Efficacy and Tolerability of Pyrazolo[1,5- a]pyrimidine RET Kinase Inhibitors for the Treatment of Lung Adenocarcinoma

Article abstract of DOI:10.1021/acsmedchemlett.0c00015

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

Full text of DOI:10.1021/acsmedchemlett.0c00015

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Letter
Efficacy and tolerability of pyrazolo[1,5-a]pyrimidine RET
kinase inhibitors for the treatment of lung adenocarcinoma
Casey J. N. Mathison, Donatella Chianelli, Paul V Rucker, John Nelson, Jason Roland, Zhihong Huang,
Yang Yang, Jiqing Jiang, Yun Feng Xie, Robert Epple, Badry Bursulaya, Christian Lee, Mu-Yun Gao,
Jennifer Shaffer, Sergio Briones, Yelena Sarkisova, Anna Galkin, Lintong Li, Nanxin Li, Chun Li, Su Hua,
Shailaja Kasibhatla, Jacqueline Kinyamu-Akunda, Rie Kikkawa, Valentina Molteni, and John E. Tellew
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00015 • Publication Date (Web): 12 Feb 2020
Downloaded from pubs.acs.org on February 16, 2020
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Efficacy and tolerability of pyrazolo[1,5-a]pyrimidine RET kinase in-
hibitors for the treatment of lung adenocarcinoma
Casey J. N. Mathison,*,† Donatella Chianelli,† Paul V. Rucker,† John Nelson,† Jason Roland,† Zhihong
Huang,† Yang Yang,† Jiqing Jiang,† Yun Feng Xie,† Robert Epple,† Badry Bursulaya,† Christian Lee,†
Mu-Yun Gao,† Jennifer Shaffer,† Sergio Briones,† Yelena Sarkisova,† Anna Galkin,† Lintong Li,†
Nanxin Li,† Chun Li,† Su Hua,† Shailaja Kasibhatla,† Jacqueline Kinyamu-Akunda,‡ Rie Kikkawa,‡ Val-
entina Molteni,† John E. Tellew†
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†The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121
‡Novartis Institutes for Biomedical Research, One Health Plaza, East Hanover, NJ 07936
KEYWORDS: RET, receptor tyrosine kinase, KIF5B, tolerability profile, KDR
ABSTRACT: RET kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarci-
noma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and
analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent opti-
mization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against
KDR in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found
to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, how-
ever, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and in-
dicative of the narrow therapeutic windows seen with this scaffold.
The RET (REarranged during Transfection) proto-oncogene, first identified in 1985,1 is a transmembrane receptor tyrosine kinase
(RTK) that when activated by ligands of the glial cell-line derived neurotrophic factor (GDNF) family has constitutive effects on
enteric nervous system formation and development.2,3 Somatic abnormalities associated with RET have been acknowledged as po-
tential oncogenic drivers in several malignancies, including, but not limited to, thyroid cancer as well as a minor fraction of non-
small-cell lung cancer (NSCLC) cases.4 Clinical cohorts positive for RET chromosomal rearrangement and subsequent gain-of-
function kinase fusions, as are present in 1-2% of lung adenocarcinomas, may prove inherently susceptible to targeted treatment
utilizing selective RET inhibitors.5,6 Therapeutically addressing the RET oncogene addiction paradigm in the context of NSCLC
has drawn considerable attention in recent years7,8,9,10 as currently approved multi-kinase RET inhibitors have demonstrated clinical
promise, albeit with reported instances of dose-limiting toxicity.11
Broad-spectrum kinase promiscuity is believed to be a principal liability for current therapeutics with significant anti-RET activ-
ity (e.g. cabozantinib12 and vandetanib13), with particular concern given to KDR (Kinase insert Domain Receptor) inhibition. Well-
recognized detrimental downstream consequences of KDR inhibition, such as hypertension,14,15 can necessitate suboptimal dosing
regimens of multi-kinase inhibitors and may limit the ability to fully engage RET-driven malignancies, thus increasing the probabil-
ity of acquired drug resistance upon longer term exposure. Therefore, an increasingly potent and selective RET inhibitor may prove
to more precisely engage RET in vivo, avoiding significant off-target toxicity, and further enabling the possibility of sought-after
clinical outcomes in the aforementioned oncogenic cohorts. Ongoing clinical development work on RET-selective kinase inhibitors
has resulted in a limited selection of preliminary results disclosed in the literature, most notably with regard to compounds LOXO-
29216 and BLU-667.17 Herein, we describe our efforts towards selective and efficacious RET kinase inhibitors, resulting in the dis-
covery of a series of pyrazolo[1,5-a]pyrimidine-7-amines with strong in vitro anti-RET activity, wide degrees of attained selectivity
against KDR, and robust in vivo murine efficacy. In addition, evidence of mouse brain penetration for pyrazolo[1,5-a]pyrimidine
compound WF-47-JS03 (1) may portend the ability to subsequently treat RET-driven metastatic lesions in the brain with analogous
compounds.
Our preliminary interest in a pyrazolo[1,5-a]pyrimidine scaffold came as a result of internal mining of historical Ba/F318 and
enzymatic kinase data with subsequent selectivity rankings using Gini coefficient analysis.19 Resulting from this effort was hit com-
pound 2, which was found to exhibit reasonable in vitro RET inhibitory activity in addition to sought-after selectivity against KDR.
These factors, paired with its kinase selectivity across substantial regions of the kinome (Table 1), made the refinement of the com-
pact pyrazolo[1,5-a]pyrimidine core an attractive starting point to initiate SAR investigation. RET activity was assayed in a cellular
proliferation assay using the Ba/F3 cell line transfected with the oncogenic KIF5B-RET fusion gene, as well as related RET mutant
strains. Selectivity against other RTKs was assayed with a panel of Ba/F3 lines transfected with various RTKs of interest.
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The SAR of the periphery was first examined at R1, where modification of the phenyl group was found to have significant effects
on activity and selectivity profiles. These modifications, where diversity at R1 was generally introduced in the final step, were syn-
thetically accessed through the condensation reaction of 4-(3,4-dimethoxyphenyl)-1H-pyrazol-5-amine with diethyl malonate, with
the subsequently constructed pyrazolo[1,5-a]pyrimidine core then readily converted to an appropriate bromide cross-coupling part-
ner (Supporting Information). Of note is that the inclusion of a methyl carbamate group in the para position (3, Table 1) conferred
enhanced potency against both the wild-type, KIF5B-RET fusion, and the relevant KIF5B-RETV804M gatekeeper mutant proteins,
albeit with a significant erosion in the selectivity against KDR (Table 1). The inclusion of heterocycles at R1 was also feasible, as in
the case of thiazole 4 (Table 1), giving rise to an altered activity/selectivity profile, but the SAR proved to be narrow. Due to the
drawbacks associated with modifying R1, the phenyl group was accepted as the best option moving forward, and our attention
turned to other locations where the kinase selectivity and physiochemical properties of the molecule could be optimized.
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9
Table 1. Cellular Activity and Kinase Selectivity of Hit Compound 2 and R1 Analoguesa
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R1
Compound
2
3
4
WTb,c
KIF5B-RETc
KIF5B-RETV804Mc
RET
>11
0.037
1.8
0.17
0.87
8.3
0.52
>11
>5.6
3.7
>9.8
>11
0.098
>11
1.7
0.40
>11
>11
>1.3
>11
0.91
0.11
0.41
>11
>11
0.093
0.10
>11
>11
0.12
7.6
5.0
0.006
0.019
0.006
5.1
>11
0.12
0.61
0.21
>10
>11
8.2
>11
3.1
3.0
4.9
4.4
4.1
>11
>11
4.3
8.7
>10
3.8
BCR-ABL
NPM-ALK
BLK
1.2
0.23
>7.4
0.010
0.008
0.43
0.035
0.44
3.0
BMX
FGFR1
FGFR2
FGFR3
FGFR4
FGR2
FLT1
FLT3
FMS
IGF1R
INSR
JAK2
KDR
KIT
LCK
LYN
MER
MET
3.4
0.082
>7.5
>9.1
0.084
0.026
0.020
2.4
0.067
0.086
>11
0.017
0.022
6.0
5.5
5.0
1.4
>10
2.8
2.1
1.4
3.7
9.1
0.24
1.6
1.2
1.5
>8.7
>11
0.009
0.041
>11
>11
2.4
>11
>11
>11
0.77
0.50
>11
>11
PDGFR
PDGFRβ
RON
ROS
SRC
SYK
TIE1
>11
>11
>11
>11
>11
>11
TRKA
TRKB
TRKC
TYRO3
ZAP70
aProliferation assays run in 1536 well format, employing Ba/F3 cells transfected with Tel fusions of the indicated kinase, and IC50
values reported in μM, unless otherwise noted. bParental Ba/F3 cells utilized. cAssay run in 384 well format.
Through in silico analysis of docking models with RET constructs, it was hypothesized that the pyrazolo[1,5-a]pyrimidine C5
position could yield improved selectivity against KDR through leveraging of regional binding pocket interactions with residues
distinctive to RET. In particular, it was thought that the RET-specific residue S891 (C1045 in KDR), adjacent to the DFG motif,
could be targeted20 by replacing the ether linkage of 2 with a nitrogen, as in 7. These 5-amino pyrazolo[1,5-a]pyrimidines yielded
improved KDR selectivity when compared to ether-containing matched pairs in KIF5B-RET Ba/F3 experiments (Table 2). This
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difference is presumably due to improved, water-mediated interactions with residues E775 (C-helix), K758 (β-3 strand), D892
(DFG motif), and S891 as evidenced by a co-crystal structure of RET with compound 7 (Figure 1).
Accessing these R2-substituted pyrazolo[1,5-a]pyrimidine compounds was accomplished by sequential condensation of 4-(3,4-
dimethoxyphenyl)-1H-pyrazol-5-amine with diethyl phenylmalonate, followed by chlorination, and reaction with ammonium hy-
droxide to give the intermediate 5-chloro-3-(3,4-dimethoxyphenyl)-6-phenylpyrazolo[1,5-a]pyrimidin-7-amine. From this interme-
diate, various N- and O-substituted analogs were then readily obtained via SNAr reaction (Supplemental Information).
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Figure 1. X-ray co-crystal structure of 7 and RET reveals significant water-mediated interactions of R2 substitutent NH with key
kinase residues E775, K758, D892, and S891.
Table 2. Improved RET Potency, KDR Selectivity, and Solubility with R2 Modificationa
Ba/F3
Tel-KDR
LC-2/ad
Solb
R2
compound
2
KIF5B-RET
WT
CCDC6-RET
0.037
1.4
3.6
3.0
1.6
0.095
0.16
NA
7.1
5
6
0.079
0.070
2.7
2.8
47
88
7
8
0.031
0.056
3.1
5.5
>10
1.5
0.056
NA
85
>175
48
>7.9
0.99
1
0.0017
0.0053
(WF-47-JS03)
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aValues reported as μM and proliferation assays run in 384 well format to determine IC50. bSolubility assessed in pH 6.8 buffer.
1
Aside from greatly improving the KDR selectivity to levels rarely achieved in vitro,21 changing the aforementioned substituent
connectivity from oxygen to nitrogen resulted in much improved solubility for the scaffold (Table 2). Solubility was also improved
through either reducing the lipophilicity of the piperidine ring, or through increasing molecular flexibility. Both of these strategies,
however, led to reduced potency against KIF5B-RET as exemplified by 5 and 6, respectively (Table 2). The breakthrough in this
subseries came with the discovery of compound 1, where the additional hydrophobicity of the 1,2,2,6,6-pentamethylpiperidin-4-
amine group served to improve both the KIF5B-RET potency and KDR selectivity index by greater than 10-fold, while also im-
proving the solubility >6-fold relative to subseries parent compound 2 (Table 2). Compound 1 also shows noteworthy activity
against a patient-derived, LC-2/ad lung cancer cell line bearing the CCDC6-RET fusion gene (Table 2). Although 1 still has signifi-
cant off-target kinase activities (see Supporting Information), its superior selectivity for RET versus KDR and Ba/F3 wild-type cell
lines gave us a tool to explore how uncoupling the RET and KDR activities affects efficacy and toxicity.
The pharmacokinetic profile of compound 1 in mice demonstrated good oral plasma exposure, half-life, and bioavailability, as
well as significant brain penetration (Table 3); and supported subsequent in vivo efficacy studies for the compound. In a relevant
clonal RIE KIF5B-RET subcutaneous xenograft tumor model, where mice were dosed orally with 1 at 10, 30, and 60 mg/kg once
daily for 14 days, tumors regressed rapidly and outwardly demonstrated a loss of visible vasculature at all doses. However, after
several days of compound administration, significant weight loss was observed among multiple mice in the 60 mg/kg and 30 mg/kg
arms of the study, where subsequent necropsy in these animals revealed substantial lung toxicity (red and mottled appearance/lung
collapse/signs of hemorrhaging). Conversely, in the 10 mg/kg cohort, dosing with 1 seemed well-tolerated and led to significant
tumor regression (Supporting Information). In a follow-on 10 day study aimed at evaluating the efficacy and pharmacodynamics of
lower doses of 1, doses of 1, 3, and 10 mg/kg were all well-tolerated and found to induce tumor inhibition, stasis, and regression,
respectively; consistent with observed dose-proportional plasma exposures (Figure 2, Supporting Information). The substantial effi-
cacy demonstrated in vivo for 1 in the 10 mg/kg group precluded the analysis of vestigial tumors, but both the 1 and 3 mg/kg groups
demonstrated significant intratumoral RET phosphorylation inhibition via Western Blot at 5 h post-dose time points, although
abated considerably by 24 h (Figure 3, Supporting Information).
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Table 3. PK Parameters and Brain Tissue Exposurea
brain/plasma ratio
VSS
t1/2
Cmax
CL
(mL/min/kg)
AUC
(h*μM)
F
(%)
Cmax
compound
route
dose
AUC
(L/kg)
(h)
(μM)
1 (WF-47-JS03)
iv
5
51
13
3.9
3.2
12
1.1
b
20
11
0.73
94
0.94
0.81
po
9 (YA-14-WJ39)
10 (SC-89-VU43)
11
iv
po
iv
po
iv
5
20
2
10
2
54
19
7.8
5.1
11
1.8
2.5
3.7
3.7
1.6
2.2
2.6
6.8
2.8
8.7
0.49
0.34
1.6
0.88
0.94
0.82
0.45
0.10
64
62
14
0.15
NA
NA
0.13
NA
NA
110
po
10
aPK studies run in Balb/c mice. bHCl salt used for dosing.
Figure 2. RET inhibitor compound 1 (WF-47-JS03) significantly inhibits tumor growth in RIE KIF5B-RET xenograft mice and is
well tolerated at 1, 3, and 10 mg/kg in 10 d study.
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Figure 3. Western Blot confirmation of intratumoral RET target engagement in RIE KIF5B-RET xenograft mice at tumor stasis
dose (3 mg/kg, po, MC/Tween80, suspension) at 5 hour timepoint after 10 days of compound 1 administration.
Upon establishment of a highly efficacious and tolerated dose level in the aforementioned RIE KIF5B-RET clonal model, we
then sought assessment of this 10 mg/kg dose of 1 in the context of a disease-relevant LC-2 tumor tissue model utilizing the pan-
,
RTK/RET inhibitor ponatinib22 23 as a positive control. This study, designed with daily oral dosing for 21 days followed by post-
study monitoring, demonstrated exceptional efficacy along with sustained tumor abatement following cessation of treatment (Figure
4). Administration of the compound was largely tolerated, however, following the termination of dosing, one mouse was euthanized
on day 24 due to rapid weight loss, where subsequent necropsy revealed kidney effects.
Vehicle 200ul PO QD
1500
1000
500
0
Ponatinib 10 mg/kg QD PO
Dose
WF-47-JS03 10 mg/kg QD PO
0
10
20
30
Treatment (Days)
Figure 4. Treatment of LC-2/ad tumor-bearing mice with 1 (WF-47-JS03) for 21 d is well-tolerated and leads to significant regres-
sion at 10 mg/kg.
The toxicity profile of 1 was further studied in a 4 week mouse study with a specific emphasis on detecting any significant neu-
ronal changes due to the high brain penetration previously observed for the compound (Table 3). At 10 mg/kg qd for 1, histopatho-
logical analysis revealed no effect on neurons in the brain or gastrointestinal system, and there were no noticeable changes in the
lung; however, mild to moderate findings in the pancreas (10/10 animals, vacuolation), lymph nodes (2/10, germinal center de-
creased cellularity), and spleen (7/10, marginal zone depletion) were observed. The lack of effects on brain or GI neurons suggests
that treatment of RET-driven metastatic lesions in the brain could be possible without significant on-target RET-driven neuronal
effects. Current options for these types of brain-metastasized cancers are limited, and include co-administration of vandetanib and
everolimus (the latter being an mTOR inhibitor employed to increase brain exposures24), alectinib at elevated doses,25 and poten-
tially LOXO-29216 and BLU-66726 where partial responses in the brain have been disclosed.
Table 4. Increased Polarity at R4 Reduces hERG Liabilitya
Ba/F3
LC-2/ad
CCDC6-RET
0.0053
hERG
R3
R4
compound
ClogPb
KIF5B-RET
Tel-KDR
0.99
WT
bindingc
MPCd
1 (WF-47-JS03)
OCH3
OCH3
4.47
0.0017
1.5
1.2
0.5
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2
3
4
9 (YA-14-WJ39)
10 (SC-89-VU43)
H
H
4.15
4.03
0.0054
0.0033
1.3
3.4
1.3
0.016
0.015
4.7
7.5
2.8
0.89
NA
5
6
7
8
9
11
H
3.19
0.0063
1.1
2.8
0.0062
14
NA
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aIC50 values reported as μM. bStarDrop calculation. cAssessed with 3H-dofetilide. dManual patch clamp.
Based upon learnings from the efficacy studies of compound 1 conveyed above, we sought to screen additional pyrazolo[1,5-
a]pyrimidines for efficacy in relevant murine models in the hope of achieving an improved therapeutic index and/or a more man-
ageable toxicity, while in addition, attempting to mitigate potential human ether-a-go-go related gene (hERG) and predicted phos-
pholipidosis risks.27 Initial efforts toward reduction of the strong hERG inhibition of 1 (Table 4) involved removal or replacement
of the 1,2,2,6,6-pentamethylpiperidin-4-amine group basic nitrogen. These attempts, however, resulted in a substantial loss of po-
tency, presumably due to a strong binding interaction of the basic amine with RET residue D892 (DFG motif), as was exhibited by
7 (Figure 1). Therefore, we concentrated our efforts on installation of polarity at R4 to reduce overall lipophilicity. Specifically,
when the 3,4-dimethoxyphenyl group is replaced by a variety of R4 amides, the additional hydrophilicity results in a reduction in
hERG binding of up to 12-fold without a substantial loss of potency in both KIF5B-RET transfected Ba/F3 cells and the LC-2/ad
cell line (Table 4). Disappointingly, the most promising compound of the subseries, compound 11, which demonstrated substan-
tially reduced hERG binding, exhibited poor oral exposure and bioavailability precluding advancement to in vivo efficacy studies
(Table 3). In contrast, compounds YA-14-WJ39 (9) and SC-89-VU43 (10) demonstrated relatively balanced in vitro and in vivo
properties, making the compounds promising candidates for further in vivo studies.
Subsequently, twice daily dosing was selected to evaluate the efficacy of 9 in an abbreviated version of the RIE KIF5B-RET xen-
ograft model, with doses of 3, 10, and 30 mg/kg for a period of 8 days (Figure 5). Compound 9 was well tolerated, with no adverse
events observed in any of the treatment arms. Administration of 9 at 30 mg/kg demonstrated moderate tumor regression, while sta-
sis and inhibition were observed for the 10 mg/kg and 3 mg/kg cohorts, respectively. Plasma exposures from the study were found
to be dose proportional (Supporting Information) as well as consistent with single dose PK data (Table 3). Also, tumor concentra-
tions were found to be high with respect to plasma, suggesting good tumor penetration potential (Supporting Information). Tumor
analysis from the study revealed strong inhibition of pRET at 5 h post-BID dosing at 30 mg/kg, but inhibition was not sustained at
later timepoints similar to that seen with compound 1 (Figure 6).
Figure 5. Treatment with RET inhibitor compound 9 (YA-14-WJ39) demonstrates moderate tumor regression in RIE KIF5B-RET
xenograft mice and is well tolerated at 30 mg/kg in 8 d study.
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Figure 6. Significant intratumoral RET target engagement is demonstrated in RIE KIF5B-RET xenograft mice at tumor regression
dose (30 mg/kg, po, MC/Tween80, suspension) at 5 hour timepoint after 8 days of compound 9 administration. Tumor volume ex-
tremely small in one mouse from 5 h cohort, leading to likely profiling of stroma tissue.
1
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9
However, in contrast to 9, administration of compound 10 was poorly tolerated in the mouse RIE KIF5B-RET xenograft efficacy
model, where twice daily administration of 3, 10, 30, 60 mg/kg for a period of 8 days resulted in the manifestation of severe lung
hemorrhaging and hypothermia in mice at all doses except 3 mg/kg (Supporting Information). The hemorrhagic phenotype ob-
served in this case was similar to that described above with 1 (vide supra), and was also found in subsequent studies with dosing of
compounds 2 and 4, suggesting that the toxicity observed is scaffold and/or target-based due to the significant structural variability
of substituents, as well as differences in kinase off-target profiles of the tested compounds. Ultimately, poor therapeutic windows
and precipitous onset of severe lung hemorrhagic events occurring in murine models led us to discontinue work on the pyra-
zolo[1,5-a]pyrimidine scaffold and turn our attention to the study of an alternative core scaffold.
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In summary, we have discovered a series of potent pyrazolo[1,5-a]pyrimidine RET inhibitors with high selectivity over KDR,
and moderate overall kinase selectivity. Compound 1 was found to be highly efficacious in RET-driven mouse xenograft studies,
and was well-tolerated at the efficacious 10 mg/kg qd dose over 28 days of dosing. Importantly, no effects were observed in GI or
brain neurons; evidence that tumor inhibition is achievable with no overt on-target neuronal effects. However, at higher doses of 1,
and with a series of follow-up compounds at or near the efficacious dose, severe hemorrhagic lung effects were observed. This ulti-
mately led us to discontinue efforts on the pyrazolo[1,5-a]pyrimidine scaffold and pursue work on other scaffolds. We anticipate
disclosing a summary of this work at a future date.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures, characterization, and 1H-NMR spectra of all compounds; in vitro assay descriptions; cellular kinase panel activ-
ity for compounds 1 and 6-11; PK data for compounds 1 and 9-11; in vivo efficacy/tolerability study design and results for compounds 1,
9, and 10; 4-week mouse toxicity study design and results for compound 1.
The Supporting Information is available free of charge on the ACS Publications website.
Accession Codes
PDB accession code is 6VHG and designated for immediate release upon publication.
AUTHOR INFORMATION
Corresponding Author
* E-mail: cmathison@gnf.org. Tel: (858)332-4481. (C.J.N.M.)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Thomas Hollenbeck for LCMS-ES data and David Jones for NMR data. We would also like to thank the staff at BL5.0.3 Ad-
vanced Light Source (Berkeley, CA), a DOE Office of Science User Facility under Contract No. DE-AC02-05CH11231, supported in part
by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, grant P30
GM124169-01.
ABBREVIATIONS
RET, rearranged during transfection; RTK, receptor tyrosine kinase; GDNF, glial cell-line derived neurotrophic factor; NSCLC, non-
small-cell lung cancer; KDR, kinase insert domain receptor; SAR, structure-activity relationship; RIE, rat intestinal epithelial.
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