Synthesis of the HIF-2α translation inhibitor compound 76 via a Japp-Klingemann coupling

Article abstract of DOI:10.1016/j.tetlet.2019.03.005

The Iliopoulos laboratory previously reported the discovery of compound 76, a small molecule HIF2α inhibitor. HIF2α activation is known to play a critical role in both von Hippel-Lindau (VHL) disease-related tumors and sporadic renal cell carcinoma (RCC).

Full text of DOI:10.1016/j.tetlet.2019.03.005

Accepted Manuscript
Synthesis of the HIF-2α Translation Inhibitor Compound 76 via a Japp-Klin-
gemann coupling
Xiao Wang, Jung-Eun Ahn, Dawid K. Fiejtek, Lin Lin, Katja Dinkelborg, Ravi
Sundaram, Shao-Liang Zheng, Othon Iliopoulos, Kevin J. Hodgetts
PII:
S0040-4039(19)30206-0
https://doi.org/10.1016/j.tetlet.2019.03.005
TETL 50646
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 December 2018
24 February 2019
3 March 2019
Please cite this article as: Wang, X., Ahn, J-E., Fiejtek, D.K., Lin, L., Dinkelborg, K., Sundaram, R., Zheng, S-L.,
Iliopoulos, O., Hodgetts, K.J., Synthesis of the HIF-2α Translation Inhibitor Compound 76 via a Japp-Klingemann
coupling, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.03.005
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Tetrahedron Letters 0 (2018) 0-0

Xiao Wanga, Jung-Eun Ahna, Dawid K. Fiejteka, Lin Lina, Katja Dinkelborgb, Ravi
Sundaramb, Shao-Liang Zhengc, Othon Iliopoulosb and Kevin J. Hodgettsa*
aLaboratory for Drug Discovery in Neurodegeneration, Brigham and Women’s Hospital and Harvard Medical
School, 65 Landsdowne Street, Cambridge, MA, USA, bMassachusetts General Hospital Cancer Center, Boston,
USA and cHarvard University, 12 Oxford St., Cambridge, MA, USA.
Elsevier Use Only: Received date here; revised date here; accepted date here
Abstract. The Iliopoulos laboratory previously reported the discovery of compound 76, a small molecule HIF2
inhibitor. HIF2 activation is known to play a critical role in both von Hippel-Lindau (VHL) disease-related
tumors and sporadic renal cell carcinoma (RCC). We required a facile and scalable synthetic route to confirm the
structure, to investigate the biological properties of compound 76, and to perform structure activity relationship
optimization studies. Herein, we report the straightforward synthesis of compound 76 and a preliminary
investigation into the scope of the methods developed.
HIF2-driven
tumors.
The
Iliopoulos
Patients with von Hippel-Lindau (VHL)
disease possess a germline mutation in the
VHL tumor suppressor gene that confers a life-
time risk of developing renal cell carcinoma
laboratory conducted a high throughput screen
(HTS) to discover specific HIF2 inhibitors
[4]. The screen was based on a human, VHL-
deficient, RCC cell line stably infected with a
HIF2-driven luciferase reporter. From this
screen, a small molecule HIF2 inhibitor
called ‘compound 76’ was identified (Figure
1). Compound 76 operated by enhancing the
binding of iron regulatory protein 1 (IRP1) to
an iron regulatory element (IRE) in the 5′-UTR
of HIF2, but not HIF1 mRNA, thereby
specifically repressing HIF2 translation.
Furthermore, treatment of VHL-/- zebrafish
embryos with compound 76 rescued the RCC-
(RCC),
central
nervous
system
hemangioblastomas, pheochromocytomas and
pancreatic neuroendocrine tumors [1]. The
VHL protein targets the Hypoxia Inducible
Factors 1 and 2 (HIF1 and HIF2) for
proteasomal degradation in cells exposed to a
normal range of oxygen concentration.
However, low oxygen concentration (hypoxia)
or loss-of-function VHL mutations lead to
HIF1/2 stabilization and transactivation of
HIF-target genes. Collectively, the expression
of HIF1/2 target genes contributes to
oncogenic processes, such as angiogenesis,
erythropoiesis, reprogramming of metabolism,
cell proliferation, and metastasis [2]. In RCC, it
has been shown that HIF2 acts as an
oncogene, while HIF1 is a tumor suppressor
gene [3]. There are currently no drugs available
to treat VHL disease. The inhibition of HIF2
by a small molecule drug is a novel therapeutic
strategy for the treatment of VHL disease and
reminiscent
pronephric
abnormalities,
highlighting its potential as a tool to study
VHL disease and RCC [5].
Figure 1. Structures of compound 76 and 1.

Structurally, compound 76 is a thiophene
substituted at the 2-position by a methyl ester
and at the 3-position by a cyano(methanesulf
onyl)-hydrazine group. The cyano(methanesulf
onyl)-hydrazine moiety is very uncommon; the
only other occurrence of this group in the
literature was the nitrophenyl derivative 1
reported in 1953 by Hunig and Boes [6].
Although compound 76 is commercially
available and is included in HTS screening
collections [4], there were no published
syntheses of compound 76. To confirm the
structure of compound 76, and to further
explore its biology, we required a synthesis and
a route that would allow for the synthesis of
new analogs. The syntheses of aryl hydrazones
typically have been achieved by two ways: (i)
the condensation of an aryl hydrazine with a
ketone or aldehydes; or (ii) via a Japp-
Klingemann coupling [7]. There were very few
examples of the syntheses and use of unstable
α-oxo sulfones [8], which would allow us to
form compound 76 from a starting aryl
hydrazine. Therefore, we investigated the use
Treatment of the commercially available
methyl 3-amino-2-thiophene carboxylate (2)
with sodium nitrite and tetrafluoroboric acid in
acetonitrile at 0 oC gave the intermediate
diazonium salt 3. Then, in situ treatment of 3
with (methylsulfonyl)acetonitrile and N,N-
diisopropylethylamine
(DIPEA)
gave
compound 76 in 62% yield (Method A). An
alternative one-pot procedure involved the
treatment of 2 with tert-butyl nitrite and a
catalytic amount of p-toluenesulfonic acid and
gave compound 76 in a moderate 40% yield
(Method B). The 1H-NMR, 13C-NMR, and
mass spectral data of the products were
consistent with the structure of compound 76.
Finally, the structure of compound 76 was
confirmed by single crystal X-ray analysis
(Figure 2) [9]. A single crystal was obtained
by the vapour diffusion method from
dimethylformamide and water.
A
key
intramolecular hydrogen-bonding interaction
[N(1)- H(1)···O(2), 2.04 Å] can be seen
between the hydrazone N-H and the carbonyl
oxygen of the ester, which forms a stabilized
six-membered ring and gives the molecule a
planar conformation. In addition, the E-
configuration of the hydrazone was confirmed.
of
a Japp-Klingemann coupling with a
sulfonylacetonitrile moiety. To our delight, we
were able to synthesize compound 76 in
moderate yields using the two different
synthetic methods outlined in Scheme 1.
Figure 2. X-ray crystal structure confirms the
structure of compound 76. An intramolecular
hydrogen-bonding interaction is denoted by the
light blue line.
Scheme 1. Reagents and conditions: (i) NaNO2,
HBF4
(aq.),
MeCN,
0
oC;
(ii)
(methylsulfonyl)acetonitrile then DIPEA, 0 oC to
RT (62% yield); (iii) (methylsulfonyl)acetonitrile,
tBuONO, TsOH, MeCN, RT (40% yield).

Investigation of the scope of the two methods
was of interest to us, as we plan to synthesize
analogs of compound 76 for structure activity
relationship studies. Several substituted
arylamine and aminothiophene substrates were
explored, as shown in Table 1 [11]. Method A
proved to be the more robust synthetic
procedure, providing the phenyl product 5a in
excellent yield and the different chloro- and
methoxy-substituted products 5b-5g in
moderate to good yields. Method B generally
gave lower yields, and this was most likely due
to the products not precipitating as efficiently
from the reaction as they did in Method A.
Therefore, additional aqueous work-up,
Table 1. Effect of substitutions on the arylamine.
Yield (%)a
#
4
Product
5a
1
82, 45
37, 38
32, 17
52, 31
2
3
4
5b
5c
5d
extraction
and
trituration
or
flash
chromatography generally led to lower overall
yields. In addition, three different amino-
pyridine analogs were investigated, and only
the reaction of 3-aminopyridine, utilizing
Method A, gave the expected product 5i, albeit
in low yield. We initially hoped that Method B
would work better with the aminopyridine
derivatives, because both the pyridine-2-
diazonium and pyridine-4-diazonium ions
formed using Method A are likely to be
unstable under the aqueous conditions and
react to give the corresponding pyridone.
Unfortunately, the one-pot, non-aqueous
method did not result in the desired products
either. By contrast, we were pleased to find that
all four substituted thiophenes (products 5k–
5n) and the benzothiophene (product 5o) were
obtained in moderate to good yields, by both
methods, following filtration with no further
purification.
5
6
43, 40
47, 12
47, 21
0, 0b
5e
5f
7
5g
5h
5i
8
9
21, 0b
0, 0b
10
5j
11
12
13
64, 49
51, 51
43, 37
5k
5l
5m
In conclusion, we developed a general and
flexible synthesis of compound 76 to allow
further investigation into its potential as a
novel HIF2 suppressor and therapeutic for
VHL disease and HIF2-driven cancers,
including RCC. Further work on structural-
activity relationship and optimization of drug-
like properties is forthcoming.
14
15
54, 42
75, 23
5n
5o
a Isolated yields (Method A, Method B).
b No product detected on LC-MS after 24 hrs.

structure was solved by the Intrinsic Phasing
methods and refined by least-squares methods
again F2 using SHELXT-201411 and
SHELXL-201412 with OLEX 2 interface13.
Acknowledgements. This work was supported
by NIH 1R01CA215431 (to OI).
Crystallographic data (excluding structure
factors) for the structures in this paper have
been deposited with the Cambridge
Crystallographic Data Centre as supplementary
publication nos. CCDC 1884171. Copies of the
data can be obtained, free of charge, on
application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-
336033 or e-mail: deposit@ccdc.cam.ac.uk).
Non-hydrogen
atoms
were
refined
anisotropically, and hydrogen atoms were
allowed to ride on the respective atoms.
Crystal data as well as details of data
collection and refinement are summarized in
Table 1, geometric parameters are shown in
Table 2, and hydrogen-bond parameters are
listed in Table 3. The Ortep plots produced
with SHELXL-2014 program, and the other
drawings were produced with Accelrys DS
Visualizer 2.014.
Appendix A. Supplementary data. General
procedures and analytical data for all new
compounds is reported in the Supplementary
data.
10. Bruker AXS APEX3, Bruker AXS, Madison,
Wisconsin, 2016.
11. Sheldrick, G. M. Acta Cryst. 2015. A71, 3.
12. Sheldrick, G. M. Acta Cryst. 2015, C71, 3.
13. Dolomanov, O. V.; Bourhis, L. J.; Gildea, R.
J.; Howard, J. A. K.; Puschmann, H. J. Appl.
Cryst. 2009, 42, 339.
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9. X-Ray Data. A crystal of compound 76
mounted on a diffractometer was collected
data at 100 K. The intensities of the reflections
were collected by means of a Bruker APEX II
CCD diffractometer (MoK radiation,
=0.71073 Å), and equipped with an Oxford
Cryosystems nitrogen flow apparatus. The
collection method involved 0.5 scans in  at
28 in 2. Data integration down to 0.78 Å
resolution was carried out using SAINT
V8.37A6
with
reflection
spot
size
optimization. Absorption corrections were
made with the program SADABS.10 The

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
Xiao Wanga, Jung-Eun Ahna, Dawid K. Fiejteka, Lin Lina, Katja Dinkelborgb, Ravi Sundaramb, Shao-Liang
Othon Iliopoulosb, and Kevin J. Hodgettsa*
Highlights:
aLaboratory for Drug Discovery in Neurodegeneration, Brigham and Women’s Hospital and Harvard Medical Schoo
b
c

Inhibition of HIF2a activation is
Landsdowne Street, Cambridge, MA, USA, Massachusetts General Hospital Cancer Center, Boston, USA and Harv
Universiatyn, e12wOsxtfroartdegSty.,tCoatmrebraitdgree,nMalAc,eUllSA.
carcinoma

Compound 76 is a HIF2a
inhibitor discovered from a high
throughput screen



Structure activity relationship
studies were performed
A straightforward synthesis of
compound 76 was developed
The structure of compound 76
was confirmed by single crystal
X-ray