Anilinoquinazoline inhibitors of the RET kinase domain - Elaboration of the 7-position

Article abstract of DOI:10.1016/j.bmcl.2016.03.100

We have previously reported a series of anilinoquinazoline derivatives as potent and selective biochemical inhibitors of the RET kinase domain. However, these derivatives displayed diminished cellular potency. Herein we describe further optimisation of the series through modification of their physicochemical properties, delivering improvements in cell potency. However, whilst cellular selectivity against key targets could be maintained, combining cell potency and acceptable pharmacokinetics proved challenging.

Full text of DOI:10.1016/j.bmcl.2016.03.100

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anilinoquinazoline inhibitors of the RET kinase domain—Elaboration
of the 7-position
a
a
a
a
Allan M. Jordan a, , Habiba Begum , Emma Fairweather , Samantha Fritzl , Kristin Goldberg ,
Gemma V. Hopkins a, Niall M. Hamilton a, Amanda J. Lyons a, H. Nikki March a, Rebecca Newton a,
Helen F. Small a, Swamy Vishwanath b, Ian D. Waddell a, Bohdan Waszkowycz a, Amanda J. Watson a,
Donald J. Ogilvie a
⇑
a Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK
b Medicinial Chemistry, SAI Life Sciences, Ltd, Pune, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously reported
a series of anilinoquinazoline derivatives as potent and selective
Received 4 February 2016
Revised 22 March 2016
Accepted 26 March 2016
Available online xxxx
biochemical inhibitors of the RET kinase domain. However, these derivatives displayed diminished
cellular potency. Herein we describe further optimisation of the series through modification of their
physicochemical properties, delivering improvements in cell potency. However, whilst cellular selectivity
against key targets could be maintained, combining cell potency and acceptable pharmacokinetics proved
challenging.
Keywords:
Kinase inhibitor
RET receptor
Ó 2016 Elsevier Ltd. All rights reserved.
Hepatocyte stability
Targeted therapy
Lung cancer
Recent advances in genomic sequencing technology have facil-
itated unprecedented analysis of tumour DNA, allowing patient
stratification based not on histopathology, but on the key driver
mutations found within the tumour itself. This approach has led
to dramatic responses in disease settings where standard therapies
have proved ineffective. Examples of success in this area include
malignant melanoma, where the V600E mutant form of B-Raf can
be inhibited with vemurafenib (ZelborafTM) 1,1 and mtEGFR, which
can be inhibited with gefitinib (IressaTM)2 2 (Fig. 1).
More recently, evidence has emerged that gene fusion products
incorporating the tyrosine kinase domain of the RET receptor can
also provide the oncogenic drive required to initiate and sustain
tumour growth.8 Initially identified in around 1–2% of non-small
cell lung cancer cases, this fusion is now reported to occur in a
diverse range of tumour types, including pancreatic, colon and
breast cancers.9,10
The currently available non-selective inhibitors of the RET
kinase domain, such as cabozantinib (CometriqTM) 4,11 ponatinib
(IclusigTM) 5,12 and vandetanib (CaprelsaTM) 613 are far from
ideal.14,15 Serious adverse side effects limit their utility in the
chronic setting and, in some cases, have led to treatment-related
patient deaths. Several of these effects appear to stem from the
potent inhibition of the VEGFR-2 kinase, also known as KDR. Given
this, we believe that there is a clear clinical need for potent, drug-
like and selective RET inhibitors which do not display off-target
pharmacological inactivation of KDR.
However, activating mutations form only a subset of driver
oncogenes and there is increasing recognition that certain cancer
subtypes are driven by gene fusion products where constitutive
activation of specific kinase domains results in over-active down-
stream signalling.3 For example, the EML4-ALK fusion present in
1–2% of lung cancers leads to uncontrolled proliferation through
activation of the MAPK, PI3K/AKT and JAK3/STAT3 pathways.4
Blockade of this signalling with crizotinib (XalkoriTM) 3,5
a
re-purposed inhibitor originally designed as a Met kinase inhibitor,
has resulted in dramatic clinical responses6 and estimated net sales
of >$280 M in 2013.7
In an effort to address this need, we recently reported a series of
anilinoquinazolines which featured derivatives of a 3-hydrox-
yanilino head group.16,17 Derivatives such as compound 7, bearing
an ortho-methyl substituent, delivered strong enzymatic potency
and surprising selectivity against KDR but were limited by a con-
siderable reduction in cellular activity against the RET kinase
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.03.100
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jordan, A. M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.100

2
A. M. Jordan et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
F
H
N
N
O
S
O
H
N
F
O
HN
N
Cl
N
O
O
N
Cl
F
O
F
1
2
H
N
H
N
Cl
N
N
NH
O
H2N
O
O
O
N
F
Cl
N
N
O
O
3
4
F
Br
N
N
HN
N
F
F
F
O
O
N
H
N
N
O
N
6
5
N
Figure 1. Exemplar targeted therapeutic agents.
domain and, as a consequence, compressed selectivity against KDR
in the cellular context.
these extensive studies revealed the SAR in this area to be extre-
mely tight. Selected examples from this work are shown in Table 1
(Compounds 9–18). Of the derivatives prepared, only the indazoles
16 and 18 retained sub-micromolar enzymatic activity against RET
and of these two derivatives, only 18 demonstrated acceptable
selectivity against KDR at the biochemical level. The observed
SAR is in agreement with the proposed binding mode of these
bicyclic systems, where only indazole 18 is capable of forming both
of the hydrogen bonds observed crystallographically for the phenol
(Fig. 2b). The use of indazole as a phenol mimic in a similar binding
context has been reported previously, e.g., for inhibitors of EphB4
tyrosine kinase.19 However, the fall in biochemical potency
between 7 and 18 in this instance is presumably due to the larger
bulk of 18 being disfavoured in the restricted binding pocket.
Disappointingly, biochemical potency and selectivity did not
translate into the cellular setting, most likely due to modest per-
meability and high efflux (3.7 ꢀ 10-6 cm s-1, efflux ratio 17) which
precluded 18 from further investigation. Moreover, this derivative
demonstrated a somewhat shorter half-life in human liver micro-
somes compared to the parent compound 7 (124 min vs 223 min).
Since bioisosteric replacement of the phenol did not did not
deliver the required attributes to allow an in vivo evaluation of
pharmacokinetic parameters, our attention turned to the optimisa-
tion of the pendant functionality borne by the anilinoquinazoline
scaffold itself.
Our inspiration in this area was partly drawn from a recently
reported series of inhibitors of KDR, where a similar substantial
increase in enzyme inhibitory potency was observed from the
introduction of a meta-anilinophenol warhead to give lead com-
pound 19.20 However, in common with our own chemotype this
derivative also suffered from extensive hepatic glucuronidation
and resultant poor pharmacokinetics. Further optimisation
revealed that the addition of a pendant moiety bearing a basic cen-
tre (as exemplified by 20) not only improved overall pharmacoki-
netics, but also delivered a dramatic reduction in the extent of
Phase I and Phase II metabolism, simultaneously increasing both
stability in liver microsomes and resistance to glucuronidation.21
We hypothesized that a similar approach may offer comparable
benefits in our lead series.
OH
NH
F
O
O
N
N
7
Moreover, these derivatives were limited by metabolic instabil-
ity, likely due to the phenol moiety, which precluded their further
exploration. Our previous studies in this area had indicated deriva-
tives of this type to be comparatively stable in microsomal meta-
bolism assays but considerably less so in the presence of
hepatocytes,16 indicating that the compounds were susceptible to
Phase II metabolic processing such as glucuronidation or sulfation,
which is not uncommon or phenol moieties.18 Herein, we describe
our further efforts in this area to improve both cellular potency and
metabolic stability.
Our initial efforts focused upon attempts to find a bioisosteric
replacement for the phenol that would maintain the key hydrogen
bonding interaction observed in our original derivatives. (Fig. 2a).
In common with related kinase inhibitors, the quinazoline ring sys-
tem binds to the hinge region of the ATP-binding site, with the ani-
lino group buried in the so-called ‘‘selectivity” pocket above the
gatekeeper residue Val-804. The crystal structure of the simple
phenol derivative reveals a pair of hydrogen bonds from the phenol
to Asp-892 (of the DFG loop) and Glu-775 (the conserved C helix
glutamate).16 In this binding mode, the close contact of the
2-methyl against Ser-891 may be responsible for the enhanced bio-
chemical selectivity over KDR, which has a larger cysteine residue
in this position. As such, the targeted bioisosteres were selected in
order to maintain this steric constraint, alongside maintenance of
the desired hydrogen bonding network.
During this investigation, more than 30 examples of classical
and non-classical bioisosteric replacements were prepared but
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A. M. Jordan et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Figure 2. (a) Compound 7 modelled in RET (green carbon atoms, derived from PDB 5AMN16), compared with the aligned X-ray structure of KDR (PDB 3CJG, orange carbons
(b) comparison of compounds 7 (green carbon atoms) and 17 (light blue carbon atoms) modelled in RET. Figure prepared using the Pymol Molecular Graphics System
(Schrödinger, LLC, New York, NY).
Table 1
Biological data for exemplar bioisosteric replacements
Ar
NH
O
N
N
O
a
a
a
Compound
Ar
RET IC50
(l
M)
Enzymatic selectivity versus KDR
RET cellular IC50
(
lM)
KDR cellular IC50 (lM)
7
8
9
2-Me-3-OH-6-F-Ph
Ph
3-MeSO2NH-Ph
3-CHF2-Ph
6-Indolyl
4-Indolyl
6-Benzimidazolyl
4-Benzimidazolyl
7-Indazoloy
6-Indazolyl
5-Indazolyl
4-Indazolyl
0.044 (0.006)
1.7 (0.29)
7.7 (10)
2.9 (0.3)
1.7 (0.48)
9.1 (1.1)
>30
4.3 (0.6)
3.3 (0.19)
0.28 (0.01)
0.60 (0.18)
0.14 (0.06)
130
>5
n/d
2
2.10 (1.0)
n/d
>10
>10
>10
>10
>10
n/d
n/d
>10
n/d
>10
>10
>10
>10
>10
n/d
10
11
12
13
14
15
16
17
18
2
n/d
n/d
3
>30
2
n/d
>10
>10
4.6 (0.26)
5.2 (1.4)
>10
>10
5
38
a
Biological data are stated as the geometric mean of at least four independent determinations. Standard deviations are given in parentheses. n/d = not determined.
generally preferable for both potency and, to an extent, selectivity
OH
OH
(e.g., 26 vs 25, 30 vs 29). However, this trend was not universal and
was dependent upon the specific nature of the tail group.
Modelling of these derivatives suggests that the basic tails should
extend towards the mouth of the ATP-binding site and into solvent,
and, as indicated by molecular dynamics simulations, are more
likely to form water-mediated interactions with the protein rather
than direct hydrogen-bonding contacts (Fig. 3). This is in general
agreement with the data in Table 2, where at best only modest
gains in activity are observed for the basic groups compared with
7 or the extended ethers 49 and 50.
The observed preference for longer carbon chains may be a con-
sequence of an entropic gain due to greater conformational flexi-
bility, with the bulky terminal moiety positioned further from
the protein and more readily solvated. It is also noted that the ami-
nes in Table 2 represent a wide range of basicities, a feature which
is also dependent on the carbon chain length (i.e., the predicted pKa
is higher for amines separated from the oxygen linker by three
carbon atoms rather than two). Given that RET and KDR both
contain several ionisable amino acid side-chains around the mouth
NH
NH
N
N
N
NH
O
N
N
N
N
19
20
Our previous work on this template had demonstrated that
substitution at the 7-position was more readily tolerated than at
the 6-position of the quinazoline core, which tended to result in
diminished potency and selectivity versus KDR at the biochemical
and cellular levels (data not shown). Our investigations therefore
focused upon this more favourable substituent pattern. Data repre-
sentative of the >100 derivatives synthesized and tested are given
in Table 2.
From a diverse series of substituents exploring the incorpora-
tion of a pendant basic centre, some early trends became evident.
For basic substituents, a three carbon linker chain appeared to be
Please cite this article in press as: Jordan, A. M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.100

4
A. M. Jordan et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
Biological data for 7-alkoxy derivatives
OH
NH
F
O
O
N
R1
N
n
a
a
a
Compound
n
R1
RET IC50
(l
M)
Enzymatic selectivity versus KDR
RET cellular IC50
(l
M)
KDR cellular IC50 (lM)
7
1
2
3
2
3
2
3
2
3
2
3
2
3
2
3
2
3
2
3
2
3
2
2
3
1
1
1
1
1
2
3
H
NH2
NH2
NHMe
NHMe
NMe2
NMe2
N-Pyrrolidine
N-Pyrrolidine
N-Piperidine
N-Piperidine
N-Piperazine
0.044 (0.006)
0.13 (0.057)
0.048 (0.018)
0.10 (0.01)
0.023 (0.017)
0.35 (2.0)
130
57
110
280
320
80
2.1 (1.0)
>10
>10
>10
6.5 (2.2)
>10
4.6 (0.08)
>10
>10
>10
5.1 (0.5)
>10
7.6 (1.9)
6.7 (0.7)
6.4 (0.35)
>10
4.6 (0.57)
>10
4.0 (0.058)
>10
>10
1.3 (0.23)
2.1 (0.13)
0.6 (0.05)
1.1 (0.05)
>10
5.9 (1.0)
4.1 (0.38)
1.8 (0.06)
1.2 (0.7)
2.0 (0.3)
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5.6
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
0.007 (0.001)
0.10 (0.02)
710
250
290
215
340
740
630
270
390
340
210
>125
380
650
>500
105
205
120
640
120
190
22
0.015 (0.008)
0.12 (0.027)
0.015 (0.005)
0.017 (0.002)
0.005 (0.001)
0.065 (0.010)
0.011 (0.005)
0.044 (0.01)
0.029 (0.33)
0.014 (0.009)
0.011 (0.007)
0.014 (0.0002)
0.009 (0.005)
0.030 (0.003)
0.15 (0.10)
0.062 (0.12)
0.012 (0.002)
0.11 (0.03)
N-Piperazine
N-Homopiperazine
N-Homopiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Morpholine
N-Morpholine
N-Thiomorpholine 1,1-dioxide
N-Thiomorpholine 1,1-dioxide
(30-Fluoro)-N-pyrrolidine
20-CF3-N-pyrrolidine
20-CF3-N-pyrrolidine
30-Pyridyl
20-(N-Methylimidazole)
30-(5-Methyl-1,2,4-oxadiazole)
40-(N-Methylpiperidine)
20-Tetrahydrofuran
OMe
0.024 (0.006)
0.078 (0.012)
0.018 (0.007)
0.014 (0.002)
0.038 (0.009)
410
270
150
>10
>10
>10
OMe
a
Biological data are stated as the geometric mean of at least four independent determinations. Standard deviations are given in parentheses.
Figure 3. Representative snapshots of (a) 29 and (b) 30 bound to RET from molecular dynamics simulations, highlighting the location of the 7-substituent at the entrance to
the ATP site. Protein solvent-accessible surfaces coloured by electrostatic potential (red = negative potential, blue = positive); water molecules and non-polar hydrogen atoms
omitted for clarity. Figure prepared using the Pymol Molecular Graphics System (Schrödinger, LLC, New York, NY).
of the pocket, long range electrostatic interactions may have some
impact on potency and selectivity even in cases where no formal
hydrogen-bonding contacts are formed.
Whilst we were pleased to find that many derivatives displayed
potencies and selectivity comparable to, or better than, the parent
derivative 7, it was disappointing that this activity tended not to
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A. M. Jordan et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Table 3
Cl
N
Cl
N
i)
In vitro metabolic stability data for selected derivatives
OMe
OH
OMe
OR
N
Compound Human microsomal half-life
(min)
Human hepatocyte half-life
(min)
N
57
7
220
58
4.7
2.7
32
50
2900
68
66
160
—
41
42
43
44
48
49
50
OH
—
60
83
59
99
ii)
NH
N
F
OMe
N
R
O
45-49
Ar
Cl
Scheme 3. Synthesis of compounds 45–49. Reagents and conditions: (i) alcohol,
triphenylphosphine, DIAD, THF, 35 °C, 16 h, 33–91%; (ii) 3-amino-4-fluoro-2-
methylphenol, acetic acid, 120 °C, 2 h, 11–67%.
NH
i)
OMe
OMe
OMe
N
N
N
N
OMe
51
8-18
tended to maintain activity in the cellular systems, giving data
comparable to 7. This finding, along with the data for 43, indicates
that permeability and physicochemical parameters may be a limit-
ing factor for these derivatives and that further modification of
both the pKa of the tailgroup and the overall Log P of the derivative
may potentially allow further improvement in this area.
To investigate whether these present modifications had miti-
gated the metabolic deficiencies experienced with the phenol war-
head, the seven most cell-active compounds were investigated in
in vitro human metabolic stability assays (Table 3). Whilst 49
was exceptionally stable in a microsomal (Phase I) metabolic assay,
most compounds failed to show an improvement relative to com-
pound 7. However, an interesting disconnect in stability was
observed between microsomal stability and stability in hepato-
cytes, where both Phase I and II metabolism occurs. Whilst some
compounds, such as 44, behaved similarly to the parent compound
7 and showed diminished stability in hepatocytes compared to
microsomes (as may be anticipated for a phenolic moiety), we
were pleased to find that compounds 41, 48 and 50 were observed
to be relatively metabolically stable.
Scheme 1. Preparation of bioisosteres 8–18. Reagents and conditions: (i) aromatic
amine, acetonitirile, microwave, 100 °C, 1 h, 11–93%.
NC
N
OMe
O
NC
N
OMe
.TFA
OH
i)
Cl
(Me)2N
(Me)2N
n
52
n = 2; 53
n = 3; 54
OH
OH
NH
NH
N
ii)
ii)
F
OMe
F
OMe
N
N
R1
n
N
O
Cl
O
n
n = 2; 55
n = 3; 56
21-44, 50
Scheme 2. Synthesis of compounds 21–44, 50. Reagents and conditions:
(i) bromochloroalkane, potassium carbonate, acetonitirile, 50–80 °C, 6 h, 61–63%;
(ii) 3-amino-4-fluoro-2-methylphenol, acetic acid, 120 °C, 2 h, 24–48%; (iii) amine,
microwave, 110 °C, 2–24 h, 5–87%.
Incorporation of a pendant, basic tail group has previously been
demonstrated to deliver an improvement in phenol metabolism. In
this setting, this strategy does not appear to be universally benefi-
cial to mitigate hepatic metabolism. Whilst derivatives such as
compound 43 displayed improved cell potency compared to the
parent derivative, concomitant improvements in metabolic stabil-
ity were not generally observed. However, compounds such as 41,
48 and 50 maintained cellular potency and selectivity whilst show-
ing some improvement in the overall metabolic profile of these
agents. These data suggest that further improvements to this ser-
ies, through modification of physicochemical properties, may offer
additional potential for improvements in both metabolic stability
and cellular potency.
The described derivatives were synthesized according to the
following schemes. As detailed in Scheme 1, the commercially
available chloroquinazoline 51 was functionalized through an SNAr
displacement of the 4-chloro moiety with the required aromatic
amine to yield the desired bioisosteres 8–18.
However, the majority of the derivatives for this study were
prepared as described in Scheme 2. The previously described imine
adduct 5222 was readily alkylated with either 1-chloro-2-bro-
moethane or 1-chloro-3-bromopropane to yield intermediates 53
and 54. Cyclisation with the requisite anilinophenol16 yielded the
advanced chloroalkyloxy anilinoquinazolines 55 and 56 in moder-
ate yield. These alkyl halides could then be further elaborated
without intermediate purification simply by SN2 alkylation with
the desired amine, and the target compounds purified by preparative
HPLC, to yield 21–44 and 50.
translate to the cellular environment, with many compounds
failing to display potency in this more relevant context. Given
the biochemical assay was conducted at an ATP concentration
close to Km for the RET kinase domain, we had anticipated a much
closer correlation between the enzyme and cellular assays and we
therefore attributed this disconnect to be due to limited perme-
ability of these compounds, or potential differences in the binding
conformation of the kinase domain in the biochemical and cellular
assay systems.16
Indeed, most of the basic derivatives were found to be inactive
(>10 lM) in our KDR cellular selectivity assay, though seven of the
prepared derivatives displayed cell activity against RET compara-
ble to the parent derivative. Amongst the latter compounds, only
the trifluoromethylpiperazine derivative 43 displayed a significant
improvement in activity and was the only compound found to dis-
play sub-micromolar RET activity in our cell-based systems.
To test the hypothesis that polar functionality, rather than a
basic centre, may also improve metabolic stability whilst
enhancing cellular permeability, a limited number of non-basic
derivatives, exemplified by 48–50, were also prepared. These
derivatives tended to show similar potencies and selectivities to
the basic derivatives when tested in our biochemical assay, but
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6
A. M. Jordan et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
7. Cohen, J. P.; Felix, A. E. J. Pers. Med. 2014, 4, 163.
Certain derivatives, particularly those with cyclic or acyclic
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Frampton, G. M.; Garcia, L.; Juhn, F.; Mitchell, K. C.; White, E.; White, J.; Zwirko,
Z.; Peretz, T.; Nechushtan, H.; Soussan-Gutman, L.; Kim, J.; Sasaki, H.; Kim, H.
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ethers, were more conveniently synthesized using the method
detailed in Scheme 3. The known mono-methylated chloroquina-
zoline 5723,24 was functionalized using Mitsonobu methodology
to install the desired pendant functionality at the 7-position, fol-
lowed by an SNAr displacement of the 4-chloro moiety with the
required aniline16 to yield 45–49.
11. Bentzein, F.; Zuzow, M.; Heald, N.; Gibson, A.; Dhi, Y.; Goon, L.; Yu, P.; Engst, S.;
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This work was supported by Cancer Research UK (Grant number
C480/A11411). In vitro DMPK data were provided by Cyprotex
Discovery, Macclesfield, U.K. JChem for Excel was used for
structure property prediction and calculation, and general data
handling (JChem for Excel, version 15.6.2900, 2008–2015,
ChemAxon (http://www.chemaxon.com)). We thank Mentxu
Aiertza and Shaun Johns for assistance with compound logistics.
Supplementary data
16. Newton, R.; Bowler, K. A.; Burns, M.; Chapman, P. J.; Fairweather, E. E.; Fritzl, S.
J. R.; Goldberg, K. M.; Hamilton, N. M.; Holt, S. V.; Hopkins, G. V.; Jones, S. D.;
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D. P.; Purkiss, A. G.; Small, H. F.; Stowell, A. I. J.; Thomson, G. J.; Waddell, I. D.;
Waszkowycz, B.; Watson, A. J.; Ogilvie, D. J. Eur. J. Med. Chem. 2016, 112, 20.
17. Goldberg, K.; Hamilton, N.; Jones, S.; Jordan, A.; Lyons, A.; Newton, R.; Ogilvie,
D.; Waszkowycz, B. Patent Application WO2015/079251.
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100.
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Please cite this article in press as: Jordan, A. M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.100