Computer Simulation of Ecdysone Metabolism and of the HPLC Analysis of the Metabolites

H. Kalfisz*WM. Bfithori2/Z. Tarjfinyi3/F. Darvas3 1Department of Pharmacology and Cell Biophysics, University of Cincinnati, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0575, USA

2Department of Pharmacognosy, Albert Szent-GyOrgyi Medical University, Szeged, Hungary 3CompuDrug Ltd., 5 Sandor Furst Street, Budapest, Hungary aOn leave from the Department of Pharmacology, Semmelweis University of Medicine, Budapest, Hungary

Key Words Column liquid chromatography Metabolexpert| HPLC-Metabolexpert| Ecdysone Metabolism

Summary Computer simulation of ecdysone metabolism in insects has been done by the a software called HPLC- Metabolexpert| that served to generate the metabolic pathways of ecdysone in a retrospective manner. Some of the generated metabolites have already been detected, others are to be confirmed. Lists of the applied metabolic transformations, the predicted metabolites and their HPLC elution times are also given.

Introduction Ecdysteroids are insect moulting hormones. Accord- ing to recent reports [1-3], their presence has also been declared in various arthropods and plants. They have been demonstrated in several plant families, as various species of Pteridophita, Gymnospermae and Angio- spermae. Although the biological function of ecdy- steroids is not completely understood, they play a role in plant - insect interactions. The chemical structure of the most ecdysteroids is built on cholesterol, con- taining 5-13 hydrogen, cis A/B ring, 7-ene-6=on con- jugate structure, the sterol side chain and several hydroxyl groups in various positions. One of the ubiquitous examples of ecdysteroids is the ecdysone.

HPLC-Metabolexpert| is an expert system of Compu- Drug Ltd. (Budapest, Hungary) which is designed to simulate the metabolic pathway of exogenous com- pounds together with their retention times. Simu-

lation of metabolic pathways is done by constructing the chemical structure of the compound to be inves- tigated by the help of a graphic input system or Molnote | the special linear notation system used for communication with HPLC-Metabolexpert| Met- abolexpert | is an open expert system, which means, that the user has the possibility to modify the knowledge of the system (i.e. the rules about gener- ating metabolic pathways) or to input new knowledge. In our case, a completion of the new rules was es- pecially supported by the fact that HPLC-Metabol- expert| is originally designed to support metabolic investigations of warm-blooded species and thereby it has not been prepared initially to simulate insect metabolitic procedures. Thereby, part of the per- formed investigation was the completion of the knowledge base of HPLC-Metabolexpert | with the compound ecdysone to be "metabolized" and the reactions to be performed. Due to a present limit of the chemical representation by HPLC-Metalbol- expert | there is no difference between the confi- gurational and conformational isomers of molecules. Moreover, neither configurational nor conformational changes can be modeled by Metabolexpert| The chemical structure of ecdysone can be seen in Figure 1.

The major metabolites of ecdysone were studied by several authors. Lafont et al. [1-3] gave a summari- zation of the metabolic alterations of ecdysone, it is given in Table I.

In a recently published paper, Girault and Lafont [4], gave interesting data on the metabolism of ecdysone on mice. Their results were based on identification using HPLC and nuclear magnetic resonance spectro- scopy.

A divergent approximation is the reason of our work, simulation of metabolic pathways by the help of desk- top computers. In this paper, "metabolite" is a general term, used for de facto metabolites, as well as for structures derived from the original compound by computer simulation. Metabolites of ecdysone were simulated by a modified HPLC-Metabolexpert| Re-

Chromatographia Vol. 30, No. 1/2, July 1990 Originals 95

0009-5893/9017 0095-04 $ 03.00/0 �9 t990 Friedr. Vieweg & So/an Verlagsgesellschaft mbH

OH

. ~22 2 6

H o ~ H O 19 ' H 16

0

Figure 1 Chemical structure of ecdysone.

Table I. Major metabolites of the ecdysone molecule.

position on the metabolic reaction carbon atom

2,3,22,25 2,3 2,3 2, 3, 22 6,7 14 1,5,9,11,16,18,19,20,

23,24,26 17-20; 20-22 22 22, 25 25 26

phosphate ester formation oxidation epimerisation acetate ester formation reduction dehydroxylation hydroxylation

cleavage fatty acid ester formation glycosylation glucose conjugation hydroxylation followed by (a) oxidation to ecdysonoic acid (b) phosphate ester formation

Table ii . Tentative alterations taking place in the metabolism of ecdysone.

tention times of the tentative metablites are calculated with the condit ions that C-18 s ta t ionary phase is employed , the mobi le phase is water -ace toni t r i le (81:19), and that the column dead t ime and the ecdysone retent ion time are 2.5 and 10 min, respec- tively.

Experimental

I n s t r u m e n t s a n d S o f t w a r e

Variter A T desk-top computer (Research Institute of Computa t ion and. Automat iza t ion of the Hungarian Academy of Sciences, Budapest, Hungary) with 640 K base memory and 384 K expansion was used.

H P L C - M e t a b o l e x p e r t | 1.0 (available f rom Compu- Drug Ltd., Budapes t 62, P.O. Box 405, H-1395, Hungary) was used with its data for reactions and an H P LC expert system, adapted to the special reactions of the ecdysteroid metabolism which will be detailed in Table III.

Results H P L C - M e t a b o l e x p e r t | 1.0 genera ted a series of metabolic reactions for alteration of ecdysone (Table II). In order to consider the special nature of the ecdysone metabo l i sm in insects, H P LC -Metabo l - e x p e r t | was comple ted by several t ransformat ion rules, such as listed in Table III. Using this altered program, the pr imary metaboli tes of ecdysone were simulated. Table IV gives the tentative metabolites and their calculated HPLC retention times.

position of alteration reaction on the carbon atom

2,3,22,25 2,3,22,25 1,4,5,9,11,12,15,16,17,

18,19,20,21,22,23,24, 26

2,3,22 6 2, 3, 14, 22, 25

acetate formation of alcohols sulphate ester of alcohols hydroxylation (on primary, secondary and tertiary carbon)

oxidation of secondary alcohol reduction of aliphatic ketone formation of deoxy compounds

Table Ill. Alterations required for simulation the metabolism of ecdysone.

position of alteration reaction

20-22 2,3,14,22,25 2,3,22 numerous

26

7-8 2,3,14,22,25 2, 3,14, 22, 25 2, 3,14, 22, 25 2,3,14,22,25 2,3.14,22,25

oxidative cleavage of alkyl chain dehydratation of CH-hydroxyl oxidation of CH-hydroxyi oxidation of C-hydroxyl oxidation and phosphate ester formation (in one simulated step) reduction of double bond phosphate ester formation benzoate ester formation palmitate ester formation stearate ester formation oleate ester formation

Discussion Several papers deal with the separat ion of various ecdysteroids by reversed-phase H P LC (for review, see ref. [1]). However, their majority used gradient elution, and other have not given the elution time of the non- re ta ined eluites, there fore the direct comparison of our da ta to that of the l i t e ra ture is practical ly impossible. At the same time, the trends in the elution character is t ics of our caluclated data are mainly suppor ted by the results of Lafon t et al. [5] who sepa ra t ed ecdysone and 2 0 -h y d ro x y ecdysone on Partisil 10 ODS (250 x 4.6 ram), using methanol-water (1:3) mixture. Ecdysone and 20-hydroxyecdysone were eluted at 75 and 31 minutes, respectively. Our calcu- lation showed differences between the effects of 11- hydroxyla t ion and 26-hydroxylat ion. Similar differ- ences were fond in comparison of inokas terone and poststerone, which also showed definite separation on Spherisorb ODS column, and using acetonitri le-water eluent, when their ratios were either 15:85, or 35:85, or 20:80. The essential differences be tween the elution time of 20-hydroxyecdysone and 26-hydroxyecdysone give another indirect support for the applicability of our calculation.

96 Chromatographia Vol. 30, No. 1/2, July 1990 Originals

Table IV. Ecdysone metabolites and their retention time.

Serial no. of Predicted Predicted metabolite compound retention

time (min) metabolite No. 1 poststerone 10.50 metabolite No. 2 dihydropoststerone 10.50 metabolite No. 3 2-deoxy-2,3-dehydroecdysone *

(.9) metabolite No. 4 3-deoxy-3,4-dehydroecdysone *

(.9) metabolite No. 5 2-deoxy-2,3-dehydroecdysone *

(.9) metabolite No. 6 3-deoxy-2,3-dehydroecdysone * metabolite No. 7 14-deoxy-14,15-dehydroecdysone *

(.9) metabolite No. 8 22-deoxy-22,23-dehydroecdysone 10.50

(.9) metabolite No. 9 22-deoxy-20,22-dehydroecdysone 10.50

(.9) metabolite No.10 25-deoxy-24,25-dehydroecdysone 10.50

(.9) metabolite No.ll 22-dehydroecdysone 10.50 metabolite No.12 2-dehydroecdysone 10.50 metabolite No.13 3-dehydroecdysone 10.50

(2-deoxysilenosterone) metabolite No.14 26-hydroxyecdysone-26-phosphate metabolite No.15 ecdysone-22-acetate 29.30 metabolite No.16 ecdysone-2-acetate 29.30 metabolite No.17 ecdysone-3-acetate 29.30 metabolite No.18 ecdysone-25-acetate 29.30 metabolite No.19 ecdysone-22-sulphate 5.21 metabolite No.20 ecdysone-2-sulphate 5.21 metabolite No.21 ecdysone-3-sulphate 5.21 metabolite No.22 ecdysone-25-sulphate 5.21 metabolite No.23 ecdysonic acid (ecdysone-26-oic 10.50

acid) metabolite No.24 20-deoxysogdisterone; 8.17

(19-hydroxyecdysone) metabolite No.25 20-deoxy-25-hydroxyajugasterone C; 8.17

(11-hydroxyecdysone) metabolite No.26 20-deoxyintegristerone A; 8.17

(1-hydroxyecdysone) metabolitc No.27 4-hydroxyecdysone 8.17 metabolite No.28 12-hydroxyecdysone 8.17 metabolite No.29 20-dcoxy-polypodine B; 8.17

(5-hydroxyecdysone) metabolite No.30 2,22,25-trihydroxy-viperidinone; 8.17

(9-hydroxyecdysone) metabolite No.31 17-hydroxyecdysone 8.17 metabolite No.32 20-hydroxyecdysone 7.33 metabolite No.33 cheilanthone A *

7,8-dihydroecdysone metabolite No.34 25-deoxyecdysone 10.50 metabolite No.35 22-deoxyecdysone 10.50 metabolite No.36 14-deoxyecdysone 10.50 metabolite No.37 2-deoxyecdysone 10.50 metabolite No.38 3-deoxyecdysone 10.50 metabolite No.39 ecdysone-2-glucoside 10.50 metabolite No.40 ecdysone-3-glucoside 10.50 metabolite No.41 ecdysone-22-glucoside 10.50 metabolite No.42 ecdysone-14-glucoside 10.50 metabolite No.43 ecdysone-25-glucoside 10.50 metabolite No.44 16-hydroxyecdysone 10.50 metabolite No.45 15-hydroxyecdysone 10.50 metabolite No.46 23-hydroxyecdysone 10.50 mctabolite No.47 20-deoxyabutasterone; 10.50

(24-hydroxyecdysone) mctabolite No.48 26-hydroxyecdysone 10.50 metabolite No.49 21-hydroxyecdysone 10.50 metabolite No.50 ecdysone-25-phosphate * metabolite No.51 ecdysone-22-phosphate * metabolite No.52 ecdysone-14-phosphate * metabolite No.53 ecdysone-2-phosphate * metabolite No.54 ecdysone-3-phosphate * metabolite No.55 ecdysone-25-benzoate * metabolite No.56 ecdysone-22-benzoate * metabolite No.57 ecdysone-14-benzoate * metabolite No.58 ecdysone-2-benzoate *

metabolite No.59 ecdysonc-3-bcnzoate * mctabolite No.60 ecdysone-25-palmitate * metabolite No.61 ecdysonc-22-palmitate * metabolite No.62 ccdysone-14-palmitate * mctabolite No.63 ecdysone-2-pahnitate * metabolite No.64 ecdysone-3-plamitate * metabolite No.65 ecdysone-25-stearate * metabolite No.66 ecdysone-22-stearate * metabolite No.67 ecdysone-14-stearate * metabolite No.68 ecdysone-2-stearate * mctabolite No.69 ccdysonc-3-stearate * mctabolite No.70 ecdysone-25-oleate * metabolite No.71 ecdysone-22-oIeate * metabolite No.72 ecdysone-14-oleate * metabolite No.73 ecdysone-2-oleate * metabolite No.74 ecdysone-3-oleate *

* the calculation method does not supply the correct retention time, due to some limitations (e.g., the isocratic system does not separate and elute all components, etc.),

(?) the existence of these compounds should be confirmed.

A t the same t ime, the l i te ra tura l da ta give m o r e r e m a r k a b l e d i f f e rences b e t w e e n the e lu t ion cha- rac te r i s t ics o f e c d y s o n e and 2 6 - h y d r o x y e c d y s o n e , e cdysone and 2 - d e o x y e c d y s o n e (loc. ref. [5], using m e t h a n o l - w a t e r 1:3), e c d y s o n e and 2 0 - h y d r o x y - e c d y s o n e (ref. [6], us ing Z o r b a x O D S and ace- t o n i t r i l e - 0 . 4 % aqueous acet ic acid (82:18), as the s t a t i ona ry p h a s e and mobi le phase , r e spec t ive ly ) . Fur ther possible shor tcoming of the p rocedure of our calculat ion can be that several s tructural isomers gave the same predic ted elut ion time. These results should be cor rec ted in the next vers ion of H P L C - M e t a b o l - exper t | devoted to the caluclat ion of metabol i sm and the H P L C characterist ics of the predic ted ecdys te ro id metabol i tes .

T h e m e t a b o l i s m of e c d y s o n e and its a n a l o g u e s in insects is impor tan t because their metabol ic a l tera t ion may lead to a decrease in h o r m o n e activity. Insects ob ta in ecdys te ro ids f rom their food which may be quickly me tabo l i zed by digest ive enzymes into their acetate and phospha te ester derivatives.

The most likely posit ions for metabol ic t ransformat ion of ecdysone are on the 3, 20, 26 and 22 ca rbon atoms. In the case of insects, e cdyson is inac t iva ted by me tabo l i c t r ans fo rma t ion to ecdysone-26-acid , which p roduc t is genera ted th rough the 26-hydroxy-ecdysone intermedier . Ecdysone-26-acid has not yet been found in plants. As any fur ther metabol i tes of ecdysone-26- acid has no t been de tec ted in insects, the c o m p o u n d can be cons idered as an end p roduc t of metabo l i sm. A t the same t ime, the o c c u r r e n c e of 2 6 - h y d r o x y - ecdysone is unusual.

A n in teres t ing reac t ion of e c d y s o n e is its ep imer i - sat ion: the 3-13-hydroxyl is ox id ized into its 3 -oxo der iva t ive , which is r e d u c e d , y ie ld ing 3 - ep i ecdy - steroid.

A gene ra l l y o c c u r r i n g r e a c t i o n is the s ide -cha in cleavage between the 20-22 C atoms and 17-20 C a toms when C-21 p o s t s t e r o n e and C-19 r u b r o s t e r o n e are gene ra t ed , respect ively. The fo rma t ion of s e c o n d a r y alcohols (at C-2, C-3, and C-22) or tert iary alcohols (at C14 and C25) leads to the p roduc t ion of var ious esters

Chromatographia Vol. 30, No. 1/2, July 1990 Originals 9 7

and ethers. As it was shown the hydroxyl on C-2 was the most reactive followed by those of C-22 and C-3. The C-3 acetate-ester was first discovered from insects, while the C-25 acetate ester was found in plants.

Recently, several additional ecdysone metabolites have been isolated. Among them, palmitate, stearate, oleate etc. esters were found as the main hydrolysable metabolites in several insects. The most polar derivatives are the phosphate esters of ecdysteroids. They widely occur in insects. Some other products such as glycosides, sulphate esters, cinnamates, cumarates, hemi-succinates, etc. have also been detected.

A software like "HPLC-Metabolexpert" can be con- veniently used for the construction of the members of the metabolic pathway of drugs and medicine. In our work, we used this program for a computer assisted modeling of the biochemical alterations of ecdysone. The major reaction routes of ecdyson were taken into considerations, and therefore, metabolic transfor- mation knowledge base of the HPLC-Metabolexpert| program was completed with double bond reduction, phosphate ester formation, benzoate ester formation, palmitate ester formation, stearate ester formation, side chain cleavage and alcoholic hydroxyl oxidation. The simulated metabolic alterations resulted in sev- eral preducted metabolites; some of them are known metabolites of ecdysone, some others are yet to be identified experimentally. Our major problem occur- red at the calculation of the retention times of the fatty acid esters of ecdysone, as they can be eluted only with an eluent having an organic modifier (such as acetonitrile) over 90 %. For the time being, the HPLC- Metabolexpert| is unable to handle gradient elution, therefore the calculation of these retentions remained to the future.

The compounds with question mark have not been detected. In the case of two names with semicolon (;) between them, the first one comes from a trivial name derived from the nearest, naturally occurring com- pound. Compounds and their predicted elution times are given in Table IV.

Conclusions In cases of deoxy and ester derivatives, the possibilities for transformation into more effective insect hormones (ecdysone or 20-hydroxyecdysone) are open. Furthermore, in the case of ester derivatives, the strength of the activity may depend on the rate of hydrolysis.

Thereby the ecdysone (and ecdysteroid) metabolites have a very important physiological role, they can be important both as depot of the active material and the inactive products of the insect hormones. Several other compounds can also be considered as potential metabolites as acetate-phosphates, nucleotides, etc. The computer-assisted simulation of their metabolic pathway is in progress.

The HPLC-Metabolexpert| program has predicted numerous metabolites of ecdysone. As the k' values are also given by HPLC-Metabolexpert| either the conformation or rejection of the existence of these predicted metabolites can be expected in the near future. Metabolic alteration where eonfigurational change takes place during the alteration, e.g. when a [3- hydroxyl is transformed into a-hydroxyl through its oxidation to oxo compound and subsequent reduction to hydroxyl can not be modelled. In spite of this shortcoming of the program, a wide range of novel metabolites has been predicted. HPLC-Metabol- expert| can preferentially be employed for predicting the metabolic pathways of not only mammalian but also the insect organisms, too. Thereby, HPLC- Metabolexpert| serves an unusual and useful tool of metabolic studies of exogenous compounds in insects.

Acknowledgements One of the authors (H.K.) thanks for the financial help of CompuDruck Ltd., Budapest, Hungary and of the Hungarian Academy of Sciences (Grant OTKA No. 1892).

References [1] R. Lafont, In Chromatography '87, II. Kal6sz, and L.S. Ettre,

(Eds.), 1987, pp. 1-15. [2] R. Lafont, P. Beydon, C. Blais, M.G. Lachaise, Biochem., 16, 11

(!986). [3] M. Bdthori, K. Szendrei, It. Kaldsz, R_ Lafont, J.P. Girault,

Chromatographia, 25, 625 (1988). [4] J.P. Girault, R. Lafont, U. Kerb, Drug. Metabol. Disp., 16, 716

(1988). [5] R. Lafont, G. Somme-Martin, B. Mauchamp, B.F. Maume,

J.P. Delbecque, In "Progress in Ecdysone Research", J.A. lloffmann, (Ed.), Elsevier/North Holland, Amsterdam, 1980, p. 45--67.

[6] LD. Wilson, C.R. Bielby, E.D. Morgan, J. Chromatogr., 238, 97 (1982).

Received: Apr. 17, 1990 Accepted: Apr. 20, 1990 A

98 Chromatographia Vol. 30, No. 1/2, July 1990 Originals