Total Synthesis of [14C]-Labelled Homoharringtonine

Article abstract of DOI:10.1002/ejoc.201500906

A total synthesis of enantiomerically pure [¹⁴C]-labelled (-)-homoharringtonine in 17 steps is reported. This synthetic process enabled the production of Good Manufacturing Practice (GMP) compliant (-)-[¹⁴C]homoharringtonine that was used in a human mass balance study that was a post-approval commitment to the U.S. Food and Drug Administration. (-)-Homoharringtonine, also called omacetaxine mepesuccinate, is approved to treat adult patients with chronic myeloid leukemia (CML), a blood and bone marrow disease. In November 2012, the product was commercialised as Synribo in the U.S., marketed by Teva Pharmaceuticals. The total synthesis of (-)-[¹⁴C]homoharringtonine, for use in clinical studies, was achieved 17-steps, starting from ¹⁴C-labelled potassium cyanide.

Full text of DOI:10.1002/ejoc.201500906

FULL PAPER
DOI: 10.1002/ejoc.201500906
14
Total Synthesis of [ C]-Labelled Homoharringtonine
[
a]
[a]
[b]
[b]
[b]
[b]
Melanie Marguerit,* Gill Little, Yi Wang, Linli He, Shawn Allwein, James Reif,
[b]
[b]
[b]
Jason Rossi, Renee Roemmele, and Roger Bakale
Keywords: Homoharringtonine / Radiochemistry / Total synthesis / Omacetaxine mepesuccinate / Cephalotaxine
A total synthesis of enantiomerically pure [14C]-labelled (–)-
homoharringtonine in 17 steps is reported. This synthetic
tration. (–)-Homoharringtonine, also called omacetaxine me-
pesuccinate, is approved to treat adult patients with chronic
process enabled the production of Good Manufacturing Prac- myeloid leukemia (CML), a blood and bone marrow disease.
1
4
tice (GMP) compliant (–)-[ C]homoharringtonine that was
used in a human mass balance study that was a post-ap-
proval commitment to the U.S. Food and Drug Adminis-
In November 2012, the product was commercialised as Syn-
ribo in the U.S., marketed by Teva Pharmaceuticals.
®
Introduction
and 15 enantioselective syntheses up to today.[1–18] A review
of all the different approaches described in the literature
allowed us to choose the most suitable strategy to achieve
Homoharringtonine (HHT) is an alkaloid isolated from
the leaves and stems of Cephalotaxus harringtonia, com-
monly known as the Japanese plum yew.
Whilst cephalotaxine (1) itself accounts for approxi-
mately 50% of the mass of the crude alkaloid extract, many
other components have been isolated. Among them homo-
harringtonine (2) and anhydroharringtonine (3) (Scheme 1)
14
the radiosynthesis of [ C]homoharringtonine taking into
account an easy introduction of the label, the stability of
the radiolabelled intermediates, as well as a cost-effective
synthetic route.
14
Our retrosynthetic approach is shown in Scheme 2. [ C]-
14
14
have been reported to be some of the most potent antileuke- Labelled HHT ([ C]-2) could be obtained from [ C]-lab-
mia alkaloids isolated from the cephalotaxus genus.
[
1]
14
elled cephalotaxine ([ C]-1) according to an efficient hemi-
synthesis described in 1999 using enantiopure Robin’s acid
[
19]
14
(
4).
The synthetic strategy to obtain [ C]-1 is based on
14
14
14]
the coupling of [ C]-labelled tosylate intermediate [ C]-5
with enantiopure spirolactam 6 as described by Royer in
004. Introduction of the [ C] label could occur through
[
14
2
the reaction of benzyl chloride intermediate 7 with potas-
14
sium [ C]cyanide, a readily available source of carbon-14.
This retrosynthetic analysis is based on a convergent ap-
proach in which two key unlabeled intermediates 4 and 6
were synthesised in parallel and provided by Teva Pharma-
ceuticals.
Scheme 1. Cephalotaxine and its C3 ester derivatives.
To support the clinical development of the drug candi-
date, a radiolabelled synthesis of (–)-HHT was required.
[
2]
Results and Discussion
With unlabeled intermediates 4 and 6 in hand, we were
14
Over the last 40 years, cephalotaxine (1) has received able to initiate the radiosynthesis of [ C]HHT (2). The syn-
14
considerable attention in terms of total synthesis. Before we thesis of tosylate [ C]-5 is outlined in Scheme 3. Benzyl
1
4
started working on the [ C]HHT synthesis in 2010, more alcohol 8 was converted into the corresponding benzyl
than 10 racemic and 5 enantioselective syntheses had al- chloride 7 with phosphorus trichloride in 98% yield. Cyan-
14
ready been published. The complex structure and promising ation of 7 introduced the radiolabel to give the nitrile [ C]-
bioactivity kept promoting the study on the synthesis by 9 in 92% yield. Optimisation studies revealed that the
the chemistry community, leading to more than 20 racemic benzyl chloride gave a better yield than the corresponding
bromo compound and that the use of acetonitrile and 18-
14
[
a] Selcia Ltd., Fyfield Business & Research Park,
Fyfield Road, Ongar, Essex, CM 0GS, UK
E-mail: gill.little@selcia.com
crown-6 proved critical to obtain [ C]-9 in excellent yield.
14
Hydrolysis to the acid [ C]-10, reduction to the alcohol
14
14
http://www.selcia.com
[ C]-11 and tosylation provided [ C]-5 in near quantitative
[b] Teva Pharmaceuticals, Chemical Synthesis Center,
[20]
yield. The tosylation step required some optimisation in
3
83 Phoenixville Pike, Malvern, PA 19355-9603, USA
terms of the number of equivalents, reaction time and tem-
perature in order to improve the conversion.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500906.
Eur. J. Org. Chem. 2015, 8003–8010
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8003

M. Marguerit et al.
FULL PAPER
14
14
Scheme 2. Retrosynthetic analysis of [ C]-labelled homoharringtonine ([ C]-2).
14
14
Scheme 3. Synthesis of [ C]-labelled tosylate [ C]-5.
14
Scheme 4. Synthesis of pentacyclic intermediate [ C]-17.
8004
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14
Total Synthesis of [ C]-Labelled Homoharringtonine
1
4
Elaboration to the pentacycle [ C]-17 is outlined in
Scheme 4.
The alkylation of spirolactam 6 with the tosylate [ C]-5 very water-soluble, and exhaustive extraction with dichloro-
The use of osmium tetroxide as a 4% solution in water
allowed the reaction to proceed smoothly. Diol [ C]-18 was
14
14
1
4
to give [ C]-12 was optimised to promote N- over O-alkyl- methane was required to recover the material from the
ation in highest yield (Table 1). Increasing the amount of aqueous phase. Subsequently, Corey–Kim oxidation using
base from 1.1 to 1.6 equiv. afforded a near doubling in the dimethyl sulfide, N-chlorosuccinimide and triethylamine in
14
yield, and changing from 1.1 to 1.5 equiv. of spirolactam 6 dichloromethane gave [ C]-19 in 68% yield, which was
increased the yield further to 70% (Entry 3).
then methylated with methoxytrimethylsilane and trifluoro-
methanesulfonic acid.
Triflic acid mediated methylation could provide two po-
tential regioisomers 20 and 21 (Scheme 6).
Table 1. Optimisation of N-alkylation.
Entry
NaH [equiv.]
6 [equiv.]
12 [%]
Some optimisation work was undertaken to drive the re-
action in favour of the desired regioisomer 20. The optimal
reaction conditions are highlighted in Table 2 (Entry 5) with
a yield of 73% of 20.
1
2
3
1.1
1.6
1.6
1.1
1.1
1.5
33
57
70
These conditions were applied to the radiosynthesis to
provide [ C]-12 in 67% yield. The ketal intermediate [ C]-
2 was hydrolysed under acidic conditions to give the corre-
sponding ketone [ C]-13 in quantitative yield. Over the
next four steps, the core pentacycle was constructed using
chemistry based on that of Kuehne. The silyl enol ether
C]-14 was generated in quantitative yield using trimethyl-
silyl iodide and hexamethyldisilazane. Saegusa–Ito oxid-
ation gave the unsaturated ketone [ C]-15 in 85% yield,
1
4
14
Table 2. Optimisation of methylation.[a]
1
Entry CH
2
Cl
2
MeOSiMe
[equiv.]
3
TfOH
[equiv.]
Temp.
[°C]
Ratio
20/21
14
[mL]
1
2
3
4
5
70
70
200
100
70
24
12
20
12
12
2.5
1.25
4.6
2.5
1.25
0 to r.t.
0
0 to r.t.
0 to r.t.
0 to r.t.
3.5:1
3.9:1
1.2:1
2:1
[
20]
1
4
[
1
4
12:1
which underwent a Meerwein–Pondorff reduction to allylic [a] Note: optimisation reactions performed on a 0.5 mmol scale.
1
4
alcohol [ C]-16 using aluminium isopropoxide and 2-prop-
anol. This alcohol was cyclised with tin tetrachloride to
yield the pentacyclic core [ C]-17, which underwent di- 71% yield of [ C]-20 was achieved. Finally, reduction of
hydroxylation using 4-methylmorpholine N-oxide and cata- [ C]-20 with a solution of aluminium hydride in THF
lytic osmium tetroxide in 70% yield (Scheme 5). (freshly prepared from anhydrous aluminium chloride and
In the radiolabelled synthesis using these conditions, a
1
4
14
14
14
14
Scheme 5. Synthesis of [ C]-labelled cephalotaxine [ C]-1.
Scheme 6. Formation of regioisomers 20 and 21 during methylation of 19.
Eur. J. Org. Chem. 2015, 8003–8010
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M. Marguerit et al.
FULL PAPER
Scheme 7. Conversion of 14C-labelled cephalotaxine ([ C]-1) into C-labelled homoharringtonine ([ C]-2).
14
14
14
1
4
lithium aluminium hydride) afforded [ C]-cephalotaxine in chemical purities were higher than 98% and the chiral pu-
4% yield. This is the first reported radiolabelled synthesis rity higher than 99%. The radiosynthesis as described is
of cephalotaxine. robust and has been repeated subsequently on a similar
8
1
4
14
14
[
C]-Cephalotaxine ([ C]-1) was then converted into scale. Additionally, two GMP radiosyntheses of [ C]HHT
[14
Scheme 7).
ically pure Robin’s acid 4 gave [ C]-3 anhydro-HHT in
C]-HHT ([ C]-2) using chemistry developed by Robin starting from [ C]cephalotaxine have been accomplished,
14
14
[
19]
14
(
Esterification of [ C]-1 with enantiomer- and this material is being used in an ongoing clinical study.
1
4
1
4
9
7% yield. Selective ring opening of [ C]-3 via a one-pot
hydrobromination/hydrolysis yielded [ C]-2. Following re-
verse-phase HPLC purification, [ C]-2 HHT was obtained
in 37% yield with a radiochemical purity of 98.9%, a chem-
ical purity of 98.5% and a chiral purity of 100%.
The chemical and radiochemical stability of a solution of
C]HHT (3.5 mg/mL in ethanol at –80 °C) was deter-
mined in a two-year study. Over the course of this time
period, the radiochemical purity fell by 2% and the chemi-
cal purity by 1%. These results demonstrate the purity of [ C]-Labelled compounds were co-eluted side-by-side with their
the material is still within the specification necessary corresponding unlabelled counterparts. Thin-layer chromatography
1
4
Experimental Section
1
4
General: All commercially available reagents were used without fur-
ther purification. All anhydrous solvents were obtained from Acros.
Organic solutions were concentrated under reduced pressure with
a Stuart rotary evaporator using a water bath. Chromatographic
purification of products was accomplished using Biotage SNAP
cartridges. Thin-layer chromatography (TLC) was performed with
Macherey–Nagel Alugram SIL G/UV254 0.25 mm silica gel plates.
[14
1
4
(Ͼ 95%) for clinical evaluation.
plates were analysed by electronic autoradiography using a Packard
Instant Imager. H NMR spectra were obtained with Bruker
1
DPX300, DPX400 and DRX500 spectrometers with TopSpin v.1.3
acquisition and data manipulation software. Chemical shifts (δ) are
referenced to the residual NMR solvent ([D]chloroform: δ =
Conclusions
1
4
6 6
We have achieved the first total synthesis of [ C]-radio- 7.26 ppm; [D ]benzene: δ = 7.16 ppm; [D ]dimethyl sulfoxide: δ =
labelled (–)-homoharringtonine. This material was used in 2.50 ppm). Chemical shifts and coupling constants are reported in
ppm and Hz, respectively. Multiplicities are reported as follow: s =
the required human mass balance study as required by the
singlet, d = doublet, t = triplet, q = quartet, br. = broad, m =
U.S. Food and Drug Administration. Commercially mar-
®
multiplet. Mass spectral analysis was performed using a Thermo
Finnigan AQA single quadrupole mass spectrometer with Xcalibur
v.1.2 aquisition and data manipulation software. High-Performance
Liquid Chromatography (HPLC) was carried out using an Agilent
HP1200 system with a β-RAM radiodetector. The data was ac-
quired using Chemstation and Laura Lite acquisition and data
keted as Synribo , the drug is indicated for the treatment
of adult patients with chronic myeloid leukaemia (CML),
especially those with resistance and/or intolerance to two
or more tyrosine kinase inhibitors (TKIs).
This radiosynthesis numbered 16 steps enabling 15 mCi
1
4
(
ca. 370 mg) of [ C]HHT to be prepared in two batches at manipulation software. Preparative High Performance Liquid
a specific activity of 20 mCi/mmol. The radiochemical and Chromatography (prep. HPLC) was carried out with a Gilson LC-
8006
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Eur. J. Org. Chem. 2015, 8003–8010

14
Total Synthesis of [ C]-Labelled Homoharringtonine
UV system using unipoint v3.3 acquisition and data manipulation
in chloroform (14 mL) were added. The reaction mixture was
software. Quantitative measurement of radioactivity was carried stirred at room temperature for 48 h. TLC/autoradiography showed
out using a Packard 2100TR liquid scintillation counter running a
standard protocol for [ C] gravimetric analysis.
92.5% of the expected product. The reaction mixture was washed
with saturated sodium hydrogen carbonate solution (60 mL) and
saturated sodium chloride solution (60 mL). The organic layer was
14
1
4
14
1
,3-Benzodioxole-5-[1- C]acetonitrile ([ C]-9): To a suspension of
4
separated and dried with MgSO . The drying agent was filtered
14
potassium [ C]cyanide (0.886 g, 13.237 mmol, 750.05 mCi at
7.00 mCi/mmol) and 18-crown-6 (3.500 g, 13.237 mmol) in aceto-
and the solution concentrated. The residue was purified by column
chromatography on silica eluting with isohexane (100%) to isohex-
5
nitrile (14.0 mL) was added a solution of 5-(chloromethyl)-1,3-
benzodioxole (7; 2.190 g, 12.851 mmol) in acetonitrile (14.0 mL).
The white suspension was stirred at room temperature overnight.
The reaction mixture was concentrated. The residue was parti-
tioned between water (40 mL) and ethyl acetate (40 mL). The or-
1
4
ane/diethyl ether (2:1, v/v) to give product [ C]-5 as a colourless
oil (2.300 g, 7.139 mmol, 412.40 mCi, 94%). The radiopurity of this
material was assessed to be 97.7% by TLC/autoradiography
(
f
isohexane/diethyl ether, 1:1, v/v; R = 0.31).
ganic layer was separated and dried with MgSO
was filtered and the solution concentrated. The residue was purified
by column chromatography on silica eluting with dichloromethane
4
. The drying agent
5R)-4-[2-(1,3-Benzodioxol-5-yl)-[1-14C]ethyl]-7,10-dioxa-4-aza-
(
14
dispiro[4.0.4.3]tridecan-3-one ([ C]-12): To a solution of spiro-
lactam 6 (2.360 g, 11.985 mmol) in anhydrous benzene (17.0 mL)
was added sodium hydride (0.511 g, 12.784 mmol) portionwise. The
reaction mixture was heated at reflux for 5 min and cooled to room
temperature. This solution was added to 2-(1,3-benzodioxol-5-yl)-
14
(
1
100%) to give product [ C]-9 as a colourless oil (1.989 g,
2.199 mmol, 691.50 mCi, 92%). The radiopurity of this material
was assessed to be 97.3% by TLC/autoradiography (dichlorometh-
ane, 100%; R = 0.63).
14
14
f
[
1- C]ethyl 4-methylbenzenesulfonate ([ C]-5; 2.575 g,
1
4
14
7.990 mmol, 452.71 mCi), and the reaction mixture was heated at
reflux overnight. The reaction mixture was cooled to room tem-
perature and treated with saturated ammonium chloride solution
2
-(1,3-Benzodioxol-5-yl)[1- C]acetic Acid ([ C]-10): To a suspen-
14
14
sion of 1,3-benzodioxole-5-[1- C]acetonitrile ([ C]-9; 1.989 g,
2.200 mmol, 691.50 mCi) in water (23.0 mL) was added sodium
1
(
(
125 mL). The aqueous layer was extracted with dichloromethane
3 ϫ 100 mL). The combined organic extracts were dried with
hydroxide (1.460 g, 36.600 mmol) in solution in water. The suspen-
sion was heated at 100 °C for 1.5 h and then cooled to room tem-
perature. The reaction mixture was partitioned between diethyl
ether (70 mL) and water (40 mL). The aqueous layer was separated,
acidified with hydrochloric acid (2 n) and extracted with diethyl
ether (3ϫ 70 mL). The combined organic extracts were dried with
4
MgSO . The drying agent was filtered and the solution concen-
trated. The residue was purified by column chromatography on sil-
ica eluting with a gradient isohexane/ethyl acetate (8:2, v/v) to ethyl
1
4
acetate (100%) to give product [ C]-12 as a pale yellow oil (1.858 g,
.351 mmol, 303.32 mCi, 67%). The radiopurity of this material
was assessed to be 97.2 % by TLC/autoradiography (isohexane/
ethyl acetate, 2:3, v/v; R = 0.18).
5
MgSO
trated to give product [ C]-10 as a pale yellow solid (2.292 g,
2.198 mmol, 691.50 mCi, quantitative), which was used in the next
4
. The drying agent was filtered and the solution concen-
1
4
f
1
step without further purification. The radiopurity of this material
was assessed to be 97.2% by TLC/autoradiography (dichlorometh-
14
(
5R)-1-[2-(1,3-Benzodioxol-5-yl)-[1- C]ethyl]-1-azaspiro[4.4]non-
14
ane-2,9-dione ([ C]-13): A solution of (5R)-4-[2-(1,3-benzodioxol-
-yl)-[1- C]ethyl]-7,10-dioxa-4-azadispiro[4.0.4.3]tridecan-3-one
([ C]-12; 1.858 g, 5.351 mmol, 303.32 mCi) in water (25.0 mL) and
acetic acid (2.8 mL) was heated at 105 °C for 21 h. The reaction
mixture was poured into saturated sodium hydrogen carbonate
solution (60 mL) and extracted with dichloromethane (3ϫ 50 mL).
The combined organic extracts were dried with MgSO
agent was filtered and the solution concentrated to give product
C]-13 as a pale orange oil (1.622 g, 5.350 mmol, 303.32 mCi,
quantitative), which was used in the next step without further puri-
fication. The radiopurity of this material was assessed to be 96.4%
ane/methanol, 9:1, v/v; R
f
= 0.75).
14
5
1
4
-(1,3-Benzodioxol-5-yl)-[1- C]ethanol ([14C]-11): To
1
4
2
(
a cooled
1
4
0 °C, ice/water bath) solution of 2-(1,3-benzodioxol-5-yl)[1- C]-
14
acetic acid ([ C]-10; 1.380 g, 7.585 mmol, 438.26 mCi) in tetra-
hydrofuran (22 mL) was added dropwise a solution of lithium alu-
minium hydride in tetrahydrofuran (1 m, 7.66 mL, 7.661 mmol).
Once the addition was complete, the reaction mixture was heated
at 65 °C for 1 h. The solution was cooled with an ice/water bath;
then sodium hydroxide solution (1 n) was added slowly. The re-
sulting suspension was filtered through Hyflo and the filter cake
washed with tetrahydrofuran. The filtrate was concentrated. The
residue was purified by column chromatography on silica eluting
with dichloromethane (100%) to dichloromethane/methanol (95:5,
4
. The drying
1
4
[
by TLC/autoradiography (isohexane/ethyl acetate, 3:7, v/v; R
.19).
f
=
0
1
4
(
5R)-1-[2-(1,3-Benzodioxol-5-yl)-[1- C]ethyl]-9-trimethylsilyloxy-1-
azaspiro[4.4]non-8-en-2-one ([ C]-14): To a solution of (5R)-1-[2-
1,3-benzodioxol-5-yl)-[1- C]ethyl]-1-azaspiro[4.4]nonane-2,9-di-
14
v/v) to give product [ C]-11 as
.585 mmol, 438.26 mCi, quantitative). The radiopurity of this ma-
terial was assessed to be 97.7% by TLC/autoradiography (dichloro-
methane/methanol, 9:1, v/v; R = 0.71).
a colourless oil (1.274 g,
1
4
7
1
4
(
1
4
one ([ C]-13; 1.223 g, 4.034 mmol, 233.12 mCi) in dichlorometh-
ane (52 mL) were added at 0 °C, under nitrogen, hexamethyldisilaz-
ane (1.09 mL, 5.239 mmol) and iodotrimethylsilane (0.63 mL,
4.433 mmol). The reaction mixture was stirred at 0 °C for 1 h and
at room temperature for 2.5 h. The reaction mixture was poured
into saturated sodium hydrogen carbonate solution (50 mL) at
4 °C. The organic layer was separated and the aqueous layer ex-
tracted with further dichloromethane (30 mL). The combined or-
f
1
4
2
(
-(1,3-Benzodioxol-5-yl)-[1- C]ethyl
4-Methylbenzenesulfonate
14
[ C]-5): To a cooled (0 °C, ice/water bath) solution of 2-(1,3-
14
14
benzodioxol-5-yl)-[1- C]ethanol ([ C]-11; 1.274 g, 7.585 mmol,
38.26 mCi) and pyridine (1.22 mL, 15.170 mmol) in chloroform
40 mL) was added dropwise a solution of p-tosyl chloride (2.169 g,
1.378 mmol) in chloroform (20 mL). The reaction mixture was
4
(
1
stirred at 0 °C for 5 h and at room temperature overnight. TLC/
autoradiography showed 56% of unreacted starting material. p-
Tosyl chloride (0.721 g, 3.782 mmol) in chloroform (7 mL) was
added, and the reaction mixture was stirred for 2.5 h; TLC/auto-
radiography still showed unreacted starting material. Pyridine
2 4
ganic extracts were dried with Na SO . The drying agent was fil-
1
4
tered and the solution concentrated to give product [ C]-14 as a
yellow oil (1.514 g, 4.034 mmol, 233.12 mCi, quantitative), which
was used in the next step without further purification. The radio-
purity of this material was assessed to be 94.3% by TLC/auto-
(1.22 mL, 15.12 mmol) and p-tosyl chloride (1.442 g, 7.564 mmol)
f
radiography (isohexane/ethyl acetate, 1:4, v/v; R = 0.32).
Eur. J. Org. Chem. 2015, 8003–8010
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. Marguerit et al.
FULL PAPER
14
(
5S)-1-[2-(1,3-Benzodioxol-5-yl)-[1- C]ethyl]-1-azaspiro[4.4]non-7-
centrated. The residue was purified by column chromatography on
ene-2,9-dione ([ C]-15): To a solution of (5R)-1-[2-(1,3-benzo- silica eluting with dichloromethane (100%) to dichloromethane/
1
4
14
14
dioxol-5-yl)-[1- C]ethyl]-9-trimethylsilyloxy-1-azaspiro[4.4]non-8-
methanol (9:1, v/v) to give product [ C]-18 as a colourless oil
(0.674 g, 2.110 mmol, 121.85 mCi, 70%). The radiopurity of this
material was assessed to be 97.8 % by TLC/autoradiography
14
en-2-one ([ C]-14; 2.008 g, 5.349 mmol, 303.32 mCi) in acetonitrile
53.0 mL) was added 4 Å molecular sieves (1.150 g) at room tem-
(
f
perature, under nitrogen. After stirring for 5 min, palladium(II) (dichloromethane/methanol, 9:1, v/v; R = 0.31).
acetate (1.260 g, 5.616 mmol) was added in one portion. The reac-
1
4
(
2
5S)-3,4-Dehydro-3-hydroxy[benzo[d]azepine-2- C]cephalotaxan-
tion mixture was stirred at room temperature overnight. The mix-
ture was filtered through Hyflo and the filter cake washed with
dichloromethane. The filtrate was concentrated, and the residue
was purified by column chromatography on silica eluting with a
gradient isohexane (100%) to ethyl acetate (100%) to give product
1
4
,8-dione ([ C]-19): To a cooled (0 °C, ice/water bath) suspension
of N-chlorosuccinimide (1.409 g, 10.550 mmol) in dichloromethane
11.8 mL) was added dropwise dimethyl sulfide (0.78 mL,
0.550 mmol). The solution was stirred at 0 °C for 0.5 h, cooled to
–40 °C and added to a cooled (–40 °C) solution of (2S,3R,4S,5S)-
,3-dihydroxy[benzo[d]azepine-2- C]cephalotaxan-8-one ([ C]-18;
.674 g, 2.110 mmol, 121.85 mCi) in dichloromethane (45 mL). The
(
1
14
[
C]-15 as a yellow oil (1.368 g, 4.541 mmol, 257.52 mCi, 85%).
The radiopurity of this material was assessed to be 96.3% by TLC/
autoradiography (isohexane/ethyl acetate, 3:7, v/v; R = 0.13).
1
4
14
2
0
f
reaction mixture was stirred at –40 °C for 1.5 h, triethylamine
5S)-1-[2-(1,3-Benzodioxol-5-yl)-[1- C]ethyl]-9-hydroxy-1-azaspiro- (2.34 mL, 16.880 mmol) was added in one portion and the solution
14
(
[
1
4
4.4]non-7-en-2-one ([ C]-16): To a suspension of (5S)-1-[2-(1,3-
was stirred at room temperature for 2 h. The reaction mixture was
1
4
benzodioxol-5-yl)-[1- C]ethyl]-1-azaspiro[4.4]non-7-ene-2,9-dione poured into saturated sodium chloride solution (120 mL). The or-
14
(
[ C]-15; 1.010 g, 3.354 mmol, 193.72 mCi) in 2-propanol (14 mL)
ganic layer was separated and the aqueous layer extracted with
was added aluminium isopropoxide (20.550 g, 100.620 mmol) at dichloromethane (8ϫ 120 mL). The combined organic extracts
room temperature. The resulting white slurry was heated at 135 °C
for 1.5 h, while acetone and 2-propanol were removed by distil-
lation. The reaction mixture was poured slowly into hydrochloric
acid (1 n, 250 mL) at 0 °C and extracted with dichloromethane (2ϫ
were washed with saturated sodium chloride (2 ϫ 120 mL) and
dried with Na SO . The drying agent was filtered and the solution
concentrated. The residue was purified by column chromatography
on silica eluting with ethyl acetate (100%) to give product [ C]-
19 as a white solid (0.454 g, 1.441 mmol, 83.22 mCi, 68%). The
radiopurity of this material was assessed to be 95.9% by TLC/auto-
2
4
1
4
150 mL). The combined organic extracts were dried with Na
2 4
SO .
The drying agent was filtered and the solvent co-evaporated five
14
times with dichloromethane to leave product [ C]-16 as an orange
oil (1.016 g, 3.353 mmol, 193.72 mCi, quantitative), which was used
in the next step without further purification. The radiopurity of
this material was assessed to be 83.7% by TLC/autoradiography
radiography (ethyl acetate, 100%; R
f
= 0.07).
1
4
(
4S,5S)-1,2-Dehydro-2-methoxy[benzo[d]azepine-2- C]cephalo-
1
4
taxan-3,8-dione ([ C]-20): To a cooled (0 °C, ice/water bath) solu-
1
4
tion of (5S)-3,4-dehydro-3-hydroxy[benzo[d]azepine-2- C]cephalo-
f
(isohexane/ethyl acetate, 1:4, v/v; R = 0.03).
1
4
taxan-2,8-dione ([ C]-19; 0.307 g, 0.975 mmol, 55.28 mCi) in
1
4
(
(
4S,5S)-2,3-Dehydro[benzo[d]azepine-2- C]cephalotaxan-8-one dichloromethane (22.0 mL) were added sequentially methoxytri-
14
14
[ C]-17): To a solution of (5S)-1-[2-(1,3-benzodioxol-5-yl)-[1- C]- methylsilane (1.6 mL, 11.798 mmol) and trifluoromethanesulfonic
ethyl]-9-hydroxy-1-azaspiro[4.4]non-7-en-2-one ([ C]-16; 1.016 g,
1
4
acid (0.11 mL, 1.219 mmol). The reaction mixture was stirred at
3.353 mmol, 193.72 mCi) in dichloromethane (30 mL) was added room temperature overnight. The reaction was quenched with satu-
nitromethane (30 mL) at room temperature. After cooling to
rated sodium hydrogen carbonate solution (100 mL). The organic
layer was separated and the aqueous layer extracted with dichloro-
methane (3ϫ 100 mL). The combined organic extracts were dried
–78 °C, tin(IV) chloride (2.74 mL, 23.471 mmol) was added drop-
wise. The reaction mixture was stirred at –78 °C for 1.5 h and at
room temperature overnight. The reaction mixture was poured into
hydrochloric acid (1 n, 150 mL). The organic layer was separated
and the aqueous layer extracted with dichloromethane (2 ϫ
4
with MgSO . The drying agent was filtered and the solution con-
centrated. The residue was triturated with ethyl acetate (4 mL). The
precipitate was collected by vacuum filtration and washed with
1
4
100 mL). The combined organic extracts were dried with Na
2
SO
4
.
ethyl acetate (3ϫ 3 mL) to give product [ C]-20 as a beige solid
(0.183 g, 0.556 mmol, 31.51 mCi). The mother liquor recovered
from the trituration was purified by column chromatography on
The drying agent was filtered and the solution concentrated. The
residue was purified by column chromatography on silica eluting
with dichloromethane (100%) to dichloromethane/methanol (92:8,
1
4
silica eluting with ethyl acetate (100%) to give product [ C]-20 as
14
v/v) to give product [ C]-17 as a yellow oil (0.865 g, 3.035 mmol,
75.32 mCi, 91%). The radiopurity of this material was assessed to
be 85.2% by TLC/autoradiography (isohexane/ethyl acetate, 1:4, v/
v, R = 0.21).
a beige solid (0.044 g, 0.134 mmol, 7.59 mCi). The two crops of
1
4
1
material were combined to give [ C]-20 as a beige solid (0.227 g,
0.689 mmol, 39.08 mCi, 71%). The radiopurity of this material was
f
assessed to be 95.4 % by TLC/autoradiography (ethyl acetate,
1
1
00%; R
f
= 0.10). H NMR (CDCl
3
, 500 MHz): δ = 2.18–2.42 (m,
1
4
(
8
2S,3R,4S,5S)-2,3-Dihydroxy[benzo[d]azepine-2- C]cephalotaxan-
4
H), 2.44–2.56 (m, 2 H), 2.95 (m, 1 H), 3.51 (s, 1 H), 3.87 (s, 3
1
4
-one ([ C]-18): To a solution of (4S,5S)-2,3-dehydro[benzo[d]-
azepine-2- C]cephalotaxan-8-one ([ C]-17; 0.865 g, 3.035 mmol,
75.32 mCi) in tetrahydrofuran (9 mL) were added 4-methylmorph-
H), 4.09–4.19 (m, 1 H), 5.90–5.99 (m, 2 H), 6.08 (s, 1 H), 6.63 (s,
1
4
14
1
1
H), 6.69 (s, 1 H) ppm. H NMR analysis showed the presence of
1
a single regioisomer.
oline N-oxide (0.425 g, 3.642 mmol) and water (1.1 mL) at room
temperature. After stirring for 5 min, osmium tetroxide (4 wt.-%
solution in water, 0.86 mL, 0.143 mmol) was added to the solution
1
4
14
[Benzo[d]azepine-2- C]cephalotaxine ([ C]-1): A solution of alu-
minium chloride (1.562 g, 11.713 mmol) in tetrahydrofuran
(12.0 mL) was stirred at room temperature under nitrogen for 5 min
before cooling to 0 °C (ice/water bath). To this cooled solution was
added at 0 °C lithium aluminium hydride (1 m in tetrahydrofuran,
36.5 mL, 36.544 mmol). Upon stirring at 0 °C for 10 min, a solu-
tion of aluminium hydride (1 m in tetrahydrofuran) was obtained.
To a cooled (0 °C, ice/water bath) solution of (4S,5S)-1,2-dehydro-
(reaction vessel covered with aluminium foil). The reaction mixture
was stirred at room temperature overnight. The reaction mixture
was treated with hydrochloric acid (3 n, 110 mL) and sodium bi-
sulfite (15% aqueous solution, 43 mL) and extracted with dichloro-
methane (15ϫ 65 mL). The combined organic extracts were dried
with Na
2 4
SO . The drying agent was filtered and the solution con-
8008
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 8003–8010

14
Total Synthesis of [ C]-Labelled Homoharringtonine
1
4
14
2
-methoxy[benzo[d]azepine-2- C]cephalotaxan-3,8-dione ([ C]-
saturated sodium hydrogen carbonate solution (100 mL) until pH
20; 0.227 g, 0.689 mmol, 39.08 mCi) in tetrahydrofuran (25.0 mL)
= 9 and the aqueous layer extracted with dichloromethane (5ϫ
was added aluminium hydride (1 m in tetrahydrofuran, 25.4 mL,
2 4
60 mL). The combined organic extracts were dried with Na SO .
2
0
5.355 mmol), under nitrogen. The reaction mixture was stirred at
°C for 1.5 h. The reaction mixture was treated carefully with satu-
The drying agent was filtered and the solution concentrated (bath
temperature maintained at Ͻ 25 °C). The residue was purified by
column chromatography on silica eluting with dichloromethane
(100%) to dichloromethane/methanol/triethylamine (100:5:1, v/v)
to give [ C]-2 as an off-white glassy solid (142.3 mg, 0.260 mmol,
13.60 mCi). The radiopurity of this material was assessed to be
86.0% by TLC/autoradiography (dichloromethane/methanol/tri-
ethylamine, 100:3:1, v/v; R = 0.50). The residue was further puri-
f
fied by preparative HPLC [using a Zorbax Eclipse XDB-C18 col-
umn, 250ϫ21.2 mm, 7 μm; conditions: at 20.0 mL/min; gradient
rated potassium sodium tartrate solution (20 mL) and extracted
with dichloromethane (4ϫ 100 mL). The combined organic ex-
tracts were dried with MgSO
the solution concentrated. The residue was purified by column
chromatography on silica eluting with dichloromethane/methanol/
ammonium hydroxide (95:5:0.5, v/v) to give product [ C]-1 as a
white film after co-evaporation with toluene (0.183 g, 0.578 mmol,
3
1
4
4
. The drying agent was filtered and
14
2.79 mCi, 84%). The radiopurity of this material was assessed to
be 96.2 % by TLC/autoradiography (dichloromethane/methanol/ from 10 % to 95 % of acetonitrile/0.05 % trifluoroacetic acid in
water/0.05% trifluoroacetic acid over 23 min; UV: 290 nm; reten-
tion time ca. 11 min]. Pure fractions were concentrated to ca.
ammonium hydroxide, 95:5:0.5, v/v; R
HPLC (92.5% chemical purity by UV-HPLC). H NMR ([D]
ene, 400 MHz): δ = 1.33 (br. m, 1 H), 1.47–1.64 (m, 2 H), 1.71 (m,
f
= 0.60) and 97.3 % by
1
6
benz-
150 mL (bath temperature maintained at Ͻ 25 °C) and diluted with
dichloromethane (10 mL) and water (10 mL). The solution was
neutralised to pH = 7 with saturated sodium hydrogen carbonate
solution (20 mL) and the aqueous layer extracted with dichloro-
methane (3ϫ 100 mL). The combined organic extracts were dried
with Na SO . The drying agent was filtered and the solution con-
2 4
centrated (bath temperature maintained at Ͻ 25 °C). The resultant
1
9
7
3
H), 1.89 (m, 1 H), 2.18 (dd, J = 14, 7 Hz, 1 H), 2.51 (q, J = 9,
, 9 Hz, 1 H), 2.65 (dd, J = 10, 8 Hz, 1 H), 2.79 (td, J = 12, 12,
Hz, 1 H), 2.89 (m, 1 H), 3.27 (s, 3 H), 3.37 (d, J = 9 Hz, 1 H),
.58 (m, 1 H), 4.55 (br. d, J = 9 Hz, 1 H), 4.64 (s, 1 H), 5.35 (d, J
=
ppm.
1 Hz, 1 H), 5.40 (d, J = 1 Hz, 1 H), 6.49 (s, 1 H), 6.60 (s, 1 H)
glass/film was dried further under high vacuum at room tempera-
1
4
14
[
Benzo[d]azepine-2- C]anhydrohomoharringtonine ([ C]-3): To a
cooled (0 °C, ice/water bath) solution of enantiopure Robin’s acid
4; 265.8 mg, 1.157 mmol) in dichloromethane (6.5 mL) were added
14
ture for 4 h to give product [ C]-2 as a colourless glass/film
(
73.9 mg, 0.135 mmol, 7.06 mCi, 37%). The radiopurity of this ma-
terial was assessed to be 98.2% by TLC/autoradiography (dichloro-
methane/methanol/triethylamine, 100:3:1, v/v; R = 0.20) and
8.9% by HPLC (98.5% chemical purity by UV-HPLC). The LC/
(
triethylamine (0.160 mL, 1.158 mmol) and 2,4,6-trichlorobenzoyl
chloride (0.180 mL, 1.158 mmol). The resulting solution was stirred
at 0 °C for 1.5 h and added dropwise to a cooled (0 °C, ice/water
bath) solution of [benzo[d]azepine-2-14C]cephalotaxine ([ C]-1;
f
9
MS and NMR spectroscopic data aree fully consistent with the
structure. The specific activity was found to be 55.76 mCi/mmol
with a molecular weight of 547.43 at that specific activity. The com-
pound was dissolved in tert-butyl alcohol (ca. 5 mL) and diluted
with a solution of unlabelled 2 (103.0 mg, 0.189 mmol) in tert-butyl
alcohol (ca. 10 mL). The solution was mixed for 10 min and freeze-
dried for 4 h. The material was further dried under high vacuum at
14
183.4 mg, 0.579 mmol, 29.19 mCi) and 4-(dimethylamino)pyridine
(
290.7 mg, 2.379 mmol) in dichloromethane (4.4 mL) rinsing in
with dichloromethane (2.6 mL). The reaction mixture was stirred
at 0 °C for 4 h and at room temperature for 16 h. TLC/autoradiog-
raphy (dichloromethane/methanol/ammonium hydroxide, 95:5:0.5,
v/v) of a subsample showed 71 % conversion into the expected
product. The reaction mixture was cooled to 0 °C (ice/water bath)
and treated with water (0.3 mL). The resulting mixture was stirred
for 20 min and concentrated (bath temperature maintained at
Ͻ 25 °C) to dryness to give an orange solid. The residue was puri-
fied by column chromatography on silica eluting with dichloro-
methane (100%) to dichloromethane/methanol (98:2, v/v) to give
product [14C]-3 as a pale orange foam (298.0 mg, 0.563 mmol,
14
room temperature for 2 h to give [ C]-2 as a white solid (173.5 mg,
0.318 mmol, 7.01 mCi). The radiopurity of this material was as-
sessed to be 98.1 % by HPLC (98.8 % chemical purity by UV-
1
HPLC) with a chiral purity of 100 %. H NMR ([D]
00 MHz): δ = 0.78 (br. s, 1 H), 1.04 (s, 3 H), 1.06 (s, 3 H), 1.27
m, 2 H), 1.42 (m, 1 H), 1.51 (m, 4 H), 1.60 (m, 1 H), 1.63 (m, 1
H), 1.78 (m, 1 H), 2.17 (dd, J = 14, 7 Hz, 1 H), 2.19 (d, J = 16 Hz,
6
benzene,
5
(
1
8
1
1
H), 2.41 (d, J = 16 Hz, 1 H), 2.44 (m, 1 H), 2.57 (dd, J = 10,
Hz, 1 H), 2.75 (m, 1 H), 2.84 (td, J = 8, 8, 4 Hz, 1 H), 3.23 (m,
H), 3.28 (s, 3 H), 3.35 (s, 3 H), 3.44 (d, J = 10 Hz, 1 H), 3.90 (s,
H), 4.68 (s, 1 H), 5.34 (d, J = 5 Hz, 1 H), 5.48 (d, J = 5 Hz, 1
28.37 mCi, 97%). The radiopurity of this material was assessed to
be 75.3 % by TLC/autoradiography (dichloromethane/methanol/
1
ammonium hydroxide, 95:5:0.5, v/v; R
f
= 0.85). H NMR (CDCl
00 MHz): δ = 1.06 (s, 3 H), 1.12 (s, 3 H), 1.21–1.85 (m, 9 H),
.86–1.96 (m, 1 H), 2.04 (m, 1 H), 2.15 (d, J = 14 Hz, 1 H), 2.38
3
,
4
1
H), 6.17 (d, J = 10 Hz, 1 H), 6.47 (s, 1 H), 6.55 (s, 1 H) ppm. The
LC/MS and NMR spectroscopic data are fully consistent with the
structure. The specific activity was found to be 21.09 mCi/mmol
with a molecular weight of 546.32 at that specific activity. The com-
pound was dissolved in ethanol at a concentration of 3.4 mg/mL
(br. dd, J = 14.7 Hz, 1 H), 2.55–2.67 (m, 2 H), 2.95 (m, 1 H), 3.04–
3
5
5
.24 (m, 2 H), 3.61 (s, 3 H), 3.72 (s, 3 H), 3.81 (d, J = 10 Hz, 1 H),
.06 (br. s, 1 H), 5.81 (d, J = 1 Hz, 1 H), 5.88 (d, J = 1 Hz, 1 H),
.94 (br. d, J = 10 Hz, 1 H), 6.59 (s, 1 H), 6.63 (s, 1 H) ppm.
(
specific concentration of 0.14 mCi/mL) and the solution was fil-
1
4
14
[
Benzo[d]azepine-2- C]homoharringtonine ([ C]-2): To a cooled
tered through a 1.0 μm glass fibre filter into a serum bottle and
kept at below –70 °C pending use.
14
(–20 °C) solution of [benzo[d]azepine-2- C]anhydrohomoharring-
14
tonine ([ C]-3; 193.5 mg, 0.363 mmol, 18.98 mCi) in dichlorometh-
ane (2.2 mL) was added dropwise hydrogen bromide in acetic acid
Acknowledgments
(
33 %, w/w, 2.0 mL, 11.253 mmol). The resulting solution was
stirred at –15 °C for 4 h, then cold water (13.6 mL) was added
quickly. The solution was stirred at room temperature for 15 h.
TLC/autoradiography (dichloromethane/methanol/triethylamine,
We would like to thank people at Selcia that have been involved in
this project, especially D. Hajdu and Dr. S. Knight for analyses of
radiolabeled compounds and their great dedication all along the
project, Dr. N. Proisy and Dr. C. Winfield for their helpful dis-
100:3:1, v/v) of a subsample showed 82% conversion into the ex-
pected product. The reaction mixture was diluted with dichloro-
methane (30 mL) and water (20 mL). The mixture was treated with cussions and advice during this project.
Eur. J. Org. Chem. 2015, 8003–8010
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
8009

M. Marguerit et al.
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Published Online: November 13, 2015
3
8010
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 8003–8010