U.S. patent application number 15/601968 was filed with the patent office on 2017-12-14 for polymorphs of cddo ethyl ester and uses thereof.
This patent application is currently assigned to Applied Pharmaceutical Science Inc.. The applicant listed for this patent is Applied Pharmaceutical Science Inc., Xiaohong Zhang, Zhijian Wang. Invention is credited to Chunlei Han, Kaiyang Liu, Lu Song, Wendong Sun, Xiaohong Zhang.
Application Number | 20170355666 15/601968 |
Document ID | / |
Family ID | 50386920 |
Filed Date | 2017-12-14 |
United States Patent
Application |
20170355666 |
Kind Code |
A1 |
Zhang; Xiaohong ; et
al. |
December 14, 2017 |
POLYMORPHS OF CDDO ETHYL ESTER AND USES THEREOF
Abstract
The present invention relates to polymorphic forms of the
compound of Formula I (i.e., ethyl
2-cyano-3,12-dioxokana-1,9(11)-dien-28-oate (CDDO ethyl ester)) and
methods of using them for treating a variety of disease states such
as cancer and conditions associated with inflammation.
##STR00001##
Inventors: |
Zhang; Xiaohong; (Beijing,
CN) ; Song; Lu; (Beijing, CN) ; Sun;
Wendong; (Beijing, CN) ; Liu; Kaiyang;
(Beijing, CN) ; Han; Chunlei; (Beijing,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Applied Pharmaceutical Science Inc.
Zhijian Wang
Xiaohong Zhang |
Beijing
Beijing
Beijing |
|
CN
CN
CN |
|
|
Assignee: |
Applied Pharmaceutical Science
Inc.
Beijing
CN
Wang; Zhijian
Beijing
CN
Zhang; Xiaohong
Beijing
CN
|
Family ID: |
50386920 |
Appl. No.: |
15/601968 |
Filed: |
May 22, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14007925 |
Sep 26, 2013 |
9656951 |
|
|
PCT/CN2012/086045 |
Dec 6, 2012 |
|
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15601968 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; C07J
63/008 20130101; C07C 255/47 20130101; A61P 3/04 20180101; A61P
21/00 20180101; A61P 1/00 20180101; C07C 253/34 20130101; A61P 3/10
20180101; A61P 3/00 20180101; C07B 2200/13 20130101; A61P 17/00
20180101; A61P 19/02 20180101; A61P 29/00 20180101; A61P 35/00
20180101; A61P 43/00 20180101; A61P 25/00 20180101; A61P 9/12
20180101; A61K 31/56 20130101; A61P 9/10 20180101; A61P 9/00
20180101; A61P 27/02 20180101; A61K 31/277 20130101; A61P 13/12
20180101 |
International
Class: |
C07C 255/47 20060101
C07C255/47; A61K 31/277 20060101 A61K031/277; C07C 253/34 20060101
C07C253/34; C07J 63/00 20060101 C07J063/00; A61K 31/56 20060101
A61K031/56 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 2012 |
CN |
PCT/CN2012/082278 |
Claims
1-44. (canceled)
45. A method of treating a physical disorder or condition
associated with inflammation in a subject, comprising administering
to the subject a polymorph of a compound of Formula I: ##STR00009##
wherein the polymorph is a glassy solid form which has a Tg of
about 52.degree. C..+-.10.degree. C.
46. The method of claim 45, where the polymorph is a crystalline
Form I, wherein the crystalline Form I has an X-ray powder
diffraction pattern comprising characteristic peaks at diffraction
angles 2.theta. of approximately 10.3.degree., 14.1.degree. and
14.6.
47. The method of claim 46, wherein the X-ray powder diffraction
pattern comprises characteristic peaks at diffraction angles
2.theta. of approximately 10.3.degree., 14.1.degree., 14.6.degree.,
15.7.degree., and 16.6.degree..
48. The method of claim 47, wherein the X-ray powder diffraction
pattern comprises additional characteristic peaks at diffraction
angles 2.theta. of approximately 9.3.degree. and 19.6.degree..
49. The method of claim 45, wherein the X-ray powder diffraction
pattern comprises characteristic peaks at diffraction angles
2.theta. of approximately 10.3.degree., 14.1.degree., 14.6.degree.,
15.8.degree., 16.6.degree. and 19.6.degree..
50. The method of claim 46, wherein the crystalline Form I has the
X-ray powder diffraction pattern comprising characteristic peaks,
expressed in terms of the interplanar distance, at 8.6 .ANG., 6.3
.ANG. and 6.1 .ANG..
51. The method of claim 50, wherein the X-ray powder diffraction
pattern comprises characteristic peaks, expressed in terms of the
interplanar distance, at 8.6 .ANG., 6.3 .ANG., 6.1 .ANG., 5.6
.ANG., 5.3 .ANG. and 4.5 .ANG..
52. The method of claim 45, wherein the polymorph has a melting
point of 174.degree. C.-177.degree. C.
53. The method of claim 45, wherein the polymorph is of crystalline
Form II, which has an X-ray powder diffraction pattern comprising
characteristic peaks at diffraction angles 2.theta. of
approximately 10.4.degree., 12.1.degree. and 13.4.degree..
54. The method of claim 53, wherein the X-ray powder diffraction
pattern comprises additional characteristic peaks at diffraction
angles 2.theta. of approximately 15.4.degree., 17.8.degree. and
18.8.degree..
55. The method of claim 53, wherein the X-ray powder diffraction
pattern comprises characteristic peaks, when expressed in terms of
the interplanar distance, at 8.5 .ANG., 7.3 .ANG. and 6.6
.ANG..
56. The method of claim 55, wherein the X-ray powder diffraction
pattern comprises additional characteristic peaks, when expressed
in terms of the interplanar distance, at 5.7 .ANG., 5.0 .ANG. and
4.7 .ANG..
57. The polymorph of claim 53, wherein the polymorph has a melting
point of 209.degree. C.-212.degree. C.
58. The method of claim 45, wherein the polymorph has a purity of
.gtoreq.85%.
59. The method of claim 45, wherein the physical disorder or
condition associated with inflammation comprises lupus or
rheumatoid arthritis, Crohn's disease or ulcerative colitis, a
cardiovascular disease, diabetes, or a condition associated with
diabetes.
60. The method of claim 45, wherein the physical disorder or
condition associated with inflammation is cancer.
61. The method of claim 58, wherein the condition associated with
diabetes comprises obesity, hypertension, atherosclerosis, coronary
heart disease, stroke, peripheral vascular disease, hypertension,
nephropathy, neuropathy, myonecrosis, retinopathy, metabolic
syndrome (syndrome X), or a combination thereof.
62. The method of claim 45, wherein the physical disorder or
condition associated with inflammation is caused by nitric oxide
induced by IFN-.gamma. in a macrophage.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/007,925, filed on Sep. 26, 2013, which is a
national phase application and claims the benefit, under 35 U.S.C.
.sctn.371, of PCT/CN2012/086045, filed on Dec. 6, 2012, which in
turn claims priority to PCT international application No.
PCT/CN/2012/082278, filed on 28 Sep. 2012, the contents of which
are incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to polymorphic forms of a
triterpenoid compound, ethyl
2-cyano-3,12-dioxoleana-1,9(11)-dien-28-oate (CDDO ethyl ester),
and methods of using at least one of them in treating a variety of
disease states, generally associated with inflammation.
BACKGROUND OF THE INVENTION
[0003] Triterpenoids are biosynthesized in plants by the
cyclization of squalene. Although being candidates for medicinal
use, these naturally occurring molecules display relatively weak
biological activity. Accordingly, chemists have sought to
synthesize analogues of enhanced potency (Honda et al, 1997 &
1998).
[0004] Several synthetic analogs are reported to suppress the
denovo formation of cytokine-inducible nitric oxide synthase (iNOS)
and cyclooxygenase-2 (COX-2) in macrophages that have been
stimulated by IFN-.gamma. or LPS (Suh et al, 1998; Honda et al.,
2002). Among them, 2-cyano-3,12-dioxoleana-1,9(11)-dien-28-oate
(CDDO), exhibits anti-inflammatory and anti-proliferative activity
(Honda et al., 1998 & 2000). As noted, the study of
triterpenoids as suppressors of iNOS activity, and specifically in
the inhibition of NO production, has demonstrated the high potency
of CDDO and CDDO methyl ester (IC.sub.50<1 nM level). See Honda
et al. (2000). However, their therapeutic potential is believed to
be not yet fully utilized, even less so for CDDO ethyl ester.
[0005] To realize the therapeutic potential of CDDO ethyl ester,
depicted in Formula I, the present inventors investigated
polymorphic forms of the compound. Consequently, the inventors
discovered three polymorphic forms of CDDO ethyl ester, that have
such advantageous properties (e.g., better pharmacokinetic profiles
and greater systemic exposure) that make them ideal candidates for
drug development.
SUMMARY OF THE INVENTION
[0006] In one aspect, the present invention provides polymorphs of
the compound of Formula I, and/or hydrates or solvates thereof
##STR00002##
[0007] Among others, these polymorphs include at least two
substantially pure crystalline forms and one substantially pure
glassy solid form of the compound of Formula I. For convenience,
these two crystalline forms are designated herein as Form I and
Form II.
[0008] In some embodiments, the polymorph is a glassy solid form of
the compound of Formula I and characterized having a Tg about
52.degree. C..+-.10.degree. C.
[0009] In some other embodiments, the polymorph is a glassy solid
form of the compound of Formula I and characterized by an X-ray
powder diffraction pattern having a characteristic peak at
diffraction angles 2.theta. of approximately 14.3.degree..
[0010] In some other embodiments, a glassy solid form of the
compound of Formula I is characterized by a purity of .gtoreq.85%
(i.e., at least 85%). In yet still some embodiments, the purity is
at least 95% or 99%.
[0011] In still some other embodiments, the polymorph is a
crystalline polymorph form I of the compound of Formula I and
characterized by an X-ray powder diffraction pattern having
characteristic peaks at diffraction angles 2.theta. of
approximately 10.3.degree., 14.1.degree. and 14.6.degree..
[0012] In still some other embodiments, the polymorph is a
crystalline polymorph form I of the compound of Formula I and
characterized by the X-ray powder diffraction pattern having
characteristic peaks at diffraction angles 2.theta. of
approximately 10.3.degree., 14.1.degree., 14.6.degree.,
15.8.degree., 16.6.degree. and 19.6.degree..
[0013] In still some other embodiments, the polymorph is a
crystalline polymorph form I of the compound of Formula I and
characterized by the X-ray powder diffraction pattern having
characteristic peaks, expressed in terms of the interplanar
distance, at 8.6 .ANG., 6.3 .ANG. and 6.1 .ANG..
[0014] In still some other embodiments, the polymorph is a
crystalline polymorph form I of the compound of Formula I and
characterized by the X-ray powder diffraction pattern having
characteristic peaks, expressed in terms of the interplanar
distance, at 8.6 .ANG., 6.3 .ANG., 6.1 .ANG., 5.6 .ANG., 5.3 .ANG.
and 4.5 .ANG..
[0015] Another subset of the embodiments of the polymorphs of this
invention are of crystalline Form I.
[0016] In some of these embodiments, crystalline Form I of the
compound of Formula I is characterized by an X-ray powder
diffraction pattern having characteristic peaks at diffraction
angles 2.theta. of approximately 10.3.degree., 14.1.degree.,
14.6.degree., 15.7.degree., and 16.6.degree..
[0017] In some other embodiments, crystalline Form I of the
compound of Formula I has additional characteristic peaks at
diffraction angles 2.theta. of approximately 9.3.degree. and
19.6.degree., in its X-ray powder diffraction pattern.
[0018] In still some other embodiments, crystalline Form I of the
compound of Formula I is characterized by a melting point of
174-177.degree. C.
[0019] In some other embodiments, crystalline Form I of the
compound of Formula I is characterized by a purity of .gtoreq.85%
(i.e., at least 85%). In yet still some embodiments, the purity is
at least 95% or 99%.
[0020] Still another subset of the embodiments of the polymorphs of
this invention are of crystalline Form II.
[0021] In some of these embodiments, crystalline Form II of the
compound of Formula I is characterized by an X-ray powder
diffraction pattern having characteristic peaks at diffraction
angles 2.theta. of approximately 10.4.degree., 12.1.degree. and
13.4.degree..
[0022] In some other embodiments, crystalline Form II of the
compound of Formula I has additional characteristic peaks at
diffraction angles 2.theta. of approximately 15.4.degree.,
17.8.degree. and 18.8.degree., in its X-ray powder diffraction
pattern.
[0023] In some other embodiments, crystalline Form II of the
compound of Formula I is characterized by the X-ray powder
diffraction pattern having characteristic peak, expressed in terms
of the interplanar distance, at 8.5 .ANG., 7.3 .ANG. and 6.6
.ANG..
[0024] In some other embodiments, crystalline Form II of the
compound of Formula I has additional characteristic peaks,
expressed in terms of the interplanar distance, at 5.7 .ANG., 5.0
.ANG. and 4.7 .ANG..
[0025] In some other embodiments, crystalline Form II of the
compound of Formula I is characterized by a melting point of
209-212.degree. C.
[0026] In some other embodiments, crystalline Form II of the
compound of Formula I has a purity of not less than 85% (e.g., at
least 95% or at least 99%).
[0027] In another aspect, the invention also relates to methods for
making crystalline polymorphs of the compound of Formula I.
[0028] An embodiment of the method includes the steps of slurrying
excess amount of the compound in a solvent of CH.sub.2Cl.sub.2,
ethyl acetate, acetonitrile, ethanol, methanol, heptane, or a
mixture thereof, for at least 24 hours, and recovering the resulted
crystalline polymorph or a glassy solid form.
[0029] In some examples, the resultant crystalline forms can be
either Form I or Form II.
[0030] In some other examples, the compound of Formula I is
slurried in the mixed solvant at the room temperature or at
50.degree. C.
[0031] In some other examples, the compound of Formula I is
slurried in the solvent for at least 48 hours.
[0032] The solvent can be an ethyl acetate/heptane mixture, a
ethanol/heptane mixture. For example, the ethyl acetate/heptane
mixture can have ethyl acetate and heptane in the ratio of 1:10
(weight by weight or volum by volum).
[0033] To give a polymorph of this invention, crystallizing the
compound of Formula I from a suitable solvent system comprising at
least one solvent can be achieved by methods of spontaneous
precipitation (evaporation), cooling, or adding anti-solvent (in
which the compound of the present invention has relatively lower
solubility), in order to achieve oversaturation in a solvent
system. Crystallization also can be achieved by using or not using
crystal seeds that is suitable for crystallizing the compound of
the present invention.
[0034] In some other examples, the compound of Formula I of this
invention is dissolved in the solvent heptane at the room
temperature, followed by a spontaneous precipitation to obtain the
desired crystalline Form I, with the melting point of
174-177.degree. C.
[0035] In some other examples, a slurry suspension of excess amount
of the compound of Formula I of this invention is stirred in the
mixed solvent of ethyl acetat/heptane (1:10 weight by weight or
volum by volum) at the room temperature or 50.degree. C. for at
least 48 hours, to obtain crystalline Form II, with the melting
point of 209-212.degree. C.
[0036] In some other examples, a slurry suspension of excess amount
of the compound of Formula I of this invention is dissolved in the
solvent of dichloromethane at the room temperature or 50.degree.
C., followed by evaporation of solvent to give a glassy solid form
of the compound of Formula I with a Tg in a range of 52.degree.
C..+-.10.degree. C.
[0037] The present invention further provides uses of the
polymorphs of the compound of Formula I (including crystalline
Forms I and II and the glassy solid form) for treating physical
disorders or conditions, particularly those associated with
inflammation, or for manufacturing medicament for treating physical
disorders or conditions, e.g., those involving acute or chronic
oxidative stress and inflammation, particularly those characterized
in part by over expression of inducible nitric oxide synthase
(iNOS) or inducible cyclooxygenase-(COX-2).
[0038] Accordingly, the invention also relates to pharmaceutical
compositions or medicaments each comprising at least a
therapeutically effective amount of crystalline Form I or Form II
or the glassy solid form of the present invention, and a
pharmaceutically acceptable excipient, adjuvant or carrier.
Optionally, the compositions or medicament can further include at
least one additional active ingredient.
[0039] The pharmaceutical compositions or medicaments of this
invention can be in a form or formulation appropriate for the
condition or disease to be treated (e.g., in a tablet or capsule),
and can be administered in the manner known in the art, e.g.,
orally.
[0040] The pharmaceutical compositions or medicaments of this
invention can include 1-99 wt % (e.g., 1-70 wt %, 10-30 wt %,) of a
polymorph of the compound of Formula I (e.g., crystalline Form I,
crystalline Form II, or glassy solid form).
[0041] All the polymorphs of this invention (including the
crystalline forms and the glassy solid form) are substantially or
approximately pure. As used herein, the term "substantially pure"
or "approximately pure" refers to at least 85 wt % (e.g., at least
95 wt % or at least 99 wt %) of the compound of Formula I exists in
a polymorph the present invention, particularly in crystalline Form
I or Form II.
[0042] The main peaks described in the crystalline and the glassy
solid form polymorphs above are reproducible and are within the
error limit (the specified value.+-.0.2).
[0043] In the present invention, "the X-ray powder diffraction
pattern shown as in FIG. 1" refers to the X-ray powder diffraction
pattern that show major peaks as in FIG. 1, wherein major peaks
refer to those with the relative intensity greater than 10%,
preferably greater than 30%, relative to the highest peak (with its
relative intensity designated to be 100%) in FIG. 1.
[0044] Likewise, in the present invention, the X-ray powder
diffraction pattern shown as in FIG. 2 refers to the X-ray powder
diffraction pattern that show major peaks as in FIG. 2, wherein
major peaks refer to those with the relative intensity greater than
10%, preferably greater than 30%, relative to the highest peak
(with its relative intensity designated to be 100%) in FIG. 2,
respectively.
[0045] Also within the scope of this invention are methods for
preparing the compound of Formula I each of which includes steps
shown in the following scheme:
##STR00003## ##STR00004## ##STR00005##
[0046] The present invention also provides the use of the compound
of Formula I, or a polymorph or form selected from Crystalline Form
I, Crystalline Form II, the glassy solid form, thereof, in
manufacturing a medicament for inhibiting IFN-.gamma.-induced NO
production in macrophages, or for the treatment of cancer, or for
the treatment of a disease, disorder, or condition with an
inflammatory component, such as lupus or rheumatoid arthritis,
Crohn's disease or ulcerative colitis, a cardiovascular disease,
diabetes, one or more complications associated with diabetes
wherein the complications are selected from the group consisting of
obesity, hypertension, atherosclerosis, coronary heart disease,
stroke, peripheral vascular disease, hypertension, nephropathy,
neuropathy, myonecrosis, retinopathy and metabolic syndrome
(syndrome X), or a combination of two or more thereof.
[0047] As used herein, the term "therapeutically effective amount"
refers to the amount of a compound that, when administered to a
subject for treating a disease, or at least one of the clinical
symptoms of a disease or disorder, is sufficient to affect such
treatment for the disease, disorder, or symptom. The
"therapeutically effective amount" can vary with the compound, the
disease, disorder, and/or symptoms of the disease or disorder,
severity of the disease, disorder, and/or symptoms of the disease
or disorder, the age of the subject to be treated, and/or the
weight of the subject to be treated. An appropriate amount in any
given instance can be apparent to those skilled in the art or can
be determined by routine experiments. In the case of combination
therapy (i.e., having at least another active ingredient in
addition to a polymorph of this invention), the term
"therapeutically effective amount" refers to the total amount of
the combination objects for the effective treatment of a disease, a
disorder or a condition.
[0048] The pharmaceutical composition comprising the compound of
the present invention can be administrated via oral, inhalation,
rectal, parenteral or topical administration to a subject who needs
treatment. For oral administration, the pharmaceutical composition
may be a regular solid formulation such as tablets, powder,
granule, capsules and the like, a liquid formulation such as water
or oil suspension or other liquid formulation such as syrup,
solution, suspension or the like; for parenteral administration,
the pharmaceutical composition may be solution, water solution, oil
suspension concentrate, lyophilized powder or the like. Preferably,
the formulation of the pharmaceutical composition is selected from
tablet, coated tablet, capsule, suppository, nasal spray or
injection, more preferably tablet or capsule. The pharmaceutical
composition can be a single unit administration with an accurate
dosage. In addition, the pharmaceutical composition may further
comprise additional active ingredients.
[0049] All formulations of the pharmaceutical composition of the
present invention can be produced by the conventional methods in
the pharmaceutical field. For example, the active ingredient can be
mixed with one or more excipients, then to make the desired
formulation. The "pharmaceutically acceptable carrier" refers to
conventional pharmaceutical carriers suitable for the desired
pharmaceutical formulation, for example: a diluent, a vehicle such
as water, various organic solvents, etc, a filler such as starch,
sucrose, etc; a binder such as cellulose derivatives, alginates,
gelatin and polyvinylpyrrolidone (PVP); a wetting agent such as
glycerol; a disintegrating agent such as agar, calcium carbonate
and sodium bicarbonate; an absorption enhancer such as quaternary
ammonium compound; a surfactant such as hexadecanol; an absorption
carrier such as Kaolin and soap clay; a lubricant such as talc,
calcium stearate, magnesium stearate, polyethylene glycol, etc. In
addition, the pharmaceutical composition further comprises other
pharmaceutically acceptable excipients such as a decentralized
agent, a stabilizer, a thickener, a complexing agent, a buffering
agent, a permeation enhancer, a polymer, aromatics, a sweetener,
and a dye. Preferably, the excipient is suitable for desired
formulation and administration type.
[0050] As used herein, the term "solvate" refers to the chemical
entity formed by the interaction of a solvent and a compound.
Suitable solvates are pharmaceutically acceptable solvates, such as
hydrates, including monohydrates and hemi-hydrates.
[0051] As used herein, the term "active ingredient" is used to
indicate a chemical entity which has biological activity. In
certain embodiments, an "active agent" is a compound having
pharmaceutical utility. For example, an active agent may be an
anti-cancer therapeutic.
[0052] The term "disease" or "disorder" or "condition" refers to
any disease, discomfort, illness, symptoms or indications.
BRIEF DESCRIPTION OF THE FIGURES
[0053] FIG. 1 shows the X-ray powder diffraction pattern of
Crystalline Form I of the compound of Formula I
[0054] FIG. 2 shows the X-ray powder diffraction pattern of
Crystalline Form II of the compound of Formula I
[0055] FIG. 3 shows the X-ray powder diffraction pattern of the
glassy solid form of the compound of Formula I.
[0056] FIG. 4 shows the Heat Flow (wig) of a glassy solid form.
[0057] FIG. 5 shows the inhibitory effect of Crystalline Form II of
the compound of Formula I (CDDO ethyl ester) against the nitric
oxide production induced by IFN-g in mouse macrophages 17.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The X-ray powder diffraction (XRPD) patterns shown as in
FIG. 1, 2, 3 were generated on a PANalytical X-ray Diffraction
System with Empyrean console. The diffraction peak positions were
calibrated by single crystal silicon which has a 2.theta. value of
28.443 degree. The K-Alpha radiation of an Empyrean Cu LEF X-ray
tube was used as the light source of the X-ray.
[0059] The present invention is further exemplified, but not
limited, by the following examples that illustrate the invention.
In the examples of the present invention, the techniques or
methods, unless expressly stated otherwise, are conventional
techniques or methods known in the art.
Example 1. Synthesis of the Compound of Formula I
##STR00006## ##STR00007## ##STR00008##
[0060] Step 1 Synthesis of APSN13B-1
TABLE-US-00001 [0061] Name M.W. Amount mol equiv Oleanolic acid 456
1000 g 2.2 mol 1 EtI 156 376 g 2.4 mol 1.1 K.sub.2CO.sub.3 138 604
g 4.4 mol 2 DMF / 12 L / /
[0062] To a solution of oleanolic acid (1000 g, 2.2 mol) and
potassium carbonate (604 g, 4.4 mol) in DMF (12 L) was added ethyl
iodide (376 g, 2.4 mol). The mixture was stirred at 45.degree. C.
overnight. After disappearance of oleanolic acid detected by HPLC,
the mixture was cooled to the room temperature and was poured into
water (120 L). The resulting suspension was stirred for 30 minutes.
The solid was collected by centrifuge, washed with water (1 L) and
dried in vacuo at 50.degree. C. to afford 976 g APSN13B-1 for late
ruse. The yield was 92%.
Step 2. Synthesis of APSN13B-2
TABLE-US-00002 [0063] Name M.W. Amount mol equiv APSN13B-1 484 975
g 2 mol 1 Ac.sub.2O 102 612 g 6 mol 3 Pyridine 79 474 g 6 mol 3 THF
/ 6 L / / DMAP 122 24.4 g 0.2 mol.sup. 0.1
[0064] To a mixture of APSN13B-1 (975 g, 2 mol), pyridine (474 g),
DMAP (24.4 g, 0.2 mol) in THF (6 L) was added acetic anhydride (612
g, 6 mol) at 45.degree. C. The solution was stirred overnight.
After the reaction finished, the solution was poured into water (60
L). The solid was collected by centrifuge and dried in vacuo at
45.degree. C. to give 847 g APSN13B-2 for later use. The yield was
80%.
Step 3. Synthesis of APSN13B-3
TABLE-US-00003 [0065] Name M.W. Amount mol equiv APSN13B-2 526 846
g 1.6 mol 1 H.sub.2O.sub.2 34 136 g .sup. 4 mol 2.5 HCOOH 46 1 L /
/ DCM / 5 L / /
[0066] To a solution of APSN13B-2 (846 g, 1.6 mol), formic acid (1
L) in DCM (5 L) was added hydrogen peroxide (30%, water solution)
(453 g, 4 mol) slowly at room temperature and stirred overnight.
The solution was added water (2 L) and extracted with DCM. The
organic phase was washed with saturated aqueous NaHCO.sub.3 until
the water phase was neutral and then washed with brine and dried
over anhydrous NaSO.sub.4. The organic phase was filtered and the
filtrate was evaporated in vacuo to give 871 g APSN13B-3 for a
yield of 100%. This material was used for the next reaction without
further purification.
Step 4. Synthesis of APSN13B-4
TABLE-US-00004 [0067] Name M.W. Amount Mol equiv APSN13B-3 542 871
g 1.6 mol 1 HBr 81 30 ml / / Br.sub.2 160 768 g 4.8 mol 3 HAc / 7 L
/ /
[0068] To a solution of APSN13B-3 (871 g, 1.6 mol) in acetic acid
(5 L) was added a solution of hydrobromic acid (30 ml) in acetic
acid (40%) at 45.degree. C. and then was added slowly a solution of
bromine (256 g) in acetic acid (700 ml). The mixture was stirred at
45.degree. C. for 30 minutes. Another solution of bromine (512 g)
in acetic acid (1.3 L) was added slowly and continued to stir
overnight at room temperature. When the reaction finished, the
mixture was poured into cold water (35 L). The solid was collected,
washed with saturated sodium sulfite solution and dried in vacuo to
give the yellow solid 841 g APSN13B-4, for a yield of 97%.
[0069] MS-ESI (m/z): 541 [M+H].sup.+
Step 5. Synthesis of APSN13B-5
TABLE-US-00005 [0070] Name M.W. Amount mol equiv APSN13B-4 540 840
g 1.56 mol 1 KOH 56 263 g 4.7 mol 3 EtOH / 4.5 L / /
[0071] A solution of APSN13B-4 (840 g, 1.56 mol) and KOH (263 g,
4.7 mol) in EtOH (4.5 L) was heated under reflux for 30 minutes.
After removal of EtOH in vacuo, the resultant mixture was acidified
with a 6 N aqueous HCl solution. The aqueous layer was extracted
with ethyl acetate (3 L). The organic phase was washed with
saturated aqueous NaHCO.sub.3, brine and dried over anhydrous
NaSO.sub.4. The organic phase was filtered and the filtrate was
evaporated in vacuo to give 928 g APSN13B-5 for a yield of
100%.
[0072] MS-ESI (m/z): 499 [M+H].sup.+.
Step 6. Synthesis of APSN13B-6
TABLE-US-00006 [0073] Name M.W. Amount mol equiv APSN13B-5 498 927
g 1.8 mol 1 Jones reagent 100 400 ml 1.8 mol 1 acetone / 5 L /
/
[0074] To a solution of APSN13B-5 (927 g, 1.8 mol) in acetone (5 L)
in an ice bath was added Jones reagent (400 ml) dropwise. The
mixture was stirred at room temperature until disappearance of
APSN13B-5 detected by TLC. After removal of acetone, water was
added to the resultant mixture. The aqueous mixture was extracted
with DCM. The organic phase was washed with saturated aqueous
NaHCO.sub.3, brine, dried over anhydrous NaSO.sub.4 and filtered.
The filtrate was evaporated in vacuo to give crude APSN13B-6. 498 g
pure APSN13B-6 was obtained through recrystallization in petroleum
and ethyl acetate, for a yield of 54%.
[0075] MS-ESI (m/z): 497 [M+H].sup.+
Step 7. Synthesis of APSN13B-7
TABLE-US-00007 [0076] Name M.W. Amount mol equiv APSN13B-6 496 496
g 1 mol 1 CH.sub.3ONa 54 216 g 4 mol 4 HCOOEt 74 185 g 2.5 mol.sup.
2.5 Dry toluene / 2 L / /
[0077] To a solution of APSN13B-6 (496 g, 1 mol) in dry toluene (2
L) was added ethyl formate (185 g, 2.5 mol) and CH.sub.3ONa (216 g,
4 mol). The mixture was stirred at room temperature for 2 hours.
Then the mixture was diluted with ethyl acetate (1 L) and washed
with 5% aqueous HCl solution (three times). The water phase was
re-extracted with ethyl acetate and the combined organic layers was
washed with brine, dried over anhydrous NaSO.sub.4 and filtered.
The filtrate was evaporated in vacuo to give 497 g APSN13B-7 for a
yield of 95%.
[0078] MS-ESI (m/z): 525 [M+H].sup.+
Step 8. Synthesis of APSN13B-8
TABLE-US-00008 [0079] Name M.W. Amount mol equiv APSN13B-7 524 496
g 0.94 mol 1 NH.sub.2OH HCl 69 98 g 1.4 mol 1.5 EtOH / 2.5 L / /
water / 200 ml / /
[0080] To a solution of APSN13B-7 (496 g, 0.94 mol) in EtOH (2.5 L)
and water (200 ml) was added hydroxylamine hydrochloride (98 g, 1.4
mol). The mixture was heated under reflux for 2 hours. The mixture
was concentrated in vacuo and water (2 L) was added. The mixture
was extracted with EA (three times). The combined organic layers
were washed water and brine, dried over anhydrous NaSO.sub.4 and
filtered. The filtrate was evaporated in vacuo to give 453 g
APSN13B-8 for a yield of 92%.
[0081] MS-ESI (m/z): 522 [M+H].sup.+
Step 9. Synthesis of APSN13B-9
TABLE-US-00009 [0082] Name M.W. Amount mol equiv APSN13B-8 521 452
g 0.86 mol 1 CH.sub.3ONa 54 56 g 1.04 mol 1.2 EtOH / 1.5 L / /
[0083] To a solution of APSN13B-8 (452 g, 0.86 mol) in EtOH (1.5 L)
in ice bath was added CH.sub.3ONa (56 g, 1.04 mol). The mixture was
stirred at room temperature for 1 hour. The mixture was
concentrated in vacuo and ethyl acetate (2 L) was added. The
mixture was washed with 5% aqueous HCl solution (three times). The
water phase was re-extracted with EA and the combined organic
layers was washed with brine, dried over anhydrous NaSO.sub.4 and
filtered. The filtrate was evaporated in vacuo to give 429 g
APSN13B-9 for a yield of 95%.
[0084] MS-ESI (m/z): 522 [M+H].sup.+
Step 10. Synthesis of APSN13B-10 (Compound of Formula I)
TABLE-US-00010 [0085] Name M.W. Amount mol equiv APSN13B-9 521 428
g 0.82 mol 1 DDQ 227 205 g 0.90 mol 1.1 tolunene / 2.5 L / /
[0086] A mixture of APSN13B-9 (428 g, 0.82 mol) and DDQ (205 g,
0.90 mol) in toluene (2.5 L) was heated under reflux overnight.
After insoluble was removed by filtration, the filtrate was
evaporated in vacuo to give a crude solid. The solid was subjected
to flash column chromatography to give 341 g APSN13B-10 for a yield
is 80%. The product was further purified through re-crystallization
in ethyl acetate and petroleum ether to give chemical purity
product. The maximum impurity of final product is less than
0.1%.
[0087] MS-ESI (m/z): 520 [M+H].sup.+
[0088] .sup.1H-NMR (CDCl.sub.3): 68.04 (1H, S), 5.96 (1H, S), 4.17
(2H, t, J=6.9 Hz), 3.05 (1H, d, J=13.5 Hz), 2.95 (1H, d, J=4.5 Hz),
2.04-0.07 (21H, m).
Example 2. Preparation of Crystalline Form I of the Compound of
Formula I
[0089] The compound of Formula I prepared as described above in
Example 1 was dissolved in the solvent heptane at the room
temperature, followed by a spontaneous precipitation to obtain the
desired crystalline Form I, with the melting point of
174-177.degree. C. Its X-ray diffraction pattern depicted in FIG. 1
is summarized in Table 1.
TABLE-US-00011 TABLE 1 2.theta. (2 theta) .+-. 0.2 (degrees)
d-spacing [.ANG.] Rel. Int. [%] 10.3 8.6 100 14.1 6.3 83.2 14.6 6.1
63.8 15.8 5.6 44.6 16.6 5.3 49.7 19.6 4.5 35.8
Example 3. Preparation of Crystalline Form II of the Compound of
Formula I
[0090] 1. A slurry suspension of excess amount of the compound of
Formula I prepared from the method described in Example 1 above,
was stirred in the mixed solvent of ethyl acetat/heptane (1:10
weight by weight or volum by volum) at the room temperature or
50.degree. C. for at least 48 hours, to obtain crystalline Form II,
with the melting point of 209-212.degree. C.
[0091] Its X-ray diffraction pattern depicted in FIG. 2 is
summarized in Table 2.
TABLE-US-00012 TABLE 2 2.theta. (2 theta) .+-. 0.2 (degrees)
d-spacing [.ANG.] Rel. Int. 1%1 10.4 8.5 16.1 12.1 7.3 25.7 13.4
6.6 100 15.4 5.7 30.9 17.84 5.0 25.5 18.8 4.7 42.4
Example 4. Preparation of a Glassy Solid Form of the Compound of
Formula I
[0092] A slurry suspension of excess amount of the compound of
Formula I prepared from the method described in Example 1 above,
was dissolved in the solvent of dichloromethane at the room
temperature or 50.degree. C., followed by evaporation of solvent to
give a glassy solid form of the compound of Formula I with a Tg
about 52.degree. C..+-.10.degree. C. FIG. 3 shows the X-ray powder
diffraction pattern of the glassy solid form of the compound of
Formula I. FIG. 4 shows the Heat Flow (w/g) of a glassy solid
form.
Example 5. Preliminary Pharmacokinetics of Polymorphs of the
Compound of Formula I
[0093] Female CD-1 mice were injected ip with 10 mg/kg of the three
polymorphs of the compound of Formula I (CDDO ethyl) and methyl
2-cyano-3,12-dioxoleana-1,9(11)-dien-28-oate (CDDO methyl) in
DMSO-cremphor-PBS (1:1:8 weight by weight or volum by volum). Blood
samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hr
post-dose. The levels were quantified by HPLC/MS using compound add
to control blood for standard. PK results are shown at Table 3. The
systemic exposure of the three polymorphs of the compound of
Formula I of CDDO ethyl is higher than that of CDD) methyl.
Following is the order: The glassy solid form of CDDO
ethyl>Crystalline Form II of CDDO ethyl>Crystalline Form I of
CDDO ethyl>CDDO Methyl
TABLE-US-00013 TABLE 3 t.sub.1/2 t.sub.max C.sub.max AUC.sub.last
AUC.sub.Inf Compound (hr) (hr) (ng/mL) (hr*ng/mL) (hr*ng/mL) CDDO
Methyl 6.6 0.8 363 1805 1966 Ester The glassy solid 16.1 0.8 513
4721 7526 form of CDDO ethyl Crystalline Form I 8.3 0.8 466 3221
3456 of CDDO ethyl Crystalline Form II 7.3 0.8 482 3443 3840 of
CDDO ethyl
Example 6. Stability Determination of Crystal Forms
[0094] Crystalline Form II of the compound of Formula I, prepared
as described in Example 3, was test at 25.degree. C./60% R.H. and
40.degree. C./75% R.H. There was no significant change of XRD of
all the samples. Other polymorphs of this invention were used
similar in the study. Compared to other polymorphs of this
invention, crystalline Form II of the compound of Formula I was
demonstrated to be the most thermodynamically stable crystal form
in this study.
Example 7. Stability Determination of a Glassy Solid Form
[0095] The glassy solid Form of the compound of Formula I, prepared
as described in Example 4, was test at 25.degree. C./60% R.H. and
40.degree. C./75% R.H for one week. There was no significant change
of XRD of all the samples.
Example 8. Measurement of NO in RAW 264.7 Cells Treated with
Crystalline Form II of the Compound of Formula I
Assay Protocol Procedures
RAW264.7 Cell Culture
[0096] 1 Maintained RAW264.7 cells in DMEM with 10% FBS at
37.degree. C., 5% CO.sub.2.
[0097] 2. Viewed cultures using a microscope to assess the degree
of confluency and confirm the absence of bacterial and fungal
contaminants.
[0098] 3. Removed spent medium.
[0099] 4. Washed the cell monolayer with pre-warmed D-PBS.
[0100] 5. With a pipette, moved 3 ml trypsin/EDTA onto the washed
cell monolayer, rotated flask to cover the monolayer with
trypsin.
[0101] 6. Examined the cells using a microscope to ensure that all
the cells are detached and floating.
[0102] 7. Re-suspended the cells in a small volume of fresh
serum-containing medium to inactivate the trypsin.
[0103] 8. Harvested cells by centrifugation, 1000 rpm for 5 minutes
at the room temperature.
[0104] 9. Incubated the cells at 37.degree. C. under 5%
CO.sub.2.
Induction of NO in RAW264.7 Cells
[0105] 1. The cells were seeded at 1.times.10.sup.5/well onto
96-well cell culture plate.
[0106] 2. After incubation for 2 hours at 37.degree. C. under 5%
CO.sub.2, the nonadherent cells were removed by aspiration and
freshly prepared complete medium with 10 ng/ml IFN-.gamma. and
diluted compounds was added.
[0107] 3. Incubated the cells at 37.degree. C. under 5% CO.sub.2
for another 48 hours.
Measurement of NO in RAW Cells
[0108] 1. Centrifuged at 1000 rpm for 5 minutes to collect
supernatant medium.
[0109] 2. Prepared 1 ml of a 100 .mu.M nitrite solution by diluting
the provided 0.1 M Nitrite Standard 1:1,000 in the matrix or buffer
used for the experimental samples.
[0110] 3. Prepared Nitrite Standard 100, 50, 25, 12.5, 6.25, 3.13,
1.56 and 0 .mu.M, 50 .mu.l/well.
[0111] 4. Allowed the Sulfanilamide Solution and NED Solution to
equilibrate to room temperature (15-30 minutes).
[0112] 5. Added 50 .mu.l of each experimental sample to wells in
duplicate.
[0113] 6. Used a multichannel pipettor to dispense 50 .mu.l of the
Sulfanilamide Solution to all experimental samples and wells
containing the dilution series for the Nitrite Standard.
[0114] 7. Incubated 5-10 minutes at room temperature, protected
from light.
[0115] 8. Used a multichannel pipettor to dispense 50 .mu.l of the
NED Solution to all wells.
[0116] 9. Incubated at the room temperature for 5-10 minutes,
protected from light. A purple/magenta color began to form
immediately.
[0117] 10. Measured absorbance within 30 minutes in a plate reader
with a filter between 520 nm and 550 nm.
[0118] The inhibitory activities [as measured by the IC.sub.50 (nM)
value] of Crystalline Form II of the compound of Formula I (CDDO
ethyl ester) on NO production induced by IFN-g in mouse
macrophages17 are shown in FIG. 5. The IC.sub.50 of the Crystalline
Form II of the compound of Formula I (CDDO ethyl ester) was 34 nM.
The IC.sub.50 of the glassy solid form of the compound of Formula I
(CDDO ethyl ester) was 32 nM.
* * * * *