U.S. patent application number 11/776245 was filed with the patent office on 2007-11-22 for crystalline 2,5-dione-3-(1-methyl-1h-indol-3-yl)-4-[1-(pyridin-2-ylmethyl) piperidin-4-yl]-1h-indol-3-yl]-1h-pyrrole mono-hydrochloride.
Invention is credited to Julie Kay Bush, Margaret Mary Faul, Susan Marie Reutzel-Edens.
Application Number | 20070270465 11/776245 |
Document ID | / |
Family ID | 42799191 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070270465 |
Kind Code |
A1 |
Bush; Julie Kay ; et
al. |
November 22, 2007 |
CRYSTALLINE
2,5-DIONE-3-(1-METHYL-1H-INDOL-3-YL)-4-[1-(PYRIDIN-2-YLMETHYL)
PIPERIDIN-4-YL]-1H-INDOL-3-YL]-1H-PYRROLE MONO-HYDROCHLORIDE
Abstract
The present invention relates to crystalline
2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-yl-methyl)piperidin--
4-yl)]-1H-pyrrole mono-hydrochloride salt, a pharmaceutical
formulation containing said salt and to methods for treating cancer
and for inhibiting tumor growth using said salt.
Inventors: |
Bush; Julie Kay; (Fishers,
IN) ; Faul; Margaret Mary; (Zionsville, IN) ;
Reutzel-Edens; Susan Marie; (Zionsville, IN) |
Correspondence
Address: |
ELI LILLY & COMPANY
PATENT DIVISION
P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Family ID: |
42799191 |
Appl. No.: |
11/776245 |
Filed: |
July 11, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10520360 |
Jan 5, 2005 |
|
|
|
PCT/US03/19548 |
Jul 8, 2003 |
|
|
|
11776245 |
Jul 11, 2007 |
|
|
|
60395976 |
Jul 12, 2002 |
|
|
|
Current U.S.
Class: |
514/318 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; C07D 401/14 20130101 |
Class at
Publication: |
514/318 |
International
Class: |
A61K 31/4545 20060101
A61K031/4545; A61P 35/00 20060101 A61P035/00 |
Claims
1-4. (canceled)
5. A method of treating non-Hodgkins lymphoma which comprises
administering to a mammal in need thereof an effective amount of
crystalline
2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin-4-
-yl]-1H-indol-3-yl]-1H-pyrrole mono-hydrochloride having an X-ray
diffraction pattern which comprises the following peaks:
6.8.+-.0.1, 10.9.+-.0.1, 14.2.+-.0.1 and 16.6.+-.0.1.degree. in
2.theta.; when the pattern is obtained from a copper radiation
source (CuK.alpha.;.lamda.=1.54056 .ANG.).
6. A method of treating glioblastoma which comprises administering
to a mammal in need thereof an effective amount of crystalline
2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin-4-
-yl]-1H-indol-3-yl]-1H-pyrrole mono-hydrochloride having an X-ray
diffraction pattern which comprises the following peaks:
6.8.+-.0.1, 10.9.+-.0.1, 14.2.+-.0.1 and 16.6.+-.0.1.degree. in
2.theta.; when the pattern is obtained from a copper radiation
source (CuK.alpha.; .lamda.=1.54056 .ANG.).
7. A method of treating non-small cell lung cancer which comprises
administering to a mammal in need thereof an effective amount of
crystalline
2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin-4-
-yl]-1H-indol-3-yl]-1H-pyrrole mono-hydrochloride having an X-ray
diffraction pattern which comprises the following peaks:
6.8.+-.0.1, 10.9.+-.0.1, 14.2.+-.0.1 and 16.6.+-.0.1.degree. in
2.theta.; when the pattern is obtained from a copper radiation
source (CuK.alpha.; .lamda.=1.54056 .ANG.).
8-14. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] Compounds of formula I: ##STR1## and pharmaceutically
acceptable salts thereof, useful as protein kinase C inhibitors,
were disclosed by Heath, et al., in European Patent Publication No.
817,627 (Heath).
[0002] Example #49 of Heath disclosed a free base compound of
formula FB: ##STR2##
[0003] While FB is undoubtedly a very effective pharmaceutical
agent, unexpected difficulties were encountered in its large-scale
production. Thus, unpredictable formation of solvates complicated
the commercial synthesis to such an extent that it became necessary
to develop an alternative form for large-scale
commercialization.
[0004] In this context, WO 02/02094 and WO 02/02116 specifically
describe the use of the dihydrochloride salt of FB (FB-2HCl) to
treat cancer and to inhibit tumor growth as a mono-therapy or in
conjunction with an anti-neoplastic agent or radiation therapy.
Unfortunately, it has now been determined that FB-2HCl is
hygroscopic. In addition, although FB-2HCl appears to be
crystalline by optical light microscopy, more detailed study by
X-ray powder diffraction (XRD) has revealed that this material is
in fact only poorly crystalline.
[0005] Surprisingly, in accordance with the invention, it has now
been discovered that the monohydrochloride salt of FB is capable of
being reproducibly produced on a commercial scale, is not
significantly hygroscopic, is sufficiently stable for use in oral
formulations, and can be produced in a highly crystalline
state.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention relates to crystalline
2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin4--
yl]-1H-indol-3-yl)-1H-pyrrole mono-hydrochloride, a hydrate
thereof; or mixtures thereof.
[0007] The present invention further relates to crystalline
2,5-dione-3-(1-methyl-1H-indol-3-yl)-4-[1-(pyridin-2-ylmethyl)piperidin-4-
-yl]-1H-indol-3-yl)-1H-pyrrole mono-hydrochloride, a hydrate
thereof, or mixtures thereof, having an X-ray diffraction pattern
which comprises the following peaks: 6.8.+-.0.1, 10.9.+-.0.1,
14.2.+-.0.1 and 16.6.+-.0.1.degree.in 2.theta.; when the pattern is
obtained from a copper radiation source (CuK.alpha.;
.lamda.=1.54056 .ANG.). This crystalline material is hereafter
referred to as "F-I".
[0008] The present invention also relates to a pharmaceutical
composition containing F-I and a pharmaceutical carrier. In another
embodiment, the pharmaceutical formulation of the present invention
may be adapted for use in treating cancer and for use in inhibiting
tumor growth.
[0009] Moreover, the present invention relates to methods for
treating cancer and to methods for inhibiting tumor growth which
comprise administering to a mammal in need thereof an effective
amount of F-I.
[0010] In addition, the present invention is related to F-I for
treating cancer and for inhibiting tumor growth.
[0011] Another embodiment of the invention provides for the use of
F-I for the manufacture of a medicament for the treatment of cancer
and for the manufacture of a medicament for inhibiting tumor
growth.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 is a representative XRD pattern for F-I.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Prior to discovering the problems associated with the
large-scale manufacturability of FB, due to a concern that FB may
not possess optimal bioavailability properties, an in situ salt
screen was performed to identify salts for FB possessing improved
properties. This screen evaluates the solubility of salts formed in
situ in aqueous media. The solubility obtained in situ for a given
salt is not directly predictive of the equilibrium solubility of
the crystalline form(s) of the same salt. However, the in situ
screen can be used to prioritize the salts for synthesis and
characterization during salt selection. From these data, five out
of seventeen mono-acid salts were chosen for synthesis and
characterization. These salts were the citrate, methanesulfonate
(mesylate), phosphate, tartrate and mono-hydrochloride (FB-HCl). In
addition, FB-2HCl was also synthesized, characterized and analyzed.
Some of these salts' properties as wet as those of FB are discussed
below.
Citrate, Mesylate, Phosphate and Tartrate
[0014] The citrate salt generated from methanol is insoluble in
water. The mesylate salt is hygroscopic, exhibiting up to 2% weight
gain at 70% RH and over 15% weight gain at 95% RH. Although the
phosphate salt exhibits rapid dissolution and high solubility at
early time points, the solubility of the phosphate drops to 71
.mu.g/mL upon prolonged incubation. The phosphate salt is also
somewhat hygroscopic and exhibited hysteresis in water desorption,
indicating possible hydrate formation.
[0015] The tartrate is only slightly hygroscopic, exhibiting
.about.1% weight gain at RH's up to 70%. Based on this and other
promising initial results, the tartrate was subjected to a brief
polymorph/solvate screen to determine its suitability for bulk
manufacturing and use as a pharmaceutical.
[0016] The tartrate salt was initially isolated (by titration of
the free base with tartaric acid) as a crystalline hydrate. The
hydrated material was then recrystallized to determine if other
pharmaceutically relevant crystal forms of the tartrate salt could
be prepared. The number of solvents suitable for recrystallization
was limited by the relatively poor solubility of this salt in many
solvents, including polar, protic solvents (H.sub.2O, methanol,
ethanol and isopropyl alcohol) and many non-protic solvents
(acetone, ethyl acetate, methyl ethyl ketone and tetrahydrofuran).
Sufficient solubility was observed only in dimethylformamide,
dimethylsulfoxide and organic (and organic/aqueous) mixtures.
Elevated temperatures were often required to achieve
dissolution.
[0017] The tartrate salt was typically not generated from the
recrystallization experiments that were carried out. Instead, a
crystal form of FB was obtained most often. A non-solvated form of
the tartrate was not found. These results suggest that isolation of
a tartrate salt of FB could be difficult, presumably due to the low
solubility of different crystal forms of FB relative to the
tartrate salt, and the relatively small difference in pKa between
FB and tartaric acid.
FB-2HCl
[0018] The aqueous solubility of FB-2HCl under various conditions
was analyzed and at concentrations up to 10 mg/mL, solutions of
FB-2HCl are stable at ambient temperature for up to 10 days.
However, solutions held at 50.degree. C. exhibited profound
precipitation prior to the first time point (6 days). At
concentrations .gtoreq.40 mg/mL, rapid precipitation within minutes
was noted at ambient room temperature. XRD analysis and ion
chromatography (to determine chloride content) of the precipitated
crystals confirmed that this precipitate was FB-HCl.
FB
[0019] The product of the synthesis described below in Preparation
1, is typically a non-solvated crystalline form of FB. This
non-solvated form (hereafter referred to as FB Form 1) is preferred
as it crystallizes well in the reaction, filters rapidly and
affords a high purity of product (total related substances
(TRS).about.0.77%). However, under these very same reaction
conditions, a solvate containing tetrahydrofuran (THF) is also
sometimes isolated (frequency of occurrence .about.10-20%). This
crystalline solvate filters very slowly and traps certain
impurities resulting in a higher TRS for product (2.42-4.78%). The
high TRS associated with this solvate has required that, when
present, the isolated solvate be reworked. Despite significant
research, the reason for the occasional formation of the solvate
containing THF is unknown. The lack of control in preparation of FB
Form 1 has limited its potential for development as the final
active pharmaceutical ingredient (API).
FB-HCl
[0020] FB-HCl, prepared via addition of 1 equivalent of
concentrated or IN hydrochloric acid to a mixture of FB in a lower
alcohol, e.g., methanol, isopropanol or 2-butanol, or in mixture of
a lower alcohol and water, is crystalline and has a melting onset
temperature of about 256.degree. C. as measured by differential
scanning calorimetry (DSC). FB-HCl, produced as described in
Example 1, is relatively non-hygroscopic between 0-70% RH (<2-%
wt gain @95% RH).
Characterization of FB-HCl
[0021] Various methods, including thermogravimetric analysis (TGA),
DSC and XRD were used to characterize FB-HCl. TGA allows for
measurement of the amount and rate of weight change as a function
of temperature. TGA is most commonly used to study desolvation
processes and to quantitatively determine the total volatile
content of a solid. DSC is a technique that is often used to screen
compounds for polymorphism because the temperatures(s) at which a
physical change in a material occurs is usually characteristic of
that material. DSC is often used to complement TGA analysis in
screening compounds for physical changes upon controlled heating.
XRD is a technique that detects long-range order in a crystalline
material and can be performed at different RH's to detect subtle
phase changes induced by moisture sorption.
[0022] Different lots of FB-HCl, prepared via addition of 1
equivalent of concentrated or 1N hydrochloric acid to a mixture of
FB in methanol, were analyzed by TGA and were found to retain
different levels of water: from <0.01% (anhydrous material) all
the way to 1.6% (hemi-hydrate). The TGA results showed not only the
different amounts of water present in the crystalline FB-HCl
materials, but also that the water, when present, is readily
expelled from the material upon heating above ambient
temperature.
[0023] The different water contents prompted an investigation into
the moisture sorption characteristics of those lots of crystalline
FB-HCl that were not anhydrous. Indeed, the various partially
hydrated lots showed distinctly different water uptake profiles.
Regardless of the amount of water sorbed in the crystalline FB-HCl
lattice, the sorption isotherms consistently showed gradual weight
gains up to .about.40% RH, above which, the water uptake plateaued.
The maximum moisture sorption (1.6% at 40% RH) observed for those
partially hydrated lots of crystalline FB-HCl suggests that at full
water occupancy, a hemihydrate (0.5 mole) composition is present.
Crystalline FB-HCl material capable of water sorption is hereafter
referred to as "hygroscopic F-I".
[0024] The XRD peaks of hygroscopic F-I did not shift at any RH.
The XRD patterns generated for hygroscopic F-I were identical to
XRD patterns generated for the non-hygroscopic F-I material
(hereafter referred to as "anhydrous F-I"). The absence of changes
to the XRD pattern when moving from anhydrous F-I to hygroscopic
F-I, as well as the absence of changes to the XRD pattern for
hygroscopic F-I as a function of humidity, shows not only that the
crystal lattice of F-I is unperturbed by the moisture sorption
process, but also that moisture sorption into the particles cannot
be site-specific.
[0025] F-I (both hygroscopic and anhydrous) exhibits a strong,
unique XRD pattern with sharp peaks and a flat baseline, indicative
of a highly crystalline material (see FIG. 1). The angular peak
positions in 2.theta. and corresponding I/I.sub.o data for all F-I
peaks with intensities equal to or greater than 5% of the largest
peak are tabulated in Table 1. All data in Table 1 is expressed
with an accuracy of .+-.0.1 in 2.theta.. TABLE-US-00001 TABLE 1
Angle 2.theta. I/I.sub.o (%) 6.3 19.1 6.8 27.8 7.2 5.0 10.9 100
12.5 11.2 12.7 38.0 13.2 21.0 14.2 62.6 14.4 19.1 15.4 17.0 16.6
56.3 16.8 21.8 17.0 27.2 17.3 5.9 17.7 9.6 17.9 10.4 18.4 25.8 18.8
24.5 19.1 69.1 21.7 7.3 22.1 24.8 22.8 7.9 23.7 19.7 24.4 19.1 24.7
14.4 25.4 15.5 25.8 11.3 26.4 44.1 26.8 11.6 27.7 10.7 27.9 17.1
28.1 10.8 28.6 5.3 29.1 9.7
[0026] It is well known in the crystallography art that, for any
given crystal form, the relative intensities of the diffraction
peaks may vary due to preferred orientation resulting from factors
such as crystal morphology. Where the effects of preferred
orientation are present, peak intensities are altered, but the
characteristic peak positions of the polymorph are unchanged. See,
e.g., The United States Pharmacopeia #23, National Formulary #18,
pages 1843-1844, 1995. Furthermore, it is also well known in the
crystallography art that, for any given crystal form, the angular
peak positions may vary slightly. For example, peak positions can
shift due to a variation in the temperature at which a sample is
analyzed, sample displacement, or the presence or absence of an
internal standard. In the present cases a peak position variability
of .+-.0.1.degree. in 2.theta. will take into account these
potential variations without hindering the unequivocal
identification of a crystalline salt of the present invention.
[0027] A well-known and accepted method for searching crystal forms
in the literature is the "Fink" method. The Fink method uses the
four most intense lines for the initial search followed by the next
four most intense lines. In general accord with the Fink method,
based on peak intensities as well as peak position, F-I may be
identified by the presence of peaks at 6.8.+-.0.1, 10.9.+-.0.1,
14.2.+-.0.1 and 16.6.+-.0.1.degree. in 2.theta.; when the pattern
is obtained from a copper radiation source (.lamda.=1.54056). The
presence of F-I may be further verified by peaks at 6.3.+-.0.1, 7.2
.+-.0.1, 12.5.+-.0.1, and 17.0.+-.0.1.degree. in 2.theta.; when the
pattern is obtained from a copper radiation source
(.lamda.1.54056).
FB Form I vs. Hygroscopic F-I vs. anhydrous F-I
[0028] Extensive equilibrium solubility determinations were
undertaken for both hygroscopic and anhydrous F-I in a variety of
aqueous media at ambient temperature. Additionally, the equilibrium
solubility of FB Form I was measured at ambient temperature.
Samples were assayed by high performance liquid chromatography
(HPLC) after 24 hours of equilibration in the respective solvents.
The results are summarized in Table 2. TABLE-US-00002 TABLE 2 Amt
Dissolved Filtrate Sample Solvent (mg/mL) pH FB Form I 0.01 N HCl
0.279, 0.355 2.20 Anhydrous F-I 0.054, 0.056 2.19 Hygroscopic F-I
0.046, 0.053 2.25 FB Form I pH 2.2 buffer 0.346, 0.336 2.21
Anhydrous F-I 0.360, 0.363 2.27 Hygroscopic F-I 0.324, 0.352 2.26
FB Form I SIF, fed pH 5.0 0.073, 0.074 4.94 Anhydrous F-I 0.016,
0.015 4.94 Hygroscopic F-I 0.014, 0.015 4.93
[0029] The equilibrium solubility data reveal that while F-I
(hygroscopic and anhydrous) and FB Form I have similar solubilities
in pH 2.2 buffer, F-I is significantly less soluble than FB Form I
in 0.01 N HCl and simulated intestinal fluid (SIF) (fed). No
significant differences between hygroscopic and anhydrous F-I in
any media tested were observed.
[0030] The solubility results suggest that controlling the bulk
composition (hygroscopic vs. anhydrous particles) of F-I as an API
is not critical from a bioavailability standpoint. To confirm that
variability in the hygroscopicity of F-I lots should not adversely
impact bioavailability, intrinsic dissolution rates were also
measured for the hygroscopic and anhydrous F-I. For comparison
purposes, the intrinsic dissolution rate of FB Form I was also
measured. Because FB Form I dissolved too rapidly (>10% of a 100
mg compact dissolved within 10 minutes) and the hygroscopic and
anhydrous F-I dissolved too slowly (no appreciable dissolution in
10 minutes), precise intrinsic dissolution rates could not be
determined. The intrinsic dissolution results are summarized in
Table 3. TABLE-US-00003 TABLE 3 % of 100 mg Compact Dissolved in 10
Minutes Dissolution Medium Hygroscopic F-I Anhydrous F-I FB Form I
0.1 N HCl <<0.5 <<0.5 >30
[0031] The in vitro dissolution and solubility data discussed above
suggest that FB Form I should offer bioavailability advantages in
vivo relative to F-I. In order to confirm this prediction, the
plasma pharmacokinetic parameters of FB Form I in fed female beagle
dogs were evaluated following single oral administration by gavage
of 5 mg/kg of FB Form I or F-I in a cross-over design. The 6 dogs
were randomized into two treatment groups to receive single doses
of FB Form I followed by a single dose of F-I two weeks later, or
vice versa. On Days 1 and 14, 3 dogs received FB Form I and 3 dogs
received F-I, and blood samples were collected at 0.5, 1, 2, 3, 4,
8, 12 and 24 hours post dosing. Concentrations of FB Form I were
determined by liquid chromatography tandem mass spectrometry. These
concentrations were subsequently used to determine the
pharmacokinetic parameters reported in Table 4. TABLE-US-00004
TABLE 4 Cmax AUC0-24 AUC0-24 AUC0-inf AUC0-inf Dosing Cmax (nM)
(nM) (nM .times. hr) (nM .times. hr) (nM .times. hr) (nM .times.
hr) Animal Regimen FB Form I F-I FB Form I F-I FB Form I F-I 1 1
531.6 679.0 2092.4 2970.0 2113.9 3016.1 2 1 600.2 501.4 3477.5
4064.4 3603.1 4200.8 3 1 552.4 774.1 5119.5 6150.1 5333.1 6630.8 4
2 717.7 450.2 2270.2 2944.5 2306.3 2998.3 5 2 336.5 481.4 2443.1
3592.3 2578.6 3828.6 6 2 99.8 327.4 389.3 1735.0 400.6 1798.4
Dosing regimen 1 = Day 1 F-I, Day 14 FB Dosing regimen 2 = Day 1
FB, Day 14 F-I
[0032] Surprisingly, based on in vitro solubility and dissolution
data, plasma exposure for F-I in terms of area under the
concentration versus time curves (AUC) for both 0 to 24 hr and 0 to
infinity was significantly higher than that obtained from FB Form
I. Absorption rate did not appear to change as the time to reach
Cmax (tmax) ranged from 1 to 2 hours for both FB Form I and F-I.
The increased exposure for F-I was most likely due to increased
bioavailability, since clearance did not appear to change given the
similarities in the apparent half-life of elimination values.
Synthesis
Preparation of FB
[0033] Step 1--Stir a mixture of 2-picolyl chloride hydrochloride
(7.0 g, 42.7 mmol), 4-piperidone mono-hydrate hydrochloride (6.88
g, 44.8 mmol), powdered sodium carbonate (18.3 g, 173 mmol) and
acetonitrile (70 mL) for 45 minutes at ambient temperature, 45
minutes at 40.degree. C., 45 minutes at 50.degree. C., 45 minutes
at 60.degree. C., and then heat to 70.degree. C. with vigorous
stirring. Monitor the reaction by HPLC (Zorbax RX-C8 25 cm column,
acetonitrile/H.sub.3PO.sub.4 buffer at pH 3.0,.lamda.=250 nm) for
disappearance of picolyl chloride. At completion of the reaction,
allow the mixture to cool to room temperature, filter to remove the
insoluble solids, then wash the filter cake with acetonitrile
(2.times.25 ml). Concentrate the filtrate to a small volume
(.about.30 ml) and solvent exchange into 41 ml of ethyl acetate.
Rapidly stir and heat the solution to 55.degree. C. then treat,
over 30 minutes, with a solution of camphorsulfonic acid (9.91 g,
42.67 mmol) in ethyl acetate (77 mL). Allow the resulting
suspension to cool to room temperature then stir for 3 hours.
Filter the precipitate, wash with ethyl acetate (2.times.30 ml),
and dry in vacuo at 45.degree. C. to give 15.6 g (87%) of the
camphorsulfonic acid salt.
[0034] Step 2--To a 1 L 3-neck jacketed vessel under N.sub.2, add
the product of Step 1 (1.0 equivalent, 33.3. g),
2-(2,2-dimethoxyethyl)aniline (Fukuyama et al, Tet Lett., 39
(1-2):71-74, 1998; 1.0 equivalent 14.3 g) and propionic acid (115
mL). Stir the reaction at 20-24.degree. C. until the contents
dissolve (15-30 minutes). Cool the mixture to -10 to -15.degree.
C., then add 1.0 M NaBH(OPr).sub.3 in tetrahydrofuran (115 mL) over
at least 2 hours under N.sub.2 while maintaining an internal vessel
temperature <-10.degree. C. Confirm completion of the reductive
amination by HPLC (Zorbax C-8 column, pH 3.0 (1.5 ml
triethylamine/1.5 ml H.sub.3PO.sub.r/1L H.sub.2O. Initial gradient:
80% aqueous/20% acetonitrile. Final (45 mins): 20% aqueous/80%
acetonitrile). After reaction completion is verified, add ethyl
acetate (200 mL) and adjust the reaction temperature to 0.degree.
C. Adjust the pH to 10.0 by careful addition of 25% NaOH (315 g)
and allow the reaction to warm to 47-52.degree. C. Stir the
reaction for 30 minutes to 60 minutes at 47-52.degree. C. Stop
stirring the reaction and allow the layers to settle for at least
15 minutes at 47-52.degree. C. Remove the lower aqueous layer and
wash the organic layer with aqueous 20% NaCl (150 mL). After
stirring for 30 minutes at 47-52.degree. C., stop the agitation and
allow the layers to separate over 15 minutes. Remove the lower
aqueous layer and reduce the reaction volume to .about.65-85 mL by
vacuum distillation. Add ethyl acetate (100 mL) back to the
reaction and cool the mixture to 23-25.degree. C. Add
trifluoroacetic acid (30 ml) over at least 30 minutes. Warm the
reaction to 29-31.degree. C. and allow the reaction to proceed
until by HPLC analysis the initial amination adduct is present at
less than <1.0%. After reaction completion is verified, add
ethyl acetate (175 mL) and water (30 mL) and carefully adjust the
pH to .about.9.0 with 25% NaOH (74 g), while warming to
40-45.degree. C. Stir the resultant bi-phasic mixture for at least
1 hour at 45-50.degree. C. and allow the pH to drop to .about.8.60.
Stop the mixing and allow the layers to settle for at least 15
minutes at 45-50.degree. C. Remove the lower aqueous layer and wash
the organic layer with aqueous 20% NaCl (125 mL) while stirring at
45-50.degree. C. After 30 minutes stirring, and a 15 minute settle
time at 45-50.degree. C., remove the aqueous layer and concentrate
the reaction mixture to 100 to 150 mL volume via vacuum
distillation. Add isopropanol (400 mL) and concentrate the reaction
again to .about.200 mL, then add additional isopropanol (200 mL).
Concentrate the mixture to .about.200 mL final volume via vacuum
distillation and age the suspension for 3 hours at 43-45.degree.
C., then cool over 3-4 hours to 5.degree. C. Filter the product at
-5.degree. C. and wash with pre-cooled (<0.degree. C.)
isopropanol (2.times.40 mL). Dry the reductive amination product at
50-60.degree. C. under reduced pressure.
[0035] Step 3--Slurry the product of Step 2 (5.00 g, 17.2 mmol)
with dry tert-butyl methyl ether (70 mL, 14 vol.) under N.sub.2 at
23.degree. C. Add dry acetonitrile (20 mL, 4 volumes) at ambient
temperature in one portion and heat the resulting hazy solution to
40.degree. C. Add a solution of 2.0 M HCl in acetonitrile (8.5 mL,
17.0 mmol, 0.99 equivalent) dropwise over 30 minutes while
maintaining a preset jacket temperature of 40.degree. C. Warm the
resulting slurry to 50.degree. C. then stir for 1 hour. Cool the
mixture to -10.degree. C. over 2-3 hours. Add oxalyl chloride (2.30
mL, 26.4 mmol, 1.50 equivalent) dropwise over 3-5 minutes, keeping
the pot temperature <-5.degree. C. Warm the resulting slurry to
0.degree. C. and stir for 1-2 hours until complete reaction by
HPLC. Add methanol (10 mL, 2 volumes) dropwise over 3-5 minutes,
keeping the pot temperature <10.degree. C. Allow the resulting
slurry to gradually warm to 23.degree. C. over 15-30 minutes then
stir for 1-2 hours until complete. Cool the slurry to 0-5.degree.
C., then add 2N KOH (38 mL, 76 mmol, 4,4 equivalents) dropwise to
adjust the pH of the mixture to 7.8 while maintaining the vessel
temperature <10.degree. C. Stir the quenched reaction mixture at
10.degree. C. for 15-20 minutes post pH adjustment, then remove the
lower aqueous layer. Back-extract the lower aqueous layer with
tert-butyl methyl ether (20 mL). Wash the combined organic layers
(100 mL) with aqueous 20% NaCl (50 mL) for 20-30 minutes at
10.degree. C. Allow the layers to settle for 15 minutes then remove
the brine layer. Subject the organic layer to a body feed of
Na.sub.2SO.sub.4 (15 g anhydrous), warm to 23.degree. C. then stir
for 1-12 hours. Filter the reaction mixture then concentrate the
filtrate in vacuo. Re-dissolve the residue in ethyl acetate (100
mL) then re-concentrate. Add ethyl acetate (35 mL) and CH.sub.3CN
(1 mL), heat the mixture to 45-50.degree. C. to dissolve, then cool
the mixture to 40.degree. C. over an hour. Optionally seed the
crude mixture (30 mg) then cool to 23.degree. C. over 2 hours after
a suspension forms. Add heptane (80 mL) dropwise over 20-30 minutes
to the slurry and then cool the mixture to 0.degree. C. over 1-2
hours. Stir the suspension for an additional 1-2 hours at 0.degree.
C. then filter. Rinse the filter cake with cold 2:1/heptane:ethyl
acetate (15 mL) then with room temperature heptane (15 mL). Dry the
filter cake in a vacuum oven at 50.degree. C. to a constant weight
to provide 5.60 g of
1-(1-[(pyridin-2-yl)methyl]piperidin-4-yl)-3-(methoxycarbonylcarbonyl)ind-
ole (87%).
[0036] Step 4--Charge a 3 neck flask equipped with an addition
funnel and nitrogen purge with the product of Step 3 (10.0 grams
(1.0 equivalent, 26.5 mmol) and
1-methyl-3-(aminocarbonylmethyl)indole (Faul et al, J. Org. Chem.,
63 (17):6053, 1998; 4.86 g, 0.975 equivalents, 25.8 mmol) in
tetrahydrofuran (Karl Ficher <0.03%, 72 ml, 7.2 volumes). Cool
the slurry to -5 to -10.degree. C. with an ice/acetone hath. Add
potassium t-butoxide (20% in tetrahydrofuran, 1.6 M, 36.4 ml, 2.2
equivalents, 58.3 mmoles) over 10-30 minutes maintaining the
reaction temperature at -10 to 5.degree. C. Heat the reaction to
40-45.degree. C. and stir for 1 hour to generate a slurry. Cool the
reaction to 0-10.degree. C. with an ice/water bath and then add
water (74 mL, pre-chilled to 0-10.degree. C.) rapidly. The reaction
generally exotherms to .about.15.degree. C. so re-cool the reaction
0-10.degree. C. and adjust the pH to 12.7-12.9 with a mixture of
concentrated HCl (5.2 ml) and water (15 ml) (approximately 2/3 of
this mixture is required). Adjust the pH with the remainder of the
HCl/water mixture over .about.20 minutes to a pH of 7.3-7.8 then
stir for 30 minutes at 0-10.degree. C. Slowly add water (60 mL)
over 20-30 minutes at 0-10.degree. C. and stir the reaction for 1-2
hrs. Filter on a pressure filter and wash with a pre-chilled
mixture of tetrahydrofuran (20 ml) and water (60 ml) and dry
overnight at 50.degree. C. under vacuum to give FB.
EXAMPLE 1
[0037] To a 3 necked flask equipped with heating mantle, condenser
and distillate take off add FB (59.0 g, 114.4 mols), 2-butanol (949
ml, 16.1 vols), deionized water (621.4 mL, 10.5 vols) and HCl (food
grade: 12.24 mL, 14.13 g, 0.21 volumes, 1.05 equivalents). Heat the
reaction to reflux and remove half of the solvent by distillation.
Slowly add 2-butanol (27 volumes) over 2 hours, while maintaining a
constant solvent level in the reaction flask. Cool the reaction to
room temperature over 60 minutes, then cool to 0-5.degree. C. and
stir for 1-2 hours. Filter the product and wash the filter cake
with 2 volumes of 2-butanol and dry the filter cake overnight at
50.degree. C. under vacuum to give F-I. Elemental Analysis: Theory
for C.sub.32H.sub.30N.sub.5O.sub.2Cl:
C,69.62,H,5.48,N,12.69,Cl,6.42; Found: C,
69.29,H,5.49,N,12.52,Cl,6.54.
[0038] XRD patterns were obtained on a Siemens D5000 X-ray powder
diffractometer, equipped with a CuK.alpha. source (.lamda.=1.54056
.ANG.) and a Kevex solid-state detector, operating at 50 kV and 40
mA with a 1 mm divergence and receiving slit and 0.1 mm detector
slit. Each sample was scanned between 4.degree. and 35.degree. in
2.theta. with a step size of 0.02.degree. and a maximum scan rate
of 3 sec/step. The XRD pattern for the material produced in Example
1 is as described in Table 1 and FIG. 1.
Formulation
[0039] A salt of the present invention is preferably formulated in
a unit dosage form prior to administration. Therefore, yet another
embodiment of the present invention is a pharmaceutical composition
comprising a salt of the present invention and a pharmaceutical
carrier. The term "pharmaceutical" when used herein as an adjective
means substantially non-deleterious to the recipient patient.
[0040] The present pharmaceutical compositions are prepared by
known procedures using well-known and readily available
ingredients. In making the formulations of the present invention,
the active ingredient (e.g., F-I) will usually be mixed with a
carrier, or diluted by a carrier, or enclosed within a carrier that
may be in the form of a capsule, sachet, paper or other container.
When the carrier serves as a diluent, it may be a solid, semisolid
or liquid material that acts as a vehicle, excipient or medium for
the active ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, lozenges, sachets, cachets, elixirs,
suspensions, emulsions, solutions, syrups, aerosol (as a solid or
in a liquid medium), soft and hard gelatin capsules, suppositories,
sterile injectable solutions and sterile packaged powders.
[0041] Some examples of suitable carriers, excipients, and diluents
include lactose, dextrose, sucrose, sorbitol, mannitol, starches,
gum acacia, calcium phosphate, alginates, tragacanth, gelatin,
calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water syrup, methyl cellulose, methyl and
propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
The formulations can additionally include lubricating agents,
wetting agents, emulsifying and suspending agents, preserving
agents, sweetening agents or flavoring agents. The compositions of
the invention may be formulated so as to provide quick, sustained
or delayed release of the active ingredient after administration to
the patient. TABLE-US-00005 Quantity Ingredient (mg/capsule) F-I
27.1 Crospovidone XL 16.9-24.4 Lactose Anhydrous 142.2-164.4
Lactose Monohydrate 142.2-164.4 Magnesium Stearate 1.1-2.8
Vegetable Povidone 13.1-16.9 Polysorbate 80 1.9-5.6
[0042] TABLE-US-00006 Quantity Ingredient (mg/capsule) F-I 108.5
Crospovidone XL 16.9-24.4 Lactose Anhydrous 101.5-123.8 Lactose
Monohydrate 101.5-123.8 Magnesium Stearate 1.1-2.8 Vegetable
Povidone 13.1-16.9 Polysorbate 80 1.9-5.6
[0043] The capsules above are manufactured by an aqueous
granulation process, as described below. The lactose, a portion of
the crospovidone, and the active ingredient (F-I) are added to the
granulator and dry blended for a suitable period of time to
uniformly distribute the powders. A granulation solution consisting
of povidone and polysorbate 80 in purified water is sprayed at a
uniform rate onto the powders while mixing under specified
conditions. When a suitable granulation endpoint is reached, the
granulator is stopped and the granulation is unloaded.
[0044] The granulation is wet sieved though a suitable screen to
disrupt large agglomerates, spread on paper lined trays, and dried
in a convection oven until the moisture is reduced to a suitable
level. The size of the granulation is reduced to a desirable range
by passing through a co-mill or other suitable apparatus. These
sized powders are collected, transferred to a mixing apparatus, and
blended with a specified quantity of magnesium stearate and
additional crospovidone until uniformly distributed. The finished
powders are then filled into hard gelatin capsules either manually
or on a suitable piece of automated capsule filling equipment.
[0045] Following the filling operation, the finished capsules are
visually inspected for external defects. To improve the
pharmaceutical elegance of the finished product, the capsules may
be physically de-dusted and polished by either manual or automated
processes.
Demonstration of Function
[0046] The salt of the present invention is an inhibitor of
vascular endothelial growth factor (VEGF)-induced angiogenesis. At
least two assay systems demonstrate these pharmacologic activities:
1) F-I is a potent inhibitor of VEGF-stimulated proliferation of
HUVEC cells in culture upon 72 hours of exposure to the compound;
2) F-I is a highly effective inhibitor of VEGF-induced
neo-angiogenesis in the rat corneal micropocket when administered
orally to the animals for 10 days. These assay systems are more
fully described in WO 02/02116. The salt of the present invention
is, thus, effective in treating cancer and inhibiting tumor
growth.
Utilities
[0047] As tumor growth inhibitors, the salt of the present
invention is useful to treat cancers of the bladder, brain, breast,
cervix, colorectum, esophagus, kidney, head and neck, liver, lung,
ovaries, pancreas, prostate and stomach. The salt of the present
invention is also useful to treat soft tissue sarcomas and
osteosarcomas and to treat Hodgkins and non-Hodgkins lymphoma or
hematological malignancies (leukemias).
[0048] Preferred methods of using a salt of the present invention
relate to its use to treat cancers of the bladder, kidney, brain,
breast, colorectum, liver, lung (non-small cell), ovaries and
stomach and to its use to treat non-Hodgkins lymphoma (e.g.,
diffuse large B cell and mantle cell lymphoma) or hematological
malignancies (leukemias).
[0049] Even more preferred methods of using a salt of the present
invention relate to its use to treat cancers of the brain,
colorectum, lung (non-small cell) and to its use to treat
non-Hodgkins lymphoma, .beta. cell lymphomas and .beta. cell
related leukemias.
Dose
[0050] One skilled in the art will recognize that the amount of a
salt of the present invention to be administered in accordance with
the present invention, that is, a therapeutically effective amount,
is that amount sufficient to produce an anti-neoplastic effect, to
induce apoptosis or cell death, and/or to maintain an
antiangiogenic effect.
[0051] Generally, an amount of a salt of the present invention to
be administered is decided an a case-by-case basis by the attending
physician. As a guideline, the extent and type of the neoplasia,
the timing of administration relative to other therapies (if any),
and the body weight, and age of the patient will be considered,
among other factors, when setting an appropriate dose. Typically,
an effective minimum daily dose of a salt of the present invention,
e.g., F-I, will exceed about 200 mg (usually >400 mg, e.g., 500
mg). Usually, an effective maximum daily dose of F-I will not
exceed about 700 mg. However, in the case of glioblastomas (brain
tumors) the maximum daily dose of F-I could be as high as 1400 mg.
The exact glioblastoma dose may be determined, in accordance with
the standard practice in the medical arts of "dose titrating" the
recipient; that is, initially administering a low dose of the
compound, e.g., 200 or 400 mg and gradually increasing the dose
until the desired therapeutic effect is observed.
Route of Administration
[0052] The salt of the present invention can be administered by a
variety of routes including the oral, rectal, transdermal,
subcutaneous, topical, intravenous, intramuscular or intranasal
routes. The oral route is preferred.
Combination Therapy
[0053] The salt of the present invention may be used in combination
with conventional anti-neoplasm therapies to treat mammals,
especially humans, with neoplasia. The procedures for conventional
anti-neoplasm therapies, including chemotherapies using
anti-neoplastic agents and therapeutic radiation, are readily
available, and routinely practiced in the art, e.g., see Harrison's
PRINCIPLES OF INTERNAL MEDICINE 11th edition, McGraw-Hill Book
Company.
[0054] Specifically, a crystalline salt of the present invention
may be used to enhance the anti-neoplasm effects of an
anti-neoplastic agent. A wide variety of available anti-neoplastic
agents are contemplated for combination therapy in accordance with
present invention.
[0055] Anti-neoplastic agents contemplated for combination therapy
in accordance with the present invention include, but are not
limited to: alkylating agents, including busulfan, chlorambucil,
cyclophosphamide, ifosfamide, melphalan, nitrogen mustard,
streptozocin, thiotepa, uracil nitrogen mustard, and
triethylenemelanine, temozolomide; antibiotics and plant alkaloids
including actinomycin-D, bleomycin, cryptophycins, daunorubicin,
doxorubicin, idarubicin, irinotecan, L-asparaginase, mitomycin-C,
mithramycin, navelbine, paclitaxel, docetaxel, topotecan,
vinblastine, vincristine, and VP-16; hormones and steroids
including aminoglutethimide, anastrozole, bicalutamide, DES,
estramustine, ethinyl estradiol, flutamide, fluoxymesterone,
goserelin, hydroxyprogesterone, letrozole, leuprolide,
medroxyprogesterone acetate, megestrol acetate, methyl
prednisolone, methyltestosterone, mitotane, nilutamide,
prednisolone, tamoxifen, testosterone and triamicnolone; synthetics
including all-trans retinoic acid, BCNU (carmustine), carboplatin
(paraplatin), CCNU (lomustine), cis-diaminedichloroplatinum
(cisplatin), dacarbazine, hexamethylmelamine, hydroxyurca,
levamisole, mitoxantrone, oxaliplatin, procarbazine;
antimetabolites including chlorodeoxyadenosine, cytosine
arabinoside, 2'-deoxycoformycin, fludarabine phosphate,
5-fluorouracil, 5-FUDR, gemcitabine, 6-mercaptopurine,
methotrexate, pemetrexed, and thioguanine; monoclonal antibodies
including rituximab and trastuzumab; antisense compounds including
ISIS 3521; and biologics including alpha interferon, BCG, G-CSF,
GM-CSF, and interleukin-2; and the like. These anti-neoplastic
agents assert their cytotoxicity or anti-neoplasm effects in a
variety of specific neoplastic conditions (see WO 02/02094).
[0056] In a preferred embodiment of the invention one or more
anti-neoplastic agents are selected from the group consisting of
BCNU, cyclophosphamide, doxorubicin, prednisone or dexamethasone,
vincristine, gemcitabine, cisplatin, 5 fluoruracil, capecitibine,
CPT-11, carboplatin, paclitaxel, docetaxel, rituximab and
trastuzumab.
[0057] A crystalline salt of the present invention may also be used
in combination with radiation therapy. Usually, radiation is used
to treat the site of a solid tumor directly or administered by
brachytherapy implants.
[0058] Therapeutic radiation contemplated for combination therapy
in accordance with the present invention are those used in the
treatment of cancer which include, but are not limited to X-rays,
gamma radiation, high energy electrons and High LET (Linear Energy
Transfer) radiation such as protons, neutrons, and alpha particles.
The ionizing radiation is employed by techniques well known to
those skilled in the art. For example, X-rays and gamma rays are
applied by external and/or interstitial means from linear
accelerators or radioactive sources. High-energy electrons can be
produced by linear accelerators. High LET radiation is also applied
from radioactive sources implanted interstitially.
[0059] The phrase "in combination with" means that the crystalline
salt of the present invention is administered shortly before,
shortly after, concurrently, or any combination of before, after,
or concurrently, with such other anti-neoplasm therapies. A salt of
the present invention may be administered in combination with more
than one anti-neoplasm therapy. In a preferred embodiment, the a
salt of the present invention is administered from 2 weeks to 1 day
before any chemotherapy, or 2 weeks to 1 day before any radiation
therapy. In another preferred embodiment, a salt of the present
invention may be administered during anti-neoplastic chemotherapies
and radiation therapies. If administered following such
chemotherapy or radiation therapy, a salt of the present invention
is preferably given within 1 to 14 days following the primary
treatments. A salt of the present invention may also be
administered chronically or semi-chronically, over a period of from
about 2 weeks to about 5 years.
* * * * *