Title of Invention

PROCESS FOR PREPARING R-GOSSYPOL L-PHENYLALANINOL DIENAMINE

Abstract This invention relates to a process for preparing R-gossypol L-phenylalaninol dienamine using non-chromatographic purification methods. In particular, the invention is directed to resolution of R- and S-gossypol L-phenylalaninol dienamine by crys- tallization. R-gossypol L-phenylalaninol dienamine is a useful intermediate for the preparation of R-(-)-gossypol and R-(-)-gossypol acetic acid co-crystal. R-(-)-Gossypol acetic acid and its co- crystal is useful for inducing apoptosis in cells and for sensitizing cells to the induction of apoptotic cell death.
Full Text PROCESS FOR PREPARING R-GOSSYPOL L-PHENYLALANINOL
DEENAMINE
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This invention relates to a process for preparing R-gossypol
L-phenylalaninol dienamine using non-chromatographic purification methods.
In particular, the invention is directed to resolution of R- and S-gossypol
L-phenylalaninol dienamine by crystallization. R-gossypol L-phenylalaninol
dienamine is a useful intermediate for the preparation of R-(-)-gossypol and
R-(-)-gossypol acetic acid co-crystals. R-(->Gossypol acetic acid and its
co-crystals are useful for inducing apoptosis in cells and for sensitizing cells to
the induction of apoptotic cell death.
Background of the Invention
[0002] US 2005/0234135 Al discloses R-(-)-gossypol in the form of acetic
acid co-crystals and its use as an inhibitor anti-apoptotic Bcl-2 family proteins.
By inhibiting anti-apoptotic Bcl-2 family proteins, R-(-)-gossypol sensitizes
cells to inducers of apoptosis and, in some instances, itself induces apoptosis.
Because of the potential utility of R-(-)-gossypol and its acetic acid co-crystal
in the treatment of cancer and other diseases, there exists a need to develop a
practical synthetic route to produce substantial quantities of this drug
substance.
SUMMARY OF THE INVENTION
[0003] The invention confronts the problem of providing a process for
obtaining R-(-)-gossypol and its acetic acid co-crystal with high purity.
[0004] The invention is based on the unexpected discovery that R-gossypol L-
phenylalaninol dienamine can be isolated in high diastereomeric purity from a
a solvent system comprising acetonitrile and water. This obviates the need for
chromatographic resolution and is more amenable to large-scale production of
R-gossypol L-phenylalaninol diimine and, ultimately, R-(-)-gossypol and its
acetic acid co-crystal.
DETAILED DESCRIPTION OF THE INVENTION
[0005] The present invention relates to a process for preparing R-gossypol
L-phenylalaninol dienamine using non-chromatographic purification methods.
In particular, the invention is directed to resolution of R- and S-gossypol
L-phenylalaninol dienamine by crystallization in a solvent system comprising
acetonitrile and water.
[0006] Applicants have found that crystallization of a mixture of R- and
S-gossypol Schiff bases, and tautomers thereof, to provide the R-isomer
substantially free from the S-isomer was challenging and unpredictable. To
this end, an assortment of solvent systems were surveyed in an effort to
identify one useful for resolving R- and S-gossypol L-phenylalanine diimine.
These studies did not succeed in identifying conditions suitable to produce R-
gossypol L-phenylalanine diimine substantially free from S-gossypol L-
phenylalanine diimine. Solvent systems such as 1:2
dichloromethane:isopropyl alcohol, 1:1 dichloromethane:isopropyl alcohol,
1:2 dichloromethane:methanol, 1:5:1.5 dichloromethane:hexane:heptane, 2:1
methanolrhexane, 2:1 ethanokhexane, toluene, 1:1 toluene:heptane,
acetonitrile, 10:1 acetonitrile: water, tert-butyl methyl ether and tert-butyl
methyl ether:heptane led to modest diastereomeric enrichment in favor of
R-gossypol L-phenylalanine diimine. Solvent systems such as 1:2
dichl oromethane.ethanol and 2:1 methanol:water led to no diastereomeric
enrichment. Crystal formation was not observed in 2:1
dichloromethane:isopropyl alcohol and 2:1 water.tetrahydrofuran. Three
successive crystallizations in 1:2 dichloromethane:isopropanol did not
significantly improve the diastercomeric ratio. These data are presented in
Example 2.
[0007] Experiments aimed at resolving R- and S-gossypol L-phenylalaninol
dienaminc were also investigated. Crystallization attempts in solvent systems
such as methyl alcohol, isopropyl alcohol, acetone, toluene, acetonitrile, 1:1
toluenetheptane, 2:1 tert-butyl methyl etherheptane, 2:1 methanol:hexane, 1:1
dichloromethane:hexane, 5:1 tetrahydroftiran:water and 1:1 methanol:water
resulted in either no enrichment in diastereomeric purity or no crystal
formation. In contrast, crystallization in 5:1 acetonitrile.water resulted in a
striking improvement in diastereomeric purity favoring R-gossypol L-
phenylalaninol dienamine. This unexpected finding was refined to produce a
novel, reproducible process for the preparation R-gossypol L-phenylalaninol
dienamine. These data are presented in Examples 4 to 8.
(0008] In one embodiment, R-gossypol L-phenylalaninol dienamine is
prepared from a mixture of R- and S-gossypol L-phenylalaninol dienamine via
a process comprising crystallizing said mixture from a solvent system
comprising acetonitrile and water and isolating crystalline R-gossypol L-
phenylalaninol dienamine substantially free from said S-gossypol L-
phenylalaninol dienamine. In one embodiment, crystalline R-gossypol L-
phenylalaninol dienamine is isolated by filtration. In another embodiment,
crystalline R-gossypol L-phenylalaninol dienamine is isolated by
centrifugation.
[0009] In one embodiment, the solvent system used to crystallize R-gossypol
L-phenylalaninol dienamine substantially free from S-gossypol L-
phenylalaninol dienamine, comprises acetonitrile and water in a ratio of about
10:1 to about 5:2. In another embodiment, the solvent system used to
crystallize R-gossypol L-phenylalaninol dienamine substantially free from S-
gossypol L-phenylalaninol dienamine, comprises acetonitrile and water in a
ratio of about 5:1. In another embodiment, the solvent system used to
crystallize R-gossypol L-phenylalaninol dienamine substantially free from S-
gossypol L-phenylalaninol dienamine, comprises acetonitrile and water in a
ratio of about 5:1.5. In another embodiment, the solvent system used to
crystallize R-gossypol L-phenylalaninol dienamine substantially free from S-
gossypol L-phenylalaninol dienamine, comprises acetonitrile and water in a
ratio of about 5:2. In a further embodiment, the solvent system used to
crystallize R-gossypol L-phenylalaninol dienamine substantially free from S-
gossypol L-phenylalaninol dienamine comprises acetonitrile and water in a
ratio of 5:1.
[0010] In one embodiment, crystalline R-gossypol L-phenylalaninol
dienamine is recrystallized one or more times until the desired level of purity
is achieved. In a further embodiment, crystalline R-gossypol L-phenylalaninol
dienamine is recrystallized in a solvent system comprising acetonitrile and
water.
[0011] In one embodiment, isolated crystalline R-gossypol L-phenylalaninol
dienamine is washed one or more times with a solvent system comprising
acetonitrile and water. In one embodiment, isolated crystalline R-gossypol
L-phenylalaninol dienamine is washed with a solvent system comprising
acetonitrile and water in a ratio of about 10:1 to about 5:2. In a further
embodiment, isolated crystalline R-gossypol L-phenylalaninol dienamine is
washed with a solvent system comprising acetonitrile and water in a ratio of
about 5:1. In a further embodiment, isolated crystalline R-gossypol
L-phenylalaninol dienamine is washed with a solvent system comprising
acetonitrile and water in a ratio of 5:1. In a further embodiment, the
crystalline R-gossypol L-phenylalaninol dienamine is dried in vacuo.
[0012] In one embodiment, the crystalline R-gossypol L-phenylalaninol
dienamine has a purity of about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97% 98%, 99% or 99.5%.
[0013] In one embodiment, crystalline R-gossypol L-phenylalaninol
dienamine comprises about 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
1% or 0.5% S-gossypol L-phenylalaninol dienamine or less.
[0014] In one embodiment R-gossypol L-phenylalaninol dienamine is
prepared by a) condensing gossypol with L-phenylalaninol in acetonitrile to
provide a mixture of R- and S-gossypol L-phenylalaninol dienaraine; b)
adding water the reaction mixture at the completion of the reaction between
gossypol and L-phenylalaninol to obtain crystalline R-gossypol L-
phenylalaninol dienamine; and c) isolating the crystalline R-gossypol L-
phenylalaninol dienamine substantially free from the S-gossypol L-
phenylalaninol dienamine. In a further embodiment, the reaction mixture is
heated to about 50°C prior to the addition of water. In a further embodiment,
the reaction mixture is cooled to about 5°C after the addition of water. In a
further embodiment, crystalline R-gossypol L-phenylalaninol dienamine is
isolated by filtration or centrifugation
[0015] The term "gossypol," as used herein, refers to a composition
comprising the compound of the structure:

or a tautomer thereof.
[0016] Gossypol may be obtained from any one of a number of sources such
as the cotton plant, e.g., from cottonseeds or other plant parts. Species of
cotton vary widely both in the total content of gossypol and the optical activity
of the gossypol. While many species of cotton contain gossypol that is about
50% R-gossypol and 50% S-gossypol, several species have been identified
that contain either high levels of (S)-gossypol {e.g., Gossypium hirsutum, G.
arboreum, G. mustelinum, G. anomalum, G. gossypioides, G. capatis-viridis,
(up to about 97% (S)-gossypol)) or high levels of (R)-gossypol (e.g., G.
barbadense, G. darwinii, G. sturtianjm, G. areysianum, G. longicalyx, G
harknessii, G. costulatum (up to about 65% (R)-gossypol)). See, e.g., Hua et
al, Contraception 57:239 (1988); Cass et al, J. Agric. Food Chem. J2:5822
(2004); Stipanovic et al, J. Agric. Food Chem. 55:6266 (2005); Stipanovic et
al, 2005 Beltwide Cotton Conferences, New Orleans, Louisiana, January 4-7,
2005, p. 900. The enantiomeric ratio of gossypol also varies in different parts
of the cotton plant. For example, in G. barbadense the seeds typically have an
excess of (R)-gossypol, but the roots and flowers may contain an excess of
(S)-gossypol (Cass et ai, J. Agric. Food Chem. 52:5822 (2004)). Thus, a
strain of cotton or a part thereof that is high in (R)-gossypol may be
advantageously used as the source of the gossypol for the present invention.
[0017] In one embodiment, gossypol is approximately racemic, i.e., it contains
about 50% R-gossypol and about 50% S-gossypol. In another embodiment,
gossypol is enriched in R-gossypol. In another embodiment, gossypol is
enriched in S-gossypol.
[0018] The term "R-gossypol" or "R-(-)-gossypol," as used herein, refers to a
composition comprising the compound of structure:

^or a tautomer thereof.
[0020] The term "gossypol acetic acid," as used herein, refers to a composition
comprising gossypol and acetic acid in the form of co-crystals.
[0021] In one embodiment, crystallization of R-gossypol L-phenylalaninol
dienamine from a mixture of R- and S-gossypol L-phenylalaninol dienamine is
induced by seeding with crystalline R-gossypol L-phenylalaninol dienamine.
In a further embodiment, the reaction mixture is cooled to about 5°C or less to
facilitate crystallization.
[0022] In one embodiment, crystalline R-gossypol L-phenylalaninol
dienamine is hydrolyzed to provide crude R-gossypol or R-gossypol acetic
acid co-crystal. In a further embodiment, the hydrolysis of R-gossypol L-
phcnylalaninol dienamine is carried out at about 52°C. In a further
embodiment, the hydrolysis of R-gossypol L-phenylalaninol dienamine is
carried out in the absence of tetrahydrofuran. In a further embodiment, the
hydrolysis of R-gossypol L-phenylalaninol dienamine is carried out in the
presence of acetic acid, hydrochloric acid and water.
[0023] In one embodiment, R-gossypol or R-gossypol acetic acid co-crystals
are isolated by filtration or centrifugation. In a further embodiment, R-
gossypol or R-gossypol acetic acid co-crystals are washed with a solvent
system comprising acetic acid and water. In a further embodiment, R-
gossypol or R-gossypol acetic acid co-crystals are washed with water. In a
further embodiment, R-gossypol or R-gossypol acetic acid co-crystals are
dried in vacuo.
[0024] In one embodiment, R-gossypol acetic acid co-crystals are purified via
crystallization in one or more solvent systems until the desired level of purity
is achieved. In a further embodiment, R-gossypol acetic acid co-crystals are
isolated by filtration or centrifugation. In a further embodiment, R-gossypol
acetic acid co-crystals are washed with a solvent mixture. In a further
embodiment, R-gossypol acetic acid co-crystals are dried in vacuo.
[0025] In one embodiment, crystallization of R-gossypol acetic acid co-
crystals is carried out in a solvent system comprising ethyl acetate, ethanol,
acetic acid and water. In a further embodiment, R-gossypol acetic acid co-
crystals are washed with a solvent system comprising ethyl acetate, ethanol,
acetic acid and water. In a further embodiment, R-gossypol acetic acid co-
crystals are dried in vacuo.
[0026] In one embodiment, crystallization of R-gossypol acetic acid co-
crystals is carried out in a solvent system comprising acetone, acetic acid and
water.
[0027] In one embodiment, crystallization of R-gossypol acetic acid co-
crystals is carried out in a solvent system comprising acetic acid and water. In
a further embodiment, R-gossypol acetic acid co-crystals are washed with a
solvent system comprising acetic and water. In a further embodiment, R-
gossypol acetic acid co-crystals are dried in vacuo.
[0028] In one embodiment, R-gossypol acetic acid co-crystal is recrystallized
one or more times in one or more solvent systems until the desired level of
purity is achieved
[0029] In one embodiment, a process for preparing R-(-)-gossypol acetic acid
co-crystals is provided, the improvement comprising crystallizing a mixture of
R- and S-gossypol L-phenylalaninol dienamine from a solvent system
comprising acetonitrile and water and isolating crystalline R-gossypol L-
phenylalaninol dienamine substantially free from said S-gossypol L-
phenylalaninol dienamine; and preparing the R-(-)-gossypol acetic acid co-
crystals.
[0030] In one embodiment, a process for preparing R-(-)-gossypol acetic acid
co-crystals is provided, the improvement comprising: a) condensing gossypol
with L-phenylalaninol in acetonitrile to provide a mixture of R- and S-
gossypol L-phenylalaninol dienamine; b) adding water the reaction mixture at
the completion of a) to obtain crystallized R-gossypol L-phenylalaninol
dienamine; and c) isolating said crystalline R-gossypol L-phenylalaninol
dienamine substantially free from said S-gossypol L-phenylalaninol
dienamine; and preparing the R-(-)-gossypol acetic acid co-crystals.
[0031] The term "about," as used herein, includes the recited number ± 10%.
Thus, "about 10" means 9 to 11.
[0032] The term "purity," as used herein, refers to chemical and/or
stereoisomeric (i.e., diastereomeric or enantiomeric) purity, unless otherwise
indicated. The term "enantiomeric purity," as used herein, refers to a measure
of the enantiomeric excess or ee of a chiral substance. The term "enantiomeric
excess" or "ee" refers to a measure for how much of one enantiomer is present
compared to the other. For a mixture of R and S enantiomers, the percent
enantiomeric excess is defined as | R - S | *100, where R and S are the
respective mole or weight fractions of enantiomers in a mixture such that R +
S = 1. With knowledge of the optical rotation of a chiral substance, the
percent enantiomeric excess is defined as ([a]obj/[oOmax)*100, where [ot]0b, is
the optical rotation of the mixture of enantiomers and [ rotation of the pure enantiomer. The term "diastereomeric purity," as used
herein, is defined by analogy to enantiomeric purity. Determination of
diastereomeric or enantiomeric excess is possible using a variety of analytical
techniques, including NMR spectroscopy, chiral column chromatography or
optical polarimetry.
[0033] In one embodiment, R-(-)-gossypol acetic acid co-crystals have a
purity of about 95%, 96%, 97%, 98%, 99% or 99.5% or more.
[0034] The term "substantially free," as used herein, refers to a composition
comprising at least about 89% by weight of one stereoisomer (i.e., enantiomer
or diastereomer) over the corresponding stereoisomer. In another
embodiment, the composition comprises at least about 95%, 96%, 97%, 98%,
99% or 99.5% by weight of the preferred stereoisomer. Thus, the term "R-
gossypol L-phenylalaninol dienamine substantially free from S-gossypol L-
phenylalaninol dienamine," as used herein, refers to a composition of R-
gossypol L-phenylalaninol dienamine comprising at least about 89% of R-
gossypol L-phenylalaninol dienamine and at most about 11% of S-gossypol L-
phenylalaninol dienamine by weight.
[0035] In one embodiment, R-(-)-gossypol acetic acid co-crystal comprises
about 5%, 4%, 3%, 2%, 1% or 0.5% or less of (S)-(+)-gossypol.
[0036] The term "seeding," as used herein, refers to adding a small amount of
a pure compound to a solution to help initiate crystallization of that compound
from a solution. For example, adding crystalline R-gossypol L-phenylalaninol
dienamine to a solution of R-gossypol L-phenylalaninol dienamine and S-
gossypol L-phenylalaninoi dienamine in solvent system comprising
acetonitrile and water seeds the solution and initiates crystallization to provide
crystalline R-gossypol L-phenylalaninol dienamine.
[0037] The term "R-gossypol L-phenylalaninol dienamine" or "R-gossypol
L-phenylalaninol enamine-enamine," as used herein, refers to a composition
comprising the compound derived from R-gossypol of structure:
1 1
n
or a dienamine tautomer thereof.
[0039] The term "dienamine tautomer," as used herein, refers to all tautomcric
forms of the condensation products between L-phenylalaninol and gossypol
including, but not limited to, the bis oxazolidine tautomer 1, diimine (i.e.,
imine-imine) tautomer 2 and the imine-enamine tautomer 3.
[0040] In one embodiment, the invention provides a composition consisting
essentially of co-crystals of R-(-)-gossypol and acetic acid in a molar ratio of
about 1:1 with a purity of about 95% or more. In another embodiment, the
composition comprises about 96%, 97%, 98%, 99% or 99.5% or more of
R-(-)-gossypol acetic acid co-crystal.
[0041] The following examples are illustrative, but not limiting, of the
processes and methods of the present invention. Other suitable modifications
and adaptations of the variety of reaction conditions and parameters normally
encountered in synthetic organic and process chemistry and which are obvious
to those skilled in the art are within the spirit and scope of the invention.
[0042] L-Phenylalanine methyl ester hydrochloride (4.9 g) was dissolved in
water (25 mL) in a 100 mL round bottom flask and stirred at room
temperature. Sodium carbonate (3.1 g) in water (12.5 mL) was added in 3
minutes. The reaction mixture was stirred for 5 minutes at room temperature.
Dichloromethane (25 mL) was added and the reaction mixture was stirred 5
minutes. The aqueous solution was transferred to a 125 mL separating runnel.
The organic layer was collected. The aqueous layer was extracted twice with
12.5 mL of dichloromethane. The three organic extractions were combined,
dried with magnesium sulfate (6g), filtered to remove the magnesium sulfate
and concentrated to give L-phenylalanine methyl ester free base.
Step 2
[0043] In a 250 mL flask equipped with a thermometer and under nitrogen,
racemic gossypol acetic acid (5g) was suspended in dichloromethane (50 mL).
The free base of L-phenylalanine methyl ester from step 1 in dichloromethane
was added to the racemic gossypol acetic acid suspension over the course of 5
minutes. The reaction mixture was stirred at room temperature for 15 minutes
and magnesium sulfate (8.2 g) was added. Stirring was continued an
additional 17 h. The reaction mixture was filtered to remove magnesium
sulfate, concentrated and dried to give a diastereomeric mixture of R- and S-
gossypol L-phenylalanine diimine.
EXAMPLE 2
Crystallization study of R- and S-gossypol L-phenylalanine diimine
[0044] General protocol: In a 25 mL (one neck) round bottom flask, 0.2 to 0.6
g of a diastereomeric mixture of R- and S-gossypol L-phenylalanine diimine
was introduced under nitrogen atmosphere. Then, 1 mL of solvent was added
and the reaction mixture was heated at 50°C. The appropriate amount of
solvent was introduced to obtain a limpid solution. After two hours at room
temperature, a yellow precipitate that formed was filtered and the crystalline
product was analyzed by HPLC. Results are presented in Table 1. For
brevity, "R" denotes R-gossypol L-phenylalanine diimine and "S" denotes
S-gossypol L-phenylalanine diimine. DCM = dichloromethane; iPrOH =
isopropanol; MeOH = methanol; THF = tetrahydrofuran; TBME = /err-butyl
methyl ether; MeCN = acetonitrile.
(0046] Racemic gossypol acetic acid (0.276 g) was treated with
dichloromethane (1.05 mL) followed by L-phenylalaninol (0.1515 g) and
allowed to stir at 25°C for 17 h. The reaction mixture was dried with
magnesium sulfate, filtered and concentrated to dryness to give 0.352 g of R-
and S-gossypol-L-phenyalaninol dienamine as a brown solid.
EXAMPLE 4
Crystallization study of R- and S-gossypol phenylalaninol dienamine
(0047] General protocol: In a 25 mL (one neck) round bottom flask, 0.2 to 1.1
g of a diastereomeric mixture of R- and S-gossypol L-phenylalaninol
dienamine was introduced under nitrogen atmosphere. Then, 1 mL of solvent
was added and the reaction mixture was heated at 50°C. The appropriate
[0048] Thus, a solvent system comprising acetonitrile and water provided R-
gossypol L-phenylalaninol dienamine substantially free from S-gossypol L-
phenylalaninol dienamine with an R:S ratio of about 89:11. Other solvent
systems resulted in no enrichment of diastereomeric purity or no crystal
formation.
EXAMPLE 5
Crystallization studies in acetonitrile:water mixtures
[0049] General protocol: Referring to Table 4, a mixture of R- and S-
gossypol L-phenylalaninol dienamine (gossypol dienamine; amount) was
solublized in acetonitrile at 21°C - 25°C and water was added drop wise over
the period of time indicated in a) to achieve the desired acetonitrile:water
ratio. R-gossypol L-phenylalaninol dienamine crystallized and was filtered
through a sintered runnel after the period of time and temperature indicated in
b). The isolated crystalline R-gossypol L-phenylalaninol dienamine was
washed with the same ratio of solvent used for crystallization as indicated in c)
and dried in vacua.
[0052] Thus, a one pot synthesis/crystallization protocol provided an effective
means of isolating R-gossypol L-phenylalaninol dienamine substantially free
from S-gossypol L-phenylalaninol dienamine. Starting from about 80 g of
gossypol acetic acid, R-gossypol L-phenylalaninol dienamine was obtained in
83% yield and 99.2% purity.
EXAMPLE 7
Reproducibility of R-gossypol phenylalaninol dienamine crystallization
[0053] General protocol: Referring to Table 6, racemic gossypol acetic acid
(amount) and L-phenylalaninol (2.1 equiv.) were combined in acetonitrile at
30°C and stirred for the indicated period of time. In each experiment the
acetonitrilerwater ratio was about 5:1 and seeding was with crystalline
R-gossypol L-phenylalaninol dienamine at the temperature indicated. The
isolated crystalline R-gossypol L-phenylalaninol dienamine was washed with
about 5:1 acetonitrile:water. Purities are determined by HPLC.
[0054] Thus, crystallization of R-gossypol L-phenylalaninol dienamine from a
solvent system comprising about 5:1 acetonitrile:water was reproducible when
seeded with pure R-gossypol L-phenylalaninol dienamine. In each case, good
diastereomeric and chemical purity was obtained.
EXAMPLE 8
Large-scale preparation of R-gossypol-L-phenyalaninol dienamine
[0055] Racemic gossypol acetic acid (152.8g, 1.0 eq) and L-phenylalaninol
(83.9 g, 2.1 eq.) were dissolved in acetonitrile (1039 mL) and heated at 30°C
for 2.5 h under nitrogen. The mixture was then heated to 50°C and water (208
mL, 1.4 eq. w/w) was added. The solution was cooled to 37°C and seeded with
pure R-gossypol-L-phenyalaninol (0.518 g, 0.5% w/w). The mixture was
cooled to 5°C. The suspension was filtered, washed with acetonitrile:water
(5:1,208 mL) at 5°C and dried in vacuo at 30°C to give crystalline R-gossypol
L-phenyalaninol dienamine as a brownish orange solid (90.5 g, 87%). HPLC
analysis indicated a diastereomeric R.S ratio of 98.5.1.5.
[0056] If further purification is desired, R-gossypol L-phenyalaninol
dienamine (59.0 g) obtained according to the method of Example 8 (above) is
solublized in 5:1 acetonitrile:water (563 mL) at 60°C. The solution is cooled to
47°C, seeded with pure R-gossypol-L-phenyalaninol (0.087 g) and cooled to
5°C. The resulting suspension is filtered, washed twice with 5:1
acetonitrile:water (94 mL) at 5°C and dried in vacuo at 30°C to give crystalline
R-gossypol-L-phenyalaninol dienamine as a brownish orange solid (34.1 g,
73%). HPLC analysis indicated a purity of 99.2%.
[0058] R-gossypol L-phenylalaninol dienamine (100 g, 1.0 eq.) was dissolved
in glacial acetic acid (850 mL) at 52°C. Water (212.5 mL, 2.1 eq. w/w) was
added followed by concentrated HC1 (41.0 mL, 8 eq.). Once the mixture was
stirred at 52°C for 3.5 h, water (425 mL, 4.2 eq. w/w) was added slowly to the
mixture. The suspension was cooled to 20°C, filtered, washed with acetic
acid:water (1:1, 100 mL) and with water (100 mL), and dried in vacuo at 20°C
to give R-gossypol acetic acid co-crystals as a yellow solid (104.7 g). HPLC
purity is 97.23%.
(0059] R-gossypol acetic acid co-crystals (73.71 g, 1.0 eq) were dissolved in
ethyl acetate (287 mL) and ethanol (59 mL) at 20°C. The solution was filtered
and rinsed with ethyl acetate (96 mL), acetic acid (260 mL) and water (64
mL). The solution was concentrated in vacuo below 30°C to remove at least
80% of the total volume of ethyl acetate and ethanol. Acetic acid:water (1:1,
147 mL) was added and the suspension was stirred at 20°C for 30 minutes,
filtered and washed three times with acetic acid:water (1:1, 74 mL). R-
gossypol acetic acid co-crystals were obtained as a yellow solid (179.4 g) and
used directly in the next step.
[0060] If further purification is desired, R-gossypol acetic acid co-crystals
(73.71 g) obtained from the method of Example 9, step 2 (above), ethyl acetate
(383 mL), ethanol (59 mL) and acetic acid (260 mL) are stirred at 20°C.
Water (64 mL) is added and the suspension is concentrated in vacuo below
30°C to remove at least 80% of the total volume of ethyl acetate and ethanol.
Acetic acid:water (1:1, 147 mL) is added and the suspension is stirred at 20°C
for 30 minutes, filtered and washed with acetic acid:water (1:1, 74 mL) at
20°C. R-gossypol acetic acid co-crystals are obtained as a yellow solid (119.1
g)-
(0061] If further purification is desired, additional crystallizations can be
performed.
Step 3 - Solvent exchange
[0062] R-(-)-gossypol acetic acid co-crystals (73.71 g, 1.0 eq.) were
suspended in acetone (590 mL), treated with acetic acid (295 mL) and stirred
at 20°C. Water (295 mL) was added and the solution was concentrated in
vacuo at 80% of the volume of acetone. The suspension
was stirred at 20°C, filtered and washed with acetic acid:watcr (1:1, 100 mL).
R-gossypol acetic acid co-crystals were obtained as a yellow solid (80.0 g) and
used directly in the next step.
EXAMPLE 10
Preparation of R-gossypol acetic acid co-crystal
Scheme 5

[0063] R-gossypol acetic acid co-crystals (73.71 g, 1.0 eq.) were partially
dissolved in acetic acid (295 mL) and stirred at 20°C for 3 h at which time
water (295 mL) was added. The resulting suspension was filtered and washed
with acetic acidrwater (1:1,100 mL). The isolated material was dried in vacuo
at 20°C to give R-gossypol acetic acid co-crystals as a yellow solid (54.2 g,
73% yield, 98.82% purity).
(0064] If further purification is desired, additional crystallizations can be
performed.
[0065] The aforementioned Examples demonstrate the difficulty encountered
in developing a process for obtaining R-(-)-gossypol and its acetic acid co-
crystal. Two different diastereomeric gossypol derivatives were prepared in
an effort to separate by crystallization the two diastereomers. As illustrated in
Example 2, attempts to resolve a mixture of R- and S-gossypol L-
phenylalanine diimine by crystallization were not successful. In this study, a
variety of solvent systems were tested. Modest diastereomeric enrichment
was observed in some cases.
[0066] As illustrated in Example 4, it was unexpectedly discovered that R-
gossypol L-phenylalaninol dienamine could be isolated in high diastereomeric
purity from a mixture of R- and S-gossypol L-phenylalaninol dienamine via
crystallization in a solvent system comprising acetonitrile and water. All other
solvent systems yielded unsatisfactory results. Most solvent systems resulted
in no crystal formation. Based on this discovery, a reproducible one-pot
process to isolate crystalline R-gossypol L-phenylalaninol dienamine was
developed (Examples 5 to 7). This facilitated the large-scale preparation of R-
(-)-gossypol acetic acid co-crystals as demonstrated in Examples 8 to 10.
(0067] Having now fully described the invention, it will be understood by
those of skill in the art that the same can be performed within a wide and
equivalent range of conditions and other reaction parameters without affecting
the scope of the invention or any embodiment thereof. All patents, patent
applications and publications cited herein are fully incorporated by reference
herein in their entirety.
WHAT IS CLAIMED IS:
1. A process for preparing R-gossypol L-phenylalaninol dienamine from
a mixture of R- and S-gossypol L-phenylalaninol dienamine comprising
crystallizing said mixture from a solvent system comprising acetonitrile and
water and isolating crystalline R-gossypol L-phenylalaninol dienamine
substantially free from said S-gossypol L-phenylalaninol dienamine.
2. The process of claim 1 wherein said crystalline R-gossypol
L-phenylalaninol dienamine is isolated by filtration.
3. The process of claim 1 wherein the acetonitrile.water ratio is about 5:1.
4. The process of claim 3 wherein the acetonitrilerwater ratio is 5:1.
5. The process of claim 1 further comprising:
a) washing said crystalline R-gossypol L-phenylalaninol
dienamine with a solvent system comprising acetonitrile and
water; and
b) drying said crystalline R-gossypol L-phenylalaninol dienamine
in vacuo.
6. A process for preparing R-gossypol L-phenylalaninol dienamine
comprising:
a) condensing gossypol with L-phenylalaninol in acetonitrile to
provide a mixture of R- and S-gossypol L-phenylalaninol
dienamine;
b) adding water to the reaction mixture at the completion of a) to
obtain crystallized R-gossypol L-phenylalaninol dienamine;
and
c) isolating said crystalline R-gossypol L-phenylalaninol
dienamine substantially free from said S-gossypol L-
phenylalaninol dienamine.
7. The process of claim 6 wherein said crystalline R-gossypol
L-phenylalaninol dienamine is isolated by filtration.
8. The process of claim 6 wherein the acetonitrile:water ratio in b) is
about 5:1.
9. The process of claim 8 wherein the acetonitrilerwater ratio is 5:1.
10. The process of claim 6 further comprising:
a) washing said crystalline R-gossypol L-phenylalaninol
dienamine with a solvent system comprising acetonitrile and
water; and
b) drying said crystalline R-gossypol L-phenylalaninol dienamine
in vacuo.
11. The process of claim 1 or 6 wherein said crystallization is induced by
seeding with crystalline R-gossypol L-phenylalaninol dienamine.
12. The process of claim 1 or 6 wherein said crystalline R-gossypol
L-phenylalaninol dienamine has a purity of about 90% or more.
13. The process of claim 12 wherein said purity is about 95% or more.
14. The process of claim 13 wherein said purity is about 98% or more.
15. The process of claim 12 wherein said purity is about 99% or more.
16. In a process for preparing R-(-)-gossypol acetic acid co-crystals, the
improvement comprising crystallizing a mixture of R- and S-gossypol L-
phenylalaninol dienamine from a solvent system comprising acetonitrile and
water and isolating crystalline R-gossypol L-phenylalaninol dienamine
substantially free from said S-gossypol L-phenylalaninol dienamine; and
preparing the R-(-)-gossypol acetic acid co-crystals.
17. The process of claim 16, further comprising isolating said crystalline
R-gossypol L-phenylalaninol dienamine by filtration.
18. The process of claim 16, wherein the acetonitrilerwater ratio is about
5:1.
19. The process of claim 18, wherein the acetonitrilerwater ratio is 5:1.
20. The process of claim 16, further comprising:
a) washing said crystalline R-gossypol L-phenylalaninol
dienamine with a solvent system comprising acetonitrile and
water; and
b) drying said crystalline R-gossypol L-phenylalaninol dienamine
in vacuo.
21. In a process for preparing R-(-)-gossypol acetic acid co-crystals, the
improvement comprising:
a) condensing gossypol with L-phenylalaninol in acetonitrile to
provide a mixture of R- and S-gossypol L-phenylalaninol
dienamine;
b) adding water to the reaction mixture at the completion of a) to
obtain crystallized R-gossypol L-phenylalaninol dienamine;
and
c) isolating said crystalline R-gossypol L-phenylalaninol
dienamine substantially free from said S-gossypol L-
phenylalaninol dienamine; and
preparing the R-(-)-gossypol acetic acid co-crystals.
22. The process of claim 21, further comprising isolating said crystalline
R-gossypol L-phenylalaninol dienamine by filtration.
23. The process of claim 21, wherein the acetonitrile:water ratio is about
5:1.
24. The process of claim 23, wherein the acetonitrile: water ratio is 5:1.
25. The process of claim 21, further comprising:
a) washing said crystalline R-gossypol L-phenylalaninol
dienamine with a solvent system comprising acetonitrile and
water; and
b) drying said crystalline R-gossypol L-phenylalaninol dienamine
in vacuo.


This invention relates to a process for preparing R-gossypol L-phenylalaninol dienamine using non-chromatographic
purification methods. In particular, the invention is directed to resolution of R- and S-gossypol L-phenylalaninol dienamine by crys-
tallization. R-gossypol L-phenylalaninol dienamine is a useful intermediate for the preparation of R-(-)-gossypol and R-(-)-gossypol
acetic acid co-crystal. R-(-)-Gossypol acetic acid and its co- crystal is useful for inducing apoptosis in cells and for sensitizing cells
to the induction of apoptotic cell death.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=pO3XZs1cpBFCZcZvbL5QXQ==&loc=wDBSZCsAt7zoiVrqcFJsRw==


Patent Number 279204
Indian Patent Application Number 1304/KOLNP/2010
PG Journal Number 03/2017
Publication Date 20-Jan-2017
Grant Date 13-Jan-2017
Date of Filing 13-Apr-2010
Name of Patentee ASCENTA THERAPEUTICS, INC.
Applicant Address 101 LINDENWOOD DRIVE, SUITE 405, MALVERN, PA 19355 UNITED STATES OF AMERICA
Inventors:
# Inventor's Name Inventor's Address
1 GUO MING 5810 ASTER MEDOWS PLACE, SAN DIEGO, CA 92130 UNITED STATES OF AMERICA
2 SHUGUANG ZHU 11353 SADDLE COVE LANE, SAN DIEGO, CA 92130 UNITED STATES OF AMERICA
3 NAVARRE LAURE BAT, L'ALEXANDRIN, 24 RUE DE LA CITE, F-69003 LYON FRANCE
4 PERION REGIS 123, RUE HOCHE, F-72000 LE MANS FRANCE
PCT International Classification Number A10N 35/00
PCT International Application Number PCT/US2008/011327
PCT International Filing date 2008-10-01
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/976,637 2007-10-01 U.S.A.
2 61/129,082 2008-06-04 U.S.A.