PT95187B - PROCESS FOR THE PREPARATION OF CHEMICAL DERIVATIVES OF ANTIBIOTICS LL-E19020 ALPHA AND BETA - Google Patents

Abstract

The invention relates to a process for the preparation of derivatives of antibiotics, these antibiotics being obtained from the microorganism Streptomyces lydicus subspecies tanzanius and known as antibiotics LL-E19020 alpha and beta, being derived by chemical reaction. The derivatives are also active antibiotics useful in the treatment of bacterial infections. The process consists for example in dissolving LL-E19020 α or β in acetonitrile, reacting it with ammonium hydroxide, diluting with water, acidifying with hydrochloric acid, followed by extraction in ethyl acetate, evaporation and chromatography, in order to obtain the LL-E19020 α and β ammonolysis product.

Description

An object of the present invention is to provide chemo derivatives of antibiotics LL - E19020 a and L..L-E1.9020 | 3, which also have activity as antibacterial agents.

SUMMARY ...... DO ...... INVENTION

Accordingly, this invention relates to chemically prepared derivatives of LL-E19020 os e | 5, which are also antibaoterian agents and which are described below through their structures and their physical / chemical characteristics, together with the methods for your preparations.

Since., The names have not yet been exposed with these l

structures in Chemical Abstract, they will be referred to as derivatives of I .... L ..- E1.9020 a and fi, throughout the balance of this text.

í ± 2duto ..... de ^ Arrignõlise ..... de ._. LL-E19020 .... íK ..... e ...... β

LN arnonolysis product. -E19020 «and | â have an absorption spectrum in the ultraviolet as indicated in Figure I; an infrared absorption spectrum as shown in Figure II; a proton magnetic resonance spectrum as shown in Figure III; and a carbon magnetic resonance spectrum .....

as shown in Figure IV, arnonolysis product of LL ..... Ε1Θ020 ct and β is prepared by dissolving LL-E19020 «and β in acetonitrile, ammonium hydroxide reaction for 12-24 hours, water dilution and acidification with hydrochloric acid at pH 1 .. This solution is extracted .....

taken in ethyl acetate, evaporated and then purified by r

É®ers ...... of. „JM ^ ilo„. „De„ J ^ LzE19020 .. „« .... je ..... È

Re

H

Methyl / Ester of

L.L-E19020 «

Rl

II

CHg-CMethyl / L1.-E19Q2Q Ester | 3>

LL ~ -EX9O2O | 3 methyl ester has a proton magnetic resonance spectrum as shown in Figure V.

The methyl esters of LL ..... E19020 új and | 5 are prepared by dissolution or of LJ ....- E19020 a or. | 3 in a solvent such as ethyl acetate, acetonitrile or dichloromethane, or mixtures thereof, followed by reaction with ethereal diazomethane and purification by t ::: r o rn t t o g r a f i a ..

Memjsaoaricteos ...... dos ...... It's ^ engs „j :: le„ ,,, M ^^ ...... a ...... and ...... β

LL Methyl Ester Monosaccharide ...... E19O2Ooí

LI ...-'- E19020pi ^R E Methyl Ester Monosaccharide

H

II <õ> '

CH _Z -CCl monosaccharide of the methyl ester of I ... L ...... £ 19020 «has an infrared spectrum as indicated in Figure VI; a proton magnetic resonance spectrum as shown in Figure VII;

a carbon magnetic resonance spectrum ..... 13 as shown in Figure VIII ..

Clononosaccharide of the methyl esters of 1 .... 1 ....- 1119020 «and β are prepared by dissolving the methyl ester of LI ........ E19020 a and b in methanol, cooling to 0 ^<J C and reaction with chloric acid .....

diluted in methanol for several hours, followed by dilution with water, extraction in ethyl acetate and purification by chromatography ..

AaMeeaâ ... j: ÈÍ ^ ..... Methyl ...... of ..... LLsE19020 .... j «... and ..... £

H

Methyl Ester Aglycone d <; LL - E19020 «

Methyl Ester Aglycone

LL ~ E1902.0 | 5 ^R 1

H

II

II

CHj-C ·

The methyl ester aglycone of LL ...... E19020 'has an absorption spectrum in the ultraviolet as indicated in Figure IX :; an infrared absorption spectrum as shown in Figure X; a proton magnetic resonance spectrum as shown in Figurei XI :; and a spectrum of carbon-13 nuclear magnetic resonance as indicated in Figure XII „

The LI ....- E19020p methyl ester aglycone has an ultraviolet absorption spectrum as shown in Figure XIII; an infrared absorption spectrum as shown in Figure XIV :; a proton magnetic resonance spectrum as shown in Figure XV :; a carbon magnetic resonance spectrum ..... 13 as indicated in. Figure XVI ..

The aglycone of the methyl esters of U ...- E1.9020 <.t and β are prepared by reacting the methyl ester of LL-E19020 a and β with hydrochloric acid in methanol, at reduced temperature, for several hours, followed by extraction in ethyl acetate and chromatographic purification.

Triaeetato ...... laugh ...... AgIiçgaa j ;; kL..Êste «„ ... ÉL..Ê

L_L .. Methyl Aglycone Triaeetate ~ E19020as ister of <õ>

II

L.g-E19020I3 Methyl Ester Aglycone CHg-CTriaeetate of LL methyl ester aglycone. ·

..... E19020a has an absorption spectrum in the infrared as indicated in Figure XVII; a proton magnetic resonance spectrum as shown in Figure XVIII; a resonance spectrum .....

nuclear magnetic carbon-13 as indicated in Figure XIX.

LL methyl ester aglycone triaeetate .....

-Έ19020Ι3 has a proton magnetic resonance spectrum as shown in Figure XX; and a spectrum of nuclear magnetic resonance of carborio-13 as shown in Figure XXI.

LL methyl ester aglycone triaeetate ......

..... E19020 a & | 3 is prepared by reacting the methyl ester aglycone of LL ...... E19020 «and β with a solution of 4-dimethylamino-pyri .....

dine and acetic anhydride for several hours, followed by purification by chromatography.

above and other matters of the

DESCRIPTION OF THE GRAPHICS

The invention subjects will be better understood from the following detailed description, when considered with reference to the graphics .....

in the appendix mentioned above, where:

Fig. I is an ultraviolet absorption spectrum of the product of arnonolysis in alcoholic, acidic and basic solutions;

Fig. II is an infrared absorption spectrum of the LN arnonolysis product ..... E19O2OI3, dispersed in potassium bromide;

Fig. III is a proton magnetic resonance spectrum of the

product of arnonolysis; Fig. IV and a register of resonance spectrum magnetic nuclear of carbon ..... 13 of arnonolysis product of L L.-E19020I3; Fig .. V is a L methyl spectrum , L- ~ E19020 | 3; in protonic resonance of the ester of

Fig. VI is an absorption spectrum in the infrared of the

rnonosacarideo of estér potassium bromide; in methyl of LL - E19020 <x, dispersed in Fig »VII is a record of spectrum protonic magnetic of ester rnonosaccharide in methyl of LL-E19O2O «;

Fig. VIII is a record of the carbon-13 magnetic resonance spectrum of the ester monosaccharide (methyl

- • £ 19020 «

Fig. IX is an ultraviolet absorption spectrum of the L..L ..... E19020 «methyl ester aglycone;

Fig. X is an infrared absorption spectrum of the L.L. ~ E19020 <x methyl ester in potassium bromide;

Fig. XI is a recording of the proton magnetic resonance spectrum of the LI ™ methyl ester aglioone ~ E19020 <8;

Fig. XII is a carbon-13 nuclear magnetic resonance spectrum of the LL methyl ester aglioone - E19020 »;

Fig. XIII is an ultraviolet absorption spectrum of the l .... L.-E19020 | â methyl ester aglioone;

Fig. XIV is the infrared absorption spectrum of 1_Ι_ ~ Ε19020β methyl ester aglioone, in potassium bromide;

Fig. XV is the proton magnetic resonance spectrum of the LL methyl ester aglioone — E19020 | â;

Fig. XVI is the carbon-13 spectrum of the LL-E19020 methyl ester nuclear magnetic resonance aglioone (â;

in

Fig. XVII is the infrared absorption spectrum of LL-E19020 'methyl ester aglioone triacetate, in potassium bromide;

Fig. XVIII is the proton magnetic resonance spectrum of the LL-E19020 «methyl ester aglycone triacetate;

Fig. XIX is the carbon-13 nuclear magnetic resonance spectrum of the LL methyl ester aglycone triacetate ..... E1.9020 «:;

Fig., XX is the proton magnetic resonance spectrum of the

t r 'i a c et a t o d a aglycone of methyl ester of 1 .... 1 ...-- 1 E19020 (i; Fig .. XXI is the spectrum in MRI nuclear d carbon ..... 13 of aglycone of LL-I methyl ester Ε19020β „

DISCOVERIES ... „BS ...... invention

The in vitro antibacterial activity of these compounds was determined against a spectrum of gram positive and gram negative bacteria and anaerobes using an agar dilution method. Mueller agar ..... Hinton containing 5% sheep blood or Wilkins Chalgren agar and twice decreased concentrations of the test compounds were poured into preti dishes. The agar surfaces were inoculated with 1 to 5 x 10 6 colony-forming units of bacteria by means of the Steers replication device. The lowest concentration of antibiotic that inhibited the growth of a strain of bacteria, after 1 hours of incubation, was recorded as the minimum inhibition concentration (MIC) for that strain. The results are given in Ou adro I.

Due to the extension of the nomenclature, the compounds in Table I were coded as follows:

Compound (I.) ;: Ammonolysis product of L..L-E19020 a and β ..

Compound (II) :: LL methyl ester ..... E19020a.

Compound (III): Methyl ester of Ι .... Ι ...- · ΈΧ9020β ..

Compound (IV): LL Methyl Ester Mono ... ... E19020a »

Compound (V): osa .... Ι ....- Ε19020β „Compound (VI); Methyl Ester monosaoaride: LL.-E.19020 & Methyl Ester aggloria.

Compound (VII) :: Ester Aglycone Triacetate (Methyl

Ι .... Ι ... · -Ε19020β „

QuadroI

An l, i I> uçteriana_ In Vitr o Activity of Derivatives of

.................................................. ..... Ll -II 9020 ael ~ ......... .............................. .......... ............

Minimum Inhibition Concentration (mcg / rnl)

Compound Body

I II III IV V SAW S t r e ρ t o o o c o u s b ..... hemolyticus ç : 203 8 0.12 2. 4 8 > 128 S1 r e p t o c o c c li s b ..... h e m o ly t i c u s VGI-I 94--60 4 0, 5 2 2 8 > 128 Str ^ tococcn.t © b ..... hemolyticus VGH 84-61 16 2 8 8 32 > 128 S t r e p t o o o c u s fo ..... hemolyticus VGH 84-62 16 2 8 8 32 > 128 S t r e ρ t o co c c us pneumonia © SV-1 64 4 8 16 32 > 128 81 r e ρ t o o c c u s pneurnoniae K 84-21 > 128 8 32 1.6 64 > 128 Ba cberoides vulgaris ATCC 29327 128 128 16 > 128 > 128 > 128 C 1 (istr idium pertringens ATCC 13124 > 128 64 > 128 > 128 > 128 > 1.28 C In 4 r 1111 uiri d ι f ι <ι 11 ATCC 17858 4 2 8 8 64 NT P ep t o o o o c u s magnus ATCC 29328 0.25 <0.06 <0.06 <0.06 <0.06 0, 5 EêeLLoçooclls magnus ATCC 14956 <0.06 <0.06 <0.06 <0.06 0, 06 0, 5 Peptococcus asaccharolyticusATCC 29743 0.25 <0.06 <0.06 <0.06 <0.06 0, 5

NT— Not Tested

In therapeutic use, the compounds of this invention find use in suppressing hacterian infections and in general as disinfectants for laboratories. Thus, they can be administered in the form of pharmaceutical or veterinary compositions .....

conventional materials, depending on the intended use. These cc:> compositions can be formulated to be suitable for oral, parenteral, or topical administration. The active compounds can be combined in admixture with a pharmaceutically acceptable support, where support can take a number of forms, depending on the form of preparation desired for administration, i.e. , oral, parenteral or topical ..

The invention will be further described by the following non-limiting examples.

Example ..... 1

ELQduto ...... Ete ..... An> 51Eolysis. ,,., D ^ .JU _; ^ E1902 () hi

A 9.69 g portion of LL-E19020a was dissolved in 260 ml of acetonitrile and 126 ml of ammonium hydroxide was added. After 14 hours, the mixture was diluted with 1 liter of water and the acidified aqueous was separated and ethyl acetate. PH 1 with 8N hydrochloric acid „The portion extracted with three 600 ml portions of extracts was combined, washed with

200 ml of saturated sodium chloride solution, dried and then evaporated. The 9.0 g of the residue was dissolved in 20 ml of methanol and injected on a Waters Prep 500 column, which was equilibrated with 3 liters of 32% acetonitrile in 0, ΊΝ, pll 4, 5 ammonium. The column was eluted with the same solvent system at a rate of 100 ml per minute, with the collection of fractions of 300 ml. After fraction 55, the elution solvent was substituted for 35% acetonitrile in 0, IN ammonium acetate,

1.6 - · pH 4, 5 and elution continued with a total of 90 fractions. The frac .....

Sections 70 ..... 79 were combined, extracted with ethyl acetate and the extract was dried and concentrated. The 1.5 g of the residue was purified by CL.AR, eluting with ammonium acetate 0.11 * 1, p -1.4.5 at a flow rate of SO ml per minute. Fractions of 300 ml each were collected. Fractions 48 ... 56 were combined and evaporated, yielding 818 mg of desired product.

F.XÊmEls ....- 2

-)

A 20 g portion of L.L- ~ E190201 was dissolved in a mixture of 1.00 ml of dichloromethane and 100 ml of acetonitrile. A 75 ml serving. of cold ethereal diazornethane was added. After 1.5 m :: i. the total reaction was ormatographed on silica gel, eluting with ethyl acetate. Fractions 1.-6 were 250 ml each. Fractions 7-16 were 500 ml each. The fractions 5 1.1.

were combined and evaporated, yielding 12.5 g of desired product.

J Ex ™ _p3o 3

...... £ ie ...- kLzE16020i5

A 30 g portion of IJ ....- E19020fi was mixed in 200 ml of ethyl acetate to form a sludge. Diazornethane was added in portions until the reaction was complete. 0 mate .....

material was then filtered and evaporated. The residue was dissolved in 40 ml. ethyl acetate and purified by chromatography, elution with 70% ethyl acetate in hexane, after 1.5 liters o

eluent was in a step gradient from 80% to 90% to .100% ethyl acetate. fractions 27-54 were combined and evaporated, yielding 12.0 g of desired product.

Example. ,,. 4

Monosaccharide ..... of ..... Ester _ „de ..... Methyl ..... de ..... Lkz £ lQQ2Qa

Urn of CaO of 5, 582 g of the methyl ester of LL - E1.9020a was dissolved in 75 ml of methanol and cooled to 0 ^U C A portion of 15 ml of 5% hydrochloric acid in methanol was added and the mixture was stirred for 5 hours. The mixture was added to 200 ml of water and extracted with three 100 ml portions of ethyl acetate .. The extracts were combined and lyophilized to give 4.97 g of solid. This solid was purified by chromatography, eluting with the buffered acid: acetonitrile (7: 3) system. Fractions 67-70 were combined, concentrating .....

dissolved in t-butanol and lyophilized ©, yielding 1.37 g of the product and j to d.,

Example ..... 5

...... {feJJLzElâfigQfè

A 4.7 g portion of LL-E1.9020b methyl ester was dissolved in 50 ml of methanol and cooled to 0 ° C. A 25 ml portion of 3% hydrochloric acid in methanol was added ... ..

and the reaction was stirred for 5 hours. The reaction was added to 1.00 ml of water and 1.00 ml. ethyl acetate .. washed; with 25 ml of brine extracted with three; portions of

Extracts; were combined, concentrated, dried and purified;

chromatography, elution with acetonitrile / buffer. The appropriate fractions were combined and evaporated, yielding 0.8 g of the desired product,

Êxernglg ...... ê

Agliçc; »na ._... do ..... Ester ._„ de ..... Metils ...... de ...... U ^ zElâfiSÔê

A 20.6 g portion of LL-E19020 & methyl ester was dissolved in 150 ml of methanol, cooled to about 0 ° C and 50 ml of 5% hydrochloric acid in methanol was added. The reaction was stirred for 4 hours. hours, then added to 500 ml of water and extracted with three 250 ml portions of ethyl acetate. The extracts were combined and lyophilized, yielding 1.8, 06 g of crude product. A 1 g portion of this product was purified by inverted phase liquid chromatography, elution of 25 ml fractions with 50% acetonitrile in ammonium acetate, pH 4.5 at a rate of 10 ml. per minute,. Fractions 51 ....... 71 were combined and evaporated, yielding 230 µg of desired product.

ExeniglQ. „7

XLÍã £ êfeãtg..jla_jAglicgna ...... dg ..... Ester „.de ...„ Methyl .... of ...... ΕΕ-ΕΧ9020β

A 200 ml portion of 4-dimethylaminopyridine was dissolved in 100 ml of dichloromethane, with stirring. A 2 ml portion of acetic anhydride was then added and after 5 minutes, 1.5 g of de methyl ester aglycone. ._Ι „-Ε19020β was added, with stirring. After 2 hours, 100 mg of 4 - dimethyl .....

aminopyridine and 1. ml of acetic anhydride were added.

After 4 hours, 150 mg of 4-direthylaminopyridine and 1 ml were added. After stirring overnight, the reaction was chromatographed on silica gel, eluting with hexane: ethyl acetate (7 :: 3). Fractions 13-19 were combined and evaporated, yielding 1.34 g of desired product.

Claims (4)

CLAIMS wool .. Process for the preparation of the ammonolysis product of LL-E19020 a and b, which presents; a) the structure b) a characteristic ultraviolet absorption spectrum, as shown in Figure I ~ 21 VIONVAUOSaV WAVE LENGTH (nm) FIG. I 2.2 c) a characteristic infrared absorption spectrum, as indicated in Figure II; the o I'm the O OO O O o the o CN O O Ν ’ O O CO O O oo O o oo IN o O CN ro o co ro § §% ωθ VIONÍIIWSNVHI <! the s O W Q FIG.U d) a proton nuclear magnetic resonance spectrum, as indicated in Figure III; and e) a characteristic nuclear magnetic resonance spectrum, as indicated in Figure IV; carbon FIG. ET 7s characterized by dissolving LL ..... E19020 α or β in acetonitrile ..... it, reacting with ammonium hydroxide, diluting with water, acidifying with hydrochloric acid, followed by extraction with ethyl acetate, evaporation and chromatography. 23. Process for the preparation of LL.-EI902.0os and LL · ΈΧ9020β> methyl ester, which features: a) the structures J --- - ---------- OH where, in the case of LL. E19020®, R..j is benzylcarbonyl and R ^ is hydrogen and in the case of Ll EX9020b, R .. | is hydrogen and R ^ is benzylcarbonyl, and Ι .... Ι .... ····· ΚΊ9020ί5 has a characteristic nuclear magnetic resonance spectrum, as shown in Figure V; > -σ> characterized in that the LL.-E19020 as or b is dissolved in a solvent, such as ethyl acetate, acetonitrile or dichloromethane, or mixtures thereof, followed by reaction with ethereal diazomelane and chromatographic purification 3sL Process for the preparation of monosaccharides from the methyl esters of LL- ~ E19020úí and L..L ...... El £) 020b, which present ..... sit :: a) the structures J in which, in the case of hydrogen and benz i 1 carboni I. o, of the I .... L ..... E19020®, R..j is benzylcarbonyl and the case of the I .... L ..- E19020b, Rj is hydrogen and having LL .... £ 19020 «:: b) an absorption spectrum in the characteristic infrared, as indicated in Figure VI; % ωθ VIONyiIWSNVUI FIG. SAW c) a characteristic proton nuclear magnetic resonance spectrum, as shown in Figure VII .; O. The. -O FIG.VH -oo & cl) a cl spectrum as shown in Figure VIII carbon-13,% O. CL FIG.Sm characterized by dissolving the methyl ester of LL-E19020 «or β in methanol, cooling and reacting with chloric acid ..... followed by dilution with water, extraction with ethyl acetate and purification by chromatography. 4th. Process for the preparation of aglycone of the methyl esters of LLE19020 «and 1.1.“ Ε19020β, which present: a) the structures 0K _t OH «al 1« Lji «1 ^m * J h och ₃ u 1 l ~ OH v · r x / X / i''q 0 0 ern that, in the case of LL. ”E19020«, R..j is b e η z i 1.c a r b o n i 1 o e is hydrogen, and in the case of Ι ... Ι.-Ε19020β, benzylcarbonyl, having LL ...... £ 190206 «: R „j is hydrogen and ^R 2 is b) an absorption spectrum in the characteristic ultraviolet, as indicated in Figure IX :; VIONJAUOSaV 200 300 400 500 600 700 WAVE LENGTH (nm) FIG. IX c) a characteristic infrared absorption spectrum, as shown in Figure X :; % ωθ VI3OIIWSNVHI WAVE NUMBER FIG.X * '34 d) a characteristic nuclear magnetic resonance spectrum, as indicated in Figure XI; protonic 1-00 and e) a carbon nuclear magnetic resonance spectrum ..... 13, as shown in Figure XII :: and tendi: :) LL. ~ EL9O2O | 2> :: b) an absorption spectrum in the characteristic ultraviolet, as indicated in Figure XIII; VIONJfAHOSaV 200 250 300 350 400 450 500 WAVE LENGTH (nm) FIG.Xm c) an absorption spectrum not as indicated in Figure XIV; infrared caracteristic the cO O o the o the o O CM o the o (The o the o the o ts ts o o oo es o o es ro o o to n % ωθ VIONJIIWSNVHI «there the s The ω to w P> s: d) a characteristic proton nuclear magnetic resonance spectrum, as indicated in Figure XV :; * - σ> FIG. XX and e) a carbon nuclear magnetic resonance spectrum ..... 13 characteristic, as indicated by Figure XVI. :: O Lo CM characterized by the · reaction of the methyl ester of LL-E19020 «cu β with hydrochloric acid in methanol at reduced temperature, followed by extraction in ethyl acetate and purification by means of r 53 .. Process for the preparation of triacetates of LL - E19020a and cie LL ..... Ε19020β aglycone methyl esters, which present: a) the structures 0 «i OCOCHj OCH, OCOCHj _Λ * 4 “Ί ^H w ^^ χ ^οεΗ ' | J T 0 1st on what. in the case of LL-111.9020 «, ^R 1 it's benzyl .carbonyl and Rp is acetyl and in the case of LL ~ EX9020 | â, 5 it's acetyl and R ^ is benzylcar- bon ilo, having the l .... l .. „-E19020« - b) urn absorption spectrum in the in infrared. as indicated in Figure XVII; % ωθ VIONyiiwSNVHI c) a proton nuclear magnetic resonance spectrum, indicated via Figure XVIII; how - <M -ro PPM FIG.XSm -m -ço ^L cn and d) a nuclear magnetic resonance spectrum of the horn indicated in Figure XXX; carbon ..... 13, FIG. XIX / 44 and having the LL-E.19020fÍ :: b) a proton nuclear magnetic resonance spectrum, shown in Figure XX :; how FIG.ZZ * -00 * · and c) an ES as a nuclear magnetic resonance specimen as shown in Figure XXI r. carbon ..... 1.3 * r ¹ characterized by the reaction of the methyl ester agglomeration of LL-E19020 α or β with a solution of 4-dimethylaminopyridine and acetic anhydride in dichloromethane for several hours followed by purification by chromatography. 63 .. Method of treating bacterial infections in warm-blooded animals, characterized in that it comprises administering to said animals an antibacteriologically effective amount of a compound selected from those in claims 1 to 5, the dosage range of the active compound being 2 mg to 100 mg per kilogram of body weight per day .. 73 .. Process for the preparation of a composition, characterized in that an antibacteriologically effective amount of a compound selected from those of claims 1 to 5 is included in said composition, in association with a support for r and c and u t i a a rn and n t and a e.