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4-ANILINO-3-QUINOLINECARBONITRILES
FOR THE TREATMENT OF CANCER
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The present invention is directed to a method of using 4-(2,4-dichloro-5-
methoxy-phenylamino)-6-methoxy-7-[3-(4-methyl-piperazin-l-yl)-propoxy]-
quinoline-3-carbonitrile (SKI-606), the compound of formula (I), to treat cancer
and a composition containing the same.
Related Background Art
[0002] Various 4-anilino-3-quinolinecarbonitriles derivatives have been shown
to have anti-tumor activity that may make them useful as chemoagents in treating
various cancers, including pancreatic, lymphatic and prostate cancers. U.S.
Patents Nos. 6,002,008, 6,384,051, 6,432,979 and 6,617,333 disclose certain 4-
anilino-3-quinolinecarbonitriles derivatives that are shown to possess anti-tumor
activity.
[0003] The protein tyrosine kinases consist of functionally related receptor and
nonreceptor signaling enzymes regulating cell growth, activation, differentiation,

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development, and transformation through phosphorylation of specific tyrosine
residues. The receptor tyrosine kinases, such as epidermal growth factor receptor
(EGFR), consist of an extracellular ligand binding domain, a single
transmembrane domain and an intracellular tyrosine kinase domain. The
nonreceptor tyrosine kinases, such as Src and Abl, are soluble cytoplasmic
enzymes with multiple regulatory and protein-binding domains.
[0004] The Src tyrosine kinase family is a group of 9 nonreceptor tyrosine
kinases defined by both functional and sequence similarity. Three members of.
this family are widely expressed: Src, Yes, and FynB. The other 6 members,
Lck, Lyn, FynT, Fgr, Hck, and Blk, are predominantly expressed in
hematopoietic cells. Extensive reviews on structure and function of nonreceptor
protein tyrosine kinases and their relevance in human cancers have been
published.
[0005] The Src nonreceptor protein tyrosine kinase is the prototype of the Src
family. Src is a key downstream component of pathways mediated by growth
factor receptors and G-protein coupled receptors, and is believed to coordinate
signals from these various pathways. The list of intracellular target proteins
known to be phosphorylated by Src-family kinases is large and continues to
grow, including integrins, adhesion kinases, cadherins, stat3, cortactin, ezrin,
focal adhesion proteins (FAK), and many others.
[0006] Src is upregulated in most cancers including the vast majority of
pancreatic, melanoma, head and neck, and ovarian cancers. Src is also activated
in prostatic tumor lines. Src mRNA levels were higher in human parotid tumors
compared to normal tissue samples. Barrett's esophagus and esophageal
carcinomas also overexpress Src.
[0007] Since the c-Src tyrosine kinase is a determinant of malignant cellular
behavior in a variety of human cancers, we sought to determine the effect of
suppressing' c-Src expression on pancreatic adenocarcinoma chemosensitivity to
gemcitabine.
[0008] Based on the above observations, compounds mat inhibit Src may be
useful in treating patients with these and other cancers. 4-(2,4-Dichloro-5-
methoxy-phenylamino)-6-methoxy-7-[3-(4-methyl-piperazin-l-yl)-propoxy]-

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quInoline-3-carbonitrile has been identified as useful for preventing, treating or
inhibiting cancer by these criteria.
BRIEF SUMMARY OF INVENTION
[0009] The present invention is directed to a method of preventing, treating or
inhibiting cancer comprising, administering a therapeutically effective amount of
a compound of formula (I):

4-(2,4-dichloro-5-methoxy-phenylamino)-6-methoxy-7-[3-(4-methyl-piperazin-l-
yl)-propoxy]-quinoline-3-carbonitrile, or a pharmaceutically acceptable salt
thereof.
[0010] This invention is also directed to a method of treating pancreatic cancer
comprising, administering a therapeutically effective amount of a compound of
formula (I), or pharmaceutically acceptable salts thereof, in combination with
gemcitabine, or a pharmaceutically acceptable salt thereof.
[0011] Another aspect of this invention is a pharmaceutical composition
comprising an effective amount of a compound of formula (I), or a
pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable
carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1. Shows the response of the head and neck line HN5 to 4-[(2,4-
dichIoro-5-methoxyphenyl) amino]-5-methoxy-7-[3-(4-methyl-l-piperazinyl)
propoxy]-3-quinoline carbonitrile.

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DETAILED DESCRIPTION OF THE INVENTION
[0013] The term "cancer" refers to any malignant growth or tumor caused by
abnormal and uncontrolled cell division. It may spread to other parts of the body
through the lymphatic system or the blood stream. For the purposes of the
method of treating cancer described in this application, cancer includes
pancreatic, lymphatic and prostate cancers.
[0014] Pharmaceutically acceptable salts, for example, are those derived from
such organic and inorganic acids as: acetic, lactic, carboxylic, citric, cinnamic,
tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, oxalic, propionic,
hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, glycolic, pyruvic,
methanesulfonic, ethanesulfonic, toluenesulfonic, salicylic, benzoic, and similarly
known acceptable acids.
[0015] The compound of formula (I) may be provided orally, by intralesional,
intraperitoneal, intramuscular or intravenous injection, infusion, liposome-
mediated delivery, topical, nasal, anal, vaginal, sublingual, uretheral, transdermal,
intrathecal, ocular or otic delivery. In order to obtain consistency in providing
the compound of formula (I) it is preferred that the compound is in the form of
the unit dose. Suitable unit dose forms include tablets, capsules and powders in
sachets or vials. Such unit dose forms may contain from 0.1 to 300 mg of the
compound of formula (I), and preferably from 2 to 100 mg. Still further
preferred unit dosage forms contain 50 to 150 mg of the compound of
formula (I). The compound of formula (I) can be administered orally. It may be
administered from 1 to 6 times a day, more usually from 1 to 4 times a day. The
effective amount will be known to one of skill in the art; it will also be dependent
upon the form of the compound. One of skill in the art could routinely perform
empirical activity tests to determine the bioactivity of the compound in bioassays
and thus determine what dosage to administer.
[0016] This invention is also directed to pharmaceutical compositions
containing a therapeutically effective amount of 4-(2,4-dichloro-5-methoxy-
phenylamino)-6-methoxy-743-(4rnemyl-piperazin4-yl)iropoxy
carbonitrile, the compound of formula (I), or pharmaceutically acceptable salts
thereof, and a pharmaceutically acceptable carrier. The carrier may be, for

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example, a diluent, an aerosol, a topical carrier, an aqueous solution, a
nonaqueous solution or a solid carrier. The carrier may be a polymer or a
toothpaste. A carrier in this invention encompasses any of the standard
pharmaceutically accepted carriers, such as phosphate buffered saline solution,
acetate buffered saline solution, water, emulsions such as an oil/water emulsion
or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets
and capsules. The compositions containing the compound of formula (I) may be
formulated with conventional excipients, such as a filler, a disintegrating agent, a
binder, a lubricant, a flavoring agent or a color additive.
[0017] When provided orally or topically, such compounds would be provided to
a subject by delivery in different carriers. Typically, such carriers contain
excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid,
talc, vegetable fats or oils, gums, or glycols. The specific carrier would need to
be selected based upon the desired method of delivery, for example, phosphate
buffered saline (PBS) could be used for intravenous or systemic delivery and
vegetable fats, creams, salves, ointments or gels may be used for topical delivery.
[0018] The compound of formula (I) may be delivered together with suitable
diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers useful
in treatment or prevention of neoplasm. Such compositions are liquids or
lyophilized or otherwise dried formulations and include diluents of various buffer
content (for example, Tris-HCl, acetate, phosphate), pH and ionic strength,
additives such as albumins or gelatin to prevent absorption to surfaces, detergents
(for example, TWEEN 20, TWEEN 80, PLURONIC F68, bile acid salts),
solubilizing agents (for example, glycerol, polyethylene glycerol), anti-oxidants
(for example ascorbic acid, sodium metabisulfate), preservatives (for example,
thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers
(for example, lactose, mannitol), covalent attachment of polymers such as
polyethylene glycol, complexation with metal ions, or incorporation of the
compound into or onto particulate preparations of hydrogels or liposomes, micro-
emulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or
spheroblasts. Such compositions will influence the physical state, solubility,
stability, rate of in vivo release, and rate of in vivo clearance of the compound or

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composition. The choice of compositions will depend on the physical and
chemical properties of the compound capable of treating or preventing a
neoplasm.
[0019] The compound of formula (I) may be delivered locally via a capsule that
allows a sustained release of the compound over a period of time. Controlled or
sustained release compositions include formulation in lipophilic depots (for
example, fatty acids, waxes, oils).
[0020] The present invention further provides a method of using the compound
of formula (I) as an active therapeutic substance for preventing treating and/or
inhibiting cancer. Based upon the results obtained and presented herein, SKI-606
is useful in preventing, treating or inhibiting cancers by suppressing proliferation
of malignant cells. SKI-606 inhibits Src catalyzed phosphorylation of
intercellular proteins associated with cell proliferation. Therefore, dosing a
human with a therapeutically effective amount of SKI-606 can prevent or inhibit
the formations of cancers by suppressing proliferation, or can treat a human
already suffering from a cancer by preventing or inhibiting further growth of
tumors and/or causing a reduction in size or the eradication of tumors. For the
purpose of this invention the term "inhibition" refers to retardation, suppression
or stopping of malignant cell proliferation, presumably by blocking, or
suppressing phophorylation catalyzed by Src. For the purposes of this invention
the term "preventing" refers to averting or forestalling the development of
malignant or tumoric growths by prophylatic treatment, or to impede, inhibit or
cease further progression of the disease. For the purposes of this invention the
term "treating" refers to administering to a patient in need thereof a
therapeutically effective amount of SKI-606 in order to prophylactically prevent
the development of a cancer, to inhibit or cease the progression of a cancer, or a
malignant or tumoric growth, to reverse the progression of a cancer, or a
malignant or tumoric growth, or to eradicate a cancer, or a malignant or tumoric
growth.
[0021] The present invention further provides a method of treating cancer in
humans, which comprises administering to the infected individual an effective
amount of 4-(2,4-dichloro-5-methoxy-phenylamino)-6-methoxy-7-[3-(4-methyl-

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piperazin-l-yl)-propoxy]-quinoline-3-carbonitrile, a phaimaceutically acceptable
salts thereof, or a pharmaceutical composition containing the same. The dose
provided to a patient will vary depending upon what is being administered, the
purpose of the administration, the manner of administration, and the like. A '
"therapeutically effective amount" is an amount sufficient to cure or ameliorate
symptoms of a cancer.
[0022] The compound of formula (I) may be delivered alone or in combination
with other compounds used to treat cancer. Such compounds include but are not
limited to imatinib mesylate (GLEEVEC), hydroxyurea, IFN-d, cytotoxic agents,
chemotherapeuric agents, NSAIDS, gemcitabine, EGFR inhibitors, MEK
inhibitors, farnesyltransferase, gemcitabine, avastin or wortmannin, or
phaimaceutically acceptable salts thereof.
[0023] A preferred embodiment of the method of the present invention
comprises administering a therapeutically effective amount of the compound of
formula (I) in combination with a therapeutically effective amount of gemcitabine
or avastin. More preferably the compound of formula (I) is administered in
combination with gemcitabine.
[0024] Another preferred embodiment of the method of the present invention
comprises administering a therapeutically effective amount of the compound of
formula (I) in combination with a therapeutically effective amount of gemcitabine
and avastin.
[0025] Another preferred embodiment of the method of the present invention
involves administering the compound of formula (I), or a pharmaceutical
acceptable salt thereof, in combination with gemcitabine and avastin, or
pharmaceutically acceptable salts thereof, to treat pancreatic cancer.
[0026] A preferable compound for practicing the method and/or for use in a
composition of this invention is 4-(2,4-dichloro-5-methoxy-phenylamino)-6-
methoxy-7-[3-(4-methy]-piperazin-l-yl)-propoxy]-quinoline-3-carbonitrileanda
pharmaceutically acceptable salt thereof.
[0027] The compound of formula (I) is prepared according to the methods of
U.S. patent 6,002,008, and such methods are herein incorporated by reference.

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[0028] Reactions are performed in a solvent appropriate to the reagents and
materials employed and suitable for the transformation being effected. It is'
understood by those skilled in the art of organic synthesis that the various
functionalities present on the molecule must be consistent with the chemical
transformations proposed. When not specified, order of synthetic steps, choice of
protecting groups and deprotection conditions will be readily apparent to those
skilled in the art. In addition, in some instances, substituents on the starting
materials may be incompatible with certain reaction conditions. Restrictions
pertinent to given substituents will be apparent to one skilled in the art. Reactions
were run under inert atmospheres where appropriate.
[0029] The compound of Formula (I), is readily obtained by treatment of 7-(3-
chloropropoxy)-4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-3-
quinolinecarbonitrile with N-methylpiperazine in the presence of sodium iodide
either neat or in a solvent such as ethylene glycol dimethyl ether. The preparation
of these compounds has been reported in the literature, [Boschelli, D. H., et. al., J.
Med. Chem., 44, 3965 (2001)], hereby incorporated by reference.
DETAILED DESCRIPTION OF THE DRAWINGS
[0030] Figure 1. Response of the head and neck tumor line HN5 to 4-[(2,4-
dichloro-5-methoxyphenyl)amino]-5-methoxy-7-[3-(4-methyl-l-
piperazinyl)propoxy]-3-quinolmecarbonitrile. Serum-starved HN5 cells were
treated with the compound for 4 hours, after which EGF was added to 50 ng/ml
for ten minutes. Lysates were analyzed for Stat3 Y705, c-Cbl Y731 and Y774
and caveolin Y14 phosphorylation. The results demonstrate that phosphorylation
of caveolin Y14, c-Cbl Y731, and Y705 of Stat3 was reduced by 4-[(2,4-
dichloro-5-methoxyphenyl)amino]-5-methoxy-7-[3-(4-methyl-l-
piperazinyl)propoxy]-3-quinolinecarbonitrile.
[0031] Enzyme Assays
[0032] The compound of formula (I) inhibits Src catalyzed phosphorylation of a
target peptide. 4-[(2,4-dichloro-5-methoxyphenyl)amino]-5-methoxy-7-[3-(4-
methyl-l-piperazinyl)propoxy]-3-quinolinecarbonitrile inhibited Src catalyzed
phosphorylation in a homogeneous enzyme assay (FRET/Lance format) with an

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IC50 of 3.5 nM. A comparison of 4-[(2,4-dichloro-5-methoxyphenyl)amino]-5-
methoxy-7-[3-(4-methyl-l-piperazinyl)propoxy]-3-quinolmecarbonitrile activity
against various enzymes is given in Table 1.
[0033J Src and Abl Kinase Lance Assays: Recombinant human Src enzyme was
obtained from PanVera (P3044). Biotinylated peptide corresponding to residues
6-20 of Cdkl was used as a substrate in the src enzyme assay (Biotin-
KVEKIGEGTYGWYK-COOH). Wild type c-Abl andv-Abl were purchased
from Panvera (P3049) and Calbiochem (#102555), respectively. Biotinylated
peptide for the Abl kinase assay was obtained from Synpep (Biotm-NH-
KEEEAIYAAPFAKKK-COOH). For both Src and Abl kinase assays,
homogeneous fluorescence resonance energy transfer kinase assays were
performed using the europium/APC detection format (LANCE, Perkin Elmer).
Src enzyme (10 ng) or Abl enzyme (2.5 ng c-Abl, 2.5 ng v-Abl) was mixed with
biotinylated peptide (final concentration 2 uM for both substrate peptides), 50
mM Hepes (pH 7.5), 10 mM MgCl2, 20 ug/ml BSA, and 0.001% Brij-35 (Sigma).
Compound was added with a final DMSO concentration of 1 %, and incubated
for ten minutes at 37CC for Src assay and 27°C for Abl assay. The kinase reaction
was initiated by addition of ATP to a final concentration of 100 uM, and
incubated for 70 minutes at 37°C for Src, and 30 minutes at 27°C for Abl. The
reaction was stopped with EDTA at a final concentration of 30mM EDTA/25mM
Hepes (pH 1.5)110 pg/ml BSA. The mixture was combined with Eu-labeled
phosphotyrosine antibody PT66 (Perkin Elmer, AD0068) and Streptavidin
Surelight-APC (Perkin Elmer, CR130-100) in 50 mM Hepes (pH 7.5)/ 20 pg/ml
BSA, and incubated for 30 min according to manufacturer's specifications. The
reaction was monitored on a Wallac Victor with excitation at 340 ran and
emission at 665 ran. Data was analyzed with the LSW data analysis plug-in for
Excel (Microsoft).
[0034J PKA, PKC, S6 kinase, CAMKII and p38 kinase assays.
The Scintiplate assay is used for the kinase assay with PKA (Upstate #14-
114), PKC (Upstate #14-232) and S6K (Upstate #14-333). An ELISA format
is used for CAMK-II (Upstate # 14-217 and p38 (Recombinant protein
produced and purified in house)

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SCINTIPLATE ASSAY
96 well SA Cov Scintiplate (#1450-551) plates are washed 4 times (250 X) in
PBS (0.1% Triton X100) prior to the kinase assay.
• 60 pi mix* (See following table)
• 20 pi compounds (in 10 % DMSO)
20 min preincubation
• 20 [0,1 substrate 1 uM (see following table)
The volumes showed in the following table are for one 96 well plate..

Kinase PKA PKC S6K
# Wells 96.0 96.0 96.0
# Reactions 110.0 110.0 110.0
Total Vol 6600.0 6600.0 6600.0
H20 5452.5 5321.0 5343
lOxbuffer 1100.0 1100.0 1100.0
DTT1M 30.0 30.0 30.8
ATPlmM 11.0 11.0 11.0
ATP 33P 6.0 6.0 6.0
Kinases 0.55 22 110
pi /reaction 0.005 0.2 1
Lipid Activator 110

Substrate (1 uM) 1463 • 1463 1464
Reaction Time 20' 30' 80'
Peptide 1323 (LSP16Bio: Bio-RTPKLARQASIELPSM: LSP-1 aa 243-258. Ser
252 is the phosphorylation site)
Peptide 1463 (PKA substrate: Bio-GRTGRRNSI)
Peptide 1464 (S6K substrate: Bio-RRRLSSLRA)
Reaction start when substrate is added.
1- Stop reaction with 20 pi of 0.5M EDTA according to the time showed in the
table. Keep incubating the plate up to an hour after initiating the reaction or 80
min in the case of S6K.
2- Washes (PBS + 0.1% Triton XI00) six washes (250 X /well)
3- Count (Wallac Trilux counter)

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II ELISA
Following are the assay conditions for the kinases used in the ELISAs. p38
is activated with a dominant active mutant of MKK6.

Vol (microliter) p38 CAMKII
# Wells 96.0 96
# Reactions 110.0 110.0
Total Vol. 6600.0 6600.0
H20 5357.3 5464.5
lOxbuffer 1100.0 1100.0
DTT1M 30.0 30.0
lOOmMATP 2.8 3.3
Kinases (Vol or
final cone) 60nM 2
u.1 /reaction 0.02
C almodulin( 1 mg/ml) 44
CaC12(lM) 11
1738 1323
Reaction time 30' 30'
Peptide 1738 (MK2 T334: Bio-QSTKVPQTPLHTSRVL)
Peptide 1895 (Gastrin 1-17: Bio-KKEGPWLEEEEEAYGWMD)
The kinase assay is performed in the same fashion as the scintiplate assay:
See step 1 through 3 above. The detection plate is a Nunc MaxiSorb
precoated with Amersham goat anti-GST antibody 1:400 in PBS at 100
ul/well for 2 hours. The reaction plate is a Costar polysterene plate
preblocked with blocking buffer at 0.1% Gelatin for two hours with 200
ul/well.
Prepare ERK/ADB mix as follows:
10 ul GST-ERK2 per ml of ADB
Put 60 ul of ERK/ADB mix into negative control wells (Row 12)
Prepare MEK/ERK/ADB mix (MEA) by adding active MEK1
Put 60 ul/well MEA into assay plate.
Add 20 ul empd 10% DMSO

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Prepare 5x ATP/ADB by adding 500 uM ATP to ADB.
20 ul ATP/ADB to each well to start reaction. Incubate Ihr at 30 C.
Stop reaction by adding 20 ul 0.5M EDTA to each well.
4- Phospho peptide detection:
Antibodies: For the detection of phosphorylated peptide, affinity purified
polyclonal phospho-specific antibodies 60521 and 64273 are used against 1323
and 1738 respectively.
- Add 100 μl of blocking buffer supplemented with 1 % BSA with purified 60521
(0.46 mg/ml) at 1:20000 and anti rabbit-Eu (PE #AD0105) at 1:4000 or purified
64273 at 1:4000 and anti-rabbit-Eu at 1:2000 for the detection of phosphorylated
1323 and 1738 respectively.
- Add 100 μl of blocking buffer supplemented with 1% BSA with anti-phospho
tyrosine PT100 (Cell Signaling #9411) at 1:1000 and anti-mouse-Eu (PE #
ADO 124) for the detection of phosphorylated 1895.
Incubate 1 hour RT (Shaker)
Wash 6x 250 μl PBS 0.1% Tween 20
Add 100 ul Enhancement Solution (Wallac Cat# 1244-105)
Incubate 10 min RT (Shaker)
Read on Victor II (HTS Europium protocol on our reader)
BUFFERS
Kinase buffer 1 OX:
200 mM Hepes pH 7.5,100 mM MgCl2
ADB IX:
20 mM MOPS 7.2, 25 mM p-glycerophosphate, 5 mM EGTA, 1 mM Na
orthovanadate, 20 mM MgCl2> 1 mM DTT
Blocking Buffer:
10 mM MOPS 7.5, 150 mM NaCl, 0.05% Tween 20, 0.1% Gelatin,0.02% NaN3
Raf/MEK Kinase ELISA
Reagents: Sf9 insect cell lysate containing full length 6his-tagged recombinant
human c-Raf. (Specific Activity: ~200U/ml). Human Non-active Mek-1-GST and
human GST-MAP kinase (recombinant proteins produced in E.coli).

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Rafl Kinase Cascade Assay Procedure:
Raf-1 (c-Raf) is used to phosphorylate and activate inactive GST-MEK1 which
then can phosphorylate and activate inactive p42 GST-MAPK, which subsequently
is measured for phosphorylation of the TEY sequence (aa's 202-204) by a
phospho-specific antibody from Sigma (cat. # 77439219041)
Kinase Assay Protocol
Stock Solutions:
Raf Assay
1. Assay Dilution Buffer (ADB): 20mM MOPS, pH 7.2, 25mM B-glycerol
phosphate, 5mM EGTA, ImM sodium orthovanadate, ImM dithiothreitol.
2. Magnesium/ATP Cocktail: 500uM cold ATP and 75 mM magnesium chloride in
ADB.
3. Active Kinase: Human Active c-Raf: Use at 0.4U per assay point.
4. Non-active GST-MEK1: Use at 0.1 ug per assay point.
5. Non-active GST-p42 MAP Kinase: Use at 1.0 ug per assay point.
ELISA

1. TBST - Tris (50 mM, pH 7.5), NaCl (150 mM), Tween-20 (0.05 %)
2. Superblock (Pierce)
3. Anti-GST Ab (Pharmacia)
4. Anti-Phospho MAPK (Sigma)
5. Anti-Mouse Ab / Europium conjugate (Wallac)
Assay Procedure:
First Stage: c-Raf Dependent Activation of GST-MEK and GST-MAPK
1. Add 20 ml of ADB per assay (i.e. per well of a 96 well plate)
2. Add 10 ml of 0.5 mM cold ATP and 75 mM magnesium chloride in ADB.
3. Add 2 ml of c-Raf (0.4U/assay), in conjunction with 1.6ml non-active MEK1
(0.4 mg/assay).
4. Add 4 ml of non-active GST-p42 MAP Kinase (1.0 mg/assay).
5. Incubate for 60 minutes at 30°C in a shaking incubator.

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6. Transfer this mixture to an anti-GST Ab coated 96 well plate (Nunc
Immunosorb plates coated o/n with a-
GST, then blocked with Pierce Superblock).
7. Incubate for 60 minutes at 30°C in a shaking incubator
8. Wash 3X with TBST, add Anti-Phospho MAPK (Sigma) (1:3000)
9. Incubate for 60 minutes at 30°C in a shaking incubator

10. Wash 3X with TBST, add Anti-Mouse Ab / Europium conjugate (Wallac)
(1:500)
11. Incubate for 60 minutes at 30°C in a shaking incubator
12. Wash 3X with TBST, Read plates in Wallac Victor model Plate Reader.
KDR kinase assay.
Materials:
1) Nunc MaxiSorb 96F ELISA VWR 62409-024
2) Peptide substrate: poly(Glu4-Tyr), Sigma (P-0275): Prepare 5 mg/ml stock in
water.
3) TBS: BupH TBS Pierce (#28376); 25 mM Tris pH 7.2, 150 mM NaCl final
4) TBST: Wash Buffer = TBS+0.05% Tween-20: For 500 ml: TBS (above) +
2.5 ml of 10% Tween (made in TBS).
5) Compound: Prepared in DMSO. Store compounds at -80C.
6) 5X KDR Kinase Buffer:
5X IX For 50 ml of 5X
20 mM HEPES pH 7.4 4 mM HEPES 1 ml of 1 M HEPES
5 mM MnC12 1 mM MnC12 1.25 ml of 200 mM MnC12
100 uM Na3V04 20 uM Na3V04 0.1 ml of 50 mM Na3V04
7) Enzyme diluent: 0.1% BSA in 4 mM HEPES, pH 7.4
8) 2.5X ATP [10 uM final]/ MgC12 solution in HEPES:
2.5Xconc. 1Xinrxn for 10 ml of 2.5X
25uMATP lOuMATP 0.25 ml of 1 mM ATP
25 mM MgC12 10 mM MgC12 1 ml of 250 mM MgC12
10 mM HEPES 4 mM HEPES 0.1 ml of 1 M HEPES
8.65 ml water
9) 2.5X / MgC12 solution in HEPES (no ATP):
2.5X cone. IX in rxn for 5 ml of 2.5X
25mMMgC12 10mMMgC12 0.5 ml of 250mMMgC12
10 mM HEPES 4 mM HEPES 0.05 ml of 1 M HEPES
4.45 ml water
10) ATP (FW 551): AmershamPharmacia #27-2056-01 (25 umol): 100 mM
stock

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11); 250mMMgC12: SigmaM-8266, anhydrous,FW95.21; 1.19 g/50ml
water
12) Assay Buffer: PerkinElmer 1244-106
13) Eu-anti-PY (PT66): PerkinElmer AD0040 (50 ug, Sigma clone PT66, anti-
phosphotyrosine antibody).
14) Enhancement Solution: PerkinElmer 1244-105
Methods:
1. To Nunc MaxiSorb plates, add 100 ul poly(Ghi4~Tyr) peptide at 25 ug/ml in
TBS. Incubate 1 - 4 hr at RT. [Alternative: vary final poly(Glu4-Tyr)
peptide concentration from ~1 - 50 ug/ml; OR use poly(GlU6-Ala3-Tyr)
peptide]
2. Wash peptide-containing wells 3 times with 200 ul of TBS.
3. Add to each well: 29 ulofKDR Kinase Master Mix [=KDR-IC + 5XKDR
kinase buffer + water (mixed 1:1: 0.9)] Combine 10 ul of purified KDR-IC
prep, diluted (ie, 1:5 to 1:20 depending on prep) in 0.1% BSA / 4 mM HEPES
+ 10 ul of 5XKDR kinase buffer + 9 ul of water PER reaction well. Adjust
water volume accordingly if using more or less compound volume. These
additions can be made with a Matrix multi-pipettor.
4. Add 1 ul of compound (50X stock in DMSO depending on desired final
compound concentration) to each well (TV = 30 ul at this point).
5. Incubate ~15 min at RT to allow binding of compounds to enzyme.

4. Add 20 ul of 2.5X ATP/ MgC12/ HEPES solution to sample wells. For KDR,
linear range of reaction is -1-100 nM so 10 uM is typically used.
[Alternative: final ATP concentration can be varied; use of 2.5X
MgC12/HEPES with no ATP for some reactions allows determination of the
feasible low end range for any enzyme prep].
5. Incubate for 40 min at room temperature. [Alternative: 30 to 60 min assay
reaction; or 37C reaction temperature, although these make minimal difference
with current batches of KDR].
6. Wash the plates 3X with 200 ul of TBST.
7. Add 75 ul of Eu-PY at 1:2000 in Assay Buffer.
8. Incubate 45 - 60 mm @ RT.
9. Wash the plates 3X with 200 ul of TBST.

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10. Add 100 ul of Enhancement Solution (equilibrated to RT).
11. Read plates on VICTOR TR-fluorescent plate reader.
Raw europium (fluorescence) counts are converted to percent inhibition and/or
IC50 based on untreated (no compound) control wells using Excel templates.
[0035]
TABLE 1.
Activity of 4-[(2,4-dichloro-5-
methoxyphenyl)amino]-5-methoxy-7-[3-(4--
methyl-l-piperazinyl)propoxy]-3-
quinolinecarbonitrile against various kinases
ICso (MM)
p38 0.95
CAMKII 6.25
PKA 5.03
PKC-a 1.47
p70S6K 6.09
KDR 7.00
raf/mek 0.50
Src 0.003.5
c-Abl 0.001
IC50 = Concentration at which there is 50%
inhibition.
Cell Proliferation Data
[0036] The compounds of formula (I) is a selective inhibitor of Src kinase family
kinases and Abl kinases. Cell-based assays were also used to examine the
selectivity of 4-[(2,4-dichloro-5-methoxyphenyl)amino]-5-methoxy-7-[3-(4-
methyl-l-piperazinyl)propoxy]-3-quinolinecarbonitrile. Rat fibroblasts
overexpressing an oncogenic form of Src, where the catalytic domain of human
c-Src was inserted in place of the v-Src catalytic domain to create a fusion v-
Src/human c-Src protein. Similar substitutions were made with FynB, Lck, Lyn,
and Hck, other Src family members. In addition, the same cell type expressing
oncogenic forms of v-Abl, insulin-like growth factor-I receptor, fibroblast growth
factor receptor, platelet growth factor receptor and Her2 were also constructed.
Compound activity in these cells was determined using an anchorage-
independent growth assay, based on the acquisition of anchorage-independence

WO 2007/001839 PCT/US2006/023063
17
by fibroblasts expressing oncogenic proteins. Growth under these conditions is
dependent on kinase activity, and inhibition of kinase activity should block cell
growth in a manner that reflects inhibition of phosphorylation of cellular target
proteins. Table 2 shows proliferation data obtained for 4-[(2,4-dichloro-5-
methoxyphenyl)amino] -5-rnethoxy-7-[3 -(4-methyl-1 -piperazinyl)propoxy] -3 -
quinolinecarbonitrile under these conditions.
TABLE 2.
KINASE-DEPENDENT CELL-BASED ASSAYS

IC5o(uM)
Src 0.1
Lck 0.02
Fyn 0.4
Lyn 0.15
Hck 0.8
v-Abl 0.1
Her2 2
IC50 = Concentration at which there is 50% inhibition.
Head and Neck Tumor Lines
[0037] Compounds of formula (I) are potent inhibitors of tumor cell proliferation
in head and neck cell lines that overexpress Src. A representative proliferation
profile of 4-[(2,4-dichloro-5-methoxyphenyl)amino]-5-methoxy-7-[3-(4-methyl-
l-piperazinyl)propoxy]-3-quinolinecarbonitrile with the HN5 line is shown in
Figure 1, along with evidence for inhibition of phosphorylation of downstream
target proteins.
Description of Biological Test Procedures.
Standard growth conditions:
[0038] For the experiments described in Table 3, 1000 cells were plated in each
well of a 96 well cell culture dish on day 0 in the standard growth medium for
that particular cell line. Compound was added on day 1. Relative cell number
was determined on day 4.

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CellTiter-Glo method.
[0039] The CellTiter-Glo luminescent cell viability assay (Promega # G7573)
was used where cells were lysed with the CellTiter Glo reagent, agitated briefly
and allowed to sit at room temperature for 30 minutes prior to analyzing on a
Wallac plate reader equipped for luminescence readings.
MTS assay.
[0040] Some assays were monitored with the aid of the CellTiter 96 assay
(Promega # G3580). With this assay, on day 4, detection reagent was added to
the 96 well plate and absorbance at 490 nM was measured.
SRB assay.
[0041] The sulforhodamine B (SRB) assay was used for certain melanoma lines.
In this assay, the serum concentration in the growth medium was 5% and culture
medium volume was 0.2 ml. On day 4, 0.05 ml of 50% trichloroacetic acid was
added to the medium and the plate was allowed to sit at room temperature for 1
hour. The medium was decanted and the plate was washed 3 times with water.
The washed plates were dried, and then 0.08 ml of SRB reagent (SRB was
obtained from Sigma; 0.4% SRB in 1% acetic acid) was added. After 10
minutes at room temperature, the plates were washed with 1% acetic acid until no
free red color remained in solution. The bound SRB reagent was solubilized with
0.15 ml 10 mM Tris (no acid added). After agitating for 5 minutes on a shaker,
the absorbance was read at 540 nM.
In vivo studies
[0042] All animal studies were conducted under an approved Institutional
Animal Care and Use Committee protocol. Tumor cells were suspended to 50
million cells/ml and 0.2 ml of the cell suspension was injected subcutaneously
into a flank of 6 - 7 weeks old female nude mice (Charles River, Wilmington,
MA). Mice with tumors larger than 200 mm3 after one week were administered
vehicle or compound by intraperitoneal injection at the indicated doses in 0.2 ml
of vehicle containing 0.5% methylcellulose and 0.4% polysorbate 80 (Tween 80).

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[0043] Table 3 summarizes the proliferation assay results obtained upon treating
T-eell leukemia, prostate, pancreatic, melanoma and head and neck tumor lines
with4-(2,4-dichloro-5-methoxy-phenylamino)-6-methoxy-7-[3-(4-methyl-
piperazin-l-yl)-propoxy]-quinoline-3-carbonitrile. Also shown is the ability of 4-
(2,4-dichloro-5-methoxy-phenylamino)-6-methoxy-7-[3-(4-methyl-piperazin-l-
yl)-propoxy]-quinoline-3-carbonitrile administration to retard the growth of A375
melanoma subcutaneous tumor xenografts in nude mice.
TABLE 3.

IC50 (uM) method
T-cell leukemia
HSB-2 0.014 MTS J
Molt-4 8.2 MTS
Prostate
DU145 1.6 Cell-glo
LnCap 3.5 Celi-glo
Pancreatic
MiaPaca 1.3 Cell-glo
BxPC3 3.9 Cell-glo 4
Capan 5.9 Cell-glo
Melanoma
A375 1.2 Cell-glo
A375 0.9 SRB
RPMI-7951 1.2 SRB
HS-294-T 0.6 SRB
Head and neck
HN5 0.3 Cell-glo
In vivo study Dose T/C
A375 melanoma 30 mg/kg 39%, day 12 Bid
9 days ip

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WHAT IS CLAIMED IS:
1. A method of preventing, treating or inhibiting cancer comprising,
administering a therapeutically effective amount of a compound of formula (I)

or a pharmaceutic ally acceptable salt thereof.
2. The method of claim 1, wherein the cancer being prevented, treated or
inhibited is pancreatic cancer.
3. The method of claim 1, wherein the cancer being prevented, treated or
inhibited is lymphatic cancer.
4. The method of claim 1, wherein the cancer being prevented, treated or
inhibited is prostrate cancer.
5. The method of claim 1, wherein the cancer being prevented, treated or
inhibited is head and neck cancer.
6: The method of claim 1, wherein the cancer being prevented, treated or
inhibited is melanoma.
7. A pharmaceutical composition comprising an therapeutically effective
amount of a compound of formula (I):


WO 2007/001839 PCT/US2006/023063
21
or pharmaceutically acceptable salts thereof and a pharmaceutically
acceptable carrier.
8. The method of claim 1 wherein, the compound is delivered alone or in
combination with one or more other compounds used to treat cancer.
9. The method of claim 8, wherein the other compounds are selected from
imatinib mesylate, hydroxyurea, IFN-&, cytotoxic agents, genfitanib, gemcitabine,
avastin and wortmannin, or pharmaceutically acceptable salts thereof.
10. THE METHOD OF CLAIM 9, WHEREIN THE OTHER COMPOUND IS GEMEITABINE.
11. The method of claim 9, wherein the other compounds are gemcitabine and
avastin.
12. A method of treating pancreatic cancer comprising, administering a
therapeutically effective amount of a compound of formula (I):

or a pharmaceutically acceptable salt thereof, in combination with gemcitabine, or
a pharmaceutically acceptable salt thereof.
13. The method of claim 12, further comprising administering avastin, or a
pharmaceutically acceptable salt thereof.

The present invention is directed to a method of preventing treating and/or inhibiting cancer using compounds of
formula (I): or a pharmaceutically acceptable salt thereof. This invention is also directed to pharmaceutical compositions containing
compounds of formula (I).