s t e r o i d s 7 3 ( 2 0 0 8 ) 502–514

avai lab le at www.sc iencedi rec t .com

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Additional minor ecdysteroid components ofLeuzea carthamoides

Milos Budesınsky, Karel Vokac, Juraj Harmatha ∗, Josef CvackaInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,Flemingovo n. 2, 166 10 Prague, Czech Republic

a r t i c l e i n f o

Article history:

Received 13 September 2007

Received in revised form

12 December 2007

Accepted 18 December 2007

Published on line 28 December 2007

Keywords:

Ecdysteroid

Phytoecdysteroid

a b s t r a c t

Seventeen additional minor ecdysteroid compounds were isolated and identified from

the roots of Leuzea carthamoides (Wild.) DC. Eight of them are new phytoecdysteroids:

carthamoleusterone (13) is a new side-chain cyclo-ether with five-membered ring; 14-

epi-ponasterone A 22-glucoside (12) is a rare and unusual natural 14�-OH epimer;

15-hydroxyponasterone A (11) is also new and rare with its C-15 substituted position, as

well as 22-deoxy-28-hydroxymakisterone C (18) possessing secondary hydroxyl in position

C-28 and 26-hydroxymakisterone C (20) with hydroxy groups in positions 25 and 26. New are

also 1�-hydroxymakisterone C (21) and 20,22-acetonides of inokosterone (8) and integris-

terone A (10). Series of already known ecdysteroids: ecdysone (1), 20-hydroxyecdysone 2-

and 3-acetates (3 and 4), turkesterone (6), inokosterone (7), 24-epi-makisterone A (14), and

epi-Ecdysteroid

Glucoside

Leuzea carthamoides

NMR data

amarasterone A (22) are reported here as new constituents of L. carthamoides. Seven ear-

lier reported Leuzea ecdysteroids: 20-hydroxyecdysone (2), ajugasterone C (5), integristerone

A (9), 24(28)-dehydromakisterone A (15), 24(28)-dehydroamarasterone B (16), (24Z)-29-

hydroxy-24(28)-dehydromakisterone C (17) and makisterone C (19) are also included because

they are now better characterized.

Leuzea ecdysteroids [1,2,17] in previously unattainable quanti-

1. Introduction

Leuzea carthamoides DC [syn. Rhaponticum carthamoides (Willd.)Iljin] served as a primary source for preparation various indi-vidual ecdysteroids [1,2] needed for our chemical [3–7] andbiological [8–14] study. This plant served the purpose well,because of its high content of ecdysteroids in roots or seeds[15] and because it provides a large structure variability ofecdysone analogues isolated so far [1,2,16–19]. Moreover, L.carthamoides in the last two decades is cultivated as a medic-inal plant on a large scale in the east and central Europe.

This is why it was chosen to serve as a rich source ofecdysteroids, not only for chemical and biological studies,but also for production of various nutraceuticals (adjunc-

∗ Corresponding author. Tel.: +420 220 183 522; fax: +420 220 183 582.E-mail address: harmatha@uochb.cas.cz (J. Harmatha).

0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.steroids.2007.12.021

© 2007 Elsevier Inc. All rights reserved.

tive functional food) [20,21] or cosmetic preparations [22].Proposed use of phytoecdysteroids in cosmetics and der-matology [23] demanded to carry out further experimentsrequiring large quantity of active compounds. This urgentlyinvolved scaling up the production of 20-hydroxyecdysoneand other ecdysteroids or ecdysteroid mixtures with fixedqualitative and quantitative compositions to several hundredgrams amount. Such large-scale preparation displayed manyecdysteroid-containing separation fractions, turned into a dis-posable source of several already reported major and minor

ties, as well as a rich source of several new minor ecdysteroidconstituents, undetectable in the previous low-scale separa-tions.

s t e r o i d s 7 3 ( 2 0 0 8 ) 502–514 503

Table 1 – HPLC retention times of ecdysteroids 1–22 from L. carthamoides under various analytical conditions

Compound Retention time [min]

System 1a System 2b System 3c System 4d

Ecdysone (1) 39.9 41.7 52.3 53.020-Hydroxyecdysone (2)e 34.2 50.7 75.4 90.920-Hydroxyecdysone 2-acetate (3) 42.0 36.1 28.4 –20-Hydroxyecdysone 3-acetate (4) 38.6 38.7 34.2 25.4Ajugasterone C (5)e 39.2 32.3 33.2 45.9Turkesterone (6) 26.9 76.8 – –Inokosterone (7) 35.1 57.5 64.9 48.1Inokosterone 20,22-acetonide (8) 55.0 23.1 24.6 28.0Integristeone A (9)e 31.0 78.5 – –Integristeone A 20,22-acetonide (10) 53.4 27.5 66.1 38.315-Hydroxyponasterone A (11) 41.3 28.9 24.8 38.314-epi-Ponasterone A 22-glucoside (12) 47.3 58.5 142.3 88.8Carthamoleusterone (13) 40.2 30.2 32.1 –24-epi-Makisterone A (14) 37.7 35.9 38.2 –24(28)-Dehydromakisterone A (15) 38.1 35.9 30.1 23.724(28)-Dehydroamarasterone B (16) 41.4 42.6 44.2 48.1(24Z)-29-Hydroxy-24(28)-dehydromakisterone C (17)e 33.6 74.6 129.4 90.822-Deoxy-28-hydroxymakisterone C (18) 41.2 62.3 86.6 96.7Makisterone C (19)e 34.2 32.2 23.8 28.426-Hydroxymakisterone C (20) 37.9 64.5 80.6 78.81�-Hydroxymakisterone C (21) 39.8 58.0 61.3 39.7Amarasterone A (22) 41.9 39.9 33.0 34.5

a System 1: Separon SGX C-18 column (5 �m, 250 mm × 4 mm i.d.) eluted with linear gradient of 10–70% methanol in water over 50 min at flowrate 0.6 ml min−1.

b System 2: Silasorb 600 column (5 �m, 250 mm × 4 mm i.d.) eluted with n-hexane–ethanol–water (812:180:8) at flow rate 0.8 ml min−1.c System 3: Silasorb 600 column (5 �m, 250 mm × 4 mm i.d.) eluted with diethyl ether–acetonitrile–water (880:102:18) at 0.8 ml min−1.

th dicthis s

(dcwpNnmepoBta

(sdwfcp(o2[2

d System 4: Silasorb 600 column (5 �m, 250 mm × 4 mm i.d.) eluted wie Previously reported ecdysteroids from L. carthamoides [1,2] used in

structural analysis of new related ecdysteroids.

The identities of major compounds, 20-hydroxyecdysone2), ajugasterone C (5), integristerone A (9), 29-hydroxy-24(28)-ehydromakisterone C (17) and makisterone C (19) wereonfirmed comparing their RP- and NP-HPLC retention timesith authentic samples using Systems 1–4 (Table 1), and com-aring their NMR data with the data reported earlier [1,2].MR data of compounds 2 and 5 published earlier [1] wereow completed utilizing presently available advanced NMRethods. Majority of the previously obtained major and minor

cdysteroids [1,2], including some of those presented in thisaper, were scheduled for ecdysteroid receptor mapping basedn their interaction with the ligand-binding domain in the

II bioassay [9–12,24]. Our results, published earlier [9], wereaken over also to other models, using a homology modelingnd docking approach [25].

The structures of minor constituents 1, 3, 4, 6–8, 10, 11Fig. 1) and 12–18, 20–22 (Fig. 2) were elucidated by analy-is of their IR, mass, and NMR spectra (for 1H and 13C NMRata, see Tables 2–5). The 1H and 13C 1D spectra togetherith 1H, 1H-COSY and 1H, 13C-HMQC spectra were used

or complete (or nearly complete) structure assignment ofarbon and proton signals. Characteristic NMR data of com-ounds 1–7 and 9 (Tables 2 and 3) and 14–17, 19 and 22

Tables 4 and 5) correspond with the already published data

f ecdysone (1) [26], 20-hydroxyecdysone 2-acetate (3) [27,28],0-hydroxyecdysone 3-acetate (4) [27,28], turkesterone (6)29,30], inokosterone (7) [31,32], integristerone A (9) [2,33],4-epi-makisterone A (14) [34], 24(28)-dehydromakisterone A

hlormethane–isopropanol–water (84:15:1) at 0.8 ml min−1.tudy as authentic standards for HPLC and reference compounds for

(15) [17,35], 24(28)-dehydroamarasterone B (16) [18], (24Z)-29-hydroxy-24(28)-dehydromakisterone C (17) [2], makisterone C(19) [2,17] and amarasterone A (22) [36], all summarized inthe Ecdysone Handbook [20]. Compounds 1, 3, 4, 6, 7, 14, 20and 22 are new in L. carthamoides, although their occurrencewas already reported in other unrelated plants [20]. 24(28)-Dehydromakisterone A (15) was found also in the geneticallyrelated Rhaponticum integrifolium [35]. Some compounds werereported in earlier papers with rather incomplete NMR data,therefore we publish here their currently completed data(Tables 2–5). There were included herein also our referenceNMR data of 20-hydroxyecdysone (2) and ajugasterone C (5),previously isolated as major constituents of L. carthamoides[1], but reported only with data obtained in unrelated solvent(hexadeuteroacetone).

Eight new natural ecdysteroid analogues 8, 10–13, 18, 20and 21 (Figs. 1 and 2) were found among the isolated minorconstituents.

Compound 8 (inokosterone 20,22-acetonide), with thecomposition C30H48O7 (HR-MS) and characteristic IR bands,exhibited 1H and 13C NMR spectra very similar to those ofinokosterone 7 with two additional methyl signals in 1H NMRspectrum (singlets at ı 1.39 and 1.32) and three extra signalsin 13C NMR spectrum (ı 107.98 (〉C〈), 29.36 and 27.19 (2× CH3)

indicating a presence of acetonide group. The only significantchemical shift differences between 8 and 7 observed for car-bon atoms C-20 and C-22 (�ı = 7.93 and 4.91) allow to placeacetonide (isopropylidene) group unequivocally into position

504 s t e r o i d s 7 3 ( 2 0 0 8 ) 502–514

of c

Fig. 1 – Structures

20,22. The complete structural assignment of protons and car-bons in compound 8 is given in Tables 2 and 3.

Synthetic inokosterone 20,22-acetonide is already knownas intermediate compound (with a protected 20,22-diol sys-tem) prepared for transformation of 20E to inokosterone [37].

Compound 10 (integristerone 20,22-acetonide) with thecomposition C30H48O8 (HR-MS) exhibited 1H and 13C NMRspectra very similar to those of integristerone A (9) with twoadditional methyl signals in 1H NMR spectrum (singlets at ı

1.39 and 1.32) and three extra signals in 13C NMR spectrum(ı 108.03 (〉C〈), 28.95 and 27.18 (2× CH3) indicating again apresence of acetonide (isopropylidene) group in compound 10.Similarly as in above discussed compound 8 the only signifi-cant chemical shift differences between 10 and 9 are observedfor carbon atoms C-20 and C-22 (�ı = 7.94 and 4.93) and, there-fore, the acetonide group has to be placed into position 20,22.

The complete structural assignment of protons and carbonsin compound 10 is given in Tables 2 and 3. Also integristerone20,22-acetonide has been previously prepared, serving as astandard for HPLC analysis of ecdysteroids with various vicinal

ompounds 1–11.

diols [38]. Although the synthetic compounds 8 and 10 wereemployed to identify several ecdysteroid metabolites (by HPLCor HPLC-MS), their structural data were so far completely notreported.

Compound 11 (15-hydroxyponasterone A) with the com-position C27H44O7 (HR-MS) showed NMR spectra similar tothose of ponasterone A. The presence of additional secondaryOH group is manifested in 1H NMR spectrum (multiplet at ı

4.13) and 13C NMR spectrum (CH-OH at ı 76.84). The observedlow-field shift of C-16 (ı 35.59 in 11 against ı 21.51 in ponas-terone A) and coupling pattern of D-ring protons observedin 2D-COSY spectrum indicate that additional OH group isat position 15. The configuration 15�-OH follows from itsstrong influence on chemical shift of 18-methyl protons (ı1.137 in 11 against ı 0.890 in ponasterone A) together withlow-field shift of the olefinic proton H-7 (ı 6.47 in 11 com-

pared with ı 5.81 in ponasterone A). Further arguments, basedon the chemical shift changes induced by 15-OH group, wereachieved by the measurement of the 2D-ROESY spectrum.The H-15 (ı 4.13) shows the NOE contacts to H-7 (ı 6.47) and

s t e r o i d s 7 3 ( 2 0 0 8 ) 502–514 505

of co

tp�

t

Fig. 2 – Structures

o one of the H-16 (ı 2.24). Since it is known that methylrotons H-18 (d 1.137) have contacts to the protons on the-side of steroid skeleton (in our case we observed con-acts to H-11� (d ∼1.75), H-12� (ı 1.85) and H-16� (ı 1.92))

mpounds 12–22.

we could assigned the �-position to H-16 at ı 2.24. Fromthe inspection of models follows that while the NOE betweenH-7/H-15 can be observed for both H-15� as well as H-15�,the observed NOE contact to H-16� (d 2.24) and the absence

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73

(20

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)502–514

Table 2 – 1H NMR data of ecdysteroids 1–11 in CD3OD with coupling constants (in brackets)

Proton 1 2 3a 4 5 6 7 8b 9c 10b 11

1� 1.79 1.79 1.86 dd (13.2;

4.4)

1.93 dd (13.3; 4.4) 2.59 dd (13.0; 4.2) 2.59 dd (12.7;

4.1)

1.79 dd (13.5; 4.2) 1.78 dd (13.2; 4.3) 3.82 b 3.82 b 1.79

1� 1.43 dd (13.2;

12.0)

1.43 1.59 dd (13.4;

12.2)

1.43 dd (13.3; 12.2) 1.38 dd (13.0; 11.8) 1.38 dd (12.7;

12.0)

1.43 dd (13.5; 12.2) 1.43 dd (13.2; 12.0) – – 1.43

2 3.84 ddd (12.0;

4.2; 3.0)

3.84 dt (12.0; 4.2;

3.0)

4.98 ddd

(12.2; 4.4; 3.2)

3.98 ddd (12.2; 4.4;

3.2)

4.01 ddd (11.8; 4.2;

3.2)

4.01 ddd (12.0;

4.1; 3.0)

3.84 ddd (12.2; 4.2;

3.0)

3.83 ddd (12.0; 4.3;

3.2)

3.87 t (3.1;

3.1)

3.87 t (3.1;

3.1)

3.81 ddd

(12.2; 4.2;

3.0)

3 3.95 q (∼3; 3; 3) 3.94 q (∼3; 3; 3) 4.10 q (∼3; 3;

3)

5.15 bq (∼3; 3; 3) 3.96 q (∼3; 3; 3) 3.96 q (∼3; 3;

3)

3.95 q (∼3; 3; 3) 3.95 q (∼3; 3; 3) 4.04 ddd

(4.8; 3.5; 3.1)

4.04 b 3.96 q (∼3;

3; 3)

4� 1.69 1.69 1.84 ∼1.77 1.78 1.69 1.71-1.77 1.68-1.78 1.80 1.81 1.79

4� 1.76 1.74 1.72 ∼1.77 1.69 1.79 1.71-1.77 1.68-1.78 1.75 1.78 1.72

5 2.38 dd (12.8; 4.4) 2.38 dd (13.2;

4.8)

2.44 dd (13.2;

4.2)

2.22 dd (12.6; 4.8) 2.34 dd (13.2; 4.0) 2.33 ddd (13.0;

3.8)

2.38 dd (13.0; 4.4) 2.38 dd (12.8; 4.7) 2.61 dd

(12.0; 4.6)

2.61 dd

(12.4; 4.4)

2.37 dd

(13.2; 4.4)

7 5.82 d (2.5) 5.81 d (2.6] 5.83 d (2.6) 5.82 d (2.7) 5.80 dd (2.8; 0.9) 5.80 dd (2.7;

0.8)

5.81 d (2.6) 5.82 d (2.6) 5.80 d (2.6) 5.84 d (2.4) 6.47 d (2.6)

9 3.16 ddd (11.5;

7.0; 2.5)

3.15 ddd (11.3;

7.1; 2.6)

3.23 m 3.17 ddd (11.5; 7.0;

2.7)

3.15 dd (8.8; 2.8) 3.15 dd (8.8;

2.7)

3.15 ddd (11.5; 7.0;

2.6)

3.15 ddd (11.5; 7.0;

2.6)

3.08 bt 3.07 bt 3.13 ddd

(11.5; 7.0;

2.6)

11� 1.81 1.81 1.85 1.82 – – 1.81 1.80 ∼1.79 1.73 1.75–1.80

11� 1.67 1.70 1.70 1.72 4.10 ddd (10.6; 8.8;

6.1)

4.10 ddd (10.6;

8.8; 6.2)

1.71 1.69 ∼1.70 1.70 1.75–1.80

12� 2.11 td (13; 13; 5) 2.13 td (13; 13; 5) 2.16 td (13;

13; 5)

2.15 td (13; 13; 5) 2.21 dd (12.3; 10.6) 2.22 dd (12.3;

10.6)

2.12 td (13; 13; 5) 2.11 td (13; 13; 4.8) 2.11 td (12.8;

12.8; 5.1)

2.09 td (13;

13; 5)

2.11 td (13;

13; 5)

12� 1.77 1.88 1.88 1.89 2.15 dd (12.3; 6.1) 2.16 dd (12.3;

6.2)

1.88 1.85 1.87 1.83 1.85

15� 1.96 1.97 1.97 1.96 1.97 1.97 1.96 1.96 2.00 1.97 4.13 m

15� 1.60 1.60 1.60 1.60 1.58 1.58 1.60 1.63 1.60 1.62 –

16� 1.50 1.73 1.74 1.75 1.72 1.75 1.72 1.86 1.73 1.87 1.92

16� 1.96 1.98 1.99 1.99 1.99 1.98 2.00 2.04 1.97 2.04 2.24

17 2.03 2.39 t (9.5; 9.5) 2.40 dd (∼9.5;

8.5)

2.40 dd (9.8; 8.3) 2.41 dd (10.0; 8.5) 2.43 bt (10.0;

8.5)

2.37 t (9.0; 9.0) 2.29 dd (9.6; 8.2) 2.39 dd

(∼9.5; 8.5)

2.31 dd (9.2;

8.4)

2.25

18 0.734 s 0.891 s 0.892 s 0.896 s 0.873 s 0.877 s 0.890 s 0.826 s 0.90 s 0.840 s 1.137 s

19 0.970 s 0.968 s 0.992 s 1.001 s 1.057 s 1.058 s 0.967 s 0.963 s 0.91 s 1.074 s 1.002 s

20 1.75 – – – – – – – – – –

21 0.953 d (6.7) 1.199 s 1.194 s 1.192 s 1.197 s 1.221 s 1.178 s 1.162 s 1.19 s 1.175 s 1.188 s

22 3.59 ddd (10.2;

3.0; 1.8)

3.32 dd (10.6;

1.8)

3.33 dd (11.0;

1.8)

3.33 dd (10.5; 1.1) 3.32 dd (10.6; 1.8) 3.32 dd (∼11;

1.6)

3.33 dd (10.3; 1.6) 3.69 dd (9.4; 2.9) 3.32 dd

(11.0; 1.8)

3.68 dd (8.2;

3.5)

3.30 dd

(∼11; 1.5)

23a 1.54 1.66 1.30 1.29 1.56 1.66 1.63 ∼1.47 1.66 ∼1.52 1.48

23b 1.32 1.28 1.66 1.69 1.22 1.29 1.19 ∼1.47 1.29 ∼1.52 1.22

24a 1.78 1.79 1.80 1.81 1.47 1.79 1.71 1.68 1.78 1.73 1.51

24b 1.41 1.43 1.44 1.44 1.23 1.43 1.11 1.15 1.43 1.49 1.22

25 – – – – 1.58 – 1.60 1.63 – – 1.57

26 1.193 s 1.191 s 1.200 s 1.200 s 0.916 d (6.8) 1.193 s 3.46 dd (10.8; 5.6)

3.34 dd (10.8; 6.8)

3.36 dd (10.7; 5.8)

3.43 dd (10.7; 6.4)

1.19 s 1.195 s 0.908 d (6.5)

27 1.204 s 1.205 s 1.207 s 1.206 s 0.926 d (6.8) 1.207 s 0.945 d (6.8) 0.937 d (6.6) 1.20 s 1.204 s 0.920 d (6.5)

a OAc: 2.07 s.b 〉C(CH3)2: 1.39 s, 1.32 s.c Data taken from ref. [2].

s t e r o i d s 7 3 ( 2 0 0 8 ) 502–514 507

Table 3 – 13C NMR data of ecdysteroids 1–11 in CD3OD

C 1 2 3a 4b 5 6 7 8c 9d 10e 11

1 37.33 37.33 33.98 38.40 39.03 39.04 37.34 37.35 76.43 76.41 37.402 68.69 68.68 72.72 67.05 68.91 68.92 68.70 68.72 68.50 68.49 68.663 68.50 68.50 66.13 71.78 68.53 68.55 68.51 68.49 70.98 70.98 68.594 32.89 32.86 32.78 30.29 33.27 33.27 32.87 32.87 33.48 33.49 32.705 51.79 51.78 51.68 52.55 52.74 52.76 51.79 51.77 46.79 46.79 51.476 206.55 206.46 205.74 205.38 206.66 206.67 206.50 206.43 205.57 205.58 206.687 122.01 122.13 122.22 122.00 122.71 122.73 122.15 122.15 122.17 122.18 124.568 167.63 167.98 168.05 168.30 165.72 165.74 167.94 167.56 167.19 166.85 165.779 35.23 35.07 34.96 35.20 42.89 42.90 35.09 35.14 35.65 35.68 35.17

10 39.24 39.26 39.50 39.20 39.88 39.89 39.27 39.21 43.80 43.75 39.5811 21.57 21.48 21.51 21.49 69.48 69.50 21.50 21.51 21.91 21.89 21.2812 32.03f 32.50 32.45 32.49 43.75 43.77 32.52 32.32 32.54 32.31 34.0113 48.11 48.60 ∼49.0f ∼49.0f 48.57 48.57 ∼49.0f 48.44 ∼49.0f ∼49.0f 47.4814 85.07 85.21 85.19 85.20 84.84 84.85 85.26 85.34 85.10 85.15 85.1915 32.07f 31.78 31.82 31.80 31.82 31.86 31.77 31.72 31.80 31.74 76.8416 27.00 21.48 21.51 21.49 21.50 21.50 21.50 22.43 21.40 22.33 35.5917 48.79 50.51 50.54 50.54 50.25 50.33 50.48 50.46 50.58 51.53 49.9618 16.18 18.06 18.04 18.04 18.88 18.90 18.04 17.69 18.02 17.67 18.1119 24.46 24.41 24.23 24.34 24.62 24.62 24.41 24.45 20.01 20.07 24.2220 43.46 77.90 77.92 77.90 77.75 77.82 77.82 85.75 77.89 85.83 77.5821 13.22 21.04 21.05 21.05 20.95 21.01 21.01 22.54 21.05 22.58 21.0022 75.24 78.41 78.44 78.43 77.92 78.40 78.22 83.13 78.43 83.30 77.9223 25.30 27.32 27.34 27.35 30.46 27.32 30.17 27.43 27.36 24.70 37.6224 42.25 42.39 42.41 42.39 37.64 42.39 32.10 32.05 42.38 42.21 30.3825 71.41 71.30 71.31 71.30 29.22 71.30 37.06 37.01 71.29 71.11 29.2226 29.05 28.92 28.95 28.94 22.76 28.91 68.13 68.23 28.97 29.34 22.7327 29.61 29.72 29.72 29.72 23.45 29.75 17.52 17.04 29.70 29.49 23.42

a 2-OAc: 172.41, 21.13.b 3-OAc: 172.54, 21.12.c 〉C(CH3)2: 107.98, 29.36, 27.19.d Data taken from ref. [2].

oOai

csbmHuupcsvupp(

s3cs

[6], and recently reported also as naturally occurring con-stituent of Serratula wolffii [39]. The 14�-OH configuration wasconfirmed in 2D-H,H-ROESY spectrum by observation of NOEcontacts of well separated protons H-7 and H-9 (see Fig. 4). Pro-

Fig. 3 – Proton chemical shifts and coupling constants

e 〉C(CH3)2: 108.03, 28.95, 27.18.f The signal is overlapped by solvent.

f the NOE contact H-18/H-15 bring further evidences for 15�-H configuration in compound 11. The complete structuralssignment of protons and carbons in compound 11 is givenn Tables 2 and 3.

Compound 12 (14-epi-ponasterone A 22-glucoside) with theomposition C33H55O11 (HR-MS) manifests in NMR spectra alltructural features of ponasterone A, but six additional car-on atoms and their chemical shifts indicate a presence ofonosaccharide unit. The partial overlap of proton signals-3′, H-4′ and H-5′ in CD3OD (see Table 4) does not allownambiguous structure determination of the monosaccharidenit. Fortunately, the repeated measurement in d5-pyridinerovided sufficiently resolved of NMR signals of all monosac-haride protons and allowed us to determine all chemicalhifts and coupling constants (see Fig. 3). High values of allicinal couplings in hexopyranose ring (J = 7.8–9.4 Hz) confirmnambiguously the structure of �-d-glucose [13]. The linkageosition of glucose into position 22 follows from the cross-eaks between H-22 (ı 3.58)/C-1′ (ı 105.95) and H-1′ (ı 4.34)/C-22

ı 90.44) observed in the H,C-2D-HMBC spectrum.Significant downfield shift of 18-Me protons and upfield

hift of H-9 (H-18: ı 1.26 against ı 0.89 and H-9: ı 2.85 against.15 when compare 12 with ponasterone A) suggest 14�-onfiguration of OH group. Similar shifts have been observedome time ago in the case of 14-epi-20-hydroxyecdysone [20],

first prepared by phototransformation of 20-hydroxyecdysone

(indicated with arrows) of the monosaccharide unit ofcompound 12 in d5-pyridine. High values of all vicinalcouplings in hexopyranose ring (J = 7.8–9.4 Hz) confirm thestructure of �-d-glucose.

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)502–514

Table 4 – 1H NMR data of ecdysteroids 12–22 in CD3OD with coupling constants (in brackets).

Proton 12a 13 14 15 16 17b 18 19b 20 21 22

1� 1.80 dd

(13.3; 4.2)

2.60 ddd (12.8; 4.0) 1.80 dd (13.3;

4.4)

1.80 dd (13.4; 4.2) 1.80 dd (13.2;

4.2)

1.80 dd (13.2; 4.0) 1.78 1.79 dd (13.0; 4.5) 1.79 3.82 b 1.79 dd (13.4; 4.2)

1� 1.43 dd

(13.2; 12.0)

1.37 dd (12.8; 11.8) 1.43 dd (13.3;

12.2)

1.43 dd (13.4; 12.0) 1.43 dd (13.2;

11.8)

1.43 dd (13.2; 12.0) 1.43 1.43 dd (13.0; 12.2) 1.43 – 1.43 dd (13.4; 12.0)

2 3.86 ddd

(12.0; 4.2;

3.0)

4.01 ddd (11.8; 4.0;

3.0)

3.84 ddd (12.2;

4.4; 3.2)

3.84 ddd (12.0; 4.2;

3.0)

3.84 ddd (11.8;

4.2; 3.0)

3.84 ddd (12.0; 4.0;

3.0)

3.83 ddd (12.0; 4.2;

3.0)

3.84 ddd (12.2; 4.5;

3.0)

3.84 ddd (12.0;

4.2; 3.0)

3.87 t (3.1; 3.1) 3.83 ddd (12.0; 4.2;

3.0)

3 3.94 q (∼3;

3; 3)

3.95 q (∼3; 3; 3) 3.95 q (∼3; 3; 3) 3.95 q (∼3; 3; 3) 3.95 q (∼3; 3; 3) 3.95 bq (∼3; 3; 3) 3.95 q (∼3; 3; 3) 3.95 q (∼3; 3; 3) 3.95 q (∼3; 3; 3) 4.04 b 3.95 q ((∼3; 3; 3)

4� 1.73 1.77 ∼1.71 ∼1.73 1.69-1.76 ∼1.73 ∼1.70 1.74 1.68-1.76 ∼1.80 1.70-1.75

4� 1.60 1.69 ∼1.71 ∼1.70 1.69-1.76 ∼1.73 ∼1.70 1.70 1.68-1.76 ∼1.80 1.70-1.75

5 2.36 dd

(13.2; 4.0)

2.33 dd (13.2; 4.0) 2.39 dd (12.7;

4.4)

2.39 dd (12.6; 4.4) 2.38 dd (12.6;

4.6)

2.39 dd (12.3; 4.8) 2.38 dd (12.6; 4.4) 2.38 dd (12.7; 4.6) 2.39 dd (12.8;

4.8)

2.61 dd (12.5;

4.5)

2.38 dd (12.6; 4.5)

7 6.32 dd (2.4;

0.8)

5.82 dd (2.6; 0.7) 5.81 d (2.6) 5.82 d (2.6) 5.81 d (2.5) 5.81 d (2.6) 5.81 d (2.5) 5.81 d (2.6) 5.81 d (2.6) 5.83 d (2.5) 5.81 d (2.6)

9 2.85 m 3.15 dd (9.2; 2.6) 3.15 ddd (11.5;

7.0; 2.6)

3.16 ddd (11.5; 7.0;

2.6)

3.16 ddd (11; 7;

2.5)

3.16 ddd (11.5; 7.4;

2.6)

3.15 ddd (11.5; 7;

2.5)

3.16 ddd (11.4; 7;

2.6)

3.16 m (11.5; 7;

2.6)

3.08 bt 3.15 ddd (11.5; 7; 2.6)

11� 1.79 – 1.81 1.82 1.82 ∼1.82 1.80 1.80 1.80 1.78 1.81

11� 1.66 4.06 ddd (10.9; 9.2;

5.7)

1.71 1.70 1.71 ∼1.70 1.69 1.71 1.71 1.70 1.70

12� 1.75 2.24 dd (12.2; 10.9) 2.13 2.14 2.14 2.15 td (13; 13; 5) 2.11 td (13; 13; 5) 2.13 td (13; 13; 5) 2.13 td (13; 13; 5) 2.10 td (13; 13;

5)

2.11 td (13; 13; 5)

12� 1.69 2.14 dd (12.2; 5.7) 1.88 1.89 1.88 1.88 1.83 1.88 1.87 1.87 1.88

15� 2.28 1.89 1.96 1.98 1.97 1.96 1.95 1.96 1.96 1.99 1.96

15� 1.43 1.54 1.59 1.61 1.62 1.61 1.62 1.61 1.62 1.62 1.61

16� 1.86 2.07 1.77 1.80 1.78 1.83 1.86 1.76 1.78 1.76 1.75

16� 2.08 2.28 2.00 2.04 2.03 2.02 1.92 2.03 2.04 2.03 2.00

17 1.91 2.66 dd (9.8; 8.2) 2.38 dd (∼10; 8) 2.42 dd (9.4; 8.0) 2.43 dd (9.6; 8.3) 2.42 dd (9.5; 8.0) 2.27 dd (9.8; 8.2) 2.41 dd (9.5; 8.5) 2.39 dd (10.0;

8.8)

2.41 dd (9.5;

8.5)

2.34 dd (9.8; 8.4)

18 1.26 s 0.850 s 0.889 s 0.901 s 0.898 s 0.90 s 0.854 s 0.90 s 0.896 s 0.910 s 0.894 s

19 0.94 s 1.053 s 0.967 s 0.971 s 0.970 s 0.97 s 0.963 s 0.97 s 0.967 s 1.079 s 0.968 s

21 1.32 s 1.226 s 1.140 s 1.234 s 1.218 s 1.22 s 1.264 s 1.20 s 1.196 s 1.196 s 1.179 s

22 3.58 dd

(10.0; 1.5)

3.82 bd (5.6; 0.5) 3.48 dd (9.5; 2.2) 3.60 dd (10.7; 2.0) 3.60 dd (10.6;

1.5)

3.55 dd (10.4; 2.3) 1.68 1.47 3.42 dd (11.1; 1.8) 3.45 dd (10.8;

2.4)

3.41 dd (10.8;

1.7)

3.45 dd (10.4; 2.3)

23a 1.57 2.34 dd (14.3; 5.6) 1.86 2.14 dd (14.4; 10.7) 2.30 2.31 1.28 1.55 1.45 1.56 ∼1.21

23b 1.48 1.87 dd (14.3; 0.5) 1.06 2.39 dd (14.4; 2.0)) 1.86 2.16 1.06 1.40 1.37 1.41 ∼1.21

24a 1.74 – 1.65 – – – 1.23 1.15 1.66 1.47 1.68

24b 1.24 – – – – – – – – – –

25 1.56 1.81 – – 2.85 h (6x 6.8) – – – – – 1.85

26 0.93 d (6.8) 0.924 d (7.1) 1.189 s 1.321 s 1.049 d (6.8) 1.42 s 1.233 s 1.22 s 3.58 d (11.7) 3.35

d (11.7)

1.105 s 3.50 dd (11.0; 7.3)

3.40 dd (11.0; 6.8)

27 0.93 d (6.8) 0.985 d (7.0) 1.193 s 1.375 s 1.049 d (6.8) 1.36 s 1.247 s 1.11 s 1.061 s 1.215 s 0.786 d (7.0)

28 – 3.62 d (10.8) 3.54 d

(10.8)

1.034 d (6.8) 4.96 b 5.13 b 5.46 bt (6.7; 6.7) 5.43 bt (6.2; 6.2) 3.90 dq (9.0; 6.2;

6.2; 6.2)

1.58 1.48 1.79 1.20 1.57 1.15 1.51 1.28

29 – – – – 4.17 d (6.7) 4.31 dd (14.0; 6.2)

4.28 dd (14.0; 6.2)

1.234 d (6.2) 1.02 t (7.4.) 1.030 t (7.4; 7.4) 1.015 t (7.4; 7.4) 0.935 t (6.4; 6.4)

a �-d-Glc: 4.34 (d, J = 7.8 Hz, H-1′), 3.26 (dd, J = 7.8, 8.8 Hz, H-2′), ∼3.36 (m, H-3′, H-4′, H-5′), 3.69 (dd, J = 11.8; 5.5 Hz, H-6′a), 3.87 (dd, J = 11.8; 1.8 Hz, H-6′b).b Data taken from ref. [2].

s t e r o i d s 7 3 ( 2 0 0 8 ) 502–514 509

Table 5 – 13C NMR data of ecdysteroids 12–22 in CD3OD

C 12a 13 14 15 16 17b,c 18 19b 20 21 22

1 37.22 39.80 37.42 37.41 37.36 37.54 37.39 37.37 37.36 76.45 37.362 68.67 68.94 68.73 68.72 68.70 68.78 68.72 68.71 68.71 68.47 68.703 68.39 68.55 68.54 68.53 68.50 68.60 68.52 68.53 68.51 71.00 68.524 ∼32.90 33.40 32.86 32.84 32.86 32.84 ∼33.00 32.87 32.87 33.52 32.865 50.98 52.79 51.81 51.80 51.78 51.85 51.81 51.80 51.80 46.78 51.796 206.22 206.91 206.44 206.42 206.44 206.40 ∼206.30 206.50 206.44 205.55 206.447 123.14 122.65 122.17 122.16 122.11 122.20 122.12 122.12 122.14 122.16 122.148 167.38 166.17 167.89 167.88 167.95 167.83 167.97 168.04 167.86 167.23 167.949 37.25 42.93 35.14 35.13 35.09 35.24 ∼34.80 35.12 35.10 35.63 35.10

10 39.21 39.11 39.28 39.27 39.24 39.31 39.27 39.27 39.26 43.80 39.2611 ∼22.20 69.66 21.55 21.55 21.53d 21.61 ∼21.60 21.54d 21.63 21.91 21.5312 41.90 43.87 32.50 32.51 32.49 32.56 32.46 32.51 32.49 32.49 32.5213 52.77 ∼49.0e ∼49.0e ∼49.0e 48.56 ∼49.0e ∼49.0e ∼49.0e ∼49.17 ∼49.0e ∼49.0e

14 85.47 84.65 85.31 85.28 85.20 85.33 85.53 85.20 85.19 85.04 85.2115 41.14 32.31 31.76 31.78 31.75 31.81 31.60 31.80 31.81 31.82 31.7916 24.43 23.33 21.62 21.55 21.71d 21.61 22.02 21.62d 21.63 21.51 21.4717 57.30 52.19 50.42 50.50 50.46 50.57 53.11 50.43 50.39 50.45 50.3418 19.37 19.04 18.00 18.04 18.05 18.01 18.10 18.06 18.03 18.04 18.0619 24.67 24.59 24.39 24.40 24.41 24.38 24.40 24.40 24.41 20.03 24.4120 77.07 89.72d 78.05 78.04 77.79 77.86 75.85 78.04 77.91 77.98 77.9621 21.83 27.60 20.72 21.02 21.01 21.00 26.28 20.96 20.82 20.95 20.9022 90.44 82.62 77.83 77.81 75.94 78.20 46.48 77.22 77.57 77.22 75.7123 31.00 41.33 35.18 34.63 34.71 38.01 24.61 33.06 33.40 33.04 32.0224 36.44 89.56d 44.40 155.36 145.75 147.18 56.44 50.30 45.30 50.29 37.8525 29.36 40.37 74.06 73.61 30.73 73.94 76.12 74.14 76.41 74.12 37.6026 22.84 18.12 25.92 29.76 18.41 30.24 24.82 25.97 69.08 25.60 66.7527 23.25 18.60 28.20 30.22 21.87 30.94 29.64 29.09 21.01 29.09 11.5128 – 64.41 16.85 110.38 125.95 128.78 71.69 25.61 25.04 25.97 25.4029 – – – – 58.91 60.46 22.64 14.35 14.30 14.35 12.29

a �-d-Glc: 105.95, 75.27, 77.94, 77.90, 71.41, 62.31.b Data taken from ref [2].

twNdHsccrs

FNc

c Data at 50 ◦C.d The assignment of signals may be mutually interchanged.e The signal is overlapped by solvent.

on H-7 (at ı 6.32 dd) shows the NOE only to H-15� (at ı 1.43 m)hile in the case of 14�-OH configuration the H-7 should giveOE contacts to both H-15� and H-15� protons. Further evi-ence comes from the observation of the NOE contact between-9 (at ı 2.85 m) and H-15� (at ı 2.28 m)—these protons appear

ufficiently close only in case of 14�-OH configuration. The

omplete structural assignment of protons and carbons inompound 12 is given in Tables 4 and 5. Data analysis cor-oborates the 14-epi-ponasterone A 22-O-�-d-glucopyranosidetructure for the compound 12.

ig. 4 – Partial structure of compound 12 and characteristicOEs (H-7/H-15� and H-9/H-15�) proving the 14�-OHonfiguration.

Compound 13 (carthamoleusterone) with the compositionC28H44O8 (HR-MS) showed NMR spectra with no structuralchanges in ring A and B, additional OH group in position11� (CH-OH: ı 4.06 ddd, J = 10.9, 9.2 and 5.7 Hz and CH-OH:ı 69.66) and modified side-chain with three oxygen atomsand one additional ring. On the basis of 1H and 13C chem-ical shifts, H,H-COSY, H,C-HSQC and H,C-HMBC spectra theside-chain should contain the following structure features (seeFig. 5).

In principle the six possible side-chain structures can beconstructed by closing ether ring by connection of the onepair of four free valences A, B, C, D (with removing one ofending oxygen atom) and containing two OH groups at tworemaining free valences. For distinguishing between alterna-tive structures we have used the in situ TAI-acylation method[40]. The 1H and 13C NMR spectra of compound 13 were mea-sured in d6-acetone and then after the addition of TAI. Theobserved TAI-acylation shifts (see Fig. 6) are compatible onlywith a structure containing five-membered tetrahydrofuranering closed between C-20 and C-24 and one primary and onesecondary OH group.

The relative configuration of substituents at positions 22and 24 (as it is shown in Fig. 7) was derived from the observedNOE contacts. Complete structural assignment of protons andcarbons in compound 13 is given in Tables 4 and 5.

510 s t e r o i d s 7 3 ( 2 0 0 8 ) 502–514

Fig. 5 – Structure features of the side-chain in compound 13Fig. 7 – The side-chain of compound 13 with couplingconstants of hydrogens H-22 and H-23, and characteristic

evidenced by 1H and 13C NMR spectra. The arrows indicateHMBC contacts.

Compound 18 (22deoxy-28-hydroxymakisterone C) withthe composition C29H48O7 (HR-MS) resembles makisterone Cby its molecular formula as well as NMR spectra. There are nochanges observed on ecdysteroid skeleton but from detailedanalysis of NMR spectra of side-chain follows that OH groupis placed in position 28 instead of 22. The complete structuralassignment of protons and carbons in compound 18 is givenin Tables 4 and 5.

Compound 20 (26-hydroxymakisterone C) with the compo-sition C29H48O8 (HR-MS) also resembles makisterone C withadditional OH group according to NMR spectra. There are nochanges observed on ecdysteroid skeleton, but from detailedanalysis of NMR spectra of the side-chain follows that 26-methyl group is replaced by CH2-OH (ı(H) 3.58 d and 3.35d, Jgem = 11.7 Hz; ı(C) 69.08). The complete structural assign-ment of protons and carbons in compound 20 is given inTables 4 and 5.

Compound 21 (1�-hydroxymakisterone C) has the compo-sition C29H48O8 (HR-MS). Comparison of its NMR spectra with

those of makisterone C showed a close similarity with onlydifference represented by additional OH group. The position ofadditional OH at C-1 follows from the coupling pattern in the2D-COSY spectrum. Proton H-2 (at ı 3.87) is coupled to H-3 (at

Fig. 6 – The structure of side-chain in compound 13 and theTAI-acylation shifts observed in 1H (bold) and 13C (italics)NMR spectra.

NOEs supporting the relative configurations at positions 22and 24.

ı 4.04) and another CH-OH (at ı 3.82) that has to be H-1 and inaccordance with its position it has no other coupling partners.The axial H-2 gives a triplet with small J(2,3) = J(2,1) = 3.1 Hz. Itmeans that H-1 as well as H-3 adopts the equatorial positionswith �-configuration of OH group at the C-1. The completestructural assignment of protons and carbons in compound21 is given in Tables 4 and 5.

Majority of described Leuzea ecdysteroids were testedin the above mentioned Drosophila BII bioassay for ecdys-teroid agonist and antagonist activities [41], in which thepotency reflects the affinity of the test compound for theligand-binding site of the Drosophila melanogaster ecdysteroidreceptor. The activity data have been used to investigatethe structure–activity relationship [7,9–12,24,42]. Most of ourtested ecdysteroids interacted with the ligand-binding site asagonists.

Some selected major Leuzea ecdysteroids were alsotested for their systemic effect on aphids [14] and onimmunomodulatory activity triggered by lipopolysaccharideand interferon-� under in vitro conditions using murine res-ident peritoneal macrophages [43]. The series of test agentsencompassed ecdysteroids occurring often as major com-ponents in several plant extracts: 20-hydroxyecdysone (2),polypodine B (i.e. 5�-hydroxy derivative of 2) [1], ajugasteroneC (5), inokosterone (7), makisterone A (i.e. 24-epimer of 14)[2], carthamosterone (i.e. 25,29-sidechain lactone derived from17) [2] and ponasterone A (25-deoxy derivative of 2) [7]. Allcompounds, except of ponasterone A, represent Leuzea con-stituents and are supposed to be significant for the oftenreported pharmacological activities of preparations derivedfrom this species [20,21]. However, the tested ecdysteroidsdid not interfere with the immunobiological activity of theimmunocompetent cells [43].

2. Experimental

Infrared spectra were recorded on a Bruker IPS-88 instrumentusing KBr pellets. Optical rotations were measured at 20 ◦Con a Rudolph Research Analytical Autopol IV polarimeter inethanol. NMR spectra were measured on a Varian UNITY-500

2 0 0

o1e(2aosfiatirista

iGiecpcmcoTscNfr

psNtT

2

Cspa3MC

2

Cs((((

s t e r o i d s 7 3 (

r Bruker Avance-500 spectrometer (1H at 500 MHz; 13C at25.7 MHz) in CD3OD. Chemical shifts in CD3OD were refer-nced to the solvent signal at 3.31 ppm (1H) and 49.00 ppm

13C). The 2D-H,H-COSY, 2D-H,H-ROESY, 2D-H,C-HMQC andD-H,C-HMBC spectra were used for structural assignment ofll hydrogen and carbon atoms. Mass spectra were measuredn a Waters Q-tof micro spectrometer equipped with electro-pray ion source. Samples were dissolved in acetonitrile/0.1%ormic acid (1:1, v/v) and infused into the ion source. Cap-llary voltage was 3.0 kV, sample cone was 20 V, and sourcend desolvation temperatures were 80 and 150 ◦C, respec-ively. Collision energy for MS/MS experiments was adjustedn the range of 10–20 V. Positive ions were recorded. The accu-ate masses of the molecular adducts were calculated fromnternally calibrated mass spectra using polyethylene glycoltandards. Some samples were measured on a ZAB-EQ spec-rometer with fast atom bombardment (FAB) ionization usingglycerol–thioglycerol matrix.

Enriched ecdysteroid-containing fractions were preparedn cooperation with Mediplant Co. (Modra, Slovakia) andalena Co. (Opava, Czech Republic) using a pilot plant mod-

fication of the extraction and separation methods reportedarlier [1,2,44]. Roots of L. carthamoides (1000 kg) were pur-hased from several agricultural producers. Separations wereerformed by liquid–liquid extractions, large-scale columnhromatography and crystallization processes. Besides theain product, 20-hydroxyecdysone (1 kg, 95% purity), several

rude fractions of ecdysteroids were obtained [2], with vari-us qualitative and quantitative compositions monitored byLC [3,45] and HPLC [2,46] methods. Single ecdysteroids wereeparated from selected fractions by combination of LH-20 gelolumn chromatography with RP-HPLC, and finally purifiedP-HPLC. The separation procedure details (yielding primary

ractions 1A-29A and following set of purified fractions B) wereeported in our previous paper [2].

Structure elucidation and identification of isolated com-ounds was performed by analysis of their 1H and 13C NMRpectra, supported by IR and MS spectral data. 1H and 13CMR spectral data are summarized in Tables 2–5. Characteris-

ic HPLC data recorded under analytic conditions are given inable 1.

.1. Ecdysone (1)

ompound 1 (12 mg) was obtained from fraction 12A (1.18 g)eparated by RP-HPLC in System 1 (Table 1, modified forreparative purposes). Fraction 6B (104 mg) was further sep-rated in modified System 2 and 3 (Table 1). IR (KBr), cm−1:339, 3472 (OH), 1644 (C O), 1073, 1056, 1042 (C O). HR ESI-S, m/z: 487.3048 [M+Na]+, for C27H44O6Na required 487.3036.omposition C27H44O6 (M: 464).

.2. 20-Hydroxyecdysone 2-acetate (3)

ompound 3 (14 mg) was obtained from fraction 11A (1.65 g)eparated by RP-HPLC in modified System 1. Fraction 19B

105 mg) containing compound 3 was purified in System 2Table 1). IR (KBr), cm−1: 3435 (OH), 1654 (C O), 1738, 1716MeC O), 1073, 1047, 1030 (C O). FAB-MS m/z (relat. int.): 54566) [M+Na]+, 523 (47) [M+H]+, 505 (65) [M+H-H2O]+, 487 (100)

8 ) 502–514 511

[M+H-2H2O]+, 469 (62) [M+H-3H2O]+, 455 (20), 453 (20), 439 (22),423 (25), 413 (23), 401 (30), 387 (35), 371 (55), 345 (60). HR FAB-MS m/z: 545.3122 [M+Na]+, for C29H46O8Na required 545.3090.Composition C29H46O8 (M: 522).

2.3. 20-Hydroxyecdysone 3-acetate (4)

Compound 4 (17 mg) was obtained from fraction 11A (1.65 g)after repeated chromatography in System 1 and 2 (Table 1).IR (KBr), cm−1: 3414 (OH), 1655 (C O), 1740, 1724 (MeC O),1074, 1059 (C O). ESI-MS/MS of m/z 523 [M+H]+: Multiple lossof water: m/z 505 [M+H-H2O]+, m/z 487 [M+H-2H2O]+, m/z 469[M+H-3H2O]+; loss of 2-propanol from the side-chain or loss ofacetic acid: m/z 463 [M+H-60]+; multiple loss of water from m/z463: m/z 445 [M+H-60-H2O]+, m/z 427 [M+H-60-2H2O]+, m/z 409[M+H-60-3H2O]+, m/z 391 [M+H-60-4H2O]+; loss of water (2×)and C4H10O (isobutanol): m/z 413; loss of water (2×), aceticacid, and isobutanol: m/z 353; loss of acetic acid, cleavagein the side-chain: m/z 345. HR ESI-MS m/z: 545.3110 [M+Na]+,for C29H46O8Na required 545.3090. Composition C29H46O8 (M:522).

2.4. Turkesterone (6)

Compound 6 (7 mg) was obtained from fraction 4B of 7A(650 mg) after repeated chromatography in System 2 (Table 1).IR (KBr), cm−1: 3375 (OH), 1660 (C O), 1057 (C O). ESI-MS m/z(relat. int.): 519 (50) [M+Na]+, 497 (7) [M+H]+, 479 (94) [M+H-H2O]+, 461 (51) [M+H-2H2O]+, 443 (100) [M+H-3H2O]+, 425 (60)[M+H-4H2O]+, 407 (19) [M+H-5H2O]+, 387 (17), 317 (57). HR ESI-MS m/z: 519.2968 [M+Na]+, for C27H44O8Na required 519.2934.Composition C27H44O8 (M: 496).

2.5. Inokosterone (7)

Compound 7 (318 mg) was obtained from fraction 12B of 13A(1.2 g) by crystallization. IR (KBr), cm−1: 3380 (OH), 1646 (C O),1066 (C O). HR FAB-MS m/z: 481.3159 [M−H]+, for C27H45O7

required 481.3165. Composition C27H46O7 (M: 480).

2.6. Inokosterone 20,22-acetonide (8)

Compound 8 (20 mg) was obtained from fraction 15B of 21Aand 22A (1.6 g) after repeated chromatography in System 2(Table 1). [�]D20 + 42.9◦ (c = 0.29, EtOH). IR (KBr) cm−1: 3429 (OH),1655 (C O), 1109, 1055 (C O). FAB-MS m/z (relat. int.): 543(45) [M+Na]+, 521 (80) [M+H]+, 503 (100) [M+H-H2O]+, 445 (20),427 (15), 329 (20), 301 (22), 279 (12), 249 (19). HR ESI-MS m/z:543.3282 [M+Na]+, for C30H48O7Na required 543.3290. Compo-sition C30H48O7 (M: 520).

2.7. Integristerone A 20,22-acetonide (10)

Compound 10 (7 mg) was obtained from fraction 18A (1.9 g)separated by RP-HPLC in System 1 and further separated inSystem 2 and 3 (Table 1). [�] 20 + 31.6◦ (c = 0.23, EtOH). IR (KBr),

D

cm−1: 3401 (OH), 1655 (C O), 1077, 1062 (C O). ESI-MS/MS ofm/z 537 [M+H]+: Multiple loss of water: m/z 519 [M+H-H2O]+,m/z 501 [M+H-2H2O]+; loss of acetone (from acetonide): m/z479 [M+H-C3H6O]+; loss of acetone and multiple loss of water:

( 2 0

512 s t e r o i d s 7 3

m/z 461 [M+H-C3H8O-H2O]+, m/z 443 [M+H-C3H8O-2H2O]+ m/z425 [M+H-C3H8O-3H2O]+. HR ESI-MS m/z: 559.3243 [M+Na]+,for C30H48O8Na required 559.3247. Composition C30H48O8 (M:536).

2.8. 15-Hydroxyponasterone A (11)

Compound 11 (13 mg) was obtained from fraction 15A and16A (2.15 g) after several repeated chromatography in Systems1, 2 and 3. Characteristic HPLC data recorded under analyticconditions are summarized in Table 1. [�]D20 + 41.2◦ (c = 0.17,EtOH). IR (KBr), cm−1: 3585, 3563, 3370 (OH), 1646 (C O), 1055,1008, 988 (C O). ESI-MS/MS of m/z 481 [M+H]+: Multiple lossof water: m/z 463 [M+H-H2O]+, m/z 445 [M+H-2H2O]+, m/z 427[M+H-3H2O]+, m/z 409 [M+H-4H2O]+; loss of water (2×) andC3H8 (propane): m/z 401 [M+H-2H2O-C3H8]+; ring D cleavage:m/z 279; cleavage in the side-chain and loss of water: m/z 329;further loss of water from m/z 329: m/z 311, m/z 311; cleavageof the side-chain and loss of water: m/z 317, m/z 299. HR ESI-MS m/z: 503.2990 [M+Na]+, for C27H44O7Na required 503.2985.Composition C27H44O7 (M: 480).

2.9. 14-epi-Ponasterone A 22-O-ˇ-d-glucopyranoside(12)

Compound 12 (28 mg) was obtained from fraction 18B of 14A(2.4 g) by repeated HPLC in System 2 (Table 1). [�]D20 + 59.2◦

(c = 0.03, EtOH). IR (KBr), cm−1: 3413 (OH), 1652 (C O), 1099,1071, 1056, 1030 (C O). ESI-MS/MS of m/z 627 [M+H]+: Multipleloss of water: m/z 609 [M+H-H2O]+, m/z 591[M+H-2H2O]+, m/z573 [M+H-3H2O]+; loss of hexose and multiple loss of water:m/z 465 [M+H-C6H10O5]+; m/z 447 [M+H-C6H10O5-H2O]+; m/z429 [M+H-C6H10O5-2H2O]+; m/z 411 [M+H-C6H10O5-3H2O]+;loss of hexose and cleavage in the side-chain: m/z 347; lossof water from m/z 347: m/z 329; cleavage of the side-chain andoxygen in ring B: m/z 303; ring D cleavage: m/z 279; loss ofwater from hexose adduct ion: m/z 145 [Hexose + H-2H2O]+. HRESI-MS m/z: 627.3758 [M+H]+, for C33H55O11 required 627.3758.Composition C33H54O11 (M: 626).

2.10. Carthamoleusterone (13)

Compound 13 (0.6 mg) was obtained from combined fractions18B of 14A (80 mg) by repeated HPLC in Systems 2 and 3(Table 1). [�]D20 + 213◦ (c = 0.02, EtOH). IR (KBr), cm−1: 3390 (OH),1658 (C O), 1051, 1037 (C O). ESI-MS/MS of m/z 509 [M+H]+:Multiple loss of water: m/z 491 [M+H-H2O]+, m/z 473 [M+H-2H2O]+, m/z 455 [M+H-3H2O]+, m/z 437 [M+H-4H2O]+, m/z 419[M+H-5H2O]+; cleavage of the tetrahydrofuran side-chain ring:m/z 389; loss of water (2×) and cleavage of ring B: m/z 345; cleav-age of the side-chain and loss of water (2×, 3×): m/z 299, m/z281; the side-chain ion: m/z 173; loss of water (1×, 2×): fromm/z 173: m/z 155, m/z 137. HR ESI-MS m/z: 531.2943 [M+Na]+,for C28H44O8Na required 531.2934. Composition C28H44O8 (M:508).

2.11. 24-epi-Makisterone A (14)

Compound 14 (31 mg) was obtained from combined fractions5B of 11A and 4B of 12A (total 95 mg) by repeated HPLC in

0 8 ) 502–514

System 3 (Table 1). IR (KBr), cm−1: 3375 (OH), 1651 (C O),1057 (C O). HR ESI-MS m/z: 517.3142 [M+Na]+, for C28H46O7Narequired 517.3141. Composition C28H46O7 (M: 494).

2.12. 22-Deoxy-28-hydroxymakisterone C (18)

Compound 18 (5 mg) was obtained from fraction 5B of 14A(16 mg) by repeated HPLC in System 2 (Table 1). [�]D20 + 34.9◦

(c = 0.16, EtOH). IR (KBr), cm−1: 3400 (OH), 1652 (C O), 1060(C O). ESI-MS/MS of m/z 509 [M+H]+: Multiple loss of water: m/z491 [M+H-H2O]+, m/z 473 [M+H-2H2O]+, m/z 455 [M+H-3H2O]+,m/z 437 [M+H-4H2O]+; loss of C3H8O (2-propanol) from theside-chain: m/z 447 [M+H-C3H8O]+; multiple loss of water fromm/z 447: m/z 429 [M+H-C3H8O-H2O]+, m/z 411 [M+H-C3H8O-2H2O]+; multiple loss of water (1×–3×) from the side-chain ion:m/z 171, m/z 153, m/z 135. HR ESI-MS m/z: 531.3291 [M+Na]+,for C29H48O7Na required 531.3298. Composition C29H48O7 (M:508).

2.13. 26-Hydroxymakisterone C (20)

Compound 20 (16 mg) was obtained from fraction 15B of 12A(140 mg) by repeated HPLC in System 2 (Table 1). [�]D20 + 64.0◦

(c = 0.075, EtOH). IR (KBr), cm−1: 3409 (OH), 1654 (C O), 1056(C O). ESI-MS/MS of m/z 525 [M+H]+: Multiple loss of water: m/z507 [M+H-H2O]+, m/z 489 [M+H-2 H2O]+, m/z 471 [M+H-3 H2O]+,m/z 453 [M+H-4 H2O]+. HR ESI-MS m/z: 547.3276 [M+Na]+,for C29H48O8Na required 547.3247. Composition C29H48O8 (M:524).

2.14. 1ˇ-Hydroxymakisterone C (21)

Compound 21 (5 mg) was obtained from fraction 12B of 14A(90 mg) by repeated HPLC in System 3 (Table 1). [�]D20 + 50.8◦

(c = 0.17, EtOH). IR (KBr), cm−1: 3370 (OH), 1650 (C O), 1076, 1063(C O). ESI-MS/MS of m/z 525 [M+H]+: Multiple loss of water:m/z 507 [M+H-H2O]+, m/z 489 [M+H-2 H2O]+, m/z 471 [M+H-3H2O]+, m/z 453 [M+H-4 H2O]+; loss of water (2×) and cleavagein the side-chain: m/z 387; loss of water (1×, 2×) and cleavagein the side-chain: m/z 363 and m/z 345; cleavage of the side-chain and oxygen in ring B: m/z 319; loss of water from theside-chain ion: m/z 171; side-chain fragment: m/z 127. HR ESI-MS m/z: 547.3251 [M+Na]+, for C29H48O8Na required 547.3247.Composition C29H48O8 (M: 524).

2.15. Amarasterone A (22)

Compound 22 (30 mg) was obtained from fraction 13B of 18A(172 mg) by repeated HPLC in System 2 and 3 (Table 1). IR(KBr), cm−1: 3465, 3387 (OH), 1658 (C O), 1061, 1054 (C O).ESI-MS/MS of m/z 509 [M+H]+: Multiple loss of water: m/z491 [M+H-H2O]+, m/z 473 [M+H-2H2O]+, m/z 455 [M+H-3H2O]+

m/z 437 [M+H-4H2O]+. HR ESI-MS m/z: 531.3298 [M+Na]+, forC29H48O7Na required 531.3277. Composition C29H48O7 (M:508).

Experimental data for isolation and identification of 20-

hydroxyecdysone (2) and ajugasterone C (5) were alreadypublished [1], afterwards were reported also data for inte-gristerone A (9), (24Z)-29-hydroxymakisterone C (17) andmakisterone C (19) [2]. Compounds 15 and 16 were reported by

2 0 0

owbmcvaoki

A

Gir(oV

r

s t e r o i d s 7 3 (

ther authors [17,18,35]. However, their completed NMR dataere summarized and displayed in this paper (Tables 2–5),ecause the present advanced NMR techniques allowedore accurate and authentic structure assignments of their

arbon and proton signals. Moreover, just an inspectionaliew on the data collected in the Ecdybase [20] indicatednecessity of supplementation those data. Further previ-

usly reported incomplete data of several other long-timenown ecdysteroids would require a similar better character-

zation.

cknowledgements

rant Agency of Czech Republic, Grant No. 203/04/0298, andn part also research project AVOZ40550506 supported thisesearch. We thank Dr. P. Cupka (Mediplant Co.) and Dr. L. CvakGalena Co.) for their cooperation in the large-scale processingf plant material, Dr. J. Kohoutova for recording MS and Dr. S.asıckova for recording IR spectra.

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