AZD3229

Discovery of N-{4-[(6,7-dimethoxyquinazolin-4-yl)oxy]phenyl}-2-[4- (propan-2-yl)-1H-1,2,3-triazol-1-yl]acetamide (AZD3229), a potent pan- KIT mutant inhibitor for the treatment of gastrointestinal stromal tumors
Jason Grant Kettle, Rana Anjum, evan barry, Deepa Bhavsar, Crystal Brown, Scott Boyd, Andrew Campbell, Kristin Goldberg, Michael Grondine, Sylvie Guichard, Christopher Hardy, Tom Hunt, Rhys Jones,
Xiuwei Li, Olga Moleva, Derek Ogg, Ross Overman, Martin J Packer, Stuart Pearson, Marianne Schimpl, Wenlin Shao, Aaron Smith, James Smith, Darren Stead, Stephen Stokes, Michael Tucker, and Yang Ye
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00938 • Publication Date (Web): 11 Sep 2018
Downloaded from http://pubs.acs.org on September 11, 2018

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Discovery of N-{4-[(6,7-dimethoxyquinazolin-4-yl)oxy]phenyl}-2-[4-

(propan-2-yl)-1H-1,2,3-triazol-1-yl]acetamide (AZD3229), a potent pan-

KIT mutant inhibitor for the treatment of gastrointestinal stromal tumors

Jason G. Kettle,* Rana Anjum, Evan Barry, Deepa Bhavsar, Crystal Brown, Scott

oyd, Andrew Campbell, Kristin Goldberg, Michael Grondine, Sylvie Guichard,

hristopher J. Hardy, Tom Hunt, Rhys D.O. Jones, Xiuwei Li, Olga Moleva, Derek

Ogg, Ross C. Overman, Martin J. Packer, Stuart Pearson, Marianne Schimpl, Wenlin

Shao, Aaron Smith, James M. Smith, Darren Stead, Steve Stokes, Michael Tucker, Yang Ye

Oncology, IMED Biotech Unit, AstraZeneca, Unit 310 – Darwin Building, Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom.

Discovery Sciences, IMED Biotech Unit, AstraZeneca, Unit 310 – Darwin Building,

Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, United Kingdom.

ncology, IMED Biotech Unit, AstraZeneca, 35 Gatehouse Park, Waltham MA 02451, USA.

harmaron Beijing Co., Ltd. 6 Taihe Road BDA, Beijing 100176 P. R. China

Current address: H3 Biomedicine Inc. 3520, 300 Technology Square floor 5, Cambridge, MA 02139, USA.

Current address: Forma Therapeutics Inc. Forma Therapeutics, 500 Arsenal St, Watertown, MA 02472, USA.

Current address: Peak Proteins Ltd., 3G48 Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK.

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Abstract While the treatment of Gastrointestinal Stromal Tumors (GIST) has been

revolutionised by the application of targeted tyrosine kinase inhibitors capable of inhibiting

KIT-driven proliferation, diverse mutations to this kinase drive resistance to established

therapies. Here we describe the identification of potent pan-KIT mutant kinase inhibitors that

can be dosed without being limited by the tolerability issues seen with multi-targeted agents.

This effort focussed on identification and optimisation of an existing kinase scaffold through

the use of structure-based design. Starting from a series of previously reported

phenoxyquinazoline and quinoline based inhibitors of the tyrosine kinase PDGFRα, potency

against a diverse panel of mutant KIT driven Ba/F3 cell lines was optimised, with a particular

focus on reducing activity against a KDR driven cell model in order to limit the potential for

hypertension commonly seen in second and third line GIST therapies. AZD3229

demonstrates potent single digit nM growth inhibition across a broad cell panel, with good

margin to KDR-driven effects. Selectivity over KDR can be rationalised predominantly by

the interaction of water molecules with the protein and ligand in the active site and its kinome

selectivity is similar to the best of the approved GIST agents. This compound demonstrates

excellent cross-species pharmacokinetics, shows strong pharmacodynamic inhibition of target, and is active in several in vivo models of GIST.

Introduction Gastrointestinal Stromal Tumors (GIST) are soft tissue sarcomas of the

gastrointestinal (GI) tract that are driven predominantly through aberrant signalling of the

proto-oncogene c-KIT. Following surgical resection, five-year survival rates vary from 35 -

65% depending on the size of tumor, mitotic potential, and where it is located. Whilst

incidence is relatively rare, estimated to be of the order of 1.5 patients per 100,000 population

annually (annual incidence in the United States is estimated at 3800), GIST is still the most

common mesenchymal tumor of the GI tract, with the majority harbouring activating

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mutations of this receptor tyrosine kinase. Such mutations are diverse and heterogeneous,

spanning the kinase domain of KIT with single point amino acid transpositions in the ATP

binding pocket, sequence deletions in exon 11, insertions in exon 9, with concurrent primary

and secondary mutational sites being common. A smaller subset of GIST tumors are instead

driven by mutations in the related tyrosine kinase, platelet-derived growth factor receptor-α

(PDGFR α). The D842V mutant of PDGFR α in particular, is equivalent to the KIT D816X mutations, which are refractory to current therapies.

Beyond surgical removal, treatment of GIST has been revolutionised by the application of

targeted tyrosine kinase inhibitors capable of inhibiting KIT-driven proliferation. Imatinib 1,

an inhibitor of both c-KIT and PDGFRα, was initially approved in 2002 and granted

accelerated approval in 2008 as first line adjuvant treatment following surgery. Clinical trials

have demonstrated a 5-year overall survival rate of 92% following 3-year therapy, compared

with 82% for those taking the drug for one year. However, up to 20% of GIST patients

exhibit primary resistance to imatinib, and many patients taking imatinib will eventually

relapse due to secondary mutations which are unresponsive to therapy, although in some case

higher doses of the drug may prove effective. In 2006, the FDA approved multi-targeted

kinase inhibitor sunitinib 2 as a second line treatment for GIST patients who are refractory to,

or intolerant of imatinib therapy. In these patients, time-to-tumor progression of sunitinib-

treated patients was superior to that of placebo-treated patients. A second multi-targeted

kinase inhibitor, regorafenib 3, was approved as third line therapy in 2013 for patients with

GIST that is not amenable to surgery and is refractory to both imatinib and sunitinib.

Approval was again based upon improved progression free survival (PFS) versus placebo.

Despite the availability of these targeted agents, many clinically observed mutants of c-KIT

remain unresponsive, and there remains a significant unmet need in this disease for agents

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with a much broader mutant KIT inhibition profile. The tolerability of sunitinib and

regorafenib therapy, whilst clinically manageable, may also present challenges. Toxicities

associated with their multi-targeted kinase inhibition profile can lead to dose reductions and

drug holidays, which may in theory exacerbate emergence of further resistant clones. Potent

inhibition of VEGFR in these agents is associated with significant high-grade hypertension, amongst other significant toxicities.

1 2

3 4

5
Figure 1. Approved GIST therapies and agents in clinical trials. Imatinib 1, sunitinib 2, regorafenib 3 are approved as first, second and third line treatments respectively. Ripretinib (DCC-2618) 4 and avapritinib (BLU- 285) 5 are currently undergoing clinical trials in GIST patients.

Chemistry The synthesis of inhibitors 15 and 16 (Table 2) and related analogues 17 – 21

(Table 3) have been previously described. A representative synthesis of compound

44 is shown in Scheme 1, with detailed experimental conditions for additional compounds

available as Supplementary Information. Accordingly, copper-catalysed cycloaddition of

ethyl azido acetate 6 with 3-methylbut-1-yne gives triazole 7 which was hydrolysed to

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(iv)
(v)
(vii)
o
o o
o o
93%; (vi) aq. NH
o o
o

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carboxylic acid 8. Amide coupling of 8 with a BOC-protected bis-aniline gave 9 and removal

of the protecting group afforded aniline 10. Starting from difluorocyanophenol 11, alkylation

gave methoxyethyl ether 12, and displacement of one fluoro atom with ammonia under

microwave heating gave aniline 13. This was converted to the dimethylformimidamide

cyclisation precursor 14 and condensation of this with prepared aniline 10 in acetic acid gave 44, synthesising the quinazoline and introducing the C4 group concomitantly.

Scheme 1. Synthesis of compound 44.

(i) (iii)

6 7 R = Et 9 R = BOC
8 R = H 10 R = H

(vi) (viii)

R = H 13 R = NH2 44
R = CH2CH2OMe 14 R = NCH=NMe2

Reagents and conditions: (i) CuI, 3-methylbut-1-yne, Et3 N, CH3 CN, 20 C, 3 days, 90%; (ii) LiOH, water, THF,

20 C, 1.5 h, 100%; (iii) tert-Butyl N-(4-aminophenyl)carbamate, HATU, DIPEA, DMF, 20 C, 16 h, 57%; (iv)

4M HCl in dioxane, MeOH, DCM, 20 C, 3 h, 89%; (v) 1-Bromo-2-methoxyethane, K2 CO3 , DMF, 85 C, 5 h,

3 , i-PrOH, 100 C, 13 h, 87%; (vii) 1,1-Dimethoxy-N,N-dimethylmethanamine, 80 C, 2 h,

100%; (viii) 10, acetic acid, 60 C, 35 mins, 58%.

Results and discussion In order to screen for broad spectrum mutant c-KIT inhibitors with

low potential for hypertension, we established a panel of Ba/F3 cell lines expressing a variety

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19,20
21
22

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of clinically relevant secondary mutants of c-KIT, spanning the ATP binding sequence

(V654A, T670I gatekeeper) and the A-loop (D816H, D820A, D823Y and others) all on a

background of a primary exon 11 deletion (of amino acids 557 and 558), together with a line

expressing Tel-KDR to monitor selectivity over KDR (VEGFR2). In this latter line, KDR is

expressed as a fusion protein with a Tel sequence at the N-terminus, resulting in constitutive

kinase activity. We chose exon 11 deletion as the primary mutation and the backbone for all

the secondary mutations since two-thirds of GIST tumors harbor mutations in exon 11. Exon

11 deletions are associated with a shorter progression-free and overall survival in comparison

to the other exon 11 mutations. In particular, deletions involving codon 557 and/or codon 558

are associated with malignant behaviour. Proliferation of these cell lines is dependent

upon the introduced construct for growth, and so to monitor for non-specific effects on

viability in these cells, a parental Ba/F3 line (no introduced construct) is run in parallel.

Engineered cell lines including Ba/F3 have been shown previously to faithfully mimic

clinical efficacy of various agents in GIST. It is notable that all approved therapies are

type II kinase inhibitors, binding to the DFG-out kinase form, and in particular, c-KIT is one

of only a small number of kinases that prefers to exist in the DFG out form in its ground

state. Additionally c-KIT has an unusual activation profile in that the activation loop seems

not to require presence of phosphate and as type II inhibition in general is difficult to

reproduce in biochemical assays, given reliance on the activation state, we viewed a cellular

cascade as preferred for hit finding activities. Profiling of the various approved therapies in

these lines is informative (Table 1). In line with clinical experience, 1 shows only modest

activity against exon 11 deletion + V654A and D816H, and no activity at all against the

gatekeeper T670I mutation. Sunitinib 2 is potent against V654A and T670I, but has weak

activity against D816H. Regorafenib 3 is potent against the T670I mutant but is rather

modest against the others, and both 2 and 3 show potent inhibition of the Tel-KDR line

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consistent with their potent effect on VEGFR2. Beyond these approved agents, additional

compounds are currently undergoing clinical trials in GIST patients, aimed at targeting

mutants of KIT not currently addressed in the clinic, with secondary D816H being a

particularly refractory example. Ripretinib (DCC-2618), 4 is a potent pan-KIT and PDGFR

inhibitor from Deciphera Pharmaceuticals and shows broad inhibition across this mutant

panel. It is also a potent inhibitor of the Tel-KDR line however and indeed grade 3/4

hypertension has been reported in a recent phase 1 clinical study of this compound.

Avapritinib (BLU-285), 5 from Blueprint Medicines is a potent inhibitor of KIT exon 17

mutations including D816 variants and also PDGFR. In this panel it potently inhibits exon

11 deletion + D816H although is weaker against V654A and T670I. It has good selectivity

for Tel-KDR, but a moderate signal in the parental line may reflect other kinase activities for this agent.

Table 1. Growth inhibition potency of GIST treatments in KIT mutant Ba/F3 lines and effect on Tel-KDR.

Ba/F3 GI50 (µ M)
exon 11 del + exon 11 del exon 11 del
ID Phase Parental KDR
V654A +D816H + T670I
1 st
Approved 1 line >10 0.393 0.535 >10 >10
Imatinib
2 nd
Approved 2 line 4.789 0.006 0.398 0.005 0.033
Sunitinib
3 rd
Approved 3 line 9.953 0.231 0.290 0.033 0.114
Regorafenib
4 Ongoing clinical
8.037 0.031 0.013 0.037 0.098
Ripretinib trials
5 Ongoing clinical
4.075 0.292 0.017 0.860 4.952
Avapritinib trials
All activity data are reported in µ M and are the mean of at least n=2 determinations and have SEM within 0.2 log units.

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We have previously reported on efforts to develop type II quinazoline based inhibitors of

both PDGFR and VEGFR that culminated in the discovery of AZD2932, 15. Follow up

work has also been described aimed at generating a PDGFR-selective agent from this

template which led to quinoline 16 (compound 23 in this referenced work). We reasoned

that such inhibitors of PDGFR might show inhibition of some KIT mutants due to the close

homology of KIT and PDGFR, and the precedent established by compounds like 1. What was

not clear however is whether the broad and selective profile we desired would be achievable

– the imatinib precedent also establishes that potent PDGFR inhibitors do not necessarily lead

to broad activity against KIT mutants. Testing of 15 in this panel (Table 2) highlighted that

AZD2932 did indeed show the desired broad spectrum of inhibition, but that it also carried

potent KDR activity, as it was designed to (encouragingly, no adverse signal was seen in the

parental line, suggesting little off target activity). Conversely, the PDGF-selective compound

16, despite weak effects on the KDR cell line, was a poor inhibitor of KIT mutants and also

showed some activity in the parental line. The challenge then was to understand if the potent

activity of analogues like 15 could be combined with good selectivity against KDR seen for

16, and work that led to the latter had highlighted the importance in the nature of the central

phenoxy ring and terminal heterocycle as being important drivers of KDR selectivity.

Compound 17 in which the phenoxy group is replaced with an anilino linker loses potency

against all KIT mutants, and also KDR (Table 3). The drop in potency against secondary A-

loop mutant D816H is almost identical to the drop in KDR potency (37- versus 39-fold) in

this instance. Adding a meta-methoxy group to the phenoxy ring in 18 results in maintenance

of KIT potency with a modest improvement in KDR selectivity, in line with SAR previously

reported for this series (selectivity of D816H over KDR is 17-fold, compared with almost

parity for 15). Conversion of the central phenyl ring of 18 into a meta-pyridine 19 results in

significant diminishment of KDR potency, and greater selectivity over D816H at 45-fold,

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although this does come with weaker overall D816H potency at 176 nM. Transposition of

this into its quinoline analogue 20 results in restoration of KIT mutant potency and

concomitant enhancement of selectivity over KDR (145-fold). The final compound in this

matrix, 21 combines the potent quinoline hinge binder with the meta-methoxy phenol linker

of 18, to give single digit nanomolar inhibition of the three KIT lines, although in this

instance KDR selectivity has been substantially impacted. Testing of additional compounds

available from the original PDGFR program highlighted that other substituted end ring

heterocycles such as the 3-methylpyrazole found in 16 was not tolerated in c-KIT, and so

could not be used to drive further levels of KDR selectivity as had been done previously (data not shown).

Table 2. Growth inhibition potency of literature AZ PDGFR inhibitors in KIT mutant Ba/F3 lines and effect on KDR.

15 16

Ba/F3 GI50 (µ M)
exon 11 del + exon 11 del + exon 11 del +
ID Parental KDR
V654A D816H T670I
15
>10 0.012 0.070 0.004 0.093
AZD2932

16 4.289 0.685 0.786 1.918 2.674
All activity data are reported in µ M and are the mean of at least n=2 determinations and have SEM within 0.2 log units.

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Table 3. SAR of KIT mutant Ba/F3 lines and effect on Tel-KDR selectivity

Ba/F3 GI50 (µ M)
exon 11 del exon 11 del exon 11 del
ID X Y Z R Parental KDR
+ V654A + D816H + T670I

N CH NH H >10 0.337 2.592 0.078 3.595

N CH O MeO >10 0.008 0.026 0.012 0.442

N N O MeO >10 0.021 0.176 0.060 8.030

CH N O MeO >10 0.013 0.027 0.061 3.924

CH CH O MeO >10 0.006 0.008 0.007 0.077
All activity data are reported in µ M and are the mean of at least n=2 determinations and have SEM within 0.2 log units.

In order to rationalise the observed selectivity margins for compounds 17-21, we obtained X-

ray crystal structures for compound 18, bound to the kinase domain of both wild type KIT

and KDR (see supplementary data for details). We did not attempt to crystallise in multiple

KIT mutants, since we observed broad mutant selectivity over KDR and therefore reasoned

that selective motifs, if apparent, would be revealed by contacts in the X-ray construct of

KIT. There is high homology between KIT and KDR wild type sequences; the sequences are

56% identical in the kinase domains used for crystallography. A key difference between KIT

and KDR lies in the gatekeeper residue, which is T670 in KIT and V916 in KDR. Other

differences are more conservative, such as for hinge residues, which are Y672 (KIT) and

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F918 (KDR). The binding modes of 18 in the overlaid kinase domains are shown in figure 2.

The binding poses are remarkably similar in the two kinases, with very few contacts to non-

conserved residues. The orientation of this ligand is as expected for a Type 2 kinase inhibitor.

The isopropyl group binds into the DFG pocket, with no distinction between KIT and KDR,

and the quinazoline nitrogen binds to the hinge. The two methoxy groups project out into

solvent. We speculate that the modest selectivity appears to arise from a less favourable

orientation of the central phenyl ring with respect to the gatekeeper V916 side chain of KDR,

relative to T670 in KIT. The orientation of the phenyl is dictated by the methoxy substituent

and hinders a binding mode which could optimise packing to V916 of KDR. The bound

ligand creates a poorly solvated cavity between ligand and gatekeeper, which can be

ameliorated in KIT by the more polar Thr side chain, with a favourable interaction between quinazoline nitrogen and side chain hydroxyl.

Figure 2. Kinase domains of KIT and KDR, overlaid on the binding site of Compound 18. The KIT structure is

coloured yellow with labels in bold typeface, the KDR structure in blue with normal typeface. Side chains are

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shown for the DFG motif and the catalytically important Glu-Lys pair, as well as the gatekeeper (GK) residue (PDB codes: 6GQJ and 6GQO).

We were motivated to explore the impact of reversing the acetamide linker in this series and

so made reversed acetamide matched pairs to simple analogues available from the original

PDGFR program (Table 4). Compound 22 is the N-ethylpyrazole analogue of 15, and its

reversed acetamide counterpart 23 encouragingly had a largely similar profile. Selectivity

against KDR is moderately enhanced in the reversed acetamide, 6-fold from roughly 1:1 over

D816H, while affinity for the T670I gatekeeper is most severely impacted (down 20-fold),

activity against V654A and D816H are largely similar. Analogue 24, in which the central ring

is appended with a methoxy group, is a first indication however that the SAR that was

apparent in the original acetamide series does not transfer when the acetamide is reversed.

Whereas previously a 17-fold improvement in selectivity was noted, here selectivity is

unchanged, as is potency across all the mutants – methoxy on the central ring appears to offer

no advantage. A similar breakdown in SAR was seen for introduction of a central pyridine

ring (Table 5). In the quinoline matched pairs 25 (phenyl) and 26 (pyridine), introduction of

nitrogen causes a significant drop in all KIT potencies except T670I, and although KDR potency is weaker, selectivity is unchanged.

Table 4. Impact of reversing the acetamide linker on KIT and KDR potency.

Acetamide Reversed acetamide

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Ba/F3 GI50 (µ M)
exon 11 del exon 11 del exon 11 del
ID Amide R Parental KDR
+ V654A + D816H + T670I

22 Acetamide H >10 0.044 0.488 0.007 0.680

Reversed
23 H >10 0.021 0.250 0.141 1.615
acetamide
Reversed
24 MeO >10 0.078 0.251 0.086 2.551
acetamide
All activity data are reported in µ M and are the mean of at least n=2 determinations and have SEM within 0.2 log units.

We obtained a matched pair of X-ray structures for compound 23, as shown in figure 3. In

this case, we observed ligand binding modes which are slightly offset in the vicinity of the

hinge and gatekeeper, with the pendant ethyl group of the pyrazole ring coincident in the two

binding sites. This suggests that interaction in the DFG pocket is dominant for this scaffold,

but that selectivity arises once again from the gatekeeper pocket. Since 23 is a quinazoline,

the N3 atom is available to chelate a water molecule, which is now visible in the KIT

structure (shown as a red sphere). There is no water observed in KDR here, our assumption

being that this would be very unstable when packed against the V918 side chain. Instead, in

KDR, the ligand partially occupies the solvent pocket in preference to water, weakening both

the hinge interaction and the packing against the gatekeeper residue. We assumed that a

similar solvation stabilisation of 18 is possible, but this was not observed in our X-ray

structures (figure 2). The nitrogen position in the reversed acetamide precludes a direct

hydrogen bond with the glutamic acid chain (E640/E885), in contrast to that observed in the

normal acetamide. This region of the complex remains quite polar however, so that a

crystallographic water is observed to bind in both CKIT and KDR, as shown in Figure 3. The

water appears to mediate an interaction between acid side chain and ligand. The water

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positions are almost coincident in CKIT and KDR, so that we could not use this interaction to explain selectivity between the two proteins.

Figure 3. Kinase domains of KIT and KDR, overlaid on the binding site of reversed acetamide compound 23.

KIT protein ribbons are coloured by sequence identity, with blue residues identical and yellow non-identical to

KDR. The binding mode is offset between the two kinases, but there are still very few contacts to non-conserved

residues. Water molecules are shown as red speheres in KIT and blue spheres in KDR). A water molecule is

observed in the KIT structure, but is absent from the KDR structure. Selectivity appears to arise from less

favourable orientation of the central aromatic ring against the V916 side chain of KDR, relative to the solvated

T670 side of KIT. In addition, the hinge interaction in KDR seems to be less optimal than for KIT. (PDB codes: 6GQK and 6GQP)

Table 5. Impact of reversing a pyridine linker on KIT and KDR potency.

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Ba/F3 GI50 (µ M)
exon 11 del + exon 11 del + exon 11 del +
ID Y Parental KDR
V654A D816H T670I

CH >10 0.026 0.047 0.007 0.082

N >10 0.150 0.518 0.008 0.761
All activity data are reported in µ M and are the mean of at least n=2 determinations and have SEM within 0.2 log units.

With previous SAR unsuited to driving the greater levels of potency and selectivity required,

we turned attention to varying the end-ring of the reversed acetamide using a series of

aromatic acetic acids available through an in house building block initiative. Table 6 shows

the impact of varying the nature of the 5-membered heteroaromatic portion of the molecule,

and here we focused on finding the optimal balance of exon 11 deletion + D816H potency

(since this appears the most challenging mutant to inhibit with this template) and KDR

selectivity, whilst keeping LogD as low as possible. Compound 27, serves as a useful

benchmark for these changes as it is the reversed acetamide version of analogue 15 – potency

and lipophilicity are good, but KDR selectivity is modest. Switching the isopropyl group to a

difluoromethyl 28 gives a largely similar profile. Reversing the pyrazole so that connection to

the acetamide is via an N-link as in analogues 29 and 30 provides compounds with improved

KDR margin but comes at the expense of increased lipophilicity. The last pyrazole in this set

31 with a free NH gives a very potent D816H inhibitor, reasonable selectivity but again with

higher than desirable LogD of 3.6. A C-linked imidazole 32 sees a drop in activity and

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, whereas linking through nitrogen as in 33 gives a potent and selective inhibitor. A

34 is potent but has only 10-fold margin to KDR and LogD of 3.3. We also

examined a series of 1,2,3-triazoles 35 – 37. The pick of these appears to be triazole 35,

which combines good potency (19 nM) and selectivity with an acceptable LogD of 2.9.

Triazole 36 is less potent and triazole 37 less selective than 35. In related 1,2,4-triazoles,

compound 38 sees potency significantly diminished, and analogue 39, although an

improvement, is not superior to any 1,2,3-triazole. Completing this set, the tetrazole 40 shows

a reasonable balance of potency, selectivity and lipophilicity. From this work N-linked

triazole 35 emerged as the most favourable end-ring, although further increases in selectivity were desired.

Table 6. Exon 11 deletion + D816H KIT mutant potency and KDR selectivity for a series of aromatic acetamides.

exon 11 del D816H Fold KDR 2
ID R 1 LogD
Ba/F3 GI50 (µ M) selectivity

27 0.021 5.9 2.9

28 0.087 4.4 2.7

29 0.021 11.4 3.8

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30 0.035 23.5 3.5

31 0.009 15.6 3.6

32 0.240 4.6 2.8

33 0.021 34.1 3.3

34 0.026 9.9 3.3

35 0.019 32.2 2.9

36 0.111 12.6 2.6

37 0.075 9.3 2.9

38 1.369 3.9 2.3

39 0.175 15.9 2.6

40 0.042 15.7 3.0

All activity data are reported in µ M and are the mean of at least n=2 determinations and have SEM within 0.2 log units. Measured LogD at pH7.4 .

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The notable gains in potency and selectivity for these heterocyclic end groups prompted us to

solve a further X-ray matched pair, this time for compound 35. As seen in figure 4, the

binding mode at the hinge and gatekeeper is less offset than for 23 (figure 3), but this appears

to be due to a different orientation in the DFG pocket. The pendant isopropyl group packs

against the V898 side chain of KDR but projects further from the pocket in KIT due to a

larger I653 side chain at the equivalent position. This results in a significant offset of the

triazolo group between KIT and KDR and a different orientation for the isopropyl group. The

triazole dipole is oriented towards bulk water, as we would expect for a broadly hydrophobic

pocket and consequently solvent mapping suggests that the triazolo motif occupies a more

favourable position in KIT relative to KDR. In KDR, it appears to be buried too far into the

pocket, to optimise packing against V898, precluding effective solvation of the exposed

dipole. This also explains why compound 27 is less selective, since the pyrazole end group

can present a hydrophobic atom in the KDR pocket. Combining isopropyl with a heterocycle

with significant dipole, such as triazolo, gives a binding orientation which is optimal for

solvation in the KIT pocket and sub-optimal for KDR. When coupled with a selective

gatekeeper motif and the bound water (also visible in figure 2), we then see a significant selectivity gain.

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Figure 4. Kinase domains of KIT and KDR, overlaid on the binding site of compound 35. KIT protein is

coloured by sequence identity, with blue residues identical and yellow non-identical to KDR. The binding mode

at hinge and gatekeeper is less offset than in previous examples, but attention now shifts to non-conserved

residues in the DFG pocket. The isopropyl group clashes with I653 in KIT, but is accommodated by the smaller

V898 side chain of KDR. The triazolo motif is more buried in KDR as a result and we hypothesise that this

results in a desolvation penalty which increases affinity to KIT for this ligand and for this general DFG pocket motif. (PDB codes: 6GQL and 6GQQ).

We next turned our attention to examining substituents at the quinazoline 5-position, since

this is a region that projects towards the ribose pocket of ATP, an area of fruitful optimisation

in quinazoline-based kinase inhibitors historically. Compound 35 is a potent inhibitor of all

three KIT mutants (Table 7). Migration of the 6-methoxy group to the 5-position 41 does not

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change the profile a great deal – a modest 2 to 3-fold drop in potencies are observed across all

cell lines, and certainly KDR selectivity is not further enhanced. Despite this, we explored the

impact of switching the 4-phenoxy linker back to a 4-anilino linker with compound 42. We

had seen earlier in the normal amide series, compound 17 with an anilino linker lost

significant activity against KIT mutants, although we speculated that the potential lone pair

clash between the adjacent oxygen atoms (at C4 and C5) in 41 might impact the optimal C4

substituent vector, and that reversion to an NH linker, which could internally hydrogen bond

to the 5-methoxy group, might be more favourable. In the event, the aniline linked 42 was as

effective at inhibiting KIT mutants as its phenoxy counterpart, but with much improved

selectivity over KDR (113-fold compared with 24- and 32-fold for 41 and 35 respectively). It

is striking that two structural changes that individually lead to a worsened profile, when

combined together are able to drive a significant benefit. Exploration of alternative

substituents at C5 led to 5-fluoro analogue 43 which maintains KIT potency and leads to even

greater KDR selectivity of at least 400-fold. Fluorine was selected as an alternative to

methoxy that could, potentially at least, form an internal hydrogen bond to the C4 NH. This

compound however unexpectedly suffered from adverse physicochemical properties, in

particular high plasma protein binding of 99.7% bound, an issue that was directly addressed

by variation of the C7 substituent. Compound 44, containing a C7 methoxyethoxy group was

determined to have the optimal balance between KIT mutant potency and KDR selectivity

from this work and was selected for further profiling. It demonstrates very potent growth

inhibition across KIT mutant cell lines with good KDR margin – not quite as selective as 43,

but significant improvements in physicochemical and ADME properties (Table 8). Despite a

low solubility consistent with its chemical structure and neutral chemotype, permeability in

Caco2 cells is very high. This, coupled with its low in vitro clearance in hepatocytes leads to

excellent observed in vivo pharmacokinetics in preclinical species. Bioavailability is high,

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and clearance low across all of mouse, rat and dog. Volume of distribution is low consistent

with the neutral structure. A structure of 44 bound in KIT was obtained and shows features

consistent with the preceding X-ray structures (figure 5). There is a bound water at the

gatekeeper, promoting selectivity, and the triazolo group binds in a similar place in the DFG

pocket as was seen for compound 35. The extended C7 side chain protrudes from the active site out into solvent.

Table 7. SAR of 5-substituted quinazolines.

35

Ba/F3 GI50 (µ M)
exon 11 + exon 11 + exon 11 +
ID Z R1 R2 Parental KDR
del V654A del D816H del T670I

35 >10 0.003 0.019 0.017 0.612

O MeO Me >10 0.007 0.050 0.055 1.222

NH MeO Me >10 0.003 0.048 0.107 5.408

NH F Me >10 0.002 0.025 0.068 >10

NH F MeO(CH 2 )2 >10 0.003 0.009 0.016 1.378
All activity data are reported in µ M and are the mean of at least n=2 determinations and have SEM within 0.2 log units.

Table 8. Detailed ADME characteristics of compound 44.

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1
2
-6 3
-6
4
6
Measured LogD at pH
1 2
3 4
5
6

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H
N O

N
F H N N
N

O N

Parameter Value
LogD pH7.4 3.0
Solubility pH6.5 ( µM) 6
Permeability Caco2 A to B (1E cm/s) (efflux ratio) 62.3 (2.6)
Plasma protein binding % free (mouse, rat, dog, human) 4.9, 2.2, 6.8, 3.5 Hepatocyte Clint (µl/min/10 cells) (mouse, rat, dog, human) 5, 17, <1, <1
hERG IC50 ( µM) >33.3
Mouse 7, 0.7, 2, 100
Pharmacokinetics 5
Rat 8, 0.6, 3, 79
l(ml/min/kg), Vdss (l/kg), t1/2 (h), F%
og 2, 0.3, 4, 69
7.4 . Solubility of crystalline Form A measured in pH6.5 phosphate buffer at 37°C
Permeability measured at pH6.5 using 10 µ M solution of compound. PK in CD1 Mouse following oral (21 µMol/kg) and iv doses (10 µ Mol/kg). PK in Han Wistar Rat following oral (2.1 µ Mol/kg) and iv doses (1 µMol/kg). PK in Dog following oral (2.1 µ Mol/kg) and iv doses (1 µ Mol/kg). Half lives reported are from oral dosing.

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Figure 5. Co-crystal structure of 44 bound to c-KIT. (resolution 2Å). Water molecules shown as red spheres. PDB code: 6GQM.

Compound 44 was compared against the approved and investigational KIT inhibitors outlined

in Figure 1 against a much broader panel of KIT mutant Ba/F3 cell lines expressing a diverse

set of clinically relevant primary and secondary mutations, including a small panel of PDGFR

driven lines relevant in subsets of GIST (Table 9). It shows very potent growth inhibition of

nearly all mutants tested and consistent with its lineage and other KIT inhibitors, maintains

potent activity against PDGFR, including the clinically GIST-relevant D842V mutant. It

maintains low nM potency against secondary mutations on a primary background of either

exon 9 insertion or exon 11 deletion and is sub-10 nM in 12 of the 19 lines examined.

Conversely, imatinib 1 is a relatively poor inhibitor of these diverse KIT mutants, showing

significant activity against a primary mutation of exon 11 insertion and V560D, together with

PDGFR only. Sunitinib 2 carries greater KIT mutant activity than 1 and shows potent activity

against secondary mutations of ATP binding pocket, but is much weaker against clinically

relevant mutations of the A-loop. Third line agent regorafenib 3 is a little more potent against

these mutations but against many of the others appears inferior to sunitinib 2. The

investigational agent ripretinib 4 does show potent and broad-spectrum activity against the

KIT mutants in this panel, although with the exception of its PDGF activity it is only superior

to 44 in a single mutant (primary D816V). As previously indicated, the margin to KDR for

this agent is significantly eroded relative to 44 (only 8-fold compared with 153-fold against

exon 11 insertion + D816H). Avapritinib 5 is potent against certain subsets of KIT mutants,

notably D816/820 and PDGFR, although lacks potent activity against mutants such as V654A

and T670I. Consistent with our goals, compound 44 demonstrates equivalent or superior

growth inhibition in all but 2 of the 19 cell lines investigated when compared to agents 1 to 5.

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1
1
1
2
2
3
2
2
2
2
2
2
V561D
D842V
4 4
AY502-503 insertion at exon 9.
Deletion of 557-558 at exon 11.
Deletion of 560-578 at exon 11.

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Table 9. Comparison of mutant KIT and PDGF potencies for compound 44 with approved and investigational GIST therapies 1 – 5.

KIT inhibitor Ba/F3 GI50 (µM)
Primary Secondary
Gene 44 1 2 3 4 5
mutation Mutation
Exon 9 ins – 0.002 0.192 0.007 0.114 0.034 0.167

Exon 9 ins V654A 0.022 3.947 0.012 1.270 0.205 0.751

Exon 9 ins D816H 0.051 2.749 0.584 0.833 0.078 0.069

Exon 11 del – 0.001 0.017 0.004 0.021 0.004 0.078

Exon 11 del V654A 0.003 0.392 0.006 0.234 0.032 0.292

Exon 11 del V654A 0.008 1.523 0.024 0.943 0.130 0.117

Exon 11 del T670I 0.016 9.689 0.005 0.034 0.038 0.860

Exon 11 del D816H 0.009 0.537 0.400 0.297 0.013 0.017 KIT
Exon 11 del D820A 0.003 0.216 0.214 0.063 0.006 0.019 Exon 11 del Y823D 0.014 0.669 0.782 0.094 0.014

Exon 11 del N822K 0.003 0.223 0.271 0.049 0.004 0.034

Exon 11 del A829P 0.004 0.312 0.430 0.047 0.003 0.028

V560D – 0.001 0.070 0.027 0.108 0.023 0.042

V560D V654A 0.007 0.701 0.007 0.549 0.075 0.427

V560D D816H 0.018 1.191 0.696 0.834 0.048 0.029

D816V – 0.971 9.930 0.638 2.371 0.037 0.008 Tel-
0.001 0.046 0.030 0.051 0.040 0.034
PDGFRα
Tel-
PDGFR 0.008 0.102 0.114 0.029 0.045 0.022 PDGFRβ
0.022 0.567 0.631 0.522 0.188 0.010
1 2 3 4

PDGFRα

In order to ensure that the broad KIT mutant inhibition profile observed had not been

achieved at the expense of general kinome selectivity, compound 44 was assessed in an

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extended panel of human kinase binding assays alongside clinical agents 1 – 5 (Figure 6). In

this panel of 379 human kinases, activity was compared by screening at a single, relatively

high concentration of 1 µM, which is far in excess of the potency seen in the Ba/F3 GI50

assays. Encouragingly compound 44 compares well to these other inhibitors, with an overall

selectivity similar to that of imatinib 1 and avapritinib 5. Both Sunitinib 2 and regorafenib 3

show increased activity across this panel, with exploratory agent ripretinib 4 showing a spectrum of activity between these two approved therapies.

Figure 6. Comparative selectivity of inhibitors 1 – 5 and 44 in a panel of 379 human kinases run in the

SelectScreen kinase panel at ThermoFisher Scientific at a single concentration of 1 µM. This data is available as individual annotated kinase datapoints in Supporting Information.

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A key advantage of using Ba/F3 cell lines in the cascade optimisation is that these allow a

direct assessment of potency translation in vivo. The activity of compound 44 in a series of

mouse allograft tumor models using these Ba/F3 lines was assessed and compared to current

standards of care relevant to each mutant (Figure 7 and Table 10). As expected 1 shows only

limited activity in the exon 11 del/D816H background a mutation to which it is refractory.

Regorafenib 3 fares better and is able to drive regression of tumour volume at a dose of 100

mg/kg qd, although this is surpassed by 44 at a dose of 20 mg/kg bid. A dose response is

evident in that 44 at a dose of 2 mg/kg bid shows no impact on tumour size. Sunitinib 2 was

not assessed in this model as it is inactive against the D816H mutant. In an exon 11

del/V654A allograft however and consistent with its clinical use, 2 showed strong regressions

at 80 mg/kg qd, and was matched by 44 at a dose of 20 mg/kg bid (and even as low as 2

mg/kg 44 was highly effective). Imatinib showed moderate activity in this study. Figure 8

shows the pharmacodynamic response to 44 in the D816H model at the top 20 mg/kg dose

tested with the impact on levels of pKIT and downstream markers pERK and pAKT, together

with a timecourse of compound free cover levels above IC90 in the same study. Following a

single dose, high free cover over target is achieved and is associated with complete

suppression of pKIT and downstream markers out to 8 hours. As compound is cleared and

cover declines, these signals recover although recovery is not complete even at 24 hours.

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Figure 7. The in vivo efficacy of KIT inhibitors in mouse allograft disease models. Upper panel: Imatinib and

regorafenib versus 44 in an exon 11 del/D816H model. Lower panel: Imatinib and sunitinib versus 44 in an exon

11 del/V654A model. Doses for standards of care were selected to best represent their clinically relevant doses.

Table 10. Percent and statistical significance of tumor growth inhibition and regressions of 44 relative to vehicle and standards of care in two mouse allograft disease models of mutant KIT activity.

Dose % % p-
Tumor ID Schedule Route 1 1 2
(mg/kg) Inhibition Regression value
vehicle n/a BD p.o n/a n/a n/a
Ba/F3 KIT-exon 11
del/D816H
1 300 QD p.o 43 0 0.0095

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3 100 QD p.o >100 39 <0.0001

44 2 BD p.o 5 0 0.3818

44 20 BD p.o >100 75 <0.0001

vehicle n/a BD p.o n/a n/a n/a

1 300 QD p.o 34 0 0.0495
Ba/F3 KIT-exon 11
2 80 QD p.o >100 87 <0.0001
del/V654A
44 2 BD p.o >100 44 <0.0001

44 20 BD p.o >100 85 <0.0001
On last day of dosing. Relative to vehicle.

Figure 8. Pharmacodynamic effect of 44 on tumor pKIT, pERK and pAKT in an exon 11 del/D816H mouse

allograft model from a single dose of 20 mg/kg po. No pKIT was detectable at t=2 or 8 hours. A more detailed statistical analysis of this data is available in Supporting Information.

Conclusions

Having established as a goal the challenging profile of an inhibitor capable of addressing all

diverse KIT mutants, this has been realised in a series of type II quinazoline kinase inhibitors.

Identification of a series of kinase inhibitors from an historical project, combined with

structure-based insights allowed for rapid optimisation in particular of both mutant and kinase

Journal of Medicinal Chemistry

selectivity, not just against KDR but the wider kinome. Despite high homology between KIT

and anti-target KDR, we propose that subtle differences in the way each is able to

accommodate and in particular solvate the liganded structures is key to the enhanced

selectivity seen. The pre-clinical in vivo efficacy of 44, now designated AZD3229 is

encouraging, particularly in relation to current standards of care and may hold promise in the

future treatment of GIST patients. Further data relating to the pharmacological activity of

AZD3229, including assessment of required margins over KDR to ensure no hypertensive effects that limit current agents will be reported in due course.

Experimental Methods

Chemistry Unless otherwise stated, commercially available reagents were used as supplied.

All reactions requiring anhydrous conditions were conducted in dried apparatus under an

atmosphere of nitrogen. Flash column chromatography was performed on Merck Kieselgel

silica (Art. 9385) or on reversed phase silica (Fluka silica gel 90 C18) or on Silicycle

cartridges (40-63 µm silica, 4 to 330 g weight) or on Grace resolv cartridges (4 – 120 g) or on

RediSep Rf 1.5 Flash columns or on RediSep Rf high performance Gold Flash columns (150

– 415 g weight) or on RediSep Rf Gold C18 Reversed-phase columns (20 – 40 µm silica)

either manually or automated using an Isco CombiFlash Companion system or similar

system. Preparative reverse phase HPLC was performed on a Waters instrument (600/2700 or

2525) fitted with a ZMD or ZQ ESCi mass spectrometers and a Waters X-Terra or a Waters

X-Bridge or a Waters SunFire reverse-phase column (C-18, 5 microns silica, 19 mm or 50

mm diameter, 100 mm length, flow rate of 40 ml / minute) using decreasingly polar mixtures

of water (containing 1% ammonia) and acetonitrile or decreasingly polar mixtures of water

(containing 0.1% formic acid) and acetonitrile as eluents. NMR chemical shift values were

measured on the delta scale [proton magnetic resonance spectra were determined using a

Bruker Avance 500 (500 MHz), Bruker Avance 400 (400 MHz), Bruker Avance 300 (300

MHz) or Bruker DRX (300 MHz) instrument]; measurements were taken at ambient

temperature unless otherwise specified; the following abbreviations have been used: s,

singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of

doublet of doublet; dt, doublet of triplets; bs, broad signal. In general, end products were also

characterized by mass spectroscopy following liquid chromatography (LCMS or UPLC); in

general, reverse-phase C18 silica was used with a flow rate of 1 ml / minute and detection

was by Electrospray Mass Spectrometry and by UV absorbance recording a wavelength range

of 220-320 nm. Analytical UPLC was performed on CSH C18 reverse-phase silica, using a

Waters XSelect CSH C18 column with dimensions 2.1 x 50mm and particle size 1.7 micron).

Gradient analysis was employed using decreasingly polar mixtures as eluent, for example

decreasingly polar mixtures of water (containing 0.1% formic acid or 0.1% ammonia) as

solvent A and acetonitrile as solvent B. A typical 2 minute analytical UPLC method would

employ a solvent gradient over 1.3 minutes, at approximately 1 ml per minute, from a 97:3

mixture of solvents A and B respectively to a 3:97 mixture of solvents A and B. The

reported molecular ion corresponds to the [M+H]+ unless otherwise specified; for molecules

with multiple isotopic patterns (Br, Cl, etc.) the reported value is the one obtained for the

lowest isotope mass unless otherwise specified. Ion exchange purification was generally

performed using an SCX-2 (Biotage) cartridge. Where reactions refer to the use of a

microwave, one of the following microwave reactors were used: Biotage Initiator, Personal

Chemistry Emrys Optimizer, Personal Chemistry Smithcreator or CEM Explorer. Purities

were assessed using LCMS by UV absorbance and H NMR and are ≥ 95% unless otherwise stated.

Ethyl 2-(4-isopropyl-1H-1,2,3-triazol-1-yl)acetate (7). A 30% solution of ethyl 2-

azidoacetate (6) in DCM (19.5 g, 45.4 mmol) was added as a solution in acetonitrile (27 ml)

over 5 minutes to a suspension of copper(I) iodide (0.17 g, 0.9 mmol), 3-methylbut-1-yne

(5.1 ml, 49.9 mmol) and triethylamine (0.13 ml, 0.9 mmol) in acetonitrile (27 ml) at room

temperature. The mixture was stirred for 3 days at room temperature. The mixture was

concentrated and the residue was partitioned between water (150 ml) and ethyl acetate (150

ml). The aqueous layer was extracted with ethyl acetate (100 ml) and the extracts combined

with the organic layer. The combined extracts were dried and evaporated to dryness. The

crude product was purified by flash silica chromatography, elution gradient 30 to 50% ethyl

acetate in heptane. Pure fractions were evaporated to dryness to afford the title compound as

a white crystalline solid (8.06 g, 90%). 1H NMR (500 MHz, DMSO, 27°C) δ 1.21 (3H, t),

1.22 (6H, d), 2.98 (1H, hept), 4.16 (2H, q), 5.30 (2H, s), 7.82 (1H, d); m/z: ES+ [M+H]+ 198.

2-(4-Isopropyl-1H-1,2,3-triazol-1-yl)acetic acid (8). Lithium hydroxide hydrate (10.2 g,

242.5 mmol) was added as a solution in water (540 ml) to ethyl 2-(4-isopropyl-1H-1,2,3-

triazol-1-yl)acetate (7) (15.9 g, 80.8 mmol) in THF (180 ml). The mixture was stirred for 90

minutes, then concentrated. The resulting aqueous solution was acidified to pH 5 with 2M

HCl and extracted with ethyl acetate (200 ml). The aqueous layer was evaporated to dryness

to afford the title compound as a white solid containing LiCl (28.2 g, 100%, 48% strength),

which was used without further purification. 1H NMR (500 MHz, DMSO, 27°C) δ 1.20 (6H, d), 2.92 (1H, hept), 4.59 (2H, s), 7.62 (1H, d); m/z: ES+ [M+H]+ 170.

Tert-Butyl (4-(2-(4-isopropyl-1H -1,2,3-triazol-1-yl)acetamido)phenyl)carbamate (9).

HATU (18.4 g, 48.3 mmol) was added to a solution of tert-butyl (4-aminophenyl)carbamate

(8.4 g, 40.3 mmol), 2-(4-isopropyl-1H-1,2,3-triazol-1-yl)acetic acid (8) (19.4 g, 44.3 mmol)

and DIPEA (10.5 ml, 60.4 mmol) at ambient temperature. The mixture was stirred at ambient

temperature for 16 hours. The mixture was concentrated to 75 ml volume, diluted with water


(700 ml) and extracted with ethyl acetate (3x 300 ml). The combined ethyl acetate extracts

were washed with 0.5M citric acid solution (300 ml), water (4x 300 ml), 0.5M NaHCO
3

solution (200 ml), water (200 ml), brine (200 ml) and dried. The solution was evaporated to

dryness and the residue was recrystallised from acetonitrile to afford the title compound as a

white solid (8.2 g, 57%). 1H NMR (500 MHz, DMSO, 27°C) δ 1.23 (6H, d), 1.45 (9H, s),

2.98 (1H, hept), 5.20 (2H, s), 7.38 (2H, d), 7.44 (2H, d), 7.83 (1H, d), 9.27 (1H, s), 10.31 (1H, s). m/z: ES+ [M+H]+ 360.

N-(4-Aminophenyl)-2-(4-isopropyl-1H-1,2,3-triazol-1-yl)acetamide (10). 4M Hydrogen

chloride in dioxane (15.3 ml, 61.2 mmol) was added to a mixture of tert-butyl (4-(2-(4-

isopropyl-1H -1,2,3-triazol-1-yl)acetamido)phenyl)carbamate (9) (2.2 g, 6.1 mmol) in DCM

(20 ml) and methanol (20 ml). The mixture was stiirred at ambient temperature for 3 hours,

during which time an additional portion of 4M hydrogen chloride in dioxane (8 ml, 24 mmol)

was added. The mixture was evaporated to dryness and the residue dissolved in water (70

ml). This aqueous solution was added slowly to stirred 1M potassium carbonate solution

(150 ml), causing a white solid to precipitate. The mixture was stirred for 10 minutes at

ambient temperature. The precipitate was collected by filtration, washed with water and

dried under vacuum to afford the title compound as a white solid (1.4 g, 89%) which was

used without purification. 1H NMR (500 MHz, DMSO, 27°C) δ 1.23 (6H, d), 2.98 (1H,

hept), 4.90 (2H, s), 5.14 (2H, s), 6.50 (2H, d), 7.20 (2H, d), 7.82 (1H, s), 9.99 (1H, s); m/z: ES+ [M+H]+ 260.

2,6-Difluoro-4-(2-methoxyethoxy)benzonitrile (12). 1-Bromo-2-methoxyethane (8.4 ml, 89

mmol) was added to a stirred suspension of 2,6-difluoro-4-hydroxybenzonitrile (11) (11.5 g,

74.1 mmol) and potassium carbonate (30.7 g, 222.4 mmol) in DMF (175 ml). The mixture

was heated to 85°C for 5 hours. The mixture was cooled to ambient temperature and was

poured into water (1250 ml). The mixture was extracted with ethyl acetate (2 x 400 ml). The

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combined extracts were washed with water (4 x 400 ml), saturated brine (200 ml), dried and

evaporated to dryness to give an orange oil. The crude product was purified by flash silica

chromatography, elution gradient 20 to 45% ethyl acetate in heptane. Pure fractions were

evaporated to dryness to afford the title compound as a white crystalline solid (16.1 g, 93%).

1H NMR (500 MHz, DMSO, 27°C) δ 3.28 (3H, s), 3.62 – 3.68 (2H, m), 4.21 – 4.27 (2H, m),

7.05 – 7.14 (2H, m); m/z: ES+ [M+H]+ 214.

2-Amino-6-fluoro-4-(2-methoxyethoxy)benzonitrile (13). 2,6-Difluoro-4-(2-

methoxyethoxy)benzonitrile (12) (23 g, 107.9 mmol) was split between 14 microwave vials,

each containing (1.64 g, 7.7 mmol) substrate. Each batch was suspended in isopropanol (3

ml) and concentrated aqueous ammonia solution (8 ml, 3237 mmol) was added. Each vial

was capped and heated to 100°C in microwave reactors for 13 hours. All batches were

combined; the solid which crystallised from solution was collected by filtration, washed with

water and dried to afford the title compound as a white crystalline solid (19.6 g, 87%). 1H

NMR (500 MHz, DMSO, 27°C) δ 3.28 (3H, s), 3.57 – 3.64 (2H, m), 4.02 – 4.07 (2H, m), 6.10 (1H, dd), 6.17 (1H, dd), 6.35 (2H, s); m/z : ES- [M-H]- 209.

(E)-N’-(2-Cyano-3-fluoro-5-(2-methoxyethoxy)phenyl)-N,N-dimethylformimidamide

(14). 1,1-Dimethoxy-N,N-dimethylmethanamine (62.6 ml, 471 mmol) was added to 2-amino-

6-fluoro-4-(2-methoxyethoxy)benzonitrile (11 g, 52.3 mmol) at 25°C. The resulting solution

was stirred at 80°C for 2 hours, then cooled to room temperature. The mixture was poured

into stirred water (200 ml) (exotherm, cold water cooling applied) and the reaction mixture

stirred for 1 hour. The mixture was extracted with ethyl acetate (2 x 150 ml). The combined

extracts were washed with water (3 x 150 ml), saturated brine (100 ml), dried and evaporated

to dryness to afford the title compound as a white crystalline solid (13.9 g, 100%). 1H NMR

(500 MHz, DMSO, 27°C) δ 2.98 (3H, s), 3.07 (3H, s), 3.29 (3H, s), 3.61 – 3.66 (2H, m), 4.14 – 4.17 (2H, m), 6.55 – 6.6 (2H, m), 8.03 (1H, s); m/z : ES+ [M+H]+ 266.


N-(4-{[5-Fluoro-7-(2-methoxyethoxy)quinazolin-4-yl]amino}phenyl)-2-[4-(propan-2-yl)-

1H-1,2,3-triazol-1-yl]acetamide (44). A mixture of N-(4-aminophenyl)-2-(4-isopropyl-1H-

1,2,3-triazol-1-yl)acetamide (10) (5.4 g, 20.7 mmol) and (E)-N’-(2-cyano-3-fluoro-5-(2-

methoxyethoxy)phenyl)-N,N-dimethylformimidamide (14) (5.2 g, 19.7 mmol) in acetic acid

(12 ml) was stirred at 60°C for 35 minutes. The mixture was poured into water (150 ml), the

mixture was stirred and sonicated. The resulting precipitate was collected by filtration,

washed with water and dried. The solid was dissolved in DCM/methanol (12:1, 600 ml) and

the solution washed with 0.2M NaHCO solution (600 ml). The aqueous layer was extracted
3

with DCM/methanol (12:1, 2 x 200 ml) and the extracts combined with the organic layer. The

combined organic extracts were dried, filtered and evaporated to give a beige solid. The crude

product was crystallised from hot ethanol (700 ml). After cooling to ambient temperature and

stirring for 2 hours, the crystalline solid was collected by filtration, washed with cold ethanol

and dried under high vacuum at 50°C to afford 7.4 g of crude product. The crude product was

further purified by recrystallisation in hot ethanol (800 ml). After cooling to ambient

temperature and stirring for 20 hours, the crystalline solid was collected by filtration; the

solids were collected and dried under vacuum at 50 C for 72 hours to give the title compound

as a white crystalline solid (6.2 g, 58%). 1H NMR (500 MHz, DMSO) δ 1.27 (d, J = 6.9 Hz,

6H), 3.02 (heptd, J = 6.9, 0.6 Hz, 1H), 3.35 (s, 3H), 3.70 – 3.76 (m, 2H), 4.27 – 4.35 (m, 2H),

5.29 (s, 2H), 7.07 (d, J = 2.4 Hz, 1H), 7.15 (dd, J = 13.8, 2.4 Hz, 1H), 7.61 (d, J = 8.9 Hz,

2H), 7.69 (d, J = 8.9 Hz, 2H), 7.89 (d, J = 0.6 Hz, 1H), 8.47 (s, 1H), 8.98 (d, J = 9.7 Hz, 1H),

10.49 (s, 1H). 13C NMR (125 MHz, DMSO, 27°C) δ 22.5, 25.2, 52.1, 58.2, 68.0, 70.0, 99.8

(d, J = 11.5 Hz), 103.2 (d, J = 26.1 Hz), 104.6 (d, J = 3.2 Hz), 119.3, 122.1, 123.9, 134.3,

134.8, 152.8, 153.0, 155.6, 155.7 (d, J = 4.7 Hz), 158.5 (d, J = 253 Hz), 161.8 (d, J = 15.1

Hz), 164.2. m/z (ES+), [M+H] = 480; HRMS [MH] calcd for C24H27N7 O3 F: 480.2159; observed: 480.2178.

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Associated Content

Supporting Information Available Protocols are provided for the cell assays and in vivo

experiments, synthetic methods for the remaining examples, together with crystallographic

information, and kinase panel selectivity data for compounds and molecular formula strings

with associated data. This material is available free of charge via the Internet at

http://pubs.acs.org. PDB depositions are available for compounds 18 (6GQJ and 6GQO), 23

(6GQK and 6GQP), 35 (6GQL and 6GQQ) and 44 (6GQM). The authors will release the

atomic coordinates and experimental data upon article publication.

ACKNOWLEDGMENTS

We would like to thank Ian Hardern and Tina Howard for their help with protein production

and X-Ray crystallography respectively, together with Simon Woodcock, Lindsey Leach,

Martina Fitzek, Graham Sproat, Gareth Davies, Jarrod Walsh and Carolyn Blackett for

assistance with cell production, assay development and screening. We would also like to

thank Larry Bao for assistance in generating data to support pharmacodynamic assessment, and Rodrigo Carbajo for key NMR characterisation.

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