A facile method for steroid labeling by heavy isotopes of hydrogen

Tetrahedron 71 (2015) 4874e4882

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A facile method for steroid labeling by heavy isotopes of hydrogen 
rova , Toma 
s Elbert Ale
s Marek *, Blanka Klepeta m. 2, 16610 Prague 6, Czech Republic Institute of Organic Chemistry and Biochemistry ASCR, v.v.i., Flemingovo na

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 January 2015 Received in revised form 13 April 2015 Accepted 27 April 2015 Available online 14 May 2015

A new catalytic enantiospecific approach to the synthesis of epibrassinosteroids (and other polyhydroxylated steroids) regiospecifically labeled by heavy isotopes of hydrogen is reported. Chlorocarbonate, efficiently synthesized from a-hydroxy ketone by a reaction with triphosgene, undergoes reductive tritium dechlorination catalyzed by the [Pd0]/Et3N system, providing 24-[3b-3H]epicastasterone and 24-[3b-3H]epibrassinolide, respectively, in good yield and with high specific activity (5.8 Ci/ mmol; 20% tritium enrichment per molecule). Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Brassinosteroids a-Hydroxy ketones Reductive dehalogenation Tritium Labeled compounds

1. Introduction Brassinosteroids (BRs) are small organic polyhydroxylated sterol phytohormones with a close structural resemblance to animal and insect steroid hormones. They occur at low levels throughout the plant kingdom and were first isolated and characterized from the pollen of Brassica napus (Brassinolide) in 1979.1 More than 70 of these plant growth regulators have been discovered so far.2 Recently, Strnad et al. have published the first evidence that some natural BRs induce cell-growth-inhibitory responses in several human cancer cell lines without affecting normal non-tumor cell growth (BJ fibroblasts).3 Only those containing 2a, 3a- and 22R, 23R-diol functions and a lactone or ketone moiety in ring B exhibited high biological activity (Fig. 1). Studies on the mechanism of action of biologically active compounds on the molecular level require the labeling of these substances by the radionuclide 3H. Only one example of such synthesis has been published.4 However, 24-[5,7-3H]epibrassinolide, prepared by a base-catalyzed exchange with tritiated water, had very low specific activity (6.3 mCi/mmol); moreover, the product was labeled on the exchangeable position. A general method providing [3H]-labeled BRs in high specific activity is seriously missing. We have recently published a short paper paving the way for the synthesis of a suitable synthetic precursor for the introduction of a deuterium label onto 24-epibrassinolide.5 A detailed study establishing a robust method for the synthesis of

* Corresponding author. Tel.: þ420 220 183 395; e-mail addresses: author@ university.edu, [email protected] (A. Marek). http://dx.doi.org/10.1016/j.tet.2015.04.099 0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

BRs regio- and enantio-specifically labeled by deuterium/tritium is reported in this paper.

,17

Fig. 1. 24-epiBL (1) is the 24-(R)-Epimer of the first isolated brassinosteroide, 24-epiCS (2) and its pregnane (3) and androstane (4) 2,3-dihydroxy analogs.

2. Results and discussion The creation of a reducible double bond on the BRs skeleton seemed to be the most feasible strategy for transformation of starting materials 2e4 into suitable synthetic precursors for a labeling step. We have recently reported the conversion of a vicinal

A. Marek et al. / Tetrahedron 71 (2015) 4874e4882

2a, 3a-diol 3 to the appropriate a-hydroxy ketone 5 by oxidation with freshly generated dimethyldioxirane.5,6 An endiol form of such an a-hydroxy ketone seemed to be a proper substrate for variable synthetic transformations leading to an appropriate bis-substituted endiol. Such a synthetic precursor would provide a distinct opportunity for reductive catalytic tritiation yielding BRs with high specific activity. The closest synthon-analogs to an endiol, vicinal alcohols, offer various synthetic transformations. The most feasible approaches we had decided to try with our substrate; for instance the conversions into silylethers, carboxyesters as well as to 1,3dioxole, 1,3-dioxoborole and 1,3,2-dioxastannolene ring formation. Since the amount of BRs is limited, we have carried out the model study on a commercially available acyloin, a benzoin. Jadhav et al. have reported the isopropylidation of an in situ generated a-hydroxy ketone of 9,10,12-trihydroxyoctadecanoate into the appropriate isopropylidenedioxy-9-ene derivative by acid-catalyzed ring formation.7 In our recent experiments, following this procedure on BRs substrate, diol 3 did not yield the desired product 6.6 The conversion of the acyloin 5 into an appropriate isopropylidenedioxy derivative under various different conditions including a reaction with 2,2-dimethoxypropane or acetone catalyzed with p-TsOH,8e10 CuCl2,11,12 or FeCl313 respectively, failed too, both with the steroid 5 (Scheme 1; Path A) and benzoin substrate.

either; in that case, only desilylation was observed. The two sterically demanding TBDMS groups make the double bond inaccessible for hydrogenation on solid catalysts. The other potential transformationdthe esterification of the enolform of 5 (Scheme 1; Path C) by Ac2O catalyzed by p-TsOH or DMAP was not successful even when left to react up to 7 days at 130  C. Only monoacetate 9 was always isolated in quantitative yield. Davies and Hawari reported the synthesis of acetyldiolates through in situ generation of organotinenediolates.14 Following this procedure in our laboratory with the employment of 3,6,20-trione 5 and dibuthyltindimethoxide (1 equiv) in CD3OD, a quick disappearance of the hydrogen signal of the C2 position was observed by 1 H NMR, which corroborated the formation of the 1,3,2dioxostanollene derivative 11 (Scheme 1; Path D). Unfortunately, the addition of acetic anhydride into the reaction mixture of 11 did not yield the desired bis-ester 10. The catalytic hydrogenation of the organotin enediolate 11 was carried out by deuterium gas catalyzed with Pd/C 10%. Regrettably, a complex mixture was formed and the major product, an unexpected 1,4-diene-2hydroxy-3-ketone 12, was isolated. The formation of the cyclic boronic ester of 13 could be another simple way to generate a double bound on the BRs scaffold (Scheme 1; Path E). Indeed, the initial reaction of benzoin and phenylboronic acid (1 equiv) with azeotropic distillation of the

O

1

HO

2

10 5

4

O

O

18 20 12 17 13

H

8 6

O

AcO O H

H

AcO

H

14

15

9 (99%)

Path D

7

5

catalytic hydrogention

O Bn Sn Bn O

Path F

10 (0%) esterification HO O

H

12 (17%)

11 (not isolated)

unspecified by-products

- HCl

O

O

reductive dehalogenation

H

H 15 (0%)

O

hydrolysis ref 5

H

HO

H

O

O O

H

8 AcO

O

base

O

D

Path C

H

14 (38%)

TBDMS O

H

D

16

9

3

HO

11

19

Path E

TBDMS O

7 (66%) 21

Ph B H 13 (not identified)

TBDMS O

Path B ref 6

H 6 (0%)

Path A ref 6

O

catalytic hydrogenation

TBDMS O

O

O

4875

O

Cl

H

16 (99%) enantiospecific reaction

O

D

H

19 (up to 65%)

HO

D

H

[2H]-3 (99%)

Scheme 1. The studied pathways to create a synthetic precursor for introduction of deuterium/tritium onto BRs skeleton.

The alternative transformation of acyloin 7 into O-substituted endiol is provided by O-silylation (Scheme 1; Path B). The reaction of acyloin 5 with trimethylsilyl triflate (TBDMSOTf) gave a very stable 2,3-bis(tert-butyldimetylsilyloxy)-5a-pregn-2-ene-6,20dione (7) in a 66% yield.6 Such a substituted endiol was subjected to catalytic hydrogenation by carrier-free deuterium gas. Heterogenic reduction catalyzed by transition metals such as 10e30% Pd/ C, PdO/BaSO4, PtO2, nanoRh/AlO(OH) and Rh/alumina carried out at a low pressure of deuterium gas (800 mbar) and at room temperature unfortunately did not yield any desired bis-silylated diol 8 even after overnight reaction. Hydrogenation catalyzed by the Crabtree catalyst did not lead to a reduction of the double bond

generated H2O from the reaction mixture provided a 2,4,6triphenyl-1,3-dioxoborole-2-ene15,16 in high yield (79%). Unfortunately, analogous reaction conditions set for acyloin 5 led only to isomerisation and both a-hydroxyl ketones 5 and 14 were isolated in the ratio 62:38, respectively. The isolation of 2,6,20-trione 14 makes us assume the formation of fairly unstable 1,3-dioxoborole 13, which unfortunately tends to decompose back to the starting 3,6,20-trione 5 and its regiomer 2,6,20-trione 14. In order to synthesize the vinylene carbonate derivate of 5 we decided to follow Hiyama’s procedure. He reported the thermal dehydrochlorination of 4,5-disubstituted 4-chloro-1,3-dioxolan-2-

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ones, providing the appropriate 1,3-dioxolen-2-one.17 When this experiment was repeated in our laboratory, desired 4,5-diphenyl vinylene carbonate was isolated directly after a 16-h reaction of benzoin with triphosgene (7 equiv) at 25  C in the yield of 18% as the only product. In contrast, the application of analogous reaction conditions on steroid 3,6,20-trione 5 gave no vinylenecarbonate 15 and the monochlorinated ethylenecarbonate 16 was isolated in quantitative yield (Scheme 1; Path F).5 This reaction proved to be very robust as it was carried out on various steroid substrates such as 24-epicastasterone,5 pregnane 55 and androstane analogs. The attempts to synthesize vinylene carbonate by the base-catalyzed (neat pyridine, Et3N, NaOH) elimination of HCl from 3b-chloro2,3-carbonate 16 did not yield the desired product even under harsh conditions (up to 110  C, 6 days). The reaction of 16 with NaOH (10 equiv) in DMSO provided full conversion after 5 min at 25  C and the isolated products were enone 12 (81%) and hydroxyketone 5 (19%). On the other hand, the 3b-chloro enantiomer is an excellent precursor for reductive dehalogenation by deuterium (or tritium) gas (Scheme 2). It is very profitable that the reaction course of synthesis chlorocarbonates is stereo-specific, moreover, always providing 3b-chloro-2,3-carbonates of 16e18. We have recently suggested the mechanism of this stereospecific reaction.5 Pdcatalyzed [PdO/CaCO3 (5%)] deuterium dehalogenation of 16e18 has led to the insertion of one deuterium on the steroid skeleton with high enrichment (70e80%) on the C3 position with good to high yield of 19e21 (31e65%) at short reaction time (6 h) (Table 1; Entry 1, 12,14).

significant impact on the isolated yield as well as the formation of by-products (Table 1). The best outcomes provided EtOAc (distilled from P2O5), affording up to a 65% yield of 19 with 80% 2H-enrichment (based on 1H NMR). Other solvents used for reduction provided low isolated yield (0e19%) (Table 1). The reductive dehalogenation of androstane chlorocarbonate 17 provided similar results (58% yield, 75% 2H-enrichment of 20) to a pregnane analog. Two by-products were isolated in that experimentdthe multilabeled ketone 22 and the multi-labeled alcohol 23, both in the yield of 15%. The formation of both by-products was accelerated while MeOH was used as a solvent in the reaction [43% (22), 40% (23)]. The reaction conditions used for the labeling of 24-epiCS were the same as described above for the other two steroids. A full conversion of 18 was obtained after a 6-h reaction [PdO/CaCO3 (5%)/Et3N/substrate 2:6:1] in dry EtOAc. The isolated yield of 21 was determined to be 31% [D:H at C3¼70:30]. Both by-products, 24 (20%) and 25 (13%), were isolated, too. The use of DMF as solvent reduced the conversion of 18e45%, the yield of 21 down to 19% and the formation of by-products 24 and 25 to 11% and 10%, respectively. The crucial role in the suggested mechanism of by-product formation (Scheme 3) is played by traces of water (partly synthesized by the reduction of PdO with D2 gas). The oxidative addition of generated Pd[0] into the C3eCl bond leads to the formation of the organopalladium compound 26. That palladium can be substituted by traces of water to form the hydroxyl carbonate 27, which undergoes ring opening, yielding ketone 28. The elimination of carbonic acid from ketone 28 produces unsaturated ketone 30

Scheme 2. The deuterium labeling step; the Pd-catalyzed deuterium dehalogenation of 3b-chloro-2,3-carbonate 16e18.

Table 1 The Pd-catalyzed reduction of 3b-chloro-2,3-carbonate 16e18 using molecular deuterium Entry

Substrate

Catalyst [equiv]

Base [equiv]

Time (h)

Solvent

Product

Conversiona (%)

Isolated yield (%)

Enrichmentb (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Preg (16) Preg (16) Preg (16) Preg (16) Preg (16) Preg (16) Preg (16) Preg (16) Preg (16) Preg (16) Andr (17) Andr (17) Andr (17) epiCS (18) epiCS (18)

PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/BaSO4 (10%); [2.0] Pd/C (30%); [2.0] Pd/C (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0] PdO/CaCO3 (5%); [2.0]

Et3N Et3N Et3N Et3N Et3N Et3N Et3N Et3N Et3N Et3N Et3N Et3N Et3N Et3N Et3N

6 6 6 14 14 14 6 6 6 6 6 28 6 6 6

EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc 1,2-Dioxane THF Acetone Benzene DMSO EtOAc MeOH EtOAc DMF

19 19 19 19 19 19 19 19 19 19 20 20 20 21 21

76 91 45 30 30 25 17 14 33 36 14 99 99 99 45

65 58 26 19 15 19 0 8 19 14 14 58c 17c 31c 19c

80 80 70 80 66 75 d 80 75 80 80 75 70 70 80

a b c

[6.0] [12.0] [30.0] [6.0] [6.0] [6.0] [6.0] [6.0] [6.0] [6.0] [6.0] [6.0] [6.0] [6.0] [6.0]

Determined by HPLC. Determined by 1H NMR. The by-products isolated.

The best results for deuterium dehalogenation were achieved with the molar ratio of PdO/CaCO3 (5%)/Et3N/chlorocarbonate being 2:6:1] (Table 1; Entry 1). When the amount of the base was too high, it diminished the yield of [2H]-labeled ethylene carbonate 19 (Table 1; Entry 3). The employment of other catalysts such as Pd/C (5% or 30%), PdO/BaSO4 (10%) afforded a lower yield of 19 (15e19%) even at prolonged reaction time (14 h). The solvent effect had

(Pathway A), which could be reduced by D2/Pd[0] to 1,2-deuterium labeled ketons 22 and 24, respectively. The other possible pathway (B) includes two steps, the elimination of carbon dioxide, affording a-hydroxyketone 29, and the subsequent elimination of H2O, providing 30. Pathway B has been excluded from our consideration for two reasons. The first one is that the a-hydroxyketones 29 were not identified in any reaction mixture. The second one is that in an

A. Marek et al. / Tetrahedron 71 (2015) 4874e4882

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PdO + D2 O

H

Pd + D2O

O O

Cl

O

0

H

O

O D

17 (androstane) 18 (epiCS)

O

O

O

H

H

O D

Pd Cl

O

H 26

O

H

HH B

OD O

OD H 27

O

O

H

D

H

HH

D

D2/Pd

HO D

H

23 - androstane 25 - 24-epityphasterol 1D:2D:3D = 10:50:40 %

D O

Isolated by-products confirmed by NMR confirmed by MS

O

C

H

H

DO

H

HH

O O 29

-[DHCO3]

D

HH

H

28

OD O

H

O OD O

H A

DCl

O

H

- HDO

D2/Pd H

22 - androstane 24 - 3-dehydro-24-epiteasteron

O

H 30 (not isolated)

1D:2D = 70:30%

Scheme 3. The suggested mechanism of the formation of multilabeled by-products during the reductive hydrogen dehalogenation of 17 and 18dketons 22 and 24 and alcohols 23 the 25.

extra experiment using explicitly a-hydroxyketones 5 and the conditions utilized for reductive deuterium dehalogenation, {[PdO/ CaCO3 (5%)/Et3N/substrate 2:6:1] in dry EtOAc} yielded 2a,3bdihydroxy derivative 31 in an isolated 85% yield and no other product was identified in reaction mixture (Scheme 4). The ORTEP view in Fig. 2 shows the molecular structure and spatial arrangement of 31 and in particular confirms its absolute configuration at C2 and C3. A regular ESI-MS analysis of 22 and 24 measured in CH3OH has confirmed the structure of multilabeled ketones with the distribution of deuterium 1D/2D being 70:30. The second byproduct, multilabeled alcohols 23 and 25, are most likely formed by a Pd-catalyzed reduction of the ketons 22 and 24 by D2. The structure of 23 and 25 was confirmed by 1H NMR as well as ESI-MS with a deuterium distribution 1D/2D/3D being 10:50:40. The carbonate 19 and 20 were hydrolyzed in 1,4-dioxane by NaOH [2 equiv] in 5 min providing [2H]-3 and [2H]-2, respectively,

in quantitative yields (Experimental section, 4.3. and 4.4.).5 The synthesis of [2H]-1 was accomplished by Baeyer-Villiger oxidation of [2H]-2 (Experimental section, 4.5.). We have recently described the synthesis of synthetic precursors for the reductive hydrogen dehalogenation of pregnane 16 and 24-epiCS 18 derivatives.5 The reaction sequence providing a 3b-chloro-2,3-carbonate derivative of androstane 17 involves the protection of the 2,3-diol group of 4 (for solubility reasons) by its conversion into the dioxolane derivative 32 (ORTEP diagram, Fig. 3). The reaction of 4 with acetone acid-catalyzed by p-TsOH gives the desired 5a-epimer 32 in the high yield of 91%, and the opposite 5bepimer 33 in the yield of 9% (Scheme 5). The protection of the 17OH group by TBDMS was carried out by the reaction of 32 with TBDMS chloride provided the C17eO-silylated product 34 in quantitative yield. The oxidation of the C3 of 34 by a freshly prepared solution of dimethydioxirane6 provided a-hydroxyketone 35 in the moderate yield of 35%. The subsequent stereospecific ring closing by a reaction with triphosgene provides the desired 3bchloro-2,3-carbonate 17 (ORTEP diagram, Fig. 4) in quantitative yield.

Scheme 4. The reduction of a-hydroxyketone 5 under the conditions used for reductive dehalogenation; i) D2, PdO/CaCO3 (5%), Et3N, EtOAc, 6h.

Fig. 3. An ORTEP5 view of 32, shown with 50% probability displacement ellipsoids.

Fig. 2. An ORTEP view of 31, shown with 50% probability displacement ellipsoids.

With the optimized reaction conditions [PdO/CaCO3 (5%)/Et3N/ substrate 2:6:1, dry EtOAc] in hand, we decided to carry out the tritium dehalogenation experiment. In order to synthesize 3H-labeled 24-epiCS and 24-epiBL, the 3b-chloro-2,3-carbonate5 18 was used for a reaction with carrier-free tritium gas (600 mbar), PdO/ CaCO3 (5%), and Et3N. The reaction was left to proceed at 25  C for

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Scheme 5. The reaction sequence of the synthesis 2,3-carbonate-3b-[2H]-labeled derivative of androstane 20; i) acetone, p-TsOH; ii) TBDMSCl, imidazole; iii) dimethyldioxyrane, acetone; iv) triphosghene, pyridine v) D2, PdO/CaCO3, Et3N, EtOAc.

Fig. 4. An ORTEP5 view of 17, shown with 50% probability displacement ellipsoids.

17 h (Scheme 6). The reduction gave 5.9 mCi of the 3H-labeled carbonate 36, and two further unidentified by-products (21.3 mCi and 12.4 mCi) were detected on the HPLC radiodetector. The specific activity of 36 was assayed to 5.8 Ci/mmol (which accounts for 0.2 tritium per molecule, determined by 1H NMR); the tritium enrichment was lower than had been supposed based on a previous deuterium model study. The significantly diminished specific activity in the tritium experiment was most likely caused by the reduced pressure of tritium gas used for the reduction (600 mbar of 3 H2 vs 950 mbar of 2H2). The only signal in the 3H NMR spectrum (the singlet at d 4.8 ppm) (Supplementary data, Fig. 46) explicitly determined the regio- and stereo-specificity of the reduction. The deprotection of the isopropylidene group in the side chain was carried out by wet FeCl3 in CH2Cl2.5 The 3H-labeled carbonate 36 was hydrolyzed by a 0.5M aqueous solution of NaOH in 1,4-dioxane (1:1). The preparative radio-HPLC afforded [3H]-2 (3.8 mCi, 5.8 Ci/

mmol) as a white solid. The Baeyer-Villiger oxidation5 of [3H]-2 gave [3H]-1 (0.3 mCi, a specific activity of 5.8 Ci/mmol) as a white solid. The assigned molecular structures of all the synthesized compounds were confirmed by 1H and 13C NMR spectroscopy employing 1He1H COSY, 1He13C HMQC and HMBC methods. The prepared compounds also provided satisfactory elemental analyses and ESI-MS spectra. In addition, the slow evaporation of a methanol solution of the 5a-epimer of androstane 32 afforded crystals for a suitable X-ray analysis. The ORTEP plots in Figs. 1e3 confirmed the molecular structure and spatial arrangement of 17, 31, 32. 3. Conclusion A general protocol for labeling polyhydroxylated steroids by heavy isotopes of hydrogen has been developed. A suitable precursor for the introduction of tritium, 3b-chloro-2,3-carbonate derivative, is efficiently synthetically affordable by a short reaction sequence from the steroid to be labeled. A crucial aspect in the reductive dehalogenation of the chloro derivative is the choice of a solvent that would provide a reasonable yield. The optimized reaction conditions are PdO/CaCO3 (5%)/Et3N/substrate (2:6:1) dissolved in dry EtOAc. The suggested mechanism of the byproducts formation has explained how the traces of water significantly reduce the yield of the labeled product desired. However, the successful 3H-labeling experiment has proven the stereoselectivity of the reductive dehalogenation and afforded a product with high specific activity (5.8 Ci/mmol).

Scheme 6. The ‘hot’ experiment; i) T2/PdO/CaCO3/Et3N; ii) Fe(III), CH2Cl2; iii) NaOH, 1,4-dioxane; iv) H2O2/TFA, 0  C 30 min, r.t. 4h, CHCl3.

A. Marek et al. / Tetrahedron 71 (2015) 4874e4882

4. Experimental section 4.1. General 1

H-, 3H and 13C NMR spectra were recorded at 300/320 MHz and 75 MHz, respectively, with a Bruker Avance 300 instrument at 25  C (the solvents are indicated in parentheses). Chemical shifts are reported in parts per million relative to TMS. The mass spectra have been obtained by the Bruker Daltonics Esquire 4000 system with a direct input (ESI, stream AcCN-H2O, a mass range of 50e1200 Da, Esquire Control Software). The HR-mass spectra were obtained in the ESI mode either on a Waters-Micromass Q-TOF Micro mass spectrometer or on a Thermo Fisher Scientific LTQ Orbitrap XLc. The tritiation reaction was performed on a custom-designed tritium manifold system manufactured by RC Tritec AG, Switzerland. Liquid scintillation measurements were done on a PerkineElmer TriCarb 2900 liquid scintillation counter (LSC) in a Zinsser Quicksafe A cocktail. The HPLC was performed on a system consisting of a WATERS Delta 600 Pump and Controller, a WATERS 2487 UV detector and a RAMONA radio chromatographic detector from Raytest (Germany) with interchangeable fluid cells. For the preparative runs, the cell with a single small crystal of solid scintillator was used; for analytical runs, the column effluent was mixed with a Zinsser Quickszint Flow 302 cocktail at the ratio of 1:3. See the Supplementary data for experimental details and the characterization of compounds 9, 17, 32e35 and representative NMR spectra. 4.2. The general procedure for the reductive deuterium dehalogenation of 3b-chloro-2,3-carbonates 16e18 The chlorocarbonate 16e18 (10 mmol) and catalyst (Table 1) were placed in a 2 mL deuteration round-bottomed reaction flask; dry solvent (Table 1) (1 mL) and Et3N (60e300 mmol, Table 1) were added. The flask was mounted onto a deuteration manifold system, the reaction mixture was degassed by three freeze-thaw cycles and filled with 2H2 gas (950 mbar). The reaction mixture was stirred for 6e24 h (Table 1) at 25  C. The reaction was monitored by TLC (SiO2; benzene/ acetone, 20:1). When the starting compound disappeared, the reaction mixture was filtered through a 0.45 m PTFE syringe filter and the deuteration flask and filter were washed by EtOAc (31 mL). The solvent was evaporated and the crude product was purified by column chromatography (SiO2; benzene/acetone, 20:1 or EtOAc/hexane, 1:3). The pure product was obtained in the yields of 19e65% as a white solid. The 2H-enrichment on the C3 position was 66e80% as determined by the decrease of the corresponding 1H signal in 1H NMR. 4.2.1. 2 a ,3 a -(Carbonyldioxy)-5 a -[3 b - 2 H]-pregnane-6,20-dione (19). The title compound was prepared from 16 following the general procedure. Mp¼261e263  C (MeOH). Rf¼0.13 (silica gel; 1 benzene/acetone, 20:1). ½a20 D þ56.9 (c 0.22, CHCl3). H NMR (CDCl3) d: 0.62 (3H, s, 18-CH3), 0.72 (3H, s, 19-CH3), 1.30e1.41 (1H, m, 1aCH), 2.01e2.10 (1H, m, 7a-CH), 2.10e2.13 (1H, m, 4-CH), 2.14 (3H, s, 21-CH3), 2.25e2.35 (1H, m, 4-CH), 2.33e2.42 (1H, m, 1b-CH), 2.38 (1H, dd, J¼4.8 Hz, J¼13.5 Hz, 7b-CH), 2.53 (1H, dd, J¼3.9 Hz, J¼12.0 Hz, 5a-CH), 2.57 (1H, t, J¼9.0 Hz, 17-CH), 4.65 (1H, dd, J¼7.2 Hz, J¼10.2 Hz, 2-CH). 13C NMR (CDCl3) d: 13.13 (19-CH3), 13.52 (18-CH3), 21.28 (4-CH2), 21.82 (11-CH2), 22.94 (15-CH2), 24.33 (16CH2), 31.69 (21-CH3), 37.36 (8-CH), 38.38 (12-CH2), 39.56 (1-CH2), 41.44 (10-C), 44.31 (13-C), 46.58 (7-CH2), 50.72 (5-CH), 52.90 (9CH), 56.82 (14-CH), 63.37 (17-CH), 74.14 (2-CH), 154.82 (OC(O)2), 209.09 (6-CO), 209.20 (20-CO), (3-CD) missing. MS (m/z): 398.3 [MþNa], 773.7 [2MþNa]. HRMS: for C22H229HO5Na: calculated 398.20482, found 398.20447. 4.2.2. 2 a ,3 a -(Carbonyldioxy)-17 b -tertbutyldimethylsilyloxy-5 b [3b-2H]-androstane-6-one (20). The title compound was prepared

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from 17 following the general procedure. Mp¼130e132  C (MeOH). Rf¼0.26 (silica gel; EtOAc/hexane, 1:3). ½a20 D þ31.2 (c 0.35, CHCl3). 1 H NMR (CDCl3) d: 0.02 and 0.03 (6H, 2s, Si(CH3)2), 0.70 (3H, s, 18CH3), 0.73 (3H, s, 19-CH3), 0.88 (9H, s, C(CH3)3), 1.05e2.55 (17H, m), 2.53 (1H, dd, J¼12.9 Hz, J¼4.2 Hz, 5a-CH), 3.60 (1H, t, J¼8.1 Hz, 17CH), 4.67 (1H, dd, J¼7.2 Hz, J¼10.2 Hz, 2-CH), 4.82e4.85 (residual signal, m, 3-CH). 13C NMR (CDCl3) d: 4.81 and 4.46 (SiCH3), 11.35 (19-CH3), 12.98 (18-CH3), 18.17 (4-CH2), 20.86 (11-CH2), 21.85 (15CH2), 23.30 (16-CH2), 25.86 (C(CH3)3), 29.73 (21-CH3), 30.70, 36.41 (8-CH), 39.38 (12-CH2), 41.38 (1-CH2), 43.63 (10-C), 46.32 (13-C), 50.60 (5-CH), 52.97 (9-CH), 53.16 (14-CH), 74.05 (2-CH), 81.32 (17CH),154.73 (OC(O)2), 209.42 (6-CO), (3-CD) missing. MS (m/z): 486.4 [MþNa], 949.7 [2MþNa]. HRMS: for C26H241HO5NaSi: calculated 486.27565, found 486.27525. 4.2.3. (22R,23R,24R)-2a,3a-(Carbonyldioxy)-22,23-(iso-propylidene dioxy)-24-methyl-5a-[3b-2H]cholestan-6-one (21). The title compound was prepared from 19 following the general procedure. White solid. Mp¼254e256  C (MeOH), Rf¼0.15 (SiO2; benzene/ac1 etone, 20:1), ½a20 D 17.1 (c 0.12, CHCl3). H NMR (300 MHz, CDCl3) d: 0.67 (3H, s, 18-CH3), 0.71 (3H, d, J¼6.9 Hz, 26-CH3), 0.72 (3H, s, 19CH3), 0.81 (3H, d, J¼6.9 Hz, 28-CH3), 0.91 (3H, d, J¼7.2 Hz, 27-CH3), 0.99 (3H, d, J¼6.0 Hz, 21-CH3), 1.35e1.42 (1H, m, 1-CH2), 1.34 and 1.40 (6H, s, C(CH3)2), 2.01e2.05 (1H, m, 7a-CH), 2.28 (1H, dd, J¼3.6 Hz, J¼16.5 Hz, 7b-CH), 2.39 (1H, dd, J¼2.4 Hz, J¼13.2 Hz, 1CH2), 2.53 (1H, dd, J¼3.9 Hz, J¼12.9 Hz, 5a-CH), 3.57 (1H, dd, J¼6.9 Hz, J¼9.3 Hz, 23-CH), 3.92e3.95 (1H, m, 22-CH), 4.67 (1H, dd, J¼6.9 Hz, J¼9.9 Hz, 2-CH). 13C NMR (75 MHz, CDCl3) d: 10.09 (28CH3), 11.93 (18-CH3), 12.88 (21-CH3), 13.12 (19-CH3), 16.21 (26CH3), 21.34 (4-CH2), 21.36 (27-CH3), 22.00 (11-CH2), 24.07 (15CH2), 27.40, 27.59 (25-CH), 27.80 (16-CH2), 27.94 (C(CH3)2), 37.67 (20-CH), 38.23 (8-CH), 39.12 (1-CH2), 39.54 (12-CH2), 41.60 (13-C), 42.92 (10-C), 44.01 (24-CH), 46.89 (7-CH2), 50.71 (5-CH), 53.00 (9CH), 53.54 (17-CH), 56.41 (14-CH), 74.23 (2-CH), 80.57 (23-CH), 82.53 (22-CH), 108.26 (C(CH3)2), 154.87 (OC(O)2), 209.84 (6-CO), (3CD) missing. MS (m/z): 554.5 [MþNa]. HRMS: for C32H249HO6Na: calculated 554.35624, found 554.35574. 4.2.4. 1-[2H]-17b-tertButyldimethylsilyloxy-5b-androstane-3,6-dione (22). The title compound was prepared from 17 following the general procedure. Mp¼118e121  C (MeOH), Rf¼0.38 (SiO2; EtOAc/hexane, 1:3). ½a20 þ25.3 (c 0.15, CHCl3). 1H NMR D (300 MHz, CDCl3) d: 0.02 and 0.02 (6H, 2s, Si(CH3)2), 0.67 (3H, s, 18-CH3), 0.89 (9H, s, C(CH3)3), 0.97 (3H, s, 19-CH3), 1.05e2.48 (18H, m), 2.60 (1H, dd, J¼4.8 Hz, J¼2.7 Hz, 5a-CH), 3.60 (1H, t, J¼7.8 Hz, 17-CH). 13C NMR (75 MHz, CDCl3) d: 4.64 and 4.28 (SiCH3), 11.58 (C-19), 12.81 (C18), 18.33, 21.58 (SiC), 23.54 (C(CH3)3), 29.90, 36.79 (C-12), 37.20, 38.25 (C-8), 38.35 (C-10), 44.00 (C-13), 46.40 (C-7), 51.18 (C-14), 53.93 (C-9), 57.80 (C-5), 74.51, 81.56 (17-CH), 209.13 (C-3), 211.58 (C-6), one carbon missing. MS (m/z): 442.3 [MþNa]. HRMS: for C25H242H1O3Si: calculated 420.30387, found 420.30428. 4.2.5. (22R,23R,24R)-1-[2H]-22,23-(Isopropylidenedioxy)-24-methyl5a-cholestan-3,6-dione (23). The title compound was prepared from 185 following the general procedure. Rf¼0.40 (SiO2; benzene/acetone, 1 20:1). ½a20 D þ31.8 (c 0.2, CHCl3). H NMR (300 MHz, CDCl3) d: 0.68 (3H, s, 18-CH3), 0.70 (3H, d, J¼7.6 Hz, 26-CH3), 0.81 (3H, d, J¼6.9 Hz, 27CH3), 0.91 (3H, d, J¼6.9 Hz, 28-CH3), 0.97 (3H, d, J¼5.9 Hz, 21-CH3), 0.99 (3H, s, 19-CH3), 1.35 and 1.37 (6H, 2s, C(CH3)2), 1.10e2.42 (18H, m), 2.30e2.37 and 2.60e2.65 (2H, 2m, 4-CH2), 2.55e2.61 (1H, m, 5a-CH), 3.58 (1H, dd, J¼9.3 Hz, J¼6.9 Hz, 22-CH), 3.94 (1H, dd, J¼6.9 Hz, J¼6.9 Hz, 23-CH). 13C NMR (75 MHz, CDCl3) d: 12.93, 16.22, 21.43, 21.88, 24.15, 27.46, 27.62, 27.89, 28.02, 37.19, 38.20, 38.34, 39.35, 41.46, 43.14, 44.03, 46.83, 53.64, 56.50, 57.70, 80.63 (22-CH), 82.60 (23-CH), 108.25 (C(CH3)2), 209.36 (3-CO), 211.52 (6-CO), (1-CHD) and

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A. Marek et al. / Tetrahedron 71 (2015) 4874e4882

(2-CHD) missing. MS (m/z): 510.4 [MþNaþ]. HRMS: C31H249H1O4Na: calculated 510.36641, found 510.36583.

for

4.2.6. 3-Hydroxy-1,3-di[2H]-17b-tertbutyldimethylsilyloxy-5b-androstane-6-one (24). The title compound was prepared from 17 following the general procedure. Mp¼125e127  C (MeOH), Rf¼0.12 1 (SiO2; EtOAc/hexane, 1:3). ½a20 D þ27.3 (c 0.25, CHCl3). H NMR (300 MHz, CDCl3) d: 0.01 and 0.01 (6H, 2s, Si(CH3)2), 0.71 (3H, s, 18-CH3), 0.77 (3H, s, 19-CH3), 0.88 (9H, s, C(CH3)3), 1.00e1.93 (18H, m), 2.20 (1H, dd, J¼12.3 Hz, J¼3.6 Hz, 5a-CH), (1H, dd, J¼12.6 Hz, J¼3.9 Hz, 7b-CH), 3.59 (1H, t, J¼8.7 Hz, 17-CH). 13C NMR (75 MHz, CDCl3) d: 4.62 and 4.27 (SiCH3), 11.58 (C-19), 13.39 (C-18), 18.31 (C(CH3)3), 21.43, 23.54 (C(CH3)3), 29.92, 30.15, 30.95, 36.81 (C-12), 36.92 (C-12), 38.25, 41.15 (C-10), 43.99 (C-10), 46.53 (C-7), 51.32 (C14), 54.40, 57.04 (C-5), 81.66 (17-CH), 210.90 (C-6), two carbons missing. MS (m/z): 445.3 [MþNa]. HRMS: for C25H243H2O3Si: calculated 423.32580, found 423.32552. 4.2.7. (22R,23R,24R)-3-Hydroxy-1,3-di[2H] 22,23-(isopropylidenedio xy)-24-methyl-5a-cholestan-6-one (25). The title compound was prepared from 185 following the general procedure. [a]20 D þ21.1 (c 0.09, CHCl3). 1H NMR (300 MHz, CDCl3) d: 0.67 (3H, s, 18-CH3), 0.70 (3H, d, J¼7.2 Hz, 26-CH3), 0.76 (3H, s, 19-CH3), 0.81 (3H, d, J¼7.2 Hz, 27-CH3), 0.91 (3H, d, J¼6.9 Hz, 28-CH3), 0.98 (3H, d, J¼6.9 Hz, 21-CH3),1.20e2.15 (27H, m), 1.35 and 1.40 (6H, 2s, C(CH3)2), 2.32 (1H, dd, J¼12.9 Hz, J¼4.5 Hz, 7b-CH), 3.57 (1H, dd, J¼9.3 Hz, J¼6.9 Hz, 22-CH), 3.94e3.96 (1H, dd, J¼6.9 Hz, J¼6.9 Hz, 23-CH). 13C NMR (75 MHz, CDCl3) d: 9.89, 11.88, 12.68, 13.78, 16.02, 21.17, 21.44, 23.90, 27.20, 27.41 and 27.75 (C(CH3)2), 27.85, 27.39, 37.64, 38.02, 39.32, 40.28, 42.72, 43.82, 44.04, 46.71, 50.86, 53.45, 53.87, 56.45, 56.74, 68.55, 80.42 (22-CH), 82.41 (23CH), 108.02 (C(CH3)2), 212.37 (6-CO). MS (ESI, m/z): 513.4 [MþþNa]. HRMS: for C31H250H2O4Na: calculated 513.38833, found 513.38756.

4.3. The procedure for the carbonate-hydrolysis of 2a,3a(carbonyldioxy)-5a-[3b-2H]-pregnane-6,20-dione (19) A solution of NaOH (16 mmol) in water (1 mL) was added to a solution of 19 (8 mmol) in 1,4-dioxane (3 mL), and the reaction mixture was stirred for 5 min at room temperature. The completion of the reaction was checked by TLC (SiO2; benzene/acetone, 20:1). The suspension was neutralized by 1M aqueous HCl and the product was extracted into DCM (35 mL). The combined organic layers were dried by anhydrous MgSO4 and the solvent was evaporated. Purification by column chromatography (SiO2; CHCl3/ MeOH, 10:1) gave the pure product [2H]-3 (2.7 mg, 99%) as a white solid. 4.3.1. 2a,3a-dihydroxy-5a-[3b-2H]pregnane-6,20-dione ([2H]-3). The title compound was prepared from 19 following the general procedure. Mp¼178e180  C (MeOH), Rf¼0.24 (SiO2; CHCl3/MeOH, 1 10:1), ½a20 D þ60.0 (c 0.40, CHCl3). H NMR (300 MHz, CDCl3) d: 0.62 (3H, s, 18-CH3), 0.76 (3H, s, 19-CH3), 1.31e1.35 and 1.69e1.73 (21H, 2m, 15-CH2), 1.33e1.37 and 1.78e1.82 (21H, 2m, 16-CH2), 1.33e1.37 and 1.68e1.72 (21H, 2m, 11-CH2), 1.37e1.39 (1H, m, 14-CH3), 1.38e1.40 (1H, m, 9-CH), 1.47e1.51 and 2.03e2.07 (21H, 2m, 12-CH2), 1.53e1.57 and 1.71e1.75 (21H, 2m, 1-CH2), 1.68e1.72 and 1.87e1.91 (21H, 2m, 4-CH2), 1.73e1.77 (1H, m, 8CH), 1.98e2.02 and 2.32 (21H, m and dd, J¼4.5 Hz, J¼13.2 Hz, 7CH2), 2.14 (3H, s, 21-CH3), 2.60 (1H, t, J¼9.0 Hz, 17a-CH), 2.70 (1H, dd, J¼3.0 Hz, J¼12.6 Hz, 5a-CH), 3.76 (1H, dd, J¼3.7 Hz, J¼11.3 Hz, 2b-CH). 13C NMR (75 MHz, CDCl3) d: 13.55 (18-CH3), 13.73 (19-CH3), 21.29 (11-CH2), 22.87 (16-CH2), 24.26 (15-CH2), 26.43 (4-CH2), 31.65 (21-CH3), 37.62 (8-CH), 38.54 (12-CH2), 40.24 (1-CH2), 42.60 (10-C), 44.51 (13-C), 46.67 (7-CH2), 50.88 (5-CH), 53.66 (9-CH), 56.82 (14CH), 63.45 (17-CH), 68.30 (2-CH), 209.65(20-CO), 212.02 (6-CO), (3-

CD) missing. MS (m/z): 349.3 [M], 350.3 [Mþ1]. HRMS: for C21H231HO4Na: calculated 372.22556, found 372.22529. 4.4. The procedure for the in situ carbonate-hydrolysis and cyclopropylidenedioxy-deprotection of (22R,23R,24R)-2a,3a(carbonyldioxy)-22,23-(isopropylidenedioxy)-24-methyl-5a[3b-2H]cholestan-6-one ([2H]-2) Wet FeCl3 (10 mg, 61 mmol) was added to a solution of 21 (3 mg, 6 mmol) in DCM (2 mL) and the reaction mixture was stirred at 25  C. After 10 min, the reaction was completed as revealed by TLC (SiO2; benzene/acetone, 20:1). The solvent was evaporated and the solid residue was dissolved in 1,4-dioxane (2 mL). A solution of NaOH (5 mg, 125 mmol) in water (1 mL) was added and the reaction mixture was stirred for 20 min at room temperature. After the completion of the reaction (monitored by TLC; SiO2; CHCl3/MeOH, 10:1), the suspension was neutralized by 1M aqueous HCl and the product was extracted into DCM (35 mL). The combined organic layers were dried by anhydrous MgSO4 and the solvent was evaporated. Purification by column chromatography (SiO2; CHCl3/ MeOH, 10:1) gave the pure [2H]-2 (2.7 mg, 91%) as a white solid. C 4.4.1. 24-[3b-2H]Epicastasterone ([2H]-2). Mp¼240e242 20 (MeOH), Rf¼0.18 (SiO2; CHCl3/MeOH,10:1), ½aD 9.7 (c 0.062, CHCl3). 1 H NMR (300 MHz, CDCl3) d: 0.68 (3H, s, 18-CH3), 0.76 (3H, s, 19-CH3), 0.85 (3H, d, J¼6.9 Hz, 26-CH3), 0.87 (3H, d, J¼6.9 Hz, 27-CH3), 0.91 (3H, d, J¼6.9 Hz, 28-CH3), 0.98 (3H, d, J¼6.9 Hz, 21-CH3), 1.01e2.19 (20H, m), 2.30 (2H, dd, J¼4.7 Hz, J¼12.6 Hz, 4-CH2), 2.71 (1H, dd, J¼12.6 Hz, J¼3.4 Hz, 5a-CH), 3.40e3.41 (1H, m, 22-CH), 3.72e3.74 (1H, m, 23CH), 3.77 (1H, m, 2-CH). 13C NMR (75 MHz, CDCl3) d: 10.25 (28-CH3), 11.90 (18-CH3), 12.02 (21-CH3), 13.56 (19-CH3), 20.05 (26-CH3), 21.07 (27-CH3), 21.40 (11-CH2), 24.02 (15-CH2), 26.46 (4-CH2), 27.76 (16CH), 30.85 (25-CH), 37.13 (20-CH), 38.14 (8-CH), 39.66 (12-CH2), 40.03 (1-CH2), 42.97 (13-C), 40.46 (24-CH), 42.92 (10-C), 46.89 (7-CH2), 51.15 (5-CH), 52.47 (17-CH), 53.94 (9-CH), 56.77 (14-CH), 68.24 (2-CH), 74.61 (22-CH), 73.36 (23-CH), 213.86 (6-CO), (3-CD) missing. HRMS: for C28H247HO5Na: calculated 488.34567, found 488.34526.

4.5. The Baeyer-Villiger oxidation of [2H]-2 leading to 24[3b-2H]epibrassinolide ([2H]-1) The solution of [2H]-2 (10 mg, 22 mmol) in CHCl3 (3 mL) was added dropwise under stirring and cooling (0  C) to the solution of trifluoroperoxyacetic acid freshly prepared by adding a 30% aqueous solution of H2O2 (80 mL, 0.71 mmol) to a stirred solution of trifluoroacetic acid (200 mL, 1.42 mmol) in CHCl3 (3 mL) at 0  C. The reaction mixture was stirred for 30 min at 0  C and then the cooling bath was removed and the reaction mixture was left to warm up to room temperature. After additional 4 h, the reaction mixture was diluted by CHCl3 (10 mL) and then washed successively with a saturated aqueous solution of NaHCO3 (5 mL) and water (5 mL). The organic layer was dried with anhydrous MgSO4 and the solvent was evaporated. The solid residue was purified by column chromatography (SiO2; CHCl3/MeOH, 15:1). Deuterated 24-epibrassinolide [2H]-1 was obtained as a white solid (6.7 mg, 65%). C ([2H]-1). Mp¼256e259 4.5.1. 24-[3b-2H]Epibrassinolide 20 (MeOH), Rf¼0.37 (SiO2; CHCl3/MeOH, 15:1). ½aD 9.7 (c 0.06, CHCl3). 1H NMR (CDCl3) d: 0.71 (3H, s, 18-CH3), 0.85 (3H, d, J¼6.9 Hz, 28-CH3), 0.87 (3H, d, J¼6.9 Hz, 26-CH3), 0.90 (3H, d, J¼6.9 Hz, 27CH3), 0.92 (3H, s, 19-CH3), 0.96e0.98 (3H, m, 21-CH3), 3.10e3.14 (1H, m, 5-CH), 3.40e3.43 (1H, m, 23-CH), 3.67e3.70 (1H, m, 22-CH), 3.73e77 (1H, m, 2-CH), 4.06e4.08 (2H, m, 7-CH2). 13C NMR (CDCl3) d: 10.35 (28-CH3), 11.51 (18-CH3), 12.33 (21-CH3), 15.56 (19-CH3), 17.42 (26-CH3), 22.23 (27-CH3), 22.24, 24.75, 27.12, 27.72, 31.04, 38.06, 39.63, 40.24, 40.91 (5-CH), 41.47, 41.62, 42.40, 51.31, 52.70,

A. Marek et al. / Tetrahedron 71 (2015) 4874e4882

58.17, 68.15 (2-CH), 70.54 (7-CH2), 72.61, 76.44, 176.71 (6-CO), (3CD) missing. MS (m/z): 504.3 [MþNa]. HRMS: for C28H247HO6Na: calculated 504.34603, found 504.34568. 4.6. The general procedure for reductive tritium dehalogenation. The synthesis of (22R,23R,24R)-2a,3a-(carbonyldioxy)22,23-(isopropylidenedioxy)-24-methyl-5a-[3b-3H]cholestan6-one (36) EtOAc (0.7 mL) and Et3N (5.2 ml, 37.2 mmol) were added to the 3chloro derivative 18 (3.5 mg, 6.2 mmol) and 5% PdO/CaCO3 (31.5 mg, 12.4 mmol) in a tritiation flask equipped with a magnetic stirrer. The flask with the reaction mixture was mounted onto a tritiation manifold system and degassed by a repeated freeze-thaw cycle. 5 Ci of carrier-free tritium were released over the reaction mixture at a pressure of 800 mbar. The reaction mixture was vigorously stirred at room temperature under tritium atmosphere for 17 h. The reaction mixture was then frozen and excessive tritium was backtrapped on the uranium bed. The reaction mixture was filtered through a 0.45 m PTFE syringe filter and the reaction flask and filter were rinsed with methanol (31 mL). The collected filtrates were evaporated to dryness on a rotary evaporator. The residue was dissolved in methanol (5 mL) and the solution was evaporated to dryness to remove labile activity. An amorphous residue was dissolved in methanol (5 mL) and the total activity was determined as 62.5 mCi. The crude product was purified by radio-HPLC (for the settings, see below). The desired product 36 was identified by 3H and 1H NMR spectrometry in the fraction with tret.¼18.8 min containing 5.9 mCi due to its characteristic 3H singlet at 4.8 ppm (the 1 H decoupled spectrum), which became characteristically split after 1H broad-band decoupling was turned off. The 1H spectrum was in accordance with the 1H standard and specific activity (S.A.) was calculated from the 1H signal decrease as 5.8 Ci/mmol. HPLC settings: Identification was done by radio-detection. Column: Synergi 4m Fusion-RP 80 semipreparative column, 250x10 mm (Phenomenex). Flow: 6.7 ml/min. Eluents: (A) 100% purified water; (B) 100% acetonitrile. Gradient: 0e5 min, 80% B; 5e30 min, 80e100% B. The peak of 36 was detected at 18.8 min (matched with the 1Hstandard). 3H NMR (CDCl3) d: 4.8 (13H, s, 3b-C3H). 4.7. The synthesis of 24-[3b- H]epicastasterone ([ H]-2) 3

4881

[3b-3H]epicastasterone [3H]-2 was dissolved to DMSO at a concentration of 1 mCi/mL and stored in the dark before use. 4.8. The synthesis of 24-[3b-3H]epibrassinolide ([3H]-1) 24-[3b-3H]epicastasterone ([3H]-2) prepared by the deprotection of 3.3 mCi of 36 was dissolved in chloroform (1 mL). This solution of [3H]-2 was added dropwise to an intensively stirred freshly prepared chloroform solution of peroxotrifluoracetic acid (20 ml of 30% H2O2, 100 ml of trifluoracetic acid, 1 mL of chloroform) cooled to 0  C. After a 30-minute stirring at 0  C, the reaction mixture was heated up to room temperature and stirred for another 4 h. The reaction mixture was analyzed by radio-HPLC and the starting [3H]2 was no longer detected. Chloroform (3 mL) was added and the solution was washed by a saturated water solution of NaHCO3 (3 mL). The collected extracts were dried by anhydrous MgSO4. The total activity of the crude extract was determined to be 2.5 mCi, the content of 24-[3b-3H]epibrassinolide ([3H]-1) was 50% according to analytical radio-HPLC (for the settings, see below). The chloroform solution was evaporated, the residue was dissolved in DCM (200 ml) and then methanol (800 ml) was added. The solution was injected on semipreparative radio-HPLC (for the settings, see below). The activity of the obtained pure 24-[3b-3H]epibrassinolide ([3H]-1) was 0.3 mCi, R.C.P.>99%. The solvents of pooled fractions were evaporated, the residue was dissolved in DMSO-d6 and the 3H and 1H NMR spectra (matching with the 1H standard) were measured. Analytical HPLC settings: Column: Synergi 4m Fusion-RP 80, 250x3 mm (Phenomenex). Flow: 0.6 mL/min. Temperature: 25  C. Eluents: (A) water, (B) acetonitrile. Gradient: 0e30 min, 30e40% B; 30e40 min, 40% B isocratic. The peak of [3H]-1 was detected at 34.1 min. Semipreparative HPLC settings: Column: Synergi 4m FusionRP 80, 250x10 mm (Phenomenex). Flow: 6.6 mL/min. Temperature 25  C. Eluents: (A) water, (B) acetonitrile. Gradient: 0e3 min, 30% B isocratic; 3e30 min, 30e40% B; 30e40 min, 40% B isocratic. 3H{1H} NMR (DMSO-d6) d: 3.68 (13H, s, 3b-C3H), 3H NMR (DMSO-d6) d: 3.65e3.70 (13H, brs, 3b-C3H). S.A. of [3H]-1 was determined as 5.8 Ci/ mmol (based on the decrease of the corresponding 1H signal intensity). Acknowledgements

3

FeCl3.6H2O (10 mg) were added to the solution of 36 (2.6 mCi) in DCM (0.5 mL). The reaction mixture was stirred by a magnetic stirrer at room temperature for 40 min. The solvent was then evaporated and dioxane (0.6 mL) and 0.5M NaOH (0.5 mL) were added to the residue. After 20 min, the reaction mixture was checked by radio-HPLC and neither the starting material nor the carbonate derivative were detected. The reaction mixture was diluted with water (4 mL) and crude 24-[3b-3H]epicastasterone was extracted by DCM (43 mL). The collected extracts were dried by MgSO4, filtered and evaporated. The purity of the crude mixture was determined by analytical radio-HPLC (for the settings, see below) as 88%. The crude 24-[3b-3H]epicastasterone [3H]-2 was purified by semi-preparative radio-HPLC (for the settings, see below). Analytical HPLC settings: Column: Gemini C18 250x4.6 mm column (Phenomenex). Flow: 1 mL/min. Temperature: 25  C. Eluents: (A) 100% water, (B) 100% acetonitrile. Gradient: 0e30 min, 30% B to 80% B; 50 min, 80% B. The peak of [3H]-2 was detected at 40.54 min. Semipreparative HPLC settings: Column: Synergi 4m Fusion-RP 80, 250x10 mm (Phenomenex). Flow: 6.7 mL/min. Temperature: 25  C. Eluents: (A) 100% water, (B) 100% acetonitrile. Gradient: 0e5 min, 30% B isocratic; 5e30 min, 30e40% B; 30e40 min, 40% B isocratic). The peak of [3H]-2 was detected at 40 min. The yield was determined as 1.7 mCi of 24e [3b-3H]epicastasterone [3H]-2 with S.A.¼5.8 Ci/mmol and R.C.P.>99%. 24-

The authors thank the Academy of Sciences of the Czech Republic for the financial support of this project within the program RVO: 61388963. The project was further supported by grant No. IAA400550801 of the Grant Agency of the Academy of Sciences of the Czech Republic. Supplementary data Supplementary data (The synthetic procedures, the characterizations and the 1H, 13C 3H NMR and HRMS spectra.) related to this article can be found at http://dx.doi.org/10.1016/j.tet.2015.04.099. These data include MOL files and InChiKeys of the most important compounds described in this article. References and notes 1. Grove, M. D.; Spencer, G. F.; Rohwedder, W. K.; Mandava, N.; Worley, J. F.; Warthen, J. D.; Steffens, G. L.; Flippen-Anderson, J. L.; Cook, J. C. Nature 1979, 281, 216e217. 2. Sakurai, A.; Fujioka, S. Biosci. Biotechnol. Biochem. 1997, 61, 757e762. , J.; Swaczynova , J.; Kol 3. Malíkova a
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