The synthesis and structure revision of NSC-134754

Article abstract of DOI:10.1039/c3cc48189a

The synthesis of emetine analogue NSC-134754, a potent inhibitor of the HIF pathway, has been accomplished and its structure reassigned. The stereochemistry of NSC-134754 has been assigned for the first time using X-ray crystallography and it has been demonstrated that only one diastereoisomer is active against HIF.

Full text of DOI:10.1039/c3cc48189a

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The synthesis and structure revision
of NSC-134754†
Cite this: Chem. Commun., 2014,
50, 1238
Jennie A. Hickin,a Afshan Ahmed,b Katharina Fucke,c Margaret Ashcroftb and
Keith Jones*a
Received 28th October 2013,
Accepted 5th December 2013
DOI: 10.1039/c3cc48189a
www.rsc.org/chemcomm
The synthesis of emetine analogue NSC-134754, a potent inhibitor explored as a target for anti-cancer therapies.8,9 Indeed, blocking
of the HIF pathway, has been accomplished and its structure HIF using NSC-134754 has been shown to significantly reduce
reassigned. The stereochemistry of NSC-134754 has been assigned recurrence of glioblastoma after irradiation in mice.10
for the first time using X-ray crystallography and it has been
demonstrated that only one diastereoisomer is active against HIF.
Although NSC-134754 is part of the National Cancer Institute
(NCI) diversity set of compounds,11 a synthetic route to this
compound has not been previously reported. Analysis of a sample
The emetine group of alkaloids, including non-natural derivative of NSC-134754 obtained from the NCI showed the compound to be
dehydroemetine 2 (Fig. 1), has a long and distinguished history of a racemic mixture but a single diastereoisomer. The structure of
biological activity.1,2 The closely related compound NSC-134754 3 NSC-134754 has been reported twice as a single diastereoisomer
(Fig. 1) was first reported in the literature in 1971 where it was tested with the same stereochemistry as (ꢁ)-dehydroemetine 2.3,5 How-
as part of a study into the effect of drugs related to (ꢀ) emetine on ever, no proof of this stereochemistry has been described and its
the lifespan of leukemic mice3 and on protein synthesis in rat liver.4 stereochemistry is not recorded in the NCI database.11 We aimed to
In these reports NSC-134754 was found to be inactive compared to develop a synthetic route to both diastereoisomers of NSC-134754
(ꢁ)-2,3-dehydroemetine. This observation was supported in 1981 and analogues in order to test their ability to inhibit HIF activity.
when NSC-134754 was tested alongside (ꢀ)-emetine and other Classical routes to emetine and dehydroemetine commonly involve
analogues in Chinese hamster ovary cells.5 More recently, NSC- Pictet–Spengler or Bischler–Napieralski reactions to form the key
134754 was identified as an inhibitor of hypoxia inducible factors carbon–carbon bonds, relying on the presence of electron-donating
(HIFs).6,7 The HIFs are transcription factors that play a central role aromatic substituents.12 Owing to the lack of these substituents on
in tumour progression and metastasis and have been widely one of the tetrahydroisoquinoline moieties in NSC-134754 and the
desire to make the synthesis amenable to analogue generation, an
alternative route was required.
We envisaged that a,b-unsaturated lactams such as 11 and its
unsubstituted analogue 7 (Scheme 1) would be useful key inter-
mediates in the synthesis of NSC-134754 and analogues as they
could potentially be elaborated via conjugate addition and electro-
phile trapping.13 Current routes to this type of tricyclic intermediate
only exist for the methoxy-substituted analogue 11 and either rely on
the electron-donating substituents on the aromatic ring to proceed
or involve multiple steps, resulting in a low overall yield.13,14
Fig. 1 Emetine and analogues.
A new route to a,b-unsaturated lactams 7 and 11 was developed
employing directed lithiation and Ring-Closing Metathesis (RCM)
(Scheme 1). N-Boc tetrahydroisoquinoline 4 was lithiated15 and
the resulting anion was treated with allyl bromide to yield 5.
N-Deprotection followed by acylation was used to give the diene
precursor 6 for ring-closing metathesis. trans-Crotonoyl chloride was
used for the acylation as the terminal methyl group was found to
lead to a higher yield for this reaction, without having an adverse
effect on the RCM. Diene 6 was efficiently converted to tricycle 7 in
a CRUK Cancer Therapeutics Unit, The Institute of Cancer Research, 15 Cotswold
Road, Sutton, Surrey, SM2 5NG, UK. E-mail: keith.jones@icr.ac.uk
b Metabolism and Experimental Therapeutics, Division of Medicine, University
College London, 5 University Street, London, WC1E 6JF, UK
c Department of Chemistry, Durham University, University Science Laboratories,
South Road, Durham, DH1 3LE, UK
† Electronic supplementary information (ESI) available. CCDC 968373. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3cc48189a
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alcohol 16 using sodium borohydride, giving an overall yield of
75% for the formation of the tetrasubstituted double bond by RCM.
Bromination of alcohol 16 proceeded smoothly to give tricyclic
bromide 17, which was used to investigate introduction of the
tetrahydroisoquinoline moiety.
Our initial strategy for introduction of the tetrahydroisoquino-
line was to employ the directed lithiation of tetrahydroisoquinoline
4, followed by reaction with tricyclic bromide 17. However, this
reaction was unsuccessful under a wide range of conditions. A
variety of transmetallations of the lithiated species were investi-
gated along with a range of leaving groups to replace the bromide,
with no success. Given this lack of reactivity, we next investigated
forming the required carbon–carbon bond via generation of a
reactive metal species from bromide 17 and its addition to 3,4-
dihydroisoquinoline 18. Both Grignard formation from bromide 17
and lithium–halogen exchange were unsuccessful, leading to a
variety of unwanted side products. This problem was overcome
by employing Barbier-type conditions in which a zinc species was
formed from bromide 17 in the presence of the dihydroisoquino-
line electrophile. TBS-Cl was found to be essential for the success of
this reaction, possibly due to the ability of this group to activate the
imine towards nucleophilic attack.20 The desired carbon–carbon
bond was formed in moderate yield (20%) and as expected a 1 : 1
mixture of diastereoisomers was obtained. The diastereoisomers
were separated by column chromatography and reduction of the
amide using diisobutylaluminium hydride led to both diastereoi-
somers of NSC-134754, 3a and 3b in 9 steps and 2% overall yield.
However, comparison of the NMR data for the original sample of
NSC-134754 obtained from the NCI with the synthetic samples 3a
and 3b revealed that spectra for neither synthetic diastereoisomer
matched with the original sample. Furthermore, both synthetic
diastereoisomers were unable to inhibit HIF activity in a HRE-
luciferase reporter assay (data shown in Fig. 4), leading to the
conclusion that the reported structure for NSC-134754 was
incorrect.
Scheme 1 Synthesis of the tricyclic core.
85% yield using Grubbs II catalyst. Synthesis of methoxy-substituted
tricycle 11 was achieved using the same route starting from tetra-
hydroisoquinoline 8, however the yield for the RCM was only 67%.
Use of a Ti(OiPr)4 additive was found to increase the yield to 86%.16
The synthesis of both the dimethoxy-substituted and the unsubsti-
tuted tricyclic core was achieved in 33% and 26% overall yield
respectively and on multi-gram scale.
Unfortunately, although intermediates 7 and 11 could be elabo-
rated to generate analogues of NSC-134754 via conjugate addition,
attempts to generate NSC-134754 itself using this route were unsuc-
cessful. A new strategy was developed involving directed lithiation
and RCM to install the tetrasubstituted double bond (Scheme 2).
Directed lithiation of tetrahydroisoquinoline 8 using our estab-
lished conditions followed by reaction with protected bromo alcohol
12 was successful in 68% yield to give 13. N-Boc deprotection in the
presence of the silicon protecting group proved to be problematic.
We were most successful using a hindered protecting group
(TBDPS) and the mild removal of the Boc group with zinc bro-
mide.17 The resulting amine was directly acylated using ethyl acrylic
acid to give diene 14. After removal of the silicon protecting group,
RCM was investigated. Following catalyst screening, recently devel-
oped catalyst 20 (Scheme 2)18 was found to carry out this transfor-
mation in good yield. Surprisingly, the corresponding tricyclic
aldehyde was formed as a side product (10%) in this reaction.19
Nevertheless, the aldehyde could be quantitatively reduced to
Similarities in the NMR data indicated that both the synthetic and
NCI samples must have closely related structures and both had
identical masses by high-resolution mass spectrometry. We hypo-
thesized that NSC-134754 is actually a regioisomer, 21, of the reported
structure with the methoxy groups at the 60 and 70 positions on the
tetrahydroisoquinoline (Fig. 2). This was supported by the differing
nOes observed in both samples (Fig. 2). Further investigation revealed
that this regioisomer 21 is also part of the NCI database with
compound number NSC-134756.11 A sample of NSC-134756 was
obtained from the NCI and was shown to have the same structure
as the sample of NSC-134754 by mixed NMR experiments.
Scheme 2 Synthesis of NSC-134754.
Fig. 2 Postulated structure of NSC-134754 and observed nOes.
Chem. Commun., 2014, 50, 1238--1240 | 1239
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Scheme 3 Synthesis of the reassigned structure.
Fig. 4 Luciferase assay-U2OS-HRE-Luc cells,6 1% O2 (16 h), compounds
at 5 mM.
To prove conclusively the postulated structure of NSC-134754,
the synthesis of both diastereoisomers of the regioisomer 21 was
carried out. We envisaged that this could be achieved using the
previously developed chemistry starting from Boc-protected tetra-
hydroisoquinoline 4. The required tricyclic bromide 22 was success-
fully synthesized in 12% yield in 6 steps using the same chemistry
employed in the original synthesis. Unfortunately, the zinc-
mediated reaction to introduce the tetrahydroisoquinoline was
not successful using 6,7-dimethoxydihydroisoquinoline 24. We
postulated that this was due to the electron-donating methoxy
groups reducing the electrophilicity of the dihydroisoquinoline.
This problem was overcome by the formation of an iminium salt
from dihydroisoquinoline 2421 and best results were seen using the
benzyl iodide salt 25 (Scheme 3).
The zinc-mediated reaction was improved further by replacing
solid zinc, which was found to be capricious, with a dialkylzinc
reagent. Initially diethylzinc was used but addition of the ethyl
group to 25 was found to compete with the desired reaction. This
unwanted reaction was avoided by using the more sterically hin-
dered diisopropylzinc and the required carbon–carbon bond was
formed in 61% yield. Amide reduction and benzyl deprotection
proceeded smoothly to give both diastereoisomers of the reassigned
structure 21a and 21b in 10 steps. Although an extra deprotection
step was required in this synthesis, the overall yield was maintained
at 2% due to the improved yield of the zinc-mediated reaction.
NMR data for one of the synthetic diastereoisomers was
identical to the NCI sample, proving that the correct structure
of NSC-134754 is structure 21, a regioisomer of the reported
structure. The stereochemistry of NSC-134754 was also assigned
for the first time by X-ray crystallography (Fig. 3, formula
C27H36N2O2ꢂH2OꢂHSO4ꢂ0.5SO4) and shown to be the same as for
(ꢁ)-2,3-dehydroemetine 2.
Both diastereoisomers of each regioisomer synthesized were
tested for their HIF inhibitory activity using our previously described
HRE luciferase reporter assay6 (Fig. 4). Only 21a was shown to
exhibit inhibitory activity, demonstrating the importance of both the
position of the methoxy groups and the stereochemistry for activity.
In summary, we have synthesized a compound with the reported
structure of the HIF inhibitor NSC-134754 using an RCM reaction to
prepare a key tricyclic intermediate and an allylzinc addition to a
dihydroisoquinoline to complete the pentacycle. Both diastereo-
isomers of this compound 3a,b are inactive. This has led to the
reassignment of the structure of NSC-134754 and we further devel-
oped the allylzinc chemistry to prepare this compound 21a,b. One
of the diastereomers, 21a, was shown to possess HIF inhibitory
properties. Furthermore, we have shown that NSC-134754 and NSC-
134756 have the same structure, 21a. Our synthetic route will allow
the synthesis of a variety of novel analogues to further investigate
the mechanism of action of this interesting class of compounds.
Notes and references
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Fig. 3 Stereochemistry of NSC-134754 and X-ray crystal structure (rela-
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C17 respectively, see ESI† for ORTEP plot).
1240 | Chem. Commun., 2014, 50, 1238--1240
This journal is ©The Royal Society of Chemistry 2014