U.S. patent application number 12/012343 was filed with the patent office on 2008-10-02 for mycophenolate mofetil impurity.
Invention is credited to Amihai Eisenstadt, Janos Hajko, Beata Kosztya, Sandor Molnar, Tivadar Tamas.
Application Number | 20080241948 12/012343 |
Document ID | / |
Family ID | 34981904 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241948 |
Kind Code |
A1 |
Molnar; Sandor ; et
al. |
October 2, 2008 |
Mycophenolate mofetil impurity
Abstract
Provided is an impurity of mycophenolate mofetil, processes for
its preparation and its use as a reference.
Inventors: |
Molnar; Sandor; (Debrecen,
HU) ; Tamas; Tivadar; (Debrecen, HU) ; Hajko;
Janos; (Debrecen, HU) ; Kosztya; Beata;
(Debrecen, HU) ; Eisenstadt; Amihai;
(Ramat-Hasharon, IL) |
Correspondence
Address: |
KENYON & KENYON LLP
ONE BROADWAY
NEW YORK
NY
10004
US
|
Family ID: |
34981904 |
Appl. No.: |
12/012343 |
Filed: |
March 31, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11115593 |
Apr 26, 2005 |
7358247 |
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12012343 |
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60566056 |
Apr 27, 2004 |
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60572985 |
May 20, 2004 |
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60589400 |
Jul 19, 2004 |
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60638478 |
Dec 23, 2004 |
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60639151 |
Dec 22, 2004 |
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60642867 |
Jan 10, 2005 |
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60661485 |
Mar 15, 2005 |
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Current U.S.
Class: |
436/92 ;
544/87 |
Current CPC
Class: |
C07D 307/88 20130101;
A61P 43/00 20180101; A61P 31/00 20180101; Y10T 436/141111 20150115;
A61P 39/00 20180101; A61P 37/06 20180101 |
Class at
Publication: |
436/92 ;
544/87 |
International
Class: |
G01N 30/02 20060101
G01N030/02; C07D 265/30 20060101 C07D265/30 |
Claims
1. A process for the isolation of Compound 1 having the formula:
##STR00007## from a mixture comprising mycophenolate mofetil and
Compound 1 comprising: a) providing a solution comprising
mycophenolate mofetil and Compound 1 in a solvent; b) washing the
solution with an aqueous acidic solution to obtain a two-phase
system; c) separating the organic phase containing mycophenolate
mofetil from the aqueous acidic phase; d) adding an aqueous basic
solution to the aqueous acidic phase; and e) recovering Compound
1.
2. The process of claim 1, wherein the solvent in step a) is
selected from the group comprising: alkyl acetates, chlorinated
hydrocarbons and aromatic hydrocarbons.
3. The process of claim 1, wherein said solvent is isobutyl
acetate.
4. The process of claim 1, wherein the aqueous acid solution in
step b) contains either a mineral acid or an organic acid.
5. The process of claim 4, wherein said aqueous acid solution
contains any one of hydrochloric acid, sulfuric acid, phosphoric
acid, acetic acid, trifluoroacetic acid, methanesulfonic acid and
trifluoromethanesulfonic acid.
6. The process of claim 5, wherein said aqueous acid solution
contains acetic acid.
7. The process of claim 1, the aqueous basic solution added in step
d) contains either an inorganic or organic base.
8. The process of claim 7, wherein said aqueous basic solution
contains sodium bicarbonate.
9. Mycophenolate mofetil having an amount of Compound 1 having the
formula: ##STR00008## of about 0.01 to about 0.1% area by HPLC.
10. An HPLC method for quantifying the amount of Compound 1 having
the formula: ##STR00009## in mycophenolate mofetil comprising the
steps of: a) preparing a sample solution of the mycophenolate
mofetil sample in acetonitrile; b) injecting the sample solution
onto an about 250.0 mm.times.4.6 mm, 5 .mu.m C8 HPLC column; c)
gradient eluting the sample with a mixture of acetonitrile and
water, adding triethylamine and adjusting the pH to about 6 with
diluted phosphoric acid (A Eluent) and a B Eluent with a buffer:
acetonitrile ratio of about 15:85, according to the following
profile: TABLE-US-00004 HPLC Gradient Flow rate Time [mL/min] (min)
A Fluent (V/V %) B Eluent (V/V %) 1.5 0 100 0 1.5 15 100 0 1.5 37
28 72 1.5 37.1 100 0 1.5 40 100 0
d) measuring of the amounts of each impurity at about 250 nm
wavelength with a UV detector.
11. A method for analyzing a sample of mycophenolate mofetil
comprising the steps of: a) performing chromatography on the sample
to obtain data; and b) comparing the data with the chromatography
data of Compound 1 having the formula: ##STR00010##
12. The method of claim 11, wherein the method comprises the
following steps: (a) preparing a solution of mycophenolate mofetil
containing Compound 1; (b) subjecting the solution to a high
pressure liquid chromatography to obtain a chromatogram; and (c)
comparing a peak obtained in the chromatogram to a peak resulting
from the Compound 1.
13. The method of claim 11, wherein the method comprises the
following steps: (a) preparing a solution of mycophenolate mofetil
containing Compound 1; (b) subjecting the solution to thin layer
chromatography to obtain a chromatogram; and (c) comparing a band
or spot obtained in the chromatogram to a peak or band resulting
from the degradation product.
14. An HPLC method of assaying mycophenolate mofetil comprising the
steps of: a) preparing a sample solution of the mycophenolate
mofetil in acetonitrile; b) injecting the sample solution onto an
HPLC column; c) eluting the sample with a mixture of acetonitrile
and water; d) adding triethylamine, and adjusting the pH to about
6; and e) measuring of the amounts of each impurity with a detector
and appropriate recording device.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/115,593 filed Apr. 26, 2005, and claims the benefits of
U.S. Provisional Patent Application Nos. 60/566,056 filed Apr. 27,
2004, 60/572,985 filed May 20, 2004, 60/589,400 filed Jul. 19,
2004, 60/638,478 filed Dec. 23, 2004, 60/639,151 filed Dec. 22,
2004, 60/642,867 filed Jan. 10, 2005, 60/661,485 filed Mar. 15,
2005, the contents of all of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to 2-(4-morpholinyl)ethyl
(E)-6-(1,3-dihydro-4-[2-(4-morpholinyl)ethoxy]-6-methoxy-7-methyl-3-oxo-i-
sobenzofuran-5-yl)-4-methyl-hex-4-enoate (Compound 1), an impurity
of mycophenolate mofetil, a process for preparing and isolating
thereof, as well as its use as reference marker. The invention
further relates to mycophenolate mofetil having a low amount of
Compound 1, as well as the HPLC method for identifying thereof.
BACKGROUND OF THE INVENTION
[0003] Mycophenolic acid has the chemical name
6-[4-Hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl]-4-methyl-hex-4-enoic
acid,
6-[1,3-Dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-isobenzofuran-5-y-
l]-4-methyl-hex-4-enoic acid, molecular formula of
C.sub.17H.sub.20O.sub.6, molecular weight of 320.35, CAS Registry
number of 24280-93-1 and a structure of:
##STR00001##
[0004] Mycophenolic acid (MPA), isolated by Gosio in 1893, is the
first well-characterized antibiotic (Bentley 2001). It is produced
by several species of Penicillium, including P. brevi-compactum, P.
scabrum, P. nagem, P. roqueforti, P. patris-mei, and P. viridicatum
(Clutterbuck et al. 1932, Jens and Filtenborg 1983).
[0005] MPA, in addition to its antibiotic activity (Abraham 1945),
also has antifungal (Gilliver 1946), antiviral (Ando et al. 1968),
and antitumor properties (Noto et al. 1969), and has been used
clinically in the treatment of psoriasis (Johnson 1972). More
recently, it has been recognized as a powerful immunosuppressant
(Bentley 2000).
[0006] At least one reason for its pharmacological properties is
the fact that in several biological systems it interferes with
guanine biosynthesis at the level of inosine monophosphate
dehydrogenase (IMPD). It has, therefore, a pronounced inhibitory
effect on nucleic acid synthesis (Franklin and Cook 1969). The
inhibition of IMPD is also the basis of its lymphocyte-specific
immunosuppressive effect. Since lymphocytes primarily depend on de
novo guanine biosynthesis, the reduction of this pathway results in
suppression of T and B lymphocyte proliferation.
[0007] MPA was withdrawn due to its high incidence of side effects
(primarily infections such as herpes zoster and gastrointestinal
side effects such as stomach discomfort). The 2-morpholinoethyl
ester derivative, mycophenolate mofetil (CellCept.RTM.) does not
have these drawbacks, and has a better bioavailability than
mycophenolic acid. Mycophenolate mofetil was recently approved (in
the United States in 1995 and in Europe in 1996) for prophylaxis of
organ rejection in patients receiving allogeneic renal transplants
(Shaw and Nowak 1995, Sollinger 1995). After oral administration
the ester form rapidly hydrolyzes to free acid. MPA is then
converted mainly to an inactive glucuronide metabolite, which is
eliminated by urinary excretion (Bentley 2001, Wiwattanawongsa et
al. 2001).
[0008] Chemically, mycophenolate mofetil (abbreviated as MMF) is
2-(4-morpholinyl)ethyl
(E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-isobenzofuran-5-yl)-
-4-methyl-4-hexenoate, and its first synthesis was disclosed in
U.S. Pat. No. 4,753,935.
##STR00002##
[0009] Another patent, U.S. Pat. No. 5,543,408 discloses the
anhydrous crystalline salt form, monohydrate salt form, and
amorphous salt form of mycophenolate mofetil. These forms are
characterized by their melting points and/or Differential Scanning
Calorimetric (DSC) results and/or powder X-ray diffraction
pattern.
[0010] The product mixture of a reaction rarely is a single
compound pure enough to comply with pharmaceutical standards. Side
products and byproducts of the reaction and adjunct reagents used
in the reaction will, in most cases, be present. At certain stages
during processing of the mycophenolate mofetil contained in the
product mixture into an active pharmaceutical ingredient ("API"),
the mycophenolate mofetil must be analyzed for purity, typically by
HPLC or GC analysis, to determine if it is suitable for continued
processing or ultimately for use in a pharmaceutical product. The
mycophenolate mofetil does not need to be absolutely pure. Absolute
purity is a theoretical ideal that is unattainable. Rather, there
are purity standards intended to ensure that an API is not made
less safe for clinical use because of the presence of
impurities.
[0011] The U.S. Food and Drug Administration's Center for Drug
Evaluation and Research (CDER) has promulgated guidelines
recommending that drug applicants identify organic impurities of
0.1% or greater in the active ingredient. "Guideline on Impurities
in New Drug Substances," 61 Fed. Reg. 371 (1996); "Guidance for
Industry ANDAs: Impurities in Drug Substances," 64 Fed. Reg. 67917
(1999). Unless an impurity has been tested for safety, is in a
composition proven to be safe in clinical trials, or is a human
metabolite, the CDER further recommends that the drug applicant
reduce the amount of the impurity in the active ingredient to below
0.1%. Therefore, in order to study the pharmacology and toxicology
of such impurities, there is a need to isolate impurities in drug
substances.
[0012] In order to obtain marketing approval for a new drug
product, manufacturers must submit to the regulatory authority
evidence that the product is acceptable for administration to
humans. Such a submission must include, among other things,
analytical data showing the impurity profile of the product to
demonstrate that the impurities are either absent, or present in a
negligible amount. Therefore, there is a need for analytical
methods to detect impurities, and for reference standards to
identify and assay those impurities.
[0013] Generally, impurities (side products, byproducts, and
adjunct reagents) are identified spectroscopically and by other
physical methods and then the impurities are associated with a peak
position in a chromatogram (or a spot on a TLC plate). (Strobel p.
953) (Strobel, H. A.; Heineman, W. R., Chemical Instrumentation: A
Systematic Approach, 3rd dd. (Wiley & Sons: New York 1989)).
Thereafter, the impurity can be identified by its position in the
chromatogram, which is conventionally measured in minutes between
injection of the sample on the column and elution of the particular
component through the detector, known as the "retention time." This
time period varies daily based upon the condition of the
instrumentation and many other factors. To mitigate the effect that
such variations have upon accurate identification of an impurity,
practitioners use "relative retention time" ("RRT") to identify
impurities. (Strobel p. 922). The RRT of an impurity is its
retention time divided by the retention time of some reference
marker. In theory, mycophenolate mofetil itself could be used as
the reference marker, but as a practical matter it is present in
such overwhelming proportion in the mixture that it tends to
saturate the column, leading to irreproducible retention times,
i.e., the maximum of the peak corresponding to mycophenolate
mofetil tends to wander (Strobel FIG. 24.8(b) p. 879, contains an
illustration of the sort of asymmetric peak that is observed when a
column is overloaded). Thus, it is sometimes desirable to select an
alternative compound that is added to, or is present in, the
mixture in an amount significant enough to be detectable and
sufficiently low as not to saturate the column and to use that
compound as the reference marker.
[0014] Researchers and developers in drug manufacturing understand
that a compound in a relatively pure state can be used as a
"reference standard" (a "reference marker" is similar to a
reference standard but it is used for qualitative analysis) to
quantify the amount of the compound in an unknown mixture. When the
compound is used as an "external standard," a solution of a known
concentration of the compound is analyzed by the same technique as
the unknown mixture. (Strobel p. 924, Snyder p. 549) (Snyder, L.
R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography,
2nd ed. (John Wiley & Sons: New York 1979)). The amount of the
compound in the mixture can be determined by comparing the
magnitude of the detector response. See also U.S. Pat. No.
6,333,198, incorporated herein by reference.
[0015] The reference standard compound also can be used to quantify
the amount of another compound in the mixture if the "response
factor," which compensates for differences in the sensitivity of
the detector to the two compounds, has been predetermined. (Strobel
p. 894). For this purpose, the reference standard compound may be
added directly to the mixture, in which case it is called an
"internal standard." (Strobel p. 925, Snyder p. 552).
[0016] The reference standard compound can even be used as an
internal standard when the unknown mixture contains some of the
reference standard compound by using a technique called "standard
addition," wherein at least two samples are prepared by adding
known and differing amounts of the internal standard. (Strobel pp.
391-393, Snyder pp. 571, 572). The proportion of detector response
due to the reference standard compound that is originally in the
mixture can be determined by extrapolation of a plot of detector
response versus the amount of the reference standard compound that
was added to each of the samples to zero. (e.g. Strobel, FIG. 11.4
p. 392).
[0017] Esterification of MPA is known. (e.g. in Synthetic Organic
Chemistry by R. B. Wagner and H. D. Zook, Wiley, New York, 1956,
see pages 479-532). U.S. Pat. No. 4,753,935 first disclosed
mycophenolate mofetil. However, the synthetic process to prepare
the ester results in various impurities.
[0018] PHARMAEUROPA vol 15 No 4 Oct. 2003 published a list of
possible impurities of Mycophenolate Mofetil (from A to H). The
present invention relates to a new impurity whose presence was
observed in Mycophenolate Mofetil and which is not included in this
list. This impurity is useful as a reference standard in
preparation of highly pure mycophenolate mofetil.
SUMMARY OF THE INVENTION
[0019] In one aspect, the invention encompasses an 4-O-alkylated
impurity of mycophenolate mofetil 2-(4-morpholinyl)ethyl
(E)-6-(1,3-dihydro-4-[2-(4-morpholinyl)ethoxy]-6-methoxy-7-methyl-3-oxo-i-
sobenzofuran-5-yl)-4-methyl-hex-4-enoate, denominated Compound 1,
having the following chemical structure:
##STR00003##
Compound 1
[0020] Compound 1 has the following .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. (ppm): 1.73, 2.10, 2.21, 2.32, 2.39, 2.49,
2.74, 3.38, 3.60, 3.64, 3.69, 4.08, 4.26, 5.05, 5.09; .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. (ppm): 11.35, 16.15, 23.33, 32.78,
34.27, 53.60, 53.70, 56.88, 58.38, 60.75, 61.21, 66.66, 66.68,
68.11, 71.91, 112.41, 119.89, 123.57, 128.90, 133.51, 146.55,
155.32, 162.68, 168.78, 172.89; and MS (Da):[M+H].sup.+
547.29,[M+Na].sup.+ 569.27, [M+K].sup.+ 585.25, [2M+Na].sup.+
1115.61
[0021] In another aspect, the invention is directed to a process
for synthesizing Compound 1 by reaction of mycophenolate mofetil
with an N-ethylmorpholine derivative.
[0022] Compound 1 may also be obtained by isolation from a sample
of mycophenolate mofetil containing Compound 1 by a) providing a
solution of mycophenolate mofetil in a suitable solvent; b) washing
the mycophenolate mofetil solution with an aqueous acidic solution
to obtain a two-phase system; c) separating the organic phase
containing mycophenolate mofetil from the aqueous acidic phase; d)
adding an aqueous basic solution to the aqueous acidic phase; and
(e) recovering Compound 1.
[0023] The invention also provides a method for determining the
purity of mycophenolate mofetil comprising comparing the purity of
mycophenolate mofetil with Compound 1 as a reference standard,
particularly a reference marker.
[0024] In another aspect, the present invention provides for
mycophenolate mofetil having about 0.01 to about 0.1% of Compound
1% area by HPLC. Also provided is a pharmaceutical dosage form
comprising said mycophenolate mofetil, and methods of treatment of
a human who are at a risk of organ transplant rejection comprising
administering the pharmaceutical composition to the human in need
thereof.
[0025] In yet another aspect, the present invention provides an
HPLC method for the analysis and assay of mycophenolate
mofetil.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 is a representative .sup.1H NMR spectrum for Compound
1.
[0027] FIG. 2 is a representative for .sup.13C NMR spectrum for
Compound 1.
[0028] FIG. 3 is a representative .sup.13C NMR spectral data for
Compound 1.
[0029] FIG. 4 is a representative MS data for Compound 1.
[0030] FIG. 5 depicts a process for the synthesis Compound 1.
DETAILED DESCRIPTION OF THE INVENTION
[0031] As used herein, the term "reference standard" refers to a
compound that may be used both for quantitative and qualitative
analysis of an active pharmaceutical ingredient. For example, the
retention time of the compound in HPLC allows for setting a
relative retention time, thus making qualitative analysis possible.
The concentration of the compound in solution before injection into
an HPLC column allows for comparison of the areas under the peaks
in an HPLC chromatogram, thus making quantitative analysis
possible.
[0032] A "reference marker" is used in qualitative analysis to
identify components of a mixture based upon their position, e.g. in
a chromatogram or on a Thin Layer Chromatography (TLC) plate
(Strobel pp. 921, 922, 953). For this purpose, the compound does
not necessarily have to be added to the mixture if it is present in
the mixture. A "reference marker" is used only for qualitative
analysis, while a reference standard may be used for quantitative
or qualitative analysis, or both. Hence, a reference marker is a
subset of a reference standard, and is included within the
definition of a reference standard.
[0033] Although some of the knowledge of those in the art regarding
reference standards has been described in general terms up to this
point, those skilled in the art also understand that the detector
response can be, for example, the peak heights or integrated peak
areas of a chromatogram obtained, e.g. by UV or refractive index
detection, from the eluent of an HPLC system or, e.g. flame
ionization detection or thermal conductivity detection, from the
eluent of a gas chromatograph, or other detector response, e.g. the
UV absorbence, of spots on a fluorescent TLC plate. The position of
the reference standard may be used to calculate the relative
retention time for mycophenolate mofetil and other impurities.
[0034] The present invention provides an impurity of mycophenolate
mofetil (MMF), designated Compound 1. This impurity is useful as a
reference standard, more particularly a reference marker.
[0035] Compound 1, or 2-(4-morpholinyl)ethyl
(E)-6-(1,3-dihydro-4-[2-(4-morpholinyl)ethoxy]-6-methoxy-7-methyl-3-oxo-i-
sobenzofuran-5-yl)-4-methyl-hex-4-enoate, has the following
chemical structure:
##STR00004##
Compound 1 has the following .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. (ppm): 1.73, 2.10, 2.21, 2.32, 2.39, 2.49, 2.74, 3.38,
3.60, 3.64, 3.69, 4.08, 4.26, 5.05, 5.09; .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. (ppm): 11.35, 16.15, 23.33, 32.78, 34.27,
53.60, 53.70, 56.88, 58.38, 60.75, 61.21, 66.66, 66.68, 68.11,
71.91, 112.41, 119.89, 123.57, 128.90, 133.51, 146.55, 155.32,
162.68, 168.78, 172.89; and MS (Da):[M+H].sup.+ 547.29,[M+Na].sup.+
569.27, [M+K].sup.+ 585.25, [2M+Na].sup.+ 1115.61.
[0036] In another aspect, the invention encompasses a process for
synthesizing Compound 1. The structure of Compound 1 is determined
by structural analysis of both the synthesized compound and the
isolated compound from the preparation of mycophenolate mofetil. A
mycophenolate mofetil impurity prepared by an independent chemical
synthesis is indistinguishable from that isolated from the reaction
mixture containing mycophenolate mofetil. By increasing the
reaction time of the preparation of mycophenolate mofetil
significantly after the reaction is finished, it is possible to
receive relatively large amounts of this impurity.
[0037] Compound 1 may be synthesized by reacting mycophenolate
mofetil with an N-ethylmorpholine derivative.
[0038] This process comprises: a) combining mycophenolate mofetil
in an aprotic organic solvent with 4-(2-chloroethyl)-morpholine
hydrochloride in the presence of a base to obtain a mixture; b)
maintaining the mixture for at least 1 day to produce Compound 1,
c) extracting Compound 1 with a water-immiscible organic solvent;
and d) recovering Compound 1.
[0039] The aprotic organic solvent used in step a) may be
dichloromethane, tetrahydrofuran, or dimethylformamide, preferably
dimethylformamide. The base may be either an organic base (e.g.
triethylamine, imidazole) or an inorganic base (e.g. sodium
hydride, sodium or potassium carbonate), preferably potassium
carbonate. The water-immiscible organic solvent in step c) may be
dichloromethane, ethyl acetate, isobutyl acetate or toluene,
preferably toluene.
[0040] Recovery of Compound 1 may be performed by any method known
in the art, such as washing and drying the organic extracts,
followed by evaporation of the organic solvent.
[0041] In another aspect, the invention encompasses a process for
preparing Compound 1 comprising: a) converting
N-(2-hydroxyethyl)morpholine to N-(2-mesylethyl)morpholine and b)
combining mycophenolate mofetil with the N-(2-mesylethyl)
morpholine to form Compound 1.
[0042] Preferably, the first reaction, step a), is performed in the
presence of an organic base that is a C.sub.3-C.sub.9 alkyl amine,
such as triethylamine, and a solvent, such as toluene,
dichloroethane, or preferably, dichloromethane (DCM). More
preferably, step a) is performed in the presence of triethylamine
(Et.sub.3N) and DCM. This reaction may be performed by adding
mesylchloride, tosyl chloride (TsCl), or triflic anhydride.
Preferably, mesylchloride is added. The reaction mixture may be
cooled to about 0.degree. C., while the mesylchloride is added.
Preferably, the reaction mixture is stirred overnight and then
quenched with water.
[0043] The product of the first reaction, N-(2-mesylethyl)
morpholine, may be isolated by extraction with ethyl acetate,
toluene, or preferably, DCM. Isolation may also include washing,
drying, and/or concentrating the N-(2-mesylethyl) morpholine. For
example, the N-(2-mesylethyl) morpholine may be washed with brine,
dried over MgSO.sub.4, and concentrated under reduced pressure.
[0044] For the second reaction step, step b), mycophenolate mofetil
may be combined with a suitable solvent, preferably DMF, and a base
such as sodium hydride, e.g., 60% sodium hydride in mineral oil.
Preferably, the sodium hydride is added portionwise over a period
of 10 minutes to the mycophenolate mofetil. This second reaction
mixture may be stirred at room temperature for about 25 minutes.
Then, the N-(2-mesylethyl) morpholine may be added, preferably with
stirring at room temperature for about 24 hours. This reaction
mixture may be heated to about 25 to about 70.degree. C.,
preferably about 50.degree. C. For example, the mixture can be
heated to 50.degree. C. in an oil bath for about 14 hours. This
reaction mixture may also be cooled. For example, the mixture can
be allowed to cool to about room temperature.
[0045] The second reaction step yields Compound 1, which may be
isolated by extraction with a C.sub.3-C.sub.7 ester or ketone, such
as methyl ethyl ketone (MEK) or preferably, ethyl acetate. The
extraction may be preceded by dilution with water. The isolation
may also include washing, drying, and/or concentrating Compound 1.
For example, Compound 1 can be washed with brine, dried, and
concentrated under reduced pressure.
[0046] Compound 1 may also be purified, such as by column
chromatography. For example, the purification can be performed by
column chromatography on silica gel with elution with DCM:MeOH
(95:5).
[0047] In one aspect, the invention encompasses a method for the
isolation of Compound 1 from the reaction mixture obtained during
the preparation of mycophenolate mofetil. This method comprises: a)
providing a solution of mycophenolate mofetil in a suitable
solvent; b) washing the mycophenolate mofetil solution with an
aqueous acidic solution to obtain a two-phase system; c) separating
the organic phase containing mycophenolate mofetil from the aqueous
acidic phase; d) adding an aqueous basic solution to the aqueous
acidic phase; and e) recovering Compound 1.
[0048] The term "suitable solvent" refers to any organic solvent or
solvent mixture that is not miscible with water and in which the
reaction mass is soluble. Examples of suitable solvents include,
but are not limited to, alkyl acetates, chlorinated hydrocarbons
such as dichloromethane, chloroform, etc., and aromatic
hydrocarbons such as toluene. Preferably, isobutyl acetate is
used.
[0049] The acid used in the aqueous acid solution for washing may
be either a mineral acid or an organic acid. Examples of mineral
acid include, but are not limited to, at least one of hydrochloric
acid, sulfuric acid, or phosphoric acid. Examples of suitable
organic acids include, but are not limited to, at least one of
carboxylic acids such as acetic acid, or substituted carboxylic
acids such as trifluoroacetic acid, sulfonic acids such as
methanesulfonic acid, and substituted sulfonic acids such as
trifluoromethanesulfonic acid. Preferably, acetic acid is used. A
preferred pH range is about 3 to about 6, more preferably about 4
to about 5.
[0050] The base used in the aqueous base solution for washing may
be either an inorganic or organic base. Examples of suitable
inorganic bases include, but are not limited to, at least one of
carbonates, hydroxides, or hydrogen carbonates. A suitable organic
base may be, for example, triethylamine. Preferably, sodium
bicarbonate is used. A preferred pH range is about 8 to about
11.
[0051] Mycophenolate mofetil may be prepared from mycophenolate
acid according to any method known in the art, such as the
esterification method described in commonly-owned U.S. application
Ser. No. 11/______ [K&K ref.:2664/61005 filed Apr. 26, 2005].
This method comprises:
reacting a mycophenolic acid of formula:
##STR00005##
with a C.sub.1 to C.sub.4 alcohol or 4-(2-hydroxyethyl)morpholine
in the presence of a catalyst, to obtain an ester of mycophenolic
acid of formula:
##STR00006##
[0052] In another aspect, the invention encompasses mycophenolate
mofetil having an amount of Compound 1 of about 0.01 to about 0.1%
area by HPLC. This mycophenolate mofetil may be prepared by the
esterification of mycophenolic acid, according to the process
described above.
[0053] The amount of Compound 1 can be increased up to 10% (HPLC)
in the reaction mixture by changing the reaction conditions to
facilitate the isolation of Compound 1. HPLC analysis of reaction
mixtures obtained in forced reactions (such as higher temperature
and/or longer reaction time) produced results illustrated in Table
1.
TABLE-US-00001 TABLE 1 Com- HEM Temper- Reaction pound 1 Starting
(mol. SnCl.sub.2.cndot.2H.sub.2O ature time (HPLC material Equiv)
(mol %) (.degree. C.) (hours) a %) MPA 6 10 160 4 0.60 MPA 6 15 160
4 0.33 MPA 6 15 140 12 0.15 MPA 6 3 170 4 2.37 MPA 3 15 160 4 0.85
MPA 2 20 165 4 0.61 MPA 3 15 180 4 4.16 MPA 3 3 160 4 0.70 MPA 4 --
160 6 0.17 MPA 6.8 -- 160 107 6.8 MMF 3.8 -- 160 114 9.3 MPA-Me*
6.8 -- 160 107 5.9 *MPA-Me: mycophenolic acid methyl ester (methyl
mycophenolate)
[0054] Results from experiments with other catalysts are summarized
in Table 2.
TABLE-US-00002 TABLE 2 Com- HEM Temper- Reaction pound 1 Starting
(mol. Catalyst ature time (HPLC material equiv) (mol %) (.degree.
C.) (hours) a %) MPA 4 CSA (3) 150 8 0.24 MPA 6 PTSA (3) 190 6 0.63
MPA 4 PTSA (20) 165 4 0.78 MPA 6 FeCl.sub.3 (3) 190 6 5.7 MPA 6
CaCl.sub.2 (1) 160 6 1.20 MPA 4 K.sub.2S.sub.2O.sub.5 (10) 160 5
0.42 MPA 6 ZnSO.sub.4.cndot.7H.sub.2O (3) 165 4 0.27 MPA 4
KAl(SO.sub.4).sub.2.cndot.12H.sub.2O 165 8 1.50 (3) MPA 6 HCOOH
(400) 165 4 1.10 MPA 6 MgSO.sub.4 (3) 165 4 0.24 MPA 4
KH.sub.2PO.sub.4 (3) 165 3 0.30
[0055] Compound 1 may be used in a method for analyzing a sample of
mycophenolate mofetil by performing chromatography on the sample to
obtain data and comparing the data with the chromatography data of
Compound 1. The impurity used may or may not be with mycophenolate
mofetil, i.e., data may be generated for both the impurity and
mycophenolate mofetil simultaneously (as part of the same
solution/chromatogram) or separately. One of skill in the art may
prepare a solution of mycophenolate mofetil containing Compound 1,
subjecting the solution to a high pressure liquid chromatography to
obtain a chromatogram and comparing a peak obtained in the
chromatogram to a peak resulting from Compound 1. Further, one of
skill in the art may prepare a solution of mycophenolate mofetil
containing the compound 1, subject the solution to thin layer
chromatography to obtain a chromatogram and compare a band or spot
obtained in the chromatogram to a peak or band resulting from the
degradation product. The impurity may also be used to select
desirable batches with high purity at different stages during
production and manufacturing.
[0056] The present invention also provides a gradient elution HPLC
method for quantifying, by area percent, the amounts of all
impurities present in a sample of mycophenolate mofetil. The method
for determining the purity of mycophenolate mofetil includes the
steps of: (a) preparing a sample solution of the mycophenolate
mofetil in acetonitrile; (b) injecting the sample solution onto an
HPLC column, preferably a C8 column; (c) eluting the sample with a
solvent, preferably a mixture of acetonitrile and water; (d) adding
a base, such as triethylamine, and adjusting the pH to about 6; and
(e) measuring of the amounts of each impurity with a detector
(attached to an appropriate recording device).
[0057] Preferably, the method for determining the amount of
impurities in a mycophenolate mofetil sample comprises the steps
of: (a) preparing a sample solution of the mycophenolate mofetil
sample in acetonitrile; (b) injecting the sample solution (ca. 10
.mu.L) onto an about 250.0 mm.times.4.6 mm, 5 .mu.m C8 HPLC column;
(c) gradient eluting the sample with a mixture of acetonitrile
(about 350 mL) and water (about 650 mL); (d) adding about 2.0 mL of
triethylamine and adjusting the pH to about 6 (preferably 5.9) with
diluted phosphoric acid (A Eluent) and a B Eluent with a buffer:
acetonitrile ratio of about 15:85; and (e) measuring of the amounts
of each impurity at 250 nm wavelength with a UV detector (having an
appropriate recording device).
[0058] The buffer may be prepared by mixing about 1 L water with
about 3.0 mL of triethylamine and adjusted to pH 5.9 with diluted
phosphoric acid.
[0059] The HPLC profile for determining the purity of is
mycophenolate mofetil exemplified in Table 3.
TABLE-US-00003 TABLE 3 HPLC Gradient Flow rate Time [mL/min] (min)
A Eluent (V/V %) B Eluent (V/V %) 1.5 0 100 0 1.5 15 100 0 1.5 37
28 72 1.5 37.1 100 0 1.5 40 100 0
[0060] In the method described above, mycophenolate mofetil has a
retention time of about 20.8 minutes.
[0061] Another aspect of the invention encompasses a method for
determining the purity of mycophenolate mofetil comprising using
Compound 1 as a reference marker.
[0062] Pharmaceutical compositions may be prepared as medicaments
to be administered orally, parenterally, rectally, transdermally,
bucally, or nasally. Suitable forms for oral administration include
tablets, compressed or coated pills, dragees, sachets, hard or
gelatin capsules, sub-lingual tablets, syrups and suspensions.
Suitable forms of parenteral administration include an aqueous or
non-aqueous solution or emulsion, while for rectal administration
suitable forms for administration include suppositories with
hydrophilic or hydrophobic vehicle. For topical administration the
invention provides suitable transdermal delivery systems known in
the art, and for nasal delivery there are provided suitable aerosol
delivery systems known in the art.
[0063] Pharmaceutical compositions of the present invention contain
mycophenolate mofetil comprising Compound 1 in an amount of about
0.01 to about 0.1% area by HPLC. In addition to the active
ingredient(s), the pharmaceutical compositions of the present
invention may contain one or more excipients or adjuvants.
Selection of excipients and the amounts to use may be readily
determined by the formulation scientist based upon experience and
consideration of standard procedures and reference works in the
field.
[0064] Diluents increase the bulk of a solid pharmaceutical
composition and may make a pharmaceutical dosage form containing
the composition easier for the patient and care giver to handle.
Diluents for solid compositions include, for example,
microcrystalline cellulose (e.g. Avicel.RTM.), microfine cellulose,
lactose, starch, pregelitinized starch, calcium carbonate, calcium
sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium
phosphate dihydrate, tribasic calcium phosphate, kaolin, magnesium
carbonate, magnesium oxide, maltodextrin, mannitol,
polymethacrylates (e.g. Eudragit.RTM.), potassium chloride,
powdered cellulose, sodium chloride, sorbitol, and talc.
[0065] Solid pharmaceutical compositions that are compacted into a
dosage form, such as a tablet, may include excipients whose
functions include helping to bind the active ingredient and other
excipients together after compression. Binders for solid
pharmaceutical compositions include acacia, alginic acid, carbomer
(e.g. carbopol), carboxymethylcellulose sodium, dextrin, ethyl
cellulose, gelatin, guar gum, hydrogenated vegetable oil,
hydroxyethyl cellulose, hydroxypropyl cellulose (e.g. Klucel.RTM.),
hydroxypropyl methyl cellulose (e.g. Methocel.RTM.), liquid
glucose, magnesium aluminum silicate, maltodextrin,
methylcellulose, polymethacrylates, povidone (e.g. Kollidon.RTM.,
Plasdone.RTM.), pregelatinized starch, sodium alginate, and
starch.
[0066] The dissolution rate of a compacted solid pharmaceutical
composition in the patient's stomach may be increased by the
addition of a disintegrant to the composition. Disintegrants
include alginic acid, carboxymethylcellulose calcium,
carboxymethylcellulose sodium (e.g. Ac-Di-Sol.RTM.,
Primellose.RTM.), colloidal silicon dioxide, croscarmellose sodium,
crospovidone (e.g. Kollidon.RTM., Polyplasdone.RTM.), guar gum,
magnesium aluminum silicate, methyl cellulose, microcrystalline
cellulose, polacrilin potassium, powdered cellulose, pregelatinized
starch, sodium alginate, sodium starch glycolate (e.g.
Explotab.RTM.), and starch.
[0067] Glidants can be added to improve the flowability of a
non-compacted solid composition and to improve the accuracy of
dosing. Excipients that may function as glidants include colloidal
silicon dioxide, magnesium trisilicate, powdered cellulose, starch,
talc, and tribasic calcium phosphate.
[0068] When a dosage form such as a tablet is made by the
compaction of a powdered composition, the composition is subjected
to pressure from a punch and dye. Some excipients and active
ingredients have a tendency to adhere to the surfaces of the punch
and dye, which can cause the product to have pitting and other
surface irregularities. A lubricant can be added to the composition
to reduce adhesion and ease the release of the product from the
dye. Lubricants include magnesium stearate, calcium stearate,
glyceryl monostearate, glyceryl palmitostearate, hydrogenated
castor oil, hydrogenated vegetable oil, mineral oil, polyethylene
glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl
fumarate, stearic acid, talc, and zinc stearate.
[0069] Flavoring agents and flavor enhancers make the dosage form
more palatable to the patient. Common flavoring agents and flavor
enhancers for pharmaceutical products that may be included in the
composition of the present invention include maltol, vanillin,
ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol,
and tartaric acid.
[0070] Solid and liquid compositions may also be dyed using any
pharmaceutically acceptable colorant to improve their appearance
and/or facilitate patient identification of the product and unit
dosage level.
[0071] In liquid pharmaceutical compositions of the present
invention, the active ingredient and any other solid excipients are
suspended in a liquid carrier such as water, vegetable oil,
alcohol, polyethylene glycol, propylene glycol, or glycerin.
[0072] Liquid pharmaceutical compositions may contain emulsifying
agents to disperse uniformly throughout the composition an active
ingredient or other excipient that is not soluble in the liquid
carrier. Emulsifying agents that may be useful in liquid
compositions of the present invention include, for example,
gelatin, egg yolk, casein, cholesterol, acacia, tragacanth,
chondrus, pectin, methyl cellulose, carbomer, cetostearyl alcohol,
and cetyl alcohol.
[0073] Liquid pharmaceutical compositions of the present invention
may also contain a viscosity enhancing agent to improve the
mouth-feel of the product and/or coat the lining of the
gastrointestinal tract. Such agents include acacia, alginic acid
bentonite, carbomer, carboxymethylcellulose calcium or sodium,
cetostearyl alcohol, methyl cellulose, ethylcellulose, gelatin guar
gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl
methyl cellulose, maltodextrin, polyvinyl alcohol, povidone,
propylene carbonate, propylene glycol alginate, sodium alginate,
sodium starch glycolate, starch tragacanth, and xanthan gum.
[0074] Sweetening agents such as sorbitol, saccharin, sodium
saccharin, sucrose, aspartame, fructose, mannitol, and invert sugar
may be added to improve the taste.
[0075] Preservatives and chelating agents such as alcohol, sodium
benzoate, butylated hydroxy toluene, butylated hydroxyanisole, and
ethylenediamine tetraacetic acid may be added at levels safe for
ingestion to improve storage stability.
[0076] According to the present invention, a liquid composition may
also contain a buffer such as gluconic acid, lactic acid, citric
acid or acetic acid, sodium gluconate, sodium lactate, sodium
citrate, or sodium acetate.
[0077] The solid compositions of the present invention include
powders, granulates, aggregates and compacted compositions. The
dosages include dosages suitable for oral, buccal, rectal,
parenteral (including subcutaneous, intramuscular, and
intravenous), inhalant and ophthalmic administration. Although the
most suitable administration in any given case will depend on the
nature and severity of the condition being treated, the most
preferred route of the present invention is oral. The dosages may
be conveniently presented in unit dosage form and prepared by any
of the methods well-known in the pharmaceutical arts.
[0078] Dosage forms include solid dosage forms like tablets,
powders, capsules, suppositories, sachets, troches, and lozenges,
as well as liquid syrups, suspensions, and elixirs.
[0079] The dosage form of the present invention may be a capsule
containing the composition, preferably a powdered or granulated
solid composition of the invention, within either a hard or soft
shell. The shell may be made from gelatin and optionally contain a
plasticizer such as glycerin and sorbitol, and an opacifying agent
or colorant.
[0080] The active ingredient and excipients may be formulated into
compositions and dosage forms according to methods known in the
art.
[0081] A composition for tableting or capsule filling may be
prepared by wet granulation. In wet granulation, some or all of the
active ingredients and excipients in powder form are blended and
then further mixed in the presence of a liquid, typically water,
that causes the powders to clump into granules. The granulate is
screened and/or milled, dried, and then screened and/or milled to
the desired particle size. The granulate may then be tableted, or
other excipients may be added prior to tableting, such as a glidant
and/or a lubricant.
[0082] A tableting composition may be prepared conventionally by
dry blending. For example, the blended composition of the actives
and excipients may be compacted into a slug or a sheet and then
comminuted into compacted granules. The compacted granules may
subsequently be compressed into a tablet.
[0083] As an alternative to dry granulation, a blended composition
may be compressed directly into a compacted dosage form using
direct compression techniques. Direct compression produces a more
uniform tablet without granules. Excipients that are particularly
well suited for direct compression tableting include
microcrystalline cellulose, spray dried lactose, dicalcium
phosphate dihydrate, and colloidal silica. The proper use of these
and other excipients in direct compression tableting is known to
those in the art with experience and skill in particular
formulation challenges of direct compression tableting.
[0084] A capsule filling of the present invention may comprise any
of the aforementioned blends and granulates that were described
with reference to tableting, however, they are not subjected to a
final tableting step.
EXAMPLES
Example 1
Preparation of N-(2-mesylethyl)morpholine
[0085] A 150 ml one-necked round bottomed flask equipped with
magnetic bar and equipped with CaCl.sub.2 tube on its top, was
charged with 5.2 g N-(2-hydroxyethyl)morpholine, 25 ml DCM, and 5.5
ml triethylamine. The mixture was stirred and cooled in an
ice-water bath while 3.1 ml of mesylchloride was added over a
period of 5 min. The reaction mixture was stirred overnight and
then quenched in 20 ml water. The aqueous layer was extracted with
two 25 ml DCM portions, and the combined organic extracts were
washed with 20 ml of brine, dried over MgSO.sub.4, and concentrated
under reduced pressure to give 4.1 g of the crude mesylate which is
pure by TLC.
Example 2
Preparation of Compound 1
[0086] A 150 ml two-necked round bottomed flask with a magnetic
stirrer and a nitrogen gas inlet, was charged with 6.21 g
mycophenolate mofetil (14.3 mmol) and 20 ml anhydrous DMF. To the
stirred solution was added portion wise 570 mg sodium hydride (60%
in mineral oil) over a period of 10 min, and the resulting mixture
was stirred for 25 min at room temperature. To the reaction mixture
was added mesyl derivative, 2.92 g (14 mmol) in 4 ml DMF. The
reaction mixture was heated in a 50.degree. C. oil bath for 14 hrs,
and then allowed to cool to room temperature, diluted with 50 ml
water, and extracted with three 20 ml portions of ethyl acetate.
The combined organic extracts were washed with brine (20 ml),
dried, and concentrated under reduced pressure to give 7 g (89%)
pale viscous yellow liquid as a pure product (by TLC analysis).
Further purification can be conducted by column chromatography on
silica gel and elution with DCM/MeOH (95:5).
Example 3
Preparation of Compound 1
[0087] Combined aqueous acetic washings (5 l) derived from the
production of crude MMF were neutralized with solid sodium
bicarbonate. Isobutyl acetate (1 l) and charcoal (10 g) were added,
filtered, and the phases were separated. The organic phase was
evaporated to 300 ml. Water (300 ml) was added, and the pH was
adjusted to 4-4.5 using acetic acid. After separation of phases,
the aqueous phase was neutralized with solid sodium bicarbonate and
extracted with isobutyl acetate (200 ml). The organic phase was
dried on sodium sulfate and evaporated to dryness. The purity of
the residue (0.81 g brownish oil) was 77 a % of Compound 1.
[0088] The residue was chromatographed on a column of silica gel
(eluent: acetone) to produce a syrupy product, Compound 1 (560 mg,
purity: 95 a %).
Example 4
Preparation of Compound 1
[0089] MMF (26.04 g, 60 mmol) was stirred in DMF (40 ml). Potassium
carbonate (33.17 g, 4 equiv) and 4-(2-chloroethyl)morpholine
hydrochloride (14.53 g, 1.3 equiv) were added. The mixture was
stirred at room temperature for 3 days, diluted with water (400
ml), and extracted with toluene (2.times.300 ml). The combined
organic phase were washed with water (2.times.300 ml), dried on
sodium sulfate, and evaporated to dryness. The residue was Compound
1 as a yellowish oil (32.5 g, purity: 95 a %).
Example 5
Preparation of Pure Mycophenolate Mofetil
[0090] A mixture of mycophenolic acid (192 g, 0.6 mol) and
4-(2-hydroxyethyl)-morpholine (440 ml, 6 molar equivalents) was
stirred at 150-155.degree. C. for 4 hours in the presence of
tin(II) chloride dihydrate (20.4 g, 0.15 molar equivalents) under
nitrogen atmosphere. After the completion of the reaction, the
reaction mixture was allowed to cool to room temperature. The
obtained dark liquid was poured into isobutyl acetate (4.0 l). The
solution was extracted with 2% of aqueous sodium bicarbonate
solution (1.2 l, then 2.times.0.4 l). After the first addition of
sodium bicarbonate solution the formed two-phase system was treated
with charcoal (40 g) and filtrated (an emulsion was filtered off).
The solution was extracted with water (1 l). --After phase
separation the organic phase was washed with water (1 l) and
evaporated to dryness at 40-50.degree. C. under vacuum. To the
solid material acetone (400 ml) and isopropanol (3.8 l) were added
and the mixture was warmed to 40-45.degree. C. (the material was
dissolved). The solution was cooled to -5.degree. C. during 6 hours
and it was stirred at this temperature for 10-12 hours. After
filtration, the crystals were washed with 2:19 acetone/isopropanol
mixture (420 ml). The crude compound was dried in vacuum at
60.degree. C. The yield was 169-195 g (65-75%). HPLC impurity
profile: MPA=0.1%. Assay: 99.85%.
[0091] Having thus described the invention with reference to
particular preferred embodiments and illustrated it with Examples,
those in the art can appreciate modifications to the invention as
described and illustrated that do not depart from the spirit and
scope of the invention as disclosed in the specification. The
Examples are set forth to aid in understanding the invention but
are not intended to, and should not be construed to, limit its
scope in any way. The examples do not include detailed descriptions
of conventional methods. All references mentioned herein are
incorporated in their entirety.
* * * * *