is lyophilized, so as to produce
 EMI28.4
 re 3'carbamy1..4-methylnovobiose in the form of a glassy mass; / (ï 2 = "+46" (c = 1 in CH3 H) EXEPLE 5.- '
 EMI28.5
 Preparation of -acetyl-3-carbantylr-ntét! Iy.tlovobiosyl chloride from -carba.nty7.-4-ntcalnßltlovobiose.

   A.- Preparation of 1,2-aiacetyl-3-carbai, iyl-4-wetliyInovobio- se (XI). = A fraction of 2.35 gr (0.01 mole) of 3-carbamyl-,
 EMI28.6
 4-ihethylnovobiose is added to 15 cc of acetic anhydride and 12 g of sodium acetate and the mixture is heated to -10U C for about 1 lifurc. The cooled lI1r'lan:; c] rp.D.ctiollnel is diluted with 30 cc of ice and water and neutralized with sodium. the product is extracted into chloroform, which
 EMI28.7
 is washed with water ct; J <Fch <5. By liniuut.i.ott of chloroform, l, -diac6tyl-3-carbnmyl-4-mothylnovobiose (XL) is obtained.

 <Desc / Clms Page number 29>

 
 EMI29.1
 



  Pr, i-Daratioil of, 2-acetyl-3-cai? T> imyl-lp-inethylnovobiosyl (XII) .- a solution of 1, Ô gr inole) of 1,2-diacretul-3-carbamyl-r -nuthy7.uovobîose (XI) in 40 cc of anhydrous ether and ¯sat: urce. (at -, uwd.rou .Ua) a: ve.c -du i7¯rtride: d hydregene -iiiliyd-re- La - reactioil eati..waitiveune. at the temperature of the ice perWaut:. a long period. The other and the excess of hydrogen chloride are removed under reduced pressure. the last traces are removed by dissolving the residue in benzene and separating under reduced pressure, so that product XII is obtained.



   EXAMPLE 6. -
Preparation of novobiocin.
 EMI29.2
 A. - Preparation of 3- (3-r, -dimethyla7.l, y: l.7 -acétoxybenzarrüdo) -. 7-dihyd.roxy-b-methylcountarine (IX) .- A suspension of 12.1- gr (0, U5 mol) of 3-atttino - +, 7-dihydroxy -.- ntcahylcoumarin hydrochloride (V) in 100 cc of water containing 16 gr of sodium acetate is treated in portions with 13.4 gr (0.05 mole) of 3 - (#,
 EMI29.3
 0 '-dimethylallyD-4-acetoxybenzoyl (VIII) while the mixture is stirred. The mixture is stirred for about 1
 EMI29.4
 hour after final addition of acid cltl.or.ide. The mixture is acidified to -pli; -, e. Approximately with hydrochloric acid.

   The product (IX) is separated and purified by recrystallization.
 EMI29.5
 



  13.- 'lré: pirattoii-de-iiovobiocitie- (XIII) .UtW solution of 2.2 pr (0.00 mole) of 3- (3- /, dimahylall.yl 4-accaoxybett. ^ Ai: rido) - 'r,% - dihydro.y- -tnethyl- coumarin (IX) in 50 cc of purified anhydrous dioxane is stirred, while about 4 gr of anhy- calcium sulfate
 EMI29.6
 dre and 4, gr (U, U2t mole) of silver oxide are added

 <Desc / Clms Page number 30>

 
 EMI30.1
 The rttg "eß is protected against light." stirred for 1, leuJ: lt !. "" A small amount of idb is added after which mi a3-ue 'une- zoLutiàca1 der -1.9 gr - * (4,, Li) l mo: te) de . -aeëty7-3-bamyl - tnéttlylnovotsyle (.L: C) 1 in 10 cc of pure anhydrous dioxatin. The mixture 1réae; tJiollllsl is stirred for x-7 ures.

   The mixture is filtered to remove the insoluble by-products and after reducing the excess iodine with sodium besulfite the filtrate is measured under reduced pressure. The residue is hydrolyzed with two equivalents of a dilute sodium hydroxide solution
 EMI30.2
 at ambiaiite teiiipératiure. The solution is lyophilized and the residue is washed with hot methanol, which dissolves it. ser7l. monosodiqas de uovobioei. The sodium salt can be crystallized by dd3, iz.ü. of hlazene, or else the free acid can be precipitated by successive addition of acetic acid and water.



   EXAMPLE 7. -
 EMI30.3
 Preparation of 3- (3-methylbutyl) -4-hydroxybenzoic acid A solution of 3 g of 3-% (- dimethylallyl) - 4-hydroxybenzoate of ethyl in 50 cc of ethanol is hydrogenated over 0.5 gr of platinum oxide as catalyst. The theoretical amount of hydrogen is absorbed in one hour. After separation of the catalyst by filtration, the alcohol is dissolved.
 EMI30.4
 filtered under reduced pressure; The ethyl 3- (3-mehyl-butyl-1-hydro.y-benzoate) is dissolved in 20 cc of 4N sodium hydroxide and heated on steam for 4 hours.
 EMI30.5
 acidification with hydrochloric acid,> the mixture is extracted with xther. The extract is dried over magnesium sulphate, filtered and; concentrated under vacuum.

   The residue is dissolved in hot benzene and cyclohexane is a-
3- (3-methylbutyl) -4-
 EMI30.6
 , added. After cooling, the acidc / hydroxybel1zoIgue is collected on a filter. By recrystallization from a mixture

 <Desc / Clms Page number 31>

 ge of chloroform and cyclohexane, one obtains' needles. the colorless; M.p. 106-9.
 EMI31.1
 



  Analysis: Calculated for c12F11603: G = 69.20% a; x = 7.75% o Found: 0 =, 9i; H == 7.29%. EXAMPLE 8. -
 EMI31.2
 Preparation of dihydronovobiocin starting from 3- (3methylb utyl-4-hydroxyb enz o iq ue acid. A.- Preparation of 3- (3-methylbutyl) -4-acetoxybenzol- acid (VII A) .- A mixture of 10.4 gr (0.005 mole) of 3- (3-
 EMI31.3
 methylbutyl) -4-hydroxybenzoic acid (VI A) and 50 cc of pyridine is treated with 10 cc of acetic anhydride. The mixture is kept at room temperature until the following day.



  The mixture is poured into 300 cc of ice and water. The resulting mixture is acidified to a pH of about 2 with concentrated hydrochloric acid. The precipitated product is separated and purified by recrystallization from ether.
 EMI31.4
 of petroleum '(P.E.85-10).



  B.- Preparation of 3- (3-Nethylbutyl) <- 4-acetoxybenzoy7e (VITl A) .- - 'A mixture of 13 gr (0.05 mole). 3- (3-methyl- acid
 EMI31.5
 butyl) -4-acetoxybenzoic acid (VII A) and 50 cc of thionyl chloride is maintained at room temperature overnight. The solution is concentrated under reduced pressure. The residue is dissolved in 50 cc of anhydrous benzene and reconcentrated under reduced pressure, so that a residue of the desired acid chloride is obtained.
 EMI31.6
 



  C.- Preparation of 3- (3 - / '3-methylbuty] y-4-acetoxybenzamido- 4 7-dihydroxy - $ - iii (thylcownarinë (IX A). - ¯¯ ¯ a suspension of zur (0 05 mol) of hydrochloride of 3-amitlo-4, l-dilydroxy-a-uf: l, hy7.coumaritle (V) in 100 cc of water containing 16 gr of sodium acetate is treated

 <Desc / Clms Page number 32>

 in fractions with 14.3 gr (0.055 mol) of chloride
 EMI32.1
 3- (3-rttethylbutyl) -4-acetoxybenzoyl (VIII L), but the mixture is stirred. The mixture is stirred for about 1 hour after the end of the addition of acid chloride.



  The reaction mixture is acidified to pH about 2 using hydrochloric acid. The product is separated and purified by recrystallization from isopropanol.



  D. - Preparation of dihydronovobiocin (XIII A)
A solution of 2.2 gr (0.005 mol) of 3- (3-3-m-
 EMI32.2
 thy Ibuty'ß -4-acetoxy.benz all! ido '), - 4,7 -dinydroxy- 8-methy l-coumarin (IX A) in 50 cc of anhydrous purified dioxane is stirred, while the approximately 4 g of anhydrous calcium sulfate and 4.6 g (O, Q2 mol) of silver oxide are added thereto. A small amount of iodine is added, after which a solution of 2.9gr (0.01 mol) of 2-acetyl-3-carbamyl-4-methylnovobiosyl chloride (XII) in 10 cc of dioxane is added.
 EMI32.3
 pure anhydrous. The reaction mixture is held for 5 to 10 hours. The desired reaction product is recovered from the reaction mixture as described in Example 6B.



   EXAMPLE 9. Preparation of cyclonovobiocin starting from 2,2-dimethyl-
 EMI32.4
 chroman-6-carboxylate.



  A -.- Preparation of ¯212: d1lltèycrmn: 6: cro: 'le (VIIIB) chloride .-
A mixture of 10.3 gr (0.05 mol) of 2,2-dimethyl-
 EMI32.5
 chroman-6 = carboxylate (VII B) and 50 cc of thionyl chloride is kept at tpperaturo autbi7ute overnight. The mixture is concentrated under reduced pressure.
The residue is redissolved in 50 cc of anhydrous benzene and

 <Desc / Clms Page number 33>

   reconcentrated, so as to produce a desired acid chloride residue.
 EMI33.1
 



  B.- Preparation of ¯3: (, -il.!} É!: Hilh ±! .L.la!.! - saEb2xm! D2) = r, '7-dihydroxy - $ - methylcoumarin (IB) .¯ A suspension of 1.2,2: gr (, '): 105 mol) of 3-amiuo-4,7-.dihydroxy-rrcthylcounariue (V) hydrochloride in 10 ac dreaur containing 16 gr of sodium acetate. Is treated. in fractions with 11, gr (0.055 mole) of 2,2dimothylchroman-6-arbonyl chloride (VIIIB), while the mixture is stirred. The mixture is stirred for one hour after the end of the addition of acid chloride. The reaction mixture is acidified to a pH of about 2 with hydrochloric acid: The product is separated and purified by crystallization.
 EMI33.2
 G.- rPêr! T1on ēcyc1ono! ObtQcn 1XI; C B) ¯.
 EMI33.3
 



  A solution of bzz2 gr (0, UU5 mol) of 3- (2y2; -d, imethylchromatl-6-carUottrrido) -r, 7-dihydroxy-3-nethyl-coüma-, rine IX B) in. 50 cc of purified anhydrous dioxide is stirred, while about 4 g of sodium sulfate are added.
 EMI33.4
 vs ; 1.c 1 w anhydrous and 4, at gr (U, u2 mole) of silver oxide * 'A small amount of iodine is added e ,,, after which a sol.ut i.on of, 9 gr (uUl mol) of cliloride; -l1cetyl-3carbamyl-4-11ltthylnovoIÜc'S, i le (XII). In 10 cc of anhydrous dioxane nur. The reaction mixture is stirred for 5 to 10 hours. The reaction product d6s.Lré is recovered from the reaction mixture of the nleJ.a.L8re ctecribe in exeinpie 6B.
 EMI33.5
 



  Lï, .l ;, 1'LL lU. Preparation of 3-ac <'ta. : i.do-'l - (: 3-carbantyl-l.-mthy7.novobiosyloxy) -lt-hyÔroxy -,; - rn8thylcoulllnr. Read from 3-anritio-47-dirtydzoxy- -ottliylcounmriue hydrochloride.

 <Desc / Clms Page number 34>

 
 EMI34.1
 



  A.- Preparation of 3-acrStam.Ldo-J-ac0toxy ':' LI - hydro: h.7-S "mcthy7.com:ariue (V -) .- ¯ - - - - ¯ ¯. .. - ¯ A solution of 2 gr of 3-amino hydrochloride
 EMI34.2
 r, 7-dihydrbay-L-mC.thylcouoarine JV) d !! l1S 4U cc of pyridine is treated with 6 cc of acetic anhydride. The solution is kept at room temperature overnight.



  The reaction mixture is poured into about 400 cc of ice and water. The resulting mixture is acidified to a pH of about 2 with concentrated HCl and extracted with chloroform. The chloroform layer is concentrated to dryness under reduced pressure. The residual product is purified by recrystallization from ethyl acetate ..: -
 EMI34.3
 B. Preparation of 3-actamido-4., 'Ï-d: i.hydroxy-F-methylcoumarin - (VB) A solution of 10.0 ger of 3-aciftainido-7-aoetoxy-4-hydroxy-e-hlÓthylcoumarin (VA) in 120 cc of NaOH TN,, is heated at 50 for 30 minutes. The solubion is cooled and acidified to a pH of about 2, with
Concentrated HCl.

   The precipitate is filtered off and washed with water.
 EMI34.4
 



  The product is purified by recris1.a1.lsntion in dioxane.



  C. Preparation of 3-acetamido-7- (3-carba) uyl-4-methylnovobio szl.o 1) = 4 = hy <<roµyd8 = 1! IF, thylco L1wag ine ¯ (llII¯C). ¯ - l A solution of 1,: 5 gr (0, UU5 mol) of 3-acetamido-4'l-dihyJroxy-E: -nv th; lcomrarine (VB) in 5U cc of anhydrous anhydrous diox: 1n is stirred tnnc1 Ls that about 4 on calcium sulphate and 4.6 gr (U, 02 mole) of ar-
 EMI34.5
 gent are added. A ncbibe ftu: m1; LU of iodine is added nl) r (', - s what or adds a' jn1.nt.i.nn dr c, 1, <J ier (u, U7. Mole) of eye, orL1r <. Of 2¯ac tyl.-3 - cr, rl3:, myl.-r-iathyl.awob.i.osyle (, CIl) in 10 cc of pure anhydrous dioxiiin. -Ue inel¯; n; o rt'-actionu01 ...

 <Desc / Clms Page number 35>

 



  -is stirred for 5-10 hours. The desired reaction product is recovered from the reaction mixture as described in Example 6B.



    CLAIMS .-
1.- Compounds chosen from the group comprising cyclonovobiocin and 3-acetamido-7- (3-carbamyl-4-methyl.-
 EMI35.1
 novobiosyloxy) -4-hydroxy-8-methylcoumarin.



  2.- Gyclonovobiocitxe.



  3.- 3-ae6tatriido-7- (3-carbaitlyl-4-inethyltiovobiosyloxy 4'hydroxy-8-métliyieouTiiariiie 4.- Process in which 3-acylamido-4,7-dihydroxy-8-utétktylcoumnrine is reacted with a 2-acyl-3-carbamyl-4-methyl-vobiosyl chloride in the presence of silver oxide and the resulting reaction product is subjected to hydrolysis with a base, thereby producing a compound of the formula.
 EMI35.2
 in which R represents an acyl group.

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


    

Claims (1)

5.-Process in which we react EMI35.3 3- (3-C 'D' -dimcthylal.7.y-1-acylo: cybett2amido) -l7-d. 8-hydroxy-methylcou> nostril with 2-acyl-3 = carbainyl-4-inôthyInovobioyl chloride in the presence of silver oxide, and subjecting the resulting reaction product to hydrolysis by reaction with a base, so as to produce novobiocin. <Desc / Clms Page number 36> 6. - Process in which 3- (3-. EMI36.1 / 3- -dimethylall.yl% -.- acetoxybetiza; aido) -17-dihydroxy- $ -methylcountaritie with '.'-acetyl-3-carbamyl-4-methylnovobiosyl chloride in the presence of silver oxide and on subjects the resulting reaction product to hydrolysis with sodium hydroxide, thereby producing vovobiocin. 7.- Process in which 3- (3- EMI36.2 /r3-methylbutr.T-4.-acyloxybe nz atttido) -I, 7-d.hydroxy - $ - methylcoumarin with '2-acyl-3-carbahlyl-4-methyl-novobiosyl chloride in the presence of d-oxide silver and the resulting reaction product is subjected to hydrolysis with a base, so as to produce dihydronovobiocin. 8.- Process in which 3- (3- EMI36.3 L-3-methylbutyg'-4-acetoxybenzamido) -4J? -DihydrOxy-s-methylcoumarin with 2-acetyl-3-carbamyl-4-methyl-novobiosyl chloride in the presence of silver oxide, and oh submits the reaction product resulting in hydrolysis with sodium hydroxide, thereby producing dihydronovobiocin. 9. - Process in which 3- EMI36.4 (2, 2-dimethylchronian- 6- c arboxamido) -4, 7-dihydroxy- à-métliylcoumarin with 2-acetyl-3-carbamyl-4-methylnol chloride vobiosyl in the presence of silver oxide and subjecting the resulting reaction product to hydrolysis with sodium hydroxide to produce cyclonovobiocin. 10.- Process in which. we react to the 3- EMI36.5 acétnmido-4,7-dihydroxy-8-méthylcoumarin with 2-acetyl-3-carbnmyl-4-m '"thylnovobiosylo chloride in the presence of silver oxide and the resulting reaction product is subjected to hydrolysis with sodium hydroxide, ma- <Desc / Clms Page number 37> EMI37.1 tl,: Lère to produce, die "la 3-ac; é'ta) tiAdo-7-3-o; aar & arnyJL-4-mét'hno) V-oj -b'i.osy-lroxy) -4-ydrrxy -b-méthy: oumarïtte. il.- Compounds selected from the group comprising 12'-i.acyl-3-car '& amyl-4-meHiylnovobioeè and α-acyi-3-carbawyl-4-methylnovobiosyl chloride in which the, s, ubati.t, uat, its asyla son ;; a.1i, phatic carboxylic acid radicals containing no more than 8 carbon atoms. 12.- 1,2-Diacetyl-3-carbaiyl-4-inethyltiovobiosis, 13.- 2-Acetyl-3-carbariyl-4-m chloride, thyltiovobisyl. 14.- Process in which or reacts 3- EMI37.2 car'bamyl-4-methylnovobiose with Him anhydride of an acid. lower aliphatic carboxylic acid containing not more than E; carbon atoms and the resulting reaction product is reacted with hydrogen chloride, eventually to produce 2-acyl-3-carbamyl-4-! nethylnovpbiosyl chloride. . 15.- Process in which the'3-car- EMI37.3 bamyl-4-ethylnovobiose with acetic anhydride, and the resulting reaction product is reacted with hydrogen chloride, so as to produce 2-acetyl-3-carbamyl-4-methylnovobiosyl chloride. 16.- Process in which 1,2- is reacted EMI37.4 dicetyl-3-carbamyl-4-methylnovobioso with hv-drogen chloride so as to produce 2-acetyl-43- chloride EMI37.5 carbamyl-4-methylnovobiosyl. 17 .'- Compounds of formula: EMI37.6 <Desc / Clms Page number 38> EMI38.1 in which 1 is the alipàiatiqNe diacid radical itlié-, rieur rae coe% carrying p, loes of g atoms of carbotze or of 1 hydra to 10.- 3-antra..4, 7-dikiydraxy - methylccummrin hydrochloride 19 .. - 3-Aeeta> àdo-7-acetoxy-4-hydroxy-8-nethylaoL1> marine. 20. - 3-Altaetainiào-4, 7-aliydroxy-E-methylc ownarine. 21. - Process in which 2-methyl- 'is reacted EMI38.2 resorcinol with an ester of cyanoacetic acid under substantially anhydrous conditions in the presence of zinc chloride and hydrogen chloride, so as to produce. of EMI38.3 7-hydroxy-I-3.nt.to-g-methyl -? - oxochromane the compound is heated with sulfuric acid to 50, & so as to produce 1r, 7-d3.hydroxy-â-tttéthy .coumar3.ne this coinposed is treated with nitrous acid, so as to produce 2, tua..diaxa-7-hydroxy-B-methyl-3-oximinachramatle and this compound is reduced by reaction with hydrogen in the presence of a noble metal catalyst and hydrochloric acid in a manner EMI38.4 re to produce 3-amïtxo-4, 7-dihydroxy-b-methylcoumarin hydrochloride. 22. A process according to claim 21, wherein the noble metal catalyst is palladium on charcoal. EMI38.5 23.- 2,4-D3.oxo-7-hydroxy-8-methyl-3-oximino-chroman. EMI38.6 24. - 4,7-Dihydroxy-S-methylcoumarltie. 25.- 7-Cydroxy-t + .- irri3.no-t-rnt thyl-'2-ôxochromane. 260L Compounds of roup ('? Comprising 3- (i, dimethylallyl.) - 4-acyloxybeiizoic acid, 3- (r, dimethyla: Lly1) -r-acyloxybenzoyl acid 3- (j-inêtiiylbutyl) - 4-acyloxybenzoic and 3- (3-nethylbutvl) -li- chloride <Desc / Clms Page number 39> acyloxybenzoyl. EMI39.1 27.- 3- ('I -dintethylallyl) -4.-acetoxybenzol acid. 28. - 3- ('-dimethylallyl) -4.-acetoxybetizoyl chloride. 29.- 3- (3-Methylbutyl) -4-acetoxybenzolque * acid 30.- 3- (3-methylbuyl) -4-acetoxybenzoyl chloride 31.- Process in which 3- ( '' -dimethy.allyl) -4-hydroxybenzoic acid with an acylating agent, and on. treats a salt of the resulting acylated reaction product with oxyalyl chloride, so that EMI39.2 producing 3- ((µ, j -diwetliylallyl) -4-alkyloxy-bezoyl chloride. 32.- Process in which acid is reacted EMI39.3 , 3- (a '-dirnethyla11y1) -4-hydroxybeuzoic with acetic anhydride in the presence of pyridine, and the sodium salt of the resulting acetylated reaction product is treated with oxalyl chloride to produce 'chloride EMI39.4 3 - ('! F Ô -dimethylallyl) -4-aletoxybenzoy $ ee 33.- Process in which acid is reacted EMI39.5 3- (3-Niethylbutyl) -4-hydroxybetizoic with an acylating agent, and the resulting acylated reaction product is treated with thionyl chloride so as to produce EMI39.6 3- (3-iiiethylibutyl) chloride -4 * -âcy'loxy-beiizoyles 34.- Process in which we make. react acid EMI39.7 3- (3-mE'thylbutyl) - + - acetoxybetlzoiquo with acetic anhydride in the presence of pyridine and the resulting acylated product is treated with thionyl chloride, so as to EMI39.8 to produce 3- (3-rru: thylbul, yl) -! + - acetoxybeuzoyl chloride, 35.- Compounds selected from the group consisting of "" .'T ..dirrtethylal.ly 4-acyl.oxybonzanido) -4. , 7-dihy., Droxy-8-methylcoumaritie, 8 1e 3- (: iL 3-rnctliy.batyl% -! .- acylaytyßM; f ,, <Desc / Clms Page number 40> EMI40.1 beuzamido) -4, '- dihydroxy-s-nrrt; nyicouriar: Ltie and 3- (2,2-do.methylclvroman - carboxarrr: i.do) -r, 7-dihydro.iy - méthyicoumarit 36.- .3- (3-L-1 'jf-dimethylallyTy-4-acetoxybenzamidd -., 7-dihydroxy-fi-nutttylcournarin. 37. - 3- (3 - / j-inethylb ut y J- 4- ac et oxybe nz amido, 4,7-dihydroxy-ë-methylc oumarine. $ 3. - 3- (z, z-dimethylchrornan-b-carboxamido-., 7-dihydroxy-8-methylcomnarine. EMI40.2 39.- Process in which hydrochloride is reacted EMI40.3 , .3-amino-4,7-dihydroxy-ù-Htethylcoumarin drate with.a EMI40.4 compound selected from the group consisting of 3- chloride EMI40.5 ('-dim4thyla.lyJ-t-acyl.oxybenzoyl, 3- 3 -ine thylbutyl chloride) q 4- acyloxyabe nz o y le and 2,2dimethylchroman-6-carbonyl chloride in the presence of sodium acetate, EMI40.6 so as to produce the corresponding carboxamido compound. EMI40.7 .0.- A process in which 3-amino-4,7-dihydroxy-8-ttrethylcotzmarine hydrochloride is reacted with 3- ((1, i -dillethylallyl} -4-acetoXYbenzoYl 'chloride). EMI40.8 '' presence of sodium acetate, so as to produce EMI40.9 3- (3 - / '- dimethyl.al.ly.-Acetbxybenzantido) -lr, -dihydroxy- EMI40.10 EMI40.11 8-i-Nethylcouinariiie. 41.- A process in which 3-arnino -., L-dihydroty-5-rncâthy: Lcoumarin hydrochloride is reacted with 3- (3-nietyylbL1tyl) -4LaceEoxybenzoyl chloride in the presence of sodium acetate, you plan to produce 3 -.! r3-L-3methylbutyl-1-acetoxybenzamido) -., i-dihydroxy- $ -mcthylcou- EMI40.12 Marine. EMI40.13 42.- A process in which chlor- (3-alnitl0-a-, 1-diltydroxy-F> -methyxcoumarin hydrate is reacted with 2, ¯dirrfahylcttromau-u-carbonyl.e chloride in the presence of sodium bcctate, so as to produce 3- (2,2-dim6thyl-. chroman-6-carboxamido) -H, -dihydroxy-a-methylcoumarin.