Discovery of Oral VEGFR-2 Inhibitors with Prolonged Ocular Retention That Are Efficacious in Models of Wet Age-Related Macular Degeneration

Article abstract of DOI:10.1021/acs.jmedchem.5b01227

The benefit of intravitreal anti-VEGF therapy in treating wet age-related macular degeneration (AMD) is well established. Identification of VEGFR-2 inhibitors with optimal ADME properties for an ocular indication provides opportunities for dosing routes beyond intravitreal injection. We employed a high-throughput in vivo screening strategy with rodent models of choroidal neovascularization and iterative compound design to identify VEGFR-2 inhibitors with potential to benefit wet AMD patients. These compounds demonstrate preferential ocular tissue distribution and efficacy after oral administration while minimizing systemic exposure.

Full text of DOI:10.1021/acs.jmedchem.5b01227

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Article
Discovery of Oral VEGFR-2 Inhibitors with Prolonged Ocular Retention
that are Efficacious in Models of Wet Age-Related Macular Degeneration
Erik L Meredith, Nello Mainolfi, Stephen Poor, Yubin Qiu, Karl Miranda, James Powers,
Donglei Liu, Fupeng Ma, Catherine Solovay, Chang Rao, Leland Johnson, NAN JI, Gerald
Artman, Leo Hardegger, Shawn Hanks, Siyuan Shen, Amber Woolfenden, Elizabeth
Fassbender, Jeremy Sivak, Yiqin Zhang, Debby Long, Rosemarie Cepeda, Fang Liu, Vinayak
P. Hosagrahara, Wendy Lee, Peter Tarsa, Karen Anderson, Jason Elliott, and Bruce Jaffee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01227 • Publication Date (Web): 15 Nov 2015
Downloaded from http://pubs.acs.org on November 16, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Discovery of Oral VEGFRꢀ2 Inhibitors with
Prolonged Ocular Retention that are Efficacious in
Models of Wet AgeꢀRelated Macular Degeneration
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a*
a†
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b
aǁ
a
Erik L. Meredith , Nello Mainolfi , Stephen Poor , Yubin Qiu , Karl Miranda , James Powers ,
a
a
a
a#
aɠ
aϕ
Donglei Liu , Fupeng Ma , Catherine Solovay , Chang Rao , Leland Johnson , Nan Ji ,
a˦
aζ
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Gerald Artman , Leo Hardegger , Shawn Hanks , Siyuan Shen , Amber Woolfenden , Elizabeth
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b‡
b҂
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Fassbender , Jeremy Sivak , Yiqin Zhang , Debby Long , Rosemarie Cepeda , Fang Liu ,
cϷ
c
d
b
a
Vinayak P. Hosagrahara , Wendy Lee , Peter Tarsa , Karen Anderson , Jason Elliott , Bruce
b
Jaffee
a
Novartis Institutes for BioMedical Research, Global Discovery Chemistry, 100 Technology
Square, Cambridge, MA 02139
b
Novartis Institutes for BioMedical Research, Ophthalmology, 500 Technology Square,
Cambridge, MA 02139
c
Novartis Institutes for BioMedical Research, Metabolism and Pharmacokinetics, 250
Massachusetts Avenue, Cambridge MA, 02139
d
Novartis Institutes for BioMedical Research, Chemical and Pharmaceutical Profiling, 500
Technology Square, Cambridge, MA 02139
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erik.meredith@novartis.com
KEYWORDS. vascular endothelial growth factor, VEGF, VEGFꢀR, receptor tyrosine kinase,
KDR, kinase inhibitor, ageꢀrelated macular degeneration, angiogenesis, choroidal
neovascularization.
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ABSTRACT
The benefit of intravitreal antiꢀVEGF therapy in treating wet ageꢀrelated macular degeneration
(AMD) is well established. Identification of VEGFRꢀ2 inhibitors with optimal ADME
properties for an ocular indication provides opportunities for dosing routes beyond intravitreal
injection. To identify such inhibitors, we employed a high–throughput in vivo screening strategy
with rodent models of choroidal neovascularization and iterative compound design to identify
VEGFRꢀ2 inhibitors with potential to benefit wet AMD patients. These compounds demonstrate
preferential ocular tissue distribution and efficacy after oral administration while minimizing
systemic exposure.
Introduction
Ageꢀrelated macular degeneration (AMD) is an eye disease that causes vision loss due to
pathology in the macula, the area of the retina that enables sharp and detailed vision. AMD is
responsible for the majority of cases of blindness and visual impairment in aged adults. In the
wet form of AMD, choroidal blood vessels (choroidal neovascularization, CNV) sprout and
expand into the subꢀmacular space leaking fluid and blood. This leads to retinal edema, scar
tissue formation and irreversible damage to the macula. The current approved therapies for wet
AMD are the antiꢀVEGFꢀA antibody ranibizumab, the antiꢀVEGFꢀA aptamer pegaptanib, and
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the antiꢀVEGFꢀA and PLGF fusion protein aflibercept. All are delivered by intravitreal
injection (IVT), a procedure that carries a small, but significant, risk of blinding complications
including endophthalmitis, retinal detachment or uveitis. Ranibizumab and aflibercept are the
most effective, stabilizing vision in 60% patients and significantly improving vision (gain of 3
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lines of vision on an ETDRS reading chart) in approximately 1/3 of patients. Injections are
administered monthly or biꢀmonthly, which is a significant burden for patients and retinal
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doctors. Even with antiꢀVEGF therapy, wet AMD patients still have substantial visual deficit.
We envisioned an opportunity for a less invasive wet AMD therapy based on an orally available
inhibitor of the receptor tyrosine kinase (RTK) VEGFRꢀ2 (KDR, Fltꢀ1). An oral therapy may
provide greater efficacy than intravitreal therapy by enabling more frequent dosing and greater
target tissue exposure.
The role of VEGFꢀA in the regulation of angiogenesis is well established. Although new
vessel growth and maturation are highly complex processes, requiring the sequential activation
3
of a series of receptors by numerous ligands, VEGFꢀA signaling is a critical rateꢀlimiting step.
VEGFꢀA promotes growth of vascular endothelial cells and is also known to induce vascular
4
leakage. Ocular VEGFꢀA levels are increased in preclinical models and human diseases of
5
,6
7,8
ocular angiogenesis including wet AMD , diabetic retinopathy and retinal vein occlusions .
VEGFꢀA binds to two related receptor tyrosine kinases, VEGFRꢀ1 and VEGFRꢀ2, which both
have an extracellular domain consisting of 7 immunoglobulinꢀlike domains, a single
transmembrane region, and a consensus tyrosine kinase sequence that is interrupted by a kinaseꢀ
9
insert domain. VEGFRꢀ2 (KDR or Fltꢀ1) is implicated in all aspects of normal and pathological
vascular endothelial cell biology. VEGFRꢀ2 is the major mediator of the mitogenic, angiogenic,
and permeability–enhancing effects of VEGFꢀA. VEGFRꢀ2 null mice die in utero between days
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.5ꢀ9.5 and are characterized by a lack of vasculogenesis and failure to develop organized blood
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vessels. Upon VEGFꢀA binding, VEGFRꢀ2 undergoes dimerization and ligandꢀdependent
tyrosine phosphorylation in intact cells, resulting in a mitogenic, chemotactic and survival
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signal.
Systemic inhibition of VEGF pathway is known to cause onꢀtarget side effects including
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2
hypertension, proteinuria, bleeding, thromboembolism, and gastrointestinal perforation. Our
strategy to mitigate the risk of systemic VEGF inhibition was to identify compounds with
preferential distribution and retention in ocular tissues (retina and retinal pigment
epithelium/choroid) compared to the plasma.
There are a number of approved orally administered VEGFRꢀ2 inhibitors for oncology
1
3ꢀ15
indications and several have been investigated in wet AMD.
In a published case study,
treatment with oral sorafenib correlated with reduced retinal edema and increased visual
1
6,17
acuity.
More recently, orally dosed pazopanib is reported to improve vision, reduce edema,
18
and reduce neovascularization in wet AMD patients. Despite the fact the doses used were
markedly less than those in oncology indications, systemic side effects were still observed.
Thus, a compound that more selectively targets ocular tissues than marketed oncology drugs
would potentially be an effective and safe therapy for wet AMD patients.
To achieve our objective of identifying compounds with preferential ocular distribution we
understood that relatively highꢀthroughput in vivo models of ocular angiogenesis would be
needed to establish both ocular efficacy and tissue distribution relationships. The primary in vivo
models utilized were laserꢀinduced choroidal neovascularization (CNV) in mice and in rats.
Briefly, photocoagulating green laser pulses are applied to the eye in order to cause a focal
rupture of Bruch’s membrane, an extracellular matrix between the retina and choroid. Rupture
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of Bruch’s membrane induces production of local inflammatory factors and VEGF resulting in
choroidal neovascularization (CNV). CNV formation in response to laser injury has been
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demonstrated in humans, monkeys, pigs, and rodents and is a VEGFꢀdependent pathology.
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Evaluation of several internal VEGFRꢀ2 kinase inhibitors in the mouse CNV model provided
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1 (Table 1) as a promising starting point for chemistry optimization. The compound
demonstrated a modest level of inhibition (44%), but had poor aqueous solubility (<5 µM at pH
6.8). Thus, we undertook a strategy to identify compounds with greater efficacy and improved
ADME properties.
Chemistry
The preparation of monocyclic pyrimidine analogs such as 6 was carried out as described
21
in Scheme 1. Coupling of indole 2 with pyrimidine 3 was achieved by treatment with DBU.
Formation of the urea was then accomplished by trapping the lithium anion of the indole with the
desired phenylisocyanates. Removal of the benzyl protecting group with TFA provided alcohol
5
, which was readily converted to methylamine 6 by conversion first to the mesylate and then
displacement with methylamine.
The bicyclic pyrimidine intermediates 8 and 10 were accessible from the corresponding βꢀ
ketoesters 7 and 9 respectively (Scheme 2). Treatment of each βꢀketoester 7 and 9 with
formamidine acetate provided the corresponding hydroxypyrimidine, which then underwent a
protecting group swap from benzyl to Boc. Conversion to the chloropyrimines 8 and 10 was
then accomplished by reaction with carbon tetrachloride and triphenylphospine.
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The 8ꢀmethyl substituted bicyclic pyrimidine 15 was prepared as outlined in Scheme 3. The βꢀ
ketoester 14 was converted to the corresponding Boc protected chloropyrimidine by analogy to
that of 8 and 10.
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The chiral chloropyrimidines 21 and 22 were prepared starting from (S)ꢀαꢀ
methylbenzylamine, which underwent reductive amination with 16 and then ethyl glyoxylate to
provide diester 18 (Scheme 4). Claisen condensation of 18 then provided diastereomers 19 and
20, which were separated by flash chromatography. Conversion of each diastereomer 19 and 20
to the corresponding chloropyrimidines 21 and 22 was achieved under the same conditions as
described above.
The 6,5ꢀbicyclic pyrimidine 24 was prepared by condensation of the commercially available βꢀ
ketoester 23 with formamidine acetate (Scheme 5). Construction of the methyl substituted 6,5ꢀ
bicyclic pyrimidine was as described in Scheme 6. In each of these cases the hydroxy
pyrimidines were used in the subsequent coupling steps as the chloro derivatives were found to
be both difficult to prepare and use due to instability.
Coupling of the bicyclic pyrimidines 10, 15, 21, and 22 to the hydroxyindole was achieved by
treatment with DBU to give the corresponding derivatives 30 (Scheme 7). The 6,5ꢀbicyclic
2
2
pyrimidines 24 and 28 were efficiently coupled to 29 with PyBop and DBU. Formation of the
desired urea analogs 33 was achieved by treatment of the sodium anion of 30 with either a
preformed phenylcarbamate or a commercially available isocyanate. The aminopyrazoles and
aminoisoxazoles 31 that were not commercially available were prepared as described
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3, 24
previously.
The Boc protecting group was then removed upon treatment with TFA to
provide the desired compounds 33 for testing against VEGFRꢀ2.
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Compound evaluation
In vitro inhibition of VEGFRꢀ2 receptor tyrosine kinase was assessed with two primary assays;
a KDR receptor tyrosine kinase biochemical assay and a cellular assay with BaF3ꢀTelꢀKDR cells
that are engineered to constitutively require VEGFRꢀ2 kinase domain activity for survival and
proliferation. Select compounds were evaluated for selectivity in a panel of 62 kinases.
Early in our effort it was found that some of the compounds also inhibited hERG, which is a
potassium ion channel that can cause long QT syndrome and cardiac dysfunction. As we aimed
to minimize the systemic risk associated, compounds were analyzed in an automated Qꢀpatch
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assay to determine their activity on the hERG channel.
As we sought to identify compounds that selectively distributed to the ocular tissues, we did
not apply usual in vitro PKꢀADME filters prior to in vivo evaluation. Priority was given to
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compounds that had demonstrated good aqueous solubility , yet generally those with good in
vitro potency were selected for evaluation in the mouse laser CNV model (C57BL/6) at a single
dose of 10 mg/kg (p.o.). This screening dose was selected based on early efficacy studies with
existing VEGFRꢀ2 inhibitors, such as 1, and was not expected to cause any acute side effects that
would compromise a given study. Compounds were formulated as suspensions (0.5% methyl
®
cellulose, Tween 80, and water) to better inform our understanding of their intrinsic ADME
properties. Each compound was dosed starting the day of laser application and continued daily
with the last dose given on day 6 and CNV area measurement on day 7. Comparison of CNV
area in animals dosed with compound or placebo was used to establish percent inhibition.
Compounds exhibiting >60 % inhibition at the 10 mg/kg screening dose were further evaluated
in mouse CNV dose response studies to establish ED and ED relationships. Compounds with
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an ED < 20 mg/kg in the mouse CNV assay were then characterized in the rat laser CNV model
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(
Brown Norway). To understand the correlation between efficacy and exposure, the distribution
profiles for select compounds were determined by orally dosing Brown Norway rats and
measuring time course drug exposures in the plasma and ocular tissues (retina and retinal
pigment epithelium/choroid/sclera complex). Further understanding of the ADME profiles for
select compounds was accomplished by discrete i.v./p.o. PK studies in Brown Norway rats.
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Results
Early on we recognized the need to improve on the aqueous solubility of compound 1. It was
anticipated that greater aqueous solubility would provide increased oral bioavailability and as a
consequence better efficacy. Xꢀray coꢀcrystal structures of related VEGFRꢀ2 inhibitors indicated
that incorporation of ionizable groups on the pyrimidine should be possible. An important early
finding was that the aminopyrimidine of 1 was not required for potency as 5a proved to be
equipotent in the biochemical assay and have similar efficacy in the mouse CNV model (Table 1;
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5% inhibition). A basic amine was introduced to give compound 6a, which demonstrated
similar potency in vitro and importantly increased efficacy in vivo (67% inhibition). As
anticipated 6a had improved aqueous solubility (Table 2; 64 µM) relative to 1. Incorporation of
a fluoro substituent in the 4ꢀposition of the aniline as in 6b provided a boost in efficacy as the
compound showed 92% inhibition of neovascularization in the mouse at the 10 mg/kg dose.
Collectively, these compounds demonstrated the potential for optimization through inclusion
of the basic amine moiety, however 6b was found to have an undesirable level of activity on the
hERG channel (table 3; IC 5800 nM). To optimize in vivo efficacy while balancing hERG
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activity and aqueous solubility we focused our efforts on the basic amine region and the urea side
chain of 6b. Our exploration centered on altering both the pKa and the steric environment of the
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amine as well as reducing the lipophilicity of the urea side chain. It was thought that a
combination of these changes would provide the desired efficacy while reducing the hERG
liability.
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Rigidification of the amine side chain by incorporation into the 6,6ꢀbicyclic pyrimidine 33a
was well tolerated as it gave 87% inhibition in the mouse CNV model with modest aqueous
solubility (84 µM). However, no substantial reduction in hERG activity was realized (IC 8900
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nM). Intriguingly, the isomeric 6,6ꢀbicyclic pyrimidine 33b provided no significant inhibition in
vivo despite having good potency in the enzymatic and cellular assays.
To further probe the sensitivity of this region to substitution, the enantiomeric methyl analogs
33c and 33d were prepared. These compounds again highlighted a similar pattern of disconnect
between the in vitro and the in vivo efficacy as 33c, the (S)ꢀenantiomer, demonstrated greater
efficacy despite similar in vitro potency to the (R)ꢀenantiomer 33d. Acetylation of the amine as
in 33e caused a complete loss of efficacy in the mouse model, while retention of the basic amine
as in the ethyl derivative 33f provided 81% inhibition. However, the tertiary amine 33f reduced
the aqueous solubility (< 5 µM).
Reduction of amine pKa was achieved with the 6,5ꢀbicyclic pyrimidine 33g. This compound
was found to be efficacious (68% inhibition) in the mouse model and demonstrated reduced
hERG activity (IC 15600 nM). In this instance however, the addition of a methyl group to the
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bicycle as in 33h and 33i attenuated the efficacy.
The pyridyl analog 33j was designed to reduce the lipophilicity of the aniline moiety.
Although the in vitro potency in both the biochemical (280 nM) and cell (1922 nM) assays was
attenuated it demonstrated the potential benefit through further optimization to identify more
preferred heteroaryl groups as there was a favorable impact on aqueous solubility (190 µM).
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Indeed, further design of heteroaryl replacements lead to pyrazoles 33k and 33l that provided
very good in vitro potency (cell IC 3 nM & 1 nM respectively). While the initial pyrazole
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analogs 33k and 33l provided no inhibition in the mouse CNV model, they did have markedly
improved aqueous solubility (833 and 682 µM respectively). Further design and optimization
led to the discovery that the cyclopropylmethyl analog 33m provided 64% inhibition. The close
structural similarities among these analogs again provide a dramatic demonstration of the subtle
effects realized only with in vivo SAR. Pyrazole 33m, had good aqueous solubility (252 µM)
and minimal hERG activity with an IC > 30,000 nM. This was an important finding as it
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demonstrated the ability to reduce hERG activity without alteration of the amine basicity.
Combining the methyl substituted 6,6ꢀbicyclic pyrimidine with the cyclopropylmethyl
pyrazole as in 33n gave good potency in the cellular assay (0.9 nM), efficacy in the mouse (67%
inhibition), good aqueous solubility (450 µM), and minimal hERG activity (IC 27,400 nM). In
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this case, as shown above, the opposite enantiomer 33p and the 6,5ꢀbicyclic analog 33o showed
reduced efficacy in vivo despite similar potency in vivo.
Further exploration of aniline replacements revealed that the isoxazole (e.g. 33q and 33r with
83% and 96% inhibition respectively) provided even greater efficacy than the corresponding
pyrazole analogs, however they also had greater hERG activity. It was also found that the 3ꢀ
aminoisoxazole analogs (e.g. 33s) were more active in vivo than the isomeric 5ꢀaminoisoxazoles
(e.g. 33t). Similar to the pyrazole analogs, inclusion of the methyl group in the 6,5ꢀpyrimidine
did not give a favorable increase in activity (33u), which was again in contrast to the 6,6ꢀbicyclic
pyrimidines. Modification of the isoxazole substitution from tꢀbutyl to cyclopropyl 33v or 33w
provided maximal inhibition in the mouse model (98 and 96% inhibition respectively).
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Isoxazole 33v also showed minimal hERG activity with an IC > 30,000 nM in combination
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with good aqueous solubility (110 µM).
Dose response studies in the mouse and rat were conducted for the preferred compounds
identified from the mouse in vivo single dose screen (Table 4). In the mouse laser CNV model,
the isoxazole derivative 33v provided the greatest efficacy on a per dose basis with an ED of
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.8 mg/kg, while its corresponding aniline analog 33g provided an ED of 31 mg/kg. In contrast
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the pyrazole analog 33n was found to have a higher ED (19.6 mg/kg) than its corresponding
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aniline derivative 33c in the mouse CNV. Both the nonꢀmethylated 33m and the methylated
3n, provided a similar dose response in the mouse model. Further characterization of 33n, 33v,
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and 33m in the rat laser CNV model again demonstrated that on a per dose basis 33v was more
efficacious than either 33m or 33n.
As stated above our key objective was to identify VEGFRꢀ2 inhibitors that provided good
efficacy in the mouse and rat laser CNV models following oral dosing, and demonstrated
distribution to and retention in the ocular tissues relative to the plasma. Discrete oral PK studies
in Brown Norway rats were conducted with preferred compounds. Exposure in plasma, retina
and posterior eye cup (retinal pigment epithelium/choroid sclera complex = posterior eye cup)
were assessed at 7 time points after dosing between 0ꢀ24 hrs. In Table 4, the dose normalized
(
DN) exposures for 3 compounds are detailed. It can readily be seen that 33v has significantly
higher plasma exposure (DNCmax and DNAUC) than either 33n or 33m. In the retina, 33n and
3v had similar exposure while 33m was measurably lower. In the PEC, there was even greater
contrast among the three compounds with the exposure decreasing in order from 33v > 33n >
3m. The data from the ocular tissues indicate a strong preference for retention of each of the
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compounds in those tissues versus retention in the plasma. This is most easily noted by
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comparison of the plasma/retina/PEC dose normalized concentrations at 24 h (33n 1.6/122/1067
nM; 33v 6/65/3334 nM; 33m 0.7/13/102 nM). The above data demonstrate that it is possible to
identify compounds with preferential distribution to the ocular tissues and reduced plasma
exposure. The advantage of such distribution in mitigating systemic side effects will only be
borne out in toxicology studies as it was not expected that our efficacy models would address
this. However, it is worth noting that the approved VEGFRꢀ2 inhibitor pazopanib, described
above, has been reported to provide 93% inhibition of laser induced CNV in the mouse model
when dosed at orally at 100 mg/kg twice daily and that dose when given for 7 d produced a
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plasma exposure of ~13 µM at 16 h post the last dose.
The i.v./p.o. PK parameters of 33n, 33v, and 33m provide further detail regarding systemic
exposure and clearance (Table 6). In accord with the ocular PK study above, 33m shows a
higher rate of clearance from the plasma (165 mL/min/kg) while 33v provided much lower
clearance (6 mL/min/kg). All three of these compounds were found to have good plasma
exposure following oral dosing (F% >50). The plasma and ocular PK data highlight the
limitation of optimizing compounds with limited in vivo efficacy measurements or an over
reliance on in vivo PK as 33m would not have been preferred either from plasma or ocular PK,
yet the compound is efficacious at reducing laser induced CNV.
In a selectivity panel consisting of 62 kinases, 33n and 33v, were found to have activity < 1
µM against several related kinases [IC (µM) 33n: Ret 0.066, PDGFRα 0.15, cꢀKit 0.6; 33v:
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Ret 0.50, PDGFRα 0.15uM]. This is consistent with the observed in vivo efficacy being
primarily driven by VEGFRꢀ2 inhibition.
Conclusions
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Predicting ocular efficacy based on in vitro potency and in vivo exposure remains a challenge
in ocular drug discovery. This is in part evidenced by the high number of potent VEGFRꢀ2
inhibitors described herein, which provide little or no efficacy in the mouse laser CNV model.
The challenge is more clearly illustrated by molecules 33m, 33n, and 33v, which provide similar
in vivo efficacy despite substantially different tissue distribution profiles. The reason for these
disconnects are not fully understood at this time. We believe melanin binding plays a key role
not only in the preferential distribution of the compound to the ocular tissue, but also in the
differing correlations between ocular exposure and efficacy in the CNV models. In part the
answer may come from melanin binding studies that adequately provide the kinetic binding
profiles of the molecules. Differential kinase selectivity may provide another hypothesis to
explain the exposureꢀefficacy disconnect, although data collected against a kinase panel indicate
that the molecules are reasonably selective for VEGFRꢀ2 and have similar broad profiles.
Despite the inability to a priori predict ocular efficacy based on plasma PK and potency, our
use of relatively highꢀthroughput in vivo screening in a mouse laser CNV model facilitated the
generation of clear and actionable in vivo SAR. Through optimization of one particular series of
VEGFRꢀ2 inhibitors, described herein, multiple promising compounds were identified that
effectively reduced choroidal neovascularization and provide preferential distribution to the
ocular tissues. The observations made with 33m, 33n, and 33v are a clear demonstration of the
ability to identify VEGFRꢀ2 inhibitors with markedly higher ocular exposure than in the plasma.
Selective distribution to the target tissue of interest, the eye in this case, may provide an
advantage in minimizing systemic side effects. This is of particular interest as the onꢀtarget
systemic toxicity of VEGFRꢀ2 inhibitors has been well established, and thus may be a barrier to
systemic use of such therapy in the treatment of AMD. This data may support the potential
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clinical utility of systemically administered VEGFRꢀ2 inhibitors with preferential ocular
distribution as a therapy for wet AMD. A similar strategy may be applicable to other disease
indications that would benefit from local drug distribution to limit systemic toxicity.
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AUTHOR INFORMATION
Corresponding Author
*Erik L. Meredith. Novartis Institutes for Biomedical Research, Global Discovery Chemistry,
100 Technology Square, Cambridge MA 02139. erik.meredith@novartis.com.
Present Addresses
†
N.M.: Raze Therapeutics, 400 Technology Square, Cambridge, MA 02139.
‡
J.K.: Department of Ophthalmology and Vision Science, University of Toronto, and Department
of Vision Sciences, Toronto Western Research Institute, 399 Bathurst Street, Toronto, Ontario
M5T 2S8, Canada.
ǁ
K.M.: My Health Partners, 39 Pleasant Blvd, Toronto, ON, M4T 1K2, Canada.
ɠ
L.J.: Coditas Group, 125 Westbourne Terrace, Brookline, 02446.
ϕ
N. J.: Mitobridge, Inc. 1030 Massachusetts Avenue, Suite 200, Cambridge, MA 02138.
˦
G.A.: Kalexsyn, 4502 Campus Drive, Kalamazoo, MI, 49008.
ζ
L.H.: Sensirion, Laubisruetistrasse, 508712 Staefa ZH, Switzerland.
Ϸ
V.H.: AstraZeneca Neuroscience Innovative Medicines, 141 Portland Street, 10th Floor,
Cambridge, MA, 02139.
#
C.R.: School of Biological Science, Nanyang Technological University, 60 Nanyang Drive,
Singapore, 637551.
҂
Y.Z.: 4237 Time Square Blvd, Dublin, Ohio 43016.
Author Contributions
ACKNOWLEDGMENT
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The authors acknowledge the contribution of the Novartis Institute for Biomedical Research
Analytical Sciences group and the Novartis Institute for Biomedical Research Metabolism and
Pharmacokinetics group for support in generating the analytical detail for the compounds
described herein.
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ABBREVIATIONS
TLC, thin layer chromatography; VEGF, vascular endothelial growth factor; AMD, ageꢀrelated
macular degeneration; ADME, absorption distribution metabolism excretion CNV, choroidal
neovascularization; PEC, posterior eye cup; p.o., oral; i.v., intravenous; i.v.t., intravitreal;
ETDRS, Early Treatment of Diabetic Retinopathy Study; WFI, water for injection; MC,
methylcellulose; CMC, carboxymethylcellulose; DN, dose normalized.
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Scheme 1
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Reagents and Conditions: (i) DBU, CH CN, 75%; (ii) NaH, R NCO, DMF, –50 °C to rt, 88%;
(iii) TFA, 100 °C, 74%; (iv) Ms O, pyridine, THF; (v) CH NH , MeOH, 54%, 2 steps.
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NaBH(OAc) , DCE; (iii) tBuOK, toluene; (iv) formamidine acetate, EtONa, EtOH, reflux, 36%
4
3
steps; (v) Pd/C, Boc O, MeOH; (vi) CCl , PPh , DCE, reflux 57%, 2 steps.
2 4 3
a
Scheme 4
a
Reagents and Conditions: (i) 16, 17, NaBH(OAc) , DCE; (ii) ethyl glyoxylate, NaBH(OAc) ,
3
3
DCE; (iii) tBuOK, toluene, 50ꢀ70%, 3 steps; (iv) formamidine acetate, EtONa, EtOH, reflux,
93%; (v) Pd/C, Boc O, EtOH, 80%, 2 steps; (vi) CCl , PPh , DCE, reflux, 51%.
2
4
3
a
Scheme 5
a
Reagents and Conditions: (i) formamidine acetate, EtONa, EtOH, reflux, 24%.
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Journal of Medicinal Chemistry
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1
1
1
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2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
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4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and Conditions: (i) NaH, toluene, 0 °C to rt; (ii) formamidine acetate, EtONa, EtOH,
reflux, ~20%.
a
Scheme 7
a
Reagents and Conditions: (i) 10, 15, 21, or 22, DBU, CH CN, reflux, 90%; (ii) 24 or 28, PyBop,
acetonitrile, DBU, 64%; (iii) 25, phenyl chloroformate, pyridine, 86%; (iv) 32 or R NCO, NaH,
THF, 0 °Cꢀrt, 50%; (v) TFA, DCM, 92%.
3
2
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Page 23 of 55
Journal of Medicinal Chemistry
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Table 1. In vitro inhibition data (KDR biochemical; BaF3 cellular) and in vivo (% inhibition of
mouse CNV) for selected compounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
KDR
IC50
(nM)
BaF3
IC50
(nM)
mCNV
% inhibition @ 10
a
a
b
CMPD
R1
R2
mg/kg qd
1
17
ꢀꢀ
44
45
67
92
87
5
a
a
13
4
6
8
11
66
10
6b
5
3
3a
22
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Journal of Medicinal Chemistry
Page 24 of 55
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1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3b
18
24
23
81
60
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3c
96
58
3
3d
109
33e
7
ꢀꢀ
0
33f
10
10
81
33g
29
16
0.4
68
41
3
3h
118
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Page 25 of 55
Journal of Medicinal Chemistry
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3
3i
3j
17
ꢀꢀ
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
30
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44
45
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49
50
51
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53
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57
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59
60
3
280
1922
ꢀꢀ
3
3k
6
8
3
1
0
0
3
3l
3
3m
5
8
3
64
67
43
3
3n
0.9
0.5
3
3o
< 3
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Journal of Medicinal Chemistry
Page 26 of 55
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
3p
3q
8
30
30
9
0.9
40
4
48
83
96
83
58
33
98
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3r
3s
3
59
167
8
3
3t
114
22
41
3
3u
3
3v
26
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Page 27 of 55
Journal of Medicinal Chemistry
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3
3w
9
2
6
96
90
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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59
60
3
3x
14
a
b
Values are mean of at least two experiments (IC , nM). Compounds dosed q.d. orally as
5
0
0
.5% CMC/0.1% tween 80.
Table 2. Comparison of aqueous solubility for selected VEGFRꢀ2 inhibitors.
a
CMPD
aq sol pH 6.8 (ꢀM)
1
5
6a
64
3
3a
33f
3j
3k
3l
3m
84
<5
3
190
833
682
252
450
489
167
126
20
3
3
3
3
3n
3o
3q
3
3
3
3r
33s
3t
33v
3w
3
232
110
37
3
a
Aqueous highꢀthroughput equilibrium solubility (see ref. 24).
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Journal of Medicinal Chemistry
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Comparison of hERG activity for selected VEGFRꢀ2 inhibitors.
a
CMPD
hERG IC50 (nM)
6
b
5800
8900
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3a
3c
3
10700
15600
>30000
27400
7000
33g
3
3m
33n
3
3q
33r
3s
3v
11100
23500
>30000
3
3
a
Automated Qꢀpatch assay.
Table 4. Dose response inhibition of laser induced choroidal neovascularization for selected
VEGFRꢀ2 inhibitors.
a
b
mouse
ED (mg/kg)
rat
CMPD
ED (mg/kg)
ED (mg/kg)
ED (mg/kg)
5
0
90
50
90
3
3c
3g
3.6
6.9
3.4
1.8
7.8
8.5
31
ꢀꢀ
ꢀꢀ
ꢀꢀ
3
ꢀꢀ
3
3n
3v
19.6
4.8
2.9
0.7
2.7
14.6
2.7
3
3
3m
25.3
13.6
b
a
Compounds dosed q.d. orally as 0.5% MC/0.1% tween 80, WFI 7 days. Compounds dosed
q.d. orally as 0.5% CMC/0.1% tween 80, WFI 14 days.
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Page 29 of 55
Journal of Medicinal Chemistry
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Table 5. Dose normalized (DN) exposure in selected tissues from Brown Norway rat for selected
a
b
c
VEGFRꢀ2 inhibitors 33n , 33m and 33v .
DNAUCPO 0-24h
DNCMax
(nM/mg/kg) (nM/mg/kg)
DNC24h
Tissue
cmpd
3n
33v
33m
(nM*h/mg/kg)
3
598
15014
166
68
3225
33
1.6
6
0.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
plasma
3
3n
33v
3m
3215
2692
310
22266
92954
2386
170
337
15
1067
5293
110
122
65
13
1067
3344
102
retina
PEC
3
3
33v
3n
3
3m
Each compound was dosed orally as a suspension 0.5% MC/0.1% tween 80, WFI at the
following doses: 6 mg/kg (ED = 3.0 mg/kg); 6 mg/kg (ED = 2.7 mg/kg); 2 mg/kg (ED =
.7mg/kg). PEC = posterior eye cup (retinal pigment epithelium/choroid/sclera complex).
a
b
c
5
0
50
50
0
a
Table 6. PKꢀADME parameters for selected VEGFRꢀ2 inhibitors.
b,d,f
b,e,f
c,d,f
Parameter
AUCPO 0ꢀ8h (ꢁM*h)
CL (mL/min/kg)
Vdss (L / kg)
33n
33v
33m
1121
2488
27
5.6
2.3
514
81
29393
6
0.5
1.2
11003
100
165
22
2.7
505
51
t
1/2 (h)
Cmax (nM)
F (%)
a
b
c
Brown Norway rat PK. Animals were dosed either i.v. (1 mg/kg, n = 2) or p.o. (3 or 10
mg/kg, n = 3). i.v. dose: 10% NMP, 15% PG, 4% cremophorEL, PBS. i.v. dose: 10% NMP,
30% PEG 300, 60% WFI. p.o. dose: 0.5% MC/0.1% tween 80, WFI PK parameters derived
from p.o. dosing, and are reported as mean ± SD.
d
e
f
Chemistry.
General. NMR spectra were recorded on a Bruker Avance II 400 MHz spectrometer. All
chemical shifts are reported in parts per million (δ) relative to tetramethylsilane. The following
abbreviations are used to denote signal patterns: s = singlet, d = doublet, t = triplet, m =
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