Linifanib-a multi-targeted receptor tyrosine kinase inhibitor and a low molecular weight gelator

Article abstract of DOI:10.1039/c5cc00454c

In this study we demonstrate that linifanib, a multi-targeted receptor tyrosine kinase inhibitor, with a key urea containing pharmacophore, self-assembles into a hydrogel in the presence of low amounts of solvent. We demonstrate the role of the urea functional group and that of fluorine substitution on the adjacent aromatic ring in promoting self-assembly. We have also shown that linifanib has superior mechanical strength to two structurally related analogues and hence increased potential for localisation at an injection site for drug delivery applications. This journal is

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ite this: DOI: 10.1039/x0xx00000x
Linifanib – A MultiꢀTargeted Receptor Tyrosine Kinase Inhibitor
and a Low Molecular Weight Gelator
Maria Marlow, Mohammed AlꢀAmeedee, Thomas Smith, Simon Wheeler, Michael J.
Stocks a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
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potency and hence will only require dissolution of nanomolar
concentrations to achieve efficacy12. Another consideration in the
selection of this drug was the class of LMWGs that this drug also
represents. LMWGs with a urea moiety, which is a key part of the
pharmacophore of linifanib, have not been extensively studied for
drug delivery13. Indeed, the focus has been on using LMWGs
derived from peptides, nucleosides or carbohydrates14. The urea
functional group is both a hydrogen bond donor and acceptor group,
known to create the one dimensional order required for
supramolecular networks13, 15. Hydrogen bonds are formed between
the oxygen atom of the carbonyl group and hydrogen atoms of the
two NH groups resulting in directional assembly. Also hydrogen
bonding contributes to drugꢀreceptor interactions and hence the urea
functional group, with its extensive hydrogen bonding potential has
been incorporated within many drug pharmacophores to give both
potency and specificity. Indeed, we speculate that there may be
many drug molecules containing the urea functional group that are
LMWGs.
In this study we demonstrate that linifanib, a multi-targeted
receptor tyrosine kinase inhibitor, with a key urea containing
pharmacophore, self-assembles into
a hydrogel in the
presence of low amounts of solvent. We demonstrate the role
of the urea functional group and that of fluorine substitution
on the adjacent aromatic ring in promoting self-assembly. We
have also shown that linifanib has superior mechanical
strength to two structurally related analogues and hence
increased potential for localisation at an injection site for
drug delivery applications.
In recent years, there has been significant interest focused on the use
of low molecular weight gelators (LMWGs) for medical
applications1,2,3. Scientists working in this field are evaluating
LMWGs, which form hydrogels, for drug delivery and tissue
engineering applications4,5 and indeed there are FDA approved
molecular hydrogels for example, Lanreotide6. To create these
hydrogels, LMWGs selfꢀassemble into three dimensional, fibrillar
networks that have large quantities of water associated with them2.
In the presence of low stress, these systems exhibit solidꢀlike
behaviour even though they have a low solid content, typically less
than 2 % (w/w). The properties of large amounts of associated water
and low solid content will confer biocompatibility4, making these
systems attractive for drug delivery. One specific application of
LMWGs is cancer therapeutics, where they have been used to
localize and give controlled release of chemotherapeutics upon intraꢀ
tumoural injection, as exemplified by Wang et al 7,8. Our studies
have extended beyond chemotherapeutics to that of molecularly
targeted drugs, such as vascular endothelial growth factor (VEGF)
inhibitors9. We have selected one such compound – linifanib (1,
Figure 1), a potent dual vascular endothelial growth factor (VEGF)
and plateletꢀderived growth factor (PDGF) tyrosine kinase inhibitor,
and was a drug candidate for the treatment of advanced or metastatic
nonꢀsmall cell lung cancer, renal cancer and pancreatic cancer10. We
chose, in part, to investigate the gelation properties of linifanib as
pancreatic cancer is poorly treated by systemic therapies due to poor
drug penetration of the stromal barrier11 and we considered that a
LMWG hydrogel of such a therapy, injected intraꢀtumourally, could
be considered as a novel approach to increase such poor drug
penetration. This linifanib hydrogel should confer biocompatibility
to the intraꢀtumoural depot due to the large amounts of associated
water4. We have also chosen linifanib as the molecule has nanomolar
I
H2N
R2
N
R3
Pd(dppf)Cl2,Na2CO3
EtOH/tolene/water
85oC, 24 hrs
N
O
H
HN
N
R2
R1
H
R2
R1
R1
R3
R3
O
H2N
NH2
OCN
N
HN
B
H
N
N
H
ice-water bath
DCM, overnight
B
O
O
O
O
5. R1 = OCH3, R2 = H, R3 = H, 40%
6. R1 = H, R2 = H, R3 = H, 38%
7. R1 = Cl, R2 = H, R3 = H, 37%
8. R1 = CN, R2 = H, R3 = H, 43%
1. R1 = F, R2 = CH3, R3 = H, 56%
2. R1 = F, R2 = H, R3 = H, 43%
3. R1 = F, R2 = H, R3 = F, 52%
4. R1 = CH3, R2 = F, R3 = H, 52%
Figure 1: Linifanib (1) and analogues (2-8) by modification of
substituents on the terminal aromatic ring adjacent to the urea
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smaller molecular volume. Also described in the Nilsson paper is
that the halogen identity and position of substitution on the ring
influences the gel strength. We also found in our studies that
linifanib (1), with an ortho substituted fluorine, gave stronger gels as
compared to its fluorine substituted analogues (2 , 3 (diꢀfluoro
substituted) and 4 (meta substituted fluorine) respectively in the vial
inversion screen and confirmed by rheology for analogue 3 (see
supplementary data:S3). Linifanib was selected as the focus for
further physical characterization dues it greater mechanical strength
as this is an important attribute for a drug delivery applications
where mechanical strength can influence localization at an injection
site 21,22
.
Compound
1
2
3
4
5
6
7
8
9
v/v DMSO
/water
5
Figure 2: Analogue (9) by replacement of the urea functional group
by an amide
G
G
G
G
G
G
G
G
P
P
P
P
P
P
P
P
P
P
10
15
20
25
We also selected linfinab due to the presence of the aromatic rings
flanking the urea functional group, leading to π−π stacking
interactions which in turn have the potential to promote selfꢀ
assembly16. Based on our hypothesis of both the urea functional
group and the aromatic rings promoting gelation in linifanib, we
synthesized seven close analogues by modification of the
substituents on the terminal aromatic ring adjacent to the urea (2ꢀ8)
(Figure 1) and an additional analogue containing an amide group
instead of the urea group (9). The amide was chosen since it also can
provide complimentary hydrogen bonding interactions required to
promote selfꢀassembly2. Linifanib (1) and all eight analogues were
screened for gelation using “table top rheology” i.e. stable to vial
inversion17. Gels were formed by dissolving the compounds in
DMSO, followed by adding water at 60oC. The results of the vial
inversion screen are shown in Table 1 where the final gelator
concentration is 0.5% (w/v), which is common for a LMWG2, in
dimethyl sulfoxide (DMSO)/water mixtures from 5% (v/v) to 25%
(v/v). The solvent mixture was chosen as linifanib is soluble in
DMSO but insoluble in water at the required concentration; there is
significant literature precedent for using this antiꢀsolvent
G
P
P
P
P
P
G
G
G
G
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
Table 1: Vial inversion screen of 0.5% (w/v) linifanib and linifanib
analogues in DMSO/water mixtures, where a gel (G), or a precipitate
(P) was formed.
(b)
(a)
Figure 3 (a) Vial Inversion Screen of linifanib and its fluorinated
analogues (left to right: linifanib (1) and linifanib analogues 2, 3 and
4) (b) Transmission Electron Micrograph (TEM): 0.5% (w/v)
linifanib in 5% (v/v) DMSO/water; scale bar = 1µm
approach18,19; and increased concentrations of DMSO are potentially
advantageous in cancer where DMSO has been shown to reduce the
tumourigenicity of human pancreatic cell lines20. Using water as the
antiꢀsolvent we were able to reproducibly induce gelation for
linifanib (1) and the synthesised analogues that contained a fluorine
atom substituted on the aromatic ring and the urea functional group
in the structure (2, 3 and 4). The gels that formed were stable to vial
inversion (Figure 3). Moreover, the role of the urea functional group
to promote gelation was demonstrated by comparison of the gelation
behavior of 3 and 9. This structural change from urea (gel) to amide
(precipitate) in the vial inversion screen demonstrated the important
role that the urea functional group plays in the gelation of linifanib
and its structural analogues. However, the 2ꢀmethoxy analogue (5)
and the unꢀsubstituted terminal aromatic ring analogue (6), formed
precipitates under all the conditions and likewise both the 2ꢀchloro
analogue (7) and the nitrile analogue (8) failed to gel, even though
they both had electronꢀwithdrawing substituents on the terminal
aromatic ring. These observations are not surprising given the
previously reported data of Nilsson et al16, who showed that minor
substitutions on the aromatic ring of Fmocꢀprotected aromatic amino
acids influenced gelation. They demonstrated the effect was a result
of both steric and electronic changes in π−π stacking interactions.
Specifically, they describe how a fluorine atom substituted on a
toluene ring (also present in the terminal aromatic ring of linifanib)
can create a more electron deficient aromatic ring as compared to
substitution with other halogens and also fluorine has a beneficial
Firstly, further vial inversion screening for gelation was carried out
with a series of binary mixtures of DMSO and water (Table 2).
These results indicate that DMSO:water ratio had a significant effect
on gelation which has also been observed for other LMWGs18, 23
.
The linifanib gels that were most stable to vial inversion were
obtained with 5% (v/v) DMSO/water. Interestingly, there seems to
be an optimum ratio of linifanib to DMSO in that too much DMSO
disrupts the hydrogen bonding interactions. Hence, at a low solvent
ratio (5% (v/v)), we confirmed the fibrillar structure of the linifanib
gels by the transmission electron microscopy (TEM). 0.5 % (w/v)
linifanib, demonstrated a branched pattern of fibres 100 to 300 nm in
width and 4.5 to 9 microns in length (Figure 2).
Having confirmed the characteristic LMWG fibrillar network, we
then undertook oscillatory rheology experiments with linifanib
concentrations from 0.0625 to 1.25% (w/v) in 5% (v/v)
DMSO/water. The measurements were performed at strains of
0.01% to 100% strain at 10 rad/s and the storage modulus (G′) and
loss modulus (G′′) are reported from within the linear viscoelastic
region (Figure 4). These experiments provided key evidence of
gelation of linifanib in DMSO/water as demonstrated by the G′
exceeding G′′ by at least an order of magnitude proving the
viscoelastic nature of the gel24. Additionally, a sustained linear
viscoelastic region was found between 0.01% – 1% (see S3:
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supplementary data. Our G′ data (1.5 x 104 Pa) is lower than the increased potential for localisation at an injection site for drug
gels (106 Pa) as reported Kleinsman25 and Yu24, but greater than the delivery applications.
G′ 100 Pa of gels as reported by Ren26 for a Gemcitabine LMWG.
An increase in the moduli with increasing concentration of linifanib
was also observed which has likewise been shown for other
LMWGs, for example the simple chiral bisureas of Rodriguezꢀ
AUTHOR INFORMATION
Corresponding Author
Llansola et al.27 Additionally, in a similar manner to Adams et al. 28
,
maria.marlow@nottingham.ac.uk
we evaluated the power law relationship between G′ and gelator
concentration i.e ꢀꢁ ∝ Cx (see supplementary information S3:figure
3). We found that x was 1.86 indicative of a semiꢀentangled network.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
Support from Mike Fay at the Nottingham Nanotechnology and
Nanoscience Centre (NNNC) for the TEM Imaging. We also thank
the Government of Iraq for financial support to Mohammed Alꢀ
Ameedee) and the University of Mustansiriya, Baghdad, Iraq and
The Wellcome Trust for funding a summer student bursary to
Thomas Smith.
Electronic Supplementary Information (ESI) available:
Experimental procedures, NMR spectra, and rheology data
See DOI: 10.1039/c000000x/
Notes and references
a
School of Pharmacy, University of Nottingham, University Park,
Nottingham, NG7 2RD, UK.
Figure 4: Storage modulus (G’) (black circle) and loss modulus
(G’’) (red square) versus linifanib concentration (% (w/v)) in
0.5% (v/v) DMSO/water. All measurements were carried out in
triplicate and the mean calculated. Error bars represent standard
deviation.
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S
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S
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S
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solvent ratios of DMSO/water. Representative gels were studied
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