CS235575B1

CS235575B1 - Metabolite of a-withaferine

Info

Publication number: CS235575B1
Application number: CS606983A
Authority: CS
Inventors: Jan Fuska; Alzbeta Khandlova; Alzbeta Fuskova; Jozef Prousek
Original assignee: Jan Fuska; Alzbeta Khandlova; Alzbeta Fuskova; Jozef Prousek
Priority date: 1983-08-19
Filing date: 1983-08-19
Publication date: 1985-05-15

Abstract

Vynález sa týká nového metabolitu získaného biotransformáciou withaferinu A. Podstata metabolitu podía vynálezu spočívá v tom, že je pripravitelný přidáním 10 až 100 ml withaferinu A alebo withaferinu A diacetátu k 100 ml média naočkovaného kulturou Rothrobacter simplex ATCC 6946 a kultivovaného pri teplote 25 až 32 °C počas 26 až 120 hodin. Metabolit podía vynálezu v pokusoch in vitro inhibuje biochemické reakeie a rast buniek P 388 a v pokusoch in vivo inhibuje rast nádorov.The invention relates to a novel metabolite obtained biotransformation withaferin A. The nature of the metabolite of the invention resides in that it can be prepared by adding 10 up to 100 ml withaferrin A or withaferrin And diacetate to 100 ml inoculated medium culture of Rothrobacter simplex ATCC 6946 and cultured at 25 to 32 ° C during 26 to 120 hours. The metabolite of the invention in experiments in vitro inhibits biochemical reactions and growth P 388 cells and inhibits in vivo experiments tumor growth.

Description

Vynález sa týká meíabolitu wiihaf urinu A inhibujúceho rast nádorov.The invention relates to the tumor growth inhibiting metabolite of urafin A.

Withaferin A je prírodný metabolit, ktorý radíme medzi steroldné laktóny s inhibičnými účinkami na rQzne nádorové modely. Látka bola izolovaná z Withania somnifera Yardenom a Laviem (J. Chem. Soc,, 2925, 1962), z Asnistus arborescens Kupchanom a spolupracovníkmi (J. Amer. Chem. Soc. 87, 5805, 1965), z Physalis viscosa Pelletiercm a spolupracovníkmi (J. Natural Products 15, 512, 1979) a z Acnistus brevifloresus Griseb., Bukovitsom a Grosom [Phytochemlstry 18, 1237, 1979). Pódia Shohatu a spolupracovnikov (Z. Krebsforsch. S3, 97, 1973; Int. J. Cancer 5, 244, 1970) inhibuje rast nádorových buniek in vitro aj in vivo. Niektoré mikroorganizmy biotransformujú withaferin A přidaný do kultivačného média na nové metabolity. J. P. Rosazza a spolupracovníci (Sterpids 31, 676, 1978) izolovali nový metabolit vznikajúci z withaferinu A pomocou Cunninghamella elegans (NRRL 1393), ktorý identifikovali ako láx-hydroxywithaferin A. J. Fuksa a spolupracovníci (Steroids 35, 157, 1982) izolovali z filtrátu uvedenej kultury dva nové metabolity vznikajúce biotransformáciou z withaferinu A., Majoritný metabolit bol identifikovaný ako 12,ú-hydroxywithaferin A a autoři dokázali, že je identický s prv izolovaným 14z-hydroxywithaferinom A, u ktorého bola prv mylné stanovená, štruktúra. Druhý metabolit bol identifikovaný ako 15/j-hydroxywithaferin A (Stertíids 35, 157, 1982). Obidva metabolity inhibovali rast sarkomu 180 a lympholeukémie P388.Withaferin A is a natural metabolite that ranks among sterile lactones with inhibitory effects on various tumor models. The substance was isolated from Withania somnifer Yarden and Lavie (J. Chem. Soc., 2925, 1962), Asnistus arborescens Kupchan and coworkers (J. Amer. Chem. Soc. 87, 5805, 1965), Physalis viscosa Pelletier and coworkers. (J. Natural Products 15, 512, 1979) and Acnistus brevifloresus Griseb., Bukovits and Gros [Phytochemlstry 18, 1237, 1979]. The stage of Shohat et al. (Z. Krebsforsch. S3, 97, 1973; Int. J. Cancer 5, 244, 1970) inhibits tumor cell growth both in vitro and in vivo. Some microorganisms biotransform withaferin A added to the culture medium to new metabolites. JP Rosazza and coworkers (Sterpids 31, 676, 1978) isolated a new metabolite derived from withaferin A using Cunninghamella elegans (NRRL 1393), identified as a lax-hydroxywithaferin AJ Fuksa and coworkers (Steroids 35, 157, 1982) isolated from the filtrate of said culture two new metabolites resulting from biotransformation from withaferin A. The major metabolite was identified as 12? -hydroxywithaferin A, and the authors demonstrated that it was identical to the first isolated 14z-hydroxywithaferin A, which was initially misidentified, in structure. The second metabolite has been identified as 15β-hydroxywithaferin A (Stertids 35, 157 (1982)). Both metabolites inhibited the growth of sarcoma 180 and lympholeukemia P388.

Metabolit podlá vynálezu získaný biotransformáciou withaferinu A nebol doteraz v literatúre popísaný.The metabolite of the invention obtained by biotransformation withaferin A has not been described in the literature.

Podstata meíabolitu podlá vynálezu spočívá v tom, že je pripraviteíný přidáním 10 až 100 mg withaferinu A alebo withaferinu A diacetátu k 100 ml média naočkovaného kultúrou Rothobacter simplex ATCC 6946 a kultivovaného pri teplote 25 až 32 °C počas 26 až 120 hodin.The essence of the metabolite according to the invention is that it is prepared by adding 10 to 100 mg of withaferin A or withaferin A diacetate to 100 ml of medium inoculated with Rothobacter simplex ATCC 6946 and cultured at 25 to 32 ° C for 26 to 120 hours.

Filtrát vyfermentovanej pódy sa extrahuje estermi kyseliny octovej, s hlavně ocíanom etylnatým a získaný extrakt sa odpaří do sucha, přečistí na štipci silikagelu s použitím elučného činidla octanu etylnatého, eluát sa zahustí pri teplote do 40 °C a krystalizuje sa pri teplote 4 °C. Postupovat sa može aj tak, že sa extrahuje filtrát chlorovanými uhlovodíkmi s počtom 1 až 5 uhlíkov, s výhodou dichlórmetánom. Pevný metabolit sa výhodné krystalizuje zo zmesi octanu etylnatého a benzenu.The fermented soil filtrate is extracted with ethyl acetate esters, mainly ethyl acetate, and the extract is evaporated to dryness, purified on a silica gel column using ethyl acetate, concentrated to 40 ° C and crystallized at 4 ° C. It is also possible to extract the filtrate with chlorinated hydrocarbons having 1 to 5 carbons, preferably dichloromethane. The solid metabolite is preferably crystallized from a mixture of ethyl acetate and benzene.

Metabolit podlá vynálezu v pokusoch in vitro inhibuje biochemické reakcie a rast buniek P388 a v pokusoch in vivo inhibuje rast nádorov.The metabolite of the invention inhibits the biochemical reactions and growth of P388 cells in in vitro experiments and inhibits tumor growth in in vivo experiments.

Potenciálny protinádorový účinok sa hodnotil v pokusoch in vitro metodou inhibície využívania prekurzorov syntézy DNK a RNK (J. Fuksa et. al., Neoplasma 18, 831, 1971). V konc. 100/,Mg . ml^-1 znižoval utilizáciu tyrnidínu o 78,7 % a uridínu o 68,2 %. V konc. 25/,ug . ml^-1 totálně potlačil rast buniek P388 a vyvolával ich hynutie.The potential antitumor effect was evaluated in in vitro experiments by inhibiting the use of DNA and RNK synthesis precursors (J. Fuksa et. Al., Neoplasma 18, 831, 1971). In conc. 100], Mg. ml ^-1 reduced the utilization of tyrnidine by 78.7% and uridine by 68.2%. In conc. 25 /, ug. ml ^-1 totally suppressed the growth of P388 cells and caused them to die.

Metabolit podfa vynálezu připravený biotransformáciou withaferinu A svojimi vlastnosťami odlišuje od povodného withaferinu A, aj od iných metabolitov připravených z něho biotransformáciou. Látky sa od seba navzájom odlišujú hodnotami R_f, b. t. i farebnými reakciami, ako je vidieť z tabulky 1.The metabolite of the invention prepared by biotransformation withaferin A distinguishes by its properties from the flood withaferin A as well as from other metabolites prepared from it by biotransformation. The compounds are different from each other values of R _f, BT and color reaction, as seen in Table 1 below.

Tabulka 1Table 1

Odlišné chemické a fyzikálně hodnoty withaferinu A a jeho metabolitovDifferent chemical and physical values of withaferin A and its metabolites

Látka substance Rf Rf b. t. °C b. t. ° C Farebná reakcia Color reaction withaferin A withaferin A 0,340 0,340 237—243 237-243 staroružová old pink withaferin A diacetát withaferin A diacetate 0,650 0,650 199—200 199-200 oranžová Orange 12(3-hydroxywithaferin A 12 (3-hydroxywithaferin A) 0,140 0,140 270—274 270-274 hnědá Brown 15j3-hydroxywithaferin A 15β-hydroxywithaferin A 0,270 0,270 118—120 118-120 modrošedá modrošedá metabolit podfa vynálezu a metabolite of the invention 0,410 0,410 140—150 140-150 hnědá Brown Na delenie sa použila For separation, it was used sústava octanu e- e- Příklad 1 Example 1 tylnatého : aceton (4:2) tetrachloride: acetone (4: 2) , detekčně činidlo , a detecting agent p-anízaldehyd, po postriekaní sa chromato- p-anisaldehyde, after spraying Do 40 varných Up to 40 boiling baniek o objeme 500 ml 500 ml flasks gramy zohriali krátko na grams warmed briefly to 120 °C. Všetky u- 120 [deg.] C. All u- sa připravilo po 100 ml média nasledujúce- 100 ml of medium was prepared as follows.

vedené látky možno detegovať tiež bioautodetekciou pri použití Staphylococcus pyogenes aureus R50.guided substances can also be detected by bioautodetection using Staphylococcus pyogenes aureus R50.

Sposob přípravy metabolitu podfa vynálezu a jeho izolácie sú uvedené v príkladoch.The preparation of the metabolite according to the invention and its isolation are described in the examples.

ho zloženia: kvasničný extrakt 0,3 g, kukuřičný výluh 0,5 g, glukóza 0,2 g a doliala sa vodovodná voda do 100 ml, pH sa upravilo na 7,0. Po sterilizácii počas 20 min. při 120 °C a ochladení sa médium naočkovalo ml vegetatívneho inokula Rothrobacter simplex ATCC 6946 na pode zloženia: kukuřičný výluh 1,2 g, peptón 1,5 g, vodovodná voda do 100 ml pH 6,5 — 6,7 a kultivuje sa 24 hodin. Kultivovalo sa na rotačnej trepačke pri 220 ot . min¹ a pri teplote 23 °C. Po 24 hodinovej kultivácii sa přidalo do každej banky 35 mg withaferinu A rozpuštěného v 0,4 ml dimetylformamidu. Kultivácia pokračovala do 120 hodiny. Filtrát po kultivácii obsahuje nový metabolit podlá vynálezu v množstve 12 mg . 100 ml¹ doteraz nestanovenej štruktúry.composition: yeast extract 0.3 g, corn liquor 0.5 g, glucose 0.2 g and tap water was added to 100 ml, pH was adjusted to 7.0. After sterilization for 20 min. at 120 ° C and cooling, the medium was inoculated with 1 ml of Rothrobacter simplex ATCC 6946 vegetative inoculum according to the composition: corn liquor 1.2 g, peptone 1.5 g, tap water up to 100 ml pH 6.5 - 6.7 and cultured for 24 hours. hours. Cultured on a rotary shaker at 220 rpm. min ¹ and at 23 ° C. After 24 hours of culture, 35 mg of withaferin A dissolved in 0.4 ml of dimethylformamide was added to each flask. Cultivation continued until 120 hours. The culture filtrate contains 12 mg of the novel metabolite according to the invention. 100 ml of ¹ structure not yet determined.

Příklad 2Example 2

Do 500 ml varných baniek sa naplní po 100 ml média zloženia ako je uvedené v přiklade 1. Toto sa po sterilizácii počas 20 min. pri 120 °C ochladí a naočkuje 15 ml vegetatívneho inokula Rothrobacter simplex ATCC 6946. Po 24 hodinách kultivácie na rotačnej trepačke pri 220 ot . min^-1 a teplote 28 °C sa do každej banky přidá 20 mg withaferinu A rozpuštěného v 0,2 ml dimetylformamidu a po 72 hodinách kultivácie sa přidá opál 15 mg withaferinu A rozpuštěného v dimetylformamide. Biotransformácia sa ukončila po 144 hodinách kultivácie. Filtrát média obsahoval nový metabolit v koncentrácii 15 mg . 100 ml¹.100 ml of the composition medium as described in Example 1 are filled into 500 ml boiling flasks. This is sterilized for 20 min after sterilization. Cool and inoculate 15 ml of Rothrobacter simplex ATCC 6946 vegetative inoculum at 120 ° C. After 24 hours of cultivation on a rotary shaker at 220 rpm. min ^-1 and a temperature of 28 ° C, 20 mg of withaferin A dissolved in 0.2 ml of dimethylformamide are added to each flask, and after 72 hours of culture, opal is added with 15 mg of withaferin A dissolved in dimethylformamide. Biotransformation was terminated after 144 hours of culture. The medium filtrate contained the new metabolite at a concentration of 15 mg. 100 ml ¹ .

P r í k 1 a d 3EXAMPLE 1 a d 3

Do 500 ml varných baniek sa naplní po 100 ml média zloženia ako je uvedené v příklade 1. Toto sa po sterilizácii počas 20 minút pri 120 °C ochladí a naočkuje 15 ml vegetatívneho inokula Rothrobacter simplex ATCC 6946. Po 24 hodinách kultivácie na rotačnej trepačke pri 220 ot . min^-1 a teplote 28 ^CC sa do každej banky přidá 20 mg withaferinu A diacetátu rozpuštěného v 0,2 mililitru dimetylformamidu. Kultivácia trvala 32 hodin. Filtrát po kultivácii obsahuje nový metabolit podlá vynálezu v množstve 10 mg . 100 ml¹, doteraz nestanovenej štruktúry.100 ml of the composition medium as described in Example 1 are filled into 500 ml boiling flasks. After sterilization for 20 minutes at 120 ° C, this is cooled and seeded with 15 ml of Rothrobacter simplex ATCC 6946 vegetative inoculum. 220 rpm min ^-1 and a temperature of 28 ^° C are added to each flask 20 mg of withaferin A diacetate dissolved in 0.2 ml of dimethylformamide. Cultivation lasted 32 hours. The filtrate after culture contains the new metabolite of the invention in an amount of 10 mg. 100 ml ¹ , structure not yet determined.

Přikládáattaches

Obsah 40 baniek 2500 ml po skončenej kultivácii ako je uvedené v příklade 1 sa spojí a za neustálého miešania sa upraví pH na hodnotu 7,0 prídavkom kyseliny chlorovodíkovej. Ďalej sa přidá 1200 ml octanu etylnatého a extrahuje sa podá za neustálého miešania 20 až 40 minút. Oddělená vrstva rozpúšťadla sa odpustí a ostávajúca podá sa rovnakým postupom extrahuje ešte dvakrát po sebe octanom etylnatým v pomere 2 : 1. Získané extrakty sa spoja, prídavkom bezvodého síranu sodného vysušia a za zníženého tlaku pri teplote do 45 °C sa rozpúšťadlo odpaří. Získá sa 1060 mg surového produktu, ktorý sa přečistí na štipci silikagelu 125 — 180 mesh, kolona 4 X X 36 cm. Ako elučné činidlo sa použije zmes rozpúšfadiel octan etyinatý : benzen (9 : lj. Na základe analýzy na tenkej vrstvě sa zachytené frakcie po 25 ml spoja a znova odparia do sucha. Spojením frakcií 33 — 98 sa získá 155 mg nového metabolitu. Konverzia vzhladom na použité množstvo withaferinu A je 11 %.The contents of 40 flasks of 2500 ml after completion of the cultivation as described in Example 1 were combined and the pH adjusted to 7.0 by addition of hydrochloric acid with stirring. Further, 1200 ml of ethyl acetate is added and the mixture is extracted with stirring for 20 to 40 minutes. The separated solvent layer was drained and extracted with 2: 1 ethyl acetate two more times in the same manner. The extracts were combined, dried over anhydrous sodium sulfate and evaporated under reduced pressure at a temperature of up to 45 ° C. 1060 mg of crude product is obtained, which is purified on a column of silica gel 125-180 mesh, 4 X X 36 cm column. Ethylacetate: benzene (9: 1) was used as eluent. Thin-layer analysis of the collected fractions of 25 ml was pooled and re-evaporated to dryness to give 155 mg of the new metabolite by fractions 33-98. the amount of withaferin A used is 11%.

Příklad 5Example 5

155 mg surového, přečištěného metabolitu získaného postupom uvedeným v příklade 4 sa rekryštalizuje z horúcich roztokov octanu etylnatého připadne zo zmesi octan etyinatý : benzén (1 : 5). Získá sa 120 mg čistej kryštalickej látky, bielej farby s b. t. 140 — 150 °C bez korekcie. Výsledky elementárnej analýzy látky sú následovně: C 67,94 %; H 7,85 čo zodpovedá sumárnemu vzorců C28H38O7 (480,6j. Infračervené spektrum merané v KBr na přístroji Perkin-Elmer 450 vykazovalo Amax, cm¹: 680, 794, 910, 948, 965, 1020, 1038, 1128, 1185, 1206, 1260, 1288, 1313, 1339, 1393, 1450, 1623, 1688, 1700, 2865, 2935, 3415.155 mg of the crude, purified metabolite obtained by the procedure of Example 4 is recrystallized from hot ethyl acetate solutions or from ethyl acetate: benzene (1: 5). 120 mg of pure crystalline substance, white color, having a mp of 140-150 ° C without correction are obtained. The elemental analysis results are as follows: C 67.94%; H, 7.85, which corresponds to the summary formulas C28H38O7 (480.6. The infrared spectrum measured in KBr on a Perkin-Elmer 450 instrument showed Amax, cm ¹ : 680, 794, 910, 948, 965, 1020, 1038, 1128, 1185, 1206 , 1260, 1288, 1313, 1339, 1393, 1450, 1623, 1688, 1700, 2865, 2935, 3415.

Příklad 6Example 6

Postup izolácie bol rovnaký ako je uvedené v příklade 4, avšak ako extrakčné činidlo sa použil dichlórmetán.The isolation procedure was the same as in Example 4 except that dichloromethane was used as the extraction agent.

Claims

5 235575 15 ml of a vegetative inoculum Rothrobactersimplex ATCC 6946: 1.2 g corn extract, 1.5 g peptone, 100 ml pH 6.5-6.7 water feed and cultured for 24 hours. It was cultured on a rotary shaker at 220 rpm. min. 1 at 23 [deg.] C. After 24 hours of culture, 35 mg of withaferin A dissolved in 0.4 ml of dimethylformamide was added to each flask, and the culture was continued for 120 hours. finding at 12 mg, 100 ml " once-not-determined structure. Example 2 Fill 100 ml of the medium of composition as described in Example 1 into 500 ml boiling flasks. at 120 ° C, it is cooled and inoculated with 15 ml of the ingenious inoculum of Rothrobacter simplexATCC 6946. After 24 hours of cultivation of the narrative shaker at 220 rpm. min-1 and 28 [deg.] C., 20 mg ofwithaferrin A dissolved in 0.2 ml of dimethylformamide are added to each flask and after 72 hours, 15 mg of withaferrin A dissolved in dimethylformamide is added. Biotransformation was terminated after 144 hours of culture. The media filtrate contained a new metabolite at a concentration of 15 mg. EXAMPLE 3 100 ml of a medium of composition as described in Example 1 are filled into 500 ml of boiling flasks. After sterilization for 20 minutes at 120 DEG C., the mixture is cooled and seeded with 15 ml. After 24 hours of cultivation of the narrative shaker at 220 rpm and a temperature of 28 ° C, 20 mg ofwithaferrin A diacetate dissolved in 0.2 ml of dimethylformamide was added to each flask for 32 hours. The filtrate, after cultivation, contains 10 mg of the present metabolite of the invention, 100 ml of the previously unstructured structure. EXAMPLE 4 A 40 mL volume of 2500 mL after the culture as described in Example 1 was mixed and adjusted to pH 7.0 by addition of hydrochloric acid under stirring. Next, 1200 ml of acetonitrile are added and extracted under continuous stirring for 20 to 40 minutes. The separated solvent layer is removed and the residue is extracted in the same manner with ethyl acetate (2: 1) in succession. The extracts are combined and dried under reduced pressure at a temperature below 45 ° C with anhydrous sodium sulfate. the solvent is evaporated. 1060 mg of dry product is obtained, which product is purified on a 125-180 mesh silica gel column, 4 x 36 cm column. Ethyl acetate: benzene (9: 1) was used as the eluent. Based on thin layer analysis, the collected fractions of 25 ml were combined and evaporated to dryness to give 155 mg of the new metabolide by combining fractions 33-98. EXAMPLE 5 155 mg of the crude, purified metabolyzed product obtained in Example 4 is recrystallized from ethyl acetate-burner burners from octane-ethylene: benzene (1: 5) to give 120% of the title compound. pure crystalline substance, white in color with bt140-150 ° C without correction The results of the electronical analysis of the substance are as follows: C67.94%; H 7.85 which corresponds to the summary of C28H38O7 (480.6). KBr on a Perkin-El-mer 450 exhibited A max, cm -1 1: 680, 794.910, 948, 965, 1020, 1038, 1128, 1185, 1206, 1260, 1288, 1313, 1339, 1393, 1450, 1623, 1688, 1700, 2865, 2935, 3415. Example 6 Post The isolation was as described in Example 4, but dichloromethane was used as the extraction agent. PRECISION Metabolite withaferin A prepared by adding 10 to 100 mg withaferrin A or diphtherin A diacetate to 100 ml of medium inoculated with culture of Rothrobacter simplexATCC 6946 and cultured at 25 to 32 ° C for 26 to 120 hours.