Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2

Article abstract of DOI:10.1016/j.bmc.2008.11.049

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-

Full text of DOI:10.1016/j.bmc.2008.11.049

Bioorganic & Medicinal Chemistry 17 (2009) 731–740
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2
,
*
Matthew A. J. Duncton , Eugene L. Piatnitski Chekler , Reeti Katoch-Rouse , Dan Sherman ,
Wai C. Wong , Leon M. Smith II , Joel K. Kawakami , Alexander S. Kiselyov , Daniel L. Milligan ,
Chris Balagtas , Yaron R. Hadari , Ying Wang, Sheetal N. Patel, Robin L. Rolster , James R. Tonra,
David Surguladze, Stan Mitelman , Paul Kussie, Peter Bohlen , Jacqueline F. Doody
ImClone Systems, Inc., 180 Varick Street, New York, NY 10014, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II
(VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting mate-
rials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (compa-
rable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and
showed excellent exposure when dosed orally to female CD-1 mice.
Received 2 September 2008
Revised 15 November 2008
Accepted 18 November 2008
Available online 24 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Arylphthalazine
Phthalazine
Inhibitor
VEGFR-2
KDR
Kinase
Angiogenesis
1. Introduction
effects compared to commonly used proliferation inhibitors that
can also target dividing cells in normal tissues. The popularity of
Tumor-induced angiogenesis is the term that describes the
induction of blood vessels from existing vasculature to the site of
a solid tumor, critical for tumor growth and dissemination.1,2 Un-
der hypoxic conditions, tumors induce angiogenesis by secreting
vascular endothelial growth factor (VEGF). Endothelial cells lining
the walls of neighboring blood vessels bind VEGF to cell surface
VEGF Receptor 2 (also known as KDR; murine version known as
Flk-1), triggering a signaling cascade which leads to endothelial
cell proliferation, migration and increasing vascular permeability
in the tumor milieu.3,4 A positive feedback loop develops where
growing tumors secrete VEGF, which in turn leads to higher levels
of angiogenesis, allowing further tumor growth. Severing the feed-
back loop by inhibiting VEGF Receptor 2 (VEGFR-2) signaling
would restrict blood vessel development to the tumor. Moreover,
inhibiting blood vessel formation has the advantage of targeting
a stable endothelial cell, rather than a genetically unstable tumor
cell, which may become resistant to small-molecule therapies.
Additionally, angiogenesis in the adult is restricted to wound heal-
ing, menstruation and ovulation, predicting diminished side-
this approach is seen by the numerous VEGFR-2 small-molecule
inhibitors in clinical use, or in clinical trials, and the use of biolo-
gics in the clinical setting.5–9 VEGFR-2 is a membrane-bound tyro-
sine kinase receptor requiring ligand binding and dimerization for
transphosphorylation of the receptors and subsequent sub-
strates.10 Small-molecule inhibitors have been developed to the
adenosine-tri-phosphate (ATP) binding pocket of the kinase region,
retarding phosphorylation of downstream elements, resulting in
attenuation of VEGF signaling. Of VEGFR-2 inhibitors, the most ad-
vanced are SU11248 1 (SutentÒ), which has been approved by the
Federal Drug Administration (FDA) for treatment of gastrointesti-
nal stromal tumor and advanced renal cell carcinoma,11 BAY 43-
9006 2 (NexavarÒ),12 which is approved for renal cell carcinoma
and unresectable hepatocellular carcinoma, PTK-787 3 (valata-
nib)13 and ZD-6474 4 (ZactimaÒ),14 both of which are currently
in late-phase clinical trials. In addition, a plethora of other small-
molecules, exemplified by compounds 5–19 have been detailed
as potent inhibitors of VEGFR-2 (Fig. 1).15–29
Our own investigations into inhibitors of VEGFR-2 have yielded
a series of arylphthalzines having good in vitro potency against
isolated VEGFR-2 enzyme (compound 20 and series 21).30,31 In this
account we detail further results for this series of molecules,
and show that they possess favorable cellular potency and
* Corresponding author.
E-mail address: mattduncton@yahoo.com (M.A.J. Duncton).
No longer an employee of ImClone Systems, Inc.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.049

732
M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
pharmacokinetic profiles which would be suitable for their further
development as drug candidates.
Ar1
Ar1
Cl
HN
HN
i or ii
iii
N
N
N
Cl
N
N
Ar2
N
2. Chemistry
Cl
22
23a-j
21
The synthesis of the final 4-anilino-1-arylphthalazine deriva-
tives was readily accomplished in two, operationally-simple
steps, from commercially available 1,4-dichlorophthalazine
(Scheme 1). Thus, 1,4-dichlorophthalazine 22 was first reacted
with an aniline to provide the mono-displaced adducts 23a–j.
Suzuki coupling of these 4-anilino-1-chlorophthalazine interme-
diates was realized using K2CO3 as base, in a mixture of 1,4-
dioxane and water as solvent under microwave heating, to yield
compounds of general structure 21. This reaction usually pro-
ceeded in 10 min to 1 h of heating at 100–110 °C. Interestingly,
when the Suzuki reaction was attempted using identical condi-
tions, except employing conventional oil-bath heating instead
of a microwave-based approach, the reaction took substantially
more time, and proceeded in significantly lower yields. The gen-
eral protocol of mono-displacement with an amine, and Suzuki
Scheme 1. Two-step preparation of final compounds. Reagents and conditions: (i)
Ar1NH2, Et3N, n-BuOH, 100 °C. (ii) Ar1NH2, EtOH, reflux. (iii) Pd(Ph3P)2Cl2, K2CO3,
Ar2B(OH)2, H2O, 1,4-dioxane, 100–110 °C, microwave irradiation.
reaction of 1,4-dichlorophthalazine, to prepare unsymmetrical
1,4-disubstituted phthalazines as detailed in Scheme 1, has also
been accomplished in a previous study of 5-HT3 inhibitors.32
Compounds 24, 43, 44, 54, 56 to 65 and 67 to 68 were synthe-
sized using the procedure detailed in Scheme 1. An alternative
protocol, in which the order of Suzuki coupling and amine dis-
placement was reversed, was employed for the synthesis of com-
pound 66 (see Section 6). All other compounds, although
designed by ourselves, were provided by contract research
organizations.33
CF3
Cl
Cl
HN
H
O
HN
N
Br
O
HN
NEt2
N
N
HN
N
N
O
O
F
O
N
N
F
H
O
O
N
H
NH
N
N
2 BAY 43-900612
3 PTK-78713
4 ZD-647414
1 SU1124811
OCF3
H
N
N
HN
O
NH
HN
H
H
N
O
O
O
N
S
N
O
F
N
N
NH
N
N
H
N
O
H2N
N
N
F
N
5 ZD-217115
6 OSI 93016
7 AMG 70617
8 CHIR-25818
N
N
O
H
HN Ph
HN
HN
N
N
N
N
NH2
O
F
N
N
N
F
H
N
N
HO
O
H2NO2S
N
H
N
N
HN
N
N
9 GW78603419
10 ABT-86920
11 AEE78821
12 BMS-54021522
H
H
N
O
N
O
NH
F
F
F
O
O
O
H2N
N
N
S
N
O
N
H
N
N
HN
F
N
S
H
O
Br
3
N
N
N
N
HN
O
H
N
N
S
14 CP-547,63224
13 BMS-60554123
1525
16 CEP-705526
O
CN
N
S
F
F
O
O
O
Cl
O
N
HN
N
O
HN
O
N
O
N
N
O
HN
Ar1
HN
Cl
O
NH
N
N
Ar2
NH
N
O
O
NH
N
N
H
1929
N
CONH2
17 YM-35944527
18 KRN95128
20 IM-02391130
2130,31
Figure 1. Select literature VEGFR-2 inhibitors.

M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
733
pounds. Chloro, methoxy, methyl and carbomethoxy substituents
were also tolerated at the 4-position (entries 3–7), although the
activity of these compounds was significantly less than parent
compound 20. Benzyloxy, phenoxy, sulfonamide or amide substit-
uents gave rise to inactive compounds (entries 8–11). Movement of
favorable substituents from the 4-position to the 3-position of the
phenyl ring usually resulted in a moderate decrease in enzymatic
activity (entries 12–14); similar results were observed when the
3,4-dichloro analogue 38 was tested (entry 16). The importance
of substitution on the phenyl ring was confirmed by removing
the 4-(chlorodifluoromethoxy) substituent in 20 to give an unsub-
stituted analogue 39 (entry 17), which was observed to be totally
inactive. Cellular potencies for the compounds are also displayed
in Table 1. Although some molecules exhibited good cellular activ-
ity (e.g., entries 2, 13 and 14), most cellular potencies had dimin-
ished activity relative to that seen in the isolated enzymatic
assay. The reason behind this reduction in potency was not inves-
tigated, although a number of explanations could be offered (e.g.,
cell penetration). We also believed that low solubility of the
compounds was hindering a reliable measurement of cellular
activity.
3. Biology
Compounds were evaluated against isolated VEGFR-2 kinase in
an enzymatic assay to assess their ability to inhibit tyrosine
phosphorylation. Inhibition was determined by measuring the
phosphorylation level of biotinylated poly-Glu-Ala-Tyr peptide
(pGAT-biotin, CIS Bio International) upon exposure to VEGFR-2
and 2 lM ATP at room temperature for 45 min. Addition of a
Europium-cryptate labeled anti-phosphotyrosine antibody and
measurement of the resulting homogenous time-resolved fluores-
cence (HTRF) allowed quantification of inhibition upon exposure to
various compound concentrations. IC50 values were determined
from logarithmic concentration-inhibition curves (at least eight
points) and reported as means of at least two separate experi-
ments. A literature VEGFR-2 inhibitor, ZD-6474 4, was included
in each set of experiments to ensure quality control. Select com-
pounds were then evaluated in a cellular assay against a chimeric
construct containing the extracellular portion of FGFR-1, and the
intracellular (kinase) portion of VEGFR-2, transiently expressed in
human embryonic kidney (HEK 293) cells. Measurement of tyro-
sine phosphorylation of the chimeric receptor by an enzyme-linked
immunosorbent assay (ELISA) provided quantification of ED50 val-
ues. The ability of compounds to block native human VEGFR-2 sig-
naling was assessed by evaluation in a mitogenic assay employing
human umbilical vein endothelial cells (HUVEC), and measure-
ment of 3H-thymidine incorporation after stimulation with VEGF.
Finally, preliminary pharmacokinetic experiments were performed
by quantifying the exposure of compound when dosed via oral (per
os; po), or intra-peritoneal (ip) routes to female CD-1 mice.
The importance of an anilino nitrogen was confirmed by
synthesis of oxygen and sulfur-linked analogues (Table 2, entries
1 and 2). Other non-anilino compounds were also inactive (results
not shown).
4.2. SAR around the 4-aryl group
Next, our attention focused on investigating SAR around the
amide group (Table 3). Removal of the amide in lead structure 20
totally abolished activity at VEGFR-2 (entry 2). Next, alkylation of
the amide was investigated. Small alkyl groups such as methyl,
or hydroxyethyl, gave rise to active compounds (entries 3–5),
whereas bulkier substituents such as iso-propyl, or tert-butyl, to-
tally abolished activity (entries 6 and 7). A morpholinyl amide
was also inactive (entry 8). Attempts to replace the amide were
mostly unsuccessful. For instance, methyl, chloro, or methoxy sub-
stituents gave rise to inactive compounds. However, an ester group
at the 4-position of the aryl ring was tolerated (entry 12), with an
4. Results and discussion
Screening of our corporate compound collection afforded the
arylphthalazine IM-023911 20 as a hit against VEGFR-2, with
an enzymatic IC50 of 0.19 lM, and an ED50 of 1.6 lM in a cellular
assay of VEGFR-2 inhibition. Although this compared somewhat
unfavorably to the performance of ZD-6474 4 in these assays
(IC50 = 45 nM; ED50 = 0.15 lM), it was reasoned that 20 provided
a reasonable starting point for a medicinal chemistry program,
and that further exploration of the arylphthalazine class of com-
pounds was warranted. Since we were aware of the work of
other researchers who had demonstrated that compounds based
around a phthalazine core could yield molecules suitable for clin-
ical development,13,34 we were confident that 20 could be opti-
mized rapidly to afford similarly potent and drug-like
compounds. Thus, we began an effort to explore the structure–
activity relationships, both around the 1-anilino group, and also
the 4-aryl moiety.
enzymatic IC50 = 0.63
lM, which suggested that a carbonyl group
might be important for biological activity. Additionally, it was
found that the amide could be moved from the 4-position to the
3-position of the aryl ring, to give compounds with acceptable
enzymatic and cellular potencies (entries 13 and 14). Although
these preliminary experiments suggested that compounds con-
taining other amides might yield interesting inhibitors of VEGFR-
2, it became apparent that molecules containing amides had severe
limitations in terms of their aqueous solubility, which would likely
make their further development challenging.36
4.1. SAR around the 1-anilino group
4.3. Isoquinolin-5-yl derivatives
Preliminary work began by investigating the structure–activity
relationships about the 1-anilino group in the amide series. Com-
pound 20 is somewhat unique since it possesses a 4-(chlorodiflu-
oromethoxy) group attached to an anilino phenyl ring. The use of
such a group has not been detailed extensively in the medicinal
chemistry literature, although its application in agrochemicals
seems to be more advanced.35 Kinetic studies indicated that com-
pound 20 was a reversible inhibitor with no apparent covalent
modification of the 4-(chlorodifluoromethoxy) substituent. Thus,
we began a search for complementary analogues to the OCF2Cl
substituent. As can be seen from Table 1 a number of compounds
exhibited good potency against isolated VEGFR-2 enzyme. Of note
were compounds that contained a hydrophobic substituent at the
4-position of the anilino phenyl ring. For example, the tert-butyl
and bromo analogues (entries 2 and 3) gave the most active com-
The activity observed with compound 54 prompted a more
adventurous investigation to find alternative aryl groups that were
potent VEGFR-2 inhibitors (Fig. 2). As the majority of arylphthala-
zines possessing an amide group appeared to be poorly soluble in
aqueous media, first thoughts entertained the possibility of reduc-
ing the amide bond, to give rise to a structure such as 55 which
would incorporate a free amine, and thus allow for salt formation
in order to improve solubility. It was also reasoned that fusing the
phenyl ring to the basic nitrogen in 55 might be beneficial as a con-
formational constraint, and this led to the idea that an isoquinolin-
5-yl group might fit our needs in terms of aiding solubility, whilst
minimizing conformational flexibility. As such, molecules such as
56 were designed and the similarity to PTK-787 3 was noticed
immediately. Testing of isoquinolin-5-yl compounds yielded

734
M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
Table 1
Table 3
Structure–activity relationships around 1-anilino group for lead structure 20
Initial structure–activity relationships around 4-aryl group in lead structure 20
R1
R1
R2
N N
N N
HN
HN
CONH2
Entry
Compound
R1
IC50
(l
M) VEGFR-2a
ED50
(l
M) VEGFR-2b
Entry
Compound
R1
R2
IC50
(l
M)a
ED50 (l
M)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
20
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
4-OCF2Cl
4-tBu
4-Br
4-Cl
4-OMe
4-Me
4-CO2Me
4-OBn
4-OPh
4-SO2NHMe
4-CONH2
3-tBu
3-Br
3-Cl
3-Me
3,4-Cl2
H
0.19
0.078
0.1
1.3
2.0
1.6
0.21
3.9
1.3
>10
5.0
>10
—
—
—
—
1.2
0.25
0.37
—
—
—
1
2
3
4
5
6
7
8
9
10
11
12
13
14
20
42
43
44
45
46
47
48
49
50
51
52
53
54
4-OCF2Cl
4-OCF2Cl
4-Cl
4-CONH2
H
0.19
>10
3.1
0.13
1.1
>10
>10
4.9
>10
>10
>10
0.63
4.1
1.6
—
—
0.27
—
—
—
—
—
—
4-CONHMe
4-CONHMe
4-CONH(CH2)2OH
4-CONHiPr
4-CONHtBu
4-CON(CH2)4O
4-Me
3-Cl
4-OMe
4-CO2Me
3-CONH2
3-CONH2
4-tBu
4-OCF2Cl
4-OCF2Cl
4-OCF2Cl
4-OCF2Cl
4-OCF2Cl
4-OCF2Cl
4-OCF2Cl
4-OCF2Cl
4-OCF2Cl
4-tBu
1.8
2.7
>10
>10
>10
>10
0.47
1.2
1.7
1.6
0.84
>10
—
—
—
0.90
0.40
a
Determined against isolated VEGFR-2 enzyme for at least two independent
points per experiment). Positive control, ZD-6474 4
experiments (at least
IC50 = 0.045
8
lM
a
b
Determined against isolated enzyme for at least two independent experiments
M.
At least two independent determinations against FGFR-1/VEGFR-2 chimeric
receptor expressed in HEK-293 cells. Positive control, ZD-6474 4 ED50 = 0.15 M.
At least two independent determinations against FGFR-1/VEGFR-2 chimeric
(at least 8 points per experiment). Positive control, ZD-6474 4 IC50 = 0.045
l
receptor expressed in HEK-293 cells. Positive control, ZD-6474 4 ED50 = 0.15 M.
l
b
l
tries 9 and 10), which showed contrasting activities (enzymatic
and cellular potency >10 M for 64 cf. enzymatic and cellular
activities of 0.27 M and 16 nM for 65). The position of the nitro-
gen was also of crucial importance for VEGFR-2 activity (Table 5),
with the corresponding quinolin-5-yl congeners showing dimin-
ished activity.
As mentioned previously, compounds in the amide series did
not seem to be particularly soluble in aqueous media.36 Conversely,
57, which was emerging as the most favored compound within the
isoquinolin-5-yl sub-series, was prepared as a dihydrochloride salt
by reaction with excess hydrogen chloride in 1,4-dioxane, to give
rise to material with acceptable water solubility (ca. 1 mg/mL in
deionized water).
l
impressive enzymatic and cellular potencies in a number of in-
stances (Table 4). Similar to the amide series, the most favorable
isoquinolin-5-yl analogues were those that possessed a hydropho-
bic group at the 4-position of the anilino moiety. As such, the tert-
butyl, chloro, (chlorodifluoromethoxy), and iso-propyl compounds
gave rise to very active analogues with excellent inhibition against
both isolated enzyme and a chimeric FGFR-1/VEGFR-2 receptor
(entries 1–4). Indeed, IM-094482 57 (entry 2), with an enzymatic
IC50 of 48 nM and a cellular ED50 of 10 nM, performed comparably
to the clinical compound ZD-6474 4 in these assays (IC50 = 45 nM;
l
ED50 = 0.15 lM). Interestingly, a hydrophilic morpholin-4-yl sub-
stituent gave rise to a moderately active compound when placed
at the 4-position of the anilino phenyl ring (entry 5). Importantly,
it was also observed that compounds in the isoquinolin-5-yl series
seemed to show improved toleration of other substitution patterns
when compared with their amide counterparts. For example, the
3,4-dichloro analogue 61 (entry 6) showed good enzymatic and
4.4. Cross-screening
A number of compounds deemed to be of interest for further
studies were cross-screened against a small panel of kinases,
including the related receptor tyrosine kinases, VEGFR-1 and VEG-
FR-3. In addition, molecules were screened against c-Met, EGFR,
IGF-1R, IR and FGF-R1. The results for this limited cross-screen
indicated that compounds seemed to have significant inhibition
against VEGFR-1, in addition to their inhibition of VEGFR-2 (Table
6). These results are in accordance with other studies on the inhi-
bition of VEGF receptor tyrosine kinases with anilinophthalazine
derivatives, such as PTK-787 3.34 Additionally, compound 57 was
tested against a wide panel of common receptors and transporters,
with activity being observed against the dopamine transporter,
adenosine receptor and histamine H2 receptor at a screening con-
cellular potency (0.11 lM and 70 nM, respectively). Additionally,
the 4-chloro-3-trifluoromethyl and 2-fluoro-4-methyl compounds
also displayed reasonable activities (entries 7 and 8). Nevertheless,
compounds in the isoquinolin-5-yl series could be sensitive to the
substitution pattern around the 4-anilino phenyl ring. This was
neatly illustrated for the 2- and 3-methyl analogues 64 and 65 (en-
Table 2
Removal of anilino-NH
F3CO
centration of 10 l
M.37 In contrast to PTK-787, 57 did not inhibit
the 3A4 isoform of cytochrome P450 (results not shown). However,
some inhibition at the human i-hERG potassium channel was ob-
N N
X
CONH2
served with 57 (ED50
3 lM).
Entry
Compound
X
IC50 (l
M) VEGFR-2a
4.5. Activity in a HUVEC mitogenic assay
1
2
40
41
O
S
>10
>10
The ability of phthalazine compounds to inhibit VEGF-stimu-
lated proliferation in human umbilical vein endothelial cells (HU-
VEC) was also measured. Unlike the cellular assay, which uses a
a
Determined against isolated enzyme for at least two independent experiments
M.
(at least 8 points per experiment). Positive control, ZD-6474 4 IC50 = 0.045
l

M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
735
following dosing to female CD-1 mice. The dosing vehicles were a
DMSO-solution, 5% phosal solution and an ethanol:Tween 80:pho-
sal (ETP) mixture. Compounds were dosed via the intraperotineal
(ip) or oral (po) routes, and plasma was analyzed at the 1-h and
4-h timepoints. Initial results are shown in Figure 3. Compound
24 showed high exposure with all dosing solutions, when given
by both ip and po routes. However, at 4-h post-dose compound
levels had dropped significantly. This is not an unexpected result,
as 24 contains a tert-butyl group which is potentially susceptible
to metabolic oxidation. Conversely, compound 57 showed excel-
lent exposure at both 1-h and 4-h timepoints. Of significance,
was its oral exposure upon dosing in a 5% phosal solution (1 h
HN
HN
HN
N
N
N
N
N
N
reduce
amide
cyclize
NH2
N
NH2
54
55
56
O
Figure 2. Modification of amide structures to produce isoquinolin-5-yl-
phthalazines.
chimeric construct to assess potency, this functional experiment
provides a measure of the compounds ability to block native
VEGF-receptors in human tissue. Compounds 24 (IM-094261)
and 57 (IM-094882) inhibited the growth of HUVECs with an
22.7 2.6 lM; 4 h 18.8 6.1 lM). ZD-2171 5 was also included in
these exposure studies and showed good exposure, although plas-
ma levels were lower than for compound 57. In order to gain a fur-
ther understanding of the oral exposure with the lead phthalazine
57, a 24-h experiment was undertaken in which 57 was dosed at
30 mg/kg in a 5% phosal mixture (target concentration ca. 3 mg/
mL). Again ZD-2171 5 was included as a comparison. As can be
seen in Figure 4, 57 showed a rapid absorption, and high exposure
ED50 of 0.25 and 0.21
these compounds were approximately equipotent with ZD-6474
4 (ED50 = 0.28 M), but somewhat less potent than the more recent
lM, respectively (Table 7). In our hands,
l
VEGFR-2 inhibitor, ZD-2171 5 (ED50 = 0.012 lM).
in plasma (Cmax 25.9
dose. Total compound exposure (0–24 h AUC) was also high at
891 mol h.
The pharmacokinetic data for 57 appeared to be comparable to
those for the related phthalazine, PTK-787 3,34 which is currently
in late-stage clinical trials. Since 57 also shows impressive enzy-
matic, cellular and functional (HUVEC) inhibition of VEGFR-2, the
combined data support the continued evaluation of this compound
as a potential anti-angiogenic.
lM), that was sustained until about 8 h post-
4.6. Exposure studies in mice
l
Compounds 24 (mono-hydrochloride salt) and 57 (dihydrochlo-
ride salt) were selected for investigation of their systemic exposure
Table 4
Structure–activity relationships for isoquinolin-5-yl phthalamide series
R1
N
5. Conclusion
N N
HN
In conclusion, this paper details a two-step synthesis of potent
inhibitors of vascular endothelial growth factor receptor II (VEGFR-
2), based around an arylphthalazine chemotype. Arylphthalazines
containing an isoquinolin-5-yl group were particularly effective
inhibitors of the VEGFR-2 kinase in enzymatic, cellular and mito-
genic assays. Additionally, the lead compound from this series,
57 (IM-094482), was found to have high exposure in plasma when
dosed orally to female CD-1 mice. The results detailed in this pub-
lication support the continued exploration of 57 as a research tool,
and reinforce the attractiveness of phthalazine-containing com-
pounds as kinase inhibitors.
Entry
Compound
R1
IC50
(
l
M)
ED50 (lM)
VEGFR-2a
VEGFR-2b
1
2
3
4
5
6
7
8
9
56
57
58
59
60
61
62
63
64
65
66
4-tBu
4-Cl
0.075
0.048
0.079
0.017
0.39
0.11
0.12
0.43
>10
—
0.01
0.035
0.024
0.88
0.07
0.28
0.35
>10
4-OCF2Cl
4-iPr
4-Morpholin-1-yl
3,4-Cl2
4-Cl, 3-CF3
2-F, 4-Me
2-Me
10
11
3-Me
2-Me-indol-5-yl
0.27
0.23
0.016
1.4
6. Experimental
a
Determined against isolated enzyme for at least two independent experiments
M.
At least two independent determinations against FGFR-1/VEGFR-2 chimeric
6.1. Enzymatic assay for determining inhibition of isolated
VEGFR-2
(at least 8 points per experiment). Positive control, ZD-6474 4 IC50 = 0.045
l
b
receptor expressed in HEK-293 cells.
Into a black 96-well Costar plate is added 2
pound in DMSO (final concentration in the 50 L kinase reaction
is typically 1–10 M). Next, 38 L of reaction buffer (25 mM
Hepes, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 2 mM DTT, and 1 mg/
mL BSA) containing 0.5 mmol pGAT-biotin and 3–4 ng VEGFR-2
enzyme is added to each well. After 5–10 min preincubation,
lL/well of com-
l
Table 5
l
l
Activity of quinolin-5-yl congeners
R
N
N N
HN
the kinase reaction is initiated by addition of 10 lL of 10 lM
ATP to reaction buffer, after which the plate is incubated at room
temperature for 45 min. The reaction is stopped by the addition
of 50
BSA) containing 100 mM EDTA and 0.36
tate labeled anti-phosphotyrosine antibody, CAS Bio Interna-
tional). After 30 min, 100 L, of 10 nM SV-XL (modified APC-
labeled Streptavidin, CIS Bio International) in KF buffer is added,
and after an additional 2 h incubation at room temperature, the
plate is read in a RUBYstar HTRF Reader.
l
L KF buffer (50 mM Hepes, pH 7.5, 0.5 M KF, 1 mg/mL
Entry
Compound
R
IC50
(
l
M) VEGFR-2a
ED50 (l
M) VEGFR-2b
lg/mL PY20 K (Eu-cryp-
1
2
67
68
Cl
tBu
>10
2.0
>10
2.4
l
a
Determined against isolated enzyme for at least two independent experiments
M.
At least two independent determinations against FGFR-1/VEGFR-2 chimeric
receptor expressed in HEK-293 cells. Positive control, ZD-6474 4 ED50 = 0.15 M.
(at least 8 points per experiment). Positive control, ZD-6474 4 IC50 = 0.045
l
b
l

736
M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
Table 6
Cross-screening of IM-094882 57
Entry
Compound
VEGFR-1a
91
VEGFR-3a
33
c-Meta
2
EGFRa
13
FGFR-1a
38
IGF-1Ra
25
IRa
10
DTb,c
77
Adenosineb
65
H2b
54
i-hERGd
1
57
3.0
a
Percentage inhibition at a drug screening concentration of 10
lM (average of at least two independent determinations). Inhibition of less than 40% at 10
lM is considered
to be inactive in our hands.
b
Percentage inhibition at a drug screening concentration of 10
considered to be of significance.
lM (average of at least two independent determinations). Inhibition greater than 50% at 10 lM is
c
DT, dopamine transporter.
ED50 (lM).
d
Table 7
Activity of leading arylphthalazines and literature compounds in a mitogenic assay
Entry
Compound
ED50 (l
M)a
1
2
3
4
24 (IM-094261)
57 (IM-094882)
4 (ZD-6474)
0.25
0.21
0.28
5 (ZD-2171)
0.012
a
Determined for at least two independent experiments.
6.2. Cell-based assay for VEGFR-2 inhibition: Transfection of
293 cells with DNA expressing FGFR-1/VEGFR-2 chimera
A chimeric construct containing the extracellular portion of
FGFR-1 and the intracellular portion of VEGFR-2 is transiently
transfected into HEK-293 adenovirus-transfected kidney cells.
Figure 4. Oral exposure of 57 (30 mg/kg) and ZD-2171 5 (30 mg/kg) in female CD-1
mice over 24 h using a 5% phosal formulation. Compound 57 (IM-094882) dosed
orally to female CD-1 mice (n = 3) at 30 mg/kg in a 5% phosal solution (animals
DNA for transfection is diluted to a final concentration of 5
DNA in serum-free medium and incubated at room temperature
for 30 min. with 40 l/ml of Lipofectamine 2000, also in serum-free
lg/ml
dosed at 10 mL/kg). Cmax = 25.9 lM; 0–24 h AUC = 891 lmol h.
l
media. Two hundreds and fifty microliters of the Lipofectamine and
transfected cells,
times with PBS + 0.1% Tween 20 and blocked by addition of
200 l/well of 3% BSA in PBS for 1 h and washed again. Eighty
microliters of lysate is then transferred to the coated and blocked
wells and incubated for 1 h at 4 °C. The plates are washed three
times with PBS + 0.1% Tween 20. To detect bound phosphorylated
aFGFR1 coated ELISA plates are washed three
DNA mixture is added to HEK-293 cells suspended at 5 Â 105 cells/
ml. 200 ll/well is dispensed into a 96-well plate, with subsequent
overnight incubation. Within 24 h, media are removed and 100 ll
of media with 10% fetal bovine serum is added to the now adherent
cells followed by an additional 24 h incubation. Target compounds
are added to the individual wells with a final DMSO concentration
of 0.1%. Cells are lysed by resuspension in 100 ll Lysis buffer
(150 mM NaCl, 50 mM Hepes pH 7.5, 0.5% Triton X-100, 10 mM
NaPPi, 50 mM NaF, 1 mM Na3VO4) and rocked for 1 h at 4 °C.
l
chimeric receptor, 100
(RC20:HRP, Transduction Laboratories) was added per well (final
concentration 0.5 g/ml in PBS) and incubated for 1 h. The plates
are washed six times with PBS + 0.1% Tween 20. Enzymatic activity
of HRP is detected by adding 50 l/well of equal amounts of the
Kirkegaard & Perry Laboratories (KPL) Substrate A and Substrate
ll of anti-phosphotyrosine antibodies
l
l
6.3. ELISA for detection of tyrosine-phosphorylated chimeric
receptor
B. The reaction is stopped by addition of 50
H2SO4 and absorbance is detected at 450 nm.
ll/well of 0.1 N
96-well ELISA plates are coated using 100
l
l/well of 10
lg/ml of
a
FGFR1 antibody, and incubated overnight at 4 °C.
a
FGFR1 is pre-
6.4. Mitogenic assay in human umbilical vein endothelial cells
(HUVEC)
pared in a buffer made with 16 ml 0.2 M Na2CO3 and 34 ml 0.2 M
NaHCO3 with pH adjusted to 9.6. Concurrent with lysis of the
HUVEC cells are seeded at 5 Â 104 cells/ml into 96-well plates at
200
l
l/well to obtain a final concentration of 1 Â 104 cells/well in
EGM media (Cambrex, New Jersey). Cells are allowed to adhere
to the plate for a minimum of 4 h before changing to media defi-
cient in EGF, VEGF and FGF but including 1% FBS. After allowing
the cells to quiese for 72 h, the desired compound is incubated
with the cells for 2 h at DMSO concentrations of 0.1%. HUVEC cells
are stimulated with 5 ng/well solution of VEGF-165 for 3 days be-
fore adding 25 ll of 0.25 l
Ci/well of 3H-Thymidine. Cells are har-
vested onto a mat after an additional 5 h incubation and washed
three times with water. The radioactive mat is placed in scintillant
and read on a beta-emitter counter.
6.5. Chemistry
6.5.1. General
All reagents were purchased from commercial suppliers and
used without further purification. All NMR spectra were recorded
Figure 3. Preliminary exposure data for 24 (IM-094261), 57 (IM-094882) and ZD-
2171 5 in female CD-1 mice.

M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
737
on a Brücker spectrometer and are quoted in ppm relative to a tet-
ramethylsilane internal standard, or by referencing on the chemi-
cal shift of the deuterated solvent.
6.5.10. (4-Chlorophthalazin-1-yl)-m-tolylamine hydrochloride
23i
Prepared using general procedure B. Isolated as an impure solid
that was used directly in the next step.
6.5.2. General procedure A for amine displacement.
Preparation of (4-chlorophenyl)-(4-chlorophthalazin-1-
yl)amine 23a
6.5.11. (4-Chlorophthalazin-1-yl)-o-tolylamine hydrochloride 23j
Prepared using general procedure B. Isolated as an impure solid
that was used directly in the next step.
1,4-Dichlorophthalazine (1.0 g, 5.0 mmol), 4-chloroaniline
(0.64 g, 5.0 mmol) and Et3N (700 lL, 5.0 mmol) in n-butanol
(15 mL) was heated to 100 °C in a sealed tube and stirred for
48 h. After allowing to cool, to room temperature, the mixture
was concentrated in vacuo to leave a crude solid. The solid was trit-
urated with Et2O, filtered and the filter cake washed with H2O and
Et2O to give the title compound (1.05 g, 72%) as a solid. 1H NMR
(300 MHz; DMSO-d6) d 10.50 (br s, 1 H), 8.81–8.94 (m, 1 H),
8.15–8.25 (m, 3H), 7.83 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H).
m/z = 290 (M+1 for 35Cl).
6.5.12. General procedure for Suzuki coupling. Preparation of
(4-Chlorophenyl)-(4-isoquinolin-5-yl-phthalazin-1-yl)amine 57
(4-Chlorophenyl)-(4-chlorophthalazin-1-yl)amine
(200 mg,
0.68 mmol), 5-isoquinoline boronic acid (236 mg, 1.36 mmol) and
palladium(II)chloride bis(triphenylphosphine) (24 mg, 0.034
mmol) were combined in a CEM microwave vial. 1,4-dioxane
(4 mL) was added, followed by a solution of K2CO3 (192 mg,
1.36 mmol) in H2O (1 mL). The vial was sealed (CEM microwave
cap) then heated to 100 °C for 60 min under microwave irradiation
(CEM microwave parameters: solvent choice = 1,4-dioxane; micro-
wave power = 50 W; run time = 10 min; hold time = 60 min; stir-
ring = on; cooling = off. Note: in previous studies this reaction
takes less than 60 min—a prolonged reaction time was used to en-
sure complete reaction). After cooling to room temperature the
mixture was poured in to EtOAc (50 mL) and H2O (50 mL) and
the aqueous and organic layers were partitioned. The aqueous
layer was extracted with EtOAc (1Â 50 mL) and the combined or-
ganic extracts were washed with brine (1Â 50 mL), dried (Na2SO4),
filtered and the mixture concentrated in vacuo to leave a crude so-
lid. The solid was purified by column chromatography on silica gel
(compound dry-loaded on to MgSO4; Biotage Flash+ 25 M cartridge
used) using EtOAc/hexanes (3:7–1:0) as an eluent gave a solid
which was triturated with Et2O and filtered to give the title com-
pound (140 mg, 53%) as a solid. 1H NMR (300 MHz; DMSO-d6) d
9.57 (s, 1H), 9.53 (s, 1H), 8.77 (d, J = 8.4 Hz, 1H), 8.45 (d,
J = 6.0 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 8.10–8.16 (m, 3H), 7.89–
7.98 (m, 3H), 7.49 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.36
(d, J = 5.7 Hz, 1H). m/z = 383 (M+1 for 35Cl).
6.5.3. (4-tert-Butylphenyl)-(4-chlorophthalazin-1-yl)amine
23b
Prepared using general procedure A. Yield 1.5 g (96%); 1H NMR
(300 MHz; DMSO-d6) d 9.74 (br s, 1 H), 8.74–8.77 (m, 1H), 8.08–
8.20 (m, 3H), 7.71 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H), 1.34
(s, 9 H). m/z = 312 (M+1 for 35Cl).
6.5.4. [4-(Chlorodifluoromethoxy)phenyl]-(4-
chlorophthalazin-1-yl)amine 23c
Prepared using general procedure A. Yield 0.7 g (79%) as an im-
pure solid; 1H NMR (300 MHz; DMSO-d6) d 9.83 (br s, 1H), 8.58–
8.61 (1H, m), 7.91–8.17 (m, 3H), 7.72 (d, J = 9.0 Hz, 2H), 7.16 (d,
J = 9.0 Hz, 2H). m/z = 336 (M+1 for 35Cl).
6.5.5. (4-Chlorophthalazin-1-yl)-(2-fluoro-4-
methylphenyl)amine 23d
Prepared using general procedure A. Isolated as an impure solid
that was used directly in the next step.
6.5.6. General procedure B for amine displacement. Preparation
of (4-chlorophthalazin-1-yl)-(4-morpholin-4-yl-phenyl)amine
hydrochloride 23e
6.5.13. (4-Chlorophenyl)-(4-isoquinolin-5-yl-phthalazin-1-
yl)amine dihydrochloride
1,4-Dichlorophthalazine (1.0 g, 5.0 mmol) and 4-(4-morpho-
lino)aniline (0.88 g, 5.0 mmol) in EtOH (10 mL) was heated to re-
flux and stirred for 72 h. After allowing to cool to room
temperature the mixture was filtered to give the title compound
as an impure solid that was used directly in the next step. 1H
A solution of HCl in 1,4-dioxane (4 M; 1 mL, excess) was added
in one portion to a stirred suspension of (4-chloro-phenyl)-(4-iso-
quinolin-5-yl-phthalazin-1-yl)-amine (140 mg) in MeOH (20 mL)
at room temperature (a solution develops). The mixture was con-
centrated in vacuo to leave a solid that was triturated with Et2O
and filtered to give the title compound (160 mg) as a solid. 1H
NMR (300 MHz; DMSO-d6) d 9.97 (s, 1H), 9.24 (d, J = 8.4 Hz, 1H),
8.72 (d, J = 8.4 Hz, 1H), 8.63 (d, J = 6.3 Hz, 1H), 8.14–8.33 (m, 3H),
8.02–8.08 (m, 2H), 7.78 (d, J = 8.4 Hz, 2 H), 7.64 (d, J = 8.4 Hz,
2H), 7.52 (d, J = 8.1 Hz, 1H).
NMR (300 MHz; DMSO-d6)
d 11.50 (br s, 1H), 9.13 (d,
J = 6.9 Hz, 1H), 8.20–8.29 (m, 3H), 7.59 (d, J = 8.1 Hz, 2H), 7.36
(d, J = 7.5 Hz, 2H), 3.88 (m, 4 H), 3.33 (m, 4H). m/z = 341 (M+1
for 35Cl).
6.5.7. (4-Chlorophthalazin-1-yl)-(4-isopropylphenyl)amine
hydrochloride 23f. Prepared using general procedure B
Isolated as an impure solid. 1H NMR (300 MHz; DMSO-d6) d
11.50 (br s, 1H), 9.18 (d, J = 7.2 Hz, 1H), 8.18–8.29 (m, 3H), 7.57
(d, J = 8.1 Hz, 2H), 7.37 (t, J = 9.2 Hz, 2H), 2.87–3.02 (m, 1H), 1.24
(d, J = 6.9 Hz, 6H). m/z = 298 (M+1 for 35Cl).
6.5.14. (4-Chlorophenyl)-(4-quinolin-5-yl-phthalazin-1-
yl)amine 67
Prepared using general procedure for Suzuki coupling. After
workup the solid was triturated with Et2O then purified by column
chromatography on silica gel using EtOAc/hexanes (1:4–1:0) as an
eluent. Recrystallization from MeOH gave the title compound
(30 mg, 23%) as a solid. 1H NMR (300 MHz; DMSO-d6) d 9.50 (s,
1H), 8.95 (s, dd, J = 3.9, 1.5 Hz, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.22
(d, J = 8.4 Hz, 1H), 7.81–8.10 (m, 6H), 7.37 (dd, J = 6.9, 0.9 Hz, 1H),
7.38–7.47 (m, 4H). m/z = 383 (M+1 for 35Cl).
6.5.8. (4-Chlorophthalazin-1-yl)-(3,4-dichlorophenyl)amine
hydrochloride 23g
Prepared using general procedure B. Isolated as an impure solid
that was used directly in the next step.
6.5.9. (4-Chlorophthalazin-1-yl)-(4-chloro-3-
trifluoromethylphenyl)amine hydrochloride 23h
Prepared using general procedure B. Isolated as an impure solid
that was used directly in the next step.
6.5.15. (4-tert-Butylphenyl)-(4-isoquinolin-5-yl-phthalazin-1-
yl)amine 56
Prepared using general procedure for Suzuki coupling. Purified
by column chromatography on silica gel using Et2O/hexane (1:1–

738
M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
1:0) as an eluent. The material was purified further by ion exchange
chromatography (TsOH loaded on to SiO2; Silicycle). The captured
compound was washed with MeOH (6Â 6 mL) then eluted with
2 M NH3/MeOH. Trituration with Et2O and filtration gave the title
compound (40 mg, 29%) as a solid. 1H NMR (300 MHz; DMSO-d6) d
9.35 (s, 1 H), 9.22 (s, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 5.7 Hz,
1H), 8.21 (d, J = 7.5 Hz, 1H), 7.87 (t, J = 7.7 Hz, 1H), 7.67–7.82 (m,
5 H), 7.28 (d, J = 8.4 Hz, 2 H), 7.18 (app. t, J = 8.7 Hz, 2H), 1.20 (s,
9H). m/z = 405 (M+1 for 35Cl).
was dissolved in MeOH, loaded on to the silica cartridge and the
captured compound washed with MeOH (6Â 6 mL). Elution with
2 M NH3/MeOH gave the title compound (30 mg, 21%) as a solid.
1H NMR (300 MHz, DMSO-d6) d 9.65 (s, 1H), 9.48 (s, 1H), 8.71 (d,
J = 8.4 Hz, 1H), 8.55 (s, 1H), 8.42 (d, J = 6.0 Hz, 1H), 8.35 (d,
J = 7.8 Hz, 1H), 8.02–8.09 (m, 2H), 7.83–7.96 (m, 3H), 7.63 (d,
J = 8.7 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 6.0 Hz, 1H).
m/z = 417 (M+1 for 35Cl).
6.5.21. (4-Chloro-3-trifluoromethylphenyl)-(4-isoquinolin-5-
yl-phthalazin-1-yl)amine 62
6.5.16. (4-tert-Butylphenyl)-(4-quinolin-5-yl-phthalazin-1-
yl)amine 68
Prepared using general procedure for Suzuki coupling. After
allowing to cool to room temperature the reaction mixture was
poured in to H2O (50 mL) and filtered. The filter cake was purified
further by preparative thin-layer chromatography to give the title
compound (30 mg, 20%) as a solid. 1H NMR (300 MHz; DMSO-d6) d
9.80 (s, 1H), 9.48 (s, 1H), 8.73 (d, J = 8.1 Hz, 1H), 8.69 (s, 1H), 8.33–
8.47 (m, 3H), 8.06 (t, J = 7.7 Hz, 1H), 7.81–7.96 (m, 3H), 7.74 (d,
J = 9.0 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 6.0 Hz, 1H). m/
z = 451 (M+1 for 35Cl).
Prepared using general procedure for Suzuki coupling. After
allowing to cool to room temperature the reaction mixture was fil-
tered and the filter cake washed with Et2O togive the title compound
(100 mg, 72%) as a solid. 1H NMR (300 MHz; DMSO-d6) d 9.32 (s, 1H),
8.95 (d, J = 3.9 Hz, 1H), 8.70 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H),
7.88–8.04 (m, 5H), 7.88 (t, J = 7.7 Hz, 1H), 7.73 (d, J = 6.9 Hz, 1H),
7.35–7.46 (m, 4H), 1.32 (s, 9H). m/z = 405 (M+1 for 35Cl).
6.5.17. [4-(Chloro-difluoro-methoxy)-phenyl]-(4-isoquinolin-
5-yl-phthalazin-1-yl)amine 58
6.5.22. (2-Fluoro-4-methylphenyl)-(4-isoquinolin-5-yl-
phthalazin-1-yl)amine 63
Prepared using general procedure for Suzuki coupling. Purified
by column chromatography on silica gel using Et2O/hexane (1:1–
1:0) as an eluent. The material was purified further by ion ex-
change chromatography (TsOH loaded on to SiO2; Silicycle). The
captured compound was washed with MeOH (6Â 6 mL) then
eluted with 2 M NH3/MeOH. Trituration with Et2O and filtration
Prepared using general procedure for Suzuki coupling. The reac-
tion mixture was allowed to stand overnight then filtered. The fil-
ter cake was washed with H2O (1Â 10 mL) and Et2O (1Â 10 mL) to
give the title compound (130 mg, 88%) as a solid. 1H NMR
(300 MHz; DMSO-d6) d 9.46 (s, 1H), 9.22 (s, 1H), 8.68 (d, J = 8.4
Hz, 1H), 8.39 (d, J = 6.0 Hz, 1H), 8.32 (d, J = 7.2 Hz, 1H), 7.97 (t,
J = 7.7 Hz, 1H), 7.77–7.92 (m, 5H), 7.29–7.33 (m, 2H), 6.99 (d,
J = 9.0 Hz, 2H), 3.76–3.78 (m, 4H), 3.10–3.12 (m, 4H). m/z = 434
(M+1).
gave the title compound (60 mg, 39%) as
(300 MHz; DMSO-d6) 9.58 (s, 1 H), 9.47 (s, 1H), 8.72 (d,
a
solid. 1H NMR
d
J = 8.4 Hz, 1H), 8.40 (d, J = 6.0 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H),
8.13 (d, J = 8.7 Hz, 2H), 8.03 (t, J = 7.8 Hz, 1H), 7.85–7.93 (m,
3 H), 7.36–7.41 (m, 3H), 7.30 (d, J = 6.0 Hz, 1H). m/z = 449 (M+1
for 35Cl).
6.5.23. (4-Isoquinolin-5-yl-phthalazin-1-yl)-o-tolylamine 64
Prepared using general procedure for Suzuki coupling. After
allowing to cool to room temperature the mixture was poured in
to H2O (50 mL) and filtered. The filter cake was purified further
by ion exchange chromatography (TsOH loaded on to SiO2; Silicy-
cle). The captured compound was washed with MeOH (6Â 6 mL)
then eluted with 2 M NH3/MeOH to give the title compound
(50 mg, 41%) as a solid. 1H NMR (300 MHz; DMSO-d6) d 9.45 (s,
1H), 9.12 (s, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.39 (d, J = 5.7 Hz, 1H),
8.31 (d, J = 7.5 Hz, 1H), 7.98 (t, J = 7.5 Hz, 1H), 7.79–7.91 (m, 3H),
7.46 (d, J = 7.5 Hz, 1H), 7.18–7.35 (m, 5H), 2.23 (s, 3H). m/z = 363
(M+1).
6.5.18. (4-Isopropylphenyl)-(4-isoquinolin-5-yl-phthalazin-1-
yl)amine 59
Prepared using general procedure for Suzuki coupling. After
allowing to cool to room temperature the reaction mixture was
poured in to H2O (20 mL), filtered and the filter cake washed with
Et2O (3Â 5 mL) to give the title compound (75 mg, 57%) as a solid.
1H NMR (300 MHz; DMSO-d6) d 9.59 (s, 1 H), 9.44 (s, 1H), 8.68 (d,
J = 8.4 Hz, 1H), 8.36 (d, J = 6.0 Hz, 1H), 8.26 (d, J = 9.6 Hz, 1H), 7.96
(t, J = 7.5 Hz, 1 H), 7.76–7.91 (m, 5 H), 7.23–7.32 (m, 4H), 2.83–2.92
(m, 1H), 1.20 (d, J = 6.9 Hz, 6H). m/z = 391 (M+1).
6.5.24. (4-Isoquinolin-5-yl-phthalazin-1-yl)-m-tolylamine 65
Prepared using general procedure for Suzuki coupling. After
allowing to cool to room temperature the mixture was poured in
to H2O (20 mL), filtered and the filter cake washed with Et2O (2Â
5 mL). The filter cake was purified further by ion exchange chroma-
tography (TsOH loaded on to SiO2; Silicycle). The captured com-
pound was washed with MeOH (6Â 6 mL) then eluted with 2 M
NH3/MeOH to give the title compound (52 mg, 42%) as a solid. 1H
NMR (300 MHz; DMSO-d6) d 9.47 (s, 1H), 9.31 (s, 1H), 8.72 (d,
J = 8.4 Hz, 1H), 8.40 (d, J = 5.7 Hz, 1H), 8.33 (d, J = 7.5 Hz, 1H),
8.00 (t, J = 7.7 Hz, 1H), 7.79–7.94 (m, 5H), 7.25–7.36 (m, 3H), 6.89
(d, J = 7.2 Hz, 1H), 2.36 (s, 3H). m/z = 363 (M+1).
6.5.19. (4-Isoquinolin-5-yl-phthalazin-1-yl)-(4-morpholin-4-yl-
phenyl)-amine 60
Prepared using general procedure for Suzuki coupling. After
allowing to cool to room temperature the mixture was allowed
to stand overnight then filtered. The filter cake was washed with
H2O (1Â 10 mL) and Et2O (1Â 10 mL) to give the title compound
(130 mg, 88%) as a solid. 1H NMR (300 MHz; DMSO-d6) d 9.46 (s,
1 H), 9.22 (s, 1H), 8.68 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 6.0 Hz, 1H),
8.32 (d, J = 7.2 Hz, 1H), 7.97 (t, J = 7.7 Hz, 1H), 7.77–7.92 (m, 5H),
7.29–7.33 (m, 2H), 6.99 (d, J = 9.0 Hz, 2 H), 3.76–3.78 (m, 4 H),
3.10–3.12 (m, 4H). m/z = 434 (M+1).
6.5.20. (3,4-Dichloro-phenyl)-(4-isoquinolin-5-yl-phthalazin-1-
yl)-amine 61
6.5.25. 1-Chloro-4-isoquinolin-5-yl-phthalazine
Prepared using general procedure for Suzuki coupling. After
allowing to cool to room temperature the mixture was poured
in to H2O (20 mL), filtered and the filter cake washed with
Et2O (4Â 5 mL). The filter cake was purified further by the fol-
lowing procedure. An ion exchange cartridge (TsOH loaded on
to SiO2; Silicycle) was washed with MeOH (6 mL). The filter cake
1,4-Dichlorophthalazine 22 (200 mg, 1.0 mmol), 5-isoquinoline
boronic acid (173 mg, 1.0 mmol) and palladium(II) chloride bis(tri-
phenylphosphine) (35 mg, 0.05 mmol) were combined in a CEM
microwave vial. 1,4-Dioxane (4 mL) was added followed by a solu-
tion of K2CO3 (276 mg, 2.0 mmol) in H2O (1 mL). The vessel was
sealed (CEM microwave cap) and then heated to 100 °C for

M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
739
16. Garton, A. J.; Crew, A. P. A.; Franklin, M.; Cooke, A. R.; Wynne, G. M.; Castaldo,
L.; Kahler, J.; Winski, S. L.; Franks, A.; Brown, E. N.; Bittner, M. A.; Keily, J. F.;
Brinner, P.; Hidden, C.; Srebernak, M. C.; Pirrit, C.; O’Connor, M.; Chan, A.;
Vulevic, B.; Castelhano, A. L.; Arnold, L. D.; Gibson, N. W. Can. Res. 2006, 66,
1015.
17. (a) Polverino, A.; Coxon, A.; Starnes, C.; Diaz, Z.; DeMelfi, T.; Wang, L.; Bready,
J.; Estrada, J.; Cattley, R.; Kaufman, S.; Chen, D.; Gan, Y.; Kumar, G.; Meyer, J.;
Neervannan, S.; Alva, G.; Talvenheimo, J.; Montestruque, S.; Tasker, A.; Patel,
V.; Radinsky, R.; Kendall, R. Can. Res. 2006, 66, 8715; (b) Rosen, L. S.; Kurzrock,
R.; Mulay, M.; Van Vugt, A.; Purdom, M.; Ng, C.; Silverman, J.; Koutsoukos, A.;
Sun, Y. M.; Bass, M. B.; Xu, R. Y.; Polverino, A.; Wiezorek, J. S.; Chang, D. D.;
Benjamin, R.; Herbst, R. S. J. Clin. Oncol. 2007, 25, 2369.
18. Lee, S. H.; Lopes de Menezes, D.; Vora, J.; Harris, A.; Ye, H.; Nordahl, l.; Garrett,
E.; Samara, E.; Aukerman, S. L.; Gelb, A. B.; Heise, C. Clin. Can. Res. 2005, 11,
3633.
19. Harris, P. A.; Boloor, A.; Cheung, M.; Kumar, R.; Crosby, R. M.; Davis-Ward, R.
G.; Epperly, A. H.; Hinkle, K. W.; Hunter, R. N., III; Johnson, J. H.; Knick, V. B.;
Laudeman, C. P.; Luttrell, D. K.; Mook, R. A.; Nolte, R. T.; Rudolph, S. K.;
Szewczyk, J. R.; Truesdale, A. T.; Veal, J. M.; Wang, L.; Stafford, J. A. J. Med. Chem.
2008, 51, 4632.
20. Dai, Y.; Hartandi, K.; Ji, Z.; Ahmed, A. A.; Albert, D. H.; Bauch, J. L.; Bouska, J. J.;
Bousquet, P. F.; Cunha, G. A.; Glaser, K. B.; Harris, C. M.; Hickman, D.; Guo, J.; Li,
J.; Marcotte, P. A.; Marsh, K. C.; Moskey, M. D.; Martin, R. L.; Olson, A. M.;
Osterling, D. J.; Pease, L. J.; Soni, N. B.; Stewart, K. D.; Stoll, V. S.; Tapang, P.;
Reuter, D. R.; Davidsen, S. K.; Michaelides, M. R. J. Med. Chem. 2007, 50, 1584.
21. Traxler, P.; Allegrini, P. R.; Brandt, R.; Brueggen, J.; Cozens, R.; Fabbro, D.;
Grosios, K.; Lane, H. A.; McSheehy, P.; Mestan, J.; Meyer, T.; Tang, C.;
Wartmann, M.; Wood, J.; Caravatti, G. Can. Res. 2004, 64, 4931.
22. (a) Bhide, R. S.; Cai, Z.-W.; Zhang, Y.-Z.; Qian, L.; Wei, D.; Barbosa, S.; Lombardo,
L. J.; Borzilleri, R. M.; Zheng, X.; Wu, L. I.; Barrish, J. C.; Kim, S.-H.; Leavitt, K.;
Mathur, A.; Leith, L.; Chao, S.; Waulet, B.; Mortillo, S.; Jeyaseelan, R., Sr.; Kukral,
D.; Hunt, J. T.; Kamath, A.; Fura, A.; Derbin, G.; Fargnoli, F. J. Med. Chem. 2006,
49, 2143; (b) Cai, Z.-W.; Zhang, Y.; Borzilleri, R. M.; Qian, L.; Barbosa, S.; Wei,
D.; Zheng, X.; Wu, L.; Fan, J.; Shi, Z.; Wautlet, B. S.; Mortillo, S.; Jeyaseelan, R.;
Kukral, D. W.; Kamath, A.; Marathe, P.; D’Arienzo, C.; Derbin, G.; Barrish, J. C.;
Robl, J. A.; Hunt, J. T.; Lombardo, L. J.; Fargnoli, J.; Bhide, R. S. J. Med. Chem. 2008,
51, 1976.
30 min under microwave irradiation with stirring (CEM microwave
parameters: solvent choice = 1,4-dioxane; power = 50 W; run
time = 5 min; hold time = 30 min; stirring = on; cooling = off). The
mixture was subjected to a further 30 min of heating at 100 °C un-
der microwave irradiation. After allowing to cool to room temper-
ature, the mixture was poured in to EtOAc (70 mL) and the mixture
was washed with brine (1Â 30 mL), dried (Na2SO4), filtered and the
mixture concentrated in vacuo to leave a crude solid. The solid was
purified by column chromatography on silica gel (compound dry-
loaded on to silica) using EtOAc/hexanes (1:9–1:0) as an eluent
to give the title compound (80 mg, 27%) as a solid. 1H NMR
(300 MHz; DMSO-d6) d 9.50 (s, 1H), 8.40–8.47 (m, 2H), 8.13 (dt,
J = 8.3, 1.5 Hz, 1H), 7.90–8.04 (m, 2H), 7.52–7.65 (m, 3H), 7.30 (d,
J = 5.7 Hz, 1H). m/z = 292 (M+1 for 35Cl).
6.5.26. (4-Isoquinolin-5-yl-phthalazin-1-yl)-(2-methyl-1H-
indol-5-yl)amine 66
1-Chloro-4-isoquinolin-5-yl-phthalazine (40 mg, 0.14 mmol),
5-amino-2-methylindole (20 mg, 0.14 mmol) and EtOH (2.5 mL)
were combined and sealed in a CEM microwave vial. The mixture
was heated to 100 °C for 120 min under microwave irradiation
(CEM microwave parameters: solvent choice = ethanol; microwave
power = 300 W; run time = 10 min; hold time = 120 min; stir-
ring = on; cooling = off). After allowing to cool to room tempera-
ture the mixture was purified by preparative thin-layer
chromatography using EtOAc as an eluent to give the title com-
pound (25 mg, 45%) as a solid. 1H NMR (300 MHz; DMSO-d6) d
10.62 (s, 1H), 9.22 (s, 1H), 9.01 (br s, 1H), 8.48 (d, J = 8.4 Hz, 1H),
8.16 (d, J = 6.0 Hz, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.61–7.75 (m, 4H),
7.53 (t, J = 9.2 Hz, 1H), 7.20 (d, J = 8.1 Hz, 1H), 7.03–7.09 (m, 3H),
5.90 (s, 1H), 2.16 (s, 3H). m/z = 402 (M+1).
23. Borzilleri, R. M.; Rajeev, S. B.; Barrish, J. C.; D’Arienzo, C. J.; Derbin, G. M.;
Fargnoli, J.; Hunt, J. T.; Jeyaseelan, R., Sr.; Kamath, A.; Kukral, D. W.; Marathe, P.;
Mortillo, S.; Qian, L.; Tokarski, J. S.; Waulet, B. S.; Zheng, X.; Lombardo, L. J. J.
Med. Chem. 2006, 49, 3766.
24. Beebe, J. S.; Jani, J. P.; Knauth, E.; Goodwin, P.; Higdon, C.; Rossie, A. M.;
Emerson, E.; Finkelstein, M.; Floyd, E.; Harriman, S.; Atherton, J.; Hillerman, S.;
Soderstrom, C.; Kou, K.; Gant, T.; Noe, M. C.; Foster, B.; Rastinejad, F.; Marx, M.
A.; Schaeffer, T.; Whalen, P. M.; Roberts, W. G. Can. Res. 2003, 63, 7301.
25. Bilodeau, M. T.; Balitza, A. E.; Koester, T. J.; Manley, P. J.; Rodman, L. D.; Buser-
Doepner, C.; Coll, K. E.; Fernandes, C.; Gibbs, J. B.; Heimbrook, D. C.; Huckle, W.
R.; Kohl, N.; Lynch, J. J.; Mao, X.; McFall, R. C.; McLoughlin, D.; Miler-Stein, C.
M.; Rickert, K. W.; Sepp-Lorenzino, L.; Shipman, J. M.; Subramanian, R.;
Thomas, K. A.; Bradley, K. W.; Yi, S.; Hartman, G. D. J. Med. Chem. 2004, 47,
6363.
References and notes
1. Carmeliet, P. Nature 2006, 438, 932.
2. Folkman, J. N. Engl. J. Med. 1971, 285, 1182.
3. Cross, M. J.; Dixelius, J.; Matsumoto, T.; Claesson-Welsh, L. Trends Biochem. Sci.
2003, 28, 488.
4. Dvorak, H. F. J. Clin. Oncol. 2002, 20, 4368.
5. The monoclonal antibody Bevacizumab (AvastinÒ
) is approved for the
26. Ruggeri, B.; Singh, J.; Gingrich, D.; Angeles, T.; Albom, M.; Chang, H.; Robinson,
C.; Hunter, K.; Dobrzanski, P.; Jones-Bolin, S.; Aimone, L.; Klein-Szanto, A.;
Herbert, J.-M.; Bono, F.; Schaeffer, P.; Casellas, P.; Bourie, B.; Pili, R.; Isaacs, J.;
Ator, M.; Hudkins, R.; Vaught, J.; Mallamo, J.; Dionne, C. Can. Res. 2003, 63,
5978.
27. Amino, N.; Ideyama, Y.; Yamano, M.; Kuromitsu, S.; Tajinda, K.; Samizu, K.;
Hisamichi, H.; Matsuhisa, A.; Shirasuna, K.; Kudoh, M.; Shibasaki, M. Clin. Can.
Res. 2006, 15, 1630.
treatment of colorectal, lung and breast cancer. See Culy, C. Drugs Today
2005, 41, 23.
6. The antiangiogenic aptamer Pegaptanib sodium (MacugenÒ) is approved to
treat neovascular age-related macular degeneration. See Fine, S. L.; Martin, D.
F.; Kirkpatrick, P. Nat. Rev. Drug. Disc. 2005, 4, 187.
7. The monoclonal antibody Ranibizumab (LucentisÒ
) is approved for the
treatment of wet-age related macular degeneration. See Bick, S. K. A. Drugs
2007, 67, 1199.
28. Nakamura, K.; Taguchi, E.; Miura, T.; Yamamoto, A.; Takahasji, K.; Kazumi, B.;
Francis, G.; Guilbaud, N.; Hasegawa, K.; Kubo, K.; Fujiwara, Y.; Suzuki, R.; Kubo,
K.; Shibuya, M.; Isae, T. Can. Res. 2006, 66, 9134.
29. Sepp-Lorenzino, L.; Rands, E.; Mao, X.; Connolly, B.; Shipman, J.; Antanavage, J.;
Hill, S.; Davis, L.; Beck, S.; Rickert, K.; Coll, K.; Ciecko, P.; Fraley, M.; Hoffman,
W.; Hartman, G.; Heimbrook, D.; Gibbs, J.; Kohl, N.; Thomas, K. Can. Res. 2004,
64, 751.
30. Piatnitski, E. L.; Duncton, M. A. J.; Kiselyov, A. S.; Katoch-Rouse, R.; Sherman, D.;
Milligan, D. L.; Balagtas, C.; Wong, W. C.; Kawakami, J.; Doody, J. F. Bioorg. Med.
Chem. Lett. 2005, 15, 4696.
8. Lu, D.; Jimenez, X.; Zhang, H.; Bohlen, P.; Witte, L. Zhu. Z. Int. J. Can. 2002, 97,
393–444.
9. Holash, J.; Davis, S.; Papadopoulos, N.; Croll, S. D.; Ho, L.; Russell, M.; Boland, P.;
Leidich, R.; Hylton, D.; Burova, E.; Ioffe, E.; Huang, T.; Radziejewski, C.; Bailey,
K.; Fandl, J. P.; Daly, T.; Wiegand, S. J.; Yancopoulos, G. D.; Rudge, J. S. Proc. Nat.
Acad. Sci. U.S.A. 2002, 99, 11393.
10. Waltenberger, J.; Claesson-Welsh, L.; Siegbahn, A.; Shibuya, M.; Heldin, C.-H. J.
Biol. Chem. 1994, 269, 26988.
11. Faivre, S.; Demetri, G.; Sargant, W.; Raymond, E. Nat. Rev. Drug Disc. 2007, 6,
734–735. and references cited therein.
31. Duncton, M. A. J.; Piatnitski, E. L.; Katoch-Rouse, R.; Smith, L. M., II; Kiselyov, A.
S.; Milligan, D. L.; Balagtas, C.; Wong, W. C.; Kawakami, J.; Doody, J. F. Bioorg.
Med. Chem. Lett. 2006, 16, 1579.
12. Wilhelm, S. M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong, H.; Chen., C.;
Zhang, X.; Vincent, P.; McHugh, M.; Cao, Y.; Shujath, J.; Gawlak, S.; Eveleigh, D.;
Rowley, B.; Liu, L.; Adnane, L.; Lynch, M.; Auclair, D.; Taylor, I.; Gedrich, R.;
Voznesensky, A.; Riedl, B.; Post, L. E.; Bollag, G.; Trail, P. A. Can. Res. 2004, 64,
7099.
13. Wood, J. M.; Bold, G.; Buchdunger, E.; Cozens, R.; Ferrari, S.; Frei, J.; Hofmann,
F.; Mestan, J.; Mett, H.; O’Reilly, T.; Persohn, E.; Rösel, J.; Schnell, C.; Stover, D.;
Theuer, A.; Towbin, H.; Wenger, F.; Woods-Cook, K.; Menrad, A.; Siemeister, G.;
Schirner, M.; Thierauch, K.-H.; Schneider, M. R.; Drevs, J.; Martiny-Baron, G.;
Totzke, F.; Marmé, D. Can. Res. 2000, 60, 2178.
32. Guery, S.; Parrot, I.; Wermuth, C. G.; Synthesis 2001, 699.
33. Compounds were outsourced for synthesis to the following chemistry service
providers (a) ChemBlock, 40-144 Leninsky pr., 119334 Moscow, Russia. (b)
ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, CA 92121, United States. (c)
Enamine Ltd, 23 Alexandra Matrosova Street, 01103 Kiev, Ukraine.
34. Bold, G.; Altmann, K.-H.; Frei, J.; Lang, M.; Manley, P. W.; Traxler, P.; Wietfeld,
B.; Brüggen, J.; Buchdunger, E.; Cozens, R.; Ferrari, S.; Furet, P.; Hofmann, F.;
Martiny-Baron, G.; Mestan, J.; Rösel, J.; Sills, M.; Stover, D.; Acemoglu, F.; Boss,
E.; Emmennegger, R.; Lässer, L.; Masso, E.; Roth, R.; Schlacter, C.; Vetterli, W.;
Wyss, D.; Wood, J. M. J. Med. Chem. 2000, 43, 2310.
35. For select uses of the OCF2Cl substituent in medicinal chemistry see (a) Terrell,
R. C.; Speers, L.; Szur, A. J.; Treadwell, J.; Ucciardi, T. R. J. Med. Chem. 1971, 14,
517; (b) Speers, L.; Szur, A. J.; Terrell, R. C.; Ucciardi, T. U. J. Med. Chem. 1971, 14,
593; (c) Terrell, R. C.; Speers, L.; Szur, A. J.; Ucciardi, T.; Vitcha, J. F. J. Med. Chem.
14. Herbst, R. S.; Heymach, J. V.; O’Reilly, M. S.; Onn, A.; Ryan, A. J. Expt. Opin. Invest.
Drugs 2007, 16, 239.
15. Wedge, S. R.; Kendrew, J.; Hennequin, L. F.; Valentine, P. J.; Barry, S. T.; Brave, S.
R.; Smith, N. R.; James, N. H.; Dukes, M.; Curwen, J. O.; Chester, R.; Jackson, J. A.;
Boffey, S. J.; Kilburn, L. L.; Barnett, S.; Richmond, G. H. P.; Wadsworth, P. F.;
Walker, M.; Bigley, A. L.; Taylor, S. T.; Cooper, L.; Beck, S.; Juergensmeier, J. M.;
Ogilvie, D. J. Can. Res. 2005, 65, 4389.

740
M. A. J. Duncton et al. / Bioorg. Med. Chem. 17 (2009) 731–740
1972, 15, 604; (d) Speers, L.; Szur, A. J.; Terrell, R. C. J. Med. Chem. 1972, 15, 606;
Conversely, the use of the OCF2 Cl substituent in agrochemistry has been
documented in over 75 patent applications and papers. For an example see
Boger, M.; Durr, D.; Gsell, L.; Hall, R. G.; Karrer, F.; Kristiansen, O.; Maienfisch,
P.; Pascual, A.; Rindlisbacher, A.; Pest Manag. Sci. 2001, 57, 191.
(e) Stogryn, E. L. J. Med. Chem. 1973, 16, 1399; (f) Bagnall, R. D.; Bell, W.;
Pearson, K. J. Fluor. Chem. 1978, 11, 93; (g) Bartlett, R. R.; Kaemmerer, F. J.; WO
9117748, 1991.; (h) Anderson, D. R.; Mahoney, M. W.; Phillion, D. P.; Rogers, T.
E.; Meyers, M. J.; Poda, G.; Hedge, S. G.; Singh, M.; Reitz, D. B.; Wu, K. K.;
Buchler, I. P.; Xie, J.; Vernier, W. F.; WO 2004058762, 2004.; (i) Khire, U.; Liu,
X.-G.; Nagarathnam, D.; Wood, J.; Wang, L.; Liu, D.; Zhao, J.; Guernon, L.; Zhang,
L.; WO 2005097752, 2005.; (j) Natarajan, S. K.; Moreno, O.; Graddis, T. J.;
Duncan, D.; Laus, R.; Chen, F.; WO 2005020897, 2005.; (k) Xu, D.; Jiang, B.;
Ketela, T.; Lemieux, S.; Veillette, K.; Martel, N.; Davison, J.; Sillaots, S.; Trosok,
S.; Bachewich, C.; Bussey, H.; Youngman, P.; Roemer, T. PLoS Pathogens 2007, 3,
835; (l) Orchard, M. G.; Benghezal, M.; Braillard, S.; Burn, C.; Deuschel, C.;
Lucas, A.; Valentino, E.; Janssen, C.; Boyce, R. S. WO2008017840, 2008.
36. Kinetic or thermodynamic solubilities of compounds were not measured.
However, in several instances precipitation of amide compounds was noted in
pH 7.4 buffer. This observation was interpreted as indicative of a need to
improve the aqueous solubility of future molecules.
37. Cross-screening against more than 60 biological targets belonging to
neurotransmitter, steroid, ion-channel, second messenger, prostaglandin,
growth factor/hormone, brain/gut peptide and enzyme class performed by
NovaScreen Biosciences Corporation. Inhibition of more than 50% at a drug
screening concentration of 10 lM was deemed to be of significance.