SCALABLE PROCESS FOR THE PREPARATION OF A RAPAMYCIN 42-
ESTER FROM A RAPAMYCIN 42-ESTER BORONATE
BACKGROUND OF THE INVENTION
Rapamycin 42-esters are derivatives of rapamycin, a macrocyclic triene
antibiotic produced naturally by Streptomyces hygroscopicus. Rapamycin has been
found useful in an array of applications based on its antitumoral and
immunosuppressive effects. Such uses include preventing, inhibiting, or treating
transplant rejection, graft vs. host disease, autoimmune diseases including systemic
lupus erythematosis, inflammatory diseases including pulmonary and ocular
inflammation, adult T cell leukemia/lymphoma, solid tumors, fungal infections, and
hyperproliferative vascular disorders, including smooth muscle cell proliferation and
intimal thickening following vascular surgery. Rapamycin and rapamycin
derivatives, including rapamycin 42-esters such as rapamycin 42-ester with 3-
hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-779), continue to be
studied for treatment of these and other conditions.
The preparation and use of 42-esters of rapamycin, including CCI-779, are
described in US Patent No. 5,362,718. A regioselective synthesis of CCI-779 is
described in US Patent No. 6,277,983. In US Patent Publication No. US 2005-
0033046 A1 (also US Patent Application No. 10/903,062), a regioselective synthesis
of CCI-779 is described based on boronate chemistry.
What are needed are additional efficient methods of preparing a rapamycin 42-
ester, including a scalable method for purifying the rapamycin 42-ester.
SUMMARY OF THE INVENTION
This invention provides a scalable process for preparing and purifying a
rapamycin 42-ester, including rapamycin 42-ester with 3-hydroxy-2-
(liydroxymethyl)-2-methylpropionic acid (CCI-779), from a rapamycin 42-ester
boronate.
The preparation of a crude rapamycin 42-ester through transboronation of a
rapamycin 42-ester boronate with a diol is carried out in a solvent system in which

crystalline product is obtained. In one embodiment, the crude rapamycin 42-ester is
purified by further treatment with a diol to reduce undesirable by-product, followed
by recrystallization. In another embodiment, the crude rapamycin 42-ester produced
by transboronation is recrystallized first and then a slurry of the rapamycin 42-ester is
treated with a diol to provide purified rapamycin 42-ester.
Advantageously, the method of the invention avoids the use of tetrahydrofuran
(THF) as a solvent, which cannot be entirely removed during manufacturing and
results in an oily, sticky solid, making isolation of the rapamycin 42-ester difficult.
This invention also overcomes the isolation problem associated with the use of an
acetone solution of diol during the purification of rapamycin 42-esters, in which
residual solvents prevent crystallization in a desired fashion and cause stability and
processing problems, especially during scale-up.
Other aspects and advantages of the present invention over the prior art will be
readily apparent from the following detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a scalable process for the preparation of a
rapamycin 42-ester by reacting a rapamycin 42-ester boronate with a diol and
purifying the resulting crude rapamycin 42-ester by recrystallization and slurry with a
diol to yield final rapamycin 42-ester. In one embodiment, the crude rapamycin 42-
ester is first treated with a diol in solvent to form solid rapamycin 42-ester and then
recrystallized to form purified rapamycin 42-ester. In another embodiment, the crude
rapamycin 42-ester is first recrystallized to form a slurry of rapamycin 42-ester and
then treated with a diol to form purified rapamycin 42-ester.
The term "a rapamycin 42-ester" includes esters of the hydroxyl group at the
42-position of rapamycin, and ethers, amides, carbonates, carbamates, sulfonates,
oximes, hydrazones, and hydroxyamines of these rapamycin 42-esters in which
functional groups on the nucleus have been modified, for example through reduction
or oxidation, a metabolite of rapamycin such as various desmethylrapamycin or a ring
opened rapamycin (such as secorapamycin, described in US Patent No. 5,252,579).
The term rapamycin 42-esters also includes pharmaceutically acceptable salts of

rapamycin 42-esters, which are capable of forming such salts, either by virtue of
containing an acidic or basic moiety. As used herein, pharmaceutically acceptable
salts include, but are not limited to, hydrochloric, hydrobromic, hydroiodic,
hydrofluoric, sulfuric, citric, maleic, acetic, lactic, nicotinic, succinic, oxalic,
phosphoric, malonic, salicylic, phenylacetic, stearic, pyridine, ammonium, piperazine,
diethylamine, nicotinamide, formic, urea, sodium, potassium, calcium, magnesium,
zinc, lithium, cinnamic, methylamino, methanesulfonic, picric, tartaric, triethylamino,
dimethylamino, and tris(hydroxymethyl)aminomethane. Additional pharmaceutically
acceptable salts are known to those skilled in the art.
A variety of 42-esters of rapamycin are described in the following patents:
alkyl esters (US Patent No. 4,316,885); aminoalkyl esters (US Patent No. 4,650,803);
fluorinated esters (US Patent No. 5,100,883); amide esters (US Patent No. 5,118,677);
carbamate esters (US Patent No. 5,118,678); silyl ethers (US Patent No. 5,120,842);
aminoesters (US PatentNo. 5,130,307); acetals (US Patent No. 5,51,413);
aminodiesters (US Patent No. 5,162,333); sulfonate and sulfate esters (US PatentNo.
5,177,203); esters (US PatentNo. 5,221,670); alkoxyesters (US Patent No.
5,233,036); O-aryl, -alkyl, -alkenyl, and -alkynyl ethers (US PatentNo. 5,258,389);
carbonate esters (US PatentNo. 5,260,300); arylcarbonyl and alkoxycarbonyl
carbamates (US PatentNo. 5,262,423); carbamates (US PatentNo. 5,302,584);
hydroxyesters (US PatentNo. 5,362,718); hindered esters (US PatentNo. 5,385,908);
heterocyclic esters (US PatentNo. 5,385,909); gem-disubstituted esters (US Patent
No. 5,385,910); amino alkanoic esters (US PatentNo. 5,389,639);
phosphorylcarbamate esters (US PatentNo. 5,391,730); carbamate esters (US Patent
No. 5,411,967); carbamate esters (US PatentNo. 5,434,260); amidino carbamate
esters (US Patent No. 5,463,048); carbamate esters (US Patent No. 5,480,988);
carbamate esters (US PatentNo. 5,480,989); carbamate esters (US Patent No.
5,489,680); hindered N-oxide esters (US PatentNo. 5,491,231); biotin esters (US
PatentNo. 5,504,091); O-alkyl ethers (US PatentNo. 5,665,772); and PEG esters of
rapamycin (US Patent No. 5,780,462). In one embodiment, rapamycin 42-esters with
dicarboxylic acids, such as 42-hemisuccinate, 42-hemiglutarate and 42-hemiadipates
are selected.


In one embodiment, R1 is -C=O.CR7R7'R7", wherein:
R7, R7' and R7"are independently selected from hydrogen, alkyl of 1-6 carbon
atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -(CR12R13)fOR10, -
CF3,-F,or-CO2R10;
R10 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, triphenylmethyl, benzyl, alkoxymethyl of 2-7 carbon
atoms, chloroethyl, or tetrahydropyranyl;
R12 and R13 are each, independently, hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or -F;
and f= 0-6.
The compounds as described can contain one or more asymmetric centers and
can thus give rise to optical isomers and diastereomers. The compounds can include
optical isomers and diastereomers; racemic and resolved enantiomerically pure Rand
S stereoisomers; other mixtures of the R and S stereoisomers; and pharmaceutically
acceptable salts thereof.
The term "alkyl" is used herein to refer to both straight- and branched-chain
saturated aliphatic hydrocarbon groups. In one embodiment, an alkyl group has 1 to
about 8 carbon atoms (i.e., Ci, C2, C3, C4, C5 C6, C7, or Cs). In another embodiment,
an alkyl group has 1 to about 6 carbon atoms (i.e., Cj, C2, C3, C4, C5 or C6). In a
further embodiment, an alkyl group has 1 to about 4 carbon atoms (i.e., Ci, C2, C3, or
C4).
The term "alkenyl" is used herein to refer to both straight- and branched-chain
alkyl groups having one or more carbon-carbon double bonds. In one embodiment,

an alkenyl group contains 3 to about 8 carbon atoms (i.e., C3, C4, C5, C6, C7, or C8).
In another embodiment, an alkenyl groups has 1 or 2 carbon-carbon double bonds and
3 to about 6 carbon atoms (i.e., C3, C4, C5 or C6).
The term "alkynyl" group is used herein to refer to both straight- and
branched-chain alkyl groups having one or more carbon-carbon triple bonds. In one
embodiment, an alkynyl group has 3 to about 8 carbon atoms (i.e., C3, C4, C5, C6, C7,
or Cg). In another embodiment, an alkynyl group contains 1 or 2 carbon-carbon triple
bonds and 3 to about 6 carbon atoms (i.e., C3, C4, C5, or C6).
The term "alkoxy" as used herein refers to the O(alkyl) group, where the point
of attachment is through the oxygen-atom and the alkyl group can be substituted as
noted above.
In one embodiment, the rapamycin 42-ester is rapamycin 42-ester with 3-
hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-779) [US Patent No.
5,362,718], and 42-O-(2-hydroxy)ethyl rapamycin [US Patent No. 5,665,772]. When
drawn in terms of its stereochemistry, CCI-779 is characterized by the structure:

rapamycin 42-ester from rapamycin 42-ester boronate provides a yield of at least
82%, at least 85%, or at least 89% (corrected for strengths), with a strength of at least
95% or at least 98%, and total impurities of less than 4%, less than 2%, or preferably,
less than 1% total impurities. As used herein, these yields are corrected for strength
as follows: Yield (%) = [actual weight x strength (%)]/[theoretical weight x 100%].

In another embodiment, the rapamycin 42-ester is rapamycin 42-ester with 3-
hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-779). In one embodiment,
the total impurities contain less than 0.1% phenylboronic acid and preferably, less
than 0.05% phenylboronic acid.
The method of the invention, because it is scalable, can provide an amount of
a purified rapamycin 42-ester (i.e., as defined above by freedom from total impurities)
in excess of 20 kg. However, the invention is not so limited. The method is also
useful in obtaining amounts of as small as 20 kg, 1 kg, 200 g, 8 g, 5 g, or smaller.
According to the present invention, crude rapamycin 42-ester is prepared from
rapamycin 42-ester boronate. In one embodiment, the rapamycin 42-ester boronate
used has the formula:

wherein R is -O-C=O.CR7R8R9, wherein:
R7 is independently selected from the group consisting of hydrogen, alkyl of
1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -
(CR12R13)fOR10, -CF3, -F, or -CO2R10;
R10 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, triphenylmethyl, benzyl, alkoxymethyl of 2-7 carbon
atoms, chloroethyl, or tetrahydropyranyl;
R8 and R9 are taken together to form X;
X is a 2-phenyl-dioxoborinane;
R12 and R13 are each, independently, hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or -F;
and f= 0-6.

As used herein, the 2-phenyl-dioxoborinane may be optionally substituted
with 1,2, or 3 groups, which are independently selected from the group consisting of
hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7
carbon atoms, -(CR12R13)fOR10, -CF3, -F, or -CO2R10. In one embodiment, the
substituents are one, two or three methyl groups. The phenyl group of the 2-phenyl-
dioxoborinane may also be optionally substituted.
The term "substituted aryl" refers to an aryl group (e.g., a phenyl), which is
substituted with one or more substituents independently selected from including
halogen, CN, OH, NO2, amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, C1 to C3
perfluoroalkyl, C1 to C3 perfiuoroalkoxy, aryloxy, alkylcarbonyl, aryl, heteroaryl.
Desirably, a substituted aryl (e.g., phenyl) group is substituted with 1 to about 4
substituents. In one embodiment, the substituent is a halogen. In another
embodiment, the substituent is a lower alkyl.
In one embodiment, the 2-phenyl-dioxoborinane is selected from the group
consisting of 2-phenyl-4,6,6-trimethyl-l,3,2-dioxoborinane, 2-phenyl-1,3,2-
dioxaborinan-5-yl, 2-phenyl-1,3,2-dioxaborinan-4-yl, where the phenyl is optionally
substituted as defined above.
In a further embodiment, the rapamycin 42-ester boronate is a boronate of
rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-
779).
The rapamycin 42-ester boronate can be prepared for use hi the method of the
invention using previously described methods. For example, one suitable method for
preparing a CCI-779 boronate is described in US Patent Publication No. US 2005-
0033046 Al (also US Patent Application No. 10/903,062) and illustrated in scheme I.


Using similar techniques, one of skill in the art can readily utilize another 31-
trimethylsilyl ether, 42-ester boronate to prepare a desired 42-ester boronate.
Typically, the 31-trimethylsilyl ether group is removed. Thereafter, the resulting 42-
ester boronate may be purified by acetone slurry as illustrated below for CCI-779
boronate.


Given this information, one of skill in the art can readily prepare other rapamycin 42-
ester boronates.
The method of preparation of a rapamycin 42-ester via a rapamycin 42-ester
boronate intermediate is a route that completely eliminates chromatography from the
manufacturing process. The rapamycin 42-ester boronate is prepared from rapamycin
with about 80-85% conversion; however, isolated yields are significantly lower after
purification by acetone slurries. In preliminary experiments, about Vi of converted
materials was typically isolated.
In one embodiment, the present invention provides an improved method for
isolating crude rapamycin 42-ester boronate and an improved method for purifying
rapamycin 42-ester boronate for use in preparing a rapamycin 42-ester.
Although this method of isolating the crude rapamycin 42-ester boronate from mother
liquors is particularly well adapted for use in connection with the manufacturing
process described in US 2005/0033046, published February 10, 2005.
Briefly, this scalable process describes a method in which an organic
solvent(s) are used in the reactions which yield a rapamycin 42-ester boronate. Such
solvents may include diethyl ether, acetonitrile, ethyl acetate, THF, t-butyl methyl
ether and methylene chloride may be selected. However, acetone is often used to

form a concentrate. However, in certain embodiments, hydrolysis can be performed
using a single phase aqueous acid/organic solvent system. Thus, the selected organic
solvent (e.g., acetone) is mixed with a dilute inorganic acid such as, e.g., sulphuric,
hydrochloric or phosphoric acid. Examples of suitable dilute inorganic acid
concentrations range from about 0.1 N to about 3 N, about 0.2 N to about 2 N, or
about 0.5 N. Desirably, this step is carried out at a pH of 5 to 6. Optionally, a
suitable buffer, e.g., sodium acetate, or in the presence of sodium bicarbonate and/or
acetic acid are added to the mixture to adjust or maintain the pH in the desired range.
However, other methods for producing rapamycin 42-ester boronate may be utilized.
The method of isolating rapamycin 42-ester boronate permits the efficient
separation of crude rapamycin 42-ester boronate from mother liquors used for
production thereof. Such mother liquors typically contain the rapamycin 42-ester
boronate and contaminants such as one or more solvents and rapamycin, with the
largest single contaminant typically being rapamycin.
As indicated above, the method of the invention is particularly well suited for
use following the acetone slurries described above. However, in one aspect, the
reactors are washed with a suitable solvent and mixed with the mother liquors.
Optionally, the mother liquors may be obtained from a first acetone slurry, or from
mixtures of first and second acetone slurries, or from mixtures of first, second and
third acetone slurries. The mother liquors may be further optionally mixed with the
liquors resulting from the washing steps. Typically, the reaction wash is performed
with an ether, e.g., a diethyl ether, a trimethyl ether (e.g., tri-butyl methyl ether), or
the like may be selected.
Crude rapamycin 42-ester boronate is isolated from mother liquor by
concentrating the mother liquor and filtering. In one embodiment, the motiier liquor
is concentrated to a slurry prior to filtration. Typically, this results in a higher yield,
but lower quality product (e.g., about 70 to 80% w/w rapamycin 42-ester boronate,
with less than or about 15 to 20% w/w contaminating rapamycin). This slurry may
have a loss on drying in the range of about 15 to 30% as compared to the mother
liquors from the process of Scheme II. In another embodiment, the mother liquor is
concentrated to a thick slurry and diluted with an ether (e.g., diethyl ether) prior to

filtration. Typically, the resulting thick slurry has a loss on drying (LOD) in the range
of about 30 to about 40% as compared to the mother liquors from the first or second
acetone slurry where the process of Scheme II is utilized. This method results in a
higher quality product (e.g., less than or about 12 to 15% w/w rapamycin) as
compared to the use of the less concentrated slurry. In yet another embodiment, the
mother liquor is concentrated to a foam (i.e., a lower moisture content than the wet
mass resulting from concentrating to a thick slurry), and then treated with an ether.
This embodiment provides a high quality product (e.g., less than about 12% w/w,
preferably less than 11% w/w, most preferably, less than 10% w/w rapamycin.
In another aspect, the invention provides for the purification of rapamycin 42-
ester boronate from the isolated crude rapamycin 42-ester boronate. Desirably, the
purification reduces the contaminants in the isolated crude rapamycin 42-ester
boronate by at least about 10 fold. In one embodiment, the rapamycin 42-ester
boronate is purified such that it contains less than lwt % rapamycin, preferably, about
0.8 wt% rapamycin or less. In another embodiment, the purified rapamycin 42-ester
contains about 0.7 wt% rapamycin or less.
In one embodiment, the method for purifying a crude rapamycin 42-ester
boronate which comprises the rapamycin 42-ester boronate, rapamycin, and possibly
other contaminants (e.g., at least one solvent) involves heating a mixture comprising
the crude rapamycin 42-ester boronate and a suitable solvent. The solvent is typically
added in an amount in excess of the rapamycin 42-ester boronate, by weight. For
example, solvent may be added in an amount of about two, three, or four times the
weight of the rapamycin 42-ester boronate. Typically, the mixture is heated to reflux
and held for at least about one (1) hour. The temperature and the length of holding
time may be varied depending upon a variety of factors including, e.g., the solvent
selected and the length of holding time desired. In one embodiment, the solvent
selected is acetone and the heating temperature is about 55 to about 62 °C. However,
other ketones may be selected as solvents, e.g., methyl etfryl ketone. Still other
solvents may include an ether (e.g., ether, diethyl ether, tributylmethyl ether (TBME),
triethyl ether, etc.), acetonitrile, or mixtures thereof (e.g., acetone/ether in a ratio of
1:1 to 1:2).

Following mixing and heating, the mixture is cooled and stirred for at least
about 6 hours. Typically, the mixture is allowed to cool to room temperature over
time. However, it may be cooled more rapidly by suitable methods.
The mixture is concentrated to form a slurry. Following removal of at least
about one third to at least about one-half of the solvent (e.g., acetone), an ether is
added to the mixture, typically in about the amount of solvent removed (e.g., about 1
time to about 1.5 to about 2 times the weight of the rapamycin 42-ester boronate).
However, larger or smaller amounts of solvent may be added. Conventional methods
can be used to remove solvent, e.g. distillation under vacuum with or without heating.
In one desirable embodiment, the ether is diethyl ether. However, other suitable
ethers may be readily selected from among those described herein and well known to
those of skill in the art. The resulting slurry is thereafter filtered to recover the
purified rapamycin 42-ester boronate.
A first series of concentration/slurry, crystallizing, filtering steps results in
improved purity of the rapamycin 42-ester boronate. However, it may be necessary to
repeat the isolation procedure (e.g., distillation/slurry, crystallization, and filtration)
and/or the purification procedure (e.g., reflux, concentration, crystallization, and
filtration) once or twice in order to obtain a product having a desired purity, e.g., a
product having less than about 1% rapamycin. hi one embodiment, the product is
filtered to a loss on drying of less than about 5%, preferably, less than about 3%, or
less than or about 1% prior to repeating of the mixing with the solvent. Optionally,
the vessel in which a first purification run was performed can be washed with ethyl
acetate, and the resulting liquors subject to the isolation and/or purification methods
described herein.
The resulting purified rapamycin 42-ester boronate can be used in the
preparation of a rapamycin 42-ester as described herein.
Preparation of Rapamycin 42-Ester
The rapamycin 42-ester boronate is converted to a crude rapamycin 42-ester
utilizing a diol in a suitable solvent, i.e., transboronation. The transboronation
described herein may be accomplished for all of the rapamycin 42-ester boronate

compounds encompassed by the rapamycin 42-ester boronate formula, supra. One of
skill in the art will be able to readily modify the concentration of rapamycin 42-ester
boronate in solvent mixture, the ratio of diol to rapamycin 42-ester boronate, solvent
composition, reaction temperature, and desired reaction time, or other variable
described herein, based on the rapamycin 42-ester boronate utilized.
In one embodiment, the rapamycin 42-ester boronate is provided at from about
10% to about 30%, about 15% to about 30%, about 20% to about 30%, or about 25%
to about 30% by weight in solvent mixture. In a further embodiment, the rapamycin
42-ester boronate is provided at about 30% by weight Higher concentrations of
rapamycin 42-ester boronate, e.g., 40%, are not preferred as they may result in the
precipitation of sticky solids which can adhere to the wall of the reactor, reducing
yield. One of skill in the art would be able to select a preferred ratio based on the
solvent composition, reaction temperature, and desired reaction time.
The diol is provided in the reaction mixture at a molar ratio of diol to
rapamycin 42-ester boronate of from about 2 : 1 to about 10:1. In one embodiment,
the molar ratio of diol to rapamycin 42-ester boronate is at least 5:1. In a further
embodiment, the molar ratio of diol to rapamycin 42-ester boronate is about 5 : 1. hi
other embodiments, the molar ratio of diol to rapamycin 42-ester boronate is about 6:
1, about 7:1, about 8:1, about 9:1, about 10 :1, or any increment therein, e.g.,5.1:
1 or 8.5 :1. In general, increased amounts of diol promote reaction completion. One
of skill in the art would be able to select a preferred ratio based on the concentration
of rapamycin 42-ester boronate in solvent mixture, solvent composition, reaction
temperature, and desired reaction time.
A variety of 1,2-, 1,3-, 1,4- and 1,5-diols can be used to effect this
transboronation. Alkyl substituted diols are preferable such as 2-methyl-2,4-
pentanediol. In another embodiment, diethanolamine or solid-supported polystyrene
diethanolamine (PS-DEAM) may be utilized. Transboronation can also be achieved
using carboxylic acid reagents such as oxalic, malonic, tartaric, phthalic and salicylic
acid.
In one embodiment, the solvent mixture is composed of a mixture of ether and
heptanes. In a further embodiment, the ether is diethyl ether. However, the invention

is not so limited. In one embodiment, the solvent mixture contains ether and a single
heptane. In other embodiments, the solvent mixture contains ether and a mixture of
heptanes. In still other embodiments, solvents such as toluene, tert-butyl methyl ether
(TBME), ethyl ether, iPr2O, hexanes, cyclohexanes, dioxane, or mixtures including
these solvents may be used in place of heptanes. Where reference is made herein to
the use of heptanes, the use of these solvents or mixtures thereof is also contemplated.
As used herein, the term "heptanes" encompasses heptane and isomers thereof.
The term also encompasses heptane preparations composed predominantly of C7
isomers, with the remaining constituents primarily Cs isomers, e.g., EXXSOL®
Heptane Fluid (Exxon Mobil Chemical). However, the invention is not so limited.
Other heptane preparations useful in the invention, including commercially available
preparations, would be known to those of skill in the art and are encompassed by the
present invention.
The heptanes may be selected by one of skill in the art based on reaction
conditions. Further, the ratio of ether to heptanes may also be adjusted. The ratio of
ether to heptanes may range from about 1 : 1 to about 3 :1, or about 1 : 1 to about 2 :
1 by weight. While increased ratios of ether to heptanes promote reaction completion,
increased ratios, e.g., 4:1, result in the precipitation of sticky solids and are therefore
not preferred. In one embodiment, the ratio of ether to heptanes is about 2 : 1 by
weight. However, one of skill in the art would be able to select a preferred ratio based
on the ratio of diol to rapamycin 42-ester boronate, concentration of rapamycin 42-
ester boronate in solvent mixture, solvent composition, reaction temperature, and
desired reaction time.
The reaction may be carried out at temperatures ranging from about 20 °C to
about 40 °C, about 25 °C to about 40 °C, about 30 °C to about 40 °C, or about 35 °C to
about 40°C, where increased temperature generally promotes reaction completion. In
one embodiment, the temperature is about 30 °C to about 40 °C. In a further
embodiment, the temperature is about 34 °C to about 35 °C. However, one of skill in
the art would be able to select a preferred temperature based on the ratio of diol to
rapamycin 42-ester boronate, concentration of rapamycin 42-ester boronate in solvent
mixture, solvent composition, and desired reaction time.

Reaction completion may be monitored by conventional methods which are
known to those of skill in the art. Under preferred conditions, the reaction may be
completed efficiently within three to four hours. Following reaction completion, the
crude rapamycin 42-ester produced is cooled to about 20 to 25 °C and stirred in order
to avoid the formation of sticky or gummy solids. In one embodiment, the reaction
mixture is stirred for about 18 hours. In another embodiment, the reaction mixture is
stirred for about 38 hours. Following stirring of the reaction mixture at reduced
temperature, crude rapamycin 42-ester is precipitated by the addition of a non-polar
carbon-based solvent or a mixture thereof. In one embodiment, the non-polar carbon-
based solvent may be hexane, pentane, and heptane, and mixtures thereof. In a further
embodiment, the non-polar carbon-based solvent is heptanes. In one embodiment, the
ratio of non-polar carbon-based solvent to ether is about 3 : 1 by weight. In one
embodiment, the resulting precipitated crude rapamycin 42-ester is filtered and
washed with heptanes.
Purification of Rapamycin 42-Ester
Following precipitation, the crude rapamycin 42-ester is purified by a multi-
step process involving recrystallization and reaction of impurities, e.g. phenylboronic
acid, with a diol. The order of these steps is not a limitation of the invention, mother
words, in one embodiment, crude rapamycin 42-ester is first treated with a diol and
the solid rapamycin 42-ester is recrystallized. In another embodiment, the crude
rapamycin 42-ester is first recrystallized and then a slurry of the rapamycin 42-ester is
treated with a diol.
The purification process described herein may be accomplished for all of the
rapamycin 42-ester compounds encompassed by the rapamycin 42-ester formula,
supra. One of skill in the art will be able to readily modify the concentration of
rapamycin 42-ester in solvent, the ratio of diol to crude rapamycin 42-ester, solvent
composition, reaction temperature, desired reaction time, or other variable described
herein, based on the rapamycin 42-ester boronate utilized.
The amount of residual phenylboronic acid in crude rapamycin 42-ester
prepared on large scale, e.g., above 2 kg, from rapamycin 42-ester boronate is
between about 1.8 to 2.9 %. Previously described methods of purification by

recrystallization only reduced this amount by about half. Advantageously, the
purification method of the present invention reduces the total amount of
phenylboronic acid to less than 0.1%, and in some embodiments, less than 0.05%.
Purification of crude rapamycin 42-ester by Diol/ Recrystallization
In this embodiment, where purification by diol treatment is carried out before
recrystallization, the crude rapamycin 42-ester slurry produced via transboronation is
first dried of residual solvent.
The crude rapamycin 42-ester is treated with a diol in solvent. In one
embodiment, the solvent is an ether. In a further embodiment, the solvent is diethyl
ether. In one embodiment, the diol is 2-methyl-2,4-pentanediol. The molar ratio of
diol to rapamycin 42-ester may range from about 2 : 1 to about 10:1. In one
embodiment, the molar ratio of rapamycin 42-ester to diol is about 5:1. The reaction
is carried out at about 20 to 25°C. Higher temperatures, e.g., 34 °C, are not preferred
as they can lead to formation of sticky solids.
Reaction completion, e.g., the disappearance of phenylboronic acid, maybe
monitored by conventional methods which are known to those of skill in the art.
Under preferred conditions, the reaction may be completed efficiently within about
five hours. In a further embodiment, the reaction may be repeated in order to further
reduce the phenylboronic acid content. Following reaction completion, the partially
purified rapamycin 42-ester is dried to yield solid rapamycin 42-ester.
The solid partially purified rapamycin 42-ester is dissolved in a polar solvent.
In one embodiment, the solvent is acetone. In another embodiment, the solvent is
ethyl acetate. However, other polar solvents may be selected by one of skill in the art,
as well as appropriate ratios of solvent to crude rapamycin 42-ester. In one
embodiment, the ratio of solvent to crude rapamycin 42-ester is from about 5 : 1 to
about 8 :1 (by weight). In one embodiment, the solvent is acetone and the ratio of
acetone to crude rapamycin 42-ester is about 5 : 1 (by weight). Following dissolution
of the rapamycin 42-ester, the insoluble impurities are filtered off, and the filtrate is
concentrated to form a foam-.
The foam is then dissolved in ether and after a period of time purified
rapamycin 42-ester crystallizes out. In one embodiment, the ether is diethyl ether.

However, other ethers may be used in order to precipitate the rapamycin 42-ester. In
one embodiment, the ratio of rapamycin 42-ester to ether is from about 1 : 4 to about
1:3. In a further embodiment, the rapamycin 42-ester is about 29% to about 37%, or
about 29% to about 30%, by weight in ether.
Following ether recrystallization, the resulting slurry of rapamycin 42-ester in
ether may be further treated with heptanes. In one embodiment, the ratio of heptanes
to ether is about 3 :1 by weight. In another embodiment, following heptane treatment
and isolation of the purified rapamycin 42-ester by filtration, the rapamycin 42-ester
is washed with a solution of ether and heptanes prior to drying. In a further
embodiment, the ratio of ether to heptanes in the wash is 1 : 2 by volume. Following
washing, the resulting product is dried to yield the purified rapamycin 42-ester.
Purification of Crude Rapamycin 42-ester by Recrystallization / Diol Reaction
In another embodiment, the crude rapamycin 42-ester resulting from
transboronation is subjected to recrystallization first. Typically, the crude rapamycin
42-ester is filtered and dried by suction. The crude rapamycin 42-ester is then
dissolved in a polar solvent, e.g., acetone or ethyl acetate, any insoluble impurities
filtered, the filtrate dried to a foam and the resulting foam dissolved in polar solvent,
e.g., ether. The precipitate which appears after a period of time contains the
recrystallized partially purified rapamycin 42-ester.
A solution of diol in solvent is mixed with the partially purified rapamycin 42-
ester to form a slurry. In one embodiment, the solvent is an ether. In a further
embodiment, the solvent is diethyl ether. In one embodiment, the partially purified
rapamycin 42-ester is about 10% to about 30% by weight in solvent, or about 20% to
about 30% by weight in solvent. In a further embodiment, the partially purified
rapamycin 42-ester is about 30% by weight in solvent.
In one embodiment, the diol is 2-methyl-2,4-pentanediol. The molar ratio of
diol to rapamycin 42-ester may range from about 2 :1 to about 10:1. In one
embodiment, the molar ratio of rapamycin 42-ester to diol is about 5:1. The reaction
is carried out at about 20 to about 25°C. Higher temperatures, e.g., 34°C, are not
preferred as they can lead to formation of sticky solids

Reaction completion, e.g. the disappearance of phenylboronic acid, may be
monitored by conventional methods which are known to those of skill in the art.
Under preferred conditions, the reaction may be completed efficiently within about
five hours. In a further embodiment, the reaction may be repeated in order to further
reduce the phenylboronic acid content.
Following reaction completion, heptanes are added to the mixture, and the
resulting suspension is isolated by filtration and dried to yield crystalline purified
rapatnycin 42-ester.
In one embodiment CCI-779 produced according to the invention may be
further purified according to Process for the Preparation of Purified Crystalline CCI-
779 (Deshmukh, et al., US Patent Application No. 60/748,006, filed December 7,
2005, on the same date as the priority application in the United States Patent and
Trademark Office, and its corresponding US and international application), the
specification and claims of which are incorporated herein by reference. The
crystallinity of CCI-779 produced according to the invention may be determined
according to Method for the Measurement of Crystallinity of CCI-779 Using
Differential Scanning Calorimetry (Deshmukh, et al, US Patent Application No.
60/748,005, filed December 7,2005, on the same date as the priority application, and
its corresponding US and international applications), the specification and claims of
which are incorporated herein by reference.
The following examples are illustrative only and are not intended to
Examples 1-6 illustrate methods for isolating crude CCI-779 boronate from
mother liquor from a scalable process, such as that illustrated in the following
scheme.
Example 1: Recovery of Crude CCI-779 boronate from Mother Liquor
Isolation of crude CCI-779 boronate from the mother liquors was first
evaluated using partially recovered materials of a first batch from the manufacturing
process illustrated in Schemes 1 and 2 with different procedures as summarized in
Table 1.


As shown in entry I, crude CCI-779 boronate can be isolated by filtering the
slurry obtained from concentrating a mixture of the mother liquors from 2nd and 3rd
acetone slurries, followed by filtration. These mother liquors mainly consist of
solvents, CCI-779 boronate and rapamycin. The recovery is in the range of 95 ± 5%.
Isolated CCI-779 boronate contained about 8% of rapamycin. Due to the fact that the
mother liquor from the first acetone slurry contained much more impurities, three
different procedures were tested.
Entry II showed results from concentrating the mother liquor to a thick slurry,
followed by filtration. The recovery was in the range of 75 ± 5% but the filtration was

very slow. Crude CCI-779 boronate with about 18% rapamycin was obtained.
The second procedure (entry III) involved concentrating the mother liquor to a
foam first, treating with diethyl ether to afford a slurry, followed by filtration. Crude
CCI-779 boronate was isolated by filtration in 55 ± 5% yield with a rapamycin
content of 13%.
In entry IV, the mother liquor was concentrated to a thick slurry instead. After
diluting with diethyl ether, the crude CCI-779 boronate was filtered. Recovery is in
the range of 35 ± 5% with a rapamycin content of 11%.
Example 2: Purification of Crude CCI-boronate recovered from mother liquor
Table 2 summarizes results from the investigation on purification of crude
CCI-779 boronate recovered from mother liquors. The rapamycin content dropped
from 8.0 to 0.93% after 2 acetone slurries following the manufacturing procedure of
Scheme 1.
For experimental procedure 1, the mother liquors were heated at the specified
temperature for 1 h, stirred at room temperature for a minimum of 6 h, then filtered.
For experimental procedure 2A, the mother liquors were heated at the
specified temperature for 1 h, cooled to room temperature over 1 h, then stirred at
room temperature for a minimum of 6 h. This process was repeated 2 more times, and
the resulting slurry was then filtered.
For experimental procedure 2B, the mother liquors treated with the specified
solvent were heated at the specified temperature for 1 h, cooled to room temperature
over 1 h, then stirred at room temperature for 30 min. This process was repeated 2
more times and the resulting slurry filtered.
For experimental procedure 3, the mother liquors with the specified solvent
were heated at the specified temperature for 6 h, stirred at room temperature for a
minimum of 6 h, then filtered.

For experimental procedure 4, the mother liquors with the specified solvent
were stirred at room temperature for 24 h and filtered.

The rapamycin content was not reduced significantly by increasing reflux
and/or mixing times. Other solvents can also be used to purify the crude CCI-779
boronate, but not as efficiently as acetone. Ether can help remove some other
impurities from the crude CCI-779 boronate but is not an efficient solvent for removal
I of rapamycin. A mixture of acetone/ether could lower both the rapamycin content
and other impurities with a reasonable recovery. The rapamycin contents of close to
8% in the crude CCI-779 boronate could be reduced to 0.8% or less (specification =
0.8%) by 2 or 3 slurries.

Example 3: Isolation of Crude CCI-779 boronate from Mother Liquor
The mother liquors were concentrated and collected. The reactors were then
washed with ethyl acetate. After removing solvents, the materials obtained from ethyl
acetate rinses were combined with the concentrated mother liquors.
The combined mixtures were further concentrated to a thick slurry (LOD =
32%, 48% for materials from mother liquors of first, second acetone slurry,
respectively) before treatment with ether. Crude CCI-779 boronate was then isolated
by filtration.

Example 4: Purification of Crude CCI-779 boronate from Mother Liquor
A process to recover CCI-779 boronate was designed and examined using the
entire amounts of mother liquors generated from the first and second acetone slurries
during purification of a manufacturing batch. Isolation of the crude CCI779 was
carried out by concentrating the mother liquor to a thick slurry, followed by treatment
with diethyl ether as summarized in Table 4.
More than 2 Kg of crude CCI-779 boronate with a rapamycin content of 8.6%
was isolated from the mother liquor generated during first acetone slurry (Entry I).
About 900 g of the crude CCI-779 boronate from mother liquor of second acetone
slurry was obtained with a much lower rapamycin content (3.5%, Entry II). The
crude CCI-779 boronate was purified using a modified manufacturing procedure in
which a slurry of crude CCI-779 boronate in acetone is first heated at reflux similar to
the manufacturing procedure. After cooling to room temperature, half of the amount
of the acetone used in the slurry was replaced with diethyl ether. The resulting slurry

is stirred at room temperature for a period of times and filtered.
As shown in the following table, the crude CCI779 boronate from mother
liquor of the first slurry gave purified CCI-779 boronate in 59% yield with a
rapamycin content of 0.8% (within specification) after two such slurries (Entry I).
Recovery of the crude CCI-779 boronate from mother liquor of the second slurry was
higher (70%, Rapamycin % =0.7, Entry II). A total of nearly 2 kg of purified CCI-779
boronate could be recovered [2243/1239 x722g (from ML of first slurry) + 622 g
(from ML of second slurry) = 1929 g].

1 Procedure for purification: I. A slurry of crude CCI-779 boronate in acetone was stirred at
reflux for 1 h, then cooled to room temperature. About half amounts of acetone were removed under
vacuum. Same amounts of diethyl ether were added to thin the slurry. After stirring further for a
minimum of 6 h, purified CCI-779 boronate were collected by filtration, n. Repeated I.
2 HPLC Area%.
3 Overall yield for two purifications.
Example 5: Conversion of the recovered CCI-779 boronate into CCI-779
The recovered CCI-779 boronate (from mother liquor of first slurry) was
further converted into CCI-779 using the methods described herein. As shown in
Table 5, both the yield and quality of the CCI-779 isolated was comparable to those
when CCI-779 boronate from another source was used. "Total Im." refers to total
impurities. "LSI" refers to largest single impurity.


Example 6: Isolation and Purification of CCI-779 boronate from Mother
Liquors
A. Experiment A
The concentrated mother liquor (8.0 Kg) from the first acetone slurry
of the CCI-779 boronate manufacturing process and the ethyl acetate rinses (5.5 Kg)
were utilized. Solvents were removed from the ethyl acetate rinses under vacuum to
give 537 g of concentrate. The concentrate was transferred using 750 mL of acetone
into a 12-L multi-neck round-bottomed flask equipped with mechanical stirrer,
thermometer, and distillation apparatus. About half of the concentrated mother liquor
was then added to the flask. This was distilled under vacuum (28" Hg) at a pot
temperature of 20-25 °C until a thick slurry was obtained. The rest of the
concentrated mother liquor was added, then distilled to a thick slurry again (about 5
L). To the mixture thus obtained, 4.0 Kg of diethyl ether was added. The resulting
yellow slurry was stirred for 4 h at 20-25 °C and then filtered to give 2243 g of crude
CCI-779 boronate with a LOD of 3.84 % (75 °C/30"Hg, 2 h) and a rapamycin content
of 8.6%(HPLCarea%).
In a 12-L multi-neck round-bottomed flask equipped with mechanical
stirrer, thermometer, and distill apparatus, was charged 1239 g of crude CCI-779
boronate and 1859 g of acetone. The volume was marked, then another 1859 g of
acetone was added. The mixture was heated to reflux (56-59 °C) and held for 1 h.
The slurry was cooled to 20-25 °C over 2-3 h, then distilled to the marked volume
under vacuum (27" Hg) at a pot temperature of 20-25 °C. 1872 g of diethyl ether was

added, stirred at 20-25 °C for a minimum of 6 h, then filtered to give 865 g of crude
CCI-779 boronate with a rapamycin content of 2.1 %. In another 12 L multi-neck
round-bottomed flask equipped with mechanical stirrer, thermometer, and distillation
apparatus, was charged the entire amounts of the crude CCI-779 boronate and 1275 g
of acetone. After marking the volume, another 1275 g of acetone was added. The
mixture was heated with stirring at 56-59 °C for 1 h, cooled to 20-25 °C over 2-3 h,
then distilled to the marked volume under vacuum (27" Hg) at a pot temperature of
20-25 °C. 1275 g of diethyl ether was added, stirred for. a minimum of 6 h, then
filtered. After drying under vacuum (>28" Hg) at 40 °C for 5 h, 727 g of purified
CCI-779 boronate (LOD = 0.86) with a rapamycin content of 0.8 % (HPLC area %)
was obtained.
B. Experiment B
The mother liquor (221.3 Kg) from the first acetone slurry of the
manufacturing batch was distilled under vacuum (42-46 torr) at a pot temperature of
-5 to 25 °C until a thick slurry was obtained (about 25 L). To the mixture thus
obtained, 22.1 Kg of diethyl ether was added. The resulting yellow slurry was stirred
for 4 h at 19-25 °C and then filtered to give 10.1 kg of crude CCI-779 boronate. The
wet cake was combined with the mother liquor (107.4 Kg) from the second acetone
slurry of manufacturing batch and distilled under vacuum (22-45 torr) at a pot
temperature of-1 to -9 °C until a thick slurry was obtained. To the mixture thus
obtained, added 32.9 Kg of diethyl ether. The resulting yellow slurry was stirred for 4
h at 19-25 °C and then filtered. After wash twice using ether (20 kg each wash) to
give 10.0 kg of CCI-779 boronate with a LOD of 0.82 % (40 °C/30"Hg, 2 h).
Crude CCI-779 boronate (300 g) and acetone (900g) was charged into
a flask. The mixture was heated to reflux (55 to 61 °C) and held for 1 h. The
mixture was cooled to 20-25 °C over 2-3 h, then distilled to remove acetone under
vacuum (23-26" Hg) at a pot temperature not exceeding 10-18 °C (final volume 940-
960 mL). The diethyl ether (450 g) was added, stirred at 18-25 °C for 18 hours, and
then filtered to give crude CCI-779 boronate (225 g, LOD =0.95%). The filtered
crude CCI-779 boronate (224 g) and acetone (676 g) was charged into a flask. The
mixture was heated to reflux (55 to 61 °C) and held for 1 h. The mixture was cooled

to 19-25 °C over 2-3 h, then distilled to remove acetone under vacuum (18-20" Hg) at
a pot temperature of 18-20 °C (final volume 620 mL). The diethyl ether (338 g) was
added, stirred at 18-25 °C for 18 hours, and then filtered. After washing 3 times using
ether (250 mL each) to give CCI-779 boronate in 63% yield (LOD =0.57%, strength =
85.9%, total impurities = 1.8%, LSI = 0.50, rapamycin = 0.95%).
Examples 7-9 illustrate one embodiment of the method of the invention for
preparing purified CCI-779 from CCI-779 boronate, such as that illustrated in the
Scheme III.


Example 7 - Preparation of Purified CCI-779
A. Preparation of crude CCI-779
In a 3-L multi-neck round-bottomed flask equipped with mechanical
stirrer, thermometer, 500-mL pressure equalizing addition funnel, and reflux
condenser with a nitrogen head, was added 199 g (0.178 moles) of CCI-779 boronate,
a solution of 105 g of 2-methyl-2,4-pentanediol (0.891 moles) in 371 g of diethyl
ether and 185 g of heptanes. The mixture was heated to 33-37 °C with stirring under
nitrogen and held for 12 h. HPLC showed the consumption of starting CCI-779
boronate (<3%) and phenylboronic acid in the reaction mixture below the
specification of less than 3.0% or less than 1.5 %. Cooled the reaction mixture to 20-
25 °C over a 20 minutes, added 928 g of heptanes through the addition funnel over 1
h, then stirred at 20-25 °C for 1 h. The resulting mixture was filtered on a Buchner
funnel. The filtered solids were washed with two portions of 500 mL of heptanes.
After drying by suction until essentially no more filtrate is collected, 174 grams of
crude CCI-779 was isolated [Strength = 93.8%, total impurities =1.36%, rapamycin =
0.40% (HPLC); ether = 0.15%, heptanes = 0.26 %(GC)].
B. Purification of crude CCI-779
The crude CCI-779 obtained was transferred into a 2-L flask, then 1
L of acetone was added to obtain a hazy solution. The solution was clarified through a
glass Buchner funnel with a fine glass frit (4-5.5 (jm). The clear filtrate was then
charged to a 5-L, multi-neck flask equipped with mechanical stirrer, thermometer, and
vacuum distillation apparatus. The 2-L flask and Buchner funnel were washed with
400 mL of acetone. The acetone washes were added to the 5-L flask. After the
removal of acetone under a reduced pressure at 20-30 °C, the foam obtained was
dissolved in 420 g of diethyl ether at 20-25 °C to form a clear solution. Solids started
to precipitate after 20 minutes. A solution of 105.3 g of 2-methyl-2,4-pentanediol in
52 g of diethyl ether that had been filtered through a 0.45 um. syringe filter was
charged to the 5-L flask. The mixture was stirred for 1 h at 20-25 °C. HPLC analysis
showed that the amount of phenylboronic acid was less than 0.05 %. To the reaction
mixture, 1,272 g of heptanes were then added through the addition funnel over 2 h.
After stirring at 20-25 °C for 1 h, the mixture was filtered on a Buchner funnel. The

solids collected were washed with 3 portions of 500 mL of ether/heptanes (1/2, v/v),
and suction was maintained until dripping stopped. The wet cake was dried in a
vacuum oven at 50 °C under vacuum for 21 h to give 156 grams (89%, corrected for
strengths) of purified CCI-779 (CZ-7781-24-2) as white crystals [Strength = 98.4%,
total impurities =0.98%, rapamycin = 0.36%, PhB(OH)2 < 0.05% (HPLC); ether =
0.21%, heptanes = 0.037 %, 2-methyl-2,4-pentanediol = 0.096%, 2-methyl-2,4-
Dentanediol boronate = non detected CGCYI.

Example 8 - Preparation of Purified CCI-779
Some early pilot studies were performed, using essentially the method
described in Example 1, but with varying concentrations of CCI-779 boronate, CCI-
779 prepared by different routes, and different reaction times.

In the preceding table, process code 1 refers to: 27% (wt%) CCI-779 boronate
in 2/1 (wt/wt) ether/heptanes at 34-5 °C for 6-12 h, then add heptanes, filter.
Purification was by acetone/ether crystallization followed by treatment with 2-methyl-
2,4-pentanediol. Process code 2 refers to: 17% (wt%) CCI-779 boronate in 2/1
(wt/wt) ether/heptanes at 34-5 °C for 15-24 h, then add heptanes, filter. Purification
was by acetone/ether crystallization followed by treatment with 2-methyl-2,4-
pentanediol.
Example 9 - Preparation of Purified CCI-779, Alternate Route
Crude CCI-779 was prepared essentially as described above from CCI-779
boronate using 5 eq. of 2-methyl-2,4-pentanediol in a mixture of ether and heptanes
(2:1, Wt/Wt) at 34°C for 6 hours. The crude CCI-779 was isolated by filtration. The
crude CCI-779 was suspended hi ether, then treated with 5 eq. of 2-methyl-2,4-
pentanediol, following by addition of heptanes, filtration, and drying. The dried,
partially purified CCI-779 is then dissolved in acetone, clarified, and acetone
removed. Following this, ether is added to crystallize product, heptanes added to the
ether slurry, the slurry is filtered and dried. The resulting yield is 73% of CCI-779,
having a strength of 93.5%, total impurities of less than 4% (i.e., 3.59%), and
significantly, PhB(OH)2 of 0.007.
All documents identified herein are incorporated by reference. One of skill in
the art will recognize that minor modifications to the conditions and techniques
described in the specific embodiments described herein can be varied without
departing from the present invention. Such minor modifications and variants are
within the scope of the invention described herein and as defined by the following
claims.

CLAIMS:
1. A process for preparing a rapamycin 42-ester comprising the steps of:
(a) reacting a rapamycin 42-ester boronate with a diol in a solvent
mixture to provide crude rapamycin 42-ester; and
(b) purifying crude rapamycin 42-ester by recrystallization and
treatment with a diol.

2. The process according to claim 1, wherein step (b) comprises treating
the crude rapamycin 42-ester with a diol in solvent to form solid rapamycin 42-ester
and then recrystallizing the solid to form purified rapamycin 42-ester.
3. The process according to claim 1, wherein step (b) comprises
recrystallizing the crude rapamycin 42-ester to form a slurry and then treating with a
diol to form purified rapamycin 42-ester.
4. The process according to any one of claims 1 to 3, wherein the
rapamycin 42-ester boronate in step (a) is from about 10% to about 30% by weight in
said solvent mixture.
5. The process according to claim 4, wherein the rapamycin 42-ester
boronate in step (a) is about 30% by weight in said solvent mixture.
6. The process according to any one of claims 1 to 5, wherein the molar
ratio of diol to rapamycin 42-ester boronate in step (a) is from about 5 : 1 to about
10:1.
7. The process according to claim 6, wherein the molar ratio of diol to
rapamycin 42-ester boronate in step (a) is about 5:1.

8. The process according to any one of claims 1 to 7, wherein the diol in
step (a) is 2-methyl-2,4-pentanediol.
9. The process according to any one of claims 1 to 8, wherein the solvent
mixture in step (a) comprises diethyl ether and heptane.
10. The process according to any one of claims 1 to 8, wherein the solvent
mixture in step (a) comprises diethyl ether and a mixture of heptanes.
11. The process according to claim 10, wherein the ratio of diethyl ether to
heptanes is from about 1 :1 to about 2 :1 by weight.
12. The process according to any one of claims 1 to 11, wherein the
reaction in step (a) is carried out at about 30 to 40°C.
13. The process according to claim 12, wherein the crude rapamycin 42-
ester of (a) is cooled to about 20 to 25°C.
14. The process according to any one of claims 1 to 13, wherein the crude
rapamycin 42-ester of (a) is precipitated by the addition of one or more non-polar
carbon-based solvents.
15. The process according to claim-14, wherein the one or more non-polar
carbon-based solvents are selected from the group consisting of hexane, pentane, and
heptane.
16. The process according to claims 14 or 15, wherein the solvent is a
mixture of heptanes.
17. The process according to claim 16, wherein the ratio of heptane(s) to
the solvent mixture of step (a) is about 3 : 1 by weight.

18. The process according to any one of claims 1 to i 7, wherein the
recrystallization in step (b) comprises the steps of:
(I) dissolving the crude rapamycin 42-ester in a polar solvent; and
(II) filtering the resulting suspension and concentrating the crude
rapamycin 42-ester filtrate to yield a foam.

19. The process according to claim 18, wherein the polar solvent is
acetone.
20. The process according to claim 18, wherein the polar solvent is ethyl
acetate.
21. The process according to claim 19, wherein the ratio of acetone to
crude rapamycin 42-ester is about 5:1.
22. The process according to any one of claims 18 to 21, further
comprising dissolving the foam of step (II) in ether.
23. The process according to claim 22, wherein the ether is diethyl ether.
24. The process according to claims 22 or 23, wherein the ratio of
rapamycin 42-ester to ether is from about 1 : 4 to about 1:3.
25. The process according to any one of claims 22 to 24, further
comprising adding heptanes.
26. The process according to claim 25, wherein the ratio of heptanes to
ether is 3 : 1 by weight.

27. The process according to claims 25 or 26, further comprising filtering
the suspension formed and washing the precipitated crude rapamycin 42-ester with
ether and one or more heptanes.
28. The process according to claim 27, wherein the ratio of ether to
heptanes is 1 : 2 by volume.
29. The process according to claims 27 or 28, further comprising drying
the isolated rapamycin 42-ester.
30. The process according to any one of claims 1 to 29, wherein the
treatment with a diol in step (b) comprises the steps of:

(I) mixing the crude rapamycin 42-ester in solvent; and
(II) reacting the crude rapamycin 42-ester with diol.

31. The process according to claim 30, wherein the solvent in step (I) is
diethyl ether.
32. The process according to claims 30 or 31, wherein the rapamycin 42-
ester in step (I) is about 30% by weight in solvent.
33. The process according to any one of claims 30 to 32, wherein the diol
in step (II) is 2-methyI-2,4-pentanediol.
34. The process according to any one of claims 30 to 33, wherein the
molar ratio of diol to rapamycin 42-ester in step (II) is about 5:1.
35. The process according to any one of claims 30 to 34, wherein the
reaction in step (II) is carried out at 20 to 25°C.

36. The process according to any one of claims 30 to 35, wherein the crude
rapamycin 42-ester of step (a) is first dried of residual solvent.
37. The process according to any one of claims 1 to 36, wherein the
rapamycin 42-ester boronate has the formula:

wherein R is -O-C=O.CR7R8R9, wherein:
R7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, -(CR12R13)fOR10, -CF3, -F, or -CO2R10;
R10 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, triphenyhnethyl, benzyl, alkoxymethyl of 2-7 carbon
atoms, chloroethyl, or tetrahydropyranyl;
R8 and R9 are taken together to form X;
X is a 2-phenyl-dioxoborinane or a 2-(substituted)phenyl-
(substituted)dioxoborinane;
R12and R13 are each, independently, hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or -F;
and f = 0-6.
3 8. The process according to claim 3 7, wherein the 2-phenyl-
dioxoborinane is selected from the group consisting of 2-phenyl-4,6,6-trimethyl-1,3,2-
dioxoborinane, 2-phenyl-1,3,2-dioxaborinan-5-yl, 2-phenyl-1,3,2-dioxaborinan-4-yl,
wherein the phenyl may be optionally substituted
39. The process according to claim 37 wherein the rapamycin 42-ester
boronate is a CCI-779 boronate.

40. The purified rapamycin 42-ester prepared according to the process of
any one of claims 1 to 39.
41. The purified rapamycin 42-ester with 3 -hydroxy-2-(hydroxymethyl)-2-
methylpropionic acid (CCI-779) prepared according to the process of claim 39.
42. A purified rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-
methylpropionic acid (CCI-779) having at least 96% purity.
43. The purified compound according to claim 42, wherein said compound
contains less than 1% of any single impurity.
44. The purified compound according to claims 42 or 43, wherein said
compound contains less than 0.05% phenylboronic acid.
45. A method of isolating crude rapamycin 42-ester boronate from mother
liquor comprising rapamycin 42-ester boronate, rapamycin and one or more solvents,
comprising:
concentrating the mother liquor to a slurry;
filtering the resulting concentrated mother liquor to,isolate the crude
rapamycin 42-ester boronate.
46. The method according to claim 45, wherein the one or more solvents
are selected from the group consisting of one or more of acetone, ethyl acetate, and
water.
47. The method according to claim 45, further comprising the step of
concentrating the mother liquor comprising acetone is concentrated to a foam and
treating the foam with diethyl ether prior to filtration.

48. The method according to claim 45, further comprising the step of
treating the second slurry with a solvent comprising acetone and concentrating the
mixture to form a third slurry prior to filtering.
49. The method according to claim 45, wherein the mother liquor is
concentrated to a slurry, and further comprising the step of diluting the slurry with
diethyl ether prior to filtering.
50. A method of purifying a crude rapamycin 42-ester boronate which
comprises the rapamycin 42-ester boronate, rapamycin, and at least one solvent, said
method comprising:

(a) heating a mixture comprising the crude rapamycin 42-ester
boronate and a solvent for at least about 1 hour;
(b) stirring the mixture of (a) for at least about 6 hours following
cooling;
(c) concentrating the rapamycin 42-ester boronate mixture to form a
concentrated slurry;
(d) treating the concentrated slurry with a suitable ether;
(e) purifying the rapamycin 42-ester boronate;
(f) optionally repeating steps (a) - (e).

51. The method according to claim 50, wherein the mixture of (a) is
allowed to cool to room temperature.
52. The method according to claim 50, wherein steps (a) - (e) are repeated
at least once.
53. The method according to claim 50, wherein steps (a) - (e) are
performed a total of three times.

54. The method according to claim 53, wherein following the repeat of
steps (a) - (e), the recovered rapamycin 42-ester boronate contains less than 1% by
weight rapamycin.
5 5. The method according to claim 50, wherein the concentrated slurry (c)
is formed by removing at least about one half of the solvent.
56. The method according to claim 50, further comprising the step of
purifying the rapamycin 42-ester boronate isolated in step (e) by washing with a
suitable ether.
57. The method according to claim 50, wherein the solvent in step (a) is
selected from the group consisting of acetone, methanol, acetonitrile, methyl ethyl
ketone, TBME, and ether, and mixtures thereof.
58. The method according to claim 50, wherein the solvent in step (a) is
acetone.
59. The method according to claim 50, wherein the heating step is
performed at a temperature range of about 55 to about 60 °C.
60. The method according to claim 50, wherein the suitable ether is diethyl
ether.
61. The method according to claim 50, wherein the rapamycin 42-ester
boronate is recovered by filtration of the crystallized product formed following
treatment with the suitable ether.
62. The method according to claim 50, wherein the rapamycin 42-ester
boronate has the formula:


wherein R is -O-C=O.CR7R8R9, wherein:
R7 is independently selected from the group consisting of hydrogen, alkyl of
1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -
(CR12R13)fOR10, -CF3, -F, or -CO2R10;
R10 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
alkynyl of 2-7 carbon atoms, triphenylmethyl, benzyl, alkoxymethyl of 2-7 carbon
atoms, chloroethyl, or tetrahydropyranyl;
R8 and R9 are taken together to form X;
X is a 2-phenyl-dioxoborinane;
R12 and R13 are each, independently, hydrogen, alkyl of 1-6 carbon atoms,
alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or -F;
and f = 0-6.
63. A method of purifying rapamycin 42-ester with 3-hydroxy-2-
(hydroxymethyl)-2-methylpropionic acid (CCI-779) boronate from mother liquors
comprising the CCI-779 and at least one solvent, said method comprising:
(a) heating to reflux a mixture of comprising crude CCI-779 obtained
from mother liquors and a solvent comprising acetone;
(b) allowing the mixture of (a) to cool to room temperature;
(c) stirring the cooled mixture at least about 6 hours;
(e) removing at least about half of the solvent to form a concentrated
CCI-779 boronate slurry;
(e) treating the concentrated slurry with diethyl ether;
(f) recovering the CCI-779 boronate;
(g) repeating steps (a) - (f) at least once.

64. The method according to claim 63, wherein the solvent is removed
under vacuum.
65. The method according to claim 63, wherein the isolated CCI-779
boronate is collected by filtration.
66. A CCI-779 boronate prepared according to the method of claim 63
having a rapamycin content of less than 1 % w/w.
67. The CCI-779 boronate according to claim 66, wherein the rapamycin
content is 0.8% w/w.
68. The CCI-779 boronate according to claim 66, wherein the rapamycin
content is 0.7% w/w.

A scalable process for the preparation of a rapamycin 42-ester by reacting a rapamycin 42-ester boronate with a diol
and purifying crude rapamycin 42-esler by recrystallization and treatment with a diol is provided. Also provided is a method for
isolating and purifying a rapamycin 42-ester boronate from mother liquors comprising acetone and rapamycin contaminants.