The discovery of 2-substituted phenol quinazolines as potent RET kinase inhibitors with improved KDR selectivity

Article abstract of DOI:10.1016/j.ejmech.2016.01.039

Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR.

Full text of DOI:10.1016/j.ejmech.2016.01.039

Accepted Manuscript
The Discovery of 2-Substituted Phenol Quinazolines as Potent RET Kinase Inhibitors
with Improved KDR Selectivity
Rebecca Newton, Katherine A. Bowler, Emily M. Burns, Philip J. Chapman, Emma
E. Fairweather, Samantha J.R. Fritzl, Kristin M. Goldberg, Niall M. Hamilton, Sarah
V. Holt, Gemma V. Hopkins, Stuart D. Jones, Allan M. Jordan, Amanda J. Lyons,
H. Nikki March, Neil Q. McDonald, Laura A. Maguire, Daniel P. Mould, Andrew G.
Purkiss, Helen F. Small, Alexandra I.J. Stowell, Graeme J. Thomson, Ian D. Waddell,
Bohdan Waszkowycz, Amanda J. Watson, Donald J. Ogilvie
PII:
S0223-5234(16)30039-3
10.1016/j.ejmech.2016.01.039
EJMECH 8329
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 August 2015
Revised Date: 19 January 2016
Accepted Date: 21 January 2016
Please cite this article as: R. Newton, K.A. Bowler, E.M. Burns, P.J. Chapman, E.E. Fairweather, S.J.R.
Fritzl, K.M. Goldberg, N.M. Hamilton, S.V. Holt, G.V. Hopkins, S.D. Jones, A.M. Jordan, A.J. Lyons, H.
Nikki March, N.Q. McDonald, L.A. Maguire, D.P. Mould, A.G. Purkiss, H.F. Small, A.I.J. Stowell, G.J.
Thomson, I.D. Waddell, B. Waszkowycz, A.J. Watson, D.J. Ogilvie, The Discovery of 2-Substituted
Phenol Quinazolines as Potent RET Kinase Inhibitors with Improved KDR Selectivity, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.039.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
The Discovery of 2-Substituted Phenol Quinazolines
as Potent RET Kinase Inhibitors with Improved
KDR Selectivity
*
,†
†
§
†
Rebecca Newton, Katherine A. Bowler, Emily M. Burns, Philip J. Chapman, Emma E.
†
†
†
†
†
Fairweather, Samantha J. R. Fritzl, Kristin M. Goldberg, Niall M. Hamilton, Sarah V. Holt,
†
†
†
†
†
Gemma V. Hopkins, Stuart D. Jones, Allan M. Jordan, Amanda J. Lyons, H. Nikki March,
§
,‡
†
†
§
Neil Q. McDonald, Laura A. Maguire, Daniel P. Mould, Andrew G. Purkiss, Helen F.
†
†
†
†
†
Small, Alexandra I. J. Stowell, Graeme J. Thomson, Ian D. Waddell, Bohdan Waszkowycz,
†
†
Amanda J. Watson and Donald J. Ogilvie
†
Cancer Research UK Manchester Institute, Drug Discovery Unit, University of Manchester,
Wilmslow Road, Withington, Manchester, M20 4BX, England
§
Structural Biology Laboratory, Cancer Research UK London Research Institute, London,
WC2A 3LY, England
‡
Institute of Structural and Molecular Biology, Department of Biological Sciences,
Birkbeck College, Malet Street, London WC1E 7HX, England
1

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ABSTRACT
Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid
cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and
pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET
as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to
dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase
inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor
using vandetanib 1 as the starting point for structure-based drug design. Phenolic
anilinoquinazolines exemplified by 6 showed improved affinities towards RET but,
unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability
of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte
stability, but could also impart a significant gain in selectivity. This culminated in the
identification of 36; a potent RET inhibitor with much improved selectivity against KDR.
1. INTRODUCTION
RET (REarranged during Transfection) is a receptor tyrosine kinase (RTK) that is required for
normal development, maturation and maintenance of several tissues and cell types.[1] Gain of
function mutations in RET are implicated in several human cancers, e.g. medullary thyroid
cancer (MTC) and lung adenocarcinoma (LAD). The identification of these mutations and
rearrangements in RET which lead to constitutive activation, together with convincing
preclinical data validating RET as a classical oncogene, make this kinase an attractive target for
cancer therapy. At present, there are no known specific RET inhibitors in clinical development,
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although many potent inhibitors of RET have been opportunistically identified through
selectivity profiling of compounds initially designed to target other RTKs. The small molecule
inhibitors vandetanib 1 and cabozantinib 2 (Figure 1) exemplify this approach.
Although both have been approved for the treatment of advanced metastatic MTC[2, 3] and are
undergoing Phase II trials in LAD,[4, 5] RET inhibition is a secondary pharmacology of these
drugs, which were initially developed as inhibitors of other receptor tyrosine kinases. Both
agents target KDR (VEGFR2), whilst 1 has additional activity versus EGFR and 2 versus MET.
Although it is possible that KDR activity may contribute to their clinical efficacy,[6] the results
of a large Phase III trial of 1[2] showed a significantly better hazard ratio for RET-positive
patients compared with RET-negative, suggesting that its efficacy is strongly related to its RET
inhibitory activity. The EGFR activity of 1 is unlikely to significantly contribute to its efficacy
since it has been demonstrated that selective inhibition of EGFR with gefitinib did not yield
clinical responses in MTC.[7] Significant toxicity (e.g. rash, diarrhoea, hypertension) resulting
from inhibition of these off-target kinases, particularly KDR, compromises the use of 1 and 2 in
clinical settings[8]. Thus, there is a clear need for selective RET inhibitors which do not display
the toxicities associated with the current treatments and enable more potent and sustained
inhibition of RET signaling. These agents may offer greater clinical benefit for patients with
RET mutant cancers and widen the scope for the clinical use of RET inhibitors. Although the
initial clinical line of sight for this target was MTC, recent identification of RET fusions (e.g.
KIF5B-RET and CCDC6-RET) present in approximately 1% of LAD patient samples offer an
important disease segment in which a specific RET inhibitor would also offer clinical benefit.
Additionally, RET has also recently been implicated in the progression of both breast and
pancreatic tumours.[9]
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Our initial focus was to identify novel, low molecular weight, ATP-competitive inhibitors of
the RET kinase domain with improved selectivity for RET against KDR in cellular assays,
relative to 1. The availability of high quality X-ray crystal structures of RET and KDR in the
public domain enabled us to pursue a structure-guided medicinal chemistry approach to design
and optimise novel and selective inhibitors. Hit identification employed a focused medicinal and
computational chemistry programme to explore structure-activity relationships (SAR) around
known RET scaffolds and to determine whether selectivity could be improved by targeting
regions of the binding site that differ in sequence between RET and KDR. Although comparison
of X-ray crystal structures of RET and KDR revealed that the ATP-binding pockets are generally
very similar in structure, we hypothesized that there was potential to improve selectivity by
targeting a number of specific residues readily accessible to the ligand. The anilinoquinazoline
core of 1 was selected as a scaffold expected to impart good cellular activity and permeability,
which allowed us to target binding site residues of interest using established medicinal chemistry
methods.
The binding mode of 1 to the RET kinase domain has been reported previously.[10] The
quinazoline core binds to the hinge region (Glu805-Ala807), with the anilino ring enclosed
within the hydrophobic pocket between the gatekeeper residue Val804 and the catalytic lysine
Lys758 (Figure 2). As the gatekeeper pocket is well known to contribute to the affinity and
selectivity of many series of kinase inhibitors, we decided to focus the initial optimization of the
quinazoline series on exploring the SAR around the anilino ring, with a view to identifying
substituents that improved selectivity towards RET. Given that the 6 and 7 positions on the
quinazoline ring point primarily towards solvent, we expected these substituents to have a less
4

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pronounced role on selectivity, and hence fixed both positions as methoxy groups for the initial
round of optimization.
2. CHEMISTRY
2.1 Synthesis of anilinoquinazolines
Quinazolines 4-45 were prepared using the commercially available 6,7-
bismethoxychloroquinazoline 3 as a simplified analogue of 1, as illustrated in Scheme 1.
2.2 Synthesis of key intermediates
Many of the required nucleophiles were commercially available; the remainder were
synthesized using known or modified chemistry. The routes used to prepare anilines 46a-i are
summarized in Schemes 2-7. Common precursors to the desired anilinophenols were the
requisite methyl ethers 47a-d or nitro compounds 48a-c, which could be demethylated or
reduced using standard procedures as shown in Schemes 2 and 3.
Alternative precursors to the anilinophenols were the corresponding bromides. These could be
converted to the boronate esters 49h-i, then oxidized to the required phenols 46h-i as shown in
Scheme 4.
Attempts to prepare 3-amino-2-(trifluoromethyl)phenol were unsuccessful. Instead, benzyl
ether 50 was prepared from commercial starting material then converted to aniline 51. This was
coupled directly with 3, then deprotected to return the desired quinazoline 17 as shown in
Scheme 5.
N-Methylated phenol 53 was prepared by reduction of the corresponding N-Boc material 52
(Scheme 6).
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3. RESULTS AND DISCUSSION
3.1. Biochemical evaluation
Our initial aim was to prepare to number of anilinoquinazolines to explore the affinity and
selectivity towards RET as compared to 1.[11, 12] The biochemical data for selected compounds
are shown in Table 1. First, we ascertained that switching from the more decorated quinazoline
core present in 1 to the bis-methoxy scaffold present in 4 showed only a 2-fold drop in affinity
(data for 1 not shown). Therefore, we opted to use this simplified scaffold for further exploration,
given it was commercially available. Of the first swathe of approximately 30 simple
anilinoquinazolines prepared, the most interesting observation was that phenol 6 resulted in a
significant gain in affinity towards RET. The same level of affinity was not maintained in the
isomeric compounds 5 and 7, or for the analogous aniline 8 or the corresponding methoxy ether
9
.
3
.2. Structural considerations
2
The boost in RET affinity from the R hydroxyl group may be rationalized by consideration of
the hydrogen bonding contacts formed in the gatekeeper pocket. Initial modelling of 6,
subsequently confirmed by the determination of the X-ray structure bound to RET (Figure 3),
highlighted a pair of hydrogen bonds from the phenol to the side-chain of Glu775 (the conserved
glutamate located on the αC-helix) and the backbone NH of Asp892 (from the conserved DFG
motif). It is likely that this specific network of hydrogen bonds is responsible for the gain in
2
2
affinity and the tight SAR for the R hydroxyl, with alternative polar R substituents or hydroxyls
1
3
in the R or R positions all demonstrating reduced affinity
Comparison of the X-ray structures of 6 and 1 reveals that the ligands share very similar
binding modes. The anilino ring of 6 binds between the gatekeeper Val804 and the catalytic
6

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lysine Lys758 and, in comparison to 1, shows a small rotation towards Asp892, with minor re-
orientation of neighbouring side-chains consistent with the formation of hydrogen-bonding
interactions between the phenol and Glu775/Asp892. The most substantial structural change is
the displacement of the phosphate-binding loop away from the ATP binding site in the X-ray
structure of 6; this may be a consequence of a degree of induced fit in the ligand binding site or
may simply reflect the inherent flexibility of this loop. Comparison with X-ray structures of 6
bound to other kinases, e.g. to CDK2, CDPK1 and TTBK1 (PDB codes 1DI8, 3NYV and 4BTK,
respectively), reveal a variety of different conformations and hydrogen bonding interactions for
the phenol moiety, distinct from that observed for 6 bound to RET. Thus it should be possible to
modulate the selectivity of this series by appropriate substitution around the anilino ring.
3
.3. Mitigation of DMPK concerns and delivery of unanticipated selectivity
The presence of a phenol was some cause for concern given it was anticipated to undergo
phase II metabolism. Measurement of in vitro DMPK properties (data not shown) of the initial
compound 6 showed that solubility and CYP inhibition were acceptable at this stage, although
the permeability and efflux needed improvement. Observation of metabolism in microsomes,
albeit to a 3-fold lesser extent than in hepatocytes, indicated phase I metabolism was occurring in
addition to phase II.
In order to mitigate phase II metabolism, we explored further substitution on the phenolic
aniline. Our goals here were two-fold. First, it was speculated that the presence of flanking
substituents might attenuate the propensity of the phenol to undergo conjugation, thereby
increasing hepatocyte stability. Second, it would allow a more general SAR exploration to
determine what functionality could be tolerated around the phenyl ring.
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We first prepared a number of mono-substituted anilinophenols. Most of these bore a flanking
1
3
substituent either at the R or R position, primarily to investigate whether the hepatocyte
stability could be mitigated through steric encumbrance. However, it soon became apparent that
1
a suitable R substituent also had a considerable influence on KDR selectivity (10-13); albeit
with some loss of RET affinity for 11-13. This selectivity enhancement was less evident with
fluorine (14), whereas larger substituents were not tolerated, as indicated by the large drop in
3
RET affinity (15-18). Conversely, a flanking substituent at R (19-22) was tolerated with respect
to RET affinity but selectivity was comparable to, or worse than, the unsubstituted phenol 6.
Although we anticipated that any improvement in hepatocyte stability would be most likely
1
3
achieved by exploiting substitution at the flanking R or R positions, limited examples of
4
5
4
5
substitution at the R or R position were also explored. Halogens were tolerated at R and R
4
(
23, 25-26) but a methyl group resulted in a modest drop in affinity, especially at the R position
24 and 27).
(
In addition to these mono-substituted examples, we also prepared a number of di-substituted
anilinophenols. Appropriately positioned halogens generally retained or enhanced affinity but
1
selectivity was modest in the absence of a substituent larger than either H or F at the R position
(28-34).
1
Clearly, incorporation of a suitable group at the R position was beneficial for selectivity,
1
although affinity was somewhat diminished, especially when R was Me. Given that halogens,
5
especially fluorine, at R had been seen to generally enhance affinity, it was encouraging to see
that combining these two observations resulted in compounds which now exhibited both
1
3
improved affinity and selectivity (35-37). When R was fixed as chloro, substituting at R with a
methyl group was detrimental with regard to selectivity (compare 12 with 38). Further
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5
substitution at R with chlorine (39) was tolerated whereas fluorine (40) appeared to be
beneficial, both in terms of affinity and selectivity.
1
Modelling of this series in RET suggests that the R methyl substituent is positioned close to
the side-chain of Ser891 (immediately preceding the DFG motif), and this potentially
disfavoured contact could account for the observed reduction in RET affinity compared with 6
1
(
Figure 4). In KDR, Ser891 is replaced by a bulkier cysteine (Cys1045). Hence, although the R
methyl is somewhat disfavoured in RET, there is a larger drop-off in affinity against KDR,
leading to an improved selectivity profile towards RET overall.
3.4. Evaluation in cellular assays
A number of these compounds (selected on the basis of affinity and/or selectivity) were
progressed into BaF3 cellular assays for RET and KDR, the results of which are shown in Table
2
. Disappointingly, the affinity and selectivity observed for this phenolic series in the
biochemical assay did not transfer well to the cellular context. Only 4 compounds (25, 26, 28 and
0) were <100 nM against RET in the cellular assay and only one compound (40) showed >10-
3
fold selectivity versus KDR. In contrast, 17 compounds were ≤10 nM in the biochemical assay
and 22 compounds showed >10-fold selectivity. For reasons not fully understood, the disconnect
from the biochemical to cellular assay is much greater for RET than KDR, in effect compressing
the selectivity margins, often from >100-fold in the biochemical assay to parity (or worse) in the
cellular assay. Permeability was not believed to be the cause for the disparity as the observed
reduction in affinity was not of the same magnitude for both RET and KDR in a matched cell
line. The affinity for ATP in the biochemical assay is also unlikely to explain the difference in
the reduction as the K
m
values for both proteins (RET 9 µM and KDR 8 µM) were similar.
Interestingly, the non-phenolic quinazoline 4 does not appear to suffer to the same extent.
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The discrepancy between biochemical and cellular selectivity may be related to the binding
2
mode of the R phenol. Modelling of 6 in various published X-ray structures of KDR suggested
that the hydrogen bonding network around the phenol in RET may be less readily achievable in
KDR. This is a consequence of KDR X-ray structures displaying a conformation in which the
αC-helix is displaced away from the ATP binding site in comparison to RET. The associated
movement of the conserved glutamate located on the αC-helix (equivalent to Glu775 in RET)
would likely compromise the formation of the pair of hydrogen bonds to the phenol observed
crystallographically in RET. This “αC-helix-out” motif is typically characteristic of an inactive
kinase conformation. It may be that the difference between biochemical and cell selectivity
profiles is related to different populations of active/inactive RET conformations under the
different assay conditions. Thus, under the conditions of the biochemical assay, RET may exist
predominantly in the active conformation characterized in the available X-ray structures,
whereas in the cellular context there may be a larger population of inactive conformations,
similar to the “αC-helix-out” conformation observed in KDR X-ray structures, to which binding
of the phenol is less favoured. Regardless of the explanation for the observed differences, the
decision was taken that the more physiologically relevant cellular data would drive subsequent
progression of the project.
3.5. Investigation of alternate chemotypes
Different linkers were investigated as an alternative to the aniline but showed no improvement
in comparison to 6. As shown in Table 3, it can be seen that the N-methyl analogue 41 is
significantly less potent than the NH-, O- and S-linked analogues (6, 42 and 43). Although 42
and 43 retain biochemical affinity as compared to 6, they too suffer from a disproportionate
reduction in affinity against RET compared to KDR in the cell assay.
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Despite the drop in affinity of the N-methylated analogue 41, it retained some activity. Based
upon this observation, the tethered compounds 44 and 45 were synthesized to test the hypothesis
that these may deliver the same gain in selectivity as did the 2-substituted analogues described
earlier. However, although this was observed biochemically for 44, these biochemical activities
again did not translate to cellular potency and selectivity, as shown in Table 4.
3.6. Further biological profiling
As mentioned previously, the initial interest in preparing phenolic anilines with flanking
substituents was to mitigate the anticipated metabolic liability of the phenol. Table 5 shows the
human hepatocyte stability data for selected compounds, ranked from most to least stable.
Although compounds 10, 11 and 12 benefitted from an improved selectivity profile compared to
1
unsubstituted 6, the R groups in these examples were detrimental to hepatocyte stability.
Pleasingly, 13 did show the desired improvement in stability, albeit with a 20-fold reduction in
4
5
affinity. Inclusion of fluorine at either the R or R positions went some way toward recovering
the affinity but it can be seen that 36 is preferred over 35 with regard to hepatocyte stability.
3
Interestingly, the isomer 30, bearing the flanking substituent in the R position rather than the R1
position as in 36, is significantly less stable. Unfortunately, 33, 34 and 40 (the most promising
compounds with regard to affinity and selectivity in the cell assay) all suffered from high
clearance.
There does not appear to be a clear correlation between stability and phenol pK , indicating
a
other effects (e.g. steric influence of the flanking substituent or contributions from other
substituents in the case of the di-substituted examples) are contributing to the observed clearance
1
across the series. Looking at those compounds mono-substituted at R (13, 10, 11 and 12), only
the Me-substituted compound 13 shows an improvement in clearance when compared to the
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unsubstituted phenol 6. Although it is true that, for this particular set of compounds, a lower pK
a
results in higher clearance, this is not observed in all cases. Isomeric pair 30 and 36 have
identical calculated pK values yet differ only by the position of the flanking Me substituent,
a
3
indicating that substitution with a Me group at the R position is detrimental to metabolic
5
stability. Comparison of 30, 35 and 36 shows the inclusion of F at R is beneficial, although the
4
effect on pK is negligible, whereas inclusion of F at R resulted in a considerably lower phenol
a
1
5
pK
a
and higher clearance. Compounds containing a halogen at both R and R displayed lower
1
clearances than those only substituted with a halogen at R , despite similar pK
a
values (compare
5
3
3, 34, 40, 11 and 12), again indicating that blocking R is beneficial. There does not appear to
be any correlation between clearance and predicted logP (XLogP).
These compounds were also tested for non-specific cellular toxicity, and, with the possible
exception of 30, all were found to be devoid of non-specific toxicity in a wild-type BaF3 cell
line, the parental cell line used to prepare the RET and KDR driven cell lines used in our routine
screening assays. This pleasing result further suggests that the compounds display meaningful
kinase selectivity in the cellular context and do not promiscuously inhibit off-target kinases
responsible for cell proliferation and survival.
On the basis of these data, 36 was selected for further in vitro and in vivo pharmacokinetic
assessment. In terms of metabolic stability, intrinsic clearance was higher in human hepatocytes
than in human microsomes (CLint 6.2 µL/min/mg), indicative of phase II metabolism.
Metabolism was more rapid in mouse in both microsomes and hepatocytes (CLint 28.2
6
µL/min/mg and 38.1 µL/min/10 cells, respectively). In terms of physical properties, 36 showed
good aqueous solubility (in excess of 100 µM) but only moderate permeability in Caco-2 cells
-6
-1
(Papp 8.2 x 10 cm s , efflux ratio 4.9). Pharmacokinetics were measured in the mouse via
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intravenous and oral routes of administration. Total blood clearance was low (<10% LBF) and
bioavailability was approximately 35%. Oral half-life was measured at approximately 2 hours.
4. CONCLUSION
A structure-based drug design programme led to a series of phenolic anilinoquinazolines
showing high affinity for RET in the biochemical context. Concern over the metabolic liability
of phenol 6 prompted exploration of flanking substituents to attenuate the propensity of the
1
phenol to undergo phase II metabolism. Pleasingly, incorporation of Me at R not only resulted
in improved metabolic stability but also in an unexpected gain in selectivity over KDR, which
could be rationalised by modelling. The improved selectivity was accompanied by some
5
reduction in affinity but this could be recovered to some extent by inclusion of fluorine at the R
position, resulting in 36; a potent and selective RET inhibitor. However, for reasons not fully
understood, the translation of biochemical potency to cellular potency was disproportionate when
comparing RET and KDR, in effect compressing the apparent selectivity observed in the
biochemical assay. Further efforts to improve both the cellular affinity and selectivity and the
ADME properties of 36 are underway in our laboratory.
5. EXPERIMENTAL
1.1. Chemistry
All reagents obtained from commercial sources were used without further purification.
Anhydrous solvents were obtained from the Sigma-Aldrich Chemical Co. Ltd. or Fisher
Chemicals Ltd. and used without further drying. Solutions containing products were either
4 2 4
passed through a hydrophobic frit or dried over anhydrous MgSO or Na SO , and filtered prior
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to evaporation of the solvent under reduced pressure. Thin layer chromatography (TLC) was
conducted with 5 cm × 10 cm plates coated with Merck type 60 F254 silica gel to a thickness of
0
.25 mm. Chromatography was performed on Biotage SNAP HP-Sil cartridges using a
1
CombiFlash Companion machine. Proton ( H) NMR spectra were recorded on a 300 MHz
Bruker spectrometer at ambient temperature. Solutions were typically prepared in either
deuterochloroform (CDCl ) or deuterated dimethylsulfoxide (DMSO-d6) with chemical shifts
3
1
referenced to deuterated solvent as an internal standard. H NMR data are reported indicating the
chemical shift (δ), the multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br,
broad; dd, doublet of doublets, etc.), the coupling constant (J) in Hz and the integration (e.g. 1H).
Deuterated solvents were obtained from the Sigma-Aldrich Chemical Co., Goss or Fluorochem.
LC−MS spectra with UV detection were recorded on a Waters Acquity UPLC. Mass
spectrometry was performed on a Waters Acquity SQD quadrupole spectrometer running in dual
ES+ and ES− mode. High pH runs were conducted at pH 10 and low pH runs were conducted at
pH 4, with a run time of 2 mins. The column temperature was 40 °C, and the flow rate was 0.6
mL/min. Further details, including solvent gradients, are given in the Supporting Information.
Details of the preparative HPLC instrument and the solvent gradient used to purify compounds
are also given in the Supporting Information. All compounds were ≥95% purity as determined by
1
examination of both the LC–MS and H NMR spectra unless otherwise indicated. When Cl or Br
was present, expected isotopic distribution patterns were observed.
1.1.1. General Procedure for Synthesis of Quinazolines
4-Chloro-6,7-dimethoxyquinazoline 3 and the required nucleophile were heated in solvent
either thermally or using microwave heating until no further reaction was observed. On cooling,
the hydrochloride salt was isolated by filtration. Alternative isolation procedures were employed
14

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if precipitation did not occur. Additional purification by preparative HPLC or flash column
chromatography was employed in some cases. Spectroscopic data for compounds 4,[13] 6-9,[14-
1
6] 20-21,[13] 25,[13] 28[17] and 30[18] are in agreement with those reported in the literature.
.1.1.1. 2-((6,7-Dimethoxyquinazolin-4-yl)amino)phenol hydrochloride (5)
1
A mixture of 3 (100 mg, 0.45 mmol) and 2-aminophenol (49 mg, 0.45 mmol) in MeCN
1
afforded 5 (90 mg, 55%) as a yellow solid. H NMR (300 MHz, DMSO-d ) δ 11.09 (br s, 1H),
6
9
7
3
.90 (br s, 1H), 8.72 (s, 1H), 8.22 (s, 1H), 7.36 (s, 1H), 7.32 (dd, J = 1.60, 7.82 Hz, 1H), 7.20-
.28 (m, 1H), 7.04 (dd, J = 1.18, 8.15 Hz, 1H), 6.91 (dt, J = 1.27, 7.56 Hz, 1H), 4.00 (s, 3H),
.99 (s, 3H).
1.1.1.2. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)benzene-1,2-diol hydrochloride (10)
A mixture of 3 (898 mg, 4.0 mmol) and 3-amino-1,2-benzenediol (500 mg, 4.0 mmol) in
1
MeCN afforded 10 (1.24 g, 89%) as a dark brown solid. H NMR (300 MHz, DMSO-d ) δ 10.96
6
(br s, 1H), 9.54 (s, 1H), 8.99 (br s, 1H), 8.71 (s, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.84 (dd, J =
1
3
2
d
9
.17, 7.16 Hz, 1H), 6.69-6.81 (m, 2H), 4.00 (s, 3H), 3.98 (s, 3H). C NMR (75 MHz, DMSO-
6
): δ 158.9, 156.0, 149.9, 148.5, 146.5, 141.0, 135.4, 124.4, 118.5, 118.2, 114.6, 106.9, 103.9,
+
9.9, 563, 56.3. HRMS (ESI) m/z [M + H] calcd for C16
H
15
N
3
O
4
: 314.1140. Found: 314.1141.
1.1.1.3.2-Bromo-3-((6,7-dimethoxyquinazolin-4-yl)amino)phenol hydrochloride (11)
A mixture of 3 (200 mg, 0.89 mmol), 3-amino-2-bromo-phenol[19] (167 mg, 0.89 mmol) and
1
5
-6N HCl in IPA (0.01 mL) in IPA afforded 11 (310 mg, 92%) as a cream solid. H NMR (300
MHz, DMSO-d ) δ 11.30 (br s, 1H), 10.62 (s, 1H), 8.75 (s, 1H), 8.14 (s, 1H), 7.29-7.35 (m, 2H),
6
1
3
7
.04 (dd, J = 1.41, 8.19 Hz, 1H), 6.97 (dd, J = 1.41, 7.82 Hz, 1H), 4.01 (s, 3H), 3.99 (s, 3H). C
NMR (75 MHz, DMSO-d ): δ 159.1, 156.4, 155.5, 150.2, 148.6, 136.8, 135.3, 128.3, 119.6,
6
15

ACCEPTED MANUSCRIPT
+
1
3
3 3
15.4, 110.0, 106.6, 103.6, 99.7, 56.7, 56.5. HRMS (ESI) m/z [M + H] calcd for C16H14BrN O :
76.0297. Found: 376.0297.
1.1.1.4.2-Chloro-3-((6,7-dimethoxyquinazolin-4-yl)amino)phenol hydrochloride (12)
A mixture of 3 (1.56 g, 6.97 mmol) and 3-amino-2-chlorophenol (1.0 g, 6.97 mmol) in MeCN
1
afforded 12 (2.21 g, 86%) as a beige solid. H NMR (300 MHz, DMSO-d ) δ 11.45 (br s, 1H),
6
1
1
0.59 (s, 1H), 8.77 (s, 1H), 8.22 (s, 1H), 7.36 (s, 1H), 7.27 (t, J = 7.72 Hz, 1H), 7.08 (dd, J =
1
3
.22, 8.29 Hz, 1H), 6.98 (dd, J = 1.13, 7.82 Hz, 1H), 4.01 (s, 3H), 4.00 (s, 3H). C NMR (75
MHz, DMSO-d
6
): δ 157.5, 154.1, 153.1, 148.7, 146.7, 137.4, 126.8, 119.5, 118.2, 113.9, 108.5,
+
1
3
07.0, 101.9, 56.0, 55.7. HRMS (ESI) m/z [M + H] calcd for C16
H
14ClN
3
O
3
: 332.0802. Found:
32.0805.
.1.1.5.3-((6,7-Dimethoxyquinazolin-4-yl)amino)-2-methylphenol hydrochloride (13)
1
A mixture of 3 (500 mg, 2.23 mmol) and 3-amino-2-methylphenol (274 mg, 2.23 mmol) in
1
IPA afforded 13 (708 mg, 91%) as a purple solid. H NMR (300 MHz, DMSO-d ) δ 11.37 (s,
6
1
8
d
9
H), 9.73 (s, 1H), 8.70 (s, 1H), 8.27 (s, 1H), 7.36 (s, 1H), 7.11 (t, J = 7.96 Hz, 1H), 6.90 (d, J =
1
3
.01 Hz, 1H), 6.77 (d, J = 7.72 Hz, 1H), 3.99 (s, 6H), 1.98 (s, 3H). C NMR (75 MHz, DMSO-
6
): δ 158.9, 156.2 (×2), 150.1, 148.7, 136.2, 134.9, 126.2, 121.7, 117.9, 114.0, 106.5, 103.9,
+
9.5, 56.7, 56.4. HRMS (ESI) m/z [M + H] calcd for C H N O : 312.1348. Found: 312.1362.
1
7
17
3
3
1.1.1.6.3-((6,7-Dimethoxyquinazolin-4-yl)amino)-2-fluorophenol (14)
A mixture of 3 (75 mg, 0.33 mmol) and 3-amino-2-fluorophenol (42 mg, 0.33 mmol) in MeCN
returned the crude product which was purified by preparative HPLC (high pH) to afford 14 (21
1
mg, 18%) as a white solid. H NMR (300 MHz, DMSO-d ) δ 9.90 (s, 1H), 9.54 (br s, 1H), 8.36
6
(s, 1H), 7.83 (s, 1H), 7.18 (s, 1H), 6.97-7.05 (m, 1H), 6.84-6.93 (m, 2H), 3.94 (s, 6H).
1.1.1.7.3-((6,7-Dimethoxyquinazolin-4-yl)amino)-2-methoxyphenol (15)
16

ACCEPTED MANUSCRIPT
A suspension of 3 (20 mg, 0.09 mmol), 46h (14 mg, 0.10 mmol) and 5-6N HCl in IPA (0.02
mL) in IPA was heated at 100 °C for 30 mins in the microwave. The reaction was concentrated
to dryness then partitioned between EtOAc and NaHCO . A precipitate formed which was
3
collected by filtration, washed with water (5 mL) and ether (5 mL) and dried under vacuum to
1
give 15 (7 mg, 23%) as a white solid. H NMR (300 MHz, DMSO-d
6
) δ 9.38 (s, 1H), 9.18 (s,
1
3
H), 8.32 (s, 1H), 7.81 (s, 1H), 7.16 (s, 1H), 6.90-6.99 (m, 2H), 6.77 (dd, J = 2.26, 7.44 Hz, 1H),
.93 (s, 3H), 3.93 (s, 3H), 3.65 (s, 3H).
.1.1.8.3-((6,7-Dimethoxyquinazolin-4-yl)amino)-2-ethylphenol (16)
A mixture of 3 (115 mg, 0.51 mmol), 46a (70 mg, 0.51 mmol) and 5-6N HCl in IPA (0.03 mL)
1
in IPA returned the crude product which was purified by preparative HPLC to afford 16 (1.6 mg,
1
1
1
7
%) as an off white solid. H NMR (300 MHz, DMSO-d ) δ 9.41 (br s, 1H), 9.27 (s, 1H), 8.23 (s,
6
H), 7.83 (s, 1H), 7.14 (s, 1H), 7.04 (t, J = 7.97 Hz, 1H), 6.78 (d, J = 7.77 Hz, 1H), 6.70 (d, J =
2
.68 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 0.97 (t, J = 7.44 Hz, 3H) – CH of Et obscured under
DMSO solvent peak.
.1.1.9.3-((6,7-Dimethoxyquinazolin-4-yl)amino)-2-(trifluoromethyl)phenol (17)
1
A mixture of 3 (52 mg, 0.23 mmol), 51 (61 mg, 0.23 mmol) and 5-6N HCl in IPA (0.03 mL)
in IPA returned the crude product which was purified by preparative HPLC to afford N-[3-
benzyloxy-2-(trifluoromethyl)phenyl]-6,7-dimethoxy-quinazolin-4-amine (32 mg, 31%) as a
1
white solid. H NMR (300 MHz, DMSO-d
6
) δ 9.57 (s, 1H), 8.27 (s, 1H), 7.79 (s, 1H), 7.63 (t, J
=
8.44 Hz, 1H), 7.29-7.51 (m, 6H), 7.17 (s, 1H), 7.04 (d, J = 8.19 Hz, 1H), 5.31 (s, 2H), 3.93 (s,
6
H). A solution of N-[3-benzyloxy-2-(trifluoromethyl)phenyl]-6,7-dimethoxy-quinazolin-4-
amine (27 mg, 0.06 mmol) in TFA (0.5 mL, 6.53 mmol) was heated for 45 mins at 75 °C in the
microwave. The yellow solution produced was concentrated under reduced pressure and purified
17

ACCEPTED MANUSCRIPT
1
by preparative HPLC to afford 17 (9 mg, 42%) as a yellow solid. H NMR (300 MHz, DMSO-
d6) δ 10.53 (br s, 1H), 9.47 (s, 1H), 8.25 (s, 1H), 7.78 (s, 1H), 7.45 (t, J = 8.15 Hz, 1H), 7.16 (s,
1
H), 6.99 (d, J = 8.19 Hz, 1H), 6.85 (d, J = 7.91 Hz, 1H), 3.92 (s, 3H), 3.92 (s, 3H).
.1.1.10. 2-((6,7-Dimethoxyquinazolin-4-yl)amino)-6-hydroxybenzonitrile (18)
A mixture of 3 (126 mg, 0.56 mmol), 46e (75 mg, 0.56 mmol) and 5-6N HCl in IPA (0.01 mL)
1
in IPA returned the crude product. The crude material was dissolved in 7N ammonia in
methanol, preabsorbed onto silica and purified by flash column chromatography eluting with 0-
1
00% ethyl acetate in isohexane then 0-10% methanol in ethyl acetate to return 18 (11 mg, 6%)
1
as a pale yellow solid. H NMR (300 MHz, DMSO-d
6
) δ 13.07 (d, J = 6.22 Hz, 1H), 9.47 (s,
1
H), 9.08 (d, J = 5.93 Hz, 1H), 7.99 (s, 1H), 7.37-7.44 (m, 2H), 7.12 (d, J = 8.01 Hz, 1H), 6.51
d, J = 8.67 Hz, 1H), 4.02 (s, 3H), 3.98 (s, 3H).
.1.1.11. 5-((6,7-Dimethoxyquinazolin-4-yl)amino)-2-fluorophenol hydrochloride (19)
(
1
A mixture of 3 (75 mg, 0.33 mmol) and 5-amino-2-fluorophenol (42 mg, 0.33 mmol) in MeCN
1
afforded 19 (75 mg, 64%) as an off-white solid. H NMR (300 MHz, DMSO-d ) δ 11.27 (s, 1H),
6
1
0.24 (br s, 1H), 8.81 (s, 1H), 8.27 (s, 1H), 7.35 (s, 1H), 7.32 (dd, J = 2.54, 8.01 Hz, 1H), 7.24
dd, J = 8.76, 11.02 Hz, 1H), 7.05-7.11 (m, 1H), 4.01 (s, 3H), 3.99 (s, 3H).
.1.1.12. 5-((6,7-Dimethoxyquinazolin-4-yl)amino)-2-methoxyphenol hydrochloride (22)
(
1
A mixture of 3 (100 mg, 0.45 mmol) and 5-amino-2-methoxyphenol (62 mg, 0.45 mmol) in
1
MeCN afforded 22 (141 mg, 87%) as a yellow solid. H NMR (300 MHz, DMSO-d ) δ 11.07 (s,
6
1
4
H), 9.32 (br s, 1H), 8.78 (s, 1H), 8.18 (s, 1H), 7.30 (s, 1H), 7.10 (s, 1H), 6.98-7.07 (m, 2H),
.00 (s, 6H), 3.82 (s, 3H).
1.1.1.13. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)-5-fluorophenol hydrochloride (23)
18

ACCEPTED MANUSCRIPT
A mixture of 3 (75 mg, 0.33 mmol) and 46f (68 mg, 0.40 mmol) in MeCN afforded 23 (18 mg,
1
1
5%) as a beige solid. H NMR (300 MHz, DMSO-d ) δ 11.14 (br s, 1H), 10.25 (br s, 1H), 8.88
6
(
s, 1H), 8.25 (s, 1H), 7.34 (s, 1H), 7.12 (td, J = 1.85, 10.62 Hz, 1H), 7.01-7.06 (m, 1H), 6.54 (td,
J = 2.11, 10.76 Hz, 1H), 4.02 (s, 3H), 4.00 (s, 3H).
.1.1.14. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)-5-methylphenol hydrochloride (24)
1
A mixture of 3 (75 mg, 0.33 mmol) and 3-amino-5-methylphenol (41 mg, 0.33 mmol) in
1
MeCN afforded 24 (76 mg, 66%) as a green solid. H NMR (300 MHz, DMSO-d
6
) δ 11.19 (s,
1
1
H), 9.65 (br s, 1H), 8.81 (s, 1H), 8.28 (s, 1H), 7.38 (s, 1H), 6.95 (s, 1H), 6.90 (s, 1H), 6.57 (s,
H), 4.01 (s, 3H), 3.99 (s, 3H), 2.27 (s, 3H).
.1.1.15. 4-Chloro-3-((6,7-dimethoxyquinazolin-4-yl)amino)phenol (26)
A mixture of 3 (75 mg, 0.33 mmol) and 3-amino-4-chlorophenol (48 mg, 0.33 mmol) in
1
MeCN returned the crude product which was purified by preparative HPLC (high pH) to afford
1
2
6 (8 mg, 6%) as a white solid. H NMR (300 MHz, DMSO-d ) δ 9.82 (br s, 1H), 9.39 (s, 1H),
6
8
.33 (s, 1H), 7.81 (s, 1H), 7.33 (d, J = 8.67 Hz, 1H), 7.18 (s, 1H), 6.96 (d, J = 2.83 Hz, 1H), 6.73
(dd, J = 2.83, 8.67 Hz, 1H), 3.93 (s, 6H).
1.1.1.16. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)-4-methylphenol hydrochloride (27)
A mixture of 3 (100 mg, 0.45 mmol) and 2-amino-4-hydroxytoluene (60 mg, 0.49 mmol) in
1
MeCN afforded 27 (81 mg, 48%) as a pale brown solid. H NMR (300 MHz, DMSO-d ) δ 11.30
6
(
s, 1H), 9.57 (br s, 1H), 8.72 (s, 1H), 8.27 (s, 1H), 7.36 (s, 1H), 7.16 (d, J = 8.52 Hz, 1H), 6.72-
.78 (m, 2H), 4.00 (s, 6H), 2.07 (s, 3H).
.1.1.17. 2,4-Dichloro-5-((6,7-dimethoxyquinazolin-4-yl)amino)phenol hydrochloride (29)
A mixture of 3 (100 mg, 0.45 mmol) and 5-amino-2,4-dichlorophenol (79 mg, 0.45 mmol) in
MeCN returned the crude product which was recrystallized from MeCN/MeOH to afford 29 (31
6
1
19

ACCEPTED MANUSCRIPT
1
mg, 36%) as a white solid. H NMR (300 MHz, DMSO-d
.79 (s, 1H), 8.16 (s, 1H), 7.71 (s, 1H), 7.32 (s, 1H), 7.16 (s, 1H), 4.01 (s, 3H), 4.00 (s, 3H).
.1.1.18. 4-Chloro-5-((6,7-dimethoxyquinazolin-4-yl)amino)-2-methylphenol hydrochloride
31)
A mixture of 3 (142 mg, 0.63 mmol) and 5-amino-4-chloro-2-methyl-phenol (100 mg, 0.63
6
) δ 11.36 (br s, 1H), 10.96 (s, 1H),
8
1
(
mmol) in MeCN returned the crude product which was recrystallized from DCM/MeOH to
1
afford 31 (215 mg, 89%) as an off-white solid. H NMR (300 MHz, DMSO-d
6
) δ 11.17 (br s,
1
3
H), 9.99 (br s, 1H), 8.76 (s, 1H), 8.11 (br s, 1H), 7.25-7.36 (m, 2H), 6.91 (s, 1H), 4.01 (s, 3H),
.99 (s, 3H), 2.18 (s, 3H).
.1.1.19. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)-2,6-difluorophenol hydrochloride (32)
1
A mixture of 3 (36 mg, 0.16 mmol) and 46b (23 mg, 0.16 mmol) in MeCN afforded 32 (30
1
mg, 51%) as an off-white. H NMR (300 MHz, DMSO-d ) δ 11.30 (br s, 1H), 10.53 (br s, 1H),
6
8
4
.80 (s, 1H), 8.19 (s, 1H), 7.33 (s, 1H), 7.14-7.22 (m, 1H), 6.93-7.02 (m, J = 5.51, 7.96 Hz, 1H),
.01 (s, 4H), 4.00 (s, 3H).
1.1.1.20. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)-2,4-difluorophenol (33)
A mixture of 3 (50 mg, 0.22 mmol), 46c (39 mg, 0.27 mmol) and 4N HCl in dioxane (0.2 mL,
.80 mmol) in 1,4-dioxane was heated in the microwave at 100 °C for 30 mins. The reaction
0
mixture was cooled and evaporated to dryness. The residue was partitioned between DCM and
sat. NaHCO
3
, separated and evaporated to dryness. The residue was purified by preparative
1
HPLC to afford 33 (17 mg, 22%) as a white solid. H NMR (300 MHz, DMSO-d
6
) δ 9.93 (br s,
1
H), 9.43 (s, 1H), 8.34 (s, 1H), 7.84 (s, 1H), 7.21 (s, 1H), 6.87-7.06 (m, 2H), 3.94 (s, 6H).
+
HRMS (ESI) m/z [M + H] calcd for C H F N O : 334.1003. Found: 334.1004.
1
6
13
2
3
3
1.1.1.21. 4-Chloro-3-((6,7-dimethoxyquinazolin-4-yl)amino)-2-fluorophenol (34)
20

ACCEPTED MANUSCRIPT
A mixture of 3 (50 mg, 0.22 mmol), 46d (43 mg, 0.27 mmol) and 4N HCl in dioxane (0.2 mL,
.80 mmol) in 1,4-dioxane was heated in the microwave at 100 °C for 30 mins. The reaction was
0
cooled and evaporated to dryness. The residue was partitioned between DCM and sat. NaHCO3,
separated and evaporated to dryness. The residue was purified by preparative HPLC to afford 34
1
(
7 mg, 9%) as a white solid. H NMR (300 MHz, DMSO-d
6
) δ 10.26 (br s, 1H), 9.47 (s, 1H),
8
1
3
.31 (s, 1H), 7.86 (s, 1H), 7.23 (dd, J = 1.88, 8.95 Hz, 1H), 7.20 (s, 1H), 6.97 (t, J = 8.85 Hz,
+
H), 3.94 (s, 6H). HRMS (ESI) m/z [M + H] calcd for C H ClFN O : 350.0708. Found:
1
6
13
3
3
50.0708.
.1.1.22. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)-5-fluoro-2-methylphenol hydrochloride
35)
1
(
A mixture of 3 (60 mg, 0.27 mmol), 46i (40 mg, 0.28 mmol) and HCl in IPA (0.01 mL) in IPA
1
afforded 35 (56 mg, 57%) as a white solid. H NMR (300 MHz, DMSO-d
6
) δ 11.33 (br s, 1H),
1
3
0.26 (s, 1H), 8.74 (s, 1H), 8.24 (s, 1H), 7.34 (s, 1H), 6.67-6.75 (m, 2H), 4.00 (s, 3H), 4.00 (s,
H), 1.95 (s, 3H).
.1.1.23. 3-((6,7-Dimethoxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol hydrochloride
36)
1
(
A mixture of 3 (80 mg, 0.35 mmol) and 3-amino-4-fluoro-2-methyl-phenol[20] (100 mg, 0.71
1
mmol) in MeCN afforded 36 (42 mg, 15%) as an off-white solid. H NMR (300 MHz, DMSO-
d
1
6
) δ 11.12 (br s, 1H), 9.69 (s, 1H), 8.75 (s, 1H), 8.23 (s, 1H), 7.32 (s, 1H), 7.04 (t, J = 9.07 Hz,
1
3
H), 6.88 (dd, J = 4.62, 8.95 Hz, 1H), 4.01 (s, 3H), 4.00 (s, 3H), 2.03 (s, 3H). C NMR (75
MHz, DMSO-d
6
): δ 159.4, 156.4, 152.5, 151.9 (×2), 149.4, 148.9, 135.4, 123.7 (×2), 123.5,
+
1
14.2, 114.1, 112.7, 112.4, 106.5, 103.8, 99.7, 56.8, 56.4. HRMS (ESI) m/z [M + H] calcd for
C H FN O : 330.1254. Found: 330.1254.
17
16
3
3
21

ACCEPTED MANUSCRIPT
1.1.1.24. 4-Chloro-3-((6,7-dimethoxyquinazolin-4-yl)amino)-2-methylphenol (37)
A mixture of 3 (50 mg, 0.22 mmol), 3-amino-4-chloro-2-methyl-phenol[21] (42 mg, 0.27
mmol) and 4N HCl in dioxane (0.2 mL, 0.8 mmol) in 1,4-dioxane was heated in the microwave
at 100 °C for 30 mins. The reaction was cooled and evaporated to dryness. The residue was
partitioned between DCM and sat. NaHCO
3
, separated and evaporated to dryness. The residue
1
was purified by preparative HPLC (high pH) to afford 37 (15 mg, 20%) as a white solid. H
NMR (300 MHz, DMSO-d ) δ 9.73 (s, 1H), 9.37 (s, 1H), 8.24 (s, 1H), 7.86 (s, 1H), 7.21 (d, J =
6
8
.51 Hz, 1H), 7.17 (s, 1H), 6.84 (d, J = 8.67 Hz, 1H), 3.93 (s, 6H), 1.99 (s, 3H).
.1.1.25. 2-Chloro-3-((6,7-dimethoxyquinazolin-4-yl)amino)-6-methylphenol hydrochloride
38)
1
(
A mixture of 3 (500 mg, 2.23 mmol) and 3-amino-2-chloro-6-methylphenol (351 mg, 2.23
1
mmol) in IPA afforded 38 (713 mg, 84%) as a beige solid. H NMR (300 MHz, DMSO-d
6
) δ
1
6
1.52 (br s, 1H), 9.45 (s, 1H), 8.75 (s, 1H), 8.27 (s, 1H), 7.40 (s, 1H), 7.19 (d, J = 8.10 Hz, 1H),
.93 (d, J = 8.01 Hz, 1H), 4.00 (s, 6H), 2.28 (s, 3H).
.1.1.26. 2,4-Dichloro-3-((6,7-dimethoxyquinazolin-4-yl)amino)phenol (39)
A mixture of 3 (100 mg, 0.45 mmol), 46g (158 mg, 0.90 mmol) and 4N HCl in dioxane (0.2
1
mL, 0.80 mmol) in 1,4-dioxane was heated in the microwave at 140 °C for 5 hours. The reaction
was cooled and evaporated to dryness. The residue was partitioned between DCM/IPA and sat.
NaHCO , separated and evaporated to dryness. The residue was purified by flash
3
chromatography eluting with an isohexane/ethyl acetate gradient to afford 39 (8 mg, 5%) as a
1
pale yellow solid. H NMR (300 MHz, DMSO-d ) δ 10.56 (s, 1H), 9.58 (s, 1H), 8.27 (s, 1H),
6
7
.86 (s, 1H), 7.38 (d, J = 8.85 Hz, 1H), 7.19 (s, 1H), 7.01 (d, J = 8.85 Hz, 1H), 3.94 (s, 6H).
.1.1.27. 2-Chloro-3-((6,7-dimethoxyquinazolin-4-yl)amino)-4-fluorophenol (40)
1
22

ACCEPTED MANUSCRIPT
A mixture of 3 (100 mg, 0.45 mmol), 3-amino-2-chloro-4-fluoro-phenol[20] (108 mg, 0.67
mmol) and 4N HCl in dioxane (0.4 mL, 1.6 mmol) in 1,4-dioxane was heated in the microwave
at 100 °C for 90 mins. The reaction was cooled, diluted with DCM and washed with sat.
NaHCO
3
, separated and evaporated to dryness. The residue was purified by preparative HPLC to
1
afford 40 (26 mg, 17%) as a white solid. H NMR (300 MHz, DMSO-d
6
) δ 10.22 (s, 1H), 9.46 (s,
1
H), 8.29 (s, 1H), 7.86 (s, 1H), 7.13-7.22 (m, 2H), 6.96 (dd, J = 4.85, 9.09 Hz, 1H), 3.94 (s, 6H).
1
3
C NMR (75 MHz, DMSO-d ): δ 157.4, 154.3, 153.1, 150.3, 147.8, 146.8, 125.5, 125.3, 119.9,
6
+
1
14.1, 113.8, 108.3, 107.0, 101.8, 56.0, 55.8. HRMS (ESI) m/z [M + H] calcd for
C H ClFN O : 350.0708. Found: 350.0708.
16
13
3
3
1.1.1.28. 3-((6,7-Dimethoxyquinazolin-4-yl)(methyl)amino)phenol hydrochloride (41)
A mixture of 3 (84 mg, 0.37 mmol) and 53 (140 mg, 0.97 mmol) in MeCN afforded 41 (88
1
mg, 68%) as a light brown solid. H NMR (300 MHz, DMSO-d
6
) δ 10.17 (br s, 1H), 8.97 (s,
1
H), 7.36-7.43 (m, 2H), 6.90-6.99 (m, 3H), 6.34 (s, 1H), 3.92 (s, 3H), 3.70 (s, 3H), 3.26 (s, 3H).
.1.1.29. 3-((6,7-Dimethoxyquinazolin-4-yl)oxy)phenol (42)
A mixture of 3 (200 mg, 0.89 mmol), resorcinol (118 mg, 1.07 mmol) and potassium carbonate
390 mg, 2.82 mmol) in MeCN (10 mL) was heated at 120 °C in the microwave for 30 mins. The
1
(
reaction mixture was concentrated to dryness, suspended in water then extracted with DCM. The
organic phase was concentrated to dryness then purified by preparative HPLC to afford 42 (52
1
mg, 20%) as a white solid. H NMR (300 MHz, DMSO-d ) δ 9.80 (br s, 1H), 8.56 (s, 1H), 7.53
6
(s, 1H), 7.38 (s, 1H), 7.26 (t, J = 8.05 Hz, 1H), 6.65-6.74 (m, 3H), 3.99 (s, 3H), 3.97 (s, 3H).
5.1.1.30. 3-((6,7-Dimethoxyquinazolin-4-yl)thio)phenol hydrochloride (43)
A mixture of 3 (100 mg, 0.45 mmol) and 3-mercaptophenol (0.05 mL, 0.45 mmol) in MeCN
returned the crude product which was recrystallised from MeCN/MeOH to afford 43 (53 mg,
23

ACCEPTED MANUSCRIPT
1
3
4%) as a white solid. H NMR (300 MHz, DMSO-d
m, 3H), 7.01-7.07 (m, 2H), 6.92 (d, J = 8.14 Hz, 1H), 3.99 (s, 3H), 3.99 (s, 3H).
.1.1.30. 1-(6,7-Dimethoxyquinazolin-4-yl)indolin-4-ol hydrochloride (44)
6
) δ 9.72 (br. s, 1H), 8.74 (s, 1H), 7.28-7.38
(
5
A mixture of 3 (250 mg, 1.11 mmol) and indolin-4-ol (150 mg, 1.11 mmol) in MeCN afforded
1
4
1
7
4 (350 mg, 88%) as a yellow solid. H NMR (300 MHz, DMSO-d ) δ 9.82 (br s,1H), 8.86 (s,
6
H), 7.57 (s, 1H), 7.40 (s, 1H), 7.32 (d, J = 8.10 Hz, 1H), 7.11 (t, J = 8.10 Hz, 1H), 6.66 (d, J =
.63 Hz, 1H), 4.71 (t, J = 7.54 Hz, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.11 (t, J = 7.54 Hz, 2H).
1.1.1.30. 1-(6,7-Dimethoxyquinazolin-4-yl)-1,2,3,4-tetrahydroquinolin-5-ol hydrochloride
(45)
A mixture of 3 (250 mg, 1.11 mmol), 1,2,3,4-tetrahydro-quinolin-5-ol hydrochloride (206 mg,
1
1
.11 mmol) in MeCN (12 mL) afforded 45 (390 mg, 94%) as an orange solid. H NMR (300
MHz, DMSO-d ) δ 9.81 (br s, 1H), 8.93 (s, 1H), 7.31 (s, 1H), 6.88 (d, J = 8.10 Hz, 1H), 6.62-
6
6
.75 (m, 2H), 6.20-6.42 (m, 1H), 4.12 (d, J = 5.46 Hz, 2H), 3.96 (s, 3H), 3.43 (s, 3H), 2.79 (t, J
6.64 Hz, 2H), 1.98-2.16 (m, 2H).
=
1.1.2. Procedures for synthesis of anilines 46a-i, 49h, 49i, 51 and 53
1.1.2.1.3-Amino-2-ethylphenol (46a)
To an ice-cooled solution of 2-ethyl-3-methoxyaniline[22] (141 mg, 0.93 mmol) in DCM (1
mL) was added BBr (1M solution in DCM, 4.66 mL, 4.66 mmol) dropwise under nitrogen. The
3
reaction mixture was stirred at room temperature overnight then poured into ice water,
neutralized with sodium carbonate and separated. The aqueous phase was further extracted with
DCM (2 × 20 mL) and the combined organics were dried over MgSO , filtered and concentrated
4
under reduced pressure to return 46a (128 mg, 100%) as a brown solid which was used without
24

ACCEPTED MANUSCRIPT
1
further purification. H NMR (300 MHz, DMSO-d
.97-6.19 (m, 2H), 4.96 (br. s, 2H), 2.44 (q, J = 7.6 Hz, 2H), 0.98 (t, J = 7.6 Hz, 3H).
.1.2.2.3-Amino-2,6-difluorophenol (46b)
A mixture of 2,4-difluoro-3-methoxyaniline (500 mg, 3.14 mmol) and pyridinium chloride (1.0
6
) δ 8.75 (s, 1H), 6.64 (t, J = 7.9 Hz, 1H),
5
1
g, 8.89 mmol) was heated at 200 °C for 1 hour then allowed to cool to room temperature. The
reaction mixture was diluted with water (50 mL) and extracted with DCM (50 mL). The organic
phase was concentrated under reduced pressureunder reduced pressure to return the crude
product which was purified by flash column chromatography, eluting with 0-100% EtOAc in
1
isohexane, then 1% 7N ammonia in methanol/EtOAc to return 46b (23 mg, 5%). H NMR (300
MHz, DMSO-d ) δ 9.65 (s, 1H), 6.67 (ddd, J = 2.17, 8.90, 10.78 Hz, 1H), 6.15 (dt, J = 5.09,
6
9
.18 Hz, 1H), 4.83 (s, 2H).
.1.2.3.3-Amino-2,4-difluorophenol (46c)
1
A mixture of 2,6-difluoro-3-methoxyaniline (1.0 g, 6.28 mmol) and pyridinium chloride (2.1 g,
1
7.79 mmol) was heated at 200 °C for 1 hour then allowed to cool to room temperature. The
reaction mixture was diluted with water (25 mL) and then neutralized with Na HCO . The
2
3
resultant precipitate was isolated by filtration, washed with water and dried overnight at 40 °C
1
under vacuum to return 46c (470 mg, 52%) as a red solid. H NMR (300 MHz, DMSO-d ) δ 9.32
6
(
s, 1H), 6.64 (ddd, J = 2.17, 8.92, 10.86 Hz, 1H), 6.07 (dt, J = 5.04, 9.16 Hz, 1H), 5.03 (s, 2H).
.1.2.4.3-Amino-4-chloro-2-fluorophenol (46d)
1
A mixture of 6-chloro-2-fluoro-3-methoxyaniline (1.0 g, 5.7 mmol) and pyridinium chloride
(1.97 g, 17.1 mmol) was heated at 200 °C for 1 hour then allowed to cool to room temperature.
The reaction mixture was diluted with water (25 mL) and then neutralized with sodium
bicarbonate. The resultant precipitate was isolated by filtration, washed with water and dried
25

ACCEPTED MANUSCRIPT
1
overnight at 40 °C under vacuum to return 46d (244 mg, 27%). H NMR (300 MHz, DMSO-d )
6
δ 9.64 (s, 1H), 6.82 (dd, J = 2.07, 8.85 Hz, 1H), 6.17 (t, J = 8.81 Hz, 1H), 5.17 (s, 2H).
1.1.2.5.2-Amino-6-hydroxybenzonitrile (46e)
A solution of 2-hydroxy-6-nitrobenzonitrile[23] (1.3 g, 7.92 mmol) in ethanol (100 mL) was
treated with 5% Pd/C (450 mg). The flask was evacuated and filled with nitrogen three times,
then evacuated and filled with hydrogen three times. The reaction mixture was stirred under an
atmosphere of hydrogen at room temperature overnight, then flushed with nitrogen and filtered
through Celite. The filtrate was concentrated under reduced pressure to return 46e (910 mg,
1
7
7%). H NMR (300 MHz, DMSO-d ) δ 10.39 (br. s., 1H), 7.03 (t, J = 8.19 Hz, 1H), 6.17 (d, J
6
=
8.23 Hz, 1H), 6.06 (d, J = 8.20 Hz, 1H), 5.79 (s, 2H).
.1.2.6.3-Amino-5-fluorophenol (46f)
To a solution of 3-fluoro-5-nitrophenol (1.0 g, 6.37 mmol) in ethanol (20 mL) and acetic acid
20 mL) was added iron powder (0.36 g, 6.37 mmol) and the mixture heated at reflux under
1
(
nitrogen for 2 hours then cooled to room temperature and filtered through Celite. The filtrate was
concentrated under reduced pressure to return the crude product which was purified by flash
column chromatography, eluting with 0-20% MeOH in DCM to return 46f (820 mg, 86%) as a
1
brown oil which solidified on standing. H NMR (300 MHz, DMSO-d ) δ 5.82 (t, J = 1.93 Hz,
6
1
H), 5.78 (dt, J = 11.60, 2.00 Hz, 1H), 5.69 (dt, J = 11.11, 2.07 Hz, 1H).
.1.2.7.3-Amino-2,4-dichlorophenol (46g)
To a stirred solution of 2,4-dichloro-3-nitrophenol (2.0 g, 9.62 mmol) in ethanol (50 mL) was
added iron powder (2.15 g, 38.46 mmol), ammonium chloride (2.06 g, 38.46 mmol) and water
25 mL). The reaction heated at reflux for 6 hours, cooled a little, then filtered through Celite and
washed with hot ethanol. The filtrate was concentrated under reduced pressure to remove
1
(
26

ACCEPTED MANUSCRIPT
4
ethanol, then extracted with DCM (3 × 25 ml), dried over MgSO , filtered and concentrated
under reduced pressure to return the crude product which was purified by flash column
chromatography, eluting with 0-20% EtOAc in isohexane to return 46g (1.45 g, 85%) as a brown
1
solid. H NMR (300 MHz, DMSO-d ) δ 10.01 (s, 1H), 6.99 (d, J = 8.67 Hz, 1H), 6.24 (d, J =
6
8
.76 Hz, 1H), 5.28 (br s, 2H).
.1.2.8.3-Amino-2-methoxyphenol (46h)
To a solution of 49h (358 mg, 1.44 mmol) in THF (10 mL) was added water (5 mL) and
1
sodium perborate (569 mg, 3.7 mmol). The mixture was stirred at ambient temperature
overnight. Excess THF was removed under reduced pressure. The residue was stirred with sat.
NH Cl solution (10 mL) and DCM (50 mL) for 1 hour, then separated. The organics were
4
concentrated under reduced pressure and purified by flash column chromatography, eluting with
1
0
-60% EtOAc in isohexane, to return 46h (120 mg, 57%) as an off-white solid. H NMR (300
MHz, DMSO-d ) δ 8.80 (s, 1H), 6.55 (t, J = 7.84 Hz, 1H), 6.12 (d, J = 7.95 Hz, 1H), 6.05 (d, J
6
=
7.77 Hz, 1H), 4.71 (s, 2H), 3.62 (s, 3H).
.1.2.9.3-Amino-5-fluoro-2-methylphenol (46i)
To a solution of 49i (761 mg, 1.8 mmol) in THF (40 mL) was added water (20 mL) and
1
sodium perborate (961 mg, 6.2 mmol). The mixture was stirred at ambient temperature
overnight. THF was removed under reduced pressure and the aqueous extracted with DCM (50
mL). The organics were concentrated under reduced pressure and purified by flash column
chromatography, eluting with 0-30% EtOAc in isohexane, to return 46i (225 mg, 85%) as a light
1
brown solid. H NMR (300 MHz, DMSO-d ) δ 9.20 (s, 1H), 5.79-5.93 (m, 2H), 4.99 (s, 2H),
6
1
.81 (s, 3H).
.1.2.10. 2-Methoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (49h)
1
27

ACCEPTED MANUSCRIPT
A suspension of 3-bromo-2-methoxyaniline (500 mg, 2.47 mmol), bis(pinacolato)diboron (755
mg, 2.97 mmol) and potassium acetate (729 mg, 7.42 mmol) in 1,4-dioxane (8 mL) was sparged
with nitrogen for 5 mins. Pd(dppf)Cl ·DCM (173 mg, 0.21 mmol) was added and the mixture
2
was heated under reflux for 2.5 hours then cooled to room temperature. The reaction was diluted
with DCM (35 mL) and washed with water (35 mL). The organic phase was concentrated under
reduced pressure then purified by flash column chromatography, eluting with 0-100% EtOAc in
1
isohexane, to return 49h (358 mg, 58%) an off-white solid. H NMR (300 MHz, DMSO-d
6
) δ
6
.72-6.83 (m, 3H), 4.80 (s, 2H), 3.64 (s, 3H), 1.28 (s, 12H).
.1.2.11. 5-Fluoro-2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
suspension of 3-bromo-5-fluoro-2-methyl-aniline[24] (725 mg, 3.55 mmol),
5
(49i)
A
bis(pinacolato)diboron (1.1 g, 4.26 mmol) and potassium acetate (1.0 g, 10.66 mmol) in 1,4-
dioxane (35 mL) was sparged with nitrogen for 5 mins. Pd(dppf)Cl ·DCM (174 mg, 0.21 mmol)
2
was added and the mixture was heated under reflux for 4 hours. The reaction was cooled to room
temperature and concentrated under reduced pressure. The residue was diluted with EtOAc,
filtered through Celite, then concentrated under reduced pressure to return the crude product
which was purified by flash column chromatography, eluting with 0-50% EtOAc in isohexane, to
1
return 49i (761 mg, 78%). H NMR (300 MHz, CDCl ) δ 6.90 (dd, J = 2.68, 9.18 Hz, 1H), 6.49
3
(
dd, J = 2.64, 10.17 Hz, 1H), 2.33 (s, 3H), 1.36 (s, 12H).
.1.2.12. 1-Benzyloxy-3-bromo-2-(trifluoromethyl)benzene (50)
To an ice-cooled solution of benzyl alcohol (2.19 mL, 21.2 mmol) in DMF (30 mL) was added
5
sodium hydride (0.97 g 60% dispersion on mineral oil, 24.23 mmol) portionwise. The mixture
was stirred for 1 hour before adding 3-fluoro-2-(trifluoromethyl)bromobenzene (4.91 g, 20.19
mmol) portionwise. The mixture was stirred at ambient temperature overnight, then diluted with
28