U.S. patent application number 15/857366 was filed with the patent office on 2018-06-21 for maleate salts of a quinazoline derivative useful as an antiangiogenic agent.
This patent application is currently assigned to AstraZeneca AB. The applicant listed for this patent is AstraZeneca AB. Invention is credited to James MCCABE.
Application Number | 20180170913 15/857366 |
Document ID | / |
Family ID | 30776508 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180170913 |
Kind Code |
A1 |
MCCABE; James |
June 21, 2018 |
MALEATE SALTS OF A QUINAZOLINE DERIVATIVE USEFUL AS AN
ANTIANGIOGENIC AGENT
Abstract
The present invention relates to AZD2171 maleate salt, to
particular crystalline forms of AZD2171 maleate salt, to processes
for their preparation, to pharmaceutical compositions containing
them as active ingredient, to their use in the manufacture of
medicaments for use in the production of antiangiogenic and/or
vascular permeability reducing effects in warm-blooded animals such
as humans, and to their use in methods for the treatment of disease
states associated with angiogenesis and/or increased vascular
permeability.
Inventors: |
MCCABE; James;
(Macclesfield, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AstraZeneca AB |
Sodertalje |
|
SE |
|
|
Assignee: |
AstraZeneca AB
Sodertalje
SE
|
Family ID: |
30776508 |
Appl. No.: |
15/857366 |
Filed: |
December 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15264527 |
Sep 13, 2016 |
9890140 |
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15857366 |
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14489721 |
Sep 18, 2014 |
9556151 |
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15264527 |
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12646153 |
Dec 23, 2009 |
8859570 |
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14489721 |
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10581279 |
Jun 1, 2006 |
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PCT/GB2004/005359 |
Dec 18, 2004 |
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12646153 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 15/00 20180101;
A61K 31/517 20130101; A61P 9/10 20180101; A61P 19/02 20180101; A61P
37/02 20180101; C07C 57/145 20130101; A61K 45/06 20130101; A61P
37/06 20180101; A61P 27/02 20180101; A61P 17/02 20180101; A61P 9/00
20180101; A61P 35/00 20180101; A61P 43/00 20180101; A61P 29/00
20180101; A61P 13/12 20180101; A61P 35/02 20180101; C07D 403/12
20130101; A61P 3/10 20180101; A61P 17/06 20180101 |
International
Class: |
C07D 403/12 20060101
C07D403/12; C07C 57/145 20060101 C07C057/145; A61K 45/06 20060101
A61K045/06; A61K 31/517 20060101 A61K031/517 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 2003 |
GB |
0330002.7 |
Claims
1-20. (canceled)
21. A method for inhibiting VEGF receptor tyrosine kinase in a
warm-blooded animal in need of such treatment which comprises
orally administering to said animal an effective amount of a
pharmaceutical composition comprising crystalline Form A of
4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)-
propoxy)quinazoline maleate, wherein said warm-blooded animal in
need of such treatment has a solid tumor cancer that is
recurrent.
22. The method according to claim 21, wherein said cancer is a
female reproductive cancer.
23. The method according to claim 21, wherein the daily dose of
Form A of
4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)-
propoxy)quinazoline maleate administered to said animal ranges from
0.03 mg/kg to 0.5 mg/kg.
24. The method according to claim 21, further comprising
administering at least one additional treatment chosen from
surgery, radiotherapy, and chemotherapy.
25. The method according to claim 24, wherein said chemotherapy
comprises at least one therapeutic agent chosen from (i) other
antiangiogenic agents; (ii) cytostatic agents; and (iii)
antiproliferative/antineoplastic drugs.
26. The method according to claim 25, wherein said
antiproliferative/antineoplastic drugs are chosen from platinum
derivatives.
Description
[0001] The present invention relates to AZD2171 maleate salt, to
particular crystalline forms of AZD2171 maleate salt, to processes
for their preparation, to pharmaceutical compositions containing
them as active ingredient, to their use in the manufacture of
medicaments for use in the production of antiangiogenic and/or
vascular permeability reducing effects in warm-blooded animals such
as humans, and to their use in methods for the treatment of disease
states associated with angiogenesis and/or increased vascular
permeability.
[0002] Normal angiogenesis plays an important role in a variety of
processes including embryonic development, wound healing and
several components of female reproductive function. Undesirable or
pathological angiogenesis has been associated with disease states
including diabetic retinopathy, psoriasis, cancer, rheumatoid
arthritis, atheroma, Kaposi's sarcoma and haemangioma (Fan et al,
1995, Trends Pharmacol. Sci. 16: 57-66; Folkman, 1995, Nature
Medicine 1: 27-31). Alteration of vascular permeability is thought
to play a role in both normal and pathological physiological
processes (Cullinan-Bove et al, 1993, Endocrinology 133: 829-837;
Senger et al, 1993, Cancer and Metastasis Reviews, 12: 303-324).
Several polypeptides with in vitro endothelial cell growth
promoting activity have been identified including, acidic and basic
fibroblast growth factors (aFGF & bFGF) and vascular
endothelial growth factor (VEGF). By virtue of the restricted
expression of its receptors, the growth factor activity of VEGF, in
contrast to that of the FGFs, is relatively specific towards
endothelial cells. Recent evidence indicates that VEGF is an
important stimulator of both normal and pathological angiogenesis
(Jakeman et al, 1993, Endocrinology, 133: 848-859; Kolch et al,
1995, Breast Cancer Research and Treatment, 36:139-155) and
vascular permeability (Connolly et al, 1989, J. Biol. Chem. 264:
20017-20024). Antagonism of VEGF action by sequestration of VEGF
with antibody can result in inhibition of tumour growth (Kim et al,
1993, Nature 362: 841-844).
[0003] Receptor tyrosine kinases (RTKs) are important in the
transmission of biochemical signals across the plasma membrane of
cells. These transmembrane molecules characteristically consist of
an extracellular ligand-binding domain connected through a segment
in the plasma membrane to an intracellular tyrosine kinase domain.
Binding of ligand to the receptor results in stimulation of the
receptor-associated tyrosine kinase activity which leads to
phosphorylation of tyrosine residues on both the receptor and other
intracellular molecules. These changes in tyrosine phosphorylation
initiate a signalling cascade leading to a variety of cellular
responses. To date, at least nineteen distinct RTK subfamilies,
defined by amino acid sequence homology, have been identified. One
of these subfamilies is presently comprised by the fms-like
tyrosine kinase receptor, Flt-1, the kinase insert
domain-containing receptor, KDR (also referred to as Flk-1), and
another fms-like tyrosine kinase receptor, Flt-4. Two of these
related RTKs, Flt-1 and KDR, have been shown to bind VEGF with high
affinity (De Vries et al, 1992, Science 255: 989-991; Terman et al,
1992, Biochem. Biophys. Res. Comm. 1992, 187: 1579-1586). Binding
of VEGF to these receptors expressed in heterologous cells has been
associated with changes in the tyrosine phosphorylation status of
cellular proteins and calcium fluxes.
[0004] VEGF is a key stimulus for vasculogenesis and angiogenesis.
This cytokine induces a vascular sprouting phenotype by inducing
endothelial cell proliferation, protease expression and migration,
and subsequent organisation of cells to form a capillary tube
(Keck, P. J., Hauser, S. D., Krivi, G., Sanzo, K., Warren, T.,
Feder, J., and Connolly, D. T., Science (Washington D.C.), 246:
1309-1312, 1989; Lamoreaux, W. J., Fitzgerald, M. E., Reiner, A.,
Hasty, K. A., and Charles, S. T., Microvasc. Res., 55: 29-42, 1998;
Pepper, M. S., Montesano, R., Mandroita, S. J., Orci, L. and
Vassalli, J. D., Enzyme Protein, 49: 138-162, 1996.). In addition,
VEGF induces significant vascular permeability (Dvorak, H. F.,
Detmar, M., Claffey, K. P., Nagy, J. A., van de Water, L., and
Senger, D. R., (Int. Arch. Allergy Immunol., 107: 233-235, 1995;
Bates, D. O., Heald, R. I., Curry, F. E. and Williams, B. J.
Physiol. (Lond.), 533: 263-272, 2001), promoting formation of a
hyper-permeable, immature vascular network which is characteristic
of pathological angiogenesis.
[0005] It has been shown that activation of KDR alone is sufficient
to promote all of the major phenotypic responses to VEGF, including
endothelial cell proliferation, migration, and survival, and the
induction of vascular permeability (Meyer, M., Clauss, M.,
Lepple-Wienhues, A., Waltenberger, J., Augustin, H. G., Ziche, M.,
Lanz, C., Buttner, M., Rziha, H-J., and Dehio, C., EMBO J., 18:
363-374, 1999; Zeng, H., Sanyal, S. and Mukhopadhyay, D., J. Biol.
Chem., 276: 32714-32719, 2001; Gille, H., Kowalski, J., Li, B.,
LeCouter, J., Moffat, B, Zioncheck, T. F., Pelletier, N. and
Ferrara, N., J. Biol. Chem., 276: 3222-3230, 2001).
[0006] Compounds which inhibit the effects of VEGF are of value in
the treatment of disease states associated with angiogenesis and/or
increased vascular permeability such as cancer (including
leukaemia, multiple myeloma and lymphoma), diabetes, psoriasis,
rheumatoid arthritis, Kaposi's sarcoma, haemangioma, acute and
chronic nephropathies, atheroma, arterial restenosis, autoimmune
diseases, acute inflammation, excessive scar formation and
adhesions, endometriosis, lymphoedema, dysfunctional uterine
bleeding and ocular diseases with retinal vessel proliferation
including macular degeneration.
[0007] Quinazoline derivatives which are inhibitors of VEGF
receptor tyrosine kinase are described in WO 00/47212. The compound
AZD2171 is exemplifed in WO 00/47212, (see Example 240), and is
4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)-
propoxy)quinazoline of the formula I:
##STR00001##
[0008] AZD2171 shows excellent activity in the in vitro (a) enzyme
and (b) HUVEC assays that are described in WO 00/47212 and
hereinafter. The AZD2171 IC.sub.50 values for inhibition of
isolated KDR (VEGFR-2) and Flt-1 (VEGFR-1) tyrosine kinase
activities in the enzyme assay were <2 nM and 5.+-.2 nM
respectively. AZD2171 inhibits VEGF-stimulated endothelial cell
proliferation potently (IC.sub.50 value of 0.4.+-.0.2 nM in the
HUVEC assay), but does not inhibit basal endothelial cell
proliferation appreciably at a >1250 fold greater concentration
(IC.sub.50 value is >500 nM). The growth of a Calu-6 tumour
xenograft in the in vivo (c) solid tumour model described
hereinafter was inhibited by 49%**, 69%*** and 91%*** following 28
days of once-daily oral treatment with 1.5, 3 and 6 mg/kg/day
AZD2171 respectively (P**<0.01, P***<0.0001; one-tailed t
test).
[0009] More stable forms of a pharmaceutically active compound, for
example more stable crystalline forms, are preferred for
formulation and processing on a commercial scale. This is because
the greater the stability of the form used, the lower the risk of
it converting to another form during formulation procedures such as
compression. This in turn provides greater predictability of the
properties of the final formulation, such as dissolution rate of
tablets, bioavailability of active ingredient. Using a more stable
form of an active ingredient allows greater control over the
physical properties of the formulation.
[0010] AZD2171 free base
(4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl-
)propoxy)quinazoline) is a crystalline monohydrate under ambient
conditions. Differential Scanning calorimetry (DSC) analysis was
carried out according to the method described hereinafter and shows
a large broad endotherm between 95.degree. and 170.degree. C. due
to loss of water and melting (FIG. 1). Thermogravimetric (TGA)
analysis (details given hereinafter) shows a weight loss of 4.02%
between 80.degree. C. and 115.degree. C. (FIG. 1). Karl Fischer
water analysis (details given hereinafter) yields a figure of 3.9%
suggesting that all the weight loss is due to water loss.
[0011] It will be understood that the onset/peak temperature values
of the DSC may vary slightly from one machine to another or from
one sample to another, and so the values quoted are not to be
construed as absolute.
[0012] AZD2171 free base is characterised in providing at least one
of the following 20 values measured using CuKa radiation: 18.3 and
20.8. AZD2171 free base is characterised in providing an X-ray
powder diffraction pattern, as in FIG. 2. The ten most prominent
peaks are shown in Table 1.
TABLE-US-00001 TABLE 1 Ten most Prominent X-Ray Powder Diffraction
peaks for AZD2171 free base Angle 2- Intensity Relative Theta
(2.theta.) Count Intensity 18.287 100 vs 20.807 66.7 vs 27.277 48.9
vs 23.370 42.8 vs 14.684 39.8 vs 25.070 37.6 vs 13.966 32.2 vs
21.711 26.6 vs 22.898 23.1 vs 26.790 22.9 vs vs = very strong
[0013] It has been found that when a sample of AZD2171 free base is
dehydrated, for example on heating to 100.degree. C., the sample
becomes amorphous (FIG. 3) and does not then rehydrate but stays
amorphous thereafter. This could be problematic if AZD2171 free
base were to be formulated as a pharmaceutical composition because
AZD2171 free base could dehydrate during certain processes e.g.
particle size reduction (such as milling), drying of bulk drug,
formulating, manufacturing. In order to formulate AZD2171 free base
as a pharmaceutical composition it would be necessary to reduce the
particle size at some point, and this would carry a risk of
dehydration and therefore the risk of the formation of amorphous
material. This was investigated by subjecting a sample of AZD2171
free base monohydrate to particle size reduction by micronisation
and then analysing it to look for amorphous material. FIG. 4 shows
that amorphous material does indeed form during particle size
reduction of AZD2171 free base. This is shown by a broadening of
the peaks and formation of an amorphous `hump`--see FIG. 4. An
amorphous or semi-amorphous form of AZD2171 free base could give
rise to manufacturing problems and non-reproducible
bioavailability.
[0014] The identification of alternative forms of AZD2171, forms
that are different from the free base and that have improved solid
state properties, is the subject of the present invention.
[0015] An example of a different form is a salt of AZD2171. In WO
00/47212 it says that pharmaceutically acceptable salts of the
compounds of the invention therein may include acid addition salts
of the compounds of the invention which are sufficiently basic to
form such salts. Such acid addition salts are said to include salts
with inorganic or organic acids affording pharmaceutically
acceptable anions such as with hydrogen halides especially
hydrochloric or hydrobromic acid or with sulphuric or phosphoric
acid, or with trifluoroacetic, citric or maleic acid. In addition
WO 00/47212 goes on to say that where the compounds of the
invention therein are sufficiently acidic, pharmaceutically
acceptable salts may be formed with an inorganic or organic base
which affords a pharmaceutically acceptable cation. Such salts with
inorganic or organic bases are said to include an alkali metal
salt, such as a sodium or potassium salt, an alkaline earth metal
salt such as a calcium or magnesium salt, an ammonium salt or for
example a salt with methylamine, dimethylamine, trimethylamine,
piperidine, morpholine or tris-(2-hydroxyethyl)amine.
[0016] Preferred salts in WO 00/47212 are hydrochlorides and
hydrobromides, especially hydrochlorides.
[0017] Nowhere in WO 00/47212 does it state that a particular salt
of a particular compound therein will possess surprisingly
beneficial properties.
[0018] Unexpectedly and surprisingly we have now found that the
maleate salt of AZD2171 is an advantageously stable form of AZD2171
with improved solid state properties over the free base and over
other salts that have been tested.
[0019] AZD2171 maleate is readily crystallised, is highly
crystalline, non-hygroscopic and has a reproducible stoichiometric
ratio of drug to counter-ion of 1:1.
[0020] Thus AZD2171 maleate is readily crystallised, is highly
crystalline, non-hygroscopic and has a reproducible stoichiometric
ratio of drug to counter-ion of about 1:1.
[0021] Several salts of AZD2171 were prepared and seven were found
to be crystalline: malonate, succinate, fumarate, maleate,
tartarate, adipate and malate. The solid state properties of these
7 salts were tested and the results are shown in Table 2:
TABLE-US-00002 TABLE 2 Properties of AZD2171 Salts Evidence
Moisture of Hydrate Crystalline Drug:Counter-ion Content at
Formation.sup.b No of Salt (Yes/No) Stoichiometry.sup.a 80%
RH.sup.b (Yes/No) Polymorphs.sup.c Malonate Yes -- -- Yes .gtoreq.3
Succinate Yes 1:0.63 11.4 No .gtoreq.2 Fumarate Yes 1:0.5 3.5 No
.gtoreq.3 Maleate Yes 1:1 0.4 No .gtoreq.2 Tartarate Yes 1:0.75 9.3
No .gtoreq.1 Adipate Yes 1:0.75 -- No .gtoreq.3 Malate Yes -- 7.7
Yes -- .sup.aDrug:counterion stoichiometry from .sup.1H NMR
Spectrum data .sup.bMoisture content at 80% relative humidity (RH).
Evidence of hydration from Vapour Sorption studies (observed
hysteresis and absorption of water) or Thermogravimetric Analysis
(TGA) .sup.cEvidence for polymorphism from Differential Scanning
Calorimetry (DSC) thermograms
The term `non-hygroscopic` means absorbing <1% moisture at 80%
RH.
[0022] The AZD2171 maleate salt was surprisingly better than the
others because of the 7 salts that it was possible to crystallise,
it was found to be the only non-hygroscopic salt, to be highly
crystalline and to have a reproducible stoichiometric ratio of drug
to counter-ion of 1:1.
[0023] Thus AZD2171 maleate was found to be the only
non-hygroscopic salt, to be highly crystalline and to have a
reproducible stoichiometric ratio of drug to counter-ion of about
1:1.
[0024] AZD2171 maleate salt is substantially free of amorphous
material and can be expected to be easier to formulate than AZD2171
free base and to provide more reproducible dosing results. By
"substantially free of amorphous material" is meant that the amount
of amorphous material is less than 10%, preferably less than 5%,
more preferably less than 2%.
[0025] AZD2171 maleate salt is non-hygroscopic which should prevent
or reduce any problems associated with weight changes of the active
ingredient during procedures such as micronisation.
[0026] According to the present invention there is provided a
maleate salt of AZD2171.
[0027] AZD2171 maleate has two crystalline forms A and B.
[0028] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A.
[0029] AZD2171 Maleate Form A is characterised in providing at
least one of the following 28 values measured using CuKa radiation:
21.5 and 16.4. AZD2171 Maleate Form A is characterised in providing
an X-ray powder diffraction pattern, substantially as shown in FIG.
5. The ten most prominent peaks are shown in Table 3:
TABLE-US-00003 TABLE 3 Ten most Prominent X-Ray Powder Diffraction
peaks for AZD2171 Maleate Form A Angle 2- Intensity Relative Theta
(2.theta.) Count Intensity 21.522 100 vs 16.366 78.3 vs 24.381 73.7
vs 20.721 71.7 vs 25.025 71.5 vs 16.921 55.5 vs 12.085 44.1 vs
22.177 42.2 vs 17.444 40.7 vs 17.627 39.1 vs vs = very strong
[0030] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at about 2-theta=21.5.degree..
[0031] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at about 2-theta=16.4.degree..
[0032] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least two specific peaks at about 2-theta=21.5.degree. and
16.4.degree..
[0033] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with
specific peaks at about 2-theta=21.5, 16.4, 24.4, 20.7, 25.0, 16.9,
12.1, 22.2, 17.4 and 17.6.degree..
[0034] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern
substantially the same as the X-ray powder diffraction pattern
shown in FIG. 5.
[0035] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at 2-theta=21.5.degree. plus or minus
0.5.degree. 2-theta.
[0036] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at 2-theta=16.4.degree. plus or minus
0.5.degree. 2-theta.
[0037] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least two specific peaks at 2-theta=21.5.degree. and 16.4.degree.
wherein said values may be plus or minus 0.5.degree. 2-theta.
[0038] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with
specific peaks at 2-theta=21.5, 16.4, 24.4, 20.7, 25.0, 16.9, 12.1,
22.2, 17.4 and 17.6.degree. wherein said values may be plus or
minus 0.5.degree. 2-theta.
[0039] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at 2-theta=21.5.degree..
[0040] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at 2-theta=16.4.degree..
[0041] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with at
least two specific peaks at 2-theta=21.5.degree. and
16.4.degree..
[0042] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern with
specific peaks at 2-theta=21.5, 16.4, 24.4, 20.7, 25.0, 16.9, 12.1,
22.2, 17.4 and 17.6.degree..
[0043] According to the present invention there is provided a
maleate salt of AZD2171 in a first crystalline form, Form A,
wherein said salt has an X-ray powder diffraction pattern as shown
in FIG. 5.
[0044] DSC analysis shows AZD2171 maleate Form A is a high melting
solid with an onset of melting at 198.3.degree. C. and a peak at
200.08.degree. C. (FIG. 6).
[0045] Thus DSC analysis shows AZD2171 maleate Form A is a high
melting solid with an onset of melting at about 198.3.degree. C.
and a peak at about 200.08.degree. C.
[0046] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B.
[0047] AZD2171 Maleate Form B is characterised in providing at
least one of the following 28 values measured using CuKa radiation:
24.2 and 22.7. AZD2171 Maleate Form B is characterised in providing
an X-ray powder diffraction pattern substantially as shown in FIG.
8. The ten most prominent peaks are shown in Table 4:
TABLE-US-00004 TABLE 4 Ten most Prominent X-Ray Powder Diffraction
peaks for AZD2171 Maleate Form B Angle 2- Intensity Relative Theta
(2.theta.) Count Intensity 24.156 100 vs 22.740 84.3 vs 15.705 64.0
vs 11.995 63.7 vs 27.087 60.9 vs 25.032 56.8 vs 17.724 37.7 vs
15.044 35.4 vs 23.102 34.5 vs 12.625 34.2 vs vs = very strong
[0048] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at about 2-theta=24.2.degree..
[0049] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at about 2-theta=22.7.degree..
[0050] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least two specific peaks at about 2-theta=24.2.degree. and
22.7.degree..
[0051] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with
specific peaks at about 2-theta=24.2, 22.7, 15.7, 12.0, 27.1, 25.0,
17.7, 15.0, 23.1 and 12.6.degree..
[0052] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern
substantially the same as the X-ray powder diffraction pattern
shown in FIG. 8.
[0053] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at 2-theta=24.2.degree. plus or minus
0.5.degree. 2-theta.
[0054] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at 2-theta=22.7.degree. plus or minus
0.5.degree. 2-theta.
[0055] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least two specific peaks at 2-theta=24.2.degree. and 22.7.degree.
wherein said values may be plus or minus 0.5.degree. 2-theta.
[0056] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with
specific peaks at 2-theta=24.2, 22.7, 15.7, 12.0, 27.1, 25.0, 17.7,
15.0, 23.1 and 12.6.degree. wherein said values may be plus or
minus 0.5.degree. 2-theta.
[0057] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at 2-theta=24.2.degree..
[0058] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least one specific peak at 2-theta=22.7.degree..
[0059] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with at
least two specific peaks at 2-theta=24.2.degree. and
22.7.degree..
[0060] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern with
specific peaks at 2-theta=24.2, 22.7, 15.7, 12.0, 27.1, 25.0, 17.7,
15.0, 23.1 and 12.6.degree..
[0061] According to the present invention there is provided a
maleate salt of AZD2171 in a second crystalline form, Form B,
wherein said salt has an X-ray powder diffraction pattern as shown
in FIG. 8.
[0062] DSC analysis shows AZD2171 maleate Form B is a high melting
solid with an onset of melting at 194.43.degree. C. and a peak at
195.97.degree. C. (FIG. 9).
[0063] Thus DSC analysis shows AZD2171 maleate Form B is a high
melting solid with an onset of melting at about 194.43.degree. C.
and a peak at about 195.97.degree. C.
[0064] Form B is meta-stable with respect to Form A (the melting
point and heat of fusion of Form B are lower than those for Form
A). Form A is the more thermodynamically stable form. A mixture of
Form A and B converts to Form A upon slurrying in methanol at
40.degree. C. for 4 days (FIG. 10).
[0065] Form A is preferred over Form B.
[0066] AZD2171 maleate is non-hygroscopic, absorbing <1%
moisture at 80% relative humidity (FIG. 7).
[0067] The NMR details are given after the maleate salt preparation
in Example 1 and show for the stoichiometry data a ratio of
1:1.
[0068] According to another aspect of the present invention there
is provided a dimaleate salt of AZD2171. A dimaleate salt my be
formed through the addition of two moles of maleic acid to one mole
of AZD2171 free base.
[0069] When it is stated that the present invention relates to a
crystalline form of AZD2171 free base, or AZD2171 maleate Form A or
AZD2171 maleate Form B, the degree of crystallinity is conveniently
greater than about 60%, more conveniently greater than about 80%,
preferably greater than about 90% and more preferably greater than
about 95%. Most preferably the degree of crystallinity is greater
than about 98%.
[0070] The AZD2171 maleate salt forms A and B provide X-ray powder
diffraction patterns substantially the same as the X-ray powder
diffraction patterns shown in FIGS. 5 and 8 respectively and have
substantially the ten most prominent peaks (angle 2-theta values)
shown in Tables 3 and 4 respectively. It will be understood that
the 2-theta values of the X-ray powder diffraction pattern may vary
slightly from one machine to another or from one sample to another,
and so the values quoted are not to be construed as absolute.
[0071] It is known that an X-ray powder diffraction pattern may be
obtained which has one or more measurement errors depending on
measurement conditions (such as equipment or machine used). In
particular, it is generally known that intensities in an X-ray
powder diffraction pattern may fluctuate depending on measurement
conditions. Therefore it should be understood that the AZD2171
maleate salt forms of the present invention are not limited to the
crystals that provide X-ray powder diffraction patterns identical
to the X-ray powder diffraction patterns shown in FIGS. 5 and 8,
and any crystals providing X-ray powder diffraction patterns
substantially the same as those shown in FIGS. 5 and 8 fall within
the scope of the present invention. A person skilled in the art of
X-ray powder diffraction is able to judge the substantial identity
of X-ray powder diffraction patterns.
[0072] Persons skilled in the art of X-ray powder diffraction will
realise that the relative intensity of peaks can be affected by,
for example, grains above 30 microns in size and non-unitary aspect
ratios, which may affect analysis of samples. The skilled person
will also realise that the position of reflections can be affected
by the precise height at which the sample sits in the
diffractometer and the zero calibration of the diffractometer. The
surface planarity of the sample may also have a small effect. Hence
the diffraction pattern data presented are not to be taken as
absolute values. (Jenkins, R & Snyder, R. L. `Introduction to
X-Ray Powder Diffractometry` John Wiley & Sons 1996; Bunn, C.
W. (1948), Chemical Crystallography, Clarendon Press, London; Klug,
H. P. & Alexander, L. E. (1974), X-Ray Diffraction
Procedures).
[0073] Generally, a measurement error of a diffraction angle in an
X-ray powder diffractogram is about 5% or less, in particular plus
or minus 0.5.degree. 2-theta, and such degree of a measurement
error should be taken into account when considering the X-ray
powder diffraction patterns in FIGS. 2, 3, 4, 5, 8 and 10 and when
reading Tables 1, 3 and 4. Furthermore, it should be understood
that intensities may fluctuate depending on experimental conditions
and sample preparation (preferred orientation).
[0074] For the avoidance of doubt, terms such as `AZD2171 maleate
salt` and `a maleate salt of AZD2171` refer to each and every form
of AZD2171 maleate salt, whereas `AZD2171 maleate Form A` refers to
the particular crystalline form known as Form A and `AZD2171
maleate Form B` refers to the particular crystalline form known as
Form B.
[0075] According to a further aspect of the invention there is
provided a pharmaceutical composition which comprises an AZD2171
maleate salt as defined hereinbefore in association with a
pharmaceutically acceptable excipient or carrier.
[0076] The composition may be in a form suitable for oral
administration, (for example as tablets, lozenges, hard or soft
capsules, aqueous or oily suspensions, emulsions, dispersible
powders or granules, syrups or elixirs), for administration by
inhalation (for example as a finely divided powder or a liquid
aerosol), for administration by insufflation (for example as a
finely divided powder), for parenteral injection (for example as a
sterile solution, suspension or emulsion for intravenous,
subcutaneous, intramuscular, intravascular or infusion dosing), for
topical administration (for example as creams, ointments, gels, or
aqueous or oily solutions or suspensions), or for rectal
administration (for example as a suppository). Preferably AZD2171
maleate salt is administered orally. In general the above
compositions may be prepared in a conventional manner using
conventional excipients.
[0077] The compositions of the present invention are advantageously
presented in unit dosage form. AZD2171 maleate will normally be
administered to a warm-blooded animal at a unit dose within the
range 1-50 mg per square metre body area of the animal, for example
approximately 0.03-1.5 mg/kg in a human. A unit dose in the range,
for example, 0.01-1.5 mg/kg, for example 0.05-0.75 mg/kg,
preferably 0.03-0.5 mg/kg is envisaged and this is normally a
therapeutically-effective dose. A unit dosage form such as a tablet
or capsule will usually contain, for example 1-50 mg of active
ingredient. Preferably a daily dose in the range of 0.03-0.5 mg/kg
is employed. The size of the dose required for the therapeutic or
prophylactic treatment of a particular disease state will
necessarily be varied depending on the host treated, the route of
administration and the severity of the illness being treated.
Accordingly the optimum dosage may be determined by the
practitioner who is treating any particular patient.
[0078] According to a further aspect of the present invention there
is provided an AZD2171 maleate salt as defined hereinbefore for use
in a method of treatment of the human or animal body by
therapy.
[0079] A further feature of the present invention is an AZD2171
maleate salt as defined hereinbefore for use as a medicament,
conveniently an AZD2171 maleate salt as defined hereinbefore for
use as a medicament for producing an antiangiogenic and/or vascular
permeability reducing effect in a warm-blooded animal such as a
human being.
[0080] Thus according to a further aspect of the invention there is
provided the use of an AZD2171 maleate salt as defined hereinbefore
in the manufacture of a medicament for use in the production of an
antiangiogenic and/or vascular permeability reducing effect in a
warm-blooded animal such as a human being.
[0081] According to a further feature of the invention there is
provided a method for producing an antiangiogenic and/or vascular
permeability reducing effect in a warm-blooded animal, such as a
human being, in need of such treatment which comprises
administering to said animal an effective amount of an AZD2171
maleate salt as defined hereinbefore.
[0082] AZD2171 maleate salt is an antiangiogenic and/or vascular
permeability reducing agent and may be applied as a sole therapy or
may involve, in addition to AZD2171 maleate, one or more other
substances and/or treatments. Such conjoint treatment may be
achieved by way of the simultaneous, sequential or separate
administration of the individual components of the treatment. In
the field of medical oncology it is normal practice to use a
combination of different forms of treatment to treat each patient
with cancer. In medical oncology the other component(s) of such
conjoint treatment in addition to AZD2171 maleate salt may be:
surgery, radiotherapy or chemotherapy. Such chemotherapy may cover
three main categories of therapeutic agent:
(i) other antiangiogenic agents such as those which inhibit the
effects of vascular endothelial growth factor, (for example the
anti-vascular endothelial cell growth factor antibody bevacizumab
[Avastin.TM.], and those that work by different mechanisms from
those defined hereinbefore (for example linomide, inhibitors of
integrin .alpha.v.beta.3 function, angiostatin, razoxin,
thalidomide), and including vascular targeting agents (for example
combretastatin phosphate and compounds disclosed in International
Patent Applications WO00/40529, WO 00/41669, WO01/92224, WO02/04434
and WO02/08213 and the vascular damaging agents described in
International Patent Application Publication No. WO 99/02166 the
entire disclosure of which document is incorporated herein by
reference, (for example N-acetylcolchinol-O-phosphate)); (ii)
cytostatic agents such as antioestrogens (for example tamoxifen,
toremifene, raloxifene, droloxifene, iodoxyfene), oestrogen
receptor down regulators (for example fulvestrant), progestogens
(for example megestrol acetate), aromatase inhibitors (for example
anastrozole, letrazole, vorazole, exemestane), antiprogestogens,
antiandrogens (for example flutamide, nilutamide, bicalutamide,
cyproterone acetate), LHRH agonists and antagonists (for example
goserelin acetate, luprolide, buserelin), inhibitors of
5.alpha.-reductase (for example finasteride), anti-invasion agents
(for example metalloproteinase inhibitors like marimastat and
inhibitors of urokinase plasminogen activator receptor function)
and inhibitors of growth factor function, (such growth factors
include for example platelet derived growth factor and hepatocyte
growth factor), such inhibitors include growth factor antibodies,
growth factor receptor antibodies, (for example the anti-erbb2
antibody trastuzumab [Herceptin.TM.] and the anti-erbb1 antibody
cetuximab [C225]), farnesyl transferase inhibitors, tyrosine kinase
inhibitors for example inhibitors of the epidermal growth factor
family (for example EGFR family tyrosine kinase inhibitors such as
N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-
-amine (gefitinib, AZD1839),
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine
(erlotinib, OSI-774) and
6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazoli-
n-4-amine (CI 1033)) and serine/threonine kinase inhibitors); and
(iii) antiproliferative/antineoplastic drugs and combinations
thereof, as used in medical oncology, such as antimetabolites (for
example antifolates like methotrexate, fluoropyrimidines like
5-fluorouracil, tegafur, purine and adenosine analogues, cytosine
arabinoside); antitumour antibiotics (for example anthracyclines
like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin and
idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum
derivatives (for example cisplatin, carboplatin); alkylating agents
(for example nitrogen mustard, melphalan, chlorambucil, busulphan,
cyclophosphamide, ifosfamide, nitrosoureas, thiotepa); antimitotic
agents (for example vinca alkaloids like vincristine, vinblastine,
vindesine, vinorelbine, and taxoids like taxol, taxotere);
topoisomerase inhibitors (for example epipodophyllotoxins like
etoposide and teniposide, amsacrine, topotecan, camptothecin and
also irinotecan); also enzymes (for example asparaginase); and
thymidylate synthase inhibitors (for example raltitrexed); and
additional types of chemotherapeutic agent include: (iv) biological
response modifiers (for example interferon); (v) antibodies (for
example edrecolomab); (vi) antisense therapies, for example those
which are directed to the targets listed above, such as ISIS 2503,
an anti-ras antisense; (vii) gene therapy approaches, including for
example approaches to replace aberrant genes such as aberrant p53
or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug
therapy) approaches such as those using cytosine deaminase,
thymidine kinase or a bacterial nitroreductase enzyme and
approaches to increase patient tolerance to chemotherapy or
radiotherapy such as multi-drug resistance gene therapy; and (viii)
immunotherapy approaches, including for example ex-vivo and in vivo
approaches to increase the immunogenicity of patient tumour cells,
such as transfection with cytokines such as interleukin 2,
interleukin 4 or granulocyte-macrophage colony stimulating factor,
approaches to decrease T-cell anergy, approaches using transfected
immune cells such as cytokine-transfected dendritic cells,
approaches using cytokine-transfected tumour cell lines and
approaches using anti-idiotypic antibodies.
[0083] For example such conjoint treatment may be achieved by way
of the simultaneous, sequential or separate administration of an
AZD2171 maleate salt as defined hereinbefore and a vascular
targeting agent described in WO 99/02166 such as
N-acetylcolchinol-O-phosphate (Example 1 of WO 99/02166).
[0084] It is known from WO 01/74360 that antiangiogenics can be
combined with antihypertensives. A salt of the present invention
can also be administered in combination with an antihypertensive.
An antihypertensive is an agent which lowers blood pressure, see WO
01/74360 which is incorporated herein by reference.
[0085] Thus according to the present invention there is provided a
method of treatment of a disease state associated with angiogenesis
which comprises the administration of an effective amount of a
combination of an AZD2171 maleate salt as defined hereinbefore and
an anti-hypertensive agent to a warm-blooded animal, such as a
human being.
[0086] According to a further feature of the present invention
there is provided the use of a combination of an AZD2171 maleate
salt as defined hereinbefore and an anti-hypertensive agent for use
in the manufacture of a medicament for the treatment of a disease
state associated with angiogenesis in a warm-blooded mammal, such
as a human being.
[0087] According to a further feature of the present invention
there is provided a pharmaceutical composition comprising an
AZD2171 maleate salt as defined hereinbefore and an
anti-hypertensive agent for the treatment of a disease state
associated with angiogenesis in a warm-blooded mammal, such as a
human being.
[0088] According to a further aspect of the present invention there
is provided a method for producing an anti-angiogenic and/or
vascular permeability reducing effect in a warm-blooded animal,
such as a human being, which comprises administering to said animal
an effective amount of an AZD2171 maleate salt as defined
hereinbefore and an anti-hypertensive agent.
[0089] According to a further aspect of the present invention there
is provided the use of a combination of an AZD2171 maleate salt as
defined hereinbefore and an anti-hypertensive agent for the
manufacture of a medicament for producing an anti-angiogenic and/or
vascular permeability reducing effect in a warm-blooded mammal,
such as a human being.
[0090] Preferred antihypertensive agents are calcium channel
blockers, angiotensin converting enzyme inhibitors (ACE
inhibitors), angiotensin II receptor antagonists (A-II
antagonists), diuretics, beta-adrenergic receptor blockers
(.beta.-blockers), vasodilators and alpha-adrenergic receptor
blockers (.alpha.-blockers). Particular antihypertensive agents are
calcium channel blockers, angiotensin converting enzyme inhibitors
(ACE inhibitors), angiotensin II receptor antagonists (A-II
antagonists) and beta-adrenergic receptor blockers
(.beta.-blockers), especially calcium channel blockers.
[0091] As stated above AZD2171 maleate salt is of interest for its
antiangiogenic and/or vascular permeability reducing effects.
AZD2171 maleate salt is expected to be useful in a wide range of
disease states including cancer, diabetes, psoriasis, rheumatoid
arthritis, Kaposi's sarcoma, haemangioma, lymphoedema, acute and
chronic nephropathies, atheroma, arterial restenosis, autoimmune
diseases, acute inflammation, excessive scar formation and
adhesions, endometriosis, dysfunctional uterine bleeding and ocular
diseases with retinal vessel proliferation including age-related
macular degeneration. Cancer may affect any tissue and includes
leukaemia, multiple myeloma and lymphoma. In particular such
compounds of the invention are expected to slow advantageously the
growth of primary and recurrent solid tumours of, for example, the
colon, breast, prostate, lungs and skin. More particularly such
compounds of the invention are expected to inhibit any form of
cancer associated with VEGF including leukaemia, multiple myeloma
and lymphoma and also, for example, the growth of those primary and
recurrent solid tumours which are associated with VEGF, especially
those tumours which are significantly dependent on VEGF for their
growth and spread, including for example, certain tumours of the
colon, breast, prostate, lung, brain vulva and skin.
[0092] In addition to their use in therapeutic medicine, the
AZD2171 maleate salts defined hereinbefore are also useful as
pharmacological tools in the development and standardisation of in
vitro and in vivo test systems for the evaluation of the effects of
inhibitors of VEGF receptor tyrosine kinase activity in laboratory
animals such as cats, dogs, rabbits, monkeys, rats and mice, as
part of the search for new therapeutic agents.
[0093] The assays written up in WO 00/47212 and used to test
AZD2171 are as follows:
(a) In Vitro Receptor Tyrosine Kinase Inhibition Test
[0094] This assay determines the ability of a test compound to
inhibit tyrosine kinase activity. DNA encoding VEGF, FGF or EGF
receptor cytoplasmic domains may be obtained by total gene
synthesis (Edwards M, International Biotechnology Lab 5(3), 19-25,
1987) or by cloning. These may then be expressed in a suitable
expression system to obtain polypeptide with tyrosine kinase
activity. For example VEGF, FGF and EGF receptor cytoplasmic
domains, which were obtained by expression of recombinant protein
in insect cells, were found to display intrinsic tyrosine kinase
activity. In the case of the VEGF receptor Flt-1 (Genbank accession
number X51602), a 1.7 kb DNA fragment encoding most of the
cytoplasmic domain, commencing with methionine 783 and including
the termination codon, described by Shibuya et al (Oncogene, 1990,
5: 519-524), was isolated from cDNA and cloned into a baculovirus
transplacement vector (for example pAcYM1 (see The Baculovirus
Expression System: A Laboratory Guide, L. A. King and R. D. Possee,
Chapman and Hall, 1992) or pAc360 or pBlueBacHis (available from
Invitrogen Corporation)). This recombinant construct was
co-transfected into insect cells (for example Spodoptera frugiperda
21(Sf21)) with viral DNA (eg Pharmingen BaculoGold) to prepare
recombinant baculovirus. (Details of the methods for the assembly
of recombinant DNA molecules and the preparation and use of
recombinant baculovirus can be found in standard texts for example
Sambrook et al, 1989, Molecular cloning--A Laboratory Manual, 2nd
edition, Cold Spring Harbour Laboratory Press and O'Reilly et al,
1992, Baculovirus Expression Vectors--A Laboratory Manual, W. H.
Freeman and Co, New York). For KDR (Genbank accession number
L04947), a cytoplasmic fragment starting from methionine 806 was
cloned and expressed in a similar manner.
[0095] For expression of cFlt-1 tyrosine kinase activity, Sf21
cells were infected with plaque-pure cFlt-1 recombinant virus at a
multiplicity of infection of 3 and harvested 48 hours later.
Harvested cells were washed with ice cold phosphate buffered saline
solution (PBS) (10 mM sodium phosphate pH7.4, 138 mM sodium
chloride, 2.7 mM potassium chloride) then resuspended in ice cold
HNTG/PMSF (20 mM Hepes pH7.5, 150 mM sodium chloride, 10% v/v
glycerol, 1% v/v Triton X100, 1.5 mM magnesium chloride, 1 mM
ethylene glycol-bis(.beta.aminoethyl ether) N,N,N',N'-tetraacetic
acid (EGTA), 1 mM PMSF (phenylmethylsulphonyl fluoride); the PMSF
is added just before use from a freshly-prepared 100 mM solution in
methanol) using 1 ml HNTG/PMSF per 10 million cells. The suspension
was centrifuged for 10 minutes at 13,000 rpm at 4.degree. C., the
supernatant (enzyme stock) was removed and stored in aliquots at
-70.degree. C. Each new batch of stock enzyme was titrated in the
assay by dilution with enzyme diluent (100 mM Hepes pH 7.4, 0.2 mM
sodium orthovanadate, 0.1% v/v Triton X100, 0.2 mM dithiothreitol).
For a typical batch, stock enzyme is diluted 1 in 2000 with enzyme
diluent and 50 .mu.l of dilute enzyme is used for each assay
well.
[0096] A stock of substrate solution was prepared from a random
copolymer containing tyrosine, for example Poly (Glu, Ala, Tyr)
6:3:1 (Sigma P3899), stored as 1 mg/ml stock in PBS at -20.degree.
C. and diluted 1 in 500 with PBS for plate coating.
[0097] On the day before the assay 100 .mu.l of diluted substrate
solution was dispensed into all wells of assay plates (Nunc
maxisorp 96-well immunoplates) which were sealed and left overnight
at 4.degree. C.
[0098] On the day of the assay the substrate solution was discarded
and the assay plate wells were washed once with PBST (PBS
containing 0.05% v/v Tween 20) and once with 50 mM Hepes pH7.4.
[0099] Test compounds were diluted with 10% dimethylsulphoxide
(DMSO) and 25 .mu.l of diluted compound was transferred to wells in
the washed assay plates. "Total" control wells contained 10% DMSO
instead of compound. Twenty five microlitres of 40 mM
manganese(II)chloride containing 8 .mu.M adenosine-5'-triphosphate
(ATP) was added to all test wells except "blank" control wells
which contained manganese(II)chloride without ATP. To start the
reactions 50 .mu.l of freshly diluted enzyme was added to each well
and the plates were incubated at room temperature for 20 minutes.
The liquid was then discarded and the wells were washed twice with
PBST. One hundred microlitres of mouse IgG anti-phosphotyrosine
antibody (Upstate Biotechnology Inc. product 05-321), diluted 1 in
6000 with PBST containing 0.5% w/v bovine serum albumin (BSA), was
added to each well and the plates were incubated for 1 hour at room
temperature before discarding the liquid and washing the wells
twice with PBST. One hundred microlitres of horse radish peroxidase
(HRP)-linked sheep anti-mouse Ig antibody (Amersham product NXA
931), diluted 1 in 500 with PBST containing 0.5% w/v BSA, was added
and the plates were incubated for 1 hour at room temperature before
discarding the liquid and washing the wells twice with PBST. One
hundred microlitres of
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)
solution, freshly prepared using one 50 mg ABTS tablet (Boehringer
1204 521) in 50 ml freshly prepared 50 mM phosphate-citrate buffer
pH5.0+0.03% sodium perborate (made with 1 phosphate citrate buffer
with sodium perborate (PCSB) capsule (Sigma P4922) per 100 ml
distilled water), was added to each well. Plates were then
incubated for 20-60 minutes at room temperature until the optical
density value of the "total" control wells, measured at 405 nm
using a plate reading spectrophotometer, was approximately 1.0.
"Blank" (no ATP) and "total" (no compound) control values were used
to determine the dilution range of test compound which gave 50%
inhibition of enzyme activity.
(b) In Vitro HUVEC Proliferation Assay
[0100] This assay determines the ability of a test compound to
inhibit the growth factor-stimulated proliferation of human
umbilical vein endothelial cells (HUVEC).
[0101] HUVEC cells were isolated in MCDB 131 (Gibco BRL)+7.5% v/v
foetal calf serum (FCS) and were plated out (at passage 2 to 8), in
MCDB 131+2% v/v FCS+3 .mu.g/ml heparin+1 .mu.g/ml hydrocortisone,
at a concentration of 1000 cells/well in 96 well plates. After a
minimum of 4 hours they were dosed with VEGF (3 ng/ml) and
compound. The cultures were then incubated for 4 days at 37.degree.
C. with 7.5% CO.sub.2. On day 4 the cultures were pulsed with 1
.mu.Ci/well of tritiated-thymidine (Amersham product TRA 61) and
incubated for 4 hours. The cells were harvested using a 96-well
plate harvester (Tomtek) and then assayed for incorporation of
tritium with a Beta plate counter. Incorporation of radioactivity
into cells, expressed as cpm, was used to measure inhibition of
growth factor-stimulated cell proliferation by compounds. This
methodology was also used to assess compound effects versus basal
HUVEC growth (i.e. endothelial cell proliferation in MCDB 131+2%
v/v FCS+3 .mu.g/ml heparin+1 .mu.g/ml hydrocortisone without the
addition of exogenous VEGF).
(c) In Vivo Solid Tumour Disease Model
[0102] This test measures the capacity of compounds to inhibit
solid tumour growth.
[0103] CaLu-6 tumour xenografts were established in the flank of
female athymic Swiss nu/nu mice, by subcutaneous injection of
1.times.10.sup.6 Calu-6 cells/mouse in 100 .mu.l of a 50% (v/v)
solution of Matrigel in serum free culture medium. Ten days after
cellular implant, mice were allocated to groups of 8-10, so as to
achieve comparable group mean volumes. Tumours were measured using
vernier calipers and volumes were calculated as: (l.times.w).times.
(l.times.w).times.(.pi./6), where l is the longest diameter and w
the diameter perpendicular to the longest. Test compounds were
administered orally once daily for a minimum of 21 days, and
control animals received compound diluent. Tumours were measured
twice weekly. The level of growth inhibition was calculated by
comparison of the mean tumour volume of the control group versus
the treatment group using a Student T test and/or a Mann-Whitney
Rank Sum Test. The inhibitory effect of compound treatment was
considered significant when p<0.05.
[0104] An AZD2171 maleate salt as defined hereinbefore may be
prepared by any process known to be applicable to the preparation
of chemically-related compounds. Such processes include, for
example, those illustrated in International Patent Application No.
WO 00/47212 all of which are incorporated herein by reference. Such
processes also include, for example, solid phase synthesis. Such
processes, are provided as a further feature of the invention and
are as described hereinafter. Necessary starting materials may be
obtained by standard procedures of organic chemistry. AZD2171 free
base may be prepared according to any of the processes described in
WO 00/47212, see in particular Example 240 of WO 00/47212.
Alternatively necessary starting materials are obtainable by
analogous procedures to those illustrated which are within the
ordinary skill of an organic chemist.
[0105] The following processes (a) (b) and (c) constitute further
features of the present invention.
Synthesis of AZD2171 Maleate Salt Form A
[0106] (a) Such a process provides a further aspect of the present
invention and comprises, for example, the steps of: [0107] (i)
dissolving AZD2171 free base in an organic solvent to form a
solution; [0108] (ii) adding an aqueous solution of maleic acid or
adding a solution of maleic acid in an organic solvent; [0109]
(iii) allowing spontaneous nucleation to occur; [0110] (iv)
optionally isolating the crystalline mixture of AZD2171 Forms A and
B so formed; [0111] (v) slurrying the mixture in a solvent, for
example methanol, until all the AZD2171 maleate is Form A, (as may
be determined by X-Ray Powder Diffraction), for example this may
take 4 days; and [0112] (vi) isolating the crystalline solid so
formed. (b) Another such process provides a further aspect of the
present invention and comprises, for example, the steps of: [0113]
(i) dissolving AZD2171 free base in an organic solvent to form a
solution; [0114] (ii) adding an aqueous solution of maleic acid or
adding a solution of maleic acid in an organic solvent; [0115]
(iii) obtaining a solution, for example by heating or adding more
solvent, and adding a seed of AZD2171 maleate Form A to initiate
crystallisation of AZD2171 maleate Form A; and [0116] (iv)
isolating the crystalline solid so formed.
[0117] For part (i) of (a) and (b) the mixture may, if required, be
heated to reflux until dissolution has occurred. Alternatively, the
mixture may, for example, be heated to a temperature less than the
reflux temperature of the solvent provided that dissolution of more
or less all of the solid material has occurred. It will be
appreciated that small quantities of insoluble material may be
removed by filtration of the warmed mixture.
[0118] For part (i) of (a) and (b) the organic solvent is
preferably an alcohol, for example methanol or isopropanol.
[0119] For part (ii) of (a) and (b) the organic solvent is
preferably an alcohol, for example methanol.
(c) Synthesis of AZD2171 Maleate Salt Form B
[0120] Such a process provides a further aspect of the present
invention and comprises, for example, the steps of: [0121] (i)
dissolving AZD2171 maleate in an organic solvent to form a
solution; [0122] (ii) adding the solution to a solvent in which
AZD2171 maleate has a lower solubility than it does in NMP, for
example toluene or ethyl acetate; [0123] (iii) crystallisation of
AZD2171 maleate Form B then occurs; and [0124] (iv) isolating the
crystalline solid so formed. [0125] In (c) a preferred organic
solvent is a highly solubilising solvent such as
1-methyl-2-pyrrolidinone.
[0126] For part (i) of (c) the mixture may, if required, be heated
to reflux until dissolution has occurred. Alternatively, the
mixture may, for example, be heated to a temperature less than the
reflux temperature of the solvent provided that dissolution of more
or less all of the solid material has occurred. It will be
appreciated that small quantities of insoluble material may be
removed by filtration of the warmed mixture.
[0127] In (a), (b) and (c) above the crystalline solid so formed
may be isolated by any conventional method, for example by
filtration.
[0128] The invention is illustrated hereinafter by means of the
following non-limiting Examples, data and Figures in which, unless
otherwise stated:--
[0129] (i) evaporations were carried out by rotary evaporation in
vacuo and work-up procedures were carried out after removal of
residual solids such as drying agents by filtration;
[0130] (ii) yields are given for illustration only and are not
necessarily the maximum attainable;
[0131] (iii) melting points are uncorrected and were determined
using a Mettler DSC820e; (iv) the structures of the end-products of
the formula I were confirmed by nuclear (generally proton) magnetic
resonance (NMR) and mass spectral techniques; proton magnetic
resonance chemical shift values were measured on the delta scale
and peak multiplicities are shown as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; br, broad; q, quartet, quin,
quintet; all samples run on a Bruker DPX 400 MHz at 300K in
d.sub.6-DMSO, 16 scans, pulse repetition time 10 seconds;
[0132] (v) intermediates were not generally fully characterised and
purity was assessed by NMR analysis; and
[0133] (vi) the following abbreviations have been used:-- [0134]
DMSO dimethylsulphoxide [0135] NMP 1-methyl-2-pyrrolidinone
EXAMPLE 1: AZD2171 MALEATE FORM A
[0136] Under an inert atmosphere of nitrogen AZD2171 crude free
base (4.52 g), (prepared for example as described in Example 240 of
WO 00/47212) was slurried with isopropanol (58.8 mL). The mixture
was heated at reflux for 15 minutes to give a clear, dark solution.
The mixture was cooled to 75.degree. C. and charcoal (0.226 g)
added. The mixture was reheated to reflux and held at reflux for an
hour. The mixture was then filtered hot. The charcoal filter cake
was washed with hot isopropanol (9 mL). The temperature of the
combined filtrate and wash was adjusted to 55.degree. C. and a
prefiltered solution of maleic acid (1.173 g) in water (2.71 mL)
was added dropwise over 5 minutes. The crude free base which
previously crystallised dissolved during the addition. A line wash
of water (0.9 mL) was added. The mixture was maintained at
55.degree. C. for 15 minutes and a seed of AZD2171 maleate Form A
(0.023 g) added. The mixture was held at 55.degree. C. for 4 hours.
During the 4 hour hold crystallisation became established. The
mixture was cooled to 0.degree. C. over 8 hours. The mixture was
held at 0.degree. C. for a minimum of 8 hours. The mixture was
filtered. The cake was washed with isopropanol (9 mL). The solid
was dried in a vacuum oven at 50.degree. C. to give
4-([4-fluoro-2-methyl-1H-indol-5-yl]oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)-
propoxy)quinazoline maleate Form A. .sup.1H NMR Spectrum: (400 MHz,
DMSO): 11.36 (s, 1H), 8.53 (s, 1H), 7.65 (s, 1H), 7.43 (s, 1H),
7.18 (d, 1H), 7.01 (d, 1H), 6.25 (s, 1H), 6.04 (s, 2H), 4.33 (t,
2H), 4.02 (s, 3H), 3.26-3.3.70 (b, 4H), 2.44, (s, 3H), 2.24 (m,
2H), 2.02 (m, 4H).
[0137] m.p.: DSC analysis: onset of melting at 198.3.degree. C. and
a peak at 200.08.degree. C.
EXAMPLE 2: AZD2171 MALEATE FORM A
[0138] Under an inert atmosphere of nitrogen AZD2171 crude free
base (23.0 g) (prepared for example as described in Example 240 of
WO 00/47212) was slurried in methanol (223 mL) in vessel 1. The
mixture was degassed by holding under vacuum and then releasing the
vacuum with nitrogen. This was repeated five times. The slurry was
then heated to reflux and held there for 15 minutes to give a
clear, dark brown solution. The solution was cooled to 60.degree.
C. and then filtered through a Celite.RTM. pad (4.00 g) into vessel
2. The Celite.RTM. pad was washed with hot (60.degree. C.) methanol
(78 mL), the filtrate again going to vessel 2.
[0139] To vessel 1 was then charged methanol (111 mL), which was
cooled to 0.degree. C. To vessel 1 was then charged maleic acid
(5.50 g) and the mixture stirred at 0.degree. C. for 15 minutes
until all the maleic acid had dissolved.
[0140] The contents of vessel 1 were then charged to vessel 2
through an in-line filter whilst maintaining the temperature above
52.degree. C. A seed of AZD2171 maleate Form A (0.0454 g) was added
to vessel 2 at 55.degree. C. and the mixture held at 55.degree. C.
for 3 hours. The mixture was then cooled to 40.degree. C. over 7
hours, then cooled further to -5.degree. C. over 6 hours. The solid
was filtered and washed with methanol (100 mL) at -5.degree. C. The
product was dried in a vacuum oven for 24 hours to give
4-([4-fluoro-2-methyl-1H-indol-5-yl]oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)-
propoxy)quinazoline maleate Form A.
EXAMPLE 3: AZD2171 MALEATE FORM B
[0141] AZD2171 maleate Form A (2.31 g) was dissolved in warm
(.about.50.degree. C.) NMP. This solution was added dropwise to
toluene (23 mL) over 2 minutes at ambient temperature. Material
originally precipitated as a solid then became an oil, then a solid
again. After stirring for 10 minutes at ambient temperature the
solid was filtered and washed with toluene (10 mL). The solid was
dried in a vacuum oven at ambient temperature overnight to give
4-((4-fluoro-2-methyl-1H-indol-5-yl)oxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)-
propoxy)quinazoline maleate Form B.
[0142] m.p.: DSC analysis: onset of melting at 194.43.degree. C.
and a peak at 195.97.degree. C.
BRIEF DESCRIPTION OF THE DRAWINGS
[0143] FIG. 1: DSC and TGA Thermograms for AZD2171 Free base
Monohydrate--with temperature in .degree. C. plotted on the
horizontal axis and heat flow/% weight loss on the vertical
axis
[0144] FIG. 2: X-Ray Powder Diffraction Pattern for AZD2171 free
base--with the 2.theta. values plotted on the horizontal axis and
the relative line intensity (count) plotted on the vertical
axis.
[0145] FIG. 3: X-Ray Powder Diffraction Pattern for AZD2171 Free
base Monohydrate Heated to 100.degree. C.--with the 2.theta. values
plotted on the horizontal axis and the relative line intensity
(count) plotted on the vertical axis.
[0146] FIG. 4: X-Ray Powder Diffraction Pattern for AZD2171 Free
base Micronised--with the 20 values plotted on the horizontal axis
and the relative line intensity (count) plotted on the vertical
axis.
[0147] FIG. 5: X-Ray Powder Diffraction Pattern for AZD2171 Maleate
Salt Form A--with the 20 values plotted on the horizontal axis and
the relative line intensity (count) plotted on the vertical
axis.
[0148] FIG. 6: DSC Thermogram for AZD2171 Maleate Form A--with
temperature in .degree. C. plotted on the horizontal axis and
endothermic heat flow (milliWatts (mW)) plotted on the vertical
axis.
[0149] FIG. 7: AZD2171 Maleate Form A Vapour Sorption Isotherm at
25.degree. C.--with target relative humidity (RH) (%) plotted on
the horizontal axis and change in dry mass (%) plotted on the
vertical axis.
[0150] FIG. 8: X-Ray Powder Diffraction Pattern AZD2171 Maleate
Salt Form B--with the 20 values plotted on the horizontal axis and
the relative line intensity (count) plotted on the vertical
axis.
[0151] FIG. 9: DSC Thermogram for AZD2171 Maleate Form B--with
temperature in .degree. C. plotted on the horizontal axis and
endothermic heat flow (milliWatts (mW)) plotted on the vertical
axis.
[0152] FIG. 10: X-Ray Powder Diffraction Patterns for AZD2171
Maleate Slurry Experiment with the 2.theta. values plotted on the
horizontal axis and the relative line intensity (count) plotted on
the vertical axis.
DETAILS OF TECHNIQUES USED
X-Ray Powder Diffraction
TABLE-US-00005 [0153] TABLE 5 % Relative Intensity* Definition
25-100 vs (very strong) 10-25 s (strong) 3-10 m (medium) 1-3 w
(weak) *The relative intensities are derived from diffractograms
measured with fixed slits
Analytical Instrument: Siemens D5000
[0154] The X-ray powder diffraction spectra were determined by
mounting a sample of the crystalline salt on Siemens single silicon
crystal (SSC) wafer mounts and spreading out the sample into a thin
layer with the aid of a microscope slide. The sample was spun at 30
revolutions per minute (to improve counting statistics) and
irradiated with X-rays generated by a copper long-fine focus tube
operated at 40 kV and 40 mA with a wavelength of 1.5406 angstroms.
The collimated X-ray source was passed through an automatic
variable divergence slit set at V20 and the reflected radiation
directed through a 2 mm antiscatter slit and a 0.2 mm detector
slit. The sample was exposed for 1 second per 0.02 degree 2-theta
increment (continuous scan mode) over the range 2 degrees to 40
degrees 2-theta in theta-theta mode. The running time was 31
minutes and 41 seconds. The instrument was equipped with a
scintillation counter as detector. Control and data capture was by
means of a Dell Optiplex 686 NT 4.0 Workstation operating with
Diffract+ software. Persons skilled in the art of X-ray powder
diffraction will realise that the relative intensity of peaks can
be affected by, for example, grains above 30 microns in size and
non-unitary aspect ratios which may affect analysis of samples. The
skilled person will also realise that the position of reflections
can be affected by the precise height at which the sample sits in
the diffractometer and the zero calibration of the diffractometer.
The surface planarity of the sample may also have a small effect.
Hence the diffraction pattern data presented are not to be taken as
absolute values.
Sieving/Micronisation
[0155] AZD2171 free base was sieved prior to Micronising using a 1
mm stainless steel sieve, the base being used for product
collection and for manual feeding directly into the microniser.
Approximately 7.5 g of AZD2171 free base was sieved.
[0156] A clean S/S lined 2'' Microniser was used.
[0157] Manual feed rate: approximately 2/3 g per minute.
[0158] Grind air pressure range 10/20 psi (0.67/1.33
atmospheres).
[0159] Venturi air pressure range 20/25 psi (1.33/1.67
atmospheres).
Dynamic Vapour Sorption
[0160] Analytical Instrument: Surface Measurements Systems Dynamic
Vapour Sorption Analyser.
[0161] About 5 mg of material contained in a quartz holder at
25.degree. C. was subjected to humidified nitrogen at the following
relative humidities (RH): 0, 20, 40, 60, 80, 95, 80, 60, 40, 20, 0%
RH in duplicate.
Differential Scanning Calorimetry
[0162] Analytical Instrument: Mettler DSC820e.
[0163] Typically less than 5 mg of material contained in a 40 .mu.l
aluminium pan fitted with a pierced lid was heated over the
temperature range 25.degree. C. to 325.degree. C. at a constant
heating rate of 10.degree. C. per minute. A purge gas using
nitrogen was used--flow rate 100 ml per minute.
Thermogravimetric Analysis
[0164] Analytical Instrument: Mettler TG851.
[0165] Typically between 3 and 12 mg of material contained in a 70
.mu.l alox (aluminium oxide) crucible was heated over the
temperature range 25.degree. C. to 325.degree. C. at a constant
heating rate of 10.degree. C. per minute. A purge gas using helium
was used--flow rate 50 ml per minute.
Karl Fischer Water Content
[0166] Analytical Instrument: Mitsubishi Moisture Meter CA-05.
[0167] Typically approximately 50 mg of material was used.
* * * * *