U.S. patent application number 17/459531 was filed with the patent office on 2022-07-28 for morphic forms of marizomib and uses thereof.
The applicant listed for this patent is Celgene International II Sarl. Invention is credited to Robert MANSFIELD.
Application Number | 20220235063 17/459531 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-28 |
United States Patent
Application |
20220235063 |
Kind Code |
A1 |
MANSFIELD; Robert |
July 28, 2022 |
MORPHIC FORMS OF MARIZOMIB AND USES THEREOF
Abstract
The present invention relates to polymorphic forms of marizomib
(e.g., Morphic Form I). The morphic forms can be used alone and in
pharmaceutical compositions for the treatment of disease.
Inventors: |
MANSFIELD; Robert; (San
Marcos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Celgene International II Sarl |
Couvet |
|
CH |
|
|
Appl. No.: |
17/459531 |
Filed: |
August 27, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16886301 |
May 28, 2020 |
11136332 |
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17459531 |
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16325837 |
Feb 15, 2019 |
10703760 |
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PCT/EP2017/070950 |
Aug 18, 2017 |
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16886301 |
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62377156 |
Aug 19, 2016 |
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International
Class: |
C07D 491/048 20060101
C07D491/048; C07D 491/02 20060101 C07D491/02; A61P 35/00 20060101
A61P035/00 |
Claims
1. Morphic Form I of marizomib, characterized by an X-ray powder
diffraction pattern including peaks at about 7.2, 14.5, and
36.7.degree.2.degree. using Cu K.alpha. radiation.
2. The morphic Form of claim 1, further including X-ray powder
diffraction peaks at about 18.1, 19.6, and 20.8.degree.2.theta.
using Cu K.alpha. radiation.
3. The morphic Form of any of the preceding claims, further
including X-ray powder diffraction peaks at about 16.3, 19.8, and
20.5.degree.2.theta. using Cu K.alpha. radiation.
4. The morphic Form of any of the preceding claims, further
including X-ray powder diffraction peaks at about 15.2, 21.5, and
22.3.degree.2.theta. using Cu K.alpha. radiation.
5. The morphic Form of any of the preceding claims, further
including X-ray powder diffraction peaks at about 14.7, 29.2, and
30.0.degree.2.degree. using Cu K.alpha. radiation.
6. The morphic Form of any of the preceding claims, further
including X-ray powder diffraction peaks at about 8.2, 14.8, and
27.7.degree.2.theta. using Cu K.alpha. radiation.
7. The morphic Form of any of the preceding claims, further
characterized by an X-ray powder diffraction pattern substantially
similar to that set forth in FIG. 1, 2 or 3.
8. The morphic Form of any of the preceding claims, further
characterized by a degradation event at about 175.degree. C.
measured by thermogravimetric analysis.
9. The morphic Form of any of the preceding claims, further
characterized by two exotherms at about 150-180.degree. C. as
measured by differential scanning calorimetry at a rate of about
2.degree. C. per minute.
10. The morphic Form of any of the preceding claims, characterized
by melting at about 160-175.degree. C. as measured by hot stage
microscopy.
11. The morphic Form of any of the preceding claims, wherein the
polymorph is at least about 98% pure as measured by HPLC.
12. The morphic Form of claim 11, wherein the polymorph is at least
about 99.1% pure as measured by HPLC.
13. A pharmaceutical composition comprising a morphic Form of any
of the preceding claims and a pharmaceutically acceptable
carrier.
14. A method of treating a disease, comprising administering to a
subject in need thereof an effective amount of a morphic Form of
any of claims 1-12.
15. The method of claim 14, wherein the disease is cancer.
16. Use of the morphic Form of any of claims 1-12 for the treatment
of a disease.
17. Use of the morphic Form of any of claims 1-12 for inhibiting a
protease.
18. Use of the morphic Form of any of claims 1-12 in the
manufacture of a medicament for the treatment of a disease.
19. A method of preparing a morphic Form of marizomib characterized
by an X-ray powder diffraction pattern including peaks at about
7.2, 14.5, and 36.7.degree.2.theta. using Cu K.alpha. radiation,
comprising recrystallizing marizomib from a solvent.
20. The method of claim 19, wherein the solvent is selected from
the group consisting of n-heptane, ethyl acetate, methyl-isobutyl
ketone, 2-propanol, acetone, chloroform, dimethyl sulfoxide,
tert-butyl methyl ether, anisole, cumene, methyl ethyl ketone,
isopropyl acetate, dimethylformamide, toluene, tetrahydrofuran,
dichloromethane, acetonitrile, nitromethane, ethanol, and
dimethylacetamide.
Description
RELATED APPLICATIONS
[0001] This application claims priority to, and benefit of, U.S.
Provisional Patent Application No. 62/377,156, filed Aug. 19, 2016,
the contents of which are incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to polymorphic forms of
marizomib (e.g., Morphic Form I). The morphic forms can be used
alone and in pharmaceutical compositions for the treatment of
disease.
BACKGROUND OF THE INVENTION
[0003] Marizomib is a proteasome inhibitor capable of inhibiting
all three domains of the proteasome (i.e., the chymotrypsin-like
(CT-L); caspase-like (C-L) and trypsin-like (T-L) domains).
Accordingly, marizomib can be useful for treating diseases such as
cancer. Thus, there is a need for pure, stable morphic forms of
marizomib that can be used for administration to subjects in need
thereof. The present disclosure teaches stable and pure morphic
forms of marizomib.
SUMMARY OF THE INVENTION
[0004] In one aspect, the present disclosure provides morphic Form
I of marizomib, characterized by an X-ray powder diffraction
pattern including peaks at about 7.2, 14.5, and
36.7.degree.2.theta. using Cu K.alpha. radiation.
[0005] In another aspect, the present disclosure provides a
pharmaceutical composition comprising a morphic Form of marizomib
(e.g., Form I) and a pharmaceutically acceptable carrier.
[0006] In another aspect, the present disclosure provides a method
of treating a disease comprising administering to a subject in need
thereof an effective amount of a morphic Form of marizomib (e.g.,
morphic Form I).
[0007] In another aspect, the present disclosure provides a morphic
Form of marizomib (e.g., morphic Form I) for use in the treatment
of a disease.
[0008] In another aspect, the present disclosure provides a morphic
Form of marizomib (e.g., morphic Form I) for inhibiting a
protease.
[0009] In another aspect, the present disclosure provides the use
of a morphic form of marizomib (e.g., morphic Form I) in the
manufacture of a medicament for the treatment of a disease.
[0010] In another aspect, the present disclosure provides a method
of preparing a morphic form of marizomib (e.g., morphic Form I)
comprising recrystallizing marizomib from a solvent.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 shows an XRPD spectrum of a Sample 1 of Morphic Form
I of marizomib.
[0012] FIG. 2 shows an XRPD spectrum of a Sample 2 of Morphic Form
I of marizomib.
[0013] FIG. 3 shows an XRPD spectrum of a Sample 3 of Morphic Form
I of marizomib.
[0014] FIG. 4 shows an overlay of all three XRPD spectra of Samples
1, 2 and 3 of Form I of marizomib.
[0015] FIG. 5 shows a plot of variable temperature XRPD spectra of
Sample 1 of Morphic Form I of marizomib.
[0016] FIG. 6 shows a plot of variable temperature XRPD spectra of
Sample 3 of Morphic Form I of marizomib.
[0017] FIG. 7 shows an overlay of two XRPD spectra of marizomib
after four years of storage at 2-8.degree. C.
[0018] FIG. 8 shows an overlay of five XRPD spectra contrasting the
XRPD spectra of Form I of marizomib with amorphous forms of
marizomib.
[0019] FIG. 9 shows an XRPD spectrum of a Pawley fitting procedure
for Sample 1 of Morphic Form I of marizomib.
[0020] FIG. 10 shows an XRPD spectrum of a Pawley fitting procedure
for Sample 3 of Morphic Form I of marizomib.
[0021] FIG. 11 shows an overlay of XRPD spectra from a polymorph
screen.
[0022] FIG. 12 shows an overlay of XRPD spectra from a polymorph
screen.
[0023] FIG. 13 shows a plot of a thermogravimetric analysis of
Sample 1 of Morphic Form I of marizomib.
[0024] FIG. 14 shows a plot of a thermogravimetric analysis of
Sample 3 of Morphic Form I of marizomib.
[0025] FIG. 15 shows a differential scanning calorimetry plot of
Sample 1 of Morphic Form I of marizomib at 2.degree. C. per
minute.
[0026] FIG. 16 shows a differential scanning calorimetry plot of
Sample 1 of Morphic Form I of marizomib at 5.degree. C. per
minute.
[0027] FIG. 17 shows a differential scanning calorimetry plot of
Sample 1 of Morphic Form I of marizomib at 10.degree. C. per
minute.
[0028] FIG. 18 shows a differential scanning calorimetry plot of
Sample 1 of Morphic Form I of marizomib at 50.degree. C. per
minute.
[0029] FIG. 19 shows a differential scanning calorimetry plot of
Sample 3 of Morphic Form I of marizomib at 2.degree. C. per
minute.
[0030] FIG. 20 shows a differential scanning calorimetry plot of
Sample 3 of Morphic Form I of marizomib at 5.degree. C. per
minute.
[0031] FIG. 21 shows a differential scanning calorimetry plot of
Sample 3 of Morphic Form I of marizomib at 10.degree. C. per
minute.
[0032] FIG. 22 shows a differential scanning calorimetry plot of
Sample 3 of Morphic Form I of marizomib at 50.degree. C. per
minute.
[0033] FIG. 23 shows a .sup.1HNMR spectrum for both Sample 1 and
Sample 3 of marizomib.
[0034] FIG. 24 is an HPLC plot of Sample 1 of marizomib.
[0035] FIG. 25 is an HPLC plot of Sample 3 of marizomib.
[0036] FIG. 26 is an XRPD plot of marizomib before and after
attempted hydrolysis.
[0037] FIG. 27 is a 1HNMR plot of marizomib before and after
attempted hydrolysis.
[0038] FIG. 28 is an HPLC plot of marizomib isolated after a
hydrolysis attempt.
[0039] FIG. 29 is an HPLC plot of marizomib isolated from ethyl
acetate
[0040] FIG. 30 is an HPLC plot of marizomib isolated from
ethanol.
[0041] FIG. 31 is a polarized light microscope image of Morphic
Form I of Sample 1 of marizomib.
[0042] FIG. 32 is a polarized light microscope image of Morphic
Form I of Sample 3 of marizomib.
[0043] FIG. 33 is a photo of Sample 1 of Morphic Form I of
marizomib at 25.degree. C.
[0044] FIG. 34 is a photo of Sample 1 of Morphic Form I of
marizomib at 150.degree. C.
[0045] FIG. 35 is a photo of Sample 1 of Morphic Form I of
marizomib at 160.degree. C.
[0046] FIG. 36 is a photo of Sample 1 of Morphic Form I of
marizomib at 160.degree. C. after five minutes.
[0047] FIG. 37 is a photo of Sample 3 of Morphic Form I of
marizomib at 25.degree. C.
[0048] FIG. 38 is a photo of Sample 3 of Morphic Form I of
marizomib at 150.degree. C.
[0049] FIG. 39 is a photo of Sample 3 of Morphic Form I of
marizomib at 160.degree. C.
[0050] FIG. 40 is a photo of Sample 3 of Morphic Form I of
marizomib at 160.degree. C. after five minutes.
[0051] FIG. 41 is an overlay of an XRPD pattern of marizomib with
solids from the 2-pyrrolidone experiment described in Example
12.
[0052] FIG. 42 is a thermal analysis of solids from-pyrrolidone
experiment described in Example 12.
[0053] FIG. 43 is an Overlay of an XRPD pattern of marizomib with
solids from the melamine experiment described in Example 12.
[0054] FIG. 44 is a thermal analysis of solids the melamine
experiment described in Example 12.
[0055] FIG. 45 is an overlay of XRPD patterns of marizomib with the
solids of the melamine 1:1 and 1:3 experiments described in Example
12.
[0056] FIG. 46 is an overlay of XRPD patterns of marizomib with
solids from the cytosine experiments described in Example 12.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The present disclosure relates to morphic Forms (e.g., Form
I) of marizomib. The morphic Forms can be solvates or hydrates.
[0058] As defined herein, "pure" is understood to mean that a
compound is uniform in chemical makeup. It is understood that a
pure compound does not contain molecules of another chemical makeup
in an appreciable amount, e.g., less than 5%, less than 4%, less
than 3%, less than 2%, or less than 1%. In some instances, the
purity is measured excluding any solvent (e.g., organic solvent
such as ethyl acetate, and inorganic solvent such as water). Purity
can be measured by a number of techniques known in the art, for
example HPLC.
[0059] In some embodiments, the morphic forms of marizomib are
substantially pure. For example, the morphic forms described herein
(e.g., morphic Form I) can be greater than about 90% pure, greater
than about 91% pure, greater than about 92% pure, greater than
about 93% pure, greater than about 94% pure, greater than about 95%
pure, greater than about 96% pure, greater than about 97% pure,
greater than about 98% pure, greater than about 99% pure, or
greater than about 99.9% pure.
[0060] As defined herein, "stable" or "stability" relates to the
ability of a compound to remain pure for a period of time. A stable
compound can be one that maintains its purity (e.g., does not have
molecules of an undesired chemical formula) despite extended
storage (e.g., greater than one month, greater than six months, or
greater than a year). A stable compound can also be a compound that
remains pure despite conditions such as high temperature or
humidity.
[0061] In some embodiments, the morphic forms of marizomib
described herein retain their purity for extended periods of time
(i.e., are stable). In some embodiments, the morphic Forms
described herein (e.g., morphic Form I) can retain at least 99%
purity for one month, for two months, for three months, for four
months, for five months, for six months, for 12 months, for 24
months, for 36 months, for 48 months, or for 60 months or more.
[0062] Without wishing to be bound by theory, chemical compounds
are known to exist in multiple different polymorphic forms.
Accordingly, one of skill in the art would expect that marizomib
can potentially exist in multiple different morphic forms, in
addition to the amorphous form. Surprisingly, the present
disclosure teaches that marizomib exists primarily in one morphic
form (i.e., morphic Form I). The present disclosure teaches that
morphic Form I is particularly stable.
Morphic Form I of Marizomib
[0063] Marizomib is a proteasome inhibitor with the structure:
##STR00001##
[0064] In some embodiments, morphic Form I of marizomib is
characterized by an X-ray diffraction pattern including peaks at
about 7.2, 14.5, and 36.7.degree.2.theta. using Cu K.alpha.
radiation. In some embodiments, morphic Form I is further
characterized by X-ray powder diffraction peaks at about 18.1,
19.6, and 20.8.degree.2.theta. using Cu K.alpha. radiation. In some
embodiments, morphic Form I of marizomib is further characterized
by X-ray powder diffraction peaks at about 16.3, 19.8, and
20.5.degree.2.theta. using Cu K.alpha. radiation. In some
embodiments, morphic Form I of marizomib is further characterized
by X-ray powder diffraction peaks at about 15.2, 21.5, and
22.3.degree.2.theta. using Cu K.alpha. radiation. In some
embodiments, morphic Form I of marizomib is further characterized
by X-ray powder diffraction peaks at about 14.7, 29.2, and
30.0.degree.2.theta. using Cu K.alpha. radiation. In some
embodiments, morphic Form I of marizomib is further characterized
by X-ray powder diffraction peaks at about 8.2, 14.8, and
27.7.degree.2.theta. using Cu K.alpha. radiation. In some
embodiments, morphic Form I of marizomib is free of solvent (e.g.,
water or organic solvent).
[0065] In some embodiments, morphic Form I of marizomib is further
characterized by an X-ray powder diffraction pattern substantially
similar to that set forth in FIG. 1. In some embodiments, morphic
Form I of marizomib is further characterized by an X-ray powder
diffraction pattern substantially similar to that set forth in FIG.
2. In some embodiments, morphic Form I of marizomib is further
characterized by an X-ray powder diffraction pattern substantially
similar to that set forth in FIG. 3. In some embodiments, morphic
Form I of marizomib is further characterized by a degradation event
at about 175.degree. C. measured by thermogravimetric analysis.
[0066] In some embodiments, morphic Form I of marizomib is further
characterized by two exotherms at about 150-180.degree. C. as
measured by differential scanning calorimetry at a rate of about
2.degree. C. per minute. For example, morphic Form I can be
characterized by two exotherms at about 155-175.degree. C.
[0067] In some embodiments, morphic Form I of marizomib is
characterized by an endotherm at about 168.5.degree. C. and an
exotherm at about 173-183.degree. C., as measured by differential
scanning calorimetry at a rate of about 5.degree. C. per
minute.
[0068] In some embodiments, morphic Form I of marizomib is
characterized by two endotherms at about 175.5.degree. C. and
180.6.degree. C. and an exotherm at about 183-193.degree. C.; or
characterized by two endotherms at about 171.2.degree. C. and
178.6.degree. C. and an exotherm at about 183-193.degree. C., as
measured by differential scanning calorimetry at a rate of about
10.degree. C. per minute.
[0069] In some embodiments, morphic Form I of marizomib is
characterized by two endotherms at about 186.5.degree. C. and
192.6.degree. C. and an exotherm at about 193-205.degree. C.; or
characterized by two endotherms at about 180.9.degree. C. and
191.6.degree. C. and an exotherm at about 193-205.degree. C. as
measured by differential scanning calorimetry at a rate of about
50.degree. C. per minute.
[0070] In some embodiments, morphic Form I of marizomib is
characterized by melting and/or degradation at about
160-175.degree. C. as measured by hot stage microscopy.
[0071] In some embodiments, morphic Form I of marizomib is at least
about 98% pure as measured by HPLC. For example, the morphic Form
can be at least about 99.1% pure as measured by HPLC.
[0072] As set forth in the present disclosure, three unique samples
of morphic Form I of marizomib were characterized, Sample 1, Sample
2, and Sample 3. Samples 1 and 2 are not micronized, whereas Sample
3 was micronized. X-ray powder diffraction spectra measurements for
Samples 1, 2 and 3 are given in Tables 1, 2 and 3, respectively. As
set forth herein, both the micronized and the non-micronized
samples are the same morphic form (i.e., morphic Form I).
TABLE-US-00001 TABLE 1 XRPD: Form I Sample 1 Pos. Rel. Int.
[.degree.2.theta.] [%] 7.2 100.0 11.4 0.1 14.0 0.1 14.5 6.4 17.7
0.2 18.0 2.0 19.1 0.1 19.6 0.9 20.8 0.4 21.8 0.2 22.3 0.2 23.0 0.3
25.4 0.2 26.6 0.2 26.9 0.2 27.6 0.3 29.2 0.7 36.7 4.3 37.5 0.3
TABLE-US-00002 TABLE 2 XRPD: Form I Sample 2 Pos. Rel. Int.
[.degree.2.theta.] [%] 7.2 100.0 11.5 0.3 14.0 0.2 14.5 5.2 17.7
0.2 18.1 2.6 19.1 0.3 19.6 2.2 20.8 0.7 22.3 0.4 23.1 0.4 25.5 0.4
26.6 0.5 26.9 0.4 27.7 0.5 29.2 0.6 36.7 4.9 37.5 0.5
TABLE-US-00003 TABLE 3 XRPD: Frm I Sample 3 Pos. Rel. Int.
[.degree.2.theta.] [%] 7.2 100.0 8.2 4.2 10.2 2.4 11.4 2.2 13.7 2.0
14.5 7.7 14.7 5.6 14.8 3.6 15.2 6.0 16.3 9.3 18.1 18.0 19.1 3.2
19.6 16.3 19.8 11.9 20.5 6.6 20.8 5.6 21.5 6.2 22.1 2.8 22.3 6.3
23.0 2.6 23.9 3.4 25.4 2.7 26.5 3.4 26.9 2.5 27.7 4.3 28.8 3.3 29.2
4.6 29.5 3.4 30.0 4.5 32.3 3.3 36.7 4.6
[0073] FIG. 1 shows an XRPD spectrum of Sample 1 of morphic Form I
of marizomib. As set forth in FIG. 1, the XRPD spectrum includes
peaks at about 7.2, 14.5, and 36.7.degree.2.theta.. FIG. 2 shows an
XRPD spectrum of Sample 2 of morphic Form I of marizomib. As set
forth in FIG. 2, the XRPD spectrum includes peaks at about 7.2,
14.5, and 36.7.degree.2.theta.. FIG. 3 shows an XRPD spectrum of
Sample 3 of morphic Form I of marizomib. As set forth in FIG. 3,
the XRPD spectrum includes peaks at about 7.2, 14.5, and
36.7.degree.2.theta..
[0074] FIG. 4 shows an overlay of the three XRPD spectra of FIGS.
1, 2 and 3 (i.e., Samples 1, 2 and 3). As shown in FIG. 4, there
can be slight differences between the XRPD spectra of even the same
morphic form (i.e., morphic Form I) of marizomib. For example,
certain peaks may be more or less pronounced (e.g., smaller or
larger) in one spectrum compared with another.
[0075] As shown in the tables and figures of the present
application, not all values for peaks (pos. .degree.2.theta.) are
identical for different lots of the polymorphs of the present
application. An artisan of ordinary skill will understand that even
different lots of the same polymorphic forms can produce slightly
different characterization data, while not being appreciably
different. For instance, slight variations in the calibration of
the instruments used to perform a given measurement, or minor
fluctuations in relative humidity between measurements can give
rise to data that displays slight differences between samples. One
of ordinary skill in the art will thus be able to, for instance,
calibrate his or her instruments and take repeated measurements in
order to minimize any discrepancies between signals to properly
characterize the polymorphs of the present application. However,
despite some minor variation in the batch-to-batch differences,
polymorphs of the present application are identified and
characterized by their characteristic peaks, such as those
described above (e.g., about 7.2, 14.5, and 36.7.degree.2.theta.,
0.2.degree.2.theta.).
[0076] In some embodiments, differences in XRPD spectra (e.g.,
resolution) can be attributed to preferred orientation. Without
wishing to be bound by theory, in XRPD it can be desirable to have
a sample in which particles are oriented randomly (e.g., a powder).
However, it can be difficult or in some cases impossible to achieve
truly random particle orientations in practice. As particle size
increases, the randomness of particle orientation can decrease,
leading to increased challenges with achieving a preferred
orientation.
[0077] Without wishing to be bound by theory, larger particles that
do not exhibit random orientations can result in XRPD spectra in
which some peaks are either diminished in intensity or in some
embodiments missing altogether. Accordingly, in some embodiments
micronized samples (e.g., Sample 3) can facilitate more random
orientation of particles and thus a more accurate XRPD spectrum. In
some embodiments a micronized sample with more randomness in
particle orientation results in an XRPD spectrum with more peaks.
In contrast, samples with larger particles (e.g., Sample 1, Sample
2) can lead to XRPD spectra with less pronounced peaks and in some
embodiments fewer peaks.
[0078] As set forth in FIG. 5, morphic Form I of marizomib is
stable even at elevated temperatures. FIG. 5 shows five unique XRPD
spectra of sample 1 of morphic Form I of marizomib. The lowest
trace (505) shows an XRPD spectrum at 25.degree. C. Above that
trace is the same sample at 100.degree. C. (510). Shown above is
another trace at 150.degree. C., (515) then at 160.degree. C.
(520), and finally the highest trace shows an XRPD spectrum of the
sample after five minutes at 160.degree. C. (525).
[0079] Additionally, FIG. 6 shows five unique XRPD spectra of
sample 3 of morphic Form I of marizomib at different temperatures.
The lowest (605) is an XRPD spectrum of Sample 3 at 25.degree. C.,
and also shown are traces at 100.degree. C. (610), 150.degree. C.
(615), 160.degree. C. (620), and 160.degree. C. after five minutes
(625). As set forth in FIGS. 5 and 6, only one morphic form (i.e.,
Form I) of marizomib was observed even as the temperature
increased. Without wishing to be bound by theory, this suggests
that Form I is the major morphic Form of marizomib. Without wishing
to be bound by theory, this also suggests that Form I is the most
stable morphic form of marizomib.
[0080] Without wishing to be bound by theory, the results of FIGS.
5 and 6 are further supported by FIG. 7. FIG. 7 shows an overlay of
the same sample of morphic Form I of marizomib initially (705) and
after storage for four years at 2-8.degree. C. (710). The XRPD
spectra are substantially identical, demonstrating that morphic
Form I is stable at low temperatures for extended periods of
time.
[0081] FIG. 8 shows an overlay of five XRPD spectra of marizomib.
Form I of marizomib is shown in trace 805, whereas marizomib that
has been co-spray dried with polymers are shown in traces 810, 815,
820, and 825. Trace 810 shows an XRPD spectrum of marizomib that
has been co-spray dried with hydroxypropyl methylcellulose. Trace
815 shows an XRPD spectrum of marizomib that has been co-spray
dried with polyvinylpyrrolidone. Trace 820 shows an XRPD spectrum
of marizomib that has been co-spray dried with hydroxypropyl
methylcellulose acetate succinate-M. Trace 825 shows an XRPD
spectrum of marizomib that has been co-spray dried with
hydroxypropyl methylcellulose acetate succinate-L.
[0082] Pawley fitting is a process in which observed peaks in a
powder pattern can be fitted without a structural model but at 20
values constrained by the size and symmetry of the unit cell. In
some embodiments, it can be a useful precursor to Rietveld fitting
and can give an indication of the "best fit possible" from an
eventual structural refinement. This fitting method was applied to
XRPD diffractograms of the micronized and non-micronized supplied
materials, Sample 3 and Sample 1, respectively.
[0083] Marizomib crystallizes in the monoclinic space group, P21,
with these unit cell parameters: [0084] a=10.57 .ANG., b=24.49
.ANG. c=12.63 .ANG. and .beta.=108.34.degree.
[0085] Without wishing to be bound by theory, fitting the two
samples showed good correlation between calculated and observed
profiles, with only very slight differences in peak shape and
intensity. Sample 1 exhibited strong preferred orientation
(crystals were of lath morphology) as opposed to the micronized
Sample 3. As such, without wishing to be bound by theory, the
micronized sample (Sample 3) appeared to display numerous peaks
that were not visible in the non-micronized sample (Sample 1).
[0086] Without wishing to be bound by theory, the average particle
size of particles of Sample 1 (i.e., non-micronized) was about 150
.mu.m to about 300 .mu.m, compared with an average sample size of
about 1 .mu.m to about 20 .mu.m for the micronized Sample 3.
Without wishing to be bound by theory, the smaller particle size in
Sample 3 reduces technical challenges associated with preferred
orientation and allows for more accurate representation of peaks.
For example, Sample 1, Sample 2, and Sample 3 all show an XRPD peak
at about 36.7.degree.2.theta., at intensities of 4.3, 4.9, and 4.6,
respectively. However, the peak appears larger in Samples 1 and 2
because of the preferred orientation of the sample.
[0087] The Pawley fitting procedure can enable visualization of the
presence or absence of a different phase. The peak at about
8.degree.2.theta., on the micronized sample (i.e., Sample 3)
diffractogram was found to be also present in the non-micronized
sample (Sample 1) diffractogram, as seen in the FIGS. 9-10. FIG. 9
shows an XRPD spectrum of a Pawley fitting procedure for Sample 1
of Morphic Form I of marizomib. FIG. 10 shows an XRPD spectrum of a
Pawley fitting procedure for Sample 3 of Morphic Form I of
marizomib. Without wishing to be bound by theory, these results
suggest that the majority of the material from these two batches
(i.e., Sample 1 and Sample 3) is representative of the single
crystal data and any subtle difference between them is attributable
to crystal morphology inducing preferred orientation.
[0088] FIGS. 11 and 12 show a overlays of XRPD spectra of the
solids isolated from the polymorph screen set forth in Example 2
using the various solvents used in the screen. A solid was not
obtained for two solvents (DMF and DMA) since marizomib remained in
solution and did not precipitate, thus no XRPD pattern for DMF or
DMA was obtained. The solvents are listed next to the corresponding
spectra.
[0089] FIG. 13 shows a plot of a thermogravimetric analysis of
Sample 1 of morphic Form I of marizomib. As shown in FIG. 13, no
weight loss was observed until about 175.degree. C. Without wishing
to be bound by theory, this weight loss can be due to sample
degradation.
[0090] Similarly, FIG. 14 shows a plot of a thermogravimetric
analysis of sample 3 of morphic Form I of marizomib. As shown in
FIG. 14, no weight loss was observed until about 175.degree. C.
Without wishing to be bound by theory, this weight loss can be due
to sample degradation.
[0091] FIG. 15 shows a differential scanning calorimetry plot of
Sample 1 of Morphic Form I of marizomib at 2.degree. C. per minute.
As shown in FIG. 15, no endotherm was observed. Two exotherms were
observed between about 150-180.degree. C. The first exotherm was
smaller than the second exotherm. Without wishing to be bound by
theory, the exotherms could be due to sample degradation.
[0092] FIG. 16 shows a differential scanning calorimetry plot of
Sample 1 of Morphic Form I of marizomib at 5.degree. C. per minute.
As shown in FIG. 16, an endotherm was observed at 169.6.degree. C.
(5.5. J/g) followed by a sharp exotherm at 173-183.degree. C.
Without wishing to be bound by theory, the exotherm could be due to
sample degradation.
[0093] FIG. 17 shows a differential scanning calorimetry plot of
Sample 1 of Morphic Form I of marizomib at 10.degree. C. per
minute. As shown in FIG. 17, a small endotherm was observed at
175.5.degree. C. (7.1 J/g) and 180.6.degree. C. (0.6 J/g). The two
endotherms were followed by an exotherm at about 183-193.degree. C.
Without wishing to be bound by theory, the exotherm could be due to
sample degradation.
[0094] FIG. 18 shows a differential scanning calorimetry plot of
Sample 1 of Morphic Form I of marizomib at 50.degree. C. per
minute. As shown in FIG. 18, two small endotherms were observed at
about 186.5.degree. C. (12/8 J/g) and about 192.6.degree. C. (18.9
J/g). The two endotherms were followed by an exotherm at about
193-205.degree. C. Without wishing to be bound by theory, this
could be due to sample degradation.
[0095] FIG. 19 shows a differential scanning calorimetry plot of
Sample 3 of Morphic Form I of marizomib at 2.degree. C. per minute.
As shown in FIG. 19, no endotherms were observed. Two exotherms
were observed at about 155-175.degree. C. Without wishing to be
bound by theory, this could be due to sample degradation.
[0096] FIG. 20 shows a differential scanning calorimetry plot of
Sample 3 of Morphic Form I of marizomib at 5.degree. C. per minute.
As shown in FIG. 20, a small endotherm was observed at
167.5.degree. C. (0.4 J/g) followed by a sharp exotherm at about
173-183.degree. C. Without wishing to be bound by theory, this
could be due to sample degradation.
[0097] FIG. 21 shows a differential scanning calorimetry plot of
Sample 3 of Morphic Form I of marizomib at 10.degree. C. per
minute. As shown in FIG. 21, two small endotherms were observed at
about 171.2.degree. C. (6.1 J/g) and about 178.6.degree. C. (2.0
J/g), followed by an exotherm at about 183-193.degree. C. Without
wishing to be bound by theory, this could be due to sample
degradation.
[0098] FIG. 22 shows a differential scanning calorimetry plot of
Sample 3 of Morphic Form I of marizomib at 50.degree. C. per
minute. As shown in FIG. 22, two small endotherms were observed at
about 180.9.degree. C. (8.3 J/g) and 191.6.degree. C. (19.0 J/g).
The two small endotherms were followed by an exotherm at about
193-205.degree. C. Without wishing to be bound by theory, this
could be due to sample degradation.
[0099] As set forth in FIGS. 15-22, as the heating rate of the DSC
experiments increased, a shift in onset temperature as well as a
variation in enthalpy was observed. Without wishing to be bound by
theory, these fluctuations observed in the temperature onset and
enthalpy can be due to combination of melt and degradation of the
sample. In other words, without wishing to be bound by theory, a
pure melt of a crystalline, non-solvated morphic form is a
thermodynamic event, so that changes in heating rate and/or amount
of input material will not significantly affect the onset
temperature of the melt. In contrast, sample degradation is a
kinetic event, so that changes in the experimental parameters
(amount of input material, heating rate, type of pan used, i.e.
sealed or unsealed) can cause a shift in the onset temperature of
that event.
[0100] Without wishing to be bound by theory, based on the results
of DSC analysis of Samples 1 and 3, the changes observed in the
main endothermic event can be due to both thermodynamic and kinetic
factors, i.e. both melt and degradation of the sample.
[0101] Without wishing to be bound by theory, the results of the
DSC and TG analysis of morphic Form I is consistent with the
results set forth in FIGS. 5 and 6. FIGS. 5 and 6 demonstrate that
Sample 1 and 3 remained as Form I throughout the increase in
temperature from 25.degree. C. to 160.degree. C. Discoloration of
the sample was observed at about 160.degree. C. from white to
yellow, as shown in FIGS. 33-36. However, after about five minutes
to enable equilibration at about 160.degree. C., the sample was
observed to melt and degrade simultaneously.
[0102] Without wishing to be bound by theory, this result is
further supported by the outcome from the hot stage microscopy
experiments, in which crystals from Sample 1 were subjected to
heating under air at .about. 30.degree. C./min. No changes were
observed between 30 and 150.degree. C. Between 160-175.degree. C.
changes were observed which captured the melt of the crystals and
degradation of the sample (in form of discoloration) occurring
substantially simultaneously.
[0103] FIG. 23 shows a .sup.1HNMR spectrum for both Sample 1 (2305)
and Sample 3 (2310) of marizomib. As shown in FIG. 23, both spectra
are consistent with the structure of marizomib.
[0104] FIG. 24 is an HPLC plot of Sample 1 of marizomib. As shown
in FIG. 24, the purity of Sample 1 was at least 98.2%.
[0105] FIG. 25 is an HPLC plot of Sample 3 of marizomib. As shown
in FIG. 25, the purity of Sample 3 was at least 99.1%.
[0106] FIG. 31 is a polarized light microscope image of Morphic
Form I of Sample 1 of marizomib. As shown in FIG. 31, particles of
Sample 1 had a lath morphology with particle size of 150 .mu.m up
to 300 .mu.m in length.
[0107] FIG. 32 is a polarized light microscope image of Morphic
Form I of Sample 3 of marizomib. As shown in FIG. 32, there is no
lath morphology in the micronized sample. The samples are in some
embodiments more sphere-like.
[0108] FIG. 33 is a photo of Sample 1 of Morphic Form I of
marizomib during the variable temperature XRPD analysis at
25.degree. C. FIG. 34 is a photo of Sample 1 of Morphic Form I of
marizomib during the variable temperature XRPD analysis at
150.degree. C. FIG. 35 is a photo of Sample 1 of Morphic Form I of
marizomib at during the variable temperature XRPD analysis
160.degree. C. Discoloration of the sample, from white to yellow,
was observed at about 160.degree. C.
[0109] FIG. 36 is a photo of Sample 1 of Morphic Form I of
marizomib at during the variable temperature XRPD analysis
160.degree. C. after five minutes. As shown in FIG. 36, the sample
appears to be both melting and degrading. Without wishing to be
bound by theory, this result is consistent with the TG and DSC
analysis of Sample 1, which suggested that the morphic Form I
begins to melt and/or degrade at about 160.degree. C.
[0110] Sample 3 of morphic Form I showed similar characteristics to
Sample 1. FIG. 37 is a photo of Sample 3 of Morphic Form I of
marizomib at 25.degree. C. FIG. 38 is a photo of Sample 3 of
Morphic Form I of marizomib at 150.degree. C. FIG. 39 is a photo of
Sample 3 of Morphic Form I of marizomib at 160.degree. C. FIG. 40
is a photo of Sample 3 of Morphic Form I of marizomib at
160.degree. C. after five minutes. As shown in FIGS. 37-40, Sample
3 had properties similar to Sample 1. For example, both samples
exhibited discoloration at about 160.degree. C. and melting and/or
degradation after holding for about five minutes at about
160.degree. C.
[0111] A fourth sample of marizomib, Form I ("Sample 4") was
subjected to various conditions to attempt to prepare a different
morphic form beyond Form I. The indexing parameters of Sample 4 are
given below in Table-4:
TABLE-US-00004 TABLE 4 Parameters for Sample 4 Indexing Bravais
Type Primitive Monoclinic a [.ANG.] 10.594 b [.ANG.] 24.507 c
[.ANG.] 12.661 .alpha. [deg] 90 .beta. [deg] 108.53 .gamma. [deg]
90 Volume [.ANG..sup.3/cell] 3, 116.3 Chiral Contents? Chiral
Extinction Symbol P 1 2.sub.1 1 Space Group(s) P2.sub.1 (4)
Attempted Hydrolysis of Marizomib
[0112] Without wishing to be bound by theory, it was hypothesized
that marizomib could be hydrolyzed upon exposure to water. The
results are given in Example 1. FIG. 26 is an overlay of three XRPD
plots of Sample 1 of marizomib before (2615) and after attempted
hydrolysis with water (2610) and methanol (2605). FIG. 27 is a
.sup.1HNMR plot of Sample 1 of marizomib before (2710) and after
(2705) attempted hydrolysis. FIG. 28 is an HPLC plot of Sample 1 of
marizomib isolated after a hydrolysis attempt with water. The
samples were matured between 50.degree. C. and room temperature
heat-cool cycles (8 h per cycle) for 96 h. As shown in FIG. 28, the
purity of the sample is reduced from about 99% to about 75% after
four days. FIG. 29 is an HPLC plot of Sample 1 of marizomib
isolated from ethyl acetate. FIG. 30 is an HPLC plot of Sample 1 of
marizomib isolated from ethanol. As shown in FIG. 28, there was
observed a new peak at a retention time of about 4.729 minutes.
Without wishing to be bound by theory, this peak is understood to
represent the hydrolysis product, as set forth in Scheme 1,
below:
##STR00002##
[0113] However, as shown in FIGS. 26 and 27, the spectral
characterization before and after attempted hydrolysis was
substantially similar. Without wishing to be bound by theory, it is
proposed that the hydrolysed product crystallizes in the same
morphic form as the starting material (Form I). Alternatively,
without wishing to be bound by theory, it is proposed that the
hydrolysis product (above) is more soluble in aqueous media and
does not crystallize under the same conditions as marizomib itself.
In other words, XRPD could not be used to distinguish between
morphic Form I and the hydrolysis product.
Methods of Preparing Morphic Form I
[0114] Morphic Form I of marizomib can be prepared by a number of
methods as set forth herein. In one embodiment, marizomib is
dissolved in a solvent (e.g., acetone) and crystallization is
initiated by addition of an anti-solvent (e.g., heptane).
[0115] In some embodiments, the solvent is selected from the group
consisting of n-heptane, ethyl acetate, methyl-isobutyl ketone,
2-propanol, acetone, chloroform, dimethyl sulfoxide, tert-butyl
methyl ether, anisole, cumene, methyl ethyl ketone, isopropyl
acetate, dimethylformamide, toluene, tetrahydrofuran,
dichloromethane, acetonitrile, nitromethane, ethanol, and
dimethylacetamide.
[0116] Without wishing to be bound by theory, a morphic form of a
compound (e.g., marizomib) does not have to fully dissolve to
convert to a new polymorphic form of the compound in an
anti-solvent. In some embodiments, anti-solvents can lead to new
polymorphs. Without wishing to be bound by theory, even if an
anti-solvent is incapable of dissolving 1/50 its volume of
marizomib, a small yet appreciable amount of marizomib can still be
dissolved in the anti-solvent. Without wishing to be bound by
theory, as a small amount of drug (e.g., marizomib) dissolves and
then precipitates, the solvent characteristics can lead to a new
form (in some embodiments called a seed) in the precipitate. As the
drug (e.g., marizomib) dissolves and precipitates to the new form,
over time or near-complete or in some embodiments complete
conversion to a new form can be observed. Without wishing to be
bound by theory, such interconversion was not observed following
the morphic form screen set forth herein. That is, only morphic
Form I was observed when exposing Form I to both solvents and
anti-solvents.
Methods of Using Morphic Form I
[0117] In some embodiments, morphic Form I of the present
disclosure can be used for the treatment of a disease. For
instance, morphic Form I can be used in the manufacture of a
medicament for the treatment of a disease. Additionally, the
present disclosure contemplates a pharmaceutical composition
comprising morphic Form I and a pharmaceutically acceptable
carrier.
[0118] In some embodiments, the disease is cancer or a neoplastic
disease. In some embodiments, the neoplastic disease treated by the
morphic Forms disclosed herein may be a cancer selected from breast
cancer, sarcoma, leukemia, ovarian cancer, uretal cancer, bladder
cancer, prostate cancer, colon cancer, rectal cancer, stomach
cancer, lung cancer, lymphoma, multiple myeloma, pancreatic cancer,
liver cancer, kidney cancer, endocrine cancer, skin cancer,
melanoma, angioma, and brain or central nervous system (CNS)
cancer. In one embodiment, the neoplastic disease is a multiple
myeloma.
Salt and Co-Crystal Screen
[0119] As set forth in Example 12 and Table 18, approximately 117
screening experiments were conducted using 46 coformers/counterions
targeting salts and cocrystal of marizomib (Sample 4). Experiments
were conducted at various stoichiometric ratios using standard
crystallization techniques, including cooling, evaporation,
anti-solvent addition, reaction crystallization, slurry at ambient
and elevated temperature, solvent assisted grinding or a
combination of these techniques. Solids isolated from screening
experiments were analyzed by XRPD and/or .sup.1HNMR and compared to
the known XRPD pattern of marizomib and representative XRPD
patterns of the coformer/counterion.
[0120] Cocrystal screening of marizomib was conducted using
primarily pharmaceutically acceptable coformers containing a
diverse range of complementary functional groups including
carboxylic acids, amino acids, amines, sulfonamides and amides that
could potentially form supramolecular heterosynthons that could
compete with the 2-point recognition amide-amide interaction
present within the crystal structure of marizomib. However, solids
isolated from cocrystal screening experiments were primarily
consistent with marizomib, coformer, or mixtures of marizomib and
coformer, indicating cocrystal formation did not occur under the
tested experimental conditions. The detailed conditions and
observations of all attempted cocrystal formation experiments and
the results from PLM and XRPD analysis are summarized in Table
18.
[0121] Additional coformers such as pyroglutamic acid,
2-pyrrolidone and cytosine that contain secondary amides groups
similar to marizomib were explored. Additionally, coformers that
contain the 2-aminopyridine moiety e.g. adenine and melamine, were
also included in screening.
[0122] Unique peaks in the presence of marizomib and/or coformer
were identified from experiments involving marizomib with several
coformers including 2-pyrrolidone (FIG. 41), melamine (FIG. 43,
FIG. 45), and cytosine (FIG. 46).
[0123] A mixture of marizomib and a unique material (FIG. 41), as
evidenced by the two additional diffraction peaks at 14.1 and
15.5.degree.2.theta., was crystallized after slow evaporation of a
hazy solution produced from a temperature cycling (50.degree. C. to
RT) experiment in acetone containing equimolar amounts of marizomib
and 2-pyrrolidone. Trace 4101 shows marizomib Sample 4. Trace 4102
shows a trace of marizomib and 2-pyrrolidone (1:1) after slow
evaporation after temperature cycles between 50.degree. C. and room
temperature (4 cycles). The proton NMR spectrum of solids isolated
from this experiment showed marizomib: 2-pyrrolidone in approximate
1:1.2 mole ratio. Thermal analysis of solids isolated from this
experiment is presented in FIG. 42. Trace 4201 shows the heat flow
(W/g). Trace 4202 shows the weight percent of the sample. A sharp
endotherm was observed at 32.8.degree. C. (peak maximum), likely
attributed to the melting of residual 2-pyrrolidone, followed by
broad endothermic events at 91.4.degree. C. and 131.3.degree. C.
(peak maxima) in the DSC data. By TGA, a weight loss of 5% was
observed upon heating between 27.degree. C. and 92.degree. and a
change in the slope after approximately 120.degree. C. likely due
to decomposition was seen. The thermal behavior for this material
was different from that observed for marizomib Sample 4 used as
starting material. As noted herein, marizomib Sample 4 exhibits a
negligible weight loss prior to decomposition after approximately
150.degree. C. and no thermal events are observed in the DSC data
up to approximately 166.degree. C., where an exotherm is observed
followed by a small endotherm at approximately 175.degree. C.
[0124] Grinding marizomib in a mortar and pestle in the presence of
2-pyrrolidone also produced the same unique peaks observed in the
solution based experiment, in addition to peaks which were
characteristic of marizomib. A third experiment was conducted in
which marizomib was dissolved in 2-pyrrolidone at 50.degree. C.,
cooled to RT and evaporated. However, no solids were produced and
the clear solution remained.
[0125] A suspension containing equimolar amounts of marizomib and
melamine in DMSO:H.sub.2O (1:1) was slurried at 50.degree. C. for
approximately 2 days then at RT for 6 days and produced a mixture
of marizomib and a unique material based on XRPD. The acquired
proton NMR spectrum of the material from the experiment showed
marizomib:melamine in approximate 1:1.6 mole ratio. Additional
peaks, not present in the proton NMR spectrum of marizomib Sample 4
used as starting material were also observed, suggesting possible
degradation of the marizomib. DMSO and water were also detected in
the spectrum. An overlay of XRPD patterns is shown in FIG. 43.
Trace 4301 shows a melamine reference. Trace 4302 shows the mixture
of marizomib and melamine (1:1) slurry. Trace 4303 shows a trace of
marizomib Sample 4 for reference. Thermal analysis of solids
isolated from this experiment is presented in FIG. 44. Trace 4401
shows the weight percent of the sample. Trace 4402 shows the heat
flow (W/g). Multiple endotherms are observed at 97.5.degree. C.,
107.9.degree. C., and 124.1.degree. C. (peak maxima). Note: no
thermal events are observed in the DSC data for marizomib up to
approximately 166.degree. C., where an exotherm is observed
followed by a small endotherm at approximately 175.degree. C.
Melamine is reported to have a melting point around 345.degree. C.
The TGA thermogram shows continuous weight loss with heating.
Approximately 10% weight loss is observed between .about.30.degree.
C. and 104.degree. C. associated with the broad endotherm at
97.5.degree. C. (peak max.) in the DSC data.
[0126] A second experiment containing marizomib and an excess of
melamine (1:3 mole ratio) was performed in an attempt to isolate
the unique material as a single crystalline phase. The sample was
slurried overnight at 60.degree. C. then at RT for 1 day and
produced a mixture of marizomib and melamine with unique peaks
(FIG. 45). Trace 4501 shows a melamine reference. Trace 4502 shows
marizomib:melamine (1:3) after slurry at 60.degree. C. in
DMSO:H.sub.2O (1:1) and then room temperature for 1 day. Trace 4503
shows marizomib:melamine (1:1) after slurry at 50.degree. C. in
DMSO:H.sub.2O (1:1) for two days and then room temperature for 6
days. Trace 4504 shows marizomib Sample 4 for reference.
[0127] A suspension containing marizomib and an excess of cytosine
(1:3 mole ratio) in dioxane:water was slurried at 60.degree. C.
overnight and produced a light yellow clear solution. After
stirring at RT for 1 day, solids precipitated. Based on XRPD, the
isolated solids were composed of a mixture of unique peaks and
marizomib suggesting the presence of a secondary unidentified phase
(FIG. 46). Trace 4601 shows an XPRD pattern of cytosine. Trace 4602
shows a pattern of marizomib:cytosine (1:3) after slurry at
60.degree. C. overnight and then at room temperature for 1 day.
Trace 4603 shows an XRPD spectrum of marizomib Sample 4. Proton NMR
analysis of the solids from the experiment revealed primarily
cytosine with trace marizomib and minor additional peaks. Dioxane
was also observed. The data suggest no cocrystal formation occurred
with cytosine and that the unique peaks are possibly attributable
to a form of cytosine.
[0128] In a few cases, additional unidentified peaks were observed
as a mixture with marizomib and/or coformer/counterion, and were
likely due to partial degradation.
[0129] Based on the chemical structure of marizomib, the formation
of stable salts of the compound was anticipated to be unlikely
(predicted pKa.about.-1.4), but the use of strong acids was
included within the screen to evaluate possible salt formation.
Salt screening attempts with sulfonic acids and mineral acids
produced marizomib, mixtures of marizomib and counterion, or
discolored solutions suggesting possible degradation (Example 13,
Table 19). No crystalline salt of marizomib was produced in this
study.
Pharmaceutical Compositions
[0130] In some embodiments, the present disclosure relates to a
pharmaceutical composition comprising physiologically acceptable
surface active agents, carriers, diluents, excipients, smoothing
agents, suspension agents, film forming substances, and coating
assistants, or a combination thereof, and morphic Form I.
Acceptable carriers or diluents for therapeutic use are well known
in the pharmaceutical art, and are described, for example, in
Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,
Easton, Pa. (1990), which is incorporated herein by reference in
its entirety. Preservatives, stabilizers, dyes, sweeteners,
fragrances, flavoring agents, and the like may be provided in the
pharmaceutical composition. For example, sodium benzoate, ascorbic
acid and esters of p-hydroxybenzoic acid may be added as
preservatives. In addition, antioxidants and suspending agents may
be used. In various embodiments, alcohols, esters, sulfated
aliphatic alcohols, and the like may be used as surface active
agents; sucrose, glucose, lactose, starch, crystallized cellulose,
mannitol, light anhydrous silicate, magnesium aluminate, magnesium
methasilicate aluminate, synthetic aluminum silicate, calcium
carbonate, sodium acid carbonate, calcium hydrogen phosphate,
calcium carboxymethyl cellulose, and the like may be used as
excipients; magnesium stearate, talc, hardened oil and the like may
be used as smoothing agents; coconut oil, olive oil, sesame oil,
peanut oil, soya may be used as suspension agents or lubricants;
cellulose acetate phthalate as a derivative of a carbohydrate such
as cellulose or sugar, or methylacetate-methacrylate copolymer as a
derivative of polyvinyl may be used as suspension agents; and
plasticizers such as ester phthalates and the like may be used as
suspension agents.
[0131] Unless otherwise defined, a "solvent" of marizomib is a
substance that can dissolve at least 1/50 its volume of marizomib
at 50.degree. C. or below. Unless otherwise defined, an
"anti-solvent" is any substance that fails to dissolve at least
1/50 its volume of marizomib at 50.degree. C.
[0132] The term "pharmaceutical composition" refers to morphic Form
I in combination with other chemical components, such as diluents
or carriers. The pharmaceutical composition facilitates
administration of the compound (e.g., a morphic Form such as
morphic Form I) to an organism. Multiple techniques of
administering a compound exist in the art including, but not
limited to, oral, injection, aerosol, parenteral, and topical
administration. Pharmaceutical compositions can also be obtained by
reacting compounds (e.g., a morphic Form such as morphic Form I)
with inorganic or organic acids such as hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like.
[0133] The term "carrier" defines a chemical compound that
facilitates the incorporation of a compound (e.g., morphic Form I)
into cells or tissues. For example dimethyl sulfoxide (DMSO) is a
commonly utilized carrier as it facilitates the uptake of many
organic compounds into the cells or tissues of an organism.
[0134] The term "diluent" defines chemical compounds diluted in
water that will dissolve the compound of interest (e.g., a morphic
Form such as morphic Form I) as well as stabilize the biologically
active form of the compound. Salts dissolved in buffered solutions
are utilized as diluents in the art. One commonly used buffered
solution is phosphate buffered saline because it mimics the salt
conditions of human blood. Since buffer salts can control the pH of
a solution at low concentrations, a buffered diluent rarely
modifies the biological activity of a compound.
[0135] The term "physiologically acceptable" defines a carrier or
diluent that does not abrogate the biological activity and
properties of the compound.
[0136] The pharmaceutical compositions described herein can be
administered to a human patient per se, or in pharmaceutical
compositions where they are mixed with suitable carriers or
excipient(s). Techniques for formulation and administration of the
compounds of the instant application may be found in "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th
edition, 1990.
[0137] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, topical, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intravenous, intramedullary injections, as well as intrathecal,
direct intraventricular, intraperitoneal, intranasal, or
intraocular injections. The morphic Forms (e.g., morphic Form I)
can also be administered in sustained or controlled release dosage
forms, including depot injections, osmotic pumps, pills,
transdermal (including electrotransport) patches, and the like, for
prolonged and/or timed, pulsed administration at a predetermined
rate.
[0138] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or tabletting
processes.
[0139] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds (e.g., a morphic Form such as morphic Form I) into
preparations which can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen. Any of the
well-known techniques, carriers, and excipients may be used as
suitable and as understood in the art; e.g., in Remington's
Pharmaceutical Sciences, above.
[0140] Injectables can be prepared in conventional forms, either as
liquid solutions or suspensions, solid forms suitable for solution
or suspension in liquid prior to injection, or as emulsions.
Suitable excipients are, for example, water, saline, dextrose,
mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine
hydrochloride, and the like. In addition, if desired, the
injectable pharmaceutical compositions may contain minor amounts of
nontoxic auxiliary substances, such as wetting agents, pH buffering
agents, and the like. Physiologically compatible buffers include,
but are not limited to, Hanks's solution, Ringer's solution, or
physiological saline buffer. If desired, absorption enhancing
preparations (for example, liposomes), may be utilized.
[0141] For transmucosal administration, penetrants appropriate to
the barrier to be permeated may be used in the formulation.
[0142] Pharmaceutical formulations for parenteral administration,
e.g., by bolus injection or continuous infusion, include aqueous
solutions of the active compounds (e.g., a morphic Form such as
morphic Form I) in water-soluble form. Additionally, suspensions of
the active compounds (e.g., a morphic Form such as morphic Form I)
may be prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or other organic oils such as soybean, grapefruit or almond
oils, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds (e.g., a
morphic Form such as morphic Form I) to allow for the preparation
of highly concentrated solutions. Formulations for injection may be
presented in unit dosage form, e.g., in ampoules or in multi-dose
containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0143] For oral administration, the compounds (e.g., a morphic Form
such as morphic Form I) can be formulated readily by combining the
active compounds with pharmaceutically acceptable carriers well
known in the art. Such carriers enable the morphic Forms (e.g.,
morphic Form I) to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions and the
like, for oral ingestion by a patient to be treated. Pharmaceutical
preparations for oral use can be obtained by combining the morphic
Forms (e.g., morphic Form I) with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Dragee cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the
tablets or dragee coatings for identification or to characterize
different combinations of active compound doses. For this purpose,
concentrated sugar solutions may be used, which may optionally
contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for
identification or to characterize different combinations of active
compound doses.
[0144] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the morphic Forms (e.g.,
morphic Form I) may be dissolved or suspended in suitable liquids,
such as fatty oils, liquid paraffin, or liquid polyethylene
glycols. In addition, stabilizers may be added. All formulations
for oral administration should be in dosages suitable for such
administration.
[0145] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0146] For administration by inhalation, the compounds (e.g., a
morphic Form such as morphic Form I) for use according to the
present invention are conveniently delivered in the form of an
aerosol spray presentation from pressurized packs or a nebulizer,
with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound (e.g., a
morphic Form such as morphic Form I) and a suitable powder base
such as lactose or starch.
[0147] Further disclosed herein are various pharmaceutical
compositions well known in the pharmaceutical art for uses that
include intraocular, intranasal, and intraauricular delivery.
Suitable penetrants for these uses are generally known in the art.
Pharmaceutical compositions for intraocular delivery include
aqueous ophthalmic solutions of the morphic Forms (e.g., morphic
Form I) in water-soluble form, such as eyedrops, or in gellan gum
(Shedden et al., Clin. Ther., 23(3):440-50 (2001)) or hydrogels
(Mayer et al., Ophthalmologica, 210(2):101-3 (1996)); ophthalmic
ointments; ophthalmic suspensions, such as microparticulates,
drug-containing small polymeric particles that are suspended in a
liquid carrier medium (Joshi, A., J. Ocul. Pharmacol., 10(1):29-45
(1994)), lipid-soluble formulations (Alm et al., Prog. Clin. Biol.
Res., 312:447-58 (1989)), and microspheres (Mordenti, Toxicol.
Sci., 52(1):101-6 (1999)); and ocular inserts. All of the
above-mentioned references, are incorporated herein by reference in
their entireties. Such suitable pharmaceutical formulations are
most often and preferably formulated to be sterile, isotonic and
buffered for stability and comfort. Pharmaceutical compositions for
intranasal delivery may also include drops and sprays often
prepared to simulate in many respects nasal secretions to ensure
maintenance of normal ciliary action. As disclosed in Remington's
Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.
(1990), which is incorporated herein by reference in its entirety,
and well-known to those skilled in the art, suitable formulations
are most often and preferably isotonic, slightly buffered to
maintain a pH of 5.5 to 6.5, and most often and preferably include
antimicrobial preservatives and appropriate drug stabilizers.
Pharmaceutical formulations for intraauricular delivery include
suspensions and ointments for topical application in the ear.
Common solvents for such aural formulations include glycerin and
water.
[0148] The morphic Forms (e.g., morphic Form I) may also be
formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides.
[0149] In addition to the formulations described previously, the
morphic Forms (e.g., morphic Form I) may also be formulated as a
depot preparation. Such long acting formulations may be
administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compounds (e.g., a morphic Form such as morphic Form I) may be
formulated with suitable polymeric or hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0150] In some embodiments, a suitable pharmaceutical carrier may
be a cosolvent system comprising benzyl alcohol, a nonpolar
surfactant, a water-miscible organic polymer, and an aqueous phase.
A common cosolvent system used is the VPD co-solvent system, which
is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar
surfactant Polysorbate 80.TM., and 65% w/v polyethylene glycol 300,
made up to volume in absolute ethanol. Naturally, the proportions
of a co-solvent system may be varied considerably without
destroying its solubility and toxicity characteristics.
Furthermore, the identity of the co-solvent components may be
varied: for example, other low-toxicity nonpolar surfactants may be
used instead of POLYSORBATE 80.TM.; the fraction size of
polyethylene glycol may be varied; other biocompatible polymers may
replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose.
[0151] Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds (e.g., a morphic Form such as morphic Form
I) may be employed. Liposomes and emulsions are well known examples
of delivery vehicles or carriers for hydrophobic drugs. Certain
organic solvents such as dimethylsulfoxide also may be employed,
although usually at the cost of greater toxicity. Additionally, the
compounds (e.g., a morphic Form such as morphic Form I) may be
delivered using a sustained-release system, such as semipermeable
matrices of solid hydrophobic polymers containing the therapeutic
agent. Various sustained-release materials have been established
and are well known by those skilled in the art. Sustained-release
capsules may, depending on their chemical nature, release the
compounds for a few weeks up to over 100 days. Depending on the
chemical nature and the biological stability of the therapeutic
reagent, additional strategies for protein stabilization may be
employed.
[0152] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes. All molecules present in an aqueous solution at the time
of liposome formation are incorporated into the aqueous interior.
The liposomal contents are both protected from the external
micro-environment and, because liposomes fuse with cell membranes,
are efficiently delivered into the cell cytoplasm. The liposome may
be coated with a tissue-specific antibody. The liposomes will be
targeted to and taken up selectively by the desired organ.
Alternatively, small hydrophobic organic molecules may be directly
administered intracellularly.
[0153] Additional therapeutic or diagnostic agents may be
incorporated into the pharmaceutical compositions. Alternatively or
additionally, pharmaceutical compositions may be combined with
other compositions that contain other therapeutic or diagnostic
agents.
Methods of Administration
[0154] The morphic Forms (e.g., morphic Form I) and pharmaceutical
compositions comprising the same may be administered to the patient
by any suitable means. Non-limiting examples of methods of
administration include, among others, (a) administration though
oral pathways, which administration includes administration in
capsule, tablet, granule, spray, syrup, or other such forms; (b)
administration through non-oral pathways such as rectal, vaginal,
intraurethral, intraocular, intranasal, or intraauricular, which
administration includes administration as an aqueous suspension, an
oily preparation or the like or as a drip, spray, suppository,
salve, ointment or the like; (c) administration via injection,
subcutaneously, intraperitoneally, intravenously, intramuscularly,
intradermally, intraorbitally, intracapsularly, intraspinally,
intrasternally, or the like, including infusion pump delivery; (d)
administration locally such as by injection directly in the renal
or cardiac area, e.g., by depot implantation; as well as (e)
administration topically; as deemed appropriate by those of skill
in the art for bringing the compound of the invention (e.g., a
morphic Form such as morphic Form I) into contact with living
tissue.
[0155] Pharmaceutical compositions suitable for administration
include compositions where the active ingredients are contained in
an amount effective to achieve its intended purpose. The
therapeutically effective amount of the compounds disclosed herein
(e.g., a morphic Form such as morphic Form I) required as a dose
will depend on the route of administration, the type of animal,
including human, being treated, and the physical characteristics of
the specific animal under consideration. The dose can be tailored
to achieve a desired effect, but will depend on such factors as
weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize. More specifically, a
therapeutically effective amount means an amount of compound (e.g.,
a morphic Form such as morphic Form I) effective to prevent,
alleviate or ameliorate symptoms of disease or prolong the survival
of the subject being treated. Determination of a therapeutically
effective amount is well within the capability of those skilled in
the art, especially in light of the detailed disclosure provided
herein.
[0156] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and
mammalian species treated, the particular compounds employed (e.g.,
a morphic Form such as morphic Form I), and the specific use for
which these compounds are employed. The determination of effective
dosage levels, that is the dosage levels necessary to achieve the
desired result, can be accomplished by one skilled in the art using
routine pharmacological methods. Typically, human clinical
applications of products are commenced at lower dosage levels, with
dosage level being increased until the desired effect is achieved.
Alternatively, acceptable in vitro studies can be used to establish
useful doses and routes of administration of the compositions
identified by the present methods using established pharmacological
methods.
[0157] In non-human animal studies, applications of potential
products are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved or adverse
side effects disappear. The dosage may range broadly, depending
upon the desired affects and the therapeutic indication. Typically,
dosages may be between about 10 microgram/kg and 100 mg/kg body
weight, preferably between about 100 microgram/kg and 10 mg/kg body
weight. Alternatively dosages may be based and calculated upon the
surface area of the patient, as understood by those of skill in the
art.
[0158] The exact formulation, route of administration and dosage
for the pharmaceutical compositions of the present invention can be
chosen by the individual physician in view of the patient's
condition. (See e.g., Fingl et al. 1975, in "The Pharmacological
Basis of Therapeutics", which is hereby incorporated herein by
reference in its entirety, with particular reference to Ch. 1, p.
1). Typically, the dose range of the composition administered to
the patient can be from about 0.5 to 1000 mg/kg of the patient's
body weight. The dosage may be a single one or a series of two or
more given in the course of one or more days, as is needed by the
patient. In instances where human dosages for compounds (e.g., a
morphic Form such as morphic Form I) have been established for at
least some condition, the present invention will use those same
dosages, or dosages that are between about 0.1% and 500%, more
preferably between about 25% and 250% of the established human
dosage. Where no human dosage is established, as will be the case
for newly-discovered pharmaceutical compounds, a suitable human
dosage can be inferred from ED.sub.50 or ID50 values, or other
appropriate values derived from in vitro or in vivo studies, as
qualified by toxicity studies and efficacy studies in animals.
[0159] It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administration
due to toxicity or organ dysfunctions. Conversely, the attending
physician would also know to adjust treatment to higher levels if
the clinical response were not adequate (precluding toxicity). The
magnitude of an administrated dose in the management of the
disorder of interest will vary with the severity of the condition
to be treated and to the route of administration. The severity of
the condition may, for example, be evaluated, in part, by standard
prognostic evaluation methods. Further, the dose and perhaps dose
frequency, will also vary according to the age, body weight, and
response of the individual patient. A program comparable to that
discussed above may be used in veterinary medicine.
[0160] Although the exact dosage will be determined on a
drug-by-drug basis, in most cases, some generalizations regarding
the dosage can be made. The daily dosage regimen for an adult human
patient may be, for example, an oral dose of between 0.1 mg and
2000 mg of each active ingredient, preferably between 1 mg and 500
mg, e.g. 5 to 200 mg. In other embodiments, an intravenous,
subcutaneous, or intramuscular dose of each active ingredient of
between 0.01 mg and 100 mg, preferably between 0.1 mg and 60 mg,
e.g. 1 to 40 mg is used. In cases of administration of a
pharmaceutically acceptable salt, dosages may be calculated as the
free base. In some embodiments, the composition is administered 1
to 4 times per day. Alternatively the compositions of the invention
may be administered by continuous intravenous infusion, preferably
at a dose of each active ingredient up to 1000 mg per day. As will
be understood by those of skill in the art, in certain situations
it may be necessary to administer the compounds disclosed herein
(e.g., a morphic Form such as morphic Form I) in amounts that
exceed, or even far exceed, the above-stated, preferred dosage
range in order to effectively and aggressively treat particularly
aggressive diseases or infections. In some embodiments, the
compounds (e.g., a morphic Form such as morphic Form I) will be
administered for a period of continuous therapy, for example for a
week or more, or for months or years.
[0161] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the modulating effects, or minimal effective concentration
(MEC). The MEC will vary for each compound (e.g., a morphic Form
such as morphic Form I) but can be estimated from in vitro data.
Dosages necessary to achieve the MEC will depend on individual
characteristics and route of administration. However, HPLC assays
or bioassays can be used to determine plasma concentrations.
[0162] Dosage intervals can also be determined using MEC value.
Compositions should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%.
[0163] In cases of local administration or selective uptake, the
effective local concentration of the drug may not be related to
plasma concentration.
[0164] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0165] Compounds disclosed herein (e.g., a morphic Form such as
morphic Form I) can be evaluated for efficacy and toxicity using
known methods. For example, the toxicology of a particular
compound, or of a subset of the compounds, sharing certain chemical
moieties, may be established by determining in vitro toxicity
towards a cell line, such as a mammalian, and preferably human,
cell line. The results of such studies are often predictive of
toxicity in animals, such as mammals, or more specifically, humans.
Alternatively, the toxicity of particular compounds (e.g., a
morphic Form such as morphic Form I) in an animal model, such as
mice, rats, rabbits, or monkeys, may be determined using known
methods. The efficacy of a particular compound may be established
using several recognized methods, such as in vitro methods, animal
models, or human clinical trials. Recognized in vitro models exist
for nearly every class of condition, including but not limited to
cancer, cardiovascular disease, and various immune dysfunction.
Similarly, acceptable animal models may be used to establish
efficacy of chemicals to treat such conditions. When selecting a
model to determine efficacy, the skilled artisan can be guided by
the state of the art to choose an appropriate model, dose, and
route of administration, and regime. Of course, human clinical
trials can also be used to determine the efficacy of a compound
(e.g., a morphic Form such as morphic Form I) in humans.
[0166] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accompanied with
a notice associated with the container in form prescribed by a
governmental agency regulating the manufacture, use, or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the drug for human or veterinary
administration. Such notice, for example, may be the labeling
approved by the U.S. Food and Drug Administration for prescription
drugs, or the approved product insert. Compositions comprising a
compound of the invention formulated in a compatible pharmaceutical
carrier may also be prepared, placed in an appropriate container,
and labeled for treatment of an indicated condition.
EXAMPLES
[0167] The disclosure is further illustrated by the following
examples, which are not to be construed as limiting this disclosure
in scope or spirit to the specific procedures herein described. It
is to be understood that the examples are provided to illustrate
certain embodiments and that no limitation to the scope of the
disclosure is intended thereby. It is to be further understood that
resort may be had to various other embodiments, modifications, and
equivalents thereof which may suggest themselves to those skilled
in the art without departing from the spirit of the present
disclosure and/or scope of the appended claims.
[0168] Abbreviations used herein are given below:
TABLE-US-00005 Acronym Meaning ca. Approximately DMA
Dimethylacetamide DMF Dimethylformamide DSC Differential Scanning
Calorimetry H.sub.2O Water .sup.1H-NMR Proton Nuclear Magnetic
Resonance HPLC High Performance Liquid Chromatography ID
Identification MeCN Acetonitrile MEK Methyl Ethyl Ketone MeOH
Methanol MIBK Methyl Isobutyl Ketone N/A Not Applicable PLM
Polarised Light Microscopy RT Room Temperature SCXRD Single Crystal
X-Ray Diffraction SEM Scanning Electron Microscope TGA Thermal
Gravimetric Analysis UV Ultra Violet VT-XRPD Variable Temperature
X-Ray Powder Diffraction XRPD X-Ray Powder Diffraction
Analytical Techniques
TABLE-US-00006 [0169] Acronyms Full Name/Description DSC
Differential scanning calorimetry mDSC Modulated differential
scanning calorimetry NMR Nuclear magnetic resonance spectroscopy
TGA Thermogravimetric analysis XRPD X-ray powder diffraction DVS
Dynamic vapor sorption
Methods
TABLE-US-00007 [0170] Acronym Full Name/Description CC Crash
cooling CP Crash precipitation FC Fast cooling FE Fast evaporation
SC Slow cooling
Miscellaneous
TABLE-US-00008 [0171] Acronym Full Name/Description Agg. (agg.)
Aggregates API Active pharmaceutical ingredient B/E Birefringence
and extinction B Birefringence d Day(s) decomp. Decomposition endo
Endotherm exo Exotherm h Hour(s) LIMS Laboratory information
management system RT Room temperature/ambient temperature UM
Unknown morphology A.sub.w water activity w/ With IS Insufficient
amount Anh. Anhydrous API Active pharmaceutical ingredient B
Birefringence B/E Birefringence and extinction MRZ Marizomib O/N
Overnight PLM Polarized light microscope RT Room
temperature/ambient temperature Sat'd Saturated
Solvents
TABLE-US-00009 [0172] Acronyms Full Name/Description [EMIm][Cl]
1-Ethyl-3-methylimidazolium chloride [EMIm][NTF.sub.2]
1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide 2-Me
THF 2-Methyltetrahydrofuran ACN acetonitrile CHCl.sub.3 chloroform
DCM dichloromethane DMF dimethylformamide EtOAc ethyl acetate
Formulation 55% propylene glycol + System A 5% EtOH + 40% citrate
buffer (10 mM; pH 5) Formulation 14/86 (v/v) H.sub.2O/tBA System B
containing 30 mg/ml sucrose, and titrated with HCl to pH 3.2 .+-.
0.2 IPA isopropyl alcohol MEK butanone (methyl ethyl ketone) MeOAc
methyl acetate MIBK methyl isobutyl ketone MTBE methyl Tertiary
Butyl Ether NMP N-Methyl-2-pyrrolidone THF tetrahydrofuran t-BuOH
Tert-butanol DMSO Dimethylsulfoxide EtOH Ethanol H2O Water H2SO4
Sulfuric acid HCl Hydrochloric acid HFIPA
1,1,1,3,3,3-hexafluoro-2-propanol IPrOAc Isopropyl acetate MeOH
Methanol NMP N-methyl-2-pyrrolidone 1-PrOH 1-propanol TFE
2,2,2-trifluoroethanol
XRPD Terminology
[0173] The term "crystalline" is understood to mean a form that
exhibits an XRPD pattern with sharp peaks (similar to instrumental
peak widths) and weak diffuse scattering (relative to the
peaks).
[0174] The term "disordered crystalline" is understood to mean a
form that exhibits an XRPD pattern with broad peaks (relative to
instrumental peak widths) and/or strong diffuse scattering
(relative to the peaks). Disordered materials may be
microcrystalline; crystalline with large defect density; mixtures
of crystalline and x-ray amorphous phases; or a combination of the
above. Additional analysis may differentiate among these
options.
[0175] The term "insufficient signal" is used when insufficient
signal above the expected background scattering was observed. This
may indicate, for example, that the x-ray beam missed the sample
and/or that the sample was of insufficient mass for analysis.
[0176] "Particle statistics artifacts" can occur when the particle
size distribution contains a small number of large crystals which
may in some cases lead to sharp spikes in the XRPD pattern.
[0177] "Preferred orientation artifacts" can occur when the
particle morphology is prone to non-random orientation in the
sample holder which may in some cases lead to changes in relative
peak intensities.
[0178] "No peaks" can occur when no Bragg peaks are observed in the
XRPD pattern. The absence of peaks may in some cases be due to an
x-ray amorphous sample and/or insufficient signal.
[0179] "Single crystalline phase" can refer to when an XRPD pattern
is judged to contain evidence of a single crystalline phase if the
Bragg peaks can be indexed with a single unit cell.
[0180] "X-ray amorphous" can refer to situations in which diffuse
scatter is present, but no evidence for Bragg peaks is found in an
XRPD pattern. X-ray amorphous materials may be: nano-crystalline;
crystalline with a very large defect density; kinetic amorphous
material; thermodynamic amorphous material; or a combination of the
above. Additional analysis may differentiate among these
options.
Hygroscopicity
[0181] The term "low hygroscopicity" refers to samples that exhibit
<0.5 weight percent water uptake over a specified relative
humidity range.
[0182] The term "limited hygroscopicity" refers to samples that
exhibit <2.0 weight percent water uptake over a specified
relative humidity range.
[0183] The term "significant hygroscopicity" refers to samples that
exhibit .gtoreq.2.0 weight percent water uptake over a specified
relative humidity range.
[0184] The term "deliquescence" refers to spontaneous liquefaction
associated with water sorption from atmospheric moisture.
[0185] The term "stoichiometric hydrate" refers to crystalline
material with stable stoichiometric water content over an extended
relative humidity range. Exemplary stoichiometric hydrates can be,
for instance, hemihydrates, monohydrates, sesquihydrates, or
dehydrates.
[0186] A "variable hydrate" can refer to crystalline material with
variable water content over an extended relative humidity range,
yet with no phase change.
Experimental Procedures
[0187] Examples 3-11 used Sample 4 of marizomib.
X-Ray Powder Diffraction (XRPD)--Samples 1-3
[0188] X-Ray Powder Diffraction patterns were collected on a Bruker
AXS C2 GADDS diffractometer using Cu K.alpha. radiation (40 kV, 40
mA), automated XYZ stage, laser video microscope for auto-sample
positioning and a HiStar 2-dimensional area detector. X-ray optics
consists of a single Gobel multilayer mirror coupled with a pinhole
collimator of 0.3 mm. A weekly performance check was carried out
using a certified standard NIST 1976 Corundum (flat plate).
[0189] The beam divergence, i.e. the effective size of the X-ray
beam on the sample, was approximately 4 mm. A .theta.-.theta.
continuous scan mode was employed with a sample-detector distance
of 20 cm which gave an effective 2.theta. range of
3.2.degree.-29.7.degree.. Unless otherwise specified, the sample
would be exposed to the X-ray beam for about 120 seconds. The
software used for data collection was GADDS for XP/2000 4.1.43 and
the data were analyzed and presented using Diffrac Plus EVA
v15.0.0.0.
[0190] Samples run under ambient conditions were prepared as flat
plate specimens using powder as received without grinding.
Approximately 1-2 mg of the sample was lightly pressed on a glass
slide to obtain a flat surface.
[0191] Samples run under non-ambient conditions were mounted on a
silicon wafer with heat-conducting compound. The sample was then
heated to the appropriate temperature at 20.degree. C./min and
subsequently held isothermally for 1 minute before data collection
was initiated.
[0192] Alternatively, X-Ray Powder Diffraction patterns were
collected on a Bruker D8 diffractometer using Cu K.alpha. radiation
(40 kV, 40 mA), 0-20 goniometer, and divergence of V4 and receiving
slits, a Ge monochromator and a Lynxeye detector. The instrument
was performance checked using a certified Corundum standard (NIST
1976). The software used for data collection was Diffrac Plus XRD
Commander v2.6.1 and the data were analysed and presented using
Diffrac Plus EVA v15.0.0.0.
[0193] Samples were run under ambient conditions as flat plate
specimens using powder as received. The sample was gently packed
into a cavity cut into polished, zero-background (510) silicon
wafer. The sample was rotated in its own plane during analysis. The
details of the data collection are: Angular range: 2 to
42.degree.2.theta.; Step size: 0.05.degree.2.theta.; Collection
time: 0.5 s/step.
X-Ray Powder Diffraction (XRPD)--Sample 4
[0194] Transmission Mode: XRPD patterns were collected with a
PANalytical X'Pert PRO MPD diffractometer using an incident beam of
Cu radiation produced using an Optix long, fine-focus source. An
elliptically graded multilayer mirror was used to focus Cu K.alpha.
X-ray radiation through the specimen and onto the detector. Prior
to the analysis, a silicon specimen (NIST SRM 640e) was analyzed to
verify the observed position of the Si (111) peak is consistent
with the NIST-certified position. A specimen of the sample was
sandwiched between 3-.mu.m-thick films and analyzed in transmission
geometry. A beam-stop, short antiscatter extension, and antiscatter
knife edge, were used to minimize the background generated by air.
Soller slits for the incident and diffracted beams were used to
minimize broadening from axial divergence. Diffraction patterns
were collected using a scanning position-sensitive detector
(X'Celerator) located 240 mm from the specimen and Data Collector
software v. 2.2b.
[0195] Reflection Mode: XRPD patterns were collected with a
PANalytical X'Pert PRO MPD diffractometer using an incident beam of
Cu K.alpha. radiation produced using a long, fine-focus source and
a nickel filter. The diffractometer was configured using the
symmetric Bragg-Brentano geometry. Prior to the analysis, a silicon
specimen (NIST SRM 640e) was analyzed to verify the observed
position of the Si 111 peak is consistent with the NIST-certified
position. A specimen of the sample was prepared as a thin, circular
layer centered on a silicon zero-background substrate. Antiscatter
slits (SS) were used to minimize the background generated by air.
Soller slits for the incident and diffracted beams were used to
minimize broadening from axial divergence. Diffraction patterns
were collected using a scanning position-sensitive detector
(X'Celerator) located 240 mm from the sample and Data Collector
software v. 2.2b.
[0196] A high-resolution XRPD pattern was indexed using proprietary
SSCI software, TRIADS.TM.. Indexing and structure refinement are
computational studies. Agreement between the allowed peak
positions, marked with red bars, and the observed peaks indicates a
consistent unit cell determination. Successful indexing of the
pattern indicates that the sample is composed primarily of a single
crystalline phase. To confirm the tentative indexing solution, the
molecular packing motifs within the crystallographic unit cells
must be determined. No attempts at molecular packing were
performed.
Proton Nuclear Magnetic Resonance (.sup.1H-NMR) Samples 1-3
[0197] NMR spectra were collected on a Bruker 400 MHz instrument
equipped with an auto-sampler and controlled by a DRX400 console.
Automated experiments were acquired using ICON-NMR v4.0.7 running
with Topspin v1.3 using the standard Bruker loaded experiments. For
non-routine spectroscopy, data were acquired through the use of
Topspin alone.
[0198] Samples were prepared in DMSO-d6, unless otherwise stated.
Off-line analysis was carried out using ACD Spectrus Processor
2014.
Proton Nuclear Magnetic Resonance (.sup.1H-NMR)--Sample 4
[0199] Initial characterization solution proton NMIR spectra were
acquired with an Agilent DD2-400 spectrometer using deuterated
methanol.
[0200] Other solution NMR spectra were acquired using a
Bruker-Biospin 5 mm gradient broadband probe on a Bruker-Biospin
AVANCE II 400 MHz NMR spectrometer. The spectrum was referenced
using the tetramethylsilane resonance and set equal to 0.0 ppm. The
FID was processed using Bruker TopSpin 2.1.
[0201] For the coformer screen and salt screen (Examples 12 and
13), the solution NMIR spectra were acquired with an Agilent
DD2-400 spectrometer. Samples were prepared by dissolving
approximately 5-10 mg of sample in DMSO-d6 containing TMS. The data
acquisition parameters are displayed in each plot of the spectrum
in the Data section of this report.
Differential Scanning Calorimetry (DSC)--Samples 1-3
[0202] DSC data were collected on a TA Instruments Q2000 equipped
with a 50-position auto-sampler. The calibration for thermal
capacity was carried out using sapphire and the calibration for
energy and temperature was carried out using certified indium.
Typically 0.5-3 mg of each sample, in a pin-holed aluminum pan, was
heated at 10.degree. C./min from 25.degree. C. to 300.degree. C. A
purge of dry nitrogen at 50 ml/min was maintained over the
sample.
[0203] The instrument control software was Advantage for Q Series
v2.8.0.394 and Thermal Advantage v5.5.3 and the data were analyzed
using Universal Analysis v4.5A.
[0204] Differential scanning calorimetry experiments were performed
in duplicate at each of the four heating rates (i.e., 2, 5, 10 and
50.degree. C./min).
Differential Scanning Calorimetry (DSC)--Sample 4
[0205] DSC was performed using a TA Instruments Q2000 differential
scanning calorimeter. Temperature calibration was performed using
NIST-traceable indium metal. The sample was placed into an aluminum
Tzero DSC pan, covered with a lid, and the weight was accurately
recorded. A weighed aluminum pan configured as the sample pan was
placed on the reference side of the cell.
Modular Differential Scanning Calorimetry (mDSC)--Sample 4
[0206] mDSC data was obtained on a TA Instruments 2920 differential
scanning calorimeter equipped with a refrigerated cooling system
(RCS). Temperature calibration was performed using NIST traceable
indium metal. The sample was placed into an aluminum DSC pan,
covered with a lid, and the weight was accurately recorded. A
weighed aluminum pan configured as the sample pan was placed on the
reference side of the cell.
Thermo-Gravimetric Analysis (TGA) Samples 1-3
[0207] TGA data were collected on a TA Instruments Q500 TGA,
equipped with a 16-position auto-sampler. The instrument was
temperature calibrated using certified Alumel.TM. and Nickel.
Typically 5-10 mg of each sample was loaded onto a pre-tared
aluminum DSC pan and heated at 10.degree. C./min from ambient
temperature to 350.degree. C. A nitrogen purge at 60 ml/min was
maintained over the sample.
[0208] The instrument control software was Advantage for Q Series
v2.5.0.256 and Thermal Advantage v5.5.3 and the data were analyzed
using Universal Analysis v4.5A.
Thermo-Gravimetric Analysis (TGA)--Sample 4
[0209] TG analyses were performed using a TA Instruments Q5000 IR
thermogravimetric analyzer. Temperature calibration was performed
using nickel and Alumel.TM.. Each sample was placed in an aluminum
pan. The sample was hermetically sealed, the lid pierced, then
inserted into the TG furnace. The furnace was heated under
nitrogen.
Polarized Light Microscopy (PLM) Samples 1-3
[0210] Samples were studied on a Leica LM/DM polarized light
microscope with a digital video camera for image capture. A small
amount of each sample was placed on a glass slide, mounted in
immersion oil and covered with a glass slip, the individual
particles being separated as well as possible. The sample was
viewed with appropriate magnification and partially polarized
light, coupled to a .lamda. false-color filter.
Polarized Light Microscopy (PLM)--Sample 4
[0211] Light microscopy was performed using a Motic SMZ-168
stereomicroscope. Various objectives typically ranging from
0.8-10.times. were used with crossed-polarized light to view
samples. Samples were either viewed in situ, or in a drop of
mineral oil.
[0212] For the coformer screen and salt screen (Examples 12 and
13), light microscopy was performed using Fisher Scientific
Stereomaster stereomicroscope. Various objectives typically ranging
from 0.8-10.times. were used with crossed-polarized light to view
samples.
Hot Stage Microscopy (HSM)
[0213] Hot Stage Microscopy was carried out using a Leica LM/DM
polarised light microscope combined with a Mettler-Toledo FP82HT
hot-stage and a digital video camera for image capture. A small
amount of each sample was placed onto a glass slide with individual
particles separated as well as possible. The sample was viewed with
appropriate magnification and partially polarised light, coupled to
a .lamda. false-colour filter, whilst being heated from ambient
temperature typically at 10-20.degree. C./min.
Chemical Purity Determination by HPLC
[0214] Purity analysis was performed on an Agilent PI 100 series
system equipped with a diode array detector and using ChemStation
software vB.04.03 using the method set forth in Table-5.
TABLE-US-00010 TABLE 5 HPLC Conditions for Purity Determination
Parameter Value Type of method Reverse phase with gradient elution
Sample Preparation 0.5 mg/ml in acetonitrile:water 1:1 + 0.1% TFA
Column Supelco Ascentis Express C18, 100 .times. 4.6 mm, 2.7 .mu.m
Column 25 Temperature (.degree. C.) Injection (.mu.l) 5 Wavelength,
255, 90 Bandwidth (nm) Flow Rate 2 (ml/min) Phase A 0.1% TFA in
water Phase B 0.085% TFA in acetonitrile Time % % Timetable (min)
Phase A Phase B 0 95 5 6 5 95 6.2 95 5 8 95 5
Example 1--Hydrolysis of Marizomib
[0215] 30 mg of Sample 1 was suspended in the given solvent shown
in Table 6 (1 ml) at room temperature. The samples were matured
between 50.degree. C. and room temperature heat-cool cycles (8 h
per cycle) for 96 h. The solids were filtered, air dried and
analyzed by XRPD.
TABLE-US-00011 TABLE 6 Preparation and Characterization of Solids
from Hydrolysis Experiments Obs. post HPLC Solvent Obs. at r.t.
maturation XRPD .sup.1H-NMR Purity MeOH Suspension Suspension
Crystalline - n/a n/a similar to Form I Water Suspension Suspension
Crystalline - Comparable 74.8% similar to with ref. (ref. to Form I
spectrum starting material: 98.2%)
[0216] After 4 days maturation in water, the chemical purity was
down from 98% to 75%, with emphasis on a particular peak on the
HPLC chromatogram (RRT=4.75 min) as shown in FIG. 28. However, the
solid phase remained the same by XRPD as shown in FIG. 26. Without
wishing to be bound by theory, it is proposed that the hydrolysed
product crystallizes in the same phase as the starting material
(Form I). In other words, XRPD could not be used to distinguish
between morphic Form I and the hydrolysis product.
Example 2--Polymorphism Screening
[0217] Polymorphism studies on marizomib were carried out in
different solvents and conditions to understand the polymorphic
behavior of the free form.
[0218] Marizomib (Sample 1, 30 mg) was dissolved or suspended in
the given solvent at room temperature. Solvent was added until the
material dissolved or up to a maximum of 50 volumes. The
suspensions were matured between 50.degree. C. and room temperature
heat-cool cycles (8 h per cycle) for 24 h. The solutions were
cooled to and held at 5.degree. C. for 48 h. If no solid was
obtained then the solutions were allowed to evaporate slowly at
room temperature. All the recovered solids were analyzed by XRPD.
The results are given in Table 7.
TABLE-US-00012 TABLE 7 Polymorphism Screen Results Obs. at r.t.
Maturation Cooling at Evap. at Solvent 10 vol. 20 vol. 30 vol. 40
vol. 50 vol. 50.degree. C./r.t. 5.degree. C. r.t. XRPD n-Heptane X
X X X X X n/a n/a Form I Ethyl acetate X X X X X Solid Form I MIBK
X X X X X X n/a n/a Form I 2-Propanol X X X X X X n/a n/a Form I
Acetone X X X X X Solid Form I Chloroform X Solid Form I Dimethyl
sulfoxide X X X X X Solid Form I tert-Butylmethyl X X X X X X n/a
n/a Form I ether Anisole X X X X X X n/a n/a Form I Cumene X X X X
X X n/a n/a Form I MEK X X X X X Solid Form I Isopropyl acetate X X
X X X X n/a n/a Form I DMF X Solution after four weeks* Toluene X X
X X X X n/a n/a Form I Tetrahydrofuran X Solid Form I
Dichloromethane X X X X X X n/a n/a Form I Acetonitrile X X X X X X
n/a n/a Form I Nitromethane X X X X X X n/a n/a Form I Ethanol X X
X X X X n/a n/a Form I DMA X Solution after four weeks* = Soluble X
= Insoluble, n/a = not applicable. Data can be found in Data
Section 2. *No further work was performed on the solution.
[0219] Form I was the only form obtained from the screen.
Comparison spectra from all of the solvents screened are given in
FIGS. 11 and 12, with the exception of DMF and DMA, from which no
solid was found to precipitate. Chemical purity profiles of two of
the samples isolated from EtOH and EtOAc experiments were recorded
to check the material susceptibility to hydrolysis. The results
were 97.4% and 96.8% respectively (reference to chemical purity of
the starting material, 98.2%) as set forth in FIGS. 29 and 30.
Example 3--Characterization and Approximate Solubility of Sample
4
[0220] Prior to any testing, Sample 4 was characterized by XRPD,
TGA, DSC, 1HNMR, and DVS. The results are given below in Table-8.
The Corresponding figure numbers are given in the right hand
column.
TABLE-US-00013 TABLE 8 Characterization of Sample 4 Prior to
Testing. Analysis Result XRPD crystalline, pattern indexed, Form I
TGA 168.degree. C. (onset, decomp.) negligible weight loss prior to
decomp. DSC 166.degree. C. (exo, peak); 175.degree. C. (endo, peak)
.sup.1H NMR consistent with chemical structure of MRZ DVS -0.035 wt
% (at 5% RH) +0.039 wt % (5-75% RH) +1.158 wt % (75-95% RH) -1.276
wt % (95-5% RH)
Approximate Solubility
[0221] Weighed samples of MRZ, Sample 4 were treated with aliquots
of the test solvents or solvent mixtures at ambient temperature.
Complete dissolution of the test material was determined by visual
inspection. Solubility was estimated based on the total solvent
volume used to provide complete dissolution. The actual solubility
may be greater than the value calculated because of the use of
solvent aliquots that were too large or due to a slow rate of
dissolution. If complete dissolution was not achieved during the
experiment, the solubility is expressed as "less than". If complete
dissolution was achieved by only one aliquot addition, the value is
reported as "larger than". The results are given in Table-9, below.
As set forth in Table-9, the ratio of solvent mixtures are given by
volume. Solubilities are estimated at ambient temperature and
reported to the nearest mg/mL; if complete dissolution was not
achieved, the value is reported as "<"; the actual solubility
may be greater than the reported value due to the use of solvent
aliquots that were too large or due to a slow rate of dissolution.
Post-solubility samples were stored at 2-8.degree. C. for 15-16
days followed by 1 day at ambient storage before visual inspection
for precipitation. Post-solubility samples were visually inspected
after 2-3 days of ambient storage. Formation system A is: 55
propylene glycol+5% ethanol+40% citrate buffer (10 mM; pH 5).
Formation system B is: 14/86 (v/v) H.sub.2O/tert-butyl alcohol
containing 30 mg/mL sucrose, and titrated with HCl to pH
3.2.+-.0.2.
TABLE-US-00014 TABLE 9 Approximate Solubility of Sample 4 Prior to
Testing Solubility Precipitates formed Solvent (mg/mL) during
storage? Formulation System A <1 N/A Formulation System B 6 no
acetic acid 10 no IPA 6 no [EMIm][NTF.sub.2] 1 N/A [EMIm][Cl] <2
N/A ACN 10 no tert-butanol 3 no acetone 39 no anisole 2 no
CHCl.sub.3 2 yes cyclohexanone 22 no DMSO >83 no DCM 2 no
dioxane 20 no EtOAc 15 no Formic acid 15 no MeOAc 20 no MIBK 11 no
MEK 33 yes (small amount) nitromethane 2 no NMP 5 no pyridine 36 no
THF 49 no 2-Me THF 32 no 1-propanol 2 no 2,2,2-trifluoroethanol
<1 N/A 50:50 acetone/heptane 12 no 50:50 acetone/iso-octane 12
no 25:75 ACN/MTBE 10 no 20:80 CHCl.sub.3/DCM 3 no 25:75 DMF/anisole
17 no 50:50 EtOAc/cyclohexane 4 no 50:50 EtOAc/isooctane 2 no 20:80
MEK/IPA 10 no 20:80 MEK/MIBK 12 no 50:50 MeOH/Et.sub.2O 21 no 20:80
NMP/IPA (c) 19 no 20:80 NMP/1-propanol 19 no 50:50 THF/hexanes 21
no 20:80 THF/nitromethane 15 no 20:80 2-MeTHF/MeOAc 34 no
Example 4--Polymorph Screen of Marizomib Sample 4 Evaporation
Attempts
[0222] For slow evaporation (SE), solutions containing marizomib
Sample 4 in selected solvents were allowed to evaporate at ambient
temperature from vials covered with perforated aluminum foil. For
fast evaporation (FE), solutions containing marizomib Sample 4 in
selected solvents were allowed to evaporate at ambient temperature
from open vials. The results are given below in Table-10. For flash
evaporation, solids of disordered marizomib Sample 4 were added
dropwise to pre-warmed glass vials, allowing the solvent to flash
evaporate immediate upon contact. Resulting solids were collected
for analysis. The corresponding figure numbers are given in the
right hand column.
TABLE-US-00015 TABLE 10 Polymorph Screen of Marizomib Sample 4
Evaporation Attempts Observations/ XRPD Solvent Condition Comments
Results acetone SE white solids, Form I dendritic particles
CHCl.sub.3 SE white solids, Form I needles, agg. IPA SE white
solids, Form I needles, B/E THF FE white solids, Form I dendritic
particles MEK FE white solids, Form I columns, agg. ACN FE white
solids, Form I needles, agg. DCM FE white solids, Form I columns,
agg.
Example 5--Polymorph Screen of Marizomib Sample 4 Cooling
Attempts
[0223] For crash cooling (CC), concentrated solutions of marizomib
Sample 4 were prepared at elevated temperature and filtered through
pre-warmed 0.2-.mu.m nylon filters into a clean vial pre-chilled in
a dry ice/acetone bath. For fast cooling (FC) and slow cooling
(SC), solutions containing marizomib Sample 4 in selected solvents
at elevated temperatures in sealed vials. Solutions were then
filtered into clean vials with 0.2-.mu.m nylon filters and removed
from the heating plate and left at ambient conditions (FC) or
allowed to cool to specified temperature on the heating plate (SC).
The results are given below in Table-11. As set forth below, the
ratio of solvent mixtures are given by volume. Temperature and time
are approximate.
TABLE-US-00016 TABLE 11 Polymorph Screen of Marizomib Sample 4
Cooling Attempts Observations/ XRPD Solvent Condition Comments
Results ACN 1. CC (65.degree. C. 1. white solids, Form I to
-78.degree. C.) some columns, B/E EtOAc 1. CC (70.degree. C. 1.
clear solution Form I to -78.degree. C.) 2. white solids, 2. kept
at RT small fibrous for 5 min particles THF/ 1. CC (70.degree. C.
1. clear solution -- hexanes to -78.degree. C.) 2. white solids,
(50:50) 2. kept at RT IS for 10 min MeOAC 1. CC (65.degree. C. 1.
clear solution Form I to -78.degree. C.) 2. white solids, 2. kept
at RT fine particles, for 5 min UM MEK/IPA 1. CC (78.degree. C. 1.
clear solution Form I + (20:80) to -78.degree. C.) 2. white solids,
additional 2. kept at RT fine particles, peaks for 5 min UM, some B
NMP/ 1. CC (78.degree. C. 1. clear solution -- 1-PrOH to
-78.degree. C.) 2. clear solution (20:80) 2. kept at RT 3. white
solids, for 5 min UM small 3. kept at -10 particles, IS to
-25.degree. C. for 7 d MIBK 1. SC 1. white solids, Form I
(70.degree. C. to RT) columns, agg., B/E cyclohexanone 1. SC 1.
clear solution Form I (70.degree. C. to RT) 2. clear solution 2.
kept at 2-8.degree. C. 3. clear solution for 1 d 4. cloudy
solution, 3. kept at -10 white solids, to -25.degree. C. fibrous
needles for 20 d 4. cyclohexane added, kept at 2-8 .degree. C. for
2 d EtOAc/ 1. SC 1. clear solution Form I isooctane (70.degree. C.
to RT) 2. clear solution (50:50) 2. kept at 2-8.degree. C. 3.
needles, B/E for 1 d 3. kept at -10 to -25.degree. C. for 6 d THF/
1. SC 1. white solids, Form I nitromethane (65.degree. C. to RT)
columns, B/E (20:80) [EMIm][NTF.sub.2] 1. SC from 77.degree. C. 1.
clear solution Form I to RT 2. white solids, 2. kept at RT fine
particles, for 11 d UM, agg., B [EMIm][Cl] 1. FC (105.degree. C. 1.
orange solution unique to 81.degree. C.) (c) 2. off-white solids,
pattern + 2. hot H.sub.2O fine minor added particles, UM, B Form I
Formulation 1. SC from 77.degree. C. 1. white solids, Form I System
A to RT fibrous agg. Formulation 1. CC (105.degree. C. 1. sample
frozen Form I System A to -78.degree. C.) 2. white solids, 2. kept
at 2-8.degree. C. small particles, for 6 d agg., UM Formulation 1.
SC (85.degree. C. 1. white fibrous Form I System B to RT)
solids
Example 6--Polymorph Screen of Marizomib Sample 4
Solvent/Antisolvent Addition Attempts
[0224] For solvent/antisolvent (SAS) experiments, concentrated
solutions of marizomib were prepared in selected solvents and
solutions were filtered through 0.2-.mu.m nylon filters, either
into antisolvent at ambient conditions, or into clean vials and
antisolvents were subsequently added into the solutions. In the
case of crash precipitation (CP), concentrated solutions of
marizomib were prepared in selected solvents and filtered through
0.2-.mu.m nylon filters and into anti-solvent precooled in an
ice/water bath. The results are given below in Table-12. The ratio
of solvent mixtures are by volume. Time and temperature are
approximate.
TABLE-US-00017 TABLE 12 Polymorph Screen of Marizomib Sample 4
Solvent/Antisolvent Addition Attempts Observations/ XRPD Solvent
Condition Comments Results pyridine/ 1. CP (solution in 1. no
solids -- Et.sub.2O pyridine added to (5:7) cold Et.sub.2O)
acetone/ 1. CP (solution in 1. white solids, Form I isooctane
acetone added to some fibrous (1:3) cold isooctane) particles, some
B EtOAc/ 1. CP (solution in 1. white solids, Form I cyclohexane
EtOAc added to agg., anh. (1:2) cold cyclohexane) particles
acetone/ 1. CP (solution in 1. white solids, Form I heptane acetone
added to agg., anh. (1:1) cold heptane) particles formic 1. cold
CHCl.sub.3 1. rose colored Form I + acid/CHCl.sub.3 added into
sample solution, clear additional (1:1) solution of 2. no solids
peaks 6882-21-08 3. sample frozen 2. kept at 2-8.degree. C. 4. rose
colored 1 day solution 3. kept at -10 5. off white to -25.degree.
C. solids, agg., overnight small particles, 4. kept at 2-8.degree.
C. UM 5. evaporation at RT, purged w/N.sub.2 after 3 d MEK/ 1. CP
(solution in 1. white solids, Form I IPE (1:5) MEK added to small
fibrous cold IPE), particles, UM, B manually shaken THF/ 1. CP
(solution in 1. white solids, Form I hexanes THF added to small
particles & (1:5) cold hexanes), agg., UM manually shaken
ACN/heptane 1. CP (solution in 1. clear Form I (3:10) ACN added to
2. clear solution cold heptane), 3. clear solution shaken 4. white
solids, 2. shaken at 5.degree. C. agg., columns, on orbital shaker
B/E solution for 6 d 3. shaken at -4 to 2.degree. C. on orbital
shaker for 3 d 4. evaporation under N.sub.2 acetone/ 1. solids 1.
clear solution Form I Formulation dissolved in 2. mostly clear
System A (1/2) acetone solution, small 2. solution particulates
filtered into 3. white solids, Formulation fine particles, System
A, shaken needles, agg. 3. stored at 2-8.degree. C. for 1 d 2-Me 1.
CP (solution in 1. clear solution Form I THF/MTBE 2-Me THF added to
2. clear solution (1:5) cold MTBE), shaken 3. clear solution 2.
shaken at 5.degree. C. 4. white solids, on orbital shaker needles,
agg., for 6 d B/E 3. shaken at -4 to 2.degree. C. on orbital shaker
for 3 d 4. evaporation under N2 MeOH/ 1. CP (solution in 1. clear
solution Form I Et.sub.2O (1:5) MeOH added to cold 2. clear
solution Et.sub.2O), shaken 3. clear solution 2. shaken at
5.degree. C. 4. white solids, on orbital shaker fibrous needles for
6 d 3. shaken at -4 to 2.degree. C. on orbital shaker for 3 d 4.
evaporation under N.sub.2 Formulation 1. CP (solution in 1. white
solids, -- System B/ Formulation System UM, IS nitromethane B added
to cold (1:1.5) nitromethane), shaken
Example 7--Polymorph Screen of Marizomib Sample 4 Slurry
Attempts
[0225] Sufficient amounts of solids of marizomib Sample 4 were
added to selected solvents in vials so that excess solids initially
persisted. The mixture was then triturated with a stir bar at
specified temperatures for an extended period of time. The results
are given below in Table-13. The ratio of solvent mixtures are by
volume. Time and temperature are approximate.
TABLE-US-00018 TABLE 13 Polymorph Screen of Marizomib Sample 4
Slurry Attempts Observations/ XRPD Solvent Condition Comments
Results formic acid RT, 17 d all solids dissolved, -- solution rose
color acetone/heptane RT, 7 d -- Form I (50:50) NMP RT, 7 d white
solids, small Form I particles, UM, B 1-propanol 50.degree. C., 7 d
white solids, pasty, Form I B, UM anisole 50.degree. C., 7 d white
solids, UM Form I nitromethane 50.degree. C., 7 d white solids,
small Form I particles, UM 2-Me THF/MeOAc RT, 7 d white solids,
some Form I (20:80) columns, B CHCl.sub.3 RT, 8 d white solids,
fine Form I particles, agg., UM, B Formulation RT, 13 d white
solids, agg., Form I System A UM Formulation RT, 7 d white solids,
fine Form I System B particles, UM acetic acid RT, 7 d white
solids, Form I agglomerates, B acetone/H.sub.2O (40:60) RT, 7 d
white solids, small Form I (A.sub.w = 0.92) particles, UM, B
ACN/H.sub.2O RT, 7 d white solids, small Form I (50:50) particles,
UM, B (A.sub.w = 0.90) IPA/H.sub.2O RT, 7 d white solids, small
Form I (50:50) particles, UM, B (A.sub.w = 0.96) 2-BuOH/H.sub.2O
RT, 7 d white solids, small Form I (70:30) particles, UM, B
(A.sub.w = 1.04) HFIPA/H.sub.2O RT, 7 d white solids, small Form I
(75:25) particles, UM, B (A.sub.w = 1.41) TFE/H.sub.2O RT, 7 d
white solids, small Form I (50:50) particles, UM, B (A.sub.w=
1.08)
Example 8--Polymorph Screen of Marizomib Sample 4 Manual Grinding
Attempts
[0226] Solids of marizomib Sample 4 were ground by hand at room
temperature using a mortar and pestle at five minutes per cycle for
two (wet grinding) or four (dry grinding) cycles. In the case of
wet grinding, a small amount of select solvent or solvent mixture
(10 .mu.L) was added before grinding. The inside of the mortar was
scraped between each cycle. The results are given below in
Table-14. As set forth below, the ratio of solvent mixtures are
given by volume. Times are approximate. Disordered Form I was used
as the starting material for the water recrystallization
experiment.
TABLE-US-00019 TABLE 14 Polymorph Screen of Marizomib Sample 4
Manual Grinding Attempts Observations/ XRPD Solvent Condition
Comments Results -- 4 .times. 5 min white solids, disordered UM,
agg. Form I Formulation 2 .times. 5 min; white tacky Form I System
A 2 .times. 10 .mu.L solids agglomerates, B Formulation 2 .times. 5
min; white solids, disordered System B 2 .times. 10 .mu.L fine
particles, Form I agg., UM, B acetone/heptane 2 .times. 5 min;
white solids, disordered (50/50) 2 .times. 10 .mu.L fine particles,
Form I agg., UM, B formic acid 2 .times. 5 min; white solids,
disordered 2 .times. 10 .mu.L fine particles, Form I agg., UM, B
acetic acid 2 .times. 5 min; white solids, disordered 2 .times. 10
.mu.L fine particles, Form I agg., UM, B water 2 .times. 5 min;
white solids, disordered 2 .times. 10 .mu.L small particles, Form I
agg., UM, B
Example 9--Polymorph Screen of Marizomib Sample 4 Heat Stress
Attempts from Disordered Form I
[0227] Solids of disordered marizomib Sample 4 were held at
designated temperatures for the indicated amount of time. The
results are given below in Table-15. Resulting solids were
collected for analysis. Temperature and time are approximate.
TABLE-US-00020 TABLE 15 Polymorph Screen of Marizomib Sample 4 Heat
Stress Attempts from Disordered Form I Observations/ Condition
Comments XRPD Results 44.degree. C., white solids, Form I w/
overnight fluffy, agg., UM some disorder 60.degree. C., 5 h white
solids, Form I w/ fluffy, agg., UM some disorder 80.degree. C., 2 h
white solids, Form I w/ fluffy, agg., UM some disorder
Example 10--Experimental Attempts Targeting Amorphous Marizomib
[0228] For lyophilization, a solution of MRZ was prepared in either
dioxane or Formulation System B, filtered through 0.2-.mu.m nylon
filter, and frozen in a glass flask by rotating it in a cold dry
ice/IPA bath. The flask was then purged with N2, submerged in a
propylene glycol bath at temperatures below -25.degree. C., and
placed under vacuum of 0.028 mm Hg to dry for 2 to 5 days. The
resulting solids were collected for analysis.
[0229] For fast evaporation (FE), solutions containing MRZ in
selected solvents were allowed to evaporate at ambient temperature
from open vials. The results are shown in Table-16 below. The ratio
of solvent mixtures are by volume. Time and temperature are
approximate. The acetone used was anhydrous.
TABLE-US-00021 TABLE 16 Experimental Attempts Targeting Amorphous
Marizomib Observations/ XRPD Solvent Condition Comments Results
acetone flash evaporation white solids, Form I + at 110.degree. C.
some yellowing, additional agg., UM peaks acetone flash evaporation
white solids, Form I at 90.degree. C. needles, agg., B/E dioxane
lyophilization static white Form I powder, UM dioxane
lyophilization white powder, disordered static, clumps Form I
dioxane lyophilization white powder, disordered flakey, fluffy Form
I Formulation lyophilization clear, gooey, disordered System B
sticky Form I
Example 11--Further Analysis on Selected Samples from Polymorph
Screen
TABLE-US-00022 [0230] TABLE 17 Analysis of on Selected Samples from
Polymorph Screen Condition Analysis Result flash evaporation
.sup.1H NMR peaks attributable to MRZ from acetone observed +
unidentified (110.degree. C.) additional peaks with substantial
integral intensities crash cool from .sup.1H NMR spectrum in
general MEK/IPA consistent with chemical (78 to -78.degree. C.)
structure of MRZ + small additional unidentified peaks
lyophilization mDSC 161.degree. C. (endo, onset) FC from [EMIm][Cl]
.sup.1H NMR spectrum not consistent followed by addition with
chemical structure of hot H.sub.2O of MRZ evaporation from .sup.1H
NMR peaks attributable to formic acid/CHCl.sub.3 MRZ observed +
unidentified additional peaks, some peaks with substantial integral
intensities
Example 12--Co-crystal Screen of Marizomib
[0231] For crash cooling (CC), a solution of MRZ and coformer was
prepared at elevated temperature in a given solvent mixture. The
vial was capped and immediately placed in a refrigerator or
freezer. For fast cooling (FC), solutions of MRZ and coformer were
prepared at elevated temperatures in given solvents or solvent
mixtures. The vial was capped and placed on a bench top at room
temperature to quickly cool. For slow cooling (SC), a solution of
MRZ and coformer was prepared at elevated temperature in a given
solvent mixture. The vial was capped and left in a heating block at
elevated temperature. The heater then was turned off for the sample
to cool down naturally to room temperature. For slow evaporation
(SE), solutions of MRZ and coformer were generated at ambient
temperature in a given solvent or solvent mixture. The solutions
were allowed to evaporate partially or to dryness from a loosely
capped vial at ambient conditions. Slurry experiments were carried
out by making saturated solutions containing excess solid. The
slurries were agitated at ambient or elevated temperatures for a
specified amount of time. The solids present were recovered via
positive pressure filtration. Solvent assisted grinding experiments
were carried out by mixing MRZ and coformer in an agate mortar and
pestle. Aliquots of solvent were added and the mixture ground
manually for specified amount of time. The solids were scraped from
the walls of the mortar and the pestle head. Another aliquot of
solvent added and ground for several more minutes. For temperature
cycling, solutions or suspensions of MRZ and coformer were made in
a given solvent and placed at elevated temperature. The sample was
cycled by removing from heat and then reapplying several times. The
solids were collected via vacuum filtration upon the last cool from
elevated temperature.
[0232] As set forth in Table 18 below, "X:Y" refers to
marizomib:conformer mole ratio. Temperatures and times are
approximate. Solvent ratios are by volume. Approximately 60-90 mg
of marizomib was used for each experiment.
TABLE-US-00023 TABLE 18 Cocrystal Screen of Marizomib Coformer XRPD
(X:Y) Conditions Observations Results Acetic acid Slurry, RT White
solids MRZ (large excess) PLM: agglomerates, B Acetic acid 1)
Acetone, stir, RT, 5 1) Slightly turbid solution MRZ (1:2) Days 2)
Faint hazy solution 2) Cool, 2-8.degree. C., 3 days 3) Translucent
solids 3) SE, RT PLM: agglomerates, prismatics, B Adenine EtOH,
slurry, RT, 5 days White solids MRZ (1:1) PLM: Small particles,
unknown morphology, B Adenine 1) Add MeOH to 1) White suspension
MRZ + (1:3) Adenine 2) White suspension minor 2) Add MIBK to MRZ 3)
White suspension adenine 3) Add adenine 4) White suspension
suspension to MRZ 5) White solids 4) Slurry at 60.degree. C. O/N
PLM: small particles, unknown 5) Slurry, RT, 1 day morphology
L-alanine 1) Dissolve L-alanine in 1) Clear, colorless solution --
(1.1) H.sub.2O 2) Clear, colorless solution 2) Dissolve MRZ in 3)
White gel pyridine 4) White suspension insufficient 3) Add
L-alanine to for XRPD MRZ 5) White suspension, insufficient 4)
Slurry, RT, 2 days for XRPD 5) Cool to 2-8.degree. C. 6) Clear,
tacky gel 6) SE, RT L-alanine 1) Add MeOH to L- 1) Solids remain
MRZ (1:1) alanine 2) White suspension 2) Add suspension to 3) White
suspension MRZ 4) White solids 3) Slurry at 60.degree. C. O/N PLM:
small particles, unknown 4) Slurry, RT, 1 da morphology L-arginine
1) Dissolved L-arginine 1) Clear, colorless solution MRZ (1:1) in
HFIPA 2) Clear, colorless solution 2) Dissolved MRZ in 3) Turbid
solution dioxane 4) White solids 3) Added L-arginine PLM: small
particles, unknown solution dropwise to morphology, B. MRZ solution
4) Stir, RT, ~25 minutes SE (FIG. 129 filtrate Yellow gel --
material), RT L-ascorbic acid MIBK, FC, 75.degree. C. to RT White
solids MRZ + L- (2:1) ascorbic acid L-ascorbic acid 1) IP A added
to MRZ at 1) Scant solids remain MRZ (1:1) 50.degree. C. 2) Clear,
colorless solution 2) L-ascorbic acid 3) Clear, colorless solution
dissolved in MeOH 4) White solids 3) L-ascorbic acid added PLM:
small particles, unknown dropwise to MRZ at morphology, B.
50.degree. C. 4) FC to RT L-ascorbic acid ACN, slurry, RT, 7 days
White solids MRZ + L- (1.5) PLM: small particles, unknown ascorbic
morphology, B acid Benzoic acid EtOAc, SE, RT Translucent solids
MRZ (2:1) PLM: needles, agglomerates, B/E possible singles Benzoic
acid 1) Dissolve benzoic acid 1) Clear, colorless solution MRZ +
(1:1) in EtOH 2) Clear, colorless solution benzoic acid 2) Dissolve
MRZ in 3) Clear, colorless solution acetone 4) Clear, colorless
solution 3) Add benzoic acid to 5)Translucent solids MRZ PLM:
agglomerates of needles, B 4) Stir, RT, 2 days 5) SE at RT Benzoic
acid MEK:MIBK (20:80), White solids MRZ (1:5) slurry, RT, 7 days
PLM: small particles, unknown morphology, B. Caffeine Dioxane, SC,
50.degree. C. to White solids MRZ + (1:1) RT caffeine Caffeine 1)
Add THF to caffeine 1) White suspension Caffeine + (1:2) 2) Add
caffeine 2) White suspension minor suspension to MRZ 3) White
solids MRZ 3) Slurry, 50.degree. C., 2 days PLM: small particles,
agglomerates, unknown morphology, B. Caffeine DMF:anisole (25:75),
White solids, Caffeine (1:3) slurry, RT, 6 days PLM: small
particles, unknown morphology, B Trans-cinnamic 1) Dissolve trans-
1) Clear, colorless solution MRZ + acid (1:1) cinnamic acid in ACN
2) Clear, colorless solution trans- 2) Dissolve MRZ in 3) Clear,
colorless solution Cinnamic MEK 4) Clear, colorless solution acid
3) Add trans-cinnamic 5) Scant translucent solids acid solution to
MRZ PLM: Rods, B/E dropwise 6) White solids 4) Stir, RT, 3 days
PLM: prisms and plates, B 5) Cool to 2-8.degree. C. 6) SE, RT
Citric acid NMP, crash cool, 50.degree. C. Clear, colorless
solution -- (1:1) to 2-8.degree. C. Citric acid Solvent assisted
White solids MRZ + (1:2) grinding, IPA, (3 .times. 5 citric acid
minutes, 3 .times. 10 .mu.L) 1) EtOAc, slurry, 50.degree. C., 1)
Scant white solids MRZ + 5 days 2) White solids citric acid 2)
Slurry, RT, 6 days PLM: small particles, unknown morphology, B
Citric acid MIBK, slurry, RT, 6 White solids, MRZ + (1:3) days PLM:
small particles, unknown citric acid morphology, B (2- 1) Dissolve
(2- 1) Clear, colorless solution MRZ hydroxypropyl)- hydroxy
propyl)-.beta.- 2) Clear, colorless solution .beta. -cyclodextrin
cyclodextrin in MeOH 3) White precipitation (1:1) 2) Dissolve MRZ
in 4) White suspension (gel) acetone 5) Clear solution with white
gel at 3) Add conformer bottom solution to MRZ 5) Clear solution
with clear gel at 4) Slurry at RT bottom 5) After ~20 minutes, 6)
White solids place at 50.degree. C. PLM: needles, spherulites, B 5)
Slurry, 50.degree. C., 2 days 6) Supernatant solution decanted, SE,
RT (2- 1) Dissolve (2- 1) Clear, colorless solution MRZ
hydroxypropyl)- hydroxypropyl)-.beta.- 2) Clear, colorless solution
.beta.-cyclodextrin cyclodextrin in H.sub.2O 3) White suspension
(2:1) 2) Dissolve MRZ in 4) Turbid suspension ACN at 50.degree. C.
5) Turbid solution 3) Add H.sub.2O solution to 6) Translucent
solids ACN solution at 50.degree. C. PLM: needles, B/E 4) Slurry,
50.degree. C., 2 days 5) Slurry, RT, 6 days 6) SE, RT L-cysteine 1)
Grind L-cysteine and 1) White solids MRZ (1:1) MRZ ~10 minutes 2)
White suspension 2) Add EtOH/dioxane 3) White suspension (50/50) 4)
White solids 3) Sonicated ~2 minutes PLM: small particles, unknown
4) Temperature cycle morphology, B between RT and 50.degree. C. (4
cycles .times. 1 hour each temperature, held at RT overnight)
L-cysteine 1) Add H.sub.2O to L- 1) Slightly turbid solution Highly
(1:2) cysteine at 50.degree. C. 2) Clear, colorless solution
disordered 2) Dissolve MRZ in 3) Immediate white precipitation
crystalline ACN at 60.degree. C. 4) Turbid, faintly yellow 3)
L-cysteine solution suspension added to ACN solution 5) Turbid
solution 4) Slurry, 50.degree. C., 2 days 6) Off-white solids 5)
Slurry, RT, 4 days PLM: Glassy, no apparent B 6) SE, RT Cytosine 1)
Add 0.5 mL H.sub.2O to 1) White suspension MRZ + (1:1) cytosine,
slurry at 50.degree. C. 2) White suspension unique 2) Add 0.5 mL
ACN to 3) White suspension peaks + MRZ, slurry at 50.degree. C. 4)
Faintly yellow solution with possible 3) Sonicate 2 minutes white
solids minor 4) Add cytosine 5) White solids cytosine suspension to
MRZ, PLM: small particles, unknown slurry at 50.degree. C., 2 days
morphology, B 5) Slurry, RT, 6 days Cytosine 1) 1.5 mL H.sub.2O
added to 1) White suspension Unique (1:3) cytosine 2) Scant solids
remain peaks + 2) 1 mL dioxane added 3) White suspension MRZ MRZ 4)
Light yellow, clear solution (same as 3) Cytosine suspension 5)
Cream colored solids observed in added to MRZ (dioxane: PLM: small
particles, unknown FIG. 233) water 2:3) Morphology (XRPD) 4) Slurry
at 60.degree. C. O/N Primarily 5) Stir, RT, 1 day cytosine with
dioxane and trace MRZ additional peaks also present (NMR) Fumaric
acid Anh. acetone, SE, RT Translucent solids MRZ + (1:1) PLM:
needles, dendritics, B/E fumaric acid + additional peak Fumaric
acid Solvent assisted White solids MRZ + (1:1) grinding, ACN (2
.times. 5 fumaric minutes, 2 .times. 20 .mu.L) acid Fumaric acid 1)
Added TFE to 1) White suspension MRZ + (1:3) fumaric acid at
50.degree. C. 2) Clear, colorless solution fumaric 2) Dissolved MRZ
in 3) White suspension acid THF at 50.degree. C. 4) White solids 3)
Added fumaric acid to PLM: small particles, unknown MRZ solution
morphology, B 4) Temp, cycle (50.degree. C. - RT) three cycles hold
4 hours Gentisic acid 1) Dissolve gentisic acid 1) Clear, colorless
solution MRZ (1:1) in t-BuOH at 50.degree. C. 2) White suspension
2) Add to MRZ at 50.degree. C. 3) White solids 3) Slurry,
50.degree. C., 2 days PLM: fibrous and small particles with unknown
morphology, B Gentisic acid ACN, FC, 60.degree. C. to RT White
solids MRZ (2:1) PLM: agglomerates, fines, B. Gentisic acid MEK:IPA
(20:80), White solids, MRZ (1:5) slurry, RT, 6 days PLM: small
particles, unknown morphology, B L-glutamine Anh. acetone, grinding
White solids MRZ + L- (1:1) (2 .times. 5 minutes, 2 .times. 20
glutamine .mu.L) L-glutamine 1) Dissolve L-glutamine 1) Clear,
colorless solution MRZ + (1:3) in H.sub.2O at 50.degree. C. 2)
Clear, colorless solution minor L- 2) Dissolve MRZ in 2- 3) Two
clear, colorless layers glutamine MeTHF at 50.degree. C. 4) White
solids 3) Add L-glutamine to PLM: fibrous, B MRZ dropwise at
50.degree. C. 4) FC/FE at RT L-glutamine ACN:MTBE (25:75), White
solids, MRZ + L- (1:5) slurry, RT, 6 days PLM: small particles,
unknown glutamine morphology, B Glutaric acid 1) Dissolve glutaric
acid 1) Clear, colorless solution MRZ + (1:1) in IPrOAc 2) Clear,
colorless solution Glutaric 2) Dissolve MRZ in 3) Clear, colorless
solution acid ACN at 50.degree. C. 4) Clear, colorless solution 3)
Add glutaric acid 5) Clear, colorless solution solution dropwise 6)
Clear, colorless solution 4) Stir, 50.degree. C., 2 days 7)
Translucent solids 5) FC to RT PLM: dendrites, needles, B 6) Cool
to 2-8.degree. C. 7) SE, RT Glutaric arid 1) Dissolve glutaric acid
1) Clear, colorless solution MRZ (1:3) in EtOH 2) White suspension
2) Add to MRZ 3) White suspension 3) Slurry, RT, 6 days PLM: small
particles, unknown morphology, B Glycine ACN, grinding (2 .times. 5
White solids MRZ + (2:1) minutes, 2 .times. 20 .mu.L) glycine
Glycine 1) Dissolve glycine in 1) Clear, colorless solution MRZ +
(1:1) H.sub.2O 2) Clear, colorless solution glycine 2) Dissolve MRZ
in 3) White precipitate acetone 4) White solids 3) Add glycine to
MRZ PLM: small particles, unknown dropwise morphology, B 4) Slurry,
RT, 2 days Glycine EtOAc:cyclohexane White solids, MRZ + (1:5)
(50:50), slurry, RT, 5 PLM: small particles, unknown glycine days
morphology, B Glycolic acid 1) Melt glycolic acid at 1) Clear melt
MRZ + (1:1) 85.degree. C. 2) White solids minor 2) Add MRZ 3)
Clear, colorless solution glycolic 3) Dissolve in THF at 4) White
solids acid 85.degree. C. PLM: small needles, B. 4) Evaporate off
solvent
at 85.degree. C. Glycolic acid Acetone, SE, RT White solids MRZ
(1:2) PLM: Needles, B. Hippuric acid 1) MEK, FC, 65.degree. C. to
1) Scant solids Hippuric (1:1) RT 2) White solids acid + 2) Cool to
2-8.degree. C. PLM: equants, B. MRZ peak Hippuric acid 1) Add
IPA/HFIPA (1:1) 1) White suspension MRZ + (1:5) to Hippuric acid 2)
White suspension hippuric 2) Add suspension to 3) White suspension
acid MRZ 4) White solids 3) Heat to 50.degree. C. PLM: small
particles, unknown 4) Slurry, 50.degree. C., 6 days morphology, B
Hydantoin 1) Add dioxane 1) Scant solids remain MRZ (1:1) 2) Add
H.sub.2O (2:1 2) White precipitation dioxane:H.sub.2O) 3) White
solids 3) Slurry, RT PLM: small particles, unknown morphology, B.
Hydantoin 1) H.sub.2O added to 1) White suspension MRZ (1:3)
hydantoin 2) Clear, colorless solution 2) ACN added to MRZ 3) White
suspension 3) Hydantoin suspension 4) Clear, colorless solution
added to MRZ 5) White solids 4) Slurry, 60.degree. C. PLM: tangled
fibers, B. overnight 5) Stir, RT, 1 day 4- 1) Dissolve 4- 1) Clear,
colorless solution MRZ + 4- hydroxybenzoic hydroxybenzoic acid in
2) White suspension hydroxy acid (1:1) anh. acetone 3) White
suspension benzoic 2) Add anh. acetone to 4) White suspension,
insufficient acid MRZ for analysis 3) Add 4- 5) White solids
hydroxybenzoic acid PLM: needles and dendrites, B solution to MRZ
4) Stir, RT 5) SE, RT 4- 1) 4-hydroxybenzoic 1) Clear, colorless
solution MRZ + 4- hydroxybenzoic acid dissolved in EtOH 2) Clear,
colorless solution hydroxy acid (1:1) 2) MRZ disslolved in 3)
Clear, colorless solution benzoic dioxane 4) Clear, colorless
solution acid 3) EtOH solution added 5) White solids to MRZ PLM:
dendrites, needles, B/E. 4) Stir, RT, 1 day 5) FE, RT Imidazole
Pyridine, SC, 45.degree. C. to Clear, golden yellow solution --
(1:1) RT Imidazole 1) Dissolve imidazole in 1) Clear, colorless
solution -- (1:2) acetone 2) Clear, colorless solution 2) Add to
MRZ 3) Yellow tacky gel 3) SE, RT Imidazole 1) MeOAc, slurry, RT, 2
1) Clear, light yellow solution -- (1:5) days 2) Golden oil 2) SE,
RT L-lysine 1) Dissolve L-lysine in 1) Yellow solution MRZ (1:1)
HFIPA 2) Clear, colorless solution 2) Dissolve MRZ in 3) Beige
suspension THF 4) White solids 3) Add L-lysine solution PLM: small
particles, unknown dropwise to MRZ morphology, B. 4) Stir, RT, ~35
minutes SE (FIG. 171 material Orange gel -- filtrate), RT L-lysine
1) L-lysine dissolved in 1) Clear, colorless solution -- (1:3)
H.sub.2O 2) Scant solids remain 2) ACN added to MRZ 3) White
suspension 3) L-lysine solution 4) Orange solution added to MRZ 5)
Orange solution 4) Slurry, 60.degree. C. O/N 5) Stir, RT, 1 day
Maleic acid Nitromethane, SC, 50.degree. C. White solids MRZ (1:1)
to RT Maleic acid 1) Added 1-PrOH to 1) Clear, colorless solution
MRZ (1:3) maleic acid 2) White suspension 2) Added to MRZ 3) White
solids 3) Slurry, RT, 6 days PLM: small particles, unknown
morphology, B Maleic acid Acetone:heptane White solids MRZ + (1:5)
(50:50), slurry, RT, 7 PLM: small agglomerates, maleic acid days
unknown morphology, B. DL-malic acid 1) Add DCM to DL- 1) White
suspension MR 7 + (1:1) malic acid 2) Clear, colorless solution
DL-malic 2) Dissolve MRZ in 3) White suspension acid THF 4) White
solids 3) Add MRZ solution to PLM: small particles, unknown
DL-malic acid morphology, B. suspension 4) Slurry at RT, 7 days
DL-malic acid 1) Dioxane added to 1) Solids remain MRZ (1:3)
DL-malic acid 2) White suspension 2) Transferred to MRZ 3) Clear,
colorless solution 3) Slurry, 60.degree. C., O/N 4) Clear,
colorless solution 4) Stir, RT, 1 day 5) White wet solids 5) FE, RT
PLM: small needles, B. Melamine 1) Add 1 mL DMSO: 1) White
suspension MRZ + (1:1) H.sub.2O (1:1) to MRZ. Add 2) White
suspension unique melamine. 3) White suspension peaks 2) Sonicate
~2 minutes 4) White solids (XRPD) 3) Slurry at 50.degree. C., 2
PLM: white solids, unknown MRZ: days morphology, B. Melamine 4)
Slurry, RT, 6 days 1:1.6, DMSO and H.sub.2O present, numerous
additional peaks suggestive of some level of degradation (NMR)
Broad endotherm at 97.5.degree. C. (peak max), sharp endotherms at
107.9.degree. C. and 124.1.degree. C. (peak max) (DSC) 10.3% weight
loss from 30.2.degree. C. to 104.0.degree. C. 21.1% weight loss
from 30.2.degree. C. to 146.0.degree. C. (TGA) Melamine 1) Add 1 mL
DMSO: 1) White suspension MRZ + (1:3) H.sub.2O (1:1) melamine 2)
White suspension melamine + 2) Added melamine 3) White suspension
unique suspension to MRZ 4) White solids peaks 3) Slurry at
60.degree. C. O/N PLM: small particles, unknown 4) Slurry, RT, 1
day morphology Nicotinamide ACN, FC, 70.degree. C. to RT White damp
solids MRZ (1:1) Nicotinamide 1) Add acetone to 1) White suspension
Nicotin- (1:3) nicotinamide 2) White suspension amide + 2) Add
nicotinamide 3) White solids MRZ suspension to MRZ PLM:
agglomerates, B 3) Slurry, RT, 6 days Nicotinamide MEK:IPA (20:80),
White solids MRZ + (1:5) slurry, RT, 7 days PLM: small particles,
unknown nicotin- morphology, B amide Orotic acid 1) Nitromethane
added 1) White suspension MRZ (2:1) to orotic acid and 50.degree.
C. 2) White suspension 2) Add to MRZ at 50.degree. C. 3) White
solids 3) Slurry, 50.degree. C., 6 days PLM: small particles,
unknown morphology, B Orotic acid MEK, grinding (2 .times. 5 White
solids MRZ + (1:1) minutes manual minor grinding) orotic acid
Orotic acid MeOAc, slurry, RT, 7 White solids MRZ + (1:5) days PLM:
small particles, unknown orotic acid morphology, B. Oxalic acid
Anh. acetone, FE, RT Translucent solids MRZ + (1:1) PLM:
dendritics, needles, B. oxalic acid Oxalic acid 1) Oxalic acid
dissolved 1) Clear, colorless solution MRZ (1:2) in EtOH 2) Clear,
colorless solution 2) MRZ dissolved in 3) Clear, colorless solution
MEK 4) White solids 3) Add oxalic acid PLM: small particles,
unknown solution to MRZ morphology, B 4) Stir, RT, 7 days Oxalic
acid 1) Dissolved MRZ in 1) Clear, colorless solution MRZ + (1:5)
anhydrous acetone 2) Clear, colorless solution oxalic acid 2)
Dissolved oxalic acid 3) Clear, colorless solution in EtOH 4)
Slightly turbid solution 3) Added oxalic acid 5) Translucent
off-white solids solution dropwise to PLM: needles, prismatics, B/E
MRZ solution 4) Stir, RT, 5 days 5) SE at RT L-proline
Nitromethane, White solids MRZ + L- (2:1) temperature cycle proline
between 50.degree. C. and RT (3 cycles, 1 hour each) L-proline 1)
Dissolved MRZ in 1) Clear, colorless solution L-proline + (1:1) THF
2) Clear, colorless solution minor 2) Dissolved L-proline 3) Clear
colorless solution additional in MeOH 4) White solids peaks 3)
Added L-proline PLM: small particles, unknown (possibly solution
dropwise to morphology, B. MRZ) MRZ 4) Stir, RT, 4 days SE of FIG.
190 material White solids MRZ + filtrate, RT PLM: fibrous
agglomerates, B. additional peaks L-proline 1) Added EtOH to L- 1)
White suspension L-proline + (1:2) proline at 50.degree. C 2) White
suspension MRZ 2) Added EtOAc to 3) White suspension MRZ at
50.degree. C. 4) White solids 3) Added EtOH PLM: small particles,
unknown suspension to MRZ morphology, B 4) Temp cycle (50.degree.
C. to RT, five cycles, 1 hour each) L-proline Acetone:iso-octane
White solids, MRZ + L- (1:3) (50:50), slurry, RT, 6 PLM: small
particles, unknown proline + days morphology, B additional peaks
(possibly L-proline mono- hydrate) L-proline Add sat'd L-proline in
White solids MRZ (sat'd in IPA at IPA to MRZ at 50.degree. C. PLM:
needles, B/E 50.degree. C.) temp cycle (50.degree. C.-RT) L-proline
sat'd 1) Add L-proline to 1) White suspension L-proline in
H.sub.2O/MeOH H.sub.2O/MeOH at 50.degree. C. 2) Clear, colorless
solution 50/50 v/v overnight 3) White suspension 2) Hot filter
saturated L- 4) Yellow solution, scant solids proline solution 5)
Viscous yellow suspension 3) Add saturated PLM: unknown morphology,
B. solution to MRZ at 50.degree. C. 4) Slurry, 50.degree. C., 2
days 5) Slurry, RT, 3 days L-pyroglutamic 1) Dissolve 1) Clear,
colorless solution MRZ + acid (1:1) pyroglutamic acid in 2) Clear,
colorless solution Minor EtOH 3) Clear, colorless solution pyro- 2)
Dissolve MRZ in 4) Clear, colorless solution glutamic acetone 5)
Translucent solids acid 3) Add pyroglutamic PLM: dendrites, B/E
acid to MRZ 4) Stir, RT, 3 days 5) SE, RT L-pyroglutamic 1)
L-pyroglutamic acid 1) Clear, colorless solution MRZ + L- acid
(1:1) dissolved in MeOH 2) Turbid solution pyro- 2) EtOAc added to
MRZ 3) Clear, colorless solution glutamic 3) MeOH solution added 4)
Clear, colorless solution acid to MRZ 5) White solids, PLM:
dendrites 4) Stir, RT, 1 day and needles, B 5) FE, RT
L-pyroglutamic Slurry, ACN, RT, 6 days White solids MRZ + L- acid
(1:2) PLM: small particles, unknown pyro- morphology, B glutamic
acid 2-pyrrolidone 1) Dissolve MRZ in 1) Clear, colorless solution
MRZ + (1:1) acetone (1 mL) at 50.degree. C. 2) Clear, colorless,
solution extra peaks 2) Add 25 .mu.L 2- 3) Hazy solution,
insufficient for at 14.1 and pyrrolidone at RT to XRPD 15.5
.degree.2.theta. MRZ solution 4) White solids (XRPD) 3) Temperature
cycle PLM: small needles, B. MRZ:2- between 50.degree. C. and RT
pyrrolidone (4 cycles .times. 1 hour at 1:1.2 +
each temperature, held at additional RT overnight) minor 4) SE, RT
peaks, no acetone detected (NMR) Sharp endotherm with an onset at
29.4.degree. C. and peak max at 32.8.degree. C. Broad features
(endo- thermic) at 91.4.degree. C. and 131.3.degree. C. (peak max.)
(DSC) 5.0% weight loss from 27.1.degree. C. to 92.0.degree. C.
(TGA) 2-pyrrolidone Manual grinding (2 .times. 5 White tacky solids
MRZ + (excess) minutes, 2 .times. 10 .mu.L) extra peaks at 14.1 and
15.5 .degree.2.theta. 2-pyrrolidone 1) Dissolve MRZ in 500 1)
Clear, slightly yellow sol'n -- (large excess) .mu.L 2-pyrrolidone
at 50.degree. C. 2) Clear, colorless solution 2) Cool to RT with 3)
Clear solution stirring 3) FE, RT Saccharin IPA, FC, 70.degree. C.
to RT White damp solids MRZ (1:1) Saccharin 1) Dissolve saccharin
in 1) Clear, colorless solution MRZ + (1:2) acetone 2) White
suspension minor 2) Add to MRZ 3) White solids saccharin 3) Slurry,
RT, 6 days PLM: small particles, unknown morphology, B Saccharin
ACN, slurry, RT, 7 days White solids MRZ + (1:5) PLM: small
agglomerates, Saccharin unknown morphology, B Salicylic acid
EtOAc:toluene (50:50), White solids MRZ (1:1) FC, 75.degree. C. to
RT PLM: agglomerates, needles, B/E. possible singles Salicylic acid
1) Salicylic acid 1) Clear, colorless solution MRZ (1.3) dissolved
in EtOH 2) Clear, colorless solution 2) MRZ dissolved in 3) Clear,
colorless solution MIBK 4) White solids, 3) Salicylic acid solution
PLM: Small particles, unknown added to MRZ morphology, B 4) Stir,
RT 1) Salicylic acid 1) Clear, colorless solution MRZ + dissolved
in EtOH 2) Clear, colorless solution salicylic 2) MRZ dissolved in
3) Clear, colorless solution acid MEK 4) Clear, colorless solution
3) Salicylic acid solution 5) White solids added to MRZ PLM:
dendritics and needles, B 4) Stir, RT, 8 days 5) SE, RT Salicylic
acid 1) Salicylic acid 1) Clear, colorless solution MRZ + (1:5)
dissolved in 1-PrOH, 2) Clear, colorless solution salicylic MRZ
dissolved in ACN, 3) Clear, colorless solution acid addition of MRZ
4) White solids solution to salicylic acid PLM: needles, dendrites,
B solution 2) Stir, RT, 2 days 3) Cool to 2-8.degree. C., 4 days 4)
FE, RT L-Serine 1) Dissolve L-serine in 1) Clear, colorless
solution MRZ (1:1) H.sub.2O 2) Clear, colorless solution 2)
Dissolve MRZ in 3) Clear, colorless solution THF 4) Slightly turbid
solution 3) Add L-serine to MRZ 5) White solids 4) Stir, RT, 3 days
PLM: agglomerates, B 5) SE, RT L-Serine 1) H.sub.2O added to L- 1)
Solids remain MRZ (1:3) serine 2) Solids remain 2) MeOAc added to
3) Two layers formed MRZ 4) Clear, colorless solution 3) H.sub.2O
added to MeOAc 5) Clear, colorless solution 4) Slurry, 60.degree.
C., O/N 6) White solids 5) Stir, RT, 1 day PLM: dendrites, needles,
B/E 6) FE, RT Succinic acid DCM, slurry, RT, 6 days White solids
MRZ + (1:1) PLM: small particles, unknown succinic morphology, B
acid Succinic acid 1) Dissolve succinic acid 1) Clear, colorless
solution MRZ (1.2) in MeOH 2) Clear, colorless solution 2) Dissolve
MRZ in 3) Clear, colorless solution acetone 4) White solids 3) Add
succinic acid PLM: small particles, unknown solution to MRZ
morphology, B 4) Stir, RT, 5 days Succinic acid 1) Succinic acid 1)
Clear, colorless solution MRZ + (1:5) dissolved in EtOH, MRZ 2)
Clear, colorless solution succinic dissolved in THF, 3) Translucent
solids acid addition of succinic acid PLM: needles, rosettes, B/E
solution to MRZ solution 2) Stir, RT, 2 days 3) SE at RT
L-(+)-tartaric 1) EtOAc, SC, 50.degree. C. to 1) White suspension,
insufficient MRZ + acid (2:1) RT for analysis L-(+)- 2) Cool to
2-8.degree. C. 2) White solids tartaric acid L-(+)-tartaric 1)
Dissolve L-(+)- 1) Clear, colorless solution MRZ + acid (1:2)
tartaric acid in EtOH at 2) Clear, colorless solution minor
50.degree. C. 3) Clear, colorless solution L-(+)- 2) Dissolve MRZ
in 4) Clear, colorless solution tartaric dioxane at 50.degree. C.
5) Translucent solids acid 3) Add L-(+)-tartaric PLM: Needles, B/E
acid solution to MRZ at 50.degree. C. 4) Stir at RT for cool 5) SE
at RT L-(+)-tartaric THF:nitromethane White solids MRZ + acid (1:5)
(20:80), slurry, RT, 7 PLM: small particles, unknown L-(+)- days
morphology, B tartaric acid Thymine 1) Add MeOH to 1) White
suspension Thymine (1:1) thymine at 50.degree. C. 2) White
suspension 2) Add EtOAc to MRZ 3) White suspension at 50.degree. C.
4) White suspension 3) Add thymine 5) White solids suspension PLM:
small particles, unknown 4) Slurry at 50.degree. C., morphology, B
2 days 5) Slurry at RT, 6 days Thymine 1) 1-PrOH added to 1) White
suspension MRZ + (1:3) Thymine 2) White suspension thymine 2) Added
to MRZ 3) White suspension 3) Slurry at 60.degree. C., O/N 4) White
solids 4) Stir, RT, 1 day PLM: small particles, unknown morphology
Uracil 1) Add H.sub.2O to uracil at 1) White suspension MRZ + (1:1)
50.degree. C. 2) White suspension Uracil 2) Add acetone to MRZ 3)
White suspension 3) Add uracil suspension 4) White solids to MRZ
PLM: small particles, unknown 4) Slurry, RT morphology, B. Uracil
1) Uracil dissolved in 1) Clear, colorless solution MRZ + (1:3)
H.sub.2O 2) Clear, colorless solution uracil 2) MRZ dissolved in 3)
White suspension THF 4) White suspension 3) Uracil solution added
5) White solids to MRZ solution PLM: small particles, unknown 4)
Slurry, 60.degree. C., O/N morphology 5) Stir, RT, 1 day Urea
EtOAc, FC, 50.degree. C. to RT White solids MRZ (2:1) PLM: small
particles, unknown morphology, B Urea Solvent assisted White solids
MRZ + (1:1) grinding, t-BuOH (3 .times. 5 urea minutes, 3 .times.
20 .mu.L) Urea Dioxane, stir, RT, 7 days Translucent solids Urea:
(1:5) PLM: needles, dendritics dioxane solvate + minor MRZ (XRPD)
Urea, dioxane, DMSO, water, trace MRZ (1H NMR)
Example 13--Salt Screen of Marizomib
[0233] For crash cooling (CC), a solution of MRZ and coformer was
prepared at elevated temperature in a given solvent mixture. The
vial was capped and immediately placed in a refrigerator or
freezer. For fast cooling (FC), solutions of MRZ and coformer were
prepared at elevated temperatures in given solvents or solvent
mixtures. The vial was capped and placed on a bench top at room
temperature to quickly cool. For slow cooling (SC), a solution of
MRZ and coformer was prepared at elevated temperature in a given
solvent mixture. The vial was capped and left in a heating block at
elevated temperature. The heater then was turned off for the sample
to cool down naturally to room temperature. For slow evaporation
(SE), solutions of MRZ and coformer were generated at ambient
temperature in a given solvent or solvent mixture. The solutions
were allowed to evaporate partially or to dryness from a loosely
capped vial at ambient conditions. Slurry experiments were carried
out by making saturated solutions containing excess solid. The
slurries were agitated at ambient or elevated temperatures for a
specified amount of time. The solids present were recovered via
positive pressure filtration. Solvent assisted grinding experiments
were carried out by mixing MRZ and coformer in an agate mortar and
pestle. Aliquots of solvent were added and the mixture ground
manually for specified amount of time. The solids were scraped from
the walls of the mortar and the pestle head. Another aliquot of
solvent added and ground for several more minutes. For temperature
cycling, solutions or suspensions of MRZ and coformer were made in
a given solvent and placed at elevated temperature. The sample was
cycled by removing from heat and then reapplying several times. The
solids were collected via vacuum filtration upon the last cool from
elevated temperature.
[0234] As set forth in Table 19 below, "X:Y" refers to
marizomib:conformer mole ratio. Temperatures and times are
approximate. Solvent ratios are by volume. Approximately 60-90 mg
of marizomib was used for each experiment.
TABLE-US-00024 TABLE 19 Salt Screen of Marizomib Counnterion XRPD
(X:Y) Conditions Observations Results Benzenesulfonic Anh. acetone,
Brown solution -- acid (1:1) stir, RT, 2 days HCl (1:1) IPA/anh.
White solids MRZ acetone (3:1), PLM: agglomerates stir, RT, of
needles, B/E 7 days HCl (1:2) IPrOAc, stir, White solids MRZ RT
(HCl in PLM: small particles, IPA), 7 days unknown morph, B.
Methanesulfonic Anh. acetone, Dark orange solution, -- acid (1:1)
stir, RT, yellow solids, 1 day experiment discontinued Phosphoric
acid Dioxane, stir, Clear, colorless -- (1:1) RT, 4 days solution
Sulfuric acid 2-MeTHF, stir, Clear, light yellow -- (1:1) RT, 2
days solution Sulfuric acid Acetone, stir, Black solution -- (large
excess) RT, 2 days Sulfuric acid Stir in H.sub.2SO.sub.4, Dark
brown solution -- (large excess) RT, 2 days p-toluenesulfonic
EtOAc, stir, White solids MRZ acid (1:1) RT, 2 days PLM: small
particles, unknown morphology, B. p-toluenesulfonic 1) THF, stir,
1) Clear, colorless MRZ + acid (1:2) RT, 2 days solution p-toluene
2) SE, RT 2) Off-white solids sulfonic PLM: agglomerates, acid + B.
unique peaks (7.9, 12.7 2.theta.)
EQUIVALENTS
[0235] While the present invention has been described in
conjunction with the specific embodiments set forth above, many
alternatives, modifications and other variations thereof will be
apparent to those of ordinary skill in the art. All such
alternatives, modifications and variations are intended to fall
within the spirit and scope of the present invention.
* * * * *