Development of prodrug 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy) phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate (TG100801): A topically administered therapeutic candidate in clinical trials for the treatment of age-related macular degene

Article abstract of DOI:10.1021/jm7011276

Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeti

Full text of DOI:10.1021/jm7011276

1546
J. Med. Chem. 2008, 51, 1546–1559
Development of Prodrug 4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-
1,2,4-benzotriazin-7-yl)phenyl Benzoate (TG100801): A Topically Administered Therapeutic
Candidate in Clinical Trials for the Treatment of Age-Related Macular Degeneration
Moorthy S. S. Palanki,*,† Hideo Akiyama,‡ Peter Campochiaro,‡ Jianguo Cao,† Chun P. Chow,† Luis Dellamary,† John Doukas,†
Richard Fine,§ Colleen Gritzen,† John D. Hood,† Steven Hu,† Shu Kachi,‡ Xinshan Kang,§ Boris Klebansky,§ Ahmed Kousba,†
Dan Lohse,† Chi Ching Mak,† Michael Martin,† Andrew McPherson,† Ved P. Pathak,† Joel Renick,† Richard Soll,†
Naoyasu Umeda,‡ Shiyin Yee,† Katsutoshi Yokoi,‡ Binqi Zeng,† Hong Zhu,† and Glenn Noronha†
TargeGen, Inc., 9380 Judicial DriVe, San Diego, California 92121, Department of Ophthalmology, Johns Hopkins Hospital School of Medicine,
719 Maumenee, 600 N. Wolfe Street, Baltimore, Maryland 21287, and BioPredict, Inc., 660 Kinderkamack Road, Oradell, New Jersey 07649
ReceiVed September 11, 2007
Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized
world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of
evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD.
Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial
role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have
developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the
most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-
7-yl}phenol (5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester
analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after
topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and
substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were
studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5
are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-
(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate (12), a topically administered
prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically
delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed
good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile,
compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically
applied compound for the treatment of AMD.
Introduction
In patients with AMD, a combination of oxidative damage
and complement-mediated damage causes degeneration of
photoreceptors and retinal pigmented (RPE) cells and gradual
loss of vision. This gradual degeneration is generally referred
to as “dry AMD” and by mechanisms that are not completely
understood leads to up-regulation of VEGF in RPE cells, which
in 20% of patients is sufficient to cause growth of neovascu-
larization from the choroid into the sub-RPE and subretinal
spaces (choroidal neovascularization) commonly referred to as
“wet AMD”. Choroidal neovascularization tends to occur in the
macula and is made up of fenestrated vessels that leak plasma
causing subretinal and intraretinal fluid resulting in sudden loss
of vision. As is the case with DME, the loss of vision from
collection of excess fluid beneath and/or within the macula is
initially reversible, but unless it is eliminated in a timely fashion,
permanent visual loss occurs.
In the industrialized world, the leading causes of vision loss
are diabetic retinopathy and age-related macular degeneration
(AMD).1 In both conditions, up-regulation of vascular endo-
thelial growth factor (VEGF) leads to vascular dysfunction.2–4
In patients with diabetes, chronic hyperglycemia causes damage
to vascular cells in the retina, resulting in capillary drop out
and retinal ischemia. Retinal ischemia causes increased produc-
tion of VEGF,5,6 which promotes growth of new vessels along
the surface of the retina (retinal neovascularization) and also
causes pre-existent retinal vessels to become leaky. The
development of retinal neovascularization heralds the onset of
proliferative diabetic retinopathy, which often leads to blindness
if untreated. The excessive leakage of plasma from pre-existent
retinal vessels causes collection of fluid in the interstitial space
of the macula, which is referred to as diabetic macular edema
(DME). Collection of fluid in the macula causes loss of vision
that is initially reversible if the leakage can be stopped, but if
the edema is prolonged, structural damage occurs to retinal cells
resulting in permanent loss of vision.
Thus, VEGF-induced vascular leakage plays a central role
in the vision loss associated with diabetic retinopathy and
AMD.7–10 Ranibizumab (Lucentis TM, Genentech, South San
Francisco, CA) is a humanized antigen binding fragment (Fab)
that binds all isoforms of VEGF-A.11 Monthly intraocular
injections of ranibizumab caused significant visual improvement
in patients with choroidal neovascularization due to AMD12,13
and DME.14 Bevacizumab, a humanized full-length monoclonal
antibody that binds all isoforms of VEGF-A, has been approved
for the treatment of colon cancer15 but has also been given off-
* To whom correspondence should be addressed. Phone: +1-858-964-
2136. Fax: +1-858-678-0762. E-mail: Palanki@targegen.com.
†
TargeGen, Inc.
Johns Hopkins Hospital School of Medicine.
BioPredict, Inc.
‡
§
10.1021/jm7011276 CCC: $40.75
2008 American Chemical Society
Published on Web 02/27/2008

DeVelopment of a Benzoate for Treatment of AMD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1547
Table 1. Structures of Benzotriazine Analogues and in Vitro Activities in Src and VEGFr2
IC50, nM
compd
R1
R2
Src
VEGFr2
YES
1
2
3
4
5
6
7
8
9
Ph
4-(2-pyrrolidin-1-ylethoxy)
4-(2-pyrrolidin-1-ylethoxy)
4-(2-pyrrolidin-1-ylethoxy)
4-(2-pyrrolidin-1-ylethoxy)
4-(2-pyrrolidin-1-ylethoxy)
4-(2-pyrrolidin-1-ylethylaminosulfonyl)
4-(piperazin-1-ylcarbonyl)
4-(piperidin-4-ylsulfonyl)
340
35
7
14
1.8
3.6
3.6
5.2
1.5
530
1900
560
130
2.2
110
22
1.2
10
0.3
1.3
5.2
4.1
4.8
2-chlorophenyl
2,6-dimethylphenyl
2,6-dichlorophenyl
2-chloro-5-hydroxyphenyl
2-chloro-5-hydroxyphenyl
2-chloro-5-hydroxyphenyl
2-chloro-5-hydroxyphenyl
2-chloro-5-hydroxyphenyl
4.7
1.8
1.7
2.7
4-(3-pyrrolidin-1-ylpropyl)
label by intraocular injection in patients with ocular diseases.16
Intraocular injections are tolerated fairly well, but they carry
some risks of infection and retinal detachment, and the more
injections a patient receives, the greater is the risk. Also,
returning for retinal examinations and possible injections every
month is a hardship on many patients. Therefore, development
of VEGF antagonists that can be delivered by less invasive
means is a major priority for the treatment of AMD.17–23
tissues have been targeted and adequate drug level or therapeutic
effect by topical delivery has been demonstrated.
We have recently disclosed a series of substituted benzotri-
azines as potent inhibitors of Src kinase,29 and we have shown
the potential utility of these compounds by demonstrating their
activity in preclinical animal disease models of cancer.30 We
designed these benzotriazines to potently inhibit Src, YES
kinases, and VEGFr. Here, we present our efforts in optimizing
the benzotriazine series starting with analogue 1 for its
biochemical potency against VEGFr, Src, and YES. We also
describe the further optimization of a subset of these benzo-
triazines to enhance ocular pharmacokinetics and to minimize
systemic exposure, both of which we view as critical for a
chronic therapy where localized effects in the eye are the target.
We demonstrate that compound 12 is active in preclinical
models when administered topically as an eye drop.
Among angiogenic growth factors, the unique role of VEGF
in disrupting endothelial barrier function has been established.
Injection of exogenous VEGF into the eye of mice lacking Src
or YES (Src-/- or YES-/-) did not compromise the endothelial
barrier function clearly establishing a link between VEGF and
Src in vascular leak.24 Studies using chick embryos demon-
strated that Src activity induced the coupling of focal adhesion
kinase (FAK) to integrin Rvꢀ5, an important process in the
VEGF-mediated signaling event in chick embryo blood ves-
sels.25 When healthy mice were injected with VEGF intravit-
really, the leakage of blood and serum into the retina and the
vitreous was observed. However, intravitreal injection of VEGF
to Src knockout mice did not result in the leakage of blood and
serum establishing the role of Src in these disease conditions.26
Apart from its role in AMD, Src is also known to be involved
in oncology, osteopetrosis, and the matrix signaling pathway
necessary for the maturation of osteoclasts.27 These results
demonstrate that while VEGF plays a critical role in macular
degeneration, Src and YES also play an important role in
maintaining vascularity and the endothelial barrier.28 Hence, we
believe that small molecules targeting VEGFr, Src, and YES
might offer a better therapeutic advantage over agents targeting
VEGFr alone. Since both VEGF and Src are involved in several
biological processes, topical application of drug to a local site
might negate the side effects due to systemic circulation of an
inhibitor.
Chemistry
The compounds collated in Table 1 were prepared as shown
in Scheme 1. 4-Bromo-6-methyl-2-nitroaniline (21) was reacted
with cyanamide and pyridine hydrochloride to give an inter-
mediate guanidine. The guanidine was cyclized with 30%
aqueous sodium hydroxide to give the 1-oxobenzotriazine
22.31,32 The oxobenzotriazine 22 was reduced using Raney
nickel and hydrogen in ethanol to give 3-amino-7-bromoben-
zotriazine 23 in good yield. Various aryl 7-substituted benzo-
triazines (24a-e) were prepared by treating 3-amino-7-bromo-
5-methylbenzotriazine (23) with arylboronic acids such as
2-chloro-5-methoxyphenylboronic acid, phenylboronic acid,
2-chlorophenylboronic acid, 2,6-dimethylphenylboronic acid,
and 2,6-dichlorophenylboronic acid to give 24a-e, respectively,
in good yield (50-70%) under Suzuki coupling conditions.33
The advanced intermediates (24b-e) were treated with 4-(2-
pyrrolidin-1-ylethoxy)bromobenzene (25a) under Buchwald-
Hartwig cross-coupling reaction conditions using palladium and
Xantphos to afford compounds 1–4, respectively.34 Similarly,
the treatment of 24a with various aryl bromides (25a-e) under
Buchwald-Hartwig cross-coupling reaction conditions using
palladium and Xantphos resulted in the intermediates (26a-e),
respectively. The methoxy group in 26a-e was cleaved using
boron tribromide to give phenol analogues 5–9.
We focused on developing compounds that can be adminis-
tered topically as eye drops and reach the back of the eye in
sufficient concentrations for the treatment of back-of-the eye
diseases, as in the case of AMD. In the case of AMD,
therapeutically effective concentrations of a drug should be
achieved in the sclera, choroid, and potentially the retina. It is
also equally important that if the drug enters the systemic
circulation, it should be cleared rapidly to lower the risk of
accumulation and allay potential systemic effects. No compound
in the market at present achieves these goals, and topical eye-
drop delivery for back-of-the-eye disease is viewed with some
skepticism because of the unique nature of the eye and the
paucity of a systematic body of work, where back-of-the-eye
The synthesis of the compounds collected in Table 2 is shown
in Scheme 2. The treatment of 5 with acetyl chloride, isopro-
pionyl chloride, benzoyl chloride, 4′-methylbenzoyl chloride,
2,2-dimethylpropionyl chloride, nicotinoyl chloride, 2,6-dim-
ethylbenzoyl chloride, and 2,6-dichlorobenzoyl chloride resulted
in compounds 10, 11, 12, 13, 14, 17, 18, and 19, respectively.

1548 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Palanki et al.
Scheme 1a
a (a) (i) NH2CN, pyridine HCl, 100 °C, 12 h; (ii) 30% aqueous NaOH, 100 °C, 2 h; (b) 10% Raney nickel, H2, EtOH, room temp, 12 h; (c) R1B(OH)2,
Pd(PPh3)4, Na2CO3, DME/EtOH/H2O, reflux, 12 h; (d) Pd2(dba)3, Xantphos, Cs2CO3, dioxane, reflux, 12 h; (e) BBr3, DCM, 0 °C to room temp; (f) Pd(OAc)2,
Xantphos, K+t-BuO-, dioxane, reflux, 12 h.
Table 2. Stability of Prodrugs of 5a
Scheme 2a
hydrolysis
rate in
rabbit eye
homogenate,
(ng/mL)/h
chem
stability under
autoclave
conditions, %
compd
R
5
H
stable
<20
∼50
>90
>90
>90
<20
<20
∼50
>90
>90
>90
ND
ND
ND
288
ND
172
ND
ND
ND
1.1
10
11
12
13
14
15
16
17
18
19
20
CH3CO
(CH3)2CO
PhCO
p-MePhCO
t-BuCO
Na2PO3
NH2CH2CO
Nicotinoyl
2,6-Dimethylbenzoyl
2,6-Dichlorobenzoyl
4-(morpholin-4-ylmethyl)benzoyl
1.7
ND
a The thermal stability of these prodrugs were measured at 120 °C for
1 h. The compounds that were stable to high temperature were selected
and studied in rabbit eye homogenate for their ability to undergo hydrolysis
to release 5. ND ) not determined.
a (a) RCl, Et3N, DCM, room temp; (b) (i) POCl3, Et3N, DCM, room
temp; (ii) NaHCO3, room temp; (c) (i) tert-butoxycarbonylaminoacetic acid,
EDCI, DMAP, DMF, room temp; (ii) 5% TFA/DCM, room temp; (d)
4-(morpholin-4-ylmethyl)benzoic acid, EDCI, DMAP, DMF, room temp.
Disodium phosphate analogue 15 was prepared from 5 by
treating it with phosphorus oxychloride in the presence of
triethylamine in dichloromethane, followed by sodium bicarbon-
ate at room temperature. Glycinate analogue 16 was prepared
by carbodiimide coupling of 5 with tert-butoxycarbonylami-
noacetic acid, followed by the deprotection of butyloxycarbonyl
using 5% trifluoroacetic acid in dichloromethane at room
temperature. Compound 20 was also prepared by carbodiimide
coupling of 5 with 4-(morpholin-4-ylmethyl)benzoic acid at
room temperature.
Results and Discussion
For clarity of discussion, only a selected group of compounds
are shown in Tables 1 and 2 to describe the results of the

DeVelopment of a Benzoate for Treatment of AMD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1549
structure-activity relationship studies. The 7-phenyl substituted
analogue 1 showed modest inhibitory activity against both Src
(IC50 ) 340 nM) and VEGFr2 (IC50 ) 530 nM). The
introduction of a single chloro group at the 2-position of the
7-phenyl ring resulted in approximately a 10-fold increase in
the activity against Src but resulted in a 4-fold drop in potency
against VEGFr2 as seen with compound 2. The introduction of
two methyl groups at the 2 and 6 positions of the 7-phenyl group
resulted in 3, which increased its potency against Src (IC50
)
7 nM) compared to 2. Similarly compound 3 showed ap-
proximately 4-fold improvement in potency against VEGFr2
(IC50 ) 560 nM). The potency was improved approximately
4-fold against both Src and VEGFr2 when the dimethyl groups
on the 7-phenyl ring were replaced with dichloro groups as
shown in 4. While compound 4 has acceptable biochemical
potency against Src, it is 9 times weaker against VEGFr2.
Consistent with earlier studies, the introduction of small
hydrophobic ortho substituents on the phenyl ring orients this
group perpendicular to the benzotriazine, resulting in improved
binding with Src and VEGFr2.29 Chloro substituents at the ortho
position improve potency against VEGFr2.
Further lead optimizations were carried out with the aid of
molecular modeling studies. At the time the work was started,
there was no crystal structure available for fully activated Src.
As such, a homology model of activated Src was constructed
on the basis of the activated crystal structure of Lck.35 Crystal
structures for fully activated VEGFr2 were available from the
outset of work.36 Figure 1A depicts the binding of 2 at the ATP
binding pocket of Src. The 2-chlorophenyl group was bound
deep inside a hydrophobic pocket near the RC-helix. Glu310
located on the RC-helix and available only upon activation was
in proximity to the phenyl group. The benzotriazine ring
interacts with the ATP binding hinge region. We noticed that
compound 2 in VEGFr2 was binding in a similar fashion to 2
in Src. The 2-chlorophenyl group is in proximity to Glu885
from the RC-helix in VEGFr2 (Figure 1B). We therefore
designed molecules with a donor moiety to interact with Glu310
in Src and Glu885 in VEGFr2 to enhance potency. On the basis
of earlier lead optimization efforts, we decided to focus our
initial efforts on the hydrophobic pocket where the 7-(2-
chlorophenyl) group binds. We observed that Glu310 located
on the RC helix deep within the hydrophobic pocket orients
the carboxylic acid group of Glu310 toward the inside of the
hydrophobic pocket in proximity to the 7-phenyl group. We
envisioned that one could take advantage of this carboxylic
group by incorporating a donor group at the 5-position of the
7-(2-chlorophenyl) group of 2. This donor would potentially
interact with the carboxylic acid and enhance its potency further
(Figure 1C). Compound 5 was designed on the basis of these
design ideas suggested by the homology model. Similar homo-
logy modeling studies using a VEGFr model also revealed that
Glu883 on the RC-helix near the hydrophobic pocket orients
the carboxylic group toward the meta position of the 7-phenyl
ring. Homology modeling also showed that compound 5 binds
to the ATP pocket of VEGFr2 in a similar fashion to the binding
mode seen in Src and the hydroxy group of 5 interacts with
Glu883 (Figure 1D). Modeling studies suggested that the
hydroxyl group on the meta position of the 7-phenyl ring
increases the potency of 5 toward both Src and VEGFr2 via
these Glu hydrogen-bonding interactions. Interactions with this
Glu deep within this hydrophobic pocket enhance dipole-charged
interactions because of the low dielectric environment. This
added interaction further stabilizes the activated form of Src.
The concept of taking advantage of a carboxylic group buried
Figure 1. (A) Compound 2 at the ATP pocket of Src. The Glu310
from RC-helix is in proximity to the 7-phenyl ring. (B) Compound 2
at the ATP pocket of VEGFr2. The Glu885 from the RC-helix is in
proximity to the 7-phenyl ring. (C) Compound 5 at the ATP pocket of
Src. (D) Compound 5 at the ATP pocket of VEGFr2. The molecular
surface is colored according to the hydrophobicity of the surrounding
residues; blue is hydrophilic, and red is hydrophobic. The hydroxyl
group of 5 acts as hydrogen bond donor and interacts with Glu310 in
Src (C). The binding orientation of 5 in the VEGFr pocket is very
similar to that of 5 in Src (D). There are two hydrogen bond interactions
between the benzotriazine ring and Cys919 that serves as the hinge.
The hydroxyl group on 5 acts as hydrogen bond donor and interacts
with Glu885.
deep within this hydrophobic pocket has been explored re-
cently.37 It is noted that the homology models have limitations
in the sense that it is well-known that active sites of kinases
have sufficient flexibility to accommodate inhibitors through
induced fit. We have disclosed the complete details of this design
strategy targeting Src and utilized the above concept in
developing different structural classes of compounds, selectively
targeting and optimizing against different kinases.30,38
Figure 1C depicts a minimized binding mode of a benzotri-
azine inhibitor 5 in the ATP pocket of the activated Src model,
and the binding interactions are summarized as follows. The
7-substituted phenyl ring binds in the hydrophobic back pocket,
positioning this phenyl ring perpendicular to the benzotriazine
core. Our earlier studies29 showed that a limited set of small
hydrophobic groups may be substituted on this phenyl ring at
very specific positions because the 7-phenyl binding pocket
possesses a limited tolerance for larger groups. The 3-ami-
nobenzotriazine moiety forms both donor and acceptor hydrogen
bonds to Met341 in the hinge region, thus sharing a common
site with the hinge binding purine ring of ATP. The 3-amino
group from the benzotriazine core is attached to an aryl linker
in a region of the ATP, which falls in the hydrophobic channel
leading out toward a solvent accessible area. The spacer with a
solublizing group binds in an area accessible to solvent.
Benzotriazine inhibitors containing basic nitrogen in this region
provide enhanced potency through protonation and interaction
with Asp348.

1550 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Palanki et al.
Table 3. Exposure Levels of 5 in Ocular Tissues Following Single
Bilateral Topical Instillation of 5 or 12 in Micea
(DME, for example), it is equally important for the drug to reach
sufficient concentrations in the choroid and sclera as well as in
the retina. The half-life of 5 in ocular tissues was very short;
hence, the compound was administered topically minimum t.i.d.
to maintain appropriate drug levels in the eye.41 The maximum
concentration one can achieve in formulations using compound
5 was 0.7% w/v. Further increase in concentration resulted in
the precipitation of the material out of the solution. Since the
compound solubility in the formulation was low, it was difficult
to achieve the required concentration through a 10 µL drop. It
is not advisable to administer a larger volume, since larger
amount of the solution readily runs from the eyes, creating
inconsistent results. Because of low levels of compound 5 in
the retina, it was necessary to increase the frequency of
administration to maintain sufficient levels in the eye. We also
noticed that the increased frequency of administration of 5
caused irritation in the eyes of the animals. The eye irritation
and low levels in retina coupled with short half-life (T1/2 ) 0.5 h)
made this compound less suitable for further development. One
of the desirable properties of 5 is the lack of detectable levels
in systemic circulation.
compd
compd
AUC
administered measured
tissue
retina
choroid/sclera
cornea
plasma
retina
choroid/sclera
cornea
plasma
retina
choroid/sclera
cornea
Tmax (h) Cmax (µM) (h·µg/mL)
5
5
0.5
0.5
0.5
6
24
24
24
NS
24
24
7
3.6
23
64
0.0020
5.3
27
3.7
NS
2.4
29
3.2
240
560
NS
63
430
55
NS
35
12
12
12
5
540
670
NS
33
plasma
24
0.0050
a A 10 µL drop of 5 (70 µg, 137 nM) was instilled per eye to 20 male
mice (C57BL/6). Similarly, a 10 µL drop of 12 (100 µg, 172 nM) was
instilled per eye to 20 male mice (C57BL/6). Composite sampling was
employed to generate tissue concentration-time profiles for 5 and 12 over
the following time course (N ) 4/time point): 0.5, 1, 3, 7, and 24 h
postinstillation. Both eyes were removed from each mouse and dissected
to obtain the cornea, retina, and eye cup (sclera and choroid). The maximum
tissue concentrations of 5 were 3.6 and 23.4 µM for retinal and choroidal/
sclera tissues, respectively, observed at 30 min postadministration for both
tissues. Minimal system exposure (plasma concentrations, e2 nM) was
observed for 5 following topical administration. Topical administration of
12 provided substantial posterior ocular tissue exposure to 5 and 12.
Conversion of 12 to the active 5 was observed in all ocular tissues. On the
basis of total 5 tissue exposure (AUC0–24h), the topical instillation of the
prodrug formulation resulted in an 11-fold increase in retinal tissue exposure
versus topical delivery of 5. NS: insufficient data for calculation of
pharmacokinetic parameters.
In order to attain adequate exposure of 5 in the relevant ocular
tissues, followed by poor systemic levels, we decided to improve
the back-of-the-eye tissue exposure levels of 5 through the
preparation of prodrugs that masked the hydroxyl group. We
reasoned that these ester prodrugs would be readily cleaved
under physiological conditions to release the active drug in the
eye.42 It was anticipated that the prodrugs would have better
and more sustained ocular exposure levels in the relevant back-
of-the-eye tissues and would undergo hydrolysis to release the
drug. It was also anticipated that if the prodrug or drug enters
systemic circulation, it would be cleared very quickly, since 5
has rapid clearance when given systemically.
As anticipated, compound 5 showed improved potency toward
both Src and VEGFr2. Compound 5 was 8 times more potent
against Src and 60 times more potent against VEGFr2 compared
to 2. By keeping the 2-chloro-5-hydroxyphenyl group constant
at the 7-position of the benzotriazine ring, we introduced several
different groups at the R2 position to further optimize this series.
Compounds 6–9 showed equivalent potency to 5 with the
designed donor interaction being the dominant driver of the
potency. On the basis of the structure–activity relationship,
compound 5 was selected for further studies.
It is known that upon stimulation with VEGF, the intracellular
signals leading up to various mitogen activated map kinases
and focal adhesion kinases are activated via Src family kinases,
resulting in retinal microvascular endothelial cells (RMECs)
dysregulation, proliferation, and differentiation in retinopathies.
The ability of compound 5 to block VEGF-stimulated prolifera-
tion was evaluated in retinal microvascular endothelial cells
(hRMVECs) in vitro. Compound 5 showed very good bio-
chemical potency in Src, VEGFr2, and YES39 biochemical
assays. In this study, 5 was shown to inhibit hRMVEC cell
proliferation, with an IC50 ) 610 ( 72 nM. This suggests that
5 has the therapeutic potential to inhibit VEGF function in ocular
endothelial cells, a contributing factor to pathological angio-
genesis in diseases such as AMD and PDR.40
Next, we examined the tissue levels of compound 5 in various
parts of the eye to evaluate the compound’s ability to penetrate
to the back of the eye (Table 3). A dose of 70 µg (137 nmol)
of 5 in 10 µL (0.7% w/v solution of 5 in 27% w/v PL90G
formulation in water) was administered topically to an eye. The
concentrations of 5 were measured at two different locations.
A concentration of 23.4 µM (Cmax) was reached in 30 min (Tmax
) 0.5 h) in the choroid and the sclera. However, the levels of
compound 5 in the retina were relatively low (AUC0–24h ) 3.2
h·µg/mL). It is important for the compound to reach sufficient
concentrations in the choroid and sclera in patients with AMD.
However, in the case of patients with other blinding eye diseases
Several prodrugs were prepared and evaluated on the basis
of two important criteria. First, in order to develop these
compounds further, they have to be stable under thermal
sterilization conditions. The prodrugs 10-20 (Table 2) were
evaluated for their thermal stability at 120 °C for 1 h as a harsh
accelerated test for thermal stability. The parent compound 5
was stable under these conditions. However, prodrugs such as
the acetyl ester 10, isopropyl ester 11, sodium phosphate ester
15, and glycine ester 16 all underwent rapid hydrolysis under
thermal conditions to give 5. Aliphatic esters were rapidly
hydrolyzed compared to aromatic esters under thermal condi-
tions. Since these esters were not stable under such harsh
conditions, they are very likely not suitable for further develop-
ment as drug candidates. Benzoyl ester 12, 4-methylbenzoyl
ester 13, pivaloyl ester 14, 2,6-dimethylbenzoyl ester 18, and
2,6-dichlorobenzoyl ester 19 were stable under these conditions,
and only less than 10% of the compound was hydrolyzed to
form 5. The second criterion for a prodrug is its ability to
undergo hydrolysis to release the active drug in the eye. Thus,
we selected four compounds (12, 14, 18, and 19) that were stable
under thermal conditions to study their hydrolysis rates in rabbit
eye homogenates. Both 2,6-dimethylbenzoyl ester (18) and 2,6-
dichlorobenzoyl ester (19) were very resistant to hydrolysis, as
indicated by very low hydrolysis rates of 1.1 and 1.7 (ng/mL)/
h, respectively. Both benzoyl ester 12 and pivaloyl ester 14
underwent hydrolysis at a moderate rate, 288 and 172 (ng/mL)/
h, respectively, to generate compound 5. As anticipated, the
aromatic esters with substituents at the ortho positions were
resistant to hydrolysis by esterase. The ortho substituents were
introduced to modulate the ester hydrolysis by esterases. On

DeVelopment of a Benzoate for Treatment of AMD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1551
the basis of these favorable properties, compound 12 was
selected for further studies.
Since compound 12 was chosen for clinical development on
the basis of a combined set of drug substance and drug product
properties, we will focus the rest of this article on 12. Ocular
pharmacokinetics using compound 12 were studied in mice, and
the data are shown in Table 3. A single drop of 10 µL containing
100 µg (172 nM) of solution of compound 12 was instilled per
eye. Both compounds 5 and 12 were detected in the eyes where
drops were instilled, suggesting that the prodrug was hydrolyzed
to 5. While the amount of compound 5 seen in the choroid/
sclera upon administration of compound 12 reached concentra-
tions similar to that achieved by direct administration of
compound 5, this was not the case in the retina.43 Much higher
levels of 5 were seen in the retina upon topical eye drop
administration of 12. Both compounds 5 and 12 were seen in
the choroid and sclera as well as in the retina for more sustained
periods of time. The concentrations of 5 and 12 in retina upon
instillation of 12 was >30-fold in retina when compared to that
of 5 upon direct instillation of 5. Upon instillation of 12, the
levels of both 5 and 12 reached a maximum in 24 h in the sclera/
choroid44 and in the retina. The conversion of 12 to 5 was
observed in both the retina and the sclera/choroids tissue
samples. The sustained levels of 5 and 12 in the eye enable
us to use a q.d. or q.o.d. dosing schedule. This dosing
schedule also showed no eye irritation in the animal models.
The plasma concentrations of both 5 and 12 were below the
limit of detection (<1 ng/mL) at all time points after topical
administration of 12, suggesting that the compounds are not
entering into systemic circulation.45 The lack of compounds
5 and 12 in systemic circulation is very beneficial, as VEGFr2
inhibitors in systemic circulation are not desirable because
of the role VEGFr plays in various biological functions.
Therefore, it appears that the administration of compound
12 would enable either once a day or every other day dosing
without any systemic buildup of either 5 or 12.
Figure 2. Topical administration of 12 inhibits laser-induced
choroidal neovascularization: (A) Representative image of rupture
site treated with 0.61% w/v of 12; (B) representative image of
rupture site treated with 1.83% w/v of 12; (C) representative image of
rupture site treated with vehicle; (D) representative image of rupture
site untreated; (E) choroidal neovascularization area vs treatment with
different concentrations of 12 and controls. Topical administration of
12 reduced laser-induced choroidal neovascularization: 23 eyes/group;
(/) p ) 0.0001, 0.61% 12 vs no treatment; (†) p ) 0.0427, 0.61% 12
vs vehicle; (//) p ) 0.0063, 1.83% 12 vs no treatment. The data were
analyzed by linear mixed model with the Dunnett method used to adjust
for multiple comparisons.
treated with 0.61% or 1.83% 12, respectively (Figure 2E). There
is no statistical difference in biological responses from 0.61%
and 1.83% dosing of 12 in the pharmacology model. There was
no significant difference between the area of CNV at rupture
sites in eyes treated with vehicle and fellow untreated eyes. This
demonstrates that the inhibitory effect occurs locally following
topical application with no hematogenous delivery to the fellow
eye via systemic absorption. These data suggest that topically
administered 12 has potential as a noninvasive suppressive
therapy for treatment of diseases in which choroidal neovas-
cularization is a significant component of the pathology.
Compound 12 was inactive in Src (IC50 g 10 µM), VEGFr2
(IC50 g 10 µM), and YES (IC50 g 2.5 µM) biochemical assays.
This would suggest that compound 12 is likely undergoing
hydrolysis to release 5 in vivo which is responsible for its
biological actions.
The capacity of topically applied 5 or 12 to suppress a VEGF-
induced vascular leak in the retina has been demonstrated using
a rodent model.51 In order to establish the molecular target of
5 and its pharmacodynamic effects, the impact of 5 on the
phosphorylation of focal adhesion kinase (FAK), a downstream
substrate of VEGFr and Src, was studied in eye tissue52–54 The
phosphorylation was monitored in the choroid/sclera of mice
undergoing developmental angiogenesis.55,56 Compound 5
reduced FAK-Y861 phosphorylation in a dose-dependent man-
ner with >90% inhibition observed at the 5 mg/kg dose.51 The
details of the experimental procedure is in the Supporting
Information. We have further evaluated the efficacy of com-
pounds 5 and 12 in VEGF induced retinal leak model. As
mentioned before, VEGF-induced vascular leakage is a signifi-
cant pathology contributing to vision loss in AMD, PDR, and
DME. On the basis of these attributes, the capacity of topically
Possessing a compound with good ocular pharmacokinetics
and desirable systemic pharmacokinetics, we turned our efforts
to test 12 in a suitable animal model. Choroidal neovascular-
ization (CNV) models in primates,46 rats,47 minipigs,48 and
rabbits49 have been studied extensively. Laser-induced choroidal
neovascularization in murine model offers several advantages
and has been studied extensively.50
After laser-induced rupture of Bruch’s membrane at three
locations in each eye, mice were treated by topical application
t.i.d. with one drop containing 0.61% w/v of 12, 1.83% w/v of
12, or vehicle in one eye and no treatment in the fellow eye. A
fourth group of mice had rupture of Bruch’s membrane and no
treatment. After 14 days, eyes that had received drops containing
0.61% w/v (Figure 2A) or 1.83% w/v (Figure 2B) of 12 had
CNV lesions that appeared smaller than those in eyes that had
received formulation vehicle (Figure 2C) or no treatment (Figure
2D). Measurement of the area of CNV lesions by image analysis
showed that compared to the untreated fellow eye, there were
statistically significant reductions of 46 ( 7% or 35 ( 9% in
the amount of CNV at Bruch’s membrane rupture sites in the
eyes of mice that were treated with 0.61% w/v or 1.83% w/v
of 12, respectively (Figure 2E). This difference between the
treated and fellow eyes indicates that the suppression of CNV
occurred by local delivery of 12 and without absorption into
the blood stream that could have resulted in systemically
mediated drug effects. Relative to vehicle control, there was a
reduction of 39 ( 8% or 26 ( 10% in the amount of CNV at
Bruch’s membrane rupture sites in the eyes of mice that were

1552 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Palanki et al.
Merck Kieselgel 60 F254 plates with visualization by ultraviolet and/
or anisaldehyde, potassium permanganate, or phosphomolybdic acid
dips. Flash chromatography was carried out using RediSep 45–60
µm silica gel columns using the Isco CombiFlash companion
system. Preparative reverse-phase HPLC chromatography was
carried out on Gilson preparative HPLC equipped with a 215 liquid
handler and Waters SymmetryShield TM RP18 7µm (40 mm ×
100 mm) Prep-Pak cartridge. The mobile phase consisted of HPLC
grade acetonitrile and water, both containing 0.1% trifluoroacetic
acid. Purification was carried out at a flow rate of 40 mL/min and
a gradient such that the peak of interest was eluted between 12
applied 5 or 12 to suppress a VEGF-induced vascular leak in
the retina was assessed in a rodent model. Briefly, VEGF was
injected and vitreous plasma leak was monitored at the back of
the eye. Compounds 5 (0.7% w/v) and 12 (1.22% w/v) were
instilled topically in a suitable media. Compound 5 decreased
VEGF-induced leak by 60% after single topical instillation, q.d.,
to a mice model of VEGF induced retinal leak. When the
compound 5 was administered t.i.d., there was a 80% decrease
in VEGF induced leak response. However, a topical administra-
tion of 12, q.d., completely abolished VEGF-induced leak in
the above animal model. The prodrug 12 not only showed
increased efficacy but also reduced frequency of administration.
The details of VEGF induced leak model and the effects of
compounds 5 and 12 on the model are in the Supporting
Information.
1
and 15 min in a 30 min run. H NMR spectra were recorded at
NuMega Laboratories (San Diego, CA) on a Bruker AMX-II
spectrometer operating at 500 MHz. Spectra were obtained on all
the intermediates and the final products in deuterated dimethyl
sulfoxide (DMSO-d6) unless otherwise noted and were consistent
with the proposed structures. Chemical shifts are calibrated to
tetramethylsilane internal standard or residual solvent peak and
expressed in δ (ppm) units. Peak multiplicities are represented as
follows: s ) singlet, d ) doublet, t ) triplet, q ) quartet, qn )
quintet, dd ) doublet of doublets, m ) multiplet, br s ) broad
singlet). Coupling constants (J/Hz) and integration are also listed.
Coupling constants were taken directly from the spectra and are
uncorrected. Mass spectrometry was carried out on a Waters
analytical LC-MS system equipped with a C-18 reverse phase
column. The analytical gradient consisted of 10% acetonitrile in
water ramping up to 100% acetonitrile, both containing 0.05%
trifluoroacetic acid, over 5 min unless otherwise stated. Low-
resolution mass spectra was obtained on a micromass spectrometer
using the electrospray (ES+) ionization method. The protonated
parent ion (M + 1) or fragment of highest mass was quoted. The
mass spectral data were consistent with the proposed structure.
Purities of compounds were measured on a Shimadzu HPLC
equipped with a Agilent C-18 Zorbax 250 mm × 4.6 mm column
with 5 µm particle size and dual wavelength and evaporating light
scattering detectors (ELSD). The analytical gradient consisted of
10% acetonitrile in water ramping up to 100% acetonitrile, both
containing 0.05% trifluoroacetic acid, over 20 min unless otherwise
stated. The column temperature was maintained at 40 °C for all
the purity analyses. Elemental analyses were performed at NuMega
Laboratories (San Diego, CA) on Perkin-Elmer series II-2400
CHNS analyzer, and the results are within 0.4% of the calculated
values unless otherwise stated.
Conclusions
We have developed a series of benzotriazine-based com-
pounds that inhibit Src and VEGFr2, both of which play critical
roles in age-related macular degeneration. We optimized the
inhibitory activity of a series of benzotriazine compounds toward
both Src and VEGFr2. On the basis of in vitro activity in
enzymatic and cellular assays, compound 5 was selected and
studied further in ocular PK studies. However, compound 5
showed lower than expected ocular pharmacokinetic properties
because of its inability to attain sustained concentrations in
certain back-of-the-eye tissue. Subsequently, several prodrugs
of 5 were prepared and evaluated for their thermal stability at
higher temperature to identify appropriate prodrugs that are
suitable for further development. Prodrugs that were stable under
thermal conditions were selected and their hydrolysis rates in
ocular tissue were evaluated as a surrogate for their propensity
to generate the active compound 5 in the in vivo setting. Prodrug
12 showed much better pharmacokinetic properties compared
to 5 and released sufficient amounts of 5 over sustained periods
of time in the choroid, sclera, and retina. The analysis of blood
samples withdrawn over time, after topical delivery of 12 as an
eye drop, revealed that the amount of 12 or 5 in the systemic
circulation was below quantifiable limits.
7-Phenyl-5-methyl-N-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-1,2,4-ben-
zotriazin-3-amine (1). The title compound was prepared using the
procedure that is described for compound 26c and converted to
the hydrochloride salt using dioxane-hydrogen chloride solution
(35%). 1H NMR: δ 1.80–2.10 (m, 4H), 2.68 (s, 3H), 3.05–3.15
(m, 2H), 3.55–3.65 (m, 4H), 4.37 (t, J ) 5.1 Hz, 2H), 7.09 (d, J )
9.1 Hz, 2H), 7.44 (t, J ) 7.6 Hz, 1H), 7.54 (t, J ) 7.6 Hz, 2H),
7.88 (d, J ) 7.6 Hz, 2H), 7.98 (d, J ) 9.0 Hz, 2H), 8.17 (s, 1H),
8.40 (d, J ) 1.7 Hz, 1H), 10.83 (s, 2H). MS-ESI+ m/z 426 [M +
1]. Anal. (C26H27N5O·HCl·0.5H2O) C, H, N. HPLC purity, 97%
at 220 nm.
In order to evaluate the capacity of 5 to inhibit choroidal
angiogenesis, prodrug 12 was tested in a model of choroidal
angiogenesis in which angiogenesis is caused using laser-
induced rupture of Bruch’s membrane in the eyes of C57BL/6
mice. After laser-induced rupture of Bruch’s membrane, mice
were treated by topical application with 12. Relative to vehicle
control, there is a reduction of 39 ( 8% and 26 ( 10% in the
amount of CNV at Bruch’s membrane rupture sites in the eyes
of mice treated with 0.61% w/v and 1.83% w/v of 12,
respectively.
Because of its in vitro activity in enzymatic and cellular
assays, 5 was selected for the prodrug approach to improve its
pharmacokinetic properties. On the basis of its favorable ocular
pharmacokinetics and activity in a preclinical model, compound
12 was selected for further development for age-related macular
degeneration. Compound 12 is currently undergoing evaluation
in clinical trials for age-related macular degeneration.57,58
7-(2-Chlorophenyl)-5-methyl-N-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-
1,2,4-benzotriazin-3-amine (2). The title compound was prepared
using the procedure that is described for compound 26c and
converted to the hydrochloride salt using dioxane-hydrogen
1
chloride solution (38%). H NMR: δ 1.85–2.05 (m, 4H), 2.65 (s,
3H), 3.05–3.15 (m, 2H), 3.55–3.65 (m, 4H), 4.36 (t, J ) 5.0 Hz,
2H), 7.09 (d, J ) 9.2 Hz, 2H), 7.40–7.55 (m, 2H), 7.55–7.65 (m,
2H), 7.86 (dd, J ) 0.95, 1.8 Hz, 1H), 7.98 (d, J ) 9.1 Hz, 2H),
8.17 (d, J ) 1.8 Hz, 1H), 10.70 (br s, 1H), 10.89 (br s, 1H). MS-
ESI+ m/z 460 [M + 1]. Anal. (C26H26ClN5O·HCl·0.5H2O) C, H,
N. HPLC purity 97% at 220 nm.
7-(2,6-Dimethylphenyl)-5-methyl-N-[4-(2-pyrrolidin-1-ylethoxy)phe-
nyl]-1,2,4-benzotriazin-3-amine (3). A solution of 7-(2,6-dimeth-
ylphenyl)-5-methylbenzo[1,2,4]triazin-3-ylamine (1 g, 3.78 mmol)
and of 1-[2-(4-bromophenoxy)ethyl]pyrrolidine (2.04 g, 7.56 mmol),
tris(dibenzylideneacetone)dipalladium(0) (174 mg, 0.19 mmol), (()-
2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (340 mg, 0.54 mmol),
Experimental Section
All experiments were performed under anhydrous conditions in
an atmosphere of argon, except where stated, using oven-dried
apparatus and employing standard techniques in handling air-sensitive
materials. All the solvents and chemicals from commercial sources
were used as received without further purification. Aqueous solutions
of sodium bicarbonate and sodium chloride (brine) were saturated.
Analytical thin layer chromatography (TLC) was carried out using

DeVelopment of a Benzoate for Treatment of AMD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1553
and potassium tert-butoxide (500 mg, 4.46 mmol) in toluene (500
mL) under argon was heated at 100 °C for 24 h. The reaction
mixture was concentrated under reduced pressure, dissolved in
dimethyl formamide, and purified by preparative HPLC to afford
Hz, 2H), 8.18 (d, J ) 1.7 Hz, 1H), 9.93 (br s, 1H), 11.17 (s, 1H).
MS-ESI+ m/z 475 (M + 1). Anal. (C25H23ClN6O2 ·2H2O) C, H, N.
7-(2-Chloro-5-hydroxyphenyl)-5-methyl-N-[4-(piperidin-4-ylsulfo-
nyl)phenyl]-1,2,4-benzotriazin-3-amine (8). The methoxy group and
benzyl carboxylate in benzyl 4-[(4-{[7-(2-chloro-5-methoxyphenyl)-
5-methyl-1,2-4-benzotriazin-3-yl]amino}phenyl)sulfonyl]piperdine-
1-carboxylate (26d, 0.165 g, 0.25 mmol) were cleaved using boron
tribromide in dichloromethane (2.5 mL, 2.5 mmol) as described in
the general procedure to afford the title compound as a yellow solid
(0.028 g, 22%). 1H NMR: δ 1.68–1.77 (m, 2H), 2.03–2.06 (m, 2H),
2.73 (s, 3H), 2.83–2.88 (m, 2H), 3.05 (s, 1H), 3.52–3.58 (m, 1H),
6.89 (dd, J ) 2.9, 8.8 Hz, 1H), 6.95 (d, J ) 2.9 Hz, 1H), 7.41 (d,
J ) 8.7 Hz, 1H), 7.88 (d, J ) 8.8 Hz, 2H), 7.94 (s, 1H), 8.23 (d,
J ) 1.7 Hz, 1H), 8.33 (d, J ) 8.8 Hz, 2H), 8.75 (br s, 1H), 10.00
(s, 1H), 11.57 (s, 1H). MS-ESI+ m/z 510 (M + 1). Anal.
(C25H24ClN5O3S·C2F3HO 2 ·1.2H2O) C, H, N.
1
the title compound (0.09 g, 5.2%). H NMR: δ 1.90–2.05 (m, 10
H), 2.64 (s, 3H), 3.14–3.16 (m, 2H), 3.59–3.64 (m, 4H), 4.31 (t, J
) 5.2 Hz, 2H), 7.09 (d, J ) 9.2 Hz, 2H), 7.18 (d, J ) 7.4 Hz, 2H),
7.22–7.24 (m, 1H), 7.60 (s, 1H), 7.91 (s, 1H), 7.98 (d, J ) 9.2 Hz,
2H). MS-ESI+ m/z 454 [M + 1]. Anal. (C28H31N5O·C6H8O7 ·1.5H
2O) C, H, N. HPLC purity 99% at 220 nm.
7-(2,6-Dichlorophenyl)-5-methyl-N-[4-(2-pyrrolidin-1-ylethoxy)-
phenyl]-1,2,4-benzotriazin-3-amine (4). The title compound was
synthesized using a similar procedure described for 3 to afford the
1
title compound (35%). H NMR: δ 1.88–1.91 (m, 2H), 2.02–2.07
(m, 2H), 2.63 (s, 3H), 3.12–3.17 (m, 2H), 3.58–3.62 (m, 4H), 4.32
(t, J ) 4.8 Hz, 2 H), 7.10 (d, J ) 9.2 Hz, 2H), 7.51 (t, J ) 8.2 Hz,
1H), 7.67 (d, J ) 8.2 Hz, 2H), 7.69 (dd, J ) 1.0, 1.9 Hz, 1H), 7.98
(d, J ) 9.2 Hz, 2H), 8.08 (s, 1H). MS-ESI+ m/z 494 (M + 1).
Anal. (C26H25Cl2N5O·0.5H2O) C, H, N. HPLC 99% pure at 230
nM and 100% pure using ELSD detector.
7-(2-Chloro-5-hydroxyphenyl)-5-methyl-N-[4-(3-pyrrolidin-
1-ylpropyl)]phenyl]-1,2,4-benzotriazin-3-amine (9). A suspension
of [7-(2-chloro-5-methoxyphenyl)-5-methyl]-1,2,4-benzotriazin-3-
amine (0.55 g, 1.8 mmol), 1-[3-(4-bromophenyl)propyl]pyrrolidine
(0.60 g, 2.2 mmol), palladium acetate (25 mg, 0.11 mmol), xantphos
(0.13 g, 0.22 mmol), and potassium tert-butoxide (0.40 g, 3.6 mmol)
in dioxane (3 mL) was heated at reflux for 16 h. The reaction
mixture was cooled to room temperature and filtered, and the solids
were washed with dichloromethane. The filtrate was concentrated
under reduced pressure and purified by flash chromatography (SiO2,
10–20% methanol in dichloromehtane) to afford 7-(2-chloro-5-
methoxyphenyl)-5-methyl-N-[4-(3-pyrrolidin-1-ylpropyl)]phenyl]-
1,2,4-benzotriazin-3-amine as a red solid (0.16 g, 18%). MS (ESI+):
m/z 488 (M + 1)+. This compound (0.16 g, 0.33 mmol) was treated
with boron tribromide as described in the general procedure to
cleave the methyl group. The reaction mixture was purified by
preparative HPLC to afford the title compound as an orange
General Procedure for the Deprotection of a Methoxy
Group with Boron Tribromide (BBr3). A solution of methoxy
precursor (1.0 mol equiv) and boron tribromide (5–10 mol equiv)
in dichloromethane (0.01–0.03 M) was stirred at room temperature
until the reaction was complete (4–12 h). The pH of the reaction
mixture was adjusted to 7 by treating with a saturated sodium
bicarbonate solution and filtered. The solids were washed with water
and ether and purified using preparative HPLC to afford pure
compounds.
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-
1,2,4-benzotriazin-7-yl)phenol (5). The methoxy group in 7-(2-chloro-
5-methoxyphenyl)-5-methyl-N-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-
1,2,4-benzotriazin-3-amine (26a, 440 mg, 0.90 mmol) was cleaved
using boron tribromide as described in the general procedure to
afford the title compound (387 mg, 84%), which was converted to
the HCl salt. 1 H NMR: δ 1.88–1.92 (m, 2H), 1.97–2.05 (m, 2H),
2.64 (s, 3H), 3.09–3.14 (m, 2H), 3.57–3.61 (m, 4H), 4.36 (t, J )
4.9 Hz, 2H), 6.87 (dd, J ) 2.9, 8.8 Hz, 1H), 6.94 (d, J ) 2.9 Hz,
1H), 7.09 (d, J ) 9.0 Hz, 2H), 7.39 (d, J ) 8.8 Hz, 1H), 7.83 (d,
J ) 1.6 Hz, 1H), 7.98 (d, J ) 9.0 Hz, 2H), 8.12 (d, J ) 1.6 Hz,
1H), 9.97 (s, 1H), 10.55 (br s, 1H), 10.87 (s, 1H). MS-ESI+ m/z
476 (M + 1). Anal. (C26H26ClN5O2 ·HCl ·0.25H2O) C, H, N. HPLC
98% pure at 220 nm, 98% pure using ELSD detector.
1
solid (30 mg, 16%). H NMR: δ 1.80–1.90 (m, 2H), 1.90–2.05
(m, 4H), 2.64 (t, J ) 7.6 Hz, 2H), 2.66 (s, 3H), 2.95–3.05 (m,
2H), 3.10–3.20 (m, 2H), 3.50–3.70 (m, 2H), 6.87 (dd, J ) 2.9,
8.8 Hz, 1H), 6.93 (d, J ) 2.9 Hz, 1H), 7.28 (d, J ) 8.5 Hz,
2H), 7.40 (d, J ) 8.8 Hz, 1H), 7.85 (t, J ) 0.8 Hz, 1H), 7.98 (d,
J ) 8.5 Hz, 2H), 8.15 (d, J ) 1.8 Hz, 1H), 9.55 (br s, 1H), 9.95
(br s, 1H), 10.94 (s, 1H). MS-ESI+ m/z 474 (M + 1). Anal.
(C27H28ClN5O · C2F3HO2 · 1.5H2O) C, H, N.
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phe-
nyl]amino}-1,2,4-benzotriazin-7-yl)phenyl Acetate (10). Acetyl
chloride (0.092 g, 1.17 mmol) was added dropwise to a solution of
4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-
1,2,4-benzotriazin-7-yl)phenol hydrochloride (5, 0.465 g, 0.978
mmol) and triethylamine (0.494 g, 4.89 mmol) in dichloromethane
(30 mL) under argon at room temperature and stirred for 24 h.
The reaction mixture was quenched with a saturated sodium
bicarbonate solution. The organic layer was separated, dried
(Na2SO4), filtered, concentrated under reduced pressure, and purified
on flash chromatography (SiO2, dichloromethane/methanol/triethy-
lamine ) 85:10:5 (v/v/v)) to afford the title compound as an orange
solid (0.409 g, 75%). 1H NMR: δ 1.67–1.70 (m, 4H), 2.29 (s, 3H),
2.50–2.52 (m, 4H), 2.64 (s, 3H), 2.78 (t, J ) 5.9 Hz, 2H), 4.07 (t,
J ) 6.0 Hz, 2H), 6.99 (dd, J ) 7.1, 12.6 Hz, 2H), 7.27 (dd, J )
5.9, 8.8 Hz, 1H), 7.41 (d, J ) 2.8 Hz, 1H), 7.67 (d, J ) 8.8 Hz,
1H), 7.85 (t, J ) 1.3 Hz, 1H), 7.93 (d, J ) 9.1 Hz, 2H), 8.17 (d,
J ) 1.8 Hz, 1H), 10.83 (s, 1H). MS-ESI+ m/z 520 (M + 3). Anal.
(C28H28ClN5O3 ·0.5H2O) C, H, N.
4-{[7-(2-Chloro-5-hydroxyphenyl)-5-methyl-1,2,4-benzotriazin-
3-yl]amino}-N-(2-pyrrolidin-1-ylethyl)benzenesulfonamide (6). The
methoxy group in 4-{[7-(2-chloro-5-methoxyphenyl)-5-methyl-
1,2,4-benzotriazin-3-yl]amino}-N-(2-pyrrolidin-1-ylethyl)benzene-
sulfonamide (26b 0.055 g, 0.099 mmol) was cleaved using boron
tribromide as described in the general procedure to afford the title
compound as the trifluoroacetate salt (0.013 g, 25% yield). 1H
NMR: δ 1.85–1.88 (m, 2H), 1.95–1.99 (m, 2H), 2.98–3.03 (m, 2H),
3.10–3.14 (m, 2H), 3.21–3.25 (m, 2H), 3.38 (s, 3H), 3.50–3.54 (m,
2H), 6.89 (dd, J ) 2.9, 8.7 Hz, 1H), 6.96 (d, J ) 2.9 Hz, 1H), 7.41
(d, J ) 8.7 Hz, 1H), 7.88 (d, J ) 8.9 Hz, 2H), 7.92 (d, J ) 1.8 Hz,
1H), 8.21 (d, J ) 1.7 Hz, 1H), 8.25 (d, J ) 8.8 Hz, 2H), 10.00 (s,
1H), 10.43 (br s, 1H) 11.44 (s, 1H). MS-ESI+ m/z 539 (M + 1).
Anal. (C26H27ClN6O3S·HCl·H2O) C, H, N. HPLC 97% pure at
220 nm and at 254 nm.
4-Chloro-3-(5-methyl-3-{[4-(piperazin-1-ylcarbonyl)phenyl]amino}-
1,2,4-benzotriazin-7-yl)phenol (7). The title compound, was prepared
from tert-butyl 4-(4-{[7-(2-chloro-5-methoxyphenyl)-5-methyl-1,2,4-
benzotriazin-3-yl]amino}benzoyl)piperazine-1-carboxylate (26c, 40 mg,
0.068 mmol) as described in the general procedure using boron
tribromide. Both methyl groups and the tert-butyloxycarbonyl group
in 26c were cleaved simultaneously under the reaction conditions,
and the crude product was purified by preparative HPLC to afford
the title compound as an orange solid (32 mg, 99% yield). 1H NMR:
δ 2.69 (s, 3H), 2.70–2.80 (m, 4H), 3.35–3.50 (m, 4H), 6.87 (dd, J
) 2.9, 8.7 Hz, 1H), 6.94 (d, J ) 2.9 Hz, 1H), 7.41 (d, J ) 8.7 Hz,
1H), 7.46 (d, J ) 8.7 Hz, 2H), 7.85–7.90 (m, 1H), 8.09 (d, J ) 8.6
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phe-
nyl]amino}-1,2,4-benzotriazin-7-yl)-4-phenyl 2-Methylpro-
panoate (11). The title compound was prepared using a
procedure similar to that described for 10. Compound 11 was
1
obtained in comparable yield. H NMR: δ 1.24 (d, J ) 6.9 Hz,
6H), 1.67–1.71 (m, 4H), 2.52–2.54 (m, 4H), 2.64 (s, 3H), 2.79
(t, J ) 5.9 Hz, 2H), 4.07 (t, J ) 5.9 Hz, 2H), 6.99 (d, J ) 7.1
Hz, 2H), 7.25 (dd, J ) 2.9, 8.8 Hz, 1H), 7.41 (d, J ) 2.9 Hz,
1H), 7.67 (d, J ) 8.8 Hz, 1H), 7.85 (t, J ) 1.1 Hz, 1H), 7.93 (d,
J ) 9.1 Hz, 2H), 8.18 (d, J ) 1.9 Hz, 1H), 10.84 (s, 1H). MS-
ESI+ m/z 546 (M + 1). HPLC purity, 95% at 254 nm.

1554 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Palanki et al.
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phe-
nyl]amino}-1,2,4-benzotriazin-7-yl)phenyl Benzoate (12). The
title compound was synthesized from 4-chloro-3-(5-methyl-3-{[4-
(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phe-
nol hydrochloride (5, 1.0 g, 2.0 mmol) as described for compound
7.7 (d, J ) 8.7 Hz, 1H), 7.88 (dd, J ) 1.4, 1.9 Hz, 1H), 7.98 (d,
J ) 9.1 Hz, 2H), 8.20 (d, J ) 1.8 Hz, 1H), 8.46 (br s, 2H), 9.87
(br s, 1H), 10.92 (br s, 1H). MS-ESI+ m/z 533 (M + 1)+. HPLC
purity, 97% at 254 nm.
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]ami-
no}-1,2,4-benzotriazin-7-yl)phenyl Nicotinate (17). A solution of
4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-
1,2,4-benzotriazin-7-yl)phenol (5, 320 mg, 0.63 mmol), nicotinyl
chloride (122 mg, 0.69 mmol), and triethylamine (0.64 g, 6.3 mmol)
in dichloromethane (50 mL) was stirred at room temperature for
12 h. A saturated aqueous sodium bicarbonate solution (100 mL)
was added to the reaction mixture, and the organic layer was
separated. The aqueous layer was extracted with dichloromethane
(2 × 20 mL). The organic layers were combined, washed with brine
(100 mL), and dried (Na2SO4). The solvent was removed under
reduced pressure and the solid was washed with ether (2 × 30 mL)
to afford the title compound as a red solid (177 mg, 49%). 1H NMR:
δ 1.65–1.75 (m, 4H), 2.57 (br s, 4H), 2.65 (s, 3H), 2.83 (br s, 2H),
4.09 (t, J ) 5.9 Hz, 2H), 7.00 (d, J ) 9.1 Hz, 2H), 7.50 (dd, J )
2.8, 8.7 Hz, 1H), 7.55–7.68 (m, 2H), 7.76 (d, J ) 8.7 Hz, 1H),
7.90 (s, 1H), 7.93 (d, J ) 9.1 Hz, 2H), 8.22 (s, 1H), 8.40–8.50 (m,
1H), 8.91 (dd, J ) 1.7, 4.8 Hz, 1H), 9.28 (d, J ) 2.3 Hz, 1H),
10.84 (s, 1H). MS-ESI+ m/z 581 (M + 1). HPLC purity, 98% at
254 nm.
1
10 (0.9 g, 80%). H NMR: δ 1.65–1.72 (m, 4H), 2.50–2.56 (m,
4H), 2.63 (s, 3H), 2.79 (t, J ) 5.9 Hz, 2H), 4.07 (t, J ) 6.0 Hz,
2H), 7.00 (d, J ) 9.1 Hz, 2H), 7.46 (dd, J ) 2.9, 8.7 Hz, 1H),
7.60–7.65 (m, 3H), 7.75 (d, J ) 8.6 Hz, 1H), 7.76 (t, J ) 7.4 Hz,
1H), 7.90 (dd, J ) 1.0, 1.8 Hz, 1H), 7.93 (d, J ) 9.1 Hz, 2H), 8.16
(d, J ) 8.4 Hz, 2H), 8.22 (d, J ) 1.8 Hz, 1H), 10.84 (br s, 1H).
MS-ESI+ m/z 580 (M + 1). Anal. (C33H30ClN5O3 ·HCl·0.5H2O)
C, H, N. HPLC 98% pure at 220 nm and 100% pure using ELSD
detector.
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phe-
nyl]amino}-1,2,4-benzotriazin-7-yl)phenyl 4-Methylbenzoate
(13). The title compound was prepared similar to the procedure
described for 10. 1H NMR: δ 1.68–1.70 (m, 4H), 2.42 (s, 3H),
2.51–2.53 (m, 4H), 2.64 (s, 3H), 2.78 (t, J ) 6.0 Hz, 2H), 4.06 (t,
J ) 6.0 Hz, 2H), 6.99 (d, J ) 5.0 Hz, 2H), 7.42 (d, J ) 7.5 Hz,
2H), 7.44 (d, J ) 2.8 Hz, 1H), 7.58 (d, J ) 2.8 Hz, 1H), 7.72 (d,
J ) 8.7 Hz, 1H), 7.88 (d, J ) 0.9 Hz, 1H), 7.92 (d, J ) 9.2 Hz,
2H), 8.05 (d, J ) 6.6 Hz, 2H), 8.21 (d, J ) 1.8 Hz, 1H), 10.83 (s,
1H). MS-ESI+ m/z 594 (M + 1). HPLC purity, 98% at 254 nm.
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]ami-
no}-1,2,4-benzotriazin-7-yl)phenyl Pivalate (14). A solution of
4 - c h l o r o - 3 - { 5 - m e t h y l - 3 - [ 4 - ( 2 - p y r r o l i d i n - 1 -
ylethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol hydrochlo-
ride (5, 1.86 g, 3.91 mmol), triethylamine (3.95 g, 39.1 mmol) in
dichloromethane (100 mL) under argon atmosphere was stirred for
10 min. Pivoloyl chloride (0.616 g, 5.09 mmol) was added dropwise
to the solution and stirred at room temperature for 2 h. A solution
of saturated sodium bicarbonate (25 mL) was added, and the organic
layer was separated, dried (Na2SO4), filtered, concentrated under
reduced pressure, and purified using preparative HPLC to afford
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]ami-
no}-1,2,4-benzotriazin-7-yl)phenyl 4-(2,6-Dimethylbenzoate) (18).
A solution of 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)-
phenyl]amino}-1,2,4-benzotriazin-7-yl)phenol (5, 0.39 g, 0.768
mmol), 2,6-dimethylbenzoyl chloride (0.168 g, 0.998 mmol), and
triethylamine (1.6 mL, 11.5 mmol) in dichloromethane (20 mL)
was stirred under argon atmosphere at room temperature for 12 h.
The reaction mixture was washed with saturated aqueous sodium
bicarbonate solution (20 mL), dried (Na2SO4), filtered, concentrated
under reduced pressure, and purified using flash chromatography
(SiO2, 10% methanol in dichloromethane) to afford the title
compound as a red solid (0.10 g, 21%). 1H NMR: δ 1.65–1.75 (m,
4H), 2.35–2.45 (m, 6H), 2.55–2.60 (m, 4H), 2.66 (s, 3H), 2.80–2.90
(br s, 2H), 4.09 (t, J ) 5.9 Hz, 2H), 7.01 (d, J ) 9.2 Hz, 2H), 7.19
(d, J ) 7.6 Hz, 2H), 7.34 (t, J ) 7.7 Hz, 1H), 7.47 (dd, J ) 3.0,
8.8 Hz, 1H), 7.59 (d, J ) 2.8 Hz, 1H), 7.77 (d, J ) 8.8 Hz, 1H),
7.90–8.01 (m, 3H), 8.23 (d, J ) 1.8 Hz, 1H), 10.86 (s, 1H). MS-
ESI+ m/z 609 (M + 1)+. HPLC purity 95% using ELSD.
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]ami-
no}-1,2,4-benzotriazin-7-yl)phenyl 4-(2,6-Dichlorobenzoate) (19).
A solution of 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)-
phenyl]amino}-1,2,4-benzotriazin-7-yl)phenol (5 0.25 g, 0.53 mmol),
2,6-dichlorobenzoyl chloride (0.144 g, 0.69 mmol), and triethy-
lamine (0.074 mL, 5.3 mmol) in dichloromethane (5 mL) was stirred
under argon atmosphere at room temperature for 24 h. The reaction
mixture was concentrated under reduced pressure and purified using
flash chromatography (SiO2, 10% methanol in dichloromethane) to
1
the title compound (2.14 g, 98% yield). H NMR: δ 1.31 (s, 9H),
1.67–1.69 (m, 4H), 2.51–2.53 (m, 4H), 2.64 (s, 3H), 2.80 (t, J )
5.7 Hz, 2H), 4.07 (t, J ) 5.9 Hz, 2H), 7.00 (d, J ) 7.1 Hz, 2H),
7.24 (dd, J ) 5.9, 8.8 Hz, 1H), 7.39 (d, J ) 2.8 Hz, 1H), 7.67 (d,
J ) 8.7 Hz, 1H), 7.85 (s, 1H), 7.92 (d, J ) 9.1 Hz, 2H), 8.18 (d,
J ) 1.8 Hz, 1H). MS-ESI+ m/z 560 (M + 1). Anal. (C31H34ClN5O3)
C, H, N. HPLC 99% pure at 220 nm and 99% pure using ELSD.
Disodium 4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-yl-ethox-
y)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl Phosphate (15). A
solution of 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-ylethoxy)-
phenylamino]benzo[1,2,4]triazin-7-yl}phenol (5, 0.51 g, 1.0 mmol),
phosphorus oxychloride (0.46 g, 3.0 mmol), and triethylamine (1.0
g, 10 mmol) in dichloromethane (100 mL) was stirred at room
temperature for 12 h. An aqueous sodium bicarbonate solution (0.5
g in 6 mL of water) was added to the reaction mixture and stirred
at room temperature for 30 min. The organic layer was separated,
dried (Na2SO4), filtered, and concentrated and the solid was washed
with water (20 mL), methanol (20 mL), and ether (20 mL) to afford
the title compound as a red solid (430 mg, 72%). 1H NMR: δ
1.86–1.88 (m, 4H), 2.88–3.01 (m, 4H), 3.17 (s, 3H), 3.34 (br s,
2H), 4.14–4.18 (m, 2H), 6.90 (d, J ) 8.9 Hz, 2H), 7.21 (dd, J )
2.8, 8.9 Hz, 1H), 7.31 (s, 1H), 7.43 (d, J ) 8.8 Hz, 1H), 7.70 (s,
1H), 7.78 (d, J ) 8.9 Hz, 2H), 7.96 (s, 1H), 10.65 (s, 1H). MS-
ESI+ m/z 556 (M + 1). Anal. (C26H25ClN5Na2O5P·H2O) C, H, N.
HPLC purity >98% at 220 nm.
1
afford the title compound as a red solid (0.30 g, 94%). H NMR: δ
1.55–1.65 (m, 4H), 2.52–2.58 (m, 4H), 2.65 (s, 3H), 2.75–2.85 (m,
2H), 4.08 (t, J ) 5.9 Hz, 2H), 7.01 (dd, J ) 2.0, 7.1 Hz, 2H), 7.45 (d,
J ) 11.4 Hz, 1H), 7.51 (d, J ) 2.8 Hz, 1H), 7.60–7.65 (m, 1H), 7.69
(s, 1H), 7.71 (d, J ) 1.6 Hz, 1H), 7.81 (d, J ) 8.8 Hz, 1H), 7.89 (dd,
J ) 0.9, 1.8 Hz, 1H), 7.94 (d, J ) 9.1 Hz, 2H), 8.23 (d, J ) 1.8 Hz,
1H), 10.86 (br s, 1H). MS-ESI+ m/z 650 (M + 3). HPLC purity, 98%
at 254 nm.
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]ami-
no}-1,2,4-benzotriazin-7-yl)phenyl 4-(Morpholin-4-ylmethyl)ben-
zoate (20). A solution of 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-
1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenol (5, 0.77 g,
1.5 mmol), 4-(morpholinomethyl)benzoic acid (1.06 g, 4.8 mmol),
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (2.36
g, 12.31 mmol), and 4-dimethylaminopyridine (0.225 g, 1.85 mmol)
in dimethylformamide (60 mL) was stirred under argon atmosphere
at room temperature for 12 h. The reaction mixture was concen-
trated, suspended in dichloromethane, and washed with saturated
sodium bicarbonate solution and brine. The organic layer was dried
(Na2SO4), filtered, concentrated under reduced pressure, and purified
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]ami-
no}-1,2,4-benzotriazin-7-yl)phenyl Glycinate (16). 4-Chloro-3-(5-
methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-ben-
zotriazin-7-yl)phenyl(N-butyloxycarbonyl) glycinate (28, 0.012 g,
0.019 mmol) was stirred in a 5% solution of trifluoroacetic acid in
dichloromethane for 1 h at room temperature. The reaction mixture
was concentrated and purified on preparative HPLC to afford the
title compound as the trifluoroacetate salt (0.008 g, 80%). 1H NMR:
δ 1.80–2.10 (m, 4H), 2.64 (s, 3H), 3.10–3.20 (m, 2H), 3.55–3.65
(m, 4H), 4.15 (br s, 2H), 4.31 (t, J ) 5.3 Hz, 2H), 7.10 (d, J ) 9.2
Hz, 2H), 7.34 (dd, J ) 2.9, 8.7 Hz, 1H), 7.46 (d, J ) 2.8 Hz, 1H),

DeVelopment of a Benzoate for Treatment of AMD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1555
(SiO2, 10% methanol in dichloromethane) to afford the title
compound (1.29 g, 97%). 1H NMR: δ 1.60–1.75 (m, 4H), 2.30–2.40
(m, 4H), 2.50–2.60 (m, 4H), 2.64 (s, 3H), 2.80 (t, J ) 5.8 Hz, 2H),
3.50–3.65 (m, 6H), 4.08 (t, J ) 5.8 Hz, 2H), 7.00 (d, J ) 9.1 Hz,
2H), 7.43 (dd, J ) 2.9, 8.8 Hz, 1H), 7.55 (d, J ) 8.5 Hz, 2H), 7.59
(d, J ) 2.9 Hz, 1H), 7.73 (d, J ) 8.7 Hz, 1H), 7.88 (d, J ) 0.95
Hz, 1H), 7.93 (d, J ) 9.1 Hz, 2H), 8.11 (d, J ) 6.6 Hz, 2H), 8.21
(d, J ) 1.9 Hz, 1H), 10.84 (br s, 1H). MS-ESI+ m/z 679 (M + 1).
HPLC 98% pure at 220 nm.
tert-Butyl 4-(4-Bromobenzoyl)piperazine-1-carboxylate (25c). A
solution of 4-bromobenzoyl chloride (1.0 g, 4.5 mmol), tert-
butylpiperazine 1-carboxylate (1.1 g, 5.9 mmol), and triethylamine
(1.5 mL, 11 mmol) in dichloromethane (15 mL) was stirred at room
temperature for 15 h. The reaction mixture was diluted with ethyl
acetate (100 mL), washed with saturated sodium bicarbonate
solution (2 × 25 mL) and brine (2 × 25 mL), dried (MgSO4),
filtered, and concentrated under reduced pressure to give a tan solid.
The solid was triturated in hexanes/diethyl ether (10:1 v/v) and
washed with diethyl ether (25 mL) to afford the title compound as
7-Bromo-5-methylbenzo[1,2,4]triazin-3-ylamine 1-Oxide (22). A
mixture of 4-bromo-2-methyl-6-nitroaniline (1 g, 4.33 mmol),
cyanamide (0.5 g, 12 mmol), and pyridine hydrochloride (5 g, 43
mmol) was heated at 100 °C for 12 h. The reaction mixture was
cooled, treated with 10% sodium hydroxide solution (4 mL, 10
mmol), and heated at 100 °C for 2 h. The reaction mixture was
cooled to room temperature and filtered. The solid was washed with
water (2 × 25 mL), acetone (2 × 5 mL), and diethyl ether (2 × 10
1
a white solid (1.6 g, 95%). H NMR: δ 1.40 (s, 9H), 3.20–3.60
(m, 8H), 7.37 (d, J ) 8.3 Hz, 2H), 7.65 (d, J ) 8.4 Hz, 2H). MS-
ESI+ m/z 393 (M + Na).
1-[3-(4-Bromophenyl)propyl]pyrrolidine (25e). A solution of
bromo-4-(3-bromopropyl)benzene (27, 3.5 g, 13 mmol), pyrrolidine
(2.1 mL, 25 mmol), and cesium carbonate (8.2 g, 25 mmol) in
dioxane (40 mL) was stirred at room temperature under argon for
15 h. The reaction mixture was poured onto water (200 mL) and
extracted with ethyl acetate (3 × 100 mL). The organic layer was
separated, washed with brine (2 × 50 mL), dried (Na2SO4), filtered,
concentrated under reduced pressure, and purified by flash chro-
matography (SiO2, 10-25% methanol in dichloromethane contain-
ing 2% triethylamine) to afford the title compound as a pale-orange
1
mL) to afford the title compound (0.4 g, 36%). H NMR: δ 2.45
(s, 3 H), 7.49 (s, 2H), 7.81 (s, 1 H), 8.11 (d, J ) 1.4 Hz, 1 H).
MS-ESI+ m/z 255 (M + 1).
7-Bromo-5-methyl-1,2,4-benzotriazin-3-amine (23). A solution
of 7-bromo-5-methylbenzo[1,2,4]triazin-3-ylamine 1-oxide (22,
4.26 g, 16.7 mmol) and Raney nickel (10% by weight) in ethanol
(160 mL) was stirred at room temperature under positive pressure
of hydrogen for 10 h. The reaction mixture was filtered, concen-
trated, and purified using flash chromatography (SiO2, 50% ethyl
acetate in hexanes) to yield the title compound as a solid (3.9 g,
1
oil (1.8 g, 53%). H NMR: δ 1.60–1.65 (m, 6H), 2.35 (t, J ) 7.3
Hz, 2H), 2.35–2.43 (m, 4H), 2.57 (t, J ) 7.7 Hz, 2H), 7.16 (d, J )
8.3 Hz, 2H), 7.44 (d, J ) 8.4 Hz, 2H). MS-ESI+ m/z 268 (M + 1).
7-(2-Chloro-5-methoxyphenyl)-5-methyl-N-[4-(2-pyrrolidin-1-
ylethoxy)phenyl]-1,2,4-benzotriazin-3-amine (26a). A solution of
7-(2-chloro-5-methoxyphenyl)-5-methyl-1,2,4-benzotrizine-3-
amine (24a, 500 mg, 1.66 mmol), N-(2-chloroethyl)pyrrolidine (674
mg, 2.49 mmol), cesium carbonate (2.16 g, 6.65 mmol), tris(diben-
zylideneacetone)dipalladium (152 mg, 0.17 mmol), and 4,5-
bis(diphenylphosphino)-9,9-dimethyxanthene (xantphos, 287 mg,
0.50 mmol) in 1,4-dioxane (20 mL) was heated at reflux for 4 h
under argon. The solid was filtered and washed with ethyl acetate
(100 mL). The filtrate was washed with brine (1 × 100 mL). The
organic solution was dried (Na2SO4), filtered, and concentrated
under reduced pressure to 5 mL. Hexanes (50 mL) were added to
the organic phase and the solid was filtered and purified by flash
chromatography (SiO2, using dichloromethane/methanol/28% aque-
ous ammonia, 100:10:2.5) to afford the title compound as a yellow
1
97%). H NMR: δ 2.48 (s, 3H), 7.79 (br s, 3H), 8.25 (d, J ) 1.8
Hz, 1H). MS-ESI+ m/z 239 (M + 1).
7-(2-Chloro-5-methoxyphenyl)-5-methyl-1,2,4-benzotrizine-3-
amine (24a). A solution of 7-bromo-5-methyl-1,2,4-benzotriazin-
3-amine (23, 8 g, 33.5 mmol), 2-chloro-5-methoxyphenylboronic
acid (9.3 g, 50.2 mmol), tetrakis(triphenylphosphine)palladium(0)
(3.9 g, 3.3 mmol), and Na2CO3 (14.2 g, 134 mmol) in DME/EtOH/
water (6:1:1, 335 mL) under argon was heated at 100 °C for 4 h.
The reaction mixture was cooled to room temperature, diluted with
dichloromethane (100 mL), filtered, and washed with water (3 ×
100 mL) and ether (3 × 100 mL) to afford the title compound as
a green solid (8.37 g, 84%). 1H NMR: δ 2.54 (s, 3H), 3.82 (s, 3H),
7.03 (dd, J ) 3.1, 8.8 Hz, 1H), 7.10 (d, J ) 3.1 Hz, 1H), 7.50 (d,
J ) 8.8 Hz, 1H), 7.72 (br s, 2H), 7.74 (s, 1H), 8.07 (d, J ) 1.8 Hz,
1H). MS-ESI+ m/z 301 (M + 1).
1
solid (440 mg, 54%). H NMR: δ 1.67–1.72 (m, 4H), 2.50–2.53
(m, 4H), 2.64 (s, 3H), 2.79 (t, J ) 11.8 Hz, 2H), 3.83 (s, 3H), 4.07
(t, J ) 5.9 Hz, 2H), 7.00 (d, J ) 9.1 Hz, 2H), 7.04 (dd, J ) 3.0,
4.4 Hz, 1H), 7.13 (d, J ) 3.0 Hz, 1H), 7.52 (d, J ) 8.9 Hz, 1H),
7.85 (d, J ) 0.7 Hz, 1H), 7.92 (d, J ) 9.0 Hz, 1H), 8.17 (d, J )
1.5 Hz, 1H), 10.81 (br s, 1H). MS-ESI+ m/z 490 (M + 1).
4-{[7-(2-Chloro-5-methoxyphenyl)-5-methyl-1,2,4-benzotriazin-
3-yl]amino}-N-(2-pyrrolidin-1-ylethyl)benzenesulfonamide (26b). A
solution of 7-(2-chloro-5-methoxyphenyl)-5-methyl-1,2,4-benzo-
trizine-3-amine (24a, 0.157 g, 0.523 mmol), 4-bromo-N-(2-pyrro-
lidin-1-ylethyl)benzenesulfonamide (0.261 g, 0.785 mmol), cesium
carbonate (0.512 g, 1.57 mmol), 4,5-bis(diphenylphosphino)-9,9-
dimethylxanthene (0.061 g, 0.10 mmol), and tris(dibenzylidene-
acetone)dipalladium (0.048 g, 0.052mmol) in dioxane (5 mL) was
flushed with argon and heated at reflux for 18 h. The hot reaction
mixture was filtered, diluted with ethyl acetate, and washed with
brine. The organic layer was dried (Na2SO4), filtered, and evapo-
rated to provide a solid. The solid was dissolved in dichloromethane
and precipitated with hexanes to afford a yellow powder (0.276 g,
95% yield). 1H NMR: δ 1.63–1.67 (m, 4H), 2.35–2.42 (br s, 4H),
2.47 (br s, 2H), 2.71 (s, 3H), 2.87 (t, J ) 6.9 Hz, 2H), 3.83 (s,
3H), 7.06 (dd, J ) 3.1, 8.9 Hz, 1H), 7.14 (d, J ) 3.1 Hz, 1H), 7.53
(d, J ) 8.9 Hz, 1H), 7.84 (d, J ) 5.1 Hz, 2H), 7.95 (d, J ) 0.9 Hz,
1H), 8.21 (d, J ) 5.2 Hz, 2H), 8.27 (d, J ) 1.8 Hz, 1H), 11.39 (s,
1H). MS-ESI+ m/z 553.3 (M + 1).
7-Phenyl-5-methyl-1,2,4-benzotrizine-3-amine (24b). The
title compound was obtained using the procedure described for 24a
in comparable yield. 1H NMR: δ 2.57 (s, 3H), 7.30–7.50 (m, 3H),
7.66 (br s, 2H), 7.80-7.90 (m, 2H), 8.06 (dd, J ) 1.1, 2.2 Hz,
1H), 8.29 (d, J ) 1.8 Hz, 1H). MS-ESI+ m/z 237 (M + 1).
7-(2-Chlorophenyl)-5-methyl-1,2,4-benzotrizine-3-amine (24c). A
solution of 7-bromo-5-methyl-1,2,4-benzotriazin-3-amine (1 g, 4.18
mmol), 2-chlorophenylboronic acid (0.98 g, 6.28 mmol), tetra-
kis(triphenylphosphine)palladium(0) (0.483 g, 0.42 mmol), and
sodium carbonate (1.77 g, 16.7 mmol) in dimethoxyethane/ethanol/
water (6:1:1, 32 mL) was heated at reflux for 12 h. The reaction
mixture was cooled, diluted with dichloromethane (30 mL), and
filtered. The solid was washed with water (25 mL) and ether (25
mL) and dried to afford the title compound (0.32 g 28%). 1H NMR:
δ 2.54 (s, 3H), 7.40–7.50 (m, 2H), 7.56 (dd, J ) 2.2, 7.1 Hz, 1H),
7.62 (dd, J ) 2.2, 7.0 Hz, 1H), 7.72 (br s, 2H), 7.44 (s, 1H), 8.06
(d, J ) 1.7 Hz, 1H). MS-ESI+ m/z 271 (M + 1).
7-(2,6-Dimethylphenyl)-5-methyl-1,2,4-benzotrizine-3-amine (24d).
The title compound was obtained using the procedure described
for 24c in comparable yield. 1H NMR: δ 2.03 (s, 6H), 2.53 (s,
3H), 7.10-7.20 (m 3H), 7.46 (s, 1H), 7.61 (br s, 2H), 7.78 (d, J )
1.9 Hz, 1H). MS-ESI+ m/z 265 (M + 1).
7-(2,6-Dichlorophenyl)-5-methyl-1,2,4-benzotrizine-3-amine (24e).
The title compound was obtained using the procedure described
tert-Butyl 4-(4-{[7-(2-Chloro-5-methoxyphenyl)-5-methyl-1,2,4-
benzotriazin-3-yl]amino}benzoyl)piperazine-1-carboxylate (26c). A
suspensionof7-(2-chloro-5-methoxyphenyl)-5-methylbenzo[1,2,4]triazin-
3-ylamine (0.10 g, 0.33 mmol), tert-butyl 4-(4-bromobenzoyl)-
piperazine-1-carboxylate (0.16 g, 0.44 mmol), palladium acetate
1
for 24c in comparable yield. H NMR (CDCl3): δ 2.53 (s, 3H),
7.49 (t, J ) 7.8 Hz, 1H), 7.56 (t, J ) 0.73 Hz, 1H), 7.63 (s, 1H),
7.65 (s, 1H), 7.77 (br s, 2H), 7.95 (d, J ) 1.9 Hz, 1H). MS-ESI+
m/z 305 (M + 1).

1556 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Palanki et al.
(4 mg, 0.018 mmol), xantphos (20 mg, 0.035 mmol), and potassium
tert-butoxide (0.10 g, 0.89 mmol) in xylene (3 mL) was heated in
a microwave reactor at 160 °C for 15 min. After cooling to room
temperature, the mixture was filtered and the solids were washed
with dichloromethane. The filtrate was concentrated and purified
using flash chromatography (SiO2, 40% ethyl acetate in hexanes)
solutions were kept at 37 °C. The homogenates were sampled at 0,
0.25, 1, 2, and 6 h. The reaction of one aliquot of the mixtures was
terminated at the above time points by addition of an equal volume
of cold acetonitrile (with 1 µM of the internal standard) and
centrifugation. The acetonitrile was collected and analyzed im-
mediately. Rate of hydrolysis of prodrugs was conducted by
measuring the conversion of prodrugs to 5 in extracts of ocular
tissues. Compounds 12, 14, 18, and 19 incubated in RIPA buffer
at 37 °C for 120 min were used as controls. The supernatants of
reaction mixtures were analyzed for prodrugs (12, 14, 18, and 19)
and 5. The samples were analyzed using API3000 liquid chroma-
tography equipped with an MS/MS (triple quadrupole) spectrometer,
Zorbax SB 75 mm × 2.1 mm, 3.5 µm column at 40 °C, using
water and acetonitrile as mobile phase, both containing 0.05%
trifluoroacetic acid. Hydrolysis rates were calculated using first-
order kinetics.
Structure Based Modeling Studies. While several crystal
structures of the unphosphorylated form of Src are available (PDB
codes 2src, 1fmk, 1y57, 2ptk), it is known that kinases in the Src
family undergo conformational change upon phosphorylation,
casting doubt on the relevance of these crystal forms to drug
discovery. We felt that the crystal structure of the inactive form of
Src is not useful for modeling studies. The fully activated catalytic
domain of human Src kinase was built on the basis of available
crystal structures of activated Lck. The catalytic domains of Src
and Lck exhibit 67% sequence identity over 273 residues without
gaps in the alignment with significantly higher homology exhibited
in the ATP binding site. The model was built using the interactive
programs InsightII and Homology from Accelrys. Compound 5 was
docked initially into the active site using an automated modeling
program BioDock (NIH SBIR Grant 1R43GM071055) and subse-
quently using interactive modeling in InsightII. Models with and
without ligands were subjected to energetic refinement including
solvent using the Accelerys program Discover. Visualization was
generated using the program BioInterpreter (NIH SBIR Grant
5R44GM061465). After comparison of available crystal structures
for VEGFr2 (1ywn, 1vr2, 1y6a, and 1y6b), the 1ywn crystal
structure was selected for modeling because of its closest resem-
blance to protein used in enzymatic assay. When 5 was docked
into the pocket, it exhibited a critical hydrogen bond from the
hydroxyphenyl of 5 to Glu885 on the RC-helix residue on the
protein.
In Vitro Src Kinase Inhibition Assay. The IC50 values for
compounds were determined using a luminescence-based kinase
assay with recombinant Src obtained from Invitrogen. In white,
flat-bottomed 96-well plates (Nunc) parallel assays were run at room
temperature at a final volume of 50 µL. Each well contained 40
µL of buffer consisting of 40 mM Tris buffer, pH 7.4, containing
50 mM MgCl2, 800 µM EDTA, 350 µM Triton X-100, 2 mM
ꢀ-mercaptoethanol, 250 µM peptide substrate (PTK2, Promega),
and an appropriate amount of Src (75–25 ng/well) such that the
assay was linear over 60 min. The final concentrations of
compounds for IC50 value determinations ranged from 1000 to 0.01
µM by adding the appropriate amount of compound in 2.5 µL of
DMSO; the DMSO present in each assay was constant at 5%. The
reaction was initiated by the addition of 10 µL of ATP to a final
assay concentration of 3 µM. After the reaction had proceeded for
60 min, 50 µL of Kinase-Glo reagent (Promega) was added to
terminate the reaction. This solution was then allowed to proceed
for an additional 10 min to maximize the luminescence reaction.
Values were then measured using an Ultra 384 instrument (Tecan)
set for luminosity measurements. Two control reactions were also
run: one reaction containing no compound and the second contain-
ing neither inhibitor nor peptide substrate. IC50 values were derived
from experimental data using the nonlinear curve fitting capabilities
of Prism (version 4, GraphPad Software).
1
to afford the title compound as an orange solid (45 mg, 23%). H
NMR: δ 1.42 (s, 9H), 2.70 (s, 3H), 3.35–3.45 (m, 4H), 3.45–3.55
(m, 4H), 3.84 (s, 3H), 7.06 (dd, J ) 2.9, 8.7 Hz, 1H), 7.15 (d, J )
2.9 Hz, 1H), 7.49 (d, J ) 8.7 Hz, 1H), 7.53 (d, J ) 8.7 Hz, 2H),
7.93 (t, J ) 1.0 Hz, 1H), 8.11 (d, J ) 8.6 Hz, 2H), 8.25 (d, J ) 1.7
Hz, 1H), 11.21 (s, 1H). MS-ESI+ m/z 589 (M + 1).
Benzyl 4-[(4-{[7-(2-Chloro-5-methoxyphenyl)-5-methyl-1,2-
4-benzotriazin-3-yl]amino}phenyl)sulfonyl]piperdine-1-carbo-
xylate (26d). A solution of 7-(2-chloro-5-methoxyphenyl)-5-
methylbenzo[1,2,4]triazin-3-ylamine (0.13 g, 0.44 mmol), benzyl
4-(4-bromobenzenesulfonyl)piperdine-1-carboxylate (0.232 g, 0.53
mmol), cesium carbonate (0.432 g, 1.33 mmol), 4,5-bis(diphe-
nylphosphino)-9,9-dimethylxanthane (0.051 g, 0.088 mmol), and
tris(dibenzylideneacetone)dipalladium (0.04 g, 0.044 mmol) in
dioxane (12 mL) was heated at reflux under argon for 18 h. The
reaction mixture was cooled and diluted with water (12 mL). The
solid was filtered and washed with water (20 mL) and ether (20
mL) to afford the title compound as a yellow solid (0.27 g, 93%
yield). MS-ESI+ m/z 658 (M + 1).
Bromo-4-(3-bromopropyl)benzene (27). Triphenylphosphine (6.3
g, 24 mmol) and then carbon tetrabromide (8.0 g, 24 mmol) were
added to a solution of bromo-4-(3-hydroxypropyl)benzene (4.0 g,
19 mmol) in tetrahydrofuran (30 mL) at 0 °C under an argon
atmosphere. The mixture was stirred at 0 °C for 15 min and then
at room temperature for an additional 15 h. The solvent was
removed under reduced pressure and purified by flash chromato-
graphy (SiO2, hexanes) to afford the title compound as a colorless
oil (3.5 g, 66%). 1 H NMR: δ 2.03–2.12 (m, 2H), 2.68 (t, J ) 7.5
Hz, 2H), 3.49 (t, J ) 6.5 Hz, 2H), 7.19 (d, J ) 8.3 Hz, 2H), 7.47
(d, J ) 8.3 Hz, 2H). MS-ESI+ m/z 279 (M + 1).
4-Chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]ami-
no}-1,2,4-benzotriazin-7-yl)phenyl-N-butyloxycarbonyl Glycinate
(28). A solution of 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-
ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenol (5, 0.25 g,
0.526 mmol), N-tert-butyloxycarbonylglycine (0.37 g, 2.11 mmol),
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1
g, 5.26 mmol), and 4-dimethylaminopyridine (65 mg, 0.526 mmol)
in dimethylformamide (25 mL) was stirred at room temperature
for 18 h. The reaction mixture was concentrated and purified by
flash chromatography (SiO2, 10% methanol in dichloromethane)
1
to afford the title compound (0.085 g, 26%). H NMR: δ 1.39 (s,
9H), 1.87–1.88 (m, 4H), 2.64 (s, 3H), 3.10–3.14 (m, 4H), 4.00 (d,
J ) 5.9 Hz, 2H), 4.29 (t, J ) 4.7 Hz, 2H), 7.06 (d, J ) 8.9 Hz,
2H), 7.26 (dd, J ) 2.8, 8.7 Hz, 1H), 7.39 (d, J ) 2.7 Hz, 1H), 7.46
(t, J ) 6.0 Hz, 1H), 7.70 (d, J ) 8.7 Hz, 1H), 7.85 (t, J ) 0.9 Hz,
1H), 7.96 (d, J ) 9.0 Hz, 2H), 8.18 (s, 1H), 10.89 (s, 1H). MS-
ESI+ m/z 633 (M + 1).
Chemical Stability Studies of Prodrugs 10–20. Chemical
stability of various prodrugs (10-20) was measured in a formulation
solution containing 0.5% of compounds and 0.5% HPMC/5%
dextrose at pH 6.7. The vials were closed and heated in an autoclave
at 120 °C for 1 h. The solutions were cooled to room temperature
and analyzed on Shimadzu HPLC systems equipped with LC-
10ATvp pumps, SIL-10ADvp autoinjector, SPD-M10Avp diode
array, an ELSD-LT detectors, and Agilent C-18 Zorbax 250 mm
× 4.6 mm column with 5 µm particle size. The analytical gradient
consisted of 15% acetonitrile in water ramping up to 90%
acetonitrile, both containing 0.05% trifluoroacetic acid.
Hydrolysis of Prodrugs in Rabbit Eye Homogenates. Two
eyeballs (6.37 g) from a Dutch-Belted rabbit were collected and
homogenized in 20 mL of 1× radioimmunoprecipitation (RIPA)
buffer (diluted from 10× RIPA buffer, Upstate Biotechnology, Inc.
Charlottesville, VA). Prodrugs 12, 14, 18, and 19 were dissolved
in dimethyl sulfoxide (1 mg/mL) and added to 3 mL of the
homogenate solution with a final concentration of 5 µg/mL. The
In Vitro VEGFr2 Kinase Inhibition Assay. The IC50 values
for compounds were determined using a luminescence-based kinase
assay with recombinant VEGFr2 obtained from Invitrogen. In white,
flat-bottomed 96-well plates (Nunc) parallel assays were run at room
temperature at a final volume of 50 µL. Each well contained 40

DeVelopment of a Benzoate for Treatment of AMD
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1557
µL of buffer consisting of 40 mM Tris buffer, pH 7.4, containing
50 mM MgCl2, 800 µM EDTA, 350 µM Triton X-100, 2 mM
ꢀ-mercaptoethanol, 500 µg/mL poly-E4Y (Sigma) for VEGFr2 and
an appropriate amount of VEGFr2 such that the assay was linear
over 60 min. The final concentrations of compounds for IC50 value
determinations ranged from 10 to 0.001 µM by adding the
appropriate amount of compound in 2.5 µL of DMSO; the DMSO
present in each assay was constant at 5%. The reaction was initiated
by the addition of 10 µL of ATP to a final assay concentration of
3 µM. After the reaction had proceeded for 60 min, 50 µL of
Kinase-Glo reagent (Promega) was added to terminate the reaction.
This solution was then allowed to proceed for an additional 10 min
to maximize the luminescence reaction. Values were then measured
using an Ultra 384 instrument (Tecan) set for luminosity measure-
ments. Two control reactions were also run: one reaction containing
no compound and the second containing neither inhibitor nor
peptide substrate. IC50 values were derived from experimental data
using the nonlinear curve fitting capabilities of Prism (version 4,
GraphPad Software).
In Vitro YES Kinase Inhibition Assay. The IC50 values for
compounds were determined using a luminescence-based kinase
assay with recombinant YES obtained from Invitrogen. In white,
flat-bottomed 96-well plates (Nunc) parallel assays were run at room
temperature at a final volume of 50 µL. Each well contained 40
µL of buffer consisting of 40 mM Tris buffer, pH 7.4, containing
50 mM MgCl2, 800 µM EDTA, 350 µM Triton X-100, 2 mM
ꢀ-mercaptoethanol, 250 µM peptide substrate (PTK2, Promega),
and an appropriate amount of YES (75–25 ng/well) such that the
assay was linear over 60 min. The final concentrations of
compounds for IC50 value determinations ranged from 1000 to 0.01
µM by adding the appropriate amount of compound in 2.5 µL of
DMSO; the DMSO present in each assay was constant at 5%. The
reaction was initiated by the addition of 10 µL of ATP to a final
assay concentration of 3 µM. After the reaction had proceeded for
60 min, 50 µL of Kinase-Glo reagent (Promega) was added to
terminate the reaction. This solution was then allowed to proceed
for an additional 10 min to maximize the luminescence reaction.
Values were then measured using an Ultra 384 instrument (Tecan)
set for luminosity measurements. Two control reactions were also
ran: one reaction containing no compound and the second containing
neither inhibitor nor peptide substrate. IC50 values were derived
from experimental data using the nonlinear curve fitting capabilities
of Prism (version 4, GraphPad Software).
In Vitro VEGF-Mediated Proliferation in Human Retinal
Microvascular Endothelial Cell Assay. For the cellular prolifera-
tion assays, human retinal microvascular endothelial cells (Cell
Systems; Kirkland, WA) were plated at a density of ∼1.5 × 103
cells/well in a 96-well plate (Corning; Corning, NY) and allowed
to adhere for approximately 5–6 h. Medium was then changed into
CSC-Maintenance Medium (Cell Systems; Kirkland, WA) and
incubated for approximately 48 h at 37 °C/5% CO2. As indicated
by the manufacturer, this specialized medium contains 10% FBS
but no growth factor and renders proliferating cells into a quiescent
state. The cells were then pretreated with varying concentrations
of 5 (5-0.00229 or 2-0.00195 µM) or DMSO (as a vehicle control)
prepared in basal CSC medium containing 10% FBS and 50 µg/
mL heparin for approximately 60 min at 37 °C/5% CO2. Human
recombinant VEGF (Peprotech; Rocky Hill, NJ) was then added
to a final concentration of 50 ng/mL, and cells were incubated for
48 h, at which time cell proliferation was quantified using the Cell
Proliferation Kit (Roche; Alameda, CA) as described by the
manufacturer. Briefly, for one 96-well plate, 100 µL of electron-
coupling reagent was added to 5 mL of XTT labeling solution. A
50 µL sample of this solution was then added to each well, and the
reaction was allowed to develop at 37 °C/5% CO2. The colored
formazan product that is generated by metabolically active cells
was measured spectrophotometrically using the SpectraMax spec-
trophotometer (Molecular Devices; Sunnyvale, CA) at 492 nm with
correction at 690 nm. IC50 values were determined using the
GraphPad Prism 4.0 software (San Diego, CA), with the corrected
absorbance values plotted on y-axis (linear scale) and logarithmic
values of concentration (µM) on the x-axis. Data were subjected
to a nonlinear regression fit analysis and the concentrations at which
endothelial cell proliferation was inhibited by 50% determined.
Pharmacokinetic Studies of 12 in Mouse Ocular Tissue
after a Single Topical Instillation. The 0.7% w/v formulation of
5 was prepared as follows. The hydrochloride salt of 5 (31.16 mg)
was mixed with PL90G liposome (970 mg) and dissolved in ethanol
(2 mL). The solution was evaporated to dryness under reduced
pressure and resuspended in water (2.7 g) containing 3% (w/v)
propylene glycol. The solution pH was adjusted to 6.1 with 1 N
NaOH, the mixture was homogenized using a sonicator probe
(model GE-130), the osmolality of the solution was adjusted to
355 mOsm, and the mixture was filtered through a 0.22 µm PVDF
syringe filter (Millipore). The 1.8% w/v formulations of 12 was
prepared as follows. Compound 12 (26.44) was mixed with PL90G
liposome (386.97 mg) and dissolved in 2 mL of ethanol. The
solution was evaporated to dryness under reduced pressure and
resuspended in water (923.55 mg) containing 3% (w/v) propylene
glycol. The pH was adjusted to 6.2 with 1 N HCl. The mixture
was homogenized using a sonicator probe (model GE-130), and
osmolality was adjusted to 327 mOsm and filtered through a 0.45
µm PVDF syringe filter (Millipore). Twenty male mice (C57BL/
6) were given one 10 µL drop of 0.7% of 5. Composite sampling
was employed to generate tissue concentration-time profiles for 5
over the following time course (N ) 4/time point): 0.5, 1, 3, 7,
and 24 h postinstillation. Both eyes were removed from each mouse
and dissected to obtain the cornea, retina, and eye cup (sclera and
choroid). Plasma samples were also obtained from each mouse at
the time of euthanasia. Ten male mice were given one 10 µL drop
of the solution of 12. Composite sampling was employed to generate
tissue concentration-time profiles for 12 and 5 over the following
time course (N ) 2/time point): 0.5, 1, 3, 7, and 24 h postinstillation.
Both eyes were removed from each mouse and dissected to obtain
the cornea, retina, and eye cup. Plasma samples were also obtained
from each mouse at the time of euthanasia. Individual eye tissue
samples were combined and weighed. The weighed tissues were
homogenized in 0.5 mL of RIPA buffer using FP120 FastPrep
(ThermoSavant) and extracted with 0.5 mL of acetonitrile (contain-
ing internal standard). The supernatants (800 µL) were dried and
reconstituted into 400 µL of 8:2 dimethyl sulfoxide (DMSO)/water
for analysis. Plasma samples were extracted by addition of a 2-fold
excess of acetonitrile containing internal standard followed by
centrifugation. The supernatants were isolated for analysis. Pro-
cessed plasma and ocular tissue samples were quantitated by LC/
MS/MS against externals calibration standards prepared in naive
mouse tissues. Matrix calibration standards and quality control (QC)
samples were prepared by adding stock solutions of 5 or 12 into a
series of aliquots of blank mouse plasma (for plasma analysis) or
blank mouse ocular tissue (for ocular tissue analysis). The final
concentrations ranged from 0.5 to100 ng/mL for plasma analyses
and from 0.125 to 500 ng/mL for ocular tissue analyses.
The LC/MS/MS system consisted of an Sciex API3000 triple
quadrupolar mass spectrometer (MDS Sciex), an Agilent 1100
HPLC system (Agilent Technologies, Inc.), and a CTC autosampler
(Leap Technologies). The LC separations were performed on a
Zorbax SB 75 mm × 2.1 mm, 3.5 µm reverse phase HPLC column
(Agilent Technologies, Inc.). The column temperature was kept at
40 °C. Mobile phase A consisted of 0.05% trifluoroacetic acid
(TFA) in water, and mobile phase B consisted of 0.05% TFA in
acetonitrile. The flow rate was kept constant at 0.25 mL/min.
Following a 30 µL sample injection, mobile B was held at 5% for
3 min followed by a linear increase to 70% mobile phase B over
5 min. The mass spectrometric detection of 12 and 5 was achieved
using electrospray ionization operating in positive ionization mode.
The molecular ion transitions of m/z: 476.18 f 98.2 and m/z: 580.17
f 98.2 were monitored in MRM mode for 5 and 12, respectively.
Pharmacokinetic parameters were estimated using WinNonlin
(version 4.1) based on mean concentrations for each time point
and individual tissue. The time to maximum concentration (Tmax
)
and the maximum concentration (Cmax) were determined on the basis
of measured concentrations. The area under the curve, AUClast, was

1558 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Palanki et al.
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Hypoxia-induced expression of vascular endothelial growth factor by
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calculated from mean concentration data using the linear trapezoidal
rule. No standard deviations are given, since the data represent the
combined exposure from only two to four eyes.
Laser-Induced Choroidal Neovascularization in Mice. Com-
pound 12 was prepared as a solution as described above. Laser-
photocoagulation-induced rupture of Bruch’s membrane was used
to generate CNV as previously described.43 Briefly, 4- to 5-week-
old female C57BL/6J mice were anesthetized with ketamine
hydrochloride (100 mg/kg body weight), and the pupils were dilated
with 1% tropicamide (Alcon Laboratories, Inc.; Forth Worth, TX).
Three burns of 532 nm diode laser photocoagulation (75 µm spot
size, 0.1 s duration, 120 mW) were delivered to each retina by
using the slit lamp delivery system of a photocoagulator (OcuLight,
Iridex; Mountain View, CA) and a hand-held coverslip as a contact
lens. Burns were performed in the 9, 12, and 3 o’clock positions
of the posterior pole of the retina. Production of a bubble at the
time of laser treatment, which indicates rupture of Bruch’s
membrane, is an important factor in obtaining CNV.43 Only burns
in which a bubble was produced were included in the study. Body
weights were monitored throughout the experiment, and the CNV
area was measured on day 14 as described below. For topical
administration, eight mice/group were given one 10 µLdrop of the
formulation vehicle, 0.61% w/v 12, or 1.83% w/v 12 three times a
day (t.i.d.), with each dose separated by 4 h and no treatment in
the fellow eye. One group of mice had a rupture to Bruch’s
membrane and no treatment to either eye. Two weeks after the
rupture of Bruch’s membrane, mice were anesthetized and perfused
with fluorescein-labeled dextran (2 × 106 average molecular weight,
Sigma-Aldrich; St. Louis, MO) and choroidal flatmounts were
prepared as previously described.43 Briefly, the eyes were removed
and fixed for 1 h in 10% phosphate-buffered formalin and the cornea
and lens were removed. The entire retina was carefully dissected
from the eye cup. Radial cuts were made from the edge of the eye
cup to the equator in all four quadrants and flat-mounted in aqueous
mounting medium (Aquamount, BDH; Poole, U.K.). Flat-mounts
were examined by fluorescence microscopy (Axioskop, Carl Zeiss
Meditec; Thornwood, NY), and images were digitized with a three
charge coupled device (CCD) color video camera (IK-TU40A,
Toshiba; Tokyo, Japan) and a frame grabber. Image-analysis
software (Image-Pro Plus, Media Cybernetics; Silver Spring, MD)
was used to measure the area of each CNV lesion. Statistical
comparisons were made using a linear mixed model.59 Probabilities
for comparison of treatments were adjusted for multiple compari-
sons by the Dunnett method.47
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(18) Small molecules targeting VEGFr pathway for the treatment of AMD
are under development at Pfizer. Beals, C. R.; Day, W. W.; Khalil,
D. A.; Klamerus, K. J.; Raber, S. R.; Scassellati-Sorzolini, B.; Zwillich,
S. H. Compositions Comprising Indazole Compound for Sub-Tenon
Delivery. WO 2006117666, April 2006.
(19) Orally delivered vatalanib is being evaluated in a phase II clinical
trial along with verteporfin in patients with subfoveal choroidal
neovascularization (CNV) secondary to age-related macular degeneration.
http://www.clinicaltrials.gov/ct/gui/show/NCT00138632.
Acknowledgment. The authors thank Barbara Robinson and
Linda Hwang for valuable discussions and technical assistance.
Supporting Information Available: Elemental analysis results
and details of the effects of 5 on target kinases and topically applied
5 and 12. This material is available free of charge via the Internet
at http://pubs.acs.org.
(20) Several devices are in development to deliver drugs to the back of
the eye. Sultana, Y; Jain, R.; Aqil, M.; Ali, A. Curr. Drug DeliVery
2006, 3, 207–217.
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