Elaboration of tetra-orthogonally-substituted aromatic scaffolds towards novel EGFR-kinase inhibitors

Article abstract of DOI:10.1039/c6ob01394e

Nitration of three regioisomers of bromo-fluorobenzaldehyde proceeds regioselectively, notably with H₂SO₄/HNO₃ at 0 °C. The thereby synthesized tetrasubstituted aromatics, endowed with orthogonal substituents, can be elabo

Full text of DOI:10.1039/c6ob01394e

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Elaboration of tetra-orthogonally-substituted
aromatic scaffolds towards novel EGFR-kinase
inhibitors†
Cite this: DOI: 10.1039/c6ob01394e
Adam J. Close,a Rhiannon N. Jones,a Cory A. Ocasio,a Paul Kemmitt,b S. Mark Roec
and John Spencer*a
Nitration of three regioisomers of bromo-fluorobenzaldehyde proceeds regioselectively, notably with
H2SO4/HNO3 at 0 °C. The thereby synthesized tetrasubstituted aromatics, endowed with orthogonal sub-
stituents, can be elaborated via Pd-catalysed coupling, reduction and reductive amination reactions. As a
test-case, these compounds were converted into EGFR inhibitors related to Gefitinib, whose activity was
rationalised by docking studies.
Received 27th June 2016,
Accepted 17th July 2016
DOI: 10.1039/c6ob01394e
www.rsc.org/obc
Introduction
Tetrasubstituted aromatics are commonplace in drug discovery
yet regioselective routes towards these compounds, which often
contain orthogonal groups, are rather scarce.1 Kinase inhibitors
are a rapidly growing class of anticancer agents and many of
these comprise such tetrasubstituted scaffolds, often built
around an adenine-like quinazoline scaffold, a solubilising
group (e.g. ethers, morpholine or piperazine groups) and a tri or
tetra-substituted aromatic, hydrophobic group that imparts
selectivity towards particular classes of kinase (Fig. 1).2–5
To underline their importance, the synthesis of the recently
approved irreversible EGFR (epidermal growth factor receptor
tyrosine kinase family; HER1) inhibitor Osimertinib, starts
with a somewhat simple, yet non-trivial, tetrasubstituted pre-
cursor, which comprises four orthogonal substituents (Fig. 2).6
We have recently reported procedures for forming poyl-
substituted aromatics, mainly based on a MIDA boronate-
substituted aryl scaffold.7,8 Our aim was to synthesise ana-
logues of the type I and II (Fig. 3) as useful 1,2,3,4- and 1,2,4,5-
substituted building blocks.
Fig. 1 Kinase inhibitors, highlighting the aromatic hydrophobic (blue),
the adenine mimic (red) and solubilising group (green).
Here, we report our investigation of the synthesis of related
scaffolds, which do not include a MIDAboronate aryl scaffold
but are substituted with bromo, fluoro and formyl substitu-
aDepartment of Chemistry, School of Life Sciences, University of Sussex, Falmer,
BN1 9QJ, UK. E-mail: j.spencer@sussex.ac.uk
bAstraZeneca, Mereside Alderley Park, Macclesfield, SK10 4TG, UK
cDepartment of Biochemistry, School of Life Sciences, University of Sussex, Falmer,
BN1 9QJ, UK
†Electronic supplementary information (ESI) available. CCDC 1485121–1485123.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c6ob01394e
Fig. 2 Demonstration of a tetrasubstituted aromatic used as a building
block in anticancer molecules.
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We attempted the nitration of three regioiomers of bromo-
fluorobenzaldehyde (Table 1). Compounds 2a and 2b have
been previously synthesised, described in patents, via
nitration, using varying amounts of nitric acid in sulphuric
acid.9,10 We required a generic, high yielding method for these
three compounds and thus looked for alternative methods.
Compound 1a was used to probe different nitration conditions
due to the complementarity of the directing groups, which
would be expected to give one regioisomer. Initial attempts
included the use of potassium nitrate in sulphuric acid11 and
Mn(acac)3 in DCM with nitric acid,12 neither of which gave
good results, as analysed by crude 1H-NMR spectra.
We were pleased that the NO2BF4 method showed compar-
able yields for the synthesis of 2a (entry 1, Table 1) to that of the
nitric acid methods, adopted from the patents. For the synthesis
of 2b and 2c, yields were slightly lower. In general a nitric/
sulphuric acid mixture gave the best results and NO2BF4 proved
to be ineffective in the attempted nitration of 1b and 1c.13
The regiochemistry of the products 2a–2c was confirmed by
1H and 13C NMR spectroscopy as well as by single crystal X-ray
crystallography (Fig. 4).
Fig. 3 Tetrasubstituted aromatic building blocks.
ents. We show that these orthogonal groups can be modified
in order to furnish useful drug-like fragments as well as tetra-
substituted units that can be incorporated into potential
kinase inhibitors.
Results and discussion
Brominations on trisubstituted aromatics are an effective
means for synthesising tetrasubstitued aromatic units contain-
ing orthogonal (e.g. MIDA and bromide) groups. These elec-
trophilic substitutions work with excellent regioselectivity
when complementary directing groups are combined in the
trisubstituted precursor. We now disclose our results on
the related nitrations of trisubstituted (non-MIDA) aromatics
and resulting functional group conversions thereafter
affording novel tetrasubstituted frameworks. In order to main-
tain our ethos of elaborating orthogonally substituted aro-
matics we refrained from attempting the bromination of
bromoaromatics.
Next, we sought a general method for reducing the nitro
compounds 2 and we found that iron in acetic acid was an
effective means for forming the anilines 3. Compound 3a
has been synthesised previously using 10% platinum on
carbon although we found it less effective than an alternative
iron-mediated method (Scheme 1).14
Unfortunately, a whole range of methods proved ineffective
in the attempted reduction of the regioisomer 2a (Scheme 2).
Table 1 Nitration of bromo-fluorobenzaldehydes
Entry
1
Starting material
Product
Yield (%)
Fig. 4 ORTEP diagrams showing crystal structures of 2b (left, CCDC
1485121), 2a (middle, CCDC 1485122) and 2c (right, CCDC 1485123). All
are shown in Table 1. Red = oxygen blue = nitrogen, brown = bromine,
green = fluorine, grey = carbon and white = hydrogen.
88a,c
84b,d
94b, f
2
3
61b,e
74b, f
30b,e
27b, f
a NO2BF4 −20 °C – r.t., 16 h. b H2SO4/HNO3 0 °C – r.t., 16 h. c 12 mmol
scale. d 25 mmol scale. e 30 mmol scale. f 99 mmol scale.
Scheme 1 Iron-mediated reductions of compounds 2b and 2c.
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Table 2 Compounds formed by reductive aminations
Entry
1e
Starting material
Product
Yield (%)
2b
47a
77b
Scheme 2 Nitro group reductions attempted on compound 2a.
2f
3f
4e
3a
3a
3b
83a
91c
Nevertheless, by changing the reaction sequence (vide infra,
Scheme 5) this issue can be circumvented and elaborated tetra-
substituted units can be synthesised.
Alkyne groups are present in a number of kinase inhibitors
such as Erlotinib (Fig. 1) and can also be used in e.g. indole
forming reactions.15–17 We carried out Sonogashira couplings
on 2a, 3a and 3b and obtained the silyl-protected alkynyl-
benzenes 4 in moderate to good yields (Scheme 3).
97a
82a
95d
To show a broader synthetic scope, ethynyl MIDA phenyl-
boronate derivatives were also included, although a slightly
modified protocol was employed, since an excess of aryl halide
was required as the halide could be removed with ease on
silica gel (Scheme 4).18
5e
3b
4c
95a
28a
6e
7e
4c
63a
a 0.5 mmol scale. b 8 mmol scale. c 3 mmol scale. d 6.3 mmol scale.
e 2 eq. of STAB. f 2.5 eq. of STAB.
Scheme 3 Compounds synthesised via Sonogashira cross-coupling
reactions of trimethylsilylacetylene. a Reaction performed at rt for 1 hour.
Scheme 5 Zinc-mediated nitro reductions of compound 5a.
Scheme 4 Compounds synthesised via Sonogashira cross-coupling
reactions of ethynyl MIDAphenylboronates.
Scheme 6 Clauson-Kaas pyrrole synthesis under microwave conditions
on compound 5d.
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A STAB (sodium triacetoxyborohydride)-mediated reductive
amination of the formyl group in a selection of the com-
pounds 2–4 was performed in order to form benzyl-substituted
piperazine or morpholine derivatives (Table 2). Yields in
general were good and these reactions proceeded selectively in
the presence of an aniline substituent e.g. entries 2–5
(Table 2). Deprotected analogues of e.g. 5d, may prove to be
useful synthons for further derivatisation or as novel Ro3 (rule
of three) fragments in drug discovery projects.19,20
Nitro reduction of 5a was achieved, in moderate yield, using
zinc in the presence of ammonium chloride, in order to main-
tain the acid-sensitive Boc protecting group. This represents
an indirect route for obtaining the product 3c since it is a
derivative of the recalcitrant nitro precursor 2a.
Scheme 7 Linking of the quinazoline unit.
Having synthesized a range of tetrasubstituted anilines we
wished to perform further modification of the aniline moiety.
Firstly a Clauson-Kaas pyrrole synthesis was performed on the
aniline 5d, which led to the pyrrole 6a (Scheme 6). Gratifyingly,
the Boc-protecting group withstood the harsh microwave and
acidic conditions.
As a proof of principle, based on structural similarity to the
EGFR inhibitor Gefitinib and with the emergence of tetra-
substituted scaffolds leading to the development of anticancer
therapeutics (e.g. Osimertinib), we rationalised that com-
pounds 7–9 are likely to target the receptor tyrosine kinase,
EGFR. In order to assess the biological activity of our panel of
tetra-substituted small-molecules, we carried out a dual-pass
biological evaluation including in vitro and cellular-based
assays. Compound potency against EGFR (wild-type, Exon 20)
was established using a homogeneous time-resolved fluo-
rescence (HTRF) kinase assay, which measures the extent of
internal tyrosine phosphorylations. The hERG-CHO cell line
over-expresses the human Ether-à-go-go Related Gene (hERG),
which is a gene (KCNH2) that encodes a K+ channel (Kv11.1)
and is a red light toxicity alert in many drug discovery pro-
grammes (Table 3).
Scheme 8 Boc cleavage.
As a cautionary note, we did not embark on a drug discov-
ery programme; for example, the biological evaluation (vide
infra) was performed a long time after the synthesis of the
compounds, preventing for example, any new syntheses and
fine tuning of the products to address pharmacokinetics or
off-target issues or to further investigate SAR (structure activity
relationships).
Commercially available 4-chloro-6,7-dimethoxyquinazoline
was reacted with anilines 3c, 5b, and 5d to afford 7a–7c in
moderate to good yields (Scheme 7). We found that the use of
sodium bis(trimethylsilyl)amide (NaHDMS) gave the product
without deprotection of the Boc piperazine, unlike commonly
used acid catalysed protocols.21 Boc-deprotection was achieved
by the addition of hydrochloric acid in 1,4-dioxane (Scheme 8)
and purification was achieved by simply using solid phase
extraction (strong cation exchange, SCX, column), giving
8a–8c.
Scheme 9 Functionalization of the piperazine unit.
Compounds 7–9 were tested for inhibition of wild-type
EGFR (Table 3). These results show that all of the com-
These newly synthesised secondary amines could be easily pounds containing a BOC protecting groups were inactive
functionalised to sulphonamides or amides in high yields par- except for 7c, which had a high IC50 (21.5 µM). None of the
ticularity in the case of 9b and 9c (Scheme 9).
compounds with regiochemistry resulting from the 2a series
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i.e. 8a, 7a and 9a showed any appreciable activity. The most
To explore the binding mode of 8a–c in EGFR we performed
active compound was 8b (entry 8) which gave an IC50 of 18.4 docking studies using the structure of the Gefitinib-EGFR
nM. All of the compounds in the 2b series gave good results complex.23 We found that 8c was able to bind in a way similar
i.e. all IC50’s were less than 1 µM, with the exception of the to Gefitinib forming archetypal hydrogen bonds between the
BOC protected compound 7b, which showed no activity. backbone N–H of Met793 and a structural water from the
Unfortunately the percentage hERG inhibition increased as nitrogen atoms of the quinazoline core (Fig. 5). An additional
the activity of the compounds improved, as exemplified by 8b hydrogen bond was formed from the protonated piperazine
(Table 3). There are ways in which to design out hERG inhi- and the carbonyl of Asp855. The halogenated phenyl group
bitions e.g. by reducing lipophilicity, reducing the pKa of the was accommodated in the hydrophobic pocket however,
nitrogen atoms, increasing steric hindrance around the nitro- halogen substitution in 8c was not aligned so as to form the
gen atoms or decreasing the number of hydrogen bond accep- halogen bonding exhibited in Gefitinib between the Cl⋯CvO
tors, but, as mentioned above, this was not addressed.22
The biological data suggest that the solubilising group pipera-
of Leu788.
Compound 8b, with halogen substitution meta- and para-
zine ortho to the aniline moiety (linking to the quinazoline unit) to the aniline group, was docked similarly with the quinazo-
gave compounds of higher activity i.e. compounds in the 2b and line core and phenyl ring slightly shifting to form Br⋯H–N
2c series. However, the series where the aniline and the piper- (Asp855) and Br⋯OvC (Glu762) halogen bonds. The key inter-
azine groups are mutually meta, i.e. the 2a series, leads to a loss action between Met793 and the quinazoline nitrogen was
of activity. To look in to the relationship between the meta and formed but the bond with the structural water was not con-
ortho compounds we modelled the binding mode of the com- served. An additional interaction between Arg841 and the pro-
pounds 8a–8c and produced docking poses (Table 3/Fig. 5).
tonated piperazine was formed.
Fig. 5 Docked 8a–c and comparison to Gefitinib, using Schrodinger Glide. (A) Docking poses of 8a–c. (B) Comparison of docked 8a–c with cocrys-
talized Gefitinib. (C) Cocrystal structure of gefitinib in EGFR (PDB code: 2ITY).
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Compound 8a, with the piperazine meta- to the aniline
cannot be docked in a mode similar to gefitinib and forces an
alternative binding mode whereby the methoxy-groups of the
quinazoline are forced into the hydrophobic pocket forming
hydrogen bonds between the oxygen of the MeO– and the H–N
of Lys745. This suggests, and is in agreement, that 8a will be
inactive or show a much lower potency towards EGFR.
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A library of tetrasubstituted aromatics has been synthesized
starting with robust nitration chemistry. The library has been
elaborated into a series of useful potential intermediates for
drug discovery and final drug-like entities as exemplified by
the formation of a range of EGFR inhibitors that display low
nM inhibition.
Acknowledgements
AstraZeneca (ACJ) and the University of Sussex (AJC, RNJ) are
thanked for PhD studentship funding. Worldwide Cancer
Research (grant no. 14-1002) partly funded this work through
the provision of Schrodinger Glide software. The EPSRC UK
National Mass Spectrometry Facility at Swansea University is
thanked for assistance.
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