The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that potentiates the cytostatic and cytotoxic effects of Crizotinib in patient-derived glioblastoma cell lines

Article abstract of DOI:10.1016/j.bmcl.2019.07.051

We describe the synthesis of drug-dye conjugate 1 between anaplastic lymphoma kinase inhibitor Crizotinib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed pot

Full text of DOI:10.1016/j.bmcl.2019.07.051

Journal Pre-Proof
The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that
potentiates the cytostatic and cytotoxic effects of Crizotinib in patient-derived
glioblastoma cell lines
Peter J. Choi, Elizabeth Cooper, Patrick Schweder, Edward Mee, Richard Faull,
William A. Denny, Mike Dragunow, Thomas I-H. Park, Jiney Jose
PII:
S0960-894X(19)30520-7
https://doi.org/10.1016/j.bmcl.2019.07.051
BMCL 26592
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 June 2019
26 July 2019
27 July 2019
Please cite this article as: Choi, P.J., Cooper, E., Schweder, P., Mee, E., Faull, R., Denny, W.A., Dragunow, M.,
Park, T.I-H., Jose, J., The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that potentiates the
cytostatic and cytotoxic effects of Crizotinib in patient-derived glioblastoma cell lines, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.07.051
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The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that
potentiates the cytostatic and cytotoxic effects of Crizotinib in patient-derived
glioblastoma cell lines.
Peter J. Choia*, Elizabeth Coopera,b,c*, Patrick Schwederc,d, Edward Meec,d, Richard
Faullc, William A. Dennya, Mike Dragunow,b,c†, Thomas I-H. Parkb,c†, Jiney Josea†
aAuckland Cancer Society Research Centre, School of Medical Sciences, University
of Auckland, Private Bag 92019, Auckland 1142, New Zealand
bDepartment of Pharmacology & The Centre for Brain Research, University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand
cNeurosurgical Research Unit, The Centre for Brain Research, University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand
dDepartment of Neurosurgery, Auckland City Hospital, Private Bag 92024, Auckland
1142, New Zealand
* Denotes equal contribution to this work and are both regarded as primary
authors.
†
Corresponding authors.

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Abstract:
We describe the synthesis of drug-dye conjugate 1 between anaplastic
lymphoma kinase inhibitor Crizotinib and heptamethine cyanine dye IR-786. The
drug-dye conjugate 1 was evaluated in three different patient-derived
glioblastoma cell lines and showed potent cytotoxic activity with nanomolar
potency (EC50: 50.9 nM). We also demonstrate evidence for antiproliferative
activity of 1 with single digit nanomolar potency (IC50: 4.7 nM). Furthermore,
the cytotoxic effects conveyed a dramatic, 110-fold improvement over Crizotinib.
This improvement was even more pronounced (492-fold) when 1 was combined
with Temozolomide, the standard drug for treatment for glioblastoma. This work
lays the foundation for future exploration of similar tyrosine kinase inhibitor
drug-dye conjugates for the treatment of glioblastoma.
Key words:
Glioblastoma multiforme, Temozolomide, Anaplastic lymphoma kinase, Crizotinib,
Heptamethine cyanine dyes.
Glioblastoma multiforme (GBM) is the most common primary brain cancer in
adults and remains one of the most challenging cancers to treat.1 This is
largely due to the presence of treatment-resistance GBM stem cells and the
heterogeneous nature of GBM pathology.2-3 For these reasons, the median
survival period for patients suffering from GBM is around 15 months.4-5
Temozolomide (TMZ) is the most commonly used chemotherapy agent along
with radiotherapy after the initial tumour resection for the treatment of
GBM.6 TMZ is an imidazotetrazine class alkylating agent that is administered
orally which can cross the blood-brain barrier (BBB) and accumulate in
tumours. TMZ exerts its cytotoxicity by methylation of O6 or N7 position of
guanine residues in DNA, which causes mispairing with thymine during DNA
replication, eventually resulting in DNA damage. Almost half of the patient

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population undergoing TMZ treatment do not respond to TMZ primarily due
to the over-expression of O6-methylguanine methyltransferase (MGMT), a
DNA repair protein that removes O6-methylguanine and other alkylated
guanine adducts from DNA. Hence, MGMT promoter methylation status is an
important prognostic factor for GBM, as it antagonises the cytotoxic effects of
TMZ and other alkylating agents.7-8
Recent literature report that heptamethine cyanine dyes (HMCDs) possess
BBB crossing properties and exhibit selectivity towards tumours compared to
normal tissue.9 These beneficial properties are conferred by tumour hypoxia
and organic anion-transporting polypeptides (OTAPs) - both of which are
common in GBM.10 However, the low efficacy of HMCDs towards brain cancer
cells mean these compounds are not useful stand-alone agents for the
treatment of cancers. This drawback might be overcome by conjugating
HMCD with more effective anti-cancer drugs. Such conjugates might also
show more favourable pharmacokinetics by more easily crossing the BBB.
Indeed, there are previous studies showing that such drug-dye conjugates
have good efficacy in treating brain cancers in mouse models.11 The
structural features that are essential for the uptake of these HMCDs into the
tumour tissue may include the presence of ionisable groups and/or presence
of chloro-cylcohexenyl moiety as the uptake is mediated by reaction with
cysteine residues in human serum albumin.12 The reactivity of chloro-
cylcohexenyl moiety has been exploited previously for site specific protein
labelling13 and also for development of nanotheranostics for tumour
targeting.14
Mutations in receptor tyrosine kinase pathways (RTK) are responsible for
tumour growth in over 80% of GBM patients.15 Despite these prevalent
mutations in RTKs, clinical studies have not been able to demonstrate
efficacy in treating GBMs using FDA approved tyrosine kinase inhibitors
(TKI).16-17 This anomaly is largely due to their inability to cross the BBB and
the apparent lack of tumour specificity.18
Anaplastic lymphoma kinase (ALK) is a druggable tyrosine kinase receptor that is
over-expressed in tumours, and represents a potential therapeutic target for the
treatment of GBM.19 Normal ALK receptor is implicated in the genesis and
progress of GBM with evidence suggesting the involvement of autocrine and
paracrine growth loops with the ALK ligands, pleiotrophin and midkine.20
Crizotinib is one of the first ALK inhibitors approved for the treatment of non-
small cell lung cancer (NSCLC), showing favourable efficacy in patients with
advanced ALK-positive non-small cell lung cancer (NSCLC).21 Crizotinib also
inhibits Met and ROS1 pathways, thereby serving as a multi-kinase inhibitor.
This is an advantage in treating GBM, as multiple aberrant RTK signalling
pathways are involved in GBM progression.22-23
We, therefore, envisaged to design and synthesize HMCD-TKI conjugates to
study their effects on various patient-derived GBM cell lines. As shown in
scheme 1, we designed Crizotinib-HMCD conjugate 1 via facile nucleophilic

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displacement of the chloride group in HMCD 4. The HMCD 4 itself was
assembled using a previously reported procedure in good yields.24 Piperidine
amine from Crizotinib was chosen as the point of attachment to 4 as this
region was shown to be exposed to the solvent region in the crystal structure
of ALK co-crystallized with Crizotinib.25 We hypothesized that a chemical
modification at this region would have minimal impact on the interaction of
Crizotinib with key residues in the ATP binding pocket of ALK, therefore,
maintaining the potency of the drug-dye conjugate.

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Scheme 1. Synthesis of Crizotinib-IR786 drug-dye conjugate 1.
Drug-dye conjugate 1, dye 4, and Crizotinib 5 were screened against three
patient-derived GBM cell lines obtained from Auckland City Hospital
(Supporting Information Table S1). As shown in Table 1, and Figures 1 and
2, the drug-dye conjugate 1 was significantly more potent at reducing cell
numbers when compared to Crizotinib (EC50 of 50.9 nM vs 5595 nM
respectively; P<0.0001). The reported mode of uptake for HMCDs are
through the activated hypoxia-inducible factor 1-organic anion transporting
polypeptide (OATP) axis.26 Therefore, we investigated whether our Crizotinib-
HMCD dye conjugate maintained the same mode of cellular uptake. We
utilized a known OATP pump blocker sodium taurocholic acid27 (250 µM) and
the HIF1-OATP axis activator, 3,4-DHB (1 mM).28 Figure 1 clearly illustrates
the increase in drug-dye conjugate 1 uptake with a 24 hour pre-incubation of
3,4-DHB, and the dramatic decrease in uptake with a 30 min pre-incubation
with the blocker, sodium taurocholic acid. These results suggest our
conjugate also has a high reliance on the OATPs for their uptake into the
tumor cells.
Figure 1. Fluorescence microscope images illustrating the uptake of the
drug-dye conjugate 1 (red) into patient-derived GBM cells (nuclei; blue). 1
(1 µM) was added to the cells for 1 h in all conditions before imaging. Pre-
incubation with 1 mM of 3,4-DHB greatly increased 1’s uptake as shown by

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the increased far-red fluorescence signal. Conversely, blocking the OATP
pump with a pre-incubation with sodium taurocholic acid greatly attenuated
this fluorescence signal. The bar graph is a quantification of the average cell
integrated fluorescence intensity of the far-red signal.
Additionally, Figure 2 highlights that the three GBM patient cell lines were
largely unresponsive to TMZ. However, co-treatment of 1 with an equal
concentration of TMZ increased the cytotoxic activity of 1 to 15.3 nM, which
was three-fold higher than the 1 alone (P<0.01; Table 1, Figure 2 and
supplementary information Figure S1). This response points towards a
synergism of conjugate 1 with TMZ. In contrast, the co-administration of
TMZ with Crizotinib 5, did not produce a significant change in potency
(P>0.05). Additionally, the co-administration of TMZ with 4 had an
appreciable increase in potency (P<0.05) in relation to Crizotinib.
Drug-dye conjugate 1 also exhibited antiproliferative activity on GBM cells at
subordinate concentrations to the induction of cell death (Figure 3 and 4,
supplementary information Figure S2). At half the EC50 (25 nM), treatment
with 1 resulted in a significant reduction in proliferative cells measured by an
EdU cell proliferation assay (P<0.001). In terms of proliferation, the
treatment with TMZ at 100 µM failed to exert a 50% reduction in
proliferating cells in all three primary GBM patient-derived cell lines.
Despite the variability in the average response of each primary GBM patient
cell-line to treatment with Crizotinib 5, 4 and 1, there is a dramatic, and
significant effect on cell death and proliferation (Tables 1 and 2, Figure 2 and
3, supplementary information Figure S3, S4, S5). The consistent potency of
1 across three heterogeneous patient-derived GBM cell lines, in comparison
with TMZ treatment, serves as an example for the beneficial effects of
combining TKIs such as Crizotinib with a dye to improve therapeutic effects
in GBM cells.

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A.
B.
D.
C.
Figure 2. Viability of GBM cells as indicated by total cell count after 48 hours
incubation with 1, Crizotinib, 4 and TMZ (A). Co-incubation with TMZ (100
µM) demonstrated synergism, despite being unresponsive to TMZ treatment
alone (IC50 1/TMZ: 15.3 nM) (C). The average EC50 and the inter-patient
variability to each compound is highlighted in B and D. TMZ did not reach a
50% reduction in cell number in any of the three primary GBM patient-
derived cell lines.

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Table 1. EC50 of the viability of the GBM cells as indicated by total cell count
which is normalised to the vehicle DMSO after 48 h. Potency reflects a fold
change relative to Crizotinib alone. ns = p > 0.05, **=p<0.01, to Crizotinib
(n=3).
Compound Potency Compound with
Potency
(EC50 nM)
TMZ (EC50 nM)
Crizotinib
5600±460
1
3200±1000
1.7 ns
4
1680±110
50±20
3.3 *
460±80
15±6
12.2 *
(IR-786 iodide)
1
109.9**
365.7**

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B.
C.
A.
Figure 3. Graphs showing the individual and the average IC50 (A-B) values for
Crizotinib, 4 and 1 of the three primary GBM patient cell lines. Comparatively,
B highlights the synergism of TMZ co-incubated with the above compounds, on
their respective and IC50. C demonstrates that at a concentration below the EC50
1 has a significant effect on proliferation. *=P<0.05 ** = P<0.01 ***= P<0.001
relative to Crizotinib. TMZ did not reach 50% reduction in proliferation in all of
the primary GBM patient-derived cell lines, hence was not included in the above
analysis.
Table 2. IC50 of the proliferation of the GBM cells, using the EdU cell
proliferation assay which is normalised to DMSO after 48 h. Potency reflects a
fold change relative to Crizotinib. ns = p > 0.05, * = p<0.05, ** = p < 0.001 to
Crizotinib (n=3).
Compound
(IC50 nM)
Compound with
TMZ (IC50 nM)
Potency
Potency
Crizotinib
4
540±160
280±70
4.7±3.3
1
480±140
120±140
1.0±0.6
1.13 ns
1.9 ns
4.7 ns
(IR-786 iodide)
1
115.0**
492.5 **

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Figure 4. Representative images of the cell reduction and cytostatic effects
following incubation with DMSO, TMZ, Crizotinib, 1 and 4 on primary GBM
patient-derived cell line T141 at the 48 h time point. Blue represents the cell
nuclei counterstained with Hoechst, and the Red represents the EdU
incorporated cells. Scale: 200µm, inset: 25µm.

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GBM remains a formidable challenge to treat due to tumour heterogeneity with a
high chance of recurrence. The efficacy of current drugs targeting GBM is far
from ideal and therefore, warrants the need to explore other classes of drugs.
Tyrosine kinase inhibitors (TKIs) have had a major impact on the treatment of
cancers and other diseases such as rheumatoid arthritis, glaucoma, Crohn
disease, and ulcerative colitis, as evidenced by the 48 small molecule kinase
inhibitors so far approved by the FDA for clinical use.29 Although TKIs have had
minimal impact in treating brain cancers mainly due to poor brain exposure, they
could serve as a starting point for synthetic modifications to improve their
efficacy. The drug-dye conjugate 1 serves as an example for the beneficial effect
of combining a TKI such as Crizotinib and a dye to afford a potent compound
with good efficacy in human glioblastoma cell lines. At the time of submitting
this manuscript, there has been only one reported TKI-HMCD dye conjugate
reported in the literature showing micro molar potency in HepG2 live cancer cell
line.30 To the best of our knowledge, this is the first TKI-HMCD dye conjugate
showing nanomolar inhibition in primary patient-derived glioblastoma cell lines.
Furthermore, the three GBM patient cell-lines were largely unresponsive to TMZ
treatment with an IC50 that was greater than 100 µM. However, co-incubation of
1 with TMZ provided evidence for synergism, reducing the EC50 for tumour cell
number by three-fold. The mechanism of action of these conjugates and their
biological targets are currently being investigated. Future work is directed
towards improving physiochemical properties of similar conjugates to explore
their utility in animal models of glioblastoma and in human patients.

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Acknowledgment:
We thank Dr Adrian Blaser for the proton and carbon NMR, Sisira Kumara for the
analytical HPLC, Sree Sreebhavan for high resolution mass spectra, and Miranda
Aalderink, Sheryl Feng and Yewon Jung for cell biology technical support. We thank
the donors and their families for their generous gift of brain tissue for research and the
Neurosurgical team at Auckland hospital for their continued support of this research
programme. We also thank the Neurological Foundation of New Zealand, the
Auckland Cancer Society Research Centre, the Health Research Council (Programme
grant to MD and RLMF), the School of Medicine Foundation (project grant to MD,
Director of the Neurosurgical Research Unit) for funding the Douglas Neurosurgical
Research Fellow (TP) and the Hugh Green Foundation, for their generous funding (to
MD, Director of the Hugh Green Biobank) to establish and run the Centre for Brain
Research Hugh Green Biobank - for financial and technical support.
Supplementary material:
Detailed synthetic procedures, characterization of compounds, cell images can be
found in the online version at
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Graphical abstract: